Abstract
This study demonstrates that the mucosal immune response to cholera toxin (CT) is compromised in old rats in comparison with young animals. The total number of immunoglobulin A (IgA)-secreting cells is similar or higher in the intestinal inductor and effector sites in old animals. However, the number of specifically induced anti-CT IgA antibody-secreting cells is lower in these tissues in comparison with those in young animals. The kinetics of this immune response in the different gut-associated lymphoid tissues studied suggests that the age-associated decline in the number of anti-CT IgA-secreting cells in the intestinal mucosa reflects impaired IgA immunoblast migration. Our data from lymphocyte adoptive transfer studies indicate that factors intrinsic to both the donor cells and the host recipient influence the migration of immunoblasts from the Peyer's patches to the effector site. For example, donor cells from old donors transferred to either young or old recipient rats migrate slower than young donor lymphocytes transferred into old host animals. In vitro studies clearly indicate that ageing does not impair antibody secretion by intestinal mucosal plasma cells. Therefore, the age-related decline in the intestinal mucosal immune response, e.g. diminished specific antibody titres in intestinal lavage, reflects fewer antibody-secreting cells in the mucosa.
Introduction
Although systemic immunosenescence is well documented, the mucosal immune response has not been well characterized in the elderly (for review see ref. 1). Epidemiological data demonstrate a correlation between ageing and an increased incidence of infectious diseases of the intestinal and respiratory tracts.2–4 Research studies reported increased serum immunoglobulin A (IgA) levels and decreased responses to antigenic challenges in a variety of species, including rodents, primates and humans, as a function of increasing age.5–9 For example, the IgA titre in the intestinal lumen declines 15–20% between maturity and senescence in mice.10 Furthermore, Schmucker et al. demonstrated significant declines in the intestinal IgA antibody responses to cholera toxin (CT) in rats and rhesus macaques.9,11
Diminished IgA titres in the intestine may reflect fewer IgA plasma cells in the intestinal mucosa, reduced local antibody production, or a decline in the transport of antibodies to the mucosal surface by the intestinal epithelial cells. Previously, we reported a six-fold age-related decline in the blood-to-bile transport of rat dimeric IgA in Fischer rats.12 This decline correlated with the concomitant loss of polymeric immunoglobulin receptors from the sinusoidal surface of hepatocytes.13 However, the absence of a similar loss of polymeric immunoglobulin receptors from the basolateral plasma membranes of either rat or monkey small intestinal enterocytes indicates that the transport mechanism for IgA across the mucosal epithelium is not impaired in old animals.9,14
The number of specific antibody-containing cells in the intestinal mucosa of old immunized rats is significantly reduced in comparison to that in young animals, substantiating our contention that ageing compromises the migration of putative IgA plasma cells to the intestinal mucosa.11,15 Furthermore, we are unaware of any data concerning the effect of ageing on antibody production/secretion by plasma cells localized to the intestinal mucosa. We suggest that intestinal immunosenescence, manifested by fewer plasma cells and diminished antibody titres at the effector site, reflects impaired IgA immunoblast migration to the intestine, reduced local antibody production, or both dysfunctions. The present study assessed the impact of ageing on these two critical events in order to determine whether or not their impairment contributes significantly to intestinal mucosal immunosenescence.
Materials and methods
Animals
Male Fischer 344/NHsd rats from the National Institute on Aging (Harlan-Sprague Dawley, Inc., Indianapolis, IN) were used throughout this study. Healthy animals were segregated into young adult (4–6 months) and old (> 24 months) age groups; the latter animals exceeded the 50% survivorship level and were considered to be senescent. The animals were housed under barrier conditions and fed standard laboratory chow and water ad libitum. Animals received intraduodenal injections of either 100 µg of CT (List Biologicals Laboratories, Campbell, CA) in 0·5 ml 0·01 m phosphate-buffered saline (PBS, pH 7·4) containing 0·2% gelatine or the vehicle alone on days 0 (primary) and 14 (boost).11 Groups of six immunized and six naı¨ve animals were fasted overnight before being killed on days 5, 7, 14 and 21 for specimen collection. Serum samples were frozen and stored at −70° until analysed. The spleen (SP), mesenteric lymph nodes (MLN) and small intestine were removed and used to prepare fresh cell suspensions.
Antibodies
Affinity-purified polyclonal sheep anti-rat IgA (The Binding Site, San Diego, CA), biotinylated goat anti-rat IgA (Rockland Immunochemicals, Gilbertsville, PA) and the IR699 rat myeloma dimeric IgA (courtesy of Dr H. Bazin, University de Louvain, Brussels) were used in enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assays (ELISA).
Preparation of cell suspensions
SP and MLN were teased through sterile 100-µm cell strainers (Falcon, Becton Dickinson, Bedford, MA) and the resulting mononuclear single-cell suspensions were centrifuged over a density separation medium (Lympholyte-Rat, Cedarlane Laboratories Limited, Ontario, Canada). The cells were washed three times and resuspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 25 mm HEPES, 2 mm glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml gentamicin (complete medium). Peyer's patches (PP) were excised from the small intestine and cell suspensions were prepared by mechanical disruption using a syringe, a 19-gauge needle and a sterile cell strainer. Tissue fragments were removed by filtering the resulting cell suspension through a glass wool column. Lamina propria (LP) lymphocytes were isolated from PP-free small intestinal segments previously flushed with calcium/magnesium-free Hanks' balanced salt solution.16 The tissue was dissociated by several consecutive incubations in complete medium containing 10 U/ml of collagenase (type VII, Sigma Chemicals, St Louis, MO). The resultant cell suspensions were diluted to final concentrations in complete medium and cell viability was determined by trypan blue exclusion.
