Ultrasensitive Confocal Fluorescence Microscopy of C-Reactive Protein Interacting With FcRIIa
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《动脉硬化血栓血管生物学》
From the Departments of Internal Medicine II–Cardiology (D.E.M., V.H., J.T.) and Biophysics (C.R., G.U.N.), University of Ulm, Germany; and the Department of Physics (G.U.N.), University of Illinois at Urbana-Champaign, Urbana, Ill.
ABSTRACT
Background— C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcRIIa may have caused false-positive results.
Methods and Results— To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcRIIa. CRP indeed binds to FcRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude.
Conclusions— Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcRIIa and explains and reconciles the diverse and conflicting data presented in the literature.
We have studied CRP binding to FcRIIa using the novel method of ultrasensitive confocal fluorescence microscopy. We unambiguously show that CRP interacts with FcRIIa and characterize this interaction quantitatively. We also provide explanations as to why controversial results were obtained previously.
Key Words: atherosclerosis ? inflammation ? receptors ? ultrasensitive fluorescence microscopy
Introduction
C-Reactive protein (CRP) is the prototype human acute-phase protein.1,2 It also is a powerful cardiovascular risk marker.3 Recent investigations suggested a pathogenic role of CRP in cardiovascular disease,4,5 which has spawned widespread interest in studies of its biological function.6–12 Importantly, it has been demonstrated that human CRP transgene expression causes accelerated aortic atherosclerosis in apolipoprotein E–deficient mice, providing first in vivo evidence of a direct involvement of CRP in atherogenesis.12
To date, ligand binding, opsonization of bioparticles,13–15 and complement activation16 are rigorously defined pathobiological CRP functions. CRP interactions with nucleated cells have gained increasing interest,6–11 and CRP binding to cellular receptors has been intensely investigated with conflicting results. Whereas some reports provided evidence of specific CRP receptors,17 other experiments demonstrated interaction with Fc receptors.18,19 The low-affinity IgG receptor FcRIIa was proposed to be the major CRP receptor.18,19 Several observations supported this concept. When coincubated with low-density lipoprotein (LDL), CRP colocalizes with clusters of FcRIIa on monocyte membranes.8 Furthermore, CRP was reported to induce FcRIIa-signaling in human promyelocytic cell line HL-60,20 and finally, experiments in FcRII- and -chain–deficient mice showed lacking CRP-mediated biological responses compared with wild-type mice.21 To demonstrate CRP binding to FcRIIa, anti-CRP antibodies were used in the initial reports18,19 because direct labeling of CRP with fluorescein isothiocyanate (FITC) or 125-I may damage the structure of the molecule and lead to ambiguous results. It was also suggested that CRP binding to FcRIIa is allele specific.19 High-affinity binding was reported for FcRIIa R/R-131, intermediate affinity binding for FcRIIa R/H-131, and low-affinity binding for FcRIIa H/H-131. Subsequently, several authors proposed that CRP may not interact with FcRIIa at all and that the observed binding of CRP to FcRIIa results from an interaction of the Fc portion of the anti-CRP antibody with FcRIIa itself.2,22,23 Indeed, using F(ab')2 fragments of anti-CRP antibodies, fluorescence-activated cell sorter (FACS) analysis revealed no CRP binding to FcRIIa-R131 on polymorphonuclear leukocytes and FcRIIa-transfected IIA.6 cells.22 Other authors have suggested that the observed binding of CRP to FcRIIa might be attributable to IgG contamination of the CRP reagent,23 and a recent review claims that CRP does not interact at all with cellular Fc receptors.2
Here we applied the novel technology of ultrasensitive confocal fluorescence microscopy to study CRP interactions with FcRIIa.24–26 Our results visually demonstrate and quantitatively show that (1) use of anti-CRP antibodies indeed affects CRP binding and leads to false-positive results; and (2) CRP, however, does bind to FcRIIa, although with lower affinity than anti-CRP antibody/CRP complexes.
Methods
Cell Culture
COS-7 cells were obtained from DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and maintained in DMEM/10% FCS with 1% penicillin/streptomycin/L-glutamine. This cell line does not express Fc receptors.
Reagents and Antibodies
Partially purified CRP was obtained from Sigma. Highly purified CRP was kindly provided by Dr T.W. Du Clos (University of New Mexico, Albuquerque). Recombinant CRP (rCRP) was obtained from Calbiochem. Western blot analysis revealed <0.1% IgG for partially purified and no detectable IgG for highly purified and rCRP. Monoclonal anti-CRP antibody, clone 2C10, was generously provided by Dr Du Clos with kind permission of Dr Larry Potempa (ImmTech, Evanston, Il).27 Anti-CD32–FITC, clone FLI8.26(2003), and unlabeled and phycoerythrin (PE)-labeled monoclonal mouse IgG1 isotype were purchased from BD Biosciences. Anti-CD32, clone KB61, was purchased from DAKO, affinity-isolated F(ab')2 PE-goat anti-mouse (GAM) from Caltag Labs, and human serum from the blood transfusion service of the University of Ulm.
FcRIIa Cloning and Transfection
Human FcRIIa cDNA (G/G genotype, coding for FcRIIa R/R-131) was generated by RT-PCR and cloned into pcDNA3.1 using the Directional TOPO Expression Kit (Invitrogen). The cDNA FcRIIa A/A genotype (coding for FcRIIa H/H-131) was generated from the FcRIIa G/G genotype using site-specific mutagenesis.28,29 Vectors were sequenced. Six-well plates were seeded at 2.5x105 cells/well. Approximately 70% to 80% confluent cells were transfected using Polyfect Reagent (Qiagen). Cells expressing heterozygous FcRIIa R/H-131 were obtained by cotransfection with FcRIIa G/G and A/A. Mock-transfected cells were treated with transfectant reagent only. Anti-CD32–FITC staining revealed similar transfection efficiency for the alleles.
