©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mitogen-activated Protein Kinase in Neutrophils and Enucleate Neutrophil Cytoplasts
EVIDENCE FOR REGULATION OF CELL-CELL ADHESION (*)

(Received for publication, June 5, 1995; and in revised form, January 10, 1996)

Michael H. Pillinger (§) Aleksander S. Feoktistov Constance Capodici Bruce Solitar Judy Levy Tommy T. Oei Mark R. Philips

From the Department of Medicine, New York University School of Medicine, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We employed neutrophils and enucleate neutrophil cytoplasts to study the activation of the mitogen-activated protein kinases (MAPKs) p44 and p42 in neutrophils by inflammatory agonists that engage G protein-linked receptors. Formyl-methionyl-leucylphenylalanine (FMLP) rapidly and transiently activated MAPK in neutrophils and cytoplasts, consistent with a role in signaling for neutrophil functions. FMLP stimulated p21activation in neutrophils and Raf-1 translocation from cytosol to plasma membrane in cytoplasts, with kinetics consistent with events upstream of MAPK activation. Insulin, a protein tyrosine kinase receptor (PTKR) agonist, stimulated neutrophil MAPK activation, demonstrating an intact system of PTKR signaling in these post-mitotic cells. FMLP- and insulin-stimulated MAPK activation in cytoplasts were inhibited by Bt(2)cAMP, consistent with signaling through Raf-1 and suggesting a mechanism for cAMP inhibition of neutrophil function. However, Bt(2)cAMP had no effect on FMLP-stimulated MAPK activation in neutrophils. The extent of MAPK activation by various chemoattractants correlated with their capacity to stimulate neutrophil and cytoplast homotypic aggregation. Consistent with its effects on MAPK, Bt(2)cAMP inhibited FMLP-stimulated aggregation in cytoplasts but not neutrophils. Insulin had no independent effect but primed neutrophils for aggregation in response to FMLP. Our studies support a p21-, Raf-1-dependent pathway for MAPK activation in neutrophils and suggest that neutrophil adhesion may be regulated, in part, by MAPK.


INTRODUCTION

The mitogen-activated protein kinases (MAPKs) (^1)p44 and p42 are serine/threonine kinases that participate in cell signaling for growth and differentiation(1) . The most completely elucidated pathway for p44 and p42 activation utilizes protein tyrosine kinase receptors (PTKR) to activate MAPK through p21 and Raf-1. In this pathway, ligation of the PTKR results in interaction of the receptor with a complex of the adaptor protein Grb2 (2) and SOS, a guanine nucleotide exchange factor(3) . These interactions bring SOS into proximity of p21(4, 5) , stimulating GTP/GDP exchange(6) . Activated p21 recruits the serine/threonine kinase Raf-1 to the plasma membrane (PM) where it is activated(7, 8, 9) . Activated Raf-1 phosphorylates a dual threonine/tyrosine kinase, MAPK or Erk kinase(10) , which in turn phosphorylates and activates p44 and p42(11) . Raf-1 appears to play a pivotal role in this pathway because its activation can be negatively regulated by cAMP-dependent protein kinase A (PKA)(12, 13) . Among other signaling pathways leading to MAPK activation are those activated by G protein-linked receptors, including receptors for lysophosphatidic acid (LPA)(14) , acetylcholine (15) , and thrombin(16) . Whereas LPA and acetylcholine activate MAPK via p21(14, 16) , the thrombin receptor can activate MAPK through a p21/Raf-1-independent pathway(17, 18) .

Circulating neutrophils are terminally differentiated, post-mitotic phagocytes that constitute the first line of host defense against microorganisms. In contrast to dividing cells that respond slowly to mitogens, neutrophils respond rapidly to inflammatory stimuli. One class of neutrophil agonists, the chemoattractants, engage seven transmembrane-spanning domain receptors that activate G(i) proteins. The only well-documented effector downstream of neutrophil G(i) is phospholipase C, a regulatory enzyme not directly linked to the MAPK pathway. Nevertheless, the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) has been shown to activate MAPK (19) and to stimulate MAPK autophosphorylation in neutrophils in a pertussis toxin-sensitive fashion(20) . MAPKs thus represent candidate effectors for the signaling pathway(s) leading from G protein activation to rapid neutrophil responses. Because currently understood mechanisms of neutrophil activation fail to explain the observation that agents that elevate intracellular cAMP inhibit some chemoattractant-stimulated neutrophil responses, the possibility that chemoattractants activate MAPKs in a p21/Raf-1-dependent fashion is an attractive hypothesis.

In the present study we utilize intact neutrophils and neutrophil cytoplasts (enucleate, granule-poor, metabolically active cell fragments) to demonstrate that MAPK activation by FMLP is associated with activation of p21 and translocation of Raf-1 to the PM and that cAMP acts via PKA to inhibit FMLP-stimulated MAPK activation in cytoplasts but not neutrophils. We also show that insulin, known to activate MAPK via p21 and Raf-1 in mitotic cells, activates MAPK in neutrophils and cytoplasts. Finally, we observed a strong correlation between MAPK activation and cell-cell adhesion in neutrophils and cytoplasts suggesting a new regulatory role for MAPK in a process critical for inflammation.


