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Role of CD8+ T cells in allergic airway disease in mice
J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 1
99 The House Dust Mite Allergen Der p 1 Modulates Respiratory Epithelial Cell Function by Activation of Protease Activated Receptors (PARs) Nithiananthan Asokananthan*, Peter Graham*, Anthony Bakker*, Karin Eidne§, Philip J ThompsonY,, Geoffrey Stewart~ *University of Western Australia, Perth, Australia §Western Australian Institute for Medical Research, Perth, Australia YAsthma & Allergy Research Institute, Perth, Australia ~Division of Inflammation and Infectious Diseases, University of Western Australia, Perth, Australia We have shown in previous studies that allergenic house dust mite proteases are potent inducers of pro-inflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved at the cellular level were unclear. In this study, we have examined the role of a novel family of G-protein coupled cell surface receptors which activate a variety of cellular functions when cleaved by proteases (PAR). Der p l, a cysteine protease, was isolated by monoclonal antibody affinity chromatography and used to stimulate cytokine release from the respiratory epithelial cells and HeLa cells. HeLa cells were transfected with either PAR-2 or PAR2/enhanced yellow fluorescent protein (EYFP) plasmids to determine the function of this receptor. Confocal microscopy was used to monitor PAR2/EYFP fusion protein expression. Changes in cytosolic Ca2+ concentration were measured using the fluorescent Ca2+ indicator, Indo- 1. Cytokine released by cells was quantified by ELISA. We showed glutathione-activated Der p 1 caused the release of IL-5, IL-6 and IL-8 from the A549 cell line in a concentration-dependent manner. This response was accompanied by changes in intracellular Ca2+ concentration. Cytokine release from the cell line in response to PAR- 1 agonist peptide was inhibited by preincubation of cells with Der p I for 2 h while the PAR-2 agonist peptide-induced cytokine release was not affected. HeLa cells transfected with PAR-2 receptor released intracellular Ca2+ when exposed to 20~tg/ml of activated Der p 1, whereas the non-transfected cells, control transfected and the non-activated Der p 1 did not. A two-fold increase in the IL-6 and IL-8 production from the PAR-2 transfected HeLa cells in response to Der p 1 was observed when compared to the non-transfected cells. Confocal microscopy demonstrated that the PAR-2/EYFP fusion protein was expressed on the plasma membrane of transfected HeLa cells. These receptors were completely internalised after 30 min incubation with activated Der p 1 but not with the nonactivated Der p 1. These data suggest that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by PAR-2 activation.
500
Role of CD8+T Cells in Allergic Airway Disease in Mice
Philippe Stock*, Tilmann Kallinich§, David Quarcoo*, Katharina Bliimchen§, Ulrich Wahn*, Eckard Hamelmann* *Charit6, Humboldt-University, Berlin, Germany §University Hospital Charit6, Berlin, Germany BACKGROUND: Whereas the functional relevance of CD4+ T cells for the development of allergic airway disease is commonly accepted, there is considerable controversy regarding the exact role of CD8+ T cells in this matter. On the one hand, CD8+ T cells are known to inhibit the production of antigen-specific IgE and the development of airway hyperreactivity (AHR) in some murine models of asthma. On the other hand, CD8+ T cells are able to promote IL-5-mediated eosinophilic airway inflammation (AI) and development of AHR following inhalative modes of sensitization. Aim of the current study was to investigate the role of CD8+ T cells independently for allergen sensitization and development of AI/AHR. M E T H O D S : Normal and CD8-depleted BALB/c mice were systemically sensitized with ovalbumine (OA) on days 1, 6 and 12 and challenged via the airways with OA on days 21 and 22. We determined a) allergic sensitization (production of immunoglobulins and cytokines); b) AI (bronchioalveolar lavage [BAL] and immunohistochemistry); and c) development of in vivo AHR (whole body plethysmography).
