Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils

The ability of human neutrophils to aid in defense against pulmonary infection with Mycobacterium tuberculosis is controversial. In this study, we have shown that neutrophils respond to and phagocytose M. tuberculosis in human lesions. Neutrophils from healthy individuals were able to kill significant fractions of an inoculum of M. tuberculosis within 1 h of phagocytosis, and this ability was enhanced by tumor necrosis factor alpha but not by gamma interferon. The mycobactericidal mechanism was nonoxidative, as inhibitors of reactive oxygen or reactive nitrogen intermediates did not interfere with killing. However, the mycobactericidal mechanism was associated with increased exposure of intracellular M. tuberculosis to neutrophil defensins. In vitro, human neutrophil peptides 1 to 3 were not able to kill the bacilli even at much higher levels. These studies support the concept that human neutrophils are directly involved in defense against infection with M. tuberculosis.     

Mycobacterium bovis BCG vaccination augments interleukin-8 mRNA expression and protein production in guinea pig alveolar macrophages infected with Mycobacterium tuberculosis

Alveolar macrophages are likely the first cell type to encounter Mycobacterium tuberculosis in a pulmonary infection, resulting in the production of chemokines. In order to evaluate this response, alveolar macrophages harvested from nonvaccinated and Mycobacterium bovis BCG-vaccinated guinea pigs were infected in vitro with live M. tuberculosis H37Ra or H37Rv (multiplicity of infection, 1:1) or cultured with lipopolysaccharide (10 micro g/ml) for 3, 12, and 24 h. Interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was determined by real-time PCR. Culture supernatants were assayed for guinea pig IL-8 protein by using a human IL-8 enzyme-linked immunosorbent assay kit. Alveolar macrophages harvested from BCG-vaccinated guinea pigs produced significantly more mRNA and protein for IL-8 than alveolar macrophages harvested from nonvaccinated guinea pigs at 12 and 24 h poststimulation or postinfection. Infection with attenuated M. tuberculosis (H37Ra) stimulated alveolar macrophages isolated from BCG-vaccinated guinea pigs to produce significantly more IL-8 mRNA than did alveolar macrophages infected with a virulent strain (H37Rv) at 12 and 24 h postinfection. Significant MCP-1 mRNA production was also detected in stimulated or infected alveolar macrophages; however, prior vaccination did not significantly affect levels of MCP-1 mRNA. Alveolar macrophages isolated from BCG-vaccinated guinea pigs produced significantly more IL-8 mRNA and protein when stimulated for 24 h with heat-killed H37Ra, heat-killed H37Rv, and H37Rv cell wall, but not mannose-capped lipoarabinomannan (ManLAM), than did cells stimulated with media alone. These observations indicate that prior vaccination may alter very early events in the M. tuberculosis-infected lung.     

Modulation of Mycobacterium lepraemurium growth in murine macrophages: beneficial effect of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor.

Mycobacterium lepraemurium grew progressively in monolayers of Proteose Peptone-elicited macrophages from C57BL/6 mice. Treatment of macrophage monolayers with gamma interferon led to an enhancement of growth of M. lepraemurium in macrophages. Treatment with tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor led to restriction of mycobacterial growth in macrophages.     

Mycobacterium tuberculosis uptake by recipient host macrophages is influenced by environmental conditions in the granuloma of the infectious individual and is associated with impaired production of interleukin-12 and tumor necrosis factor alpha

