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The Journal of Immunology, 2001, 167: 5355-5361.
Copyright © 2001 by The American Association of Immunologists

Loss of Pentameric Symmetry of C-Reactive Protein Is Associated with Promotion of Neutrophil-Endothelial Cell Adhesion1

Christine Zouki*, Barbara Haas*, John S. D. Chan{dagger}, Lawrence A. Potempa{ddagger} and János G. Filep2,*

* Research Center, Maisonneuve-Rosemont Hospital and Department of Medicine, University of Montréal, Montréal, Québec, Canada; {dagger} Centre de Recherche, Centre Hospitalier de l’Université de Montreal-Hôtel Dieu, University of Montréal, Montréal, Québec, Canada; and {ddagger} NextEra Therapeutics, Vernon Hills, IL 60061


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The classic acute-phase reactant C-reactive protein (CRP) is a cyclic pentameric protein that diminishes neutrophil accumulation in inflamed tissues. When the pentamer is dissociated, CRP subunits undergo conformational rearrangement that results in expression of a distinctive isomer with unique antigenic and physicochemical characteristics (termed modified CRP (mCRP)). Recently, mCRP was detected in the wall of normal human blood vessels. We studied the impact and mechanisms of action of mCRP on expression of adhesion molecules on human neutrophils and their adhesion to human coronary artery endothelial cells. Both CRP and mCRP (0.1–200 µg/ml) down-regulated neutrophil L-selectin expression in a concentration-dependent fashion. Furthermore, mCRP, but not CRP, up-regulated CD11b/CD18 expression and stimulated neutrophil extracellular signal-regulated kinase activity, which was accompanied by activation of p21ras oncoprotein, Raf-1, and mitogen-activated protein kinase kinase. These actions of mCRP were sensitive to the mitogen-activated protein kinase kinase inhibitor PD98059. mCRP markedly enhanced attachment of neutrophils to LPS-activated human coronary artery endothelial when added together with neutrophils. This effect of mCRP was attenuated by an anti-CD18 mAb. Thus, loss of pentameric symmetry in CRP is associated with appearance of novel bioactivities in mCRP that enhance neutrophil localization and activation at inflamed or injured vascular sites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C-reactive protein (CRP),3 a prototypical acute phase reactant, is a member of the pentraxin family of highly conserved cyclic pentameric proteins (1). In healthy subjects, serum concentration of CRP is <1 µg/ml; it can increase as much as 1000-fold within 24 h after the onset of inflammation or tissue damage (1, 2). Elevated plasma levels of CRP have been noted in a variety of pathological conditions (1, 2), they are of prognostic value in some diseases such as rheumatoid arthritis (3), and they have been reported to be predictive of subsequent acute cardiovascular events among apparently healthy men and women (4, 5) and patients with stable and unstable angina (6). Despite extensive studies spanning several decades, the exact role and mechanisms of action of CRP as a modulator of inflammation has not been well defined. CRP functions as an opsonin for certain microorganisms (7, 8), it can activate the complement system (9), and it can induce release of proinflammatory cytokines from monocytes (10). However, its net effect is anti-inflammatory in transgenic mice that produce large amounts of CRP (11, 12). Such an effect of CRP may be explained by its ability to inhibit neutrophil chemotaxis (13), adhesion to endothelial cells (14), migration into tissues (15), and superoxide production and degranulation (16). Furthermore, CRP binds to the surface membranes of intact apoptotic cells and protects the cells from assembly of the terminal membrane attack complex (17), thereby promoting noninflammatory clearance of apoptotic cells. Once tissue necrosis has occurred, CRP does not exert an anti-inflammatory effect (18).

To reconcile the pro- and anti-inflammatory actions of CRP, it has been proposed that distinct species of CRP are formed during inflammation. Indeed, conformationally altered and/or proteolytic forms of CRP express several epitopes that are not present on native CRP (19) and display properties distinct from those of native CRP (20, 21). Pentameric CRP can be dissociated into free subunits through various chemical manipulations in vitro (20). These subunits expressing several neoepitopes are referred to as modified or monomeric CRP (mCRP). mCRP Ags were detected in inflamed rabbit liver and muscle (22), as well as in the wall of human normal blood vessels (23). We have previously reported that native CRP prevents neutrophil adhesion to endothelial cells by inducing L-selectin shedding from neutrophils without inducing cell activation (14). Although native CRP binds primarily to the low-affinity IgG Fc{gamma}RIIa (CD32), and to some extent to the high-affinity IgG Fc{gamma}RI (CD64) (24, 25, 26), mCRP binds to the low-affinity IgG immune complex Fc{gamma}RIIIb (CD16) (27). We attempted to define mCRP’s role in the regulation of neutrophil adhesion. In this study, we report that mCRP stimulates the proinflammatory responses of up-regulating CD11b/CD18 expression on human neutrophils via activation of the p21ras oncoprotein (Ras)/Raf-1/mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (Erk) signaling pathway, and promoting {beta}2-integrin-dependent adhesion of neutrophils to endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

