Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.     

Purification of guinea pig tumor necrosis factor (TNF): comparison of its antiproliferative and differentiative activities for myeloid leukemic cell lines with those of recombinant human TNF

Recently, we suggested that the effect of differentiation inducing factor (D-factor) which is found in the supernatant of macrophages, and induced the differentiation of a mouse myeloid leukemic cell line, M1, into macrophage-like cells, may be a result of the cooperative effects of tumor necrosis factor (TNF) and interleukin 1 (IL-1). In this study, we purified guinea pig (G.P.) TNF secreted from peritoneal macrophages and compared the antiproliferative and differentiative effects of the G.P. TNF with those of recombinant human TNF (rHuTNF). The purification scheme consisted of ultrafiltration, gel filtration-high performance liquid chromatography (HPLC), DEAE-HPLC, and reverse-phase HPLC. The cytotoxic activity of the purified substance was approximately 1.5 x 10(8) U/mg. The isoelectric point was 5.2. The molecular weight was 40 to 45 kDa as estimated by gel filtration and 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence was determined to be Ser-Ala-Ser-Gln-Asn-Asp . . . . Approximately 76 or 71% homology between G.P. TNF and mouse or human TNF exists in the NH2-terminal 21 residues. The purified G.P. TNF and rHuTNF demonstrated D-factor activity only in the presence of recombinant human IL-1 alpha in M1 cells. We also determined the effect of TNF on two human myeloid leukemic cell lines (THP-1 and U937). The purified G.P. TNF and rHuTNF inhibited the growth of U937 cells, but did not induce their differentiation. In THP-1 cells, TNF slightly inhibited the growth and induced differentiation. In mouse cell lines G.P. TNF was more effective than rHuTNF for differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of diet with and without exercise training on markers of inflammation and fat distribution in overweight women

The independent effects of exercise and weight loss on markers of inflammation (MOI) in obese individuals have not been clearly characterized. The objectives of this study were to: (i) identify the independent effects of exercise and weight loss on MOI and (ii) determine whether changes in MOI were associated with changes in fat distribution. Subjects were 126 healthy, premenopausal women, BMI 27–30 kg/m². They were randomized to one of three groups: diet only, diet + aerobic‐, or diet + resistance training until a BMI <25 kg/m² was achieved. Fat distribution was measured with computed tomography, and body composition with dual‐energy X‐ray absorptiometry. Serum concentrations of tumor necrosis factor (TNF)‐α, soluble TNF receptor 1 (sTNF‐R1), soluble TNF receptor 2 (sTNF‐R2), C‐reactive protein (CRP), and interleukin (IL)‐6 were assessed. Results of repeated‐measures ANOVA indicated a significant effect of time on MOI, such that MOI decreased with weight loss. Results of mixed‐model analysis indicated that adjusting for intra‐abdominal adipose tissue (IAAT) and total fat mass explained the decreases in TNF‐α and sTNF‐R1, whereas only total fat mass explained the decreases in sTNF‐R2, IL‐6, and CRP. In conclusion, weight loss was associated with decreases in MOI. The effect of weight loss appeared to be mediated by changes in total fat mass or IAAT. Addition of exercise did not alter the response, suggesting that weight loss has a more profound impact for reducing MOI in overweight women than exercise.     

TNF is necessary for castration-induced prostate regression, whereas TRAIL and FasL are dispensable

TNF, a proinflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. However, there have been few examples of a physiologic role for TNF-induced apoptosis in vivo. Here, we describe a novel role for TNF in prostate epithelial cell apoptosis after androgen withdrawal. Employing high-resolution serial magnetic resonance imaging to measure mouse prostate volume changes over time, we demonstrate that the extent of castration-induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes but not mice deficient for TNF-related apoptosis-inducing ligand or Fas signaling. Wild-type mice receiving soluble TNF (sTNF) receptor 2 (to bind TNF and block signaling) before castration exhibit an identical reduction of prostate regression. Together, these data indicate that uniquely among known extrinsic death signals, TNF is required for castration-induced prostate regression. Additionally, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate after castration. This is consistent with a paracrine role for TNF in prostate regression. When injected into the peritoneum of Tnf(-/-) mice at the time of castration, sTNF restores normal levels of prostate regression. However, wild-type mice receiving sTNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient but acts in the context of additional castration-induced signals. These findings support a physiologic role for TNF in prostate regression after androgen withdrawal. Understanding this role may lead to novel therapies for prostate cancer.     

