For revision of the English text, I am grateful to Carol Ann Pelli,. HonBSc ..... diseases including chronic lung diseases (Burnett et al., 1988, 1994; Palmgren et.
Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki
Matrix Metalloproteinases as Markers of Inflammation in Equine Chronic Obstructive Pulmonary Disease (COPD) With Special Reference to Gelatinases and Collagenases
Saara M. Raulo
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Veterinary Medicine, University of Helsinki, for public criticism in the Auditorium Maximum, Hämeentie 57, Helsinki on 7 April 2001 at 12 noon.
Helsinki 2001
SUPERVISED BY:
Päivi Maisi, DVM, PhD Department of Clinical Veterinary Sciences Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
REVIEWED BY:
Fiona M. Cunningham, DVM, PhD Department of Veterinary Basic Sciences, The Royal Veterinary College, North Mymms, Herfordshire, UK
and Veli-Matti Kähäri, MD, PhD Center for Biotechnology, University of Turku, Turku, Finland
OPPONENT:
Nungavarum S. Ramamurthy, DVM, PhD Department of Oral Biology & Pathology, School of Dental Medicine, State University of New York, Stony Brook, USA
Cover. (photo) Bronchoalveolar cells from a COPD horse expressing MMP- 8 mRNA.
ISBN 952-91-3304-9 (printed) ISBN 951-45-9904-7 (PDF) Gummerus Saarijärvi, 2001
This work is dedicated to honor the memory of my co-workers
Anna-Liisa Koivunen, DVM, and Juhani Hirvonen, DVM. And to the memory of Professor Markus Sandholm, DVM, PhD .
Contents
CONTENTS ACKNOWLEDGMENTS
9
ABBREVIATIONS
10
ABSTRACT
11
LIST OF ORIGINAL PUBLICATIONS
13
INTRODUCTION
14
1.
16
REVIEW OF THE LITERATURE
16
1.1. Equine COPD
1.1.1.
General aspects of equine COPD
16
1.1.2.
Clinical findings
16
1.1.3.
Cytology of respiratory secretions
17
1.1.4.
Inflammation
17
1.1.5.
Controlling inflammation in COPD
19
1.2. Degradation of extracellular matrix and basement membrane
19
1.3. Matrix Metalloproteinases
22
1.3.1.
Structure and function
22
1.3.2.
Interstitial collagenases
24
MMP-1 and MMP-8
24
MMP-13
25
Gelatinases
25
MMP-2
25
MMP-9
26
Stromelysins, matrilysins, metalloelastase
26
1.3.3.
1.3.4.
4
Contents
1.3.5.
Membrane-type matrix metalloproteinases
27
MMP-14
27
1.3.6.
Other MMPs
27
1.3.7.
Regulation of MMPs
27
28
1.3.7.1. Activation
1.3.7.1.1. Natural activators
28
Oxidative stress
28
Proteolysis
28
1.3.7.1.2. Chemical activators
28
28
APMA
29
1.3.7.2. Inhibition
1.3.7.2.1. Natural inhibitors
29
Alpha-2-macroglobulin
29
TIMPs
29
NGAL
29
1.3.7.2.2. Chemical inhibitors
29
EDTA
29
Tetracyclines and their chemically modified analogs
29
Other synthetic inhibitors
30
2. AIMS OF THE STUDY
31
3. MATERIALS AND METHODS
32
32
3.1. Horses
3.1.1. Challenge design
33
Miscellaneous challenges
33
Time-course study
34 5
Contents 34
3.2. Samples
3.2.1.
TELF
34
3.2.2.
BALF
35
BALF cells
35
3.2.3.
Blood neutrophils
35
3.2.4.
Serum
35
3.2.5.
Lung tissue
36 36
3.3. Assays
3.3.1.
Enzyme activity assays
36 36
3.3.1.1. Zymography
Gelatin and elastin zymography
36
APMA activation
37
EDTA inhibition
37
CMT-3 inhibition
37 38
3.3.1.2. Fluorometry
Determination of elastinolytic activity in TELF
38
Inhibition of elastinolytic activity
38
3.3.1.3. Type I collagen degradation
39
Immuno assays
39
3.3.2.1. Western blotting
39
3.3.2.
Specific MMP-antibodies
39
Immunoblotting
40
3.3.2.2. Immunocyto- and immunohistochemistry
3.3.3.
40 41
In situ hybridization
41
3.4. Statistical analysis
6
Contents
43
4. RESULTS
43
4.1. Sample collection
4.1.1.
TELF and BALF
43
4.1.2.
Blood neutrophil separation
43 44
4.2. Zymography
4.2.1.
Standardization
44
4.2.2.
Gelatinolytic and elastinolytic activity
45
4.2.2.1. TELF
45
4.2.2.2. BALF
45
Clinical samples
45
Miscellaneous challenges
45
Time-course study
47 47
4.2.2.3. Blood
Serum
47
Neutrophils and lymphocytes
47
4.2.3.
4.2.4.
Gelatinolytic and elastinolytic activities at different
47
molecular weight ranges
47
MMP-9 activation degree
48 50
4.3. Fluorometry
4.3.1. 4.3.2.
Standardization
50
Elastinolytic activity
50 50
4.4. Activation and inhibition
4.4.1.
Activation effects of APMA
50
4.4.2.
Inhibitory effects of EDTA
50
4.4.3.
Inhibitory effects of CMT-3
51
7
Contents
4.5. Type I collagen degradation
51
4.6. Correlation of neutrophils with other measured parameters
51
4.7. Western blotting
52
4.7.1.
MMP-9 and NGAL
52
4.7.2.
MMP-2 and MMP-14
52
4.7.3.
MMP-8
53
4.7.4.
MMP-13
53
4.7.5.
Effects of CMT-3 inhibition
53
4.8. Identification of cellular sources of MMPs
53
4.8.1
Immunocyto- and immunohistochemistry
53
4.8.2.
In situ hybridization
54
5.
DISCUSSION
56
6.
CONCLUSIONS
67
7.
REFERENCES
70
ORIGINAL ARTICLES
91
8
Acnowledgments ACNOWLEDGMENTS This study was carried out at the Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki from 1995 to 2000. Financial support provided by the Finnish Academy of Science, the Faculty of Veterinary Medicine, the Finnish Veterinary Foundation, and the Eemil Aaltonen Foundation is gratefully acknowledged. In 1992, Dr. Päivi Maisi and my co-worker Anna-Liisa Koivunen, DVM, founded the basis for the research on proteolysis in connection with equine COPD at the College of Veterinary Medicine (now the Faculty of Veterinary Medicine, University of Helsinki). Unfortunately, Anna-Liisa was not given the time to complete her work. I mourn her loss yet am most grateful for her efforts and for the fundamental work she conducted, without which this thesis would not have been carried out. I express my deep gratitude to my supervisor Päivi Maisi, DVM, PhD, for her enthusiasm for the subject, for her guidance, and for overcoming many human tragedies these past few years. My gratitude to Timo Sorsa, DDM, PhD, for scientific and technical expertise as well as much needed support. It requires a team to achieve results in the scientific world. My special appreciation to my co-workers: Martti Nevalainen, DVM, Emma Pirilä, BSc, Minna Rajamäki, DVM, Leena Räsänen, DVM, PhD, Kaiju Prik, MD, and Taina Tervahartiala, DDM. For technical support, my sincere thanks to Merja Ranta, Kirsi Kauppila, and Satu Sankari, DVM, PhD, for handling the samples, and to Juha Alhgren and Martti Attila, DF, PhD, for software assistance. Matti Järvinen, in addition to handling samples, is acknowledged for his contribution in solving technical problems with the software. Warm thanks to Arto Ketola for helping me with the statistical analyses. For revision of the English text, I am grateful to Carol Ann Pelli, HonBSc, from the Helsinki University Language Center. The Equine Research Center, Ypäjä, the Riding Police, Helsinki, and all the horse owners are thanked for providing their horses. In addition, my thanks to Dr. Bruce McGorum and his research group for providing material for study III. My appriciation to Vetcare and Orion Animal health for their kind sponsorship.
Saara Raulo
9
Abbreviations ABBREVIATIONS
APMA
4-aminophenylmercuric acetate
BALF
bronchoalveolar lavage fluid
BE
bronchiectasis
BM
MME
macrophage metalloelastase
MMP
matrix metalloproteinase
MT-MMP
membrane-type matrix metalloproteinase
basement membrane
MW
molecular weight
COPD
chronic obstructive pulmonary disease
NG
neutrophilic granulocyte
NGAL CMT
chemically modified tetracycline
neutrophil gelatinaseassociated lipocaline
NSAID DEPC
diethylpyrocarbonate
nonsteroidal anti-inflammatory drug
DIC
digoxigenin
PAF
platelet-activating factor
ECM
extracellular matrix
PAGE
EDTA
ethylenediaminetetraacetic acid
polyacrylamide gel electrophoresis
PBS
phosphate-buffered saline
FITC
fluorescein-isothiocyanate
PMN
polymorphonuclear leukocyte
HBSS
Hanks’ balanced salt solution
RS
respiratory secretion
SDS
sodium dodecyl sulfate
HDS
hay-dust suspension challenge
TELF
tracheal epithelial lining fluid
HS
hay-straw challenge
TC
tetracycline
Ig
immunoglobulin
Th
T-helper lymphocyte
IL
interleukin
TIMP
tissue inhibitor of matrix metalloproteinases
kDa
kilodalton TNF
tumor necrosis factor
LPS
lipopolysaccharide WBC
white blood cell count
10
Abstract ABSTRACT Chronic obstructive pulmonary disease (COPD) or heaves is a common condition in horses of northern countries. COPD is a chronic inflammatory condition of the lung due to a constant hypersensitivity reaction resulting from recurrent irritant inhalation. Clinically, repeated coughing, nasal discharge, and excess mucus with abundant neutrophils in respiratory secretions (RSs) are observed. Proteolytic processes with matrix metalloproteinase (MMP) participation have been connected to chronic pulmonary diseases. Thus, the aim of this thesis was to determine major MMP involvement in proteinase activities in the lungs of COPD horses to enhance understanding of proteolytic features in the pathogenesis of equine COPD. In addition, possible pharmacological inhibition of excess MMP activity was studied. Data indicate that tracheal epithelial lining fluid (TELF) is representative of the entire respiratory tract and, thus, can be used along with bronchoalveolar lavage as diagnostic material to evaluate proteinase activities in the lungs of horses. Measurements of serum, blood neutrophil, or blood lymphocyte gelatinolytic activity were of limited value in distinguishing horses with COPD from healthy horses. Therefore, analysis of gelatinolytic activity in blood yields little information regarding the proteolytic processes in the lungs of horses. Elastinolytic activity is detectable in TELF obtained from horses and seems to be attributable to metalloproteinases. This activity was significantly higher in TELF from horses with COPD than in TELF obtained from healthy horses, suggesting participation of elastases in the destruction of lung tissue of horses with COPD. Markedly increased elastinolytic activity in TELF from horses with COPD was detected by means of zymography and fluorometry. The fluorometric assay has the potential to be used as a diagnostic method to distinguish between healthy horses and those with respiratory disease. Elevated collagenase activity in TELF evidently reflects lung tissue destruction in COPD and indicates an active disease stage. Pathologically elevated collagenolytic activities were found to be associated with equine COPD and with severity of disease. Two inductive MMP-type collagenases, namely MMP-8 (Collagenase-2) and MMP13 (Collagenase-3), were identified. Elevated MMP-8 and MMP-13 immunoreactivities were found in TELF of COPD horses as compared with healthy TELF. The active form of MMP-13 was clearly evident in all COPD horses, but was virtually absent in healthy horses, thus differentiating well between healthy and COPD TELF.
11
Abstract Among the gelatinases, MMP-9 (Gelatinase B) was found to be the most frequently detected MMP in RSs of COPD horses, indicating an ongoing inflammatory condition in their lungs. MMP-9, in particular, marked increased and was converted to active forms. In addition, levels of immunoreactive gelatinase-associated lipocaline (NGAL) were increased in the RSs of COPD horses, and NGAL bound to MMP9 could also partly explain the increased levels of complexed MMP-9 in COPD RSs. In contrast, MMP-2 (Gelatinase A) levels did not differ between healthy to COPD samples and thus, do not seem to play a role in equine COPD. Unchanged levels of MMP-2-associated MMP-14 (MT1-MMP), lend greater support to MMP2 not participating in equine COPD. The reaction of the equine lung to inhaled irritants appears to be reflected by increased MMP-9 activities, which can be detected both by means of gelatinolytic and elastinolytic zymographs. MMP-9 is thus suggested to serve as a potential diagnostic marker for the active phase of equine COPD. Lung macrophage and epithelial cells were found to be major cellular sources of MMP-2, MMP-9, MMP-8, and MMP-13, with neutrophils being another source of MMP-9. MMP-9 activity could be inactivated by means of MMP inhibitors such as chemically modified tetracyclines (CMT). For all samples, CMT-3 inhibited elastinolytic and gelatinolytic activity. CMTs, such as CMT-3, may provide an additional treatment possibility for horses with COPD.
12
List of original publications LIST OF ORIGINAL PUBLICATIONS This work is based on the following original articles referred to in the text by Roman numerals I–VI: I.
Raulo, S.M., Maisi, P. (1998) Gelatinolytic activity in tracheal epithelial lining fluid and in blood from horses with chronic obstructive pulmonary disease. American Journal of Veterinary Research 59, 818-823.
II.
Raulo, S.M., Sorsa, T., Tervahartiala, T., Pirilä, E., Maisi, P. (2001) MMP-9 as a marker of inflammation in tracheal epithelial lining fluid (TELF) and in bronchoalveolar lavage fluid (BALF) of COPD horses. Equine Veterinary Journal 33, 128-136.
III.
Nevalainen M., Raulo, S.M., Brazil, T.J., Pirie, R.S., Sorsa, T., McGorum B.C., Maisi, P. (2001) Inhalation of organic dusts and lipopolysaccharide increases gelatinolytic matrix metalloproteinases (MMPs) in the lungs of heaves horses. Equine Veterinary Journal (accepted).
IV.
Maisi, P., Kiili, M., Raulo, S.M., Pirilä, E., Sorsa, T. (1999) MMP inhibition by chemically modified tetracycline-3 (CMT-3) in equine pulmonary epithelial lining fluid. Annals of the New York Academy of Sciences 878, 675-677.
V.
Raulo, S.M., Sorsa, T., Maisi, P. (2000). Concentrations of elastinolytic metalloproteinases in respiratory tract secretions of healthy horses and horses with chronic obstructive pulmonary disease. American Journal of Veterinary Research 61, 10671073.
VI.
Raulo, S.M., Sorsa, T., Kiili, M., Maisi, P. (2000) Increased collagenase activity with characteristics of MMP-8 and –13 in equine COPD. American Journal of Veterinary Research (accepted).
13
Introduction INTRODUCTION Chronic obstructive pulmonary disease (COPD) is one of the most common diseases that equine veterinary practitioners treat in Finland due to exceptionally long, cold winters, when horses are kept stabled for extended periods. Incidence of COPD in horses in Switzerland is reported to be 54% (Bracher et al., 1991) and in northern United States 12% (Larson and Busch, 1985). Although no prevalence statistics exist on the disease in Finland, it is assumed to be at least as common as in Switzerland due to similar climatic and environmental factors. Besides being frequent, equine COPD causes considerable financial losses. In horses, COPD eventually leads to decreased performance capacity, to early retirement from sports use, and finally, to affected horses being euthanazed. Chronic obstructive pulmonary disease, also known as heaves, haysickness, chronic bronchiolitis, chronic obstructive lung disease, and several other names, has been a common ailment in domesticated horses since antiquity. Clinical signs are not only restricted to domestic horses, however, since similar signs have been seen in zebras and Mongolian horses in zoos (Beech, 1991). A condition closely resembling COPD, called summer pasture associated obstructive pulmonary disease, is found especially in hot climatic regions (Beech, 1991), but is also well documented in more temperate climates. Dixon et al. (1995) reported that nearly 10% of examined COPD horses in Scotland also suffered from summer pasture associated obstructive pulmonary disease . Poor stable management and the low quality of inhaled air with an excess of dust from hay, bedding, and feeding stuffs as well as increased amounts of mold spores and ticks are thought to be some of the factors causing symptoms and acute onset of the disease (Derksen et al., 1988; Robinson et al., 1996). Chronic obstructive pulmonary disease was recognized already in the late 1800s to be an inflammatory condition as indicated by Gresswell and Gresswell (1885), who stated that “broken wind is a nervous, inflammatory disease, characterized by difficult and spasmic breathing, the inspiratory act being easily performed, the expiratory being very prolonged, and accomplished by two apparent efforts”. Today, the disease is recognized as an allergic condition and an “occupational” disease of horses, known to be a hypersensitivity reaction (McGorum et al., 1993b; Robinson et al., 1996), with chronic lung inflammation by nature, characterized by pulmonary neutrophilia (Derksen et al., 1985).
