Leishmania major Amastigotes Induce p50/c-Rel NF-κΒ Transcription Factor in Human Macrophages: Involvement in Cytokine Synthesis

Abstract

Invasion of a host by pathogens is frequently associated with activation of nuclear factor kappa B (NF-κB), which is implicated in various aspects of immune function required for resistance to infection. However, pathogens may also subdue these mechanisms to secure their survival. Here we describe the effect of Leishmania major infection on NF-κB transcription factor activation in both promonocytic human cell line U937 and fresh human monocytes. Infection by L. major amastigotes blocked nuclear translocation of a phorbol-12 myristate-13 acetate (PMA)-induced p50/p65 NF-κB complex in PMA-treated differentiated U937 cells and triggered expression of p50- and c-Rel-containing complexes in both U937 cells and fresh human monocytes. These p50/c-Rel complexes, triggered by direct cell-parasite interactions, were detectable within 30 min after the interaction and were transcriptionally active. The NF-κΒ inhibitor caffeic acid phenethyl ester inhibited production of both tumor necrosis factor alpha and interleukin-10 (IL-10) induced by Leishmania amastigotes in differentiated U937 cells. Similar results for IL-10 induction were observed with amastigote-infected human monocytes. Our results indicate that L. major amastigotes activate NF-κB by specifically inducing p50- and c-Rel-containing complexes which are likely involved in the regulation of cytokine synthesis.

Leishmania spp. are obligate intracellular parasites which reside and multiply in their mammalian hosts within macrophages. Macrophages are one of the first lines of defense in the response against pathogenic infection. Infection of a macrophage leads to induction of numerous cellular genes, several of which encode cytokines that stimulate an inflammatory response and resistance to pathogens. Hence, control of a Leishmania infection requires activation of macrophages to a microbicidal state. To escape the host immune defense and to survive, Leishmania parasites have developed different strategies and inhibit several macrophage functions, including phagocytosis, nitric oxide generation, interleukin-12 (IL-12) production, and major histocompatibility complex class II expression (7, 12).

Pathogen invasion frequently induces activation of the nuclear factor kappa B (NF-κB) transcription factor, which plays an important role in initiation of innate immune responses. The NF-κB-Rel family is composed of five different members, NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel (15). These proteins are present in resting cells as inactive complexes sequestered in the cytoplasm by tight binding to the inhibitory protein IκB (5). A wide variety of signaling pathways lead to degradation of IκB, which allows release and nuclear translocation of the NF-κB dimers.

The importance of NF-κB in resistance to infection has been clearly established by studies in which mice deficient in different NF-κB family members were shown to be susceptible to several viral, bacterial, and parasitic infections (45). Several microbial pathogens overcome the host immune response by targeting several parts of NF-κB pathways. Thus, the extracellular bacterium Yersinia inoculates into the target cells its virulence factors, which have the capacity to inhibit activation of kinases that act upstream of IκB phosphorylation or to interfere with degradation of the inhibitory protein (35, 36, 40). More recently, it has been reported that infection of macrophages by Toxoplasma gondii results in degradation of IκB but does not lead to nuclear translocation of NF-κB (43). Mycobacterium ulcerans, which also inhibits NF-κB activation, may act either by blocking phosphorylation of RelA and subsequently nuclear translocation or by directly interfering with NF-κB DNA binding (37). Microorganisms can also take advantage of the NF-κB system, and activation of NF-κB may be a strategy that protects the host cells from apoptosis, allowing pathogens to survive, replicate, and disseminate (20, 24).

Several reports have indicated that Leishmania parasites interfere with signal transduction pathways. Indeed, Leishmania donovani infection has been shown to induce alterations in signaling dependent on protein kinase C and Ca²⁺ and to inhibit the gamma interferon signaling pathway (11, 32, 34). L. donovani has also been reported to fail to induce phosphorylation of p38MAPK, c-Jun N-terminal kinase, and extracellular signal-regulated kinase 1/2. The same report also showed that L. donovani infection does not lead to degradation of IκBα (39). However, no previous studies have dealt with the direct effect of Leishmania amastigote infection on NF-κB activation in human macrophages. In this study we investigated the effects of Leishmania major amastigotes on the host NF-κΒ transcription factor in both the human U937 cell line and fresh human monocytes and macrophages.

