(Received for publication, November 30, 1994; and in revised form, January 13, 1995)
From the
We previously showed that cultured human umbilical vein
endothelial cells (HEC) exposed to the inflammatory cytokines tumor
necrosis factor- or interleukin-1 display increased activity of
-galactoside
2,6-sialyltransferase. This is associated with
enhanced expression of ligands for the B cell receptor CD22
, which
recognizes
2-6-linked sialic acids (Hanasaki, K., Varki, A.,
Stamenkovic, I., and Bevilacqua, M. P.(1994) J. Biol. Chem. 269, 10637-10643). Here we report that increased expression
of CD22 ligands is a feature of dermal microvascular endothelial cells
as well, and is also observed in response to the cytokine
interleukin-4. Tumor necrosis factor-
stimulation of HEC causes no
change in the profile of endothelial glycoproteins recognized by CD22,
but doubles the proportion of total cellular N-linked
oligosaccharides capable of binding tightly to CD22. This modest change
is sufficient to cause a marked increase in
2-6-linked
sialic acid-dependent binding of Chinese hamster ovary (CHO) cells
expressing recombinant human CD22. In contrast, B lymphoma cell lines
expressing higher levels of cell surface CD22 do not show such sialic
acid-dependent binding to activated HEC. Since B lymphoma cells
themselves also express high levels of
2-6-linked sialic
acids, their CD22 molecules might be rendered nonfunctional by
endogenous ligands. In support of this, the lectin function of CD22 can
be directly detected on transfected CHO cells, but not on B lymphoma
cells. Furthermore, coexpression of
-galactoside
2,6-sialyltransferase with CD22 in the CHO cells abrogates sialic
acid-dependent binding to cytokine-activated HEC. However, such
co-transfected cells can bind to B lymphoma cells in a manner
apparently less dependent upon
2-6-linked sialic acid,
suggesting CD22-mediated interactions that may not be directly
dependent on its lectin function. Thus, CD22-mediated interactions
between B cells and activated vascular endothelium may be positively
regulated by induction of
2-6-linked sialic acid-bearing
endothelial cell ligands, but negatively regulated by such ligands on
the B cells expressing CD22. Since expression of both CD22 and
-galactoside
2,6-sialyltransferase are regulated during B
cell ontogeny, these findings could be of importance in B cell function
and/or trafficking.
Sialic acids (Sias) ()are a family of nine-carbon
carboxylated sugars found at terminal positions of mammalian cell
surface sugar chains(1, 2) . Because of their location
and negative charge, Sias can inhibit cell-cell interactions by
nonspecific (2, 3) or specific (4) mechanisms.
However, they can also serve as ligands for specific cell-cell
recognition molecules such as the selectins, sialoadhesin, and
CD22(5, 6, 7, 8, 9, 10, 11, 12) .
CD22 is a B cell-restricted glycoprotein whose sequence defines it as a
member of the immunoglobulin (Ig) superfamily. CD22
is the larger
of two human CD22 isoforms identified, containing two additional Ig
domains (numbers 3 and 4) not present in the shorter isoform
CD22
(13) . Early studies indicated that CD22 may function
in B cell activation(14, 15) , and its cytoplasmic
domain becomes rapidly phosphorylated following B cell stimulation with
anti-µ(16) . Additionally, CD22 can function as an adhesion
molecule, mediating interactions with activated blood cells and
accessory cells(13, 17, 18, 19) . A
soluble chimeric form of CD22
(CD22Rg), containing the three
amino-terminal Ig-like domains of human CD22
fused to the
COOH-terminal Fc domains of human IgG can bind and precipitate
potential glycoprotein ligands on activated T and B cells, one of which
is the tyrosine phosphatase
CD45(9, 13, 20, 21) . Sialic acid
(Sia) is an essential component of recognition by CD22(13) .
Several studies have established that the structural motif specifically
recognized is Sia
2-6Gal
1-4GlcNAc- (9, 13, 19, 21) (see also the
accompanying paper(22) ), which is found in varying numbers on
the antennae of N-linked oligosaccharides of some cell surface
glycoproteins(1, 23) . In contrast,
2-3-linked Sia-containing structures found on the same
oligosaccharides are not recognized.
Many different
sialyltransferases have been identified that can transfer Sia residues
onto glycoprotein oligosaccharides(23, 24) . Each
shows specificity not only for the linkage formed (2-3,
2-6, or
2-8), but also for the acceptor
structure. The enzyme responsible for synthesizing the structure
recognized by CD22 is
-galactoside
2,6-sialyltransferase
(ST6N), which catalyzes the reaction: CMP-Sia +
Gal
1-4GlcNAc
1-
CMP +
Sia
2-6Gal
1-4GlcNAc
1-(25, 26) .