In this study, the mAbs used included FITC-conjugated mouse anti-human L-selectin mAb DREG-56 (BD PharMingen, San Diego, CA), R-PE-conjugated mouse anti-human CD11b mAb Leu-15 and FITC-labeled anti-human CD11a mAb G-25.2 (BD Immunocytometry Systems, Mountain View, CA), and R-PE-conjugated mouse anti-human CD18 mAb MEM-48 (Monosan, Uden, The Netherlands). The following function-blocking murine mAbs were used in neutrophil-endothelial cell adhesion assays: anti-L-selectin mAb DREG-56 (IgG1, BD PharMingen), anti-E-selectin mAb ENA-2 (IgG1, purified F(ab')2, Monosan), and anti-CD18 mAb L130 (IgG1, BD Immunocytometry Systems). The irrelevant mAb MOPC-21 (IgG1) was used as a negative control. Antisera specific for Raf-1 (C-12) and normal rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). [{gamma}-32P]ATP was from DuPont-New England Nuclear (Boston, MA).

Human CRP was obtained from Calbiochem (La Jolla, CA). Purity of the protein was ascertained as a single silver-stained protein band of 23 kDa, and by using mAbs that distinguish antigenicity differences in CRP and mCRP (19). High-purity native CRP was stored in buffers containing CaCl2 to prevent the spontaneous formation of mCRP from the native CRP pentamer. Modified CRP was made from native CRP by treatment with 8 M urea in the presence of 10 mM EDTA for 1 h at 37°C, followed by exhaustive dialysis into 25 mM Tris-HCl (pH 8.3). A recombinant form of mCRP (rmCRP) with both cysteine residues mutated to alanine residues (i.e., C36->A; C97->A) was expressed in Escherichia coli and was isolated from inclusion bodies to >95% purity (28). To enhance solubility, rmCRP was reacted with maleic anhydride under conditions that allowed for selective reaction with nucleophilic amine groups (29). Cysteine-mutated rmCRP was directly comparable with mCRP produced from the native CRP pentamer in terms of SDS-PAGE size, solubility, antigenicity, and in vitro activities, including actions on L-selectin and CD11b/CD18 expression on neutrophils. Therefore, with the exception of the studies on neutrophil adhesion molecules, the results obtained with solubilized, maleylated rm CRP are described in this report.

Neutrophil isolation and activation

Polymorphonuclear leukocytes (PMNs) were isolated from human peripheral blood as described previously (14). PMNs (107 cells/ml, purity >95%) were incubated with PD98059, SB 203580, wortmannin, or genistein for 20 min at 37°C, challenged with mCRP for 30 min, and surface expression of L-selectin, CD11b, CD11a, or CD18 was analyzed. To assess whether mCRP is active in the microenvironment of whole blood, in separate experiments, whole blood aliquots were challenged with mCRP or native CRP and adhesion molecule expression was then analyzed. In other experiments, PMNs were lysed at the end of the incubation period in ice-cold lysis buffer (20 mM Tris, 1 mM EGTA, 2 mM Na3VO4, 25 mM NaF, 0.5% (v/v) Triton X-100, 2 mM PMSF, 40 µg/ml aprotinin, and 10 µg/ml each of chymostatin, leupeptin, and pepstatin A, pH 7.4). Cell lysates were used for further analysis.

Analysis of surface Ag expression

Direct immunofluorescence labeling of resting and treated PMNs was performed as described (14). Cells were stained with a saturating concentration of fluorescence dye-conjugated anti-human L-selectin, CD11a, CD11b, or CD18 mAb. Nonspecific binding was evaluated using appropriately labeled mouse IgG1. Double- or single-color immunofluorescence staining was analyzed by a flow cytometer (FACScan, BD Immunocytometry Systems) with Lysis II software. The results are presented as relative fluorescence units (RFU): RFU = (FUexperimental - FUisotype) x 100/(FUcontrol - FUisotype), where FUexperimental and FUcontrol are the L-selectin and CD11b mean fluorescence intensity of treated cells and cells cultured in medium only, respectively, and FUisotype is the mean fluorescence intensity of class-matched irrelevant Ab.

