INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The development of late responses and airway hyperresponsiveness (AHR) following airway antigen challenge in asthmatic patients has been associated with increased numbers of eosinophils and T lymphocytes in the airways of these patients (1, 2). One recognized adherence pathway that these cells might utilize to infiltrate the airways is binding to vascular cell adhesion molecule-1 (VCAM-1), which can be induced to be expressed on endothelial cells (3). Cell binding to VCAM-1 is mediated by very late antigen-4 (VLA-4), an ₄₁ heterodimeric integrin (CD49d/CD29). VLA-4 is expressed on T lymphocytes, eosinophils, monocytes, and mast cells, but not on neutrophils (4). VLA-4 also mediates binding of these cells to a variant of fibronectin (FN) at a binding site that expresses a 25-amino-acid sequence termed CS-1 (8, 9). Blockade of the ₄ chain (CD49d) of VLA-4 with specific monoclonal antibodies (mAbs) can inhibit adherence of eosinophils, lymphocytes, and monocytes to VCAM-1 and to the CS-1 region of FN (4, 6, 7, 10, 11). In addition to participation of ₄ integrins in cellular adhesion, engagement of ₄ integrins may be involved in cell-activation pathways (12, 13). For example, inhibition of VLA-4-mediated mast-cell binding to FN by a CS-1 ligand mimic results in decreased degranulation (14). Similarly, treatment of ovine eosinophils with an -specific mAb, HP1/2, reduced platelet-activating factor (PAF)-induced eosinophil peroxidase release (15). Thus, ₄ integrins are potentially involved by varied mechanisms in pathways of cell recruitment and cell activation.

In a previous study, we utilized an ₄-specific mAb, HP1/2, which blocks ₄-integrin-dependent cellular adhesion and activation (11, 13) to demonstrate that inhibition of ₄ integrin functions has a protective effect against the development of antigen-induced late-phase bronchial responses and AHR (15). Subsequently, we showed that the antigen-induced late response and AHR could also be blocked with an aerosolized soluble VCAM-Ig fusion protein that binds to ₄-integrins in their high-affinity state (16). These results with sheep, in conjunction with similar findings in other laboratories (17), suggest that VLA-4-dependent pathways are important for the later inflammatory events that occur in the airways after antigen challenge.

Because CS-1 variants of FN are increased during inflammatory states (18), inhibitors of this alternative pathway for VLA-4 binding might also block antigen-induced responses. To formally test this hypothesis, we have used the CS-1 ligand mimic phenylacetyl-L-leucyl-L-aspartyl-L-phenylalanyl-D-prolineamide (18), and determined its effect on allergen-induced early airway responses, late airway responses, AHR, and lung inflammation in allergic sheep. Our results demonstrate that the CS-1 ligand mimic blocks adhesion of sheep alveolar macrophages (AM) to FN in vitro and in vivo, blocks antigen- induced late airway responses and AHR. Unexpectedly, however, we observed that the CS-1 ligand mimic also provided significant inhibition of the early airway response. These changes in airway function were associated with a reduction in the combined numbers of VLA-4-positive cells in airway biopsies obtained 24 h after challenge.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal Preparation

A total of six allergic sheep, weighing 27 to 36 kg (mean: 31 kg), were used to study the airway effects of the test compounds. Another group of 11 sheep, weighing 21 to 54 kg, were used for additional biopsy studies. All sheep had previously been shown to develop both early and late bronchial responses to inhaled Ascaris summ antigen. The sheep were conscious and were restrained in a modified shopping cart in the prone position, with their heads immobilized. After topical anesthesia of the nasal passages with 2% lidocaine, a balloon catheter was advanced through one nostril into the lower esophagus. The animals were intubated with a cuffed endotracheal tube through the other nostril, with a flexible fiberoptic bronchoscope used as a guide. All protocols used in this study were approved by the Mount Sinai Medical Center Animal Research Committee, which is responsible for assuring the humane care and use of experimental animals.

