Download Identification of the hemoglobin scavenger receptor

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Brief report
Identification of the hemoglobin scavenger receptor/CD163 as a natural
soluble protein in plasma
Holger Jon Møller, Niels Anker Peterslund, Jonas Heilskov Graversen, and Søren Kragh Moestrup
The hemoglobin scavenger receptor
(HbSR/CD163) is an interleukin-6– and
glucocorticoid-regulated macrophage/
monocyte receptor for uptake of haptoglobin-hemoglobin complexes. Moreover,
there are strong indications that HbSR
serves an anti-inflammatory function. Immunoprecipitation and immunoblotting
enabled identification of a soluble plasma
form of HbSR (sHbSR) having an electrophoretic mobility equal to that of recombinant HbSR consisting of the extracellular
domain (scavenger receptor cysteinerich 1-9). A sandwich enzyme-linked immunosorbent assay was established and
used to measure the sHbSR level in 130
healthy subjects (median, 1.87 mg/L;
range, 0.73-4.69 mg/L). To evaluate the
sHbSR levels in conditions with increased
leukocyte stimulation and proliferation,
140 patients admitted to a hematological
department were screened. Several patients, with a broad spectrum of diagnoses, had a level of sHbSR above the
range of healthy persons. Patients with
myelomonocytic leukemias and pneumonia/sepsis exhibited the highest levels
(up to 67.3 mg/L). In conclusion, sHbSR is
an abundant plasma protein potentially
valuable in monitoring patients with infections and myelomonocytic leukemia.
(Blood. 2002;99:378-380)
© 2002 by The American Society of Hematology
Introduction
The hemoglobin scavenger receptor (HbSR), also known as CD163
and M130, is the recently identified receptor scavenging haptoglobin-hemoglobin complexes.1 HbSR is a member of the scavenger
receptor cysteine-rich (SRCR) family2 and consists of 9 extracellular group B SRCR domains, a transmembrane segment, and a short
cytoplasmic tail3 with several potential phosphorylation sites.
HbSR is expressed exclusively in cells of the monocytic lineage,
and high expression is seen in phagocytic tissue macrophages.4
There are strong indications that HbSR and its ligand may play a
direct active role in the inflammatory response, and HbSR is tightly
regulated by proinflammatory and anti-inflammatory mediators.
Glucocorticoids, interleukin (IL)-6 and IL-10 (but not IL-4 or
IL-13) induce increased expression of both messenger RNA and
surface receptor protein.5-7 Furthermore, antibody-mediated HbSR
cross-linkage induces intracellular signaling and secretion of IL-6
and granulocyte-macrophage colony-stimulating factor.8,9 Probably, the antibody mimics a ligand-induced signaling.
Shedding of a soluble form of HbSR from in vitro cultured
cells10 and detection of CD163 immunoreactivity in plasma suggest
the existence of a natural soluble form of HbSR.11 We identified
this soluble receptor (sHbSR) in plasma and established a sensitive
assay showing high concentrations in healthy individuals. We then
explored whether the concentration of sHbSR varied in a panel of
patients with various hematological diseases. These data indicate
that the level of sHbSR is dependent on leukocyte stimulation and
proliferation.
Study design
From the Department of Clinical Biochemistry and the Department of
Hematology, AAS Aarhus University Hospital, and Department of Medical
Biochemistry, University of Aarhus, Denmark.
Reprints: Holger J. Møller, Department of Clinical Biochemistry, Aarhus
University Hospital (Amtssygehuset), Tage Hansens Gade 2, DK-8000, Århus
C, Denmark; e-mail: [email protected]; or Søren K. Moestrup, Department of
Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark; e-mail:
[email protected].
Submitted June 11, 2001; accepted August 24, 2001.
Supported by Aarhus University Research Foundation, The Novo Nordisk
Foundation, and the Danish Medical Research Council.
S.K.M. and J.H.G. have declared a financial interest in ProteoPharmaAps,
whose product was studied in the present work.
