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Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using the monoclonal anti-C1qRP to affinity purify this surface protein, we obtained 10 peptide sequences, designed oligonucleotide primers and probes based on those sequences, and cloned and sequenced a cDNA that encodes a novel type 1 membrane protein (GenBank Accession U94333). However, the amino acid sequence deduced from this cDNA indicates that the mature protein is composed of 631 amino acids, which is calculated to be 66,495 Da 14 , while previous characterization of C1qRP demonstrated that it migrates in SDS-PAGE gels with a relative mobility of 100,000, which shifts upon reduction to 126,000. While it was known that the molecule recognized by R139 and R3 contained some sialic acid 15 , the basis for the large discrepancy between the predicted molecular mass and the migration of the protein in SDS-PAGE had not been discerned.
The ability to up-regulate this powerful effector mechanism of the innate immune response at early stages of infection before the development of adaptive responses would be a potentially useful prophylactic and/or therapeutic approach. Thus, it is critical to characterize the molecular interaction sites both on the ligand and the receptor. To initiate these studies, we generated new data to validate that the molecule recognized by the mAb, R139 and R3, which we designate C1qRP, plays a critical role in inducing this enhancement of phagocytic function. The data presented document that SPA is also able to trigger enhanced phagocytosis through C1qRP, and that cross-linking of C1qRP via the IgM mAb, R3, was sufficient to induce this cellular response. Furthermore, the structural features contributing to the migration of the mature C1qRP in SDS-PAGE were characterized by comparing the electrophoretic mobility of the purified C1qRP with the recombinantly expressed C1qRP, with glycosidase-treated C1qRP, and with C1qRP synthesized in an in vitro system lacking the ability to posttranslationally modify the protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RPMI 1640 medium, F12 Nutrient Mixture (HAM), SuperScript Preamplification System for First Strand cDNA Synthesis Kit, Lipofectin Reagent, G418, and Taq DNA polymerase were purchased from Life Technologies (Grand Island, NY). C1q was isolated from plasma-derived human serum by the method of Tenner et al. 16 and modified as described 17 . The preparations used were active, as determined by hemolytic titration, and homogeneous, as assessed by SDS-PAGE. Protein concentration was determined using an extinction coefficient (E1%) at 280 nm of 6.82 for C1q 18 . Human SPA isolated from alveolar proteinosis patients 5 was a generous gift from Dr. Jo Rae Wright, Duke University (Durham, NC). Except where noted otherwise, all other reagents were purchased in the highest quality from Sigma (St. Louis, MO).
Anti-C1qRP mAbs R139 (IgG2b) and R3 (IgM), generated using C1q-binding proteins as the immunogen 13 , were purified before use, as previously described 14 . The rabbit polyclonal anti-C1qRP Ab, QR1, was generated using R3-purified C1qRP, as described 14 .
COS-1 cells and the human histiocytic cell line, U937, were grown in RPMI 1640 medium containing 10% supplemented bovine calf serum (HyClone, Logan, UT) and 10 mM HEPES, pH 7.4. CHO-K1 cells were grown in F12 Nutrient Mixture containing 10% FCS (HyClone). Human peripheral blood monocytes were isolated from blood units collected into CPDA1 blood collection bags (Baxter, Deerfield, IL) at the UCI Medical Plaza (Irvine, CA). Monocytes were isolated by counterflow elutriation using a modification of the technique of Lionetti et al. 19 , as described 20 . Greater than 95% of the cells in each preparation were monocytes according to size analysis on a Coulter Channelyzer (Hialeah, FL). Previous analysis has substantiated that such populations are nonspecific esterase positive and >98% viable 20 .
