MUC18, a member of the immunoglobulin superfamily ...

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 1

Department 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 hemopoiesis, the profile of surface molecule expression is relatively poorly understood. Mice were immunized with cultured human bone marrow stromal cells in order to raise monoclonal antibodies to novel cell surface molecules, which might be involved in interactions with hemopoietic cells. Three antibodies, WM85, CC9 and EB4 were produced, and were found to identify a 100–110 kDa antigen on bone marrow fibroblasts. Molecular cloning revealed the molecule to be MUC18 (CD146), a member of the immunoglobulin superfamily, previously described as a marker of metastatic melanoma. In addition to the expected expression on melanoma cell lines and endothelial cells, a number of human leukemic cell lines were found to express MUC18, including all six T leukemia lines tested, one of five B lineage lines and one of four myeloid lines. Analysis of bone marrow samples from patients revealed positivity in 20% of B lineage ALL (n = 20), one of three T-ALL, 15% of AML (n = 13) and 43% of various B lymphoproliferative disorders (n = 7). No apparent reactivity was observed with mononuclear cells from normal peripheral blood or bone marrow, including candidate hemopoietic stem cells characterized by their expression of the CD34 antigen. However, positive selection of bone marrow mononuclear cells labeled with MUC18 antibody revealed a rare subpopulation (⬍1%) containing more than 90% of the stromal precursors identified in fibroblast colony-forming assays. The structure and tissue distribution of MUC18 suggest a functional role in regulation of hemopoiesis. Keywords: MUC18; immunoglobulin superfamily; stroma; leukemia

Introduction Hemopoiesis is a complex process, requiring the generation of large numbers of mature, differentiated cells from a relatively small stem cell pool. In the adult this takes place within the bone marrow microenvironment in close association with various extracellular matrix proteins and a number of specialized cell types, including fibroblasts, endothelial cells, macrophages and adipocytes.1–4 These cells are likely to influence hemopoiesis through secretion of soluble or membraneassociated growth factors and regulatory cytokines5 as well as contact-mediated signalling pathways. Adhesion molecules on the surface of developing hemopoietic cells of different lineages and stages of differentiation influence their localization in relation to bone marrow stromal cells and matrix proteins.6,7 The importance of stromal cells can be demonstrated in vitro where stromal cultures are capable of supporting hemopoiesis for several weeks.8 Studies involving physical separation of hemopoietic progenitors from stromal layers or the addition of monoclonal antibodies to adhesion molecules such as CD44 suggest that contact is important.7,9 It therefore follows that molecules expressed on the cell surface of bone marrow stromal cells are likely to play an important role in

Correspondence: KF Bradstock, Dept of Haematology, Westmead Hospital, Westmead, NSW 2145, Australia; Fax: 612 9689 2331 Received 20 June 1997; accepted 25 September 1997

the maintenance and regulation of hemopoiesis. Bone marrow fibroblasts (BMF) are a major constituent of these cultures, and under certain culture conditions can be grown as a virtually homogenous population.10 BMF secrete fibronectin and collagens types I, III and IV, express the cell surface antigens CD10, CD13, CD44, CD71 and contain smooth muscle cytoskeletal components.1,4,11 A number of cellular ligands for integrin-mediated adhesive interactions belong to the large immunoglobulin superfamily (IgSF). These molecules share a sequence of approximately 100 amino acids, featuring a centrally placed disulphide bridge which stabilizes a series of anti-parallel b-strands.12 The final molecular weight of IgSF molecules varies depending on the number of Ig-like domains and the extent of post-translational glycosylation.13 Several IgSF molecules including ICAM-1 (CD54), PECAM-1 (CD31) and VCAM-1 (CD106) are known to be expressed on endothelial cells,13–15 while VCAM-1 is also expressed on BMF and is subject to upregulation by cytokines such as tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1) and IL-4.16 In order to define other stromal cell surface molecules which may be important in hemopoiesis, monoclonal antibodies (mAbs) were raised by immunizing mice with cultured human marrow fibroblasts or stromal cells. We report on three antibodies which were found to recognize another IgSF molecule, MUC18 (CD146), which has previously been identified on metastatic melanoma cells, endothelial cells and smooth muscle, but not on bone marrow stromal cells.17–20 Materials and methods