14
Introduction Excessive proteolytic action is considered to be one of the major pathogenic factors in human allergic pulmonary diseases (Cambell et al., 1987; Sibille and Reynolds, 1990). Increased amounts of lysosomal enzymes (Sandholm et al., 1990; Maisi et al., 1994) and proteolytic activity (Grünig et al., 1985; Sandholm et al., 1990; Koivunen et al., 1996) have previously been detected in COPD horses’ respiratory secretions (RS). In addition, a correlation between increased proteolytic activity in RSs and decreased performance capacity has been reported (Maisi et al., 1995). The metalloproteinase enzyme family has been proposed to have a significant role in the development of equine COPD (Koivunen et al., 1997a, 1997b). Deeper understanding of these features and their possible interactions by downregulating specific pathologically elevated proteinases could lead to new clinical, diagnostic, and pharmacological procedures and interventions for equine COPD in veterinary science.
15
Review of the literature 1. REVIEW OF THE LITERATURE 1.1. Equine COPD 1.1.1. General aspects of equine COPD Equine COPD is often compared with human emphysema and/or asthma. Pulmonary emphysema is a common pathological sequela of human respiratory disease characterized by neutrophilia. However, as emphysema is not a major feature of equine COPD, the condition perhaps more closely resembles human asthma. Nevertheless, neutrophils, rather than the increased eosinophils of human asthma (Bradley et al., 1991), are the predominant polymorphonuclear leukocytes (PMN) in RSs from horses with COPD. The absence of emphysema in equine as compared with human neutrophil-mediated respiratory disease is explained by variable pathogenesis in different species. While neutrophil elastase levels and mediator release functions are found to be similar, other factors, such as differences in the abundance and function of intra- and extracellular proteinase inhibitors, may explain the absence of emphysema (Dubin et al., 1994; Dagleish et al., 1999). Pathologically, only minor structural changes, such as mild fibrosis or emphysema, are found (Kaup et al., 1990b), and the majority of these changes are fully reversible. Despite extensive efforts, the precise pathogenesis of equine COPD remains obscure. Several etiological factors have been proposed to be associated with the disease. In addition to allergic response to environmental factors, such as respirable stable dust and mold spores (Derksen et al., 1988; Robinson et al., 1996), previous viral respiratory infections (Gerber, 1973) and inherited genetic susceptibility (Marti et al., 1995) have been suggested.
1.1.2. Clinical findings In horses with COPD, there is prominent airway inflammation (Dixon, 1992; Derksen, 1993) characterized by recurrent hypersensitivity, mucus secretion, and bronchospasm, resulting in airway obstruction and altered gas exchange (Robinson et al., 1996). An extensive increase in neutrophil number is found in RSs; neutrophils invade the lung and accumulate in the lumen of airways, especially bronchioles (Robinson et al., 1996). Coughing is a sensitive clinical indicator, with slight serous nasal discharge and increased respiratory rate accompanied by labored expiratory breathing effort and flaring nostrils also being characteristic (Dixon et al., 1995).
16
Review of the literature The changes in lung function observed in COPD are indicative of diffuse airway obstruction with higher pulmonary resistance and lower dynamic compliance, resulting in changes in intrapleural pressure, uneven distribution of ventilation, and finally, hypoxaemia (Thompson and McPherson, 1984; Robinson et al., 1996). The obstruction of airways as a result of nonspecific hyperresponsiveness together with increased mucus and exudate production, which is found throughout the respiratory tract (Robinson et al., 1996), leads to abnormal findings on thoracic or tracheal auscultation such as expiratory dyspnea, wheezing, and crackling lung sounds due to gas trapped in alveoli. Because of the increased respiratory effort, horses adopt the characteristic breathing pattern involving abdominal muscles in exhalation. The symptoms, which are reversible with periodic remissions, are variations from only slight coughing and slightly altered performance to severe dyspnea at rest. In most cases, symptoms are seasonally associated with wintertime, when horses are kept stabled (Derksen et al., 1985), but in advanced cases, they may remain permanent. The condition tends to affect horses above the age of 4 years and symptoms often deteriorate with age.
1.1.3. Cytology of respiratory secretions Tracheal epithelial lining fluid (TELF) and bronchoalveolar lavage fluid (BALF) from a healthy horse contains macrophages, lymphocytes, a few epithelial cells, and very few neutrophils. In contrast, numbers of polymorphonuclear leukocytes are markedly increased in TELF and BALF from horses with COPD (Beech, 1975; Roszel et al., 1985; Mair et al., 1987; Roberts, 1992; Tremblay et al., 1993).
1.1.4. Inflammation A hypersensitivity reaction to allergens, such as airborne dust, and a response to endotoxins contribute to the development of airway inflammation and dysfunction (McGorum et al., 1998) in the lungs of COPD horses. The ratio of B-lymphocytes in BALF of COPD horses is significantly increased by allergen exposure (McGorum, 1995), suggesting the involvement of antibodymediated mechanisms in COPD lung inflammation. Local immunoglobulins (Ig) IgE, IgA, IgM, and IgG-mediated late-phase immunoreactive hypersensitivity reactions in the lung are a predisposing condition and are involved in the immunopathology leading to development of COPD in the horse (Halliwell et al., 1993; McGorum et al., 1993c; Schmallenbach et al., 1998). The Ig interacts with the inhaled antigen to form a complex that activates the complement, which then induces infiltration of inflammatory cells such as macrophages and neutrophils. Local IgE also contributes 17
Review of the literature to the anaphylactic type of hypersensitivity reaction. As a result, mast cells and basophils are degranulated to release locally acting inflammatory mediators including histamine, leukotrienes, platelet-activating factor (PAF), and proteases (McGorum, 1995). Considerable evidence suggests lymphocytes to have a central role in pulmonary immunoresponses. Among these cells, the subgroup of CD4-surface antigen-bearing T-helper (Th) cells is significantly increased in BALF of severely affected COPD horses (Watson et al., 1997; Kleiber et al., 1999). In contrast, exposure to hay and straw reduces the T-suppressor lymphocyte population (McGorum et al., 1993e), but increases the expression of major histocompatability (MHC) class II antigens on the Th cells, indicating their activation (McGorum, 1995). Activated Th cells are pro-inflammatory cells that induce both cellular and humoral inflammatory responses. It has been postulated that if COPD is an allergic condition the cytokine response profile in the BALF of COPD horses would more closely resemble the Th2-like profile as in human asthma (Krug et al., 1999), which suggests a hypersensitivity rather than a secondary immune-response reaction (Lavoie et al., 2000a). However, further evidence does not support this assumption, as the cytokine profile neither displays the typical Th1- nor Th2-type cytokine profile (Ainsworth et al., 2000; Giguére et al., 2000). Furthermore, the percentage of CD8+ cells has recently been detected to be higher in BALF of COPD horses (Kleiber et al., 1999), indicating involvement of both CD4+ and CD8+ T-cell types in the pathogenesis of equine COPD. Recruitment of neutrophils in the lung is a typical feature of equine COPD (Fairbairn et al., 1993, 1996). Bronchiolar neutrophilia in COPD is speculated to be a result of stimulation or even overstimulation of pulmonary macrophages, leading to expression of cytokines that are chemotactic for neutrophilic granulocytes (Franchini et al., 1998). Neutrophils are found to being activated (Marr et al., 1997) and to accumulate from peripheral blood into the lung after exposure to allergens (Fairbairn et al., 1993). Pulmonary macrophages and lymphocytes are capable of producing chemotactic substances, which induce neutrophil migration and recruitment to the lung. Leukotrienes have been reported to induce an early recruitment of neutrophils to the lungs of horses persisting for more than 5 hours, with no effect on peripheral blood leukocyte numbers being observed (Marr et al., 1998). This suggests that leukotrienes released due to antigen challenge are involved in the pathogenesis of COPD. Moreover, COPD horses have a stronger expression of IL-8, a potent chemoattractant for neutrophils (Lavoie et al., 2000b). After exposure to allergens, the responsiveness of platelets is altered (Ablett et al., 1997). However, the PAF, a mediator for acute inflammatory responses and a known chemoattractant for equine neutrophils (Dawson et al., 1988; Fairbairn et al., 1996), seems to have a minor role in antigen response in COPD-affected horses’ 18
Review of the literature lungs (Marr et al., 1996). Neutrophils and macrophages have the capacity to damage host tissues by releasing potentially histotoxic products, and they have been shown to contribute to clinical symptoms and tissue injury in several pulmonary inflammatory disorders (Sibill and Reynolds, 1990). Moreover, inflammatory mediators, such as cytokines and leukotrienes, are known to induce matrix metalloproteinase (MMP) release/production at sites of inflammation (Esteve et al., 1998; Siwik et al., 2000). Inhibition of these MMPs has prevented allergen-induced airway inflammation in an asthma model (Kumagai et al., 1999). Mast cells, and perhaps basophils, accumulating in the lung are also believed to be involved in airway nonspecific hyperreactivity in equine COPD (Kaup et al., 1990a; McGorum et al., 1993c; Hoffman et al., 1998). Thus, equine COPD seems to be a pulmonary hypersensitivity rather than a nonspecific toxic response (McGorum et al., 1993a). Deficient regulation of systemic immune response in COPD-affected horses with several different immunological mechanisms appear to be involved. Continuous inhalation of allergens/irritants leads to persistent inflammation and eventually to a chronic inflammatory condition, which may result in irreversible changes to the lung tissue, finally affecting the mechanical function of the lung.
1.1.5. Controlling inflammation in COPD At present, corticosteroids are the only effective anti-inflammatory drugs used to control inflammationin COPD. In human asthma, corticosteroid treatment has been suggested to involve modulation of the balance between MMPs and their natural inhibitors (Hashino et al., 1999; Tanaka et al., 2000). The use of nonsteroidal anti inflammatory drugs (NSAID) has not been successful in controlling signs and course of COPD.
1.2. Degradation of extracellular matrix and basement membrane The extracellular matrix (ECM) of connective tissues in the lung is highly organized and consists of tightly bound collagen and elastic fibers, and various glycoproteins. The ECM provides mechanical support for tissues and separates the tissue compartments from each other. This complex structure of connective tissue is highly resistant to hydrolysis. ECM remodeling and degradation is controlled not only by structural resistance but also by a delicate balance between several different proteolytic enzymes and their activators and inhibitors. Proteolysis of the ECM is a key component of the inflammatory response, acting as the primary effector in the 19
Review of the literature modulation of cell-cell and cell-ECM interactions underlying both disease and repair processes. Several different proteinase types participate in physiological and pathological ECM remodeling and degradation. These can be assigned to four major groups: aspartics, cysteines, serine proteinases, and MMPs. The first two are mostly active at acidic pH, the latter two at neutral pH. Elastin, the major component of elastic fibers, is essential for proper structural and functional integrity of the lungs. Elastin is an insoluble hydrophobic molecule that is highly resistant to proteolysis. Its degradation and improper repair are pivotal in the development of pulmonary emphysema (Janoff, 1985) and pulmonary dysfunction. Certain serine proteinases, cysteine proteinases, and MMPs exert elastinolytic activity. Neutrophil elastase is a serine proteinase with broad substrate specificity including elastin. Cathepsin L, a cysteine proteinase, degrades elastin at acidic pH (Shapiro et al., 1991a). Among the MMPs, several family members, including (MMP-3 (Stromelysin-1), -7 (Matrilysin–1), -9 (Gelatinase B), -10 (Stormelysin-2), and – 12 (Metalloelastase)), are found to degrade elastin. Collagen is a molecule composed of three polypeptide α-chains arranged in a rod-like triple helical conformation. Its distinct structure is resistant to proteolytic attack by all other enzymes except collagenases. At the moment, there are three members of collagenase-MMPs, (MMP-1 (Collagenase-1), -8 (Collagenase-2), and –13 (Collagenase-3)), that cleave fibrillar collagens in a distinctive manner, producing fragments of approximately ¾ (N-terminal) and ¼ (C-terminal) the size of the original molecule. These spontaneously denature at body temperature into gelatin peptides which are rapidly cleaved by a variety of proteolytic enzymes. Thus, collagen degradation initiated by collagenases has been considered to be an important and evidently rate-limiting step in tissue degradation (Weiss, 1989; Woessner, 1991). Increased elastinolytic activity induced by serine proteinases and MMPs has been identified in the BALF of human smokers (Janoff et al., 1983a; Niederman et al., 1984) and patients with emphysema (Finlay et al., 1997a) or asthma (Lemjabbar et al., 1999). Tracheal epithelial lining fluid obtained from horses with COPD expresses MMP activity with characteristics of gelatinolytic (Koivunen et al., 1997a) and collagenolytic activity (Koivunen et al., 1997b). According to published literature, no elastinolytic activity has been reported in samples of equine pulmonary tissues or respiratory tract secretions. Alveolar macrophages produce increased amounts of matrix-degrading enzymes that attack both elastin and collagens (Gibbs et al., 1999a). Neutrophils in bronchioles and alveoli cause tissue damage by secreting destructive enzymes (Sibile and Reynolds, 1990), such as gelatinases belonging to the MMP family. 20
Review of the literature Infiltrating inflammatory cells (neutrophils, monocytes, and macrophages), fibroblasts, other resident connective tissue cells, and epithelial cells secrete elastinolytic, collagenolytic or gelatinolytic proteinases. In addition to neutrophil elastase, equine neutrophils contain another elastinolytic serine proteinase, proteinase 3 (Dubin et al., 1994). All of these proteinases are potential participants in destruction of lung tissue. In neutrophils, MMPs are stored in intracellular granules and are released when cells are stimulated (O’Connor and FitzGerald, 1994) often at the site of inflammation. Cells other than infiltrating neutrophils (von Fellenberg et al., 1985) have also been suggested as sources for elastinolytic activity in the RSs. These include the macrophages (Gibbs et al., 1999b), epithelial cells, and endothelial cells present in the lungs of humans and rats. In humans, monocytes contain and secrete serine proteinases, elastase, and cathepsin G, but have little capacity to produce MMPs (Shapiro et al., 1991b), with the expression of MMP being associated with cellular differentiation toward the macrophage (Shapiro et al., 1991b). With regard to MMPs, human mononuclear phagocytes express a changing profile of proteinases that participate in elastin degradation. Immature mononuclear phagocytes synthesize, store, and release neutrophil elastase, a serine proteinase, whereas mature monocytes store, but do not synthesize, this type of elastase. After development to mature macrophages, they neither synthesize nor store neutrophil-type elastase (Shapiro et al., 1991a); instead, they express neutrophil elastase-like activity that can be inhibited by MMP inhibitors. As monocytes differentiate into macrophages, they lose the ability to secrete MMP-7 (Matrilysin), but acquire the capacity to produce large amounts of other MMPs such as MMP-2 (Gelatinase A), -9, and –12 (Shapiro, 1994). Macrophage MMPs can directly degrade insoluble elastin (Senior et al., 1989). Human alveolar macrophages also synthesize cysteine-type proteinases, such as cathepsin L, with elastinolytic activity (Shapiro et al., 1991a). Degradative action of tissue destructive serine proteinases and MMPs leads to connective tissue and basement membrane (BM) destruction in many inflammatory diseases including chronic lung diseases (Burnett et al., 1988, 1994; Palmgren et al., 1992; Finlay et al., 1997a, 1997b; Ohno et al., 1997). In equine COPD, the role of MMPs has been suggested to be more significant than the role of the serine proteinases (Koivunen et al., 1996), although it is very evident that MMPs and tissue-degrading serine proteinases act in concert and form cascades (Sorsa et al., 1997). Total gelatinolytic activity in TELF is shown to be significantly higher in horses with COPD than in healthy horses (Koivunen et al., 1997a). In addition, total proteolytic, collagenolytic, and gelatinolytic activities in TELF from horses with COPD are related to stage and severity of the disease (Koivunen et al., 1996; Koivunen et al., 1997a), but it is not yet fully understood what components in RS and lung tissue are responsible for these activities. Koivunen et al. (1996) suggested 21
Review of the literature that proteolytic activity in the respiratory tract of horses with COPD was of mixed activity, indicating that different enzyme groups and cellular sources may be involved.
