HIV infection of placental macrophages: effect on the secretion of HIV stimulatory cytokines.

Vertical transmission of HIV-1 can occur at three different stages: during gestation, delivery and breast feeding. To determine the role of cytokines in vertical transmission of HIV during gestation, we studied the secretion of IL-1beta, TNF-alpha and IL-6 from in vitro infected and Mock-infected placental macrophages (Hofbauer cells) in comparison to blood monocyte derived macrophages (MDM). Hofbauer cells stimulated with lipopolysaccharide (LPS) secreted lower levels of HIV stimulatory cytokines (6-8 ng/ml) in the supernatants than MDM (26 ng/ml, p<0.005). Cytokine levels in MDM decreased upon HIV infection to 7 ng/ml. IL-6 was the major cytokine produced after LPS stimulation by the two cell populations (p<0.005), being MDM the major cytokine producer. In vitro infection studies with a M-tropic virus (HIV-BaL) indicated that MDM were 10x more susceptible to HIV than placental macrophages (p=0.001). Our results indicate that although macrophages from term placenta secrete lower amount of HIV stimulatory cytokines than MDM, there was no correlation between the levels of cytokines and HIV production by both cells.     

A functional C5a anaphylatoxin receptor in a teleost species

The anaphylatoxins are potent, complement-derived low m.w. proteins that bind to specific seven-transmembrane receptors to elicit and amplify a variety of inflammatory reactions. C5a is the most potent of these phlogistic peptides and is a strong chemoattractant for neutrophils and macrophages/monocytes. Although lower vertebrates possess complement systems that are believed to function similarly to those of mammals, anaphylatoxin receptors have not previously been characterized in any nonmammalian vertebrate. To study the functions of C5a in teleost fish, we generated recombinant C5a of the rainbow trout, Oncorhynchus mykiss (tC5a), and used fluoresceinated tC5a (tC5aF) and flow cytometry to identify the C5a receptor (C5aR) on trout leukocytes. Granulocytes/Macrophages present in cell suspensions of the head kidney (HKL), the main hemopoietic organ in teleosts, showed a univariate type of receptor expression, whereas those from the peripheral blood demonstrated either a low or high level of expression. The binding of tC5aF was inhibited by excess amounts of unlabeled tC5a or tC5a(desArg), demonstrating that sites other than the C-terminal of tC5a interact with the C5aR. Both tC5a and tC5a(desArg) were able to induce chemotactic responses in granulocytes in a concentration-dependent manner, but the desArg derivative was at least 10-fold less active. Homologous desensitization occurred after HKL were exposed to continuous or high concentrations of tC5a, with a loss of tC5aF binding and an 80% reduction in chemotactic responses toward tC5a. Pertussis toxin reduced the migration of HKL toward tC5a by 40%, suggesting only a partial involvement of pertussis toxin-sensitive G(i) proteins in tC5a-mediated chemotaxis.     

Human C3a and C3a desArg anaphylatoxins have conserved structures,in contrast to C5a and C5a desArg

Complement is a part of innate immunity that has a critical role in the protection against microbial infections, bridges the innate with the adaptive immunity and initiates inflammation. Activation of the complement, by specific recognition of molecular patterns presented by an activator, for example, a pathogen cell, in the classical and lectin pathways or spontaneously in the alternative pathway, leads to the opsonization of the activator and the production of pro‐inflammatory molecules such as the C3a anaphylatoxin. The biological function of this anaphylatoxin is regulated by carboxypeptidase B, a plasma protease that cleaves off the C‐terminal arginine yielding C3a desArg, an inactive form. While functional assays demonstrate strikingly different physiological effects between C3a and C3a desArg, no structural information is available on the possible conformational differences between the two proteins. Here, we report a novel and simple expression and purification protocol for recombinant human C3a and C3a desArg anaphylatoxins, as well as their crystal structures at 2.3 and 2.6 Å, respectively. Structural analysis revealed no significant conformational differences between the two anaphylatoxins in contrast to what has been reported for C5a and C5a desArg. We compare the structures of different anaphylatoxins and discuss the relevance of their observed conformations to complement activation and binding of the anaphylatoxins to their cognate receptors.     