ST6N expression is increased in the liver during inflammatory responses
and shows a regulated expression in other
tissues(24, 27, 28) . This results at least
in part from the activity of different 5` promoter elements, including
a lymphocyte-specific promotor(29) . Some cell surface epitopes (e.g. CD75 and CD76) defined by monoclonal antibodies which
demonstrate specific histological patterns in lymphoid tissues have
also been shown to depend upon the presence of
2-6-Sias and
the expression of
ST6N(30, 31, 32, 33, 34) .
Activation of vascular endothelium by cytokines and bacterial
products results in a coordinated display of new cell-surface
glycoproteins including the cell adhesion molecules E-selectin, ICAM-1,
and VCAM-1 (35, 36, 37) . We have previously
shown that inflammatory cytokines, tumor necrosis factor-
(TNF-
) and interleukin-1 (IL-1), as well as lipopolysaccharide
(LPS), act on human umbilical vein endothelial cells (HEC) to induce
increased expression of cellular ST6N(38) . This is accompanied
with enhanced expression of
2-6-linked Sias (detected by Sambucus nigra agglutinin, SNA) and an increase of CD22
ligands (detected by
2-6-linked Sia-dependent binding of
soluble CD22Rg)(38) . Here, we explore if this increased
binding of CD22Rg to cytokine-activated HEC is mediated by newly
synthesized glycoproteins or by an increase in CD22 binding to N-linked oligosaccharides. Also, while CD22-dependent cell
adhesion events involving cytokine-activated HEC can be positively
regulated by induction of
2-6-linked Sia-bearing endothelial
cell ligands, we demonstrate negative regulation of CD22 by
2-6-sialylated ligands on B cells expressing CD22. The
latter fits well with the recent work of Braesch-Andersen and
Stamenkovic(39) , who showed that CD22 molecules bearing
2-6-linked Sia are functionally inactive.
The CD22 binding activity of oligosaccharides was
performed as described
previously(21, 42, 43) . Briefly, CD22Rg
columns were constructed by adsorbing 1 mg of CD22Rg to 0.75 ml of
protein A-Sepharose in a siliconized 1-ml polystyrene pipette. Samples
of H-labeled oligosaccharides (originating from 18 µg
of endothelial cell protein) were mixed with the non-binding marker
[
C]ManNAc, and then applied to the column at 4
°C in Tris-buffered saline (20 mM Tris-HCl, pH 7.3, 140
mM NaCl, 0.02% sodium azide). Three drop fractions (about 80
µl) were collected for 19 fractions (as indicated in the text), the
column warmed to room temperature for 15 min and then eluted for a
further 21 fractions with buffer at room temperature.
For some blocking experiments with
2-6-sialyllactose (
2-6-sLac), an alternative
quantitative assay was performed. Transfected CHO cells were cultured
in a 48-well plate to confluency, and washed three times with PBS
before assay. Daudi cells (2
10
/ml) were cultured
in RPMI, 10% FCS supplemented with 5 µCi/ml
[methyl-
H]thymidine (ICN Biomedicals,
Inc.) for 48 h. After washing extensively with PBS, 2 mM EDTA,
1% BSA,
H-labeled Daudi cells were resuspended in PBS, 0.5%
BSA, 2 mM EDTA, 4 mM MgCl
. Daudi cell
suspensions were mixed with various concentrations of
2-6-sLac, added to the CHO cells and incubated under
rotation (100 rpm) for 30 min at 4 °C. After washing 4 times with
ice-cold PBS, the bound cells were solubilized with 0.1% Nonidet
P-40/PBS, and their radioactivity was counted.
HEC are commonly used model cells for
the study of endothelial cell biology. To establish that the biological
responses we observed are a more general feature of vascular
endothelial cells, we examined microvascular endothelial cells derived
from human neonatal foreskin. As shown in Fig. 1, treatment of
these cells with TNF- resulted in increased CD22 ligand expression
on these cells. As with HEC, induction of CD22Rg ligands is both dose-
and time-dependent, with a half-maximal effect at a similar
concentration (
2 units/ml) of TNF-
(38) . Thus,
increased CD22 ligand expression in response to cytokines is not a
unique feature of human HEC, but may be a general feature of
endothelial cell activation.
