Binding and Internalization of C-Reactive Protein by Fcgamma Receptors on Human Aortic Endothelial Cells Mediates Biological Effects
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动脉硬化血栓血管生物学 2005年第7期
From the Laboratory for Atherosclerosis and Metabolic Research, Department of Pathology (S.D., I.J.), UC Davis Medical Center, Sacramento, Calif; and the Department of Medicine (T.W.D.C.), University of New Mexico and Veterans Affairs Medical Center, Albuquerque, N.M.
Correspondence to Ishwarlal Jialal, MD, PhD, Director of the Laboratory for Atherosclerosis and Metabolic Research, 4635 II Avenue, Res 1 Bldg, Rm 3000, Sacramento, CA 95817. E-mail ishwarlal.jialal@ucdmc.ucdavis.edu
Abstract
Objective— In addition to being a cardiovascular risk marker, recent studies support a role for CRP in atherothrombosis. Several investigators have reported that CRP binds to Fcgamma receptors on leukocytes. The aim of the study is to determine the processing of CRP by human aortic endothelial cells (HAECs).
Methods and Results— Binding studies were performed by incubation of HAECs with biotinylated CRP (B-CRP, 25 to 200 μg/mL) for 30 to 180 minutes at 4°C. B-CRP binding was quantitated using streptavidin-fluorescein isothiocyanate followed by flow cytometry. Saturable binding of CRP was obtained at 60 minutes with a CRP concentration between 100 to 150 μg/mL and Kd of 88 nM. CRP binding was inhibited by 10x cold CRP (58%). CRP (100 μg/mL) significantly upregulated surface expression of Fcgamma receptors, CD32, as well as CD64 on HAECs (P<0.01). Also, preincubation with anti-CD32 and CD64 antibodies significantly inhibited maximal binding of CRP to HAECs 64% and 30%, respectively, whereas antibodies to CD16 had no effect. Internalization of CRP, as determined by loss of surface expression, was 50%. Also, binding and internalization of biotinylated CRP was confirmed by confocal microscopy and CRP colocalized with CD32 and CD64. Most importantly, the increase in interleukin-8, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1 and the decrease in eNOS and prostacyclin induced by CRP was abrogated with antibodies to CD32 and CD64.
Conclusions— We demonstrate that CRP mediates its biological effects on HAECs via binding and internalization through Fcgamma receptors, CD32 and CD64.
In addition to being a risk marker, CRP exerts atherothrombotic effects in endothelial cells. In this study, using flow cytometry and fluorescence microscopy, we show that CRP binds to CD32 and CD64 on HAECs, is internalized, and exerts its biological effects. Antibodies to CD32 and CD64 abrogated the biological effects of CRP, whereas antibodies to CD16 had no effect.
Key Words: inflammation ? C-reactive protein ? Fcgamma receptors ? endothelial cells ? atherothrombosis
Introduction
Inflammation is pivotal in all stages of atherosclerosis.1 Numerous prospective studies have confirmed that high levels of C-reactive protein (CRP), the prototypic marker of inflammation, predicts cardiovascular events and is a risk marker.2,3 Also recent studies support a role for CRP in atherothrombosis.4–6 To date, it has been shown that in monocytes, CRP induces the production of inflammatory cytokines, promotes monocyte chemotaxis, uptake of oxidized LDL, and tissue factor expression. In endothelial cells (ECs), CRP increases the expression of cell adhesion molecules, monocyte-chemotactic protein-1 and endothelin-1 (ET-1), interleukin (IL)-8, and plasminogen activator inhibitor (PAI)-1 and decreases eNOS expression and activity and prostacyclin release.4–6 In smooth muscle cells (SMCs), CRP has been shown to activate angiotensin-1 type receptors.5
There are at least 3 types of human Fcgamma receptors, the high affinity receptor CD64 and the 2 low affinity receptors, CD32 and CD16.7 Several investigators have reported the presence of receptors for CRP on mononuclear cells and neutrophils. Tebo et al8 and Zahedi et al9 concluded that 2 receptors for CRP were present on mononuclear cells in humans. It was noted that there was some possible association with Fc receptors (FcRs) as IgG could inhibit binding. Bharadwaj et al10 have reported that the major receptor for CRP on leukocytes is Fcgamma receptor II (CD32). In transfected COS cells, CRP has been shown to bind to Fcgamma receptor 1 (CD64).11 However, to date, no reports have documented a CRP receptor on ECs. Data available so far favor CD32 and CD64 as the most likely candidates because all EC subtypes investigated thus far are negative or weakly positive for CD16.12 CD32 has been localized to placental and dermal microvascular ECs as well as liver sinusoidal ECs.13,14 Immune complexes from vasculitis patients bind to CD32 of ECs.15In addition, we have shown previously in a preliminary report that incubation of human aortic ECs (HAECs) with anti-CD32 antibodies significantly reversed the stimulation of PAI-1 by CRP.16 In this study, we demonstrate that the major receptors for CRP in ECs are CD32 and CD64, and they orchestrate its biological effects.