Poliovirus protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface

Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-kappaB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.     

Induction of differentiation of human monoblastic and myeloblastic leukemia cell lines by TNF muteins

The differentiation inducing activity of five recombinant human tumor necrosis factor (rHuTNF) muteins, prepared by protein engineering techniques, was compared by measuring the ability to reduce nitroblue tetrazolium dye of human myelogenous leukemia cells. TNF(C-Phe), in which the C-terminal leucine of TNF molecule was replaced by phenylalanine, was 20-times as potent in induction of differentiation of U-937 cells as the parent TNF(N-Met), in which methionine was added to N-terminal sequence of TNF. The differentiation inducing activity of TNF muteins was not always proportional to their binding activity to the receptors nor to their cytotoxicity on U-937 cells.     

Effects of a new antibiotic, isohematinic acid, on the resistance of mice to experimental infections

Isohematinic acid, an antibiotic newly isolated from the culture broth of Actinoplanes philippinensis SANK 61681, was assessed for its ability to enhance nonspecific resistance to bacterial infections against Escherichia coli and Pseudomonas aeruginosa in mice. This agent, as well as BM 12,531 (Azimexon), was found to prolong the survival of normal mice infected with E. coli and also of compromised mice infected with either E. coli or P. aeruginosa, whose defense system had been deteriorated by treatment with carboquone, an alkylating agent. Like BM 12,531, isohematinic acid administered to normal mice significantly increased the nitroblue tetrazolium reducing potency of polymorphonuclear leucocytes (PMN), indicating that the microbicidal activity of PMN was enhanced by these agents. In addition, in the compromised mice these agents were able to restore the number of peripheral blood leucocytes, which had been reduced to about 30% of the normal level by carboquone. These results suggest that isohematinic acid, like BM 12,531, enhances nonspecific resistance to these bacterial infections by stimulating the microbicidal activity of PMN and inducing leucocytosis.     

Tumour necrosis factor alpha receptors: role in the physiopathology of protozoan parasite infections

Tumour necrosis factor alpha (TNF alpha) is an important cytokine in immune regulation and resistance to various micro-organisms. It provides signals to the target cells through two different receptors: TNFR1 and TNFR2. The present report reviews the role of TNF receptors (TNFRs) in the immune response against protozoan parasite infections of medical interest (Toxoplasma gondii, Leishmania major, Trypanosoma cruzi, Plasmodium spp.). TNF alpha has been regarded as a modulator cytokine in host defence against protozoans infections and recent findings on experimental gene-deficient mice have showed that TNF alpha/TNFRs pathway may be beneficial for host protection during these infections.     

Induction of resistance in mice against murine cytomegalovirus by cellular components of Lactobacillus casei

Recently, we reported that heat-killed Lactobacillus casei (LC) protected mice from murine cytomegalovirus (MCMV) infection by augmentation of natural killer (NK) cell activity. In the present study, we examined which components of LC cell induce the nonspecific resistance most effectively. Whole cell preparation of original LC, susceptible to bacteriophages SG-T and J1, was more effective than its mutants resistant to either bacteriophage. Although the activity of LC cells decreased upon fractionation, cell wall fractions were more active than cytoplasmic fractions. Glycoprotein (GP), a cell wall constituent, was a potent inducer of the resistance. The relative activity of cellular components to induce the resistance was evaluated by a protection index, a ratio of plaque-forming units (PFU) per 50% lethal dose (LD50) for treated mice to that for untreated mice. The protection indices of LC cells and GP were approximately 80 and 28, respectively. The protective effect of GP was evidenced by a decrease in titers of infectious viruses replicated in the target organs. Not only LC cells but also GP, although to a lesser degree, enhanced NK cell activity both in uninfected mice and MCMV-infected mice. The activity of LC cells and GP to augment NK cell activity correlated with the protection index. GP treatment did not modify interferon (IFN) production during MCMV infection. Thus, GP of LC cells seems to be the active principle to endow mice with resistance to MCMV.     