Phagocytosis assay
Opsonized target particles for the phagocytosis assay were sheep erythrocytes (E) bearing either IgG anti-SRBC (EAIgG) or IgM anti-SRBC and C4b (EAIgMC4b) (prepared as previously described 21) to assess FcR- and CR1-mediated phagocytosis, respectively. Eight-well Lab Tek chambers (Nalgene, Naperville, IL) were coated with varying concentrations of C1q, SPA, HSA, iron-saturated transferrin, control IgM, or R3 1 . Monocytes (6.25 x 104 cells/well) were added to each chamber, and the cells were centrifuged at 700 rpm (RT6000; DuPont Sorvall, Newtown, CT) for 3 min and subsequently placed at 37°C in 5% CO2 for 45 min. Targets were then added (107/100 µl) and the slides were again subjected to centrifugation (700 rpm, 3 min) and incubated for 30 min at 37°C. For CR1-mediated phagocytosis, 10 ng/ml phorbol dibutyrate was added with the opsonized targets, as it is well known that while monocytes can bind to targets via CR1, this receptor must be activated in these less differentiated myeloid cells to mediate phagocytosis of complement-opsonized targets 1 . After removing unbound targets by washing, bound, noningested targets were removed by hypotonic lysis 1 . Cells were then fixed in 1% glutaraldehyde and stained with Giemsa. Phagocytosis was quantitated using light microscopy. The number of E targets ingested per 100 effector cells was defined as the phagocytic index (PI), whereas the percentage of effector cells ingesting at least one E target was defined as the percent phagocytosis. Each experiment, performed on separate days with different donors, used duplicate sample wells per condition. Controls of unopsonized E were not ingested by monocytes or macrophages under any conditions. Statistical analysis was performed using the paired Student’s t test.
rC1qRP expression
Reverse-transcriptase PCR was performed using RNA isolated from U937 cells with the SuperScript Preamplification System for First Strand cDNA Synthesis Kit, according to manufacturer’s instructions. A 2034-bp cDNA was amplified that contains the entire C1qRP coding region, using Pfu polymerase (Stratagene, La Jolla, CA), and 5'-GCAGAGGGCCACACAGAGACCG-3' as the forward primer, and the oligonucleotide 5'-GCTCTGAGGATGGTGGCTGGTG-3' as the reverse primer. After purification of the C1qRP cDNA using the Geneclean DNA purification kit (Bio101, La Jolla, CA), according to manufacturer’s instructions, 2.5 U Taq polymerase, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, and 0.2 mM dATP were added in a 100 µl reaction and incubated for 1 h at 37°C to add terminal A’s for cloning into the pGEM-T vector (Promega, Madison, WI). The PCR product was then gel purified and cloned into pGEM-T. Insert-containing plasmids were screened for orientation by restriction digest mapping and were subcloned into pcDNA3.1(+) (Invitrogen, Carlsbad, CA). The plasmid insert was sequenced at both ends to ensure it contained the full-length coding region and for proper orientation for expression.
For transient expression of the receptor, plasmid constructs were transfected into COS-1 cells grown in six-well culture plates (Corning Costar, Encinitas, CA) using 2 µg of DNA per well and the liposome formulation, Lipofectin Reagent, according to manufacturer’s instructions. After 48–72 h, the transfected cells were washed with PBS, then lysed in 1 ml/well of extraction buffer (10 mM triethanolamine, pH 7.4, 1 mM CaCl2, 1 mM MgCl2, 0.15 M NaCl, 1% Nonidet P-40, 2 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF). C1qRP immunoprecipitation with the R139 mAb, SDS-PAGE (7.5%), and Western blotting with biotinylated R3 mAb were performed as described previously 14 , and developed by enhanced chemiluminescence with the HRPL kit (National Diagnostics, Atlanta, GA).
For stable transfections, constructs were transfected into CHO-K1 cells grown in 100-mm culture dishes (Corning Costar) using 5.5 µg of DNA per dish, as described above. After 72 h, the transfected cells were split into medium containing 400 µg/ml G418. Individual colonies were selected and tested for C1qRP surface expression by FACS analysis, as described 13 . For additional analysis of the rC1qRP, 1 x 107 cells were washed with PBS, then lysed, immunoprecipitated, and detected, as described above.
