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Departments of
*
Medicine and
Pediatrics, and
Program in Molecular Signal Transduction, National Jewish Medical and Research Center, Denver, CO 80206; and
§
Division of Pulmonary Science and Critical Care Medicine, University of Colorado School of Medicine, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and macrophage
inflammatory protein-2 (MIP-2) release, and eliminated migration of
murine neutrophils toward the chemokines MIP-2 and KC. In contrast,
alveolar macrophages required a 1000-fold greater concentration of M39
to block release of TNF- and MIP-2. Systemic inhibition of p38 MAPK
resulted in significant decreases in the release of TNF- and
neutrophil accumulation in the airspaces following intratracheal
administration of LPS. Recovery of MIP-2 and KC from the airspaces was
not affected by inhibition of p38 MAPK, and accumulation of mononuclear
cells was not significantly reduced. When KC was instilled as a
proinflammatory stimulus, neutrophil accumulation was significantly
decreased by p38 MAPK inhibition independent of TNF- or LPS.
Together, these results demonstrate a much greater dependence on the
p38 MAPK cascade in the neutrophil when compared with other leukocytes,
and suggest a means of selectively studying and potentially modulating
early inflammation in the lung. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of particular interest is the ability of LPS to induce lung
inflammation, as local or systemic endotoxin release is an important
feature of many diseases, including focal pneumonias, cystic fibrosis,
and the acute respiratory distress syndrome. LPS is not an effective
chemoattractant for neutrophils, but can trigger an inflammatory
cascade via the synthesis of cytokines and other proinflammatory
mediators by resident alveolar macrophages
(AM),3 local mast
cells, fibroblasts, epithelia, and endothelial cells. The release of
TNF- and neutrophil-directed chemokines such as IL-8 are essential
to early LPS-mediated neutrophil recruitment.
The combined effects of TNF- and IL-8 on neutrophil recruitment are
complex and incompletely understood. Known roles of TNF- include
activation of endothelial cells to express adherence proteins,
induction of an array of secondary inflammatory mediators, and
"priming" of neutrophils for enhanced phagocytic and bactericidal
activity (7). Through studies with specific Abs and
genetically modified mice, the requirement for TNF- in the
pathogenesis of LPS-induced shock and tissue injury has been confirmed.
However, these techniques do not allow for selective reduction of the
release of TNF- by a cell type, nor modulation of the ability of
neutrophils to respond to TNF-. As a single agent, TNF- also does
not induce chemotaxis of neutrophils. However, the ELR(+)CXC chemokine
IL-8 is one of the most specific neutrophil chemoattractants yet
described (8, 9, 10). IL-8 has not been identified in mice,
but macrophage inflammatory protein-2 (MIP-2) and KC share the same
ELR(+)CXC structure and act as functional homologue of human IL-8
(11, 12).
Selective responses of cells to external stimuli may be understood
through differential activation of intracellular signaling mechanisms.
The mitogen-activated protein kinase (MAPK) superfamily are highly
conserved signaling kinases that regulate cell growth, differentiation,
and stress responses (13). At least three distinct
families of MAPKs exist in mammalian cells: the p42/44 extracellular
signal-regulated kinase (ERK) MAPKs, c-Jun
NH2-terminal kinases (JNKs), and p38 MAPK
(14, 15, 16). Both the coordinated release of cytokines by
host defense cells and the functional response of neutrophils to
cytokines and other proinflammatory agents are to varying degrees
regulated by p38 MAPK. In the neutrophil, p38 MAPK is activated in
response to many stimuli, including LPS and TNF- (17, 18). Once activated, p38 MAPK is capable of modulating
functional responses through phosphorylation of transcription factors
and activation of other kinases. In LPS-stimulated neutrophils, p38
MAPK regulates distinctly different functions, including adhesion,
activation of NF-
B, and the synthesis of TNF- and IL-8
(19, 20, 21). In neutrophils stimulated with the
chemoattractant FMLP, activation of p38 MAPK regulates both
superoxide anion release and chemotaxis (19, 20). In the
LPS-stimulated monocyte/macrophage, inhibition of p38 MAPK blocks
TNF- and IL-8 release (16, 22). In cells other than
leukocytes, p38 MAPK also regulates stress-responses, including release
of IL-8 by bronchial epithelial cells in response to TNF- or other
inflammatory stimuli (23). LPS stimulation also causes
activation of p38 MAPK in endothelial cells, resulting in up-regulation
of the ICAM-1 adhesion molecule (24).
