| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Laboratories of
*
Tumor Immunology and
Immunobiology, Pacific Northwest Research Institute, Seattle, WA 98122
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Isolation of cDNA encoding CD83 revealed that it is a 45-kDa type 1 membrane glycoprotein member of the Ig superfamily (2). It is composed of a single extracellular V-type Ig-like domain, a transmembrane region, and a 40-aa short cytoplasmic domain. The CD83 structure is similar to that of several other members of the Ig superfamily. CD83 shows highly restricted cellular expression, it shares 23% overall identity with myelin protein Po, the most abundant glycoprotein in the peripheral myelin of mammals (6, 7), and has significant homologies with the B7 ancestral gene family that includes B-G, butyrophilin, MOG, BT, BT2, B7c, B7-1, and B7-2 (8, 9, 10, 11). However, its function is not known. We now present data suggesting that CD83 mediates adhesion of DC to circulating monocytes and to a fraction of activated T cells or stressed T cells by a specific binding of CD83 to a 72-kDa counterreceptor (ligand). We further show that CD83Ig binding to its ligand is eliminated by neuraminidase, an enzyme specific for the most common sialic acid, N-acetylneuraminic acid. Thus, CD83Ig binds to a carbohydrate epitope that depends on sialic acid residues. This classifies CD83 as a sialic acid-binding Ig-like lectin (Siglec; Ref. 12). Our data further suggest that the formation of the carbohydrate epitope recognized by CD83 is influenced by cell growth conditions and can be rapidly altered by cellular stress and early transition to apoptosis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A population highly enriched for DC was isolated from 200 ml of human peripheral blood by discontinuous Nycodenz gradient centrifugation, as described elsewhere (13). Nycodenz was purchased as Nycoprep (13% (w/v) Nycodenz, 0.58% (w/v) NaCl, 5 mM Tris-HCl, pH 7.2, density = 1.068 ± 0.001, 335 ± 5 mOsm/kg) from Nycomed Pharma (Oslo, Norway). At the end of the purification procedure, RNA was directly extracted from DC by TRIzol (Life Technologies, Grand Island, NY) and reverse transcripted (Superscript II; Life Technologies). cDNA from DC was amplified with PCR primers containing a 5' HindIII site: gaataagctt atg tcg cgc ggc ctc cag ctt ctg ctc c and a 3' BglII site in the antisense primer: gag cca gca gca gga gaagatctt ccg ctc tgt att tc. The PCR product (457 bp) was cloned into pCDNA1 human IgG1 (a gift from Robert Peach, Bristol Myers Squibb Pharmaceutical Institute, Princeton, NJ). DNA from recombinant colonies was amplified by Qiagen plasmid maxi kit (Qiagen, Valencia, CA), sequenced, and transfected into COS7 cells. After 3 days, the presence of soluble protein in cell supernatant was checked by Western blot analysis and the fusion protein was purified by protein A-Sepharose 4B affinity chromatography (Zymed, South San Francisco, CA). Stable transfectants were generated in Chinese hamster ovaries cells by using CD83Ig cDNA cloned into pD18 (14).
CD83 retrovirus construction and generation of transfected cell line
CD83 cDNA was cloned into pLNCX vector (15). DNA from recombinant colonies was amplified by Qiagen plasmid maxi kit and transfected into ecotropic packaging cells (PE501) by using a calcium phosphate method (16). PE501 viral supernatant was used to infect PG13 cells, a primate-specific packaging line. PG13 supernatant was harvested, filtered, and used to infect 1C, a colon carcinoma line derived in our laboratory. Recombinant colonies were selected by G418 (Life Technologies).
Media for cell culture and flow cytometry
Cells were cultured with a standard medium (referred to as RPMI medium), which consisted of RPMI 1640 (Life Technologies) supplemented with glutamine (1%; Life Technologies), penicillin/streptomycin (1%; Life Technologies), and 10% FCS (Atlanta Biological, Norcross, GA). All labeling for flow cytometry was conducted at 4°C in a medium that consisted of DMEM (Life Technologies) supplemented with 5% FCS without azide (referred to as DMEM medium). In some experiments, this medium was supplemented with 0.6 M sucrose (Sigma-Aldrich, St. Louis, MO).
