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,**,¶
Departments of
* Molecular Immunology,
Medical Immunology, and
General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan;
PRESTO, Tokyo, Japan;
¶ CREST Japan Science and Technology Corp., Tokyo, Japan;
|| Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, Tokyo, Japan;
# Department of Tropical Medicine, Jikei University School of Medicine, Tokyo, Japan; and
** Section of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Medical College, National Cheng Kung University, Tainan, Taiwan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and Th2 cells
produce IL-4, IL-5, and IL-13. The development of Th1 and Th2 cells is
a central issue in understanding Th cell-dependent immune responses in
infectious, allergic, and autoimmune diseases (2, 3, 4). Th1
cells play a central role in cellular immunity, whereas Th2 cells are
crucial for humoral immunity and are involved in allergic and
helminthic diseases. The direction of differentiation toward Th1 and Th2 cells is dependent on the exogenous cytokines present during primary antigenic stimulation of naive T cells (4, 5, 6). IL-12-induced STAT4 activation promotes Th1 cell differentiation, whereas IL-4R-mediated signaling, including STAT6 activation, is required for Th2 cell differentiation. Ag recognition by TCR is also indispensable for both Th1 and Th2 cell differentiation (4). Flavell and colleagues (7, 8, 9) showed that Th1 cell differentiation and Th1 cytokine production are dependent on c-Jun N-terminal kinase and the p38 mitogen-activated protein kinase (MAPK)3 cascade, respectively. We have reported that there is a preferential requirement for the TCR-induced activation of p56lck, calcineurin, and the Ras/MAPK cascade in the differentiation of Th2 cells (10, 11, 12). We analyzed the efficiency of Th1/Th2 cell differentiation in naive T cells from H-ras-dominant-negative Ras (dnRas) transgenic (Tg) mice, in which enzymatically inactive Ras molecules were overexpressed, and found that TCR-induced MAPK cascade activation was strongly compromised. Severely impaired Th2 cell differentiation and increased Th1 cell differentiation were observed, whereas other functions, such as anti-TCR-induced proliferative responses and IL-2 production, were within normal ranges (12). In addition, in vivo Ag-induced Th2 responses, such as Ag-specific IgG1 and IgE production, were impaired, whereas Th1-dependent IgG2a immune responses were enhanced. However, it has not been elucidated whether IL-5 production and Th2-dependent eosinophilic inflammation are also dependent on the activation of the Ras/MAPK cascade in T cells.
Airway inflammation is a central issue in the pathogenesis of asthma. The airway of asthma patients demonstrates chronic inflammation characterized by leukocyte infiltration in the peribronchiolar and perivascular regions of the lung, hypersecretion of mucus, obstruction of airways, epithelial damage, and basement membrane thickening (13). Recent clinical and experimental investigations revealed crucial roles for CD4+ Th2 cells and eosinophils in allergic airway inflammation and hyperresponsiveness (14, 15, 16). Th2 cytokines, including IL-4, IL-5, and IL-13, appear to play a crucial role in the development of allergic airway inflammation (17, 18). IL-4 has been suggested to be important for the generation of allergen-specific Th2 cells during sensitization (19), whereas IL-13 is thought to be more important in the induction of airway hyperresponsiveness (20, 21, 22). In contrast, IL-5 appears to be critical for the induction of eosinophilic inflammation in bronchial tissues (23, 24, 25). However, the differential roles for these Th2 cytokines in the development of allergic asthma remain ambiguous.
Here, we used dnRas Tg mice to assess the role of Ras activation in OVA-induced allergic airway inflammation in a mouse asthma model. Our results suggest that the levels of Ras activation in T cells determine the development of Th2-dependent eosinophilic inflammation and Ag-induced airway hyperresponsiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A Tg mouse with T cell-specific dnRas expression driven by the lck proximal promoter was characterized previously (12, 26). In this report, 6- to 8-wk-old heterozygous (Tg+/-) dnRas Tg mice with a (C56BL/6 (B6) x BALB/c)F1 background were used. All mice used in this study were maintained under specific pathogen-free conditions. Animal care was in accordance with the guidelines of Chiba University (Chiba, Japan).
Immunization and airway challenge with OVA
dnRas Tg mice 6–8 wk old were immunized i.p. with 100 µg of OVA (chicken egg albumin; Sigma-Aldrich, St. Louis, MO) in 1 mg of aluminum hydroxide gel (alum) on days 0 and 7. On days 14, 15, and 16, the sensitized mice inhaled aerosolized OVA for 30 min in a chamber (31 x 23 x13 cm) connected to a nebulizer that generates a 1% w/v OVA aerosol mist. Control mice inhaled 0.9% saline.