1.3. Matrix Metalloproteinases MMPs are a group of proteolytic enzymes participating in ECM and BM degradation and turnover (O’Connor and FitzGerald, 1994; Woessner, 1994; Sepper et al., 1995; Finlay et al., 1997b). The imbalance between MMP expression, activation, and inhibition is associated with tissue destruction in inflammatory lung diseases (Campbell et al., 1987). Matrix metalloproteinases, an endopeptidase family, play an important role in destruction and remodeling of BM and connective tissues. They have an essential role in the homeostasis of the ECM in physiological conditions, such as wound healing (Woessner, 1991), and in pathological processes including inflammation, rheumatoid arthritis, tumor metastatic invasion, etc. (Birkedal-Hansen et al., 1993a; Birkedal-Hansen, 1995). 1.3.1. Structure and function MMPs are a family of at least 20 genetically distinct but structurally related neutral proteinases. Characteristic features for most MMPs are intracellular inductive expression, extracellular secretion in latent zymogen form, activation via proteolytic cleavage of the N-terminal propeptide or by nonproteolytic agents, and inhibition by endogenous proteinase inhibitors. In principle, all MMP members consist of three common domain structures, as illustrated in Figure 1. These include (i) a proenzyme with a hydrophobic N-terminal signal peptide sequence connected to the propeptide domain, a part that is essential for maintaining the proenzyme in a latent form and which is lost upon proteolytic or nonproteolytic activation; (ii) a catalytic domain containing the zinc-binding site. The catalytic Zn interacts with the highly conserved cysteine (C) in the proenzyme domain to maintain the proenzyme in an inactive conformation; and (iii) all except matrilysin contain a C-terminal hemopexin- or vitronectin-like domain connected to the catalytic domain with a proline-rich hinge region. The C-terminal domain regulates substrate specificity and participates in binding and inhibition by tissue inhibitors of metalloproteinases (TIMPs). The C-terminal of the membrane-type MMPs (MT-MMPs) also contains a transmembrane domain. In general, the MMPs are divided into the following five subgroups by structure and different substrate specificity: 1) interstitial collagenases; 2) gelatinases; 3) 22
Review of the literature stromelysins, matrilysins, and metalloelastase; 4) membrane-type MMPs; and 5) other MMPs.
PROTOTYPE
I
Signal peptide Propeptide
C Zn II
Zinc-binding site
III Collagenases,
C C
Stromelysins
Hinge region
I C Zn II
III
C C
Gelatinases
Fibronectin type II inserts
I C Zn
Matrilysins
II I C Zn II
III
C C
MT-MMPs
Transmembrane domain
Figure 1: Matrix metalloproteinase domain structure. MMPs share three common domains: a proenzyme domain (I), a catalytic domain (II), and a Cterminal domain (III) (modified from Birkedal-Hansen, 1995 and Shapiro and Senior, 1999).
23
Review of the literature 1.3.2. Interstitial collagenases Increased type I collagen degradation indicates the action of collagenases. Degradation of collagen I has previously been found to be increased in TELF of horses suffering from COPD (Koivunen et al., 1997b). Collagenase activity has been detected in several noninfectious lung diseases, such as fibrosis, allergic alveolitis, and chronic lung inflammation, and in most of these tissue-destructive pathological processes, the collagenase identified exerted characteristics of the neutrophil-type enzyme (MMP-8) (O’Connor and FitzGerald, 1994; Sepper et al., 1995; Finlay et al., 1997b). MMP-1 and MMP-8 MMP-1 (Collagenase-1) and MMP-8 (Collagenase-2) are virtually identical in structure, but MMP-8 has a larger molecular size due to its higher glycosylation degree. MMP-1 is expressed in a 52-kDa latent form that is converted to a 42-kDa active form (Woessner, 1991). MMP-8 is secreted in a latent 75 to 80-kDa form (Birkedal-Hansen, 1995) to be converted to its 66-kDa active form (Woessner, 1991). Collagenases MMP-1, MMP-8, and MMP-13, represent a MMP subgroup that can initiate the degradation of native type I, II, and III collagens under neutral conditions, which are further degraded by other MMPs and proteolytic enzymes (O’Connor and FitzGerald, 1994; Woessner, 1994). MMP-1 preferentially degrades type III collagen and is generally believed to be constitutively expressed and involved in normal tissue turnover (Woessner, 1991). Besides fibroblasts, several other cells, e.g. keratinocytes, endothelial cells, and macrophages (Birkedal-Hansen, 1993a), also produce MMP-1, initially called fibroblast-type collagenase. MMP-8 prefers type I collagen and is primarily thought to be synthesized by PMNs during their maturation in bone marrow, stored in intracellular granules, and expressed by triggered degranulating PMNs at the site of inflammation (Hasty et al., 1990; Ding et al., 1997). MMP-8, also called neutrophil-type collagenase, has recently been found to be expressed by certain non-PMN lineage cells such as endothelial cells (Hanemaaijer et al., 1997). Moreover, MMP-8 has been linked to bronchial ciliated epithelial cells, glandular cells, and monocyte-macrophage-like cells in human bronchiectatic (BE) lungs, and its elevated expression was associated with significant active MMP-8 presence (Prikk et al., 2001). Alveolar epithelial cells have previously been found to synthesize interstitial collagenases in vitro (Pardo et al., 1997). Initial preliminary characterization, based on a collagenase-specific doxycyclineinhibition test of collagenolytic activity in TELF from COPD horses (Suomalainen et al., 1992; Greenwald et al., 1998), revealed that collagenase activity consisted predominantly of neutrophil–type MMP-8, with a lesser amount of fibroblast-type 24
Review of the literature MMP-1 (Koivunen et al., 1997b). This is in accordance with the general concept which indicates MMP-8 to be inducible expressed at the site of inflammation and MMP-1 to be constitutively expressed for normal tissue remodeling (Woessner, 1991; Romanelli et al., 1999). MMP-13 MMP-13 (Collagenase–3) is secreted in a latent 60-kDa form (Freije et al., 1994). During the activation process, an intermediate 50-kDa form has been found, which is then further cleaved to form a 48-kDa active enzyme (Knäuper et al., 1996a). In contrast to MMP-1 and -8, MMP- 13 clearly has a wider substrate specificity, degrading types I, II, and III collagens as well as basement membrane type IV collagen, proteoglycans, fibronectin, fibrillin, versican, and tenascin (Knäuper et al., 1996b). Of the various collagen types, MMP-13 most effectively cleaves type II collagen. MMP-13 is primarily suggested to be related to tumor invasion (Freije et al., 1994; Johansson et al., 1997, 1999), but has also been found to occur in chronic dermal ulcers (Vaalamo et al., 1997) and periodontal inflammation (Uitto et al., 1998). For instance, MMP-13 expression is induced in rat lung fibroblasts during inflammatory lung injury (Mariani et al., 1998). MMP-13 enhances wound healing of the alveolar epithelial monolayer by modulating the repair process by decreasing cell adhesion and stiffness, and by increasing cell migration (Planus et al., 1999). 1.3.3. Gelatinases The gelatinase MMPs have an additional domain with three tandem fibronectin type II repeats in their catalytic domain (Figure 1) that provide these enzymes with the ability to bind to collagen and gelatin (Wallon and Overall, 1997). Their substrate profile consists of denatured collagens, i.e., gelatin (Birkhedal-Hansen, 1995). In addition, gelatinases degrade type IV, V, VII, X, XI, and XII collagens, elastin, and fibronectin, among others (Reynolds, 1996). Gelatinases have also been suggested to degrade native type I collagen in an acidic environment, thus participating in remodeling of collagenous ECM (Johansson et al., 2000). T-Lymphocytes from healthy human beings also express gelatinases A and B, but their expression is differently regulated; secretion of gelatinase B is constitutive, whereas secretion of gelatinase A is inducible (Romanic and Madri, 1994; Leppert et al., 1995). MMP-2 MMP-2 (Gelatinase A / type IV gelatinase) is secreted in a latent 72-kDa form and is converted into 62 to 59-kDa forms during proteolytic activation (BirkedalHansen et al., 1993a). MMP-2 is widely expressed by most cell types (Zaoui et 25
Review of the literature al., 1996). MMP-2, has the ability to degrade elastin (Senior et al., 1991), and is produced predominantly by fibroblasts and other resident connective tissue cells (Janoff et al., 1983a). MMP-9 MMP-9 (Gelatinase B / type IV gelatinase) is secreted as a 92-kDa latent form to be converted into 82 to 68-kDa active forms during activation processes (Birkedal-Hansen et al., 1993b). It has the ability to degrade components such as gelatin, elastin, collagens IV, V, VII, X, XI, XIV, and fibronectin (Senior et al., 1991). A considerable amount of proMMP-9 in tissue and body fluids occurs in complexed forms (Woessener, 1991; Birkedal-Hansen, 1993a; Kjeldsen et al., 1993), such as the approximately 220-kDa dimeric form of the molecule (Triebel et al., 1992). MMP-9 is a secretory component of neutrophil primary granules and a major inducible macrophage product which is also found in other PMN leukocytes (O’Connor and FitzGerald, 1994). Lymphocytes have been suggested to regulate MMP-9 production of neutrophils, monocytes (Seki et al., 1995), and macrophages (Lacraz et al., 1995). Transmigrating neutrophils are found to secrete MMP-9 and to degrade type IV endothelial BM collagen (Murphy and Crabbe, 1995). Koivunen et al. (1997a) found that in TELF of horses with COPD gelatinolytic activity of a 90 to 110-kDa gelatinase, suspected to be MMP-9, correlated with neutrophil score, suggesting that neutrophils were the source of MMP-9 in these samples. 1.3.4. Stromelysins, matrilysin, metalloelastase Among matrix metalloproteinases, MMP-3 and -10 (Stromelysin-1 and -2) (Murphy et al., 1991), MMP-7 (Matrilysin-1) (Woessner, 1995), and MMP-12 (Macrophage metalloelastase, MME) (Senior et al., 1989; Belaaouaj et al., 1995) have the ability to degrade elastin, laminin, and fibronectin. Additionally, MMP-3 and MMP-12 have the ability to degrade gelatin. MMP-3 also cleaves α 1-proteinase inhibitor and TNF- α precursor (Chandler et al., 1997), and inactivates IL-1 β (Ito et al., 1996). MMP-3 has been localized in several lung cells (Dahlen et al., 1999), fibroblastic cells and epithelial cells. The smallest MMPs, MMP-7 and MMP-26 (Matrilysin-2) (de Coignac et al., 2000), lack the hemopexin domain (Figure 1), have a wide substrate profile, and are secreted by normal airway epithelial cells (Dunsmore et al., 1998). MMP-12 is expressed at least by alveolar macrophages (Shapiro et al., 1993).
26
Review of the literature 1.3.5. Membrane-type matrix metalloproteinases In addition to the basic MMP structure, the membrane-type matrix metalloproteinases (MT-MMPs) contain an additional sequence between the proand catalytic domains (Basset et al., 1990; Takino et al., 1995; Will and Hinzmann, 1995), providing the basis for furin-activation of latent enzyme (Johansson et al., 2000). MT-MMPs are bound to cellular membrane by a specific transmembrane domain that is situated at the C-terminal end of the hemopexin domain (Figure 1), which is followed by the intracellular part of the domain. Six different MT-MMPs have been identified, these being MMP-14, -15, 16, -17(Murphy et al., 1999), –24 (Llano et al., 1999), and –25 (Pei, 1999). At least, MMP-14, -15, and -16 are known to activate proMMP-2. MMP-14 MMP-14 (MT1-MMP) is secreted as a 63-kDa form (Knäuper et al., 1996b). It activates by hydrolyzing latent MMP-2 at the cell membrane, involving interaction of proMMP-2 with the MMP-14/TIMP-2 complex (Stanton et al., 1998). As a result of the proteolytic action of MMP-14, proMMP-2 is shown to be activated (Sato et al., 1994; Lee et al., 1997). Besides acting as an initiator of an activation cascade for MMP-2, MMP-14 has a direct proteolytic effect on substrates such as collagen types I, II, and III, gelatin (D’Ortho et al., 1998), fibronectin, and laminin. 1.3.6. Other MMPs At least 20 different MMP enzymes are known today, with their number constantly growing as new MMPs are found. The substrate profiles of the most recently found MMPs, namely MMP-19 (Pendas et al., 1997), -20 (Enamelysin)(Llano et al., 1997), –23 (Velasco et al., 1999), and –27 (Epilysin)(Lohi et al., 2000), have only been partly determined.
1.3.7. Regulation of MMPs MMP activity, secretion, and cellular expression are regulated by a delicate balance of MMP activators and inhibitors (Woessner, 1991). Their action and expression are controlled by cytokines, several other proinflammatory mediators, such as TNF-α and IL-1β (Hanemaaijer et al., 1997), growth factors, hormones, (O’Connor and FitzGerald, 1994; Shapiro, 1998) and cell-matrix and cell-cell contacts. Regulation of the synthesis, expression, and activation of MMPs involves the following steps: transcriptional regulation, intra - and extracellular activation of the zymogen form of proMMPs as a result of losing/cleaving the 10 to 20-kDa 27
Review of the literature propeptide domain, and inhibition by specific endogenous inhibitors (Birkedal-Hansen et al., 1993a; Birkedal-Hansen, 1995; Murphy and Knäuper, 1997).
1.3.7.1. Activation 1.3.7.1.1. Natural activators Oxidative stress Free radicals, such as H2 O2 or NO, were noted to increase expression of MMP mRNA and to decrease TIMP mRNA (Brenneisen et al., 1997; Tanaka et al., 1998), providing evidence for the involvement of oxidative stress in the regulation of MMPs. Oxidative stress, measured by glutathione redox ratio, increased significantly in pulmonary epithelial lining fluid of horses with acute crisis of recurrent airway obstruction as compared with healthy horses, indicating an association between oxidative stress and equine lower airway disorders (Art et al., 1999). Proteolysis Proteinases, such as neutrophil-derived elastase, which is one of the pathophysiologic activators of MMP-9 (Ferry et al., 1997), or MMP-14, which is shown to activate proMMP-2 intracellularly (Lee et al., 1997) and extracellularly on the cell surface (Sato et al., 1994; Strongin et al., 1995; Knäuper et al., 1996a), are examples of proteolytic activators of MMPs. Furthermore, MMP-13 activates latent gelatinases MMP-2 and MMP-9 (Knäuper et al., 1996b). Another proteinase that can cleave the propeptide of most MMPs is plasmin.
1.3.7.1.2. Chemical activators Nonproteolytic activators include organomercurials, metal ions, thiol reagents, oxidants, chaotropic agents, and low pH. APMA The action of 4-aminophenylmercuric acetate (APMA), the most commonly used organomercurial activator of proMMPs in vitro, is based on the probable interaction of APMA with the cysteine residue, converting it to a nonbinding form, thereby releasing the active site. The covalent interaction between cysteine and Zn retains latent MMPs in their proform.
28
Review of the literature 1.3.7.2. Inhibition 1.3.7.2.1. Natural inhibitors Two major endogenous inhibitors are known for MMPs, the nonspecific inhibitors, such as α 2 -macroglobulin, and the specific inhibitors, called TIMPs. Alpha–2-macroglobulin The α 2 -macroglobulin (Nagase et al., 1994) is a serum protein which is mainly responsible for regulating MMPs and serine proteinases in serum and body fluids (Sottrup-Jensen and Birkedal-Hansen, 1989). TIMPs Tissue inhibitors of metalloproteinases are the specific endogenous inhibitors of MMPs that are widely distributed in tissue and body fluids (Birkedal-Hansen et al., 1993a). The action of TIMPs on MMPs includes inhibition of catalytically active forms of the enzymes and prevention or delay of conversion of proMMPs to their active forms. The inhibition is based on TIMP binding to the Zn-binding site or to the hemopexin domain. Four different TIMPs have been recognized thus far, all sharing common structural features and being secreted by a variety of cell types. NGAL Neutrophil gelatinase-associated lipocaline (NGAL) forms heterodimers with MMP-9 by covalent binding (Kjeldsen et al., 1993). The NGAL serves as a negative feedback in inflammation by binding to lipophilic substrates and neutralizing the inflammatory mediators (Kjeldsen, 1995; Westerlund et al., 1996).