EXPERIMENTAL PROCEDURES

Materials

Except where otherwise noted, reagents were purchased from Sigma. Accuprep was from Accurate Scientific, Inc. Dextran T500, acrylamide/bis solution, TEMED, and ammonium persulfate were from Pharmacia Biotech Inc. Human recombinant interleukin-8 (Il-8) and myelin basic protein (MBP) substrate peptide (MBPp) were from Upstate Biotechnology Inc. MBPp control peptide (valine substituted for threonine) was synthesized by Chiron Mimotopes. Anti-p21 monoclonal antibody Y13-259 was from Oncogene Science. Anti-Raf-1 monoclonal antibody was from Transduction Laboratories. Antisera specific for SOS1/2, Raf-1 (catalog sc-133), p44 (sc-93), p42 (sc-154), and p44/p42 (sc-94) were from Santa Cruz Biotechnology. Rabbit anti-mouse and rabbit anti-rat antisera were from Organon Teknika Corporation (Cappel). ATP, GTPS, and human recombinant insulin were from Boehringer Mannheim. [-P]ATP was from Amersham Corp. [alpha-P]GTP was from DuPont NEN. I-protein A was from ICN. KT5720 was from Kamiya Biomedical Company. 5`N-Ethylcarboxamidoadenosine (NECA) was from Research Biochemicals Int. Anti-phosphotyrosine antibody was the kind gift of Dr. Benjamin Margolis.

Neutrophil Isolation, Cytoplast Preparation, and Subcellular Fractionation

Neutrophils were prepared by the method of Boyum (21) . Cytoplasts were prepared by the method of Roos et al.(22) .

Gel Renaturation MAPK Activity Assay

Neutrophils (5 times 10^7/ml) or cytoplasts (5 times 10^8/ml) were suspended in cell buffer and incubated in the absence or presence of agonists and/or inhibitors. Neutrophil lysates were prepared by the method of Torres et al.(19) . Cytoplast incubations were stopped by addition of SDS sample buffer. Neutrophil lysate (10^6 cell eq) or cytoplast lysate (10^7 cell eq) was analyzed using the gel renaturation method of Kameshita and Fujisawa (23) except that prepared gels contained 0.25 mg/ml myelin basic protein (MBP) or bovine serum albumin (BSA), and phosphorylation buffer contained 2.5 µCi/ml [P]ATP. MAPK activity was analyzed by phosphorimaging and quantitated as MBP phosphorylation of the p44/p42 region of each gel.

MBP Peptide Substrate (MBPp) Kinase Activity Assay

Neutrophils (2 times 10^8/ml) or cytoplasts (10^9/ml) were incubated in the absence or presence of Bt(2)cAMP for 10 min at 37 °C, followed by incubation in the absence or presence of 100 nM FMLP for 1 min. Reactions were stopped by addition of lysis buffer (20 mM Tris, pH 7.4, 1 mM NaEGTA, 2 mM sodium vanadate, 25 mM sodium fluoride, 0.5% Triton X, 2 mM PMSF, 10 trypsin inhibitor units/ml aprotinin, and 10 µg/ml each of chymostatin, antipain, and pepstatin). Neutrophil lysates were centrifuged (14,000 times g for 10 min at 4 °C) to remove nuclei. Lysates were kept on ice for 15 min, followed by incubation for 15 min at 37 °C in a buffer (25 mM Tris, pH 7.4, 12.5 mM MgCl(2), 125 mM NaEGTA, 1.25 mM sodium fluoride, 2 mM dithiothreitol, 220 µM ATP, and 25 µCi/ml [P]ATP) containing 500 µM MBPp or a control peptide in which valine was substituted for threonine. Reactions were stopped by the addition of 15% formic acid. The lysates were spotted onto phosphocellulose papers that were washed thoroughly with distilled water and quantitated by scintillation counting. Duplicate assays in the absence of MBP peptide were performed to determine non-MAPK background kinase activities.

p44/p42 Immunoprecipitation

Cytoplasts (2 times 10^8/condition) were suspended in cell buffer and incubated for 1 min at 37 °C in the presence or absence of 100 nM FMLP. Reactions were stopped by addition of SDS lysis buffer (0.5% SDS, 1 mM dithiothreitol, 2 mM sodium vanadate, 25 mM sodium fluoride, 2 mM PMSF, 10 trypsin inhibitor units/ml aprotinin, and 10 µg/ml each chymostatin, antipain, and pepstatin). Lysates were heated for 5 min at 100 °C and then diluted 1:5 in ice-cold Nonidet P-40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2 mM sodium vanadate, 25 mM sodium fluoride, 2 mM PMSF, 10 trypsin inhibitor units/ml aprotinin, and 10 µg/ml each chymostatin, antipain, and pepstatin) and kept on ice for 30 min, followed by immunoprecipitation with antiserum specific for p44, p42, both, or isotype control, and capture on protein A-Sepharose beads. The beads were washed 3 times in Nonidet P-40 lysis buffer, resuspended in SDS sample buffer, heated (100 °C for 3 min), and the supernatants analyzed for MAP kinase activity using the gel renaturation MAPK kinase activity assay. Alternatively, samples were analyzed for p44/p42 by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with an antiserum recognizing both p44 and p42.

