Pharmacokinetics of Murine Tumor Necrosis Factor

The in vivo administration of tumor necrosis factor (TNF) provides a new approach to the immunotherapeutic treatment of tumors. We evaluated the pharmacokinetics of murine tumor necrosis factor in mice as a model for application in the human system. TNF had a clearance of 0.013 ml/min/g and a serum half life of 10.5 minutes. Its volume of distribution was consistant with the extracellular space. This information can provide parameters by which to select optimal modes of treatment for eradication of in vivo neoplasms.     

Self-Induction of Defense Against Tumor Necrosis Factor Cytotoxicity in Tumor Cells and Normal Cells

Investigation on the effect of TNF on RNA and protein synthesis by tumorigenic and normal cell lines showed their synthesis in tumor cells to be increased at 12 h and to peak at 24 h of incubation with TNF, while that in normal diploid fibroblast (HEL) cells was apparently unaffected by the presence of TNF. The increase correlated with cell susceptibility to cytotoxic effect by TNF. Artificial inhibition of either RNA or protein synthesis by L-M cells. by addition of artinomycin D or cycloheximide, increased the cytotoxic effect of TNF and thus suggested that the elevated RNA and protrin synthesis is related not, to the cytotoxic reaction itself but rather to a defense mechanism. Similar incubaticin of HEL cells with TVF in the presence of either inhibtor resulted in the occurrence of cytotoxicity not observed with TNF alone, thus suggesting the existence of a defense mechanism in normal, TNF-resistant rolls which is absent, or greatly wealtend in tumor cells.     

鼠腹腔巨噬细胞诱生TNF的条件探讨

本文研究了用鼠腹腔巨噬细胞诱生TNF的适合条件。结果显示LFS刺激8～30h内TNF的产量最高,5～10μg/ml的LPS为合适的刺激剂量;较好的巨噬细胞浓度在2×10~6/ml左右和静止的腹腔定居巨噬细胞不能产生明显的TNF。特别引人注意的是有效的TNF诱生有赖于巨噬细胞培养中LPS的持续存在以及培养液中的小牛血清对TNF的产生有明显的抑制作用。本实验为进一步研究TNF及巨噬细胞的免疫学提供了有用的数据。     

A Tumor Necrosis Factor Mimetic Peptide Activates a Murine Macrophage Cell Line To Inhibit Mycobacterial Growth in a Nitric Oxide-Dependent Fashion

