The intracellular molecular events involved in the ␤-cell death process are complex but poorly understood. Cytokines, e.g., interleukin (IL)-1␤, may play a crucial role in inducing this process. Protein synthesis is necessary for the deleterious effect of IL-1, and induction of both protective and deleterious proteins has been described. To characterize the rather complex pattern of islet protein expression in rat islets in response to IL-1, we have attempted to identify proteins of altered expression level after IL-1 exposure by 2D gel electrophoresis and mass spectrometry. Of 105 significantly changed (i.e., up-or downregulated or de novo-induced) protein spots, we obtained positive protein identification for 60 protein spots. The 60 identifications corresponded to 57 different proteins. Of these, 10 proteins were present in two to four spots, suggesting that posttranslatory modifications had occurred. In addition, 11 spots contained more than one protein. The proteins could be classified according to their function into the following groups: 1) energy transduction; 2) glycolytic pathway; 3) protein synthesis, chaperones, and protein folding; and 4) signal transduction, regulation, differentiation, and apoptosis. In conclusion, valuable information about the molecular mechanisms involved in cytokine-mediated ␤-cell destruction was obtained by this approach. Protein synthesis inhibitors (e.g., cycloheximide) effectively protect IL-1-exposed islets from destruction (12). Hence, protein synthesis is necessary for the deleterious effect of IL-1. Previous studies have shown that IL-1 induces the synthesis of members of the heat shock protein (HSP) family, like heme oxygenase (13) and HSPs 70 and 90 (14,15), and hyperexpression of HSPs in islets is partially protective against cytokine-induced ␤-cell destruction (16). Furthermore, it has been reported that IL-1 may induce the synthesis of unknown proteins with molecular weights of 45,50, 75, 85, 95, and 120 kDa (17). It has previously been shown that rat islets exposed to IL-1 release NO into the culture media (18). iNOS has been cloned from islets (19) in which it has been shown to be inducible in ␤-cells only (20). We have further shown that IL-1 also upregulates IL-1 converting enzyme mRNA transcription (21) in rat islets. Also, SOD is shown to be upregulated in islets by cytokines (15,22).Based on ␤-cell-selective toxic effects, we hypothesized that IL-1 induces a rather complex pattern of both protective and deleterious events and mechanisms in islets cells, and that in ␤-cells the deleterious events seem to prevail (23). We further suggested that this might be reflected at the level of islet protein expression (24). To examine this hypothesis, we used 2D gel electrophoresis to produce a database of rat islet proteins containing about 2,200 protein spots characterized by molecular weight and isoelectric point (pI). The data presented here provide the first global assessment of the IL-1-mediated ␤-cell-damaging processes at the protein level. We could demonstra...

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Background: Successful allergen–specific immunotherapy is achieved with progressively increasing doses of allergen or allergoid. In order to gain further insight into the mechanism of action of allergoids several in vitro investigations were conducted. Methods: Peripheral blood mononuclear cells (PBMC) from grass pollen allergic and nonallergic subjects were stimulated with either grass pollen extract or allergoid and the proliferation and cytokine production (IL–5, IFN–γ) were measured. Similar investigations were performed with Phl p 5–specific T cell lines (TCL) and clones (TCC). Dendritic cells and PBMC were compared in terms of their relative efficacies as antigen–presenting cells. Results: Both allergen and allergoid induced proliferation and Th2 and Th1 cytokine synthesis by PBMC of allergic subjects, whereas PBMC of nonallergic subjects did not produce IL–5. The maximum level of IL–5 was obtained with a lower concentration than was necessary for maximal IFN–γ production. Higher stimulation doses of allergen and allergoid shifted the cytokine profiles towards a Th1 phenotype. TCL and TCC clearly showed reactivity with both allergen and allergoid when using autologous PBMC for antigen presentation, but compared with the native allergen the reactivity of the allergoid was reduced with most of the TCC. Using dendritic cells for antigen presentation a pronounced increase of stimulation of the TCC especially for the allergoids becomes obvious. Conclusion: In common with grass pollen allergen the corresponding allergoids possess a strong allergen–specific T cell–stimulating capacity. However, the degree of T cell stimulation by the allergoid seems to be dependent on the type of the antigen–presenting cell. Both, allergen and allergoid, can modulate T cell responses in a dose–dependent manner.

At PFC concentrations comparable with those in blood during liquid ventilation, PFC liquids did not induce variables associated with inflammation. In the presence of high PFC concentrations, simulating the condition in which bronchoalveolar cells are exposed to PFC, monocytes may be induced by PFB to produce reactive oxygen species, and blood leukocytes induced by PFB to express CD11b and by PFD to secrete interleukin-8; the presence of either PFC attenuated tumor necrosis factor-alpha production after lipopolysaccharide stimulation.

Increased production of oxidants subsequent to phagocyte stimulation has been associated with tissue damage in lung inflammatory disorders. The overall oxidative burden of the lung may vary with inflammatory cell composition.Flow cytometry using three different dyes, dihydroethidium (DHE), dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine 123 (DHR), all compounds that by interaction with oxidants are transformed to fluorescent products, was used to examine the production of intracellular oxidants in alveolar macrophages (AMs), including size-defined subpopulations, monocytes (Ms) and polymorphonuclear neutrophils (PMNs) during in vitro incubation in the presence or absence of phorbol myristate acetate (PMA).PMA stimulation led to slightly increased (two-fold) (p<0.05) DHE-induced fluorescence in AMs, whereas it was greatly increased in Ms and PMNs (13-fold and 113-fold, respectively). The levels of DCFH-DA-and DHR-induced fluorescence were significantly (p<0.05) increased (four-fold and 110-fold, respectively) by PMA stimulation of PMNs, but not of AMs and Ms. Significant differences (p<0.05) in the levels of DHE-and DCFH-DA-induced fluorescence in small and large AMs were also demonstrated.The results show that the potential to increase the generation of various oxidants upon stimulation was: PMNs> Ms>AMs, suggesting that the total oxidative burden of the lungs is dependent on the type of inflammatory cells present, as well as on their state of activation.

To elucidate the innate immune responses to group A streptococci (GAS) important in the pathophysiology of sepsis, flow cytometric techniques were applied to study the effects of live and heat-inactivated GAS, including their particulate and soluble components, on the expression of leukocyte adhesion molecules CD11b (Mac-1) and CD62L (L-selectin), and leukocyte production of reactive oxygen species (ROS) in human whole blood. GAS caused marked time- and concentration-dependent increases in CD11b and ROS, while CD62L was downregulated. Live and heat-inactivated GAS induced similar changes in leukocyte adhesion molecules, whereas ROS production induced by heat-inactivated GAS (and its particulate fraction) was 4 (2.5)-fold higher than with live GAS. Leukocyte nitric oxide production (24 h) was not enhanced. Although GAS proved a more potent inducer of ROS production, leukocyte responses to GAS were similar to those reported for lipolysaccharides, indicating that Gram-positive and Gram-negative bacteria activate common pathways in the inflammatory response. High ROS production may contribute to tissue damage caused by GAS.

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