Transmission of Mycobacterium tuberculosis from one individual to another usually is associated with episodes of coughing. The bacteria leave the environment of the lung cavity of the infected person and travel in droplets to reach the recipient's respiratory tract. Therefore, at the time that the bacteria encounter alveolar cells (macrophages and epithelial cells) in the new host, they express virulence determinants that are regulated by the environmental conditions in the infected person. To determine if those environmental conditions encountered in the lung cavity (hyperosmolarity, acidic pH, and low oxygen tension, among others) would influence the uptake of M. tuberculosis by the recipient's alveolar macrophages, M. tuberculosis H37Rv was incubated under several conditions for different periods of time, washed at 4 degrees C, and used to infect human monocyte-derived macrophages. While increased osmolarity had no effect on M. tuberculosis uptake compared to the uptake of bacteria grown on 7H10 Middlebrook medium, both acidic pH and anaerobiosis increased the uptake of the H37Rv strain four- to sixfold. Using anti-CD11b receptor blocking antibodies or mannoside to inhibit the uptake of M. tuberculosis by macrophages, we determined that while uptake of M. tuberculosis cultured on 7H10 medium was inhibited 77% +/- 6% in the presence of anti-CD11b antibody, the antibody had no effect on the uptake of M. tuberculosis incubated at pH 6.0 and was associated with 27% inhibition of M. tuberculosis previously exposed to anaerobic conditions. The mannose receptor was also not involved with invasion after exposure to acidic conditions, and mannoside resulted in only 32% inhibition of uptake by macrophages of M. tuberculosis exposed to anaerobiosis. Uptake by macrophages also resulted in the secretion of significantly lower amounts of interleukin-12 and tumor necrosis factor alpha than that by macrophages infected with a strain cultured under laboratory conditions. M. tuberculosis cultured under the pH and oxygen concentration found in the granuloma expresses a large number of proteins that are different from the proteins expressed by bacteria grown under laboratory conditions. The results suggest that M. tuberculosis in vivo may be adapted to gain access to the intracellular environment in a very efficient fashion and may do so by using different receptors from the complement and mannose receptors.     

Streptococcal histone induces murine macrophages To produce interleukin-1 and tumor necrosis factor alpha

The histone-like protein (HlpA) is highly conserved among streptococci. After lysis of streptococci in infected tissues, HlpA can enter the bloodstream and bind to proteoglycans in the glomerular capillaries of kidneys, where it can react with antibodies or stimulate host cell receptors. Deposits of streptococcal antigens in tissues have been associated with localized acute inflammation. In this study, we measured the ability of purified HlpA (5 to 100 microg/ml), from Streptococcus mitis, to induce the production of proinflammatory cytokines by cultured, murine peritoneal macrophages. The release of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) was time and concentration dependent and was not diminished by the presence of polymyxin B. Exposure of macrophages to a mixture of HlpA and lipoteichoic acid resulted in a synergistic response in the production of both TNF-alpha and IL-1. Stimulation with a mixture of HlpA and heparin resulted in reduced cytokine production (50% less IL-1 and 76% less TNF-alpha) compared to that by cells incubated with HlpA alone. The inclusion of antibodies specific to HlpA in macrophage cultures during stimulation with HlpA did not affect the quantity of TNF-alpha or IL-1 produced. These observations suggest that streptococcal histone may contribute to tissue injury at infection sites by promoting monocytes/macrophages to synthesize and release cytokines that initiate and exacerbate inflammation. Streptococcus pyogenes, which can infect tissues in enormous numbers, may release sufficient amounts of HlpA to reach the kidneys and cause acute poststreptococcal glomerulonephritis.     

Mycobacterium tuberculosis lipomannan induces apoptosis and interleukin-12 production in macrophages

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as a virulence factor of Mycobacterium tuberculosis, and modification of the terminal arabinan residues of this compound with mannose caps (producing mannosyl-capped LAM [ManLAM]) in M. tuberculosis or with phosphoinositol caps (producing phosphoinositol-capped LAM [PILAM]) in Mycobacterium smegmatis has been implicated in various functions associated with these lipoglycans. A structure-function analysis was performed by using LAMs and their biosynthetic precursor lipomannans (LMs) isolated from different mycobacterial species on the basis of their capacity to induce the production of interleukin-12 (IL-12) and/or apoptosis of macrophage cell lines. Independent of the mycobacterial species, ManLAMs did not induce IL-12 gene expression or apoptosis of macrophages, whereas PILAMs induced IL-12 secretion and apoptosis. Interestingly, uncapped LAM purified from Mycobacterium chelonae did not induce IL-12 secretion or apoptosis. Furthermore, LMs, independent of their mycobacterial origins, were potent inducers of IL-12 and apoptosis. The precursor of LM, phosphatidyl-myo-inositol dimannoside, had no activity, suggesting that the mannan core of LM was required for the activity of LM. The specific interaction of LM with Toll-like receptor 2 (TLR-2) but not with TLR-4 suggested that these responses were mediated via the TLR-2 signaling pathway. Our experiments revealed an important immunostimulatory activity of the biosynthetic LAM precursor LM. The ratio of LAM to LM in the cell wall of mycobacteria may be an important determinant of virulence, and enzymes that modify LM could provide targets for development of antituberculosis drugs and for derivation of attenuated strains of M. tuberculosis.     