The control of mycobacterial infections depends on the cytokine-mediated activation of mononuclear phagocytes to inhibit the growth of intracellular mycobacteria. Optimal activation requires the presence of T-cell-derived gamma interferon (IFN-γ) and other signals, including tumor necrosis factor (TNF). Recently, an 11-mer peptide based on amino acids 70 to 80 of the human TNF sequence, TNF_(70-80), was found to have TNF mimetic properties, which include the activation of human and mouse neutrophils to kill Plasmodia spp. Therefore, we investigated the capacity of TNF_(70-80) to activate the murine macrophage cell line RAW264.7 infected with the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG). When RAW264.7 cells were pretreated with human TNF or TNF_(70-80) in the presence of IFN-γ, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of ³H-labeled uracil and a concomitant release of nitric oxide as measured by the nitrite in the culture supernatants. TNF- or TNF_(70-80)-induced macrophage activation was dependent on IFN-γ and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-γ antisera. Both nitrite release and BCG growth inhibition were abrogated by competitive inhibitors of l-arginine, which blocked the activation of inducible nitric oxide synthase. A soluble form of the Type 1 TNF receptor blocked the activation of BCG-infected macrophages by human TNF and TNF_(70-80), demonstrating that the effect of TNF_(70-80) is dependent on signaling through TNF receptor I. The mimetic effects of TNF_(70-80) on macrophage activation in vitro suggest that treatment with TNF_(70-80) may modulate mycobacterial infections in vivo.Mycobacteria are intracellular parasites which replicate within the shielded environment of monocyte-derived tissue macrophages. Activation of antibacterial killing mechanisms within these cells by cytokines is essential for the control of mycobacterial infections (24). Gamma interferon (IFN-γ) plays a central role in this since it is produced by a variety of lymphocytes responding to mycobacterial infections, including CD4⁺ and CD8⁺ αβ T cells and γδ T cells. Administration of recombinant IFN-γ protects mice against lethal Mycobacterium tuberculosis infection in some but not all experimental models (13, 20), whereas neutralization with anti-IFN-γ antibodies exacerbates the infection (13). The failure of mice deficient in IFN-γ or IFN-γ receptors to control M. tuberculosis infection confirms that this cytokine is essential for killing M. tuberculosis (12, 20). Studies with human and murine macrophages, however, have demonstrated that additional signals are required to fully activate mycobacterial killing (24). Potential activators include other cytokines, such as tumor necrosis factor (TNF), interleukin-4 (IL-4), IL-6, and granulocyte-macrophage colony-stimulating factor (15, 18, 19), and in humans 1,25-dihydroxy-vitamin D₃, the biologically active form of vitamin D₃ (14). TNF alone cannot activate macrophages sufficiently to kill mycobacteria, but it does synergize with IFN-γ to increase the antimycobacterial activity of infected macrophages in vitro (18). Administration of anti-TNF antibodies decreases the resistance of mice to infection with M. bovis bacillus Calmette-Guérin (BCG) (25) and M. tuberculosis (21). TNF is a necessary requirement for effective antimycobacterial immunity, since mice deficient in the 55-kDa TNF receptor I (TNFRI) develop progressive M. tuberculosis infection (21). The protective effects of TNF and the lethal consequences of anti-TNF antibodies have been observed in other models of intracellular bacterial infection, including infections by Listeria monocytogenes, Salmonella typhimurium, and Legionella spp. (37). Although in mycobacterial infections, such as leprosy, high levels of TNF have been associated with tissue damage and systemic toxicity, local TNF synthesis is essential for the control of mycobacterial infections (35).Studies with neutralizing anti-human TNF monoclonal antibodies (MAb) demonstrated that the sequence from amino acids 65 to 85 of the TNF molecule was involved in binding to the TNF receptor (32). By use of truncated peptides, amino acids 70 to 80 were identified as essential for TNF activity (33). When this peptide sequence was modified by substitution of leucine-76 for isoleucine, the subsequent peptide TNF_(70-80) had increased stability in vitro in the presence of serum (32a) and possessed TNF mimetic properties both in vitro and in vivo (27). TNF_(70-80) stimulated a reactive oxygen burst in human and murine neutrophils (27) and activated human neutrophils to kill Plasmodium falciparum (27). In a murine model of Plasmodium chabaudi infection, systemic therapy with TNF_(70-80) increased the rate of recovery and clearance of parasites (27). More recently, TNF_(70-80) was found to reduce the weight loss and systemic effects in mice chronically infected with Pseudomonas aeruginosa (32a). The demonstrated properties of TNF_(70-80) and the known requirement of TNF for activating macrophages led us to examine whether this mimetic peptide would have antimycobacterial activity on a murine macrophage cell line. We now report that TNF_(70-80) synergizes with IFN-γ to activate murine macrophages to inhibit the growth of M. bovis BCG and that this property is dependent on its activation of inducible nitric oxide synthase (iNOS).     

Lung Bacterial Clearance in Murine Pneumococcal Pneumonia

We studied the bactericidal capacity of the rat lung during the development of pneumococcal pneumonia. Pneumonia was produced in a lower lobe by the intrabronchial instillation of 10(4)Streptococcus pneumoniae cells in buffer. Lung bacterial counts progressively increased, reaching 10(7) per lung within 48 h, and the increase was associated with localized atelectasis and consolidation. Bacterial multiplication was inhibited with tetracycline at various intervals after infection, and the subsequent clearance of pneumococci was determined. Viable pneumococci were rapidly killed by lung defenses if bacterial multiplication was inhibited within 12 h of the onset of infection. No change occurred in the bacterial populationif tetracycline was delayed until 24 h after infection, indicating that pneumococcal killing by lung defenses had ceased. This effect could be reproduced with the addition of pneumococcal capsular polysaccharide to the inoculum, which produced a dose-related inhibition of pneumococcal clearance. The clearance of S. epidermidis was not impaired in the presence of pneumococcal pneumonia or by administration of exogenous capsular polysaccharide. These data indicate that pneumococcal pneumonia causes a marked impairment in lung antipneumococcal defenses within 24 h of the onset of infection. This acquired defect in antibacterial defenses may be due to the accumulation of pneumococcal capsular material in the lungs of infected animals.     

