Pulmonary and Hematological Inflammatory Responses to Intravenous Cellulose Micro-Particles in Broilers1 W. Wang,2 R. F. Wideman, Jr., T. K. Bersi, and G. F. Erf Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 inflammatory response within the surrounding lung parenchyma. Monocytes and basophilic granulocytes closely surrounded the particles. CD4, CD8, TCR1, TCR2, and TCR3 subsets of T cells and B cells were present in the outer rim of the granuloma/lymphocyte aggregates. Circulating total white blood cell (WBC, leukocytes) concentrations were similar in both groups at all times postinjection, whereas at 48 h post-injection the percentages of eosinophils and basophils among circulating WBC were higher in the particle group than in the control group (P ≤ 0.05). The circulating monocyte concentration also increased within 24 h postinjection (P ≤ 0.05). These observations demonstrate that cellulose micro-particles trapped in the pulmonary vasculature initiated acute focal inflammatory responses in the lungs and that the proportions of WBCs in the blood are modulated within 48 h postinjection.
ABSTRACT When injected intravenously, cellulose micro-particles become lodged in pulmonary arterioles. The current study investigated the systemic and pulmonary inflammatory responses triggered by cellulose micro-particles at 3, 24, and 48 h post-injection in 6-wk-old broilers. Proportions and concentrations of circulating white blood cells were assessed in saline-injected (control group) and cellulose-injected (particle group) birds. Hematoxylin-eosin (HE)-stained cross-sections of the lungs were used to count the number of granuloma/lymphocyte aggregates, which is indicative of the severity of the inflammatory response to the trapped particles. The cellular components of the aggregates were identified by immunohistochemical staining of frozen cross sections of the lungs. Results showed that cellulose micro-particles trapped in the pulmonary vasculature initiated a dynamic, localized
(Key words: intravenous micro-particle, pulmonary hypertension syndrome, pulmonary inflammatory response, leukocyte, broiler chicken) 2003 Poultry Science 82:771–780
of particles injected and by initiating an acute focal inflammatory response in the lung parenchyma (Wang et al., 2002; Wideman and Erf, 2002). The intrapulmonary inflammatory response to micro-particles is believed to contribute to the onset of pulmonary hypertension through the release of vasoconstrictors such as endothelin-1, thromboxane, and serotonin (Wideman and Erf, 2002). In addition, the inflammatory response may damage the gas exchange surfaces within the lung parenchyma and contribute to the onset of hypoxemia associated with pulmonary hypertension syndrome (PHS, ascites) (Wideman, 2000, 2001). The hemodynamic changes initiated by i.v. micro-particle injections are similar to those triggered by surgical occlusion of one pulmonary artery, which was proven to be a successful procedure for selecting broilers that are genetically resistant to PHS (Wideman et al., 1996; Wideman and French, 1999, 2000; Wideman and Erf, 2002). When compared with the surgi-
INTRODUCTION Recent studies in broilers demonstrated that i.v. injections of micro-particles, having a size suitable to occlude the pulmonary precapillary arterioles, can cause sustained pulmonary hypertension by increasing the pulmonary vascular resistance (Wideman and Erf, 2002; Wideman et al., 2002). After injection into a wing vein, microparticles are carried to the lungs by the returning venous blood, where they become lodged in the pulmonary vasculature. Intravenously injected cellulose micro-particles trigger pulmonary hypertension by physically occluding the pulmonary vasculature in proportion to the numbers
2003 Poultry Science Association, Inc. Received for publication October 28, 2002. Accepted for publication February 6, 2003. 1 U.S. patent pending (File No. 09-913,774) protects the exclusive rights of the University of Arkansas to all uses of the intravenous microparticle injection technology within the context of evaluating or affecting pulmonary vascular capacity, pulmonary vascular resistance, pulmonary hypertension, cardio-pulmonary hemodynamics, and susceptibility to pulmonary hypertension and pulmonary hypertension syndrome (ascites) in domesticated animal species. 2 To whom correspondence should be addressed:
[email protected].
