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Soluble L-selectin is present in human plasma at high levels and retains functional activity

L-selectin expressed by granulocytes, lymphocytes, and monocytes is responsible for initial leukocyte attachment to inflamed endothelium and high endothelial venules of peripheral lymph nodes. After leukocyte activation in vitro, L-selectin is rapidly shed from the cell surface. In this study, shed L-selectin (sL-selectin) from both lymphocytes and neutrophils was demonstrated to be present in high levels in human plasma by Western blot analysis and using a quantitative ELISA. In serum from normal human blood donors, a mean sL-selectin level of 1.6 +/- 0.8 micrograms/ml (n = 63) was found by ELISA. In addition, semipurified sL-selectin from plasma inhibited L-selectin-specific attachment of lymphocytes to cytokine-activated endothelium in a dose- dependent manner. L-selectin-dependent leukocyte attachment was completely inhibited at sL-selectin concentrations of 8-15 micrograms/ml, while physiological concentrations of sL-selectin caused a small but consistent inhibition of lymphocyte attachment. sL-selectin in plasma also inhibited anti-L-selectin mAb (2-5 micrograms/ml) binding to the surface of leukocytes. Interestingly, one epitope present within the EGF-like domain of L-selectin was lost in sL- selectin, suggesting a conformational change in the structure of the receptor after shedding. The presence of serum sL-selectin with functional activity indicates a potential role for sL-selectin in the regulation of leukocyte attachment to endothelium.

phocytes, and monocytes is responsible for initial leukocyte attachment to inflamed endothelium and high endothelial venules of peripheral lymph nodes. After leukocyte activation in vitro, L-selectin is rapidly shed from the cell surface. In this study, shed L-selectin (sL-selectin) from both lymphocytes and neutrophils was demonstrated to be present in high levels in human plasma by Western blot analysis and using a quantitative ELISA. In serum from normal human blood donors, a mean sL-selectin level of 1.6 ___ 0.8 #g/ml (n = 63) was found by ELISA. In addition, semipurified sL-selectin from plasma inhibited L-selectinspecific attachment of lymphocytes to cytokine-activated endothelium in a dose-dependent manner. L-selectin-dependent leukocyte attachment was completely inhibited at sL-selectin concentrations of 8-15 /zg/ml, while physiological concentrations of sLselectin caused a small but consistent inhibition of lymphocyte attachment, sL-selectin in plasma also inhibited anti-L-selectin mAb (2-5 #g/ml) binding to the surface of leukocytes. Interestingly, one epitope present within the EGF-like domain of L-selectin was lost in sL-selectin, suggesting a conformational change in the structure of the receptor after shedding. The presence of serum sL-selectin with functional activity indicates a potential role for sL-selectin in the regulation of leukocyte attachment to endothelium.
L-selectin is expressed on the surface of most leukocytes, including lymphocytes, neutrophils, monocytes, eosinophils, Olivier Spertini's present address is Division of Hematology, University Hospital, CHUV, Lausanne, Switzerland.
A unique feature of L-selectin is that it is shed from the cell surface after cellular activation in vitro (14,19,22,23,40). It has been proposed, at least in the mouse model, that shedding of MEL-14 from leukocytes might be necessary to enable leukocytes to transmigrate through endothelium into sites of inflammation in vivo (20,22). This would provide a rapid means for the regulation of leukocyte adhesion and deadhesion to endothelium. Although the subsequent fate and possible function of the shed L-selectin (sL-selectin) molecule is not known, elegant studies by Woodruff and colleagues have previously demonstrated the presence of a soluble factor in rat thoracic duct lymph capable of inhibiting lymphocyte binding to HEV (4). Furthermore, they demonstrated that this factor was antigenically related to a structure(s) present on lymphocytes and speculated that this cell-surface molecule might play a role in adherence of lymphocytes to HEV of lymph nodes (4,5,6).
A number of surface molecules present on cells of various lineages are now known to be shed and thereby released into the extracellular milieu (48). These include the receptors for interleukin-1, intedeukin-2 (CD25), transferrin (CD71), insulin, growth hormone, tumor necrosis factor (34), colonystimulation factor-1 (10), and nerve growth factor (8), as well as CD8 and CD14. These proteins are quite diverse in structure and amino acid sequence and have no unifying functional characteristics that are currently appreciated. In most cases, proteases cleave the receptor near the membrane, releasing a nearly intact extracellular domain with ligandbinding activity (8,10,22,40). Thus, receptor function may not only be regulated by proteolytic cleavage of the receptor from the cell surface, but also by the presence of shed receptor in the extracellular environment. The present study shows that sL-selectin from neutrophils and lymphocytes is found in plasma at high levels and that it may retain functional activity. Since sL-selectin inhibited L-selectin-mediated adhesion in vitro, it may modulate leukocyte binding to endothelium in vivo.

