| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Oral Biology and
Microbiology, Immunobiology Vaccine Center, University of Alabama, Birmingham, AL 35294; and
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, in both
peripheral (i.e., spleen) and mucosal (i.e., Peyer’s patches (PP))
lymphoid compartments (10, 11). Selective induction of Th2
cells was associated with mucosal secretory IgA and serum IgG1, IgG2b,
and potent IgE Ab responses (12).
The T cells which occur in the elderly are often characterized by
altered phenotypes, reduced responses to mitogens and impaired cytokine
production (13, 14, 15). Further, it has been reported that
senescent T cells, which were of the memory type, showed decreased
intracellular phosphorylation of CD3
(16). Thus, it is
likely that these age-related T cell responses exhibit altered help for
B cell and Ab responses. Indeed, it was shown that T cells from aged
mice down-regulate B cell responsiveness. Increased numbers of splenic
CD5+ B cells producing IL-10 were found in aged
mice (17). Additionally, recent studies showed the
development of stable clonal B cell populations in aged mice detected
by Ig heavy chain mRNA CDR3 size analysis (18, 19).
Despite these well known age-associated changes as well as recent observations made possible by advances in cellular and molecular analysis of the induction and regulation of mucosal immune responses, the precise nature of mucosal immune responses which occur in the elderly remain poorly defined. To gain more information on this important issue, we have compared mucosal and systemic immune responses of aged (either 12–14 or 24 mo) and young adult (6–8 wk) C57BL/6 mice by using our well-characterized oral immunization regimen with OVA and CT as mucosal adjuvant.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 mice were purchased from the Frederick Cancer Research
Facility (National Cancer Institute, Frederick, MD). Upon receipt, mice
were transferred to microisolators, maintained in horizontal flow
cabinets, and provided with sterile food and water ad libitum.
Experiments were performed using young adult mice between 6 and 8 wk of
age or aged mice either between 12 and 14 mo (
1 year), or mice over
2 years of age.
Immunization
Aged and young adult mice were immunized three times at weekly intervals with oral doses of 1 mg OVA (Fraction V; Sigma, St. Louis, MO) and 10 µg CT (List Biological Laboratories, Campbell, CA) in PBS (10, 20). Serum and mucosal secretions were collected on days 7, 14, and 21. In one series, mice were immunized on day 35 and serum and mucosal secretions were collected 1 wk later. In some experiments, mice were immunized s.c. on days 0 and 7 with 100 µg OVA and 1 µg CT (11). The mice were sacrificed 7 days after the last immunization. Spleen as well as PP and intestinal lamina propria (LP) were extracted as examples of systemic and mucosal tissues, respectively, and subjected to OVA-specific enzyme-linked immunospot (ELISPOT) assays. Further, CD4+ T cells isolated from spleen and/or PP were examined for the analysis of Ag-specific T cell-derived cytokine.
Ab assays
Ab titers in serum and fecal extracts were determined by ELISA
(21, 22). Falcon Microtest assay plates (Becton Dickinson,
Oxnard, CA) were coated with 100 µl of an optimal concentration of
OVA (1 mg/ml) or 100 µl rCT-B (5 µg/ml; List Biological
Laboratories) in PBS. To detect Ag-specific Ab levels, HRP-conjugated
goat anti-mouse µ, , or 
heavy chain-specific Abs (Southern
Biotechnology Associates, Birmingham, AL) were employed. For IgG
subclass determination, biotinylated mAb specific for IgG1 and IgG2a
(PharMingen, San Diego, CA) and peroxidase-conjugated goat
anti-biotin Ab were employed. Endpoint titers were expressed as the
last dilution yielding an OD at 414 nm (OD414) of
>0.1 U above negative control values after a 15-min incubation. In
some experiments, the plates were coated with 100 µl/well goat
anti-mouse Igs (Southern Biotechnology Associates) at 2 µg/ml.
Total IgM, IgG, and IgA levels in serum and fecal extracts were
estimated by comparison with serial dilutions of mouse IgM, IgG, and
IgA standards (Southern Biotechnology Associates).
