Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 204 (11), 2719-31

LPS-induced Down-Regulation of Signal Regulatory Protein {Alpha} Contributes to Innate Immune Activation in Macrophages

Affiliations

LPS-induced Down-Regulation of Signal Regulatory Protein {Alpha} Contributes to Innate Immune Activation in Macrophages

Xiao-Ni Kong et al. J Exp Med.

Abstract

Activation of the mitogen-activated protein kinases (MAPKs) and nuclear factor kappaB (NF-kappaB) cascades after Toll-like receptor (TLR) stimulation contributes to innate immune responses. Signal regulatory protein (SIRP) alpha, a member of the SIRP family that is abundantly expressed in macrophages, has been implicated in regulating MAPK and NF-kappaB signaling pathways. In addition, SIRPalpha can negatively regulate the phagocytosis of host cells by macrophages, indicating an inhibitory role of SIRPalpha in innate immunity. We provide evidences that SIRPalpha is an essential endogenous regulator of the innate immune activation upon lipopolysaccharide (LPS) exposure. SIRPalpha expression was promptly reduced in macrophages after LPS stimulation. The decrease in SIRPalpha expression levels was required for initiation of LPS-induced innate immune responses because overexpression of SIRPalpha reduced macrophage responses to LPS. Knockdown of SIRPalpha caused prolonged activation of MAPKs and NF-kappaB pathways and augmented production of proinflammatory cytokines and type I interferon (IFN). Mice transferred with SIRPalpha-depleted macrophages were highly susceptible to endotoxic shock, developing multiple organ failure and exhibiting a remarkable increase in mortality. SIRPalpha may accomplish this mainly through its association and sequestration of the LPS signal transducer SHP-2. Thus, SIRPalpha functions as a biologically important modulator of TLR signaling and innate immunity.

