Hajime Nagahata, Hiromi Nochi, Koichi Tamoto, Hiroshi Noda and Gary J. Kociba. ABSTRACT. RESUME. INTRODUCTION. Expression of CD18 on bovine.
Expression and Role of Adhesion Molecule CD1 8 on Bovine Neutrophils Hajime Nagahata, Hiromi Nochi, Koichi Tamoto, Hiroshi Noda and Gary J. Kociba
ABSTRACT
RESUME
INTRODUCTION
Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.
L'expression du CD18 sur les neutrophiles bovins suite a une stimulation au serum active par le zymosan (SAZ) ou par l'acetate myristate de phorbol (AMP) ainsi que les effets d'anticorps monoclonaux (AcM) diriges contre le CD18 ou les antigenes de surface des neutrophiles (S2G8 et le S5F8G10) ont ete evalues sur le pouvoir adherent, la reponse chemotactique et la phagocytose de ces derniers. L'expression du CD18 des neutrophiles a ete augmentee apries traitement au SAZ et 'a l'AMP de 12,2 et 54,2 %. Aucune difference de l'expression du CD18 par les neutrophiles d'une taure Holstein affectee d'une deflcience des facteurs d'adhesion les leucocytes apres stimulation au SAZ et 'a l'AMP n'a etet notee. L'adherence, la reponse chemotactique et la phagocytose etaient diminuees (p < 0,01) chez les neutrophiles traites continuellement aux anti-CD18. L'adherence etait aussi diminuee de faqon significative chez les neutrophiles pretraites avec l'anti-CD18. Pour les neutrophiles pretraites avec les anti-CD18, une difference significative (p < 0,01) a ete notee avec les neutrophiles traites continuellement. Les autres AcM diriges contre la surface des neutrophiles n'ont pas influence l'adherence des neutrophiles ni la phagocytose. Cette tetude demontre les roles positifs du SAZ et de l'AMP sur l'expression du CD18 sur les neutrophiles de bovins et demontre que le CD18 joue un role important dans les fonctions requerant l'adhesion des neutrophiles. (Traduit par Dr Pascal Dubreuil)
Bovine leukocyte adhesion deficiency (BLAD) is a hereditary disorder which is characterized by recurrent bacterial infections, progressive periodontitis, ulcers of oral mucosa and impaired inflammatory response, associated with persistent marked neutrophilia (1-6). Deficient CD18 expression on neutrophils has been clearly demonstrated by flow cytometric analysis (1,4-6). Activities of adherence, chemotactic movements, phagocytosis and zymosan-stimulated, luminol-dependent chemiluminescence (CL) response of neutrophils from affected cattle with BLAD were severely impaired (1,4,5). The pathogenesis of BLAD has been considered to be identical to that recognized in human leukocyte adhesion deficiency
(7-9).
Integrins, adhesion molecules, have been shown to play a role in cell-cell or cell-matrix interactions (7-10). The CD1 1/CD 18 complex consists of three heterodimeric receptors, also known as LFA-1, Mac-l and p150,95, each with a separate alpha chain (CDl la,b,c) and a common beta chain (CD18)(9,10). The importance of CDlla/CD18 and CDllb/CD18 has been demonstrated in a wide variety of granulocyte adhesion reactions (7-10). However, limited studies concerning the expression and role of CD18 on bovine neutrophils have been performed (11-14). It is of interest to characterize the CD18 expression on leukocyte surfaces not only to understand the characteristics of the functions of neutrophils from affected cattle with BLAD, but also the roles of CD18 on normal bovine neutrophils. The purpose of this study was
Animal Health Laboratory, Department of Veterinary Medicine, Rakuno Gakuen University, Bunkyodai-Midori 582, Ebetsu, Hokkaido 069, Japan (Nagahata, Noda), Department of Microbiology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-02, Japan (Nochi, Tamoto) and Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, Ohio 43210 (Kociba). Submitted January 4, 1994.