Inhibition of glycosylation
U937 cells were grown in the absence or presence of 1–5 mM
benzyl 2-acetamido-2-deoxy-
-D-galactopyranoside (BAG) or
6, 12, and 18 µg/ml tunicamycin for 24–72 h in a 37°C, 5%
CO2 incubator. The cells were then washed once with PBS,
lysed with extraction buffer above, and immunoprecipitated using the
R139 mAb. After SDS-PAGE under reducing conditions and transfer to
nitrocellulose, the receptor was detected by Western blotting with the
polyclonal QR1 Ab, then developed by enhanced chemiluminescence.
O-Glycosidase digestion
U937 cells (5 x 108) growing in log phase were harvested and lysed with extraction buffer. The lysate was precleared using packed Sepharose CL-40, applied to a R3-affinity column, and eluted with 0.1 M glycine, pH 2.4, 0.5 M NaCl, 0.05% Nonidet P-40, and 1 mM PMSF. The eluate was concentrated by Centricon (Amicon, Beverly, MA) and boiled with 0.5% SDS for 2 min. After cooling to room temperature, cacodylate buffer was added (20 mM NaCacodylate, pH 6, 0.5% Nonidet P-40) and boiled again for 2 min. The sample, cooled to room temperature, was divided into five equal aliquots and incubated at 37°C for 18 h in presence of O-glycosidase (1 mU; Boehringer Mannheim, Indianapolis, IN) alone, neuraminidase (2 mU; Calbiochem, La Jolla, CA) alone, O-glycosidase and neuraminidase (1 mU, 2 mU, respectively), or with no enzymes added. A parallel sample with no enzyme added was left on ice for the duration of digestion to insure there was no protein modification in absence of enzymes during the incubation at 37°C. After the incubation, all of the samples were analyzed by SDS-PAGE under reducing conditions, followed by Western blotting, as described above with the QR1 Ab.
In vitro expression
TNT Coupled Reticulocyte Lysate System (Promega) was used according to manufacturer’s instructions. The 1956-bp C1qRP cDNA subcloned into the pcDNA3.1(+) plasmid and the cDNA encoding the mature C1qRP protein excluding the 21 amino acid signal sequence, which was amplified from the C1qRP-containing pcDNA3.1(+) plasmid as described above using Pfu polymerase and cloned into the pGEM-T vector, were used as DNA templates. One microgram of DNA template was added to the rabbit reticulocyte reaction mixture containing 20 mM amino acid mixture minus cysteine, 10 U RNA polymerase, [35S]cysteine (Amersham, Arlington Heights, IL), and 40 U RNasin ribonuclease inhibitor (Promega), and incubated at 30°C for 60 min. The reaction mixture was then analyzed by SDS-PAGE under reducing conditions, followed by exposure to Fuji RX Medical x-ray film (Stamford, CT) overnight (12–16 h) at -70°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous experiments had shown that the anti-C1qRP
mAb, R139, was able to inhibit both C1q- and MBL-mediated but not
fibronectin-mediated enhancement of phagocytosis 4, 15 . Since SPA
also enhances FcR- and CR1-mediated phagocytosis 3 , the effect of
preincubation of isolated monocytes with anti-C1qRP mAb
on the response triggered by SPA was tested. Fig. 1
presents data from four experiments in
which monocytes pretreated with buffer or mAb were added to wells that
had been coated with HSA, transferrin, SPA, or C1q, and phagocytosis
assessed. Anti-C1qRP inhibited both the C1q and SPA
enhancement of CR1-mediated phagocytosis by human monocytes in these
experiments and others, with an average percent inhibition of PI of
50 ± 17% for C1q (n = 5, p <
0.02) and 55 ± 14% for SPA (n = 5,
p < 0.02) when coated at 8 µg/ml, and 77 ±
10% for C1q and 48 ± 22% for SPA when the C1q/SPA concentration
used for coating was 4 µg/ml (n = 3). Inhibition of
C1q- or SPA-enhanced phagocytosis was not observed in parallel samples
incubated with control mouse IgG2b.
