Martín A. Isturiz

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.

Fibrinogen Promotes Neutrophil Activation and Delays Apoptosis

The acute phase of the inflammatory response involves an increase in the concentrations of different plasma proteins that include fibrinogen (Fbg) and multiple proinflammatory mediators. In parallel, neutrophil activation is thought to play a crucial role in several inflammatory conditions, and it has been recently demonstrated that Fbg specifically binds to the α-subunit of CD11b/CD18 on neutrophil surface. Although several reports have shown that CD11b engagement modulates neutrophil responses, the effect of human Fbg (hFbg), one of CD11b physiologic ligands, has not been exhaustively investigated. We have now shown that incubation of purified neutrophils with hFbg induces a transient and rapid elevation of free intracellular Ca2+. This early intracellular signal is accompanied by changes in the expression of neutrophil activation markers, including enhancement of CD11b and CD66b, and down-regulation of FcγRIII. In addition, we have evaluated the effect of hFbg on two functional events related to expression and resolution of inflammation: cytotoxic capacity and rate of neutrophil apoptosis. We have found that activation of neutrophils by hFbg resulted in both enhancement of phagocytosis and Ab-dependent cellular cytotoxicity, and delay of apoptosis. We conclude that during inflammatory processes, soluble Fbg could influence neutrophil responses, increasing and prolonging their functional capacity.

Pretreatment of mice with lipopolysaccharide (LPS) or IL-1beta exerts dose-dependent opposite effects on Shiga toxin-2 lethality.

Haemolytic uraemic syndrome (HUS) has been closely associated with infection with a group of Shiga toxin‐producing enterohaemorrhagic Eschericchia coli in young children. Shiga toxins (Stx) have been implicated as pathogenic agents of HUS by binding to the surface receptor of endothelial cells. LPS is a central product of the Gram‐negative bacteria and several reports have documented that both LPS and Stx are important for disease development. In this study the reciprocal interactions between LPS and Stx2 are analysed in a mouse model. The results demonstrated that LPS was able to reduce or enhance Stx2 toxicity, depending on the dose and the timing of the injection. The involvement of the main early cytokines induced by LPS, tumour necrosis factor alpha (TNF‐α) and IL‐1β, in those LPS opposite effects on Stx2 toxicity was evaluated. Stx2 toxicity was enhanced by in vivo injection of murine TNF‐α and low doses of murine IL‐1β. However, at higher doses of IL‐1β which induced corticosteroid increase in serum, Stx2 lethality was decreased. Considering that dexamethasone and IL‐1β reproduce the LPS protective effects, it is suggested that endogenous corticosteroids secondary to the inflammatory response induced by LPS, mediate the protection against Stx2. It can be concluded that the fine equilibrium between proinflammatory and anti‐inflammatory activities strongly influences Stx2 toxicity.

Fibrinogen-CD11b/CD18 interaction activates the NF-κB pathway and delays apoptosis in human neutrophils

The regulation of neutrophil half‐life by members of the coagulation cascade is critical for the resolution of the inflammatory response. We have demonstrated that soluble fibrinogen (sFbg) delays human neutrophil (PMN) apoptosis through a mechanism that involves CD11b interactions, and phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated kinase 1/2 (ERK1/2).Since NF‐κB is a key element in the regulation of apoptotic mechanisms in several immune cells, we investigated whether NF‐κB is involved in the control of PMN survival by sFbg. We showthat sFbg triggers inhibitor protein κB (IκB‐α) degradation and NF‐κB activation. Furthermore, pharmacological inhibition of NF‐κB abrogates sFbg effects on apoptosis. In addition, specific inhibition of MAPK ERK1/2 significantly reduces NF‐κB translocation by sFbg, suggesting a relationship between ERK1/2 and NF‐κB activation. Similar results are obtained when granulocytic‐differentiated HL‐60 cells are treated with sFbg, making this model highly attractive for integrin‐induced gene expression studies. It can be concluded that NF‐κB participates in the prevention of apoptosis induced by sFbg with the participation of MAPK ERK1/2. These results shed light on the molecular mechanisms that control human granulocyte apoptosis, and suggest that NF‐κB regulation may be of benefit for the resolution of the inflammatory response.

Soluble fibrinogen modulates neutrophil functionality through the activation of an extracellular signal-regulated kinase-dependent pathway.

The integrin family not only mediates the recruitment of polymorphonuclear leukocytes (PMN) to sites of inflammation but also regulates several effector functions by binding to specific ligands. We have recently demonstrated that soluble fibrinogen (sFbg) is able to trigger an activating signal in PMN through an integrin-dependent mechanism. This activation results in degranulation, phagocytosis enhancement, and apoptosis delay. The aim of the present work was to further elucidate the molecular events that follow sFbg interaction with CD11b in human PMN, and the participation of this signaling pathway in the regulation of neutrophil functionality. We demonstrate that sFbg triggers a cascade of intracellular signals that lead to focal adhesion kinase and extracellular signal-regulated kinase 1/2 tyrosine phosphorylation. The activation of this mitogen-activated protein kinase pathway plays a central role in the sFbg modulation of secondary granule degranulation, Ab-dependent phagocytosis, and apoptosis. However, fibrinogen-induced secretory vesicle degranulation occurs independently of the signaling transduction pathways investigated herein. In the context of an inflammatory process, the intracellular signal pathway activated by sFbg may be an early event influencing the functionality of PMN.

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.

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.

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‐α) and IL‐1β 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.

Impaired neutrophils in children with the typical form of hemolytic uremic syndrome

Experimental and clinical evidence suggest that activated neutrophils (PMN) could contribute to endothelial damage in Hemolytic Uremic Syndrome (D+HUS). Additionally, while PMN-activating cytokines and PMN-derived products have been found in D+HUS sera, we have demonstrated phenotypic alterations in D+HUS PMN compatible with a deactivation state. Here, we investigated whether D+HUS PMN were actually hyporesponsive, and explored some of the mechanisms probably involved in their derangement. Twenty-two D+HUS children were bled in the acute period, and blood samples from healthy, acute uremic and neutrophilic children were obtained as controls. We evaluated degranulation markers in response to cytokines, intracellular granule content, and reactive oxygen species (ROS) generation in circulating D+HUS and control PMN. The influence of D+HUS-derived plasma and the direct effects of Stx in vitro were evaluated on healthy donors’ PMN. We found that D+HUS PMN presented reduced degranulatory capacity in response to cytokines and intracellular granule content, and decreased ROS generation. D+HUS plasma or Stx did not affect the phenotype and function of healthy donors’ PMN. These results suggest that upon hospitalization D+HUS PMN are functionally impaired and show features of previous degranulation, indicating a preceding process of activation with release of ROS and proteases involved in endothelial damage.