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National Public Health Institute and MediCity Research Laboratory, Turku University, Turku, Finland;
Liver Research Laboratories, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom; and
Department of Internal Medicine, Turku University Central Hospital, Turku, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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10 kDa) higher than that of transmembrane VAP-1 under nonreducing
conditions. In contrast, the electrophoretic mobilities of monomeric
sVAP-1 and transmembrane VAP-1 were similar after reduction and
boiling. Adhesion assays showed that the circulating sVAP-1 modulates
lymphocyte binding to endothelial cells. Inflammation can cause an
elevation of serum sVAP-1 levels, because sVAP-1 concentrations in
patients with certain liver diseases were two- to fourfold higher than
those in normal individuals. In contrast, rheumatoid arthritis and
inflammatory bowel diseases were not associated with elevated levels of
sVAP-1. These findings indicate that there is a functionally active,
soluble form of VAP-1 in circulation and suggest that the serum level
of sVAP-1 might be a useful marker of disease activity in inflammatory
liver diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over recent years, many of the leukocyte-endothelial cell adhesion molecules, such as ICAM-1, VCAM-1, platelet endothelial cell adhesion molecule-1, selectins, and CD44, have been shown to circulate in soluble forms (10, 11, 12, 13, 14, 15, 16, 17, 18). Extracellular parts of CD44, L- and E-selectin, VCAM-1, and ICAM-1 are cleaved enzymatically from the cell surface and released into the bloodstream (19). This process of shedding is one mechanism by which soluble forms of adhesion molecules can be produced. In addition, circulating molecules can arise at the level of RNA processing, as has been demonstrated for P-selectin and platelet endothelial cell adhesion molecule-1, which have alternatively spliced mRNAs lacking the transmembrane domains (14, 20, 21). Soluble adhesion molecules may have distinct and contrasting physiologic effects. They may function as inhibitors of cell to cell adhesion by competing with their membrane-bound forms. For example, soluble L-selectin and soluble ICAM-1 reportedly inhibit lymphocyte adhesion in vitro (16, 22). Alternatively, soluble molecules can trigger responses in cells that bear their ligands (23). Furthermore, because the serum levels of these circulating adhesion molecules alter in different disease states (10, 12, 13, 15, 24, 25), the determination of their concentrations can be useful for the diagnosis and monitoring of certain diseases.
We hypothesized that VAP-1 might exist in a soluble form. In this study, we have identified and quantitated such a circulating form of VAP-1 in normal human serum (NHS) and have characterized the biochemical structure of soluble VAP-1 (sVAP-1). We also show that the level of sVAP-1 is elevated in patients with certain liver diseases but not in patients with rheumatoid arthritis (RA) or inflammatory bowel diseases (IBDs), raising the possibility that the determination of sVAP-1 may have clinical significance in the follow-up of inflammatory liver diseases. Moreover, our studies indicate that the presence of sVAP-1 enhances the binding of lymphocytes to VAP-1 transfectants, which suggests an adhesion-modulating function for sVAP-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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New anti-VAP-1 mAbs were produced by immunizing mice with
affinity-purified VAP-1. In brief, VAP-1 was immunoprecipitated from
precleared 1% Nonidet P-40 lysates of tonsil stroma using the original
anti-VAP-1 mAb 1B2; this original mAb had been immobilized on
cyanogen bromide (CNBr)-activated Sepharose 4B beads as an affinity
matrix, as described below for other mAbs. After extensive washings,
the beads were mixed with IFA and used to immunize specific
pathogen-free BALB/c mice. After three s.c. injections into foot pads
at 1-wk intervals, mice were sacrificed, and the popliteal lymph nodes
were collected. The isolated lymphocytes were then fused with
nonsecreting SP-2 myeloma cells (American Type Culture Collection,
Manassas, VA) using standard procedures. Hybridomas were screened using
immunofluorescence staining of stable VAP-1 cDNA transfectants, and
positive hybridomas were subcloned at least twice by limiting dilution.
