Biosynthesis and processing of the cell adhesion molecule PECAM-1 includes production of a soluble form.

PECAM-1 (CD31) is a 130-kDa glycoprotein found on platelets, endothelial cells, granulocytes, and monocytes, as well as on certain myelomonocytic cell lines. Recent studies have shown that PECAM-1 may be involved in activation of leukocyte integrins and may also be involved in adhesive interactions of circulating leukocytes and the vessel wall. In spite of the important functional role that PECAM-1 plays in these processes, little is known about the biosynthesis, processing, and turnover of PECAM-1 on the cell surface. We have studied the biosynthesis of PECAM-1 in the promonocytic cell line U937, and in endothelial cells, by pulse-chase labeling and immunoprecipitation. PECAM-1 was synthesized as a 110-kDa precursor form, which was processed into the 130-kDa mature form within 1-3 h, during which time it began to move to the cell surface. The protein disappeared from the cell surface in both cell types about 48 h after labeling. A soluble form of PECAM-1, which is 5-10 kDa smaller than cell-associated PECAM-1 and contains the cytoplasmic tail, was observed in the culture media of HUVECs and phorbol ester-treated U937 cells. This form of soluble PECAM-1 is encoded by an alternatively spliced mRNA from which the exon containing the transmembrane domain has been removed. Soluble PECAM-1 was also detected in normal human plasma at levels of 10-25 ng/ml. Two isoforms of plasma PECAM-1, which differed in the presence of the cytoplasmic tail, were observed by Western blot analysis. In parallel with soluble forms of other cell adhesion molecules, soluble PECAM-1 may play a role in modulating the inflammatory response.

ll Established Investigators of the American Heart Association.
The abbreviations used are: PECAM-1, plateleUendothelia1 cell adhesion molecule 1; SPECAM-1, soluble PECA"1 produced by cultured cells; srPECAM-1, soluble recombinant PECA"1 containing only the extracellular domain; ACT, polyclonal antibody against the cytoplasmic tail of PECA"1; CHO cells, Chinese hamster ovary cells; D-PBS, Dulbecco's phosphate-buffered saline; GPIIIa, platelet glycoprotein IIIa; HEL cells, human erythroleukemia cells; HUVECs, human umbilical vein endothelial cells; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; FCS, fetal calf serum; bp, base pair(s); ELISA, enzyme-linked immunosorbent assay; FPLC, fast protein liquid chro- (1) that was originally characterized as a myelomonocytic differentiation antigen (2,3) present on monocytes, granulocytes, and several myeloid leukemia cell lines (4)(5)(6). More recent studies have demonstrated the existence of this membrane glycoprotein on the surface of human platelets and at the border of endothelial cells in contact with each other (1,7,8). Molecular cloning studies have revealed the presence of six externally exposed immunoglobulin-like domains, a short transmembrane domain, and a relatively long cytoplasmic tail of 118 amino acids containing potential sites for phosphorylation, lipid modification, and other post-translation modifications (1). Approximately 40% of the molecular mass of PECA"1 is comprised of carbohydrate residues whose influence on the adhesive properties of PECAM-1 is as yet unknown.
A great deal has been learned in the last several years about the role of PECA"1 in mediating cellular interactions. PECA"1 is strongly localized to the intercellular junctions of endothelial cells and other monolayer cells in which it is expressed (1,(7)(8)(9) and appears to function in initiating cell-cell contact (9,10) and in mediating cell migration (11). PECA"1 is also present at 50,000-100,000 molecules/cell on circulating monocytes and granulocytes (121, and it has been thought for some time that PECA"1 might therefore be involved in mediating either homophilic (9)  Muller et al. (15) have provided evidence that PECA"1 plays a role in leukocyte trafficking in vitro, and Vaporciyan et al. (16) have recently shown that antibodies to PECA"1 block neutrophil recruitment in vivo in three different animal models.
PECA"1 may also serve as a signaling molecule in a number of different cellular adhesion cascades. PECA"1 becomes phosphorylated in response to a number of different agonists (17,18) and has been shown to associate with the platelet cytoskeleton following cellular activation (18). Interestingly, a number of different laboratories have shown that engagement of PECAM-1 within the plasma membrane can modulate the adhesive functions of p l integrins in selected T-cell subsets (19) and p2 integrins in lymphokine-activated killer cells (20). The signaling pathways by which PECA"1 induces integrin upregulation are not clear, but may nonetheless have widespread cellular consequences, as evidenced by the reported effects of anti-PECAM-1 antibodies on neutrophil and monocyte chemotaxis (2) and on the generation of reactive metabolites (21,22).
In spite of the importance of PECA"1 in mediating the interactions between circulating leukocytes and the vascular wall, virtually nothing is known about its biogenesis within the matography; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TBS-T, Tris-buffered saline containing 0.05% Tween 20. cell, its half-life on the cell surface, or potential alternative forms of the molecule that could modulate the process of leukocyte transmigration. Changes in the rate of synthesis or turnover, for example, could affect the stability or integrity of the endothelial cell barrier. In this report, we examine the biosynthesis and processing of PECA"1 in a leukocyte model, as well as in endothelial cells. Our results provide evidence that the half-life of PECA"1 on the endothelial cell surface is surprisingly short and demonstrate the existence of a circulating form of soluble PECA"1 that may function to regulate PECAM-1-mediated cellular interactions.