p21 GTP/GDP Exchange

-Neutrophils (5 times 10^7/ml) suspended in buffer (10 mM HEPES, 1 mM EGTA, 0.193 mM CaCl(2), 140 mM KCl, 2 mM MgCl(2), 1 mM ATP, and 20-50 µCi/ml [alpha-P]GTP (3000 Ci/mmol)) were electroporated (3 times at 380 mV administered at 2-min intervals, Cell-Porator, Life Technologies, Inc.). Electroporated neutrophils were incubated for 5 min at 4 °C and 5 min at 37 °C, followed by incubation at 37 °C in the absence or presence of 100 nM FMLP. Reactions were stopped by addition of ice-cold cell buffer. Neutrophils were washed twice in cell buffer and lysed in 50 mM Tris, pH 7.5, 20 mM MgCl(2), 150 mM NaCl, and 1% Nonidet P-40 for 20 min at 4 °C. Lysate supernatants (16,000 rpm for 15 min) were cleared of unbound nucleotide in a slurry of BSA-coated activated charcoal for 1 h at 4 °C, and the supernatants were recovered (16,000 rpm for 15 min). p21 was immunoprecipitated overnight from lysate supernatants with the anti-p21 antibody Y13-259 or an isotype control monoclonal antibody and captured on protein A-Sepharose beads pre-exposed to rabbit anti-rat antiserum. Beads were washed three times in lysis buffer and twice in wash buffer containing 50 mM Tris, pH 7.5, 20 mM MgCl(2), and 150 mM NaCl. Nucleotides were eluted from immunoprecipitated p21 in 20 mM Tris, pH 7.5, 20 mM EDTA, 2% SDS, 0.5 mM GDP, and 0.5 mM GTP for 5 min at 68 °C. Supernatants were spotted onto polyethyleneimine-cellulose plates and analyzed by thin layer chromatography using 0.75 mM KH(2)PO(4) as a solvent. GTP and GDP migration was monitored by ultraviolet fluorescence. [alpha-P]GTP and [alpha-P]GDP so analyzed were visualized and quantitated by phosphorimaging.

Membrane Translocation of Raf-1, p44/p42, and SOS

Assays for FMLP-stimulated translocation of proteins from cytosol (CS) to PM in cytoplasts were performed as described previously(24) . Localization of proteins was determined by SDS-polyacrylamide gel electrophoresis and immunoblotting of CS and PM fractions with appropriate antisera.

Neutrophil Aggregation

Neutrophil (1.25 times 10^7/ml) and cytoplast (4 times 10^8/ml) aggregation were monitored as described previously(25) . Aggregation curves were quantitated as the area under the curve in the first 2 min following stimulation.


RESULTS

FMLP Stimulates MAPK Activation in Neutrophils and Enucleate Neutrophil Cytoplasts

MAPK activity was detected in lysates of intact neutrophils as discrete MBP kinase activities using a gel renaturation assay (Fig. 1A). FMLP-stimulated MBP kinase activity was detected only in the 42-44-kDa region, consistent with the molecular weight of the MAPKs, p44 and p42. Because the presence of nucleic acids and granular proteases in SDS lysates of intact neutrophils impaired the resolving capacity of MBP-impregnated gels, we also analyzed MAPK activation by this method in enucleate, granule-depleted cytoplasts. In cytoplasts as in neutrophils, FMLP stimulated MBP kinase activity in the 42-44-kDa region of the gel (Fig. 1B). In some but not all gels resolution of cytoplast proteins was sufficient to allow identification of two discrete FMLP-sensitive MBP kinase activities, consistent with the molecular weight of p44 and p42. Duplicate gels in which BSA was substituted for MBP revealed no phosphoproteins, excluding autophosphorylation and confirming MBP kinase activity for each band. Thus, the only MBP kinases stimulated by FMLP had molecular weights consistent with the MAPKs p44 and p42.


Figure 1: FMLP stimulates MAPK activation in neutrophils and enucleate neutrophil cytoplasts. Neutrophils (A) and cytoplasts (B) were incubated for 1 min at 37 °C in the absence or presence of 100 nM FMLP and analyzed for MAP kinase activity by gel renaturation MAP kinase activity assay as described under ``Experimental Procedures.'' Neutrophils (C) and cytoplasts (D) were incubated for 1 min at 37 °C in the absence or presence of 100 nM FMLP, lysed with 1% Nonidet P-40, and analyzed for kinase activity toward an MBP-derived peptide substrate specific for MAPK (MBPp). Results shown are representative of eight (A and B), or are the mean ± S.E. (C and D) of three, experiments for each condition.



To establish that the FMLP-sensitive MBP kinase activity observed in the gel renaturation assay corresponded to MAPK-type phosphorylation of the MBP molecule, which has numerous non-MAPK phosphorylation sites, we tested the ability of lysates of FMLP-stimulated cells or cytoplasts to phosphorylate MBPp, a synthetic peptide containing only the MBP amino acid sequence specifically phosphorylated on threonine by Erk (PRTP)(26) . Neutrophil and cytoplast lysates both contained MBPp kinase activity that was markedly stimulated by FMLP (Fig. 1, C and D). A peptide in which valine was substituted for threonine gave only background counts. The fold-increase stimulated by FMLP was greater in neutrophils than cytoplasts, consistent with the results obtained in the gel renaturation assay.

We confirmed the identity of cytoplast FMLP-sensitive MBP kinases as p44 and p42 by immunoprecipitating p44 and p42 from unstimulated or FMLP-stimulated cytoplasts and analyzing the precipitates by immunoblot and gel renaturation MBP kinase assays. Coimmunoprecipitation of p44 and p42 followed by immunoblot using a third antiserum recognizing both Erks revealed two polypeptides of expected molecular weight that were unaffected by FMLP stimulation (Fig. 2A). Neither protein was precipitated by a control antiserum. In contrast, p44/p42 antisera precipitated MBP kinase activity in the 42-44-kDa region of the gel only from lysates of cytoplasts that had been stimulated with FMLP (Fig. 2B). The resolving power of the MBP-impregnated gels in these experiments was inadequate to distinguish p44 from p42 kinase activity. However, when p44 and p42 were immunoprecipitated separately from FMLP-stimulated lysates each precipitate contained 42-44-kDa MBP kinase activity, although somewhat less than the coprecipitate (Fig. 2B). FMLP-stimulated lysates immunoprecipitated with control antisera contained no 42-44-kDa MBP kinase activity, although higher molecular weight activities were pulled down nonspecifically. Thus, the measurement of radioactivity in immunoprecipitates in the absence of simultaneous assessment of the molecular weight of the kinase is inadequate for measuring MAPK activity specifically. Accordingly, these data are the clearest demonstration to date that FMLP, acting through a G protein-linked receptor, activates p44 and p42 in neutrophils. Moreover, they demonstrate that the signaling pathway for formyl peptide receptor-stimulated MAPK activation does not depend on nuclear or granular elements since it is retained in neutrophil cytoplasts.


