INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The development of late airway responses (LAR) and airway hyperresponsiveness (AHR) after airway antigen challenge (1- 4) in asthmatic patients has been associated with increased numbers of eosinophils and T lymphocytes in the airways of these patients (5). One recognized adherence pathway that eosinophils and T lymphocytes utilize in common to infiltrate the airways is binding to vascular cell adhesion molecule-1 (VCAM-1), which can be induced to be expressed on endothelial cells (11). Cell binding to VCAM-1 is mediated by very late antigen-4, (VLA-4), the ₄₁ heterodimeric integrin (CD49d/CD29). VLA-4 is a key surface receptor which is now known to be expressed at high levels not only on T lymphocytes and eosinophils as originally thought (12), but on mast cells, basophils, and macrophages (17). In addition, recent data suggest that neutrophils have low level expression of the ₄₁ integrin, which is functionally active (18). The ₄₁ integrin also mediates binding of these cells to a variant of fibronectin at a binding site which expresses a 25 amino acid sequence termed CS-1 (19). Blockade of the ₄ chain, CD49d, with specific monoclonal antibodies (mAb) can inhibit adherence of eosinophils, lymphocytes, and monocytes to VCAM-1 and to the CS-1 region of fibronectin (12, 13, 16, 20, 21). In addition to participation of ₄ integrins in cellular adhesion, engagement of ₄ integrins may be involved in cellular activation pathways (22). Thus, ₄ integrins are potentially involved by varied mechanisms in pathways of cell recruitment and activation.

Allergic sheep undergoing airway challenge with Ascaris suum antigen demonstrate many of the pathophysiologic and inflammatory airway responses seen in patients after airway provocation with specific antigen, including the development of LAR, prolonged AHR, and infiltration of VLA-4-positive expressing cells into the airway (27, 28). Using this model, we showed that an ₄-specific mAb, HP1/2, that blocks ₄ integrin-dependent cellular adhesion and activation (20, 26) inhibited the development of LAR and AHR (28). Subsequently, we showed that the antigen-induced LAR and the AHR could be blocked using an aerosolized soluble VCAM- Ig fusion protein, which binds to ₄-integrins in their high- affinity state (29), or a small-molecule inhibitor (CS-1 ligand mimic) which blocks VLA-4-mediated binding to fibronectin. In the latter study, the inhibition of the pathophysiologic responses was associated with a reduction in the VLA-4-expressing cells recruited to the airway. Our results, in conjunction with similar findings from other laboratories (30), add support for the hypothesis that activation and recruitment of VLA-4-expressing leukocytes to the airways contribute to the later pathophysiologic events (i.e., LAR and AHR) that occur after antigen challenge and, so, make this pathway a potential therapeutic target for the treatment of allergic airway disease.

With few exceptions, the aforementioned studies used mAb to VLA-4 or VCAM-1 to block allergen-induced inflammatory events. Although the mAb studies provide evidence that the pathways in question participate in the allergic response, there may be problems associated with the therapeutic use of such agents including, but not limited to, antigenicity. Such problems can be avoided by the use of small-molecule inhibitors. BIO-1211 (N-[[4-[[[(2-methylphenyl)amino] carbonyl]amino]- phenyl]acetyl]-L-leucyl-L--aspartyl-L-valyl-L-proline), is a noncovalent, small-molecule, tight-binding inhibitor (k_off = 1.4 × 10⁴ s¹, dissociation constant [K_d] = 70 pM) of VLA-4 (34). The in vitro inhibitory concentration of 50% (IC₅₀) of BIO-1211 was 1 to 2 nM (34), which is approximately 10-fold more potent than the CS-1 ligand mimic used previously in sheep (27). Given these characteristics, one might expect BIO-1211 to show better inhibitory activity in vivo than the aforementioned CS-1 ligand mimic.

