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*
Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021; and
Merck Research Laboratories, Rahway, NJ 07065
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adhesion and production of ROI are interrelated, since adhesion is a prerequisite for a large oxidative burst in response to cytokines (4). A distinctive feature of this response is a significant lag period (20–60 min) between the addition of agonist and the beginning of ROI production. During this time PMN adhere to the ligand-coated substrate and spread. The adhesion-dependent oxidative burst requires ligation of leukocyte integrins, as demonstrated by blockade of the response by Abs against CD18 and failure of cells from leukocyte adhesion-deficient patients to respond (5). The participation of the microfilamentous cytoskeleton is also necessary, since dihydrocytochalasin B interferes with both cell spreading and ROI production (4).
Low concentrations (<1 ng/ml) of bacterial LPS act through the glycosylphosphatidyl inositol-anchored protein CD14 on the plasma membrane of PMN (mCD14) to directly elicit leukocyte integrin-dependent adhesion (6). In serum, LPS is delivered to mCD14 through the actions of the transfer protein, LPS binding protein, and the shuttle protein, soluble CD14 (sCD14) (7). The requirement for LPS binding protein to deliver LPS to mCD14 can be circumvented by forming complexes of LPS with sCD14. These complexes have a ratio of 1 to 2 LPS per sCD14 and are capable of stimulating leukocytes in an mCD14-dependent fashion (8). Adhesion in response to LPS exhibits an initial 10- to 15-min lag period before onset, during which time internalization of LPS may be required to initiate signaling for adhesion (9). Phosphatidylinositol 3-kinase (PI3K) is rapidly activated by LPS in PMN, and the PI3K inhibitors, wortmannin and LY294002, block PMN adhesion to fibrinogen in response to LPS, suggesting that PI3K activity is required at an early stage of signaling (9). However, the identity of other signaling components in this pathway remains to be elucidated.
The stress-activated MAP kinase p38 has been identified as participating in the LPS-mediated production of cytokines by monocytes (10). LPS stimulation leads to phosphorylation and activation of p38 (11, 12), and specific inhibitors of p38 abrogate cytokine production in response to LPS (13, 14).
PMN also phosphorylate and activate p38 in response to LPS (15), suggesting that LPS signaling pathways are similar in PMN and monocytes. Here we present evidence that p38 is phosphorylated in PMN with a time course and concentration dependence similar to those for LPS-stimulated cell adhesion and that a specific inhibitor of p38 kinase activity blocks LPS-stimulated PMN adhesion and the adhesion-dependent oxidative burst.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formyl-norleucyl-leucyl-phenylalanine (fNLLP), aprotinin, PMA,
di-isopropyl fluorophosphate, PMSF, scopoletin, horseradish
peroxidase (HRP), Dulbecco’s PBS, and cation-deficient Dulbecco’s PBS
(PD) were purchased from Sigma (St. Louis, MO). FBS was obtained from
HyClone (Logan, UT). Pyrogen-free human serum albumin (HSA) was
purchased from Centeon, Armour & Berring (Kankakee, IL). SB203580,
antipain, leupeptin, benzamidine, chymostatin, pepstatin A, and human
fibrinogen were purchased from Calbiochem-Novabiochem (La Jolla, CA).
Monoclonal anti-phosphotyrosine, anti-p38, anti-Erk-1, and
anti-Erk-2 Abs were obtained from Upstate Biotechnology (Lake
Placid, NY). mAbs against CD14, 60b (16) and 26ic (16), were purified
from ascites by chromatography on protein A-Sepharose (Sigma). LPS Ra
(R60) from Salmonella minnesota was obtained from List
Biologics (Campbell, CA). TNF-
(recombinant, human) was purchased
from Genzyme (Cambridge, MA). Complexes of monomeric LPS and
recombinant soluble human CD14 (sCD14), expressed and purified as
described below, were made by incubating 5 µg/ml LPS with 500 µg/ml
sCD14 for 16 h at 37°C. These were conditions sufficient to
assure that all the LPS was bound by sCD14 (7).
Expression and purification of recombinant human sCD14
The cDNA encoding the complete open reading frame for human CD14 (17) was cloned into the multiple cloning site adjacent to the metallotheonein promoter of expression vector pRmHa3 (18), resulting in the construct pRmHa3-hCD14. Schneider-2 insect cells, 2 x 106 in T75 tissue culture flasks (19), were cotransfected with 10 µg of salmon sperm DNA, 9 µg of pRmHa3-hCD14, and pUChsneo (20) using the calcium phosphate transfection method with a commercially available kit as outlined by the manufacturer (Life Technologies, Gaithersburg, MD). After 48-h growth at 27°C, transfected cells were selected with 0.5 mg/ml G418 sulfate in Schneider insect medium (Sigma) supplemented with 10% heat-inactivated FBS (Life Technologies). Selected transfectants were adapted to growth in serum-free Ex-cell 401 medium (JRH Biosciences, Lenexa, KS) supplemented with 2 mM L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin, and 0.5 mg/ml G418 sulfate. Expression of CD14 in the pRmHa3-hCD14 transfected cells was induced by growing the cells (2 x 106/ml) in serum-free medium containing 1 mM CuSO4. CD14 expression on the cell surface was documented by flow cytometry using FITC-labeled anti-CD14 (clone MY4; Coulter, Hialeah, FL; data not shown). Soluble CD14 secreted into the medium was detected as the dominant band at 45 to 48 kDa in SDS-PAGE and by Western blotting with a polyclonal anti-CD14. Maximal expression was obtained after 3-day growth at 27°C, yielding approximately 5 mg/l of sCD14.
