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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2469-2475
Published online before print August 17, 2006, doi: 10.1161/01.ATV.0000241572.05292.fb
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2469.)
© 2006 American Heart Association, Inc.

Vascular Biology

C-Reactive Protein Decreases Interleukin-10 Secretion in Activated Human Monocyte-Derived Macrophages via Inhibition of Cyclic AMP Production

Uma Singh; Sridevi Devaraj; Mohan R. Dasu; Dana Ciobanu; Jane Reusch; Ishwarlal Jialal

From the Laboratory for Atherosclerosis and Metabolic Research (U.S., S.D., M.R.D., D.C., I.J.), University of California, Davis Medical Center, Sacramento; and VA Medical Center, Mather (I.J.) and Department of Medicine (J.R.), University of Colorado Health Sciences Center, Denver.

Correspondence to I. Jialal, MD, PhD, Director, Laboratory for Atherosclerosis and Metabolic Research, University of California, Davis Medical Center, 4635, 2nd Ave, Res Bldg 1, Sacramento, CA 95817. E-mail ishwarlal.jialal{at}ucdmc.ucdavis.edu


*    Abstract
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 *Abstract
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Objective— C-Reactive protein (CRP), a cardiovascular risk marker, could also participate in atherosclerosis. Atherosclerotic plaques express CRP and interleukin (IL)-10, a major antiinflammatory cytokine. IL-10 deficiency results in increased lesion formation, whereas IL-10 delivery attenuates lesions. We tested the effect of CRP on lipopolysaccharide (LPS)-induced IL-10 secretion in human monocyte-derived macrophages (HMDMs).

Methods and Results— Incubation of HMDMs with CRP significantly decreased LPS-induced IL-10 mRNA and intracellular and secreted IL-10 protein and destabilized IL-10 mRNA. Also, CRP alone increased secretion of IL-8, IL-6, and tumor necrosis factor from HMDMs and did not inhibit LPS-induced secretion of these cytokines. Fc {gamma} receptor I antibodies significantly reversed CRP-mediated IL-10 inhibition. CRP significantly decreased intracellular cAMP, phospho-cAMP response element binding protein (pCREB), and adenyl cyclase activity. cAMP agonists reversed CRP-mediated IL-10 inhibition. Overexpression of wild-type and constitutively active CREB in THP-1 cells revealed attenuation of the inhibitory effect of CRP on LPS-induced IL-10 levels. CRP also inhibited hemoglobin:haptoglobin-induced IL-10 and heme oxygenase-1. Furthermore, administration of human CRP to rats significantly decreased IL-10 levels.

Conclusions— This study provides novel evidence that CRP, by decreasing IL-10 alters the antiinflammatory/proinflammatory balance, accentuating inflammation, which is pivotal in atherothrombosis.

CRP treatment of HMDMs significantly decreased LPS-induced IL-10 mRNA and intracellular and secreted IL-10 protein and destabilized IL-10 mRNA. CRP significantly decreased intracellular cAMP and pCREB. Thus, CRP inhibits LPS-induced IL-10 secretion via inhibition of cAMP production.


Key Words: CRP • antiinflammatory cytokine • IL-10 • HMDM • atherosclerosis


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
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 down arrowDiscussion
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Increasing evidence supports the involvement of inflammation in the pathogenesis of atherosclerosis and acute coronary syndromes (ACS).1 Monocyte-macrophages are pivotal cells in atherosclerosis and participate in all stages from initiation of the fatty streak to plaque rupture. C-Reactive protein (CRP), the prototypic marker of inflammation, in addition to being a risk marker, appears to be an active participant in atherosclerosis.2

CRP is known to be present in atherosclerotic lesions and is significantly higher in the plaque of patients with unstable angina pectoris than in those with stable angina pectoris.3 CRP induces proinflammatory cytokines in the major cells involved in atherosclerosis.2,4–7 However, the effect of CRP on the antiinflammatory cascade has not been explored. This can be important in atherogenesis because an imbalance between proinflammatory and antiinflammatory cytokine production could enhance the inflammatory process.8,9

Interleukin (IL)-10 is a potent antiinflammatory cytokine that is expressed in human atherosclerotic plaques,10 primarily in macrophages. IL-10 is secreted by lymphocytes of the T-helper cell type 2 subtype as well as in large amounts by macrophages.11 Although the role of IL-10 in human atherosclerosis is evolving, in mice, overexpression reduces lesion progression.12–14 Furthermore, IL-10 deficiency in leukocytes is reported to induce a 2-fold increase in lesion development in the thoracic aorta compared with controls. Also, IL-10 deficiency led to a marked increase in lymphocyte and macrophage accumulation associated with a significant reduction in collagen accumulation.12 Heme oxygenase-1 (HO-1) overexpression in LDLR–/– mice reduces plaque formation.15 Collectively, these data suggest that IL-10 may influence the local inflammatory process within the atherosclerotic lesion.