Nonspecific immunomodulation influences resistance of mice to experimental infection with Mesocestoides corti and Ascaris suum

The influence of nonspecific immunomodulation on the course of experimental infection was examined in larval cestodosis (Mesocestoides corti) and ascaridosis (Ascaris suum) in mice. Immunosuppressive treatment (with azathioprine or hydrocortisone) resulted in a decrease of resistance in both models. The subsequent administration of T-activin to immunosuppressed mice led to the restoration of resistance to a level equal to that of untreated control mice. The administration of different immunomodulators partially protected mice against M. corti (T-activin, thymomodulin) or A. suum (T-activin, thymomodulin, thymosin fr.5, bursa-activin) infection. The protective effect of different treatments did not correlate with the level of specific antibody in the sera of infected mice. These results, which confirmed the decisive role of T-cell immunity in the resistance to the helminth infections, raise the possibility of the use of immunomodulators (thymic preparations) in the immunoprophylaxis of helminthoses.     

Modulation of murine macrophage responses stimulated with influenza glycoproteins.

Previously it was reported that influenza virus stimulated, nonspecific resistance was largely due to its glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The enhancement of natural killer cell activity was the intrinsic property of NA and HA. In the present study, the stimulatory effect of these glycoproteins on the murine peritoneal macrophages was studied. Electrophoretically purified glycoproteins, NA and HA, of influenza virus A/USSR/90/77 (H1N1) were administered intraperitoneally to C3H/HeN mice, with or without stearyl tyrosine (ST). Macrophages were isolated and were restimulated with phorbol myristate acetate. H2O2 secretion was determined by horseradish peroxidase dependent oxidation of phenol red assay. HA enhanced H2O2 secretion only in the presence of ST (60 nmol.mg-1.h-1), whereas NA alone stimulated H2O2 secretion (83 nmol.mg-1.h-1), by 6-fold over control (13 nmol.mg-1.h-1), and this stimulation was further increased (136 nmol.mg-1.h-1) in the presence of ST. Interleukin 1 (IL-1) activity was determined by using D10.G4.1 cells. There was a little stimulation of IL-1 activity (less than 1 U/mL) of macrophages isolated from HA-primed of HA+ST-primed mice restimulated with HA. On the other hand, IL-1 activity of macrophages isolated from NA-primed mice restimulated with NA significantly increased (102 U/mL) over control (less than 1 U/mL), and an additional 2-fold increase (231 U/mL) resulted when macrophages from NA+ST-primed mice were used. Tumor necrosis factor (TNF) activity was examined by using L929 cells. Negligible TNF activity was observed in macrophages isolated from either HA-primed or HA+ST-primed mice restimulated with HA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible Role of Macrophage Mediated Nonspecific Cytotoxicity in Tumour Resistance

IN addition to their role in specific tumour immunity^1–3, macrophages seem to be the effector cells in mediating specific and nonspecific resistance to intracellular organisms in mice^4–7, the critical factor being the development of a population of activated macrophages which resist destruction by and are able to destroy intracellular pathogens^4,8. Because resistance to neoplasia seems to be primarily cell mediated, we have investigated whether the nonspecific cell-mediated resistance conferred by chronic intracellular protozoal infection provides protection against tumours. These infections conferred resistance to a variety of autochthonous and transplantable murine tumours^9,10. Since the mechanism of resistance to tumours and unrelated infectious agents in the protozoan-infected mice might be similar, the effect of activated macrophages on target cells was evaluated. The results demonstrated that peritoneal macrophages from mice chronically infected with Toxoplasma have the capacity to non-specifically destroy target cells in vitro by a nonphagocytic mechanism.     

A potent endocytosis inhibitor Ikarugamycin up-regulates TNF production

Ikarugamycin (IK) is an antibiotic which has been reported to have a variety of functions, such as inhibition of clathrin-mediated endocytosis (CME), anti-tumor effects and regulation of the immune system. Whether IK influences cytokine production is poorly understood. We have investigated the relationship between IK and production of tumor necrosis factor-α (TNF). TNF plays a pivotal role in pathogenesis of many diseases. Although the dynamics of soluble TNF (sTNF) has been widely explored so far, the functions of the membrane form of TNF (mTNF) have not been fully elucidated. We demonstrated that IK increases the amount of mTNF and prolongs the duration of TNF expression. This effect is unrelated to the shedding activity of disintegrin and metalloproteinase domain-containing protein 17 (ADAM 17). Our results revealed that there is a mechanism to terminate inflammation at the cellular level which IK dysregulates. Furthermore, IK can be a tool to study TNF signaling due to its effect of increasing mTNF expression.     

