C-reactive Protein Inhibits Chemotactic Peptide-induced p38 Mitogen-activated Protein Kinase Activity and Human Neutrophil Movement*

Rita M. HeuertzDagger §, Sally M. TricomiDagger , Uthayashanker R. Ezekielparallel , and Robert O. WebsterDagger §

From the Departments of Dagger  Internal Medicine and § Molecular Microbiology and Immunology, Saint Louis University School of Medicine and the parallel  Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Serum levels of the acute-phase reactant, C-reactive protein (CRP), increase dramatically during acute inflammatory episodes. CRP inhibits migration of neutrophils toward the chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an anti-inflammatory agent. Since tyrosine kinases are involved in neutrophil migration and CRP has been shown to decrease phosphorylation of some neutrophil proteins, we hypothesized that CRP inhibits neutrophil chemotaxis via inhibition of MAP kinase activity. The importance of p38 MAP kinase in neutrophil movement was determined by use of the specific p38 MAP kinase inhibitor, SB203580. CRP and SB203580 both blocked random and fMLP-directed neutrophil movement in a concentration-dependent manner. Additionally, extracellular signal-regulated MAP kinase (ERK) was not involved in fMLP-induced neutrophil movement as determined by use of the MEK-specific inhibitor, PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor did it alter the ability of CRP or SB203580 to inhibit fMLP-induced chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase activity in a concentration-dependent manner as measured by an in vitro kinase assay. Impressively, CRP-mediated inhibition of p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis (r = -0.7144). These data show that signal transduction through p38 MAP kinase is necessary for neutrophil chemotaxis and that CRP intercedes through this pathway in inhibiting neutrophil movement.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Upon the onset of inflammation, there is an increase in the concentration of a number of plasma proteins which are collectively termed acute-phase reactants. C-reactive protein (CRP),1 the classic acute-phase reactant in man, has a normal serum concentration of less than 1 µg/ml; however, within 24 h after onset of inflammation, levels can increase as much as 1000-fold (1). This dramatic increase in CRP suggests an important role for CRP in inflammation and the fact that CRP is highly conserved throughout evolution indicates an important biological role for CRP. Besides this humoral acute-phase response, there are also cellular components of acute inflammation. Neutrophils are among the first leukocytic cells to migrate into tissues in response to invading pathogens or other initiators of inflammatory injury. One of the first steps of neutrophil involvement in acute inflammation is chemotaxis, directed movement toward chemotactic agents, such as complement fragments (C5a and C5a des-Arg), cytokines (interleukin-8), and bacteria-derived peptides such as formyl-methionine-leucine-phenylalanine (fMLP).

CRP is present at sites of inflammation in association with neutrophils. Selective localization of CRP to sites of inflammation in vivo has been shown by double staining rabbit muscle tissue sections to visualize necrotic muscle associated with CRP (2). Du Clos et al. (3) induced allergic encephalomyelitis in rabbits and found increased neutrophil numbers in the spinal cord sections that correlated with elevated spinal fluid CRP levels. Parish showed that serum CRP levels in patients with chronic neutrophil vasculitis lesions were double that seen in patients with chronic mononuclear cell vasculitis lesions (4). In humans, neutrophils have been implicated in the pathogenesis of several acute inflammatory diseases such as acute myocardial infarction, bacterial infections, and acute respiratory distress syndrome (5-7). Acute respiratory distress syndrome is a noncardiogenic pulmonary edema with characteristics of an acute inflammatory reaction located primarily in the lung causing gross alterations in lung structure and function. In addition to massive influx of neutrophils into the alveolar space, significant elevations of CRP have been found in serum (8) and bronchoalveolar lavage fluid (9) from acute respiratory distress syndrome patients. CRP plays a role in modulating inflammatory and immune responses as a result of its ability to activate the classical complement pathway (10, 11), to modify platelet-activating factor-induced platelet aggregation (12), to bind T lymphocytes and suppress certain lymphocyte functions (13), and to enhance phagocytosis (14, 15). We have shown that CRP inhibits neutrophil chemotaxis in vitro (8) and neutrophil alveolitis in vivo (16-18).

