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Application of live and heat killed BCG but not purified protein derivative from Mycobacterium tuberculosis inhibits allergen-induced airway eosinophilia
J ALLERGY CLIN IMMUNOL
Abstracts
$317
VOLUME 109, NUMBER 1
" 7 ~ Analysis of Differential Gene Expression in Human Peripheral
' 7 " 7 Lung Inflammation Induced by Mixed T Cell Responses: Critical
9 JF ~JI Blood T Cells From Patients With Atopic Dermatitis
9 E/Mediators
Yoshiko Matsumoto*, Tadahiro Oshida*, Yukiho lmai*, Ning Lu Yoshida*, Keiko Matsui*, Yuji Sugita*, Akira Akasawa§, Hirohisa Saito¥ *Genox
Janice Hwang*, David Yen§, Iris Shahar¥, Donna Rennick§, Debra D Donaldson~, Naftali Kaminski¥, Gabriele Grunig* *Columbia Universi-
Research, Incorporated, Kawasaki, Kanagawa, Japan §National Children's Medical Research Center, 3-35-31, Taishido, Setagaya, Tokyo, Japan ~qational Children's Hospital, 3-35-3 l, Taishido, Setagaya, Tokyo, Japan Analysis of genes that are differentially expressed between patients with atopic dermatitis and normal volunteers will provide important information on the underlying molecular mechanisms for the development of the disease. Transcripts in peripheral T cells from 59 subjects were analyzed by a fluorescent differential display method. Approximately 100 differentially amplified DNA fragments were sequenced. The sequences were matched to known genes, full-length cDNAs, ESTs and genome sequences in public databases. The 80 known genes could be classified into many functionally different categories: cytokines, receptors, signal transduction factors, ribosomal factors and others. To confirm the results of the differential display experiments, expression levels of the selected genes were measured by a real-time quantitative RT-PCR method. A number of known and novel genes, including heat shock protein 40 and vasopressin-activated calciummobilizing receptor 1 (VACM-1, cullin5), were shown to be more highly expressed in the patients than in the normal volunteers. These genes may be involved in the pathogenesis of atopic dermatitis.
ty, New York, NY §DNAX Research Institute for Molecular and Cellular Biology, Pain Alto, CA ¥Sheba Medical Center, Tel Hashomer, Israel ~Genetics Institute, Cambridge, MA Asthma is an epidemic of world-wide dimensions. The immunopathogenesis has been shown to be strongly dependent on the development ofT helper 2 (Th2) responses to inhaled antigen. Recently, however, multiple reports have shown that T helper 1 (Thl) responses appear to be critical too. For example, cytokines of the Th2 and of the Th 1 type are simultaneously present in the airways of asthmatic subjects. The expression of these cytokines is increased relative to control subjects. Furthermore, a desensitization protocol with specific antigen that reduced the immediate and the late phase asthmatic response to the antigen led to the simultaneous decrease of Th2 and Thl cytokine production in response to the antigen. Some of the asthma-mouse models depend on the simultaneous presence of Thl and Th2 cytokines for the full development of the asthma phenotype, including airway hyperreactivity. However, mechanistic studies designed to identify critical mediators that are elaborated in the lungs exposed to cytokines produced in the course of mixed T cell responses are scarce. We have shown that IFNy (the signature Thl cytokine) and IL-13 (a Th2 cytokine that is critically important for the elaboration of the asthma phenotype) when used simultaneously "for intranasal challenge of mice, induces complex inflammation in the lungs. The inflammatory index is higher in these mice as compared to mice given either IL- 13 or IFNyalone or control protein. Goblet cell hyperplasia is inhibited when compared to mice given IL-13 alone. IL-6 levels are much increased when compared to mice given either IL- 13 or IFNy alone or control protein. The composition of the inflammatory cells in the airways is significantly altered, eosinophils are decreased (relative to mice given IL-13), NK cells and cells with a phenotype reminiscent of dendritic cells are increased (relative to mice given either IL-13 or IFNy alone or control protein). This indicates a complex regulation of gene expression in mice given IL-13 and 1FNy as compared to mice given either IL- 13 or IFNy alone or control protein. Therefore, lungs of groups of mice were examined for gene expression by real time PCR and by microarray analysis. The role of selected chemokines for the migration of airway inflammatory cells was then analyzed in vitro.