CpG oligodeoxynucleotides protect mice from lethal challenge with Candida albicans via a pathway involving tumor necrosis factor-alpha-dependent interleukin-12 induction

In this study, we have attempted to determine whether the systemic administration of CpG oligodeoxynucleotide (CpG-ODN) 1826 would protect mice against systemic lethal Candida albicans infection. CpG-ODNs were found completely to protect mice from death and also reduced the growth of C. albicans in the kidneys. The administration of CpG-ODNs resulted in early interleukin (IL)-12 mRNA expression in the kidneys and an increase in serum IL-12 levels. The protective activity of CpG-ODN was abolished in IL-12-deficient (IL-12-/-) mice, thereby indicating the IL-12-dependency inherent to the effects of CpG-ODN. The protective effect of CpG-ODN was not associated with the activity of NF-kappaB. Interestingly, in tumor necrosis factor (TNF)-alpha-deficient (TNF-/-) mice CpG-ODN neither exerted protective effects nor induced IL-12 expression. These data indicate that CpG-ODN protects animals against lethal C. albicans challenge via a pathway that involves the TNF-alpha-dependent induction of IL-12.     

Human recombinant TNF suppresses lipoprotein lipase activity and stimulates lipolysis in 3T3-L1 cells

Tumor necrosis factor (TNF) has been reported to be identical to "cachectin," a monokine which we have previously proposed as a mediator of the enhanced catabolism observed in patients or animals responding to various invasive stimuli such as infections. Detailed quantitative studies were conducted on the effects of TNF on fatty acid metabolism in 3T3-L1 cells in order to explore the extent of the catabolic effects exerted by TNF compared with those by the crude cachectin. 3T3-L1 adipocytes responded to recombinant human TNF, showing a decrease in LPL activity and an increase in intracellular lipolysis. When TNF in the crude cachectin preparation was completely neutralized with anti-TNF antibody, about 75% of LPL suppression activity in the crude cachectin was absorbed, indicating that most of the mediator responsible for LPL suppression in the crude preparation is TNF. In contrast to the above effect on LPL, TNF markedly increased the lipolysis of stored fat in the cells. The effect on LPL was observed as early as 2 h after the addition of TNF, but enhancement of lipolysis required a time lag of at least 3 h before any increase of glycerol release became apparent. The effective concentrations of TNF for the stimulation of lipolysis were much higher (2.5 to 49 nM) than those for LPL suppression (50 pM to 50 nM), but both were in the same range as the concentration required for tumoricidal effect. These results demonstrate that cachectin is synonymous with TNF and that it plays an important role in the pathophysiology of deranged lipid metabolism through both suppression of LPL and enhancement of lipolysis in patients coping with invasive conditions such as infections.     

Babesia: the protective effects of killed Propionibacterium acnes on the infections of two rodent Babesia parasites in mice

In the present study, we investigated the protective effects of killed Propionibacterium acnes on the infections of two rodent Babesia parasites in mice. Pre-treatment with "EqStim" (a commercially available immunostimulant containing killed P. acnes) showed significant resistance to both infections. To elucidate the immunological status in the mice, the concentrations of multiple cytokines were measured in serum collected from infected mice. After B. microti infection, the levels of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12p70, and tumor necrosis factor (TNF)-alpha in the treated group were significantly lower than in the control group. In contrast, after B. rodhaini infection, only IL-12p70 and TNF-alpha were detectable at significantly higher levels in the treated group than in the control group. The present findings indicated the protective effects of killed P. acnes on rodent babesiosis even with different immune responses between the B. microti and B. rodhaini infections. Killed P. acnes might be a powerful tool for the control of serious livestock babesiosis.     

Protective effect of curcumin on pulmonary and cardiovascular effects induced by repeated exposure to diesel exhaust particles in mice

