Histopathologic aspects of experimental cysticercosis and in situ cytokines profile in C57BL/6 mice / Aspectos histopatológicos da cisticercose experimental e perfil de citocinas in situ em camundongos C57BL/6

ABSTRACT Neurocysticercosis (NCC) is one of the parasitic infections that most affects the central nervous system. The knowledge regarding its immunopathogenesis and pathophysiology needs broadening. Taenia crassiceps cysticerci are used as the NCC experimental model. The aim of this work was to describe the general pathological processes and the in situ cytokine profile in C57BL/6 mice inoculated intracranially with viable T. crassiceps cysticerci. The histopathology analysis showed cysticerci in the extraparenchymal and intraventricular region, mononuclear inflammatory infiltration surrounding the parasite, microgliosis and meningitis. The analysis of the in situ immune profiles showed a predominance of the Th2 response. The IL-4 and IL-10 dosages were significantly increased in the infected group. The decrease in the INF-gamma dosage reflects the immunomodulation from the cysticerci. In conclusion, a T. crassiceps NCC infection in C57BL/6 mice triggers an inflammatory response, a predominance of Th2 type in situ profile, with mononuclear inflammatory cell infiltration, meningitis and microgliosis.

RESUMO Neurocisticercose (NCC) é uma das doenças parasitárias que mais afeta o sistema nervoso central. É necessário aprofundar o conhecimento em relação à sua imunopatogênese e patofisiologia. Os cisticercos de Taenia crassiceps são utilizados como modelo experimental para estudos da NCC. O objetivo deste trabalho foi descrever os processos patológicos gerais e o perfil de citocinas in situ em camundongos C57BL/6 inoculados via intracerebral com cisticercos viáveis de T. crassiceps. A análise histopatológica demonstrou cisticercos nas regiões extra-parenquimatosa e intraventricular, infiltrado inflamatório de células mononucleares ao redor do parasita, microgliose e meningite. A análise in situ do perfil de citocinas mostrou uma predominância da resposta Th2. As dosagens de IL-4 e IL-10 foram significativamente maiores no grupo infectado. Conclui-se que a NCC por T. crassiceps em camundongos C57BL/6 induz uma resposta inflamatória com predominância in situ de citocinas do perfil Th2, com infiltrado inflamatório de células mononucleares, meningite e microgliose.

Prolonged expression of MHC class I - peptide expression in bone marrow derived retrovirus transfected matured dendritic cells by continuous centrifugation in the presence of IL-4.

Background & objectives: Dendritic cells (DCs) are potent antigen presenting cells which proceed from immature to a mature stage during their differentiation. There are several methods of obtaining long lasting mature antigen expressing DCs and different methods show different levels of antigen expressions. We investigated bone marrow derived DCs for the degree of maturation and genetically engineered antigen presentation in the presence of interleukin-4 (IL-4) as a maturity enhancer. Methods: DCs and transfected retrovirus were cultured together in the presence of granulocyte-macrophage colony stimulating factor (GMCSF)-IL4, GMCSF +IL4, lipopolysaccharide (LPS). B 7.1, B7.2 and CD11c were measured by the degree of immune fluorescence using enhanced green fluorescent protein (EGFP) shuttled retrovirus transfected antigen. Degree of MHC class I molecule with antigen presentation of antigen was also evaluated by fluorescence activated cell sorting. The antigen presenting capacity of transfected DCs was investigated. Bone marrow DCs were generated in the presence of GMCSF and IL-4 in vitro. Dividing bone marrow cells were infected with EGFP shuttled retrovirus expressing SSP2 by prolonged centrifugation for three consecutive days from day 5, 6 and 7 and continued to culture in the presence of GMSCF and IL-4 until day 8. Results: IL-4 as a cytokine increased the maturation of retrovirus transfected DCs by high expression of B 7-1 and B 7-2. Also, IL-4 induced DC enhanced by the prolonged centrifugation and it was shown by increased antigen presentation of these dendric cells as antigen presenting cell (APC). Cytolytic effects were significantly higher in cytotoxic T cell response (CTLs) mixed with transfected DCs than CTLs mixed with pulsed DCs. Interpretation & conclusions: There was an enhanced antigen presentation by prolonged expression of antigen loaded MHC class I receptors in DCs in the presence of IL-4 by prolonged centrifugation.