Given this central role of p38 MAPK as a regulator of multiple
inflammatory responses in many diverse cell types, we questioned the
effect of in vivo p38 MAPK inhibition on neutrophil accumulation in the
lung. For these studies, we employed a murine model of mild pulmonary
inflammation in response to a single administration of LPS in the
airspace. Inhibition of p38 MAPK was accomplished with the novel
compound M39, which is the most highly selective and potent inhibitor
of p38 MAPK described to date (25). We studied the effects
of selective inhibition of p38 MAPK on several events critical in the
pathogenesis of the early inflammatory response of the murine lung.
Herein we report that in vitro inhibition of p38 MAPK resulted in a
significant decrease in murine neutrophil function, but a limited
effect on other inflammatory responses. In vivo, this resulted in the
loss of initial neutrophil recruitment to the airspaces, while later
accumulation of mononuclear cells remained largely intact. Together,
these data indicate the potential for relatively selective in vivo
inhibition of neutrophilic responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endotoxin-free reagents and plastics were used in all
experiments. Aprotinin, leupeptin, Tris-HCl, Triton X-100, igepal,
PMSF, EDTA, EGTA, Nonidet P-40, and protein A-Sepharose were purchased
from Sigma (St. Louis, MO), and [
-32P]ATP
was purchased from Amersham (Arlington Heights, IL). M39
[(S)-5-[2-(1-phenylethylamino)pyrimidin-4-yl]-1-methyl-4-(3-trifluoromethylphenyl)-2-(4-piperidinyl)
imidazole] was provided by Merck (Rahway, NJ) and stored in DMSO at
-20°C. LPS was purified from Escherichia coli 0111:B4
(List Biological Laboratories, Campbell, CA). Recombinant KC and MIP-2
were purchased from R&D Systems (Minneapolis, MN). Activated
transcription factor (ATF)-21–110 was prepared
as previously described (17, 19).
Animals
Female C57BL/6 mice (Harlan Sprague Dawley, Indianapolis, IN), 6–12 wk of age and weighing 16–20 g, were used in all experiments. They were given commercial pellet food and water ad libitum. All experiments were performed in accordance with the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals after review of the protocol by the Animal Care and Use Committee of the National Jewish Medical and Research Center. Anesthesia was provided by a single i.p. injection of 333 mg/kg avertin. Avertin was prepared by mixing 10 g tribromoethyl alcohol (Aldrich, Milwaukee, WI) with 10 ml teriary amyl alcohol (Aldrich) and diluting this stock to a 2.5% solution in sterile saline.
Murine bronchial alveolar lavage (BAL)
BAL was performed immediately following sacrifice of the animals by pentobarbital overdose or cervical dislocation. The procedure was performed with the lungs in situ but with the chest cavity opened by midline incision. The trachea was intubated orally or directly through a small cut-down of the skin overlying the trachea with a 20-g angiocath (Baxter Quik-Cath, Baxter Health Care, Deerfield, IL). Two to four 0.8-ml aliquots of saline with 20 U/ml heparin were instilled and sequentially removed by gentle hand suction with a 1-ml syringe. The volume of BAL recovered was quantified and cells recovered were counted in a hemocytometer. Cell types were determined by Wright staining of a spun sample. All slides were counted twice by different observers blinded to the status of the animal. Samples for cytokine analysis were immediately frozen in a dry ice/ethanol bath and stored at -70°C.
