Cytokines play an important regulatory role during immune responses against pathogens. The outcome of an induced cytokine pattern is determined by many factors. It strongly depends on the nature of the pathogen and the host’s ability to control the quality and strength of cytokine signals. In pulmonary infection with Cryptococcus neoformans T helper (Th) 1 and Th17 cell subsets and their associated cytokines confer protection, whereas a Th2-biased response with production of interleukin (IL) -4 confers susceptibility. Since inappropriate Th responses often lead to death in immunosuppressed human patients, especially HIV-1 infected patients, this work aimed to elucidate mechanisms of Th2 induction and regulation by assessing the Th2 hallmark cytokine IL-4 in an experimental model of cryptococcosis. Therefore, a kinetic study of IL-4 expression during 70 days after intranasal infection was performed in susceptible mice. The analyses included characterization of pulmonary leukocytes and Th cell cytokine profiling. IL-4 profiling revealed Cryptococcus-specific IL-4 production not before six weeks after infection. This unexpected finding was further validated by equal results observed in a kinetic study done in IL-4 reporter mice. These mice express a green fluorescent protein simultaneously to IL-4 expression in the same cell and this protein can be detected by flow cytometry. Two cellular sources of IL-4 were identified: Th2 cells were found as expected, but also, as shown for the first time, eosinophilic granulocytes could be demonstrated to secrete IL-4. Next, the influence of eosinophils on pulmonary inflammation and disease development was investigated using ΔdblGATA-1 mice constitutively devoid of eosinophilic granulocytes. Experiments with infected ΔdblGATA-1 mice revealed novel regulatory functions of eosinophils in cryptococcosis. In the absence of eosinophils pulmonary Th cell recruitment was significantly diminished. In addition, Th2 polarization was reduced in ΔdblGATA-1 mice as shown by reduced numbers of Th2 cells expressing the Th2-related surface marker T1/ST2 and reduced albeit not absent IL-4 production by Th cells. In addition to reduced IL-4 production, in the absence of eosinophils Th cells with enhanced interferon-γ and IL-17 production were observed. However, control of pulmonary fungal growth was only slightly enhanced in the absence of eosinophils and dissemination of cryptococci to the brain was unaltered. This may be related to the shared IL-4 production by not only eosinophils but also Th2 cells. Blocking more than one cellular source of IL-4 could be required to prevent immunopathology. To test the hypothesis of gradual IL-4-dependent immunopathology, experiments were conducted using mice expressing only one allele of the IL-4receptor (R) alpha (α) chain (+/-) instead of two (+/+). Indeed, mono-allelic expression of the IL-4Rα resulted in an intermediate expression of the IL-4R on the surface of myeloid and lymphoid cells indicating a gene-dosage effect for IL-4R expression. Infected IL-4Rα+/- mice displayed reduced susceptibility as compared with IL-4Rα+/+ mice, and IL-4Rα-/- mice completely lacking IL-4R expression were found to be protected with survival for the complete time period of the experiment (i.e. up to 275 days). Reduced susceptibility found in infected IL-4Rα+/- mice was associated with decreased serum levels of immunoglobulin E, reduced mucus production by airway epithelia, attenuation of airway hyper-reactivity, and reduced formation of alternatively activated macrophages in lung parenchyma – pathophysiological features, which are typically found in experimental models of asthma but also in asthma of humans and animals. Since no up-regulation of IL-4R by the infection with Cryptococcus neoformans was found, the experimental pulmonary infection model used appears to be a very sensitive low-level IL-4 system. This work highlights the outstanding role of IL-4 and its different cellular sources as well as its receptor in cryptococcosis and provides novel insights into pathogenesis. Moreover, a cellular (i.e. eosinophils) and a molecular (i.e. IL-4R) target for treatment of this mycosis and possibly of asthma is provided.

La modification post-traductionnelle des chimiokines par la dipeptidyl peptidase-4 (DPP4 ou CD26) régule négativement le trafic des lymphocytes, et son inhibition améliore la migration des lymphocytes T et l'immunité anti-tumorale en préservant la forme fonctionnelle de CXCL10. En étendant ces résultats initiaux aux humains et à un modèle préclinique de carcinome hépatocellulaire, nous avons découvert un nouveau mécanisme par lequel l'inhibition de DPP4 améliore les réponses anti-tumorales par le recrutement des éosinophiles. Plus précisément, l'administration d'inhibiteurs de DPP4 (DPP4i) conduit à des concentrations tumorales plus élevées de CCL11 (ou eotaxine) et à une augmentation de la migration des éosinophiles exprimant CCR3 dans les tumeurs. Un meilleur contrôle de la croissance tumorale a été observé lors du traitement par DPP4i, un effet conservé chez les souris Rag2–/– mais abrogé uniquement lors de la déplétion des éosinophiles ou de l'inhibition de leur dégranulation. Nous avons également démontré que l'expression tumorale d’IL-33 était nécessaire et suffisante pour une réponse anti-tumorale médiée par les éosinophiles et que ce mécanisme contribuait à l'efficacité des inhibiteurs de points de contrôle immunitaires. Ces résultats révèlent un nouveau mécanisme par lequel le contrôle tumoral est médiée par IL-33 et les éosinophiles, mécanisme ici révélé lorsque les mécanismes endogènes de régulation immunitaire par DPP4 sont inhibés. / Dipeptidyl peptidase-4 (DPP4 or CD26)–mediated post-translational modification of chemokines has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional CXCL10. In extending these initial findings to humans and pre-clinical hepatocellular carcinoma models, we discovered a new mechanism whereby DPP4 inhibition improves anti-tumor responses by eosinophil recruitment. Specifically, administration of DPP4 inhibitors (DPP4i) resulted in higher concentrations of CCL11 (or eotaxin) and increased CCR3-mediated eosinophil migration into mouse tumors. Enhanced tumor control was observed upon treatment with DPP4i, an effect strikingly preserved in Rag2–/– mice, and abrogated only upon depletion of eosinophils or inhibition of their degranulation. We further demonstrated that tumor expression of IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses, and that this mechanism contributed to checkpoint inhibitor efficacy. These findings provide new insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immune regulation are inhibited.