EXPERIMENTAL PROCEDURES Cells-The U937 and HEL cell lines were obtained from the American Type Culture Collection and were maintained in RPMI 1640 media (Life Technologies, Inc.) containing 10% heat-inactivated FCS (HyClone Laboratories) and 40 pg/ml gentamicin (Sigma). Human umbilical vein endothelial cells (HUVECs) were prepared and cultured as previously described (23).
All cells had attached to the culture dish when labeling was initiated. The cell monolayer was washed with D-PBS and preincubated with methionine-free RPMI media containing 10% dialyzed FCS for 30 min at 37 "C. Addition of [36S]methionine (DuPont NEN) at 200 pCi/ml initiated the pulse, which was continued at 37 "C for 20 min or 1 h. The labeling media were removed, the cells were washed with D-PBS, and the chase was begun by addition of RPMI/10% FCS containing 1 mg/ml methionine. After incubation at 37 "C for the indicated times, cells were washed with cold D-PBS/2 mM phenylmethylsulfonyl fluoride and lysed by scraping into 800 p1 of lysis buffer. The lysates were incubated for 30 min on ice with mixing and centrifuged a t 13,000 x g for 15 min at 4 "C. Aliquots of the supernatants were precipitated with ice-cold trichloroacetic acid to quantitate newly synthesized protein. All lysates were stored at -70 "C prior to immunoprecipitation analysis. HUVECs were labeled in a similar manner.
To detect the appearance of newly synthesized PECA"1 on the cell surface, the cells were labeled as described above. At the end of the chase, the cells were washed with D-PBS and placed in RPMI/10% FCS containing 10 pg/ml polyclonal anti-PECAM-1 IgG and 0.02% NaN, for 2 h a t 4 "C. The cells were then washed extensively with ice-cold D-PBS containing 10% FCS and lysed a s described above.
Preparation of Cell Culture Media for Detection of Soluble PECA" 1-Media were removed from the cells after the various chase times. Following low speed centrifugation, the media were concentrated 2-3fold using a Centricon 30 (Amicon). The supernatants were processed through 0.2-pm syringe filters (Corning) and then centrifuged at 100,000 x g for 3 h a t 4 "C. The supernatants were stored at -70 "C.
Immunoprecipitation of PECAM-I-Equal volumes of lysate (about 10% of the total) and all of the culture media from each time point of a pulse/chase experiment were immunoprecipitated. Samples were precleared by addition of 10 pg of normal rabbit IgG (Sigma) and 50 p1 of a 10% slurry of Pansorbin (Calbiochem) a t 4 "C for 1 h. After removal of the Pansorbin, the supernatants were incubated with 10 pg of polyclonal anti-PECAM-1 IgG or normal rabbit IgG for 16 h at 4 "C. Immune complexes were collected by addition of 50 pl of a 50% slurry of protein A-Sepharose (Sigma) for 2 h a t 4 "C, followed by centrifugation and extensive washing. Lysates of cells which were incubated with anti-PECAM-1 antibodies prior to lysis to detect cell-surface PECA"1 were immunoprecipitated by the addition of protein A-Sepharose without preclearing. The samples were prepared for gel electrophoresis by boiling for 5 min in 80 pl of SDS sample buffer containing 5% 2-mercaptoethanol, and the supernatants were separated on 7% polyacrylamide gels followed by fluorography.
Glycosidase Deatment of PECAM-1-Samples were immunoprecipitated as described above. PECA"1 was eluted from the beads and treated with several glycosidases essentially as described by Johnston et al. (24).
PECA"1 absorbed to protein A-Sepharose beads was treated with 50 milliunits/ml of endo-P-N-acetylglucosaminidase (endo H; Boehringer Mannheim) for 16 h a t 37 "C. For treatment with Nglycosidase F (N-glycanase; Genzyme), the beads were washed and boiled for 5 min in 10 pl of 0.5% SDS, 1% 2-mercaptoethanol. The volumes were adjusted to final detergent concentrations of 0.1% SDS and 1.25% Triton X-100 and incubated for 5 h at 37 "C in the presence of 15 unitdm1 N-glycanase. Samples treated with neuraminidase (Gen-zyme) were boiled for 5 min prior to incubation with 1 unit/ml neuraminidase for l h at 37 "C. Some samples treated with neuraminidase were then incubated with 60 milliunits/ml of endo-a-N-acetylgalactosaminidase (0-glycanase, Genzyme) for 16 h at 37 "C. After treatment with N-glycanase as described above, some samples were treated with neuraminidase for 1 h, followed by incubation with 0-glycanase for 16 h. After enzyme treatments, SDS sample buffer was added to all reactions.