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Pretreatment of human monocytes with R3, a IgM mAb that recognizes
C1qRP, blocked the C1q-mediated enhancement of phagocytosis
13 triggered by the adherence of monocytes to C1q-coated wells,
similar to the inhibition by R139. However, since R3, as a IgM mAb, has
the potential for cross-linking multiple C1q receptors without engaging
Fc receptors, the ability of surface-bound R3 alone to modulate
monocyte phagocytic capacity was tested. Monocytes were added to wells
that had been coated with 5–50 µg/ml of R3 or a control irrelevant
mouse IgM or 8 µg/ml of C1q, and phagocytosis of EAIgMC4b
targets was assessed. Fig. 2 shows
results from a representative experiment in which R3 mediates
enhancement of phagocytosis in a concentration-dependent manner. When
data from four separate experiments were averaged, the PI for cells
adhered to wells coated with 25 and 50 µg/ml R3 was 69 ± 20 and
132 ± 34, respectively, while the average PI for cells adhered to
wells coated with 25 and 50 µg/ml control mouse IgM was 25 ± 21
and 52 ± 19, respectively (n = 4). While the
absolute values varied with the donors tested, the differences between
R3 and IgM are significant (p < 0.003) using
the Student’s paired t test for analysis. The same results
were observed when EAIgG targets were used (data not
shown). Again, no such effect was seen with several distinct irrelevant
mouse IgM run in parallel, demonstrating the specificity of this
response for anti-C1qRP. These data suggest that
immobilized R3 via the ligation of C1qRP mimics the
C1q-/MBL-/SPA-mediated enhancement of phagocytosis.
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The 631 deduced amino acid coding region of the mature
C1qRP predicts a polypeptide with a molecular mass of
66,495 Da, which is substantially smaller than the 126,000 relative
mobility of the reduced native protein as assessed by SDS-PAGE. Thus,
it was necessary to determine whether the expressed product of the
cloned cDNA for C1qRP would migrate similarly to verify
that the entire coding region has been determined and that no errors
were made during the DNA sequencing that would result in a false,
premature stop codon. Flanking primers and Pfu polymerase were used to
amplify the coding region by reverse-transcriptase PCR from U937 total
RNA. The resulting cDNA was cloned into the mammalian expression
vector, pcDNA3.1(+). The plasmid construct was transiently
transfected into COS cells, and the transfected cells were lysed with
detergent. R139 mAb was added for immunoprecipitation, and the
resulting protein complexes were separated by SDS-PAGE under reducing
conditions, and transferred to nitrocellulose. C1qRP was
detected with the anti-C1qRP polyclonal Ab, QR1. As
shown in Fig. 3A, cells that
were transfected with the C1qRP construct in the correct
orientation express a protein that is indistinguishable in size from
native C1qRP immunoprecipitated from control U937 cells.
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) demonstrates that
successful expression and transport to the plasma membrane had
occurred. Cells were then lysed with detergent, and the expressed
protein was immunoprecipitated with the R139 mAb. The protein complexes
were separated by SDS-PAGE under reducing and nonreducing conditions,
and transferred to nitrocellulose. C1qRP was detected with
biotinylated R3 mAb for the nonreduced protein, and with QR1 for the
reduced protein. As shown in Fig. 3
, B and C,
only cells that were transfected with the C1qRP construct
express a protein that is indistinguishable in size from native
C1qRP immunoprecipitated from control U937 cells.
Untransfected cells, as well as cells transfected with the pcDNA vector
only, do not express anti-C1qRP Ab-reactive proteins
(Fig. 3, B and C, lanes 3,
4 and 7, 8). The fact that the
recombinant receptor is recognized by both the R139 and R3 mAbs (Fig. 3B) whose epitopes are conformation dependent demonstrates
that at least some proper folding of the expressed receptor occurs and
the correct disulfide bonds are formed. Additionally, the recombinantly
expressed protein migrates in SDS-PAGE gels with the same
characteristic shift as the native receptor, from the 100,000
Mr nonreduced form (Fig. 3B) to the
126,000 Mr reduced form (Fig. 3C).
