Attenuation of allergic inflammation by neutralization of IGF-I in murine asthmatic models

Abstracts $341

J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2

092 Attenuation of Allergic Inflammation by Neutralization of 1094 Expressionof uPAR(CD87)on Airway Eosinophils(EOS)FollowIGF-I in Murine Asthmatic Models ing SegmentalBronchoprovocationwith Allergen(SBP-AG) N. Yamashita j, H. Tashimo 1, M. Nakajima I , H. Ishida I, M. KuramochP, E Kaneko 1, R. KawashimaJ, T. Horiuchi 2, K. Ohtal; ITeikyo University School of Medicine, Tokyo, JAPAN, 2Kanto Central Hospital, Tokyo, JAPAN. RATIONALE: It has been suggested that airway wall thickening in asthma involves the production of extracellular matrix (ECM) components, including collagen, by myofibroblasts. In this study, we tried to clarify the role of IGF-I, a progression factor of fibroblast, in asthma. METHODS: Mice were immunized with ovalubmin + alum and challenged with ovalbumin inhalation, Anti-lGF-I neutralizing antibody was continuously introduced using osmotic pump during challenge. Pulmonary function was analyzed using whole body plethysmography before and after achetylcholine inhalation. RESULTS: Anti-IGF-I neutralizing antibody significantly inhibited the elevation of airway resistance and the increase of the airway wall thickening induced with OA inhalation. Airway inflammation was also inhibited by IGF-! neutralizing antibody, confirming by BALF cell analysis and histological examination, cDNA array analysis revealed that the neutralization of IGF-1 induced the activation of caspase- 1 and the reduction of anti-apoptotic Bag- 1 mRNA. CONCLUSIONS: In conclusion, not only induction of fibroblast proliferation but also anti-apoptotic effects of IGF-I may play a role in the process of airway inflammation and remodeling of asthma. Funding: Se!f-funded

" 093 ,

Chromosomal Substitutionsbetween Rat Strains with Xhlor Th2-biased Responses: A Novel Strategy for Physiogenomic Studies of Allergic Inflammation

R. E Lemanske, Jr. I, L. D. Mikus j , A. Tuffaha ], R. L. Sorkness L2, A. W. Cowley, Jr. 3, H. J. Jacob 3, L. A. RosenthaP; IUniversity of Wisconsin Medical School, Madison, WI, 2University of Wisconsin School of Pharmacy, Madison, WI, 3Medical College of Wisconsin, Milwaukee, WI. RATIONALE: Because the BN rat is an important model of Th2-biased responses, we investigated whether consomic strains constructed between BN and Dahl Salt Sensitive (SS) rats would be useful for studying the physiogenomics of allergic inflammation. METHODS: Consomic rat strains are being constructed by introgression of individual BN chromosomes into the SS genetic background (http://pga.mcw.edu). SS.BN9, SS.BN 16, SS.BN18, and SS.BN20 rats have BN chromosomes 9, 16, 18, and 20 substituted into the SS background, respectively. Splenocytes were incubated with Con A for 24 h; IFN-7, IL-4, and IL-13 were measured by ELISA. Two-way mixed lymphocyte cultures of splenocytes from each strain were incubated for 72 h; IL-2 and 1FN-y were measured by ELISA. RESULTS: Con A-stimulated SS, compared with BN, splenocytes produced significantly more IFN-~/, significantly less IL-4 and IL-13, and significantly elevated IFN-'~/IL-4 and IFN-)'/IL- 13 ratios (p < 0.001 ), demonstrating a Thl-biased SS response. Histoincompatibility, detected by IL-2 and IFN- 7 production in two-way mixed lymphocyte cultures, was present only between splenocytes of.strains that would be predicted to be histoincompatible based on their expected MHC haplotypes, which are encoded on chromosome 20. SS, SS.BN9, SS.BN16, and SS.BNI8 splenocytes were histocompatible with each other, but histoincompatible with BN and SS.BN20 splenocytes. Thus, the consomic rat immune systems developed as predicted, and BN and SS.BN20 rats may be suitable lbr reciprocal cell and organ transplants. CONCLUSIONS: Chromosomal substitution between rat strains with Th 1- or Th2-biased responses represents a potentially powerful strategy for identifying chromosomal regions regulating allergic inflammation and Th 1/Th2 balance. Funding: NIH/NIAID

