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Receptor Signaling, but Supports Toll-Like Receptor 4 Signaling in Murine Peritoneal Macrophages1
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* Biophysics Program,
Department of Molecular Genetics,
Department of Internal Medicine/Division of Pulmonary and Critical Care Medicine,
Dorothy and M. Davis Heart and Lung Research Institute, and
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Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210
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Abstract |
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R clustering in macrophages activates signaling events that result in phagocytosis. Phagocytosis is accompanied by the generation harmful byproducts such as reactive oxygen radicals and production of inflammatory cytokines, which mandate that the phagocytic process be subject to a tight regulation. The molecular mechanisms involved in this regulation are not fully understood. In this study, we have examined the role of the inositol 3-phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in FcR-induced macrophage function. We demonstrate that in ex vivo murine peritoneal macrophages that are deficient in PTEN expression, FcR-induced Akt and extracellular signal-regulated kinase phosphorylation are enhanced. Notably, PTEN−/− macrophages showed constitutively high phosphorylation of Akt. However, PTEN did not seem to influence tyrosine phosphorylation events induced by FcR clustering. Furthermore, PTEN−/− macrophages displayed enhanced phagocytic ability. Likewise, FcR-induced production of TNF-
, IL-6, and IL-10 was significantly elevated in PTEN−/− macrophages. Surprisingly, LPS-induced TNF- production was down-regulated in PTEN−/− macrophages. Analyzing the molecular events leading to PTEN influence on LPS/Toll-like receptor 4 (TLR4) signaling, we found that LPS-induced activation of mitogen-activated protein kinases is suppressed in PTEN−/− cells. Previous reports indicated that LPS-induced mitogen-activated protein kinase activation is down-regulated by phosphatidylinositol 3-kinase through the activation of Akt. Our observation that Akt activation is basally enhanced in PTEN−/− cells suggests that PTEN supports TLR4-induced inflammatory responses by suppressing the activation of Akt. Thus, we conclude that PTEN is a negative regulator of FcR signaling, but a positive regulator of TLR4 signaling. These findings are the first to demonstrate a role for PTEN in FcR- and TLR4-mediated macrophage inflammatory response. |
Introduction |
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R clustering by immune complexes initiates a signaling cascade that results in phagocytosis of the immune complex (1). Accompanying phagocytosis is the generation of reactive oxygen and nitrogen radicals as well as the production of inflammatory cytokines, which can result in tissue damage were it left unregulated. Accordingly, recent advances in this area demonstrated that phagocytosis is a highly regulated process involving negative regulation by protein tyrosine phosphatases as well as inositol phosphatases (2, 3, 4, 5, 6).
Murine macrophages express FcRI, FcRIIb, and FcRIIIa (7). FcRI and IIIa are activating receptors, and are associated with low m.w. -subunit homodimers, which contain an immunoreceptor tyrosine-based activation motif (ITAM).4 Upon receptor clustering by immune complexes, the tyrosines in the ITAM are phosphorylated by the membrane-associated Src family tyrosine kinases (8). The phosphorylated ITAMs serve as docking sites for Src homology 2 domain-containing cytosolic proteins, including the tyrosine kinase Syk, the Ras adapter Shc, and the p85 subunit of phosphatidylinositol (PtdIns) 3-kinase. Activation of both Syk and PtdIns3-kinase has been shown to be critical for phagocytosis (9, 10, 11). PtdIn3,4,5P3, the lipid product of PtdIns3-kinase, is an important second messenger that is necessary for the activation of pleckstrin homology domain-containing enzymes such as the serine/threonine kinase Akt, the guanine nucleotide exchange factor Vav, and the Tec family tyrosine kinase Btk, involved in intracellular calcium mobilization (12). The Ras/extracellular signal-regulated kinase (ERK) pathway has been shown to be important for activation of transcription factors that promote cytokine gene expression in response FcR clustering (13). Paradoxically, ITAM-associated FcR also recruit phosphatases, such as the inositol phsophatases Src homology 2-containing inositol phosphatase-1 (SHIP-1) and SHIP-2, and the protein tyrosine phosphatase Src homology protein-1, thereby ensuring a tempered response to immune complex stimulation (3, 4, 5, 6).
