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(Journal of Leukocyte Biology. 2002;71:133-140.)
© 2002 by Society for Leukocyte Biology

Identification of human CD93 as the phagocytic C1q receptor (C1qRp) by expression cloning

Peter Steinberger*, Andreas Szekeres{dagger}, Stefan Wille*, Johannes Stöckl*, Nicole Selenko*, Elisabeth Prager{dagger}, Günther Staffler{dagger}, Otto Madic*, Hannes Stockinger{dagger} and Walter Knapp*,{dagger}

* Institute of Immunology, University of Vienna, A-1090 Vienna, Austria; and
{dagger} Institute of Immunology— Vienna International Research Cooperation Center at NFI, University of Vienna, A-1235 Vienna, Austria

Correspondence: Dr. Peter Steinberger, Institute of Immunology, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria. E-mail: peter.steinberger{at}univie.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD93 is a ~120 kDa O-sialoglycoprotein that within the hematopoietic system is selectively expressed on cells of the myeloid lineage. So far, its primary structure and function were unknown. We used retroviral-expression cloning to isolate the CD93 cDNA. Sequence analysis revealed that CD93 is identical to a protein on human phagocytes termed C1q receptor (C1qRp). C1qRp was shown previously to mediate enhancement of phagocytosis in monocytes and was suggested to be a receptor of C1q and two other structurally related molecules. When studying CD93 transductants and control cells, we found that cells expressing CD93 have enhanced capacity to bind C1q. Furthermore, we show that immature dendritic cells (DC) express CD93/C1qRp, and mature DC, known to have reduced capacity for antigen uptake and to have lost the ability to phagocytose, show weak-to-negative CD93/C1qRp expression.

Key Words: dendritic cells • cell-surface molecules • complement • molecular biology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In search for informative differentiation antigens, which allow the analysis and classification of normal and malignant lymphohematopoietic precursor cells and their progeny, we established almost 20 years ago a panel of monoclonal antibodies (mAb) with cell lineage and differentiation stage-restricted reactivity [1 ]. Among them was mAb VIMD2, which reacted preferentially with monocytic cells, showed weak binding to granulocytes, and was negative with lymphoid cells [1 ]. The antibody was already evaluated at the First International Workshop on Human Leukocyte Differentiation Antigens [2 ] but could only be clustered at the Fifth Workshop together with three other mAb (mAb X2 from J. H. Peters, Göttingen; mAb WDS4.B4 from W. De Smet, Sint-Genesius-Rhode; and mAb VIMD2b from our laboratory) on the basis of a highly concordant cellular reaction profile [3 ]. Further, we could demonstrate that all four mAb recognized a leukocyte surface protein with the same molecular mass of 120 kDa and with the characteristic features of an O-sialoglycoprotein [4 ]. On the basis of these findings, the cluster designation CD93 was assigned to these mAb and the antigen recognized by them [3 , 4 ].

Still, the primary structure as well as the function of the CD93 molecule remained unknown. We used a previously described retrovirus-based cDNA expression library constructed from a human myeloid cell line, KG1a (unpublished results), to clone the CD93 cDNA. Sequence analysis and database comparison revealed that CD93 is identical to the C1qRp (C1q receptor), which was cloned recently by Tenner and co-workers [5 ]. Studies by the same group suggest that three structurally similar proteins, the complement protein C1q, the mannose-binding lectin (MBL), and the pulmonary surfactant protein A (SPA), are able to enhance immunoglobulin G (IgG)- or complement opsonization-mediated phagocytosis by human monocytes [6 7 8 ]. Furthermore, they found that antibodies to C1qRp are able to inhibit this phagocytosis-stimulating effect. This would indicate that the C1qRp protein is interacting directly with the phagocytosis-enhancing ligands or is a common component of a phagocytosis-enhancing receptor complex. Here, we therefore analyzed the binding of C1q to cells expressing CD93/C1qRp.

Because of their unique ability to prime naive T cells, dendritic cells (DC) have recently attracted considerable interest, and they are now thought the most potent antigen-presenting cells (APC) of the immune system [9 ]. During their development, DC go through an immature stage where they have a high capacity to take up soluble and particulate matter. Upon maturation, DC almost completely lose this capacity but show increased potency to stimulate T cells [10 , 11 ]. We show here that immature DC express CD93/C1qRp and down-regulate this surface marker upon maturation, which points to a potential role of this receptor in uptake of particles by DC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and cell culture
The CD93 mAb VIMD2 was obtained from Balb/c mice immunized with KG-1 cells, a cell line of acute myeloblast leukemia (AML) origin, using standard hybridoma technology. The following murine mAb were also generated in our laboratory: VIAP (isotype-control mAb), VIM13 (CD14), VIPl-1(CD41), and VIMD2b (CD93). The CD93 mAb X2 and WDS4.B4 were from the Fifth International Workshop on Leukocyte Differentiation Antigens, and the CD40 mAb G28-5 was a kind gift from J. A. Ledbetter (Seattle, WA). CD3 mAb (UCHT-1) and CD14 mAb (MEM18) were provided by An der Grub (Kaumberg, Austria). mAb specific for CD11b (BEARI) and CD19 (J4.119) were purchased from Immunotech (Marseille, France). CD16 mAb (3G8), CD34 mAb (581), CD56 mAb (MEM 188), and CD86 mAb (BU63) were obtained from Caltag (San Francisco, CA).

