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*
MediCity Research Laboratory, University of Turku, and National Public Health Institute, Department in Turku, and
Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In humans, two endothelial molecules appear to be crucial in the first recognition steps of PLN HEV by lymphocytes. Peripheral node addressins (PNAd) mediate HEV adhesion and rolling under flow conditions by binding to their lymphocyte counterreceptor known as L-selectin (5, 6, 7, 8, 9, 10, 11). Interaction between L-selectin and PNAd is characteristically oligosaccharide dependent. Sulfate, sialic acid, and fucose modifications of PNAd are essential for binding to occur (12, 13, 14). PNAd are defined by a mAb MECA-79 that detects a sulfation-dependent glycotope on multiple proteins. At least six different bands are seen with mAb MECA-79 in immunoblotting and immunoprecipitation experiments, and they all may represent molecules with distinct protein backbones (5, 12, 14). The 50-kDa form of PNAd is a secreted mucin-type molecule called glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1, also known as Sgp-50 (15)). One of the protein backbones of the 90- to 105-kDa species (or Sgp-90) is identical to CD34 (16), a widely expressed transmembrane protein. However, in CD34 null mice, a MECA-79-reactive 90-kDa band was still visible, indicating that another uncharacterized protein in addition to CD34 at this m.w. range is also included in the PNAd Ag family (17). The 200-kDa form of PNAd is an inducible and sulfated L-selectin ligand that may be important in lymphocyte homing at sites of inflammation (18). The 170-kDa species of PNAd is a sulfated protein that also binds to L-selectin (14). The molecular identities of these other forms of PNAd remain to be elucidated.
Vascular adhesion protein-1 (VAP-1) is an endothelial molecule that supports lymphocyte binding to HEV in humans under nonstatic conditions (19). A rabbit homologue of VAP-1 has been shown to function under in vivo flow conditions during the formation of the initial transient contacts between the endothelial and lymphoid cells (20). VAP-1 mediates HEV adhesion of CD8-positive and CD16-positive lymphocytes. In contrast, Th cells, B cells, and monocytes do not bind via VAP-1 (20). VAP-1 is defined by a mAb 1B2 that immunoprecipitates 90- and 170-kDa glycoproteins. The adhesive function of VAP-1 is critically dependent on proper oligosaccharide modifications, since desialylated VAP-1 does not bind lymphocytes (21). The lymphocyte ligand of VAP-1 is currently unknown.
VAP-1 and PNAd have in common the expression in PLN-type HEV and inducibility in venules at extralymphatic sites upon inflammation. Both molecules also include 90- and 170-kDa sialoglycoprotein species. Moreover, both VAP-1 and PNAd mediate oligosaccharide-dependent lymphocyte adherence under shear conditions. Finally, combined blocking of VAP-1 and PNAd has only marginal additive effects in HEV adhesion assays using unfractionated PBL (5, 7, 12, 19, 20, 21, 22, 23). For these reasons, it has remained possible that VAP-1 is comprised of the 90- and 170-kDa forms of PNAd. In this study, we show that this is not the case but, rather, these two glycoproteins represent two molecularly independent ways for lymphocyte binding to PLN HEV.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Function-blocking mAbs 1B2 against human VAP-1 and MECA-79 against mouse and human PNAd have been described (7, 19). mAb TK8-14 is a new IgG2a anti-VAP-1 Ab that was produced by immunizing specific pathogen-free BALB/c mice with affinity-purified VAP-1 obtained from tonsil stromal lysate using 1B2-coupled CnBr-activated Sepharose 4B beads as an affinity matrix. Immunization, hybridoma production, subcloning, and testing were done using previously described techniques (19), except that SP-2/0 mouse cells were used as the myeloma partner. The specificity of TK8-14 against VAP-1 was shown by positive staining with VAP-1 (but not with mock) transfectants and by an identical staining pattern with the prototype anti-VAP-1 mAb 1B2 in tissue sections. In reciprocal immunoprecipitations of tonsil lysate, mAbs 1B2 and TK8-14 both depleted all VAP-1 reactivity from the sample when analyzed by immunoblotting. In gel analyses, it was evident that mAb TK8-14 reacts with the dimeric 170-kDa VAP-1 and, under reducing conditions, with the monomeric 90-kDa form of VAP-1 (data not shown). FITC-conjugated mAbs against CD4 and CD8 were from Becton Dickinson (Palo Alto, CA). mAb 7C7 is a mouse mAb against epithelial cells of bursa of Fabricius in chicken, and mAb 3G6 is a mouse mAb against an Ag expressed on chicken peripheral T cells (produced by Drs. Maarit Wuorela and Olli Vainio, respectively (University of Turku)). TIB146 (from American Type Culture Collection, Rockville, MD) is a rat mAb against mouse B cell-specific B220 Ag (24). Mouse mAbs 2C8 against human CD31 (25) and Hermes-3 against human CD44 (26) have been described. In this study, mAbs 7C7, 3G6, and TIB146 served as isotype-matched nonbinding control mAbs, and mAbs 2C8 and Hermes-3 as isotype-matched binding control mAbs.
