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Chemokine receptors as RSV receptors: Roles in viral entry and immunopathology of the lung
$302 Abstracts
J ALLERGY CLIN IMMUNOL FEBRUARY 2003
31 Effect of FLT3-LigandTreatment on BALF of Ova Sensitized and Challenged Mice
33 Chemokine Receptors as RSV Receptors: Roles in Viral Entry and Immunopathologyof the Lung
J. H. Edwan 1, D. K. Agrawal2; :Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, 2Center for Allergy, Asthma and Immunology, Creighton University School of Medicine, Omaha, NE. We have previously reported that Flt-3 Ligand (FIt3-L), a cytokine that affects growth and differentiation of hematopoietic cells, prevents and reverses established allergic airway inflammation in mouse model. In this study, we investigated the effect of FIt3-L on the expansion of lung dendritic cells and cytokine levels in bronchoalveolar lavage fluid (BALF). Airway hyperresponsiveness to methacholine was established in Balb/c mice sensitized and challenged with ovalbumin (OVA) followed by ten days treatment with 6 I.t g FIt3-L per day (i.p.). BALF was collected and lungs were removed and digested in collagenase D. Dendritic cells (DCs) were isolated using CDI lc micromagnetic beads by positive selection. Isolated CD1 lc+ ceils were stained with anti-CDl lc-PE, anti-CDl IbFITC, and anti-CD8e~-FITC and analyzed by flow cytometry. DCs were gated on high forward and 90 ~ light scatter and majority of lymphocytes and debris were gated out. There was a significant increase in CD1 lb+ cells (46.2 +_ 3.3%) and decrease in CD8ot + cells (5.1 -+ 0.7%) in OVAsensitized and challenged lungs as compared to controls (CDI lb+ cells 11.2 -+ 0.6%; CD8ot + cells 8.2 +_0.2%). Flt3-L treatment did not significantly affect the number of lung CDI lb+ or CD8~ + cells in the lungs. However, CD1 lc+ CDI lb- cells and CDI lc+ CD8~ - were significantly increased after FLT-3 treatment. FLT-3 treatment significantly decreased the levels of BALF cytokines (TNF~, IL-2, IL-4, and IL-5). IFN- 7 was not detectable in BALE These data suggest Flt3-L decreases inflammatory cytokines in the lungs with no significant effect on lung DCs.
F. Bellessort, V. We]lemans, B. Lamkhioued; [mmunologie et Pathologie Respiratoires, Universit6 de Montr6al, Montr6al, PQ, CANADA. RATIONALE: This study is based on emerging new findings suggesting that chemokines play a critical role in the containment of viral infections and act as major virus-suppressive factors. The present study is based on the hypothesis that RSV infection of airway epithelial cells is mediated by chemokine receptor(s). The RSV-G attachment protein (gp90) is required for infection to occur and is most likely the binding protein for RSV on its target cells. The purpose of this study is to identify the cellular receptor(s) of the RSV-gp90. METHODS: ]mmunostaining and RT-PCR were employed to assess the expression of chemokine receptors mRNA and protein by respiratory epithelial cells. Eotaxin and anti-CCR-3 Abs were tested for their abilities to inhibit RSV A-2 infectivity during attachment. For plaque reduction neutralization assays, eotaxin or anti-CCR-3 antibodies were during the attachment of RSV. To provide biochemical evidence showing a physical association between RSV gp90 and CCR-3, a combination of immunoprecipitation and western blotting techniques was used. RESULTS: We demonstrate that Chemokine receptors (CCR3, CCR5 and CXCR4) are expressed constitutively on epithelial cells in asthmatic airways. In the airways of patients with asthma, chemokine receptors were found predominantly on epithelial cells. Eotaxin, a specific ligand for CCR3, and anti-CCR3 Abs were able to inhibit in vitro infection of respiratory epithelial cells by RSV. Transfection of CCR-3 into cells permits infection by RSV. CONCLUSIONS: CCR-3 represents a potential important therapeutic target for pharmacological intervention in RSV infection in bronchiolitis and asthma.