Soluble CD14 was purified from the conditioned medium by a simple three-step procedure. Briefly, protein was precipitated by addition of ammonium sulfate to a final concentration of 60% (w/v). The pellet was dissolved in 0.25x PBS (BioWhittaker, Walkersville, MD) and 1 mM EDTA (buffer A), with 1 mM PMSF and 10 mM HEPES, pH 7.3, and then dialyzed against buffer A with 1 mM PMSF. The dialyzed sample was loaded on a MonoQ column (Pharmacia Biotech, Piscataway, NJ) equilibrated in buffer A with 0.42 mM Pefabloc SC (Boehringer Mannheim, Indianapolis, IN). Protein was eluted with a continuous gradient of 38 to 300 mM NaCl in buffer A with Pefabloc SC. Soluble CD14 was identified in the fractions by SDS-PAGE and Western blotting, as described above. Fractions containing sCD14 were pooled, diluted fivefold in buffer B (25 mM 2-[N-morpholino]ethanesulfonic acids (pH 5.8) and 0.42 mM Pefabloc SC), and loaded on a HiTrap SP column (Pharmacia Biotech) equilibrated in buffer B. Protein was eluted from the column by a continuous gradient of 25 to 150 mM NaCl. Fractions containing sCD14 were pooled and dialyzed against PD. All purification steps were performed at 4°C. Soluble CD14 obtained by this protocol was at least 95% pure as judged by SDS-PAGE. Recombinant sCD14 appeared as a mixture of differently glycosylated forms between 45 and 48 kDa, similar to the pattern described by Haziot et al. (21).
All reagents and solutions used for production and purification of sCD14 were of cell culture quality, if available. Endotoxin contamination of the sCD14 preparation was <0.3 ng LPS/mg CD14 as determined by the colorimetric Limulus amebocyte lysate assay (QCL-1000, BioWhittaker, Walkersville, MD) using ReLPS from Salmonella minnesota (List Biologics) as the standard. The identity and activity of sCD14 were confirmed by numerous assays, including immunoblots, LPS transfer assays with boron dipyrromethane-labeled LPS as previously described (22), and various cell-based assays (this manuscript and data not shown).
Cell preparation
PMN were prepared from freshly drawn human blood on Neutrophil Isolation Medium (Cardinal Associates, Sante Fe, NM) exactly as previously described (23). Contaminating erythrocytes were removed by hypotonic lysis, and PMN were suspended in Dulbecco’s PBS with 0.5 mg/ml HSA, 0.3 U/ml aprotinin, and 3 mM glucose (HAP buffer) for adhesion assays or in Krebs-Ringer phosphate buffer with 5.5 mM glucose (pH 7.35; KRPG) (24) for assays of the oxidative burst.
Electrophoresis and Western blotting of PMN lysates
For each condition, 1 ml of PMN at 5 x 106 cells/ml was used. At the end of the experimental treatment, the cells were washed twice with Dulbecco’s PBS with 1 mM Na3VO3 and 3 mM di-isopropyl fluorophosphate on ice. They were lysed by incubation on ice for 20 min in 0.1 ml 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO3, 50 mM NaF, 0.3 U/ml aprotinin, 2 mM PMSF, 50 µg/ml benzamidine, and 5 µg/ml each of antipain, leupeptin, chymostatin, and pepstatin A. Lysates were centrifuged for 5 min at 12,000 x g, and the supernatants were prepared for SDS-PAGE under reducing conditions.
SDS-PAGE was run on 10% gels (Novex, San Diego, CA) using standard Tris-glycine buffers. Transfer of proteins to nitrocellulose was accomplished in 1.5 h at 300 mA. The filters were blocked overnight with PBS and 1% nonfat dry milk and washed twice with PBS before sequential staining with primary Ab in PBS and 0.1% dry milk (1 µg/ml anti-phosphotyrosine, 0.2 µg/ml anti-p38, anti-Erk1, and anti-Erk2) for 2 h, two washes with PBS, and HRP-conjugated goat anti-mouse IgG diluted 1/100 in PBS and 0.1% dry milk for 2 h. Bound Ab was detected with an ECL kit (Amersham, Arlington Heights, IL) according to the manufacturer’s directions.
Assay for MAPKAP kinase activity
Lysates of PMN, made as described above, were diluted 10-fold in
50 mM HEPES (pH 7.6), 100 mM NaCl, 1% Triton X-100, 2 mM EDTA, 5 µM
pyrophosphate, 0.5 µM okadaic acid, 1 mM
Na3VO4 and 10x protease Inhibitor Mixture
(Calbiochem, 539131). A 21-µl aliquot of diluted cell lysate was
mixed with 3 µl of 250 mM HEPES (pH 7.6), 200 mM MgCl2, 1
mM Na3VO4, 20 mM DTT, 200 mM ß-glycerol
phosphate, and 50 mM NaF for 10 min at ambient temperature. To this, 6
µl of 125 µg/ml recombinant human hsp27 (StressGen Biotechnologies,
Victoria, Canada), 100 µM ATP, and 1 mCi/ml
[
-33P]ATP were added. The reaction mixture was mixed
gently and incubated at ambient temperature for 30 min. The reaction
was stopped by adding 15 µl of 3x SDS-PAGE gel sample buffer to the
reaction and boiling for 3 min. Phosphorylated hsp27 was resolved by
SDS-PAGE (12%), and the gel was dried and subjected to
phosphorimager analysis for quantitation.