Airway Mechanics

The techniques used for investigating airway mechanics have been described previously (15). Pleural pressure was estimated with an esophageal balloon catheter (filled with 1 ml of air), which was positioned 5 to 10 cm from the gastroesophageal junction. With the catheter in this position the end-expiratory pleural pressure ranged from 2 cm H₂O to 5 cm H₂O. Once the balloon was placed, it was secured so that it remained in the same position for the duration of the experiment. Lateral pressure in the trachea was measured with a sidehole catheter (I.D. = 2.5 mm) advanced through and positioned distal to the tip of the endotracheal tube. The tracheal and pleural-pressure catheters were connected to a differential pressure transducer (MP45; Validyne, Northridge, CA) for the measurement of transpulmonary pressure, which was defined as the difference between tracheal and pleural pressure. Airflow was measured by connecting the proximal end of the endotracheal tube to a pneumotachograph (Fleisch No. 1; Dyna Sciences, Inc., Blue Bell, PA). The transpulmonary pressure and flow signals were recorded on a multichannel physiologic recorder, which was linked to a 80-386 DOS Personal Computer (CCI Inc., Miami, FL) for on-line calculation of mean pulmonary flow resistance (RL) by dividing the change in transpulmonary pressure by the change in flow at mid-tidal volume (VT) (obtained by digital integration). The mean of at least five breaths, free of swallowing artifact, was used to obtain RL in cm H₂O/L/s. Immediately after the measurement of RL, thoracic gas volume (Vtg) was measured in a constant-volume body plethysmograph to obtain specific lung resistance (SRL = RL × Vtg) in L × cm H₂O/L/s.

Aerosol Delivery Systems

All aerosols were generated with a disposable medical nebulizer (Raindrop; Puritan Bennett, Lenexa, KS) that provided an aerosol with a mass median aerodynamic diameter of 3.2 µm as determined with an Andersen cascade impactor. The nebulizer was connected to a dosimeter system consisting of a solenoid valve and a source of compressed air (20 psi). The output of the nebulizer was directed into a plastic T-piece, one end of which was connected to the inspiratory port of a piston respirator (Harvard Apparatus, Mills, MA). The solenoid valve was activated for 1 s at the beginning of the inspiratory cycle of the respirator. Aerosols were delivered at VT of 500 ml and a rate of 20 breaths/min, as previously described (15).

Concentration Response Curves to Carbachol Aerosol

To assess bronchial responsiveness, we generated cumulative concentration-response curves to carbachol by measuring SRL immediately after inhalation of buffer and after each consecutive administration of 10 breaths of increasing concentrations of carbachol (0.25, 0.5, 1.0, 2.0, and 4.0% [wt/vol] in buffered saline). The provocation test was discontinued when SRL increased by more than 400% from the postsaline value, or after the highest carbachol concentration had been administered. Bronchial responsiveness was assessed as previously described (15) by determining the cumulative carbachol concentration (in breath units [BU]) that increased SRL by 400% over the postsaline value (PC₄₀₀) through interpolation from the dose-response curve. One BU was defined as one breath of a 1% (wt/vol) carbachol aerosol solution.

Bronchial Biopsies

Bronchial biopsies were done before the initiation of treatment and 24 h after antigen challenge. Pre- and postchallenge biopsy specimens were obtained from opposite lungs, and at least three specimens were obtained from each lung at each time point. Biopsy specimens were fixed in 10% buffered formalin and processed routinely for paraffin embedding. Tissue sections (4 µm) were stained with Giemsa, using the microwave method of Churukian (19), which gives more uniform staining and better contrast between nuclei and cytoplasm. Parallel sections were stained with toluidine blue for identification of metachromatic-staining cells (mast cells/basophils). Slides were examined with a BH2 light microscope (Olympus Corp., Tokyo, Japan) equipped with differential interference contrast optics, using a calibrated eyepiece grid (10 × 10), which covered 1,600 µm² with a ×40 objective. The number and distribution of inflammatory cells (polymorphonuclear leukocytes [PMN], lymphocytes, eosinophils, and mast cells/basophils) was assessed in bronchial epithelium and lamina propria. A minimum of five fields from each biopsy were examined. The number of cells for each cell type were averaged for the five fields, and the results were expressed as number of cells/grid.

