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Cellular component of lavage fluid from broilers with normal versus aerosol-primed airways
Cellular component of lavage fluid from broilers with normal versus aerosol-primed airways A. G. Lorenzoni,*1 G. F. Erf,* N. C. Rath,† and R. F. Wideman Jr.* *University of Arkansas, Department of Poultry Science, Fayetteville 72701; and †University of Arkansas, USDA, Agricultural Research Service, Poultry Production and Product Safety Research Unit, Fayetteville 72701 cyte concentration in blood did not differ between groups, but the percentage of blood lymphocytes was lower in broilers from the Red#3+PG group compared with birds from the control group (52.4 ± 2.9 and 56.9 ± 2.9%, respectively). Cells recovered from the lavage fluid from both groups were primarily heterophils. The concentration of leukocytes was greater in the lavage fluid of broilers from the Red#3+PG group compared with broilers from the control group (763.2 ± 158.7 and 402.9 ± 62.6 white blood cells/µL, respectively), but the proportions among leukocytes were not different between the 2 groups. We propose that the increased concentration of leukocytes present within the airways was one of the components that enabled broilers pretreated with aerosolized Red#3+PG to exhibit PH responses to intratracheal lipopolysaccharide.
Key words: mucosal immune system, airway, lipopolysaccharide 2009 Poultry Science 88:303–308 doi:10.3382/ps.2008-00379
INTRODUCTION
We hypothesize that sensitization of the airways may be attributable to 3 factors. First, inhalation of Red#3+PG may interfere with the protective role of surfactant proteins in the conductive airways. Surfactant-associated proteins present in the respiratory airways of all vertebrates have the ability to modulate the mucosal immune system of the airways. For example, surfactant protein D is able to bind LPS molecules within the airways, decreasing the amount of free LPS able to stimulate respiratory resident macrophages in rats (van Rozendaal et al., 1999). In addition, surfactant protein A inhibits the production of proinflammatory cytokines after intratracheal administration of LPS in mice (Borron et al., 2000). The inability of surfactant proteins to bind incoming antigens efficiently may result from chemical interactions with Red#3+PG, which may permit greater quantities of LPS and other antigens to stimulate immune cells. Second, aerosolized Red#3+PG may elicit inflammation of the mucosa of the conducting airways, thereby causing immune cells to migrate to the submucosa of the airways. In ducks, nonciliated epithelial cells from the
Previously, we reported that broilers reared under commercial conditions exhibit pulmonary hypertension (PH) in response to intratracheal lipopolysaccharide (LPS) administration. In addition, broilers grown in environmental chambers under optimal conditions (low density, low humidity, fresh and dry litter) do not exhibit PH in response to an intratracheal challenge with LPS. However, 24 h after inhalation of an aerosolized mixture of propylene glycol and the food colorant red dye#3 (Red#3+PG), broilers reared in environmental chambers do develop PH responses when challenged with intratracheal LPS (Lorenzoni and Wideman, 2008). These findings indicate that the inhalation of aerosolized Red#3+PG primes or sensitizes the airways to intratracheal LPS.
©2009 Poultry Science Association Inc. Received September 3, 2008. Accepted October 24, 2008. 1 Corresponding author: [email protected]
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ABSTRACT Previously, we reported that intratracheal administration of lipopolysaccharide elicited pulmonary hypertension (PH) in broilers reared under commercial conditions and in broilers reared in environmental chambers and pretreated with aerosolized red food colorant # 3 and propylene glycol (Red#3+PG), but not in control broilers reared in environmental chambers. The objective of the present experiment was to determine possible changes in the number or proportion of airway leukocytes that could contribute to the magnitude of the PH responses elicited in broilers. Birds were aerosolized for 40 min with a saturated mixture of Red#3+PG. After 24 h, a blood sample was taken, the broilers were killed, and a pulmonary lavage process was conducted in each bird. Leukocyte concentration (white blood cells/µL) and differential leukocyte counts (%) were measured in blood and lavage fluid. Leuko-
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MATERIALS AND METHODS Bird Management Chicks from a commercial broiler line (Cobb-Vantress) were wing-banded and reared on clean wood shavings litter in environmental chambers (8 m2 of floor space) within the Poultry Environmental Research Laboratory on the University of Arkansas Poultry Research Farm. The birds were brooded at 33°C from d 1 to 3, 31°C from d 4 to 6, 29°C from d 7 to 10, 26°C from d 11 to 14, and 24°C thereafter. Birds were fed a 23% CP corn- and soybean meal-based diet formulated to meet the NRC (1994) standards for all ingredients. Birds received feed and water ad libitum. Feed was provided as crumbles throughout the experiment. Lights were on for 24 h/d through d 4 and for 16 h/d thereafter.
Airway Priming A CompMist Piston Compressor Nebulizer (Mabis Healthcare, Waukegan, IL) was used to vaporize the Red#3+PG solution into aerosol droplets ranging in diameter from 0.5 to 10 µm (Tiano and Dalby, 1996; Rau, 2002). Four 9-wk-old broilers at a time were placed inside an 80-L3 plastic box (50 × 40 × 40 cm), and the outlet tube from the nebulizer was inserted through a 2-cm hole in the uppermost portion of one end of the box. The nebulizer was loaded with a saturated solution of Red#3+PG, which was allowed to suffuse the entire box for up to 40 min. The CO2 accumulation inside the box promoted vigorous panting, which facilitated deep inhalation of the fog-like vapor into the respira-
tory tract. Panting inside the plastic box or aerosolization with water, blue, and yellow food colorant failed to induce hypertensive responses to intratracheal LPS (Lorenzoni and Wideman, 2008). Accordingly, control birds did not undergo aerosol pretreatment.