The isotype of the mAbs was determined using ImmunoType strips (Sigma,
St. Louis, MO). All new anti-VAP-1 mAbs, which were designated
TK8–14, TK8–18, and 2D10, showed an identical tissue-staining pattern
with the mAb 1B2, and these mAbs reacted with a 170-kDa molecule during
the immunoblotting of tonsil stroma under nonreducing conditions. The
mAbs 3G6 and Hermes-3 were used as negative controls in these
experiments (see Table I
).
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Serum samples
Serum samples from 74 normal healthy volunteers between the ages of 21 and 68 (mean ± SD, 35 ± 13) were obtained and stored at -70°C. Of the donors, 34 were males and 40 were females. Blood was drawn into Terumo Venoject serum tubes (Leuven, Belgium) with a 20-gauge needle and allowed to clot for at least 30 min. After centrifugation at 3000 rpm for 10 min, serum was collected, aliquoted, and kept frozen at -70°C until use in the sVAP-1 ELISA.
Serum samples from patients with different inflammatory diseases were also measured by the sVAP-1 ELISA. There were 11 patients with RA, 9 with IBDs (6 with ulcerative colitis and 3 with Crohn’s disease), and 41 with different kinds of liver diseases. The liver disease group contained patients with alcoholic liver disease (ALD) (n = 11), primary biliary cirrhosis (PBC) (n = 11), primary sclerosing cholangitis (PSC) (n = 8), hepatocellular carcinoma (HCC) arising in the noncirrhotic liver (n = 8) and colorectal hepatic metastases (CHM) (n = 3).
sVAP-1 ELISA
Wells of microtiter plates (96-well, flat-bottom, white Cliniplate EB; Labsystems, Helsinki, Finland) were coated with 100 µl of the anti-VAP-1 mAb TK8–18 at 10 µg/ml in 0.1 M NaHCO3 buffer (pH 9.6) at 4°C overnight and then at 37°C for 1 h. The wells were washed six times with 0.1% Tween 20 in PBS (Tween/PBS) and then blocked to prevent nonspecific adsorption by the addition of 200 µl of PBS containing 1% gelatin and 1% nonfat milk powder (blocking solution) for 45 min at room temperature. After washing the wells six times with Tween/PBS, 175 µl of each serum sample (at 1:25 dilution in the blocking solution) was added into the wells, and the plates were left at room temperature for 1 h. The wells were then washed six times with Tween/PBS and incubated with 100 µl of the biotinylated anti-VAP-1 mAb TK8–14 or biotinylated control mAb Hermes-3 (10 µg/ml in the blocking solution) at room temperature for 1 h. After six washes with Tween/PBS, 100 µl of streptavidin-horseradish peroxidase (Amersham Int., Buckinghamshire, U.K.; diluted 1:1000 in the blocking solution) was added into the wells, and the plates were allowed to incubate at room temperature again for 1 h. Thereafter, plates were washed six times with Tween/PBS and finally developed with chemiluminescence ELISA reagent (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions. The intensities of the chemiluminescence reactions in the wells were always measured after a 3-min incubation time with a Luminoskan 390 luminometer (Labsystems).
All serum samples were initially measured at a 1:25 dilution. However, some of the samples gave values that were out of the linear range of the assay when diluted to 1:25; therefore, those samples were remeasured after dilution so that the obtained values fit into the linear range. Each sample was measured in triplicate with both the anti-VAP-1 mAb and the negative control mAb. The specific VAP-1 value was calculated by subtracting the mean background value of the negative control from the mean value of VAP-1.
Each assay included a titration of the previously quantified tonsil lysate; this titration was used to generate a standard curve. To obtain a protein milieu similar to the serum samples, tonsil lysate standards were diluted in blocking solution that contained as much sVAP-1-depleted human serum as the test samples contained patient serum. Calculations of the amount of sVAP-1 in the serum samples were made by comparing the specific VAP-1 values with a standard curve of titrated tonsil lysate using linear regression analysis.
