Fas (CD95) induces macrophage proinflammatory chemokine production via a MyD88-dependent, caspase-independent pathway

Abstract

Activation of the prototypical death receptor, Fas (CD95), can induce both caspase-dependent cell death and production of proinflammatory chemokines, leading to neutrophil recruitment and end-organ injury. The precise mechanism(s) by which Fas up-regulates chemokine production and release, is currently unclear. We hypothesized that Fas-induced chemokine release by macrophages is dependent on the MyD88 adaptor molecule and independent of caspase activity. To test this hypothesis, we measured chemokine response to Fas activation both in RAW 264.7 cells with RNAi-attenuated MyD88 expression and in MyD88-deficient primary macrophages. We found that Fas-induced chemokine release was abrogated in the absence of MyD88. In vivo, MyD88(-/-) mice had impaired CXCL1/KC release and polymorphonuclear cell recruitment in response to intratracheal treatment with the Fas-activating monoclonal antibody, Jo-2. Furthermore, Fas-induced chemokine release was not dependent on either IL-1 receptor signaling or on caspase activity. We conclude that MyD88 plays an integral role in Fas-induced macrophage-mediated inflammation.

Fig 1

Effect of Fas activation by either mAb Jo-2 or rhFasL in the RAW 264.7 murine macrophage-like cell line. Alamar Blue assay determination of cell viability normalized to untreated cells (A) and production and release of CXCL2/MIP-2 (B). Data are presented as mean ± SEM of four independent experiments. * p ≤ 0.05.

Fig 2

Disruption of MyD88-dependent signaling and Fas-induced chemokine expression. Transfection of RAW 264.7 cells with MyD88-specific siRNA resulted in attenuation of MyD88 protein by Western blot (A) and in reduced CXCL2/MIP-2 after mAb Jo-2 treatment (B). Data are presented as mean ± SEM of three independent experiments. * p ≤ 0.05; † p ≤ 0.01.

Fig 3

Expression of CXCL1/KC (A) and CXCL2/MIP-2 (B) in peritoneal macrophages derived from wild-type and MyD88^−/− mice. Expression of CXCL1/KC (C) in bone marrow-derived macrophages (BMDM) from wild-type and MyD88^−/− mice. Data are presented as mean ± SEM of three independent experiments, n=3/group. * p ≤ 0.05; † p ≤ 0.01.

Fig 4

Expression of CXCL2/MIP-2 in BMDM following stimulation with nothing, isotype IgG control Ab, mAb Jo-2, poly(I:C) (MyD88-independent TLR-3 agonist), or Pam₃CSK (MyD88-dependent TLR-2 agonist). Data are presented as mean ± SEM of four independent experiments. Note that data are presented on a log scale to accommodate the large range of CXCL2/MIP-2 concentrations. Comparisons are made between BMDM from wild-type and MyD88^−/− mice for each treatment. † p ≤ 0.01

Fig 5

Bronchoalveolar lavage fluid concentrations of CXCL1/KC (A) and CXCL2/MIP-2 (B) and polymorphonuclear (C) and total leukocyte (D) cell counts from wild-type and MyD88^−/− mice eighteen hours after intratracheal treatment with either mAb Jo-2 (n=6/genotype) or isotype IgG (n=6/genotype) antibody. * p ≤ 0.05; † p ≤ 0.01.

Fig 6

Effect of IL-1 receptor blockade on Fas-induced production of CXCL2/MIP-2. (A) RAW 264.7 cells untreated or treated for 18-hr with either mAb Jo-2 or rhFasL in the presence or absence of IL-1 receptor antagonist (IL-1ra). n=3/group. (B) BMDM isolated from either wild-type or MyD88^−/− mice and treated for 18-hr with mAb Jo-2 in the presence or absence of IL-1ra. n=4/group. Data are presented as mean ± SEM of three independent experiments. * p ≤ 0.05 compared with untreated cells.

Fig 7

Role of caspase function in Fas-induced chemokine expression. Caspase activity was inhibited in bone marrow derived macrophages by zVAD pre-treatment (A) or in RAW 264.7 cells by expression of CrmA (B), and chemokines were measured eighteen hours after stimulation with either mAb Jo-2 or rhFasL. Data are presented as mean ± SEM of three independent experiments. * p ≤ 0.05; † p ≤ 0.01.