Binding Assays With Nonlabeled CRP and Anti-CRP Antibodies Using FACS
CRP-binding assays were performed with COS-7 cells 48 hours after transfection as described.18 Detached cells were incubated with different preparations of CRP at a concentration of 1.74 μmol/L, which corresponds to 200 μg/mL. This concentration was used by Bharadway et al18 and found to induce optimal signaling by Chi et al20 in ice-cold PBS containing 0.05% azide and 0.1% BSA (PAB) for 1 hour on ice. After washing with PAB, cells were stained with anti-CRP (2C10) for 0.5 hours on ice. Cells were washed and stained with PE-GAM–F(ab')2 for 0.5 hours on ice in dark. Cells were washed and analyzed using FACS with CellQuest software (BD Biosciences). A total of 30 000 cells were gated by fluorescence-1 (green) and fluorescence-2 (red). A total of 95% mock-transfected cells stained in the absence of CRP were assessed as background. Cell viability assays (trypan blue) revealed 99% viable transfected cells. Staining with IgG1-PE isotype antibody was used as a control. Because of identical results for the different CRP preparations, rCRP was used for all subsequent analyses. Results are expressed as mean±SD. Scores were compared using Student paired t test (Microsoft Excel 2000). A P<0.05 was considered statistically significant.
Confocal Imaging and Analysis
Confocal images were collected using a laser scanning confocal fluorescence microscope with single-fluorophore sensitivity.24–26 An Ar+/Kr+ ion laser (Spectra Physics 164) and an HeNe laser (Polytec) were used for fluorescence excitation at 514.5 nm and 632.8 nm, respectively. The excitation light was focused into the sample, and the resulting fluorescence emission was collected by a water immersion objective (C-Apochromat 63x/1.2 W; Zeiss). Highly efficient detection in 2 spectral channels (green 557 to 607 nm; red 665 to 850 nm) was accomplished by splitting the fluorescence light using custom-made bandpass filters in conjunction with dichroic mirrors (AHF) and subsequent detection with single-photon counting detectors (AQR-14; Perkin–Elmer). The whole instrument is controlled by our homemade software.
Confocal fluorescence images consisting of 128x128 pixels were acquired in a field of 90x90 μm2 with a depth resolution of 2 μm for both excitation wavelengths with 0.5-μW laser power incident on an area of 0.3 μm2.
For quantitative analysis, the fluorescence emitted by membranes of selected cells was examined as a function of time after incubation with fluorescently labeled CRP or anti-CD32 antibodies or after equilibration with these proteins at different concentrations. Within 1 series of measurements, the same number (typically 100) of the brightest pixels from the membrane of a chosen cell was analyzed.
Fluorescence Labeling
CRP and monoclonal anti-CRP antibodies were labeled with Cy3-N-hydroxy-Succinimidyl ester (NHS) (Amersham) by coupling the succinimidyl ester derivative of the dye to amine groups in phosphate buffer at pH 8.2. Anti-CD32 (KB61) antibodies were conjugated with Alexa Fluor 647 dye (Molecular Probes) using maleimide coupling to free thiol groups. Unreacted dye was removed by gel filtration. The degree of labeling was kept low (1 to 2 fluorescent labels per protein molecule, as quantified by optical absorption spectroscopy) to minimize dye interactions. Pseudonative SDS-PAGE revealed identical bands for nonlabeled and Cy3-NHS–labeled CRP.
Colocalization Measurements
Solutions containing FcRIIa-transfected COS-7 cells were transferred to a sandwich chamber consisting of 2 glass cover slips separated by mylar spacers (thickness 200 μm). After 15 minutes, cells were exposed to solutions of CRP–Cy3 at 0.87 μM (100 μg/mL, shown to induce optimal signaling20) or anti-CRP antibody/CRP–Cy3 complexes at 6.7 nM (1 μg/mL, for anti-CRP antibody 2C10). After washing, receptor staining was performed using solutions with 0.5 μM Alexa 647-labeled anti-CD32 antibodies, and subsequently, confocal images were taken.
Association Kinetics and Equilibrium Binding
The kinetics of association of Cy3-labeled CRP to FcRIIa-transfected COS-7 cells was studied by acquiring confocal images as a function of time. For the kinetics of the anti-CRP antibody/CRP complex, a constant concentration of 0.87 μM unlabeled CRP was used in combination with different concentrations of Cy3-labeled anti-CRP antibodies.
For studies of equilibrium binding, incubation times were adjusted in accordance with the kinetic data. COS-7 cells were exposed to different concentrations of Cy3-labeled CRP and CRP/anti-CRP–Cy3 complexes. Confocal images were analyzed to assess the degree of saturation of the receptors.
Results
FACS Analysis Using Nonlabeled CRP and Anti-CRP Antibodies
FACS analysis of FcRIIa- and mock-transfected COS-7 cells was performed in the presence and absence of CRP. FcRIIa–131R/R-transfected cells showed 54.2% positivity (PE staining) after incubation with 1.74 μM CRP (Figure 1a). This experiment confirmed the original data,18,19 which led to the interpretation of high-affinity binding of CRP to FcRIIa. Three different CRP preparations (partially purified, highly purified, and rCRP) yielded identical results (data not shown).
Figure 1. a, FACS analysis using nonlabeled CRP and anti-CRP antibodies. Cells were incubated with 1.74 μM CRP (200 μg/mL) and stained with anti-CRP antibody (2C10) and F(ab)2–PE-GAM. High PE staining (54.2%) for FcRIIa–131R/R-transfected cells in the presence of CRP. b, FACS analysis of alleles showing changes in the staining of cells expressing different FcRIIa-alleles. c, Role of anti-CRP antibody/CRP complexes. Identical results for FACS analysis of FcRIIa–131R/R-transfected cells after treatment with preformed anti-CRP antibody/CRP complexes and after sequential addition of CRP and anti-CRP antibodies.