Funding: Creighton University
932 RSVG Glycoprotein is a Selective Th2 Chemoa.ractant V. WeUemans, F. Bellessort, B. Lamkhioued; Immunologie et Pathologie Respiratoires, Universit6 de Montr6al, Montr6al, PQ, CANADA. RATIONALE: RSV enters target cells by forming a complex between the viral attachment (G) glycoprotein (gp90) and CCR3 chemokine receptor. Recent evidence suggests that the secreted form of gp 90 (sgp90) can induce IL-5 and IL-13, producing atypical pulmonary eosinophilia and enhanced illness in RSV-challenged mice. Therefore, the goal of this study was to dissect out the molecular mechanisms underlying the immunopathology of RSV gp90. METHODS: Secreted gp90 and eotaxin were tested for their ability to attract human eosinophils and cord blood differentiated Th]/Th 2 cells. Cell migration in the presence or absence of anti-CCR3 Nabs was evaluated using microchemotaxis chamber assay. The mucus production induced by sgp90 was visualized by AB/PAS staining of respiratory epithelial cells (A549). RESULTS: We found that eosinophils Th I and Th 2 cells support RSV replication. The number of RSV infected Th i cells is less than RSV infected Th 2 cells. The addition of anti-CCR3 Abs to culture medium was able to inhibit RSV-infection most effectively during virus adsorption to eosinophils and Th 2 cells, whereas in Th] cells anti-CCR3 Abs have no effect. In addition we found that sgp90 attract eosinophils and Th 2 cells, whereas, it had a weak chemotactic activity on Th I cells when compared to Th 2 cells. Both eotaxin and gp90 are able to induce the mucus production by respiratory cells as assessed by PAS staining. CONCLUSIONS: CCR3 may represent a potential important therapeutic target for pharmacological intervention in bronchiolitis and asthma.
Funding: Grant Monies
Funding: Grant Monies
934 Suplatast Tosilute (ST)Inhibits Thymus-and Activation-Regulated Chemokine (TARC) Production by Antigen-Specific Human T Helper 2 (Th2) Cells N. O d a 1,2, K. Minoguchi ], A. Tanaka I, T. Yokoe I, H. Matsuo ], S. K. Huang 2, M. AdachP ; t Showa University, Tokyo, JAPAN, 2Johns Hopkins University, Baltimore, MD. RATIONALE: Suplatast tosilate is an anti-allergic agent that suppresses IL-4 and IL-5 production. TARC/CCLI7 is important in Th2 cell recruitment and is up-regulated in airway epithelial cells of asthmatics following allergen challenge. The objective of this study was to investigate modulation of suplatast tosilate on the production of TARC by T cells. METHODS: Purified protein derivative (PPD)-specific Thl and Dermatophagiodes farinae (Der f)-specific Th2 cell lines were established from nine patients with house dust mite-allergic asthma. The effect of ST on mRNA expression and protein production of TARC from Thl or Th2 cell lines was investigated after stimulation with relevant antigens or phytohemagglutinin (PHA). In addition, the effect of IL-4, IL-10, and IFN-g on TARC production by Der f-specific Th2 cell lines in the presence or absence of ST was studied. RESULTS: Although PPD-specific Thl cell lines did not produce TARC after stimulation with either PPD or PHA, stimulation of Der f-specific Th2 cell lines with Der f or PHA increased the production of TARC. ST at 10 I.tg/ml or higher inhibited the production of TARC by Der f-specific Th2 cell lines stimulated with either Der f or PHA (p<0.05). TARC production by Der f-specific Th2 cell lines was increased only by activation with IL-4 but not with IL-10 or 1FN-g; this increase in TARC production was inhibited by ST at 10 p.g/ml or higher (p<0.05). CONCLUSIONS: These results demonstrate that ST is able to inhibit TARC production by human Th2 cells, suggesting its clinical utility in allergic diseases.