Isolation of Alveolar Macrophages

The distal tip of a specially designed, 80-cm fiberoptic bronchoscope was wedged into a randomly selected subsegmental bronchus. Subsegments on each side of the lung were lavaged with 180 ml of phosphate-buffered saline (PBS) in 30-ml aliquots. The bronchoalveolar lavage fluid (BALF) was strained through two layers of sterile gauze into 50-ml conical polypropylene tubes and centrifuged at 200 × g for 10 min. The supernatants were decanted, the cells resuspended in calcium- and magnesium-free Dulbecco's PBS (D-PBS), and again centrifuged. The procedure was repeated three times in total. To separate alveolar macrophages from the other lung cells, a Percoll density-gradient centrifugation was performed, using isosmotic Percoll. The washed cells were resuspended in 2 ml of Hank's balanced salt solution (HBSS) with 100 U of penicillin and 0.1 mg of streptomycin per ml. The cell suspension was then carefully layered on top of two layers of Percoll (densities of 1.075 g/ml and 1.070 g/ml, respectively) in sterile, 15-ml polypropylene tubes, and was centrifuged at 500 × g for 30 min at room temperature. Alveolar macrophages were obtained from the 1.070 g/ml Percoll layer because they were of sufficient number, with a high degree of purity, in this layer. This ring was collected, resuspended in D-PBS, and washed twice by centrifugation at 300 × g at room temperature in conical 50-ml polypropylene tubes. A cell count with a hemocytometer, a viability test with 0.5% trypan blue solution, and a cytospin centrifugation for cell differentiation were performed. The viability was usually between 90 and 95%, and the cell differentiation showed a purity between 80 and 90%, with mainly lymphocytes as contaminating cells.

Agents

Ascaris suum extract (Greer Diagnostics, Lenoir, NC) was diluted with PBS to a concentration of 82,000 protein nitrogen units/ml and delivered as an aerosol (20 breaths/min × 20 min). This crude preparation has an endotoxin level of 50 EU/ml, which does not have an effect on pulmonary responses in sheep (20). Carbamylcholine (Carbachol; Sigma Chemical Co., St. Louis, MO) was dissolved in buffered saline at concentrations of 0.25, 0.50, 1.0, 2.0, and 4.0% wt/vol and delivered as an aerosol. Compound A (the CS-1 ligand mimic phenylacetyl-L-leucyl-L-aspartyl-L-phenylalanyl-D-prolineamide, active agent) and Compound B phenylacetyl-L-aspartyl-L-leucyl-L-phenylalanyl-D-prolineamide, inactive control) were dissolved in 1 ml dimethylsulfoxide (DMSO) and 2 ml PBS at a concentration of 30 mg/3 ml, and the total volume of 3 ml was delivered by aerosolization. As previously described, the inactive control does not inhibit VLA-4 dependent cell adhesion in vitro (18).

RPMI 1640, penicillin 10,000 U/ml, streptomycin 10 mg/ml, PBS powder, Percoll, FN, from bovine plasma, bovine serum albumin (BSA), and ethylenediamine tetraacetic acid (EDTA) were all obtained from Sigma Chemical Co. Trypan blue was obtained from MCB Manufacturing Chemists (Cincinnati, OH), Hemacolor was obtained from Harleco (Philadelphia, PA), sterile calcium- and magnesium-free D-PBS and sterile calcium- and magnesium-free HBSS were obtained from Gibco Laboratories (Grand Island, NY), and endotoxin-free sterile water was obtained from Baxter Healthcare Corporation (Deerfield, IL). The mAb HP1/2 was a gift from R. Lobb (Biogen, Inc., Cambridge, MA).

Protocols

In vitro studies: adhesion of alveolar macrophages. Sterile, 24-well polystyrene tissue-culture plates with lids were used after coating with FN. The FN was dissolved in D-PBS at a concentration of 40 mg/ml, after which 200 µl of the FN solution used to coat each well. The plates were shaken at 120 rpm for 15 min at room temperature, placed in a humidified CO₂ incubator at 38.5° C for 4 h, and then turned upside down and dried overnight. The wells were subsequently washed twice with 1 ml of warm D-PBS and incubated at 37° C with 200 µl of 1% denatured BSA for 2 h, to block nonspecific binding sites. The plates were rinsed with warm RPMI 1640 immediately before use.

Alveolar macrophages from normal sheep were isolated as described earlier and resuspended in RPMI 1640 at a concentration of 1 × 10⁶ viable cells/ml. Cell suspensions were incubated at room temperature for 15 min with 50 and 100 µg/ml of the active CS-1 ligand mimic (Compound A), the control peptide (Compound B), or 100 µg/ml HP1/2. One milliliter of the cell suspension was added to each well and was incubated for 1 h in a humidified CO₂ incubator at 38.5° C. Prior to incubation, a cell count was repeated, using phase-contrast microscopy, to determine the number of cells/ml delivered to each well. After the incubation, the RPMI 1640 containing the nonadherent cells was aspirated from each well and transferred into 6-ml polypropylene tubes that contained 1 ml of D-PBS with 0.028% EDTA to prevent aggregation. The wells were washed twice with 1 ml of warm RPMI 1640 to remove all nonadherent cells. The total number of cells aspirated was then counted through a phase-contrast microscopy, and the percentage of adherent cells was calculated.