In the present study, we formally test the hypothesis that the small-molecule inhibitor of VLA-4, BIO-1211, blocks antigen-induced LAR, antigen-induced AHR, and antigen- induced recruitment of VLA-4-expressing cells to the airways of allergic sheep. Our results show that either aerosol or intravenous administration of BIO-1211 blocks these events. In addition, we show for the first time that a specific VLA-4 inhibitor can reverse allergen-induced AHR.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal Preparation

Allergic sheep weighing 27 to 50 kg were used. All sheep had previously been shown to develop both early and late bronchial responses to inhaled A. suum antigen (35). 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 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

Breath-by-breath determination of mean pulmonary flow resistance (RL) was measured with the esophageal balloon technique described extensively by us (27, 28). The mean of at least 5 breaths, free of swallowing artifact, were 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 cm H₂O · s¹.

Aerosol Delivery Systems

All aerosols were generated using 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 by 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 a tidal volume of 500 ml and a rate of 20 breaths/min as previously described (27, 28).

Concentration Response Curves to Carbachol Aerosol

Airway responsiveness was determined from cumulative concentration response curves to carbachol inhaled as previously described. SRL was measured immediately after inhalation of phosphate-buffered saline (PBS) 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 PBS). The provocation test was discontinued when SRL increased over 400% from the post-PBS value or after the highest carbachol concentration had been administered. The cumulative carbachol concentration (in breath units [BU]) that increased SRL by 400% over the post-PBS value (PC₄₀₀) was calculated by interpolation from the dose-response curve. One BU was defined as one breath of a 1% wt/vol carbachol aerosol solution (27, 28).

Bronchoalveolar Lavage (BAL)

The distal tip of a specially designed 80-cm fiberoptic bronchoscope was wedged into a randomly selected subsegmental bronchus. Lung lavage was performed by infusion and aspiration of 30-ml aliquots of PBS (pH 7.4) at 39° C. An aliquot of 30 ml was infused in each of three different airways (total 90 ml) at each time point. The effluent collected was strained through two layers of gauze to remove mucus and then centrifuged at 250 × g for 15 min at 4° C. The cell pellet was resuspended in PBS, and an aliquot of this resuspension was transferred to a hemocytometer chamber to determine total cells. Total viable cells were assessed by trypan blue exclusion. A second aliquot of the cell suspension was spun in a cytospin and stained with Diff-Quik (Baxter, McGraw Park, IL) for a differential cell count (×100; oil objective) and parallel slides were stained with 0.1% Toluidine-Blue-O (St. Louis, MO) for identification of metachromatic staining cells (mast cells/basophils). BAL supernatants saved for tissue kallikrein activity assays (36) were centrifuged at 300 × g for 15 min before being frozen at 70° C.

Tissue Kallikrein

Tissue kallikrein (TK) activity was determined in unconcentrated samples from BAL using a microtiter assay. Frozen BAL supernatants were thawed and then centrifuged at 13,400 × g for 15 min at 4° C. Then, 150 µl BAL was incubated with 25 µl of trypsin (20 µg/ml in trizma buffer 0.05 M pH 8.2) for 15 min at 37° C. Then, 25 µl of soy bean trypsin inhibitor (4 mg/ml in 0.1 M trizma buffer pH 8.2) was added, followed by 100 µl of substrate DL Val-Leu-Arg p-nitroanilide dissolved in trizma buffer 0.05 M pH 8.2 with 0.05% albumin, and incubated for 24 h in a humidified CO₂ incubator. The values were reported as the change in optical density between zero and 1 h, measured at a wavelength of 405 nM. All assays were done in duplicate.

