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*
Second Division, Department of Internal Medicine, and
Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan; and
Department of Internal Medicine, National Sanatorium of Tenryu Hospital, Hamakita, Shizuoka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) mRNA, whereas cells from young animals expressed
more Th2 type cytokine mRNAs including those for IL-4 and IL-5.
Decreased expression of Th2 type cytokine transcripts in aged animals
was further confirmed by quantitative analysis, competitive RT-PCR of
BAL cells, and in situ hybridization. The age-associated changes of
cytokine profile were not restricted to BAL cells but were a general
feature of lymphocytes, as shown by examination of popliteal lymph
nodes draining the site of sensitization. These findings suggest
that decreased allergic inflammation in aged animals is attributable to
age-dependent impairment of Th2 generation in response to
Ag. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a representative Th1
cell cytokine, is known to have an inhibitory effect on Th2-related
responses (8, 12). Thus, the balance between Th1 and Th2
cytokine profiles could modify the immune response at sites of
inflammation (3, 13).
The severity of allergic disorders such as bronchial asthma, allergic
rhinitis, and atopic dermatitis is considered to decline with age
(14, 15). Possible explanations for this change include
our previous observation that aged animals failed to produce
Ag-specific IgE Ab and to generate eosinophil influx into sites of
inflammation in response to Ag stimulation (16). However,
the mechanisms remain to be elucidated. In this study, we investigated
whether age-associated alterations in the production of cytokines,
including IL-4, IL-5, and IFN-, were involved in the decreased
allergic response of aged rats with OVA-induced asthma. We studied
differences of cytokine mRNA expression in the bronchoalveolar lavage
(BAL)2 cells of young
and aged rats using the RT-PCR method and found that expression of mRNA
for Th2 type cytokines (IL-4 and IL-5) was selectively decreased in the
aged rats compared with young animals. We also confirmed histologically
that the number of IL-5 mRNA-expressing cells was decreased in the
lungs of aged rats using the in situ hybridization technique.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Young (8- to 10-wk-old) and aged (100- to 120-wk-old) male Brown Norway (BN) rats were obtained from Charles River (Tokyo, Japan). The rats were kept at the animal research facility of Hamamatsu University School of Medicine in an air-filtered, specific-pathogen-free environment during these experiments.
Sensitization with OVA and inhalation challenge
Both young and aged rats were sensitized by a s.c. injection into the foot pads of 0.25 mg/kg OVA (Sigma, St. Louis, MO) and alum containing 20 mg/kg Al(OH)3 (provided by Dr. H. Nagai, Gifu Pharmaceutical University, Gifu, Japan) in borate-buffered saline. At the same time, 0.5 ml of Bordetella pertussis vaccine (Wako Pure Chemical Industries, Osaka, Japan) containing 1 x 1010 heat-killed bacilli in PBS (pH 7.4) was administered i.p. as an adjuvant. After 7 days, 0.125 mg/kg OVA in PBS was injected s.c. Fourteen days after the first sensitization, rats were challenged with an aerosol of 5% OVA for 10 min under pentobarbital sodium anesthesia. We also conducted experiments using challenge with 5% BSA (Sigma) to confirm that OVA-induced responses were Ag specific.
Harvesting of BAL fluid and cell analysis
Rats were sacrificed 24 h after OVA challenge by exsanguination under ether anesthesia. The lungs were removed intact from rats, and BAL was performed with 5 x 3 ml of HBSS (Sigma) through a polyethylene tube introduced into the trachea. The BAL fluid was centrifuged at 200 x g for 10 min, and cell pellets were resuspended in 1 ml of HBSS. BAL fluid supernatants were studied for ELISA. The total cell count was determined, and aliquots of the suspensions were used for preparation of cytospin slides to assess the cell differentiation. The rest of each cell suspension was recentrifuged, and the cell pellet was prepared for RNA extraction. In another set of experiments, the lungs were harvested, cut into small pieces, fixed with 4% paraformaldehyde in PBS for 16 h, and immersed in 30% sucrose in PBS for 8 h. Sections were cut, embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan), and stored at -80°C until use for in situ hybridization.