In vivo studies. At 3 to 4 d before treatment was begun, baseline airway responsiveness (i.e., PC₄₀₀) was determined and a baseline bronchial biopsy performed. Then at 4 d before antigen challenge, the animals began treatment with 30 mg Compound A (the CS-1 ligand mimic) or Compound B (the inactive control). The animals were treated two times a day for 3 d and then, on the fourth day, at 0.5 h before antigen challenge and again at 4 h after challenge. On the antigen-challenge day, SRL was measured and the animals were then treated with the designated compound. SRL was remeasured 0.5 h after treatment (just before challenge) and the animals were then challenged with antigen. SRL was then remeasured immediately after, hourly from 1 to 6 h after, and half-hourly from 6.5 to 8 h after antigen challenge, as previously described (15). Postchallenge determinations of PC₄₀₀ were made at 24 h after antigen challenge, after which a second biopsy was performed. These studies were done in six sheep in a blinded, randomized crossover fashion. The trials with Compounds A and B were separated from one another by at least 3 wk. Our previous studies have shown that this time interval is sufficient for the sheep to recover from prior challenges (21). For these studies, peripheral blood was drawn for differential analysis at baseline, 30 min after drug administration at the beginning of antigen challenge, and 1, 2, 3, 4, 5, 8, and 24 h after antigen challenge.

In a second experimental series, bronchial biopsies were obtained from an additional set of 11 sheep, before and 24 h after antigen challenge. These sheep underwent the same treatment protocol as described earlier, except that PBS aerosol rather than Compounds A or B was used for the treatments.

Statistics

Statistical analysis was done with statistical programs in SYSTAT for Windows (Version 5; SYSTAT Inc., Evanston, IL). Analysis of the airway mechanics data was first performed through a multifactorial analysis of variance (ANOVA) with repeated measures to determine overall effects of the treatment protocols. Individual values at each specific time point were compared with paired t tests. A paired t test was also used to compare peak early and late responses (i.e., an animal's maximum response between 5 and 8 h after antigen challenge). Significance was accepted when p < 0.05 with a two-tailed analysis. Cell counts were compared through Wilcoxon's signed rank test. A value of p < 0.05 was considered significant in a two-tailed test for the experiment used to demonstrate an increase in VLA-4-positive cells after antigen challenge. Because the results demonstrated an increase in VLA-4-positive cells after challenge (expected result), we used a one-tailed test to determine significance (p < 0.05) in the treatment trials (22). Values in the text and figures are reported as mean ± SE. Cell data are reported as median and range.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In Vitro Adhesion Studies

Figure 1 illustrates that Compound A (the active compound) inhibited binding of sheep alveolar macrophages to FN, whereas the scrambled peptide (Compound B) did not. Compound A at 100 µg/ml inhibited the binding of sheep alveolar macrophages by 43% (p < 0.05). This inhibitory effect was lost if the peptide concentration was reduced to 50 µg/ml. The response was specific for the active peptide because there was no inhibition seen with equivalent doses of the inactive peptide (Compound B). Figure 1 also shows that the inhibition seen with the active peptide in these experiments was similar to that (36%, p < 0.05) achieved with 100 µg/ml of the mAb HP1/2, which has previously been shown to inhibit VLA-4- dependent binding of sheep cells.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Effect of Compound A (active agent), Compound B (inactive control), and mAb HP1/2 on macrophage adherence to fibronectin. Values are mean ± SE for four or five experiments (*p < 0.05 versus control [0 µg/ml]).