Can J Vet Res 1995; 59: 1-7
I
TABLE I. Comparison of CD18 expression on neutrophils from healthy Holstein heifers and a Holstein heifer affected with leukocyte adhesion deficiency after stimulation with ZAS and PMA Untreated ZAS-stimulated PMA-stimulated 13.1 ± 9.9(7) 14.7 ± 1 1.3(7)h 20.2 ± 13.8(6)b (I00)c (1 12.2),.d (154.2)C e BLADa 0.305 0.347 0.621 Negative control = Fluorescence intensity 0.5 ± 0.2 (mean ± SD) NOR = Normal Holstein heifers. BLAD = A Holstein heifer affected with leukocyte adhesion deficiency. Untreated = unstimulated neutrophils. ZAS = Stimulated by zymosan activated serum. PMA = Stimulated by phorbol myristate acetate 'Data show the fluorescence intensity (mean ± SD) without subtraction of the negative control bNumber of heifers cResults are expressed as the mean percentage fluorescent intensity as compared to that of untreated neutrophils dSignificantly (p < 0.05) different from untreated neutrophils Significantly (p < 0.01) different from untreated neutrophils NOR-
antigens (22), S2G8 (mouse MAB of isotype IgGI) and S5F8G10 MAB (mouse MAB of isotype IgG2a) were kindly provided by Dr. Max J. Paape (Animal Science Institute, Beltsville, Maryland). PRETREATED AND CONTINUOUSLY TREATED NEUTROPHILS WITH MONOCLONAL ANTIBODIES ANTI-CD 18, S2G8 AND S5F8G10 Eighty ,L (4 X 106 cells) of neu-
trophil suspension were incubated with 20 puL of anti-CD18 MAB (MHM 23) for 20 min at 4°C. Neutrophils (4 X 106 cells) were incubated with 40 jiL of S2G8 or S5F8G10 to evaluate the expression of CD 18 on were resuspended in Hanks' balanced MAB for 20 min at 4°C. These antibovine neutrophils after stimulation salt solution (HBSS, Nissui Pharma- CDl8, S2G8 and S5F8G10 MAB with zymosan activated serum (ZAS) ceutical Co., Ltd, Tokyo, Japan) to a treated cells were washed twice with and phorbol myristate acetate(PMA) concentration of 5 X 106 cells/mL, and HBSS at 200 X g for 5 min. The cells and to elucidate the effects of anti- were kept in an ice-cold bath. were resuspended in HBSS at a conCD18 monoclonal antibody (MAB, Neutrophils comprised more than 93% centration of 4 X 106 cells/mL and MHM 23) on adherence, chemotactic of the resulting cell population were used as the pretreated group. responses and phagocytosis of bovine by Wright-Giemsa stain, and more Neutrophil suspensions containing neutrophils in comparison with those than 99% of cells were viable anti-CD18, S2G8, or S5F8G10 MAB of S2G8 and S5F8G10 MAB directed when assessed by trypan blue dye were also utilized in each assay and against other cell surface antigens of exclusion. were referred to as continuously bovine neutrophils. treated groups. In some experiments, STIMULATION OF NEUTROPHILS neutrophils (3 X 106 cells) were incuZymosan A (Sigma Chemical Co., bated with unconjugated anti-CD18 MATERIALS AND METHODS St. Louis, Missouri) was added to a MAB (MHM 23) at saturating confreshly pooled bovine serum (10 mg/ centrations. After incubation for ANIMALS mL) and mixed thoroughly. The mix- 30 min at 4°C, cells were washed ture was shaken by a rotating rack at twice with PBS, and then cells were Seven clinically healthy HolsteinFriesian heifers from 7-11 months old 37°C for 1 h. The supernatant was incubated with fluorescein isothiowere used as controls. A Holstein- collected by centrifugation (700 X g, cyanate (FITC)-conjugated anti-CD18 Friesian heifer diagnosed as BLAD 10 min) and used as ZAS (16). MAB for 30 min at 4°C. The cells based on flow cytometric analysis of Phorbol myristate acetate (Sigma were washed twice with PBS and then CD18 expression and DNA-polymerase Chemical Co., St. Louis, Missouri) fixed in 1% paraformaldehyde for chain reaction test (6) was the source of was dissolved in dimethyl sulfoxide at flow cytometric analysis. a concentration of 1 mg/mL and was BLAD neutrophils. diluted to 1 ,ug/mL in HBSS. Five ADHERENCE ASSAY BLOOD SAMPLES hundred ,LL of neutrophil suspension The adherence of neutrophils to Twenty mL of blood were collected (3-4 X 106 cells) were incubated with nylon fibers was examined according from the jugular vein into a heparin either ZAS or PMA at final