| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Laboratory of Experimental Internal Medicine and
Department of Infectious Diseases, Tropical Medicine, and AIDS, Academic Medical Center, and
CLB and Laboratory for Clinical and Experimental Immunology, University of Amsterdam, Amsterdam, The Netherlands;
Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877; and
¶ Boehringer Ingelheim Pharma KG, Biberach, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
,
IL-6, IL-10, and IL-1R antagonist) was strongly inhibited by both low
and high dose p38 MAPK inhibitor. In addition, p38 MAPK inhibition
diminished leukocyte responses, including neutrophilia, release of
elastase-1-antitrypsin complexes, and up-regulation of
CD11b with down-regulation of L-selectin. Finally, blocking p38 MAPK
decreased C-reactive protein release. These data identify p38 MAPK as a
principal mediator of the inflammatory response to LPS in humans.
Furthermore, the anti-inflammatory potential of an oral p38 MAPK
inhibitor in humans in vivo suggests that p38 MAPK inhibitors may
provide a new therapeutic option in the treatment of inflammatory
diseases. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1, growth factors, and stresses such
as heat shock, hypoxia, and ischemia/reperfusion (6, 7).
In addition, p38 MAPK positively regulates a variety of genes involved
in inflammation, such as TNF-, IL-1, IL-6, IL-8, cyclooxygenase-2,
and collagenase-1 and -3 (7).
Because of the broad proinflammatory role of p38 MAPK in several in
vitro systems, inhibition of this pathway has been advocated as a novel
therapeutic strategy for inflammatory diseases (8).
However, the effect of p38 MAPK inhibition on in vivo models of
inflammation has only been examined in a limited number of studies with
equivocal results. The p38 MAPK inhibitors have been found to reduce
LPS-induced TNF- production in mice and rats (9, 10, 11).
This result could not be duplicated with one of these inhibitors
(SB203580) in mice despite almost completely abolishing the p38 MAPK
activity in spleen cells harvested from these animals
(12). Furthermore, inhibition of p38 MAPK was associated
with a decrease in neutrophil recruitment and TNF- release in
bronchoalveolar lavage fluid in mice after intratracheal administration
of LPS (13), but with elevated TNF- concentrations in
lungs during murine pneumococcal pneumonia and tuberculosis
(12). In a murine model of peritonitis induced by cecal
ligation and puncture, delayed administration of SB203580 improved
survival and prevented the enhanced release of IL-10 by macrophages
harvested from mice with peritonitis while concurrently improving the
reduced IL-12 release by these cells (14).
Knowledge of the activation of p38 MAPK and its role in inflammation in humans in vivo is limited despite current interest in p38 MAPK inhibition in the treatment of human inflammatory disease. In addition, there are conflicting animal data. Therefore, in the present study we used the well-characterized model of human inflammation produced by i.v. injection of low dose LPS (15) to evaluate the activation of p38 MAPK and the effect of a new orally administered p38 MAPK inhibitor.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The p38 MAPK inhibitor used in this study (BIRB 796 BS) was developed by Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT). BIRB 796 BS is 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)-naphtalen-1-yl]-urea (empirical formula, C31H37N5O3; m.w., 527.6), a water-soluble, orally bioavailable molecule. Details about the structure and specificity of BIRB 796 BS for p38 MAPK will be published in a separate manuscript (16). BIRB 796 BS has a >330-fold selectivity for p38 MAPK compared with 12 other protein kinases studied. In contrast to other p38 MAPK inhibitors (e.g., SB203580), BIRB 796 BS prevents both the phosphorylation and kinetic activity of p38 MAPK by binding to a novel allosteric binding site as well as to the ATP pocket of p38 MAPK.
Effect of BIRB 796 BS on p38 MAPK activation in vitro
Blood from healthy volunteers was collected with 10 U/ml heparin
(Leo Pharmaceutical Products, Weesp, The Netherlands). PBMCs were
isolated by density gradient sedimentation on Ficoll-Paque (Pharmacia
Biotech, Uppsala, Sweden) from blood diluted 1/1 with PBS. PBMCs were
washed twice with PBS and resuspended in RPMI 1640 supplemented
with 10% heat-inactivated FCS (both from Life Technologies, Grand
Island, NY) at a concentration of 
5 x
106 cells/ml in 15-ml tubes (BD Biosciences,
Franklin Lakes, NJ). After preincubation for 1 h with BIRB 796 BS
(1, 10, 100, or 1000 nM) or DMSO as the solvent control, samples
(containing 5 x 106 cells each) were
stimulated with LPS from Escherichia coli serotype 0111:B4
(25 ng/ml; Sigma-Aldrich, St. Louis, MO). After 15 min, 12 ml ice-cold
PBS was added to each 15-ml tube, and cells were centrifuged at
400 x g for 5 min at 4°C. The cell pellets were
lysed in 100 µl 3x SDS-sample buffer; this mixture was briefly
sonicated twice (10 s each time) and boiled for 5 min, followed by
brief centrifugation and storage at -20°C until further analysis. As
for equal loading during Western blotting (see below), equal amounts of
sample (25 µl; containing 1.25 x 106
cells) were analyzed. Furthermore, after immunoblotting the blots were
subjected to Amido Black staining to assess equal loading.