Orientador: Sonia Jancar / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T23:40:55Z (GMT). No. of bitstreams: 1 Landgraf_RichardtGama_D.pdf: 1629843 bytes, checksum: d732dae7c868194b49b83ed52b21a953 (MD5) Previous issue date: 2006 / Resumo: A asma é uma doença inflamatória crônica que envolve, simultaneamente, a participação de diversos tipos celulares, citocinas e mediadores inflamatórios. Apesar do grande número de estudos sobre o papel da bradicinina e do óxido nítrico (NO) na asma, vários aspectos não foram abordados e outros são controversos. Os objetivos deste trabalho foram, em modelo murino de asma: 1) caracterizar alguns parâmetros da inflamação pulmonar como infiltrado de eosinófilos e linfócitos (CD4 +, CD8 +, B, T?d e NK1.1) no espaço aéreo, reatividade das vias aéreas à metacolina e produção de muco e avaliar o efeito de antagonistas de receptor induzível (B1) e constitutivo (B2) da bradicinina nestes parâmetros; 2) comparar o efeito da supressão aguda da síntese de NO (por tratamento com inibidores da síntese) com a ausência constitucional da iNOS (utilizando animais deficientes para este gene) nestes parâmetros; 3) avaliar a cinética da expressão da iNOS no pulmão e o tipo celular responsável pela produção de NO na fase inicial da inflamação alérgica pulmonar. Camundongos foram sensibilizados por injeção intraperitoneal de ovalbumina utilizando hidróxido de alumínio como adjuvante. Uma dose de reforço foi administrada após 7 dias e o desafio antigênico realizado no 14º e 21º dia por aerosol. Os antagonistas de receptores da bradicinina e os inibidores de NO foram administrados por via intraperitoneal 30 minutos antes de cada desafio antigênico. Os resultados obtidos mostram que o tratamento com o antagonista do receptor induzível B1 da bradicinina (R-954) reduziu significativamente o número de eosinófilos no BAL, a hiperreatividade das vias aéreas e a secreção de muco, entretanto não alterou o número de linfócitos no BAL. Por outro lado, o tratamento com o antagonista do receptor constitutivo B2 da bradicinina (HOE-140) aumentou o número de eosinófilos e linfócitos no BAL, porém, não alterou a hiperreatividade das vias aéreas nem a secreção de muco. Estes efeitos dos antagonistas foram semelhantes nas linhagens C57Bl/6 e BALB/c. Estes resultados sugerem que, neste modelo de inflamação alérgica pulmonar, a ativação dos receptores constitutivos da bradicinina regula negativamente a inflamação enquanto que a ativação dos receptores induzíveis regula positivamente. O tratamento com os inibidores de NO, L-NAME ou aminoguanidina, diminuiu o número de eosinófilos e linfócitos no BAL, a hiperreatividade das vias aéreas e a secreção de muco. Surpreendentemente, camundongos deficientes para o gene da iNOS apresentaram inflamação das vias aéreas semelhantes aos camundongos não deficientes, e o tratamento destes camundongos com os inibidores de NO não afetou nenhum dos parâmetros avaliados. Estes dados mostram que a inibição aguda da iNOS tem efeito anti-inflamatório neste modelo de asma, enquanto que a ausência constitutiva desta enzima não tem efeito. Observamos ainda que no BAL obtido 12 horas após o desafio antigênico, as células que expressam marcador fluorescente de NO são células com núcleo polimórfico. Isto coincidiu com o pico de infiltração de neutrófilos para o BAL e com o pico de expressão da iNOS no pulmão. Estes resultados sugerem que neutrófilos do BAL produzem NO na fase inicial da asma murina. Assim, nossos estudos contribuíram para uma melhor compreensão do papel dos receptores constitutivos e induzidos da bradicinina, na asma e sugeriram que a utilização de antagonistas do receptor B1 poderia ter um efeito protetor. Além disso, a demonstração da produção de NO por neutrófilos que migraram para o BAL na fase inicial da inflamação alérgica sugere que inibidores da síntese de NO possam ter efeito protetor quando utilizados em fase precoce da estimulação antigênica / Absract: Asthma is a chronic inflammatory disease that involves simultaneously several cell types, cytokines and inflammatory mediators. Despite the large number of publications dealing with the role of nitric oxide (NO) and bradykinin in asthma, several aspects of the disease were not addressed and some are controvertial. In the present study, using a murine model of asthma, we: 1) characterized some parameters of pulmonary inflammation such as airways infiltration of eosinophil and lymphocytes (CD4 +, CD8 +, B, T?d e NK1.1), airways reactivity to methacholine and mucus secretion and evaluated the effect of antagonists of the inducible (B1) and constitutive (B2) bradikinin receptors in these parameters; 2) compared the effect of acute suppression of NO synthesis (by treatment with NO inhibitors) with the absence of iNOS (using mice deficient for this gene) on these parameters; 3) evaluated the temporal profile of iNOS expression in the lungs and the cell type responsible for lung NO production in the initial phase of lung inflammation. Mice were