The amount of VAP-1 present in the standard tonsil lysate was determined as follows: Immunoaffinity-purified VAP-1 was obtained from tonsil lysate by column chromatography using CNBr-activated Sepharose 4B beads coupled with anti-VAP-1 mAb as described below. Aliquots of the affinity-purified sample were electrophoresed on 5 to 12.5% SDS-PAGE gels. The resolved proteins were then stained with two different protein dyes: AgNO3 and Cypro Orange (Bio-Rad, Hercules, CA). The silver-stained gel was scanned using the Microcomputer Imaging device (Imaging Research, Ontario, Canada) and the Cypro orange-stained gel was scanned using a Fluoro Imager (Fluoro Imager 595, Molecular Dynamics, Grefeld, Germany). The densities of the VAP-1 bands were quantitated against the protein standard curves that had been generated with BSA, and an average of the two results obtained by staining with different protein dyes was calculated. The concentration of VAP-1 in the standard tonsil lysate was finally determined by comparing the ELISA signal from the same batch of immunoaffinity-purified VAP-1 with the signal from the titration of standard tonsil lysate.
Immunoaffinity purification and immunoblotting of VAP-1
sVAP-1 was purified by immunoaffinity column chromatography using AB serum obtained from the Finnish Red Cross Blood Bank. For purification, 200 ml of AB serum was precipitated with 25% (NH4)2SO4 overnight at 4°C. After centrifugation at 10,000 rpm for 30 min at 4°C, the precipitate was resuspended in lysis buffer (10 mM Tris-base (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, and 1% Nonidet P-40). Next, we precleared the precipitate by passing it sequentially through Sepharose CL-4B beads (Pharmacia Biotech AB, Uppsala, Sweden) and three columns of CNBr-activated Sepharose 4B beads (Pharmacia Biotech) that had been coupled with normal rat serum, BSA, and irrelevant mAbs 3G6 or Hermes-3. The precleared serum was then passed through 1 ml of CNBr-activated Sepharose 4B beads that had been coupled with the anti-VAP-1 mAb TK8–18. After washing the beads overnight with lysis buffer and subsequently with 10 ml of ice-cold water, the bound VAP-1 was eluted in three fractions of 1 ml using 50 mM triethylamine (pH 11.4), and the fractions were stored at -70°C. The affinity-purified samples were subsequently dried with a HetoVac concentrator (Heto-Holten A/S, Allerød, Denmark) and resuspended in 150 µl of lysis buffer. Contaminating Igs were then depleted by incubating the samples with protein G-Sepharose beads (Pharmacia Biotech) at room temperature for 2 h and then discarding the beads. We subjected 50-µl aliquots of the samples to sialidase treatment that was performed with 5 mU of Vibrio cholerae neuraminidase (Behringwerke AG, Marburg, Germany) at 37°C for 2 h. For gel electrophoresis, the samples were mixed with Laemmli sample buffer with or without reduction (5% 2-ME), incubated at either 37°C for 20 min (nonreduced) or 95°C for 5 min (reduced), and loaded on 5 to 12.5% SDS-PAGE gels. The resolved proteins were transferred onto nitrocellulose membranes (Hybond/enhanced chemiluminescence (ECL), Amersham) with a Hoefer electroblotter (San Francisco, CA). The nitrocellulose sheets were then developed using the ECL detection system (ECL kit for Western blotting, Amersham) according to the manufacturer’s recommendations. In brief, the membranes were blocked with PBS containing 10% nonfat milk powder and 0.3% Tween 20, and the primary Abs were used at 2 µg/ml.
VAP-1 Ag was also immunoaffinity-isolated from tonsil. For purification, tonsil stroma was lysed in lysis buffer supplemented with 1 mM EDTA, 1% aprotinin, and 1 mM PMSF as protease inhibitors, and insoluble material was removed by centrifugation. The supernatants were then precleared, passed over affinity columns, depleted of Igs, digested with sialidase, electrophoresed, and immunoblotted as described above. The tonsil lysate as such was also used as the source of membrane-bound VAP-1 Ag for immunoblotting. Lysate supernatants were depleted of Igs with protein G beads before electrophoresing on an SDS-PAGE gel, as described above for serum samples.