Fungal beta-glucan interacts with vitronectin and stimulates tumor necrosis factor alpha release from macrophages.

beta-Glucans are polymers of D-glucose which represent major structural components of fungal cell walls. It was shown previously that fungi interact with macrophages through beta-glucan receptors, thereby inducing release of tumor necrosis factor alpha (TNF-alpha). Additional studies demonstrated that vitronectin, a host adhesive glycoprotein, binds to fungi and enhances macrophage recognition of these organisms. Since vitronectin contains a carbohydrate-binding region, we postulated that vitronectin binds fungal beta-glucans and subsequently augments macrophage TNF-alpha release in response to this fungal component. To study this, we first determined the release of TNF-alpha from alveolar macrophages stimulated with fungal beta-glucan. Maximal TNF-alpha release occurred with moderate concentrations of beta-glucan (100 to 200 micrograms/ml), whereas higher concentrations of beta-glucan (> or = 500 micrograms/ml) caused apparent suppression of the TNF-alpha activity released. This suppression of TNF-alpha activity by high concentrations of beta-glucan was mediated by the particulate beta-glucan binding soluble TNF-alpha, through the lectin-binding domain of the cytokine, rendering the TNF-alpha less available for measurement. Next, we assessed the interaction of vitronectin with beta-glucan. Binding of 125I-vitronectin to particulate fungal beta-glucan was dose dependent and specifically inhibitable by unlabeled vitronectin. Furthermore, treatment of beta-glucan with vitronectin substantially augmented macrophage TNF-alpha release in response to this fungal component. These findings demonstrate that fungal beta-glucan can directly modulate TNF-alpha release from macrophages. Further, these studies indicate that the host adhesive glycoprotein vitronectin specifically binds beta-glucan and augments macrophage cytokine release in response to this fungal element.     

Taxol, a microtubule-stabilizing antineoplastic agent, induces expression of tumor necrosis factor alpha and interleukin-1 in macrophages.

Taxol, a naturally occurring diterpene with antitumor activity, induces tubulin polymerization to generate abnormally stable and nonfunctional microtubules. Previously, we showed that taxol has lipopolysaccharide (LPS)-like effects on macrophages. As LPS is a potent inducer of macrophage cytokine production, we investigated whether a similar effect is exerted by taxol. In a dose-dependent manner, LPS-free taxol induced release of biologically active tumor necrosis factor alpha (TNF) by inflammatory murine macrophages. Taxol-induced production of TNF was inhibitable by interleukin-10. By Northern blot, taxol (10 and 1 microM) induced TNF mRNA expression to an extent similar to LPS. Induction of TNF mRNA by 10 microM taxol was detectable at 45 min of stimulation, maximal at 90 min, and evident for at least 8 h. The same low concentration of taxol also induced interleukin 1 (IL-1) alpha and beta mRNA expression. We conclude that taxol triggers macrophages for TNF and IL-1 production. These LPS-like effects of taxol might contribute to its antitumor activity.     

Temporal effect of tumor necrosis factor alpha on murine macrophages infected with Mycobacterium avium.

Members of the Mycobacterium avium complex are a family of bacteria that persist within macrophages in the face of an immune response. Elimination of these organisms is likely due to cytokine-induced macrophage activation. Because macrophage activation by tumor necrosis factor alpha (TNF-alpha) appears critical for killing of intracellular M. avium, early downregulation of TNF-alpha levels in infected macrophages has been suggested as a survival mechanism for virulent strains of M. avium. We examined the relationship between TNF-alpha and growth of M. avium strains of differing virulence, as measured by their ability to grow in murine bone marrow-derived macrophages. When exogenous TNF-alpha was added immediately following macrophage infection, significant growth inhibition of virulent M. avium strains was observed. If TNF-alpha addition was delayed by 24 h or more, growth inhibition was abrogated. To determine if early downregulation of TNF-alpha levels could explain the differential growth of virulent and avirulent strains, levels of TNF-alpha and prostaglandin E2 (PGE2), which has been shown to suppress TNF-alpha production in uninfected macrophages, were quantified over time. Upregulation of both TNF-alpha and PGE2, as measured by enzyme-linked immunosorbent assay, was evident by 6 h postinfection, indicating that the ability of M. avium to replicate in macrophages was not directly correlated with early downregulation of TNF-alpha production. However, TNF-alpha bioactivity, as measured by cytotoxicity, was significantly decreased in virulent M. avium strains at all time periods examined. Treatment of infected macrophages with gamma interferon immediately after infection resulted in significantly increased levels of nitric oxide but did not affect the growth of virulent M. avium strains. These results suggest that while significant levels of TNF-alpha are present in supernatants from all M. avium strains, levels of biologically active TNF-alpha are significantly reduced in supernatants from virulent M. avium strains. Preliminary results suggest that upregulation of the soluble p75 TNF receptor may be one mechanism by which TNF-alpha bioactivity reduction occurs.     

