The β2 integrin cell adhesion molecules (CAM) mediate polymorphonuclear leukocyte (PMNL) emigration in most inflamed tissues, but, in the lung, other yet to be identified CAMs appear to be involved. In Lewis rats, the intratracheal injection of Escherichia coli-LPS induced acute (6-h) PMNL accumulation in the lung parenchyma (280 × 106 by myeloperoxidase assay; PBS control = 35 × 106) and bronchoalveolar lavage fluid (BALF = 27 × 106; PBS = 0.1 × 106). Parenchymal accumulation was not inhibited by a blocking Ab to β2 integrins and only minimally inhibited (20.5%; p < 0.05) in BALF. We examined the role of α4β1 and α5β1 integrins and of selectins in this PMNL recruitment. Treatment with mAbs to α4β1 or α5β1, even in combination, had no effect on PMNL accumulation induced by intratracheal LPS. However, anti-α4 combined with anti-β2 mAbs inhibited PMNL recruitment to the parenchyma by 56% (p < 0.001) and to BALF by 58% (p < 0.01). The addition of anti-α5 mAb to β2 plus α4 blockade inhibited PMNL accumulation further (by 79%; p < 0.05). In contrast, blockade of L-, P-, and E-selectins in combination or together with β2, α4, and α5 integrins had no effect. LPS-induced BALF protein accumulation was not inhibited by treatment with anti-β2 plus α4 mAbs, but was prevented when α5β1 was also blocked. Thus, while selectins appear to play no role, α4β1 and α5β1 function as major alternate CAMs to the β2 integrins in mediating PMNL migration to lung and to pulmonary vascular and epithelial permeability.

A key pathological event during acute inflammation is the recruitment of leukocytes such as polymorphonuclear leukocytes (PMNLs)3 to sites of inflammation. The infiltration of these cells into areas of inflammation involves several families of cell adhesion molecules (CAMs) including selectins, integrins, and their ligands, the Ig superfamily adhesion molecules (1, 2). The selectins (L-, P-, and E-selectin) mediate the initial tethering of leukocytes to the vascular endothelium, which sequesters them from the blood flow and brings them into contact with the endothelium CAMs. This leads to a firm adhesion of the PMNL to the endothelial wall, mediated by interactions of integrins, especially of the β2 (CD18) family (LFA-1, CD11a/CD18 and Mac-1, and CD11b/CD18) with the Ig superfamily (ICAM-1 and ICAM-2) present on the endothelium. This is followed by transendothelial migration of the PMNL out of the vessel and migration of the cell to the site of inflammation via additional integrin interactions with the extracellular matrix (1, 3, 4). This multistep paradigm is based primarily on studies conducted on peripheral tissues involved by inflammation.

Several studies have examined leukocyte recruitment mechanisms in acute lung inflammation. The adhesion molecules implicated in PMNL recruitment have been varied depending on the stimulus (LPS, C5a, bacteria, immune complexes, etc.) used. For example, pulmonary IgG immune complex injury induces PMNL infiltration that is in part β2 integrin dependent (5), whereas the response to intrapulmonary C5a deposition is largely β2 integrin independent (6). In vitro studies of PMNL migration across pulmonary endothelium have also indicated that the adhesion molecule mechanisms are in part chemotactic factor dependent, IL-8 in particular inducing β2 integrin-independent PMNL transmigration (7, 8). It has also been shown that the mechanisms involved vary with the chronicity of reactions. For example, PMNL recruitment to lung inflammation induced by a single infection with Pseudomonas aeruginosa is primarily β2 integrin dependent but becomes β2 integrin independent with repeated infection (9). Furthermore, PMNL infiltration induced by Streptococcus pneumoniae, Staphylococcus aureus, or HCl aspiration was shown to be β2 integrin independent even on initial exposure (10, 11, 12). These findings are in marked contrast to PMNL infiltration in other tissues, which is highly β2 integrin-dependent (12, 13, 14). Despite the accumulated evidence that PMNL recruitment to lung inflammation involves β2 integrin-independent mechanisms, the alternate mechanisms remain unidentified.

