María C. Pistoresi-Palencia

Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice

We have previously demonstrated that subcutaneously administered ovalbumin (OVA) plus synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG‐ODN) as adjuvant stimulate cellular and humoral immunity and promote T helper cell type 1 differentiation in aged mice. The present study assessed the ability of CpG‐ODN to induce an OVA‐specific immune response after oral immunization in young (3‐month‐old) and aged (18‐month‐old) BALB/c mice. Oral OVA/CpG‐ODN immunization induces a similar OVA‐specific T cell‐proliferative response (in mucosal and systemic tissues), immunoglobulin G (IgG) in plasma, and IgA in intestinal washes in both groups of ages. The OVA‐specific humoral immune response observed in aged mice was similar to the one observed in young mice, peaking at day 7 after the last oral immunization and was present over 40 days after the last oral immunization. The pattern of cytokines released in culture supernatants in both groups of mice was similar, with specific interferon‐γ secretion in the absence of interleukin‐5 responses. These results provide evidence that orally administered OVA/CpG‐ODN induces a young‐like, specific, immune response against OVA in aged mice, showing that CpG‐ODN might be used as a mucosal adjuvant during aging.

Neutrophils Exhibit Differential Requirements for Homing Molecules in Their Lymphatic and Blood Trafficking into Draining Lymph Nodes

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

Involvement of accessory cells in the Trypanosoma cruzi-induced inhibition of the polyclonal response of T lymphocytes.

Infection with Trypanosoma cruzi is characterized by hyporesponsiveness of the immune system during the acute phase of infection. To better understand the immunological mechanisms affected by T. cruzi, we studied if a reduced T cell proliferative response could originate from an inability of T cells to proliferate or a functional deficiency at the level of accessory cells (AC). The inhibitory effect exerted by T. cruzi was during the induction phase of the lymphoproliferative response, suggesting the participation of AC in the hyporesponse. Then we further investigated the potential of the parasite to interfere with accessory cell‐dependent and ‐independent pathways of human T cell proliferation. Peripheral blood mononuclear cells and peripheral blood lymphocytes from healthy individuals, enriched for T cells, were analysed with regard to their proliferative capacity using: phytohaemagglutinin, immobilized anti‐CD3 monoclonal antibody (MoAb) and MoAb to the CD28 antigen, anti‐CD3 MoAb and recombinant IL‐2 and anti‐CD3 MoAb plus phorbol myristate acetate in the presence of parasites. Significant suppression of the proliferative response was caused by the parasite only when AC were present. The parasite markedly reduced the surface expression of HLA‐DR and CD11b antigens, key molecules in PHA‐induced proliferation. Addition of indomethacin to the culture failed to reverse the inhibitory effect of the parasites, suggesting that prostaglandin E2 was not involved. These data suggest that AC in contact with T. cruzi become incompetent as antigen presenting cell because they are unable to induce a normal proliferative response in T lymphocytes.

Interferon-γ priming is involved in the activation of arginase by oligodeoxinucleotides containing CpG motifs in murine macrophages

Recognition of microbial products by macrophages (Mφ) stimulates an inflammatory response and plays a critical role in directing the host immune response against infection. In the present work, we showed for the first time that synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG) are able to stimulate, in the presence of interferon‐γ (IFN‐γ), both arginase and inducible nitric oxide synthase (iNOS) in murine Mφ. Unexpectedly, IFN‐γ, a cytokine believed to be an inhibitor of arginase activity, intervened in the activation of this enzyme. A significant increase in arginase activity was observed upon a short pre‐incubation (1 hr) with IFN‐γ and subsequent CpG stimulation. Therefore, a very interesting observation of this study was that the CpG‐mediated arginase activity is dependent on IFN‐γ priming. The increase in arginase activity as a result of stimulation with CpG plus IFN‐γwas correlated with augmented expression of the arginase II isoform. The use of pharmacological specific inhibitors revealed that arginase activity was dependent on p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated protein kinase (ERK), but independent of c‐Jun N‐terminal kinase (JNK) activation. This report reveals a singular effect of the combination of CpG and IFN‐γ, one of the mayor cytokines produced in response to CpG administration in vivo.

Age-associated changes in lymphoid and antigen-presenting cell functions in mice immunized with Trypanosoma cruzi antigens.

The purpose of these studies was to analyze the role of different immune cell populations in the immune response against Trypanosoma cruzi antigens in aged mice. Mice of different ages (3 and 12 months old) were immunized i.d. with S-105 plus Bordetella pertussis as adjuvant and we compared the activities of the lymph node cells taken from 3- and 12-month-old donor animals to transfer DTH or antibody production to 3-month-old recipients. This study revealed that adherent and non-adherent immune lymph node cells of aged donor animals did not transfer response against the foreign antigen (S-105) whereas 3-month-old non-adherent lymph node cells transferred a DTH response as well as helped the specific antibody production. When total lymph node cells from 3- and 12-month-old mice were mixed, we observed an inhibition of S-105 transferred response indicating a suppressive effect of aged cells on the 3-month-old mice cells. Furthermore, we analyzed the participation of antigen-presenting cells (APC) in the immune response changes related to the previously described aged mice. Peritoneal cavities cells (PC), pulsed in vivo with S-105, obtained from 3- and 12-month-old mice were transferred to normal recipients and a DTH response to S-105 was studied. We observed that the DTH response was lower in the recipients of aged PC with respect to recipients of young PC. The results suggest that APC from aged mice are involved in controlling the cellular immune response to S-105. Age-related changes in immune T cell and APC are discussed in the context of these observations.

Humoral autoimmune response to rat male accessory glands. Effect of castration on the humoral response.