Purification of PBL and of monocytes
PBMCs (5–10 x 107) were isolated from 50–100 ml fresh blood from healthy donors by sedimentation in Ficoll-Paque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden) and washed twice in RPMI medium. For experiments involving T cell activation, the PBMCs were resuspended in RPMI medium and stimulated with Abs or with Ab-conjugated beads as described below.
Induction of cellular stress and apoptosis
Induction of cellular stress of HPB-ALL cells was conducted in four different ways: 1) by incubating the cells in a T75 culture flask with an airtight lid for 2–4 days in RPMI medium without HEPES buffer; 2) by suspending 2–5 x106 cells in 1 ml of RPMI medium, seeding them into six-well plates, and exposing them for 4 min to UV irradiation by an antimicrobial UV lamp placed 50 cm above the cells inside a biosafety cabinet; 3) by incubating the cells in culture medium at different pH (6–7.4) or in culture medium supplemented with 25 mM HEPES buffer (Life Technologies); or 4) by exposing the cells to oxidative stress. This was accomplished by adding to the medium 2-fold serial dilutions of hydrogen peroxide (10 mM, 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM) and incubating 2–5 x106 cells for 10 min at 37°C. After two washes, the cells were incubated in RPMI medium at 37°C until labeling 1 h to 7 days later.
Jurkat cell apoptosis was induced by anti-CD95 (Fas) mAb from Beckman Coulter (Palantine, IL). Petri dishes were coated with 1 µg/ml anti-murine IgM mAb from Beckman Coulter in bicarbonate buffer (Sigma) for 2 h at 37°C. Jurkat cells were washed three times with PBS and incubated for 1 h with 4-fold serial dilutions anti-Fas mAb. After two washes with culture medium, anti-Fas-coated Jurkat cells were incubated in the anti-murine IgM-coated petri dish in culture medium at 37°C overnight.
Flow cytometry analysis
Monoclonal Abs recognizing the following Ags were used: CD83 (HB15A), IgG1 and IgG2a isotype controls from Immunotech, and CD11b (17), CD4, CD8, and CD3 from BD PharMingen (Lexington, KY). To detect apoptosis, we used an annexin V kit (Beckman Coulter) according to the manufacturer’s instructions. CD83Ig, CD80Ig (14), and CD40Ig (18) fusion proteins were biotinylated with EZ-Link N-hydroxy-succinimi-biotin (Pierce, Rockford, IL) kit according to the manufacturer’s procedure. In some experiments cells, the HPB-ALL line were incubated with neuraminidase (Sigma) for 15 min at 37°C (1 U/5 x 106cells) or with neuraminidase together with 2.5 mg/ml of a sialidase inhibitor (2,3-dehydro-2-deoxy-N-acetylneuraminic acid, Sigma). Cells were washed twice in DMEM medium and labeled at 4°C for 45 min with 1 µg/ml biotinylated CD83Ig followed by two washes in DMEM medium and labeled for 15 min at 4°C with 3 µl/100 µl of PE streptavidin (BD PharMingen).
Cold competition experiments
Cells or beads were washed twice and incubated for 15–30 min at 4°C with 50 µg/ml unlabeled CD83Ig or with 20 µg/ml anti-CD83 mAb in DMEM medium. The cells then were labeled with PE-conjugated anti-CD83 mAb or with 1 µg/ml biotin-conjugated CD83Ig as described previously in DMEM medium. In some experiments, incubations were carried in DMEM medium that had been supplemented with 2-fold serial dilutions of sucrose (from 0.6 to 0.1 M).
Labeling of beads
A total of 50 µg of material to be labeled (CD83Ig, anti-CD83 mAb, and a mixture of anti-CD28 mAb and anti-CD3 mAb) were conjugated to magnetic beads (Tosylativated Dynabeads M-450; Dynal, Lake Success, NY) according to a published protocol (19). Beads conjugated with CD83Ig or anti-CD83 mAb were used to test the specificity of CD83Ig fusion protein. Anti-CD28/anti-CD3 mAb-conjugated beads were used to stimulate T cells.
