hi”,\” Pnntrd

112G‘wwr/r FLU Im- Resadl. in Great Bntam. All rights

Vol. 3, pp. 355-368, reserved.

0955--1235’9? t’ 1993 Pergamon

1992

Prw

$1 5.00 Ltd

NATURAL KILLER CELL STIMULATORY FACTOR (NKSF) OR INTERLEUKIN-12 IS A KEY REGULATOR OF IMMUNE RESPONSE AND INFLAMMATION Giorgio Trinchieri,* Maria Wysocka,” Annalisa D’Andrea,* Manthrasalam Rengaraju,* Miguel Aste-Amezaga,* Marek Kubin,* Nicholas M. Valiante* and Jihed Chehimit

tchildren’s

*The Wistar Institute. Philadelphia, PA Hospital of Philadelphia. Philadelphia. PA

3601 Spruce Street 19104. U.S.A. 34th and Civic Center Boulevard 19104, U.S.A.

Nutural Killer cell Stimulatory Factor (NKSF) or interleukin-12 (IL-12) is a hrterodimeric cytokine of 70 kDa formed by a heavy chain of 40 kDa (~40) and a light chain of 3.5 kDa (~35). Although it was originally identified and purtfied from the supernatant qf’Epstein-Barr virus-transformed B cell lines, it has been shown that among peripheral blood cells NKSFIIL-12 is predominantlv produced by3monocytes, with loww production by B cells and other accessory cells. The most powerful inducers of NKSFjILI.? production are bacteria, bacterial products and parasites. In uddition to the hiologically~ active p70 heterodimer, the cells producing NKSFIIL-I2 also secrete u large cwess qfmonomericp40, a molecule with no demonstrable biological activity. NKSFjIL13 is active on T lymphocytes and NK cells on which it induces production qf’lymphokines. enhancement qf‘cytotoxic activity and mitogenic effects. NKSFIIL-I2 induces Tand NK cells to produce IFN-y and synergizes with other IFN-y inducers in this eflect. In vitro. and probably in vivo, NKSFIIL-I2 is required for optimal IFN-y production. When human lymphocytes are stimulated with antigens in vitro, addition of exogenous NKSF! IL-12 to the culture induces differentiation qf T helper type 1 I Thl) cells, whereas neutralization of endogenous NKSFIIL-12 with antibodies,favors dt~ermtiation of Th2 cells. IFN-y. a product of Thl cells, enhances NKSFIIL-I2 production by mononucleur cells. whereus IL-IO and IL-4. products of Th2 cells, eficiently inhibit it. Therefore, NKSFIIL-12 appears to be an important inducer of Thl responses produced by uccessory~ cells during early antigenic stitnulation and its production is regulated bv a positive leedbuck mechanism mediated by Thl cells through IFN-)I and a negative one by Th2 cells through IL-IO and IL-4. The balance of IL-12 production versus IL-IO and IL-4 production ear!), during an immune response might therefore be instrumental in determining Thl-type versus Th2-type immune responses. Because of’ this potential role qf IL-12 during immune responses, our results demonstrating the impaired ability of HIV seropoLGtive patients to produce NKSFIIL-12 in response to bacterial stimulation suggest that thi,v clefecf in NKSFIIL-12 production might be a ,factor contributing to their immune depression. Keywords:

cells.

Natural

killer cell stimulatory

factor. interleukin-I

2. interferon-y.

T helper

356

G. Trinchieri et al. INTRODUCTION

Natural killer cell stimulatory factor (NKSF) or interleukin-12 (IL-12) was identified as a factor secreted by human Epstein-Barr virus (EBV)-transformed B cell lines, and mediating several biological activities on human T and NK cells, including induction of interferon-gamma (IFN-y) production, enhancement of cell-mediated cytotoxicity and comitogenic effects on resting T cells [2 13.NKSF/IL- 12 was purified to homogeneity from the conditioned medium of the phorbol diester-stimulated RPMI8866 EBV-transformed cell line and, unlike other cytokines, was shown to have a heterodimeric structure [21]. The genes encoding the two polypeptide chains of NKSF/ IL- 12 were cloned on the basis of the partial amino acid sequences obtained from the purified proteins and biologically active recombinant NKSF/IL-12 was produced in eukaryotic cells transfected with the cDNA for both NKSF/IL-12 chains [42]. A Cytotoxic Lymphocyte Maturation Factor (CLMF) was also identified in the conditioned medium of an EBV-transformed B cell line (NC37 line) on the basis of its ability to synergize with IL-2 in inducing the generation of Lymphokine Activated Killer (LAK) cells [36]. Purification and cloning of the genes encoding CLMF demonstrated that NKSF and CLMF are the same cytokine [21, 22, 36, 421 and the unifying term of IL- 12 [ 181 is now widely accepted. Recent studies from our group and others have shown that NKSF/IL-12 in viva is likely to play important physiological roles in the regulation of immune responses and inflammation and that this cytokine offers promising possibilities for therapeutical use. Figure 1 schematically depicts some of the known functions of NKSF/IL- 12 described in this review.

