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Originally published online as doi:10.1189/jlb.1006652 on June 18, 2007

Published online before print June 18, 2007
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(Journal of Leukocyte Biology. 2007;82:721-728.)
© 2007 by Society for Leukocyte Biology

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

William A. Altemeier*,1,2, Xiaodong Zhu*,2, William R. Berrington*, John M. Harlan* and W. Conrad Liles*,{dagger}

* Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA; and
{dagger} Department of Medicine, Toronto General Research Institute, McLaughlin-Rotman Centre for Global Health, McLaughlin Centre for Molecular Medicine, University of Toronto, Toronto, Ontario, Canada

1 Correspondence: Box 356522, University of Washington, 1959 NE Pacific St., Seattle, WA 98105-6522, USA. E-mail: billa{at}u.washington.edu


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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.

Key Words: inflammation • apoptosis • lung injury • neutrophil


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INTRODUCTION
 
Fas (CD95), a member of the tumor necrosis factor receptor super family, is expressed on a wide variety of cell types and is activated by binding to Fas ligand (FasL or CD178) [1 , 2 ]. Fas activation prototypically depends on the adaptor molecule, Fas-associated protein with death domain (FADD), to initiate caspase-8 proteolytic autocleavage, leading ultimately to activation of the effecter caspase, caspase-3, and cell death. Fas activation is implicated in the pathogenesis of a variety of disease conditions involving multiple organs, including the lungs [3 4 5 ], heart [6 7 8 ], and kidneys [9 10 11 12 ]. These findings have led to intense interest in pursuing anti-Fas and anti-apoptosis treatments as possible therapeutic interventions in clinical diseases associated with tissue injury.

Although Fas activation is often associated with caspase-mediated cell death, increasing evidence implicates Fas in chemokine expression and neutrophil recruitment as well. Mice treated with intratracheal Jo-2, a Fas-activating monoclonal antibody (mAb), up-regulate expression of multiple proinflammatory chemokines, leading to lung neutrophilic infiltration [5 , 13 ]. Neutrophilic peritonitis was also induced in mice injected with Fas ligand-expressing tumor cells [14 ]. The exact mechanism by which Fas induces expression of proinflammatory chemokines is incompletely understood; however, it has been previously shown that Fas activation can induce nuclear translocation of the proinflammatory transcription factor, nuclear factor-{kappa}B (NF-{kappa}B) [15 , 16 ]. Furthermore, resident macrophages are a likely source of Fas-induced chemokine expression in vivo, as macrophages in vitro are resistant to Fas-induced cell death and secrete chemokines following Fas activation [16 17 18 ].

We hypothesized that Fas-induced chemokine expression by macrophages is dependent on the MyD88 adaptor protein. MyD88 is a widely expressed protein, which is required for initiation of intracellular signaling following ligand binding by the IL-1 receptor family and by all Toll-like receptors (TLR) except TLR-3 [19 , 20 ]. Furthermore, MyD88 contains a death domain that enables intracellular association with FADD under certain circumstances [21 , 22 ]. To test this hypothesis, chemokine response to the Fas-activating mAb, Jo-2, was measured in the presence or absence of functional MyD88 in both isolated macrophage cell culture and in intact mouse lungs in vivo. Chemokine response to Fas activation was also measured in the presence of IL-1 receptor blockade and caspase inhibition to determine whether MyD88-dependent signaling required IL-1 signaling and/or caspase activity.


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MATERIALS AND METHODS
 
Reagents
mAb Jo-2 and isotype IgG control antibody were obtained from BD PharMingen (San Diego, CA, USA). Recombinant human Fas ligand (rhFasL – the extracellular domain fused at the N terminus to a linker peptide and a Flag tag) and a cross-linking enhancer were obtained from Alexis Biochemicals (prod# ALX-850-014-KI02, San Diego, CA, USA). Lipopolysaccharide (LPS) was obtained from List Biological Laboratories (Campbell, CA, USA) and Pam3CSK and poly(I:C) were from Invivogen Corp. (San Diego, CA, USA) IL-1 receptor antagonist (IL-1ra), recombinant murine TNF-{alpha}, and antibodies for CXCL1 (KC) and CXCL2 (MIP-2) were obtained from R and D Systems (Minneapolis, MN, USA). The pan-caspase inhibitor, Z-Val-Ala-Asp(OMe)-FMK (zVAD) was obtained from MP Biomedicals, Inc. (Aurora, OH, USA). Rabbit anti-mouse MyD88 polyclonal antibody was purchased from ProSci Inc. (Poway, CA, USA). Superscript first-strand cDNA synthesis kit and cell culture media were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). MyD88 primer-probe set and master mix for qPCR measurement of MyD88 were purchased from Applied Biosystems (Foster City, CA, USA). Enhanced chemiluminescent reagents for Western blot detection were from Cell Signaling Technology (Danvers, MA, USA). Alamar Blue reagent was from Trek Diagnostic Systems (Cleveland, OH, USA).