Popliteal lymph node cell culture
To investigate age-associated changes in the systemic response of lymphocytes to immunization, popliteal lymph nodes (pLN), which were the draining nodes for the OVA administration site, were excised 2 wk after the first sensitization. pLN cells were prepared at 1 x 106/ml in RPMI 1640 (Sigma) containing 5% FCS (Life Technologies, Gaithersburg, MD). Then 2-ml aliquots of the cell suspensions were cultured in a 24-well flat-bottom culture plate at 37°C under a humidified atmosphere of 5% CO2 in air with or without OVA at a final concentration of 1 mg/ml. Cells were recovered at 3, 6, 9, 12, 18, and 24 h after the start of incubation; washed twice; and used for preparation of mRNA. Cell viability was tested by trypan blue exclusion and always exceeded 90%. The supernatants of cultured pLN cells of 24 h of incubation were obtained for ELISA study.
Flow cytometric analysis of popliteal lymph node cells
An aliquot of the pLN cell suspension was used for analysis of cell surface Ag expression by direct immunofluorescence method with a flow cytometer (Epics Profile II, Coulter, Hialeah, FL). The number of fluorescence-positive cells per 5000 cells was determined. Briefly, pLN cells (1 x 106/ml) were incubated for 30 min in the dark with a saturating concentration of FITC-labeled anti-rat CD5 (OX-19) or PE-labeled anti-rat CD45RA (OX-33) to detect CD5+ T cells and B cells, respectively. Then the cells were washed twice with HBSS containing 0.5% BSA (Sigma). The mAbs were purchased from PharMingen (San Diego, CA).
RNA preparation and cDNA synthesis
Total cellular RNA was isolated from BAL cells and pLN cells by the acid guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (17). The RNA was precipitated by centrifugation at 4°C, washed once with 70% ethanol, air-dried, and suspended in diethylpirocarbonate-treated water. The amounts of RNA was determined by spectrophotometry, and 2 µg total cellular RNA were used for the first-strand cDNA synthesis with Moloney murine leukemia virus reverse transcriptase (200 U, Life Technologies) and random hexanucleotide (100 nM). After terminating the reaction by heating at 70°C for 5 min, distilled water was added up to 100 µl.
PCR amplification
The expression of IL-2, IL-4, IL-5, IL-10, and IFN- mRNA was
studied by PCR amplification. ß-Actin was also amplified as a control
for constitutive gene expression. Gene-specific oligonucleotide primers
were designed using the published sequence information on the rat
cDNAs for the individual cytokines and ß-actin obtained from GenBank.
The primer sequences were as follows: IL-2, sense,
5'-CATTGTTGAGATGATGC TTTGACA, antisense, 5'-GTCAACAGCGCACCCACTTCAAGC
(408- bp product); IL-4, sense, 5'-ACCTTGCTGTCACCCTGTTCTGC,
antisense, 5'-GTTGTGAGCGTGGACTCATTCACG (352-bp product); IL-5,
sense, 5'-TGCTTCTGTGCTTGAACGTTCTAAC, antisense,
5'-TTCTCTTTTTGTCCGTCAATGTATTTC (298-bp product) ; IL-10, sense,
5'-GACTTTAAGGGTTACTTGGGTTGC, antisense, 5'-CACTGCCTTGCTTTTATTCTCACA
(200-bp product); IFN-, sense, 5'-GAAAGCCTAGAAAGTCTGAAGAAC,
antisense, 5'-GCACCGACTCCTTTTCCGCTTCCT (387-bp product);
ß-actin, sense, 5'-TCCTGTGGCATCCATGAAACT, antisense,
5'-GAAGCATTTGCGGTGCACGAT (300-bp product).
PCR amplification was performed by adding 2.5-µl aliquots of each
cDNA sample to 22.5 µl of reaction mixture (50 mM KCl, 10 mM Tris-HCl
(pH 8.8), 0.1% Triton X-100, 0.4 mM concentrations each of dNTP, 3.0
mM MgCl2 for IL-5, and 1.5 mM for other
cytokines, 0.2 µM forward and reverse gene-specific primers)
containing 1.25 U of Taq DNA polymerase (Nippongene, Tokyo,
Japan). Amplification was done in a DNA Thermal Cycler (Perkin-Elmer
Cetus, Norwalk, CT) under the following conditions: denaturation at
95°C for 1 min, annealing at 58°C for 1 min, and extension at
72°C for 2 min, with final extension at 72°C for 4 min. We
determined the optimum number of cycles for each primer set so that the
specific product was amplified during the exponential phase of
amplification. Based on the results of preliminary studies, 40 cycles
were used for IL-2 and IL-4, whereas IL-5, IL-10, and IFN- were run
for 32 cycles, and ß-actin was amplified for 26 cycles. Negative
control reactions performed without RT yielded no detectable fragments
with any primer pair.