Enumeration of Ab-forming cells (AFCs)
The spleen was removed aseptically and single-cell suspensions were prepared as described (21, 22). After being carefully excised from the intestinal wall, PP were dissociated using 0.5 mg/ml collagenase type V (Sigma) to obtain single-cell preparations (10, 20). Mononuclear cells in the LP were isolated after removal of PP from the small intestine using a combination of enzymatic dissociation and discontinuous Percoll gradients (Pharmacia, Uppsala, Sweden; Refs. 10 and 21). Mononuclear cells in the interface between the 40 and 75% layers were removed, washed, and resuspended in RPMI 1640 (Cellgro Mediatech, Washington, DC) supplemented with 15 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (complete medium). An ELISPOT assay was employed to detect cells producing IgM, IgG, and IgA Abs. Ninety-six-well nitrocellulose plates (Millititer HA, Millipore, Bedford, MA) were coated with 1 mg/ml OVA (100 µl/well) for anti-OVA-specific AFCs; or 5 µg/ml CT-B (100 µl/well) for anti-CT-B-specific AFCs (10, 20, 21, 22).
Ag-specific CD4+ T cell responses
CD4+ T cells were purified by the magnetic activated cell sorter system (Miltenyi Biotec, Sunnyvale, CA) as described previously (21, 22). Briefly, cells were incubated in a nylon wool column (Polysciences, Warrington, PA) to remove B cells and macrophages. Enriched T cell populations were then incubated with biotinylated anti-CD4 (GK 1.5) mAb followed by streptavidin-conjugated microbeads and passed through a magnetized column. The purified T cell fractions were >97% CD4+ and were >99% viable. Cells were resuspended in complete medium and purified CD4+ T cells (4 x 106 cells/ml) were cultured with or without 1 mg/ml OVA in the presence of T cell-depleted, irradiated (3000 rad) splenic APCs. These APCs were derived from naive mice and were placed in 96- or 24-well tissue culture plates (Corning, Corning, NY.) for 5 days at 37°C in a moist atmosphere of 5% CO2 in air. To assess Ag-specific T cell responses, 0.5 µCi of [3H]thymidine (Amersham, Arlington Heights, IL) was added for the final 18 h of incubation. The cells were harvested and the degree of [3H]thymidine incorporation was determined by scintillation counting. In some experiments, culture supernatants were harvested after 2 or 5 days of incubation and were then subjected to cytokine-specific ELISA. For cytokine-specific mRNA analysis, CD4+ T cells were harvested after 2 days of incubation and were then subjected to cytokine-specific, semiquantitative RT-PCR assay.
Cytokine-specific ELISA
Levels of cytokines in culture supernatants were measured by
ELISA. The details of the ELISA for IFN-, IL-2, IL-4, IL-5, IL-6,
and IL-10 have been described previously (21, 22). For
coating and detection, the following mAbs were used: for
anti-IFN-, R4-6A2 and XMG 1.2 mAbs; for anti-IL-2, JES6-1A12
and JES6-5H4 mAbs; for anti-IL-4, BVD4-1D11 and BVD6-24G2 mAbs; for
anti-IL-5, TRFK-5 and TRFK-4 mAbs; for anti-IL-6, MP5-20F3 and
MP5-32C11 mAbs; and for anti-IL-10, JES5-2A5 and JES5-16E3 mAbs.
The levels of cytokines produced by Ag-specific T cells were calculated
by subtracting the results of control cultures (e.g., without Ag
stimulation) from those of Ag-stimulated cultures. This ELISA was
capable of detecting 0.78 ng/ml IFN-, 8 pg/ml IL-2, 23.4 pg/ml IL-4,
0.78 U/ml IL-5, 200 pg/ml IL-6, and 0.4 ng/ml IL-10.
Quantitative analysis of cytokine-specific mRNA
For evaluation of cytokine-specific mRNA levels in OVA-stimulated CD4+ T cells, a competitive RT-PCR was employed (21). Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction procedure. Aliquots of extracted RNA (25 µg/ml) were spiked with a cytokine-specific rRNA standard and subjected to reverse transcriptase reaction using Superscript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD). For the amplification of cDNA, 35 cycles of reaction programmed at 1 min at 95°C and 1 min at 60°C were performed. The PCR product was quantitated by capillary electrophoresis with the laser fluorescence detection system (Laser Induced Fluorescence (LIF), Preparative/Analytical Capillary Electrophoresis (P/ACE); Beckman Coulter, Fullerton, CA) as described previously (21). The fluorescence content of each cytokine-specific RT-PCR product was expressed as the peak area of relative fluorescence light units. The levels of cytokine-specific mRNA were calculated based upon the levels of cytokine-specific rRNA.
