Interleukin-1beta induces in vivo tolerance to lipopolysaccharide in mice.

Endotoxin or lipopolysaccharide (LPS) tolerance may be partially due to the secretion of potent anti-inflammatory cytokines following severe Gram-negative infections, or by low doses of LPS. In this work, we describe the effects of interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF-), two early cytokines secreted after LPS exposure, in the induction of LPS tolerance. Our results demonstrate that mice treated with three daily doses of 100 ng of IL-1 were tolerant to LPS-induced shock. However, TNF- was unable to induce an LPS refractory state. Given the fact that 100 ng of IL-1 increase the plasma levels of glucocorticoids, we evaluated whether a daily injection of dexamethasone (DEX) alone was able to reproduce the LPS-like tolerant state. However, no signs of LPS refractoriness were detected, except when DEX was administered concomitantly with a dose of IL-1 that does not induce corticosterone secretion (12 ng/mouse). This dose was found to induce in vitro up-regulation of the glucocorticoid receptors (GcR) of peritoneal macrophages following 24 h of treatment. In addition, we demonstrate that IL-1 is capable of inducing the down-regulation of Toll-like receptor 4 (TLR4), a crucial molecule in the signal transduction of LPS. Taken together, our results indicate that IL-1 can generate tolerance to LPS in vivo, and suggest that the regulation of mechanisms of the down-regulation of TLR4, as well as those involved in the expression of GcR and/or in the secretion of glucocorticoids, would be crucial for these effects.

Dendritic cells as a major source of macrophage-derived chemokine/CCL22 in vitro and in vivo.

Macrophage-derived chemokine (MDC)/CCL22 is a CC chemokine active on dendritic cells (DC), NK cells and Th2 lymphocytes. The present study was aimed at comprehensively investigating MDC production in vitro and in vivo. DC were the most potent producers of MDC among leukocytes tested. Endothelial cells did not produce MDC under a variety of conditions. Signals that induce maturation (lipopolysaccharide, IL-1, TNF, CD40 ligand, recognition of bacteria and yeast) dramatically augmented MDC production, and dexamethasone and vitamin D3 blocked it. Prostaglandin E(2), which blocked the acquisition of IL-12 production and the capacity to promote Th1 generation, did not affect MDC production. Using mass spectrometry-based techniques, DC supernatants were found to contain N-terminally truncated forms of MDC [MDC(3-69), MDC(5-69) and MD(C7-69)] as well as the full-length molecule. In vivo, CD1a(+), CD83(+), MDC(+) DC were found in reactive lymph nodes, and in Langerhans' cell histiocytosis. Skin lesions of atopic dermatitis patients showed that CD1a(+) or CD1b(+) DC, and DC with a CD83(+) phenotype were responsible for MDC production in this Th2-oriented disorder. Thus, DC are the predominant source of MDC in vitro and in vivo under a variety of experimental and clinical conditions. Processing of MDC to MDC(3-69) and shorter forms which do not recognize CCR4 is likely to represent a feedback mechanism of negative regulation.

Deferoxamine reduces tissue injury and lethality in LPS-treated mice.

We studied the effect of deferoxamine (DFX), an iron chelator, which can also act as a free radical scavenger, in an experimental murine model of sepsis. In vivo studies demonstrated that pretreatment of mice with DFX reduces tumor necrosis factor alpha (TNF-alpha) serum levels and increases the rate of survival of mice inoculated with lethal doses of lipopolysaccharide (LPS) or Escherichia coli O111:B4. By using the iron chelated form of DFX (ferrioxamine) the same results were obtained, suggesting that in this model, DFX could act as a free radical scavenger. On the other hand, DFX prevents mortality induced either by LPS or murine recombinant TNF-alpha in D(+)-galactosamine (GalN)-sensitized mice. These protective actions of DFX correlate with an attenuated tissue damage observed in lungs, livers and kidneys of LPS-treated animals and GalN-sensitized mice inoculated with TNF-alpha.

The down-regulation of FcgammaRII and FcgammaRIIIB by N-formyl-methionyl-leucyl-phenylalanine (FMLP) depends on secretory events in human neutrophils.

