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Originally published online as doi:10.1189/jlb.1005602 on January 24, 2006

Published online before print January 24, 2006
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(Journal of Leukocyte Biology. 2006;79:837-845.)
© 2006 by Society for Leukocyte Biology

The macrophage scavenger receptor CD163: endocytic properties of cytoplasmic tail variants

Marianne Jensby Nielsen*, Mette Madsen*, Holger J. Møller{dagger} and Søren K. Moestrup*,{dagger},1

{dagger} Departments of Clinical Biochemistry, Aarhus Hospital, and
* Medical Biochemistry, University of Aarhus, Denmark

1Correspondence: Department of Medical Biochemistry, University of Aarhus, Ole Worms Allé, Building 170, 8000 Aarhus C, Denmark. E-mail: skm{at}biokemi.au.dk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD163 is the monocyte/macrophage-specific receptor for haptoglobin-hemoglobin (Hp-Hb) complexes. The cytoplasmic tail of human CD163 exists as a short tail variant and two long tail variants. Reverse transcriptase-polymerase chain reaction analysis indicated that all three CD163 variants are substantially expressed in blood, liver, and spleen, and the short tail variant is the predominant mRNA species. Using cell transfectants in which cDNA encoding the CD163 variants was inserted at the same site in the genome, we evaluated the expression and endocytic properties of the tail variants. Ligand uptake analysis showed that cells expressing the CD163 short tail variant exhibited a higher capacity for ligand endocytosis than cells expressing the CD163 long tail variants. The difference in endocytic activity was explained by confocal microscopic analysis, showing marked deviations in subcellular distribution. Surface expression was far most pronounced for the CD163 short tail variant, whereas the long tail variants were most abundant in the Golgi region/endosomes. Mutational change of a putative signal for endocytosis (Tyr-Arg-Glu-Met), present in a common part of the cytoplasmic tail of the variants, almost completely inactivated the endocytic activity of the short tail variant. In conclusion, the three physiological tail variants of CD163 may contribute to Hp-Hb endocytosis by means of the common ligand-binding region and endocytic signal. However, the high mRNA expression level and relatively high endocytic capacity of the short tail variant suggest that it accounts for the majority of Hp-Hb uptake from the circulation, whereas the long tail variants may have yet-unknown intracellular roles.

Key Words: haptoglobin • haemoglobin • endocytic signal • transfection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rapid elimination of hemoglobin (Hb), released into plasma during intravascular hemolysis, is crucial to avoid the toxicity of the oxidative heme group of Hb. In plasma, Hb instantly binds the acute-phase protein haptoglobin (Hp), which is depleted from plasma in patients with accelerated hemolysis. The Hp-Hb complex is subsequently taken up by tissue macrophages, which degrade the globin part in lysosomes and convert heme to bilirubin and iron. In this process, the acute phase-regulated protein CD163 acts as the scavenger receptor internalizing the Hp-Hb complex [1 ]. The high affinity for CD163 is specific for the Hp-Hb complex, whereas Hp or Hb alone displays no or low affinity toward CD163.

CD163, which is expressed exclusively in monocytes/macrophages, is a relatively newly identified member of the macrophage scavenger receptor family (reviewed in ref. [2 ]). In addition to its function as a Hb scavenger, CD163 appears to be involved in intracellular signaling. This signaling function is triggered by ligand binding to CD163 at the cell surface and results in a protein tyrosine kinase-dependent signal and secretion of interleukin-6 (IL-6) and IL-10 [3 , 4 ]. The CD163-mediated IL-6 and IL-10 secretion points to an immunomodulatory function. Furthermore, as IL-6 and IL-10 are known to up-regulate CD163 (refs. [5 , 6 ] and reviewed in ref. [7 ]), these results suggest the existence of a positive feedback mechanism for CD163 induction. A role for CD163 in immunomodulation is also indicated by the finding that degradation of heme, internalized via CD163, results in the production of metabolites with suggested anti-inflammatory effects (reviewed in ref. [8 ]).

CD163 also exists as a soluble protein in normal human plasma, resulting from proteolytic shedding of the membrane-bound form [9 , 10 ]. Phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS), and cross-linking of the Fc receptor for immunoglobulin G (IgG) have been reported to induce shedding of CD163 [11 12 13 ], but the biological function of soluble CD163 is not yet defined.

CD163 comprises a large, extracellular region with nine scavenger receptor cysteine-rich (SRCR) class B domains, a transmembrane segment, and a short cytoplasmic tail [14 ]. Several different CD163 mRNAs, all arising from alternative splicing of a single CD163 gene, have been described [14 , 15 ]. Three of these encode CD163 proteins with different C-terminal cytoplasmic tails: the CD163 short tail variant, previously demonstrated to mediate endocytosis of Hp-Hb [1 ], and two not-yet characterized forms (the CD163 long tail variant 1 and the CD163 long tail variant 2).