Mice and cell culture
Breeding pairs of MyD88–/– mice on a C57Bl/6 background (>10 backcrosses) were generously provided by Dr. Thomas Hawn (University of Washington). C57BL/6 control mice were purchased from Jackson Laboratories (Hollister, CA, USA). The University of Washington institutional animal care and use committee approved all experiments.

Bone marrow-derived macrophages (BMDM) were prepared from femora of wild-type and MyD88–/– mice. After euthanasia by cervical dislocation under isoflurane anesthesia, femora were aseptically removed and dissected free of adhering tissues. Marrow cavities were flushed by injection of RPMI-1640 medium. Collected bone marrow cells were incubated in a 100 x 15 mm Petri dish in a medium mixture containing 60% RPMI-1640 complete medium (supplemented with 20% heat-inactivated fetal bovine serum ({Delta}FBS), 1% L-glutamine, 1% HEPES, 1% Pen-Strep) and 40% L929 cell-conditioned medium as a source of M-CSF for 7 days in 5% CO2 at 37°C as described previously [23 ].

Thioglycollate-elicited peritoneal macrophages were obtained from C57Bl/6 and MyD88–/– mice by peritoneal washing 5 days after intraperitoneal injection of 1 ml of 3% Brewer thioglycollate broth. The cells were washed twice with Dulbecco’s modified Eagle's medium (DMEM) and resuspended in DMEM supplemented with 10% {Delta}FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, 1.5 g/l sodium carbonate, 4.5 g/l D-(+)-glucose and 1% Pen-Strep and incubated at 5% CO2, 37°C overnight. Adherent cells were used the next day in experiments.

The mouse macrophage cell line, RAW 264.7, was obtained from American Type Culture Collection (Manassas, VA, USA). Cells were grown and maintained in DMEM supplemented with 10% {Delta}FBS, penicillin 100 U/ml, and streptomycin 100 µg/ml.

RNA interference (RNAi) in RAW 264.7 cells
Pre-designed small interfering RNA (siRNA) oligonucleotides targeting endogenous MyD88 were purchased from Ambion (Austin, TX, USA). The MyD88 target sequences used were A, sense GCAUUUUAAAGCAACCUGGtt, antisense CCAGGUUGCUUUAAAAUGCtc; and B, sense CGUUCUCUACCAUAGAGGCtt, antisense GCCUCUAUGGUAGAGAACGtg. The siRNA duplexes were transfected using lipofectamine 2000 (Invitrogen) into RAW 264.7 cells following the manufacturer’s protocol. Briefly, cells were plated at 2.5 x 105 cell/well in a 6-well plate. After 24 h, cells were treated with 100 nM of MyD88-siRNAs in a transfection mixture containing lipofectamine 2000 12 µl/well and 1 ml DMEM without serum and penicillin/streptomycin overnight in 5% CO2, at 37°C. The following day, the transfection mixture was replaced with fresh growth medium. Transfected cells were harvested at 48 h after transfection to assess MyD88 gene knockdown by quantitative real-time PCR and at 72 h to assess MyD88 protein level by Western blot analysis and for use in subsequent Fas-stimulation experiments. siRNA-negative lipofectamine treatment and transfection with siRNA targeting GAPDH (Silencer GAPDH siRNA kit, Ambion) were used to control for nonspecific effects.

Cloning and stable expression of CrmA cDNA construct
The construct containing cDNA encoding for cytokine response modifier A (CrmA) on the pBM-IRES-Puro vector was provided by Dr. Daniel Bowen-Pope (University of Washington) [24 ]. Retrovirus was prepared from the Phoenix amphotropic packaging cell line (ATCC) transfected with 24 µg of the expression plasmid by calcium phosphate precipitation [25 ]. Retroviral supernatants were collected 48 h after transfection and filtered through a 0.45 µm sterile syringe filter (Pall Corporation, Ann Arbor, MI, USA). For infection, RAW 264.7 cells of 1 x 106/well were plated in a 6-well culture plate and incubated with retroviral supernatant and DMEM/10%{Delta}FBS mixture supplemented with 15 µg/ml polybrene and 10 mM HEPES. Cells were centrifuged at 800 g and 35°C for 2 h and then incubated overnight in 5% CO2, at 37°C. The following day, retroviral supernatant mixture was replaced with growth medium. Cells were analyzed and sorted by puromycin selection (10 µg/ml for 48 h). Stably transduced cells were used for subsequent experiments. Protein expression of transfected cDNA was confirmed by Western blot analysis.

