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Characterization of Responses of Human Nasal Epithelial Cells to Pro-inflammatory Mediators and Secretagogues
S72 Abstracts
SATURDAY
280
Nares: Local Cytokine Pattern in Nasal Secretions
C. Klemens, D. Liebl, E. Pfrogner, F. Jund, M. Kramer; Klinikum Grosshadern, Oto-Rhino-Laryngology, Head and Neck Surgery,LudwigMaximilians-University Munich, Munich, GERMANY. RATIONALE: Patients with NARES (non-allergic rhinitis with eosinophilia syndrome) complain about typical symptoms of persistent allergic rhinitis, mainly nasal congestion and rhinorrhea. Nevertheless, the underlying pathophysiology of NARES still remains unknown. The objective of the study was to investigate cytokines and chemokines in nasal secretions of patients with NARES and persistent allergic rhinitis to further elucidate the pathophysiology of NARES. METHODS: Nasal secretions of 31 patients suffering from NARES, 21 patients with persistent allergic rhinitis (PAR) and 20 healthy controls were gained by the cotton wool method and analyzed for IL-1b, IL-2, IL4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, GM-CSF, G-CSF, IFN- , TNF-, MCP-1, and MIP-1 (Bio-Plex Cytokine Assay). ECP and Tryptase as mediators for cellular activation were analyzed by ELISA. RESULTS: We found high and significantly elevated levels of proinflammatory cytokines IL-1b, IL-6, IFN-g and TNF- as well as of IL-4, IL-17 and MCP-1 in nasal secretions of patients with NARES compared to healthy controls and PAR. Concentrations of IL-7, G-CSF and GMCSF were higher in NARES than in controls, while there was no significant elevation of GM-CSF in patients with PAR. IL-5 and MIP-1b showed likewise elevated levels in PAR and NARES compared to controls. CONCLUSIONS: The elevated levels of pro-inflammatory cytokines in NARES are an indicator for the chronic process of inflammation in NARES. IL-4, IL-5 and GM-CSF may be released by eosinophils to sustain eosinophilic migration and retention in the inflamed mucosa. IL-17 may play a role in remodeling processes of the nasal mucosa in patients with NARES. Funding: Bio-Rad
281
Local Activation of the Complement System in Nasal Polyps
T. Van Zele, G. Holtappels, P. Van Cauwenberge, C. Bachert; ENT department 1P1, University hospital of Ghent, Gent, BELGIUM. RATIONALE: Complement factors C3a and C5a exert pro-inflammatory effects like increasing the vascular permeability and the recruitment and activation of inflammatory cells. Nasal polyps are characterised by infiltrating eosinophils, neutrophils and a profound oedema in the nasal polyp tissue. We aimed to study the presence of complement activation in NP therefore the systemic and local levels of C5a, C3a, ECP, MPO, albumin and 2 macroglobulin in nasal secretions and serum were investigated. METHODS: Levels of C3a, C5a, ECP, MPO (ELISA) and albumin, 2 macroglobulin (nefelometry) were determined on nasal secretions and serum from 12 chronic rhinosinusitis patients with nasal polyps and 10 control patients. Tissue cryo-sections were stained for C3, CR2, membrane attack complex, C3aR and C5aR. RESULTS: C3a and C5a concentrations were significant higher in nasal secretions of NP compared to controls. Secondly serum levels of C3a and C5a were in NP significantly lower compared to levels in nasal secretions. C5a levels in nasal secretion were positively correlated with ECP and 2 macroglobulin levels while C3a was strongly correlated with albumin and 2 macroglobulin. Tissue cryo-sections showed clearly the presence of local C3, CR2, membrane attack complex, C3aR and C5aR in NP tissue. CONCLUSIONS: The elevated levels of C3a and C5a in nasal secretions of NP patients point towards a local complement activation which is confirmed on immunohistochemistry. Secondly levels C3a and C5a correlate with markers for eosinophilic inflammation and plasma-exudation, which are characteristic for NP. Therefore the complement system might play a role in the eosinophilic inflammation and plasma exudation of NP. Funding: BOF and FWO grant