Production of Soluble Recombinant PECAM-1 (srPECAM-1)-A recombinant construct encoding only the extracellular domain of PECA"1 (residues 1-574) was produced by placing 2 stop codons just 5' of the sequence encoding the transmembrane domain in the PE-CAM-1 cDNA, leading to deletion of the transmembrane domain and the cytoplasmic tail. A cassette was first generated by PCR which utilized a sense oligonucleotide beginning at base 1429 of the cDNA (5'-CGTTGCGAATCGATCAGTGGA-3') and an antisense oligonucleotide complementary to the cDNA sequence from bases 1927-1944 with 2 stop codons (underlined) and a n XbaI restriction site in place of the transmembrane domain sequence (5'-C'l"I'GCCTGGAAGAAATGATA-ATCTAGACCGGA-3'). PCR amplification was performed from the fulllength PECAM-1 cDNA i n pGEM7 (Promega), resulting in a 531-bp product. This DNA was cut with Aut11 and XbaI to yield the 396-bp cassette, which was gel-purified. PECA"1 cDNA in pGEM7 was cut with the same enzymes, and a 1.5-kilobase 5' XbaI-Aut11 fragment extending from the beginning of the open reading frame to base 1556 was isolated. The two fragments were ligated into the mammalian expression vector EMC-3 (kind gift of Dr. Glenn Larsen, Genetics Institute, Boston, MA) linearized with XbaI. The vector EMC-3 results from the modification of pMT2 (25), in which the sequence encoding dihydrofolate reductase was placed under the control of a separate promoter. Clones were analyzed for the presence of an insert of the correct size and orientation with the restriction enzyme EcoRI. The sequence of the cassette generated by PCR was confirmed by nucleotide sequence analysis.
Transient expression of the CsC1-purified plasmid i n COS cells followed by immunoprecipitation confirmed that srPECAM-1 was secreted into the culture media and showed that srPECAM-1 migrates a t about 90 kDa on SDS gels, as compared to cellular PECA"1 at 130 kDa. Stable lines were created by transfection of the expression vector into dihydrofolate reductase-deficient CHO (line DG44, obtained from Lawrence Chasin, Columbia University) using calcium phosphate co-precipitation (26). Selection and amplification of PECA"1 expression was carried out in the presence of methotrexate. The culture media were assayed for the presence of srPECA"1 by ELISAand on Western blots. The cells were then cloned by limiting dilution, and clones secreting the highest levels of PECA"1 were chosen for expansion following screening of culture media by antigen capture ELISA. The srPECA"1 was metabolically labeled with by incubation of the cells with 200 pCi/ml of [35S]methionine for 16 h, after which the culture media were removed and stored in aliquots at -70 "C.
For purification of srPECA"1, the cells were expanded into roller bottles and grown in serum-free medium. Culture media were passed over a 5-ml column of wheat germ agglutinin-Sepharose (Sigma). The column was washed with 0.02 M Tris-HC1, pH 7.4, 0.5 M NaCl, and bound proteins, including srPECAM-1, were then eluted by the addition of 0.5 M a-methyl mannoside in the same buffer. The eluate was concentrated, buffer-exchanged into 0.05 M NaH,PO,, pH 7.4, 0.15 M NaC1, and further purified by molecular sieve FPLC over two Superose-12 columns connected in tandem. Fractions were screened by SDS-PAGE followed by silver stain and Western blot analysis. Protein content was quantitated by absorbance a t 280 nm.
Production ofAnti-human PECAM-1 Qtoplasmic Tail (ACT) Specific Polyclonal Antibody-An antibody reactive with only the cytoplasmic domain of human PECA"1 was produced by generating a recombinant bacterial fusion protein containing the PECA"1 cytoplasmic tail (amino acid residues 594-711) with 6 histidine residues added at the N terminus. The appropriate cDNA was generated by PCR, utilizing a sense oligonucleotide complementary to the first 15 bases of the sequence encoding the cytoplasmic tail, as well as a 5"mismatched extension carrying a BamHI restriction site which would be in-frame when ligated into the expression vector (5"CA"GGATCCAAATGT-TATTTTCTG-3'). The antisense primer was complementary to a 15base cDNA sequence flanking the stop codon and included a 5'-mismatched extension containing a Hind111 restriction site (5'-GCCTAAGCTTCTAAGTTTCATCAAG-3'). The single PCR amplification product was gel-purified, double-digested with BamHI and HindIII, and ligated into the BamHI and HindIII sites present in the polylinker region of pDS566xHis E. coli expression vector (27,28). The recombinant protein was expressed in E. coli strain SG13009 as previously described (29) and purified using a nickel-chelate affinity resin (Ni"-NTA agarose, Qiagen) (30,31). which bound tightly to the histidine residues. Purified recombinant protein was then injected subcutaneously into a rabbit a t 14-day intervals, followed by bleeding 10 days after the third injection. Immunoprecipitation of ["Slmethionine-labeled or cell surface biotinylated protein extracts from platelets, endothelial cells, U937 cells, and 3T3 cell lines transfected with full-length PECA"1 cDNA or with mutant constructs missing the entire cytoplasmic domain confirmed the specificity of the antiserum for the cytoplasmic tail of PECA"1.
