Anomalous role of tumor necrosis factor alpha in experimental enterococcal infection

The murine D-galactosamine (D-gal) model of tumor necrosis factor alpha (TNF-alpha) hypersensitization was used as an initial tool to investigate the potential contribution of TNF-alpha to lethal intraperitoneal (i.p.) infection with Enterococcus faecalis. D-gal sensitized mice to lethal E. faecalis infection, whereas dexamethasone and neutralizing anti-TNF-alpha antibody protected D-gal-treated, E. faecalis-infected mice, implicating TNF-alpha in the lethal response to E. faecalis infection in D-gal-treated mice. Circulating TNF-alpha was undetectable for at least 8 h following i.p. E. faecalis infection, although low peritoneal levels of TNF-alpha were detected within 3 h, suggesting that localized TNF-alpha production contributed to the lethal response to E. faecalis infection in D-gal-treated mice. Although i.p. E. faecalis infection failed to induce a detectable systemic TNF-alpha response, circulating Interleukin-6 (IL-6) was detected within 3 h of infection. IL-6 was also detected in the peritoneum within an hour of infection, prior to the appearance of peritoneal TNF-alpha. In striking contrast to in vivo results, E. faecalis induced a potent and rapid TNF-alpha response from both mouse peritoneal macrophages and the RAW 264.7 cell line in vitro. This led us to hypothesize that TNF-alpha production in response to E. faecalis infection is suppressed by IL-6 in vivo. In vitro experiments demonstrated a statistically significant, but modest, inhibitory effect of IL-6 on TNF-alpha production by RAW cells stimulated with E. faecalis. Collectively, these data indicate that acute, lethal E. faecalis infection appears to induce an unusual cytokine response that differs in character from that previously described for most other gram-positive and gram-negative bacteria.     

Bacterial-lipopolysaccharide-induced release of lactoferrin from human polymorphonuclear leukocytes: role of monocyte-derived tumor necrosis factor alpha.

We have examined the role played by human peripheral blood monocytes in mediating responses of human polymorphonuclear leukocytes (PMN) to bacterial lipopolysaccharide (LPS) in vitro. When incubated with Salmonella typhimurium LPS at 37 degrees C, human PMN suspended in serum-free buffer released the specific granule constituent lactoferrin into the surrounding medium. Release of lactoferrin from PMN varied with the concentration of LPS (1 to 1,000 ng/ml) as well as with the duration of incubation (2 to 60 min) and was not accompanied by significant release of the cytoplasmic enzyme lactate dehydrogenase. LPS-induced release of lactoferrin from PMN was augmented significantly when cell suspensions were supplemented with additional monocytes and lymphocytes. Only monocytes, however, secreted significant amounts of lactoferrin-releasing activity (in a time- and concentration-dependent manner) when incubated separately with LPS. Lactoferrin-releasing activity was heat (80 degrees C for 15 min) labile, eluted after chromatography on Sephadex G-100 with an apparent molecular weight of approximately 60,000, and was inhibited by antibodies to tumor necrosis factor alpha. Thus, LPS-induced noncytotoxic release of lactoferrin from human PMN suspended in serum-free buffer is mediated, at least in part, by tumor necrosis factor alpha derived from contaminating monocytes.     

Induction of tumor necrosis factor alpha by the group- and type-specific polysaccharides from type III group B streptococci.

