Human C-Reactive Protein Activates Monocyte-Derived Dendritic Cells and Induces Dendritic Cell-Mediated T-Cell Activation
Objective— Recent studies proposed a pathogenic role for C-reactive protein (CRP), an independent predictor of cardiovascular disease (CVD), in atherosclerosis. Therefore, we tested whether CRP may modulate dendritic cell (DC) function, because these professional antigen-presenting cells have been implicated in atherogenesis.
Methods and Results— Human monocyte-derived immature DCs were cultured with human CRP (0 to 60 μg/mL) for 24 hours. Thereafter, activation markers were measured by flow-cytometry and DCs were cocultured with CFSE-labeled lymphocytes to measure T-cell proliferation and interferon (IFN)-γ secretion after 8 days. Exposure to 60 μg/mL CRP (n=5) induced an activated cell morphology and significant (CD40 increase MFI 5.23±0.28, P<0.01 paired t test; CD80 6.18±0.51, P<0.01) to modest (CD83 1.38±0.17, P<0.05, CCR7 1.60±0.29, P=0.05) upregulation of DC activation markers. The expression of CD86 and HLA-DR was high, but not affected. T-lymphocytes incubated with CRP-pulsed DCs displayed increased IFN-γ secretion and proliferation (P<0.001). DC activation was concentration-dependent and detected from 2 μg/mL CRP; the maximum effect was equivalent to that seen with 0.1 μg/mL lipopolysaccharide (LPS). Polymyxin B abolished the LPS response, without influencing CRP effects. Finally, immunohistochemistry could demonstrate DC/CRP colocalization in human atherosclerotic lesions.
Conclusions— These findings suggest that CRP in plaques or found circulating in CVD patients can influence DC function during atherogenesis.
The recruitment of immune cells, macrophages, and T-cells plays a key role in the initiation and progression of atherosclerosis. Later on, activation of these inflammatory cells may elicit plaque rupture resulting in acute cardiovascular events.1 It has been suggested that dendritic cells (DCs) mediate T-cell activation in atherosclerosis.2 DCs are potent antigen-presenting cells required for initiation of innate and adaptive immune responses.3 Indeed some proatherogenic factors such as oxidized low density lipoproteins4,5 and nicotine6 were reported to induce DC activation resulting in T-cell stimulatory capacities. Recently we7 and others8 demonstrated that numbers of DC precursors were lower in the circulation of patients with coronary artery disease in comparison to healthy subjects. Interestingly, the numbers of circulating DC precursors showed a negative relationship with serum C-reactive protein (CRP) levels.7
Serum CRP levels slightly above normal have been put forward as indicator of mild inflammation associated with cardiovascular disease (CVD) and an independent predictor of CVD.9,10 CRP is the prototypical acute phase protein in humans, rising rapidly in response to inflammation.11–13 It is a member of the pentraxin family of proteins and consists of a cyclic arrangement of 5 identical 23-kDa subunits.11–13 CRP is mainly expressed by hepatocytes, but can be synthesized in other tissues,11–13 and is often abundantly present in human atherosclerotic lesions.14,15 The major function of CRP includes its ability to bind ligands exposed on damaged membranes or bacteria (opsonization) for activation of the complement pathway via binding to C1q or for enhancement of phagocytosis.11–13 As a well-known ligand of phosphorylcholine residues, CRP binds avidly to oxidized low-density lipoproteins16 and may induce foam cell formation in atherosclerosis.17 Indeed in atherosclerotic lesions colocalization of CRP with apolipoprotein B was demonstrated14 and foam cells underneath the endothelium were shown to stain positive for CRP.15 By binding to Fcγ receptors I and II (FcγRI/CD64 and FcγRII/CD32)11,13,18 CRP facilitates the uptake of opsonized particles.17
There is accumulating evidence that CRP is not only a risk marker of CVD, but actively contributes to atherosclerosis. It has been reported that CRP induces endothelial cell activation and dysfunction.17,19 Other in vitro studies show effects on vascular smooth muscle cells, monocytes, and macrophages suggesting that CRP may promote atherosclerotic plaque formation, plaque maturation, plaque destabilization, and eventually plaque rupture, as reviewed by Verma et al.17
Very recent in vitro studies showed that CRP attenuates the very early steps of DC differentiation, mainly through interaction with FcγRII.20 We addressed the hypothesis that CRP may affect the function of DCs after their differentiation by exposing human monocyte-derived DCs to CRP for 6 to 48 hours. Thereafter, the expression of activation markers was studied by means of flow cytometry; their effects on autologous T-cells were investigated in a mixed lymphocyte reaction (MLR). Finally, to examine whether in situ interactions between CRP and DCs in atherosclerotic plaques are possible, the presence of CRP and DC markers S100 and fascin2 was examined in human carotid arteries.