Fas activation
In a 96-well cell culture plate, bone marrow-derived or peritoneal macrophages were plated at 1 x 105 cells/200 µl/well in RPMI-1640 plus10% {Delta}FBS, and RAW 264.7 cells were plated at 5 x 104 cells/200 µl/well in DMEM-10% {Delta}FBS. After 24 h, unattached cells were washed out and 200 µl/well of fresh medium + 2% {Delta}FBS was added. Cells were stimulated with mAb Jo2 (1 µg/ml), isotype control antibody (1 µg/ml), rhFasL (10 ng/ml) + enhancer (0.5–1 µg/ml), or nothing (control). In a subset of experiments, cells were stimulated with ligands that signal via MyD88-dependent pathways (Pam3CSK 100 ng/ml, LPS 50 ng/ml) or by MyD88-independent pathways (poly(I:C) 25 µg/ml, TNF-{alpha} 10 ng/ml) as controls to confirm the presence or absence of functioning MyD88 protein. In certain experiments, cells were incubated with either zVAD or DMSO control 30 min prior to Fas stimulation or with IL-1ra 60 min prior to Fas stimulation. After 18 h, culture supernatant was collected for measurement of CXCL1/KC, and CXCL2/MIP-2 by ELISA.

Cell viability following Fas activation was measured by Alamar Blue assay according to the manufacturer’s instructions. Briefly, after medium was removed for chemokine measurement, freshly prepared phenol-free medium containing 5% FBS and 10% Alamar Blue reagent was added, and the cells were incubated for 4 h at 37°C and 5% CO2. Cell viability was determined by absorbance at 570 nm corrected for absorbance at 600 nm.

For in vivo experiments, mice were briefly anesthetized with isoflurane, and either mAb Jo-2 or isotype control antibody (2.5 µg/g body weight) was administered by oropharyngeal aspiration, as described previously [26 ]. Mice were returned to their cage to recover with free access to food and water. After 18 h, mice were anesthetized with isoflurane and killed by cervical dislocation. Both lungs were lavaged with three 1-ml aliquots of PBS containing 0.6 mM EDTA at 37°C. Aliquots were pooled. Total and differential cell counts were determined with a hemacytometer and by a cytospin prep with a modified Wright stain, respectively. The remaining fluid was spun at 1500 g and 4°C for 10-min, and the supernatant was stored in aliquots for later protein determination by Bradford assay and for chemokine measurement by ELISA.

Statistical analysis
All data are presented as mean values ± SEM. Comparisons between two groups were made by paired t test. Comparison among multiple groups was done by ANOVA with post-hoc comparisons using the Tukey HSD test. Statistical significance was identified at a P value ≤ 0.05.


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RESULTS
 
Fas activation in macrophages results in chemokine expression but not cell death
To assess the response of the murine macrophage-like cell line, RAW 264.7, to Fas activation, cells were treated with control IgG antibody, the Fas-activating monoclonal antibody, mAb Jo-2, or rhFasL for 18 h. Cell viability was assessed by the Alamar Blue assay according to the manufacturer’s instructions and normalized to untreated cells. CXCL1/KC and CXCL2/MIP-2 were measured in cell culture supernatants. Fas activation by either mAb Jo-2 or rhFasL did not significantly affect cell viability (Fig. 1A ). Consistent with previous reports, Fas activation with mAb Jo-2 resulted in production and release of CXCL2/MIP-2 (Fig. 1B) . This was not the result of a nonspecific IgG response, as supernatant collected from cells treated with control IgG antibody did not have elevated CXCL2/MIP-2 concentration compared with supernatant from untreated cells.


Figure 1
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  		Figure 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 means ± SEM of four independent experiments. *P ≤ 0.05.

A similar response was observed with Fas activation by treatment with rhFasL. Fas activation of RAW 264.7 cells produced minimal CXCL1/KC following stimulation with either Jo-2 or rhFasL. This attenuated CXCL1/KC response was not limited to Fas activation but was also observed following stimulation with lipopolysaccharide (data not shown). These results confirm in a murine macrophage cell line the previously reported finding that Fas activation of human macrophages results in chemokine release but not cell death [16 , 18 ].

MyD88 is required for Fas-induced chemokine expression by macrophages
To evaluate the role of MyD88 in Fas-induced chemokine expression, native MyD88 expression was attenuated in RAW 264.7 cells by RNAi (Fig. 2A ). Compared with control cells treated with transfection reagent alone, MyD88 knockdown resulted in reduced CXCL2/MIP-2 protein expression at baseline (54.2±22.5 vs. 6.8±2.1 pg/ml, P=0.03) and after Fas activation by mAb Jo-2 (1180±295 vs. 516.4±170.5 pg/ml, P=0.001, Fig. 2B ). There was no difference in CXCL2/MIP-2 production between nontransfected cells and cells with GAPDH knockdown under any treatment conditions, thereby excluding the possibility of nonspecific siRNA attenuation of Fas-induced chemokine expression (Fig. 2B) . Cell viability by Alamar Blue assay did not differ among cells treated with lipofectamine with or without siRNA (data not shown).


Figure 2
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  		Figure 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 analysis (A) and in reduced CXCL2/MIP-2 after mAb Jo-2 treatment (B). Data are presented as means ± SEM of three independent experiments. *P ≤ 0.05; {dagger}P ≤ 0.01.