Cell culture Normal blood and bone marrow mononuclear cells collected from volunteers were separated by centrifugation on Ficoll– Paque (Pharmacia, Uppsala, Sweden). Mononuclear cells were washed in medium before immunofluorescence analysis. To prepare BMF, mononuclear cells separated from bone marrow samples (BMMNC) taken from allogeneic bone marrow donors were cultured in McCoys 5A culture medium (ICN Biomedicals, Costa Mesa, CA, USA) supplemented with 10% fetal calf serum (FCS; ICN), L-glutamine and antibiotics. After removal of non-adherent cells, adherent layers consisted of homogenous populations of elongated spindle-shaped cells. When confluent, adherent layers were passaged by trypsinEDTA treatment and replated in 75 cm2 culture flasks (Costar, Cambridge, MA, USA). For immunization of mice or immunofluorescence studies, flasks were treated with 5 mM EDTA (20 min at 37°C), and washed twice in RPMI-1640 medium prior to use. Cells for injection into mice were then resuspended in serum-free medium. Fibroblasts were also cultured in a similar fashion from sterile biopsies of articular cartilage, chorionic villus, bronchial mucosa and amniotic fluid. Human leukemic cell lines were maintained in log growth

MUC18 expression on bone marrow stroma RJA Filshie et al

phase in medium supplemented with 10% FCS. Human umbilical vein endothelial cells (HUVECs) were cultured as described previously.21 For activation studies, endothelial cells were incubated with recombinant TNF-␣ (Genzyme, Cambridge, MA, USA) at 200 U/ml for 24 h at 37°C. Similar studies on BMF were performed using IL-4 (Genzyme) or TNF␣ at 200 U/ml except that exposure to cytokines was varied from 30 min to 24 h.

Fibroblast colony-forming cell assays (CFU-F) This in vitro assay of stromal progenitors was performed as previously described.22,23 Briefly, BMMNC were plated at a concentration of 1 × 105/ml in 24-well tissue culture plates in alpha-modified Eagles’s medium (␣-MEM; Flow Laboratories, Irvine, UK) containing 20% FCS, L-glutamine, ␤-mercaptoethanol and antibiotics. After 14 days culture at 37°C in 5% CO2, cultures were fixed in 1% paraformaldehyde and stained with 0.1% toluidine blue. Clusters of more than 50 cells were scored as CFU-F. BMMNC were dual labeled with the mAbs CC9 and STRO-1, which is known to identify stromal precursors,24 using a double indirect method with fluorochromeconjugated heavy chain-specific second layer antibodies. Cells were sorted into CC9+ and CD9− fractions of the STRO1+ population and plated as above.

Generation of monoclonal antibodies BALB/c mice were injected intraperitoneally with 2.5 × 106 washed fresh BMF or human bone marrow stromal cultures (HBMSC)25 and boosted twice at monthly intervals. Five days after the third injection animals were sacrificed, spleens removed and splenic lymphocyte suspensions prepared. These were fused with NS1-Ag4 murine myeloma cells according to standard procedures. Fusion products were plated in 96-well flat bottom tissue culture plates (Costar). After approximately 2 weeks wells containing clones were examined for monoclonal antibody secretion by indirect immunofluorescence staining of BMF or by ELISA against HBMSC.26 Selected positive clones were sub-cloned three times by limiting dilution to generate stable hybridoma lines.

Immunofluorescence and flow cytometry Aliquots of 1–2 × 105 cells were incubated with test antibody, or appropriate isotype-matched non-binding control antibody, for 10 min at room temperature (RT), then washed twice in phosphate-buffered saline containing 0.2% sodium azide (PBSA). Cells were then incubated with sheep antibody to mouse Ig, conjugated with fluorescein isothiocyanate (Silenus, Melbourne, Australia) for a further 10 min at RT. After two further washes in PBSA cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA).

