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*
First Department of Internal Medicine and
Department of Microbiology, Nihon University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mAb, anti-IL-1ß mAb,
anti-TNF- mAb, anti-IL-8 mAb, anti-IFN-ß mAb,
anti-RANTES mAb, and a combination of these mAbs did not affect IV
infection-induced increases in p38 MAP kinase, Erk, and JNK
phosphorylation, indicating that each cytokine neutralized by
corresponding Ab was not involved in IV infection-induced
phosphorylation of MAP kinases. N-acetylcysteine (NAC)
did not affect IV infection-induced increases in MAP kinase
phosphorylation, whereas NAC attenuated RANTES production by 18.2%,
indicating that reactive oxygen species may act as a second messenger
leading to RANTES production via p38 MAP kinase- and JNK-independent
pathway. These results indicate that p38 MAP kinase and JNK, at least
in part, regulate RANTES production by bronchial epithelial
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many extracellular stimuli elicit specific biological responses through activation of mitogen-activated protein (MAP) kinase cascades (9). Mammalian MAP kinase superfamily has been molecularly characterized: extracellular signal-regulated kinase (Erk), p38 MAP kinase, and c-Jun-NH2-terminal kinase (JNK). p38 MAP kinase and JNK are activated by environmental stresses such as hyperosmotic shock, heat shock, cold shock, UV irradiation, and inflammatory cytokines and play an important role in apoptosis and cytokine expression (10, 11, 12, 13, 14, 15), whereas Erk is activated by mitogenic stimuli and plays a central role in cell proliferation and differentiation (16, 17); however, recent studies have suggested that Erk and JNK also play an important role in the signal cascades of induction of various inflammatory mediators including cytokines and chemical mediators (18, 19, 20, 21, 22, 23).
Airway epithelial cells are well known to produce various cytokines that are possibly involved in the production of airway inflammation (24, 25). RANTES that exhibits a chemotactic activity for eosinophils has been shown to play an important role in the production of airway inflammation of asthmatics through the recruitment of eosinophils into the site of airway inflammation (26). It has been shown that IV infection stimulates airway epithelial cells to produce various cytokines including RANTES (6, 7, 8); however, the intracellular signal regulating RANTES production by IV-infected airway epithelial cells has not been determined. Therefore, in the present study we attempted to examine the role of MAP kinase superfamily in RANTES production by IV-infected human bronchial epithelial cells (BEC). To this end, we examined MAP kinase phosphorylation and activity in IV-infected BEC, and the effect of SB 203580 as the specific inhibitor of p38 MAP kinase (27), PD 98059 as the specific inhibitor of MAP/Erk kinase kinase-1 (MEK-1) on Erk activity (28), and CEP-1347 as the specific inhibitor of JNK signaling pathway (29) on RANTES production by IV-infected BEC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IV strain A/Udon/307/72 (H3N2) was grown in Madin-Darby canine kidney cells (American Type Culture Collection, Manassas, VA) in DMEM (Nissui, Tokyo, Japan) and semipurified by two cycles of differential centrifugation from the infected culture supernatants. Virus stock was stored at -80°C.
Cells and reagents
BEC line NCI-H292 was obtained from American Type Culture
Collection. NCI-H292 were grown in culture medium which is RPMI 1640
(Nissui) supplemented with 10% heat-inactivated FCS (Mitsubishikasei,
Tokyo, Japan), streptomycin and penicillin (Meiji Pharmaceutical,
Tokyo, Japan). SB 203580 and PD 98059 were obtained from
Calbiochem-Novabiochem (La Jolla, CA) and New England Biolabs (Beverly,
MA), respectively. CEP-1347 was kindly provided by Cephalon (West
Chester, PA). SB 203580, PD 98059 and CEP-1347 were dissolved in DMSO.
N-acetylcysteine (NAC) was obtained from Sigma (St. Louis,
MO). Anti-human IL-1 mAb, anti-human IL-1ß mAb,
anti-human TNF- mAb, and anti-human IL-8 mAb were obtained
from Genzyme (Cambridge, MA). Polyclonal rabbit anti-human IFN-ß
Ab and anti-human RANTES mAb were obtained from Chemicon (Temecula,
CA) and PeproTech (London, U.K.), respectively.
