Loss of Pentameric Symmetry in C-Reactive Protein Induces Interleukin-8 Secretion Through Peroxynitrite Signaling in Human Neutrophils
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循环研究杂志 2005年第9期
the Research Center, Maisonneuve-Rosemont Hospital (T.K., L.J., J.G.F.), University of Montreeal, Montreeal, Queebec, Canada
Immtech International, Inc (L.A.P.), Vernon Hills, Ill.
Abstract
Plasma levels of C-reactive protein (CRP), nitrotyrosine, and interleukin-8 (IL-8) are known predictors of acute cardiovascular events. Peroxynitrite (ONOOeC) may function as an intracellular signal for the production of IL-8; however, it is not known whether CRP regulates these events. Emerging evidence suggests that some bioactivities of CRP are expressed only when the pentameric structure of CRP is lost, resulting in formation of monomeric or modified CRP (mCRP). We studied the impact of human native CRP and bioengineered mCRP that cannot rearrange into the pentameric structure on ONOOeC formation and ONOOeC-mediated IL-8 gene expression in human leukocytes. Incubation of human whole blood or isolated neutrophils with mCRP (0.1 to 100 e蘥/mL) for 4 hours increased IL-8 gene expression and secretion that was blocked 70% by the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME). In neutrophils, mCRP simultaneously increased superoxide production and endothelial nitric oxide synthase-mediated NO formation, leading to enhanced ONOOeC formation, and consequently activation of nuclear factor-B and activator protein-1. Native CRP had no detectable effect at 4 hours, whereas it enhanced IL-8 release after a 24-hour incubation that was blocked by L-NAME. An anti-CD16 antibody, but not an anti-CD32 antibody, produced 60% to 70% reductions in mCRP-stimulated NO formation and IL-8 release (both P<0.05). These results suggest that loss of the pentameric symmetry in CRP, resulting in formation of mCRP, leads to IL-8 release from human neutrophils via peroxynitrite-mediated activation of nuclear factor-B and activator protein-1.
Key Words: C-reactive protein leukocytes interleukins signal transduction inflammation
Introduction
Epidemiological and clinical studies have shown strong and consistent relationships between circulating markers of inflammation and risk prediction of future coronary artery disease (CAD). Among these markers, elevated plasma levels of C-reactive protein (CRP) are predictive for subsequent acute coronary events among apparently healthy men and women and patients with stable or unstable angina.1,2 The C-X-C chemokine interleukin-8 (IL-8) is a sensitive marker of unstable CAD.3 Increases in IL-8 levels may coincide with increased CRP levels,4 though IL-8 does not directly stimulate CRP gene expression.5,6 Increased plasma levels of nitrotyrosine, a "molecular fingerprint" for nitric oxide-derived oxidants,7 are also associated with the presence of CAD.8 Statin therapy produced reductions in nitrotyrosine concentrations similar to those in total cholesterol and CRP.8 It is uncertain, however, whether changes in these inflammatory markers occur independently or whether they are interrelated.
Widespread neutrophil activation has also been detected in patients with acute CAD, though the existence of a correlation between neutrophil activation and plasma CRP remains controversial.9,10 Neutrophil infiltration into plaques was found to be actively associated with acute coronary events.11 Recent results suggest that peroxynitrite (ONOOeC), formed in a reaction of superoxide with NO,7 functions as an intracellular messenger to mediate IL-8 gene expression in lipopolysaccharide (LPS) or cytokine-stimulated human leukocytes through activation of the transcription factors nuclear factor (NF)-B and activator protein-1 (AP-1).12eC15 CRP induces cytokine release from endothelial cells.16eC18 Recent results suggest that conformational rearrangement in native CRP, leading to the formation of monomeric or modified CRP (mCRP), is required for activation of endothelial cells.19 CRP and mCRP bind to the distinct receptors FcRIIa (CD32) and FcRIII (CD16), respectively, on human neutrophils20eC22 and exert opposing actions. For instance, native CRP inhibits neutrophil activation, adherence, and trafficking,5,23eC25 whereas mCRP promotes neutrophil adhesion to endothelial cells19 and suppresses neutrophil apoptosis.26
In the present study, we investigated the impact of native pentameric CRP and mCRP on IL-8 production in human whole blood and isolated neutrophils. To gain insight into the underlying molecular mechanisms in neutrophils, we characterized the immunoglobulin (Ig) G receptor subtype (ie, the CRP receptor) involved and examined whether induction of IL-8 gene and protein expression is mediated through stimulation of ONOOeC-dependent activation of NF-B and AP-1.
Materials and Methods
CRP Isoforms
High purity (>99%) human native CRP (Calbiochem) was stored in NaN3-free 20 mmol/L Tris, 150 mmol/L NaCl buffer (pH 7.5) containing 2 mmol/L CaCl2 to prevent spontaneous formation of mCRP from the native pentamer. A recombinant form of mCRP (rmCRP, purity >97%) that cannot rearrange into a pentameric structure was engineered, characterized, and compared with mCRP prepared from native CRP by urea chelation as described.26 Native CRP was distinguished from mCRP by binding and antigenicity differences27 and by their secondary structure.26 The endotoxin level of all protein solutions was below the detection limit (0.125 endotoxin units/mL, corresponding to 0.01 ng/mL LPS) of the Limulus assay (Sigma).
Cell Stimulation
Venous blood (anticoagulated with sodium heparin, 50 U/mL) was obtained from 24 healthy volunteers who had denied taking any medication for at least 2 weeks. The Clinical Research Committee approved the experimental protocols. Neutrophil granulocytes (purity >95%, viability >97%) were isolated as described.23 Whole blood aliquots or isolated neutrophils (5x106 cells/mL) in microcentrifuge tubes were placed on a rotator and challenged with native CRP or mCRP with or without the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) at 37°C in 5% CO2 atmosphere. In additional experiments, the responses to CRP and mCRP were studied in the presence of the specific NF-B inhibitor pyrrolidine dithiocarbamate (PDTC, 100 e蘭ol/L), function-blocking anti-CD16 monoclonal antibody (mAb) 3G8 (isotype: IgG1), anti-CD32 mAb FLI-8.26 (isotype: IgG1), or the irrelevant class-matched MOPC-21 antibody (all at 2.5 e蘥/mL, BD PharMingen). At the designated time points, the plasma was harvested and stored at eC20°C for later cytokine analysis. Cells were processed as described below.
Measurements of IL-8 mRNA Expression and Secretion
Levels of IL-8 in plasma or culture medium were determined by a selective enzyme immunoassay (OptEIA, BD Biosciences). Intra-assay and interassay coefficients of variation were typically <4% and <6%, respectively. IL-8 and GAPDH mRNA expression was assayed in RNase protection assays with the Direct Protect kit (Ambion) as described previously.12
Nitric Oxide and Superoxide Formation
Intracellular formation of NO was monitored by flow cytometry after incorporation of diaminofluorescein diacetate (5 e蘭ol/L, Clontech) into neutrophils.15 Superoxide production was determined as superoxide dismutase-inhibitable reduction of ferricytochrome c.15
Expression of NO Synthase Isoforms
Total RNA was isolated from 1x107 neutrophils using TriZol reagent (Invitrogen). cDNA was prepared with superscript reverse transcriptase (Invitrogen). The following primers were used for subsequent PCR analysis: human inducible NOS (iNOS), sense 5'-TCTCTCGGCCACCTTTGATGAG-3' and antisense 5'-GGTTGCATCCAGCTTGACCAG-3'; human endothelial NOS (eNOS), sense 5'-GTGATGGCGAAGCGAGTGAAG-3' and antisense 5'-CCGAGCCCGAACACACAGAAC-3'; human neuronal NOS, sense 5'-CTTCTGGCAACAGCGGCAATTTG-3' and antisense 5'-TGGACTCAGATCTAAGGCGGTTG-3'; -actin, sense 5'-ATGCCATCCTGCGTCTGGAC-3' and antisense 5'-AGCATTTGCGGTGCACGATGG-3'.28 The corresponding 412-base pair, 421-base pair, 458-base pair, and 500-base pair fragments were amplified enzymatically by 40, 35, 40, and 25 repeated cycles, respectively, and subjected to electrophoresis on 1.2% agarose containing ethidium bromide.
Detection of Peroxynitrite
NO-dependent fluorescence of rhodamine, an oxidation product of dihydrorhodamine 123 (DHR 123),29 and nitrotyrosine formation were measured as markers of ONOOeC formation. DHR 123 (20 e蘭ol/L, Molecular Probes) was added to some samples during the last 60 minutes of incubation in the presence or absence of L-NAME (1 mmol/L), and fluorescence was analyzed by a flow cytometer (FACScan, Becton Dickinson).12 The NO synthase blocker-inhibitable proportion of DHR oxidation can be attributed to ONOOeC because ONOOeC readily oxidizes DHR 123, whereas NO does not.29 DHR 123 oxidation by mCRP was also determined in neutrophils pretreated with the calmodulin inhibitor W7 (20 e蘭ol/L), the broad PKC inhibitor GF109203X (200 nM), the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin (100 nM), or the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI, 5 e蘭ol/L). In additional experiments, neutrophil extracts (prepared as in reference15) were analyzed for the presence of nitrotyrosine, a "molecular fingerprint"7 of ONOOeC by an enzyme immunoassay (detection limit: 2 ng/mL) (Cayman Chemicals) using nitrotyrosine as standard.15 Intra-assay and interassay coefficients of variation were typically <6%.