Materials and Methods
Antibodies L-selectin directed mAbs were the anti-LAM1-3, -4, -6, -7, -8, -10, -11, and -12 mAbs directed against epitopes within the lectin domain, anti-LAMl-1, -5 and -15 reactive with epitopes within the EGF-like domain and anti-LAMl-14 which reacts with the SCR regions of L-selectin, all of the IgGl isotype (42). The anti-L-selectin mAbs were purified by salt fractionation followed by anion exchange chromatography, with the mAb concentration determined by light absorption. The anti-LAM1-3 mAb was bound to CNBr-activated Sepharose 4B (Pharmacia LKB Biotechnology Inc., Piscaraway, N J) at 2.5 nag of mAb bound per 1 ml of beads (anti-LAM-Sepbarose) using the methods of the manufacturer.

Isolation of Blood Mononuclear Cells
Heparinized blood was obtained according to protocols approved by the Human Protection Committee of Dana-Farber Cancer Institute, Boston, MA. Mononuclear cells were isolated by Ficoll Hypaque density gradient centrifugation. Cells were immediately resuspended in RPMI 1640 (Gibco-BRL, Gaithersburg, MD) containing 10% FCS and kept at 4~ until use. In most instances, these cells will be referred to as lymphocytes since 85-95 % of the cell population was lymphocytes as determined by morphology (Wright's stain) and flow cytometry analysis. Neutrophils were purified by centrifugation on a cushion of Mono-Poly Resolving Medium (Flow Laboratories, Inc., McLean, VA) followed by lysis of the red cells with icecold hypotonic 0.2% (wt/vol) NaCI solution. Neutrophils were finally resuspended in HBSS containing 5 % FCS (Sigma Chemical Co., St. Louis, MO). When sL-selectin was obtained for Western blot analysis, the mononuclear cells were first incubated in plastic dishes for 30 min in RPMI/10% FCS at 37~ to remove adherent monocytes. Nonadherent cells retained surface L-selectin and were predominantly lymphocytes (•99 %).

Indirect Immunofluorescence Analysis
Indirect immunofluorescence analysis was carried out after washing the cells three times. Cells (1 x 106 cells) were resuspended in 100/zl of media containing various concentrations of the indicated mAb, and incubated for 60 rain at 4"C. After washing, the cells were treated with FITCconjugated goat anti-mouse Ig antibodies (Southern Biotechnology Associates, Birmingham, AL) for 20 min at 4~ The cells were washed, and fixed (1% paraformaldehyde in PBS), and single color fluorescence was determined on a flow cytometer (ELITEr'; Coulter Immunology, Hialeah, FL). 10,000 cells were analyzed for each sample and the relative mean fluorescence intensity of L-selectin + cells was determined on a linear scale.
In some experiments, cells were stained in the presence of human plasma, sL-selectin was precleared from an aliquot of the same plasma by immunoprecipitation with anti-LAM-Sepharose (I ml of beads per 4 ml of plasma). The efficiency of the immunoprecipitations was tested by ELISA (<20 ng/ml). Lymphocytes (I x 106) were resuspended either in 100/zl of plasma or precleared plasma containing various concentrations of the purified mAb (added 1:100) as indicated. The cells were washed twice, stained, and analyzed as above.

sL-selectin Purification
sL-selectin was semipurified from plasma obtained from heparinized human blood. Plasma was salt-fractionated with Na2SO4 (18% wt/vol) before the sL-selectin containing supernatant fraction was dialyzed against 0.02 M Tris buffer, pH 8.0, 0.5 M NaCI. The sL-selectin preparation was further purified by affinity column chromatography using anfi-LAM-Sepharose. sL-selectin was eluted from the column with 0.1 M Na a~etate buffer, pH 3.5, 0.15 M NaC1, and the low pH of the eluate was immediately raised by the addition of 2.0 M Tris buffer, pH 9.0. The pooled fractions of the eluate peak were concentrated and transferred into PBS by ultrafiltration. The concentration of sL-selectin was determined by ELISA and subsequently adjusted to ,~15 #g/ml in PBS. At this sL-selectin concentration, the total protein concentration of the samples varied between 130 and 220/~g/ml, and sL-selectin represented "~6-10 % of total protein. In general, this procedure gave a 2,200-3,700-fold enrichment for sL-selectin. For use in lymphocyteendothelial adhesion assays, semipurified sL-selectin was transferred into RPMI 1640/10% FCS by further ultraliltration.

