Effect of a very late antigen-4 receptor antagonist on allergen-induced airway responses and inflammation in asthma.

BACKGROUND: Very late antigen-4 (VLA(4)) plays a key role in the recruitment of eosinophils in allergic responses in animal studies. OBJECTIVE: We investigated whether pretreatment with multiple doses of a VLA(4) receptor antagonist, HMR 1031, protects against allergen-induced airway responses and airway inflammation in humans. METHODS: Fourteen asthmatics (7F/7M), 18-49 years, PC(20) forced expiratory volume in 1 s (FEV(1)) methacholine (M) (<8 mg/mL; FEV(1) 82.3-116.1% predicted) with dual responses to inhaled allergen participated in a double-blind, placebo-controlled, cross-over study. Each treatment period consisted of 9 days, separated by >or=2 weeks. Exhaled nitric oxide (eNO), PC(20)FEV(1)(M) and hypertonic saline-induced sputum was obtained on Days 1, 7 and 9. Subjects inhaled HMR 1031 (20 mg b.i.d.) or placebo (P) on Days 1--8. On Day 8, an allergen bronchoprovocation test was performed, the airway response was measured by FEV(1), and expressed as %fall from baseline. Data from 12 evaluable subjects are presented here. RESULTS: Both treatments were well tolerated. There was no significant difference between HMR 1031 and P in the early asthamatic response: mean AUC (0-3 h)+/-SEM (%fall h): 26.01+/-4.26 and 17.41+/-4.26, respectively (P=0.18), nor in the late response: mean AUC (3-9 h)+/-SEM (%fall h): 97.09+/-8.63 and 97.61+/-8.63, respectively, P=0.97. This corresponded to the absence of significant allergen-induced changes in PC(20)FEV(1)(M), eNO, sputum eosinophils and soluble inflammation markers between both treatment periods. CONCLUSIONS: Treatment with multiple inhaled doses of the VLA(4) antagonist, HMR 1031, did not result in detectable protection against allergen-induced airway responses or airway inflammation in asthma.

Design and synthesis of potent and selective inhibitors of integrin VLA-4.

The synthesis and identification of a novel series of inhibitors of integrin VLA-4 are described. Their in vitro activity and selectivity against closely related integrins are also presented.

1,25-Dihydroxyvitamin D3 suppresses the expression of the VCAM-1 receptor, VLA-4 in human leukemic HL-60 cells.

Very late antigen-4 (VLA-4) is the complex with alpha4 and beta1 integrins, which is the receptors to fibronectin and VCAM-1. We evaluate the effect of 1,25(OH)2D3 on the expression of VLA-4 in human leukemic HL-60, U937 cells and human melanoma A375 cells. Flow cytometric analysis demonstrate that the expression of alpha4 integrin is negatively regulated in the cell lines we studied. The expression of beta1 integrin is also decreased in HL-60 and U937 cells. The mRNA expression of alpha4 integrin is significantly decreased by the treatment with 1,25(OH)2D3, whereas 1,25(OH)2D3 does not alter the expression of beta1 mRNA. The adhesion assay demonstrate that the number of adherent cells treated with 1, 25(OH)2D3 is significantly lower than that untreated on VCAM-1-coated wells. Because VCAM-1 is highly expressed in the endothelial cells, it is possible that 1,25(OH)2D3 prevents the attachment of the cells from the endothelial cells in vivo.

Novel inhibitors of alpha 4 beta 1 integrin receptor interactions through library synthesis and screening.

A library of 2302 small molecule beta-turn mimetics was screened for inhibition of the alpha 4 beta 1 integrin-CS1 splice variant binding interaction. Preliminary data revealed several active ligands, and validation with purified material culminated in the identification of some of the first small molecule ligands (1, IC50 = 5 microM, and 2, IC50 = 8 microM) to be reported for this class of integrins.

Bone marrow stromal cells induce myeloid and lymphoid development of the sorted hematopoietic stem cells in vitro.