NF-B and AP-1
Intranuclear, DNA bound, NF-B/p65 and AP-1/c-Fos were measured with a flow cytometry assay13,15 and were used as estimates of NF-B and AP-1 activation, respectively. In brief, leukocyte nuclei prepared with the Cycletest Plus DNA reagent kit (Becton Dickinson) were stained with rabbit polyclonal anti-human NF-B/p65 or c-Fos antibodies or with normal rabbit IgG (to assess nonspecific binding of IgG to nuclei) and then with fluorescein isothiocyanateeCconjugated anti-rabbit IgG antibody (all from Santa Cruz Biotechnology) and propidium iodide. Singlet neutrophil nuclei were gated using the doublet-discrimination module and fluorescence intensity was analyzed with a FACScan flow cytometer using the Cell Quest Pro software.
Calcium Mobilization Assay
Intracellular Ca2+ concentration was monitored in Fura-2/AM (1 e蘭ol/L)-loaded neutrophils in a Perkin-Elmer spectrofluorometer (excitation: 340 nm, emission: 510 nm) as previously described.30
Western Blot Analysis
Protein extracts were prepared by lysing 2x106 neutrophils in 100 e蘈 of lysis buffer, and immunoblot analysis of phosphorylated and total Akt was performed using the Phospho Plus Akt antibody kit (New England Biolabs) as described.26
Neutrophil Viability
Neutrophil viability was assessed by flow cytometry immediately after staining with propidium iodide (0.5 e蘥/mL).
Statistical Analysis
Results are expressed as mean±SEM. Statistical comparisons were made by ANOVA using ranks (Kruskal-Wallis test) followed by Dunn’s multiple contrast hypothesis tests to identify differences between various treatments. Repeated measures were analyzed by the Friedman test followed by the Wilcoxon-Wilcon test. Values of P<0.05 were considered significant.
Results
Effects of CRP and mCRP on IL-8 Production and IL-8 mRNA Expression
Incubation of human whole blood with mCRP for 4 hours resulted in concentration-dependent IL-8 release, whereas native CRP was without effect (Figure 1). Significant induction was detected with 5 e蘥/mL, which peaked at 100 e蘥/mL mCRP (Figure 1B). Native CRP started to increase IL-8 release only after 8 hours of incubation; however, it was a considerable less potent inducer of IL-8 production than mCRP at any time points studied (Figure 1A). Both mCRP-and CRP-induced IL-8 release were markedly, though never completely, inhibited by L-NAME (Figure 1A). Likewise, mCRP, but not CRP, induced concentration-dependent IL-8 release from isolated neutrophils after 4 hours of incubation (Figure 1C). On a molar basis, bioengineered mCRP and mCRP prepared from native CRP evoked similar IL-8 release (Figure 1D). Recombinant mCRP-induced (50 e蘥/mL) IL-8 release (5.6±0.7 ng/mL) was unaffected by the formyl-peptide receptor antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe (50 e蘭ol/L) (5.4±0.5 ng/mL, n=6, P>0.1). Therefore, because of the enhanced solubility, bioengineered mCRP was used in subsequent experiments. Heat inactivation of native CRP (60 minutes at 100°C) resulted in a complete loss of its activity (data not shown). Also, LPS (Escherichia coli serotype O111:B4) at 0.02 ng/mL, a concentration 2-fold higher than the maximum level of LPS contamination in our protein solutions, did not induce detectable IL-8 release (0.24±0.03 ng/mL versus 0.23±0.03 ng/mL in unstimulated neutrophils, n=4, P>0.1).
We performed RNase protection assays on RNA extracted from leukocytes after 4 hours of incubation with mCRP. Consistent with the observations at the protein level, mCRP evoked concentration-dependent increases in IL-8 mRNA expression, which were suppressed by L-NAME (Figure 2). Native CRP at 100 e蘥/mL did not produce detectable changes (Figure 2).
mCRP Induces NO, Superoxide, and Peroxynitrite Formation in Neutrophils
Incubation of neutrophils for 4 hours with mCRP led to simultaneous increases in O2·eC and NO production. Significant increases were detected with 5 e蘥/mL and peaked at 100 e蘥/mL mCRP (Figure 3). The increases in O2·eC and NO production coincided with increases in NO-dependent DHR123 oxidation, indicating enhanced ONOOeC formation (Figure 3). Addition of the NO donor spermine NONOate (0.5 mmol/L) to L-NAME-treated neutrophils restored DHR 123 oxidation (rhodamine fluorescence in relative fluorescence units, control: 22±4; mCRP: 50±4; L-NAME+mCRP: 29±3; spermine NONOate+L-NAME+mCRP: 48±4; n=4; P>0.1 compared with mCRP). To confirm ONOOeC formation, we measured cellular nitrotyrosine content. Neutrophils exposed to mCRP for 4 hours contained significantly higher amounts of nitrotyrosine than unchallenged cells, and the increases in nitrotyrosine correlated with those in DHR 123 oxidation (Figure 3).
To identify the NOS isoform(s) responsible for enhanced NO production, we performed reverse transcription polymerase chain reaction on cDNA prepared from neutrophils. These assays resulted in the amplification of eNOS mRNA, but not neuronal NOS and iNOS, in freshly isolated cells and in response to mCRP at 2 and 4 hours after addition of the protein (Figure 4). mCRP did not affect eNOS mRNA expression (Figure 4).
To characterize the proximal signaling events associated with mCRP-induced ONOOeC formation, we monitored calcium mobilization. mCRP evoked a rapid increase in intracellular Ca2+ similar to that observed with fMLP (Figure 5A), and transiently enhanced (peak at around 2 minutes) phosphorylation of Akt (Figure 5B), indicating phosphatidylinositol 3-kinase activation. Consistently, calmodulin blockade with W7 or inhibition of PI3-kinase with wortmannin significantly reduced mCRP-induced DHR 123 oxidation, and these actions were not additive with L-NAME (Figure 5C). Inhibition of NADPH oxidase with DPI effectively attenuated mCRP-evoked DHR 123 oxidation to the same degree in the absence and presence of L-NAME (Figure 5C). By contrast, the PKC inhibitor GF109203X produced only modest decreases in mCRP-induced DHR 123 oxidation that were further enhanced by L-NAME (Figure 5C).
mCRP Stimulates Nuclear Accumulation of AP-1 and NF-B in Neutrophils
Because transcription of IL-8 gene requires activation of NF-B and AP-1,31 we examined whether mCRP can activate these pathways in neutrophils. We prepared nuclei from unstimulated and mCRP-stimulated neutrophils and analyzed nuclear accumulation of NF-B and AP-1 using flow cytometry. Figure 6A shows representative results illustrating mobilization of NF-B/p65 and AP-1/c-Fos to the nucleus (ie, the increased fluorescence represents increased amounts of NF-B or AP-1 bound to DNA) in response to mCRP. Nuclear immunostaining was completely blocked by preincubation of the antibodies with the appropriate blocking peptides. The actions of mCRP were concentration-dependent and were markedly attenuated by L-NAME (Figure 6A and 6B), consistent with inhibition of IL-8 mRNA expression and production.
Preincubation of neutrophils with PDTC markedly attenuated, though never completely inhibited, mCRP-induced IL-8 production (Figure 7), confirming that increased nuclear accumulation of NF-B correlated with induction of IL-8 production.
Involvement of CD16 in mCRP Signaling
Previous studies identified CD16 (FcRIII) as a neutrophil receptor for mCRP.22,26 We used function-blocking mAbs to CD16 and CD32 (FcRIIa) as competitors to confirm whether the mCRP actions described above were also mediated via CD16. The anti-CD16 mAb partially prevented mCRP-induced NO formation assessed at 4 hours, and IL-8 release assessed at 4 and 24 hours after addition of mCRP, whereas the anti-CD32 or the irrelevant MOPC-21 antibody had no detectable effects (Figure 8). Increasing the concentration of the anti-CD16 mAb did not result in further inhibition (data not shown). At 24-hours culture, native CRP-induced IL-8 release was also attenuated by the anti-CD16 mAb, but not by the anti-CD32 mAb or MOPC-21 (Figure 8B).
Effect of mCRP on Neutrophil Viability
Because increased NO and ONOOeC formation could affect cell viability,7,32 we assessed neutrophil survival. Consistent with previous studies,26 after 4 hours culture in vitro, the percentage of viable neutrophils was slightly higher in the presence of mCRP (95±2%, n=5, P<0.05) than in untreated (control) samples (89±2%) or in the presence of native CRP (88±3%, n=5, P<0.05 versus mCRP).