These results indicate that the cloned cDNA encodes the polypeptide
sequence sufficient for complete expression and posttranslational
modifications of the mature C1qRP protein.
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To begin to investigate the possible posttranslational
modifications that may contribute to the altered mobility of the
C1qRP polypeptide in SDS-PAGE, U937 cells were cultured in
the presence of the protein glycosylation inhibitors tunicamycin (for
N-linked glycosylation) or BAG (for O-linked
glycosylation). C1qRP immunoprecipitated from cells grown
for 24 h in up to 18 µg/ml of tunicamycin did not show a
detectable shift in mobility in SDS-PAGE gels compared with receptor
from untreated cells (data not shown). However, since the predicted
amino acid sequence of the receptor indicates only one Asn-X-Ser/Thr
N-linked glycosylation site 14 , it is possible that
preventing glycosylation at this single site would not result in a
significantly different migration pattern of the protein. Use of the
O-Glyci.bas program, developed by Elhammer et al. for predicting
O-glycosylation sites based on amino acid sequences
surrounding potential serine and threonine acceptor sites 22 ,
suggested a relatively high number of sites favorable for
O-linked glycosylation in C1qRP (Fig. 5). As predicted, treatment of U937 cells
with BAG did result in a marked change in the mobility of
C1qRP. As shown in Fig. 6,
C1qRP immunoprecipitated from cells grown in 3 mM BAG had a
significant difference in mobility, 113,000 ± 1,400
Mr (n = 5), relative to the
native C1qRP (126–128,000 Mr).
Additional experiments with cultures grown in 5 mM BAG for up to
72 h (not shown) showed no further increase in mobility.
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). Importantly, while these
experiments indicate that glycosylation does not account for the entire
discrepancy between the predicted and observed size of the receptor,
they do indicate significant glycosylation and exclude the possibility
that the 126,000 Mr protein is a dimer of
the 66,495-kDa polypeptide.
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C1qRP cDNA was transcribed and translated in an in
vitro system, employing rabbit reticulocyte components that lack the
capacity for posttranslational modification. The major polypeptide
produced in this in vitro translation reaction migrates in SDS-PAGE as
85,500 ± 3,900 Mr (n = 6).
A slightly larger product is detected, as expected, when the coding
region, including the signal peptide, is used as a template (Fig. 8, lane 2 versus lane
1). It is expected that the signal sequence would be cleaved in
the mature protein, and thus it is the smaller
85,500-Mr band that represents the peptide
without other posttranslational modifications. Both the major
85,500-Mr product and the 65,000 minor
product seen in Fig. 8, lane 1, are seen when the in vitro
translation reaction is immunoprecipitated with polyclonal
antiC1qRP, suggesting that the lower band represents a
truncated translation product. Since the molecular mass deduced
from the amino acid sequence of the mature protein is 66,495 Da, these
data demonstrate that the composition of the amino acid sequence of the
protein itself dictates altered mobility of SDS-PAGE 23 . In addition,
although significantly reduced from Mr of
126,000 mature protein, there remains a discrepancy between the
neuraminidase/O-glycosidase-treated protein (107,900
Mr) and the 85,500 Mr of
the unmodified nascent polypeptide chain, suggesting that some
posttranslational modification other than O-glycosylation on
serine and threonine may contribute to the mature protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The degree of inhibition by the anti-C1qRP mAb of
ligand-induced enhancement of phagocytic function rarely is greater
than 80% (Fig. 1), similar to that seen in previous studies with C1q
and MBL 4, 13, 15 . This would be consistent with a requirement for
continuous inhibition of receptor-ligand interactions, rather than an
all or none signaling mechanism. In addition, it is possible that the
avidity of soluble Ab for C1qRP is lower than that of
immobilized ligand for the C1qRP or receptor complex.
