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IP008 Activation of neutrophil NADPH oxidase complex during infection — regulation by endothelial cells
Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Poster
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0
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Modeling antigen presentation of Listeria monocytogenes-derived CD8 T cell epitopes Janda, J}; Lindenstruth, V.2; Geginat, G. 1 ZFakult~t f[~r klinische Medizin Mannheim der Universit~t Heidelberg; Institut fElr Medizinische Mikrobiologie 2Universit~t Heidelberg; Kirchhoff-Institut f6r Physik
A mathematical model was developed to simulate the dynamics of antigen processing and presentation of CD8 T cell epitopes derived from Listeria monocytogenes. The model predicts a minor influence of the stability of MHC/peptide complexes on the peptide presentation pattern of infected cells while in the case of cross-presented antigens the stability of MHC/peptide complexes governs the antigen presentation pattern. Experimental data obtained with antigen presenting cells that present L. monocytogenes-derived antigenic peptides support this model. The distinct antigen presentation patterns of professional antigen presenting cells versus infected target cells suggests an explanation for the general observation that the quantity of naturally processed antigenic peptides in infected cells does not necessarily correlate with the strength of a peptide-specific T cell response.
Activation of neutrophil NADPH oxidase complex during infection - regulation by endothelial cells K6nig, B.1; K~nig, W. 1 Otto - yon - Guericke - University; Medical Microbiology
Introduction: In granulocytes, NADPH oxidase is involved in the antimicrobial defense. The NADPH oxidase consists of several subunits which are either integral membrane proteins (p22phox, gp91phox) or are located in the cytosol (p47phox, p67phox). However, radical oxygen formation by the NADPH oxidase complex may also lead to tissue destruction. Methods and Data: We focussed on the NADPH oxidase complex in neutrophils exposed a) towards various host derived chemotactic compounds; b) towards mucoid and nonmucoid P. aeruginosa strains. For this purpose a) various neutrophil-relevant chemotactic factors (TL-8, LTB4, C5a) at total concentrations of 100-, 10-, lng per 8001JI volume; b) clinical P, aeruginosa strains (mucoid, non-mucoid) were added to the lower compartment of a double chamber (51Jm pore size); neutrophils (lx105) were added to the upper compartment for 4-6 hours. The chemotactic factors IL-8, LTB4, and C5a attracted 26.33%+8.2% %; 21+5%; 12+4% of the neutrophils. The mucoid P. aeruginosa phenotypes dosedependently attracted up to 16% of the initial neutrophils; the non-mucoid phenotype was significantly less potent. Similar results were obtained with a layer of epithelial or endothelial cells on the membrane. The attracted neutrophils differed markedly in their characteristics as was assessed by qualitative
http://www.dghm.org
209
Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Poster
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and quantitative RT-PCR (p22phox, p47phox, p67phox and gp91phox). IL-8 and C5a-attracted neutrophils expressed dose-dependently p22phox and p47phox mRNA. In inverse dose-dependency was observed in LTB4 attracted neutrophils. Only the mucoid P. aeruginosa phenotype induced p22phox and p47phox specific mRNA. However, the results were only obtained in case of an endothelial cell layer between lower and upper compartment of the chamber system. Conclusion: We hypothesize that endothelial cells direct the inflammatory signals which then lead to overwhelming responses by neutrophils in dependence of the respective stimulus.
The bacterial superantigen staphylococcal enterotoxin B (SEB) modulates chemokine receptor expression on CD4+ T lymphocytes Fang, J ) , K0nig, W.1; K0nig, B }
~Otto-von-Guericke-University Magdeburg, Medical Microbiology Introduction: The bacterial superantigen Staphylococcal enterotoxin B (SEB) can be frequently isolated from the skin of patients with atopic dermatitis. Epicutaneous application of intradermal injection of staphylococcal enterotoxins induces inflammation of the skin, which is T-cell dependent. For the migration of the T-Cells towards the inflammation chemokines and chemokine receptors (CKRs) seem to be essential. We systematically investigated the association of 14 CKRs (CCR1-9, CXCR1-6) with CD4+ T-cells from peripheral blood mononuclear cells (PBMC) in the presence of SEB. Methods and Data: CKRs expression on CD4+ T-cells were analyzed by flow cytometry and RT-PCR. Cytokine secretion was assessed by ELISA in the supernatants from SEBstimulated PBMC. In a first set of experiments PBMC from healthy donors were treated with SEB (10ng/ml) for up to 7 days. SEB increased surface antigen expression (CD11b, CD25, CD31, CD45RO, CD54, CD69 and CD95) and is a strong inducer of CLA on T-cells, the homing receptor for skin T-cells. SEB-treated PBMC released significant amounts of IL-8, RANTES, MIP-1alpha, MIP-l-beta, Eotaxin, MCP-1. SEB predominantly upregulated the chemokine receptors CCR3, CCR4 and CXCR3 on CD4+ T-cells. The maximum effects were observed after 5 days of incubation. Furthermore PBMC were polarized towards T h l or Th2 cytokine expressing T-cells through incubation in the presence of II-12/antiIL-4 or IL-4/anti-IL-12. In Th2 polarized CD4+ T-cells from PBMC SEB induced an upregulation of surface CCR4 (Th2-associated CKRs). Similarly, SEB induced an upregulation of CCR4- mRNA on MACS-isolated CD4+ T-cells from an atopic donor. In T h l polarized CD4+ Tcells from PBMC SEB led to an downregulation of CXCR3 (Thl-associated CKRs).
