Pergamon Developmental and Comparative Immunology, Vol. 20, No. 1, pp. 61-75, 1996 Copyright 0 1996 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0145-305X/96 515.00+0.00 0145-305X(95)OOO38-O A BURSAL STROMAL DERIVED CYTOKINE INDUCES PROLIFERATION OF MHC CLASS II BEARING CELLS Tania D. Obranovich and Richard L. Boyd Department of Pathology and Immunology, Monash Medical School, Commercial Road, Prahran, 3181 Victoria, Australia (Submitted June 1995; Accepted October 1995) nAbstract-To delineate the mechanisms of early embryonic chicken bursapoiesis, we developed several virally transformed stromal cell lines. Extensive phenotyping of one of these lines, BSL.2, with a panel of mAb mapped it as deriving from the El1 bursal surface epithelium. The functional properties of BSL.2 were assessed in vitro by culture of its conditioned medium with either embryonic bursal cells (BC) or adherent cell depleted bone marrow (BM). In both these populations there was an induction of prolif- eration during the first 72 h of culture, fol- lowed by a subsequent loss of proliferative activity coincident with the appearance of phagocytic and non-specific esterase positive adherent cells, which remained viable in cul- ture for up to 3 weeks. Flow cytometric ana- lysis of the proliferating cells demonstrated an increase in the number of MHC Class II+ (B-L+) cells for both BM and BC. There was, however, an overall decrease in total viable cell number during the period of prolifera- tion, demonstrating the stimulation of a specific subpopulation of cells. FACS separation of the unstimulated adherent depleted BM and BC populations demon- strated that only a subpopulation of the B-L+ cells proliferated in response to BSL.2 CM in addition to a subpopulation of B-L- BM. BSL.2 CM stimulated B-L+ BM and BC co-expressed the antigen detected by 68.1, which has been described as a MO marker, Address correspondence to: Dr T. Obrano- vich, Department of Pathology and Immu- nology, Monash Medical School, Commercial Road, Prahran, 3181 Victoria, Australia. Tel.: 61-3-276-2746; Fax.: 61-39-529-6484. but is also detectable on bursal lymphocytes. In ovo administration, at Ell, of concen- trated BSL.2 conditioned medium resulted in a significant increase, by E18, of bursal weight and Bu-1 and B-L+ cell number. There were no simultaneous alterations to the spleen. Preliminary MW analysis of the BSL.2 supematant has revealed an active factor of approximately 90 kDa. Copyright 0 1996 Published by Elsevier Science Ltd. q lKeywords-Chicken; Bursa of Fabricius; Stromal cells; Cytokines; B cells; Macrophages. Nomenclature cMGF BC BMAE CM E FCM FCS M0 NSE SE chicken myelomonocytic growth factor bursal cells bone marrow associated epithelium conditioned medium days of embryogenesis flow cytometry fetal calf serum macrophage non-specific esterase surface epithelium Introduction The commitment, growth and differentia- tion of multipotential stem cells into mature B lymphocytes and myeloid cells has been repeatedly shown to depend upon the influences of a functional stromal microenvironment (1). Analyses of the mammalian bone marrow stromal microenvironment have revealed that one 61