Western blotting
Samples mixed with SDS-sample buffer were loaded on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Subsequently, membranes were blocked in 5% BSA in PBS supplemented with 0.1% Tween 20 and washed in 0.2% BSA in PBS supplemented with 0.1% Tween 20. The extent of p38 MAPK activation was determined using Abs against phosphorylated (Thr180/Tyr182) p38 MAPK (New England Biolabs, Beverly, MA), used at a 1/1000 dilution incubated overnight. After three washes for 10 min each, secondary Ab incubation was performed for 1 h with HRP-conjugated goat anti-rabbit Ig (DAKO, Glostrup, Denmark) at a 1/2000 dilution in 10% human serum in PBS supplemented with 0.1% Tween 20. After ECL using Lumilight+ substrate, Ab binding was visualized using a Lumi-imager (Roche, Mannheim, Germany).
LPS administration to humans in vivo
The study was performed as a randomized, double-blind, placebo-controlled experiment. The study was approved by the institutional scientific and ethics committees, and written informed consent was obtained from each subject before the start of the study. Twenty-four healthy male volunteers (mean age, 22 years; range, 19–29 years) participated in the investigation. All subjects were in good health as documented by history, physical examination, electrocardiogram, and routine laboratory screening. Tests for HIV and hepatitis B and C were negative. The participants did not use any medication. All participants were nonsmokers. The subjects fasted overnight before LPS administration. On the study day, two i.v. cannulas were inserted, one for LPS administration and one for blood collection. Eight subjects received 600 mg BIRB 796 BS (high dose), eight subjects received 50 mg BIRB 796 BS (low dose), and eight subjects received placebo. BIRB 796 BS was given as an oral solution in 15 ml polyethylene glycol 400. The placebo solution consisted of 15 ml polyethylene glycol 400. The study drug and placebo were administered orally 3 h before infusion of LPS. LPS (E. coli LPS, lot G1; United States Pharmacopeial Convention, Rockville, MD) was administered as a bolus i.v. injection at a dose of 4 ng/kg body weight. Oral temperature, blood pressure, and heart rate were measured every 30 min during the first 2 h after LPS challenge, thereafter at 1-h intervals for 4 h, then at a decreased frequency. Clinical symptoms, such as headache, chills, myalgia, nausea, vomiting, abdominal pain, and backache, were recorded throughout the study using a graded scale (0 = absent, 1 = mild, 2 = moderate, 3 = severe). Blood was obtained from an i.v. cannula before administration of BIRB 796 BS or placebo (-3 h), directly before LPS administration (0 h), and at 5, 15, 30, 60, and 90 min and 2, 3, 4, 5, 6, 8, 10, and 24 h thereafter.
Measurement of BIRB 796 BS plasma levels
Plasma samples were analyzed for BIRB 796 BS concentrations using a validated HPLC method with electrospray ionization mass spectrometry/mass spectrometry detection. Following the solid phase extraction of the analyte from plasma, BIRB 796 BS and the internal standard (d8-BIRB 796 BS) were separated chromatographically, followed by detection via the Tandem mass spectrometer. The linear range was established with calibration standards from 0.1 to 1000 ng/ml using peak height ratios. The lower limit of quantitation was 0.1 ng/ml using 500 ml plasma.