Auto-Antibodies to Tumour Necrosis Factor a in Healthy Humans and Patients with Inflammatory Diseases and Gram-Negative Bacterial Infections

A semi-quantitative immunoblotting method was developed to screen for serum auto-antibodies against tumour necrosis factor alpha (TNF alpha). Forty nitrocellulose strips containing identical amounts of human recombinant TNF alpha (rTNF alpha) were prepared for each set-up, and the anti-TNF alpha antibody immunoreactivities were scored according to the density of the resulting colour reaction. A significant number of sera from apparently healthy donors contained detectable auto-antibodies to TNF alpha (40%), while the strongest reaction was observed in 8%. A higher prevalence of anti-TNF alpha antibodies was found in sera from patients with Gram-negative bacterial septicaemia (66%), cystic fibrosis with chronic Pseudomonas aeruginosa lung infection (72%), and various rheumatic diseases (61%). The antibodies in sera from these patients belonged primarily to the IgG and IgM classes, the latter exhibiting the strongest response. Longitudinally collected serum samples from patients in septic endotoxin shock revealed that the anti-TNF alpha antibodies were induced initially during septicaemia, reaching maximum reactivities within the first week and returning to low or undetectable levels on days 9-20.     

Soluble Tumor Necrosis Factor Receptor in Serum and Urine Throughout Normal Pregnancy and at Delivery

PROBLEM : To determine the concentration of the two soluble tumor necrosis factor receptors (sTNFR), sp55 and sp75, in healthy pregnant women. METHOD : Serum and urine samples were longitudinally collected from a group of pregnant women (N=53) five times throughout pregnancy. Maternal and umbilical sera were obtained from some of the deliveries (N=31). The samples were analysed using ELISA based on two monoclonal antibodies (IV4E and 3H5) against the soluble tumor necrosis factor receptors (sp55 and sp75). RESULTS : Serum concentration of sp55 and sp75 were increased in pregnant women compared to that of nonpregnant controls. Concentration of both sTNFRs increased towards term. Labor was associated with further increase of sp55. Concentrations of sp55 and sp75 in umbilical serum were significantly higher than those of maternal serum. Significant correlations were observed between maternal and umbilical sTNFR concentrations. CONCLUSIONS : The current study suggests that pregnancy is associated with an activation of mechanisms regulating the biological activities of TNF.     

Phagocytic and Tumor Necrosis Factor Alpha Response of Human Mast Cells following Exposure to Gram-Negative and Gram-Positive Bacteria

Recent studies have implicated rodent mast cells in the innate immune response to infectious bacteria. We report that cord blood-derived human mast cells (CBHMC) obtained from culture of cord blood progenitors phagocytozed and killed various gram-negative and gram-positive bacteria and simultaneously released considerable amounts of tumor necrosis factor alpha. Overall, the extent of the endocytic and exocytic response of CBHMC correlated with the number of adherent bacteria. Thus, human mast cells are intrinsically capable of mediating microbial recognition and of actively contributing to the host defense against bacteria.     

Increased Levels of Soluble Tumor Necrosis Factor Receptors in Atherosclerosis: No Clear Relationship with Levels of Tumor Necrosis Factor

Inflammatory mediators, such as tumor necrosis factor alpha (TNF), may be important in the pathogenesis of atherosclerosis. Interactions between TNF and its target cell(s) requires the presence of specific receptors on the latter. Plasma levels of the two soluble forms of these receptors (tumor necrosis factor receptors, (sTNFr)) and TNF itself were measured by ELISA in 20 patients with peripheral vascular disease (PVD), 20 survivors of a myocardial infarction, and 20 age and sex matched controls. Levels of the p55 variant of the sTNFr were unchanged but levels of the p75 variant were increased in both groups of patients (ANOVA both P < 0.01). TNF was also raised in both groups of patients (both P < 0.05) but levels did not correlate with either sTNFr. In atherosclerosis, increased levels of p75 sTNFr may be further evidence of inappropriate leukocyte activation but unlikely to modulate the effect of free plasma TNF.     

Increased Leptin Expression in Mice with Bacterial Peritonitis is Partially Regulated by Tumor Necrosis Factor Alpha

Plasma leptin and ob gene mRNA levels were increased in mice following bacterial peritonitis, and blocking an endogenous tumor necrosis factor alpha (TNF-α) response blunted the increase. However, plasma leptin concentrations did not correlate with the associated anorexia. We conclude that leptin expression is under partial regulatory control of TNF-α in peritonitis, but the anorexia is not dependent on increased leptin production.     

