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C1qRP, the C1q Receptor That Enhances
Phagocytosis, Is Detected Specifically in Human
Cells of Myeloid Lineage, Endothelial Cells, and
Platelets
Ronald R. Nepomuceno and Andrea J. Tenner
J Immunol 1998; 160:1929-1935; ;
http://www.jimmunol.org/content/160/4/1929
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Copyright © 1998 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
C1qRP, the C1q Receptor That Enhances Phagocytosis, Is
Detected Specifically in Human Cells of Myeloid Lineage,
Endothelial Cells, and Platelets1
Ronald R. Nepomuceno2 and Andrea J. Tenner3
T
he archetypal function for C1q has been its participation
in the effector arm of the humoral immune response as the
recognition subunit of C1, the first component of the classical complement cascade (CCP).4 However, although immune
complexes are often considered the major activator of C1 via the
binding of C1q to Ab Fc domains, it is known that C1q in C1 is
able to bind directly to various substances, including certain viruses and bacteria, and activate CCP in an Ab-independent manner
(1, 2; reviewed in Ref. 3). In addition, C1q has been shown to
interact with cells of the immune system, including monocytes (4),
neutrophils (4), B cells (5), and eosinophils (6), as well as with
endothelial cells (7–9), platelets (10), smooth muscle cells (11),
epithelial cells (11), and fibroblasts (12), initiating a variety of
cellular responses, including the stimulation or enhancement of
different host defense mechanisms, depending on the cell type to
Department of Molecular Biology and Biochemistry, University of California, Irvine,
CA 92697
Received for publication September 9, 1997. Accepted for publication October
30, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Arthritis Foundation, the American
Heart Association, the National Institutes of Health (Grant AI 41090) to A.J.T., and
by National Institutes of Health/National Cancer Institute Training Program in Carcinogenesis Grant 5T32-CA09054 to R.R.N.
2
Current address: Stanford University School of Medicine, MSLS, 3rd Floor, MC:
5487, Stanford, CA 94305.
3
Address correspondence and reprint requests to Dr. Andrea J. Tenner, Department
of Molecular Biology and Biochemistry, University of California-Irvine, 3205 Biological Sciences II, Irvine, CA 92697. E-mail address: [email protected]
4
Abbreviations used in this paper: CCP, classical complement pathway; CR, complement receptor; MBL, mannose binding lectin; SPA, pulmonary surfactant protein
A; C1q-CLR, collagen-like fragment of C1q; PE, phycoerythrin; SSPE, standard saline-phosphate-EDTA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Copyright © 1998 by The American Association of Immunologists
which binds (reviewed in Ref. 13). Specifically, C1q enhances
both FcR- and CR1-mediated phagocytosis by human monocytes
and macrophages in vitro (14, 15) and phagocytosis of immune
complexes by human neutrophils (16), and it triggers or enhances
the generation of toxic oxygen species such as superoxide anions
by human neutrophils (17), eosinophils (6), and vascular smooth
muscle cells (18). C1q has also been shown to enhance neutrophil
killing of the microbe Dirofilaria immitis (19), as well as eosinophil killing of Shistosoma mansoni (6). It also enhances phagocytosis of Salmonella minnesota by pulmonary endothelial cells and
enhances superoxide anion release by these cells during phagocytosis of these bacteria (20). A number of other in vitro functions
have been attributed to the interaction of C1q with cells including
stimulation Ca21-activated K1 channels and initiation of chemotaxis in mouse fibroblasts (21), enhancement of the uptake of
Trypanosoma cruzi trypomastigotes by human fibroblasts (22),
and stimulation of synthesis of Ig by human B lymphocytes (5,
23). Thus, the interaction of C1q with various cells may play an
important role in host defense during the critical period before a
specific immune response can be generated.
C1q-binding proteins were purified from cell surface proteins
extracted from monocytes, U937 cells, or neutrophils via affinity
chromatography using a matrix to which the collagen-like fragment of C1q (C1q-CLR) was immobilized (24). Proteins that
bound C1q-CLR were eluted from this matrix and used as immunogens to generate mAbs that were then screened for the ability of
the Ab to inhibit the C1q-induced enhancement of phagocytosis.