'7~
T-Helper and T-Suppressor Lymphecytes in Bronchoalveelar
Space During Efferent Phase of Lung Delayed Type Hypersensi9 A F U tivity Reaction Induced With Single Antigen Inhalation Hanna Grubek-Jaworska*, Grazyna Hoser§, Pawel Droszcz*, Ryszarda Chazan* *Medical University of Warsaw, Warsaw, Poland §Center for Postgraduate Medical Education, Warsaw, Poland The precise mechanism(s) involved in pathogenesis of hypersensitivity pneumonitis (HP) has not been determined. HP is characterised by inflammatory lymphocytic alveolitis and remarkable T-lymphocyte increase detected in the bronchoalveolar lavage fluid (BALF). It is suggested that both helper (CD4+) and suppressor (CD8+) T cells might contribute to the pathogenesis of HP. Experiments on animal models suggest that more important for pathogenesis of HP is cell mediated immunity with expression of delayed type hypersensitivity (dth) than complex-AgAb-mediated immunity, but the relationship between the subsets of BALF lymphocytes and humoral or cell-mediated allergic reactions is still not clear. The aim of our study was distinction CD4+ and CD8+ T cells in BALF lymphocyte during experimental lung dth reaction. The experiment was performed on male Dunkin Hartley (Charles River) guinea pigs sensitised with BCG vaccine and inhaled with tubercle bacilli antigens (tuberculin).The presence of spcific IgE and IgG antibodies against mycobacterial antigen in the serum of BCG immunized animals was excluded using PCA method. 24 h after tuberculin inhalation (at the time of max influx of lymphocyte into alveolar space in sensitised guinea pigs), in sensitised and non-sensitised (control) animals bronchoalveolar lavage was performed. Total cell number estimation, differential microscopical examination of BAL-fluid cells and phenotyping of BAL-fluid lymphocyte (by flow cytometry) were performed. In BALF of sensitised guinea pigs, as compared to the control, statistically significant increase of percent and absolute number of T, CD4+ and CD8+ lymphocytes were observed. BALF lymphocytes in sensitized animals showed a slightly more intense expression of specific phenotype markers. The CD4/CD8 ratio in both studied groups did not differ significantly and was individually variable (2.94 +/- 0.72 SEM in experimental vs 4.41 +/1.29 SEM in control group). We conclude that both CD4+ and CD8+ lymphocytes (with some predominance of helper cells) particpate in the efferent phase of lung delayed type hypersensitivity reaction induced by antigen inhalation.
" 7 g Application of Live and Heat Killed BCG but Not Purified Protein Derivative From MycobacteriumTuberculosisInhibits AllergenInduced Airway Eosinophilia
9 /U
Tamgls Major*, Gisela Wohlleben§, Klaus J Erb§ *Department of Pulmonology, Semmelweis University Budapest, Hungary, Budapest, Hungary §Center for Infectious Diseases, University of Wuerzburg, Wuerzburg, Germany The increased prevalence of asthma over the past decades has become a major public health issue for the industrialised world. It has been proposed that this increase is due to the steady decline of infectious diseases such as tuberculosis. Supporting this view was our previously published finding, that an infection of the lung with Mycobacterium bovis-Bacillus CalmetteGuerin (BCG) inhibited ovalbumin (OVA) induced airway eosinophilia in a murine model of asthma. Next we investigated, if the BCG had to be alive to mediate this effect. For this purpose we treated mice intranasally with either heat killed BCG (HKBCG) or purified protein derivative of M. tuberculosis (PPD) and analysed if the treated mice developed airway eosinophilia after intraperitoneal OVA-priming and subsequent OVA airway-challenge. Our results clearly showed that HKBCG but not PPD (given 4 or 8 weeks prior to allergen airway challenge) resulted in a strong suppression of airway eosinophilia. The inhibition of airway eosinophilia correlated with a reduction of Th2 type cells in the lung. Furthermore, HKBCG-induced suppression of airway eosinophilia was strongly reduced in IFN-y deficient mice. Taken together, our data clearly shows that the application of HKBCG but not PPD can protected mice from developing allergen-induced airway eosinophilia. HKBCG in contrast to live BCG may also be a promising candidate for a prospective asthma vaccine in humans since negative side effects due to mycobacterial infection can be ruled out.
