973 Th1 and Th2 Cytokine-mediated CXC Chemokine Production in Human Lung Microvascular Endothelial Cells in Vitro A. Matsuda, H. Saito, K. Matsumoto; Department of Allergy and Immunology, National Research Institute for Child Health and Develop- ment, Tokyo, JAPAN. RATIONALE: Increased vascularity is an important element of airway remodeling in patients with bronchial asthma. However, the molecular mechanism involved in this process remains unclear. In order to clarify it, in vitro responses of human lung microvascular endothelial cell (HMVEC-L) to several cytokines were examined. METHODS: HMVEC-L was incubated with a combination of TNF- alpha and IL-4 (Th2 condition) or a combination of TNF-alpha and IFN- gamma (Th1 condition) in the presence or absence of VEGF and FGF2 for 48 hours. Cell proliferation was measured by 3 H-thymidine uptake. Tube formation was determined by light microscopy after cells were seeded on Matrigel for 18 hours. Comprehensive gene expression profiles of HMVEC-L in Th1 or Th2 condition were analyzed by using high-density oligonucleotide probe arrays (GeneChip) and real-time PCR. RESULTS: Both Th1 and Th2 conditions significantly reduced VEGF- or FGF2-mediated HMVEC-L proliferation. However, Th2 condition but not Th1 condition markedly promotes tube formation of HMVEC-L both in the presence and absence of VEGF or FGF2. GeneChip analysis revealed that CXCL1, CXCL2, CXCL5, CXCL6 and CXCL8 which are known to bind CXCR2 and to possess angiogenic activity, were predominantly up- regulated in the Th2 condition. In contrast, CXCL9, CXCL10 and CXCL11 which bind to CXCR3 and possess angiostatic activity, were significantly up-regulated only in the Th1 condition. We also found that the induction of these chemokines was not inhibited by dexamethasone treatment at all. CONCLUSIONS: Our results suggest a possible role of CXC chemokines in the pathogenesis of airway remodeling in asthma through their angiogenic/angiostatic activities. Funding: National Institute of Biomedical Innovation 974 Differential IL-2-Induced Accumulation of IL-13 + and IFN- Gamma + Peripheral Blood T Cells (PBL) between Asthmatic and Non-Asthmatic Subjects M. J. Loza, S. Foster, S. P. Peters, R. B. Penn; Internal Medicine/Center for Human Genomics, Wake Forest University School of Medicine, Win- ston-Salem, NC. RATIONALE: IL-13 is an important effector molecule in asthma pathol- ogy. Contrary to initial characterizations of asthma as a “Th2” disease, both IL-13 and IFN-gamma levels are also elevated in the airways of asthmatics. In PBL, numbers of IL-13 + cells can be increased by anti- gen/CD3-mediated stimulation or independent of antigen in response to IL-2 or IL-15, whereas increases in IFN-gamma + T cells generally require co-stimulation with IL-12. We tested whether asthmatic lymphocytes dif- fer in their response to antigen -dependent or -independent stimulation for accumulation of IL-13 + and IFN-gamma + T cells. METHODS: PBL obtained from asthmatic and control, non-asthmatic subjects were stimulated for cytokine production with PMA, Calcimycin, and monensin before and after 5-d culture with CD3+CD28 mAb and IL-2, or 6-d culture with IL-2. Expression of IL-13 and IFN-gamma in gated T cells was analyzed by flow cytometry. RESULTS: There were no significant differences in day 0 proportions of IL-13 + and IFN-gamma + T cells between asthmatics and controls. Mean increase in IL-13 + T cells was significantly greater in asthmatics than con- trols in response to IL-2 (2-fold greater) but not CD3-mediated stimula- tion. Although IFN-gamma + cells did not increase in response to IL-2 in controls, in asthmatics, IFN-gamma + cells increased significantly in mean. The difference between asthmatics and controls for mean numbers of IFN-gamma + cells after CD3-mediated stimulation was not significant. CONCLUSIONS: IL-2-induced accumulation of IL-13 + and IFN- gamma + T cells is greater in asthmatics than control subjects. These results may implicate IL-2/IL-15-mediated allergen-independent processes in the seemingly paradoxical co-elevation of IL-13 and IFN-gamma levels in asthmatics. 