Gel analysis
Ten microliters of each PCR product were subjected to electrophoresis on 6% polyacrylamide gel in Tris borate-EDTA buffer and stained with ethidium bromide. Gels were viewed, and the images were stored digitally using an image analysis system consisting of an UV light transilluminator and a charge-coupled device camera. The images were analyzed densitometrically using Densitograph software (AE-6900 MF, ATTO, Tokyo, Japan). For each cDNA sample, the densitometric units of the amplified cDNA fragments were counted for semiquantitative evaluation by normalization with the ß-actin band.
Competitive PCR for IL-4 and IL-5 mRNA
To ensure the level of transcripts of IL-4 and IL-5 mRNA, we performed competitive PCR using cDNA internal standards. DNA competitors were synthesized with the PCR MIMIC Construction Kit (Clontech, Palo Alto, CA). The IL-4 and IL-5 competitors were 540 and 440 bp long, respectively, and their sequences were complementary to the cytokine primers. The competitors were adjusted to 1 x 10-1 amol/µl. One microliter of a 5-fold stepwise dilution of the competitors and the same amount of cDNA were amplified by 36 cycles in the same tubes. Subsequently, gel analysis was done, and the cDNA concentrations of the samples were determined by detecting an equal band intensity of the competitor.
RNA probe synthesis and in situ hybridization
Digoxygenin-labeled rat IL-5 cRNA probes (sense and anti-sense) were generated as follows. The IL-5 PCR product was subcloned into pGEM-T Easy Vector (Promega, Madison, WI). In vitro transcription was performed using a DIG-RNA Labeling Kit (Boehringer Mannheim, Amsterdam, The Netherlands) in the presence of T7 (sense) or SP6 (antisense) RNA polymerase and digoxygenin-labeled UTP. We then prepared 5-µm cryostat sections of the lungs and performed in situ hybridization as described previously (18). In brief, hybridization was done for 16 h at 45°C using sense or antisense IL-5 cRNA probes. Posthybridization was conducted under highly stringent conditions, and RNase A treatment was performed. The signals were detected using a DIG detection kit (Boehringer Mannheim). IL-5 mRNA positive cells around the airways were quantified with an analyzing software (MacSCOPE, Mitani, Toyama, Japan), and the number of the positive cells was expressed per millimeter of length of basement membrane.
Measurement of antigenic levels of IL-4 and IFN-
To confirm the difference of cytokine profile in between young
and aged rats, we also measured the antigenic levels of IL-4 and
IFN- in BAL fluid supernatants and supernatants of cultured pLN
cells with ELISA test kits (Biosource International, Camarillo, CA);
the detection limit of the kit was 2 or 15 pg/ml for IL-4 or IFN-,
respectively. BAL fluid supernatants were concentrated 10 times with an
ultrafiltration membrane (molecular limit,10,000) (Millipore, Tokyo,
Japan).
Statistical analysis
All values are expressed as the mean ± SEM. To compare values between groups, data were subjected to one-way ANOVA plus the Mann-Whitney U test. Significance was accepted at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the optimal conditions, we conducted preliminary time
course study of cellular differentiation and cytokine mRNA levels in
BAL cells after OVA inhalation challenge in young rats (Table I
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Semiquantitative RT-PCR analysis of BAL cells showed that the
expression of IL-4 and IL-5 mRNA increased up to 24 h after Ag
challenge and then decreased at 48 h (Table II). Whereas transcripts of IL-10 and
IFN- reached a plateau at 24 through 48 h, there was a
nonsignificant change of IL-2 mRNA levels after OVA provocation. The
BSA inhalation failed to induce significant changes in cytokine mRNA
expression compared with saline inhalation (Table II). On the basis of
these data, we performed subsequent analysis of BAL cell cytokine mRNAs
at 24 h after OVA inhalation challenge.
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We confirmed that there was no increase in the number of
eosinophils in aged rats 24 h after OVA challenge (Fig. 1), a finding consistent with our
previous observation (16). The number of eosinophils in
aged rats was approximately one-tenth of that seen in young rats (Fig. 1). The number of neutrophils and macrophages was also higher in the
young group, whereas there was no significant difference of lymphocytes
between the two groups (Fig. 1).
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Expression of mRNA.
Fig. 2 shows cytokine mRNA expression in
BAL cells from young and aged rats at 24 h after Ag exposure.
