Reprogramming of lipopolysaccharide-primed macrophages is controlled by a counterbalanced production of IL-10 and IL-12

We studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-alpha and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-alpha and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naive macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-gamma dose dependently and differentially regulated subsequent LPS-induced production of TNF-alpha, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-alpha and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-alpha responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-alpha response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-alpha production. We also found that the initial LPS priming of naive macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-gamma in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-alpha and NO responses in LPS-primed macrophages.     

IL-12, but not IFN-gamma, plays a major role in sustaining the chronic phase of colitis in IL-10-deficient mice

IL-10-deficient (IL-10(-/-)) mice develop chronic enterocolitis mediated by CD4+ Th1 cells producing IFN-gamma. Because IL-12 can promote Th1 development and IFN-gamma production, the ability of neutralizing anti-IL-12 mAb to modulate colitis in IL-10(-/-) mice was investigated. Anti-IL-12 mAb treatment completely prevented disease development in young IL-10(-/-) mice. Treatment of adult mice resulted in significant amelioration of established disease accompanied by reduced numbers of mesenteric lymph node and colonic CD4+ T cells and of mesenteric lymph node T cells spontaneously producing IFN-gamma. In contrast, anti-IFN-gamma mAb had minimal effect on disease reversal, despite a significant preventative effect in young mice. These findings suggested that IL-12 sustains colitis by supporting the expansion of differentiated Th1 cells that mediate disease independently of their IFN-gamma production. This conclusion was supported by the finding that anti-IL-12 mAb greatly diminished the ability of a limited number of CD4+ T cells expressing high levels of CD45RB from diseased IL-10(-/-) mice to expand and cause colitis in recombination-activating gene-2(-/-) recipients, while anti-IFN-gamma mAb had no effect. Furthermore, IL-12 could support pathogenic IL-10(-/-) T cells stimulated in vitro in the absence of IL-2. While these studies show that IL-12 plays an important role in sustaining activated Th1 cells during the chronic phase of disease, the inability of anti-IL-12 mAb to abolish established colitis or completely prevent disease transfer by Thl cells suggests that additional factors contribute to disease maintenance.     

MCP-1 protects mice in lethal endotoxemia

The overzealous production of proinflammatory cytokines in sepsis can result in shock, multiorgan dysfunction, and even death. In this study, we assessed the role of monocyte chemoattractant protein-1 (MCP-1) as a mediator of sepsis in endotoxin-challenged mice. Intraperitoneal administration of LPS to CD-1 mice induced a substantial time-dependent increase in MCP-1 in plasma, lung, and liver. The passive immunization of mice with rabbit antimurine MCP-1 antiserum 2 h before endotoxin administration resulted in a striking increase in LPS-induced mortality from 10% in control animals to 65% in anti-MCP-1-treated animals. Importantly, the administration of anti-MCP-1 antibodies to endotoxin-challenged mice resulted in increases in peak TNF-alpha and IL-12 levels, and also in a trend toward decreased serum levels of IL-10. Conversely, the administration of recombinant murine MCP-1 intraperitoneally significantly protected mice from endotoxin-induced lethality, and resulted in an increase in IL-10 levels, a decrease in IL-12 levels, and a trend toward decreased levels of TNF. In conclusion, our findings indicate that MCP-1 is a protective cytokine expressed in murine endotoxemia, and does so by shifting the balance in favor of antiinflammatory cytokine expression in endotoxin-challenged animals.     