Little is known about CRP-induced signal transduction in the neutrophil. A specific CRP receptor is present on the surface of neutrophils (19, 20) but the identity of the receptor remains elusive. CRP pretreatment of human neutrophils stimulated with fMLP results in a significant reduction in the degree of phosphorylation of several neutrophil proteins (21). Stimulation of the fMLP receptor on the neutrophil results in activation of two known members of the MAP kinase family, extracellular signal-regulated kinase (ERK) and p38 MAP kinase (22, 23). Activation of p38 MAP kinase, but not ERK, is indispensable for fMLP-induced chemotaxis of human neutrophils (22). Inhibition of p38 MAP kinase blocks transforming growth factor-beta -induced neutrophil chemotaxis and actin polymerization (24) and beta 2-integrin mobilization from cytoplasmic granules to the plasma membrane (25). Activation of p38 MAP kinase leads to the activation of mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2 (26) which phosphorylates the small heat shock protein, hsp27 (27, 28). Hsp27 has chaperone-like properties and acts as an inhibitor of actin polymerization (29, 30). Phosphorylation of hsp27 stimulates the polymerization of actin (31) and so help to repair the actin microfilament network disrupted during cellular stress thereby maintaining cellular integrity and aiding cell survival during stress.

In the current study, we hypothesized that CRP inhibits neutrophil movement by blocking the p38 MAP kinase signal transduction pathway. Here we show that inhibition of p38 MAP kinase with the specific inhibitor, SB203580, inhibits directed and random movement of neutrophils similar to that seen with CRP. Additionally, we show that CRP inhibits p38 MAP kinase activity and that inhibition of p38 MAP kinase activity correlates with inhibition of chemotaxis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human C-reactive Protein-- Human CRP was obtained from a commercial source (Biodesign International, Kennebunk, ME). SDS-PAGE of the human CRP followed by silver stain revealed a single band at 23 kDa and was ascertained as pure by densitometry (Molecular Dynamics, Sunnyvale, CA). The concentration of endotoxin in the purified CRP was 132 endotoxin units/ml (11 ng/ml) as determined by the Limulus amebocyte assay (Associates of Cape Cod, Woods Hole, MA). Immunoblotting for serum amyloid P, a common contaminant found in CRP preparations, was negative. Concentration of the purified CRP was determined by the BCA protein assay (Pierce Chemical Co., Rockford, IL). In all experiments, CRP concentrations were expressed as micrograms/ml. Using 125 kDa as the molecular mass of native human CRP, 100 µg/ml corresponded to 0.8 µM native molecule.

Isolation of Neutrophils-- Venous blood obtained from normal healthy human volunteers was treated with EDTA (0.15%) to prevent coagulation and layered onto NIMTM isolation media (Cardinal Associates, Santa Fe, NM) using a ratio of 1 part NIMTM to 1.4 parts whole blood. After centrifugation (630 × g, 22 °C, 40 min), the granulocyte-rich fraction was collected and recentrifuged (1000 × g, 4 °C, 10 min). The cell pellet was resuspended in sterile phosphate-buffered (pH 7.3) saline and hypotonic lysis performed to remove contaminating erythrocytes. Neutrophils were pelleted and washed once with sterile phosphate-buffered saline. Neutrophil yields were routinely >95% with >98% viability.

Neutrophil Chemotaxis-- Chemotaxis was quantified by the leading front method of Zigmond and Hirsch (32) utilizing a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD) and nitrocellulose filters with a pore size of 3 µm (Neuroprobe). All samples were prepared in chemotaxis buffer consisting of Hanks' balanced salt solution containing 10 mM Hepes (pH 7.4, Sigma) and 1% bovine serum albumin (Sigma). Neutrophils were pretreated with the p38 MAP kinase inhibitor, SB203580 (Calbiochem, San Diego, CA), the MEK1/MEK2 inhibitor, PD98059 (Calbiochem) at a concentration (100 µM) that blocks ERK1 and ERK2 activities (33), human CRP or normal human plasma with or without addition of purified human CRP for 10 min at 23 °C. Buffer or the chemoattractant fMLP (1 × 10-7 M) was added in duplicate to the lower chambers and neutrophils (200,000/well) were added to the upper chambers. After incubation for 35 min at 37 °C, the filters were removed, fixed in isopropyl alcohol, and stained with Harris acid hematoxylin. Either net neutrophil migration (distance moved in response to treatment minus distance moved in response to buffer (µm/35 min)) or percent inhibition of chemotaxis ([1 - net migration of treatment group divided by net migration of fMLP] × 100) was reported. In order to study mechanisms by which CRP inhibits neutrophil chemotaxis, an optimal concentration of fMLP (0.1 µM) was used. At this concentration of fMLP, concentrations of SB203580 severalfold higher than the IC50 were used.