Endothelial-Leukocyte Attachment Assay
Lymphocyte adhesion to cytoldne-activated endothelium under nonstatic conditions was determined in a test system adapted from the Stamper/ Woodruff assay for frozen tissue sections (46) exactly as described (43). Briefly, human umbilical vein endothelial cells (HUVEC) were isolated from cord veins, and grown in M199 medium supplemented with 10% FCS, endothelial cell growth factor (50 t~g/ral, Biomedical Technologies, Inc., Stoughton, MA) and porcine intestinal beparin (50 mg/ml; Sigma Chemical Co.) as described (43). Endothelial cells were grown to confluence on gelatin (0.1%)-coated glass slides and stimulated with TNF-a (100 U/ml) at 37~ for the times indicated. The monolayers were carefully washed and incubated at 4~ for 15 rain with 75/zl of RPMI/10% FCS alone or containing semipurified sL-selectin. As a control, in some instances media containing semipurified sL-selectin were precleared by immunoprecipitation with anti-LAM-Sepharose. Without further washing, 5 • 106 lymphoeytes in 75 t21 of the respective media were added. After 20 rain of incubation at 4~ with rotation at 64 rpm, the slides were fixed overnight in glutaraldehyde (1% [vol/vol] in PBS; Polysciences, Warrington, PA), and stained with hematoxylin. The number of adherent leukooytes was determined by counting six microscopic fields (0.09 mm2/field) and the results were expressed as means + SD.

Production of the L-selectin and IgG Chimera cDNA and Protein
Generation of the L-selectin and IgG chimeric cDNA will be described in detail elsewhere (44a). Basically, the 1400-bp BanlI fragment from a eDNA encoding the CH1 through CH3 domains of the human IgGl constant region was inserted at a BanlI site introduced into pLAM-1 cDNA (49) (amino acid number 370 in the membrane proximal region of the mature protein) by oligonucleotide directed mutngenesis. The recombinant DNA was sequenced and the conservation of L-selectin and IgG~ restriction sites in the pLAM-IgG DNA was contirmed by restriction mapping. The LAM-IgG DNA was subeloned into the AprM8 expression vector (provided by Dr. Lloyd Klickstein, Center for Blood Research, Boston, MA) and used to transiently transfect COS cells by the DEAE dextran method. The transfected COS cells were cultured in AIM-V serum-free media (Gibeo-BRL) and the supernatant fluid containing the chimeric LAM-IgG fusion protein was harvested after 3 d.

sL-selectin ELISA
The ELISA used to quantitate sL-selectin levels in biological fluids will be described in detail elsewhere (44a). Briefly, wells of microtiter plates (96 welt, flat bottom, E.I.A./R.I.A. plate, Costar, Cambridge, MA) were coated with anti-L-selectin rnAb as indicated. After washing with TBS, the wells were blocked with 2% BSA and 1% gelatin in TBS. The wells were washed, and the test samples were added to triplicate wells. Each assay included the titration of a previously quantified plasma sample that was used to generate a standard curve. After washing, the plates were incubated with biotinylated anti-LAM1-3 mAb (1 /~g/ml) for 60 rain at 20~ The wells were washed, and avidin-HRP (0.1/zg/ml, Pierce Chemical Co., Rockford, IL) was added for 30 rain at 20~ Again, the plates were washed and finally developed using o-phenylenediamine (0.125% wt/vol, Sigma Chemical Co.) as a substrate in 0.1 M citrate buffer, pH 4.5, in the presence of H202. The OD of the reaction mixture was quantitated using an ELISA-reader (Vmax kinetic microplate reader; Molecular Devices, Menlo Park, CA). Results were obtained when the OD for the well containing the highest concentration of standard plasma was ,00.8 at 495 nm. The relative concentralion of sL-selectin in individual samples was calculated by comparing the mean OD obtained for triplicate wells with a semilog standard curve of titrated plasma using linear regression analysis (r ~ 0.97).
The amount of sL-selectin present in the standard plasma was quantitated in two ways. First, K562-LAM-1 cells ('011 liters of cultured ceils, "01.1 x 10 l~ cells) were resuspended in PBS (1 x 107 cells/ml) and were stimulated with PMA (100 ng/ml) for 2 h at 37~ The supernatant fluid was collected, concentrated by ultrafiltration, and affinity purified by column chromatography using anti-LAM-Sepharose as described above. The semipurified sample was electrophoresed on a 10% SDS-polyacrylamide gel that was subsequently stained by Coomassie blue to reveal a prominent 71-kD band and additional bands of eul80, 57, 47, and 22 kD. The gel was scanned using a Hewlett Packard Desk Scanner and the density of the 7 l-kD band was quantitated against a standard curve generated with BSA using the Enlmnce TM program (Microsystems, Des Moines, IA) on an Apple Macintosh Ilcx computer. The concentration of sL-selectin in the standard plasma was calculated to be "01.3 t~g/ml by comparing the signal from semipurified sL-selectin to the L-selectin-ELISA titration curve of standard plasma with linear regression analysis. The detection limit of the L-selectin-ELISA was determined to be ;~5 ng/ml. In a second set of experiments, COS cells were grown in serum-free medium after transient transfection with the LAM-IgG chimera eDNA. Supernatant fluid was collected from the cells and run over a Protein A-Sepharose (Pharmacia LKB Biotechnology) affinity chromatography column and the fusion protein was duted from the column by high salt-tow pH buffer. The purified fusion protein was quantitated after SDS-PAGE analysis by comparison of the stained protein band with a standard curve of BSA. From this analysis, it appeared that OD values for standard plasma would be equivalent to "01.9 t~g/ml of LAM-IgG fusion protein.
Since the dimeric nature of the fusion protein might double the intensity of staining in our sandwich ELISA, the amount of sL-setectin in the standard plasma may be half the value of ,ol.9/~g/mt.