Materials and Methods
CRP was procured from 3 different sources: CRP purified from plasma (Sigma Chemicals), recombinant human CRP (Calbiochem), and purified CRP from human pleural fluid in the laboratory of Dr T.W. Du Clos. All CRP preparations were purified under sterile conditions using Endotoxin-removal columns (Pierce Biochemicals) and used only if the concentration of endotoxin was <0.125 EU/mL.17 All cell culture media were endotoxin-free. Furthermore, Western blotting using HRP-conjugated IgG antibodies failed to reveal any contamination of purified CRP with IgG. Antibodies to CD16 (clone 3G8-IgG1) were purchased from Pharmingen, Serotec, CD32 (FLI8.26-IgG2b and AT10-IgG1) were purchased from Pharmingen, Santa Cruz, and CD64 (10.1, IgG1) was purchased from Pharmingen, Santa Cruz, respectively, along with their respective isotype controls.
HAECs were cultured in EGM-2MV media (Biowhittaker), and confluent cultures below the 6th passage were used for all experiments.17
CRP Binding
CRP was biotinylated using reagents from Pierce (EZ Sulfo NHS biotinylation reagents) at a molar ratio of biotin to CRP of 20 to 1. Biotinylation was checked using EZ biotin quantitation reagents from Pierce Biotechnologies. Biotinylated CRP (25 to 200 μg/mL) was then added to confluent human aortic ECs (1x106 in PAB–PBS with 1 mmol/L CaCl2/MgCl2 containing 1 mg/mL BSA, azide 10 mmol/L, HEPES 20 mmol/L) at 4°C for a time course of 180 minutes. CRP binding was quantitated by tagging the biotinylated CRP that is bound to the cell surface of HAECs using streptavidin–FITC followed by flow cytometry. Nonspecific binding was assessed using a 10- to 20-fold excess cold CRP. Kinetics of binding was determined using Graph Pad Prism 4 Software. Inhibition studies were undertaken with monoclonal antibodies to CD32, CD64, and CD16 (as a negative control). Unlabeled CRP with streptavidin–FITC was also used as negative control. CRP internalization was measured after incubating at 4°C to attain maximum binding. The media was aspirated, and fresh media without azide was added to the cells. The cells were then incubated at 37°C for a period of 30 to 180 minutes to determine internalization of CRP at 37°C. Furthermore, cells were preincubated with monoclonal antibodies to CD16, CD32, and CD64 (20 μg/mL) to saturate the receptors to see whether this would prevent internalization of CRP to HAECs/HCAECs.
Cell Surface Expression of Fcgamma Receptors by Flow Cytometry
Cell surface expression of FcR was assessed by incubating 1x105 cells in PBS with 25 to 200 μg/mL CRP for 30 to 180 minutes at 4°C in PBS with calcium and magnesium with 10 mmol/L azide which blocks internalization. Thereafter, specific fluorochrome-labeled antibodies to CD16, CD32, and CD64 were added and incubated for another 30 minutes. After detachment with PBS–EDTA, cells were analyzed by flow cytometry to determine the abundance of each of these receptors. Isotype controls were used with each experiment and expressed as mfi/1x105 cells.
Colocalization of Fcgamma Receptors with CRP by Fluorescence Microscopy
For immunofluorescence studies, HAECs were cultured on cover slips and incubated at 4°C for binding studies and at 37°C in PBS for 1 hour with 100 μg of biotinylated CRP for internalization. Cells were washed with PBS, fixed with 3.5% paraformaldehyde, and permeabilized with 0.5% Triton-X 100 in PBS and then incubated with streptavidin–FITC. Cells were washed in PBS and then observed under a confocal microscope. Colocalization of FITC–CRP and specific fluorochrome-labeled CD32/CD64 was also examined by confocal microscopy. Control incubations included unlabeled CRP with streptavidin–FITC.
Biological Effects of CRP
To determine whether CRP binding to the FcR orchestrates its proinflammatory prothrombotic biological effects (inhibition of prostacyclin synthase and eNOS and stimulation of IL-8, ICAM-1, and VCAM-1), HAECs were preincubated with excess monoclonal antibodies to CD16, CD32, and CD64 alone and in combination followed by the addition of different concentrations of CRP 0 to 50 μg/mL. Biological effects of CRP were examined as described previously.17,18 We tested whether the biological effects of CRP can be blocked using piceatannol (a Syk kinase inhibitor, 50 μmol/L for 30 minutes).
Statistical Analyses
All data are expressed as mean±SD. Comparison of the biological effects of CRP was assessed by paired t tests, and significance was set at P<0.05.
Results
Binding studies with CRP at doses of 25 to 200 μg/mL showed that CRP bound in a dose-dependent saturable fashion (Figure 1a). Saturable binding of CRP was obtained at 60 minutes with a predicted CRP concentration of 104 μg/mL, and a Kd of 88.2 nM was computed. Time course experiments conducted for 30 to 180 minutes revealed maximal binding at 60 minutes and this was used for all further experiments.
