MUC18, a Member of the Immunoglobulin Superfamily, Is

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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. Fusion products were mercaptoethanol, and run on a 7% polyacrylamide gel. Elec- plated in 96-well flat bottom tissue culture plates (Costar). trophoresed proteins were blotted on to nitrocellulose mem- After approximately 2 weeks wells containing clones were branes (Biorad, North Ryde, Australia) which were then examined for monoclonal antibody secretion by indirect washed twice in TBS (20 mM Tris and 0.5 M NaCl, pH 7.5), immunofluorescence staining of BMF or by ELISA against followed by incubation in blocking buffer consisting of 5% HBMSC.26 Selected positive clones were sub-cloned three (w/v) skim milk powder in TBS with 0.05% Tween-20 (TTBS). times by limiting dilution to generate stable hybridoma lines. 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 Immunofluorescence and flow cytometry in TTBS an enhanced chemiluminescence kit (ECL; Amersham) was used as recommended by the manufacturer Aliquots of 1–2 × 105 cells were incubated with test antibody, to allow visualisation of bands on X-ray film. 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 Adhesion assays (PBSA). Cells were then incubated with sheep antibody to mouse Ig, conjugated with fluorescein isothiocyanate (Silenus, For adhesion assays, NALM-6 or K-562 cells were labeled 51 27 Melbourne, Australia) for a further 10 min at RT. After two with Na2Cr O4 as previously described. Labeled cells were further washes in PBSA cells were analyzed on a FACScan then added to confluent BMF layers in 96-well microtiter flow cytometer (Becton Dickinson, Mountain View, CA, USA). plates which had been incubated (30 min at 37°C) with satu- rating concentrations of WM85 ± CD29 blocking antibody (4B4; Coulter), or appropriate negative and positive control Immunogold staining of bone marrow antibodies. After allowing contact for 60 min at 37°C, non- adherent cells were removed by washing with RPMI + 10% Specimens of formalin-fixed, paraffin-embedded human fetal FCS, and the remaining adherent cells and BMF were solubil- bone marrow were generously provided by Dr Yee Kong ized using 2% Triton in H2O, and quantitated in a gamma (Department of Pathology, Women and Children’s Hospital, counter. In some experiments, marrow stromal layers were Adelaide, Australia). Sections were cut at 5 mm, de-waxed in labeled with antibody and binding of purified marrow CD34+ xylene and rehydrated through a graded series of ethanol into cells was tested. Labeling was performed at 4°C in this case MUC18 expression on bone marrow stroma RJA Filshie et al 416 and incubation after addition of CD34+ cells was limited to three out of seven with assorted B cell chronic lymphoprolifer- 30 min. ative 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 Molecular cloning of stromal cell surface antigens emerging. Three cases of precursor-B ALL were tested at diagnosis and relapse, and while all three were negative at The gene encoding the cell surface antigen identified by the diagnosis, one case became positive at relapse. monoclonal antibodies was isolated from a human bone marrow stromal cell cDNA library in the retroviral vector, pRUF- neo as recently described.26 Briefly, cDNA synthesized from Immunogold staining of bone marrow mRNA from HBMSC cultures was directionally cloned into the retroviral vector pRUFneo. Plasmid DNA from the library Sections of fetal bone marrow showed moderately strong was used to transfect the amphotropic virus packaging line, staining of marrow sinusoidal endothelial cells as well as mar- PA317. Virus-containing supernatant from these cells was row stromal cells (Figure 2). Sections of rib demonstrated simi- ⌿ used to infect the ecotropic packaging cell line 2, which in lar findings, but with the additional observation of vascular turn was used to infect the murine factor-dependent cell line smooth muscle staining (data not shown). 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 Fibroblast colony-forming assays WM85, CC9 and EB4, and expanded into clonal populations using multiple rounds of immuno-magnetic bead (Dynabead) It has previously been shown that BMMNC cultured in appro- selection followed by fluorescence activated cell sorting priate conditions give rise to small numbers of colonies with (FACS). Genomic DNA prepared from FDC-P1 cells was used fibroblastoid features (CFU-F).22 Studies with the murine IgM in a polymerase chain reaction using retroviral-specific mAb STRO-1 revealed that the STRO-1+ subset of BMMNC primers to recover proviral cDNA inserts. Gel-purified DNA contain all the clonogenic stromal precursors responsible for was partially sequenced and data analyzed by referring to the generation of CFU-F, or indeed many of the adherent cell available data bases. 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 Results 29 ± 2/105 cells plated (Table 3). Analysis of BMMNC for expression of WM85 and CC9 by flow cytometry showed no Immunophenotypic data apparent reactivity when gated around any of the predomi- nant cell populations. However, analysis of ungated cells Three hybridoma clones were isolated which secreted mono- revealed the presence of a rare subpopulation of positively clonal antibody against bone marrow stromal cells. Although labeled cells with high forward and side light scattering the antibodies WM85, CC9 and EB4 were independently properties. Studies were conducted to determine the pro- derived, they were subsequently shown to identify the same portion of CFU-F expressing the antigen identified by CC9. protein, and are considered together. We have previously demonstrated that the Mab STRO-1 ident- Antibodies typically showed a weak staining pattern on ifies all assayable CFU-F in BM.23 Two-colour FACS analysis BMF (Figure 1). Fibroblastoid cells cultured from amniotic was performed using STRO-1 and CC9 to determine whether fluid, chorionic villus and lung were also reactive, while CC9 subdivided the STRO-1-positive population. The cells fibroblasts from articular cartilage were negative. Although the were then sorted based on CC9 and STRO-1 expression. The intensity of staining varied, the strongest binding appeared to frequency of CFU-F obtained in the CC9+/STRO-1+ population be with amniocytic cells. BMF exposed to TNF-␣ or IL-4 for was found to be almost nine-fold greater than in the periods ranging from 30 min to 24 h showed no changes in CC9−/STRO-1+ sorted cells (Table 3). the level of antigen expression as assessed by flow cytometry (data not shown). Testing on HUVECs showed strong positivity. Normal lym- Immunoprecipitation and SDS-PAGE phocytes and monocytes from peripheral blood and normal bone marrow mononuclear cells were consistently negative. To ascertain the nature of the molecule identified by the Purified CD34+ marrow cells were prepared using Dynabeads monoclonal antibodies immunoprecipitation was performed as described previously28 and these also showed no reactivity using NALM-6 cells. Surface proteins on intact cells were bio- (data not shown). All T lineage leukemic cell lines tested, and tinylated prior to lysis. WM85 immunoprecipitated a broad one of four myeloid lines (K562), were weakly reactive (Table band of approximately 100–110 kDa under reduced and non- 1). One of six B lineage leukemic cell lines (NALM-6) was reduced conditions (Figure 3). 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 Adhesion assays positive but the breast adenocarcinoma cell line T47D was negative. The ability of WM85 to block adhesion of leukemic cells to Following the results on human leukemic cell lines, a num- BMF was tested using chromate-labeled NALM-6 cells. No ␤ ber of samples from patients with hematological malignancies inhibition of binding was noted, even after blocking of 1- were studied (Table 2 and Figure 1). One out of three cases mediated adhesion with a blocking CD29 monoclonal anti- of T-ALL, four out of 20 cases of B lineage ALL (mainly precur- body. Similar results were found using the myeloid cell line sor-B and pre-B phenotype), two out of seven with AML and K562 (data not shown). MUC18 expression on bone marrow stroma RJA Filshie et al 417