Discussion
The present results provide evidence for a novel mechanism by which CRP may affect the inflammatory process by stimulating ONOOeC formation and signaling in human neutrophils. These actions are expressed when the pentameric structure undergoes a conformational rearrangement, however, leading to formation of mCRP. Our results also suggest a potential link between CRP, IL-8, nitrotyrosine, and neutrophil activation, as it may occur in the blood of patients with acute CAD.
Our study using human whole blood and isolated neutrophils demonstrates that mCRP stimulates a rapid (within 4 hours) and potent synthesis of IL-8. This is a primary response to mCRP that requires de novo protein synthesis and transcription of the IL-8 gene. Comparison of plasma and culture medium IL-8 levels suggests that 70% of IL-8 release was of neutrophil origin in blood. Native CRP did not evoke detectable changes at 4 hours. This was unexpected, for native CRP induced rapid (within minutes) shedding of L-selectin from the surface of neutrophils,23 and cannot be explained by the slight differences in the viability of mCRP or CRP-treated neutrophils. Human blood contains all the yet unidentified serum cofactors that were required for CRP activation of endothelial cells.16 The CRP stimulation became detectable only after 8 hours of incubation, coinciding with in vitro kinetics of dissociation into subunits.33 Although CRP clearly enhanced IL-8 production at 8 to 24 hours of incubation, it was a considerably less potent inducer of IL-8 release than mCRP. These observations suggest that conformational rearrangement of CRP is required to induce IL-8 production in neutrophils, and that the amounts of mCRP generated from CRP within 4 hours are not sufficient to evoke detectable increases in IL-8. The time course of neutrophil activation by native CRP appears to be similar to that of CRP activation of endothelial cells.19 Neither LPS (at a concentration 2-fold higher than might be present in our protein preparations) nor heat-inactivated CRP evoked detectable IL-8 release, indicating that CRP and mCRP signaling was responsible for the observed effects.
Blockade of NO synthesis with L-NAME inhibited to a similar degree mCRP-induced IL-8 release and nuclear accumulation of NF-B and AP-1 in neutrophils, coinciding with suppression of IL-8 mRNA expression. Interestingly, L-NAME also attenuated native CRP-induced IL-8 release, and the degree of inhibition was comparable to that detected with mCRP. These results pointed to the involvement of NO in mediating these responses. Previous studies have shown that ONOOeC rather than NO by itself mediates IL-8 release from human neutrophils in response to LPS or cytokines.12,34,35 Accordingly, we found that mCRP simultaneously enhanced superoxide and NO formation, coinciding with increases in NO-dependent oxidation of DHR123 and nitration of protein tyrosine residues. A significant portion of rhodamine fluorescence in mCRP-stimulated neutrophils can be attributed to ONOOeC, because it depends on NO-related species (it can be inhibited by L-NAME), whereas NO per se does not oxidize DHR 123.29 Further, the NO donor spermine NONOate restored DHR 123 oxidation in L-NAMEeCtreated neutrophils, indicating that L-NAME does not inhibit NADPH oxidase activation. Although nitrotyrosine is often considered as a distinct "molecular fingerprint" of ONOOeC formation,7 peroxidase-dependent tyrosine nitration has also been described.36,37 Interestingly, in human neutrophils, ONOOeC appears to be the predominant mechanism for tyrosine nitration.38
Reverse-transcriptase polymerase chain reaction amplified eNOS, but not iNOS and neuronal NOS-specific products, in unchallenged neutrophils and in neutrophils treated with mCRP for up to 4 hours. These results point toward increased eNOS activity as the source for enhanced NO production in response to mCRP. Previous studies on constitutive NOS expression in human neutrophils yielded contradictory results, as both the absence and presence of eNOS and neuronal NOS have been reported.28,39eC41 Contaminating cells or differences in the NOS assays used might account for this apparent discrepancy. Unstimulated human neutrophils do not express iNOS, whereas iNOS-positive neutrophils have been detected in tissue exudates28,41 and after more than 16 hours incubation with cytokines.41 Whether mCRP could induce iNOS expression after prolonged incubation periods remains to be investigated. There is compelling evidence that eNOS-derived NO contributes to ONOOeC formation in amounts sufficient to activate signaling mechanisms and even to induce cell damage.32
Ca2+ mobilization and activation of calmodulin and PI3-kinase appears to be required for mCRP-induced ONOOeC formation. Ca2+ transients control calmodulin-mediated eNOS activation and the activity of PLC1 and PLC2, which through formation of diacylglycerol and activation of PI3-kinase lead to activation of NADPH oxidase.42 The inhibitory actions of W7, wortmannin, and DPI on DHR 123 oxidation were not additive with those of L-NAME, suggesting that these compounds inhibited the same reaction, ie, the ONOOeC-dependent oxidation through suppression of either NO or O2·eC formation. PKC appears to play a minor role in mCRP signaling, for GF109203X inhibited only a small portion of DHR 123 oxidation by mCRP.
The IL-8 gene contains cis-regulatory elements for NF-B, AP-1, and NF-IL-6.31 Of these transcription factors, NF-B plays a key role in the induced expression of IL-8. Accordingly, we found that mCRP stimulates nuclear accumulation of NF-B and AP-1, and inhibition of NF-B activation with PDTC decreased IL-8 production by 66%. The mechanism of action of PDTC has not been fully defined, but likely involves inhibition of formation of oxidants that would result in activation of IB- kinase and/or enhancing phosphorylation of I-B.43,44 Thus, NF-B activation could be attributed to decomposition products of ONOOeC rather than the parent molecule itself.
Our results indicate that mCRP-induced IL-8 gene expression and release are predominantly mediated through the low affinity immune complex binding IgG receptor CD16, for the function-blocking anti-CD16 mAb 3G8, but not anti-CD32 mAb, markedly, though never completely, inhibited NO formation and IL-8 production in neutrophils. Furthermore, only the anti-CD16 mAb attenuated native CRP-induced IL-8 release at 24 hours incubation. These observations imply that during 24-hours culture, conformational rearrangement might have occurred in native CRP, yielding mCRP, because native CRP does not bind to CD16.20eC22 We cannot exclude the possibility that mCRP may bind different sites from IgG on CD16, for blockade of the epitope defined by the mAb 3G8 reduced 80% of mCRP binding at 0°C.22 Alternatively, mCRP might interact with other as yet unidentified cell surface molecules. These may include direct binding to positively charged residues on proteins and direct interaction with the lipid membrane (Potempa et al, unpublished observations, 2005). The slight inhibition of mCRP activation of endothelial cells by anti-CD16 mAb19 lends support to the existence of mCRP receptor(s) other than CD16.
The mechanisms that induce conformational rearrangement in native CRP in vivo are still unknown. Native, pentameric CRP dissociates into free subunits after binding to plasma membranes33 or in denaturating or oxidative environment,27 yielding mCRP. The percentage of CRP that might have dissociated into subunits during a 24-hour incubation period, however, remains to be determined. Unlike CRP, mCRP appears to be predominantly membrane-bound,45,46 therefore making its detection rather difficult. Because the commercial CRP antibody (clone 8) mainly recognizes mCRP,47 tissue immunostaining attributed to native CRP in previous studies might have been due to mCRP. Neutrophils may come into contact with mCRP expressed in the arterial wall at sites of endothelial injury45 or perhaps on other leukocytes, thereby aggravating the inflammatory response and contributing to plaque destabilization.11 Stimulated neutrophils release ONOOeC, though the ratio of intracellularly retained and released ONOOeC cannot be deduced.12 Increased ONOOeC release is consistent with enhanced nitration of plasma proteins and may contribute to tissue damage. Finally, our results do not exclude contribution of other cells to elevated plasma levels of nitrotyrosine and IL-8. Indeed, mononuclear leukocytes and endothelial cells also release IL-8 in response to native CRP or mCRP, thereby increasing plasma cytokine levels in patients with CAD.
In summary, the present results indicate that loss of pentameric symmetry in CRP, resulting in formation of mCRP, stimulates IL-8 production by human neutrophils through ONOOeC-mediated activation of NF-B and AP-1. These observations point toward neutrophils as a major source of nitrosative stress and IL-8 and may provide a potential link between CRP, neutrophil activation, plasma nitrotyrosine, and IL-8, all implicated in predicting future acute CAD.
Acknowledgments
This study was supported by grants (to J.G.F.) and Doctoral Research Awards (to T.K. and L.J.) from the Canadian Institutes of Health Research (MOP-64283) and the Heart and Stroke Foundation of Quebec.
Dr Potempa was employed by NextEra Therapeutics, and is currently employed by Immtech International Inc, of Vernon Hills, Ill. Immtech is a minority owner of the modified C-reactive protein technology. Dr Potempa received support for his work on modified C-reactive protein from Immtech (until 1999) and from NextEra (from 1998 to 2002).
Both authors contributed equally to this study.
References
Bassuk SS, Rifai N, Ridker PM High-sensitivity C-reactive protein: clinical importance. Curr Probl Cardiol. 2004; 29: 439eC493.
Danesh J, Wheeler JG, Hirschfield G, Eda S, Eiriksdottir G, Rumley A, Lowe GD, Pepys MB, Gudnason V. C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med. 2004; 350: 1387eC1397.