Abstracts
$317
VOLUME 109, NUMBER 1
" 7 ~ Analysis of Differential Gene Expression in Human Peripheral
' 7 " 7 Lung Inflammation Induced by Mixed T Cell Responses: Critical
9 JF ~JI Blood T Cells From Patients With Atopic Dermatitis
9 E/Mediators
Yoshiko Matsumoto*, Tadahiro Oshida*, Yukiho lmai*, Ning Lu Yoshida*, Keiko Matsui*, Yuji Sugita*, Akira Akasawa§, Hirohisa Saito¥ *Genox
Janice Hwang*, David Yen§, Iris Shahar¥, Donna Rennick§, Debra D Donaldson~, Naftali Kaminski¥, Gabriele Grunig* *Columbia Universi-
Research, Incorporated, Kawasaki, Kanagawa, Japan §National Children's Medical Research Center, 3-35-31, Taishido, Setagaya, Tokyo, Japan ~qational Children's Hospital, 3-35-3 l, Taishido, Setagaya, Tokyo, Japan Analysis of genes that are differentially expressed between patients with atopic dermatitis and normal volunteers will provide important information on the underlying molecular mechanisms for the development of the disease. Transcripts in peripheral T cells from 59 subjects were analyzed by a fluorescent differential display method. Approximately 100 differentially amplified DNA fragments were sequenced. The sequences were matched to known genes, full-length cDNAs, ESTs and genome sequences in public databases. The 80 known genes could be classified into many functionally different categories: cytokines, receptors, signal transduction factors, ribosomal factors and others. To confirm the results of the differential display experiments, expression levels of the selected genes were measured by a real-time quantitative RT-PCR method. A number of known and novel genes, including heat shock protein 40 and vasopressin-activated calciummobilizing receptor 1 (VACM-1, cullin5), were shown to be more highly expressed in the patients than in the normal volunteers. These genes may be involved in the pathogenesis of atopic dermatitis.
ty, New York, NY §DNAX Research Institute for Molecular and Cellular Biology, Pain Alto, CA ¥Sheba Medical Center, Tel Hashomer, Israel ~Genetics Institute, Cambridge, MA Asthma is an epidemic of world-wide dimensions. The immunopathogenesis has been shown to be strongly dependent on the development ofT helper 2 (Th2) responses to inhaled antigen. Recently, however, multiple reports have shown that T helper 1 (Thl) responses appear to be critical too. For example, cytokines of the Th2 and of the Th 1 type are simultaneously present in the airways of asthmatic subjects. The expression of these cytokines is increased relative to control subjects. Furthermore, a desensitization protocol with specific antigen that reduced the immediate and the late phase asthmatic response to the antigen led to the simultaneous decrease of Th2 and Thl cytokine production in response to the antigen. Some of the asthma-mouse models depend on the simultaneous presence of Thl and Th2 cytokines for the full development of the asthma phenotype, including airway hyperreactivity. However, mechanistic studies designed to identify critical mediators that are elaborated in the lungs exposed to cytokines produced in the course of mixed T cell responses are scarce. We have shown that IFNy (the signature Thl cytokine) and IL-13 (a Th2 cytokine that is critically important for the elaboration of the asthma phenotype) when used simultaneously "for intranasal challenge of mice, induces complex inflammation in the lungs. The inflammatory index is higher in these mice as compared to mice given either IL- 13 or IFNyalone or control protein. Goblet cell hyperplasia is inhibited when compared to mice given IL-13 alone. IL-6 levels are much increased when compared to mice given either IL- 13 or IFNy alone or control protein. The composition of the inflammatory cells in the airways is significantly altered, eosinophils are decreased (relative to mice given IL-13), NK cells and cells with a phenotype reminiscent of dendritic cells are increased (relative to mice given either IL-13 or IFNy alone or control protein). This indicates a complex regulation of gene expression in mice given IL-13 and 1FNy as compared to mice given either IL- 13 or IFNy alone or control protein. Therefore, lungs of groups of mice were examined for gene expression by real time PCR and by microarray analysis. The role of selected chemokines for the migration of airway inflammatory cells was then analyzed in vitro.
