Imunofenotipagem e remodelamento da matriz extracelular na sarcoidose pulmonar e extrapulmonar / Immunophenotyping and extracellular matrix remodeling in pulmonary and extrapulmonary sarcoidosis

OBJETIVO: Investigar o significado de marcadores de imunidade celular e de componentes elásticos/colágeno da matriz extracelular em estruturas granulomatosas em biópsias de pacientes com sarcoidose pulmonar ou extrapulmonar. MÉTODOS: Determinações qualitativas e quantitativas de células inflamatórias, de fibras de colágeno e de fibras elásticas em estruturas granulomatosas em biópsias cirúrgicas de 40 pacientes com sarcoidose pulmonar e extrapulmonar foram realizadas por histomorfometria, imuno-histoquímica, e técnicas de coloração com picrosirius e resorcina-fucsina de Weigert. RESULTADOS: A densidade de linfócitos, macrófagos e neutrófilos nas biópsias extrapulmonares foi significativamente maior do que nas biópsias pulmonares. Os granulomas pulmonares apresentaram uma quantidade significativamente maior de fibras de colágeno e menor densidade de fibras elásticas que os granulomas extrapulmonares. A quantidade de macrófagos nos granulomas pulmonares correlacionou-se com CVF (p < 0,05), ao passo que as quantidades de linfócitos CD3+, CD4+ e CD8+ correlacionaram-se com a relação VEF1/CVF e com CV. Houve correlações negativas entre CPT e contagem de células CD1a+ (p < 0,05) e entre DLCO e densidade de fibras colágenas/elásticas (r = -0,90; p = 0,04). CONCLUSÕES: A imunofenotipagem e o remodelamento apresentaram características diferentes nas biópsias dos pacientes com sarcoidose pulmonar e extrapulmonar. Essas diferenças correlacionaram-se com os dados clínicos e espirométricos dos pacientes, sugerindo que há duas vias envolvidas no mecanismo de depuração de antígenos, que foi mais eficaz nos pulmões e linfonodos.

OBJECTIVE: To investigate the significance of cellular immune markers, as well as that of collagen and elastic components of the extracellular matrix, within granulomatous structures in biopsies of patients with pulmonary or extrapulmonary sarcoidosis. METHODS: We carried out qualitative and quantitative evaluations of inflammatory cells, collagen fibers, and elastic fibers in granulomatous structures in surgical biopsies of 40 patients with pulmonary and extrapulmonary sarcoidosis using histomorphometry, immunohistochemistry, picrosirius red staining, and Weigert's resorcin-fuchsin staining. RESULTS: The extrapulmonary tissue biopsies presented significantly higher densities of lymphocytes, macrophages, and neutrophils than did the lung tissue biopsies. Pulmonary granulomas showed a significantly higher number of collagen fibers and a lower density of elastic fibers than did extrapulmonary granulomas. The amount of macrophages in the lung samples correlated with FVC (p < 0.05), whereas the amount of CD3+, CD4+, and CD8+ lymphocytes correlated with the FEV1/FVC ratio and VC. There were inverse correlations between TLC and the CD1a+ cell count (p < 0.05), as well as between DLCO and collagen/elastic fiber density (r = -0.90; p = 0.04). CONCLUSIONS: Immunophenotyping and remodeling both showed differences between pulmonary and extrapulmonary sarcoidosis in terms of the characteristics of the biopsy samples. These differences correlated with the clinical and spirometric data obtained for the patients, suggesting that two different pathways are involved in the mechanism of antigen clearance, which was more effective in the lungs and lymph nodes.

Alveolar macrophages modulate allergic inflammation in a murine model of asthma

The role of alveolar macrophages (AMs) in the pathogenesis of asthma is still unknown. The aim of the present study was to investigate the effects of AM in the murine model of asthma. AMs were selectively depleted by liposomes containing clodronate just before allergen challenges, and changes in inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid were measured. AMs were then adoptively transferred to AM-depleted sensitized mice and changes were measured. Phenotypic changes in AMs were evaluated after in vitro allergen stimulation. AM-depletion after sensitization significantly increased the number of eosinophils and lymphocytes and the concentrations of IL-4, IL-5 and GM-CSF in BAL fluid. These changes were significantly ameliorated only by adoptive transfer of unsensitized AMs, not by sensitized AMs. In addition, in vitro allergen stimulation of AMs resulted in their gaining the ability to produce inflammatory cytokines, such as IL-1beta, IL-6 and TNF-alpha, and losing the ability to suppress GM-CSF concentrations in BAL fluid. These findings suggested that AMs worked probably through GM-CSF-dependent mechanisms, although further confirmatory experiments are needed. Our results indicate that the role of AMs in the context of airway inflammation should be re-examined.

