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(Journal of Leukocyte Biology. 2001;70:793-800.)
© 2001 by Society for Leukocyte Biology

C1qRP, a myeloid cell receptor in blood, is predominantly expressed on endothelial cells in human tissue

Maria I. Fonseca, Philip M. Carpenter*, Minha Park, Gail Palmarini, Edward L. Nelson{dagger} and Andrea J. Tenner

Departments of Molecular Biology and Biochemistry,
* Pathology, and
{dagger} Medicine, University of California, Irvine, Irvine, CA 92697


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
C1qRP is a type I cell surface glycoprotein that has been shown to enhance ingestion of suboptimally opsonized targets by phagocytes in vitro. In this study, we developed and characterized polyclonal antibodies to study the tissue distribution of this receptor targeted to either the N- or C-terminal portion of the molecule. C1qRP was detected in vascular endothelial cells and in a subset of pyramidal neurons in the brain, as well as neutrophils, but it was absent in most tissue macrophages. Analysis of in vitro differentiation of blood monocytes to dendritic cells demonstrated a down-regulation of the receptor as monocytes differentiate to dendritic cells, providing a possible explanation for the lack of reactivity of these cells in tissue. The predominant presence of C1qRP in endothelial cells, while compatible with a phagocytic role in host defense and/or clearance of cellular material, suggests other possible novel roles for this receptor.

Key Words: complement • phagocytosis • dendritic cells • receptor • monocytes • neutrophils


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
C1q, a serum protein first recognized for its role in activating the classical complement pathway, belongs to a family of soluble defense collagens and, as such, interacts in a receptor-mediated way to stimulate a variety of cell functions involved in host defense mechanisms [1 ]. These functions include enhancement of phagocytosis by monocytes, macrophages, and neutrophils; superoxide production by neutrophils; and induction of expression of adhesion molecules in endothelial cells (reviewed in [2 ]). C1q has also been shown to bind to apoptotic cells, which suggests that it can help rapidly clear these cells and thereby contribute to avoiding the autoimmunity involving intracellular autoantigens [3 , 4 ].

Several binding proteins have been isolated and proposed as candidate receptors for C1q (reviewed in refs 5 6 ). Monoclonal antibodies developed against a cell surface glycoprotein isolated from U937 cells and monocytes inhibited the enhancement of phagocytosis mediated by C1q [7 ], as well as that mediated by other members of the defense collagen family [8 ]. These results indicate that this polypeptide is a common functional receptor/receptor subunit shared by these ligands that can modulate phagocytosis in early stages of infection or trauma when little or no antibody is present. This Clq receptor that enhances phagocytosis, designated C1qRP, has been cloned and characterized as a novel, heavily glycosylated, type I membrane glycoprotein [8 , 9 ]. The molecule displays strong homology (67–87 % identity) across humans, mice, and rats [10 11 12 13 14 ]. C1qRP was first detected on professional phagocytic cells such as peripheral blood monocytes, neutrophils [2 ], microglia [15 ], and myeloid cell lines [12 ]. It has also been expressed in other cell types, such as endothelial cells [13 , 16 ], human primitive stem cell populations [16a ], early hematopoietic cells in fetal mouse [11 ], and NK cells in rat [13 , 14 ]. Thus, at least in these species, this receptor may be involved in functions other than modulation of phagocytic activity, such as cell adhesion (e.g., in early hematopoietic stem cell development) or as yet undefined roles in nonphagocytic innate NK cells.