Materials and Methods
Chemicals and Reagents
CRP purified protein (98% purity by SDS-PAGE according to manufacturer) was obtained from Chemicon International Inc (Biognost, Belgium). Its purity and pentameric conformation was confirmed with Native gel electrophoresis (NativePage Novex Bis-Tris gel 4% to 16%, Invitrogen), followed by Coomassie and silver staining (please refer to http://atvb.ahajournals.org, supplemental Figure IA and IB). For other reagents please refer to supplemental materials.
Preparation of DCs
Human DCs were prepared as previously described.21 Briefly, peripheral blood mononuclear cells were isolated by density gradient centrifugation (Lymphocyte separation medium, ICN Biomedicals) of fresh buffy coats from healthy blood donors (Blood Transfusion Centre, Antwerp). From these cells highly purified monocytes were isolated using magnetic CD14 microbeads (Miltenyi Biotec, purity >90%). CD14+ monocytes were cultured at 37°C in a humidified atmosphere supplemented with 5% CO2 in 6-well plates (2 to 3×106 cells) for 6 days in 3 mL complete IMDM medium containing 2.5% human serum, 20 ng/mL granulocyte/macrophage colony stimulating factor (CSF) and 25 ng/mL interleukin (IL)-4. GM-CSF and IL-4 were additionally added to the cell cultures on day three. The remaining peripheral blood lymphocytes (PBL) were cryopreserved.22
Pulsation of DCs
On day 6 of culture, monocyte-derived DCs were collected, plated in IMDM with 2.5% serum (106 cells/mL, 24 well plates) and stimulated with CRP (0.6 to 60 μg/mL), or LPS (0.1 μg/mL) as positive control. Negative control samples contained control solvent (10 mmol/L Tris-HCl, 140 mmol/L NaCl, pH 8.0 with 0.1% sodium azide). Stimulation of DCs was studied after 24 hours unless mentioned otherwise. To test the specificity of the CRP response, moAbs (20 μg/mL) against FcγRII/CD32 and FcγRIIa/CD32a were added to the cell culture. Moreover, the LPS binding antibiotic polymyxin B was added to the cell cultures unless they were exposed to LPS.
Expression of FcγRI and FcγRII by immature DCs was analyzed with CD64-fluorescein isothiocyanate (FITC) and CD32-PE, respectively. After incubation of immature DCs with CRP, LPS, or control solvent, morphology of DCs was microscopically analyzed. For immunophenotyping fluorescently labeled mouse anti-human moAbs were used: CD80-FITC, HLA-DR-PerCP, CD83-FITC, CD40-PE, CD14-FITC, and CCR7-PE. Cell fluorescence intensities were compared with isotype-matched control antibodies IgG1-FITC, IgG1-PE, and IgG2a-PE. 10 000 events were measured by flow cytometry (FACScan, Becton Dickinson), and data were analyzed with WinMDI software.
Mixed Lymphocyte Reaction (MLR)
After 24-hour pulsation with 60 μg/mL CRP, DCs were washed and plated in fresh IMDM supplemented with 5% human serum at 105 cells per well in 48-well plates with 9×105 autologous PBL labeled with CFSE. With each cell division CFSE fluorescence intensity of the cells is reduced.23 After 8 days of coculture, cells were harvested, labeled with mouse anti-human CD3-PE and propidium iodide; CFSE fluorescence of propidium iodide -negative and CD3-positive T-cells was determined by fluorescence-activated-cell sorter (FACS). The supernatants were collected for measurement of IFN-γ by ELISA.
For Immunohistochemistry please refer to supplemental materials.
All results were expressed as mean±SEM, n represents the number of buffy coats. Statistical analyses were performed by paired Student t test or Repeated Measures ANOVA combined with Dunnett Multiple Comparison Test, using GraphPad Prism5. Variables showing heterogeneity of variances were logarithmically transformed. P<0.05 was considered statistically significant.
