Systemic lupus erythematosus is a multifactorial autoimmune disease with an as yet unknown etiopathogenesis. It is widely thought that self-immunization in systemic lupus is driven by defective clearance of dead and dying cells. In lupus patients, large numbers of apoptotic cells accumulate in various tissues including germinal centers. In the present review, we discuss the danger signals released by apoptotic cells, their triggering of inflammatory responses, and the breakdown of B-cell tolerance. We also review the pathogenic role of apoptotic cell clearance in systemic lupus erythematosus.

CXCR4 is a G protein-coupled receptor (GPCR) that has multiple critical functions in normal and pathologic physiology that include regulation of the metastatic behavior of mammary carcinoma, and utilization as a coreceptor for infection by T-tropic strains of human immunodeficiency virus-1. Molecular dynamic simulations of the rhodopsin-based homology model of CXCR4 were performed in a solvated lipid bilayer to reproduce the microenvironment of this integral membrane protein.The amino acids in CXCR4 necessary for interaction with an inverse agonist, T140, and a weak partial agonist, AMD3100, identified by alanine scanning mutants, were spatially consistent when computationally docked. Whereas T140 binds residues in extracellular domains and regions of the hydrophobic core proximal to the cell surface, amino acids in the central hydrophobic core are critical to binding of AMD3100. The physical localization of T140 binding to CXCR4 by biochemical analyses corroborated the molecular and computational approaches. The structural basis for the interaction of T140 and AMD3100 with CXCR4 confirms that the mechanisms used by these agents are different. This complementary utilization of molecular, physical, and computation analysis provides a powerful approach to elucidate GPCR conformation.

Leukotriene B 4 (LTB 4 ) activates the G-protein-coupled receptor leukotriene B 4 receptor 1 (BLT1) to mediate a diverse array of cellular responses in leukocytes including chemotaxis, calcium mobilization, degranulation, and gene expression. To determine the role of phosphorylation in BLT1 regulation, we generated mutants of BLT1 in which all of the serine/threonine residues in the C-tail are converted to alanine or to aspartate/glutamate. These mutants expressed in rat basophilic leukemia RBL-2H3 cells bound LTB 4 with similar affinity and activated all of the known functional activities of BLT1, albeit at different levels. The conversion of phosphorylation sites to alanine resulted in enhanced Gprotein-mediated activities, whereas conversion to aspartate/glutamate resulted in reduced responses and a right shift in dose response, indicating that receptor phosphorylation is a critical regulator of G-protein-mediated pathways. Surprisingly, translocation of ␤-arrestin and receptor internalization was completely independent of BLT1 phosphorylation. Real-time analysis of ␤-arrestin translocation and receptor internalization using digital fluorescence video microscopy in cells expressing a red fluorescent protein labeled BLT1 and a green fluorescent protein-tagged ␤-arrestin confirmed phosphorylation-independent ␤-arrestin translocation and internalization of BLT1. In ␤-arrestin-deficient mouse embryo fibroblasts, the BLT1 receptors failed to display endosomal localization upon stimulation. In these cells, co-expression of ␤-arrestin-green fluorescent protein with BLT1-red fluorescent protein resulted in co-localization of BLT1 and ␤-arrestin upon activation. Thus, receptor phosphorylation-dependent mechanisms regulate G-protein-mediated pathways; however, phosphorylation-independent mechanisms regulate ␤-arrestin association and internalization of BLT1.Leukotriene B 4 (LTB 4 ), 1 a lipid mediator derived from arachidonic acid metabolism, is a potent chemoattractant for neutrophils (1). LTB 4 also activates a wide range of other biological responses in leukocytes including the respiratory burst, granule release, and changes in gene expression (2-4). Two distinct G-protein-coupled receptors, BLT1 and BLT2, were identified from several mammalian sources (3, 5). Whereas the high affinity receptor BLT1 is expressed mainly in phagocytic leukocytes and T-lymphocytes, the low affinity receptor BLT2 is more ubiquitously expressed. A number of studies using gene-targeted mice and antagonists and genetic analysis in mice and humans have implicated the importance of leukotrienes and their receptors in inflammatory diseases such as asthma, rheumatoid arthritis, and atherosclerosis (6, 7). However, the molecular mechanisms of signaling and regulation of leukotriene receptor functions largely remain unexplored.The activation of BLT1 in primary leukocytes or in cell lines expressing BLT1 results in G-protein activation, phosphoinositide hydrolysis, calcium mobilization, exocytosis, and chemotaxis (3, 8). Although BLT1 couples to ...

Glomerulonephritis is one of the most severe manifestations of systemic lupus erythematosus, with considerable morbidity and mortality. There remains a major unmet need for successful management of lupus nephritis. TAM family receptor tyrosine kinases (Mer and Axl) play an important role in the maintenance of immune homeostasis in the kidney. Mer is constitutively expressed in the glomeruli; Axl expression is inducible in glomeruli under inflammatory conditions. To investigate the distinct functions of Axl and Mer in lupus nephritis, we compared the severity of nephrotoxic serum glomerulonephritis in WT, Axl-KO, Mer-KO, and Axl/Mer-KO mice. Mer-KO mice developed severe glomerulonephritis, with significantly decreased survival and increased blood urea nitrogen levels, compared to WT mice given the same treatment. However, nephrotoxic serum-treated Axl-KO mice had significantly increased survival rates and improved renal function as compared to similarly treated WT, Mer-KO, and Axl/Mer-KO mice. Interestingly, mice lacking both Axl and Mer developed kidney inflammation comparable to WT mice. Western blot analysis revealed significantly increased Stat3 phosphorylation and caspase-1 activation in the kidneys of nephritic Mer-KO mice. In contrast, Axl deficient nephrotoxic serum-injected mice showed decreased Akt phosphorylation and Bcl-xl upregulation. Thus, the reciprocal activation of Axl and Mer receptor tyrosine kinases has a major impact on the outcome of renal inflammation.

The Mer receptor tyrosine kinase is both an important mediator of apoptotic cell phagocytosis and a regulator of macrophage and DC cytokine production. Since phenotypically distinguishable macrophages are known to have different functions, we have examined Mer expression of murine splenic macrophages. We also used serum deficient in the Mer ligand, growth arrest-specific protein 6 (Gas6) to define better the role of this Mer ligand in macrophage function. By immunofluorescence staining, we found Mer to be strongly expressed in splenic red pulp, largely on platelets. We also found Mer expression on marginal zone macrophages. Strikingly, all tingible body macrophages bore Mer. In functional phagocytosis assays of apoptotic cells, Gas6 appeared to be the sole ligand for Mer, and this system accounted for about 30% of splenic macrophage phagocytosis of apoptotic cells. Taken together, the expression pattern of mer on macrophage subpopulations in the spleen and its Gas6-dependent role in macrophage phagocytosis suggest an important role for Mer in the modulation of immune responses.