Isolation of cells
Human neutrophils were isolated by the plasma Percoll method
(26) and suspended in Krebs-Ringer phosphate buffer with
0.2% dextrose at pH 7.2 or in RPMI 1640 culture medium (BioWhittaker,
Walkersville, MD). Mature murine bone marrow neutrophils were isolated
from mouse femurs and tibias. Animals were sacrificed by cervical
dislocation, and the bones were dissected. Both ends of each bone were
removed, and a 25-gauge needle on a 5-ml syringe containing HBSS
(without calcium, magnesium, bicarbonate, or phenol red) was employed
to express marrow from the bones. Marrow cords were collected in a
50-ml polypropylene conical tube (Becton Dickinson, Bedford, MA) and
subsequently resuspended by gentle aspiration of the suspension through
a 19-gauge needle. The marrow cells were pelleted by centrifugation at
112 x g for 6 min, washed once with HBSS, and
resuspended in HBSS to a final volume of 2 ml in preparation for
density gradient centrifugation. A stock solution of Percoll (100%
fine grade; Pharmacia Fine Chemicals, Piscataway, NJ) was prepared in
10x HBSS in a ratio of 9:1 (v/v) Percoll:10x HBSS. A 3 x 2-ml
Percoll discontinuous density gradient (72, 64, and 52% with 1x HBSS)
was prepared in a 15-ml polypropylene conical tube (Becton Dickinson).
The marrow suspension was layered on top of the Percoll gradient and
centrifuged at 1060 x g for 30 min. Morphologically
mature appearing neutrophils at a concentration of >95% formed a band
at the interface of the 64 and 72% Percoll layers. This band was
carefully aspirated and mixed with 12 ml of 1x HBSS in a 15-ml conical
tube, centrifuged at 112 x g for 6 min, washed twice
with 1x HBSS, and resuspended in 1x HBSS to a volume of 2 ml and
counted by hemocytometer. Typical yields were 
1–2 x
107 mature bone marrow neutrophils per mouse. In
separate studies, the marrow neutrophils were shown to have equivalent
functional responses and recirculation patterns when compared with
peripheral murine neutrophils (B.T.S., unpublished observations).
Murine peripheral blood neutrophils were isolated by modification of
methods previously reported (27) for the purification of
rabbit peripheral neutrophils. Mice were volume expanded and
exsanguinated into a 3.8% citrate solution followed by centrifugation
at 300 x g for 20 min. The cell pellet was resuspended
in 6% dextran and 0.9% NaCl solution (in a ratio of 1:5.25,
dextran:saline) to a final volume of 150% the original blood volume
and sedimented at unity gravity for 30 min. The leukocyte-rich
supernatant was aspirated, washed once in HBSS, layered over a Percoll
gradient (78, 66, and 54%) and centrifuged at 1060 x
g for 30 min. Cytospun samples of the dense band revealed
>90% neutrophils. Following lysis with hypotonic saline, typical
yields were 2–4 x 105 peripheral blood
neutrophils per mouse. Trypan blue dye exclusion showed the cells to be
>97% viable following purification. Murine AM were isolated by two
sequential BALs. Typical yields were 2 x
105 cells per mouse and were 97–99% AM as
assessed by Wright staining of spun samples.
Neutrophil functional assays
All experiments were done in the presence of 1% human or murine
heat-inactivated platelet-poor plasma. Cytokine release assays were
performed with murine neutrophils isolated from peripheral blood or
murine AM resuspended in RPMI 1640 containing 2% murine
heat-inactivated platelet-poor plasma at a concentration of 5 x
106 cells/ml. One milliliter of cells suspension
was added per well of a 12-well flat-bottom tissue culture-treated
polystyrene plate (Costar, Corning, NY). Cells were allowed to settle
without agitation for 60 min at 37°C (in the presence or absence of
the p38 MAPK inhibitor), followed by addition of stimuli for the
designated periods. At the end of the stimulation, the supernatant was
removed for quantification of KC, MIP-2, or TNF- by immunoassay (R&D
Systems). In vitro inhibition of p38 MAPK was performed by incubation
of neutrophils or macrophages with M39 over a range of concentrations
for 60 min at 37°C. Collagen gel migration assays were performed as
previously described (27) with minor modifications.