To gain further insight into the in vivo function of the human C1qRP, we studied its tissue distribution by immunocytochemistry, using newly developed and characterized polyclonal antibodies. The antibodies specifically recognized C1qRP and were reactive with myeloid and endothelial cells in culture. In normal human tissues, C1qRP staining was associated with essentially all vascular endothelium, a subset of pyramidal neurons in the brain and tissue neutrophils, but, surprisingly, it was absent in myeloid cells of liver, spleen, and lymph node tissue. Fluorescein-activated cell sorter (FACS) analysis demonstrated a loss of C1qRP expression when monocytes were differentiated to dendritic cells in vitro and further activated with TNF-{alpha}. These findings indicate that the role of this receptor in phagocytic cells may be stage-specific and suggest that additional functional roles for C1qRP remain to be discovered.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
U937 cells (human histiocytic cell line) were obtained from American Type Culture Collection (Rockville, MD) and grown in suspension in RPMI-1640 media (Gibco BRL, Rockville, MD) containing 10% fetal calf serum (FCS) (Hyclone, Logan,UT). HUVEC (human umbilical vascular endothelial cells) and HUCE (human capillary endothelial cells) were obtained from Dr. C. Hughes (UCI) and cultured in M199 supplemented with 20% FCS, endothelial cell growth supplement (Collaborative Biomedical, Bedford, MA) and heparin (Sigma, St. Louis, MO). Transfected CHO (Chinese hamster ovary) cells were cultured in HAM’s F12/10% FCS with 200 µg/mL G418 (Gibco BRL). SF9 cells were grown in Ex-CellTM 401 serum-free media (JRH Bioscience, Lenexa, KS). Rat microglia, isolated as previously described [15 ], were cultured on coverslips in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS. Mouse peritoneal macrophages (nonactivated) were obtained from Dr. E. Peterson (UCI) from mouse peritoneal lavage with cold phosphate-buffered saline (PBS) and cultured in 10% FCS/DMEM.

Human peripheral blood monocytes were purified from the blood of normal healthy donors by gradient centrifugation followed by elutriation, as previously described [16 ]. To induce dendritic cell differentiation, monocytes were plated at 1 x 106 cells/mL in RPMI supplemented with 10% low-endotoxin FCS (Summit Biotechnology, Forth Collins, CO), 1% penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1X nonessential amino acid mixture (Gibco), 50 µM 2- mercaptoethanol (Sigma), 100 U/mL granulocyte-macrophage colony stimulating factor (GM-CSF) (Immunex, Seattle, WA), and 20 ng/mL interleukin-4 (PeproTek Inc., Rocky Hill, NJ), following the procedure of Nelson et al. [17 ]. Cultures were fed every 6–7 days by removing half of the culture volume and adding an equal volume of fresh media containing sufficient GM-CSF and IL-4 for the entire culture volume. TNF-{alpha} (PeproTek Inc.) (20 ng/mL) was added at day 12 or 13 for 48 h to obtain activated dendritic cells with the "mature" phenotype. A portion of elutriated monocytes were cryopreserved in the vapor phase of liquid nitrogen suspended in 90% FCS (Summit) and 10% dimethyl sulfoxide (DMSO; Fisher Chemicals, Fair Lawn, NJ).

Tissue
Sections of human tissue were obtained from the Department of Pathology at UCI Medical Center, the Cooperative Human Tissue Network (Case Western Reserve University), and the Tissue Repository at the UCI Institute of Brain Aging and Dementia.

Generation of antibodies against C1qRP
Rabbits were immunized with soluble C1qRP (sC1qRP) produced recombinantly by CHO cells transfected with a pcDNA3.1 plasmid containing the 1.7-Kb fragment corresponding to the extracellular domain of C1qRP [9 ] (rabbit #1125) or after infection of SF9 insect cells with Baculovirus containing the 1.7-Kb coding region of sC1qRP (rabbit #1157). sC1qRP was purified from cell media by affinity chromatography using a column prepared by coupling monoclonal anti-C1qRP antibody [18 ] to Reactigel 6x (Pierce, Rockford, IL) [9 ] and subsequent sodium dodecyl sulfate-polyacrylimide gel electrophoresis (SDS-PAGE) gel purification prior to immunization following standard procedures. A third polyclonal antibody (rabbit #1150) was generated by immunizing with keyhole limpet hemocyanin (KLH) coupled to a synthetic peptide sequence [9 ] (C11 = NQYSPTPGTDC) from the carboxy terminus of C1qRP. IgG from serum was purified by octanoic acid/ammonium sulfate precipitation [19 ].

To characterize the reactivity and specificity of these antibodies, sC1qRP, U937, or HUVEC cell lysates were either directly run in 7.5% SDS polyacrylamide gels or first immunoprecipitated with the monoclonal antibody R139 [7 ]. Samples were then transferred to nitrocellulose and probed with antisera (1:500–1:1000 dilution) or purified IgG (5 µg/mL) of each of the three antibodies. After incubation with horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (Jackson Immunoresearch, West Grove, PA), antibody reactivity was detected using diaminobenzidine (DAB) as a substrate (Vector Laboratories, Burlingame, CA) or enhanced chemiluminescence (ECL) (Amersham Pharmaceutical Biotechnology, Piscataway, NJ). When one monoclonal was used to immunoprecipitate the receptor and another was used to probe it, the probing monoclonal was biotinylated and detected with Streptavidin-HRP (Jackson) and DAB as indicated above.