PBL were isolated from healthy volunteers or from buffy coats. PLN were unaffected nodes removed during surgical operations performed for diagnostic purposes. Tonsils were from tonsillectomies.
A rat PLN HEV-derived endothelial cell line, Ax, was a kind gift from Dr. M. Miyasaka (University of Osaka, Osaka, Japan), and the cells were maintained as described (27). Stable VAP-1 transfectants were produced by transfecting the Ax cells with a VAP-1 cDNA35 in an eukaryotic expression vector pcDNA3, by lipofection and selecting geneticin-resistant colonies. Mock transfectants were done identically using the VAP-1 cDNA in an inverse orientation in the vector.
Immunostainings
Normal indirect immunoperoxidase staining of acetone-fixed
frozen sections was done exactly as described (19). For two-color
immunofluorescence stainings, the sections were sequentially overlaid
with mAb 1B2, FITC-conjugated sheep anti-mouse Ig supplemented with
5% AB serum, mAb MECA-79, and finally, phycoerythrin-conjugated mouse
anti-rat 
-light chain-specific mAb supplemented with 5% normal
mouse serum. In a negative control staining, isotype-matched nonbinding
mAbs were used in the first and third stage. Between each incubation,
the slides were washed twice in PBS. Coverslips were attached with
Fluoromount. Sections were analyzed under epiillumination and GR filter
block (Leica), allowing simultaneous visualization of both
fluorochromes.
Ax transfectants were harvested from tissue culture flasks by a trypsin-EDTA incubation. After washings, the cells were sequentially incubated with primary Abs (10 µg/ml) and then with an appropriate FITC-conjugated anti-mouse or anti-rat second stage reagent. Cells were analyzed using FACScan and CellQuest software (Becton Dickinson).
Metabolic labelings
Metabolic labelings of tonsil in an in vitro culture system were done essentially as described (21). Briefly, small tonsil cubes were starved in an appropriate medium, labeled with Na2H35SO4 or [35S]methionine/cysteine, and lysed in the lysis buffer (150 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-base (pH 7.2), 1% Nonidet P-40, 1% aprotinin, and 1 mM PMSF). The Ags were immunoprecipitated from the precleared lysates with protein A beads armed with mAbs 1B2, 3G6, 2C8, MECA-79, and TIB146. After washings, the Ags were eluted in reducing Laemmli’s sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.01% bromphenol blue containing 5% 2-ME) and loaded in 5 to 12.5% gradient SDS-PAGE gels. All samples bearing the same label were analyzed in parallel lanes of one gel. After electrophoresis, the gels were fixed, soaked in Enlightning (DuPont, Wilmington, DE), dried, and subjected to autoradiography.
Immunoblottings and cross-precipitations
Immunoblottings were done essentially as described (21). Briefly, Ig-depleted Nonidet P-40 lysate from tonsil stroma was mixed with nonreducing Laemmli’s sample buffer. After mild heating (30 min at 37°C), the samples were resolved in SDS-PAGE and the Ags were transferred onto Hybond enhanced chemoluminescence (ECL) nitrocellulose filters for immunoblotting with the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
For cross-precipitations, mAbs 1B2, TK8-14, MECA-79, 2C8, and TIB146 were coupled to rabbit anti-mouse- or rabbit anti-rat-conjugated protein A-Sepharose. Ig-depleted tonsil lysate was subjected to two rounds of preclearing with the specific protein A beads. Thereafter, the beads were discarded and the supernatants were depleted of residual Ig molecules by a 2-h incubation with protein G beads. Finally, the supernatants were filtered to remove any residual Ig-containing beads, mixed with Laemmli’s sample buffer, and subjected to SDS-PAGE and immunoblotting, as described above.