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
Characterization of Responses of Human Nasal Epithelial Cells to Pro-inflammatory Mediators and Secretagogues J. L. Stahl, E. B. Cook, F. M. Graziano; Medicine, University of Wisconsin-Madison, Madison, WI. RATIONALE: A potential immunoregulatory role of epithelial cells has been demonstrated in a variety of tissues based on responses, in vitro, to pro-inflammatory mediators, chemical secretagogues and bacterial cell wall components. This study examined the potential immunoregulatory role of human nasal epithelial cells (HNE). METHODS: Primary cultures of HNE (n=3-6; derived from individual donors) were obtained from PromoCell GmBH, or Oligene GmBH, Germany. HNE were cultured in 24-well plates and stimulated with a panel of biologically relevant pro-inflammatory mediators and secretagogues (histamine, dimaprit [H1 agonist], calcium ionophore [CaI], phorbal ester [PMA], TNF, IFN , PAF, Staphylococcus aureus cell wall extract [SACWE], Escherichia coli LPS) (3 concentrations each to determine dose responses) for 24 hours. Supernates were harvested to measure IL-6, IL8, GM-CSF and TNF secretion by ELISA and cells were harvested with trypsin-EDTA to determine ICAM-1 expression by flow cytometry. RESULTS: HNE released significant amounts of IL-6, IL-8 and GMCSF in response to TNF (p< 0.05). Significant IL-6 and IL-8 release resulted from stimulation with SA-CWE (p< 0.05). Release of IL-8 was also significant with PAF stimulation (p< 0.05). PMA, CaI, and IFN stimulated IL-6, IL-8, and GM-CSF release in a dose dependent manner, but concentrations were not statistically greater than unstimulated (p<0.1). TNF release was minimal. Significant ICAM-1 upregulation resulted from stimulation with CaI, TNF, IFN , and SA-CWE. HNE were unresponsive to histamine, dimaprit and LPS. CONCLUSIONS: HNE have the potential to promote cell migration, activation, and maintenance of pro-inflammatory cells in the upper airway. Funding: NIH
282
283
Ultrastructural Findings In Patients with Allergic Rhinitis
S. Knipping; ENT Department, Martin Luther University, Halle/ Saale, GERMANY. RATIONALE: The symptoms of allergic rhinitis are considered to be a result of accumulation and activation of inflammatory cells. Furthermore, some neuropeptides like Calcitonin Gene Related Peptide (CGRP) and Substance P (SP) play an additional role in pathophysiology of allergic rhinitis. METHODS: Tissue samples from 38 human turbinates of patients with perennial rhinitis were taken during nasal surgery and preserved in phosphate-buffered glutaraldehyde. Ultrathin sections were cut and electron microscopy was performed. Additionally, samples were dehydrated and embedded in Araldit. Primary antibodies against CGRP and Substance P were applied and an immunocytochemical staining technique using a gold labeled antibody was carried out. Immunostained structures were photodocumented by using a transmission electron microscope. RESULTS: In the nasal mucosa an extensive edema and inflammatory cells like lymphocytes, plasma cells, eosinophiles and macrophages was found. The capillaries showed an activated endothelium. Immunoreactive nerve fibers were detected perivascular and in the periglandular tissue around the acini and ducts. Neuroglandular synapses with dense core vesicles and positive immunoreactions to CGRP and SP could be observed. CONCLUSIONS: Using electron microscopical techniques nerval structures near the submucosal glands could be demonstrated. Immunoreactions to the vasoactive neuropeptides CGRP and SP were detected in periglandular and perivascular nerves. These findings demonstrate the direct nerval influence on nasal mucosa in allergic rhinitis. CGRP and SP may induce vascular permeability and glandular secretion. These findings further elucidate pathomorphological mechanisms in allergic rhinitis. Funding: German Research Society
