Leukemia (1998) 12, 414–421 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies RJA Filshie1, ACW Zannettino2, V Makrynikola1, S Gronthos2, AJ Henniker1, LJ Bendall1, DJ Gottlieb1, PJ Simmons2 and KF Bradstock1 1Department of Haematology, Westmead Hospital, Westmead, New South Wales; and 2Hanson Centre for Cancer Research, Leukaemia Research Unit, Division of Haematology, IMVS, Adelaide, South Australia, Australia Despite the importance of bone marrow stromal cells in hemo- the maintenance and regulation of hemopoiesis. Bone marrow poiesis, the profile of surface molecule expression is relatively fibroblasts (BMF) are a major constituent of these cultures, and poorly understood. Mice were immunized with cultured human under certain culture conditions can be grown as a virtually bone marrow stromal cells in order to raise monoclonal anti- 10 bodies to novel cell surface molecules, which might be homogenous population. BMF secrete fibronectin and col- involved in interactions with hemopoietic cells. Three anti- lagens types I, III and IV, express the cell surface antigens bodies, WM85, CC9 and EB4 were produced, and were found CD10, CD13, CD44, CD71 and contain smooth muscle cyto- to identify a 100–110 kDa antigen on bone marrow fibroblasts. skeletal components.1,4,11 A number of cellular ligands for Molecular cloning revealed the molecule to be MUC18 (CD146), integrin-mediated adhesive interactions belong to the large a member of the immunoglobulin superfamily, previously described as a marker of metastatic melanoma. In addition to immunoglobulin superfamily (IgSF). These molecules share a the expected expression on melanoma cell lines and endo- sequence of approximately 100 amino acids, featuring a cen- thelial cells, a number of human leukemic cell lines were found trally placed disulphide bridge which stabilizes a series of to express MUC18, including all six T leukemia lines tested, one anti-parallel b-strands.12 The final molecular weight of IgSF of five B lineage lines and one of four myeloid lines. Analysis molecules varies depending on the number of Ig-like domains of bone marrow samples from patients revealed positivity in and the extent of post-translational glycosylation.13 Several 20% of B lineage ALL (n = 20), one of three T-ALL, 15% of AML (n = 13) and 43% of various B lymphoproliferative disorders IgSF molecules including ICAM-1 (CD54), PECAM-1 (CD31) (n = 7). No apparent reactivity was observed with mononuclear and VCAM-1 (CD106) are known to be expressed on endo- cells from normal peripheral blood or bone marrow, including thelial cells,13–15 while VCAM-1 is also expressed on BMF and candidate hemopoietic stem cells characterized by their is subject to upregulation by cytokines such as tumor necrosis expression of the CD34 antigen. However, positive selection of factor-␣ (TNF-␣), interleukin-1 (IL-1) and IL-4.16 In order to bone marrow mononuclear cells labeled with MUC18 antibody define other stromal cell surface molecules which may be revealed a rare subpopulation (Ͻ1%) containing more than 90% of the stromal precursors identified in fibroblast colony-for- important in hemopoiesis, monoclonal antibodies (mAbs) ming assays. The structure and tissue distribution of MUC18 were raised by immunizing mice with cultured human mar- suggest a functional role in regulation of hemopoiesis. row fibroblasts or stromal cells. We report on three antibodies Keywords: MUC18; immunoglobulin superfamily; stroma; leukemia which were found to recognize another IgSF molecule, MUC18 (CD146), which has previously been identified on metastatic melanoma cells, endothelial cells and smooth mus- Introduction cle, but not on bone marrow stromal cells.17–20 Hemopoiesis is a complex process, requiring the generation of large numbers of mature, differentiated cells from a rela- Materials and methods tively small stem cell pool. In the adult this takes place within the bone marrow microenvironment in close association with Cell culture various extracellular matrix proteins and a number of special- ized cell types, including fibroblasts, endothelial cells, macro- Normal blood and bone marrow mononuclear cells collected phages and adipocytes.1–4 These cells are likely to influence from volunteers were separated by centrifugation on Ficoll– hemopoiesis through secretion of soluble or membrane- Paque (Pharmacia, Uppsala, Sweden). Mononuclear cells associated growth factors and regulatory cytokines5 as well as were washed in medium before immunofluorescence analy- contact-mediated signalling pathways. Adhesion molecules sis. To prepare BMF, mononuclear cells separated from bone on the surface of developing hemopoietic cells of different marrow samples (BMMNC) taken from allogeneic bone mar- lineages and stages of differentiation influence their localiz- row donors were cultured in McCoys 5A culture medium (ICN ation in relation to bone marrow stromal cells and matrix pro- Biomedicals, Costa Mesa, CA, USA) supplemented with 10% teins.6,7 The importance