Previous studies suggested that circulating tumor necrosis factor alpha (TNF-alpha) may have a pathophysiologic role in experimental neonatal sepsis induced by group B streptococci (GBS). This study was undertaken to investigate the ability of the type III and group-specific polysaccharides of GBS to induce TNF-alpha production and TNF-alpha-dependent lethality in neonatal rats. The cytokine was detected in plasma samples by the L929 cytotoxicity assay. Intracardiac injections of either polysaccharide induced dose-dependent, transient elevations in plasma TNF-alpha levels that returned to baseline values after 5 h. The group-specific antigen induced significantly higher mean peak TNF-alpha levels than the type III antigen (125 +/- 47 versus 44 +/- 15 U/ml with 70 mg/kg of body weight). Glycogen (70 mg/kg), used as a negative control, did not induce TNF-alpha. The lipopolysaccharide-neutralizing agent polymyxin B did not decrease TNF-alpha levels induced by either polysaccharide, ruling out contamination with endotoxin as a possible cause of TNF-alpha induction. Fifty percent lethal doses of the type III and group-specific antigens given as intracardiac injections were 105 and 16 mg/kg, respectively. Salmonella endotoxin, used as a positive control, had a 50% lethal dose of 0.1 mg/kg. The lethal activities of GBS polysaccharides, as well as endotoxin, were completely prevented by pretreatment of neonatal rats with the respective specific antibodies or anti-murine TNF-alpha serum. To assess the relative importance of the type-specific substance in TNF-alpha induction by whole bacteria, two unrelated GBS transposon mutants devoid of only the type-specific capsular polysaccharide (COH1-13 and COH31-15) were employed. Each of the heat-killed unencapsulated mutants was able to produce plasma TNF-alpha level elevations or TNF-alpha-dependent lethality but was significantly less efficient in these activities than the corresponding encapsulated wild-type strain. These data suggest that the presence of type-specific material on GBS is not necessary for the stimulation of TNF-alpha production. Type III capsular polysaccharide, however, can significantly increase the ability of GBS to induce TNF-alpha. Further studies will be needed to assess the importance of TNF-alpha induction by the group- and type-specific antigens in the pathophysiology of GBS disease.     

类风湿关节炎患者血清sTNF-R、TNF-α的变化

目的为探讨类风湿关节炎 (RA)与可溶性肿瘤坏死因子受体 (s TNF- R)、TNF- α、TNF- α/ s TNF- R比值的关系。方法应用双抗体夹心 EL ISA法检测了活动期 RA(2 8例 ) ,稳定期 RA (12例 )及健康人 (30例 )血清中 s TNF- R 、s TNF- R 、TNF-α的水平。结果活动期 RA患者血清 s TNF- R 、s TNF- R 、TNF-α水平明显高于健康人及稳定期 RA组 ,P均 <0 .0 1。稳定期 RA患者血清 s TNF- R 、s TNF- R 、TNF-α水平亦明显高于健康人 ,P<0 .0 1。在 RA患者中 ,血清 s TNF- R 、s TNF- R 水平与 ESR、CRP、Ritchie index呈显著正相关 ,与类风湿因子 (RF)水平无相关性。RA患者治疗 3个月后 s TNF- R 、s TNF- R 及 TNF- α/ s TNF R比值显著下降。结论 RA患者血清 s TNF- R 、s TNFR- 水平显著增高 ,且与疾病活动度呈正相关。测定血清 s TNF- R 、s TNF- R 、TNF- α/ s TNF- R水平可作为 RA诊断 ,监测疾病活动、治疗及判断预后的一项有意义的实验室指标     

Role of tumor necrosis factor alpha in asthma

Asthma is a heterogeneous disease in which various cytokines orchestrate airway inflammation. Tumor necrosis alpha (TNF-alpha) is a proinflammatory cytokine that has been implicated in the modulation of inflammation in various diseases, including asthma. Although TNF-alpha blocking strategies have been an effective therapeutic modality in diseases such as rheumatoid arthritis, their role in asthma and the effects of the blockade in asthma is poorly understood. This article examines the role of TNF-alpha in asthma and the effects of blocking TNF-alpha as a possible therapeutic option in patients with severe corticosteroid-dependent asthma.     

Activated Langerhans cells release tumor necrosis factor

Langerhans cells act as antigen-presenting cells in immune reactions in the skin. What other roles they may play in inflammation is less well defined. We have tested whether these cells can produce TNF-alpha, an important mediator of inflammation. Resting Langerhans cells produce less than 0.1 U TNF-alpha/ml. Langerhans cells stimulated with phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) release 4-5 U TNF-alpha/ml. Specificity of the released TNF-alpha in an L929 cytotoxicity assay was confirmed by using neutralizing anti-TNF-alpha monoclonal antibodies, and the identity of TNF-alpha was further confirmed by Northern blot hybridization with an TNF-alpha oligomer DNA probe. Activated Langerhans cells may contribute to inflammation in the skin by releasing TNF-alpha, which is known to effect fibroblast growth, endothelial cell activation, and lymphocyte function.     