Abbreviation Key: ABC-Px = Avidin-biotin complex-peroxidase; HE = hematoxylin and eosin; HS = horse serum; IHC = Immunohistochemical staining; mAbs = monoclonal antibodies; PHS = pulmonary hypertension syndrome; RBC = red blood cells; RT = room temperature; TCR = T cell receptor; WBC = white blood cells.
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cal technique, the i.v. micro-particle injection method is much more time- and labor-efficient, and the selection pressure can be finely controlled by adjusting the numbers and sizes of particles injected. Therefore, i.v. microparticle injections are well suited for selection of PHSresistant broilers in large scale breeding programs (Wideman et al., 2002). Preliminary histological examinations of lung tissues obtained from broilers injected with cellulose micro-particles revealed progressive inflammation-associated changes in the lung parenchyma surrounding the microparticles. Within 2 d postinjection, the micro-particles were surrounded by granulomatous tissue consisting primarily of mononuclear giant cells and lymphocyte aggregates. The inflammatory response subsided by 18 d postinjection when the micro-particles had been cleared from the lungs (Wideman et al., 2002). Considering the reaction of the immune system to the entrapped micro-particles, it is possible that using intravenous micro-particle injections to select for PHS resistance may result in co-selection for certain pulmonary and systemic immune profiles. For example, broilers are more likely to survive this selection technique if their immunological responses are particularly effective in clearing or walling off the entrapped micro-particles and restoring a normal lung capacity. Alternatively, broilers may be eliminated from the population if vasoconstrictors released during the immunological response substantially amplify the physical occlusion caused by micro-particle entrapment. Unintended shifts in immune profiles may not be beneficial when broilers are forced to deal with the multitude of disease challenges that are typical of commercial poultry production. Hence, as an initial approach toward achieving a better understanding of the potential immunological consequences of using the micro-particle technique for selecting broiler lines, the current study was designed to evaluate the cellular components of the pulmonary and systemic inflammatory responses to injected cellulose micro-particles within 48 h postinjection. The extent and type of pulmonary inflammatory responses triggered by the trapped particles was assessed by counting the numbers of granuloma/lymphocyte aggregates in lung sections and by determining the type of immune cells present in the aggregates using conventional histology and immunohistochemistry.
MATERIALS AND METHODS
search Farm. Chicks were wing banded and placed on fresh wood shavings in environmental chambers (8 m2 floor space). They were brooded at 33°C on d 1 to 5, 29°C on d 6 to 10, 27°C on d 11 to 17, and 21°C thereafter. Water and a corn-soybean meal broiler ration (22.7% CP, 3,059 kcal ME/kg), formulated to meet or exceed the minimum NRC (1994) standards for all ingredients, were provided ad libitum. The lights were on for 24 h/d through d 5, and for 23 h/d thereafter.
Experimental Design At 35 d of age, 42 birds were randomly selected and divided into two groups of 21 birds (the control group and the particle group). Fresh heparinized saline (150 units ammonium heparin4/mL of 0.9% NaCl) was prepared. Microgranular CM-32 ion exchange cellulose5 (30 µm average particle dimension) was suspended at 0.02 g/mL in heparinized saline solution, and this cellulose suspension was vortexed continuously on a magnetic stirring plate to keep the particles evenly distributed. The suspension was drawn into a 1-mL tuberculin syringe through an attached 22-gauge needle and injected into birds of the particle group via the left basilica (wing) vein at a dosage of 0.25 mL per bird. Birds of the control group were injected with 0.25 mL of heparinized saline. Each group was further divided into three subsets of seven birds, and 2-mL blood samples were collected from the right basilica vein into heparinized syringes at 3, 24, and 48 h postinjection from birds of each subset, respectively. The blood samples were used for determination of proportions and concentrations of various types of blood cells, as described below. The birds were euthanized with carbon dioxide immediately after the blood samples were taken. The lungs were collected and the portion of the left lung between the first and second anterior rib indentations was fixed in 10% phosphate buffered formalin,5 embedded in paraffin, and sectioned (5 µm) in the transverse plane. The sections were stained with hematoxylin and eosin (HE) for histological examination (Luna, 1968). The portion of the right lung between the first and second anterior rib indentations was embedded in Tissue-Tek OCT6 freezing medium, snap frozen in liquid nitrogen, and stored at −80°C until cryostat sectioning in the transverse plane for immunohistochemistry- and Wrightstaining (described below) to identify and localize cellular components involved in the inflammatory response to the micro-particles lodged in the blood vessels of the lungs.