Immunocytochemistry
After 1–2 days of culture at a density of 1–2 x 105 cells/mL, cells were fixed in 4% formaldehyde in PBS; incubated with blocking solution (2% bovine serum albumin [BSA]/1% normal donkey serum in PBS); and labeled with either 1125, 1150, 1157, or control rabbit IgG (from preimmunization bleeds) at 5–20 µg/mL for 1 h at room temperature, followed by CY3-conjugated donkey antirabbit IgG (Jackson) (1:500). For live cell staining, antibodies were added to cells in culture media for 30 min at 37°C, washed three times, and fixed with 4% formaldehyde before the secondary antibody was added. Cells were mounted in Vectashield (Vector, Burlingame, CA). For tissue staining, paraffin sections were dewaxed and rehydrated. Tissue was microwaved in antigen unmasking solution (Vector). Endogenous peroxidase was blocked by treatment with 1% H2O2/3% methanol/Tris buffer saline. Sections were permeabilized with Tris A (0.1%Triton X-100 in Tris buffer saline), blocked with 2% BSA/Tris A containing 1% normal goat serum, and incubated (1 h at room temperature or overnight at 4°C) with 5–20 µg/mL rabbit IgG control; 1125, 1150, or 1157 IgG; or 1:100 dilution of anti-CD31 (PECAM-1, an endothelial cell marker, Dako, Carpinteria, CA) or 1:50 anti CD68 (a macrophage cell marker, Dako). Label was detected by a biotinylated goat antirabbit or antimouse antibody ABC peroxidase kit (Vector), using DAB as a substrate. Sections were counterstained with hematoxylin (Vector). Free floating sections (brain) were stained as previously described [20 ].

FACS analysis of cell surface markers
Cells were incubated for 30 min on ice in Hanks’ balanced saline solution (containing 1 mM CaCl2, 1 mM Mg Cl2); 0.2% BSA; 0.2% sodium azide with FITC- or PE- conjugated monoclonal antibodies to B7.2 (CD86), B7.1, and CD83 (CD80) (BD-Pharmingen, San Diego, CA); CD14 (Becton Dickinson, San Jose, CA) or with unconjugated monoclonal antibody to C1qRP (R139); or with cell supernatant to anti-ICAM (CD54) (Developmental Studies Hybridoma Bank, Iowa City, IA), followed by FITC-conjugated F(ab')2 goat-antimouse IgG (Jackson ImmunoResearch Laboratories). In all cases, matched isotype control antibodies (Sigma) were used to determine background fluorescence. After washing, relative cell fluorescence was analyzed on FACScan (Becton Dickinson) as described [17 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Polyclonal antibodies 1125 , 1150, and 1157 specifically recognize C1qRP
Antibodies generated against the extracellular domain of C1qRP (1125, 1157) were reactive with sC1qRP in enzyme-linked immunosorbent assay (ELISA) and with the cell surface of myeloid cells by flow cytometry, but they showed no reactivity against peripheral blood lymphocytes or epithelial cell lines (Hela, HEK293) (data not shown). Immunization with a peptide sequence corresponding to the carboxy terminal domain of the C1qRP [9 ] generated a polyclonal antibody (1150, anti C11) that recognized specifically the synthetic peptide, as assayed by ELISA (data not shown). Specificity of each antibody immunoreactivity was tested by Western blot against both the recombinantly expressed receptor and U937 cell extracts (Fig. 1 ). As shown in Figure 1A , lane 2, 1125 antibody was reactive with a 97,000 Mr band in a purified sC1qRP preparation run under reducing conditions. The smear of bands at 68,000 Mr and lower appear to be incompletely processed but secreted molecules. In U937 cell lysates run under nonreducing (Fig. 1B , lanes 2–4) or reducing (not shown) conditions, C1qRP was recognized by all three antibodies. In contrast, the 1150 antibody, anti C11, displayed no reactivity to the soluble C1qRP (lacking the carboxy terminal sequence) (data not shown) as anticipated. Preimmune IgG (Fig. 1A and 1B , lane 1) was not reactive with sC1qRP or U937 lysates.



