S74 Abstracts J ALLERGY CLIN IMMUNOL JANUARY 2002 1 ~ PBMC Proliferative Responsesand Cytokine mRNA Expression: 1,111- The Effect of Costimulationby Two Allergens E Hadley, Farida Y Patel, L Farid, EC McKie, RB Gore, N Sehgal, Adnan Custovic, Ashley A Woodcock Wythenshawe Hospital, Manchester, UK We have studied how allergens interact in their effect on PBMC's from Asthmatics with mite sensitisation (MAA, n=8), Asthmatics with sensitisa- tion to both cat and mite (MCAA, n=8), compared to control subjects (Non- atopic, Non-asthmatic, NN, n=8). Cells were cultured at 1 x 105 (prolifera- tion) and 5 × 106 (cytokine) cells/well in Aim V serum-free medium and stimulated with PHA (ll.tg/ml), dust mite (30~tg/ml) and Cat extracts (30,000SQU/ml), dust mite and Cat combined or negative control (Aim V). Proliferation was assessed by 3H-thymidine uptake on day 6 (ll.tCi well). IL-4, IL-5, IFN-~' and TGF-I] mRNA expression was quantified by Taqman Real-Time RT-PCR (3 days PHA, 6 days for other stimulants). RNA was extracted by Trizol LSTM/acid-phenol chloroform and mRNA expressed in orders of magnitude less or greater than the negative control. AA PBMC proliferated more than NN after stimulation with mite extract only: [MAA: median: 34.1 (range: 1.61- 81.97]; [MCAA: 39.96 (18.23- 176.42)]; [NN: 16.37 (2.97- 27.68)]; [Mann-Whitney, p<0.05 for both]. HDM+Cat co- stimulation produced a greater effect in PBMC proliferation across all 3 clinical groups, compared to the sum of their separate effects: [HDM+Cat co-stimulation: 71.53 (6.52- 206.34), Sum (HDM and Cat): 43.49 (4.86- 191.09), p=0.009]; in the NN group alone: [HDM+Cat co-stimulation: 61.5 (6.52- 206.34), Sum (HDM and Cat): 31.96 (8.64- 44.36), p=0.01]. IL-4 mRNA expression in PBMC was significantly higher post HDM+Cat co- stimulation for MAA [4.28 (0.77-57.4)] and MCAA [1.58 (0.51-5.74)] than NN [0.57 (0.12-4.65)] (p=0.029 and 0.038 respectively); and post cat stimulation for MAA than NN [1.28 (0.21-15.2); 0.16 (0.05-1.01); p=0.021]. IL-5 mRNA expression was significantly higher post HDM+Cat costimulation for MAA [82.05 (13.4-255)] and MCAA [52.85 (5.55-576)] than NN [3.53 (0.002-15.5)] (p=0.001 and 0.007 respectively); post cat stimulation for MAA [3.04 (1.17-57.6)] and MCAA [15.55 (0.91-301)] than NN [0.62 (0.01-66.1)] (p=0.029 and 0.010); post HDM stimulation for MCAA than NN [158 (1.05-391); 17 (0.02-48.8); p=0.007]; and post PHA stimulation for MCAA than NN [157.5 (12.2-423); 15.5 (0.22-168); p=0.038]. IFN- 7 mRNA was incrementally decreased by costimulation in the MAA group: [HDM+Cat co-stimulation: 8.07 (2.2-15.5), Sum (HDM and Cat): 42.13 (7.56-102.36), p=0.015] and TGF- 13mRNA expression in the MCAA group: [HDM+Cat co-stimulation: 1.76 (0.84-3.34), Sum (HDM and Cat): 3.49 (1.45-8.34), p=0.021]. In conclusion: (a) Both prolif- erative responses and cytokine mRNA expression are significantly differ- ent in AA versus NN. (b) Co-stimulation by allergens (HDM+Cat) appears to have a positive synergistic effect on PBMC proliferation in both AA and NN, but down-regulates IFN-'/and TGF-I] mRNA expression only in AA. 83 IL-9 Induces IL-13 Expression In Vitro, and In Vivo Jamila Louahed*, Peter J O'Brien§, Yuhong Zhou§, Christine Weiss¥, Jean-Christoph Renauld*, Roy C Levitt¥ *Ludwig Institute for Cancer Research, Brussels, Belgium §Genaera Corporation, Plymouth Meeting, PA ¥Genaera Corporation, Plymouth Meeting, PE Interleukin-9 (IL-9) and Interleukin-13 (IL-13) are cytokines expressed in inflammatory immune responses that are controlled by T-Helper-2 (TH- 2) type T lymphocytes. Recently it has been suggested that these cytokines play a central role in the pathogenesis of asthma. Specifically, transgenic expression of IL-9 or IL-13 in the lung produced an asthmatic like response. IL-13 expression is also found to be elevated in asthma patients and asthma has been genetically linked to chromosome 5q, a region that contains both the IL-13 and the IL-9 genes. With the knowledge that IL-9 is important in asthma and other inflammatory diseases and reports that IL-13 is also up- regulated in these conditions it we sought to determine if one cytokine could control the other. Here we present in vivo and in vitro data to show that IL-9 controls the levels of IL-13 in inflammatory diseases such as asthma. We examined IL-13 gene expression in the murine mast cell line L138 and BW51.47 murine thymic lymphoma cells in response to rlL-4, rlL-9, or rlL- 3. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) show that IL-13 mRNA is upregulated in response to rlL-9, but not rlL-4 or rlL-3. Northern analysis ofcytokine expression in Tg5 and Tg54 mice, which over- express an IL-9 transgene, was compared to expression in isogenic control mice. These results show that IL- 13 is specifically overexpressed in the glan- dular stomach and lung of IL-9 transgenic mice, but not in control mice. These results suggest that IL-9 is a regulator of IL- 13 production, and con- firm the pleitropic effects of IL-9 as a key regulator in asthma. 184 Characterization of Chemokine Responses in Purified Human Basophils R Schleirner*, Carol Bickel§, John White~, Lina Lim§, Bruce Scott Boch- nerff, Donald Macglashan§ *Johns Hopkins University, Baltimore, MD §Johns Hopkins University School of Medicine, Baltimore, MD ¥Glaxo- SmithKline, King of Prussia, PA Recognition of the importance of basophils in asthma and other allergic diseases in humans is expanding at a rapid rate. New studies indicate sub- stantial basophil infiltration of the airways in autopsies of lungs from indi- viduals that died of asthma. Since basophils are capable of producing sig- nificant quantities of peptide leukotrienes, IL-4 and IL- 13, their presence in diseased airways is likely to be of pathological relevance. In order to better understand the mechanisms by which basophils infiltrate the airways, we have screened over 30 chemokines for their ability to induce basophil migration in microchemotaxis chambers. Basophils were purified from leukapheresis packs using elutriation and negative selection (Miltenyi). The procedure included an overnight incubation with 30 pg/ml of IL-3: Responses to chemokines were tested at concentrations of 1, 10 and 100 nM. Among the tested CC (beta) chemokines, the greatest responses were observed with (MCP-4 = eotaxin-1 = eotaxin-2 = eotaxin-3 = MIP-3) > [HCC-4 = RANTES > MCP-2 > LEC] > MCP-3 = SLC = ELC = TECK. Among the tested CXC (alpha) chemokines, the greatest responses were observed with (SDF- 1c~> IL-8) > GCP-2. Chemokines shown in parenthe- ses were effective at concentrations of 1 nM and higher, those in brackets were effective at 10 nM and higher and others required 100 nM. Data are from n>2 experiments in all cases. Flow cytometric evaluation of human basophils revealed expression of CCR 1, CCR2, CCR3, CCR7, CXCR1 and CXCR4. Preliminary results with a specific antibody against CCR3 indi- cate that the response to HCC-4 may be mediated by CCR3, a known recep- tor for eotaxins 1-3, MCP-4 and RANTES. A single study with purified human eosinophils indicated that HCC-4 is also active as an eosinophil chemoattractant and that its action is blocked by anti-CCR3. Based upon the results to date, it appears that basophil chemotaxis can be mediated by CXCRI, CXCR4, CCRI, CCR2, CCR3 and possibly CCR7. Ongoing studies are designed to definitively determine the chemokine receptors expressed on basophils which mediate the observed responses. In conclu- sion, our studies have identified several chemokines not previously known to induce basophil chemotaxis and suggest that a number of chemokines are capable of mediating basophil recruitment to sites of allergic inflamma- tion. These findings will help focus in vivo studies to assess the chemokines and chemokine receptors relevant to basophil recruitment in vivo. 185 Prevalence of AtOpyand At°pic Diseases in Icelandic sch°°'" c h i l d r e n Michael Clausen*, Bengt Bjorksten§, Asgeir Haraldsson*, Sigurdur Kristjansson* *Landspitali University Hospital, Reykjavik, Iceland §Cen- ter for Allergy Research, Karolinska Institute, Stockholm, Sweden BACKGROUND: The prevalence of adult atopic diseases in Iceland is lower than in Western Europe but there are no data concerning children. OBJECTIVE: To investigate the prevalence of atopic diseases and sen- sitivity to common allergens, in 10-11 year old schoolchildren as part of the phase-2 of the International Study of Asthma and Allergy in Children (ISAAC). METHODS: 946 children answered a questionnaire about atopic dis- eases. Skin prick tests (SPT) with 6 allergens were performed on 775 chil- dren and they were also examined for signs of atopic eczema. RESULTS: The prevalence of allergic rhinitis was 11% and asthma 9%. The reported prevalence to eczema was 26.9%, but only 10.3% had signs