Clelia M. Riera

Evidence of a role for galectin‐1 in acute inflammation

Galectin‐1 (Gal‐1), a member of a family of β‐galactoside‐binding proteins, has been suggested to play key roles in immunological and inflammatory processes. The present study deals with the concept of an in vivo role for Gal‐1 in acute inflammation by using the rat hind paw edema test. Local administration of Gal‐1 (0.5, 2, 4 and 8 μg / ml) inhibited acute inflammation induced by bee venom phospholipase A2 (PLA2) when it was injected 30 min before the enzyme or co‐injected together with PLA2. The anti‐inflammatory effect was prevented by a specific antibody, but independent of its carbohydrate‐binding properties. In contrast, Gal‐1 failed to inhibit histamine‐induced edema. Histopathological studies showed a clear reduction of the inflammatory process when Gal‐1 was injected before PLA2, evidenced by a diminished number of infiltrated polymorphonuclear neutrophils and scarce degranulated mast cells. The anti‐inflammatory effect was also assessed in vitro, showing that Gal‐1 treatment reduced prostaglandin E2 secretion and arachidonic acid release from stimulated peritoneal macrophages. Results presented here provide the first evidence for a role of Gal‐1 in acute inflammation and suggest that the anti‐inflammatory effect involves the inhibition of both soluble and cellular mediators of the inflammatory response.

Chitosan induces different L-arginine metabolic pathways in resting and inflammatory macrophages.

Chitosan is a linear polymer of N-acetyl-D-glucosamine and deacetylated glucosamine widely used as a wound-healing accelerator in clinical and veterinary medicine. Chitosan enhances the functions of inflammatory cells such as macrophages (Mphi), inducing the production of cytokines as well as the expression of activation markers, Fc receptors and mannose receptor. In this work we studied the effects of chitosan on the arginine metabolic pathways of both resident and inflammatory (proteose-peptone elicited) rat Mphi. Our results show that low molecular weight (LMW) chitosan activated moderately both the inducible nitric oxide synthase (iNOS) and arginase pathways in resident Mphi. In inflammatory Mphi treated with chitosan instead, the arginase activity was strongly enhanced. Supernatants of chitosan-stimulated Mphi enhanced the proliferation of the rat cell line C6. These findings suggest that the healing activity of chitosan could rely on the enhanced arginase activity observed in a wound-associated inflammatory milieu.

Regulated expression of a 16-kd galectin-like protein in activated rat macrophages.

We investigated the presence of a galectin‐like protein in rat mononuclear cells using a polyclonal antibody raised against a soluble lactose‐binding lectin purified from adult chicken liver that immunoreacted strongly with a broad protein band of about 16 kd in Western blot assays. Immunochemical studies revealed a constitutive expression of this protein in mononuclear cells mainly in the macrophage (Mφ) population. Subcellular localization was assessed by Western blot assays of the cytosolic and membrane fractions of different cell populations studied: (1) spleen mononuclear cells, (2) T cell‐enriched, (3) B cell‐ and Mφ‐enriched populations, and (4) peritoneal cells, processed in the presence of lactose. In broad agreement with immunocytochemical studies of nonpermeabilized and permeabilized cells, Western blot assays suggest that this protein is localized mainly in the cytoplasmic compartment but also associated with the cell surface. By flow cytometric analyses we detected about a 14% of ED1 double‐positive cells corresponding to Mφs that constitutively express this galectin‐like protein associated with their cell surface. The cytosolic fraction obtained from the Mφ‐enriched cell population showed hemagglutinating activity specifically inhibited by β‐galactoside–related sugars. Moreover, this galectin‐like protein was retained in a lactosyl‐Sepharose matrix and specifically eluted with lactose. In this work, evidence is also provided to show that different stimuli are able to modulate the expression of the galectin‐like protein. Expression was upregulated in inflammatory and activated Mφs, revealing a significant increase in phorbol ester– and formylmethionine oligopeptide–treated cells. Both stimuli involving protein kinase C activation pathway have been able not only to up‐regulate the total expression of this protein but also to modulate its subcellular localization.