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  		Figure 1. Antibodies against sC1qRP and carboxy terminal peptide sequence of C1qRP specifically recognize C1qRP. (A) Western blot of purified recombinant sC1qRP under reducing conditions probed with purified IgG from preimmunization bleed (PB) (lane 1) or from polyclonal antisera #1125 (lane 2). (B) Western blot of U937 cell extracts under nonreducing conditions probed with purified IgG from preimmunization bleed (PB) (lane 1) or from polyclonal antipeptide antibody 1150 (lane 2) and polyclonal antibodies against sC1qRP 1125 (lane 3) and 1157 (lane 4). (C) Western blot of U937 and HUVEC (human umbilical vascular endothelial cells) immunoprecipitated with monoclonal R139 or IgG2b control and probed with biotinylated monoclonal antibody R3.

 
To establish the utility of the antibodies for use in immunohistochemistry and across species, reactivity was assessed on both live and fixed cells. All three anti-C1qRP antibodies reacted with CHO cells transfected with the full-length receptor when permeabilized by fixation prior to staining (Fig. 2E F and data not shown), whereas only 1125 (Fig. 2D) and 1157 (not shown) polyclonal antibodies stained cells under live (nonpermeabilized) conditions. The lack of reactivity of the antipeptide antibody (1150), directed against the carboxy terminus, in nonpermeabilized cells (Fig. 2C) confirmed the predicted intracellular location of the C-terminal portion of this transmembrane protein, as shown previously by FACS for the mouse and rat C1qRP [10 , 15 ]. No reactivity of any of the antibodies under any condition was seen with cells transfected with vector alone.



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  		Figure 2. Anti-C1qRP antibodies recognize the receptor expressed in transfected Chinese hamster ovary (CHO) cells and demonstrate the intracellular location of the carboxy terminus. CHO cells transfected with the coding region only for the full-length C1qRP were immunostained before (A–D) or after (E–F) formaldehyde fixation using antibody 1125 (D, F) or 1150 (C, E). A, B are phase fields of C, D. Label was detected by CY3-donkey antirabbit antibody. Anti-sC1qRP antibodies (1125) react with fixed (F) and live (D) cells. Antipeptide antibody against carboxy terminal sequence of the receptor (1150) does not stain live nonpermeabilized cells (C) confirming the intracellular location of the C-terminus of the receptor. Scale bar: 25 µm.

 
Further analysis demonstrated that these antibodies stained human monocytes and cultured endothelial cells, as expected from previous FACS analysis with monoclonal anti-C1qRP. It is interesting that immunocytochemistry using the polyclonal antibodies showed that in monocytes, the pattern of staining was membrane-associated and punctuated with clusters (Fig. 3F ), whereas in HUVEC (Fig. 3B and 3C) and HUCE cells (Fig. 3D) , the label was both membrane-associated and present as an apparent intracellular receptor pool. IgG from the preimmunization bleed gave very low background in monocytes (Fig. 3E) , HUVEC (Fig. 3A) , or HUCE (data not shown) cells. Although these data indicate a diverse cellular staining pattern, Western blot analysis of endothelial cell lysates demonstrated no detectable size difference between endothelial and myeloid cell C1qRP (Fig. 1C) . The polyclonal antibodies, in addition, which are strongly reactive with rat microglia (Fig. 4B ) and murine peritoneal macrophages (Fig. 4D) , showing robust staining across species.



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  		Figure 3. Antibodies against C1qRP are reactive with human myeloid and endothelial cells. HUVEC (A–C), HUCE (human capillary endothelial cells) (D), or human monocytes (E, F) were stained either before (C) or after (A, B, D, E, F) fixation with purified IgG from rabbit 1125 (B, C, F) or 1150 (D), or control rabbit IgG (preimmunization bleed) (A, E) followed by fluorescently labeled CY3 donkey antirabbit IgG. Scale bar: 25 µm.

 


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  		Figure 4. Anti-sC1qRP is reactive with rat and murine myeloid cells. Rat microglia (A, B) or mouse peritoneal macrophages (C, D) fixed with formaldehyde were stained with 1125 IgG (B, D) or prebleed IgG (A, C) followed by fluorescently labeled CY3 donkey antirabbit IgG. Scale bar: 25 µm.