378
Purification of HbSR and production of recombinant sHbSR
The membrane form of human HbSR was purified as recently described.1
The exact concentration of HbSR was determined by amino acid analysis of
hydrolyzed protein.12 A recombinant sHbSR derivative consisting of the
extracellular domain (SRCR 1-9) without transmembrane segment and
cytoplasmic tail was expressed in Chinese hamster ovary (CHO) cells
stably transfected with an HbSR complementary DNA (cDNA) fragment
encoding amino acids 1 through 1045 of human HbSR. The cDNA plasmid
was generated by the following procedure: A cDNA fragment corresponding to the bases 3045 to 3135 with the addition of a stop codon and a Not I
site was created by polymerase chain reaction (PCR). The PCR-generated
DNA fragment was ligated into the internal Pst I site (position 3056-3061)
and the Not cloning site of the previously described full-length HbSR
pcDNA⫹ plasmid.1 This procedure substituted bases 3136 to 3351,
encoding the transmembrane region and the cytoplasmatic tail of HbSR,
with a stop codon. The expression product from the transfected CHO cells
was secreted into the medium as a soluble protein. The recombinant protein
representing the extracellular domain of HbSR was purified from the
medium by haptoglobin-hemoglobin affinity chromatography.1
Patient samples
Serum was obtained from 130 blood donors at least 3 months after the last
donation (65 men and 65 women), and surplus plasma and/or serum was
obtained from blood samples from 140 patients taken for routine analysis at
the Department of Hematology, Aarhus University Hospital (Denmark).
Clinical data were obtained from the medical files of 131 of the patients.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
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BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
CD163 IS A NATURAL SOLUBLE PROTEIN IN PLASMA
379
Results and discussion
Figure 1. Identification of sHbSR in plasma. Immunoblotting with the monoclonal
GHI/61 antibody of purified membrane-bound HbSR, recombinant sHbSR (extracellular domain expressed in CHO cells), and sHbSR immunoprecipitated (with
polyclonal anti-HbSR IgG) from plasma. Because of hyperglycosylation of the sHbSR
expressed in CHO cells, the electrophoretic mobility was also compared after
deglycosation with PNGase F. (B) Immunoblotting with the monoclonal GHI/61
antibody of sHbSR immunoprecipitated from different plasma samples with different
levels of sHbSR as measured by the sandwich enzyme-linked immunosorbent assay
(ELISA). Enhanced chemiluminescence was used as the detection system.
Figure 1 shows the identification of a soluble form of HbSR by
immunoprecipitation of plasma with polyclonal anti-HbSR-IgGsepharose and subsequent detection by immunoblotting. A single
band with an apparent molecular weight close to that of the
full-length membrane form of HbSR was detected. No soluble forms of
HbSR with lower molecular weights were detected. The solubility
and the size of the protein suggest that this protein constitutes the
extracellular HbSR domain consisting of 9 SRCR protein modules.
Accordingly, the soluble deglycosylated HbSR protein displays
electrophoretic mobility similar to that of deglycosylated recombinant soluble HbSR consisting of SRCR modules 1 through 9.
The anti-HbSR polyclonal antibody used for immunoprecitation and the monoclonal antibody used for immunodetection were
then applied for antigen-immobilization and detection in a sandwich ELISA for measuring sHbSR (with purified HbSR used as a
standard) in plasma. The specificity of the assay measuring
increased levels of sHbSR was validated by immunoprecipitation/
immunoblotting of plasma from hematological patients (see below)
with various levels of HbSR (Figure 1B). The median concentration of sHbSR in 130 blood donors was measured to 1.87 mg/L
(range, 0.73-4.69 mg/L), yielding a reference interval of 0.89 to
3.95 mg/L (Figure 2A). This high level is comparable to that of
soluble transferrin receptor13 and is several fold higher than that of
Precipitation and identification of sHbSR in plasma
Plasma samples (100 ␮L) from blood donors and patients were dialyzed
overnight against 10 mM Hepes (Sigma, St Louis, MO), 140 mM NaCl, 2
mM CaCl2, and 1 mM MgCl2, and incubated 2 hours at room temperature
with 30 ␮g polyclonal rabbit anti-HbSR immunoglobulin (IgG)1 coupled to
cyanogen bromide–activated sepharose (Pharmacia). The beads were
washed 6 times in phosphate-buffered saline (PBS), and bound protein was
released by incubation in a sodium dodecyl sulfate (SDS)–Tris sample
buffer for 4% to 16% SDS–polyacrylamide gel electrophoris. HbSR protein
was then detected by nonreducing immunoblotting with the use of
monoclonal anti-CD163 antibody (GHI/61) (PharMingen, Franklin Lakes,
NJ) as primary antibody (2 ␮g/mL). Deglycosylation of purified HbSR,
recombinant sHbSR, and plasma sHbSR was carried out with the use of
PNGase F (Boehringer, Mannheim, Germany).