Binding assays with CRP (1.74 μM) were performed in cells transfected with the FcRIIa alleles (131R/R, 131R/H, and 131H/H). A decrease in staining was seen in the order 131R/R131R/H131H/H in the presence (Figure 1b, black) (RR:RH:HH=1.6:1.2:1.0, RR/RH, RH/HH, RR/HH); and absence (Figure 1b, dotted) of CRP, (RR:RH:HH=2.8:2.0:1.0, RR/RH, RR/HH, RH/HH; and also for a mouse IgG1 isotype control (Figure 1b, hatched; (RR:RH:HH=2.5:1.8:1.0, RR/RH, RR/HH, RH/HH). The differences in staining reflect the differences in binding of IgG1 to the "high" (131R/R) and "low responder" (131H/H) forms of FcRIIa.29 Treatment of FcRIIa–131R/R-transfected cells with preformed anti-CRP antibody/CRP complex led to the same positive results (Figure 1c, right) as obtained by addition of CRP to cells and subsequent incubation with anti-CRP antibodies (Figure 1c, left).
Confocal Imaging
After incubation of FcRIIa-transfected COS-7 cells with CRP–Cy3 and subsequent washing with PBS, green fluorescence from the cell membrane was observed by confocal imaging. As an example, Figure 2a shows CRP–Cy3 binding to a cell that strongly expresses FcRIIa. The fluorescence shows a focal pattern. The signal was much stronger when the cells were coincubated with unlabeled anti-CRP antibodies (Figure 2c). During incubation of cells that had bound CRP–Cy3 (Figure 2a) with anti-CD32–alexa 647 (Figure 2b), strict colocalization of binding sites for CRP–Cy3 and anti-CD32–alexa-647 was observed. Incubation of CRP–Cy3/ anti-CRP antibody complex binding cells (Figure 2c) with anti-CD32–alexa 647 (Figure 2b) also showed strict colocalization of binding sites for CRP–Cy3 and anti-CD32–alexa-647. Competitive binding was evident from the ratio of red to green membrane fluorescence compared with the ratio obtained when incubating anti-CD32 antibodies before the immune complexes (data not shown). Figure 2e and 2f displays control cells from a different area of the sample shown in Figure 2a and 2b that do not express FcRIIa and do not bind CRP.
Figure 2. Colocalization of CRP and FcRIIa. Representative examples of confocal scan images of COS-7 cells are shown. Left (a, c, and e), Staining with Cy3-labeled CRP (green detection channel). Right (b, d, and f), staining with anti-CD32–alexa-647 (red detection channel). a and b, Single cell binding CRP–Cy3 (a) and subsequently, stained with anti-CD32–alexa-647 (b). c and d, Single cell binding CRP–Cy3/anti-CRP (c) and subsequently, stained with anti-CD32–alexa-647 (d). e and f, Four cells from a different area of the sample shown in a and b that display only cellular autofluorescence but neither binding of CRP–Cy3 (e) nor anti-CD32–alexa-647 (f). The intensity of both images is scaled by a factor of 10 with respect to a and b to enable visualization of the weak autofluorescence (Bar=10 μm).
To further confirm the interaction of CRP with FcRIIa in the absence of anti-CRP antibody, we imaged cells before and during equilibration with Cy3–CRP (Figure 3a and 3b). Incubation of Cy3–CRP (4.2 μM) cells with high concentrations of unlabeled CRP (100 μM) revealed a moderate decrease in fluorescence on the hour time scale, suggesting competitive inhibition (data not shown). Figure 3c and 3d shows consecutive incubation of Cy3–CRP-incubated cells with anti-CRP antibody, which caused a pronounced increase of membrane fluorescence (Figure 3d). Apparently, residual CRP–Cy3 diffusing freely in solution was trapped by FcRIIa on the membranes mediated by anti-CRP antibody. Experiments with isotype-matched control antibodies at identical concentrations did not show any enhancement of CRP binding to the cells (data not shown). After addition of 10% human AB serum, a slight increase in CRP binding (at 50% receptor saturation) was observed, possibly attributable to affinity enhancement by ligand (eg, lipoprotein) binding to CRP.
Figure 3. Scan images of Cy3–CRP binding to FcRIIa. The same cells before (a) and 10 minutes after (b) incubation with Cy3-labeled CRP (4.2 μM). The autofluorescence of individual cells is similar (left). In the presence of Cy3–CRP (right), all cells show negligible fluorescence in the interior, whereas 2 cells with high receptor expression show enhanced fluorescence at the cell membrane because of specific receptor interactions. Image a is scaled to 100-fold higher sensitivity to visualize the much weaker autofluorescence. The membrane fluorescence observed in the absence of antibodies (c) increases strongly after addition of anti-CRP antibodies (d), as observed 20 minutes after infusion (Bar=10 μm).
Association Kinetics
The association rate of CRP and anti-CRP antibody/CRP with FcRIIa was determined from an analysis of the membrane-located fluorescence as a function of incubation time (Figure 4). The data in Figure 4a show that equilibration with Cy3-labeled CRP at a concentration of 0.87 μM takes >1 hour. The kinetics are observed to speed up in proportion to the CRP concentration, and consequently, at 5.2 μM CRP, equilibration takes only a few minutes. Assuming that the dissociation rate coefficient is much smaller than the association rate coefficient, an exponential fit of the kinetic data yields a second-order association coefficient of (370±100) M–1s–1. The assumption is justified because we did not observe significant CRP dissociation from FcRIIa on the hour time scale. Kinetic experiments on the anti-CRP antibody/CRP complex were performed at fixed CRP concentration of 0.87 μM and varying the concentration of Cy3-labeled anti-CRP antibodies (0.67 nM, 2 nM, 67 nM; Figure 4b). A linear increase of the association rate with anti-CRP antibody concentration was observed in the low concentration range, yielding a second-order association rate coefficient of (1.1±0.3)x106 M–1s–1 (at 0.87 μM CRP).
Figure 4. Association kinetics of CRP and anti-CRP antibody/CRP complexes to FcRIIa. Kinetics of CRP (a) and anti-CRP (b) antibody/CRP association with the COS-7 membrane, as determined from the analysis of the membrane fluorescence (symbols). The lines are exponential fits to the experimental data.