In contrast, FcRIIb is an inhibitory receptor that contains a tyrosine-based inhibitory motif, which when phosphorylated predominantly recruits phosphatases such as SHIP-1 and SHIP-2 (14, 15). SHIP-1 and SHIP-2 are inositol 5-phosphatases that consume PtdIns3,4,5P3 and dampen activation events (16). Consistent with this, SHIP-deficient macrophages show enhanced ability to phagocytose IgG-coated particles (2). In addition, we, and others, have demonstrated that SHIP-1 and SHIP-2 down-regulate NF-
B-dependent gene transcription (3, 4, 17). Likewise, the inositol 3-phosphatase and tensin homologue deleted on chromosome 10 (PTEN) recently has been shown to down-regulate phagocytosis in COS-7 fibroblasts transfected to express the human FcRIIa (18). However, there have been no studies examining the role of PTEN in FcR-mediated activation of macrophages.
Macrophages also respond to the bacterial endotoxin LPS through the Toll-like receptor (TLR) 4 (19). LPS engagement of TLR4 initiates a series of signaling events that culminate in the production of inflammatory cytokines (20). LPS induces the activation of the mitogen-activated protein (MAP) kinases ERK, p38, and c-Jun N-terminal kinase, followed by the activation of downstream transcription factors NF-B, AP-1, and Egr-1, promoting cytokine gene transcription. In addition, LPS induces the activation of PtdIns3-kinase and its downstream target Akt (21). In contrast to the activating role that PtdIns3-kinase and Akt play in immune receptor, cytokine receptor, and growth factor receptor signaling, these enzymes are reported to inhibit LPS-induced activation of MAP kinases and gene transcription driven by NF-B, AP-1, and Egr-1 (22).
In this study, we have investigated the role of PTEN in FcR and LPS-mediated signaling using peritoneal macrophages derived from macrophage-specific PTEN knockout and control animals. Our data indicate that FcR-induced signaling events such as Akt phosphorylation and ERK phosphorylation are up-regulated in PTEN−/− macrophages. Notably, Akt phosphorylation was constitutively elevated in PTEN−/− macrophages. Other experiments showed the FcR expression levels and upstream tyrosine phosphorylation events were unaffected by the presence or absence of PTEN. Analyzing the functional consequence of PTEN involvement in FcR signaling, we found that phagocytic ability was significantly enhanced in the PTEN−/− macrophages. Likewise, PTEN−/− macrophages made significantly higher levels of TNF-, IL-6, and IL-10 in response to FcR clustering. In contrast to the enhanced response to FcR clustering, PTEN−/− macrophages were less responsive to LPS stimulation than PTEN+/+ macrophages. Thus, PTEN−/− macrophages displayed diminished TNF- production upon LPS treatment, when compared with PTEN+/+ macrophages. Examining the molecular mechanism of PTEN influence on LPS-induced TNF- production, we found that activation of MAP kinases is suppressed in LPS-stimulated PTEN−/− cells, indicating that PTEN influences TLR4 signaling upstream of the MAP kinases ERK and p38. TLR4-induced activation of MAP kinases has been shown to be suppressed by Akt. Our current data indicate that PTEN−/− cells have enhanced basal level of Akt activation, suggesting that PTEN promotes LPS-induced inflammatory response by suppressing Akt. Taken together, these data indicate that PTEN suppresses FcR signaling, but promotes TLR4 signaling.
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Materials and Methods |
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Abs specific for phospho-ERK, phospho-Akt, phospho-p38, and PTEN were purchased from Cell Signaling Technology (Beverly, MA). Actin and TLR4 Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse CD16/32 (FcRIII/II) was purchased from BD PharMingen (San Diego, CA). LPS from Escherichia coli strain 0127:B8 was obtained from Difco (Detroit, MI).