975 Regulation Of IL-5R On Bone Marrow Eosinophils and CD34 + Progenitors L. D. Saleh, G. M. Gauvreau, M. Duong, K. Howie, A. Cordova, R. Sung, T. Rerecich, P. M. O’Byrne; McMaster University, Hamilton, ON, CANADA. RATIONALE: Interleukin-5 (IL-5) is a critical hematopoietic growth factor for eosinophil differentiation, activation and survival. Previous studies have demonstrated that IL-5R levels on peripheral blood eosinophils are downregulated following exposure to IL-5. METHODS: Peripheral blood (PB) and bone marrow (BM) samples were collected from 8 allergic asthmatics for collection of eosinophils and non-adherent mononuclear cells. Cells were incubated with IL-5 (1ng/ml) for 10 min and 120 min, stained for IL-5R, and analyzed by flow cytom- etry. Eosinophils were identified by gating on the CD16- population, and CD34 + progenitors were identified using a sequential gating strategy. RESULTS: BM and PB eosinophils expressing IL-5R decreased to 22.30±15.70% and 18.38±26.34%, respectively, after 120 min incubation with IL-5, compared to control 54.96±10.54% and 70.41±19.51%, respec- tively (p=0.002 and 0.001). There was a trend towards significance in the fall in IL-5R after 120 min between BM and PB eosinophils (p=0.056). BM CD34 + progenitor expression of IL-5R was 7.70±11.00%, lower than BM or PB eosinophils, being 54.96±10.54% and 70.41±19.51%, respectively (p<0.001). There was no effect of IL-5 on BM CD34+ prog- enitors after 10 min (p=0.227) or 120 min (p=0.251) compared to control. CONCLUSIONS: BM and PB eosinophils downregulate IL-5R after IL-5 incubation, but BM CD34+ progenitors, do not. This suggests that IL-5R is regulated differently in BM CD34+ progenitors. There is very low expression of IL-5R on BM CD34+. 976 Brain Derived Neurotrophic Factor (BDNF), B-Lymphocyte Chemoattractant (CXCL13), and Macrophage Inhibitory Pro- tein 3 (MIP-3) are Increased in Sputum of Severe and Non- severe Asthmatics with Elevated Neutrophil Percentages A. T. Hastie, W. C. Moore, J. Krings, R. Smith, P. Vestal, M. Wu, D. A. Meyers, S. P. Peters, E. Bleecker; Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem, NC. RATIONALE: Qualitative protein array analysis indicates that patients with severe asthma (i.e. those with persistent symptoms and frequent medical encounters despite high dose, inhaled corticosteroids [ICS]) have apparently greater amounts of BDNF, CXCL13, and MIP-3 in their spu- tum compared with patients with mild persistent asthma. Whether these increased mediator levels are associated with increased inflammation is not known. METHODS: After informed consent, 173 asthmatics (37 characterized as “severe” on high dose ICS [SA], 136 “nonsevere” [NSA] by NHLBI & SARP criteria) underwent assessment of cells from induced sputum and ELISAs on sputum supernates for BDNF, CXCL13, BMP-4 and MIP-3. Statistical analysis was performed with Sigmastat (ANOVA and t-test as appropriate). RESULTS: The total cell count/gram sputum and the % neutrophils (PMNs) were significantly greater in SA than in NSA, (median total cell count/gm: SA= 2.8x10 6 vs NSA= 2.2 x 10 6 , p=0.04; % PMNs: SA=46% vs NSA=35%, p=0.045, ANOVA). BDNF, CXCL13 and MIP-3 were increased in sputa of SA compared to NSA, but did not reach significance. However, BDNF, CXCL13 and MIP-3 were significantly increased in those asthmatics with greater than 40% PMNs in sputum compared to asthmatics with less than 40% PMNs (BDNF: >40%PMN=18.5 vs <40%PMN=11.5 pg/ml, p<0.001; CXCL13: >40%PMN=94 vs <40%PMN=38 pg/ml, p<0.001; MIP-3: >40%PMN=706 vs <40%PMN=302 pg/ml, p<0.001). CONCLUSIONS: Severe asthmatics have increased total cell count/gm sputum and increased % PMNs present in their sputum. Asthmatics with increased % PMNs have concommitantly elevated levels of BDNF, CXCL13 and MIP-3 in their sputum compared to asthmatics with fewer PMNs. Funding: NHLBI 069167, 067663, and Wake Forest University General Clinical Research Center (GCRC) M01-RR07122 S252 Abstracts J ALLERGY CLIN IMMUNOL FEBRUARY 2006 MONDAY