The human cell line KG1a and the murine cell lines EL-4, BW5147{alpha}ß-, Sp2/6, and P815 that were used as target cells for retrovirus infection were maintained in RPMI-1640 medium supplemented with 2 mM glutamine and 10% fetal bovine serum (FBS). The ecotropic retroviral packaging cell line Phoenix-E [12 , 13 ] (a kind gift from G. P. Nolan and colleagues) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM glutamine and 10% FBS. For activation of lymphocytes, peripheral blood mononuclear cells (PBMC) were cultivated in the presence of phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma Chemical Co., St. Louis, MO) and ionomycin (1 µM; Sigma Chemical Co.) for 2 days. Monocytes were separated from freshly isolated PBMC by magnetic cell sorting (MACS) using the mAb specific to CD14 (VIM13 and MEM18 as described [14 ]). Monocyte-derived DC were generated by culturing monocytes in RPMI containing 10% FBS in the presence of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 50 ng/ml) and rh interleukin (IL)-4 (100 U/ml) for 8 days. Both reagents were kind gifts of the Novartis Research Institute (Vienna, Austria). In some experiments, DC maturation was induced by cultivating day 6 monocyte-derived DC for another 2 days in the presence of lipopolysaccharides (LPS; 1 µg/ml; Sigma Chemical Co.) or ionomycin (1 µM). Human peripheral DC were isolated from PBMC as follows: Cells were labeled with a mixture of biotinylated mAb against the lineage-specific markers CD3, CD14, CD16, CD56, CD19, CD34, CD41, and CD11b. The cells were incubated with streptavidin-immunomagnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany), and labeled cells were removed by MACS. The resulting cells lacked lineage markers and were identified as LIN- HLA-DR+ peripheral DC. For maturation of peripheral DC, these cells were cultured in RPMI-1640 containing 10% FBS in the presence of rhIL-3 (100 U/ml; R&D Systems, Minneapolis, MN) and rh tumor necrosis factor {alpha} (TNF-{alpha}; 100 U/ml; Ernst Boehringer Institut f. Arzneimittelforschung, Vienna, Austria) for 2 days.

Retroviral cDNA expression library
The generation of a retroviral expression library derived from KG1a cDNA is described elsewhere (unpublished results). Recombinant retroviral vector DNA (60 µg) representing the cDNA library was mixed with 50 ml DMEM containing Nuserum IV culture supplement (Becton Dickinson, San Jose, CA) and glutamine (2 mM). In a second tube, 50 ml DMEM containing Nuserum and glutamine was mixed with 1 ml (diethylamino)ethyl (DEAE)-dextran (10 mg/ml) and with 0.5 ml chloroquine diphosphate (10 mM). Both mixtures were incubated for 10 min (37°C), combined, and added to 4 x 107 ecotropic packaging cells (Phoenix-E). After incubation for 2 h at 37°C, cells were spun down and resuspended in 20 ml DMEM containing 10% FBS and seeded in two 175 cm2 culture dishes. After 72 h of culture at 32°C, the supernatant was harvested. The filtered (45 µm filter) supernatant was supplemented with 50 µl polybrene (4 mg/ml) and added to 2 x 107 target cells in 20 ml RPMI containing 10% FBS, and cells were incubated for 24 h at 32°C. Subsequently, the cells were spun down and resuspended in 100 ml RPMI containing 10% FBS, incubated for 24 h (37°C), and then used for the selection procedure.

Isolation of CD93-expressing cells from the retrovirus-based cDNA library-transduced cell pool
The retrovirus-infected cell pool (4x107 cells) was washed with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.02% NaN3. The cells were then incubated with VIMD2 (10 µg/ml) for 20 min on ice in a volume of 400 µl in PBS containing 1% BSA and 0.02% NaN3. After two washing steps, cells were resuspended in 320 µl PBS with 1% BSA and 0.02% NaN3. Magnetically labeled goat anti-mouse IgG MicroBeats (80 µl, Miltenyi Biotech) were added, and cells were incubated for 20 min at 4°C. Cells were washed twice in degassed PBE [PBS containing 0.5% BSA and 2 mM ethylenediaminetetraacetate (EDTA)] and resuspended in 500 µl PBE. A MACS-RS+ separation column (Miltenyi Biotech) was equilibrated with 500 µl PBE, and the cell suspension was applied to the column attached to a magnetic cell separator (Miltenyi Biotech). The column was washed three times with 500 µl PBE. Subsequently, the column was removed from the cell separator, and the retained cells were flushed out with 1 ml PBE, using the plunger supplied with the column. The eluted cells were put into culture and expanded. When the cell number reached approximately 4 x 107, the selection procedure was repeated. Four rounds of MACS were done. After the fourth cycle of selection, single-cell clones were obtained by limiting dilution culturing.