Figure 1:
Increase in CD22Rg binding upon
activation of human dermal microvascular endothelial cells with
TNF-. Confluent microvascular endothelial cells, stimulated for 48
h with various concentrations of TNF-
as indicated, were incubated
with CD22 mRg. After washing, cells were incubated with
peroxidase-conjugated goat anti-mouse IgG Ab and binding was detected
as described under ``Experimental Procedures.'' The data are
the mean ± S.E. of triplicate
determinations.
Figure 2:
Identification of CD22-binding proteins in
quiescent and cytokine-activated HEC by affinity precipitation. HEC
were incubated with or without 200 units/ml TNF- in the medium
containing [6-
H]glucosamine for 72 h. The cell
lysates were precipitated with CD22Rg or P-selectin-Rg and PAS. Bound
proteins were resolved by SDS-polyacrylamide gel (4-20%)
electrophoresis and detected by fluorography. CD22Rg precipitates of
lysates derived from unstimulated (lane 1) and
TNF-
-stimulated HEC (lane 2), and P-selectin-Rg
precipitates derived from unstimulated (lane 3) and
TNF-
-stimulated HEC (lane 4) were shown. Molecular mass
markers (kDa) are indicated.
Figure 3:
CD22Rg column affinity chromatography of N-linked oligosaccharides isolated from
[H]glucosamine-labeled HEC
glycoproteins. HEC were incubated with or without 200 units/ml
TNF-
in the medium containing
[6-
H]glucosamine for 72 h. N-Linked
oligosaccharides were released from total glycoproteins using peptide N-glycosidase F, purified, desalted, and concentrated.
Aliquots corresponding to material derived from 18 µg of protein
were mixed with 300 cpm of [
C]ManNAc, and
applied to a CD22Rg-PAS column. Fractions (80 µl) were collected
and their radioactivity monitored. The arrow indicates the
point at which the column was warmed from 4 °C to ambient
temperature. A, unstimulated HEC. B, TNF-
-stimulated HEC.
Figure 4:
Binding of CD22-transfected CHO cells to
HEC. A, binding of wild-type (WT-CHO) and CD22-expressing
(CD22-CHO) CHO cells to resting (open bars) and
TNF--stimulated (closed bars) HEC. Confluent HEC were
stimulated with or without 200 units/ml TNF-
for 48 h.
EDTA-harvested CHO and CD22-CHO cells were added to HEC monolayers (1
10
cells/well) and incubated for 30 min at 4
°C. After washing, the adherent cells were fixed and counted
microscopically. B, effect of
2-6-sLac on
CD22-transfected CHO cell binding to TNF-
-stimulated HEC.
TNF-
-stimulated HEC were pretreated with or without anti-VCAM-mAb,
and incubated with CD22-transfected CHO cells in the absence or
presence of 1.5 mM
2-6-sLac. For A and B, the data are the mean ± S.E. of triplicates of a
representative experiment (n = 3). The level of
expression of CD22 on these CHO transfectants is indicated in Fig. 5.
Figure 5: Flow cytometry analysis of CD22 on transfected CHO and Daudi cells. Wild-type and CD22-transfected CHO cells (EDTA-harvested) and Daudi cells were stained with PE-conjugated anti-CD22 mAb BL22 and analyzed as described under ``Experimental Procedures.'' Similar staining patterns were obtained using another anti-CD22 mAb Leu-14.
Figure 6:
Adhesion of Daudi cells to
TNF--stimulated HEC. Effects of anti-VCAM-mAb and
2-6-sLac on Daudi cell adhesion to TNF-
-activated HEC.
After stimulation with or without 200 units/ml TNF-
for 48 h, HEC
were pretreated with or without anti-VCAM-mAb, and incubated with Daudi
cells (1
10
cells) in the absence or presence of
1.5 mM
2-6-sLac for 30 min at 4 °C. After
washing, the adherent cells were fixed and counted microscopically. The
data are the mean ± S.E. of triplicates of a representative
experiment (n = 3).
Figure 7: Detection of lectin activity of cell-surface expressed CD22. FITC-tagged AGP was used to stain CD22-CHO cells (panel A), Daudi cells (panel B), or WT-CHO cells (panel C). The expression of cell-surface CD22 on the CD22-CHO cells used here is similar to those of Daudi cells (data not shown), although this particular clone contained a subpopulation that are negative, see panel D. This likely explains the incomplete staining seen in panel A.