The p38 MAPK measurements during the in vivo study
Blood for measurement of p38 MAPK was collected in
heparin-containing Vacutainer tubes (BD Vacutainer Systems,
Plymouth, U.K.) at 0, 5, 15, 30, and 60 min, and at 4, 8, and 24 h
relative to LPS injection. After collection, erythrocytes from 4.5-ml
aliquots were lysed by adding 40 ml ice-cold isotonic
NH4Cl solution (155 mmol/L
NH4Cl, 10 mmol/L KHCO3, 0.1
mmol/L EDTA (pH 7.4), and 1 mM Pefabloc; Merck, Darmstadt,
Germany) for 30 min. The remaining leukocytes were centrifuged for 5
min at 400 x g at 4°C, washed twice with ice-cold
PBS, and resuspended in 400 µl PBS. Two hundred microliters of the
cell suspension was added to 125 µl 3x SDS-sample buffer; this
mixture was briefly sonicated twice (10 s each time) and boiled for 5
min, followed by brief centrifugation and storage at -20°C. To the
remaining cell suspension, which was intended for the kinase assay, 800
µl ice-cold cell lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM
Na3VO4, 1 µg/ml
leupeptin, and 1 mM Pefabloc) were added. Samples were sonicated four
times for 5 s each time on ice and centrifuged at 7000 x
g for 10 min at 4°C. Protein content in the clear
supernatant was determined with a bicinchoninic acid protein assay kit
(Pierce, Rockford, IL), using BSA as the standard, and the supernatant
was stored at -80°C.
For all subjects phosphorylation of p38 MAPK was measured by Western blot. Ab binding was quantified using image analysis software (EFM Software, Rotterdam, The Netherlands), and measurements were corrected for the amount of protein loaded to allow quantitative comparison of p38 phosphorylation between blood samples, which may contain different amounts of leukocyte protein as a consequence of the LPS challenge. The sample obtained at -3 h was not adequately treated and was omitted from the analysis. All other samples were compared with one random sample set at an arbitrary value of 100 U for comparing p38 MAPK phosphorylation between subjects. In addition, a minimum measured activity was considered necessary for use as an internal reference (i.e., 25 on a 28 digital gray scale). Two subjects failed to meet this requirement (one from the low dose and one from the high dose BIRB 796 BS-treated volunteers), possibly due to low protein yield in these samples, and were not included in the analysis.
The p38 MAPK enzymatic activity was measured using a kinase assay (New
England Biolabs). White blood cell lysates were prepared as described
above. A once-diluted slurry of agarose hydrazide-bound Abs to
phosphorylated
(Thr180/Tyr182) p38 MAPK
(40 µl) was used to selectively immunoprecipitate active p38 MAPK
from the cell lysate by gently shaking overnight at 4°C. To assure
equal loading, a fixed amount of lysate was used per sample (80 µg
in 340 µl cell lysis buffer). The immunoprecipitate was washed twice
with 500 µl ice-cold cell lysis buffer and twice with 500 µl
ice-cold kinase buffer (25 mM Tris (pH 7.5), 5 mM
-glycerolphosphate, 2 mM DTT, 0.1 mM
Na3VO4, and 10 mM
MgCl2) at 4°C. The kinase reactions were
conducted in the presence of 200 µM ATP and 2 µg activating
transcription factor-2 (ATF-2) fusion protein at 30°C for 30 min.
After the reaction had been terminated by the addition of 3x
SDS-sample buffer, the mixture was boiled for 5 min, followed by brief
centrifugation. ATF-2 phosphorylation was selectively measured by
Western immunoblotting as described previously using specific Abs
against phosphorylated (Thr71) ATF-2.
Other assays
Cytokine concentrations were determined in
EDTA-anti-coagulated plasma by specific ELISAs according to the
manufacturers’ instructions (with detection limits). These ELISAs were
as follows: TNF- (2.8 pg/ml), IL-6 (1.2 pg/ml), and IL-10 (2.4
pg/ml) (all from Central Laboratory of The Netherlands Red Cross Blood
Transfusion Service, Amsterdam, The Netherlands), and IL-1R antagonist
(IL-1ra; 410 pg/ml; R&D Systems, Minneapolis, MN).
Elastase-1-antitrypsin complexes in EDTA
plasma were measured with an ELISA modified from a previously described
RIA procedure (17). Briefly, ELISA plates (Maxisorp; Nunc,
Roskilde, Denmark) were coated with polyclonal rabbit Abs against human
elastase and incubated with the samples to be tested. Bound complexes
were detected by incubation with biotinylated mAbs against complexed
1-antitrypsin and streptavidin-peroxidase.
Results were referred to a standard curve consisting of pooled human
plasma supplemented with purified elastase and expressed as nanograms
of elastase per milliliter. C-reactive protein (CRP) was measured in
serum by ELISA (detection limit, 3 mg/L) according to the
manufacturer’s instructions (Roche).