 
C1qRP is predominantly present in endothelial cells in human tissue
The polyclonal antibodies were used to map the distribution of the C1qRP in a wide variety of human tissue, including kidney, liver, spleen, aorta, heart, brain, lung, lymph nodes, small bowel, appendix, placenta, and skin. C1qRP was predominantly present in vascular endothelial cells in all tissues. In the kidney, the antibodies labeled interstitial and glomerular capillary endothelium (Fig. 5B ). Although the level of staining was low, there was a clear difference over the background staining obtained with the control IgG (Fig. 5A) . In the placenta, the staining was particularly strong in the endothelial cells of capillaries of the villi (Fig. 5C) . Capillaries of the sinuses in the red pulp in spleen, as well as endothelium of trabecular vessels, were C1qRP positive (Fig. 5D) . In the heart, C1qRP was present in the endothelium of blood vessels of connective tissue and in the delicate capillaries associated with individual cardiomyocytes (Fig. 5E) . In coronary artery atherosclerotic plaque, the receptor was present in the thin layer of endothelium in the tunica intima (Fig. 5F) . The endothelium of the aorta and aortic vasa vasorum were also stained by all antibodies (not shown). Colocalization of C1qRP with CD31, a platelet/endothelial cell adhesion molecule, was evident when adjacent sections in all tissues were stained. For example, in liver, CD31 positive endothelial cells of portal tracts (portal veins and hepatic artery) were stained by anti-C1qRP (Fig. 6A B ), but no label is seen in capillaries from sinusoids that also lack CD31. In lymph nodes, staining is associated with all the CD31-staining endothelium, including endothelial venules (data not shown).



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  		Figure 5. Localization of the C1qRP in human tissue. Kidney (A, B), placenta (C), spleen (D), heart (E), coronary artery (F), and brain frontal cortex (G) were incubated with 1157 (E), 1150 (D) or 1125 (B, C, F, G) IgG or control prebleed IgG (A). Antibody binding was detected by peroxidase-conjugated antirabbit IgG using DAB as the chromogen. Slides were counterstained with hematoxylin. C1qRP is localized to vascular endothelial cells. Scale bar: 25 µm.

 


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  		Figure 6. C1qRP colocalizes with an endothelial cell marker. Serial sections of liver were stained with anti CD31 (A), 1125 IgG (B), or anti-CD68 (C), as described in Figure 5 . C1qRP labeling pattern is coincident with the endothelial marker CD31 but not with the macrophage marker CD68. Scale bar: 25 µm.

 
As anticipated from the previously documented expression of C1qRP by blood neutrophils, infiltrating neutrophils were detected in some tissues by staining with anti-C1qRP antibodies (not shown). In contrast, C1qRP was not detectably colocalized with CD68, a marker for macrophages, in the liver (Fig. 6B 6C) , the lung, or most other tissues. In skin, however, the single detected exception, some CD68-positive cells were stained positively for C1qRP, as well as endothelial cells and a subset of keratinocytes (striatum spinosum layer) (data not shown). It is interesting that in normal brain, anti-C1qRP antibodies labeled endothelial cells in capillaries but also a subset of pyramidal neurons (Fig. 5G) . Future studies are required to further assess potential differences in proteins associated with C1qRP in these diverse cell types and to determine the functional role(s) of the receptor in these cells.

C1qRP expression is down-regulated when monocytes differentiate into dendritic cells
Given the paucity of myeloid cell staining in tissues, experiments were performed to determine whether the expression of C1qRP was modulated as primarily phagocytic monocytes differentiated to mature antigen-presenting dendritic cells. Human monocytes isolated from peripheral blood were differentiated to dendritic cells by culturing in the presence of GM-CSF and IL-4. After 12–13 days in culture, cells were activated with TNF-{alpha}. Differentiation and activation were followed by surface markers. As expected, freshly purified monocytes expressing a high level of CD14 also expressed high levels of C1qRP. Up-regulation of B7.2 (CD86) (Fig. 7 ) and B7.1 (CD80) (data not shown), and the disappearance of CD14 expression as monocytes differentiate into dendritic cells by culturing in GM-CSF and IL-4, was concomitant with down-regulation of C1qRP expression. Induction of a mature or activated dendritic cell phenotype by TNF-{alpha}, characterized by CD83 expression (Fig. 7) and up-regulation of ICAM-1 (CD54, not shown), was accompanied by a further down-regulation of C1qRP (Fig. 7) . Similar results were seen with polyclonal anti-C1qRP antibodies (data not shown). These data are consistent with the lack of myeloid C1qRP staining in dendritic cell compartments in tissue.