Abstracts
S169
RESULTS: Systemic sensitization followed by intranasal allergen challenges leads to interstitial (12h after single challenge) and peribronchial (60h) infiltration of CD8+ T cells into lung tissues. CD8-depletion prior to allergic sensitization resulted in markedly increased serum levels of total IgE and OA-specific IgE and IgG1. Antigen-specific stimulation of peribronchial lymph node cells of sensitized CDS-depleted animals induced markedly enhanced IL-4 and IL-5 production compared to cells of sensitized undepleted controls, whereas levels of IFN-y and IL-12 were decreased. CD8-depletion prior to sensitization resulted in markedly increased numbers of leukocytes and eosinophils in BAL-fluids and enhanced development of in vivo AHR following allergen airway challenge, compared to undepleted controls. CD8-depletion of sensitized animals prior to airway challenges did not significantly alter development of AI and AHR, compard to undepleted controls. CONCLUSION: Depletion of CD8+ T cells prior to allergic sensitization resulted in a) enhanced allergic sensitization and Th2 responses, b) increased cell numbers in BAL-fluids and c) increased in vivo AHR. These data show a critical involvement of CD8+ T cells in the development of allergic AI in this model, indicating a protective mechanism against development of allergen sensitization, AI and AHR.
501 The Relationship oflnfiltrating Eosinophilsto Markers of Repnir and Resolution (Myofibroblasts, TGF-~ and Tenascin) in Allergen-Induced Late-Phase Reactions in Human Skin Simon Phipps, Sun Ying, A Barry Kay NHLI Division, Faculty of Medicine, Imperial College, London, UK The allergen-induced late-phase skin reaction provides a useful model for studying repair processes associated with allergic inflammation. We have used this to test the hypothesis that infiltrating eosinophils play a role in induction of myofibroblast formation and expression of tenascin (Tn)-C by providing substantial repositories of transforming growth factor (TGF)[~ during resolution and repair. Myofibroblasts are a feature of allergeninduced late asthmatic reactions and may contribute to the thickened subepithelial reticular basement membrane (RBM) due to their close proximity. TGF-[3, is an inducer of myofibroblast formation both in vivo and in vitro. To investigate the kinetics of, and relationship between, eosinophil infiltration (congo red+), the formation of myofibroblast-like (a-smooth muscle actin+) cells, TGF-~ immunoreactivity and the expression of tenascin-C (a matricellular protein expressed during development and in response to injury), skin biopsies were obtained from atopic individuals (n = 10) at 1, 3, 6, 24 (including diluent control), 48 and 72 hr following intradermal allergen challenge and the tissue examined by immunohistochemistry. Eosinophilic recruitment was evident within 1 hr following allergen-challenge, in contrast to the diluent-challenged site where tissue eosinophils were minimal. The number of congo red+ eosinophils infiltrating the skin peaked at 6 hr and remained elevated out to 72 hr (a time when the late phase reaction had virtually resolved). In contrast, the number of myofibroblast-like cells increased progressively, peaking between 24 and 48 hr. By 72 hr, the number of these cells had declined rapidly. The temporal pattern of TGF-[3 immunoreactive cells was similar to that observed for eosinophil infiltration. Furthermore, approximately half of the TGF-[~+ cell population at 24 hr were eosinophils. Expression of Tn-C was observed at later time points only. Single Tn-C+ cells, predominantly located in the lower dermis and fusiformic in shape, were evident at 6 hr, peaked at 24 hr and remained elevated out to 72 hr. In addition, immunoflourescence of both Tn-C and et-SM actin+ registered a number of dual positive cells. Together, these data suggest that the formation of myofibroblast-like cells may be partly under the control of infiltrating TGF-[3+ leukocytes, in particular the eosinophil. Moreover, expression of the highly regulated matricellular protein Tn-C indicates that this protein may serve as a useful marker in repair and resolution of allergen-induced inflammation. Furthermore, the cutaneous late-phase reaction may be a useful model to study tissue remodelling following allergic inflammation in other sites such as the airways.
