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CD74 mediates MHC class-II antigenic peptide loading and presentation and plays an important role in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus. C57BL/6 Faslpr mice that develop spontaneous lupus-like autoimmunity and pathology showed elevated CD74 expression in the inflammatory cell infiltrates and the adjacent tubular epithelial cells (TECs) in kidneys affected by lupus nephritis but negligible levels in kidneys from age-matched wild-type mice. The inflammatory cytokine IFN-γ or IL-6 induced CD74 expression in kidney TECs in vitro. The presence of kidney TECs from Faslpr mice, rather than from wild-type mice, produced significantly stronger histones, dsDNA, and ribonucleoprotein-Smith Ag complex-induced CD4+ T cell activation. Splenocytes from CD74-deficient FaslprCd74-/- mice had muted responses in a MLR and to the autoantigen histones. Compared with FasIprCd74+/+ mice, FasIprCd74-/- mice had reduced kidney and spleen sizes, splenic activated T cells and B cells, serum IgG and autoantibodies, urine albumin/creatinine ratio, kidney Periodic acid-Schiff score, IgG and C3 deposition, and serum IL-6 and IL-17A levels, but serum IL-2 and TGF-β levels were increased. Study of chronic graft-versus-host C57BL/6 mice that received donor splenocytes from B6.C-H2bm12/KhEg mice and those that received syngeneic donor splenocytes yielded similar observations. CD74 deficiency reduced lupus-like autoimmunity and kidney pathology in chronic graft-versus-host mice. This investigation establishes the direct participation of CD74 in autoimmunity and highlights a potential role for CD74 in kidney TECs, together with professional APCs in systemic lupus erythematosus. [ABSTRACT FROM AUTHOR]

Cysteinyl cathepsin K (CatK) is expressed in osteoclasts to mediate bone resorption, but is also inducible under inflammatory conditions. Faslpr mice on a C57BL/6 background develop spontaneous systemic lupus erythematosus-like manifestations. Although normal mouse kidneys expressed negligible CatK, those from Faslpr mice showed elevated CatK expression in the glomeruli and tubulointerstitial space. Faslpr mice also showed elevated serum CatK levels. CatK deficiency in Faslpr mice reduced all tested kidney pathologies, including glomerulus and tubulointerstitial scores, glomerulus complement C3 and IgG deposition, chemokine expression and macrophage infiltration, and serum autoantibodies. CatK contributed to Faslpr mouse autoimmunity and pathology in part by its activity in TLR-7 proteolytic processing and consequent regulatory T (Treg) cell biology. Elevated TLR7 expression and proteolytic processing in Faslpr mouse kidneys and Tregs showed significantly reduced levels in CatK-deficient mice, leading to increased spleen and kidney Treg content. Purified CD4+CD25highFoxp3+ Tregs from CatK-deficient mice doubled their immunosuppressive activity against Teffector cells, compared with those from CatK-sufficient mice. In Faslpr mice, repopulation of purified Tregs from CatK-sufficient mice reduced spleen sizes, autoantibody titers, and glomerulus C3 and IgG deposition, and increased splenic and kidney Treg contents. Tregs from CatK-deficient mice had significantly more potency than CatK-sufficient Tregs in reducing spleen sizes, serum autoantibody titers, and glomerulus C3 deposition, and in increasing splenic and kidney Treg content. This study established a possible role of CatK in TLR7 proteolytic activation, Treg immunosuppressive activity, and lupus autoimmunity and pathology. [ABSTRACT FROM AUTHOR]

Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B-deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia. [ABSTRACT FROM AUTHOR]

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease. [ABSTRACT FROM AUTHOR]

