Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis.

Galectin-1 (GAL-1), a member of a family of conserved beta-galactoside-binding proteins, has been shown to induce in vitro apoptosis of activated T cells and immature thymocytes. We assessed the therapeutic effects and mechanisms of action of delivery of GAL-1 in a collagen-induced arthritis model. A single injection of syngeneic DBA/1 fibroblasts engineered to secrete GAL-1 at the day of disease onset was able to abrogate clinical and histopathological manifestations of arthritis. This effect was reproduced by daily administration of recombinant GAL-1. GAL-1 treatment resulted in reduction in anticollagen immunoglobulin (Ig)G levels. The cytokine profile in draining lymph node cells and the anticollagen IgG isotypes in mice sera at the end of the treatment clearly showed inhibition of the proinflammatory response and skewing towards a type 2-polarized immune reaction. Lymph node cells from mice engaged in the gene therapy protocol increased their susceptibility to antigen-induced apoptosis. Moreover, GAL-1-expressing fibroblasts and recombinant GAL-1 revealed a specific dose-dependent inhibitory effect in vitro in antigen-dependent interleukin 2 production to an A(q)-restricted, collagen type 2-specific T cell hybridoma clone. Thus, a correlation between the apoptotic properties of GAL-1 in vitro and its immunomodulatory properties in vivo supports its therapeutic potential in the treatment of T helper cell type 1-mediated autoimmune disorders.

Molecular mechanisms implicated in galectin-1-induced apoptosis: activation of the AP-1 transcription factor and downregulation of Bcl-2.

Galectins are emerging as a new class of bioactive molecules with specific immunomodulatory properties. Galectin-1 (Gal-1), a member of this family, has been shown to induce apoptosis of mature T cells and immature thymocytes. To gain insight into the intracellular signals transduced by Gal-1 upon binding to mature T cells, we investigated whether this protein triggered activation of the dimeric AP-1 transcription factor. A marked increase in the binding of nuclear extracts to synthetic oligonucleotides containing the AP-1 consensus sequence, could be detected by an electrophoretic mobility shift assay, when T cells were cultured for 30 min in the presence of Gal-1. This DNA-binding activity was preceded by a rapid increase in the levels of c-Jun mRNA, as determined by Northern blot analysis. Requirement of AP-1 for Gal-1-induced apoptosis was confirmed by the dose-dependent reduction on the level of DNA fragmentation observed when cells were pre-treated with curcumin (an inhibitor of AP-1 activation) before exposure to Gal-1. Finally, evidence is also provided by Western blot analysis, showing that Gal-1 inhibits Concanavalin A (Con A) induction of Bcl-2 protein. Results presented in this study provide the first experimental evidence regarding AP-1 and Bcl-2 as targets of the signal transduction pathway triggered by Gal-1 and set the basis for a more in depth understanding of the molecular mechanisms of T-cell death regulation.

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 (M phi) 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 M phi-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 macrophages that constitutively express this galectin-like protein associated with their cell surface. The cytosolic fraction obtained from the M phi-enriched cell population showed hemagglutinating activity specifically inhibited by beta-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 macrophages, 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.

A simple two-step method for purification of secretory IgA from human colostrum.

This paper describes an extremely simple method for the purification of secretory IgA in two steps from a colostrum pool. The first step of this technique utilizes the agar transparency effect produced by high concentrations of secretory IgA contained in colostrum pool when it is subjected to electrophoresis in this support. The secretory IgA is obtained by extraction and elution of the transparent zone adjacent to the well. In the second step, in order to obtain the maximum degree of purity, the eluate is subjected to precipitation with ammonium sulfate (1.33 M) or chromatographed through Sephadex G-200 gel. The simplicity of the method and the purity of the protein obtained make this procedure suitable for purification of macromolecular substances in concentrated state.

A simple method for detection of IgG rheumatoid factor.

A simple method for detection of IgG rheumatoid factor (RF) in sera and joint fluid is described. The technique is based on the action of 0.1 M 2-mercaptoethanol mixed directly with the material. After 2 h of incubation determinations of human anti-IgG were performed by latex agglutination test. Comparison with data obtained by using the conventional method, sequential 24 h treatment with 0.1 M 2-mercaptoethanol and 0.01 M iodoacetamide, shows similar results for both methods. A correlation was observed between the presence of IgGRF in synovial fluid and a severe clinical course with invalidating forms in patients suffering rheumatoid arthritis.