Figure 1. a, Binding curve of C-reactive protein on HAECs. HAECs were incubated with different Biotinylated-CRP (25 to 250 μg/mL) in PAB at 4°C for 60 minutes followed by addition of streptavidin FITC as described in Methods. Results are mean of 7 different experiments. Kd was plotted using Graph Pad Prism software. b, Inhibition of biotinylated CRP binding to HAECs. HAECs were preincubated with antibodies to CD32, CD64, CD16, or the combination or 10x cold CRP for 1 hour before addition of Biotinylated CRP (100 μg/mL), and inhibition of maximal binding of B-CRP was assessed by flow cytometry as described in Methods. Results are mean of 6 different experiments.
CRP binding was inhibited by 10x cold CRP (58%) (Figure 1b). Furthermore, binding of CRP to HAECs was significantly inhibited when HAECs were preincubated for 1 hour with antibodies to CD32 (64%) and CD64 (30%) as well as the combination (59%) before addition of Biotinylated CRP (100 μg/mL). The combination of antibodies to CD32 and CD64 was not additive to either alone. Preincubation with anti-CD16 antibody failed to have any significant effect on binding (Figure 1b).
Internalization of CRP was assessed by flow cytometry as a decrease in surface expression and was performed with CRP (100 μg/mL) for a period of 240 minutes at 37°C. As seen in Figure 2, maximal internalization of CRP was obtained by 60 minutes, although only 50% of the CRP appeared to be internalized. Preincubation with antibodies to CD32 and CD64 reduced CRP internalization by 24% and 29%, respectively.
Figure 2. Internalization of CRP. Internalization of B-CRP was examined by flow cytometry as loss of surface expression after incubation of B-CRP for a period of 30 to 240 minutes at 37°C and addition of streptavidin FITC as described in Methods. Results are mean of 5 different experiments.
We then examined surface expression of Fcgamma receptors on HAECs in presence and absence of CRP. Although there was minimal surface expression of CD16, HAECs expressed CD32 and CD64, both of which were increased 5-fold and 2-fold, respectively, with CRP (Figure 3).
Figure 3. Expression of Fcgamma receptors on HAECs. HAECs were incubated in absence and presence of CRP (100 μg/mL) and cells (100 000) were analyzed by flow cytometry with respective isotype controls as described in Methods. Results are mean of 5 different experiments. *P<0.001 compared with control
To confirm the presence of the receptors, we used fluorescence microscopy. When cells were incubated at 4°C, fluorescent staining for CRP was observed on the cell surface, and the same cells were positive for CD32–PE fluorescence. Although there was weak fluorescent staining for CD64 on some cells, no fluorescent staining was observed for CD16 (Figure 4a). Furthermore, CRP internalization was observed with perinuclear staining at 37°C, with strong colocalization with CD32 (Figure 4b) and also with CD64 (Figure 4c).
Figure 4. a, Visualization of B-CRP, CD32, and CD64 by confocal microscopy. Cells were incubated for 60 minutes at 4°C for maximal binding along with CRP (B-CRP followed by streptavidin-FITC), and expression of CD32 (PE) and CD64 (PE) were assessed. b and c, Intracellular localization of CRP (FITC), CD32 (PE, b), and CD64 (PE, c) after internalization with CRP (100 μg/mL) for 120 minutes at 37°C by fluorescence microscopy.
Finally, to determine whether CRP binding to CD32 and CD64 orchestrates its proinflammatory prothrombotic biological effects in HAECs, cells were preincubated with excess monoclonal antibodies to CD16, CD32, and CD64 followed by the addition of CRP 50 μg/mL. Thereafter, biological effects of CRP were tested. We have previously shown that the most significant effects of CRP are increased secretion of IL-8 and inhibition of prostacyclin release in HAECs.17,18 As seen from Figure 5a and 5b, the effects of CRP 50 μg/mL were reversed with antibodies to CD32 and CD64 but not by CD16 antibodies. Also, we tested whether the biological effects of CRP on IL-8 can be blocked using piceatannol, a Syk kinase inhibitor, because signaling through Fcgamma receptors requires activation of Syk kinase.19 The Syk inhibitor, through which Fcgamma receptors signal, was able to reverse the stimulatory effect of CRP on IL-8 (C-6.8±1.9 nmol/mg pr; CRP 50 μg/mL –16.4±2.6 nmol/mg pr; CRP50 μg/mL+Piceatannol –10.7±1.4 nmol/mg pr). Also, antibodies to CD32 and CD64 inhibited the stimulatory effect of CRP on secreted ICAM-1, VCAM-1, and eNOS as evaluated by cGMP assay (Table).
Figure 5. a and b, Effect of antibodies to Fcgamma receptors on CRP-induced IL-8 release (a) and CRP-induced inhibition of prostacyclin (b). Results are mean of 6 different experiments *P<0.001 compared with control, #P<0.05 compared with CRP.
Effect of Antibodies to CD32 and CD64 on the Biological Effects of CRP in HAEC
Discussion
In addition to being a risk marker, several recent lines of evidence point to a proatherogenic role for CRP.2–6 Several groups have examined the processing of CRP by Fcgamma receptors on leukocytes, and knockout mice lacking all 3 Fcgamma receptors, CD16, CD32, and CD64, fail to bind CRP.8–11,20–22 However, there is no report to date on how CRP is processed by ECs and mediates its biological effects. In this report, we demonstrate using 2 strategies, flow cytometry and fluorescence microscopy, that human aortic ECs express CD32 and CD64, which mediate the biological effects of CRP in these cells.