IL-4 Independent Nuclear Translocalization of STAT6 in HeLa Cells by Entry of Toxoplasma gondii

Toxoplasma gondii provokes rapid and sustained nuclear translocation of the signal transducer and activator of transcription 6 (STAT6) in HeLa cells. We observed activation of STAT6 as early as 2 hr after infection with T. gondii by the nuclear translocation of fluorescence expressed from exogenously transfected pDsRed2-STAT6 plasmid and by the detection of phosphotyrosine-STAT6 in Western blot. STAT6 activation occurred only by infection with live tachyzoites but not by co-culture with killed tachyzoites or soluble T. gondii extracts. STAT6 phosphorylation was inhibited by small interfering RNA of STAT6 (siSTAT6). In view of the fact that STAT6 is a central mediator of IL-4 induced gene expression, activation of STAT6 by T. gondii infection resembles that infected host cells has been stimulated by IL-4 treatment. STAT1 was affected to increase the transcription and expression by the treatment of siSTAT6. STAT6 activation was not affected by any excess SOCS's whereas that with IL-4 was inhibited by SOCS-1 and SOCS-3. T. gondii infection induced Eotaxin-3 gene expression which was reduced by IFN-gamma. These results demonstrate that T. gondii exploits host STAT6 to take away various harmful reactions by IFN-gamma. This shows, for the first time, IL-4-like action by T. gondii infection modulates microbicidal action by IFN-gamma in infected cells.

The presence of CD8+ invariant NKT cells in mice

Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8+ iNKT cells in mice raised the question of whether CD8+ iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8+ iNKT cells. To address this question, we analyzed iNKT cell-specific TCR Valpha14+ transgenic mice, where the Valpha14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8+ iNKT cells which respond to the NKT cell-specific glycolipid ligand alpha-galactosylceramide. Unlike conventional iNKT cells, CD8+ iNKT cells produce predominantly IFN-gamma but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8+ iNKT cells in wild type mice. Our results suggest that CD8+ NKT cells do exist in mice, although their population size is quite small. Their Th1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.

Additive Effect of Diesel Exhaust Particulates and Ozone on Airway Hyperresponsiveness and Inflammation in a Mouse Model of Asthma

Allergic airway diseases are related to exposure to atmospheric pollutants, which have been suggested to be one factor in the increasing prevalence of asthma. Little is known about the effect of ozone and diesel exhaust particulates (DEP) on the development or aggravation of asthma. We have used a mouse asthma model to determine the effect of ozone and DEP on airway hyperresponsiveness and inflammation. Methacholine enhanced pause (P(enh)) was measured. Levels of IL-4 and IFN-gamma were quantified in bronchoalveolar lavage fluids by enzyme immunoassays. The OVA-sensitized-challenged and ozone and DEP exposure group had higher P(enh) than the OVA-sensitized-challenged group and the OVA-sensitized-challenged and DEP exposure group, and the OVA-sensitized-challenged and ozone exposure group. Levels of IFN-gamma were decreased in the OVA-sensitized-challenged and DEP exposure group and the OVA-sensitized-challenged and ozone and DEP exposure group compared to the OVA-sensitized-challenged and ozone exposure group. Levels of IL-4 were increased in the OVA-sensitized-challenged and ozone exposure group and the OVA-sensitized-challenged and DEP exposure group, and the OVA-sensitized-challenged and ozone and DEP exposure group compared to OVA-sensitized-challenged group. Co-exposure of ozone and DEP has additive effect on airway hyperresponsiveness by modulation of IL-4 and IFN-gamma suggesting that DEP amplify Th2 immune response.

Somatropin promotes dermatophagoides farinae-specific IgE generation independently of IL-4 and IL-10.

Interleukin (IL)-10 accelerates the IgE production of anti-CD40- and IL-4-stimulated PBMC by enhancing the IL-6 production of T lymphocytes or antigen-primed spleen cells, in addition to its role as a regulator of the inflammatory responses. To further investigate the mechanisms enhancing IgE synthesis, we determined the effect of somatropin as well as IL-10 on the secretion of Dermatophagoides farinae (Df)-specific IgE by K7 cells, which originate from an EBV-immortalized cell line. Df-pulsed autologous T cells, as well as the supernatants of these cultures, increased the synthesis of Df-specific IgE. Antigen-specific IgE was also enhanced when K7 cells were treated with anti-CD40 antibody and with both IL-4 and IL-10, or with IL-4 and IL-10 without anti-CD40 antibody. The treatment of K7 cells with anti-CD40 antibody and IL-4, or anti-CD40 antibody and IL-10 did not increase IgE production. The Df-specific IgE activity of the supernatants of K7 cells treated with somatropin alone was increased significantly although somatropin did not show any additive effect on the IgE production of anti-CD40 antibody-treated cells. The results indicate that IL-10, a Th2-type cytokine, directly affects the mature B cells that produce IgE, and that the secretion of IgE is increased by treatment with IL-10 in cells that are stimulated with anti-CD40 and IL-4 at the level of the EBV-immortalized cell line, which has already switched to IgE production. Somatropin similarly stimulates activated mature B cells to enhance their production of antigen-specific IgE without class switching, independently of IL-4 and IL-10.

