Human Endomucin
2002; Elsevier BV; Volume: 160; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)61114-5
ISSN1525-2191
AutoresUlrike Samulowitz, Annegret Kuhn, Gertrud Brachtendorf, Roman Nawroth, Attila Braun, Ágnes Bánkfalvi, W. Böcker, Dietmar Vestweber,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoEndomucin is a typical sialomucin that we recently identified on the surface of mouse endothelial cells and on putative hematopoetic clusters of the dorsal aorta in the embryo. We have generated a panel of monoclonal antibodies (mAbs) against the extracellular part of human endomucin and polyclonal antibodies against the cytoplasmic part. Using immunohistochemistry endomucin was specifically detected on endothelial cells of blood and lymphatic vessels of all analyzed human tissues. In addition, the polyclonal antibodies stained the epithelium of the epidermis as well as epithelial and myoepithelial cells of the eccrine and apocrine glands in the skin. This nonendothelial staining could only be seen with a subset of mAbs if the staining procedure was amplified. Although high endothelial venules (HEVs) were not significantly stained with mAbs against endomucin, the polyclonal antibodies clearly detected endomucin on HEVs in lymphatic organs of the mouse and human, suggesting HEV-specific glycosylation affecting recognition by the mAbs. Indeed, endomucin isolated from human and mouse lymphoid organs carried the MECA-79 epitope that defines a set of L-selectin ligands on HEVs called peripheral node addressins. We conclude that human and mouse endomucin are endothelial sialomucins with the potential to function as L-selectin ligands. Endomucin is a typical sialomucin that we recently identified on the surface of mouse endothelial cells and on putative hematopoetic clusters of the dorsal aorta in the embryo. We have generated a panel of monoclonal antibodies (mAbs) against the extracellular part of human endomucin and polyclonal antibodies against the cytoplasmic part. Using immunohistochemistry endomucin was specifically detected on endothelial cells of blood and lymphatic vessels of all analyzed human tissues. In addition, the polyclonal antibodies stained the epithelium of the epidermis as well as epithelial and myoepithelial cells of the eccrine and apocrine glands in the skin. This nonendothelial staining could only be seen with a subset of mAbs if the staining procedure was amplified. Although high endothelial venules (HEVs) were not significantly stained with mAbs against endomucin, the polyclonal antibodies clearly detected endomucin on HEVs in lymphatic organs of the mouse and human, suggesting HEV-specific glycosylation affecting recognition by the mAbs. Indeed, endomucin isolated from human and mouse lymphoid organs carried the MECA-79 epitope that defines a set of L-selectin ligands on HEVs called peripheral node addressins. We conclude that human and mouse endomucin are endothelial sialomucins with the potential to function as L-selectin ligands. Sialomucins form a heterogeneous class of highly O-glycosylated sialic acid-rich glycoproteins that adopt an extended rod-like structure because of their extensive O-glycosylation. This feature enables them to be highly accessible on the cell surface and, therefore, allows some of them either to support or to prevent cell adhesion. Several examples for each of these two functions are known and some sialomucins have been found to support both functions depending on the way they are glycosylated. One of the first sialomucins that was demonstrated to have anti-adhesive activity was the primarily epithelial mucin MUC-1/episialin that can inhibit the cell adhesive activity of integrins1Wesseling J van der Valk S Vos H Sonnenberg A Hilkens J Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components.J Cell Biol. 1995; 129: 255-265Crossref PubMed Scopus (451) Google Scholar and of E-cadherin.2Wesseling J van der Valk SW Hilkens J A mechanism for inhibition of E-cadherin-mediated cell-cell adhesion by the membrane-associated mucin episialin/MUC1.Mol Biol Cell. 1996; 7: 565-577Crossref PubMed Scopus (362) Google Scholar Analyzing gene ablated mice allowed to show that T cells devoid of CD43 entered secondary lymphoid organs more readily than T cells with CD43, indirectly suggesting a function for this sialomucin in cell repulsion.3Stockton BM Cheng G Manjunath N Ardman B von Andrian UH Negative regulation of T cell homing by CD43.Immunity. 