Expression of IL-4 and IL-5 mRNAs was significantly higher in the young
group than in the aged group, whereas IFN- mRNA was higher in the
aged rats. The expression of IL-2 and IL-10 mRNAs did not differ
between the groups.
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in BAL fluids.
ELISA analysis confirmed that the protein levels of IL-4 in BAL fluid
supernatants were significantly higher in young rats than aged rats
(young vs aged, 18.36 ± 2.19 vs 3.26 ± 0.43,
p < 0.01). However, we could not detect IFN- with
ELISA in BAL fluid supernatants from both groups. Histological localization of IL-5 mRNA expression
We performed in situ hybridization to detect localization of the
cells expressing IL-5 mRNA. There was a substantial number of
IL-5-positive cells in the peripheral airways of lungs from the young
rats (Figs. 4, A and
B). In contrast, there were few IL-5-positive cells in the
lungs of the aged rats (Figs. 4, C and D). The
quantitative analysis of the in situ hybridization showed that the
number of IL-5 mRNA-positive cells per length of basement membrane was
significantly larger in the tissue from young rats than those from aged
rats (young vs aged, 40.74 ± 4.58 vs 6.35 ± 0.73, p <
0.01).
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Expression of cytokine mRNA. To further analyze the age-related differences of cytokine expression shown by Ag-stimulated lymphocytes, we tested in vitro cytokine mRNA expression by pLN cells. These pLN cells showed no significant difference in the proportion of OX-19-positive cells between young and aged animals (young vs aged, 49.0 ± 4.0% vs 40.8 ± 1.3%) by flow cytometric analysis. In addition, May-Giemsa staining of pLN cells revealed that there were few eosinophils among these cells in both groups of animals.
Sensitized pLN cells were incubated in the presence or absence of OVA,
and cytokine mRNAs were studied by semiquantitative RT-PCR analysis.
When the cells were cultured without OVA, there was no significant
increase of IL-5 mRNA expression during the 24-h incubation period
(Fig. 5A). However, incubation
with OVA caused a significant increase of IL-5 mRNA expression in the
young rats, but not in the aged rats. In contrast, there was no
age-dependent difference in the expression of ß-actin mRNA (Figs. 5A and 6).
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, freshly harvested cells expressed the
mRNAs at a certain level in both groups (Figs. 5
, B and
C). However, when the cells were incubated with OVA,
enhanced expression of IL-4 mRNA was seen in the young rats, and
increased IFN- mRNA expression was observed in the aged rats after
24 h of incubation (Figs. 5, B and C). In
contrast, the cells lost their expression of these mRNAs during
incubation without OVA.
ELISA analysis for IL-4 and IFN- in the supernatants of cultured
pLN cells.
Results of ELISA study for IL-4 and IFN- in supernatants of cultured
pLN cells were shown in Table III. In
supernatants from young rats, the elevated levels of IL-4 were observed
when the cells were cultured with OVA, whereas we did not detect IL-4
in those from aged animals. In contrast, the levels of IFN- were
significantly higher in the supernatants of the cells cultured with OVA
in aged rats than in those in young rats.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that OVA-sensitized aged rats failed to exhibit
Ag-specific eosinophilic bronchial inflammation with decreased airway
reactivity to Ag exposure. In addition, serum OVA-specific IgE levels
were also significantly lower in aged rats than in young ones when both
groups were sensitized in the same way (young vs aged, 0.698 ±
0.10 vs 0.446 ± 0.06 (absorbance unit), p <
0.05) (16). In the present study, we focused on
age-related changes of cytokine profile in BN rats. We observed that
mRNAs for Th2 type cytokines (IL-4 and IL-5) were decreased in BAL
cells from aged rats, whereas the level of a Th1 type cytokine
(IFN-) was higher in the aged rats than in the young rats after Ag
challenge. Furthermore, our in vitro study of cytokine profile in
OVA-stimulated pLN cells showed that cells from aged rats
preferentially expressed Th1 type cytokine in response to Ag
stimulation, whereas young rats predominantly expressed Th2 type
cytokines. These findings were consistent with our previous in vitro
observations that BCG-primed splenocytes from aged Sprague Dawley rats
produced more IFN- than those from the young animals in response to
BCG restimulation (26). Based on these findings, it is
suggested that age-related changes of cytokine profile can be
attributable to the decreased allergic reaction with aging.
