Attenuation of leukocyte sequestration by selective blockade of PECAM-1 or VCAM-1 in murine endotoxemia

Background: Molecular mechanisms regulating leukocyte sequestration into the tissue during endotoxemia and/or sepsis are still poorly understood. This in vivo study investigates the biological role of murine PECAM-1 and VCAM-1 for leukocyte sequestration into the lung, liver and striated skin muscle. Methods: Male BALB/c mice were injected intravenously with murine PECAM-1 IgG chimera or monoclonal antibody (mAb) to VCAM-1 ( 3 mg/kg body weight); controls received equivalent doses of IgG2a ( n = 6 per group). Fifteen minutes thereafter, 2 mg/kg body weight of Salmonella abortus equi endotoxin was injected intravenously. At 24 h after the endotoxin challenge, lungs, livers and striated muscle of skin were analyzed for their myeloperoxidase activity. To monitor intravital leukocyte-endothelial cell interactions, fluorescence videomicroscopy was performed in the skin fold chamber model of the BALB/c mouse at 3, 8 and 24 h after injection of endotoxin. Results: Myeloperoxidase activity at 24 h after the endotoxin challenge in lungs (12,171 +/- 2,357 mU/g tissue), livers ( 2,204 +/- 238 mU/g) and striated muscle of the skin ( 1,161 +/- 110 mU/g) was significantly reduced in both treatment groups as compared to controls, with strongest attenuation in the PECAM-1 IgG treatment group. Arteriolar leukocyte sticking at 3 h after endotoxin (230 +/- 46 cells x mm(-2)) was significantly reduced in both treatment groups. Leukocyte sticking in postcapillary venules at 8 h after endotoxin ( 343 +/- 69 cells/mm(2)) was found reduced only in the VCAM-1-mAb-treated animals ( 215 +/- 53 cells/mm(2)), while it was enhanced in animals treated with PECAM-1 IgG ( 572 +/- 126 cells/mm(2)). Conclusion: These data show that both PECAM-1 and VCAM-1 are involved in endotoxin-induced leukocyte sequestration in the lung, liver and muscle, presumably through interference with arteriolar and/or venular leukocyte sticking. Copyright (C) 2004 S. Karger AG, Basel

Signaling via interleukin-4, receptor alpha chain is required for successful vaccination against schistosomiasis in BALB/c mice

Radiation-attenuated (RA) schistosome larvae are potent stimulators of innate immune responses at the skin site of exposure (pinna) that are likely to be important factors in the development of Th1-mediated protective immunity. In addition to causing an influx of neutrophils, macrophages, and dendritic cells (DCs) into the dermis, RA larvae induced a cascade of chemokine and cytokine secretion following in vitro culture of pinna biopsy samples. While macrophage inflammatory protein 1 and interleukin-1 (IL-1) were produced transiently within the first few days, the Th1-promoting cytokines IL-12 and IL-18 were secreted at high levels until at least day 14. Assay of C3H/HeJ mice confirmed that IL-12 secretion was not due to lipopolysaccharide contaminants binding Toll-like receptor 4. Significantly, IL-12 p40 secretion was sustained in pinnae from vaccinated mice but not in those from nonprotected infected mice. In contrast, IL-10 was produced from both vaccinated and infected mice. This cytokine regulates IL-12-associated dermal inflammation, since in vaccinated IL-10/ mice, pinna thickness was greatly increased concurrent with elevated levels of IL-12 p40. A significant number of IL-12 p40 cells were detected as emigrants from in vitro-cultured pinnae, and most were within a population of rare large granular cells that were Ia, consistent with their being antigen-presenting cells. Labeling of IL-12 cells for CD11c, CD205, CD8, CD11b, and F4/80 indicated that the majority were myeloid DCs, although a proportion were CD11c F4/80, suggesting that macrophages were an additional source of IL-12 in the skin

The proteasome inhibitor bortezomib controls indoleamine 2,3-dioxygenase 1 breakdown and restores immune regulation in autoimmune diabetes