Interferon-alpha prevents endotoxin-induced mortality in mice

Endotoxins, the lipopolysaccharide (LPS) moieties on the bacterial cell wall, cause many of the pathological features of Gram-negative septicemia. Tumor necrosis factor (TNF), primarily a product of monocyte/macrophages, has been shown to mediate many of the pathophysiological effects of endotoxin. Kupffer cells, the largest macrophage population in the body, release TNF when stimulated by LPS in vitro. A recombinant human hybrid interferon-alpha A/D (rIFN-alpha) markedly inhibited this LPS-elicited TNF production by Kupffer cells. The effects of rIFN-alpha were further tested in C57BL/6 mice receiving a lethal dose (400 micrograms/mouse) of LPS. All LPS-treated mice died within 2 days. Pretreatment with rIFN-alpha 1 h before LPS challenge improved the survival at 3 days to 22% (5/23, p < 0.04). In contrast, rIFN-alpha was more effective when administered 20 min after LPS injection, increasing the survival rate to 81% (13/16, p < 0.0001). TNF mRNA expression in the liver and spleen 50 min after LPS challenge, and plasma TNF 1.5 h after LPS were also reduced by either pretreatment or post-treatment with rIFN-alpha. Subsequently, experiments were carried out to test the efficacy of delayed rIFN-alpha treatment. A significant protective effect was still apparent when rIFN-alpha was administered 6, 10 and even 14 h (81%, 62% and 28% survival, respectively) after LPS challenge when serum TNF levels had already returned to near baseline. These experimental results suggest that rIFN-alpha might have a therapeutic potential for the prevention and treatment of the deleterious effects associated with endotoxemia besides mechanisms initially blocking TNF production.     

Anti-IL-12 antibody prevents the development and progression of collagen-induced arthritis in IFN-gamma receptor-deficient mice

In several models of inflammation, including collagen-induced arthritis (CIA), the disease-promoting effect of IL-12 has been attributed to its well-known ability to produce IFN-gamma. However, IFN-gamma receptor knockout (IFN-gammaR KO) mice of the DBA/1 strain have been reported to be more susceptible to CIA than corresponding wild-type mice, indicating the existence of an IFN-gamma-mediated protective pathway in this model. In the present study the development of CIA was found to be completely prevented by pretreatment with a neutralizing anti-IL-12 antibody, not only in wild-type, but significantly also in IFN-gammaR KO mice. In both strains of mice, the protective effect of anti-IL-12 was associated with lower production of anti-collagen type II antibodies. In vivo stimulation with anti-CD3 antibody in arthritic IFN-gammaR KO mice resulted in production of higher levels of circulating IFN-gamma, TNF and IL-2 than in corresponding control mice that had not received the arthritis-inducing immunization. This was not the case in arthritis-developing wild-type mice. Furthermore, the protective effect of anti-IL-12 antibody in mutant, but not in wild-type mice, was associated with lower circulating IFN-gamma, TNF and IL-2 and higher IL-4 and IL-5 cytokine levels following an anti-CD3 challenge. The data indicate that IL-12 promotes the development of arthritis independently of its ability to induce or favor production of IFN-gamma. In fact, any IFN-gamma produced in the course of the disease process rather exerts a protective effect. Furthermore, our study suggests that, in the absence of a functional IFN-gamma system, endogenous IL-12 exerts its disease-promoting effect by favoring production of other Th1-associated cytokines (IL-2 and TNF), by inhibiting development of IL-4- and IL-5-producing T cells and by stimulating production of anti-collagen autoantibodies.     

Role of xanthine oxidase and reactive oxygen intermediates in LPS- and TNF-induced pulmonary edema

We studied the role of reactive oxygen intermediates (ROI) in lipopolysaccharide (LPS)-induced pulmonary edema. LPS treatment (600 micrograms/mouse, IP) was associated with a marked induction of the superoxide-generating enzyme xanthine oxidase (XO) in serum and lung. Pretreatment with the antioxidant N-acetylcysteine (NAC)--1 gm/kg orally, 45 minutes before LPS--or with the XO inhibitor allopurinol (AP)--50 mg/kg orally at -1 hour and +3 hours--was protective. On the other hand nonsteroidal antiinflammatory drugs (ibuprofen, indomethacin, and nordihydroguaiaretic acid) were ineffective. These data suggested that XO might be involved in the induction of pulmonary damage by LPS. However, treatment with the interferon inducer polyriboinosylic-polyribocytidylic acid, although inducing XO to the same extent as LPS, did not cause any pulmonary edema, indicating that XO is not sufficient for this toxicity of LPS. To define the possible role of cytokines, we studied the effect of direct administration of LPS (600 micrograms/mouse, IP), tumor necrosis factor (TNF, 2.5 or 50 micrograms/mouse, IV), interleukin-1 (IL-1 beta, 2.5 micrograms/mouse, IV), interferon-gamma (IFN-gamma, 2.5 micrograms/mouse, IV), or their combination at 2.5 micrograms each. In addition to LPS, only TNF at the highest dose induced pulmonary edema 24 hours later. LPS-induced pulmonary edema was partially inhibited by anti-IFN-gamma antibodies but not by anti-TNF antibodies, anti-IL-1 beta antibodies, or IL-1 receptor antagonist (IL-1Ra).     

