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IL-13+ T Cells in (Human) Asthma: Is There a Link to Steroid-Resistance
S242 Abstracts
Number and Phenotypes of Dendritic Cells in the Bronchial Mucosa of Asthmatics and Controls A. V. H. Jayaratnam, S. Ying, Q. Meng, C. Corrigan; Department of Asthma, Allergy and Respiratory Sciences, King’s College London School of Medicine at Guy’s, King’s College and St. Thomas’s Hospitals, London, UNITED KINGDOM. RATIONALE: Dendritic cells (DC) regulate cytokine production by T cells at mucosal surfaces and have recently become identifiable in tissue sections by the development of phenotype specific antibodies against the markers BDCA-1/3 (expressed on myeloid DC) and BDCA-2/4 (plasmacytoid DC). As a prelude to investigating chemokine and other mediator expression by mucosal DC in asthma, we hypothesized that DC of all phenotypes are identifiable in the bronchial mucosa of asthmatics compared with controls, but their numbers differ. METHODS: We used immunohistochemistry with validated antibodies (anti-BDCA-1,2,3 and 4) and digital image analysis to measure the numbers of DC in the submucosa in sections of endobronchial biopsies from 9 asthmatics (mean FEV1 87 ± 9.44% predicted, age 21 - 59 years) and 9 controls (mean FEV1 95 ± 4.20% predicted, age 19 - 39 years). RESULTS: Mean (±SEM) numbers of cell/mm2 of submucosa expressing BDCA-1, 2,3,4 were 8.04 ± 3.03, 31.90 ± 11.98, 20.77 ± 5.77 and 26.71 ±10.64 respectively in the asthmatics and 45.97 ± 14.37, 28.43 ± 7.03, 49.87 ± 7.84 and 38.95 ± 11.57 in the controls. Mean total numbers of BDCA-3 and BDCA-1 positive cells were significantly lower in asthmatics when compared to controls, whereas those of BDCA-2 and BDCA-4 positive cells were statistically equivalent. CONCLUSIONS: DC of all four BDCA phenotypes can be identified in the bronchial mucosa of asthmatics and controls. There is selective depletion of myeloid cells in asthma. Funding: GlaxoSmithKline UK Limited
935
Toll-like Receptor Agonists and PGE2 Differentially Regulate CysLT1 Receptor Expression and Function in Human Dendritic Cells M. Rola-Pleszczynski, M. Thivierge, J. Stankova; Immunology Division, ˜ de Sherbrooke, Sherbrooke, PQ, CANADA. UniversitA© RATIONALE: Dendritic cells (DC) are key players in the development of immune responses, but the role of cysteinyl-leukotrienes in human DC function remain unclear. We studied the expression and function of the CysLT receptors during DC differentiation from monocytes and their subsequent maturation. METHODS: Receptor expression was measured by flow cytometry and real-time PCR. Responsiveness to LTD4 stimulation was assessed by calcium flux and chemotaxis. RESULTS: Maturation of DC with lipopolysaccharide (LPS), a classical Toll-like receptor (TLR)4 agonist, reduced CysLT1 expression by 50%, whereas CysLT2 expression was increased. In contrast, the TLR3 agonist poly I:C had no effect on receptor expression. Downregulation of CysLT1 expression by LPS could not be mimicked by TNFalpha alone or in combination with IL-1beta or IL-6. It was, however, prevented by inhibitors of cyclooxygenase and could be reproduced by a combination of TNFalpha and PGE2. Immature DC and DC matured with poly I:C, but not with LPS, responded to LTD4 with a robust cytosolic calcium flux, which was prevented by the CysLT1 antagonist montelukast. LTD4 induced DC chemotaxis and enhanced DC migration in response to CCL19 in DC matured with poly I:C, but only weakly in DC matured with LPS. CONCLUSIONS: Our data suggest that responsiveness of human DC to LTD4 depends on their maturational stimuli, which can differentially modulate DC involvement in allergic inflammation. Funding: Canadian Institutes of Health Research
936
MONDAY
CD8+/BLT1+/IL-13+ T Cells in (Human) Asthma: Is There a Link to Steroid-Resistance E. W. Gelfand; Department of Pediatrics, National Jewish Medical and Research Centr, Denver, CO.