Importantly, decreased levels of IL-10 and increased levels of CRP have been demonstrated in patients with ACS or chronic heart failure or patients undergoing hemodialysis,16–18 and the levels of CRP as well as IL-10 are prognostic for the development and outcome of ACS.3,19 Also, decreased IL-10 production has been reported from monocytes after lipopolysaccharide (LPS) stimulation in patients with ACS.16 However, there are no mechanistic studies exploring the relation between CRP and IL-10. Accordingly, we tested the effect of CRP on LPS-induced IL-10 production in human monocyte-derived macrophages (HMDMs).


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Please see details in online supplement, available at http://atvb.ahajournals.org.1–14

CRP was purified from human ascitic or pleural fluids by the method of Duclos et al,1 and HMDMs were cultured as described.2,3 The cells were stimulated with LPS or hemoglobin:haptoglobin (Hp:Hb)4 to secrete IL-10. The culture medium was used to measure IL-10 and other cytokines/chemokines. Also, intracellular IL-10 was assayed by flow cytometry. CRP-specific effects on IL-10 secretion were delineated as described earlier.5 IL-10 gene expression6 and mRNA stability7 were also assessed. The surface expression of Fc {gamma} receptors by flow cytometry as well as cAMP response element binding protein (CREB) and phospho-CREB (pCREB) expression by Western blotting and ELISA were also quantitated. In addition, the involvement of specific Fc {gamma} receptor8 and cAMP pathway9,10 was assessed. Furthermore, transfection of various plasmid constructs of CREB was performed11 in THP-1 cells. Also, the effect of CRP administration on IL-10 in vivo was tested in rats as reported.12 Human CRP13 and rat IL-10 were measured in the sera. Furthermore, the effect of CRP on isolated rat macrophages14 was also assessed in terms of the binding8 of human CRP and the involvement of cAMP pathway on the inhibitory effect of CRP on IL-10.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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The basal levels of IL-10 were low and were significantly upregulated by LPS treatment (P<0.001). CRP pretreatment significantly decreased LPS-induced IL-10 secretion (P<0.01) (Figure 1a). The use of boiled CRP preparation as well as CRP that had been preincubated with anti–CRP-IgG–coated plates failed to inhibit IL-10 secretion (Figure 1a). CRP significantly inhibited LPS-induced intracellular IL-10 levels quantitated by flow cytometry (P<0.05) (Figure 1b). Furthermore, we tested the effect of CRP on IL-10 using the Hp:Hb complex as an agonist. CRP inhibited Hp:Hb-induced IL-10 release (Hp:Hb, 4925±1305; CRP+Hp:Hb, 1935±642 pg/mg cell protein [n=6 experiments, P<0.001]) and intracellular HO-1 (Hp:Hb, 358±56; CRP+Hp:Hb, 157±45 ng/mg cell protein [P<0.05]).


Figure 1
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  		Figure 1. a, Effect of native CRP, boiled CRP, and CRP preincubated to anti-CRP IgG plates on LPS-induced IL-10 secretion. Seven-day-old HMDMs were incubated with CRP (0 and 25 µg/mL) for 12 hours followed by LPS (500 ng/mL) challenge for 24 hours. IL-10 secretion was measured in supernatants. Data are presented as mean±SD of 5 experiments in duplicate (from 15 donors). §P<0.001 as compared with control, *P<0.01 as compared with LPS-induced IL-10. b, Effect of CRP on LPS-induced intracellular IL-10. Seven-day-old HMDMs were incubated with CRP (25 µg/mL) for 12 hours followed by LPS (500 ng/mL) challenge for 12 hours. IL-10 accumulation in the cells was measured by flow cytometry. Data are presented as mean±SD of 3 experiments in duplicate. §P<0.01 as compared with control, *P<0.05 as compared with LPS alone.