Phosphorylation of MEK and Erk, and Erk activity assay

Western blot analysis of phosphorylated MEK and Erk 1/2 (p44/42 MAPK) was performed as described previously (30) using the Phospho Plus MEK 1/2 and Erk 1/2 MAPK Ab kits (New England Biolabs, Beverly, MA).

Erk 1/2 activity was measured with the p44/42 MAPK Assay kit (New England Biolabs) using Elk-1 fusion protein, a specific target for Erk 1/2 (31) following immunoprecipitation of Erk 1/2 with an immobilized anti-phospho-p44/42 MAPK mAb. Phosphorylation of Elk-1 at Ser383 wasquantitated by densitometry following immunoblotting using an anti-phospho-Elk-1 polyclonal Ab and chemiluminescence detection.

Raf-1 kinase and Ras activation assays

Raf-1 activity was determined by a modification of the method of Gardner et al. (32). Raf-1 was immunoprecipitated, Ag-Ab complexes were isolated with protein A-Sepharose CL-4B, and Raf-1 activity was measured using the Raf-1 Kinase Cascade Assay kit (Upstate Biotechnology, Lake Placid, NY) by adding inactive MEK1, inactive Erk-2, and ATP (200 µM), and then myelin basic protein (20 µg/assay) and [{gamma}-32P]ATP (10 µCi/assay) to the samples in accordance with the manufacturer’s protocols. The reaction mixtures were spotted onto phosphocellulose squares, which were washed thoroughly, and were counted for radioactivity (phosphorylated myelin basic protein).

Activated p21Ras (Ras-GTP) from neutrophil lysates was affinity precipitated by using GST-Ras binding domain of Raf-1 (residues 1–149) fusion protein conjugated to agarose (Upstate Biotechnology) (33). The beads were washed extensively and boiled in reducing sample buffer. The eluted proteins were resolved on a 10% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, probed with a mouse anti-Ras mAb (clone RAS10, Upstate Biotechnology), and visualized using a goat anti-mouse secondary Ab conjugated to horseradish peroxidase (Bio-Rad, Mississauga, Ontario) and a chemiluminescence detection system.

Culture of endothelial cells

Normal human coronary artery endothelial cells (HCAEC) obtained from Clonetics (San Diego, CA) were cultured as described (14). HCAEC (passages 3–6), seeded into 96-well microplates and grown to confluence, were used in the experiments.

Neutrophil-endothelial cell adhesion assay

The adhesion assay was performed using 51Cr-labeled neutrophils as described (14). In brief, monolayers of HCAEC in 96-well microplates were cultured with LPS (1 µg/ml), a well-known activator of endothelial cells, for 4 h at 37°C in a 5% CO2 atmosphere. In some experiments, mCRP was added to HCAEC during the last 30 min of incubation with LPS. The monolayers were then washed, and 2 x 105 neutrophils in 100 µl were added. In some experiments, mCRP was added together with neutrophils to HCAEC treated with LPS for 4 h. In another set of experiments, LPS-activated HCAEC were incubated for 15 min with ENA-2 mAb (10 µg/ml) or MOPC-21 mAb (20 µg/ml) before the addition of neutrophils. Radiolabeled neutrophils were incubated with DREG-56 mAb (20 µg/ml), L130 mAb (10 µg/ml), or MOPC-21 mAb for 15 min before addition to HCAEC. After incubation of HCAEC with neutrophils with or without mCRP for 30 min at 37°C on an orbital shaker at 90 rpm, loosely adherent or unattached neutrophils were removed by washing, and the endothelial monolayer plus the adherent neutrophils were lysed in 150 µl of 0.1% Triton X-100. The number of adhered neutrophils in each experiment was estimated from the radioactivity of a control sample.