Bird Management Male broilers from a commercial hatchery3 were transported on the day of hatch to the Poultry Environmental Research Lab at the University of Arkansas Poultry Re-
3
Hubbard ISA, Hot Spring, AR. Sigma Chemical Co., St. Louis, MO. 5 Fisher Scientific, St. Louis, MO. 6 Sakura Finetek, Inc., Torrance, CA. 7 Abbott Laboratories, Abbott Park, IL. 4
Differential Blood Cell Counts Concentrations of red blood cells (RBC), thrombocytes, and total white blood cells (WBC, leukocytes) were determined using an automated hematology analyzer (CellDyn 3500 System7) which had been standardized for analysis of domestic fowl blood cell populations. For each blood sample, two blood monolayers (blood smears) were prepared, stained with Wright stain (Lucas and Jamroz, 1961; Erf and Smyth, 1996), and examined using a bright
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INFLAMMATORY RESPONSES TO INTRAVENOUS MICRO-PARTICLES TABLE 1. Effects of i.v. cellulose micro-particles (particle) or heparinized saline (control) on the concentrations of various blood cells in 6-wk-old male broiler chickens Concentration of blood cells1 RBC (106/µL) Time post-injection 3h 24 h 48 h
WBC (103/µL)
Thrombocyte (103/µL)
Control
Particle
Control
Particle
Control
Particle
2.50 ± 0.13 2.80 ± 0.08 2.65 ± 0.10
2.66 ± 0.05 2.75 ± 0.15 2.70 ± 0.03
10.40 ± 1.06 12.95 ± 1.52 11.05 ± 1.39
10.33 ± 0.97 14.50 ± 1.55 14.02 ± 2.86
32.01 ± 4.11 23.89 ± 3.59† 34.43 ± 3.12
33.01 ± 3.88 23.81 ± 4.39† 31.71 ± 2.23
1 Means ± SEM, n = 7. For all the variables, there is no difference between the control and particle groups at any time postinjection or among either group across time (P > 0.05). †The main effect of time was significant whereby the average thrombocyte concentration of the two groups at 24 h postinjection was lower than those at 3 and 48 h post-injection (P ≤ 0.05).
field microscope at 1000 × magnification. For each blood smear slide, a total of at least 400 WBC and at least one cell of each differential WBC type (lymphocytes, heterophils, monocytes, eosinophils, basophils) were counted, and percentages of differential WBC were calculated by using numbers of WBC of a given type divided by the total WBC multiplied by 100. The differential WBC concentrations were calculated using the proportions of differential WBC multiplied by the total WBC concentration.
Immunohistochemistry and Wright Staining of the Lung Tissue Imunohistochemical analysis of the lung tissue was performed following a standard protocol (Bucy et al., 1988). Briefly, serial transverse sections (7 µm) of frozen lung tissues were cut at −24°C using a Cryostat.8 The sections were mounted on poly-lysine-coated slides, and fixed in cold acetone (4°C) for 5 min and air-dried. Then the sections were incubated overnight at room temperature (RT) with blocking buffer (10% HS–PBS) consisting of 10% horse serum (HS) and 90% phosphate buffered saline (PBS, 0.01 M, pH 7.2) to inhibit nonspecific binding of immuno-reagents. Following the overnight incubation, tissue sections were incubated for 1 h at RT with primary antibodies (separate sections for different types of antibodies). Primary antibodies were mouse monoclonal antibodies (mAbs) IgG9 specific for one of the following mononuclear cell surface markers: chicken TCR1 (γ/δ T cell receptor, Chen et al., 1988; Chen et al., 1994), TCR2 (α/ β1 T cell receptor, Chen et al., 1988; Chen et al., 1994), TCR3 (α/β2 T cell receptor, Chen et al., 1989), CD4 (Chan et al., 1988), CD8 (Chan et al., 1988), Bu-1(Rothwell et al., 1996; Tregaskes et al., 1996) and K110 (Kaspers et al., 1993). These markers were used to determine the presence of TCR defined T cell subsets, T helper lymphocytes, cyto-
8 Microm Laborgera¨te GmbH, Robert-Bosch-Strasse 49, Waldorf, Germany. 9 Southern Biotechnology Assoc., Inc., Birmingham, AL. 10 Hyun Lillehoj, U.S. Department of Agriculture, BARC-East, Beltsville, MD. 11 Vextor Laboratories, Inc., Burlingame, CA. 12 Olympus Inc., Melville, NY.