sensitized by ip injection of ovalbumin using alum as adjuvant. One booster was administered after 7 days and antigen challenge performed on day 14 and 21 by aerosol. The bradykinin receptor antagonists and the NO inhibitors were given ip, 30 min before each challenge. The results obtained show that treatment with the B1 receptor antagonist, R-954 significantly reduced the number of eosinophils in the BAL, airways hyperreactivity and mucus secretion but did not alter the number of BAL lymphocytes. Treatment with the constitutive (B2) bradykinin receptor,HOE-140), however, increased eosinophils and BAL lymphocytes number, but had no effect on airways hyperreactivity nor in mucus secretion. These effects of the antagonists were observed in both, C57Bl/6 e BALB/c mice. These results suggest that, in this model of lung inflammation, activation of constitutive bradykinin receptors would down-regulate the inflammation whereas activation of the inducible receptor would have a pro-inflammatory effect. Treatment with NO inhibitors, L-NAME or aminoguanidine, reduced eosinophils and lymphocytes in BAL, airways hyperreactivity and mucus secretion. Surprisingly, in mice deficient of iNOS gene the airways inflammation was similar to that observed in iNOS sufficient mice and treatment of the deficient mice with the NO inhibitors did not affect the parameters analyzed. These results show that acute inhibition of iNOS has anti-inflammatory effect in this model of asthma whereas the absence of this enzyme has no effect. It was also observed that in the BAL collected 12h after antigen challenge, the cells that expressed a fluorescent NO marker were cells with polymorphic nucleous. This was coincident with the peak of neutrophils infiltration to the BAL and with the peak expression of lung iNOS. These results suggest that BAL neutrophils produce NO in the initial phase of murine asthma. Thus, our studies contributed for a better understanding of the role of the constitutive and inducible bradykinin in asthma and suggest that antagonists of B1 receptor could have a beneficial effect on asthma. Moreover, the demonstration of NO production by neutrófilos that had migrated to the BAL suggests that NO synthesis inhibitors would have a beneficial effect when administered early after antigenic stimulation / Doutorado / Doutor em Farmacologia

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Eosinophils are commonly described as cells from innate immunity that act on parasitic infections and lung diseases. However, in recent years, new functions are being added to these cells, among them, the maintenance of reproductive homeostasis. In addition, since the 1960s, studies have shown the estrogen relationship and the selective eosinophils migration to the uterus of castrated rats. The elucidation of the mechanism for the migration of these cells appears to be based on findings showing that the CCR3 receptor and its CCL11 chemokine are expressed in the human endometrium. Objective: The objective of this study was to evaluate the CCR3 participation in the eosinophils migration to the uterus of castrated mice, induced by 17-β-estradiol (E2). Methods: C57/BL6 mice, which received subcutaneous E2 injection, were used to determine the time and dose response of E2 (times 6, 12, 24 and 48 hours and doses of 0.1, 1, 3, 10, 30, 100 and 300 μg/kg) that promotes uterine weight gain and eosinophil migration. Subsequently, using the air bubble model, we sought to standardize CCL11- induced eosinophil recruitment, and the dose of the CCR3 antagonist (SB 328437 at doses of 1, 3 and 10 mg/kg) which promotes reduction in eosinophils migration. In addition, in the same model, the effect of the uterine extract with and without the administration of E2 (at the times of 6, 12 and 24 h) on the leukocyte migration was evaluated. Finally, an investigation was carried out on the effect of SB 328437 on uterine weight and eosinophil recruitment. The eosinophil peroxidase (EPO) absorbance and histology were used to evaluate the eosinophil migration, in which the uterus was stained with orcein specific for eosinophils. Results: The results demonstrated that the dose of 100 μg/kg E2 in the 24 hour period promoted a 40% increase in uterine weight, accompanied by eosinophil migration. In the air bubble model, it was observed that CCL11 recruited eosinophils, and SB 328437 promoted a 55% reduction in migration of these cells. The extract of the uterus caused the migration of total and eosinophilic leukocytes, but there was no difference between the groups with and without the administration of E2. Corroborating the results of SB 328437 in the air bubble experiment, the evaluation of its effect on uterine weight and eosinophils recruitment demonstrated that dose of 3 mg/kg reduced both parameters (approximately 45% and 56%, respectively) when stimulated with E2. The results were analyzed using ANOVA with Tukey post-test (more than two groups), and t test (two groups). Conclusion: Based on the results, it