DNA antibodies detected by microhemagglutination test in the diagnosis of systemic lupus erythematosus.

A method of tanned erythrocyte agglutination of wide applicability is described for the study of DNA antibodies. The practical aspects of the method have been explored in estimation of antibodies to DNA in human cases of systemic lupus erythematosus, discoid lupus erythematosus, rheumatoid arthritis, other collagenoses and not related diseases. This method was found to be of high sensitivity and specificity, detecting DNA antibodies only in sera of systemic lupus erythematosus. Results were compared with those obtained by immunofluorescence test using rat liver hepatocytes as source of nuclei.

Peritoneal antigen-presenting cells pulsed in vivo with myelin basic protein induce the suppression of experimental autoimmune encephalomyelitis (EAE) in Wistar rats.

Suppression of Experimental Autoimmune Encephalomyelitis (EAE) can be achieved by i.p. administration of soluble myelin basic protein (MBP) in adult Wistar rats before the immunization. In the present work, we analyze the role of peritoneal antigen-presenting cells (APC) in the induction of tolerance to EAE. Peritoneal cells (PC) pulsed in vivo with MBP were obtained from rats that had been intraperitoneally injected 2 h previously with soluble MBP (MBP-PC) and then inoculated in recipient rats before the induction of EAE. Our findings show that the i.p. treatment of the animals with MBP-PC before the immunization was able to diminish the incidence and severity of the disease, reduce the histological alterations, abrogate the proliferative response against MBP and change the pattern of the humoral response to MBP. Moreover, when spleen mononuclear cells (MNC) from tolerant animals were cultured together with spleen MNC from sick animals, a dose-dependent inhibition of the proliferative response was observed, arguing for the presence of a regulatory cell population in the tolerant animals. It is also demonstrated that the MBP-PC are activated and their capability of inducing suppression of EAE is highly associated with the enhanced expression of MHC class II IA molecule. Our results show that peritoneal cells pulsed in vivo with MBP are able to induce tolerance and suggest that the up-regulation of MHC class II on MBP-PC is a necessary event for tolerance induction in our model.

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.

Adjuvant effect of liposomes in the autoimmune response to rat male accessory glands.

Wistar rats submitted to 3 intravenous (i.v.) or intraperitoneal (i.p.) immunizations with saline extract of rat male accessory glands (RAG) associated to liposomes developed a delayed type hypersensitivity (DTH) response to RAG after the first immunization, a remission state after the second immunization and a specific DTH response after the third injection. In a further study we transferred spleen mononuclear (SpM) cells from i.p. immunized rats taken 10 days after the second immunization (DTH negative) to normal or immunized recipients 24 h before or 10 days after the first immunization with RAG liposomes, respectively. The DTH response was reduced only in recipients previously immunized. Besides, it was possible to show that the transfer of SpM cells present when the response increased after the third injection in i.p. immunized donors reduced the suppression observed after the second injection. Rat accessory gland biopsies taken 10 days after the last immunization showed in nearly all cases mast cells, plasma cells and eosinophils with scarce lymphoid elements and increased acinar desquamation. This kind of infiltration had characteristics of cutaneous basophil hypersensitivity.

Evaluation of the immunosuppression in an experimental model of autoimmunity: suppressor activity of spleen cells from cyclophosphamide-treated rats.

Rats immunized with chemically modified rat male accessory glands (MRAG) and injected 3 days later with cyclophosphamide (CY) were unable to develop humoral and cellular immune response to the autoantigen of MRAG. The present report demonstrates that the spleen mononuclear (SpM) cells transference from rats injected with CY 3 days after the antigen to normal male or female syngeneic animals before immunization with MRAG did not suppress the immune response to this antigen, whereas the transference of SpM cells from suppressed animals to animals previously immunized, depressed the delayed type hypersensitivity (DTH) response against MRAG (suppression of the expression) only in male rats. Similar results were obtained by transference of purified T cells. SpM cells did not suppress an established humoral immune response induced in male or female rats. The results suggest that non-adherent cells present in the spleen of male suppressed rats might be one of the responsible mechanisms for suppression of the efferent phase of the cellular autoimmune response to MRAG.