Statistics
Statistical analysis used the paired or unpaired t test appropriately.

Detection of sL-selectin in Plasma by Western Blot Analysis
In vitro, leukocytes and L-selectin cDNA-transfected cells shed L-selectin from the cell surface that can be detected in the culture supernatant fluid. To determine whether this process also occurs in vivo, the anti-LAM1-3 mAb, which identifies an epitope within the lectin domain, was used to immunoprecipitate reactive materials from normal human plasma. The precipitated materials were then analyzed by SDS-PAGE, transferred to nitrocellulose, and the presence of a soluble form of L-selectin was visualized by Western blot analysis using the anti-LAMl-14 mAh that hinds to a L-selectin epitope located within the SCR region. Two predominant isoforms of sL-selectin were observed, a '~62-kD isoform and a 75-100-kD isoform (Fig. 1). Analysis of the culture supernatant fluid from PMA-stirnulated lymphocytes, neutrophils, and K562 cells transfected with L-selectin eDNA (K562-LAM) revealed different, cell-specific isoforms of sL-selectin (Fig. 1). Neutrophils shed a 75-100-kD isoform of sL-selectin that traveled as a broad band with an ill-defined upper border most likely due to heavy glycosylation (33). Lymphocytes generated a 62-kD isoform and K562-LAM cells shed a 71-kD isoform of sL-selectin. No specific proteins were isolated from the supernatant fluid of untransfected K562 cells using the above assays, and preclearing the plasma or supernatant fluid with the anti-LAM1-3 mAb eliminated subsequent Western blot results (data not shown). Therefore, it is likely that the two predominant isoforms of sL-selectin identified in plasma derived from both lymphocytes and neutrophils. Furthermore, sL-selectin in serum contained the lectin, EGF, and SCR domains as it was visualized in these experiments using mAb reactive with the lec- Figure 1. Different isoforms of sL-selectin detected by Western blot analysis. Plasma from a normal donor was salt fractionated, purified by affinity chromatography, and immunoprecipitation using anti-LAM1-3 mAb bound to Sepharose beads. Lymphocytes, neutrophils, and K562-LAM cells (1 x 107/ml) were incubated with PMA, and the culture supernatant fluids were isolated and concentrated tenfold by ultratiltration. The harvested materials were analyzed by SDS-PAGE (7.5%), blotted onto nitrocellulose, and stained with the anti-LAMI-14 mAb for Western blot analysis.
tin (anti-LAM1-3) and SCR (anti-LAMl-14) domains, but the relative molecular mass of sL-selectin is smaller than that for L-selectin isolated from detergent solubilized cells as previously described (27,40).

Quantitation of sL-selectin in Plasma and Serum by ELISA
The amount of sL-selectin found in human plasma was quantitated with a sandwich ELISA using the anti-LAM1-5 mAb as a capture antibody and biotinylated anti-LAM1-3 mAb as a detecting antibody. This specific combination of mAb provided the highest level of sensitivity so that "~5 ng of sLselectin could be easily detected. The level of sL-selectin determined by ELISA in the sera of a population of healthy normal blood donors was found to be 1.6 5:0.8 #g/ml, n = 63 (Fig. 2). In some donors, sL-selectin levels were simultaneously quantitated in plasma and serum with similar results obtained (1.9 5:1.0 #g/ml, n = 18). It is unlikely that sLselectin in plasma or serum might still be membrane bound, as the same levels of sL-selectin were found in serum and plasma before and after ultracentrifugation, sL-selectin was stable in whole blood left to stand at 20~ before separation of serum or plasma for at least 24 h. Storage of serum or plasma at 4~ for up to three months in the presence of azide or repeated thawing and freezing (up to 10 times) did not affect the ability to detect sL-selectin in serum. In conclusion, it appears that sL-selectin is stable and present at relatively high levels in human plasma and serum.