was possible to confirm the hypothesis that the CCR3 receptor participates in the eosinophils migration to the uterus of C57/BL6 mice after E2 induction. Furthermore, it represents an important pathway to be considered in studies aimed at elucidating mechanisms in in physiological and pathological processes involving the eosinophils recruitment. / Os eosinófilos são comumente descritos como células pertencentes à imunidade inata que agem em infecções parasitárias e nas doenças pulmonares. Porém, nos últimos anos, novas funções estão sendo acrescidas a essas células, dentre as quais, de manutenção da homeostase reprodutiva. Além disso, desde a década de 60, estudos demonstraram a relação do estrogênio com a migração seletiva de eosinófilos para o útero de ratas castradas. A elucidação do mecanismo para a migração dessas células baseia-se em achados evidenciando que o receptor CCR3 e sua quimiocina CCL11 estão expressos no endométrio humano. Objetivo: Diante do exposto, o estudo objetivou avaliar a participação do CCR3 na migração de eosinófilos para o útero de camundongos castrados, induzida por 17-β-estradiol (E2). Metodologia: Camundongos C57/BL6 receberam a injeção de E2 subcutânea, objetivando determinar o tempo e a dose resposta do E2 (tempos de 6, 12, 24 e 48 horas e doses de 0,1, 1, 3, 10, 30, 100 e 300 μg/kg) que promove aumento do peso do útero e migração de eosinófilos. Posteriormente, utilizando o modelo de bolha de ar, buscou-se padronizar o recrutamento de eosinófilos induzido por CCL11, e a dose do antagonista do CCR3 (SB 328437, nas doses de 1, 3 e 10 mg/kg) que promove redução da migração de eosinófilos. Ademais, no mesmo modelo, avaliou-se o efeito do extrato do útero com e sem a administração de E2 (nos tempos de 6, 12 e 24 h), na migração de leucócitos. Por último, foi realizada uma investigação sobre o efeito do SB 328437 no peso do útero e no recrutamento de eosinófilos. Para avaliação da migração de eosinófilos foi utilizada a absorbância da Peroxidase de Eosinófilos (EPO) e a histologia, no qual, os úteros foram corados com orceína, específico para eosinófilos. Resultados: Os resultados demonstraram que a dose de 100 μg/kg de E2 no tempo de 24 horas promoveu aumento de 40% no peso do útero, acompanhado da migração de eosinófilos. No modelo de bolha de ar, observou-se que a CCL11 recrutou os eosinófilos, e o SB 328437 promoveu redução de 55% na migração dessas células. O extrato do útero provocou a migração de leucócitos totais e de eosinófilos, porém não houve diferença entre os grupos com ou sem a administração de E2. Corroborando os resultados do SB 328437 no experimento da bolha de ar, a avaliação do efeito do antagonista no peso do útero e no recrutamento de eosinófilos, demonstrou que a dose de 3 mg/kg reduziu ambos os parâmetros (aproximadamente em 45% e 56%, respectivamente) quando estimulados com E2. Os resultados foram analisados utilizando o ANOVA com pós- teste de Tukey (mais de dois grupos), e o teste t (em dois grupos). Conclusão: Perante os resultados encontrados, foi possível confirmar a hipótese de que o receptor CCR3 participa da migração de eosinófilos para o útero de camundongos C57/BL6, após indução com E2. Outrossim, representa uma importante via a ser considerada em estudos que visam a elucidação de mecanismos em processos fisiológicos e patológicos envolvendo o recrutamento de eosinófilos. / São Cristóvão, SE

Introdução: Há crescentes evidências que sinalizam o papel do estresse crônico como desencadeante e mesmo perpetuador de crises asmáticas, bem como a importância da produção de muco e do transporte mucociliar na fisiopatologia da asma brônquica. Objetivos: Deste modo, consideramos relevante avaliar em cobaias com inflamação alérgica crônica pulmonar como o estresse físico repetido, induzido pela natação forçada, modula as respostas de transporte mucociliar, as propriedades do muco, o infiltrado eosinofílico e a expressão de IL-13 no epitélio e nas células inflamatórias na parede das vias aéreas. Métodos: Os animais receberam inalações duas vezes por semana durante quatro semanas com doses crescentes de ovoalbumina (grupo OVA e OVA-NAT) ou solução fisiológica (grupo SAL e SAL-NAT). Após 24 horas da quarta inalação os animais dos grupos denominados SAL-NAT e OVA-NAT foram submetidos ao protocolo de natação forçada por dez dias com intervalo de dois dias, para a indução do estresse. Após 72 horas da última inalação, os animais foram anestesiados, sendo testadas: a velocidade de transporte mucociliar (VTM), a freqüência de batimento ciliar (FBC), e a diferença de potencial transepitelial (PD). Após estas medidas foram coletadas amostras de muco para avaliação do ângulo de contato (AC) e da transportabilidade pela tosse (TT). Foi coletada uma amostra de sangue para a dosagem de cortisol sérico. Os pulmões foram retirados, fixados e submetidos à coloração de LUNA, para identificação de eosinófilos, e à técnica imunohistoquímica para detecção da expressão de IL-13 no epitélio brônquico e