Throughout all migration assays, Krebs-Ringer-phosphate dextrose with
0.25% human serum albumin was used as the buffer. Between 2 and 5
x 106 neutrophils were loaded per gel for each
condition studied. Gels were inverted onto mounting media containing
0.1% p-phenylenediamine and 70% glycerol with 1 drop of
defined diameter fluorescent beads (DNA-Check; Coulter, Hialeati, FL)
to establish the scale. All gels were examined with a x40 dry
objective and numerical aperture 0.55. Diagrams of cells in each gel
section were made at 5-µm intervals, and the number of cells at each
depth were recorded. A minimum of three gel sections were examined for
each condition, and averages were calculated for each depth. Values for
cell distribution at each depth were expressed as a percent of total
cells observed in the entire vertical section.
P38 MAPK immunoprecipitation assays
Kinase activity of p38 MAPK was assayed from
immunoprecipitated samples by the ability to phosphorylate
ATF-21–110 as previously described
(19).
Intratracheal instillation of proinflammatory stimuli
Following anesthesia with avertin, a 300-ng aliquot of LPS or 1 µg KC dissolved in 50 µl saline containing 0.1% human serum albumin was injected into the mouse airways by passing a 22-gauge bent feeding needle with a 1.25-mm ball diameter (Popper & Sons, New Hyde Park, NY) through the oropharynx into the trachea.
In vivo inhibition of p38 MAPK
Anesthetized mice were administered M39 by gastric intubation of a 22-gauge straight feeding needle with a 2.25-mm ball diameter (Popper & Sons). Fasting mice were placed in a semiupright position, and M39 suspended in 100 µl hydroxypropylmethylcellulose (Abbott Laboratories, Abbott Park, IL) was instilled at a dose of 3 mg/kg. The M39 was administered 2 h before and 12 h following intratracheal instillation of KC or LPS, except for time points earlier than 12 h, in which a single dose of M39 was administered.
Histological examination and quantification of neutrophil accumulation of murine lung tissue
Animals were treated with saline or LPS in the presence or absence of M39. At 24 h, the animals were sacrificed by pentobarbital overdose and a midline incision was performed. A 20-g catheter (Baxter Health Care) was inserted into the trachea and secured by tying with 2–0 silk followed by careful dissection to remove the lungs from the thoracic cavity. After full inflation with air to 25 cm water pressure, the trachea was tied and the lungs submerged in 1.5% glutaraldehyde solution in sodium cacodylate buffer for 24 h. Sections (4 µm) taken across the entire lung were embedded in paraffin. Sagittal sections were stained with hematoxylin-eosin and examined by light microscopy. Between two and four animals were studied for each condition, and representative sections of lungs were chosen by two independent observers blinded to the treatment status of the animals. Photomicrographs were taken at x400 magnification. Quantification of neutrophil accumulation in the whole lung excluding the airspaces was performed by myeloperoxidase (MPO) assay as previously described (28) with minor modifications. Following BAL, isolated whole lungs were frozen in liquid nitrogen, weighed, and then homogenized. Following centrifugation at 20,000 x g for 30 min, the insoluble pellet was resuspended in 50 mM potassium phosphate buffer, pH 6.0, with 0.5% hexadecyltrimethylammonium bromide. Samples were sonicated, incubated at 60°C for 2 h, and assayed for activity in a hydrogen peroxide/o-dianisidine buffer at 460 nm. Results are expressed as units of MPO activity per gram of lung tissue.
