ICAM-3 is constitutively expressed on human lymphocytes and lymphocytic cell lines and contributes to their activation when it is engaged by β2 integrins on other target leukocytes. ICAM-3 is also expressed at high levels on myeloid leukocytes, but its function on these cells is unknown. We tested the hypothesis that ICAM-3 mediates proinflammatory signaling in myeloid leukocytes in early inflammation using immobilized monoclonal antibodies to engage ICAM-3 on freshly isolated human monocytes or neutrophils. Supernatants were analyzed by ELISA for three chemokines (MIP-1α, MCP-1, and IL-8 - products of immediate-early genes that are induced when myeloid cells are activated). Immobilized monoclonal antibodies against ICAM-3 induced chemokine secretion depending on the domain of ICAM-3 that they recognized. This chemokine secretion was greater than that induced by immobilized antibodies against ICAM-1 or immobilized control proteins. Coengagement of ICAM-3 and Fc receptors (FcγRI or FcγRII) was required for maximal secretion of MIP-1α (3.5-5.5x baseline, n=6) and IL-8 (7-13x baseline, n=6). When engaged separately, ICAM-3 (via F(ab′)2) and FcγRII did not cause chemokine secretion, and FcγRI induced MIP-1α and IL-8 secretion at lower levels than those observed with coengagement. In parallel with monocytes, neutrophils also secreted IL-8 when ICAM-3 and Fc receptors were coengaged. Microscopy demonstrated dramatic spreading of monocytes engaged by antibodies to ICAM-3. Spreading was induced by F(ab′)2 fragments of an antibody against ICAM-3, demonstrating direct signaling, but was most rapid with coengagement of Fc receptors. Disruption of this spreading with cytochalasin D decreased chemokine secretion induced by coengagement, suggesting that leukocyte spreading is important in the induction of MIP-1α and IL-8 in this system. We conclude that ICAM-3 may signal chemokine gene expression in myeloid cell-cell interactions in inflammatory lesions. Binding of Fc receptors by IgG in the local environment may amplify the response, an event that occurs at lower levels or not at all if ICAM-3 is not engaged.