Neutrophil Treatment and Preparation of Whole Cell Lysates-- Purified neutrophils were diluted to 1 × 108/ml in Hank's balanced salt solution with 0.25% bovine serum albumin (HBSA: Sigma) and 2 × 107 cells were pretreated with CRP by incubation for 10 min at 23 °C. In some experiments, cells were treated with 50% normal human plasma with or without the addition of purified human CRP. Cells were activated by the addition of fMLP (1 × 10-7 M) followed by incubation at 37 °C for 1 min after which all tubes were immediately centrifuged (200 × g, 10 min, 4 °C) to pellet the neutrophils. Supernatants were aspirated and pellets resuspended in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin) containing 1% protease inhibitor mixture (Sigma). Reaction mixtures were sonicated on wet ice at 35% power for ten 1-s pulses using a Sonic Dismembrator 300 (Fisher Scientific, Pittsburgh, PA). Reactions were then centrifuged (15,000 × g, 2 min, 4 °C) and supernatants quantitated for protein content using the BCA assay (Pierce Chemical Co.). Neutrophil cell lysates were frozen at -70 °C until assayed.

Immunoprecipitation of p38 MAP Kinase and Phosphorylation of p38 MAP Kinase Substrate, ATF2-- Neutrophil lysates (200 µg) were incubated overnight at 4 °C with 4 µl of rabbit anti-human p38 MAP kinase polyclonal antibody (New England Biolabs, Beverly, MA) followed by adsorption with protein A-Sepharose (1 h, 4 °C: Pharmacia Biotech Inc., Piscataway, NJ). Samples were centrifuged (15,000 × g, 1 min, 4 °C), washed twice with lysis buffer, and twice with kinase reaction buffer (25 mM Tris, pH 7.5, 5 mM beta -glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2). Each pellet was resuspended and incubated (30 min, 30 °C) in kinase buffer containing 200 µM ATP (New England Biolabs) and 2 µg of ATF-2 fusion protein (New England Biolabs). Reactions were stopped by addition of 6 × Laemmli buffer and boiling for 5 min. Cell lysates (50 µg of protein/lane) were resolved on 12.75% SDS-PAGE gels. Proteins were then transferred to nitrocellulose membranes (0.45 µm pore size: Micron Separations Inc., Westboro, MA) using a semidry transfer unit (Fisher Scientific). Each membrane was stained with Ponceau S (Sigma) to verify efficient and equal transfer of protein samples. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST: 1 h, 23 °C) followed by overnight incubation at 4 °C with rabbit anti-human phospho-ATF2 (Thr71) peptide (New England Biolabs) in TBST with 5% bovine serum albumin (1:1000). The secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG diluted 1:2000. To detect the phosphorylated ATF2-antibody-peroxidase complex, enhanced chemiluminescence was used (NEN Life Science Products, Boston, MA). Band intensity was quantitated by densitometry (Molecular Dynamics, Sunnyvale, CA). Results were expressed as the ratio of ATF-2 intensity units to IgG intensity units (treatment/load control) or as the percent of the positive control (activity of treatment group divided by activity of fMLP group × 100).

