benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Autoimmune-Diseases

To assess the effect of caspase 3 inhibition, in the expression of intracellular antigens induced by apoptosis.. Skin explants of neonatal Balb/c mice were used to assess the autoantigen expression. Skin was obtained by punch biopsies, tissues were cultured in DMEM; cell death was induced by chemicals and assessed by TUNEL. The expression of La, Ro, Sm, RNP, Cajal Bodies and NuMa antigens were monitored by immunohistochemistry using autoantibodies or monoclonal antibodies against these antigens.. Chemicals used to induce cell death, successfully produced apoptosis or necrosis in more than 60% of keratinocytes, and viability was significantly decreased when it was compared with those in controls. An increased expression of all skin intracellular antigens in skin biopsies treated with chemicals, major antigenic expression was detected with anti-La and anti-Ro antibodies. The caspase 3 inhibitor DEVD-CMK significantly decreased the expression of antigens induced by chemicals.. By this result we can infer that caspase inhibitors modify apoptosis and decrease the autoantigens associated to cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Autoantigens; Autoimmune Diseases; Biopsy; Camptothecin; Caspase Inhibitors; Cells, Cultured; Cycloheximide; Cysteine Proteinase Inhibitors; Drug Evaluation, Preclinical; Hydrogen Peroxide; In Situ Nick-End Labeling; Mercuric Chloride; Mice; Mice, Inbred BALB C; Organ Culture Techniques; Skin

Neutrophils represent an important source of autoantigens for antineutrophil cytoplasmic antibody associated with vasculitis. To date, two cytoskeletal proteins, vinculin and vimentin, have been reported to be expressed on the cell surfaces of activated macrophages, platelets, and apoptotic T lymphocytes. However, such cell surface expression has never been studied in human neutrophils. As we recently demonstrated that different cytoskeletal proteins were cleaved in apoptotic neutrophils, we hypothesized that some of these were expressed on the cell surface of apoptotic neutrophils. Herein, we found that among vinculin, paxillin, gelsolin, vimentin, lamin B1, alpha-tubulin, and beta-tubulin, only the two intermediate filament (INFIL) proteins, vimentin and lamin B1, are expressed on the cell surface of 24-h aged neutrophils [spontaneous apoptosis (SA)]. By monitoring intracellular expression of vimentin and lamin B1 during SA, we found that these two proteins were cleaved and that such cleavage was reversed by the pan caspase inhibitor N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD-fmk). When neutrophil apoptosis was delayed or suppressed by lipopolysaccharide or the cytokines granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, or interleukin-4, the loss of intracellular expression of vimentin and lamin B1 was prevented. The INFIL proteins were absent from the cell surface when neutrophil apoptosis was delayed. Addition of z-VAD-fmk significantly decreased the cell surface expression of vimentin and lamin B1 during SA. This study provides the first evidence that apoptotic neutrophils express cytoskeletal proteins on their surface, opening the possibility that these cells may participate in the development of autoantibodies directed against cytoskeletal proteins, a condition frequently reported in several inflammatory diseases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Autoantibodies; Autoimmune Diseases; Caspase Inhibitors; Caspases; Cell Membrane; Cells, Cultured; Down-Regulation; Enzyme Inhibitors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Lamin Type B; Lipopolysaccharides; Membrane Proteins; Neutrophils; Vimentin

Recent studies have indicated that cells undergoing apoptosis are the source of autoantigens which drive autoimmune responses in systemic lupus erythematosus (SLE). It has been recognized for many years that in vitro stimulation of T cells with irradiated major histocompatibility complex (MHC) class II-bearing autologous cells results in T-cell proliferation with immunological specificity and memory, namely the autologous mixed lymphocyte reaction (AMLR). The nature of the major stimulants in the AMLR is still unclear. We investigated whether apoptotic fragments from irradiated cells act as antigenic stimulators for AMLR or nucleohistone-primed T cells. T-cell proliferation in the primary AMLR was significantly suppressed by the presence of a caspase inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD.fmk), indicating that apoptotic antigens released from irradiated autologous feeder cells act as stimulators of AMLR T cells. This inhibitory effect of Z-VAD was not caused by toxic effects, because the T-cell response to the mitogen phytohaemagglutinin (PHA) was not inhibited by Z-VAD. A nucleohistone preparation was shown to contain antigens that are important in the AMLR, as culture with nucleohistone (but not with thyroglobulin or hen-egg lysozyme) primed T cells to respond with secondary kinetics in a subsequent AMLR that was also suppressed by Z-VAD. Our data provide evidence that the AMLR constitutes a model for the evaluation of cellular and molecular mechanisms that may be relevant to the pathogenesis of SLE and similar autoimmune diseases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Autoantigens; Autoimmune Diseases; Autoimmunity; Caspase Inhibitors; Cell Division; Coculture Techniques; Cysteine Proteinase Inhibitors; Gamma Rays; Humans; Lupus Erythematosus, Systemic; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Models, Immunological; T-Lymphocytes

