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C-reactive protein enhances the respiratory burst of neutrophils-induced by antineutrophil cytoplasmic antibody
Molecular Immunology 52 (2012) 148–154
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C-reactive protein enhances the respiratory burst of neutrophils-induced by antineutrophil cytoplasmic antibody Peng-cheng Xu a,b,c,d,e , Jian Hao a,b,c,d , Xiao-wei Yang a,b,c,d , Dong-yuan Chang a,b,c,d , Min Chen a,b,c,d,∗ , Ming-hui Zhao a,b,c,d a
Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034, China Institute of Nephrology, Peking University, Beijing 100034, China c Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 100034, China d Key Laboratory of CKD, Prevention and Treatment, Ministry of Education of China, Beijing 100034, China e Department of Nephrology, General Hospital of Tianjin Medical University, Tianjin 300052, China b
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 14 April 2012 Accepted 15 May 2012 Available online 7 June 2012 Keywords: C-reactive protein Neutrophil Anti-neutrophil cytoplasmic antibody Vasculitis
a b s t r a c t Serum C-reactive protein (CRP) was one of the useful biomarkers for evaluating the disease activity in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Cumulating studies proved that CRP was pathogenic in a variety of diseases. In the current study, the in vitro effects of CRP to prime neutrophils for ANCA-induced respiratory burst were investigated with flow cytometry. Without TNF-␣ in the reactive system, ANCA could only induce a slight level of respiratory burst of neutrophils. CRP could enhance the respiratory burst of neutrophils induced by ANCA against myeloperoxidse [mean fluorescence intensity (MFI, 68.45 ± 16.87 vs. 58.65 ± 15.09, P < 0.05) or by ANCA against proteinase 3 (MFI, 79.51 ± 15.90 vs. 61.73 ± 14.89, P < 0.05). Although CRP (50 g/mL, incubating for 30 min) could not active neutrophils alone, after incubation with neutrophils for 10 min, CRP (50 g/mL) could increase the expression of membrane proteinase 3 of neutrophils (MFI, 365.27 ± 143.50 vs. 235.32 ± 124.65, P < 0.05). Heat-treated CRP could not enhance the levels of neutrophils respiratory burst induced by ANCA or increase the expression of membrane proteinase 3 of neutrophils. So CRP can prime neutrophils and enhance the respiratory burst induced by ANCA and might be pathogenic in AAV. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), serum C-reactive protein (CRP) was one of the useful biomarkers for evaluating the disease activity. The concentration of serum CRP was often elevated in active phase of AAV, and fell rapidly with remission of the disease (Hind et al., 1984; Kälsch et al., 2010). CRP is synthesized and catabolized in hepatocytes and is a highly soluble serum protein of the pentraxin family. During the onset of inflammation or tissue injury, serum CRP is always dramatically elevated up to 1000-fold or more within 24–72 h (Ballou and Kushner, 1992). Actually, after binding to plasma membranes or in a denaturating or oxidative environment, CRP might dissociate into free subunits yielding insoluble modified/monomeric CRP (mCRP) (Potempa et al., 1983; Devaraj et al., 2006).
∗ Corresponding author at: Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034, China. Tel.: +86 10 66551736; fax: +86 10 66551055. E-mail address: [email protected] (M. Chen). 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.05.012
Cumulating studies suggested the pathogenic role of CRP in a variety of diseases, such as metabolic syndrome, atherothrombosis, acute myocardial infarction, cerebral infarction (Gill et al., 2004) and kidney diseases (Trachtman et al., 2006). For example, CRP could increase the expression of adhesion molecules in endothelial cells, inhibit nitric oxide in aortic endothelial cells (Venugopal et al., 2002) and rat mesangial cells (Trachtman et al., 2006), stimulate IL-8 release from several cell types, increase plasminogen activator inhibitor-1 expression, and increase the release of IL-1, IL-6, IL-18, and TNF-␣ from monocytes (Eisenhardt et al., 2009). The most direct clinical evidence was that activation of inflammation in healthy volunteers was observed after infusion of recombinant human CRP (Bisoendial et al., 2005). Serum CRP levels were of prognostic value in some diseases such as bacterial infection, hypertension, metabolic syndrome, acute cardiovascular events, and rheumatoid arthritis (Haverkate et al., 1997; Ridker et al., 1997; Jones, 1995; Jialal et al., 2004). In AAV, several previous studies have reported that serum CRP level correlated closely with disease activity (Hind et al., 1984). However, whether CRP is pathogenic in AAV is still unclear yet. In AAV, ANCA can enhance the degranulation of neutrophils and increase the production of superoxide and pro-inflammatory
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cytokines of neutrophils. High level of CRP is a characteristic of active AAV and in vitro studies have documented specific receptors for CRP on neutrophils. pCRP binds to Fc␥RI (CD64) and Fc␥RIIa (CD32), while mCRP binds to Fc␥RIII (CD16) on human neutrophils, respectively (Khreiss et al., 2005; Kettritz et al., 1997; Falk et al., 1990). Since the neutrophils respiratory burst has been proved to play a pivotal role in AAV, the in vitro effects of CRP on the respiratory burst of neutrophils induced by ANCA were investigated in the current study. 2. Methods 2.1. Patients and plasma Patients’ plasma was obtained from 5 AAV patients with positive ANCA against myeloperoxidase (MPO-ANCA) and 5 AAV patients with positive ANCA against proteinase 3 (PR3-ANCA) at presentation. All the 10 patients were diagnosed in Peking University First Hospital and fulfilled the Chapel Hill Consensus Conference classification criteria of AAV (Jennette et al., 1994). All patients with MPO-ANCA and 1 out of 5 patients with PR3-ANCA had biopsyproven glomerulonephritis. Plasma from five healthy blood donors was collected as normal controls. All the plasma was stored at −20 ◦ C until use. The research was in compliance of the declaration of Helsinki and approved by the ethic committee of the hospital. Inform consent was obtained from each participant. 2.2. Purification of IgG fractions IgG fractions containing anti-MPO antibodies, anti-PR3 antibodies or IgG fractions of normal people were purified by protein G affinity column (Amersham Pharmacia, Sweden) with 0.01 mol/L phosphate-buffered saline (PBS, pH 7.4) as starting buffer and 0.1 mol/L glycine, pH 2.7 as eluting buffer, at a flow rate of 1 mL/min at room temperature. IgG was eluted and neutralized to pH 7.0 by 2 mol/L Tris–HCl, pH 9.0 immediately, and dialyzed against PBS.
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Table 1 General data of the patients with AAV. Patient no.
Gender
Age (years)
Titer of ANCA (lgT)
ANCA type
1 2 3 4 5 6 7 8 9 10
F M M F F M M M F M
56 59 62 67 62 72 67 68 21 50
3.51 3.51 2.60 4.41 3.81 2.0 2.0 2.6 2.0 2.9
MPO-ANCA MPO-ANCA MPO-ANCA MPO-ANCA MPO-ANCA PR3-ANCA PR3-ANCA PR3-ANCA PR3-ANCA PR3-ANCA
Abbreviations: ANCA, anti-neutrophil cytoplasm antibodies; MPO, myeloperoxidase; PR3, proteinase 3.
incubated with cytochalasin B (5 g/mL, Sigma) for 5 min at 37 ◦ C to enhance the oxygen radical production. Then, neutrophils were loaded with 0.05 mM DHR (Sigma) and 2 mM sodium azide (NaN3 ) at 37 ◦ C and primed with TNF-␣ (2 ng/mL) for 15 min at 37 ◦ C. The CRP or heat-treated CRP was added with a final concentration of 50 g/mL. The normal IgG, MPO-ANCA-containing IgG or PR3-ANCA-containing IgG was added with a final concentration of 100 g/mL. The reaction was stopped after 20 min by addition of 1 mL of ice-cold HBSS/1%BSA. We analyzed samples using Calibur flow cytometer (BD FACSCalibur). Data were collected from 20,000 cells per sample. The mean fluorescence intensity (MFI), representing the amount of generated oxygen radicals, was reported. All assays were taken in triplicate.
2.3. CRP preparation for in vitro study High purity (>99%) human native CRP was purchased from Sigma (C-4063; St. Louis, MO). Modified CRP was generated with native CRP by heating at 70 ◦ C for 30 min. After heating, the white deposit formed and was resuspended for the following experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 1/20 of standard SDS and Coomassie staining were used to confirm cleavage of CRP (Taylor and van den Berg, 2007). The endotoxin level of CRP was below the detection limit (0.125 EU/mL) of the Limulus assay (Sigma). 2.4. Neutrophils isolation Neutrophils from healthy donors were isolated from heparinized blood by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway) as previously described (Van Rossum et al., 2005), with some minor modifications. Briefly, erythrocytes were removed twice by hypotonic lysis with ice-cold ammonium chloride. Thereafter, cells were washed with ice-cold Hanks balanced salt solution (HBSS) without Ca2+ and Mg2+ , and resuspended in HBSS with Ca2+ and Mg2+ (2.5 × 106 /mL). 2.5. Measurement of respiratory burst by oxidation of dihydrorhodamine (DHR) to rhodamine The generation of reactive oxygen radicals using DHR was assessed as described previously (Kettritz et al., 2001), with some minor modifications. In brief, neutrophils (2.5 × 106 /mL HBSS) were
Fig. 1. Electrophoretic analysis of CRP and heat-treated CRP. All samples were subjected to PAGE with 1/20 SDS. Gel was stained with Coomassie brilliant blue. 1: Native CRP; 2: modified CRP produced with CRP by heating at 70 ◦ C for 30 min.