Figure 1 Flow cytometry analysis of WM85 reactivity with BMF (a), a melanoma cell line, NM16 (b), leukemic blasts from a case of precursor- B 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 WM85, CC9 and EB4 to MUC18 was confirmed by immuno- bone marrow stromal layers was unaffected by the addition fluorescent labeling of the transfected FDC-P1 cells. of the antibodies CC9 or EB4 (data not shown).

Discussion Molecular cloning Many cell surface molecules on hemopoietic cells have been Partial sequencing of the human cDNA insert recovered from identiﬁed, and include adhesion receptors, growth factor FDC-P1 cells and subsequent FASTA alignment analysis receptors, and others whose functions remain unclear. While revealed 100% homology with the human MUC18 gene (data interactions with the bone marrow microenvironment are not shown). recognized as being of vital importance, relatively little is In order to validate the results, the recovered cDNA was known about the surface molecules on stromal cells. By recloned into the retroviral vector and transfected into FDC- immunizing mice with cultured human marrow stromal cells P1 cells as previously described.26 Speciﬁcity of the antibodies we have generated a number of monoclonal antibodies which MUC18 expression on bone marrow stroma RJA Filshie et al 418 Table 1 Flow cytometric analysis of MUC18 expression on leukemic cell lines and other cell types

Cell type % Positive Fluorescence intensity

B lymphoid leukemic cell lines KM3 −ve Raji −ve NALM-6 98 +++ Reh −ve Daudi −ve WM-ALL-1 −ve T lymphoid leukemic cell lines CEM 95 + to ++ HSB-2 65 + Jurkatt 92 ++ Molt-4 54 + 8402 64 + T-ALL-1 52 + Myeloid leukemic cell lines K562 69 − KG1 ve + HL60 −ve U937 −ve Fibroblastoid cells BMF 41 + to ++ Amniocytes 87 ++ Articular cartilage −ve Chorionic villus 24 + Pulmonary 72 + Melanoma Miller # 42 + WMM175 # 97 ++++ NM16 # 96 ++++ NM200 # 96 +++ Other HUVEC # 60 +++ PBMNC −ve BMMNC −ve T47D #−ve Mesothelioma # 95 ++

The antibody WM85 was used in these experiments. Results represent mean of at least two experiments except those indicated by # = test + ++ +++ ++++ Ͼ (n 1). Fluorescence intensity: (mean ﬂuorescence of /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 No. cases % Positive