937
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
RATIONALE: In murine models of allergen-induced airway hyperresponsiveness and inflammation, a subset of CD8 T cells expresses the high affinity receptor for leukotriene B4 (BLT1) and produce IL-13. BLT1 is required for CD8 T-cell accumulation in the lungs. METHODS: To determine if this unique subset of CD8 T cells is present in human asthmatics, BAL cells and lung tissue were stained (3-color fluorescence) with antibodies against CD8, BLT1, and IL-13. BAL macrophages were examined for spontaneous release of LTB4. RESULTS: In lung tissue from asthmatics, the number of CD8+/BLT1+ T cells was increased with no such cells in normal tissue. Triple-stained CD8+/BLT1+/IL-13+ T cells were demonstrated in BAL fluid of asthmatics and not from normals. Moreover, the number of these cells appeared higher in corticosteroid-resistant individuals compared to sensitive individuals. Macrophages from resistant individuals possess unique features when compared to cells from sensitive individuals. Strikingly, when spontaneous LTB4 release from BAL macrophages was examined, steroid-resistant macrophages released 3-fold more LTB4 than steroidsensitive cells. CONCLUSIONS: We identified a unique and important subset of IL-13producing CD8+T cells in both allergic mice and asthmatics. Expression of the high affinity receptor for LTB4 is apparently critical for their accumulation in the lungs and airways. Increased production of LTB4 may be correlated with increased numbers of these CD8+T cells. Corticosteroidresistance and increased LTB4 release from airway macrophages in these patients may lead to the increased accumulation of these pathologic CD8+/BLT1+/IL-13+ in the airways contributing to persistent and refractory asthma. Supported by NIH grants HL-36577, HL-61005, and EPA grant R825702. Funding: NIH and EPA Regulatory Role of CD4+CD25+ T Cells in the Antigen Specific Induction of Tolerance in a Murine Model of Asthma C. Boudousquié, C. Longaretti, N. Barbier, F. Spertini; Centre Hospitalier Universitaire Vaudois, LAUSANNE, SWITZERLAND. RATIONALE: CD4+CD25+ natural regulatory T cells (Treg) are key players in controlling the development of asthma and allergic disease. The role of CD25+ T cells in the mechanisms leading to tolerance in an established model of asthma has not yet been defined. METHODS: OVA-sensitized BALB/c mice (PC61-OVA) were depleted of CD25+ cells by intraperitoneal injection of mAb PC61 four days (D31) before intranasal treatment (INT) with OVA for three days (D35-37) and challenged thrice by OVA aerosols 10 days later (D45-47-49). T cell subsets were characterized by flow cytometry and suppressor activity by cell proliferation assay in coculture systems. RESULTS: After INT, CD25 non-depleted mice (INT-OVA) were protected from OVA aerosol exposure as shown by a strongly limited influx of eosinophils into bronchoalveolar lavage fluid (BALF) and induction of T cell hyporesponsiveness. OVA specific CD4+IL-10+ TR1-type T cells peaked at D43. In contrast, in PC61-OVA mice, CD25 depletion severely hampered tolerance induction as indicated by a marked enhancement in eosinophil recruitment into BALF and a vigorous antigen specific T cell response to OVA upon allergen challenge. Both in bronchial lymph nodes and lungs, CD4+CD25+Foxp3+ T cells were downregulated after INT (D37, 0.02%) and prior to challenge (D43, 1%) as compared to INT-OVA mice (3% and 2.1% respectively). CONCLUSIONS: CD4+CD25+ T cell depletion of OVA sensitized mice was able to reverse tolerance to OVA, suggesting the depletion of cells with regulatory potentials. The relationship between CD25+ natural Treg and inducible IL-10+ TR1-type Treg will have to be defined. Funding: Swiss National Science Foundation
938