Figure 8:
Binding of CHO cells transfected with CD22
and/or ST6N to TNF--stimulated HEC. Confluent HEC were stimulated
with 200 units/ml TNF-
for 48 h. EDTA-harvested WT-CHO, ST6N-CHO,
CD22-CHO, and CD22/ST6N-CHO cells were added to HEC monolayers (4
10
cells/well) and incubated for 30 min at 4
°C. After washing, the adherent cells were fixed and counted
microscopically. The data are the mean ± S.E. of triplicates of
a representative experiment (n =
3).
Figure 9:
Binding
of transfected CHO cells to Daudi cells. A, binding of
transfected CHO cells to Daudi cells. Daudi cells were added to
monolayers of parental or transfected CHO cells, and incubated for 30
min at 4 °C. After washing, the adherent cells were fixed and the
total number of bound Daudi cells to 200 CHO cells counted
microscopically. The data are the mean ± S.E. of triplicates of
a representative experiments (n = 3). B, effect of 2-6-sLac on binding of Daudi cells to
transfected CHO cells. [
H]Thymidine-labeled Daudi
cells were incubated with transfected CHO cells in the presence of
various concentrations of
2-6-sLac for 30 min at 4 °C.
After washing, the bound cells were solubilized and their radioactivity
was counted. Results are expressed as percent of total binding obtained
in the absence of
2-6-sLac after subtracting the background
levels (obtained in the binding of
H-labeled Daudi cells to
CHO cells). The data are the mean ± S.E. of triplicate
determinations.
Figure 10:
Cross-binding patterns between different
CHO sublines transfected with CD22 and/or ST6N. Using the wild-type CHO
cells and the three sublines stably transfected to express ST6N
(``ST''), CD22 (``22''), or both
(``22/ST''), a series of binding assays was
performed. The four different cell lines, labeled with
[H]thymidine for 18 h and detached with PBS-EDTA,
were added to confluent monolayer cultures of the four different cell
lines. After adhesion for 30 min on ice, nonadherent cells were washed
away and bound cells quantitated by scintillation counting as described
under ``Experimental Procedures.'' Equal numbers of cells of
approximately equal cpm (±10%) were added; each bar represents the average of triplicate determinations ±
S.D.
The sequential steps of leukocyte rolling, activation,
adhesion, and extravasation into inflammed tissues involves several
receptor-ligand pairs, which are themselves often under control of
several different cytokines(37, 50) . Cultured
endothelial cells such as HEC are frequently employed for studies of
both leukocyte adhesion and cytokine regulation. When exposed to
TNF-, IL-1, or LPS, these cells respond with increased levels of
expression of VCAM-1, ICAM-1, P-selectin, and
E-selectin(37, 50) , and an as yet unidentified
L-selectin ligand(51, 52) . In parallel, stimulation
often results in increased levels and/or activity of adhesion molecules
on monocytes, lymphocytes, and granulocytes(37, 50) .
Although IL-4 is recognized as a lymphocyte cytokine, recent evidence
indicates that it can also activate endothelial cells (45) in a
manner significantly different from that by TNF-
, IL-1, or LPS
(induction of just VCAM-1 and L-selectin ligand, but not the other
adhesion molecules). Moreover, IL-4 stimulation of HEC increases the
adhesion of lymphocytes, basophils, eosinophils, and monocytes, but not
of neutrophils(45) . Thus, the coordinate actions of different
cytokines serves to regulate these essential steps in the inflammation
pathway. Much of this work has focused on the trafficking of
neutrophils or T cells into inflammed tissues, and the migration of T
or B lymphocytes into normal lymphoid organs(37, 50) .
We recently demonstrated that TNF-, IL-1, or LPS stimulation of
HEC cells results in increased levels of expression of ST6N (both mRNA
and enzyme activity) and of total cellular
2-6-linked Sia
residues (38) . The latter can be detected by increased levels
of binding of the lectin SNA, as well as by soluble recombinant CD22Rg,
both of which require
2-6-linked Sia residues for
recognition. Here, we show that the cytokine IL-4 can also induce such
a response, and generalize these results by showing ST6N responses to
cytokines in human dermal microvasculature endothelial cells as well.