CRP treatment significantly decreased (P<0.05) LPS-induced IL-10 mRNA expression (Figure 2a). This was confirmed using real-time RT-PCR, which also revealed significantly increased IL-10 mRNA (P<0.02) following LPS stimulation as compared with control (Figure 2b). These events preceded the secretion of maximum levels of IL-10 secretion in macrophages supernatants by several hours (data not shown). Furthermore, CRP significantly decreased IL-10 mRNA stability (t1/2: without CRP, 210±75 minutes; with CRP, 160±50 minutes; P<0.05).


Figure 2
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  		Figure 2. a, Effect of CRP on LPS-induced IL-10 mRNA by RT-PCR. HMDMs were incubated with CRP for 12 hours followed by LPS challenge for 4 hours. RT-PCR for IL-10 mRNA or GAPDH mRNA (as loading control) was performed. Lane 1, control; lane 2, LPS; lane 3, CRP+LPS. The gel is representative of n=3 experiments. b, Effect of CRP on LPS-induced IL-10 mRNA by real-time RT-PCR. HMDMs were incubated with CRP for 12 hours followed by LPS challenge for 4 hours. IL-10 mRNA was determined by real-time PCR as described in Materials and Methods. The results are normalized with GAPDH mRNA and are presented as arbitrary units derived from {Delta}Ct values. The results are means±SD of 3 experiments. §P<0.02 as compared with control, *P<0.05 as compared with LPS.

CRP has been reported to bind to the family of Fc {gamma} receptors in monocytes, such as Fc {gamma} receptor I (Fc {gamma} RI) (CD64)20 and Fc {gamma} RII (CD32).21 The surface expression of CD64 in HMDMs was the greatest, with negligible CD16 (Table I in the online data supplement, available at http://atvb.ahajournals.org). Use of neutralizing antibodies to CD64 but not CD32 resulted in significant reversal (ANOVA, P<0.05) of CRP-mediated IL-10 inhibition (Figure 3). Anti-CD16 (Fc {gamma} RIII) or isotype control antibody failed to have any effect on CRP-mediated IL-10 inhibition.


Figure 3
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  		Figure 3. Effect of Fc {gamma} receptor–neutralizing antibodies on LPS-induced IL-10 in the presence of CRP. HMDMs were preincubated with respective antibodies for 1 hour before CRP treatment. ANOVA, P<0.01. Post hoc Tukey test: §P<0.001 as compared with control; *P<0.04 for CRP pretreated cells as compared with LPS alone; **P<0.05 as compared with CRP+LPS-treated cells. The results are mean±SD of 3 experiments.

CRP has been reported to induce inflammatory effects, ie, secretion of various proinflammatory cytokines and chemokines. However, because of the recent controversy of contaminants22,23 present in CRP preparations, we also tested the effect of our CRP preparation (azide free and passed through detoxigel column) in HMDMs. The incubation of CRP (12.5 and 25 µg/mL) with HMDMs for 24 hours led to a significant increase in IL-6 and tumor necrosis factor (TNF) (ANOVA, P<0.02) and IL-8 (ANOVA, P<0.001) secretion (supplemental Figure Ia and Ib). Also, the secretion of these cytokines/chemokine was significantly (P<0.001) induced by LPS (supplemental Figure Ia and Ic). CRP pretreatment followed by LPS challenge did not inhibit LPS-induced proinflammatory cytokines/chemokines (supplemental Figure Ia and Ic).

IL-10 is known to be regulated by cAMP levels in cells of monocytic lineage.24 CRP significantly inhibited intracellular cAMP levels in LPS-activated cells by inhibiting adenyl cyclase (AC) activity (Figure 4a). To evaluate whether inhibition of cAMP was the mechanism for the inhibitory effect of CRP on LPS-induced IL-10, cells were pretreated with cAMP agonists, 8Br-cAMP and DbcAMP, before CRP treatment. Treatment with cAMP agonists led to a significant (P<0.01) attenuation of CRP-mediated IL-10 inhibition (Figure 4b).