MATERIALS AND METHODS

Reagents and antibodies.

Phorbol-12 myristate-13 acetate (PMA), Geneticin, cytochalasin D, and caffeic acid phenethyl ester (CAPE) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Anti-NF-κΒ isoform antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). NF-κB oligonucleotides were obtained from Genset SA (Paris, France). A construct containing three NF-κΒ target sequences linked to the luciferase gene (3X-NF-κΒ-Luc) was kindly provided by J. Pierre (INSERM, Chatenay-Malabry, France). All cell culture media and reagents were purchased from Sigma-Aldrich Chemie GmbH. Cytokine enzyme-linked immunosorbent assay (ELISA) kits were obtained from Becton-Dickinson Int. (Le Pont de Claix, France).

Parasites.

A Tunisian strain of L. major amastigotes (MHOM/TN/95/GLC94 zymodeme MON25) was maintained by serial inoculation of 2 × 10⁷ amastigotes into the hind footpads of female BALB/c mice. After 4 to 6 weeks, the mice were sacrificed, and the amastigotes were purified from the lesions. Briefly, the infected feet were rubbed over a sieve, and the sieved material was centrifuged at 100 × g for 5 min to remove large cell debris. The supernatant was centrifuged at 2,000 × g for 10 min. The pellet was washed with sterile medium, and the amastigotes were resuspended and counted. Peanut agglutinin-negative (PNA⁻) promastigotes (41) from the same Tunisian strain of L. major were grown at 26°C in RPMI 1640 supplemented with 5 mM l-glutamine, 10% heat-inactivated fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). To rule out the possibility that serum components from infected mice played any role, in some experiments promastigotes were incubated with serum from Leishmania-infected BALB/c mice.

Culture, differentiation, and infection of U937.

The promonocytic human cell line U937 was cultured at 37°C in RPMI 1640 supplemented with 5 mM l-glutamine, 10% heat-inactivated fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Prior to infection, PMA (10 ng/ml) was added to the culture medium for 24 h to allow macrophage differentiation of U937 cells, which were then incubated with freshly isolated amastigotes or PNA⁻ promastigotes of L. major at a parasite-to-cell ratio of approximately 10:1. After the desired time of incubation at 37°C in 5% CO₂, noningested amastigotes were removed, and the cells were harvested to prepare samples for an electrophoretic mobility shift assay (EMSA).

Human monocyte culture and stimulation.

Peripheral blood mononuclear cells were prepared from consenting healthy donors by Ficoll/Hypaque density gradient centrifugation (Amersham Pharmacia Biotech, Buckinghamshire, England). The peripheral blood mononuclear cells were allowed to adhere to six-well tissue culture plates (Costar) for 1 h at 37°C in the presence of 5% CO₂ in RPMI 1640 lipopolysaccharide (LPS)-free medium (Sigma-Aldrich Chemie GmbH) containing 10% autologous serum, 5 mM l-glutamine, and antibiotics. The wells were washed with prewarmed medium to remove nonadherent cells. Adherent cells were resuspended in RPMI 1640 medium and incubated with freshly isolated amastigotes or PNA⁻ promastigotes of L. major at a parasite-to-cell ratio of 5:1.

EMSA.

Nuclear proteins were prepared from 10⁷ cells by a modification of the method of Dignam et al. (13). Briefly, cells were washed with phosphate-buffered saline and resuspended in buffer A (10 mM HEPES [pH 7.8], 15 mM KCl, 2 mM MgCl₂, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol). The cells were incubated for 10 min on ice, mixed, and centrifuged for 5 min at 1,000 × g. The supernatants were removed, and the pellets were resuspended in buffer A supplemented with 0.2% NP-40 and incubated on ice for 5 min. After centrifugation, the nuclear pellets were resuspended in buffer A, and 3 M KCl was added dropwise until the KCl concentration was 0.39 M. The nuclei were incubated for 5 min at 4°C and then centrifuged at 14,000 × g. The protein concentration in the nuclear extract was determined by the micro-BCA assay (Pierce, Rockford, Ill.), and aliquots were stored at −80°C until they were used.