The cytoplasmic tails of these three CD163 isoforms share a common, 42 amino acid membrane proximal region, whereas the length and sequence of the extreme C-terminal region are specific for each variant. The membrane proximal region includes a potential internalization motif of the type YXX{phi} (where {phi} represents a bulky hydrophobic residue), situated 24 residues downstream from the transmembrane segment (reviewed in refs. [16 , 17 ]).

We recently characterized the ligand-binding properties of the extracellular region of CD163 and found that SRCR domain 3 of CD163 is critically involved in the binding of Hp-Hb [18 ]. In this study, the molecular characterization of CD163 concerns the endocytic properties of the intracellular region of the CD163 cytoplasmic isoforms stably expressed in Chinese hamster ovary (CHO) cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Measurement of CD163 mRNA
Buffy coats and EDTA-stabilized peripheral full blood were obtained from blood donors. Peripheral blood mononuclear cells (PBMC) were purified from buffy coats by Histopaque-1077 (Sigma Chemical Co., Brøndby, Denmark) gradient separation and cultured at 0.43 x 106/ml in RPMI 1640 (Invitrogen, Taastrup, Denmark), with or without dexamethasone (5x10–4 mol/l) for 48 h. Total RNA was isolated from mononuclear cells and 1 ml blood using the QIAamp® RNA blood mini kit from Qiagen (Albertslund, Denmark). Human spleen total RNA and human liver total RNA were purchased at Clontech (Palo Alto, CA). In addition, total RNA was extracted from Flp-In CHO cells stably transfected with the CD163 short tail variant, the CD163 long tail variant 1, or the CD163 long tail variant 2 (see below) for use as calibrator materials in quantitative polymerase chain reactions (qPCR).

cDNA was synthesized by incubation of 1 µl RNA, 6.3 mM MgCl2, 0.3 mM deoxy-unspecified nucleoside 5'-triphosphate, 2.5 mM oligo dT primer, 20 U RNase inhibitor, and 50 U murine leukemia virus reverse transcriptase (RT) in a total volume of 20 µl (Applied Biosystems, Nærum, Denmark) at 42°C for 30 min, followed by 99°C for 5 min.

Real-time qPCR was performed for each splice variant in a LightCycler® system (Roche Diagnostics, Hvidovre, Denmark) by incubating 1 µl synthesized cDNA, 0.5 µl (5 pmol) of each primer (CD163 short tail variant forward primer: 5'-gtccaccaaattcaataccg-3', reverse primer: 5'-tcagaatggcctcctgagga-3'; CD163 long tail variant 1 forward primer: 5'-gtccaccaaattcaataccg-3', reverse primer: 5'-tccattccagaaataggaag-3'; CD163 long tail variant 2 forward primer: 5'-gtccaccaaattcaataccg-3', reverse primer: 5'-caaagtagaatgtctgtgcc-3'), 5 µl platinum CYBR Green qPCR SuperMix uracil DNA glycosylase (Roche Diagnostics), and 0.5 µl bovine serum albumin (BSA; 1 mg/ml) in a total volume of 10 µl (cycling conditions were 50°C for 2 min and 95°C for 2 min, followed by 50 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 10 s). The PCR threshold cycle was determined by the LightCycler® software Version 3.5. A standard curve was included in each run by inclusion of a series of dilutions of RNA from specifically transfected cells. By RT-PCR analysis (using the primers 5'-acatagatcatgcatctgtcatttg-3' and 5'-cattctccttggaatctcacttcta-3', which amplify all three variants), the CD163-transfected cells were shown to contain similar amounts of the respective mRNA variants. The relative mRNA abundance of the three cytoplasmic CD163 variants in blood, liver, and spleen was then compared by normalizing to the measured level in the short tail variant.