of stromal cells can be demonstrated fetal calf serum (FCS; ICN), L-glutamine and antibiotics. After in vitro where stromal cultures are capable of supporting hem- removal of non-adherent cells, adherent layers consisted of opoiesis for several weeks.8 Studies involving physical separ- homogenous populations of elongated spindle-shaped cells. ation of hemopoietic progenitors from stromal layers or the When confluent, adherent layers were passaged by trypsin- addition of monoclonal antibodies to adhesion molecules EDTA treatment and replated in 75 cm2 culture flasks (Costar, such as CD44 suggest that contact is important.7,9 It therefore Cambridge, MA, USA). For immunization of mice or immuno- follows that molecules expressed on the cell surface of bone fluorescence studies, flasks were treated with 5 mM EDTA marrow stromal cells are likely to play an important role in (20 min at 37°C), and washed twice in RPMI-1640 medium prior to use. Cells for injection into mice were then resus- pended in serum-free medium. Fibroblasts were also cultured Correspondence: KF Bradstock, Dept of Haematology, Westmead in a similar fashion from sterile biopsies of articular cartilage, Hospital, Westmead, NSW 2145, Australia; Fax: 612 9689 2331 chorionic villus, bronchial mucosa and amniotic fluid. Received 20 June 1997; accepted 25 September 1997 Human leukemic cell lines were maintained in log growth MUC18 expression on bone marrow stroma RJA Filshie et al 415 phase in medium supplemented with 10% FCS. Human PBS. Prior to incubation with monoclonal antibody the sec- umbilical vein endothelial cells (HUVECs) were cultured as tions were incubated with 5% normal goat serum (NGS) in described previously.21 For activation studies, endothelial PBS supplemented with 0.05% Tween 20 (Sigma, St Louis, cells were incubated with recombinant TNF-␣ (Genzyme, MO, USA) for 30 min to block potential non-specific binding. Cambridge, MA, USA) at 200 U/ml for 24 h at 37°C. Similar Sections were then incubated for 1 h at RT with mAb CC9 or studies on BMF were performed using IL-4 (Genzyme) or TNF- appropriate isotype-matched negative control, after which ␣ at 200 U/ml except that exposure to cytokines was varied they were washed three times in PBS + Tween 20 (PBST) over from 30 min to 24 h. a 30 min period. Specifically bound mAb was identified by incubation for 2 h at RT in biotinylated goat anti-mouse Ig (Caltag, Camperdown, Australia) diluted 1:200 in PBST + NGS Fibroblast colony-forming cell assays (CFU-F) followed by further washes in PBST as described above. Fol- lowing incubation in blocking solution containing PBS + NGS This in vitro assay of stromal progenitors was performed as supplemented with 0.8% bovine serum albumin (BSA) and previously described.22,23 Briefly, BMMNC were plated at a 0.1% IGSS quality gelatin (Amersham, Bucks, UK) sections concentration of 1 × 105/ml in 24-well tissue culture plates in were incubated in AuroProbe One streptavidin (Amersham) alpha-modified Eagles’s medium (␣-MEM; Flow Laboratories, diluted 1:80 in PBS supplemented with 1% NGS, BSA and Irvine, UK) containing 20% FCS, L-glutamine, ␤-mercapto- gelatin according to the manufacturer’s recommendations. ethanol and antibiotics. After 14 days culture at 37°Cin5% After three washes in PBS/BSA/gelatin, specimens were post- CO2, cultures were fixed in 1% paraformaldehyde and stained fixed for 10 min in 2% glutaraldehyde, washed twice with dis- with 0.1% toluidine blue. Clusters of more than 50 cells were tilled water and subjected to silver enhancement using the scored as CFU-F. BMMNC were dual labeled with the mAbs IntenSE M kit (Amersham). Finally, sections were coun- CC9 and STRO-1, which is known to identify stromal precur- terstained with Mayer’s hematoxylin prior to mounting. sors,24 using a double indirect method with fluorochrome- conjugated heavy chain-specific second layer antibodies. Cells were sorted into CC9+ and CD9− fractions of the STRO- Immunoprecipitation and SDS-PAGE 1+ population and plated as above. Approximately 5 × 107 NALM-6 cells were incubated with NHSS-Biotin reagent (Amersham) for 30 min at RT, followed Generation of monoclonal antibodies by washing in PBS and lysis with 1% Triton X-100 in PBS containing 0.2 mg/ml leupeptin, 0.5 mM PMSF and 5 mM BALB/c mice were injected intraperitoneally with 2.5 × 106 EDTA (20 min at 4°C). Lysates were pre-cleared by incubation washed fresh BMF or human bone marrow stromal cultures with Pansorbin cells (Calbiochem, La Jolla, CA, USA) for 16 h (HBMSC)25 and boosted twice at monthly intervals. Five days at 4°C, then added to Dynabeads (Dynal, Oslo, Norway) pre- after the third injection animals were sacrificed, spleens viously labeled with approximately 10 mg of monoclonal anti- removed and splenic lymphocyte suspensions prepared. body, and incubated for a further 2 h at 4°C. Bound proteins These were fused with NS1-Ag4 murine myeloma cells were dissolved in SDS running buffer with or without ␤- according to standard procedures.