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TNF-alpha and Dibutyryl cAMP Stimulate Maturation of Monocyte-derived Dendritic Cells
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Role of MAPK signaling in Dendritic Cells Maturation in Allergic Patients to Amoxicillin S. Lo´pez1, C. Mayorga1, M. Torres2, C. Antu´nez1, T. Ferna´ndez1, E. Go´mez1, P. Chaves1, M. Blanca2; 1Fundacion IMABIS, Ma´laga, SPAIN, 2 Carlos Haya Hospital, Ma´laga, SPAIN. RATIONALE: Dendritic cells (DC) are essential antigen-presenting cells in immune responses. It has been demonstrated that, in some individuals, haptens can induce DC maturation, in similar ways as some bacterial (LPS) or inflammatory (TNFa) molecules do. The role of mitogen-activated protein kinase (MAPK) in DC maturation induced by TNFa, LPS and contact sensitizers has been recently shown. The aim of this study was to analyze the role of MAPK in in vitro DC maturation induced by amoxicillin (AX), TNFa and LPS in 7 patients with a non-immediate exanthematic reaction to AX and in 6 healthy controls who tolerate this drug. METHODS: Immature monocyte-derived DC were incubated with AX, LPS or TNFa at optimal period of time for each one, and pre-treated with or without SB203580 or PD98056, which are the specific inhibitors of p38MAPK and ERK, respectively. Intracellular DC activation was determined by flow cytometry using phosphorylated p38MAPK and ERK antibodies. RESULTS: In immature-DC of patients, significant phosphorylation was found only for ERK, which was specifically inhibited by PD98056, although AX induced a semimature phenotype with HLA-DR, CD80 and CD86 expression. Controls did not show any maturation or activation with AX. TNFa and LPS induced phosphorylation of the two MAPK in both patients and controls immature-DC. CONCLUSIONS: In allergic subjects, AX by itself induces the DC maturation and ERK phosphorilation. Although it is known that ERK pathway does not affect or negatively regulate the DC maturation, this AX-induced maturation may be due to other pathways effect. Funding: SAS 5406
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Increases in Plasmacytoid but Not Myeloid Dendritic Cells (DCs) Parallel the Time Course of Allergen-Induced Cutaneous Late Responses K. T. Nouri-Aria, M. R. Jacobson, K. Furukido, D. Lee, G. Banfield, S. R. Durham; National Heart & Lung Institute, Imperial College London, London, UNITED KINGDOM. RATIONALE: DCs transport antigens from the periphery to lymphoid tissues and contribute to T cell activation and differentiation. We investigated the numbers and phenotypes of plasmacytoid (pDCs) and myeloid dendritic cells (mDCs) in cutaneous biopsies before and after allergen challenge. METHODS: 3 mm punch skin biopsies were obtained at 8 hr and 48 hr after intradermal allergen (10 biological units in 0.02 ml, ALK Abello, Denmark) and after challenge with allergen diluent from 8 Atopic (AT) and 6 nonatopic subjects (NAC). Dual immunofluorescence microscopy was used to enumerate mDCs (CD1c/BDCA-1) and pDCs (CD303/BDCA-2) and to colocalize DCs to IgE and IFN-a. RESULTS: In cutaneous biopsies from AT, pDC numbers were significantly greater at 8 hrs after allergen compared with Dil (p 5 0.03). There was also a trend for significance between AT and NAC at 8 hrs (p 5 0.08). No changes in mDCs were observed at 8 hr whereas mDCs were markedly increased at 48 hrs compared with Dil (p 5 0.02). There was also a trend for significance between AT and NAC at 48 hrs (p 5 0.07). Approximately 10% of cutaneous pDCs were IgE-positive after allergen compared to 2% in Dil. In contrast, no allergen-induced changes in IgE-positive mDCs were detected. Similarly, IFN-a expression (a hallmark of pDCs) was elevated on pDCs but not mDCs after challenge. CONCLUSIONS: The peak increase in pDC numbers (but not mDCs) that express FceRI and IFN-a coincides with the cutaneous LPR and supports the involvement of pDCs in Th2 cell polarization in man, whereas delayed (48 hr) recruitment of mDCs may possibly be involved in resolution of LPR.