All samples for immunoblottings were analyzed in parallel lanes of the same gel whenever possible. In all cases, m.w. markers (Rainbow; Amersham) were included in all strips to allow precise juxtaposition of the lanes.
Peptide maps of VAP-1
VAP-1 was purified from precleared tonsil lysate by immunoaffinity chromatography using cyanogen bromide-activated Sepharose 4CL beads (Pharmacia, Piscataway, NJ), as suggested by the manufacturer. Bound VAP-1 Ag was eluted from the matrix with triethylamine (pH 11.5) and lyophilized. The purified Ag was resuspended in nonreducing Laemmli’s sample buffer, and a portion of it was loaded in SDS-PAGE gels that were subsequently silver stained for analyzing the purity of the preparation. The rest of the sample was separated in another SDS-PAGE gel, electroblotted onto Problott polyvinylidine difluoride membrane (Applied Biosystems, Foster City, CA), and stained with Coomassie brilliant blue. The specific 90- and 170-kDa bands were excised separately, treated with polyvinylpyrrolidone (PVP-40; Sigma, St. Louis, MO), and subjected to digestion with 1 µg of sequencing-grade trypsin (Boehringer Mannheim, Indianapolis, IN). The on membrane digestion was performed overnight using the protocol of Fernandez (28). The generated trypsin peptides were separated by HPLC equipped with a Vydac C18 column (2.1 mm x 150 mm) reverse-phase column (The Separations Group, Hesperia, CA). The solvent gradient used was 2 to 30% B (0–63 min), (A = 0.1% trifluoroacetic acid/H2O, B = 0.08% trifluoroacetic acid/acetonitrile), 30 to 60% B (63–95 min), 60 to 80% B (95–105 min), 80% B (105–110 min), and 80 to 2% B (110–115 min), with a flow rate of 150 µl/min. The detection wavelength was 220 nm.
Immunomagnetic cell separations and adhesion assays
CD4-positive and CD8-positive PBL were isolated using MACS (Miltenyi Biotechs). A total of 100 to 300 x 106 cells was incubated with anti-CD4 or anti-CD8 MACS superparamagnetic microbeads and isolated in a BS-type column in a MACS apparatus, according to the manufacturer’s instructions. Specificity of the isolation was always confirmed by staining the cells with FITC-conjugated anti-CD4 and anti-CD8 mAbs against different epitopes.
The HEV adhesion assays were done as described (29). Briefly, the target tissue was pretreated with the anti-endothelial or control mAbs for 30 min, and then the PBL subpopulations (3 x 106 cells/section) were added. The whole assay was done under nonstatic (rotation) conditions on an orbital shaker (60 rpm). After 30 min, the nonadherent cells were tilted off and the adherent cells were fixed to the sections by an overnight incubation in ice-cold PBS containing 1% glutaraldehyde. The sections were analyzed under dark-field microscopy, and the number of cells adherent to HEV was scored. Each adhesion assay was done two to five times, and at least 120 HEV per target tissue from at least two different donors were counted. The results are expressed as percentage of control binding, which is defined by the number of HEV-bound lymphocytes in control mAb-treated sections.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In PLN, both VAP-1 and PNAd are expressed in most HEV (data not
shown). Therefore tonsil, which represents a mixed-type lymphatic organ
containing both PLN- and mucosa-type HEV, was analyzed to study the
colocalization of these two Ags. In many areas of tonsil, the vessels
were positive for both Ags, but, in the same specimens,
VAP-1+PNAd- and
VAP-1-PNAd+ HEV were also detected (Figs. 1
and 2).
In certain HEV, both VAP-1 and PNAd were preferentially expressed
luminally, and in others, expression was found throughout the whole
thickness of the HEV cells. VAP-1 was also found in the dendritic cells
of germinal centers and in the smooth muscle cells of vessels, whereas
MECA-79 epitopes were absent from these cell types. Instead, fine
irregular septae within lymphatic tissue stained positively with mAb
MECA-79, but not with an anti-VAP-1 mAb 1B2. Thus, VAP-1 and PNAd
are coexpressed prominently in many paracortical HEV that support most
lymphocyte trafficking in vivo. Nevertheless, unique expression of one
or the other of these molecules in certain HEV indicates that even
adjacent HEV can have different adhesive functions.
