IL-10–producing B cells, also known as regulatory B cells (Bregs), play a key role in controlling autoimmunity. In this study, we report that chimeric mice specifically lacking IL-10–producing B cells (IL-10−/−B cell) developed an exacerbated arthritis compared with chimeric wild-type (WT) B cell mice. A significant decrease in the absolute numbers of Foxp3 regulatory T cells (Tregs), in their expression level of Foxp3, and a marked increase in inflammatory Th1 and Th17 cells were detected in IL-10−/− B cell mice compared with WT B cell mice. Reconstitution of arthritic B cell deficient (μMT) mice with different B cell subsets revealed that the ability to modulate Treg frequencies in vivo is exclusively restricted to transitional 2 marginal zone precursor Bregs. Moreover, transfer of WT transitional 2 marginal zone precursor Bregs to arthritic IL-10−/− mice increased Foxp3+ Tregs and reduced Th1 and Th17 cell frequencies to levels measured in arthritic WT mice and inhibited inflammation. In vitro, IL-10+/+ B cells established longer contact times with arthritogenic CD4+CD25− T cells compared with IL-10−/− B cells in response to Ag stimulation, and using the same culture conditions, we observed upregulation of Foxp3 on CD4+ T cells. Thus, IL-10–producing B cells restrain inflammation by promoting differentiation of immunoregulatory over proinflammatory T cells.

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D−/− hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function.

An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1␣, and the CX 3 C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxinsensitive increase in the concentration of cytosolic free Ca 2؉ . Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G q/11 -dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. Human cytomegalovirus (HCMV), a member of the ␤-herpesvirus family, is a slowly growing virus which causes severe birth defects and a wide spectrum of disorders in older children and adults. While HCMV infections of immunocompetent individuals are usually asymptomatic, HCMV infection of immunocompromised patients can result in disseminated disease characterized by fever and leukopenia, hepatitis, pneumonitis, esophagitis, gastritis, colitis, and retinitis (62). HCMV is one of several related species-specific viruses that cause similar diseases in various animals. In vivo, HCMV appears to replicate in a variety of cell types, including fibroblasts, epithelial cells, endothelial cells, and smooth muscle cells (75). HCMV has evolved several mechanisms to evade detection by the host immune system (47) and can latently persist in the host lifelong.HCMV infection has long been known to be associated with changes in the activity of many cellular signaling pathways (reviewed in references 3 and 29). The cellular responses triggered by HCMV infection can be subdivided into changes emerging within minutes after the initial contact between virus and host cell, likely to be caused by the binding of the virus to and its passage through the h...

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Cathepsin E is an aspartic endosomal proteinase, expressed at high levels in some epithelial and haemopoetic cells. The enzyme has been implicated in a variety of functions, including antigen processing. This study documents strain-specific variation in expression of cathepsin E in mice. The levels of cathepsin E protein and message are profoundly decreased in haemopoetic cells from C57BL/6J mice, compared to levels in 129S2/Sv or Balb/c. The deficiency is cell-type-specific, as protein levels in gut are not affected. Deficiency affects B cell, T cells, macrophages and dendritic cells. The low cathepsin E phenotype cosegregates with the C57BL/6J genotype in a panel of C57BL/6J x 129S2/Sv F2 mice. Analysis of the promoter region of cathepsin E reveals a polymorphism which destroys a previously described functional PU.1 transcription binding consensus sequence in the C57BL/6J genome. Antigen processing of ovalbumin by dendritic cells, which has previously been shown to require cathepsin E, is impaired in C57BL/6J-derived dendritic cells. C57BL/6J mice thus exhibit a profound tissue-specific deficiency in cathepsin E expression, which may have important implications for the immune phenotype of this mouse strain.