Assay for inhibition of cellular adhesion
Adhesion of PMN to fibrinogen-coated Terasaki plates was performed exactly as previously described (25). Briefly, PMN were labeled with the fluorescent dye carboxyfluorescein diacetate succiminidyl ester (Molecular Probes, Eugene, OR), and 104 cells were added to each fibrinogen-coated Terasaki well. After addition of inhibitor, the plates were incubated for 10 min at 37°C before addition of agonists and continued incubation at 37°C. Adhesion was quantitated by measuring the fluorescence in each well before and after washing in a Cytofluor 2300 (PerSeptive Biosystems, Framingham, MA). Percent adhesion was calculated as: (fluorescence after washing/fluorescence before washing) x 100. Samples for each condition were run in triplicate, and the data are presented as the mean ± SD. Experiments representative of at least three repetitions are shown.
Adhesion-dependent oxidative burst
Oxidative burst was measured using the previously described assay of HRP-catalyzed oxidation of scopoletin by H2O2 (24). Polystyrene Primaria 96-well tissue culture plates (Becton Dickinson, Lincoln Park, NJ) were coated with FBS for 30 min at 37°C. Each well was then washed vigorously three times by forceful squirting of 3 ml of 0.9% NaCl to prevent blockade of the reaction by soluble BSA (26). To each well was added 50 µl scopoletin (39 µM in KRPG), 10 µl HRP (0.5 U/ml), 13 µl NaN3 (1 mM), and 10 µl of inhibitor with or without agonist (TNF, fNLLP, LPS/sCD14 complexes, or PMA). The reaction was started by adding 3 x 104 PMN/well in 20 µl. The plates were maintained at 37°C, and the scopoletin fluorescence was read every 10 min at an excitation of 360 nm and an emission of 460 nm in a Cytofluor 4000 fluorescence plate reader equipped with a temperature control device (PerSeptive Biosystems). Samples were read in quadruplicate, and the data are presented ± SD for a representative experiment of at least three performed with the same results. The nanomoles of H2O2 produced per 30,000 PMN were calculated exactly as previously described (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dual phosphorylation of p38 on tyrosine and threonine confers
enhanced enzymatic activity (27). We confirmed reports that p38 is
tyrosine phosphorylated in response to LPS under conditions that
stimulate PMN adhesion. Tyrosine phosphorylation of p38 was tested
after treatment of PMN with 10 ng/ml LPS in the presence of 1% serum
for 20 min at 37°C. These are conditions previously reported to
stimulate phosphorylation of p38 (15, 28), and 20 min falls after the
initial lag period in the adhesive response to LPS (9). We confirmed
tyrosine phosphorylation of a 42-kDa band that migrated with p38 by
Western blot of cell lysates (data not shown). The appearance of
phosphorylated tyrosine in p38 was entirely blocked when PMN were
incubated with mAb 60b, an Ab that blocks CD14-mediated responses to
LPS (29) (Fig. 1
). On the other hand, mAb
26ic, which binds CD14 but does not block CD14-mediated responses to
LPS (29), had no effect on phosphorylation of p38. These data support
the previously reported observation that stimulation of p38
phosphorylation and kinase activity are dependent on mCD14 (15).
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A). Enhanced
phosphorylation was detected after 20 min with concentrations of LPS as
low as 0.3 ng/ml, and a maximal response occurred at 1.25 ng/ml LPS. A
similar dose response was observed for LPS-dependent stimulation of the
activity of MAPKAP kinase, the downstream substrate of p38, although
this activity was observed at concentrations of LPS as low as 0.15
ng/ml (data not shown). The concentration dependence for stimulation of
PMN adhesion was also similar to those for p38 phosphorylation and
MAPKAP kinase activity (Fig. 2B). Like MAPKAP kinase
activity, adhesion could be detected at concentrations of LPS below
those that produced a visible band of tyrosine phosphorylation on p38,
suggesting that the Western blot assay was less sensitive than the
enzymatic and cell-based assays.
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). The peak activity of MAPKAP kinase
lagged 20 min behind the peak for p38 phosphorylation and declined
after 40 min (Fig. 4A).
Adhesion in response to LPS exhibits at least a 10-min lag before onset
and declines after 40 min (9) (Fig. 6A). Tyrosine
phosphorylation of p38 was therefore apparent before adhesion was
observed, and both dephosphorylation of p38 and the decline in MAPKAP
kinase activity commenced before or contemporaneously with the decline
in adhesion. These kinetics suggest that activation of p38 and its
downstream effector MAPKAP kinase could participate in signaling for
adhesion. Additional studies with an inhibitor of p38 were conducted to
test this hypothesis for LPS and other agonists of PMN function.
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The compound SB203580 is reported to be a specific inhibitor of
p38 kinase activity (30). It blocked LPS-stimulated MAPKAP kinase
activity in PMN by about 50% at concentrations of 0.3 µM and above
(Fig. 4
B), demonstrating its activity on p38 under our
experimental conditions. SB203580 was therefore used to test whether
p38 functions in ß2-integrin-dependent PMN adhesion in
response to LPS, TNF, fNLLP, or PMA. Fibrinogen is a ligand for the
leukocyte integrin CD11b/CD18 (31) and was therefore chosen as an
appropriate substrate for the assay.