Figures

Figure 1.
Figure 1.
Increased signaling in SIRPα knockdown macrophages upon LPS stimulation. (A) RAW cells were stably transfected with empty vector or constructs containing shRNA specific for SIRPα or Myc-tagged SIRPα, and SIRPα expression levels were detected by Western blotting. (B) Cell proliferation of stable RAW cells was measured using CCK-8 assay at the indicated times. Data are the mean ± the SEM of triplicates from an experiment that was repeated a total of three times with similar results. (C) Strongly increased signaling in SIRPα-KD macrophage cell lines. 5 × 105 cells/well SIRPα-KD, -OV, and -VT RAW cells were stimulated with 10 ng/ml LPS for the indicated times. Cell lysates were prepared and blotted with the indicated antibodies. (D) Peritoneal macrophages were transiently transfected with siRNA (D10 and/or D12) targeting SIRPα or irrelevant control siRNA. The reduction of SIRPα expression was demonstrated by Western blotting. (E) 1.5 × 105 cells/well peritoneal macrophages from C57BL/6 mice were transfected with control or SIRPα siRNA, and then stimulated with 10 ng/ml of LPS for the indicated minutes. Cell lysates were blotted as mentioned in C. (F) 1 × 105 SIRPα-KD, -OV, and -VT cells were transfected with the NF-κB or AP-1 reporter plasmids (0.2 μg), together with the control plasmid pRL-TK (0.02 μg), and treated with various doses of LPS for 6 h, and then luciferase activities were detected. Data are expressed as relative fold activation to that of nonstimulated (−) sets. *, P < 0.05; **, P < 0.01 (OV or KD different from VT).
Figure 2.
Figure 2.
Increased cytokine production of SIRPα knockdown macrophages upon LPS stimulation in vitro. (A) 5 × 105 cells/well SIRPα-KD, -OV, and -VT cells were treated with different amounts of LPS for 12 or 24 h, after which culture supernatants were harvested for measurement of TNFα, IL-6, and NO production. (B) Cytokine production by LPS-challenged peritoneal macrophages. 3 × 105 cells/well transfected with SIRPα siRNA or negative control oligonucleotides. (C) Cytokine antibody array analysis of LPS-treated cytokine production in SIRPα-KD and -OV cells. Cells were primed with 100 ng/ml LPS for 12 h, after which culture supernatants were harvested for the cytokine antibody array analysis. The density value of each test sample was normalized to control spots on the same membrane graphed. (D) 5 × 105 cells/well SIRPα-KD, -OV, and -VT cells were primed with different amount of LPS for 12 h, after which culture supernatants were harvested for measurement of MIP-1α and RANTES by ELISA. (E) IFN-β production after LPS challenge in stable RAW cell lines (left) or in peritoneal macrophages transfected with SIRPα siRNA (right) were analyzed as mentioned above. (F) Cells were incubated with 100 ng/ml of LPS for the indicated time, and IFN-β mRNA levels were determined by quantitative real-time PCR. (G) Cells were transfected with the ISRE reporter plasmid (0.2 μg), together with the control plasmid pRL-TK (0.02 μg), and treated with various doses of LPS for 6 h, and then luciferase activities were detected. The data are representative of three independent experiments with similar results. (H) Peritoneal macrophages from C57BL/6 mice were transfected with control or SIRPα siRNA and then stimulated with 10 ng/ml of LPS for the indicated minutes. Cell lysates were blotted with anti–phospho-IRF3 and anti-GAPDH antibodies. Data are the mean ± the SEM of three independent experiments. *, P < 0.05; **, P < 0.01 (OV or KD different from VT in A, D, F, and G).
Figure 3.
Figure 3.
Increased response in mice transfered with SIRPα knockdown macrophages upon LPS stimulation in vivo. (A) Reconstitution of macrophage-depleted mice with RAW264.7 cells. Fluorescent dye-labeled RAW264.7 cells were injected i.v. into GdCl3-treated Balb/C mice. Mouse organs (spleen, liver, and lung) were preserved for fluorescence microscopy analysis. Numerous injected cells are detectable in lung, spleen, and liver. Tissue morphology is visualized by hematoxylin staining. (B) Similar lethality in mice reconstituted with RAW264.7 cells challenged with LPS. Age- and sex-matched cohorts of mice (n = 6) were pretreated with GdCl3 (10 mg/kg of body weight) or PBS and, 24 h later, were i.v. injected with RAW264.7 cells (107/each) or PBS, respectively. Another 24 h later, mice were i.p. administered with 25 mg LPS/kg of body weight, and lethality was observed over 60 h after this challenge. The data are representative of two independent experiments with similar results. (C) More lethality in mice transfered with SIRPα-KD cells challenged with LPS. Age- and sex-matched cohorts of mice (n = 7) were pretreated with GdCl3 (10 mg/kg of body weight), and 24 h later were i.v. injected with SIRPα-KD, -OV, and -VT cells (107/each). Another 24 h later, mice were i.p. administered with PBS or 20 mg LPS/kg of body weight, and lethality was observed over 120 h after this challenge. The data are representative of two independent experiments with similar results. (D) Sera from mice pretreated as described in A and injected with PBS or LPS (10 mg/kg of body weight) were collected at 3 h after the challenge, and IL-6 and TNFα levels were measured by ELISA. Data show the mean ± the SEM for three mice from each group. *, P < 0.05; **, P < 0.01 (OV or KD different from VT). (E) Severe multiple organ failure in mice transfered with SIRPα-KD cells challenged with LPS. Representative images of lung and liver with histological sections from mice pretreated as described in C and killed at 24 h after LPS administration (10 mg/kg weight body). Bars, 100 μm.
Figure 4.
Figure 4.