FACS analysis
Leukocyte counts and differentials were assessed in EDTA-anti-coagulated blood using a Stekker analyzer (counter STKS; Coulter, Bedfordshire, U.K.). Expression of CD11b (Mac-1) and L-selectin (CD62L) on circulating granulocytes was determined in heparinized blood obtained at -3, 0, 2, 4, 6, and 24 h relative to LPS injection. All blood samples were placed on ice immediately after blood drawing. After lysis of erythrocytes in isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.4) for 10 min, samples were centrifuged at 400 x g for 5 min. The remaining cells were washed and subsequently kept in PBS containing 0.5% BSA, 1.5 mM sodium azide, and 0.35 mM EDTA at a final concentration of 5 x 106 cells/ml. All procedures were performed at 4°C. The following Abs were used: FITC-labeled mouse anti-human L-selectin (Immunotech, Marseilles, France) and PE-labeled mouse anti-human CD11b (Immunotech). All FACS reagents were used in concentrations recommended by the manufacturer. To correct for nonspecific staining, all analyses were also conducted with the appropriate control Abs (FITC- and PE-labeled murine IgG1 (CLB, Amsterdam, The Netherlands)). At least 10,000 granulocytes were counted in each assay. Mean cell fluorescence of forward and side angle scatter-gated granulocytes was assessed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Data are presented as the difference between mean cell fluorescence intensities of specifically and nonspecifically stained cells.
Statistical analysis
All laboratory-based values are given as the mean ± SEM. Differences in results among the three treatment groups were tested by repeated measurements analysis of variance. A value of p < 0.05 was considered to represent a statistically significant difference.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To assess the effect of BIRB 796 BS on p38 MAPK activity, we
stimulated PBMCs with LPS in the presence of increasing concentrations
of BIRB 796 BS or diluent (Fig. 1
). BIRB
796 BS inhibited LPS-induced phosphorylation of p38 MAPK in a
dose-related fashion.
|
Plasma levels of BIRB 796 BS determined after oral ingestion of
the p38 MAPK inhibitor peaked at 0.5–2 h after LPS injection (low dose
group, 0.74 ± 0.25 µM; high dose group, 7.38 ± 1.64 µM;
i.e., within the same range as the concentrations used in the in vitro
experiment). Although there is abundant evidence that active p38 MAPK
is involved in LPS-induced cytokine production in vitro (13, 18, 19), it is not known whether p38 MAPK has a similar role in
humans in vivo. To investigate the activation of p38 MAPK in human
endotoxemia and the effectiveness of BIRB 796 BS in inhibition of p38
MAPK phosphorylation, we measured p38 MAPK activation using
phosphospecific Abs at various time points before and after LPS
injection in healthy human subjects. As shown in Fig. 2A, administration of LPS
resulted in p38 MAPK activation in subjects who did not receive BIRB
796 BS, peaking at 60 min. Both low dose and high dose BIRB 796 BS
significantly inhibited p38 MAPK activation (both p <
0.05 vs placebo). To further establish the effectiveness of BIRB 796 BS
in inhibiting p38 MAPK enzymatic activity in vivo, a kinase assay was
performed on white blood cell lysates (one example of three is shown in
Fig. 2B). In subjects who did not receive BIRB 796 BS,
enhanced p38 MAPK enzymatic activity was observed 60 min after LPS
administration relative to 0 h. In contrast, treatment with BIRB
796 BS almost completely prevented p38 MAPK activation at 60 min. These
results are, to our knowledge, the first demonstration of p38 MAPK
activation in a human model of inflammation and show that BIRB 796
effectively inhibits p38 MAPK in vivo.
|
LPS injection induced symptoms consisting of fever, chills,
myalgia, headache, nausea, vomiting, abdominal pain, and backache. In
subjects treated with LPS and placebo, mean body temperatures peaked
after 3 h (38.3 ± 0.2°C). Although both doses of BIRB 796
BS tended to reduce the febrile response, this effect did not reach
statistical significance (Fig. 3). BIRB
796 BS treatment attenuated LPS-induced symptoms in both incidence and
severity and delayed the time of maximal presentation (Table I). Apparently, activation of p38 MAPK is
involved in the generation of LPS-induced clinical signs and
symptoms.
|
|
The p38 MAPK positively regulates a number of cytokine genes in
vitro (6, 7). Therefore, the effect of pharmacological p38
MAPK inhibition on cytokine release during endotoxemia in humans was
examined. Administration of LPS to subjects not treated with BIRB 796
BS elicited transient rises in the plasma concentrations of TNF-,
IL-6, IL-10, and IL-1ra (Fig. 4). All
these cytokine responses were strongly inhibited by both doses of BIRB
796 BS (all p < 0.05 vs placebo, except for IL-1ra at
the low dose). In addition, the inhibitory effect of BIRB 796 BS
appeared dose dependent, although the low dose diminished cytokine
release to a statistically significant extent. Thus, p38 MAPK
activation is required for cytokine release during human
inflammation.
|
The p38 MAPK phosphorylation results in activation of several
proinflammatory neutrophil functions in vitro (6, 7).