Bronchial Biopsies

Bronchial biopsies were obtained and processed as previously described (27). The initial biopsy was obtained in one lung, then the next (second) biopsy was obtained from the alternate lung. This process was followed throughout the protocol so that sequential biopsies were always obtained from opposite lungs. Biopsies were obtained from the fourth-generation bronchi and above (excluding the carina). From 1 to 3 specimens were obtained at each biopsy time point. Specimens were fixed in 10% formalin and then processed routinely for paraffin embedding. Tissue sections (4 µm) were stained with hematoxylin- eosin and parallel sections with Giemsa using the microwave method of Churukian (37), as described previously (27) 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 ×40 objective. The number and distribution of inflammatory cells (neutrophils, lymphocytes, eosinophils, and mast cells/basophils) was assessed in bronchial epithelium and the lamina propria. A minimum of three fields from each biopsy were examined. The number of cells for each cell type were averaged for the three fields, and the results were expressed as a number of cells per high-power field (hpf). The reader was blinded as to the group (BIO-1211 treated or placebo) from which the biopsies were taken (27).

Agents

A. 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). Carbamylcholine (Carbachol; Sigma Chemical Co., St. Louis, MO) was dissolved in PBS at concentrations of 0.25, 0.50, 1.0, 2.0, and 4.0% wt/vol and delivered as an aerosol. BIO-1211 was dissolved in either sodium phosphate or Tris buffer. When using Tris, any required dilution was performed using normal (0.9%) saline. Doses were prepared in 3 to 5 ml total volume depending on the route of administration (aerosol or intravenously) for the study.

Protocols

A series of protocols were used to examine the effects of BIO-1211 on antigen-induced early airway response (EAR), LAR, and AHR. The same general protocol was used for all studies, except that the dosage and time of BIO-1211 administration varied. The basic protocol consisted of measuring baseline airway responsiveness (i.e., PC₄₀₀) 1 to 4 d before antigen challenge. On the antigen challenge day, SRL was measured just before antigen challenge and then the animals were challenged with A. suum. SRL was 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 (35, 38). Postchallenge determinations of airway responsiveness (PC₄₀₀) were made at 24 h after antigen challenge. For the single-dose pretreatment studies, SRL was measured (baseline) and then the animals were treated with BIO-1211 or placebo, 0.5 h or 2 h before antigen challenge. SRL was then remeasured just before antigen challenge and then after challenge (as described previously). For the multiple-dose pretreatment studies, animals were treated once daily for 4 d and then challenged either 0.5 h or 24 h after the last dose of drug.

For the studies examining the reversal of post-antigen-induced airway AHR, baseline airway responsiveness was determined and then the animals were challenged with antigen and measurements of SRL were obtained over 8 h as previously described. On the next day, a postchallenge PC₄₀₀ was measured to ensure that the animals developed AHR. Then, 2 h after determining the postchallenge PC₄₀₀, the animals were treated with either PBS (placebo) or 3 mg BIO-1211. This procedure (determination of PC₄₀₀ followed by treatment) was continued 48 h and 72 h after antigen challenge. Measurements of PC₄₀₀ were also made at 96 h and 216 h (9 d) after antigen challenge.

In five unchallenged allergic sheep, we determined if treatment with BIO-1211 affected baseline airway responsiveness. To do this, PC₄₀₀ was determined 30 min after treatment with saline or 3 mg BIO-1211. Experiments were separated by at least 48 h and randomized.

Effects of BIO-1211 on Airway Markers of Inflammation

Protocol. The study was designed as a randomized cross-over study using 10 sheep. Each sheep had previously been shown to have developed EAR and LAR to A. suum. For this study, a baseline BAL and bronchial biopsy were performed before initiating treatment. Then the sheep began a 4-d, once-a-day treatment protocol with 3 mg (1 mg/ ml) aerosol of BIO-1211 or PBS (control). A second "baseline" BAL and biopsy were obtained 2 h after treatment on the third day. On the fourth day treatment was given and then 0.5 h later the sheep were challenged with antigen. Postchallenge BALs were performed 1 h, 4 h, 6.5 to 8 h, and 24 h after challenge. Postchallenge biopsies were obtained 6.5 to 8 h and 24 h after challenge. Sequential biopsies were obtained from alternate lungs so that no one lung was biopsied at consecutive time points. Sheep were randomized to receive either drug or placebo. The alternate treatment was given  3 wk later.