Quantitation of sL-selectin in Supernatants of Activated Leukocytes
Stimulation or the culturing of leukocytes has been associated with shedding of L-selectin from the cell surface. fluid obtained from untransfected cells cultured at the same density (Fig. 3 a). Similarly, medium from freshly isolated neutrophils cultured at 37~ for 60 min also contained detectable sL-selectin (Fig. 3 b). Incubation of the neutrophils with stimuli that do not affect cell surface L-selectin expression, granulocyte-colony stimulating factor, interleukin 1, monocyte-colony stimulating factor, interleukin 6, interferon % and interleuldn 4, did not induce an increase in sL-selectin in these experiments. However, stimulation with formylated methionine-leucine-phenylalanine, lipopolysaccharide, granulocyte/monocyte-colony stimulating factor, interleukin 8, and tumor necrosis factor (TNF) induced L-selectin shedding corresponding to their potency to stimulate cell surface shedding (Fig. 3 b and data not shown). Culturing lymphocytes for 60 rain at 37~ caused some shedding of L-selectin, which was greatly enhanced by PMA treatment (Fig. 3 c).
In similar experiments, quantitation of the amount of sLselectin found in the supernatant fluid after activation of neutrophils and lymphocytes (1 x l07 cells/ml) varied between ~10-30 ng/ml. However, PMA-activation in PBS oflymphocytes for >25 min, and neutrophils for >10 min, resulted in the gradual degradation of sL-selectin. Degradation was not observed in the supernatant from K562-LAM cells, where sL-selectin was consistently found at concentrations of 22 5= 6 ng/ml (1.7 5: 0.5% of standard plasma; n = 14). In experiments not shown, elevated levels of sL-selectin were also detected in the culture supernatant fluid of lymphocytes cultured with phytohemagglutinin, concanavalin A, and pokeweed mitogen for 3-6 d at 37~ These mitogens are known to cause cell surface loss of L-selectin (51). Thus, loss of L-selectin from the cell surface directly correlates with an increase of sL-selectin in the culture medium.

Inhibition of Lymphocyte Binding to Activated Endothelium by sL-selectin
sL-selectin was semipurified from plasma by salt fractionation followed by affinity chromatography with the anti-LAM1-3 mAb. The column eluate was concentrated, and the level of sL-selectin present was quantitated by ELISA and adjusted to ~15 t~g/ml (6-10% of total eluate protein), sLselectin, at different concentrations, in RPMI 1640/10% FCS was incubated (15 rain, 4~ with cytokine-activated endothelial cells before examining lymphocyte attachment to endothelium through L-selectin. While sL-selectin at physiological concentrations caused only partial inhibition of lymphocyte attachment (32.1 + 15.9%, n = 10), it was found to inhibit most L-selectin-dependent lymphocyte binding at concentrations of 8/~g/ml (52-100 %) and caused almost total inhibition of L-selectin-mediated binding at 12-15 #g/ml (93-100%; Fig. 4 depicts a typical dose response curve). Lymphocyte binding mediated by L-selectin was calculated as the difference between total binding at any given concentration of sL-selectin and lymphocyte binding found in the presence of anti-LAM1-3 mAb, which blocks all L-selectin mediated adhesion (43). Importantly, sLselectin at 15 pg/ml caused no additional inhibition of lymphocyte binding beyond that obtained with anti-LAM1-3 mAb alone. When the sL-selectin samples were precleared with the anti-LAMl-15 mAb (which does not block L-selectin adhesion) before the assays, the preparation was not able to inhibit lymphocyte attachment, demonstrating that sL-  selectin mediated the inhibition (Fig. 4). Thus, it appears that sL-selectin is capable of inhibiting L-selectin-specific lymphocyte adhesion to endothelium by binding to its putative endothelial ligand. The inhibitory capacity of sL-selectin at normal physiological concentrations (1.5 #g/ml) was further evaluated to determine if it could alter the interaction of lymphocytes with endothelium. Endothelium was activated with TNF (100 U/ml) for different periods of time to induce the L-selectin ligand in a manner similar to what might happen during the initiation of an inflammatory response. During the course of induction of the L-selectin ligand, sL-selectin treatment of the endothelium caused a consistent, but small, inhibition of L-selectin-dependent lymphocyte attachment (30% at 2 h, 43 % at 3 h, 41% at 4 h, 15 % at 5 h, and 2 % at 6 h) (Fig. 5). Again, the combination of sL-selectin with the anti-LAM1-3 mAb did not cause a greater inhibition than that observed with the mAb alone. Thus, during the development of an inflammatory response, it is likely that sL-selectin will be able to alter the course of leukocyte attachment, although the influence appears to be small in this in vitro assay.

