In general terms, both αVβ3 and αXβ2 appeared to regulate IL-8 release acutely, whereas αVβ5 could have a role in inhibiting MIP-1β synthesis and/or release. There does not appear to be a hierarchy either between or within sCD23-binding integrin families with respect to control of cytokine

release. Integrins are best understood in terms of their adhesion-like activities, characterized by binding to linear sequences such as RGD in matrix proteins.32 However, it is increasingly clear that other ligands that lack RGD sequences ABT 263 bind integrins, and many such ligands use stretches of basic residues to bind target integrins. Examples include the binding of HIV-TAT to αVβ5,36 association of the snake venom jararhagin with the I-domain of α2β1 via an RKKH motif,37 the interaction of the angiogenic factor CCN1 with αMβ2 that is dependent on a pair of adjacent lysines,38 and the binding of the γC fragment of fibrinogen to αIIbβ3 which is also dependent on two pairs KU-60019 of lysine groups.38 Our own data demonstrate that sCD23 interacted

with αVβ5 using a basic motif (RKC) to bind the integrin at a site that did not recognize RGD sequences.15 Therefore, anti-integrin antibodies directed to distinct epitopes on the four integrins, including mAbs that either inhibited or failed to impede adhesion-dependent activities of the target integrins, were tested for effects on cytokine release. The responses were assessed in ELISA of supernatants from THP-1 cells, representative of an immature monocyte, and U937 cells, representative of a more differentiated macrophage-type cell. In all cases, none of IgG1, Vn or soluble RGDS tetrapeptide provoked release of IL-8, MIP-1β or RANTES to any degree greater

than that found in supernatants of untreated cells (Fig. 3a,b). For αVβ5 integrins, both the P1F6 and 15F11 reagents promoted release of IL-8 and MIP-1β from THP-1 cells, though the P1F6 reagent, which inhibits RGD-mediated functions of αVβ5, is by far the more effective stimulus (Fig. 3a). Neither antibody had any effect on RANTES release. By contrast, however, anti-αVβ5-specific mAbs failed to drive release of either Cell Penetrating Peptide IL-8 or MIP-1β from the more mature U937 cell line (Fig. 3b). As expected, and consistent with the data from THP-1 cells, there was no effect on release of RANTES from U937 cells (Fig. 3b, black bars). For the αVβ3-directed mAbs, only the 23C6 reagent promoted release of IL-8 and MIP-1β from THP-1 cells; the LM609 mAb had no effect (Fig. 3a,b). Neither reagent promoted RANTES release in THP-1 or U937 cells, and both were ineffective in promoting IL-8 or MIP-1β release in the latter cell line. The 23C6 reagent did, however, retain the capacity to elicit MIP-1β release from U937 cells. The AMF7 and LM142 anti-αV mAbs showed stimulatory effects on IL-8 and MIP-1β release in THP-1 cells, but generally not in U937 cells (Fig. 3a,b).