- Email: [email protected]
Modulation of nerve growth factor release from human nasal fibroblasts: Possible role in airway inflammatory diseases
S72
Abstracts
"7 ~ Increased Proinflammatory Cytokines Present in Breast Milk of /~bJI Women With Atopic/Autoimmune Disorders May Induce Gastrointestinal Epithelial Cell Apoptosis Victoria Maria Diokno*, Maya Srivastava§, Alton Melton*, Edward Conrod§ *Cleveland Clinic Federation, Cleveland, OH §Metro Health Medical Center, Cleveland, OH Previous studies have documented the presence of various cytokines, growth factors, and soluble receptors in human breast milk that may affect the neonatal mucosal immune system. Wide inter-individual differences in concentration are observed. In this study, breast milk donors (n=38) were classified into two groups: those with atopic or autoimmune disorders, and those without. Colostrum, transitional, and mature breast milk were collected into sterile 15 cc tubes, centrifuged for 15 minutes at 3000 G. The whey faction was used in ELISA assays for HGE IL-1RA, IL-2Ralpha, IL8, M-CSE TNF-alpha, TNF-beta, TGF-betal, TGF-beta2, MCP-1, RANTES, and GRO-alpha (R&D, Biosource). RNA was extracted from breast milk ceils using Qiagen kit and cryopreserved for semiquantitative RT-PCR. During sample collection donors completed a questionnaire regarding their/their infant's history of atopic/autoimmune disorders. Results showed increased TNF-alpha in breast milk of women with atopic/antoimmune disease (x=44.2 pg/ml vs. 0.8 pg/ml). In mature milk from atopic mothers, mean IL-IRA was also three times controls (x=1969 pg/ml versus 753 pg/ml), mean concentrations of IL-8 (x= 269 pg/ml versus 98 pg/ml), M-CSF (x=29, 717 pg/ml versus 15,090 pg/ml) and TNFalpha (x=404 pg/ml versus 185 pg/ml), were approximately twice as high in the atopic or autoimmune group. RT-PCR confirmed the presence of mRNA for TNF-alpha and TGF-betal, as well as NFKB p65 and p50, and strong expression of IKBa mRNA in breast milk. To determine potential significance of the increased proinflammatory cytokines on the infant, HT29 intestinal epithelial cells were used as an in vitro model. Cells were cultured with and without 4, 40, and 400 pg/ml TNF-alpha with and without 1000 pg/ml TGF-betal for 6-24 hours in RPMI1640 10%FCS/l%glutamine/penicillin/streptomycin at 370C in a 5% CO2 incubator. Cells were harvested after trypsinization and stained with Apoptosis Detection Kit, Annexin V-CY3 (Sigma). TNF-alpha at 400 pg/ml lead to significant cell apoptosis by 24 hours, but this damage was prevented by simultaneous culture with TGF-betal. Results suggest that elevated TNF-alpha at levels found in milk from some mothers with autoimmunity/allergy may be sufficient to cause epithelial destruction, and thus increase gut antigen exposure. This may be a potential mechanism of atopy transmission in some breastfed infants. High amounts of free TGF-beta may ameliorate this effect. If immune modulating formulas become available, such as those containing TGF-beta, supplementation may be a possible therapeutic strategy. Further experiments to determine effect of increased breast milk IL-8, TNF-alpha and TNF-beta together on HT-29 and Coco-2 cell cytokine expression are ongoing.
