CCR5, CXCR4, and CD4 are clustered and closely apposed on microvilli of human macrophages and T cells

The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an o rdered multistep mechanism to allow the binding and entry of human immunode ficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechani sm depends on the spatial distribution of the participating molecules on th e cell surface. Immunoelectron microscopy was performed to address the subc ellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were doubl e labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then sta ined with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predomi nantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixe d microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the d iameter of an HIV-1 virion. Such distributions are likely to facilitate coo perative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of t herapeutically useful inhibitors of the entry process. Although the mechani sm underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synt hesis and well before insertion into the cellular membrane. Chemokine recep tors normally act as sensors, detecting concentration gradients of their li gands and thus providing directional information for cellular migration dur ing both normal homeostasis and inflammatory responses. Localization of the se sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, si nce selectins, some integrins, and actin are also located on or in the micr ovillus, this organelle has many of the major elements required for chemota xis.