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Human mast cells (MC) were derived from umbilical cord blood and bone marrow progenitors cultured in the presence of a conditioned medium from a human mastocytosis cell strain and recombinant human kit ligand (rhKL). KL induced MC of predominantly two immunophenotypes, MCT and MCTC. In contrast, the conditioned medium induced MC subtypes MCTC and a third subtype, MCC, positive for chymase but negative for tryptase. This study clearly demonstrates that a third type of MC, MCC, can be induced in vitro from normal human progenitors. Correspondence to: Prof. S.A. Krilis, Department of Immunology. Allergy and Infectious Disease, St. George Hospital, Kogarah, New South Wales, 2217 (Australia), Tel. 61 2 93502955, Fax 61 2 93503981, E-Mail [email protected] Introduction Distinct subclasses of human mast cells (MC) have been described [1]. The MCT expresses one or more tryptases, whereas the MCTC expresses tryptase, chymase, cathepsin G and carboxypeptidase A. Recently, studies using imrau-nostaining with antibodies to tryptase and chymase in tissue MC have indeed suggested that a tryptase-negative MC might exist in humans [2]. Recombinant human kit ligand (rhKL) induces MC in vitro from a variety of sources of human hematopoietic progenitor cells, but the MC are immature [3]. Our group has recently demonstrated that conditioned medium derived from a human mastocytosis cell line (HBM-M) can also induce normal bone marrow progenitor cells to become morphologically mature cells that express FcεRI and the MC-specifíc proteases tryptase, chymase and carboxypeptidase [4]. In this study, MC subtypes derived from human bone marrow and umbilical cord blood were assessed by a systematic sequential double immu-noenzymatic analysis. Materials and Methods HBM-M-conditioned medium (HBM-M-CM) was prepared and cultures of human bone marrow and umbilical cord blood cells were performed as described previously [4]. Mouse antitryptase monoclonal and rabbit anti-chymase polyclonal antibodies were used for double immunostaining. Briefly, cytospun cells were incubated with antitryptase antibody after fixation in Carnoy’s fixative followed by rabbit antimouse IgG and APAAP complex. Slides were developed in naph-thol AS-MX phosphate solution containing fast red. Subsequently, cells were incubated with antichymase antibody (kindly provided by Dr. N. Schecter, USA) followed by D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /3 0/ 20 17 1 1: 43 :2 1 P M sheep antirabbit-HRP. The slides were then treated with a freshly prepared 3,3-diaminobenzidine tetra-hydrochloride solution. In these immunohistochemical reactions, MCT are stained red, whereas MCC are stained yellow-brown. MC-reappear as both colors when stained. KARGER E-Mail karger¢ä karger.ch Fax+41 61 306 12 34 http://www.karger.ch © 1997 S.KargerAG, Basel 1018-2438/97/1133-0289 $12.00/0 This article is also accessible online at: http://BioMedNet.com/karger Results and Discussion Bone marrow cells cultured with HBM-M-CM contained predominantly MCTC and MCt subtypes. The percentage of MCC in total MC decreased overtime from 100% at day 10 to 56.5% at day 21, when the percentage of MCTC was maximal and represented 37.4% of total MC in the culture. When human MC were derived by culturing umbilical cord blood cells in the presence of rhKL alone, the MCT cells were the major MC population at the early time points. The numbers of MCTC were increased slightly after 4 weeks of culture. After 5 weeks, 63, 30 and 7% (n = 5) of the total MC in the umbilical cord blood cultures were of the MCT, MCTC and MCC subtypes, respectively. In contrast, cultures supplemented with both HBM-M-CM and rhKL resulted in 57.9% MCTC at day 49 and 61.3% MCC at day 21. However, the number of MCT cells on day 49 was only 26% (n = 6) (fig. 1). Using the conditioned media and cytokine combination, we derived a novel population of human MC that does not express tryptase. The HBM-M-CM induced MCC and MCTC cells with downregulation of the MCC cells and the appearance of MCT cells when rhKL was added to the cultures. Based on ultrastructure and differences in protease composition between the MCT and MCTC types of MC, it has been postulated that there are distinct developmental pathways for these cells. The fact that the expression of MCC and MCT can be reversibly altered by culturing these MC in the presence and absence of HBM-M-CM indicates that in the human the phenotype of MC with respect to protease expression is not fixed. This has been elegantly demonstrated in vitro with bonemarrow-derived rodent MC cultured in the presence of different combinations of cytokines [5]. The results of this study would suggest that huFig.1. The percentage of MC subtypes of total MC in umbilical cord blood cultures with HBMM-CM and KL at different time points. D ow nl oa de d by : 54.70.40.11-10/30/201711:43:21PM man MC in culture are in a dynamic state and their protease phenotype is regulated by cytokinesin the microenviron-ment.AcknowledgmentsWe thank Dr N. Schecter for providing polyclonal antibody to chy-mase. This study wassupported by a grant from the National Health and Medical Research Council of Australia andthe St. George Hospital Cancer Research Fund.ReferencesIrani AA, Schechter NM, Craig SS, DeBlois G, Schwartz LB: Two types of human mast cellsthat have distinct neutral protease compositions. Proc Natl Acad Sci USA 1986;83:4464-4468.Weidner N, Austen KF: Heterogeneity of mast cells at multiple body sites. Fluorescentdetermination of avidin binding and immunofluores-cent determination of chymase, tryptase, andcarboxypeptidase content. Pathol Res Pract 1993;189:156-162.Li L, Zhang XT, Krilis SA: Factors that affect human mast cell and basophil growth; in Razin E,Pecht I, Rivera .1 (eds): Signal Transduction in Mast Cells and Basophils. Austin, Landes, inpress.Li L, Macpherson JJ, Adelstein S, Bunn CL, Atkinson K, Phadke K, Krilis SA: Conditionedmedia from a cell strain derived from a patient with mastocytosis induces preferentialdevelopment of cells that possess high affinity IgE receptors and the granule protease phenotypeof mature cutaneous mast cells. J BiolChem 1995; 270:2258-2263.Eklund KK, Ghildyal N, Austen KF, Stevens RL: Induction by IL-9 and suppression by 1L-3 and1L-4 of the levels of chromosome 14-dc-ríved transcripts that encode late-expressed mouse mastcell proteases. J Immunol 1993; 151:4266-4273. 290Int Arch Allergy Immunol 1997;113:289-290Li/Krilisinternational Archives ofAllergy ‚n < , Immunology Downloadedby: 54.70.40.11-10/30/201711:43:21PM