Regulation of development of hematopoietic stem cells was examined by culturing Lin- c-Kit+ Sca1+ stem cells sorted from bone marrow (BM) cells by fluorescence-activated cell sorting on a layer of TBR59, a BM stromal cell line established from simian virus 40 T-antigen gene transgenic mice. The sorted stem cells did not show self-renewal, but two waves (at 7 and 13 days) of a cobblestone formation were induced by the stromal cell layer. The cobblestones were formed by finite cell division (eight divisions on average) of sorted Lin- c-Kit+ Sca1+ stem cells, and divided cells were still immature. The c-Kithigh stem cell population was induced to form the first wave of cobblestone formation committed to myeloid lineage, whereas c-Kitlow population was induced to form the second wave of this formation committed to lymphoid lineage. Both cobblestone formations require c-Kit function, but very late activation antigen-4-vascular cell adhesion molecule-1 interaction plays different parts in the two lineages.

In vivo blood monocyte migration to acute inflammatory reactions, IL-1 alpha, TNF-alpha, IFN-gamma, and C5a utilizes LFA-1, Mac-1, and VLA-4. The relative importance of each integrin.

UNLABELLED: The role of the monocyte integrins, Mac-1, LFA-1, and VLA-4, on the adhesion of rat blood monocytes to rat microvascular endothelial cells in vitro and the importance of these receptors in monocyte migration to inflammation in vivo were evaluated. Monocyte adhesion to cytokine (IL-1, IFN-gamma, and TNF-alpha)-stimulated endothelial cells was mediated by Mac-1, LFA-1, and VLA-4, but Mac-1 appeared to be less important than LFA-1 or VLA-4. After i.v. injection, large numbers of 51Cr-labeled blood monocytes migrated within 2 h to dermal inflammatory sites induced by C5a, IL-1 alpha, IFN-gamma, TNF-alpha, LPS, and poly inosinic:cytidylic acid. Anti-Mac-1 mAb treatment had no effect, whereas anti-LFA-1 inhibited migration to C5a and the cytokines by 20 to 40%. Blocking both Mac-1 and LFA-1 decreased monocyte accumulation by 50 to 70% to all stimuli. Anti-VLA-4 inhibited monocyte migration to IL-1 alpha, IFN-gamma, TNF-alpha, and LPS, but not to C5a. Combining anti-Mac-1 with anti-VLA-4 did not increase this inhibition, whereas blocking VLA-4 and LFA-1 together further suppressed (60-85%) migration. Combined treatment with mAb to all three integrins inhibited > 98% of the monocyte migration to the inflammatory stimuli. IN CONCLUSION: 1) 51Cr blood monocytes can be used to quantify monocyte migration to inflammatory reactions in the rat. 2) Monocytes use Mac-1, LFA-1, and VLA-4 for in vitro adhesion and in vivo migration to cutaneous inflammation, and these integrins are essential for normal migration because blockade of all three virtually abolishes monocyte accumulation. 3) Mac-1 plays a less important role than LFA-1, as LFA-1 appears to substitute for Mac-1, and VLA-4 and LFA-1 can mediate much of the adhesion and migration. 4) The initiating inflammatory stimulus also modifies monocyte integrin usage, supporting the multistep combinatorial model of leukocyte extravasation.

Involvement of the very late antigen 4 integrin on melanoma in interleukin 1-augmented experimental metastases.

We have previously reported that treatment with interleukin 1 (IL-1) induced the augmentation of lung tumor colonies by a human melanoma in nude mice. Here we have investigated the involvement of the alpha 4 beta 1 integrin, the very late antigen 4 (VLA-4) in this augmentation. A375M melanoma cells expressed high levels of VLA-4 and preferentially adhered to a surface coated with vascular cell adhesion molecule 1 (VCAM-1), the ligand for VLA-4 on activated endothelial cells. This adhesion was inhibited by treating tumor cells with saturating concentrations of mAb to VLA-4. The production of lung colonies was significantly enhanced in nude mice given an injection of IL-1 before A375M melanoma cells. Immunoperoxidase staining showed that VCAM-1 could be expressed on lung vascular endothelium of mice in response to IL-1. Pretreatment of melanoma cells with a mAb to VLA-4 completely abrogated the IL-1-induced augmentation of lung colonies. Using two metastatic melanoma clones (clones 2/4 and 2/60) that expressed different levels of VLA-4, we found that only VLA-4-bearing cells adhered to a VCAM-1-coated surface and formed enhanced numbers of lung colonies in IL-1-treated nude mice. This augmentation was inhibited by pretreating the tumor cells with anti-VLA-4 mAb. These results demonstrate, in vivo, the functional involvement of VLA-4 on melanoma cells in IL-1-mediated lung colony augmentation, most probably involving the interaction of tumor cells with VCAM-1 on activated endothelial cells.

