A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera

Barry D. Hock, Masato Kato,1, Judith L. McKenzie and Derek N. J. Hart,1 Haematology/Immunology Research Group, Christchurch Hospital and Christchurch School of Medicine, Christchurch, New Zealand

Correspondence to: D. N. J. Hart, Mater Medical Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, Queensland 4101, Australia


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD83 is an inducible glycoprotein expressed predominantly by dendritic cells (DC) and B lymphocytes. Expression of membrane CD83 (mCD83) is widely used as a marker of differentiated/activated DC but its function and ligand(s) are presently unknown. We report the existence of a soluble form of CD83 (sCD83). Using both a sCD83-specific ELISA and Western blotting, we could demonstrate the release of sCD83 by mCD83+ B cell and Hodgkin's disease-derived cell lines, but not mCD83 cells. Inhibition of de novo protein synthesis did not affect the release of sCD83 during short-term (2 h) culture of cell lines although mCD83 expression was significantly reduced, suggesting sCD83 is generated by the release of mCD83. Isolated tonsillar B lymphocytes and monocyte-derived DC, which are mCD83low, released only low levels of sCD83 during culture. However, the differentiation/activation of these populations both up-regulated mCD83 and increased sCD83 release significantly. Analysis of sera from normal donors demonstrated the presence of low levels (121 ± 3.6 pg/ml) of circulating sCD83. Further studies utilizing purified sCD83 and the analysis of sCD83 levels in disease may provide clues to the function and ligand(s) of CD83.

Keywords: CD83, dendritic cell, soluble


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD83 antigen is an inducible glycoprotein member of the Ig superfamily whose cell membrane expression is now widely used as a marker of differentiated or activated human dendritic cell (DC) populations (1,2). The cellular expression of CD83 in vivo is associated predominantly with cells of the dendritic lineage(s) including Langerhans cells and dermal DC within skin (1,3), a subpopulation of circulating blood DC (4) and interdigitating DC within lymphoid tissue (1). DC play a central role in the initiation of immune responses and DC maturation, with its associated phenotypic and functional changes, is crucial to this role (5,6). The majority of DC populations, either isolated from tissue (4,7,8) or generated by in vitro culture with granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 (9), are CD83 but rapidly up-regulate expression following in vitro culture or activation. CD83 expression is, however, not strictly DC specific, as it is expressed at low levels by germinal center B lymphocytes in vivo and is inducible, following activation, on isolated B and T lymphocytes (1,8). A recent study in mice has reported the expression of a CD83 ligand by B cells and demonstrated that the presence of recombinant CD83–Ig inhibits T cell activation (10). However, the function of human CD83 and its ligand has yet to be established.

An increasing number of studies have described soluble(s) forms of a structurally and functionally diverse range of cell-surface molecules including adhesion molecules, cytokine receptors, activation antigens and apoptosis-associated antigens (1113). Elevated levels of many of these molecules have been associated with different disease states and there is increasing evidence that they play an important role in the regulation of cellular interactions. A number of these molecules including sIL-6R, sBaff, sSLAM, sCD21 and sFas have been shown to both retain ligand-binding activity and to mediate functional effects (12,1417).

Soluble forms of a number of Ig superfamily members have been described (1821) and a preliminary study had reported that human cells can release a soluble form of CD83 (sCD83) (22). As in vivo generated sCD83 may provide information relevant to CD83 function and any potential ligand(s), we investigated the potential release of sCD83 by human cells. We describe the development of an ELISA for the detection and quantitation of sCD83, and demonstrate the presence of sCD83 in the supernatants of CD83+ cultured cells and normal human sera.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies and immunolabeling
The mAb CMRF-31 (CD14) and CMRF-28 (HLA-DR) were produced in our laboratory. The mAb HB15a (CD83, IgG2b) and TP1.55.3 (CD69, IgG2b) were obtained from Coulter Immunotech (Marseilles, France). The CD19 mAb FMC63 (IgG2a), and the isotype control mAb X63 (IgG1), Sal4 (IgG2b) and Sal5 (IgG2a) were a gift from Professor H. Zola (Child Health Research Institute, North Adelaide, SA, Australia). OKT3 (CD3), HNK-1 (CD57) and L243 (HLA-DR) were produced from hybridomas obtained from the ATCC (Rockville, MD). CD20 mAb, and phycoerythrin (PE)-conjugated antibodies to CD3, CD14, CD16, CD19 and HLA-DR antigens were purchased from Becton Dickinson (San Jose, CA). PEconjugated CD1a (VIT6B) was purchased from Caltag (Burlingame, CA). FITC- and PE-conjugated sheep anti-mouse Ig (FITC–SAM and PE–SAM respectively) were purchased from Silenus (Boronia, Victoria, Australia). Biotinylated goat anti-rabbit Ig (biotin–GAR), horseradish peroxidase (HRP)-conjugated goat anti-mouse Ig (GAM) and HRP-conjugated goat anti-rabbit Ig (GAR) were purchased from Dako (Carpenteria, CA). HRP-conjugated goat anti-human IgG-Fc (GAH) was purchased from ICN (Costa Mesa, CA). Purified human IgG1- and Fc-specific goat anti-human IgG (GAH) were purchased from Sigma (St Louis, MO), and streptavidin–HRP from Amersham Pharmacia Biotech (Auckland, New Zealand).