Cloning of the CD93 cDNA insert from the selected cell pool—polymerase chain reaction (PCR) recovery of the expressed proviral cDNA insert
After four rounds of selection, 1 x 107 target cells were used for total RNA preparation with the TRI Reagent (Sigma Chemical Co.) following the manufacturer’s instructions. Oligo TT-primed first-strand cDNA was generated from 5 µg total RNA using the first-strand cDNA synthesis kit from Life Technologies (Paisley, UK), according to the provided protocol. The retrovirus-encoded cDNA insert was PCR-amplified from 1 µl first-strand cDNA with the oligonucleotide primers LIB S: 5'-GCTCACTTACAGGCTCTCTA-3' and LIB A: 5'-CAGGTGGGGTCTTTCATTCC-3', specific for the flanking retroviral sequences. The Expand PCR system (Roche Molecular Biochemicals, Indianapolis, IN) was used for the PCR amplification under standard conditions. The obtained PCR products were gel-purified and digested using the restriction endonuclease EcoRI. The digested PCR product was ligated into appropriately cut pBabeMN vector DNA ([15 ]; a kind gift of G. P. Nolan and colleagues) and introduced into the Escherichi coli strain DH5a by electroporation. Plasmid DNA obtained from transformed clones was analyzed by restriction endonuclease digest, and selected plasmids were transfected into Phoenix-E cells using LipofectAmine (Life Technologies), according to the manufacturer’s instructions to confirm that the transfected cells react with the CD93 mAb VIMD2.

DNA sequence analysis
Plasmid DNA was prepared from selected clones (Plasmid Maxi Kit; Qiagen, Chatsworth, CA) and used for sequence analysis (VBC Genomics, Vienna, Austria). To minimize the risk of sequence errors introduced during PCR amplification, another DNA fragment generated from a separate PCR amplification was cloned into the pBabeMN vector DNA, and the resulting plasmid was also used for DNA sequencing.

Analysis of CD93/C1qRp interaction with C1q
Purified C1q (200 µg; Calbiochem, San Diego, CA) was incubated with 1 µg Biotin-X-NHS (Calbiochem) for 40 min at room temperature with intermittent agitation. We stopped the reaction by adding glycine to a final concentration of 50 mM. The reaction mixture was subsequently dialyzed against PBS. The biotinylated C1q (C1qbio) was stored at -70°C in aliquots. After thawing, the C1qbio was stored at 4°C for up to 3 days. For flow cytometric-binding studies with C1qbio, a low ionic-strength buffer [70% Hanks’ balanced saline solution without Mg++ and Ca++ and 30% of a 5% glucose-0.2% gelatin solution] was used throughout the staining. Cells (1.5x105) were incubated with C1qbio in a total volume of 20 µl for 10 min at room temperature. Cells were washed and incubated with a streptavidin-phycoerythrin (PE) conjugate (Becton Dickinson; final concentration, 0.1 µg/ml).

Flow cytometric analysis using antibodies
For flow cytometric analysis using murine mAb, cells (1x107/ml) were incubated with primary antibody (10 µg/ml) for 20 min on ice, washed, and incubated with fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of sheep anti-mouse Ig Abs (An der Grub) as described [16 ]. In some experiments, an Oregon Green-labeled anti-mouse Ig conjugate (Molecular Probes, Eugene, OR) was used as a secondary reagent.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of CD93-expressing transductants
The retroviral vector containing the KG1a cDNA library was introduced into the packaging cell line Phoenix-E by transfection, and the retrovirus containing supernatant was used to infect mouse target cells. The transduced cell pool was subjected to four rounds of selection for binding to mAb VIMD2 using MACS. After two rounds of selection, a small number (<1%) of cells reacted with VIMD2 by immunofluorescence (data not shown). After rounds three and four, 12.8% and 63.2%, respectively, of the selected cell pool stained positively with VIMD2 by flow cytometry. The background staining with untransduced control cells was below 1% (Fig. 1 ).



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  		Figure 1. Enrichment of VIMD2-reactive target cells during activated MACS. Untransduced target cells (control) and the cell pools obtained after three and four rounds of selection of the target cells transduced with the retroviral KG1a expression library were probed with isotype-control mAb (open histograms) or CD93 mAb VIMD2 (shaded histograms). Cells were stained with an FITC-conjugate and analyzed by flow cytometry.

 
PCR recovery and cloning of the CD93-encoding cDNA
Target cells obtained after four rounds of selection with VIMD2 were used for total RNA isolation. First-strand cDNA synthesis was generated from the RNA and was PCR-amplified using oligonucleotide primers specific for the flanking retroviral sequences. The ~3.7 kb PCR-product was EcoRI-cut and cloned into the retroviral vector pBabeMN and transformed into E. coli cells. Plasmid DNA was prepared from the transformed E. coli cells and introduced into Phoenix-E cells by transfection to confirm that it contained the insert of interest. The resulting transfectants were strongly reacting with mAb VIMD2 by flow cytometry. No reactivity was seen with cells transfected with the control vector pBabeMN-lacZ that contained the ß-galactosidase cDNA (Fig. 2 ).



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  		Figure 2. CD93 mAb VIMD2 binds to cells transfected with plasmid DNA containing the isolated cDNA. Phoenix-E cells were transfected with control plasmid DNA (left) or with plasmid DNA containing cDNA that was isolated from the sorted target cells (right). Two days post-transfection, cells were probed with isotype-control mAb (open histograms) or with VIMD2 (shaded histograms). Cells were stained with FITC-conjugate and analyzed by flow cytometry.