T cell stimulation with anti-CD28/anti-CD3 mAb-coated beads
PBMC were incubated 5 days with conjugated or nonconjugated beads (control) in RPMI medium at 37°C. After that time, the beads were magnetically removed and the cells resuspended in RPMI medium supplemented with 10 IU/ml of rIL-2 (Roche Molecular Biochemicals, Indianapolis, IN) and cultivated for 2 wk.
Cell lysate preparation
HPB-ALL (5 x 107–10 8) cells were washed three times in ice-cold PBS, resuspended in 4 ml of lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail tablets, complete, mini; Roche Molecular Biochemicals), and incubated on ice for 30 min. Lysates were centrifuged for 30 min at 12,000 x g at 4°C, and the supernatants were harvested. In some experiments, HPB-ALL cells were treated by neuraminidase before lysis, as described in the flow cytometry analysis section.
Immunoprecipitation
Supernatants of cell lysates were incubated for 1 h with 50 µl of streptavidin-Sepharose 4B conjugate (Zymed) at 4°C. Streptavidin-Sepharose was removed by centrifugation, and 1 ml of lysate was incubated overnight at 4°C with 50 µg of biotinylated CD83Ig. Immunoprecipitated proteins were separated by 3 h of incubation with streptavidin-Sepharose followed by six washes with 1 ml of lysis buffer and one wash with PBS. Streptavidin-Sepharose was then resuspended in 2x SDS sample buffer (Novex, San Diego, CA) with 1% 2-ME and boiled for 10 min, after which 20 µl of the supernatant was loaded on a 4–12% gradient gel (Novex). Subsequently, the migration gel was blotted and probed as described in the following section.
Western blotting analysis
Supernatants of cell lysates, streptavidin-Sepharose-purified samples, and CD83Ig were eluted in SDS-PAGE sample buffer containing 2-ME and then boiled. CD83Ig (10 µg) was also reduced with 10 mM DL-DTT (Sigma) for 30 min at 37°C and free sulfhydryl residues were alkylated with 25 mM of iodoacetamide, pH 8 (Sigma), for 1 h at 37°C. Samples were run on a tris-glycine 4–12% gradient gel (Novex). The amount of proteins in cell lysates was quantified with micro bicinchoninic acid protein reagent kit (Pierce) according to the manufacturer’s instructions. After migration proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Novex), they first were probed with biotinylated CD83Ig 0.5 µg/ml, washed four times (WesternBreeze; Invitrogen, Carlsbad, CA), and then probed with 1:5000 streptavidin-HRP (BD PharMingen). The signal was detected by ECL (Amersham Pharmacia Biotech) according to the manufacturer’s protocol and quantified by using OptiQuant version 03.00 (Packard Instruments, Meriden, CT).
Adhesion assays
Wild-type 1C cultured human carcinoma cells and CD83-transfected
cells obtained from these (1C/CD83) were seeded at 5 x
105 cells/well into a 48-well plate (Costar,
Cambridge, MA) and incubated in culture medium at 37°C for 24 h
to allow their adhesion. After 24 h, the plates were washed one
time with fresh medium to remove nonadherent cells. Nonstressed or
HPB-ALL cells stressed by growth at low oxygen level were labeled with
50 µCi of 51Cr (Amersham Pharmacia Biotech) for
45 min at 37°C then washed twice and resuspended in RPMI medium at
2.5 x 106 cells/ml in the presence of 10
µg/ml anti-
2 integrin mAb 60.3
(20) or with 10 µg/ml anti-CD83 mAb. Next, 2-fold
serial dilutions of 51Cr-labeled HPB-ALL cells
were distributed and incubated for 1–4 h at 37°C with 1C or 1C/CD83
cells. Finally, the cells were washed once with PBS and once according
to a procedure derived from gravity flow wash (21).