Bortezomib (BTZ) is a first-in-class proteasome inhibitor approved for the therapy of multiple myeloma that also displays unique regulatory activities on immune cells. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme exerting potent immunoregulatory effects when expressed in dendritic cells (DCs), the most potent antigen-presenting cells capable of promoting either immunity or tolerance. We previously demonstrated that, in inflammatory conditions, IDO1 is subjected to proteasomal degradation in DCs, turning these cells from immunoregulatory to immunostimulatory. In non-obese diabetic (NOD) mice, an experimental model of autoimmune diabetes, we also identified an IDO1 defect such that the DCs do not develop tolerance toward pancreatic islet autoantigens. We found that BTZ rescues IDO1 protein expression in vitro in a particular subset of DCs, i.e., plasmacytoid DCs (pDCs) from NOD mice. When administered in vivo to prediabetic mice, the drug prevented diabetes onset through IDO1- and pDC-dependent mechanisms. Although the drug showed no therapeutic activity when administered alone to overtly diabetic mice, its combination with otherwise suboptimal dosages of autoimmune-preventive anti-CD3 antibody resulted in disease reversal in 70% diabetic mice, a therapeutic effect similar to that afforded by full-dosage anti-CD3. Thus, our data indicate a potential for BTZ in the immunotherapy of autoimmune diabetes and further underline the importance of IDO1-mediated immune regulation in such disease

Proteasomal protein degradation: adaptation of cellular proteolysis with impact on virus-and cytokine-mediated damage of heart tissue during myocarditis

Viral myocarditis is an inflammation of the heart muscle triggered by direct virus-induced cytolysis and immune response mechanisms with most severe consequences during early childhood. Acute and long-term manifestation of damaged heart tissue and disturbances of cardiac performance involve virus-triggered adverse activation of the immune response and both immunopathology, as well as, autoimmunity account for such immune-destructive processes. It is a matter of ongoing debate to what extent subclinical virus infection contributes to the debilitating sequela of the acute disease. In this review, we conceptualize the many functions of the proteasome in viral myocarditis and discuss the adaptation of this multi-catalytic protease complex together with its implications on the course of disease. Inhibition of proteasome function is already highly relevant as a strategy in treating various malignancies. However, cardiotoxicity and immune-related adverse effects have proven significant hurdles, representative of the target's wide-ranging functions. Thus, we further discuss the molecular details of proteasome-mediated activity of the immune response for virus-mediated inflammatory heart disease. We summarize how the spatiotemporal flexibility of the proteasome might be tackled for therapeutic purposes aiming to mitigate virus-mediated adverse activation of the immune response in the heart

Argyrin B a non-competitive inhibitor of the human immunoproteasome exhibiting preference for β1i

Inhibitors of the proteasome have found broad therapeutic applications however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non-competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the β5i and β1i sites of the immunoproteasome over the β5c and β1c sites of the constitutive proteasome with nearly 20-fold selective inhibition of β1i over the homologous β1c. Molecular modelling attributes the β1i over β1c selectivity to the small hydrophobic S1 pocket of β1i and β5i over β5c to site-specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity

Immunoproteasome LMP2 60HH Variant Alters MBP Epitope Generation and Reduces the Risk to Develop Multiple Sclerosis in Italian Female Population

Background: Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis. Methodology/Principal Findings: Immunoproteasomes and PA28-ab regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP111\u2013119. Conclusion/Significance: The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLAA* 02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis

The role of the proteasome in the generation of MHC class I ligands and immune responses

The ubiquitin–proteasome system (UPS) degrades intracellular proteins into peptide fragments that can be presented by major histocompatibility complex (MHC) class I molecules. While the UPS is functional in all mammalian cells, its subunit composition differs depending on cell type and stimuli received. Thus, cells of the hematopoietic lineage and cells exposed to (pro)inflammatory cytokines express three proteasome immunosubunits, which form the catalytic centers of immunoproteasomes, and the proteasome activator PA28. Cortical thymic epithelial cells express a thymus-specific proteasome subunit that induces the assembly of thymoproteasomes. We here review new developments regarding the role of these different proteasome components in MHC class I antigen processing, T cell repertoire selection and CD8 T cell responses. We further discuss recently discovered functions of proteasomes in peptide splicing, lymphocyte survival and the regulation of cytokine production and inflammatory responses