Macarena Beigier-Bompadre

A Novel Function for Galectin-1 at the Crossroad of Innate and Adaptive Immunity: Galectin-1 Regulates Monocyte/Macrophage Physiology through a Nonapoptotic ERK-Dependent Pathway

Several environmental factors can differentially regulate monocyte and macrophage response patterns, resulting in the display of distinct functional phenotypes. Galectin-1, an endogenous lectin found at peripheral lymphoid organs and inflammatory sites, has shown immunoregulatory activity in vivo in experimental models of autoimmunity and cancer. Whereas compelling evidence has been accumulated regarding the effects of galectin-1 on T cell fate, limited information is available on how galectin-1 may impact other immune cell types. In the present study, we report a novel role for galectin-1 in the regulation of monocyte and macrophage physiology. Treatment with galectin-1 in vitro differentially regulates constitutive and inducible FcγRI expression on human monocytes and FcγRI-dependent phagocytosis. In addition, galectin-1 inhibits IFN-γ-induced MHC class II (MHC-II) expression and MHC-II-dependent Ag presentation in a dose-dependent manner. These regulatory effects were also evident in mouse macrophages recruited in response to inflammatory stimuli following treatment with recombinant galectin-1 and further confirmed in galectin-1-deficient mice. Investigation of the mechanisms involved in these functions showed that galectin-1 does not affect survival of human monocytes, but rather influences FcγRI- and MHC-II-dependent functions through active mechanisms involving modulation of an ERK1/2-dependent pathway. Our results provide evidence of a novel unrecognized role for galectin-1 in the control of monocyte/macrophage physiology with potential implications at the crossroad of innate and adaptive immunity.

Increased Susceptibility to Apoptosis of CD56dimCD16+ NK Cells Induces the Enrichment of IFN-γ-Producing CD56bright Cells in Tuberculous Pleurisy

Tuberculous pleuritis is a good model for the study of specific cells at the site of active Mycobacterium tuberculosis (Mtb) infection. We investigated the frequency and phenotype of NK cells in paired samples of peripheral blood and pleural fluid (PF) from patients with tuberculosis (TB) or parapneumonic infection. We demonstrated for the first time a reduction of NK cells in PF from TB with an enrichment in the CD56brightCD16− subset. In agreement, in PF NK cells we observed an increased expression of CD94, NKG2A, CD62L, and CCR7 molecules and lower expression of Bcl-2 and perforin. The activation markers CD69 and HLA-DR were also increased. The enrichment in the CD56bright subset was due to an increased susceptibility to apoptosis of CD56+CD16+ NK cells mediated by heat-labile and stable soluble factors present in tuberculous effusions and not in PF from other etiologies. Furthermore, in TB patients, Mtb-induced IFN-γ production by PF NK cells was not dependent on the presence of CD3+, CD19+, and CD14+ cells, suggesting a direct interaction of CD56bright cells with Mtb and/or the involvement of other accessory cells present at the site of Mtb infection.

Interleukin‐1β 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‐1β (IL‐1β) and tumour necrosis factor alpha (TNF‐α), 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‐1β were tolerant to LPS‐induced shock. However, TNF‐α was unable to induce an LPS refractory state. Given the fact that 100 ng of IL‐1β 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‐1β 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‐1β 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‐1β 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.

Immune Complexes Inhibit Differentiation, Maturation, and Function of Human Monocyte-Derived Dendritic Cells