According to this procedure, the plates were immersed in PBS in a large
container and suspended upside down above the bottom of the container
for 15 min to allow nonadherent cells to detach. They then were turned
slowly right side up, were removed from the container, and the PBS in
each well was removed by aspiration. The adherent cells remaining after
this procedure were lysed by 100 µl of PBS plus 0.2% Triton X-100
(Fisher Scientific, Fairlawn, NJ). Lysates (40 µl) were transferred
into LumaPlate-96 plates (Packard Instruments) and counted with a
Top-Count NXT (Packard Instruments).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We constructed a CD83Ig fusion protein as described in
Materials and Methods (Fig. 1
A). It was engineered without
an Ig hinge region between the coding sequence for CD83
extracytoplasmic domain (432 bp) and the CH2 and CH3 domains and
contains two mutations, one at 231 bp, transforming valine to proline,
and the other at 531 bp, transforming a proline to a serine. These
structural modifications eliminated the binding to FcR. CD83Ig did not
bind to cells expressing Fc
RI (U937), FcRII (normal B cells and B
cell leukemia lines, Raji, Ramos, Bjab), or FcRIII (blood CD16 plus
NK cells; data not shown). To check the specificity and proper folding
of the CD83Ig fusion protein, experiments were performed that showed
that PE-labeled anti-CD83 mAb bound to CD83Ig-conjugated beads and
that a PE-labeled isotype control mAb did not (Fig. 1B). The
binding of PE-labeled anti-CD83 mAb to the CD83Ig-conjugated beads
was partially blocked by preincubation with an unlabeled anti-CD83
mAb (20 µg/ml) for 15 min at 4°C (Fig. 1B). Conversely,
CD83Ig bound to anti-CD83 mAb-conjugated beads whereas CD40Ig did
not bind (Fig. 1C). The binding of CD83Ig to beads
conjugated with anti-CD83 mAb was completely blocked by
preincubation with an unlabeled anti-CD83 mAb (20 µg/ml) for 15
min at 4°C (Fig. 1C, picture 2). 2-ME
incompletely reduced CD83Ig, which migrated as a mixture of a 60-kDa
monomer and a 120-kDa dimer (Fig. 1D, lane 1).
However, after reduction with DTT and alkylation with iodoacetamide,
CD83Ig migrated as a single band of an 
98-kDa monomer (Fig. 1D, lane 2).
|
According to flow cytometry analysis of fresh PBMC, biotinylated
CD83Ig was found to bind to <1% of CD3+ cells
and to 4% of CD3- cells in the
lymphocyte-scatter gate (gate 1), whereas biotinylated CD40Ig used as
control did not bind (Fig. 2, B and C). In the larger cell-scatter gate (gate
2), biotinylated CD83Ig bound to >75% of cells that expressed CD11b
(Fig. 2D), CD4 low+ (data not shown),
and CD14 (Fig. 2E), i.e., cells with the distinctive
characteristics of circulating monocytes. When CD83Ig labeling was
performed in the presence of anti-CD83 mAb (HB15A), the binding of
CD83Ig to the CD14+ cells consistently increased
up to 90% (Fig. 2F), whereas an isotype control mAb did not
increase the binding of CD83Ig to these cells. The binding of CD83Ig to
monocytes was specific, because biotinylated CD40Ig did not bind at all
(data not shown). This suggests that the HB15A mAb binding epitope is
distinct from the active binding site of CD83 ligand, consistent with
the lack of function described for this anti-CD83 mAb. The CD83Ig
binding to monocytes decreased after 90 min of culture (Fig. 3, A and B). This
decrease was less in the presence of the anti-CD18
(2 integrin) Ab 60.3 (Fig. 3, C and
D), which blocks the adhesion of monocytes to plastic
(22, 23, 24); because adhesion induces monocyte activation,
this suggests that expression of the CD83 ligand on monocyte correlates
with a resting stage. In contrast, the binding of CD83Ig to
CD3+ T lymphocytes increased from <1% for
resting cells (Fig. 2C) to 6% for cells activated by 2 wk
of culture with anti-CD3/anti-CD28-conjugated beads (Fig. 4B).