Kanda T, Hirao Y, Oshima S, Yuasa K, Taniguchi K, Nagai R, Kobayashi I. Interleukin-8 as a sensitive marker of unstable coronary artery disease. Am J Cardiol. 1996; 77: 304eC307.
Kushner I, Broder ML, Karp D. Control of the acute-phase response. Serum C-reactive protein kinetics after acute myocardial infarction. J Clin Invest. 1978; 61: 235eC242.
Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med. 1999; 340: 448eC454.
Black S, Kushner I, Samols D. C-reactive protein. J Biol Chem. 2004; 279: 48487eC48490.
Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol. 1996; 271: C1424eCC1437.
Shishehbor MH, Aviles RJ, Brennan ML, Fu X, Goormastic M, Pearce GL, Gokce N, Keaney JF, Penn MS, Sprecher DL, Vita JA, Hazen SL. Association of nitrotyrosine levels with cardiovascular disease and modulation by statin therapy. J Am Med Assoc. 2003; 289: 1675eC1680.
Buffon A, Biasucci LM, Liuzzo G, D’Onofrio G, Crea F, Maseri A. Widespread coronary inflammation in unstable angina. N Engl J Med. 2002; 347: 5eC12.
Baldus S, Heeschen C, Meinertz T, Zeiher AM, Eiserich JP, Munzel T, Simoons ML, Hamm CW. CAPTURE Investigators. Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation. 2003; 108: 1440eC1445.
Naruko N, Ueda M, Haze K, van der Wal AC, van der Loos CM, Itoh A, Komatsu R, Ikura Y, Ogami M, Shimada Y, Ehara S, Yishiyama M, Takeuchi K, Yoshikawa J, Becker AE. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation. 2002; 106: 2894eC2900.
Filep JG, Beauchamp M, Baron C, Paquette Y. Peroxynitrite mediates IL-8 gene expression and production in lipopolysaccharide-stimulated human whole blood. J Immunol. 1998; 161: 5656eC5662.
Zouki C, Je畓sef L, Ouellet S, Paquette Y, Filep JG. Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes. J Leuk Biol. 2001; 69: 815eC824.
Matata BM, Galinanes M. Peroxynitrite is an essential component of cytokines production mechanism in human monocytes through modulation of nuclear factor-kappa B DNA binding activity. J Biol Chem. 2002; 277: 2330eC2335.
Je畓sef L, Zouki C, Petasis NA, Serhan CN, Filep JG. Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-B and AP-1 activation, and IL-8 gene expression in human leukocytes. Proc Natl Acad Sci U S A. 2002; 99: 13266eC13271.
Pasceri V, Willerson JT, Yeh ETH. Direct proinflammatory effects of C-reactive protein on human endothelial cells. Circulation. 2000; 102: 2165eC2168.
Pasceri V, Chang J, Willerson JT, Yeh ETH. Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerotic drugs. Circulation. 2001; 103: 2531eC2534.
Verma S, Li SH, Badiwala MV, Weisel RD, Fedak PW, Li RK, Dhillon B, Mickle DA. Endothelin antagonism and interleukin-6 inhibition attenuate the proatherogenic effects of C-reactive protein. Circulation. 2002; 105: 1890eC1896.
Khreiss T, Je畓sef L, Potempa LA, Filep JG. Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells. Circulation. 2004; 109: 2016eC2022.
Bharadwaj D, Stein MP, Volzer M, Mold C, Du Clos TW. The major receptor for C-reactive protein on leukocytes is Fc receptor type II. J Exp Med. 1999; 190: 585eC590.
Stein MP, Edberg JC, Kimberly RP, Mangan EK, Bharadwaj D, Mold C, Du Clos TW. C-reactive protein binding to FcRIIa on human monocytes and neutrophils is allele-specific. J Clin Invest. 2001; 105: 369eC376.
Heuertz RM, Schneider GP, Potempa LA, Webster RO. Native and modified C-reactive protein bind different receptors on human neutrophils. Int J Biochem Cell Biol. 2005; 37: 320eC3335.
Zouki C, Beauchamp M, Baron C, Filep JG. Prevention of in vitro neutrophil adhesion to endothelial cells through shedding of L-selectin by C-reactive protein and peptides derived from C-reactive protein. J Clin Invest. 1997; 100: 522eC529.
Heuertz RM, Webster RO. Role of C-reactive protein in acute lung injury. Mol Med Today. 1997; 3: 539eC545.
Xia D, Samols D. Transgenic mice expressing rabbit C-reactive protein (CRP) are resistant to endotoxemia. Proc Natl Acad Sci U S A. 1997; 94: 2575eC2580.
Khreiss T, Je畓sef L, Hossain S, Chan JSD, Potempa LA, Filep JG. Loss of pentameric symmetry of C-reactive protein is associated with delayed apoptosis of human neutrophils. J Biol Chem. 2002; 277: 40775eC40781.
Potempa LA, Siegel NJ, Fiedel BA, Potempa RT, Gewurz H. Expression, detection and assay of neoantigen (neo-CRP) associated with a free C-reactive protein subunit. Mol Immunol. 1987; 24: 531eC541.
Wheeler MA, Smith SD, Garcia-Cardena G, Nathan CF, Weiss RM, Sessa WC. Bacterial infection induces nitric oxide synthase in human neutrophils. J Clin Invest. 1997; 99: 110eC116.
Kooy NW, Royall JA, Ischiropoulos H, Beckman JS. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Rad Biol Med. 1994; 16: 149eC156.
Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with generally improved fluorescence properties. J Biol Chem. 1985; 260: 3440eC3450.
Mukaida N, Okamoto S, Ishikawa Y, Matsushima K. Molecular mechanism of interleukin-8 gene expression. J Leuk Biol. 1994; 56: 554eC558.
Szabe?C. The role of peroxynitrite in the pathophysiology of shock, inflammation and ischemia-reperfusion injury. Shock. 1996; 6: 79eC88.
Wang HW, Sui SF. Dissociation and subunit rearrangement of membrane-bound human C-reactive proteins. Biochem Biophys Res Commun. 2001; 288: 75eC79.
Cuthbertson BH, Galley HF, Webster NR. The effects of nitric oxide and peroxynitrite on interleukin-8 and elastase from lipopolysaccharide-stimulated whole blood. Anesth Analg. 1998; 86: 427eC431.
Iho S, Tanaka Y, Takauji R, Kobayashi C, Muramatsu I, Iwasaki H, Nakamura K, Sasaki Y, Nakao K, Takahashi T. Nicotine induces human neutrophils to produce IL-8 through the generation of peroxynitrite and subsequent activation of NF-B. J Leukoc Biol. 2003; 74: 942eC951.
Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B, van der Vliet A. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature. 1998; 391: 393eC397.
Brennan ML, Wu W, Fu X, Shen Z, Song W, Frost H, Vadseth C, Narine L, Lenkiewicz E, Borchers MT, Lusis AJ, Lee JJ, Lee NA, Abu-Soud HM, Ischiropoulos H, Hazen SL. A tale of two controversies: defining the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species. J Biol Chem. 2002; 277: 17415eC17427.
Galinanes M, Matata BM. Protein nitration is predominantly mediated by a peroxynitrite-dependent pathway in cultured human leucocytes. Biochem J. 2002; 367: 467eC473.
Schmidt HHHW, Seifert R, Bhme E. Formation and release of nitric oxide from human neutrophils and Hl-60 cells induced by a chemotactic peptide, platelet-activating factor and leukotriene B4. FEBS Lett. 1989; 244: 357eC360.
Wallerath T, Gath I, Aulitzky WE, Pollock JS, Kleinert H, Frstermann U. Identification of the NO synthase isoforms expressed in human neutrophil granulocytes, megakaryocytes and platelets. Thromb Haemostasis. 1997; 77: 163eC167.
Evans TJ, Buttery LDK, Carpenter A, Springall DR, Polak JM, Cohen J. Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingested bacteria. Proc Natl Acad Sci U S A. 1996; 93: 9553eC9558.
Sechez-Mejorada G, Rosales C. Signal transduction by immunoglobulin Fc receptors. J Leukoc Biol. 1998; 63: 521eC533.
Schreck R, Meier B, Mannel DN, Drge W, Bauerle PA. Dithiocarbamates as potent inhibitors of nuclear factor B activation in intact cells. J Exp Med. 1992; 175: 1181eC1194.
Suzuki YJ, Mitzuni M, Packer L. Signal transduction for nuclear factor-B activation. Proposed location of antioxidant-inhibitable step. J Immunol. 1994; 153: 5008eC5015.
Diehl EE, Haines GK, Radosevich JA, Potempa LA. Immunuohistochemical localization of modified C-reactive protein antigen in normal human vascular tissue. Am J Med Sci. 2000; 319: 79eC83.
Rees RF, Gewurz H, Siegel JN, Coon J, Potempa LA. Expression of a C-reactive protein neo-antigen (neo-CRP) in inflamed rabbit liver and muscle. Clin Immunol Immunopathol. 1988; 48: 95eC107.