Lavage Procedure Twenty-four hours after the airway priming, the aerosolized and the control broilers were anesthetized with an intramuscular injection of 1 mL of allobarbital (5,5-diallyl-barbituric acid; 25 mg/mL; Sigma Chemical Co., St. Louis, MO) and 1 mL of ketamine HCl (100 mg/mL), and they were fastened in dorsal recumbency on a surgical board. The proximal end of a PE-50 tubing filled with heparinized saline (200 IU of heparin/mL of 0.9% NaCl) was inserted into the basilica vein and 10 mg of thiopental (20 mg/mL; Sigma Chemical Co.) was administered. One milliliter of blood was collected in a heparinized tube for further analysis of white blood cell (WBC) concentrations and proportions among WBC populations. Next, the bird was killed by bleeding via the venous cannula. In pilot studies, this method of euthanasia under deep anesthesia was found to reduce terminal struggling and thereby dramatically reduce the incidence of blood contaminating the lavage fluid after a broiler was killed. The skin of the neck was cut and the trachea was exposed and freed from connective tissue. A small incision was made in the mid trachea to allow the insertion of the tip of a 30-cm length of silicone tubing with an outer diameter (3 mm) slightly smaller than the inner diameter of the trachea. The tubing was fastened airtight inside the trachea with suture thread. The distal end of the silicone tubing was attached to a 3-way valve connected to two 20-mL syringes (modified from the method of Toth and Siegel, 1986). One of the syringes was used to create vacuum to collapse the air sacs (indicated by the retraction and collapse of the trachea) to minimize the amount of fluid that could remain trapped in the air sacs. The other syringe was used to gently administer 20 mL of roomtemperature saline (NaCl 0.9%). The thoracic cavity was massaged for 3 min while the bird was gently rotated laterally and dorsoventrally. The fluid was aspirated back into the syringe, and then fresh saline was administered and the lavage process was repeated a total of 3 times in each bird. The collected fluid from each bird was pooled, filtered through a coarse mesh filter, and vortexed. Immediately thereafter, 40 mL of the combined fluid was kept for analysis and the remainder was discarded. With this procedure, it was assumed that the lung volume of birds from the same flock did not vary significantly (Wideman and Bottje 1993) and that the lavage fluid within the lungs followed a similar distribution and suspended a proportional number of leukocytes in each bird. The fluid from each bird was labeled and kept at 4°C until analysis. An alternating schedule was used to analyze the samples from the Red#3+PG and control groups.
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lower respiratory system (atria and infundibula of the gas exchange region) take up and translocate particles (antigens) to the interstitium, where they can bind to receptors on the surface of monocytes, endothelial cells, and dendritic cells (Stearns et al., 1987; Brown et al., 1997). A change in the proportion or concentration of submucosal immune cells may change the outcome of chemical signals produced after the interaction with the imported antigens, thereby directly affecting physiological-immune responses. Migration of immune cells into the submucosa of the airways may pave the way for the third possibility, which is migration of immune cells directly into the airways. Chicken macrophages can be recruited into the airways after appropriate stimulation. In fact, 5-fold increases in free respiratory macrophages were reported 24 h after vaccination with Pasteurella multocida (Toth et al., 1988). The objective of the present study was to evaluate changes in the concentration and proportion of immune cells within the airways of broilers after a treatment with aerosolized Red#3+PG. An increase in leukocyte numbers or a shift in the proportions of leukocytes within the conducting airways may provide a clue to the increased PH responsiveness to intratracheal administration of LPS.
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Cellular Analysis of Lavage Fluid and Blood
Statistical Analysis
The lavage fluid was centrifuged at 250 × g for 10 min, the supernatant fluid was discarded, and the pellet was suspended in 10 mL of Dulbecco’s PBS (Sigma Chemical Co.). Next, 200 µL of the resuspended pellet was placed into a Cytopro 7620 cytocentrifuge (Wesco Inc., UT) and centrifuged at 50 × g for 3 min to transfer the cells to the surface of a microscope slide. Blood smears were prepared on microscope slides to evaluate the proportions (%) among leukocytes (lymphocytes, heterophils, monocytes, eosinophils, and basophils) in blood. The slides were stained with Wright stain and a minimum of 300 leukocytes were identified and counted for each slide at 1,000× magnification using a brightfield microscope (Lucas and Jamroz, 1961). To determine the concentrations of leukocytes in the lavage fluid and blood, a drop of 1:5 PBS dilution of lavage fluid and a drop of 1:100 PBS dilution of blood were placed into a hemocytometer. A 10-min period was allowed to elapse for thrombocytes to adhere to the glass surface and thereby induce a morphometric change for effective differentiation of thrombocytes from other lymphocytes. The red blood cells (RBC) and leukocytes were counted at 400× magnification using a bright-field microscope.
Differences between the Red#3+PG and control groups in RBC and WBC concentrations (RBC/ µL and WBC/µL), blood leukocyte proportions (%), lavage fluid cell concentrations (RBC/µL and WBC/ µL), and lavage fluid leukocyte proportions (%) were evaluated using Student’s t-test. Statistical differences were declared when P ≤ 0.05. The RBC:WBC ratio (hereafter called the correction factor) from the blood was used to adjust the leukocyte concentration from the lavage fluid for blood contamination with the following formula (Table 1): (WBC/µL in lavage fluid) – (RBC in lavage fluid/ correction factor).
Macroscopic Evaluation of Lavage Fluid Immediately after being extracted from the respiratory airways, the lavage fluid samples appeared to be clear. However, after centrifugation, all samples presented some degree of blood contamination that was seen as a thin red layer above a larger white pellet.
Table 1. Red blood cell (RBC) and white blood cell (WBC) count in blood and lavage fluid from control broilers and broilers that had inhaled a saturated solution of aerosolized red dye #3 and propylene glycol (Red#3+PG)1 Blood Bird no.