Integrins are a large family of heterodimeric, transmembrane glycoproteins. Members of this family of CAMs have been found in all tissues and on all leukocytes. There are two types of integrin subunits (α and β), which associate to form over 20 different αβ heterodimers (15). PMNLs are known to express several integrins in addition to the β2 integrins (CD11/CD18). For example, PMNLs also express several β1 integrins, including α2β1 (very late Ag (VLA)-2), α4β1 (VLA-4), α5β1 (VLA-5), α6β1 (VLA-6), and α9β1 (16, 17, 18, 19, 20, 21, 22, 23). Although expressed at low levels on PMNLs, α4β1 and α9β1 have been shown to mediate human PMNL rolling or transendothelial migration in vitro, and α4β1 can contribute to PMNL migration in rat models of inflammation (19, 22, 24). The α2β1, α4β1, α5β1, α6β1, and α9β1 integrins may also mediate extravascular migration of PMNL (17, 20, 22).

In the analysis of the mechanisms of PMNL migration to lung inflammation, studies to date have examined the effect of blocking specific CAMs with Ab or knocking out CAMs by gene deletion. This strategy has eliminated the function of one or, in a few cases, two or three adhesion molecules. However, studies have not examined the role of multiple β1 integrins possibly functioning in concert with β2 integrins and selectins. In this study, we examined the role of these families of CAMs in PMNL recruitment to the lung in response to intratracheal (i.t.) injection of bacterial LPS in the rat, which, in this species, induces marked β2 integrin-independent PMNL infiltration into the parenchyma with alveoli. Our results demonstrate a major role for α4β1 and α5β1 integrins, in addition to β2 integrins, in this process.

Male Lewis rats, weighing 250–350 g, were purchased from Charles River Canada (St.-Contant, Quebec, Canada) and used in all experiments. The experimental protocols were approved by the Dalhousie University committee of laboratory animal care.

LPS (Escherichia coli 0111:B4) was obtained from List Biological Laboratories (Campbell, CA). Cetyltrimethylammonium chloride (CTAC) and 3,5,5-tetramethylbenzidine-dihydrochloride were obtained from Sigma (St. Louis, MO).

The mouse IgG1 mAb WT.3 (a gift from M. Miyasaka, Osaka University, Osaka, Japan) recognizes and functionally blocks the rat β2-chain of the leukocyte CD11/CD18 integrins (25). The mouse IgG1 mAb TA-2 was generated in our laboratory and has been shown to react with and functionally block the rat α4-chain of VLA-4 (26). The hamster IgG mAb HMα5 reacts with and blocks the rat α5 of VLA-5 (27, 28) and HRL-3 (a gift from D. Anderson, Pharmacia Upjohn, Kalamazoo, MI, and M. Miyasaka, Osaka University) reacts with and blocks rat L-selectin (29). The mouse IgG2a mAb RMP-1 recognizes and blocks rat P-selectin, and the mouse IgG1 mAb RME-1 blocks rat E-selectin (30, 31). These were generated in our laboratory. All mAbs to α-subunits and to selectins were used as F(ab′)2 generated by pepsin digestion, as previously described (32). The mouse mAb B9 (IgG1) anti-pertussis toxin was used as a control mAb, as previously described (14, 19). Rats received 1 mg of each mAb i.v. as indicated. None of the mAb treatments used, even in combination, caused a decrease in the blood PMNL count. Measurement by immunofluorescent titration and flow cytometry analysis of the concentration of Ab in the serum of recipient animals at sacrifice indicated that this dose is sufficient to maintain serum levels of the Abs at more than five times saturation throughout the 6-h experiment.

Anesthetized rats (by Ketamine and Innovar i.v.) were given an i.p injection of 0.2 mg/kg atropine sulfate to reduce reflex coughing following LPS instillation. Then, the trachea was isolated, and 50 μg of LPS in PBS (total volume, 0.2 ml) or PBS alone (control) was injected via a 27-gauge butterfly needle. Within 5 min of i.t. administration of the LPS, the indicated mAbs were injected i.v.

Before sacrifice at 6 h, rats were pretreated with 80 mg/kg pentoxifylline in 0.45% NaCl i.p. 15–20 min before vascular perfusion. The perfusion was performed as previously described (33) with minor modifications. Briefly, a 25-gauge butterfly needle was inserted into the inferior vena cava below the diaphragm of anesthetized animals (Ketamine, 50 mg/kg, and Xylazine, 10 mg/kg, i.p.). The abdominal aorta was transected to allow perfusate to drain, and 25 ml Tyrode’s solution with Ca2+ and Mg2+ was infused. Then, the butterfly needle was inserted into the inferior vena cava above the diaphragm, and 25 ml additional Tyrode’s solution with Ca2+ and Mg2+ was infused. The perfusion was completed with the infusion of 10 ml PBS-0.1% EDTA into the pulmonary artery. Bronchoalveolar lavage was then performed by inserting a polyethylene catheter into the trachea, and the lungs were lavaged four times with a total of 28 ml cold PBS-0.1% EDTA. In all animals, 20–25 ml of BALF was recovered, and the cell concentration was determined by hemocytometer counting with crystal violet staining. BALF protein concentration was determined by UV absorbance at 280 μm using albumin as standard. The lungs were removed and immediately frozen at −70°C until myeloperoxidase (MPO) extraction was performed.