Equilibrium Binding Studies
The affinities of CRP and anti-CRP antibody/CRP to FcRIIa were examined quantitatively by confocal imaging. The saturation of receptors with ligands was determined from the membrane-located fluorescence as a function of the free ligand concentration. Figure 5 shows the data as symbols; lines are best-fit model calculations assuming simple receptor/ligand equilibria. For CRP–Cy3, the fit yields an equilibrium dissociation coefficient KD=3.7±1 μM for the interaction with FcRIIa. For affinity studies of the anti-CRP antibody/CRP complex, unlabeled CRP was present at a concentration of 0.87 μM, and the concentration of Cy3-labeled anti-CRP antibodies was varied to obtain the saturation curve. From the data in Figure 5, an almost 2 orders of magnitude higher affinity of the complexes is apparent. Quantitative analysis of the data yields KD=45±20 nM.
Figure 5. Equilibrium binding curves of CRP and anti-CRP antibody/CRP complexes to FcRIIa. The degree of saturation of the receptors with ligands was determined from the membrane fluorescence and plotted logarithmically as a function of the protein concentration in the solution. The curves were normalized to their extrapolated saturation values. Experimental data are represented by symbols. Lines are theoretical binding curves calculated for a bimolecular process.
Discussion
In this study, we investigated binding of CRP to FcRIIa in transfected COS-7 cells. FACS analysis of COS-7 cells transfected with FcRIIa alleles in the presence and absence of CRP (Figure 1b) strongly suggested a critical involvement of anti-CRP antibody/FcRIIa interactions in the detection of CRP binding. Thus, we applied ultrasensitive confocal fluorescence microscopy to clarify CRP interactions with FcRIIa. This novel technique enables us to apply a very gentle labeling (1 to 2 fluorescent labels per protein molecule), which ensures that the protein is still in its functionally competent state.30 Despite the low emission level, the single-molecule sensitivity of the microscope still allows direct visual interpretation of the images. Two major observations were made: (1) CRP indeed binds to FcRIIa, and (2) addition of anti-CRP antibodies leads to anti-CRP antibody/CRP complex formation and clustering of the ligand. This is obvious already from qualitative inspection of the confocal images: Cy3-labeled CRP colocalizes with FcRIIa on the membrane surface. This fluorescence is absent for cells not expressing FcRIIa and shows a focal pattern. Addition of excess unlabeled CRP results in a moderate decrease in the fluorescence emission on the hour time scale, suggesting competitive inhibition. Addition of unlabeled anti-CRP antibodies significantly increases the fluorescence emission (Figures 2 and 3).
From the quantitative analysis of the membrane fluorescence (Figures 4 and 5), the following results were obtained: (1) Dissociation of Cy3-CRP and anti-CRP antibody/CRP from FcRIIa receptors was not observed after flushing the samples with buffer solution, which implies that both ligands are tightly bound, with dissociation times on the hour time scale. (2) In contrast, binding of labeled anti-CRP antibody alone to FcRIIa receptors can only be observed as long as the antibody is present in solution. After purging with buffer solution, the enhanced fluorescence from the cell surface vanishes immediately, which is in agreement with off-rates of 1 s–1 reported for the interaction of mouse IgGs with FcRII receptors.31 (3) The binding equilibrium of CRP–Cy3 with FcRIIa is established very slowly (minutes to hours at micromolar concentrations) and can be quantified by a second-order association rate coefficient of 370 M–1s–1. This low value strongly suggests that persistent association to cell membranes can only be achieved by interaction with multiple FcRIIa receptors. CRP is known to be pentameric,1,2 and a low probability of interacting with >1 receptor may explain the small association rate. Competitive inhibition by unlabeled CRP and the focal pattern of fluorescence also suggest receptor clustering in response to Cy3-labeled CRP. CRP interaction with multiple FcRIIa receptors may be a prerequisite for kinase activity and FcRIIa signaling.20,32 (4) The association of CRP is observed to speed up markedly in the presence of anti-CRP antibodies. This observation implies that the additional interaction of the anti-CRP antibody with FcRIIa greatly assists in forming a persistent bond between CRP and the receptors. The bimolecular association rate coefficient of 106 M–1s–1 determined for the anti-CRP antibody/CRP complexes (at fixed CRP concentration) is close to the value of 0.4x106 M–1s–1 reported for the association of mouse IgGs with FcRIIa.31 Interestingly, properties of the 2 components are combined in the immune complex, which shows an association rate coefficient typical of an antibody and a very small dissociation rate coefficient, as with CRP alone. (5) The KD for CRP dissociation from FcRIIa is 3.7 μM, whereas it is 80-fold smaller for the anti-CRP antibody/CRP complex. Qualitatively, this decrease is expected from the behavior of the association rates (Figure 4). We note that our KD of 45 nM for the anti-CRP antibody/CRP complex is identical within the experimental error to the previously reported value of 66 nM for CRP binding, which was determined by antibody-dependent assays on transfected COS-7 cells.18
The visual demonstration that use of anti-CRP antibodies indeed affects CRP binding and leads to false-positive results supports the criticism22,23 directed toward the original observations.18,19 In view of the data presented in this report, some conclusions drawn on CRP interactions with Fc receptors may have to be reconsidered. It is of utmost importance to apply antibody-independent methods to the study of CRP interactions with Fc receptors. In view of the high sensitivity of the assay resulting from selective analysis of receptor-located fluorescence, the gentleness of single-fluorophore labeling of CRP, and the possibility of quantitative analysis, ultra-sensitive confocal fluorescence microscopy may be the method of choice to answer some of the most intriguing questions concerning CRP interactions with cellular receptors, such as: (1) What is the affinity of CRP to FcRI compared with FcRIIa?33 (2) Does ligand (for example, LDL) binding to CRP increase its affinity to Fc receptors? (3) Is clustering of Fc receptors with other cell surface molecules involved in CRP binding to leukocytes?
To conclude, ultrasensitive confocal fluorescence microscopy may significantly contribute to the understanding of CRP binding to nucleated cells, with the potential aim of developing CRP receptor blockers for the treatment of atherosclerosis and its sequelae.