We have previously demonstrated that N-formyl-methionyl-leucyl-phenylalanine (FMLP) induces down-regulation of FcgammaRs on human neutrophils (PMN) modifying different FcgammaR-dependent functions. The aim of this work was to assess the cellular mechanisms by which FMLP exerts this effect on FcgammaRs. The role of the microfilament and cytoskeletal apparatus in this process was evaluated using cytochalasin B (CB), an inhibitor of microfilament functions. The expression of FcgammaRIIIB and FcgammaRII after CB + FMLP treatment was drastically diminished when compared to FMLP-treated cells. Neutrophil degranulation induced by FMLP affect only 22% of the cells in response to FMLP. However, the FcgammaRs of the whole PMN population were reduced, suggesting that secretory products could be responsible for the down-regulation induced by FMLP or FMLP + CB. In fact, supernatants from FMLP-treated PMN also induced FcyRs down-regulation on naive neutrophils. Moreover, supernatants from FMLP + CB-treated PMNs exerted a higher effect. Data obtained from permeabilized PMN show that after FMLP treatment there is an intracellular depletion of both FcgammaRIIIB and FcgammaRII. In addition, the FcgammaR down-regulation is abrogated by phenyl methyl sulfonyl fluoride (PMSF) but not by other protease inhibitors such as pepstatin, thiorphan, phosphoramidon and leupeptin, suggesting a role for serine protease(s) in this process.

Interleukin-2 restores natural killer activity inhibited by sera from HIV+ hemophilic patients.

Natural killer (NK) activity is impaired in patients with positive serology for the human immunodeficiency virus (HIV). We previously found an inhibitory effect of sera from hemophilic (He) HIV+ patients on normal NK activity. In the present study, we have further characterized this effect by studying its reversibility, temperature and time incubation dependence. Since interleukin 2 (IL-2) is able to enhance NK levels, we analyzed the capacity of this lymphokine to reverse the effect of He HIV+ sera. We found that when IL-2 activation of NK activity occurred simultaneously or after HIV+ serum-treatment, a significant restoration of NK function was observed. In contrast, preincubation with IL-2 did not affect the inhibitory effect exerted by HIV+ sera.

Interleukin-2 restores natural killer activity inhibited by sera from HIV+ hemophilic patients.

Natural killer (NK) activity is impaired in patients with positive serology for the human immunodeficiency virus (HIV). We previously found an inhibitory effect of sera from hemophilic (He) HIV+ patients on normal NK activity. In the present study, we have further characterized this effect by studying its reversibility, temperature and time incubation dependence. Since interleukin 2 (IL-2) is able to enhance NK levels, we analyzed the capacity of this lymphokine to reverse the effect of He HIV+ sera. We found that when IL-2 activation of NK activity occurred simultaneously or after HIV+ serum-treatment, a significant restoration of NK function was observed. In contrast, preincubation with IL-2 did not affect the inhibitory effect exerted by HIV+ sera.

N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibits tumour necrosis factor-alpha (TNF-alpha) production on lipopolysaccharide (LPS)-stimulated human neutrophils.

During gram-negative infections bacterial components, such as LPS and formylated peptides, exert profound physiological effects on polymorphonuclear neutrophils (PMN) resulting in increased neutrophil effector activities, including the generation of oxidative metabolites, degranulation, phagocytosis and cytokine release. There is not enough evidence about the relationships between LPS and formylated bacterial peptides in the triggering and regulation of the immune inflammatory response. In this study, we present evidence indicating that pretreatment of human PMN with a prototype formylated peptide such as fMLP results in the inhibition of TNF-alpha secretion, a key molecule that plays a central role in the pathogenesis of septic shock. This inhibitory effect of fMLP does not appear to alter the expression of LPS receptors or the transcriptional pathway of the TNF-alpha mRNA, but instead, fMLP reduces the expression of the membrane form of TNF-alpha on the PMN surface. These findings indicate that fMLP, a typical proinflammatory agent, could play, at least in determined conditions, an anti-inflammatory role.

[Human polymorphonuclear neutrophils (PMN) induce lipopolysaccharide (LPS) liberation from gram-negative bacteria]. / Neutrófilos humanos (PMN) inducen la liberación de lipopolisacáridos (LPS) de bacterias Gram negativas.