Immunogold staining of bone marrow Specimens of formalin-fixed, paraffin-embedded human fetal bone marrow were generously provided by Dr Yee Kong (Department of Pathology, Women and Children’s Hospital, Adelaide, Australia). Sections were cut at 5 mm, de-waxed in xylene and rehydrated through a graded series of ethanol into

PBS. Prior to incubation with monoclonal antibody the sections were incubated with 5% normal goat serum (NGS) in PBS supplemented with 0.05% Tween 20 (Sigma, St Louis, MO, USA) for 30 min to block potential non-specific binding. Sections were then incubated for 1 h at RT with mAb CC9 or appropriate isotype-matched negative control, after which they were washed three times in PBS + Tween 20 (PBST) over 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 followed by further washes in PBST as described above. Following incubation in blocking solution containing PBS + NGS supplemented with 0.8% bovine serum albumin (BSA) and 0.1% IGSS quality gelatin (Amersham, Bucks, UK) sections were incubated in AuroProbe One streptavidin (Amersham) diluted 1:80 in PBS supplemented with 1% NGS, BSA and gelatin according to the manufacturer’s recommendations. After three washes in PBS/BSA/gelatin, specimens were postfixed for 10 min in 2% glutaraldehyde, washed twice with distilled water and subjected to silver enhancement using the IntenSE M kit (Amersham). Finally, sections were counterstained with Mayer’s hematoxylin prior to mounting.

Immunoprecipitation and SDS-PAGE Approximately 5 × 107 NALM-6 cells were incubated with NHSS-Biotin reagent (Amersham) for 30 min at RT, followed 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 EDTA (20 min at 4°C). Lysates were pre-cleared by incubation with Pansorbin cells (Calbiochem, La Jolla, CA, USA) for 16 h at 4°C, then added to Dynabeads (Dynal, Oslo, Norway) previously labeled with approximately 10 mg of monoclonal antibody, and incubated for a further 2 h at 4°C. Bound proteins were dissolved in SDS running buffer with or without ␤mercaptoethanol, and run on a 7% polyacrylamide gel. Electrophoresed proteins were blotted on to nitrocellulose membranes (Biorad, North Ryde, Australia) which were then washed twice in TBS (20 mM Tris and 0.5 M NaCl, pH 7.5), followed by incubation in blocking buffer consisting of 5% (w/v) skim milk powder in TBS with 0.05% Tween-20 (TTBS). After two further washes in TTBS membranes were incubated with streptavidin conjugated to horse radish peroxidase (1:2000 in TTBS for 1 h). Following additional washing steps in TTBS an enhanced chemiluminescence kit (ECL; Amersham) was used as recommended by the manufacturer to allow visualisation of bands on X-ray film.

Adhesion assays For adhesion assays, NALM-6 or K-562 cells were labeled with Na2Cr51O4 as previously described.27 Labeled cells were then added to confluent BMF layers in 96-well microtiter plates which had been incubated (30 min at 37°C) with saturating concentrations of WM85 ± CD29 blocking antibody (4B4; Coulter), or appropriate negative and positive control antibodies. After allowing contact for 60 min at 37°C, nonadherent cells were removed by washing with RPMI + 10% FCS, and the remaining adherent cells and BMF were solubilized using 2% Triton in H2O, and quantitated in a gamma counter. In some experiments, marrow stromal layers were labeled with antibody and binding of purified marrow CD34+ cells was tested. Labeling was performed at 4°C in this case

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and incubation after addition of CD34+ cells was limited to 30 min.

Molecular cloning of stromal cell surface antigens The gene encoding the cell surface antigen identified by the monoclonal antibodies was isolated from a human bone marrow stromal cell cDNA library in the retroviral vector, pRUFneo as recently described.26 Briefly, cDNA synthesized from mRNA from HBMSC cultures was directionally cloned into the retroviral vector pRUFneo. Plasmid DNA from the library was used to transfect the amphotropic virus packaging line, PA317. Virus-containing supernatant from these cells was used to infect the ecotropic packaging cell line ⌿2, which in turn was used to infect the murine factor-dependent cell line FDC-P1 by co-cultivation. Infected cells were selected for G418 resistance, and cells expressing genes encoding HBMSC antigens were selected using the monoclonal antibodies WM85, CC9 and EB4, and expanded into clonal populations using multiple rounds of immuno-magnetic bead (Dynabead) selection followed by fluorescence activated cell sorting (FACS). Genomic DNA prepared from FDC-P1 cells was used in a polymerase chain reaction using retroviral-specific primers to recover proviral cDNA inserts. Gel-purified DNA was partially sequenced and data analyzed by referring to available data bases. Results