Particulate air pollution has been associated with increased risk of cardiopulmonary diseases. However, the underlying mechanisms are not fully understood. We have previously demonstrated that single dose exposure to diesel exhaust particle (DEP) causes lung inflammation and peripheral thrombotic events. Here, we exposed mice with repeated doses of DEP (15 μg/animal) every 2(nd) day for 6 days (a total of 4 exposures), and measured several cardiopulmonary endpoints 48 h after the end of the treatments. Moreover, the potential protective effect of curcumin (the yellow pigment isolated from turmeric) on DEP-induced cardiopulmonary toxicity was assessed. DEP exposure increased macrophage and neutrophil numbers, tumor necrosis factor α (TNF α) in the bronchoalveolar lavage (BAL) fluid, and enhanced airway resistance to methacoline measured invasively using Flexivent. DEP also significantly increased plasma C-reactive protein (CRP) and TNF α concentrations, systolic blood pressure (SBP) as well as the pial arteriolar thrombosis. It also significantly enhanced the plasma D-dimer and plasminogen activator inhibitor-1 (PAI-1). Pretreatment with curcumin by oral gavage (45 mg/kg) 1 h before exposure to DEP significantly prevented the influx of inflammatory cells and the increase of TNF α in BAL, and the increased airway resistance caused by DEP. Likewise, curcumin prevented the increase of SBP, CRP, TNF α, D-dimer and PAI-1. The thrombosis was partially but significantly mitigated. In conclusion, repeated exposure to DEP induced lung and systemic inflammation characterized by TNFα release, increased SBP, and accelerated coagulation. Our findings indicate that curcumin is a potent anti-inflammatory agent that prevents the release of TNFα and protects against the pulmonary and cardiovascular effects of DEP.     

Synergism for cytokine-mediated disease during concurrent endotoxin and viral challenges: roles for NK and T cell IFN-gamma production

Viral infections in humans or mice can result in increased sensitivity to challenges with bacteria, bacterial products, or cytokine administration. During lymphocytic choriomeningitis virus infections, mice are more sensitive to the lethal effects of bacterial endotoxin LPS, and in the experiments reported here, were observed at up to 10-fold lower doses in infected than in uninfected mice. The mechanisms responsible for heightened susceptibility under these conditions were evaluated. Kinetic studies demonstrated that virus-infected mice had 3- to 50-fold increases over uninfected mice in peak serum TNF, IL-12, and IFN-gamma levels after LPS administration. All three cytokines contributed to lethality during dual challenge, because neutralization of any one of the factors protected from death. Production of TNF was not dependent on either NK or T cells. In contrast, these populations were the predominant sources of IFN-gamma, as determined by lack of detectable IFN-gamma production in NK and T cell-deficient mice and by intracellular cytokine expression in the cell subsets. Concordant with the demonstrations that both cell populations produced IFN-gamma and that this factor was critical for lethality, removal of either subset alone was not sufficient to protect mice from death resulting from dual challenges. Increased resistance required absence of both cell subsets. Taken together, the data show that during viral infections, the normally protective immune responses can profoundly modify reactions to secondary heterologous challenges, to result in dysregulated cytokine expression and consequent heightened detrimental effects.     

Increase of resistance to Pseudomonas aeruginosa infection in mice caused by Staphylococcus aureus]

In our study of opportunistic pathogens, we have some indication that Staphylococcus aureus can increase resistance in mice against Pseudomonas aeruginosa. Intraperitoneal injections of sublethal doses of S. aureus had a protective effect in mice against lethal doses of P. aeruginosa, more so if living and coagulase-positive S. aureus strains were injected. This protective effect was obtained both with laboratory and freshly isolated hospital strains. The interval between these infections can be extended from 2 h up to 1 week and it is still possible to observe the resistance phenomenon. The increased resistance was accompanied by a decrease in viable units of P. aeruginosa in the peritoneal cavity of mice 6 h after the injection of this species. There was no protection by S. aureus against Candida albicans in similar experimental conditions. These observations indicate that intermicrobial ecology, understood here as the previous presence of another species in a host, may be a significant factor in the resistance to infection with opportunistic pathogens such as P. aeruginosa.     

Immunity to tapeworms: acquired resistance to Hymenolepis citelli in the mouse

The dynamics of secondary infections with Hymenolepis citelli in mice are described. A primary infection of one and six cysticercoids for 21 days sensitized CFLP male mice against homologous challenge infections. Acquired resistance was manifested mainly as stunting/destrobilation of secondary worms. The severity of stunting depended on the intensity of the primary infection. Secondary worms were not expelled more rapidly than primary worms but the protective response retards growth early in challenge infections. Sensitization of mice for seven days with six or 24 cysticercoids did not confer a measurable protective response, whereas priming by the same regime for 21 days induced a significant protective response. Acquired resistance to challenge waned with time in the absence of the primary worms. The growth and survival of a six-cysticercoid primary infection was enhanced by the administration of the immunosuppressant drug cortisone acetate. Worms from cortisone-treated mice were heavier than those from untreated controls. Acquired resistance to homologous challenge was also partially ablated in cortisone-treated mice. It is suggested that rejection of primary infections and stunting/destrobilation of secondary worms may be immunologically mediated.