1998; 8: 373-381Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Most striking is the anti-adhesive function of the highly charged sialoprotein podocalyxin that was originally identified on the foot processes of podocytes in kidney glomeruli.4Kerjaschki D Sharkey DJ Farquhar MG Identification and characterization of podocalyxin—the major sialoprotein of the renal glomerular epithelial cell.J Cell Biol. 1984; 98: 1591-1596Crossref PubMed Scopus (391) Google Scholar At these sites, podocalyxin is involved in maintaining the spacing of these subcellular structures, most likely by charge repulsion, and deficiency of the corresponding gene leads to defects in the filtration activity of glomeruli.5Doyonnas R Kershaw DB Duhme C Merkens H Chelliah S Graf T McNagny KM Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin.J Exp Med. 2001; 194: 13-27Crossref PubMed Scopus (267) Google Scholar Besides anti-adhesive activities, some sialomucins clearly serve proadhesive functions. Most proadhesive sialomucins, although not all, serve as ligands for the selectins, cell adhesion molecules that recognize carbohydrate structures and function as initiators of cell contacts between leukocytes and endothelial cells.6McEver RP Selectin-carbohydrate interactions during inflammation and metastasis.Glycoconj J. 1997; 14: 585-591Crossref PubMed Scopus (241) Google Scholar, 7Vestweber D Blanks JE Mechanisms that regulate the function of the selectins and their ligands.Physiol Rev. 1999; 79: 181-213Crossref PubMed Scopus (834) Google Scholar The best functionally characterized selectin ligand is the sialomucin P-selectin glycoprotein ligand-1 that is essential for leukocyte extravasation under most inflammatory conditions and is able to bind to the two endothelial selectins, E- and P-selectin, as well as to L-selectin on leukocytes.8McEver RP Role of PSGL-1 binding to selectins in leukocyte recruitment.J Clin Invest. 1997; 100: 485-491Crossref PubMed Google Scholar L-selectin mediates the entry of leukocytes into inflamed tissue as well as the homing of lymphocytes into lymphatic tissue, a process that occurs in the specialized blood vessels known as high endothelial venules (HEVs).9Gallatin WM Weissman IL Butcher EC A cell surface molecule involved in organ-specific homing of lymphocytes.Nature. 1983; 304: 30-34Crossref PubMed Scopus (1241) Google Scholar, 10Rosen SD Bertozzi CR The selectins and their ligands.Curr Opin Cell Biol. 1994; 6: 663-673Crossref PubMed Scopus (448) Google Scholar L-selectin initiates the contact between lymphocytes and high endothelial cells by binding to certain carbohydrate-presenting glycoproteins. Searching for HEV-specific antigens that would support L-selectin-mediated binding of lymphocytes to HEVs, the monoclonal antibody (mAb) MECA-79 was found that blocked this interaction and the homing of lymphocytes into lymph nodes.11Streeter PR Rouse BTN Butcher EC Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.J Cell Biol. 1988; 107: 1853-1862Crossref PubMed Scopus (543) Google Scholar This antibody binds exclusively to endothelial cells of HEVs as well as to activated endothelium in chronically inflamed sites12Michie SA Streeter PR Bolt PA Butcher EC Picker LJ The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing.Am J Pathol. 1993; 143: 1688-1698PubMed Google Scholar, 13Onrust SV Hartl PM Rosen SD Hanahan D Modulation of L-selectin ligand expression during an immune response accompanying tumorigenesis in transgenic mice.J Clin Invest. 1996; 97: 54-64Crossref PubMed Scopus (90) Google Scholar and in rejected human heart transplants.14Toppila S Paavonen T Nieminen MS Hayry P Renkonen R Endothelial L-selectin ligands are likely to recruit lymphocytes into rejecting human heart transplants.Am J Pathol. 