Synthetic oligonucleotides containing the consensus κΒ sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) or mutant κΒ sequence (5′-AGTTGAGGCGACTTTCCCAGGC-3′) were annealed, and the consensus κΒ double-stranded oligonucleotides were end labeled with [γ³²P]ATP by using T4 polynucleotide kinase (Amersham-Pharmacia Biotech). The binding reaction mixtures containing 0.5 ng of DNA probe, 2 μg of poly(dI-dC), and binding buffer (10 mM HEPES [pH 7.5], 50 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol) were incubated for 20 min at room temperature. The protein-DNA complexes were separated from the free probe by electrophoresis. The gels were then dried and autoradiographed. To assess the specificity of the reaction, competition assays were performed with a 100-fold excess of unlabeled consensus NF-κΒ or mutant NF-κΒ oligonucleotides. For antibody supershift experiments, the nuclear extracts were incubated for 1 h with 1 μg of the specific antibody (Santa Cruz Biotechnology) before addition of the labeled probe.

Transfection and luciferase assay.

U937 cells were stably transfected by electroporation with the 3X-NF-κΒ-Luc NF-κΒ-luciferase reporter construct. Differentiated cells either were not infected or were infected with L. major parasites, harvested, and lysed. The protein concentration was determined by the micro-BCA assay. Luciferase activity was determined with a luminometer (TD-20/20; Promega, Charbonnieres, France). The level of luciferase induction was calculated by determining the ratio of the specific luciferase activity in the stimulated or infected cells to the specific luciferase activity in unstimulated cells.

Cytokine assays.

Differentiated U937 cells or freshly purified monocytes were infected with Leishmania amastigotes in the presence or absence of CAPE in six-well tissue culture plates. After 24 h, the amounts of cytokines released into the supernatants were measured by using human tumor necrosis factor alpha (TNF-α) and IL-10 ELISA kits (BD Biosciences, Le Pont de Claix, France). The assays were performed according to the manufacturer's instructions.

Statistical analysis.

Student's t test was employed to assess the significance of the differences in cytokine synthesis. A P value less than 0.05 was considered statistically significant.

RESULTS

NF-κΒ expression in L. major-infected U937 cells.

Several studies have shown that NF-κB is activated after exposure to a range of pathogens, including viruses, bacteria, and parasites. We investigated whether L. major amastigotes interfere with NF-κB activation in the differentiated U937 cell line. As parasite infection of U937 cells requires prior differentiation of the cells into macrophages stimulated by PMA, NF-κB DNA binding activity was first analyzed in undifferentiated cells, as well as in differentiated cells treated with PMA. Nuclear extracts prepared from untreated cells revealed that there was constitutive expression of a κB DNA binding protein mediating the formation of a nucleoprotein complex, designated complex I (Fig. 1A, lane 1). Compared to the results obtained with undifferentiated cells, PMA treatment decreased the intensity of complex I and induced the formation of a new complex, designated complex II, which was characterized by less electrophoretic mobility (Fig. 1A, lane 2).

FIG. 1.

(A) NF-κΒ activation in the human U937 cell line in response to Leishmania infection. Activation of NF-κΒ was measured by EMSA by using a radiolabeled oligonucleotide encompassing the NF-κΒ consensus motif. Nuclear extracts were prepared from unstimulated cells (lanes 1, 4, and 7), PMA-treated differentiated cells (lanes 2, 5, and 8), or cells treated with PMA and infected with L. major amastigotes (lanes 3, 6, and 9). Lane P contained free probe. Nuclear extraction and EMSA were performed as described in Materials and Methods. The specificity of binding was determined by competition with a 100-fold excess of unlabeled NF-κΒ oligonucleotide (NF-κΒ) (lanes 4, 5, and 6) or a 100-fold excess of unlabeled NF-κΒ mutant oligonucleotide (NF-κΒm) (lanes 7, 8, and 9). The arrowheads indicate the positions of NF-κΒ complexes I, II, and III. (B) Kinetic of appearance of Leishmania-induced NF-κB complex III. PMA-treated differentiated U937 cells were incubated without (lane PMA) or with amastigotes for different times (30 min and 1, 2, 4, and 6 h). Untreated cells were used as a control (lane NS). Lane P contained free probe.