RBC/µL
Red#3+PG 1 Red#3+PG 2 Red#3+PG 3 Red#3+PG 4 Red#3+PG 5 Red#3+PG 6 Red#3+PG 7 Red#3+PG 8 Red#3+PG 9 Red#3+PG 10 Red#3+PG average SE Control 1 Control 2 Control 3 Control 4 Control 5 Control 6 Control 7 Control 8 Control 9 Control 10 Control average SE
2.5 3.1 3.6 3.3 3.5 4.1 2.9 3.0 3.7 3.6 3.3 0.2 3.0 2.7 3.1 2.7 2.9 2.9 3.2 3.7 3.2 3.2 3.1 0.2
a,b
× × × × × × × × × × × × × × × × × × × × × × × ×
106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106
Lavage fluid
WBC/µL 3.7 5 4 2.8 3.3 4.1 4.3 3.6 4.2 3.2 3.8 0.2 3.4 4.6 2.7 2.2 3.4 3.8 3.7 4 4.2 3.6 3.6 0.2
× × × × × × × × × × × × × × × × × × × × × × × ×
104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104
CF2
RBC/µL
WBC/µL
WBC st lav3
67.57 61.91 91.56 118.30 106.44 100.82 65.77 83.26 88.69 110.57 89.49 6.27 87.38 56.79 113.87 124.43 84.65 75.00 86.72 93.13 75.00 88.89 88.59 6.10
810 1,370 1,400 1,560 678 359 6 315.63 96.88 96.88 669.3 187.2 1,930 350 305 568 34 96 38 68 204 243 383.5 179.8
1,110 1,600 1,483 1,010 807 500 252 303 321 321 771.0a 161.0 890 542 236 250 213 425 471 279 442 323 407.3b 64.3
1,098.0 1,577.9 1,468.0 996.8 800.8 496.4 252.7 299.3 320.8 321.0 763.2a 158.7 867.9 536.7 233.7 245.4 212.1 423.7 470.4 278.7 439.6 320.6 402.9b 62.6
Difference between the corresponding column of the 2 treatments. Red#3+PG birds were treated with a vaporized solution of food colorant red # 3 and propylene glycol 24 h before lavage fluid collection. Control birds received no pretreatment. 2 CF = correction factor = RBC in blood/WBC in blood. 3 WBC st lav = standardized WBC in lavage fluid = (WBC/µL in lav) − (RBC in lav/CF). 1
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RESULTS
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Microscopic Evaluation of Lavage Fluid and Blood
The objective of the present experiment was to determine possible changes in the number or proportion of airway leukocytes that could contribute to the magnitude of the PH responses elicited in broilers by an intratracheal challenge with LPS (Lorenzoni and Wideman, 2008). One of the most difficult aspects to address when studying cells derived from the airways of birds is the intricate anatomy of the lungs, which have conducting airways that connect with several air sacs. The intercommunication between airways and air sacs complicates the process of lung lavage and fluid collection. Several techniques have been described to collect lavage fluid from birds (Toth and Siegel, 1986; Toth et al., 1988; Fulton et al., 1990, 1993; Klika et al., 1996; Nganpiep and Maina, 2002; Holt et al., 2005). However, during pilot studies, we were unable to collect bloodfree lavage fluid in a consistent manner. A possible explanation for this observation is that blood capillaries within the lungs of different broiler lines may have different degrees of fragility. Apparently, the method of euthanasia also plays an important role in the collection of suitable samples. Terminal struggles caused by euthanasia via cervical dislocation or CO2 inhalation may contribute to the rupture of lung capillaries. We found that gradually bleeding deeply anesthetized birds was the optimal way to obtain grossly clear lavage fluid consistently. To strengthen the calculations presented in this experiment, leukocyte concentrations in the lavage fluid were normalized to adjust for variable levels of blood contamination among the samples. Our results showed that the number of leukocytes (Table 1), but not the proportion among leukocytes present within the airways, was greater in broilers from the Red#3+PG group than in birds from the control group. We also found that the proportion of lymphocytes within the blood was lower in the Red#3+PG group than in the control group. These results support the theory that the number of local cells interacting directly with environmental antigens may have an impact in the subsequent mucosal immune response, and suggest that the mobilization of lymphocytes from the blood may be involved as well. In fact, preliminary histological observations of lung sections derived from the same birds that were included in the present study suggest that lymphocytes may be sequestered within the submucosa of the intrapulmonary conducting airways. In mammals, neutrophils migrate into the small conducting airways and alveoli through preexisting holes surrounded by fibroblasts in the endothelial and epithelial basal laminae. This migration seems to be mediated by the expression of intercellular adhesion molecule-1 on the pulmonary endothelium (Behzad et al., 1995) and occurs within the pulmonary capillaries rather than within postcapillary venules (Doerschuk, 2000). It is possible that a similar mechanism operates in birds.
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Concentration of RBC and Leukocytes in Lavage Fluid and Blood. As shown in Table 1, the standardized leukocyte concentration in the lavage fluid was greater in the Red#3+PG group than in the control group (763.2 ± 158.7 and 402.9 ± 62.6 leukocytes/µL, respectively), but no differences between these groups were found for the concentration of WBC or RBC in blood. Blood contamination, measured as the number of RBC/µL of lavage fluid, was not different between the groups; however, birds within the Red#3+PG group tended to exhibit more blood contamination in lavage fluid than did birds in the control group (Table 1). Proportions Among Cell Populations in the Lavage Fluid. Heterophils, lymphocytes, and macrophages were the predominant immune cells within the lavage fluid. Ciliated and nonciliated epithelial cells were often present in the lavage fluid but they were not included in the differential leukocyte counting. Heterophils were the predominant cell type in the lavage fluid from both groups. Heterophils very often presented a clear cytoplasm filled with engorged and elongated granules. The heterophilic granules were typically larger than the granules of blood heterophils, and usually a round dark dot was present in the center of each granule. Basophils and eosinophils were very scarce (≤1 per 300 cells) and thus were included with the heterophils in the more general category of granulocytes, which did not differ significantly between the Red#3+PG and control groups (92.2 ± 1.8 and 89.4 ± 2.1%, respectively). Lymphocytes were recognized as small round cells with a relatively large nucleus and a rim of dark-blue cytoplasm. Macrophages were larger than lymphocytes and their nuclei were also larger, but not as round as the nuclei of the lymphocytes. Additionally, the cytoplasm of macrophages was more abundant and paler than the lymphocytic cytoplasm. Because of the abundant number of cells that morphologically did not correspond to macrophages or lymphocytes, but rather shared common characteristics, a general mononuclear leukocytes category was created, which corresponded to the sum of both cell populations. The mononuclear leukocytes percentage did not differ between the Red#3+PG and control groups (7.8 ± 1.8 and 10.6 ± 2.1%, respectively). Proportions Among Cell Populations in Blood. The percentage of lymphocytes in the blood was lower (P = 0.02) in the Red#3+PG group than in the control group (52.8 ± 2.9 and 56.9 ± 2.9%, respectively). Values for heterophils (32.0 ± 2.8 and 28.7 ± 2.5), monocytes (9.0 ± 1.4 and 7.0 ± 1.0), basophils (4.8 ± 0.7 and 5.4 ± 0.5), and eosinophils (1.8 ± 0.3 and 2.1 ± 0.4) were not different between the Red#3+PG and control groups, respectively.