99 The House Dust Mite Allergen Der p 1 Modulates Respiratory Epithelial Cell Function by Activation of Protease Activated Receptors (PARs) Nithiananthan Asokananthan*, Peter Graham*, Anthony Bakker*, Karin Eidne§, Philip J ThompsonY,, Geoffrey Stewart~ *University of Western Australia, Perth, Australia §Western Australian Institute for Medical Research, Perth, Australia YAsthma & Allergy Research Institute, Perth, Australia ~Division of Inflammation and Infectious Diseases, University of Western Australia, Perth, Australia We have shown in previous studies that allergenic house dust mite proteases are potent inducers of pro-inflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved at the cellular level were unclear. In this study, we have examined the role of a novel family of G-protein coupled cell surface receptors which activate a variety of cellular functions when cleaved by proteases (PAR). Der p l, a cysteine protease, was isolated by monoclonal antibody affinity chromatography and used to stimulate cytokine release from the respiratory epithelial cells and HeLa cells. HeLa cells were transfected with either PAR-2 or PAR2/enhanced yellow fluorescent protein (EYFP) plasmids to determine the function of this receptor. Confocal microscopy was used to monitor PAR2/EYFP fusion protein expression. Changes in cytosolic Ca2+ concentration were measured using the fluorescent Ca2+ indicator, Indo- 1. Cytokine released by cells was quantified by ELISA. We showed glutathione-activated Der p 1 caused the release of IL-5, IL-6 and IL-8 from the A549 cell line in a concentration-dependent manner. This response was accompanied by changes in intracellular Ca2+ concentration. Cytokine release from the cell line in response to PAR- 1 agonist peptide was inhibited by preincubation of cells with Der p I for 2 h while the PAR-2 agonist peptide-induced cytokine release was not affected. HeLa cells transfected with PAR-2 receptor released intracellular Ca2+ when exposed to 20~tg/ml of activated Der p 1, whereas the non-transfected cells, control transfected and the non-activated Der p 1 did not. A two-fold increase in the IL-6 and IL-8 production from the PAR-2 transfected HeLa cells in response to Der p 1 was observed when compared to the non-transfected cells. Confocal microscopy demonstrated that the PAR-2/EYFP fusion protein was expressed on the plasma membrane of transfected HeLa cells. These receptors were completely internalised after 30 min incubation with activated Der p 1 but not with the nonactivated Der p 1. These data suggest that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by PAR-2 activation.
500
Role of CD8+T Cells in Allergic Airway Disease in Mice
Philippe Stock*, Tilmann Kallinich§, David Quarcoo*, Katharina Bliimchen§, Ulrich Wahn*, Eckard Hamelmann* *Charit6, Humboldt-University, Berlin, Germany §University Hospital Charit6, Berlin, Germany BACKGROUND: Whereas the functional relevance of CD4+ T cells for the development of allergic airway disease is commonly accepted, there is considerable controversy regarding the exact role of CD8+ T cells in this matter. On the one hand, CD8+ T cells are known to inhibit the production of antigen-specific IgE and the development of airway hyperreactivity (AHR) in some murine models of asthma. On the other hand, CD8+ T cells are able to promote IL-5-mediated eosinophilic airway inflammation (AI) and development of AHR following inhalative modes of sensitization. Aim of the current study was to investigate the role of CD8+ T cells independently for allergen sensitization and development of AI/AHR. M E T H O D S : Normal and CD8-depleted BALB/c mice were systemically sensitized with ovalbumine (OA) on days 1, 6 and 12 and challenged via the airways with OA on days 21 and 22. We determined a) allergic sensitization (production of immunoglobulins and cytokines); b) AI (bronchioalveolar lavage [BAL] and immunohistochemistry); and c) development of in vivo AHR (whole body plethysmography).