DC Morphology and Maturation
After 24-hour incubation with CRP (60 μg/mL), DCs (n=5) showed an activated phenotype consisting of multiple enlarged clusters of elongated cell morphology compared with DCs incubated with control solvent containing 0.1% sodium azide (Figure 1). CRP induced DC activation as shown by the 2- to 4-fold upregulation of the median fluorescence intensity (MFI) of CD40, CD80, CD83, and CCR7 when compared with control (Figure 2). This was also reflected by the significant increase of cells positive for these markers: CD40 (control 3±2%, CRP 33±12%; P<0.05), CD80 (control 2±1%, CRP 45±3%; P<0.01), CD83 (control 4±1%, CRP 12±4%; P=0.07), and CCR7 (control 2±1%, CRP 10±3%; P<0.05). In contrast, expression for HLA-DR and CD86 was strong, but neither MFI (Figure 2) nor percentage of positive cells (not shown) was influenced by CRP.
The maturation of CRP-stimulated DCs was equivalent to activation with 0.1 μg/mL LPS (Figure 3). However, the effects of LPS were completely suppressed by polymyxin B, whereas the effects of CRP were not influenced by polymyxin B (Figure 3A). Therefore in all subsequent experiments the LPS-binding antibiotic was added to avoid interferences attributable to possible contamination with endotoxins. Because CD40 and CD80 showed the most robust upregulation, they were selected for further study.
Effect of CRP Concentration and Specificity
Upregulation of CD80 and CD40 by DCs was evident from 24 hours and increased further after 48-hour incubation with 60 μg/mL (please see supplemental Figure II). However, because of increasing variability for CD40, 24 hours was selected as time point. CD80 and CD40 expression gradually increased with rising CRP concentrations (Figure 4), starting at 2 μg/mL and 6 μg/mL CRP, respectively. The maximum expression was reached at 60 (CD80) or 20 μg/mL CRP (CD40) and was equivalent to that seen with 0.1 μg/mL LPS. Staining with propidium iodide or annexin V showed low rates of cell death that were not different between DCs treated with increasing CRP concentrations, LPS or control buffer (data not shown).
Consistent with previous reports,24,25 FcγRII/CD32 was expressed by most (79±6%, n=9) immature monocyte-derived DCs, whereas expression of FcγRI/CD64 was less common (10±3%, n=9). Therefore, specificity of the CRP effect was tested by blocking the FcγRII/CD32 on immature DCs. Preincubation of DCs with moAbs (20 μg/mL) against the FcγRII or IIa reduced CD80 MFI (Figure 3B), and the numbers of CD80-positive cells with 48±8 and 59±9%, respectively, compared with cells exposed to CRP in the absence of blockers. The antibodies themselves were without effects (Figure 3B).
CRP Augments the Ability of DCs to Stimulate T-Cells
The DC activation by CRP was further evaluated by analysis of their ability to stimulate autologous T-cell proliferation. DCs incubated with control buffer or CRP for 24 hours were thoroughly washed and then added to T-cells for 8 days. Control DCs hardly induced T-cell proliferation. In contrast, prepulsation of DCs with CRP evoked a strong proliferative T-cell response, as indicated by the reduced CFSE fluorescence (Figure 5). Furthermore, T-cell activation was confirmed by the significant increase of IFN-γ in the supernatant, compared with T-cells incubated with control DCs.
In Vivo Location of CRP and DCs in Human Arteries
To test whether CRP could potentially interact with DCs in atherosclerotic lesions, we performed double staining for CRP and DCs (Figure 6). All specimens contained S100-positive DCs, as well as diffuse CRP deposits of variable size and density in the intima beneath the endothelium or close to the medial border. Figure 6A through 6D demonstrates regions with colocalization of CRP and S100-positive DCs, but also areas where the markers did not overlap. Colocalization of fascin and S100 (Figure 6E) illustrates specificity of the S100 antibody for DCs. Also CD3-positive T-cells and CRP deposits could be found together.
Recently we and others reported a decrease in circulating DCs in patients with atherosclerotic coronary artery disease.7,8 This decline in blood DCs could have been caused by an active recruitment of DCs into the vascular wall, possibly in response to atherogenic factors. Because the blood DC count was inversely correlated with serum CRP levels, we addressed the hypothesis that CRP could be an activator of DCs.