CD3+CD8+ T lymphocytes
bound to CD83Ig (Fig. 4C), whereas
CD3+CD4+ T lymphocytes did
not (Fig. 4D). A total of 90% of the cells binding to
CD83Ig were costained with annexin V (Fig. 4E) as compared
with <6% of the cells binding to CD80Ig, used as control (Fig. 4F). Altogether, these data indicate that CD83Ig binds to a
ligand the expression of which is regulated by cell activation and
apoptosis. No binding of CD83Ig was found on CD56-positive NK cells,
nor to granulocytes or erythrocytes (data not shown).
|
|
|
The binding of CD83Ig to B, T, myeloid, and monocyte cell lines
was examined. CD83Ig did not bind to any of four B cell lines (Nalm6,
Reh, Bjab, DHL10), or to two myeloid cell lines (Thp1, HL60) or to the
monocyte cell line U937 (data not shown). Similarly, no binding was
observed to Molt4, CEM, Hut78, or Jurkat cells in exponential growth.
However, CD83Ig did bind to HPB-ALL cells (25) in
light-scatter gates 1 and 2 (Fig. 5, A and B). Interestingly, we found that CD83Ig
binding increased after 24 h of incubation in a flask with a
tightly closed lid, and this increase was greater for cells in gate 2
than for cells in gate 1 (Fig. 5, C–F). This
suggested that expression of the CD83 ligand might be regulated by cell
growth conditions and/or by cellular stress and led us to study the
effect of cellular stress, including apoptotic events, on CD83Ig
binding. The increase of CD83Ig binding on HPB-ALL cells in gate 2 was
not induced by UV irradiation, by hydrogen peroxide, or by incubation
in a tight-close lid flask in the presence of HEPES (data not shown).
This suggested that an additional condition was required to increase
the binding of CD83Ig. Additional experiments demonstrated that the
CD83Ig binding increase in gate 2 was correlated with the pH of the
culture medium. When cells were incubated in a tight-close lid flask at
pH 7, 10% of HPB-ALL cells bound to CD83Ig in gate 2 (Fig. 5E), whereas at pH 6.5 the binding increased up to 44%
(Fig. 5F). In contrast, in gate 1 the binding of CD83Ig to
HPB-ALL cells did not significantly vary with the pH (Fig. 5, C and D). Finally, 5–6% of CD83Ig-bearing cells
in gate 1 (Fig. 5, C and D) and none of the
CD83Ig-bearing cells in gate 2 (Fig. 5, E and F)
were labeled by annexin V. Thus, CD83Ig binding to HPB-ALL cells
depends on the pH of the cell medium and is not dependent on apoptosis,
because the cells that bound CD83Ig did not bind annexin V.
|
, G–I). Unlabeled CD83Ig blocks biotinylated CD83Ig binding in hypertonic medium but not in DMEM medium
We were unable to block the binding of biotin-conjugated CD83Ig to
HPB-ALL cells with unconjugated CD83Ig and therefore hypothesized that
there could be a rapid, receptor-mediated internalization of the fusion
protein, as described in some other systems (27). Thus, we
tested CD83 ligand endocytosis in the presence of a high sucrose
hypertonic medium known to block internalization by preventing
clathrin-coated pit formation (28). Fig. 6 shows that it was possible to block the
binding of biotin-conjugated CD83Ig with unlabeled CD83Ig in the
presence of 0.6 M sucrose and that the blocking was proportional to the
sucrose concentration.
|
Lysates of HPB-ALL cells were immunoprecipitated with
biotin-conjugated CD83Ig and collected on streptavidin-Sepharose beads.
After washing, the beads were eluted with SDS sample buffer and
separated on tris-glycine 4–12% gradient gels (Fig. 7). After transfer, filters were blotted
with biotinylated CD83Ig. Fig. 7 shows that a 72-kDa molecule binds to
CD83Ig in HPB-ALL cell lysates, which were immunoprecipitated with
biotinylated CD83Ig (lane 1) or directly blotted
without immunoprecipitation (lanes 3–6, 2-fold
serial dilutions). Biotinylated CD83Ig was detected in the control lane
(lane 2). Thus, the ligand epitope detected by CD83Ig
is not destroyed by boiling in SDS.