Efecto inmunosupresor de Trypanosoma lewisi (Kinetoplastidae) sobre la multiplicación de Toxoplasma gondii (Sarcocystidae) en macrófagos alveolares y peritoneales de rata blanca

The immunosuppressant effect of T. lewisi (Kinetoplastidae) infection on the multiplication of Toxoplasma gondii (Sarcocystidae) on alveolar and peritoneal macrophages of the white rat. The immunosuppressant effect of T. lewisi infection on the multiplication of T. gondii was compared in peritoneal (MP) and alveolar macrophages (MA) of white rat. Two animal groups were infected with T. lewisi and sacrificed after four days and seven days post infection. A group without infection was maintained as a control. The number of intracellular parasites (tachyzoites) (IT) was counted by light microscopy, calculating the rate infection rate per 100 total cells (TC) and per infected cells (IC) for each group of phagocyte cells. The relation quotient IT, TC or IC multiplied percent, provided a statistical ratio (RE) of the relative number of parasites in both cellular types for each time interval. MA as well as MP obtained after 4 days showed a significant increase in the multiplication of T. gondii with respect to the control. Unlike the MP (which had an increase in the multiplication of T. gondii the fourth day of infection with T. lewisi diminishing towards the seventh day), the MA had an increase in the multiplication of the parasite from the fourth to the seventh day. This difference can be related to the route of infection used for the experiments, that affect the MP directly with a greater effect in comparison with the MA of the lungs. Lung compartment will be affected later, when the infection becomes systemic between the fourth and sixth day of infection. The immunity against T. gondii is similar between both phagocytes, but the time of infection and the compartment where the cells are located, makes the difference in the response time against T. gondii. Supernatants from macrophage cultures or T. lewisi by rat did not induced any immunosuppression. Rev. Biol. Trop. 57 (1-2): 13-22. Epub 2009 June 30.

El efecto inmunosupresor de la infección de T. lewisi sobre la multiplicación de T. gondii fue comparado en macrófagos peritoneales (MP) y alveolares (MA) de rata. El número de parásitos (taquizoitos) intracelulares (TI) fue contado por microscopía de luz. Los macrófagos alveolares y peritoneales (MP) de animales con 4 días de infección con T. lewisi muestran un aumento significativo en la multiplicación de T. gondii. A diferencia de los MP (que muestran un aumento en la multiplicación de T. gondii al cuarto día de infección con T. lewisi disminuyendo hacia el séptimo día), los MA mantienen un aumento en la multiplicación del parásito desde el cuarto, aumentando hacia el séptimo día de infección. Esta diferencia se puede deber a la ruta de infección utilizada para los experimentos que afectan directamente los MP donde se observa un efecto mayor y más temprano en comparación con los MA aislados de los pulmones, compartimiento afectado cuando la infección se vuelve sistémica entre el cuarto y sexto día de infección. La inmunidad contra T. gondii es similar entre ambas células fagocíticas, pero el tiempo de infección y el compartimiento donde se encuentren las células hace la diferencia en el tiempo de respuesta contra un parásito dado, en nuestro caso T. gondii. No hubo evidencia de que los sobrenadantes de cultivos de macrófagos provenientes de ratas infectadas ni el lisado de tripanosomas indujeran el efecto inmunosupresor.

