Predominant Expression of CD44 Splice Variant v10 in Malignant and Reactive Human Skin Lymphocytes
1998; Elsevier BV; Volume: 111; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.1998.00302.x
ISSN1523-1747
AutoresStephan N. Wagner, Christine Wagner, Uwe Reinhold, Renate Funk, Margot Zöller, Manfred Goos,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoThe remarkable functional diversity of the cell surface receptor CD44 may be due to expression of multiple variant isoforms generated by alternative splicing of variant exons. Functional and correlative data implicate a role of CD44 variant isoforms in adhesion dependent processes such as lymphocyte recirculation and tumor progression and metastasis. We have analyzed 25 primary cutaneous lymphomas and 35 reactive lymphoid cell skin infiltrates or T cell-mediated skin diseases for the expression of CD44 variant isoforms. Irrespective of histologic typing, staging, and grading, cutaneous lymphomas as well as nonmalignant skin-infiltrating CD3+CD4+ and CD8+ T and CD19+ B lymphocytes exhibited a strong expression of CD44v10 and a moderate expression of CD44v3 as determined by immunohistochemistry, immunofluorescence microscopy, and mRNA analysis. Expression of v5, v6, v7, and v9-containing CD44 variant isoforms was not detected. Furthermore, flow cytometry revealed expression of CD44v10 on a significant proportion of peripheral blood lymphocytes from Sézary’s syndrome patients and a remarkable coexpression with cutaneous lymphocyte antigen. These results indicate a distinct CD44 variant isoform expression pattern associated with skin-homing lymphocytes different to lymphatic cells at noncutaneous sites. This differential expression pattern of CD44 variant isoforms may contribute to the development of lymphocyte skin infiltrates and/or the unique biologic behavior of cutaneous lymphomas. The remarkable functional diversity of the cell surface receptor CD44 may be due to expression of multiple variant isoforms generated by alternative splicing of variant exons. Functional and correlative data implicate a role of CD44 variant isoforms in adhesion dependent processes such as lymphocyte recirculation and tumor progression and metastasis. We have analyzed 25 primary cutaneous lymphomas and 35 reactive lymphoid cell skin infiltrates or T cell-mediated skin diseases for the expression of CD44 variant isoforms. Irrespective of histologic typing, staging, and grading, cutaneous lymphomas as well as nonmalignant skin-infiltrating CD3+CD4+ and CD8+ T and CD19+ B lymphocytes exhibited a strong expression of CD44v10 and a moderate expression of CD44v3 as determined by immunohistochemistry, immunofluorescence microscopy, and mRNA analysis. Expression of v5, v6, v7, and v9-containing CD44 variant isoforms was not detected. Furthermore, flow cytometry revealed expression of CD44v10 on a significant proportion of peripheral blood lymphocytes from Sézary’s syndrome patients and a remarkable coexpression with cutaneous lymphocyte antigen. These results indicate a distinct CD44 variant isoform expression pattern associated with skin-homing lymphocytes different to lymphatic cells at noncutaneous sites. This differential expression pattern of CD44 variant isoforms may contribute to the development of lymphocyte skin infiltrates and/or the unique biologic behavior of cutaneous lymphomas. CD44 standard isoform CD44 variant isoform primary cutaneous lymphoma cutaneous lymphocyte-associated antigen/carbohydrated P-selectin glycoprotein ligand-1 T cell lymphoma CD44 represents a heterogenous class of glycosylated proteins with molecular masses ranging from 80 to over 250 kDa in size. In its most widely expressed 80–90 kDa “standard or hematopoietic” form (CD44s/CD44H), CD44 has been shown to represent the principle cell surface receptor for hyaluronan (Aruffo et al., 1990Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. CD44 is the principle cell surface receptor for hyaluronate.Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2062) Google Scholar;Miyake et al., 1990Miyake K. Underhill C.B. Lesley J. Kincade P.W. 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Possible involvement in lymphoid cell adherence and activation.J Biol Chem. 1995; 270: 7437-7444Crossref PubMed Scopus (157) Google Scholar;Ehnis et al., 1996Ehnis T. Dieterich W. Bauer M. Lampe B. Schuppan D. A chondrotin/dermatan sulfate form of CD44 is a receptor for collagen XIV (undulin).Exp Cell Res. 1996; 229: 388-397Crossref PubMed Scopus (65) Google Scholar;Weber et al., 1996Weber G.F. Ashkar S. Glimcher M.J. Cantor H. Receptor–ligand interaction between CD44 and osteopontin (eta-1).Science. 1996; 271: 509-512Crossref PubMed Scopus (775) Google Scholar) with lower affinity. CD44 has been implicated originally as a lymph node homing receptor, directing lymphocyte adhesion to high endothelial venules and the extracellular matrix (Jalkanen et al., 1986Jalkanen S.T. Bargatze R.F. Herron L.R. Butcher E.C. A lymphoid cell surface glycoprotein involved in endothelial cell recognition and lymphocyte homing in man.Eur J Immunol. 1986; 16: 1195-1202Crossref PubMed Scopus (344) Google Scholar), and may also participate in multiple adhesion-dependent cellular processes such as prothymocyte homing, macrophage and T cell activation, natural killer cell-mediated cytotoxicity, hemopoiesis, and tumor metastasis (Haynes et al., 1989Haynes B.F. Telen M.J. Hale L.P. Denning S.M. CD44-a molecule involved in leukocyte adherence and T cell activation.Immunol Today. 1989; 10: 423-428Abstract Full Text PDF PubMed Scopus (493) Google Scholar;Underhill, 1992Underhill C. CD44: the hyaluronan receptor.J Cell Sci. 1992; 103: 293-298Crossref PubMed Google Scholar;Günthert, 1993Günthert U. CD44: a multitude of isoforms with diverse functions.Curr Top Microbiol Immunol. 1993; 184: 47-63Crossref PubMed Scopus (240) Google Scholar;Lesley et al., 1993Lesley J. Hyman R. Kincade P.W. 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Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44show differences in glycosylation.J Exp Med. 1995; 182: 431-437Crossref PubMed Scopus (173) Google Scholar), phosphorylation of the cytoplasmic domain (Pure et al., 1995Pure E. Camp R.L. Perrit D. Panettieri R.A. Lazaar A.L. Nayak S. Defective phosphorylation and hyaluronate binding of CD44 with point mutations in the cytoplasmic domain.J Exp Med. 1995; 181: 55-62Crossref PubMed Scopus (82) Google Scholar), and binding of CD44 to the cytoskeleton (Bourguignon et al., 1993Bourguignon L.Y.W. Lokeshwar V.B. Chen X. Kerrick W.G.L. Hyaluronic acid-induced lymphocyte signal transduction and HA receptor (GP 85/CD44) –cytoskeleton interaction.J Immunol. 1993; 151: 6634-6644PubMed Google Scholar), or to multiple CD44 isoforms generated by alternative splicing of at least 10 “variant” exons (designated as v1–v10) inserted into a specific site within a membrane-proximal segment of the extracellular domain (Screaton et al., 1992Screaton G.R. Bell M.V. Jackson D.G. Cornelis F.B. Gerth U. Bell J.I. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons.Proc Natl Acad Sci USA. 1992; 89: 12160-12164Crossref PubMed Scopus (955) Google Scholar). These so-called “variant” isoforms (CD44v) are generated by selective usage of different variant exons or exon combinations. The subsequently inserted peptide sequences are remarkably hydrophilic and provide further glycosylation and chondroitin sulfate attachment sites, which may endow the CD44 molecule with additional binding properties (Bennett et al., 1995bBennett K.L. Modrell B. Greenfield B. et al.Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons.J Cell Biol. 1995 b; 131: 1623-1633Crossref PubMed Scopus (141) Google Scholar;Günthert et al., 1995Günthert U. Stauder R. Mayer B. Terpe H. Finke L. Friedrichs K. Are CD44 variant isoforms involved in human tumor progression?.Cancer Surv. 1995; 24: 19-42PubMed Google Scholar). Generation of CD44v isoforms may provide an additional molecular basis for the functional diversity of the CD44 molecule as indicated by experimental and correlative data in tumor progression and metastasis. Thus, CD44v isoforms containing variable exon v4–7 or v6–7 have been shown to confer metastatic behavior in a rat pancreas adenocarcinoma cell line in vivo (Günthert et al., 1991Günthert U. Hofmann M. Rudy W. et al.A new variant of glykoprotein CD44 confers metastatic potential to rat carcinoma cells.Cell. 1991; 65: 13-24Abstract Full Text PDF PubMed Scopus (1550) Google Scholar;Rudy et al., 1993Rudy W. Hoffmann M. Schwartz-Albiez R. Zöller M. Heider K-H Ponta H. Herrlich P. Two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants: each one individually suffices to confer metastatic behavior.Cancer Res. 1993; 53: 1262-1268PubMed Google Scholar), and lymphogenic spread of this cell line could be prevented by anti-CD44v6 antibody (Reber et al., 1990Reber S. Matzku S. Günthert U. Ponta H. Herrlich P. Zöller M. Retardation of metastatic tumor growth after immunization with metastasis-specific monoclonal antibodies.Int J Cancer. 1990; 46: 919-927Crossref PubMed Scopus (77) Google Scholar;Seiter et al., 1993Seiter S. Arch R. Reber S. et al.Prevention of tumor metastasis formation by anti-variant CD44.J Exp Med. 1993; 177: 443-455Crossref PubMed Scopus (324) Google Scholar). Similarly, transfection of CD44v8–v10 into a murine fibrosarcoma cell line increased tumor cell growth and metastasis formation (Dougherty et al., 1992Dougherty G.J. Dougherty S.T. Eaves C.J. McBride W.H. Expression of human CD44R1 enhances the metastatic capacity of murine fibrosarcoma cells.Proc Am Assoc Cancer Res. 1992; 33: 35-42Google Scholar), and transfection of CD44v3 into lymphocytic cells resulted in bone marrow tumor formation in vivo (Bartolazzi et al., 1995Bartolazzi A. Jackson D. Bennett K. et al.Regulation of growth and dissemination of a human lymphoma by CD44splice variants.J Cell Sci. 1995; 108: 1723-1733Crossref PubMed Google Scholar). Expression of some variant exon combinations has been linked to defined tissues (i.e., CD44v8–v10 to epithelial tissue) (Stamenkovic et al., 1991Stamenkovic I. Aruffo A. Amiot M. Seed B. The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate bearing cells.Embo J. 1991; 10: 343-348Crossref PubMed Scopus (512) Google Scholar;Günthert, 1993Günthert U. CD44: a multitude of isoforms with diverse functions.Curr Top Microbiol Immunol. 1993; 184: 47-63Crossref PubMed Scopus (240) Google Scholar), and in some human malignancies CD44v isoform expression profiles are correlated with malignant phenotype, tumor progression, or prognosis (Fox et al., 1993Fox S.B. Gatter K.C. Jackson D.G. et al.CD44 and cancer screening.Lancet. 1993; 342: 548-549Abstract PubMed Scopus (88) Google Scholar;Günthert et al., 1995Günthert U. Stauder R. Mayer B. Terpe H. Finke L. Friedrichs K. Are CD44 variant isoforms involved in human tumor progression?.Cancer Surv. 1995; 24: 19-42PubMed Google Scholar;Zöller, 1995Zöller M. CD44: physiological expression of distinct isoforms as evidence for organ-specific metastasis formation.J Mol Med. 1995; 73: 425-438Crossref PubMed Scopus (66) Google Scholar). In a similar fashion, activation as well as infiltrative or aggressive behavior of lymphocytes or lymphoid neoplasms may be related to CD44v isoform expression as suggested by CD44v6 expression during antigenic stimulation of T lymphocytes (Arch et al., 1992Arch R. Wirth K. Hofmann M. Ponta H. Matzku S. Herrlich P. Zöller M. Participation in normal immune responses of a metastasis-inducing splice variant of CD44.Sci (Wash DC). 