Statistics. For the airway studies the area under the curve (AUC) for antigen-induced EAR (0 to 4 h) and LAR (4 to 8 h) was computed using the trapezoid rule and these values were used for comparisons between placebo and drug trials. Comparisons of prechallenge and postchallenge PC₄₀₀ were performed from ratios of post-/prechallenge values of PC₄₀₀. For the later analysis, a ratio of 1 indicates that there was no change in the airway responsiveness, whereas a ratio below 1 indicates the development of AHR. Comparisons to placebo responses were made with a one-way analysis of variance (ANOVA) followed by a Tukey-Kramer or Newman-Keuls test (2-tailed) or by an unpaired t test when appropriate.

Comparisons of changes in baseline SRL before and after drug treatment (Table 1) were made with paired t test. Likewise, a paired t test was used to determine if BIO-1211 affected the PC₄₀₀ in unchallenged sheep.

Data for BAL cell responses and TK levels were not normally distributed and, therefore, analyzed using Friedman's two-way ANOVA. If the null hypothesis was rejected, then a post hoc test using Wilcoxon's paired test was used to determine at specific times which variables were different from each other. For these analyses, the two "baseline" values, i.e., baseline 1 and baseline 2, were averaged and this value was then used as the baseline for statistical purposes. One set of BAL samples was lost, therefore only data from nine sheep are reported.

Biopsy results were analyzed using Friedman's two-way ANOVA because the data also failed the normality test. If the null hypothesis was rejected, then a post hoc test using Wilcoxon's paired test was used to determine at specific times which variables were different from each other. The biopsy taken before treatment, i.e., baseline 1, and that obtained after treatment, baseline 2, were averaged and this value used as baseline for statistical purposes. This averaging of the baseline biopsies was considered justified because there were no statistical differences between the baseline 1 and 2 biopsies for any of the cell variables measured in either trial. Two biopsies were lost from the drug-treated group at 6.5 h. To correct for this, we used a missing value formula (39) and inserted the calculated value before statistical analysis.

All values in the text and figures are reported as mean ± SE. Significance was accepted when p < 0.05 using a two-tailed analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Airway Responses

Acute treatments. Table 1 shows the effects of BIO-1211 for the different treatment protocols on baseline SRL, and Table 2 provides a summary of the statistical analysis for the antigen-induced responses for the airway studies described. As seen in Table 1, treatments with doses of up to 30 mg BIO-1211 did not significantly affect baseline airway tone. Figure 1 shows the time course of the changes in SRL after antigen challenge with doses of 1 or 3 mg of aerosolized BIO-1211 given 0.5 h before antigen challenge. Also illustrated are the changes in airway responsiveness with each of these doses expressed as a ratio of the postchallenge/prechallenge value. For these trials, effective protection against the LAR and AHR was achieved with a single 3-mg dose of BIO-1211. This dose also reduced the EAR. Increasing the dose to either 10 or 30 mg did not significantly increase the protection against the EAR, the LAR, or the post- antigen-induced AHR (Figure 2, Table 2). Intravenous treatment with BIO-1211 given 0.5 h before antigen challenge, produced results similar to the aerosol treatments (Table 2).

View larger version (24K): [in this window] [in a new window]   Figure 1.   Effect of treatment with aerosol BIO-1211 on the time course of antigen-induced changes in specific lung resistance (SRL, cm H2O · s1) in allergic sheep (top). BIO-1211 was administered 0.5 h before challenge. The bottom figure represents the change in airway responsiveness to inhaled carbachol expressed as a ratio of the post (performed 24 h after antigen challenge) to prechallenge PC400. A ratio of 1 indicates that there was no change in the airway responsiveness, whereas a ratio below 1 indicates the development of AHR. The 1-mg BIO-1211 dose was ineffective, whereas the 3-mg dose maximally inhibited the LAR and the AHR. Values are man ± SE for 5 to 17 sheep (see Table 2 for statistical analysis). *p < 0.05 versus placebo.

