05-168 Sigma-AldrichAnti-TNFα Antibody, clone MPG-XT3

The impact of environmental stressors on the magnitude of specific chemokine gene expression was examined in mouse bone marrow-derived macrophages stimulated through various TLRs. Levels of TLR-stimulated CXCL1 and CXCL2 but not CXCL10 or CCL5 mRNAs were selectively enhanced (greater than 10-fold) in stressed macrophages. The amplification was also manifested for other proinflammatory cytokines, including TNF-α, IL-1α, and IL-6. Responses through TLR3 and TLR4 exhibited the greatest sensitivity, reflecting a requirement for Toll/IL-IR domain-containing adaptor-inducing IFN-β (TRIF), the adaptor protein selectively associated with these TLRs. IFN regulatory factor 3, a transcription factor that is downstream of TLR4/TRIF signaling, was not required for sensitivity to stress-induced chemokine amplification. c/EBP homologous protein and X box binding protein 1 have been reported to enhance inflammatory cytokine responses but are not required for amplification of TLR3/4-induced CXCL1 expression. Rather, receptor-interacting protein kinase 1, a kinase also linked with TLR3/4/TRIF signaling, is required and involves a stress-dependent increase in its abundance and ubiquitination. Whereas NF-κB activation is necessary for TLR-induced chemokine gene transcription, this factor does not appear to be the primary mechanistic target of environmental stress. The application of stress also enhanced chemokine expression in macrophages infiltrating the peritoneal cavity but was not observed in the resident peritoneal cells or in the liver. These findings identify novel mechanisms for modulating the magnitude and duration of selective TLR-induced chemokine and cytokine gene expression and further establish the importance of cell stress pathways in coordinating the outcomes of cellular and tissue injury.

Psoriasis is one of the most frequent skin diseases world-wide. The disease impacts enormously on affected patients and poses a huge financial burden on health care providers. Several lines of evidence suggest that the nuclear hormone receptor peroxisome proliferator activator (PPAR) beta/delta, known to regulate epithelial differentiation and wound healing, contributes to psoriasis pathogenesis. It is unclear, however, whether activation of PPARbeta/delta is sufficient to trigger psoriasis-like changes in vivo.Using immunohistochemistry, we define the distribution of PPARbeta/delta in the skin lesions of psoriasis. By expression profiling, we confirm that PPARbeta/delta is overexpressed in the vast majority of psoriasis patients. We further establish a transgenic model allowing inducible activation of PPARbeta/delta in murine epidermis mimicking its distribution in psoriasis lesions. Upon activation of PPARbeta/delta, transgenic mice sustain an inflammatory skin disease strikingly similar to psoriasis, featuring hyperproliferation of keratinocytes, dendritic cell accumulation, and endothelial activation. Development of this phenotype requires the activation of the Th17 subset of T cells, shown previously to be central to psoriasis. Moreover, gene dysregulation in the transgenic mice is highly similar to that in psoriasis. Key transcriptional programs activated in psoriasis, including IL1-related signalling and cholesterol biosynthesis, are replicated in the mouse model, suggesting that PPARbeta/delta regulates these transcriptional changes in psoriasis. Finally, we identify phosphorylation of STAT3 as a novel pathway activated by PPARbeta/delta and show that inhibition of STAT3 phosphorylation blocks disease development.Activation of PPARbeta/delta in the epidermis is sufficient to trigger inflammatory changes, immune activation, and signalling, and gene dysregulation characteristic of psoriasis.