Exposure to ultraviolet radiation exacerbates the skin lesions of autoimmune diseases, and is known to induce cell surface expression of SS-A/Ro antigen on keratinocytes in vitro. Following up on recent reports on ultraviolet-B (UVB)-induced oxidative stress, we examined the role of oxidative stress in the surface expression of SS-A/Ro antigen on human keratinocytes. First, the exclusive induction by UVB irradiation of the 52-kDa protein (Ro52) but not of the 60-kDa protein (Ro60) of SS-A/Ro antigen was demonstrated by means of indirect immunofluorescence. The surface expression of Ro52 induced by UVB irradiation was concentration-dependently inhibited by N-acetyl-L-cysteine, an antioxidant. Furthermore, surface expression of Ro52 was similarly induced by diamide, a chemical oxidant. We next used Hoechst 33342 staining and the TUNEL assay to demonstrate that a low dose (20 mJ/cm(2)) of UVB did not induce apoptosis but induced the surface expression of Ro52. Moreover, zVAD-fmk, a pan-caspase inhibitor, did not inhibit UVB-induced surface expression of Ro52 even at a high dose (200 mJ/cm(2)) of UVB, which was sufficient to induce apoptosis in keratinocytes in the absence of zVAD-fmk. Taken together, we concluded that UVB-induced surface expression of Ro52 on keratinocytes is mediated by oxidative stress through a pathway other than apoptosis.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antigens, Surface; Apoptosis; Autoantigens; Autoimmune Diseases; Benzimidazoles; Blotting, Western; Caspase Inhibitors; Cells, Cultured; Diamide; Enzyme Inhibitors; Fluorescent Dyes; Free Radical Scavengers; Humans; In Situ Nick-End Labeling; Infant, Newborn; Keratinocytes; Oxidative Stress; Ribonucleoproteins; RNA, Small Cytoplasmic; Ultraviolet Rays

T cell-mediated inflammation is considered to play a key role in the pathogenic mechanisms sustaining multiple sclerosis (MS). Caspase-1, formerly designated IL-1beta-converting enzyme, is crucially involved in immune-mediated inflammation because of its pivotal role in regulating the cellular export of IL-1beta and IL-18. We studied the role of caspase-1 in experimental autoimmune encephalomyelitis (EAE), the animal model for MS. Caspase-1 is transcriptionally induced during EAE, and its levels correlate with the clinical course and transcription rate of proinflammatory cytokines such as TNF-alpha, IL-1beta, IFN-gamma, and IL-6. A reduction of EAE incidence and severity is observed in caspase-1-deficient mice, depending on the immunogenicity and on the amount of the encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide used. In caspase-1-deficient mice, reduced EAE incidence correlates with defective development of anti-MOG IFN-gamma-producing Th1 cells. Finally, pharmacological blockade of caspase-1 in Biozzi AB/H mice, immunized with spinal cord homogenate or MOG35-55 peptide, by the caspase-1-inhibitor Z-Val-Ala-dl -Asp-fluoromethylketone, significantly reduces EAE incidence in a preventive but not in a therapeutic protocol. These results indicate that caspase-1 plays an important role in the early stage of the immune-mediated inflammatory process leading to EAE, thus representing a possible therapeutic target in the acute phase of relapsing remitting MS.

Topics: Amino Acid Chloromethyl Ketones; Animals; Autoimmune Diseases; Caspase 1; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Disease Susceptibility; Encephalomyelitis, Autoimmune, Experimental; Female; Immunosuppressive Agents; Infusion Pumps, Implantable; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Sheath; Neuroprotective Agents; RNA, Messenger; Spinal Cord; Th1 Cells; Up-Regulation