View larger version (29K): [in this window] [in a new window]   Figure 2.   Dose-response data for aerosol BIO-1211 (top). Airway responses to antigen are expressed as AUC for the EAR (0 to 4 h) and LAR (4 to 8 h). AHR data express as PC400 ratio (bottom). Increasing the BIO-1211 dose provided increasing inhibition of EAR. Values are mean ± SE for 3 to 7 sheep. *p < 0.05 versus placebo (see Table 2 for statistical analysis).

If the pretreatment time was extended to 2 h, the 3-mg aerosol dose was still maximally effective in blocking the LAR and the postchallenge AHR (Table 2), but there was greater (71%) inhibition of the EAR than was seen with the acute dosing. However, intravenous administration of the drug 2 h before challenge was not as effective as the aerosol treatment against the EAR and LAR and completely failed to protect against the postchallenge AHR (Table 2).

To ensure that the protective effects on the LAR and the postchallenge AHR seen with BIO-1211 were not due to interference with the EAR, animals were treated 1.5 h after antigen challenge with either 3 mg aerosol BIO-1211 or with 3 mg BIO-1211 intravenously. Both treatments significantly blocked the LAR and the associated AHR (Figure 3).

View larger version (32K): [in this window] [in a new window]   Figure 3.   Effects of a single 3-mg aerosol or intravenous treatments with BIO-1211 given 1.5 h after antigen challenge. Both aerosolized and intravenously administered BIO-1211 significantly inhibited the LAR and the post-antigen-induced AHR. Values are mean ± SE for 2 to 4 sheep. *p < 0.05 versus placebo (see Table 2 for statistical analysis).

Multiple treatments. Figure 4 illustrates the effects of a once a day treatment for 4 d (last dose given 0.5 h before antigen) with 1 mg aerosol BIO-1211. In contrast to the earlier results with an acute dosing (Figure 1), multiple treatments with this 1-mg dose gave 86% protection against the EAR, 86% protection against the LAR, and completely inhibited the post-antigen-induced AHR. Increasing the dose to 3 mg only slightly improved the protective effect of BIO-1211 on the LAR using this 4-d dosing regimen. However, the effectiveness of the 3-mg dose was not diminished if the antigen challenge occurred 24 h after the last dose of a 4-d dosing regimen (Table 2).

View larger version (28K): [in this window] [in a new window]   Figure 4.   Effects of multiple treatments with aerosol BIO-1211 given once daily for 4 d. Antigen challenge was given 0.5 h after the last dose. Although the 1-mg aerosol dose was ineffective when given as a single treatment, the 4-d repeat dosing markedly inhibited the antigen-induced responses. Values are mean ± SE for 3 to 7 sheep. *p < 0.05 versus placebo (see Table 2 for statistical analysis).

Reversal of post-antigen-induced hyperresponsiveness. We had previously shown that postchallenge AHR is maintained for up to 2 wk after a single provocation in sheep that develop LARs (28). Figure 5 illustrates that after this post-antigen- induced AHR is established, it can be reversed by treatment with BIO-1211 (3 mg). In the control trial, the post-/prechallenge PC₄₀₀ ratio was 0.39 ± 0.04 one day after antigen challenge and the PC₄₀₀ ratio remained at this level for the remaining 9 d. In the BIO-1211 trial, the PC₄₀₀ ratio steadily improved from the initial value of 0.47 ± 0.08 one day after challenge, to a value of 1.03 ± 0.10 on the fourth day after antigen challenge (1 d after the last drug treatment). This reversal was maintained up to the 9-d measurement.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Reversal of existing AHR by once-daily dosing with 3 mg aerosol BIO-1211, for 3 d. The sheep were allowed to develop post- antigen-induced AHR (i.e., decreased PC400 ratio) 1 d after antigen challenge. After (0.5 h) the determination of the sheep's PC400, the sheep were dosed with placebo or 3-mg aerosol BIO-1211. BIO-1211 treatment normalized airway responsiveness to carbachol within 3 d; responsiveness remained normal thereafter. Values are mean ± SE for four sheep. *p < 0.05 versus placebo.

