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Interactions among leukocytes and leukocytes with immune-associated auxiliary cells represent an essential feature of the immune response that requires the involvement of cell adhesion molecules (CAMs). In the immune system, CAMs include a wide range of members pertaining to different structural and functional families involved in cell development, activation, differentiation and migration. Among them, β 2 integrins (LFA-1, Mac-1, p150,95 and α D β 2 ) are predominantly involved in homotypic and heterotypic leukocyte adhesion. β2 integrins bind to intercellular (I)CAMs, actin cytoskeleton-linked receptors belonging to immunoglobulin superfamily (IgSF)-CAMs expressed by leukocytes and vascular endothelial cells, enabling leukocyte activation and transendothelial migration. β2 integrins have long been viewed as the most important ICAMs partners, propagating intracellular signalling from β2 integrin-ICAM adhesion receptor interaction. In this review, we present previous evidence from pioneering studies and more recent findings supporting an important role for ICAMs in signal transduction. We also discuss the contribution of immune ICAMs (ICAM-1, -2, and -3) to reciprocal cell signalling and function in processes in which β2 integrins supposedly take the lead, paying particular attention to T cell activation, differentiation and migration.

Activation of the integrin phagocytic receptors CR3 (α M β 2 , CD11b/CD18) and CR4 (α X β 2 , CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β 2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (α M ), ITGAX (α X ) and ITGB2 (β 2 ) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored α M expression. In general, the expression of α X was less responsive to jasplakinolide treatment than α M , indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling α M β 2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored α M expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of α M β 2 and α X β 2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Torres-Gomez, Fiyouzi, Guerra-Espinosa, Cardeñes, Clares, Toribio, Reche, Cabañas and Lafuente.)