Generation of PTEN conditional knockout animals
PTEN conditional knockout mice (PTENfl/fl) were generated as follows. Two loxP sites were introduced in two HpaI sites in introns 3 and 5. This resulted in two loxP sites flanking exons 4 and 5. Exon 5 of PTEN encodes lipid phosphatase domain (23, 24). Tissue-specific expression of cre removes PTEN exons 4 and 5, resulting in the loss of lipid phosphatase activity. Lys-cre mice decribed by Clausen et al. (25) were mated with PTENfl/fl mice. Macrophages from PTENfl/fl mice were used as wild-type controls. All mice were on 129/Bl6 background.
Isolation of peritoneal macrophages
Thioglycolate-elicited murine peritoneal macrophages were harvested, as described previously (26). Briefly, 1 ml of 2.9% Brewer’s complete thioglycolate broth was injected i.p., and macrophages were harvested after 5 days. Adherent cells were used in experiments.
Cell stimulation, lysis, immunoprecipitation, and Western blotting
Peritoneal macrophages were serum starved overnight, and activated by clustering FcR with mAb 2.4G2 and mouse F(ab')2 anti-rat IgG secondary cross-linking Ab or with 500 ng/ml LPS. Resting and activated cells were lysed in TN1 buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 10 mM Na4P2O7, 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 10 mM Na3VO4, and 10 µg/ml each aprotinin and leupeptin), and postnuclear lysates were incubated overnight with the Ab of interest and protein G-agarose beads (Life Technologies, Carlsbad, CA). Immune complexes bound to beads were washed in TN1 and boiled in SDS sample buffer (60 mM Tris, pH 6.8, 2.3% SDS, 10% glycerol, 0.01% bromphenol blue, and 1% 2-ME) for 5 min. Proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, probed with the Ab of interest, and developed by ECL.
Immunoblot data quantitation
The ECL signal was quantitated using a scanner and a densitometry program (Scion Image; Scion, Frederick, MD). To quantitate the phosphospecific signal in the activated samples, we first subtracted background, normalized the signal to the amount of actin in the lysate, and plotted the values as fold increase over unstimulated samples (wild type), as previously described (5).
Flow cytometry analysis of FcR expression
Peritoneal macrophages were tested for expression of FcR by incubating with anti-murine FcRIII/II mAb 2.4G2, at a concentration of 10 µg/ml for 30 min at 4°C. The cells were washed and incubated with FITC-labeled mouse F(ab')2 anti-rat IgG secondary Ab for 30 min at 4°C. Cells were subsequently washed, fixed in 1% paraformaldehyde, and analyzed by flow cytometry on an Elite EPICS FACS (Corixa, Hialeah, FL).
Preparation of IgG-coated SRBCs
SRBCs (Colorado Serum, Denver, CO) were washed in PBS, and labeled with PKH26 Red (Sigma-Aldrich, St. Louis, MO). Labeled cells were then washed in PBS and incubated with a subagglutinating dose of rabbit anti-SRBC IgG (Diamedix, Miami, FL) at 37°C for 1 h. Unbound IgG was removed by washing the cells with PBS.
Phagocytosis assays
IgG-coated SRBCs described above were added to the peritoneal macrophages. The cells were pelleted by low speed centrifugation to increase contact between SRBCs and phagocytes. The samples were prepared in duplicate and incubated for 1 h at either 4°C to study binding, or 37°C to study phagocytosis. All cells were fixed in 1% paraformaldehyde and mounted on slides to be viewed under a fluorescence microscope. For the phagocytosis assay, cells were subjected to brief hypotonic lysis with water to get rid of externally bound RBCs before fixation in paraformaldehyde. Binding index represents the number of bound SRBC in 100 macrophages. That the binding was via the transfected Fc receptors was confirmed by the lack of binding observed in samples incubated with fluoresceinated RBCs that were not opsonized with IgG. No binding or phagocytosis was seen in any of the samples treated with nonopsonized RBCs. Phagocytosis was measured by counting the total number of RBCs ingested by 100 macrophages (phagocytic index). Of note, only 25–30% of the macrophages displayed binding and phagocytic ability. The experiment was repeated three times.
Preparation of heat-aggregated IgG
Heat-aggregated IgG was prepared according to methods described previously. In brief, Chromopure mouse IgG at a concentration of 750 µg/ml was heated at 62°C for 30 min and then cooled on ice immediately and used directly to stimulate the cells.