To explore the role of antigen-specific CD4؉ T cells in glomerulonephritis, we administered ovalbumin 323-339 peptide conjugated to glomerular-binding polyclonal antibody and induced disease in RAG1 ؊/؊ mice with CD4 ؉ T cells from OT2 ؋ RAG1 ؊/؊ mice. These OT2 ؋ RAG1 ؊/؊ mice have a transgenic T-cell receptor specific for this peptide. When CD4؉ T cells were primed in vivo, crescentic glomerulonephritis developed after 21 days in mice given peptide-conjugated glomerular-binding antibody but not unconjugated antibody control. We then investigated the relative roles of T H 1 and T H 17 cells, using Fab 2 fragments of glomerular-binding antibody to exclude a role for antibody in this model. T cells from OT2 ؋ RAG1 ؊/؊ mice were polarized in vitro, and T H 1 or T H 17 cell lines were injected into mice that were also given peptideconjugated Fab 2 or unconjugated Fab 2 control, giving four experimental groups. After 21 days crescentic glomerulonephritis was seen in mice receiving T H 17 cells and peptide-conjugated Fab 2 but in none of the other three groups. These results suggest that T H 17 but not T H 1 cells can induce crescentic glomerulonephritis.

Background: Cathepsin D is a well-characterized aspartic protease expressed ubiquitously in lysosomes. Cathepsin D deficiency is associated with a spectrum of pathologies leading ultimately to death. Cathepsin D is expressed at high levels in many cells of the immune system, but its role in immune function is not well understood. This study examines the reconstitution and function of the immune system in the absence of cathepsin D, using bone marrow radiation chimaeras in which all haematopoietic cells are derived from cathepsin D deficient mice.

Improved chemical inhibitors are required to dissect the role of specific antigen processing enzymes and to complement genetic models. In this study we explore the in vitro and in vivo properties of a novel class of targeted inhibitor of aspartic proteinases, in which pepstatin is coupled to mannosylated albumin (MPC6), creating an inhibitor with improved solubility and the potential for selective cell tropism. Using these compounds, we have demonstrated that MPC6 is taken up via mannose receptor facilitated endocytosis, leading to a slow but continuous accumulation of inhibitor within large endocytic vesicles within dendritic cells, and a parallel inhibition of intracellular aspartic proteinase activity. Inhibition of intracellular proteinase activity is associated with reduction in antigen processing activity, but this is epitope specific, preferentially inhibiting processing of T cell epitopes buried within compact proteinase-resistant protein domains. Unexpectedly, we have also demonstrated, using quenched fluorescent substrates, that little or no cleavage of the disulfide linker takes place within dendritic cells, but this does not appear to affect the activity of MPC6 as an inhibitor of cathepsins D and E in vitro and in vivo. Finally, we have shown that MPC6 selectively targets dendritic cells and macrophages in spleen in vivo. Access to non-lymphoid tissues is very limited in the steady state, but is strongly enhanced at local sites of inflammation. The strategy adopted for MPC6 synthesis may therefore represent a more general way to deliver chemical inhibitors to cells of the innate immune system, especially at sites of inflammation.Antigen presenting cells (including dendritic cells (DC) and macrophages) are equipped with an array of membrane and cytoplasmic receptors known as Pattern Recognition Receptors (PRR), with which they bind to microbial components (Pathogen Associated Molecular Patterns, PAMPs). Once internalised, any PAMP-associated proteins are subject to regulated proteolysis (the exogenous antigen processing pathway), producing peptides which bind to Class II Major Histocompatibility Complex (MHC) receptors, and hence stimulate T cell adaptive immunity 1 . The interaction between antigen presenting cell and T cell is widely recognised as being one of the key steps regulating both the magnitude and the A number of studies have used antibodies to deliver antigens to antigen presenting cells in vitro or in vivo 2 . This has achieved some significant successes. However, a wealth of experience from the field of tumour biology has shown that delivery of drugs via antibody conjugates poses formidable technical problems. An alternative approach is to target DC using ligands of lectins such as mannose receptors, themselves a family of PRRs 3,4 . We have explored this targeting strategy in the context of using the selective inhibitor pepstatin 1 to identify the role of aspartic proteinases cathepsins D and E in the proteolysis of antigen 5,6 . Pepstatin itself is a very potent inhibitor (IC...