1
" 7 ~ IL-4 Production in Responseto Allergen Stimulation and CliniJl' I,]I col Symptoms of Allergic Rhinitis
Eric Howell*, Paul Mehlhop*, Anastasia Sigounas*, George Sigounas*, Dhavalkumar Patel§ *Brody School of Medicine at East Carolina University, Greenville, NC §Duke University Medical Center, Durham, NC IL-4 has been implicated as a major cytokine involved in the mechanism of allergic rhinitis. As such, we investigated the relationship between IL-4 production by peripheral blood mononuclear cells (PBMCs) in response to dust mite allergen and the clinical symptoms exhibited by patients with positive skin prick results to dust mite allergen. PBMCs were isolated from peripheral blood donated by human subjects and the frequency of IL-4 and 1NF-~/producing cells was measured by ELISPOT after in vitro challenge with dust mite allergen. Clinical symptoms of allergic rhinitis experienced by the study participants were obtained by administration of the Rhinitis Outcomes Questionnaire (ROQ), a validated symptom survey for allergic rhinitis. Additional questions were added to gauge the environmental factors that participants identified as triggers of their allergy symptoms. The group that tested positive
J ALLERGY CLIN IMMUNOL JANUARY 2002
by skin prick to dust mite allergen had elevated numbers of IL-4 producing cells compared to those who were not skin test positive for dust mite allergen (4.0/200,000 cells vs. 0.67/200,000 cells, respectively). The perception of dust as a trigger of allergic symptoms was also higher in the dust mite positive group compared to the dust mite negative group (average score of 3.3 in dust mite positive group, 1.25 in the dust mite negative group). Further, the participants that both tested positive for dust mite allergen and perceived dust as a significant trigger of their allergic symptoms had an average total symptom score of 34.29 upon symptom survey, in contrast to the average total score of 6.67 for those who were negative for dust mite and did not identify dust as a trigger of their symptoms. We found that in addition to a positive correlation between IL-4 production and skin test results, IL-4 production also correlates with clinical symptoms and the perception of dust as a trigger of allergic rhinitis, suggesting a link between IL-4 production and the clinical manifestation of allergic rhinitis upon exposure to dust mite allergen.
177 Upregulatioofn IL-9 and ,nterleukin-9-Associated
CalciumActivated Chloride Channel (ICACC) in Nasal Epithelium Following In Vivo Allergen Challenge
Mario Kontolemos*, Masao Toda*, Roy C Levitt§, Qutayba A Hamid* *Meakins-Christie Laboratory, McGill University, Montreal, QC, Canada §Genaera Corporation, Plymouth Meeting, PA We have previously shown that the Th2-associated cytokine IL-9 is upregulated in allergic airway diseases including asthma. IL-9 has been demonstrated to induce the release of a number of inflammatory mediators, and has more recently been shown to be a potent stimulus for the production and secretion of mucus from airway epithelial cells. ICACC is a chloride channel associated with the differential expression of several mucus genes. In this study we set out to examine the expression of IL-9 following local specific antigen challenge and the association of expression with ICACC mRNA. Nasal biopsies were obtained from allergic individuals out of season both before (baseline) and after local antigen challenge with either ragweed or diluent (control). Immunocytochemistry and in-situ hybridization were used to assess IL-9 and ICACC protein and mRNA levels, while mucus-secreting epithelial cells were identified using PAS staining. Although we were able to detect IL-9 and ICACC immunoreactivity in most baseline specimens, their expression was significantly upregulated following ragweed challenge only. The majority of the IL-9-positivity was localized to infiltrating inflammatory cells, while ICACC expression was localized to mucus-producing epithelial cells. This study demonstrates the relationship between specific antigen challenge and expression of both IL9 and ICACC, suggesting a possible mechanism for the increased production of mucus from airway epithelial cells during inflammation. Supported by a grant from Genaera Corporation.
