Immune complex-FcgammaR interaction modulates monocyte/macrophage molecules involved in inflammation and immune response.

The interaction between receptors for the Fc portion of IgG (FcgammaRs) from monocytes/macrophages and immune complexes (IC) triggers regulatory and effector functions. Recently, we have demonstrated that IC exert a drastic inhibition of basal and IFN-gamma-induced expression of MHC class II on human monocytes. Taking into account that the regulation of MHC class II molecules is a crucial event in the immune response, in this report we extend our previous studies analysing the effect of STAT-1 phosphorylation in the down-regulatory process, the fate of the intracellular pool of MHC class II molecules and the effect of complement on MHC class II down-regulation induced by IC. We also studied the effect of IC on the expression of MHC class II (I-A(d)) in macrophages using a mouse model of chronic inflammation. We demonstrate that IC induce a depletion not only on surface expressed but also on intracellular MHC class II content and that IC-induced down-regulation of MHC class II is not mediated by the inhibition of STAT-1 phosphorylation. On the other hand, the effect of IC is not specific for the down-regulation of MHC class II, for it could be restricted to other molecules involved in inflammatory processes. Our experiments also show that the activation of the complement system could be a crucial step on the regulation of the effect of IC on MHC class II expression. In agreement with our in vitro experiments using human monocytes, IC treatment reduces the expression of MHC class II in a mouse model of chronic inflammation.

Immune complexes (IC) down-regulate the basal and interferon-gamma-induced expression of MHC class II on human monocytes.

The interaction of Fc receptors for IgG (FcgammaRs) on monocytes/macrophages with immune complexes (IC) triggers regulatory and effector functions. Previous studies have shown that FcgammaR-IC interactions inhibit the IFN-gamma-induced expression of MHC class II in murine macrophages. However, the mechanism(s) responsible for these effects have not been elucidated. In addition, whether this IC-dependent effect also occurs in human cells is not known. Taking into account the fact that IC and IFN-gamma are frequently found in infections and autoimmune disorders, together with the crucial role MHC class II molecules play in the regulation of immune response, we explored the effect and mechanism of IC-induced MHC class II down-regulation in human peripheral blood mononuclear cells (PBMC). This effect was studied either in the presence or absence of IFN-gamma. We demonstrate that IC exert a drastic inhibition of basal and IFN-gamma-induced expression of MHC class II on human monocytes. This effect was mediated through the interaction of IC with both FcgammaRI and FcgammaRII. Moreover, similar results were obtained using supernatants from IC-treated PBMC. The IC-induced down-regulation of MHC class II is abrogated by pepstatin and phosphoramidon, supporting the role of aspartic protease(s) and metalloprotease(s) in this process. In parallel with MHC class II expression, antigen presentation was markedly inhibited in the presence of IC.

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.

Depletion of liver and splenic macrophages reduces the lethality of Shiga toxin-2 in a mouse model.

The haemolytic uraemic syndrome (HUS) is a clinical syndrome consisting of haemolytic anaemia, thrombocytopenia, and acute renal insufficiency. HUS is the most frequent cause of acute renal failure in childhood. It has been previously suggested that the presence of Shiga toxin (Stx) is necessary but not sufficient for HUS development, and cytokines such as tumour necrosis factor-alpha (TNF-alpha) and IL-1beta appear to be necessary to develop the syndrome. Since the mononuclear phagocytic system (MPS) is the major source of these cytokines, macrophages might be one of the relevant targets for Stx action in the pathophysiology of HUS. In this study our objective was to examine the role of the hepatic and splenic macrophages in a mouse model of HUS induced by injection of Shiga toxin type-2 (Stx2) or Stx2 plus lipopolysaccharide (LPS). For this purpose, depletion of mice macrophages by liposome-encapsulated clodronate (lip-clod), followed by injection of STx2 or Stx2 plus LPS, was assayed. In this study we show that depletion of hepatic and splenic macrophages by clodronate treatment induces a survival of 50% in animals treated with Stx2 alone or in presence of LPS. This maximal effect was observed when lip-clod was injected 48-72 h before Stx2 injection. Biochemical and histological parameters show characteristics of the lesion produced by Stx2, discarding non-specific damage due to LPS or lip-clod. In addition, we determined that the toxic action of Stx2 is similar in BALB/c and N:NIH nude mice, indicating the T cell compartment is not involved in the Stx2 toxicity. Briefly, we demonstrate that macrophages play a central role in the pathophysiology of HUS, and that the systemic production of cytokines by liver and/or spleen is for Stx2 to manifest its full cytotoxic effect. In addition, the toxicity of Stx2 alone, or in presence of LPS, is independent of the T cell compartment.

Enhancement of immune complex clearance by TNF-alpha in a murine model.

Recently, we presented evidence that lipopolysaccharide (LPS) treatment of BALB/c mice induces an enhancement on mononuclear phagocytic system functions, leading to a more efficient clearance of immune complexes (IC). In the present study we analyzed the role of tumor necrosis factor alpha (TNF-alpha), one of the earliest mediators released after LPS injection, in the clearance of IC. Our results show that the enhancing effect of LPS on clearance can be partially reproduced by intravenous injection of sera from mice injected with LPS 1 h before. At this time point, the levels of TNF-alpha reach a maximal peak of 240 +/- 73 U50%/ml [TNF-alpha (+) serum]. However, sera obtained after 4 h of LPS injection, with a TNF-alpha activity of 3.5 U50%/ml [TNF-alpha (-) serum], did not exert any relevant effect on IC clearance. In addition, the effect of TNF-alpha (+) serum was completely blocked by preincubation with rabbit anti-TNF-alpha antibody. Moreover, the enhancement of IC clearance can be similarly induced by administering murine recombinant TNF-alpha. Furthermore, the LPS-insensitive C3H/HeJ mice, which do not secrete TNF-alpha in response to LPS, showed a normal IC clearance after LPS injection. Taken together, these results strongly suggest that the enhancement of IC clearance by LPS treatment could be mediated, at least in part, by TNF-alpha.

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.

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.

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.