DISCUSSION
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tory factors such as IL-6 in chicken heterophils (Rath et al., 1998). In conclusion, the respiratory tract of birds with primed airways had a greater number of leukocytes, primarily granulocytes (heterophils), within the airways and a lower proportion of circulating lymphocytes in blood compared with control birds. Regardless of the lack of a hypertensive response in control birds from a comparable study in our laboratory (Lorenzoni and Wideman, 2008), a considerable number of WBC were found within the airways of control birds. This could mean that the different components of the mucosal immune system (surfactant proteins, epithelial cells, or signals from submucosal leukocytes) may serve as a mechanism of reciprocal modulation and that imbalance of this system of modulation may be necessary to mount an immune response.
ACKNOWLEDGMENTS This research was supported by an Animal Health grant from the University of Arkansas Division of Agriculture, Agricultural Experimental Station (Fayetteville).
REFERENCES Behzad, A. R., F. Chu, and D. Walker. 1995. Fibroblasts are in position to provide directional information to migrating neutrophils during pneumonia in rabbit lungs. Microvasc. Res. 51:303–316. Borron, P., J. C. McIntosh, T. R. Korfhagen, J. A. Whitsett, J. Taylor, and J. R. Wright. 2000. Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo. Am. J. Physiol. Lung Cell. Mol. Physiol. 278:L840–L847. Brown, R. E., J. D. Brain, and N. Wang. 1997. The avian respiratory system: A unique model for studies of respiratory toxicosis and for monitoring air quality. Environ. Health Perspect. 105:188–200. Doerschuk, C. M. 2000. Leukocyte trafficking in alveoli and airway passages. Respir. Res. 1:136–140. Fulton, R. M., W. M. Reed, and D. B. DeNicola. 1990. Light microscopic and ultrastructural characterization of cells recovered by respiratory-tract lavage of 2- and 6-week old chickens. Avian Dis. 34:87–98. Fulton, R.M., W.M. Reed, and H.L. Thacker. 1993. Cellular response of the respiratory tract of chickens to infection with Massachusetts 41 and Australian T infectious bronchitis viruses. Avian Dis. 37:951–960. Harmon, B. G. 1998. Avian heterophils in inflammation and disease resistance. Poult. Sci. 77:972–977. Holt, P. G., J. Oliver, N. Bilyk, C. McMenamin, P. G. McMenamin, G. Kraal, and T. Thephen. 1993. Downregulation of the antigen presenting cell function of pulmonary dendritic cells in vivo by resident alveolar macrophages. J. Exp. Med. 177:397–407. Holt, P. S., H. D. Stone, R. W. Moore, and R. K. Gast. 2005. Development of a lavage procedure to collect lung secretions from chickens for evaluating respiratory humoral immunity. Avian Pathol. 34:396–398. Klika, E., D. W. Scheuermann, M. H. A. De Groodt-Lasseel, I. Bazantova, and A. Switka. 1996. Pulmonary macrophages in birds (barn owl, Tyto tyto alba), domestic fowl (Gallus gallus f. domestica), quail (Coturnix coturnix), and pigeons (Columbia livia). Anat. Rec. 246:87–97. Lorenzoni, A. G., and R. F. Wideman. 2008. Intratracheal administration of bacterial lipopolysaccharide elicits pulmonary hypertension in broilers with primed airways. Poult. Sci. 87:645–654.
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Sentinel immune cells are present in the airways of birds (Fulton et al., 1990; Klika et al., 1996; Nganpiep and Maina, 2002; present study). These cells may be able to respond to an intratracheal LPS challenge under normal conditions. However, it seems clear that mucosal immunity evolved to allow a degree of tolerance to environmental antigens and avoid repeated and massive life-threatening responses (Xavier and Podolsky, 2000). The existence of momentarily underresponsive sentinel cells suggests that at least 2 of the 3 proposed mechanisms (airway immune cells, surfactant proteins, or submucosal immune cells) act in concert to mount a mucosal immune response. Supporting this theory, surfactant protein A downregulates proinflammatory cytokine production evoked by Candida albicans in human alveolar macrophages (Rosseau et al., 1999) and, in turn, rat pulmonary alveolar macrophages can secrete nitric oxide-dependant soluble mediators that suppress the activation of dendritic cells located within the submucosa (Holt et al., 1993). With regard to the number of leukocytes within the airways of control birds, it could be argued that control birds in this experiment were not raised in a pathogenfree environment; thus, control birds also were challenged with low but constant doses of aerosolized litter dust. On the other hand, under the experimental conditions of this study, the air and bed quality in the environmental chambers are far superior to those found in commercial poultry production. In fact, we have shown that birds raised under standard commercial conditions have their airways already primed and ready to respond to intratracheal LPS challenges (Lorenzoni and Wideman, 2008). A primed state of the airways may mean that the surfactant fluid is undergoing chemical changes, or that immune cells within airways and submucosa are already stimulated with a cascade of proinflammatory factors (such as IL-6) and thus are ready to respond. The idea that deficient management practices are responsible for the high incidence of respiratory diseases in broilers has already been proposed (Nganpiep and Maina, 2002). We agree with this theory and further propose a mechanism that explains the immune consequences of deficient management practices. Under commercial conditions, the respiratory tract of birds is already primed; thus, large numbers of inflammatory cells may already be present within the lungs. Inflammatory factors derived from these cells may aggravate the integrity of the respiratory endothelium, allowing for the easier colonization and spread of pathogens within a flock. Degranulation of heterophils, the major cellular component found in the present study, leads to the release of cationic peptides, acid phosphatase, cathepsin, oxidative factors, and several lysosomal enzymes, including metalloproteinase, which are known to disrupt connective tissue (Harmon, 1998). Interestingly, LPS is known to upregulate the production and release of metalloproteinase, oxidative factors, and proinflamma-
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Lucas, A. M., and C. Jamroz. 1961. Atlas of Avian Hematology. Agric. Monogr. USDA, Washington, DC. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Nganpiep, L. N., and J. N. Maina. 2002. Composite cellular defense stratagem in the avian respiratory system: Functional morphology of the free (surface) macrophages and specialized pulmonary epithelia. J. Anat. 200:499–516. Rath, N. C., G. R. Huff, J. M. Balog, and W. E. Huff. 1998. Fluorescein isothiocyanate staining and characterization of avian heterophils. Vet. Immunol. Immunopathol. 64:83–95. Rau, J. L. 2002. Design principles of liquid nebulization devices currently in use. Respir. Care 47:1257–1275. Rosseau, S., P. Hammerl, U. Maus, A. Gunter, W. Seeger, F. Grimminger, and J. Lohmeyer. 1999. Surfactant protein A down-regulates proinflammatory cytokine production evoked by Candida albicans in human alveolar macrophages and monocytes. Immunology 163:4495–4502. Stearns, R. C., G. M. Barnas, M. Walski, and J. D. Brain. 1987. Deposition and phagocytosis of inhaled particles in the gas exchange region of the duck, Anas platyrhynchos. Respir. Physiol. 67:23–36.