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  		Figure 7. C1qRP protein expression is down-regulated during monocyte differentiation into dendritic cells. Flow cytometry of elutriated peripheral blood monocytes, autologous derived dendritic cells and TNF-{alpha} activated dendritic cells. Cells were labeled with antibodies (open histograms) for CD14 (monocyte marker), CD86 (marker of dendritic differentiation), CD83 (TNF-{alpha} inducible marker of mature dendritic cells), and C1qRP (R139) or with isotype control of each antibody (solid histograms).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study describes the cell-specific expression of C1qRP in human tissue. Three polyclonal antibodies reactive with C1qRP were generated, one using the peptide sequence of the 11 carboxy terminal amino acids (C11) of the protein as the immunogen and two using purified soluble extracellular (N-terminal) portion of C1qRP recombinantly expressed by CHO cells or in a Baculovirus system. These antibodies, with demonstrated specificity for C1qRP via ELISA and Western blot analysis, were deemed useful for detecting the receptor in tissues, in contrast to the available monoclonal antibodies, known to require intact disulfide bonds [18 ], which were not reactive. In the human tissues examined here (liver, spleen, kidney, brain, and others), the receptor was selectively detected in all (CD31-positive) vascular endothelial cells, infiltrating neutrophils, in a subset of keratinocytes in skin, and in a subset of pyramidal neurons in the brain. Although Dean et al. published in situ hybridization analysis of rat heart and lung sections [13 ], this is the first extensive examination of C1qRP expression in multiple tissues and demonstrates that the apparent constitutive synthesis of this molecule in all tissues [13 ] is due to its presence on endothelial cells in all tissues rather than ubiquitous expression on all cells.

The lack of detectable receptor expression in tissue macrophages expressing the macrophage specific CD68 (macrosialin) in histologic sections of the liver, kidney, and spleen was unexpected because of the prevalence of the receptor on peripheral blood monocytes and with the exception of HL-60 cells, all myeloid cell lines tested [7 , 16 ]. However, the lack of staining was confirmed in tissue from different sources and with all three antibodies. This observation suggested that the receptor levels might be down-regulated when monocytes migrate from the circulation and differentiate into macrophages. In fact, we observed a down-regulation of the receptor when monocytes in culture were differentiated into dendritic cells and further activated with TNF-{alpha}. This result supports the hypothesis that down-regulation occurs in vivo upon certain differentiation and activation pathways. The down-regulation of this receptor in differentiated professional phagocytic cells would be in agreement with a less active phagocytic role of these cells as they become antigen-presenting cells. However, in vitro, rat neonatal microglia, even those with reactive characteristics, display C1qRP antigen and phagocytosis enhancing function [15 ], and murine peritoneal macrophages also express the receptor (Fig. 4D) . Lovik et al. [14 ] also detected differences in C1qRP expression levels, depending on the activation state of macrophage populations in rat. Similarly, whereas the expression was detected here on some tissue neutrophils consistent with previous descriptions of expression on peripheral neutrophils in human [16 , 18 ] and rat [14 ], Lovik and colleagues showed little expression on rat peritoneal granulocytes. These differences are either a function of the specificity of the monoclonal antibodies used to detect rat C1qRP or, more likely, suggest modulation of expression of this surface glycoprotein in neutrophils as well as monocyte-derived cells. Thus, future investigations of the diverse "activation" states of macrophages and neutrophils, including studies in inflamed tissue, should determine how this receptor is modulated in resident and activated myeloid lineage cells.

The apparent reactivity of anti C1qRP with neurons and keratinocytes was unexpected. Although we considered the possibility of a novel cross-reacting epitope, this explanation is unlikely as it would require cross-reactivity with both antibody to the extracellular domain (#1125 and 1157) and antibody to the intracellular domain (#1150) of C1qRP. Since C1q has been synthesized in the brain in response to multiple kinds of injuries [21 22 23 24 25 26 ], the presence of C1qRP on pyramidal neurons could indicate endocytic activity of those cells. Alternatively, these cells may represent a stem cell population recently shown to exist in the brain [27 ]. Future studies are needed to determine if the presence of this receptor in a selective neuronal population is of significance in neurodegenerative diseases such as Alzheimer’s disease.