nas células inflamatórias presentes na parede das vias aéreas. As adrenais também foram retiradas, imediatamente pesadas e submetidas à coloração de hematoxilina e eosina para avaliação histopatológica, utilizando a morfometria para determinação das áreas de cada uma de suas camadas. Resultados: Os Resumo animais do grupo OVA apresentaram uma redução da VTM, do PD, da TT e aumento do AC, da área de epitélio de vias aéreas, de muco ácido, do número de eosinófilos e da expressão de IL-13, no epitélio brônquico e nas células inflamatórias presentes ao redor das vias aéreas, comparativamente aos controles (p<0,05 para todas as comparações). Houve redução da área total da adrenal e de cada uma de suas camadas comparativamente aos controles (p<0,05 para todas as comparações). Os animais submetidos ao protocolo de estresse (grupos SAL-NAT e OVA-NAT) apresentaram aumento nos níveis de cortisol sérico (p<0,05), no peso das adrenais comparativamente aos grupos e OVA (p<0,05). Os animais submetidos ao protocolo de estresse e expostos à ovoalbumina (grupo OVA-NAT) mostraram redução na VTM, aumento no AC, na área de muco ácido do epitélio brônquico e em todas as camadas das adrenais, comparativamente aos resultados obtidos nos animais do grupo OVA (p<0,05 para todas as comparações). Conclusão: O processo inflamatório crônico pulmonar altera o transporte mucociliar e as propriedades reológicas do muco, o que se associou ao recrutamento eosinofílico, aumento da expressão de IL-13 no epitélio brônquico e nas células inflamatórias presentes na parede brônquica. Além disto, observamos a redução de todas as camadas da adrenal. Neste modelo experimental, o estresse físico repetido reduz o transporte mucociliar devido a alterações das propriedades do muco, particularmente potencializando o aumento de muco ácido e de sua hidrofobicidade e adesividade. Estes efeitos parecem estar relacionados à ativação adrenal, mas independentes do recrutamento eosinofílico e da resposta Th2 mediada pela IL-13 / Background: It has increasing evidence linking the role of stress in the onset of asthma exacerbation and in the maintenance of asthmatic crises, as well as the importance of mucus production and the mucociliary clearance in the asthma physiopathology. Objectives: So, we consider relevant to evaluate in guinea pigs with pulmonary chronic allergic inflammation how the induced repeated physical stress, caused by forced swimming, modulates the mucociliary clearance, the mucus properties, the eosinophilic infiltration and the IL-13 expression on bronchial epithelial cells in the airway wall. Methods: The animals had received inhalations two times per week during four weeks with increasing doses of ovalbumin (OVA and OVA-S groups) or saline solution (SAL and SAL-S groups). After twenty four hours of the 4th inhalation, the animals (named as SAL-S and OVA-S groups) had been submitted to the protocol of forced swimming, per ten days with an interval of two days, for the stress induction. After 72 hours of the last inhalation, the animals were anaesthetized and the tracheal mucus clearance (TMC), the ciliary beat frequency (CBF), and the difference of transepithelial potential difference (PD) were measured. After these measurements had been done mucus samples were collected for evaluation of the contact angle (CA) and cough transportability (CT). To serum cortisol dosage a little amount of blood was collected. The lungs were dissected, fixed and submitted to histological techniques, like LUNA stain for identification of eosinophils and the IL-13 immunohistochemistry technique for its detection in the bronchial epithelium and in the inflammatory cells present in the airways walls. The adrenal glands were excised, immediately weighed and submitted to haematoxylin and eosin stain for histopathological evaluation using the morphometry to determine the areas of each of their layers. Results: The animals of OVA Summary group presented a reduction of the TMC, the PD, and the CT. On the other hand they presented an increase of the CA, of the airways epithelial area, of the acid mucus, of the eosinophils number as well as on the IL-13 expression in the airways epithelium and on the inflammatory cells around the airways, compared to the controls (p<0.05 for all comparisons). There was a reduction of the adrenal gland total area and on each one of its layers compared to the controls (p<0.05 for all comparisons). The animals submitted to the forced swim stress protocol (SAL-S and OVA-S groups) showed an increase on serum cortisol levels, on adrenal gland weight compared to OVA group (p<0.05). The animals submitted to the forced swim stress protocol and to the sensitization protocol with ovalbumin (OVA-S) showed a reduction in the TMC and an increase on the CA, bronchial epithelial acid mucus area and in all adrenal gland layers compared to the results obtained in the OVA group animals (p<0.05 for all comparisons). Conclusions: The chronic inflammatory pulmonary process alters the mucociliary transport and mucus rheological properties which was associated to eosinophilic recruitment, increases in the IL-13 expression on bronchial epithelial cells and inflammatory cells in airway walls. In addition, we observed a reduction in all adrenal zones. In this experimental model the repeated physical stress reduces the mucociliary clearance due to the mucus rheological properties alterations, particularly increasing the amount of acid mucus and its wettability and adhesivity. These effects seem to be related to the adrenal activation but independently of the eosinophilic recruitment and of Th2 responses mediated by IL-13