p38 MAP Kinase-induced MAPKAP Kinase 2 Phosphorylation of Hsp27-- Neutrophils were treated with CRP or HBSA buffer for 10 min at 23 °C followed by exposure to fMLP (1 × 10-7 M) or HBSA buffer for 1 min at 37 °C. Cells were centrifuged (200 × g, 4 °C, 10 min) and pellets suspended vigorously in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Igepal CA-630, 20 mM sodium fluoride, 2 mM Na3VO4, 5 mM phenylmethylsulfonyl fluoride, and 100 µl of antiprotease mixture (Sigma)). Reaction mixtures were centrifuged (3000 × g, 4 °C, 10 min) and equal volumes of supernatant and kinase buffer (30 mM HEPES, pH 7.3, 20 mM MgCl2, 2 mM EGTA, 5 µM okadaic acid, 30 µM H-7 protein kinase C inhibitor, 10 µM Na3VO4, 4 mM dithiothreitol, 30 µg/ml hsp27, and 60 µCi/ml [gamma -32P]ATP (NEN Life Science Products Inc.)) were mixed. Reactions were incubated (30 °C, 10 min) and stopped by the addition of 6 × Laemmli buffer and boiling (100 °C, 5 min). Reactants were separated by SDS-PAGE and protein bands stained with Coomassie Blue to ensure equal protein loading in each lane. Gels were dried and band intensity quantitated using a PhosphorImager (Molecular Dynamics). Results were expressed as percent of unstimulated control.

Statistical Analysis-- Data are reported as the mean ± S.E. Comparisons of sample means were analyzed using a repeated measures ANOVA followed by a Tukey multiple range test. To determine if a difference existed between treatments (CRP or SB203580 ± PD98059), a two-way ANOVA was performed. A correlation coefficient was obtained by linear regression of percent inhibition of neutrophil chemotaxis on p38 MAP kinase activity displayed as percent of positive control. Differences with p < 0.05 were considered significant.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CRP Inhibits Neutrophil Movement-- Since chemotaxis of neutrophils is an indicator of the cellular inflammatory response, the effect of purified human CRP on neutrophil chemotaxis to the bacterial chemotactic peptide, fMLP, was examined. To gain insight into mechanistic aspects of CRP on neutrophil motility, random movement was also assessed. CRP inhibited fMLP-induced chemotaxis and random movement of neutrophils in a concentration-dependent manner (Fig. 1) similar to what has been reported (8, 18). CRP significantly inhibited neutrophil chemotaxis from 100 to 500 µg/ml and random movement at 100 and 200 µg/ml. The 50% inhibitory concentration (IC50) for neutrophil chemotaxis to fMLP was 90 µg/ml and for random movement was 80 µg/ml.


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Fig. 1.   Effect of C-reactive protein on random and fMLP-induced neutrophil movement. Human neutrophils were pretreated with CRP or buffer for 10 min at 23 °C followed by evaluation of movement toward fMLP (chemotaxis, ) or buffer (random movement, black-square). Buffer or chemoattractant (fMLP, 1 × 10-7 M) was added in duplicate to the lower chambers and neutrophils (200,000/well) were added to the upper chambers. Movement was reported as net neutrophil migration (distance moved in response to treatment minus distance moved in response to buffer (µm/35 min)). n = 3. *, p < 0.05 versus no CRP control.

Blockade of p38 MAP Kinase Inhibits Neutrophil Movement-- Since it has been reported that activation of p38 MAP kinase is necessary for fMLP-induced chemotaxis of human neutrophils (22), we wanted to determine whether random movement also required p38 MAP kinase and to verify that the p38 MAP kinase specific inhibitor, SB203580, blocked fMLP-induced chemotactic movement. As shown in Fig. 2, blockade of p38 MAP kinase inhibited fMLP-induced chemotaxis and random movement of neutrophils in a concentration-dependent manner. SB203580 effectively inhibited fMLP-induced chemotaxis from 39.5 ± 5.3 µm/35 min (fMLP) to 31.3 ± 7.2, 15.3 ± 6.7, and 6.0 ± 6.6 µm/35 min (30, 50, and 100 µM SB203580, respectively) with the maximal degree of inhibition being 85%. Additionally, SB203580 blocked random movement concentration dependently from 0 to -22.3 ± 7.0 µm/35 min (100 µM SB203580). These results indicate p38 MAP kinase is involved in both random and fMLP-induced neutrophil movement. The IC50 for chemotaxis to fMLP was 45 µM.


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Fig. 2.   Effect of p38 MAP kinase on random and fMLP-induced neutrophil movement. Human neutrophils were isolated and pretreated with the p38 MAP kinase specific inhibitor, SB203580, or buffer for 10 min at 23 °C followed by evaluation of movement toward fMLP (1 × 10-7 M) (chemotaxis, ) or buffer (random movement, black-square) as described in the legend of Fig. 1. n = 4-6. *, p < 0.05 versus the buffer control.