Aims: Regulatory T cells (Tregs) protect mice from angiotensin II (Ang-II)-induced abdominal aortic aneurysms (AAA). This study tested whether AAA patients are Treg-insufficient and the Treg molecular mechanisms that control AAA pathogenesis. Methods and results: ELISA determined the Foxp3 concentration in blood cell lysates from 485 AAA patients and 204 age- and sex-matched controls. AAA patients exhibited lower blood cell Foxp3 expression than controls (P < 0.0001). Pearson's correlation test demonstrated a significant but negative correlation between Foxp3 and AAA annual expansion rate before (r = -0.147, P = 0.007) and after (r = -0.153, P = 0.006) adjustment for AAA risk factors. AAA in apolipoprotein E-deficient (Apoe-/-) mice that received different doses of Ang-II exhibited a negative correlation of lesion Foxp3+ Treg numbers with AAA size (r = -0.883, P < 0.0001). Adoptive transfer of Tregs from wild-type (WT) and IL10-deficient (Il10-/-) mice increased AAA lesion Treg content, but only WT mice Tregs reduced AAA size, AAA incidence, blood pressure, lesion macrophage and CD4+ and CD8+ T-cell accumulation, and angiogenesis with concurrent increase of lesion collagen content. Both AAA lesion immunostaining and plasma ELISA demonstrated that adoptive transfer of WT Tregs, but not Il10-/- Tregs, reduced the expression of MCP-1. In vitro cell culture and aortic ring assay demonstrated that only Tregs from WT mice, but not those from Il10-/- mice, reduced macrophage MCP-1 secretion, macrophage and vascular cell protease expression and activity, and aortic ring microvessel formation. Conclusion: This study supports a protective role of Tregs in human and experimental AAA by releasing IL10 to suppress inflammatory cell chemotaxis, arterial wall remodelling, and angiogenesis. [ABSTRACT FROM AUTHOR]

Background: This study was undertaken to evaluate the feasibility, safety, and therapeutic effects of percutaneous renal cryoablation under local anesthesia with conscious sedation for patients who have unresectable stage 1 (T1NoMo) renal cell carcinoma (RCC) in high surgical risk. Methods: Eighteen patients who were not candidates for surgery underwent primary cryosurgery guided by gray-scale ultrasound. Contrast-enhanced ultrasonography (CEUS) and contrast-enhanced computed tomography (CT) were performed to evaluate treatment at completion. Results: The mean follow-up period was 26.8 months (range, 12-56 months). All tumors were biopsied before cryosurgery. Seventeen tumors remained free of enhancement during follow-up period. No major complications associated with cryoablation procedures were found though two instances of subcapsular hematomas, one of retroperitoneal errhysis and one of nausea, were seen after cryoablation. One patient had a local recurrence of tumor and received additional cryoablation. Local tumor control rate was 100 % of T1NoMo tumors including the recurrence case who underwent additional cryoablation. Conclusions: Percutaneous cryoablation can be recommended as a feasible, safe, and promising therapy for the treatment of renal tumor, especially those unresectable stage 1 RCC, with a low risk of complications. [ABSTRACT FROM AUTHOR]

The cysteine protease cathepsin K has been implicated in pathogenesis of cardiovascular disease. We hypothesized that ablation of cathepsin K protects against obesity-associated cardiac dysfunction. Wild-type mice fed a high-fat diet exhibited elevated heart weight, enlarged cardiomyocytes, increased left ventricular wall thickness, and decreased fractional shortening. All these changes were reconciled in cathepsin K knockout mice. Cathepsin K knockout partly reversed the impaired cardiomyocyte contractility and dysregulated calcium handling associated with high-fat diet. Additionally, cathepsin K knockout alleviated whole-body glucose intolerance and improved insulin-stimulated Akt phosphorylation in high-fat diet--fed mice. High-fat feeding increased the expression of cardiac hypertrophic proteins and apoptotic markers, which were inhibited by cathepsin K knockout. Furthermore, high-fat feeding resulted in cathepsin K release from lysosomes into the cytoplasm. In H9c2 myoblasts, silencing of cathepsin K inhibited palmitic acid--induced release of cytochrome c from mitochondria and expression of proapoptotic signaling molecules. Collectively, our data indicate that cathepsin K contributes to the development of obesity-associated cardiac hypertrophy and may represent a potential target for the treatment to obesity-associated cardiac anomalies. [ABSTRACT FROM AUTHOR]