Tiano, S. L., and R. N. Dalby. 1996. Comparison of a respiratory suspension aerosolized by an air-jet and an ultrasonic nebulizer. Pharm. Dev. Technol. 1:261–268. Toth, T. E., R. H. Pyle, T. Caceci, P. B. Siegel, and D. Ochs. 1988. Cellular defense of the avian respiratory system: Influx and nonopsonic phagocytes activated by Pasteurella multocida. Infect. Immun. 56:1171–1179. Toth, T. E., and P. B. Siegel. 1986. Cellular defense of the avian respiratory tract: Paucity of free-residing macrophages in the normal chicken. Avian Dis. 30:67–75. van Rozendaal, B. A. W., H. A. Chris, M. van Eijk, L. M. G. van Golde, W. F. Voorhout, H. P. M. van Helden, and H. P. Haagsman. 1999. Aerosolized endotoxin is immediately bound by pulmonary surfactant protein D in vivo. Biochim. Biophys. Acta 1454:261–269. Wideman, R. F., and W. G. Bottje. 1993. Current understanding of the ascites syndrome and future research directions. Pages 1–20 in Nutr. Techn,ical Symp. Proc. Novus International Inc., St. Louis, MO. Xavier, R. J., and D. K. Podolsky. 2000. How to get along—Friendly microbes in a hostile world. Science 289:1483–1484. Downloaded from http://ps.oxfordjournals.org/ at Gazi Universitesi on May 3, 2015
Key words: mucosal immune system, airway, lipopolysaccharide 2009 Poultry Science 88:303–308 doi:10.3382/ps.2008-00379
INTRODUCTION
We hypothesize that sensitization of the airways may be attributable to 3 factors. First, inhalation of Red#3+PG may interfere with the protective role of surfactant proteins in the conductive airways. Surfactant-associated proteins present in the respiratory airways of all vertebrates have the ability to modulate the mucosal immune system of the airways. For example, surfactant protein D is able to bind LPS molecules within the airways, decreasing the amount of free LPS able to stimulate respiratory resident macrophages in rats (van Rozendaal et al., 1999). In addition, surfactant protein A inhibits the production of proinflammatory cytokines after intratracheal administration of LPS in mice (Borron et al., 2000). The inability of surfactant proteins to bind incoming antigens efficiently may result from chemical interactions with Red#3+PG, which may permit greater quantities of LPS and other antigens to stimulate immune cells. Second, aerosolized Red#3+PG may elicit inflammation of the mucosa of the conducting airways, thereby causing immune cells to migrate to the submucosa of the airways. In ducks, nonciliated epithelial cells from the
Previously, we reported that broilers reared under commercial conditions exhibit pulmonary hypertension (PH) in response to intratracheal lipopolysaccharide (LPS) administration. In addition, broilers grown in environmental chambers under optimal conditions (low density, low humidity, fresh and dry litter) do not exhibit PH in response to an intratracheal challenge with LPS. However, 24 h after inhalation of an aerosolized mixture of propylene glycol and the food colorant red dye#3 (Red#3+PG), broilers reared in environmental chambers do develop PH responses when challenged with intratracheal LPS (Lorenzoni and Wideman, 2008). These findings indicate that the inhalation of aerosolized Red#3+PG primes or sensitizes the airways to intratracheal LPS.
©2009 Poultry Science Association Inc. Received September 3, 2008. Accepted October 24, 2008. 1 Corresponding author: [email protected]
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ABSTRACT Previously, we reported that intratracheal administration of lipopolysaccharide elicited pulmonary hypertension (PH) in broilers reared under commercial conditions and in broilers reared in environmental chambers and pretreated with aerosolized red food colorant # 3 and propylene glycol (Red#3+PG), but not in control broilers reared in environmental chambers. The objective of the present experiment was to determine possible changes in the number or proportion of airway leukocytes that could contribute to the magnitude of the PH responses elicited in broilers. Birds were aerosolized for 40 min with a saturated mixture of Red#3+PG. After 24 h, a blood sample was taken, the broilers were killed, and a pulmonary lavage process was conducted in each bird. Leukocyte concentration (white blood cells/µL) and differential leukocyte counts (%) were measured in blood and lavage fluid. Leuko-
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MATERIALS AND METHODS Bird Management Chicks from a commercial broiler line (Cobb-Vantress) were wing-banded and reared on clean wood shavings litter in environmental chambers (8 m2 of floor space) within the Poultry Environmental Research Laboratory on the University of Arkansas Poultry Research Farm. The birds were brooded at 33°C from d 1 to 3, 31°C from d 4 to 6, 29°C from d 7 to 10, 26°C from d 11 to 14, and 24°C thereafter. Birds were fed a 23% CP corn- and soybean meal-based diet formulated to meet the NRC (1994) standards for all ingredients. Birds received feed and water ad libitum. Feed was provided as crumbles throughout the experiment. Lights were on for 24 h/d through d 4 and for 16 h/d thereafter.
Airway Priming A CompMist Piston Compressor Nebulizer (Mabis Healthcare, Waukegan, IL) was used to vaporize the Red#3+PG solution into aerosol droplets ranging in diameter from 0.5 to 10 µm (Tiano and Dalby, 1996; Rau, 2002). Four 9-wk-old broilers at a time were placed inside an 80-L3 plastic box (50 × 40 × 40 cm), and the outlet tube from the nebulizer was inserted through a 2-cm hole in the uppermost portion of one end of the box. The nebulizer was loaded with a saturated solution of Red#3+PG, which was allowed to suffuse the entire box for up to 40 min. The CO2 accumulation inside the box promoted vigorous panting, which facilitated deep inhalation of the fog-like vapor into the respira-
tory tract. Panting inside the plastic box or aerosolization with water, blue, and yellow food colorant failed to induce hypertensive responses to intratracheal LPS (Lorenzoni and Wideman, 2008). Accordingly, control birds did not undergo aerosol pretreatment.
