Abstract
Apoptotic cells are rapidly engulfed by adjacent tissue cells or macrophages before they can release pro-inflammatory/proimmunogenic intracellular contents. In addition, recognition of the apoptotic cells is actively anti-inflammatory and anti-immunogenic with generation of anti-inflammatory mediators such as transforming growth factor-beta (TGF-beta) and anti-inflammatory eicosanoids. Here, we have investigated the role played by the induction of TGF-beta in the coordinate expression of anti-inflammatory eicosanoids or peroxisome proliferator-activated receptor-gamma and in the suppression of pro-inflammatory lipid mediators and nitric oxide (NO). By use of a dominant negative TGFbetaII receptor, TGF-beta signaling was blocked, and its participation in the consequences of apoptotic cell stimulation was determined. The induction of TGF-beta itself could be attributed to exposed phosphatidylserine on the apoptotic cells, which therefore appears to drive the balanced inflammatory mediator responses. Arachidonic acid release, COX-2, and prostaglandin synthase expression were shown to be significantly dependent on the TGF-beta production. On the other hand, a requirement for TGF-beta was also shown in the inhibition of thromboxane synthase and thromboxanes, of 5-lipoxygenase and sulfidopeptide leukotrienes, as well as of inducible nitric-oxide synthase and NO. TGF-beta-dependent induction of arginase was also found and would further limit the NO generation. Finally, apoptotic cells stimulated production of 15-lipoxygenase and 15-hydroxyeicosatetraenoic acid, a potentially anti-inflammatory pathway acting through peroxisome proliferator-activated receptor-gamma, and lipoxin A(4) production, which were also up-regulated by a TGF-beta-dependent pathway in this system. These results strongly suggest that the apoptotic cell inhibition of pro-inflammatory mediator production is pleiotropic and significantly dependent on the stimulation of TGF-beta production.
Highlights
The interaction and recognition are triggered by surface changes on the apoptotic cells
The stimulation of TGF- production by apoptotic cells in this system was blocked by 30 min and incubated with viable or apoptotic Jurkat cells for 18 h. *, significantly different from controls. #, significantly different from apoJ alone
We show that the effect of the apoptotic cells is to drive a complex coordinated inhibition of potentially inflammatory mediators along with induction of potentially anti-inflammatory molecules in macrophages that are orchestrated by the production of TGF-
Summary
Antibodies and Reagents—TGF- was purchased from R&D Systems. Lipopolysaccharide (LPS, Escherichia coli 0111:B4) was from List Biological Laboratories, Inc. MAb217 is an IgM monoclonal antibody that was originally raised against PS-recognizing macrophages It was obtained from concentrated hybridoma supernatants. Cell Culture, Stimulation and Measurement of Pro-inflammatory Mediators by ELISA—Murine peritoneal macrophages were obtained from BALB/c mice 4 days after intraperitoneal injection of 1 ml of thioglycollate. The cells were cultured in serum-free DMEM in the absence or presence of LPS, IFN-␥, factor Va, or cyclosporin A for 18 h with 3 ϫ 106 apoptotic Jurkat cells (apoJ), viable Jurkat cells (ViableJ), 50 g/ml mAb217, 50 g/ml control isotype IgM, or 100 M liposomes (containing 30:70 molar ratios of PS:PC or PC alone). The cells were washed and incubated in serum-free DMEM containing 0.1% human serum albumin and stimulated with LPS or apoJ or ViableJ or mAb217 or isotype control IgM. All data were analyzed using JMP statistical software (version 5; SAS Institute) for the Macintosh computer
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