Local role for tumor necrosis factor alpha in the pulmonary inflammatory response to Mycobacterium tuberculosis infection

The local intrapulmonary role of tumor necrosis factor alpha (TNF-alpha) in a protective host response during acute and chronic infection with Mycobacterium tuberculosis is incompletely understood. To directly assess its role in the intrapulmonary immune response, we compared the responses of transgenic mice with a local pulmonary blockade of TNF-alpha (SPCTNFRIIFc mice) to mice with globally inhibited TNF-alpha (TNFRKO mice) and mice with normal immune systems (control mice). Consistent with previous reports, 100% of TNFRKO mice died by 28 days after aerosol infection, and these mice had markedly increased numbers of bacteria and widespread tissue necrosis in their lungs compared to controls. The median survival time of the SPCTNFRIIFc mice was 142 days, and 75% died by 180 days. Even though the numbers of bacteria in the lungs of the SPCTNFRIIFc mice were marginally increased compared to controls, these mice had a persistent neutrophilic inflammatory response and increased expression of proinflammatory cytokines (interleukin-1 alpha/beta [IL-1 alpha/beta], IL-18, gamma interferon, IL-6, and macrophage migration inhibitory factor) and chemokines (eotaxin, macrophage inflammatory protein 1 alpha/beta, gamma interferon-inducible protein 10, macrophage chemotaxic protein 1, and TCA-3) in their lungs. These studies with the SPCTNFRIIFc mice provide direct evidence for the local importance of TNF-alpha in the proper regulation of host defense to M. tuberculosis. The studies also suggest that when the local actions of TNF-alpha are selectively impaired in the lungs, tissue destruction and death ensue, at least in part, due to persistent expression of proinflammatory mediators that would normally be downregulated.     

Babesia bovis-stimulated macrophages express interleukin-1beta, interleukin-12, tumor necrosis factor alpha, and nitric oxide and inhibit parasite replication in vitro

The tick-transmitted hemoparasite Babesia bovis causes an acute infection that results in persistence and immunity against challenge infection in cattle that control the initial parasitemia. Resolution of acute infection with this protozoal pathogen is believed to be dependent on products of activated macrophages (Mphi), including inflammatory cytokines and nitric oxide (NO) and its derivatives. B. bovis stimulates inducible nitric oxide synthase (iNOS) and production of NO in bovine Mphi, and chemical donors of NO inhibit the growth of B. bovis in vitro. However, the induction of inflammatory cytokines in Mphi by babesial parasites has not been described, and the antiparasitic activity of NO produced by B. bovis-stimulated Mphi has not been definitively demonstrated. We report that monocyte-derived Mphi activated by B. bovis expressed enhanced levels of inflammatory cytokines interleukin-1beta (IL-1beta), IL-12, and tumor necrosis factor alpha that are important for stimulating innate and acquired immunity against protozoal pathogens. Furthermore, a lipid fraction of B. bovis-infected erythrocytes stimulated iNOS expression and NO production by Mphi. Cocultures of Mphi and B. bovis-infected erythrocytes either in contact or physically separated resulted in reduced parasite viability. However, NO produced by bovine Mphi in response to B. bovis-infected erythrocytes was only partially responsible for parasite growth inhibition, suggesting that additional factors contribute to the inhibition of B. bovis replication. These findings demonstrate that B. bovis induces an innate immune response that is capable of controlling parasite replication and that could potentially result in host survival and parasite persistence.     