Background-The interplay between the immune system and abnormal metabolic conditions sustains and propagates a vicious feedback cycle of chronic inflammation and metabolic dysfunction that is critical for atherosclerotic progression. It is well established that abnormal metabolic conditions, such as dyslipidemia and hyperglycemia. cause various cellular stress responses that induce tissue inflammation and immune cell activation, which in turn exacerbate the metabolic dysfunction. However, molecular events linking these processes are not well understood. Methods and Results--Tissues and organs of humans and mice with hyperglycemia and hyperlipidemia were examined for expression of ligands for NKG2D, a potent immune-activating receptor expressed by several types of immune cells, and the role of NKG2D in atherosclerosis and metabolic diseases was probed with the use of mice lacking NKG2D or by blocking NKG2D with monoclonal antibodies. NKG2D ligands were upregulated in multiple organs, particularly atherosclerotic aortas and inflamed livers. Ligand upregulation was induced in vitro by abnormal metabolites associated with metabolic dysfunctions. Using apolipoprotein E-deficient mouse models, we demonstrated that preventing NKG2D functions resulted in a dramatic reduction in plaque formation, suppressed systemic and organ inflammation mediated by multiple immune cell types, and alleviated abnormal metabolic conditions. Conclusions--The NKG2D/ligand interaction is a critical molecular link in the vicious cycle of chronic inflammation and metabolic dysfunction that promotes atherosclerosis and might be a useful target for therapeutic intervention in the disease. [ABSTRACT FROM AUTHOR]

Background: Recent studies have suggested that mast-cell activation and inflammation are important in obesity and diabetes. Plasma levels of mast cell proteases and the mast cell activator immunoglobulin E (IgE) may serve as novel inflammatory markers that associate with the risk of pre-diabetes and diabetes mellitus. Methods and Results: A total of 340 subjects 55 to 75 years of age were grouped according to the American Diabetes Association 2003 criteria of normal glucose tolerance, pre-diabetes, and diabetes mellitus. The Kruskal-Wallis test demonstrated significant differences in plasma IgE levels (P = 0.008) among groups with different glucose tolerance status. Linear regression analysis revealed significant correlations between plasma levels of chymase (P = 0.030) or IgE (P = 0.022) and diabetes mellitus. Ordinal logistic regression analysis showed that IgE was a significant risk factor of pre-diabetes and diabetes mellitus (odds ratio [OR]: 1.674, P = 0.034). After adjustment for common diabetes risk factors, including age, sex, hypertension, body-mass index, cholesterol, homeostatic model assessment (HOMA) index, high-sensitivity C-reactive protein (hs-CRP), and mast cell chymase and tryptase, IgE remained a significant risk factor (OR: 1.866, P = 0.015). Twovariable ordinal logistic analysis indicated that interactions between hs-CRP and IgE, or between IgE and chymase, increased further the risks of developing pre-diabetes and diabetes mellitus before (OR: 2.204, P = 0.044; OR: 2.479, P = 0.033) and after (OR: 2.251, P = 0.040; OR: 2.594, P = 0.026) adjustment for common diabetes risk factors. Conclusions: Both IgE and chymase associate with diabetes status. While IgE and hs-CRP are individual risk factors of prediabetes and diabetes mellitus, interactions of IgE with hs-CRP or with chymase further increased the risk of pre-diabetes and diabetes mellitus. [ABSTRACT FROM AUTHOR]