Figure 2: Anti-Erk antisera immunoprecipitate active and inactive p44 and p42 from cytoplast lysates. A, cytoplasts (1.5 times 10^8/condition) were incubated in the absence (lane 1) or presence of 100 nM FMLP (lanes 2 and 3) for 1 min at 37 °C, lysed, and immunoprecipitated with antisera to p44 and p42 together (lanes 1 and 2) or control antiserum (lane 3) as described under ``Experimental Procedures'' and then analyzed by SDS-polyacrylamide gel electrophoresis and Western blot with a third antibody directed against both p44 and p42. B, cytoplasts (1.5 times 10^8/condition) were incubated in the absence (lane 1) or presence of 100 nM FMLP (lanes 2-5) for 1 min at 37 °C, lysed, and immunoprecipitated with antisera to p44 and p42 together (lanes 1 and 2), p44 alone (lane 3), p42 alone (lane 4), or control antiserum (lane 5) as described under ``Experimental Procedures'' and then analyzed by gel renaturation MAP kinase assay. Results shown are representative of four experiments.



Because neutrophils required post-stimulation processing that made precise kinetic measurements difficult, we studied the kinetics of FMLP-stimulated MAPK activation in cytoplasts that could be rapidly lysed in SDS sample buffer without releasing nucleic acids and granular proteases (Fig. 3A and Fig. 4). FMLP-stimulated activation of p44 and p42 was transient, peaking at 1 to 2 min and returning to base line by 10 min. Because MAPK activity is associated with tyrosine phosphorylation of p44 and p42, we compared MBP kinase activities with tyrosine phosphorylation of cytoplast proteins following stimulation with FMLP (Fig. 3B). Cytoplast lysates contained a prominent 42-kDa tyrosine phosphoprotein whose phosphorylation was stimulated by FMLP with kinetics identical to those of the p42 kinase activity. When the blot was stripped and reprobed with an antiserum directed to p44/p42 (Fig. 3C), the 42-kDa phosphoprotein aligned precisely with p42. The amount of p44 and p42 detected by immunoblot did not change with FMLP stimulation, confirming that the antiphosphotyrosine blot detected changes in phosphotyrosine content, not amount of protein. Since the anti-Erk antiserum used has greater affinity for p44 than p42, the immunoblot results revealing a darker p42 band suggest that more p42 than p44 is expressed in human neutrophils. This could explain why the anti-phosphotyrosine antibody was apparently only sensitive enough to detect phosphorylated p42, a result consistent with earlier studies(27) . These data confirm that enucleate neutrophil cytoplasts retain the signaling molecules necessary to respond to FMLP stimulation by phosphorylating and then dephosphorylating Erk on tyrosine and transiently activating MAPK activity.


Figure 3: Kinetics of MAPK activation and p42 phosphorylation/dephosphorylation in FMLP-stimulated cytoplasts. Cytoplasts stimulated with 100 nM FMLP at 37 °C for the times indicated were analyzed for MAP kinase activity by gel renaturation assay (A) and immunoblotting for phosphotyrosine-containing proteins (B) as described under ``Experimental Procedures.'' C, the nitrocellulose from B was stripped (19) and reprobed with an anti-p44/p42 antiserum. Results shown are representative of two experiments.




Figure 4: Insulin stimulates MAPK activity in cytoplasts and neutrophils. Cytoplasts or neutrophils were stimulated for the times indicated in the absence or presence of FMLP (100 nM) or insulin (200 nM) and analyzed for MAP kinase activity by gel renaturation assay quantitated by phosphorimaging. Results shown are the mean ± S.E. for 4 (FMLP, cytoplasts, insulin, and neutrophils) or 10 (insulin and cytoplasts) experiments.



Pathways of MAPK Activation in Neutrophils and Cytoplasts

G protein-stimulated MAPK activation has been shown to proceed via both p21/Raf-1-dependent and independent pathways. We therefore studied whether MAPK activation by FMLP in neutrophils and cytoplasts was preceded by p21 and Raf-1 activation. We first determined by immunoblot analysis that terminally differentiated neutrophils retain all of the elements of the classical p21/Raf-1-dependent MAPK pathway, including Grb2, SOS, Raf-1, MAPK or Erk kinase, and p44 and p42 in CS and p21 in PM (not shown). To confirm that neutrophils retain the capacity to respond to stimuli that activate p21/Raf-1-dependent MAPK pathways, we examined the effect of PTKR agonists on neutrophils. Epidermal growth factor had no effect on neutrophil or cytoplast MAPK activity, suggesting a lack of expression of EGF receptor on myeloid cells. In contrast, insulin, at concentrations (20-200 nM) required to maximally activate MAPK in mitotic cells expressing insulin receptors(28) , activated MAPK in neutrophils (312 ± 113% control) and cytoplasts (148 ± 21% control) with kinetics distinct from those of FMLP stimulation (Fig. 4). Thus at least one PTKR known to activate MAPK in a p21/Raf-1-dependent fashion is functionally expressed in human neutrophils suggesting that p21/Raf-1 signaling is intact.