 
Reactivity of CD93 mAb with cells cloned from the selected cell pool and with KG1a cells
After four rounds of selection, single-cell clones were generated by limiting dilution and analyzed for binding of VIMD2 by flow cytometry. Eleven out of 12 cell clones analyzed were specifically reacting with VIMD2 (data not shown). One cell clone, Bw-93-4, was selected for further experiments. Bw-93-4-cells, the myeloid cell line KG1a that was used as a source for the cDNA library, and negative control cells (untransfected target cells) were probed with CD93 mAb, VIMD2, VIMD2b, X2, WDS4.B4, and with a control mAb. B-93-4 cells as well as KG1a cells reacted strongly with all four CD93 mAb, which were described to recognize three distinct epitopes [4 ]. None of the mAb bound the untransduced target cells (Fig. 3 ).



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  		Figure 3. Reactivity of CD93 mAb with Bw-93-4 cells and KG1a cells. Bw-93-4, a cell clone derived from the transduced target cell pool obtained after four rounds of selection, was probed with four different CD93 mAb and isotype-control mAb (left). Untransduced target cells (middle) and the myeloid cell line KG1a (right) were also analyzed. The shaded histograms show binding of CD93 mAb (VIMD2, VIMD2b, WDS4.84, and X2). The open histograms represent the reactivity of the isotype-control mAb.

 
Sequence analysis
Retroviral vector DNA, containing the 3.3 kb insert obtained from the selected target cell pool, was subjected to DNA sequence analysis. To minimize the risk of sequence errors introduced during PCR amplification of the cDNA insert, two cloned DNA fragments that were amplified separately were subjected to sequence analysis. The final cDNA insert had a total length of 3337 bases and did not contain a poly-A tail. An open reading frame corresponding to a 652 amino acid polypeptide beginning with an ATG start codon was found. The nucleotide sequence and the deduced amino acid sequence are shown in Figure 4 . The nucleotide sequence is deposited in the EMBL Data Library under accession number AJ295142. Database searches revealed that the cloned cDNA was almost identical to the cDNA of the human phagocytic C1qRp described previously [5 ]. C1qRp-CD93 is a type 1 membrane protein with the following putative structural elements: a C-type carbohydrate recognition domain (CRD), five epidermal growth factor (EGF)-like domains, a transmembrane domain, and a short cytoplasmic tail [5 ]. The difference between the molecular weight predicted from the polypeptide chain (66.5 kDa) and its mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis is probably a result of the heavy glycosylation and its primary sequence that is high on alanine, proline, and charged amino acids [17 ]. The CD93 cDNA was compared with the C1qRp cDNA sequence and a genomic sequence containing the gene of C1qRp, GenBank accession numbers U94333 and AL118508, respectively. In codon 318, the final CD93 cDNA sequence obtained by us differed from both C1qRp sequences used for comparison. This leads to an amino acid substitution (V to A) in the gene product of the CD93 cDNA sequence isolated from the retroviral library. This is most likely a result of a sequence polymorphism in the KG1a cell line, which was the source of our cDNA library.



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  		Figure 4. cDNA and deduced amino acid sequence of CD93. The cDNA insert derived from the retrovirus library transduced target cells after screening with the CD93 mAb VIMD2 was subjected to DNA sequence analysis. Numbering of bases and amino acids is shown on the left and right, respectively.

 
Analysis of C1q binding to cells by flow cytometry
Although the C1qRp was implicated in C1q binding and thought to mediate enhanced phagocytosis upon interaction with this complement protein, binding of C1q to C1qRp has not been demonstrated directly, and despite its name, it is not generally accepted that C1q binds the C1qRp [18 , 19 ]. Therefore, we investigated the binding of C1q to CD93/C1qRp transductants. As a control, we used untransduced cells from the same cell line. Purified human C1q was biotinylated "lightly" to avoid aggregation as described [20 ]. Cells were incubated with increasing amounts of C1qbio and stained with a streptavidin-PE conjugate. As in previous studies where interaction of C1q with cell-surface receptors was investigated [20 21 22 23 ], the interaction was analyzed in a low ionic-strength buffer. For CD93/C1qRp and untransduced control cells, increasing cell-fluorescence intensity was measured when increasing amounts of C1qbio were added to the cells, indicating that both cell types are able to bind C1q (Fig. 5 ). However, with all C1q concentrations tested, the CD93/C1qRp-expressing cells showed higher fluorescence intensity compared with the control cells. Both cell types did not bind biotinylated control protein (BSAbio). However, we were unable to detect specific binding of C1q to CD93/C1qRp transductants at physiological ionic strength (data not shown). At our experimental conditions, we did not see a significant reduction of C1qbio binding to the cells upon preincubation with unlabeled C1q (data not shown). None of the four CD93 mAb (VIMD2, VIMD2b, X2, and WDS4.B4) inhibited the binding of C1q to CD93 transductants (data not shown).



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  		Figure 5. Interaction of C1q with CD93/C1qRp-transduced cells. The binding of C1qbio to CD93/C1qRp transductants was analyzed by flow cytometry. Cells were incubated with buffer only or with increasing concentrations of C1qbio as indicated. A low ionic-strength buffer was used throughout the staining. Cell-bound C1qbio was detected with a streptavidin-PE conjugate. The shaded histograms show C1q reactivity with CD93/C1qRp transductants, and the open histograms represent the control staining—reactivity of C1q with untransduced cells derived from the same cell line. BSAbio did not bind to the cells. The binding experiments were repeated several times with a similar outcome.