Experimental autoimmune encephalomyelitis (EAE) is the rodent model of multiple sclerosis (MS), a chronic autoimmune neuroinflammatory disease that has a devastating impact on various neurological functions of the patients. The hallmarks of both, MS and EAE, are neuroinflammation, demyelination and neuroaxonal degeneration. Various types of lymphoid and myeloid cells were shown to infiltrate the central nervous system and to participate in disease pathology. However, the role of eosinophil granulocytes has been less explored thus far. An early study showed that eosinophils infiltrate into the spinal cord of EAE mice and suggested their role in the disease progression. Recently, it was reported that eosinophils can play a protective role against EAE when mice are treated with an extract from helminths. Furthermore, it was shown that EAE development is not altered in mice deficient for interleukin-5, an important eosinophil pro-survival factor. Taken together, the role of eosinophils in EAE is currently unclear and needs to be investigated in detail. In the present study, we use the active model of EAE, whereby we immunized the C57BL/6 mouse strain with MOG35-55 peptide emulsified in the complete Freund’s adjuvant, in order to study a possible contribution of eosinophils to the disease pathology. Using the flow cytometry and RT-qPCR analysis of the spinal cord, we show that eosinophils infiltrate into the tissue in the course of EAE. The infiltration is likely driven by eosinophil chemoattractants, such as eotaxin-1, as the concentration of the latter was increased in the spinal cord during EAE, as shown on mRNA and protein level. Moreover, detailed flow cytometry analysis of spinal cord eosinophils revealed that they show signs of activation, namely an increase in CD11b and decrease in CCR-3 surface expression. Furthermore, we observed signs of degranulation of spinal cord eosinophils in EAE which was measured as a decrease of the side scatter parameter and an upregulation of CD63 surface expression. These data suggest a potential role of eosinophils in the pathology of EAE. In order to elucidate whether eosinophils are important for the disease development, eosinophil-deficient mice were subjected to EAE and the clinical development of the disease was observed. For this purpose, we used two independent models of eosinophil deficiency - ΔdblGATA1 and interleukin-5-depleted mice. ΔdblGATA1 mice are a genetically manipulated mouse strain bearing a deletion in GATA1 promoter that causes a specific depletion of eosinophils. Interestingly, clinical development of EAE was not affected in these mice when compared to their wild-type controls. As a next step, we depleted eosinophils by injecting wild-type mice with an antibody against the eosinophil pro-survival factor interleukin-5 in order to reduce eosinophil numbers in the effector phase of EAE. In accordance with the result from the experiment with ΔdblGATA1 mice, EAE progression was not altered in the eosinophil-depleted mice when compared to mice that were injected with an isotype control antibody. Further, we analyzed the neuroinflammation and demyelination in the spinal cord of 4ΔdblGATA1 mice subjected to EAE. Specifically, the infiltration of inflammatory cell populations, including CD4 and CD8 T cells, neutrophils and macrophages, was assessed by flow cytometry. In agreement with the unchanged clinical EAE development, inflammatory cell infiltration was not affected in ΔdblGATA1 mice. Furthermore, we analyzed expression of pro-inflammatory cytokines in the spinal cord of ΔdblGATA1 mice subjected to EAE in order to better characterize the inflammatory status. No significant changes were detected further confirming that eosinophils do not contribute to neuroinflammation in EAE. Finally, we assessed the demyelination in the spinal cord of ΔdblGATA1 EAE mice using luxol fast blue staining to detect myelin. In accordance with the unaffected clinical development and inflammatory status, we did not observe any difference in the spinal cord demyelination in ΔdblGATA1 mice when compared to their wild-type littermates. Taken together, although eosinophils infiltrate into the spinal cord of EAE mice and are activated and degranulate therein, they are dispensable for EAE development.

No description available.

The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively.