To confirm the role of MyD88 in Fas-induced chemokine release, peritoneal macrophages isolated from both wild-type and MyD88–/– mice were stimulated with mAb Jo-2, or rhFasL. Wild-type peritoneal macrophages treated with mAb Jo-2 produced increased CXCL1/KC compared with PBS-treated macrophages (777.3±601.2 vs. 207.8±157.3, P=0.03, Fig. 3A ). A similar trend was observed with rhFasL treatment (546.3±391.8 vs. 207.8±157.3 pg/ml, P=0.057). Similarly, Fas activation by treatment with either mAb Jo-2 or rhFasL tended to increase CXCL2/MIP-2 production (P=0.06 and P=0.14, respectively, Fig. 3B ). In contrast to peritoneal macrophages isolated from wild-type mice, macrophages from MyD88–/– mice did not produce CXCL1/KC or CXCL2/MIP-2 in response to Fas-activation by treatment with either mAb Jo-2 or rhFasL (Fig. 3A and 3B) . The observed differential response to Fas activation by either Jo-2 or rhFasL in peritoneal macrophages isolated from wild-type or MyD88–/– mice did not result from differences in cell viability (82.2±2.7% vs. 82.8±6.7%, P=0.89 for Jo-2 and 75.7±7.5% vs. 80.0±2.1%, P=0.42 for rhFasL).


Figure 3
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  		Figure 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 means ± SEM of 3 independent experiments, n = 3/group. *P ≤ 0.05; {dagger}P ≤ 0.01.

Because the response of macrophages is in part determined by tissue-specific differentiation, CXCL1/KC production after Fas activation was measured in bone marrow-derived macrophages (BMDM) isolated from wild-type and MyD88–/– mice. Treatment with mAb Jo-2 increased CXCL1/KC production (110.4±27.6 vs. 40.0±7.2 pg/ml, P=0.007) in BMDM from wild-type mice (Fig. 3C) . Similarly, rhFasL increased CXCL1/KC release (232.8±63.1 vs. 40.0±7.2 pg/ml, P=0.01) from wild-type BMDM. No increase in CXCL1/KC was observed following Fas activation in BMDM obtained from MyD88–/– mice (Fig. 3C) . There was no difference between wild-type and MyD88–/– macrophage viability after stimulation with either mAb Jo-2 (83.5±10.5% vs. 88.9±6.3%, P=0.41) or rhFasL (71.0±13.8% vs. 84.9±6.6%, P=0.14).

To ensure that absence of MyD88 specifically down-regulates Fas-induced chemokine production as opposed to a more general suppression of chemokine production, we repeated studies in BMDM collected from wild-type and MyD88–/– using poly(I:C) (a synthetic double-strand RNA, which signals via TLR-3) as a MyD88-independent control and Pam3CSK (a synthetic lipoprotein, which signals via TLR-2) as a MyD88-dependent control. These results confirmed that MyD88 was required for Fas-induced and TLR-2-induced CXCL2/MIP-2 production but not for TLR-3-induced CXCL2/MIP-2 production (Fig. 4 ).


Figure 4
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  		Figure 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 means ± 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. {dagger}P ≤ 0.01.

To determine whether Fas activation in vivo resulted in MyD88-dependent expression of CXC chemokines, we compared intratracheal mAb Jo-2 instillation with instillation of a control isotype IgG antibody in mice. Eighteen hours following mAb Jo-2 instillation, CXCL1/KC was increased in bronchoalveolar lavage fluid from wild-type mice (1894±691 vs. 57±42 pg/ml, P=0.006) but not from MyD88–/– mice (134±36 vs. 103±18, Fig. 5A ). In contrast, CXCL2/MIP-2 concentration was not significantly increased 18 h following mAb Jo2 instillation in either wild-type or MyD88–/– mice (Fig. 5B) . In addition to increased CXCL1/KC production, wild-type mice had more polymorphonuclear cells in bronchoalveolar lavage fluid 18 h following mAb Jo-2 instillation (2.5±2.1x104 vs. 0.09±0.03x104 cells, P=0.02, Fig. 5C ). Treatment with mAb Jo-2 did not increase polymorphonuclear cell recruitment to the alveolar space in MyD88–/– mice (0.13±0.05 vs. 0.09±0.02x104 cells, P=0.8).


Figure 5
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  		Figure 5. Bronchoalveolar lavage fluid concentrations of CXCL1/KC (A) and CXCL2/MIP-2 (B) and polymorphonuclear (C) and total leukocytes (D) cell counts from wild-type and MyD88–/– mice 18 h after intratracheal treatment with either mAb Jo-2 (n=6/genotype) or isotype IgG (n=6/genotype) antibody. *P ≤ 0.05; {dagger}P ≤ 0.01.