Based on this evidence, a 126,000 Mr (reduced) protein eluted
from the C1q-CLR matrix and recognized by an inhibiting mAb
was then described as a receptor or receptor component for C1q.
This C1q receptor, which modulates monocyte phagocytosis
(C1qRP), has been purified and cloned from the U937 monocytelike cell line (25). C1qRP is a type I membrane glycoprotein and in
U937 cells is encoded by a single mRNA species of 6.7 kb. R139
0022-1767/98/$02.00
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The complement component C1q can interact with a variety of different cells, resulting in multiple functional consequences
depending on the cell type. mAbs R3 and R139, which recognize a 126,000 Mr (reduced) cell surface protein, are able to abrogate
the C1q-mediated enhancement of monocyte phagocytosis. The cDNA encoding this C1q receptor that modulates phagocytosis,
C1qRP, has recently been cloned. Using a DNA probe based on the coding region of the receptor, Northern blot and RT-PCR
analysis of RNA isolated from different cell types showed C1qRP expression in cells of myeloid origin and in endothelial cells, but
not in cells of lymphoid origin nor in the HeLa epithelial-like cell line or iliac artery smooth muscle cells. FACS analysis of cell
surface expression of C1qRP, as detected by mAb R139 and R3, corresponded in all cases to the mRNA levels detected. Using the
anti-C1qRP mAb, the 126,000 Mr receptor was also detected in lysates of human platelets. Interestingly, C1qRP is not expressed
by the promyelocytic leukemia cell line HL-60, and differentiation of these cells with various chemical compounds did not induce
C1qRP expression. It has been reported that C1q can induce specific receptor-mediated responses in fibroblasts. However, RNA
and cell surface expression analysis for C1qRP indicate that this particular C1q receptor is not expressed by either human gingival
or human skin fibroblasts. These data demonstrate selective expression of C1qRP in specific cell types and support the hypothesis
that there is more than one C1q receptor mediating the diverse responses triggered by C1q. The Journal of Immunology, 1998,
160: 1929 –1935.
1930
Materials and Methods
Media, reagents, and Abs
RPMI 1640 medium, DMEM, SuperScript preamplification system for
first-strand cDNA synthesis kit, TRIZOL reagent, RadPrime DNA labeling
system, Taq DNA polymerase, and salmon sperm DNA were purchased
from Life Technologies (Grand Island, NY). Human serum albumin, manufactured by Baxter Healthcare Corporation (Glendale, CA), was obtained
from the American Red Cross Blood Services (Washington, DC). mAbs to
CD19, CD3, and CD33 were purchased from Becton Dickinson (Richmond, CA). Except where noted otherwise, all other reagents were purchased in the highest quality from Sigma Chemical Co. (St. Louis, MO).
Monoclonal anti-C1qRP Abs R139 (IgG2b) and R3 (IgM) and polyclonal
Ab QR1 were generated as previously described (25, 26). Purified R3 IgM
was obtained from ascites fluid using the ImmunoPure IgM purification kit
(Pierce Chemical Co., Rockford, IL) and biotinylated using ImmunoPure
NHS-LC-biotin (Pierce Chemical Co.) according to the manufacturer’s instructions. The R139 and QR1 Abs were purified by sequential octanoic
acid and ammonium sulfate precipitations as described (33) and resuspended in PBS, pH 7.4.
Cells and cell culture
The U937, THP-1, and HL-60 human cell lines were obtained from the
American Type Culture Collection (Rockville, MD). CEM cells were a
kind gift from Dr. E. Remold-O’Donnell (Center for Blood Research, Boston, MA). U937, K562, and Raji cells were cultured in RPMI 1640 medium
containing 10% supplemented bovine calf serum (HyClone Laboratories,
Logan, UT). Human skin and gingival fibroblasts were biopsied from normal volunteers and, like the HeLa cell line, were cultured in DMEM sup5
R. R. Nepomuceno, S. Ruiz, M. Park, and A. J. Tenner. C1qRP is a heavily Oglycosylated cell surface protein involved in the regulation of phagocytic activity.