Immunophenotypic data Three hybridoma clones were isolated which secreted monoclonal antibody against bone marrow stromal cells. Although the antibodies WM85, CC9 and EB4 were independently derived, they were subsequently shown to identify the same protein, and are considered together. Antibodies typically showed a weak staining pattern on BMF (Figure 1). Fibroblastoid cells cultured from amniotic fluid, chorionic villus and lung were also reactive, while fibroblasts from articular cartilage were negative. Although the intensity of staining varied, the strongest binding appeared to be with amniocytic cells. BMF exposed to TNF-␣ or IL-4 for periods ranging from 30 min to 24 h showed no changes in the level of antigen expression as assessed by flow cytometry (data not shown). Testing on HUVECs showed strong positivity. Normal lymphocytes and monocytes from peripheral blood and normal bone marrow mononuclear cells were consistently negative. Purified CD34+ marrow cells were prepared using Dynabeads as described previously28 and these also showed no reactivity (data not shown). All T lineage leukemic cell lines tested, and one of four myeloid lines (K562), were weakly reactive (Table 1). One of six B lineage leukemic cell lines (NALM-6) was strongly positive. All four metastatic melanoma cell lines were reactive (Figure 1), with three showing very strong positivity. Cultured malignant pleural mesothelial cells were also positive but the breast adenocarcinoma cell line T47D was negative. Following the results on human leukemic cell lines, a number of samples from patients with hematological malignancies were studied (Table 2 and Figure 1). One out of three cases of T-ALL, four out of 20 cases of B lineage ALL (mainly precursor-B and pre-B phenotype), two out of seven with AML and

three out of seven with assorted B cell chronic lymphoproliferative disorders were found to react with the antibodies. The percentage of positive cells, as well as the intensity of staining, varied considerably between cases, with no particular pattern emerging. Three cases of precursor-B ALL were tested at diagnosis and relapse, and while all three were negative at diagnosis, one case became positive at relapse.

Immunogold staining of bone marrow Sections of fetal bone marrow showed moderately strong staining of marrow sinusoidal endothelial cells as well as marrow stromal cells (Figure 2). Sections of rib demonstrated similar findings, but with the additional observation of vascular smooth muscle staining (data not shown).

Fibroblast colony-forming assays It has previously been shown that BMMNC cultured in appropriate conditions give rise to small numbers of colonies with fibroblastoid features (CFU-F).22 Studies with the murine IgM mAb STRO-1 revealed that the STRO-1+ subset of BMMNC contain all the clonogenic stromal precursors responsible for the generation of CFU-F, or indeed many of the adherent cell types identified in human long-term bone marrow cultures.24 In three experiments the frequency of clonogenic stromal precursors (CFU-F) in unsorted BMMNC was found to average 29 ± 2/105 cells plated (Table 3). Analysis of BMMNC for expression of WM85 and CC9 by flow cytometry showed no apparent reactivity when gated around any of the predominant cell populations. However, analysis of ungated cells revealed the presence of a rare subpopulation of positively labeled cells with high forward and side light scattering properties. Studies were conducted to determine the proportion of CFU-F expressing the antigen identified by CC9. We have previously demonstrated that the Mab STRO-1 identifies all assayable CFU-F in BM.23 Two-colour FACS analysis was performed using STRO-1 and CC9 to determine whether CC9 subdivided the STRO-1-positive population. The cells were then sorted based on CC9 and STRO-1 expression. The frequency of CFU-F obtained in the CC9+/STRO-1+ population was found to be almost nine-fold greater than in the CC9−/STRO-1+ sorted cells (Table 3).

Immunoprecipitation and SDS-PAGE To ascertain the nature of the molecule identified by the monoclonal antibodies immunoprecipitation was performed using NALM-6 cells. Surface proteins on intact cells were biotinylated prior to lysis. WM85 immunoprecipitated a broad band of approximately 100–110 kDa under reduced and nonreduced conditions (Figure 3).

Adhesion assays The ability of WM85 to block adhesion of leukemic cells to BMF was tested using chromate-labeled NALM-6 cells. No inhibition of binding was noted, even after blocking of ␤1mediated adhesion with a blocking CD29 monoclonal antibody. Similar results were found using the myeloid cell line K562 (data not shown).