Establishment of stable CD163 transfectant cells
cDNA encoding the three human cytoplasmic isoforms of CD163 [14 ] was ligated into the KpnI and NotI sites of the mammalian expression vector pcDNA5/FRT (F1p recombination target) (Invitrogen). The CD163 short tail variant Y1091A mutant cDNA construct was generated by a two-step PCR using Pfu Turbo polymerase (Stratagene, La Jolla, CA). The first step produced a megaprimer using a mutated forward primer 5'-gtccaccaaattcaagcccgggagatgaattcttgcc-3' and reverse primer 5'-ccgctcgagtcagtgtggctcagaatggc-3', and the second step produced the total CD163 short tail variant Y1091A mutant cDNA using the megaprimer as reverse primer and the forward primer 5'-cccaagcttgaattcttagttgttttc-3'. In both reactions, the wild-type CD163 short tail variant cDNA was used as template. The CD163 short tail variant Y1091A mutant PCR product was purified with the QIAEX II gel extraction kit (Qiagen) and ligated into the XhoI and HindIII sites of the pcDNA5/FRT vector. The pcDNA5/FRT constructs were transfected into Flp-In CHO cells (Invitrogen) using FuGENE 6 (Roche Diagnostics). Stable transfectants were selected with 500–750 µg/ml Hygromycin B (Invitrogen), and expression products were analyzed by immunoblotting of cell lysates using a rabbit polyclonal anti-CD163 antibody [1 ]. Stably transfected clones were grown in serum-free CHO medium (HyQ-CCM, HyClone, Logan, UT) containing 300 µg/ml Hygromycin B.

Binding of recombinant CD163 to cyanogen bromide (CNBr)-activated Sepharose 4B beads coupled with Hp-Hb
Hp (mixed phenotypes) and Hb A0 were from Sigma Chemical Co. CNBr-activated Sepharose 4B beads (Amersham Biosciences, Hillerød, Denmark) coupled with Hp-Hb were washed twice with a solution containing 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, and 140 mM NaCl, pH 7.8, before incubation with cell lysates from CD163-transfected Flp-In CHO cells. After an overnight incubation at 4°C, beads were washed six times in the above solution. Bound CD163 was eluted by addition of sodium dodecyl sulfate (SDS)-containing sample buffer and visualized by Western blotting using the rabbit polyclonal anti-CD163 antibody.

Confocal immunofluorescence microscopy of CHO cells
We examined the cellular distribution of the CD163 variants in transfected Flp-In CHO cells by confocal immunofluorescent analysis. Cells were grown on chamber slides (Nunc, Roskilde, Denmark) or coverslips (Hounisen, Risskov, Denmark). For the surface-staining experiments, living cells were incubated in serum-free CHO medium containing 10 µg/ml rabbit polyclonal anti-CD163 antibody for 1 h at 4°C. After washing three times in phosphate-buffered saline (PBS) buffer (10 mM NaH2PO4, 0.15 M NaCl, 0.6 mM CaCl2), pH 7.4, the cells were fixed in 4% formaldehyde for 1 h at 4°C, washed three times in PBS, pH 7.4, containing 0.05% Triton X-100, followed by a 1-h incubation at room temperature with Alexa 488-conjugated secondary goat anti-rabbit IgG (Molecular Probes, Leiden, The Netherlands), diluted 1:200 in PBS buffer, pH 7.4, containing 0.05% Triton X-100.

To analyze the total cellular distribution of CD163 and to compare the CD163 localization with Vti1b/GS28 localization, fixed, permeabilized cells (fixed in 4% formaldehyde for 30 min at room temperature and washed three times in PBS buffer, pH 7.4, containing 0.05% Triton X-100) were incubated for 1 h at room temperature with rabbit polyclonal anti-CD163 antibody (10 µg/ml) and a mouse antibody against Vti1b or GS28 (both 1 µg/ml; Catalog No. 611434, BD Biosciences PharMingen, San Diego, CA) in PBS, pH 7.4, containing 0.05% Triton X-100. After washing three times in PBS, pH 7.4, with 0.05% Triton X-100, cells were incubated at room temperature for 1 h with the fluorescence-labeled secondary antibodies Alexa 488-conjugated goat anti-rabbit IgG and Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes), both diluted 1:200 in PBS, pH 7.4, with 0.05% Triton X-100.

To visualize internalization of Hp-Hb, complexes of Hp(2-2) and Hb A0 (both from Sigma Chemical Co.) were labeled with Alexa fluor 488 (Molecular Probes). Cells were washed with PBS, pH 7.4, prior to incubation with Alexa-488-labeled Hp(2-2)-Hb [diluted in serum-free medium for CHO cells supplemented with 1% BSA, 100 µM chloroquine (Fluka, Buchs, Switzerland), and 100 µM leupeptin (Sigma Chemical Co.)] for 30 min at 37°C. At the end of the incubation period, the cells were washed in PBS, pH 7.4. Subsequently, 10 µg/ml rabbit polyclonal anti-CD163 (diluted in serum-free CHO medium) was added to the cells and allowed to bind CD163, exposed on the cell surface during a 1-h incubation period at 4°C. After washing in PBS, pH 7.4, the cells were fixed in 4% formaldehyde for 1 h at 4°C, followed by washing in PBS, pH 7.4, containing 0.05% Triton X-100, and incubation with Alexa-594-conjugated goat anti-rabbit IgG diluted 1:200 in PBS, pH 7.4, containing 0.05% Triton X-100 for 1 h at room temperature.