To study the rapid FMLP-stimulated p21 activation predicted by the kinetics of MAPK activation, we studied FMLP-stimulated guanine nucleotide exchange on p21. Because isolated neutrophils had a bench life (<6 h by lactate dehydrogenase release assay) inadequate to ensure equilibrium labeling of nucleotide pools by metabolic labeling with [P]orthophosphate, we permeabilized neutrophils by electroporation in the presence of [alpha-P]GTP to rapidly label intracellular pools of GTP. In this system p21 activation is measured as total [alpha-P]guanine nucleotide loading (guanine nucleotide exchange with or without GTPase activation). Electroporated neutrophils were stimulated with FMLP (100 nM), p21 was immunoprecipitated from cell lysates, and the amount of [alpha-P]guanine nucleotide associated with p21 was determined by thin layer chromatography (Table 1). The amount of [alpha-P]guanine nucleotide associated with p21 following FMLP stimulation was 164 ± 20% control at 30 s and remained stable for as long as 5 min, suggesting that FMLP-stimulated guanine nucleotide exchange on p21 in neutrophils peaks no later than 30 s after stimulation. Since hydrolysis of [alpha-P]GTP on p21 results in p21bullet[alpha-P]GDP, total [alpha-P]guanine nucleotide associated with p21 cannot distinguish transient from persistent p21 activation. However, the percentage of [alpha-P]guanine nucleotide associated with p21 as [alpha-P]GTP declined after 1 min, suggesting a GTPase-activating protein activity limiting activation.



Membrane association of Raf-1 appears to be required for its activation by PTKR(7, 8, 9) . Since Raf-1 is recruited to the PM of cells transfected with oncogenic, activated p21(7, 9) , we tested whether FMLP, in addition to activating p21, can stimulate Raf-1 translocation from the CS to PM of cytoplasts. When unstimulated cytoplasts were sonicated and separated by centrifugation into soluble and insoluble fractions, 14 ± 3% of total immunodetected Raf-1 (supernatant + pellet) was associated with the pellet. This analysis is likely to overestimate the true membrane-associated pool since cytoplast disruption may not have been complete and vesiculated cytoplast membrane is likely to sequester CS. FMLP stimulated a 2.2-fold increase in the amount of Raf-1 associated with cytoplast membranes. Thus, despite the potential overestimation of basal membrane-associated Raf-1, our system was sensitive enough to detect membrane translocation of this molecule. Kinetic analysis (Fig. 5) revealed that Raf-1 translocation to the membrane peaked at 30 s to 1 min and remained stable for at least 10 min following FMLP stimulation. In contrast to Raf-1, we observed no FMLP-stimulated translocation of p44 or p42 to PM in cytoplasts. Although neutrophil CS contained an abundant supply of SOS, this molecule also did not translocate from cytoplast CS to PM in response to FMLP. Thus, both FMLP-stimulated p21 activation and Raf-1 translocation preceded MAPK activation, consistent with a role for both events upstream of Erk activation in the FMLP-stimulated pathway.


Figure 5: FMLP stimulates translocation of Raf-1 from CS to PM in cytoplasts. Cytoplasts were incubated at 37 °C in the absence or presence of 100 nM FMLP for the times indicated, sonicated, and separated into membrane and soluble fractions as described under ``Experimental Procedures.'' Fractions were assayed for Raf-1 by immunoblot quantitated by phosphorimaging. Results shown are the mean ± S.E. for four experiments.



PTKR activation of MAPK via p21 and Raf-1 may be down-regulated by cAMP-dependent, PKA-mediated phosphorylation of Raf-1, resulting in impaired interactions between p21 and Raf-1(12, 13) . We therefore tested whether FMLP-stimulated MAPK activity in neutrophil cytoplasts can be similarly inhibited by cAMP. The membrane-permeable, phosphodiesterase-resistant cAMP analog dibutyryl cAMP (Bt(2)cAMP) (1 mM) completely inhibited insulin-stimulated cytoplast MAPK activity and inhibited FMLP-stimulated cytoplast MAPK activity by 46.4 ± 11.7% (Fig. 6A). Agents that raise intracellular cAMP by indirect mechanisms, including isobutrylmethylxanthine (50 µM), forskolin (50 µM), isoproterenol (10 µM), and the adenosine A(2) receptor agonist NECA (10 µM) also inhibited FMLP-stimulated MAPK activity by approximately 50% (Fig. 6B). To confirm that cAMP inhibition of FMLP-stimulated MAPK in cytoplasts is PKA-dependent, we tested the effect of KT5720 that, at concentrations below 2 µM, is a specific inhibitor of PKA (29) . KT5720 (1 µM) reversed the inhibitory effect of Bt(2)cAMP on FMLP-stimulated MAPK activation (Fig. 6C). These data demonstrate that FMLP-stimulated MAPK activity in cytoplasts is inhibited by cAMP in a PKA-dependent manner and support a requirement for p21/Raf-1 interactions in FMLP-stimulated MAPK activation.


Figure 6: cAMP inhibits FMLP-stimulated MAPK activity in cytoplasts but not neutrophils. A, cytoplasts were incubated for 5 min in the absence or presence of 1 mM Bt(2)cAMP, stimulated (100 nM FMLP for 1 min or 200 nM insulin for 10 min), and analyzed for MAP kinase activity by gel renaturation assay quantitated by phosphorimaging. B, cytoplasts were incubated for 5 min in the presence of isobutyrylmethylxanthine (50 µM), forskolin (50 µM), isoproterenol (10 µM), or NECA (10 µM) and stimulated for 1 min with 100 nM FMLP and analyzed by gel renaturation assay and phosphorimaging. C, cytoplasts were incubated for 5 min in the absence or presence of the specific PKA inhibitor KT5720 (1 µM), followed by 5-min incubation with Bt(2)cAMP (1 mM) and 1-min stimulation with 100 nM FMLP, and analyzed by gel renaturation assay and phosphorimaging. D, neutrophils or cytoplasts were incubated for 10 min with 1 mM Bt(2)cAMP, stimulated with 100 nM FMLP for 1 min, and analyzed for MBPp kinase activity. Results are expressed as the percent of stimulated MAPK activity in the absence of drugs and are given as the mean ± S.E. of three experiments.