CD22Rg specificially precipitates a family of glycoproteins from HEC
cells, including several in the molecular mass range of 130-150
kDa, and some above 200 kDa. The increased binding of CD22 to activated
HEC is not explained by synthesis of new and superior ligands, because
the pattern of precipitated glycoproteins remain unchanged. This
observation contrasts with our previous result that when glycoproteins
of TNF- stimulated HEC cells are stained with the lectin SNA, only
a few new protein bands appear(38) . Notably, while VCAM-1
represents a dominant TNF-
-induced SNA-staining
glycoprotein(38) , it is not prominent in CD22Rg precipitates
of metabolically-labeled glycoproteins. Thus, while identity of the
proteins precipitated from HEC cells by CD22Rg remain unknown, they
seem to represent a subset of the total proteins bearing
2-6-linked Sia residues, and are not primarily the
previously known cytokine-inducible adhesion molecules. This fits well
with our previous observations that some
2-6-sialylated
serum glycoproteins exhibited unexpectedly poor binding affinity to
CD22Rg(42) , and that only certain
2-6-sialylated
glycoproteins from activated lymphocytes or ST6N-expressing COS cells
are precipitated by CD22Rg(9) . Furthermore, as we show in the
accompanying paper(49) , IgM and haptoglobin are the dominant
CD22-binding proteins in human serum, despite the fact that this is a
rich source of many other abundant
2-6-sialylated
glycoproteins. The structural basis for the selective recognition of
2-6-sialylated glycoproteins by CD22 is partly explored in
the accompanying paper(49) .
Since no new
CD22Rg-precipitable glycoproteins are identified after TNF-
stimulation, the increase in CD22Rg binding could be due to increased
levels of constitutively expressed sialylated glycoproteins and/or to
changes in their sialylation. In support of the latter, we observed a
doubling of the content of N-linked oligosaccharides
containing two or more
2-6-linked Sia residues. Direct
binding studies presented in the accompanying paper (22) indicate that such bi-
2-6-sialylated
biantennary oligosaccharides bind to CD22Rg 30-fold better than do
mono-
2-6-sialylated structures, probably owing to the
interaction of the two
2-6-Sia binding sites present on the
bivalent CD22Rg chimera. The increased level of
bi-
2-6-sialylated structures in the activated HEC fits well
with the previously reported increase in ST6N (both activity and
mRNA)(38) . Of course, other factors may also affect the actual
content of
2-6-Sia residues (e.g. competing
glycosyltransferases and oligosaccharide branching), and have not been
examined here.
It is difficult to isolate pure populations of truly
unactivated mature CD22-positive B lymphocytes from humans, and no
cultured cell lines properly recapitulate this phenotype. In
particular, rapidly growing CD22-positive B cell lines are, by their
very nature, ``activated'' and coexpress high levels of
2-6-linked
Sia(30, 32, 33, 34, 53) .
While the latter phenotype is relevant to the biology of the activated
B cell, it is necessary to use transfected cell lines to study the
adhesion properties of CD22 in isolation. Others have previously done
this using transient expression of CD22 in COS cells(39) .
While this can give useful results, the rather high and unpredictable
levels of cell-surface CD22 generated could potentially produce
artifacts. Therefore, we prepared stable transfectants with CHO cells
expressing either the full-length human CD22, the human ST6N enzyme, or
both. To ensure comparability, specific clones were selected that
express CD22 at levels (by flow cytometry analysis) similar to those
seen in B lymphoma cell lines.
As expected from the results using
soluble CD22Rg, CD22-expressing CHO cells bind well to
TNF--stimulated HEC. However, the improvement in binding seen over
the unactivated HEC is much more dramatic than might be expected from
the modest (2-fold) increase in bi-
2-6-sialylated
oligosaccharides (compare Fig. 3with Fig. 4). This may
be because cell adhesion assays are more dependent upon the absolute
density of given receptor-ligand pairs than are enzyme-linked
immunosorbent assay analyses using soluble receptors, i.e. the
``threshold effect''(54) . The inhibition of adhesion
by
2-6-sLac and the lack of binding of nontransfected
wild-type CHO cells to TNF-
-stimulated HEC indicates that other
adhesion molecules expressed by CHO cells are not involved. Thus, the
increase in ST6N in activated endothelium might be sufficient to exceed
the threshold for B cells to bind in vivo.
In the normal
immune system, early activated B cells are expected to carry both CD22
and CD22 ligands on their cell surfaces(13, 55) .