Statistics
The significance of the difference (e.g., p values) between groups was evaluated by the Mann Whitney U test using a Statview II program designed for Macintosh computers.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To compare the Ag-specific mucosal immune responses of aged and
young adult mice, it was important to first determine total Ig levels
in mucosal secretions. Aged mice showed higher serum IgM levels than
did young adult mice (Table I
). However,
both serum IgG and IgA levels in aged mice were comparable to those of
young adult mice (Table I). Higher levels of IgG were associated with
fecal extracts of aged when compared with young adult mice. In
contrast, IgM and IgA in fecal extracts showed no significant
differences between aged mice and young adults (Table I). These
results show that although increased levels of serum IgM and fecal
IgG were noted, no significant Ig isotype deficiency occurred with
aging.
|
We next examined OVA-specific immune responses in aged mice. The
young adult and both groups of aged mice were given oral OVA and CT as
adjuvant, and OVA-specific Ab responses in fecal extracts were
examined. Significant IgA Ab responses occurred in the fecal extracts
of young adult mice; however, these Ab responses were low in both of
those groups of aged mice (Fig. 1A). Because it has been shown
that oral immunization with protein Ag and CT induces Ag-specific
responses in systemic as well as mucosal sites, OVA-specific
serum Ab levels were also tested. Levels of OVA-specific IgG Abs were
elevated in the serum of young adult mice by day 14, with peak
responses seen 7 days later. In contrast, aged mice showed no
detectable levels of Ag-specific mucosal or systemic Ab responses at
day 14. Significant levels of OVA-specific serum IgG Ab responses were
detected in both groups of aged mice by day 21; however, these
responses were significantly lower than those seen in young adult mice
(Fig. 1B, p < 0.05). Further, OVA-specific
serum IgA Ab responses were also less marked in aged than in young
adult mice (Fig. 1B, p < 0.05). These
responses remained low and did not increase when mice were boosted with
an additional oral dose at day 35 (data not shown). When IgG subclass
responses were examined, aged mice showed lower levels of serum IgG1 Ab
responses than young adult mice (Fig. 1C, p
< 0.05). As expected, low OVA-specific IgG2a Ab responses were seen in
young adult as well as in all aged mice, and this is due to the
propensity of CT to induce Th2-type responses when used as mucosal
adjuvant. These results indicate that aged mice possess lower
Ag-specific IgG and IgA Ab responses in serum and in mucosal secretions
than do young adult mice.
|
These results were further confirmed at the B cell level by using
an Ag-specific ELISPOT assay (Fig. 2).
Mononuclear cells from LP, PP, and spleen taken from young adult or
both groups of aged mice were subjected to OVA-specific ELISPOT assay
to determine the numbers of AFCs. Both groups of aged mice possessed
8- to 9-fold lower numbers of IgA AFCs in LP than did young adult
mice (700/106). Similarly, aged mice showed
reductions in both OVA-specific IgA AFCs in PP and in anti-OVA IgG
and IgA AFCs in the spleen. In contrast, both groups of aged mice
showed increased numbers of OVA-specific IgM AFCs in spleen,
suggesting that switches to both IgG and IgA were impaired. These
results show that immunity is comparably impaired in 1- and 2-year-old
mice given oral OVA and CT as mucosal adjuvant.
|
It was important to test whether immune responses to CT were also
reduced in aged mice because CT is a potent mucosal Ag as well as an
adjuvant. Young adult mice given oral OVA and CT three times at weekly
intervals showed significantly high levels of CT-B-specific IgA and IgG
Ab responses in fecal extracts and serum samples, respectively. In
contrast, these mucosal and systemic anti-CT-B Ab responses were
reduced in both 1-year-old mice and in those over 2 years of age (Fig. 3, A and B;
p < 0.05). High levels of CT-B-specific serum IgG1 Ab
responses were noted in young adult mice; however, these responses were
markedly reduced in both groups of aged mice (Fig. 3C,
p < 0.05). Furthermore, significantly reduced numbers
of CT-B-specific IgA AFCs were seen in the PP and LP of both groups of
aged mice. In addition, the splenic CT-B-specific IgG AFCs were also
reduced (Fig. 4). These results show that
even strong immunogens such as CT fail to elicit serum or mucosal Ab
responses in mice aged beyond the first year of life.
|
|
Because both 1- and 2-year-old mice showed impaired Ab responses
when OVA and CT were administered orally, it was important to test
whether similar immune dysregulation occurs in both groups of aged mice
immunized by the perenteral route. Both groups of aged mice and young
adult mice were immunized s.c. with OVA plus CT. When OVA-specific IgG
Ab responses in serum and AFCs in spleen were examined, both groups of
aged mice showed reduced IgG Ab levels as well as lower AFC responses
(Fig. 5, A and B).