Labeling for flow cytometry was carried out on ice as described previously (8).

Cell lines
The human cell lines Jurkat (T cell), Mann (Epstein–Barr virus-transformed B cell line), Raji (Burkitt's lymphoma), K562 (myelo-erythroid) and U937 (monocytoid leukemia) were grown in RPMI 1640 (Life Technologies, Auckland, New Zealand) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% FCS (Irvine Scientific, Santa Anna, CA). The Hodgkin's disease (HD)-derived cell line L428 was obtained from Dr V. Diehl (Clinik for Innere Medizine, Cologne, Germany), and the HD-derived cell lines KM-H2 and HDLM-2 (grown in 20% FCS) were obtained from Dr H. G. Drexler (German Collection of Micro-organisms and Cell Cultures, Braunschweig, Germany). In a number of experiments, cell lines were activated by culture in media supplemented with phorbol myristate acetate (PMA; Sigma) at 25 ng/ml.

Cell preparation
Blood was obtained from volunteer donors and tonsils obtained at routine tonsillectomy with appropriate informed consent, according to Ethical Committee guidelines. Peripheral blood mononuclear cells (PBMC) and tonsillar lymphocytes were isolated over a Ficoll-Paque (Amersham Pharmacia Biotech) density gradient, and non-T lymphocyte (ER) and T lymphocyte (ER+)-enriched fractions prepared as described previously (23).

Tonsillar B lymphocytes were activated by culture of the ER fraction (2.5x106 cells/ml) in either media (10% FCS in RPMI 1640) alone or media supplemented with PMA at 25 ng/ml. T lymphocytes were purified from ER+ PBMC fractions by incubation with MHC class II mAb and human complement. Purified preparations were >90% CD3+ as determined by flow cytometry and were cultured (2x106 cells/ml) in either media or media supplemented with PMA at 25 ng/ml. Lymphocyte activation was monitored by immunolabeling with CD25 mAb.

Monocyte derived DC (Mo-DC) were generated from the adherent fraction of PBMC by 6 day culture in recombinant human GM-CSF and IL-4 as described previously (8), then harvested and immature Mo-DC purified by immunodepletion. Briefly, cells were labeled with a mix of CD3, CD19, CD20 and CD57 mAb, and labeled cells removed by magnetic immunodepletion (BioMag magnetic microspheres; PerSeptive Biosystems, Framingham, MA) and FACS sorting (following labeling with FITC–SAM). Purified Mo-DC were then cultured (106 cells/ml in Falcon 24-well plates) for 72 h in media containing GM-CSF (50 ng/ml) and IL-4 (50 ng/ml), and supplemented with either nil, tumor necrosis factor (TNF)-{alpha} (20 ng/ml; Hoffman-La Roche, Basel, Switzerland) or lipopolysaccharide (LPS, 1 µg/ml; Sigma).

Recombinant proteins
To prepare CD83–His, a 390-bp PCR fragment containing the human CD83 extracellular domain was amplified from pHB15 (a gift from T. Tedder, Duke University Medical Center, Durham, NC) (1) with a pair of CD83 gene-specific primers 5'-GAAGATCTACGCCGGAGGTGAAGGTG-3' (at nucleotide 68–85) and 5'-GAAGATCTCTCCGCTCTGTATTTCTT-3' (at nucleotide 425–442) with engineered BglII sites (underlined) using Taq polymerase (Roche Molecular Biochemicals, Auckland, New Zealand). The fragment was digested with BglII and cloned into pQE12 vector (Qiagen, Clifton Hill, Victoria). A CD83-containing clone (pCD83–His) was selected and the DNA sequence confirmed. A pCD83–His transformed XL1-blue (Stratagene, La Jolla, CA) was cultured in the presence of 1 mM isopropyl-ß-D-thiogalactopyranoside for 5 h, and the cells were extracted with 1% Triton X-100 in PBS and CD83–His purified using Ni-NTA agarose (Qiagen) in a non-denaturing condition.