Co-Addition of CRP and SB203580 Does Not Augment the Inhibitory Effect-- To aid in understanding whether CRP and SB203580 inhibit neutrophil chemotaxis through similar signal transduction pathways, the effect of co-addition of CRP and SB203580 on fMLP-induced movement was assessed. When CRP and SB203580 were both added at their IC50 concentrations, no additive nor synergistic augmentation of inhibition of fMLP-induced neutrophil chemotaxis was observed over that seen with CRP alone (Fig. 3). These results suggest that both CRP and SB203580 inhibit neutrophil movement via the p38 MAP kinase pathway.


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Fig. 3.   Effect of co-addition of CRP and SB203580 on neutrophil chemotaxis. Human neutrophils were isolated and pretreated with IC50 amounts of CRP (90 µg/ml), SB203580 (45 µM), or CRP (90 µg/ml) plus SB203580 (45 µM) for 10 min at 23 °C followed by evaluation of movement toward fMLP (1 × 10-7 M). n = 4. *, p < 0.001 versus fMLP control.

ERK Is Not Necessary for fMLP-induced Neutrophil Movement-- It has been reported that p38 MAP kinase, but not ERK is required for chemotaxis of neutrophils (22). We sought to determine whether blockade of ERK1 and ERK2 affected the ability of CRP and SB203580 to inhibit chemotaxis. PD98059 specifically inhibits ERK1 and ERK2 by blocking their upstream activators, MEK1 and MEK2 (34). We confirmed the findings of Zu et al. (22) that PD98059 did not block fMLP-induced chemotaxis at any concentration tested (Fig. 4A). Additionally, inhibition of ERK with PD98059 at a concentration (100 µM) that completely blocks MEK-induced activation of ERK in neutrophils (33) did not affect the ability of CRP (Fig. 4B) or SB203580 (Fig. 4C) to inhibit fMLP-induced neutrophil chemotaxis. These results suggest that CRP and blockade of p38 MAP kinase inhibit chemotaxis through a pathway separate from ERK.


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Fig. 4.   Effect of PD98059 on fMLP-induced neutrophil chemotaxis. Human neutrophils were isolated and pretreated with PD98059 alone (A), with PD98059 and CRP (B) or with PD98059 and SB203580 (C) for 10 min at 23 °C followed by evaluation of movement toward fMLP (1 × 10-7 M). The concentration of PD98059 used in the combination experiments was 100 µM. Filled squares, PD98059 added; Open squares, no PD98059. n = 4.

Inhibition of fMLP-induced p38 MAP Kinase Activity by CRP-- In view of the above results, a likely mechanism that explains the inhibitory action of CRP on neutrophil chemotaxis is that CRP inhibits p38 MAP kinase. For this reason, the effect of CRP on p38 MAP kinase activity was examined. When p38 MAP kinase was immunoprecipitated and an in vitro kinase assay performed to detect p38 MAP kinase phosphorylation of its substrate, activating transcription factor-2 (ATF2), CRP was found to inhibit fMLP-induced p38 MAP kinase activity of neutrophils, especially at concentrations >= 100 µg/ml (Fig. 5A). The presence of two predominant bands was noted, one of the phosphorylated ATF2 substrate and the other of the immunoprecipitating antibody (IgG) reacting with the anti-IgG secondary antibody. Stimulation of neutrophils with fMLP significantly activated p38 MAP kinase over unstimulated (vehicle) cells as previously reported (23, 35) (Fig. 5B). Analysis of CRP inhibition of p38 MAP kinase activity revealed that CRP inhibited substrate phosphorylation by p38 MAP kinase in a concentration-dependent fashion with significance observed at 100 and 200 µg/ml CRP.