1999; 155: 1303-1310Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar MECA-79 defines a carbohydrate epitope15Hemmerich S Butcher EC Rosen SD Sulfation-dependent recognition of high endothelial venules (HEV)-ligands by L-selectin and MECA 79, an adhesion-blocking monoclonal antibody.J Exp Med. 1994; 180: 2219-2226Crossref PubMed Scopus (249) Google Scholar, 16Yeh JC Hiraoka N Petryniak B Nakayama J Ellies LG Rabuka D Hindsgaul O Marth JD Lowe JB Fukuda M Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase.Cell. 2001; 105: 957-969Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 17Bruehl RE Bertozzi CR Rosen SD Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody.J Biol Chem. 2001; 275: 32642-32648Crossref Scopus (42) Google Scholar that is specifically found on a small number of glycoproteins expressed on the surface of endothelial cells in HEVs. Because of the blocking function of MECA-79, these antigens were defined as peripheral node addressins (PNAds). Independent of this approach, several glycoprotein ligands of L-selectin were identified by using L-selectin as an affinity probe. First, four major protein bands were affinity-isolated from detergent extracts of [35S]-sulfate-labeled mouse lymph nodes.18Imai Y Singer MS Fennie C Lasky LA Rosen SD Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor.J Cell Biol. 1991; 113: 1213-1221Crossref PubMed Scopus (255) Google Scholar All four of these bands reacted with MECA-79 and two of them were later identified as GlyCAM-119Lasky LA Singer MS Dowbenko D Imai Y Henzel WJ Grimley C Fennie C Gillett N Watson SR Rosen SD An endothelial ligand for L-selectin is a novel mucin-like molecule.Cell. 1992; 69: 927-938Abstract Full Text PDF PubMed Scopus (587) Google Scholar and CD34.20Baumhueter S Singer MS Henzel W Hemmerich S Renz M Rosen SD Lasky LA Binding of L-selectin to the vascular sialomucin CD34.Science. 1993; 262: 436-438Crossref PubMed Scopus (595) Google Scholar In human tonsils, MECA-79 also recognizes four major bands of 60, 105, 165, and 205 kd with almost similar electrophoretic mobility as those isolated from mouse lymph nodes21Berg EL Robinson MK Warnock RA Butcher EC The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor.J Cell Biol. 1991; 114: 343-349Crossref PubMed Scopus (274) Google Scholar, 22Puri KD Finger EB Gaudernack G Springer TA Sialomucin CD34 is the major ligand in human tonsil high endothelial venules.J Cell Biol. 1995; 131: 261-270Crossref PubMed Scopus (143) Google Scholar, 23Sassetti C Tangemann K Singer MS Kershaw DB Rosen SD Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34.J Exp Med. 1998; 187: 1965-1975Crossref PubMed Scopus (222) Google Scholar and two of them were identified as CD34 (105 kd)22Puri KD Finger EB Gaudernack G Springer TA Sialomucin CD34 is the major ligand in human tonsil high endothelial venules.J Cell Biol. 1995; 131: 261-270Crossref PubMed Scopus (143) Google Scholar and podocalyxin (160 kd),23Sassetti C Tangemann K Singer MS Kershaw DB Rosen SD Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34.J Exp Med. 1998; 187: 1965-1975Crossref PubMed Scopus (222) Google Scholar respectively. Interestingly, another protein band migrating close to CD34 became detectable on complete depletion of CD34 from a purified PNAd fraction.22Puri KD Finger EB Gaudernack G Springer TA Sialomucin CD34 is the major ligand in human tonsil high endothelial venules.J Cell Biol. 1995; 131: 261-270Crossref PubMed Scopus (143) Google Scholar The identity of this protein is unknown. Mouse endomucin was originally identified by expression cloning with the help of three mAbs that had been raised against mouse endothelial cells.24Morgan SM Samulowitz U Darley L Simmons DL Vestweber D Biochemical characterization and molecular cloning of a novel endothelial specific sialomucin.Blood. 1999; 93: 165-175Crossref PubMed Google Scholar Analyzing the tissue distribution of this sialomucin in the adult mouse, we detected the antigen exclusively on endothelial cells of any tissue or organ that was tested.24Morgan SM Samulowitz U Darley L Simmons DL Vestweber D Biochemical characterization and molecular cloning of a novel endothelial specific sialomucin.Blood. 