|
Experiments were performed to test whether the epitope detected by
CD83Ig on the 72-kDa protein was part of the protein itself or was a
carbohydrate attached to the protein. We found that a treatment of
HPB-ALL cells with neuraminidase diminished the CD83Ig binding (Fig. 8B), although treatment with
sialidase usually enhances cell-cell interactions (29, 30)
by removing negatively charged sialic acids. Inhibition of
neuraminidase by a sialidase inhibitor,
2,3-dehydro-2-deoxy-N-acetylneuraminic acid, prevented the
reduction of CD83Ig binding to HPB-ALL (Fig. 8C) as well as
to fresh monocytes (data not shown). Lysates were prepared from HPB-ALL
cells before or after treatment by neuraminidase, and their protein
concentrations were quantified. Even though the protein concentration
of the cell lysates was unchanged, the CD83Ig reactivity with the
72-kDa protein was reduced by 31–37% (Fig. 8D) with
neuraminidase treatment of HPB-ALL cells, as measured by OptiQuant
program (Packard, Meriden, CT). This indicates that the ability of the
CD83 ligand to bind to CD83 is dependent on glycosylation by sialic
acid residues.
|
We used cells from 1C colon cancer line transfected with CD83
retrovirus (1C/CD83; Fig. 9A)
to test their adhesion to HPB-ALL (which express the CD83Ig ligand);
wild-type 1C cells were used as a control. HPB-ALL cells were incubated
for 3 days at pH 7.4 or at pH 6.5, harvested, and labeled with chromium
51. After two washes to remove unincorporated radioactivity,
51Cr-labeled cells were incubated for 1–4 h with
adherent 1C or 1C/CD83 in RPMI medium (pH 7.4) supplemented with either
10 µg/ml of the anti-integrin mAb 60.3 to avoid nonspecific
adherence or with 10 µg/ml of an anti-CD83 mAb. The strongest
binding was observed after 3 h of incubation (Fig. 9, B
and C). After three washes with PBS, adherent cells were
lysed with 100 µl of lysate buffer, and 40 µl was harvested and
counted. HPB-ALL cells showed a higher level of adhesion to 1C/CD83
than to 1C, both in medium (data not shown) and in the presence of 60.3
(Fig. 9B). The anti-CD83 mAb blocked the adhesion of
HPB-ALL cells incubated at pH 6.5 to 1C/CD83 (Fig. 9C). This
adhesion pattern exactly followed the pattern of the CD83 ligand,
indicating that the CD83-CD83 ligand interaction mediates adhesion.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DC can be derived from monocytes by stimulation with IL4 and GM-CSF and/or Ag (32, 33, 34). Because the CD83 ligand disappears quickly during monocyte culture, adhesion between CD83 on DC and its ligand on monocytes may represent a regulatory mechanism to control APC maturation. Conversely, mature DC may stimulate the monocytes to release chemokines and/or cytokines to amplify immune responses.
In cultures of lymphocytes, binding of CD83Ig was seen on 6% of CD8+ T cells after 2 wk of activation through their CD3 and CD28 receptors. This binding increased 2- to 3-fold in the absence of APC, and addition of mature DC but not CD83Ig to the T cell cultures down-regulated the expression of CD83 ligand (data not shown). This suggests that CD83-specific interactions between subpopulations of T cells and DC may be important when the T cells are at a specific stage of maturation. It is not yet known whether or not the activated T cells that expresses the CD83 ligand represent subpopulations of stressed cells; however, activated cells that bound CD83Ig were also annexin V positive. Neither is it known whether interaction of the ligand-positive stressed cells with DC is a mechanism for removal or rescue of such cells.
A recent publication by Cramer et al. (35) suggests that mouse CD83 ligand is expressed by B cells labeled with an anti-CD45R mAb (B220), because mouse CD83Ig bound to B220+ splenocytes from normal BALB/c mice but not to splenocytes from µMT (B cell knockout) mice. In contrast, in human peripheral blood we did not detect any CD83Ig binding to B cells labeled with an anti-CD19 mAb, while in mouse blood we could detect human CD83Ig binding to circulating monocytes (data not shown). In our study, we demonstrated that cellular stress including cell growth at low pH or progression into apoptosis after anti-Fas stimulation can increases the binding of CD83Ig. It is possible that the CD83Ig binding to mouse splenocytes described by Cramer et al. may be related to the stage of B cell activation or cell stress. It is also possible that there are differences between mouse and human leukocytes and that the mutation in human CMP-sialic acid hydroxylase (36) may alter the sialic acid recognition by CD83Ig.