Protective effect of a single interleukin-12 (IL-12) predose against the toxicity of subsequent chronic IL-12 in mice: role of cytokines and glucocorticoids

The mechanisms of interleukin-12 (IL-12) toxicity were studied in mice using a schedule (murine rIL-12, 400 ng/mouse, intraperitoneally [IP] once daily for 5 days) that markedly reduced body weight and food intake. On day 5, IL-12-treated mice had elevated serum and spleen IFN-gamma and tumor necrosis factor (TNF). Serum sTNFR-P75 and corticosterone (CS) were also elevated. IL-12 toxicity was partially prevented by anti-IFN-gamma antibodies or dexamethasone (DEX). A pre-dose of IL-12 (200 ng/mouse on day -14) completely prevented the toxicity of subsequent IL-12. The IL-12 predose also inhibited IL-12-induced IFN-gamma levels, but did not modify IL-12-induced CS, TNF or sTNFR-P75. A protective effect was observed with a predose of lipopolysaccharide (LPS) or murine recombinant (r)IL-10. The protective effect of the IL-12 predose was reduced by coadministration of anti-IFN-gamma, but a predose of murine rIFN-gamma was not protective, suggesting that IFN-gamma is necessary but not sufficient for the protective effect of IL-12. The IL-12 predose specifically protected against IL-12 toxicity and did not modify LPS toxicity. These data indicate that IL-12 can induce tolerance to its own toxicity, probably through a downregulation of IL-12-induced IFN-gamma but independently of endogenous glucocorticoids. IFN-gamma, and possibly IL-10, might be important in this tolerance.     

IL-13 protects mice from lipopolysaccharide-induced lethal endotoxemia: correlation with down-modulation of TNF-alpha, IFN-gamma, and IL-12 production

IL-13 is a potent down-modulator of macrophage proinflammatory activity in vitro, similar in this context to the anti-inflammatory cytokines IL-4 and IL-10. Since IL-10 effectively confers protection to mice from LPS-induced lethal endotoxemia through inhibition of proinflammatory cytokine production, we investigated whether IL-13 may also be capable of providing protection in this experimental model of endotoxic shock. A single injection of recombinant murine IL-13 (rmIL-13; 0.5-10 microg) significantly increased survival in a dose-dependent manner when a lethal i.p. injection of endotoxin was administered to BALB/c mice. This effect appeared to be IL-13 specific, since survival was not affected in mice that received heat-inactivated rmIL-13. rmIL-13 provided significant protection to mice even when given 30 min after LPS injection; however, this protection decreased in a time-dependent manner as the administration of rmIL-13 was delayed by 1, 2, and 5 h following LPS injection. The protective effect of IL-13 was correlated with significant decreases in the production of the inflammatory mediators TNF-alpha, IFN-gamma, and IL-12 as well as a decrease in the anti-inflammatory mediator IL-10. Our data suggest that IL-13 provides protection from LPS-induced lethal endotoxemia in a manner that is similar to but independent from that of IL-10, and therefore can be added to the list of cytokine immunomodulators that might be beneficial in the treatment of septic shock.     

Interleukin-10 inhibits activation of coagulation and fibrinolysis during human endotoxemia