Statistical analysis
Data were analyzed using JMP statistical software (SAS
Institute, Cary, NC). Student’s unpaired t test
(two-tailed) was use to determine significance of p38 MAPK inhibition
(Fig. 1
) and neutrophil accumulation and MPO content (Fig. 8) for a
single time point. Differences in chemotaxis (Fig. 2) were analyzed by
a 
2 test. One-way ANOVA was used to analyze
the effect of LPS-induced cytokine release and leukocyte accumulation
over time (Figs. 3 and 4). Differences in in vivo cell accumulation and
cytokine release over time in the presence and absence of p38 MAPK
inhibition (Figs. 5, 6, and 9) were analyzed by two-way ANOVA. When a
significant interaction between inhibition and time existed, the effect
of inhibition was analyzed separately for each time point. For all
tests, p < 0.01 was considered significant unless
otherwise indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MAPK activation in murine neutrophils
Previous reports have demonstrated phosphorylation and activation
of p38 MAPK in human neutrophils following stimulation with LPS
(17, 18). To determine whether activation of p38 MAPK
occurs in murine neutrophils in response to LPS, and assess the ability
of M39 to inhibit p38 MAPK activity, murine bone marrow neutrophils
were stimulated with LPS in the presence and absence of M39. Activity
and phosphorylation of p38 MAPK were assessed simultaneously by
immunoprecipitation of the kinase from neutrophil lysates stimulated
with LPS or left unstimulated. The p38 MAPK was resolved by
SDS-PAGE, and the Western blot was stained with an Ab capable of
detecting tyrosine phosphorylation of p38 MAPK (Fig. 1
A). For comparison, an equal
number of human neutrophils were stimulated and analyzed in the
identical manner (Fig. 1A). The blot was then reprobed with
a second Ab against p38 MAPK, confirming that equivalent amounts of
kinase were immunoprecipitated for each condition (data not shown).
Activity of p38 MAPK was determined by combining immunoprecipitated
p38 MAPK with ATF-21–110, a known substrate
(29), in the presence of [32P]ATP
(Fig. 1B). LPS stimulation resulted in robust tyrosine
phosphorylation of p38 MAPK in both murine and human neutrophils.
However, p38 MAPK isolated from LPS-stimulated cells treated with
M39 had significantly reduced kinase activity. Inhibition of p38 MAPK
by M39 may result in varying degrees of decreased tyrosine
phosphorylation between different cell types and species (our
unpublished observations). These results demonstrate phosphorylation
and activation of p38 MAPK in the murine neutrophil and the ability
of M39 to inhibit LPS-induced activation of p38 MAPK.
Inhibition of p38 MAPK blocks chemokine-induced chemotaxis of murine neutrophils
Chemotaxis is a complex response involving coordination of
adhesion and actin assembly. We have reported previously that
inhibition of p38 MAPK results in loss of chemotaxis response by human
neutrophils to FMLP (19). We tested the effect of p38 MAPK
inhibition on migration of murine neutrophils toward the
chemoattractants MIP-2 and KC. Neutrophil chemotaxis through a
three-dimensional collagen matrix was quantified by counting the number
of cells within a series of 5-µm intervals after 75 min of exposure
to the chemokines. In the presence of M39, neutrophil chemotaxis to
MIP-2 (Fig. 2A) and KC (Fig. 2B) was blocked.
Effect of p38 MAPK inhibition on cytokine release of LPS-stimulated murine neutrophils and AM
An important role of AM is cytokines release in response to LPS,
thus triggering and coordinating early inflammation. Neutrophils also
have the capability to synthesize and release a limited number of
cytokines (30) and under certain conditions may be
important in perpetuating the inflammatory response. Activation of p38
MAPK has been associated with cytokine production by both
monocytes/macrophages and neutrophils. We tested the effect of p38 MAPK
inhibition with M39 on LPS-induced release of TNF-, MIP-2, and KC
from adherent neutrophils and AM (see Materials and
Methods). An IC50 of M39 inhibition of
TNF- and MIP-2 release by LPS-stimulated neutrophils was achieved
with a concentration of M39 <0.1 nM (Fig. 3A). In contrast, the
IC50 of M39 for LPS-activated AM to achieve
inhibition of TNF- and MIP-2 release was >1000-fold higher (Fig. 3B). Neither LPS-stimulated neutrophils nor AM were found to
release significant quantities of KC under the conditions studied (Fig. 3, A and B). Viability of neutrophils and
macrophages treated with 10 µM M39 ranged from 97 to 99%, equal to
the viability of the untreated cells (data not shown). These results
support the conclusion that in vitro inhibition of p38 MAPK may result
in a relatively greater loss of functional response by the neutrophil
than by the AM.
