Eur. J. Immunol. 1992. 22: 1461-1466

Christine Couturier, Gerlinde JahnsO, Michel D. Kazatchkine and Nicole Haeffner-Cavaillon INSERM U28, H8pital Broussais, Paris

LPS receptors involved in cytokine release

1461

Membrane molecules which trigger the production of interleukin-1 and tumor necrosis factor-a by lipopolysaccharide-stimulated human monocytes* We have investigated the role of the membrane molecules CD11/CD18 and CD14 which may mediate the binding of lipopolysaccharide (LPS) to human monocytes, in the induction of the production and release of interleukin (1L)-1 and tumor necrosis factor-a (TNF-a) by LPS-stimulated cells. Blockade of CD1la, C D l l b and CD18 with saturating concentrations of specific mAb did not inhibit the release of cytokines from LPS-stimulated monocytes. In contrast, inhibition of the release of IL-1fi and TNF-a occurred in monocytes cultures that had been pretreated with either of two monoclonal antibodies (mAb) recognizing different epitopes on the CD14 molecule.The binding of LPS to CD14 has been previously shown to require serum factors. In the present study, we found that serum had an enhancing effect on the release of IL-1 and TNF-a from LPS-stimulated cultures of normal human monocytes. The inhibitory effect of antLCD14 mAb was, however, observed in cultures performed in the presence or in the absence of serum, suggesting that triggering of IL-1ITNF-a release by CD14 is independent of LPS-binding proteins or other serum proteins. IL-1fi and TNF-a were also released from LPS-stimulated cultures of monocytes from patients with paroxysmal nocturnal hemoglobinuria lacking expression of CD14. Thus, CD14 but not CDllICD18 can trigger serum-dependent and independent cytokine release from endotoxin-stimulated normal human monocytes; CD14 is not, however, the only LPS receptor that is involved in the secretory response of endotoxin-stimulated cells.

1 Introduction Stimulation of normal human monocytes with lipopolysaccharide (LPS) results in the synthesis and release of interleukin-1 (IL-l), tumor necrosis factor-a (TNF-a), IL-6, and in a wide range of other secondary responses [l]. Induction of these activities is mediated by the interaction of the polysaccharide and/or lipid A of LPS with membrane components of the monocyte [2, 31. Using radiolabeled LPS, three sites mediating the binding of LPS have been identified on human monocytes: (a) the glycolipidanchored CD14 molecule [4] which serves as a specific binding site for intact LPS in the presence of serum protein(s) [5]. A serum protein that may be involved in this interaction is the recently identified LPS-binding protein (LBP) which mediates the attachment of complexes of LPS with LBP to CD14 [6, 71; (b) an as-yet-unidentified molecule which mediates the serum-dependent binding of the

[I 102441

purified polysaccharide of LPS from Salmonella minnesota R7 and (c) nonspecific (i.e. non-saturable) binding sites which mediate hydrophobic interactions of LPS with the monocyte membrane [4]. Other experimental approches have indicated a role for the CD11ICD18 Leu-CAM complex in the binding of LPS to monocytes [8], have led to the isolation of an 80-kDa molecule which serves as a receptor for intact LPS and lipid-A [9, 101 and to the characterization of a 38-kDa molecule of the monocyte membrane which recognizes the KDO residues of LPS”. The present study was aimed at investigating the role of CDllICD18 and of CD14 in the production of IL-1 and TNF-a by LPS-stimulated monocytes. The results indicate that CD14 but not CD11ICD18 may trigger the production and release of the cytokines by LPS-stimulated cells. Since intact LPS also induces cytokine release from cells lacking CD14 and from normal cells cultured in the absence of serum, the serum protein-dependent interaction of LPS with CD14 is not, however, the only pathway of cytokine induction by endotoxins in human monocytes.