The CD83–Ig construct used a 442-bp PCR fragment amplified from pHB15 with a pair of CD83 gene-specific primers 5'-CCCAAGCTTATGTCGCGCGGCCTCCAG-3' (at nucleotides 11–28) and 5'-GCGAATTCACTTACCTCTCTCCGCTCTGTATTTCTT-3' (at nucleotide 425–442) containing a HindIII and BamHI site (underlined) respectively, and a splicing donor sequence (in italics). The fragment was digested with HindIII and BamHI, then cloned into the pIg-1 vector (24). The resultant plasmid (pCD83–Ig) was transfected into COS-7 cells using Fugene 6 (Roche Molecular Biochemicals), the conditioned medium collected 5–7 days after transfection and the CD83–Ig fusion protein purified using a HiTrap Protein A column (Amersham Pharmacia Biotech). The identity of the CD83–Ig was confirmed by N-terminus amino acid sequencing (Protein Microchemistry Facility, University of Otago, New Zealand).

The IgG equivalent protein concentration of purified CD83–Ig was determined by quantitative ELISA using purified human IgG1 as the standard. Briefly 96-well plates (Maxisorb; Nunc, Roskilde, Denmark) were coated with GAH then, following blocking (2% BSA in PBS), incubated with dilutions of CD83–Ig or purified hIgG1. Following washing and incubation with HRP–GAH, plates were developed using o-phenylenediamine dihydrochloride (OPD; Sigma), the reaction was stopped with H2SO4 and the absorbance read at 492nm.

Recombinant CD40–Ig was kindly provided by Dr M. Widmer (Immunex, Seattle, WA).

Preparation of polyclonal rabbit anti-CD83
A New Zealand white rabbit was immunized with recombinant CD83. After repeated immunizations with CD83–His (n = 2) and CD83–Ig (n = 4) the rabbit was given a final boost with CD83–Ig and the sera collected 12 days later. The Ig fraction was then purified by chromatography using a HiTrap Protein A column. Cross-reactive Ig within the purified fraction was then depleted by passage over human Ig-conjugated Sepharose (Sigma), bovine Ig-conjugated Sepharose (Sigma) and FCS-conjugated Sepharose (HiTrap NHS-activated; Amersham Pharmacia Biotech). The non-absorbed Ig fraction was then used as rabbit anti-CD83 (RA83) in all experiments described below. Control rabbit Ig was prepared from the sera of a non-immunized rabbit in the same manner. The specificity of RA83 was analyzed by ELISA. Briefly 96-well plates were coated with human Ig or Ig constructs (5 µg/ml) then following blocking (5% non-fat dried milk in PBS) incubated with mAb (5 µg/ml) or rabbit Ig (10 µg/ml) diluted in 0.1% BSA in PBS. Following washing, wells were incubated with either HRP–GAM, –GAR or –GAH (1:2000 dilution in 0.1% BSA in PBS), then following further washing, developed with OPD as described above.

sCD83 ELISA
sCD83 levels were determined using a sandwich ELISA. Ninety-six-well plates (Maxisorb; Nunc) were pre-coated for 1 h at 37°C with HB15a mAb (5 µg/ml) then blocked with 10% goat serum (Life Technologies) in PBS prior to application of samples. Following overnight incubation on ice, wells were incubated (1 h, 37°C) with RA83 at 10 µg/ml in diluent (5% goat serum, 5% mouse serum, 5% FCS and 1% non-fat dry milk in PBS) prior to incubation (1 h, 37°C) with biotin–GAR (1:5000 dilution). Streptavidin–HRP (1:2000 dilution) was then added in 1% BSA in PBS and incubated for 1 h at 37°C. Between each step plates were thoroughly washed with 0.1% Tween 20 in PBS. Plates were developed using OPD as above.

In all analyses of human serum, isotype-matched control mAb (CD69)-coated wells were used to provide a measure of the non-specific background for each individual sample. In a number of experiments serum was immunodepleted by overnight incubation at 4°C with HB15 or isotype-matched control mAb-coated immunomagnetic beads prior to analysis. All serum samples were obtained from normal donors and stored for 24–48 h at 4°C prior to centrifugation at 10,000 g for 30 min and analysis of the non-pelleted material.

