Interleukin-4 (IL-4) stimulation of cells leads to the activation of multiple signaling pathways, one of which involves Stat6. We have generated Stat6-deficient mice by gene targeting in embryonic stem cells to determine the role of this transcription factor in mediating the biologic functions of IL-4. IL-4-induced increases in the cell surface expression of both MHC class II antigens and IL-4 receptor are completely abrogated, and lymphocytes from Stat6-deficient animals fail to proliferate in response to IL-4. Stat6-deficient B cells do not produce IgE following in vivo immunization with anti-IgD. In addition, Stat6-deficient T lymphocytes fail to differentiate into Th2 cells in response to either IL-4 or Il-13. These results demonstrate that, despite the existence of multiple signaling pathways activated by IL-4, Stat6 is essential for mediating responses to IL-4 lymphocytes.

By using a potential-dependent J-aggregate-forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1), we find that membrane potentials across mitochondria in a living cell can be heterogeneous. Remarkably, even within a long contiguous mitochondrion, regional heterogeneity in membrane potentials appears to be possible.

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-γ and differentiation with vitamin D3, respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.

Chemokines provide signals for activation and recruitment of effector cells into sites of inflammation, acting via specific G protein–coupled receptors. However, in vitro data demonstrating the presence of multiple ligands for a given chemokine receptor, and often multiple receptors for a given chemokine, have led to concerns of biologic redundancy. Here we show that acute cardiac allograft rejection is accompanied by progressive intragraft production of the chemokines interferon (IFN)-γ–inducible protein of 10 kD (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T cell α chemoattractant (I-TAC), and by infiltration of activated T cells bearing the corresponding chemokine receptor, CXCR3. We used three in vivo models to demonstrate a role for CXCR3 in the development of transplant rejection. First, CXCR3-deficient (CXCR3−/−) mice showed profound resistance to development of acute allograft rejection. Second, CXCR3−/− allograft recipients treated with a brief, subtherapeutic course of cyclosporin A maintained their allografts permanently and without evidence of chronic rejection. Third, CXCR+/+ mice treated with an anti-CXCR3 monoclonal antibody showed prolongation of allograft survival, even if begun after the onset of rejection. Taken in conjunction with our findings of CXCR3 expression in rejecting human cardiac allografts, we conclude that CXCR3 plays a key role in T cell activation, recruitment, and allograft destruction.

No abstract

A lymphocyte population that expresses surface markers found on T cells and natural killer (NK) cells secretes large amounts of interleukin-4 (IL-4) immediately after T cell receptor ligation. These NK-like T cells are thus thought to be important for the initiation of type 2 T helper cell (TH2) responses. CD1-deficient mice were found to lack this lymphocyte subset, but they could nevertheless mount a protypical TH2 response; after immunization with antibody to immunoglobulin D (IgD), CD1-deficient mice produced IgE. Thus, although dependent on CD1 for their development, IL-4-secreting NK-like T cells are not required for TH2 responses.

Extravascular coagulation leading to fibrin deposition accompanies many immune and inflammatory responses. Although recognized by pathologists for decades, and probably pathologic under certain conditions, the physiologic functions of extravascular coagulation remain to be fully defined. This study demonstrates that thrombin can activate macrophage adhesion and prompt interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production in vivo. Peritoneal macrophages were elicited with thioglycollate (TG) and then activated in situ, either by intraperitoneal injection of lipopolysaccharide (LPS) or by injection of antigen into mice bearing antigen-primed T cells. Others previously established that such treatments stimulate macrophage adhesion to the mesothelial lining of the peritoneal cavity. The present study demonstrates that thrombin functions in this process, as macrophage adhesion was suppressed by Refludan, a highly specific thrombin antagonist, and induced by direct peritoneal administration of purified thrombin. Although recent studies established that protease activated receptor 1 (PAR-1) mediates some of thrombin's proinflammatory activities macrophage adhesion occurred normally in PAR-1-deficient mice. However, adhesion was suppressed in fibrin(ogen)-deficient mice, suggesting that fibrin formation stimulates macrophage adhesion in vivo. This study also suggests that fibrin regulates chemokine/cytokine production in vivo, as direct injection of thrombin stimulated peritoneal accumulation of IL-6 and MCP-1 in a fibrin(ogen)-dependent manner. Given that prior studies have clearly established inflammatory roles for PAR-1, thrombin probably has pleiotropic functions during inflammation, stimulating vasodilation and mast cell degranulation via PAR-1, and activating cytokine/chemokine production and macrophage adhesion via fibrin(ogen). IntroductionVasodilation and increased vascular permeability are among the earliest signs of inflammation. These events stimulate the extravasation of inactive coagulant precursors, which become activated upon exposure to extravascular tissues. The ensuing coagulation cascade culminates with the generation of thrombin, a protease that cleaves extravasated fibrinogen, prompting its polymerization and deposition as fibrin. Accordingly, localized extravascular fibrin deposition accompanies many type 1 T helper cell (Th1)-associated responses, including autoimmune neuropathologies, 1-4 glomerulonephritis, 5,6 rheumatoid arthritis, 7-9 Crohn's disease, 10,11 allograft rejection, 12,13 delayed-type hypersensitivity, 14-19 and viral infections. 20,21 For some time, it has been appreciated that such Th1-associated coagulation has physiologic consequences, as the swelling that accompanies delayed-type hypersensitivity responses is suppressed in anticoagulated or fibrinogen-deficient subjects. [14][15][16][17][18][19] However, the full significance of immune-associated extravascular coagulation remains to be defined.Recent studies suggest that thrombin is a physiologic m...

Summary IL-10 is an anti-inflammatory mediator, important in limiting immunopathology. Its impact is influenced both by the timing and localization of its release. Here we show that NK cells rapidly express IL-10 during acute infection with the rapidly disseminating pathogens Toxoplasma gondii, Listeria monocytogenes or Yersinia pestis. Direct IL-12 signals proved necessary and sufficient for NK induction of IL-10. NK cells from T. gondii-infected mice inhibited dendritic cell release of IL-12 in an IL-10-dependent manner and NK cell depletion resulted in elevated serum IL-12. Together these data suggest an innate, negative feedback loop, in which IL-12 limits its own production by eliciting IL-10 from NK cells. In contrast to the systemic pathogens, NK cell IL-10 was not elicited by locally restricted infection with influenza virus or with a Y. pestis strain attenuated to prevent dissemination. Thus, systemic infections uniquely engage NK cells in an IL-10-mediated immunoregulatory circuit that functions to alleviate inflammation during sepsis.