Cell cultures
The cells were placed onto tissue culture plate (no. 1007,
Falcon, Oxnard, CA) for Western blot analysis and in vitro kinase assay
and 24-well flat-bottom tissue culture plate (Corning, Corning, NY) for
cytokine production, and cultured using culture medium at 37°C in
humidified 5% CO2 atmosphere. When the cells
were grown in subconfluent conditions, the culture medium was replaced
with serum-free RPMI 1640, and the cells were cultured for 16 h.
To examine IV infection-induced Erk, p38 MAP kinase, and JNK
phosphorylation and activity and cytokine production, the cells were
infected with IV at a multiplicity of infection (moi) of 2. The cells
for analysis of p38 MAP kinase, Erk, and JNK phosphorylation were lysed
at the desired times as indicted after IV infection. To examine the
effect of SB 203580, PD 98059 and CEP-1347 on p38 MAP kinase activity,
Erk activity, and JNK activity, respectively, and RANTES production by
IV-infected BEC, the cells that had been incubated with SB 203580, PD
98059, or CEP-1347 or a combination of these inhibitors for 1 h
were infected with IV. The cells were cultured for 6 h for the
analysis of MAP kinase activity and for 24 h for the determination
of cytokine production. At the end of 24-h cultivation, the culture
supernatants were harvested and centrifuged, and the supernatants were
collected, filtrated with a Millipore filter, and stored at -80°C
until assay. To examine the effect of anti-cytokine Ab on IV
infection-induced p38 MAP kinase, Erk and JNK phosphorylation,
anti-IL-1ß mAb, anti-IL-1 mAb, anti-TNF- mAb,
anti-IFN-ß Ab, anti-IL-8 mAb, anti-RANTES mAb, or a
combination of these Abs were added to the cultures. Then the cells
were infected with IV. The cells were cultured for 6 h for
analysis of p38 MAP kinase, Erk, and JNK phosphorylation. To examine
the effect of NAC on IV infection-induced p38 MAP kinase, Erk and JNK
phosphorylation, and RANTES production, the cells that had been
incubated with NAC (10 mM) for 1 h were infected with IV. The
cells were cultured for 6 h for the analysis of MAP kinase
phosphorylation and for 24 h for the determination of cytokine
production.
Western blot analysis of p38 MAP kinase, Erk, and JNK
Analysis of threonine and tyrosine phosphorylation of p38 MAP kinase was performed using an anti-phosphorylated threonine and tyrosine of p38 MAP kinase Ab (anti-phospho-specific p38 MAP kinase Ab, New England Biolabs), which is specific for active p38 MAP kinase and does not cross-react with Erk and JNK. Analysis of threonine and tyrosine phosphorylation of Erk was performed using an anti-phosphorylated threonine and tyrosine of p42/p44 MAP kinase Ab (anti-phospho-specific p42/p44 MAP kinase Ab, New England Biolabs), which is specific for active p42/p44 MAP kinase and does not cross-react with p38 MAP kinase and JNK. Analysis of threonine and tyrosine phosphorylation of JNK was performed using an anti-phosphorylated threonine and tyrosine of JNK Ab (anti-phospho-specific JNK Ab, New England Biolabs), which is specific for active JNK and does not cross-react with p38 MAP kinase and Erk. Analysis of p38 MAP kinase, Erk, and JNK was performed according to manufacture’s instructions as described previously (14). Briefly, after separating proteins from cell lysate by a 15% SDS-PAGE, the cell lysate containing 10 µg of protein was electrophoretically transferred to membrane, and the membrane was incubated with specific Ab to phosphorylated threonine and tyrosine of p38 MAP kinase (affinity-purified rabbit polyclonal IgG), specific Ab to phosphorylated threonine and tyrosine of Erk (affinity-purified rabbit polyclonal IgG), or specific Ab to phosphorylated threonine and tyrosine of JNK (affinity-purified rabbit polyclonal IgG) for analysis of JNK. Then it was incubated with the HRP-conjugated anti-rabbit IgG Ab and HRP-conjugated anti-biotin Ab to detect biotinylated protein markers. Blots were incubated with ECL (enhanced chemiluminescence) solution for 1 min and exposed on Kodak (Rochester, NY) XAR film. To show the amounts of p38 MAP kinase, Erk, and JNK precipitated, blots were stripped and reprobed using phosphorylation-state independent p38 MAP kinase-specific Ab (affinity-purified rabbit polyclonal IgG) to determine total p38 MAP kinase levels, phosphorylation-state independent p42/p44 MAP kinase-specific Ab (affinity purified rabbit polyclonal IgG) to determine total p42/p44 MAP kinase levels, or phosphorylation-state independent JNK-specific Ab (affinity purified rabbit polyclonal IgG) to determine total JNK levels, respectively.