Acknowledgments
This work was supported by Deutsche Forschungsgemeinschaft (SFB 451 and 569).
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ABSTRACT
Background— C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcRIIa may have caused false-positive results.
Methods and Results— To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcRIIa. CRP indeed binds to FcRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude.
Conclusions— Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcRIIa and explains and reconciles the diverse and conflicting data presented in the literature.
We have studied CRP binding to FcRIIa using the novel method of ultrasensitive confocal fluorescence microscopy. We unambiguously show that CRP interacts with FcRIIa and characterize this interaction quantitatively. We also provide explanations as to why controversial results were obtained previously.
Key Words: atherosclerosis ? inflammation ? receptors ? ultrasensitive fluorescence microscopy
Introduction
C-Reactive protein (CRP) is the prototype human acute-phase protein.1,2 It also is a powerful cardiovascular risk marker.3 Recent investigations suggested a pathogenic role of CRP in cardiovascular disease,4,5 which has spawned widespread interest in studies of its biological function.6–12 Importantly, it has been demonstrated that human CRP transgene expression causes accelerated aortic atherosclerosis in apolipoprotein E–deficient mice, providing first in vivo evidence of a direct involvement of CRP in atherogenesis.12
To date, ligand binding, opsonization of bioparticles,13–15 and complement activation16 are rigorously defined pathobiological CRP functions. CRP interactions with nucleated cells have gained increasing interest,6–11 and CRP binding to cellular receptors has been intensely investigated with conflicting results. Whereas some reports provided evidence of specific CRP receptors,17 other experiments demonstrated interaction with Fc receptors.18,19 The low-affinity IgG receptor FcRIIa was proposed to be the major CRP receptor.18,19 Several observations supported this concept. When coincubated with low-density lipoprotein (LDL), CRP colocalizes with clusters of FcRIIa on monocyte membranes.8 Furthermore, CRP was reported to induce FcRIIa-signaling in human promyelocytic cell line HL-60,20 and finally, experiments in FcRII- and -chain–deficient mice showed lacking CRP-mediated biological responses compared with wild-type mice.21 To demonstrate CRP binding to FcRIIa, anti-CRP antibodies were used in the initial reports18,19 because direct labeling of CRP with fluorescein isothiocyanate (FITC) or 125-I may damage the structure of the molecule and lead to ambiguous results. It was also suggested that CRP binding to FcRIIa is allele specific.19 High-affinity binding was reported for FcRIIa R/R-131, intermediate affinity binding for FcRIIa R/H-131, and low-affinity binding for FcRIIa H/H-131. Subsequently, several authors proposed that CRP may not interact with FcRIIa at all and that the observed binding of CRP to FcRIIa results from an interaction of the Fc portion of the anti-CRP antibody with FcRIIa itself.2,22,23 Indeed, using F(ab')2 fragments of anti-CRP antibodies, fluorescence-activated cell sorter (FACS) analysis revealed no CRP binding to FcRIIa-R131 on polymorphonuclear leukocytes and FcRIIa-transfected IIA.6 cells.22 Other authors have suggested that the observed binding of CRP to FcRIIa might be attributable to IgG contamination of the CRP reagent,23 and a recent review claims that CRP does not interact at all with cellular Fc receptors.2
Here we applied the novel technology of ultrasensitive confocal fluorescence microscopy to study CRP interactions with FcRIIa.24–26 Our results visually demonstrate and quantitatively show that (1) use of anti-CRP antibodies indeed affects CRP binding and leads to false-positive results; and (2) CRP, however, does bind to FcRIIa, although with lower affinity than anti-CRP antibody/CRP complexes.
Methods
Cell Culture
COS-7 cells were obtained from DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and maintained in DMEM/10% FCS with 1% penicillin/streptomycin/L-glutamine. This cell line does not express Fc receptors.
Reagents and Antibodies
Partially purified CRP was obtained from Sigma. Highly purified CRP was kindly provided by Dr T.W. Du Clos (University of New Mexico, Albuquerque). Recombinant CRP (rCRP) was obtained from Calbiochem. Western blot analysis revealed <0.1% IgG for partially purified and no detectable IgG for highly purified and rCRP. Monoclonal anti-CRP antibody, clone 2C10, was generously provided by Dr Du Clos with kind permission of Dr Larry Potempa (ImmTech, Evanston, Il).27 Anti-CD32–FITC, clone FLI8.26(2003), and unlabeled and phycoerythrin (PE)-labeled monoclonal mouse IgG1 isotype were purchased from BD Biosciences. Anti-CD32, clone KB61, was purchased from DAKO, affinity-isolated F(ab')2 PE-goat anti-mouse (GAM) from Caltag Labs, and human serum from the blood transfusion service of the University of Ulm.
FcRIIa Cloning and Transfection
Human FcRIIa cDNA (G/G genotype, coding for FcRIIa R/R-131) was generated by RT-PCR and cloned into pcDNA3.1 using the Directional TOPO Expression Kit (Invitrogen). The cDNA FcRIIa A/A genotype (coding for FcRIIa H/H-131) was generated from the FcRIIa G/G genotype using site-specific mutagenesis.28,29 Vectors were sequenced. Six-well plates were seeded at 2.5x105 cells/well. Approximately 70% to 80% confluent cells were transfected using Polyfect Reagent (Qiagen). Cells expressing heterozygous FcRIIa R/H-131 were obtained by cotransfection with FcRIIa G/G and A/A. Mock-transfected cells were treated with transfectant reagent only. Anti-CD32–FITC staining revealed similar transfection efficiency for the alleles.
