372 Mononuclear eosinophils developed in vitro from hybrid eosinophil/basophil granulocytes produce GM-CSF

J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 1, PART 3

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GM-CSF R e g u l a t i o n in H u m a n P e r i p h e r a l B l o o d E o s i n o p h l l s . RJ H o r w i t z M D P h D , W W B u s s e M D , JS Malter M D . M a d i s o n , W I

Abstracts

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E o s i n o p h i l s ( E O S ) B i n d R h i n o v i r u s (RV) v i a I C A M - I a n d P r e s e n t A n t i g e n to R e - S p e c i f i c T C e l l s . __Z_T IJ=,rt,el/1~ ~am: ~AR I~elly: RR Rnd~'i~la~: I1~ g~l~w;cle ~nd V ~ BnSS~ University of Wisconsin, Madison, Wl, USA. RV infection is a major trigger of asthma exacerbations, and is associated with increased recruitment of EOS to the airway. To establish potential RV-EOS interactions, the following /n v/fro experiments were cotxktcted to determine whether RV binds directly to EOS via ICA.lvld and whether EOS can present RV antigens to RVspecific T-cells. Blood EOS were isolated by negative immunomagnetic selection. To measure RV binding to EOS, the cells were incubated for one hr with rsdiolabeled RV16. Little binding to unstimulated EOS was demonstrated; however, if the EOS were incubated overnight with GMCSF (to promote ICAM-1 expression), binding of radiolabeled virus was demonstrated. RV binding was blocked (60%) by anti-ICAM-1 ant~ody. In addition, EOS induced vigorous proliferation in homologous RV-specific T-coil clones only when co-cultured in the presence of RV16. Proliferation was not triggered by aLlogeneie EOS. These findings demonstrate that EOS can bind RV via the ICAM-1 receptor, and can mediate MItC-restricted proliferation of RV-specific T cells. These findings suggest that RV-EOS interactions contribute to airway inflammation during RV infections and enhance the likelihood for wheezing in asthma.

Interleukin-5-Mediated Eosinophilia in Hodgkin's D i s e a s e (HD). M Sfinchez-Borges, E Di Biagio, J D e s e n n e , R Sufirez, R S o m o z a , G A c q u a t e l l a . Caracas, V e n e z u e l a . We have studied cell-mediated immunity and eosinophils (Eos) in 18 HD patients and 16 controls. Significant eosinophilia was present in 5 patients and in none of the controls (p--0.03). Eos levels were lower in advanced disease and correlated with CD4+ T cell numbers in peripheral blood (PB) (r=0.5,13=0.03). Mononucleat cells from HD patients had decreased responses to PHA and reduced in vitro production of IL-2, yIFN, GM-CSF and sCD23. GM-CSF secretion was 82_+/-31pg/ml for patients with eosinophilia and 162+/-67pg/ml in those without eosinophilia. An eosinophil-survivabenhancing activity was present in sera and culture supematants (CS) from controls and patients; this activity was significantly higher for patients and was stronger for those with eosinophilia. Such activity was completely neutralized by preincubation with mAbs to IL-5 but not with normal mouse serum. Our results suggest a decreased Till function with TH2 predominance in patients with HD. IL-5 would be responsible for the eosinophilia observed in HD.

GM-CSF is an important eosinophil growth factor and is produced in vitro by eosinophils in response to various stimuli, including ionomycin and fibronectin. Studies designed to aseet'tain the mode of regulation of GM-CSF have been hampe/cd by the high intraeellular RNAse content and the inability to effectively transfect human eosinophils. Consequently, we examined ionomycin-indneed expression of GM-CSF in human peripheral blood eosinophils. Steady-state levels of GM-CSF mRNA c,an be detected by Northern blot analysis as early as two hrs after exposure to I uM ionomycin. Maximal accumulation of GM-CSF mRNA is seen between 8-16 Ins of culture with ionomycin, while overall cell viability remains greater than 90 %. No accumulation is seen in control eosinoplfil cultures. In order to determine if GM-CSF transcription contributed to the observed upregulation of message, we have developed techniques to transfect DNA constructs into human peripheral blood eosinophils using panicle-mediated gene transfer ("gene gun"). Preliminary experiments with cytomegalovims-lneiferase (CMV-luc) expression vectors has resulted in expression of assayable iuciferase activity. Therefore, we are now in a position to utilize this technology to investigate GM-CSF expression in eosinophils.

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M o n o n u c l e a r e o s i n o p h i l s d e v e l o p e d in vitro f r o m hybrid eosinophil/basophil granulocytes produce GMCSF J.A. Bo'¢ce, M.D., D. Friend, M.D., M, F. Gurish, Ph.D., K. F. Austen, M.D., W. F. Owen, M.D. Boston, MA We recently described the development in vitro of 90% pure hybrid eosinophil/hasophil granulocytes (HG) from human cord blood mononuclear cells (CBMNC) cultured for 14 d in the presence of IL3, IL-5 and Matrigel T M (MG). To characterize the dcvelopmcntal progression of HG, they were maintained for an additional 14 d of culture. After 28 d of culture, 73 + 1% (mean + SEM, n = 6) of the non-adherent cclls were mononuclear eosinophils (MNE), 13 + 3% were eosinophils with 2 or more nuclear lobes, 13 + 4% were residual HG, and 0.2 + 0.1% were basophils. More than 90% of these 28-d MNE were hypodensc. After an additional 5 d of culture in RPMI 1640 with 10% FCS lacking cytokines, 65 + 4% (n = 5) of these 28 d CB-dcrivcd MNE were viable, compared to 84 + 3% of the 14-d HG (n = 5). Only 2 + 1% of peripheral blood eosinophils (PBE) survived 5 d of culture without exogenous cytokines (n=5). 50% conditioned medium (CNO prepared from 2g-d MN-E and 14-d HG, respectively, maintained the survival of 60 + 7% and 77 + 7% of freshly isolated PBE over 72 h of culture; 20:1:8 % survived in RPMI 1640 with 10% FCS alone (n = 3). The eosinophil viabilitysustaining activity of both 50% MNE-CM and HG-CM was neutralized with an anti-GM-CSF antibody. 88% of these 28-