The interaction between immune complexes (IC) and the receptors for the Fc portion of IgG (FcγRs) triggers regulatory and effector functions in the immune system. In this study, we investigated the effects of IC on differentiation, maturation, and functions of human monocyte-derived dendritic cells (DC). When IC were added on day 0, DC generated on day 6 (IC-DC) showed lower levels of CD1a and increased expression of CD14, MHC class II, and the macrophage marker CD68, as compared with normally differentiated DC. The use of specific blocking FcγR mAbs indicated that the effect of IC was exerted mainly through their interaction with FcγRI and to a lesser extend with FcγRII. Immature IC-DC also expressed higher levels of CD83, CD86, and CD40 and the expression of these maturation markers was not further regulated by LPS. The apparent lack of maturation following TLR stimulation was associated with a decreased production of IL-12, normal secretion of IL-10 and CCL22, and increased production of CXCL8 and CCL2. IC-DC displayed low endocytic activity and a reduced ability to induce allogeneic T cell proliferation both at basal and LPS-stimulated conditions. Altogether, these data reveal that IC strongly affect DC differentiation and maturation. Skewing of DC function from Ag presentation to a proinflammatory phenotype by IC resembles the state of activation observed in DC obtained from patients with chronic inflammatory autoimmune disorders, such as systemic lupus erythematosus disease and arthritis. Therefore, the altered maturation of DC induced by IC may be involved in the pathogenesis of autoimmune diseases.

Immune complex–FcγR interaction modulates monocyte/macrophage molecules involved in inflammation and immune response

The interaction between receptors for the Fc portion of IgG (FcγRs) 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‐γ‐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‐Ad) 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.

Tolerance to lipopolysaccharide (LPS) regulates the endotoxin effects on Shiga toxin-2 lethality.

It has been suggested that Shiga toxin (Stx) is necessary but not sufficient for hemolytic uremic syndrome (HUS) development, and pro-inflammatory stimuli such as lipopolysaccharide (LPS) from Gram negative bacteria are needed. Taking into account that LPS is present in the natural infection during HUS development, detoxification or regulation of LPS activity could be crucial to define the course of the disease. The objective of the present study was to investigate whether tolerance to LPS and/or antibodies to LPS, are able to modify the LPS-induced modulation of Stx type-2 (Stx2) lethality in a mouse model. Our results demonstrate that the high levels of IgG anti-LPS antibodies in immunized mice did not modify the dual effects of LPS (enhancement or protection) on Stx2 action. This could be attributed to the fact that antibodies do not recognize the active portion of LPS molecule (lipid A). However, the enhancement of Stx2 toxicity exerted by LPS was inhibited in tolerant mice. This effect could be ascribed to the inhibition of LPS-induced TNF-alpha and IL-1beta secretion in tolerant animals, two cytokines known to be involved in the overexpression of Stx receptors. The phenomenon of LPS-induced protection on Stx2 toxicity was also inhibited in tolerant animals, although the mechanism involved in this effect is not clear. This is the first description which shows the influence of endotoxin tolerance on the evolution of experimental HUS. However, like in Gram negative infections, further knowledge on tolerance mechanism is necessary in order to achieve a comprehensive view of this phenomenon.

Immune complexes (IC) down‐regulate the basal and interferon‐γ‐induced expression of MHC Class II on human monocytes

The interaction of Fc receptors for IgG (FcγRs) on monocytes/macrophages with immune complexes (IC) triggers regulatory and effector functions. Previous studies have shown that FcγR–IC interactions inhibit the IFN‐γ‐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‐γ 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‐γ. We demonstrate that IC exert a drastic inhibition of basal and IFN‐γ‐induced expression of MHC class II on human monocytes. This effect was mediated through the interaction of IC with both FcγRI and FcγRII. 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 formyl peptide N-formyl-methionyl-leucyl-phenylalanine downregulates the expression of FcgammaRs in interferon-gamma-activated monocytes/macrophages in vitro and in vivo.

N‐Formyl peptides are cleavage products of bacterial and mitochondrial proteins that have pro‐inflammatory activities and play an important role in antibacterial host defence. FcγRI is a receptor for the Fc portion of immunoglobulin G expressed in monocytes that mediates cytotoxicity and is upregulated by interferon‐γ (IFN‐γ) and interleukin‐10 (IL‐10). In this report, we demonstrate that N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) downregulates the expression of FcγRI in IFN‐γ‐treated monocytes, but not in IL‐10‐treated monocytes. We determine that supernatants obtained from monocytes treated with IFN‐γ and then exposed to FMLP induce the downregulation of FcγRI in naïve monocytes. This effect is abrogated by the protease inhibitors phenylmethylsulphonyl fluoride and phosphoramidon, which inhibit serine and metalloproteases, respectively. Supernatants from FMLP‐treated neutrophils also induce the downregulation of FcγRI, when added to naïve monocytes. Similar observations were obtained in vivo in a mouse model of chronic inflammation. In vivo, FMLP also downregulates the expression of FcγRs in IFN‐γ‐activated macrophages. Our results support the existence of a new mechanism through which FMLP could modulate the activity of monocytes/macrophages during bacterial infections.