sL-selectin in Serum Blocks Anti-L-selectin mAb Binding
The presence of circulating sL-selectin was further verified by demonstrating that the reactivity of anti-L-selectin mAb with L-selectin § ceils could be inhibited by plasma. When lymphocytes were stained using anti-L-selectin mAb in undiluted human plasma, no significant staining was obtained at concentrations of mAb that were saturating in RPMI/FCS (data not shown). Next, lymphocytes (1 • 106/100 #1) were incubated with various concentrations of L-selectin-directed mAb diluted (1:100) in autologous plasma or autologous plasma which had been precleared of sL-selectin by immunoprecipitation. After completion of indirect immunofluorescence staining, antibody binding was assessed by flow cytometry. In most cases, the presence of plasma inhibited anti-L-selectin mAb staining when the mAbs were used at 2 to 5 #g/ml (Fig. 6). Thus, ,x,13-33 pM IgG~ (150 kD) bound to "~21 pM sL-selectin (75 kD) based on the concentration of sL-selectin present in plasma (1.6/zg/ml) determined as outlined above, sL-selectin-dependent inhibition of mAb binding was seen in plasma for mAb which recognize the lectin domain (anti-LAM1-3, -4, and -10) and the EGFlike domain (anti-LAM1-5, and -15). In contrast, binding of the anti-LAMl-1 mAb, which binds an epitope in the EGFlike domain, was not significantly inhibited by the presence of plasma (Fig. 6 D).

The LAMI-1 Epitope was not Detected on sL-selectin from Human Plasma
Since anti-LAMl-1 mAb binding to lymphocytes was not inhibited by plasma, the epitope identified by this mAb may not be present on sL-selectin. To examine this further, ELISA were performed in which different L-selectin-directed mAbs were bound to an ELISA plate as capture antibodies with the anti-LAM1-3 mAb as the detecting antibody. Lectin domain-specific antibodies (anti-LAM1-6, -7, -10, and -11) and EGF-like domain specific mAb (anti-LAM1-5 and -15) gave strong, easily detectable positive signals (Fig. 7). Wells coated with the anti-LAM1-3 and -4 mAb, which cross-block the binding of the detecting antibody (anti-LAM1-3), served as internal controls for background reactivity. In contrast to all other mAb, anti-LAMl-1 gave a signal that was not significantly different from background, as defined by using BSA as the capture reagent. Thus, the epitope located within the EGF-like region which is specifically recognized by the anti-LAMl-1 mAb appears to be lost from L-selectin upon shedding. As L-selectin was easily recognized by all L-selectin-specific mAb in immunofluorescence staining of lymphocytes and all three extracellular domains are preserved on sL-selectin found in plasma, conformational changes in the L-selectin protein may lead to the loss of the EGF-like domain related LAMI-1 epitope.
To determine whether the transmembrane or cytoplasmic regions of L-selectin are necessary to uphold the complete tertiary structure of its extraceUular domains, a second form of soluble L-selectin, a LAM-IgG fusion protein was generated and epitope-mapped by ELISA. The chimeric eDNA used to generate the fusion protein was constructed so that it contained essentially the entire extracellular region of L-selectin. Culture supernatant fluid of COS cells that were transiently transfected with the chimeric eDNA were tested for the production of LAM-IgG by ELISA. The anti-LAM1-5, -7, and -15 mAb, which detected sL-selectin in plasma and in supernatant fluid from PMA-stimulated K562-LAM cells, also bound the LAM-IgG chimera at similar levels (Fig. 8).
In contrast, while the anti-LAMl-1 mAb failed to generate a significant signal with sL-selectin from plasma and K562-LAM transfectants, it readily bound the LAM-IgG fusion protein (Fig. 8). Thus, it is likely that sL-selectin generated in vivo or in vitro loses a conformational determinant that is required for anti-LAMl-1 mAb binding.