1999; 93: 165-175Crossref PubMed Google Scholar Tissues from various stages of mouse embryo development revealed early expression of endomucin on endothelia at E8.0 as well as on clustered putative hematopoetic cells associated with the luminal surface of the endothelium of the dorsal aorta.25Brachtendorf G Kuhn A Samulowitz U Knorr R Gustafsson E Potocnik AJ Fässler R Vestweber D Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoetic clusters in the dorsal aorta.Dev Dyn. 2001; 222: 410-419Crossref PubMed Scopus (46) Google Scholar In addition, endomucin is clearly expressed on endothelium of the aorta at stage E11.5, but only weakly detectable as patchy, focal staining on aortic endothelium at stage E15.5 and on the adult aorta.25Brachtendorf G Kuhn A Samulowitz U Knorr R Gustafsson E Potocnik AJ Fässler R Vestweber D Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoetic clusters in the dorsal aorta.Dev Dyn. 2001; 222: 410-419Crossref PubMed Scopus (46) Google Scholar In combination with anti-adhesive effects of endomucin ectopically expressed in transfected human embryonal kidney cells26Kinoshita M Nakamura T Ihara M Haraguchi T Hiraoka Y Tashiro K Noda M Identification of human endomucin-1 and -2 as membrane-bound O-sialoglycoproteins with anti-adhesive activity.FEBS Lett. 2001; 499: 121-126Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar it has been speculated that endomucin may play a role in the detachment of hematopoetic cells from endothelium during early hematopoiesis.27Ueno M Igarashi K Kimura N Okita K Takizawa M Nobuhisa I Kojima T Kitamura T Samulowitz U Vestweber D Shimomura T Suda T Nakashima K Taga T Endomucin is expressed in embryonic dorsal aorta and is able to inhibit cell adhesion.Biochem Biophys Res Commun. 2001; 287: 501-506Crossref PubMed Scopus (8) Google Scholar The tissue distribution of human endomucin protein was unknown and no antibodies against this antigen were available. To identify and analyze human endomucin, we cloned the full-length cDNA and generated an endomucin-IgG fusion protein that we used to raise a panel of mAbs. Each of these antibodies recognized the extracellular domain of native endomucin on human umbilical vein endothelial cells (HUVECs) and most bound also to the unglycosylated, in vitro translated antigen. In addition, we raised polyclonal antibodies against the cytoplasmic tail of the protein that react with both the murine and the human antigen. Similar to the tissue distribution of the mouse orthologue, the mAbs against human endomucin stained specifically and selectively endothelial cells in any human tissue that was analyzed. However, using the polyclonal antibodies, we found two important differences. First, these antibodies additionally detected the antigen on epithelium of the skin epidermis as well as in eccrine and apocrine glands. Only a subset of mAbs could verify this staining, and only if amplifying detection systems were used, suggesting that the epitopes were masked on most endomucin moieties on these epithelial cells. Second, similar to the distribution pattern for the mouse orthologue, human endomucin was not readily detectable on HEVs of lymphatic tissue with any of the mAbs. However, the polyclonal antibodies clearly detected endomucin on HEVs in human tonsils as well as in mouse lymph nodes. This demonstrates that endomucin is not absent from HEVs and suggests that endomucin on HEVs is differently modified than on other endothelial cells. Indeed, we found that endomucin isolated from lymphatic tissue carries the PNAd-specific carbohydrate epitope MECA-79, demonstrating that endomucin belongs to the group of glycoproteins defined as PNAds. Searching the human Expressed Sequence Tag (EST) database with BLAST at National Center for Biotechnology Information we found several clones with homology to the transmembrane and cytoplasmic region of mouse endomucin. AA426230, AA426155, and AA464807 matched between bp 572 to 945 of mouse endomucin (AF060883) corresponding to the 19 membrane proximal extracellular amino acids, the transmembrane and the cytoplasmic region, as well as part of