Figure 4
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  		Figure 4. A, i, Effect of CRP on intracellular cAMP levels in HMDMs. The cells were incubated with LPS (500 ng/mL for 24 hours) in the absence or presence of CRP (25 µg/mL) for 12 hours. The intracellular cAMP levels were measured by ELISA. ANOVA, P<0.01; post hoc Tukey test: *P<0.001 vs control; **P<0.035 vs LPS alone. The results are mean±SD of 3 experiments in duplicate. A, ii, Effect of CRP on AC activity in HMDMs. HMDMs on day 7 were pretreated with CRP for 12 hours followed by LPS challenge for 24 hours in serum-free medium. Membrane fractions were used to measure AC activity by the conversion of 32P-ATP to 32P-cAMP. Data are presented as mean±SD of 3 experiments in duplicate. The results are expressed as 32P-cAMP counts/mg protein. *P<0.05 vs control, **P<0.05 vs LPS alone. B, Effect of cAMP agonists on CRP-mediated IL-10 inhibition. Cells were treated with Db-cAMP (50 µmol/L) or Br-cAMP (50 µmol/L) 1 hour before CRP treatment (12 hours) followed by LPS challenge for 24 hours. IL-10 was measured in supernatants. The results are mean±SD of 3 experiments in duplicate. ANOVA, §P<0.01; post hoc Tukey test: P<0.001 vs control; *P<0.01 vs LPS; **P<0.01 vs CRP+LPS. C, Effect of CRP on pCREB/CREB levels. Cells were treated with cAMP agonists 30 minutes before cell harvesting following CRP+LPS treatment. Cell lysates were prepared as described in Materials and Methods. C, i, Effect of CRP on ratio of pCREB/CREB. Protein (50 µg) was used for measurement of CREB and pCREB by ELISA and ratio of pCREB/CREB was calculated. ANOVA, P<0.05; post hoc Tukey test: §P<0.02 vs control; *P<0.03 vs LPS alone; **P<0.01 vs CRP+LPS. The results are mean±SD of 3 experiments in duplicate. C, ii, Western blot analysis depicting effect of CRP on pCREB and CREB done in cell lysates as described in Materials and Methods. Lane 1, control; lane 2, LPS; lane 3, CRP+LPS; lane 4, CRP+LPS+ Db-cAMP; lane 5, CRP+LPS+Br-cAMP. §P<0.04 vs control, *P<0.05 vs LPS alone, **P<0.002 vs CRP+LPS (n=3 experiments for densitometric ratios).

Because CREB is a downstream protein target to cAMP-PKA pathway,25 we further explored whether CRP had any effect on activation of CREB by affecting its phosphorylated form. LPS treatment resulted in significantly increased pCREB levels, with no change in CREB levels. However, CRP treatment led to significantly decreased levels of pCREB without any effect on CREB levels. The ratio of pCREB/CREB was significantly increased (P<0.02) in LPS-stimulated cells compared with controls and was significantly decreased (P<0.03) in cells pretreated with CRP as compared with LPS-treated cells alone (Figure 4c). The Western blot analysis revealed significantly decreased expression of pCREB with minimal change in CREB with CRP treatment (Figure 4c). Furthermore, these levels were significantly increased (P<0.002) following treatment with cAMP agonists as compared with CRP treatment.

The transcription factor CREB is an established regulator of IL-10 production.24 We therefore examined whether CREB was mechanistically important for the effect of CRP in LPS induction of IL-10. THP-1 cells were transfected with plasmids having cDNA for CREB (wild type, constitutively active [DCREB] and dominant negative [KCREB and ACREB]). Overexpression of wild type and DCREB in THP-1 cells restored LPS-induced IL-10 secretion, even in the presence of CRP. Consistent with this observation, plasmid transfection of dominant negative forms of CREB resulted in almost complete inhibition of IL-10 secretion (Figure 5a). This suggests that CREB is important for LPS-stimulated IL-10 secretion. To confirm the efficacy of the transiently transfected plasmid in affecting CREB specific activity, we assessed CREB-activation state. We observed increased pCREB/CREB levels in cells with overexpression of wild-type and constitutively active CREB as compared with nontransfected cells; however, minimal CREB activity was detected in cells overexpressing dominant negative form of CREB (Figure 5b).


Figure 5
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  		Figure 5. Effect of transfection in THP-1 with indicated CREB plasmids on CRP-mediated IL-10 inhibition. THP-1 cells were grown in 12-well plates and transfected with indicated CREB plasmids as detailed in Materials and Methods. Forty-eight hours after transfection, THP-1 cells were pretreated with CRP (25 µg/mL) for 12 hours followed by LPS (500 ng/mL) for 24 hours. A, Supernatants were used for IL-10 measurement *P<0.05 vs control, **P<0.05 vs LPS alone, Figure 5P<0.01 vs control in transfected cells. B, Cells were used for the measurement of total and pCREB. The results are expressed as pCREB/CREB ratio. *P<0.03 vs control, **P<0.045 vs LPS alone, Figure 5P<0.029 vs control in transfected cells.