'7~
T-Helper and T-Suppressor Lymphecytes in Bronchoalveelar
Space During Efferent Phase of Lung Delayed Type Hypersensi9 A F U tivity Reaction Induced With Single Antigen Inhalation Hanna Grubek-Jaworska*, Grazyna Hoser§, Pawel Droszcz*, Ryszarda Chazan* *Medical University of Warsaw, Warsaw, Poland §Center for Postgraduate Medical Education, Warsaw, Poland The precise mechanism(s) involved in pathogenesis of hypersensitivity pneumonitis (HP) has not been determined. HP is characterised by inflammatory lymphocytic alveolitis and remarkable T-lymphocyte increase detected in the bronchoalveolar lavage fluid (BALF). It is suggested that both helper (CD4+) and suppressor (CD8+) T cells might contribute to the pathogenesis of HP. Experiments on animal models suggest that more important for pathogenesis of HP is cell mediated immunity with expression of delayed type hypersensitivity (dth) than complex-AgAb-mediated immunity, but the relationship between the subsets of BALF lymphocytes and humoral or cell-mediated allergic reactions is still not clear. The aim of our study was distinction CD4+ and CD8+ T cells in BALF lymphocyte during experimental lung dth reaction. The experiment was performed on male Dunkin Hartley (Charles River) guinea pigs sensitised with BCG vaccine and inhaled with tubercle bacilli antigens (tuberculin).The presence of spcific IgE and IgG antibodies against mycobacterial antigen in the serum of BCG immunized animals was excluded using PCA method. 24 h after tuberculin inhalation (at the time of max influx of lymphocyte into alveolar space in sensitised guinea pigs), in sensitised and non-sensitised (control) animals bronchoalveolar lavage was performed. Total cell number estimation, differential microscopical examination of BAL-fluid cells and phenotyping of BAL-fluid lymphocyte (by flow cytometry) were performed. In BALF of sensitised guinea pigs, as compared to the control, statistically significant increase of percent and absolute number of T, CD4+ and CD8+ lymphocytes were observed. BALF lymphocytes in sensitized animals showed a slightly more intense expression of specific phenotype markers. The CD4/CD8 ratio in both studied groups did not differ significantly and was individually variable (2.94 +/- 0.72 SEM in experimental vs 4.41 +/1.29 SEM in control group). We conclude that both CD4+ and CD8+ lymphocytes (with some predominance of helper cells) particpate in the efferent phase of lung delayed type hypersensitivity reaction induced by antigen inhalation.
" 7 g Application of Live and Heat Killed BCG but Not Purified Protein Derivative From MycobacteriumTuberculosisInhibits AllergenInduced Airway Eosinophilia
9 /U
Tamgls Major*, Gisela Wohlleben§, Klaus J Erb§ *Department of Pulmonology, Semmelweis University Budapest, Hungary, Budapest, Hungary §Center for Infectious Diseases, University of Wuerzburg, Wuerzburg, Germany The increased prevalence of asthma over the past decades has become a major public health issue for the industrialised world. It has been proposed that this increase is due to the steady decline of infectious diseases such as tuberculosis. Supporting this view was our previously published finding, that an infection of the lung with Mycobacterium bovis-Bacillus CalmetteGuerin (BCG) inhibited ovalbumin (OVA) induced airway eosinophilia in a murine model of asthma. Next we investigated, if the BCG had to be alive to mediate this effect. For this purpose we treated mice intranasally with either heat killed BCG (HKBCG) or purified protein derivative of M. tuberculosis (PPD) and analysed if the treated mice developed airway eosinophilia after intraperitoneal OVA-priming and subsequent OVA airway-challenge. Our results clearly showed that HKBCG but not PPD (given 4 or 8 weeks prior to allergen airway challenge) resulted in a strong suppression of airway eosinophilia. The inhibition of airway eosinophilia correlated with a reduction of Th2 type cells in the lung. Furthermore, HKBCG-induced suppression of airway eosinophilia was strongly reduced in IFN-y deficient mice. Taken together, our data clearly shows that the application of HKBCG but not PPD can protected mice from developing allergen-induced airway eosinophilia. HKBCG in contrast to live BCG may also be a promising candidate for a prospective asthma vaccine in humans since negative side effects due to mycobacterial infection can be ruled out.