Role of Tumor Necrosis Factor Alpha in the Host Response of Mice to Bacteremia Caused by Pneumolysin-Deficient Streptococcus pneumoniae

Pneumolysin-deficient mutant strains of Streptococcus pneumoniae are known to cause less-severe sepsis than wild-type pneumococcal strains that produce pneumolysin. This difference is associated with greater host resistance in mice infected with the pneumolysin-deficient strains. These studies show that the host resistance developed during the first 1 to 2 days after infection with a pneumolysin-deficient mutant strain is dependent on tumor necrosis factor alpha but is apparently independent of interleukin 1β (IL-1β) or IL-6. Survival beyond 5 days appeared to depend on the ability of the mice to produce IL-1β.     

Serum Tumor Necrosis Factor Activity in Inflammatory Bowel Disease

Serum tumor necrosis factor (TNF) levels in 33 patients with inflammatory bowel disease (IBD) were measured by using a sensitive enzyme immunoassay. Four of five Crohn's diseases (CD) and nine of twenty eight ulcerative colitis (UC) had elevated levels of serum TNF. In active CD or UC, a greater fraction of patients studied had significantly increased serum TNF levels (3/3 for CD and 8/11 for UC). Production of TNF by peripheral blood monocytes when stimulated by lipopolysaccharide was also increased in these patients and correlated with their serum TNF levels. These rusults suggest that TNF may have some pathoetiological meaning in IBD.     

The Endogenous Balance of Soluble Tumor Necrosis Factor Receptors and Tumor Necrosis Factor Modulates Cachexia and Mortality in Mice Acutely Infected with Trypanosoma cruzi

To better understand the role of tumor necrosis factor (TNF) during Trypanosoma cruzi infection in BALB/c mice, we have investigated the kinetics of circulating tumor necrosis factor (TNF), soluble TNF receptor 1 (sTNR1), and sTNFR2 levels, as well as the interactions between such factors, in relation to parasitemia, cachexia, and mortality of acutely infected animals. Our data show that the parasitemic phase of T. cruzi infection in mice is associated with high levels of circulating TNF and sTNFR2, resulting in the formation of cytokine-receptor complexes and some degree of neutralization of TNF bioactivity. Although sTNR2 levels always exceeded TNF levels, low sTNFR/TNF circulating ratios were associated with cachexia in all infected mice, whereas the lowest ratios were observed in dying animals harboring the highest parasitemia. We also studied the modulation of sTNFR/TNF ratios induced by anti-TNF antibodies administered to infected animals and their consequences on the outcome of the infection. The injection of anti-TNF monoclonal antibody (MAb) TN3 into infected mice resulted in a paradoxical overproduction of TNF (associated with a higher parasitemia), lowered the sTNFR/TNF circulating ratios, and considerably worsened cachexia and mortality of animals. Another anti-TNF MAb (1F3F3) decreased the in vivo availability of TNF as well as parasite levels and reduced cachexia. Altogether, such results highlight that, besides playing a beneficial role early in infection, TNF also triggers harmful effects in the parasitemic phase, which are limited by the in vivo simultaneous endogenous production of soluble receptors.     

Heat-Killed Streptococcus suis Capsular Type 2 Strains Stimulate Tumor Necrosis Factor Alpha and Interleukin-6 Production by Murine Macrophages

Streptococcus suis capsular type 2 is an important etiological agent of swine meningitis, and it is also a zoonotic agent. Since mononuclear phagocytes have been suggested to play a central role in the pathogenesis of meningitis, the objective of the present study was to evaluate the capacity of whole killed S. suis type 2 organisms to induce the release of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) by murine macrophages. Induction of cytokines was evaluated in the presence or absence of phorbol ester (phorbol 12-myristate 13-acetate [PMA]) costimulation. Results showed that S. suis type 2 stimulated the production of both cytokines in a concentration- and time-dependent fashion. Although large doses of bacteria were required for maximal cytokine release, titers were similar to those obtained with the lipopolysaccharide (LPS) positive control. An increase in cytokine release was observed with both S. suis and LPS with PMA costimulation. Experiments with cytochalasin-treated macrophages showed that the stimulation of cytokine production was phagocytosis independent. When macrophages were stimulated with an unencapsulated mutant, an increase in TNF production was observed, but the absence of the capsule had no effect on IL-6 production. In fact, whereas purified capsular polysaccharide of S. suis failed to induce cytokine release, purified S. suis cell wall induced both TNF and, to a lesser extent, IL-6. IL-6 secretion probably requires some distinct stimuli which differ from those of TNF. Finally, the S. suis putative virulence factors suilysin and extracellular protein EF showed no cytokine-stimulating activity. The ability of S. suis to trigger macrophages to produce proinflammatory cytokines may have an important role in the initiation and development of meningitis caused by this microorganism.     