Antigen-induced inhibition of the autoimmune response to rat male accessory glands: bone marrow dependence of the enhancement of IA+ but not IE+ antigen-presenting cells.

IE+ peritoneal cells (PC), involved in the induction of suppression of autoimmune response to rat male accessory glands (RAG), are obtained from rats 2 h after i.p. injection of a purified fraction (FI) of RAG (FI-PC2h). In contrast, IA+ PC, involved in the induction of autoimmune response to RAG, are obtained from rats 24 h after FI of RAG injection (FI-PC24h). The present report analyzes the effect of irradiation or irradiation/bone marrow reconstitution on the induction of both populations of PC. Peritoneal cell donor rats were irradiated in a telegamma therapeutic Cs137. Twenty hours later half of them were i.v. reconstituted with 40 x 10(7) bone marrow cells. Six days later rats were i.p. injected with 200 micrograms of FI of RAG and 10(7) resident PC. The PC were harvested 2 h or 24 h later. The ability of resident PC to yield IE+ FI-PC2h involved in the induction of suppression is not impaired by irradiation, but the ability of resident PC to yield IA+ FI-PC24h involved in the induction of a positive response is impaired by irradiation and restored by bone marrow reconstitution of irradiated rats. Culture of normal PC with FI of RAG for 2 h or 24 h shows a selective increase in IE+ cells able to induce suppression to RAG.(ABSTRACT TRUNCATED AT 250 WORDS)

Adoptive transfer of suppression of the autoimmune response to rat male accessory glands: characterization of the suppressor cells.

We have examined the mechanism of suppression of autoimmunity to rat male accessory glands (RAG) by T suppressor cells. This suppression was accomplished by transfer to syngeneic rats of spleen mononuclear (SpM) cells from rats rendered unresponsive by pretreatment with low doses of a purified fraction of RAG (containing the autoantigen). The experiments demonstrated that the suppressor cells that act on the inducer phase of the suppression are cyclophosphamide (Cy) sensitive and that they can be positively selected on antigen-coated plates. On the other hand, the inducer phase T suppressor cells present on spleens coming from antigen-pretreated rats did not suppress the autoimmune response in normal recipients that had been irradiated (850 rad 137Cs) just prior to receiving the cells or injected with Cy 14 days after transfer. The results indicate that the regulation of immune response to the autoantigen of RAG is complex and that it involves the interaction of many cell types.

Effects of thymus supernatants on the phenotype of different subsets of peritoneum antigen-presenting cells.

Peritoneal cells (PC) obtained 2 h subsequent to intraperitoneal (i.p.) injection of low doses (200 micrograms) of a purified fraction of rat male accessory glands (FI-RAG) are phenotypically and functionally different from those obtained 24 h after i.p. injection. In fast, PC obtained 2 h after FI-RAG injection are mainly IE+ and are involved in inducing specific suppression to RAG. In contrast, PC obtained 24 h after FI-RAG injection are mainly IA+ and capable of inducing specific response to RAG. For their induction, IA+ PC require cells within or recently derived from bone marrow. In order to analyze the mechanisms involved in IA+ bone marrow-dependent cell generation in the peritoneum, we studied the distribution of FI-RAG following i.p. injection. It was established that FI-RAG is found mainly in the thymus 2 h after injection and remains there for at least 24 h. Subsequently, we analyzed, in 4 groups of rats, the influence of thymic culture supernatants on the phenotype of cells appearing in the peritoneal cavity 2 h after FI-RAG injection. An increase in IA+ PC was observed 2 h after i.p. injection of FI-RAG in animals that had received either supernatants from normal thymic cells cultured with FI-RAG or those from thymic cells taken from animals injected with FI-RAG 2 h prior to being killed. Supernatants of thymic cells from animals injected with FI-RAG 24 h prior to being killed or from normal thymic cells do not increase the percentage of IA+ PC.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-induced inhibition of autoimmune response to rat male accessory glands. Role of macrophages in the induction of suppressor cells.