Interleukin-10 (IL-10) has been found to inhibit lipopolysaccharide (LPS)-induced tissue factor expression by monocytes in vitro. To determine the effects of IL-10 on LPS-induced activation of the hemostatic mechanisms in vivo, we performed a placebo-controlled, cross-over study of human endotoxemia. Two groups of eight volunteers were challenged with LPS (4 ng/kg) on two occasions: once in conjunction with placebo, and once with recombinant human IL-10 (rhIL-10; 25 microg/kg). In group 1, placebo or rhIL-10 was given 2 minutes before LPS challenge, group 2 received placebo or rhIL-10 1 hour after LPS administration. Pretreatment with rhIL-10 reduced both LPS-induced activation of the fibrinolytic system (plasma concentrations of tissue type plasminogen activator, plasmin-alpha2-antiplasmin complexes, and D-dimer), and inhibition of fibrinolysis (plasma levels of plasminogen activator inhibitor 1), whereas posttreatment only inhibited the latter response. Both IL-10 pre- and posttreatment attenuated activation of the coagulation system (plasma levels of prothrombin fragment F1 + 2 and thrombin-antithrombin complexes). These results indicate that rhIL-10, besides its well-described inhibitory effects on cytokine release, potently modulates the fibrinolytic system and inhibits the coagulant responses during endotoxemia.     

Alleviation of lipopolysaccharide-induced acute liver injury in Propionibacterium acnes-primed IFN-gamma-deficient mice by a concomitant reduction of TNF-alpha, IL-12, and IL-18 production

The present study was designed to investigate the role of IFN-gamma in LPS-induced liver injury following priming with Propionibacterium acnes. At 1 week after priming BALB/c mice with P. acnes, a large number of macrophages (Mphi) and lymphocytes predominantly infiltrated the portal area, resulting in the intrahepatic formation of granulomas consisting of epithelioid and lymphoid cells. In comparison, in IFN-gamma gene-disrupted BALB/c mice (IFN-gamma knockout mice), the number of infiltrated Mphi was decreased, with a significant reduction in the number and size of granulomas. Subsequent elicitation with a low dose of LPS induced massive hepatic necrosis in wild-type BALB/c mice, with a marked increase in the serum levels of TNF-alpha, IL-12, and IL-18 and subsequently of alanine transferase. In contrast, IFN-gamma knockout mice developed scattered focal necrosis of the liver with significantly lower levels of serum alanine transferase as well as drastic decreases in TNF-alpha, IL-12, and IL-18 production. The administration of an anti-IFN-gamma neutralizing mAb at the eliciting phase significantly alleviated liver injury and reduced serum IL-12 and IL-18 levels. Thus, endogenously produced IFN-gamma is involved in the pathogenesis of this liver injury model by regulating Mphi infiltration and granuloma formation in the priming phase as well as cytokine production in the eliciting phase.     

Benzydamine protection in a mouse model of endotoxemia

OBJECTIVE: Previous studies have shown that benzydamine (40 mg/kg s.c.) is able to inhibit tumor necrosis factor (TNF) production and to reduce mouse lethality when administered before or concomitantly with LPS. The present study was designed to further investigate benzydamine activity against LPS-induced toxicity in terms of potency and therapeutic effects. METHODS: Female Balb/c mice were used. A dose-response curve of animal lethality versus endotoxin dose was performed (LD50 = 45 micrograms/mouse). Therapeutic effects were studied selecting the dose of LPS to achieve an LD100 (160 micrograms/mouse). Mortality was assessed daily and mice were followed for 8 days. The potential mode of action of therapeutically administered benzydamine was also investigated. TNF alpha and IL-1 beta levels were measured, at 5 h after LPS injection, both in sera and in lungs. Moreover, the drug was assayed in a TNF-dependent cytoxicity test. RESULTS: Benzydamine, administered at 20 mg/kg s.c. simultaneously with the endotoxin, significantly increased LPS LD50 up to 230 micrograms/mouse (p < 0.05). Moreover, the drug significantly protected mice against LPS-induced lethality when administered either 30 min or 4 h after endotoxin injection (p < 0.001). Benzydamine, therapeutically administered at 20 mg/kg s.c., significantly reduced TNF alpha and IL-1 beta production induced by LPS both in serum and lungs and it was shown to inhibit TNF-dependent cytoxicity on L929 cells. CONCLUSIONS: These results clearly demonstrate the therapeutic activity of benzydamine in a simple model of endotoxic shock. Available data confirm the potential role of benzydamine as an anti-cytokine agent and provide suggestions for novel therapeutic applications of this anti-inflammatory drug.     