Previous studies reported that anti-C1qRP R139 does not
inhibit C1q binding to cells, and R3, a IgM anti-C1qRP,
only partially inhibits the C1q binding to monocytes, to the
monocytic-like cell line U937, and to neutrophils 13 . Thus, the
mechanisms by which the Abs inhibit the ligand-induced response are not
known at this time. One possibility is that the Abs block a
conformational alteration critical for signaling. Studies using the
recombinantly expressed C1qRP will define whether the
component of the phagocytosis-modulating receptor that is recognized by
the mAbs R139 and R3 actually binds the ligand or if it is involved in
the signaling function of the receptor or both.
Previous studies have shown that multivalent presentation of C1q or a conformational change induced by aggregation or immobilization of the C1q monomer seems to be required for the generation of a functional response (reviewed in Refs. 5, 26, and 27). The mAb R3 is a IgM pentamer with the ability to cross-link multiple receptors, and thus to potentially mimic the response triggered by multivalent C1q. The data presented in this work clearly show that cross-linking of C1qRP by immobilized R3 increases the monocyte phagocytic capacity in a dose-dependent manner, identifying C1qRP, the polypeptide recognized by the mAb, as a critical component of this important cellular response, and confirming the requirement for a multivalent presentation to activate C1q receptors at least in monocytes.
Characterization of the structural features of this novel cell surface
molecule is critical to understand the ligand-receptor interactions and
subsequent signal transduction mechanisms involved in this host
response. While the mAb that inhibited the enhancement of phagocytosis
consistently immunoprecipitated a molecule of 126,000
Mr (C1qRP) from monocytes,
neutrophils, U937 cells 15 , and platelets 28 , the primary amino
acid sequence deduced from the cloned cDNA predicted a protein with a
molecular mass of 66,495 Da. Translation in an in vitro system (Fig. 8)
that lacks translational modification demonstrated that the amino acid
sequence itself results in an aberrant migration in SDS-PAGE, similar
to that reported for other molecules with a high percentage of alanine,
proline, and charged amino acids in their primary sequence 23, 29 .
The results of the experiments investigating glycosylation of this
surface molecule are consistent with multiple O-linked
glycosylation sites. Indeed, the extracellular portion of the molecule
proximal to the transmembrane domain has an abnormally high percentage
of serine, threonine, and proline residues, providing high probability
for O-glycosylation 14 (Fig. 5). While some of the
O-linked oligosaccharides contain N-acetyl
galactosamine, as indicated by the inhibitory effect of BAG, which acts
as an acceptor for UDP-gal:galNAc-ß1,3 galactosyltransferase 30 ,
the smaller apparent size of
neuraminidase/O-glycosidase-treated C1qRP
(107,000 Mr) suggests that either there are
other O-linked sugars that are not inhibited by BAG,
or that the inhibitor is not completely effective. In addition, as
mentioned above, there remains a discrepancy between the in vitro
translated product of 85,500 Mr and the
glycosidase-treated protein (107,000 Mr),
suggesting additional posttranslational modifications, such as other
forms of O-glycosylation, phosphorylation, and/or sulfation.
While future experiments will be necessary to further define the
functional significance of this glycosylation, earlier experiments
suggested that at least sialic acid was not critical for the
enhancement of phagocytosis 15 . It has been suggested that
O-linked glycosylation produces an elongated polypeptide
structure 31 . In some surface proteins, a short glycosylated domain
separates the functional portion of the protein from the transmembrane
domain, possibly serving to extend the functional domain of the
molecule well beyond the cell surface for interaction with
extracellular matrix proteins or other cell or particle surfaces. This
may be the function of this serine/threonine-rich region in
C1qRP.