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Data from different laboratories show that mAb MECA-79 recognizes
several different protein cores, including a 
150 to 170- and a
90-kDa form (5, 9), which have a similar m.w. as we have reported for
VAP-1 (21). We first wanted to see whether the electrophoretic
mobilities of PNAd and VAP-1 are identical when analyzed in parallel
under the same conditions. Avid incorporation of sulfate is a
characteristic of many forms of PNAd (12), and therefore, small pieces
of human tonsil were labeled in vitro with inorganic
35S sulfate. From these lysates, immunoprecipitation
with mAb MECA-79 yielded prominent smeared bands of about 50, 105, and
170 kDa, and weaker, diffuse, 40- and 200-kDa bands (Fig. 3A). In contrast, no
labeled molecules were precipitated with mAb 1B2.
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B). From
[35S]methionine/cysteine lysates, the 90- and 170-kDa
forms of VAP-1 were clearly seen. The unavoidable immunoprecipitation
of nonspecific components at the 105- and 50-kDa range (similar in size
to the CD34/Sgp90 and GlyCAM-1 components of PNAd) with an
isotype-matched control mAb TIB146 rendered
[35S]methionine/cysteine labeling unfeasible for further
experimentation.
Since metabolic labelings can selectively label different species and
since neither label gave equally strong signals for VAP-1 and PNAd
glycoproteins, we sought to determine the m.w. by another method.
To that end, tonsil lysate was separated in SDS-PAGE and probed with
mAbs MECA-79 and 1B2 in immunoblotting experiments. mAb 1B2
specifically reacted with a 170-kDa molecule that was indistinguishable
in size from one of those seen by mAb MECA-79 (Fig. 3C). The 90-kDa form of VAP-1 was not detectable in
immunoblots with mAb 1B2. Large-scale immunoisolation of tonsil VAP-1
yielded enough material from both the 90- and 170-kDa forms of VAP-1
for peptide mapping (Fig. 4A). The HPLC elution
profiles include peptides that are released from the membrane during
trypsin digestion. Especially large peptides are efficiently retained
by the membrane and are not detected in the chromatogram, which reduces
the complexity of the tryptic peptide mixtures. The positions and
intensities of separated selected peptides, as shown in Figure 4B, strongly suggest that they originate from molecules with
identical primary structures. Thus, the 170-kDa mature VAP-1 is a
homodimer and the monomeric 90-kDa form of VAP-1 loses the 1B2
epitope.
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mAb MECA-79 does not recognize VAP-1 transfectants
The staining and m.w. analyses would be compatible with the
possibility that mAb 1B2 recognizes 90- and 170-kDa forms of PNAd. To
further elucidate the molecular identity of VAP-1 and PNAd, we used
novel VAP-1 cDNA expression transfectants made in a rat HEV-like
endothelial cell line Ax. As shown in Figure 5, the original anti-VAP-1 mAb 1B2
and a novel anti-VAP-1 mAb TK8-14 brightly stain VAP-1, but not
mock, transfectants, whereas control mAbs were negative. In contrast,
the anti-PNAd mAb MECA-79 did not recognize VAP-1 transfectants.
These results suggest that VAP-1 and PNAd are not identical molecules.
However, the transfectant data do not prove the distinct molecular
nature of VAP-1 and PNAd, since mAb MECA-79 recognizes an
oligosaccharide-dependent epitope (14) and Ax cells may not be able to
decorate rVAP-1 with the same modifications as occur in high
endothelial cells in vivo.
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To show directly that natural VAP-1 is not identical with the 170-
and 90-kDa species of PNAd, cross-precipitations were performed. As
shown in Figure 6, the 170-kDa species of
VAP-1 was effectively and specifically depleted from the lysate with
the protein A-Sepharose-1B2 beads (lane 13),
whereas no change in MECA-79 reactivity (lane
5) was observed. In a complementary set of experiments,
protein A-MECA-79 beads were used to deplete tonsil lysates of PNAd,
and the precleared lysate was used for immunoblotting. The efficacy of
preclearing was shown by the fact that all MECA-79-reactive bands were
absent from this lysate (Fig. 6, lane 7).
Importantly, the 170-kDa species of VAP-1 remained intact after removal
of all forms of PNAd (Fig. 6, lane 15).
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, lane 8), but the 90-
to 100-kDa MECA-79 molecules remained intact (lane
4). In a reciprocal setup, depletion of all
MECA-79-reactive species (lane 2) did not
affect the detection of the 90-kDa VAP-1 molecule (lane
6). Control precipitations with irrelevant isotype-matched
mAbs did not affect the reactivity of either VAP-1 or PNAd.