All cell-free supernatants were centrifuged at 10,000 g for 30 min and supplemented with 40 mM HEPES (pH 7.4) prior to analysis. Where required samples were diluted in media. Media alone was used as a measure of background and wells coated with an isotype-matched control capture mAb (CD69) were used to confirm ELISA specificity. In a number of experiments an additional centrifugation step at 100,000 g for 1 h was also carried out.

Standard curves for the estimation of sCD83 concentration were generated using serial dilutions of CD83–Ig prepared in media and the results were expressed as a protein concentration based on the IgG1 standard.

Immunoprecipitation and Western blotting
The HD-derived cell line KM-H2 was cultured (5x106 cells/ml, 5 h) in RPMI 1640 supplemented with PMA (25 ng/ml) prior to recovery of the cells and the conditioned cell supernatant. Following washing, cell lysates (0.25% CHAPS/0.5% Triton X-100) were prepared either directly or following cell-surface biotinylation using sulfo-NHS-biotin (Pierce, Rockford, IL) as described previously (25). Recovered supernatant was supplemented with protease inhibitors (complete Tm; Roche Molecular Biochemicals) prior to centrifugation at 10,000 g for 30 min and subsequent concentration of non-pelleted material with a Centricon 10 (Millipore, Bedford, MA). Immunoprecipitation from cell lysates or concentrated supernatant was performed using Protein G–Sepharose (Sigma) preabsorbed with either HB15a or control mAb. Following washing with 0.1% Tween 20 in PBS, bound material was eluted with SDS–PAGE sample buffer. Biotin-labeled lysate was analyzed by immunoabsorption using mAb-coated ELISA plates as described previously (26). Eluted material was subjected to SDS–PAGE (10%, reducing) prior to transfer to nitrocellulose membrane (Hybond C; Amersham Pharmacia). The membrane was incubated at 37°C overnight in PBS then blocked for 1 h with 5% non-fat dry milk prior to incubation for 1 h at room temperature with rabbit anti-CD83 at 5 µg/ml in 10% goat serum, 10% FCS and 1% non-fat dry milk in PBS. In a number of experiments either CD83–Ig or human Ig at 35 µg/ml was also present during incubation with RA83. The membrane was incubated for 1 h at room temperature with HRP–GAR (1:2000 dilution) prior to washing and visualization of protein bands by chemiluminescence using Super Signal (Pierce, Rockford, IL). Biotin-labeled molecular mass standards (BioRad, Hercules, CA) and biotinylated immunoprecipitates were detected as described previously (27), then visualized by chemiluminescence as above.

Inhibition studies
The effect of cycloheximide (Sigma) on soluble and cell-surface CD83 was analyzed by ELISA and flow cytometry. Cell lines were cultured (5x106 cells/ml) in either media alone or media supplemented with PMA (25 µg/ml), cycloheximide (10 µg/ml) or PMA plus cycloheximide. Following incubation (2 h, 37°C) the supernatant and cells were recovered, and sCD83 and mCD83 levels determined by ELISA and flow cytometry respectively. For each treatment the levels of sCD83 and the MFI of cell-surface CD83 was expressed as a percentage of the corresponding samples incubated in media alone.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Establishing an ELISA for sCD83
Polyclonal rabbit anti-CD83 (RA83) was prepared for the detection of sCD83. As shown in Fig. 1Go(a), RA83 and the commercially available CD83 mAb HB15 both recognized CD83–Ig specifically but did not bind human IgG or the control CD40–Ig protein. A sandwich ELISA specific for the detection of sCD83 antigen was developed using HB15a as the capture antibody and RA83 as the detection reagent. The sensitivity limit of the sCD83 ELISA was 50 pg/ml as determined using CD83–Ig as an internal standard (Fig. 1bGo) and the standard curve was linear in the range 50–1000 pg/ml. The specificity of the ELISA was verified using an irrelevant substrate (human Ig, Fig. 1bGo), irrelevant isotype-matched capture mAb or irrelevant rabbit Ig as the detection reagent (data not shown).



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Fig. 1. Specificity and sensitivity of the sCD83 ELISA. (A) The reactivity of HB15, RA83 and GAH with the target proteins human Ig, CD83–Ig and CD40–Ig was analyzed by ELISA as described in Methods. (B) Standard curve for the sCD83 ELISA. Various amounts of CD83–Ig or human Ig diluted in media were assayed by sCD83 ELISA as described in Methods. Data are shown as mean OD ± SEM of triplicate samples and are from representative experiments.