1.3.7.2.2. Chemical inhibitors EDTA Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that attracts Ca+ and Zn+ ions, thus interfering with the action of the catalytic domain of MMP enzymes (Auld, 1995). Tetracyclines and their chemically modified analogs Chemically modified tetracyclines (CMTs) are modified non-antimicrobial tetracycline (TC)-derivatives (Golub et al., 1987) that are widely used in periodontal disease research to inhibit the proteolytic action of MMPs. CMTs are specially designed to lack antimicrobial ability but preserve the antiproteinase activity. Tetracyclines act as cation-chelators, and it seems that their action on MMPs is a non-competitive inhibition which is associated with the binding of the molecule to the secondary zinc, resulting in conformational change and loss of activity (Sorsa et al., 1994). Additionally, TCs inhibit the oxidative activation of proMMPs (Lauhio et al., 1992; Ramamurthy et al., 1993; Sorsa et al., 1994; Golub et al., 1995). 29
Review of the literature These actions can affect MMP activity already intracellularly in PMNs, where increased levels of TCs have been found (Gabler et al., 1992). Part of the inhibitory effect is also suspected to be due to down-regulation of gene expression of MMPs, especially MMP-2, thus affecting MMP protein production (Uitto et al., 1994). Other synthetic inhibitors While the list of new synthetic MMP inhibitors is rapidly expanding, only a few are mentioned here. Bisphosphonates, such as clodronate, alendronate, and pamidronate, have been shown to inhibit metalloproteinases (Teronen, 1998), but the mechanism of their action on MMPs remains obscure. The MMP-inhibition mechanism may involve the ability of the bisphosphonates to act as cation-chelators, similar to the mechanism of TCs (Sorsa et al., 1994; Golub et al., 1997). Among the hydroxamate inhibitors, batimastat has been shown to inhibit equine gelatinolytic MMPs in vitro (Pollitt et al., 1998). This inhibitor’s hydroxamate group binds to the catalytic domains zinc, resulting in a potent but reversible inhibition of the MMP. Similarly to hydroxamatates, carboxyalkyl inhibitors act as chelator compounds (Brown and Giavazzi, 1995). Other small molecular peptides have also been developed, such as the novel tissue-permeable cyclic peptide, which is a highly selective MMP-2 inhibitor (Koivunen et al., 1999). In the search for more effective treatments and potential therapeutics, the investigation has extended from peptidelike broad-spectrum MMP inhibitors towards advanced synthesis of protein structure-based, small molecular, nonpeptidic, selectively targeted MMP-inhibitors (Michaelides and Curtin, 1999).
30
Aims of the study 2.
AIMS OF THE STUDY
This thesis focused on MMPs detected from respiratory secretions of COPD horses. The main objectives were the following: 1.
to determine whether the major proteolytic activity in RSs of horses represents MMPs;
2.
to study whether increased gelatinolytic MMP activity in the inflamed organ is reflected in the blood;
3.
to detect elastinolytic activity in RSs of horses and to determine whether an association exists between this activity and equine COPD;
4.
to characterize in detail gelatinase and collagenase MMPs in horse RSs;
5.
to identify cellular sources of gelatinase and collagenase MMPs in horse RSs;
6.
to study the possible use of gelatinolytic, elastinolytic, and collagenolytic MMPs as potential diagnostic markers of chronic respiratory tract inflammation;
7.
to study possible inhibitors of gelatinases and elastases in vitro, with special reference to their use as future drugs.
31
Materials and methods 3. MATERIALS AND METHODS 3.1. Horses Clinical history, physical examination, neutrophil content in TELF and BALF samples, and blood cytology and chemistry were used as the basis for grouping of horses (Nylor et al., 1992; Dixon et al., 1995) summarized in Table 1. A total of 19 clinically healthy horses owned by the Horse Research Center, Ypäjä or by the Riding Police, Helsinki were used as controls. These horses had no history or signs of respiratory disease based on clinical examination, and laboratory findings were within reference values. All healthy horses were sampled at the end of a pasture period in the autumn of 1996 and 1997. Twenty-nine horses were classified as COPD-affected according to clinical history and signs of chronic recurrent pulmonary disease. All COPD horses had been coughing repeatedly for at least the last two years, and on physical examination, they were identified to be at the acute inflammatory phase. Sampling was performed during wintertime, when the horses were stabled most of the day. Twenty-four horses were patients with respiratory disease assigned to the Large Animal Clinic of the Faculty of Veterinary Medicine, Helsinki University during the winter of 1995, 1996, and 1997. In addition, five COPD horses (study VI) owned by the Horse Research Center, Ypäjä, were repeatedly sampled. These five recurrently symptomatic COPD horses were sampled twice, first during stabling while exhibiting symptoms and then after a summer at pasture while they were clinically in remission. On clinical evaluation, all 29 COPDaffected horses had labored breathing and changes in sound on auscultation at rest. Endoscopic findings from the upper airways and trachea revealed increased amounts of mucopurulent secretions. Neutrophil numbers were elevated both in TELF and BALF. In TELF, the number of neutrophils was judged by a semiquantitative scoring system from 0 to 4. Analysis of EDTA anticoagulated blood (hemoglobin, white blood cell counts (WBCs), and fibrinogen) and arterial blood gas tension showed values within reference values for all horses. None of the horses had received any medication during the two weeks prior to evaluation and sampling of the respiratory tract. For the inhibition trial (study IV), TELF samples from COPD horses in study I were used.
32
Materials and methods Healthy controls
COPD
Study
I
II
V
VI
total
I
II
V
VI
total
n= Sex
4
15
10
12
19
7
17
10
17
29
7/2/6
4/4/2
5/2/5
7/6/6
5/0/2
7/8/2
4/1/5
10/6/1
18/7/4
0/4
8/7
3/7
6/6
8/11
2/5
6/11
6/4
9/8
13/16
average years
11.8
6.6
6.9
6.7
7.7 (3-16)
8
8.2
9.7
10.4
8.9 (5-17)
TELF NG-score
0-1
0-1
0.5-1
0-1
0.5-1
2-3
2-4
2-4
2-4
2-4*
BALF NG %
-
2.2
2.3
2.3
2.2 (0-5.4)
-
29.4
28.3
37
29.4* (9.4-72.7)
pO2 mmHg
-
103.5
102.5
103.6
-
79.8
66.3
73.5
fibrinogen g/l
3.4
3.3
3.4
3.1
3.8
3.3
3.6
3.7
lymphocytes 9 x 10 /ml
6.3
9.2
9.1
9.0
9.6
9.2
9.5
9.4
hemoglobin g/l
-
140.5
140
143.4
140.3
135.5
137.2
126.1
mare / gelding/ stallion 0/3/1
Breed finnhorse / standard
Age
BLOOD 103.5 (90.5-109.6) 3.4 (2.6-3.9) 8.6 (4.9-14.3) 140.5 (101-178)
74.1* (48.2-101) 3.6 (2.5-5.8) 9.4 (5.6-14.4) 137.5 (115-174)
Table 1. Summary of clinical values of horses used in studies I,II,V, and VI. * values in bold differ significantly between healthy and COPD horse. Age, neutrophil (NG) -score / %, pO2, fibrinogen, lymphocytes, and hemoglobin are expressed as averages (ranges).
3.1.1. Challenge design (III) Experimental respiratory challenge of COPD and healthy horses was performed at the University of Edinburgh in Scotland, UK by Dr. McGorum and collaborators. The experiment was part of a larger study. A standardized challenge protocol previously described by McGorum et al. (1993c,d) and Pirie et al. (2001a,b) was conducted. Frozen cell-free BALF samples were transported to Finland for analysis. Miscellaneous challenges (III a) Seven COPD and six healthy horses underwent a series of four different challenge treatments. Challenges involved a 5-hour exposure to saline as control, to lipopolysaccharide (2000 µg soluble Salmonella typhimurium Ra60) (LPS), to natural hay-straw (HS), and to hay-dust suspension (HDS). All horses were kept at pasture or housed in a lowdust environment between challenges, and an appropriate time interval 33
Materials and methods (Pirie et al., 2001b) was maintained between successive challenges. Horses were shown to be in remission prior to challenging, assessed by clinical examination, arterial blood gas analyses, as well as by bronchoscopy and BALF cytology, all of which were whitin normal values. BALF samples were collected 6 hours after each challenge. Time-course study (III b) Six COPD horses were challenged with natural hay-straw for 5 hours followed by sequential BALF collection (Pirie et al., 2001a). BALF samples were collected prior to challenge and at 5 hours, 24 hours, 4 days, 7 days, and 14 days postchallenge.
3.2. Samples 3.2.1. TELF (I, II, IV,V,VI) Prior to TELF and BALF collection, horses were sedated with demetedomidine and butorphanol (i.v.). Tracheal wash was performed according to a standard procedure by inserting a sterile catheter through the biopsy channel of an endoscope (Olympus Optical Co., Tokyo, Japan). Ten milliliters of sterile physiologic saline was infused into the mid-trachea, and the pool formed in the lower tracheal floor was aspirated immediately. Recovered TELF was cytologically evaluated and stored at –70o C until further analysis. Between 5 and 15 milliliters of TELF was obtained from each horse. Neutrophil count in TELF was determined from air-dried smears that were stained by a rapid-staining kit (Hemacolor No. 16661, Merck, Heidelberg, Germany) and Wright’s stain. Cell density in the smear was evaluated at 200X magnification, and the neutrophil count in the observation field was graded from 0 to 4 (with 0 corresponding to 0 - 2 neutrophils, 1 corresponding to few observed neutrophils but no aggregates, 2 corresponding to a moderate increase in neutrophils and aggregates, 3 corresponding to an abundant increase in neutrophils and aggregates, and 4 corresponding to a field full of neutrophil aggregates) (Winder et al., 1990; Tulamo and Maisi, 1997). Dilution of TELF samples by tracheal wash fluid was corrected by measuring the urea concentration in serum and in the recovered tracheal fluid (Guttmann and Bergmeyer, 1994). Finally, to express results in equal dilutions to original RSs, the dilution effect was calculated according to Rennard et al. (1986).
34
Materials and methods 3.2.2. BALF (II, III, VI) Following TELF collection, the endoscope was passed further. Local anesthetic (Carbocain 1%, Astra, Södertälje, Sweden) was infused into the lower airway walls to prevent coughing. Finally, the tip of the endoscope was wedged to a bronchius and the obstructed lung lobe was lavaged with 300 milliliters of warmed (37o C) sterile physiologic saline solution that was administered in 50-milliliter aliquots. Immediately after introduction, the lavage fluid was gently aspirated, set on ice, and processed further. The harvested BALF was passed through a sterile gauze to remove excess mucus. One portion of native BALF was separated and stored at –70o C until analysis (II). The remaining fluid was centrifuged and the supernatant (referred to as the cellfree BALF) was stored at –70o C (II, III). BALF cells (II, VI) The remaining cells were washed with PBS, centrifuged, and the cell pellet was dissolved in PBS. Cytocentrifuge slides of 40 000 cells/slide were prepared by Cytospin 3 (Shandon Scientific Ltd., Cheshire, UK) on silanated SUPER FROST++ (Menzel, Sybron Co., Germany) microscopy slides. Total and differential cell counts (300 cells counted) were performed (McGorum and Dixon, 1994). The rest of the slides were stored at -70o C. 3.2.3. Blood neutrophils (I, II, VI) Blood samples were collected by means of jugular venipuncture into tubes containing EDTA and stored on ice. Total and differential WBC counts were determined. Blood leukocytes were separated into two fractions on discontinuous Percoll (Pharmacia & Upjohn, Stockholm, Sweden) gradients by centrifuging at 20o C (Pycock et al., 1987). Both the neutrophilic fraction and the lymphocytic fraction were washed with Hanks´ balanced salt solution (HBSS) and centrifuged. Pellets were suspended in HBSS, and total and differential cell counts were determined using a Bürker chamber. Separated cells were stored at -20o C until further analysis. Lysis of cells was verified by means of microscopy. For immunohistochemistry (II, VI), part of the neutrophils were washed, cytocentrifuged, and stored similarly to BALF cells. 3.2.4. Serum (I) Blood serum was collected from the jugular vein blood samples simultaneously with blood cell collection. Serum was separated from blood by centrifugation and the pellet was stored at -20o C. 35
Materials and methods 3.2.5. Lung tissue (II) Frozen sections (10 µm) of lung tissue from a euthanazed COPD horse were cut with a freezing microtome. Sections were immediately fixed in 4% paraformaldehyde (in PBS) and dehydrated in a graded series of 50%, 70%, and absolute ethanol and stored at –70o C until used.