For histological examination, bronchial lavage was omitted and, after vascular perfusion, 10 ml buffered formalin (3.7%) was infused via the pulmonary artery to fix the lung. Wedge segments from each lobe were paraffin embedded, and 5-μm sections were taken and stained with hematoxylin and eosin.

Lungs were extracted as previously described (33, 34). Briefly, samples of lung tissue from each lobe were pooled (350–450 mg, ∼10–15% of total lung weight) and freeze-dried. These samples were then homogenized in 50 mM HEPES (pH 8.0) at 0.5% dry w/v with a pestle homogenizer (Talboys Engineering, Emerson, NJ) in glass tubes at setting 30–40 until fully homogenized. Samples were then centrifuged at 10,000 × g for 30 min at 4°C, and the supernatant was discarded. The pellet was then rehomogenized in the same volume of 50 mM HEPES (pH 8.0) and centrifuged again at 10,000 × g for 30 min. The resulting pellet was homogenized again in the same volume of 0.5% CTAC in distilled water and clarified by repeat centrifugation. The resultant clear supernatant was analyzed for MPO activity. The same protocol was used to extract MPO from BALF cell pellets.

The collected supernatant from the HEPES/CTAC extracts of lung tissue was diluted 10-fold with 10 mM citrate buffer (pH 5.0), and 75 μl of this solution was pipetted into 4 wells of a 96-well plate. As negative control, “stop solution” (4 N H2SO4) was added to two of the wells. Then, 75 μl of the substrate solution (3 mM 3,5,5-tetramethylbenzidine-dihydrochloride, 120 mM resorcinol, and 2.2 mM hydrogen peroxide in distilled water (H2O2)) was added to all the wells, and the reaction was stopped after 2 min at 22°C by adding stop solution to the test wells.

A standard curve of MPO activity vs PMNL number was developed by homogenizing a known number of Percoll-purified blood PMNLs using the same technique used in MPO extraction from lung and also from BALF cells, as previously reported (34). The extract from these PMNLs was titrated, and a standard curve relating MPO activity to PMNL number was plotted. Aliquots of this standard extract were stored at −70°C, and a sample with known MPO content was tested in each assay as an internal standard and for calculation of PMNL concentration as previously described (34).

All data reported are arithmetic means. Error bars represent one SEM. Differences between means of indicated groups were analyzed by ANOVA with Bonferoni corrections for multiple comparisons.

Rats received an i.t. injection of LPS or PBS as a control and were sacrificed 6 h later. In rats receiving i.t. LPS (control), the lung parenchyma PMNL content increased 9-fold over PBS control to 2.8 ± 0.1 × 108 PMNL (Fig. 1). The PMNL content in BALF in PBS control animals was consistently <1 × 106, but in rats receiving i.t. LPS, the PMNL content reached 2.7 ± 0.1 × 107 (Fig. 2). The i.t. injection of LPS also led to a significant increase in BALF protein concentration compared with PBS control animals (Fig. 3). Protein concentration in the PBS control was 0.14 ± 0.03 mg/ml, and, in rats receiving i.t. LPS, the protein concentration increased to 0.30 ± 0.03 mg/ml (p < 0.001).

FIGURE 1.

Effect of anti-integrin and anti-selectin treatment on either i.t. PBS (left), i.t. LPS plus control mAb (B9), or LPS-induced PMNL accumulation in the lung. Rats were given anti-β2 mAb WT.3 or F(ab′)2 mAbs to α4 (TA-2), α5 (HMa5), or L- (HRL-3), P-(RMP-1), and E-(RME-1) selectins. Total lung PMNL content was determined from MPO content of the parenchyma as described in Materials and Methods. Shown are means ± SEM of four to six animals per group.

FIGURE 1.