Therefore, the effect of BIRB 796 BS on neutrophil activation induced
by LPS in vivo was assessed by measuring neutrophil counts, release of
elastase, and expression of CD11b and L-selectin (Fig. 5). LPS injection in subjects treated
with placebo was associated with a biphasic change in neutrophil
numbers in peripheral blood, characterized by initial neutropenia with
a nadir at 1 h, followed by neutrophilia peaking at 8 h. LPS
administration also induced a transient rise in the plasma
concentrations of elastase-1-antitrypsin
complexes, reflecting neutrophil degranulation (17) and an
up-regulation of CD11b at the surface of circulating granulocytes with
a concurrent down-modulation of L-selectin, indicative of cellular
activation (20). Whereas the lower BIRB 796 BS dose tended
to attenuate these LPS-induced neutrophil responses
(p < 0.05 for L-selectin; p 
0.05 for other parameters vs placebo), the higher dose of the p38 MAPK
inhibitor resulted in significant reduction of all parameters of
neutrophil activation (all p < 0.05 vs placebo),
suggesting an essential role for p38 MAPK in mediating leukocyte
responses in vivo.
|
To obtain insight into the role of p38 MAPK on more subacute
consequences of LPS-induced inflammation, CRP concentrations were
measured 3 h before and 24 h after LPS injection (Fig. 6). LPS induced a profound increase in
the serum levels of this acute phase reactant, which was significantly
lower in the group receiving BIRB 796 BS (p <
0.05 for both doses vs placebo).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Intravenous administration of LPS induced activation of p38 MAPK in blood, peaking after 1 h, as demonstrated by two different methods, Western blotting with Abs against phosphorylated (Thr180/Tyr182) p38 MAPK and a kinase assay. Using similar methods it has been previously demonstrated that there is transient activation of p38 MAPK in splenocytes of mice peaking within 15 min of i.p. injection of LPS (12). The later activation of p38 MAPK in the human model compared with the mouse may be related to differences in the route of LPS administration, the LPS dose administered, and the cell types analyzed.
The p38 MAPK activation was measured in lysates of all white blood cells obtained from whole blood, rather than in isolated cell fractions, because we argued that the isolation procedures might affect the activation status of p38 MAPK. Several papers have described the in vitro effects of LPS on different leukocyte cell fractions without showing apparent effects of the different separation methods on basal p38 MAPK activation. However, in contrast to these in vitro studies, in vivo endotoxemia studies do not allow cell separation before exposure to LPS. Furthermore, in our experience more elaborate sample handling can lead to activation of kinases (data not shown). Therefore, to accurately assess the activation state of p38 MAPK at several time points during human endotoxemia, we felt it warranted to process the in vivo stimulated whole blood samples quickly and with minimal intervention. Unfortunately, no information concerning the relative contributions of the several leukocyte subsets to the beneficial effect of MAPK inhibition can be gathered in this fashion. In the future, intracellular FACS analysis of MAPK activation, a method that is currently being evaluated in our laboratory, might solve this problem.
BIRB 796 BS, given orally at either 50 or 600 mg, strongly reduced p38
MAPK activation in vivo (Fig. 2
). Both BIRB 796 BS doses attenuated
LPS-induced inflammatory responses, although the higher dose appeared
to exert stronger inhibitory effects. Of note, inflammatory effects
produced by LPS were not abrogated completely. BIRB 796 BS at the doses
used in this study may not completely inhibit p38 MAPK activity, either
in the blood or in immunocompetent cells not present in the circulation
(endothelial cells, fibroblasts, and tissue macrophages). In addition,
p38 MAPK-independent pathways of inflammation may contribute to some of
the effects seen.
Inhibition of p38 MAPK was associated with a profound reduction in the
release of both pro- and anti-inflammatory members of the cytokine
network. The strong reduction in LPS-induced TNF- release with the
p38 MAPK inhibitor BIRB 796 BS corresponds to the in vitro effect of
p38 MAPK inhibition in stimulated monocytic cells and neutrophils
(4, 10, 13, 19, 21, 22). In addition, p38 MAPK inhibitors
diminished LPS-induced TNF- production in mice and rats in vivo
(9, 10, 11), and mice deficient in MAPK-activated protein
kinase-2, a downstream substrate kinase for p38 MAPK, proved to be
resistant to LPS-induced shock (23). A previous study in
mice was not able to demonstrate an inhibitory effect of the p38 MAPK
inhibitor SB203580 on TNF- release after i.p. administration of LPS
(12). This may be the result not only of species
differences, but also of differences in the mechanism of inhibition of
the two drugs. In vitro data suggest that besides p38 MAPK other stress
signaling pathways, e.g., MAPK family members p42/44 and c-Jun
N-terminal kinase and the NF-
B pathway, are involved in LPS-induced
TNF- release (24, 25). Indeed, all three major MAPK
family members and NF-B can be activated upon stimulation with LPS
(26, 27, 28). Furthermore, the TNF- promotor has binding
sites for NF-B as well as for transcription factors under control of
the MAPK family (e.g., AP-1) (26, 29). Thus, full
expression of the TNF gene seems to involve activation of several of
the above-mentioned stress pathways. However, the relative importance
of each of these pathways may vary under different conditions.