PMA-treated differentiated cells were then incubated with L. major amastigotes for 2 h, and nuclear proteins were extracted for EMSA. As shown in Fig. 1A, lane 3, L. major infection had two prominent effects. It eliminated expression of the constitutive NF-κB DNA binding proteins, as well as the PMA-induced NF-κB DNA binding proteins, as shown by the disappearance of both complex I and complex II. In addition, it induced the formation of a new protein NF-κB DNA binding complex (designated complex III), which had greater electrophoretic mobility than complexes I and II.

The specificity of NF-κB binding was confirmed by the complete displacement of all three NF-κB DNA binding complexes in the presence of a 100-fold excess of unlabeled wild-type oligonucleotide (Fig. 1A, lanes 4 to 6) but not in the presence of a 100-fold excess of the mutant oligonucleotide (Fig. 1A, lanes 7 to 9). This result was probably not due to the presence of residual murine cells, since amastigote preparations obtained by Percoll purification (22) induced the same NF-κB complex. Moreover, similar results were obtained with PNA⁻ promastigotes (data not shown).

Kinetic experiments performed with PMA-treated differentiated U937 cells infected by L. major amastigotes showed that elimination of complexes I and II and induction of the new complex III occurred 30 min after infection. These effects persisted for at least 6 h (Fig. 1B).

The role of parasite internalization in NF-κB DNA binding activity was then assessed by using cytochalasin D, an inhibitor of actin polymerization. PMA-treated differentiated U937 cells were treated with 40 μM cytochalasin D for 1 h prior to incubation with amastigotes (Fig. 2, lane 6). An EMSA revealed that cytochalasin D-treated cells still down-expressed complexes I and II and formed complex III. Thus, the effects of Leishmania infection on NF-κB DNA binding complex formation do not require parasite internalization but are probably triggered by surface interactions between parasites and macrophages.

FIG. 2.

Effect of cytochalasin D treatment of U937 cells on NF-κΒ activation. Lanes 2 and 3 contained extracts of unstimulated and PMA-treated differentiated U937 cells, respectively. PMA-treated differentiated cells were infected with L. major (lane 4) or treated with cytochalasin D (CytD) for 1 h prior to infection (lane 6). As a control, PMA-treated differentiated cells were washed twice and then incubated for 2 h in medium without PMA (lane 5). Lane 1 contained an extract of amastigotes. A, amastigotes.

It seems unlikely that the disappearance of complexes I and II was a consequence of PMA withdrawal; rather, it appeared to be due to the parasite infection. Indeed, EMSA performed with PMA-treated differentiated U937 cells that were washed twice and rested in medium alone for 2 h showed that both complex I and complex II persisted (Fig. 2, lane 5). Furthermore, incubation of parasite extract with radiolabeled NF-κB oligonucleotide did not result in any complex formation (Fig. 2, lane 1), indicating that the complexes were not due to amastigote-derived proteins.

Characterization of Leishmania-induced NF-κΒ complexes.

In order to identify which members of the NF-κB family are involved in the NF-κB nucleoprotein complexes induced by parasite infection, we performed antibody supershift experiments using antibodies specific for p50, p52, p65, or c-Rel proteins. The supershift assays showed that the p50 homodimer was constitutively expressed in the nuclei of nonstimulated U937 cells and that p50 and p65 subunits of NF-κB translocated from cytoplasm to the nucleus upon PMA stimulation (data not shown). As shown in Fig. 3, addition of anti-p65 monoclonal antibody did not affect the mobility of the parasite-induced complex III. The latter complex was completely supershifted by the anti-p50 antibodies and was partially supershifted by c-Rel antibodies. The NF-κB complexes induced by promastigotes also contained p50 and c-Rel proteins (data not shown). These results show that Leishmania parasites modulate the pattern of NF-κB expression in the host cells; they inhibit the DNA binding activity of p50/p65 heterodimers and induce a complex composed of p50/p50 homodimers and p50/c-Rel heterodimers.

FIG. 3.

Characterization of Leishmania-induced NF-κB components by supershift experiments. Complexes formed between the NF-κB probe and nuclear proteins extracted from PMA-treated differentiated U937 cells infected by amastigotes were analyzed by EMSA. The reaction mixtures contained either no antibody (−) (lane 2) or antibody directed against p50 (lane 3), p65 (lane 4), c-Rel (lane 5), or p50 and c-Rel (lane 6). The arrowheads indicate the positions of complexes supershifted by antibody binding.