Data analysis

Results are expressed as means ± SEM. Statistical comparisons were made by ANOVA using ranks (Kruskal-Wallis test) followed by Dunn’s multiple contrast hypothesis test to identify differences between various treatments, or by the Wilcoxon signed rank test and Mann-Whitney U test for paired and unpaired observations, respectively. Values of p <0.05 were considered significant for all tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Modified CRP, but not native CRP, up-regulates expression of CD11b/CD18 on neutrophils

Fig. 1Go reports a representative result illustrating the impact of mCRP on adhesion molecule expression on PMNs. Incubation with mCRP down-regulated the expression of L-selectin, and up-regulated the expression of CD11b. mCRP induced similar changes in adhesion molecule expression in both isolated PMNs (Fig. 1GoA) and in neutrophils in whole blood (Fig. 1GoB). Both mCRP and CRP reduced surface expression of L-selectin on isolated neutrophils in a concentration-dependent manner (Fig. 1GoC). mCRP was approximately two orders of magnitude more potent than CRP, inducing a 50% loss of surface L-selectin at ~1 µg/ml (p < 0.05). Unlike native CRP (1–200 µg/ml), mCRP also increased expression of CD11b with an apparent EC50 value of 8 µg/ml (Fig. 1GoD). The addition of mCRP caused similar increases in the expression of CD18 (data not shown). The maximum changes in L-selectin and CD11b that could be achieved with mCRP were similar to those evoked by 1 µM platelet-activating factor (PAF; 89 ± 5 and 73 ± 5% decreases in L-selectin expression, and 95 ± 16 and 85 ± 4% increases in CD11b expression by mCRP and PAF, respectively, n = 6, both p > 0.1). Neutrophil viability was not affected by mCRP at the concentrations tested (data not shown).



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  		FIGURE 1. Modified CRP-induced changes in adhesion molecule expression on human neutrophils. Isolated neutrophils (A) or whole blood aliquots (B) were incubated with 100 µg/ml mCRP for 30 min at 37°C. In each histogram is also displayed the negative control of immunostaining with class-matched irrelevant Abs (C). Shown is a representative of six experiments using blood from different donors. Concentration-dependent effects of native CRP and mCRP on surface expression of L-selectin (C) and CD11b (D). Isolated neutrophils were challenged with native CRP or mCRP for 30 min. RFUs are presented as percentage of control, i.e., the mean fluorescence intensity of PMNs incubated in medium only. Results are the mean ± SEM for six experiments using neutrophils from different donors.*, p < 0.05; **, p < 0.01; ***, p < 0.001 vs control.

 
The capacity of mCRP to affect the affinity of {beta}2 integrins was investigated using the mAb G-25.2, an Ab that recognizes the {beta} propeller domain in LFA-1 (34). Conformational changes and association of the I domain with the {beta} propeller domain are thought to be required for formation of a higher affinity form of LFA-1 and Mac-1 (34, 35). Incubation of PMNs with mCRP did not induce a detectable increase in staining with mAb G-25.2 (data not shown).

Inhibition of MAPK kinase reverses mCRP-induced changes in CD11b/CD18 expression on neutrophils

The MEK inhibitor PD98059 effectively prevented mCRP-induced up-regulation of CD11b/CD18 expression, whereas it was a less effective inhibitor in reversing mCRP-induced down-regulation of L-selectin expression (Fig. 2Go). Although wortmannin alone down-regulated L-selectin expression, neither it nor genistein significantly affected mCRP-induced changes in L-selectin and CD11b/CD18 expression. The selective p38 MAPK inhibitor SB 203580 (36) had little effect on mCRP-induced changes in either L-selectin or CD11b/CD18 expression (Fig. 2Go).



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  		FIGURE 2. Effects of MEK, p38 MAPK, and tyrosine kinase inhibitors on expression of neutrophil adhesion molecules. PMNs were incubated with PD98059 (an inhibitor of MEK), SB; 203580 (p38 MAPK inhibitor), wortmannin (phosphatidyl inositol-3 kinase inhibitor), or genistein (protein tyrosine kinase inhibitor) for 20 min at 37°C and were then challenged with 100 µg/ml mCRP for 30 min. Adhesion molecule expression is presented as the percentage of control mean fluorescence intensity that was 78 ± 4 for L-selectin and 1016 ± 37 for CD11b. Values are the mean ± SEM of five independent experiments.

 
Modified CRP, but not native CRP, activates Erk via the Ras/Raf-1/MEK signaling cascade

Incubation of PMNs with mCRP induced a time- and concentration-dependent increase in phosphorylation of Erk relative to unstimulated controls (Fig. 3GoA). Phosphorylation of Erk was rapid in onset, reaching a peak at around 2 min. The relative degree of Erk phosphorylation induced by 1 µM PAF is shown for comparison. We also assayed Erk activity by measuring the ability of neutrophil lysates to phosphorylate Elk-1, a specific target for Erk (31). Stimulation of PMNs with mCRP resulted in concentration-dependent increases in Elk-1 phosphorylation that were sensitive to PD98059 (Fig. 3GoC).