We show a strong dose-dependent saturable binding curve for CRP with a Kd of 88 nM. Previously, CRP binding to K-562 cells (erythroleukemia cell line), which have only CD32, was reported to have a Kd of 38 nM, and the Kd for CD32-transfected COS cells was 66 nM.10 There are few reports of Fcgamma receptors on ECs. Using immunohistochemistry on frozen tissue samples, CD32 antigens were expressed on placental villous capillary endothelium.14 CD16 was not expressed on umbilical vein or arterial endothelium. However, they failed to observe any antibody positivity for umbilical cord arterial and venous endothelium. Also, by RT-PCR and Southern blotting, CD32 has been shown to be expressed in dermal microvascular ECs, whereas CD16 and CD64 are not.13 CD32 receptors have been shown to be upregulated with phorbol esters and interferon gamma in monocyte cell lines.23 In this study, we report saturable uptake of CRP in HAECs via CD32 and CD64. Importantly, binding was inhibited by 10x cold CRP and by preincubation with antibodies to CD32 and CD64 but not by antibodies to CD16. Furthermore, there was no additive effect of antibodies to CD32 and CD64 with regards to inhibition of maximal binding. This may be because these receptors exhibit positive cooperativity in HAECs accounting for similar inhibition in maximal binding of CRP or because of antibody–antibody interaction preventing optimum binding of the antibodies to their respective receptors.
Using fluorescence microscopy we confirmed that at 4°C, CRP was bound at the cell surface; furthermore, both CD32 and CD64 were also evident. At 37°C, CRP was internalized and colocalized mainly with CD32. A recent report showed by confocal microscopy that CD32 receptors are weakly expressed in ECs and upregulated by incubation with tumor necrosis factor (TNF) and CRP but not TNF alone.24 Furthermore, as seen in this study, they showed perinuclear cytoplasmic localization of CD32. The presence of Fcgamma receptors on human ECs and their upregulation with cytokines has been previously reported by Pan et al,25 who showed a dose-dependent increase in surface CD32 expression by fluorescent and confocal microscopy. They also reported that that CD32 and CD64 expression in native ECs was low and that it was upregulated by cytokines. In addition, Western blotting for CD32 revealed a band that was increased after incubation with CRP (data not shown), providing further evidence for the presence of CD32 on HAECs.
Ligand-receptor engagement results in orchestration of biological activity. We showed that incubation of HAECs with monoclonal antibodies to CD32 and CD64 before addition of CRP markedly reversed the proatherogenic effects of CRP on prostacyclin synthase and IL-8. Similar results were observed with ICAM-1, VCAM-1, cGMP, and PAI-1. Blocking antibodies to CD32 and CD64 were able to reverse the biological effects of CRP on ECs, demonstrating that CRP indeed mediates its proatherogenic effects by binding to CD32 and CD64 on HAECs. Previously, incubation of monocytes with antibodies to CD32 have been shown to attenuate the CRP-induced increase in CD11b and subsequent adhesion to ECs.26 Williams et al27 showed that CRP-stimulated matrix metalloproteinase (MMP)-1 expression by U937 cells could be blocked with antibodies to CD32. Also, in THP-1 cells, CRP-stimulated CCR-2 expression was abrogated in presence of antibodies to CD64.28 We have shown previously that preincubation with CD32 antibodies reverses the upregulation of PAI-1 in HAECs with CRP. Recently, Li et al29 showed that CRP enhances LOX-1 expression in HAECs and that this effect was reversed with antibodies to CD32 and CD64, with the combination being more effective. The present study is the first comprehensive report demonstrating CRP binding and internalization in HAECs using flow cytometry and confocal microscopy. Both CD32 and CD64 inhibited CRP binding to HAECs. In addition, we show colocalization of CRP with both CD32 and CD64. Most significantly, the biological effects of CRP were prevented by antibodies to CD32 and CD64, supporting the above preliminary observations with respect to CRP inducing PAI-1 and LOX-1 via Fcgamma receptors in HAECs. Furthermore, the Syk inhibitor piceatannol was able to reverse the upregulation of IL-8 by CRP. It has been previously shown that signaling through CD32 requires activation of Syk. Although CRP could trigger tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of CD32 and Syk kinase and thereby upregulate IL-8 release, these possibilities will be explored in future studies. Alternatively, the decrease in prostacyclin with CRP is probably mediated through induction of ROS, upregulation iNOS, and nitration of prostaglandin synthase.30
Thus, we make the novel observation that CRP binds to Fcgamma receptors, mainly CD32, and also CD64 on HAECs to mediate its biological activity. Future studies will attempt to define the subtypes of the Fcgamma receptors that mediate the biological effects of CRP, elucidate the signaling pathways of CD32 and CD64 activation by CRP, and examine CRP uptake by other cells in the vasculature such as the smooth muscle cells and cholesterol-loaded macrophages.