Immunostained cells were analyzed by confocal immunofluorescence microscopy using a Zeiss LSM-510 confocal microscope (Zeiss, Jena, Germany).

Endocytosis experiments
Endocytic analysis was performed with CD163-transfected or nontransfected Flp-In CHO cells grown to confluence in 24-well plates (0.07–0.09 mg protein/well) essentially as described [19 ]. In brief, complexes of Hp(2-2) and Hb A0 (both from Sigma Chemical Co.) were labeled with 125I using the chloramine-T method, and triplicates of cells were incubated in serum-free CHO medium containing 4000 counts per minute 125I-labeled Hp(2-2)-Hb for various time intervals at 37°C. At the end of the incubation period, the medium was removed and precipitated with 12.5% trichloroacetic acid (TCA) to separate soluble fragments of degraded Hp(2-2)-Hb from intact Hp(2-2)-Hb. Degradation of ligand was determined as the cell-mediated increase in TCA-soluble radioactivity in the medium. Finally, the washed cell layers were lysed with 0.5 M NaOH, and cell-associated radioactivity was determined by counting the radioactivity of the lysate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The CD163 short tail variant is the predominant CD163 mRNA species
Figure 1 shows a schematic presentation of the three CD163 variants investigated in the present study. The different C-terminal tail sequences are displayed.


Figure 1
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  		Figure 1. Schematic presentation of the CD163 cytoplasmic tail variants. SRCR domains are indicated by oval symbols. The variants have amino acids (aa.) 1–1109 in common. This part encodes the extracellular domain, the transmembrane region, and the first 42 amino acids of the cytoplasmic tail. The amino acid sequences of the different C-terminal tails are displayed. Numbers denote amino acid positions.

 
In agreement with previous data on blood monocytes [14 , 20 ], RT-PCR analysis performed on RNA from blood, liver, and spleen demonstrated expression of all three CD163 tail variants (Fig. 2A 2C and 2D ). The CD163 short tail variant mRNA was the predominant isoform detected in blood (accounting for ~50% of the total mRNA amount of CD163 cytoplasmic variants; see Fig. 2A ) as well as in liver and spleen (accounting for ~70% of the total mRNA amount of CD163 cytoplasmic variants in both tissues; see Fig. 2C and D ). The CD163 long tail variant 1 was the least abundant mRNA species in blood, whereas in liver and spleen, the least abundant variant was the CD163 long tail variant 2. We also investigated the CD163 mRNA expression profile in mononuclear cells and found the relative abundance of the variants to be similar to that in blood (Fig. 2B) .


Figure 2
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  		Figure 2. mRNA expression of CD163 cytoplasmic tail variants in human blood and tissues. The relative abundance of the CD163 short tail variant, the CD163 long tail variant 1, and the CD163 long tail variant 2 was determined by qPCR in blood (A), mononuclear cells with (+) or without (–) stimulation with dexamethasone (B), liver (C), and spleen (D). RNA from Flp-In CHO cells stably transfected with each variant was used as calibrators in qPCR reactions, and results were normalized to the short tail variant (=1).

 
CD163 expression is well known to be induced by glucocorticoids [21 ]. We therefore examined the effect of dexamethasone on mRNA expression of the individual CD163 cytoplasmic tail variants in mononuclear cells. It is interesting that dexamethasone treatment of mononuclear cells gave rise to an approximate fivefold and fourfold up-regulation of the mRNA of the CD163 short tail variant and the CD163 long tail variant 1, respectively, whereas the CD163 long tail variant 2 mRNA amount was less dramatically affected (~1.7-fold up-regulation).

The cytoplasmic tail variants of CD163 bind Hp-Hb
To analyze functional properties of the CD163 cytoplasmic tail isoforms, we established stably transfected Flp-In CHO cell lines expressing the three variants (Fig. 3A ). The Flp-In system, which allows the CD163 cDNAs to be inserted at the same site in the genome, was chosen to avoid substantial differences in transcriptional activity. Even so, protein expression of the CD163 short tail variant was ~50% of the CD163 long tail variant 1 expression and ~200% of the CD163 long tail variant 2 expression, as determined by Western blot analysis, thus suggesting that the tail sequences affect the overall expression levels.