Background: Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined. Methods and Results: Using bone marrow-derived mast cells from wild-type, Tnf-/-, Ifng-/-, Il6-/- mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC. Conclusion: Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: Cysteine protease cathepsins are important in extracellular matrix protein degradation, cell apoptosis, and angiogenesis. Mice lacking cathepsins are protected from tumor progression in several animal models, suggesting that the regulation of cathepsin activities controls the growth of various malignant tumors. Methods and Results: We tested the role of cathepsins using a mouse model of multistage epithelial carcinogenesis, in which the human keratin-14 promoter/enhancer drove the expression of human papillomavirus type 16 (HPV16) early region E6/E7 transgenes. During the progression of premalignant dysplasia, we observed increased expression of cysteine protease cathepsin S, but concomitantly reduced expression of cathepsin endogenous inhibitor cystatin C in the skin tissue extract. Absence of cystatin C in these transgenic mice resulted in more progression of dysplasia to carcinoma in situ on the face, ear, chest, and tail. Chest and ear skin extract real time PCR and immunoblot analysis, mouse serum sample ELISA, tissue immunohistological analysis, and tissue extract-mediated in vitro elastinolysis and collagenolysis assays demonstrated that cystatin C deficiency significantly increased cathepsin expression and activity. In skin from both the chest and ear, we found that the absence of cystatin C reduced epithelial cell apoptosis but increased proliferation. From the same tissue preparations, we detected significantly higher levels of pro-angiogenic laminin 5-derived γ2 peptides and concurrently increased neovascularization in cystatin C-deficient mice, compared to those from wild-type control mice. Conclusion: Enhanced cathepsin expression and activity in cystatin C-deficient mice contributed to the progression of dysplasia by altering premalignant tissue epithelial proliferation, apoptosis, and neovascularization. [ABSTRACT FROM AUTHOR]

Abdominal aortic aneurysm (AAA), an inflammatory disease, involves leukocyte recruitment, immune responses, inflammatory cytokine production, vascular remodeling, neovascularization, and vascular cell apoptosis, all of which contribute to aortic dilatation. This study demonstrates that mast cells, key participants in human allergic immunity, participate in AAA pathogenesis in mice. Mast cells were found to accumulate in murine AAA lesions. Mast cell-deficient KitW-sh/KitW-sh mice failed to develop AAA elicited by elastase perfusion or periaortic chemical injury. KitW-sh/KitW-sh mice had reduced aortic expansion and internal elastic lamina degradation; decreased numbers of macrophages, CD3+ T lymphocytes, SMCs, apoptotic cells, and CD31+ microvessels; and decreased levels of aortic tissue IL-6 and IFN-γ. Activation of mast cells in WT mice via C48/80 injection resulted in enhanced AAA growth while mast cell stabilization with disodium cromoglycate diminished AAA formation. Mechanistic studies demonstrated that mast cells participated in angiogenesis, aortic SMC apoptosis, and matrix-degrading protease expression. Reconstitution of KitW-sh/KitW-sh mice with bone marrow-derived mast cells from WT or TNF-α-/- mice, but not from IL-6-/- or IFN-α-/- mice, caused susceptibility to AAA formation to be regained. These results demonstrate that mast cells participate in AAA pathogenesis in mice by releasing proinflammatory cytokines IL-6 and IFN-α, which may induce aortic SMC apoptosis, matrix-degrading protease expression, and vascular wall remodeling, important hallmarks of arterial aneurysms. [ABSTRACT FROM AUTHOR]

Cysteine proteases play an important part in human pathobiology. This report shows the participation of cathepsin L (CatL) in adipogenesis and glucose intolerance. In vitro studies demonstrate the role of CatL in the degradation of the matrix protein fibronectin, insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R), essential molecules for adipogenesis and glucose metabolism. CatL inhibition leads to the reduction of human and murine pre-adipocyte adipogenesis or lipid accumulation, protection of fibronectin from degradation, accumulation of IR and IGF-1R β-subunits, and an increase in glucose uptake. CatL-deficient mice are lean and have reduced levels of serum glucose and insulin but increased levels of muscle IR β-subunits, fibronectin and glucose transporter (Glut)-4, although food/water intake and energy expenditure of these mice are no less than their wild-type littermates. Importantly, the pharmacological inhibition of CatL also demonstrates reduced body weight gain and serum insulin levels, and increased glucose tolerance, probably due to increased levels of muscle IR β-subunits, fibronectin and Glut-4 in both diet-induced obese mice and ob/ob mice. Increased levels of CatL in obese and diabetic patients suggest that this protease is a novel target for these metabolic disorders. [ABSTRACT FROM AUTHOR]

