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* Department of Internal Medicine,
Molecular, Cellular, and Developmental Biology Program,
Biophysics Program, and
Dorothy and M. Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH 43210
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Abstract |
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B-dependent gene transcription in response to LPS stimulation. These results suggest that SHIP positively regulates LPS-induced activation of Raw 264.7 cells. To test the validity of these observations in primary macrophages, LPS-induced events were compared in bone marrow macrophages derived from SHIP+/+ and SHIP–/– mice. Results indicated that LPS-induced MAPK phosphorylation is enhanced in SHIP+/+ cells, whereas Akt phosphorylation is enhanced in SHIP–/– cells compared with SHIP+/+ cells. Finally, LPS-induced TNF-
and IL-6 production was significantly lower in SHIP–/– bone marrow-derived macrophages. These results are the first to demonstrate a role for SHIP in TLR4 signaling, and propose that SHIP is a positive regulator of LPS-induced inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1, 2). The receptor proximal signaling events include the association of the adapter MyD88 with TLR4, followed by the recruitment and activation of IL-1R-associated kinase 1 (IRAK-1).4 More distal to the receptor, the MAPKs ERK1/2, p38, and JNK and the transcription factors NF-B and AP-1 become activated and promote gene transcription. The signaling events bridging receptor proximal events to the activation of MAPKs are not fully understood.
PI3K is activated in response to LPS stimulation and has been shown to play a role in the signaling cascades triggered by LPS (3). In a recent study, Guha and Mackman (4) demonstrated that inhibition of PI3K results in enhanced activation of LPS-induced MAPK activation and gene transcription driven by the transcription factors NF-B, AP-1, and Egr-1. The inhibitory effect of PI3K was shown to be mediated via the serine/threonine kinase Akt. In other reports, it has been shown that PI3K negatively regulates the stability of cyclo-oxygenase 2 mRNA in LPS-stimulated human alveolar macrophages (5), LPS-induced NO synthase production in glial cells (6), and LPS-induced NO production from murine peritoneal macrophages (7). Finally, in a recent study, Williams and colleagues (8) demonstrated that inhibition of PI3K in vivo resulted in increased inflammatory cytokine levels in the serum, as well as early mortality in septic mice, in a murine model of cecal ligation and puncture. Interestingly, while the above reports all indicate that PI3K dampens LPS signaling, other groups have found that PI3K activity promotes LPS signaling (reviewed in Ref. 9). These disparate observations may reflect cell type-specific influences of PI3K, which are currently not fully understood (reviewed in Ref. 9).
SHIP is specifically expressed in hemopoietic cells and is capable of reversing the effects of PI3K by hydrolyzing the 5' phosphate of its product phosphatidylinositol-3,4,5-trisphosphate (PtdIns3,4,5P3) (10). The inhibitory role of SHIP in immune receptor and growth factor/cytokine receptor signaling is well established (reviewed in Ref. 10). Thus, the catalytic activity of SHIP results in the consumption of PtdIns3,4,5P3 and leads to inhibition of downstream pleckstrin homology domain-containing enzymes that are dependent on PtdIns3,4,5P3 for their activation (11, 12, 13). In addition, the noncatalytic regions of SHIP have been shown to influence signaling pathways by association with critical proteins that regulate these pathways (14, 15, 16). However, it is not known whether SHIP is involved in regulating LPS signaling.
In this study, we have analyzed the influence of SHIP in LPS-induced activation of murine macrophages. We report that SHIP becomes tyrosine phosphorylated upon LPS stimulation in Raw 264.7 cells, and translocates to lipid rafts along with IRAK. Transient cotransfection experiments testing the functional consequence of SHIP revealed that NF-B-driven gene transcription in response to LPS is up-regulated in the presence of wild-type SHIP but not an isolated Src homology 2 (SH2) domain of SHIP lacking in enzyme activity. These results suggest that SHIP is a positive regulator of LPS-induced activation of macrophages. Consistent with this notion, bone marrow-derived macrophages (BMM) from SHIP-deficient mice stimulated with LPS displayed reduced activation of ERK and p38 MAPKs in comparison to wild-type BMM. Likewise, LPS-induced TNF- and IL-6 production was lower in SHIP-deficient BMM. In contrast, Akt phosphorylation was enhanced in SHIP–/– BMM. Taken together, these data demonstrate that SHIP promotes LPS-induced macrophage inflammatory response, at least in part through the inactivation of Akt. These results are in contrast to the previously reported inhibitory role of SHIP in immune receptor and growth factor receptor signaling (10). However, these findings are consistent with the reported negative regulatory role of PI3K in TLR4 signaling (4, 5, 6, 7).