Characterization of murine pulmonary inflammation in response to intratracheal LPS
To study the role of p38 MAPK activation in the lungs, a model of
mild pulmonary inflammation was developed. Following intratracheal
administration of LPS, leukocytes and selected cytokines were
quantified from BAL samples over a series of time points. A dose of LPS
was selected that would elicit an exuberant neutrophil influx, followed
by a secondary accumulation of mononuclear cells (primarily macrophages
and monocytes), with near complete resolution by 72 h (Fig. 4A). The maximal neutrophil
accumulation in the airspaces occurred at 24 h following LPS
installation (Fig. 4A). In association with administration
of LPS, production of TNF-, MIP-2, and KC peaked within 4
h, returning to baseline by 12 h (Fig. 4B). Cytokine
recovery was negligible by 24 h, the point at which neutrophil
influx was greatest, suggesting that cytokine release by neutrophils is
minimal in this model.
Inhibition of p38 MAPK in vivo results in decreased leukocyte accumulation in the airspaces
To quantify changes in inflammation observed in the setting of in
vivo p38 MAPK inhibition, we conducted BAL studies over 72 h
following administration of LPS. Numbers of neutrophils and mononuclear
cells recovered by BAL in mice following intratracheal LPS were
counted. Administration of M39 resulted in significant reduction of
neutrophil accumulation from 4 to 24 h (Fig. 5A). By 48 h, neutrophils
were no longer present in the airways, but were replaced by
monocytes/macrophages. When total mononuclear cells were evaluated, the
effect of systemic p38 MAPK inhibition was not statistically
significant (Fig. 5B). Together, these plots support the
conclusion that in vivo inhibition of p38 MAPK result primarily in
reduction of the early neutrophil accumulation, with little effect on
the later recruitment of monocytes/macrophages.
Inhibition of p38 MAPK in vivo results in decreased TNF- release
in the airspaces
BAL studies of mice 0–72 h following administration of LPS were
analyzed for TNF-, MIP-2, and KC. Only TNF- was found to be
significantly reduced by in vivo inhibition of p38 MAPK with M39 (Fig. 6A), with no detectable change
in the release of MIP-2 (Fig. 5B) or KC (Fig. 5C). This data suggests that systemic inhibition of p38 MAPK
can have divergent effects on cytokine release, and that release of the
KC and MIP-2 chemokines by resident pulmonary immune cells in the mouse
is relatively less dependent on p38 MAPK signaling than TNF- under
the conditions studied.
Inhibition of p38 MAPK in vivo results in diminished histological evidence of pulmonary inflammation
The effect of in vivo p38 MAPK inhibition on the histological
changes of mild LPS-induced pulmonary inflammation were evaluated.
Animals were administered LPS intratracheally in the presence and
absence of M39and compared with saline-treated controls. After 24
h, LPS-treated mice (Fig. 7B)
demonstrated a significant interstitial and intraalveolar accumulation
of leukocytes and edema when compared with saline treated animals (Fig. 7A). In the presence of 38 MAPK inhibition, inflammatory
changes were evident, but to a lesser extent (Fig. 7C).
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Decreased neutrophil accumulation in the airspaces in response to
LPS following inhibition of p38 MAPK could possibly be due to decreased
retention of neutrophils in the pulmonary vasculature or lung
interstitum, or by loss of the ability of the cells to migrate into the
alveoli. The MPO assay was used to quantify the neutrophil burden in
the pulmonary vasculature and interstitium. Animals were administered
LPS intratracheally in the presence and absence of M39 and compared
with saline-treated controls at 24 h, identical with the
conditions depicted in Fig. 7, A–C. Neutrophil accumulation
in the airspaces was determined by BAL (Fig. 8A), and following BAL the
isolated lungs were subjected to the MPO assay (Fig. 8B).
Although significant reduction in neutrophil accumulation is observed
in the setting of in vivo p38 MAPK inhibition (Fig. 8A), the
amount of neutrophils present in the isolated lungs with or without p38
MAPK inhibition are equivalent (Fig. 8B). These results
support the conclusion that systemic inhibition of p38 MAPK results in
a loss of migration of neutrophils into the airways, consistent with
the effects of in vitro p38 MAPK inhibition on neutrophil chemotaxis
(Fig. 2).