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Figure 1 Flow cytometry analysis of WM85 reactivity with BMF (a), a melanoma cell line, NM16 (b), leukemic blasts from a case of precursorB ALL (c), and a case of T-ALL (d). Histograms overlaid with appropriate isotype-matched negative control.

Binding of purified bone marrow CD34+ cells to human bone marrow stromal layers was unaffected by the addition of the antibodies CC9 or EB4 (data not shown).

WM85, CC9 and EB4 to MUC18 was confirmed by immunofluorescent labeling of the transfected FDC-P1 cells. Discussion

Molecular cloning Partial sequencing of the human cDNA insert recovered from FDC-P1 cells and subsequent FASTA alignment analysis revealed 100% homology with the human MUC18 gene (data not shown). In order to validate the results, the recovered cDNA was recloned into the retroviral vector and transfected into FDCP1 cells as previously described.26 Specificity of the antibodies

Many cell surface molecules on hemopoietic cells have been identified, and include adhesion receptors, growth factor receptors, and others whose functions remain unclear. While interactions with the bone marrow microenvironment are recognized as being of vital importance, relatively little is known about the surface molecules on stromal cells. By immunizing mice with cultured human marrow stromal cells we have generated a number of monoclonal antibodies which


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Table 1

Flow cytometric analysis of MUC18 expression on leukemic cell lines and other cell types

Cell type

% Positive

B lymphoid leukemic cell lines

KM3 Raji NALM-6 Reh Daudi WM-ALL-1 CEM HSB-2 Jurkatt Molt-4 8402 T-ALL-1 K562 KG1 HL60 U937 BMF Amniocytes Articular cartilage Chorionic villus Pulmonary Miller # WMM175 # NM16 # NM200 # HUVEC # PBMNC BMMNC T47D # Mesothelioma #

T lymphoid leukemic cell lines

Myeloid leukemic cell lines

Fibroblastoid cells

Melanoma

Other

−ve −ve 98 −ve −ve −ve 95 65 92 54 64 52 69 −ve −ve −ve 41 87 −ve 24 72 42 97 96 96 60 −ve −ve −ve 95

Fluorescence intensity

+++

+ to ++ + ++ + + + + + to ++ ++ + + + ++++ ++++ +++ +++

++

The antibody WM85 was used in these experiments. Results represent mean of at least two experiments except those indicated by # (n = 1). Fluorescence intensity: (mean fluorescence of test/control): +, 1–10-fold, ++, 10–20-fold, +++, 20–50-fold, ++++ ⬎ 50-fold. T47D, breast adenocarcinoma cell line; BMF, bone marrow fibroblasts; HUVEC, human umbilical vein endothelial cells; PBMNC, peripheral blood mononuclear cells; BMMNC, bone marrow mononuclear cells. Table 2 Reactivity of MUC18 with marrow samples from patients with hematological disease

Disease ALL (B lineage) T-ALL AML B lymphoproliferative CLL Other

No. cases

% Positive

20 3 13 7 4 25

20 33 15 43 0 0

BMMNC were tested for WM85 expression by flow cytometry. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; Other, samples from patients undergoing hematological investigation where results were either normal or non-diagnostic of hematological malignancy.

recognize previously undescribed stromal cell surface molecules. Date presented here describe the generation of three mAbs, WM85, CC9 and EB4, which following expression cloning and subsequent sequence analysis were found to identify MUC18 (CD146), a member of the immunoglobulin superfamily.29 The molecule contains five homologous extracellular immunoglobulin domains, a transmembrane anchoring motif and a short cytoplasmic tail. Cloning of MUC18 predicted a core protein with a molecular weight of 63 kDa, and showed the presence of a number of potential N-linked glycosylation sites, in common with other IgSF proteins.29 This is the first

Figure 2 Immunogold staining of section of fetal bone marrow with CC9. Darkened areas show positive staining. Thin layer of staining around marrow sinusoids represents endothelial cell labeling (small arrows), while polygonal shaped cells (large arrows) are typical of stromal cells.