the 3′UTR. AF464807 and AF121324 further extended the 3′UTR but with lower homology. Based on these EST sequences, we created the oligonucleotide HEM777–750 GCCTAGGTGTGGAGAGAATTCCTCAAGC as 3′ oligo for a 5′-rapid amplification of cDNA ends (5′RACE) on an adaptor-ligated human heart cDNA library (Marathon-Ready cDNA; Clontech, Palo Alto, CA), which was performed according to the manufacturer's instructions. The resulting fragment of ∼900 bp was cloned and sequenced and the open reading frame of 783 bp was verified by comparison with an reverse transcriptase-polymerase chain reaction (PCR) product obtained with total RNA from HUVECs using the oligonucleotides HEM-5-15 GCACCATGGAACTGCTTCAA and HEM777-758 GCCTAGGTGTGGAGAGAATT. The 5′-untranslated region was cloned with the 5′RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Gibco Life Technologies, Karlsruhe, Germany) according to the manufacturer's instructions. Briefly, total RNA from HUVECs was reverse-transcribed with the oligonucleotide HEM457-439 TACCAGTTTTAGAAGGT, TdT-tailed and the first PCR was performed with the oligonucleotides HEM449-425 TTAGAAGGTGATGCATCTGGTTGTA and AAP from the kit. As the second nested PCR yielded no fragment, the previous PCR fragment was cloned and sequenced and predicted a 5′-untranslated region of 108 bp. The 3′-untranslated region was cloned by reverse transcription of HUVEC total RNA with the oligonucleotide 3′RACE-Adapter primer (Gibco Life Technologies) followed by a PCR with the oligonucleotides HEM 641-660 GAATGTCTGGAAGGCAGAT and the Abridged Universal Amplification Primer (Gibco Life Technologies). A DNA fragment corresponding to nucleotides 553 to 1803 of GenBank entry J00228 spanning the genomic sequence of the hinge and the constant region 2 and 3 of the heavy chain of human immunoglobulin γ was amplified from hIgG:pCMV528Hahne M Jäger U Isenmann S Hallmann R Vestweber D Five TNF-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.J Cell Biol. 1993; 121: 655-664Crossref PubMed Scopus (201) Google Scholar and cloned into pcDNA3 (Invitrogen, Gronigen, The Netherlands) resulting in the vector hIgG:pcDNA3. A cDNA fragment coding for the complete extracellular domain of human endomucin with the triplet for the membrane proximal serine residue AGT replaced by the serine-coding triplet TCA creating a splice consensus site was amplified with the oligonucleotides HEM5-IgG CGGGATCCATGGAACTGCTTCAAGTG and HEM3-IgG GGAATTCACTTACCTGAGGAATAAGACCGGCTGGTTG and cloned into hIgG:pcDNA3 in analogy to Hahne and colleagues.28Hahne M Jäger U Isenmann S Hallmann R Vestweber D Five TNF-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.J Cell Biol. 1993; 121: 655-664Crossref PubMed Scopus (201) Google Scholar The resulting expression vector was stably transfected into CHO-Pro−5 cells and the recombinant fusion protein was isolated as described.28Hahne M Jäger U Isenmann S Hallmann R Vestweber D Five TNF-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.J Cell Biol. 1993; 121: 655-664Crossref PubMed Scopus (201) Google Scholar To generate mAbs, rats were immunized seven times within a time period of 36 days subcutaneously with 30 μg of purified human endomucin-IgG fusion protein and Freund's adjuvant per injection. Hybridoma fusions were done as described29Borges E Eytner R Moll T Steegmaier M Matthew A Campbell P Ley K Mossmann H Vestweber D The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum.Blood. 1997; 90: 1934-1942Crossref PubMed Google Scholar using the mouse myeloma SP2/0. Supernatants were screened for antibody binding to immobilized endomucin IgG in enzyme-linked immunosorbent assays.30Bosse R Vestweber D Only simultaneous blocking of L- and P-selectin completely inhibits neutrophil migration into mouse peritoneum.Eur J Immunol. 