Transcriptional activity of Leishmania-induced NF-κΒ complexes.

It was demonstrated previously that the c-Rel transcription factor can act as a positive or negative regulator of transcription in macrophages (18). The transactivation function of the parasite-induced NF-κB dimers was investigated. U937 cells were stably transfected with a plasmid containing three NF-κB target sequences linked upstream of the luciferase reporter gene. As shown in Fig. 4, L. major amastigotes induced significant (P < 0.05) transactivation of the reporter gene, as determined by measuring luciferase activity in cell lysates. This effect could result in up to a sixfold increase in the level of luciferase activity compared with the level induced by PMA 6 h following infection.

FIG. 4.

Effect of Leishmania infection on transactivation mediated by NF-κΒ in U937 cells. U937 cells were stably transfected with the NF-κΒ-luciferase construct. Transfected cells were either not stimulated (NS), differentiated by PMA treatment (PMA), or stimulated and left in contact with L. major amastigotes for 6 h (PMA+A). Luciferase activity was determined in cell lysates after the desired time of incubation and was normalized to the protein content. The results are representative of the results of three different experiments and are expressed as the fold increases in luciferase activity induced by the experimental conditions relative to the luciferase activity measured in U937 resting cells.

Effect of L. major infection on cytokine production by U937 cells.

The ability of Leishmania parasites to modulate NF-κB activation in macrophages suggests that this effect may interfere with host gene activity. Thus, we investigated the involvement of NF-κB in Leishmania-induced monokine production.

We first checked the capacity of Leishmania parasites to induce IL-10 and TNF-α production by U937 cells. Differentiated U937 cells were incubated with amastigotes for 18 h, and the levels of IL-10 and TNF-α in the supernatants were determined by ELISA. The spontaneous secretion of IL-10 by U937 cells was low and was weakly induced after PMA treatment. However, upon infection by Leishmania amastigotes, there was a threefold increase in IL-10 secretion (P < 0.01) (Fig. 5). The baseline TNF-α secretion by U937 cells was undetectable. PMA stimulation resulted in a significant increase in TNF-α synthesis, which was further amplified (P < 0.05) by infection of U937 cells by amastigotes (Fig. 5).

FIG. 5.

Effects of L. major infection and the NF-κΒ inhibitor CAPE on in vitro production of IL-10 and TNF-α by U937 cells. U937 cells were either not stimulated (NS) or were differentiated by PMA treatment (PMA). PMA-treated differentiated U937 cells were infected by amastigotes (A) for 18 h or were treated with CAPE prior to infection with L. major amastigotes (A+CAPE). Cytokine levels were then measured by specific ELISA. The results are representative of the results of three different experiments.

Finally, we investigated the involvement of the p50/c-Rel NF-κB complex in Leishmania amastigote-induced cytokine production by U937 cells. Treatment of cells with different nontoxic concentrations of CAPE, a specific NF-κB inhibitor, showed that there were dose-dependent (data not shown) and significant decreases in the amounts of IL-10 and TNF-α (P < 0.01 and P < 0.05, respectively) produced by L. major amastigote-infected U937 cells (Fig. 5). These results suggest that NF-κB complex induction following L. major amastigote infection is involved in the triggering of cytokine synthesis.

NF-kB expression in L. major-infected human monocytes.

To extend the results obtained with the human-derived U937 cell line, the effect of L. major infection on NF-κB binding activity and its involvement in IL-10 synthesis were examined with freshly isolated human peripheral blood monocytes.

Monocyte monolayers were incubated for 2 h with freshly isolated amastigotes at a parasite-to-cell ratio of 5:1, and then nuclear extracts were prepared and an EMSA was performed. As shown in Fig. 6, human monocytes infected with Leishmania amastigotes induced the appearance of a DNA-protein complex with an electrophoretic mobility similar to that of complex III observed in PMA-treated differentiated U937 cells. These complexes were neither specific to amastigotes alone nor due to murine serum components since similar results were obtained with PNA⁻ or PNA⁻ immunoglobulin G-coated promastigotes (data not shown). Incubation with anti-c-Rel and incubation with anti-p50 (but not incubation with anti-p65) resulted in partial depletion and total depletion, respectively, of the specific NF-κB binding band and the appearance of supershifted bands. These data strongly suggest that p50- and c-Rel-containing complexes are selectively induced in human monocytes in response to Leishmania infection.