Cytokine production in vitro
Two weeks after the last OVA or Nippostrongylus
brasiliensis injection, the spleens were removed. Whole spleen
cell populations were cultured for 3 days with 6.25–50 µg/ml
DNP-N. brasiliensis Ag or 3–100 µg/ml OVA
(27). The concentrations of cytokines (IL-4, IL-5, IL-13,
and IFN-) in the culture supernatant were quantified by standard
ELISA as described previously (28).
Collection and analysis of bronchoalveolar lavage (BAL) fluid
Two days after the last OVA inhalation, BAL was performed as described (29). Total BAL fluid was collected, and the cells in 100-µl aliquots were counted. One hundred thousand viable BAL cells were cytocentrifuged onto slides by a Cytospin 3 (Shandon, Pittsburgh, PA), and stained with May-Grünwald-Giemsa solution (Merck, Rahway, NJ) as described (24). Two hundred leukocytes were counted on each slide. Cell types were identified based on morphological criteria. The percentages and absolute numbers of each cell type were calculated.
Lung histology
Two days after the last OVA inhalation, the lungs were fixed in 4% paraformaldehyde-PBS, dehydrated in 50–100% ethanol-propanol-xylene, and embedded in paraffin. Then the samples were sectioned and stained with H&E or Luna (30) and examined for pathological changes under a light microscope at x80 and x160.
Measurement of airway responsiveness
Airway responsiveness was assessed by methacholine-induced airflow obstruction of conscious mice placed in a whole body plethysmograph (model PLY3211; Buxco Electronics, Troy, NY) as described (29). The respiratory parameters were obtained by exposing mice to 0.9% saline aerosol, followed by incremental doses (3–50 mg/ml) of aerosolized methacholine. Airflow obstruction was monitored and analyzed by system XA software (model SFT1610; Buxco Electronics). Results are expressed as the percentage of baseline enhanced pause values after 0.9% saline exposure.
Helminthic infection and the measurement of Th2 responses.
dnRas Tg mice were injected s.c. with 750 third-stage N. brasiliensis larvae on days 0 and 21 or inoculated orally with 300 third-stage Heligmosomoides polygyrus larvae (27, 31). To determine the number of eosinophils in the blood, mice were bled 14 days after primary and 7 days after secondary N. brasiliensis infection and 21 days after H. polygyrus infection. The number of eosinophils was counted under a microscope after staining with Hinkelmann’s solution. The number of eosinophils before primary or secondary infection was <50/ml.
Statistical analysis.
Student’s t test was used to evaluate the significance of the differences.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The goal of this study was to evaluate the roles for Ras
activation in T cells in allergic airway inflammation and airway
hyperresponsiveness. We initiated the analysis by assessing Ag-induced
Th2 cytokine production in dnRas Tg mice, where Ras activation is
inhibited specifically in T cells. Eight-week-old dnRas Tg mice with a
(B6 x BALB/c)F1 background were immunized
with OVA in alum. Two weeks later, spleen cells were individually
prepared, and in vitro Ag-induced cytokine production was measured
(Fig. 1
). As can be seen, OVA Ag
dosage-dependent increases in the production of Th2 cytokines (IL-4,
IL-5, and IL-13) were observed in littermate (LM) mice and, as
expected, significantly reduced responses in the dnRas Tg cultures were
detected. In contrast, a slight enhancement of IFN- production was
detected. The production of IL-2 appeared to be slightly decreased.
These results are consistent with the previous finding that Th2 cell
differentiation is impaired in dnRas Tg mice (12), as is
Ag-specific IgE production. Here, Ag-induced IL-5 and IL-13 production
are revealed to be attenuated in dnRas Tg mice.
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Two weeks after OVA immunization, dnRas Tg mice were exposed to
inhaled OVA for 3 consecutive days, and a further 2 days later BAL
fluid was examined for eosinophilic infiltration. As can be seen in
Fig. 2A, the majority of
infiltrated cells were eosinophils in LM mice. The percentages and
absolute numbers of eosinophils, macrophages, and neutrophils were
calculated as described in Materials and Methods. Fig. 2B shows significantly decreased frequencies of eosinophils
and increased frequencies of macrophages. As shown in Fig. 2C, a substantial reduction in the absolute number of
eosinophils was revealed. The number of leukocytes in the BAL fluid was
significantly reduced in dnRas Tg mice (see total). According to
morphological criteria, the number of lymphocytes was negligible in the
cells designated as "others." They appeared to be blast cells (data
not shown). We also measured cytokines (IL-2, IL-4, IFN-, and IL-5)
in the BAL fluid. Under our conditions, only IL-5 was detectable by
ELISA, and slightly decreased concentrations were consistently detected
in the dnRas Tg groups (Fig. 2D).