1994; 24: 3019-3024Crossref PubMed Scopus (192) Google Scholar Binding to an immobilized selectin IgG28Hahne M Jäger U Isenmann S Hallmann R Vestweber D Five TNF-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.J Cell Biol. 1993; 121: 655-664Crossref PubMed Scopus (201) Google Scholar was used as a negative control. Positive clones were tested in a second screen for binding to HUVECs using an established cell surface enzyme-linked immunosorbent assay.28Hahne M Jäger U Isenmann S Hallmann R Vestweber D Five TNF-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.J Cell Biol. 1993; 121: 655-664Crossref PubMed Scopus (201) Google Scholar To generate anti-endomucin antibodies against glycosylation-independent epitopes, a polyclonal rabbit antiserum was raised against a peptide covering the 15 C-terminal amino acids and containing an additional N-terminal cysteine residue for coupling with the bifunctional crosslinker 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) (Sigma, Munich, Germany). Immunization was performed with bovine serum albumin-conjugated peptide and affinity purification of the antibodies with the ovalbumin-conjugated peptide as described.31Borges E Tietz W Steegmaier M Moll T Hallmann R Hamann A Vestweber D P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin.J Exp Med. 1997; 185: 573-578Crossref PubMed Scopus (230) Google Scholar For specificity controls of the immunohistochemistry signals obtained with these antibodies, we depleted specific antibodies from the batches of affinity-isolated antibodies by reincubating them with the endomucin peptide column. Mock depletion was done by a similar incubation with an analogous MBS-ovalbumin peptide column carrying a peptide covering the last 15 C-terminal amino acids of mouse N-cadherin. The mAb MECA-7911Streeter PR Rouse BTN Butcher EC Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.J Cell Biol. 1988; 107: 1853-1862Crossref PubMed Scopus (543) Google Scholar against a carbohydrate epitope on PNAds was purified from the corresponding hybridoma obtained from the American Type Culture Collection (Rockville, MD). For immunoperoxidase staining, 2-μm paraffin-embedded sections of various 4% paraformaldehyde-fixed human tissues were cut on a microtome. After mounting the slides on poly-l-lysine-coated slides (Menzel-Gläser, Nuβloch, Germany), dewaxed specimens were immersed in 10 mmol/L of sodium citrate buffer, pH 6.0, in plastic Coplin jars and microwave-treated twice for 8 minutes. After cooling to room temperature, endogenous peroxidase was blocked by incubation with 0.1% H2O2/0.02 mol/L NaN3 in phosphate-buffered saline (PBS) for 30 minutes at room temperature. Nonspecific binding was blocked by incubation with 2% bovine serum albumin (BSA), fraction V (Sigma) in PBS, pH 7.4. Tissue sections were incubated with primary antibodies, either as tissue culture supernatant or diluted in 1% BSA in PBS, pH 7.4, for 1 hour followed by incubation with affinity-purified peroxidase-conjugated donkey anti-rat IgG (H+L), goat anti-rat IgG+IgM, or goat anti-rabbit IgG (Dianova, Hamburg, Germany) diluted 1:1000 in 1% BSA in PBS, pH 7.4. After the reaction was visualized with 3-amino-9-ethylcarbazole, tissue sections were counterstained with Mayer's hematoxylin and mounted. For control purposes, the first antibodies were either omitted or replaced by an irrelevant isotype-matched reagent. Anti-human CD34, clone QBEnd10 (DAKO, Hamburg, Germany), was used for positive controls, and V.5C7 or V.7C7 against mouse endomucin,24Morgan SM Samulowitz U Darley L Simmons DL Vestweber D Biochemical characterization and molecular cloning of a novel endothelial specific sialomucin.Blood. 1999; 93: 165-175Crossref PubMed Google Scholar which do not cross-react with the human antigen, were used as negative controls on human tissues. Tissues were derived from the archive of the Gerhard Domagk Institute for Pathology. For staining of frozen tissues, 7-μm cryostat sections were cut and mounted on poly-l-lysine-coated slides and fixed in acetone for 10 minutes at 4°C, followed by blocking of endogenous peroxidase activity for 30 minutes at room temperature. Nonspecific binding was blocked with 2% BSA in PBS, pH 7.4, for 30 minutes. Tissue sections were incubated for 1 hour with hybridoma supernatant followed by incubation with affinity-purified peroxidase-labeled donkey anti-rat IgG+IgM (dilution 1:1000, Dianova). After visualization with 3-amino-9-ethylcarbazole, tissue sections were counterstained