Effects of BIO-1211 on baseline airway responsiveness to carbachol. To ensure that the protective effects of BIO-1211 were not due to alterations in smooth muscle responsiveness, we determined the PC₄₀₀ before and after a single 3-mg dose of BIO-1211 in five unchallenged allergic sheep. Before treatment, the PC₄₀₀ was 15.4 ± 2.3 BU and after treatment, PC₄₀₀ was 16.7 ± 2.5 BU. Thus, the PC₄₀₀ ratio in these unchallenged animals (1.09 ± 0.04) was not significantly altered by BIO-1211.

Effect of BIO-1211 on Airway Markers of Inflammation

BAL cell response. Overall, there were no significant differences detected between drug and placebo trials for total cells/ml recovered by BAL (Figure 6). When the specific cell types were analyzed, the only significant overall difference in response to treatment was seen with the neutrophils (p < 0.01) and eosinophils (p < 0.02). In both instances, the recovery of both cell types by BAL was reduced in the drug arm. Considering all time points, the mean reduction in the neutrophil and eosinophil response with treatment was 53% and 77%, respectively. Consistent with this decreased cell response we found that, overall, the antigen-induced increase in TK activity was significantly reduced in the drug treatment arm (p < 0.01; Figure 7).

View larger version (29K): [in this window] [in a new window]   Figure 6.   Effect of 4-d dosing with 3 mg BIO-1211 on antigen-induced changes in BAL cells. BIO-1211 did not affect the total number of cells or the number of lymphocytes recovered by BAL. There was, however, an overall significant inhibition of both neutrophils (p < 0.01) and eosinophils (p < 0.02) in the BIO-1211-treated groups. Values are mean ± SE for nine sheep.

View larger version (29K): [in this window] [in a new window]   Figure 7.   Effect of 4-d dosing with 3 mg BIO-1211 on antigen-induced changes in tissue kallikrein (TK) activity (expressed as optical density [OD] units). Overall, BIO-1211 significantly inhibited TK activity (p < 0.001). Values are mean ± SE for nine sheep.

Bronchial biopsies. Overall treatment with BIO-1211 significantly reduced the inflammatory cell response in the airways. Figure 8 illustrates the effect of the drug on the cellular response. Overall differences were seen for lymphocytes (p < 0.03), eosinophils (p < 0.03), and neutrophils (p < 0.01). However, part of the eosinophil and neutrophil response could be related to the decreased baseline values in the drug trial relative to the placebo trial. Given the responses of the individual cell types, it is not surprising that the total inflammatory cell response (i.e., lymphocytes, metachromatic staining cells, eosinophils, and neutrophils) was reduced in the BIO-1211 treatment arm.

View larger version (14K): [in this window] [in a new window]   Figure 8.   Effect of 4-d dosing with 3 mg BIO-1211 on antigen-induced changes in cell responses in bronchial biopsies. Total VLA-4 cells = sum of eosinophils, lymphocytes, metachromatic staining cells, and neutrophils. Total change = (6.5 h + 24 h)  BSL. Values are mean ± SE for 10 sheep. Lymphocytes, eosinophils, neutrophils, and total VLA-4 cells showed overall differences between drug-treated and placebo arm. *p < 0.05 versus placebo at specific time points.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of this study indicate that a tight-binding, small-molecule inhibitor of VLA-4, BIO-1211, can modify antigen-induced EAR, LAR, and the prolonged AHR that follow an acute antigen challenge in the allergic sheep model. The molecule was protective when given either as an aerosol or intravenously in single doses or equivalent protective effects could be obtained by reducing the dose of the compound and using multiple treatments. The protection against the LAR and the post-antigen-induced AHR seen in the pretreatment protocols was not the result of inhibition of the EAR because these events were blocked if the drug was given after the EAR had resolved. The protective effect against the LAR and the post- antigen-induced AHR was associated with a reduction in the recruitment of VLA-4-expressing cells (i.e., eosinophils, lymphocytes, metachromatic staining cells, and neutrophils) to the airways after challenge. Finally, we describe for the first time that a VLA-4 inhibitor can reverse the persistent antigen- induced AHR seen in this animal model. This novel observation provides additional support for the role of VLA-4 in the chronic inflammatory events that follow antigen challenge. The fact that VLA-4 inhibitors can protect against one or more of the later pathophysiologic events that follow antigen provocation is not in and of itself a novel finding. However, several aspects of the data presented here are new and, therefore, extend the previous observations not only in this animal model, but observations obtained in other models as well (40).