CRP Activates DCs In Vitro
The present study shows that CRP can indeed activate monocyte-derived DCs in vitro. This was evidenced by an increased expression of DC maturation markers. The costimulatory molecules CD80 and CD40 were significantly, the lymph node homing receptor CCR7 and the activation marker CD83 were modestly upregulated after 24-hour incubation with CRP. The maximum expression of all markers was equivalent to that seen with LPS. In contrast, the expression of HLA-DR and CD86 was high, but was not different between controls and CRP-pulsed DCs. The CRP effect was concentration-dependent and became apparent at 2 μg/mL. This is below the Kd for CRP dissociation from FCγRIIa (3.7 μmol/L ≈450 μg/mL)18 and the numbers of activated DC were significantly suppressed by moAbs against FCγRII. However, complete inhibition was not reached, and other mechanisms involved in CRP activation of DCs remain to be established.
Cell morphology pointed to DC activation as well: the CRP-stimulated DCs became more elongated and were arranged in cell clusters compared with nonstimulated DCs. In addition, the CRP-stimulated DCs acquired the capacity to activate T-cells, as indicated by the induction of cell proliferation and the secretion of IFN-γ.
Our results seem to be at variance with a recent report showing that CRP impairs early differentiation of monocytes to DCs, leading to inhibition of LPS-induced maturation, antigen uptake, and presentation abilities.20 Those CRP effects were also mediated by FcγRII/CD32, but were mainly seen at the earliest time points of DC differentiation when the cells showed high expression of FcγRIIa.20 There are 3 important distinctions between that report and the present study. In contrast to our and other24,25 observations, the immature DCs lost FcγRIIa expression during differentiation. Secondly, Zhang et al did not analyze the direct effects of CRP in the absence of LPS. Thirdly, they studied the effects of prolonged incubation (1 to 5 days) of CRP on monocyte-derived precursor DCs. Indeed, at late stages of DC differentiation CRP lost its inhibitory effect on LPS stimulation.20 In another study Tobiasova et al26 did not show an increase of CD83 and CD86 when DCs were exposed to CRP (5 μg/mL). It should be noted that we also failed to see upregulation of CD86. Though CD83 is a specific DC marker, its upregulation was also less pronounced compared with CD80 or CD40 in our hands. Moreover differences between the experimental protocol (serum free X-VIVO 15 medium, another source of CRP, allogeneic T-cells in MLR, … ) could also have led to different observations. Yet, Tobiasova et al do mention a modest proliferative response in T cells that were cocultured with 5 μg/mL CRP-treated DCs.26
Several studies point to an antigen-driven T-cell response in atherosclerosis, most likely directed against antigens present in the culprit atherosclerotic plaques.27 As professional antigen-presenting and immune-regulating cells, DCs are therefore likely to play an important role in the development of atherosclerosis. Some proatherogenic factors that have already been shown to activate DCs and to induce DC-mediated T-cell activation are oxidized LDL,4,5 lysophosphatidylcholine,28 advanced glycation end products,29 and nicotine.6 Our in vitro data indicate that CRP could also behave as one of those stimuli. The interactions between DCs and CRP could occur systemically and locally. The latter possibility was examined by means of immunohistochemistry of human atherosclerotic plaques and is discussed first.
Local Interaction Between DCs and CRP in Human Arteries
Western blot proved the specificity of the primary antibody used for immunohistochemistry of CRP. CRP deposits were found in plaques close to the luminal or medial border. The selective staining of specific regions confirms results of others, who also found that CRP protein was invariably present in atherosclerotic lesions, regardless of lesion type (early versus advanced) or nature (foam cell–rich versus extracellular matrix rich).14,30 The CRP depositions could be attributable to infiltration from the blood,14,15 or local biosynthesis in the plaque,31 in response to inflammatory cytokines.17 Whatever mechanism, it is clear from the immunohistochemical data that microdomains are created in the plaque with high CRP concentrations, far in excess of blood levels.