For an alternative analysis of sVAP-1, serum was depleted of most albumin by three successive incubations with Blue Sepharose beads (Pharmacia Biotech) at 4°C overnight. Thereafter, the serum was incubated with protein G beads to remove Igs and mixed with Laemmli sample buffer. After heating at 37°C for 20 min, the samples were run on a 5 to 12.5% SDS-PAGE gel, and the resolved proteins were transferred onto a nitrocellulose membrane. Nitrocellulose strips were then developed using biotinylated primary mAbs and the ECL kit for Western blotting. Blocking was performed with 0.3% Tween 20 in PBS, the biotinylated mAbs were used at 1 µg/ml, and streptavidin-horseradish peroxidase was used as the secondary detection reagent.
Adhesion assays
Stable VAP-1 transfectants were generated by transfecting a eukaryotic expression vector, pcDNA3, containing the VAP-1 cDNA into a VAP-1- endothelial cell line, Ax, as described elsewhere (40). Mock transfectants were transfected similarly using pcDNA3 containing VAP-1 cDNA in an inverse orientation. The expression of VAP-1 on these cells was determined by immunofluorescence stainings with anti-VAP-1 and control mAbs and by FACS analyses using standard techniques.
Adhesion assays were performed on microscopic slides. A wax pen circle (2 cm in diameter) was drawn on the slide, and the area delineated by the circle was precoated with 1% gelatin in PBS for 3 h at 37°C. A total of 20,000 Ax VAP-1 or mock transfectants were seeded within the circles in RPMI 1640 medium containing 10% FCS, penicillin (100 IU/ml) and streptomycin (100 µg/ml), and 10 mM HEPES and grown into confluence.
A serum sample containing a high concentration of sVAP-1 was divided into two 0.5 ml aliquots. sVAP-1 was specifically depleted from one aliquot by two sequential incubations with CNBr-beads that had been coupled to the mAb TK8–14. The other aliquot was treated identically with beads that contained the nonbinding control mAb 3G6. The efficacy of the treatment was controlled by analyzing the sVAP-1 concentrations in both samples using the sandwich ELISA.
Freshly isolated PBLs were obtained from normal volunteers using Ficoll centrifugation. PBLs were preincubated with 50% serum (2 x 106 PBLs diluted in 25 µl of RPMI 1640 containing 25 µl serum) with or without sVAP-1 for 20 min at room temperature. Meanwhile, the microscopic slides containing VAP-1 or control transfectants were washed in PBS, and 100 µl of RPMI 1640 was added within each wax pen circle to evenly cover the adherent monolayer. Next, 50 µl of the PBL suspension in serum was added onto each wax pen circle without washing, and the PBLs were allowed to bind to the transfectants for 30 min under constant rotation (60 rpm on an orbital shaker) at 7°C. Thereafter, the nonadherent cells were decanted off, the slides were dipped twice in RPMI 1640, and finally, the adherent PBLs were fixed to the transfectants by an overnight incubation in ice-cold PBS containing 1% glutaraldehyde.
The number of adherent cells was counted under a microscope using an ocular grid that covered an area of 0.25 mm2 at x200 magnification. Nine predefined areas at the center of the circle were counted in each circle. Three slides per sample (with a total area of 6.75 mm2) were counted in each of three independent experiments. The results are expressed as the number of bound cells/mm2.
Statistical methods
Differences in the levels of circulating VAP-1 between two groups were analyzed using the Mann-Whitney U test. A correlation between age and serum sVAP-1 level was tested using the Spearman’s rank correlation method. p values of <0.01 were considered significant. All p values are two-tailed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A quantitative sandwich ELISA was developed, using newly generated
anti-VAP-1 mAbs, to determine the amount of sVAP-1 in serum
samples. The highest level of sensitivity was obtained using the
anti-VAP-1 mAb TK8–18 as a capture Ab and biotinylated
anti-VAP-1 mAb TK8–14 as a detecting Ab. Tonsil detergent lysate
and pooled human AB serum were used as positive controls in the
assay. The chemiluminescence signals obtained with 1:10 dilutions
of tonsil lysate and AB serum were 3000 and 1500 U,
respectively.