Acknowledgments
Grant support from the National Institutes of Health (NIH K24 AT00596, NIH RO1 HL74360) is gratefully acknowledged. We thank Bryce Autret and Dana Ciobanu for technical assistance.
References
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Devaraj S, Kumaresan PR, Jialal I. Effect of C-reactive protein on chemokine expression in human aortic endothelial cells. J Mol Cell Cardiol. 2004; 36: 405–410.
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Correspondence to Ishwarlal Jialal, MD, PhD, Director of the Laboratory for Atherosclerosis and Metabolic Research, 4635 II Avenue, Res 1 Bldg, Rm 3000, Sacramento, CA 95817. E-mail ishwarlal.jialal@ucdmc.ucdavis.edu
Abstract
Objective— In addition to being a cardiovascular risk marker, recent studies support a role for CRP in atherothrombosis. Several investigators have reported that CRP binds to Fcgamma receptors on leukocytes. The aim of the study is to determine the processing of CRP by human aortic endothelial cells (HAECs).
Methods and Results— Binding studies were performed by incubation of HAECs with biotinylated CRP (B-CRP, 25 to 200 μg/mL) for 30 to 180 minutes at 4°C. B-CRP binding was quantitated using streptavidin-fluorescein isothiocyanate followed by flow cytometry. Saturable binding of CRP was obtained at 60 minutes with a CRP concentration between 100 to 150 μg/mL and Kd of 88 nM. CRP binding was inhibited by 10x cold CRP (58%). CRP (100 μg/mL) significantly upregulated surface expression of Fcgamma receptors, CD32, as well as CD64 on HAECs (P<0.01). Also, preincubation with anti-CD32 and CD64 antibodies significantly inhibited maximal binding of CRP to HAECs 64% and 30%, respectively, whereas antibodies to CD16 had no effect. Internalization of CRP, as determined by loss of surface expression, was 50%. Also, binding and internalization of biotinylated CRP was confirmed by confocal microscopy and CRP colocalized with CD32 and CD64. Most importantly, the increase in interleukin-8, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1 and the decrease in eNOS and prostacyclin induced by CRP was abrogated with antibodies to CD32 and CD64.
Conclusions— We demonstrate that CRP mediates its biological effects on HAECs via binding and internalization through Fcgamma receptors, CD32 and CD64.
In addition to being a risk marker, CRP exerts atherothrombotic effects in endothelial cells. In this study, using flow cytometry and fluorescence microscopy, we show that CRP binds to CD32 and CD64 on HAECs, is internalized, and exerts its biological effects. Antibodies to CD32 and CD64 abrogated the biological effects of CRP, whereas antibodies to CD16 had no effect.
Key Words: inflammation ? C-reactive protein ? Fcgamma receptors ? endothelial cells ? atherothrombosis
Introduction
Inflammation is pivotal in all stages of atherosclerosis.1 Numerous prospective studies have confirmed that high levels of C-reactive protein (CRP), the prototypic marker of inflammation, predicts cardiovascular events and is a risk marker.2,3 Also recent studies support a role for CRP in atherothrombosis.4–6 To date, it has been shown that in monocytes, CRP induces the production of inflammatory cytokines, promotes monocyte chemotaxis, uptake of oxidized LDL, and tissue factor expression. In endothelial cells (ECs), CRP increases the expression of cell adhesion molecules, monocyte-chemotactic protein-1 and endothelin-1 (ET-1), interleukin (IL)-8, and plasminogen activator inhibitor (PAI)-1 and decreases eNOS expression and activity and prostacyclin release.4–6 In smooth muscle cells (SMCs), CRP has been shown to activate angiotensin-1 type receptors.5
There are at least 3 types of human Fcgamma receptors, the high affinity receptor CD64 and the 2 low affinity receptors, CD32 and CD16.7 Several investigators have reported the presence of receptors for CRP on mononuclear cells and neutrophils. Tebo et al8 and Zahedi et al9 concluded that 2 receptors for CRP were present on mononuclear cells in humans. It was noted that there was some possible association with Fc receptors (FcRs) as IgG could inhibit binding. Bharadwaj et al10 have reported that the major receptor for CRP on leukocytes is Fcgamma receptor II (CD32). In transfected COS cells, CRP has been shown to bind to Fcgamma receptor 1 (CD64).11 However, to date, no reports have documented a CRP receptor on ECs. Data available so far favor CD32 and CD64 as the most likely candidates because all EC subtypes investigated thus far are negative or weakly positive for CD16.12 CD32 has been localized to placental and dermal microvascular ECs as well as liver sinusoidal ECs.13,14 Immune complexes from vasculitis patients bind to CD32 of ECs.15In addition, we have shown previously in a preliminary report that incubation of human aortic ECs (HAECs) with anti-CD32 antibodies significantly reversed the stimulation of PAI-1 by CRP.16 In this study, we demonstrate that the major receptors for CRP in ECs are CD32 and CD64, and they orchestrate its biological effects.
