Carolina Rubel

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.

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.

Development of DNA Vaccines against Hemolytic-Uremic Syndrome in a Murine Model

ABSTRACT Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2ΔA). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.

Endogenous glucocorticoids attenuate Shiga toxin-2-induced toxicity in a mouse model of haemolytic uraemic syndrome.

The concept that during an immune challenge the release of glucocorticoids (GC) provides feedback inhibition on evolving immune responses has been drawn primarily from studies of autoimmune and/or inflammatory processes in animal models. The epidemic form of haemolytic uraemic syndrome (HUS) occurs secondary to infection with Gram‐negative bacteria that produce Shiga toxin (Stx). Although Stx binding to the specific receptors present on renal tissue is the primary pathogenic mechanism, inflammatory or immune interactions are necessary for the development of the complete form of HUS. The aim of this study was to investigate the influence of endogenous GC on Stx‐toxicity in a mouse model. Stx2 was injected into GC‐deprived mice and survival rate, renal damage and serum urea levels were evaluated. Plasma corticosterone and cytosolic GC receptor (GR) concentration were also determined at multiple intervals post‐Stx2 treatment. Higher sensitivity to Stx2 was observed in mice lacking endogenous GC, evidenced by an increase in mortality rates, circulating urea levels and renal histological damage. Moreover, Stx2 injection was associated with a transient but significant rise in corticosterone secretion. Interestingly, 24 h after Stx inoculation significant increases in total GR were detected in circulating neutrophils. These results indicate that interactions between the neuroendocrine and immune systems can modulate the level of damage significantly during a bacterial infection.

Phenotype markers and function of neutrophils in children with hemolytic uremic syndrome

Abstract The hemolytic uremic syndrome (HUS) is the most-common cause of acute renal failure in children. Several researchers have reported the presence of neutrophil (PMN) activating cytokines, such as interleukin-8 and tumor necrosis factor-α, in the sera of HUS patients. Moreover, PMN-derived products, such as elastase, were increased. These observations have lead to the hypothesis that activated PMN could act as mediators of endothelial damage. The objective of this investigation was to directly evaluate the activation status of peripheral PMN from children with HUS. For this purpose, 12 children with typical HUS were bled during the acute period, before dialysis and/or transfusion, and 8 of them were also bled after 1 month follow-up. Additionally, blood samples from healthy control children admitted for routine surgical procedures, chronic uremic children, and neutrophilic children with acute infections not related to HUS were collected and processed in an identical manner. The function and membrane activation markers of PMN from these groups were evaluated.We found that during the acute period of HUS, PMN had reduced expression of FcγRIII (CD16) and CD11b, were degranulated, and exhibited an impaired antibody-dependent cellular cytotoxicity. These parameters returned to normal after clinical recuperation. We conclude that PMN activation in HUS patients is a very early and transient event, and upon hospitalization before dialysis PMN show a phenotype and functional pattern of partial deactivation.

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.