Bacterial lipopolysacharride (LPS) is the major membrane component of Gram negative bacteria. It is a potent pleiotropic stimulus for the immune system frequently associated with septic syndrome or septic shock. The detoxification of LPS in Gram negative sepsis is one of the important problems to resolve in clinical treatments. In this study we compare the capacity of polymorphonuclear neutrophils (PMN) in LPS detoxification in two different situations: a) when LPS is offered to PMN as an isolated molecule; b) when the LPS offered is part of the whole Gram negative bacteria (E. coli 0111:B4). Our results show that PMN are able to inhibit the capacity of LPS to produce TNF-alpha. However, when whole bacteria, instead of LPS, are incubated with PMN, an enhancement in the production of tumor necrosis factor alpha (TNF-alpha) is observed. The bacterial overburden of PMN is not the reason for the spread of LPS after PMN incubation. Our conclusion is that PMN have a dual capacity to deal with LPS, either inactivating or releasing it depending on how it is offered.

Hydroxyl radical scavengers inhibit TNF-alpha production in mononuclear cells but not in polymorphonuclear leukocytes.

The hydroxyl radical (HO*) scavengers dimethylthiourea (DMTU), tetramethylthiourea (TMTU), dimethylsulfoxide (DMSO) and deferoxamine (DFX), the latter being an iron chelator which prevents HO* formation by blocking the Fenton reaction, were found to inhibit TNF-alpha production in LPS-stimulated human PBMC but not in PMN. Furthermore, this effect was not LPS-specific, as TNF-alpha production was reduced by HO* radical scavengers to a similar extent upon stimulation of PBMC with immune complexes (IC), concanavalin A (Con A) and phorbol myristate acetate (PMA). Other scavengers such as glutathione (GSH), N-acetylcysteine (NAC), ascorbic acid (ASC) and mannitol (MAN) do not have effect on the production of TNF-alpha either in PBMC or PMN. These results provide evidence that the participation of ROI in the regulation of TNF-alpha production differ in different cell types. Particularly, the data presented in this work indicate that HO* radicals have a central role in the production of this inflammatory cytokine by human PBMC.

Inhibition of Fc gamma R-dependent functions by N-formylmethionylleucylphenylalanine in human neutrophils.

Human polymorphonuclear neutrophils (PMN) participate in different cellular functions, including phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and release of reactive oxygen intermediates. Each of these functions can be triggered by receptors for the Fc portion of IgG molecules (Fc gamma R). Normal resting neutrophils possess Fc gamma RII and Fc gamma RIIIB receptors. They also have specific membrane receptors for formylated peptides such as the prototype N-formylmethionylleucylphenylalanine (FMLP). In this report, we present evidence that preincubation of PMN with FMLP inhibits different PMN Fc gamma R-dependent functions such as phagocytosis, ADCC, and immune complex-dependent cytotoxicity. These inhibitory effects can be explained, at least in part, by downregulation of both Fc gamma RII and Fc gamma RIII. Unexpectedly, preincubation of FMLP with PMN was not necessary for ADCC inhibition. Taking into account that the FMLP-dependent Fc gamma R downregulation is not observed before 30 min of incubation, and the onset of ADCC occurs rapidly (seconds), it is possible that FMLP can modify this function by altering early intracellular events.

Variations in the platelet arginine/nitric oxide pathway during the ovarian cycle in females affected by menstrual migraine.

Previous studies have reported the existence of an arginine/nitric oxide (NO) pathway and the involvement of a Ca2+, NADPH-dependent nitric oxide synthase enzyme (NOS) in the generation of NO in human platelets. In the present research, we determined the rate of production of NO and cGMP in the cytosol of platelets stimulated by collagen in 20 females with menstrual migraine (MM), (age range 24-40 years), assessed in the follicular and luteal phases, interictally and ictally in the latter period. The same patients were also assessed at mid-cycle. At the same time, the variations in the collagen response of platelets were evaluated. Moreover, these parameters were determined in the same periods in 20 age-matched control females and in 20 females affected by non-menstrually related migraine (nMM). The collagen-stimulated production of NO in the cytosol of the platelet cytosol was significantly higher in migraine patients with MM than in the control subjects. In MM patients, the increase was greater in the luteal phase of the cycle than during the follicular phase (p < 0.005). A rise in NO production in platelets was also present, although to a lesser extent, in females affected by nMM compared to the healthy females, but this rise was most evident at ovulation (p < 0.001). A slight but significant increase was also observed at mid-cycle in control women, but this increase did not reach the values determined in the migraine groups (p < 0.02). NO production in platelets stimulated by collagen was significantly increased during attacks with respect to the interictal period in both patient groups. Similar variations were observed in the production of cGMP in MM and nMM patients. The increase in NO production was accompanied by a decrease in platelet aggregation in the migraine groups compared with the control group; this decrease was most evident at mid-cycle in nMM patients and in the luteal phase in MM patients. These data suggest an activation of the L-arginine/ NO pathway in MM and nMM patients which could explain the modifications in the platelet response to collagen evidenced in migraine-free periods and during attacks. The activation of this pathway is more accentuated in the luteal phase in MM patients, and this could be the cause of the increased susceptibility to migraine attacks in perimenstrual and menstrual periods in these patients.