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-alpha, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-gamma-inducing cytokines, IL-12 and IFN-alpha, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-gamma production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-alpha, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-alpha and IL-18 play a significant role in enhancing IFN-gamma synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Metabolism of nitric oxide by Neisseria meningitidis modifies release of NO-regulated cytokines and chemokines by human macrophages

Macrophages produce nitric oxide (NO) via the inducible nitric oxide synthase as part of a successful response to infection. The gene norB of Neisseria meningitidis encodes a NO reductase which enables utilization and consumption of NO during microaerobic respiration and confers resistance to nitrosative stress-related killing by human monocyte-derived macrophages (MDM). In this study we confirmed that NO regulates cytokine and chemokine release by resting MDM: accumulation of TNF-alpha, IL-12, IL-10, CCL5 (RANTES) and CXCL8 (IL-8) in MDM supernatants was significantly modified by the NO-donor S-nitroso-N-penicillamine (SNAP). Using a protein array, infection of MDM with N. meningitidis was shown to be associated with secretion of a wide range of cytokines and chemokines. To test whether NO metabolism by N. meningitidis modifies release of NO-regulated cytokines, we infected MDM with wild-type organisms and an isogenic norB strain. Resulting expression of the cytokines TNF-alpha and IL-12, and the chemokine CXCL8 was increased and production of the cytokine IL-10 and the chemokine CCL5 was decreased in norB-infected MDM, in comparison to wild-type. Addition of SNAP to cultures infected with wild-type mimicked the effect observed in cultures infected with the norB mutant. In conclusion, NorB-catalysed removal of NO modifies cellular release of NO-regulated cytokines and chemokines.     

Highly pathogenic avian influenza virus H5N1 infects alveolar macrophages without virus production or excessive TNF-alpha induction

Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 causes severe, often fatal pneumonia in humans. The pathogenesis of HPAIV H5N1 infection is not completely understood, although the alveolar macrophage (AM) is thought to play an important role. HPAIV H5N1 infection of macrophages cultured from monocytes leads to high percentages of infection accompanied by virus production and an excessive pro-inflammatory immune response. However, macrophages cultured from monocytes are different from AM, both in phenotype and in response to seasonal influenza virus infection. Consequently, it remains unclear whether the results of studies with macrophages cultured from monocytes are valid for AM. Therefore we infected AM and for comparison macrophages cultured from monocytes with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus, and determined the percentage of cells infected, virus production and induction of TNF-alpha, a pro-inflammatory cytokine. In vitro HPAIV H5N1 infection of AM compared to that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% vs up to 84%), lower virus production and lower TNF-alpha induction. In vitro infection of AM with H3N2 or H1N1 virus resulted in even lower percentages of infected cells (up to 7%) than with HPAIV H5N1, while virus production and TNF-alpha induction were comparable. In conclusion, this study reveals that macrophages cultured from monocytes are not a good model to study the interaction between AM and these influenza virus strains. Furthermore, the interaction between HPAIV H5N1 and AM could contribute to the pathogenicity of this virus in humans, due to the relative high percentage of infected cells rather than virus production or an excessive TNF-alpha induction.     

Acceleration of R5 HIV replication by polymorphonuclear neutrophils in cultures of macrophages