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  		FIGURE 3. Activation of neutrophil MEK and Erk by mCRP. Isolated neutrophils were challenged with mCRP (100 µg/ml) or PAF (1 µM) for the indicated times at 37°C (A) or with various concentrations of mCRP or CRP for 2 min (B). PMNs were lysed and the phosphorylation of kinases was assessed in immunoblots using polyclonal phospho-specific Abs. The results are representative of four independent experiments. C, Stimulation of Erk activity by mCRP. PMNs were preincubated with PD98059 (100 µM) for 10 min and were then challenged with mCRP for 2 min at 37°C. Active Erk 1/2 was immunoprecipitated from cell lysates, and Erk activity was measured by immunoblotting phosphorylated Elk-1, an Erk-selective substrate. Relative staining intensity was quantitated by densitometry. Results shown are the mean ± SEM of four independent experiments. *, p < 0.05; **, p < 0.01 vs unstimulated.

 
Because Erk activity in response to growth factors depends upon the sequential activation of Ras and the kinases Raf-1 and MEK (37, 38, 39), we examined whether mCRP stimulation of Erk is similarly mediated via this cascade. Phosphorylation of MEK was rapid in onset and maximal within 2 min after addition of mCRP to PMNs, and occurred in a concentration-dependent fashion (Fig. 3GoA). Native CRP, shown for comparison, did not evoke MEK phosphorylation (Fig. 3GoB). Within 2 min after its addition to neutrophils, mCRP also evoked increases in neutrophil Raf-1 kinase activity (Fig. 4GoA) and induced a concentration- and time-dependent association of Ras with the GST-Ras binding domain of Raf-1 (Fig. 4Go, B and C, respectively), indicating Ras activation.



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  		FIGURE 4. Activation of neutrophil Raf-1 and Ras by mCRP (A). Raf-1 kinase activity was measured following immunoprecipitation of Raf-1 as described in Materials and Methods. Control experiments performed in the absence of substrate or using a nonspecific control antiserum resulted in levels of Raf-1 activity below those of unstimulated lysate. Results shown are the mean ± SEM of four experiments. *, p < 0.05; **, p < 0.01 vs unstimulated. PMNs were challenged with various concentrations of mCRP for 2 min (B), or with 100 µg/ml mCRP for the indicated times (C). GTP-bound active Ras was isolated from neutrophil lysates by affinity precipitation with a GST-Ras binding domain fusion protein followed by immunoblot analysis with an anti-Ras Ab. Shown is a representative result; the experiment was repeated four times.

 
To provide further evidence that mCRP stimulation of neutrophil Erk is mediated via Raf-1 and MEK, we tested whether PD98059 has an effect on Erk activation. PD98059 specifically inhibits MEK and prevents its phosphorylation and activation by Raf-1 or other kinases (40). Preincubation of PMNs with 100 µM PD98059 resulted in 89 ± 2% and 81 ± 8% inhibition of mCRP-stimulated Erk activity and up-regulation of CD11b expression, respectively.

Modified CRP promotes neutrophil adhesion to HCAEC

Only a few PMNs were able to bind to unstimulated HCAEC. Neutrophil adherence was enhanced 13-fold by activation of HCAEC with 1 µg/ml LPS (Fig. 5GoA). The number of adherent neutrophils was further enhanced when PMNs were added together with mCRP to LPS-activated HCAEC. No further increases in neutrophil adherence were detected when PMNs were added to HCAEC that were cultured with LPS for 3.5 h and then with LPS plus 100 µg/ml mCRP for an additional 30 min (LPS alone: 5.0 ± 0.1 x 104 adherent PMNs per well; LPS + mCRP: 5.2 ± 0.2 x 104 adherent PMNs per well, n = 5, p > 0.1). Furthermore, incubation of HCAEC with 100 µg/ml mCRP for 30 min did not increase adhesion of unstimulated PMNs (0.41 ± 0.08 x 104 adherent PMNs per well vs 0.37 ± 0.07 x 104 adherent PMNs per well to unstimulated HCAEC, n = 4, p > 0.1), and mCRP did not enhance neutrophil adhesiveness when it was added together with PMNs to untreated HCAEC (0.34 ± 0.03 x 104 adherent PMNs per well, n = 4, p > 0.1).



