XXXIV CONGRESSO BRASILEIRO DE PNEUMOLOGIA E TISIOLOGIA APLICAÇÕES CLÍNICAS DA PESQUISA BÁSICA NA ASMA José Roberto Lapa e Silva Instituto de Doenças do Tórax/Hospital Universitário Clementino Fraga Filho Universidade Federal do Rio de Janeiro DELINEAMENTO Impacto da pesquisa básica no conhecimento da patogenia da asma - Cascata inflamatória - Definição dos alvos para terapia Desenvolvimento de modelo experimentais em inflamação alérgica Modulação da resposta inflamatória por biofármacos Queda da incidência das moléstias infecciosas e aumento da incidência das moléstias imunitarias em meio século J.F. Bach N Engl J Med 37 9-9
NIHLB: EXPERT PANEL REPORT 3: GUIDELINES OF ASTHMA, 7 NIHLB: EXPERT PANEL REPORT 3: GUIDELINES OF ASTHMA, 7 NIHLB: EXPERT PANEL REPORT 3: GUIDELINES OF ASTHMA, 7
NIHLB: EXPERT PANEL REPORT 3: GUIDELINES OF ASTHMA, 7 Proposed T cell imbalance in atopy Early infections e.g. TB, measles, endotoxins IL- IL- IFN-γ Th cells Allergen Certain infections (RSV) Cigarette smoking? IL- Th cells IL-, IL-3, IL- From P. Barnes, Asthma models may involve the induction of acute or protracted bronchoconstriction by antigen and / or of subsequent lung inflammation and functional airways disturbances. Markers are: mast cell activation and release of mediators production and release of bronchoactive eicosanoids early reactions (bronchoconstriction, airways edema) airways infiltration by eosinophils, lymphocytes and basophils and their subsequent activation production of IL-, IL-, TNF-α, and other cytokines late reactions (chronic inflammation, hyperreactivity) mucosal metaplasia very late reactions (anatomical rearrangements) 3
Protocol for the study of the interactions between Inflammation of lungs and of airways, bronchopulmonary hyperreactivity and mucosal metaplasia Immunisation Allergy starts Booster Mice become allergic but remain asymptomatic Provocation Mice show asthma phenotypes Day Day 7 Day and later. hyperreativity. augmented gene expression and production of mediators 3. recruitment of inflammatory cells. Mucosal metaplasia Main morphological differences between inflamed human and murine bronchial wall Human Murine Ciliostasis Epithelial ulceration Submembrane fibrosis Eosinophils Lymphocytes Mast cells Smooth muscle hypertrophy Submucosal gland hyperplasia Ciliated cells Granulocytes Clara cells Lymphocytes Mucus cells Basal cells Mast cells A longitudinal section of the murine airways Extrapulmonary bronchi (with cartilage) Bronchioles Major intrapulmonary bronchi (partially with cartilage) Blood vessels Alveoli Pharmacologie cellulaire, INSERM U8, Institut Pasteur, 998
Epithelial changes and mucus production in bronchi and bronchioles of BP mice after one intranasal ovalbumin provocation Normal and 3-6 hours after provocation after hours after 8 hours after 7 hours to 6 days Epithelial cell size and cross-sectional width of smooth muscle in the major bronchi of mice 7 hours after one ovalbumin intra-nasal provocation Size in Micrometres 3 3 Epithelial cell length p = <, Saline Ovalbumin 8 6 8 Width of smooth muscle p =,98 Saline Ovalbumin The murine model: evaluation of hyperreactivity Penh Penh 3 3 Saline hour 6 hours - Ovalbumin 3 hours hours Mus. Al. 9 6 8-6 8 Time (minutes)
controls Time-dependent cell infiltration after a single intranasal provocation with ovalbumin in BP mice N of cells/mm length of the epithelium Percentage of mucus secreting cells 3 Eosinophils CD CD8 3h 6h h 8h 7h 6d d Time after the intranasal provocation Mucosa brônquica normal de cobaia Mucosa brônquica de cobaia alérgica 6
Mucosa brônquica de cobaia sensibilizada mas não provocada Mucosa brônquica de cobaia sensibilizada e provocada BPNaive 7