IL-5 is considered to be a key effector cytokine in eosinophilic infiltration of the allergic inflammatory sites (9, 27). This would be consistent with our previous observation that pretreatment of young rats with an anti-IL-5 Ab suppressed pulmonary eosinophilia (16). The present study further indicated that IL-5 mRNA expression in BAL cells and the number of IL-5-positive cells in the lungs were decreased in aged animals along with the failure to elicit allergic inflammation. Although previous reports have indicated that IL-5 is detected not only in CD4+ T cells (known to be the major source in allergic inflammation (4, 28)) but also in mast cells (29) and eosinophils (30), we did not assess exactly how each of these cell types contributed to IL-5 mRNA expression in the present study. However, our in vitro experiments with Ag-primed pLN cells, which contained few eosinophils and mast cells, provided evidence that an age-associated decline of the capacity in lymphocytes to express IL-5 mRNA was involved in the decrease of IL-5 mRNA expression in BAL cells from aged rats.
IL-2 is known to be essential for the proliferation and clonal expansion of Ag-specific T cells (31). On the other hand, IL-10 was originally considered to be a predominantly Th2 type cytokine in mice (3), although its role in allergic reaction remains unclear. We found no significant changes in the expression of IL-2 and IL-10 mRNA in BAL cells from young and aged rats, suggesting that these cytokines are not intimately involved in age-associated alterations of allergic inflammation.
Some investigators have suggested that cytokines produced by CD8+ T cells have suppressive effect on allergic inflammation (32). In this regard, we have previously demonstrated that there was no difference in the CD4:CD8 ratio of BAL cells from young and aged rats (16). Thus, it seems unlikely that a change in the CD4:CD8 balance was involved in the producing different cytokine profiles of the young and aged animals, although this observation by no means excludes the possibility that aged rats show functional dominance of CD8+ T cells.
It has been widely recognized that the alterations in immune system
during the aging process are mainly characterized by quantitative and
qualitative changes of lymphocytes, especially T cells (33, 34). There is a large body of literature showing that T cells
from aged humans and rodents have a decreased ability to proliferate
and to yield clonal expansion (31, 33), which appear to be
partially caused by the impairment of calcium-dependent signal
transduction in aged T cells (35). With respect to
age-related alterations of cytokine profiles, Hobbs et al.
(36) have reported that T cells from the spleens of aged
mice expressed higher levels of not only IFN- mRNA but also IL-4 and
IL-5 mRNAs, when compared with young animals after stimulation with a
plate-bound anti-CD3 Ab. In contrast, our data clearly demonstrated
that IL-4 and IL-5 transcripts were selectively suppressed in aged
animals. This discrepancy between their study and ours may be explained
by the different methods used. We used an in vivo model with
stimulation by a specific Ag (OVA), whereas they used an in vitro model
with nonspecific stimulation signals (anti-CD3 Ab). Because it has
been shown that the cytokine profiles are markedly influenced by the
priming status in vivo (37, 38), we believe that our model
is more suitable for the analysis of alterations in Ag-specific
cytokine profile generated in the physiological changes associated with
the aging process.
The molecular mechanism underlying age-associated changes of the
cytokine profile has proved elusive. The early decision toward Th1 and
Th2 cells in immune response depends on the balance between IL-12 or
IFN- (39, 40), which favors Th1 responses, and IL-4 or
IL-10, which favors Th2 responses (41). Thus, Th1
differentiation begins with the production of IL-12 by macrophages and
the subsequent induction of IFN- production by NK cells
(40). However, the sources of the initial IL-4 needed to
induce Th2 development have not been clearly defined, although
candidate sources of IL-4 include
NK+CD4+ T cells,
CD4+ memory T cells, eosinophils, and mast
cell/basophil lineage. It is therefore conceivable that dysfunction of
IL-4 production by these cells during sensitization may be involved in
age-associated changes of the cytokine profile. Taken together, our
findings suggest that decreased Th2 type cytokine might be responsible
for the suppression of allergic reactions in aged animals.
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Footnotes |
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2 Abbreviations used in this paper: BAL, bronchoalveolar lavage; pLN, popliteal lymph node.
Received for publication August 31, 1998. Accepted for publication April 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Immunology 79:562.[Medline]
in immune regulation. I. IFN- inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol. 140:4245.[Abstract]
in ovalbumin-exposed sensitized Brown-Norway rats. Immunology 88:247.[Medline]
in murine collagen-induced arthritis. Eur. J. Immunol. 25:1184.[Medline]
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, macrophages.