In Vivo Studies

Figure 2 shows the time course of the airway responses before and after antigen challenge when the animals were treated with Compound A (active agent) and Compound B (inactive agent). There was a highly significant difference between the treatments (p < 0.001, ANOVA). When the animals received Compound B, SRL increased by 269 ± 23% (p < 0.05) from a baseline value of 1.01 ± 0.03 L × cm H₂O/L/s immediately after challenge. SRL returned to baseline values by 4 h after challenge, but then began to increase again by 5 h after challenge. The maximum increase during this late response (i.e., 5 to 8 h) was 198 ± 34% (p < 0.05). Treatment with Compound A provided partial protection against the immediate bronchoconstrictor response to inhaled antigen. After treatment with Compound A, SRL increased by 162 ± 18% (p < 0.05 versus baseline) from a baseline value of 1.01 ± 0.03 L × cm H₂O/L/s. This increase is SRL was significantly smaller (p < 0.05) than the increase seen in the control trial. SRL remained significantly elevated above baseline at 1 h after challenge, but then returned to prechallenge values and did not increase significantly above baseline throughout the remainder of the 8 h measurement period. The maximum increase in SRL during the late response was 26 ± 7% (p < 0.05 versus that with Compound B). Neither compound affected baseline airway tone.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Top: Effect of Compound A (active agent) and Compound B (inactive agent) on antigen-induced early and late bronchial responses. Compounds were given once a day for 3 d and then on the 4th day 30 min before and 4 h after antigen challenge. Compound A reduced the acute increase in specific lung resistance (SRL) and blocked the late response as compared with control (Compound B). (*p < 0.05 versus Compound A). Bottom: Effect of Compounds A and B on postchallenge airway responsiveness. When the animals were treated with Compound B (control trial), PC400 decreased after antigen challenge (i.e., the sheep became hyperresponsive). Treatment with Compound A prevented this effect (*p < 0.05 versus baseline; +p < 0.05 versus Compound A). All values are mean ± SE for six sheep.

Airway Responsiveness

SRL returned to baseline values 24 h after challenge in the trials with both Compound A and Compound B. Assessment of airway responsiveness at this time showed the sheep treated with Compound B to be hyperresponsive to inhaled carbachol. Postchallenge PC₄₀₀ decreased to 11.6 ± 1.5 BU from a prechallenge value of 20.3 ± 3.5 BU (Figure 2). The AHR in the control trial contrasted sharply with the results obtained when the sheep were treated with Compound A, in which no significant change in airway responsiveness was observed. PC₄₀₀ was 22.1 ± 2.8 BU before challenge and 25.7 ± 5.2 BU at 24 h after challenge (Figure 2).

Peripheral Leukocytes

In general, the changes in numbers of peripheral eosinophils, lymphocytes, and neutrophils were similar when the animals were treated with Compound A and Compound B. Both groups showed a steady decrease in eosinophils and lymphocytes and an increase in neutrophils after challenge (Figure 3).

View larger version (18K): [in this window] [in a new window]   Figure 3.   Effect of Compounds A and B on the number of peripheral eosinophil, lymphocytes, and neutrophils before and after antigen challenge. Responses of these cells were similar in both groups. Values are mean ± SE for six sheep.

Bronchial Biopsies

Results of analysis of bronchial biopsy specimens from untreated sheep (n = 11) before and 24 h after antigen challenge are given in Table 1. The airway responses of these sheep to inhaled antigen were similar to those seen in the animals treated with the inactive peptide (early-response SRL = 256 ± 24%, late-response SRL = 180 ± 11%, and AHR PC₄₀₀ prechallenge = 27 ± 2 BU and postchallenge = 13 ± 1 BU). The results with these animals indicate that the numbers of both VLA-4-positive cells (eosinophils, lymphocytes, and metachromatic cells) and VLA-4-negative cells (neutrophils) increased in the biopsy specimens obtained 24 h after antigen challenge. Furthermore, the increase in numbers of the individual VLA-4-positive cells resulted in a significant increase in the total number of VLA-4-positive (T-VLA-4) cells in the biopsy specimens.