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Materials and Methods |
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Raw 264.7 murine macrophage cell line was obtained from American Type Culture Collection (Manassas, VA), and maintained in RPMI 1640 with 3.5% FBS. Abs specific for phospho-ERK, phospho-Akt, and phospho-p38 were purchased from Cell Signaling Technology (Beverly, MA). Actin, Akt, and TLR4 Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). SHIP Ab and IRAK Ab were from Upstate Biotechnology (Lake Placid, NY). Anti-mouse CD16/32 (Fc
RIII/II) was purchased from BD Pharmingen (San Diego, CA). Rabbit polyclonal SHIP Ab was a generous gift from Dr. K. M. Coggeshall (Oklahoma Medical Research Foundation, Oklahoma City, OK). LPS from Escherichia coli strain 0127:B8 was obtained from Difco (Detroit, MI).
Culture of murine BMM
BMM were derived from SHIP+/+ and SHIP–/– male littermates as previously described (17). Briefly, bone marrow cells were cultured in RPMI 1640 containing 5% FBS, 10 µg/ml polymixin B, and supplemented with 20 ng/ml CSF-1 for 7 days, before they were used in the experiments.
Immunoprecipitation and Western blotting
Macrophages were stimulated with or without 500 ng/ml LPS, or by clustering FcR as previously described (17). 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, 10 µg/ml each aprotinin and leupeptin). Proteins were separated by SDS/PAGE, transferred to nitrocellulose membranes, and probed with the Ab of interest and appropriate HRP-conjugated secondary Abs. The filters were then 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 phospho-specific 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, as previously described (17).
Transfection and luciferase assays
Raw 264.7 cells were transfected with the appropriate plasmid DNA using the Amaxa Nucleofector apparatus (Amaxa Biosystems, Cologne, Germany). Briefly, 2 x 106 cells were resuspended in 100 µl of Cell Line Nucleofector Solution V (Amaxa Biosystems) and were nucleofected with 1 µg of NF-B-luciferase (NF-B-luc) and/or 5 µg of wild-type SHIP or SHIP SH2 domain alone. Immediately after nucleofection, 500 µl of prewarmed RPMI 1640 was added to the transfection mix, before transferring to 12-well plates containing 1.5 ml of prewarmed RPMI 1640 per well. Plates were incubated for 24 h at 37°C.
Transfected cells were left untreated or stimulated with 500 ng/ml LPS for 2 h. The cells were lysed in 100 µl of Luciferase Cell Culture Lysis 5x Reagent (Promega, Madison, WI). Luciferase activity was then measured using Luciferase Assay Reagent (Promega), as previously described (17).
Sucrose density gradients
Triton-soluble and Triton-insoluble cell fractions were prepared using slight modifications of methods described previously (18). Briefly, Raw 264.7 cells starved in serum-free medium were left untreated or stimulated with 100 ng/ml LPS for 30 min and then lysed in TN1 buffer containing 0.5% Triton X-100. One milliliter of the lysate was mixed with 1 ml of 85% sucrose and loaded at the bottom of a Beckman centrifuge tube (Beckman Coulter, Fullerton, CA), and overlaid with 7 ml of 30% sucrose followed by 3.5 ml of 5% sucrose. The gradients were centrifuged for 17 h at 38,000 rpm at 4°C in a Beckman SW40Ti rotor (Beckman Coulter). Nine fractions (
1.4 ml each) were collected from the top of the gradient and were used for analysis by Western blotting.
ELISA determination of cytokine production
Cells were cultured for varying time points ranging from 2 to 14.5 h (overnight) in the presence or absence of 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.
Measurement of receptor expression by flow cytometry
Murine BMM were tested for expression of FcRs by incubating with anti-FcRII/III mAb 2.4G2 (BD Pharmingen) at a concentration of 10 µg/ml for 30 min at 4°C. The cells were washed and incubated with FITC-labeled goat anti-rat Ig 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 (Coulter, Hialeah, FL). Data from 10,000 cells per condition were recorded to yield the percentage of cells expressing receptors (see Fig. 6B).