NKSF/IL-12:

MOLECULAR

STRUCTURE

AND RECEPTOR

STUDIES

NKSF/IL-12 is a heterodimer of 70,000 daltons (~70) formed by two covalentlylinked glycosylated chains ofapproximately 40,000 (~40) and 35,000 (~35) daltons [2 l]. Operationally, 1 U/ml of NKSF/IL- 12 was defined as the concentration of NKSF/IL12 able to induce half-maximal stimulation of IFN-y production in 18 h from human peripheral blood lymphocytes and corresponds to a 3.6 PM concentration of the purified natural NKSF/IL-12 [21]. The ~35 cDNA sequence encodes a 253 amino acid polypeptide [ 18,421 corresponding to a mature secreted protein with a calculated M, of 27,500 containing 7 cysteine residues and three possible N-glycosylation sites. The p40 cDNA sequence encodes a 378 amino acid polypeptide with a 22 amino acid hydrophobic signal sequence, corresponding to a mature protein of calculated M, of 34,700 and 10 cysteine residues, 4 possible N-linked glycosylation sites and one consensus heparin binding site [18, 421. Transient transfection of COS cells or stable transfection of CHO cells with either p40 or ~35 cDNA induces secretion of the respective NKSF/IL-12 chains, but co-transfection with both cDNA is required for secretion of the biologically active p70 heterodimer [ 18,421. Murine B cell lines also produce NKSF/IL-12 1251and the two genes encoding the murine p40 and ~35 chains were cloned based on the cross-hybridization with human cDNA clones and found to have 70 and 60% sequence homology, respectively, with the corresponding human genes [32]. Human NKSF/IL- 12 is not active on mouse cells, but

pEq GM-CSF

Proliferrtion (stimulation and inhibition);

B cells

(CD8

NK cells + T cells.

IFN-y IL-2

T cells CTL)

B cells

Ncutrophilr

Monocytcs Mwophqes

FIGURE 1. Production and major biological effects of NKSF/IL-12. The beterodimeric cytokine NKSF/lL-12 is mostly produced by pbagocytic cells, B cells and possibly other peripheral blood accessory cells in response to bacteria, parasites or their products 191. The target cells of NKSF/IL-12 are T and NK cells on which NKSF/IL12 mediates the indicated biological effects (induction of cytokine secretion, mitogenic effects, enhancement of cell-mediated cytotoxicity) 121, 431. NKSF/IL-12 has also an inductive role on Thl cell differentiation and prevents differentiation of IL-4producing Tb2 cells 1241. NKSF/IL-12 is a potent inhibitor of IgE production, probably not acting directly on B cells, but indirectly on Th cells (201. Primordial interleukin

Il.-6

1-q

IL-12

IL-I 2-R gp 130.like?

FIGURE 2. Possible evolutionary origin of NKSF/IL-12. Tbe NKSF/IL-12 p35 chain has homology to IL-6, whereas the NKSF/IL-12 p40 chain belongs to the hematopoietic cytokine receptor family and is homologous to the extracellular position of the IL&R. The IL-6R is shed from the cells in a soluble form. Unlike other soluble cytokine receptors, soluble IL-6R is tmt competing for biii of the membrane receptor to IL-6, hut co&ii in solution with IL-6 to form a complex that interacts with the second chain of the BAR, gpl30, arul through this molecule induces signal tramduction. Because of the analogy of NKSF/ILlZ with IL6 and ILdR, it has been postulated 1171 that IL-12 may be derived from a primordial cytokii (p-IL) and one chain of its receptor (p-IL-Ra). See text for details.

358

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et ul.