ELISA determination of cytokine production
Cells were cultured overnight in the presence or absence of heat-aggregated IgG or 500 ng/ml LPS. Cell supernatants were harvested, centrifuged to remove dead cells, and analyzed by ELISA using cytokine-specific kits from R&D Systems (Minneapolis, MN). Data were analyzed using a paired Student’s t test.
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Results |
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R-induced signaling events are enhanced in PTEN−/− macrophages
FcR clustering induces the activation of multiple signaling enzymes, including the serine/threonine kinases Akt and ERK (7). To examine the role of PTEN in FcR signaling, PTEN+/+ and PTEN−/− peritoneal macrophages were serum starved overnight and subsequently stimulated for 5 min by clustering FcR. First, Akt phosphorylation was analyzed by Western blotting protein-matched whole cell lysates (WCL). The results shown in Fig. 1A, upper panel, indicate that serine phosphorylation of Akt is enhanced in PTEN−/− macrophages. As seen in lane 3, PTEN−/− macrophages also have constitutively high Akt phosphorylation. The lower panel of Fig. 1A is a reprobe of the same membrane with actin Ab, to ensure that the differences in Akt phosphorylation observed were not due to unequal loading of protein in the different lanes.
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R-induced ERK phosphorylation was examined by Western blotting with phospho-ERK Ab (Fig. 1B, upper panel). Results indicated that ERK phosphorylation in PTEN−/− macrophages is also enhanced in comparison with that in PTEN+/+ macrophages.
FcR expression and FcR-induced proximal signaling are not influenced by PTEN
To test whether the signaling differences observed in PTEN+/+ and PTEN−/− macrophages could be due to a difference in the level of FcR expression in the two cell types, FcR expression was analyzed. For this, cells were first labeled with the murine FcRIII/II mAb 2.4G2, followed by FITC-labeled F(ab')2 mouse anti-rat IgG. The cells were then analyzed by flow cytometry. Results shown in Fig. 2A indicate that both PTEN+/+ and PTEN−/− macrophages expressed comparable levels of FcR.
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R clustering are the phosphorylation of the receptor-associated ITAM, activation of the tyrosine kinase Syk, and tyrosine phosphorylation of cellular proteins by Syk (8, 27). The inositol phosphatase activity of PTEN results in the consumption of the 3-phosphorylated products of PtdIns3-kinase that are produced more distal to the receptor. However, PTEN also has the ability to dephosphorylate tyrosine-phosphorylated proteins, although no specific substrates have yet been identified (28). Thus, we next examined whether PTEN might influence overall cellular tyrosine phosphorylation induced by FcR clustering. In this study, protein-matched WCL from PTEN+/+ and PTEN−/− macrophages, resting or stimulated by clustering FcR, were analyzed by Western blotting with anti-phosphotyrosine Ab. As seen in Fig. 2B, there was no major, detectable influence of PTEN on FcR-induced tyrosine phosphorylation. Western blotting WCL with PTEN Ab confirmed the absence of PTEN in the PTEN−/− macrophages (Fig. 2C). PTEN−/− macrophages display enhanced phagocytic ability
FcR-mediated phagocytosis is critically dependent on the activation of PtdIns3-kinase and the downstream PtdIns3,4,5P3-dependent events such as the activation of Rac through Vav (9, 10). PTEN consumption of PtdIns3,4,5P3 suggests that PTEN can down-regulate phagocytosis. Consistent with this, PTEN has been shown to suppress phagocytosis by human FcRIIa in a transfected COS-7 fibroblast model (18). To determine whether PTEN regulates phagocytosis in macrophages, phagocytosis assays were performed with PTEN+/+ and PTEN−/− peritoneal macrophages, as we have previously described (29). Thus, the macrophages were incubated with IgG-coated, PKH26 Red-labeled SRBCs for 1 h at 4°C to measure binding ability, or at 37°C to measure phagocytic ability. Three independent experiments were performed, each time analyzing 100 macrophages. The results are shown in Fig. 3. Consistent with equivalent expression of FcR, both PTEN+/+ and PTEN−/− macrophages displayed comparable ability to bind the opsonized SRBCs (Fig. 3A). In contrast, PTEN−/− macrophages showed significantly enhanced (p value 0.048) ability to phagocytose the SRBCs (Fig. 3B), indicating that PTEN negatively influences phagocytosis. Of note, the percentage of PTEN+/+ or PTEN−/− macrophages actually binding the SRBC, or ingesting the SRBC was equivalent, indicating that in the absence of PTEN more SRBCs are being ingested by the same number of macrophages.