Table 1 also provides the median changes and Figure 4 the individual changes in the cell responses based on the specimens taken from animals treated with Compounds A and B. Although there was considerable variability in the individual responses, the directional changes following antigen challenge were consistent with those reported for the larger group of untreated animals. The specimens obtained from animals treated with Compound A showed a decrease in the number of metachromatic-staining cells and no increases in any other cell type (Table 1). Thus, there was no significant increase in the number of T-VLA-4-containing cells. When these animals were treated with Compound B, the pattern of response was similar to that in the larger group of untreated animals (i.e., all of the VLA-4-positive cells increased after antigen challenge, and therefore so did the T-VLA-4-positive cells; Table 1). As was seen with Compound A, there was no significant change in VLA-4-negative cells.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Effect of Compounds A and B on the number of polymorphonuclear leukocytes (PMN), lymphocytes (LYM), eosinophils (E), metachromatic-staining cells (MC), and total VLA-4-positive cells (VLA4) at baseline (BSL) and 24 h after antigen challenge (PC) for individual sheep. Median values and statistics can be found in Table 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of this study indicate that a ligand that blocks an alternative adherence site of ₄ integrins can modify antigen-induced late bronchial responses and the prolonged AHR that follow an acute antigen challenge in the allergic sheep model. Furthermore, the increase in VLA-4-positive cells (eosinophils, lymphocytes, and metachromatic-staining cells) seen in biopsy specimens from unprotected animals at 24 h after antigen challenge was absent in the animals treated with the active ligand. Thus, the airway-protective effect seen in the animals treated with the active compound was associated with a reduced inflammatory response. The peptide constituting the active compound was effective when given as an aerosol in multiple doses before and then once after antigen challenge. These results extend our previous studies, which showed that an mAb against ₄ integrins (15) and a newly synthesized compound with anti-VLA-4 activity block these same events (23). Collectively, these data provide strong evidence that the ₄ adhesion pathway participates in pathophysiologic responses associated with the prolonged inflammatory events that follow antigen challenge in the allergic sheep model.

Recent studies have supported a role for ₄-expressing leukocytes in the pathophysiology of allergen-induced airway responses (17). However, in most instances, effects of inhibiting these responses by blocking ₄ have been seen with antibodies to ₄. The present studies differ in that the blocking agent was directed at the CS-1 binding site within the extracellular matrix. Because CS-1 binding can be distinguished from VCAM-1 binding (6, 18), our current findings suggests that blocking the interaction of ₄-expressing leukocytes with either type of counterreceptor interferes with the migration and/or activation of these cells, resulting in protection against the allergen-induced late response and AHR.

The positive results obtained with mAb HP1/2 against ₄-expressing leukocytes in antigen-induced abnormalities in the airways made it reasonable to predict that a small-molecule ligand inhibitor of ₄ would be equally effective. Such a hypothesis was supported by previous studies showing that a CS-1 peptide mimic, as well as an anti-₄ mAb, blocked ₄-mediated binding of Jurkat T-lymphoblastoid cells to endothelium from the synovium of patients with rheumatoid arthritis (18). Interestingly, T-cell binding was not inhibited by monoclonal anti-VCAM-1 antibodies, indicating that the CS-1 sequence of FN, and not VCAM-1, was the counterligand for these cells. Although there is evidence for upregulation of VCAM-1 in asthmatic airways with airflow limitation (24), little is known about CS-1-containing variants of FN. However, it is likely that such variants of FN are also increased in asthmatic airways, because interleukin-1 (IL-1), which is increased in BALF from asthmatic subjects (25), can induce human umbilical-vein endothelial cells to generate splicing variants of fibronectin containing CS-1 (18). Such a mechanism may be responsible for reports of selective expression of CS-1 on endothelium from inflammatory sites in the lung (18). Thus, it may be that ₄-expressing leukocytes have two distinct pathways with which to migrate into the airway, and that interruption of either pathway can protect against antigen-induced airway dysfunction.

That VLA-4-positive cells play an important role in the late response and AHR in this model is consistent with previous data showing that these airway changes are associated with increased eosinophils in BALF (21) and biopsy specimens (26), and with the ability of different agents with anti-VLA-4 properties (i.e., the anti-₄ mAb HP1/2 and TYB-2285a newly synthesized agent) to inhibit these antigen-induced airway responses as well as eosinophil recruitment and activation seen in BALF (15, 23). In this study we extended our previous observations by examining biopsies from a larger pool of allergic animals and determining not only the numbers of eosinophils that were recruited to the airway, but also the numbers of mast cells and lymphocytes that appeared 24 h after antigen challenge. The appearance of these other cell types was expected, since they also express VLA-4, and because our previous findings indicated that antigen challenge induces a peripheral lymphocytopenia (probably representing mobilization) and an increase in the number of mast cells in the airways (27). Biopsies from the untreated animals in the present study confirmed that individual VLA-4-positive cells increased 24 h after antigen challenge. Given the individual cell results, it is not surprising that the total number of VLA-4-positive cells (T-VLA-4) increased. We used these criteria, then, to assess the response of the smaller group of animals treated with the test compounds. When the animals were treated with Compound A, there was no increase in any cell type. There was, however, a small but significant decrease in the number of metachromatic-staining cells found in the specimens after challenge. Thus, the T-VLA-4-positive cells did not change in the animals treated with the active agent. In contrast, when the animals were treated with the control peptide, the individual VLA-4-positive cells increased, and the T-VLA-4-positive cells therefore also increased. There was no significant increase in PMNs in either trial, indicating that the VLA-4-negative cells did not respond differently. The collective data support the hypothesis that interference with the appearance of VLA-4-positive cells in the airways of sheep with airway hypersensitivity to A. suum antigen after antigen challenge is associated with protection against the late response and AHR.