Discussion
The presence of different isoforms of intact sL-selectin in plasma and serum was revealed by Western blot analysis (Fig. 1). sL-selectin in serum appeared to be derived from both lymphocytes and neutrophils since the analysis of sLselectin isolated from neutrophils (95-105 kD) and lymphocytes (74 kd) produced bands of corresponding relative molecular mass. The differences in relative molecular mass between lymphocyte and neutrophil sL-selectin most likely result from differences in glycosylation of a single protein species since only a single L-selectin mRNA species has been identified (33), and K562 cells transfected with the pLAM-1 eDNA that is complementary to this mRNA also produced a different isoform of sL-selectin. Cleavage of L-selectin is likely to be proximal to the membrane spanning region encoded by exon VHI; cleavage in this region would account for the small difference in relative molecular mass between the intact cellular L-selectin molecule and its shed form (23,28,40). This is supported by the extraordinarily high degree of conservation of exon VIII between human L-selectin and the corresponding region of mouse L-selectin (9,33). In addition, anti-L-selectin mAb binding studies demonstrated that the lectin, EGF-like, and SCR domains are retained by sL-selectin ( Figs. 1 and 7). It is therefore possible that the factor in rat lymph characterized by Chin and Woodruff as being capable of inhibiting lymphocyte attachment to HEV was in part sL-selectin (4)(5)(6).
While the direct demonstration of sL-selectin in plasma and supernatant fluid from activated neutrophils and lymphocytes confirms that the in vivo and in vitro loss of L-selectin surface expression is due to shedding, development of the sL-selectin-ELISA allowed for the first time an easy, efficient, and sensitive method for quantification of sL-selectin. Remarkably, in a population of normal healthy blood donors the mean serum level of sL-selectin was determined to be 1.6 • 0.8/~g/ml. The concentration of sL-selectin was determined by comparison with a reference plasma sample that had been standardized by two independent means, and ranged between 0.85 and 1.9 #g/ml. The mean of this range of values of 1.3/~g/ml was therefore chosen as the basis of all calculations. Experimental data derived from shedding experiments in K562-LAM transfectants further substantiate the validity of this estimate (44a). K562-LAM cells express 30,000-43,000 L-selectin molecules on each cell as determined by Scatchard analysis. After PMA stimulation of these cells, all L-selectin molecules (71 kD) are shed. Thereby, the concentration of sL-selectin in plasma can be calculated by comparing the concentration of sL-selectin in medium (13% the concentration of standard plasma) shed from K562 transfectants (1 x 107 cells/ml) to a titration curve of standard plasma. According to this calculation, plasma concentrations would lie in the range of 2.1-3.0 #g/ml. Similar considerations make it highly unlikely that the amount of sL-selectin found in serum or plasma is generated from the shedding of cell surface L-selectin by blood leukocytes present during the preparation of the samples: the quantity of sL-selectin actually found in serum is 10-25-fold higher than the quantity of L-selectin that could maximally be shed from the cell surface of leukocytes (~4 x 10~/ml) present in the samples, since most leukocytes express 50,000-100,000 receptors/cell as determined by Scatchard analysis (44a). Therefore, sL-selectin must be generated by the ongoing shedding of cell surface L-selectin from leukocytes in vivo.
Another adhesion molecule, ICAM-1, was recently shown to circulate in plasma at a concentration of 0.16 #g/ml (35), a concentration 10-fold lower than that found for sL-selectin. Functional activity of soluble ICAM-1, however, could only be demonstrated when it was immobilized to plastic (35). In contrast, sL-selectin semipurified from plasma inhibited L-selectin-mediated lymphocyte attachment at suboptimal levels of ligand expression on TNF-activated endothelium in a dose-dependent manner with complete inhibition at ,x,8-15 #g/ml (Fig. 4). At concentrations of sL-selectin similar to those found in the plasma of normal blood donors, a small but significant (p < 0.005) inhibition of leukocyte endothelial binding was observed at various levels of ligand expression (Fig. 5). Similarly, using a soluble fusion protein composed of the Fe portion of human IgG and the extracellular domains of MEL-14 at 1 t~g per tissue section, Vvhtson et al. (53) have found •75 % inhibition of lymphocyte binding to HEV. Preliminary studies suggest that circulating sL-selectin may be functional in vivo since immunohistochemical staining of tissues with anti-L-selectin mAb revealed sL-selectin specifically bound to the luminal surface of endothelial cells, both on HEV endothelium and at sites of inflammation (Munro, M., B. Schleiffenbaum, and T. E Tedder, manuscript in preparation). This is not unexpected, as the MEL-14-IgG fusion protein also binds to HEV in vitro (53).