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TNF-alpha and Dibutyryl cAMP Stimulate Maturation of Monocyte-derived Dendritic Cells L. M. DuBuske1, A. Y. Hancharou2, L. P. Titov2; 1Immunology Research Institute of New England, Gardner, MA, 2Research Institute for Epidemiology and Microbiology, Minsk, BELARUS. RATIONALE: Impaired monocyte-derived dendritic cells (mdDC) in patients with chronic hepatitis have been associated with reduced maturational responses. This study investigates agents to mature mdDC to produce 90% CD801/CD831 mdDC in culture. METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from whole blood of 7 patients with chronic hepatitis B and C by density centrifugation. Monocytes isolated from PBMC using adhesion to plastic were cultured for 6 days in medium with GM-CSF/IL4 to obtain immature DC (iDC) which were cultured with TNF-alpha (50 ng/ml), or dibutyryl cAMP (db-cAMP) in different concentrations or TNF-alpha plus db-cAMP (0.5 mg/ml) for 24 hours to produce mature DC (mDC). Cell phenotypes were assessed using a ‘‘FACSCalibur’’ flow cytometer with monoclonal antibodies to CD80, CD83, CD86 and HLA-DR. RESULTS: TNF-alpha induced maturation of mdDC producing 40.02 6 8.22% mature CD801/CD831 cells. Although db-cAMP at various concentrations up-regulated expression of CD80 and CD83 by mdDC up to 94.32 6 4.32% and 30.50 6 14.03% respectively, this effect was variable, seen best using 0.5 mg/ml. Combination of TNF-alpha and dbcAMP induced significant up-regulation of percent expression and relative fluorescent intensity of CD80, CD83, and CD86 expression by mdDC compared with TNF-alpha (p < 0.05), producing 90.09 6 1.30% mature mdDC. CONCLUSIONS: Combination of TNF-alpha and db-cAMP induces 90% maturation of mdDC derived from patients with chronic hepatitis.
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The Role of Mannose Receptor in Allergen Recognition by Human Dendritic Cells (DC) A. M. Ghaemmaghami, F. Shakib, L. Martinez-Pomares, C. Yang; The University of Nottingham, Nottingham, UNITED KINGDOM. RATIONALE: It is thought that the mannose receptor (MR) is partially involved in the uptake of the major house dust mite allergen Der p 1 by human DC. The aim of this study was to elucidate MR-Der p 1 interaction with a view to establishing the role of MR in Der p 1-specific Th2 responses. METHODS: Immature DC were generated from peripheral blood monocytes and MR expression was down-regulated by gene silencing (siRNA). Der p 1 uptake was analysed using flow cytometry and the effect of MR down-regulation on T cell differentiation was assessed in DC-T cell cocultures. RESULTS: We have shown that MR is the main receptor for Der p 1 on DC and that the CTLD-4-7 domain is the Der p 1 binding site on MR. Using memory and naı¨ve T cells from Der p 1 sensitized individuals, we have shown that in the absence of MR there is preferential Th1, rather Th2, polaraization (n 5 3; P < 0.05). CONCLUSIONS: MR plays a major role in recognition/uptake of Der p 1 by human DC, and this recognition appears to play an important role in the induction of Der p 1-specific Th2 responses. We therefore suggest that MR is a potential target for the treatment of allergic conditions. Funding: Asthma UK
SATURDAY
Abstracts S11
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Role of MAPK signaling in Dendritic Cells Maturation in Allergic Patients to Amoxicillin S. Lo´pez1, C. Mayorga1, M. Torres2, C. Antu´nez1, T. Ferna´ndez1, E. Go´mez1, P. Chaves1, M. Blanca2; 1Fundacion IMABIS, Ma´laga, SPAIN, 2 Carlos Haya Hospital, Ma´laga, SPAIN. RATIONALE: Dendritic cells (DC) are essential antigen-presenting cells in immune responses. It has been demonstrated that, in some individuals, haptens can induce DC maturation, in similar ways as some bacterial (LPS) or inflammatory (TNFa) molecules do. The role of mitogen-activated protein kinase (MAPK) in DC maturation induced by TNFa, LPS and contact sensitizers has been recently shown. The aim of this study was to analyze the role of MAPK in in vitro DC maturation induced by amoxicillin (AX), TNFa and LPS in 7 patients with a non-immediate exanthematic reaction to AX and in 6 healthy controls who tolerate this drug. METHODS: Immature monocyte-derived DC were incubated with AX, LPS or TNFa at optimal period of time for each one, and pre-treated with or without SB203580 or PD98056, which are the specific inhibitors of p38MAPK and ERK, respectively. Intracellular DC activation was determined by flow cytometry using phosphorylated p38MAPK and ERK antibodies. RESULTS: In immature-DC of patients, significant phosphorylation was found only for ERK, which was specifically inhibited by PD98056, although AX induced a semimature phenotype with HLA-DR, CD80 and CD86 expression. Controls did not show any maturation or activation with AX. TNFa and LPS induced phosphorylation of the two MAPK in both patients and controls immature-DC. CONCLUSIONS: In allergic subjects, AX by itself induces the DC maturation and ERK phosphorilation. Although it is known that ERK pathway does not affect or negatively regulate the DC maturation, this AX-induced maturation may be due to other pathways effect. Funding: SAS 5406
