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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 peptide-conjugated 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.

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. 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. Preliminary results suggest that access to nonlymphoid 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.

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.

Merozoite Surface Protein 1 is expressed on the surface of malaria merozoites and is important for invasion of the malaria parasite into erythrocytes. MSP1-specific CD4 T cell responses and antibody can confer protective immunity in experimental models of malaria. In this study we explore the contributions of cathepsins D and E, two aspartic proteinases previously implicated in antigen processing, to generating MSP1 CD4 T-cell epitopes for presentation. The absence of cathepsin D, a late endosome/lysosomal enzyme, is associated with a reduced presentation of MSP1 both following in vitro processing of the epitope MSP1 from infected erythrocytes by bone marrow-derived dendritic cells, and following in vivo processing by splenic CD11c+ dendritic cells. By contrast, processing and presentation of the soluble recombinant protein fragment of MSP1 is unaffected by the absence of cathepsin D, but is inhibited when both cathepsin D and E are absent. The role of different proteinases in generating the CD4 T cell repertoire, therefore, depends on the context in which an antigen is introduced to the immune system.

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., Author Summary Tissue macrophages frequently undergo a program of cell death, termed apoptosis, following sustained ingestion and killing of bacteria. In macrophages, induction of apoptosis enhances bacterial killing when macrophages' initial killing capacity is exhausted. We have investigated the mechanism of apoptosis in macrophages exposed to pneumococci, the commonest cause of bacterial pneumonia. We show that the cell structure containing ingested bacteria, the phagolysosome, becomes permeabilized early in the death process. Pneumococcal exposure activates a phagolysosomal enzyme, cathepsin D, which induces apoptosis. Cathepsin D activation is required for permeabilization of mitochondria, an organelle implicated in apoptosis induction. Cathepsin D reduces levels of a negative regulator of apoptosis in macrophages, Mcl-1, by enhancing its association with an enzyme, which mediates its degradation. The importance of these findings was confirmed in a bone marrow transplant model in which mice either received bone marrow from mice containing or lacking the cathepsin D gene. This model showed that reduced apoptosis of alveolar macrophages occurred when cathepsin D was lacking, and that this impaired clearance of pneumococci in the mouse lung. We conclude that during bacterial challenge, lysosomal permeabilization and cathepsin D activation triggers a novel death pathway, in a timely fashion, linking bacterial killing to apoptosis induction.

Expression of the human cytomegalovirus (HCMV)-encoded chemokine receptor homologue pUS28 in mammalian cells results in ligand-dependent and -independent changes in the activity of multiple cellular signal transduction pathways. The ligand-dependent signalling activity of pUS28 has been shown to be predominantly mediated by heterotrimeric G proteins of the G(i/o) and G(12/13) subfamilies. Ligand-independent constitutive activity of pUS28 causing stimulation of inositol phosphate formation has been correlated with the coupling of pUS28 to G proteins of the G(q) family. It is well known that activation of G(q) proteins by cell surface receptors is coupled to activation of the Rho GTPase RhoA. Activated RhoA regulates numerous cellular functions, including the activity of the transcription factor serum response factor (SRF). The marked activation of G(q) proteins by pUS28 in transfected and HCMV-infected cells prompted us to investigate its effect on SRF activity. The results presented herein demonstrate that expression of pUS28 in COS-7 cells caused a vigorous induction of SRF activity. This effect was observed in the absence of chemokines known to interact with pUS28, and was specifically mediated by endogenous G(q) and/or G(11) as well as RhoA and/or a closely related Rho GTPase. The stimulatory effect of pUS28 and Galpha(q/11) was independent of phospholipase C-beta (PLCbeta) activation and was markedly sensitive to inhibition by wild-type, but not by constitutively active Galpha(16), thus identifying Galpha(16) as a modulator of Galpha(q/11) function likely to act by competing with Galpha(q/11) for and thus uncoupling Galpha(q/11) from activation by pUS28.

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-1α, and the CX 3 C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive 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. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5′-O-(3-[ 35 S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.

Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.