Figure 3
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  		Figure 3. Expression of recombinant CD163 cytoplasmic tail variants in Flp-In CHO cells. (A) Detection of recombinant CD163 splice variants in lysates of stably transfected Flp-In CHO cells. Triton X-100 solubilized cells were subjected to nonreducing SDS-polyacrylamide gel electrophoresis (4–16% polyacrylamide) followed by electroblotting and immunodetection with rabbit polyclonal anti-CD163 antibody. Positions of molecular size standard markers are indicated. The protein expression of the CD163 short tail variant was ~50% of the CD163 long tail variant 1 expression and ~200% of the CD163 long tail variant 2 expression. (B) Hp-Hb binding ability of the expressed CD163 variants. Cell lysates were incubated with Hp-Hb-sepharose beads or blank sepharose beads. After extensive washing, bound material was eluted and subjected to immunoblotting as described in A.

 
In accordance with the CD163 isoforms having identical extracellular domains, the three CD163 variants from lysate of transfected CHO cells could be precipitated by Hp-Hb-sepharose beads (Fig. 3B) , thus demonstrating that the CD163 variants are all active in terms of Hp-Hb binding.

Transfected cells expressing the CD163 short tail variant have a higher capacity for ligand internalization and degradation than transfected cells expressing the CD163 long tail variants 1 and 2
To compare the endocytic properties of the CD163 variants, Flp-In CHO cells transfected with the three cytoplasmic tail isoforms of CD163 were incubated with 125I-labeled Hp-Hb. Figure 4A shows the time course of cell-associated radioactivity and TCA-soluble radioactivity (representing degraded Hp-Hb) in the medium. As previously seen [1 ], the cell-associated radioactivity in cells expressing the CD163 short tail variant reached a plateau after 1 h of incubation (left panel). A similar time course of cell-associated radioactivity was observed for cells expressing the CD163 long tail variants 1 or 2, although the plateau reached in the cells expressing the CD163 long tail variant 1 was at a lower level (Fig. 4A , middle panels).


Figure 4
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  		Figure 4. Effect of the cytoplasmic tails on Hp-Hb endocytosis in stably transfected Flp-In CHO cells. (A) Time course for cell-associated ({square}) and degraded ({blacksquare}) 125I-Hp-Hb in CD163-transfected or nontransfected Flp-In CHO cells. Confluent cell layers (0.07–0.09 mg protein/well) were incubated with 125I-Hp-Hb at 37°C for various time intervals. Degradation was measured as the cell-mediated increase of TCA-soluble radioactivity in the growth medium, and cell-associated ligand was determined by counting the radioactivity of cell lysate. Values are mean ± 1 SD of triplicate samples. The experiments shown are representative of three independently performed experiments. As a result of the different expression levels of the CD163 variants (see Fig. 3A ), the values measured in cells expressing the CD163 long tail variants 1 and 2 were standardized to the values of the CD163 short tail variant-expressing cells by multiplication, by 0.5 and 2, respectively. (B) Effect of the lysosomal inhibitors chloroquine and leupeptin (both 100 µM) on Hp-Hb endocytosis. The amount of cell-associated (open bars) and degraded (solid bars) 125I-Hp-Hb in the absence or presence of chloroquine and leupeptin after a 4-h incubation period at 37°C is indicated. Values are mean ± 1 SD of triplicate samples. The experiments shown are representative of three independently performed experiments.

 
The uptake of 125I-labeled Hp-Hb in all three cell lines was accompanied by degradation of the ligand, as determined by the appearance of TCA-soluble radioactivity in the medium (Fig. 4A) . However, when related to CD163 protein expression levels, the amount of degraded Hp-Hb ligand measured in cells expressing the CD163 short tail variant was ~2.5-fold and ~1.6-fold higher than in cells expressing the CD163 long tail variants 1 and 2, respectively. Only a low degree of cell-associated radioactivity and TCA-soluble radioactivity was detected in nontransfected Flp-In CHO cells (Fig. 4A , right panel).

The lysosomal inhibitors leupeptin and chloroquine (both 100 µM) strongly inhibited degradation of the 125I-Hp-Hb complex in all three transfected cell lines (Fig. 4B) , and accordingly, the amount of cell-associated radioactivity was increased. We conclude from this set of experiments that the CD163 cytoplasmic tail isoforms are all able to mediate endocytosis of the Hp-Hb complex and that cellular uptake is accompanied by lysosomal degradation of the cargo. However, in relation to the overall CD163 protein expression level, the CD163 short tail variant has a substantially higher endocytic efficacy than the CD163 long tail variants 1 and 2.

