Targeted Gene Deletion Demonstrates That Adhesion Molecule ICAM-4 Is Critical for Erythroblastic Island Formation.

Erythroid progenitors proliferate, differentiate and enucleate within specialized bone marrow subcompartments, termed erythroblastic islands, which are comprised of developing erythroblasts surrounding a central macrophage. Growing evidence suggests that within erythroblastic islands adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. We are exploring the potential function of erythroid ICAM-4, a recently characterized member of the immunoglobulin superfamily, in erythroblastic island formation. We earlier identified α4β1 and αV integrins as ICAM-4 binding partners. Since erythroblasts express α4β1 and ICAM-4 and macrophages exhibit αV, ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast and erythroblast-macrophage attachments. Indeed, two synthetic peptides that block ICAM-4/αV adhesion caused a marked decrease in the percentage of islands formed. To more definitively test whether ICAM-4 attachments are active in erythroblastic islands we generated ICAM-4 knockout mice and compared the capacity of single cell suspensions from freshly harvested ICAM-4 null and wild type bone marrow to form erythroblastic islands in vitro, using a reproducible live cell island reconstitution assay that we have established. Islands and their cellular components were identified and quantitated by three-color immunofluorescent microscopy employing fluoresceinated erythroid-specific TER119 antibody, macrophage-specific F4/80 antibody and a DNA probe. Strikingly, we observed a 47% decrease in the percentage of islands formed from bone marrow of ICAM-4 null mice compared to wild type littermates (n=10 and n=10, respectively). We also studied the ability of ICAM-4 null erythroblasts to form islands in vivo by analyzing intact islands freshly harvested from mouse bone marrow. Similar to the in vitro data we found a marked decrease in the percentage of islands formed in the bone marrow of ICAM-4 null mice compared to wild type littermates. The null mice had 44% fewer islands than wild type mice. Taken together, the results of this phenotypic analysis provide convincing evidence that ICAM-4 is one of the adhesion molecules critical for erythroblastic island formation. We postulate that this newly identified erythroblast receptor may be important not only for adhesive integrity of the island structure but also for initiating intracellular signaling essential for normal erythroid terminal differentiation.