concentra- to the method of MacGregor et al (17) containing tube (20 IU/mL, VT-100, tions of 30% and 100 ng/mL respec- with slight modifications (5). One mL Terumo Co., Ltd, Tokyo, Japan). tively for 20 min at 37°C. Neutrophils of each untreated, pretreated, and conTotal and differential leukocyte which were incubated in HBSS alone tinuously treated neutrophil suspencounts were performed according to for 20 min at 37°C were referred to as sion (4 x 106 cells) containing 10% standard hematological methods (15). unstimulated neutrophils. autologous plasma was applied to fiber columns (75 mg, 3 denier, nylon ISOLATION OF NEUTROPHILS MONOCLONAL ANTIBODIES (MAB) type 200, Fenwal Laboratories, Antihuman CD18 MAB (MHM 23, Deerfield, Illinois). Neutrophil counts Neutrophils were isolated from heparinized peripheral blood by Ficoll- code No. M783, lot No. 118) and were performed after the samples Conray gradients, followed by fluorescein isothiocyanate(FITC)- were allowed to percolate through the antihuman CD18 nylon fiber columns by gravity flow hypotonic red blood cell lysis conjugated described previously (16). Isolated (MHM 23, code No. 839, lot No. 032) in a 37°C humidified 5% CO, atomoneutrophils were washed once with were purchased from Dako A/S, sphere. Neutrophil adherence was calphosphate-buffered saline solution Glostrup, Denmark. Antibodies rec- culated from the formula: percentage (PBS, pH 7.2) without Ca'2 and Mg2', ognizing bovine neutrophil surface of neutrophil adherence = 100 -
2
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FITC
CHEMOTACTIC ASSAY
The chemotaxis under agarose performed according to the method of Nelson et al (18) with slight modifications (5). Zymosan A was added to freshly pooled bovine serum (10 mg/mL) and mixed on a rotating rack at 37°C for 1 h. The supernatant was collected after centrifugation (700 X g, 10 min) and used as the zymosan treated chemotactic factor. Three sets of three wells each, 2.5 mm in diameter and 2.5 mm apart were made in agarose gel. The middle well of each set of three wells received 10 ,uL of each untreated, pretreated and continuously treated neutrophil suspension (1 X 106 cells), respectively. Ten ,uL of the control medium were added to the inner well. After incubation for 2 h at 37°C and 5% CO, in a humidified incubator, the plates were flooded with 5 mL of assay was
Y*
0
CD18 (4)
S2G8 S5F8G10 (2) (2)
CD18 S2G8 S5F8Gl 0 (4) (2) (2) Continuous incubation with Antibody
Pre-incubated with Antibody
Fig. 2. Effects of preincubation and continuous incubation of neutrophils with anti-CD18 MAB (MHM 23), S2G8 or S5F8G1O MAB on adherence activity. Untreated = Neutrophils were held at 4°C for 20 min in HBSS medium. Preincubated = Neutrophils were incubated with anti-CD18 MAB (MHM 23) at 4°C for 20 min and then washed. Continuous incubation = Neutrophils were incubated together with anti-CD18 MAB (MHM 23), S2G8 or S5F8G10 MAB, respectively. Results are expressed as mean percentage (±SD) relative to untreated neutrophils. Parentheses show the number of assays. * Significantly (p < 0.01) different from that of untreated. ** Significantly (p < 0.01) different from those of untreated and preincubated.
methanol and placed in room
[neutrophils/mL of effluent sample/ neutrophils/mL of initial sample) x 100].
*
20-~
Relative Fluorescence Intensity (Log scale)
Fig. 1. Flow cytometric analysis of CD18 on bovine neutrophils. Neutrophils were reacted with FITC-conjugated normal mouse IgG or FITC-conjugated anti-CD18 MAB and analyzed on a flow cytometer. Gated histograms of neutrophils from normal and affected animals with BLAD are shown. I = Affected Holstein heifer with BLAD, neutrophils were reacted with FITC-conjugated anti-CD18 MAB (MHM 23). 2 = Normal Holstein heifer, neutrophils were reacted with FITC-conjugated normal mouse IgG. 3 = Normal Holstein heifer, neutrophils were reacted with unconjugated anti-CD18 MAB (MHM 23) and followed by FITC-conjugated anti-CD18 MAB (MHM 23). 4 = Normal Holstein heifer, neutrophils were reacted with FITC-labeled anti-CD18 MAB (MHM 23). D = Negative area, E = Positive area.