Schwedler S, Guderian F, Dammrich J, Potempa LA, Wanner C. Tubular staining of modified C-reactive protein in diabetic chronic kidney disease. Nephrol Dial Transplant. 2003; 18: 2300eC2307.(Tarek Khreiss, Levente Je)
Immtech International, Inc (L.A.P.), Vernon Hills, Ill.
Abstract
Plasma levels of C-reactive protein (CRP), nitrotyrosine, and interleukin-8 (IL-8) are known predictors of acute cardiovascular events. Peroxynitrite (ONOOeC) may function as an intracellular signal for the production of IL-8; however, it is not known whether CRP regulates these events. Emerging evidence suggests that some bioactivities of CRP are expressed only when the pentameric structure of CRP is lost, resulting in formation of monomeric or modified CRP (mCRP). We studied the impact of human native CRP and bioengineered mCRP that cannot rearrange into the pentameric structure on ONOOeC formation and ONOOeC-mediated IL-8 gene expression in human leukocytes. Incubation of human whole blood or isolated neutrophils with mCRP (0.1 to 100 e蘥/mL) for 4 hours increased IL-8 gene expression and secretion that was blocked 70% by the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME). In neutrophils, mCRP simultaneously increased superoxide production and endothelial nitric oxide synthase-mediated NO formation, leading to enhanced ONOOeC formation, and consequently activation of nuclear factor-B and activator protein-1. Native CRP had no detectable effect at 4 hours, whereas it enhanced IL-8 release after a 24-hour incubation that was blocked by L-NAME. An anti-CD16 antibody, but not an anti-CD32 antibody, produced 60% to 70% reductions in mCRP-stimulated NO formation and IL-8 release (both P<0.05). These results suggest that loss of the pentameric symmetry in CRP, resulting in formation of mCRP, leads to IL-8 release from human neutrophils via peroxynitrite-mediated activation of nuclear factor-B and activator protein-1.
Key Words: C-reactive protein leukocytes interleukins signal transduction inflammation
Introduction
Epidemiological and clinical studies have shown strong and consistent relationships between circulating markers of inflammation and risk prediction of future coronary artery disease (CAD). Among these markers, elevated plasma levels of C-reactive protein (CRP) are predictive for subsequent acute coronary events among apparently healthy men and women and patients with stable or unstable angina.1,2 The C-X-C chemokine interleukin-8 (IL-8) is a sensitive marker of unstable CAD.3 Increases in IL-8 levels may coincide with increased CRP levels,4 though IL-8 does not directly stimulate CRP gene expression.5,6 Increased plasma levels of nitrotyrosine, a "molecular fingerprint" for nitric oxide-derived oxidants,7 are also associated with the presence of CAD.8 Statin therapy produced reductions in nitrotyrosine concentrations similar to those in total cholesterol and CRP.8 It is uncertain, however, whether changes in these inflammatory markers occur independently or whether they are interrelated.
Widespread neutrophil activation has also been detected in patients with acute CAD, though the existence of a correlation between neutrophil activation and plasma CRP remains controversial.9,10 Neutrophil infiltration into plaques was found to be actively associated with acute coronary events.11 Recent results suggest that peroxynitrite (ONOOeC), formed in a reaction of superoxide with NO,7 functions as an intracellular messenger to mediate IL-8 gene expression in lipopolysaccharide (LPS) or cytokine-stimulated human leukocytes through activation of the transcription factors nuclear factor (NF)-B and activator protein-1 (AP-1).12eC15 CRP induces cytokine release from endothelial cells.16eC18 Recent results suggest that conformational rearrangement in native CRP, leading to the formation of monomeric or modified CRP (mCRP), is required for activation of endothelial cells.19 CRP and mCRP bind to the distinct receptors FcRIIa (CD32) and FcRIII (CD16), respectively, on human neutrophils20eC22 and exert opposing actions. For instance, native CRP inhibits neutrophil activation, adherence, and trafficking,5,23eC25 whereas mCRP promotes neutrophil adhesion to endothelial cells19 and suppresses neutrophil apoptosis.26
In the present study, we investigated the impact of native pentameric CRP and mCRP on IL-8 production in human whole blood and isolated neutrophils. To gain insight into the underlying molecular mechanisms in neutrophils, we characterized the immunoglobulin (Ig) G receptor subtype (ie, the CRP receptor) involved and examined whether induction of IL-8 gene and protein expression is mediated through stimulation of ONOOeC-dependent activation of NF-B and AP-1.
Materials and Methods
CRP Isoforms
High purity (>99%) human native CRP (Calbiochem) was stored in NaN3-free 20 mmol/L Tris, 150 mmol/L NaCl buffer (pH 7.5) containing 2 mmol/L CaCl2 to prevent spontaneous formation of mCRP from the native pentamer. A recombinant form of mCRP (rmCRP, purity >97%) that cannot rearrange into a pentameric structure was engineered, characterized, and compared with mCRP prepared from native CRP by urea chelation as described.26 Native CRP was distinguished from mCRP by binding and antigenicity differences27 and by their secondary structure.26 The endotoxin level of all protein solutions was below the detection limit (0.125 endotoxin units/mL, corresponding to 0.01 ng/mL LPS) of the Limulus assay (Sigma).
Cell Stimulation
Venous blood (anticoagulated with sodium heparin, 50 U/mL) was obtained from 24 healthy volunteers who had denied taking any medication for at least 2 weeks. The Clinical Research Committee approved the experimental protocols. Neutrophil granulocytes (purity >95%, viability >97%) were isolated as described.23 Whole blood aliquots or isolated neutrophils (5x106 cells/mL) in microcentrifuge tubes were placed on a rotator and challenged with native CRP or mCRP with or without the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) at 37°C in 5% CO2 atmosphere. In additional experiments, the responses to CRP and mCRP were studied in the presence of the specific NF-B inhibitor pyrrolidine dithiocarbamate (PDTC, 100 e蘭ol/L), function-blocking anti-CD16 monoclonal antibody (mAb) 3G8 (isotype: IgG1), anti-CD32 mAb FLI-8.26 (isotype: IgG1), or the irrelevant class-matched MOPC-21 antibody (all at 2.5 e蘥/mL, BD PharMingen). At the designated time points, the plasma was harvested and stored at eC20°C for later cytokine analysis. Cells were processed as described below.
Measurements of IL-8 mRNA Expression and Secretion
Levels of IL-8 in plasma or culture medium were determined by a selective enzyme immunoassay (OptEIA, BD Biosciences). Intra-assay and interassay coefficients of variation were typically <4% and <6%, respectively. IL-8 and GAPDH mRNA expression was assayed in RNase protection assays with the Direct Protect kit (Ambion) as described previously.12
Nitric Oxide and Superoxide Formation
Intracellular formation of NO was monitored by flow cytometry after incorporation of diaminofluorescein diacetate (5 e蘭ol/L, Clontech) into neutrophils.15 Superoxide production was determined as superoxide dismutase-inhibitable reduction of ferricytochrome c.15
Expression of NO Synthase Isoforms
Total RNA was isolated from 1x107 neutrophils using TriZol reagent (Invitrogen). cDNA was prepared with superscript reverse transcriptase (Invitrogen). The following primers were used for subsequent PCR analysis: human inducible NOS (iNOS), sense 5'-TCTCTCGGCCACCTTTGATGAG-3' and antisense 5'-GGTTGCATCCAGCTTGACCAG-3'; human endothelial NOS (eNOS), sense 5'-GTGATGGCGAAGCGAGTGAAG-3' and antisense 5'-CCGAGCCCGAACACACAGAAC-3'; human neuronal NOS, sense 5'-CTTCTGGCAACAGCGGCAATTTG-3' and antisense 5'-TGGACTCAGATCTAAGGCGGTTG-3'; -actin, sense 5'-ATGCCATCCTGCGTCTGGAC-3' and antisense 5'-AGCATTTGCGGTGCACGATGG-3'.28 The corresponding 412-base pair, 421-base pair, 458-base pair, and 500-base pair fragments were amplified enzymatically by 40, 35, 40, and 25 repeated cycles, respectively, and subjected to electrophoresis on 1.2% agarose containing ethidium bromide.
Detection of Peroxynitrite
NO-dependent fluorescence of rhodamine, an oxidation product of dihydrorhodamine 123 (DHR 123),29 and nitrotyrosine formation were measured as markers of ONOOeC formation. DHR 123 (20 e蘭ol/L, Molecular Probes) was added to some samples during the last 60 minutes of incubation in the presence or absence of L-NAME (1 mmol/L), and fluorescence was analyzed by a flow cytometer (FACScan, Becton Dickinson).12 The NO synthase blocker-inhibitable proportion of DHR oxidation can be attributed to ONOOeC because ONOOeC readily oxidizes DHR 123, whereas NO does not.29 DHR 123 oxidation by mCRP was also determined in neutrophils pretreated with the calmodulin inhibitor W7 (20 e蘭ol/L), the broad PKC inhibitor GF109203X (200 nM), the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin (100 nM), or the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI, 5 e蘭ol/L). In additional experiments, neutrophil extracts (prepared as in reference15) were analyzed for the presence of nitrotyrosine, a "molecular fingerprint"7 of ONOOeC by an enzyme immunoassay (detection limit: 2 ng/mL) (Cayman Chemicals) using nitrotyrosine as standard.15 Intra-assay and interassay coefficients of variation were typically <6%.