Manuscript in preparation.
plemented with 10% FCS, 1 mM sodium pyruvate (BioWhittaker, Walkersville, MD), and 0.1 mM nonessential amino acids solution (Life
Technologies). HL-60 and CEM cells were cultured in RPMI 1640 containing 20 or 10%, respectively, FCS (HyClone). THP-1 cells were grown
in RPMI 1640, 10% FCS, and 5 3 1025 M b-mercaptoethanol. Human
peripheral blood monocytes and lymphocytes were isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (34)
as described (4). Blood units were collected into CPDA1 blood-pack units
(Baxter Healthcare Corp., Deerfield, IL) at the UCI Medical Plaza, Irvine,
CA. Cells in each preparation were checked for homogeneity according to
size analysis on a Coulter Channelyzer (Miami, FL). Neutrophils were
isolated from blood drawn from normal volunteers into EDTA-containing
syringes. After centrifugation on lymphocyte separation medium (Organon
Teknika Corp., Durham, NC) cushions and dextran sedimentation as described (15), the residual RBC were removed by hypotonic lysis.
FACS analysis
Cells were washed in HBSS containing 1 mM Ca21, 1 mM Mg21, 0.2%
sodium azide, and 0.2% human serum albumin (FACS buffer), and subsequently incubated for 30 min on ice with mAbs R139, biotinylated R3, or
isotype-matched control Abs. Cells were washed three times in FACS
buffer, then incubated with either FITC-conjugated F(ab9)2 anti-mouse IgG
(Tago, Burlingame, CA), or PE-conjugated streptavidin (Becton Dickinson, San Jose, CA) for 30 min on ice. In some experiments, cells were then
incubated with FITC-anti-CD3, PE-anti-CD19, or PE-anti-CD33 (Becton
Dickinson). After washing three times in FACS buffer, the cell-associated
fluorescence was measured using a FACScan or FACSCalibur (Becton
Dickinson).
Northern blot analysis
HUVEC total RNA designated BC was a kind gift from Dr. Bruce Cronstein (New York University Medical Center, New York, NY). HUVEC
total RNA designated JW and human iliac artery smooth muscle cell total
RNA were kind gifts from Dr. Jeff Winkles (American Red Cross Holland
Laboratory, Rockville, MD). Total RNA from monocytes and neutrophils
was isolated using the RNA isolation kit purchased from Stratagene (La
Jolla, CA). For other cell types, mRNA was isolated using the MicroFastTrack mRNA isolation kit (Invitrogen, San Diego, CA) and total RNA was
isolated using TRIZOL reagent (Life Technologies) according to manufacturer’s instructions. RNA was heat denatured for 15 min at 65°C and
then separated by electrophoresis through 1% agarose gels containing
formaldehyde. The RNA was transferred overnight in 103 SSPE to maximum strength nytran (Schleicher & Schuell, Keene, NH) and UV crosslinked to the membrane. A C1qRP-specific probe, based on a 1458 bp
cDNA insert corresponding to 74.5% of the coding region of C1qRP, was
generated by the random priming method using the RadPrime DNA labeling system (Life Technologies) and 32P-labeled dCTP (DuPont-NEN Research Products, Boston, MA). A similarly generated probe specific for
human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (a kind gift
from Dr. Hung Fan, University of California, Irvine, CA) was used as a
control for monitoring the level of RNA loaded. Hybridization of the
probes were conducted for 16 to 20 h at 42°C in 50% formamide, 53
SSPE, 53 Denhardt’s reagent, 0.1% SDS, and 0.1 mg/ml salmon sperm
DNA. After washing twice in 63 SSPE, 0.1% SDS for 15 min each at
room temperature, twice in 13 SSPE, 0.1% SDS for 15 min at 37°C, and
once in 13 SSPE, 0.1% SDS for 30 min at 60°C, bound probes were
detected by autoradiography.