Effect of matrine on mouse hepatitis and tumor necrosis factor production induced by Propionibacterium acnes/lipopolysaccharides

The effect of matrine (Mat) on lipopolysaccharides (LPS)-induced fatal hepatitis and tumor necrosis factor (TNF) production in Propionibacterium acnes (PA)-primed mice were studied. Mice were injected i.p. LPS (10 micrograms/mouse) 7 d after i.p. PA (0.5 ml/mouse) to induce fatal hepatitis. After i.p. LPS, serum TNF activity rose to 1657 +/- 406 kU.L-1 at 1.5 h and ALT activity increased up to 1,496 +/- 890 U.L-1 at 5 h. Six of 8 mice died within 5 h and the massive hemorrhagic necrosis of the liver was observed in all mice. Administration of Mat (10, 50 mg.kg-1, i.p., bid x 3 d) before the LPS injection markedly reduced the elevation of serum TNF and ALT activity in a dose-dependent manner, and diminished the mortality induced by LPS. Liver congestion and necrosis induced by LPS in PA-primed mice were ameliorated markedly by Mat pretreatment. Mat (62.5-250 mg.L-1) inhibited LPS-induced TNF release from PA-primed mouse peritoneal macrophage in vitro in a concentration-dependent manner. These results seggest that Mat protected PA-primed mice from the development of fatal hepatitis induced by LPS due to inhibition of TNF production.     

IL-18 accounts for both TNF-alpha- and Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice

When LPS is administered to heat-killed Propionibacterium acnes-primed BALB/c nude mice, they develop endotoxin-induced liver injury. As previously reported, this liver injury can be prevented by treatment with an Ab against IL-18, a novel cytokine with the ability to induce IFN-gamma production and up-regulate functional Fas ligand (FasL) expression. To identify the pathologic role of IL-18 in this liver injury, we investigated the hepatic cytokine network and FasL induction after LPS challenge. After LPS challenge to BALB/c nude mice, their livers expressed IL-12 mRNA, followed by the induction of IFN-gamma and FasL mRNA and then by the late elevation of TNF-alpha mRNA, but stably expressed IL-18 mRNA. The TNF-alpha induction curve had two peaks. The first peak was the result of the direct reaction to LPS, and the late peak might have been induced, since P. acnes-elicited Kupffer cells showed one-peak TNF-alpha kinetics in response to LPS stimulation in vitro. LPS-activated P. acnes-elicited Kupffer cells secreted both IL-12 and IL-18, as determined by ELISA and bioassay, respectively. The in vivo administration of anti-IL-18 just before an LPS challenge suppressed not only the induction of IFN-gamma and the late TNF-alpha elevation, but also the FasL induction, resulting in the total prevention of liver injury, whereas such an anti-IL-12 treatment did not. Anti-IFN-gamma treatment reduced the late increase in TNF-alpha, but not FasL, resulting in a partial prevention of the liver injury. The administration of anti-TNF-alpha just before elevation of the late TNF-alpha peak also markedly, but incompletely, suppressed the LPS-induced liver injury. These data suggested that IL-18 activates both TNF-alpha- and FasL-mediated hepatocytotoxic pathways in endotoxin-induced liver injury.     

A pathogenic role of IL-12 in blood-stage murine malaria lethal strain Plasmodium berghei NK65 infection

We studied whether the infection with a blood-stage murine malaria lethal Plasmodium berghei NK65 induces IL-12 production, and if so, how the IL-12 production is involved in the protection or pathogenesis. The infection of C57BL/6 mice enhanced mRNA expression of IL-12 p40 and also IFN-gamma, IL-4, and IL-10 in both spleen and liver during the early course of the infection. It also enhanced the mRNA expression of TNF-alpha, Fas ligand, and cytokine-inducible nitric oxide synthase. Increased IL-12 p40 production was also observed in the culture supernatant of spleen cells and in sera of infected mice. In addition, the infection caused massive liver injury with elevated serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and body weight loss. Treatment of these infected mice with neutralizing mAb against IL-12 prolonged the survival and diminished the liver injury with reduced elevation of serum serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and decreased body weight loss. However, the anti-IL-12 treatment did not affect parasitemia, and all these mice eventually died. Similar results were obtained when infected mice were treated with neutralizing mAb against IFN-gamma. Moreover, anti-IL-12 treatment greatly reduced the secretion and mRNA expression of IFN-gamma in both spleen and liver. These results suggest that the lethal P. berghei NK65 infection induces IL-12 production and that the IL-12 is involved in the pathogenesis of liver injury via IFN-gamma production rather than the protection.     