3.3. Assays 3.3.1. Enzyme activity assays 3.3.1.1. Zymography The catalytic action of MMP enzymes is dependent on the zinc ion in the active site and the enzymes are stabilized by calcium ions. Each MMP has a distinct protein substrate profile with partially overlapping profiles. In the present study, gelatin and elastin were used as substrates in the SDS-PAGE gels. With the zymographic method, sample proteins are first separated electrophoretically according to their specific molecular weight (MW) and electrical charge. After electrophoresis, the gels are renatured and the degradation of the substrate being studied is visualized by incubating the gels in standardized buffer with Ca+ and Zn+ ions added. TELF supernatant was separated by centrifugation for 4 minutes at 170 g. Before zymography, the supernatant was diluted on the basis of the serum-to-tracheal fluid urea concentration ratio to a standard dilution of 1:700 for gelatin zymography and 1:6 for elastin-zymography with TNC buffer (pH 7.5). The most diluted TELF samples were freeze-dried before dilution. Native BALF and cell-free BALF were not further diluted. Gelatin and elastin zymography (detailed description of zymography in studies I and II) Gelatin zymography (I, II, III, and IV) and elastin zymography (V) were used to determine gelatinolytic and elastinolytic activity, respectively. The assays were performed essentially as described by Sepper et al. (1994) and Murphy and Crabbe (1995). All samples were first mixed in a 2:1 ratio with sample buffer containing Brom Phenol Blue and protein denaturing 6% SDS (pH 6.8), and pre-incubated for 2 hours at 20o C. Samples were then loaded into 10-12% SDS-polyacrylamide gels containing 1 mg/ml porcine skin gelatin (G-2625, Sigma Chemicals Co., St Louis, MO, USA) or 1 mg/ml κ-Elastin (ETNA-Elastin, Elastin Products Co., Owensville, MO, USA) as substrate. The zymograms were run at 4o C to avoid spontaneous activation. Prestained SDS-PAGE high-range MW standard (Bio-Rad, Richmond, CA, USA) and a standard suspension of lyzed equine neutrophils (prepared as 36
Materials and methods described in blood neutrophils) as well as reference gelatinases of recombinant human MMP-9 (Westerlund et al., 1996) (II, III, IV, V) and recombinant human MMP2 (Sorsa et al., 1997) (III, V) were analyzed in parallel. After incubation at 37o C in conditions favoring enzyme activity, 17 - 21 hours and 66 hours for gelatinases and elastases, respectively, gelatinolyitic/elastinolytic activity was visualized, by staining with Coomassie blue, as clear bands against a blue background. The band densities were determined by scanning and analyzing using an image analysis and processing system (Cream, Kem-En-Tek, Copenhagen, Denmark). Densitometric results were calculated in area mode after subtraction of background gray values. To enable densitometric comparison between zymograms, all bands were related to the corresponding equine neutrophil lysate standard on the same gel. Zymograms were analyzed for total gelatinolytic or elastinolytic activity, complex-, proMMP-9-, active MMP-9-, and MMP-2 forms (Mellanen et al., 1998). APMA activation To determine effects of APMA activation on gelatinolytic activity (I), samples were incubated for 1 hour at 37o C with 2 mM APMA (Murphy and Crabbe, 1995) in Tris-HCl before preincubation in sample buffer, followed by zymography as described. EDTA inhibition To determine inhibitory effect of EDTA on gelatinolytic (I, II) and elastinolytic (V) activity, zymography was performed as described, except that 10 mM EDTA, a sufficient concentration to remove the metal from most metallopeptidases (Auld, 1995), was added to the preincubation sample buffer. To refold SDS-denatured proteins, SDS was removed from gels and proteins by washing the gels 3 times in two different wash solutions. The initial wash solution contained Tween-80 and 10 mM EDTA, and the second wash also contained Zn and Ca. Finally, gels were incubated at 37o C in 50 mM Tris-HCl including Zn, Ca, and 10 mM EDTA, for 17 hours and 66 hours for gelatinases and elastases, respectively. CMT-3 inhibition To determine inhibitory effect of chemically modified tetracycline, CMT-3 (Metastat, CollGenex Pharmaceuticals Inc, Newtown, PA. USA), on gelatinolytic (IV) and elastinolytic (V) activities, zymography was performed as described, except that TELF samples were first incubated for 1 hour at 37o C with 25 µM, 50 µM, or 150 µM CMT-3 for gelatin zymography and with 50 µM, 150 µM, or 500 µM CMT-3 for elastin zymography before preincubation with sample buffer. To remove SDS, gels were washed 3 times in two renaturing wash solutions after zymography. The initial wash solution contained Tween-80, and the second wash solution was supplemented with one of the forementioned consentrations of CMT-3 for gelatinases 37
Materials and methods and elastases. Gels were then incubated at 37o C in Tris-HCl supplemented with corresponding consentration of CMT-3 for 17 hours for gelatinases and for 66 hours for elastases. 3.3.1.2. Fluorometry Degradation of elastin was detected by fluorometric elastase assay modified from Quesada et al. (1997) and introduced in study V. Detection of elastinolytic activity from TELF was performed in microtitration plates with an elastin substrate ligated with fluorogenic fluorescein-isothiocyanate (FITC) (No. E80, Elastin Products Co., Owensvill, MO, USA). As the substrate elastin is degraded, the fluorogenic FITC is released and can be monitored at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Elastin degradation was monitored every 10 min with a fluorometer (Fluoroscan II, Labsystems, Helsinki, Finland) connected to an IBM-PC computer. Reaction was carried out in clear 96-well microplates (Cliniplate, Labsystems, Helsinki, Finland) for 12 hours at 37o C. To assess a suitable substrate concentration for the assay, two pooled samples containing equal amounts of TELF were prepared. Elastin-fluorescein substrate was dissolved to yield a 2 mg/ml substrate concentration and further diluted in double dilution steps. Two replicates of each dilution were assayed and results were expressed as mean values. Finally, the most suitable concentration for studying elastinolytic activity was selected. Determination of elastinolytic activity in TELF (V) TELF from four healthy horses and from four COPD horses studied for gelatinolytic activity in study I were selected. Fluorometric measurements were performed at two intervals: immediately after mixing of the sample, buffer, and fluorogenic substrate, one portion of the mixture was pipetted on to the microplate, and fluorogenic activity was measured for a period of 6 hours. The remainder of the mixture was incubated in sealed Eppendorf tubes on a shaker at 37o C for 66 hours, followed by a second 6-hour measurement period. Three replicates of each sample were studied at both times, and corresponding blanks without enzyme or sample were included to evaluate the spontaneous hydrolysis of the substrate and were subtracted to determine net values. After subtraction of blank values, results were corrected according to dilution of serum-to-tracheal fluid urea concentration ratio. Inhibition of elastinolytic activity To determine effects of EDTA and CMT-3 on elastinolytic activity (V), fluorometry was performed as previously described, except that TELF samples were incubated for 1 hour at 37o C with 1 mM or 2.5 mM EDTA, or 25 µM, 50 µM, or 150 µM CMT-3 prior to fluorometry. Inhibitory effects were studied only in the first measurement period. 38
Materials and methods 3.3.1.3. Type I collagen degradation (VI) The type I degradation assay is based on the unique ability of interstitial collagenases among other MMPs to degrade the triple helical structure of native type I, II, and III collagens between the Gly775 -Ile776 or Gly775 -Leu776 bonds, thus generating the characteristic αA (3/4) and αB (1/4) degradation products (Hasty et al., 1990; Knäuper et al., 1996a). Because MMP-8 favors type I collagen, mainly MMP-8 activity is established with this assay based on substrate specificity (Knäuper et al., 1996a; Lindy et al., 1997). For the collagen I assay, TELF supernatant was separated by centrifuging for 4 minutes at 170 g, and diluted 1:100 based on the blood serum-tracheal wash urea ratio with sterile 0.9% saline. Purified type I collagen from a rat tail was used as a substrate (Miller and Rhodes, 1982; Konttinen et al., 1994). The urea-adjusted TELF supernatants were incubated with 1.5 µM triple helical type I collagen monomers for four days in dark at 20o C. Collagen degradation was stopped by heating, and the reaction products were separated electrophoretically from intact collagen I on 10% SDS-PAGE gels. As a control, one lane was loaded with a standard that included pure collagen I. Intact type I collagen α 1 and α 2 chains and the ¾ α 1A and α 2A collagen degradation products were visualized as four blue bands against a clear background on the electrophoretic gels that were recorded by scanning and analyzing with an image analysis and processing system. Densitometric results were calculated in area mode after subtraction of background gray values. The value representing ¾-cleavage fragments of α A chains was multiplied by 4/3 to obtain the total quantity of degradation products. The proportion of degradation products from total type I collagen was calculated as a percentage to measure the collagenase activity in the samples. 3.3.2. Immuno assays 3.3.2.1. Western blotting (detailed procedure in studies II and VI) Specific MMP- antibodies Specific rabbit polyclonal anti-human MMP-2 (II, III), MMP-8 (VI), MMP9 (II, III), MMP-13 (VI), MMP-14 (II), and NGAL (II) antibodies were used. The polyclonal anti-human MMP-2, -8, -9 and –13 antibodies have been previously used to detect various and distinct multiple molecular weight forms/species of human and rat MMPs (Lauhio et al., 1994; Sorsa et al., 1994; Westerlund et al., 1996; Hanemaaijer et al., 1997; Pirilä et al., 2001). Thus the specificity of the antibodies and their ability to detect nonhuman MMPs has been tested. The polyclonal anti MMP-8 and MMP–13 antibodies are not commercially available. The rabbit antihuman polyclonal antibody against MMP-8(Michaelis et al., 1990) was kindly 39
Materials and methods prepared and donated by Dr. Jurgen Michaelis, Department of Pathology, Christohnoch Medical School, Christohnoch, New Zealand and the anti MMP-13 antibody (Freije et al., 1994) was kindly prepared and donated by Dr.Carlos LopezOtin, Department of Molecular Biology, University of Quiedo, Quiedo, Spain. In addition, rabbit polyclonal antihuman MMP-2, MMP-9 (Sorsa et al., 1994), NGAL (Kjeldsen et al., 1993) and MMP-14 (Biogenesis Ltd., Poole, England) antibodies were used. Accurate antibody dilutions were chosen according to preliminary studies. Purified human neutrophil MMP-8 (Sorsa et al., 1994) and lyzed equine blood neutrophils were analyzed in parallel for neutrophil type MMP-8. Purified human MMP-9 (Westerlund et al., 1996) and MMP-13 (Lindy et al., 1997) were used as positive controls on MMP-9 and MMP–13 blots. Immunoblotting Gelatinolytic bands detected by zymography (II, III), collagenases (VI), and related MMPs (II) were identified by Western blotting. TELF samples were diluted 1:6 according to the urea blood concentration; the most diluted TELF and BALF samples were concentrated 10-fold by freeze-drying and dissolving in distilled aqua. After electrophoretic separation on SDS-PAGE gels, the proteins were transferred on to nitrocellulose membranes (Schleicher & Schull, Dassel, Germany). Nonspecific binding was blocked with gelatin and membranes were then incubated with specific antibodies against MMP-2 (1:500 dilution), MMP-8 (1:500), MMP9 (1:200), MMP-13 (1:700), MMP-14 (1:300), or NGAL (1:500) for 12 hours at 22 o C. This was followed by peroxidase conjugation with goat antirabbit immunoglobulins (Sigma Chemicals Co., St. Louis, MO, USA) for 1 hour at 22o C. After washing, the blots were developed with nitroblue tetrazolium and 5-bromochloro-3-indolyl-phosphate in N-N-dimethylformamide. For each specific MMP, purified human MMP was run in parallel as a positive control. Specific immunoreactivities were visualized as dark bands. As in zymography, a scanner linked to the image analysis and processing systemwas used to record membranes. The background gray level was subtracted, and densitometric results were calculated in area mode. Western blots of MMP-9 and MMP-2 were analyzed for total immunoreactivity, consisting of complex-, pro-, and active forms and degradation products (Mellanen et al., 1998). NGAL was analyzed for total immunoreactivity, consisting of complex-, dimer-, and monomer forms. Western blots of collagenases MMP-8 and MMP-13 were analyzed for total immunoreactivity and for different MW (kDa) forms. 3.3.2.2. Immunocyto- and immunohistochemistry (detailed procedure in studies II, III, and VI) The previously mentioned specific rabbit polyclonal antihuman MMP-2 (II, 40
Materials and methods III), MMP-8 (VI), MMP-9 (II, III), and MMP-13 (VI) antibodies were used. Cytospin cell slides or frozen lung tissue sections were melted at room temperature, air dried, and fixed in 4o C acetone and were thereafter washed in PBS. For immunostaining, a commercial kit (Dako, Glostrup, Germany) was used according to manufacturers’ instructions. Nonspecific binding was blocked with goat serum (1:50) for 20 minutes at 20o C. Cells were incubated with the specific antibodies for MMP-2 (1:700), MMP-8 (1:250), MMP-9 (1:500), or MMP-13 (1:500) for 30 minutes at 37o C and thereafter at 4o C for 17 hours. After treatment with antirabbit secondary serum and streptavidine, the slides were stained with Cromogen-AEC stain (Dako, Glostrup, Germany) for 10 minutes and counterstained with Mayer’s hematoxylin. For negative controls, the specific antibodies were replaced with 2 µg/ml rabbit immunoglobulin G or with 3% BSA in PBS. 3.3.3. In situ hybridization (detailed description in studies II and VI) For detection of MMP-8 and -9 mRNA in the cytosol of BALF cells and lung tissue cells, in situ hybridization was basically performed as described by Mäkelä et al. (1999). All solutions were treated with 0.1% diethylpyrocarbonate (DEPC). RNA probes produced in vitro by transcription from human cDNA for MMP-8 (VI) and MMP-9 (II). Digoxigenin (DIC)-conjugated UTP (Boehringer, Mannheim, Germany) was incorporated into the RNA probes during the synthesis phase, and corresponding sense probes were used as controls for nonspecific hybridization. Before hybridization, the frozen sections were thawed, rehydrated, incubated in 0.2 M HCl for chemical proteolysis, and washed twice in DEPC water. Sections immersed in glycine were acetylated in acetic anhydride in triethanolamine for background reduction and equilibrated in standardized saline citrate before blocking prehybridization at 58o C for MMP-9 (II) and at 57 o C for MMP-8 (VI). Hybridization buffer containing DIC-labeled antisense or sense probe was applied and hybridization occurred overnight at 58o C. Hybridization and posthybridization washes were done under stringent conditions. Digoxigenin was detected with alkaline phosphatase substrate Fast Red chromogen (Boehringer, Mannheim, Germany) after using alkaline phosphatase-conjugated anti-DIC Fab fragments. Finally, the sections were dipped in tap water and counterstained with Mayer’s hematoxylin.
3.4. Statistical analysis A computerized statistical program (Prizm, Graph Pad Software Inc., San Diego, USA) was used to analyze the data. The nonparametric Mann-Whitney test 41
Materials and methods was used to define differences between TELF and BALF samples, between healthy and COPD horses, and between values after miscellaneous challenges (III a). The paired Wilcoxon signed-rank test was used to test correlations between gelatinolytic activities in TELF or BALF, and between immunoreactivities of MMP-2 and MMP14 or between MMP-9 and monomer NGAL in TELF or BALF of COPD and healthy horses. Friedman two-way analysis of variance for ranks, followed by Dunn’s multiple comparison tests, with baseline contrasted to other time points, were used to test for the effect of time on the three analyzed MMP activities and neutrophil content in COPD horses’ BALF in the time-course study (III b). A two-tailed Pearson linear regression analysis was used to test elastinolytic activity analyzed with the two methods introduced in study IV. After pooling data of healthy and COPD horses, using the nonparametric Spearman rank correlation test, the correlation between neutrophil (scores in TELF/counts in BALF) and other measured parameters (levels of gelatinolysis/elastinolysis/proMMP-9/active MMP-9/MMP2 and collagen degradation %) or correlations between collagen I degradation and MMP-8/MMP-13 immunoreactivity in TELF were tested. Significance was assumed at P < 0.05.
42
Results 4. RESULTS
4.1. Sample collection 4.1.1. TELF and BALF (I, II, V, VI) Mean percentages of TELF and average recovery of BALF are summarized in Table 2. Mean percentages of TELF adjusted according to urea concentration were significantly higher for COPD horses (P < 0.05). No significant difference was observed in recovery volume of BALF or in total number of cells recovered in BALF between COPD and healthy horses. Neutrophil scores in TELF and neutrophil percentages in BALF were used to group COPD horses. The neutrophil content of TELF/BALF was markedly higher (P < 0.01) in COPD horses as compared with healthy horses. Relative neutrophil scores in TELF showed a high correlation with absolute neutrophil counts in BALF (R = 0.81, P < 0.001). The lymphocyte number in BALF was significantly lower (P < 0.05) in COPD horses. Macrophage, mast cell, and eosinophil numbers did not differ significantly between COPD and healthy horses. Neutrophil contents in BALF were statistically higher (P < 0.05) only in COPD horses after challenges with HS or HDS as compared with saline challenge. In contrast, LPS inhalation increased (P < 0.05) BALF neutrophil content both in COPD and healthy control horses. In the time-course study, BALF neutrophil content increased significantly (P < 0.01) at 5 hours post-challenge (III). 4.1.2. Blood neutrophil separation (I) The purity of the collected blood neutrophil and lymphocyte fractions was 97.5% and 96.4%, respectively.
43
Results
Healthy controls
COPD
Study
I
II
V
VI
total
I
II
V
VI
total
n=
4
15
10
12
19
7
17
10
17
29
1.26
3.43
4.42
3.4
2.97 (1.06-13.11)
35.6
39.4
48.5
39.9
38.7* (4.95- 83.33))
recovery (ml)
170.5
166.3
163.4
170.5 (138–221)
156.9
147.3
155
156.9 (102-202)
Neutrophils X 1000 cells/ml
8.4
9.6
8.6
8.4 ( 0-17)
168.3
240.6
193.6
168.3* (25.4 -765)
Lymphocytes X 1000 cells/ml
187
214.6
192
187* (75–385)
125.6
118.1
116
126 (30–229)
Macrophages X 1000 cells/ml
219
241.2
222.2
219 (117–356)
163.6
123.7
135.4
164 (18–360)
Eosinophils X 1000 cells/ml
3.1
4.2
3.6
3 ( 0–19)
0.4
0.4
0.3
0.3 (0–2)
TELF TELF %
BALF
Table 2. Summary of sample collection in studies I,II,V, and VI, with values expressed as averages (ranges). *values in bold are significantly different between COPD and healthy horses.