Interestingly, deletion of one of the NF-B binding sites from the
TNF promoter had little effect on LPS-induced TNF- production
(29). We observed that inhibition of p38 MAPK decreased
endotoxemia-induced plasma TNF- levels up to 97%. These data
suggest that in the human endotoxemia model the p38 MAPK pathway has
little redundancy with respect to TNF- release. The inhibition of
cytokine release in subjects treated with BIRB 796 BS could have been
related to the reduction in TNF- secondary to p38 MAPK inhibition.
Elimination of endogenous TNF- activity with an anti-TNF Ab or a
TNF receptor fusion protein in the human LPS model was accompanied by a
marked reduction in the release of other cytokines and cytokine
inhibitors, including IL-6, IL-10, and IL-1ra (30, 31, 32, 33). In
addition, anti-TNF administration resulted in reduced IL-10 release
by LPS-stimulated monocytes, whereas p38 MAPK inhibition attenuated
both TNF- and IL-10 production in this in vitro system
(22). Thus, together with our in vivo findings these data
indicate that p38 MAPK is involved in the production not only of
proinflammatory cytokines, but also of anti-inflammatory cytokines.
Yet the subjects treated with BIRB 796 BS demonstrated evidence of an
overall anti-inflammatory effect, suggesting that p38 MAPK
inhibition predominantly influences proinflammatory pathways.
The p38 MAPK is considered important for many different proinflammatory neutrophil functions (13, 21, 34, 35, 36, 37, 38, 39). Although the anti-inflammatory effects of p38 MAPK inhibitors on neutrophils are well known in vitro, little is known about the in vivo relevance of these findings. Inhibition of p38 MAPK has been reported to reduce neutrophil influx into bronchoalveolar lavage fluid after intratracheal administration of LPS in mice (13). Furthermore, local application of a p38 MAPK inhibitor in the lumen of an ileal loop before administration of Clostridium difficile toxin A at the same location has been associated with a strong reduction in both neutrophil recruitment and the severity of the resulting enteritis in mice (40). This study demonstrates that p38 MAPK inhibition attenuates the neutrophil response to i.v. LPS in humans in vivo and reduces the activation of neutrophils, as indicated by inhibition of degranulation, up-regulation of CD11b, and down-modulation of L-selectin. These data correspond with previous in vitro reports demonstrating that p38 MAPK inhibition reduces neutrophil degranulation (36, 37), the shedding of L-selectin (35), and the up-regulation of CD11b (37, 38). BIRB 796 BS also reduced the incidence and severity of clinical symptoms and delayed the time point of maximal presentation, an effect that probably would have been more clear-cut if more subjects had been studied.
Intravenous injection of LPS induces a reproducible transient inflammatory state in normal subjects that is considered relevant for the investigation of pathophysiologic pathways operative in inflammatory conditions. As such, this model of inflammation in man offers an opportunity to obtain proof of the principle for the action of anti-inflammatory compounds. It should be noted that the human endotoxemia model is less suitable to investigate the efficacy of postponed treatment with an anti-inflammatory compound, because the inflammatory response to i.v. LPS is very rapid and transient. Nonetheless, the current findings establish that inhibition of p38 MAPK by the oral administration of BIRB 796 BS exerts anti-inflammatory effects during experimental endotoxemia. These effects are comparable to those seen in this model with anti-TNF Abs, TNFR fusion protein, and IL-10 (30, 33, 41, 42) drugs presently used clinically or in trials in the management of chronic inflammatory diseases such as rheumatoid arthritis and Crohn’s disease (43, 44, 45, 46). Clearly, the greatest advantages of compounds such as BIRB 796 BS would be their oral availability and lack of immunogenicity compared with the biological products. Taken together with (limited) animal data, these results provide hope for the future use of oral p38 MAPK inhibitors in patients with inflammatory diseases.
|
Footnotes |
|---|
2 J.B. and B.v.d.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Judith Branger, Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Room G2-105, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail address: j.branger{at}amc.uva.nl
4 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ATF-2, activating transcription factor-2; CRP, C-reactive protein; IL-1ra, IL-1R antagonist.