* This work was supported by Institut National de la santC et de la Recherche Medicale (INSERM), Agence Nationale de Recherches sur le Sida (ANRS), Association pour la recherche sur le Cancer (ARC-6766) and Groupement d’Inter&t Public “Therapeutiques Substitutives”. Recipient of a grant from Association pour I’Utilisation du Rein Artificiel (Paris) and from the Deutsche Gesellschaft fur Klinische Chemie. Correspondence: Nicole Haeffner-Cavaillon, INSERM U28, HBpital Broussais, 96 rue Didot, F-75674 Paris CEDEX 14, France Abbreviations: LBP: Lipopolysaccharide-binding PNH: Paroxysmal nocturnal hemoglobinuria

protein

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992

2 Materials and methods 2.1 Reagents and antibodies Indomethacin was purchased from Sigma Chemical, St. Louis, MO. LPS from Neisseria meningitidis (N.m. LPS) was a gift from D. Shulz, Institut MCrieux, Lyon, France.

*

Morrison, D. C., Lei, M. G , Chen,T. Y., Flebbe, L. M., Halling, G., Field, S. Dwyer, D. and Baykousheva, S., Characterization of endotoxin-specific receptors on mammalian lymphoreticular cells, Circ. shock 1991. 34: abstract 116.

0014-2980/92/0606-1461$3.50+ .25/0

1462

C. Couturier, G. Jahiis, M. D. Kazatchkinc and N. Haeffncr-Cavaillon

The following anti-CD1 l/CD18 monoclonal antibodies (mAb) were used: anti-CDllb mAb MN41 (a gift from Dr. G. D. Ross, Louisville, KE) and 4B3 (Dr. E . Brown, St. Louis, MO), and anti-CD 18 mAb BL5 (Dr. J. Brochier, Montpellier, France). Anti-CD14 inAb were: anti-Leu-M3 (Becton Dickinson, Mountain view, CA), My4 (Coultronics, Hialeah, FL) and TOM2 (Immunotech, Luminy, France). The non-overlaping character of the epitopes rccognized by mAb anti-Lcu-M3, My4 and IOM2 on the CD14 molecule was assessed by competitive binding studies using cytofluorometry. Anti-HLA class I mAb IOT2 (clone BY. 12.1) was purchased from Immunotech. Anti-CR1 (CD35) mAb J3D3 was prepared as described [ l l ] . Noncommercially obtained mAb were purified from ascites fluid by contraprecipitation with octanoic acid [12]. 2.2 Monocytes and monocyte cultures Peripheral blood mononuclear cells (PBMC) were prepared by centrifugation of citrated venous blood from healthy adult donors and from seven patients with paroxysmal nocturnal hemoglobinuria (PNH) on MSL (Milieu de Skparation dcs Lymphocytes; Eurobio, Paris, France). Each experiment dealing with normal monocytes was pcrforrned with cells from at least four healthy donors. PBMC were enriched in monocytes by allowing the cells to adhere to plastic culture dishes (Costar, Cambridge, MA) for 45 min at 37°C in the absence of serum. Under these conditions, adherent cclls contained >85 YOmonocytes as assessed by morphological analysis by phase contrast microscopy and histochemical staining for nonspecific estcrase activity (NSE) using a-naphtyl acetate as a substrate [MI. Mononuclear adherent cells (5 x lo5 NSE+ cells/well) were cultured in RPMI 1640 medium (Whittaker MA Bioproducts, Walkersville, MD) containing 100 IU/ml penicillin, 100 pg/ml streptomycin, and 1 pg/ml indomethacin in the prcsence or absence of cytokine inducers and in thc presence or absence of normal human AB serum (NHS). RPMI 1640 contained no detectable LPS as assessed by the Limulus amebocyte lysate assay and by the lack ot induction of IL-1 in cultures of monocytes in mcdium alone [14]. 2.3 Assays for 1L-la, IL-16 and TNF-a IL-1 activity was quantitated by means of a conventional Con A co-mitogenic assay using C3H/HeJ thymocytes [15]. Extracellular TL-1 activity was measured in culture supernatants. To dctermine cell-associated IL-1, mononuclear adhcrent cells werc lyzed by three freeze-thaw cycles at -80°C in RPMI 1161; monocyte lysates were centrifuged at 2000 rpm for 10 min and TL-I activity was assessed in the supernatants. Concentrations of IL-la and IL-lP antigens were determined using an ELISA from Immunotech. The concentration of TNF-a was determined using a radioimmunoassay from Amersham (Les Ulis, France). 2.4 Cytoflnorometry Expression of CD14 on monocytes was assessed by direct immunofluorescence and cytotluorometry using a FACScan analyzer (Becton Dickinson, Mountain View, CA).