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Fig. 5.   Effect of CRP on fMLP-induced p38 MAP kinase activity (phosphorylation of activating transcription factor-2). Purified human neutrophils were pretreated with CRP or vehicle (Veh) for 10 min at 23 °C. Cells were then activated by incubation with fMLP (1 × 10-7 M, 37 °C, 1 min), centrifuged, and cell pellets lysed. Neutrophil lysates (200 µg) were incubated with anti-p38 MAP kinase antibody followed by adsorption with protein A-Sepharose. An in vitro kinase activity assay was performed on immunoprecipitated p38 MAP kinase using ATF2 as substrate. Reactants were separated by SDS-PAGE, immunoblotted using anti-phosphorylated-ATF-2 antibody, and visualized by enhanced chemiluminescence. The presence of two bands was noted: phosphorylated ATF2 substrate and immunoprecipitating antibody (IgG) reacting with the anti-IgG secondary antibody. A representative immunoblot is shown in A. Band intensity was quantitated by densitometry and results were expressed in B as percent of the positive control (activity of treatment group divided by activity of fMLP group × 100) after normalization of the data as the ratio of ATF-2 intensity units to IgG intensity units (treatment/load control). n = 7. *, p < 0.05 versus the fMLP control. **, p < 0.01 versus the fMLP control.

CRP also inhibited p38 MAP kinase activity in an in vitro kinase activity assay assessing MAPKAP kinase-2 phosphorylation of hsp27. MAPKAP kinase-2 is a physiologic target of p38 MAP kinase (26) and phosphorylates hsp27 (28). In neutrophils, fMLP activates MAPKAP kinase-2 which phosphorylates and activates hsp27 (36). CRP inhibited phosphorylation of hsp27 by MAPKAP kinase-2 in a concentration-dependent manner with significant inhibition evident at 500 µg/ml (Fig. 6). This result confirms that CRP inhibits p38 MAP kinase activity.


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Fig. 6.   Effect of CRP on fMLP-induced p38 MAP kinase activity (phosphorylation of hsp27 by MAPKAP kinase-2 after substrate phosphorylation by p38 MAP kinase). Purified human neutrophils were pretreated with CRP or vehicle for 10 min at 23 °C. Cells were activated with fMLP (1 × 10-7 M, 37 °C, 1 min), centrifuged, and cell pellets lysed. Cell supernatants were reacted with hsp27 and [gamma -32P]ATP. Reactions were stopped by boiling and reactants separated by SDS-PAGE. Gels were dried and band intensity quantitated using a PhosphorImager (Molecular Dynamics). Results were expressed as percent of unstimulated control. n = 5. *, p < 0.01 versus fMLP control.

Correlation of Inhibition of Neutrophil Chemotaxis with Inhibition of p38 MAP Kinase Activity-- Interestingly, CRP concentrations inhibitory for fMLP-induced p38 MAP kinase activity were also inhibitory for fMLP-induced chemotaxis (compare Figs. 1 and 5B). As shown in Fig. 7, CRP-mediated inhibition of fMLP-induced p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis with a correlation coefficient of -0.7144.


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Fig. 7.   Comparison of the effect of CRP on fMLP-induced p38 MAP kinase activity with the percent inhibition of fMLP-induced chemotaxis by CRP. Percent inhibition of fMLP-induced chemotaxis was regressed onto p38 MAP kinase activity (percent positive control). Concentrations of human CRP (µg/ml) are shown next to each point on the regression line. The correlation coefficient was obtained by linear regression of percent inhibition of neutrophil chemotaxis on p38 MAP kinase activity. n = 7 and r = -0.7144.

Inhibition of fMLP-induced Neutrophil Chemotaxis and p38 MAP Kinase Activity in Normal Plasma Spiked with CRP-- To examine the physiological relevance of CRP inhibition of p38 MAP kinase activity and correlative inhibition of neutrophil movement, the effects of normal human plasma spiked with purified human CRP on neutrophil chemotaxis and p38 MAP kinase activity were assessed. CRP addition to normal plasma resulted in a concentration-dependent inhibition of fMLP-directed movement of neutrophils with significant inhibition seen at 200 µg/ml CRP (Fig. 8A). Similarly, CRP addition to normal plasma resulted in inhibition of fMLP-induced p38 MAP kinase activity with significance observed at 200 µg/ml CRP (Fig. 8B). These results indicate that in the presence of normal plasma, CRP inhibits both fMLP-directed neutrophil movement and p38 MAP kinase activity.