Number and Phenotypes of Dendritic Cells in the Bronchial Mucosa of Asthmatics and Controls A. V. H. Jayaratnam, S. Ying, Q. Meng, C. Corrigan; Department of Asthma, Allergy and Respiratory Sciences, King’s College London School of Medicine at Guy’s, King’s College and St. Thomas’s Hospitals, London, UNITED KINGDOM. RATIONALE: Dendritic cells (DC) regulate cytokine production by T cells at mucosal surfaces and have recently become identifiable in tissue sections by the development of phenotype specific antibodies against the markers BDCA-1/3 (expressed on myeloid DC) and BDCA-2/4 (plasmacytoid DC). As a prelude to investigating chemokine and other mediator expression by mucosal DC in asthma, we hypothesized that DC of all phenotypes are identifiable in the bronchial mucosa of asthmatics compared with controls, but their numbers differ. METHODS: We used immunohistochemistry with validated antibodies (anti-BDCA-1,2,3 and 4) and digital image analysis to measure the numbers of DC in the submucosa in sections of endobronchial biopsies from 9 asthmatics (mean FEV1 87 ± 9.44% predicted, age 21 - 59 years) and 9 controls (mean FEV1 95 ± 4.20% predicted, age 19 - 39 years). RESULTS: Mean (±SEM) numbers of cell/mm2 of submucosa expressing BDCA-1, 2,3,4 were 8.04 ± 3.03, 31.90 ± 11.98, 20.77 ± 5.77 and 26.71 ±10.64 respectively in the asthmatics and 45.97 ± 14.37, 28.43 ± 7.03, 49.87 ± 7.84 and 38.95 ± 11.57 in the controls. Mean total numbers of BDCA-3 and BDCA-1 positive cells were significantly lower in asthmatics when compared to controls, whereas those of BDCA-2 and BDCA-4 positive cells were statistically equivalent. CONCLUSIONS: DC of all four BDCA phenotypes can be identified in the bronchial mucosa of asthmatics and controls. There is selective depletion of myeloid cells in asthma. Funding: GlaxoSmithKline UK Limited
935
Toll-like Receptor Agonists and PGE2 Differentially Regulate CysLT1 Receptor Expression and Function in Human Dendritic Cells M. Rola-Pleszczynski, M. Thivierge, J. Stankova; Immunology Division, ˜ de Sherbrooke, Sherbrooke, PQ, CANADA. UniversitA© RATIONALE: Dendritic cells (DC) are key players in the development of immune responses, but the role of cysteinyl-leukotrienes in human DC function remain unclear. We studied the expression and function of the CysLT receptors during DC differentiation from monocytes and their subsequent maturation. METHODS: Receptor expression was measured by flow cytometry and real-time PCR. Responsiveness to LTD4 stimulation was assessed by calcium flux and chemotaxis. RESULTS: Maturation of DC with lipopolysaccharide (LPS), a classical Toll-like receptor (TLR)4 agonist, reduced CysLT1 expression by 50%, whereas CysLT2 expression was increased. In contrast, the TLR3 agonist poly I:C had no effect on receptor expression. Downregulation of CysLT1 expression by LPS could not be mimicked by TNFalpha alone or in combination with IL-1beta or IL-6. It was, however, prevented by inhibitors of cyclooxygenase and could be reproduced by a combination of TNFalpha and PGE2. Immature DC and DC matured with poly I:C, but not with LPS, responded to LTD4 with a robust cytosolic calcium flux, which was prevented by the CysLT1 antagonist montelukast. LTD4 induced DC chemotaxis and enhanced DC migration in response to CCL19 in DC matured with poly I:C, but only weakly in DC matured with LPS. CONCLUSIONS: Our data suggest that responsiveness of human DC to LTD4 depends on their maturational stimuli, which can differentially modulate DC involvement in allergic inflammation. Funding: Canadian Institutes of Health Research
936
MONDAY
CD8+/BLT1+/IL-13+ T Cells in (Human) Asthma: Is There a Link to Steroid-Resistance E. W. Gelfand; Department of Pediatrics, National Jewish Medical and Research Centr, Denver, CO.