Mast cells contribute importantly to allergic and innate immune responses by releasing various preformed and newly synthesized mediators. Previous studies have shown mast cell accumulation in human atherosclerotic lesions. This report establishes the direct participation of mast cells in atherogenesis in low-density lipoprotein receptor–deficient (Ldlr−/−) mice. Atheromata from compound mutant Ldlr−/− KitW-sh/W-sh mice showed decreased lesion size, lipid deposition, T-cell and macrophage numbers, cell proliferation and apoptosis, but increased collagen content and fibrous cap development. In vivo, adoptive transfer of syngeneic wild-type or tumor necrosis factor (TNF)-α-deficient mast cells restored atherogenesis to Ldlr−/−KitW-sh/W-sh mice. Notably, neither interleukin (IL)-6- nor interferon (IFN)-γ-deficient mast cells did so, indicating that the inhibition of atherogenesis in Ldlr−/−KitW-sh/W-sh mice resulted from the absence of mast cells and mast cell–derived IL-6 and IFN-γ. Compared with wild-type or TNF-α-deficient mast cells, those lacking IL-6 or IFN-γ did not induce expression of proatherogenic cysteine proteinase cathepsins from vascular cells in vitro or affect cathepsin and matrix metalloproteinase activities in atherosclerotic lesions, implying that mast cell–derived IL-6 and IFN-γ promote atherogenesis by augmenting the expression of matrix-degrading proteases. These observations establish direct participation of mast cells and mast cell–derived IL-6 and IFN-γ in mouse atherogenesis and provide new mechanistic insight into the pathogenesis of this common disease. [ABSTRACT FROM AUTHOR]

The cysteine protease cathepsin S is highly expressed in malignant tissues. By using a mouse model of multistage murine pancreatic islet cell carcinogenesis in which cysteine cathepsin activity has been functionally implicated, we demonstrated that selective cathepsin S deficiency impaired angiogenesis and tumor cell proliferation, thereby impairing angiogenic islet formation and the growth of solid tumors, whereas the absence of its endogenous inhibitor cystatin C resulted in opposite phenotypes. Although mitogenic vascular endothelial growth factor, transforming growth factor-β1, and the anti-angiogenic endostatin levels in either serum or carcinoma tissue extracts did not change in cathepsin S- or cystatin C-null mice, tumor tissue basic fibroblast growth factor and serum type 1 insulin growth factor levels were higher in cystatin C-null mice, and serum type I insulin growth factor levels were also increased in cathepsin S-null mice. Furthermore, cathepsin S affected the production of type IV collagen-derived anti-angiogenic peptides and the generation of bioactive pro-angiogenic γ2 fragments from laminin-5, revealing a functional role for cathepsin S in angiogenesis and neoplastic progression. [ABSTRACT FROM AUTHOR]

Presents information on a study that determined the effect of cathepsin S deficiency on LDL receptor-deficient mice. Methodologies of the study; Results of the study; Discussion on the study.

Major histocompatibility complex class II antigen presentation requires the participation of lysosomal proteases in two convergent processes. First, the antigens endocytosed by the antigen-presenting cells must be broken down into antigenic peptides. Second, class II molecules are synthesized with their peptide-binding site blocked by invariant chain (Ii), and they acquire the capacity to bind antigens only after Ii has been degraded in the compartments where peptides reside. The study of genetically modified mice deficient in single lysosomal proteases has allowed us to determine their role in these processes. Cathepsins (Cat) B and D, previously considered major players in MHC class II antigen presentation, are dispensable for degradation of Ii and for generation of several antigenic determinants. By contrast, Cat S plays an essential role in removal of Ii in B cells and dendritic cells, whereas Cat L apparently does so in thymic epithelial cells. Accordingly, the absence of Cat S and L have major consequences for the onset of humoral immune responses and for T-cell selection, respectively. It is likely that other as yet uncharacterized lysosomal enzymes also play a role in Ii degradation and in generation of antigenic determinants. Experiments involving drugs that interfere with protein traffic suggest that more than one mechanism for Ii removal, probably involving different proteases, can co-exist in the same antigen-presenting cell. These findings may allow the development of protease inhibitors with possible therapeutic applications. [ABSTRACT FROM AUTHOR]