Determination of the concentration of sHbSR in plasma
Polyclonal rabbit anti-HbSR IgG (0.004 g/L [4 mg/L]) was coated in
microtiter wells. After being washed, 100 ␮L sample (diluted 1:50 in PBS
with albumin, pH 7.2) was added and incubated for 1 hour. The wells were
washed, and 100 ␮L monoclonal anti-CD163 antibody (GHI/61, 2 ␮g/mL)
was added and incubated for 1 hour. After being washed, 100 ␮L
peroxidase-labeled antibody (goat antimouse immunoglobulins, DAKO
P447 [Carpinteria, CA], diluted 1:4000) was added and incubated for 1
hour. The wells were washed, and 100 ␮L orthophenyldiamine/H2O2
substrate solution was added. After 15 minutes, 50 ␮L of 1 M H2SO4 was
added, and the plates were read at 492/620 nm. Control samples and
standards of purified HbSR were coanalyzed in each run.
Determination of haptoglobin phenotypes
The haptoglobin phenotypes (1-1, 2-1, or 2-2) were determined by
nonreducing immunoblotting of 12.5 nL serum or plasma with the use of a
polyclonal rabbit antihaptoglobin antibody (DAKO) as primary antibody.
Figure 2. Determination of sHbSR in plasma of hematological patients.
(A) Determination of sHbSR in plasma of hematological patients by sandwich ELISA
with polyclonal anti-HbSR used as capture-antibody and with monoclonal anti-CD163
antibody (GHI/61) used as detector antibody. Concentration of sHbSR in controls
(CTRL) (n ⫽ 130) and hematological patients (n ⫽ 140) with various diagnoses. LYM
indicates lymphoma; MM, myeloma; LL, lymphatic leukemia; ML, myelomonocytic
leukemia; AN, anemia from various causes (hemolytic anemia, aplastic anemia,
iron-deficiency anemia, vitamin B12–deficiency anemia, sickle cell anemia, and
thalassemia); MISC, miscellaneous hematological diagnosis (malignant histiocytosis, myelodysplasia, lymphocytosis, essential thrombocytosis, polycytemia, amyloidosis). The right column (INF) shows the sHbSR values in the hematological patients
with infections (pneumonia/sepsis). Solid lines indicate 2.5 and 97.5 percentiles
of the log-Gauss–transformed distribution of the sHbSR values in blood donors.
(B) Patient with newly diagnosed AML type M4 and in chemotherapy. The patient
developed an infection on day 14. Solid line indicates 97.5 percentile of blood donors.
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380
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
MØLLER et al
soluble CD5, which is another member of the SRCR subfamily
reported to be present in plasma.14 The concentration of sHbSR was
not related to the haptoglobin phenotype: the median level was 1.8
mg/L in persons with the 1-1 phenotype (n ⫽ 9) and 1.9 mg/L in
persons with the multimeric 2-1 (n ⫽ 27) and 2-2 phenotype
(n ⫽ 16).
To evaluate whether the concentration of sHbSR might be
affected in conditions with increased leukocyte stimulation and
proliferation, we screened 140 patients admitted to a hematological
department. Several of the patients in this group had sHbSR levels
strikingly above the range measured in healthy blood donors
(Figure 2A). Highest levels of sHbSR were detected in patients
with myelomonocytic leukemia (mean, 9.8 mg/L) (Figure 2A),
especially among those with newly diagnosed nontreated disease or
infection. One patient with newly diagnosed acute monocytic
leukemia (French-American-British M5b, expressing CD13, CD14,
CD33, CD38, and HLADR) had an sHbSR concentration of 67.3
mg/L. A prospective analysis of the sHbSR level in 2 newly
diagnosed AML M4 and M5 patients starting chemotherapy
showed a parallel decrease in the high leukocyte count and sHbSR
concentration (Figure 2B shows the course of the M4 patient).
Interestingly, the sHbSR level increased again shortly after an
infection was diagnosed. Overall, there was a positive correlation
between total leukocyte counts and sHbSR (r2 ⫽ 0.12; P ⬍ .0001;
n ⫽ 129), and between monocyte counts and sHbSR (r2 ⫽ 0.10;
P ⫽ .0003; n ⫽ 122).