178
Modulationof NerveGrowthFactorReleaseFromHumanNasal Fibroblasts: Possible Role in Airway Inflammatory Diseases
Gerald Hanf*, Oliver Noga*, Ulrich Gerd Ohnemus*, Bettina Rost*, Christian Paschen§, Gert Kunkel* *Charitr, Humboldt-University, Berlin, Germany §ENT-Department, Charite, Humboldt University Berlin, Berlin, Germany The neurotrophin nerve growth factor (NGF) plays an important role for cells of the nervous, endocrine and immune system. High serum levels of NGF were found in patients with inflammatory airway diseases, including allergic rhinitis and bronchial asthma. Elevated levels of NGF have been detected in human airways after segmental allergen provocation. Recently we found that inhaled corticosteroids decrease NGF serum levels in subjects suffering from bronchial asthma. Fibroblasts play an important role in inflammatory processes of the airways like chronic rhinitis, polyposis nasi or asthma bronchiale, especially in the processes of "airway remodeling". In this study we have investigated whether structural cells like nasal fibroblasts might be a possible source of increased NGF levels and whether this release can be modulated by lipopolysaccharide (LPS), interleukin- 1[~(ILl!3), basic fibroblast growth factor (bFGF) and dexamethasone. Human
J ALLERGYCLIN IMMUNOL VOLUME 109, NUMBER 1
nasal fibroblasts, isolated from inferior nasal mucosa samples were cultured in RPMI 1640 medium containing 2% FCS, penicillin/streptomycin, amphotericin B and glutamine in a humidified atmosphere at 37°C with 5% CO 2. Experiments were performed after the 3rd passage. At confluence the cells were were stimulated with LPS, IL-1]3, bFGF and dexamethasone. NGF was measured in cellculture supernatants by ELISA. Under basal conditions, fibroblasts secreted NGF in time-dependent fashion. This effect was significantly augmented by treatment with LPS, IL-1~ and bFGF and inhibited by dexamethasone. Our results suggest that NGF released from human nasal fibroblasts participates in inflammatory airway diseases and probably is another target for the anti-inflammatory effects of steroids.
" 7 0 Regulation of Human Conjunctival Epithelial Cell Surface Receptor Expression and Chemokine Release by TNFa, IL-lJ3 and IFN'y
1 Iill
James L Stahl, Ellen B Cook, Neal P Barney, Frank M Graziano University of Wisconsin, Madison, WI Conjunctival epithelial cells (EC) play an important role in ocular allergic inflammation through release of chemokines, resulting in recruitment of inflammatory cells which in turn can interact directly with the epithelium via adhesion and MHC class I! receptors. These events must be discreetly regulated because different patterns of expression and cellular infiltration exist in acute versus chronic allergic disease, where different proinflammatory cytokines predominate. To better understand EC responses to pro-inflammatory cytokines known to be present in ocular allergic inflammation, we examined the individual and combined effects of TNFot, IL-1 and IFNy on chemokine release (IL-8, RANTES) and surface receptor expression (ICAM-I, HLA-DR, DP, DQ) in primary cultures of EC. EC were isolated from cadaveric conjunctival tissues and cultured in 24-well plates until almost confluent (3 to 5 passages). Cytokines (0.005 - 50 ng/ml) were added, alone or in various combinations, 24 hrs prior to harvesting of supernates for ELISAs and cells (with Trypsin-EDTA) for flow cytometry using PE-conjugated anti-lCAM-1 and FITC-conjugated pan-HLA-DR, DP, DQ. EC released IL-8 constitutively and IL-8 release was increased in a dose dependent manner by IL-113 p<0.0001, to a lesser extent by TNFct p<0.0001) but not by IFNy. An additive effect was observed when IL-113 and T N F a were combined (p<0.0001). In contrast, RANTES was more tightly regulated. RANTES was not constitutively released and not induced by either cytokine alone, yet any combination resulted in dose dependent RANTES release with TNFa and IFNy being the most potent combination (p<0.0001 for all combinations). IFNy was the strongest inducer of HLA expression (p<0.01), which was not expressed constitutively. ICAM-1 was expressed constitutively and dramatically upregulated in a dose dependent manner by IFNy (p<0.0001), but to a lesser extent by IL- 1[3 (p<0.001) and T N F a (p<0.0001) which produced equivalent effects. These results indicate that IFNy is critical for upregulation of surface receptors required for cell to cell interactions including migration (ICAM-1) and antigen presentation (HLA-DR, DP, DQ). Furthermore, in conditions of chronic inflammation, such as atopic keratoconjunctivitis, the combined presence of IFN? and either TNFa or IL-I~, resulting in RANTES release, might help explain why T-cell infiltration persists in chronic ocular allergic conditions.