80-
T
a chamber at temperature over night. After
removing the methanol, 5 mL of PBS10% formalin were added and incubated for 60 min at room temperature. After removing the formalin, the agarose was removed and the cells were stained with Wright's stain. Random migration and chemotaxis were measured with an ocular micrometer. PHAGOCYTOSIS ASSAY
Phagocytic ability of neutrophils assessed by the yeast ingestion method (16). Briefly, 200 [uL of each untreated, pretreated and continuously treated neutrophils (3 X 106 cells), 100 [uL of normal pooled serum (n = 3) and 200 ,uL of yeast suspension (heatkilled, 2.5 X 108/mL) were mixed in each tube, and incubated for 20 min at 37°C with gentle shaking. Thereafter, 2 mL of ice-cold physiological saline were added and the material was centrifuged at 400 X g for 10 min. Two drops of 0.01% fuchsin solution were added to the precipitate and ingested particles were enumerated under microscope. Results were expressed was
the percentage of phagocytic neutrophils.
as
FLOW CYTOMETRIC ANALYSIS OF CD18
Immunofluorescent analysis of neutrophils was performed (4). Neutrophil suspension (3 X 106 cells) was incubated with FITC-conjugated antihuman CD18 MAB (MHM 23) at saturating concentrations. Fluorescein isothiocyanate-conjugated normal mouse IgG (Health Sciences, University of Hokkaido, Japan) was used as a control. After incubation at 4°C for 30 min, cells were washed twice with PBS, fixed in 1% paraformaldehyde, and analyzed on a flow cytometer (Coulter Epics Elite, Coulter Electronics, Hialeah, Florida) using a logarithmic amplifier. Logarithmic values of specific fluorescent intensity were calculated. Neutrophils were characterized by forward and orthogonal light-scattering and gated on cytogram, and the percentage of neutrophils expressing CD18 was quantitated by measurement of fluorescent intensity. Background fluorescence after labeling with IgG control
FITC-conjugated anti-CD 18 MAB as determined by flow cytometric analysis. After treatment, the relative binding of FITC-conjugated anti-CD18 MAB was 9.6% in comparison to 5untreated controls. The effects of preincubation and continuous incubation of bovine neua 46. trophils with anti-CD18, S2G8, or S5F8G1O MAB on adherence, chemotactic response and phagocytosis of .I neutrophils were determined. The (a. 112. adherence activities of neutrophils preincubated or continuously incubated with anti-CD18 MAB were significantly different from controls (p < 2 0.01) with 46.3 ± 5.1 and 32.8 + 20. 1% of mean adherence activities to nylon fibers, respectively (Fig. 2). The mean adherence of neutrophils preincubated with anti-CD18 MAB was statistically different (p < 0.01) from that of neutrophils continuously incubated with anti-CD18 MAB. Mean adherence activities of neutrophils preincubated and continuously incubated with S2G8 MAB were 103.5 + ProContinuousi 14.6 and 115.5 + 2.4%, respectively, and those with S5F8G1O MAB were Uttd with 3~~ncuzbated. Untreated anti -incubationwith anti 107.2 ± 5.0 and 98.3 ± 7.6% of those with untreated neutrophils. CDt18 CD1 8AB The random migration and chemoFig. 3. Effects of preincubation and continuous incubation of neutrophils with anti-CD18 taxis of neutrophils preincubated with MAB (MHM 23) on chemotactic responses. anti-CD18 MAB were not signifiUntreated = Neutrophils were held at 4°C for 20 min in HBSS medium. different from that of untreated cantly (O): Random migration, (El): Chemotaxis. Preincubated = Neutrophils were incubated with anti-CD18 MAB (MHM 23) at 4°C for neutrophils, whereas the random migration and chemotaxis of neu20 min and then washed. Continuous incubation = Neutrophils were incubated together with anti-CD18 MAB trophils incubated continuously with (MHM 23). anti-CD18 MAB were significantly Results are expressed as mm (mean ± SD). (p < 0.01) diminished as compared Parentheses show the number of assays. * Values of continuous incubation were significantly (p < 0.01) different from those of with that of untreated neutrophils untreated and preincubated. (Fig. 3). A significant (p < 0.01) difference was found in chemotactic responses between pretreated and treated neutrophils. continuously of PMA with that and as compared antibody was -0.7% of specific Phagocytic ability of neutrophils untreated neutrophils. The mean fluorescence. increases of CD18 expression by ZAS was unaffected by preincubation with STATISTICAL ANALYSIS and PMA were 12.2 and 54.2%, anti-CD18 MAB, whereas a signifiIn neutrophils from a cant (p < 0.01) difference was found respectively. were Differences between groups compared by use of the Duncan's heifer with BLAD, the expression of in neutrophils continuously incubated multiple range test. Values of p < 0.05 CD18 was deficient and within nega- with anti-CD18 MAB as compared with that of untreated neutrophils tive controls. were regarded as significant. The effect of preincubation of neu- (Fig. 4). The difference in phagocytic RESULTS trophils with anti-CD18 MAB on the ability between pretreated and continExpression of CD 18 on bovine neu- detection of CD18 on bovine neutro- uously treated neutrophils was signifitrophils associated with ZAS and phils was determined (Fig. 1). Bind- cant (p < 0.01). Mean phagocytic abilPMA stimulation was determined ing of FITC-conjugated anti-CD18 ities of neutrophils continuously (Table I). Significant (p < 0.05, p < MAB was barely detected with incubated with S2G8 and S5F8G1O 0.01) increases of CD18 expression neutrophils that were preincubated MAB were 114.3 ± 2.2 and 11 1.2 + were observed in neutrophils from with unconjugated anti-CD 18 4.5% of that with untreated neuhealthy controls stimulated with ZAS MAB followed by incubated with trophils, respectively.