Materials and Methods
CRP was procured from 3 different sources: CRP purified from plasma (Sigma Chemicals), recombinant human CRP (Calbiochem), and purified CRP from human pleural fluid in the laboratory of Dr T.W. Du Clos. All CRP preparations were purified under sterile conditions using Endotoxin-removal columns (Pierce Biochemicals) and used only if the concentration of endotoxin was <0.125 EU/mL.17 All cell culture media were endotoxin-free. Furthermore, Western blotting using HRP-conjugated IgG antibodies failed to reveal any contamination of purified CRP with IgG. Antibodies to CD16 (clone 3G8-IgG1) were purchased from Pharmingen, Serotec, CD32 (FLI8.26-IgG2b and AT10-IgG1) were purchased from Pharmingen, Santa Cruz, and CD64 (10.1, IgG1) was purchased from Pharmingen, Santa Cruz, respectively, along with their respective isotype controls.
HAECs were cultured in EGM-2MV media (Biowhittaker), and confluent cultures below the 6th passage were used for all experiments.17
CRP Binding
CRP was biotinylated using reagents from Pierce (EZ Sulfo NHS biotinylation reagents) at a molar ratio of biotin to CRP of 20 to 1. Biotinylation was checked using EZ biotin quantitation reagents from Pierce Biotechnologies. Biotinylated CRP (25 to 200 μg/mL) was then added to confluent human aortic ECs (1x106 in PAB–PBS with 1 mmol/L CaCl2/MgCl2 containing 1 mg/mL BSA, azide 10 mmol/L, HEPES 20 mmol/L) at 4°C for a time course of 180 minutes. CRP binding was quantitated by tagging the biotinylated CRP that is bound to the cell surface of HAECs using streptavidin–FITC followed by flow cytometry. Nonspecific binding was assessed using a 10- to 20-fold excess cold CRP. Kinetics of binding was determined using Graph Pad Prism 4 Software. Inhibition studies were undertaken with monoclonal antibodies to CD32, CD64, and CD16 (as a negative control). Unlabeled CRP with streptavidin–FITC was also used as negative control. CRP internalization was measured after incubating at 4°C to attain maximum binding. The media was aspirated, and fresh media without azide was added to the cells. The cells were then incubated at 37°C for a period of 30 to 180 minutes to determine internalization of CRP at 37°C. Furthermore, cells were preincubated with monoclonal antibodies to CD16, CD32, and CD64 (20 μg/mL) to saturate the receptors to see whether this would prevent internalization of CRP to HAECs/HCAECs.
Cell Surface Expression of Fcgamma Receptors by Flow Cytometry
Cell surface expression of FcR was assessed by incubating 1x105 cells in PBS with 25 to 200 μg/mL CRP for 30 to 180 minutes at 4°C in PBS with calcium and magnesium with 10 mmol/L azide which blocks internalization. Thereafter, specific fluorochrome-labeled antibodies to CD16, CD32, and CD64 were added and incubated for another 30 minutes. After detachment with PBS–EDTA, cells were analyzed by flow cytometry to determine the abundance of each of these receptors. Isotype controls were used with each experiment and expressed as mfi/1x105 cells.
Colocalization of Fcgamma Receptors with CRP by Fluorescence Microscopy
For immunofluorescence studies, HAECs were cultured on cover slips and incubated at 4°C for binding studies and at 37°C in PBS for 1 hour with 100 μg of biotinylated CRP for internalization. Cells were washed with PBS, fixed with 3.5% paraformaldehyde, and permeabilized with 0.5% Triton-X 100 in PBS and then incubated with streptavidin–FITC. Cells were washed in PBS and then observed under a confocal microscope. Colocalization of FITC–CRP and specific fluorochrome-labeled CD32/CD64 was also examined by confocal microscopy. Control incubations included unlabeled CRP with streptavidin–FITC.
Biological Effects of CRP
To determine whether CRP binding to the FcR orchestrates its proinflammatory prothrombotic biological effects (inhibition of prostacyclin synthase and eNOS and stimulation of IL-8, ICAM-1, and VCAM-1), HAECs were preincubated with excess monoclonal antibodies to CD16, CD32, and CD64 alone and in combination followed by the addition of different concentrations of CRP 0 to 50 μg/mL. Biological effects of CRP were examined as described previously.17,18 We tested whether the biological effects of CRP can be blocked using piceatannol (a Syk kinase inhibitor, 50 μmol/L for 30 minutes).
Statistical Analyses
All data are expressed as mean±SD. Comparison of the biological effects of CRP was assessed by paired t tests, and significance was set at P<0.05.
Results
Binding studies with CRP at doses of 25 to 200 μg/mL showed that CRP bound in a dose-dependent saturable fashion (Figure 1a). Saturable binding of CRP was obtained at 60 minutes with a predicted CRP concentration of 104 μg/mL, and a Kd of 88.2 nM was computed. Time course experiments conducted for 30 to 180 minutes revealed maximal binding at 60 minutes and this was used for all further experiments.
Figure 1. a, Binding curve of C-reactive protein on HAECs. HAECs were incubated with different Biotinylated-CRP (25 to 250 μg/mL) in PAB at 4°C for 60 minutes followed by addition of streptavidin FITC as described in Methods. Results are mean of 7 different experiments. Kd was plotted using Graph Pad Prism software. b, Inhibition of biotinylated CRP binding to HAECs. HAECs were preincubated with antibodies to CD32, CD64, CD16, or the combination or 10x cold CRP for 1 hour before addition of Biotinylated CRP (100 μg/mL), and inhibition of maximal binding of B-CRP was assessed by flow cytometry as described in Methods. Results are mean of 6 different experiments.