1 80
Decreased Serum IL-12 in Wheezing Infants
Laurens Pieter Koopman*; Huub Savelkoul§, Jorrit Gerritsen~,, lnesz Van Benten*, Bert Brunekreef~, Herman Neijens* *Erasmus University, Rotterdam, Netherlands §Wageningen University, Wageningen, Netherlands ¥Beatrix Children's Hospital, Groningen, Netherlands ~University of Utrecht, Utrecht, Netherlands OBJECTIVE: To investigate the association between serum markers and atopic symptoms in the first year of life, and to evaluate the prognostic value of these markers for the development of wheezing and skin rash in the second year of life.
Abstracts
S73
METHODS: Data of 86 children on the development of wheezing and skin rash in the first two years of life was collected prospectively, using parental completed questionnaires, weekly symptom cards, structured interview, and physical examination. Serum markers [interleukin (IL)-10, IL- 12, IL- 13, eotaxin, soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble IL-2 receptor (slL-2R)] and total and specific IgE were determined at age 1. RESULTS: Children who developed wheezing in the first year of life had lower IL-12 serum levels than symptom-free children (median 40.3 pg/ml versus 49.0 pg/ml, p = 0.01) and had a higher serum IL-10/IL-12 ratio (median 0.41 versus 0.31, p = 0.001) at age 1. The IL-10/IL-12 ratio increased with an increasing number of wheezing episodes. Levels of sEselectin in children with wheezing and in children with itchy skin rash in the first year of life were higher than in symptom-free children (median 6.1 ng/ml and 5.9 ng/ml versus 4.9 ng/ml, p = 0.01 and p = 0.03 respectively). Children who developed wheezing in the second year of life already had increased slCAM- 1 levels at age 1. CONCLUSION: Children who developed wheezing in the first year of life showed a serum cytokine response skewed towards a T-helper 2 profile, with lower IL-12 levels and an increased IL-10/IL-12 ratio. Children who developed wheezing in the second year of life had elevated slCAM- 1 levels at age 1. Follow-up is needed to evaluate the prognostic value of various serum markers for the development of allergic disease in later childhood.
181 Model Increase Scar Formation in an Ovalbumin-lnduced Mouse of Allergic Airway Disease May Be Influenced by Epithelial Cell TGF[~Synthesis Roger S Thrall, Linda Guernsey, Laura Manzo, Susan Kindel, Michelle M Cloutier University of Connecticut, Farmington, CT The acute allergic inflammatory response induced by aerosolized ovalbumin in previously sensitized mice has been well characterized and is remarkably similar to allergic airway disease in humans. In this study we begin to characterize the chronic stages of this model after 6 weeks of continuous daily ovalbumin inhalation in C57BI/6J mice. Mice sacrificed at this 6 week time point have significant increased levels of lung collagen (total lung hydroxyproline: control mice - 138 _+22,6 wk asthma mice - 213 •+35, p<.03) but little evidence of existing allergic airway disease. Lung eosinophils and serum IgE levels elevated in the acute stage (10 days of ovalbumin inhalation) have returned to almost normal levels by 6 weeks. Conditioned media (CM) from isolated and cultured bronchial and tracheal epithelial cells from naive mice, 10 day acute asthma mice, and 6 week chronic asthma mice were compared for TGF~ levels. There was a dramatic increase in TGF~ levels in CM from bronchial epithelial cells at 10 day and 6 weeks as compared to epithelial cells isolated from naive animals (see Table below). The CM from tracheal epithelial cell cultures did not demonstrate a similar increase. The results of this study imply a regulatory role for bronchial epithelial cells in this model. TGF[~ is an important cytokine in the regulation of collagen metabolism and scar formation and inluences airway remodeling. Expression of TGFI3 in CM of bronchial and tracheal epithelial cell cultures