Cell-to-cell contact between monocyte-derived macrophages (MDM) and endothelial cells has resulted in the increased proliferation of CC chemokine receptor 5/M-tropic (R5) human immunodeficiency virus (HIV) in MDM. In the present study, R5 HIV replication was shown to be increased by polymorphonuclear neutrophils (PMN) in MDM cultures through the soluble factors released from these PMN. The replication of R5 HIV in MDM was greatly enhanced when PMN were added to cultures. An increase in the replication of R5 HIV was also demonstrated when the virus was replicated in MDM cultured in a double chamber transwell with PMN. Chemokine ligand (CCL) 2 and interleukin (IL)-10 were detected in culture fluids of PMN exposed to R5 HIV. The replication of R5 HIV was not accelerated in cultures of MDM and PMN in a double chamber transwell supplemented with a mixture of monoclonal antibodies directed against CCL2 and IL-10. Similarly, the replication of R5 HIV was accelerated in MDM cultures supplemented with a mixture of recombinant CCL2 and IL-10. These results indicated that, in response to the viral stimulation, PMN produce CCL2 and IL-10 and enhance the replication of R5 HIV in MDM cultures.     

Over-expression of hsp-70 inhibits bacterial lipopolysaccharide-induced production of cytokines in human monocyte-derived macrophages

Cytokines released from monocytes and macrophages are major mediators of inflammation. Heat shock significantly inhibits cytokine production from these cells. To investigate whether this inhibitory effect was mediated by heat-shock proteins (HSP), we transfected human peripheral blood monocyte-derived macrophages (MDM) with HSP-70 cDNA and examined Brucella melitensis lipopolysaccharide (LPS)-induced cytokine production in transfected cells. Over-expression of HSP-70 protein in the gene-transfected MDM had no effect on cytokine synthesis unless LPS was added. LPS-induced increases in production of tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-10 and IL-12 were significantly inhibited by the over-expression of HSP-70. However, over-expression of HSP-70 did not block LPS-induced increase in IL-6 synthesis. To further confirm these results, an antisense HSP-70 DNA oligomer was used to block HSP-70 synthesis. The inhibitory effect of HSP-70 on LPS-induced cytokine production in gene- transfected cells was completely reversed after treatment of cells with 5 microM antisense HSP-70. The same concentration of antisense HSP-70 also partially reversed heat-shock-induced inhibition of LPS-stimulated cytokine production. These results suggest that HSP-70 is involved in the regulation of LPS-induced cytokine production and that this family of proteins plays a role in mitigating adverse effects of endotoxin during infection or other pathological stresses.     

Cytokine Regulation of Human Immunodeficiency Virus Type 1 Entry and Replication in Human Monocytes/Macrophages through Modulation of CCR5 Expression

Human macrophages express chemokine receptors that act as coreceptors for human immunodeficiency virus type 1 (HIV-1) and are major targets for HIV-1 infection in vivo. The effects of cytokines on HIV-1 infection of macrophages and on the expression of CCR5, the principal coreceptor for macrophage-tropic viruses, have now been investigated. Expression of CCR5 on the surface of freshly isolated human monocytes was virtually undetectable by flow cytometry with the monoclonal antibody 5C7. However, after culture of monocytes for 48 h in serum-free medium, approximately 30% of the resulting macrophages expressed CCR5 and the cells were susceptible to infection by macrophage-tropic HIV-1. Addition of either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) to the cultures markedly increased both the extent of HIV-1 entry and replication as well as surface expression of CCR5. In contrast, addition of the T-helper 2 (Th2) cell-derived cytokine interleukin-4 (IL-4) or IL-13 prevented the expression of CCR5 induced by culture in medium alone, and IL-4 inhibited virus entry, replication, and cytopathicity under these conditions. IL-4 or IL-13 also prevented the stimulatory effects of M-CSF or GM-CSF on CCR5 expression as well as HIV-1 entry and replication. In addition, IL-4 reversed the increase in CCR5 expression induced by pretreatment of cells with M-CSF. Although IL-10 also inhibits HIV-1 replication in macrophages, it did not suppress surface CCR5 expression induced by colony-stimulating factors. These results indicate that the cytokine environment determines the susceptibility of macrophages to HIV-1 infection by various mechanisms, one of which is the regulation of HIV-1 coreceptor expression.     

Induction of interleukin-10 by human immunodeficiency virus type 1 and its gp120 protein in human monocytes/macrophages.