The ability to induce antibodies to rat male accessory glands in male and female rats was demonstrated, but a higher response with wider specificity was revealed in female animals. In order to investigate whether this different response may be influenced by sexual hormones we castrated male and female rats at 4 or 30 days after birth. After that we studied the course and specificity of their humoral response to male accessory glands comparing them with that of sham-operated, sex-matched, littermate controls. Orchidectomy in male or oophorectomy in female rats changed neither the course nor specificity of humoral immune response. We conclude, therefore, that hormonal factors do not play any important role in the experimental model under study.

Diminished percentage of antigen bearing cells in the lymph nodes of immune aged rats.

We have demonstrated previously that during experimental autoimmune prostatitis (EAP), aged rats show a diminished humoral autoimmune response. In the present paper we have studied the transport of the autoantigen from the site of injection toward lymphatic organs in rats of different ages with or without EAP. We used as autoantigen prostatic components (rat accessory glands (RAG)) conjugated with fluorescein isothiocyanate (FITC). Studies of flow cytometry, fluorescent microscopy and confocal microscopy show no differences in the percentage of RAG-FITC positive cells or in the localization of the cells in the popliteal lymph nodes of not-immunized young and aged rats. On the other hand, in 18-month-old rats immunized with either RAG or Ovalbumin there were lower levels of specific IgG antibodies and fewer antigen containing cells in the draining lymph nodes than those of 3- or 12-month-old rats. In all groups fluorescent cells were MHC class II positive and some were IgM positive. Our results demonstrate that in immunized 18-month-old rats there is a diminished percentage of cells bearing the antigen in the draining lymph nodes after antigen injection in the skin, related to the levels of specific antibodies able to form antigen-antibody complexes in the periphery.

Age-related alterations in inflammatory response during experimental autoimmune prostatitis

Experimental autoimmune prostatitis (EAP) is an experimental model of autoimmune disease, developed in Wistar rats against prostatic components. The 12-and 18-month-old rats with EAP show a higher cellular autoimmune response and lower humoral autoimmune response compared to 3-month-old rats. The analysis of NO(.) and O(2)(-) production by peritoneal exudate cells (PECs) resulted in a higher NO(.) and O(2)(-) production in EAP rats at all ages, compared to control animals. PECs from 12- and 18-month-old rats produced more NO(.) and less O(2)(-) than PECs from 3-month-old rats. However, lipopolysacharide (LPS) did not stimulate PECs from aged rats for NO(.) production as much as in 3-month-old rats and thus, turning out in a lower index of LPS-stimulation of PECs from aged rats, compared to 3-month-old rats. Furthermore, the mast cells number in prostates of EAP rats, especially the number of degranulated cells, was higher than in control animals, but no significant differences were found between 3- and 12-month-old control rats. In conclusion, these results show that aging affects differentially the inflammation mediators during EAP.

Decreased expression of intercellular adhesion molecule-1 (ICAM-1) and urokinase-type plasminogen activator receptor (uPAR) is associated with tumor cell spreading in vivo

The development of an effective antitumor immune response to control tumor growth is influenced by the tumor cell itself and/or by the tumor microenvironment. Tumor invasion and tumor cell spreading require a finely tuned regulation of the formation and loosening of adhesive contacts of tumor cells with the extracellular matrix (ECM). In our laboratory, a rat tumor cell line derived from a spontaneous rat sarcoma revealed, by flow cytometry, a high frequency of intercellular adhesion molecule-1 (ICAM-1, 70.1 ± 8.7%) and urokinase-type plaminogen activator receptor (uPAR, 51.2 ± 5.2%) positive cells, while a weak expression of MHC class II (IA, 2.2±0.2% and IE, 17.4±3.7%) and B7 (12.1±2.2%) antigens was detected. In our tumor experimental model, after implantation of tumor cells, visible tumor masses were present at days 5–7 with a relatively fast tumor growth until day 15 (progressive phase) followed by a suppression of the tumor growth (regressive phase). Here we present data that correlates a significant decrease in the frequency of ICAM-1 and uPAR expressing tumor cells with the appearance of tumor cells in sites distant from that of the primary tumor. In addition we describe the development of a cellular immune response which controls the tumor progression and is associated with an increase in the expression of major histocompatibility complex (MHC) class II IA antigen during tumor development. The histological examination at tumor progressive and regressive time points revealed the relevant presence of polymorphonuclear neutrophils (PMNs) evidencing colliquative necrosis in tumor growth areas. Taken together, these results support the idea that the balance between adhesive interactions, proteolytic activity and tumorigenicity may lead to a tumor invasive phenotype.

Changes in the development of experimental autoimmune prostatitis (EAP) by castration in aged rats.

During Experimental Autoimmune Prostatitis (EAP), 12-month-old rats show a higher cellular autoimmune response and lower humoral autoimmune response against prostatic components than 3-month-old rats subjected to the same antigen stimulus. We analyzed if thymus recovery by orchidectomy could affect the development of EAP in 12-month-old rats. Thirty days after gonadectomy, 12-month-old rats showed an increment in the thymic mass and in the thymocytes absolute number, with percentages of the four main cell subpopulations (defined by CD4-CD8 molecules expression) similar to the 3-month-old rats. The DTH response of castrated 12-month-old with EAP were diminished in comparison with sham-castrated 12-month-old rats with EAP, resembling the values observed in 3-month-old rats with EAP. The prostates of castrated 12-month-old rats with EAP did not show inflammatory mononuclear cell infiltration, as did control 3- and 12-month-old rats with EAP. Castration seems to modulate negatively EAP in 12-month-old rats, possibly through the regeneration of thymus after testosterone deprivation.