SATURDAY
280
Nares: Local Cytokine Pattern in Nasal Secretions
C. Klemens, D. Liebl, E. Pfrogner, F. Jund, M. Kramer; Klinikum Grosshadern, Oto-Rhino-Laryngology, Head and Neck Surgery,LudwigMaximilians-University Munich, Munich, GERMANY. RATIONALE: Patients with NARES (non-allergic rhinitis with eosinophilia syndrome) complain about typical symptoms of persistent allergic rhinitis, mainly nasal congestion and rhinorrhea. Nevertheless, the underlying pathophysiology of NARES still remains unknown. The objective of the study was to investigate cytokines and chemokines in nasal secretions of patients with NARES and persistent allergic rhinitis to further elucidate the pathophysiology of NARES. METHODS: Nasal secretions of 31 patients suffering from NARES, 21 patients with persistent allergic rhinitis (PAR) and 20 healthy controls were gained by the cotton wool method and analyzed for IL-1b, IL-2, IL4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, GM-CSF, G-CSF, IFN- , TNF-, MCP-1, and MIP-1 (Bio-Plex Cytokine Assay). ECP and Tryptase as mediators for cellular activation were analyzed by ELISA. RESULTS: We found high and significantly elevated levels of proinflammatory cytokines IL-1b, IL-6, IFN-g and TNF- as well as of IL-4, IL-17 and MCP-1 in nasal secretions of patients with NARES compared to healthy controls and PAR. Concentrations of IL-7, G-CSF and GMCSF were higher in NARES than in controls, while there was no significant elevation of GM-CSF in patients with PAR. IL-5 and MIP-1b showed likewise elevated levels in PAR and NARES compared to controls. CONCLUSIONS: The elevated levels of pro-inflammatory cytokines in NARES are an indicator for the chronic process of inflammation in NARES. IL-4, IL-5 and GM-CSF may be released by eosinophils to sustain eosinophilic migration and retention in the inflamed mucosa. IL-17 may play a role in remodeling processes of the nasal mucosa in patients with NARES. Funding: Bio-Rad
281
Local Activation of the Complement System in Nasal Polyps
T. Van Zele, G. Holtappels, P. Van Cauwenberge, C. Bachert; ENT department 1P1, University hospital of Ghent, Gent, BELGIUM. RATIONALE: Complement factors C3a and C5a exert pro-inflammatory effects like increasing the vascular permeability and the recruitment and activation of inflammatory cells. Nasal polyps are characterised by infiltrating eosinophils, neutrophils and a profound oedema in the nasal polyp tissue. We aimed to study the presence of complement activation in NP therefore the systemic and local levels of C5a, C3a, ECP, MPO, albumin and 2 macroglobulin in nasal secretions and serum were investigated. METHODS: Levels of C3a, C5a, ECP, MPO (ELISA) and albumin, 2 macroglobulin (nefelometry) were determined on nasal secretions and serum from 12 chronic rhinosinusitis patients with nasal polyps and 10 control patients. Tissue cryo-sections were stained for C3, CR2, membrane attack complex, C3aR and C5aR. RESULTS: C3a and C5a concentrations were significant higher in nasal secretions of NP compared to controls. Secondly serum levels of C3a and C5a were in NP significantly lower compared to levels in nasal secretions. C5a levels in nasal secretion were positively correlated with ECP and 2 macroglobulin levels while C3a was strongly correlated with albumin and 2 macroglobulin. Tissue cryo-sections showed clearly the presence of local C3, CR2, membrane attack complex, C3aR and C5aR in NP tissue. CONCLUSIONS: The elevated levels of C3a and C5a in nasal secretions of NP patients point towards a local complement activation which is confirmed on immunohistochemistry. Secondly levels C3a and C5a correlate with markers for eosinophilic inflammation and plasma-exudation, which are characteristic for NP. Therefore the complement system might play a role in the eosinophilic inflammation and plasma exudation of NP. Funding: BOF and FWO grant