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Fig. 8.   Effect of purified CRP addition to normal plasma on fMLP-induced neutrophil movement and p38 MAP kinase activity. Human neutrophils were pretreated with different concentrations of buffer or CRP in 50% normal plasma for 10 min at 23 °C (chemotaxis) or 1 min at 37 °C (p38 activity assay) followed by evaluation of fMLP (1 × 10-7 M)-induced movement (A) or p38 MAP kinase activity (B). Assays were performed as described in the legend to Figs. 1 and 5. Control, normal human plasma diluted 1:2 with chemotaxis buffer; CRP (50, 100, or 200), normal human plasma diluted 1:2 with chemotaxis buffer containing purified human CRP (50, 100, or 200 µg/ml). n = 3. *, p < 0.05 versus normal plasma control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stimulation of neutrophils by chemotactic peptides leads to the phosphorylation of several intracellular proteins believed to participate in activation of the microbicidal function of these cells. In the present study we described the inhibition of p38 MAP kinase by CRP and correlated inhibition of kinase with arrest of movement. The specific p38 MAP kinase inhibitor, SB203580, inhibited both fMLP-directed and random movement of neutrophils similar to that seen with CRP. Additionally, inhibition of ERK1 and ERK2 with the specific inhibitor, PD98059, revealed no significant difference from control in chemotaxis thereby supporting the hypothesis that ERK1 and ERK2 are not involved in neutrophil movement. The role of ERK in neutrophil migration remains unclear, however, since it has been reported that inhibition of ERK with PD98059 does not inhibit fMLP-induced chemotaxis (22, 37, 38) and that PD98059 potentiates the chemotactic response of neutrophils (39). Finally, addition of purified CRP to normal plasma resulted in inhibition of neutrophil chemotaxis and p38 MAP kinase activity indicating that these results have physiological significance.

To our knowledge, this is the first report of an endogenous serum protein which inhibits cell movement by blockade of an intracellular signal transduction pathway. The precise kinase inhibited by CRP in this cascade remains to be elucidated. Since CRP inhibits phosphorylation of p38 MAP kinase substrates, the expectation is that the site of CRP action is upstream of or at the point of p38 MAP kinase activation. Additionally, the precise mechanism by which CRP inhibits p38 MAP kinase (or an upstream activator) remains undetermined. It is known that CRP binds a specific receptor on neutrophils (19, 20) which to date has not been cloned. After binding, CRP may activate its own signal transduction pathway which intercepts and alters the fMLP-induced signaling cascade. Alternatively, CRP may directly intercept the signaling pathway of fMLP. Conceivably, CRP in its native or one of its altered forms could enter the cell and act directly on a member of the p38 MAP kinase cascade. Finally, CRP may inhibit signal transduction through receptors with similar structural motifs, for example, the seven transmembrane-spanning receptors of chemoattractants such as fMLP, complement fragment (C5a), and interleukin-8 (reviewed in Ref. 40). It has been reported that fMLP and C5a receptors are highly conserved in the transmembrane and cytoplasmic domains while being maximally divergent in the extracellular loops where ligand binds (41). It is interesting to speculate that the ability of CRP to inhibit neutrophil movement to several different chemoattractants (16-18) may be by alteration of signaling through the conserved cytoplasmic domains.

It has been proposed that a specific signaling pathway for chemoattractant-induced neutrophil movement exists (42). This hypothesis has been suggested based upon the observation that both respiratory burst and migration of neutrophils require tyrosine phosphorylation in the signaling pathway; however, the former needs phospholipase D activation and the latter does not (42). Signal transduction therefore appears to be similar for respiratory burst and migration until the pathway reaches phospholipase D, at which point there is a split in the signaling paths. CRP-induced signal transduction may intercept fMLP-induced signaling at a point after phospholipase D activation and before p38 MAP kinase substrate phosphorylation. In addition, SB203580 inhibits neutrophil chemotaxis but not superoxide production whereas PD98059 potentiates superoxide and has no effect on chemotaxis (22) indicating that p38 MAP kinase is necessary for movement and ERK may be an important pathway for the respiratory burst response.