Ação dos leucotrienos na fagocitose via receptores Fc`GAMA´ e manose em macrófagos alveolares e mecanismos moleculares envolvidos / Importance of leukotrienes en phagocytosis of Fc`GAMA´ and mannose receptor in alveolar macrophages and molecular mecanismes involved

Os leucotrienos aumentam a fagocitose e a atividade microbicida contra uma série de patógenos. Em macrófagos alveolares, LTB`IND.4´ e LTD`IND.4´ aumentam a fagocitose via Fc`GAMA´R de modo dependente de PKC. Entretanto, o papel das isoformas específicas da PKC, das MAPK, e da Pi3K neste processo, ainda não é conhecido. Além disso, pouco se sabe sobre a importância dos leucotrienos na fagocitose via outros receptores. Os objetivos deste trabalho são: a) ampliar o conhecimento sobre as vias de sinalização ativadas pelos leucotrienos durante a fagocitose de hemácias opsonizadas por IgG; b) avaliar o efeito dos leucotrienos na fagocitose de Candida albicans, por macrófagos alveolares e as vias de sinalização intracelular envolvidas. Observou-se que os leucotrienos endogenamente produzidos ou adicionados aos macrófagos alveolares, aumentam a fagocitose via Fc`GAMA´R e para isso utilizam distintas vias de sinalização intracelular. A ação do LTB`IND.4´ envolveu predominantemente a via ERK1/2 e PKC`ALFA´ e com menor intensidade da PKC`DELTA´...

Atividade fungicida e secretora de macrófagos alveolares de camundongos susceptíveis e resistentes infectados pelo P. brasiliensis / Fungicidal and secretory ability of alveolar macrophages from susceptible and resistant mice infected with P. brasiliensis

Estudos em nosso laboratório carcterizaram camundongos B10.A e A/J, respectivamente, como susceptíveis e resistentes à infecção pulmonar pelo fungo Paracoccidioides brasiliensis. A imunidade inata desempenha papel fundamental no controle inicial dos patógenos e na regulação da resposta imune adquirida. Como os macrófagos alveolares são as primeiras células do hospedeiro a interagir com o fungo, propusemo-nos a estudar a capacidade fungicida e secretora dos macrófagos alveolares de camundongos susceptíveis e resistentes ao P. brasiliensis para melhor compreender a PCM pulmonar. Camundongos B10.A e A/J normais (n: 10-15) foram submetidos a lavagem bronco-alveolar (LBA) e a suspensão celular obtida (2x'10 POT. 5' células/poço)...

Previous studies in our laboratory characterized B10.A and A/J mice as susceptible and resistant strains to pulmonary Paracoccidioides brasiliensis infection. Innate immunity plays a fundamental role on the control of the initial growth of pathogens as well as in the acquired immunity that subsequently develops. As alveolar macrophages are the first host cells to interact with P. brasiliensis we decided to study the fungicidal and secretory ability of alveolar macrophages from resistant and susceptible mice to P. brasiliensis to better understand the pulmonary model of paracoccidioidomycosis. Normal B10.A and A/J mice (n=10-15) were submitted to bronchoalveolar lavage (BAL) and cell suspensions (2x105 cells/well) were pre-activated ovemight with IFN-γ, IL-12 or the combination of these two cytokines (50,000, 10,000 and 2,000 pg/rnL). After that, macrophages were in vitro challenged with P. brasiliensis yeasts (1:50 fungus: macrophage ratio) and 72h later fungicidal activity was determined by colony forming units counts (CFU). Nitrite and cytokines production were determined in culture supematants by Griess reaction and ELISA, respectively. Data were expressed as means ± SE and analyzed by Student's t test. Our results showed that B10.A macrophages pre-activated with the different assayed concentrations of IFNγ, IL-12 or both cytokines presented elevated fungicidal ability (51¬-97%) concomitant with the presence of high levels of NO, IL-12 and MCP-l and low amounts of IL-10 and GM-CSF. NO synthesis occurred in low levels but high concentrations of IL-10 and GM-CSF associated to low amounts of IL-12 and MCP-1 were detected in the co-cultures supematants. NO synthesis inhibition by aminoguanidine clearly showed that the fungicidal ability of B10.A but not of A/J macrophages was NO¬ dependent. Treatment ...

Immunology of tuberculosis.