In this study, we evaluated the effects of human immunodeficiency virus type 1 (HIV-1) and its gp120 protein on interleukin-10 (IL-10) expression in cultured human monocytes/macrophages. Infection of either 1-day monocytes or 7-day monocyte-derived macrophages with HIV-1 strain Ba-L resulted in clear-cut accumulation of IL-10 mRNA at 4 and 24 h. Likewise, treatment of these cells with recombinant gp120 induced IL-10 mRNA expression and caused a marked increase in IL-10 secretion. Monoclonal antibodies to gp120 strongly inhibited recombinant gp120-induced IL-10 secretion by monocytes/macrophages. Moreover, the addition of IL-10 to monocytes/macrophages resulted in a significant inhibition of HIV-1 replication 7 and 14 days after infection. On the whole, these results indicate that HIV-1 (possibly through its gp120 protein) up-regulates IL-10 expression in monocytes/macrophages. We suggest that in vivo production of IL-10 by HIV-primed monocytes/macrophages can play an important role in the early response to HIV-1 infection.     

Quantification of PPAR-gamma protein in monocyte/macrophages from healthy smokers and non-smokers: a possible direct effect of nicotine

Previous observations demonstrated that Peroxisome Proliferator-Activated Receptor-gamma (PPAR-gamma), a key regulator of adipocyte differentiation, is expressed in a large variety of cells, including cells of the monocyte/macrophage lineage. This study was aimed to quantify both the constitutive and ligand-induced PPAR-gamma expression in monocytes and monocyte-derived macrophages (MDM) isolated from healthy smokers and non-smokers, and to evaluate the possible direct effect of nicotine. PPAR-gamma protein was detected by Western blot and quantification was performed by calculating the ratio between PPAR-gamma and beta-actin protein expression. Cytokine release was measured with enzyme-linked immunoassay kits. Constitutive PPAR-gamma protein was detected in human monocytes and its expression was up-regulated along with differentiation to MDM. The endogenous ligand 15-deoxy-delta(12,14)-prostaglandin J(2) and the synthetic agonist ciglitazone enhanced PPAR-gamma expression, the former being effective also at low micromolar concentrations. Both agonists significantly inhibited the basal secretion of pro-inflammatory cytokines (e.g., TNF-alpha, IL-6), ciglitazone being more potent. Monocytes and MDM from healthy smokers presented a significantly enhanced (4-fold and 2.5-fold, respectively) constitutive PPAR-gamma expression, as compared to those from healthy non-smokers. However, ligand-induced PPAR-gamma expression and inhibition of cytokine secretion were similar in healthy smokers and non-smokers. Nicotine dose-dependently enhanced PPAR-gamma expression with a maximum at 10 muM, and inhibited release of pro-inflammatory cytokines; these effects were reversed by alpha-bungarotoxin. Nicotine and PPAR-gamma agonists did not exert synergistic effects. In conclusion, monocytes and MDM from healthy smokers present a constitutively enhanced PPAR-gamma expression; this effect is reproduced, to some extent, by nicotine in vitro.     

Persistent CCR5 utilization and enhanced macrophage tropism by primary blood human immunodeficiency virus type 1 isolates from advanced stages of disease and comparison to tissue-derived isolates