 
Analysis of human cell lines
A panel of cell-free supernatants from cultured human cell lines was analyzed for the presence of sCD83 (Table 1Go). The cell lines U937 and K562 did not express detectable levels of either cell-surface or sCD83 following culture in either media or media plus PMA. The B lymphoid and HD-derived cell lines analyzed express cell membrane CD83 (mCD83) constitutively, although the levels of expression fluctuate during normal tissue culture. Supernatants obtained following culture (4 h) of these cell lines contained sCD83 at concentrations >=100 pg/ml with the HD cell lines, in particular KM-H2, releasing the highest levels of sCD83. The presence of PMA during culture induced a significant increase in sCD83 release by the B cell lines analyzed and to a lesser extent the HD-derived cell line KM-H2. In contrast, sCD83 release by the HD-derived cell line L428 was not significantly increased by PMA. Analysis of the kinetics of sCD83 release by the cell lines Raji (Fig. 2aGo) and KM-H2 (n = 3, data not shown) over a 5-h period demonstrated that sCD83 levels increased steadily during culture in either media or media plus PMA and that there was a lag phase of 2–3 h before significant PMA induced increases in sCD83 were observed. Although culture in the presence of PMA did not significantly alter mCD83 expression by KM-H2, PMA induced small increases in the level of mCD83 expressed by the B lymphoid lines Raji (130 ± 4%, n = 4) and Mann (148 ± 16%, n = 2) during short-term culture (2 h, data not shown).


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Table 1. sCD83 release by human cell lines during culture
 


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Fig. 2. Soluble CD83 release by human cell lines. (A) The cell line Raji was cultured in media or media plus PMA and sCD83 levels determined by ELISA at the time points indicated. Data are shown as mean sCD83 concentration ± SEM and are from a representative experiment of three performed (B), the cell lines Raji and KM-H2 were cultured (4 h) in either media or RPMI and the supernatants analyzed. Supernatants from cell lines cultured in media were analyzed both before and after ultracentrifugation (100,000 g). Triplicate samples were analyzed by ELISA using either CD83 or CD69 (control) as the capture mAb and the OD obtained shown as mean ± SEM. Data are from a representative experiment of three performed.

 
The specificity of the sCD83 ELISA signal obtained using cell supernatants was confirmed in all experiments using an irrelevant capture mAb (Fig. 2bGo and data not shown). Ultracentrifugation (100,000 g, 1 h) did not significantly diminish the observed levels of CD83 confirming the soluble nature of the detected material (Fig. 2bGo). Similar levels of sCD83 were detected when the cell lines were cultured in either media (RPMI 1640 with 10% FCS) or RPMI 1640 (Fig. 2bGo), demonstrating that serum components are not involved in the release of sCD83.

Inhibition studies
The requirement for de novo protein synthesis in both the spontaneous and PMA-induced release of sCD83 was analyzed in short-term cultures (2 h) using the protein synthesis inhibitor cycloheximide. As shown in Fig. 3Go, the presence of cycloheximide during culture of the cell lines KM-H2 and Raji resulted in a significant reduction in mCD83 levels. In contrast, levels of sCD83 in the same cultures were slightly increased in the presence of cycloheximide. Cycloheximide had a similar effect on cultures, which were additionally supplemented with PMA although, as described above, PMA-induced increases in mCD83 and sCD83 levels are limited in culture times <3 h. These results demonstrate that the release of sCD83 does not require de novo protein synthesis and that the release of sCD83 is associated with a concomitant decrease in the levels of mCD83. Similar results were obtained with the cell lines L428 and Mann (n = 2, data not shown).



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Fig. 3. Effect of cycloheximide on soluble and cell-surface CD83. The cell lines KM-H2 (a) and Raji (b) were cultured for 2 h in either media alone (nil) or media supplemented with cycloheximide (CHX), PMA or a combination of both. Conditioned media and cells were then collected, and levels of sCD83 (pg/ml) and mCD83 (MFI) following each treatment determined. For each treatment the level of sCD83 and the MFI was expressed as a percentage normalized relative to the sample cultured in media alone (100%). Data from four representative experiments performed on each cell line are shown as mean percentage ± SEM.