 
Analysis of CD93/C1qRp expression and C1q binding of human leukocytes
Human monocytes, lymphocytes, and granulocytes were analyzed for expression of CD93/C1qRp using mAb VIMD2. In agreement with previous studies, monocytes as well as granulocytes expressed CD93/C1qRp. However, compared with monocytes, granulocytes express less CD93/C1qRp. Human peripheral lymphocytes including natural killer (NK) cells were consistently negative for this marker. This is in contrast to the rat homolog of CD93/C1qRp that was shown to be expressed on all circulating NK cells [24 , 25 ]. PMA and ionomycin-activated lymphocytes were also CD93/C1qRp-negative (data not shown).

Using low ionic-strength conditions, we analyzed the binding of C1q to these cells. We found that monocytes as well as granulocytes bind C1q, whereas only a small subpopulation of lymphocytes binds to C1q (Fig. 6 ). These findings show that cells, which express CD93, are also binding C1q. Because lymphocytes are CD93-, but a small subset of these cells binds C1q, our results also indicate that there are other C1q binding structures on human leukocytes.



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  		Figure 6. Human leukocytes expressing CD93/ClqRp are binding Clq. (A) Human lymphocytes, granulocytes, and monocytes were analyzed for CD93/C1qRp expression. Cells were probed with the CD93 mAb VIMD2 (shaded histograms) or with isotype-control mAb (open histograms). (B) Human lymphocytes, granulocytes, and monocytes were probed with C1qbio (30 µg/ml, shaded histograms) and control protein (30 µg/ml BSAbio, open histograms shown in bold).

 
Expression of CD93/C1qRp on monocyte-derived DC is down-regulated upon maturation and correlates with binding of C1q
DC can be generated from monocytes in vitro by supplementing cultures with IL-4 and GM-CSF. These cells have the characteristics of immature DC and can be induced to mature by inflammatory stimuli such as IL-1, LPS, or ionomycin [26 , 27 ]. It is well established that maturation of DC, which leads to enhancement of their T-cell stimulatory capacity, is accompanied by a significant down-regulation of phagocytosis, endocytosis, and pinocytosis [10 , 28 ]. DC generated from monocytes with IL-4 and GM-CSF express CD93/C1qRp on their surface (Fig. 7 ). When we cultivated these DC in the presence of ionomycin, they obtained the phenotype of mature DC, as indicated by the up-regulation of major histocompatibility complex (MHC) class II. It is interesting that at the same time, CD93/C1qRp density was strongly reduced on these cells (Fig. 7A) . Similar results were obtained when we used LPS as maturation stimulus (data not shown). Using low ionic-strength conditions, we also compared the binding of C1q with immature and mature DC by flow cytometry. Our results indicate that there is a correlation between CD93 expression and C1q binding in monocyte-derived DC: Mature DC, which compared with immature DC have a strongly reduced CD93/C1qRp expression, also bind less C1q (Fig. 7B) . Our data show that mature DC, which express very little CD93, still bind considerable amounts of C1q. Therefore, it is likely that there are other C1q-binding structures on DC.



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  		Figure 7. Maturation of monocyte-derived DC leads to down-regulation of CD93/C1qRp and to a reduced binding of C1qbio. Monocytes were isolated from peripheral blood leukocytes and cultivated in the presence of IL-4 and GM-CSF. On day 6, the maturation stimulus ionomycin was added to immature DC. On day 8, both cell types were analyzed for reactivity with antibodies or with C1q by flow cytometry. (A) Immature DC (shaded histograms) and mature DC (open histograms shown in bold) were probed with CD93 mAb VIMD2 or with CD86-specific mAb as indicated. The open histograms shown in a thin line represent the reactivity of the isotype-control mAb. (B) Immature DC (shaded histograms) and mature DC (open histograms shown in bold) were probed with C1qbio or with control protein (BSAbio) as indicated.

 
Human peripheral DC express CD93/C1qRp; in vitro maturation leads to a down-regulation of this receptor
Human peripheral DC were enriched from PBMC by MACS and were analyzed for expression of CD93/C1qRp by flow cytometry. The freshly isolated peripheral DC were expressing relatively high levels of CD93/C1qRp (Fig. 8 ). IL-3 and TNF-{alpha} were described to induce maturation of human peripheral DC [29 ]. When we cultivated the isolated cells in the presence of these cytokines, we found up-regulation of maturation markers such as CD40. It is interesting that as with monocyte-derived DC, we found a strong reduction of CD93/C1qRp expression on these mature peripheral DC.



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  		Figure 8. Peripheral DC down-regulate CD93/C1qRp upon maturation. Peripheral DC were isolated from PBMC and were analyzed for reactivity with CD93 mAb VIMD2 or with CD40-specific mAb as indicated (shaded histograms). The isolated cells were also cultivated in the presence of the maturation-inducing cytokines IL-3 and TNF-{alpha} and probed with mAb as described above (open histograms shown in bold). The open histograms shown in a thin line represent the reactivity of the isotype-control mAb.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
So far, the primary structure of the antigen recognized by the mAb VIMD2, which together with three other mAb has been clustered as CD93, was unknown. A retroviral-cloning approach allowed us to identify this molecule as the human phagocytic C1qRp, which was first described by Tenner and colleagues [5 , 30 ]. The rat and the mouse homolog of this protein have been cloned recently [19 , 24 , 25 , 31 ]. All three proteins are heavily glycosylated type I transmembrane proteins with an N-terminal C-type lectin-like domain (CTLD) adjacent to a cysteine-rich domain composed of a tandem array of five EGF-like repeats. The transmembrane domain is followed by a short cytoplasmic tail.

