Nucleotide and corrected amino acid sequence of the functional recombinant rat anaphylatoxin C5a

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Although monocytes can be directed to develop into dendritic cells (DC) in vitro, the molecular mechanisms that induce their transformation in vivo are largely unknown. In the present study we employed an in vivo SCID mouse model to investigate the impact of two proinflammatory chemotaxins, the anaphylatoxin C5a and the chemokine macrophage inflammatory protein-1α (CCL3), on the differentiation of human monocytes and immature DC generated from monocytes in the presence of GM-CSF and IL-4. Both C5a and macrophage inflammatory protein-1α recruited human monocytes and immature DC into the peritoneal cavity of SCID mice, but only C5a induced their differentiation into phenotypically mature DC by 48 h after injection. Macrophages derived from monocytes by in vitro culture were resistant to C5a-mediated transformation in vivo. The effect of C5a was indirect, since C5a-stimulated TNF-α and PGE2 were found to be obligatory as well as sufficient to induce differentiation of monocytes. In contrast to monocytes, in vitro generated immature DC required TNF-α, but not PGE2, for their C5a-mediated maturation in vivo. C5a-transformed monocytes represented an inflammatory type of DC, as they constitutively secreted high amounts of TNF-α, but also retained the capacity to release the Th1 cytokine IL-12 p70 upon stimulation with CD40 ligand. In summary, we identified for the first time a cascade of inflammatory signals that can induce the transformation of monocytes into DC in vivo. This novel function emphasizes the important immunoregulatory role of C5a at the interface of innate and adaptive immunity.

Section: Reagentsmentioning

confidence: 99%

Anaphylatoxin C5a Induces Monocyte Recruitment and Differentiation into Dendritic Cells by TNF-α and Prostaglandin E2-Dependent Mechanisms

Soruri¹,

Riggert

Schlott³

et al. 2003

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“…X91892, ID: RNC5AARPT) (4) as described previously (4,16). RrC5a contained, in addition to the original sequence of amino acids 1-77, the N-terminal sequence MRGSHHHHHHGS used for its purification from bacterial lysates by Ni 2ϩ -chelate chromatography and was depleted of endotoxins by affinity chromatography on polymyxin B agarose (Sigma).…”

Section: Preparation Of Rrc5amentioning

confidence: 99%

“…C5a, a glycopeptide of 77 (rat) and 74 (human) amino acids, respectively (1)(2)(3)(4), is the N-terminal cleavage product of the ␣-chain of the precursor protein C5. C5a is involved in systemic defense reactions by causing contraction of smooth muscle cells, an increased vascular permeability, degranulation of mast cells, and activation, chemotaxis, and margination of neutrophils (5)(6)(7).…”

mentioning

confidence: 99%

Anaphylatoxin C5a Actions in Rat Liver: Synergistic Enhancement by C5a of Lipopolysaccharide-Dependent α2-Macroglobulin Gene Expression in Hepatocytes Via IL-6 Release from Kupffer Cells

Mack¹,

Jungermann²,

Götze

et al. 2001

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The effects of the anaphylatoxins C5a and C3a on the liver are only poorly characterized in contrast to their well known systemic actions. Recently, it has been demonstrated that the anaphylatoxin C5a enhanced glucose output from hepatocytes (HC) indirectly via prostanoid release from Kupffer cells (KC). In the present study, it is shown that recombinant rat C5a (rrC5a), together with LPS, activated the gene of the acute phase protein α2-macroglobulin (α2MG) in HC also indirectly via IL-6 release from KC. RrC5a alone increased neither IL-6 mRNA in nor IL-6 release from KC, whereas LPS alone did so. However, rrC5a synergistically enhanced the LPS-dependent increase in IL-6 mRNA and IL-6 release. Only rIL-6, but not TNF-α or IL-1β, enhanced α2MG mRNA in HC. In line with the actions of rrC5a and LPS on KC, conditioned medium of KC stimulated only with rrC5a did not increase α2MG mRNA in HC. However, medium of KC stimulated with rrC5a plus LPS induced α2MG mRNA expression in HC more strongly than medium from cells stimulated only with LPS; thus, C5a acted synergistically with LPS. The stimulatory effects of KC-conditioned medium could partially be inhibited by a neutralizing anti-IL-6 Ab, indicating that KC-derived IL-6 was a major mediator in C5a- plus LPS-elicited α2MG gene expression. These results suggest that C5a, besides enhancing glucose output via prostanoids, is involved in the initiation of the acute phase response in HC via proinflammatory cytokines from KC. This provides evidence for another important function of C5a in the regulation of hepatocellular defense reactions.

“…C5a, a glycopeptide of 77 and 74 aa in rat and human, respectively (1)(2)(3)(4), causes the degranulation of mast cells, the contraction of smooth muscle cells, an increase in vascular permeability, and the chemotaxis and activation of neutrophils with local release of reactive oxygen species, eicosanoids, and cytokines (5-7). C5a receptors (C5aR) 2 have been cloned in various species including human (8,9), mouse (10), dog (11), guinea pig (12), and rat (13,14).…”

Section: Induction Of Functional Anaphylatoxin C5a Receptors On Hepatmentioning

confidence: 99%

“…X91892, ID: RNC5AARPT) (4) as described previously (4,24). RrC5a contained in addition to the original sequence of amino acids 1-77 the N-terminal sequence MRGSHHHHHHGS used for its purification from bacterial lysates by Ni 2ϩ -chelate chromatography and was depleted of endotoxins by affinity chromatography on polymyxin B agarose.…”

Section: Preparation Of Rrc5amentioning

confidence: 99%

Induction of Functional Anaphylatoxin C5a Receptors on Hepatocytes by In Vivo Treatment of Rats with IL-6

Schieferdecker¹,

Schlaf

Koleva³

et al. 2000

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In normal rat liver, anaphylatoxin C5a receptors (C5aR) are only expressed by nonparenchymal cells, mainly Kupffer cells and hepatic stellate cells, but not by parenchymal cells, i.e., hepatocytes (HC). Nevertheless, C5a stimulates glucose output by HC. This HC-specific defense reaction is induced indirectly via prostanoids secreted by the C5aR-expressing Kupffer cells and hepatic stellate cells. It is shown here that under inflammatory conditions simulated by in vivo treatment of rats with IL-6 C5aR mRNA and protein were induced in HC in a time-dependent manner. Maximal mRNA and protein expression were observed at 4–8 h and 8–10 h, respectively, after IL-6 injection. The newly expressed receptors were functional, because recombinant rat C5a significantly activated glycogen phosphorylase in HC isolated from IL-6-treated but not in HC from control rats. In perfused livers of IL-6-treated animals in contrast to control animals, recombinant rat C5a-induced glucose output was not impaired by inhibition of prostanoid synthesis and function with the cyclooxygenase inhibitor indomethacin and the thromboxane receptor antagonist daltroban. These results indicate that HC-specific defense reactions might be differently regulated under normal and inflammatory conditions as shown here for the indirect prostanoid-dependent or direct C5a-induced activation of hepatocellular glycogen phyosphorylase and glucose output in control or IL-6-treated rats, respectively.