Funding: NIH
J ALLERGY CLIN IMMUNOL FEBRUARY 2003
31 Effect of FLT3-LigandTreatment on BALF of Ova Sensitized and Challenged Mice
33 Chemokine Receptors as RSV Receptors: Roles in Viral Entry and Immunopathologyof the Lung
J. H. Edwan 1, D. K. Agrawal2; :Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE, 2Center for Allergy, Asthma and Immunology, Creighton University School of Medicine, Omaha, NE. We have previously reported that Flt-3 Ligand (FIt3-L), a cytokine that affects growth and differentiation of hematopoietic cells, prevents and reverses established allergic airway inflammation in mouse model. In this study, we investigated the effect of FIt3-L on the expansion of lung dendritic cells and cytokine levels in bronchoalveolar lavage fluid (BALF). Airway hyperresponsiveness to methacholine was established in Balb/c mice sensitized and challenged with ovalbumin (OVA) followed by ten days treatment with 6 I.t g FIt3-L per day (i.p.). BALF was collected and lungs were removed and digested in collagenase D. Dendritic cells (DCs) were isolated using CDI lc micromagnetic beads by positive selection. Isolated CD1 lc+ ceils were stained with anti-CDl lc-PE, anti-CDl IbFITC, and anti-CD8e~-FITC and analyzed by flow cytometry. DCs were gated on high forward and 90 ~ light scatter and majority of lymphocytes and debris were gated out. There was a significant increase in CD1 lb+ cells (46.2 +_ 3.3%) and decrease in CD8ot + cells (5.1 -+ 0.7%) in OVAsensitized and challenged lungs as compared to controls (CDI lb+ cells 11.2 -+ 0.6%; CD8ot + cells 8.2 +_0.2%). Flt3-L treatment did not significantly affect the number of lung CDI lb+ or CD8~ + cells in the lungs. However, CD1 lc+ CDI lb- cells and CDI lc+ CD8~ - were significantly increased after FLT-3 treatment. FLT-3 treatment significantly decreased the levels of BALF cytokines (TNF~, IL-2, IL-4, and IL-5). IFN- 7 was not detectable in BALE These data suggest Flt3-L decreases inflammatory cytokines in the lungs with no significant effect on lung DCs.
F. Bellessort, V. We]lemans, B. Lamkhioued; [mmunologie et Pathologie Respiratoires, Universit6 de Montr6al, Montr6al, PQ, CANADA. RATIONALE: This study is based on emerging new findings suggesting that chemokines play a critical role in the containment of viral infections and act as major virus-suppressive factors. The present study is based on the hypothesis that RSV infection of airway epithelial cells is mediated by chemokine receptor(s). The RSV-G attachment protein (gp90) is required for infection to occur and is most likely the binding protein for RSV on its target cells. The purpose of this study is to identify the cellular receptor(s) of the RSV-gp90. METHODS: ]mmunostaining and RT-PCR were employed to assess the expression of chemokine receptors mRNA and protein by respiratory epithelial cells. Eotaxin and anti-CCR-3 Abs were tested for their abilities to inhibit RSV A-2 infectivity during attachment. For plaque reduction neutralization assays, eotaxin or anti-CCR-3 antibodies were during the attachment of RSV. To provide biochemical evidence showing a physical association between RSV gp90 and CCR-3, a combination of immunoprecipitation and western blotting techniques was used. RESULTS: We demonstrate that Chemokine receptors (CCR3, CCR5 and CXCR4) are expressed constitutively on epithelial cells in asthmatic airways. In the airways of patients with asthma, chemokine receptors were found predominantly on epithelial cells. Eotaxin, a specific ligand for CCR3, and anti-CCR3 Abs were able to inhibit in vitro infection of respiratory epithelial cells by RSV. Transfection of CCR-3 into cells permits infection by RSV. CONCLUSIONS: CCR-3 represents a potential important therapeutic target for pharmacological intervention in RSV infection in bronchiolitis and asthma.