FIG. 6.

(A) Effect of L. major infection on NF-κΒ activation in human monocyte cells. Activation of NF-κΒ was measured by EMSA by using a radiolabeled oligonucleotide encompassing the NF-κΒ consensus motif. Nuclear extracts were prepared from unstimulated cells (NS) (lane 2) and amastigote-infected cells (lane 3). For supershift experiments, extracts from amastigote-infected cells were incubated with either antibody directed against p50 (lane 4), antibody directed against p65 (lane 5), antibody directed against c-Rel (lane 6), antibody directed against p50 and c-Rel (lane 7), antibody directed against p50 and p65 (lane 8), or antibody directed against p65 and c-Rel. (lane 9). Lane 1 contained free probe (P). (B) Effect of L. major infection on IL-10 production by human monocytes and macrophages. Monocytes were either not stimulated (NS), infected with amastigotes (A), or treated for 1 h with CAPE before incubation of the cells with amastigotes (A+CAPE). The IL-10 level was measured 18 h after stimulation by ELISA.

Similarly, the effect of the NF-κB inhibitor CAPE on IL-10 production was tested with fresh human monocytes. CAPE-treated or control untreated monocytes were incubated with amastigotes for 18 h, and the amounts of IL-10 in the supernatant were then determined by ELISA. As a positive control, monocytes were also treated with LPS, a known inducer of IL-10 secretion (data not shown). After 18 h of infection, the IL-10 levels in the culture supernatant were increased compared to the levels obtained with uninfected control monocytes. Treatment of monocytes with CAPE partially and significantly (P < 0.01) inhibited the production of IL-10 (Fig. 6). This result indicates that the p50/c-Rel NF-κB heterodimer expressed upon infection by L. major amastigotes is involved in IL-10 secretion by freshly isolated human monocytes.

DISCUSSION

Leishmania parasites have the ability to survive and to multiply within macrophages. Their capacity to circumvent leishmanicidal activities of macrophages have been linked to alterations in key signaling cascades (11, 31, 32). To further characterize the mechanisms by which Leishmania interferes with signal transduction pathways, we investigated activation of the NF-κB pathway in the U937 cell line and in human monocytes exposed to L. major amastigotes.

Our results show that amastigotes inhibit the translocation of the p50/p65 heterodimer triggered by PMA in differentiated U937 cells. This effect likely reflects inhibition of Ca²⁺-dependent protein kinase C translocation and its down-modulation by Leishmania, as reported by Descoteaux et al. (11). More interestingly, L. major amastigotes induce a complex composed of p50/p50 homodimers and p50/c-Rel NF-κB heterodimers both in PMA-treated differentiated U937 cells and in freshly isolated human monocytes. Similar results were obtained when U937 cells were infected with promastigotes. Using a luciferase reporter construct driven by the NF-κB transcription factor, we showed that this newly induced p50/c-Rel complex is transcriptionally active. Activation of the NF-κB transcription factor by Leishmania parasites is not fully understood. Mattner and coworkers previously reported that murine peritoneal macrophages infected by L. major parasites contain only a few NF-κB proteins that could bind to the NF-κB binding site (29). Studies of IκBα degradation or IκBα mRNA expression as a reliable indicator of NF-κB activation have yielded contradictory results. Indeed, whereas L. donovani promastigotes do not induce IκB degradation in murine bone marrow-derived macrophages, an increase in IκB mRNA expression was detected by in situ hybridization in the footpads of L. donovani-infected mice (14, 39). Our results showing that Leishmania induced NF-κB activation are in accordance with the latter findings.