Cellular distribution of the CD163 cytoplasmic tail variants
To further characterize the CD163 cytoplasmic tail variants, we investigated their subcellular distribution in transfected Flp-In CHO cells by confocal immunofluorescence microscopy. Analysis of CD163 cell-surface expression disclosed important differences among the cell lines expressing the three CD163 variants. As seen in Figure 5 and confirmed by semiquantitative assessment of the fluorescence intensity by use of the LSM-510 software, cells transfected with the CD163 short tail variant exhibited a pronounced cell-surface staining (left panel), whereas cells expressing the CD163 long tail variants 1 or 2 displayed a significantly lower degree of cell-surface staining (Fig. 5 , middle and right panels). The surface staining of the CD163 variants was not affected by incubation with a saturating concentration (1 µM) of the ligand Hp-Hb (data not shown). This suggests a constitutive recycling of the CD163 variants to the plasma membrane.


Figure 5
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  		Figure 5. Cell-surface immunostaining of CD163 cytoplasmic tail variants in transfected Flp-In CHO cells. Detection of CD163 at the cell surface by confocal immunofluorescence microscopy. Living cells were incubated with rabbit polyclonal anti-CD163 antibody at 4°C. After fixation, CD163 was visualized using Alexa-488-labeled goat anti-rabbit antibody.

 
Staining of fixed, permeabilized cells showed that cells expressing the CD163 long tail variants 1 and 2 displayed a proportionally higher degree of intracellular staining compared with the cell peripheral staining (Fig. 6 , left panels). When related to the overall CD163 protein expression level, the amount of intracellular staining appeared particularly prominent in cells expressing the CD163 long tail variant 1. Using antibodies against proteins resident in the Golgi-apparatus/endosomes, we determined the intracellular localization of the CD163 splice variants in more detail. As seen in Figure 6 , the most intense intracellular staining for CD163 in all three cell lines coincided with the staining for Vti1b, a protein located in the trans-Golgi network (TGN) area and endosomes [22 , 23 ] (Fig. 6 , middle panels). However, use of antibody against the cis-Golgi protein GS28 [24 , 25 ] revealed a distinct difference in distribution pattern (Fig. 6 , right panels). Whereas the CD163 long tail variant 2 localization displayed some overlap with GS28, there was poor colocalization between this cis-Golgi marker and the CD163 short tail variant as well as the CD163 long tail variant 1.


Figure 6
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  		Figure 6. Subcellular distribution of CD163 cytoplasmic tail variants in transfected Flp-In CHO cells. Detection of CD163 in stably transfected Flp-In CHO cells by confocal immunofluorescence microscopy. Fixed and permeabilized cells were incubated with rabbit polyclonal anti-CD163 antibody and mouse antibodies against Vti1b or GS28. CD163 variants were visualized using Alexa-488-labeled goat anti-rabbit antibody (green). Vti1b/GS28 was visualized with Alexa-594-labeled goat anti-mouse antibody (red).

 
Mutation of the candidate internalization signal inhibits internalization
To investigate the role of the putative internalization signal (1091YREM1094) in the common membrane proximal part of the CD163 cytoplasmic domain, we established a stably transfected Flp-In CHO cell line, which expresses the CD163 short tail variant carrying a mutation in the candidate internalization signal (denoted CD163 short tail variant Y1091A mutant; see Fig. 7A ). Western blot analysis of cell lysates showed that the expression level of the CD163 short tail variant Y1091A mutant was similar to that of the wild-type receptor in transfected cells (data not shown).


Figure 7
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  		Figure 7. Inhibition of Hp-Hb endocytosis by mutation of the candidate internalization signal 1091YREM1094. (A) Sequence of the cytoplasmic tail of the CD163 short tail variant. The potential internalization signal is highlighted in green. The position of the introduced mutation is indicated. (B) Cell-associated (open bars) and degraded (solid bars) 125I-Hp-Hb in Flp-In CHO cells expressing the CD163 short tail variant wild-type receptor or the CD163 short tail variant Y1091A mutant form. Cells were incubated with 125I-Hp-Hb at 37°C, and the amount of cell-associated and degraded 125I-Hp-Hb after 4 h was determined as described in Figure 4 . Values are mean ± 1 SD of triplicate samples. The experiments are representative of three independent experiments. (C) Vesicular uptake of fluorescent Hp-Hb complexes (green) in transfected Flp-In CHO cells and cell-surface immunostaining of CD163 (red). Cells were incubated with Alexa-488-labeled Hp-Hb for 30 min at 37°C in the presence of the lysosomal inhibitors chloroquine and leupeptin (both 100 µM), followed by incubation with rabbit polyclonal anti-CD163 antibody at 4°C. After fixation, cells were incubated with Alexa-594-labeled goat anti-rabbit antibody to visualize CD163 at the cell surface. Notice the low accumulation of Alexa-488-labeled Hp-Hb in the cells expressing the receptor mutant.