N-Formyl-Methionyl-Leucyl-Phenylalanine Inhibits both Gamma Interferon- and Interleukin-10-Induced Expression of FcγRI on Human Monocytes

ABSTRACT Three different classes of receptors for the Fc portion of immunoglobulin G (FcγRs), FcγRI, FcγRII, and FcγRIII, have been identified on human leukocytes. One of them, FcγRI, is a high-affinity receptor capable of induction of functions that include phagocytosis, respiratory burst, antibody-dependent cell-mediated cytotoxicity (ADCC), and secretion of cytokines. This receptor is expressed on mononuclear phagocytes, and this expression is regulated by cytokines and hormones such as gamma interferon (IFN-γ), IFN-β, interleukin-10 (IL-10), and glucocorticoids. We have recently demonstrated that the chemotactic peptideN-formyl-methionyl-leucyl-phenylalanine (FMLP) is capable of inducing a time-dependent downregulation of both FcγRIIIB and FcγRII in human neutrophils, altering FcγR-dependent functions. Considering the biological relevance of the regulation of FcγRI, we investigated the effect of FMLP on the overexpression of FcγRI induced by both IFN-γ and IL-10 on human monocytes. We demonstrate that FMLP significantly abrogated IFN-γ- and IL-10-induced FcγRI expression, although its basal level of expression was not altered. However, other IFN-γ-mediated effects such as the overexpression of the major histocompatibility complex class II antigens and the enhancement of lipopolysaccharide-induced secretion of tumor necrosis factor alpha were not affected by FMLP treatment. The formyl peptide completely inhibited the IFN-γ- and IL-10-induced enhancement of ADCC and phagocytosis carried out by adherent cells. The inhibitory effect of FMLP on FcγRI upregulation could exert an important regulatory effect during the evolution of bacterial infections.

Monocytes and neutrophils from tuberculosis patients are insensitive to anti-inflammatory effects triggered by the prototypic formyl peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP)

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis where formyl peptides, which are cleavage products of bacterial and mitochondrial proteins, are present. In this study, we demonstrated that interferon gamma (IFN)‐γ and interleukin (IL)‐10 induced the overexpression of the receptor for the Fc portion of IgG I (FcγRI) in monocytes from tuberculosis (TB) patients, showing that these cells respond to IFN‐γ and IL‐10 signals. We also demonstrated that lower doses of IL‐10 render monocytes from TB patients less responsive to higher doses of the cytokine. Although the prototypic formyl peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) is a well‐known proinflammatory agonist, we have demonstrated previously that preincubation of monocytes with FMLP inhibited the up‐regulation of FcγRI induced by IFN‐γ or IL‐10. This effect was not observed in monocytes from TB patientes. FMLP also induced the down‐regulation of the expression of FcγRI in monocytes that had been activated already with IFN‐γ. However, this effect of FMLP was not observed in monocytes from TB patients and supernatants from monocytes obtained from these patients were incapable of inducing the down‐regulation of FcγRI. In contrast to normal donors, supernatants from FMLP‐treated neutrophils from TB patients did not modify the basal level of expression of FcγRI in monocytes from normal donors. In conclusion, in this study we demonstrated the existence of two novel mechanisms that may contribute to the pathological effects generated by M. tuberculosis: the enhancement of FcγRI in response to IFN‐γ and IL‐10, and the unresponsiveness to the anti‐inflammatory effects induced by formyl peptides.