Interestingly, 1-year-old mice immunized with OVA and CT s.c. contained
high levels of CT-B-specific serum IgG Abs that were identical with
those of young adult mice (Fig. 6A). In contrast, mice over 2
years of age showed markedly reduced anti-CT-B IgG Ab responses
(Fig. 6A, p < 0.05). Similarly,
CT-B-specific splenic IgG AFCs were seen in 1-year-old mice but not in
the 2-year-old mouse group (Fig. 6B). Although these results
show that aging alters immune responses in the systemic compartment,
significant immunity is retained through the first year of
life.
|
|
Our findings to this point suggest that early age-associated
changes occur in the mucosal immune system which precede similar
alterations in the systemic immune system. To determine whether this
occurs at the level of T cell help, we next assessed T
cell-proliferative responses in young adult and aged mice.
CD4+ T cells from spleen or PP were cultured with
OVA in the presence of irradiated T cell-depleted spleen cells.
CD4+ T cells from both spleen and PP of aged mice
immunized orally with OVA and CT showed lower proliferative responses
than did those of young adult mice (Fig. 7A). After systemic
immunization, most of the 1-year-old mice showed reduced splenic
CD4+ T cell proliferation, but 3 of 16 1-year-old
mice showed T cell-proliferative responses which were comparable to
those of young adult mice (Fig. 7B). Similarly, and in
marked contrast to impaired responses seen in mice over 2 years of age
(data not shown), CT-B-specific proliferative responses were not
reduced in 1-year-old mice. These results further support our
observations that systemic immune responses in 1-year-old mice are in a
transition period, and that mucosal age-associated alterations have
already taken place.
|
We next examined Ag-specific Th1 and Th2 cytokine responses in
young adult and aged mice. The culture supernatants from splenic
CD4+ T cells of young adult mice given oral OVA
plus CT exhibited high levels of OVA-specific IL-4 and IL-5 production
(Table II). Further,
CD4+ T cells from PP of these mice produced
significant levels of IL-4 after OVA re-stimulation (Table II). In
contrast, CD4+ T cell cultures of spleen or PP
from both groups of aged mice orally immunized with OVA plus CT had no
detectable levels of Th1 or Th2 cytokines. OVA-specific Th1- and
Th2-type cytokine responses were also examined in systemically
immunized young adult and aged mice. As seen in orally immunized mice,
splenic CD4+ T cells from young adult mice
produced high levels of IL-4 and IL-5. In contrast, synthesis of these
cytokines in 1-year-old mice was much less marked than in older mice.
Thus, significant levels of IL-4 (55.3 ± 26.2 pg/ml) and IL-5
(5.3 ± 4.0 U/ml) synthesis were detected in
CD4+ T cell cultures from 1-year-old mice (Table II). These results were further confirmed at the mRNA level by using
IL-4-specific quantitative RT-PCR analysis (Table III). Thus, orally or systemically
immunized young adult mice showed high levels of IL-4-specific mRNA in
spleen and PP. With the exception of 1-year-old mice immunized
systemically, all groups of aged mice given OVA and CT showed no
detectable IL-4 mRNA. Taken together, our results indicate that
age-associated dysfunction occurs in the mucosal immune system well
before it is seen in the systemic immune compartment.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has been shown that aging is associated with several dysfunctional stages in lymphocyte activation, particularly with progression of lymphocytes to a state of immune unresponsiveness to Ags and to an increased incidence of autoimmune disease (23, 24). Especially affected are T cell responses including IL-2 production, IL-2 receptor expression, signal transduction, and programmed cell death, all of which have been reported in the elderly (25, 26, 27, 28, 29, 30, 31). Our previous studies have shown that mucosal immunization with protein Ags coadministered with CT as mucosal adjuvant elicited Th2-type cytokines, especially IL-4, and mediated mucosal and systemic immune responses to protein Ag as well as to CT itself (10, 20, 32, 33). This mucosal immunization regimen is thus ideal to address the effects of aging on the mucosal immune system in mice. This study confirmed previous observations that Ag-specific B and T cell responses occur in 2-year-old mice. More importantly, our findings are the first to show that age-associated immune dysregulation occurs in the mucosal system at both cellular and molecular levels much earlier than occurs in the systemic lymphoid system.