Effect of Mycobacterium bovis BCG vaccination on Mycobacterium-specific cellular proliferation and tumor necrosis factor alpha production from distinct guinea pig leukocyte populations

In this study, we focused on three leukocyte-rich guinea pig cell populations, bronchoalveolar lavage (BAL) cells, resident peritoneal cells (PC), and splenocytes (SPC). BAL cells, SPC, and PC were stimulated either with live attenuated Mycobacterium tuberculosis H37Ra or with live or heat-killed virulent M. tuberculosis H37Rv (multiplicity of infection of 1:100). Each cell population was determined to proliferate in response to heat-killed virulent H37Rv, whereas no measurable proliferative response could be detected upon stimulation with live mycobacteria. Additionally, this proliferative capacity (in SPC and PC populations) was significantly enhanced upon prior vaccination with Mycobacterium bovis BCG. Accordingly, in a parallel set of experiments we found a strong positive correlation between production of antigen-specific bioactive tumor necrosis factor alpha (TNF-alpha) and prior vaccination with BCG. A nonspecific stimulus, lipopolysaccharide, failed to induce this effect on BAL cells, SPC, and PC. These results showed that production of bioactive TNF-alpha from mycobacterium-stimulated guinea pig cell cultures positively correlates with the vaccination status of the host and with the virulence of the mycobacterial strain.     

Neutralization of tumor necrosis factor alpha suppresses antigen-specific type 1 cytokine responses and reverses the inhibition of mycobacterial survival in cocultures of immune guinea pig T lymphocytes and infected macrophages

Neutralization of tumor necrosis factor alpha (TNF-alpha) significantly down-regulated antigen-induced lymphoproliferation and the expression of interleukin-12 p40 and gamma interferon mRNA and enhanced the viability of intracellular attenuated and virulent mycobacteria in cocultures of immune T cells and macrophages obtained from Mycobacterium bovis BCG-vaccinated guinea pigs. This suggests the crucial role of TNF-alpha in the activation of a type 1 T-cell response against Mycobacterium tuberculosis infection.     

Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin-1 beta by human mononuclear phagocytes.

Human peripheral blood monocytes and a human monocyte cell line were exposed to the toxin pneumolysin. Pneumolysin-exposed cells produced significantly larger amounts of tumor necrosis factor alpha and interleukin-1 beta than cells not exposed to the toxin. The viability of cells was not affected by the concentrations of pneumolysin used in the experiments.     

Bikunin inhibits lipopolysaccharide-induced tumor necrosis factor alpha induction in macrophages

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-alpha) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-alpha expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-alpha protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 microM). (iii) Inhibition by bikunin of TNF-alpha induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-alpha release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-alpha production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.     

Recombinant tumor necrosis factor alpha does not inhibit the growth of African trypanosomes in axenic cultures

Mice whose tumor necrosis factor alpha (TNF-alpha) genes were disrupted developed higher levels of parasitemia than wild-type mice following infection with Trypanosoma congolense IL1180 or T. brucei brucei GUTat3.1, confirming the results of earlier studies. To determine whether TNF-alpha directly affects the growth of these and other bloodstream forms of African trypanosomes, we studied the effects of recombinant mouse, human, and bovine TNF-alpha on the growth of two isolates of T. congolense, IL1180 and IL3338, and two isolates of T. brucei brucei, GUTat3.1 and ILTat1.1, under axenic culture conditions. The preparations of recombinant TNF-alpha used were biologically active as determined by their capacity to kill L929 cells. Of five recombinant TNF-alpha lots tested, one lot of mouse TNF-alpha inhibited the growth of both isolates of T. brucei brucei and one lot of bovine TNF-alpha inhibited the growth of T. brucei brucei ILTat1.1 but only at very high concentrations and without causing detectable killing of the parasites. The other lots of mouse recombinant TNF-alpha, as well as human TNF-alpha, did not affect the growth of any of the test trypanosomes even at maximal concentrations that could be attained in the culture systems (3,000 to 15,000 U of TNF-alpha/ml of medium). These results suggest that exogenously added recombinant TNF-alpha generally does not inhibit the growth of African trypanosomes under the culture conditions we used. The impact of TNF-alpha on trypanosome parasitemia may be indirect, at least with respect to the four strains of trypanosomes reported here.     