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R-induced cytokine production is enhanced in PTEN−/− macrophages
FcR-mediated phagocytosis is accompanied by the production of cytokines such as TNF-, IL-6, and IL-10 (7). Previous reports indicated that transcription of these cytokine genes is dependent on NF-B activation (13). We, and others, have recently reported that the inositol phosphatases SHIP-1 and SHIP-2, which consume PtdIns3,4,5P3, down-regulate NF-B-dependent gene transcription (3, 4, 17). We, therefore, examined the influence of PTEN on FcR-induced cytokine production. For this, PTEN+/+ and PTEN−/− peritoneal macrophages were stimulated overnight with heat-aggregated IgG. Cell supernatants were harvested, and the production of cytokines was measured by ELISA. Cells were lysed and estimated for protein content, and the ELISA results were further normalized to the amount of protein in each well. Results indicated that production of TNF-, IL-6, and IL-10 was all significantly higher (p values 0.01, 0.0012, and 0.0029, respectively) in PTEN−/− macrophages (Fig. 4).
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R-mediated macrophage activation is negatively regulated by PTEN.
LPS-induced TNF- production is down-regulated in PTEN−/− macrophages
To determine whether PTEN plays a role in regulating LPS-mediated activation of macrophages, we examined LPS-induced inflammatory response in PTEN+/+ and PTEN−/− murine peritoneal macrophages. For this, PTEN+/+ and PTEN−/− cells were stimulated overnight with 500 ng/ml LPS, and cell supernatants were analyzed by ELISA for the production of TNF-. Surprisingly, results indicated that LPS-stimulated PTEN−/− macrophages make significantly lower amounts of TNF- (p value 0.016), in comparison with PTEN+/+ macrophages (Fig. 5A). To ensure that the difference in the inflammatory response is not due to a difference in the level of TLR4 expression, we next assessed TLR4 expression in the PTEN+/+ and PTEN−/− macrophages. The results shown in Fig. 5B demonstrate that TLR4 is equivalently expressed in both cell types.
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We next analyzed the molecular mechanism by which PTEN influences TLR4 signaling. Because TNF- production by LPS-stimulated macrophages is dependent on the activation of MAP kinases, we analyzed the effect of PTEN on LPS-induced activation of ERK and p38. In this study, PTEN+/+ and PTEN−/− macrophages were stimulated with 500 ng/ml LPS for varying time points, and phosphorylation of ERK and p38 was analyzed by Western blotting with phosphospecific Abs. Results indicated that both ERK phosphorylation and p38 phosphorylation were suppressed in PTEN−/− macrophages (Fig. 6, A and B, upper panels). A reprobe of the same membranes with actin Ab indicated that the phosphorylation differences were not due to unequal loading of protein in the lanes (Fig. 6, A and B, middle panels). These data indicate that PTEN influences TLR4 signaling upstream of the MAP kinases ERK and p38. Previous reports have indicated that during TLR4 signaling, Akt serves to down-regulate MAP kinase activation and the downstream induction of cytokine gene expression. Thus, in parallel experiments, we analyzed Akt phosphorylation. As seen in Fig. 6C, serine phosphorylation of Akt is induced by LPS treatment in PTEN+/+ cells. In contrast, PTEN−/− cells displayed constitutively elevated serine phosphorylation of Akt. These observations suggest that PTEN may support LPS signaling by suppressing Akt.