Introdução: A dermatite atópica (DA) é uma doença cutânea inflamatória de caráter crônico, recidivante, em que o prurido intenso e a xerose cutânea são frequentes. A etiopatogenia da DA é multifatorial, envolvendo fatores genéticos, ambientais e imunológicos. Eosinófilos são leucócitos polimorfonucleares multifuncionais que estão implicados na patogênese de diversos processos inflamatórios, incluindo a DA. Além da produção e secreção de diversas proteínas presentes nos grânulos citoplasmáticos, os eosinófilos também apresentam potencial para secretar metaloproteinases, enzimas proteolíticas que degradam vários componentes da matriz extracelular, e estão presentes em diversos processos fisiológicos e patológicos. Objetivo: Avaliar: 1) o perfil fenotípico dos eosinófilos na dermatite atópica do adulto, através da expressão das moléculas CCR3, CD23, CD38, CD69 e CD62L; 2) o perfil funcional, a partir da secreção de metaloproteinases, inibidores teciduais de metaloproteinases e RANTES por eosinófilos purificados. Métodos: Foram incluídos 41 adultos diagnosticados com DA, de acordo com os critérios de Hanifin & Rajka e 45 controles adultos sadios. A gravidade da doença foi mensurada através do escore de gravidade EASI (Eczema Area and Severity Index). Eosinófilos (LIN 1- CCR3+) do sangue periférico foram analisados para os marcadores CCR3, CD38, CD69, CD23 e CD62L através da citometria de fluxo (LSRFortessa, BD Biosciences) a análise foi realizada com o FlowJo 7.5.6 software. Eosinófilos purificados de indivíduos com DA e indivíduos controles foram estimulados com enterotoxina de Staphylococcus aureus B (SEB) e FSL-1 (agonista de receptores Toll-like 2 e 6), e os sobrenadantes foram coletados para dosagem de metaloproteinases (MMPs), inibidores teciduais de metaloproteinases 1 e 2 (TIMP-1 e TIMP-2) e RANTES por ELISA e por Cytometric bead array. Resultados: Indivíduos com DA apresentaram maior frequência de eosinófilos (LIN1- CCR3+), relacionada à gravidade da doença. Observou-se também, que a frequência de CD62L (L-selectina) e de CD23 (receptor de baixa afinidade para IgE) em eosinófilos (LIN1- CCR3+) diminui em pacientes com DA. Os receptores de ativação precoce (CD69) e tardio (CD38) não mostraram diferença estatística entre os grupos analisados. Os níveis séricos de MMPs e de TIMPs foram similares entre os controles e pacientes. Ao analisarmos a secreção de MMPs e de (TIMPs), a partir de eosinófilos purificados de pacientes com dermatite atópica, observamos diminuição dos níveis basais de TIMP-1 e TIMP-2 e de RANTES. Conclusões: Na DA do adulto, o perfil fenotípico e funcional dos eosinófilos mostrou: perfil de ativação da fase aguda, com expressão aumentada de CCR3; potencial de migração elevado, em decorrência da diminuição da expressão de CD62L; falhas no processo de ativação dos eosinófilos via CD23, bem como, no remodelamento tecidual mediado por TIMP-1 e TIMP-2 e na quimotaxia mediada por RANTES / Introduction: Atopic dermatitis (AD) is an inflammatory, chronic and recurrent skin disease characterized by intense pruritus and xerosis. AD has a complex etiopathogenesis, which involves the influence of genetics, environment, and immunological disorders, among others. Eosinophils are multifunctional polymorphonuclear leukocytes that contribute to the pathogenesis of several inflammatory processes, such as AD. In addition to the production and secretion of diverse proteins of the cytoplasmic granules, eosinophils have also the potential to secrete metalloproteinases (MMPs), proteolytic enzymes with a primary role for degrading several extracellular matrix components, present in distinct physiological and pathological processes. Objective: To evaluate:1) the phenotypic profile of eosinophils in adults with atopic dermatitis through the expression of CCR3, CD23, CD38, CD69 and CD62L molecules; 2) the functional profile through secretion of MMPs, tissue inhibitors of metalloproteinases 1 and 2 ( TIMP-1 and TIMP-2) and RANTES by purified eosinophils. Methods: This work enrolled 41 patients with AD, diagnosed according to Hanifin & Rajka\'s criteria) and 45 healthy controls. Severity of the disease was established utilizing EASI (Eczema Area and Severity Index). Eosinophils (Lineage cocktail 1- CCR3+) from peripheral blood were analyzed for CCR3, CD38, CD69, CD23 and CD62L by flow cytometry (LSRFortessa, BD Biosciences), and analysis was performed using the FlowJo 7.5.6 software. Purified eosinophils were stimulated with Staphylococcus aureus enterotoxin B (SEB) FSL-1 (Toll-like receptor 2/6 agonist), and supernatants were collected for MMPs, TIMPs and RANTES secretion, evaluated by ELISA and cytometric bead array (CBA). Results: Patients with AD have a higher frequency of eosinophils (LIN1- CCR3+), related to disease severity. Moreover, the frequency of CD62L (L-selectin) and CD23 (low-affinity receptor for IgE) in (LIN1- CCR3+) eosinophils was reduced in individuals with AD. CD69 and CD38 (early and late activation receptors) did not show significant difference in the studied groups. Serum levels of MMPs and of TIMP-1 and TIMP-2 were similar in healthy controls and AD patients. When analyzing secretion of MMPs and TIMPs by purified eosinophils from AD individuals, we detected a decrease in baseline levels of TIMP-1, TIMP-2, and reduced RANTES-mediated chemotaxis. Conclusions: Eosinophils in AD exhibit an activation profile of acute phase, with enhanced CCR3 expression, high potential for migration due to reduced expression of CD62, defective activation mechanisms via CD23, altered tissue remodeling process mediated by TIMP-1 and TIMP-2 and reduced RANTES-mediated chemotaxis