CELLULAR SOURCE BRONCHIAL Naive 10-day Asthma 6-week Asthma
TGF~ IN CM (pg/me) 392 2867 4371
TRACHEAL Na'fve 10-day Asthma 6-week Asthma
1271 563 1273
Abstracts
"7 ~ Increased Proinflammatory Cytokines Present in Breast Milk of /~bJI Women With Atopic/Autoimmune Disorders May Induce Gastrointestinal Epithelial Cell Apoptosis Victoria Maria Diokno*, Maya Srivastava§, Alton Melton*, Edward Conrod§ *Cleveland Clinic Federation, Cleveland, OH §Metro Health Medical Center, Cleveland, OH Previous studies have documented the presence of various cytokines, growth factors, and soluble receptors in human breast milk that may affect the neonatal mucosal immune system. Wide inter-individual differences in concentration are observed. In this study, breast milk donors (n=38) were classified into two groups: those with atopic or autoimmune disorders, and those without. Colostrum, transitional, and mature breast milk were collected into sterile 15 cc tubes, centrifuged for 15 minutes at 3000 G. The whey faction was used in ELISA assays for HGE IL-1RA, IL-2Ralpha, IL8, M-CSE TNF-alpha, TNF-beta, TGF-betal, TGF-beta2, MCP-1, RANTES, and GRO-alpha (R&D, Biosource). RNA was extracted from breast milk ceils using Qiagen kit and cryopreserved for semiquantitative RT-PCR. During sample collection donors completed a questionnaire regarding their/their infant's history of atopic/autoimmune disorders. Results showed increased TNF-alpha in breast milk of women with atopic/antoimmune disease (x=44.2 pg/ml vs. 0.8 pg/ml). In mature milk from atopic mothers, mean IL-IRA was also three times controls (x=1969 pg/ml versus 753 pg/ml), mean concentrations of IL-8 (x= 269 pg/ml versus 98 pg/ml), M-CSF (x=29, 717 pg/ml versus 15,090 pg/ml) and TNFalpha (x=404 pg/ml versus 185 pg/ml), were approximately twice as high in the atopic or autoimmune group. RT-PCR confirmed the presence of mRNA for TNF-alpha and TGF-betal, as well as NFKB p65 and p50, and strong expression of IKBa mRNA in breast milk. To determine potential significance of the increased proinflammatory cytokines on the infant, HT29 intestinal epithelial cells were used as an in vitro model. Cells were cultured with and without 4, 40, and 400 pg/ml TNF-alpha with and without 1000 pg/ml TGF-betal for 6-24 hours in RPMI1640 10%FCS/l%glutamine/penicillin/streptomycin at 370C in a 5% CO2 incubator. Cells were harvested after trypsinization and stained with Apoptosis Detection Kit, Annexin V-CY3 (Sigma). TNF-alpha at 400 pg/ml lead to significant cell apoptosis by 24 hours, but this damage was prevented by simultaneous culture with TGF-betal. Results suggest that elevated TNF-alpha at levels found in milk from some mothers with autoimmunity/allergy may be sufficient to cause epithelial destruction, and thus increase gut antigen exposure. This may be a potential mechanism of atopy transmission in some breastfed infants. High amounts of free TGF-beta may ameliorate this effect. If immune modulating formulas become available, such as those containing TGF-beta, supplementation may be a possible therapeutic strategy. Further experiments to determine effect of increased breast milk IL-8, TNF-alpha and TNF-beta together on HT-29 and Coco-2 cell cytokine expression are ongoing.