CD83 is structurally related to the B7 ancestral gene family, and its closest homology is 23% of identity with the myelin protein Po, which is an I-type lectin that recognizes a sulfated carbohydrate. Therefore, it is highly interesting that the CD83 ligand contains a sialic acid, classifying CD83 as a siglec i.e., it belongs to a subfamily of I-type lectins that can bind sialic acids and presently includes nine members. All members of the siglec family share a restricted cell expression, to the hemopoietic and immune systems for most of them (37), and to the nervous system for myelin-associated glycoprotein (siglec 4A; Ref. 38). Although four of eight siglecs that have been characterized today share >60% protein sequence homology and are closely linked to 19q13.333–41, the binding of four other siglecs, siglec 3 (CD33; Ref. 39), siglec 5 (40), siglec 8 (41), and siglec 9 (42, 43), varies with respect to both the nature of the sialic acid and its linkage to subterminal sugars (37). The extracellular regions of these siglecs are made up of an N-terminal V-set Ig-like domain followed by a variable number of C2-set domains. Importantly, sialic acid binding depends on the N-terminal V-set domain (44).
We conclude that CD83 is an adhesion receptor that belongs to the siglec family and that its binding site depends on glycosylation on sialic acids of a protein of 72 kDa. We further conclude that formation of the carbohydrate epitope recognized by CD83 is highly sensitive to cell-growth conditions and can be rapidly altered by cell stress and/or early transition to apoptosis. Our data suggest that interaction between CD83 and its ligand plays a role in intracellular communications involving DC, circulating monocytes, and certain populations of activated and/or stressed T lymphocytes.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Nathalie Scholler, Laboratory of Tumor Immunology, Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122.
3 Abbreviations used in this paper: DC, dendritic cell; Siglec, sialic acid-binding Ig-like lectin; PVDF, polyvinylidene difluoride.
Received for publication September 26, 2000. Accepted for publication January 8, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
for use in cancer immunotherapy. Ann. Surg. 226:6.[Medline]
This article has been cited by other articles:
|
|
 |
|
  W. K. Decker, D. Xing, S. Li, S. N. Robinson, H. Yang, D. Steiner, K. V. Komanduri, and E. J. Shpall Th-1 polarization is regulated by dendritic-cell comparison of MHC class I and class II antigens Blood, April 30, 2009; 113(18): 4213 - 4223. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Luthje, B. Kretschmer, B. Fleischer, and M. Breloer CD83 regulates splenic B cell maturation and peripheral B cell homeostasis Int. Immunol., August 1, 2008; 20(8): 949 - 960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Prazma, N. Yazawa, Y. Fujimoto, M. Fujimoto, and T. F. Tedder CD83 Expression Is a Sensitive Marker of Activation Required for B Cell and CD4+ T Cell Longevity In Vivo J. Immunol., October 1, 2007; 179(7): 4550 - 4562. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kuwano, C. M. Prazma, N. Yazawa, R. Watanabe, N. Ishiura, A. Kumanogoh, H. Okochi, K. Tamaki, M. Fujimoto, and T. F. Tedder CD83 influences cell-surface MHC class II expression on B cells and other antigen-presenting cells Int. Immunol., August 1, 2007; 19(8): 977 - 992. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. T. Prechtel, N. M. Turza, A. A. Theodoridis, and A. Steinkasserer CD83 Knockdown in Monocyte-Derived Dendritic Cells by Small Interfering RNA Leads to a Diminished T Cell Stimulation J. Immunol., May 1, 2007; 178(9): 5454 - 5464. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Ding, H. Mehta, W. J. McCune, and M. J. Kaplan Aberrant Phenotype and Function of Myeloid Dendritic Cells in Systemic Lupus Erythematosus J. Immunol., November 1, 2006; 177(9): 5878 - 5889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Hirano, M. O. Butler, Z. Xia, S. Ansen, M. S. von Bergwelt-Baildon, D. Neuberg, G. J. Freeman, and L. M. Nadler Engagement of CD83 ligand induces prolonged expansion of CD8+ T cells and preferential enrichment for antigen specificity Blood, February 15, 2006; 107(4): 1528 - 1536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Rughetti, I. Pellicciotta, M. Biffoni, M. Backstrom, T. Link, E. P. Bennet, H. Clausen, T. Noll, G. C. Hansson, J. M. Burchell, et al. Recombinant Tumor-Associated MUC1 Glycoprotein Impairs the Differentiation and Function of Dendritic Cells J. Immunol., June 15, 2005; 174(12): 7764 - 7772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Klein, S. Koch, B. Borm, J. Neumann, V. Herzog, N. Koch, and T. Bieber CD83 localization in a recycling compartment of immature human monocyte-derived dendritic cells Int. Immunol., April 1, 2005; 17(4): 477 - 487. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ohta, E. Landis, T. Boulay, R. B. Phillips, B. Collet, C. J. Secombes, M. F. Flajnik, and J. D. Hansen Homologs of CD83 from Elasmobranch and Teleost Fish J. Immunol., October 1, 2004; 173(7): 4553 - 4560. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. F. Garcia-Martinez, M. W. Appleby, K. Staehling-Hampton, D. M. Andrews, Y. Chen, M. McEuen, P. Tang, R. L. Rhinehart, S. Proll, B. Paeper, et al. A Novel Mutation in CD83 Results in the Development of a Unique Population of CD4+ T Cells J. Immunol., September 1, 2004; 173(5): 2995 - 3001. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Price, P. S. Sharp, M. E. North, S. J. Rainbow, and S. C. Knight Advanced Glycation End Products Modulate the Maturation and Function of Peripheral Blood Dendritic Cells Diabetes, June 1, 2004; 53(6): 1452 - 1458. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Senechal, A. M. Boruchov, J. L. Reagan, D. N. J. Hart, and J. W. Young Infection of mature monocyte-derived dendritic cells with human cytomegalovirus inhibits stimulation of T-cell proliferation via the release of soluble CD83 Blood, June 1, 2004; 103(11): 4207 - 4215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yang, Y. Yang, J. Raycraft, H. Zhang, S. Kanan, Y. Guo, Z. Ronai, I. Hellstrom, and K. E. Hellstrom Melanoma cells transfected to express CD83 induce antitumor immunity that can be increased by also engaging CD137 PNAS, April 6, 2004; 101(14): 4990 - 4995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Munster, K. P. A. MacDonald, M. Kato, and D. J. N. Hart Human T lymphoblasts and activated dendritic cells in the allogeneic mixed leukocyte reaction are susceptible to NK cell-mediated anti-CD83-dependent cytotoxicity Int. Immunol., January 1, 2004; 16(1): 33 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Schmitz, L. K. Clayton, and E. L. Reinherz Gene expression analysis of thymocyte selection in vivo Int. Immunol., October 1, 2003; 15(10): 1237 - 1248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hinz, P. Lemke, I. Anagnostopoulos, C. Hacker, D. Krappmann, S. Mathas, B. Dorken, M. Zenke, H. Stein, and C. Scheidereit Nuclear Factor {kappa}B-dependent Gene Expression Profiling of Hodgkin's Disease Tumor Cells, Pathogenetic Significance, and Link to Constitutive Signal Transducer and Activator of Transcription 5a Activity J. Exp. Med., September 2, 2002; 196(5): 605 - 617. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Zimmer, A. T. Larregina, C. M. Castillo, S. Capuano III, L. D. Falo Jr, M. Murphey-Corb, T. A. Reinhart, and S. M. Barratt-Boyes Disrupted homeostasis of Langerhans cells and interdigitating dendritic cells in monkeys with AIDS Blood, April 15, 2002; 99(8): 2859 - 2868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Scholler, M. Hayden-Ledbetter, A. Dahlin, I. Hellstrom, K. E. Hellstrom, and J. A. Ledbetter Cutting Edge: CD83 Regulates the Development of Cellular Immunity J. Immunol., March 15, 2002; 168(6): 2599 - 2602. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Arrode, C. Boccaccio, J.-P. Abastado, and C. Davrinche Cross-Presentation of Human Cytomegalovirus pp65 (UL83) to CD8+ T Cells Is Regulated by Virus-Induced, Soluble-Mediator-Dependent Maturation of Dendritic Cells J. Virol., January 1, 2002; 76(1): 142 - 150. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