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). Leukocyte infiltration
in peribronchiolar regions was significantly milder in dnRas Tg mice
than in control mice (Fig. 3A, compare a and
b). Bronchiolar mucus hyperproduction and airway obstruction
were often seen in littermate controls (Fig. 3Aa). A summary
of infiltrated leukocyte numbers is shown in Fig. 3B, where
a significant reduction was noted. Simultaneously, the sections were
stained with Luna, and perivascular and peribronchiolar regions were
assessed for eosinophilic infiltration (Fig. 3C). Although
some perivascular and peribronchiolar regions showed the presence of
significant numbers of leukocytes in dnRas Tg lung (Fig. 3C,
c and d), the number of eosinophils was clearly
fewer in dnRas Tg mice than in LM controls. Taken together, these
results suggest that OVA-induced IL-5 and eosinophilic inflammation in
the lung is dependent on the levels of Ras activation in T cells.
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To assess the levels of airway hyperresponsiveness in
OVA-sensitized mice, dnRas Tg mice were immunized and exposed to
inhaled OVA. Then airway hyperresponsiveness was assessed by measuring
methacholine-induced airflow obstruction in a whole body
plethysmograph. As shown in Fig. 4A, airway hyperresponsiveness
was diminished in the dnRas Tg mice. The levels of airway
hyperresponsiveness in nonsensitized dnRas Tg mice were similarly
assessed and did not reveal a significant difference between normal and
dnRas Tg mice (Fig. 4B), suggesting that the baseline level
of airway hyperresponsiveness was not altered in dnRas Tg mice. Thus,
the Ras activation level was found to control the development of airway
hyperresponsiveness in a mouse OVA-induced asthma model.
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Finally, we assessed the role of Ras activation in eosinophilia
induced by nematode infection. Eight-week-old dnRas Tg mice were
infected with N. brasiliensis or H.
polygyrus, both of which are known to induce eosinophilic
inflammation in experimental mice (32, 33). The number of
eosinophils in the peripheral blood of uninfected normal mice was lower
than 50/mm3 (data not shown). As shown in Fig. 5A, the number of eosinophils
increased greatly after primary or secondary infection with N.
brasiliensis in wild-type LM (B6 x
BALB/c)F1 mice. The levels of eosinophilia were
significantly lower in the dnRas Tg groups. In H.
polygyrus infection, the numbers of eosinophils in the blood
of dnRas Tg mice were reduced to about one-half to one-third of those
in LM controls (experiment 1 or 2). Concurrently, we prepared spleen
cells from N. brasiliensis-infected animals, and N.
brasiliensis Ag-induced cytokine production in vitro was assessed.
As can be seen in Fig. 5B, the production of IL-5 and IL-13
was significantly reduced in dnRas Tg cultures. A slight increase in
the production of IFN- was observed. The decrease in the production
of IL-4 in dnRas groups was not as prominent (Fig. 5B, lL-4
panel). The reason for this apparent disparity from the OVA challenge
data (see earlier) is not know, but the N. brasiliensis
Ag-specific response may have been masked by a high background level of
IL-4 production (
50 pg/ml) by spleen cells from N.
brasiliensis-infected mice (see Fig. 5B, IL-4 panel,
extreme left). Only at the highest in vitro DNP-N.
brasiliensis Ag dose of 50 µg/ml does the difference in IL-4
production become evident where the highest level of IL-4 detected
reached 150 pg/ml. In any event, IL-5 production and eosinophilia
induced by nematode infection also appear to be dependent on the levels
of activation of Ras in T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Thus, it is conceivable that the
block in the development of eosinophilic airway inflammation and airway
hyperresponsiveness is a consequence of the reduced generation of
Ag-specific Th2 cells during OVA sensitization. Undoubtedly, however,
there are effects of dnRas downstream of Th2 priming.
IL-5 is critical for the development of eosinophilic inflammation
(34). Airway eosinophilic inflammation and airway
hyperresponsiveness have been demonstrated to be reduced by
anti-IL-5 Ab treatment and IL-5 gene deletion in mice (24, 25). Furthermore, ectopic Tg expression of IL-5 in the lung
epithelium results in eosinophilic invasion of airways comparable with
that seen in asthmatic patients (35). Interestingly, the
IL-5 Tg mice displayed airway hyperresponsiveness in the absence of
Ag-induced inflammation. Thus, IL-5 appears to be most critical for the
development of airway eosinophilic inflammation and also the
development of airway hyperresponsiveness. In dnRas Tg mice,
significantly reduced IL-5 levels were seen (Fig. 1). Because the
production of IL-4 and IL-13 was also reduced in dnRas Tg mice, it is
not so easy to estimate the contribution of the IL-5 deficit to the
attenuated airway inflammation. However, it is now clear that the
activation of Ras is involved in the pathogenesis of both eosinophilic
inflammation and airway hyperresponsiveness.