RT-PCR analysis for C1qRP expression
Total RNA isolated as described above was treated with RQ1 RNase-free
DNase (Promega, Madison, WI) according to the manufacturer’s instructions. First-strand cDNA synthesis from 2 mg of total RNA was conducted
using the SuperScript preamplification system (Life Technologies) with
random hexamers as primers. As a control for the presence of residual
genomic DNA in the RNA preparations, parallel cDNA synthesis reactions
were conducted in the absence of the reverse transcriptase. Two microliters
of the 20-ml cDNA synthesis reaction were used in 50-ml PCR reactions
containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2
mM of each dNTP, 0.1 mM of each primer, and 2 U of Taq DNA polymerase (Life Technologies). The C1qRP primers that amplify a 538-bp
cDNA are 59-AGCTGAGCGCTGCCGAGGCCCA-39 for the sense primer
and 59-TTGGCCGCAGAGGCAAAGGGCA-39 for the antisense primer.
RT-PCR control amplimer sets for human b-actin and transferrin receptor
were purchased from Clontech (Palo Alto, CA). After an initial 2-min
denaturing at 94°C, PCR amplification was conducted for 30 cycles of
denaturing for 30 s at 94°C, annealing for 30 s at 60°C, and extension for
1 min at 72°C in a Perkin-Elmer DNA thermal cycler (Norwalk, CT). PCR
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and R3, the mAbs that recognize C1qRP, have been used to demonstrate surface expression of the receptor on cells of myeloid
origin, such as U937 cells, monocytes, and neutrophils (26), and
on human endothelial cells (27), but the receptor is not found on
cells of lymphoid origin, including lymphocytes, the Raji B cell,
and CEM T cell lines (26). Interestingly, the mAbs R139 and R3
inhibit the C1q-mediated enhancement of monocyte phagocytosis,
but are not able to affect the superoxide production triggered by
C1q in neutrophils, suggesting that the receptors that mediate these
two responses differ in some way. However, since C1qRP is
present on the surface of neutrophils, the possibility of this receptor playing a role in the C1q triggering of superoxide anion release
cannot be completely excluded.
Two other proteins with a macromolecular structure strikingly
similar to C1q are also able to mediate enhanced phagocytosis by
human monocytes through C1qRP: mannose-binding lectin (MBL)
and pulmonary surfactant protein A (SPA) (28, 29).5 However,
unlike C1q, neither MBL nor SPA is able to trigger the neutrophil
superoxide production (30). These data combined with the lack of
inhibition of C1q-mediated superoxide production by the antiC1qRP mAbs, have led us to propose that there are at least two
distinct receptors on myeloid cells: C1qRP, which modulates
phagocytosis, and C1qRO2, which triggers superoxide anion generation. The presence of more than one C1q receptor on the different cell types, as well as different C1q receptors on the same cell
type, would be similar to that seen for IgG and C3 receptors (31,
32). To further characterize the cell type-specific expression of
C1qRP and to verify that receptor expression correlates with surface mAb reactivity, molecular probes based on the recently
cloned C1qRP cDNA, as well as the R139 and R3 Abs, were used
to determine the expression of C1qRP in various human cell types
at both the protein and mRNA level. These studies provide a critical, initial step in determining whether C1qRP, in addition to enhancing monocyte phagocytosis, participates in some of the other
functions triggered by C1q.
ANALYSIS OF C1qRP EXPRESSION
The Journal of Immunology
1931
products were resolved on 1% agarose gels run in TAE according to standard protocols (35) and stained with ethidium bromide.
Platelet C1qRP Immunoprecipitation
Normal human platelet lysate (in 20 mM Tris-HCl, pH 8, 0.1 M NaCl, 1%
Triton X-100, 10 mM N-ethylmaleimide, 0.01% leupeptin) was kindly provided by Dr. Diane Nugent of Children’s Hospital of Orange County, Orange, CA. The total protein concentration of the lysate was 8.25 mg/ml.