Furthermore, mAb MECA-79 does not recognize purified VAP-1 Ag (data not
shown). Thus, these studies unambiguously show that the 170- and 90-kDa
species of PNAd and VAP-1 are indeed distinct molecular species.
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We have shown recently that VAP-1 mediates the binding of
CD8-positive and CD16-positive lymphocytes, but not that of
CD4-positive or CD19-positive lymphocytes or CD14-positive
monocytes, to PLN HEV (20). We therefore wanted to compare whether the
prototype peripheral lymph node addressin, PNAd, would also display
lymphocyte subtype-selective adhesion pattern. To this end, binding of
immunomagnetically purified CD4-positive and CD8-positive PBL to PLN
HEV was analyzed. The purity of the CD4-positive and CD8-positive cells
was >97 to 100% when analyzed by FACS. Anti-VAP-1 mAb 1B2 very
efficiently blocked the adhesion of CD8-positive cells to PLN HEV, but
it did not affect binding of CD4-positive cells (Fig. 8). The anti-PNAd mAb, MECA-79,
inhibited by 60% the adherence of CD8-positive cells to PLN HEV, but
it also efficiently reduced interactions between PLN HEV and
CD4-positive cells. In those experiments in which 1B2, MECA-79, and
1B2 + MECA-79 inhibitions were done in parallel using the same
donor cells and the same target tissues, combined blocking of VAP-1 and
PNAd did not augment the inhibition of PLN HEV adhesion of CD4-positive
cells seen in the presence of mAb MECA-79. In these assays, 1B2 and
MECA-79 showed marginal additive effects in PLN HEV binding of
CD8-positive cells. This tendency, however, failed to reach statistical
significance and, when the results from other experiments are combined,
it is no longer detectable due to interindividual variation in the
adhesion (Fig. 8). Thus, these data strongly indicate that mAbs 1B2 and
MECA-79 recognize separate endothelial adhesion molecules and that CD4
cells only use PNAd. In contrast, both molecules are needed for the
interactions between CD8-positive cells and PLN HEV, suggesting that
these molecules mediate successive or overlapping steps in the adhesion
cascade.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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90- and 170-kDa protein species.
It has been shown earlier that these two adhesion molecules display
similar characteristics both in tissue distribution and inducibility at
similar sites during chronic inflammation (22, 23). Moreover, both
molecules are sialoglycoproteins, the adhesive function of these two
endothelial adhesion molecules depends on proper sialic acid
modifications, and they both mediate early interactions between
lymphocytes and the vessel wall (11, 14, 20, 21). Based on these
similarities, we tested a hypothesis that VAP-1 would consist of the
same 90- and 170-kDa molecules that are included among the six or more
glycoproteins comprising the PNAd Ags. Our results show, however, that
the Ags recognized by anti-VAP-1 and anti-PNAd Abs are
molecularly distinct. We acknowledge that our present experiments
cannot formally rule out the possibility that anti-VAP-1 mAbs would
recognize an activation-dependent epitope of PNAd (if such exists).
Thus, most likely, the present results reveal two molecularly
independent adhesion mechanisms that mediate the binding of distinct
lymphocyte subpopulations to PLN HEV in humans. Both mAbs 1B2 and MECA-79 detect abundantly sialylated 90- and 170-kDa glycoprotein species from endothelial cells. The 170- and 90-kDa forms of PNAd are sulfated, and at least the 90-kDa form is also fucosylated, whereas VAP-1 apparently lacks these modifications (14, 21). The 170-kDa form of PNAd has not been molecularly cloned, but the 90-kDa species of PNAd was shown to be identical to CD34. Surprisingly, however, generation of CD34 null mice revealed that a 90-kDa MECA-79-reactive glycoprotein was still found in these animals (17). Thus, the 90-kDa form of PNAd consists of at least two distinct molecular species, CD34 and an uncharacterized sialoglycoprotein. In this study, we have used reciprocal immunoprecipitations and staining of VAP-1 transfectants to prove that the 170- and 90-kDa Ags of VAP-1 have different protein scaffolds from the PNAd glycoforms of similar size that react with mAb MECA-79.