Funding: Creighton University
932 RSVG Glycoprotein is a Selective Th2 Chemoa.ractant V. WeUemans, F. Bellessort, B. Lamkhioued; Immunologie et Pathologie Respiratoires, Universit6 de Montr6al, Montr6al, PQ, CANADA. RATIONALE: RSV enters target cells by forming a complex between the viral attachment (G) glycoprotein (gp90) and CCR3 chemokine receptor. Recent evidence suggests that the secreted form of gp 90 (sgp90) can induce IL-5 and IL-13, producing atypical pulmonary eosinophilia and enhanced illness in RSV-challenged mice. Therefore, the goal of this study was to dissect out the molecular mechanisms underlying the immunopathology of RSV gp90. METHODS: Secreted gp90 and eotaxin were tested for their ability to attract human eosinophils and cord blood differentiated Th]/Th 2 cells. Cell migration in the presence or absence of anti-CCR3 Nabs was evaluated using microchemotaxis chamber assay. The mucus production induced by sgp90 was visualized by AB/PAS staining of respiratory epithelial cells (A549). RESULTS: We found that eosinophils Th I and Th 2 cells support RSV replication. The number of RSV infected Th i cells is less than RSV infected Th 2 cells. The addition of anti-CCR3 Abs to culture medium was able to inhibit RSV-infection most effectively during virus adsorption to eosinophils and Th 2 cells, whereas in Th] cells anti-CCR3 Abs have no effect. In addition we found that sgp90 attract eosinophils and Th 2 cells, whereas, it had a weak chemotactic activity on Th I cells when compared to Th 2 cells. Both eotaxin and gp90 are able to induce the mucus production by respiratory cells as assessed by PAS staining. CONCLUSIONS: CCR3 may represent a potential important therapeutic target for pharmacological intervention in bronchiolitis and asthma.
Funding: Grant Monies
Funding: Grant Monies
934 Suplatast Tosilute (ST)Inhibits Thymus-and Activation-Regulated Chemokine (TARC) Production by Antigen-Specific Human T Helper 2 (Th2) Cells N. O d a 1,2, K. Minoguchi ], A. Tanaka I, T. Yokoe I, H. Matsuo ], S. K. Huang 2, M. AdachP ; t Showa University, Tokyo, JAPAN, 2Johns Hopkins University, Baltimore, MD. RATIONALE: Suplatast tosilate is an anti-allergic agent that suppresses IL-4 and IL-5 production. TARC/CCLI7 is important in Th2 cell recruitment and is up-regulated in airway epithelial cells of asthmatics following allergen challenge. The objective of this study was to investigate modulation of suplatast tosilate on the production of TARC by T cells. METHODS: Purified protein derivative (PPD)-specific Thl and Dermatophagiodes farinae (Der f)-specific Th2 cell lines were established from nine patients with house dust mite-allergic asthma. The effect of ST on mRNA expression and protein production of TARC from Thl or Th2 cell lines was investigated after stimulation with relevant antigens or phytohemagglutinin (PHA). In addition, the effect of IL-4, IL-10, and IFN-g on TARC production by Der f-specific Th2 cell lines in the presence or absence of ST was studied. RESULTS: Although PPD-specific Thl cell lines did not produce TARC after stimulation with either PPD or PHA, stimulation of Der f-specific Th2 cell lines with Der f or PHA increased the production of TARC. ST at 10 I.tg/ml or higher inhibited the production of TARC by Der f-specific Th2 cell lines stimulated with either Der f or PHA (p<0.05). TARC production by Der f-specific Th2 cell lines was increased only by activation with IL-4 but not with IL-10 or 1FN-g; this increase in TARC production was inhibited by ST at 10 p.g/ml or higher (p<0.05). CONCLUSIONS: These results demonstrate that ST is able to inhibit TARC production by human Th2 cells, suggesting its clinical utility in allergic diseases.
Funding: NIH