1992; 257: 682-685Crossref PubMed Scopus (398) Google Scholar;Koopmann et al., 1993Koopmann G. Heider K-H Horst E. et al.Activated human lymphocytes and aggressive non Hodgkins’ lymphomas express a homologue of the rat metastasis-associated variant of CD44.J Exp Med. 1993; 177: 897-904Crossref Scopus (315) Google Scholar;Stauder et al., 1995Stauder R. Eisterer W. Thaler J. Günthert U. CD44 variant isoforms in Non-Hodgkin’s lymphoma: a new independent prognostic factor.Blood. 1995; 85: 2885-2899Crossref PubMed Google Scholar) and correlation of CD44v6 expression with grading and prognosis in primary nodal non-Hodgkin’s lymphomas (Koopmann et al., 1993Koopmann G. Heider K-H Horst E. et al.Activated human lymphocytes and aggressive non Hodgkins’ lymphomas express a homologue of the rat metastasis-associated variant of CD44.J Exp Med. 1993; 177: 897-904Crossref Scopus (315) Google Scholar;Ristamäki et al., 1995Ristamäki R. Joensuu H. Söderström K-O Jalkanen S. CD44v6 expression in Non-Hodgkin’s lymphoma: an association with low histological grade and poor prognosis.J Pathol. 1995; 176: 259-267Crossref PubMed Scopus (64) Google Scholar;Stauder et al., 1995Stauder R. Eisterer W. Thaler J. Günthert U. CD44 variant isoforms in Non-Hodgkin’s lymphoma: a new independent prognostic factor.Blood. 1995; 85: 2885-2899Crossref PubMed Google Scholar). Infiltration of a specific subset of peripheral blood lymphocytes into the skin requires a specific multistep process of lymphocyte adhesion to and migration through vascular endothelium (Butcher, 1991Butcher E.C. Leukocyte-endothelial cell recognition: three (or more) steps to specifity and diversity.Cell. 1991; 67: 1033-1036Abstract Full Text PDF PubMed Scopus (2459) Google Scholar;Springer, 1994Springer T.A. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6255) Google Scholar;DeGrendele et al., 1996DeGrendele H.C. Estess P. Picker L.J. Siegelman M.H. CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyte-endothelial cell primary adhesion pathway.J Exp Med. 1996; 183: 1119-1130Crossref PubMed Scopus (350) Google Scholar;Uksila et al., 1997Uksila J. Salmi M. Butcher E.C. Tarkkanen J. Jalkanen S. Function of lymphocyte homing-associated adhesion molecules on human natural killer and lymphokine-activated killer cells.J Immunol. 1997; 158: 1610-1617PubMed Google Scholar). Furthermore, primary cutaneous lymphomas represent clinicopathologic entities with a biologic behavior significantly different to primary nodal lymphomas (Willemze et al., 1997Willemze R. Kerl H. Sterry W. et al.EORTC classification for primary cutaneous lymphomas: a proposal from the cutaneous lymphoma study group of the european organization for research and treatment of cancer.Blood. 1997; 90: 354-371PubMed Google Scholar). Because adhesion properties of lymphocytes as well as tumor cell proliferation, growth, and metastasis have been shown to be determined significantly by the composition of variant exons spliced into the CD44H frame work (Stamenkovic et al., 1991Stamenkovic I. Aruffo A. Amiot M. Seed B. The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate bearing cells.Embo J. 1991; 10: 343-348Crossref PubMed Scopus (512) Google Scholar;Sy et al., 1991Sy M.S. Guo Y-J Stamenkovic I. Distinct effects of two CD44 isoforms on tumor growth in vivo.J Exp Med. 1991; 174: 859-866Crossref PubMed Scopus (300) Google Scholar;Dougherty et al., 1992Dougherty G.J. Dougherty S.T. Eaves C.J. McBride W.H. Expression of human CD44R1 enhances the metastatic capacity of murine fibrosarcoma cells.Proc Am Assoc Cancer Res. 1992; 33: 35-42Google Scholar, Dougherty et al., 1994Dougherty G.J. Cooper D.L. Memory J.F. Chiu R.K. Ligand binding specificity of alternatively spliced CD44 isoforms.J Biol Chem. 1994; 269: 9074-9078Abstract Full Text PDF PubMed Google Scholar;Bartolazzi et al., 1995Bartolazzi A. Jackson D. Bennett K. et al.Regulation of growth and dissemination of a human lymphoma by CD44splice variants.J Cell Sci. 1995; 108: 1723-1733Crossref PubMed Google Scholar;Droll et al., 1995Droll A. Dougherty S.T. Chiu R.K. Dirks J.F. McBride W.H. Cooper D.L. Dougherty G.J. Adhesive interactions between alternatively spliced CD44 isoforms.J Biol Chem. 1995; 270: 11567-11573Crossref PubMed Scopus (62) Google Scholar;Jackson et al., 1995Jackson D.G. Bell J.I. Dickinson R. Timans J. Shields J. Whittle N. Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon.J Cell Biol. 1995; 128: 673-685Crossref PubMed Scopus (208) Google Scholar), the expression pattern of CD44v isoforms on cutaneous reactive or malignant lymphocytes may not necessarily correlate with that observed in nodal lymphomas or on activated peripheral blood lymphocytes. In this study, we provide evidence that skin-infiltrating lymphocytes lack CD44v6 expression but strongly upregulate CD44v10 expression during both the manifestation of an immune response and the development of cutaneous lymphoid malignancies. Human tissue specimens represented 25 primary cutaneous lymphomas (CL; including two cases of Sézary’s syndrome with respective lymph node samples), 10 lymphocyte skin infiltrates of atopic dermatitis and allergic contact dermatitis, respectively, 25 tissue samples of diverse reactive or T cell-mediated cutaneous inflammatory diseases, including a pseudolymphoma, and two cases of inflammatory skin-draining lymph nodes (Table I, Table II). Extirpated samples were halved, one part being immediately frozen in liquid nitrogen for immunohistochemical staining and RNA extraction. The other part was fixed in 10% buffered formalin (pH 7.0) and embedded in paraffin wax for histologic examination. Diagnosis was made independently by two of the authors according to standard histologic criteria (Willemze et al., 1997Willemze R. Kerl H. Sterry W. et al.EORTC classification for primary cutaneous lymphomas: a proposal from the cutaneous lymphoma study group of the european organization for research and treatment of cancer.Blood. 1997; 90: 354-371PubMed Google Scholar), and confirmed by immunophenotyping (CL) and intralocus T cell receptor γ rearrangement analysis [T cell lymphoma (TCL)].Table IClinicohistopathologic features of primary cutaneous lymphomasClinicopathologic classificationnGradingTypingStagingPatch/plaque-stage mycosis fungoides1indolentT cellI APatch/plaque-stage mycosis fungoides5indolentT cellI BPatch/plaque-stage mycosis fungoides1indolentT cellII ATumor-stage mycosis fungoides5indolentT cellII BErythroderma-stage mycosis fungoides1indolentT cellIIILarge cell anaplastic (CD30+) TCL2indolentT cellII BLarge cell anaplastic (CD30+) TCL1indolentT cellIV ALymphomatoid papulosis3indolentT cellSubcutaneous panniculitis-like TCL1aggressiveT cellII BSézary’s syndrome2aggressiveT cellIV ALarge cell pleomorphic (CD30–) TCL1aggressiveT cellII BImmunocytoma1indolentB cellII BImmunocytoma1indolentB cellIV B Open table in a new tab Table IIExpression of CD44v on lymphocytes within various tissue samplesLymphocytes withinnsv3v5v6v7v9v10Primary cutaneous lymphomas Patch/plaque-stage mycosis fungoides7+++a–, negative; (+), very weak/equivocal; +, weak; ++, moderate; +++, strong immunostaining.++––––+++ Tumor-stage mycosis fungoides5+++++––––+++ Erythroderma-stage mycosis fungoides1+++++––––+++ Large cell anaplastic (CD30+) TCL3+++++––––+++ Large cell pleomorphic (CD30–) TCL1+++++––––+++ Subcutaneous panniculitis-like TCL1+++++––––+++ Lymphomatoid papulosis3+++++––––+++ Immunocytoma2+++++––––+++ Sézary’s syndrome2+++++––––+++Pseudolymphoma1+++++––––+++Allergic contact dermatitis7+++++––––+++Atopic dermatitis3+++++––––+++Psoriasis vulgaris4+++++––––+++Lichen planus3+++(+)––––++Alopecia areata2+++(+)––––++Toxic epidermal necrolysis4++++––––+++Pityriasis lichenoides et varioliformis acuta1+++(+)––––++Granuloma anulare2+++(+)––––++Dermatitis herpetiformis1++++––––+++Lupus erythematodes4++++––––+++Malignant