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  		FIGURE 5. mCRP enhances CD18-dependent adhesion of human neutrophils to HCAEC. Confluent HCAEC monolayers were cultured in medium only (control) or activated with LPS (1 µg/ml) for 4 h, as indicated. Radiolabeled PMNs or PMNs together with mCRP (A) were then added and incubated with HCAEC for 30 min at 37°C on an orbital shaker. PMNs were added alone (B) or together with 100 µg/ml mCRP (C) to LPS-activated HCAEC and were incubated for 30 min in the absence (medium) or presence of function blocking mAbs directed against CD18, CD62E, and CD62L as indicated on the y axis. The irrelevant mAb MOPC-21 (IgG1) was used as a negative control. Results are expressed as the mean ± SEM of six experiments in triplicate using neutrophils from different donors. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs LPS-activated HCAEC; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with adhesion of mCRP-treated PMNs to LPS-activated HCAEC.

 
Because multiple receptors are involved in neutrophil adhesion to LPS-stimulated HCAEC even under nonstatic conditions (14), and mCRP affected expression of both L-selectin and CD11/CD18 on PMNs, we assayed the contribution of L-selectin, {beta}2 integrins, and E-selectin to the binding interaction. A significant proportion of neutrophil attachment to LPS-activated HCAEC was blocked by mAbs binding to E-selectin (31 ± 2%, n = 4), L-selectin (24 ± 2%), and CD18 (30 ± 2%). The combination of these mAbs inhibited neutrophil adhesion by ~95% (p < 0.001; Fig. 5GoB). The mCRP-stimulation of PMN adhesion to LPS-activated HCAEC was blocked by mAbs binding to E-selectin (44 ± 4%, n = 6), L-selectin (11 ± 4%), and CD18 (59 ± 4%; Fig. 5GoC). The large inhibition noted with Ab blocking CD18 suggests the mCRP effect on neutrophil-endothelial cell attachment is predominantly promoted in a CD18-dependent manner. The combination of these three mAbs inhibited PMN adhesion by ~90% (p < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we describe that there is a "hidden" bioactivity in CRP that is expressed when the pentameric structure dissociates and undergoes a conformational rearrangement. Furthermore, this hidden CRP bioactivity appears to promote pro-inflammatory neutrophil reactions, whereas the pentameric CRP contributes anti-inflammatory activities (11, 12, 13, 14, 15, 16).

The conformationally altered CRP, which we refer to as modified or mCRP, can be expressed by removing calcium ions from the pentameric CRP under denaturing conditions (20, 21). No proteolytic reaction is required to express mCRP from CRP; however, mCRP is more susceptible to proteolysis than is the native CRP molecule (41). mCRP has much reduced solubility, it self aggregates into lattice-like structures (42), and it expresses neoantigens distinct from the native CRP pentamer (19). mCRP selectively binds immune complexes (42) and enhances leukocyte oxidative metabolism stimulated by aggregated IgG (43).

In these studies we show that mCRP, unlike CRP, up-regulates the surface expression of CD11b/CD18 adhesion molecules on human neutrophils, leading to increased adhesion of PMNs to the activated endothelium. We also examined the mechanisms of mCRP signaling in PMNs, observing a possible role for the Ras/Raf-1/MEK/Erk signaling pathway in mCRP-stimulated neutrophil responses.

At low microgram per milliliter concentrations, mCRP down-regulated L-selectin and up-regulated CD11b/CD18 expression on isolated PMNs as well as on neutrophils in the microenvironment of whole blood. Our results suggest that this activation is most likely mediated via activation of Erk. mCRP stimulation of Erk was rapid, and was concentration and time dependent. Erk activation was detected by immunoblotting using anti-phospho-Erk Abs and was confirmed by direct measurements of Erk activity using the selective Erk target, Elk-1. The kinetics of Erk activation by mCRP in PMNs and mitotic cells appears to be distinct, rapidly peaking at 2–5 min for mCRP, and 5–10 min for mitotic cells (44). Erk stimulation in response to protein tyrosine kinase receptors and some G protein-linked receptors proceeds via Ras, Raf-1, and MEK (37, 38, 39). Our data indicate that mCRP stimulation of Erk in neutrophils also involves this pathway because mCRP stimulated Ras and Raf-1 kinase activity and evoked phosphorylation of MEK; and the specific MEK inhibitor PD98059 inhibited Erk activation by mCRP, although this inhibition was incomplete (~90%).