Lavage Procedure Twenty-four hours after the airway priming, the aerosolized and the control broilers were anesthetized with an intramuscular injection of 1 mL of allobarbital (5,5-diallyl-barbituric acid; 25 mg/mL; Sigma Chemical Co., St. Louis, MO) and 1 mL of ketamine HCl (100 mg/mL), and they were fastened in dorsal recumbency on a surgical board. The proximal end of a PE-50 tubing filled with heparinized saline (200 IU of heparin/mL of 0.9% NaCl) was inserted into the basilica vein and 10 mg of thiopental (20 mg/mL; Sigma Chemical Co.) was administered. One milliliter of blood was collected in a heparinized tube for further analysis of white blood cell (WBC) concentrations and proportions among WBC populations. Next, the bird was killed by bleeding via the venous cannula. In pilot studies, this method of euthanasia under deep anesthesia was found to reduce terminal struggling and thereby dramatically reduce the incidence of blood contaminating the lavage fluid after a broiler was killed. The skin of the neck was cut and the trachea was exposed and freed from connective tissue. A small incision was made in the mid trachea to allow the insertion of the tip of a 30-cm length of silicone tubing with an outer diameter (3 mm) slightly smaller than the inner diameter of the trachea. The tubing was fastened airtight inside the trachea with suture thread. The distal end of the silicone tubing was attached to a 3-way valve connected to two 20-mL syringes (modified from the method of Toth and Siegel, 1986). One of the syringes was used to create vacuum to collapse the air sacs (indicated by the retraction and collapse of the trachea) to minimize the amount of fluid that could remain trapped in the air sacs. The other syringe was used to gently administer 20 mL of roomtemperature saline (NaCl 0.9%). The thoracic cavity was massaged for 3 min while the bird was gently rotated laterally and dorsoventrally. The fluid was aspirated back into the syringe, and then fresh saline was administered and the lavage process was repeated a total of 3 times in each bird. The collected fluid from each bird was pooled, filtered through a coarse mesh filter, and vortexed. Immediately thereafter, 40 mL of the combined fluid was kept for analysis and the remainder was discarded. With this procedure, it was assumed that the lung volume of birds from the same flock did not vary significantly (Wideman and Bottje 1993) and that the lavage fluid within the lungs followed a similar distribution and suspended a proportional number of leukocytes in each bird. The fluid from each bird was labeled and kept at 4°C until analysis. An alternating schedule was used to analyze the samples from the Red#3+PG and control groups.
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lower respiratory system (atria and infundibula of the gas exchange region) take up and translocate particles (antigens) to the interstitium, where they can bind to receptors on the surface of monocytes, endothelial cells, and dendritic cells (Stearns et al., 1987; Brown et al., 1997). A change in the proportion or concentration of submucosal immune cells may change the outcome of chemical signals produced after the interaction with the imported antigens, thereby directly affecting physiological-immune responses. Migration of immune cells into the submucosa of the airways may pave the way for the third possibility, which is migration of immune cells directly into the airways. Chicken macrophages can be recruited into the airways after appropriate stimulation. In fact, 5-fold increases in free respiratory macrophages were reported 24 h after vaccination with Pasteurella multocida (Toth et al., 1988). The objective of the present study was to evaluate changes in the concentration and proportion of immune cells within the airways of broilers after a treatment with aerosolized Red#3+PG. An increase in leukocyte numbers or a shift in the proportions of leukocytes within the conducting airways may provide a clue to the increased PH responsiveness to intratracheal administration of LPS.
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Cellular Analysis of Lavage Fluid and Blood
Statistical Analysis
The lavage fluid was centrifuged at 250 × g for 10 min, the supernatant fluid was discarded, and the pellet was suspended in 10 mL of Dulbecco’s PBS (Sigma Chemical Co.). Next, 200 µL of the resuspended pellet was placed into a Cytopro 7620 cytocentrifuge (Wesco Inc., UT) and centrifuged at 50 × g for 3 min to transfer the cells to the surface of a microscope slide. Blood smears were prepared on microscope slides to evaluate the proportions (%) among leukocytes (lymphocytes, heterophils, monocytes, eosinophils, and basophils) in blood. The slides were stained with Wright stain and a minimum of 300 leukocytes were identified and counted for each slide at 1,000× magnification using a brightfield microscope (Lucas and Jamroz, 1961). To determine the concentrations of leukocytes in the lavage fluid and blood, a drop of 1:5 PBS dilution of lavage fluid and a drop of 1:100 PBS dilution of blood were placed into a hemocytometer. A 10-min period was allowed to elapse for thrombocytes to adhere to the glass surface and thereby induce a morphometric change for effective differentiation of thrombocytes from other lymphocytes. The red blood cells (RBC) and leukocytes were counted at 400× magnification using a bright-field microscope.
Differences between the Red#3+PG and control groups in RBC and WBC concentrations (RBC/ µL and WBC/µL), blood leukocyte proportions (%), lavage fluid cell concentrations (RBC/µL and WBC/ µL), and lavage fluid leukocyte proportions (%) were evaluated using Student’s t-test. Statistical differences were declared when P ≤ 0.05. The RBC:WBC ratio (hereafter called the correction factor) from the blood was used to adjust the leukocyte concentration from the lavage fluid for blood contamination with the following formula (Table 1): (WBC/µL in lavage fluid) – (RBC in lavage fluid/ correction factor).
Macroscopic Evaluation of Lavage Fluid Immediately after being extracted from the respiratory airways, the lavage fluid samples appeared to be clear. However, after centrifugation, all samples presented some degree of blood contamination that was seen as a thin red layer above a larger white pellet.
Table 1. Red blood cell (RBC) and white blood cell (WBC) count in blood and lavage fluid from control broilers and broilers that had inhaled a saturated solution of aerosolized red dye #3 and propylene glycol (Red#3+PG)1 Blood Bird no.