p38 MAP kinase and Erk kinase assay
The activity of p38 MAP kinase was analyzed by commercially available kits (p38 MAP Kinase Assay Kit, New England Biolabs). The kit employs two different Abs, anti-p38 MAP kinase Ab which is specific for p38 MAP kinase and does not cross-react with ERK1/2 or JNK, and anti-phospho-specific activating transcription factor-2 (ATF-2) Ab to detect p38 MAP kinase-induced phosphorylation of ATF-2. p38 MAP kinase activity was analyzed by a specific immunoprecipitation with anti-phospho-specific p38 MAP kinase Ab followed by an in vitro kinase assay of its substrate, ATF-2, according to the manufacturer’s instruction, as described previously (14). The activity of Erk was analyzed by commercially available kits (MAP Kinase Assay Kit, New England Biolabs). The kit employs two different Abs, anti-phospho-specific p42/p44 MAP kinase Ab which is specific for active p42/p44 MAP kinase and does not cross-react with p38 MAP kinase or JNK, and anti-phospho-specific Elk-1 Ab to detect p42/p44-induced phosphorylation of Elk-1. Erk activity was analyzed by a specific immunoprecipitation with anti-phospho-specific p42/p44 kinase Ab followed by an in vitro kinase assay of its substrate, Elk-1, according to manufacture’s instructions, as described previously (30). Briefly, the cell lysate containing 200 µg of protein was incubated with anti-p38 MAP kinase Ab to selectively immunoprecipitate p38 MAP kinase or anti-phospho-specific p42/p44 MAP kinase Ab to selectively immunoprecipitate active p42/p44 MAP kinase from cell lysates, and the immunoprecipitates were incubated with ATF-2 fusion protein or Elk-1 fusion protein in the presence of ATP, a process which allowed immunoprecipitated active p38 MAP kinase to phosphorylate its substrate, ATF-2, and immunoprecipitated active p42/p44 to phosphorylate its substrate, Elk-1. The samples were separated by a 15% SDS-PAGE, transferred to membranes, and blotted with anti-phospho-specific ATF-2 Ab or anti-phospho-specific Elk-1 Ab. The membrane was incubated with HRP-conjugated anti-rabbit IgG Ab and HRP-conjugated anti-biotin Ab, and then the membrane was incubated with 10 ml of the ECL solution and exposed on Kodak XAR film for 1 min.
JNK kinase assay
The activity of JNK was analyzed by commercially available kits (SAPK/JNK Assay Kit, New England Biolabs). The kit employs an N-terminal c-Jun fusion protein bound to Sepharose beads to selectively pull down JNK from cell lysates, after which the kinase reaction is conducted in the presence of unlabeled ATP. c-Jun phosphorylation is selectively measured using phospho-specific c-Jun Ab that specifically measures JNK-induced phosphorylation of c-Jun. Analysis of activity of JNK was performed according to the manufacturer’s instruction, as described previously (14). Briefly, the cell lysate containing 200 µg of protein was mixed with 2 µg of c-Jun fusion protein beads and then incubated. After microcentrifugation, the pellet was washed and suspended in kinase buffer with ATP. Equal amounts of protein (5 µg/lane) were separated by 15% SDS-PAGE, transferred to a membrane, and blotted with anti-phospho-c-Jun Ab. The membrane was incubated with HRP-conjugated anti-rabbit IgG Ab and HRP-conjugated anti-biotin Ab and then the membrane was incubated with 10 ml of the ECL solution and exposed on Kodak XAR film for 1 min.