Identification of the Alternatively Spliced RNA Species Encoding Soluble PECAM-1-RNA was extracted from HUVECs and from both untreated and PMA-treated HEL cells by the method of Chomczynski and Sacchi (32). The synthesis of cDNA was primed from 1 pg of RNA using random hexamers (Pharmacia Biotech, Inc.) in the presence of Moloney murine leukemia virus reverse transcriptase (Life Technologies). The primary PCR was performed using the sense primer a (see Fig. 6A) from base 1754 to 1773 (5'-GAGAMAAGAGGGCAAACCC-3'), and the antisense primer c (5'-TCGTCAGGTGAAGACT-3'), which extends from base 2077 to 2060. The primary PCR reaction was then diluted 1:lOO and reamplified in a nested PCR using primers b and c (base 1865-1882; 5'-TCAACAGAGCCAACCACG-3'). One-half of the PCR reaction was separated by gel electrophoresis on a 1.9% agarose gel, blotted to a nylon membrane (Genescreen Plus, DuPont NEN), and UV-cross-linked. The blot was then hybridized to a "*P-labeled PE-CAM-1 cDNA probe, washed to high stringency (0.1 x SSC, 0.1% SDS, 68 "C), and exposed to Kodak XAR film a t -70 "C with an intensifying screen. The 215-bp product was obtained by preparative agarose gel electrophoresis of the entire 1' PCR reaction, followed by isolation using Geneclean (Bio 101). The ends of the purified PCR product were flushed using the Klenow fragment of DNA polymerase, followed by ligation into pGEM-5Zf (Promega) which had been cut with EcoRV to yield blunt ends. Three clones generated from HUVEC RNA and 3 clones from HEL cell RNA were sequenced using Sequenase (U. S. Biochemical Corp.).
Detection of PECAM-1 in Normal Human Plasma-Plasma from citrated whole blood was ultracentrifuged a t 100,000 x g for 3 h and processed through a 0.2-pm filter to remove debris and membrane fragments. Plasma PECA"1 was absorbed out of some plasma samples using monoclonal and polyclonal anti-PECAM-1 antibodies which had been coupled a t a ratio of 4 mg of IgG/ml to cyanogen bromide-activated Sepharose (Pharmacia) according to the manufacturer's instructions. Parallel plasma samples were incubated with preimmune rabbit and mouse IgG-Sepharose beads, as well as an anti-GPIIIa antibody (AP-3) coupled to Sepharose. Complete absorption was achieved by incubating plasma samples with 25 pl of antibody-coupled Sepharose beads a t 4 "C for 16 h with mixing.
PECA"1 in plasma was measured using an antigen-capture ELISA. A rabbit polyclonal antibody directed against PECA"1, diluted to 25 pg/ml as IgG in carbonate buffer, pH 9.5, was adsorbed onto flat-bottomed microtiter plates (Immulon 2, Dynatech Laboratories) for 16 h a t 4 "C. The wells were rinsed with TBS containing 0.05% Tween 20 (TBS-T) and blocked with 2% bovine serum albumin in TBS-T for 1 h a t 22°C. Plasma samples (100 PI), processed as described above, were added to triplicate wells and incubated for 1 h a t 37 "C. Plasma PECAM-1 was quantitated by comparison to a standard curve of purified srPECA"1 serially diluted in plasma which had been depleted of PECA"1 by repeated absorptions with anti-PECAM-1 IgG-Sepharose. Unbound proteins were removed by extensive washing with TBS-T, after which the plates were incubated with biotinylated monoclonal anti-PECAM-1 IgG (5 pg/ml) for 1 h a t 37 "C. ABC reagent with alkaline phosphatase as the detection enzyme (Vectastain kit, Vector Laboratories) was added for 15 min a t 37 "C. Finally, the wells were rinsed, and color was developed by the addition of 2 mg/ml p-nitrophenyl phosphate in diethanolamine buffer, pH 9.8, containing 0.5 mhl MgCI,. The absorbance a t 405 nm was quantitated a t 20-min intervals on an automated ELISA reader (Molecular Devices). Negative controls included P E C A " 1-depleted plasma, capture with normal rabbit IgG, and omission of detection antibodies.
Soluble forms of PECA"1 from plasma and H W E C culture media were characterized qualitatively by immunoprecipitation and Western blot analysis, followed by chemiluminescent detection. Human plasma samples were diluted 1:lO with TBS, ultracentrifuged a t 100,000 x g for 3 h, and processed through a 0.2-pm filter. Tissue culture media were concentrated 4-6-fold prior to ultracentrifugation and filtration as previously described. Samples were immunoprecipitated using antibodies that had been directly conjugated to Sepharose beads. Bound proteins To detect cell-surface PECA"1, intact cells were incubated with an anti-PECAM-1 polyclonal antibody (10 pg/ml) for 2 h at 4 "C, washed five times, and lysed in Triton X-100. Total cellular PECAM-1 was detected by immunoprecipitation of detergent-lysed cells. Both sets of immunoprecipitates were analyzed by SDS-PAGE and fluorography. Molecular weight standards ( x 10.' ) are indicated on the right. Note that PECA"1 reaches the cell surface quickly after synthesis (3 h), but remains there less than 48 h.
were separated by SDS-PAGE on 7'7; gels and transferred to nylon membranes (Immobilon, Millipore). After blocking of unbound sites with 2% bovine serum albumin in TBS-T, PECA"1 was detected with an anti-PECAM-1 monoclonal antibody, followed by goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson Laboratories). Finally, the blots were incubated with chemiluminescent substrate IECL kit, Amersham) and exposed to film (Hyperfilm-ECL, Amersham).
