RESUMEN
Drak2-deficient (Drak2-/-) mice are resistant to multiple models of autoimmunity yet effectively eliminate pathogens and tumors. Thus, DRAK2 represents a potential target to treat autoimmune diseases. However, the mechanisms by which DRAK2 contributes to autoimmunity, particularly type 1 diabetes (T1D), remain unresolved. Here, we demonstrate that resistance to T1D in non-obese diabetic (NOD) mice is due to the absence of Drak2 in T cells and requires the presence of regulatory T cells (Tregs). Contrary to previous hypotheses, we show that DRAK2 does not limit TCR signaling. Rather, DRAK2 regulates IL-2 signaling by inhibiting STAT5A phosphorylation. We further demonstrate that enhanced sensitivity to IL-2 in the absence of Drak2 augments thymic Treg development. Overall, our data indicate that DRAK2 contributes to autoimmunity in multiple ways by regulating thymic Treg development and by impacting the sensitivity of conventional T cells to Treg-mediated suppression.
Asunto(s)
Enfermedades Autoinmunes , Diabetes Mellitus Tipo 1 , Ratones , Animales , Interleucina-2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T Reguladores/metabolismo , Ratones Endogámicos NODRESUMEN
TLR activation is an important component of innate immunity but also contributes to the severity of inflammatory diseases. Cysteine cathepsins (Cat) B, L and S, which are endosomal and lysosomal proteases, participate in numerous physiological systems and are upregulated during various inflammatory disorders and cancers. Macrophages have the highest cathepsin expression and are major contributors to inflammation and tissue damage during chronic inflammatory diseases. We investigated the impact of TLR activation on macrophage Cat B, L and S activities using live-cell enzymatic assays. TLR2, TLR3 and TLR4 ligands increased intracellular activities of these cathepsins in a differential manner. TLR4-induced cytokines increased proteolytic activities without changing mRNA expression of cathepsins or their endogenous inhibitors. Neutralizing antibodies recognizing TNF-α, IL-1ß and IFN-ß differentially eliminated cathepsin upregulation. These findings indicate cytokines induced by MyD88-dependent and -independent signaling cascades regulate cathepsin activities during macrophage responses to TLR stimulation.
Asunto(s)
Catepsinas/metabolismo , Interferón beta/inmunología , Interleucina-1beta/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Catepsinas/genética , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Ligandos , Lipopolisacáridos/inmunología , Macrófagos/enzimología , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Cathepsins are endosomal/lysosomal proteases that play important roles in regulating cell physiological processes in cardiovascular, neurological, musculoskeletal, and immunological systems. Pathophysiological processes are often associated with a change in cathepsin expression and activity, leading to the possibility of using cathepsins as disease markers for diagnosis and prognosis. METHODS: We describe a new assay utilizing an argon laser flow cytometer to measure activities of cysteine cathepsins B, L, and S in live cells using cell permeable fluorogenic cresyl violet-conjugated peptides as selective substrates. Substrate concentration dependency and time kinetics studies were performed. The activity assay was combined with immunofluorescence staining to detect cell lineage-specific molecules and assess cathepsin activities in a heterogeneous cell population. RESULTS: Substrate concentrations utilized were not limiting, because MFI significantly increased in a macrophage cell line stimulated with bacterial lipopolysaccharide. Selective cathepsin inhibitors demonstrated the selectivity of substrate cleavage. Cells fixed and stored before analysis had no loss of fluorescence product. Activities of cathepsins B, L and S in splenic B cells, T cells and macrophages identified by immunofluorescence staining were analyzed. CONCLUSION: This novel technique determines cathepsin activities on a per cell basis without requiring purification of different cell types from a heterogeneous cell population.
Asunto(s)
Catepsinas/metabolismo , Colorantes Fluorescentes , Animales , Línea Celular , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Macrófagos/enzimología , Ratones , Bazo/citología , Bazo/enzimología , Coloración y EtiquetadoRESUMEN
Interleukin-3 (IL-3), which is derived from T cells and other sources, can promote the differentiation, proliferation, and migration of mast cells, basophils, and eosinophils. However, little is known about the ability of IL-3 to regulate the function of these cells in IgE-dependent and -independent allergic responses in vivo. Therefore, we sought to investigate the extent to which endogenously produced IL-3 can influence mast cell secretory function, the expression of local and systemic anaphylactic responses, and ragweed-induced eosinophilic peritonitis. We found that peritoneal mast cells from IL-3 deficient (IL-3 -/-) mice released less serotonin following challenge with low doses of anti-IgE antibody or antigen ex vivo than do cells isolated from corresponding wild-type (IL-3 +/+) mice. Both IL-3 -/- and +/+ mice expressed equivalent IgE-dependent passive cutaneous anaphylaxis responses following challenge with specific antigen and exhibited equivalent active systemic anaphylaxis responses to ovalbumin as assessed by changes in body temperature, death rates, total IgE production, and histamine release. In contrast, ragweed allergen immunization and peritoneal allergen challenge resulted in eosinophil recruitment that was greater in IL-3 -/- mice than in IL-3 +/+ mice. Our data demonstrates that IL-3 does not appear to be essential for local or systemic anaphylaxis. However, IL-3 production in vivo was found to enhance the mediator release from freshly isolated peritoneal mast cells stimulated ex vivo, and, unexpectedly, to inhibit the accumulation of eosinophils associated with a ragweed-induced allergic peritonitis model.