Another cellular response triggered by C1q is the production of superoxide by neutrophils and eosinophils. However, several lines of evidence suggest that this response may be mediated by a receptor, C1qRO2-, distinct from C1qRP. First, none of the anti-C1qRP mAbs inhibit 13 or mimic (unpublished data) C1q-mediated oxidative burst in neutrophils. In addition, neither immobilized SPA nor MBL triggers superoxide production in neutrophils 32 , suggesting that the interaction motif on C1q required for the receptor that mediates superoxide generation differs from that required for enhancing phagocytosis. Finally, the region of the C1q molecule required to stimulate superoxide production (just above the kink in the collagen-like region) has little homology (beyond the GXY collagen motif) with any region of SPA, and only minimal homology with MBL 33 . These data indicate that the neutrophil C1qR/receptor complex differs in some way from C1qRP. Similarly, a recent report has provided evidence supporting earlier suggestions that there is an additional SPA receptor distinct from C1qRP that modulates phospholipid secretion 34 . This apparent multiplicity of receptors for these multidomain proteins may permit the primitive orchestration of more than one type of function in response to a variety of stimuli. It should be noted that Tino and Wright 8 demonstrated that SPA stimulation of phagocytosis of specific pathogens is inhibited in monocytes adhered to surface-bound C1q, but not in alveolar macrophages similarly treated. Thus, the differentiation state of the macrophage may dictate the surface distribution of C1qRP and/or may induce additional functional receptors for these ligands.
The stimulation of phagocytic function may be particularly beneficial as a prophylactic treatment for individuals at risk for infection, such as individuals with genetic immunodeficiencies or infected with HIV, patients undergoing cancer chemotherapy, or patients undergoing high risk surgery. Other agents that influence the activation state of phagocytes often trigger the production of proinflammatory cytokines that can directly cause unwanted side effects, including, but not limited to, the up-regulation of HIV production. In contrast, recent studies by Jasinskiene and coworkers6 demonstrate that C1q and SPA do not trigger the release of proinflammatory cytokines by human monocytes. The ability to stimulate clearance of cellular debris, pathogens, or immune complexes without triggering the production of cytokines 35 6 that in some instances could be detrimental for the host, makes C1qRP an ideal candidate for therapeutic manipulation. Knowledge of the specific ligand-receptor interactions involved in mediating particular functions should facilitate selective modulation of desired responses (enhanced phagocytic capacity via C1qRP) without the induction of proinflammatory cytokines or generation of toxic oxygen radicals.
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Acknowledgments |
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Footnotes |
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2 Current address: Stanford University, MSLS, 3rd Floor, MC:5492, Stanford, CA 94305.
3 Current address: C. Nal. Farmacobiologia, Instituto de Salud Carlos III, Madrid, Spain.
4 Address correspondence and reprint requests to Dr. Andrea J. Tenner, 3205 Biological Sciences II, Dept. Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900.
5 Abbreviations used in this paper: MBL, mannose-binding lectin; BAG, benzyl 2-acetamido-2-deoxy--D-galactopyranoside; CHO, Chinese hamster ovary; E, sheep erythrocyte; HSA, human serum albumin; PI, phagocytic index; SPA, pulmonary surfactant protein A.
6 N. Jasinskiene, R. Rochford, S. Ruiz, and A. J. Tenner. Complement component C1q selectively enhances phagocytosis, but not the production of proinflammatory cytokines. Submitted for publication.
Received for publication September 25, 1998. Accepted for publication December 18, 1998.