1
" 7 ~ IL-4 Production in Responseto Allergen Stimulation and CliniJl' I,]I col Symptoms of Allergic Rhinitis
Eric Howell*, Paul Mehlhop*, Anastasia Sigounas*, George Sigounas*, Dhavalkumar Patel§ *Brody School of Medicine at East Carolina University, Greenville, NC §Duke University Medical Center, Durham, NC IL-4 has been implicated as a major cytokine involved in the mechanism of allergic rhinitis. As such, we investigated the relationship between IL-4 production by peripheral blood mononuclear cells (PBMCs) in response to dust mite allergen and the clinical symptoms exhibited by patients with positive skin prick results to dust mite allergen. PBMCs were isolated from peripheral blood donated by human subjects and the frequency of IL-4 and 1NF-~/producing cells was measured by ELISPOT after in vitro challenge with dust mite allergen. Clinical symptoms of allergic rhinitis experienced by the study participants were obtained by administration of the Rhinitis Outcomes Questionnaire (ROQ), a validated symptom survey for allergic rhinitis. Additional questions were added to gauge the environmental factors that participants identified as triggers of their allergy symptoms. The group that tested positive
J ALLERGY CLIN IMMUNOL JANUARY 2002
by skin prick to dust mite allergen had elevated numbers of IL-4 producing cells compared to those who were not skin test positive for dust mite allergen (4.0/200,000 cells vs. 0.67/200,000 cells, respectively). The perception of dust as a trigger of allergic symptoms was also higher in the dust mite positive group compared to the dust mite negative group (average score of 3.3 in dust mite positive group, 1.25 in the dust mite negative group). Further, the participants that both tested positive for dust mite allergen and perceived dust as a significant trigger of their allergic symptoms had an average total symptom score of 34.29 upon symptom survey, in contrast to the average total score of 6.67 for those who were negative for dust mite and did not identify dust as a trigger of their symptoms. We found that in addition to a positive correlation between IL-4 production and skin test results, IL-4 production also correlates with clinical symptoms and the perception of dust as a trigger of allergic rhinitis, suggesting a link between IL-4 production and the clinical manifestation of allergic rhinitis upon exposure to dust mite allergen.
177 Upregulatioofn IL-9 and ,nterleukin-9-Associated
CalciumActivated Chloride Channel (ICACC) in Nasal Epithelium Following In Vivo Allergen Challenge
Mario Kontolemos*, Masao Toda*, Roy C Levitt§, Qutayba A Hamid* *Meakins-Christie Laboratory, McGill University, Montreal, QC, Canada §Genaera Corporation, Plymouth Meeting, PA We have previously shown that the Th2-associated cytokine IL-9 is upregulated in allergic airway diseases including asthma. IL-9 has been demonstrated to induce the release of a number of inflammatory mediators, and has more recently been shown to be a potent stimulus for the production and secretion of mucus from airway epithelial cells. ICACC is a chloride channel associated with the differential expression of several mucus genes. In this study we set out to examine the expression of IL-9 following local specific antigen challenge and the association of expression with ICACC mRNA. Nasal biopsies were obtained from allergic individuals out of season both before (baseline) and after local antigen challenge with either ragweed or diluent (control). Immunocytochemistry and in-situ hybridization were used to assess IL-9 and ICACC protein and mRNA levels, while mucus-secreting epithelial cells were identified using PAS staining. Although we were able to detect IL-9 and ICACC immunoreactivity in most baseline specimens, their expression was significantly upregulated following ragweed challenge only. The majority of the IL-9-positivity was localized to infiltrating inflammatory cells, while ICACC expression was localized to mucus-producing epithelial cells. This study demonstrates the relationship between specific antigen challenge and expression of both IL9 and ICACC, suggesting a possible mechanism for the increased production of mucus from airway epithelial cells during inflammation. Supported by a grant from Genaera Corporation.