http://www.dghm.org
210
©
0
~D ~ °~
Modeling antigen presentation of Listeria monocytogenes-derived CD8 T cell epitopes Janda, J}; Lindenstruth, V.2; Geginat, G. 1 ZFakult~t f[~r klinische Medizin Mannheim der Universit~t Heidelberg; Institut fElr Medizinische Mikrobiologie 2Universit~t Heidelberg; Kirchhoff-Institut f6r Physik
A mathematical model was developed to simulate the dynamics of antigen processing and presentation of CD8 T cell epitopes derived from Listeria monocytogenes. The model predicts a minor influence of the stability of MHC/peptide complexes on the peptide presentation pattern of infected cells while in the case of cross-presented antigens the stability of MHC/peptide complexes governs the antigen presentation pattern. Experimental data obtained with antigen presenting cells that present L. monocytogenes-derived antigenic peptides support this model. The distinct antigen presentation patterns of professional antigen presenting cells versus infected target cells suggests an explanation for the general observation that the quantity of naturally processed antigenic peptides in infected cells does not necessarily correlate with the strength of a peptide-specific T cell response.
Activation of neutrophil NADPH oxidase complex during infection - regulation by endothelial cells K6nig, B.1; K~nig, W. 1 Otto - yon - Guericke - University; Medical Microbiology
Introduction: In granulocytes, NADPH oxidase is involved in the antimicrobial defense. The NADPH oxidase consists of several subunits which are either integral membrane proteins (p22phox, gp91phox) or are located in the cytosol (p47phox, p67phox). However, radical oxygen formation by the NADPH oxidase complex may also lead to tissue destruction. Methods and Data: We focussed on the NADPH oxidase complex in neutrophils exposed a) towards various host derived chemotactic compounds; b) towards mucoid and nonmucoid P. aeruginosa strains. For this purpose a) various neutrophil-relevant chemotactic factors (TL-8, LTB4, C5a) at total concentrations of 100-, 10-, lng per 8001JI volume; b) clinical P, aeruginosa strains (mucoid, non-mucoid) were added to the lower compartment of a double chamber (51Jm pore size); neutrophils (lx105) were added to the upper compartment for 4-6 hours. The chemotactic factors IL-8, LTB4, and C5a attracted 26.33%+8.2% %; 21+5%; 12+4% of the neutrophils. The mucoid P. aeruginosa phenotypes dosedependently attracted up to 16% of the initial neutrophils; the non-mucoid phenotype was significantly less potent. Similar results were obtained with a layer of epithelial or endothelial cells on the membrane. The attracted neutrophils differed markedly in their characteristics as was assessed by qualitative
http://www.dghm.org
209
Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Poster
C~ GESE~C.
~
%
g
3
and quantitative RT-PCR (p22phox, p47phox, p67phox and gp91phox). IL-8 and C5a-attracted neutrophils expressed dose-dependently p22phox and p47phox mRNA. In inverse dose-dependency was observed in LTB4 attracted neutrophils. Only the mucoid P. aeruginosa phenotype induced p22phox and p47phox specific mRNA. However, the results were only obtained in case of an endothelial cell layer between lower and upper compartment of the chamber system. Conclusion: We hypothesize that endothelial cells direct the inflammatory signals which then lead to overwhelming responses by neutrophils in dependence of the respective stimulus.
The bacterial superantigen staphylococcal enterotoxin B (SEB) modulates chemokine receptor expression on CD4+ T lymphocytes Fang, J ) , K0nig, W.1; K0nig, B }
~Otto-von-Guericke-University Magdeburg, Medical Microbiology Introduction: The bacterial superantigen Staphylococcal enterotoxin B (SEB) can be frequently isolated from the skin of patients with atopic dermatitis. Epicutaneous application of intradermal injection of staphylococcal enterotoxins induces inflammation of the skin, which is T-cell dependent. For the migration of the T-Cells towards the inflammation chemokines and chemokine receptors (CKRs) seem to be essential. We systematically investigated the association of 14 CKRs (CCR1-9, CXCR1-6) with CD4+ T-cells from peripheral blood mononuclear cells (PBMC) in the presence of SEB. Methods and Data: CKRs expression on CD4+ T-cells were analyzed by flow cytometry and RT-PCR. Cytokine secretion was assessed by ELISA in the supernatants from SEBstimulated PBMC. In a first set of experiments PBMC from healthy donors were treated with SEB (10ng/ml) for up to 7 days. SEB increased surface antigen expression (CD11b, CD25, CD31, CD45RO, CD54, CD69 and CD95) and is a strong inducer of CLA on T-cells, the homing receptor for skin T-cells. SEB-treated PBMC released significant amounts of IL-8, RANTES, MIP-1alpha, MIP-l-beta, Eotaxin, MCP-1. SEB predominantly upregulated the chemokine receptors CCR3, CCR4 and CXCR3 on CD4+ T-cells. The maximum effects were observed after 5 days of incubation. Furthermore PBMC were polarized towards T h l or Th2 cytokine expressing T-cells through incubation in the presence of II-12/antiIL-4 or IL-4/anti-IL-12. In Th2 polarized CD4+ T-cells from PBMC SEB induced an upregulation of surface CCR4 (Th2-associated CKRs). Similarly, SEB induced an upregulation of CCR4- mRNA on MACS-isolated CD4+ T-cells from an atopic donor. In T h l polarized CD4+ Tcells from PBMC SEB led to an downregulation of CXCR3 (Thl-associated CKRs).
http://www.dghm.org
210