Tuberculosis is a major health problem throughout the world causing large number of deaths, more than that from any other single infectious disease. The review attempts to summarize the information available on host immune response to Mycobacterium tuberculosis. Since the main route of entry of the causative agent is the respiratory route, alveolar macrophages are the important cell types, which combat the pathogen. Various aspects of macrophage-mycobacterium interactions and the role of macrophage in host response such as binding of M. tuberculosis to macrophages via surface receptors, phagosome-lysosome fusion, mycobacterial growth inhibition/killing through free radical based mechanisms such as reactive oxygen and nitrogen intermediates; cytokine-mediated mechanisms; recruitment of accessory immune cells for local inflammatory response and presentation of antigens to T cells for development of acquired immunity have been described. The role of macrophage apoptosis in containing the growth of the bacilli is also discussed. The role of other components of innate immune response such as natural resistance associated macrophage protein (Nramp), neutrophils, and natural killer cells has been discussed. The specific acquired immune response through CD4 T cells, mainly responsible for protective Th1 cytokines and through CD8 cells bringing about cytotoxicity, also has been described. The role of CD-1 restricted CD8(+) T cells and non-MHC restricted gamma/deltaT cells has been described although it is incompletely understood at the present time. Humoral immune response is seen though not implicated in protection. The value of cytokine therapy has also been reviewed. Influence of the host human leucocyte antigens (HLA) on the susceptibility to disease is discussed. Mycobacteria are endowed with mechanisms through which they can evade the onslaught of host defense response. These mechanisms are discussed including diminishing the ability of antigen presenting cells to present antigens to CD4(+) T cells; production of suppressive cytokines; escape from fused phagosomes and inducing T cell apoptosis. The review brings out the complexity of the host-pathogen interaction and underlines the importance of identifying the mechanisms involved in protection, in order to design vaccine strategies and find out surrogate markers to be measured as in vitro correlate of protective immunity.

Mac-1-mediated Uptake and Killing of Bordetella bronchiseptica by Porcine Alveolar Macrophages

The role of Mac-1 as a receptor for Bordetella bronchiseptica infection of alveolar macrophages (AMphi) was examined using 6 strains (2 ATCC and 4 pathogenic field isolates) to assess B. bronchiseptica binding, uptake and replication in primary porcine AMphi. All B. bronchiseptica strains were rapidly killed by porcine serum in a dose- and time-dependent manner. However, heat-inactivated porcine serum (HIS) did not demonstrate any bacterial-killing activity, suggesting that complement may have a direct killing activity. All field isolates were more resistant to direct complement-mediated B. bronchiseptica killing. The uptake of B. bronchiseptica into AMphi was inhibited approximately 50% by antiMac-1 monoclonal antibodies in the medium. However, B. bronchiseptica phagocytosed in the presence of serum or HIS was not altered by anti-Mac-1 antibodies although more bacteria were internalized by addition of serum or HIS. These data suggest that Mac-1 is a target for direct uptake of B. bronchiseptica via opsoninindependent binding. The phagocytosed B. bronchiseptica, either via direct or serum-mediated binding, were efficiently killed by AMphi within 10 hr postinfection. This demonstrates that Mac-1-mediated B. bronchiseptica uptake is a bacterial killing pathway not leading to productive infections in AMphi.

Interleukin-5 production by peripheral blood monocytes from asthmatic patients: effect of alveolar macrophages

This study aimed to assess the role of ALM in the pathogenesis of bronchial asthma by studying the effect of ALM derived from bronchoalveolar lavage [BAL] on IL-5 production by peripheral blood monocytes PBMC [including T lymphocytes] in cocultures in patients with bronchial asthma as compared to that in non asthmatic individuals. Nineteen patients with bronchial asthma were enrolled in this study. Ten normal non-smoker subjects were considered as controls. Patients were subjected to broncoalveolar lavage [BAL]. The lavage fluid was cultured in 3 wells; one with peripheral blood monocytes [PBMC], another with PBMC with mitogen stimulation and the third with PBMC with BAL cells and stimulation. Cultures were incubated and the supernatants were assayed for IL-5 by EL1SA. The mean [SD] age for the asthmatic patients was 37.67[9.66] years with a mean [SD] body mass index of 28.3[6.12]. Male constituted 53% [11/19] of the studied asthmatic patients. The levels of 1L-5 in the supernatant of resting PBMN cultures were significantly higher in patients with asthma in all three states [basal state, after PHA stimulation, in cocultures with ALM [mean [SD] = 219.45[68.34] ng/ml, range [100-320 ng/ml], 484.85[115048.01], range [170-670 ng/ml], 1118 [336.59], range [530-1800 ng/ml], respectively. The respective levels in the nonatopic normal subjects was [mean [SD]: 21.20[8.97], 26.8[10.10], 29.30[7.87]]. The differences between the three states in the asthmatic patients were highly significant. The changes between the three states in the non-asthmatic patients were insignificant. IL5 production by PBMN is markedly increased in asthmatic patients versus non-asthmatic subjects, furthermore, IL-5 production was markedly amplified by co-culturing PBMN with autologous ALM derived from BAL in the asthmatics patients. This is in contrast to the finidings in non-asthmatic subjects where IL5 production was not augmented by autologous ALM. The fact that ALM from non-asthmatic subjects functioned poorly as APC may represent a local inhibitory protective mechanism in the airways