Another novel finding of the present study is that parenteral immune responses in 1-year-old mice immunized s.c. with OVA and CT showed less marked immune deficiency than did those of mice over 2 years of age. In this regard, serum anti-CT-B IgG Ab responses in 1-year-old mice were comparable to those of young adult mice, even though anti-OVA IgG Ab responses were essentially absent. Further, intact OVA-specific CD4+ T cell-proliferative responses were seen in 3 of 16 1-year-old mice used in this study. Although we cannot yet explain how intact OVA-specific T cell responses occur in the absence of anti-OVA IgG Ab responses, additional studies of cytokine synthesis by these T cells, as well as of costimulatory molecule expression and B cell characterization in 1-year-old mice is underway in our laboratory. Our previous study showed that CD40 ligand-deficient mice immunized s.c. with OVA and CFA revealed significantly elevated OVA-specific CD4+ T cell-proliferative but not Ab responses (34). Furthermore, a recent study indicated that CT enhanced CD86 expression on APCs and these effects were not influenced by CD40-CD40 ligand interactions (35). These results suggest that age-associated alterations in CD40 ligand expression by splenic CD4+ T cells may occur in 1-year-old mice. Based upon the present study, we suggest that the parenteral immune system in 1-year-old mice may be in a transitional stage between normal and age-associated deficiency. Thus, Ab responses to the weak Ag OVA, which always requires an adjuvant for the induction of immunity was impaired in 1-year-old mice; however, CT, a potent Ag, induced normal immune responses in these mice. Indeed, the majority of past studies have used 24-mo-old mice to investigate age-associated dysregulation in T and B cell immunity. Another possible explanation for the unique pattern of parenteral immune responses in 1-year-old mice is that the adjuvant activity of CT may be lost; however, the molecule may retain full antigenicity. Thus, 1-year-old mice may be an ideal model to investigate the cellular and molecular mechanisms for CT adjuvanticity when compared with immunogenicity.
In contrast, mucosal immune responses including both OVA- and CT-B-specific Ab and cytokine responses induced by oral OVA and CT in 1-year-old mice were markedly reduced and were comparable to those seen in 2-year-old mice. In addition, systemic immune responses were also diminished in 1-year-old mice given oral OVA and CT. When one considers the common mucosal immune system, mucosal inductive sites such as the PP play a central role in the induction of Ag-specific immune responses in both mucosal and systemic tissues. Thus, the decreased IgG Ab responses in 1-year-old mice immunized with oral OVA plus CT may simply be due to the impaired mucosal immune system in these mice. To support this, systemic immunization resulted in intact OVA-specific T cell responses as well as CT-B-specific Ab responses in 1-year-old mice. These results indicate age-associated alterations arise in the mucosal immune system earlier than in the parenteral immune compartment.
The central importance of the mucosal immune system as a first line of defense against numerous pathogens has been well established. For example, pathogens that are encountered after ingestion or inhalation may subsequently colonize the gastrointestinal or upper respiratory tracts (1, 2). Both oral and nasal immunizations have been shown to effectively induce mucosal immune responses at the mucosal barrier itself (1, 2). We feel that our findings presented in this report may be of importance in efforts to develop effective mucosal vaccines for the elderly. For example vaccines to prevent influenza and Streptococcus pneumoniae pulmonary pneumonia are less effective in the elderly. Thus, one may postulate that induction of mucosal immunity in middle-aged as well as aged individuals would be difficult to achieve and may explain vaccine failures. In addition, innate immunity also plays important roles in host defense as well as maintaining immune homeostasis. Because adoptive immunity is impaired in aged mice, it is possible that the innate immune system such as natural IgA Ab responses may provide compensatory functions for protection. Indeed, our results showed that total mucosal IgA Ab levels were normal in aged mice. The exact mechanisms which resulted in enhanced innate and diminished adoptive immunity in aged mice are currently under investigation in our laboratory by using recombinant Salmonella expressing OVA or the Tox C fragment of tetanus toxin as a mucosal Ag delivery system. Furthermore, oral tolerance is an important host mechanism to maintain immune homeostasis against normal flora Ags as well as environmental Ags. Thus, our current efforts are also focused on elucidation of the precise cellular and molecular mechanisms that regulate oral tolerance in aged mice.
