Eur. J. Immunol. 1990. 20: 253-257 C5a enhances LPS-induced IL 1 and TNF release 253 zy Jean-Marc Cavaillono, Catherine Fittingo zyxwvuts and Nicole Haeffner-CavaillonA Unit6 d'Immuno-Allergic, Institut PasteurO and INSERM U28, H6pital Broussaisa, Paris 1 Introduction Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocyt es and macrophages Lipopolysaccharide (LPS) is a potent inducer of interleukin 1 (IL 1) synthesisand release, and of tumor necrosis factor (TNF) secretion. Many signals can enhance the LPS-induced production of these cytokines.Wehave previously observed that addition of low amounts of normal human serum to the culture medium enhances IL 1 production. Among serum factors, anaphylatoxinsC3a and C5a and/or their desArg derivatives have been shown to enhance LPS-induced IL 1 and TNF production. However, the capacity of natural anaphylatoxins to induce by themselves the production of cytokines remains a controversal issue. We have investigated the capacity of human recombinant C5a (hrC5a) to induce IL 1 and TNF production. Despite its lack of direct triggering, hrC5a was able to act synergistically with LPS, leading to higher IL 1 and TNF release by human monocytes and mouse peritoneal macrophages. As assessed by the comitogenic assay, hrC5a increased IL 1 release, whereas cell-associated IL 1 activity was not significantlymodified. Measurement by enzyme-linked immunosorbent assay of human IL lp led to similar conclusions, whereas measurement of IL la by radioimmunoassay indicated, in addition, an increase in intracellular IL la. LPS of Gram-negative bacteria is one of the most potent activatorsof monocytes and Ma, and many of its biological activities are due to its capacity to trigger the production and release of IL 1 and TNF-a by monocytes/M<P. Conse- quently, many others cytokines such as IL 2 [l], IL 4 [2], IL 6 [3], CSF [4, zyxwvutsr 51 and platelet-derived growth factor [6] are produced by IL 1-stimulated cells. Some of these cytokines are able to up-regulate or down-regulate the production of IL 1 induced by LPS. Serum [7] and serum components, such as anaphylatoxins, have been reported to act synergistically with LPS. We reported that native C3dC3adesArg induces IL 1 production by human mono- cytes and enhances IL 1 release induced by LPS or its polysaccharide moiety [8]. Native C5a was also shown to induce the production of IL 1 and TNF by human mono- cytes and to amplify their production induced by LPS [9-111. The synergistic action of anaphylatoxins and LPS might be relevant to Gram-negative infections since LPS are known to lead to complement activation. However, Arend et al. [12] proposed that activities of the anaphyla- toxins are due to LPS contamination. Since all reported data have been obtained with native purified anaphylatox- ins, we decided to investigate the capacity of human rC5a (hrC5a) to induce the production of I L 1 and TNF by human monocytes and mouse peritoneal M@ as well as its ability to act synergistically with LPS. [I 78791 Correspondence: Jean-Marc Cavaillon zyxwvutsr , Unit6 d'Immuno-Allergie , Institut Pasteur, 28, rue Dr. Roux, F-75724 Pans Cedex 15, France Abbreviations: hrCsS: Human recombinant C5a NSE: Non- specific esterase 2 Materials and methods 2.1 Reagents hrC5a was purchased from Sigma (St. Louis, MO), recon- stituted in saline, aliquoted and kept in liquid nitrogen. As judged by colorimetric limulus assay, the LPS contamina- tion was 1 pg/pg rC5a. Calcium ionophore was purchased from Sigma. IL la and p were a generous gift of Rh8ne Poulenc (Vitry/Seine, France). LPS were prepared by phenol water procedure [13] ; zyx Neisseria meningitidis LPS was kindly provided by Dr. M. Caroff (Inst. Biochimie, Orsay) and Escherichia coli 0111: B4 LPS was obtained from Sigma. 2.2 Histamine release Histamine release was performed as described by Le Mao et al. [14]. Briefly, human leucocytes obtained by glucose dextran sedimentationwere washed, resuspended inTris/al- bumin/Ca2+ ,Mg2+ buffer and incubated for 40 min at 37 "C with or without histamine release inducers. Cells were centrifuged, and SN treated with 4 N perchloric acid. The histamine content was measured by fluorimetric assay as described by Lebel et al. [15]. 2.3 Cell cultures and IL 1 assays Human monocytes were selected by adherence from PBMC obtained after centrifugation on Ficoll-Hypaque (MSL; Eurobio, Paris, France) as previously described [7]. Briefly, 5 x 105 nonspecific esterase-positive cells (NSE+) were allowed to adhere in 24-well multidish plates (Nunclon, Roskilde, Denmark). Adherent cells were cultured for 24 h in 0.5 ml FWMI 1640medium (Gibco, Uxbridge, GB) in the absence of serum and in the absence of presence of IL 1 z 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990 0014-2980/90/0202-0253$02.50/0