4.2. Zymography 4.2.1. Standardization (I) Total neutrophil number fitted well (r2 = 0.97) to a logarithmic curve when a twofold dilution series of the equine blood neutrophil fraction which was used as a standard was assayed. Total gelatinolytic activity in 885 equine neutrophils was equivalent to gelatinolytic activity in 0.04 µg of bacterial collagenase type IV (Clostridium histolyticum E.C.3.4.24.3, digestion activity 445 units/ng, Merck, Darmstadt, Germany) (I, Figure 1). An equal amount of neutrophils was used as a positive interassay standard that was run in parallel for each zymography gel. Gelatinolytic activity was expressed in relation to activity of the neutrophil standard value. The lower limit of detection for the assay was gelatinolytic activity in 20 equine neutrophils, which corresponded to gelatinolytic activity of 0.9 ng of bacterial collagenase type IV. 44
Results 4.2.2. Gelatinolytic and elastinolytic activity 4.2.2.1. TELF All TELF samples were found to exhibit gelatinolytic activity (Table 3). Activity in COPD horses was significantly higher (P < 0.01 (I), P < 0.001 (II)) than in healthy horses for all forms analyzed. The activity in TELF was significantly higher (P < 0.0001) than activity in an equivalent volume of blood serum (I, Figure 4). TELF was also found to degrade κ-elastin (V). Elastinolytic activity was significantly higher (P < 0.001) in TELF of COPD horses than that of healthy horses (Table 3). 4.2.2.2. BALF Clinical samples Similar to TELF samples (Table 3), corresponding findings regarding gelatinases were recorded in BALF samples (II). BALF of COPD horses showed significantly increased (P < 0.0001) total gelatinolytic activity as well as significantly increased activities of high MW gelatinase forms (P < 0.0001), proMMP-9 (P < 0.0001), and active MMP-9 (P < 0.0001), both in native BALF and in cell-free BALF. Only in cell-free BALF, proMMP-2 activity was significantly elevated (P < 0.0001) in COPD horses in relation to healthy horses. Low MW gelatinases were mostly observed in cell-free BALF in COPD. High MW gelatinase complexes were detected only in low concentrations in TELF/BALF from healthy horses. The elevation in total gelatinolytic activity, in high MW gelatinase forms as well as in pro- and active MMP-9 forms, showed a significantly high correlation (P < 0.0001) between TELF and BALF of COPD horses. Miscellaneous challenges (III a) No statistically significant differences were present between gelatinolytic activities in BALF samples collected 6 hours post saline challenge from COPD or healthy horses. Values were identical to baseline values analyzed at prechallenge in the timecourse study (Table 3). Thus, saline challenge activities were considered as control values. BALF samples collected 6 hours after LPS challenge revealed significantly increased (P < 0.05) total gelatinolytic activity for both COPD and healthy horses. In contrast, challenge with HS and HDS showed significantly increased (P < 0.05) total gelatinolytic activity for only COPD horses. The increase in total gelatinolytic activity was due to MMP-9 and its activation forms, whereas MMP-2 activities remained statistically unchanged. 45
Results
Healthy controls
COPD
study
I
II
III a
V
VI
I
II
III a
n=
4
15
6
10
12
7
17
6
S
challenge sampling time
LPS
HS
HDS
S
LPS
HS
III b
V
VI
VI
7
10
17
5
HDS
HS 0h
6h
5.6
24.1*
pasture stabling
ZYMOGRAPHY (total relative activity) TELF gelatinolysis BALF gelatinolysis TELF elastinolysis
5.9
6.8 7.0
49.2* 34.4* 12.8 25.4* 12.1 9.4
38.2* 12.7 29.5* 28.2* 28.2* 10.4
114.4*
11.9
162.6*
FLUOROMETRY (total relative activity) TELF elastinolysis
COLLAGEN I degradation (%) TELF
1.8
12.4*
3.5
27.7*
Table 3. Summary of average results of zymographic, fluorometric, and collagen I degradation assays. Zymography results of TELF are adjusted according to urea blood concentration and both TELF and BALF are related to the neutrophil standard on each zymograph. Fluorometry results of TELF are adjusted according to urea blood concentration. HS = hay-straw, LPS = lipopolysaccharide, HDS = hay-dust suspension, and S = saline control challenge. *values in bold are significantly different between COPD and healthy horses.
46
Results Time-course study (III b) Total gelatinolytic activity was significantly increased 5 hours (Table 3) and 24 hours postchallenge. The increase was due to MMP-9. At 5 hours postchallenge, both pro- and active MMP-9 forms were significantly elevated (P < 0.01 and P < 0.05, respectively). At 24 hours postchallenge, the active MMP-9 form declined to its prechallenge level, but the proMMP-9 form remained significantly increased (P < 0.05). Total gelatinolytic activity declined to prechallenge level in four days. No significant differences were detected for MMP-2 activities. 4.2.2.3. Blood Serum (I) No statistically significant difference was found in total gelatinolytic activity in serum between healthy horses and horses with COPD. Neutrophils and lymphocytes (I,V) Both neutrophils and lymphocytes were found to express gelatinolytic activity, with gelatinolytic activity in neutrophils being significantly higher (P < 0.001) than in lymphocytes. For individual horses, gelatinolytic activity in neutrophils did not correlate with activity in lymphocytes. 4.2.3. Gelatinolytic and elastinolytic activities at different molecular weight ranges The sites of the two major gelatinases, as detected by zymography, were identified by parallel runs of purified human MMP-2, purified human MMP-9, and MW standards on the gel (Figure 2). Gelatinolytic activities both in TELF and BALF represented altogether eight distinct molecular sizes, two of these representing high MW gelatinase complex forms (> 110 kDa), one evidently representing proMMP-9 (range 90 – 110 kDa), two evidently representing active MMP-9 (range 75 – 85 kDa), one evidently representing proMMP-2 (range 65 – 75 kDa), and two representing lower (< 50 kDa) MW gelatinolytic species. All samples from COPD and healthy horses revealed gelatinases corresponding to proMMP-9 and proMMP-2 sizes. Most activity on elastin substrate gels (V) was expressed at 80 to 95 kDa, which was equal to the activity of purified human MMP-9. Purified human MMP-2 was expressed at ~72 kDa, but corresponding elastinolytic activity was not found for TELF of that MW (Figure 2). In addition, slight elastinolytic activity at 25 kDa in TELF of COPD horses was found. Neutrophils did not express gelatinolytic/elastinolytic activity at 65 to 75 kDa, 47
Results
Gelatin substrate MMP-2 MMP-9 TELF
Elastin substrate BALF
L
PMN
MW
MMP-2 MMP-9
TELF
PMN
104 kDa 81 kDa
47.7 kDa
34.6 kDa
Figure 2. Gelatin and elastin zymographies of purified MMP-2 and -9, of TELF and BALF samples, and of separated blood lymphocytes (L) and blood neutrophils (PMN).
but expressed activity at other MW ranges detected in TELF/BALF. Lymphocytes expressed gelatinolytic activity corresponding to both MMP-2 and MMP-9. For all samples, the range corresponding to proMMP-9 expressed the highest gelatinolytic activity. 4.2.4. MMP-9 activation degree (II, III) Precentage of activated MMP-9 was calculated from zymograms of TELF and BALF. For COPD horses, the median activation values were in TELF 41% (range 14.9 - 76.6%) (Figure 3A), in native BALF 34.1% (range 16.7 - 48.1%), and in cell-free BALF 31.4% (range 15.9 - 60.9%) (Figure 3B). For most healthy samples, no active MMP-9 could be detected. Five hours post HS challenge, MMP9 activation % increased significantly (P < 0.05) compared with prechallenge values (Figure 3D); however, for subsequent samples, the difference disappeared. When compared with the saline control, for COPD horses only, the MMP-9 activation % was significantly increased after HS (P < 0.01) and after HDS (P < 0.01) challenge, but not after LPS challenge, (Figure 3C). For healthy horses, none of the challenges increased MMP-9 activation as compared with the saline control.
48
Results
A
B
MMP-9 activation %
100
***
80
***
60 40 20 0 Healthy
COPD
Healthy
C
COPD
D
MMP-9 activation %
100 80
* 60
*
*
40 20 0 Saline
LPS
HS
HDS
0h
5h
Figure 3. Activation % of MMP-9 in TELF (A), Cell-free BALF (B), after saline, LPS, HD, HDS challanges of COPD horses (C), and pre- (0 h) and post- (5 h) HS challenge of COPD horses (D). ***P < 0.001, *P < 0.05.
49
Results 4.3. Fluorometry (V) 4.3.1. Standardization Determination of elastinolytic activity in TELF was optimal with 2 mg of substrate/ml. Only results obtained during the initial 6 hours of both measurement periods were used because decreased sample volume as a result of evaporation started to disturb fluorescence values after 6 hours of incubation. The effect of evaporation was detectable as variable fluorescence values, especially with lower substrate concentrations (V, Figure 1). Therefore, the area under the curve was used to study differences in elastinolytic activities during the first 6 hours of measurement in native TELF, and only during the first 4 hours to study effects of inhibitor supplementation on the elastinolytic activities in TELF. 4.3.2. Elastinolytic activity Elastinolytic activities in TELF obtained from horses with COPD were significantly higher (P < 0.05) than those obtained from healthy horses during the 6hour measurement period (Table 3). After 66 hours of incubation, the corresponding activities for COPD horses ranged from 442 to 2 467 (median 1 207); however, no elastinolytic activity was detectable for healthy horses. Elastinolytic activity in TELF determined by fluorometry correlated significantly (R = 0.868, P = 0.005) to elastinolytic activity determined by zymography after 66 hours of incubation.
4.4. Activation and inhibition 4.4.1. Activation effect of APMA (I) Zymography revealed that APMA activation transferred gelatinolytic activities towards smaller MW proteins. A 10 to 15-kDa change towards smaller MW products was detected. The most obvious changes were scanned at ranges above 110 kDa, and between 103 and 106 kDa. In addition, total gelatinolytic activity decreased by 24%. 4.4.2. Inhibitory effects of EDTA (I, II, V) All gelatinolytic activity in TELF, BALF, blood neutrophils, and blood lymphocytes was completely inhibited by 10 mM of EDTA (I, II). Elastinolytic activity in TELF was also completely inhibited by 10 mM EDTA as revealed by elastin zymography (V). In contrast, using fluorometry, elastinolytic activity was only 50
Results partly but significantly inhibited during the first 4 hours of measurement, by 1 mM EDTA (median 31%, range18 - 54%, P < 0.01) and by 2.5 mM EDTA (median 42%, range 20 - 70 %, P < 0.05) in TELF samples from horses with COPD (V). Inhibitory effects were already detectable after one hour by 1 mM EDTA (median 29%, range 22 - 39%, P < 0.05) and by 2.5 mM EDTA (median 44%, range 25 56 %, P < 0.05) in TELF samples from horses with COPD. Higher concentrations of EDTA interfered with the substrate in fluorometry. 4.4.3. Inhibitory effects of CMT-3 (IV, V) No inhibition was detected if CMT-3 was used only at preincubation of samples, but inhibition was achieved when CMT-3 was also added to washing and incubation solutions in zymography (IV). For gelatinolytic activity, inhibition rates of 86% for TELF and 100% for human recombinant MMP-9 were achieved with 150 µM CMT-3 (IV, Figure 1). Elastinolytic activity was totally inhibited by 500 µM CMT3, and visible inhibition was observed with 150 µM CMT-3 (V). For gelatin zymography of TELF, the IC50 with CMT-3 was between 20-90 µM (IV). In fluorometry, 25 µM CMT-3 significantly inhibited (median 26%, range 11 39%, P < 0.01) elastinolytic activity in TELF samples of horses with COPD during the 4-hour measurement period (V). An inhibitory effect was already detectable after one hour (median 43%, range 19 - 66%, P < 0.05). Higher CMT-3 concentrations interfered with the substrate.
4.5. Type I collagen degradation (VI) The average degradation percentage of collagen I was significantly higher (P 0.5), 51
Results in BALF. After pooling data from all challenges (III a), absolute neutrophil counts were revealed to be correlated with BALF levels of proMMP-9 (R = 0.83, P < 0.0001) and active MMP-9 (R= 0.67, P < 0.0001), but not MMP-2 (R = 0.14, P > 0.5). Relative neutrophil score in TELF correlated significantly with total elastinolytic activity determined by zymography (R = 0.708, P < 0.001) and fluorometry (R = 0.9662, P < 0.001). Collagen I degradation percentages correlated both with relative neutrophil scores in TELF (R = 0.79, P < 0.0001) and absolute neutrophil counts in BALF (R = 0.79, P < 0.0001).
4.7. Western blotting 4.7.1. MMP-9 and NGAL Western blots from equine TELF (II) and BALF (II, III) showed immunoreactivity for high MW complex forms of MMP-9, proMMP-9, and four different active forms of MMP-9. Moreover, low MW (< 40 kDa) immunoreactivities marked as degradation products of MMP-9 were detected. Levels of immunoreactive MMP-9 (II) in TELF from COPD horses were significantly higher (P < 0.0001) than those from healthy horses for all forms of MMP-9 analyzed. Total immunoreactivity for MMP-9 (II) was increased more in native BALF than in cell-free BALF. The increase in native BALF was mainly due to a significant increase (P < 0.0001) in high MW and pro-forms of MMP-9, whereas in cell-free BALF especially the active forms of MMP-9 were clearly significantly increased (P < 0.0001). In addition, low MW MMP-9 immunoreactivities showed a greater increase in TELF (P < 0.0001) and BALF (P < 0.001) from COPD horses than healthy horses. Increased levels of 25-kDa monomer NGAL immunoreactivity (II) were detected in TELF of COPD horses (P < 0.01), and this increase coexisted with increased levels of active MMP-9 immunoreactivity (P < 0.05). 4.7.2. MMP-2 (II, III) and MMP-14 (II) Western blot using an antibody specific to MMP-2 confirmed 65 to 75-kDa gelatinases to represent MMP-2. No significant differences for levels of MMP-2 immunoreactivities (II) in TELF or cell-free BALF were detected between COPD horses and healthy horses. Native BALF showed a significant increase (P < 0.01) for high MW forms of MMP-2. Total relative immunoreactivity of MMP-14 was not elevated in TELF of COPD horses (4628 + 1094) as compared with that of healthy horses (4164 + 1008) (P = 0.54).
52
Results 4.7.3. MMP-8 (VI) Western blot analyses of TELF for MMP-8 showed immunoreactivity at four MW sizes: at approximately 80 and 70 kDa, and at approximately 40 and 30 kDa. Furthermore, some small 20-kDa forms assumed to be degradation products, and complexed forms exceeding 80 kDa were detected. All of these forms were also present in equine neutrophil lysates. A statistically significant (P < 0.001) 13-fold increase in total immunoreactivity for MMP-8 was detected from TELF of COPD horses as compared with TELF from healthy horses. The highest increases (P < 0.001) in MMP-8 immunoreactivities in COPD horses were detected at 40 kDa and at 20 kDa. A significant difference (P < 0.01) between TELF of COPD and healthy horses was also found in MMP8 immunoreactivity at the 70 to 80-kDa range. 4.7.4. MMP-13 (VI) Western blot for MMP-13 showed the most prominent immunoreactivity at two MW sizes: at approximately 30 to 35 kDa, and at approximately 20 to 25 kDa. In addition, a slight reaction was observed at approximately 55 kDa. A statistically significant (P< 0.001) 26-fold increase was found in total immunoreactivity for MMP13 in the TELF of COPD horses as compared with TELF of healthy horses. Immunoreactivities of both main detected forms of MMP-13 were significantly different (P < 0.001) between TELF from those two groups. Total immunoreactivity of MMP-8 and MMP-13 correlated with collagen I degradation percentage (R = 0.639, P = 0.01 and R = 0.682, P < 0.01, respectively). 4.7.5. Effects of CMT-3 inhibition (IV) Western blot analyses showed no inducible fragmentation of MMP-8 or MMP9 by CMT-3 treatment of TELF or human recombinant MMP-9.
4.8. Identification of cellular sources of MMPs 4.8.1. Immunocyto- and immunohistochemistry Immunocytochemistry of BALF cells revealed MMP-9 protein in macrophages, neutrophils, and lymphocytes. Separated blood neutrophils expressed MMP-9 protein intensely. In addition, epithelial and macrophage-like cells showed MMP-2 immunoreactivity.
53
Results Clear immunoreactivities to MMP-8 and MMP-13 were detected in BALF epithelial cells and in macrophages. MMP-9 protein was expressed in a lung tissue section from a COPD horse. (Figure 4) 4.8.2. In situ hybridization In situ hybridization revealed MMP-9 mRNA expression in bronchial epithelial cells of a lung tissue section from a COPD horse. Furthermore, BALF macrophages were shown to express MMP-8 mRNA (Figure 4).