PMN were preincubated for 10 min at 37°C with increasing
concentrations of SB203580 before addition of agonists for 15 to 20 min
at 37°C and subsequent measurement of adhesion. SB203580 blocked PMN
adhesion to fibrinogen in response to LPS in a concentration-dependent
fashion, with a concentration of about 0.1 µM having a half-maximal
effect (Fig. 5A). In the same
experiment, SB203580 inhibited adhesion in response to TNF by almost
50% at a concentration of 0.6 µM and above (Fig. 5B).
This is consistent with the reported IC50 for SB203580 of
about 0.6 µM for the inhibition of p38 activity in vitro and 1 µM
for IL-6 production in response to TNF by L929 cells (30, 32). It is
also consistent with the concentration dependence observed for SB203580
inhibition of MAPKAP kinase activity in LPS-stimulated PMN (Fig. 4B). For neither MAPKAP kinase activity nor adhesion in
response to LPS or TNF was complete inhibition by SB203580 observed.
However, MAPKAP kinase activity was inhibited to the same extent as
adhesion, suggesting that the effect of SB203580 on adhesion was
through its inhibition of p38. The compound inhibited adhesion in
response to fNLLP by only about 20% and failed to inhibit adhesion in
response to PMA at concentrations up to 2.4 µM (Fig. 5, C
and D), indicating that the compound was not toxic to the
cells and had no direct effect on the interaction of leukocyte
integrins with the ligand-coated substrate. These results suggest that
p38 function is required for PMN adhesion in response to LPS or TNF,
but is not strongly required for adhesion in response to the
alternative agonist fNLLP. TNF and LPS elicit many of the same
responses from cells, causing PMN adhesion (6, 33) and synthesis of a
similar spectrum of cytokines by both monocytes and endothelial cells.
The signaling pathways leading from TNF and LPS to PMN adhesion may
share p38 as a common element.
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The effect of SB203580 on adhesion over time was tested to
determine whether the rate of adhesion was slowed by blockade of p38.
PMN were preincubated for 10 min at 37°C with 1 µM SB203580, and
then agonists were added for different times at 37°C before
measurement of adhesion. The results, shown in Figure 6, demonstrate that in the presence of
the p38 inhibitor, the initial rate of adhesion was much slower in
response to LPS or TNF but was only slightly decreased in response to
fNLLP. In addition, the overall extent of adhesion was depressed for
both LPS and TNF when p38 was blocked; 1 µM SB203580 inhibited peak
adhesion in response to LPS or TNF by 40 to 50%. SB203580 had no
effect on either the rate or the extent of adhesion in response to PMA
(Fig. 6), which bypasses cell surface receptors to stimulate PKC
directly.
The p38 inhibitor also blocks the adhesion-dependent oxidative burst
Since cytokine-stimulated, adherent PMN exhibit a massive
oxidative response, we tested whether this secondary response to
adhesion was also abrogated by an inhibitor of p38. PMN were
preincubated for 10 min at 37°C with increasing concentrations of
SB203580 before addition of agonist and further incubation at 37°C.
Production of ROI was monitored over time using HRP-catalyzed oxidation
of scopoletin by H2O2. As was the case for
adhesion, SB203580 slowed the initial rate of the adhesion-dependent
oxidative burst in response to LPS and TNF in a concentration-dependent
manner (Fig. 7, A and
B). This led to a large reduction in the amount of
H2O2 produced by 60 min, with 50% inhibition
observed at about 1 µM SB203580. There was also a
concentration-dependent decrease in the initial rate of
H2O2 production in response to fNLLP, but the
compound was less potent in blocking the response to fNLLP than it was
in blocking that for LPS or TNF (Fig. 7C). Fifty
percent inhibition of the fNLLP response was not achieved at
concentrations up to 2 µM. As was the case with adhesion, the
compound had no effect on the response to PMA (Fig. 7D),
indicating that the compound was not toxic to the cells and had no
direct effect on the assay. These results suggest that the p38
inhibitor may have its primary effect on the oxidative burst by
blocking adhesion. However, we cannot rule out the possibility that p38
participates in additional ways to signal the oxidative burst. Further
studies were conducted to determine at what time after addition of
agonist p38 function was required for the adhesion-dependent oxidative
burst.
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To test when p38 function was required for the oxidative burst, 1
µM SB203580 was added either at the same time as the agonist (LPS or
TNF) or at intervals after the addition of agonist, and
H2O2 production was monitored over time at
37°C. The inhibitory effect of SB203580 on the oxidative burst in
response to LPS or TNF was gradually lost with the increase in time of
addition following stimulation (Fig. 8).