 
Biochemistry
The relative molecular mass of cell-associated and sCD83 was analyzed by immunoprecipitation and Western blotting. A previous study using surface-iodinated CD83+ transfectants demonstrated that HB15 specifically immunoprecipitates proteins that migrate as a single broad band centered at ~45 kDa (1). There was, however, some variation in the mol. wt of the band obtained from other cell types, presumably as a result of post-translational differences in glycosylation. As shown in Fig. 4Go(a) a similarly broad, single band of cell-surface proteins (~53 kDa) was specifically immunoprecipitated by HB15a from cell-surface biotinylated KM-H2. Immunostaining with RA83 demonstrated that HB15a specifically immunoprecipitates a 50 kDa doublet from the supernatant of KM-H2 cultures and no additional bands were detected even following prolonged exposure times. In contrast, HB15a specifically immunoprecipitated a range (32–57 kDa) of proteins from whole-cell lysates of KM-H2 that could be detected in immunoblots using RA83. The specificity of this staining was further confirmed in inhibition studies. As shown in Fig. 4Go(b), the presence of CD83–Ig, but not human Ig, abrogated the binding of RA83 to the HB15a immunoprecipitated proteins within KM-H2 lysates. As the protein core of CD83 has an estimated mol. wt of only 21 kDa and the CD83 molecule appears to be heavily glycosylated, the protein bands immunoprecipitated from whole-cell lysates probably represent CD83 at different stages of post-translational processing. The absence of these additional protein bands in immunoprecipitates obtained from supernatant further confirmed that the detection of sCD83 is not due to the non-specific release of cellular CD83 by necrotic cells.



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Fig. 4. Detection of sCD83 by immunoblotting. (A) Concentrated cell-free supernatant, whole-cell lysates and cell-surface biotin-labeled lysates were prepared from the cell line KM-H2 and immunoprecipitated with either control or CD83 mAb as described in Methods. Following Western blotting immunoprecipitated material from whole-cell lysates and supernatants was detected with RA83 and biotinylated cell-surface proteins were detected with streptavidin–HRP. Samples are from a representative experiment of three performed and were analyzed on the same gel. The panels shown are the optimal exposures for each set of samples. (B) Whole-cell lysates of KM-H2 were immunoprecipitated with CD83 mAb and following Western blotting immunoprecipitated material was detected by staining with RA83 either alone (nil) or in the presence of saturating amounts of human Ig or CD83–Ig. The immunoblot shown is representative of two separate experiments performed.

 
sCD83 generation by Mo-DC and B cells
Cell-surface CD83 expression is restricted predominantly to activated DC and B lymphocytes. To determine whether these populations also release sCD83, cell-free supernatants prepared from cultures of Mo-DC and B lymphocytes were analyzed by ELISA. As shown in Fig. 5Go(a), a subpopulation of isolated tonsillar B lymphocytes weakly expresses cell-surface CD83 and only moderate increases in expression are induced by culture in media alone. Culture in the presence of PMA, however, induces cell-surface CD83 expression on the majority of B lymphocytes within 24 h. Similarly, only low levels of sCD83 were detectable in the supernatants of B lymphocytes cultured in media alone, whilst PMA-activated B lymphocytes released significantly higher levels with the level of sCD83 increasing steadily in a time-dependent manner (Fig. 5bGo). Ultracentrifugation of supernatants did not significantly reduce the observed levels of sCD83.



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Fig. 5. Release of sCD83 by tonsil B lymphocytes. Tonsillar B lymphocytes were cultured for 0–72 h in the presence or absence of PMA. At the time points indicated the supernatant and cells were harvested, and the levels of sCD83 and cell-surface CD83 determined. (A) Dot-plots of tonsil B lymphocytes double labeled with the mAb indicated at T = 0 h or following 24 h culture in media or media plus PMA. Gates delineating positive staining were set on the basis of negative control staining. (B) Concentrations of sCD83 in culture supernatant at the time points indicated as determined by ELISA. Supernatants from 72 h cultures were analyzed before and after (post) ultracentrifugation. Triplicate cultures were analyzed and data are shown as mean sCD83 concentration ± SEM. Data are from a representative experiment of three performed.

 
Mo-DC derived from adherent PBMC by culture in GM-CSF/IL-4 were purified and then cultured for 3 days in the presence of GM-CSF/IL-4, either alone or supplemented with the maturation agents TNF-{alpha} or LPS. As shown in Fig. 6Go(a), DC cultured in GM-CSF/IL-4 alone did not express detectable mCD83, whilst the addition of TNF-{alpha} induced mCD83 expression on the majority of DC and LPS induced mCD83 expression on the entire DC population. The levels of sCD83 in these cultures mirrored the levels of mCD83 expression with GM-CSF/IL-4-cultured DC releasing only low levels of sCD83, TNF-{alpha}-matured DC releasing moderate levels and LPS-matured DC releasing the highest levels of sCD83 (Fig. 6bGo). Ultracentrifugation did not significantly reduce the observed levels of sCD83—again confirming the soluble nature of the detected material.