Effect of anti-integrin and anti-selectin treatment on either i.t. PBS (left), i.t. LPS plus control mAb (B9), or LPS-induced PMNL accumulation in the lung. Rats were given anti-β2 mAb WT.3 or F(ab′)2 mAbs to α4 (TA-2), α5 (HMa5), or L- (HRL-3), P-(RMP-1), and E-(RME-1) selectins. Total lung PMNL content was determined from MPO content of the parenchyma as described in Materials and Methods. Shown are means ± SEM of four to six animals per group.

Close modal
FIGURE 2.

Effect of anti-integrin and anti-selectin treatment on LPS-induced PMNL accumulation in BALF. Rats were treated as in Fig. 1, and PMNL content of BALF was determined by hemocytometer counting. Shown are means ± SEM of four to six animals per group.

FIGURE 2.

Effect of anti-integrin and anti-selectin treatment on LPS-induced PMNL accumulation in BALF. Rats were treated as in Fig. 1, and PMNL content of BALF was determined by hemocytometer counting. Shown are means ± SEM of four to six animals per group.

Close modal
FIGURE 3.

Effect of anti-integrin and anti-selectin treatment on protein concentration in the BALF following i.t. LPS. Rats were treated as in Fig. 1 by i.t. PBS (left) or LPS plus treatments in Fig. 1 as indicated. Protein concentration was determined in BALF supernatant. Shown are means ± SEM of four to six animals per group.

FIGURE 3.

Effect of anti-integrin and anti-selectin treatment on protein concentration in the BALF following i.t. LPS. Rats were treated as in Fig. 1 by i.t. PBS (left) or LPS plus treatments in Fig. 1 as indicated. Protein concentration was determined in BALF supernatant. Shown are means ± SEM of four to six animals per group.

Close modal

To assess whether migration out of the vessel and through the lung parenchyma into the airspaces induced significant PMNL degranulation and release of cellular MPO, which may influence assessment of parenchymal PMNL content using the MPO assay, we assayed the MPO activity of PMNL in the BALF of rats 6 h following i.t. LPS and compared this to MPO in blood PMNL. As Fig. 4 shows, the BALF PMNL number predicted by MPO extraction, using blood PMNL MPO content as a standard, was not significantly different from the PMNL number in BALF determined by a visual hemocytometer quantitation of PMNL in BALF.

FIGURE 4.

Comparison of PMNL content determined by hemocytometer counting with that predicted using MPO extraction. Rats were given F(ab′)2 anti-α4, anti-α5, L-, P-, and E-selectin mAbs as indicated. Shown are means ± SEM of three experiments.

FIGURE 4.

Comparison of PMNL content determined by hemocytometer counting with that predicted using MPO extraction. Rats were given F(ab′)2 anti-α4, anti-α5, L-, P-, and E-selectin mAbs as indicated. Shown are means ± SEM of three experiments.

Close modal

The role of β2, α4β1 (VLA-4), and α5β1 (VLA-5) integrins and of L-, P-, and E-selectins in PMNL migration into the lung during acute LPS-induced inflammation was studied using blocking mAbs. As Fig. 1 illustrates, mAbs to β2 integrins, or α4 plus α5 integrins separately or to L-, P-, and E-selectins as a group, had no effect on PMNL accumulation in the parenchyma after i.t. LPS administration. In contrast, mAbs to β2 in combination with α4β1 integrin inhibited by >50% the PMNL accumulation in the parenchyma (1.34 ± 0.1 × 108 PMNLs; p < 0.001) as compared with LPS controls. Treatment of rats with mAbs to β2 plus α4β1 and also α5β1 in combination inhibited PMNL accumulation significantly more (8.8 ± 1.0 × 107; p < 0.05) when compared with the β2 plus α4β1 mAb-treated group. To determine whether selectins may be involved in the remaining PMNL infiltration, rats were additionally treated with mAbs to L-, P-, and E-selectin in combination. However, no additional inhibition of PMNL accumulation was observed (7.2 ± 0.5 × 107) compared with rats with β2, α4β1, and α5β1 blockade.

As shown in Fig. 2, under nearly all conditions of integrin and selectin blockade, PMNL accumulation in BALF mirrored the changes in PMNL accumulation in the parenchyma. However, unlike the parenchyma, the pretreatment of rats with mAb to β2 integrins slightly but significantly inhibited the migration of PMNLs into the BALF of rats receiving i.t. LPS as compared with control treated rats (21 ± 1.5 × 106; p < 0.05). This inhibition was further enhanced by the addition of mAb to α4 to the mAb treatment (11 ± 1.1 × 106 PMNL; p < 0.05, as compared with anti-β2 treatment alone). When anti-α5 mAb was added to the β2 and α4 mAbs, inhibition was further and significantly increased (5.6 ± 0.8 × 106; p < 0.05, as compared with anti-β2 and anti-α4 treatment). As with PMNL accumulation in the parenchyma, no additional inhibition of PMNL accumulation was observed when L-,P-, and E-selectins were blocked in concert with β2, α4, and α5 integrins (5.6 ± 0.01 × 106).

