Extracellular protein disulfide isomerase regulates ligand-binding activity of αMß2 integrin and neutrophil recruitment during vascular inflammation.

ß2 integrins play a crucial role during neutrophil recruitment into the site of vascular inflammation. However, it remains unknown how ligand-binding activity of the integrin is regulated. Using fluorescence intravital microscopy in mice generated by crossing protein disulfide isomerase (PDI) floxed mice with lysozyme-Cre transgenic mice, we demonstrate that neutrophil PDI is required for neutrophil adhesion and crawling during tumor necrosis factor-α-induced vascular inflammation in vivo. Rescue experiments show that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. Studies with blocking anti-PDI antibodies and αLß2 or αMß2 null mice suggest that extracellular PDI regulates αMß2 integrin-mediated adhesive function of neutrophils during vascular inflammation. Consistently, we show that neutrophil surface PDI is important for αMß2 integrin-mediated adhesion of human neutrophils under shear and static conditions and for binding of soluble fibrinogen to activated αMß2 integrin. Confocal microscopy and biochemical studies reveal that neutrophil surface PDI interacts with αMß2 integrin in lipid rafts of stimulated neutrophils and regulates αMß2 integrin clustering, presumably by changing the redox state of the integrin. Thus, our results provide the first evidence that extracellular PDI could be a novel therapeutic target for preventing and treating inappropriate neutrophil sequestration.

Functions of metallothionein generating interleukin-10-producing regulatory CD4+ T cells potentiate suppression of collagen-induced arthritis.

Metallothionein, a cysteine-rich stress response protein that is naturally induced by a variety of immunologic stressors, has been shown to suppress autoimmune disorders through mechanisms not yet fully defined. In the present study, we examined the underlying mechanisms by which metallothionein might mediate such regulation of autoimmunity. Naïve CD4+ T cells from metallothionein-deficient mice differentiated to produce significantly less IL-10, TGF-gamma, and repressor of GATA, but more IFN-gamma and T-bet, when compared with those from wild-type mice. The levels of IL-4 and GATA-3 production were not different between the two groups of mice. Conversely, treatment with exogenous metallothionein during the priming phase drove naïve wild-type CD4+ T cells to differentiate into cells producing more IL-10 and TGF-beta, but less IFN-gamma than untreated cells. Metallothionein-primed cells were hyporesponsive to restimulation, and suppressive to T cell proliferation in an IL-10-dependent manner. Lymphocytes from metallothionein-deficient mice displayed significantly elevated levels of AP-1 and JNK activities in response to stimulation compared with those from wild-type controls. Importantly, transgenic mice overexpressing metallothionein exhibited significantly reduced susceptibility to collagen-induced arthritis and enhanced IL-10 level in the serum, relative to their nontransgenic littermates. Taken together, these data suggest that metallothionein is able to promote the generation of IL-10- and TGF-beta-producing type 1 regulatory T-like cells by downregulating JNK-dependent AP-1 activity. Thus, metallothionein may play an important role in the regulation of Th1-dependent autoimmune arthritis, and may represent both a potential target for therapeutic manipulation and a critical element in the diagnostic assessment of disease potential.

The time-dependent serial gene response to Zeocin treatment involves caspase-dependent apoptosis in HeLa cells.

Zeocin, a member of the bleomycin/phleomycin family of antibiotics, is known to bind DNA and to induce apoptosis in cervical cancer cells, but the mechanism underlying this apoptotic response is poorly understood. The present study was undertaken to elucidate time-dependent serial transcript patterns in the HeLa cervical carcinoma cell line, following treatment with Zeocin. The HeLa cell proliferation rate was found to gradually decrease following Zeocin exposure, in a time-and dose-dependent manner. RNA transcript level measurements, for time-dependent serial gene expression profiling, were determined at 0, 6, 12, 18 and 24 hr using a 0.5 k apoptosis functional microarray chip. Further statistical analysis, using a significance test at a 95% confidence level, for transcripts with a greater than 2-fold change on the array chips, identified 49 up-regulated and 57 down-regulated genes. Our gene expression profile data indicate that Zeocin treatment induces an initial release of cytochrome c, the down-regulation of Bcl-X (L), ENDOG, DAXX and MDM2, and the up-regulation of CASP and BID. This suggests that a p53-independent mitochondrial caspase cascade pathway is primarily involved in Zeocin-induced apoptosis. Such caspasedependent cytotoxic activity also implies that this cell death pathway occurs via the caspase 8 and BID genes. However, disruption of either FAS or TNFR1 signaling did not interfere with the Zeocin induced apoptotic response in our experimental system. We hypothesize that Zeocin could be active against cervical cancer cell resistance to conventional chemotherapy and postulate that Zeocin is a novel candidate for the development of new chemotherapeutic treatments of gynecological cancers.

Regulation of GRB2 and FLICE2 expression by TNF-alpha in rheumatoid synovium.

Chronic rheumatoid arthritis (RA) is characterized by the hyperplasia of synovial tissue, which results from dysregulation of proliferative and antiapoptotic signals transduced in the synovial cells by unknown mechanisms. To identify candidate factors involved in the regulation of synovial hyperplasia, the expression profile of 205 apoptosis-related genes was compared between tissues from patients with RA and osteoarthritis (OA) using a cDNA microarray. Upregulated genes in the RA synovium included TNFR2, FLICE2, and signaling molecules involved in a MAP kinase pathway (GRB2, MAPK p38). In contrast, genes encoding SARP1 and various cell cycle regulators were down-regulated in the RA synovium relative to OA. Importantly, the expression levels of GRB2 and FLICE2 genes were remarkably enhanced in RA synoviocytes but not in OA synoviocytes in response to tumor necrosis factor (TNF)-alpha treatment. Thus, these results suggest that over-expression of GRB2 and FLICE2 in RA synovium is caused by TNF-alpha inducibility differentially regulated in RA synoviocytes and provide potential pathogenic roles of these genes in the hyperplasia of the RA synovium.

Beneficial effects of rosmarinic acid on suppression of collagen induced arthritis.

OBJECTIVE: To assess the therapeutic potential of rosmarinic acid (RosA) in an inflammatory autoimmune arthritis model. METHODS: Collagen induced arthritis is established in male DBA/1 mice. Mice were administered daily with 50 mg/kg/day of RosA for 15 days from Day 21 post-immunization and inspected daily to determine the progression of arthritis. After termination of injection, affected hindpaws were subjected to histopathological analyses and immunohistochemical assays for cyclooxygenase-2 (COX-2) expression. RESULTS: Repeated administration of RosA dramatically reduced the arthritic index and number of affected paws. Histopathologic observations closely paralleled clinical data, showing that RosA treated mice retained nearly normal architecture of synovial tissues, whereas control mice exhibited severe synovitis. Synovial tissues from RosA treated mice exhibited remarkably reduced frequency of COX-2-expressing cells, compared to those from untreated mice. CONCLUSION: RosA suppressed synovitis in a murine collagen induced arthritis model; this effect may be beneficial for treatment of rheumatoid arthritis in clinical settings.