6-
T
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.
-
T
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(4)
(4)
NAB
4
(4)
resting neutrophils contain CD18 in intracellular vesicles, and that ZAS and PMA stimulate transport of this stored CD 18 to the cell surface, as has 100been documented for human neutrophils (23). The CD18 expression of human neutrophils associated with 80to Irl stimulation was increased five to tenn 02 * fold (8,23,25). The phenomenon of XW 60upregulation of adhesion molecules on human neutrophils was considered 0 to be the result of translocation of ° 40intracellular pools to the cell surface. Although the extent of intracellular reserve of CD18 molecules in bovine 20neutrophils remains to be elucidated, it appears to be lower than that of oL human granulocytes. In contrast, defiCD18 CD1 8 S2G8 S5F8G1O cient expression of CD18 on neutrophils from a Holstein heifer with (4) (5) (2) (2) BLAD was detected, and increased CD18 expression was not associated Pre-incubated Continuous incubation with stimulation with ZAS and PMA. with NAB with NAB the value obtained at PMAAlthough Fig. 4. Effects of preincubation and continuous incubation of neutrophils with anti-CD18 stimulated was twofold higher comMAB (MHM 23), S2G8 or S5F8G1O MAB on phagocytosis responses. Untreated = Neutrophils were held at 4°C for 20 min in HBSS medium. pared with those of untreated and Preincubated = Neutrophils were incubated with anti-CD18 MAB (MHM 23) at 40C for ZAS-treated neutrophils, value at 20 min and then washed. Continuous incubation = Neutrophils were incubated together with anti-CD18 MAB (MHM PMA-stimulated neutrophils was within the negative region. This may 23), S2G8 or S5F8G1O MAB, respectively. Yeast phagocytosis was performed at 37°C for 30 min. be related to the nonspecific aggregaResults are expressed as mean percentage (±SD) relative to untreated. Parentheses show the tion after treatment of PMA, as would number of assays. * Values of continuous incubation were significantly (p < 0.01) different from those of be observed by the light-scattering profiles on flow cytometry. These untreated and preincubated. findings demonstrated that neutrophils from affected cattle with sion of CD18 on normal neutrophils BLAD were deficient in upregulation DISCUSSION was increased significantly by the of CD18 molecules when stimulated Cell surface adherence reactions exposure of neutrophils to ZAS or by ZAS and PMA stimulation and are of central importance in a wide PMA in our study. Zymosan activated were consistent with the results with spectrum of leukocyte functions serum increased CD18 expression by ZAS stimulation in the recent report which contribute to host defenses to 12.2%, and this value was consistent of Olchowy et al (14). Aggregation of bovine neutrophils infections (7-10). Adhesion mole- with a previous report (11). Although cules expressed by both the leuko- increased expression of CD18 after has been reported to be inhibited by cytes and the endothelial cells are stimulation of bovine neutrophils with use of MAB (MAB 60.3) against important in regulating these adhesion PMA has been reported, considerable human CD18 multimeric cell surface interactions (7,8,19-21). The impor- variation of CD18 expression has glycoproteins (11). In transendothetance of Mac- 1 and LFA- 1 has been been observed in neutrophils stimu- lial migration of bovine neutrophils in demonstrated in a wide variety of lated with PMA (11,12). The mean vitro recently reported by Boschsler granulocyte adhesion reactions relative increase in CD18 expression et al (13) and Olchowy et al (14), the (7-10). In our study, the identification by PMA stimulation was 54.2% in the addition of an anti-CD 18 MAB (MAB of bovine CD18 on neutrophils was present study. The magnitude of 60.3) caused significantly decreased performed using an antihuman CD18 increase in CD18 expression stimu- migration of bovine neutrophils. MAB (MHM 23). In regard to the lated with PMA was close to that of a In our study, differences in adherence, specificity of employing antihuman previous report (11). The reason for chemotaxis and phagocytosis of CD18 MAB (MHM 23) in this study, differences in CD18 expression in neutrophils with untreated, preSalgar et al (22) reported that more response to PMA may be due to assay treated, and continuously treated with than 99% of cross-reactivity of this conditions such as preparation of neu- anti-CD18 MAB (MHM 23) were eviCD18 MAB (MHM 23) with bovine trophils and PMA, and individual dif- dent. Significantly decreased neuneutrophils, and the CD18 complex ference of animals. Although some trophil functions were found in assays on bovine neutrophils has been docu- variations of CD18 expression were for adherence with both pretreated mented (4,5,12,22). Surface expres- found, these findings suggest that and continuously treated neutrophils,