ALL (B lineage) 20 20 T-ALL 3 33 AML 13 15 B lymphoproliferative 7 43 CLL 4 0 Other 25 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. Figure 2 Immunogold staining of section of fetal bone marrow with CC9. Darkened areas show positive staining. Thin layer of stain- recognize previously undescribed stromal cell surface ing around marrow sinusoids represents endothelial cell labeling molecules. (small arrows), while polygonal shaped cells (large arrows) are typical Date presented here describe the generation of three mAbs, of stromal cells. WM85, CC9 and EB4, which following expression cloning and subsequent sequence analysis were found to identify report of MUC18 expression on bone marrow fibroblasts, MUC18 (CD146), a member of the immunoglobulin super- stromal precursors and cells from patients with hemopoietic family.29 The molecule contains five homologous extracellular malignancies. immunoglobulin domains, a transmembrane anchoring motif MUC18 was originally described as a tumor marker on and a short cytoplasmic tail. Cloning of MUC18 predicted a advanced primary or metastatic melanoma, being positive in core protein with a molecular weight of 63 kDa, and showed 80% of such cases, but not present on cells from benign the presence of a number of potential N-linked glycosylation naevi.20 Correlation between vertical thickness of primary sites, in common with other IgSF proteins.29 This is the first melanoma lesions (which in turn correlates with risk of met- MUC18 expression on bone marrow stroma RJA Filshie et al 419 Table 3 Expression of CC9 on clonogenic bone marrow stromal In addition to cloning data, we were able to immunoprecip- precursors (CFU-F) itate a 100–110 kDa protein from the pre-B cell line NALM- 6 which is comparable to the 113 kDa protein of MUC18 pre- BMMNC fraction Clonogenic frequency cipitated from a melanoma cell line.29 This suggests that the (CFU-F/1 × 105 cells) mature protein exists in a glycosylated form, as is the case for other members of the IgSF such as VCAM-113 and NCAM.31 Unsorted 29 ± 2 STRO-1 + CC9+ 172 ± 8 Flow cytometric analysis of cultured HUVECs showed positive STRO-1 + CC9− 20 ± 1 reactivity as expected, although it is of interest that in situ staining of human umbilical cord failed to demonstrate any 18 Sorted BMMNC were plated after dual labeling with STRO-1 and endothelial staining. This difference, as well as the reported CC9. Results represent mean of three experiments ± 1 s.d. 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 immuno- fluorescent 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 detec- tion 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 ICAM- 1 (CD54),13 PECAM-1 (CD31)15 and VCAM-1 (CD106),14 BMF Figure 3 Immunoprecipitation from the pre-B cell line NALM-6 express VCAM-1,16 and NCAM is found on murine stromal with WM85 shows a single broad band of approximately 100 kDa cells.32 While the ␤ and ␤ integrins have been shown to be under reducing and non-reducing conditions (reduced only shown). 1 2 involved in mediating adhesion of normal and leukemic cells to BMF, blocking of known stromal ligands is usually incom- astasis and ultimately survival) and MUC18 expression has plete, leading to speculation about additional novel stromal also been noted.29 Northern blot analysis of mRNA from a cell surface molecules with adhesive functions.27,33 It was not range of normal human tissues with a MUC18 cDNA probe possible to show blocking of adhesion of two leukemic cell demonstrated a detectable signal in almost all tested.18 Immu- lines to BMF using the mAb WM85, although this does not nohistochemical analysis of tissue sections showed staining exclude an adhesive function for the protein. Indeed, evi- localized to smooth muscle cells, cerebellar cortex and endo- dence for an adhesive role comes from Shih et al,17 who were thelial cells of high endothelial venules and capillaries.17–19 able to show binding of MUC18-positive melanoma cells to Sections from a number of vascular tumors have also been immobilized, purified MUC18 protein. This binding was shown to stain positively for MUC18.19 While early studies blocked by an antibody to the carbohydrate HNK-1 or by sol- suggested a lack of expression on normal peripheral blood uble MUC18 