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Results |
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Previous studies indicated that LPS stimulation results in the movement of TLR4 into the cholesterol-enriched detergent-insoluble lipid rafts, where the receptor complex is assembled (18). SHIP is a cytosolic enzyme that must translocate to the plasma membrane to access its lipid substrates (10). Thus, to test whether SHIP is involved in LPS signaling, Triton-soluble and Triton-insoluble fractions were isolated from Raw 264.7 murine macrophage cells using sucrose density gradients. The fractions were separated by SDS/PAGE and examined for the presence of SHIP by Western blotting. As previously reported (18), results indicated that IRAK becomes associated with lipid rafts following LPS stimulation (Fig. 1A, lower panel). Likewise, SHIP translocated to the lipid rafts upon LPS stimulation (Fig. 1B, lower panel). Neither SHIP nor IRAK were detected in the lipid rafts in unstimulated cells (Fig. 1, A and B, upper panels)
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SHIP positively regulates NF-B-dependent gene transcription in response to LPS stimulation
LPS stimulation of macrophages results in the activation of transcription factors such as NF-B. We, and others, have previously reported that SHIP negatively regulates FcR- and growth factor receptor-mediated activation of NF-B (17, 19, 20). Therefore, we asked whether SHIP influenced gene transcription driven by NF-B in response to LPS stimulation. In these experiments, Raw 264.7 cells were transiently transfected with luciferase reporter plasmids that were dependent on NF-B binding (NF-B-luc). Stimulation of these transfected cells with LPS induced NF-B-dependent transcription of luciferase as previously reported (Fig. 2A) (4). Results indicated that the induction of luciferase gene transcription was significantly enhanced in the presence of overexpressed wild-type SHIP (p = 0.04). In contrast, the overexpression of SHIP SH2 domain alone that lacks the catalytic domain failed to influence NF-B-driven luciferase gene transcription (p = 0.25). Cell lysates from the transfectants were analyzed by Western blotting to ensure that the transfected wild-type SHIP and the SHIP SH2 domain were indeed expressed (Fig. 2B). These results suggest that SHIP enzyme activity positively regulates TLR4-induced activation of the NF-B transcription factor. Because SHIP and PI3K are opposing enzymes, these results are consistent with the findings of Guha and Mackman (4), demonstrating that PI3K negatively regulates LPS-induced NF-B activity.
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To examine the molecular details of SHIP influence on TLR4 signaling, we next assessed MAPK activation in BMM derived from SHIP+/+ and SHIP–/– littermates. In these experiments, BMM were serum-starved overnight and then stimulated with 500 ng/ml LPS for varying time points. Phosphorylation of the MAPKs ERK and p38 was analyzed by Western blotting protein-matched lysates with phospho-specific ERK and p38 Abs (Fig. 3, A and B, upper panels). Results indicated that robust ERK and p38 phosphorylation was induced in SHIP+/+ BMM. In contrast, ERK and p38 phosphorylation were reduced in SHIP–/– BMM. The same membranes were reprobed for actin, to ensure equal loading in all lanes (Fig. 3, A and B, middle panels). In parallel experiments to confirm that the SHIP–/– cells were indeed deficient in SHIP expression, whole cell lysates were probed with anti-SHIP Ab (Fig. 3C). To ensure that the signaling differences observed in the SHIP+/+ and SHIP–/– cells were not due to a difference in the expression of TLR4, TLR4 immunoprecipitates were probed with anti-TLR4 Ab (Fig. 3D). These results indicate that the influence of SHIP on TLR4-induced signaling occurs upstream of ERK and p38.
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Recent studies indicated that Akt activation leads to a down-regulation of LPS-induced MAPK activation (4). Overexpression of a dominant-negative Akt in these studies led to enhanced transcriptional activity driven by NF-B, AP-1, and Egr-1. To test whether the suppression of MAPK phosphorylation in the SHIP–/– macrophages is accompanied by enhanced Akt activation, serine phosphorylation of Akt was compared in SHIP+/+ and SHIP–/– BMM stimulated with LPS. The results shown in Fig. 4A indicate that Akt phosphorylation is induced by LPS in SHIP+/+ BMM by 15 min after stimulation. In contrast, Akt phosphorylation in SHIP–/– BMM is constitutively high, and remains enhanced after LPS stimulation. These results suggest that SHIP may positively regulate LPS-induced MAPK activation by suppressing the activation of Akt.
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LPS-induced inflammatory cytokine production is down-regulated in SHIP-deficient macrophages
To address whether the suppression of LPS-induced signaling events in SHIP–/– BMM is reflected in functional outcomes, we next assessed cytokine production induced by LPS. For this, SHIP+/+ and SHIP–/– BMM were stimulated for varying time points with 500 ng/ml LPS. Cell supernatants were harvested and analyzed for TNF- by ELISA. Results indicated that TNF- production was suppressed significantly in SHIP–/– BMM compared with SHIP+/+ BMM (Fig. 5A). Parallel experiments revealed that LPS-induced IL-6 production was also significantly lower in SHIP–/– macrophages than in SHIP+/+ macrophages (Fig. 5B). These results are consistent with our above observations that SHIP serves to positively regulate TLR4 function.