The immunoprecipitation was performed as described (26) using 333 ml of
the platelet lysate or 8.3 3 106 cell equivalents of freshly elutriated (4)
human monocytes (in the above lysis buffer) per sample. Precipitated material was separated by SDS-PAGE, transferred to nitrocellulose (Schleicher & Schuell) as described (25), and probed with the biotinylated R3
mAb. Bound Ab was detected chemiluminescently with streptavidin-horseradish peroxidase and the HRPL Western kit (National Diagnostics,
Atlanta, GA).
Results
FACS analysis of C1qRP cell surface expression
Northern blot analysis
Because the mAbs are specific for distinct epitopes on the receptor
protein, it is possible that C1qRP may be present on different cell
lines but that the mAb-binding epitopes may be missing, altered, or
in a different conformation, such that they are no longer accessible
to the Abs. Therefore, RNA isolated from the different cell types
was subjected to Northern blot analysis using a 1458-bp cDNA
fragment corresponding to approximately 75% of the receptor’s
coding region to test for the presence of C1qRP RNA. C1qRP
mRNA from U937 cells migrates as a single species of 6.7 kb in
denaturing agarose gels (25) and is easily detected (Fig. 2). The
single 6.7-kb RNA transcript was found in all cell types in which
FIGURE 1. FACS analysis for detection of C1qRP surface expression.
Cells were stained with R3 and R139 anti-C1qRP mAbs and with the appropriate PE- or FITC-labeled secondary Ab, respectively. Relative fluorescence intensity for R3 is shown on the vertical axis, while relative fluorescence intensity for R139 is shown on the horizontal axis. Quadrant
markers were set according to cells stained with isotype-matched control
Abs.
receptor expression was detected by the mAbs, including U937
cells, monocytes, endothelial cells, THP-1 cells, and neutrophils
(Fig. 2, A and B). Receptor RNA was detected, albeit at very low
levels, in mRNA isolated from K562 cells (Fig. 2A), consistent
with the level of protein expression detected by the anti-C1qRP
Abs. Conversely, C1qRP expression was not detected in mRNA
isolated from skin or gingival fibroblasts, HeLa, Raji, or CEM cells
(Fig. 2A), consistent with the lack of detectable surface immunoreactivity with R139 and R3 (Fig. 1 and Ref. 26). In total RNA
isolated from elutriated lymphocytes, HL-60 cells, and iliac artery
smooth muscle cells, C1q receptor expression was also not detected (Fig. 2B).
RT-PCR analysis
To verify the results of the Northern blot analysis, RT-PCR, a
more sensitive method for the detection of C1qRP RNA transcripts
in the various cells, was employed. Total RNA was isolated from
the cells and used to generate cDNA using random hexamers as
primers. The resulting cDNA was then used as the template in PCR
reactions to amplify a 538-bp fragment of C1qRP that corresponds
to 27.5% of the protein coding region. As shown in Figure 3, U937
cells, monocytes, neutrophils, THP-1, and endothelial cells express
the receptor. Again, K562 cells express a lower level of the receptor RNA. Consistent with the pattern of receptor expression
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The mAbs R139 (IgG2b) and R3 (IgM) recognize the extracellular
domain of C1qRP, which was previously shown to be expressed on
human monocytes, neutrophils, the U937 monocyte-like cell line,
and endothelial cells (26, 27), but not on lymphocytes or the Raji
and CEM lymphoblastoid cell lines (26). To further examine the
cell surface expression of C1qRP on other cell types, the two mAb
were used in FACS analysis of various primary and established
cell lines. Of the cells examined in this study, the acute monocytic
leukemia cell line THP-1 and the chronic myelogenous leukemia
cell line K562 were found to express C1qRP, at substantially lower
levels, however, compared with U937 cells (Fig. 1). The epitheliallike HeLa cell line was not reactive with either of the anti-C1qRP
mAbs. Human fibroblasts have been reported to bind and respond
functionally to C1q (21, 22, 36, 37); however, neither human skin
or gingival fibroblasts express C1qRP as determined by R139 and
R3 reactivity (Fig. 1).