murine NKSF/IL-12 is active on both murine and human lymphocytes [32]. Interspeciesheterodimers are active on both human and murine lymphocytes when the ~3.5chain is of murine origin, but active only on human cells when the p35 chain is of human origin, regardlessof the origin of the p40 chain, suggestingthat the ~35 chain has a determining effect on the speciesspecificity of the heterodimer [32]. The primary amino acid sequenceof the NKSF/IL-12 p35 chain indicates an a-helixrich structure, similar to most cytokines. When the ~35 chain sequencewas compared with the sequencesof the cytokines IL-6 and G-CSF, it was observed that many of the amino acids at conserved positions in these two cytokines are also present in the ~35 sequence[26]. The p40 sequencewas found not to be homologous with any cytokines, but rather to belong to the hematopoietic cytokine receptor family, which is characterized by 4 cysteine and 1 tryptophan residue in conserved positions in the extracellular portions and by a WSXWS motif [ 171.The p40 sequencehas a significant sequencehomology with the extracellular portions of the IL-6 receptor and the ciliary neurotrophic factor (CNTF) receptor [ 17,321. IL-6R, CNTF-R and NKSF/IL- 12 p40 have an N-terminal immunoglobulin-like domain followed by the sequencecharacteristics of the receptor family; the WSXWS motif (that in the p40 sequenceis modified by the insertion of an alanine) is near the C-terminus in the p40 molecule. Most cytokine receptors can be releasedby cells in soluble forms, which usually have a C-terminus immediately following the WSXWS motif and are produced either by proteolytic digestion of the transmembrane form or by alternative splicing of the messagewith elimination of the exons coding for the transmembrane and cytoplasmic portions [38]. The binding of IL-6 to the IL-6R is a low affinity interaction. but upon association of gp 130,a non-ligand binding signal-transducing transmembrane protein, a high affinity complex is produced and signal transduction through gp130 is triggered [37]. The soluble form of the IL-6R, unlike other soluble receptors, doesnot compete for binding of IL-6 to the cellular receptors; rather, the soluble IL-6R binds in solution with IL-6 and this complex can bind to gpl30 on the cell surface. mediating signal transduction and IL-6 biological activities [37, 381. It is therefore possible to hypothesize that heterodimeric NKSFjIL- 12 is evolutionarily derived from a primordial cytokine (~35 equivalent), that similarly to IL-6, had a multichain receptor (Fig. 2). The transmembrane form of one chain of the receptor (~40 equivalent) was lost, but an efficient association of the primitive cytokine and the primitive soluble receptor was maintained by introducing a covalent linkage between the two chains. The heterodimeric complex. similar to the soluble IL-6R-IL-6 complex, would still be able to bind with high affinity to the one or more remaining transmembrane chains of the receptor, inducing signal transduction and biological activity. If this hypothesis on the evolutionary origin of NKSF/IL-12 is correct, one would assumethat, similar to IL-6 and IL-6R, the ~35 and p40 chains of NKSF/IL-I2 have maintained a ligand--receptor-like affinity for each other, even in the absenceof the covalent linkage between the two chains. Indeed, when monomeric recombinant NKSF/IL-12 p40 and ~35 are added together to responsive cells, all the biological activities of NKSF/IL-12 can be demonstrated (M. Rengaraju, A. D-Andrea, G. Trinchieri, unpublished results), although at concentrations from 2 to 5 orders of magnitude higher than those effective for the covalently linked heterodimer. Analysis of steady state binding data of IL-12 by Scatchard analysis identified a single binding site on PHA-activated lymphoblasts with an equilibrium dissociation constant of 100-600 PM and 1000-9000 sites/cell [6]. Crosslinking and immunoprecipitation experiments with anti-NKSF/IL-12 antibodies identified a single protein

Ii~tcrldin-

3.5Y

I.?

of approximately 110 kDa [6]. The cellular distribution of IL-12R was analyzed by identifying cell-bound IL- 12 with fluorescent anti-IL- 12antibodies: the presenceof the receptor was detected on activated T or NK cells, but not on B cells nor on resting T or NK cells [lo]. The available data on NKSF/IL-I2R only in part explain the cellular specificity and biological activity of NKSF/IL- 12. Some of the biological activities. e.g. the proliferative effect or the induction of IFN-y, can be demonstrated at concentrations lower than I PM [3, 21, 281. If the dissociation constant of the identified receptor is more than 100PM. it is necessaryto assumeeither that signal transduction takes place at minimal occupancy of the receptors or that additional unidentified chains of the receptor are required for determining a low number of high affinity binding sites.The other discrepancy with the functional data is that receptorscannot be identified on resting T and NK cells, whereascertain biological activities of NKSFI L12.e.g. enhancement of cell-mediated cytotoxicity or induction of IFN-yproduction, Liremediated with a similar dose-responsecurve on both resting and activated NK and T cells [3]. It is not clear yet whether the p40 chain, the ~35 chain, or both are directly involved in binding to the receptor. The observations that antibodies [7] and sitespecific chemical modifications of a tryptophan residue on the p40 chain [30] block NKSFIL-I2 binding to the receptor and that ~35 is responsible for determining the species-specificity of NKSF/IL-12 [32] suggest,however. that both chains may play ;I role.

INDUCTION

OF LYMPHOKINE

PRODUCTiON

BY NKSF/IL-12

NKSF/IL-12 was originally identified and purified on the basis of its ability to induce IFN-y production by both resting and activated T and NK cells [21]. All Tcell subsets.specifically CD4’ and CD8’ T cellsaswell asT cellsexpressingeither the 31/jar y6T cell receptor (TCR) chains, respond to NKSF/IL- 12with IFN-yproduction [3]. Production of IFN-y by resting but not activated T and NK cells, in responseto either NKSF,IIL-12 or IL-2, requires the participation of non-adherent MHC class II positive cells. distinct from monocytes and B cells [3]. Both the nature of the accessory cells and the mechanism by which they facilitate production of IFN- y remain to be elucidated. The induction of IFN-y production by NKSF/IL-12 is characterized by a strong synergistic effect with IL-2 and with several other stimuli able to induce IFN-y production (e.g. mitogenic lectins, alloantigens, phorbol diesters, anti-CD3 and anti-CD28 antibodies on T cells, anti-CD1 6 antibodies, immunocomplexes, target cells and phorbol diesters on NK cells) [3]. Although NKSF/IL-12 up-regulates IFN- y gene expression at a transcriptional level, the synergistic effect of NKSFjIL-I2 and IL-2 is mostly due to a post-transcriptional effect that increases stability of IFN-y mRNA [2]. NKSF/IL-I2 synergizes with suboptimal doses of other IFN- y inducing stimuli at concentrations of less than I PM, suggesting that the induction of IFN- ymay represent one of the principal physiologic functions of NKSF 1L- 12 irz viva [27,28]. Other lymphokines, including at least TN F-cwand GM-CSF. are also induced by NKSF/IL-12 [27, 281: in the case of these two lymphokines. no synergistic effect is observed by co-stimulation of lymphocytes with both NKSF,:IL- I2 and IL-2 [2X].