Fas-induced chemokine expression does not require interleukin-1 receptor signaling or caspase activation
Fas-associated chemokine expression has been reported to be associated with IL-1 receptor signaling via either caspase-dependent processing and release of IL-1ß [14 ] or autocrine amplification of IL-1 receptor signaling [27 ]. To assess the role of IL-1 receptor signaling in Fas-induced chemokine expression, RAW 264.7 cells were stimulated with either rhFasL or mAb Jo-2 following preincubation with or without recombinant IL-1 receptor antagonist (IL-1ra). Pretreatment with IL-1ra did not affect CXCL2/MIP-2 production and release in response to Fas activation by either rhFasL or mAb Jo-2 (Fig. 6A ). Similar findings were observed in primary wild-type BMDM treated with mAb Jo-2 in the presence or absence of IL-1ra (Fig. 6B) . BMDM isolated from MyD88–/– did not produce chemokines in response to mAb Jo-2 regardless of whether cells were pretreated with IL-1ra.


Figure 6
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  		Figure 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 h 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 h with mAb Jo-2 in the presence or absence of IL-1ra; n = 4/group. Data are presented as means ± SEM of three independent experiments. *P ≤ 0.05 compared with untreated cells.

To investigate the role of caspase activation in Fas-induced chemokine release, BMDM were preincubated with zVAD, a synthetic pan-caspase inhibitor, or DMSO carrier prior to Fas activation. Compared with DMSO, treatment with zVAD enhanced CXCL1/KC release following Fas activation by mAb Jo-2 (1468±520 vs. 489±143 pg/ml, P=0.017) and by rhFasL (873±377 vs. 535±204 pg/ml, P=0.019) (Fig. 7A ). zVAD pretreatment resulted in a trend toward increased CXCL1/KC release in cells in the absence of Fas activation as well (228±8 vs. 63±17 pg/ml, P=0.06). Because pharmacologic inhibitors can have nonspecific effects, we performed a second experiment in which RAW 264.7 cells were transfected with either CrmA, a proteinase inhibitor gene product of cowpox, which targets multiple caspases, including interleukin-1ß-converting enzyme (caspase-1) [28 ], or vector alone, followed by stimulation with mAb Jo-2. Inhibition of caspase activity by CrmA expression did not reduce CXCL2 release in response to mAb Jo-2; in fact, a small increase in CXCL2 was observed as compared with transfection of vector alone (2011±144 vs. 1601±181 pg/ml, P=0.008, Fig. 7B ). A similar pattern was observed with rhFasL stimulation. Thus, caspase inhibition by two different methods, using both primary macrophages and a macrophage cell line, failed to attenuate chemokine release in response to Fas activation.


Figure 7
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  		Figure 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 18 h after stimulation with either mAb Jo-2 or rhFasL. Data are presented as means ± SEM of three independent experiments. *P ≤ 0.05; {dagger}P ≤ 0.01.


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DISCUSSION
 
For this study, we hypothesized that Fas-induced chemokine release by macrophages requires MyD88. The important findings were that: 1) CXC chemokine production and release following Fas activation was absent in macrophages lacking MyD88 (Figs. 2 and 3) , 2) intratracheal mAb Jo-2 instillation into MyD88–/– mice did not increase BAL fluid CXCL1/KC or CXCL2/MIP-2 concentration or recruitment of PMN cells to the alveolar compartment (Fig. 5) , and 3) Fas-induced chemokine release does not require signaling via the IL-1 receptor or caspase activity (Figs. 6 and 7) .

Numerous studies have now demonstrated that, in addition to causing caspase-mediated cell death or apoptosis, Fas activation can also lead to expression of proinflammatory cytokines and, in particular, chemokines, resulting in neutrophilic infiltration [5 , 14 , 16 , 17 , 29 ]. This duality of both proapoptotic and proinflammatory signaling is common to other members of the TNF receptor family [30 ]. However, as opposed to TNF receptors, the current study demonstrates that proinflammatory signaling by Fas is MyD88-dependent.

Previous studies have suggested a critical role for signaling via the MyD88-dependent IL-1 receptor in Fas-associated inflammatory cytokine expression. Miwa et al. found that neutrophilic peritoneal inflammation caused by inoculation with Fas-expressing tumor cells was abrogated in IL-1{alpha}/ß knockout mice or by treatment with a broad caspase inhibitor, thereby implicating caspase processing of IL-1 in Fas-induced inflammation [14 ]. More recently, Ma et al. reported that Fas activation, resulting in release of MyD88 from FADD, amplified signaling via either IL-1 receptor or TLR-4 [27 ]. In the current study, Fas-induced chemokine production did not require costimulation by either IL-1{alpha}/ß or by TLR ligands. Additionally, chemokine response to Fas activation was not attenuated in the presence of IL-1 receptor antagonist (Fig. 6) . Consistent with previous reports [31 , 32 ], caspase inhibition by either zVAD or CrmA augmented Fas-induced chemokine expression, suggesting that caspase-dependent processing of IL-1 was not necessary (Fig. 7) .