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Fig. 2. Effects to induce the respiratory burst of neutrophils of CRP without TNF-␣ in reactive system. (A) Effects to induce the respiratory burst of neutrophils of CRP. Hollow squares: without CRP or heat-treated CRP in reactive system; solid squares: with CRP in reactive system; gray squares: with heat-treated CRP in reactive system. htCRP: heat-treated CRP. *P < 0.05. (B and C) Representative flow cytometric analyses of respiratory burst of neutrophils induced by CRP and ANCA. The dashed curve represents baseline burst of neutrophils with normal IgG alone. The solid curve represents burst of neutrophils induced by MPO-ANCA-IgG alone (B) or PR3-ANCAIgG alone (C). The shadowed curve represents burst of neutrophils induced by CRP plus MPO-ANCA-IgG (B) or PR3-ANCA-IgG (C).
2.6. Membrane expression of ANCA target antigens on neutrophils after priming Membrane expression of PR3 and MPO were assessed using flow cytometry as previously described (Rarok et al., 2002) with some minor modifications. All steps were performed on ice. Briefly, samples containing 2.5 × 106 neutrophils/mL HBSS were incubated with 2 ng/mL TNF-␣ or 50 g/mL CRP or heat-treated CRP for 10 min at 37 ◦ C, then washed with HBSS/1% BSA by centrifugation at 1800 × g, 4 ◦ C for 5 min, and incubated with 0.5 mg/mL heatedaggregated goat IgG (Sigma) for 15 min to saturate Fc␥ receptors. Next, cells were stained with a saturation dose of mouse monoclonal IgG1 antibodies directed against human PR3 or MPO (R&D Systems Antibodies) or with an irrelevant IgG1 control antibody for 30 min. Next, non-bound antibodies were washed off with PBS/1%BSA. This step was followed by 30 min incubation with phycoerythrin (PE)-conjugated goat anti-mouse antibody (Southern Biotechnology Associates, Birmingham, AL, USA). MFI was calculated to represent the level of PR3- or MPO-expression on neutrophils. All assays were tested in triplicate. To study which receptors were involved in the CRP-mediated priming effects on neutrophils, the neutrophils were preincubated with 10 g/mL F(ab)2 fragments of mouse anti-human CD64 (Fc␥RI inhibitor, Ancell, Bayport, Minn), 10 g/mL F(ab)2 fragments of mouse anti-human CD32 (Fc␥RIIa inhibitor, Ancell) or 10 g/mL F(ab)2 fragments of mouse anti-human CD16 (Fc␥RIII inhibitor, Ancell) before addition of CRP.
Fig. 3. Effects to induce the respiratory burst of neutrophils of CRP with TNF-␣ in reactive system. (A) Effects to induce the respiratory burst of neutrophils of CRP and heat-treated CRP. Hollow squares: without CRP or heat-treated CRP in reactive system; solid squares: with CRP in reactive system; gray squares: with heat-treated CRP in reactive system. htCRP: heat-treated CRP. *P < 0.05. (B and C) Representative flow cytometric analyses of respiratory burst of neutrophils induced by CRP and ANCA. The dashed curve represents baseline burst of neutrophils with normal IgG alone. The solid curve represents burst of neutrophils induced by MPO-ANCA-IgG alone (B) or PR3-ANCA-IgG alone (C). The shadowed curve represents burst of neutrophils induced by CRP plus MPO-ANCA-IgG (B) or PR3-ANCA-IgG (C).
2.7. Measurement of cytokine with enzyme-linked immunosorbent assay (ELISA) After stimulation, the culture supernatants were collected, centrifuged, and processed for IL-8 quantification by commercially available ELISA kits (Thermo Scientific Pierce, Rockford, IL USA). The sensitivity was 2 pg/mL 2.8. Statistical analysis Variables were expressed as mean ± SD and were evaluated using independent t-test or one-way ANOVA analysis as appropriate. It was considered significant difference if the P-value was less than 0.05. Analysis was performed with SPSS statistical software package (version 15, Chicago, IL, USA). 3. Results Demographic and clinical data of the patients are shown in Table 1. 3.1. Electrophoretic analysis of CRP and heat-treated CRP in PAGE with 1/20 standard SDS Since CRP will dissociate in standard PAGE, PAGE with 1/20 standard SDS was taken. As Fig. 1 indicated, after heated at 70 ◦ C
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Fig. 4. Time- and concentration-dependent effects on neutrophils of CRP. (A) Time- and concentration-dependent effects on neutrophils of CRP. (B) Time- and concentrationdependent effects on neutrophils of heat-treated CRP. Hollow squares: 0 g/mL CRP or heat-treated CRP; gray squares: 25 g/mL CRP or heat-treated CRP; solid squares: 50 g/mL CRP or heat-treated CRP. (C) Representative flow cytometric analyses of the spontaneous activation of neutrophils after isolation. The dashed curve: 30 min; the solid curve: 1 h; the shadowed curve: 3 h. (D) Representative flow cytometric analyses of the effect of CRP on the spontaneous activation of neutrophils at 3 h. The dashed curve: 0 g/mL CRP; the solid curve: 25 g/mL CRP; the shadowed curve: 50 g/mL CRP.
for 30 min, CRP dissociated to small molecules. In PAGE with 1/20 standard SDS, CRP was in the position of larger than 250 kD, while heat-treated CRP was in the position of nearly 15 kD. 3.2. Effects of CRP and heat-treated CRP to induce neutrophils respiratory burst Without TNF-␣ in the reactive system, and when normal IgG (100 g/mL, incubating for 30 min) or CRP (50 g/mL, incubating for 30 min) was added alone, the level of MFI was 52.12 ± 12.23 and 53.45 ± 14.65 respectively. So CRP alone (50 g/mL, incubating for 30 min) could not activate neutrophils. Without TNF-␣ in the reactive system, both MPO-ANCA-IgG and PR3-ANCA-IgG could only induce a slight level of respiratory burst of neutrophils [MFI, 58.65 ± 15.09 for MPO-ANCA-IgG (P < 0.05, compared with normal IgG) and 61.73 ± 14.89 for PR3-ANCA-IgG (P < 0.05, compared with normal IgG), respectively]. However, when CRP and ANCA were added together, the level of respiratory burst of neutrophils was increased significantly [MFI, 68.45 ± 16.87 for MPO-ANCAIgG plus CRP (P < 0.05, compared with MPO-ANCA-IgG alone) and 79.51 ± 15.90 for PR3-ANCA-IgG plus CRP (P < 0.05, compared with PR3-ANCA-IgG alone), respectively] (Fig. 2). The LPS blocker Polymyxin B (2 g/mL) did not influence the results significantly (MFI, 70.34 ± 23.46 for MPO-ANCA-IgG plus CRP plus Polymyxin B and 75.23 ± 24.70 for PR3-ANCA-IgG plus CRP plus Polymyxin B). When there was TNF-␣ (2 ng/mL) in the reactive system, CRP alone (50 g/mL, incubating for 30 min) could not induce neutrophils respiratory burst either (57.45 ± 17.39, P > 0.05, compared with normal IgG plus TNF-␣), but both MPO-ANCA-IgG and PR3-ANCA-IgG could induce higher level of respiratory burst of neutrophils significantly [76.49 ± 26.41 for MPO-ANCA-IgG plus
TNF-␣ (P < 0.05, compared with MPO-ANCA-IgG alone without TNF-␣) and 97.27 ± 34.52 for PR3-ANCA-IgG plus TNF-␣ (P < 0.05, compared with PR3-ANCA-IgG alone without TNF-␣), respectively]. CRP could enhance the respiratory burst of neutrophils induced by ANCA plus TNF-␣ (92.27 ± 37.47 for MPO-ANCA-IgG plus TNF␣ plus CRP, P < 0.05, compared with MPO-ANCA-IgG plus TNF-␣; and 124.26 ± 31.47 for PR3-ANCA-IgG plus TNF-␣ plus CRP, P < 0.05, compared with PR3-ANCA-IgG plus TNF-␣, respectively) (Fig. 3). Heated-treated CRP could neither induce neutrophils respiratory burst alone nor enhance the respiratory burst of neutrophils induced by ANCA, with or without TNF-␣ in the reactive system (Figs. 2 and 3). 3.3. Time- and concentration-dependent effects on neutrophils of CRP Without TNF-␣ and CRP added in the system, the baseline level of respiratory burst of neutrophils was 52.12 ± 12.23, 68.52 ± 19.54 and 86.67 ± 33.22 (expressed as MFI) after neutrophils were isolated 30 min, 1 h and 3 h respectively, which indicated spontaneous activation occurred after neutrophils were isolated. When the concentration of CRP was 25 g/mL, there was no obvious change of the level of respiratory burst of neutrophils at 3 h (MFI, 96.45 ± 26.86, P > 0.05, compared with the baseline level at 3 h). When the concentration of CRP was increased to 50 g/mL, the level of respiratory burst of neutrophils at 3 h was increased (MFI, 113.34 ± 40.67, P < 0.05, compared with the baseline level at 3 h). CRP could not influence the level of respiratory burst of neutrophils at 30 min or 1 h at any concentration (Fig. 4). Heat-treated CRP could not influence the level of respiratory burst of neutrophils at any time and at any concentration (Fig. 4).
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Fig. 5. Effects to prime neutrophils of CRP. (A) Membrane expression of PR3 of neutrophils. (B) Membrane expression of MPO of neutrophils. htCRP: heat-treated CRP. *P < 0.05. (C) Representative flow cytometric analyses of the membrane expression of PR3 of neutrophils. The dashed curve represents the membrane expression of PR3 of neutrophils without priming. The solid curve represents the membrane expression of PR3 of neutrophils primed by 50 g/mL CRP. The shadowed curve represents the membrane expression of PR3 of neutrophils primed by 2 ng/mL TNF-␣. (D) Preincubation of neutrophils with function-blocking antibodies and LPS blocker Polymyxin B. *P < 0.05.