The specificity of the ELISA was confirmed by three different procedures: coating the wells with an irrelevant mAb 3G6, using serum samples that had been depleted of sVAP-1 by immunoaffinity chromatography, and detecting with an irrelevant biotinylated mAb, Hermes-3. No significant reactivity was observed in any of these controls (chemiluminescence values of 0.3–4 U). Since as many as 1 to 3% of normal individuals may have anti-mouse IgG Abs in their serum that can cross-link the capture and biotinylated detecting murine Abs in the absence of Ag, we chose to use the murine mAb Hermes-3 as a negative control for every serum sample in all subsequent experiments to eliminate all possible false positive results.
The obtained chemiluminescence signals were converted into nanogram/milliliter values by including a titration of a previously quantitated standard of tonsil lysate in each performed assay and using linear regression analysis (see Materials and Methods). Using one batch of tonsil lysate as a standard was found to be the best alternative in the absence of rsVAP-1. This ELISA proved to be linear up to 1.1 ng of VAP-1/well, and as little as 0.005 ng of VAP-1/well was reliably detected with this assay. In addition, the inter- and intraassay variations were usually <10%. The amount of immunoreactive sVAP-1 in serum was found to be unaffected by five freeze-thaw cycles.
Detection of sVAP-1 in NHS
Sera from 74 healthy individuals between the ages of 21 and 68
(mean 35) were tested for the amount of sVAP-1 by this ELISA. The
sVAP-1 concentrations in this population of 34 males and 40 females
varied from 49 to 138 ng/ml (Fig. 1A), and the mean ± SD
level for this group of normal controls was 88 ± 20 ng/ml. The
sVAP-1 values did not correlate with the age (r =
-0.076; p = 0.52) or sex (p =
0.30) of the donors. In addition, 12 of the blood donors were retested
several months after the first measurement to evaluate the
intra-individual variability of the sVAP-1 level. The observed changes
in their values were between 0.1% and 20% (mean 7%) (see Table II). In 8 of the 12 cases, the changes
were within the variation limits of the assay; in the remaining 4
cases, the changes were <20%. Thus, the level of sVAP-1 in the serum
of healthy individuals seems to be quite stable over time. Moreover, to
study diurnal variations of the sVAP-1 level, four of the normal blood
donors were tested for their sVAP-1 levels at four different time
points: at 6 p.m., at 12 a.m., at 6 a.m., and at 12
p.m. As seen in Figure 1B, sVAP-1 concentrations tended to
be higher in the evening than in the morning, but the differences were
quite minor (<25%).
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SDS-PAGE and the immunoblotting analysis of affinity-purified
sVAP-1 were performed to determine the molecular mass of the
circulating VAP-1 molecule and to compare it with the membrane-bound
form. Initially, nonreducing conditions and mild heating of samples
were chosen because the mature dimeric VAP-1, which consists of two
identical subunits held together by disulfide bonds, dissociates into
its monomeric subunits by reduction and boiling (9). Two different
anti-VAP-1 mAbs, 2D10 and TK8–18, were used as primary Abs in
immunoblottings. 2D10 is an Ab that recognizes an epitope only in the
intact dimeric form of VAP-1, whereas the epitope recognized by TK8–18
is present both in dimeric and monomeric forms (see below). When
detected with the mAb 2D10, a specific, 180-kDa band was seen in
purified sVAP-1 samples (Fig. 2A, lane 3). The
apparent molecular mass of circulating sVAP-1 (lane
3) was slightly higher (10 kDa) than that of the membrane-bound
tonsil form of VAP-1 (lanes 1 and 2). When
the mAb TK8–18 was used to detect sVAP-1 in immunoblotting, a
180-kDa band (Fig. 2A, lane 6) was observed as
well. Hence, the electrophoretic mobility of dimeric sVAP-1 is slightly
slower than that of transmembrane VAP-1 under nonreducing, nonboiling
conditions.
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B, lanes 4–6). Thus, the monomeric
subunits of membrane VAP-1 and sVAP-1 have similar molecular masses
(see Discussion). No detection was seen with the mAb 2D10 in
these reduced and boiled samples (Fig. 2B, lanes
1–3), indicating that the epitope recognized by this Ab was
destroyed in monomeric VAP-1.