360

G. Trinchieri

EFFECT OF NKSF/IL-12

ON LYMPHOCYTE-MEDIATED

et al.

CYTOTOXICITY

NKSF/IL-12 rapidly enhances (4-18 h) the cytotoxic ability of resting or activated NK cells [4, 5, 211. This activity of NKSF/IL-12 is shared with a small group of other cytokines, including IFN-a [40] and IL-2 [39]. Although the maximum cytotoxic activity induced by NKSF/IL-12 is usually lower than that mediated by NK cells stimulated with optimal doses of IL-2, NKSF/IL-12 significantly enhances NK cellmediated cytotoxicity at doses of < 1 pM that are IOO- to I OOO-fold lower than the concentration of IFN-aor IL-2 required to induce a similar effect [21]. The mechanism by which NKSF/IL- 12 enhances NK cell-mediated cytotoxicity includes increased expression of surface adhesion molecules, as well as an increased number of NK cell granules containing molecules involved in the cytotoxic process [4, 5, 3 11. NKSF/IL- 12 also induces LAK cell generation in 3- to 5-day cultures and synergizes with IL-2 in this effect [15, 18, 27, 351. A role for TNF in the NKSF/IL-12 induced generation of LAK cells is suggested by the inhibition mediated by anti-TNF antibodies added to the cultures [27]; however, a similar role for TNF could not be demonstrated in the short-term enhancement of NK cell cytotoxic activity induced by NKSF/IL- 12 [5]. NKSF/IL-12 enhances the cytotoxic activity not only of NK cells but also of T cells: this is indicated by the ability of NKSF/IL- 12 to enhance the generation of alloreactive cytotoxic T lymphocytes (CTL) [15] and to enhance the ability of T cells to mediate anti-CD3 antibody redirected killing [5]. REGULATION

OF T AND NK CELL PROLIFERATION

BY NKSF/IL-12

NKSF/IL- 12 has only a minimal effect, if any, on the proliferation of resting T and NK cells, but enhances the proliferation of T cells induced by various mitogenic stimuli, including lectins, alloantigens, anti-CD3 antibodies and phorbol diesters [21, 281. Co-culture of PBL with certain irradiated EBV-transformed cell lines induces a preferential growth of NK cells [29]: although the NKSF/IL-12 production by the cell lines does not, by itself, explain the preferential proliferative effect on NK cells, addition of neutralizing anti-NKSF/IL12 antibodies at the beginning of the culture significantly inhibited the proliferation and the cytotoxic activity of NK cells, whereas addition of NKSF/IL-12 enhanced these activities in the cultures [41]. On both activated T and NK lymphoblasts NKSF/IL- 12 at concentrations of less than 1 pM has a direct mitogenic effect [I 8,28, 36.421. However, NKSF/IL- 12 cannot induce proliferation at the same level as that induced by optimal levels of IL-2 and lymphocytes cannot be maintained in long-term culture in the presence of NKSF/IL12. When NKSF/IL-12 and IL-2 are added together, an additive effect is observed on the proliferation of T lymphoblasts with TCR @, whereas NKSF/IL-12 inhibits the proliferative effects of high doses of IL-2 on T lymphoblasts with TCR yS and on NK lymphoblasts [28]. NKSF/IL- 12 also inhibits the mitogenic effect of IL-2 on resting NK cells [3 11.This paradoxical effect of NKSF/IL- 12 is also observed with cell lines: the IL2-induced proliferation of a T a$ leukemia cell line is not affected by NKSF/IL-12, whereas that of a T yS line is inhibited by NKSF/IL- 12 [28]. However, this inhibition probably depends on the state of activation of the cells and/or on the relative concentration of the two cytokines, as indicated by the ability of NKSF/IL-12 to enhance the proliferation of NK cells in the co-cultures of PBL with B cell lines [4 l] and

Interleukin-

36 I

I?

by its inability to inhibit IL-2- induced proliferationof NK cells purified from these cocultures at the time of maximum proliferation and activation [28]. The mechanism by which NKSF/IL- 12 inhibits IL-2 induced proliferation in certain cell types appears to be partially due to TNF production, because anti-TNF antibodies prevent the inhibition, suggesting that TNF is required, although not sufficient, for this inhibitory effect [28]. PRODUCTION