Citoquinas: la internet biológica ¿futura terapia en el asma bronquial? / Cytokines: the biological internet, the future therapy of bronchial asthma?

Las citoquinas son polipéptidos producidos por variadas células nucleadas que actúan como intercomunicadores celulares. Participan en funciones de defensa y reparación del adño del organismo y restablecimiento de la homeostasis. En los últimos años y gracias al desarrollo de la biología molecular, ha sido posible identificar y producir en el laboratorio numerosas citoquinas disponibles en el tratamiento de diversas enfermedades. En el asma bronquial existe un desbalance de algunas citoquinas con predominio de la producción de las interleuquinas (ILs) dependientes de los linfocitos tipo Th-2, como IL-4 e IL-5, las cuales inducen la producción de IgE y la eosinofilia, respectivamente. Actualmente están en marcha estudios clínicos tendientes a bloquear o impedir la acción de la IL-4 e IL-5 mediante anticuerpos monoclonales anti-IL o mediante la acción inhibidora sobre estas citoquinas que ejerce la IL-12. En esta revisión bibliográfica se analiza el estado actual de esta nueva futura terapia del asma

Immune responses of IL-5 transgenic mice to parasites and aeroallergens

Eosinophils have long been thought to be effectors of immunity to helminth but have also been implicated in the pathogenesis of asthma. Patterns of cytokine production in the host may influence the pathogenesis of these diseases by regulating the activities of eosinophils and other components of the immune response. Mice which constitutively over-express IL-5 have profound and life-long eosinophilia in a restricted number of tissues. Although eosinophils from IL-5 transgenics are funtionally competent for a number of parameters considered to be important in inflammation, untreated animals are overtly normal and free of disease. In addition, the responses of these animals when exposed to aeroallergens and helminth present a number of apparent paradoxes. Eosinophil accumulation in tissue adjacent to major airways is rapid and extensive in transgenics exposed to the aeroallergen, but even after treatment with antigen over many months these mice show no evidence of respiratory distress or pathology. Helminth-infected IL-5 transgenics and their non-transgenic littermates develop similar inflammatory responses at mucosal sites and are comparable for a number of T cell and antibody responses, but they differ considerably in their ability to clear some parasite species. The life-cycle of Nippostrongylus brasilensis is significantly inhibited in IL-5 transgenics, but that of Toxocara canis is not. Our results suggest that eosinophilia and/or over-expression of IL-5 may actually impair host resistance to Schistosoma mansoni and Trichinella spiralis. The pathogenesis of diseases in which eosinophils are involved may therefore be more complex than previously thought.

Interleukin-4 and interleukin-5 as targets for inhibition of eosinophilic inflammation and allergic airways hyperreactivity

Clinical and experimental investigations suggest that allergen-specific CD4+ T-cells, IgE and the cytokines IL-4 and IL-5 play central roles in initiating and sustaining an asthmatic response by regulating the recruitment and/or activation of airways mast cells and eosinophils. IL-5 plays a unique role in eosinophil development and activation and has been strongly implicated in the aetiology of asthma. The present paper summarizes our recent investigations on the role of these cytokines using cytokines knockout mice and a mouse aeroallergen model. Investigations in IL-5-/- mice indicate that this cytokines is critical for regulating aeroallergen-induced eosinophilia, the onset of lung damage and airways hyperreactivity during allergic airways inflammation. While IL-4 and allergen-specific IgE play important roles in the regulation of allergic disease, recent investigations in IL-4-/- mice suggest that allergic airways inflammation can occur via pathways which operate independently of these molecules. Activation of these IL-4 independent pathways are also intimately associated with CDA+ T-cells, IL-5 signal transduction and eosinophilic inflammation. Such IL-5 regulated pathways may also play a substantive role in the aetiology of asthma. Thus, evidence is now emerging that allergic airways disease is regulated by humoral and cell mediated pocesses. The central role of IL-5 in both components of allergic disease highlights the requirements for highly specific therapeutic agents which inhibit the production or action of this cytokines.