Both VAP-1 and PNAd are preferentially expressed in the same set of HEV in vivo under normal conditions. Most PLN-type HEV synthesize both VAP-1 and PNAd, but exclusive expression of VAP-1 or PNAd in certain HEV offers a possibility to fine tune tissue-selective lymphocyte-endothelial cell interactions, since the lymphocyte counterreceptors of these two molecules are separate. L-selectin is the only known lymphocyte receptor for PNAd (5), whereas the lymphocyte receptor for VAP-1 is currently unknown. Nevertheless, we have shown that L-selectin is neither sufficient nor necessary to mediate the adhesion of PBL to PLN HEV via VAP-1 (20). Thus, in two morphologically indistinguishable venules, L-selectin-positive lymphocytes can bind either to VAP-1- or PNAd-positive HEV, whereas L-selectin-negative cells cannot interact via PNAd.
VAP-1 mediates T cell subtype-selective recognition of endothelial cells, since anti-VAP-1 mAb largely abolished PLN HEV adhesion of CD8-positive T cells, but had no effect on the binding of CD4-positive cells. In contrast, PNAd is important in PLN HEV recognition of both T cell subtypes, although adhesion of CD8-positive T cells was more efficiently blocked with mAb MECA-79 than that of CD4-positive cells. In the case of PNAd-mediated adhesion, this may relate to the fact that L-selectin is expressed at significantly higher levels on CD8-positive cells than on CD4-positive lymphocytes (30). Recently, data have been emerging that different T cell subpopulations may rely on quite distinctive endothelial adhesion molecules during the first steps of the adhesion cascade. Th1-type CD4 cells, but not Th2-type T cells, have been shown to express a functionally intact P-selectin glycoprotein ligand-1 that allows them to bind to P-selectin and E-selectin and invade inflammatory sites in which a Th1-type response predominates (31, 32). On the other hand, memory-type RO-positive CD4-positive T cells, but not naive CD4-positive cells, have functionally active adhesion receptors that allow them to bind to E- and P-selectin and VCAM-1 under flow conditions (33). Moreover, our results directly imply that CD4-positive cells have to utilize an additional distinct adhesion pathway for PLN HEV binding since the binding of CD4-positive cells was only partially inhibited by anti-PNAd treatment and it was unaffected by mAbs against VAP-1. Together these data show that the specificity of the adhesion cascade may be critically controlled at the lymphocyte subpopulation level, possibly by selective expression of functional receptors for endothelial adhesion ligands.
Based on our results, we propose that during the multistep adhesion cascade most L-selectin-positive CD8-positive cells make simultaneous or successive contacts in PLN HEV between L-selectin and PNAd, and between a non-L-selectin receptor and VAP-1. Normal L-selectin-positive CD8-positive cells probably need both of these contacts for successful adhesion, since inhibition of either interaction leads to pronounced abrogation of PLN adhesion. On the other hand, L-selectin-negative effector cells (recently activated cells that have shed L-selectin and a subpopulation of memory cells (34)) may rely directly on VAP-1-dependent mechanisms in PLN HEV recognition. There is direct evidence that both VAP-1 and PNAd are involved in the early steps of the adhesion cascade that take place under conditions of shear (11, 20). Currently, we believe that PNAd may initiate the contacts between the blood-borne CD8-positive L-selectin-positive cells and endothelium more efficiently than VAP-1, but that VAP-1 is also brought into the play very quickly before the signaling step.
In conclusion, we have shown that VAP-1 and PNAd are distinct adhesion molecules. However, these molecules act in concert to mediate PLN HEV recognition of CD8-positive PBL in humans. These findings cast new light on the regulation of tissue-selective endothelial binding of distinct lymphocyte subpopulations during the adhesion cascade.
Note added in proof. The 170-kDa form of PNAd is podocalyxin (Sassetti, C., K. Tangemann, M. S. Singer, D. B. Kershaw, and S. D. Rosen. 1998. Identification of podocalyxin-like protein as an HEV ligand for L-selectin [abstr.]. In Molecular Mechanisms of Leukocyte Trafficking. Keystone Symposia, Lake Tahoe, NV, p. 41 (Abstr. 214)).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marko Salmi, MediCity Research Laboratory, Tykistökatu 6A, FIN-20520 Turku, Finland. E-mail address:
3 Abbreviations used in this paper: HEV, high endothelial venule; GlyCAM-1, glycosylation-dependent cell adhesion molecule-1; MACS, magnetic activated cell sorting; PLN, peripheral lymph node; PNAd, peripheral lymph node addressin; VAP-1, vascular adhesion protein-1.
Received for publication September 18, 1997. Accepted for publication February 3, 1998.
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