toxic lymphocytes, B lymphocytes, and monocytes/macrophages and thrombocytes, respectively. All commercial antibodies were used at a final concentration of 5 µg/mL in the blocking buffer. After incubation with primary antibodies, the sections were incubated for 30 min at RT with secondary antibody (biotinylated polyclonal horse anti-mouse IgG,11 15 µg/mL in the blocking buffer) to detect bound primary antibodies, and then for 30 min at RT with Avidin-biotin complex-peroxidase (ABC-Px)11 to label bound secondary antibody. Each of the above incubation steps was followed by washing the sections with running PBS for 1 min. The ABC-Px activity was detected by incubating the sections with the substrate 3, 3′-diaminobenzidine4 (DAB, brown end product deposited at the primary antibody-binding site on the section) at RT for 5 to 10 min, depending on color development. The reaction was stopped by washing the sections with running PBS once sufficient color had developed. The ABC-Px stained sections were counterstained with methyl-green stain, dehydrated, and covered with cover slips for microscopic observation. One section from each lung also was stained using the procedure for Wright-staining of chicken blood smears (Lucas and Jamroz, 1961).
Evaluation of Tissue Sections HE-stained sections were used to determine the number of granuloma/lymphocyte aggregates per cm2 lung section. The number of granuloma/lymphocyte aggregates per cm2 lung section was determined by counting all granuloma/lymphocyte aggregates in the section using a microscope (bright field, 100×) and measuring the size of the section using a 100 mm2 ocular grid12 divided into 100 small squares (1 mm2 each) (Erf et al., 1997). The cell types within the granuloma/lymphocyte aggregates were identified qualitatively by examining immunohistochemically-stained tissue sections for T cell subsets (CD4, CD8, TCR1, TCR2, and TCR3), B cells, and macrophages and Wright-stained sections for basophils, eosinophils, and heterophils.
Statistical Analysis The SigmaStat two-way ANOVA was used to evaluate the main effects of intravenous injection of cellulose mi-
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FIGURE 2. Effect of cellulose micro-particle injection on number of lymphocyte aggregates in the lung tissue at 3, 24, and 48 h post-injection (HE-stained cross-sections). Top panel: Number of lymphocytes aggregates per cm2 lung section in the cellulose-injected broilers (particle, n = 5, mean ± SEM, 6 wk old) and heparinized saline-treated broilers (control, n = 5, mean ± SEM, 6-wk-old) post-injection. Asterisk (*) designates values of the particle group that are different from those of the control group within a time point (P ≤ 0.05). The values within either group did not change over time (P ≤ 0.05). Bottom panel: Number of lymphocyte aggregates per cm2 lung section associated with microscopically visible particles (particle group only, n = 5, mean ± SEM) over time post-injection. Values with no shared letters (a, b) are different (P ≤ 0.05).
cro-particles, time postinjection, and the interactions between the two factors on the percentages and concentrations of circulating total and differential WBC, on the concentrations of circulating RBC and thrombocytes, and on the number of granuloma/lymphocyte aggregates per cm2 lung section. Means were differentiated at P ≤ 0.05 by the Student-Newman-Keuls method (Jandel Scientific, 1994).