Inhibition of interferon gamma induced interleukin 12 production: a potential mechanism for the anti-inflammatory activities of tumor necrosis factor

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) gamma production. IFN-gamma, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-gamma inhibits production of chemokines (macrophage inflammatory proteins MIP-1alpha and MIP-1beta). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-gamma priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-gamma-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF+/+ and TNF-/- mice injected with Corynebacterium parvum were compared. TNF-/- mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF-/- mice compared with TNF+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF-/- mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-gamma inhibition of chemokine production and inhibition of IFN-gamma-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).     

Protective effects of niacinamide in staphylococcal enterotoxin-B-induced toxicity

Staphylococcal enterotoxins (SE) interact with major histocompatibility complex (MHC) class II cell-surface receptors, eliciting signal transduction in antigen-presenting cells (APC). Subsequent toxin-class II complex interaction with specific T-cell receptors induces T-cell activation. We investigated the effect of niacinamide and interleukin (IL)-10 on SEB-induced responses. In a macrophage cell line, niacinamide (ED50--2mM) and IL-10 (ED50--7U/ml) inhibited interferon (IFN)-gamma-induced MHC class II expression in a dose-dependent manner. Also, niacinamide was a potent inhibitor of T-cell proliferation induced by SEB (ED50-- 1 mM) while IL-10 has minimal effects. In mice, the temporal responses of IL-1alpha, tumor necrosis factor (TNF)-alpha, IL-2, and IFN-gamma evoked by SEB were synergistically potentiated by lipopolysaccharide (LPS). Lethality occurred only when SEB was potentiated by LPS. Niacinamide or IL-10 improved survival of mice after lethal SEB challenge. Niacinamide reduced cytokine serum levels, although the pattern differed from that of IL-10. Niacinamide primarily reduced IL-2 and IFN-gamma, while IL-10 predominantly reduced IL-1alpha and TNF-alpha. The immunomodulatory effects of niacinamide observed on SEB-induced activation of APC and T-cells in vitro and in the LPS potentiated murine model for SEB-induced toxicity suggest it may have therapeutic value.     

Involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist in LPS-induced rabbit uveitis

The objective of the study was to investigate involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist (IL-1Ra) in lipopolysaccharide (LPS)-induced uveitis. Intravitreal injection of LPS (100 ng) to rabbits induced a massive leukocyte infiltration and protein leakage into the aqueous humor. Aqueous leukocyte counts and protein levels reached a peak 24 hr after this injection. The peak concentrations of aqueous TNF alpha (230 +/- 37 pg ml-1, at 9 hr) and IL-1 beta (185 +/- 80 pg ml-1, at 18 hr) preceded peak levels of aqueous leukocyte counts and protein levels. In contrast, the levels of aqueous IL-1Ra peaked at 48 hr (12,239 +/- 1964 pg ml-1) and a fairly high concentration of IL-1Ra remained when the inflammatory reactions subsided. Immunohistochemistry and leukocyte-depletion studies showed that infiltrating leukocytes were the major cellular sources of aqueous TNF alpha, IL-1 beta and IL-1Ra. Intravitreal injection of homologous TNF alpha (0.1-1.5 micrograms) or IL-1 beta (0.5-5 ng) reproduced a rapid leukocyte infiltration and protein leakage. Administration of anti-TNF alpha mAb (10 micrograms) suppressed the number of LPS-induced infiltrating neutrophils by 50%, mononuclear cells by 58%, and protein leakage by 42%. Administration of rabbit IL-1Ra (10 micrograms) also suppressed neutrophil influx by 78%, however, neither mononuclear cell influx nor protein leakage was inhibited by rabbit IL-1Ra. Co-administration of the two inhibitors enhanced inhibition of neutrophil infiltration to 88%, and protein leakage to 64%. We conclude that TNF alpha and IL-1 beta are the principal mediators of LPS-induced uveitis. Our observations also suggest that endogenous IL-1Ra may down-regulate inflammatory reactions.     