 
As expected, the mutation did not affect binding of Hp-Hb, as determined by Hp-Hb affinity precipitation of the receptor present in lysate of transfected cells (data not shown). However, the endocytic properties of the CD163 short tail variant Y1091A mutant were strongly compromised as shown in Figure 7B . After a 4-h incubation period with 125I-Hp-Hb, the amount of ligand degradation measured in cells expressing the CD163 short tail variant Y1091A mutant was ~2.5-fold lower than in cells expressing the wild-type receptor. To evaluate the effect of the mutation at the single-cell level, the endocytic properties were also investigated by confocal immunofluorescence microscopic analysis after incubation with fluorescent Hp-Hb complexes (Fig. 7C) . Although the cell lines showed a similar level of surface staining (data not shown), the vesicular uptake of fluorescent Hp-Hb was reduced greatly in cells expressing the CD163 short tail variant Y1091A mutant compared with cells expressing the wild-type receptor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have analyzed endocytic properties of three CD163 splice variants with different cytoplasmic tails using stably transfected cell lines. We demonstrate that the three variants are able to bind and internalize the Hp-Hb complex and that cellular uptake is accompanied by lysosomal degradation of the cargo. However, as a consequence of a higher cell-surface expression, the CD163 short tail variant had a higher endocytic efficacy than the CD163 long tail variants 1 and 2, which predominated in TGN and endosomes. The transfection of cells was done using a technology inserting the cDNAs at the same site of the genome. Therefore, it seems reasonable to suggest that the observed relative differences in CD163 variant subcellular distribution also apply to the various macrophage populations in vivo.

We furthermore investigated the mRNA expression profile of the CD163 cytoplasmic tail isoforms and found that all three variants are expressed in blood, liver, and spleen. It is important that our results demonstrate that the CD163 short tail variant is the predominant mRNA species in blood and both tissues examined. Previous work on CD163 splice variant expression is restricted to studies on the CD163-expressing SU-DHL-1 cell line and human monocytes (LPS-stimulated, glucocorticoid-stimulated, or nonstimulated) [14 , 20 ]. In these studies, the CD163 short tail variant mRNA was also shown to represent the major CD163 mRNA species.

The ability of the three CD163 variants to mediate internalization of surface-bound ligand appears to be a result of a YXX{phi} internalization motif [26 ] present in the common part of the cytoplasmic tail region. Accordingly, substitution of the Tyr residue of the 1091YREM1094 motif in the CD163 short tail variant strongly impaired endocytosis. The low endocytic activity of the CD163 long tail variants relative to the CD163 short tail variant is therefore more likely to be a consequence of differences in cell-surface expression level than differences in endogenous endocytic potential.

It is interesting that alternative splicing of cytoplasmic domains as a means of modulating receptor subcellular distribution has also been reported for the human SorCs1 receptor and several members of the integrin family (ref. [27 ] and reviewed in ref. [28 ]). The fact that the bulk of the CD163 long tail variants concentrates on TGN/endosomal compartments at steady-state in transfected cells suggests alternative functions for these variants. One possible function for CD163 in the TGN is sorting of proteins transported via the secretory pathway. However, it is also feasible that the large pool of CD163 in the TGN area serves as a temporary storage, which can be released and transported to the cell surface in response to specific stimuli.

Of the three variants studied, the CD163 long tail variant 1 was particularly enriched inside the cell, where it displayed complete colocalization with Vti1b. It is intriguing that an acidic region containing a candidate casein kinase II (CKII) phosphorylation site is found in the cytoplasmic tail of the CD163 long tail variant 1 but is absent in the other tail variants (Fig. 8 ). AC motifs containing CKII phosphorylation sites are found in several transmembrane proteins, including the endoprotease furin, which localize to the TGN at steady-state (reviewed in ref. [26 ]). Upon budding from the TGN, furin can be retrieved from a post-TGN endosome to the TGN by a mechanism dependent on CKII phosphorylation of the AC motif [29 30 31 ]. By analogy with this, it is tempting to believe that the AC motif residing in the CD163 long tail variant 1 serves as a TGN-retrieval signal that mediates high accumulation of the CD163 long tail variant 1 in the endosomal/TGN compartment.


Figure 8
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  		Figure 8. Multiple species alignment of the CD163 cytoplasmic domains. Alignment of the cytoplasmic tail of human CD163 cytoplasmic tail variants (short tail variant, GenBank Accession No. Z22968; long tail variant 1, GenBank Accession No. Z22969; long tail variant 2, GenBank Accession No. Z22970) and the cytoplasmic region of pig CD163 (GenBank Accession No. NM_213976), rat CD163 (GenBank Accession No. XM_232342), chimpanzee CD163 (GenBank Accession No. XM_508986), dog CD163 (GenBank Accession No. XM_534898), mouse CD163 (GenBank Accession No. AF274883), and cow CD163 (GenBank Accession No. XM_613380). Please note that only an incomplete version of the cow CD163 sequence (lacking the most C-terminal residues) is available. The acidic cluster (AC) of human CD163 long tail variant 1 is indicated by a horizontal line. Putative CKII and protein kinase C (PKC) phosphorylation motifs in the three human variants are shown.