O carcinoma espinocelular de lábio (CEC) é uma patologia relativamente freqüente e acomete preferencialmente indivíduos de pele clara e com antecedentes de exposição solar. A localização labial do CEC está associada ao comportamento clínico mais agressivo. Algumas características clínicas e histológicas, como tamanho do tumor, grau de diferenciação, espessura, invasão perineural e perivascular, atuam como fatores prognósticos do CEC de lábio. A presença do infiltrado inflamatório vem sendo associada ao comportamento de diversos tumores, dentre eles o CEC. Esse infiltrado é composto por linfócitos T, B, células natural killer, macrófagos e eosinófilos. O papel do infiltrado eosinofílico no CEC é bastante controverso, mostrando associação tanto com melhor como com pior prognóstico. Neste trabalho, tentamos relacionar os infiltrado eosinofílico e linfocítico entre si e estabelecer uma possível associação com o prognóstico do tumor. Foram avaliados 29 casos de CEC de lábio atendidos no ambulatório de cirurgia dermatológica do HC-FMUSP, no período de 1980 a 1989. Foram colhidos dados clínicos provenientes dos prontuários (sexo, idade, tabagismo, duração da lesão, presença de recidiva e metástase) e dados histológicos (grau de diferenciação, espessura, invasão perineural e perivascular, comprometimento da camada muscular, contagem de eosinófilos peritumoral e intratumoral, contagem de linfócitos) em lâminas coradas pela hematoxilia-eosina. A comparação entre a quantificação dos eosinófilos na área intra e peritumoral mostrou-se semelhante. Não foi encontrada significância estatística entre a contagem de eosinófilos tumorais e fatores que funcionam como prognósticos do CEC de lábio (espessura, duração da lesão, infiltrado perivascular e perineural, comprometimento da camada muscular, grau de diferenciação, presença de recidiva e metástase). O infiltrado linfocítico desempenha fator prognóstico nos casos de melanoma, porém, no CEC, ainda é controverso. Foi realizada a classificação semiquantitativa do infiltrado linfocítico e comparada às mesmas variáveis estudadas com os eosinófilos. Foi encontrada relação estatisticamente significativa entre maior infiltrado linfocítico e menor grau de diferenciação das células tumorais, sugerindo um papel importante dos antígenos de superfície presentes no desencadeamento da resposta imune linfocitária anti-tumoral. As outras comparações não se mostraram significativas. Tentamos relacionar ainda a quantidade de eosinófilos e linfócitos no infiltrado, e observamos que as duas populações de células se comportam de maneira independente. Concluímos então que os infiltrados eosinofílicos peritumoral e intratumoral apresentam quantidades semelhantes de eosinófilos; o infiltrado eosinofílico não tem relação com prognóstico do CEC de lábio; o número de linfócitos no infiltrado tumoral é maior em tumores mais indiferenciados; o infiltrado linfocítico não influencia no restante dos fatores que funcionam como prognóstico do CEC de lábio; a quantidade de eosinófilos e linfócitos no CEC de lábio comportam-se de maneira independentes / The squamous-cell carcinoma (SCC) is a pathology quite frequent in people in general, mostly in white persons with history of excessive exposition to the sun rays. The fact of the SCC is located on the lips is associated with the most aggressive clinical behavior. Some clinical and histological characteristics, as the size of the tumor, degree of differentiation, density, perineural and perivascular invasion, act as prognostic factors of SCC of the lip. The presence of the tumor-infiltrating has being associated with the behavior of several tumors, including SCC. This infiltrating is composed by lymphocytes T, B, natural killer cells, macrophages and eosinophils. The role of the eosinophilict infiltrating in SCC is controversial enough because it shows association with both best and worst prognosis. In this study, we tried to stablish relations between the eosinophilic and the lymphocytic infiltratings, to stablish a possibility of association with the tumor prognosis. In order to achieve our objective, we have analized 29 cases of SCC os lip observed in the ambulatory of dermatologic surgery of HC-FMUSP from 1980 to 1989. We have also collected clinical data from the patient\'s promptuaries (sex, age, tobaccoism, time of lesion, presence of recurrence and metastasis) and histological data (degree of differentiation, depth, perineural and perivascular invasion, infiltration of the muscle layer, counting of peritumoral and intratumoral eosinophils, counting of lymphocytes) in microscopic features stained with hematoxylin and eosin. When comparing quantities of eosinophils in the intra and peritumoral areas we found them similar. We have not found any significant differences between the counting of tumoral eosinophils and factors that work as SCC of lips prognosis (depth, time of lesion, perivascular and perineural invasion, infiltration of the muscle layer, degree of differentiation, presence of recurrence and metastasis). The lymphocytic infiltrating acts as prognostic factor in the cases involving melanoma, however, in CEC it is still controversial. We have proceeded a classification of the lymphocytic infiltrating and compared with the same fluctuations studied with the eosinophils. We have found a significant relationship between the higher lymphocytic infiltrating and a minor degree of differentiation of tumoral cells, which suggests that the surface antigens in the lymphocytic anti-tumor immune response have an important role. Other comparisons were not significant. We tried to list the quantity of eosinophils and lyphocytes in the infiltrating and we noted that two groups of cells had differents behaviors. We conclude, therefore, that: the peritumoral and intratumoral eosinophilic infiltrating present similar quantities of eosinophils; the eosinophilic infiltrating is not related to the SCC of lips prognosis; the number of lyphocytes in the tumoral infiltrating is higher in less differentiated tumors; the lymphcytic infiltrating has no influence in the other factors that work as lips\' CEC prognosis; and the quantity of eosinophils and lymphocites in the SCC of lips have independent behaviors