The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation. PS/2, R1-2, or MK2 significantly inhibited the recruitment of eosinophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid and decreased inflammation in the lung tissues. Escalating in vivo doses of PS/2 or MK2 increased circulating levels of rat immunoglobulin G in the plasma. The binding of phycoerytherin-labeled anti-alpha4 mAb to blood T cells from PS/2-treated mice was reduced, implying that alpha4 sites were already occupied. T cells and eosinophils in BAL fluid from mice treated with PS/2 or MK2 were phenotypically different from controls. Selective decreases of alpha4+ T cells in the BAL fluid after PS/2 or MK2 treatment were coupled with changes in CD8+, CD11a, and CD62L expression. The alpha4-integrin and VCAM-1 may have important roles in the antigen-induced recruitment of T cells and eosinophils during OA-induced airway inflammation. The data suggest that these adhesion molecules may be suitable targets for therapeutic intervention in certain conditions of pulmonary inflammation.

Cytokines released from CD4+ T lymphocytes contribute to the pathogenesis of asthma by influencing the differentiation and function of eosinophils, the primary effector cells that cause airway epithelial damage. Using a model of ovalbumin (OA)-induced, eosinophil-rich chronic lung inflammation in sensitized mice, we have defined the role of T lymphocytes further by using three-color flow cytometry to characterize the adhesion and activation antigens that may be associated with the migration of these cells into the lung and airway lumen. OA inhalation in OA-sensitized C57BL/6 mice resulted in an early (6 to 24 h) influx of neutrophils into the bronchial lumen as enumerated by bronchoalveolar lavage (BAL), which was followed by a marked accumulation of lymphocytes and eosinophils between 24 to 72 h. Phenotypic analysis of BAL or lung tissue T cells showed that most Thy-1 CD3+ T cells were CD4+ (CD4: CD8 ratio of 3 to 4:1). The majority (90%) of the T cells in lung or BAL fluid expressed alpha beta T-cell receptors (TCR). Only 3 to 7% of the T cells were gamma delta TCR+ even though almost 25% of the T cells were CD4- CD8-. There were very few natural killer (NK) or B cells in BAL fluid compared with 15% B cells in dissagregated lung tissue. In contrast to T cells in spleen, almost all the lung and BAL T cells were of the memory phenotype, as ascertained by the expression of high levels of CD44 and by the absence of L-selectin and CD45RB on the cell surface. Fifty to ninety percent of lung and BAL T cells from vehicle-sensitized or OA-sensitized and challenged mice expressed the adhesion molecules CD11a (LFA-1), CD54 (ICAM-1), and CD49d (VLA-4). The early T-cell activation marker CD69 was upregulated on 30% of the lung and BAL T cells in OA-sensitized mice after antigen inhalation. When BAL fluid T cells from OA-sensitized and challenged mice were analyzed for their coexpression of adhesion and/or activation molecules, 75% of the cells that expressed one of three adhesion molecules, CD54, CD49d, or CD11a, also expressed at least one of the other two antigens. At least 15% of BAL T cells had all three of these molecules on their cell surfaces. The OA-dependent, temporally regulated emigration of T cells into the bronchial lumen after exposure to aerosolized antigen may be correlated with the accumulation of cells that express the memory phenotype with enhanced expression of adhesion molecules.

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.

We studied the adhesion of human peripheral blood T lymphocytes to human synovial fibroblasts stimulated with interferon-y (IFNy), tumor necrosis factor a (TNFcu), interleukin-lp (IL-lp), or combinations of these cytokines. T lymphocytes bound poorly to untreated human synovial fibroblasts. IFN y treatment resulted id the largest increase in adhesion, followed by TNFa and IL-1p. Combinations of IFNy + TNFa and IFNy + I L -l p had a synergistic effect on intercellular adhesion molecule 1 (ICAM-1) expression and adhesion. The increase in cellular adhesion induced by cytokines correlated with the up-regulation of the number of cells expressing ICAM-1 and the density of antigedcell. There was no synergistic effect on leukocyte functionassociated antigen 3 (LFA-3) or on HLA class I or class I1 antigen expression. Adhesion was only partially inhibited by anti-ICAM-1, anti-LFA-1, or anti-CD18. These findings suggest the existence of ICAM-1-independent and CDll/CDlS-independent adhesion mechanisms. Anti-LFA-3 was completely ineffective as an inhibitor of adhesion. There was no additive or synergistic advantage of using combinations of antibod-~