Because our results showed that CD4+ T cells from aged mice exhibited lower proliferative responses than those taken from young adult mice, it is possible that the age-associated reductions in Ag-specific Ab and T cell-proliferative responses could involve an alteration in responsiveness to T cell growth factors such as IL-2. Indeed, a reduced frequency of IL-2-producing CD4+ T cells and a low IL-2 receptor expression by this T cell population (28, 29) are one result of aging. Mucosal IL-2 treatment may allow us to overcome age-impaired mucosal immune responses by enhancing mucosal immunity or abrogating unresponsiveness in aged mice. Our previous study showed that oral or nasal administration of rIL-12 successfully converted Th2-type responses induced by OVA and CT to Th1-type responses with intact mucosal IgA Abs (36, 37). Furthermore, recent work showed that the effects of aging on IL-2 production can be abrogated by exogenous IL-2 delivery (38). Thus, it is possible that pretreatment of mice with mucosal rIL-2 could induce Ag-specific mucosal IgA and IgG responses in aged mice immunized orally with OVA and CT. Studies to address this possibility are currently underway in our group.
With regard to IL-4 synthesis, it has been reported that anti-CD3 mAb-stimulated CD4+ T cells elicited higher levels of IL-4 synthesis in senescent than in young adult mice (39, 40, 41, 42). Indeed, spontaneous IL-4 production by CD4+ T cells from PP of 1-year-old mice was higher than for PP CD4+ T cells from young adult mice (aged mice, 510.5 ± 220.8 pg/ml vs young mice, 344.9 ± 98.1 pg/ml); however, these differences were not statistically significant. In contrast to these findings, cytokine analyses at both the protein and mRNA levels revealed low or no IL-4 production by Ag-stimulated CD4+ T cells from either PP or spleen of aged mice. The discrepancies between our results and those of others can be explained simply by the lack of Ag-specific memory CD4+ T cell induction in aged mice. The polyclonal stimulation signals such as anti-CD3 or the combination of anti-CD3 and anti-CD28 could be of sufficient potency to trigger CD45RBlow and CD44high, CD4+ memory T cells, the major T cell population occurring in senescence, to produce IL-4. In contrast, Ag-induced IL-4 synthesis may require the generation of effector memory cells from naive CD4+ T cells, a pathway which appears to be defective in aged mice (38). Thus, low levels of Ag-specific IgA and IgG Ab responses in aged mice immunized orally with OVA and CT may be explained by a lack of these Th2-type cytokine-producing cells. To support this contention, it was shown that impaired mucosal IgA Ab responses were detected in IL-4-deficient mice immunized with protein Ag and CT as mucosal adjuvant (32, 43). Further, it has been shown that both IL-4 and IL-5 are important B cell stimulating and growth factors (44, 45).
The presence of a high frequency of 
T cells in the epithelium of
the intestine is one of the unique characteristics of the mucosal
immune system (46, 47). It was shown that a reduced
frequency of intraepithelial T cells occurs in the small
intestine of senescent mice (48). We have also confirmed
this finding in 1-year-old mice using flow cytometry and histological
analysis (data not shown). Previous studies showed that intraepithelial
T cells were essential for the maintenance of the biological
functions of intestinal epithelial cells (49). Thus, a
reduced frequency of intraepithelial T cells may result in
leakage of LP Abs into the lumen of the small intestine. Indeed,
elevated levels of total IgG were seen in fecal extracts of aged mice.
Our previous study has further shown that these T cells play an
important role in the induction and regulation of mucosal IgA responses
(20). For example, TCR-deficient mice, which lack
T cells, exhibit impaired levels of both total and Ag-specific
IgA Ab responses when immunized orally with TT and CT
(20). Thus, a low frequency of T cells in the
epithelium of aged mice may partially explain why impaired mucosal IgA
Ab responses are seen. To directly test this idea, we are currently
investigating mucosal immune responses in senescent TCR knockout
mice as well as in anti- mAb-treated, aged mice.
In summary, the present study has shown impaired mucosal B and T cell immunity for Ag-specific Ab and CD4+ T cell responses at the protein, cellular, and molecular levels. Our results are especially important in that they show that age-associated alterations possibly arise first in the mucosal immune system. To provide improved immunity in the elderly against various infectious diseases, it will be essential to elucidate the precise cellular and molecular mechanisms at work in the senescent mucosal immune system.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Kohtaro Fujihashi, Department of Oral Biology, Immunobiology Vaccine Center, University of Alabama, Medical Center, BBRB 761, Birmingham, AL 35294-2170.