The mechanism by which Fas induces chemokine expression via MyD88-dependent signaling remains unknown. Two possibilities are that 1) Fas signals NF-{kappa}B activation through the MyD88-dependent activation of IRAK-4 leading ultimately to phosphorylation of I{kappa}B{alpha}; and/or 2) MyD88 regulates Fas-induced chemokine expression at a post-transcriptional level. Several studies support the latter possibility. First, MyD88 has recently been shown to mediate post-transcriptional stabilization of mRNA induced by interferon-{gamma} receptor signaling [33 ]. Additionally, two recent studies have described MyD88-independent pathways by which Fas activation leads to chemokine expression. Interestingly, both of these papers, which study Fas activation in lymphocytes, implicated an important role for caspase-8. The first study implicated a noncatalytic function of caspase-8, enhanced by the broad caspase inhibitor zVAD, in chemokine expression [31 ]. In the second study, caspase-8 mediated cleavage of cFLIP was reported to be required for NF-{kappa}B activation [34 ]. One important consideration is that our current study was conducted using macrophages, which may respond differently to Fas activation as compared with lymphocytes, which were the cell type studied in the previous reports.

These results must be interpreted within the context of several points. Only CXCL1/KC and CXCL2/MIP-2 release were measured. Although other molecules can recruit neutrophils to inflamed tissue, we chose to focus on CXCL1/KC and CXCL2/MIP-2 because both signal via CXCR2, which has been shown to play a central role in neutrophil recruitment and tissue injury in a variety of disease models [35 36 37 38 39 ] and because both are known to be transcriptionally regulated by NF-{kappa}B, which is activated following Fas stimulation [15 , 16 ]. This study did not address whether the Fas-MyD88 interaction is direct or indirect. Three possibilities are that 1) Fas interacts directly with MyD88, 2) activated Fas clusters with other trans-membrane receptors, which signal via MyD88 (e.g., toll-like receptors), resulting in their activation and initiation of signaling, or 3) release of MyD88 during recruitment of FADD to Fas results in MyD88 being available for either association with IRAK-4 and initiation of gene transcription or post-transcriptional mRNA stabilization. This last possibility is reasonable given that FADD interacts with both MyD88 and Fas via common death domain regions [22 ] and that Fas is not known to contain the TIR domain to which MyD88 is recruited on both the IL-1 receptor and Toll-like receptors.

In conclusion, this is the first study to implicate MyD88 in Fas-induced inflammation. MyD88-dependent signaling by Fas contrasts with other death receptors such as the TNF family of receptors, which initiate inflammatory responses independent of MyD88. This conclusion is supported by the fact that Fas-induced proinflammatory signaling via MyD88 is not dependent on a simple caspase-mediated IL-1 response. Disrupting MyD88 leads to reduced chemokine expression and, in vivo, to attenuated neutrophil recruitment. Thus, disruption of MyD88-dependent signaling may provide a new avenue for developing novel pharmacologic therapies for diseases associated with activation of the Fas-Fas ligand system.


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ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grants HL-71020 (W.A.A.), HL-73996 (W.C.L.), HL-080623 (J.M.H.) and by an American Lung Association Biomedical Research grant (W.A.A.), and a Canada Research Chair in Inflammation and Infectious Diseases (W.C.L.)


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FOOTNOTES
 
2 These authors contributed equally to this work. Back

Received October 31, 2006; revised April 30, 2007; accepted May 22, 2007.