BIO-1211 is a tight-binding inhibitor to ₄₁. BIO-1211 binding to Jurket cells could be inhibited by VCAM-Ig or CS-1, suggesting that ₄₁ ligand occupy an overlapping site on the integrin (34). The molecule has a K_d at equilibrium of 70 pM. In vitro, the IC₅₀ of BIO-1211 was 1 to 2 nM (34), which is approximately 10-fold more potent than the CS-1 ligand mimic used previously by us (27). Given these data, it was expected that BIO-1211 should be more potent in vivo than the small-molecule peptide inhibitor previously used in the sheep model. Our results confirm that expectation.

Our own work as well as that of others has supported a role for ₄-expressing leukocytes in the pathophysiology of allergen-induced airway responses (40). However, in most instances the effects have been seen with antibodies to ₄. We recently showed that similar results could be obtained with a small-molecule inhibitor of the cell matrix adhesion CS-1 binding site for VLA-4 (27). Because CS-1 binding can be distinguished from VCAM-1 binding (41), blocking ₄-expressing leukocyte interaction with either counter receptor interferes with the cell migration, or activation, or both, resulting in protection against the allergen-induced LAR and AHR.

The results of the present study support our hypothesis and extend our previous findings with the CS-1 peptide mimic indicating that small molecule inhibitors are effective in modifying VLA-4-mediated pathophysiologic events in the airways. Of importance here, as compared with previous studies, are the fact that single treatments with low doses of compound were shown to be effective, the ability of the inhibitor to work by both intravenous and aerosol routes, and most importantly, the new data showing that the VLA-4 antagonists can reverse AHR.

As in our previous study with the anti-VLA-4 mAb HP1/2 (28), we found that BIO-1211 was effective against antigen- induced LAR and AHR when given either intravenously or by aerosol. Our results are different from those reported by Henderson and coworkers (42) in which parenteral administration of an anti-₄ mAb did not protect against AHR in a mouse model of late-phase pulmonary inflammation. The reasons for the divergent responses are unclear, and except for species differences, any other attempt at an explanation would be highly speculative.

We used both BAL and bronchial biopsies to assess the anti-inflammatory potential of BIO-1211. Both measurements showed similar trends, i.e., the recruitment of VLA-4 cells being reduced with treatment. In conjunction with the BAL cellular response, we found that TK, which we have previously shown to be a marker of inflammation and AHR (36), was reduced in the BIO-1211-treated animals. Thus, the anti-inflammatory profile of BIO-1211 is consistent with its functional protection in this animal model.