3.4. Effects to prime neutrophils of CRP
3.5. Effects of CRP on IL-8 release of neutrophils
Then we tested whether CRP could prime neutrophils for ANCA-induced respiratory burst. Without priming, the baseline membrane PR3 expression of neutrophils was 235.32 ± 124.65 (MFI). CRP (50 g/mL, incubating for 10 min) could prime neutrophils (MFI, 365.27 ± 143.50, P < 0.05, compared with the baseline level), but the effect was weaker than TNF-␣ (2 ng/mL, incubating for 10 min) (MFI, 456.32 ± 153.84, P < 0.05, compared with CRP). Although a slight increased membrane PR3 expression was observed when TNF-␣ was added together with CRP (MFI, 523.36 ± 165.75), the difference between TNF-␣ alone and TNF␣ plus CRP was not significant (P > 0.05). Interestingly, although we could not detect significant change of the membrane MPO expression of neutrophils after adding CRP alone or TNF-␣ alone, a significant increased membrane MPO expression was detected when TNF-␣ was added together with CRP (MFI, 294.86 ± 138.05, P < 0.05, compared with baseline level 202.17 ± 136.83). Heattreated CRP could not prime neutrophils (Fig. 5). Then we used function-blocking antibodies as competitors to assess the possible involvement of the receptors on neutrophils in mediating the actions of CRP. Preincubation of neutrophils with anti-CD16 resulted in attenuation of the increased membrane expression of PR3 which was induced by CRP (MFI, from 365.27 ± 143.50 to 289.03 ± 194.79). Neither antiCD32 nor anti-CD64 affected the membrane expression of PR3 significantly. The LPS blocker Polymyxin B (2 g/mL) did not significantly influence the results (MFI, 347.75 ± 148.65 for CRP plus Polymyxin B, P > 0.05, compared with CRP) (Fig. 5).
Without CRP and ANCA added in the system, the baseline concentration of IL-8 at different time was 0.23 ± 0.07, 0.24 ± 0.08, 0.55 ± 0.08 and 0.78 ± 0.14 (ng/mL) after neutrophils were isolated 0 min, 30 min, 60 min and 180 min respectively. After CRP (50 g/mL) was added, there was no obvious change of the concentration of IL-8 at 30 min (0.29 ± 0.07 ng/mL, P > 0.05, compared with the baseline at 30 min), but the concentration of IL-8 at both 60 min and 180 min were increased (0.86 ± 0.12 ng/mL at 60 min, P < 0.05, compared with the baseline at 60 min and 1.27 ± 0.18 ng/mL at 180 min, P < 0.05, compared with the baseline at 180 min) (Fig. 6). Heat-treated CRP could not influence the concentration of IL-8 at any time (data not shown). Without lone time incubation, CRP alone (50 g/mL, incubating for 30 min) could not stimulate the IL-8 release from neutrophils [0.28 ± 0.04 ng/mL, P > 0.05, compared with normal IgG (0.26 ± 0.06 ng/mL)], while ANCA alone could not enhance the IL-8 release from neutrophils either (0.34 ± 0.03 ng/mL for MPO-ANCAIgG, P > 0.05, compared with normal IgG and 0.39 ± 0.07 ng/mL for PR3-ANCA-IgG, P > 0.05, compared with normal IgG). When CRP and ANCA were added together, the concentration of IL-8 increased significantly (0.49 ± 0.04 ng/mL for CRP plus MPO-ANCA-IgG, P < 0.05, compared with normal IgG and 0.61 ± 0.08 ng/mL for CRP plus PR3ANCA-IgG, P < 0.05, compared with normal IgG) (Fig. 6). 4. Discussion CRP induces oxidative stress in various cells including endothelial cells, smooth muscle cells and monocyte-macrophages in vitro
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Fig. 6. Effects of CRP on IL-8 production of neutrophils. (A) Time-dependent release of IL-8 from neutrophils in response to CRP. (B) Amplification of ANCA-induced IL-8 release after preincubation with CRP. *P < 0.05.
(Ryu et al., 2007; Venugopal et al., 2003). However, the effect of human CRP on neutrophils was far from clear (Khreiss et al., 2004; Devaraj et al., 2006). Some previous findings suggested an antipriming role for CRP (Dobrinich and Spagnuolo, 1991; Mortensen and Zhong, 2000), while other studies speculated that CRP could boost the respiratory burst of neutrophils (Prasad, 2004; Zeller et al., 1986; Zeller and Sullivan, 1992; Lu et al., 2011). In the current study, we demonstrated that CRP could hardly activate neutrophils directly but could prime neutrophils and enhance the ANCA-induced activation of neutrophils. In the present study, CRP primed neutrophils obviously at a concentration of 50 g/mL. Since such level of CRP was very common in patients with AAV, we thought that the priming of neutrophils by CRP was also common in AAV. In the current study, we could not detect increased membrane-bound MPO expression of neutrophils after adding CRP alone or TNF-␣ alone. Similar phenomenon has also been reported previously (Reumaux et al., 2006). However, when CRP and TNF-␣ were added together, increased membrane MPO was detected. We speculated that it was because upon activation, PR3 remained mainly membrane-bound, while MPO were mainly released into the extracellular medium (Witko-Sarsat et al., 1999). So without strong stimulation, the change of membranebound MPO might be not enough to be detected. Based on the primary structure of the composing protomers, pentraxins are divided into two groups, i.e. short and long pentraxins. CRP and serum amyloid P component (SAP) are prototypic short pentraxins, whereas pentraxin 3 (PTX3) and other subsequently identified proteins are prototypic long pentraxins (Breviario, 1992). All these three members can interact with neutrophils with different effects (Bharadwaj et al., 2001; Gershov et al., 2000; Lu et al.,
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2008). SAP and CRP can bind to apoptotic neutrophils then result in increased phagocytosis of apoptotic neutrophils by macrophages (Bijl et al., 2003; Van Rossum et al., 2004). In contrast, PTX3 can bind only to late apoptotic neutrophils, inhibit instead of facilitate uptake of apoptotic cells (Van Rossum et al., 2004). Besides, CRP and PTX3 have been reported to influence the rolling and adhesion ability of neutrophils (McEver, 2010; Deban et al., 2010; El Kebir et al., 2011). However, to the best of our knowledge, CRP is the only pentraxin which has been found to be able to influence the generation of reactive oxygen species of neutrophils. Recent studies have suggested that monomeric CRP might be a more potent activator of various cells than native CRP and the pathogenic role of CRP was partially attributed to monomeric CRP (Khreiss et al., 2004, 2005). Although all the procedures in the current study were administrated in calcium-containing buffer which could prevent the generation of monomeric CRP theoretically, we could not exclude the possibility that the effect of CRP on neutrophils was due to the structure change of the CRP, because preincubation of neutrophils with anti-CD16, not anti-CD32 or anti-CD64, resulted in attenuation of the increased membrane expression of PR3 which was induced by CRP. Actually, calciumindependent binding of pCRP to membranes has been reported to lead to a rapid but partial structural change, producing molecules that express CRP subunit antigenicity but with retained native pentameric conformation (Wang and Sui, 2001; Ji et al., 2007). In the current study, a high concentration 50 g/mL, which was a very common concentration of CRP in AAV, was needed for CRP to exhibit obvious neutrophils-priming effect, so we speculated that when the concentration was high enough, pCRP could bind the membrane of neutrophils and then conformational change of pCRP happened. When CRP subunit antigenicity was exposed, such “modified” CRP would bind Fc␥RIII and then prime neutrophils. Interestingly, we did not detect neutrophils-priming effects of monomeric CRP which was produced by heating native CRP. We thought it might be attributed to the very low solubility of these monomeric CRP. 5. Conclusion In conclusion, CRP could prime neutrophils and enhance the respiratory burst induced by ANCA, which indicated that priming neutrophils and enhancing the respiratory burst induced by ANCA might be one of the pathogenic roles of CRP in AAV. Conflict of interest statement The authors have declared no conflicts of interest. Acknowledgements This study is supported by a grant of Chinese 973 project (No. 2012CB517702) and two grants of the National Natural Science Fund (Nos. 30972733 and 81021004). References Ballou, S.P., Kushner, I., 1992. C-reactive protein and the acute phase response. Advances in Internal Medicine 37, 313–336. Bharadwaj, D., Mold, C., Markham, E., Du Clos, T.W., 2001. Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis. Journal of Immunology 166, 6735–6741. Bijl, M., Horst, G., Bijzet, J., Bootsma, H., Limburg, P.C., Kallenberg, C.G., 2003. Serum amyloid P component binds to late apoptotic cells and mediates their uptake by monocyte-derived macrophages. Arthritis and Rheumatism 48, 248–254. Bisoendial, R.J., Kastelein, J.J., Levels, J.H., Zwaginga, J.J., van den Bogaard, B., Reitsma, P.H., Meijers, J.C., Hartman, D., Levi, M., Stroes, E.S., 2005. Activation of inflammation and coagulation after infusion of C-reactive protein in humans. Circulation Research 96, 714–716. Breviario, F., d‘Aniello, E.M., Golay, J., Peri, G., Bottazzi, B., Bairoch, A., Saccone, S., Marzella, R., Predazzi, V., Rocchi, M., Della Valle, G., Dejana, E., Mantovani, A.,