120-
5
random migration and chemotaxis with continuously treated neutrophils, and phagocytosis with continuously treated neutrophils. The reasons for these differences found in preincubated and continuously incubated groups in each assay were likely to be related to the expression of CD18 on neutrophil surface. Assays for phagocytosis and chemotactic response of neutrophils require a relatively longer period of time than that required for adherence in a column (1-2 min). The upregulation of CD18 could occur continuously in chemotaxis and phagocytosis assays during the relatively longer period of incubation time. Interestingly, significant differences of chemotactic responses and phagocytosis of neutrophils from pretreated and continuously treated neutrophils were observed. This indicates that newly upregulated CD18 on neutrophils is expressed on neutrophils during incubation and participates in the adherence-related functions of neutrophils. In contrast, inhibition of neutrophil adherence in this study was considered to be due to the blocking of CD18 by anti-CD 18 (MHM 23), with little upregulation during the relatively short period of incubation. This indicates that the upregulation of CDl 1/CD 18 adhesion molecules is an initial step in adherence-dependent neutrophil functions. The most profound inhibition of adherence-related functions was observed in neutrophils incubated continuously in the presence of anti-CD18 MAB (MHM 23). Our results confirmed the recent reports of Bochsler et al (13) and Olchowy et al (14) which showed that transendothelial migration of normal bovine neutrophils to ZAS was reduced to a level comparable to the BLAD neutrophils by treatment with anti-CD18 MAB (MAB 60.3). Salgar et al (22) produced a panel of 14 MABs directed against bovine neutrophil surface antigens and reported that these MAB did not show the binding to the epitope that CD18 recognized. In our study, neutrophils incubated with S2G8 and S5F8G10 MABs showed enhanced or unchanged adherence and phagocytic activities. It may be due to the upregulation, rather than down regulation, as observed by Salgar et al (22). These results support that significant inhibitory effects observed in neutrophil function when 6
continuously incubated with antiCD18 MAB (MHM 23) were due to the inhibition of upregulation of CD1 1/CD 18 adhesion molecules. The complete block of adherencerelated functions of healthy neutrophils by anti-CD18 MAB (MHM 23) was not found, indicating that CD18independent adhesion molecules such as very late antigens (VLA antigens), L-selectin or others also play an important role for cell adhesion (21,26). Adherence-related neutrophil functions after blocking the CD18 molecules by anti-CD18 MAB treatment were likely to be greatly dependent on the magnitude of intracellular reserve and upregulations of CD11/ CD18 molecules. Further studies are required to elucidate this possibility. These results demonstrated that CDl8 expression on bovine neutrophils increased significantly by adequate stimulation and supported the concept that CD1l/CD18 adhesion molecules are important for a broad range of adherence-related functions. ACKNOWLEDGMENTS The authors wish to thank Dr. Max J. Paape, Milk Secretion and Mastitis Laboratory, Animal Science Institute, Beltsville, Maryland, for his generous gift of monoclonal antibodies S2G8 and S5F8G10. Thanks are also due to reviewers for their comments on the manuscript, and Takeo Miura, Animal Health Laboratory, Department of Veterinary Medicine, Rakuno Gakuen University for performing the blood collection and for preparation of the figures. This study was supported in part by a Grant in Aid for Scientific Research Fund (No.05660372) from the Ministry of Education, Science and Culture, Japan.
3.
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