RESULTS FIGURE 1. Percentage of eosinophils (top panel), basophils (second panel), monocytes (third panel), heterophils (fourth panel), and lymphocytes (bottom panel) within the circulating white blood cell population over time postinjection in the cellulose micro-particle-injected group (particle, mean ± SEM, n = 7, 6-wk-old) and heparinized saline-treated group (control, mean ± SEM, n = 7, 6 wk old). Asterisk (*) designates values of the particle group that are different from those of the corresponding control group. Within the particle group, values with no shared letters (a, b) are different (P ≤ 0.05).
The RBC and total WBC concentrations in the circulation were not affected by intravenous cellulose microparticle injections at any time postinjection (P > 0.05, Table 1). Blood thrombocyte concentrations did not differ between the particle and control groups at any time postinjection; however, the average blood thrombocyte concentration for both groups was lower at 24 h postinjection when compared with the 3 and 48 h post-injection values (P ≤ 0.05, Table 1). In the control group, the differential WBC concentrations and percentages in the circulation
0.31 ± 0.06 0.27 ± 0.02 0.25 ± 0.05 0.23 ± 0.05 0.24 ± 0.03 0.24 ± 0.03 0.21 ± 0.08 0.15 ± 0.03 0.32 ± 0.05* Means within a column with no common superscript letter (a, b) are different (P ≤ 0.05). Means ± SEM, n = 7. *The particle-injected group is different from the corresponding heparinized saline-treated control (P ≤ 0.05). 1
0.18 ± 0.02 0.52 ± 0.08a 0.77 ± 0.30a 0.39 ± 0.08 0.47 ± 0.12 0.42 ± 0.06 2.34 ± 0.54 2.84 ± 0.42 2.69 ± 0.93 1.90 ± 0.43 2.44 ± 0.53 2.00 ± 0.34 7.41 ± 0.60 10.62 ± 1.05 9.67 ± 1.46 7.80 ± 0.67 9.70 ± 0.92 8.40 ± 1.04 3h 24 h 48 h
a,b
0.20 ± 0.05 0.37 ± 0.08 0.59 ± 0.26* 0.15 ± 0.02 0.21 ± 0.06 0.12 ± 0.02
0.17 ± 0.04 0.13 ± 0.02 0.11 ± 0.02
Particle Particle Control Particle Control Particle Control Particle Control Particle Control
Time postinjection
Lymphocytes
Heterophils
Monocytes
b
Eosinophils
Basophils
Control
H:L Ratio Differential WBC concentration (103/µL)1
TABLE 2. Effects of i.v. cellulose micro-particles (particle) or heparinized saline (control) on the concentrations of differential white blood cells (WBC) and on heterophil to lymphocyte concentration ratio (H:L) in 6-wk-old male broiler chickens
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did not change over time (Table 2, Figure 1). Within the particle group, the monocyte concentration was higher at 24 and 48 h post-injection when compared with 3 h postinjection (Table 2), and the percentage of monocytes among WBC at 48 h postinjection, though not at 24 h postinjection, was higher than that at 3 h postinjection (Figure 1). The percentage of monocytes was lower in the particle group than in the control group at 3 h postinjection, while no difference in monocyte percentage was found between the two groups at 24 or 48 h postinjection (Figure 1). The concentration of monocytes in the blood was not different between the two groups at 3 and 24 h postinjection (Table 2). By 48 h postinjection, the monocyte concentration was marginally higher in the particle group than the control group (P = 0.07, Table 2). In the particle group, the eosinophil concentration in the blood and the percentage of eosinophils among circulating WBC increased in response to i.v. particles and were higher than those of the control group by 48 h postinjection (Table 2, Figure 1). The particle group also had a higher basophil concentration in the blood and a higher percentage of basophils among WBC than the control group at 48 h postinjection (Table 2, Figure 1). The percentage of basophils among WBC of the particle group at 48 h postinjection was higher than that at 24 h postinjection, though not higher than that at 3 h postinjection. Intravenous