Measurement of RANTES
The concentration of RANTES in the culture supernatants from BEC were measured by commercially available ELISA kits (Amersham International, Aylesbury, U.K.). ELISA was performed according to the manufacturer’s instructions. All samples were assayed in duplicate.
Statistical analysis
Statistical significance was analyzed by using ANOVA. A p value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether IV infection could induce the
threonine and tyrosine phosphorylation of p38 MAP kinase, Erk, and JNK,
BEC were infected with IV, and p38 MAP kinase, Erk, and JNK in the
cells were immunoblotted. Amounts of phosphorylated threonine and
tyrosine of p38 MAP kinase in IV-infected cells increased at 1 h,
were maximal at 6 h, and sustained between 8 and 12 h;
thereafter, they returned to near basal levels at 24 h (Fig. 1
a, upper panel).
Amounts of phosphorylated threonine and tyrosine of Erk in IV-infected
cells increased at 30 min, were maximal at 6 h, and sustained
between 8 and 12 h; thereafter they returned to near basal levels
at 24 h (Fig. 1b, upper panel). Amounts of
phosphorylated threonine and tyrosine of JNK in IV-infected cells
increased at 4 h and were maximal at 6 h; thereafter they
sustained between 8 to 12 h and returned to near basal levels at
24 h (Fig. 1c, upper panel). The lower
panels of Fig. 1a showed that equal amounts of p38 MAP
kinase protein were immunoblotted with phosphorylation-independent p38
MAP kinase-specific Ab regardless of time of culture periods,
indicating that IV infection-induced p38 MAP kinase phosphorylation
occurred in the absence of changes in p38 MAP kinase protein levels.
Similarly, IV infection induced in Erk and JNK phosphorylation occurred
in the absence of changes in Erk protein levels and JNK protein levels
(lower panels of Fig. 1, b and
c).
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Activation of p38 MAP kinase, Erk, and JNK is mediated by dual
phosphorylation of the threonine and tyrosine residues of p38 MAP
kinase, Erk, and JNK (9, 13, 31), respectively. Increases
in the threonine and tyrosine phosphorylation of p38 MAP kinase, Erk,
and JNK in IV-infected BEC shown in Fig. 1
reflect activation state of
p38 MAP kinase, Erk, and JNK. In addition to analysis of the threonine
and tyrosine phosphorylation of p38 MAP kinase, Erk, and JNK, we next
examined whether IV infection could induce p38 MAP kinase, Erk, and JNK
activity, and the effect of SB 20380 on p38 MAP kinase activity, the
effect of PD 98059 on Erk activity and the effect of CEP-1347 on JNK
activity in IV-infected BEC. IV infection induced p38 MAP kinase
activity as demonstrated by the increased phosphorylation of its
substrate, ATF-2 (Fig. 2a),
Erk activity as demonstrated by the increased phosphorylation of its
substrate, Elk-1 (Fig. 2b), and JNK activity as demonstrated
by the increased phosphorylation of c-Jun (Fig. 2c).
SB 203580, PD 98059, and CEP-1347 attenuated IV infection-induced
increases in p38 MAP kinase activity (Fig. 2a), Erk activity
(Fig. 2b), and JNK activity (Fig. 2c),
respectively. SB 203580, PD 98059, and CEP-1347 did not attenuate IV
infection-induced increases in Erk and JNK activity, p38 MAP
kinase and JNK activity, and p38 MAP kinase and Erk activity,
respectively (data not shown). Addition of DMSO vehicle alone did not
attenuate IV infection-induced increases in p38 MAP kinase, Erk, and
JNK activity (data not shown).