Figure 4. (right) (a-e) Immunocytochemistry on bronchoalveolar cells from a COPD horse: (a) MMP-2, (b) MMP-8, (c) MMP-9, (d) MMP-13, and (e) negative control. Immunohistochemistry on lung tissue from a COPD horse: (f) MMP-9, and (g) negative control. In situ hybridization: (h) MMP-8 mRNA in bronchoalveolar cells from a COPD horse, and (i) MMP-9 mRNA in lung tissue from a COPD horse. Scale bar 100µM. Õ neutrophil, ê macrophage, Þ epithelial cell.
54
Results
b
a
e c
d
f
g
h
i
55
Discussion 5. DISCUSSION Characteristic to equine COPD is a chronic inflammatory condition of the lung as a result of a delayed hypersensitivity response to continual antigen inhalation. As an inflammatory response, neutrophils invade the lung and accumulate in the lumen of terminal airways (Robinson et al., 1996). Thus an increased number of neutrophils is typically revealed in the epithelial lining fluids of lungs of COPD horses by means of both tracheal epithelial lavage and bronchoalveolar lavage (Roszel et al., 1985; Mair et al., 1987; Roberts, 1992; Tremblay et al., 1993). The lung-invading neutrophils are believed to originate from blood (Fairbairn et al., 1993). Our study indicated elevated neutrophil counts for all COPD horses examined, both in TELF representing upper and lower airways, and in BALF representing lower airways. Increased proteolysis (von Fellenberg, 1987, Koivunen et al., 1996), expressing collagenolytic (Koivunen et al., 1997b) and gelatinolytic (Koivunen et al., 1997a) activities, has recently been found in TELF of COPD horses. Increased collagen degradation has also been shown to occur during development of lung emphysema in guinea pigs and in man (Selman et al., 1996; Ohnishi et al., 1998), and to be associated with tissue destruction of rat lungs exposed to 100% oxygen (Pardo et al., 1996). These findings indicate a clear association between BALF MMPs and respiratory disease activity and progression. Increased elastinolytic activity has traditionally been considered to be the main cause of proteolytic lung destruction in various chronic lung diseases (Janoff, 1983b). Increased amounts of elastinolytic activity have been detected in human respiratory tract secretions of smokers and those with α 1 -antitrypsin deficiency (Janoff et al. 1983a, Janoff, 1985). However, despite attempts (Grünig et al., 1985), elastinolytic activity has not been successfully detected and characterized in RS of horses. Disturbances in the delicate balance between proteinases and proteinase inhibitors have been suggested to be important in the pathogenesis of chronic lung diseases, including equine chronic noninfectious lung inflammatory diseases (von Fellenberg, 1987). For a long time, interest was focused on the changes in the molecular ratio of elastases and its inhibitors as the aetiopathogenic cause of human asthma and emphysema (Gadec et al., 1984). From elastases, the investigation extended to other proteinases, including matrix metalloproteinases. The most recent interest in the pathogenesis of chronic lung diseases has focused on gelatinases. An imbalance between MMP-9 and its natural inhibitor TIMP-1, with an excess of MMP-9, has been found in the lungs of asthma patients (Hoshino et al., 1998; Vignola et al., 1998; Mautino et al., 1999a,b; Tanaka et al., 2000). The imbalance 56
Discussion of MMP-9/TIMP-1 has been related to airway inflammation, hyperresponsiveness, and abnormal remodeling in asthma (Hoshino et al., 1998). Several proteinolytic enzymes are capable of degrading collagen, gelatin, and elastin. The MMP family members are zinc-dependent enzymes that cleave almost all major components of the extracellular matrix and BM (Birkedal-Hansen et al., 1993a; O’Connor and FitzGerald, 1994; Backett et al., 1996). All MMPs, including extracellular and membrane-associated MMPs, are produced and secreted in a proenzyme form (O’Connor and FitzGerald, 1994; Backett et al., 1996) and require extracellular (Zaoui et al., 1996), or cell surface-associated, activation to be catalytically competent. Besides being linked to normal physiological turnover and remodeling of connective tissues, MMP activity has been related to tumor metastasis and invasion, infiltration and migration of inflammatory cells, inflammatory tissue destruction, and wound healing. In addition, it has been suggested to be an important mediator in the development of allergic inflammation (Holgate, 1997). In lungs, increased metalloproteinase-type activity was first detected in humans with idiopathic pulmonary fibrosis (Gadek et al., 1979) and subsequently was reported in connection with other respiratory tract diseases such as BE, emphysema, and asthma (Sepper et al., 1995; Finlay et al., 1997a; Lemjabbar et al., 1999). In a model of asthma in mice, MMPs have been suggested to be crucial for the infiltration of inflammatory cells and the induction of airway hyperresponsiveness (Kumagai et al., 1999). The definition of metalloproteinase activity is based on the requirement of MMPs for cations in specimens. Inhibitory effects of the chelating agent EDTA (Zaoui et al., 1996) verified that all detected activities, as determined by κ-elastin and gelatin zymographies, were attributable to metalloproteinases. In the fluorometric assay, EDTA inhibited up to 70% of elastinolytic activity in TELF, suggesting a major involvement of MMP-type elastases in TELF. APMA activation is based on the characteristic feature of MMPs losing their propeptide domain, thereby leading to conversion of proMMP to its active forms in a stepwise manner (Zaoui et al., 1996). APMA generates a reduction of 10 to 15 kDa in MW of detected gelatinolytic activities, a decrease that typically resembles conversion of MMP-proenzyme to its active forms (Leppert et al., 1995). Mainly based on these properties, the presence of metalloproteinase activity was confirmed in horse TELF and BALF. MMPs are usually subgrouped into collagenases, gelatinases, stromelysins, matrilysins, and other MMPs. The involvement of two MMP subgroups, collagenases and gelatinases, have subsequently been characterized in TELF and BALF. Among collagenases, especially the inductive MMP-8 and MMP-13 have been 57
Discussion connected to pathological tissue destruction associated with inflammation (Weiss, 1989; Vaalamo et al., 1997; Uitto et al., 1998; Romanelli et al., 1999; Prikk et al., 2001), while constitutively expressed MMP-1 is mainly involved in physiological tissue remodeling, e.g. re-epithelialization of dermal wounds (Saarialho-Kere et al., 1993). MMP-8 has been suspected to be the major interstitial collagenase species behind the increased collagenase activity in lungs of emphysematous and BE patients (Sepper et al., 1995; Finlay et al., 1997b). In this work, a 7-fold increase in autoactive collagenase, as measured by a type I collagen degradation assay, was detected in TELF of COPD horses. The increase in collagenolytic activity in TELF was associated with the symptomatic disease stage. The apparent decrease in collagenolytic activity at remission, strongly suggests that collagenases participate in lung tissue destruction during exacerbation, the symptomatic phase of equine COPD. Gelatinolytic MMP activity, determined by gelatin zymography from upper RSs by TELF, and from lower RSs by BALF, seemed to be markedly higher along the entire respiratory tract of COPD horses as compared with healthy horses. The elevated gelatinolytic activities from TELF and BALF were highly correlated in COPD horses. The main gelatinolytic MMPs detected by means of zymography included MMP-2 and MMP-9, as well as their aggregates and break-down products (Sepper et al., 1994; Westerlund et al., 1996). Besides direct degradation of elastin, elastases including MMPs have a role in biological cascades that indirectly result in degradation of elastin. Furthermore, elastinolytic MME promotes elastinolysis by cleaving α 1 -antiproteinase, thus destroying the body’s endogenous neutrophil anti-elastase shield (Shapiro et al., 1991a). Equine neutrophil elastase and α 1 -antiproteinase are both detected in equine neutrophils (Dagleish et al., 1999). In addition to being gelatinolytic, gelatinase MMPs, particularly MMP-2 and MMP-9, are known to degrade elastin (Senior et al., 1991). The fluorometric elastase assay indicated a clear difference in TELF elastinolytic activity between healthy horses and horses with COPD, with higher activities for COPD. With fluorometry, total elastinolytic activity is measured, apparently including neutrophil elastase, MME, and other elastinolytic MMPs (Chandler et al., 1996). Elevated levels of elastinolytic activity in TELF of COPD horses were also visualized by κ-elastin zymography, with the major activity corresponding to human MMP-9. The involvement of collagenase- and gelatinase type MMPs was thus evident. To identify specific collagenase and gelatinase subspecies, Western immunoblotting using specific antibodies was performed. Several immunoreactivities for the collagenases MMP-8 and MMP-13 were revealed. A 13-fold higher immunoreactivity for MMP-8 was found for TELF of COPD as compared with TELF of healthy horses. According to MW, immunoreactivities were presumed to 58
Discussion correspond to pro- and active PMN-type MMP-8, and to pro- and active mesenchymal type MMP-8 (Hanemaaijer et al., 1997). In addition, a small, approximately 20-kDa, immunoreactivity was assumed to represent a fragmented lower MW form of MMP-8. High MW (> 80 kDa) forms of MMP-8 most probably represented complex forms of MMP-8 such as activated MMP-8 species bound to endogenous inhibitors like α 2 -macroglobulin and/or tissue inhibitors of metalloproteinases (Nagase et al., 1994). A 26-fold higher immunoreactivity for MMP-13 was found in TELF of COPD horses than in TELF of healthy horses. Only the presumed active and fragmented species of MMP-13 were present according to MW; the active 35-kDa MMP-13 form significantly differentiated healthy TELF from COPD TELF, as it was clearly evident in fluid of all COPD horses but virtually absent in that of healthy horses. The human proMMP-13 is displayed in a glycosylated 60-kDa form that is converted to the 48-kDa active form (Knäuper et al., 1996a). An intermediate species of approximately 55 kDa was slightly detected in a few of the COPD TELF samples and may represent the proMMP-13 form. All detected forms were of smaller MW than those of humans, which may represent differences between species. Furthermore, MMP-13 has been shown to participate in the activation cascade of MMP-9 (Knäuper et al., 1997). Since MMP-9 was found to be activated in TELF of COPD horses, the two detected forms of MMP-13 may well represent intermediates or fragments resulting from that activation process. Besides the two detected collagenase MMPs, a the third collagenase-type MMP, MMP-1, has also been suggested (Koivunen et al., 1997) to be partly involved in the collagenolytic activity in equine RSs. Analysis of zymography results revealed the main elastinolytic and gelatinolytic activities as having the same MW as pure human MMP-9, but only weak activity was found having the same MW as pure human MMP-2. The main gelatinolytic activity was also converted by APMA into lower MW forms, similar to conversion of MMP-9 (Leppert et al., 1995). These results indicated that the main activity was evidently attributable to MMP-9, representing pro- and active forms. The additional 25-kDa elastinolytic activity detected for COPD horses may represent MME (MMP12) or equine neutrophil elastase, the most abundant elastase in neutrophils (Dubin et al., 1994; Dagleish et al., 1999). Both of these elastases can cleave insoluble elastin (Chandler et al., 1996), and have been associated with pulmonary emphysema (Janoff, 1985). Equine neutrophils express and release elastinolytic neutrophil elastase and certain other neutral serine proteinases, although the activities are considerably lower than those reported for human neutrophils (von Fellenberg et al., 1985). Furthermore, the weak activity in the range suspected to represent the constitutive MMP-2 was detected in lymphocytes, TELF, and BALF, but not in neutrophils. Neutrophils have previously been reported not to produce MMP-2 (Shapiro et al., 59
Discussion 1991b; O’Connor and FitzGerald, 1994), while human T-lymphocytes have been shown to express both gelatinase A and B (Leppert et al., 1995). Finally, MMP-2 and MMP-9 were identified and quantified with the Western immunoblot technique using specific antibodies (Westerlund et al., 1996). A positive signal for MMP-9 was immunologically identified at approximately 92 kDa, confirming zymographic findings (Koivunen et al. 1997a). Quantitation revealed that both MMP-9 immunoreactivities and gelatinolytic activities were clearly increased in TELF and BALF from symptomatic COPD horses and in BALF from challenged COPD horses. Interestingly, after LPS challenge, for both COPD and healthy horses, increased MMP-9 and total gelatinolytic activity levels were found. In contrast, after hay challenge, increased levels were detected only for COPD, whereas healthy horses’ levels showed no significant changes. In addition to increased total immunoreactivity and MMP-9 activity, MMP-9 was found to be extensively activated in RS of COPD horses, while healthy horses mostly exhibited only low levels of proMMP-9. High MW forms of MMP-9 immunoreactivities were clearly increased in RS of COPD horses. These forms evidently represent dimeric forms of MMP-9 (Triebel et al., 1992), complexes of MMP-9 with different MMPs, complexes of MMP-9 with MMP inhibitors such as TIMP and/or with other molecules including NGAL (Kjeldsen et al., 1993; Westerlund et al., 1996). After activation, MMP-9 readily forms complexes with TIMP (Reynolds, 1996). Gelatinolytic activity of approximately 72 kDa was identified to represent MMP2, and quantitation revealed that MMP-2 levels were only slightly elevated, if at all, in COPD as compared with healthy samples. MMP-2 has been considered to be constitutively expressed (Romanic and Madri, 1994; Leppert et al., 1995), and thus, its induction in inflammation has been detected infrequently. Nonetheless, in inflammation involving endotoxin, MMP-2 expressed and secreted locally from the epithelium has recently been shown to be increased during acute or early inflammation (Mäkelä et al., 1999). In addition, levels of membrane type 1 matrix metalloproteinase (MMP-14), an initiator of an activation cascade for MMP-2 (Sato et al., 1994), were not increased in COPD samples. This suggests that MMP-2 and its activation seem to have no active role in tissue destruction associated with equine COPD. While emphysema is not a characteristic feature of equine COPD (Robinson et al., 1996), it is characteristic of human lung emphysema, where increased MMP-2 activity levels and an increased ratio of active forms of MMP-2, together with increased MMP-14 levels, are found (Ohnishi et al., 1998). These findings concerning MMP-2 suggest different gelatinase-type participation in different inflammatory lung diseases with a different pathogenesis. Increased MMP-9 and unchanged MMP-2 levels may confirm the inducible nature of MMP-9 and the constitutive secretion of MMP-2, i.e. the manner by which they are secreted from e.g. human airway smooth muscle cells (Foda et al., 60
Discussion 1999). They may also reveal the specific nature of the equine lung to react to inhaled irritants by secreting gelatinase B. Significantly increased immunoreactivities of NGAL that correlated with increased MMP-9 immunoreactivities in COPD horse TELF samples were observed. NGAL is a 24-kDa protein secreted from secondary granules of activated neutrophils (Kjeldsen et al., 1993) which forms 130-kDa complexes with active MMP-9 (Hibbs et al., 1985). These complexes have been shown to increase in human gingival inflammation (Westerlund et al., 1996). The high MW gelatinolytic forms (exceeding 110 kDa), significantly increased in zymographs of COPD RS samples, most likely partly consisted of complexed MMP-9/NGAL, both of which were shown to exist in increased amounts in TELF of COPD horses by immunoblotting. A parallel increase in MMP-9 and NGAL further points to the involvement and activation of neutrophils in the inflamed lung. MMPs originating from infiltrated blood neutrophils may at least partly explain the increase in collagenolytic, elastinolytic and gelatinolytic activities in RSs of horses with COPD (Dent et al., 1995). Other possible sources of MMPs include lymphocytes (Leppert et al., 1995), macrophages (Senior et al., 1991), mast cells (Kanbe et al., 1999), and epithelial cells (Yao et al., 1996). All of these cells have been found in equine pulmonary epithelial lining fluid (Roszel et al., 1985). The synthesis of MMPs, MMP-8 and MMP-9 originating from PMNs, is believed to already be completed before PMNs emigrate from bone marrow (Hasty et al., 1990; Mainradi et al., 1991), and the mature enzymes are stored in the PMN intracellular granules. Therefore, the action of PMN MMPs is believed to be regulated only by the factors that affect PMN-degranulation and concomitant proteinase release, not by their de novo mRNA and protein biosyntheses (Weiss, 1989; Hasty et al., 1990; Schettler et al., 1991). Unlike PMN MMPs, most other MMPs, such as MMP-2, are not stored in cells, and their synthesis and secretion are regulated mainly at the transcription level (Page, 1991; Birkedal-Hansen et al., 1993b). Among the PMN MMPs, MMP-9 is thought to be important for neutrophil migration across the BM (Delclaux et al., 1996). This indicates a strong possibility of neutrophils contributing to the detected increase in MMP-9 enzyme activities in TELF and BALF of COPD horses. Active MMP-9 in BALF correlated well with neutrophil content during the follow-up after HS challenge, suggesting a clear relationship between MMP-9 and neutrophils. Multiple forms of immunoreactive MMP-8 were found. The subspecies suspected to initiate PMN-type MMP-8 correlated well with neutrophil score in TELF and with the neutrophil % in BALF of COPD horses, whereas other subspecies 61