particles had no effect on either the concentrations of heterophils and lymphocytes in the blood or their percentages among WBC at any time postinjection, nor was the heterophil to lymphocyte (H:L) ratio affected by the particle injection throughout the 48 h postinjection period (Table 2, Figure 1). Lymphocyte aggregates were occasionally observed in the lung parenchyma of normal, healthy, vehicle-injected broilers. Based on immunohistochemical staining, such lymphocyte aggregates consisted of B cells and all subsets of TCR-defined T cells (TCR1, TCR2, and TCR3 T cells) that were either CD4 T helper cells or CD8 cytotoxic cells (not shown). There were fewer lymphocyte aggregates per cm2 lung section in the control group than in the particle group at all postinjection intervals (Figure 2). The number of lymphocyte aggregates associated with microscopically visible particles in the lung sections of the particle group increased over time, and the number at 48 h postinjection was higher than that at 3 h postinjection (Figure 2). The intravenously injected micro-particles of the size used in this study became lodged in inter- and intra-parabronchial arterioles. A micro-particle trapped in an intra-parabronchial arteriole is shown in Figure 3A. The micro-particles triggered dynamic localized inflammatory reactions in the lung parenchyma throughout the 3 to 48 h postinjection period (Figures 3A and 3B). At 3 h postinjection, a variety of immune cells including macrophages, B cells, T helper cells, cytotoxic T cells, and basophilic cells surrounded the blood vessels in which the micro-particles became trapped, and individual cells of these types also appeared to adhere to the surface of the micro-particles inside the vessels (Figures 3C, 3E, 3I, 3M, and 3O). Multitudes of immune cells were mobilized
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FIGURE 3. Lung sections from 6-wk-old male broilers showing granuloma/lymphocyte aggregates developed around the i.v. injected cellulose micro-particles. The cellular components in the aggregates were identified by Wright stain and an indirect immunoperoxidase staining method (Immunohistochemical staining, IHC). A. Cellulose micro-particle (arrow) surrounded by mononuclear cells, 3 h postinjection, paraffin section, HE stain, 100×; B. Cellulose micro-particle (arrow) and the surrounding granuloma/lymphocyte aggregates, 48 h postinjection, paraffin section, HE stain, 100×; C. Macrophage/monocyte (m, characteristic shapes of nuclei), mast cells (b, dark blue granules, confirmed by Toluidine Blue staining method—not shown) and an eosinophil (e, red round granules) in the vicinity of a micro-particle (P), 3 h postinjection, frozen section, Wright stain 1,000×; D. Monocytes/macrophages (m) and mast cells (b) in the vicinity of a micro-particle (P), 48 h postinjection, frozen section, Wright stain, 600×; E. Monocytes/macrophages (dark reaction product near the micro-particle, K1 mAbs), 3 h postinjection, frozen section, IHC, 200×; F. Monocytes/macrophages, 24 h postinjection, frozen section, IHC, 200×; G. Monocytes/macrophages, 48 h postinjection, frozen section, IHC, 200×; H. Endogenous peroxidase activity (unlabelled control, no primary mAbs), 3 h postinjection, frozen section, IHC, 100×; I. B cells (Bu-1 mAbs), 3 h postinjection, frozen section, IHC, 100×; J. B cells, 48 h postinjection, frozen section, IHC, 100×; K. TCR2 T cells (TCR2 mAbs), 3 h postinjection, frozen section, IHC, 100×; L. TCR2 T cells, 48 h postinjection, frozen section, IHC, 100×; M. CD4 T cells (CD4 mAbs), 3 h postinjection, frozen section, IHC, 100×; N. CD4 T cells, 48 h postinjection, frozen section, IHC, 100×; O. CD8 T cells (CD8 mAbs), 3 h postinjection, IHC, 100×; P. CD8 T cells, 48 h postinjection, frozen section, IHC, 100×.