The present data further suggest that Erk activation is required for mCRP stimulation of CD11b/CD18 expression and consequently of PMN adhesion to HCAEC. Previous studies reported a tight correlation between Erk activation and neutrophil aggregation in response to chemoattractants (45) and arachidonic acid (46), as well as between Erk activation and neutrophil adhesion to HCAEC in response to peroxynitrite (30). We also found that the degree of Erk inhibition by PD98059 was similar to the degree of inhibition of CD11b/CD18 expression. However, PD98059 appeared to be a less potent inhibitor of the mCRP down-regulation of L-selectin expression, indicating the involvement of other, Erk-independent pathway(s) in adhesion signaling. Physiological stimuli regulate adhesion by either altering the affinity of the individual integrin molecule or by inducing clustering of {beta}2 integrins (i.e., increasing avidity) (34, 35). mCRP did not affect mAb G25.2 binding to the {beta} propeller domain of LFA1, whose expression is associated with formation of a higher affinity LFA-1 binding reaction (34, 35). These results suggest that mCRP does not alter the affinity of LFA-1, and probably of Mac-1, rather it induces clustering of {beta}2 integrins, thereby increasing the overall strength of binding. However, our results do not preclude the possibility that in the presence of integrin ligands, mCRP might affect a ligand-induced affinity increase secondary to integrin clustering.

Leukocyte extravasation into tissues involves a multistep interaction of leukocytes and endothelial cells via regulated expression of surface adhesion molecules (47, 48). The initial capture and tethering of circulating neutrophils to the endothelium is mediated by selectins expressed on leukocytes (L-selectin) or on activated endothelial cells (P- and E-selectins). L-selectin is rapidly shed after cell activation with a concomitant up-regulation of CD11b/CD18 (Mac-1) (49). The {beta}2 integrins, Mac-1 and LFA-1 (CD11a/CD18), are largely responsible for subsequent tightening of the adhesion and transendothelial migration of neutrophils via their endothelial counterligands, ICAM-1 and ICAM-2 (47, 48).

Induction of selective L-selectin shedding from neutrophils by native CRP (14) or nonsteroid anti-inflammatory drugs (50) was found to attenuate PMN-HCAEC attachment. This down-regulation of L-selectin expression occurred in the absence of cell activation. Although both native CRP and mCRP induced L-selectin shedding, native CRP inhibited (14), whereas mCRP actually promoted neutrophil adhesion to HCAEC. This augmentation was largely due to its action on PMNs rather than on HCAEC in this interaction, because addition of mCRP for the last 30 min of culture with LPS or culture of HCAEC with mCRP in the absence of LPS for 30 min did not produce significant increases in the number of adherent PMNs. Furthermore, mCRP did not enhance neutrophil adhesiveness if HCAEC were not activated (i.e., pretreated with LPS). No adhesion experiments were performed with neutrophils preincubated with mCRP because by up-regulating CD11b/CD18 expression, mCRP may induce neutrophil aggregation, therefore making interpretation of the results difficult. In our PMN-HCAEC binding assay, P-selectin-dependent adhesion was not studied because endothelial P-selectin expression occurs within 10–20 min after application of inflammatory stimuli and is sustained for ~60 min (51).

Based on the present and previous results, we propose a model to explain the opposite role for native CRP and mCRP in the regulation of neutrophil trafficking (Fig. 6Go). We propose that the opposite actions of native CRP and mCRP on neutrophil adhesion could be attributed to activation of different receptors. Native CRP binds primarily to the low-affinity IgG Fc{gamma}RIIa (CD32) and to some extent to the high-affinity IgG Fc{gamma}RI (CD64) (24, 25, 26). Such binding is associated with shedding of L-selectin without neutrophil activation, and subsequent attenuation of PMN adhesion to activated endothelial cells (14). When pentameric CRP is dissociated into its modified monomeric form, binding to neutrophils is mediated through the low-affinity immune complex binding IgG FcR, Fc{gamma}RIIIb (CD16) (27). Fc{gamma}RIIIb is a glycosylphosphatidyl inositol-linked receptor (52) that requires other molecules on the cell surface to initiate phagocytosis. mCRP, either alone or associated with immune-complexed IgG (42), could augment such receptor-mediated triggering of neutrophil activation, contributing to the strength and speed of the neutrophil response during an acute phase. The binding of mCRP to the low-affinity Fc{gamma}-immune complex receptor may lead to activation of Src kinases (52), which then initiate Erk activation via Ras, Raf-1, and MEK, as previously observed in other systems. It should be noted that in these studies, we did not evaluate the direct role of the Fc{gamma}RIIIb receptor in the mCRP regulation of neutrophil adhesion molecules. Previous studies have shown that immune complexes acting at Fc{gamma}RIIIb on neutrophils stimulate expression of total and functional CD11b/CD18, but have little effect on L-selectin expression (53, 54). Heterotypic cross-linking of Fc{gamma}RIIIb markedly reduces L-selectin expression (54). Therefore, it is possible that the mCRP effect may be mediated through some yet undefined cell surface receptor or triggering process that transmits the activation signal to the Ras/Raf-1/MEK/Erk pathway. The activated pathway regulates CD11b/CD18 expression and/or other signaling events, which, in addition to Erk, contribute to down-regulation of L-selectin expression. Intriguingly, the effects of mCRP on neutrophil L-selectin and CD11b/CD18 expression more closely mimic the activation of G-protein-linked receptors (e.g., receptors for IL-8 and fMLP) than that of homotypic cross-linking of Fc{gamma}RIIIb (53).