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Introna, M., 1992. Interleukin-1-inducible genes in endothelial cells. Cloning of a new gene related to C-reactive protein and serum amyloid P component. Journal of Biological Chemistry 267, 22190–22197. Deban, L., Russo, R.C., Sironi, M., Moalli, F., Scanziani, M., Zambelli, V., Cuccovillo, I., Bastone, A., Gobbi, M., Valentino, S., Doni, A., Garlanda, C., Danese, S., Salvatori, G., Sassano, M., Evangelista, V., Rossi, B., Zenaro, E., Constantin, G., Laudanna, C., Bottazzi, B., Mantovani, A., 2010. Regulation of leukocyte recruitment by the long pentraxin PTX3. Nature Immunology 11, 328–334. Devaraj, S., Venugopal, S., Jialal, I., 2006. Native pentameric C-reactive protein displays more potent pro-atherogenic activities in human aortic endothelial cells than modified C-reactive protein. Atherosclerosis 184, 48–52. Dobrinich, R., Spagnuolo, P.J., 1991. Binding of C-reactive protein to human neutrophils inhibition of respiratory burst activity. Arthritis and Rheumatism 34, 1031–1038. Eisenhardt, S.U., Thiele, J.R., Bannasch, H., Stark, G.B., Peter, K., 2009. C-reactive protein: how conformational changes influence inflammatory properties. Cell Cycle 8, 3885–3892. El Kebir, D., Zhang, Y., Potempa, L.A., Wu, Y., Fournier, A., Filep, J.G., 2011. C-reactive protein-derived peptide 201–206 inhibits neutrophil adhesion to endothelial cells and platelets through CD32. Journal of Leukocyte Biology 90, 1167–1175. Falk, R.J., Terrell, R.S., Charles, L.A., Jennette, J.C., 1990. Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proceedings of the National Academy of Sciences of the United States of America 87, 4115–4119. Gershov, D., Kim, S., Brot, N., Elkon, K.B., 2000. C-reactive protein binds to apoptotic cells, protects the cells from assembly of the terminal complement components, and sustains an anti-inflammatory innate immune response: implications for systemic autoimmunity. Journal of Experimental Medicine 192, 1353–1364. Gill, R., Kemp, J.A., Sabin, C., Pepys, M.B., 2004. Human C-reactive protein increases cerebral infarct size after middle cerebral artery occlusion in adult rats. Journal of Cerebral Blood Flow and Metabolism 24, 1214–1218. Haverkate, F., Thompson, S.G., Pyke, S.D., Gallimore, J.R., Pepys, M.B., 1997. Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. Lancet 349, 462–466. Hind, C.R., Winearls, C.G., Lockwood, C.M., Rees, A.J., Pepys, M.B., 1984. Objective monitoring of activity in Wegener’s granulomatosis by measurement of serum C-reactive protein concentration. Clinical Nephrology 21, 341–345. Jennette, J.C., Falk, R.J., Andrassy, K., Bacon, P.A., Churg, J., Gross, W.L., Hagen, E.C., Hoffman, G.S., Hunder, G.G., Kallenberg, C.G., et al., 1994. Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis and Rheumatism 37, 187–192. Jialal, I., Devaraj, S., Venugopal, S.K., 2004. C-reactive protein: risk marker or mediator in atherothrombosis. Hypertension 44, 6–11. Ji, S.R., Wu, Y., Zhu, L., Potempa, L.A., Sheng, F.L., Lu, W., Zhao, J., 2007. Cell membranes and liposomes dissociate C-reactive protein (CRP) to form a new, biologically active structural intermediate: mCRP(m). FASEB Journal 21, 284–294. Jones, J.G., 1995. Interrelationship of outcome measures and process variables in early rheumatoid arthritis. Journal of Rheumatology 22, 186. Kälsch, A.I., Csernok, E., Münch, D., Birck, R., Yard, B.A., Gross, W., Kälsch, T., Schmitt, W.H., 2010. Use of highly sensitive C-reactive protein for followup of Wegener’s granulomatosis. Journal of Rheumatology 37, 2319–2325. Kettritz, R., Jennette, J.C., Falk, R.J., 1997. Crosslinking of ANCA-antigens stimulates superoxide release by human neutrophils. Journal of the American Society of Nephrology 8, 386–394. Kettritz, R., Schreiber, A., Luft, F.C., Haller, H., 2001. Role of mitogen-activated protein kinases in activation of human neutrophils by antineutrophil cytoplasmic antibodies. Journal of the American Society of Nephrology 12, 37–46. Khreiss, T., József, L., Potempa, L.A., Filep, J.G., 2004. Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells. Circulation 109, 2016–2022. Khreiss, T., József, L., Potempa, L.A., Filep, J.G., 2005. Loss of pentameric symmetry in C-reactive protein induces interleukin-8 secretion through peroxynitrite signaling in human neutrophils. Circulation Research 97, 690–697.
Lu, J., Marjon, K.D., Marnell, L.L., Wang, R., Mold, C., Du Clos, T.W., Sun, P., 2011. Recognition and functional activation of the human IgA receptor (FcalphaRI) by C-reactive protein. Proceedings of the National Academy of Sciences of the United States of America 108, 4974–4979. Lu, J., Marnell, L.L., Marjon, K.D., Mold, C., Du Clos, T.W., Sun, P.D., 2008. Structural recognition and functional activation of FcgammaR by innate pentraxins. Nature 456, 989–992. McEver, R.P., 2010. Rolling back neutrophil adhesion. Nature Immunology 11, 282–284. Mortensen, R.F., Zhong, W., 2000. Regulation of phagocytic leukocyte activities by C-reactive protein. Journal of Leukocyte Biology 67, 495–500. Potempa, L.A., Maldonado, B.A., Laurent, P., Zemel, E.S., Antigenic, Gewurz H., 1983. Electrophoretic and binding alterations of human C-reactive protein modified selectively in the absence of calcium. Molecular Immunology 20, 1165–1175. Prasad, K., 2004. C-reactive protein increases oxygen radical generation by neutrophils. Journal of Cardiovascular Pharmacology and Therapeutics 9, 203–209. Rarok, A.A., Stegeman, C.A., Limburg, P.C., Kallenberg, C.G., 2002. Neutrophil membrane expression of proteinase 3 (PR3) is related to relapse in PR3ANCA-associated vasculitis. Journal of the American Society of Nephrology 13, 2232–2238. Reumaux, D., Hordijk, P.L., Duthilleul, P., Roos, D., 2006. Priming by tumor necrosis factor-alpha of human neutrophil NADPH-oxidase activity induced by antiproteinase-3 or anti-myeloperoxidase antibodies. Journal of Leukocyte Biology 80, 1424–1433. Ridker, P.M., Cushman, M., Stampfer, M.J., Tracy, R.P., Hennekens, C.H., 1997. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. New England Journal of Medicine 336, 973–979. Ryu, J., Lee, C.W., Shin, J.A., Park, C.S., Kim, J.J., Park, S.J., Han, K.H., 2007. FcgammaRIIa mediates C-reactive protein-induced inflammatory responses of human vascular smooth muscle cells by activating NADPH oxidase 4. Cardiovascular Research 75, 555–565. Taylor, K.E., van den Berg, C.W., 2007. Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein. Immunology 120, 404–411. Trachtman, H., Futterweit, S., Arzberger, C., Bod, J., Goldschmiedt, J., Gorman, H., Reddy, K., Franki, N., Singhal, P.C., 2006. Nitric oxide and super-oxide in rat mesangial cells: modulation by C-reactive protein. Pediatric Nephrology 21, 619–626. Van Rossum, A.P., Fazzini, F., Limburg, P.C., Manfredi, A.A., Rovere-Querini, P., Mantovani, A., Kallenberg, C.G., 2004. The prototypic tissue pentraxin PTX3, in contrast to the short pentraxin serum amyloid P, inhibits phagocytosis of late apoptotic neutrophils by macrophages. Arthritis and Rheumatism 50, 2667–2674. Van Rossum, A.P., van der Geld, Y.M., Limburg, P.C., Kallenberg, C.G., 2005. Human anti-neutrophil cytoplasm autoantibodies to proteinase 3 (PR3-ANCA) bind to neutrophils. Kidney International 68, 537–541. Venugopal, S.K., Devaraj, S., Jialal, I., 2003. C-reactive protein decreases prostacyclin release from human aortic endothelial cells. Circulation 108, 1676–1678. Venugopal, S.K., Devaraj, S., Yuhanna, I., Shaul, P., Jialal, I., 2002. Demonstration that C-reactive protein decreases eNOS expression and bioactivity in human aortic endothelial cells. Circulation 106, 1439–1441. Wang, H.W., Sui, S.F., 2001. Dissociation and subunit rearrangement of membranebound human C-reactive proteins. Biochemical and Biophysical Research Communications 288, 75–79. Witko-Sarsat, V., Cramer, E.M., Hieblot, C., Guichard, J., Nusbaum, P., Lopez, S., Lesavre, P., Halbwachs-Mecarelli, L., 1999. Presence of proteinase 3 in secretory vesicles: evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules. Blood 94, 2487–2496. Zeller, J.M., Landay, A.L., Lint, T.F., Gewurz, H., 1986. Aggregated C-reactive protein binds to human polymorphonuclear leukocytes and potentiates Fc receptormediated chemiluminescence. Journal of Laboratory and Clinical Medicine 108, 567–576. Zeller, J.M., Sullivan, B.L., 1992. C-reactive protein selectively enhances the intracellular generation of reactive oxygen products by IgG-stimulated monocytes and neutrophils. Journal of Leukocyte Biology 52, 449–455.