Additional immunoblotting experiments under nonreducing conditions were
performed to determine whether the affinity purification process
affects the structure of the sVAP-1 molecule. Normal serum that had
been depleted of most albumin and Igs was used as the source of sVAP-1
Ag in SDS-PAGE and immunoblotting. The primary Ab used in
immunoblotting was the biotinylated anti-VAP-1 mAb TK8–14. This
Ab, like the mAb TK8–18, recognizes an epitope that is present in both
the dimeric and monomeric forms of VAP-1 (our unpublished
observations). The reason for using mAb TK8–14 instead of mAb TK8–18
was that biotinylated TK8–18 did not work properly in these
immunoblotting experiments. One specific band was detected by mAb
TK8–14 in the serum sample. The band was in the range of 180 kDa (Fig. 3), as was the case in affinity-purified
sVAP-1 samples. Thus, these data indicate that the slower
electrophoretic mobility of sVAP-1 compared with membrane VAP-1 under
nonreducing conditions is not caused by effects of the affinity
purification process (e.g. possible rearrangements of disulfide bonds)
on the structure of the sVAP-1 molecule. Moreover, this experiment
implies that there is only one naturally occurring form of VAP-1 in the
circulation, which is the dimeric, 180-kDa species. To rule out the
possibility that a small portion of sVAP-1 might be in a monomeric
form, we confirmed the immunoblotting result with a more sensitive
method. To that end, AB serum was depleted of dimeric VAP-1 by CNBr
beads that had been coupled with the anti-VAP-1 mAb 2D10, and this
serum sample was tested by the sVAP-1 ELISA in which both the capturing
(TK8–18) and detecting (TK8–14) mAbs recognize both the dimeric and
monomeric form of VAP-1. No reactivity was observed in this sample.
Thus, monomeric-circulating VAP-1 was not detected by this method
either. Consequently, most, if not all, sVAP-1 is dimeric.
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180
kDa), we suspected that the dimeric sVAP-1 may not contain any sialic
acid residues. To test this hypothesis experimentally,
immunoaffinity-isolated sVAP-1 was subjected to sialidase treatment
before SDS-PAGE and immunoblotting. The sialidase digestion caused a
remarkable change in the electrophoretic mobility of sVAP-1 samples
(data not shown), indicating that sVAP-1 also contains abundant sialic
acid decorations. Increased levels of sVAP-1 in patients with inflammatory liver diseases
We hypothesized that patients with inflammatory disorders may show
elevated levels of sVAP-1 because endothelial VAP-1 is up-regulated
upon inflammation and is translocated to the luminal surface of vessels
(7). Therefore, we tested samples from patients with different systemic
inflammatory conditions, including RA and IBDs, by the sVAP-1 ELISA.
However, the sVAP-1 values obtained from these patients did not differ
significantly from the values of healthy controls (Fig. 4A). Because of the strong
constitutive expression of VAP-1 on hepatic endothelia (8) we also
tested sera from 41 patients with different liver diseases by the
sVAP-1 ELISA. In contrast to other patients tested, many of the liver
disease patients had sVAP-1 values that were clearly elevated when
compared with healthy controls (Fig. 4A). The mean
level ± SD for this liver disease group was 140 ± 61 ng/ml,
and the range was 50 to 315 ng/ml. The difference of sVAP-1
concentrations between the liver patient group and normal controls was
statistically highly significant (p < 0.0001).
Moreover, when comparisons were made according to the nature of the
liver disease, differences within the liver disease group became
apparent. The highest values were found in patients with active
cirrhosis due to ALD (Fig. 4B). Patients with PBC showed a
less pronounced but statistically significant increase in the levels of
sVAP-1 (p < 0.001), whereas the levels in
patients with PSC did not differ significantly from those of normal
controls. The increase in sVAP-1 levels in patients with HCC did not
approach statistical significance, although some of these patients had
elevated levels. In contrast, all of the patients with CHM had values
within the normal range. Taken together, serum sVAP-1 concentrations
can be modulated by certain inflammatory liver diseases.
