A murine model to study the in vivo T helper cell polarizing capacity of pollen-associated lipid mediators (PALMs) during primary sensitization

A Murine Model to Study the In Vivo T Helper Cell Polarizing Capacity of Pollen-Associated Lipid Mediators (PALMs) During Primary Sensitization J. Gutermuth1, C. Traidl-Hoffmann1, J. Ring2, H. Behrendt1, T. Jakob1,2; 1Division of Environmental Dermatology and Allergy GSF/TUM and ZAUM - Center for Allergy and Environ., Technical University Munich, Munich, GERMANY, 2Department of Dermatology and Allergy Biederstein, Technical University Munich, Munich, GERMANY. RATIONALE: Pollen grains not only function as allergen carriers, but also release bioactive lipid mediators (PALMs) upon contact with the aqueous phase. PALMs modulate human dendritic cells function in a fashion that results in a Th2 polarization of human naïve T cells in vitro. To address, whether PALMs show similar effects in vivo, we analyzed events of early T cell polarization during primary sensitization in mice. METHODS: To overcome the low frequency of naïve antigen-specific T cells in wild type mice, we established a DO11.10/BALB/c chimera using adoptive transfer, thus ensuring a defined number of OVA-peptide-specific CD4 cells in regional lymph nodes. 48 hours after intranasal instillation of OVA-peptide alone or OVA-peptide plus aqueous birch pollen extract (APE) to DO11.10/BALB/c chimeras, cells were obtained from draining lymph nodes, restimulated with peptide in vitro and on day 6 intracellular IL-4 and IFN- were analyzed by flow cytometry. RESULTS: Transfer of 5x106 T cells led to 1-2% of OVA-peptide TCR transgenic T Cells in regional lymph nodes. 48 hours after intranasal instillation of OVA-peptide, proliferation of antigen specific T cells was detected in draining, but not in non-draining lymph nodes. In comparison to exposure with OVA-peptide alone, intranasal instillation of OVApeptide plus APE lead to an increase in IL-4 - and a decrease of IFN- producing antigen specific T cells, as determined after peptide restimulation. CONCLUSIONS: PALMs appear to exert immunomodulatory activities on the early phase of primary sensitization in vivo, that result in a bias towards Th2 polarization. Funding: German Ministery of Science/ Faculty of Medicine, TU Munich

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Effect of Toll-Like Receptor Ligands on Survival of Human Bronchial Epithelial Cells H. Nagase1, K. Hirai2, T. Adachi1, J. Nakano1, K. Yamamoto3, N. Yamashita1, K. Ohta1; 1Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, JAPAN, 2Department of Bioregulatory Function, University of Tokyo Graduate School of Medicine, Tokyo, JAPAN, 3Department of Allergy and Rheumatology, University of Tokyo Graduate School of Medicine, Tokyo, JAPAN. RATIONALE: Viral infection can cause the exacerbation of asthma and virally infected bronchial epithelial cells (BEC) are reported to undergo apoptosis. Although cell death of infected BEC might have a role in worsening of airway damage, its precise molecular mechanism remains unclear. Recently, human Toll-like receptors (TLRs) are identified as receptors for pathogen recognition. To date, there is no comprehensive study about the effect of TLR ligands on the cell survival of human BEC. METHODS: We determined the effect of ligands for TLR2, 3, 4, 5, 7/8, and 9 on survival of BEAS-2B and primary cultured human bronchial epithelial cells (PBEC) by Annexin V and Propidium iodide by using flow cytometry. Cell survival was also determined by TUNEL assay. We also investigated the effect of TLR ligands on IL-8 and IP-10 generation by using ELISA. RESULTS: Among various TLR ligands, only TLR3 ligand, Poly I:C specifically up-regulated IL-8 generation and induced IP-10 generation in both BEAS-2B and PBEC. Also in case of the effect on cell survival, only Poly I:C induced significant decrease of survival, increase of apoptosis in BEAS-2B (n=9, p<0.01) and PBEC (n=4, p<0.01). Poly I:C-induced death of BEAS-2B was partially but significantly reversed by the treatment by anti-TLR3 mAb (n=4, p<0.05). CONCLUSIONS: TLR3 is reported to recognize viral double-stranded RNA. Our results suggested the prominent role of TLR3 in inducing the cell-death of virally infected BEC and apoptosis of BEC might work against the dissemination of virus and the worsening of airway damage. Funding: Grants-in-Aid from the Ministry of Education, Science, Sport