Interestingly, the promyelocytic leukemia cell line HL-60,
which consists primarily of neutrophilic promyelocytes, does not
express C1qRP, despite the fact that human neutrophils express the
receptor at relatively high levels (26). This lack of receptor expression may be due to the difference in differentiation state of the
cell line compared with circulating neutrophils (38). To test this
hypothesis, HL-60 cells were induced to both a monocytic differentiation (with 1a,25-dihydroxy-vitamin D3 and PMA) and granulocytic differentiation (with DMSO and retinoic acid) over a
6-day period. FACS analysis of the differentiated cells showed no
increase in R139 and R3 reactivity over untreated HL-60 cells
(data not shown). Differentiation was verified by the changes in
morphology and adherence of the cells, by the expected increases
in CD14 expression for monocytic differentiation (39), and in CR3
expression for granulocytic and monocytic differentiation (40), as
determined by FACS analysis and by an increase in phorbol 12,13dibutyrate-induced superoxide production by DMSO and retinoic
acid differentiated cells.
1932
determined by the other methods, HL-60, HeLa, Raji, CEM, and
both skin and gingival fibroblast RNA do not contain a detectable
amount of C1qRP transcripts. While C1qRP mRNA was not detected in these cell lines, either the b-actin gene or the transferrin
receptor gene, each of which is expressed at low levels in these
cells, was detected, verifying the presence of RNA from the different cell types in the RT-PCR reactions. In all cases, control
reactions in the absence of reverse transcriptase to check for
genomic DNA contamination were negative (data not shown).
Surprisingly, cells from the elutriated lymphocyte population
consistently contained a low level of C1qRP message detectable by
RT-PCR. Although these cells appear to be negative by Northern
blot (Fig. 2B), and FACS analysis of the lymphocyte population
stained with the R139 mAb are distinctly negative, cells stained
with the R3 mAb occasionally demonstrate a very slight increase
in their mean fluorescence intensity vs control cells (26). While the
population did not contain any monocytes as determined by size
analysis on a Coulter Channelyzer (data not shown), it is possible
that the isolated population does contain a small number of myeloid progenitor cells, such as peripheral blood dendritic cells (41),
that express C1qRP, even in an undifferentiated state, similar to
FIGURE 3. RT-PCR for detecting C1qRP RNA. Two micrograms of
DNase-treated total RNA from each cell type was used to generate cDNA
using random primers and SuperScript II reverse transcriptase at 42°C.
After separate PCR reactions with primers specific for C1qRP cDNA or the
human b-actin or human transferrin receptor gene products, equal volumes
of reaction products were subjected to agarose gel electrophoresis, stained
with ethidium bromide, and photographed under UV light. Control reactions for genomic DNA contamination were conducted in the absence of
the reverse transcriptase and were consistently negative (data not shown).
SMC, smooth muscle cell.
U937 and K562 cells. Cells of myeloid lineage have the cell surface marker CD33, which distinguishes these cells from other
blood leukocytes. However, FACS analysis of elutriated lymphocytes stained with an anti-CD33 Ab did not detect a positive population of myeloid cells (data not shown). Additional FACS analyses demonstrate that the elutriated lymphocyte population
contained neither CD31 T cells nor CD191 B cells, which also
stained positive with the anti-C1qRP Abs (data not shown), and
thus the source of the RT-PCR signal remains unknown.
Human platelets express C1qRP
The reported C1q-mediated effects on human platelets include inhibition of the collagen-induced platelet aggregation (10, 42, 43)
and induction of aIIb/b3 integrins, expression of P-selectin, and
procoagulant activity (44). These functional observations, along
with the demonstration that free C1q binds specifically to human
platelets (10), suggest that surface C1q receptors exist on these
cells. To determine whether C1qRP could have a role in platelet
function in response to C1q, detergent extracts of isolated human
platelets were subjected to immunoprecipitation and detection with
the anti-C1qRP mAbs. As shown in Figure 4, a 100,000 Mr (nonreduced) band similar in size to C1qRP precipitated from U937
detergent extracts is detected in the platelet lysate. Additionally,
this platelet molecule reacts with both the R139 and R3 mAbs,
indicating that platelets do in fact express C1qRP.