Dual inhibition of VLA-4 and LFA-1 maximally inhibits cutaneous delayed-type hypersensitivity-induced inflammation.

Lymphocytes express surface receptors that mediate adhesion to endothelial cells and control T cell migration into inflammatory sites. Lymphocyte VLA-4 and LFA-1 mediate adhesion to cytokine-activated endothelium, but the contribution of these molecules to in vivo migration and lymphocyte mediated inflammation is not clear. Here we show that both VLA-4 and LFA-1 contribute to not only lymphocyte adhesion but to in vivo lymphocyte migration in the rat and that nearly complete inhibition of lymphocyte accumulation is observed when both integrins are blocked. Furthermore, inhibition of delayed-type hypersensitivity-induced inflammation, as quantified by skin induration and fibrin deposition, is observed with either anti-VLA-4 or anti-LFA-1, but much stronger inhibition is observed with a blockade of both integrins. Thus, dual inhibition of the VLA-4 and LFA-1 pathways is required for a maximal anti-inflammatory effect in some types of T cell-mediated inflammation.

Inhibition of insulitis and prevention of diabetes in nonobese diabetic mice by blocking L-selectin and very late antigen 4 adhesion receptors.

Leukocyte adhesion to the endothelial venules in the pancreatic islets is thought to be one of the initial steps in the development of insulin-dependent diabetes mellitus. It has been suggested that leukocyte adhesion to endothelium is a sequential multistep process involving various different homing receptors. We report here that blocking different homing receptors--namely, L-selectin and very late antigen 4 (VLA-4)--which function during different stages of the adhesion process, by specific monoclonal antibodies inhibits insulitis and prevents diabetes in mice. Moreover, leukocyte attachment to the inflamed vessels within pancreatic sections could be inhibited by anti-L-selectin and anti-VLA-4 antibodies. Interestingly, anti-L-selectin or anti-VLA-4 antibody did not appear to influence the autoimmune response to a panel of pancreatic beta-cell autoantigens. These data suggest that L-selectin and VLA-4 receptors are involved in mediating leukocyte homing to the islets and that intervention of these two adhesion pathways may provide a novel approach for treatment of autoimmune diseases such as insulin-dependent diabetes mellitus.

Inhibition of the degradation of the precursor and of the mature beta 1 integrin subunit by different protein synthesis inhibitors and by ATP depletion.

A stabilization of both the precursor and the mature beta 1 integrin subunit was observed in metabolically labeled human skin fibroblasts and Molt-4 T lymphocytes upon addition of protein synthesis inhibitors or by ATP depletion. Differential effects of protein synthesis inhibitors are reported since the slow degradation of the mature beta 1 subunit was sensitive to cycloheximide but not to puromycin. We also show that the half-life of the mature subunit was not dependent on intracellular lysosomal degradation or on ubiquitination suggesting that VLA turn-over occurs at the cell surface and might involve proteins or proteases with short half-life.

Platelet adhesion to laminin: role of Ca2+ and Mg2+ ions, shear rate, and platelet membrane glycoproteins.

The adhesion of platelets to purified laminin under flow conditions was investigated. Adhesion to laminin was strongly dependent on the presence of divalent cations. In the absence of cations platelet adhesion (8% coverage in 5 minutes) was maximal at a shear rate of 100/s and no adhesion could be detected at shear rates above 800/s. In the presence of 0.8 mmol/L Mg2+ and 2 mmol/L Ca2+ platelet adhesion reached its maximum (30% coverage) around 800/s. At 1,800/s platelets still adhered to purified laminin (coverage of 6%). Antibodies against the E8 domain of laminin and antibodies against the alpha 6 and beta 1 chains of platelet membrane glycoprotein very late activation antigen-6 (VLA-6), completely inhibited adhesion. No inhibition was found with antibodies against glycoprotein IIb:IIIa, against the alpha 2 chain of VLA-2, and against the alpha 5 chain of VLA-5. Fibronectin and von Willebrand factor were not involved in laminin-dependent adhesion. Anti-VLA-6 partly inhibited platelet adhesion to the extracellular matrix of endothelial cells at shear rates below 800/s. Preincubation of the matrices with antilaminin E8 antibodies did not influence the adhesion. These results show that purified laminin supports platelet adhesion and that the presence of VLA-6 is important for platelet adhesion under flow conditions. The protein in the matrix with which VLA-6 interacts is currently unknown.