Time-dependent mucosal metaplasia after a single intranasal provocation with ovalbumin in BP mice Infiltração brônquica de linfócitos T CD+ Citocinas pró- e anti-inflamatórias Pró-inflamatórias IL- TNF IL-6 IL-8 (quimiocina) IL- IL-/ IL-7 IL-8 Interferon-γγ (IFN-γγ) Anti-inflamatórias IL-Ra; stnf-r; sil-rii IL- IL-6 IL- IL-//3 IL- IL-3 Transforming growth factor-β β (TGF β ) 8
- IL- etc TH TH TH 3 IL- IL- - B + Eosinófilos IgE + RESPOSTA ALÉRGICA TGFβ e IL- TH INFγ B IgG Modificado de Durham, 3 e Aun, Pereira e Melo, 3 Estratégias para reduzir as citocinas. Redução das células produtoras (com citostáticos) Citocinas inibidoras (IL ) Inibidores da transdução do sinal Regulação da expressão gênica (glicocorticosteróides) Inibidores da liberação Redução das citocinas circulantes (anticorpos monoclonais, receptores solúveis) Bloqueadores de receptor (antagonistas ou anticorpos monoclonais). Mycobacterium bovis, BCG vivo atenuado Induz uma resposta imune de tipo Th (IFN-γ). Induz uma hipersensibilidade retardada à tuberculina. Associação inversa entre a intensidade da hipersensibilidade retardada e a asma (Shirakawa, 997). Estudos no camundongo mostram que a indução de uma resposta de tipo Th pode contrabalançar a resposta de tipo Th induzida pela ovalbumina. Papel das células apresentadoras de antígeno na iniciação da resposta de tipo Th após a administração de BCG. 9
Produção de IFN-γ pelos linfócitos T do gânglio linfático regional pg/ml 3 Newborns Adults 8 8 Weeks after BCG immunization Control Rectal route s.c. route Produção de IL- pelos linfócitos T do gânglio linfático regional Newborns Adults pg/ml 8 8 Weeks after BCG immunization Control Rectal route s.c. route Produção de IFN-γ pelo esplenócitos IFN-γ production by splenocytes pg/ml Newborns Adults 8 8 Weeks after BCG immunization Control Rectal route s.c. route
Produção de IL- pelos esplenócitos Newborns Adults pg/ml 8 8 Weeks after BCG immunization Control Rectal route s.c. route Conclusões O BCG induz o recrutamento células dendríticas no reto e nos gânglios mesentéricos. Este recrutamento, assim como sua capacidade de produzir IL-, são menores nos camundongos recémnascidos. As vias retal e s.c. permitem o recrutamento de células T de memória nos gânglions. O BCG induz uma resposta mista Th/Th. Effects of Mycobacterium bovis BCG on the development of allergic inflammation and bronchial hyperresponsiveness in hyper-ige BP mice vaccinated as newborns. Vaccine ; 9:8-9 Nahori M-A, Lagranderie M, Lefort J, Thouron F, Joseph D, Winter N, Gicquel B, Lapa e Silva JR, Vargaftig BB. Unités de Pharmacologie Cellulaire and Génétique Micobactérienne, Institut Pasteur, Paris, France Division of Pulmonary Medicine, Institute of Thoracic Diseases, Clementino Fraga Filho University Hospital Federal University of Rio de Janeiro, Brazil
6 l 6 6 6 6 Figure : Inhibition of BHR by the intra-nasal vaccination with BCG of days old BP mice immunized with ovalbumin at different intervals 8 saline OA BCG + OVA 8 weeks post BCG immunization weeks post BCG immunization 6 weeks post BCG immunization Penh 6 3 3-3 - - 3 6 minutes 7 8 9-3 - - 3 6 minutes 7 8 9-3 - - 3 6 minutes 7 8 9 BCG 73P - weeks injections at week interval of Al(OH)3 + OVA µg/kg µ s.c. week hours provocation OVA µg or saline i.n. BHR Figure : Interference with antigen-induced cytokine production in the BALF by the BCG vaccination of days old BP mice immunized with ovalbumin after different intervals Control BCG 73P IL- 3 IL- IFN γ Pg/ml 7 3 8 6 8 6 Weeks post BCG immunization 8 6 BCG 73P i.n. 8, or 6 wks Al(OH)3 + OVA. wk s.c. injections at wk interval hours I.n.provocation BALF µg OVA or cytokines saline Figure 3: Interference with ex vivo cytokine production of the i.n. vaccination with BCG in days old BP mice immunized with OVA after 8, or 6 weeks pg/m Control IL- BCG 73P IFN-γ 3 8 6 8 6 BCG 73P i.n. 8, or 6 wks Al(OH)3 + OVA. wk s.c. injections at wk interval I.n.provocation µg OVA or saline Culture of hours tracheobronchial ganglia cells, OVA stimulation at 7 h