Avaliaçäo do estado funcional dos macrófagos alveolares de ratos tratados com amiodarona / Evaluation of alveolar mascrophage functions of amiodarone treated rats

Embora a amiodarona seja um antiarrítmico efetivo, seu uso está associado a vários efeitos colaterais. Entre esses, o mais sério é a toxicidade pulmonar caracterizada por pneumonite intersticial e fibrose. A patogênese da toxicidade pulmonar näo está determinada. Contudo, o aspecto morfológico proeminente induzido pela amiodarona é a fosfolipidose. O efeito da fosfolipidose sobre as funçöes macrofágicas, permanece ainda obscuro. A hipótese levantada por este trabalho é que macrófagos alveolares de ratos tratados com amiodarona apresentam alteraçöes funcionais que contribuem para o aparecimento da pneumonite. Para testar esta hipótese, estudou-se o LBA e a histologia pulmonar em três linhagens de ratos (Wistar, Wistar-Kyoto e SHR), de ambos os sexos, avaliando a tipagem e quantificaçäo de células (Experimento 1). Em ratos machos da linhagem Wistar tratados com amiodarona, cujos macrófagos alveolares apresentaram acentuada fosfolipidose, estudou-se as funçöes macrofágicas básicas (espraiamento e liberaçäo de H2O2) e a expressäo de moléculas de adesäo (CD18 e e CD54) em sua superfície, por citometria de fluxo (Experimento 2). Os resultados destes experimentos mostraram que o uso da linhagem Wistar para estudo da intoxicaçäo por amiodarona é eficiente em simular como o modelo experimental, as alteraçöes na celularidade do LBA e a fosfolipidose pulmonar. A intoxicaçäo por amiodarona produzida em animais também apresenta predileçäo pelo sexo masculino. As linhages de ratos Wistar-Kyoto e SHR, apesar de serem isogênicas, näo servem como modelo de pneumonite por amiodarona devido à grande suceptibilidade à infecçöes e hemorragia. Os estudos funcionais dos macrófagos alveolares revelaram que estas células encontram-se ativadas durante todo o período de estudo. O pico de maior ativaçäo macrofágica ocorreu com seis semanas de tratamento com amiodarona, provavelmente por maior recrutamento de monócitos mostrado pelo aumento de expressäo das moléculas de adesäo. A fosfolipidose näo inibe as funçöes macrofágicas e, neste modelo, os macrófagos alveolares estäo ativados pelo amiodarona e/ou fosfolipidose, gerando uma resposta inflamatória após seis semanas do uso da droga; resposta esta näo mais observada na 12ª semana.

Inmunología y patogénesis de tuberculosis / Immunology and phatogenesis of tuberculosis

La resistencia adquirida a tuberculosis se realiza por mecanismos de inmunidad medida por células, siendo los principales factores los macrófagos y linfocitos T. Ambos son indispensables para el desarrollo de inmunidad, pero también contribuyen en la patogénesis

Tabaquismo: alteraciones producidas por el uretano en los receptores de macrófagos y el sistema del complemento / Smoking: alterations by urethano in macrophages, complement system

Se comunican los resultados de un análisis de la acción del uretano sobre las células inmunocompetentes (macrófagos), el sistema del complemento y también en los enterocitos. El estudio de los receptores de macrófagos por medio de la formación de rosetas puso de manifiesto que concentraciones altas de uretano destruyen a los macrófagos y disminuyen la unión con IgG y el tercer componente del complemento. Los resultados obtenidos experimentalmente in vitro sugieren que de acuerdo con el grado y tiempo de la exposición al humo de tabaco se puede romper el equilibrio del sistema inmunitario y de otras poblaciones celulares que desembocan en los procesos patológicos que se relacionan con tabaquismo