The present paper describes a mechanism responsible for the induction of inducer-phase suppressor cells effective to suppress the autoimmune response to rat male accessory glands (RAG). In fact, we reported here that marked suppression of delayed type hypersensitivity (DTH) reaction and humoral response to chemically modified rat male accessory glands (MRAG) can be obtained when previously to be immunized with MRAG in complete Freund's adjuvant (CFA) syngeneic rats were pretreated with peritoneal cells (PC) coupled with a purified fraction of RAG (containing the autoantigen). The involvement of MRAG-specific inducer-phase suppressor cells was demonstrated by adoptive transfer experiments of spleen mononuclear cells from unresponsive donors to normal syngeneic rats 24 h prior to immunization of the recipients with MRAG-CFA. The PC used to treat the animals show a large proportion of non-specific-esterase positive, Ox-41 bearing macrophage-like cells. Moreover, the antigen-coupled PC able to trigger the suppressor cells showed the presence of the autoantigen of RAG on their surface. The role of the antigen presenting cells in the induction of MRAG-specific inducer-phase suppressor cells is discussed.

Humoral response against myelin antigens in two strains of rats with different susceptibility to experimental allergic encephalomyelitis (EAE).

Lewis (Lw) rats are susceptible and Wistar (Wr) rats are usually resistant to the induction of experimental allergic encephalomyelitis (EAE). In this study we analyze the humoral response to myelin antigens, providing evidence for different B cell response to myelin basic protein (MBP) and other myelin proteins between these two strains of rats with different susceptibility to EAE. In fact, IgG anti-MBP titers in Wr rats were markedly higher than in Lw ones. Moreover, an inverse relationship between the amount of antigen injected to induced EAE and the level of anti-MBP antibodies was observed in Wr rats, while IgG anti-MBP varied in a positive dose-depending manner in sera from Lw rats. Also, sera from Wr rats analyzed by immunoblotting showed a strong reactivity with MBP and other myelin proteins, but sera from Lw rats reacted only with MBP. Evaluation of IgA and IgM against MBP in Wr rats showed again higher titers of these isotypes when compared with the titers observed in Lw rats. The distribution of IgG subclasses in sera from both strains indicated that Wr produced low titers of specific IgG1, while Lw rats did not produce specific IgG1. However, Wr rats showed high levels of IgG2a, IgG2b and IgG2c subclasses while lesser titers of these isotypes were observed in Lw animals. These findings indicate that both strains have the capacity to develop antibodies against portions of the MBP molecule, but antibody production is greater in the resistant Wistar rats suggesting a B cell activation in these animals, that could be related to their lower susceptibility.

Potentiation of autoimmune response in rats infected with Toxoplasma gondii. Inhibition of suppressor system and impairment of thymic cellular populations.

In the present work we studied the influence that an infection with Toxoplasma gondii in thymus proximity produces on the suppression of autoimmune response to autoantigen of rat male accessory glands (RAG). The suppression was achieved injecting syngeneic animals with low doses of autoantigen of RAG previous to the immunization with chemically modified rat male accessory glands (MRAG). Rats were infected in thymus proximity with 3 x 10(3) trophozoites of T. gondii before or after to be suppressed. Controls were rats only infected or only suppressed. The delayed hypersensitivity response against MRAG, (DTH test), was significantly potentiated in rats only infected and in the animals suppressed before the infection (p less than 0.001). The suppression was not inhibited in the animals suppressed after infection. The suppression of humoral response against MRAG studied by ELISA and passive hemagglutination test was prevented in rats infected before as well as in rats infected after the induction of suppression (p less than 0.001). Decrease of CD4+ CD8+ and Ox18+ (class I MHC antigen) and increase of CD4+ CD8- and CD4- CD8- thymocytes was observed in the rats where the DTH response was potentiated. These results indicate that the infection with T. gondii in thymus proximity was able to inhibit the suppression of response to autoantigen of RAG producing selective impairment in thymic suppressor influence.

Rheumatoid arthritis and its association with HLA-DR antigens. II. Antibodies to native connective tissue antigens detected by enzyme linked immunosorbent assay.