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IL-16 C-Terminal Peptide Expression In Vivo Decreases Inflammation, IL-4, and Mucus Production in a Murine Model of Asthma N. A. Parada, J. Urdaneta-Jaimes, M. Pollman, J. Lasky, M. Friedman, G. W. Hoyle; Section Pulmonary, Critical Care and Environmental Medicine, Tulane University Health Sciences Center, New Orleans, LA. RATIONALE: IL-16 has been shown to decrease Th2 cytokines in vitro by decreasing antigen driven activation and to decrease inflammation in an animal model. A 16 amino acid peptide from the C-terminus of IL-16 blocks IL-16 mediated CD4+ lymphocyte migration in vitro and has been shown to decrease airway hyperresponsiveness in an animal model. We hypothesized that expression of this IL-16 peptide in vivo would decrease inflammation and Th2 cytokines in a murine asthma model. METHODS: Transgenic (TG) mice expressing the IL-16 C-terminal peptide from the airway epithelial-specific Clara cell secretory protein promoter were generated. The IL-16 peptide was fused to a signal sequence and a FLAG epitope tag. Transgene expression was detected in the lungs by Northern blot analysis and FLAG immunohistochemistry. Untreated TG mice exhibited normal lung histology. TG mice and non-transgenic (NTG) controls were sensitized with ovalbumin (OVA) intraperitoneally and challenged with aerosolized OVA. RESULTS: Histological scores for tissue inflammation in OVA mice were significantly lower in TG mice compared to NTG mice (p<0.05). There was no significant difference in lavage fluid eosinophils between TG and NTG mice. IL-4 levels in lavage fluid were significantly lower from OVA TG mice compared to OVA NTG (p<0.05). IL-5 and IL-13 levels were not significantly different. Quantitation of PAS staining indicated that mucus production was significantly lower in the OVA TG mice compared with OVA NTG mice (p<0.01). CONCLUSIONS: Expression of IL-16 peptide in vivo inhibits allergic tissue inflammation, IL-4 production, and mucus production in a murine model of allergic inflammation. Funding: NIH NIAID K08 AI49790, ALA RG-043-N

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TSG-6 Deficiency Impairs Airway Inflammation and Hyaluronan Deposition in an Ova-Induced Pulmonary Inflammation Model S. Swaidani1, C. de la Motte1, C. Fulop2, T. G. Glant3, M. A. Aronica4; 1Immunology, Cleveland Clinic Foundation, Cleveland, OH, 2Biomedical Engineering, Cleveland Clinic Foundation, Cleveland, OH, 3Orthopedic Surgery and Biochemistry, Rush University, Chicago, IL, 4Pulmonary, Allergy and Critical Care Medicine, Cleveland Clinic Foundation, Clevland, OH. RATIONALE: The glycoprotein Tumor necrosis factor-stimulated gene6 (TSG-6) contains a hyaluronan-binding site and is an important component of the extracellular matrix (ECM). TSG-6 is not constitutively present in tissues but is up regulated by various cytokines and growth factors and may play a role in lymphocyte adhesion and migration. To test the hypothesis that TSG-6 interacts with hyaluronan (HA) and is necessary for ECM organization and leukocyte recruitment, we investigated the function of TSG-6 using an ovalbumin (OVA) sensitized murine model of allergic pulmonary inflammation. METHODS: Measurements of airway hyperresponsiveness (AHR), collection of bronchoalveolar lavage (BAL), lung histology and confocal microscopy to determine HA deposition were performed on TSG-6-/- and wild-type BALB/c littermates sensitized and challenged to OVA and in un-challenged controls. RESULTS: Comparison of OVA sensitized and challenged TSG-6-/- mice to wild-type BALB/c littermates demonstrated: 1) a significant decrease in inflammatory cell recovery form bronchoalveolar lavage fluid 2) evidence of inflammation in the peribronchial and perivascular areas yet diminished inflammation in the lung parenchyma and 3) a significant reduction in hyaluronan deposition in the lung and 4) no difference in the development of AHR. CONCLUSIONS: These results suggest that TSG-6 may play an important role in the recruitment and/or migration of inflammatory cells in vivo and TSG-6 may play a role in the increase in HA deposition seen in allergic pulmonary inflammation. Taken together these results suggest that TSG-6 may be an important link between inflammation and structural changes that can occur in the asthmatic airway. Funding: NIH/NHLBI K08 HL04449-01

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Abstracts S45

J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2