protein, but not with a number of MUC18 mAbs. lymphocytes or human leukemic cell lines,18 MUC18 has The MUC18-negative myeloid leukemic cell line, U937, did recently been recognized as an activation antigen on human not bind in this system even after being transfected with T lymphocytes.30 MUC18 cDNA, suggesting the binding may not be homo- MUC18 expression on bone marrow stroma RJA Filshie et al 420 philic, or alternatively that adhesive function is dependent on 9 Miyake K, Medina KL, Hayashi S-I, Ono S, Hamaoka T, Kincade post-translational modification. In addition to the postulated PW. Monoclonal antibodies to Pgp-1/CD44 block lympho-hemo- role in cell–cell binding, a possible function in cell–matrix poiesis in long-term bone marrow cultures. J Exp Med 1990; 171: 477–488. binding has been suggested. The MUC18 antigen contains an 10 Liesveld JL, Abboud CN, Duerst RE, Ryan DH, Brennan JK, Licht- amino acid sequence in the second Ig loop very similar to the man MA. Characterization of human marrow stromal cells: role glycosaminoglycan recognition sequence found at the same in progenitor cell binding and granulopoiesis. Blood 1989; 73: position in the related IgSF molecules, NCAM and PECAM- 1794–1800. 1.17,34,35 Verfaillie et al36 have demonstrated binding of early 11 Keating A, Whalen CR, Singer JW. Cultured marrow stromal cells hemopoietic progenitors to fibronectin via heparan sulphate express common acute lymphoblastic leukemia (CALLA): impli- cations for marrow transplantation. Br J Haematol 1983; 55: on hemopoietic cells, and others have shown that stromal- 623–628. associated proteoglycans are also likely to have an adhesive 12 Hunkapiller T, Hood L. Diversity of the immunoglobulin gene role.37,38 Thus, binding to glycosaminoglycans may provide a superfamily. Adv Immunol 1989; 44: 1–63. direct mechanism for binding of hemopoietic progenitors or 13 Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. perhaps an indirect mechanism for co-localization of stromal Blood 1994; 84: 2068–2101. and hemopoietic cells. Although MUC18 and PCAM-1 are 14 Osborn L, Hession C, Tizard R, Vassallo C, Lyhowskyj S, Chi- both expressed on cells within the bone marrow the pattern Rosso G, Lobb R. Direct expression cloning of vascular cell adhesion molecule-1, a cytokine-induced protein that binds lym- of distribution is quite different. MUC18 expression appears phocytes. Cell 1989; 59: 1203–1211. to be restricted to fibroblasts and endothelial cells, while 15 Simmons DL, Walker C, Power C, Pigott R. Molecular cloning of PECAM-1 is present on endothelial cells, some hemopoietic CD31, a putative intercellular adhesion molecule closely related cells and stromal macrophages but not fibroblasts.15,39 to carcinoembryonic antigen. J Exp Med 1990; 171: 2147–2152. PECAM-1 has been shown to be involved in both homotypic 16 Simmons PJ, Masinovsky B, Longenecker BM, Berenson R, Torok- and heterotypic adhesion,34,35 and more recently found to Storb B, Gallatin WM. Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of have a role in the up-regulation of VLA-4-mediated binding of + 28 hematopoietic progenitor cells. Blood 1992; 80: 388–395. CD34 marrow cells to VCAM-1. The possibility of a similar 17 Shih I-M, Elder DE, Speicher D, Johnson JP, Herlyn M. Isolation function for MUC18 may deserve further investigation. It is of and functional characterisation of the A32 melanoma-associated interest that both endothelial cells and BMF express MUC18 antigen. Cancer Res 1994; 54: 2514–2520. suggesting a likely interaction with hemopoietic cells. The 18 Sers C, Riethmuller G, Johnson JP, MUC18, a melanoma-pro- functional significance of MUC18 expression on a proportion gression molecule, and its potential role in tumor vascularization of leukemic cell lines and some cases of leukemia is and hematogenous spread. Cancer Res 1994; 54: 5689–5694. 19 Kuzu I, Bicknell R, Fletcher CDM, Gatter KC. Expression of unknown. Further studies will be required to identify the role adhesion molecules on the endothelium of normal tissue vessels of this molecule in the hemopoietic system. and vascular tumors. Lab Invest 1993; 69: 322–328. 20 Lehmann JM, Holzmann B, Breitbart EW, Schiegelow P, Riethmuller G, Johnson JP. 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