A Role for Interleukin-6 in Host Defense against Murine Chlamydia trachomatis Infection

Interleukin-6-deficient (IL-6^−/−) knockout mice had significantly increased Chlamydia trachomatis levels in lung tissue and increased mortality compared to B6129F₂/J controls early after intranasal infection. Gamma interferon production and chlamydia-specific antibody levels were consistent with a decreased but reversible Th1-like response in IL-6^−/− mice. IL-6 is needed for an optimal early host response to this infection.     

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-α) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-α 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-α protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 μM). (iii) Inhibition by bikunin of TNF-α 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-α target molecules interleukin-1β (IL-1β) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-α release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-α 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.     

Bacterial Clearance and Cytokine Profiles in a Murine Model of Postsurgical Nosocomial Pneumonia

The development of a nosocomial pneumonia is facilitated by alterations in host innate pulmonary antibacterial defenses following surgical trauma, which can result in decreased pulmonary bacterial clearance and increased morbidity and mortality. In a murine model of postoperative nosocomial infection, surgical stress (laparotomy) decreased Escherichia coli clearance from the lungs of animals that underwent surgery. Consistent with previous studies, (i) pulmonary levels of tumor necrosis factor alpha at 6 h and of interleukin-1β (IL-1β), IL-6, and gamma interferon (IFN-γ) at 24 h post-bacterial infection (PBI) were decreased in animals that underwent laparotomy 24 h prior to E. coli infection (LAP/E. coli) compared to animals that received E. coli only; (ii) KC and macrophage inhibitory protein 2 were elevated at 6 h PBI in LAP/E. coli animals compared to E. coli-only animals; however, at 24 h PBI, levels were higher in the E. coli-only group; (iii) at 24 h PBI, monocyte chemoattractant protein 1 was lower in the LAP/E. coli group compared to the E. coli-only group; (iv) IL-10 levels were unaffected at all time points evaluated; and (v) the total number of neutrophils present in the lungs of LAP/E. coli animals at 6 h PBI was decreased in comparison to that in E. coli-only animals, resulting in decreased bacterial clearance and increased mortality in LAP/E. coli animals by 24 h PBI. Similar changes in cytokine profiles, pulmonary bacterial clearance, and mortality were consistent with reported findings in patients following surgical trauma. This model, therefore, provides a clinically relevant system in which the molecular and cellular mechanisms that lead to the development of nosocomial pneumonia can be further explored.     

Autotransporter and Two-Partner Secretion: Delivery of Large-Size Virulence Factors by Gram-Negative Bacterial Pathogens

A number of protein secretion mechanisms have been identified in gram-negative pathogens. Many of these secretion systems are dependent upon the Sec translocase for protein export from the cytoplasm into the periplasm and then utilize other mechanisms for transport from the periplasm through the outer membrane. In this article, we review secretion similarities between autotransporter and two-partner secretion systems, and we report similarities between the autotransporter secretion mechanism with that of intimin/invasins. Considering that many secreted proteins are virulence factors, a better understanding of their secretion mechanisms will aid in the development of disease treatments and new bacterial vaccines.     

Augmentation of Innate Host Defense by Expression of a Cathelicidin Antimicrobial Peptide

Antimicrobial peptides, such as defensins or cathelicidins, are effector substances of the innate immune system and are thought to have antimicrobial properties that contribute to host defense. The evidence that vertebrate antimicrobial peptides contribute to innate immunity in vivo is based on their expression pattern and in vitro activity against microorganisms. The goal of this study was to investigate whether the overexpression of an antimicrobial peptide results in augmented protection against bacterial infection. C57BL/6 mice were given an adenovirus vector containing the cDNA for LL-37/hCAP-18, a human cathelicidin antimicrobial peptide. Mice treated with intratracheal LL-37/hCAP-18 vector had a lower bacterial load and a smaller inflammatory response than did untreated mice following pulmonary challenge with Pseudomonas aeruginosa PAO1. Systemic expression of LL-37/hCAP-18 after intravenous injection of recombinant adenovirus resulted in improved survival rates following intravenous injection of lipopolysaccharide with galactosamine or Escherichia coli CP9. In conclusion, the data demonstrate that expression of an antimicrobial peptide by gene transfer results in augmentation of the innate immune response, providing support for the hypothesis that vertebrate antimicrobial peptides protect against microorganisms in vivo.