OF NKSF/IL-12

Analysis of NKSF/IL- 12 mRNA accumulation in different cell types indicated that almost all of them, including B, T and NK cells, monocytes. leukemic cell lines derived from the above cell types as well as different malignant tumor cell lines (e.g. the large majority of melanoma cell lines), express transcripts of the p35 gene [9]. By contrast. expression of the p40 mRNA was more restricted and present only in cell types and lines in which we could also demonstrate the production of the NKSF/IL-12 heterodimer and biological activity [9]. For detection of NKSF/IL-12 proteins, we established radioimmunoassays (RIA) with different pairs of monoclonal antibodies, specifically measuring the free p40 chain, the free p35 chain, or the p70 heterodimer [9]. When production of NKSF/IL-12 by different cell lines was analyzed, only EBVtransformed B cell lines produced NKSF/IL-12. Most of these lines constitutively produced NKSF/IL- 12 and both mRNA accumulation and protein production were significantly enhanced by treatment with phorbol diesters [9]. As previously observed during the purification of NKSF/IL-12 from supernatant fluid of the RPMI-8866 and NC37 cell lines [21,36], all the EBV-transformed cell lines secreted the free p40 chain in large excess over the biologically active heterodimer [9]. Although phorbol diesteractivation mostly regulated the expression of the p40 mRNA, increased secretion of both the free p40 chain and the heterodimer was observed 191.Significant secretion of the free p35 chain was never detected in B cells or other cell lines, although the p35 mRNA was expressed in most of the lines [9]. These data suggest that p35 requires linkage with the p40 chain for efficient secretion, and that the intracellular coupling ot the two chains is not very effective, resulting in secretion of an excess of free p40 chains. Peripheral blood mononuclear cells (PBMC) spontaneously produce in vitro up to 1 rig/ml of the free p40 chain and lower concentrations of the heterodimer: this production was not enhanced by phorbol diesters, but was significantly increased by stimulation with bacteria or bacterial products such as fixed S~uphvlococcus aurtw. M~dxxteria tuberculosis or bacterial lipopolysaccharide (LPS) [9]. The constitutivc production of NKSF/IL-12 by PBMC observed in early experiments [9] was probably due to endotoxin contamination in the tissue culture medium. S. uureus was found to be the best in vitro stimulus for NKSF/IL- 12. Both an excess of the free p40 chain and up to 0.5 rig/ml of the p70 heterodimer were secreted by PBMC stimulated by S. cIureu,s [9]. Both S. aureus and LPS induced a many-fold but transient increase of p40 mRNA with a peak around 3 h after stimulation [9 and M. Aste, A. D’Andrea and G. Trinchieri, unpublished results]. The p70 heterodimer produced by PBMC was demonstrated to mediate all the characteristic activities of NKSF/IL-12, i.e. induction of IFN-y, enhancement of cytotoxicity and mitogenic effects on T and NK lymphoblasts [9]. Within PBMC, adherent monocytes are responsible for most NKSF/IL-12 production,

362

G. Trinchieri

et al.

Monocyte-derived macrophages and, to a lesser extent. neutrophils (M. Cassatella, A. D’Andrea and G. Trinchieri, unpublished results) are also induced to produce NKSF/IL12 in response to LPS or S. uureus. Non-adherent PBL, carefully depleted of monocytes, can produce NKSF/IL-12 in response to S. aureus; the non-adherent producer cells are both B cells and other not yet identified MHC class II positive cells [9]. Neither production of NKSF/IL12 nor accumulation of p40 mRNA was observed in purified preparations of T or NK cells [9]. Human myeloid leukemia cell lines were not found to produce NKSF/IL-12 either constitutively or in response to phorbol-diesters [9], although these compounds induced them to produce other cytokines, e.g. TNF. However, when LPS or S. uureus were used as stimuli, the macrophage cell line THP-1 produced both the free p40 chain and the p70 heterodimer. Two other lines, the promyelocytic HL-60 and ML3, produced NKSF/IL-12 in response to LPS only after pretreatment with inducers of differentiation such as dimethylsulfoxide (M. Kubin and G. Trinchieri, unpublished observations).

POSSIBLE PHYSIOLOGICAL ROLES OF NKSF/IL-12: IFN-y PRODUCTION AND INDUCTION OF A T HELPER RESPONSE

REGULATION OF CELL TYPE 1 (Thl)

Neutralizing anti-NKSF/IL-12 antibodies almost completely suppress IFN-y production stimulated in PBMC by NKSF/IL12-inducers such as S. aureus or LPS [9]. However, when IFN-y production was induced by stimuli such as IL-2, anti-CD3 antibodies or mitogenic lectins that have no significant effect on NKSF/IL-12 production, anti-NKSF/IL-12 antibodies still mediated a significant inhibition (> 60%) of IFN-yproduction [9]. Unlike PBMC, no inhibition of IFN-yproduction by anti-NKSF/IL-12 antibodies was observed when purified T or NK cell preparations were used in the absence of cell types able to produce NKSF/IL-12 [9]. These results indicate that NKSF/IL-12 is required in vitro for optimal production of IFN-1/ with most stimuli, and that for some stimuli (S. aureus or LPS) it is the major factor responsible for the production. In order to analyze the role of NKSF/IL-12 in IFN-yproduction in viva, we studied the ability of LPS to induce NKSF/IL-12 and IFN-y production in C57BL/6 or BALB/c mice, primed or not two weeks earlier with Bacille Calmette-G&in (BCG). In BCG-primed mice, high titers of TNF-a and IFN-y were detected in the serum of the mice within 1 and 5 h, respectively, from LPS, i.v. injection, whereas in non-primed mice much lower cytokine concentrations, if any, were detected. Induction of NKSF/ IL-12 by LPS was evaluated by analyzing mRNA accumulation in the spleen and NKSF/IL-12 (~40 + ~70) concentrations in the serum, using a RIA utilizing two rat anti-murine NKSF/IL-12 monoclonal antibodies (M. Wysocka and G. Trinchieri, unpublished results). In both BCG-primed and unprimed animals p40 mRNA was induced in the spleen by LPS within 1 h and NKSF/IL-12 (~40 + ~70) reached a peak of lo-20 rig/ml in the serum 3 h after LPS injection. Because only BCG-primed mice produced high levels of IFN-y. this result was unexpected. However, treatment of the mice 24 h before and at the time of LPS injection with neutralizing rat anti-murine NKSF/IL12 monoclonal antibodies efficiently suppressed IFN-y production, confirming that NKSF/IL-12 is required in viva for IFN-yproduction in response to LPS