CRP administration to rats resulted in significantly increased (P<0.001) hCRP levels (18.9±2.3 mg/L); however, the levels were undetectable in the rats administered human serum albumin (hSA) (Figure 6). The levels of rat IL-10 in sera were significantly decreased in hCRP-injected rats (median value- 42 pg/mL) (P<0.05) as compared with hSA-injected rats (median value, 390 pg/mL) in vivo. The levels of IL-10 in hSA-injected rats were similar to saline-injected rats (data not shown).


Figure 6
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  		Figure 6. Scatter plot depicting the levels of IL-10 in sera of individual rats following hCRP or hSA administration (20 mg/kg body weight per day for 3 days IP) to Sprague–Dawley rats (n=6/group). Rat IL-10 was measured by a BD fluorescence-activated cell sorter array. P<0.05 compared with hSA-administered rats.

The binding experiments with human CRP to rat macrophages revealed that human CRP does bind to these cells in a dose-dependent manner (supplemental Figure II). The binding was significantly inhibited in the presence of 10x cold human CRP, pretreatment with anti-rat CD32 IgG as well as anti-rat IgG (because monomeric IgG blocks the high-affinity receptor CD64) (n=3 experiments).

Pretreatment of rat macrophages in vitro with human CRP (25 µg/mL) for 12 hours followed by LPS challenge (500 ng/mL) for 24 hours led to significant inhibition in IL-10 secretion (supplemental Figure III; P<0.02). Furthermore, the use of cAMP agonists significantly (P<0.01) reversed CRP-mediated inhibition of LPS-induced IL-10 release (supplemental Figure III). Thus, CRP inhibits IL-10 secretion in both HMDMs and rat macrophages by inhibiting cAMP.


*    Discussion
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 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we evaluated the effect of CRP on IL-10 secretion in HMDMs following stimulation with LPS. The rationale for using LPS as the chosen agonist is that LPS is a known inducer of macrophage activation, and our study would allow us to separate an effect of CRP independent of LPS contamination. Furthermore, LPS is reported to signal through TLR-4 pathway, and LPS receptors (Toll receptors- 4 [TLR-4]) are pathogenically related to atherosclerosis.26,27 Importantly, previous studies have reported decreased IL-10 release from circulating monocytes of ACS patients following LPS activation.16 Thus, we examined the effect of CRP on LPS-induced IL-10 release from HMDMs.

CRP treatment resulted in a significant decrease in LPS-induced IL-10 mRNA, intracellular IL-10, as well as IL-10 secretion. We also explored the effect of CRP on IL-10 secretion using a natural physiological ligand, Hp:Hb complex.28 The latter induces IL-1029 via CD163 receptor, a scavenger receptor for Hp:Hb complexes. CRP also inhibited Hp:Hb induced IL-10 secretion. Also, CD163-mediated clearance of modified forms of hemoglobin is reported to be important in atherogenesis in diabetic vasculopathies.28 As reported previously, we confirmed that purified CRP stimulated IL-6, IL-8, and TNF secretion. CRP treatment before LPS challenge did not inhibit LPS-induced proinflammatory cytokine secretion but significantly inhibited IL-10. These data provide further evidence that CRP shifts the balance toward a proinflammatory phenotype by inhibiting IL-10 secretion induced by LPS but not TNF and IL-6. Importantly, elevated serum levels of IL-10 are associated with a significantly improved outcome19 of patients with ACS, supporting the concept that the balance between pro- and antiinflammatory cytokines is a major determinant of outcome in these patients.

The mechanistic link between CRP and atherosclerosis has been the focus of much research. There have been some recent reports22,23 arguing that CRP-induced inflammatory changes are attributable to possible contaminants, such as LPS and azide. However, in this report, we have used an in-house CRP preparation (azide free and passed through detoxigel column). Furthermore, our data clearly separate a proinflammatory effect of CRP independent of LPS because CRP inhibited IL-10 release induced by LPS. Additionally, by using different strategies, we show that IL-10 inhibition is CRP specific. Furthermore, the concentration of CRP used in this study has been recorded in patients with ACS.3,16 Therefore, it is likely that local CRP levels in atherosclerotic lesions, either from the circulation or its local synthesis by cells present in arterial lesions, negatively affect production of IL-10 by macrophages.2 We provide novel evidence that CRP inhibits a potent antiinflammatory cytokine and further show that this effect is mediated via downregulation of cAMP. The treatment of HMDMs with cAMP agonists before CRP led to a reversal of CRP-mediated IL-10 inhibition, confirming the role of cAMP-mediated pathway in CRP-mediated IL-10 inhibition. Also, we show here that AC activity was significantly inhibited in cells pretreated with CRP as compared with LPS treatment alone. Our finding of increased intracellular cAMP and AC activity in LPS-treated macrophages is consistent with other reports in the literature,30–32 in which LPS is known to induce sensitization of AC. Additionally, we also observed significant inhibition of HO-1 by CRP. Thus, it is possible that CRP is inhibiting IL-10 and HO-1 via inhibition of cAMP. Interestingly, the HO-1 gene, like IL-10 has cAMP response elements in its promoter.33 In future studies, we will elucidate the interrelationship between cAMP and HO-1 on IL-10 secretion.