Adhesion to novel basement membrane component BM180 in the presence of laminin-1 promotes stimulus-secretion coupling in lacrimal acinar cells [G. W. Laurie, J. D. Glass, R. A. Ogle, C. M. Stone, J. R. Sluss, and L. Chen. Am. J. Physiol. 270 (Cell Physiol. 39): C1743-C1750, 1996]. The identity of the active laminin-1 site and the possibility that other promoters of coupling are present in the acinar cell microenvironment were probed by use of different substrates, media, neutralizing antibodies and cell numbers. Regulated peroxidase secretion was unaffected by basement membrane coat concentration and was detectable at reduced levels in serum-free medium. Anti-laminin-1 antibodies, particularly against sites in the beta1 and gamma1 chains, but not alpha1 chains, partially suppressed regulated secretion, as did an anti-collagen IV antibody. Without effect were RGD peptide and antibodies against entactin, the beta1-integrin subunit, and several growth factors. Increasing cell number in serum-free medium revealed an unknown, serum-maskable, secretion-enhancing activity with a remarkable specificity for regulated secretion. Stimulus-secretion coupling, therefore, appears to be modulated by several extracellular factors whose relative contributions remain to be determined.

Dermatophagoides farinae (Der f) is one of the most common species of dust mites that induce asthma and allergic rhinitis. We have reported that Der f challenge on sensitized mice elicited a distinct type of hypersensitivity, called early-type hypersensitivity (ETH), in subcutaneous tissues and in airways. The airway ETH was accompanied by a series of inflammatory and immunological events including cytokine production, adhesion molecule expression, inflammatory cell infiltration, eosinophilia, and airway hyperreactivity. In the present study, we further defined the course of the Der-f-induced eosinophilia and examined the local cytokine gene expression and the roles of cytokines, mast-cell-derived vasoactive amines, and corticosteroids in the development of pulmonary eosinophilia. BALB/c mice were sensitized with crude extract of Der f in complete Freund's adjuvant and were intranasally challenged with Der f on day 14 after sensitization. The number of blood eosinophils, total and differential leukocyte counts in bronchoalveolar lavage (BAL) fluids, and the expression of cytokine genes in BAL cells were assessed at various time points after challenge for up to 12 days. The total number of leukocytes in the BAL fluids was increased 6 h after challenge (AC) and peaked at 72 h. The early cellular response in the BAL fluids was dominated by neutrophils which were subsequently replaced by a marked infiltration of eosinophils. The number of eosinophils in BAL fluids increased at 24 h and peaked at 72 h, making up 43% of all cells recovered by BAL. BAL eosinophils declined gradually to normal background levels around day 12. Concurrently, there was a significant reduction in the number of eosinophils in blood 24 h AC. The number of blood eosinophils increased thereafter, reached a peak at 72 h, and remained above baseline level for up to 10 days. Saline challenge did not induce eosinophilia in BAL fluids and blood of sensitized mice. Histopathological examination revealed a mixed granulocytic, monocytic pulmonary inflammation with a large number of eosinophils accumulating within the submucosa of the airways and blood vessels of sensitized mice after challenge. Der f challenge induced a sequential expression pattern of eight cytokine genes in BAL cells. The mRNA of interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha strongly expressed throughout the course of the experiment. The IL-6 mRNA expression peaked at 0.5-72 h, IL-10 at 1-6 and 48-72 h, IL-4 at 6-72 h, IL-2 at 6-96 h, IL-5 at 24-72 h, and interferon-gamma at 24-96 h. Intraperitoneal injection of sensitized mice with monoclonal antibody (mAb) to murine IL-5 (TRFK5, 300 micrograms/mouse) 1 h before challenge caused 62% suppression of eosinophils in the BAL fluids. The concomitant accumulation of neutrophils and mononuclear cells, however, was not affected by this treatment. On the other hand, intranasal administration of mAb to murine TNF-alpha (MP6-XT3, 20 micrograms/ mouse), but not IL-5, 1 h before challenge and 24 h AC significantly reduced the numbers of eosinophils, neutrophils, and lymphocytes in the BAL fluids. The intraperitoneal injection of dexamethasone (50 mg/kg) for a total of four times resulted in total inhibition of the Der-f-induced cellular responses, whereas vasoactive amine antagonists (diphenhydramine, ketanserin and cyprohepatidine) did not show any effect.