L-arginine/nitric oxide pathway activation in platelets of migraine patients with and without aura.

Nitric oxide (NO) in platelets has been proposed as a promising tool for studying NO variations in migraine. In the present research the platelet response to collagen and the basal and collagen-induced production of NO and cGMP in platelet cytosol were assessed in migraine patients (25 with aura and 35 without aura) both interictally and ictally, and compared with the same parameters in 30 age-matched control subjects. A reduced responsiveness to collagen was found in migraine patients, particularly those with aura, and this was more marked during attacks (ANOVA interictal periods: p < 0.01, attacks: p < 0.02) The basal and collagen-stimulated production of NO and cGMP in the platelet cytosol was significantly higher in migraine patients with aura assessed in interictal periods than in control subjects, and this production was further increased during attacks (interictal period: NO ANOVA: p < 0.001, ictal period: p < 0.01; cGMP: interictal period p < 0.01, ictal period: p < 0.02). The increase in platelet NO and cGMP production was also evident, though to a lesser extent, in migraine patients without aura. The present research supports the hypothesis of an activation of the L-arginine/NO pathway in migraine patients, especially those with aura, and confirms the findings of a previous study of increased levels of L-arginine in platelets of migraine patients studied in headache free-periods, and decreased collagen aggregation in whole blood.

Anti-oxidants inhibit the enhancement of the immune response caused by oil adjuvants.

Adjuvants are agents that can induce strong immunity to different antigens. They are thought to act mainly by stimulating macrophages, causing the release of cytokines, which in turn induce an inflammatory focus necessary for the adjuvant action. The authors found that catalase, ascorbic acid, N-acetylcysteine and glutathione are able to inhibit the enhancing effect of incomplete Freund adjuvant (IFA) and polyoxyethylated castor oil upon the humoral immune response to sheep red blood cells (SRBC). None of the anti-oxidants tested inhibited the basal immune response to the antigen. In addition, mice inoculated with different concentrations of hydrogen peroxide showed an enhanced response against SRBC, mimicking the effect observed with adjuvants. Delayed type hypersensitivity induced by SRBC in the presence of IFA was also inhibited by catalase. In conclusion, the report indicates that oxygen radicals are crucial molecules involved in the adjuvant effect observed in SRBC immunized mice.

Inhibition of normal natural killer cytotoxicity by sera from hemophilic patients.

In this study we searched for circulating antibodies or other serum factors that could account for the natural killer (NK) defect observed in hemophiliacs (He) infected with the human immunodeficiency virus (HIV). We analyzed the effect of negative or positive sera for HIV from He on normal NK activity. We showed that sera from He interfered with normal NK cytotoxicity. The inhibitory activity was higher in HIV+ sera and increased as the HIV disease progressed. HIV- sera also inhibited NK function, although to a lesser extent than HIV+, and it was probably due to isoimmunization through replacement treatment with plasma-derived concentrates. For each individual, no direct correlation was found between NK inhibition (NK-INH) of sera and the NK activity of He peripheral blood mononuclear cells (PBMC). Furthermore, He serum was poorly inhibitory on autologous PBMC. Preincubation of allogenic effector or target cells with He sera revealed that the inhibitory effect was the result of the reaction with these cells. A positive correlation was found by comparing NK-INH of whole He sera with the serum levels of circulating immune complexes. When the NK-INH assay was performed using the same concentration of DEAE-purified IgG from N, HIV- or HIV+, we found that HIV+ AIDS IgG was more inhibitory than the others.