e cels a rea Figure : Interference with eosinophil counts in the BALF and with IgE titers by the i.n. vaccination with BCG in days old BP mice immunized with OVA after 8, or 6 weeks Eosinophil counts (x ) Control Eosinophils in BALF 8 6 units/ml BCG 73P OA-specific IgE 8 6 Wks post BCG BCG 73P i.n. 8, or 6 wks Al(OH)3 + OVA. s.c. injections at wk interval wk I.n.provocation µg OVA or saline hours BALF eosinophil counts and serum IgE determination Figure 6 : Recruitment of immunocompetent cells to the lungs after i.n. vaccination with BCG in days old BP mice after 6 h and 3 days Control BCG 73P CD86 8 CD * 6 * * * e ls/u 6h 3d 6h 3d CD C CD 8 Class II (Ia) * * 3 * 3 * * * 6h 3d 6h 3d 6h 3d Number of + cells per unit area/time post BCG immunization / u Dendritic cells recruited to the lung shortly after intranasal delivery of Mycobacterium bovis BCG drive the primary immune response towards a Th-type cytokine production. Immunology 3: 8:-3 Lagranderie M, Nahori MA, Balazuc AM, Kiefer-Biasizzo H, Lapa e Silva JR, Milon G, Marchal G, Vargaftig BB. Unités de Pharmacologie Cellulaire, Institut Pasteur, Paris, France Division of Pulmonary Medicine, Institute of Thoracic Diseases, Clementino Fraga Filho University Hospital Federal University of Rio de Janeiro, Brazil 3
Murine model of asthma BP mice (Th background) s.c. i.n. aerosol d d 7 d d Alum - OVA ( µg) Sensitization Alum - OVA ( µg) OVA ( µg) Challenge Methacholine (6 mm) Protocol for lung cells isolation 6 BCG ( CFU). i.n. Bronchoalveolar lavages Lung digestion (Collagenase, Dnase, EDTA) Separation (MACS) Alveolar Mø (AMs) Gr-, F/8+, CDb- CDb + Interstitial Mø (IMs) F/8+, CDc- CDc + Dendritic cells (DCs) F/8+, CDb- Left: phagocytosis of BCG by lung DC (A, control non-labeled BCG; B, labeled BCG). Morphology of lung DC (C,D). Right: CDc marker in lung sections (A, naive BP; B and C, BP and C7BL/6, both 8 h after i.n. BCG)
Phagocytic capacity of APCs recruited to the lungs after the i.n. delivery of labeled BCG % of phagocytic cells 8 6 I Ms AMs DCs 6 8 Hours after BCG delivery Cell surface phenotype of lung DCs CD8 CD86 MHC Class II CD8 α CDb MHC Class II and CD86 Produção de IL- p por células apresentadoras de antigeno provenientes dos pulmões IMs AMs DCs Pg / ml 6 8 96 Horas após BCG
Produção de IFN-γ por explantes pulmonares estimulados in vitro com anti-cd3 semanas apos a administração de BCG Control BCG immunized Pg / lung 8 6 8 6 9 6 Days after BCG Mycobacterium bovis BCG killed by extended freeze-drying reduced bronchial hyperresponsiveness in two animal models. J. Allergy Clin. Immunol. 8: :7-8 Lagranderie M, Abolhassani M, Vanoirbeek JA, Lefort J, Nahori MA, Lapa e Silva JR, Huerre M, Vargaftig BB, Marchal G. Unités de Pharmacologie Cellulaire, Institut Pasteur, Paris, France Division of Pulmonary Medicine, Institute of Thoracic Diseases, Clementino Fraga Filho University Hospital Federal University of Rio de Janeiro, Brazil 6
BPOVA DILUENT PAS BP OVA EFD PAS CONCLUSÕES A pesquisa básica é o eixo para a compreensão dos mecanismos envolvidos na patogenia da asma, para a identificação de novos alvos moleculares e para o desenvolvimento de novas estratégias farmacológicas, inclusive de biofármacos. jrlapa.ntg@terra.com.br 7