Some of the patients with lymphoma, lymphatic leukemia, and
myelomatosis also had increased concentration of sHbSR. One
patient with lymphoma and a high sHbSR level (23.2 mg/L) had a
fatal sepsis. Two patients with myelomatosis and a high sHbSR
concentration (8.5 and 6.4 mg/L) also had an infection. As seen in
the right column of Figure 2A, the majority of hematological
patients with infections had elevated levels of sHbSR. This may
suggest that the high sHbSR level in some hematological patients is
due to the infection rather than the primary disease. A significant
correlation with plasma-C-reactive protein (P-CRP) was not observed (r2 ⫽ 0.03; P ⫽ .11; n ⫽ 88), but it seemed that patients
with increased levels of sHbSR represented a fraction of the
patients with increased levels of P-CRP (not shown). Most patients
with anemia and other nonmalignant diseases had sHbSR levels
within the reference range (Figure 2A). Although one patient with
intravascular hemolysis (due to artificial heart valves) had a high
level of sHbSR (11.3 mg/L), no correlation between sHbSR and
biochemical parameters of hemolysis (plasma-haptoglobin, blood
reticulocyte counts, or Coombs test) was registered.
In conclusion, sHbSR represents a novel, abundant natural
protein in human plasma in accordance with a physiological
shedding of HbSR. Highly increased levels of sHbSR are seen in
patients with myelomonocytic leukemias and infections. This
suggests that the plasma level of sHbSR reflects the total pool of
membrane-bound HbSR, which may be increased in case of
proliferation of cells of myelomonocytic origin or in case of
upregulation of HbSR expression by acute phase mediators.
Prospective studies of various patient groups have now been
initiated to further evaluate sHbSR as a diagnostic parameter in
hematological and inflammatory diseases.
Acknowledgments
Our thanks to Kirsten Hald and Kirsten Lassen for excellent
technical assistance.
References
1. Kristiansen M, Graversen JH, Jacobsen C, et al.
Identification of the haemoglobin scavenger receptor. Nature. 2001:409;198-201.
2. Ritter M, Buechler C, Langmann T, Schmitz G.
Genomic organization and chromosomal localization of the human CD163 (M130) gene: a member of the scavenger receptor cysteine-rich superfamily. Biochem Biophys Res Commun. 1999:
260;466-474.
3. Law SK, Micklem KJ, Shaw JM, et al. A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily. Eur
J Immunol. 1993:9;2320-2325.
4. Zwadlo G, Voegeli R, Osthoff KS, Sorg C. A mAb
to a novel differentiation antigen on human macrophages associated with the down-regulatory
phase of the inflammatory process. Exp Cell Biol.
1987;55:295-304.
5. Högger P, Dreier J, Droste A, Buck F, Sorg C.
Identification of the integral membrane protein
RM3/1 on human monocytes as a glucocorticoidinducible member of the scavenger receptor cysteine-rich family (CD163). J Immunol. 1998;161:
1883-1890.
6. Sulahian TH, Hogger P, Wahner AE, et al. Human
monocytes express CD163, which is upregulated
by IL-10 and identical to p155. Cytokine. 2000;12:
1312-1321.
7. Buechler C, Ritter M, Orso E, Langmann T,
Klucken J, Schmitz G. Regulation of scavenger
receptor CD163 expression in human monocytes
and macrophages by pro- and antiinflammatory
stimuli. J Leukoc Biol. 2000;67:97-103.
CD163 signaling on CKII and protein kinase C.
Eur J Immunol. 2001;31:999-1009.
10. Droste A, Sorg C, Högger P. Shedding of CD163,
a novel regulatory mechanism for a member of
the scavenger receptor cysteine-rich family. Biochem Biophys Res Commun. 1999;256:110-113.
11. Sulahian TH, Hintz KA, Wardwell K, Guyre PM.
Development of an ELISA to measure soluble
CD163 in biological fluids. J Immunol Methods.
2001;252:25-31.
12. Sottrup-Jensen L. Determination of halfcystine in
proteins as cysteine from reducing hydrolyzates.
Biochem Mol Biol Int. 1993;30:789-794.
8. Van den Heuvel MM, Tensen CP, van As JH, et al.
Regulation of CD 163 on human macrophages:
crosslinking of CD163 induces signaling and activtion. J Leukoc Biol. 1999:66:858-866.
13. Feelders RA, Kuiper-Kramer EPA, van Eijk HG.
Structure, function and clinical significance of
transferrin receptors. Clin Chem Lab Med. 1999;
37:1-10.
9. Ritter M, Buechler C, Kapinsky M, Schmitz G.
Interaction of CD163 with the regulatory subunit
of casein kinase II (CKII) and dependence of
14. Calvo J, Places L, Espinosa G, et al. Identification
of a natural soluble form of human CD5. Tissue
Antigens. 1999;54:128-137.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
2002 99: 378-380
doi:10.1182/blood.V99.1.378
Identification of the hemoglobin scavenger receptor/CD163 as a natural
soluble protein in plasma
Holger Jon Møller, Niels Anker Peterslund, Jonas Heilskov Graversen and Søren Kragh Moestrup
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