Similar coexpression of both CD22 and its ligands is found with B
lymphoma cells, such as Daudi and Raji cells(13) . Since the B
cells of the mantle zone of secondary follicles in lymph nodes are in
an activated state, express CD22(55) , and also express
2-6-sialylated structures which are potentially ligands for
CD22(30) , it is important to know if CD22 can still mediate
cell adhesion under such circumstances. In this regard, the recent
report of Braesch-Anderson and Stamenkovic (39) demonstrated
that when CD22 is transiently coexpressed with ST6N in COS cells, a
loss of binding to sialylated structures results. Our results here with
CHO cell lines stably expressing CD22 and/or ST6N have confirmed and
extended this observation. In addition, direct probing of the lectin
function of CD22 on cell surface by AGP staining indicates a loss of
function in the Daudi B lymphoma cells. Thus, ST6N expression can
regulate CD22-mediated adhesion both negatively (if expressed on cells
expressing CD22) and positively (if expressed on potential target
cells).
A summary of most of the cell binding results from this
paper is presented in Table 1. In general, the data are
internally consistent, with ST6N expression correlating with binding by
CD22-expressing cells, and doubly positive cells showing lack of
lectin-mediated binding. The only unexpected results were obtained with
Daudi B lymphoma cells, which express both CD22 and ST6N, and do not
demonstrate CD22 function on the surface by direct probing. As expected
from all other results, they do not bind to HEC in a sialic
acid-dependent manner. Despite this, they bind to CHO cells
coexpressing CD22 and ST6N, which are themselves deficient in the
lectin activity (see Table 1). This interaction is dependent upon
the presence of CD22 on the CHO cells (there is no binding to CHO cells
expressing ST6N only). While this interaction was inhibited by high
concentrations of 2-6-sLac, it was not affected by the
coexpression of ST6N with CD22 on the CHO cells. Three possible
explanations of these observations are suggested. First, Daudi cells
may express such high levels of
2-6-sialylation that they
can efficiently compete with the endogenous
2-6-sialylated
structures expressed on the ST6N/CD22-CHO cells. Second, Daudi cells
may express a lymphocyte-restricted sialoglycoprotein ligand capable of
superior binding to very small numbers of active CD22 molecules on the
CD22/ST6N-CHO cells that are not masked by endogenous ligands.
Additionally, Daudi cells are known to express other adhesion molecules
not found on CHO cells(45, 56) , which could be
contributing. Finally, the previously suggested homotypic interaction
between CD22-positive cells (18) might depend upon endogenous
2-6-sialylation (causing a conformational change in the CD22
molecules), which can presumably be blocked by high concentrations of
exogenously added
2-6-sLac. The available data provides
partial support for each of the hypotheses. First, a higher percentage
of N-linked oligosaccharide purified from Daudi cells contain
two and three
2-6-linked Sia residues than N-linked
oligosaccharides from ST6N-expressing CHO cells (Fig. 3). (
)Second, Daudi cells express CD45, which is known to be a
high affinity ligand for CD22, while CHO cells do not. Finally, Daudi
cells do show significant clumping among themselves in undisturbed
cultures. These observations point to the complexities of cell adhesion
processes, and the need for further studies. Regardless, they clearly
establish that cell activation status can regulate CD22-dependent
adhesion events by affecting expression of both CD22 and ST6N.
The
``autoinactivation'' of CD22 lectin activity by endogenous
oligosaccharide ligands reported here and elsewhere (39) is not
without precedent in vertebrate lectin biology. In early studies of the
hepatocyte asialoglycoprotein receptor, it was noted that sialidase
treatment of hepatocytes causes loss of receptor activity because of
binding to newly generated endogenous ligands(57) ; activity
could then be restored by resialylation(58) . It was
subsequently suggested that this might be a natural mechanism to
regulate the activity of this receptor(59) . It is noteworthy
that there are other situations where this could potentially occur. For
example, neutrophils constitutively express L-selectin as well as large
amounts of cell surface sialyl-Lewis, which is considered
to be a low affinity ligand for this receptor(60) . The
possibility that this selectin is partly occupied by these endogenous
ligands has not been explored.
Finally, the relevance of the interaction of CD22-positive cells with activated endothelium needs to be explored. Resting B cells possess CD22 on the cell surface without high level expression of ST6N. During inflammatory processes, these cells could bind to activated vascular endothelium via recognition of CD22 ligands, perhaps to obtain partially processed antigen from tissues, and/or to traffic into the inflammed tissues sites. While these two processes are not part of current dogma concerning B cell trafficking and function, they are now worthy of consideration. However, as discussed in the accompanying paper(49) , such interactions would have to take place in the presence of blood plasma which contains many sialylated glycoproteins, some of which are potent inhibitors of CD22. It appears likely that other pairs of adhesion molecules (e.g. L-selectin and its ligand on activated endothelium) would have to contribute substantially toward an initial binding event.