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Fig. 6. Release of sCD83 by Mo-DC. Purified GM-CSF/IL-4 generated Mo-DC were cultured in GM-CSF/IL-4 supplemented with either nil, TNF or LPS. At 72 h the supernatant and cells were harvested, and the levels of sCD83 and cell-surface CD83 determined. (A) Dot-plots of Mo-DC double labeled with HB15 and CD1a following culture in the conditions indicated. Gates delineating positive staining were set on the basis of negative control staining. (B) Concentration of sCD83 in supernatants of Mo-DC cultured in the conditions indicated. Triplicate samples were analyzed and data are shown as mean ± SEM. Data are from representative experiments of four performed.

 
Purified T cells did not release detectable levels of sCD83 following culture in either media or media plus PMA (n = 4, data not shown)

Serum sCD83
Having observed sCD83 in the supernatants of activated B lymphocytes and DC cultures we investigated whether sCD83 could be detected in normal human sera. All serum samples were analyzed using both CD83 mAb and an isotype-matched CD69 mAb as capture reagents, and the reading obtained in the sCD83 ELISA using CD69 mAb was used as a measure of the background for each sample. As shown in Fig. 7Go(a), sCD83 could be detected in normal serum samples and the observed level of sCD83 was not significantly reduced by ultracentrifugation. The specificity of the ELISA for the detection of sCD83 in serum samples was further confirmed by immunodepletion. As shown in Fig. 7Go(a), a single round of immunodepletion with CD83 mAb prior to analysis of sera significantly reduced the observed levels of sCD83 but the levels were not affected by immunodepletion with an isotype-matched control mAb. Immunodepletion did not completely remove all detectable sCD83, but in view of the extremely low levels of material remaining (<80 pg/ml) following this procedure this probably reflects the lower limit at which immunodepletion is effective. No signal was detected in this ELISA when normal rabbit Ig was used as the detection agent, further confirming the specificity of the ELISA.



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Fig. 7. sCD83 in human serum. (A) A serum sample was analyzed by sCD83 ELISA either directly or following ultracentrifugation, immunodepletion with CD83 mAb or immunodepletion with CD69 mAb. Data are shown as mean ± SEM of duplicate samples and are from a representative experiment of four performed. (B) Sera from normal donors were assayed by ELISA for sCD83. Duplicate samples of each sera was analyzed and the data shown as a scattergram of the mean sCD83 concentration for each sample.

 
Sera from a total of 44 donors were analyzed by ELISA (Fig. 7bGo). We found detectable levels of sCD83 in all serum samples analyzed with a mean concentration of 121 ± 3.6 pg/ml. Analysis of a serum pool prepared from the same group of donors gave 132 ± 2.5 pg/ml.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell membrane expression of CD83 (mCD83) is an early event in both DC and B lymphocyte activation, and mCD83 is widely utilized as a marker of differentiated/activated DC populations (1,2,5,6). We have identified an additional soluble form of CD83, which is released from mCD83+ cell populations during culture. Analysis of human cell lines demonstrated that mCD83+ B cell and HD-derived cell lines release sCD83, and the levels of sCD83 release were directly correlated with the levels of mCD83 expression. The PMA-induced activation of B cell but not HD-derived cell cultures resulted in significant increases in the levels of both mCD83 and sCD83. Isolated tonsillar B lymphocytes and immature DC, which express only low levels of mCD83, released only low levels of sCD83 during culture. Activation/maturation of these populations both up-regulated expression of mCD83 and resulted in increased release of sCD83. We also identified sCD83 in normal human sera and this may be derived from either DC or B lymphocytes as a result of in vivo immune activity.

A number of studies have reported that DC and activated B cell populations actively secrete exosomes that express a number of cell-surface molecules (28,29). The finding in this study that the levels of detected sCD83 are not significantly reduced by ultracentrifugation indicates that little, if any, CD83 is released as part of these exosomal fractions. Soluble forms of membrane molecules are typically released as either the product of differential splicing of mRNA, enzymatic cleavage of the cognate membrane form or, as observed with sIL-6R, a combination of both (12,13,30). Inhibition of de novo protein synthesis by cycloheximide did not prevent the release of sCD83 by cultured cell lines although a significant decrease in mCD83 was observed. These observations demonstrate that the release of at least a significant proportion of sCD83 is not the result of de novo synthesis of sCD86 from alternatively spliced mRNA. This and the decrease in mCD83 levels observed which accompanied the release of sCD83 from the cycloheximide-treated cell lines strongly suggests that, at least in this system, sCD83 is generated primarily by proteolytic shedding of the ectodomain of mCD83. Further studies using specific protease inhibitors will, however, be required to confirm this hypothesis. This data does not, however, preclude the possibility that some cell types under particular conditions may in addition also generate sCD83 by differential splicing as has been observed previously with sIL-6 (30). The direct correlation between the levels of sCD83 and mCD83 observed in this study together with previously reported Southern blotting data (31) also strongly suggest that the detected sCD83 is not the product of another distinct gene.