In contrast to proinflammatory cytokines that are mainly regulated by transcription factors such as nuclear factor {kappa}B and activator protein-1, the transcription of the antiinflammatory cytokine IL-10 in monocytic cells is activated by cAMP24 via transcription factors such as CREB and C/EBP. CREB binding to enhancer upstream of IL-10 is primarily known to be involved in transcriptional regulation of IL-10 gene. In addition to decreasing IL-10 levels, we go further to show that CRP results in decreased activation of the downstream transcription factor pCREB. Furthermore, we also show here that overexpression of CREB by wild-type and constitutively active plasmids, attenuate the inhibitory effect of CRP on LPS-induced IL-10 levels. Together, these results point to CREB as a novel target for LPS-induced IL-10. In macrophages, CRP disarms the cellular defense mechanism of IL-10 release by interfering with CREB function. Our results are in support of the previously reported well-evident role of CREB in other model systems of vascular diseases.34,35 Thus it strengthens our hypothesis that CRP inhibits LPS-induced IL-10 secretion via downregulation of cAMP and pCREB in HMDMs.

Additionally, immunocytochemical detection of Fc {gamma} receptors in human atherosclerotic lesions36 has been demonstrated. We have previously shown that blocking of CD32/CD64 in HAEC abrogates effects of CRP.37 In addition, in human leukocytes, CRP has been shown to induce CCR2 expression38 via CD64 and matrix metalloproteinase (MMP)-1 expression39 as well as low-density lipoprotein uptake40 via CD32. Our experiments revealed that the blocking of CD64 significantly reversed CRP-mediated IL-10 inhibition. Previously, ligation of Fc {gamma} RI (CD64) has been shown to inhibit IL-10,41 and this is in support of our findings.

Furthermore, a valid animal model to test the effect of hCRP has been an issue in the literature. As reviewed recently,2 CRP is not a major acute-phase protein in mice. Delivery of human/rabbit CRP to mice infected with pathogens/endotoxin resulted in decreased lethality; this protection appeared to be attributable to a paradoxical antiinflammatory effect (an increased IL-10 production) in mice that needs to be further elucidated.42 However, as reviewed,22 in a rat model, human CRP administration increased myocardial and cerebral infarction following coronary and cerebral artery ligation, respectively. Recently, Pepys et al22 further validated the rat as an appropriate model to test the effect of human CRP. By blocking the effects of CRP with an inhibitor, they prevented the increase in infarct size. Additionally, human CRP works in the rat by activating complement; however, rat CRP does not have this ability.22 Thus, we believe that the rat is an appropriate and relevant model to study the effect of human CRP. Circulating levels of human CRP were significantly increased and IL-10 were significantly decreased in hCRP compared with hSA administered in Sprague–Dawley rats. Furthermore, in vitro treatment of isolated rat macrophages with CRP followed by LPS challenge resulted in an inhibition of IL-10. Additionally, pretreatment of rat macrophages with cAMP agonists reversed CRP-mediated IL-10 inhibition. Hence, our findings confirm that CRP indeed leads to a decrease in IL-10 production both in an in vitro model (HMDMs and rat macrophages) as well as in vivo. We also showed that human CRP binds to Fc {gamma} receptors on rat macrophages.

In conclusion, this study makes the novel observation that CRP inhibits IL-10 production via inhibition of AC. We confirmed the effect of CRP on IL-10 inhibition both in vivo as well as in vitro. Because IL-10 is an antiinflammatory cytokine, this study further underscores the role of CRP in promoting atherothrombosis via accentuating the inflammatory process by inhibiting IL-10 and stimulating proinflammatory cytokines.


*    Acknowledgments
 
Sources of Funding

This work was supported by National Institutes of Health grants NIH K24 AT00596 and RO1 HL074360 (to I.J.).

Disclosure(s)

None.


*    Footnotes
 
Original received May 31, 2006; final version accepted August 4, 2006.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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