Effect of interferon-gamma on nitric oxide hemoglobin production in endotoxin-treated rats and its synergism with interleukin 1 or tumor necrosis factor

We studied the in vivo effect of interferon-gamma (IFN-gamma) on nitric oxide (NO) generation. ESR spectra of nitric oxide hemoglobin (HbNO) appeared after a lag time of 2h in the blood of rats treated with Escherichia coli lipopolysaccharide (LPS). IFN-gamma enhanced LPS-induced HbNO formation in rats without modifying the time lag, although IFN-gamma alone did not induce HbNO formation. The plasma nitrate concentration was approximately one order of magnitude higher than the HbNO concentration. On treatment with LPS alone, the amount of tumor necrosis factor (TNF) released decreased after 2 h. Simultaneous addition of IFN-gamma and LPS increased TNF release for at least 8 h. Interleukin 1 (IL-1) release was detected only at 2 h in both groups. We also investigated the in vivo interactions of these cytokines. TNF plus IL-1 induced the greatest HbNO generation, followed by TNF plus IFN-gamma, and then IL-1 plus IFN-gamma. These results suggest that increase of TNF release by IFN-gamma plays a key role in NO generation in LPS-treated rats.     

Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice

We investigated the mechanisms by which treatment with anti-IL-12 Ab prevents cure of infection with Leishmania major in resistant C57BL/6 mice. Consistent with delayed production of IL-12, anti-IL-12 Abs could be administered as late as 2 wk after infection to exacerbate disease. Starting at 2 wk of infection, the cultured lymph node cells from mice treated with either polyclonal or monoclonal anti-IL-12 Abs persistently generated 3- to 10-fold more IL-4 and IL-10 in response to L. major Ag compared with cells from mice receiving preimmune goat IgG. Reciprocal decreases in Ag-specific IFN-gamma production were observed in mice receiving anti-IL-12 Abs. A similar reversal of IFN-gamma and IL-4 production accompanied progressive disease induced by pretreatment with a single dose of anti-IFN-gamma mAb. Although IFN-gamma production was suppressed for up to 4 wk in mice treated with monoclonal anti-IL-12 or anti-IFN-gamma, coadministration of neutralizing anti-IL-4 IgG reversed progressive illness. These findings demonstrate that IL-12 produced in vivo is necessary for both the emergence of IFN-gamma producing cells and the down-regulation of Th2 cell responses during murine leishmaniasis. Furthermore, the uninhibited production of IL-4 was required to sustain progressive infection initiated by the decreased IFN-gamma synthesis observed in anti-IL-12 and anti-IFN-gamma-treated mice.     

IL-18 (IFN-gamma-inducing factor) regulates early cytokine production in, and promotes resolution of, bacterial infection in mice

IL-12-induced IFN-gamma production is essential for clearance of Yersinia enterocolitica infection. Similar to IL-12, the recently described cytokine IL-18 (IFN-gamma-inducing factor) is produced by macrophages and induces IFN-gamma production in spleen cells. Therefore, we have investigated the role of IL-18 in Yersinia infection of mice. Heat-killed yersinia-triggered IL-18-promoted IFN-gamma production of splenocytes was predominantly dependent on endogenous IL-12 production, whereas IL-12-promoted IFN-gamma production was not IL-18 dependent. IL-18-induced IFN-gamma production was to a higher degree dependent on IFN-gammaR-mediated mechanisms and in synergism with IL-2 resulted in at least fivefold higher IFN-gamma levels as compared with the combination of IL-12 plus IL-2. Analysis of the effect of IL-18 on IL-12 production of LPS-stimulated peritoneal macrophages revealed that IL-18 decreased LPS-induced IL-12 production, indicating that IL-18 might be involved in negative regulation of IL-12 production. In vivo studies revealed that Yersinia-resistant C57BL/6 mice expressed fourfold higher IL-18 mRNA levels than did susceptible BALB/c mice. Administration of anti-IL-18 Abs caused a 100- to 1000-fold increase in bacterial counts in the spleen of infected mice but did not change IFN-gamma production levels. Taken together, our data demonstrate that IL-18 is involved in regulation of cytokine production during the early phase of bacterial infections as well as in clearance of Yersinia infection.