melanoma3+++(+)––––++Lymph node, inflammatory skin draining2+++++–(+)––+++a –, negative; (+), very weak/equivocal; +, weak; ++, moderate; +++, strong immunostaining. Open table in a new tab Peripheral blood mononuclear cells were isolated from whole blood by Ficoll-Hypaque density centrifugation. T cell enriched populations were obtained from peripheral blood mononuclear cells by passage through a nylon wool column (Wako, Neuss, Germany). The proportion of residual CD14+ monocytes and CD19+ B cells was <1%. Immunohistochemistry was performed as described previously (Heider et al., 1993Heider K-H Hofmann M. Hors E. van den Berg F. Ponta H. Herrlich P. Pals S.T. A human homologue of the rat metastasis-associated variant of CD44 is expressed i colorectal carcinomas and adenomatous polyps.J Cell Biol. 1993; 120: 227-233Crossref PubMed Scopus (361) Google Scholar;Koopmann et al., 1993Koopmann G. Heider K-H Horst E. et al.Activated human lymphocytes and aggressive non Hodgkins’ lymphomas express a homologue of the rat metastasis-associated variant of CD44.J Exp Med. 1993; 177: 897-904Crossref Scopus (315) Google Scholar). Briefly, 8 μm cryosections were fixed in acetone for 10 min, washed in phosphate-buffered saline, and preincubated with normal goat serum (10% in phosphate-buffered saline). After incubation with the primary antibody for 1 h, immunoreaction was visualized by APAAP staining (Cordell et al., 1984Cordell J.L. Falini B. Erber W.N. et al.Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase (APAAP complexes).J Histochem Cytochem. 1984; 32: 219-229Crossref PubMed Scopus (2831) Google Scholar). All primary antibodies were titrated to give optimal results. Immunostaining of human skin keratinocytes, which express variant exon sequences v2–v10 (Hudson et al., 1995Hudson D.L. Sleeman J. Watt F.M. CD44 is the major peanut lectin-binding glycoprotein of human epidermal keratinocytes and plays a role in intercellular adhesion.J Cell Sci. 1995; 108: 1959-1970PubMed Google Scholar), served as internal quality standard. Negative controls included omission of the first, second, and/or third antibody layer and substitution of the primary antibody by rabbit preimmune or nonimmune IgG or monoclonal mouse IgG of irrelevant specificity. Primary antibodies included variant exon-specific monoclonal BBA11 (R&D Systems, Wiesbaden, Germany) directed against v3, VFF8 (v5), VFF7 (v6), VFF9 (v7), VFF16 (v10), SFF2 directed against CD44 frame work (Bender, Vienna, Austria), and FW11.24 (anti-v9, kindly provided by U. Günthert, Basel Institute for Immunology, Basel, Switzerland). For double labeling immunofluorescence microscopy, cryosections were fixed in acetone for 10 min at room temperature and blocked with phosphate-buffered saline containing 1% (wt/vol) bovine serum albumin. Incubation of tissue sections with monoclonal antibody VFF16 recognized by dichlorotriazinyl amino fluorescein-conjugated goat anti-mouse IgG (Dianova, Hamburg, Germany) was followed by incubation with monoclonal mouse antibodies directed against antigens (clones) CD1a (Na1/34), CD3 (UCHT1), CD4 (MT310), CD8 (DK25), CD16 (DJ130c), CD19 (HD37), and CD68 (KP1) (DAKO, Hamburg, Germany) detected by biotinylated rabbit anti-mouse immunoglobulins (DAKO) and subsequently labeled with streptavidin-Texas Red (Amersham Buchler, Braunschweig, Germany). Flow cytometric analyses were performed by one- or two-color immunofluorescence using fluorescein isothiocyanate- and phycoerythrin-conjugated antibodies. For indirect immunofluorescence, 5 × 105 cells were incubated with unconjugated primary antibody followed by fluorescein isothiocyanate- or phycoerythrin-conjugated affinity purified goat anti-mouse IgG F(ab′)2 fragments (Coulter-Immunotech, Hamburg, Germany). Stained cells were subsequently subjected to flow cytometry on a Coulter Epics XL (Coulter-Immunotech) and fluorescence emissions collected by