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RANTES production was reportedly induced by IV infection
(7). IV infection induced p38 MAP kinase, Erk, and JNK
phosphorylation and activity, and SB 203580, PD 98059, and CEP-1347
attenuated IV infection-induced increases in p38 MAP kinase, Erk, and
JNK activity, respectively (Figs. 1 and 2). These observations
suggested that IV infection-induced RANTES production might be mediated
through p38 MAP kinase-, Erk-, and JNK-dependent pathway. To test this
possibility, BEC that had been incubated with various concentrations of
SB 203580, PD 98059 or CEP-1347 for 1 h were infected with IV, and
the concentrations of RANTES in the culture supernatants were
determined at 24 h after IV infection. SB 203580 attenuated RANTES
production in a dose-dependent manner (Fig. 3a). A total of 10 µM SB
203580 attenuated RANTES production by 45.3%. PD 98059 did
not affect RANTES production (Fig. 3b). CEP-1347 attenuated
RANTES production in a dose-dependent manner (Fig. 3c). A
total of 1 µM CEP-1347 attenuated RANTES production by 45.2%.
Addition of DMSO vehicle alone did not affect RANTES production by
IV-infected BEC (data not shown).
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SB 203580 and CEP-1347 partially attenuated IV infection-induced
RANTES production. From these results, we next examined the effect of a
combination of SB 203580 (10 µM) and CEP-1347 (1 µM) on RANTES
production. SB 203580 and CEP-1347 additively attenuated RANTES
production by 69.1%, but a complete inhibition was not seen (Fig. 4). Addition of DMSO vehicle alone did
not affect RANTES production by IV-infected BEC (data not shown).
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Because IV infection has been shown to induce the production of
various cytokines by BEC (6, 7, 8), it might be possible that
cytokine produced by IV-infected BEC is responsible for the induction
of p38 MAP kinase, Erk, and JNK phosphorylation. To test this
possibility, anti-IL-1 mAb, anti-IL-1ß mAb,
anti-TNF- mAb, anti-IL-8 mAb, anti-IFN-ß Ab, and
anti-RANTES mAb were added to the culture, and then BEC were
infected with IV. The threonine and tyrosine phosphorylation of p38 MAP
kinase, Erk, and JNK were analyzed at 6 h after IV infection. As
shown in Fig. 5, each Ab and a
combination of all Abs did not affect IV infection-induced increases in
p38 MAP, Erk, and JNK phosphorylation. In addition, we measured the
concentrations of IL-1, IL-1ß, TNF-, IL-8, IFN-ß, and RANTES
in the culture supernatants from IV-infected BEC at 6 h after IV
infection by a specific ELISA. The concentrations of these cytokines
were below the assay sensitivity limit. Each Ab used in this study had
sufficient Ab concentrations for neutralizing corresponding cytokine
activity of assay sensitivity limit.
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Because it has been shown that IV infection induces the generation
of reactive oxygen species (ROS) (32) and that ROS can
induce MAP kinase phosphorylation (33, 34, 35), we examined
the effect of NAC on the threonine and tyrosine phosphorylation of p38
MAP kinase, Erk, and JNK to clarify the involvement of ROS in IV
infection-induced p38 MAP kinase, Erk, and JNK phosphorylation. The
results showed that NAC did not affect of IV infection-induced
increases in p38 MAP kinase, Erk, and JNK phosphorylation, indicating
that ROS which can be scavenged by NAC were not involved in IV
infection-induced phosphorylation of p38 MAP kinase, Erk, and JNK
phosphorylation (Fig. 6). Finally,
because it has been shown that IV infection activates NF-
B leading
to IL-8 expression and antioxidants inhibit IV infection-induced
NF-B activation (32), we examined the effect of NAC on
IV infection-induced RANTES production. The results showed that NAC
attenuated RANTES production by 18.2% (Fig. 7). The total number of the cells and
cell viability at the end of the culture period of each experiment,
determined by trypan blue exclusion dye, did not differ with culture
conditions, suggesting that IV infection-induced RANTES production and
the attenuation by inhibitors and NAC of RANTES production did not
result from cell cytotoxicity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, TNF-, IL-8, IFN-ß, and
RANTES in the culture supernatants from IV-infected BEC at 6 h
postinfection were not detected by specific ELISA; and 3) NAC did not
affect IV infection-induced increases in p38 MAP kinase, Erk, and JNK
phosphorylation. These results indicated that cytokines as described
above and ROS were not involved in IV infection-induced phosphorylation
of p38 MAP kinase, Erk, and JNK. However, a role of cell-associated and
intracellular cytokines in the induction of MAP kinase phosphorylation
remains to be determined. It is also of interest to identify
responsible IV-specific molecule for the induction of MAP kinase
phosphorylation and determine a relationship between IV growth and the
induction of MAP kinase phosphorylation. It is likely that IV
replication is more directly related to the induction of MAP kinase
phosphorylation, because a time-course of the induction of MAP kinase
phosphorylation is closely correlated with a time-course of IV growth.