SIRPα is not required for endotoxin tolerance. (A) 5 × 105 cells/well SIRPα-KD, -OV, and -VT cells were untreated or tolerized with 100 ng/ml LPS for 24 h, washed, and rechallenged with 100 ng/ml LPS. After 12 h of incubation, the media were assessed for TNFα and IL-6, and for NO levels after 24 h. Data are presented as the mean ± the SEM of 3–8 independent experiments. (B) 5 × 105 cells/well SIRPα-KD, -OV, and -VT cells were tolerized with LPS or CpG for 24 h, washed, and rechallenged with the indicated doses of LPS or CpG. TNFα, IL-6, and NO levels were determined as described in A. (C) 3 × 105 cells/well peritoneal macrophages from C57BL/6 mice were transfected with either control or SIRPα siRNA. 24 h later, cells were tolerized with LPS or CpG for 24 h, washed, and rechallenged with the indicated doses of LPS or CpG. TNFα, IL-6, and NO levels were determined as described in A.
Figure 5.
Figure 5.
LPS-induced SIRPα reduction contributes to macrophage activation. (A) Stable RAW-derived cell lines (left) and peritoneal macrophages transiently transfected with either control or SIRPα siRNA (right) were incubated with or without 100 ng/ml LPS for 12 h. Immunoblots with a TLR4-specific antibody were performed. (B) Down-regulation of SIRPα protein levels in macrophages after LPS treatment. Mouse macrophage cell lines RAW264.7 and J774A.1, as well as peritoneal macrophages from C57BL/6 mice, were incubated with 100 ng/ml LPS for the indicated times (left) or with the indicated LPS doses for 12 h (right), and SIRPα protein expression was determined by Western blotting. PWC, peritoneal macrophages. (C) Down-regulation of SIRPα mRNA levels in macrophages after LPS treatment. RAW264.7 macrophages were incubated with 100 ng/ml of LPS for the indicated time (left), or with the indicated LPS doses for 12 h (right), and SIRPα mRNA levels were determined by quantitative real-time PCR. (D) The reduction of SIRPα protein level involves protein degradation. RAW264.7 cells were incubated for 2 h at 37°C in the absence or presence of 100 μM chloroquine, 10 μM NH4Cl, 40 μM MG132, or 50 μg/ml CHX, after which they were treated with or without 100 ng/ml LPS for 5 h and subjected to Western blot analysis using SIRPα-specific antibody. (E) TLR4 is essential for the down-regulation of SIRPα. Peritoneal macrophages from TLR4 KO (C57BL/10ScCr) and littermate WT1 (C57BL/10snj) mice or TLR4 mutant (C3H/HeJ) and littermate WT2 (C3H/Heouj) mice were incubated with 100 ng/ml LPS for the indicated times, and SIRPα protein expression was determined by Western blot analysis.
Figure 6.
Figure 6.
SIRPα inhibits LPS signaling mainly through sequestration of SHP-2. (A) RAW264.7 cells were stimulated with LPS for the indicated time, immunoprecipitated for endogenous SIRPα, and probed with an antiphosphotyrosine antibody. Immunoblots were also probed with anti-SIRPα, –SHP-1, and –SHP-2 antibodies. (B) Basal association of SHP proteins is dependent on SIRPα phosphorylation. RAW264.7 cells were treated with vehicle (DMSO), 10 μM PP2, or 50 μM genistein for 1 h, and then subjected to immunoprecipitation and immunoblot, as in A. (C) TLR4-induced SIRPα phosphorylation is mediated by Src kinases. RAW264.7 cells were stimulated with LPS for 15 min in the absence or presence of DMSO, PP2, or genistein, and then subjected to immunoprecipitation and immunoblotting as in A. (D) Western blot analysis demonstrates the effects of siRNAs that specifically down-regulate SHP-1 or -2 expression in peritoneal macrophages. (E) Peritoneal macrophages transfected with SHP-1– or -2–specific siRNAs were stimulated with 100 ng/ml LPS for 12 h, and the production of TNFα and IL-6 was determined using ELISA. Data are presented as the mean ± the SEM of 3–6 independent experiments. *, P < 0.05; **, P < 0.01 (SHP-1 or SHP2 siRNA different from negative control siRNA). (F) IFN-β production after LPS challenge in peritoneal macrophages. 3 × 105 cells/well transfected with SHP-1– or -2–specific siRNAs was analyzed as in E. *, P < 0.05; **, P < 0.01 (SHP-1 or -2 siRNA different from negative control siRNA). (G) Peritoneal macrophages transfected with SHP-1 or -2 siRNAs or negative control oligonucleotides were stimulated with 10 ng/ml of LPS for the indicated times. Cell lysates were blotted with the indicated antibodies. (H) Peritoneal macrophages were transfected with SIRPα-specific siRNA alone or together with SHP-1– or -2–specific siRNAs with subsequent LPS stimulation. The production of TNFα and IL-6 were determined as described in E. *, P < 0.05; **, P < 0.01 (SIRP1α/SHP-1 or SHP-2 double-knockdown is different from SIRP1α knockdown alone). (I) Rescue of cytokine production by reintroduction of WT SIRPα. 2 × 106 cells/well SIRPα-KD cells transfected with GFP, WT SIRPα (WT), or its mutant form SIRPα-4F (4F) were treated with 100 ng/ml LPS. The amounts of secreted TNFα and IL-6 in supernatants were determined by ELISA as described in E. Data are the mean ± the SEM of three independent experiments. **, P < 0.01 (WT different from GFP).
Figure 7.
Figure 7.
SIRPα prevents LPS-induced SHP-2–IKK complex formation. (A) RAW264.7 cells were treated with 100 ng/ml LPS for the indicated time period. Equal amounts of cell lysates were immunoprecipitated with SHP-2–specific antibody. Precipitated proteins and cell lysates were blotted with anti–SHP-2, anti-IKKβ, and anti-TBK1 antibodies. (B) SIRPα-KD, -OV, and -VT RAW cells were stimulated with 100 ng/ml LPS for the indicated minutes, immunoprecipitated for endogenous SHP-2, and probed with anti-IKKβ, -TBK1, and −SHP-2 antibodies.

Similar articles

See all similar articles

Cited by 47 PubMed Central articles

See all "Cited by" articles

References

    1. Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2:675–680. - PubMed
    1. Takeda, K., and S. Akira. 2005. Toll-like receptors in innate immunity. Int. Immunol. 17:1–14. - PubMed
    1. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21:335–376. - PubMed
    1. Beutler, B. 2000. Tlr4: central component of the sole mammalian LPS sensor. Curr. Opin. Immunol. 12:20–26. - PubMed
    1. Kawai, T., and S. Akira. 2006. TLR signaling. Cell Death Differ. 13:816–825. - PubMed

Publication types

MeSH terms

Feedback