NF-B and AP-1
Intranuclear, DNA bound, NF-B/p65 and AP-1/c-Fos were measured with a flow cytometry assay13,15 and were used as estimates of NF-B and AP-1 activation, respectively. In brief, leukocyte nuclei prepared with the Cycletest Plus DNA reagent kit (Becton Dickinson) were stained with rabbit polyclonal anti-human NF-B/p65 or c-Fos antibodies or with normal rabbit IgG (to assess nonspecific binding of IgG to nuclei) and then with fluorescein isothiocyanateeCconjugated anti-rabbit IgG antibody (all from Santa Cruz Biotechnology) and propidium iodide. Singlet neutrophil nuclei were gated using the doublet-discrimination module and fluorescence intensity was analyzed with a FACScan flow cytometer using the Cell Quest Pro software.
Calcium Mobilization Assay
Intracellular Ca2+ concentration was monitored in Fura-2/AM (1 e蘭ol/L)-loaded neutrophils in a Perkin-Elmer spectrofluorometer (excitation: 340 nm, emission: 510 nm) as previously described.30
Western Blot Analysis
Protein extracts were prepared by lysing 2x106 neutrophils in 100 e蘈 of lysis buffer, and immunoblot analysis of phosphorylated and total Akt was performed using the Phospho Plus Akt antibody kit (New England Biolabs) as described.26
Neutrophil Viability
Neutrophil viability was assessed by flow cytometry immediately after staining with propidium iodide (0.5 e蘥/mL).
Statistical Analysis
Results are expressed as mean±SEM. Statistical comparisons were made by ANOVA using ranks (Kruskal-Wallis test) followed by Dunn’s multiple contrast hypothesis tests to identify differences between various treatments. Repeated measures were analyzed by the Friedman test followed by the Wilcoxon-Wilcon test. Values of P<0.05 were considered significant.
Results
Effects of CRP and mCRP on IL-8 Production and IL-8 mRNA Expression
Incubation of human whole blood with mCRP for 4 hours resulted in concentration-dependent IL-8 release, whereas native CRP was without effect (Figure 1). Significant induction was detected with 5 e蘥/mL, which peaked at 100 e蘥/mL mCRP (Figure 1B). Native CRP started to increase IL-8 release only after 8 hours of incubation; however, it was a considerable less potent inducer of IL-8 production than mCRP at any time points studied (Figure 1A). Both mCRP-and CRP-induced IL-8 release were markedly, though never completely, inhibited by L-NAME (Figure 1A). Likewise, mCRP, but not CRP, induced concentration-dependent IL-8 release from isolated neutrophils after 4 hours of incubation (Figure 1C). On a molar basis, bioengineered mCRP and mCRP prepared from native CRP evoked similar IL-8 release (Figure 1D). Recombinant mCRP-induced (50 e蘥/mL) IL-8 release (5.6±0.7 ng/mL) was unaffected by the formyl-peptide receptor antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe (50 e蘭ol/L) (5.4±0.5 ng/mL, n=6, P>0.1). Therefore, because of the enhanced solubility, bioengineered mCRP was used in subsequent experiments. Heat inactivation of native CRP (60 minutes at 100°C) resulted in a complete loss of its activity (data not shown). Also, LPS (Escherichia coli serotype O111:B4) at 0.02 ng/mL, a concentration 2-fold higher than the maximum level of LPS contamination in our protein solutions, did not induce detectable IL-8 release (0.24±0.03 ng/mL versus 0.23±0.03 ng/mL in unstimulated neutrophils, n=4, P>0.1).
We performed RNase protection assays on RNA extracted from leukocytes after 4 hours of incubation with mCRP. Consistent with the observations at the protein level, mCRP evoked concentration-dependent increases in IL-8 mRNA expression, which were suppressed by L-NAME (Figure 2). Native CRP at 100 e蘥/mL did not produce detectable changes (Figure 2).
mCRP Induces NO, Superoxide, and Peroxynitrite Formation in Neutrophils
Incubation of neutrophils for 4 hours with mCRP led to simultaneous increases in O2·eC and NO production. Significant increases were detected with 5 e蘥/mL and peaked at 100 e蘥/mL mCRP (Figure 3). The increases in O2·eC and NO production coincided with increases in NO-dependent DHR123 oxidation, indicating enhanced ONOOeC formation (Figure 3). Addition of the NO donor spermine NONOate (0.5 mmol/L) to L-NAME-treated neutrophils restored DHR 123 oxidation (rhodamine fluorescence in relative fluorescence units, control: 22±4; mCRP: 50±4; L-NAME+mCRP: 29±3; spermine NONOate+L-NAME+mCRP: 48±4; n=4; P>0.1 compared with mCRP). To confirm ONOOeC formation, we measured cellular nitrotyrosine content. Neutrophils exposed to mCRP for 4 hours contained significantly higher amounts of nitrotyrosine than unchallenged cells, and the increases in nitrotyrosine correlated with those in DHR 123 oxidation (Figure 3).
To identify the NOS isoform(s) responsible for enhanced NO production, we performed reverse transcription polymerase chain reaction on cDNA prepared from neutrophils. These assays resulted in the amplification of eNOS mRNA, but not neuronal NOS and iNOS, in freshly isolated cells and in response to mCRP at 2 and 4 hours after addition of the protein (Figure 4). mCRP did not affect eNOS mRNA expression (Figure 4).
To characterize the proximal signaling events associated with mCRP-induced ONOOeC formation, we monitored calcium mobilization. mCRP evoked a rapid increase in intracellular Ca2+ similar to that observed with fMLP (Figure 5A), and transiently enhanced (peak at around 2 minutes) phosphorylation of Akt (Figure 5B), indicating phosphatidylinositol 3-kinase activation. Consistently, calmodulin blockade with W7 or inhibition of PI3-kinase with wortmannin significantly reduced mCRP-induced DHR 123 oxidation, and these actions were not additive with L-NAME (Figure 5C). Inhibition of NADPH oxidase with DPI effectively attenuated mCRP-evoked DHR 123 oxidation to the same degree in the absence and presence of L-NAME (Figure 5C). By contrast, the PKC inhibitor GF109203X produced only modest decreases in mCRP-induced DHR 123 oxidation that were further enhanced by L-NAME (Figure 5C).
mCRP Stimulates Nuclear Accumulation of AP-1 and NF-B in Neutrophils
Because transcription of IL-8 gene requires activation of NF-B and AP-1,31 we examined whether mCRP can activate these pathways in neutrophils. We prepared nuclei from unstimulated and mCRP-stimulated neutrophils and analyzed nuclear accumulation of NF-B and AP-1 using flow cytometry. Figure 6A shows representative results illustrating mobilization of NF-B/p65 and AP-1/c-Fos to the nucleus (ie, the increased fluorescence represents increased amounts of NF-B or AP-1 bound to DNA) in response to mCRP. Nuclear immunostaining was completely blocked by preincubation of the antibodies with the appropriate blocking peptides. The actions of mCRP were concentration-dependent and were markedly attenuated by L-NAME (Figure 6A and 6B), consistent with inhibition of IL-8 mRNA expression and production.
Preincubation of neutrophils with PDTC markedly attenuated, though never completely inhibited, mCRP-induced IL-8 production (Figure 7), confirming that increased nuclear accumulation of NF-B correlated with induction of IL-8 production.
Involvement of CD16 in mCRP Signaling
Previous studies identified CD16 (FcRIII) as a neutrophil receptor for mCRP.22,26 We used function-blocking mAbs to CD16 and CD32 (FcRIIa) as competitors to confirm whether the mCRP actions described above were also mediated via CD16. The anti-CD16 mAb partially prevented mCRP-induced NO formation assessed at 4 hours, and IL-8 release assessed at 4 and 24 hours after addition of mCRP, whereas the anti-CD32 or the irrelevant MOPC-21 antibody had no detectable effects (Figure 8). Increasing the concentration of the anti-CD16 mAb did not result in further inhibition (data not shown). At 24-hours culture, native CRP-induced IL-8 release was also attenuated by the anti-CD16 mAb, but not by the anti-CD32 mAb or MOPC-21 (Figure 8B).
Effect of mCRP on Neutrophil Viability
Because increased NO and ONOOeC formation could affect cell viability,7,32 we assessed neutrophil survival. Consistent with previous studies,26 after 4 hours culture in vitro, the percentage of viable neutrophils was slightly higher in the presence of mCRP (95±2%, n=5, P<0.05) than in untreated (control) samples (89±2%) or in the presence of native CRP (88±3%, n=5, P<0.05 versus mCRP).
Discussion
The present results provide evidence for a novel mechanism by which CRP may affect the inflammatory process by stimulating ONOOeC formation and signaling in human neutrophils. These actions are expressed when the pentameric structure undergoes a conformational rearrangement, however, leading to formation of mCRP. Our results also suggest a potential link between CRP, IL-8, nitrotyrosine, and neutrophil activation, as it may occur in the blood of patients with acute CAD.