High levels of relatively low avidity sL-selectin circulating in plasma might serve as a biological adhesion buffer system to prevent the attachment of leukocytes at sites where low level upregulation of the L-selectin ligand has occurred on endothelial cells, similar to the function proposed for soluble P-selectin (12). This hypothesis is in keeping with the small but consistent inhibitory effect of physiological concentrations of sL-selectin on L-selectin specific lymphocyte attachment to HUVEC during the course of ligand induction (Fig.  5). The inhibitory effect of sL-selectin may be biologically most relevant at low levels of ligand expression. Future studies on the binding affinity of sL-selectin for its endothelial ligand will have to be carried out to determine whether sL-selectin can efficiently compete with cell-bound receptor for ligand. Since the number of sL-selectin molecules in blood is much higher than the number of cell-surface receptors in the same volume of blood, it is likely that sLselectin is of a lower affinity. Alternatively, their affinities may be equivalent, but cell-surface receptors may function in a cooperative manner or the localized relative density of receptors on the ceil surface may be higher than the corresponding relative density in solution. It is also possible that the increase in affinity of L-selectin which follows cellular activation may allow the competitive displacement of sLselectin from the endothelial surface (41).
An interesting finding was that sL-selectin did not contain the epitope wihhin the EGF-like domain identified by the anti-LAMl-1 mAb, while this epitope was readily demonstrated on the LAM-IgG fusion protein (Fig. 8) and is found on cell-surface L-selectin (21,42,51). This was revealed both by ELISA (Fig. 7) and by a lack of competitive inhibition in immunofluorescence staining of leukocytes (Fig. 6). Since the EGF-like domain is retained in sL-selectin, altera-t_ions in the tertiary structure of L-selectin may have occurred after shedding that result in a lack of anti-LAMl-1 mAb binding. It is possible that the Fc portion of IgG fused to the L-selectin extracellular domain was sufficient to stabilize the tertiary structure of L-selectin, so that anti-LAMlq mAb is still able to recognize its epitope on this soluble form of the molecule. Anti-LAMI-1 mAb binding to L-selectin not only inhibits leukocyte attachment to endothelium, but also induces an increase in the binding of PPME and fucoidin (21,42), carbohydrates which define L-selectin lectin activity (16,21,43,47,56,57). Since conformationai changes involving the LAMlq epitope may be important for the increase in L-selectin affinity that follows leukocyte activation (41), it is conceivable that the loss of the LAMI-1 epitope results in a lower affinity of sL-selectin for ligand.
The levels of sL-selectin normally found in human serum competitively inhibited the in vitro binding of L-selectindirected mAbs to leukocytes. This finding has important implications for future in vivo studies both in animals and humans. Functionally active mAbs will have to be introduced into animals in large enough quantities to saturate binding to sL-selectin in blood to allow efficient binding of mAb to leukocytes and subsequent modulation of leukocyte function. In addition, the formation of immune complexes between sL-selectin and L-selectin directed mAb will have to be considered. The use of F(ab): or F(ab) fragments instead of whole immunoglobulin might also be preferable, especially when neutrophil functions are to be evaluated. Therefore, inhibitory mAb such as anti-LAMl-1 which recognizes epitopes on L-selectin that are lost on sL-selectin might also prove helpful.
It has been proposed that L-selectin shedding is necessary for the transmigration of leukocytes through endothelium allowing rapid deadhesion of the leukocytes from endothelial cell surfaces. However, immunohistochemical staining of lymph nodes and inflamed tissues demonstrated variable proportions of L-selectin + extravascular lymphocytes, neutrophils, and monocytes (Munro, M., B. Schleiffenbaum, and T. E Tedder, manuscript in preparation). Therefore, it is possible that once leukocytes localize in tissues, they continue to express L-selectin, but this receptor is continually shed. As only a small proportion of the body's leukocytes are normally found in the circulation, it might be hypothesized that tissue localized leukocytes represent a major source of sL-selectin. The in situ production of sL-selectin by tissuelocalized leukocytes would help explain the unpredicted high level of sL-selectin found in the circulation. However, the L-selectin ligand expressed by HEV of peripheral lymph nodes is shed in vitro and may also be shed in vivo (28). Thereby, if the ligand is shed at high enough levels in vivo, it might adhere to sL-selectin, thus inhibiting its ability to affect cell adhesion in vivo. In addition, significant levels of the shed ligand may also serve as an inhibitor of L-selectinmediated adhesion to endothelium. Nonetheless, the presence of a soluble isoform of L-selectin with functional activity in serum indicates a new potential mechanism for in vivo regulation of leukocyte attachment to endothelium.