Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to the proteasome in dendritic cells

Dendritic cells (DCs) capture and internalize human immunodeficiency virus (HIV)-1 through C-type lectins, including DC-SIGN. These cells mediate efficient infection of T cells by concentrating the delivery of virus through the infectious synapse, a process dependent on the cytoplasmic domain of DC-SIGN. Here, we identify a cellular protein that binds specifically to the cytoplasmic region of DC-SIGN and directs internalized virus to the proteasome. This cellular protein, leukocyte-specific protein 1 (LSP1), was defined biochemically by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LSP1 is an F-actin binding protein involved in leukocyte motility and found on the cytoplasmic surface of the plasma membrane. LSP1 interacted specifically with DC-SIGN and other C-type lectins, but not the inactive mutant DC-SIGNΔ35, which lacks a cytoplasmic domain and shows altered virus transport in DCs. LSP1 diverts HIV-1 to the proteasome. Down-regulation of LSP1 with specific small interfering RNAs in human DCs enhanced HIV-1 transfer to T cells, and bone marrow DCs from lsp1−/− mice also showed an increase in transfer of HIV-1BaL to a human T cell line. Proteasome inhibitors increased retention of viral proteins in lsp1+/+ DCs, and substantial colocalization of virus to the proteasome was observed in wild-type compared with LSP1-deficient cells. Collectively, these data suggest that LSP1 protein facilitates virus transport into the proteasome after its interaction with DC-SIGN through its interaction with cytoskeletal proteins

LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1−/−) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-α and interleukin-1β) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1−/− chimeric mice using bone marrow transplantation revealed that in mice with Lsp1−/− endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1−/− neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1−/− animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1−/− mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration

Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis

Atherosclerotic lesions develop in regions of arterial curvature and branch points, which are exposed to disturbed blood flow and have unique gene expression patterns. The cellular and molecular basis for atherosclerosis susceptibility in these regions is not completely understood. In the intima of atherosclerosis-predisposed regions of the wild-type C57BL/6 mouse aorta, we quantified increased expression of several proinflammatory genes that have been implicated in atherogenesis, including vascular cell adhesion molecule–1 (VCAM-1) and a relative abundance of dendritic cells, but only occasional T cells. In contrast, very few intimal leukocytes were detected in regions resistant to atherosclerosis; however, abundant macrophages, including T cells, were found throughout the adventitia (Adv). Considerably lower numbers of intimal CD68+ leukocytes were found in inbred atherosclerosis-resistant C3H and BALB/c mouse strains relative to C57BL/6 and 129; however, leukocyte distribution throughout the Adv of all strains was similar. The predominant mechanism for the accumulation of intimal CD68+ cells was continued recruitment of bone marrow–derived blood monocytes, suggestive of low-grade chronic inflammation. Local proliferation of intimal leukocytes was low. Intimal CD68+ leukocytes were reduced in VCAM-1–deficient mice, suggesting that mechanisms of leukocyte accumulation in the intima of normal aorta are analogous to those in atherosclerosis

GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions

The contribution of intimal cell proliferation to the formation of early atherosclerotic lesions is poorly understood. We combined 5-bromo-2′-deoxyuridine pulse labeling with sensitive en face immunoconfocal microscopy analysis, and quantified intimal cell proliferation and Ly-6Chigh monocyte recruitment in low density lipoprotein receptor–null mice. Cell proliferation begins in nascent lesions preferentially at their periphery, and proliferating cells accumulate in lesions over time. Although intimal cell proliferation increases in parallel to monocyte recruitment as lesions grow, proliferation continues when monocyte recruitment is inhibited. The majority of proliferating intimal cells are dendritic cells expressing CD11c and major histocompatibility complex class II and 33D1, but not CD11b. Systemic injection of granulocyte/macrophage colony-stimulating factor (GM-CSF) markedly increased cell proliferation in early lesions, whereas function-blocking anti–GM-CSF antibody inhibited proliferation. These findings establish GM-CSF as a key regulator of intimal cell proliferation in lesions, and demonstrate that both proliferation and monocyte recruitment contribute to the inception of atherosclerosis

The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1

AbstractBackground: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway.Results: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis.Conclusions: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylating CD72, SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity

Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response.  </p