As an indicator of the increase in lung vascular and epithelial permeability resulting from inflammatory injury, BALF protein concentration was quantified. As illustrated in Fig. 3, protein concentration in the BALF of LPS-injected control mAb-treated rats was twice that of rats receiving i.t. PBS (LPS group = 0.3 ± 0.03 mg/ml; PBS = 0.14 ± 0.03 mg/ml; p < 0.001). Protein concentration in BALF from rats treated with anti-β2 mAbs alone or in combination with anti-α4 mAb was not significantly different from LPS control (0.27 ± 0.01 mg/ml). Inhibition of protein leak into the BALF was only observed when animals received mAb to β2, α4, and also α5 integrin, irrespective of selectin blockade (0.15 ± 0.01 mg/ml; p < 0.001).

Examination of lung sections from animals receiving either i.t. PBS or LPS indicated that LPS induced a marked increase in leukocyte infiltration into the lung interstitium (Fig. 5). The predominant cells in the infiltrate at 6 h were PMNLs. In the lungs of animals treated with mAbs to β2 and α4 plus α5 integrins, a marked decrease of PMNL infiltration was observed (Fig. 5 c).

FIGURE 5.

Histology of lung 6 h after i.t. LPS injection. Partially inflated, perfusion-fixed left lung lobes were paraffin-embedded, and sections were stained with hematoxylin and eosin. A, From an animal which received i.t. PBS; B, received i.t. LPS; and C, received i.t. LPS and anti-β2 plus α4 plus α5 mAb treatment. Magnification, ×125.

FIGURE 5.

Histology of lung 6 h after i.t. LPS injection. Partially inflated, perfusion-fixed left lung lobes were paraffin-embedded, and sections were stained with hematoxylin and eosin. A, From an animal which received i.t. PBS; B, received i.t. LPS; and C, received i.t. LPS and anti-β2 plus α4 plus α5 mAb treatment. Magnification, ×125.

Close modal

The major objective of this study was to define the mechanisms of PMNL emigration in acute lung inflammation, because most studies have concluded that there are yet unidentified β2 integrin-independent mechanisms involved in this process. Our data are consistent with these previous reports. We found that PMNL recruitment into the parenchyma during LPS-induced acute lung inflammation in the rat was not inhibited by function blocking mAb to the β2 integrins, and PMNL accumulation in the BALF was only slightly, albeit significantly, inhibited (by 20%). This is despite the fact that the anti-β2 mAb used (WT.3) has been shown by us to inhibit PMNL recruitment to inflammation in skin by >80% and arthritic joints by >60% (19).

Here we show that multiple integrins participate in PMNL migration into lung parenchyma and into the bronchoalveolar space in the rat during LPS-induced lung inflammation. The α4β1 integrin has recently been recognized on activated PMNL and to contribute to PMNL capture, rolling, and migration (19, 24, 35). Our findings demonstrate that it contributes to β2-independent PMNL recruitment, because α4 blockade in conjunction with β2 blockade significantly inhibited PMNL accumulation in both the parenchyma and BALF (Figs. 1 and 2). Furthermore, the α5β1 integrin, which is also expressed on PMNL at relatively low levels (19, 24, 36), also contributes to PMNL recruitment to the lung when β2 and α4 integrins are nonfunctional, because anti-α5 mAb inhibited PMNL accumulation in the parenchyma and BALF significantly more than observed with β2 plus α4 blockade (Figs. 1 and 2). These mechanisms appear to be functionally redundant alternative pathways for PMNL recruitment to the lung, because neither β2 blockade nor α4 plus α5 blockade reduced PMNL accumulation in the parenchyma and only minimally in the airspace (Figs. 1 and 2). The lack of effect of treatment with mAb to β2 or to α4 plus α5 integrins or of a nonbinding control mAb (B9) on PMNL accumulation in parenchyma supports the conclusion that the inhibitory effects were not due to nonspecific effects.

