Anti-Integrin alpha 5 Antibody [5H10-27 (MFR5)] (A121794)

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

A class of adhesion molecules, the VLA integrins, are expressed on thymocytes and have been shown to affect immature thymocyte differentiation in vitro. This study examines the ability of cAMP to regulate VLA receptor function in thymocytes. Pharmacologic agents that raise intracellular cAMP enhanced the binding of immature CD4- CD8- and CD4+ CD8+ thymocytes to fibronectin while having no effect on the binding of the more mature JIId- thymocytes. PGE2, a hormone produced by thymic epithelial cells and known to raise intracellular cAMP levels in thymocytes, also increased the binding of immature thymocytes to fibronectin. In contrast, activation of protein kinase C via PMA enhanced the binding of all three thymocyte subsets. The cAMP-induced binding was blocked by mAbs to the VLA integrin chains alpha 4 and alpha 5 and by the protein kinase A (PKA) inhibitor, (Rp)-cAMPS, indicating that activation of PKA enhances VLA-4 and VLA-5 receptor function. Activation of PKA was induced in all three thymocyte subsets following addition of cAMPa or forskolin, indicating that the inability of cAMP to enhance the binding of JIId- thymocytes was not due to an inability to activate PKA. Thus, cAMP enhances integrin function in thymocytes in a maturation stage-specific manner.

Implanting mouse blastocysts adhere through their abembryonic surfaces to the endometrial extracellular matrix. Because blastocysts cultured on fibronectin in vitro dissociate to form trophoblast outgrowths, it is unclear whether this adhesion is initially mediated by fibronectin receptors on the apical or basolateral surface of the trophectoderm. Intact blastocysts were examined in a ligand binding assay utilizing the fibronectin cell binding domain attached to fluorescent microspheres. Fibronectin binding activity on the apical surface of the trophectoderm was confined to the abembryonic pole of the blastocyst, where trophoblast differentiation initiates, and was regulated temporally in accordance with blastocyst outgrowth. Soluble fibronectin (IC50 = 0.2 microM) or Gly-Arg-Gly-Asp-Ser-Pro, but not laminin, competitively inhibited fibronectin binding activity. Addition of antibodies against the alpha v, alpha 5, beta 1, or beta 3 integrin subunits also inhibited binding activity. Blastocysts cultured in the absence of an adhesive substratum exhibited fibronectin binding activity only after exposure to immobilized or soluble ligand. Potentiation of binding activity by ligand was unaffected by cycloheximide but was sensitive to brefeldin A inhibition of protein trafficking. These findings suggest that the interaction of fibronectin with the trophectoderm induces a translocation event that up-regulates fibronectin binding beta 1- and beta 3-class integrins on the apical surface.

Interaction with stromal cells is known to be crucial for growth and differentiation of hematopoietic cells. To characterize adhesion molecules involved in this interaction, we examined adhesion of a panel of lymphoid, myeloid, and mast cell lines with stromal cells. We found that very late antigen-4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) were major adhesion molecules in lymphoid and myeloid cells, whereas myeloma cells adhered to stromal cells through hyaluronate. We investigated regulation of VLA-4 during differentiation of myeloid cells using a neutrophil precursor cell line, L-G3. Differentiation of neutrophils induced by granulocyte colony-stimulating factor was accompanied with down-regulation of VLA-4. Induced L-G3 cells adhered to stromal cells in proportion to the expression of VLA-4. Mast cells used two mechanisms to adhere to fibroblasts and stromal cells. They adhered to fibronectin through VLA-5 when stimulated with steel factor and also directly to membrane-anchored steel factor through c-kit.