 
The present data furthermore demonstrate that the protein expression of the CD163 long tail variant 1 was approximately twofold higher than that of the CD163 short tail variant and fourfold higher than that of the CD163 long tail variant 2 in transfected cells. This was an unexpected observation, as the Flp-In expression system was used to eliminate substantial differences in transcriptional activity. These expression levels do of course not reflect the in vivo expression in macrophages but may indicate intrinsic differences in protein stability of the receptor variants. It is interesting that mutation of CKII sites in the AC of the duck Gp180 cytoplasmic domain significantly reduces protein half-life, thus indicating that the CKII sites of the AC have a positive effect on protein stability [32 ]. A similar function for the CKII site of the AC in the CD163 long tail variant 1 could explain the high protein expression level of the CD163 long tail variant 1 relative to the CD163 short tail variant and the CD163 long tail variant 2 in transfected cells.

Sequence alignments of human CD163 with mouse CD163 reveals yet an interesting feature of the CD163 long tail variant 1 tail sequence. Whereas the 39 most N-terminal residues of the mouse CD163 cytoplasmic domain show considerable homology with the common cytoplasmic region of the human CD163 cytoplasmic tail variants, including a potential internalization signal of the YXX{phi} type (Fig. 8) , the remaining part of the mouse CD163 C-terminal sequence displays high homology to the extreme C terminus of the CD163 long tail variant 1 (Fig. 8) . No corresponding region is found in the tails of the CD163 short tail variant or the CD163 long tail variant 2. The available dog CD163 sequence also reveals overall high homology to the human CD163 long tail variant 1 sequence. However, in this case, the homology stems from the 25 residues immediately following the membrane proximal region of the long tail variant 1 in addition to the membrane proximal region itself. It is intriguing that the part corresponding to the extreme C-terminal of the human CD163 long tail variant 1 and the mouse CD163 lacks in the dog protein. Analysis of the available CD163 sequence from chimpanzee reveals almost complete sequence identity between the chimpanzee CD163 cytoplasmic domain and the entire cytoplasmic domain of the human CD163 long tail variant 1.

Available species-specific CD163 sequences also include the pig, rat, and cow CD163 sequences. Whereas the pig and rat sequences most closely resemble the human CD163 short tail variant sequence, the cow CD163 is more related to the human CD163 long tail variant 2 (Fig. 8) . The finding that close homologues of all three human CD163 cytoplasmic tail isoforms are found in various species underscores the physiological relevance of the human CD163 cytoplasmic tail variants.

The common cytoplasmic domain sequence of the human CD163 variants encompasses motifs complying with the consensus for phosphorylation by PKC and CKII (Fig. 8) . Accordingly, all three variants are phosphorylated by PKC-{alpha} in vitro, whereas only the CD163 short tail variant and the CD163 long tail variant 1 are phosphorylated by CKII in vitro [33 ]. As described previously [33 ], the cytoplasmic domain sequence specific for the CD163 long tail variant 1 contains additional, potential phosphorylation sites for PKC and CKII (Fig. 8) , thus suggesting that the CD163 long tail variant 1 could be subject to a higher degree of phosphorylation than its tail variants. Alternative splicing of the CD163 cytoplasmic region may therefore modulate the phosphorylation status of the intracellular tail and possibly, intracellular signaling mediated by CD163, as has been reported for the homologous SRCR-containing protein CD6 [34 ]. In this way, CD163 might contribute to immunological functions of the macrophage [35 ]. However, this aspect of the CD163 variants remains to be investigated further.

In conclusion, the present study shows that despite a common signal conveying endocytosis, the CD163 cytoplasmic tail variants display divergent endocytic activities as a result of differences in their subcellular localization patterns. The high endocytic efficacy of the CD163 short tail variant, together with its relatively high mRNA expression level, suggests it to be the main contributor to Hp-Hb uptake from the circulation. The high content of the long tail variants in the Golgi area indicates that these variants represent an intracellular CD163 pool for mobilization and/or a pool with specific intracellular functions.


   ACKNOWLEDGEMENTS
 
This work was supported by the Lundbeck Foundation and The Danish National Health Research Council. We thank Gitte Ratz and Kirsten Bank Petersen for technical assistance and S. K. Alex Law for providing the cDNAs encoding the CD163 cytoplasmic tail variants.

Received October 24, 2005; accepted November 17, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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