The CTLD of these proteins bear distant homology to the carbohydrate recognition domains (CRD) of the C-type lectin family, which includes endocytosis-mediating receptors such as the mannose receptor (MR) and DEC-205 [32 33 34 ]. These proteins bind and internalize carbohydrate-bearing ligands by receptor-mediated endocytosis [34 ]. However, CD93/C1qRp seems to clearly differ from these receptors. We did not observe internalization of CD93/C1qRp upon its ligation with bivalent antibody or with its natural ligand Clq as was seen for endocytic receptors such as the MR and DEC-205. We also found no evidence of binding of mannose-coupled BSA to CD93/C1qRp-expressing transductants (data not shown).

Others have speculated that CD93/C1qRp and its homologs might play a role in cell-cell interaction [19 , 25 ]. However, to date, there is no experimental evidence for such a function. An entire series of studies by Tenner and colleagues points toward a role of CD93/C1qRp in the regulation of phagocytosis [6 7 8 , 30 ].

The authors demonstrated that the addition of C1q as well as of SPA or MBL could enhance phagocytosis of opsonized RBC (red blood cells) by human monocytes. C1q, SPA, and MBL are structurally similar proteins that are known to recognize pathogen-associated molecular patterns (PAMPS) [35 ]. Tenner and colleagues demonstrated that C1qRp-specific antibodies are able to inhibit this enhancement of phagocytosis. Taken together, these data suggest that this mAb-defined monocyte-surface structure, which seems to be involved in the C1q-mediated regulation of phagocytosis, is a receptor for C1q or physically or functionally linked with a molecule recognizing C1q.

The identification of C1q receptors is notoriously difficult. The analysis of C1q interaction with whole cells is hampered by the stickiness and delicacy of C1q and by the presence of more than one C1q binding protein. A number of C1q binding proteins have been described, but their nature as specific C1q receptors is often not generally accepted [18 , 19 ]. This also holds true for the C1qRp described by Tenner and co-workers [5 , 30 ], which was also doubted to actually bind C1q.

The availability of CD93/C1qRp transfectants allowed us to compare directly C1q binding of CD93/C1qRp-expressing cells with CD93/C1qRp-negative control cells of the same cell type. In this system, we could detect enhanced binding of C1q to CD93/C1qRp transductants using low ionic-strength conditions. We think that these results support the notion that the effect of C1q on the enhancement of monocyte phagocytosis is mediated through binding of C1q to CD93/C1qRp as suggested by Tenner and co-workers [30 ]. Because we were unable to detect specific binding of soluble C1q to CD93/C1qRp-expressing cells at physiological conditions, we could not show that CD93/C1qRp is indeed a receptor of C1q. Tenner and co-workers [23 ] argue that in vivo, C1q would generally be presented to CD93/C1qRp-bearing cells as aggregates because most C1-binding substances are multivalent in structure. Therefore, it is conceivable that in vivo, CD93/C1qRp binds to clusters of C1q at normal ionic strength. However, the identity of C1qRp as a receptor for C1q is still controversial, and even the research group that coined the term C1qRp recently referred to this receptor as the "C1q receptor/receptor component" [36 ]. Furthermore, the rat as well as the mouse homolog of this protein was termed AA4 rather than C1qRp by some groups [19 , 25 ]. We hope that the CD designation will offer a way out of the confusion regarding the nomenclature of this molecule.

Recently, a phagocytosis-enhancing effect of C1q was also shown in murine cells that express the mouse homolog of the C1qRp [36 ]. This effect was inhibited by intracellular application of a polyclonal antibody that binds to the cytoplasmic tail of the murine as well as the human C1qRp [36 , 37 ]. This indicates that the intracellular domain, which is highly conserved in those two species, plays a role in transducing a signal that leads to enhancement of phagocytic function. The cytoplasmic tail of the human CD93/C1qRp and the rat homolog comprise a carboxy-terminal tyrosine phosphorylation motif [5 , 24 , 25 ], and coprecipitation studies showed evidence that CD93 is associated with protein kinases [38 ].

We found an interesting correlation of the expression pattern of CD93/C1qRp in DC with their functional state. So-called immature DC, known to be particularly potent in the uptake of antigens by phago-, endo-, and pinocytosis but relatively inefficient in antigen presentation [10 , 26 ], are strongly CD93-positive. In contrast, mature DC, which are good T-cell stimulators but barely take up antigen and have lost the capacity to phagocytose [28 , 39 ], have also lost expression of the phagocytic CD93/C1qRp (Figs. 7 and 8) . This concomitant reduction of phagocytic capacity and CD93/C1qRp density during DC maturation would be in line with a potentially relevant role of CD93/C1qRp in DC phagocytosis in general but does not prove such a linkage. It can be envisaged, however, that in vivo, the CD93/C1qRp molecules we found on immature DC contribute to an enhanced uptake of antigen if complexed with C1q. This could help to direct the adaptive immune response toward C1q-activating structures such as foreign antigen complexed by IgG- or IgM-opsonized particles.