In terleukin- 12

363

(M. Wysocka and G. Trinchieri, unpublished results). The paradoxical in vivo production of NKSF/IL-12 in the absence of IFN-y production could be due to production of the p70 heterodimer in primed but not in unprimed mice, or, more likely, by the requirement for IFN-y production of factors other than NKSF/IL-12. As discussed below, TNF rapidly produced in BCG-primed but not in unprimed mice after LPS injection could be a candidate co-factor with NKSF/IL-12 for IFN-y induction. Pathogens such as Listeria monocytogenes [1] and Toxoplasma go&ii [34] that infect macrophages typically induce strong T and NK lymphocyte responses leading to the production of IFN-y. Soluble mediators released from macrophage accessory cells upon exposure to microbial products are the apparent stimulus for the NK cell IFN-y response. TNF-a is essential for this response, but in itself is not sufficient to induce IFN-y production [l, 341. NKSF/IL- 12 was shown to be essential for stimulation of IFN-7 synthesis in response to in vitro T. gondii infection and macrophages are the likely source of NKSF/IL-12 in this system [16]. A major implication of these findings is that induction of NKSF/IL-12 by bacteria and parasites, especially intracellular pathogens, may be a key element in their early stimulation of cell-mediated host defenses. In particular, NKSF/IL- 12, by inducing the production of IFN-yand other lymphokines, has the ability to activate phagocytic cells and to induce other inflammatory responses. Also, as discussed below, production of JL-12 early in the infection might have a major regulatory effect in the subsequent antigen-specific adaptive immune responses. It has recently been investigated (A. D’Andrea, M. Aste, M. Kubin, X.-J. Ma and G. Trinchieri, submitted for publication) whether the cytokines IL-10 and IL-4 that are potent inhibitors of IFN-yproduction by lymphocytes [13,19] and of several cytokines produced by monocytes [1 1, 141, were able to inhibit NKSF/IL- 12 production and, through this effect, suppress IFN-y production. Interestingly, the mechanism of inhibition of IFN-I/ by IL-10 has been shown to be mediated indirectly through an effect on accessory cells and not directly on the IFN- y-producing lymphocytes [ 191. IL10 and IL-4 suppressed NKSF/IL-12 production by monocytes and by PBMC, induced by either S. aureus or LPS. IL-10 and IL-4 prevented the induced accumulation of p40 mRNA, and inhibited secretion of both the free p40 chain and of the p70 heterodimer. A similar inhibition by IL-10 and IL-4 was observed for TNF-cr mRNA and protein as previously reported [1 1, 141. Although TNF-CY, by itself or in combination with other stimuli, is not an effective inducer of IFN-yproduction, TNF is required for optimal IFN-y production, as has been demonstrated both in vitro and in rive [ 1, 341. The ability of several different stimuli to induce IFN-y production by human PBMC was almost completely suppressed by treatment with IL-lo. As discussed above, anti-NKSF/IL-12 inhibited IFN-y production from 60 to 90%. depending on the stimulus [9] and a significant, although lower, inhibition was observed with every IFN-y stimulus in culture in the presence of anti-TNF-cr antibodies. Importantly, a mixture of anti-NKSF/IL-12 and anti-TNF antibodies almost completely suppressed IFN-)/ production to levels comparable to those obtained with IL-IO (A. D’Andrea and G. Trinchieri, unpublished results). NKSF/IL12 with or without TNF greatly diminished the ability of IL-10 to innibit IFN-y production induced by various stimuli. These results suggest that the inhibition of both NKSF/IL-12 and TNF production by IL-10 is the major mechanism by which this cytokine mediates the inhibition of IFN-yproduction by lymphocytes, in the presence of accessory cells. However, the ability of IL-IO to inhibit to a certain extent IFN-y

364

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et ul.