Eur. J. Immunol. 1992. 22: 1461-1466

PBMC from normal donors and from patients with PNH were labeled with phycoerythrin-labeled mAb anti-Leu-M3 or tluorescein-labeled mAb IOM2. Analysis of CD14+ cells was performed on monocytes gated by light scatter.

3 Results 3.1 Role of CD11/CD18 in the release of IL-1 and TNF-a by LPS-stimulated normal human monocytes We investigated the release of IL-1 and TNF-a by LPSstimulated normal human monocytes from four donors on which CD11/CD18 sites had been blocked by mAb directed against the a and (3 chains of the Leu-CAM complex. Cultured monocytes were incubated with either antiC D l l a mAb 25.3.1, anti-CDllb mAb MN41 or antLCD18 mAb BL-5 for 1 h at room temperature before addition of LPS and further culture of the cells in the presence or absence of 0.2 % (dv) NHS for 24 h.The mAb were used at saturating concentrations that had been determined in preliminary binding experiments of radiolabeled antibodies to adherent monocytes [17]. Binding of MN41 totally inhibited the formation of C3bi-dependent rosettes by adherent monocytes (data not shown). Pretreatment of the cells with anti-CDll/CDlS mAb did not inhibit the release of IL-1 and TNF-a by LPS-treated monocytes (Table 1). Rather, pretreatment with the mAb resulted in a slightly enhanced IL-1 and TNF-a response by the cells. 3.2 Role of CD14 in the release of IL-1 and TNF-a by LPS-stimulated monocytes from normal individuals and from patients with PNH Two antLCD14 mAb, My4 and IOM2 which recognize distinct epitopes on the CD14 molecule were used in these experiments. Previous experiments had shown that preincubation of monocytes with saturating concentrations of the antibodies (1 pg/106 cells) inhibited the specific serumdependent binding of radiolabeled N.m. LPS to the cells [4]. None of the antibodies alone induced IL-I nor TNF-a release from cultured monocytes. To investigate the role of CD14 in mediating LPS-induced release of IL-1 and TNF-a, adherent monocytes fromfour healthy donors were preincubated with saturating concentrations of mAb My4 and IOM2 and then cultured in the presence of LPS and of 0.2% NHS for 24 h. As shown in Fig. 1, mAb My4 and IOM2 inhibited the release of IL-lP induced by LPS from monocytes. Isotype-matched control mAb directed against monocyte surface molecules (i.e. anti-CD35 IgGl J3D3 and anti-HLA class I IgGza IOT2) did not exhibit any inhibitory activity. Pretreatment of monocytes with mAb My4 and IOM2 also inhibited the release of TNF-a by cells stimulated with low doses of LPS ( < I pg/5 x lo5 cells; Fig. 2). The inhibitory effect of the mAb on the release of TNF-a was consistently observed using cells from different donors (Table2). Control mAb J3D3 and IOT2 had no effect (data not shown). The inhibitory effect of mAb My4 was also observed in cultures of LPS-stimulated monocytes performed under serum-free conditions (data not shown). The role of CD14 in the induction of cytokine production was further investigated using CDl4-deficient monocytes

Eur. J. Immunol. 1992. 22: 1461-1466

LPS receptors involved in cytokine release

1463

Table 1. Release of IL-1 and TNF-a by LPS-stimulated monocytes pretreated with a nti-CD lK D 18 mAba) Monoclonal antibody

Cytokine release No LPS

LPS LPS (0.01 pg/5 x 105 cells) (0.1 pg/5 x 105 cells) IL-1 TNF-a IL- 1 TNF-a IL- 1 TNF-a (cpm x 1W) (pglml) (cpm x 10-3) (pglml) (cpm x W ) (pg/mI)

a) Adherent monocytes ( 5 x 10’ cells/well) were incubated with saturating concentrations (10 pg/5 x 105 No antibody 1400