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Discussion |
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R and TLR4 signaling, but in entirely opposing ways. Our results indicate that FcR signaling and functional outcomes such as phagocytosis and cytokine generation are down-regulated by PTEN, most likely by virtue of the ability of PTEN to consume PtdIns3,4,5P3 (Fig. 7A). These findings are the first to demonstrate a role for PTEN in FcR function in murine macrophages, and are consistent with previous work from our laboratory and others demonstrating that inositol 5-phosphatases that consume PtdIns3,4,5P3 likewise down-regulate FcR-induced macrophage function (2, 3, 4, 6).
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production are promoted by the presence of PTEN. These findings suggest that PtdIns3,4,5P3-dependent downstream enzymes may be suppressing the LPS-induced MAP kinase pathways. Consistent with this latter notion, previous studies indicated that PtdIns3-kinase activation of Akt results in the suppression of LPS-induced MAP kinase activation, as well as the activation of the transcription factors (22) (Fig. 7B). Thus, Guha and Mackman (22) demonstrated that inhibition of PtdIns3-kinase results in enhanced activation of LPS-induced MAP kinase activation, and gene transcription driven by the transcription factors NF-B, AP-1, and Egr-1. Furthermore, overexpression of constitutively active Akt was sufficient to mediate these inhibitory events. Similarly, in other reports, it has been shown that PtdIns3-kinase negatively regulates the stability of COX2 mRNA in LPS-stimulated human alveolar macrophages (30), LPS-induced NO synthase production in glial cells (31), and LPS-induced NO production from murine peritoneal macrophages (32). Our data show that PTEN−/− macrophages have constitutively high Akt phosphorylation (Fig. 6C), perhaps accounting for the reduced MAP kinase activation in these cells in response to LPS stimulation. In support of an inhibitory role for Akt and a positive role for inositol phosphatases, which suppress Akt activation, in LPS-induced macrophage activation, Rauh et al. (33) recently reported that LPS-treated SHIP−/− peritoneal macrophages make lower levels of inflammatory cytokines in comparison with SHIP+/+ peritoneal macrophages. Likewise, these investigators found that LPS-induced NO production was also lower in SHIP−/− peritoneal macrophages. Based on these findings, an elegant model was proposed wherein chronically elevated levels of PtdIns3,4,5P3 in the SHIP−/− macrophages may drive these macrophages to an anti-inflammatory phenotype, reminiscent of endotoxin tolerance.
The serine/threonine kinase Akt/PKB has been extensively studied (34). In addition to promoting cell survival through the phosphorylation of Bad, caspase 9, Forkhead family proteins, and IB kinase, Akt is also reported to influence gene transcription through transcription factors such as NF-B, and promote cytoskeletal rearrangements and cell migration through the activation of p70S6K (35). Paradoxically, Akt appears to promote NF-B activation in some cell systems (34) and suppress NF-B in others (22). Likewise, Akt has been shown to down-regulate MAP kinase activation in some cases (36, 37, 38), but not in others. The opposing effects of Akt may be due to the activation of distinct downstream effectors, of which there are many. The specificity of Akt effects have been proposed to be dictated by its protein-protein interactions, which may in turn depend on cell type, type of stimulus, subcellular compartment, and/or posttranslational modifications of the interacting proteins (34).
In summary, the novel findings of this study are that the inositol 3-phosphatase PTEN regulates FcR and TLR4 signaling in peritoneal macrophages in opposing ways. PTEN negatively regulates FcR-mediated signaling events such as activation of Akt and ERK, phagocytosis, and cytokine production. In contrast, PTEN supports LPS-induced TNF- production. Our data also indicate that the influence of PTEN on LPS-induced TNF- production is upstream of the MAP kinases ERK and p38, and perhaps through the activation of Akt. Thus, we propose that macrophage innate immune responses are tightly regulated by the combined actions of lipid kinases and phosphatases.
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Footnotes |
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2 X.C. and G.W. have contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Susheela Tridandapani, 473 West 12th Avenue, Columbus, OH 43210. E-mail address: tridandapani.2{at}osu.edu
4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; PtdIns, phosphatidylinositol; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SHIP, Src homology 2-containing inositol phosphatase; TLR, Toll-like receptor; WCL, whole cell lysate.
Received for publication December 29, 2003. Accepted for publication February 12, 2004.
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