Roles of the bacterial cell wall and capsule in induction of tumor necrosis factor alpha by type III group B streptococci.

Group B streptococci (GBS) are the major cause of sepsis and fatal shock in neonates in the United States. The precise role of tumor necrosis factor alpha (TNF-alpha) in the development of human GBS sepsis has not been defined; however, whole GBS have been shown to induce the production of this inflammatory cytokine. We sought to determine which bacterial cell wall components of GBS are responsible for triggering TNF-alpha production. Human cord blood monocytes were stimulated with encapsulated (COH1) or unencapsulated (COH1-13) whole type III GBS or with purified bacterial components, including type III capsular polysaccharide (III-PS), group B polysaccharide (GB-PS), lipoteichoic acid (LTA), or peptidoglycan (PG). Lipopolysaccharide from Escherichia coli served as a control. Supernatants were harvested at specific timed intervals, and TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Monocytes exposed to COH1 and COH1-13 induced similar amounts of TNF-alpha. III-PS, GB-PS, LTA, and PG each induced TNF-alpha in a time- and concentration-dependent manner. However, TNF-alpha release was significantly greater after stimulation by the GB-PS or PG than after stimulation by III-PS or LTA (P < 0.05). Our findings indicate that GB-PS and PG are the bacterial cell wall components primarily evoking TNF-alpha release. These, alone or in concert with other factors, may be responsible for septic shock accompanying GBS sepsis.     

Regulation of the processing and release of tumor necrosis factor alpha in a human macrophage cell line

The proteolytic cleavage of a variety of membrane proteins, including pro-tumor necrosis factor alpha (pro-TNF-alpha), is induced by various reagents such as phorbol 12-myristate 13-acetate (PMA). In this study we generated a human macrophage cell line that constitutively produces TNF-alpha, and examined how the processing and release of TNF-alpha are regulated. The processing and release of TNF-alpha were enhanced by PMA through a protein kinase C-dependent pathway. Although only hydroxamate matrix metalloproteinase (MMP) inhibitors inhibited the basal processing and release of TNF-alpha, the PMA-induced processing and release of TNF-alpha were inhibited not only by MMP inhibitors but also by 1,10-phenanthroline, 3,4-dichloroisocoumarin (3,4-DCI), iodoacetamide, and Nalpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK). Hydroxamate MMP inhibitors and 1,10-phenanthroline inhibited the processing of TNF-alpha on the cell surface, whereas 3,4-DCI, iodoacetamide, and TPCK inhibited the transport of TNF-alpha to the cell surface. These results suggest that serine and/or cysteine protease(s) may be involved in PMA-induced processing and/or transport to the cell surface.     

Role of tumor necrosis factor alpha in Helicobacter pylori gastritis in tumor necrosis factor receptor 1-deficient mice

Increased gastric production of interleukin 8 and tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of Helicobacter pylori-associated gastroduodenal disease. In the present study we used a mouse model to demonstrate whether loss of the tumor necrosis factor receptor 1 (TNF-R1) function leads to differences in gastric inflammation or the systemic immune response in H. pylori infection. Six different clinical isolates of H. pylori (three cytotoxin-positive and three cytotoxin-negative strains) were adapted to C57BL/6 mice. TNF-R1-deficient (TNF-R1(-/-)) mice (n = 19) and isogenetic controls (n = 24) were infected and sacrificed after 4 weeks of infection. Inflammation of the stomach and the humoral immune response to H. pylori were evaluated by histological, immunohistochemical, and serological methods. There was no detectable difference in the grade or activity of gastritis in TNF-R1(-/-) mice when they were compared with wild-type mice, but the number of lymphoid aggregates was slightly reduced in the gastric mucosa of TNF-R1(-/-) mice. Interestingly, total immunoglobulin G (IgG), as well as IgG1, IgG2b, and IgG3, H. pylori-specific antibody titers were significantly higher in wild-type mice. As revealed by immunoblot analysis, the difference in reactivity against H. pylori antigens was not based on a failure to recognize single H. pylori antigens in TNF-R1(-/-) mice. We therefore suggest that TNF-R1-mediated TNF-alpha signals might support a systemic humoral immune response against H. pylori and that the gastric inflammatory response to H. pylori infection seems to be independent of TNF-R1-mediated signals.     