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FIGURE 2. Northern blot for detection of C1qRP RNA. mRNA (2 mg
per lane) isolated using oligo(dT) cellulose (A) or total RNA (8.75 mg
HUVEC, 10 mg all others) (B) was subjected to agarose gel electrophoresis
under denaturing conditions. The RNA was transferred to nylon membranes, then hybridized with a 32P-labeled C1qRP-specific probe generated
by the random priming method. After washing and exposing to autoradiographic film, the same membrane was stripped, hybridized to a 32P-labeled
GAPDH probe, washed, and exposed to film. HUVEC were obtained from
Drs. Bruce Cronstein (BC) and Jeff Winkles (JW).
ANALYSIS OF C1qRP EXPRESSION
The Journal of Immunology
FIGURE 4. Immunoprecipitation of C1qRP from human platelet lysate.
U937 cell lysate (lanes 1 and 2) and human platelet lysate (lanes 3 and 4)
were immunoprecipitated with 5 mg of isotype-matched control IgG2b
mAb (lanes 1 and 3) or the R139 mAb (lanes 2 and 4). After separation
by SDS-PAGE under nonreducing conditions, the precipitated proteins
were transferred to nitrocellulose and probed with biotinylated R3 mAb.
Positions of the m.w. standards are indicated on the right.
Expression of C1qRP, the receptor that modulates phagocytosis in
vitro upon ligation with C1q, was found to be limited to cell types
that actively phagocytose (with the exception of platelets), supporting the hypothesis that this particular C1q receptor plays a
specific role in modulating this important host defense mechanism.
C1qRP is detected on human cells of myeloid lineage, including
monocytes, neutrophils, and the U937, THP-1, and K562 cell lines,
and HUVECs as determined by FACS, Northern blot, and RTPCR analyses. Interestingly, C1qRP is also expressed on human
platelets, although the function of this receptor on platelets has yet
to be analyzed. For the most part, the receptor was not detected in
cells of lymphoid lineage, including PBL and the Raji and CEM
lymphoblastoid cell lines, the exception being a low level of
C1qRP message detected by RT-PCR in RNA isolated from elutriated lymphocyte populations. However, because of the sensitivity of the RT-PCR technique, it may be possible that the detected
C1qRP message is the result of amplification of RNA from myeloid progenitor cells, which are similar in size to the lymphocyte
population and thus copurify with these cells. The fact that cells
gated on CD31 or CD191 fluorescence were not stained with the
anti-C1qRP Abs supports the hypothesis that peripheral lymphocytes do not express this C1q receptor. However, future studies
with either more highly purified subpopulations of cells or immunohistochemical staining of tissues should identify the cell population responsible for this C1qRP mRNA expression. C1qRP is also
not expressed by fibroblasts or the HeLa epithelial-like cell line.
Thus, importantly, these data demonstrate that this particular C1q
receptor does not participate in the binding and responses of fibroblasts to C1q and that the binding activity of C1q to cells such
as Raji is not mediated by C1qRP. Iliac artery smooth muscle cells
also do not express C1qRP (Figs. 2B and 3). However, C1q can
still bind (11) and trigger superoxide release from these cells (45),
consistent with the hypothesis that the receptors modulating C1qmediated superoxide generation and enhanced phagocytosis are
distinct.
The promyelocytic cell line HL-60, in which C1qRP was not
detected at either the protein or RNA level, is one exception in the
pattern of C1qRP expression in cells of myeloid lineage. These
cells consist primarily of neutrophilic promyelocytes. Thus, since
blood neutrophils that express C1qRP are terminally differentiated,
the HL-60 cells may not be at a differentiation state in which the
receptor is expressed. Thus far, our studies using various chemical
agents to induce differentiation of these cells have not resulted in
C1qRP expression. We conclude that additional factors or differ-
entiation parameters must be present to induce C1qRP expression
in the HL-60 cell line.