RBC/µL
Red#3+PG 1 Red#3+PG 2 Red#3+PG 3 Red#3+PG 4 Red#3+PG 5 Red#3+PG 6 Red#3+PG 7 Red#3+PG 8 Red#3+PG 9 Red#3+PG 10 Red#3+PG average SE Control 1 Control 2 Control 3 Control 4 Control 5 Control 6 Control 7 Control 8 Control 9 Control 10 Control average SE
2.5 3.1 3.6 3.3 3.5 4.1 2.9 3.0 3.7 3.6 3.3 0.2 3.0 2.7 3.1 2.7 2.9 2.9 3.2 3.7 3.2 3.2 3.1 0.2
a,b
× × × × × × × × × × × × × × × × × × × × × × × ×
106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106
Lavage fluid
WBC/µL 3.7 5 4 2.8 3.3 4.1 4.3 3.6 4.2 3.2 3.8 0.2 3.4 4.6 2.7 2.2 3.4 3.8 3.7 4 4.2 3.6 3.6 0.2
× × × × × × × × × × × × × × × × × × × × × × × ×
104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104 104
CF2
RBC/µL
WBC/µL
WBC st lav3
67.57 61.91 91.56 118.30 106.44 100.82 65.77 83.26 88.69 110.57 89.49 6.27 87.38 56.79 113.87 124.43 84.65 75.00 86.72 93.13 75.00 88.89 88.59 6.10
810 1,370 1,400 1,560 678 359 6 315.63 96.88 96.88 669.3 187.2 1,930 350 305 568 34 96 38 68 204 243 383.5 179.8
1,110 1,600 1,483 1,010 807 500 252 303 321 321 771.0a 161.0 890 542 236 250 213 425 471 279 442 323 407.3b 64.3
1,098.0 1,577.9 1,468.0 996.8 800.8 496.4 252.7 299.3 320.8 321.0 763.2a 158.7 867.9 536.7 233.7 245.4 212.1 423.7 470.4 278.7 439.6 320.6 402.9b 62.6
Difference between the corresponding column of the 2 treatments. Red#3+PG birds were treated with a vaporized solution of food colorant red # 3 and propylene glycol 24 h before lavage fluid collection. Control birds received no pretreatment. 2 CF = correction factor = RBC in blood/WBC in blood. 3 WBC st lav = standardized WBC in lavage fluid = (WBC/µL in lav) − (RBC in lav/CF). 1
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RESULTS
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Microscopic Evaluation of Lavage Fluid and Blood
The objective of the present experiment was to determine possible changes in the number or proportion of airway leukocytes that could contribute to the magnitude of the PH responses elicited in broilers by an intratracheal challenge with LPS (Lorenzoni and Wideman, 2008). One of the most difficult aspects to address when studying cells derived from the airways of birds is the intricate anatomy of the lungs, which have conducting airways that connect with several air sacs. The intercommunication between airways and air sacs complicates the process of lung lavage and fluid collection. Several techniques have been described to collect lavage fluid from birds (Toth and Siegel, 1986; Toth et al., 1988; Fulton et al., 1990, 1993; Klika et al., 1996; Nganpiep and Maina, 2002; Holt et al., 2005). However, during pilot studies, we were unable to collect bloodfree lavage fluid in a consistent manner. A possible explanation for this observation is that blood capillaries within the lungs of different broiler lines may have different degrees of fragility. Apparently, the method of euthanasia also plays an important role in the collection of suitable samples. Terminal struggles caused by euthanasia via cervical dislocation or CO2 inhalation may contribute to the rupture of lung capillaries. We found that gradually bleeding deeply anesthetized birds was the optimal way to obtain grossly clear lavage fluid consistently. To strengthen the calculations presented in this experiment, leukocyte concentrations in the lavage fluid were normalized to adjust for variable levels of blood contamination among the samples. Our results showed that the number of leukocytes (Table 1), but not the proportion among leukocytes present within the airways, was greater in broilers from the Red#3+PG group than in birds from the control group. We also found that the proportion of lymphocytes within the blood was lower in the Red#3+PG group than in the control group. These results support the theory that the number of local cells interacting directly with environmental antigens may have an impact in the subsequent mucosal immune response, and suggest that the mobilization of lymphocytes from the blood may be involved as well. In fact, preliminary histological observations of lung sections derived from the same birds that were included in the present study suggest that lymphocytes may be sequestered within the submucosa of the intrapulmonary conducting airways. In mammals, neutrophils migrate into the small conducting airways and alveoli through preexisting holes surrounded by fibroblasts in the endothelial and epithelial basal laminae. This migration seems to be mediated by the expression of intercellular adhesion molecule-1 on the pulmonary endothelium (Behzad et al., 1995) and occurs within the pulmonary capillaries rather than within postcapillary venules (Doerschuk, 2000). It is possible that a similar mechanism operates in birds.
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Concentration of RBC and Leukocytes in Lavage Fluid and Blood. As shown in Table 1, the standardized leukocyte concentration in the lavage fluid was greater in the Red#3+PG group than in the control group (763.2 ± 158.7 and 402.9 ± 62.6 leukocytes/µL, respectively), but no differences between these groups were found for the concentration of WBC or RBC in blood. Blood contamination, measured as the number of RBC/µL of lavage fluid, was not different between the groups; however, birds within the Red#3+PG group tended to exhibit more blood contamination in lavage fluid than did birds in the control group (Table 1). Proportions Among Cell Populations in the Lavage Fluid. Heterophils, lymphocytes, and macrophages were the predominant immune cells within the lavage fluid. Ciliated and nonciliated epithelial cells were often present in the lavage fluid but they were not included in the differential leukocyte counting. Heterophils were the predominant cell type in the lavage fluid from both groups. Heterophils very often presented a clear cytoplasm filled with engorged and elongated granules. The heterophilic granules were typically larger than the granules of blood heterophils, and usually a round dark dot was present in the center of each granule. Basophils and eosinophils were very scarce (≤1 per 300 cells) and thus were included with the heterophils in the more general category of granulocytes, which did not differ significantly between the Red#3+PG and control groups (92.2 ± 1.8 and 89.4 ± 2.1%, respectively). Lymphocytes were recognized as small round cells with a relatively large nucleus and a rim of dark-blue cytoplasm. Macrophages were larger than lymphocytes and their nuclei were also larger, but not as round as the nuclei of the lymphocytes. Additionally, the cytoplasm of macrophages was more abundant and paler than the lymphocytic cytoplasm. Because of the abundant number of cells that morphologically did not correspond to macrophages or lymphocytes, but rather shared common characteristics, a general mononuclear leukocytes category was created, which corresponded to the sum of both cell populations. The mononuclear leukocytes percentage did not differ between the Red#3+PG and control groups (7.8 ± 1.8 and 10.6 ± 2.1%, respectively). Proportions Among Cell Populations in Blood. The percentage of lymphocytes in the blood was lower (P = 0.02) in the Red#3+PG group than in the control group (52.8 ± 2.9 and 56.9 ± 2.9%, respectively). Values for heterophils (32.0 ± 2.8 and 28.7 ± 2.5), monocytes (9.0 ± 1.4 and 7.0 ± 1.0), basophils (4.8 ± 0.7 and 5.4 ± 0.5), and eosinophils (1.8 ± 0.3 and 2.1 ± 0.4) were not different between the Red#3+PG and control groups, respectively.