Binding Assays With Nonlabeled CRP and Anti-CRP Antibodies Using FACS
CRP-binding assays were performed with COS-7 cells 48 hours after transfection as described.18 Detached cells were incubated with different preparations of CRP at a concentration of 1.74 μmol/L, which corresponds to 200 μg/mL. This concentration was used by Bharadway et al18 and found to induce optimal signaling by Chi et al20 in ice-cold PBS containing 0.05% azide and 0.1% BSA (PAB) for 1 hour on ice. After washing with PAB, cells were stained with anti-CRP (2C10) for 0.5 hours on ice. Cells were washed and stained with PE-GAM–F(ab')2 for 0.5 hours on ice in dark. Cells were washed and analyzed using FACS with CellQuest software (BD Biosciences). A total of 30 000 cells were gated by fluorescence-1 (green) and fluorescence-2 (red). A total of 95% mock-transfected cells stained in the absence of CRP were assessed as background. Cell viability assays (trypan blue) revealed 99% viable transfected cells. Staining with IgG1-PE isotype antibody was used as a control. Because of identical results for the different CRP preparations, rCRP was used for all subsequent analyses. Results are expressed as mean±SD. Scores were compared using Student paired t test (Microsoft Excel 2000). A P<0.05 was considered statistically significant.
Confocal Imaging and Analysis
Confocal images were collected using a laser scanning confocal fluorescence microscope with single-fluorophore sensitivity.24–26 An Ar+/Kr+ ion laser (Spectra Physics 164) and an HeNe laser (Polytec) were used for fluorescence excitation at 514.5 nm and 632.8 nm, respectively. The excitation light was focused into the sample, and the resulting fluorescence emission was collected by a water immersion objective (C-Apochromat 63x/1.2 W; Zeiss). Highly efficient detection in 2 spectral channels (green 557 to 607 nm; red 665 to 850 nm) was accomplished by splitting the fluorescence light using custom-made bandpass filters in conjunction with dichroic mirrors (AHF) and subsequent detection with single-photon counting detectors (AQR-14; Perkin–Elmer). The whole instrument is controlled by our homemade software.
Confocal fluorescence images consisting of 128x128 pixels were acquired in a field of 90x90 μm2 with a depth resolution of 2 μm for both excitation wavelengths with 0.5-μW laser power incident on an area of 0.3 μm2.
For quantitative analysis, the fluorescence emitted by membranes of selected cells was examined as a function of time after incubation with fluorescently labeled CRP or anti-CD32 antibodies or after equilibration with these proteins at different concentrations. Within 1 series of measurements, the same number (typically 100) of the brightest pixels from the membrane of a chosen cell was analyzed.
Fluorescence Labeling
CRP and monoclonal anti-CRP antibodies were labeled with Cy3-N-hydroxy-Succinimidyl ester (NHS) (Amersham) by coupling the succinimidyl ester derivative of the dye to amine groups in phosphate buffer at pH 8.2. Anti-CD32 (KB61) antibodies were conjugated with Alexa Fluor 647 dye (Molecular Probes) using maleimide coupling to free thiol groups. Unreacted dye was removed by gel filtration. The degree of labeling was kept low (1 to 2 fluorescent labels per protein molecule, as quantified by optical absorption spectroscopy) to minimize dye interactions. Pseudonative SDS-PAGE revealed identical bands for nonlabeled and Cy3-NHS–labeled CRP.
Colocalization Measurements
Solutions containing FcRIIa-transfected COS-7 cells were transferred to a sandwich chamber consisting of 2 glass cover slips separated by mylar spacers (thickness 200 μm). After 15 minutes, cells were exposed to solutions of CRP–Cy3 at 0.87 μM (100 μg/mL, shown to induce optimal signaling20) or anti-CRP antibody/CRP–Cy3 complexes at 6.7 nM (1 μg/mL, for anti-CRP antibody 2C10). After washing, receptor staining was performed using solutions with 0.5 μM Alexa 647-labeled anti-CD32 antibodies, and subsequently, confocal images were taken.
Association Kinetics and Equilibrium Binding
The kinetics of association of Cy3-labeled CRP to FcRIIa-transfected COS-7 cells was studied by acquiring confocal images as a function of time. For the kinetics of the anti-CRP antibody/CRP complex, a constant concentration of 0.87 μM unlabeled CRP was used in combination with different concentrations of Cy3-labeled anti-CRP antibodies.
For studies of equilibrium binding, incubation times were adjusted in accordance with the kinetic data. COS-7 cells were exposed to different concentrations of Cy3-labeled CRP and CRP/anti-CRP–Cy3 complexes. Confocal images were analyzed to assess the degree of saturation of the receptors.
Results
FACS Analysis Using Nonlabeled CRP and Anti-CRP Antibodies
FACS analysis of FcRIIa- and mock-transfected COS-7 cells was performed in the presence and absence of CRP. FcRIIa–131R/R-transfected cells showed 54.2% positivity (PE staining) after incubation with 1.74 μM CRP (Figure 1a). This experiment confirmed the original data,18,19 which led to the interpretation of high-affinity binding of CRP to FcRIIa. Three different CRP preparations (partially purified, highly purified, and rCRP) yielded identical results (data not shown).
Figure 1. a, FACS analysis using nonlabeled CRP and anti-CRP antibodies. Cells were incubated with 1.74 μM CRP (200 μg/mL) and stained with anti-CRP antibody (2C10) and F(ab)2–PE-GAM. High PE staining (54.2%) for FcRIIa–131R/R-transfected cells in the presence of CRP. b, FACS analysis of alleles showing changes in the staining of cells expressing different FcRIIa-alleles. c, Role of anti-CRP antibody/CRP complexes. Identical results for FACS analysis of FcRIIa–131R/R-transfected cells after treatment with preformed anti-CRP antibody/CRP complexes and after sequential addition of CRP and anti-CRP antibodies.