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C-reactive protein enhances the respiratory burst of neutrophils-induced by antineutrophil cytoplasmic antibody Peng-cheng Xu a,b,c,d,e , Jian Hao a,b,c,d , Xiao-wei Yang a,b,c,d , Dong-yuan Chang a,b,c,d , Min Chen a,b,c,d,∗ , Ming-hui Zhao a,b,c,d a
Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034, China Institute of Nephrology, Peking University, Beijing 100034, China c Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 100034, China d Key Laboratory of CKD, Prevention and Treatment, Ministry of Education of China, Beijing 100034, China e Department of Nephrology, General Hospital of Tianjin Medical University, Tianjin 300052, China b
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 14 April 2012 Accepted 15 May 2012 Available online 7 June 2012 Keywords: C-reactive protein Neutrophil Anti-neutrophil cytoplasmic antibody Vasculitis
a b s t r a c t Serum C-reactive protein (CRP) was one of the useful biomarkers for evaluating the disease activity in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Cumulating studies proved that CRP was pathogenic in a variety of diseases. In the current study, the in vitro effects of CRP to prime neutrophils for ANCA-induced respiratory burst were investigated with flow cytometry. Without TNF-␣ in the reactive system, ANCA could only induce a slight level of respiratory burst of neutrophils. CRP could enhance the respiratory burst of neutrophils induced by ANCA against myeloperoxidse [mean fluorescence intensity (MFI, 68.45 ± 16.87 vs. 58.65 ± 15.09, P < 0.05) or by ANCA against proteinase 3 (MFI, 79.51 ± 15.90 vs. 61.73 ± 14.89, P < 0.05). Although CRP (50 g/mL, incubating for 30 min) could not active neutrophils alone, after incubation with neutrophils for 10 min, CRP (50 g/mL) could increase the expression of membrane proteinase 3 of neutrophils (MFI, 365.27 ± 143.50 vs. 235.32 ± 124.65, P < 0.05). Heat-treated CRP could not enhance the levels of neutrophils respiratory burst induced by ANCA or increase the expression of membrane proteinase 3 of neutrophils. So CRP can prime neutrophils and enhance the respiratory burst induced by ANCA and might be pathogenic in AAV. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), serum C-reactive protein (CRP) was one of the useful biomarkers for evaluating the disease activity. The concentration of serum CRP was often elevated in active phase of AAV, and fell rapidly with remission of the disease (Hind et al., 1984; Kälsch et al., 2010). CRP is synthesized and catabolized in hepatocytes and is a highly soluble serum protein of the pentraxin family. During the onset of inflammation or tissue injury, serum CRP is always dramatically elevated up to 1000-fold or more within 24–72 h (Ballou and Kushner, 1992). Actually, after binding to plasma membranes or in a denaturating or oxidative environment, CRP might dissociate into free subunits yielding insoluble modified/monomeric CRP (mCRP) (Potempa et al., 1983; Devaraj et al., 2006).
∗ Corresponding author at: Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034, China. Tel.: +86 10 66551736; fax: +86 10 66551055. E-mail address: [email protected] (M. Chen). 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.05.012
Cumulating studies suggested the pathogenic role of CRP in a variety of diseases, such as metabolic syndrome, atherothrombosis, acute myocardial infarction, cerebral infarction (Gill et al., 2004) and kidney diseases (Trachtman et al., 2006). For example, CRP could increase the expression of adhesion molecules in endothelial cells, inhibit nitric oxide in aortic endothelial cells (Venugopal et al., 2002) and rat mesangial cells (Trachtman et al., 2006), stimulate IL-8 release from several cell types, increase plasminogen activator inhibitor-1 expression, and increase the release of IL-1, IL-6, IL-18, and TNF-␣ from monocytes (Eisenhardt et al., 2009). The most direct clinical evidence was that activation of inflammation in healthy volunteers was observed after infusion of recombinant human CRP (Bisoendial et al., 2005). Serum CRP levels were of prognostic value in some diseases such as bacterial infection, hypertension, metabolic syndrome, acute cardiovascular events, and rheumatoid arthritis (Haverkate et al., 1997; Ridker et al., 1997; Jones, 1995; Jialal et al., 2004). In AAV, several previous studies have reported that serum CRP level correlated closely with disease activity (Hind et al., 1984). However, whether CRP is pathogenic in AAV is still unclear yet. In AAV, ANCA can enhance the degranulation of neutrophils and increase the production of superoxide and pro-inflammatory
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cytokines of neutrophils. High level of CRP is a characteristic of active AAV and in vitro studies have documented specific receptors for CRP on neutrophils. pCRP binds to Fc␥RI (CD64) and Fc␥RIIa (CD32), while mCRP binds to Fc␥RIII (CD16) on human neutrophils, respectively (Khreiss et al., 2005; Kettritz et al., 1997; Falk et al., 1990). Since the neutrophils respiratory burst has been proved to play a pivotal role in AAV, the in vitro effects of CRP on the respiratory burst of neutrophils induced by ANCA were investigated in the current study. 2. Methods 2.1. Patients and plasma Patients’ plasma was obtained from 5 AAV patients with positive ANCA against myeloperoxidase (MPO-ANCA) and 5 AAV patients with positive ANCA against proteinase 3 (PR3-ANCA) at presentation. All the 10 patients were diagnosed in Peking University First Hospital and fulfilled the Chapel Hill Consensus Conference classification criteria of AAV (Jennette et al., 1994). All patients with MPO-ANCA and 1 out of 5 patients with PR3-ANCA had biopsyproven glomerulonephritis. Plasma from five healthy blood donors was collected as normal controls. All the plasma was stored at −20 ◦ C until use. The research was in compliance of the declaration of Helsinki and approved by the ethic committee of the hospital. Inform consent was obtained from each participant. 2.2. Purification of IgG fractions IgG fractions containing anti-MPO antibodies, anti-PR3 antibodies or IgG fractions of normal people were purified by protein G affinity column (Amersham Pharmacia, Sweden) with 0.01 mol/L phosphate-buffered saline (PBS, pH 7.4) as starting buffer and 0.1 mol/L glycine, pH 2.7 as eluting buffer, at a flow rate of 1 mL/min at room temperature. IgG was eluted and neutralized to pH 7.0 by 2 mol/L Tris–HCl, pH 9.0 immediately, and dialyzed against PBS.
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Table 1 General data of the patients with AAV. Patient no.
Gender
Age (years)
Titer of ANCA (lgT)
ANCA type
1 2 3 4 5 6 7 8 9 10
F M M F F M M M F M
56 59 62 67 62 72 67 68 21 50
3.51 3.51 2.60 4.41 3.81 2.0 2.0 2.6 2.0 2.9
MPO-ANCA MPO-ANCA MPO-ANCA MPO-ANCA MPO-ANCA PR3-ANCA PR3-ANCA PR3-ANCA PR3-ANCA PR3-ANCA
Abbreviations: ANCA, anti-neutrophil cytoplasm antibodies; MPO, myeloperoxidase; PR3, proteinase 3.
incubated with cytochalasin B (5 g/mL, Sigma) for 5 min at 37 ◦ C to enhance the oxygen radical production. Then, neutrophils were loaded with 0.05 mM DHR (Sigma) and 2 mM sodium azide (NaN3 ) at 37 ◦ C and primed with TNF-␣ (2 ng/mL) for 15 min at 37 ◦ C. The CRP or heat-treated CRP was added with a final concentration of 50 g/mL. The normal IgG, MPO-ANCA-containing IgG or PR3-ANCA-containing IgG was added with a final concentration of 100 g/mL. The reaction was stopped after 20 min by addition of 1 mL of ice-cold HBSS/1%BSA. We analyzed samples using Calibur flow cytometer (BD FACSCalibur). Data were collected from 20,000 cells per sample. The mean fluorescence intensity (MFI), representing the amount of generated oxygen radicals, was reported. All assays were taken in triplicate.
2.3. CRP preparation for in vitro study High purity (>99%) human native CRP was purchased from Sigma (C-4063; St. Louis, MO). Modified CRP was generated with native CRP by heating at 70 ◦ C for 30 min. After heating, the white deposit formed and was resuspended for the following experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 1/20 of standard SDS and Coomassie staining were used to confirm cleavage of CRP (Taylor and van den Berg, 2007). The endotoxin level of CRP was below the detection limit (0.125 EU/mL) of the Limulus assay (Sigma). 2.4. Neutrophils isolation Neutrophils from healthy donors were isolated from heparinized blood by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway) as previously described (Van Rossum et al., 2005), with some minor modifications. Briefly, erythrocytes were removed twice by hypotonic lysis with ice-cold ammonium chloride. Thereafter, cells were washed with ice-cold Hanks balanced salt solution (HBSS) without Ca2+ and Mg2+ , and resuspended in HBSS with Ca2+ and Mg2+ (2.5 × 106 /mL). 2.5. Measurement of respiratory burst by oxidation of dihydrorhodamine (DHR) to rhodamine The generation of reactive oxygen radicals using DHR was assessed as described previously (Kettritz et al., 2001), with some minor modifications. In brief, neutrophils (2.5 × 106 /mL HBSS) were
Fig. 1. Electrophoretic analysis of CRP and heat-treated CRP. All samples were subjected to PAGE with 1/20 SDS. Gel was stained with Coomassie brilliant blue. 1: Native CRP; 2: modified CRP produced with CRP by heating at 70 ◦ C for 30 min.