CRP binding was inhibited by 10x cold CRP (58%) (Figure 1b). Furthermore, binding of CRP to HAECs was significantly inhibited when HAECs were preincubated for 1 hour with antibodies to CD32 (64%) and CD64 (30%) as well as the combination (59%) before addition of Biotinylated CRP (100 μg/mL). The combination of antibodies to CD32 and CD64 was not additive to either alone. Preincubation with anti-CD16 antibody failed to have any significant effect on binding (Figure 1b).
Internalization of CRP was assessed by flow cytometry as a decrease in surface expression and was performed with CRP (100 μg/mL) for a period of 240 minutes at 37°C. As seen in Figure 2, maximal internalization of CRP was obtained by 60 minutes, although only 50% of the CRP appeared to be internalized. Preincubation with antibodies to CD32 and CD64 reduced CRP internalization by 24% and 29%, respectively.
Figure 2. Internalization of CRP. Internalization of B-CRP was examined by flow cytometry as loss of surface expression after incubation of B-CRP for a period of 30 to 240 minutes at 37°C and addition of streptavidin FITC as described in Methods. Results are mean of 5 different experiments.
We then examined surface expression of Fcgamma receptors on HAECs in presence and absence of CRP. Although there was minimal surface expression of CD16, HAECs expressed CD32 and CD64, both of which were increased 5-fold and 2-fold, respectively, with CRP (Figure 3).
Figure 3. Expression of Fcgamma receptors on HAECs. HAECs were incubated in absence and presence of CRP (100 μg/mL) and cells (100 000) were analyzed by flow cytometry with respective isotype controls as described in Methods. Results are mean of 5 different experiments. *P<0.001 compared with control
To confirm the presence of the receptors, we used fluorescence microscopy. When cells were incubated at 4°C, fluorescent staining for CRP was observed on the cell surface, and the same cells were positive for CD32–PE fluorescence. Although there was weak fluorescent staining for CD64 on some cells, no fluorescent staining was observed for CD16 (Figure 4a). Furthermore, CRP internalization was observed with perinuclear staining at 37°C, with strong colocalization with CD32 (Figure 4b) and also with CD64 (Figure 4c).
Figure 4. a, Visualization of B-CRP, CD32, and CD64 by confocal microscopy. Cells were incubated for 60 minutes at 4°C for maximal binding along with CRP (B-CRP followed by streptavidin-FITC), and expression of CD32 (PE) and CD64 (PE) were assessed. b and c, Intracellular localization of CRP (FITC), CD32 (PE, b), and CD64 (PE, c) after internalization with CRP (100 μg/mL) for 120 minutes at 37°C by fluorescence microscopy.
Finally, to determine whether CRP binding to CD32 and CD64 orchestrates its proinflammatory prothrombotic biological effects in HAECs, cells were preincubated with excess monoclonal antibodies to CD16, CD32, and CD64 followed by the addition of CRP 50 μg/mL. Thereafter, biological effects of CRP were tested. We have previously shown that the most significant effects of CRP are increased secretion of IL-8 and inhibition of prostacyclin release in HAECs.17,18 As seen from Figure 5a and 5b, the effects of CRP 50 μg/mL were reversed with antibodies to CD32 and CD64 but not by CD16 antibodies. Also, we tested whether the biological effects of CRP on IL-8 can be blocked using piceatannol, a Syk kinase inhibitor, because signaling through Fcgamma receptors requires activation of Syk kinase.19 The Syk inhibitor, through which Fcgamma receptors signal, was able to reverse the stimulatory effect of CRP on IL-8 (C-6.8±1.9 nmol/mg pr; CRP 50 μg/mL –16.4±2.6 nmol/mg pr; CRP50 μg/mL+Piceatannol –10.7±1.4 nmol/mg pr). Also, antibodies to CD32 and CD64 inhibited the stimulatory effect of CRP on secreted ICAM-1, VCAM-1, and eNOS as evaluated by cGMP assay (Table).
Figure 5. a and b, Effect of antibodies to Fcgamma receptors on CRP-induced IL-8 release (a) and CRP-induced inhibition of prostacyclin (b). Results are mean of 6 different experiments *P<0.001 compared with control, #P<0.05 compared with CRP.
Effect of Antibodies to CD32 and CD64 on the Biological Effects of CRP in HAEC
Discussion
In addition to being a risk marker, several recent lines of evidence point to a proatherogenic role for CRP.2–6 Several groups have examined the processing of CRP by Fcgamma receptors on leukocytes, and knockout mice lacking all 3 Fcgamma receptors, CD16, CD32, and CD64, fail to bind CRP.8–11,20–22 However, there is no report to date on how CRP is processed by ECs and mediates its biological effects. In this report, we demonstrate using 2 strategies, flow cytometry and fluorescence microscopy, that human aortic ECs express CD32 and CD64, which mediate the biological effects of CRP in these cells.