Binding assays with CRP (1.74 μM) were performed in cells transfected with the FcRIIa alleles (131R/R, 131R/H, and 131H/H). A decrease in staining was seen in the order 131R/R131R/H131H/H in the presence (Figure 1b, black) (RR:RH:HH=1.6:1.2:1.0, RR/RH, RH/HH, RR/HH); and absence (Figure 1b, dotted) of CRP, (RR:RH:HH=2.8:2.0:1.0, RR/RH, RR/HH, RH/HH; and also for a mouse IgG1 isotype control (Figure 1b, hatched; (RR:RH:HH=2.5:1.8:1.0, RR/RH, RR/HH, RH/HH). The differences in staining reflect the differences in binding of IgG1 to the "high" (131R/R) and "low responder" (131H/H) forms of FcRIIa.29 Treatment of FcRIIa–131R/R-transfected cells with preformed anti-CRP antibody/CRP complex led to the same positive results (Figure 1c, right) as obtained by addition of CRP to cells and subsequent incubation with anti-CRP antibodies (Figure 1c, left).
Confocal Imaging
After incubation of FcRIIa-transfected COS-7 cells with CRP–Cy3 and subsequent washing with PBS, green fluorescence from the cell membrane was observed by confocal imaging. As an example, Figure 2a shows CRP–Cy3 binding to a cell that strongly expresses FcRIIa. The fluorescence shows a focal pattern. The signal was much stronger when the cells were coincubated with unlabeled anti-CRP antibodies (Figure 2c). During incubation of cells that had bound CRP–Cy3 (Figure 2a) with anti-CD32–alexa 647 (Figure 2b), strict colocalization of binding sites for CRP–Cy3 and anti-CD32–alexa-647 was observed. Incubation of CRP–Cy3/ anti-CRP antibody complex binding cells (Figure 2c) with anti-CD32–alexa 647 (Figure 2b) also showed strict colocalization of binding sites for CRP–Cy3 and anti-CD32–alexa-647. Competitive binding was evident from the ratio of red to green membrane fluorescence compared with the ratio obtained when incubating anti-CD32 antibodies before the immune complexes (data not shown). Figure 2e and 2f displays control cells from a different area of the sample shown in Figure 2a and 2b that do not express FcRIIa and do not bind CRP.
Figure 2. Colocalization of CRP and FcRIIa. Representative examples of confocal scan images of COS-7 cells are shown. Left (a, c, and e), Staining with Cy3-labeled CRP (green detection channel). Right (b, d, and f), staining with anti-CD32–alexa-647 (red detection channel). a and b, Single cell binding CRP–Cy3 (a) and subsequently, stained with anti-CD32–alexa-647 (b). c and d, Single cell binding CRP–Cy3/anti-CRP (c) and subsequently, stained with anti-CD32–alexa-647 (d). e and f, Four cells from a different area of the sample shown in a and b that display only cellular autofluorescence but neither binding of CRP–Cy3 (e) nor anti-CD32–alexa-647 (f). The intensity of both images is scaled by a factor of 10 with respect to a and b to enable visualization of the weak autofluorescence (Bar=10 μm).
To further confirm the interaction of CRP with FcRIIa in the absence of anti-CRP antibody, we imaged cells before and during equilibration with Cy3–CRP (Figure 3a and 3b). Incubation of Cy3–CRP (4.2 μM) cells with high concentrations of unlabeled CRP (100 μM) revealed a moderate decrease in fluorescence on the hour time scale, suggesting competitive inhibition (data not shown). Figure 3c and 3d shows consecutive incubation of Cy3–CRP-incubated cells with anti-CRP antibody, which caused a pronounced increase of membrane fluorescence (Figure 3d). Apparently, residual CRP–Cy3 diffusing freely in solution was trapped by FcRIIa on the membranes mediated by anti-CRP antibody. Experiments with isotype-matched control antibodies at identical concentrations did not show any enhancement of CRP binding to the cells (data not shown). After addition of 10% human AB serum, a slight increase in CRP binding (at 50% receptor saturation) was observed, possibly attributable to affinity enhancement by ligand (eg, lipoprotein) binding to CRP.
Figure 3. Scan images of Cy3–CRP binding to FcRIIa. The same cells before (a) and 10 minutes after (b) incubation with Cy3-labeled CRP (4.2 μM). The autofluorescence of individual cells is similar (left). In the presence of Cy3–CRP (right), all cells show negligible fluorescence in the interior, whereas 2 cells with high receptor expression show enhanced fluorescence at the cell membrane because of specific receptor interactions. Image a is scaled to 100-fold higher sensitivity to visualize the much weaker autofluorescence. The membrane fluorescence observed in the absence of antibodies (c) increases strongly after addition of anti-CRP antibodies (d), as observed 20 minutes after infusion (Bar=10 μm).
Association Kinetics
The association rate of CRP and anti-CRP antibody/CRP with FcRIIa was determined from an analysis of the membrane-located fluorescence as a function of incubation time (Figure 4). The data in Figure 4a show that equilibration with Cy3-labeled CRP at a concentration of 0.87 μM takes >1 hour. The kinetics are observed to speed up in proportion to the CRP concentration, and consequently, at 5.2 μM CRP, equilibration takes only a few minutes. Assuming that the dissociation rate coefficient is much smaller than the association rate coefficient, an exponential fit of the kinetic data yields a second-order association coefficient of (370±100) M–1s–1. The assumption is justified because we did not observe significant CRP dissociation from FcRIIa on the hour time scale. Kinetic experiments on the anti-CRP antibody/CRP complex were performed at fixed CRP concentration of 0.87 μM and varying the concentration of Cy3-labeled anti-CRP antibodies (0.67 nM, 2 nM, 67 nM; Figure 4b). A linear increase of the association rate with anti-CRP antibody concentration was observed in the low concentration range, yielding a second-order association rate coefficient of (1.1±0.3)x106 M–1s–1 (at 0.87 μM CRP).
Figure 4. Association kinetics of CRP and anti-CRP antibody/CRP complexes to FcRIIa. Kinetics of CRP (a) and anti-CRP (b) antibody/CRP association with the COS-7 membrane, as determined from the analysis of the membrane fluorescence (symbols). The lines are exponential fits to the experimental data.