Received for publication October 30, 2001. Accepted for publication February 15, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1-antitrypsin and lactoferrin in sepsis: evidence for neutrophils as mediators in fatal sepsis. J. Lab. Clin. Med. 119:159.[Medline]
gene expression in endotoxin-stimulated primary glial cultures. J. Neurosci. 18:1633. mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils. J. Clin. Invest. 103:851.[Medline]
: role of the p38 and p42/44 mitogen-activated protein kinases. J. Immunol. 60:920.
biosynthesis. Nat. Cell Biol. 1:94.[Medline]
production by human monocytes involves the raf-1/MEK1-MEK2/ERK1-ERK2 pathway. Infect. Immun. 67:3824.B pathway by inflammatory stimuli in human neutrophils. Blood 9:3421.
gene regulation by virus and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 87:9769. or FMLP stimulation. J. Immunol. 60:1982.
2-integrin upregulation is blocked by a p38 MAP kinase inhibitor. Biochem. Biophys. Res. Commun. 270:858.[Medline]
monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial: ATTRACT study group. Lancet 354:1932.[Medline]
for Crohn’s disease: Crohn’s disease cA2 study group. N. Engl. J. Med. 337:1029.This article has been cited by other articles:
|
|
 |
|
  T. Thalhamer, M. A. McGrath, and M. M. Harnett MAPKs and their relevance to arthritis and inflammation Rheumatology, April 1, 2008; 47(4): 409 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-T. Jia, W. Wei, B. Ma, Y. Xu, W.-J. Liu, Y. Wang, K.-Y. Lv, H.-T. Tang, D. Wei, and Z.-F. Xia Activation of p38 MAPK by Reactive Oxygen Species Is Essential in a Rat Model of Stress-Induced Gastric Mucosal Injury J. Immunol., December 1, 2007; 179(11): 7808 - 7819. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. O'Keefe, J. S. Mudgett, S. Cupo, J. N. Parsons, N. A. Chartrain, C. Fitzgerald, S.-L. Chen, K. Lowitz, C. Rasa, D. Visco, et al. Chemical Genetics Define the Roles of p38{alpha} and p38 in Acute and Chronic Inflammation J. Biol. Chem., November 30, 2007; 282(48): 34663 - 34671. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. van 't Veer, P. S. van den Pangaart, M. A. D. van Zoelen, M. de Kruif, R. S. Birjmohun, E. S. Stroes, A. F. de Vos, and T. van der Poll Induction of IRAK-M Is Associated with Lipopolysaccharide Tolerance in a Human Endotoxemia Model J. Immunol., November 15, 2007; 179(10): 7110 - 7120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bahador and A. S. Cross Review: From therapy to experimental model: a hundred years of endotoxin administration to human subjects Innate Immunity, October 1, 2007; 13(5): 251 - 279. [Abstract] [PDF]
|
|
|
|
 |
|
 S. Bhattacharyya, D. E. Brown, J. A. Brewer, S. K. Vogt, and L. J. Muglia Macrophage glucocorticoid receptors regulate Toll-like receptor 4-mediated inflammatory responses by selective inhibition of p38 MAP kinase Blood, May 15, 2007; 109(10): 4313 - 4319. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-K. Park, R. Fischer, R. Dechend, E. Shagdarsuren, A. Gapeljuk, M. Wellner, S. Meiners, P. Gratze, N. Al-Saadi, S. Feldt, et al. p38 Mitogen-Activated Protein Kinase Inhibition Ameliorates Angiotensin II-Induced Target Organ Damage Hypertension, March 1, 2007; 49(3): 481 - 489. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Renckens, J. M. Pater, and T. v. d. Poll Plasminogen Activator Inhibitor Type-1-Deficient Mice Have an Enhanced IFN-{gamma} Response to Lipopolysaccharide and Staphylococcal Enterotoxin B J. Immunol., December 1, 2006; 177(11): 8171 - 8176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S E Sweeney and G S Firestein Mitogen activated protein kinase inhibitors: where are we now and where are we going? Ann Rheum Dis, November 1, 2006; 65(suppl_3): iii83 - iii88. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Wood, L. M. Nail, N. A. Perrin, C. R. Elsea, A. Fischer, and B. J. Druker The Cancer Chemotherapy Drug Etoposide (VP-16) Induces Proinflammatory Cytokine Production and Sickness Behavior-like Symptoms in a Mouse Model of Cancer Chemotherapy-Related Symptoms. Biol Res Nurs, October 1, 2006; 8(2): 157 - 169. [Abstract] [PDF]
|
|
|
|
 |
|