Although these observations support the hypothesis that the active compound interfered with the appearance of VLA-4-positive cells in sheep airways following antigen challenge, the conclusion drawn from these results are tempered by the small number of test specimens analyzed. Two points are noteworthy. The first is that there was considerable variability in the median prechallenge number of the lymphocytes contained in specimens when the sheep were treated with Compound A (8.1 cells/grid) as compared with the number (2.8 cells/grid) when they were treated with Compound B. Although this difference appeared large, these values were not significantly different. Furthermore, despite this difference in baseline lymphocyte numbers, the changes seen after challenge appear to have followed the appropriate course (i.e., lymphocytes increased in the animals treated with Compound B, whereas there was no change in lymphocyte number when the animals were treated with the CS-1 ligand mimic, Compound A). Thus, although the difference in prechallenge lymphocyte numbers may be somewhat disconcerting, we would suggest that the directional changes may in fact be more important than the absolute prechallenge number of a specific cell type. This suggestion is based on the results reported for the untreated sheep, in which antigen challenge produced airway responses (early, late, and AHR) that were comparable with those seen in the sheep treated with Compound B. This occurred despite the large difference in prechallenge lymphocyte numbers between these two groups, thus making it unlikely that the prechallenge lymphocyte number affected the results of this study.

The second point of concern involves the neutrophil responses in both groups of treated animals. Again, on the basis of the results with the untreated group, we would suggest that the small sample size, rather than a nonspecific effect of the treatment compound, is likely to have been the reason for the lack of a significant increase in neutrophil numbers in the specimens from this group after challenge. Furthermore, as was discussed for the lymphocytes, if suppressing the neutrophil response was important for the protection seen with the active compound, then the animals treated with inactive agent should not have responded to antigen.

It was of interest that the sheep with the active peptide showed a significant reduction in the early bronchial response. This in vivo finding is consistent with that in reports of in vitro studies which indicated that adherence of rat peritoneal mast cells to FN caused enhanced IgE-mediated exocytosis, which was blocked by CS-1 peptide (14). The inhibitory action was related in part to the ability of CS-1 to block the adherence of mast cells to FN. In the present studies, we confirmed that the active compound and the anti-₄ mAb HP1/2, which had previously been shown to inhibit VLA-4-mediated binding of sheep blood mononuclear leukocytes, inhibited macrophage binding to FN. These results are consistent with the hypothesis that the active peptide interferes with VLA-4-mediated processes in the allergic sheep. Thus, the reduction in the early response seen in these experiments may reflect the interaction of VLA-4 inhibitor (Compound A) with the putative ₄ integrin on mast cells, thereby causing a reduced response to antigen stimulation.

Several studies have examined the effects of treatments with antibodies to VLA-4 or VCAM-1 on the responses to antigen provocation (17). However, the role of a CS-1 ligand mimic has only been examined in one other study, using a rabbit model of allergic bronchoconstriction (28). In that study, VLA-4 inhibitor (Compound A) was given as an aerosol in a single dose of 100 mg/kg at 90 min before challenge. This dose of Compound A was reported to reduce the antigen-induced decrease in dynamic compliance by 94% for the early response and 87% for the late response. However, in contrast to the present study, in which Compound A gave 100% protection against AHR following antigen-challenge, the single-dose treatment in rabbits resulted in only 28% protection. The increased protection seen in the present study was probably the result of the additional dose of Compound A given 4 h after antigen challenge. In accord with the finding in the present study, the single-dose treatment in rabbits did tend to reduce the numbers of eosinophils recovered by bronchoalveolar lavage (BAL) (28).

In conclusion, these results support a role for ₄ integrins in the production of antigen-induced late-phase airway obstruction and hyperresponsiveness. Furthermore, the results support the potential utility of inhalational approaches with antiadhesion therapy to modulating the airway-cell activation that contributes to the pathophysiology of asthma.