J. B. Sedgwick, A. M. Brooks, E. A. Hazel, W. W. Busse: Medicine, University of Wisconsin, Madison, WI. RATIONALE: Urokinase-type plasminogen activator receptor (uPAR, CD87) is constitutively expressed on peripheral blood neutrophils, is upregulated upon cell activation, and participates in neutrophil adhesion, transmigration and degranulation through ]31 and [~2 integrins, uPAR binding to uPA and activation of plasminogen also provides a mechanism of cell surface degradation of matrix proteins thus allowing cells to migrate into and through inflammatory tissues. Since expression of this receptor on EOS could provide a similar mechanism for the recruitment of airway EOS in asthma, we hypothesized that airway EOS have enhanced expression of uPAR. METHODS: Peripheral EOS and neutrophils were isolated from subjects with mild-to-moderate asthma and uPAR expression was measured by FACS. RESULTS: Peripheral blood EOS expressed less uPAR compared to neutrophils when measured as either percentage positive cells (13-+5.8 vs 46.1+14.7, p=0.038, n=6) or mean fluorescence units (MFU: 4.1_1.2 vs 15_+4.5, p=0.022). EOS were also isolated from blood and bronchoalveolar (BAL) fluid 48 h after SBP-AG of asthma subjects. Again, circulating EOS expressed little CD87, whereas, BAL EOS had increased uPAR expression (15.6 MFU and 51.5% positive cells) comparable to circulating neutrophils. CONCLUSIONS: These data suggest that peripheral EOS do not utilize uPAR for vascular adhesion and transmigration but its increased expression on airway EOS may be a mechanism of tissue adhesion, migration and functional activation following allergen challenge of asthma subjects. Funding: Supported by NIH grants MOI RR03186 and P50 HL56396

1095 ness Blockade of L-Selectin EnhancesAirway Hyperresponsive(AHR) Through Inhibition of Thl ResponsesFollowing Allergen Sensitization and Challenge K. Takeda, N. Miyahara, T. Kodama, A. Joetham, C. Taube, A. Balhorn, A. Dakhama, E. W. Gelfand; Pediatrics, National Jewish Medical and Research Center, Denver, CO. RATIONALE: L-selectin plays a major role in granulocyte adhesion and serves as a T cell homing receptor. It has been shown to be important for development of airway allergic inflammation in studies of L-selectin-deficient mice. We investigated the effect of anti-L-selectin Ab (MEL- 14) in a model of allergen-induced airway inflammation and hyperresponsiveness. METHODS: Ovalbumin (OVA) sensitized and challenged BALB/c mice were treated with MEL-14 just before challenge, and mice were analyzed after the last challenge. RESULTS: MEL-14 treatment markedly inhibited T lymphocyte homing to local (peribronchial) lymph nodes (p<0.001), whereas AHR was significantly upregulated (p=0.02). Serum levels of OVA-specific IgE, IgGl and cell composition in bronchoalveolar lavage (BAL) fluid were comparable to contruls receiving rat IgG. Cytokine analysis revealed a decrease in IFN-y levels in BAL fluid (p=0.003). Also, in vitro IFN- 7 and IL-12 production in lung mononuclear cells (MNCs) was decreased in MEL-14-treated mice (p=0.04 and 0.02). Furthermore, adoptive transfer of lung MNCs from sensitized and challenged mice after co-culture with MEL-14 triggered the development of greater AHR compared to cells from control mice (p=0.009). CONCLUSIONS: T cells migrate to allergic inflammatory sites utilizing L-selectin. Prevention of this migration/accumulation with anti-L-selectin antibody is associated with decreased Thl cytokine production and enhanced AHR. Funding: NIH grants HL-36577, HL-61005 and EPA grant R825702