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. 2013 Jul;43(7):1907-13.
doi: 10.1002/eji.201243075. Epub 2013 May 13.

The interaction between C5a and both C5aR and C5L2 receptors is required for production of G-CSF during acute inflammation

Markus Bosmann  1 , Mikel D Haggadone, Firas S Zetoune, J Vidya Sarma, Peter A Ward
Affiliations

The interaction between C5a and both C5aR and C5L2 receptors is required for production of G-CSF during acute inflammation

Markus Bosmann et al. Eur J Immunol. 2013 Jul.

Abstract

The complement activation product, C5a, is a key factor for regulation of inflammatory responses. C5a and C5adesArg bind to their receptors, C5aR and C5L2, but the functional roles of C5L2 remain controversial. We screened the patterns of 23 inflammatory mediators in cultures of LPS-activated mouse peritoneal elicited macrophages (PEMs) in the presence or absence of recombinant mouse C5a. Production of most mediators studied was suppressed by C5a, whereas G-CSF production was enhanced. G-CSF gene expression and secretion from PEMs was amplified two- to threefold by C5a in a dose- and time-dependent fashion. The degradation product C5adesArg promoted lower levels of G-CSF. The effects of C5a on G-CSF were associated with activation of PI3K/Akt and MEK1/2 signaling pathways. C5a did not enhance G-CSF production in cultures of PEMs from either C5aR- or C5L2-deficient mice, indicating that both C5a receptors are indispensable for mediating the effects of C5a in the production of G-CSF. Finally, G-CSF levels in plasma during polymicrobial sepsis after cecal ligation and puncture were substantially lower in C5aR- or C5L2-deficient mice as compared with that in C57BL/6J WT mice. These findings elucidate the functional characteristics of the C5L2 receptor during the acute inflammatory response.

Keywords: Akt; Cecal ligation and puncture; MEK1/2; Macrophages; Sepsis.

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Conflict of interest statement

Conflict of interest: The authors declare no financial or commercial conflict of interest.

Figures

Figure 1
Figure 1
C5a-induced amplification of G-CSF production from macrophages. (A) Peritoneal elicited macrophages (PEMs) from C57BL/6J mice were stimulated for 8 h with LPS (1 μg/ml) alone or in combination with different doses of recombinant mouse C5a (10–100 nM) followed by detection of G-CSF by ELISA in supernatant fluids. (B) Time course of G-CSF secretion from PEMs after LPS (1 μg/ml) alone or together with rmC5a (100 nM), ELISA. (C) Comparison of activity of rmC5a (100 nM) and rmC5adesArg (100nM) to enhance G-CSF in supernatants from LPS-activated PEMs, 8h, ELISA. (D) Real time PCR of mRNA for G-CSF from PEMs left as untreated controls (Ctrl), after LPS (1 μg/ml), LPS plus C5a (100 nM) or C5a alone, 6 h.(E) Phospho-MEK1 (serine 217/serine 221)in PEMs after incubation with C5a (100 nM) and LPS (1 μg/ml) alone or in combination. Phosphorylation levels in untreated cells were used as 1-fold, bead-based assay. (F) Phospho-Akt (threonine 308) after stimulation with C5a, LPS or LPS plus C5a, bead-based assay. (G) Relative release of G-CSF from LPS-activated PEMs after blockade of PI3K/Akt with LY294002 (25 μM) or MEK1/2 with PD98059 (50 μM) or U0126 (1 μM), 8 h, ELISA. All experiments were done with cells from C57BL/6J mice. (H) PEMs were treated with combinations of LPS, C5a and LY294002 as indicated followed by quantitation of G-CSF, 8 h, ELISA.(I)PEMs (C57BL/6J) were incubated for 8 h with LPS alone or LPS plus rmIL-10 (10 ng/ml) followed by detection in supernatants of IL-1β, IL-12(p40), G-CSF, INFγ and TNFα. Amounts of cytokines after LPS alone were used as 100% values, bead-based assays.(J)Relative release of G-CSF in PEMs from C57BL/6J mice compared to PEMs from IL-10−/− mice after LPS ± C5a, 8h, ELISA. Data shown in each frame are from three independent experiments, student’s two-tailed t-test;*P<0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2
Studies on the production of G-CSF with genetic absence of the C5aR receptor or C5L2 receptor.(A) Relative release of G-CSF from PEMs from C57BL/6J (Wt), C5aR−/− or C5L2−/− mice after LPS (1 μg/ml) alone or together with C5a (100 nM), 8 h, ELISA. Results with LPS alone were used as the 100% value for each mouse strain.(B) Detection of G-CSF in plasma of C57BL/6J mice 12 h after endotoxic shock (LPS 10 mg/kg bodyweight i.p.). Mice were pretreated with neutralizing polyclonal anti-C5a antiserum or control (Ctrl) serum (500 μl i.p., n≥7/group), ELISA.(C) Plasma concentrations of G-CSF in C57BL/6J (Wt) mice after sham-OP (n=7) or cecal ligation and puncture (CLP, n=15) compared to concentrations after CLP in C5aR−/− mice (n=14) or C5L2−/− mice (n=11). Sham-OP was performed only with Wt mice, 24 h, ELISA. Data shown in frame A were from three independent experiments each done in duplicate wells, and data in frame B and C were analyzed using numbers of mice as indicated, student’s two-tailed t-test;*P<0.05, **P<0.01.
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References

    1. Bosmann M, Ward PA. Role of C3, C5 and anaphylatoxin receptors in acute lung injury and in sepsis. Adv Exp Med Biol. 2012;946:147–159. - PMC - PubMed
    1. Gerard C, Gerard NP. C5A anaphylatoxin and its seven transmembrane-segment receptor. Annual Review of Immunology. 1994;12:775–808. - PubMed
    1. Gerard NP, Gerard C. The chemotactic receptor for human C5a anaphylatoxin. Nature. 1991;349:614–617. - PubMed
    1. Wetsel RA. Expression of the complement C5a anaphylatoxin receptor (C5aR) on non-myeloid cells. Immunol Lett. 1995;44:183–187. - PubMed
    1. Bosmann M, Haggadone MD, Hemmila MR, Zetoune FS, Sarma JV, Ward PA. Complement activation product C5a is a selective suppressor of TLR4-induced, but not TLR3-induced, production of IL-27(p28) from macrophages. J Immunol. 2012;188:5086–5093. - PMC - PubMed

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