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To investigate the physiologic role of circulating VAP-1, adhesion assays were performed. Since we were unable to isolate enough functionally intact VAP-1 from serum samples, and because we do not have rsVAP-1, the effect of native serum VAP-1 on the binding of PBLs to VAP-1 transfectant monolayers was analyzed. We have shown that PBLs bind >25 times better to VAP-1-transfected Ax cells than to mock-transfected controls (40). PBLs were preincubated with a serum sample containing high levels of sVAP-1 (120 ng/ml) and with an identical serum sample from which the sVAP-1 was specifically depleted (0 ng/ml) by sequential immunoprecipitations. The rationale was that under these conditions sVAP-1 has the possibility to bind to its counter-receptor on lymphocytes and either inhibit adhesion to VAP-1 transfectants (if sVAP-1 is has an inhibitory role) or enhance binding to VAP-1 transfectants (if sVAP-1 has a triggering function).
In three independent assays using PBLs from different blood
donors, PBL binding to VAP-1 transfectants was increased when sVAP-1
was present during the preincubation step (Fig. 5). Binding to mock transfectants was
always negligible (<5 cells/mm2). Thus, these data
indicate that circulating VAP-1 does not serve an adhesion-blocking
function. Rather, sVAP-1 most likely enhances lymphocyte binding to
endothelial cells by triggering the functional up-regulation of other
adhesion molecules on lymphocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunoblotting studies revealed that the apparent molecular mass
of affinity-purified sVAP-1 is slightly higher than that of
transmembrane VAP-1 under gentle, nonreducing conditions. The affinity
purification process may affect the structure of VAP-1, because large
aggregates were often seen in affinity-isolated tonsil VAP-1 samples
(see Fig. 2A, lane 5). These aggregates are
thought to be multimers of the VAP-1 molecule that have arisen during
the purification process. However, the structure of affinity-isolated
sVAP-1 was not modified by the purification process, because the same
180-kDa band that was seen in affinity-isolated sVAP-1 samples was
observed in the serum sample that had only been depleted of most
albumin and Igs (Fig. 3). The relatively weak intensity of the specific
sVAP-1 band in Figure 3 is due to very small amount of Ag in the
sample. After multiple preclearings, there is <4 ng of sVAP-1
in the 50-µl serum sample that can be loaded per well, and the
blotting procedure is never very efficient with large molecules such as
dimeric sVAP-1. In contrast to the size difference between sVAP-1 and
membrane-bound VAP-1 that was seen under nonboiling, nonreducing
conditions, the electrophoretic mobilities of VAP-1 from these sources
were identical after reduction and boiling. Taken together, these data
suggest that the monomeric subunits of these two forms of VAP-1 are
very similar, if not identical, but that different rearrangements in
the structure of dimeric membrane bound VAP-1 and sVAP-1 occur under
nonreducing conditions, resulting in minor shifts in apparent SDS-PAGE
mobilities. We also showed that there is no monomeric sVAP-1 in the
circulation, because monomeric sVAP-1 was undetectable either by
immunoblotting of sVAP-1 in serum or by ELISA in serum samples that had
been depleted specifically of dimeric VAP-1. Based on these data, it
seems probable that the dimeric 180-kDa species is the only naturally
occurring form of sVAP-1 in the circulation.
It is likely that sVAP-1 is derived from the transmembrane form of
VAP-1 by proteolytic cleaving. The location of the cleavage site just
proximal to the membrane-spanning region would explain the similar
molecular masses of sVAP-1 and membrane VAP-1 under reducing
conditions. Our sequence data (40) indicate that deletion of the
transmembrane and cytoplasmic domains would cause only an 2 kDa
decrease in the molecular mass of VAP-1, which would not be clearly
demonstrated by SDS-PAGE. However, we cannot formally exclude the
possibility that sVAP-1 is encoded by an alternatively spliced mRNA
lacking the transmembrane sequence, or even that it is a secreted
protein encoded by an independent gene.
Like the membrane-bound endothelial VAP-1 (9), circulating VAP-1 contains abundant sialic acid decorations. The other posttranslational modifications are likely to be very similar if not identical in these two forms of VAP-1 as well, since monomeric membrane-bound VAP-1 and sVAP-1 had apparently identical molecular masses. The finding of abundant sialic acid residues in sVAP-1 is particularly important, because the adhesive function of VAP-1 is dependent upon its sialic acids (9). Thus, our observation that sVAP-1 is heavily sialylated suggests that it might be functionally active.