Note added in proof: The construction of the retroviral cDNA library that was the source of the CD93-cDNA has been published recently: Wille, S., Szekeres, A., Majdic, O., Prager, E., Staffler, G., Stöckl, J., Kunthalert, D., Prieschl, E. E., Baumruker, T., Burtscher, H., Zlabinger, G. J., Knapp, W., Stockinger, H. (2001) Characterization of CDw92 as a Member of the Choline Transporter-Like Protein Family Regulated Specifically on Dendritic Cells. J. Immunol. 167, 5795–5804.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Austrian Science Fund (SFB005.2) and by the Competence Center "Bio-Molecular Therapeutics." We thank Dr. Garry P. Nolan and colleagues for providing the retroviral vector pBabeMN-Z and the packaging cell line Phoenix-E and Prof. Dr. Johannes Menzl for helpful discussion. We appreciate the expert technical assistance of Saro Küng and Petra Kohl.

Received April 1, 2001; revised August 26, 2001; accepted August 27, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Knapp, W., Bettelheim, P., Majdic, O., Liszka, K., Schmidmeier, W., Lutz, D. (1984) Diagnostic value of monoclonal antibodies to leucocyte-differentiation antigens in lymphoid and nonlymphoid leukemias Bernard, A. Boumsell, L. Dausset, J. Milstein, C. Schlossman, S. F. eds. Leucocyte Typing ,564-573 Springer-Verlag Berlin.
  2. Bernard, A., Boumsell, L., Hill, C. (1984) Joint report of the first international workshop on human leucocyte differentiation antigens by the investigators of the participating laboratories Bernard, A. Boumsell, L. Dausset, J. Milstein, C. Schlossman, S. F. eds. Leucocyte Typing ,9-142 Springer-Verlag Berlin.
  3. Shaw, S., Luce, G. G., Gilks, W. R., Anderson, K., Ault, K., Bochner, B. S., Boumsell, L., Denning, S. M., Engleman, E. G., Fleisher, T., et al (1995) Leucocyte differentiation antigen database Schlossman, S. F. Boumsell, L. Gilks, W. Harlan, J. M. Kishimoto, T. Morimoto, C. Ritz, J. Shaw, S. Silverstein, R. Springer, T. Tedder, T. F. Todd, R. F. eds. Leukocyte Typing V ,16-198 Oxford University Press Oxford.
  4. Mai, I., Pickl, W. F., Stöckl, J., Majdic, O., Mellors, A., De Smet, W., Xu, H., Peters, J. H., Knapp, W. (1995) CD93 (group 11) cluster workshop report Schlossman, S. F. Boumsell, L. Gilks, W. Harlan, J. M. Kishimoto, T. Morimoto, C. Ritz, J. Shaw, S. Silverstein, R. Springer, T. Tedder, T. F. Todd, R. F. eds. Leucocyte Typing V ,986-988 Oxford University Press Oxford.
  5. Nepomuceno, R. R., Henschen, E. A., Burgess, W. H., Tenner, A. J. (1997) cDNA cloning and primary structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro Immunity 6,119-129
  6. Tenner, A. J., Robinson, S. L., Borchelt, J., Wright, J. R. (1989) Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis J. Biol. Chem. 264,13923-13928
  7. Tenner, A. J., Robinson, S. L., Ezekowitz, R. A. (1995) Mannose binding protein (MBP) enhances mononuclear phagocyte function via a receptor that contains the 126,000 M(r) component of the C1q receptor Immunity 3,485-493
  8. Guan, E. N., Burgess, W. H., Robinson, S. L., Goodman, E. B., McTigue, K. J., Tenner, A. J. (1991) Phagocytic cell molecules that bind the collagen-like region of C1q. Involvement in the C. 1q-mediated enhancement of phagocytosis J. Biol. Chem. 266,20345-20355
  9. Banchereau, J., Steinman, R. M. (1998) Dendritic cells and the control of immunity Nature 392,245-252
  10. Sallusto, F., Cella, M., Danieli, C., Lanzavecchia, A. (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products J. Exp. Med. 182,389-400
  11. Engering, A. J., Cella, M., Fluitsma, D., Brockhaus, M., Hoefsmit, E. C., Lanzavecchia, A., Pieters, J. (1997) The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells Eur. J. Immunol. 27,2417-2425
  12. Costa, G. L., Benson, J. M., Seroogy, C. M., Achacoso, P., Fathman, C. G., Nolan, G. P. (2000) Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease J. Immunol. 164,3581-3590
  13. Hitoshi, Y., Lorens, J., Kitada, S. I., Fisher, J., LaBarge, M., Ring, H. Z., Francke, U., Reed, J. C., Kinoshita, S., Nolan, G. P. (1998) Toso, a cell surface, specific regulator of Fas-induced apoptosis in T cells Immunity 8,461-471
  14. Pickl, W. F., Majdic, O., Kohl, P., Stockl, J., Riedl, E., Scheinecker, C., Bello, F. C., Knapp, W. (1996) Molecular and functional characteristics of dendritic cells generated from highly purified CD14+ peripheral blood monocytes J. Immunol. 157,3850-3859
  15. Kinoshita, S., Su, L., Amano, M., Timmerman, L. A., Kaneshima, H., Nolan, G. P. (1997) The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells Immunity 6,235-244