937
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
RATIONALE: In murine models of allergen-induced airway hyperresponsiveness and inflammation, a subset of CD8 T cells expresses the high affinity receptor for leukotriene B4 (BLT1) and produce IL-13. BLT1 is required for CD8 T-cell accumulation in the lungs. METHODS: To determine if this unique subset of CD8 T cells is present in human asthmatics, BAL cells and lung tissue were stained (3-color fluorescence) with antibodies against CD8, BLT1, and IL-13. BAL macrophages were examined for spontaneous release of LTB4. RESULTS: In lung tissue from asthmatics, the number of CD8+/BLT1+ T cells was increased with no such cells in normal tissue. Triple-stained CD8+/BLT1+/IL-13+ T cells were demonstrated in BAL fluid of asthmatics and not from normals. Moreover, the number of these cells appeared higher in corticosteroid-resistant individuals compared to sensitive individuals. Macrophages from resistant individuals possess unique features when compared to cells from sensitive individuals. Strikingly, when spontaneous LTB4 release from BAL macrophages was examined, steroid-resistant macrophages released 3-fold more LTB4 than steroidsensitive cells. CONCLUSIONS: We identified a unique and important subset of IL-13producing CD8+T cells in both allergic mice and asthmatics. Expression of the high affinity receptor for LTB4 is apparently critical for their accumulation in the lungs and airways. Increased production of LTB4 may be correlated with increased numbers of these CD8+T cells. Corticosteroidresistance and increased LTB4 release from airway macrophages in these patients may lead to the increased accumulation of these pathologic CD8+/BLT1+/IL-13+ in the airways contributing to persistent and refractory asthma. Supported by NIH grants HL-36577, HL-61005, and EPA grant R825702. Funding: NIH and EPA Regulatory Role of CD4+CD25+ T Cells in the Antigen Specific Induction of Tolerance in a Murine Model of Asthma C. Boudousquié, C. Longaretti, N. Barbier, F. Spertini; Centre Hospitalier Universitaire Vaudois, LAUSANNE, SWITZERLAND. RATIONALE: CD4+CD25+ natural regulatory T cells (Treg) are key players in controlling the development of asthma and allergic disease. The role of CD25+ T cells in the mechanisms leading to tolerance in an established model of asthma has not yet been defined. METHODS: OVA-sensitized BALB/c mice (PC61-OVA) were depleted of CD25+ cells by intraperitoneal injection of mAb PC61 four days (D31) before intranasal treatment (INT) with OVA for three days (D35-37) and challenged thrice by OVA aerosols 10 days later (D45-47-49). T cell subsets were characterized by flow cytometry and suppressor activity by cell proliferation assay in coculture systems. RESULTS: After INT, CD25 non-depleted mice (INT-OVA) were protected from OVA aerosol exposure as shown by a strongly limited influx of eosinophils into bronchoalveolar lavage fluid (BALF) and induction of T cell hyporesponsiveness. OVA specific CD4+IL-10+ TR1-type T cells peaked at D43. In contrast, in PC61-OVA mice, CD25 depletion severely hampered tolerance induction as indicated by a marked enhancement in eosinophil recruitment into BALF and a vigorous antigen specific T cell response to OVA upon allergen challenge. Both in bronchial lymph nodes and lungs, CD4+CD25+Foxp3+ T cells were downregulated after INT (D37, 0.02%) and prior to challenge (D43, 1%) as compared to INT-OVA mice (3% and 2.1% respectively). CONCLUSIONS: CD4+CD25+ T cell depletion of OVA sensitized mice was able to reverse tolerance to OVA, suggesting the depletion of cells with regulatory potentials. The relationship between CD25+ natural Treg and inducible IL-10+ TR1-type Treg will have to be defined. Funding: Swiss National Science Foundation
938