Yu et al.(37) have recently reported that cAMP failed to inhibit FMLP-stimulated MAPK activity in human neutrophils. To explore this discrepancy we compared the effect of Bt(2)cAMP on FMLP-stimulated MAPK activity in neutrophils and cytoplasts (Fig. 6D). As measured by the MBPp kinase activity assay, 1 mM Bt(2)cAMP inhibited FMLP-stimulated cytoplast MAPK activity by 53 ± 13% but had no effect on FMLP-stimulated MAPK activity in neutrophils. Thus, a regulatory role for cAMP in MAPK activation can be observed in vitro in lysates from cytoplasts but not from intact neutrophils suggesting that a factor(s) derived from the nucleus and/or cytoplasmic granules masks the effect.

Role of MAPK in Neutrophil Homotypic Aggregation

To determine a potential role for MAPK activation in neutrophil function, we compared MAPK activation with neutrophil adhesiveness as measured by homotypic aggregation in neutrophils and cytoplasts. Although each of the best-studied neutrophil chemoattractants (FMLP, LTB(4), C5a, and Il-8) acts through a similar seven transmembrane-spanning receptor linked to a similar or identical G protein (likely G), their efficacies at stimulating neutrophil functions vary dramatically. We therefore compared the ability of each chemoattractant to stimulate MAPK activity with its effect on aggregation. Like FMLP, saturating concentrations of LTB(4), C5a, and Il-8 stimulated MAPK activation in intact neutrophils by 1 min (Table 2); however, LTB(4), C5a, and Il-8 were significantly less efficacious than FMLP in activating MAPK. The extent of aggregation stimulated by each agonist correlated well (R^2 = 0.92) with the degree of MAPK activation (Fig. 7, A and C). LPA, which acts via a G protein-linked receptor and p21 to stimulate MAPK in cells in culture(14) , had no effect on neutrophil MAPK activity or homotypic aggregation (not shown). In cytoplasts, whereas FMLP stimulated MAPK activity (318 ± 34% control), the other chemoattractants demonstrated little or no effect on MAPK activity (Table 2). As in intact neutrophils, the ability of the chemoattractants to stimulate cytoplast aggregation correlated well (R^2 = 0.99) with their ability to stimulate cytoplast MAPK activity (Fig. 7, B and D). These data confirm divergent signaling from distinct G protein-linked receptors and argue strongly for an association between MAPK activation and adhesion. The ability of Bt(2)cAMP to inhibit FMLP-stimulated MAPK activity in cytoplasts but not neutrophils suggested that similar effects might be observed on homotypic aggregation. Indeed, Bt(2)cAMP (1 mM) had no effect on FMLP-stimulated neutrophil aggregation but inhibited FMLP-stimulated cytoplast aggregation by 33 ± 2% (Fig. 8). This observation further supports a role for MAPK activation in neutrophil adhesion.




Figure 7: Chemoattractant-induced homotypic aggregation and MAPK activation in neutrophils and cytoplasts. Neutrophils (10^6/ml) (A) and cytoplasts (5 times 10^7/ml) (B) prepared from the same donor were analyzed for homotypic aggregation in response to FMLP (100 nM), LTB(4) (300 nM), C5a (100 nM), and Il-8 (100 nM). The correlations between MAPK activation (abscissae) and aggregation (ordinate) in neutrophils (C) and cytoplasts (D) in response to various chemoattractants were plotted, and regression coefficients were calculated. Results shown are representative (A and B) or the means (C and D) of three experiments.




Figure 8: Bt(2)cAMP inhibits FMLP-stimulated homotypic aggregation in cytoplasts but not in neutrophils. Neutrophils or cytoplasts were incubated for 15 min at 37 °C in the absence or presence of 1 mM Bt(2)cAMP and then assayed for homotypic aggregation in response to 100 nM FMLP. Results shown are the means ± S.E. for three experiments.



In contrast to the chemoattractants, insulin activated neutrophil and cytoplast MAPK but failed to stimulate neutrophil aggregation, suggesting that MAPK activation may be necessary but not sufficient to support aggregation. We therefore tested whether insulin could prime neutrophils for chemoattractant-stimulated functions. Preincubation of neutrophils with insulin had little or no effect on homotypic aggregation stimulated by concentrations of chemoattractants inducing maximal aggregation responses. However, preincubation with insulin for 10 min primed neutrophils for aggregation in response to concentrations of FMLP and LTB(4) that induced submaximal aggregation responses (Fig. 9). Shorter incubations (i.e. times at which insulin failed to stimulate MAPK activity) had no effect on aggregation. The priming effect of insulin on FMLP- and LTB(4)-stimulated aggregation was dose-dependent, peaking at 200 nM. A trend toward insulin priming of neutrophils for C5a-stimulated aggregation was observed but did not achieve statistical significance. Insulin had no effect on Il-8-stimulated aggregation. Thus, the effect of 200 nM insulin on neutrophil homotypic aggregation by submaximal concentrations of chemoattractants was proportional to the ability of these chemoattractants to stimulate MAPK activity in neutrophils (FMLP>LTB(4)>C5a>Il-8). Insulin potentiation of FMLP- and LTB(4)-stimulated homotypic aggregation was glucose-independent. In contrast, insulin had no direct effect on FMLP-induced O(2) generation and beta-glucuronidase release and potentiated these responses only in the presence of glucose, presumably by increasing glucose transport and affecting metabolism.