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
Characterization of Responses of Human Nasal Epithelial Cells to Pro-inflammatory Mediators and Secretagogues J. L. Stahl, E. B. Cook, F. M. Graziano; Medicine, University of Wisconsin-Madison, Madison, WI. RATIONALE: A potential immunoregulatory role of epithelial cells has been demonstrated in a variety of tissues based on responses, in vitro, to pro-inflammatory mediators, chemical secretagogues and bacterial cell wall components. This study examined the potential immunoregulatory role of human nasal epithelial cells (HNE). METHODS: Primary cultures of HNE (n=3-6; derived from individual donors) were obtained from PromoCell GmBH, or Oligene GmBH, Germany. HNE were cultured in 24-well plates and stimulated with a panel of biologically relevant pro-inflammatory mediators and secretagogues (histamine, dimaprit [H1 agonist], calcium ionophore [CaI], phorbal ester [PMA], TNF, IFN , PAF, Staphylococcus aureus cell wall extract [SACWE], Escherichia coli LPS) (3 concentrations each to determine dose responses) for 24 hours. Supernates were harvested to measure IL-6, IL8, GM-CSF and TNF secretion by ELISA and cells were harvested with trypsin-EDTA to determine ICAM-1 expression by flow cytometry. RESULTS: HNE released significant amounts of IL-6, IL-8 and GMCSF in response to TNF (p< 0.05). Significant IL-6 and IL-8 release resulted from stimulation with SA-CWE (p< 0.05). Release of IL-8 was also significant with PAF stimulation (p< 0.05). PMA, CaI, and IFN stimulated IL-6, IL-8, and GM-CSF release in a dose dependent manner, but concentrations were not statistically greater than unstimulated (p<0.1). TNF release was minimal. Significant ICAM-1 upregulation resulted from stimulation with CaI, TNF, IFN , and SA-CWE. HNE were unresponsive to histamine, dimaprit and LPS. CONCLUSIONS: HNE have the potential to promote cell migration, activation, and maintenance of pro-inflammatory cells in the upper airway. Funding: NIH
282
283
Ultrastructural Findings In Patients with Allergic Rhinitis
S. Knipping; ENT Department, Martin Luther University, Halle/ Saale, GERMANY. RATIONALE: The symptoms of allergic rhinitis are considered to be a result of accumulation and activation of inflammatory cells. Furthermore, some neuropeptides like Calcitonin Gene Related Peptide (CGRP) and Substance P (SP) play an additional role in pathophysiology of allergic rhinitis. METHODS: Tissue samples from 38 human turbinates of patients with perennial rhinitis were taken during nasal surgery and preserved in phosphate-buffered glutaraldehyde. Ultrathin sections were cut and electron microscopy was performed. Additionally, samples were dehydrated and embedded in Araldit. Primary antibodies against CGRP and Substance P were applied and an immunocytochemical staining technique using a gold labeled antibody was carried out. Immunostained structures were photodocumented by using a transmission electron microscope. RESULTS: In the nasal mucosa an extensive edema and inflammatory cells like lymphocytes, plasma cells, eosinophiles and macrophages was found. The capillaries showed an activated endothelium. Immunoreactive nerve fibers were detected perivascular and in the periglandular tissue around the acini and ducts. Neuroglandular synapses with dense core vesicles and positive immunoreactions to CGRP and SP could be observed. CONCLUSIONS: Using electron microscopical techniques nerval structures near the submucosal glands could be demonstrated. Immunoreactions to the vasoactive neuropeptides CGRP and SP were detected in periglandular and perivascular nerves. These findings demonstrate the direct nerval influence on nasal mucosa in allergic rhinitis. CGRP and SP may induce vascular permeability and glandular secretion. These findings further elucidate pathomorphological mechanisms in allergic rhinitis. Funding: German Research Society