Cell movement is of the utmost importance in phagocytic cells such as neutrophils since migration to the site of infection is an important early step in host defense against microbial invasion. The recruitment of neutrophils and their accumulation at extravascular sites of inflammation is stimulated by soluble chemotactic factors and is accompanied by several biochemical events related to activation of the cells. As is seen in many cell types, phosphorylation of p38 MAP kinase initiates a cascade of activation events associated with the cytoskeleton. Phosphorylation of p38 MAP kinase in neutrophils results in the activation of MAPKAP kinase-2 which phosphorylates hsp27 and lymphocyte-specific protein 1 (LSP1), both of which are F-actin-binding proteins (31, 43). Additionally, LSP1 is reported to be the major substrate for MAPKAP kinase-2 in human neutrophils (36, 43). Neutrophils isolated from patients with neutrophil actin dysfunction are defective in motility and fMLP-induced actin polymerization and overexpress LSP1 (44). It is conceivable that inhibition of p38 MAP kinase by CRP leads to decreased phosphorylation of LSP1 with resultant loss of actin polymerization and cell motility.

It has also been shown that transient increases in intracellular calcium are required for cellular release from previous sites of attachment but does not prevent cell polarization, ruffling, or other processes associated with motility (45). Interestingly, CRP has no effect on transient calcium increases in stimulated neutrophils2 thereby suggesting that CRP is not acting at the point of cellular release from attachment sites but rather at the step of ruffling or polarization. Phosphorylation of hsp27 by MAPKAP kinase-2 may play a role in membrane ruffling since a nonphosphorylatable hsp27 mutant inhibits membrane ruffles (reviewed in Ref. 46). CRP inhibition of p38 MAP kinase may be eliciting its inhibitory effect on neutrophil movement by inhibiting the p38 MAP kinase substrate, MAPKAP kinase-2, and its substrate, hsp27. By inhibiting phosphorylation of hsp27, CRP may be preventing the formation of membrane ruffles necessary for movement. Finally, microtubules are necessary for neutrophil polarization and microtubule inhibitors decrease neutrophil motility (47), revealing the importance of polarization to the movement process. Activation of p38 MAP kinase may play an important role in proper arrangement of microtubules and CRP may be inhibiting movement at this step.

The basis for the current study was the observation that CRP pretreatment of fMLP-stimulated human neutrophils resulted in a significant reduction in the degree of phosphorylation of several neutrophil proteins (21). These included proteins that were not identified but had molecular weights similar to p38 MAP kinase and other members of the MAP kinase family. Since ERK1 and ERK2 are not involved in F-actin polymerization of neutrophils (38) nor are they required for the activation of MAPKAP kinase-2 or hsp27 (27) or other actin-binding proteins, a likely candidate was p38 MAP kinase.

Of importance to this discussion is the report that inhibition of p38 MAP kinase prevents the progression of inflammation-mediated arthritis in an animal model (48). Arthritis patients have increased serum CRP levels (49) and interestingly, transgenic mice expressing a rabbit CRP transgene had mild or no inflammation compared with animals not expressing the transgene in an antigen-induced arthritis model (50). Also, overexpression of rabbit CRP by these transgenic mice prevented neutrophil movement into chemoattractant-instilled lungs in an acute lung injury animal model (16). Therefore, elucidation of the mechanism of inhibition of neutrophil movement is of great importance in models of inflammation. The current findings support inhibition of p38 MAP kinase as a mechanism by which CRP inhibits neutrophil movement. Additionally, CRP may play important roles in regulation of other neutrophil functions, thereby halting the acute inflammation process. Future work will focus on determining the precise mechanism by which CRP inhibits p38 MAP kinase.

    ACKNOWLEDGEMENTS

We thank Tradina R. Pichon and Deborah A. Williams for technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL51199 (to R. O. W.) and the American Heart Association, Missouri Affiliate (to R. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Pulmonary Medicine, Saint Louis University School of Medicine, 3635 Vista Ave. at Grand Blvd., St. Louis, MO 63110. Tel.: 314-577-8732; Fax: 314-577-8859; E-mail: heuertzr{at}wpogate.slu.edu.

2 R. M. Heuertz, unpublished data.

    ABBREVIATIONS

The abbreviations used are: CRP, C-reactive protein; fMLP, f-Met-Leu-Phe; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MAPKAP, mitogen-activated protein kinase-activated protein; PAGE, polyacrylamide gel electrophoresis; ATF, activating transcription factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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