DISCUSSION
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tory factors such as IL-6 in chicken heterophils (Rath et al., 1998). In conclusion, the respiratory tract of birds with primed airways had a greater number of leukocytes, primarily granulocytes (heterophils), within the airways and a lower proportion of circulating lymphocytes in blood compared with control birds. Regardless of the lack of a hypertensive response in control birds from a comparable study in our laboratory (Lorenzoni and Wideman, 2008), a considerable number of WBC were found within the airways of control birds. This could mean that the different components of the mucosal immune system (surfactant proteins, epithelial cells, or signals from submucosal leukocytes) may serve as a mechanism of reciprocal modulation and that imbalance of this system of modulation may be necessary to mount an immune response.
ACKNOWLEDGMENTS This research was supported by an Animal Health grant from the University of Arkansas Division of Agriculture, Agricultural Experimental Station (Fayetteville).
REFERENCES Behzad, A. R., F. Chu, and D. Walker. 1995. Fibroblasts are in position to provide directional information to migrating neutrophils during pneumonia in rabbit lungs. Microvasc. Res. 51:303–316. Borron, P., J. C. McIntosh, T. R. Korfhagen, J. A. Whitsett, J. Taylor, and J. R. Wright. 2000. Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo. Am. J. Physiol. Lung Cell. Mol. Physiol. 278:L840–L847. Brown, R. E., J. D. Brain, and N. Wang. 1997. The avian respiratory system: A unique model for studies of respiratory toxicosis and for monitoring air quality. Environ. Health Perspect. 105:188–200. Doerschuk, C. M. 2000. Leukocyte trafficking in alveoli and airway passages. Respir. Res. 1:136–140. Fulton, R. M., W. M. Reed, and D. B. DeNicola. 1990. Light microscopic and ultrastructural characterization of cells recovered by respiratory-tract lavage of 2- and 6-week old chickens. Avian Dis. 34:87–98. Fulton, R.M., W.M. Reed, and H.L. Thacker. 1993. Cellular response of the respiratory tract of chickens to infection with Massachusetts 41 and Australian T infectious bronchitis viruses. Avian Dis. 37:951–960. Harmon, B. G. 1998. Avian heterophils in inflammation and disease resistance. Poult. Sci. 77:972–977. Holt, P. G., J. Oliver, N. Bilyk, C. McMenamin, P. G. McMenamin, G. Kraal, and T. Thephen. 1993. Downregulation of the antigen presenting cell function of pulmonary dendritic cells in vivo by resident alveolar macrophages. J. Exp. Med. 177:397–407. Holt, P. S., H. D. Stone, R. W. Moore, and R. K. Gast. 2005. Development of a lavage procedure to collect lung secretions from chickens for evaluating respiratory humoral immunity. Avian Pathol. 34:396–398. Klika, E., D. W. Scheuermann, M. H. A. De Groodt-Lasseel, I. Bazantova, and A. Switka. 1996. Pulmonary macrophages in birds (barn owl, Tyto tyto alba), domestic fowl (Gallus gallus f. domestica), quail (Coturnix coturnix), and pigeons (Columbia livia). Anat. Rec. 246:87–97. Lorenzoni, A. G., and R. F. Wideman. 2008. Intratracheal administration of bacterial lipopolysaccharide elicits pulmonary hypertension in broilers with primed airways. Poult. Sci. 87:645–654.
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Sentinel immune cells are present in the airways of birds (Fulton et al., 1990; Klika et al., 1996; Nganpiep and Maina, 2002; present study). These cells may be able to respond to an intratracheal LPS challenge under normal conditions. However, it seems clear that mucosal immunity evolved to allow a degree of tolerance to environmental antigens and avoid repeated and massive life-threatening responses (Xavier and Podolsky, 2000). The existence of momentarily underresponsive sentinel cells suggests that at least 2 of the 3 proposed mechanisms (airway immune cells, surfactant proteins, or submucosal immune cells) act in concert to mount a mucosal immune response. Supporting this theory, surfactant protein A downregulates proinflammatory cytokine production evoked by Candida albicans in human alveolar macrophages (Rosseau et al., 1999) and, in turn, rat pulmonary alveolar macrophages can secrete nitric oxide-dependant soluble mediators that suppress the activation of dendritic cells located within the submucosa (Holt et al., 1993). With regard to the number of leukocytes within the airways of control birds, it could be argued that control birds in this experiment were not raised in a pathogenfree environment; thus, control birds also were challenged with low but constant doses of aerosolized litter dust. On the other hand, under the experimental conditions of this study, the air and bed quality in the environmental chambers are far superior to those found in commercial poultry production. In fact, we have shown that birds raised under standard commercial conditions have their airways already primed and ready to respond to intratracheal LPS challenges (Lorenzoni and Wideman, 2008). A primed state of the airways may mean that the surfactant fluid is undergoing chemical changes, or that immune cells within airways and submucosa are already stimulated with a cascade of proinflammatory factors (such as IL-6) and thus are ready to respond. The idea that deficient management practices are responsible for the high incidence of respiratory diseases in broilers has already been proposed (Nganpiep and Maina, 2002). We agree with this theory and further propose a mechanism that explains the immune consequences of deficient management practices. Under commercial conditions, the respiratory tract of birds is already primed; thus, large numbers of inflammatory cells may already be present within the lungs. Inflammatory factors derived from these cells may aggravate the integrity of the respiratory endothelium, allowing for the easier colonization and spread of pathogens within a flock. Degranulation of heterophils, the major cellular component found in the present study, leads to the release of cationic peptides, acid phosphatase, cathepsin, oxidative factors, and several lysosomal enzymes, including metalloproteinase, which are known to disrupt connective tissue (Harmon, 1998). Interestingly, LPS is known to upregulate the production and release of metalloproteinase, oxidative factors, and proinflamma-
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