report of MUC18 expression on bone marrow fibroblasts, stromal precursors and cells from patients with hemopoietic malignancies. MUC18 was originally described as a tumor marker on advanced primary or metastatic melanoma, being positive in 80% of such cases, but not present on cells from benign naevi.20 Correlation between vertical thickness of primary melanoma lesions (which in turn correlates with risk of met-


Table 3 Expression of CC9 on clonogenic bone marrow stromal precursors (CFU-F)

BMMNC fraction

Clonogenic frequency (CFU-F/1 × 105 cells)

Unsorted STRO-1 + CC9+ STRO-1 + CC9−

29 ± 2 172 ± 8 20 ± 1

Sorted BMMNC were plated after dual labeling with STRO-1 and CC9. Results represent mean of three experiments ± 1 s.d.

Figure 3 Immunoprecipitation from the pre-B cell line NALM-6 with WM85 shows a single broad band of approximately 100 kDa under reducing and non-reducing conditions (reduced only shown).

astasis and ultimately survival) and MUC18 expression has also been noted.29 Northern blot analysis of mRNA from a range of normal human tissues with a MUC18 cDNA probe demonstrated a detectable signal in almost all tested.18 Immunohistochemical analysis of tissue sections showed staining localized to smooth muscle cells, cerebellar cortex and endothelial cells of high endothelial venules and capillaries.17–19 Sections from a number of vascular tumors have also been shown to stain positively for MUC18.19 While early studies suggested a lack of expression on normal peripheral blood lymphocytes or human leukemic cell lines,18 MUC18 has recently been recognized as an activation antigen on human T lymphocytes.30

In addition to cloning data, we were able to immunoprecipitate a 100–110 kDa protein from the pre-B cell line NALM6 which is comparable to the 113 kDa protein of MUC18 precipitated from a melanoma cell line.29 This suggests that the mature protein exists in a glycosylated form, as is the case for other members of the IgSF such as VCAM-113 and NCAM.31 Flow cytometric analysis of cultured HUVECs showed positive reactivity as expected, although it is of interest that in situ staining of human umbilical cord failed to demonstrate any endothelial staining.18 This difference, as well as the reported reduction in expression on cultured smooth muscle cells over time, suggests other factors may regulate MUC18 expression. Some variation in positivity on cultured BMF was noted, however cells were always MUC18+, and no modulation by IL-4 or TNF-␣ could be demonstrated. In addition to the immunofluorescent labeling of mature cultures of BMF, the observation that rare FACS-sorted MUC18+ BMMNC contain almost all the cells giving rise to CFU-F indicates that MUC18 is also expressed on stromal progenitor cells. The mAb STRO-1 is similarly able to identify stromal precursors, although this antibody also labels erythroid precursors.24 After labeling with STRO-1 and the MUC18 mAb CC9, most of the CFU-F were found in the dual positive fraction, confirming that both antibodies label the same stromal cell precursor population. In contrast to Sers et al18 who were unable to demonstrate MUC18 positivity on any human leukemic cell lines, we were able to show MUC18 positivity on all leukemic T cell lines tested as well as on a minority of B lineage and myeloid leukemic cell lines. However, the expression by T cell lines is in keeping with a recent report that MUC18 is expressed on activated T cells.30 Our findings are reinforced by the detection of MUC18 on cells from patients with acute leukemia of lymphoid (T or B) or myeloid lineage, as well as some with chronic B cell lymphoproliferative disorders. The cells tested came from patients of various ages and at different stages of disease progression. While numbers were small, no obvious clinical correlations were apparent. However, in three cases of precursor-B ALL it was possible to test cells from diagnosis and relapse, and in one case a change from a negative to a positive phenotype was seen, suggesting a possible correlation with disease progression as has been described in malignant melanoma. The discovery that MUC18 is expressed on BMF from normal marrow suggests that this molecule is likely to have a function in normal hemopoiesis. Being a member of the IgSF it would be reasonable to speculate on an adhesive role. Endothelial cells express other IgSF members, namely ICAM1 (CD54),13 PECAM-1 (CD31)15 and VCAM-1 (CD106),14 BMF express VCAM-1,16 and NCAM is found on murine stromal cells.32 While the ␤1 and ␤2 integrins have been shown to be involved in mediating adhesion of normal and leukemic cells to BMF, blocking of known stromal ligands is usually incomplete, leading to speculation about additional novel stromal cell surface molecules with adhesive functions.27,33 It was not possible to show blocking of adhesion of two leukemic cell lines to BMF using the mAb WM85, although this does not exclude an adhesive function for the protein. Indeed, evidence for an adhesive role comes from Shih et al,17 who were able to show binding of MUC18-positive melanoma cells to immobilized, purified MUC18 protein. This binding was blocked by an antibody to the carbohydrate HNK-1 or by soluble MUC18 protein, but not with a number of MUC18 mAbs. The MUC18-negative myeloid leukemic cell line, U937, did not bind in this system even after being transfected with MUC18 cDNA, suggesting the binding may not be homo-