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A. Haddad, M. R. Nokhbeh, D. A. Alexander, S. J. Dawe, C. Grise, N. Gulzar, and K. Dimock Binding to Decay-Accelerating Factor Is Not Required for Infection of Human Leukocyte Cell Lines by Enterovirus 70 J. Virol., March 15, 2004; 78(6): 2674 - 2681. [Abstract] [Full Text] [PDF]
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P. J. Norsworthy, L. Fossati-Jimack, J. Cortes-Hernandez, P. R. Taylor, A. E. Bygrave, R. D. Thompson, S. Nourshargh, M. J. Walport, and M. Botto Murine CD93 (C1qRp) Contributes to the Removal of Apoptotic Cells In Vivo but Is Not Required for C1q-Mediated Enhancement of Phagocytosis J. Immunol., March 15, 2004; 172(6): 3406 - 3414. [Abstract] [Full Text] [PDF]
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 W. T. Watford, M. B. Smithers, M. M. Frank, and J. R. Wright Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L1011 - L1022. [Abstract] [Full Text] [PDF]
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E. P. McGreal, N. Ikewaki, H. Akatsu, B. P. Morgan, and P. Gasque Human C1qRp Is Identical with CD93 and the mNI-11 Antigen But Does Not Bind C1q J. Immunol., May 15, 2002; 168(10): 5222 - 5232. [Abstract] [Full Text] [PDF]
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X. Feng, M. G. Tonnesen, E. I. B. Peerschke, and B. Ghebrehiwet Cooperation of C1q Receptors and Integrins in C1q-Mediated Endothelial Cell Adhesion and Spreading J. Immunol., March 1, 2002; 168(5): 2441 - 2448. [Abstract] [Full Text] [PDF]
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P. Steinberger, A. Szekeres, S. Wille, J. Stockl, N. Selenko, E. Prager, G. Staffler, O. Madic, H. Stockinger, and W. Knapp Identification of human CD93 as the phagocytic C1q receptor (C1qRp) by expression cloning J. Leukoc. Biol., January 1, 2002; 71(1): 133 - 140. [Abstract] [Full Text] [PDF]
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W. T. Watford, J. R. Wright, C. G. Hester, H. Jiang, and M. M. Frank Surfactant Protein A Regulates Complement Activation J. Immunol., December 1, 2001; 167(11): 6593 - 6600. [Abstract] [Full Text] [PDF]
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 M. Arora, E. Munoz, and A. J. Tenner Identification of a Site on Mannan-binding Lectin Critical for Enhancement of Phagocytosis J. Biol. Chem., November 9, 2001; 276(46): 43087 - 43094. [Abstract] [Full Text] [PDF]
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M. I. Fonseca, P. M. Carpenter, M. Park, G. Palmarini, E. L. Nelson, and A. J. Tenner C1qRP, a myeloid cell receptor in blood, is predominantly expressed on endothelial cells in human tissue J. Leukoc. Biol., November 1, 2001; 70(5): 793 - 800. [Abstract] [Full Text] [PDF]
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R. Opavsky, P. Haviernik, D. Jurkovicova, M. T. Garin, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, J. Bies, S. Garfield, S. Pastorekova, et al. Molecular Characterization of the Mouse Tem1/endosialin Gene Regulated by Cell Density in Vitro and Expressed in Normal Tissues in Vivo J. Biol. Chem., October 12, 2001; 276(42): 38795 - 38807. [Abstract] [Full Text] [PDF]
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S. D. Webster, M. D. Galvan, E. Ferran, W. Garzon-Rodriguez, C. G. Glabe, and A. J. Tenner Antibody-Mediated Phagocytosis of the Amyloid {{beta}}-Peptide in Microglia Is Differentially Modulated by C1q J. Immunol., June 15, 2001; 166(12): 7496 - 7503. [Abstract] [Full Text] [PDF]
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 F. E. Barr, H. Pedigo, T. R. Johnson, and V. L. Shepherd Surfactant Protein-A Enhances Uptake of Respiratory Syncytial Virus by Monocytes and U937 Macrophages Am. J. Respir. Cell Mol. Biol., November 1, 2000; 23(5): 586 - 592. [Abstract] [Full Text]
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 T. Yokota, K. Oritani, I. Takahashi, J. Ishikawa, A. Matsuyama, N. Ouchi, S. Kihara, T. Funahashi, A. J. Tenner, Y. Tomiyama, et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages Blood, September 1, 2000; 96(5): 1723 - 1732. [Abstract] [Full Text] [PDF]
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Y. D. Dean, E. P. McGreal, H. Akatsu, and P. Gasque Molecular and Cellular Properties of the Rat AA4 Antigen, a C-type Lectin-like Receptor with Structural Homology to Thrombomodulin J. Biol. Chem., October 27, 2000; 275(44): 34382 - 34392. [Abstract] [Full Text] [PDF]
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