  16. Majdic, O., Stockl, J., Pickl, W. F., Bohuslav, J., Strobl, H., Scheinecker, C., Stockinger, H., Knapp, W. (1994) Signaling and induction of enhanced cytoadhesiveness via the hematopoietic progenitor cell surface molecule CD34 Blood 83,1226-1234
  17. Nepomuceno, R. R., Ruiz, S., Park, M., Tenner, A. J. (1999) C1qRP is a heavily O-glycosylated cell surface protein involved in the regulation of phagocytic activity J. Immunol. 162,3583-3589
  18. Nicholson, W. A., Klickstein, L. B. (1999) C1q-binding proteins and C1q receptors Curr. Opin. Immunol. 11,42-46
  19. Petrenko, O., Beavis, A., Klaine, M., Kittappa, R., Godin, I., Lemischka, I. R. (1999) The molecular characterization of the fetal stem cell marker AA4 Immunity 10,691-700
  20. Klickstein, L. B., Barbashov, S. F., Liu, T., Jack, R. M., Nicholson, W. A. (1997) Complement receptor type 1 (CR1, CD35) is a receptor for C1q Immunity 7,345-355
  21. Bordin, S., Kolb, W. P., Page, R. C. (1983) C1Q receptors on cultured human gingival fibroblasts: analysis of binding properties J. Immunol. 130,1871-1875
  22. Tenner, A. J., Cooper, N. R. (1981) Identification of types of cells in human peripheral blood that bind C1q J. Immunol. 126,1174-1179
  23. Tenner, A. J., Cooper, N. R. (1980) Analysis of receptor-mediated C1q binding to human peripheral blood mononuclear cells J. Immunol. 125,1658-1664
  24. Lovik, G., Vaage, J. T., Dissen, E., Szpirer, C., Ryan, J. C., Rolstad, B. (2000) Characterization and molecular cloning of rat C1qRp, a receptor on NK cells Eur. J. Immunol. 30,3355-3362
  25. Dean, Y. D., McGreal, E. P., Akatsu, H., Gasque, P. (2000) Molecular and cellular properties of the rat AA4 antigen, a C-type lectin-like receptor with structural homology to thrombomodulin J. Biol. Chem. 275,34382-34392
  26. Sallusto, F., Lanzavecchia, A. (1994) Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha J. Exp. Med. 179,1109-1118
  27. Lyakh, L. A., Koski, G. K., Telford, W., Gress, R. E., Cohen, P. A., Rice, N. R. (2000) Bacterial lipopolysaccharide, TNF-alpha, and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in CD14+ monocytes through distinct pathways that activate NK-kappaB J. Immunol. 165,3647-3655
  28. Steinman, R. M., Swanson, J. (1995) The endocytic activity of dendritic cells J. Exp. Med. 182,283-288
  29. Kohrgruber, N., Halanek, N., Groger, M., Winter, D., Rappersberger, K., Schmitt, E. M., Stingl, G., Maurer, D. (1999) Survival, maturation, and function of CD11c- and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines J. Immunol. 163,3250-3259
  30. Guan, E., Robinson, S. L., Goodman, E. B., Tenner, A. J. (1994) Cell-surface protein identified on phagocytic cells modulates the C1q-mediated enhancement of phagocytosis J. Immunol. 152,4005-4016
  31. Norsworthy, P. J., Taylor, P. R., Walport, M. J., Botto, M. (1999) Cloning of the mouse homolog of the 126-kDa human C1q/MBL/SP-A receptor, C1qR(p) Mamm. Genome 10,789-793
  32. Jiang, W., Swiggard, W. J., Heufler, C., Peng, M., Mirza, A., Steinman, R. M., Nussenzweig, M. C. (1995) The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing Nature 375,151-155
  33. Ezekowitz, R. A., Sastry, K., Bailly, P., Warner, A. (1990) Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells J. Exp. Med. 172,1785-1794
  34. Stahl, P. D., Ezekowitz, R. A. (1998) The mannose receptor is a pattern recognition receptor involved in host defense Curr. Opin. Immunol. 10,50-55
  35. Medzhitov, R., Janeway, C. J. (2000) Innate immune recognition: mechanisms and pathways Immunol. Rev. 173,89-97
  36. Kim, T. S., Park, M., Nepomuceno, R. R., Palmarini, G., Winokur, S., Cotman, C. A., Bengtsson, U., Tenner, A. J. (2000) Characterization of the murine homolog of C1qR(P): identical cellular expression pattern, chromosomal location and functional activity of the human and murine C1qR(P) Mol. Immunol. 37,377-389
  37. Webster, S. D., Park, M., Fonseca, M. I., Tenner, A. J. (2000) Structural and functional evidence for microglial expression of C1qR(P), the C1q receptor that enhances phagocytosis J. Leukoc. Biol. 67,109-116
  38. Angelisova, P., Cinek, T., Josek, J., Horejsi, V. (1995) Association of protein kinases with the myeloid cell antigens Schlossman, S. F. Boumsell, L. Gilks, W. Harlan, J. M. Kishimoto, T. Morimoto, C. Ritz, J. Shaw, S. Silverstein, R. Springer, T. Tedder, T. F. Todd, R. F. eds. Leucocyte Typing V ,997-999 Oxford University Press Oxford.
  39. Reis e Sousa, C., Stahl, P. D., Austyn, J. M. (1993) Phagocytosis of antigens by Langerhans cells in vitro J. Exp. Med. 178,509-519



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