In vitro inhibition of group B streptococcus-induced polymorphonuclear leukocyte aggregation

Evidence suggests that part of the pathophysiologic response seen in group B streptococcal (GBS) sepsis may be due to polymorphonuclear leukocyte (PMN) activation. Indomethacin (INDO), which inhibits eicosanoid metabolism, attenuates the pathophysiologic response stimulated by GBS, possibly due to inhibition of PMN aggregation. We examined the capability of two eicosanoid metabolism inhibitors, INDO and nordihydroguaiaretic acid (NDGA), to inhibit PMN aggregation induced by heat-inactivated opsonized GBS and GBS-activated plasma. Opsonized GBS-induced PMN aggregation was inhibited by INDO (50–500M) and NDGA (1–100M). Over similar concentration ranges, INDO and NDGA had no significant eifect on PMN aggregation induced by GBS-activated plasma. PMNs in plasma aggregate in response to unopsonized GBS. The stimuli for aggregation are opsonized GBS and GBS-activated plasma. INDO (50–500M) was unable to inhibit aggregation under this condition. Over the same concentration range in which INDO inhibited opsonized GBS-induced PMN aggregation, INDO was unable to inhibit opsonized GBS-induced superoxide production in PMNs. NDGA was examined but was found to interfere with the assay. The above evidence suggests PMN aggregation via eicosanoid metabolism may play a role in GBS-induced sepsis, which may be attenuated by agents such as INDO and NDGA.     

Human monocyte receptors involved in tumor necrosis factor responses to group B streptococcal products

Several group B streptococcal products have been previously found to stimulate human monocytes to produce tumor necrosis factor alpha. In order to identify the receptors involved in these responses, monocytes were stimulated with purified group- or type-specific carbohydrates or lipoteichoic acid in the presence of anti-receptor monoclonal antibodies, soluble CD14, or lipopolysaccharide-binding protein. Results indicate that CD14 plays an important role in tumor necrosis factor alpha responses to all of the stimuli tested. Moreover, both CD14 and complement receptor type 3 may be involved in responses to the group-antigen.     

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.     

The role of complement, antibody, and tumor necrosis factor alpha in the killing of Plasmodium falciparum by the monocytic cell line THP-1.

Killing of Plasmodium falciparum blood forms by the differentiated human myelomonocytic THP-1Mo cell line was studied by a radiometric assay. Results showed that parasite killing was promoted by complement, antimalarial antibody, and the cytokines tumor necrosis factor alpha and gamma interferon. Differentiated THP-1Mo appears to be a useful monocytic cell line for the study of mechanisms of immunity to Plasmodium.     

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.     

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.     

Critical role of peripheral blood phagocytes and the involvement of complement in tumour necrosis factor enhancement of passive collagen-arthritis.

Studies have implicated tumour necrosis factor-alpha (TNF-alpha) in type-II collagen (CII)-induced arthritis (CIA), a well established animal model of human rheumatoid arthritis. Precisely how TNF is involved in CIA is not yet clear. In this study the effects of TNF on CIA were examined, independent of its potential effects on the immune response, by performing peri-articular injection of TNF in combination with passive immunization of rats. A sub-arthritic dose (5 mg) of affinity-purified anti-CII IgG, which alone was insufficient to induce spontaneous clinical arthritis, was used throughout the study. Obvious clinical arthritis that persisted for several days was rapidly induced by injections of 100 ng TNF into hindpaws of rats that were passively immunized shortly before the TNF injection. Injections of TNF in non-immunized control rats did not induce clinical arthritis, nor did buffer-only injections in passively immunized controls. The clinical arthritic response was a local phenomenon, limited only to the TNF-injected hindpaws. No swelling was observed in the opposite, buffer-injected hindpaws, indicating the effects of TNF were not systemic. Depletion of peripheral blood phagocytes with anti-rat neutrophil antiserum before passive immunization completely abolished the ability of TNF to induce clinical arthritis, identifying phagocytic cells as the essential target cells in evoking this arthritic response. A role for complement activation was also demonstrated in this model through the use of a soluble recombinant version of CD35, the cell surface complement receptor type-1 (sCR1, BRL55730), which significantly reduced TNF-induced arthritis in phagocyte-replete rats.