selective filtration (530 nm for fluorescein isothiocyanate, 575 nm for phycoerythrin). Each antibody was titrated to obtain optimal results, staining with irrelevant appropriate isotype-matched antibodies served as control. Primary antibodies used were SFF2, VFF16, and directly fluorescein isothiocyanate-conjugated anti-CLA (cutaneous lymphocyte-associated antigen/carbohydrated P-selectin glycoprotein ligand-1) HECA-452 (Pharmingen, San Diego, CA). Total cellular RNA was prepared from tissue specimens by guanidinium thiocyanate extraction and cesium chloride centrifugation (Chirgwin et al., 1979Chirgwin J.M. Przybyla A.E. McDonald R.J. Rutter W.J. Isolation of biologically active RNA from sources enriched in ribonuclease.Biochemistry. 1979; 18: 5294-5297Crossref PubMed Scopus (16527) Google Scholar). RNA samples (10 μg total cellular RNA) were size fractionated on 1% agarose, 2 M formaldehyde gels and transferred to nylon membranes by vacuum blotting. Membranes were UV-cross-linked (Stratalinker, Stratagene, Heidelberg, Germany) and hybridized to random primed [α-32P] dATP-labeled cDNA probes at 42°C as described (Wagner et al., 1992Wagner S.N. Ruhri C. Kunth K. Holecek B.U. Goos M. Höfler H. Atkinson M.J. Expression of stromelysin 3 in the stromal elements of human basal cell carcinoma.Diagn Mol Pathol. 1992; 1: 200-205Crossref PubMed Google Scholar). After high stringency washing, finally two times for 60 min in 0.1 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate at 55°C for CD44 and 68°C for β-actin, filters were subjected to autoradiography for 12–48 h. The probe used comprised nucleotides from position 270–761 of the published human CD44 cDNA sequence (Stamenkovic et al., 1989Stamenkovic I. Amiot M. Pesando J.M. Seed B. A lymphocyte molecule implicated in lymph node homimg is a member of the cartilage link protein family.Cell. 1989; 56: 1057-1062Abstract Full Text PDF PubMed Scopus (554) Google Scholar). The probe was radiolabeled to a specific activity of 2 × 109 cpm per μg using a T7 DNA polymerase random priming kit (Stratagene, Heidelberg, Germany). Five micrograms total cellular RNA of each case were subjected to reverse transcription using random hexanucleotide primers and mouse mammary leukemia virus reverse transcriptase (Promega, Heidelberg, Germany). First strand cDNA syntheses were carried out for 1.5 h at 42°C in a total volume of 50 μl. Five microliters of first strand cDNA were amplified by Taq polymerase (Stratagene, Heidelberg, Germany) essentially as described byMackay et al., 1994Mackay C.R. Terpe H-J Stauder R. Marston W.L. Stark H. Günthert U. Expression and modulation of CD44 variant isoforms in humans.J Cell Biol. 1994; 124: 71-82Crossref PubMed Scopus (396) Google Scholar. Oligonucleotide primers used were AAGGAGAAGCTGTGCTACGTCG, 5′ sequence, and ATCCACACGGAGTACTTGCG, 3′ sequence, for β-actin, and GCACAGACAGAATCCCTGCTACC, 5′ sequence, and GGGGTGGAATGTGTCTTGGTCTC, 3′ sequence, according to positions 759–781 and 805–783 (directly flanking CD44 variant exons), respectively, of the published human CD44 cDNA sequence (Stamenkovic et al., 1989Stamenkovic I. Amiot M. Pesando J.M. Seed B. A lymphocyte molecule implicated in lymph node homimg is a member of the cartilage link protein family.Cell. 1989; 56: 1057-1062Abstract Full Text PDF PubMed Scopus (554) Google Scholar). PCR reaction parameters were 94°C, 1 min; 55°C, 1 min; 72°C, 1 min over 22 cycles for β-actin. Based on densitometric scanning of β-actin RT-PCR products, the amount of cDNA for CD44v RT-PCR reaction was adjusted. PCR reaction parameters for CD44 were 96°C, 5 s; 58°C, 15 s; 72°C, 1 min over 30 cycles. Under these conditions the increase of PCR products was confirmed to be linear in both PCR reactions. Furthermore, titration experiments excluded competitive inhibition of generation of l
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