Macrophages release tumor necrosis factor alpha and interleukin-12 in response to intracellular Bacillus anthracis spores

Herein we report that infection of a murine macrophage cell line with Bacillus anthracis results in the production of tumor necrosis factor alpha and interleukin-12 (IL-12). When infected with B. anthracis spores in combination with lipopolysaccharide, macrophages release increased amounts of IL-12. We found no evidence of inhibition of cytokine responses in macrophages infected with B. anthracis spores.     

Arabinofuranosyl-terminated and mannosylated lipoarabinomannans from Mycobacterium tuberculosis induce different levels of interleukin-12 expression in murine macrophages.

Lipoarabinomannan (LAM) is a major surface lipoglycan of Mycobacterium tuberculosis. In the present study, we demonstrated that arabinofuranosyl-terminated LAM (AraLAM) derived from a rapidly growing Mycobacterium sp., but not extensively mannosylated LAM derived from the Erdman strain, is capable of inducing interleukin-12 (IL-12) expression in murine macrophages. Since IL-12 is known to drive the differentiation of naive T cells toward T-helper type 1 (Th1) cell development, AraLAM may be an effective adjuvant in vaccines and immunotherapies that need Th1 responses.     

Differential expression of tumor necrosis factor and interleukin-6 by peritoneal macrophages in vivo and in culture.

To investigate the differences in cytokine regulation in vitro as compared to in vivo, we examined the synthesis of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by peritoneal macrophages in response to lipopolysaccharide (LPS). Mice (CBA/J) were primed with an intraperitoneal injection of complete Freund's adjuvant and after 2 weeks, peritoneal cells were harvested for culture or mice were injected intraperitoneally with LPS for in vivo studies. In ascites fluid, TNF-alpha peaked 1 hour after LPS and returned to baseline levels by 4 hours. In contrast, TNF-alpha in the media reached maximum at 7 hours. Expression of TNF-alpha messenger (m)RNA in vivo was rapid but transient, as levels peaked at 15 minutes and returned to baseline 1 hour after LPS. In contrast, TNF-alpha mRNA in vitro became maximal at 1 hour, but remained elevated to 5 hours after LPS. In vivo, IL-6 in ascites fluid peaked at 2 hours, whereas in vitro, IL-6 continued increasing to 24 hours. In vivo, IL-6 mRNA reached maximum at 30 minutes, but fell below baseline by 1.5 hours after LPS. In contrast, IL-6 mRNA in vitro was sustained at maximal expression between 5 to 9 hours after LPS. These results demonstrate that both TNF-alpha and IL-6 synthesis is more rapid in vivo than in vitro. The rapid kinetics of cytokine expression in vivo must considered when designing strategies to inhibit cytokine action in vivo.     

Influence of Yersinia pseudotuberculosis outer proteins (Yops) on interleukin-12, tumor necrosis factor alpha and nitric oxide production by peritoneal macrophages

An essential key to pathogenicity in Yersinia is the presence of a 70 kb plasmid (pYV) which encodes a type-III secretion system and several virulence outer proteins whose main function is to enable the bacteria to survive in the host. Thus, a specific immune response is needed in which cytokines are engaged. The aim of this study was to assess the influence of Yersinia outer proteins (Yops) released by Yersinia pseudotuberculosis on the production of the proinflammatory cytokines, interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO) by murine peritoneal macrophages. To this end, female Swiss mice were infected intravenously with wild-type Y. pseudotuberculosis or with mutant strains unable to secrete specific Yops (YopE, YopH, YopJ, YopM, and YpkA). On the 7th, 14th, 21st, and 28th days after infection, the animals were sacrificed and the cytokines and NO were assayed in the peritoneal macrophages culture supernatants. A fall in NO production was observed during the course of infection with all the strains tested, though during the infection with the strains that did not secrete YopE and YopH, the suppression occurred later. There was, in general, an unchanged or sometimes increased production of TNF-alpha between the 7th and the 21st day after infection, compared to the control group, followed by an abrupt decrease on the last day of infection. The IL-12 production was also suppressed during the infection, with most of the strains tested, except with those that did not secrete YopJ and YopE. The results suggest that Yops may suppress IL-12, TNF-alpha, and NO production and that the most important proteins involved in this suppression are YopE and YopH.