To our knowledge, this is the first report that PMNLs use α4β1 for migration to lung inflammation. This leukocyte can be added to the lymphocyte, monocyte, and eosinophil, which use α4 in conjunction with β2 integrins for migration to lung inflammation (33, 37). Further studies will be required to ascertain the major counterligands used by α4β1 on PMNL. VCAM-1 is one candidate, because it is up-regulated in the pulmonary vasculature during inflammation (38, 39), although up-regulated expression on endothelium of ligands such as the connecting segment-1 (CS-1) isoform of fibronectin may also contribute (40). However, recent studies indicate that other counterligands yet to be defined may play a predominant role in vivo (41).

Our findings are the first evidence for a role of α5 in PMNL recruitment in vivo. Previous studies have demonstrated the role of α5β1 in PMNL adhesion to fibronectin and PMNL migration across fibroblast barriers (36, 42). The current in vivo results suggest that α5β1 may also contribute to PMNL emigration from blood to some extent, because α5 blockade inhibited PMNL accumulation in the parenchyma in the presence of β2 and α4 blockade. These ligands in vivo may involve cell-associated as well as extracellular matrix-bound fibronectin or possibly also the Ig superfamily member L1, which in the mouse is recognized by α5β1 (43).

Another important finding is that these integrins may play a role in PMNL-mediated lung injury, as reflected in increased BALF protein accumulation. These integrins likely contribute to this process in concert, because blockade of β2 alone or α4 plus α5 had no effect, but the protein leakage was prevented with simultaneous blockade of β2, α4, and α5 integrins (Fig. 3). The α5β1 integrin may be as important as β2 or α4β1 integrins in this response, because no attenuation of the protein exudation was observed unless α5 was blocked.

The analysis of BALF protein accumulation revealed that this lung inflammatory injury may require a threshold of PMNL infiltration to develop or that it is a PMNL-independent response, because we observed either a maximum BALF protein concentration of 0.3 mg/ml or the normal PBS control level of 0.15 mg/ml, despite various degrees of inhibition of PMNL recruitment. For example, although β2 plus α4 blockade inhibited PMNL recruitment by 56–58%, this had no effect on BALF protein, whereas further inhibition of PMNL accumulation (up to 80%) with blockade of also α5β1 eliminated the protein leakage ( Figs. 1–3). Whether a critical threshold for PMNL transendothelial migration, activation, mediator release, and transepithelial migration are required to induce the vascular and epithelial permeability increase deserves further investigation because of the therapeutic implications.

Studies of the role of selectins in PMNL recruitment to lung inflammation have indicated either a partial or no role in PMNL recruitment to the lung, depending on the inflammatory stimulus (45, 46, 47, 48). Because the mechanisms and mediators involved in different pulmonary reactions are likely to be varied, the results have not been directly comparable. Our finding that blockade of L-, P-, and E-selectin had no effect on PMNL accumulation or on BALF protein increase, even in rats treated with mAbs to β2, α4, and α5 integrins, suggests that the selectins are not required for these responses to LPS, at least in the lungs of rats. This is likely because the predominant site of PMNL emigration in this model is at the alveolar level where close contact between PMNL and pulmonary endothelium occurs due to the small diameter of alveolar capillaries. This may minimize the requirement for selectin-mediated tethering and rolling (1, 49, 50, 51).

In summary, our studies indicate a major role for the β2 family, α4β1, and α5β1 integrins, but no detectable role for selectins in PMNL migration into the lung during acute LPS-induced inflammation. Further studies examining the role of these integrins in other models of lung inflammation are required to define their contribution to PMNL trafficking to the lung, activation, and the associated endothelial and epithelial injury during pulmonary inflammation.

We gratefully acknowledge the gifts of Abs and compounds from colleagues listed and the excellent technical assistance of Carol Jordan and Derek Rowter. We are also grateful to the IWK Grace Health Center Department of Pathology, in particular to Marlene Henry and Connie Isenor for preparation of histology and photographs.

1

This work was supported by Grants MT-7684 and GR-13298 from the Medical Research Council of Canada. J.A.B. is the recipient of studentships from Dalhousie University Faculty of Graduate Studies and the Dalhousie Inflammation Group.

3

Abbreviations used in this paper: PMNL, polymorphonuclear leukocyte; CAM, cell adhesion molecule; BALF, bronchoalveolar lavage fluid; VLA, very late Ag; i.t., intratracheal; CTAC, cetyltrimethylammonium chloride; MPO, myeloperoxidase.

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