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Fig. 2. Effects to induce the respiratory burst of neutrophils of CRP without TNF-␣ in reactive system. (A) Effects to induce the respiratory burst of neutrophils of CRP. Hollow squares: without CRP or heat-treated CRP in reactive system; solid squares: with CRP in reactive system; gray squares: with heat-treated CRP in reactive system. htCRP: heat-treated CRP. *P < 0.05. (B and C) Representative flow cytometric analyses of respiratory burst of neutrophils induced by CRP and ANCA. The dashed curve represents baseline burst of neutrophils with normal IgG alone. The solid curve represents burst of neutrophils induced by MPO-ANCA-IgG alone (B) or PR3-ANCAIgG alone (C). The shadowed curve represents burst of neutrophils induced by CRP plus MPO-ANCA-IgG (B) or PR3-ANCA-IgG (C).
2.6. Membrane expression of ANCA target antigens on neutrophils after priming Membrane expression of PR3 and MPO were assessed using flow cytometry as previously described (Rarok et al., 2002) with some minor modifications. All steps were performed on ice. Briefly, samples containing 2.5 × 106 neutrophils/mL HBSS were incubated with 2 ng/mL TNF-␣ or 50 g/mL CRP or heat-treated CRP for 10 min at 37 ◦ C, then washed with HBSS/1% BSA by centrifugation at 1800 × g, 4 ◦ C for 5 min, and incubated with 0.5 mg/mL heatedaggregated goat IgG (Sigma) for 15 min to saturate Fc␥ receptors. Next, cells were stained with a saturation dose of mouse monoclonal IgG1 antibodies directed against human PR3 or MPO (R&D Systems Antibodies) or with an irrelevant IgG1 control antibody for 30 min. Next, non-bound antibodies were washed off with PBS/1%BSA. This step was followed by 30 min incubation with phycoerythrin (PE)-conjugated goat anti-mouse antibody (Southern Biotechnology Associates, Birmingham, AL, USA). MFI was calculated to represent the level of PR3- or MPO-expression on neutrophils. All assays were tested in triplicate. To study which receptors were involved in the CRP-mediated priming effects on neutrophils, the neutrophils were preincubated with 10 g/mL F(ab)2 fragments of mouse anti-human CD64 (Fc␥RI inhibitor, Ancell, Bayport, Minn), 10 g/mL F(ab)2 fragments of mouse anti-human CD32 (Fc␥RIIa inhibitor, Ancell) or 10 g/mL F(ab)2 fragments of mouse anti-human CD16 (Fc␥RIII inhibitor, Ancell) before addition of CRP.
Fig. 3. Effects to induce the respiratory burst of neutrophils of CRP with TNF-␣ in reactive system. (A) Effects to induce the respiratory burst of neutrophils of CRP and heat-treated CRP. Hollow squares: without CRP or heat-treated CRP in reactive system; solid squares: with CRP in reactive system; gray squares: with heat-treated CRP in reactive system. htCRP: heat-treated CRP. *P < 0.05. (B and C) Representative flow cytometric analyses of respiratory burst of neutrophils induced by CRP and ANCA. The dashed curve represents baseline burst of neutrophils with normal IgG alone. The solid curve represents burst of neutrophils induced by MPO-ANCA-IgG alone (B) or PR3-ANCA-IgG alone (C). The shadowed curve represents burst of neutrophils induced by CRP plus MPO-ANCA-IgG (B) or PR3-ANCA-IgG (C).
2.7. Measurement of cytokine with enzyme-linked immunosorbent assay (ELISA) After stimulation, the culture supernatants were collected, centrifuged, and processed for IL-8 quantification by commercially available ELISA kits (Thermo Scientific Pierce, Rockford, IL USA). The sensitivity was 2 pg/mL 2.8. Statistical analysis Variables were expressed as mean ± SD and were evaluated using independent t-test or one-way ANOVA analysis as appropriate. It was considered significant difference if the P-value was less than 0.05. Analysis was performed with SPSS statistical software package (version 15, Chicago, IL, USA). 3. Results Demographic and clinical data of the patients are shown in Table 1. 3.1. Electrophoretic analysis of CRP and heat-treated CRP in PAGE with 1/20 standard SDS Since CRP will dissociate in standard PAGE, PAGE with 1/20 standard SDS was taken. As Fig. 1 indicated, after heated at 70 ◦ C
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Fig. 4. Time- and concentration-dependent effects on neutrophils of CRP. (A) Time- and concentration-dependent effects on neutrophils of CRP. (B) Time- and concentrationdependent effects on neutrophils of heat-treated CRP. Hollow squares: 0 g/mL CRP or heat-treated CRP; gray squares: 25 g/mL CRP or heat-treated CRP; solid squares: 50 g/mL CRP or heat-treated CRP. (C) Representative flow cytometric analyses of the spontaneous activation of neutrophils after isolation. The dashed curve: 30 min; the solid curve: 1 h; the shadowed curve: 3 h. (D) Representative flow cytometric analyses of the effect of CRP on the spontaneous activation of neutrophils at 3 h. The dashed curve: 0 g/mL CRP; the solid curve: 25 g/mL CRP; the shadowed curve: 50 g/mL CRP.
for 30 min, CRP dissociated to small molecules. In PAGE with 1/20 standard SDS, CRP was in the position of larger than 250 kD, while heat-treated CRP was in the position of nearly 15 kD. 3.2. Effects of CRP and heat-treated CRP to induce neutrophils respiratory burst Without TNF-␣ in the reactive system, and when normal IgG (100 g/mL, incubating for 30 min) or CRP (50 g/mL, incubating for 30 min) was added alone, the level of MFI was 52.12 ± 12.23 and 53.45 ± 14.65 respectively. So CRP alone (50 g/mL, incubating for 30 min) could not activate neutrophils. Without TNF-␣ in the reactive system, both MPO-ANCA-IgG and PR3-ANCA-IgG could only induce a slight level of respiratory burst of neutrophils [MFI, 58.65 ± 15.09 for MPO-ANCA-IgG (P < 0.05, compared with normal IgG) and 61.73 ± 14.89 for PR3-ANCA-IgG (P < 0.05, compared with normal IgG), respectively]. However, when CRP and ANCA were added together, the level of respiratory burst of neutrophils was increased significantly [MFI, 68.45 ± 16.87 for MPO-ANCAIgG plus CRP (P < 0.05, compared with MPO-ANCA-IgG alone) and 79.51 ± 15.90 for PR3-ANCA-IgG plus CRP (P < 0.05, compared with PR3-ANCA-IgG alone), respectively] (Fig. 2). The LPS blocker Polymyxin B (2 g/mL) did not influence the results significantly (MFI, 70.34 ± 23.46 for MPO-ANCA-IgG plus CRP plus Polymyxin B and 75.23 ± 24.70 for PR3-ANCA-IgG plus CRP plus Polymyxin B). When there was TNF-␣ (2 ng/mL) in the reactive system, CRP alone (50 g/mL, incubating for 30 min) could not induce neutrophils respiratory burst either (57.45 ± 17.39, P > 0.05, compared with normal IgG plus TNF-␣), but both MPO-ANCA-IgG and PR3-ANCA-IgG could induce higher level of respiratory burst of neutrophils significantly [76.49 ± 26.41 for MPO-ANCA-IgG plus
TNF-␣ (P < 0.05, compared with MPO-ANCA-IgG alone without TNF-␣) and 97.27 ± 34.52 for PR3-ANCA-IgG plus TNF-␣ (P < 0.05, compared with PR3-ANCA-IgG alone without TNF-␣), respectively]. CRP could enhance the respiratory burst of neutrophils induced by ANCA plus TNF-␣ (92.27 ± 37.47 for MPO-ANCA-IgG plus TNF␣ plus CRP, P < 0.05, compared with MPO-ANCA-IgG plus TNF-␣; and 124.26 ± 31.47 for PR3-ANCA-IgG plus TNF-␣ plus CRP, P < 0.05, compared with PR3-ANCA-IgG plus TNF-␣, respectively) (Fig. 3). Heated-treated CRP could neither induce neutrophils respiratory burst alone nor enhance the respiratory burst of neutrophils induced by ANCA, with or without TNF-␣ in the reactive system (Figs. 2 and 3). 3.3. Time- and concentration-dependent effects on neutrophils of CRP Without TNF-␣ and CRP added in the system, the baseline level of respiratory burst of neutrophils was 52.12 ± 12.23, 68.52 ± 19.54 and 86.67 ± 33.22 (expressed as MFI) after neutrophils were isolated 30 min, 1 h and 3 h respectively, which indicated spontaneous activation occurred after neutrophils were isolated. When the concentration of CRP was 25 g/mL, there was no obvious change of the level of respiratory burst of neutrophils at 3 h (MFI, 96.45 ± 26.86, P > 0.05, compared with the baseline level at 3 h). When the concentration of CRP was increased to 50 g/mL, the level of respiratory burst of neutrophils at 3 h was increased (MFI, 113.34 ± 40.67, P < 0.05, compared with the baseline level at 3 h). CRP could not influence the level of respiratory burst of neutrophils at 30 min or 1 h at any concentration (Fig. 4). Heat-treated CRP could not influence the level of respiratory burst of neutrophils at any time and at any concentration (Fig. 4).
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Fig. 5. Effects to prime neutrophils of CRP. (A) Membrane expression of PR3 of neutrophils. (B) Membrane expression of MPO of neutrophils. htCRP: heat-treated CRP. *P < 0.05. (C) Representative flow cytometric analyses of the membrane expression of PR3 of neutrophils. The dashed curve represents the membrane expression of PR3 of neutrophils without priming. The solid curve represents the membrane expression of PR3 of neutrophils primed by 50 g/mL CRP. The shadowed curve represents the membrane expression of PR3 of neutrophils primed by 2 ng/mL TNF-␣. (D) Preincubation of neutrophils with function-blocking antibodies and LPS blocker Polymyxin B. *P < 0.05.