We show a strong dose-dependent saturable binding curve for CRP with a Kd of 88 nM. Previously, CRP binding to K-562 cells (erythroleukemia cell line), which have only CD32, was reported to have a Kd of 38 nM, and the Kd for CD32-transfected COS cells was 66 nM.10 There are few reports of Fcgamma receptors on ECs. Using immunohistochemistry on frozen tissue samples, CD32 antigens were expressed on placental villous capillary endothelium.14 CD16 was not expressed on umbilical vein or arterial endothelium. However, they failed to observe any antibody positivity for umbilical cord arterial and venous endothelium. Also, by RT-PCR and Southern blotting, CD32 has been shown to be expressed in dermal microvascular ECs, whereas CD16 and CD64 are not.13 CD32 receptors have been shown to be upregulated with phorbol esters and interferon gamma in monocyte cell lines.23 In this study, we report saturable uptake of CRP in HAECs via CD32 and CD64. Importantly, binding was inhibited by 10x cold CRP and by preincubation with antibodies to CD32 and CD64 but not by antibodies to CD16. Furthermore, there was no additive effect of antibodies to CD32 and CD64 with regards to inhibition of maximal binding. This may be because these receptors exhibit positive cooperativity in HAECs accounting for similar inhibition in maximal binding of CRP or because of antibody–antibody interaction preventing optimum binding of the antibodies to their respective receptors.
Using fluorescence microscopy we confirmed that at 4°C, CRP was bound at the cell surface; furthermore, both CD32 and CD64 were also evident. At 37°C, CRP was internalized and colocalized mainly with CD32. A recent report showed by confocal microscopy that CD32 receptors are weakly expressed in ECs and upregulated by incubation with tumor necrosis factor (TNF) and CRP but not TNF alone.24 Furthermore, as seen in this study, they showed perinuclear cytoplasmic localization of CD32. The presence of Fcgamma receptors on human ECs and their upregulation with cytokines has been previously reported by Pan et al,25 who showed a dose-dependent increase in surface CD32 expression by fluorescent and confocal microscopy. They also reported that that CD32 and CD64 expression in native ECs was low and that it was upregulated by cytokines. In addition, Western blotting for CD32 revealed a band that was increased after incubation with CRP (data not shown), providing further evidence for the presence of CD32 on HAECs.
Ligand-receptor engagement results in orchestration of biological activity. We showed that incubation of HAECs with monoclonal antibodies to CD32 and CD64 before addition of CRP markedly reversed the proatherogenic effects of CRP on prostacyclin synthase and IL-8. Similar results were observed with ICAM-1, VCAM-1, cGMP, and PAI-1. Blocking antibodies to CD32 and CD64 were able to reverse the biological effects of CRP on ECs, demonstrating that CRP indeed mediates its proatherogenic effects by binding to CD32 and CD64 on HAECs. Previously, incubation of monocytes with antibodies to CD32 have been shown to attenuate the CRP-induced increase in CD11b and subsequent adhesion to ECs.26 Williams et al27 showed that CRP-stimulated matrix metalloproteinase (MMP)-1 expression by U937 cells could be blocked with antibodies to CD32. Also, in THP-1 cells, CRP-stimulated CCR-2 expression was abrogated in presence of antibodies to CD64.28 We have shown previously that preincubation with CD32 antibodies reverses the upregulation of PAI-1 in HAECs with CRP. Recently, Li et al29 showed that CRP enhances LOX-1 expression in HAECs and that this effect was reversed with antibodies to CD32 and CD64, with the combination being more effective. The present study is the first comprehensive report demonstrating CRP binding and internalization in HAECs using flow cytometry and confocal microscopy. Both CD32 and CD64 inhibited CRP binding to HAECs. In addition, we show colocalization of CRP with both CD32 and CD64. Most significantly, the biological effects of CRP were prevented by antibodies to CD32 and CD64, supporting the above preliminary observations with respect to CRP inducing PAI-1 and LOX-1 via Fcgamma receptors in HAECs. Furthermore, the Syk inhibitor piceatannol was able to reverse the upregulation of IL-8 by CRP. It has been previously shown that signaling through CD32 requires activation of Syk. Although CRP could trigger tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of CD32 and Syk kinase and thereby upregulate IL-8 release, these possibilities will be explored in future studies. Alternatively, the decrease in prostacyclin with CRP is probably mediated through induction of ROS, upregulation iNOS, and nitration of prostaglandin synthase.30
Thus, we make the novel observation that CRP binds to Fcgamma receptors, mainly CD32, and also CD64 on HAECs to mediate its biological activity. Future studies will attempt to define the subtypes of the Fcgamma receptors that mediate the biological effects of CRP, elucidate the signaling pathways of CD32 and CD64 activation by CRP, and examine CRP uptake by other cells in the vasculature such as the smooth muscle cells and cholesterol-loaded macrophages.
Acknowledgments
Grant support from the National Institutes of Health (NIH K24 AT00596, NIH RO1 HL74360) is gratefully acknowledged. We thank Bryce Autret and Dana Ciobanu for technical assistance.
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