L-Cell Aggregation Assay-Aggregation of PECAM-1-transfected Lcells was performed as previously described (9,13). The ability of recombinant soluble PECA"1 to block the interaction was determined by incubating aliquots of the cell suspension with the indicated amounts of FPLC-purified protein during the aggregation assay.

Kinetics of PECAM-1 Synthesis and
Cell Surface Expression-The ability to induce de novo synthesis in PMAtreated U937 cells (6) allowed the development of an attractive model system for studying the biosynthesis and processing of PECA"1 in leukocyte precursors. To determine the kinetics of PECA"1 synthesis, cells were treated with PMA for 2 days to up-regulate PECA"1 expression and were then metabolically labeled with [%]methionine for different pulse and chase times. The results of such an experiment are shown in Fig. 1. When U937 cells were labeled for 20 min and chased for 20 min, a pre-PECAM-1 species with M , = 110,000 became extensively labeled (Fig. L4, left panel). At 60 min of chase, some label began to appear in the mature form of PECAM-1 ( M , = 130,000). After a 3-h chase, most of the PECA"1 detected was in the mature form. To determine the stability of the newly synthesized protein, the cells were pulsed for 1 h and then chased for longer periods of time (Fig. lB, left punel). PECA"1 remained present within the cell for 24 h, after which levels of labeled protein dropped significantly.
To detect the appearance of PECAM-1 on the cell surface, labeled U937 cells were incubated with anti-PECAM-1 antibodies prior to cell lysis. Antibody incubation was performed at 4 "C in the presence of sodium azide to prevent internalization. Following extensive washing, the cells were lysed and immune complexes were collected with protein A-Sepharose. As shown in Fig. lA (  detected in HUVECs within 20 min, and significant levels of the mature form appeared within 1 h after labeling (Fig. 2 A ) . Mature PECA"1 persisted in the cells for 48 h postsynthesis, after which most of the protein disappeared. Detection of PECAM-1 on the cell surface of HUVECs (Fig. 2 B ) indicated that most of the newly synthesized protein had disappeared from the cell surface within 2 days, similar to that observed in the myeloid U937 cells.
Analysis of PECA"1 Glycosylation-Although there are 9 potential sites for N-linked carbohydrate modification in the amino acid sequence of PECA"1(1), the precise glycosylation pattern has not yet been determined. Similar to other membrane glycoproteins, however, the "pre-PECAM-1" protein detected early in its biogenesis (Figs. l and 2) likely represents a precursor containing high-mannose carbohydrate moieties which may be further processed into complex forms in the Golgi apparatus, resulting in the mature form of PECA"1. To analyze the glycosylation pattern of PECAM-l, U937 cells were labeled for 20 min and chased for 1 h to obtain approximately equal amounts of pre-PECAM-1 and mature PECA"1 (Fig. 3, lane 1 ). Detergent lysates were prepared, immunoprecipitated with anti-PECAM-1 antibody, and finally treated with several glycosidases. As shown in Fig. 3 (lane 21, only pre-PECAM-1 was sensitive to endo H, which selectively removes high mannose residues. When the immunoprecipitates were treated with N-glycanase, which cleaves most forms of N-linked carbohydrates, both pre-and mature PECA"1 decreased in M, to about 100,000 (Fig. 3, lane 4 ) . Further characterization of the complex carbohydrate moieties of PECA"1 by evaluating sensitivity to neuraminidase treatment indicated that the mobility of mature PECAM-1, but not pre-PECAM-1, was increased by neuraminidase (Fig. 3, lanes 5 and 6). Treatment of PECA"1 with neuraminidase followed by 0-glycanase did not increase the mobility of PECA"1 any more than neuraminidase alone (Fig. 3, lanes 5-8). This observation was confirmed by treatment with N-glycanase, neuraminidase, and 0-glycanase (Fig.   3, lanes 9 and IO). PECA"1 from HEL cells and HUVECs gave identical patterns of sensitivity to glycosidase treatment (not shown). Chemical deglycosylation of PECA"1 with trifluoromethanesulfonic acid did not result in any further change in the mobility of PECAM-1 (not shown), suggesting that enzymatic deglycosylation had been complete.

Myeloid Cell Lines and HUVECs Secrete a Soluble Form of PECA"1 Which Contains the
Cytoplasmic Tail-Recently, several laboratories have reported the existence of soluble, biologically active, forms of L-selectin (331, ICA"1 (34, 351, and P-selectin (36). In order to examine whether a soluble form of PECA"1 might be synthesized by either endothelial or myeloid cells in culture, U937 cells were labeled with [35Slmethionine for 1 h and chased for various lengths of time. Culture media from these cells were processed through a 0.2-pm filter and centrifuged a t 100,000 x g for 3 h, and the top 112 to 314 of the supernatant was carefully removed for immunoprecipitation analysis using antibodies specific for the extracellular domain of PECA"1. As shown in Fig. 4, soluble PECA"1 could be detected at low levels in the culture media within 3 h after labeling and continued to accumulate in the media for 2 days. The soluble form migrated slightly more rapidly than the membrane-associated form. A soluble form of PECA"1 was also observed in the culture media of HUVECs (not shown) and migrated slightly more rapidly than the corresponding cellassociated form. When the spun and filtered culture media were phase-extracted with Triton X-114 (37, 381, sPECA"1 partitioned completely into the aqueous phase (data not shown), suggesting that the soluble form of PECA"1 secreted into the media of cultured cells does not appear to contain a transmembrane domain.