Viral phenotype, tropism, coreceptor usage, and envelope gene diversity were examined in blood isolates collected from 27 individuals at different stages of human immunodeficiency virus type 1 (HIV-1) disease and tissue derived isolates from 10 individuals with AIDS. The majority (89%) of blood and all tissue HIV-1 isolates from all stages of infection were non-syncytium inducing and macrophage (M) tropic. Tropism and productive infection by HIV isolates in both monocytes and monocyte-derived macrophages (MDM) increased in advanced disease (HIV tropism for monocytes, 1 of 6 from categories I and II versus 11 of 21 [P = 0.05] from category IV and II [CD4 < 250]; and high-level replication in MDM, 1 of 6 from categories I and II versus 16 of 21 from categories IV and II [P = 0. 015]). There was a high level of replication of blood and tissue isolates in T lymphocytes without restriction at any stage. Overall, the level of replication in MDM was 5- to 10-fold greater than in monocytes, with restriction in the latter occurring mainly at entry and later stages of replication. Only three blood isolates were identified as syncytium inducing, and all had a dualtropic phenotype. There was a significant increase of HIV envelope gene diversity, as shown by a heteroduplex mobility assay, in advanced disease; this may partly underlie the increase of HIV replication in MDM. Unlike blood isolates (even those from patients with advanced disease), tissue isolates displayed greater similarities (90%) in productive infection between MDM and monocytes. The majority (87%) of all isolates, including those from patients with advanced disease, used CCR5, and only 5 of 37 isolates showed expanded coreceptor usage. These results indicate that in the late stage of disease with increasing viral load and diversity, CCR5 utilization and M-tropism persist in blood and tissue and the replicative ability in macrophages increases. This suggests that these characteristics are advantageous to HIV and are important to disease progression.     

Anaphylatoxin C5a induces monocyte recruitment and differentiation into dendritic cells by TNF-alpha and prostaglandin E2-dependent mechanisms

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-1alpha (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-1alpha 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-alpha and PGE(2) 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-alpha, but not PGE(2), for their C5a-mediated maturation in vivo. C5a-transformed monocytes represented an inflammatory type of DC, as they constitutively secreted high amounts of TNF-alpha, 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.     

Macrophages exposed to lymphotropic and monocytotropic HIV induce similar CTL responses despite differences in productive infection

Macrophages are accessory cells that are vulnerable to infection by HIV-1. HTLV-IIIB, a lymphotropic strain of HIV, infects macrophages poorly resulting in either no or low levels of virus expression compared to high levels of productive infection after exposure of macrophages to the monocytotropic HIV strain Ada-M. Whether this results in an impaired ability of HTLV-IIIB-exposed macrophages to initiate protective cytotoxic T lymphocyte (CTL) immune responses against these strains is not well defined. We investigated the ability of monocyte-derived macrophages (MDM) exposed to lymphotropic and monocytotropic HIV strains to initiate primary CTL responses in vitro. MDM exposed to HTLV-IIIB induced a specific primary CTL response that was comparable to MDM exposed to the monocytotropic strain Ada-M despite marked differences in productive HIV infection in MDM between the two strains. CTL generated in this model were MHC-restricted, strain-specific, and CD8+. These data demonstrate that high levels of productive HIV infection in accessory cells are not a prerequisite for the generation of a primary CTL response, suggesting a novel immunologic interaction between MDM and lymphotropic HIV strains.     

Human cytomegalovirus infection reduces surface CCR5 expression in human microglial cells,astrocytes and monocyte-derived macrophages

Within the brain, glial cells are target cells for human cytomegalovirus (HCMV) and HIV. We infected cultures of unstimulated human microglial cells and astrocytes of embryonic origin and of monocyte-derived macrophages (MDM) with HCMV strain AD169 and observed down-regulation of the plasma membrane expression of CCR5 in the three cell types, and of CXCR4 and CD4 in microglial cells only. Cells were then coinfected simultaneously or at a 24-h interval with both AD169 and two different HIV-1 monocytotropic strains. HCMV late antigens and HIV-1 tat protein colocalized in the cytoplasm of 5-10% of microglia and MDM. p24 antigen levels decreased 10- to 40-fold in supernatants of MDM and the reduction was greater when HCMV infection was performed 24 h before HIV-1 infection. These data suggest that HCMV-induced reduction in the cell-surface expression of the primary co-receptor of HIV-1 monocytotropic strains may impair the ability of HIV to infect these cells.     

Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila infection

Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor-alpha (TNF-alpha) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF-alpha restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) alpha/beta increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody-treated cultures corresponded with enhanced levels of TNF-alpha in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF-alpha is important in controlling L. pneumophila replication, and IL-1 can regulate TNF-alpha levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.