Producción de interferón-gamma e interleuquina 4 por células mononucleadas de sangre periférica infectadas con virus respiratorio sincicial y adenovirus / Interferon-gamma and interleukin-4 production by respiratory synctytial virus and adenovirus infected peripheral blood mononuclear cells

Las infecciones respiratorias por virus respiratorio sincicial (VRS) y adenovirus (Ad) son la principal causa de morbimortalidad entre las infecciones respiratorias agudas bajas (RAB) de la infancia. Ambos virus pueden dejas secuelas, al VRS se le ha atribuido desencadenar obstrucción bronquial persistente y al Ad, especialmente el Ad7h, bronquectasias, fibrosis y daño pulmonar crónico. Los mecanismos por los que estos virus pueden producir estas secuelas no se conocen, pero hay evidencias que sugieren que ésta sea causada por un mecanismo inmunológico, dependiente del tipo de virus y de la respuesta del huésped. El objetivo de este trabajo fue determinar el tipo de respuesta inmune frente a la infección por VRS y Ad mediante la cuantificación de interferón-gamma (IFN-gamma) e interleuquina-4 (IL-4), citoquinas marcadas de respuesta inmune celular y humoral respectivamente. Las ILs fueron cuantificadas en el sobrenadante de cultivo de células mononucleadas de sangre periférica infectadas o no infectadas in vitro con VRS, Ad3, Ad7h y control con mitógeno (PHA) y de células mononucleadas de lactantes con infección natural por VRS y grupo control estimuladas o no con mitógenos (PHA y PWN). Los lactantes con IRAB por VRS presentaron una disminución significativa en la producción de IFN-gamma e IL-4 por células mononucleadas no estimuladas y estimuladas con PHA. Esta disminución fue mayor para el IFN-gamma que para la IL-4, por lo que la relación IFN eta/IL-4 fue menor en estos lactantes. La producción de IL-4 pero no la de IFN-eta de los lactantes infectados con VRS que tenían antecedentes de atopia (p< 0,02). Las células mononucleadas de niños sanos infectadas in vitro con Ad estimulan la producción de IFN-gamma pero no la de IL-4 y con Ad7h, responden con una producción de IFN-gamma en este modelo in vitro. Estos resultados sugieren que la respuesta del sistema inmune frente a la infección viral dependerá del tipo de virus infectante y de la variabilidad genética del individuo. Esto demuestra la importancia de estudiar la respuesta inmune de cada virus en los diferentes individuos ya que el resultado de protección o daño dependerá de la interrelación huésped-virus

Anti-IL-4 antibody inhibits antigen specific IgE response but fails to prevent chicken gamma globulin-induced active systemic anaphylaxis: evidence for the involvement of IgG antibodies

It has recently been reported that interleukin-4 (IL-4) is required for the production of IgE, and anti-IL-4 monoclonal antibody (mAb) inhibits in vivo IgE responses. These suggest that blocking of IL-4 activity may be useful for the prevention or treatment of immediate hypersensitivity disorders. In this study we investigated whether anti-IL-4 has a regulatory role in chicken-gamma globulin (CGG)-induced active systemic anaphylaxis. Multiple injections of anti-IL-4 (up to 40 mg/mouse) failed to protect the mice from fatal anaphylaxis. Anti-IL-4 strongly suppressed CGG-specific IgE response (>90%) without any suppressive effect on CGG-specific IgG (IgG1, IgG2a, IgG2b, and IgG3) responses. Because these data suggest the possibility that fatal anaphylaxis could be induced by IgG antibodies, we examined the possibility using anti-CGG polyclonal and the subclasses of IgG monoclonal antibodies. Passive sensitization of mice with polyclonal antibodies elicited severe and fatal anaphylactic shock; about 50% of the mice died. The activity of antibodies was not diminished by heat treatment (56 degrees C, 2h), suggesting that the anaphylaxis was not mediated by IgE. Shock was also elicited by each subclass of IgG mAb; of these, IgG1 was the most effective. Combination of the IgG subclasses elicited more exaggerated shock; about 30% of mice died. These data indicate that IgG antibodies are themselves sufficient to induce systemic anaphylaxis. Therefore, the failure of anti-IL-4 to prevent active anaphylaxis is probably due to the inability of anti-IL-4 to suppress the production of IgG antibodies.