Discussion did not, suggesting that this MMP form originated from neutrophils during the course of equine COPD. As for the other forms of MMP-8, they seemed to represent the nonPMNtype and thus, apparently, originated from other lung cell types. The expression of nonPMNtype MMP-8 subspecies has been shown to be derived from cells, such as endothelial cells, chondrocytes, and fibroblasts, after induction by proinflammatory mediators such as TNF-α and IL-1β (Hanemaaijer et al., 1997). Increased MMP-8 immunoreactivity has also been found in BALF of BE patients, with multiple forms of activation representing both PMN- and nonPMNtypes as compared with healthy subjects who demonstrated only low levels of immunoreactivity for latent forms of MMP-8 (Prikk et al., 2001). The neutrophiltype MMP-8, identified in BALF of BE patients, correlated with severity of disease (Sepper et al., 1995). The most evident difference in immunoreactivity was detected with the suspected mesenchymal-type MMP-8, suggesting involvement of cells besides neutrophils in COPD pathogenesis. The 7-fold increase in collagenase activity in TELF of COPD horses paralleled the increased percentage of neutrophils in BALF and the increased neutrophil score in TELF. A comparable parallel increase in BALF neutrophils and collagenase activity has been reported for human adult respiratory disease syndrome patients (Christner et al., 1985). Gelatin zymographs revealed what seemed to be MMP-2 and MMP-9 in blood lymphocytes, TELF, and BALF, and only MMP-9 in blood neutrophils. Total gelatinolytic activity in peripheral blood lymphocytes was less than one-third of that in neutrophils, with the main activity matching MMP-9 for both cell types. This result corresponds well with the considerable higher MMP-9 content found in human blood neutrophils than in other human blood leukocytes (Fujisawa et al., 1999). No difference in gelatinolytic activity was observed in peripheral blood leukocytes of healthy and COPD horses, corresponding to MMP-9 content in peripheral blood leukocytes of healthy and asthmatic patients (Fujisawa et al., 1999). Gelatinases in lymphocytes were only slightly activated by APMA, while in neutrophils they were clearly activated, suggesting a different MMP activation mechanism for lymphocytes than for neutrophils. Elastinolytic activity has previously been detected in equine neutrophils (von Fellenberg et al., 1985). Equine blood neutrophil lysates expressed elastinolysis only at the same MW as that for pure MMP-9, indicating that neutrophils are a potential source for elastinolytic MMP-9. Equine neutrophils apparently contain MMP-9, but not MMP-2, similar to human neutrophils (O’Connor and FitzGerald, 1994), and MMP-9 has been shown to be the dominant type of gelatinase in neutrophils (O’Connor and FitzGerald, 1994). Total elastinolytic activity in both 62
Discussion assays used correlated with the neutrophil score in TELF samples, which further supports neutrophils being a predominant source of elastinolytic activity. Macrophages and connective tissue cells are other possible sources for elastinolytic activity. Shapiro (1994) estimated that human MME and MMP-9 each accounted for approximately one-half of macrophage-derived metalloelastase activity in cultured alveolar macrophages. Cellular origins of MMP-2, -8, -9, and -13 in BALF were identified by immunocytochemistry. MMP-9 immunoreactivity was observed in macrophages, lymphocytes, and neutrophils, whereas MMP-2 immunoreactivity was observed in epithelial cells and macrophages. Bronchial epithelial cells of COPD horses also expressed MMP-9 mRNA, in agreement with reports of human bronchial epithelial cells in interstitial lung disease (Fakuda et al., 1998). MMP-8 was detected by immunocytochemistry and in situ hybridization, in equine BALF epithelial cells and macrophages. MMP-8 was also detected in equine blood neutrophils by Western blotting; however, no evident signal for MMP-8 was found by immunocytochemistry in BALF neutrophils. An explanation for this might be that neutrophils at the site of inflammation may release their MMP-8 upon triggered degranulation (Westerlund et al., 1996). A strong signal for MMP-13 was observed in BALF epithelial cells, with an exceptionally strong signal being found at the apical area of these cells. Previous studies have shown that BALF inflammatory cells (neutrophils and macrophages) from asthmatic patients (Mautino et al., 1997) and resident bronchial epithelial cells contain and evidently release MMP-9 and MMP-2 (Hayashi et al., 1996; Yao et al., 1996). Moreover, MMP-8 and -9 have been localized to lung neutrophils (Dahlen et al., 1999; Segura–Valdez et al., 2000). It has also been documented in vitro that alveolar epithelial cells synthesize several MMPs (Pardo et al., 1997) such as MMP-2 and MMP-9 (Hayashi et al., 1996; D’Ortho et al., 1997). Pharmacological application of steroidal anti-inflammatory drugs, i.e. glucocorticosteroids, reduces expression of inflammatory mediators, among them expression of MMP-9 in asthma (Hoshino et al., 1999). Natural inhibitors of metalloproteinases and a synthetic MMP inhibitor have been observed to inhibit cellular infiltration to the airway lumen and to reduce airway hyperresponsiveness in a murine model of asthma (Kumagai et al., 1999). Regarding the therapeutic implications of blocking the pathologically excessive gelatinolytic and collagenolytic cascades, especially in tissue-destructive inflammatory diseases, it has been reported (Golub et al., 1985) that TCs inhibit the catalytic activities of mammalian MMPs, both gelatinases and collagenases. Because of the adverse effects of long-term administration of TCs to horses, chemically modified TC, an agent that lacks the antimicrobial properties of TCs but maintains the ability to inhibit mammalian MMPs 63
Discussion (Golub et al., 1992), was selected to be tested in vitro. CMT-3, which is the most effective TC-derived MMP inhibitor and is known to inhibit human MMP-9 (Sorsa et al., 1998), inhibited all gelatinolytic and elastinolytic activity in TELF as determined by zymography. Moreover, CMT-3 significantly reduced elastinolytic activity determined by fluorometry. Recombinant elastase inhibitors, such as horse blood leukocyte inhibitor, have been suggested for use in treatment of emphysema in horses (Kordula et al., 1993). Total gelatinolytic activity in blood neutrophils, blood lymphocytes, and serum from horses with COPD did not differ from that for healthy horses. Thus, gelatinolytic activity differed between healthy horses and horses with COPD only in the diseased organ itself, the lung. Similar differences in phagocytic activity of lung neutrophils (Klucinski et al., 1994) and IgG levels (Halliwell et al., 1993) have been reported; phagocytic activity of lung neutrophils was significantly higher for horses with COPD than for healthy horses, but no significant difference was observed in phagocytic activity of blood neutrophils. Increased extracellular gelatinolytic activity in BALF and immunoreactivity for MMP-9 detected in the extracellular matrix of asthmatic lungs (Ohno et al., 1997) suggest extracellular secretion of gelatinolytic MMPs during lung disease. In the present study, elevated gelatinolytic activities were found in both native BALF, representing both intra- and extracellular compartments of the fluid, and cell-free BALF, representing only the extracellular compartment. Total gelatinolytic activities in BALF were low for healthy horses, being lower for cell-free BALF than for native BALF, indicating that healthy horses have hardly any gelatinolytic activity released extracellularly. Thus, the gelatinolytic activity in BALF of healthy horses is located mainly intracellularly, suggesting a low degree of activation of inflammatory cells such as neutrophils and monocytes/macrophages. Unlike in healthy horses, the gelatinolytic activity of cell-free BALF from COPD horses was higher than that of native BALF, indicating increased gelatinolytic activity released into the extracellular milieu of the lower airways of COPD horses. In addition, BALF cells evidently contain natural MMP inhibitors, but their extracellular levels are insufficient to provide protection by down-regulating concomitantly released and activated MMPs. Despite the small number of samples studied by fluorometry, there seemed to be a specific inhibitory value for each sample; these results resemble previous findings of inhibition of caseinolytic activities in equine TELF (Koivunen et al., 1996) and suggest a mixed type of elastinolysis, which varies among horses. These findings suggest involvement of other elastases in addition to metalloproteinase-type gelatinases. Noteworthy is that degradation of elastin and perhaps collagen are predisposing to the airspace enlargement that is characteristic of emphysema, whereas 64
Discussion degradation of BM proteins, especially by MMPs, might promote inflammatory cell accumulation and distort epithelial architecture (Shapiro and Senior, 1999), features connected to equine COPD. At remission, active MMP-9 levels in COPD horses, expressed in relation to prechallenge values, were found to be decreased similar to levels of active MMP-9 in RSs of stable asthmatic patients (Vigola et al., 1998). The absence of active MMP-9 in BALF of stable asthmatics was suggested to be due to excessive levels of TIMP-1, the natural inhibitor of MMP-9, that overexceeded MMP-9 (Mautino et al., 1999b). While TIMP-1 and NGAL cannot completely abolish MMP-9 activation they can clearly retard it (Sorsa et al., 1997). Although levels of TIMP1 have not yet been studied in RS of horses, the imbalance between MMP-9 and TIMP-1, similar to human asthma (Tanaka et al., 2000), could explain the increased MMP-9 activation found in COPD horses undergoing active inflammation. This observation is compatible with the observed reduction in collagen I degradation capacity in RS of COPD horses after a period at pasture, with the collagenolytic values for COPD horses approaching values of healthy horses. One of the key observations in this study was the increased level and activation of MMP-9. All samples collected from symptomatic COPD patients and from COPD horses subjected to HS or HDS challenge showed increased levels of MMP-9 that were highly converted to active forms. In contrast, healthy horses showed no increase in MMP-9 levels, not even following HS or HDS challenge. Interestingly, the LPS challenge, both for COPD and healthy horses, significantly increased levels of MMP9, but not the degree of MMP-9 activation. This indicates that both COPD and healthy horses seem to react similarly to substances such as LPS, but differently to respiratory irritation by substances such as hay-dust, in which active MMP-9 seems to play a significant role. Our data suggest that constant respiratory irritation, corresponding to HS or HDS challenge, evidently leads to permanently elevated active MMP-9 levels in the lungs of COPD horses. Thus, active MMP-9 could be one of the endogenous factors mediating lung ECM and bronchial epithelial injury (Holgate et al., 1999) deriving to the chronic nature of equine COPD. Increased levels of complexed MMP-9 may also reflect the elevated degree of MMP-9 activation at the site of inflammation (Bosse et al., 1999). The ratio of MMP-9/TIMP-1 has been used to predict the effectiveness of corticosteroid therapy for asthmatic patients (Bosse et al., 1999). We suggest that MMP-9 levels and MMP-9 activation could similarly serve as diagnostic and predictive clinical indicators of the severity and activity of the lung disease prior to planning treatment of a COPD horse. More generally, MMP-9 and its activation products could serve as specific clinical markers of hypersensitivity inflammation in the equine lung (Brazil and McGorum, 2001). 65
Discussion As Shapiro and Senior (1999) stated,“to determine the role of MMPs in health and disease is to find an association between MMP expression and a biological process or a disease state”. An attempt has been made by these studies to uncover the relationship between gelatinases and collagenases and the pathogenesis and state of disease in equine COPD.
66
Conclusions 6. CONCLUSIONS
1.
APMA activation and EDTA inhibition indicated that the gelatinolytic activity detected by gelatin and elastin zymographs in TELF and BALF from horses is metalloproteinase activity. Apparently, MMPs are also crucial in elastinolysis, similar to their role in gelatinolysis, collagenolysis, and caseinolysis (Koivunen et al., 1996, 1997a, 1997b).
2.
Gelatinolytic activity was increased in RS of COPD horses, but not in serum, blood neutrophils, or blood lymphocytes. Thus, not unexpectedly, measurements of serum, blood neutrophil, or blood lymphocyte gelatinolytic activity were of little diagnostic value in distinguishing horses with COPD from healthy horses. As for human pleural effusions, MMPs are suggested to be from local or localized cells, not from subsequent ultrafiltration of blood (Hurewitz et al., 1992). This supports the concept of local production and control of inflammation, rather than systemic disturbance, at least for gelatinolytic MMPs. Therefore, analysis of MMPs in blood gives little information as to the proteolytic processes in the lung of horses with COPD.
3.
Elastinolytic activity is detectable in equine TELF and seems to be at least partly attributable to metalloproteinases. Elastinolytic activity was found in TELF from all horses, and this activity was significantly higher in TELF obtained from horses with COPD than in TELF obtained from healthy horses. Elastinolytic activity in TELF can be detected by means of κ-elastin zymography or fluorescein-labeled elastin fluorometry.
4.
Two inducible collagenases, MMP-8 and MMP-13, were identified in equine TELF. Immunoreactivities for both gelatinases MMP-2 and MMP9 were also found.
5.
Neutrophils and lymphocytes are possible sources of MMP-9 in RSs. In addition, lung macrophages and epithelial cells were found to be potential sources of MMP-9 and major sources of MMP-8 and MMP-13.
6.
Data obtained indicates that TELF is representative of the entire respiratory tract and might therefore be used besides bronchoalveolar lavage samples 67
Conclusions as diagnostic material to evaluate proteinase activities in the lungs of horses with COPD. Increased gelatinolytic, elastinolytic, and collagenolytic activities possibly reflect active ongoing inflammation and destruction of pulmonary tissue in horses with COPD. Pathologically elevated collagenolytic activity was associated with equine COPD and with the activity of the disease. Immunoreactivities of both MMP-8 and MMP-13 were elevated in TELF of COPD horses as compared with healthy TELF. The active form of MMP-13 differentiated well healthy TELF from COPD TELF, being clearly evident in all TELF of COPD horses but virtually absent in TELF of healthy horses. Assessment of MMP-8 and –13 may provide an additional diagnostic tool for identifying active disease phases of equine COPD. MMP-type elastinolysis is increased in horses with COPD, thus, suggesting that excessive elastinolysis is involved in the pathogenesis of equine COPD. The fluorometric elastin assay has the potential to be used as a diagnostic method for distinguishing healthy horses from horses with respiratory tract disease. Advantages of this method are fast detection and simultaneous analyses of high numbers of samples. Among the gelatinases, MMP-9 was found to be the most prominent MMP in RSs of COPD horses. MMP-9 markedly increases in equine COPD and is converted to active forms. Equine airways appear to react to inhaled allergens and irritants by increasing MMP-9 levels and activation in their RSs. Elevated active MMP-9 activity was also demonstrated in RS of COPD horses after inducing an hypersensitivity reaction by different challenges. These findings further suggest that MMP-9 and its activation plays a significant role in equine COPD that is similar to that found in human asthma (Lemjabbar et al., 1999). Total MMP-9 and activated MMP-9 immunoreactivities, as well as the relationship between proMMP-9 and activated MMP-9, may well be of diagnostic value when estimating the ongoing active phase of inflammation in the lungs of COPD horses. Longitudinal studies are needed to assess MMP-9 as a potential diagnostic marker in indicating active and destructive phases of equine COPD and in reflecting the environmental load on the airways. In addition, levels of immunoreactive NGAL increase in RS of COPD horses, and NGAL complexed to MMP-9 could explain, at least in part, the increased levels of complexed MMP-9 in RS of COPD horses. The MMP-2 level did not seem to vary between RSs from healthy and COPD horses.
68
Conclusions 7.
Results obtained indicated that MMP-9 activity could be inactivated by means of chemically modified TCs. Gelatinolytic and elastinolytic MMP activities were inhibited by CMT-3. Therefore, a targeted and efficient MMP inhibitor, such as CMT-3, may provide an additional treatment possibility and a valuable drug for use in diminishing MMP activity and lung tissue destruction in the respiratory tract of horses with COPD. In vivo experiments are needed to verify the inhibitory effect of MMP inhibitors on levels of MMPs in the lungs and RSs as well as on lung tissue destruction.
69
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