Autoimmune Prostatitis: State of the Art

The prostate is one of the main male sex accessory glands and the target of many pathological conditions affecting men of all ages. Pathological conditions of the prostate gland range from infections, chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) of a still unknown aetiology to benign hyperplasia and cancer. CP/CPPS is one of the most prevalent diseases in the urologic clinic and affects men younger than 50 years old. A significant advance in the understanding of CP/CPPS was made when an autoimmune response against prostate antigens was revealed in a considerable number of patients. During the last 30 years, extensive work has been done regarding the development and characterization of different rodent models of experimental autoimmune prostatitis (EAP). It has been demonstrated that tolerance to prostate antigens can be disrupted in some strains of rats and mice and cellular and humoral responses to prostate antigens are elicited. A Th1 pattern has been described and the cellular response seems to be the major pathogenic mechanism involved. Immune cells infiltrate the gland and induce prostate lesions. The genetic background and hormonal imbalance are factors that could contribute to the onset of the disease in susceptible young males. Moreover, spontaneous autoimmune prostatitis could also occur with advanced age in susceptible strains. In this review, we summarize the current knowledge regarding rodent models of EAP and the immunological alterations present in CP/CPPS patients. We also discuss the reliability of these experimental approaches as genuine tools for the study of human disease.

Prostatein (or rat prostatic steroid binding protein) is a major autoantigen in experimental autoimmune prostatitis

Experimental autoimmune prostatitis (EAP) is a disease that could be considered an experimental model of human non‐bacterial prostatitis. In this experimental model, male rats are intradermally immunized with a saline extract of male sex accessory glands (RAG) in an adequate adjuvant. The prostatitis observed in the immunized animals develops as a consequence of the immune response against RAG antigens, and the histological lesion is strikingly similar to the pattern of prostatic inflammation observed in the human disease. In this study, we purified one of the prostatic autoantigens recognized by the autoantibodies in our model. Amino acid sequence analysis identified the purified protein as prostatein or rat prostatic steroid binding protein, a member of the uteroglobin superfamily. Prostatein was recognized not only by the humoral autoimmune response, but also by the cellular autoimmune response. Certainly, the DTH response and lymph node cell proliferative assays against prostatein in immunized animals yielded positive results. Prostatein is not only the target of the autoimmune response in animals immunized with the whole extract, but also an inducing antigen of the disease. Purified prostatein, when incorporated to an adequate adjuvant, elicited cellular and humoral autoimmune response and lesion in the prostate gland. The identification of one of the target antigens in autoimmune prostatitis has provided a further refinement and characterization of our model, which could serve for a better understanding of the aetiology, pathogenesis and pathophysiology of non‐bacterial prostatitis.

Granulocyte-macrophage colony-stimulating factor protects dendritic cells from liposome-encapsulated dichloromethylene diphosphonate-induced apoptosis through a Bcl-2-mediated pathway.

Liposome‐encapsulated dichloromethylene diphosphonate (L‐MDP) has been used for depleting cells of the monocyte‐macrophage lineage. We have undertaken this study to investigate whether dendritic cells are susceptible to this liposome‐encapsulated compound. Dendritic cells were cultured in the presence of L‐MDP and further processed for apoptosis detection. The highly characteristic DNA cleavage into oligonucleosome‐sized fragments, incorporation of biotinylated dUTP into DNA strand breaks and the typical ultrastructural features of apoptosis were evident in dendritic cells exposed to the drug. More importantly, we demonstrated that granulocyte‐macrophage colony‐stimulating factor protects dendritic cells not only from apoptosis induced by the exogenous compound but also from spontaneous apoptosis. Western blot analysis revealed that this protection was tightly correlated with the activation of a Bcl‐2‐mediated pathway. Regulation of the apoptotic threshold of dendritic cells will be advantageous for the generation of new insights in immunotherapy.

Modulation of the inflammatory response by corticotropin-releasing factor.

Peptides of the corticotropin-releasing factor (CRF) family have been shown to have either pro- or anti-inflammatory activities. CRF (10-30 micrograms/kg) administered subcutaneously or intravenously could inhibit edema and dye leakage in the rat paw produced by several injuries. These findings are opposed to some results suggesting a predominantly pro-inflammatory effect of CRF mainly in arthritic processes. The purpose of this work was to identify in vivo and in vitro the conditions for the pro- or anti-inflammatory actions of CRF in order to clarify its physiological and pharmacological function. Using the rat paw edema test we observed that only the highest doses of CRF employed (5 micrograms) induced a moderate and sustained swelling. Pre-treatment with low doses of CRF (0.5-5 ng) was able to inhibit the edema induced by Naja naja phospholipase A2, carrageenin or histamine. Higher doses (50 ng-5 micrograms) had no anti-inflammatory activity. When co-inhibited with Naja naja phospholipase A2 or histamine the peptide did not modify the swelling at doses up to 500 ng, showing at 5 micrograms an additive edema with Naja naja phospholipase A2. In vitro, CRF did not modify the release of histamine but slightly increased the release of arachidonic acid to the medium. Our findings show a clear dose dependence on the local effects of CRF in inflammatory responses. These results suggest that the mechanisms of the two dose-related phenomena may be distinct.