The decline in inhibition was observed as early as 10 min for LPS and 5
min for TNF, and by 20 min there was no effect on the oxidative burst
(Fig. 8, A and B). Thus, addition of SB203580
after the end of the lag period was not inhibitory. Inhibition during
the lag was time dependent, suggesting that p38 kinase activity is
important at a very early stage in signaling for the oxidative burst
and, further, that its function is required throughout the lag to
achieve maximal generation of ROI.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main focus in PMN has been on the kinase activity of p38 itself and on identifying proteins downstream of the kinase, although some functional correlates have also been made. Phosphorylation and activation of p38 in response to LPS (15, 28), TNF (36), formyl peptide (36, 37), and PMA (15) have been reported. In addition, the immediate downstream effector of p38, MAPKAP kinase-2, is phosphorylated in PMN in response to granulocyte-macrophage CSF (38) and formyl peptide (37, 39), and SB203580 blocks activation of MAPKAP kinase-2 in PMN (37). Functional studies have suggested that p38 may play a role in signaling arachidonic acid release and the production of ROI. SB203580 partially blocks phosphorylation of cPLA2 and diminishes its activity in PMN stimulated with TNF (40). A competitive inhibitory peptide of hsp27, a protein phosphorylated by MAPKAP kinase-2 (41, 42), introduced into PMN has been reported to diminish the small oxidative burst of cells in suspension in response to FMLP and PMA (39). SB203580 has also been reported to inhibit adhesion of PMN to endothelial cells in response to FMLP and the oxidative burst of cells in suspension in response to the same agonist (43).
Our results both confirm that p38 is phosphorylated by PMN in response to LPS in a mCD14-dependent manner and go beyond previous reports to describe an important functional significance of p38 activation in PMN. Here we demonstrated that blockade of p38 kinase activity with a specific inhibitor diminished two physiologically important functions of PMN. Integrin-dependent adhesion of PMN to fibrinogen and the adhesion-dependent oxidative burst in response to LPS and TNF were both inhibited by SB203580 at concentrations consistent with the IC50 of the compound for p38 inhibition (30, 32). These results define a common component in the signaling pathways for LPS and TNF. LPS and TNF also share PI3K as a common signaling component, since wortmannin effectively blocks adhesion in response to these agonists (9). Preliminary experiments suggest that wortmannin at concentrations that inhibit adhesion does not block phosphorylation of p38 in response to LPS (P. A. Detmers and D. Zhou, unpublished observations). However, published results suggest that wortmannin, but not the alternative PI3K inhibitor LY29002, partially blocks p38 activation in response to formyl peptide (37, 44). Further work will be necessary to determine the order of p38 and PI3K in a cascade or, alternatively, whether they lie on separate pathways.
The inhibitor also partially blocked both adhesion and the oxidative burst in response to fNLLP. It is likely that SB203580 was more effective in blocking ROI production than adhesion in response to fNLLP, because the substrates used for the assays were different. Serum offers fewer ligand binding sites for ß2-integrins than does fibrinogen, and adhesion to serum would thus be easier to inhibit. Formyl peptide-stimulated adhesion of PMN to endothelial cells is blocked 50% by 10 µM SKF86002 (43), a p38 inhibitor with an IC50 for the enzyme around 1 µM (45). This suggests that although formyl peptide causes activation of the kinase (36, 37, 43) and MAPKAP kinase 2 (37, 39), adhesion to different substrates in response to formyl peptide may be differentially p38 dependent. SB203580 had no inhibitory effect on PMN responses to PMA, although it was previously reported that PMA can activate p38 (15). Apparently this activity is incidental to the ability of PMA to stimulate adhesion and an oxidative burst.
Several lines of evidence suggest that p38 participates in signaling the production of ROI primarily through its role in signaling integrin-dependent adhesion. SB203580 diminished integrin-dependent adhesion, and it has been previously shown that blockade of leukocyte integrin-ligand interactions by Abs abrogates the production of ROI by PMN in response to TNF (5). Further, p38 kinase activity was required at an early stage in the lag period before the production of ROI, during which adhesion, cell spreading, and assembly of the oxidase components occurs. When SB203580 was added at times longer than 5 or 10 min after addition of LPS or TNF, the inhibitory effectiveness for ROI production declined. Thus, while we cannot rule out the possibility that p38 functions in both signaling for adhesion and in another process necessary for oxidase assembly, blockade of adhesion alone by SB203580 appears sufficient to explain inhibition of the oxidative burst.
Two observations suggest that p38 has little role in directly regulating the function of the oxidase complex. When SB203580 was added to PMN as late as 20 min after addition of the agonist, at the end of the lag period, ROI production proceeded unimpeded, suggesting that once assembly of the oxidase complex is accomplished, p38 function is no longer required. In addition, there was no inhibition by SB203580 of ROI production in response to PMA, an agonist that bypasses both receptors on the cell surface and the requirement for adhesion to produce a massive oxidative burst. In previous reports an inhibitory peptide of hsp27 partially inhibited the oxidative burst by PMN in suspension in response to PMA (39), and SKF86002 substantially blocked the response to formyl peptide by cells in suspension (43). The apparent difference in our results may be a function of the type of inhibitor used and the magnitude of the response measured.