Figure 9: Insulin primes neutrophils for chemoattractant-stimulated homotypic aggregation. Neutrophils were incubated in the presence of the indicated concentrations of insulin for 10 min at 37 °C, stimulated with FMLP (10 nM), LTB(4) (3 nM), C5a (10 nM), or IL-8 (10 nM) at concentrations determined to induce submaximal neutrophil aggregation and analyzed for homotypic aggregation. Results shown are the means ± S.E. for three or more experiments.




DISCUSSION

Although well established, the link between chemoattractant-stimulated G protein signaling pathways and the MAPK cascade is poorly elucidated. Neutrophils are a good system in which to study G protein-mediated signaling because the cellular responses are rapid and easily quantitated. Enucleate, granule-depleted neutrophil cytoplasts retain the capacity to respond to chemoattractants (30) and thus represent a simplified system useful in studying chemoattractant signaling through G proteins. We employed neutrophils and cytoplasts to study the kinetics of chemoattractant-stimulated activation of p21, Raf-1, and MAPK and observed an association between MAPK activation and cell-cell adhesion.

The analysis of MAPK activity in cytoplast lysates by an MBP kinase gel renaturation assay offered distinct advantages over similar studies of lysates of intact neutrophils, including resolution of two MAPKs in cytoplast lysates, identified by immunoprecipitation as p44 and p42. Moreover, the ability to terminate stimulation by direct addition of SDS sample buffer permitted more accurate kinetic analysis of cytoplasts than of neutrophils. FMLP-stimulated cytoplast MAPK activation was rapid and transient, consistent with a role for MAPK in signaling pathways for neutrophil functions such as O(2) generation, degranulation, and cell-cell adhesion but slower than the previously reported kinetics of neutrophil MAPK activation(19, 27, 31) . The greater precision afforded by kinetic analysis of MAPK in cytoplasts thus allowed comparison with the kinetics of activation of other putative elements in the FMLP-stimulated MAPK cascade, such as p21 and Raf-1. The use of cytoplasts also permitted observation of MAPK signaling in the absence of nuclear or granular elements. Thus, phosphorylation and dephosphorylation on tyrosine residues of p42 in cytoplasts, with kinetics paralleling those of MAPK activation, indicate that the molecular machinery required for regulating MAPK activity by Erk kinase and phosphatase activities is retained in cytoplasts and so independent of any nuclear factors that may regulate MAPK. This observation may distinguish neutrophils from proliferating cells, in which activated MAPK translocates to the nucleus where it is down-regulated by dual phosphothreonine/phosphotyrosine phosphatases such as PAC-1(32) .

Although a wide variety of G protein-linked and non-G protein-linked receptors have been demonstrated on neutrophils, none of the classical PTKRs have been reported. Insulin, however, has been shown to bind to human neutrophils(33) , stimulate chemokinesis(34) , and prime for chemotaxis to FMLP(35) , indicating that PTKRs for insulin are expressed on these cells. Our observation that insulin activated MAPK in neutrophils and cytoplasts suggests that a p21/Raf-1 pathway is functionally intact and can be engaged by at least one PTKR. Thus, the neutrophil formyl peptide receptor may also activate MAPK through the p21/Raf-1 pathway. However, the longer latency for insulin- than for FMLP-stimulated MAPK activation indicates that the pathways to p21 activation may be distinct.

Our observation that FMLP activated p21 in neutrophils supports p21/Raf-1 signaling. Although a previous study came to the same conclusion using neutrophils metabolically labeled with [P]orthophosphate(36) , we found the bench life of neutrophils insufficient to label nucleotide pools to equilibrium, an absolute requirement for interpreting GTP/GDP ratios of GTPase-bound nucleotide as an indicator of p21 activation. We therefore analyzed total labeled guanine nucleotide associated with immunoprecipitated p21 from lysates of cells electroporated in the presence of [alpha-P]GTP and found maximal increase after 30 s of exposure to FMLP (i.e. preceding peak MAPK activity). Concordant with a prior report(36) , the proportion of [alpha-P]GTP associated with p21 declined by 5 min, suggesting sequential guanine nucleotide exchange factor and GTPase-activating protein activities following FMLP stimulation.

Raf-1 has been shown to translocate from CS to PM in cultured cells exposed to serum(7) . However, Raf-1 translocation in response to ligation of neither a specific PTKR nor a G protein-linked receptor has been demonstrated in any cell type. We have shown that cytoplasts are an ideal system with which to assay PM translocation of cytosolic proteins(24) . Using this system, we now report FMLP-stimulated translocation of Raf-1 to the PM. These data complement those of Worthen et al.(36) who reported FMLP-stimulated Raf-1 kinase activity. Like p21 activation, FMLP-stimulated Raf-1 translocation preceded MAPK activation. Our failure to observe SOS translocation in response to FMLP suggests that SOS may not participate in G protein activation of p21. Alternatively, translocation of SOS may not be necessary for its activity, or the kinetics of SOS translocation may be too rapid to have been appreciated in our assay.

Our observation that MAPK activity in cytoplasts was inhibited by agents that raise intracellular cAMP and that a PKA antagonist reversed this inhibition is also consistent with p21/Raf-1-dependent signaling since cAMP has been shown to down-regulate MAPK activation by PKA-dependent phosphorylation of Raf-1, inhibiting p21/Raf-1 interactions(12, 13) . Bt(2)cAMP has been shown to inhibit neutrophil Raf-1 kinase activity(36) , supporting an effect of cAMP in neutrophils at the level of Raf-1. However, Bt(2)cAMP inhibition of FMLP-stimulated MAPK activation has not previously been demonstrated. Indeed, Yu et al.(37) reported that Bt(2)cAMP does not inhibit FMLP-stimulated MAPK in cytochalasin B-treated neutrophils. Our data confirm this observation in intact neutrophils but show that cytoplasts express a cAMP-sensitive pathway. The exposure of a cAMP-sensitive pathway in cytoplasts may be explained by increased phosphodiesterase or Raf-1 phosphatase activities in detergent lysates of granule-replete, nucleated neutrophils. Alternatively, neutrophils may possess both cAMP-sensitive and -insensitive G protein-linked pathways of MAPK activation, the latter preferentially inactivated during cytoplast preparation. Indeed, Faure and Bourne (38) have recently shown that cell lines in which stimulation of MAPK activity by LPA is cAMP-insensitive nevertheless demonstrate cAMP inhibition of Raf, suggesting a Raf-independent pathway of MAPK activation.