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R-mediated signaling
The above findings are in contrast to previous reports demonstrating a negative regulatory role for SHIP in immune receptor and growth factor receptor signaling (10, 16, 17, 21, 22, 23). Thus, to test whether the macrophages used in our experiments would respond differently to other stimuli, we stimulated the cells by clustering FcRs. To cluster FcR, BMM were first incubated with mAb 2.4G2 anti-murine FcRII/III, followed by cross-linking with mouse F(ab')2 anti-rat IgG secondary Ab, and the resultant ERK phosphorylation was analyzed. Consistent with other reports (24), results indicated that ERK phosphorylation is higher in SHIP–/– macrophages in comparison to SHIP+/+ macrophages (Fig. 6A). Both wild-type and knockout macrophages showed equivalent FcR expression as determined by flow cytometry using mAb 2.4G2 by methods described previously (Fig. 6B) (17). These findings indicate that the role of SHIP as a positive or negative regulator of signaling events is stimulus specific.
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Discussion |
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PI3K is reported to suppress the LPS-induced inflammatory response through the activation of Akt (4). Akt is a serine/threonine kinase that is activated in a PtdIns3,4,5P3-dependent manner (26) and appears to exert its inhibitory influence on LPS-induced activation events at two different levels (Fig. 7). First, Akt activation leads to the suppression of MAPK activation. Thus, Akt has been shown to phosphorylate and inactivate MEK kinase 3, resulting in the suppression of p38 (27, 28). Other studies indicate that Akt suppresses ERK by inactivating the upstream Raf-1 kinase (29). A second mechanism of inhibition by Akt is reported to occur through the phosphorylation and inactivation of GSK-3
, resulting in down-regulation of NF-B transcriptional activity (4). Our current observations that in SHIP–/– BMM, where Akt activation is constitutively enhanced, LPS-induced MAPK phosphorylation and inflammatory cytokine production are down-regulated support these previous reports.
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Rs. Other studies have indicated an essential role for the carboxyl-terminal region of SHIP in membrane association and function of SHIP (31, 32). Our findings that overexpression of SHIP SH2 domain had no influence on LPS-induced NF-B activation suggest that the SH2 domain may not be involved in membrane translocation of SHIP during TLR4 signaling (Fig. 2). The same SHIP SH2 domain construct has been shown by our group and others to function in a dominant-negative manner in influencing FcR-mediated function. In the latter case, the SH2 domain is critical for membrane translocation of SHIP. Further studies are needed to understand the molecular mechanism of SHIP translocation to the membrane in response to LPS stimulation.
We, and others, have previously reported a negative regulatory role for SHIP in the activation of NF-B and the Ras/ERK pathway by immune receptors and growth factor receptors (14, 17, 19, 33, 34). Paradoxically, our current findings show that SHIP positively regulates MAPK and NF-B activation during LPS signaling. Indeed, a positive role for SHIP has been previously reported by Rothman and colleagues (35) in 32D myeloid cells stimulated with IL-4. These investigators found that IL-4-induced proliferative response is enhanced in the presence of SHIP. Thus, it would seem that the role of SHIP as a negative or a positive regulator of signaling events is stimulus specific, and likely involves as yet unidentified downstream targets that are distinct for the various stimuli.
In summary, we have investigated the role of the inositol phosphatase SHIP in LPS-mediated activation of macrophages. Our results demonstrate that SHIP serves to positively regulate LPS-induced MAPK activation and inflammatory cytokine production. These results demonstrate a role for inositol phosphatases in LPS-induced inflammatory response, and are consistent with a recent review article by Rauh et al. (36) that proposed a role for SHIP in LPS-induced macrophage responses based on their unpublished observations. Also consistent with this, we have recently observed that the inositol 3-phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which like SHIP consumes PtdIns3,4,5P3 and suppresses Akt activation, promotes LPS-induced signaling in macrophages (37). In contrast to the negative regulatory role of inositol phosphatases in immune receptor signaling, our data indicate that SHIP positively regulates TLR4 signaling. Based on our observations, we propose that macrophage inflammatory responses to bacterial LPS are finely tuned by the concerted actions of lipid kinases and phosphatases.
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Acknowledgments |
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Footnotes |
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2 H.F. and R.P. 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: IRAK, IL-1R-associated kinase; BMM, bone marrow-derived macrophages; NF-B-luc, NF-B luciferase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; PtdIns3,4,5P3, phosphatidylinositol-3,4,5-trisphosphate; SH2, Src homology 2.
Received for publication December 8, 2003. Accepted for publication April 19, 2004.
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J. P. Luyendyk, G. A. Schabbauer, M. Tencati, T. Holscher, R. Pawlinski, and N. Mackman Genetic Analysis of the Role of the PI3K-Akt Pathway in Lipopolysaccharide-Induced Cytokine and Tissue Factor Gene Expression in Monocytes/Macrophages J. Immunol., March 15, 2008; 180(6): 4218 - 4226. [Abstract] [Full Text] [PDF]
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