The modulation of NF-κB expression by Leishmania parasites is cytochalasin D resistant, suggesting that parasite uptake is not an absolute requirement for inhibition of the DNA binding activity of p50/p65 and induction of p50/c-Rel. Both effects are likely triggered by surface interactions between parasites and macrophages. A previous report showed that some inhibitory effects of Leishmania parasites on macrophages were probably initiated by ligation of specific cell surface receptors rather than by disruption of downstream events (27). It is well known that the internalization of L. major amastigotes by phagocytic cells occurs primarily through the Fcγ receptors and CR3 (19). However, antibody cross-linking of Fcγ receptors and stimulation of CR3 were shown to induce p50/p65 NF-κB expression in a human monocytic cell line (2, 46). Our results show that Leishmania amastigotes induce p50- and c-Rel-containing complexes. Moreover, infection by L. major promastigotes (immunoglobulin G free) induces a complex composed of p50/p50 homodimers and p50/c-Rel heterodimers. These results suggest that some macrophage surface molecules other than Fcγ and CR3 are likely implicated in the cell-amastigote interactions and transduce signals to monocytes and macrophages which result in nuclear translocation of the newly described p50/c-Rel complex rather than the canonical p50/p65 complex. Such putative receptors have not been identified yet. Toll-like receptors (TLRs) are tempting candidates, although no proof has been presented to date to implicate TLRs in a macrophage-Leishmania interaction (1). Recently, however, upregulation and stimulation of TLR-2 by L. major lipophosphoglycan have been observed with human NK cells (6).

Gene deletion studies have clearly established that NF-κB is required for resistance to a variety of viral, bacterial, and parasitic infections, including Leishmania infections (45). These studies provide indirect evidence for the unique function of each member of the Rel family in L. major infection. NF-κB1 (p50) plays a key role in the adaptive immune response to L. major and is required for CD4 T-cell proliferation and development of Th1 responses (4). In contrast, NF-κB2 (p52) and c-Rel are involved in the innate responses to this intracellular pathogen. Thus, p52^−/− mice exhibit a specific impairment in CD40-induced IL-12 production by macrophages (44), and c-Rel^−/− mice with the genetic background of a resistant strain develop progressive experimental leishmaniasis (25). The increased susceptibility to L. major is correlated with a defect in macrophage function. Resident macrophages from c-Rel-deficient mice showed a marked reduction in NO production and parasite killing compared to the results obtained with macrophages from wild-type mice (18). Thus, our finding that L. major selectively induces p50/c-Rel in human monocytes is consistent with the results described above and further illustrates the ability of a given cell to use different components of the NF-κB family under various physiological or pathological conditions to fine-tune the gene activation response to the stimulus.

Macrophages exploit NF-κB-dependent pathways to produce several molecules associated with the host inflammatory response, including cytokines (10, 26), chemokines (21), inducible oxide synthase (47), and adhesion molecules. In the present study we investigated the effects of amastigotes on the anti-inflammatory IL-10 cytokine and the proinflammatory TNF-α cytokine. We showed that L. major amastigotes induce TNF-α production in differentiated U937 cells, as previously reported for promastigotes (3, 16). We also showed that IL-10 is induced by L. major amastigotes in both differentiated U937 cells and freshly isolated monocytes. Similar results were reported by Kane and coworkers, although IL-10 production in their hands required costimulation with LPS (23).

The role of NF-κB in IL-10 and TNF-α gene activation has been demonstrated previously (10, 30). Using the specific NF-κΒ inhibitor CAPE (33), we showed that NF-κB is also involved in Leishmania amastigote-induced IL-10 and TNF-α production. Interestingly, macrophages from c-Rel^−/− mice produce reduced levels of TNF-α in response to Leishmania infection (18). On the other hand, c-Rel-containing complexes play a significant role in the regulation of other cytokine genes in macrophages, especially the IL-12p40 gene (17, 42), and a recent study showed that c-Rel is required for IL-12p40 production by macrophages in response to several microbial product-transducing activation signals through a TLR (28). The fact that Leishmania infection induces both inhibition of IL-12 production and p50/c-Rel complex formation suggests that the inhibition probably does not act through NF-κB pathways (38).

Our findings show that L. major-induced p50/c-Rel NF-κB complexes are involved in the production of the anti- or proinflammatory cytokines; TNF-α is clearly involved in control of inflammation at the site of parasite inoculation (9), while IL-10 has a potent immunosuppressive role (23) and is implicated in the long-term survival of L. major (8). However, the role of IL-10 might not be only to promote disease, since it could act as a feedback regulator of the inflammatory process triggered by the parasite in order to limit tissue pathology.

The mechanism by which L. major amastigotes inhibit p50/p65 translocation and induce p50/c-Rel complex formation and production of IL-10 and TNF-α in human macrophages remains to be explored.