3.4. Effects to prime neutrophils of CRP
3.5. Effects of CRP on IL-8 release of neutrophils
Then we tested whether CRP could prime neutrophils for ANCA-induced respiratory burst. Without priming, the baseline membrane PR3 expression of neutrophils was 235.32 ± 124.65 (MFI). CRP (50 g/mL, incubating for 10 min) could prime neutrophils (MFI, 365.27 ± 143.50, P < 0.05, compared with the baseline level), but the effect was weaker than TNF-␣ (2 ng/mL, incubating for 10 min) (MFI, 456.32 ± 153.84, P < 0.05, compared with CRP). Although a slight increased membrane PR3 expression was observed when TNF-␣ was added together with CRP (MFI, 523.36 ± 165.75), the difference between TNF-␣ alone and TNF␣ plus CRP was not significant (P > 0.05). Interestingly, although we could not detect significant change of the membrane MPO expression of neutrophils after adding CRP alone or TNF-␣ alone, a significant increased membrane MPO expression was detected when TNF-␣ was added together with CRP (MFI, 294.86 ± 138.05, P < 0.05, compared with baseline level 202.17 ± 136.83). Heattreated CRP could not prime neutrophils (Fig. 5). Then we used function-blocking antibodies as competitors to assess the possible involvement of the receptors on neutrophils in mediating the actions of CRP. Preincubation of neutrophils with anti-CD16 resulted in attenuation of the increased membrane expression of PR3 which was induced by CRP (MFI, from 365.27 ± 143.50 to 289.03 ± 194.79). Neither antiCD32 nor anti-CD64 affected the membrane expression of PR3 significantly. The LPS blocker Polymyxin B (2 g/mL) did not significantly influence the results (MFI, 347.75 ± 148.65 for CRP plus Polymyxin B, P > 0.05, compared with CRP) (Fig. 5).
Without CRP and ANCA added in the system, the baseline concentration of IL-8 at different time was 0.23 ± 0.07, 0.24 ± 0.08, 0.55 ± 0.08 and 0.78 ± 0.14 (ng/mL) after neutrophils were isolated 0 min, 30 min, 60 min and 180 min respectively. After CRP (50 g/mL) was added, there was no obvious change of the concentration of IL-8 at 30 min (0.29 ± 0.07 ng/mL, P > 0.05, compared with the baseline at 30 min), but the concentration of IL-8 at both 60 min and 180 min were increased (0.86 ± 0.12 ng/mL at 60 min, P < 0.05, compared with the baseline at 60 min and 1.27 ± 0.18 ng/mL at 180 min, P < 0.05, compared with the baseline at 180 min) (Fig. 6). Heat-treated CRP could not influence the concentration of IL-8 at any time (data not shown). Without lone time incubation, CRP alone (50 g/mL, incubating for 30 min) could not stimulate the IL-8 release from neutrophils [0.28 ± 0.04 ng/mL, P > 0.05, compared with normal IgG (0.26 ± 0.06 ng/mL)], while ANCA alone could not enhance the IL-8 release from neutrophils either (0.34 ± 0.03 ng/mL for MPO-ANCAIgG, P > 0.05, compared with normal IgG and 0.39 ± 0.07 ng/mL for PR3-ANCA-IgG, P > 0.05, compared with normal IgG). When CRP and ANCA were added together, the concentration of IL-8 increased significantly (0.49 ± 0.04 ng/mL for CRP plus MPO-ANCA-IgG, P < 0.05, compared with normal IgG and 0.61 ± 0.08 ng/mL for CRP plus PR3ANCA-IgG, P < 0.05, compared with normal IgG) (Fig. 6). 4. Discussion CRP induces oxidative stress in various cells including endothelial cells, smooth muscle cells and monocyte-macrophages in vitro
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Fig. 6. Effects of CRP on IL-8 production of neutrophils. (A) Time-dependent release of IL-8 from neutrophils in response to CRP. (B) Amplification of ANCA-induced IL-8 release after preincubation with CRP. *P < 0.05.
(Ryu et al., 2007; Venugopal et al., 2003). However, the effect of human CRP on neutrophils was far from clear (Khreiss et al., 2004; Devaraj et al., 2006). Some previous findings suggested an antipriming role for CRP (Dobrinich and Spagnuolo, 1991; Mortensen and Zhong, 2000), while other studies speculated that CRP could boost the respiratory burst of neutrophils (Prasad, 2004; Zeller et al., 1986; Zeller and Sullivan, 1992; Lu et al., 2011). In the current study, we demonstrated that CRP could hardly activate neutrophils directly but could prime neutrophils and enhance the ANCA-induced activation of neutrophils. In the present study, CRP primed neutrophils obviously at a concentration of 50 g/mL. Since such level of CRP was very common in patients with AAV, we thought that the priming of neutrophils by CRP was also common in AAV. In the current study, we could not detect increased membrane-bound MPO expression of neutrophils after adding CRP alone or TNF-␣ alone. Similar phenomenon has also been reported previously (Reumaux et al., 2006). However, when CRP and TNF-␣ were added together, increased membrane MPO was detected. We speculated that it was because upon activation, PR3 remained mainly membrane-bound, while MPO were mainly released into the extracellular medium (Witko-Sarsat et al., 1999). So without strong stimulation, the change of membranebound MPO might be not enough to be detected. Based on the primary structure of the composing protomers, pentraxins are divided into two groups, i.e. short and long pentraxins. CRP and serum amyloid P component (SAP) are prototypic short pentraxins, whereas pentraxin 3 (PTX3) and other subsequently identified proteins are prototypic long pentraxins (Breviario, 1992). All these three members can interact with neutrophils with different effects (Bharadwaj et al., 2001; Gershov et al., 2000; Lu et al.,
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2008). SAP and CRP can bind to apoptotic neutrophils then result in increased phagocytosis of apoptotic neutrophils by macrophages (Bijl et al., 2003; Van Rossum et al., 2004). In contrast, PTX3 can bind only to late apoptotic neutrophils, inhibit instead of facilitate uptake of apoptotic cells (Van Rossum et al., 2004). Besides, CRP and PTX3 have been reported to influence the rolling and adhesion ability of neutrophils (McEver, 2010; Deban et al., 2010; El Kebir et al., 2011). However, to the best of our knowledge, CRP is the only pentraxin which has been found to be able to influence the generation of reactive oxygen species of neutrophils. Recent studies have suggested that monomeric CRP might be a more potent activator of various cells than native CRP and the pathogenic role of CRP was partially attributed to monomeric CRP (Khreiss et al., 2004, 2005). Although all the procedures in the current study were administrated in calcium-containing buffer which could prevent the generation of monomeric CRP theoretically, we could not exclude the possibility that the effect of CRP on neutrophils was due to the structure change of the CRP, because preincubation of neutrophils with anti-CD16, not anti-CD32 or anti-CD64, resulted in attenuation of the increased membrane expression of PR3 which was induced by CRP. Actually, calciumindependent binding of pCRP to membranes has been reported to lead to a rapid but partial structural change, producing molecules that express CRP subunit antigenicity but with retained native pentameric conformation (Wang and Sui, 2001; Ji et al., 2007). In the current study, a high concentration 50 g/mL, which was a very common concentration of CRP in AAV, was needed for CRP to exhibit obvious neutrophils-priming effect, so we speculated that when the concentration was high enough, pCRP could bind the membrane of neutrophils and then conformational change of pCRP happened. When CRP subunit antigenicity was exposed, such “modified” CRP would bind Fc␥RIII and then prime neutrophils. Interestingly, we did not detect neutrophils-priming effects of monomeric CRP which was produced by heating native CRP. We thought it might be attributed to the very low solubility of these monomeric CRP. 5. Conclusion In conclusion, CRP could prime neutrophils and enhance the respiratory burst induced by ANCA, which indicated that priming neutrophils and enhancing the respiratory burst induced by ANCA might be one of the pathogenic roles of CRP in AAV. Conflict of interest statement The authors have declared no conflicts of interest. Acknowledgements This study is supported by a grant of Chinese 973 project (No. 2012CB517702) and two grants of the National Natural Science Fund (Nos. 30972733 and 81021004). References Ballou, S.P., Kushner, I., 1992. C-reactive protein and the acute phase response. Advances in Internal Medicine 37, 313–336. Bharadwaj, D., Mold, C., Markham, E., Du Clos, T.W., 2001. Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis. Journal of Immunology 166, 6735–6741. Bijl, M., Horst, G., Bijzet, J., Bootsma, H., Limburg, P.C., Kallenberg, C.G., 2003. Serum amyloid P component binds to late apoptotic cells and mediates their uptake by monocyte-derived macrophages. Arthritis and Rheumatism 48, 248–254. Bisoendial, R.J., Kastelein, J.J., Levels, J.H., Zwaginga, J.J., van den Bogaard, B., Reitsma, P.H., Meijers, J.C., Hartman, D., Levi, M., Stroes, E.S., 2005. Activation of inflammation and coagulation after infusion of C-reactive protein in humans. Circulation Research 96, 714–716. Breviario, F., d‘Aniello, E.M., Golay, J., Peri, G., Bottazzi, B., Bairoch, A., Saccone, S., Marzella, R., Predazzi, V., Rocchi, M., Della Valle, G., Dejana, E., Mantovani, A.,