Several groups have proposed a role for MAPK in neutrophil O(2) generation(39) . However, Yu et al.(37) have recently demonstrated that MAPK activation and O(2) generation can be dissociated in neutrophils. In contrast, we observed a good correlation between MAPK activation and cell-cell adhesion in both neutrophils and cytoplasts. The extent of MAPK activation correlated closely with the degree of homotypic aggregation stimulated by each of four chemoattractants in both cells and cytoplasts. Interestingly, both cell-cell adhesion and MAPK responsiveness to LTB(4), C5a, and Il-8 were preferentially lost in the process of preparing cytoplasts relative to their responsiveness toward FMLP. Whereas Yu et al.(37) observed a discordance between the marked inhibition of O(2) generation by Bt(2)cAMP(37, 40) and its failure to inhibit MAPK activation in FMLP-stimulated neutrophils(37) , in our studies Bt(2)cAMP inhibited neither FMLP-stimulated MAPK activation nor FMLP-stimulated homotypic aggregation in neutrophils but significantly inhibited both of these responses in cytoplasts. Thus the effect of Bt(2)cAMP on FMLP-stimulated MAPK activity in both neutrophils and cytoplasts correlated with its effect on cell-cell adhesion. Our discovery that insulin both activated MAPK in human neutrophils and primed these cells for homotypic aggregation in response to chemoattractants demonstrates a further correlation between MAPK activation and cell-cell adhesion. The inability of insulin to directly stimulate aggregation suggests that MAPK may be necessary but not sufficient to directly or indirectly regulate adhesion molecules. The failure of insulin to stimulate or prime neutrophils for O(2) generation or degranulation in the absence of extracellular glucose supports the hypothesis that MAPK regulates some but not all neutrophil functions. Thus, although O(2) generation can be dissociated from MAPK activation, our studies of neutrophils and cytoplasts support a role for MAPK activation in cell-cell adhesion.

Neutrophil homotypic aggregation is mediated by activation of the beta(2) integrin CD11b/CD18(25) . The activation states of integrins appear to be regulated by interactions of the cytoplasmic domains of these heterodimeric transmembrane glycoproteins with the actin cytoskeleton through focal adhesion plaques(41) . Thus, MAPK might regulate cell-cell adhesion through phosphorylation of molecules regulating focal adhesion plaques. In addition to a hypothetical role in regulating the actin cytoskeleton, MAPKs have a well-established role in regulating the microtubule cytoskeleton by associating with and phosphorylating microtubule-associated proteins(42) . The relationship between MAPK activation and leukocyte adhesion suggests new targets for anti-inflammatory drugs since leukocyte adhesion to vascular endothelium is the first committed step in the inflammatory response. Furthermore, insulin stimulation of neutrophil MAPK and priming for chemoattractant-stimulated adhesion suggest a molecular mechanism for impaired neutrophil function in type I diabetes, a state of insulin deficiency associated with increased susceptibility to bacterial infection.

Since neutrophils are terminally differentiated, non-mitotic cells, the effects of MAPK on transcription factors related to growth and differentiation are unlikely to be relevant. Our studies with enucleate cytoplasts support this view. Marshall (43) has recently proposed that the outcome of MAPK signaling is dependent largely on its duration of activation. If so, rapid MAPK activation in neutrophils may represent a distinct category of signaling. Alternatively, differentiated cells might also be distinguished by their complement of MAPK substrates. The only well-defined MAPK substrate also implicated in neutrophil activation is cytoplasmic phospholipase A(2)(44) . Further studies are likely to identify other MAPK substrates involved in rapid neutrophil responses.


FOOTNOTES

*
This work was supported by grants from the National Arthritis Foundation (to M. H. P. and M. R. P.), the New York Arthritis Foundation (to M. H. P., M. R. P., and T. T. O.), from the Skirball Institute of Biomolecular Medicine (to M. H. P.), and by National Institutes of Health Grants AI36224 (to M. R. P.), AR11949-28 (to M. H. P. and C. C.), HL19721-19 (to M. H. P. and M. R. P.), and AR07176 (to C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Rheumatology NB16N1, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-6404; Fax: 212-263-8804.

(^1)
The abbreviations used are: MAPK, mitogen-activated protein kinase; PTKR, protein tyrosine kinase receptor; PM, plasma membrane; PKA, protein kinase A; LPA, lysophosphatidic acid; FMLP, formyl-methionyl-leucyl-phenylalanine; Il-8, interleukin-8; NECA, 5`N-ethylcarboxamidoadenosine; CS, cytosol; MBP, myelin basic protein; MBPp, myelin basic protein peptide substrate; BSA, bovine serum albumin; LTB(4), leukotriene B(4); Bt(2)cAMP, dibutyryl cAMP; TEMED, N,N,N`,N`-tetramethylethylenediamine.


ACKNOWLEDGEMENTS

We thank Gerald Weissmann for his guidance and support, Bruce Cronstein for advice and for critically reviewing the manuscript, Marianna Feoktistov for expert technical assistance, and Martine Torres, Roland Staud, and Martin Carroll for helpful discussions.


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