INFLAMMATORY RESPONSES TO INTRAVENOUS MICRO-PARTICLES
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Figure 3 continued.
toward the micro-particles in an effort to wall them off within 3 hr postinjection. By 24 h postinjection, immune cell aggregates were more organized. The periphery of each micro-particle was completely and intensively surrounded by monocytes/macrophages (Figure 3F), clusters of mast cells were found within close vicinity to the micro-particles, and lymphocytes were located toward the outer rim of the immune cell aggregates surrounding
the micro-particles (not shown). Mast cells were recognized by the presence of dark blue granules in their cytoplasm in Wright-stained sections. These granules display a metachromatic purple color when stained with Toluidine Blue (Toluidine Blue staining was used to cross check that mast cells were identified correctly in Wrightstained sections). By 48 h postinjection, the arrangement of the immune cells around the micro-particles clearly
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suggested a foreign-body type granulomatous response in the lungs (Figure 3B). The occluded arterioles appeared edematous at the sites adjacent to the micro-particles, and the boundaries of the endothelium could not be clearly defined (Figure 3D). Tissues immediately surrounding the micro-particles were granulomatous, consisting primarily of monocytes/macrophages and basophilic cells (Figures 3D and 3G), and the smooth muscle cells from the arterial wall characterized by elongated nuclei appeared mingled among the macrophages and basophilic cells (Figure 3D). It appeared that monocytes/macrophages, basophilic cells, and individual lymphocytes infiltrated the arterial walls and caused granulomatous arteritis around the micro-particles (Figures 3B, 3D). Intense lymphocyte aggregates were present around the periphery of the granulomas. Immunohistochemical staining of cell surface markers indicated that most of these lymphocytes were T cells, with B cells comprising a minor component (Figures 3I to 3P). The T cells were either CD4 helper T cells or CD8 cytotoxic cells (Figure 3M to 3P). When classified according to T cell receptor (TCR), all three sublineages of T cells (TCR1, TCR2, or TCR3 T cells) were present. Microscopic observation (cell numbers were not counted) showed that TCR2 T cells (Figures 3K and 3L) were more abundant than TCR1 or TCR3 T cells in the lymphocyte aggregates, and TCR3 T cells appeared to be as abundant as TCR1 T cells; both B cells and all subtypes of T lymphocytes increased in number in the aggregates over time postinjection. The absence of heterophils and the extremely rare occurrence of eosinophils in the immune cell aggregates surrounding the micro-particles were particularly noteworthy. By 48 h postinjection, the periphery of most micro-particles was still intact, although the outer margins of some micro-particles appeared to be partially digested (not shown).
DISCUSSION The pulmonary granulomatous response to cellulose micro-particles in the current study is characteristic of the walling off response by the host immune system toward a foreign particle caught in animal tissues. Pulmonary granulomatous inflammation has been experimentally induced in mice by i.v. injections of parasitic larvae, Sephadex micro-beads, or other slowly degradable and poorly soluble substances such as divinyl copolymer beads (Hood et al., 1984; Weinstock et al., 1988; Cook, 1990). Indeed, intravenous injections of Sephadex particles have been used in guinea pigs and rats to induce pulmonary immune activities which serve as animal models of asthma and other inflammatory lung diseases (Laycock et al., 1986; Cook et al., 1989; Das et al., 1995; Namovic, et al., 1996; Tramontana et al., 2002). Studies in rats demonstrated that a single i.v. injection of Sephadex G200 resulted in a foreign-body type granulomatous reaction in the lung tissue, with the granulomas reaching their maximum size 2 to 7 d after injection and persisting for 84 d (Cook et al., 1989; Cook, 1990). Our previous study in broilers demonstrated that a single i.v. injection of
Sephadex or cellulose micro-particles induced granuloma/lymphocyte aggregates in the lungs which reached maximum size 5 to 7 d postinjection and persisted for less than 18 d. Thereafter, except a few small areas of fibrosis, no histological evidence of lung damage, inflammation, or entrapped micro-particles was observed (Wideman et al., 2002). These comparisons might indicate that broilers elicit a more rigorous inflammatory response toward foreign particles trapped in the lung parenchyma than do mammals. Indeed, different cellular components appear to be involved in the immune reaction to the particles when broilers and rats are compared. It has been reported that from 2 d onward after Sephadex injection, the granulomas in the lungs of rats were typified by large numbers of macrophages and small percentages of lymphocytes (Cook et al., 1989; Cook, 1990). Few eosinophils were found in