40
Increases in Plasmacytoid but Not Myeloid Dendritic Cells (DCs) Parallel the Time Course of Allergen-Induced Cutaneous Late Responses K. T. Nouri-Aria, M. R. Jacobson, K. Furukido, D. Lee, G. Banfield, S. R. Durham; National Heart & Lung Institute, Imperial College London, London, UNITED KINGDOM. RATIONALE: DCs transport antigens from the periphery to lymphoid tissues and contribute to T cell activation and differentiation. We investigated the numbers and phenotypes of plasmacytoid (pDCs) and myeloid dendritic cells (mDCs) in cutaneous biopsies before and after allergen challenge. METHODS: 3 mm punch skin biopsies were obtained at 8 hr and 48 hr after intradermal allergen (10 biological units in 0.02 ml, ALK Abello, Denmark) and after challenge with allergen diluent from 8 Atopic (AT) and 6 nonatopic subjects (NAC). Dual immunofluorescence microscopy was used to enumerate mDCs (CD1c/BDCA-1) and pDCs (CD303/BDCA-2) and to colocalize DCs to IgE and IFN-a. RESULTS: In cutaneous biopsies from AT, pDC numbers were significantly greater at 8 hrs after allergen compared with Dil (p 5 0.03). There was also a trend for significance between AT and NAC at 8 hrs (p 5 0.08). No changes in mDCs were observed at 8 hr whereas mDCs were markedly increased at 48 hrs compared with Dil (p 5 0.02). There was also a trend for significance between AT and NAC at 48 hrs (p 5 0.07). Approximately 10% of cutaneous pDCs were IgE-positive after allergen compared to 2% in Dil. In contrast, no allergen-induced changes in IgE-positive mDCs were detected. Similarly, IFN-a expression (a hallmark of pDCs) was elevated on pDCs but not mDCs after challenge. CONCLUSIONS: The peak increase in pDC numbers (but not mDCs) that express FceRI and IFN-a coincides with the cutaneous LPR and supports the involvement of pDCs in Th2 cell polarization in man, whereas delayed (48 hr) recruitment of mDCs may possibly be involved in resolution of LPR.
41
TNF-alpha and Dibutyryl cAMP Stimulate Maturation of Monocyte-derived Dendritic Cells L. M. DuBuske1, A. Y. Hancharou2, L. P. Titov2; 1Immunology Research Institute of New England, Gardner, MA, 2Research Institute for Epidemiology and Microbiology, Minsk, BELARUS. RATIONALE: Impaired monocyte-derived dendritic cells (mdDC) in patients with chronic hepatitis have been associated with reduced maturational responses. This study investigates agents to mature mdDC to produce 90% CD801/CD831 mdDC in culture. METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from whole blood of 7 patients with chronic hepatitis B and C by density centrifugation. Monocytes isolated from PBMC using adhesion to plastic were cultured for 6 days in medium with GM-CSF/IL4 to obtain immature DC (iDC) which were cultured with TNF-alpha (50 ng/ml), or dibutyryl cAMP (db-cAMP) in different concentrations or TNF-alpha plus db-cAMP (0.5 mg/ml) for 24 hours to produce mature DC (mDC). Cell phenotypes were assessed using a ‘‘FACSCalibur’’ flow cytometer with monoclonal antibodies to CD80, CD83, CD86 and HLA-DR. RESULTS: TNF-alpha induced maturation of mdDC producing 40.02 6 8.22% mature CD801/CD831 cells. Although db-cAMP at various concentrations up-regulated expression of CD80 and CD83 by mdDC up to 94.32 6 4.32% and 30.50 6 14.03% respectively, this effect was variable, seen best using 0.5 mg/ml. Combination of TNF-alpha and dbcAMP induced significant up-regulation of percent expression and relative fluorescent intensity of CD80, CD83, and CD86 expression by mdDC compared with TNF-alpha (p < 0.05), producing 90.09 6 1.30% mature mdDC. CONCLUSIONS: Combination of TNF-alpha and db-cAMP induces 90% maturation of mdDC derived from patients with chronic hepatitis.
42
The Role of Mannose Receptor in Allergen Recognition by Human Dendritic Cells (DC) A. M. Ghaemmaghami, F. Shakib, L. Martinez-Pomares, C. Yang; The University of Nottingham, Nottingham, UNITED KINGDOM. RATIONALE: It is thought that the mannose receptor (MR) is partially involved in the uptake of the major house dust mite allergen Der p 1 by human DC. The aim of this study was to elucidate MR-Der p 1 interaction with a view to establishing the role of MR in Der p 1-specific Th2 responses. METHODS: Immature DC were generated from peripheral blood monocytes and MR expression was down-regulated by gene silencing (siRNA). Der p 1 uptake was analysed using flow cytometry and the effect of MR down-regulation on T cell differentiation was assessed in DC-T cell cocultures. RESULTS: We have shown that MR is the main receptor for Der p 1 on DC and that the CTLD-4-7 domain is the Der p 1 binding site on MR. Using memory and naı¨ve T cells from Der p 1 sensitized individuals, we have shown that in the absence of MR there is preferential Th1, rather Th2, polaraization (n 5 3; P < 0.05). CONCLUSIONS: MR plays a major role in recognition/uptake of Der p 1 by human DC, and this recognition appears to play an important role in the induction of Der p 1-specific Th2 responses. We therefore suggest that MR is a potential target for the treatment of allergic conditions. Funding: Asthma UK
SATURDAY
Abstracts S11
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2