with Mayer's hematoxylin and mounted. Alternatively, staining of serial sections of lymph nodes with primary antibodies was detected with tetramethylrhodamine B isothiocyanate-labeled goat anti-rat IgG or dichlortriazinyl amino fluorescein (DTAF)-labeled goat anti-rat IgG+IgM (dilution 1:100, Dianova) and visualized in a Zeiss-Axioscope fluorescence microscope. For double staining of mouse lymph node tissue sections were first incubated with mAb MECA-79 followed by incubation with a DTAF-labeled goat anti-rat IgM (μ chain, dilution 1:100; Dianova). Subsequently, the same sections were incubated with the polyclonal rabbit antiserum against endomucin followed by incubation with a Cy3-labeled donkey anti-rabbit IgG (dilution 1:500, Dianova). For amplifying the peroxidase-staining procedure, the avidin-biotin-based Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA), was used. Briefly, paraffin sections were fixed and pretreated as described above. The sections were then incubated with appropriate primary antibodies diluted in PBS containing 1% BSA, followed by biotinylated secondary antibody. The sections were then washed with PBS and incubated with an avidin-biotin-peroxidase complex. The reactions were visualized and counterstained as described above. Fresh human peripheral blood samples were fractionated on Ficoll-Histopaque (1.077 g/ml and 1.119 g/ml; Sigma Chemical Co., St. Louis, MO) gradients according to standard procedures. Mononuclear cells and granulocytes were collected and washed twice before staining. Before staining, Fc-receptors of the cells were blocked with mAb 3G8 (mouse anti-human FcγRIII) and IV.3 (anti-human FcγRII) (MEDAREX Inc., Annandale, NJ) at doses of 1 μg per 106 cells for 10 to 15 minutes at 4°C. For two-color staining of the peripheral blood leukocytes the following mouse antibody conjugates, coupled with fluorescein isothiocyanate (FITC) or phycoerythrin were used: CD3 FITC (clone HIT3a, IgG2a), CD14 FITC (clone MπP9, IgG2b), CD19 FITC (clone SJ25C1, IgG1), CD56 FITC (clone NCAM16.1, IgG2b), CD45 FITC or phycoerythrin (clone HI30, IgG1), CD15 FITC (clone HI98, IgM). Directly conjugated antibodies were purchased from Becton Dickinson, San Jose, CA. All antibodies were used at 10 μg/ml per 107 cells/ml. For triple-stage staining, a biotinylated donkey anti-rat IgG, (Fab′2) fragment (Dianova) was used at a dilution of 1:400. Streptavidin-phycoerythrin (Dianova) was used at a dilution of 1:100. Antibody incubations were done in fluorescence-activated cell sorting (FACS) buffer (PBS, pH 7.4, 0.5% dialyzed fetal calf serum, 0.02% NaN3) for 20 to 30 minutes on ice. Cells were analyzed on a FACScalibur using Cellquest software (Becton Dickinson, Heidelberg, Germany). Cell surface biotinylation of HUVECs and immunoprecipitations and immunoblots were essentially done as described.24Morgan SM Samulowitz U Darley L Simmons DL Vestweber D Biochemical characterization and molecular cloning of a novel endothelial specific sialomucin.Blood. 1999; 93: 165-175Crossref PubMed Google Scholar, 31Borges E Tietz W Steegmaier M Moll T Hallmann R Hamann A Vestweber D P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin.J Exp Med. 1997; 185: 573-578Crossref PubMed Scopus (230) Google Scholar Human endomucin lacking posttranslational modifications was synthesized by a coupled in vitro transcription/translation reaction in a reticulocyte lysate using the TNT Quick Coupled Transcription/Translation System (Promega, Mannheim, Germany). Based on the manufacturer's instructions, each reaction containing 8 μl of TNT Quick Master Mix, 164 KBq of 35S-methionine (Amersham, Freiburg, Germany), and 0.04 μg of vector DNA (either pcDNA3 containing full-length mouse endomucin cDNA or vector without insert) was filled up to a final volume of 10 μl with diethyl pyrocarbonate-H2O and incubated for 90 minutes at 37°C. Subsequently reactions were subjected to immunoprecipitations as described above. As negative or positive standards, either 10 μl of a reaction containing the vector without insert or 2 μl of a reaction containing the
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