Decreased neutrophil accumulation by inhibition of p38 MAPK in vivo occurs due to reduced neutrophil chemotaxic response
Observed decreases in pulmonary inflammation following systemic
inhibition of p38 MAPK might occur due to diminished response of
neutrophils to LPS, as a result of decreased TNF- release (Fig. 6A) or as a selective inhibition of neutrophil chemotaxis
(Fig. 2B). To evaluate the effect of systemic p38 MAPK
inhibition on neutrophil chemotaxis independent of LPS and TNF-, a
model using KC-induced pulmonary inflammation was studied. KC is a
potent and selective chemoattractant for murine neutrophils that
triggers little of the inflammatory cascade. KC was administered
intratracheally in mice in the presence and absence of M39, and BAL
studies were performed from 0 to 48 h. Under the conditions
studied, KC induced a rapid and self-limited migration of neutrophils
that was significantly decreased by in vivo p38 MAPK inhibition (Fig. 9A). Unlike LPS, a substantial
later accumulation of mononuclear cells did not occur in response to KC
(Fig. 9B). BAL analysis demonstrated no measurable release
of MIP-2 and TNF- and levels of KC within the airspaces decreased
rapidly following administration (data not shown). These results
suggest that decreases in LPS-induced neutrophil accumulation following
systemic inhibition of p38 MAPK can occur due to reduced neutrophil
response, independent of TNF- production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Through in vitro studies of various cell lines and primary cells, p38
MAPK has been linked to a variety of inflammatory responses. With the
recent development of potent and specific inhibitors, the role of p38
MAPK in both in vitro systems and complex in vivo models of
inflammation can be studied. Murine neutrophils were found to have
nearly identical activation of p38 MAPK in response to stimulation
by LPS with what has previously been reported in human neutrophils.
Treatment of murine neutrophils with the novel p38 MAPK inhibitor M39
resulted in significant inhibition of p38 MAPK activity. Important
functional effects of p38 MAPK inhibition in the murine neutrophil were
the loss of chemotaxis toward MIP-2 and KC and the loss of TNF- and
MIP-2 release in response to LPS. Unexpectedly, parallel studies of
murine AM demonstrated a 1000-fold greater concentration of M39 is
required to block release of TNF-, MIP-2, or KC. The greater
sensitivity of neutrophils to inhibition of the p38 MAPK cascade was
also observed in vivo. In response to intratracheal administration of
LPS, the influx of neutrophils, but not mononuclear cells, was
significantly decreased in the setting of systemic p38 MAPK inhibition.
Quantification of the neutrophil accumulation in the whole lung
demonstrated that under the conditions studied only the airspaces have
a reduction of the influx of neutrophils, supporting the in vitro
analysis of the dependence of neutrophil chemotaxis on p38 MAPK
activation. Recovery of TNF- in the airspaces was reduced through
p38 MAPK inhibition, but quantities of MIP-2 and KC were not affected.
When KC was used as a primary neutrophil chemoattractant, secondary
release of TNF- and MIP-2 was not evoked, but neutrophil influx was
significantly blocked by systemic p38 MAPK inhibition.
Although the MAPK cascades are highly conserved, it is now understood
that specific utilization of the MAPK cascades differs between
neutrophils, macrophages, and other cells. In monocytes or macrophage
cell lines, LPS has been reported to activate p42/44 (ERK) MAPK and JNK
as well as the p38 MAPK cascade (31, 32, 33). Release of
TNF- by monocytes or macrophage cell lines can be blocked through
selective inhibition of either the JNK (32), p38 MAPK
(16), or the p42/44 (ERK) MAPK cascade (34).
Disruption of a component of the p38 MAPK cascade in
MKK3-/- mice failed to reduce TNF-
release by peritoneal macrophages in response to LPS (35).
In T cells, inhibition of p38 MAPK has less of an effect on TNF-
release than does inhibition of the p42/44 (ERK) cascade
(36). In contrast, LPS stimulation of neutrophils does not
result in activation of the p42/44 (ERK) MAPKs or the JNKs (17, 18, 37). As a short-lived, terminally differentiated primary
cell, the neutrophil possesses a more limited synthetic capability and,
in response to LPS, uses fewer of the available intracellular signal
transduction mechanisms. Thus, the selective loss of neutrophil
function in the setting of systemic p38 MAPK inhibition suggests that
neutrophils are relatively more dependent on signal transduction via
the p38 MAPK cascade than AM.