Our previous results indicate that the efficiency of Th2 cell
differentiation depends on the activation level of the
Ras/extracellular signal-regulated kinase (ERK) MAPK cascade
(12). Consistent with this observation, the production of
Th2 cytokines (IL-4, IL-5, and IL-13) was impaired in dnRas Tg T cells
(Fig. 1). In naive CD4 T cells, stimulation with a very large amount of
IL-4 (100 U/ml) did not induce detectable levels of ERK MAPK cascade
activation (our unpublished observations). In contrast, TCR
stimulation induced a substantial increase in the level of ERK
phosphorylation (12). These results suggest that
activation of the Ras/ERK MAPK cascade in naive CD4 T cells is a
consequence of TCR-mediated signaling. The downstream functional target
molecules of ERK-MAPK in Th2 cell differentiation are not clarified
very well at this time. One candidate is the IL-4R signaling complex
(12). IL-4-induced tyrosine phosphorylation of Jak1 and
STAT6, which reflects the strength of IL-4R signaling, was enhanced
when naive T cells were exposed to short term treatment with PMA (an
agonistic of the Ras/MAPK cascade), suggesting that activation of
Ras/MAPK cascade up-regulated IL-4R function. Another candidate is
NF-
B. A role for NF-B in GATA3 expression and Th2 cell
differentiation was demonstrated in an allergic airway inflammation
system using mice that lack the p50 subunit of NF-B
(36). Although we observed weak activation of NF-B
signaling in purified naive CD4 T cells after stimulation with
anti-TCR mAb, the activation was not inhibited by treatment with
PD98059 (our unpublished observations), suggesting that this NF-B
activation is not dependent on the ERK MAPK cascade. Thus, NF-B
regulation of GATA3 expression may not be involved in the observed ERK
MAPK-mediated regulation of allergic airway inflammation.
The accumulated results implicating the involvement of the activation
of Th2 cells in the development of asthma support several new
strategies that could attenuate Th2-induced airway inflammation in the
asthmatic airway. Most of the strategies aim to block Th2 cell
differentiation and the production of Th2 cytokines (37, 38). One of the potential strategies is to develop drugs that
block the signal transduction pathway required for Th2 cell
differentiation. Inhibition of the Ras pathway, as shown in the present
study, could alter the cytokine pattern by suppressing IL-4, IL-5 and
IL-13 and enhancing IFN- production. In fact, a mitogen-activated
protein/extracellular signal-related kinase kinase inhibitor, U0126,
has been shown to reduce IL-13-induced cell stiffness in cultured human
airway smooth muscle cells, a model of airway narrowing observed in
patients with asthma (39). In addition, several other
independent pathways downstream of Ras have been reported and appear to
mediate the distinct biological functions of Ras. The activation of Ras
results in the phosphorylation of p70/S6 kinases (40).
Rapamycin, a p70/S6 kinase inhibitor, has been reported to inhibit the
proliferation of T lymphocytes from patients with chronic asthma, and
rapamycin has been suggested to be useful in clinical trials for
asthmatic patients given their modes of action in T cells
(41). Thus, specific blockade of the Ras-Raf/ERK MAPK
pathway may have therapeutic value in asthma.
In summary, in a mouse allergic asthma model, the activation of Ras in T cells controls the development of Th2-dependent eosinophilic airway inflammation and airway hyperresponsiveness. Thus, a search for specific inhibitors focusing on Ras-mediated signaling pathways would be helpful for establishing a new approach to the treatment of inflammatory diseases such as bronchial asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Toshinori Nakayama, Department of Medical Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: nakayama{at}med.m.chiba-u.ac.jp
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; BAL, bronchoalveolar lavage; dnRas, H-ras-dominant negative Ras; LM, littermate; Tg, transgenic; ERK, extracellular signal-regulated kinase.
Received for publication February 19, 2002. Accepted for publication June 7, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in GATA3 expression and Th2 differentiation in allergic airway inflammation. Nat. Immunol. 2:45.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, LM
mice; 
, dnRas Tg mice. Values of p were determined by
Student’s t test and were as follows: IL-4, 0 µg/ml:
0.5<, 6.25 µg/ml: 0.5<, 12.5 µg/ml: 0.5<, 25 µg/ml: 0.5<, 50
µg/ml: 0.004. IL-5, 6.25 µg/ml: 0.001, 12.5 µg/ml: <0.0001, 25
µg/ml: <0.0001, 50 µg/ml: 0.05. IL-13, 6.25 µg/ml: 0.04, 12.5
µg/ml: 0.005, 25 µg/ml: <0.001, 50 µg/ml: 0.5<. IFN-