Since PMN adhesion does not require protein synthesis, it is unlikely that the signaling pathway leading from p38 is identical with that used for cytokine production by monocytes. At least two cytoplasmic proteins downstream of p38 present themselves as potential candidates that could participate in promoting adhesion. The p38 substrate MAPKAP kinase-2 phosphorylates both hsp27 and LSP1 in PMN (28, 46), although LSP1 appears to be the preferred substrate (46). LSP1 functions as an actin binding protein, and patients who overexpress LSP1 exhibit a defect in PMN locomotion and enhanced production of superoxide (47). It is not clear yet, however, whether phosphorylation controls the actin-binding activity of LSP1. Phosphorylation of hsp27 causes oligomerization (48, 49), and the oligomers can interact with microfilaments to stabilize actin in a polymerized form (49, 50). When hsp27 was overexpressed in a Chinese hamster cell line, pinocytic activity was enhanced along with the concentration of polymerized actin in the cell cortex (51). On the other hand, pinocytosis and cortical filamentous actin were decreased in cells expressing a mutant form of the protein that could not be phosphorylated (51). Since internalization of LPS may be required to complete the signaling necessary for PMN adhesion (9, 52) it is possible that control of hsp27 phosphorylation may regulate the internalization process. In this model p38 phosphorylation in response to LPS would occur very early, before internalization, and the downstream phosphorylation of hsp27 and perhaps LSP1 would promote more rapid internalization of LPS and lead to further steps in the signaling cascade. Further work will be required to test this hypothesis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patricia A. Detmers, Merck Research Laboratories, 126 East Lincoln Ave., R80W-250, Rahway, NJ 07065.
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; ROI, reactive oxygen intermediates; mCD14, membrane CD14; sCD14, recombinant soluble human CD14; PI3K, phosphatidylinositol 3-kinase; fNLLP, formyl-norleucyl-leucyl-phenylalanine; HRP, horseradish peroxidase; PD, cation-deficient Dulbecco’s PBS; HSA, human serum albumin; HAP, Dulbecco’s PBS with 0.5 mg/ml human serum albumin, 0.3 U/ml aprotinin, and 3 mM glucose; KRPG, Krebs-Ringer phosphate buffer with 5.5 mM glucose MAPKAP, mitogen-activated protein kinase-activated protein; hsp27, heat shock protein-27; LSP1, lymphocyte-specific protein-1.
Received for publication October 14, 1997. Accepted for publication April 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by cells in whole blood. J. Immunol. 152:5868.[Abstract]
chain of fibrinogen. Proc. Natl. Acad. Sci. USA 88:1044. or FMLP stimulation. J. Immunol. 160:1982.-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils. Biochem. J. 319:17.
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 E. Pluskota, O. I. Stenina, I. Krukovets, D. Szpak, E. J. Topol, and E. F. Plow Mechanism and effect of thrombospondin-4 polymorphisms on neutrophil function Blood, December 1, 2005; 106(12): 3970 - 3978. [Abstract] [Full Text] [PDF]
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L. C. Parker, M. K. B. Whyte, S. K. Dower, and I. Sabroe The expression and roles of Toll-like receptors in the biology of the human neutrophil J. Leukoc. Biol., June 1, 2005; 77(6): 886 - 892. [Abstract] [Full Text] [PDF]
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 R. A. Clemens, S. A. Newbrough, E. Y. Chung, S. Gheith, A. L. Singer, G. A. Koretzky, and E. J. Peterson PRAM-1 Is Required for Optimal Integrin-Dependent Neutrophil Function Mol. Cell. Biol., December 15, 2004; 24(24): 10923 - 10932. [Abstract] [Full Text] [PDF]
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M. Bouaouina, E. Blouin, L. Halbwachs-Mecarelli, P. Lesavre, and P. Rieu TNF-Induced {beta}2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK J. Immunol., July 15, 2004; 173(2): 1313 - 1320. [Abstract] [Full Text] [PDF]
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B. Kasper, E. Brandt, S. Bulfone-Paus, and F. Petersen Platelet factor 4 (PF-4)-induced neutrophil adhesion is controlled by src-kinases, whereas PF-4-mediated exocytosis requires the additional activation of p38 MAP kinase and phosphatidylinositol 3-kinase Blood, March 1, 2004; 103(5): 1602 - 1610. [Abstract] [Full Text] [PDF]
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 H. Kutsuna, K. Suzuki, N. Kamata, T. Kato, F. Hato, K. Mizuno, H. Kobayashi, M. Ishii, and S. Kitagawa Actin reorganization and morphological changes in human neutrophils stimulated by TNF, GM-CSF, and G-CSF: the role of MAP kinases Am J Physiol Cell Physiol, January 1, 2004; 286(1): C55 - C64. [Abstract] [Full Text] [PDF]
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S. Singh, D. W. Powell, M. J. Rane, T. H. Millard, J. O. Trent, W. M. Pierce, J. B. Klein, L. M. Machesky, and K. R. McLeish Identification of the p16-Arc Subunit of the Arp 2/3 Complex as a Substrate of MAPK-activated Protein Kinase 2 by Proteomic Analysis J. Biol. Chem., September 19, 2003; 278(38): 36410 - 36417. [Abstract] [Full Text] [PDF]