178
Modulationof NerveGrowthFactorReleaseFromHumanNasal Fibroblasts: Possible Role in Airway Inflammatory Diseases
Gerald Hanf*, Oliver Noga*, Ulrich Gerd Ohnemus*, Bettina Rost*, Christian Paschen§, Gert Kunkel* *Charitr, Humboldt-University, Berlin, Germany §ENT-Department, Charite, Humboldt University Berlin, Berlin, Germany The neurotrophin nerve growth factor (NGF) plays an important role for cells of the nervous, endocrine and immune system. High serum levels of NGF were found in patients with inflammatory airway diseases, including allergic rhinitis and bronchial asthma. Elevated levels of NGF have been detected in human airways after segmental allergen provocation. Recently we found that inhaled corticosteroids decrease NGF serum levels in subjects suffering from bronchial asthma. Fibroblasts play an important role in inflammatory processes of the airways like chronic rhinitis, polyposis nasi or asthma bronchiale, especially in the processes of "airway remodeling". In this study we have investigated whether structural cells like nasal fibroblasts might be a possible source of increased NGF levels and whether this release can be modulated by lipopolysaccharide (LPS), interleukin- 1[~(ILl!3), basic fibroblast growth factor (bFGF) and dexamethasone. Human
J ALLERGYCLIN IMMUNOL VOLUME 109, NUMBER 1
nasal fibroblasts, isolated from inferior nasal mucosa samples were cultured in RPMI 1640 medium containing 2% FCS, penicillin/streptomycin, amphotericin B and glutamine in a humidified atmosphere at 37°C with 5% CO 2. Experiments were performed after the 3rd passage. At confluence the cells were were stimulated with LPS, IL-1]3, bFGF and dexamethasone. NGF was measured in cellculture supernatants by ELISA. Under basal conditions, fibroblasts secreted NGF in time-dependent fashion. This effect was significantly augmented by treatment with LPS, IL-1~ and bFGF and inhibited by dexamethasone. Our results suggest that NGF released from human nasal fibroblasts participates in inflammatory airway diseases and probably is another target for the anti-inflammatory effects of steroids.
" 7 0 Regulation of Human Conjunctival Epithelial Cell Surface Receptor Expression and Chemokine Release by TNFa, IL-lJ3 and IFN'y
1 Iill
James L Stahl, Ellen B Cook, Neal P Barney, Frank M Graziano University of Wisconsin, Madison, WI Conjunctival epithelial cells (EC) play an important role in ocular allergic inflammation through release of chemokines, resulting in recruitment of inflammatory cells which in turn can interact directly with the epithelium via adhesion and MHC class I! receptors. These events must be discreetly regulated because different patterns of expression and cellular infiltration exist in acute versus chronic allergic disease, where different proinflammatory cytokines predominate. To better understand EC responses to pro-inflammatory cytokines known to be present in ocular allergic inflammation, we examined the individual and combined effects of TNFot, IL-1 and IFNy on chemokine release (IL-8, RANTES) and surface receptor expression (ICAM-I, HLA-DR, DP, DQ) in primary cultures of EC. EC were isolated from cadaveric conjunctival tissues and cultured in 24-well plates until almost confluent (3 to 5 passages). Cytokines (0.005 - 50 ng/ml) were added, alone or in various combinations, 24 hrs prior to harvesting of supernates for ELISAs and cells (with Trypsin-EDTA) for flow cytometry using PE-conjugated anti-lCAM-1 and FITC-conjugated pan-HLA-DR, DP, DQ. EC released IL-8 constitutively and IL-8 release was increased in a dose dependent manner by IL-113 p<0.0001, to a lesser extent by TNFct p<0.0001) but not by IFNy. An additive effect was observed when IL-113 and T N F a were combined (p<0.0001). In contrast, RANTES was more tightly regulated. RANTES was not constitutively released and not induced by either cytokine alone, yet any combination resulted in dose dependent RANTES release with TNFa and IFNy being the most potent combination (p<0.0001 for all combinations). IFNy was the strongest inducer of HLA expression (p<0.01), which was not expressed constitutively. ICAM-1 was expressed constitutively and dramatically upregulated in a dose dependent manner by IFNy (p<0.0001), but to a lesser extent by IL- 1[3 (p<0.001) and T N F a (p<0.0001) which produced equivalent effects. These results indicate that IFNy is critical for upregulation of surface receptors required for cell to cell interactions including migration (ICAM-1) and antigen presentation (HLA-DR, DP, DQ). Furthermore, in conditions of chronic inflammation, such as atopic keratoconjunctivitis, the combined presence of IFN? and either TNFa or IL-I~, resulting in RANTES release, might help explain why T-cell infiltration persists in chronic ocular allergic conditions.