Equilibrium Binding Studies
The affinities of CRP and anti-CRP antibody/CRP to FcRIIa were examined quantitatively by confocal imaging. The saturation of receptors with ligands was determined from the membrane-located fluorescence as a function of the free ligand concentration. Figure 5 shows the data as symbols; lines are best-fit model calculations assuming simple receptor/ligand equilibria. For CRP–Cy3, the fit yields an equilibrium dissociation coefficient KD=3.7±1 μM for the interaction with FcRIIa. For affinity studies of the anti-CRP antibody/CRP complex, unlabeled CRP was present at a concentration of 0.87 μM, and the concentration of Cy3-labeled anti-CRP antibodies was varied to obtain the saturation curve. From the data in Figure 5, an almost 2 orders of magnitude higher affinity of the complexes is apparent. Quantitative analysis of the data yields KD=45±20 nM.
Figure 5. Equilibrium binding curves of CRP and anti-CRP antibody/CRP complexes to FcRIIa. The degree of saturation of the receptors with ligands was determined from the membrane fluorescence and plotted logarithmically as a function of the protein concentration in the solution. The curves were normalized to their extrapolated saturation values. Experimental data are represented by symbols. Lines are theoretical binding curves calculated for a bimolecular process.
Discussion
In this study, we investigated binding of CRP to FcRIIa in transfected COS-7 cells. FACS analysis of COS-7 cells transfected with FcRIIa alleles in the presence and absence of CRP (Figure 1b) strongly suggested a critical involvement of anti-CRP antibody/FcRIIa interactions in the detection of CRP binding. Thus, we applied ultrasensitive confocal fluorescence microscopy to clarify CRP interactions with FcRIIa. This novel technique enables us to apply a very gentle labeling (1 to 2 fluorescent labels per protein molecule), which ensures that the protein is still in its functionally competent state.30 Despite the low emission level, the single-molecule sensitivity of the microscope still allows direct visual interpretation of the images. Two major observations were made: (1) CRP indeed binds to FcRIIa, and (2) addition of anti-CRP antibodies leads to anti-CRP antibody/CRP complex formation and clustering of the ligand. This is obvious already from qualitative inspection of the confocal images: Cy3-labeled CRP colocalizes with FcRIIa on the membrane surface. This fluorescence is absent for cells not expressing FcRIIa and shows a focal pattern. Addition of excess unlabeled CRP results in a moderate decrease in the fluorescence emission on the hour time scale, suggesting competitive inhibition. Addition of unlabeled anti-CRP antibodies significantly increases the fluorescence emission (Figures 2 and 3).
From the quantitative analysis of the membrane fluorescence (Figures 4 and 5), the following results were obtained: (1) Dissociation of Cy3-CRP and anti-CRP antibody/CRP from FcRIIa receptors was not observed after flushing the samples with buffer solution, which implies that both ligands are tightly bound, with dissociation times on the hour time scale. (2) In contrast, binding of labeled anti-CRP antibody alone to FcRIIa receptors can only be observed as long as the antibody is present in solution. After purging with buffer solution, the enhanced fluorescence from the cell surface vanishes immediately, which is in agreement with off-rates of 1 s–1 reported for the interaction of mouse IgGs with FcRII receptors.31 (3) The binding equilibrium of CRP–Cy3 with FcRIIa is established very slowly (minutes to hours at micromolar concentrations) and can be quantified by a second-order association rate coefficient of 370 M–1s–1. This low value strongly suggests that persistent association to cell membranes can only be achieved by interaction with multiple FcRIIa receptors. CRP is known to be pentameric,1,2 and a low probability of interacting with >1 receptor may explain the small association rate. Competitive inhibition by unlabeled CRP and the focal pattern of fluorescence also suggest receptor clustering in response to Cy3-labeled CRP. CRP interaction with multiple FcRIIa receptors may be a prerequisite for kinase activity and FcRIIa signaling.20,32 (4) The association of CRP is observed to speed up markedly in the presence of anti-CRP antibodies. This observation implies that the additional interaction of the anti-CRP antibody with FcRIIa greatly assists in forming a persistent bond between CRP and the receptors. The bimolecular association rate coefficient of 106 M–1s–1 determined for the anti-CRP antibody/CRP complexes (at fixed CRP concentration) is close to the value of 0.4x106 M–1s–1 reported for the association of mouse IgGs with FcRIIa.31 Interestingly, properties of the 2 components are combined in the immune complex, which shows an association rate coefficient typical of an antibody and a very small dissociation rate coefficient, as with CRP alone. (5) The KD for CRP dissociation from FcRIIa is 3.7 μM, whereas it is 80-fold smaller for the anti-CRP antibody/CRP complex. Qualitatively, this decrease is expected from the behavior of the association rates (Figure 4). We note that our KD of 45 nM for the anti-CRP antibody/CRP complex is identical within the experimental error to the previously reported value of 66 nM for CRP binding, which was determined by antibody-dependent assays on transfected COS-7 cells.18
The visual demonstration that use of anti-CRP antibodies indeed affects CRP binding and leads to false-positive results supports the criticism22,23 directed toward the original observations.18,19 In view of the data presented in this report, some conclusions drawn on CRP interactions with Fc receptors may have to be reconsidered. It is of utmost importance to apply antibody-independent methods to the study of CRP interactions with Fc receptors. In view of the high sensitivity of the assay resulting from selective analysis of receptor-located fluorescence, the gentleness of single-fluorophore labeling of CRP, and the possibility of quantitative analysis, ultra-sensitive confocal fluorescence microscopy may be the method of choice to answer some of the most intriguing questions concerning CRP interactions with cellular receptors, such as: (1) What is the affinity of CRP to FcRI compared with FcRIIa?33 (2) Does ligand (for example, LDL) binding to CRP increase its affinity to Fc receptors? (3) Is clustering of Fc receptors with other cell surface molecules involved in CRP binding to leukocytes?
To conclude, ultrasensitive confocal fluorescence microscopy may significantly contribute to the understanding of CRP binding to nucleated cells, with the potential aim of developing CRP receptor blockers for the treatment of atherosclerosis and its sequelae.
Acknowledgments
This work was supported by Deutsche Forschungsgemeinschaft (SFB 451 and 569).
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