association with the Sephadex particles; however, large numbers of eosinophils presented in the adventitial layers between the pulmonary blood vessels and the bronchioles (Cook et al., 1989; Cook, 1990). In contrast, a large population of lymphocytes was found in the granuloma/lymphocyte aggregates around the trapped particles in the lungs of broilers examined in the current study at 48 h postinjection. In addition, basophils were present together with macrophages in close association with the particles in broiler lungs. Collectively, these observations suggested that different immune mechanisms might be involved in the mammalian and avian responses to polysaccharide micro-particles trapped in pulmonary arterioles. The inflammatory responses in the lungs were accompanied by changes in concentrations of differential white blood cells in the blood. When compared with the salineinjected controls, a drop in monocyte concentration in the circulation was detected at 3 h postinjection of the cellulose micro-particles, which coincided with appearance of monocytes/macrophages around the trapped micro-particles in the lung parenchyma. This suggested that monocytes started migrating from the blood stream toward the micro-particles within a few hours postinjection. The circulating monocyte concentration also increased over time from 3 to 24 h postinjection, which was accompanied by increasing numbers of monocytes/macrophages surrounding the micro-particles. These observations indicated that monocytes/macrophages were the first line of defense. In addition to monocytes/macrophages, basophils appeared in close association with the micro-particles in the lungs. This participation of basophils in the inflammatory response also was reflected by a higher basophil concentration in the circulation of the particle group at 48 h post-injection when compared with the control group (Table 2). Though lymphocytes appeared in abundant numbers in the lungs in response to the micro-particles, surprisingly no change in lymphocyte concentration in the blood was found. Eosinophils only occasionally were noticed in close vicinity to the particles (Figure 3C); however, a higher eosinophil concentration was detected in the blood of the particle group at 48 h postinjection when compared with the control group.
INFLAMMATORY RESPONSES TO INTRAVENOUS MICRO-PARTICLES
This experiment showed that several types of immune cells in the circulation were stimulated by i.v. cellulose micro-particle injections in broilers. In contrast, the systemic response to i.v. injection of parasitic larvae or Sephadex beads in rats were typified by blood eosinophilia (increases in eosinophil numbers) (Cook et al., 1989; Cook, 1990; Das et al., 1995). Based on these results, it appears that i.v. injections of insoluble polysaccharide micro-particles trigger different inflammatory responses in broilers when compared with mammals. Although all broilers examined in the current study responded similarly to the i.v. micro-particle injection in terms of pulmonary and systemic inflammatory responses, the magnitude of the responses was quite variable among individual birds. For example, at 48 h postinjection, the coefficient of variation (CV) for concentrations of circulatory monocytes, eosinophils, and basophils in particle-injected birds were 102, 114, and 45%, respectively. The CV for the numbers of lymphocyte aggregates/cm2 lung section in the particle-injected birds at 48 h postinjection was 24%. The possible relationship between this variability in inflammatory responsiveness and the variable susceptibility of particle-injected broilers to PHS (Wideman et al., 2002) awaits further investigation. In conclusion, cellulose micro-particles trapped in the pulmonary vasculature initiated acute focal inflammatory responses within surrounding lung parenchyma and triggered systemic inflammatory responses that resulted in increases in the concentrations of blood monocytes, eosinophils, and basophils within 48 h postinjection. The pulmonary and systemic inflammatory responses triggered by cellulose micro-particles trapped in the lungs of broilers in this experiment were different from those elicited in mammals treated with Sephadex particles. Further studies are required to investigate the mechanism by which the inflammatory responses triggered by i.v. inert foreign particles might contribute to the onset of PHS in broilers, as well as the potential impact of micro-particle injection on changing the immunological profiles of selected broilers.
ACKNOWLEDGMENTS The authors gratefully acknowledge Joe N. Beasley for his expertise in identifying mast cells in the lung sections, and David Cross, Xiaoli Wang, Vjollca H. Konjufca, and J. Chad Johnson for their technical assistance in this project. This research was supported by a grant from Hubbard ISA, Walpole, NH 03608, and an Animal Health Research award from the Agricultural Experiment Station of the University of Arkansas Division of Agriculture.
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