1 80
Decreased Serum IL-12 in Wheezing Infants
Laurens Pieter Koopman*; Huub Savelkoul§, Jorrit Gerritsen~,, lnesz Van Benten*, Bert Brunekreef~, Herman Neijens* *Erasmus University, Rotterdam, Netherlands §Wageningen University, Wageningen, Netherlands ¥Beatrix Children's Hospital, Groningen, Netherlands ~University of Utrecht, Utrecht, Netherlands OBJECTIVE: To investigate the association between serum markers and atopic symptoms in the first year of life, and to evaluate the prognostic value of these markers for the development of wheezing and skin rash in the second year of life.
Abstracts
S73
METHODS: Data of 86 children on the development of wheezing and skin rash in the first two years of life was collected prospectively, using parental completed questionnaires, weekly symptom cards, structured interview, and physical examination. Serum markers [interleukin (IL)-10, IL- 12, IL- 13, eotaxin, soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble IL-2 receptor (slL-2R)] and total and specific IgE were determined at age 1. RESULTS: Children who developed wheezing in the first year of life had lower IL-12 serum levels than symptom-free children (median 40.3 pg/ml versus 49.0 pg/ml, p = 0.01) and had a higher serum IL-10/IL-12 ratio (median 0.41 versus 0.31, p = 0.001) at age 1. The IL-10/IL-12 ratio increased with an increasing number of wheezing episodes. Levels of sEselectin in children with wheezing and in children with itchy skin rash in the first year of life were higher than in symptom-free children (median 6.1 ng/ml and 5.9 ng/ml versus 4.9 ng/ml, p = 0.01 and p = 0.03 respectively). Children who developed wheezing in the second year of life already had increased slCAM- 1 levels at age 1. CONCLUSION: Children who developed wheezing in the first year of life showed a serum cytokine response skewed towards a T-helper 2 profile, with lower IL-12 levels and an increased IL-10/IL-12 ratio. Children who developed wheezing in the second year of life had elevated slCAM- 1 levels at age 1. Follow-up is needed to evaluate the prognostic value of various serum markers for the development of allergic disease in later childhood.
181 Model Increase Scar Formation in an Ovalbumin-lnduced Mouse of Allergic Airway Disease May Be Influenced by Epithelial Cell TGF[~Synthesis Roger S Thrall, Linda Guernsey, Laura Manzo, Susan Kindel, Michelle M Cloutier University of Connecticut, Farmington, CT The acute allergic inflammatory response induced by aerosolized ovalbumin in previously sensitized mice has been well characterized and is remarkably similar to allergic airway disease in humans. In this study we begin to characterize the chronic stages of this model after 6 weeks of continuous daily ovalbumin inhalation in C57BI/6J mice. Mice sacrificed at this 6 week time point have significant increased levels of lung collagen (total lung hydroxyproline: control mice - 138 _+22,6 wk asthma mice - 213 •+35, p<.03) but little evidence of existing allergic airway disease. Lung eosinophils and serum IgE levels elevated in the acute stage (10 days of ovalbumin inhalation) have returned to almost normal levels by 6 weeks. Conditioned media (CM) from isolated and cultured bronchial and tracheal epithelial cells from naive mice, 10 day acute asthma mice, and 6 week chronic asthma mice were compared for TGF~ levels. There was a dramatic increase in TGF~ levels in CM from bronchial epithelial cells at 10 day and 6 weeks as compared to epithelial cells isolated from naive animals (see Table below). The CM from tracheal epithelial cell cultures did not demonstrate a similar increase. The results of this study imply a regulatory role for bronchial epithelial cells in this model. TGF[~ is an important cytokine in the regulation of collagen metabolism and scar formation and inluences airway remodeling. Expression of TGFI3 in CM of bronchial and tracheal epithelial cell cultures
CELLULAR SOURCE BRONCHIAL Naive 10-day Asthma 6-week Asthma
TGF~ IN CM (pg/me) 392 2867 4371
TRACHEAL Na'fve 10-day Asthma 6-week Asthma
1271 563 1273