Specific immunotherapy effect on peripheral blood T1/T2 lymphocytes in atopic patients

Manuela Rebordão¹

Luís Delgado²

Helena Pinto³

Augusto Remédios⁴

L Taborda-Barata⁵

A coordenação das características humorais e celulares da resposta alérgica, sabe-se hoje, está dependente da regulação por linfócitos T. As vacinas de alergénios são uma terapêutica que consegue modular a resposta das células T, e cujos mecanismos imunológicos permanecem incompletamente esclarecidos.

Objectivo: Avaliar o efeito da imunoterapia, após um ano de tratamento, na expressão de citocinas de perfil T1 e T2 em linfócitos de sangue periférico de doentes atópicos.

Material e métodos: Estudaram-se dez doentes atópicos sensibilizados a aeroalergénios comuns a fazerem vacinas de alergénios num período médio de um ano. Dentre estes, seis foram estudados antes e após a vacina. Como controlo estudou-se um grupo atópico sem imunoterapia constituído por 14 doentes também sensibilizados a aeroalergénios comuns e um grupo de indivíduos não atópicos, saudáveis, constituído por 7 elementos. A activação dos linfócitos T fez-se com PMA, ionomicina e brefeldina e estudaram-se as citocinas intracitoplasmáticas IFN-g, IL-4, IL-5 e IL-10 por citometria de fluxo. Procedeu-se a análise estatística por testes não paramétricos (Teste de Mann-Whitney U e Wilcoxon), considerando-se significativo p£0,05.

Resultados: A expressão de IL-4 e IL-5 nas células T, caracteristicamente aumentada nos doentes atópicos, respectivamente 13,8 (3,1-31,8) e 6,7% (1,0-20,4), é significativamente mais baixa no grupo que realizou a imunoterapia [5,4 (2,9-15,6) p= 0,007 e 2,1% (0,6-4,8) p=0,035] não diferindo do grupo controlo não atópico [5,1 (4,1-6,9) e 1,0 (0,4-2,1)]. Os níveis de IFN-g não variaram significativamente entre os três grupos estudados, mas a razão IFN-g//IL-4 nos linfócitos T CD4 aumentou significativamente nos doentes submetidos a imunoterapia. Por outro lado, houve um aumento da expressão de IL-10 nas células T circulantes do grupo sob imunoterapia, comparativamente a controlos não atópicos [1,9 (1,0-4,9) versus 1,4% (0,9-1,4) p=0,02], sendo mais evidente nos linfócitos T CD8. A IL-10 correlacionou-se de forma significativa com todas as citocinas de perfil T2 (IL-4 e IL-5) e com o fenótipo Tc2.

Conclusão: Após um ano de imunoterapia, a resposta das células T do sangue periférico a uma estimulação policlonal evidenciou uma diminuição da expressão das citocinas (IL-4 e IL-5), caracteristicamente aumentadas na doença alérgica. O aumento da IL-10, que também verificámos, sugere a existência de uma população reguladora de perfil T2, sendo mais evidente nos linfócitos T CD8.

Palavras chave: Imunoterapia, linfócitos T1 e T2, razão IFN-g / IL-4, IL-10.

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Abstract

Allergen-specific immunotherapy has been used for successful treatment of atopic diseases. They may act by modifying the patterns of cytokines produced by T cells. However, the precise mechanism by which it accomplishes these effects is still incompletely understood.

Objective: To evaluate the effect of one year immunotherapy on cytokines profiles T1 and T2 of peripheral blood lymphocytes in atopic patients.

Methods: We studied 10 atopic patients sensitised to common environmental allergens receiving immunotherapy over one year mean period. Six of these patients were studied before and after immunotherapy. Fourteen atopic patients untreated and 7 non-atopic subjects were used as control groups. Intracellular cytokine production (IFN-g; IL-4; IL-5; IL-10) was determined by flow cytometry following stimulation with phorbol myristate acetate (PMA), ionomycin and brefeldin. Mann-Whitney U and Wilcoxon non-parametric tests were utilized for the statistical analysis.

Results: The expression of IL-4 and IL-5 in T cells, characteristically increased in atopic patients, respectively 13.8 (3.1 31.8) and 6.7% (1.0 -20.4), was significantly lower in the immunotherapy group [5.4 (2.9 -15.6) p=0.007 and 2.1% (0,6 4.8) p=0.035] and similar in the non-atopic control group. The levels of IFN-g did not differ between the studied groups but the ratio IFN-g / IL-4 produced by CD4+ T lymphocytes increased significantly in the patients receiving immunotherapy. In addition, there was an increase in the expression of IL-10 by T cells of the immunotherapy group compared to the non-atopic controls [1.9 (1.0 4.9) versus 1.4% (0.9 1.4) p=0.02], being more evident in CD8+ T lymphocytes. IL-10 correlated significantly with all the profile T2 cytokines (IL-4 and IL-5) and with the phenotype Tc2.

Conclusion: After one year of immunotherapy the peripheral T cells response to a polyclonal stimulation revealed a reduction in IL-4 and IL-5 production, characteristically increased in atopic disease. The increase of IL-10 that we found in our study suggested the existence of a profile T2 regulatory population, more evident in CD8+T lymphocytes.

Keywords: Immunotherapy, T1 and T2 lymphocytes, ratio IFN-g / IL-4, IL-10.

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^* Trabalho vencedor ex-aequo (Secção A)

¹ Técnica Superior de Saúde. Assistente principal. Serviço de Análises Clínicas do Hospital Militar de Belém/Senior Health Technician. Senior assistant. Belém Military Hospital Analysis Clinic

² Professor Associadoda Faculdade de Medicina da Universidade do Porto. Serviço e Laboratório de Imunologia, Faculdade de Medicina da Universidade do Porto, Hospital de São João, Porto/Associate Professor at Porto University Medical School. Immunology Unit and Laboratory, Porto University Medical School, Hospital de São João, Porto

³ Tenente-coronel médica Directora do Serviço de Pneumologia do Hospital Militar de Belém/Medical Lieutenant Colonel Director of Pulmonology Unit, Belém Military Hospital

⁴ Tenente-coronel farmacêutico Director do Serviço de Análises Clínicas do Hospital Militar de Belém/Pharmacist Lieutenant Colonel Director of Analysis Clinic, Belém Military Hospital

⁵ Professor Auxiliar de imunologia clínica, Faculdade de Ciências da Saúde, Universidade da Beira Interior. Director do Serviço de Imunoalergologia do Hospital Pêro Covilhã/Assistant Professor Immunology Unit, Beira Interior University Health Sciences School. Immunoallergologist, Director of Immunoallergology Unit, Pêro Covilhã Hospital

Recebido/aceite para publicação/received/accepted for publication: 05.11.12