The pattern of expression of C1qRP reported here sharply distinguishes this receptor from two other reported C1q-binding proteins, which have been termed cC1qR and gC1qR. Ghebrehiwet
and colleagues, and later others, isolated a 60,000 Mr protein
termed cC1qR, since it was originally characterized as binding to
the collagen-like region of C1q. More recent reports suggest that it
may be identical to calreticulin (46), a Ca21-binding protein found
in several cellular compartments, with many postulated functions
(47, 48). In contrast to the restricted expression of C1qRP, the
60,000 Mr protein is present on human B cells, Raji cells, and
human T cells (49 –52) as well as on most other cell types tested
(11, 53–56). There is some in vitro evidence that cC1qR is involved in C1q-induced platelet aggregation (reviewed in Ref. 57).
However, a C1q response mediated by this 60,000 Mr protein has
yet to be demonstrated on myeloid or endothelial cells.
gC1qR is another protein identified as binding the globular
heads of C1q (58). While originally reported to be a 33-kDa glycoprotein expressed on the surfaces of vascular and blood cells
(58 – 60), the apparent lack of a transmembrane domain or a consensus sequence for glycosylphosphatidylinositol anchoring does
not make it obvious how gC1qR attaches to the membrane for the
reported surface detection of this protein. Recent studies by van
den Berg and coworkers and Muller-Esterl and colleagues demonstrate that gC1qR is not detected significantly on the surfaces of
most of these cells by FACS analysis (61, 62). Rather, they propose that this 33-kDa protein is a soluble, intracellular or vesicular
protein that can be secreted, but not as cell surface receptor. Additionally, Herwald and coworkers and Lim et al. have demonstrated kininogen-binding (63) and vitronectin-binding (64) activities for this 33-kDa protein independent of C1q. A cellular
response to C1q mediated by gC1qR has not yet been
demonstrated.
The relatively high levels of expression of C1qRP on endothelial
cells is quite interesting in that C1q has been shown to bind to
endothelial cells in a receptor-mediated fashion (7, 8) and has been
shown to mediate binding of immune complexes and aggregates to
endothelial cells in vitro (9). In addition, C1q bound to immune
complexes induces an increase in the adhesive properties of endothelial cells for leukocytes (27) and, alone, mediates the binding of
the S. minnesota Re mutant to pulmonary endothelial cells and
elevates the level of superoxide anion release from these cells in
response to the bacteria (20). While both the 60,000 Mr cC1qRand 33,000 Mr gC1qR-binding proteins have been shown to be
expressed by endothelial cells (27, 55, 62), thus far, the only function demonstrated by either cC1qR or gC1qR derived from endothelial cells is the inhibition of complement-mediated lysis of C1qsensitized erythrocytes by cC1qR (65, 66). Whether or not C1qRP,
cC1qR, or gC1qR is involved in the C1q-mediated increase in
endothelial cell adhesiveness for leukocytes, or the binding and
destruction of bacteria by endothelial cells triggered by C1q, remains to be investigated.
The ability to selectively enhance a defense mechanism such as
phagocytosis with limited, if any, additional effects on other systemic functions in immunocompromised patients, including those
with AIDS, individuals with genetic immunodeficiencies, or patients undergoing surgery with high risk of infection, may provide
a highly effective means of promoting positive clinical outcomes
for these patients. In addition to C1q, MBL, and SPA, other agents
such as cytokines (67, 68) and specific extracellular matrix proteins (69) have also been shown to enhance phagocyte function.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Discussion
1933
1934
Acknowledgments
The authors thank Angela C. Taylor and Lisa Salazar-Murphy for their
assistance with the cell culture and FACS analysis experiments; Dr. Bruce
Cronstein (NYU Medical Center, New York, NY) for HUVEC RNA; Dr.
Jeff Winkles (American Red Cross Holland Laboratory, Rockville, MD)
for HUVEC and smooth muscle cell RNAs; and Dr. Diane Nugent (Children’s Hospital of Orange County, Orange, CA) for the platelet lysate.
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