Whereas the eosinophil and its products have been the major focus in the development of the LAR and AHR, other studies have identified roles for metachromatic staining cells (43) and lymphocytes (47), both of which express VLA-4. Other studies have suggested that the neutrophil can contribute as well (50). In a previous study (27), we introduced the concept that, the total numbers of VLA-4-positive cells, rather than just eosinophils, may be an important determinant of these pathophysiologic events. At that time, we included only metachromatic staining cells, eosinophils, and lymphocytes in the VLA-4-expressing cell group. However, in view of recent data (see the subsequent discussion), it appears that neutrophils may also express VLA-4. Our previous studies with HP1/2, a mAb for VLA-4 (28) and the CS-1 ligand mimic (27), indicated that neutrophil responses were not different between the treated and untreated groups after antigen challenge. This would be expected given that flow cytometric analysis showed that resting sheep neutrophils did not bind HP1/2 (28). However, in the present study, both BAL and biopsy results indicated an apparent protection against neutrophil movement after antigen challenge. The reduction in neutrophils associated with VLA-4 treatment in the biopsy specimens might, in part, be explained by the increased number of neutrophils in the sections at baseline in placebo trial. However, baseline BAL neutrophil counts were not different and, yet, BIO-1211 still reduced the neutrophil influx. These findings suggest that BIO-1211, unlike its weaker predecessors, had an effect on neutrophils as well. Findings of VLA-4-mediated neutrophil migration have been reported for rat neutrophils in models of adjuvant arthritis and dermal inflammation (53), and in activated human neutrophil migration through connective tissue fibroblast barriers (54).

Studies with human neutrophils also indicate that VLA-4-dependent adhesion occurs under certain flow conditions. For example, neutrophils stimulated with dihydrocytochalasin B and either C5a or FMLP can tether and roll on either VCAM-1-expressing endothelial cells or VCAM-1-transfected cells in an ₄-dependent fashion (17). Therefore, given our present findings, it is possible that sheep neutrophils, like rat and human neutrophils, express low levels of VLA-4 which are functional and that the reduced neutrophil response seen in the present study was a direct effect of BIO-1211 inhibition owing to its increased binding affinity for ₄₁.

The mechanism leading to the persistent AHR that follows antigen provocation in these sheep is still uncertain. However, our recent data strongly suggest that metachromatic staining cells may play a role in this event, possibly by releasing low concentrations of inflammatory mediators, including cytokines that do not alter airway tone, but that heighten the responsiveness of the airways to exogenous stimuli. This hypothesis is based on the collective data from three different studies. First, we showed that metachromatic staining cells increased 2-fold in bronchial sections obtained 24 h after challenge from allergic sheep that exhibited LAR (55). Second, we showed that mast cell tryptase can cause histamine release, presumably by causing degranulation of metachromatic staining cells (56). Third, we found that the tryptase inhibitor, APC-366, could also reverse this post-antigen-induced AHR (57). Although we cannot conclusively say which metachromatic staining cells (mast cells or basophils) are the primary cell responsible for this post-antigen-induced AHR, these data would indicate that they are important effector cells.

It is important to note that, although BIO-1211 can block and reverse antigen-induced AHR, it does not affect airway tone or baseline airway responsiveness. Thus, neither baseline SRL nor PC₄₀₀ values in unchallenged animals were altered by BIO-1211 treatment and, so, the compound does not appear to act as a smooth muscle relaxant. This would suggest that the mechanism by which BIO-1211 affects antigen-induced AHR is by reducing metachromatic cell recruitment to the airways, or interfering with cell degranulation, or by both mechanisms.

Whereas the cell responses in the presence of BIO-1211 seen in the present study support the inhibition of cell recruitment, the reduction in the EAR seen with BIO-1211 treatment provides evidence that binding VLA-4 can modify mediator release. Although we did not specifically measure mediators in this study, our own studies with selectin inhibitors (52) and findings from other laboratories using VLA-4 antibodies support this view. For example, the EAR was reduced in rats treated with an anti-VLA-4 antibody (31) and this protection was associated with a reduction in mast cell mediators (52). Consistent with this is a report indicating that rat peritoneal mast cell IgE-mediated exocytosis (degranulation) was blocked by anti-VLA-4 agents (58). Thus, the reduction in the EAR seen in these experiments may reflect the interaction of BIO-1211 with the mast cell VLA-4 causing a reduced response to antigen stimulation.

In conclusion, these results provide further evidence for the involvement of ₄ integrins in the production of antigen-induced LAR and AHR. In addition, our results support the potential utility of inhalational approaches with anti-adhesion therapy to mediate airway cell activation that contributes to asthma pathophysiology.