Galectins: a key intersection between glycobiology and immunology

Galectins are a family of evolutionarily conserved animal lectins, widely distributed from lower invertebrates to mammals. They share sequence and structure similarities in the carbohydrate recognition domain and specificity for polylactosamine-enriched glycoconjugates. In the last few years significant experimental data have been accumulated concerning their participation in different biological processes requiring carbohydrate recognition such as cell adhesion, cell growth regulation, inflammation, immunomodulation, apoptosis and metastasis. In the present review we will discuss some exciting questions and advances in galectin research, highlighting the significance of these proteins in immunological processes and their implications in biomedical research, disease diagnosis and clinical intervention. Designing novel therapeutic strategies based on carbohydrate recognition will provide answers for the treatment of autoimmune disorders, inflammatory processes, allergic reactions and tumor spreading.

Suppression of experimental autoimmune encephalomyelitis (EAE) by intraperitoneal administration of soluble myelin antigens in Wistar rats

Intraperitoneal (i.p.) treatment of Wistar rats with bovine myelin (BM) or myelin basic protein (MBP) previously to immunization with BM-CFA showed a diminished incidence and severity of experimental autoimmune encephalomyelitis (EAE) (2/13 and 0/7, respectively) when compared with rats immunized with BM-CFA (11/17) or i.p. treated with ovalbumin (2/4). Concomitantly, animals treated with BM or MBP exhibited a marked reduction of proliferative response to MBP which was highly positive when spleen mononuclear cells from nontreated and ovalbumin treated animals were assayed. Rats that were treated with MBP before immunization produce IgA, IgM, total IgG and subclasses of IgG, IgG2a, IgG2b, IgG2c specific for MBP in similar levels than those observed in nontreated immunized animals. However, a higher incidence and level of IgG1 was observed in MBP treated rats, meanwhile rats i.p. treated with total BM showed a highly reduced humoral response. The herein presented results show that i.p. treatment with low amounts of soluble forms of myelin antigens markedly reduced the clinical symptoms of the disease, the histological alterations, the cellular proliferative response to MBP, and produced changes in the autoimmune humoral response.

Identification of rat prostatic steroid binding protein (PSBP) as an immunosuppressive factor.

Prostatic steroid binding protein (PSBP) is the major protein produced ( approximately 20% of the total cytosolic protein) and secreted into the seminal fluid by the rat ventral prostate but its physiological function has not been elucidated yet. Since PSBP is secreted into the seminal fluid (which is itself a potent immunosuppressor) and has strong homology with uteroglobin (which possess an important anti-inflammatory function) our aim was to determine what effect, if any, PSBP would have on the immune system. With that purpose in mind we performed mononuclear cell cultures in the presence or absence of purified PSBP and analysed the effect of this protein on different functional parameters. PSBP inhibits the mitogen-induced proliferation of normal rat spleen mononuclear cells (MNC) specifically and in a dose-dependent manner. It reduces the production of IL-2 and the expression of its receptor (analysed by flow cytometry) which are important events for lymphocyte proliferation. Also, PSBP was able to inhibit OVA-specific proliferation of lymph node cells from previously primed animals. The immunosuppressive effect of PSBP is not due to an inherent toxic effect to the cells, since the cell viability was kept intact at the different times of culture studied. We also analysed the effect of rat PSBP on mitogen-induced proliferation of mouse spleen and human blood MNC. The proliferation was strongly abolished in a dose-dependent and non-species specific fashion. Moreover, PSBP strongly inhibits the human mixed lymphocyte reaction. Taken together, the present data support evidence for a new type of function for PSBP. We report that PSBP is a potent immunosuppressor factor and we describe its effect on the immune function in vitro. Here, we discuss the possible implications of these findings in the protection of sperm from immunologic damage in the feminine reproductive tract.