Abstracts
S169
RESULTS: Systemic sensitization followed by intranasal allergen challenges leads to interstitial (12h after single challenge) and peribronchial (60h) infiltration of CD8+ T cells into lung tissues. CD8-depletion prior to allergic sensitization resulted in markedly increased serum levels of total IgE and OA-specific IgE and IgG1. Antigen-specific stimulation of peribronchial lymph node cells of sensitized CDS-depleted animals induced markedly enhanced IL-4 and IL-5 production compared to cells of sensitized undepleted controls, whereas levels of IFN-y and IL-12 were decreased. CD8-depletion prior to sensitization resulted in markedly increased numbers of leukocytes and eosinophils in BAL-fluids and enhanced development of in vivo AHR following allergen airway challenge, compared to undepleted controls. CD8-depletion of sensitized animals prior to airway challenges did not significantly alter development of AI and AHR, compard to undepleted controls. CONCLUSION: Depletion of CD8+ T cells prior to allergic sensitization resulted in a) enhanced allergic sensitization and Th2 responses, b) increased cell numbers in BAL-fluids and c) increased in vivo AHR. These data show a critical involvement of CD8+ T cells in the development of allergic AI in this model, indicating a protective mechanism against development of allergen sensitization, AI and AHR.
501 The Relationship oflnfiltrating Eosinophilsto Markers of Repnir and Resolution (Myofibroblasts, TGF-~ and Tenascin) in Allergen-Induced Late-Phase Reactions in Human Skin Simon Phipps, Sun Ying, A Barry Kay NHLI Division, Faculty of Medicine, Imperial College, London, UK The allergen-induced late-phase skin reaction provides a useful model for studying repair processes associated with allergic inflammation. We have used this to test the hypothesis that infiltrating eosinophils play a role in induction of myofibroblast formation and expression of tenascin (Tn)-C by providing substantial repositories of transforming growth factor (TGF)[~ during resolution and repair. Myofibroblasts are a feature of allergeninduced late asthmatic reactions and may contribute to the thickened subepithelial reticular basement membrane (RBM) due to their close proximity. TGF-[3, is an inducer of myofibroblast formation both in vivo and in vitro. To investigate the kinetics of, and relationship between, eosinophil infiltration (congo red+), the formation of myofibroblast-like (a-smooth muscle actin+) cells, TGF-~ immunoreactivity and the expression of tenascin-C (a matricellular protein expressed during development and in response to injury), skin biopsies were obtained from atopic individuals (n = 10) at 1, 3, 6, 24 (including diluent control), 48 and 72 hr following intradermal allergen challenge and the tissue examined by immunohistochemistry. Eosinophilic recruitment was evident within 1 hr following allergen-challenge, in contrast to the diluent-challenged site where tissue eosinophils were minimal. The number of congo red+ eosinophils infiltrating the skin peaked at 6 hr and remained elevated out to 72 hr (a time when the late phase reaction had virtually resolved). In contrast, the number of myofibroblast-like cells increased progressively, peaking between 24 and 48 hr. By 72 hr, the number of these cells had declined rapidly. The temporal pattern of TGF-[3 immunoreactive cells was similar to that observed for eosinophil infiltration. Furthermore, approximately half of the TGF-[~+ cell population at 24 hr were eosinophils. Expression of Tn-C was observed at later time points only. Single Tn-C+ cells, predominantly located in the lower dermis and fusiformic in shape, were evident at 6 hr, peaked at 24 hr and remained elevated out to 72 hr. In addition, immunoflourescence of both Tn-C and et-SM actin+ registered a number of dual positive cells. Together, these data suggest that the formation of myofibroblast-like cells may be partly under the control of infiltrating TGF-[3+ leukocytes, in particular the eosinophil. Moreover, expression of the highly regulated matricellular protein Tn-C indicates that this protein may serve as a useful marker in repair and resolution of allergen-induced inflammation. Furthermore, the cutaneous late-phase reaction may be a useful model to study tissue remodelling following allergic inflammation in other sites such as the airways.