154
P.-c. Xu et al. / Molecular Immunology 52 (2012) 148–154
Introna, M., 1992. Interleukin-1-inducible genes in endothelial cells. Cloning of a new gene related to C-reactive protein and serum amyloid P component. Journal of Biological Chemistry 267, 22190–22197. Deban, L., Russo, R.C., Sironi, M., Moalli, F., Scanziani, M., Zambelli, V., Cuccovillo, I., Bastone, A., Gobbi, M., Valentino, S., Doni, A., Garlanda, C., Danese, S., Salvatori, G., Sassano, M., Evangelista, V., Rossi, B., Zenaro, E., Constantin, G., Laudanna, C., Bottazzi, B., Mantovani, A., 2010. Regulation of leukocyte recruitment by the long pentraxin PTX3. Nature Immunology 11, 328–334. Devaraj, S., Venugopal, S., Jialal, I., 2006. Native pentameric C-reactive protein displays more potent pro-atherogenic activities in human aortic endothelial cells than modified C-reactive protein. Atherosclerosis 184, 48–52. Dobrinich, R., Spagnuolo, P.J., 1991. Binding of C-reactive protein to human neutrophils inhibition of respiratory burst activity. Arthritis and Rheumatism 34, 1031–1038. Eisenhardt, S.U., Thiele, J.R., Bannasch, H., Stark, G.B., Peter, K., 2009. C-reactive protein: how conformational changes influence inflammatory properties. Cell Cycle 8, 3885–3892. El Kebir, D., Zhang, Y., Potempa, L.A., Wu, Y., Fournier, A., Filep, J.G., 2011. C-reactive protein-derived peptide 201–206 inhibits neutrophil adhesion to endothelial cells and platelets through CD32. Journal of Leukocyte Biology 90, 1167–1175. Falk, R.J., Terrell, R.S., Charles, L.A., Jennette, J.C., 1990. Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proceedings of the National Academy of Sciences of the United States of America 87, 4115–4119. Gershov, D., Kim, S., Brot, N., Elkon, K.B., 2000. C-reactive protein binds to apoptotic cells, protects the cells from assembly of the terminal complement components, and sustains an anti-inflammatory innate immune response: implications for systemic autoimmunity. Journal of Experimental Medicine 192, 1353–1364. Gill, R., Kemp, J.A., Sabin, C., Pepys, M.B., 2004. Human C-reactive protein increases cerebral infarct size after middle cerebral artery occlusion in adult rats. Journal of Cerebral Blood Flow and Metabolism 24, 1214–1218. Haverkate, F., Thompson, S.G., Pyke, S.D., Gallimore, J.R., Pepys, M.B., 1997. Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. Lancet 349, 462–466. Hind, C.R., Winearls, C.G., Lockwood, C.M., Rees, A.J., Pepys, M.B., 1984. Objective monitoring of activity in Wegener’s granulomatosis by measurement of serum C-reactive protein concentration. Clinical Nephrology 21, 341–345. Jennette, J.C., Falk, R.J., Andrassy, K., Bacon, P.A., Churg, J., Gross, W.L., Hagen, E.C., Hoffman, G.S., Hunder, G.G., Kallenberg, C.G., et al., 1994. Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis and Rheumatism 37, 187–192. Jialal, I., Devaraj, S., Venugopal, S.K., 2004. C-reactive protein: risk marker or mediator in atherothrombosis. Hypertension 44, 6–11. Ji, S.R., Wu, Y., Zhu, L., Potempa, L.A., Sheng, F.L., Lu, W., Zhao, J., 2007. Cell membranes and liposomes dissociate C-reactive protein (CRP) to form a new, biologically active structural intermediate: mCRP(m). FASEB Journal 21, 284–294. Jones, J.G., 1995. Interrelationship of outcome measures and process variables in early rheumatoid arthritis. Journal of Rheumatology 22, 186. Kälsch, A.I., Csernok, E., Münch, D., Birck, R., Yard, B.A., Gross, W., Kälsch, T., Schmitt, W.H., 2010. Use of highly sensitive C-reactive protein for followup of Wegener’s granulomatosis. Journal of Rheumatology 37, 2319–2325. Kettritz, R., Jennette, J.C., Falk, R.J., 1997. Crosslinking of ANCA-antigens stimulates superoxide release by human neutrophils. Journal of the American Society of Nephrology 8, 386–394. Kettritz, R., Schreiber, A., Luft, F.C., Haller, H., 2001. Role of mitogen-activated protein kinases in activation of human neutrophils by antineutrophil cytoplasmic antibodies. Journal of the American Society of Nephrology 12, 37–46. Khreiss, T., József, L., Potempa, L.A., Filep, J.G., 2004. Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells. Circulation 109, 2016–2022. Khreiss, T., József, L., Potempa, L.A., Filep, J.G., 2005. Loss of pentameric symmetry in C-reactive protein induces interleukin-8 secretion through peroxynitrite signaling in human neutrophils. Circulation Research 97, 690–697.
Lu, J., Marjon, K.D., Marnell, L.L., Wang, R., Mold, C., Du Clos, T.W., Sun, P., 2011. Recognition and functional activation of the human IgA receptor (FcalphaRI) by C-reactive protein. Proceedings of the National Academy of Sciences of the United States of America 108, 4974–4979. Lu, J., Marnell, L.L., Marjon, K.D., Mold, C., Du Clos, T.W., Sun, P.D., 2008. Structural recognition and functional activation of FcgammaR by innate pentraxins. Nature 456, 989–992. McEver, R.P., 2010. Rolling back neutrophil adhesion. Nature Immunology 11, 282–284. Mortensen, R.F., Zhong, W., 2000. Regulation of phagocytic leukocyte activities by C-reactive protein. Journal of Leukocyte Biology 67, 495–500. Potempa, L.A., Maldonado, B.A., Laurent, P., Zemel, E.S., Antigenic, Gewurz H., 1983. Electrophoretic and binding alterations of human C-reactive protein modified selectively in the absence of calcium. Molecular Immunology 20, 1165–1175. Prasad, K., 2004. C-reactive protein increases oxygen radical generation by neutrophils. Journal of Cardiovascular Pharmacology and Therapeutics 9, 203–209. Rarok, A.A., Stegeman, C.A., Limburg, P.C., Kallenberg, C.G., 2002. Neutrophil membrane expression of proteinase 3 (PR3) is related to relapse in PR3ANCA-associated vasculitis. Journal of the American Society of Nephrology 13, 2232–2238. Reumaux, D., Hordijk, P.L., Duthilleul, P., Roos, D., 2006. Priming by tumor necrosis factor-alpha of human neutrophil NADPH-oxidase activity induced by antiproteinase-3 or anti-myeloperoxidase antibodies. Journal of Leukocyte Biology 80, 1424–1433. Ridker, P.M., Cushman, M., Stampfer, M.J., Tracy, R.P., Hennekens, C.H., 1997. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. New England Journal of Medicine 336, 973–979. Ryu, J., Lee, C.W., Shin, J.A., Park, C.S., Kim, J.J., Park, S.J., Han, K.H., 2007. FcgammaRIIa mediates C-reactive protein-induced inflammatory responses of human vascular smooth muscle cells by activating NADPH oxidase 4. Cardiovascular Research 75, 555–565. Taylor, K.E., van den Berg, C.W., 2007. Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein. Immunology 120, 404–411. Trachtman, H., Futterweit, S., Arzberger, C., Bod, J., Goldschmiedt, J., Gorman, H., Reddy, K., Franki, N., Singhal, P.C., 2006. Nitric oxide and super-oxide in rat mesangial cells: modulation by C-reactive protein. Pediatric Nephrology 21, 619–626. Van Rossum, A.P., Fazzini, F., Limburg, P.C., Manfredi, A.A., Rovere-Querini, P., Mantovani, A., Kallenberg, C.G., 2004. The prototypic tissue pentraxin PTX3, in contrast to the short pentraxin serum amyloid P, inhibits phagocytosis of late apoptotic neutrophils by macrophages. Arthritis and Rheumatism 50, 2667–2674. Van Rossum, A.P., van der Geld, Y.M., Limburg, P.C., Kallenberg, C.G., 2005. Human anti-neutrophil cytoplasm autoantibodies to proteinase 3 (PR3-ANCA) bind to neutrophils. Kidney International 68, 537–541. Venugopal, S.K., Devaraj, S., Jialal, I., 2003. C-reactive protein decreases prostacyclin release from human aortic endothelial cells. Circulation 108, 1676–1678. Venugopal, S.K., Devaraj, S., Yuhanna, I., Shaul, P., Jialal, I., 2002. Demonstration that C-reactive protein decreases eNOS expression and bioactivity in human aortic endothelial cells. Circulation 106, 1439–1441. Wang, H.W., Sui, S.F., 2001. Dissociation and subunit rearrangement of membranebound human C-reactive proteins. Biochemical and Biophysical Research Communications 288, 75–79. Witko-Sarsat, V., Cramer, E.M., Hieblot, C., Guichard, J., Nusbaum, P., Lopez, S., Lesavre, P., Halbwachs-Mecarelli, L., 1999. Presence of proteinase 3 in secretory vesicles: evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules. Blood 94, 2487–2496. Zeller, J.M., Landay, A.L., Lint, T.F., Gewurz, H., 1986. Aggregated C-reactive protein binds to human polymorphonuclear leukocytes and potentiates Fc receptormediated chemiluminescence. Journal of Laboratory and Clinical Medicine 108, 567–576. Zeller, J.M., Sullivan, B.L., 1992. C-reactive protein selectively enhances the intracellular generation of reactive oxygen products by IgG-stimulated monocytes and neutrophils. Journal of Leukocyte Biology 52, 449–455.