We are currently investigating to clarify this point. The specific inhibitors of p38 MAP kinase and Erk signaling pathway have been identified, providing effective tools for investigating the role of p38 MAP kinase and Erk in cellular signaling (27, 28). In addition, we used CEP-1347, which is a novel inhibitor of the JNK signaling pathway (29). SB 203580, PD 98059, and CEP-1347 were used as the specific inhibitor of p38 MAP kinase activity, MEK-1 activity, and JNK signaling pathway, respectively, to elucidate the biological functions of different p38 MAP kinase, Erk, and/or JNK. A total of 10 µM SB 203580 and 50 µM PD 98059 were used in this study to examine the inhibitory effect of these inhibitor on RANTES production, because the previous studies with analysis of the role of p38 MAP kinase and Erk in eliciting various biological responses including cytokine expression showed that these concentrations of inhibitors almost completely inhibited (27, 28, 36, 37). A total of 1 µM CEP-1347 can rescue motoneurons undergoing apoptosis and inhibit JNK activity (29). Consequently, 10 µM SB 203580, 50 µM PD 98050, and 1 µM CEP-1347 employed in this study were sufficient concentrations to examine the signal transduction pathway. IV infection induced simultaneously p38 MAP kinase, Erk, and JNK activity. A total of 10 µM SB 203580, 50 µM PD 98059, and 1 µM CEP-1347 almost completely attenuated IV infection-induced increases in p38 MAP kinase, Erk and JNK activity, respectively. In the production of RANTES, 10 µM SB 203580 caused a 45.3% decrease and 1 µM CEP-1347 caused a 45.2% decrease in RANTES production, whereas 50 µM PD 98059 did not inhibit RANTES production. In addition, SB 203580 and CEP-1347 additively attenuated RANTES production by 69.1%. The inhibition by a combination of these inhibitors was statistically significant compared with that by SB 203580 or CEP-1347 only. These results indicated that p38 MAP kinase and JNK, but not Erk, at least in part participate in regulating RANTES production. The incomplete inhibition of RANTES production induced by a combination of SB 203580 and CEP-1347 observed in this study might suggest that parallel pathways which are p38 MAP kinase- and JNK-independent pathway may regulate RANTES production. One possible signal that may regulate IV infection-induced RANTES production by BEC is ROS.
It has been shown that IV infection activates NF-B leading to IL-8
expression and antioxidants inhibit IV infection-induced NF-B
activation (32). In this study, NAC did not affect IV
infection-induced increases in p38 MAP kinase and JNK phosphorylation,
whereas NAC attenuated RANTES production by 18.2%. Collectively, these
results indicated that ROS generated by IV infection might act as a
second messenger leading to the induction of RANTES production via p38
MAP kinase- and JNK-independent pathway.