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REFERENCES
 
    1
  1. Fine, A., Anderson, N. L., Rothstein, T. L., Williams, M. C., Gochuico, B. R. (1997) Fas expression in pulmonary alveolar type II cells Am. J. Physiol. 273,L64-L71[Medline]
    2. 2
  2. Curtin, J. F., Cotter, T. G. (2003) Live and let die: regulatory mechanisms in Fas-mediated apoptosis Cell. Signal. 15,983-992[CrossRef][Medline]
    3. 3
  3. Matute-Bello, G., Lee, J. S., Liles, W. C., Frevert, C. W., Mongovin, S., Wong, V., Ballman, K., Sutlief, S., Martin, T. R. (2005) Fas-mediated acute lung injury requires fas expression on nonmyeloid cells of the lung J. Immunol. 175,4069-4075[Abstract/Free Full Text]
    4. 4
  4. Matute-Bello, G., Liles, W. C., Frevert, C. W., Nakamura, M., Ballman, K., Vathanaprida, C., Kiener, P. A., Martin, T. R. (2001) Recombinant human Fas ligand induces alveolar epithelial cell apoptosis and lung injury in rabbits Am. J. Physiol. Lung Cell. Mol. Physiol. 281,L328-L335[Abstract/Free Full Text]
    5. 5
  5. Matute-Bello, G., Winn, R. K., Jonas, M., Chi, E. Y., Martin, T. R., Liles, W. C. (2001) Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation Am. J. Pathol. 158,153-161[Abstract/Free Full Text]
    6. 6
  6. Li, Y., Takemura, G., Kosai, K., Takahashi, T., Okada, H., Miyata, S., Yuge, K., Nagano, S., Esaki, M., Khai, N. C., et al (2004) Critical roles for the Fas/Fas ligand system in postinfarction ventricular remodeling and heart failure Circ. Res. 95,627-636[Abstract/Free Full Text]
    7. 7
  7. Lee, P., Sata, M., Lefer, D. J., Factor, S. M., Walsh, K., Kitsis, R. N. (2003) Fas pathway is a critical mediator of cardiac myocyte death and MI during ischemia-reperfusion in vivo Am. J. Physiol. Heart Circ. Physiol. 284,H456-H463[Abstract/Free Full Text]
    8. 8
  8. Jeremias, I., Kupatt, C., Martin-Villalba, A., Habazettl, H., Schenkel, J., Boekstegers, P., Debatin, K. M. (2000) Involvement of CD95/Apo1/Fas in cell death after myocardial ischemia Circulation 102,915-920[Abstract/Free Full Text]
    9. 9
  9. Uguz, A., Gonlusen, G., Ergin, M., Tuncer, I. (2005) Expression of Fas, Bcl-2 and p53 molecules in glomerulonephritis and their correlations with clinical and laboratory findings Nephrology 10,311-316[CrossRef][Medline]
    10. 10
  10. Erkan, E., Garcia, C. D., Patterson, L. T., Mishra, J., Mitsnefes, M. M., Kaskel, F. J., Devarajan, P. (2005) Induction of renal tubular cell apoptosis in focal segmental glomerulosclerosis: roles of proteinuria and Fas-dependent pathways J. Am. Soc. Nephrol. 16,398-407[Abstract/Free Full Text]
    11. 11
  11. Hamar, P., Song, E., Kokeny, G., Chen, A., Ouyang, N., Lieberman, J. (2004) Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury Proc. Natl. Acad. Sci. USA 101,14883-14888[Abstract/Free Full Text]
    12. 12
  12. Du, C., Jiang, J., Guan, Q., Yin, Z., Masterson, M., Parbtani, A., Zhong, R., Jevnikar, A. M. (2004) Renal tubular epithelial cell self-injury through Fas/Fas ligand interaction promotes renal allograft injury Am. J. Transplant. 4,1583-1594[CrossRef][Medline]
    13. 13
  13. Wortinger, M. A., Foley, J. W., Larocque, P., Witcher, D. R., Lahn, M., Jakubowski, J. A., Glasebrook, A., Song, H. Y. (2003) Fas ligand-induced murine pulmonary inflammation is reduced by a stable decoy receptor 3 analogue Immunology 110,225-233[CrossRef][Medline]
    14. 14
  14. Miwa, K., Asano, M., Horai, R., Iwakura, Y., Nagata, S., Suda, T. (1998) Caspase 1-independent IL-1beta release and inflammation induced by the apoptosis inducer Fas ligand Nat. Med. 4,1287-1292[CrossRef][Medline]
    15. 15
  15. Ponton, A., Clement, M. V., Stamenkovic, I. (1996) The CD95 (APO-1/Fas) receptor activates NF-kappaB independently of its cytotoxic function J. Biol. Chem. 271,8991-8995[Abstract/Free Full Text]
    16. 16
  16. Park, D. R., Thomsen, A. R., Frevert, C. W., Pham, U., Skerrett, S. J., Kiener, P. A., Liles, W. C. (2003) Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages J. Immunol. 170,6209-6216[Abstract/Free Full Text]
    17. 17
  17. Neff, T. A., Guo, R. F., Neff, S. B., Sarma, J. V., Speyer, C. L., Gao, H., Bernacki, K. D., Huber-Lang, M., McGuire, S., Hoesel, L. M., et al (2005) Relationship of acute lung inflammatory injury to Fas/FasL system Am. J. Pathol. 166,685-694[Abstract/Free Full Text]
    18. 18
  18. Shimizu, M., Yoshimoto, T., Sato, M., Morimoto, J., Matsuzawa, A., Takeda, Y. (2005) Roles of CXC chemokines and macrophages in the recruitment of inflammatory cells and tumor rejection induced by Fas/Apo-1 (CD95) ligand-expressing tumor Int. J. Cancer 114,926-935[CrossRef][Medline]
    19. 19
  19. Takeuchi, O., Akira, S. (2002) MyD88 as a bottle neck in Toll/IL-1 signaling Curr. Top. Microbiol. Immunol. 270,155-167[Medline]