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  		FIGURE 6. A model for neutrophil activation by mCRP. See text for explanation. Broken arrows indicate an undefined pathway. GPI, glycosylphosphatidyl inositol.

 
This model represents a best fit for the available data; it fails to provide a mechanism by which mCRP, either alone or associated with the Fc{gamma}RIIIb, leads to the activation of Ras and/or Raf-1, and to the activation of the neutrophil response. What these data do clearly show, however, is that the apparently contradictory pro- and anti-inflammatory activities attributed to CRP over the years may be manifest in two isomeric conformations of the CRP molecule. Each has the same primary sequence and would not be easily discernible using standard analytical assays. The formation of mCRP from CRP is nonproteolytic and irreversible (55). We have noted that prolonged storage of purified CRP in the absence of calcium will cause a spontaneous conversion of CRP to mCRP (our unpublished observation). These observations raise the possibility that previous studies may have used CRP test samples that were contaminated with higher amounts of the mCRP conformer. Of note, when various polyclonal anti-"CRP" antisera were tested for specific reactivity to each of native CRP and mCRP Ags, all antisera tested showed from 3 to 16% specificity to the mCRP Ag (56).

Our results may have relevance to excessive leukocyte trafficking in inflammation. It is tempting to speculate that endothelial injury may result in exposure of mCRP that is naturally expressed in the intima (23). Contact with PMNs loosely attached to the site would result in PMN activation, firm attachment, and consequent emigration into injured/inflamed tissues. Alternatively, mCRP may be also formed at sites of injury or infection as part of the activation of the acute inflammatory response. The present and previous findings lend strong experimental support to the hypothesis that conformationally altered forms of CRP such as mCRP display potent pro-inflammatory activities potentiating activated responses of neutrophils, monocytes, and platelets (43, 57), whereas native CRP has activities mainly associated with the resolution of inflammation.

In summary, our data indicate that loss of pentameric symmetry in CRP is associated with the appearance of novel pro-inflammatory biomodalities in mCRP. mCRP activates neutrophil Erk via the Ras/Raf-1/MEK signaling cascade, leading to up-regulation of CD11b/CD18 expression and promoting neutrophil adhesion to HCAEC via a CD18-dependent mechanism. Thus, native CRP and mCRP play an opposite role in the regulation of leukocyte trafficking during inflammation.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada/Canadian Institutes of Health Research (MT-12573 to J.G.F. and MT-15070 to J.S.D.C. and J.G.F.). Back

2 Address correspondence and reprint requests to Dr. János G. Filep, Research Center, Maisonneuve-Rosemont Hospital, 5415 boulevard de l’Assomption, Montréal, Québec, Canada H1T 2 M4. E-mail address: janos.g.filep{at}umontreal.ca Back

3 Abbreviations used in this paper: CRP, C-reactive protein; Erk, extracellular signal-regulated kinase; HCAEC, human coronary artery endothelial cells; mCRP, modified or monomeric CRP; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; PAF, platelet-activating factor; PMNs, polymorphonuclear leukocytes; rmCRP, recombinant mCRP; Ras, p21ras oncoprotein; PMN, polymorphonuclear leukocyte; RFU, relative fluorescence units. Back

Received for publication May 29, 2001. Accepted for publication August 20, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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