3 Abbreviations used in this paper: CT, cholera toxin; TT, tetanus toxoid; PP, Peyer’s patches; LP, lamina propria; AFC, Ab-forming cell; ELISPOT, enzyme-linked immunospot; CT-B, CT B subunit.
Received for publication June 28, 2000. Accepted for publication August 9, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression. J. Immunol. 151:575.[Abstract]
/ T cell-deficient mice have impaired mucosal immunoglobulin A responses. J. Exp. Med. 183:1929. T cells regulate mucosally induced tolerance in a dose dependent fashion. Intern. Immunol. 11:1907.-chain-binding cytokines, can reverse the defect in generation of CD4 effector T cells from naive T cells of aged mice. J. Exp. Med. 190:1013. T-cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nature 333:855.[Medline]
T cells. Nature 336:479.[Medline]
T cells. Proc. Natl. Acad. Sci. USA 92:6147.This article has been cited by other articles:
|
|
 |
|
  H. Kiyohara, T. Nagai, K. Munakata, K. Nonaka, T. Hanawa, S. J. Kim, and H. Yamada Stimulating Effect of Japanese Herbal (Kampo) Medicine, Hochuekkito on Upper Respiratory Mucosal Immune System Evid. Based Complement. Altern. Med., December 1, 2006; 3(4): 459 - 467. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hagiwara, Y. I. Kawamura, K. Kataoka, B. Rahima, R. J. Jackson, K. Komase, T. Dohi, P. N. Boyaka, Y. Takeda, H. Kiyono, et al. A Second Generation of Double Mutant Cholera Toxin Adjuvants: Enhanced Immunity without Intracellular Trafficking. J. Immunol., September 1, 2006; 177(5): 3045 - 3054. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Alignani, B. Maletto, M. Liscovsky, A. Ropolo, G. Moron, and M. C. Pistoresi-Palencia Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice J. Leukoc. Biol., June 1, 2005; 77(6): 898 - 905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Kobayashi, T. Kohda, K. Kataoka, H. Ihara, S. Kozaki, D. W. Pascual, H. F. Staats, H. Kiyono, J. R. McGhee, and K. Fujihashi A Novel Neurotoxoid Vaccine Prevents Mucosal Botulism J. Immunol., February 15, 2005; 174(4): 2190 - 2195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Fayad, H. Zhang, D. Quinn, Y. Huang, and L. Qiao Oral Administration with Papillomavirus Pseudovirus Encoding IL-2 Fully Restores Mucosal and Systemic Immune Responses to Vaccinations in Aged Mice J. Immunol., August 15, 2004; 173(4): 2692 - 2698. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kataoka, J. R. McGhee, R. Kobayashi, K. Fujihashi, S. Shizukuishi, and K. Fujihashi Nasal Flt3 Ligand cDNA Elicits CD11c+CD8+ Dendritic Cells for Enhanced Mucosal Immunity J. Immunol., March 15, 2004; 172(6): 3612 - 3619. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hagiwara, J. R. McGhee, K. Fujihashi, R. Kobayashi, N. Yoshino, K. Kataoka, Y. Etani, M.-N. Kweon, S. Tamura, T. Kurata, et al. Protective Mucosal Immunity in Aging Is Associated with Functional CD4+ T Cells in Nasopharyngeal-Associated Lymphoreticular Tissue J. Immunol., February 15, 2003; 170(4): 1754 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kato, K. Fujihashi, R. Kato, T. Dohi, K. Fujihashi, Y. Hagiwara, K. Kataoka, R. Kobayashi, and J. R. McGhee Lack of oral tolerance in aging is due to sequential loss of Peyer's patch cell interactions Int. Immunol., February 1, 2003; 15(2): 145 - 158. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), over
2-year-old (
), and young adult (
) mice. Each group of mice was
immunized once a week for 3 wk with oral doses of 1 mg OVA and 10 µg
CT as mucosal adjuvant. Seven days after the last oral immunization,
IgM, IgG, and IgA Ab levels in fecal extracts (A), serum
(B), and IgG subclass levels in serum (C)
were determined by OVA-specific ELISA. Values shown are the mean
± SEM for 15 mice in each experimental group.