    20. 20
  20. Janssens, S., Beyaert, R. (2002) A universal role for MyD88 in TLR/IL-1R-mediated signaling Trends Biochem. Sci. 27,474-482[CrossRef][Medline]
    21. 21
  21. Hardiman, G., Rock, F. L., Balasubramanian, S., Kastelein, R. A., Bazan, J. F. (1996) Molecular characterization and modular analysis of human MyD88 Oncogene 13,2467-2475[Medline]
    22. 22
  22. Bannerman, D. D., Tupper, J. C., Kelly, J. D., Winn, R. K., Harlan, J. M. (2002) The Fas-associated death domain protein suppresses activation of NF-kappa B by LPS and IL-1 beta J. Clin. Invest. 109,419-425[CrossRef][Medline]
    23. 23
  23. Marr, K. A., Balajee, S. A., Hawn, T. R., Ozinsky, A., Pham, U., Akira, S., Aderem, A., Liles, W. C. (2003) Differential role of MyD88 in macrophage-mediated responses to opportunistic fungal pathogens Infect. Immun. 71,5280-5286[Abstract/Free Full Text]
    24. 24
  24. Schaub, F. J., Liles, W. C., Ferri, N., Sayson, K., Seifert, R. A., Bowen-Pope, D. F. (2003) Fas and Fas-associated death domain protein regulate monocyte chemoattractant protein-1 expression by human smooth muscle cells through caspase- and calpain-dependent release of interleukin-1alpha Circ. Res. 93,515-522[Abstract/Free Full Text]
    25. 25
  25. Kinoshita, S., Su, L., Amano, M., Timmerman, L. A., Kaneshima, H., Nolan, G. P. (1997) The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells Immunity 6,235-244[CrossRef][Medline]
    26. 26
  26. Altemeier, W. A., Matute-Bello, G., Gharib, S. A., Glenny, R. W., Martin, T. R., Liles, W. C. (2005) Modulation of lipopolysaccharide-induced gene transcription and promotion of lung injury by mechanical ventilation J. Immunol. 175,3369-3376[Abstract/Free Full Text]
    27. 27
  27. Ma, Y., Liu, H., Tu-Rapp, H., Thiesen, H. J., Ibrahim, S. M., Cole, S. M., Pope, R. M. (2004) Fas ligation on macrophages enhances IL-1R1-Toll-like receptor 4 signaling and promotes chronic inflammation Nat. Immunol. 5,380-387[CrossRef][Medline]
    28. 28
  28. Zhou, Q., Snipas, S., Orth, K., Muzio, M., Dixit, V. M., Salvesen, G. S. (1997) Target protease specificity of the viral serpin CrmA. Analysis of five caspases J. Biol. Chem. 272,7797-7800[Abstract/Free Full Text]
    29. 29
  29. Kang, S. M., Schneider, D. B., Lin, Z., Hanahan, D., Dichek, D. A., Stock, P. G., Baekkeskov, S. (1997) Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction Nat. Med. 3,738-743[CrossRef][Medline]
    30. 30
  30. Hehlgans, T., Mannel, D. N. (2002) The TNF-TNF receptor system Biol. Chem. 383,1581-1585[CrossRef][Medline]
    31. 31
  31. Kreuz, S., Siegmund, D., Rumpf, J. J., Samel, D., Leverkus, M., Janssen, O., Hacker, G., Dittrich-Breiholz, O., Kracht, M., Scheurich, P., et al (2004) NF{kappa}B activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP J. Cell Biol. 166,369-380[Abstract/Free Full Text]
    32. 32
  32. Wajant, H., Haas, E., Schwenzer, R., Muhlenbeck, F., Kreuz, S., Schubert, G., Grell, M., Smith, C., Scheurich, P. (2000) Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD) J. Biol. Chem. 275,24357-24366[Abstract/Free Full Text]
    33. 33
  33. Sun, D., Ding, A. (2006) MyD88-mediated stabilization of interferon-gamma-induced cytokine and chemokine mRNA Nat. Immunol. 7,375-381[CrossRef][Medline]
    34. 34
  34. Golks, A., Brenner, D., Krammer, P. H., Lavrik, I. N. (2006) The c-FLIP-NH2 terminus (p22-FLIP) induces NF-{kappa}B activation J. Exp. Med. 203,1295-1305[Abstract/Free Full Text]
    35. 35
  35. Belperio, J. A., Keane, M. P., Burdick, M. D., Londhe, V., Xue, Y. Y., Li, K., Phillips, R. J., Strieter, R. M. (2002) Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury J. Clin. Invest. 110,1703-1716[CrossRef][Medline]
    36. 36
  36. Reutershan, J., Morris, M. A., Burcin, T. L., Smith, D. F., Chang, D., Saprito, M. S., Ley, K. (2006) Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung J. Clin. Invest. 116,695-702[CrossRef][Medline]
    37. 37
  37. Ness, T. L., Hogaboam, C. M., Strieter, R. M., Kunkel, S. L. (2003) Immunomodulatory role of CXCR2 during experimental septic peritonitis J. Immunol. 171,3775-3784[Abstract/Free Full Text]
    38. 38
  38. Bhatia, M., Hegde, A. (2007) Treatment with antileukinate, a CXCR2 chemokine receptor antagonist, protects mice against acute pancreatitis and associated lung injury Regul. Pept. 138,40-48[CrossRef][Medline]
    39. 39
  39. Tarzami, S. T., Miao, W., Mani, K., Lopez, L., Factor, S. M., Berman, J. W., Kitsis, R. N. (2003) Opposing effects mediated by the chemokine receptor CXCR2 on myocardial ischemia-reperfusion injury: recruitment of potentially damaging neutrophils and direct myocardial protection Circulation 108,2387-2392[Abstract/Free Full Text]



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