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philic, or alternatively that adhesive function is dependent on post-translational modification. In addition to the postulated role in cell–cell binding, a possible function in cell–matrix binding has been suggested. The MUC18 antigen contains an amino acid sequence in the second Ig loop very similar to the glycosaminoglycan recognition sequence found at the same position in the related IgSF molecules, NCAM and PECAM1.17,34,35 Verfaillie et al36 have demonstrated binding of early hemopoietic progenitors to fibronectin via heparan sulphate on hemopoietic cells, and others have shown that stromalassociated proteoglycans are also likely to have an adhesive role.37,38 Thus, binding to glycosaminoglycans may provide a direct mechanism for binding of hemopoietic progenitors or perhaps an indirect mechanism for co-localization of stromal and hemopoietic cells. Although MUC18 and PCAM-1 are both expressed on cells within the bone marrow the pattern of distribution is quite different. MUC18 expression appears to be restricted to fibroblasts and endothelial cells, while PECAM-1 is present on endothelial cells, some hemopoietic cells and stromal macrophages but not fibroblasts.15,39 PECAM-1 has been shown to be involved in both homotypic and heterotypic adhesion,34,35 and more recently found to have a role in the up-regulation of VLA-4-mediated binding of CD34+ marrow cells to VCAM-1.28 The possibility of a similar function for MUC18 may deserve further investigation. It is of interest that both endothelial cells and BMF express MUC18 suggesting a likely interaction with hemopoietic cells. The functional significance of MUC18 expression on a proportion of leukemic cell lines and some cases of leukemia is unknown. Further studies will be required to identify the role of this molecule in the hemopoietic system. Acknowledgements This work was supported by The NSW Cancer Council, The Anti-Cancer Foundation of The Universities of South Australia, The National Health and Medical Research Council of Australia and the University of Adelaide. We wish to thank Mary Sartor for assistance with flow cytometric testing. References 1 Wilkins BS, Jones DB. Immunohistochemical characterization of intact stromal layers in long-term cultures of human bone marrow. Br J Haematol 1995; 90: 757–766. 2 Weiss L. The hematopoietic microenvironment of the bone marrow: an ultrastructural study of the stroma in rats. Anat Rec 1976; 186: 161–184. 3 Zuckerman KS, Wicha MS. Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures. Blood 1983; 61: 540–547. 4 Chen Z-Z, Van Bockstaele DR, Buyssens N, Hendrics D,De Meester I, Vanhoof G, Scharpe SL, Peetermans ME, Berneman ZN. Stromal populations and fibrosis in long-term bone marrow cultures. Leukemia 1991; 5: 772–781. ¨ 5 Thalmeier K, Meißner P, Reisbach G, Hultner L, Mortensen BT, ¨ Brechtel A, Oostendorp RAJ, Dormer P. Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines. Exp Hematol 1996; 24: 1–10. 6 Kincade PW. Cell interaction molecules and cytokines which par` ticipate in B lymphopoiesis. In: Hindley S (ed). Baillieres’s Clinical Haematology, 5. WB Saunders: London, 1992, pp 575–598. 7 Clark BR, Gallagher JT, Dexter TM. Cell adhesion in the stromal ` regulation of haemopoiesis. In: Hindley S (ed). Baillieres’s Clinical Haematology, 5. WB Saunders: London, 1992, pp 619–652. 8 Gartner S, Kaplan HS. Long-term culture of human bone marrow cells. Proc Natl Acad Sci USA 1980; 78: 4756–4759.

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