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R. El Bekay, M. Alvarez, J. Monteseirin, G. Alba, P. Chacon, A. Vega, J. Martin-Nieto, J. Jimenez, E. Pintado, F. J. Bedoya, et al. Oxidative stress is a critical mediator of the angiotensin II signal in human neutrophils: involvement of mitogen-activated protein kinase, calcineurin, and the transcription factor NF-{kappa}B Blood, July 15, 2003; 102(2): 662 - 671. [Abstract] [Full Text] [PDF]
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K. C. Malcolm and G. S. Worthen Lipopolysaccharide Stimulates p38-dependent Induction of Antiviral Genes in Neutrophils Independently of Paracrine Factors J. Biol. Chem., April 25, 2003; 278(18): 15693 - 15701. [Abstract] [Full Text] [PDF]
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 V. S. Bansal, S. Vaidya, E. P. Somers, M. Kanuga, D. Shevell, R. Weikel, and P. A. Detmers Small Molecule Antagonists of Complement Receptor Type 3 Block Adhesion and Adhesion-Dependent Oxidative Burst in Human Polymorphonuclear Leukocytes J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1016 - 1024. [Abstract] [Full Text] [PDF]
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J. A. Nick, S. K. Young, P. G. Arndt, J. G. Lieber, B. T. Suratt, K. R. Poch, N. J. Avdi, K. C. Malcolm, C. Taube, P. M. Henson, et al. Selective Suppression of Neutrophil Accumulation in Ongoing Pulmonary Inflammation by Systemic Inhibition of p38 Mitogen-Activated Protein Kinase J. Immunol., November 1, 2002; 169(9): 5260 - 5269. [Abstract] [Full Text] [PDF]
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 E. M. Mtairag, X. Houard, S. Rais, C. Pasquier, M. Oudghiri, M.-P. Jacob, O. Meilhac, and J.-B. Michel Pharmacological Potentiation of Natriuretic Peptide Limits Polymorphonuclear Neutrophil-Vascular Cell Interactions Arterioscler Thromb Vasc Biol, November 1, 2002; 22(11): 1824 - 1831. [Abstract] [Full Text] [PDF]
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 S. R. Yan, W. Al-Hertani, D. Byers, and R. Bortolussi Lipopolysaccharide-Binding Protein- and CD14-Dependent Activation of Mitogen-Activated Protein Kinase p38 by Lipopolysaccharide in Human Neutrophils Is Associated with Priming of Respiratory Burst Infect. Immun., August 1, 2002; 70(8): 4068 - 4074. [Abstract] [Full Text] [PDF]
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T. Orlic, W. H. Loomis, A. Shreve, S. Namiki, and W. G. Junger Hypertonicity increases cAMP in PMN and blocks oxidative burst by PKA-dependent and -independent mechanisms Am J Physiol Cell Physiol, June 1, 2002; 282(6): C1261 - C1269. [Abstract] [Full Text] [PDF]
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J. Hua, K. Sakamoto, and I. Nagaoka Inhibitory actions of glucosamine, a therapeutic agent for osteoarthritis, on the functions of neutrophils J. Leukoc. Biol., April 1, 2002; 71(4): 632 - 640. [Abstract] [Full Text] [PDF]
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T. Vasselon and P. A. Detmers Toll Receptors: a Central Element in Innate Immune Responses Infect. Immun., March 1, 2002; 70(3): 1033 - 1041. [Full Text] [PDF]
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T. Vasselon, W. A Hanlon, S. D Wright, and P. A. Detmers Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38 J. Leukoc. Biol., March 1, 2002; 71(3): 503 - 510. [Abstract] [Full Text] [PDF]
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 F. Gao, T.-L. Yue, D.-W. Shi, T. A Christopher, B. L Lopez, E. H Ohlstein, F. C Barone, and X. L Ma p38 MAPK inhibition reduces myocardial reperfusion injury via inhibition of endothelial adhesion molecule expression and blockade of PMN accumulation Cardiovasc Res, February 1, 2002; 53(2): 414 - 422. [Abstract] [Full Text] [PDF]
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D. C. Cara, J. Kaur, M. Forster, D.-M. McCafferty, and P. Kubes Role of p38 Mitogen-Activated Protein Kinase in Chemokine-Induced Emigration and Chemotaxis In Vivo J. Immunol., December 1, 2001; 167(11): 6552 - 6558. [Abstract] [Full Text] [PDF]
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M. O. Hannigan, L. Zhan, Y. Ai, A. Kotlyarov, M. Gaestel, and C.-K. Huang Abnormal Migration Phenotype of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2-/- Neutrophils in Zigmond Chambers Containing Formyl-Methionyl-Leucyl-Phenylalanine Gradients J. Immunol., October 1, 2001; 167(7): 3953 - 3961. [Abstract] [Full Text] [PDF]
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M. Thiel, F. Buessecker, K. Eberhardt, A. Chouker, F. Setzer, U. Kreimeier, K.-E. Arfors, K. Peter, and K. Messmer Effects of hypertonic saline on expression of human polymorphonuclear leukocyte adhesion molecules J. Leukoc. Biol., August 1, 2001; 70(2): 261 - 273. [Abstract] [Full Text] [PDF]
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P. Piccardoni, R. Sideri, S. Manarini, A. Piccoli, N. Martelli, G. de Gaetano, C. Cerletti, and V. Evangelista Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin Blood, July 1, 2001; 98(1): 108 - 116. [Abstract] [Full Text] [PDF]
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A. Schmidt, E. Caron, and A. Hall Lipopolysaccharide-Induced Activation of {beta}2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase Mol. Cell. Biol., January 15, 2001; 21(2): 438 - 448. [Abstract] [Full Text]