Our study using human whole blood and isolated neutrophils demonstrates that mCRP stimulates a rapid (within 4 hours) and potent synthesis of IL-8. This is a primary response to mCRP that requires de novo protein synthesis and transcription of the IL-8 gene. Comparison of plasma and culture medium IL-8 levels suggests that 70% of IL-8 release was of neutrophil origin in blood. Native CRP did not evoke detectable changes at 4 hours. This was unexpected, for native CRP induced rapid (within minutes) shedding of L-selectin from the surface of neutrophils,23 and cannot be explained by the slight differences in the viability of mCRP or CRP-treated neutrophils. Human blood contains all the yet unidentified serum cofactors that were required for CRP activation of endothelial cells.16 The CRP stimulation became detectable only after 8 hours of incubation, coinciding with in vitro kinetics of dissociation into subunits.33 Although CRP clearly enhanced IL-8 production at 8 to 24 hours of incubation, it was a considerably less potent inducer of IL-8 release than mCRP. These observations suggest that conformational rearrangement of CRP is required to induce IL-8 production in neutrophils, and that the amounts of mCRP generated from CRP within 4 hours are not sufficient to evoke detectable increases in IL-8. The time course of neutrophil activation by native CRP appears to be similar to that of CRP activation of endothelial cells.19 Neither LPS (at a concentration 2-fold higher than might be present in our protein preparations) nor heat-inactivated CRP evoked detectable IL-8 release, indicating that CRP and mCRP signaling was responsible for the observed effects.
Blockade of NO synthesis with L-NAME inhibited to a similar degree mCRP-induced IL-8 release and nuclear accumulation of NF-B and AP-1 in neutrophils, coinciding with suppression of IL-8 mRNA expression. Interestingly, L-NAME also attenuated native CRP-induced IL-8 release, and the degree of inhibition was comparable to that detected with mCRP. These results pointed to the involvement of NO in mediating these responses. Previous studies have shown that ONOOeC rather than NO by itself mediates IL-8 release from human neutrophils in response to LPS or cytokines.12,34,35 Accordingly, we found that mCRP simultaneously enhanced superoxide and NO formation, coinciding with increases in NO-dependent oxidation of DHR123 and nitration of protein tyrosine residues. A significant portion of rhodamine fluorescence in mCRP-stimulated neutrophils can be attributed to ONOOeC, because it depends on NO-related species (it can be inhibited by L-NAME), whereas NO per se does not oxidize DHR 123.29 Further, the NO donor spermine NONOate restored DHR 123 oxidation in L-NAMEeCtreated neutrophils, indicating that L-NAME does not inhibit NADPH oxidase activation. Although nitrotyrosine is often considered as a distinct "molecular fingerprint" of ONOOeC formation,7 peroxidase-dependent tyrosine nitration has also been described.36,37 Interestingly, in human neutrophils, ONOOeC appears to be the predominant mechanism for tyrosine nitration.38
Reverse-transcriptase polymerase chain reaction amplified eNOS, but not iNOS and neuronal NOS-specific products, in unchallenged neutrophils and in neutrophils treated with mCRP for up to 4 hours. These results point toward increased eNOS activity as the source for enhanced NO production in response to mCRP. Previous studies on constitutive NOS expression in human neutrophils yielded contradictory results, as both the absence and presence of eNOS and neuronal NOS have been reported.28,39eC41 Contaminating cells or differences in the NOS assays used might account for this apparent discrepancy. Unstimulated human neutrophils do not express iNOS, whereas iNOS-positive neutrophils have been detected in tissue exudates28,41 and after more than 16 hours incubation with cytokines.41 Whether mCRP could induce iNOS expression after prolonged incubation periods remains to be investigated. There is compelling evidence that eNOS-derived NO contributes to ONOOeC formation in amounts sufficient to activate signaling mechanisms and even to induce cell damage.32
Ca2+ mobilization and activation of calmodulin and PI3-kinase appears to be required for mCRP-induced ONOOeC formation. Ca2+ transients control calmodulin-mediated eNOS activation and the activity of PLC1 and PLC2, which through formation of diacylglycerol and activation of PI3-kinase lead to activation of NADPH oxidase.42 The inhibitory actions of W7, wortmannin, and DPI on DHR 123 oxidation were not additive with those of L-NAME, suggesting that these compounds inhibited the same reaction, ie, the ONOOeC-dependent oxidation through suppression of either NO or O2·eC formation. PKC appears to play a minor role in mCRP signaling, for GF109203X inhibited only a small portion of DHR 123 oxidation by mCRP.
The IL-8 gene contains cis-regulatory elements for NF-B, AP-1, and NF-IL-6.31 Of these transcription factors, NF-B plays a key role in the induced expression of IL-8. Accordingly, we found that mCRP stimulates nuclear accumulation of NF-B and AP-1, and inhibition of NF-B activation with PDTC decreased IL-8 production by 66%. The mechanism of action of PDTC has not been fully defined, but likely involves inhibition of formation of oxidants that would result in activation of IB- kinase and/or enhancing phosphorylation of I-B.43,44 Thus, NF-B activation could be attributed to decomposition products of ONOOeC rather than the parent molecule itself.
Our results indicate that mCRP-induced IL-8 gene expression and release are predominantly mediated through the low affinity immune complex binding IgG receptor CD16, for the function-blocking anti-CD16 mAb 3G8, but not anti-CD32 mAb, markedly, though never completely, inhibited NO formation and IL-8 production in neutrophils. Furthermore, only the anti-CD16 mAb attenuated native CRP-induced IL-8 release at 24 hours incubation. These observations imply that during 24-hours culture, conformational rearrangement might have occurred in native CRP, yielding mCRP, because native CRP does not bind to CD16.20eC22 We cannot exclude the possibility that mCRP may bind different sites from IgG on CD16, for blockade of the epitope defined by the mAb 3G8 reduced 80% of mCRP binding at 0°C.22 Alternatively, mCRP might interact with other as yet unidentified cell surface molecules. These may include direct binding to positively charged residues on proteins and direct interaction with the lipid membrane (Potempa et al, unpublished observations, 2005). The slight inhibition of mCRP activation of endothelial cells by anti-CD16 mAb19 lends support to the existence of mCRP receptor(s) other than CD16.
The mechanisms that induce conformational rearrangement in native CRP in vivo are still unknown. Native, pentameric CRP dissociates into free subunits after binding to plasma membranes33 or in denaturating or oxidative environment,27 yielding mCRP. The percentage of CRP that might have dissociated into subunits during a 24-hour incubation period, however, remains to be determined. Unlike CRP, mCRP appears to be predominantly membrane-bound,45,46 therefore making its detection rather difficult. Because the commercial CRP antibody (clone 8) mainly recognizes mCRP,47 tissue immunostaining attributed to native CRP in previous studies might have been due to mCRP. Neutrophils may come into contact with mCRP expressed in the arterial wall at sites of endothelial injury45 or perhaps on other leukocytes, thereby aggravating the inflammatory response and contributing to plaque destabilization.11 Stimulated neutrophils release ONOOeC, though the ratio of intracellularly retained and released ONOOeC cannot be deduced.12 Increased ONOOeC release is consistent with enhanced nitration of plasma proteins and may contribute to tissue damage. Finally, our results do not exclude contribution of other cells to elevated plasma levels of nitrotyrosine and IL-8. Indeed, mononuclear leukocytes and endothelial cells also release IL-8 in response to native CRP or mCRP, thereby increasing plasma cytokine levels in patients with CAD.
In summary, the present results indicate that loss of pentameric symmetry in CRP, resulting in formation of mCRP, stimulates IL-8 production by human neutrophils through ONOOeC-mediated activation of NF-B and AP-1. These observations point toward neutrophils as a major source of nitrosative stress and IL-8 and may provide a potential link between CRP, neutrophil activation, plasma nitrotyrosine, and IL-8, all implicated in predicting future acute CAD.
Acknowledgments
This study was supported by grants (to J.G.F.) and Doctoral Research Awards (to T.K. and L.J.) from the Canadian Institutes of Health Research (MOP-64283) and the Heart and Stroke Foundation of Quebec.
Dr Potempa was employed by NextEra Therapeutics, and is currently employed by Immtech International Inc, of Vernon Hills, Ill. Immtech is a minority owner of the modified C-reactive protein technology. Dr Potempa received support for his work on modified C-reactive protein from Immtech (until 1999) and from NextEra (from 1998 to 2002).
Both authors contributed equally to this study.
References
Bassuk SS, Rifai N, Ridker PM High-sensitivity C-reactive protein: clinical importance. Curr Probl Cardiol. 2004; 29: 439eC493.
Danesh J, Wheeler JG, Hirschfield G, Eda S, Eiriksdottir G, Rumley A, Lowe GD, Pepys MB, Gudnason V. C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med. 2004; 350: 1387eC1397.
Kanda T, Hirao Y, Oshima S, Yuasa K, Taniguchi K, Nagai R, Kobayashi I. Interleukin-8 as a sensitive marker of unstable coronary artery disease. Am J Cardiol. 1996; 77: 304eC307.