IL-10 IL-4 IL-5 IL-6

IL-2 IFN-7

FIGURE 3. Immunoregulatory function of NKSF/IL-12. NKSF/IL-12 favors differentiation of the Thl cells by a mechanism that is in part dependent on its effect on NK cells, but that does not necessarily require production of IFN-1 1241. Thl cells, by producing IFN-y that augments production of NKSF/IL-12 by macrophages, are involved in a positive feedback loop, whereas Th2 cells, by produciug IL-10 and IL-4 that suppress production of NKSF/IL-12, are part of a negative feedback loop.

production, even in the presenceof NKSF/IL- 12and TNF or of anti-NKSF/IL- I2 and anti-TNF antibodies, suggeststhat IL-IO might also affect other required factors for IFN- y-production. Similarly to lymphocyte proliferation [ 121, IL- IO might downregulate the expression of surface molecules required for optimal functions of accessorycells. BecauseNKSF/IL-I 2 induces secretion of JFN- y, a potent promoter of the differentiation of Th 1 cells, and becausethe major products of Th2 cells, IL-1 0 and IL-4, suppressNKSF/IL-12 production, the effects exerted on the in vitro development of antigen specific cell lines and T cell clones by addition or neutralization of IL- 12 in lymphocyte bulk culture were analyzed [24]. T cell lines specific for Dermatophugoides pteronyssinus Group 1 (Der p.I), derived in the presence of IL-12 during the initial antigenic stimulation, exhibited reduced ability to produce IL-4 and increased ability to produce IFN-y. These lines developed into Der p.J-specific CD4’ T cell clones showing a ThO- (producing both JL-4 and IFN-y) or Thl- (producing IFN-1/ phenotype) instead of dispiaying a Th2-like (producing IL-4) cytokine profile [24]. By contrast, purified protein derivative (PPD)-specific T cell lines, derived in the presence of anti-IL-12 antibody during the initial antigenic stimulation, exhibited an increased ability to produce IL-4 and developed into PPD-specific CD4’ T cell clones showing a ThO instead of a Thl-like profile [24]. The influence of IL-12 on the cytokine secretion profile of Der p.I-specific cell lines was not prevented by addition to lymphocyte bulk culture of anti-IFN-y antibodies, but could be at least partially inhibited by removal from bulk cultures of CD16+ NK cells 1241.Thus, NKSF/IL-12 and NK cellsappear to have inhibitory effects on the development of IL-4-producing cellsand to play an active role in promoting Thl-like response. NKSF/IL-12 has been shown to profoundly inhibit in vitro the IgE production by PBL cultured in the presenceof IL-4 [20]. The results obtained in this experimental system [20], together with the lack of evidence of NKSF/IL- 12 receptors in resting or activated B cells [IO], suggestthat NKSF/IL- 12 is not acting directly on the B cells, but rather possibly potentiating type 1 and

fnterleukin-12

36.F

suppressing type 2 helper T cells. The inductive role of NKSF/IL-12 on Thl-like responses suggests that this cytokine may have a key role, depending on its constitutive level of production in the host or on the ability of the antigen or infectious agents to induce its production, in determining the type of immune response. Because Th 1-like responses are often protective, e.g. in bacteria or parasite infection, whereas Th2- like responses are less efficient in protection from infection and are prevalent in pathological situations such as allergy and autoimmunity, this inductive role of NKSF/TL- 12 could have a major physiological relevance. Once the immune responses are established, negative or positive feedback mechanisms involving NKSF/IL- 12 might be involved in order to stabilize the type of response. For example, Thl cells could have a positive feedback by producing IFN-ythat potentiates the ability of phagocytic cells to produce NKSF/IL- 12. whereas Th2 cells could mediate a negative feedback by producing IL- IO and IL-4, potent inhibitors of NKSF/IL-12 production (Fig. 3). Preliminary studies in mice infected with Leishmania (P. Scott, T. Scharton. M. Wysocka and G. Trinchieri, unpublished data) suggest that NKSF/IL-I2 might have itz vivo in the murine system the same Thl-inducing ability as shown in vitro with human cells. It remains to be determined in the mouse, or for possible therapeutical applications in humans, whether NKSF/IL-12 or NKSF/IL-12 antagonists are able to alter the Th I or Th2 characteristics of an ongoing immune response. HIV SEROPOSITIVE HAVE A REDUCED

AND AIDS PATIENTS ABILITY TO PRODUCE

RESPOND TO NKSF/IL-12, BUT IT IN RESPONSE TO S. AUREUS

NKSF/IL-12 enhances the cytotoxic activity of NK cells from HIV’ patients and in particular the very depressed activity observed in a group of advanced patients was augmented within the range of activity observed with NK cells from healthy donors. However, this increase was less than that of healthy donors’ lymphocytes stimulated with NKSF/IL-12 [4]. Interestingly, NKSF/IL-12 was shown to enhance the NK cytotoxicity not only against tumor-derived cell lines, but also against cells infected with different types of viruses including herpesviruses and HIV [S]. Production of IFNy by lymphocytes from HIV’ patients was 5- to IO-fold lower than that of lymphocytes from healthy donors; however, lymphocytes from HIV’ patients produced IFN-y in response to either NKSF/IL-I2 or IL-2 and, similarly to healthy donors’ lymphocytes, were synergistically stimulated by the two inducers together [4]. When NKSF/IL-12 production in response to S. aureu.s in vitro was analyzed. PBMC from over 50 HIV’ patients showed more than a 1O-fold decrease in production of both the free p40 chain and the p70 heterodimer (J. Chehimi and G. Trinchieri. unpublished results). Although patients with advanced disease and low numbers of CD4’ cells had the lowest ability to produce NKSF/IL-12, a depressed production NKSF/IL-12 was also shown with cells from most patients even at an early stage of the disease and with an almost normal number of CD4’ cells. Unlike NKSF/IL-12. PBMC from the HIV+ patients produced normal or slightly higher than normal levels of both TNF-r and IL- 1/I, two cytokines with at least partially overlapping producer cell types similar to NKSF/IL-12. HIV’ patients, early after infection, have a depressed T helper cell function and a shift toward a prevalently Th2-like response with production of IL-4 and IL-lo. These two cytokines that are powerful inhibitors of NKSF/IL- 12 production, are probably not responsible for the decreased production of