Two possible mechanisms could lead to the appearance of a soluble form of PECA"1 in the culture media (Fig. 5A). First, alternative splicing out of the exon encoding the transmem- . The culture media were collected a t each time point, processed as described under "Experimental Procedures," and immunoprecipitated with a PECAM-1-specific polyclonal antibody. The "culture media" lanes were exposed to x-ray film twice as long as the "cell lysates" lanes.  2 and 4 ) . Note the specificity of ACT for PECA"1 molecules that contain the cytoplasmic tail and its inability to react with the truncated recombinant form of PECA"1. C, reactivity of soluble PECA"1 with ACT. Culture media from '"S-labeled H W E C s was processed as de- Southern blot analysis of the PCR products. Ten percent of the indicated PCR reactions were separated on a 1.99 agarose gel, blotted onto a nylon membrane, and hybridized with a "P-labeled PECA"1 cDNA probe. Note the presence of PCR products 108 bp smaller than the predicted size of the full-length product. C, sequencing of the alternatively spliced species. The 215-bp PCR product of primer pair a + c was eluted from a preparative agarose gel, subcloned, and subjected to nucleotide sequence analysis. The gel shows the sequence of the antisense strand of the cDNA synthesized from HUVEC RNA and demonstrates the deletion of exon 9, which encodes the transmembrane domain. The arrow indicates the splice junction between exons 8 and 10.
bound a s well as soluble transmembraneless PECA"1, but not with truncated PECA"1 (Fig. 5A). We found that ACT bound full-length PECA"1 in detergent lysates of U937 cells (Fig.  5B, lane 21, but not a truncated form of PECA"1 derived from the culture media of CHO cells transfected with a PECA"1 construct encoding residues 1-574 (srPECAM-1) (Fig. 5B, Lane  4 ) , as predicted. None of the many other labeled proteins which are present in the CHO cell culture media were recognized by ACT, demonstrating its specificity. To determine whether the cytoplasmic tail is present on the soluble form of PECA"1 found in conditioned media, culture media from HUVECs were immunoprecipitated with ACT. As shown in Fig. 5C, the soluble form of PECAM-1 secreted from cells was recognized as efficiently by ACT as by an anti-PECAM-1 polyclonal antibody directed against the extracellular domain of the molecule, indicating that the cytoplasmic tail is retained in the soluble form of PECAM-1. Similar results were obtained with culture media from PMA-treated U937 cells (not shown).
Detection of a n Alternatively Spliced mRNA Species Lacking the Exon Encoding the Dansmembrane Domain-The presence in cell culture media of a soluble form of PECA"1 containing an intact cytoplasmic tail is consistent with the possibility that alternative splicing of the exon encoding the transmembrane domain leads to secretion of a subpopulation of PECAM-1 from the cells. Characterization of the PECA"1 gene in our labo-ratory2 has shown that the transmembrane domain is encoded by an exon of 108 base pairs. To determine whether an alternatively spliced species lacking the exon encoding the transmembrane domain might exist, RNA from HUVECs was reverse-transcribed with random hexamers and PCR-amplified using primer pairs which flank the exon encoding the transmembrane domain (Fig. 6A) were predicted for full-length and transmembraneless PECAM-1, respectively, from primer pair b + c. The PCR products were separated by agarose gel electrophoresis, Southernblotted, and probed with a 32P-labeled PECA"1 cDNA probe (Fig. 6B). All 4 predicted bands were detected, with the 215and 104-bp species present at a n estimated 1-5% of total transcript levels. To show conclusively that the exon encoding the transmembrane domain had been spliced out, the 215-bp fragment was gel-purified, subcloned, and sequenced. As shown in Fig. 6C, the sequence contained the predicted splice junction of exon 8 (..AGTCTC), encoding Ig loop 6, immediately followed by exon 10 (..GGTTCG), which encodes the first exon of the cytoplasmic tail. Several clones of the 215-bp PCR products derived from HuvECs and from PMA-treated HEL cells contained the same sequence. Thus, an alternatively spliced RNA species which encodes the soluble form of PECA"1 is present in HU-VECs and HEL cells. PECAM-I Is Present in Normal Plasma-Detection of a soluble form of PECAM-1 in the culture media of HUVECs and myeloid cell lines prompted us to look for soluble PECA"1 in normal human plasma. Samples of plasma from several donors were ultracentrifuged and sterile-filtered to remove cell debris and then tested in an antigen-capture ELISA for the presence of PECAM-1. The assay was calibrated by a standard curve employing purified srPECAM-1, which indicated that PECA"1 could be detected at levels as low as 5 ng/ml (Fig.  7A). Screening of several plasmas (Fig. 7 B ) demonstrated that a soluble form or forms of PECA"1 were present at approximately 10-25 ng/ml. The specificity of detection was verified by solid phase absorption of the plasmas with antibodies specific for either PECA"1 or GPIIIa, as well as with control rabbit and mouse IgGs. Monoclonal as well as polyclonal anti-PECA"1 IgGs were able to specifically remove PECA"1 from the plasmas, while control rabbit and mouse IgGs, as well as a GPIIIa monoclonal antibody, did not reduce detection of PECA"1 in the plasma samples (Fig. 7B). Since GPIIIa is carried on platelets, leukocytes, and endothelial cells, cell types which also express PECAM-1, the results with the anti-GPIIIa monoclonal antibody indicate that the PECA"1 detected in plasma is not associated with cellular debris or platelet microparticles. Similar levels of PECA"1 were detected in plasma samples from 8 other normal individuals.