Cell-surface CD83 is heavily glycosylated and because of glycosylation heterogeneity migrates as a broad band of proteins during SDS–PAGE. In contrast, the sCD83 detected by Western blotting migrated as a more discrete lower mol. wt doublet, suggesting that mCD83 and sCD83 differ in their level/heterogeneity of glycosylation. Interestingly, similar differences in the band patterns of the respective cell-surface and soluble forms have been observed in studies on ICAM-3 (32) and CD89 (18), which are, in common with CD83, heavily glycosylated Ig superfamily members. Studies on murine CD83 have also shown that the COS-7 expressed mCD83 was considerably more heterogeneous in molecular mass than the COS-7 expressed of the CD83 ectodomain (33).

CD83 expression is restricted predominantly to DC and B lymphocytes, which up-regulate it rapidly following activation. Although the up-regulation of CD83 is associated with concurrent up-regulation of co-stimulatory and adhesion molecules (34,35), the function of human CD83 is unknown. Increased release of sCD83 is, in common with a number of other activation antigens such as CD25 (36) and CD89 (18), associated with the concurrent up-regulation of mCD83 and so does not appear to represent a mechanism for down-regulating mCD83 expression. A recent study on mouse CD83 expression and function (10) has now reported the presence of a CD83 ligand on B cells, and demonstrated that the presence of CD83–Ig inhibits T cell activation. An increasing number of studies have described immunoregulatory roles for the soluble counterparts of membrane molecules including FasL, IL-6R and CD21 (12,14,37). These include acting as carrier proteins and either blocking or enhancing the activity of their membrane bound counterparts (12,13). The finding that a soluble recombinant form of CD83 inhibits mouse T cell activation (10) suggests that in vivo generated sCD83 may have an immunoregulatory role.

The soluble forms of many molecules have been reported to circulate at detectable levels in human sera (12). Although mCD83 is expressed by a relatively low number of cells in vivo, low levels of sCD83 were detected circulating in normal human sera. Elevated serum levels of a number of other soluble activation-associated antigens such as CD25, FasL, CD30 and adhesion molecules have been described in numerous disease states (19,20,36). The comparatively restricted expression of CD83, together with its rapid up-regulation following DC and B cell activation, make serum sCD83 levels a potentially selective means of detecting the initiation of immune responses such as those involved in solid organ transplant rejection or graft-versus-host disease. The high levels of sCD83 release by HD-derived cell lines together with the observation that most Reed Sternberg/Hodgkin's cells in patients are CD83+ (38) also suggest that serum sCD83 levels may provide a useful prognostic marker in this disease. The measurement of serum levels of sCD83 in different disease states may also provide information relevant to the function of this molecule and these studies are currently being undertaken.

Although CD83 ligand expression has been detected on mouse B cells utilizing a mouse CD83–Ig construct (10), studies utilizing human CD83–Ig have so far failed to identify CD83 ligand-expressing cells amongst human hemopoietic populations. As CD83 is heavily glycosylated it is possible that human recombinant CD83 does not bear the correct carbohydrate moieties for ligand binding. Purification of in vivo generated sCD83 may therefore provide a useful additional means of identifying its ligands.

In summary, our results demonstrate the existence of a soluble form of CD83, which is released by activated DC and B lymphocytes and circulates in normal sera. Further studies utilizing purified sCD83 and analysis of sCD83 levels in disease may provide clues to the function and ligand(s) of CD83.


    Acknowledgments
 
The authors acknowledge the generosity of Professor Tom Tedder in providing pHB15, which made this work possible.


    Abbreviations
 
DC dendritic cells
GM-CSF granulocyte macrophage colony stimulating factor
HD Hodgkin's disease
HRP horseradish peroxidase
mCD83 membrane bound CD83
Mo-DC monocyte-derived dendritic cells
OPD o-phenylenediamine dihydrochloride
PBMC peripheral blood mononuclear cell
PE phycoerythrin
PMA phorbol myristate acetate
sCD83 soluble CD83
TNF tumor necrosis factor

    Notes
 
1 Present address: Mater Medical Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, Queensland 4101, Australia Back

Transmitting editor: J. Banchereau

Received 8 November 2000, accepted 13 April 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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