The distribution of frequencies of HLA-DR alloantigens in HLA-DR4 negative subjects was determined in patients with Rheumatoid arthritis (RA) and normal individuals. An increased incidence of HLA-DR1 alloantigen in DR4 negative RA patients (45.9%) compared with DR4 negative healthy controls (23.6%) was found. The difference became significant when the incidence of DR1 was compared between patients with severe disease stages (III-IV) (75%) in contrast to 32% of incidence in patients of the milder stages (I-II) (p less than 0.05). Using Enzyme Linked Immunosorbent Assay we have determined the incidence of serum antibodies to native bovine type I and type II collagens and proteoglycans in patients with RA. Presence of serum antibodies to native type I collagen was detected in 59% of patients with RA, 60% of sera exhibited reactivity to type II collagen and 12% had antibodies to proteoglycans. There was no correlation between the presence of antibodies to type I and II collagens and disease stages, however, the incidence of serum antibodies to proteoglycans was increased in severe disease stages. On the other hand, the presence of high levels of antibodies to type I collagen was associated to HLA-DR1 antigen, (p less than 0.05).

Antigen induced inhibition of autoimmune response to rat male accessory glands: role of thymocytes on the efferent phase of the suppression.

In the present study, we report that Cy-sensitive, MRAG-adherent spleen mononuclear (SpM) inductor-phase T suppressor (Ts) cells obtained from rats pretreated with low doses of a purified fraction (FI) of rat male accessory gland antigens (RAG) are mainly OX19+ and W3/25+. Furthermore, thymocytes from rats pretreated with FI of RAG restore the suppression of the autoimmune response to RAG autoantigens in irradiated recipients of SpM inductor-phase Ts cells. In contrast, thymocytes from rats pretreated with rat heart saline extract (unrelated antigen) did not recuperate the suppression of the autoimmune response detected by macrophage migration inhibitory factor (MIF) and delayed-type hypersensitivity. The suppressor thymocytes did not directly exert their inhibitory effect because they were not effective to suppress the autoimmune response to RAG autoantigens when irradiated recipients did not receive SpM inductor-phase Ts cells. The effect of these thymocytes was found in PNA--but not in PNA+ thymic cell population. The perithymic injection of Toxoplasma gondii did block their suppressor activity. The present report clearly shows an active participation of thymus in the efferent phase of the suppressor circuit that controls the autoimmune response to MRAG. The implications of these findings are discussed.

Effect of gangliosides in the autoimmune response induced by liposome-associated antigens.

A model of autoimmunity to rat male accessory glands (RAG) was recently developed by intraperitoneal administration of three doses of native RAG associated with liposomes. In this work we analysed the effects of gangliosides in the cellular response to RAG when they were intraperitoneally administrated prior to the second dose of liposome-associated RAG. Results show that the ganglioside treatment could modify an established DTH response. Also, gangliosides markedly reduced the number of Ia antigen-positive peritoneal exudated cells (PEC). However, they modified neither the processing of liposomes through PEC nor their viability. Moreover, we obtained cellular response by transferring PEC from immunized donors into naive receptors.

Delayed hypersensitivity and lesions following isoimmunization with modified rat male accessory glands: kinetics of induction.

The kinetics of the cellular immune response to rat male accessory glands were studied in Wistar rats isoimmunized with modified rat male accessory glands extract and complete Freund's adjuvant at 0, 30 and 45 days. The animals were divided into seven groups, and each group was sacrificed weekly. One immunization was sufficient for the induction of 2-, 6- and 24-h footpad reactivity. The reaction increased until 21 days post-immunization. After the second injection the reaction decreased and was negative 12 days later. Migration inhibitory factor (MIF) activity monitored by a mixed-direct assay was demonstrated in rats from all groups except in the animals studied at day 42 in which macrophage migration was markedly stimulated. The absence of MIF activity correlated with a lack of delayed type hypersensitivity (DTH) response. The humoral response was studied and detected by passive hemagglutination in a few sera after the first immunization. A second injection was necessary to obtain a more frequent occurrence and higher titres of antibodies. Histological modifications in the target organs started to appear in the group of animals studied at 35 days and were characterized by a mononuclear infiltrate in the prostate, coagulating glands and seminal vesicles. In several cases there was also infiltration of polymorphonuclear cells. Specimens obtained at 35 days showed the most severe lesions.