Analysis of Plasma PECAM-1-To assess the biochemical properties of human plasma PECA"1, several samples were analyzed by immunoprecipitation using polyclonal anti-PECAM-1 or ACT antibodies after 10-fold dilution of plasma and ultracentrifugation a t 100,000 x g for 3 h. The immunoprecipitated proteins were separated by SDS-PAGE, Westernblotted, and detected with an anti-PECAM-1 monoclonal antibody followed by peroxidase-labeled goat anti-mouse IgG and a chemiluminescent substrate. As shown in Fig. 8A, soluble PECA"1 from plasma samples is present as two distinct species with apparent molecular weights of 120,000 and 90,000, the latter being significantly smaller than the cell-associated form immunoprecipitated from a HUVEC lysate (cf: lane 2 with lanes 5 , 8, 11, and 13). The 120-kDa soluble isoform was recognized by the ACT antibody (lanes 6,9,12, and 141, indicating that the cytoplasmic tail is present; however, the lower molecular weight species was not recognized by ACT (compare lanes 5,  8, and 11 with lanes 6,9, and 12). To confirm that the 90-kDa band was indeed a form of PECA"1 lacking the cytoplasmic tail, a sequential absorption experiment was performed. An aliquot of plasma was incubated with ACT-Sepharose, the beads were removed, and the sample was then incubated with anti-PECAM-1-Sepharose. As shown in Fig. 8A, lanes 13-15, ACT bound only the 120-kDa species of soluble PECAM-1, but failed to immunoprecipitate the lower band, which continued to be specifically recognized by the antiPECAM-1 antibody. Therefore, it would appear that circulating plasma PECA"1 exists as two isoforms that differ by the presence of the cytoplasmic tail.
In order to determine the relationship between plasma PECAM-1 and that observed in cell culture media, immunoprecipitates from both sources were analyzed by Western blot analysis in the same gel. As shown in Fig. 8B, the 120-kDa isoform of SPECAM-1 is clearly the major form found in cell culture media (see Figs. 4, 5C, and 8B), although we occasionally observe traces of lower molecular mass products (Fig. 8B, second  lane from the left). In contrast, plasma PECA"1 is relatively evenly split between the 120-and 90-kDa isoforms. The results of immunoprecipitation of SPECAM-1 from both plasma and cell culture media with ACT (Fig. 8B, third and sixth lanes) indicate that only the 120-kDa form is derived by alternative splicing and thereby retains the cytoplasmic tail.
Soluble PECA"1 Can Block Adhesive Interactions of Membrane-bound PECAM-I-Recent studies have shown that PECA"1 mediates heterophilic, Ca2+-dependent aggregation of transfected mouse L-cells (9,10) and plays a key role in leukocyte transmigration through endothelial cell monolayers, both in cell culture (15) and in the intact vessel wall (16). To determine whether soluble forms of PECA"1 similar to those in plasma might retain adhesive function and potentially modulate this process, we prepared large amounts of soluble recombinant PECA"1 (see Fig. 5 , panels A and B ) , which corresponds in size and composition to the 90-kDa form found in cell culture media and in plasma. Using the L-cell aggregation assay as a measure of functional activity (Fig. 91, we found that soluble PECA"1 at a concentration of 10 pg/ml was able to inhibit PECAM-1-specific cellular interactions almost completely, with moderate inhibition of intermediate concentrations. Thus, it would appear that soluble PECA"1 is functional and may potentially be involved in modulation of the inflammatory process in vivo if high local concentrations of these PECA"1 isoforms were present. DISCUSSION PECA"1 is widely distributed on cells of the vascular system, as it is found on endothelial cells, platelets, neutrophils, monocytes, and some T-cell subsets. The protein localizes to the intercellular junctions of endothelial cells and transfected monolayer cells. In addition, PECA"1 seems to serve as a "trigger" molecule in activating integrins on circulating leukocytes and may also be involved in the transendothelial migration of leukocytes during the inflammatory process. It is intriguing that the synthesis of PECA"1 is so tightly regulated, as it is not found outside the vasculature. Little is known about the biochemical events involved in PECA"1 synthesis and processing in those cell types that express it which may affect its subsequent functional activities. Like other membrane glycoproteins, PECAM-1 has been shown to be synthesized as a smaller molecular weight precursor molecule that is further processed into a mature form (8). The kinetics of intracellular trafficking and turnover of PECA"1 on the cell surface, however, are important parameters that have not yet been examined. Therefore, pulse-chase labeling of U937 cells and HUVECs was used to further characterize the synthesis and processing of PECAM-1. As shown in Figs. 1 and 2, pre-PECAM-1 could be detected in as little as 20 min following the addition of radiolabeled methionine, and mature PECA"1 was expressed on the cell surface within 3 h.