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K. Suzuki, T. Hasegawa, C. Sakamoto, Y.-M. Zhou, F. Hato, M. Hino, N. Tatsumi, and S. Kitagawa Cleavage of Mitogen-Activated Protein Kinases in Human Neutrophils Undergoing Apoptosis: Role in Decreased Responsiveness to Inflammatory Cytokines J. Immunol., January 15, 2001; 166(2): 1185 - 1192. [Abstract] [Full Text] [PDF]
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A. Mocsai, Z. Jakus, T. Vantus, G. Berton, C. A. Lowell, and E. Ligeti Kinase Pathways in Chemoattractant-Induced Degranulation of Neutrophils: The Role of p38 Mitogen-Activated Protein Kinase Activated by Src Family Kinases J. Immunol., April 15, 2000; 164(8): 4321 - 4331. [Abstract] [Full Text] [PDF]
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E. Paine, R. Palmantier, S. K. Akiyama, K. Olden, and J. D. Roberts Arachidonic Acid Activates Mitogen-activated Protein (MAP) Kinase-activated Protein Kinase 2 and Mediates Adhesion of a Human Breast Carcinoma Cell Line to Collagen Type IV through a p38 MAP Kinase-dependent Pathway J. Biol. Chem., April 6, 2000; 275(15): 11284 - 11290. [Abstract] [Full Text] [PDF]
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J. A. Nick, S. K. Young, K. K. Brown, N. J. Avdi, P. G. Arndt, B. T. Suratt, M. S. Janes, P. M. Henson, and G. S. Worthen Role of p38 Mitogen-Activated Protein Kinase in a Murine Model of Pulmonary Inflammation J. Immunol., February 15, 2000; 164(4): 2151 - 2159. [Abstract] [Full Text] [PDF]
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 D. A. Partrick, E. E. Moore, P. J. Offner, D. R. Meldrum, D. Y. Tamura, J. L. Johnson, and C. C. Silliman Maximal Human Neutrophil Priming for Superoxide Production and Elastase Release Requires p38 Mitogen-Activated Protein Kinase Activation Arch Surg, February 1, 2000; 135(2): 219 - 225. [Abstract] [Full Text] [PDF]
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C. T. Park and S. D. Wright Fibrinogen is a component of a novel lipoprotein particle: Factor H-related protein (FHRP)-associated lipoprotein particle (FALP) Blood, January 1, 2000; 95(1): 198 - 204. [Abstract] [Full Text] [PDF]
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S. Vaidya, E. P. Somers, S. D. Wright, P. A. Detmers, and V. S. Bansal 15-Deoxy-{Delta}12,1412,14-prostaglandin J2 Inhibits the {beta}2 Integrin-Dependent Oxidative Burst: Involvement of a Mechanism Distinct from Peroxisome Proliferator-Activated Receptor {gamma} Ligation J. Immunol., December 1, 1999; 163(11): 6187 - 6192. [Abstract] [Full Text] [PDF]
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L. Liu, P. Moesner, N. L. Kovach, R. Bailey, A. D. Hamilton, S. M. Sebti, and J. M. Harlan Integrin-dependent Leukocyte Adhesion Involves Geranylgeranylated Protein(s) J. Biol. Chem., November 19, 1999; 274(47): 33334 - 33340. [Abstract] [Full Text] [PDF]
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J. P. Lian, R. Huang, D. Robinson, and J. A. Badwey Activation of p90RSK and cAMP Response Element Binding Protein in Stimulated Neutrophils: Novel Effects of the Pyridinyl Imidazole SB 203580 on Activation of the Extracellular Signal-Regulated Kinase Cascade J. Immunol., October 15, 1999; 163(8): 4527 - 4536. [Abstract] [Full Text] [PDF]
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 T. Vasselon, E. Hailman, R. Thieringer, and P. A. Detmers Internalization of Monomeric Lipopolysaccharide Occurs after Transfer Out of Cell Surface Cd14 J. Exp. Med., August 16, 1999; 190(4): 509 - 522. [Abstract] [Full Text] [PDF]
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H. Kankaanranta, P. M. De Souza, P. J. Barnes, M. Salmon, M. A. Giembycz, and M. A. Lindsay SB 203580, an Inhibitor of p38 Mitogen-Activated Protein Kinase, Enhances Constitutive Apoptosis of Cytokine-Deprived Human Eosinophils J. Pharmacol. Exp. Ther., August 1, 1999; 290(2): 621 - 628. [Abstract] [Full Text]
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S. B. Rizoli, O. D. Rotstein, and A. Kapus Cell Volume-dependent Regulation of L-selectin Shedding in Neutrophils. A ROLE FOR p38 MITOGEN-ACTIVATED PROTEIN KINASE J. Biol. Chem., July 30, 1999; 274(31): 22072 - 22080. [Abstract] [Full Text] [PDF]
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R. A. Ward, M. Nakamura, and K. R. McLeish Priming of the Neutrophil Respiratory Burst Involves p38 Mitogen-activated Protein Kinase-dependent Exocytosis of Flavocytochrome b558-containing Granules J. Biol. Chem., November 17, 2000; 275(47): 36713 - 36719. [Abstract] [Full Text] [PDF]
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J. E. Smolen, T. K. Petersen, C. Koch, S. J. O'Keefe, W. A. Hanlon, S. Seo, D. Pearson, M. C. Fossett, and S. I. Simon L-Selectin Signaling of Neutrophil Adhesion and Degranulation Involves p38 Mitogen-activated Protein Kinase J. Biol. Chem., May 19, 2000; 275(21): 15876 - 15884. [Abstract] [Full Text] [PDF]
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) and continued incubation
at 37°C. H2O2 produced was measured at 10-min
intervals as described in Materials and Methods. The
figure is representative of four separate experiments performed on
quadruplicate samples.