The functional role for sVAP-1 was actually shown in adhesion assays. When sVAP-1 had been depleted from the serum in which PBLs were preincubated, lymphocyte binding to VAP-1-transfected endothelial cells decreased. These results suggest that sVAP-1 binds to its (uncharacterized) lymphocyte counter-receptor during the preincubation step and causes a change in the adhesive status of the lymphocyte. The mechanism of enhanced binding after sVAP-1 preincubation remains to be established. However, ligation of VAP-1R on lymphocytes might trigger a signal that results in the up-regulation of the expression and/or avidity of some other lymphocyte adhesion molecule(s). This could then lead to increased PBL adherence to endothelial monolayers by binding of lymphocyte adhesion receptors to their cognate counter-receptors, which are expressed on these endothelial transfectants. Since the very low level of binding of PBLs to mock transfectants apparently did not increase after preincubation in VAP-1+ serum, PBLs most likely recognize endothelial adhesion molecules whose expression is directly or indirectly dependent upon VAP-1 expression in these cells. Our results suggest that increased levels of sVAP-1 may lead to an increased adhesion of PBLs to vascular endothelia locally at sites of inflammation. This mechanism could serve to augment the immune response at the scene of inflammation, where the other components of the multistep adhesion cascade are also active. It should be noted that sVAP-1 may have a normal physiologic role in maintaining a proadhesive status of PBLs toward endothelia, since its concentration in normal individuals is within the range used in these adhesion experiments; the complete depletion of sVAP-1 resulting in decreased adhesion is an artificial condition used in this experimental setup.
Significantly elevated levels of sVAP-1 were found in patients with liver diseases. When these sVAP-1 values were correlated with the diagnosis of the patients, differences between the disease groups were observed. Patients with ALD had the most marked elevations in systemic circulation. Indeed, levels in these patients were high enough to suggest that local concentrations of sVAP-1 could cause biologic effects. Patients with PBC also had elevated levels, although levels in patients with PSC were not elevated when compared with controls. This finding is relevant, because these patients have marked cholestasis, which suggests that impaired bile flow per se does not cause the elevation of sVAP-1 levels. It was also interesting to note that the patients with HCC had higher sVAP-1 values than the patients with CHM, which fits well with the finding of high VAP-1 expression on primary liver tumors but not in liver metastases (28). The other nonhepatic inflammatory diseases we studied (i.e., RA and IBDs) were not associated with elevated levels of sVAP-1. These data suggest that the elevation of sVAP-1 concentrations is specific for certain inflammatory diseases, such as ALD. In contrast, levels of other soluble adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, have been demonstrated to be elevated not only in ALD but in practically all liver diseases (29, 30, 31, 32, 33, 34). Moreover, elevated levels of these other circulating adhesion molecules have been reported in many other inflammatory disorders (24), including RA (35, 36, 37) and IBDs (38, 39). Thus, the specificity of elevated sVAP-1 levels for certain liver diseases warrants further study to determine the potential role of sVAP-1 in the follow-up of the inflammatory activity of these diseases.
In conclusion, we have shown that dimeric sVAP-1 circulates in the serum of healthy individuals at a level that is comparable with those of most other soluble endothelial adhesion molecules (24). Moreover, the level of sVAP-1 is increased in certain inflammatory liver diseases in a more disease-specific manner than the levels of other known circulating endothelial adhesion molecules (24). sVAP-1 may exert its biologic function by regulating the leukocyte-endothelial cell adhesion cascade.
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2 Address correspondence and reprint requests to Dr. Riikka Kurkijärvi, MediCity Research Laboratory, Turku University, Tykistökatu 6 A, 20520 Turku, Finland. E-mail address:
3 Abbreviations used in this paper: VAP-1, vascular adhesion protein-1; sVAP-1, soluble vascular adhesion protein-1; NHS, normal human serum; CNBr, cyanogen bromide; RA, rheumatoid arthritis; IBD, inflammatory bowel disease; ALD, alcoholic liver disease; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; HCC, hepatocellular carcinoma; CHM, colorectal hepatic metastases; ECL, enhanced chemiluminescence.
Received for publication November 3, 1998. Accepted for publication March 31, 1998.
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