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NKSF/IL- 12 by the patients because they are also inhibitors of production of TNF-a! and IL- 1p, cytokines that are produced in normal concentrations by the patients. The dominance of the ThZlike immune response in the I-IIV’ patients probably depresses even further their ability to fight opportunistic infection [23, 331, and it has been proposed as one of the reasons why HIV’ patients are unable to mount an efficient immune response against the HIV infection [8]. It will therefore be important to determine whether the reduced production of NKSF/IL-12 is directly induced by HIV and plays a role in further depressing the immune responsiveness of the patients, or whether it is itself the consequence of the altered immune status of the patients. It would also be important to determine whether NKSF/IL- 12 treatment could shift the immune system more toward a Thl-like response, possibly improving its effectiveness against opportunistic infectious agents or even against HIV. REFERENCES 1. Bancroft GJ, Sheehan KCF, Schreiber RD, Unanue ER. Tumor necrosis factor is involved in the T cell-independent pathway of rnacrophage activation in scid mice. J Immunol. 1989; 143: 127-130. 2. Chan SH, Kobayashi M, Santoli D, Perussia B, Trinchieri G. Mechanisms of IFN-y induction by natural killer cell stimulatory factor (NKSF/IL-12): Role of transcription and mRNA stability in the synergistic interaction between NKSF and IL-2. J Immunol. 1992; 148: 92-98. 3. Chan SH, Perussia B, Gupta JW, Kobayashi M, Pospisil M, Young HA, Wolf SF, Young D, Clark SC, Trinchieri G. Induction of IFN-yproduction by NK cell stimulatory factor (NKSF): Characterization of the responder cells and synergy with other inducers. J Exp Med. 1991; 173: 869879. 4. Chehimi J, Starr S, Frank I, Rengaraju M, Jackson SJ, Llanes C, Kobayashi M, Perussia B, Young D, Nickbarg E, Wolf SF, Trinchieri G. Natural killer cell stimulatory factor (NKSF) increases the cytotoxic activity of NK cells from both healthy donors and HIV-infected patients. J Exp Med. 1992; 175: 789-796. 5. Chehimi J, Valiante NM, D’Andrea A, Rengaraju M, Rosado Z, Kobayashi M, Perussia B, Wolf S, Starr SE, Trinchieri G. Enhancing effect of natural killer cell stimulatory factor (NKSF/IL-12) on cellmediated cytotoxicity against tumor-derived and virus-infected cells. Eur J Immunol. 1993; in press. 6. Chizzonite R, Truitt T, Desai BB, Nunes P, Podlaski FJ, Stern AS, Gately MK, IL-12 receptor. I. Characterization of the receptor on PHA-activated human lymphoblasts. J Immunol. 1992; 148: 31173124. 7. Chizzonite R, Truitt T, Podlaski FJ, Wolitzky AG, Quinn PM, Nunes P, Stem AS, Gately MK. IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts. J Immunol. 1991; 147: 1548-1556. 8. Clerici M, Herkin FT, Venzon DJ, Blatt S, Hendrix GW, Wynn TA, Shearer G. Changes in interleukin-2 and interleukin-4 production in asymptomatic, human immunodeficiency virusseropositive individuals. J CIin Invest. 1993; in press. 9. D’Andrea A, Rengaraju M, Valiante NM, Chehimi J, Kubin M, Aste M, Chan Sh, Kobayashi M, Young D, Nickbarg E, Chizzonite R, Wolf SF, Trinchieri G. Production of natural killer cell stimulatory factor (NKSF/IL- 12) by peripheral blood mononuclear cells. J Exp Med. 1992; 176: 13871398. 10. Desai BB, Quinn PM, Wolitzky AG, Mongini PKA, Chizzonite R, Gately MK. The IL-12 receptor. II. Distribution and regulation of receptor expression. J Immunol. 1992; 148: 3125-3132. Il. de Waal Malefyt R, Abrams J, Bennett B, Figdor C, de Vries J. IL-10 inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med. 1991; 174: 1209-1220. 12. Ding L, Shevach EM. IL-10 inhibit mitogen-induced T cell proliferation by selectively inhibiting macrophage costimulatory function. J Immunol. 1992; 148: 3133-3139. 13 Fiorentino DF, Bond MW, Mosmann TR. Two types of mouSe helper T cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Thl clones. J Exp Med. 1989; 170: 2081-2095. 14. Fiorentino DF, Zlotnik A, Mosmann TR, Howard MH, O’Garra A. IL-IO inhibits cytokine

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