The predominant presence of C1qRP in endothelial cells is interesting in light of the ability of these cells to respond to immune complexes, to phagocytose certain pathogens, and to clear apoptotic cells [28 ]. Whether the interaction of ligand with this receptor promotes a proinflammatory response or modulates the inflammatory response remains to be seen. For example, Cronstein and colleagues reported that C1q bound to immune complexes stimulated the expression of endothelial adhesion molecules [29 ], usually considered an indication of proinflammatory response, although no evidence for the involvement of C1qRP was detected. Subsequently, independent studies by van den Berg et al. demonstrated that partially aggregated C1q triggered proinflammatory cytokine production [30 ]. More recently, Jarvis et al., using C1q-bearing immune complexes, documented a sustained release of IL-8 by cultured endothelial cells that could be mimicked by the IgM monoclonal anti-C1qRP antibody, R3 [31 ]. It turns out that antibodies reactive with the collagen-like region of C1q (C1q-CLR), the domain that contains the receptor interaction site [32 ], have been found in sera from 100% of HUVS patients [33 , 34 ], in sera of many patients with rheumatoid vasculitis [35 ], and in sera of many patients with systemic lupus erythematosus (SLE) [36 37 38 ]. Whether these antibodies to the ligand C1q may block its binding to C1qRP or promote binding of complexes to the endothelium through interactions with both FcR and C1qRP is unknown. Elucidating these interactions and correlating them with either detrimental or the inflammatory clinical presentation of these individuals will clarify the role of this receptor on endothelial cells and may suggest possible clinically advantageous therapeutic interventions.

In addition to the phagocytic function, other distinct roles have been attributed to C1qRP. Petrenko et al. [11 ] reported the presence of the murine homolog of C1qRP early in development in hematopoietic progenitor cells, and Bonnet and colleagues have observed C1qRP on CD34- and CD34+ primitive stem cell populations that are SRC (SCID-repopulating cells) [16a ], raising the possibility of a role for this receptor in hematopoietic development and angiogenesis. However, preliminary reports of the survival of a C1qRP knock-out mouse indicates that either there are functionally redundant molecules or the lack of these functions does not lead to embryonic lethality [39 ].

The predominant location of the human C1qRP on endothelial cells in tissue reported in our work, and in rat heart and lung tissue [13 ], as well as the similarity of motifs shared with other molecules thought to be involved in cell–cell or cell–substrate interactions [9 , 11 , 13 ] suggests that, apart from its modulation of phagocytic activity, this receptor may have a role in cell adhesion. In addition, its the presence on nonphagocytic cells, such as a distinct neuronal population in brain seen here and in rat NK cells [14 ], raises the possibility that this receptor might also have other novel roles. The antibodies described here react with C1qRP expressed in human tissue and on rat and mouse myeloid cells. The use of these reagents in both human culture systems and rodent models should facilitate the identification of the role of this surface receptor in a variety of developmental and pathological pathways.


   ACKNOWLEDGEMENTS
 
These studies were supported by a grant from NIH AI41090 (AJT). Support for obtaining human blood products used in this study was provided in part by Public Health Service research grant M01 RR00827 from the National Center for Research Resources. Tissue resources provided by the Neuropathology Core of the ADRC at the University of California Irvine were supported by 1 P50 AG16573 from NIH. Authors thank Drs C. Hughes (UCI) and E. Peterson (UCI) for reagents and helpful discussions.

Note added in press: During the submission of this manuscript, two studies relevant to our work were published. They report data on the expression (Dean et al., 2001, Eur. J. Immunol, 31,1370–1361) and function of C1qRP (Lovik et al, 2001, Scand. J. Immunol.,53, 410–415).


   FOOTNOTES
 
Correspondence: Dr. A. J. Tenner, Dept. Molecular Biology & Biochemistry, 3205 Biological Sciences II, University of California, Irvine, Irvine, CA 92697-3900. E-mail: atenner{at}uci.edu

Received March 1, 2001; revised July 8, 2001; accepted July 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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