Kushner I, Broder ML, Karp D. Control of the acute-phase response. Serum C-reactive protein kinetics after acute myocardial infarction. J Clin Invest. 1978; 61: 235eC242.
Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med. 1999; 340: 448eC454.
Black S, Kushner I, Samols D. C-reactive protein. J Biol Chem. 2004; 279: 48487eC48490.
Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol. 1996; 271: C1424eCC1437.
Shishehbor MH, Aviles RJ, Brennan ML, Fu X, Goormastic M, Pearce GL, Gokce N, Keaney JF, Penn MS, Sprecher DL, Vita JA, Hazen SL. Association of nitrotyrosine levels with cardiovascular disease and modulation by statin therapy. J Am Med Assoc. 2003; 289: 1675eC1680.
Buffon A, Biasucci LM, Liuzzo G, D’Onofrio G, Crea F, Maseri A. Widespread coronary inflammation in unstable angina. N Engl J Med. 2002; 347: 5eC12.
Baldus S, Heeschen C, Meinertz T, Zeiher AM, Eiserich JP, Munzel T, Simoons ML, Hamm CW. CAPTURE Investigators. Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation. 2003; 108: 1440eC1445.
Naruko N, Ueda M, Haze K, van der Wal AC, van der Loos CM, Itoh A, Komatsu R, Ikura Y, Ogami M, Shimada Y, Ehara S, Yishiyama M, Takeuchi K, Yoshikawa J, Becker AE. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation. 2002; 106: 2894eC2900.
Filep JG, Beauchamp M, Baron C, Paquette Y. Peroxynitrite mediates IL-8 gene expression and production in lipopolysaccharide-stimulated human whole blood. J Immunol. 1998; 161: 5656eC5662.
Zouki C, Je畓sef L, Ouellet S, Paquette Y, Filep JG. Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes. J Leuk Biol. 2001; 69: 815eC824.
Matata BM, Galinanes M. Peroxynitrite is an essential component of cytokines production mechanism in human monocytes through modulation of nuclear factor-kappa B DNA binding activity. J Biol Chem. 2002; 277: 2330eC2335.
Je畓sef L, Zouki C, Petasis NA, Serhan CN, Filep JG. Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-B and AP-1 activation, and IL-8 gene expression in human leukocytes. Proc Natl Acad Sci U S A. 2002; 99: 13266eC13271.
Pasceri V, Willerson JT, Yeh ETH. Direct proinflammatory effects of C-reactive protein on human endothelial cells. Circulation. 2000; 102: 2165eC2168.
Pasceri V, Chang J, Willerson JT, Yeh ETH. Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerotic drugs. Circulation. 2001; 103: 2531eC2534.
Verma S, Li SH, Badiwala MV, Weisel RD, Fedak PW, Li RK, Dhillon B, Mickle DA. Endothelin antagonism and interleukin-6 inhibition attenuate the proatherogenic effects of C-reactive protein. Circulation. 2002; 105: 1890eC1896.
Khreiss T, Je畓sef L, Potempa LA, Filep JG. Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells. Circulation. 2004; 109: 2016eC2022.
Bharadwaj D, Stein MP, Volzer M, Mold C, Du Clos TW. The major receptor for C-reactive protein on leukocytes is Fc receptor type II. J Exp Med. 1999; 190: 585eC590.
Stein MP, Edberg JC, Kimberly RP, Mangan EK, Bharadwaj D, Mold C, Du Clos TW. C-reactive protein binding to FcRIIa on human monocytes and neutrophils is allele-specific. J Clin Invest. 2001; 105: 369eC376.
Heuertz RM, Schneider GP, Potempa LA, Webster RO. Native and modified C-reactive protein bind different receptors on human neutrophils. Int J Biochem Cell Biol. 2005; 37: 320eC3335.
Zouki C, Beauchamp M, Baron C, Filep JG. Prevention of in vitro neutrophil adhesion to endothelial cells through shedding of L-selectin by C-reactive protein and peptides derived from C-reactive protein. J Clin Invest. 1997; 100: 522eC529.
Heuertz RM, Webster RO. Role of C-reactive protein in acute lung injury. Mol Med Today. 1997; 3: 539eC545.
Xia D, Samols D. Transgenic mice expressing rabbit C-reactive protein (CRP) are resistant to endotoxemia. Proc Natl Acad Sci U S A. 1997; 94: 2575eC2580.
Khreiss T, Je畓sef L, Hossain S, Chan JSD, Potempa LA, Filep JG. Loss of pentameric symmetry of C-reactive protein is associated with delayed apoptosis of human neutrophils. J Biol Chem. 2002; 277: 40775eC40781.
Potempa LA, Siegel NJ, Fiedel BA, Potempa RT, Gewurz H. Expression, detection and assay of neoantigen (neo-CRP) associated with a free C-reactive protein subunit. Mol Immunol. 1987; 24: 531eC541.
Wheeler MA, Smith SD, Garcia-Cardena G, Nathan CF, Weiss RM, Sessa WC. Bacterial infection induces nitric oxide synthase in human neutrophils. J Clin Invest. 1997; 99: 110eC116.
Kooy NW, Royall JA, Ischiropoulos H, Beckman JS. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Rad Biol Med. 1994; 16: 149eC156.
Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with generally improved fluorescence properties. J Biol Chem. 1985; 260: 3440eC3450.
Mukaida N, Okamoto S, Ishikawa Y, Matsushima K. Molecular mechanism of interleukin-8 gene expression. J Leuk Biol. 1994; 56: 554eC558.
Szabe?C. The role of peroxynitrite in the pathophysiology of shock, inflammation and ischemia-reperfusion injury. Shock. 1996; 6: 79eC88.
Wang HW, Sui SF. Dissociation and subunit rearrangement of membrane-bound human C-reactive proteins. Biochem Biophys Res Commun. 2001; 288: 75eC79.
Cuthbertson BH, Galley HF, Webster NR. The effects of nitric oxide and peroxynitrite on interleukin-8 and elastase from lipopolysaccharide-stimulated whole blood. Anesth Analg. 1998; 86: 427eC431.
Iho S, Tanaka Y, Takauji R, Kobayashi C, Muramatsu I, Iwasaki H, Nakamura K, Sasaki Y, Nakao K, Takahashi T. Nicotine induces human neutrophils to produce IL-8 through the generation of peroxynitrite and subsequent activation of NF-B. J Leukoc Biol. 2003; 74: 942eC951.
Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B, van der Vliet A. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature. 1998; 391: 393eC397.
Brennan ML, Wu W, Fu X, Shen Z, Song W, Frost H, Vadseth C, Narine L, Lenkiewicz E, Borchers MT, Lusis AJ, Lee JJ, Lee NA, Abu-Soud HM, Ischiropoulos H, Hazen SL. A tale of two controversies: defining the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species. J Biol Chem. 2002; 277: 17415eC17427.
Galinanes M, Matata BM. Protein nitration is predominantly mediated by a peroxynitrite-dependent pathway in cultured human leucocytes. Biochem J. 2002; 367: 467eC473.
Schmidt HHHW, Seifert R, Bhme E. Formation and release of nitric oxide from human neutrophils and Hl-60 cells induced by a chemotactic peptide, platelet-activating factor and leukotriene B4. FEBS Lett. 1989; 244: 357eC360.
Wallerath T, Gath I, Aulitzky WE, Pollock JS, Kleinert H, Frstermann U. Identification of the NO synthase isoforms expressed in human neutrophil granulocytes, megakaryocytes and platelets. Thromb Haemostasis. 1997; 77: 163eC167.
Evans TJ, Buttery LDK, Carpenter A, Springall DR, Polak JM, Cohen J. Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingested bacteria. Proc Natl Acad Sci U S A. 1996; 93: 9553eC9558.
Sechez-Mejorada G, Rosales C. Signal transduction by immunoglobulin Fc receptors. J Leukoc Biol. 1998; 63: 521eC533.
Schreck R, Meier B, Mannel DN, Drge W, Bauerle PA. Dithiocarbamates as potent inhibitors of nuclear factor B activation in intact cells. J Exp Med. 1992; 175: 1181eC1194.
Suzuki YJ, Mitzuni M, Packer L. Signal transduction for nuclear factor-B activation. Proposed location of antioxidant-inhibitable step. J Immunol. 1994; 153: 5008eC5015.
Diehl EE, Haines GK, Radosevich JA, Potempa LA. Immunuohistochemical localization of modified C-reactive protein antigen in normal human vascular tissue. Am J Med Sci. 2000; 319: 79eC83.
Rees RF, Gewurz H, Siegel JN, Coon J, Potempa LA. Expression of a C-reactive protein neo-antigen (neo-CRP) in inflamed rabbit liver and muscle. Clin Immunol Immunopathol. 1988; 48: 95eC107.
Schwedler S, Guderian F, Dammrich J, Potempa LA, Wanner C. Tubular staining of modified C-reactive protein in diabetic chronic kidney disease. Nephrol Dial Transplant. 2003; 18: 2300eC2307.(Tarek Khreiss, Levente Je)