Although considerable recent interest has been focused on the
activation and function of p38 MAPK, there are few reports of in vivo
inhibition of this signaling pathway. The pyridinyl imadazole
compounds, including SB203580 and SK&F86002, are the most widely
studied p38 MAPK inhibitors and have been shown to possess
antiinflammatory properties in animal models. These early p38 MAPK
inhibitors were shown to reduce neutrophil influx in response to
monosodium urate- or carrageenan-induced peritonitis (38)
and collagen-induced arthritis (39, 40). In response to
i.p. injection of LPS, administration of these compounds resulted in
decreased recovery of TNF- by peritoneal washout (41)
and decreased serum TNF- and mortality in a murine model of
endotoxin shock (40, 42, 43). The antiinflammatory effects
of these p38 MAPK inhibitors occurred in the absence of generalized
immunosupression (40, 41, 44, 45). The effects of systemic
p38 MAPK inhibition on pulmonary inflammation has not been described.
To date, nearly all studies of the functional role of p38 MAPK have
used the compound SB203580, which has an IC50 =
39 ± 11 nM for p38 MAPK as well as considerable inhibitory
effects toward c-Raf (IC50 = 330 ± 155 nM)
and JNK21 (IC50 = 290 ± 110 nM). In
comparison, M39 has an IC50 = 0.11 ± 0.046
nM for p38 MAPK and is significantly less active toward c-Raf
(IC50 = >1000 nM) or JNK21
(IC50 = 675 nM) (25). As a more
potent and selective p38 MAPK inhibitor, M39 is better suited for in
vivo studies than previously available compounds.
In the murine model of mild LPS-induced pulmonary inflammation, the
predominant effect of in vivo p38 MAPK inhibition was to reduce
recruitment of neutrophils. Based on in vitro chemotaxis assays to
MIP-2 and KC, it would appear that this effect can occur as a result of
decreased neutrophil response. BAL measurement of chemokine levels
support this conclusion, as neither KC nor MIP-2 release was decreased
in animals treated with M39. In addition, in vivo inhibition of p38
MAPK blocked neutrophil accumulation in response to intratracheal
administration of KC, independent of LPS or TNF-. Although decreased
TNF- release following LPS stimulation was detected in animals
treated with M39, this effect appears to be of secondary importance
under the conditions studied. The apparently greater dependence of
neutrophils on p38 MAPK signaling when compared with resident cells of
the lung suggests the potential for selective analysis and modulation
of neutrophil influx in pulmonary inflammation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerry A. Nick, National Jewish Medical and Research Center, D403, 1400 Jackson Street, Denver, CO 80206. E-mail address:
3 Abbreviations used in this paper: AM, alveolar macrophages; ATF-2, activated transcription factor-2; BAL, bronchial alveolar lavage; ERK, extracellular signal-regulated kinases; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MIP-2, macrophage inflammatory protein-2; MPO, myeloperoxidase.
Received for publication June 11, 1999. Accepted for publication December 9, 1999.
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in endotoxin-challenged lung tissue. Am. J. Pathol. 143:1009.[Abstract]
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production in a manner distinct from LPS activation of monocytes. J. Immunol. 162:659.This article has been cited by other articles:
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) or left untreated (
) for 60 min at 37°C and
then subjected to an in vitro collagen gel migration assay (see
Materials and Methods) toward MIP-2 (10-7
M) for 75 min at 37°C. Each plot depicts distance of migration in
10-µm intervals, expressed in mean percent of total cells
counted ± SEM from three consecutive experiments, with three
independent readings at each depth. Cells that failed to penetrate the
gel were quantified in the interval labeled "surface." The failure
of neutrophils to migrate beyond the gel surface following inhibition
of p38 MAPK was significant (p < 0.0001) by
) released was quantified. Each plot depicts
mean cytokine release (pg per 106 cells) ± SEM for
each concentration of M39 from three consecutive experiments. The M39
concentration-dependent inhibition of cytokine release is significant
for TNF-