The newly synthesized protein was found to be only moderately stable, disappearing from the cell surface within 2-3 days and

Antibody Used for lmmunoab~~rption
from intracellular pools with approximately the same kinetics however, that the rate of PECAM-1 synthesis and decay is ( Figs. 1 and 2). slower in intact vessels, where endothelial cells are known to regulation in HUVECs was similar to that observed in U937 Analysis of the glycosylation pattern of PECAM-1 indicated cells, which seems to indicate that the homotypic interactions that the precursor form of PECAM-1 contains a number of high of PECAM-1 at endothelial cell junctions do not stabilize the mannose carbohydrate residues, as demonstrated by its susturnover rate of the protein. We cannot rule out the possibility, ceptibility to endo H digestion (Fig. 3). It would appear that all Interestingly, the kinetics of PECAM-1 synthesis and down-divide as slowly as once every 2 years. media PECA"1. Samples of HUVEC media represent 12 ml of culture media which were processed a s described under "Experimental Procedures." Note that the 120-kDa soluble PECA"1 isoforms derived from both cell culture media and normal human plasma contain the cytoplasmic tail, as evidenced by reactivity with ACT. of these are modified in the Golgi to complex carbohydrate residues, as the mature form of PECAM-1, while sensitive to N-glycanase and neuraminidase, was no longer affected by endo H. Treatment of either form of PECA"1 with N-glycanase led to a decrease in apparent molecular weight to about 100,000. The predicted size of the mature polypeptide chain, however, is about 80 kDa (l), suggesting that the remaining 20 kDa may be composed of 0-linked carbohydrate moieties. Treatment with 0-glycanase, however, did not cause a shift in the mobility of PECAM-1. It is possible that the enzyme failed to cleave due to the presence of fucosylated residues at the nonreducing terminal polylactosamine moiety of the 0-linked carbohydrate (39). Alternatively, PECA"1 may simply migrate anomalously on SDS-PAGE gels, as chemical deglycosylation of PECAM-1 also did not increase the mobility of the protein any more than was observed with N-glycanase. The sites of glycosylation and the precise structures of the carbohydrate moieties on PECAM-1 remain to be elucidated.
PECAM-1 joins a number of adhesion proteins existing in both membrane-bound and soluble forms, among which are P-selectin f36), Lselectin (33,. and ICAM-I (34, 3 5 ) . Soluble PECAM-1 in cell culture media contains the c-ytoplasmic tail and appears to be encoded by an alternatively spliced mRNA species from which the exon encoding the transmembrane domain has been removed. The loss of the amino acids encoded by exon 9 would predict the loss of about 4 kDa from the molecular mass of mature PECAM-1 and appears to correspond to the size of SPECAM-1 that we found to be secreted by U937 cells and H w F C s (Figs. 4 and 5). Both the transcript for SPECAM-1 and the resulting soluble protein are significantly less abundant than full-length membrane-associated PECAM-1.
Discovery of a soluble form of PECAM-1 in culture media prompted us to look for soluble PECAM-1 in plasma, and the results indicate that PECAM-1 is present in normal human plasma at levels of 10-25 nglml. Although these levels are low, continuous production of soluble PECA"1 from the minor transmembraneless PECAM-1 mRNA species by the 5,000 square meters of endothelial cells that are present in the body could easily account for the concentration detected. Interestingly, two isoforms of PECAM-1, which migrated a t 120 kDa and 90 kDa on SDS-PAGE, were observed in plasma. The-120 kDa form appears to correspond to the major soluble PECAM-1 observed in HUVEC culture media and retains the c-ytoplasmic tail, while the 90-kDa form does not carry the c-ytoplasmic tail and appears to correspond to srPECAM-1 which is truncated at the transmembrane domain. It is not known whether 90-kDa soluble PECAM-1 is specifically cleaved from the cell surface or results from proteolytic cleavage of the 120-kDa form following its secretion from the cell.
Although the role of plasma PECAM-1 in c,ioo remains to be determined, we have shown that soluble PECAM-I truncated at the transmembrane domain can inhibit the heterotypic adhesive interactions mediated by cell-surface PECA..-1 expressed on transfected L-cells. The recent studies of Muller et al. (15), which showed that PECAM-1 is required for transendothelial migration of leukocytes, presumably mediated through homotypic interactions between PECAM-I on the leukocytes and endothelial cells, are also relevant. These authors were able to inhibit transendothelial migration of leukoc-ytes with soluble recombinant PECAM-1 which is truncated within the sixth Ig domain. Thus, the presence of low levels of circulating PECA"1 may help to counteract the tendency of leukocytes to leave the vasculature under normal conditions, whereas higher circulating levels present during inflammatory conditions, if found to exist, might be involved in limiting further transmigration as part of a negative feedback loop. The potential role of the cytoplasmic tail in modifying the functions of the two isoforms of soluble PECAM-1 is also of interest.
The widespread presence of PECAM-1 in the vascular system predicts that PECA"1 has important functions in each of the cell types in which it is expressed. We have elucidated some of the events involved in the biosynthesis and processing of PECAM-1, including the synthesis of a soluble form. Further characterization of the cellular and molecular properties of PECAM-1 in each of these cells types should aid in our understanding of its role in inflammation and the immune response.