 T. Inoue, D. L. Boyle, M. Corr, D. Hammaker, R. J. Davis, R. A. Flavell, and G. S. Firestein Mitogen-activated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis PNAS, April 4, 2006; 103(14): 5484 - 5489. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Bijangi-Vishehsaraei, M. R. Saadatzadeh, A. Werne, K. A. W. McKenzie, R. Kapur, H. Ichijo, and L. S. Haneline Enhanced TNF-{alpha}-induced apoptosis in Fanconi anemia type C-deficient cells is dependent on apoptosis signal-regulating kinase 1 Blood, December 15, 2005; 106(13): 4124 - 4130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Maris, A. F. de Vos, M. C. Dessing, C. A. Spek, R. Lutter, H. M. Jansen, J. S. van der Zee, P. Bresser, and T. van der Poll Antiinflammatory Effects of Salmeterol after Inhalation of Lipopolysaccharide by Healthy Volunteers Am. J. Respir. Crit. Care Med., October 1, 2005; 172(7): 878 - 884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Pruett, Q. Zheng, C. Schwab, and R. Fan Sodium Methyldithiocarbamate Inhibits MAP Kinase Activation through Toll-like Receptor 4, Alters Cytokine Production by Mouse Peritoneal Macrophages, and Suppresses Innate Immunity Toxicol. Sci., September 1, 2005; 87(1): 75 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kuma, G. Sabio, J. Bain, N. Shpiro, R. Marquez, and A. Cuenda BIRB796 Inhibits All p38 MAPK Isoforms in Vitro and in Vivo J. Biol. Chem., May 20, 2005; 280(20): 19472 - 19479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Shiratsuchi and M. D. Basson Activation of p38 MAPK{alpha} by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure Am J Physiol Cell Physiol, May 1, 2005; 288(5): C1083 - C1093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. E. Burdon, C. Martin, and S. M. Rankin The CXC chemokine MIP-2 stimulates neutrophil mobilization from the rat bone marrow in a CD49d-dependent manner Blood, March 15, 2005; 105(6): 2543 - 2548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J Westra, P C Limburg, P de Boer, and M H van Rijswijk Effects of RWJ 67657, a p38 mitogen activated protein kinase (MAPK) inhibitor, on the production of inflammatory mediators by rheumatoid synovial fibroblasts Ann Rheum Dis, November 1, 2004; 63(11): 1453 - 1459. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Schabbauer, M. Tencati, B. Pedersen, R. Pawlinski, and N. Mackman PI3K-Akt Pathway Suppresses Coagulation and Inflammation in Endotoxemic Mice Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1963 - 1969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
R. Renckens, S. Weijer, A. F. de Vos, J. M. Pater, J. C. Meijers, C. E. Hack, M. Levi, and T. van der Poll Inhibition of Plasmin Activity by Tranexamic Acid Does Not Influence Inflammatory Pathways During Human Endotoxemia Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 483 - 488. [Abstract] [Full Text]
|
|
|
|
|
|
J. Branger, B. van den Blink, S. Weijer, A. Gupta, S. J.H. van Deventer, C. E. Hack, M. P. Peppelenbosch, and T. van der Poll Inhibition of coagulation, fibrinolysis, and endothelial cell activation by a p38 mitogen-activated protein kinase inhibitor during human endotoxemia Blood, June 1, 2003; 101(11): 4446 - 4448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. C. Aird The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome Blood, May 15, 2003; 101(10): 3765 - 3777. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 50 mg BIRB 796 BS (
), or 600 mg
BIRB 796 BS (
). Venous blood was obtained at the indicated time
points, erythrocytes were lysed, and the total white blood cell
fraction was lysed and assayed for p38 MAPK phosphorylation and
enzymatic activity. A, p38 MAPK phosphorylation in
leukocytes. Results (mean ± SEM) were obtained by Western
blotting with phosphospecific Abs and expressed relative to a random
sample, which was set on 100 arbitrary units. B,
Inhibition of p38 MAPK enzymatic activity by BIRB 796 BS. p38 MAPK
enzymatic activity was determined by measuring phosphorylation of ATF-2
(pATF-2) in an in vitro kinase assay using immunoprecipitated
phosphorylated p38 MAPK from leukocyte lysates. Results were analyzed
using image analysis software and are represented as bars (arbitrary
units).