As described by others2 we detected S100- and fascin-positive DCs in the intima of human arteries of both early and advanced atherosclerotic plaques. Because the arterial intima is not innervated and the antibody does not cross-react with other vascular cells, S100 is a convenient marker for vascular DCs at this location.2 DCs in normal arteries presumably act as sentinels to detect noxious agents in the vessel wall. A sentinel network of vascular DCs may sample and process exogenous and endogenous antigens that can trigger an inflammatory nidus within the arterial wall.32 Increased vascular DC frequency is seen as plaques develop, and the proportion of activated DCs increases as well.2 In vulnerable plaques, mature DCs are detected in clusters with T-cells, mostly in plaque shoulders.33
The present study documented for the first time colocalization of CRP and DCs by means of immunohistochemical double staining. In analogy with a previous study of macrophages,14 this was not a universal phenomenon. Yet the double staining showed unequivocally that interactions between CRP and DCs can occur locally in arteries. The detection of DC activation markers (eg, CD86, CD80, HLA-DR) would be interesting, but requires more extensive histological research since these markers are not exclusively expressed by DCs.
It remains to be established whether DCs are able to secrete CRP or to induce CRP production by other vascular cells.
Others had already proposed that the presence of CRP in plaques could recruit circulating monocytes.17,30 Indeed, it was described that CRP is chemotactic for monocytes, induces monocyte CCR2 expression, activates endothelial cells, and raises expression of their adhesion molecules.17,30 After infiltrating the plaque these monocytes can, depending on micro-environmental stimuli, differentiate into macrophages or DCs. The dual immunohistochemistry illustrated colocalization of DCs and CRP, but does not prove functional relationships. Yet, the in vitro data show that CRP could activate immature DCs locally in the plaque once they happen to be exposed to CRP.
Systemic Interaction Between DCs and CRP in the Circulation
Elevated CRP concentrations are seen in patients with risk factors such as smoking, diabetes, metabolic syndrome, hypertension, or renal failure and in acute coronary syndromes. In addition, increased levels of serum CRP may predict future cardiovascular events, independently of the presence of those risk factors.9,10 The recently reported decrease in dendritic precursor cell numbers in patients with severe coronary artery disease7,8 could therefore reflect their activation in response to circulating CRP, leading to an active recruitment into atherosclerotic lesions. Although circulating CRP levels under 10 μg/mL have historically been regarded as clinically insignificant, slightly elevated CRP plasma levels (1 to 3 μg/mL) have been associated with moderate, and 3 to 10 μg/mL with high risk of developing cardiovascular disease.9–12 The dose-response study showed that DC activation occurred from 2 μg/mL, which is in the range of above-mentioned concentrations. Moreover, the maximum activation occurred at concentrations (20 to 60 μg/mL) that are found in patients suffering from infectious or inflammatory diseases (10 to 200 μg/mL).11,12 In those pathological settings, elevated CRP concentrations in response to inflammation and infection may also provoke DC activation and subsequently induce adaptive immune responses that could accelerate the process of atherosclerosis.
Lately controversy has arisen as to whether CRP itself or confounding factors in commercially available preparations such as endotoxins or the preservative sodium azide exert proatherogenic effects.34,35 In the present study each experiment included buffer solution containing sodium azide at the same concentration as in the CRP preparation as a negative control and LPS as a positive control. Secondly, incubation of DCs with CRP in the presence of the LPS-binding antibiotic polymyxin B completely abolished the effects of a high concentration of LPS, whereas activation by CRP was not affected at all. Hence, it seems unlikely that contaminating endotoxins contributed to the DC activation. Thirdly the specificity was further indicated by the fact that the activation by CRP was at least partly receptor-mediated: the effects of CRP were suppressed by anti-CD32, a CRP receptor13,18 expressed on DCs.24,25 Finally, gel electrophoresis failed to detect contaminating proteins in the CRP preparation and confirmed its pentameric structure. Taken together, this implies a CRP-dependent mechanism for the DC activation and subsequent T-cell activation seen in the present study. Other recent studies reconfirm CRP as a proatherogenic protein, independent of contaminants.17,19,36,37
These findings indicate that increased CRP concentrations as found in the blood of patients with CVD or in atherosclerotic arteries can promote inflammatory responses in DCs. Moreover, CRP and DCs can be found together in human atherosclerotic plaques, pointing to the possibility for local interactions. Because atherosclerosis is generally accepted as an immune-driven process,1,2 these systemic or local interactions could contribute to an acceleration of atherosclerosis and its complications.
Sources of Funding
E.V.V. is supported by a UA-BOF fund. H.B. was supported by the Interuniversity Attraction Poles Programme – Belgian State – Federal Office for Scientific, Technical and Cultural Affairs, P5/02. V.F.I.V.T. is a postdoctoral fellow of the Fund for Scientific Research Flanders.
Original received February 6, 2007; final version accepted December 20, 2007.
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