The mechanism of activation and the function of MAP kinases have been extensively studied. A variety of extracellular stimuli activate p38 MAP kinase and elicit a variety of cellular functions. p38 MAP kinase-mediated cytokine expression has been well documented (14, 15, 36, 37). Recent studies have indicated the involvement of Erk and the coordinate regulation by p38 MAP kinase and Erk in cytokine expression in various cells (18, 19, 20, 21). However, our results showed that Erk was not involved in IV infection-induced RANTES production by BEC. Phosphorylation and catalytic activation of JNK in cells has been implicated in the intracellular signal promoting apoptosis (9, 38). In addition, the JNK pathway was recently shown to play a role in cytokine expression (22, 23). However, little is known about the role of JNK in IV-induced RANTES production in BEC. Our results with JNK-mediated RANTES production by IV-infected BEC indicate new evidence on the role of JNK in airway epithelial cells, which is the regulation of RANTES production. RANTES are produced by a variety of cells (39, 40, 41). In human umbilical vein endothelial cells, Erk has been shown to regulate RANTES production (42). Collectively, the signal transduction pathway regulating RANTES production may be cell type-specific.
IV infection induces the expression of various cytokines including RANTES in airway epithelial cells. Eosinophilia, neutrophilia, and high levels of IL-8 and eosinophilic cationic protein are seen in induced sputum from bronchial asthmatics infected with IV (43). RANTES that exhibits a chemotactic activity for eosinophils has been shown to play an important role in the production of airway inflammation of asthmatics through the recruitment of eosinophils into the site of airway inflammation (26). Our results with the role of p38 MAP kinase- and JNK-dependent pathway in RANTES production by IV-infected BEC are important in understanding the pathogenesis of IV-induced asthma exacerbation.
Finally, MAP kinase cascades are connected with the activation of
various transcription factors that participate to various extents in
the inducible expression of gene-encoding cytokine. The promoter of the
gene-encoding RANTES contains sequences for the binding several nuclear
transcription factors including NF-B and AP-1 (44, 45).
These transcription factors participate to various extents in the
inducible expression of the gene encoding RANTES. p38 MAP kinase has
been implicated in the activation of multiple transcription factors,
including NF-B (46). JNK has been implicated in the
activation of multiple transcription factors, including AP-1 (9, 47). Recently, Hiura et al. (48) have shown that
JNK and NF-B response elements are involved in RANTES gene
activation in macrophage cell line, RAW 264.7 cells, stimulated by LPS.
We have not investigated and identified downstream targets of p38 MAP
kinase and JNK leading to RANTES gene expression (47, 49).
Therefore, further study should be done to clarify the links between
the activation of these kinases and the expression of RANTES
gene.
From the data presented in this paper, we conclude that p38 MAP kinase and JNK, at least in part, regulate RANTES production by IV-infected BEC.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shu Hashimoto, First Department of Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamimachi, Itabashi-ku, Tokyo 173-8610, Japan. E-mail address:
3 Abbreviations used in this paper: IV, influenza virus; MAP, mitogen-activated protein; Erk, extracellular signal-regulated kinase, JNK, c-Jun-NH2-terminal kinase; BEC, human bronchial epithelial cells; NAC, N-acetylcysteine; MEK-1, MAP/Erk kinase kinase-1; ROS, reactive oxygen species.
Received for publication October 6, 1999. Accepted for publication December 30, 1999.
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S. HASHIMOTO, Y. GON, I. TAKESHITA, K. MATSUMOTO, S. MARUOKA, and T. HORIE Transforming Growth Factor-{beta}1 Induces Phenotypic Modulation of Human Lung Fibroblasts to Myofibroblast Through a c-Jun-NH2-Terminal Kinase-Dependent Pathway Am. J. Respir. Crit. Care Med., January 1, 2001; 163(1): 152 - 157. [Abstract] [Full Text]
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S. D. Griego, C. B. Weston, J. L. Adams, R. Tal-Singer, and S. B. Dillon Role of p38 Mitogen-Activated Protein Kinase in Rhinovirus-Induced Cytokine Production by Bronchial Epithelial Cells J. Immunol., November 1, 2000; 165(9): 5211 - 5220. [Abstract] [Full Text] [PDF]
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) or infected with IV (•)
(a). The concentrations of RANTES in the culture
supernatants were determined at 24 h after IV infection. The
results are expressed as the mean ± SD in three different
experiments. *1, p < 0.05 compared with RANTES
concentrations in BEC cultured without an inhibitor; *2,
p < 0.01 compared with RANTES concentrations in
BEC cultured without an inhibitor.