In vitro cell cultures
Suspensions of SP, MLN, PP and LP mononuclear cells (1 × 104 and 1 × 106 cells/well) were incubated in complete medium in 96-well round-bottom culture plates for 5 days at 37° in a 5% CO2 environment.
Detection of antibody-secreting cells by ELISPOT
The numbers of IgA- and anti-CT IgA-secreting cells were determined by ELISPOT assay.17 Nitrocellulose plates (Millititer HA, Millipore Corp., Bedford, MA) were coated overnight with either sheep anti-rat IgA (5 µg/ml) for IgA-secreting cells or monosialoganglioside-GM1 followed by CT (5 µg/ml) for anti-CT IgA-secreting cells. The plates were blocked with complete medium (3 hr at 37°) and inoculated with 100 µl of diluted cell suspensions. After incubating overnight (37°), the cells were washed ten times with PBS containing 0·05%Tween-20. IgA-secreting cells were detected by incubating in 100 µl of biotinylated goat anti-rat IgA (2 mg/ml at 1 : 2000 dilution) in PBS–Tween-20 (2 hr), followed by avidin–horseradish peroxidase (A-HRP, 1 : 1000) for 1 hr at room temperature. The ELISPOT was developed by adding 100 µl of 1·6 mm 3-amino-9-ethylcarbazole in 0·1 m sodium acetate buffer containing 0·015% H2O2 (Kit AEC, Sigma) to each well. After the reaction mixture was washed off with water, the secreting cells were counted using a stereomicroscope and the data were expressed as the mean ± SEM of IgA- or anti-CT IgA-secreting cells per 106 cells.
Detection of antibodies by ELISA
Total IgA and anti-CT IgA antibody concentrations in the culture medium and serum were measured in quadruplicate using ELISA.11 Microtitre wells were coated with either CT (10 µg/ml) or sheep anti-rat IgA (2·5 µg/ml) and were incubated sequentially with 2% bovine serum albumin (BSA), 75 µl of serially diluted serum or culture supernate, biotinylated goat anti-rat IgA (1 : 5000) and A-HRP (1 : 1000) and then reacted with o-phenylenediamine (Zymed Laboratories, South San Francisco, CA). Animals receiving the vehicle alone were used as negative controls. Values for anti CT-IgA and total IgA titres were calculated from the linear portion of a rat dimeric IgA standard curve and expressed as either µg immunoglobulin/ml of serum or ng immunoglobulin/106 cells.
Adoptive transfer of PKH26-labelled cells
Viable mononuclear cells isolated from donor MLN and labelled in vitro with the PKH26-fluorescent molecule (PKH26 Kit, Sigma) were injected into the femoral veins of recipient rats (30 × 106 in 0·5 ml sterile PBS). The animals were killed 20 hr after transfer and segments of the small intestine were frozen for quantitative fluorescence microscopy. Negative control tissues were obtained from untreated animals, i.e. rats receiving neither CT nor vehicle. Frozen sections of small intestine were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) and the number of PKH26-positive cells in the intestinal mucosa was counted using an ocular grid (0·0144 mm2) and a double-blind protocol. Five cross-sections of intestine from four separate segments were evaluated per animal and the values were expressed as the number of PKH26-positive cells per mm2 of small intestinal mucosa.
Statistical analysis
All of the data were expressed as the mean ± the standard error of the mean (SEM). Differences between groups were assessed using the Mann–Whitney U-test and Student's t-test (non-parametric). Values of P < 0·05 were considered statistically significant.
Results
Cell yields
Intraduodenal administration of cholera holotoxin did not cause any obvious untoward effects in the rats, e.g. diarrhoea, morbidity, or weight loss. Cell recoveries per organ (mean ± SEM, n = 40) from young and old rats, respectively, were as follows: SP, 228 ± 11 and 315 ± 23 (× 106); MLN, 66·8 ± 4·3 and 57·9 ± 4·0 (× 106); PP, 16·6 ± 1·2 and 13·6 ± 1·15 (× 106); LP, 21·5 ± 2·3 and 33·2 ± 4·8 (× 106) (n = 40). The yields of SP and LP cells recovered from old rats were significantly greater than from young animals (P < 0·05).
Serum IgA and antibody levels
The total serum IgA levels were significantly higher in both immunized and naı¨ve old rats in comparison to young animals on days 7 and 21 (P < 0·05; Table 1). In contrast, the anti-CT IgA response was significantly lower in the sera of the old rats versus the young animals at both post-immunization intervals. Furthermore, when the specific antibody levels were expressed as a percentage of the total serum IgA, the difference was even greater.
Table 1.
Serum concentrations of total IgA and anti-CT IgA (µg/ml)
| Young | Old | |||
|---|---|---|---|---|
| Day 7 | Day 21 | Day 7 | Day 21 | |
| Total IgA | 210 ± 71 | 132 ± 20 | 441 ± 250* | 951 ± 405* |
| Anti-CT IgA | 16·3 ± 2·2 | 54 ± 24 | 2·5 ± 0·8* | 37 ± 5* |
| %Anti-CT of total IgA | 7·5 ± 0·1 | 41 ± 3 | 0·56 ± 0·01* | 8·2 ± 8* |
Values represent the mean ± SEM for 10 animals.
Significantly different compared to values from young animals (P < 0·05).
In vitro antibody secretion
Anti-CT IgA secretion was measured in mononuclear cells isolated from SP and gut-associated lymphoid tissues (GALT) on day 21 (Fig. 1). Antibody secretion was significantly lower in SP (50%), PP (63%), and LP (61%) cells isolated from old rats in comparison to cells from young animals (P < 0·05). However, when antibody secretion was expressed relative to the number of anti-CT IgA-secreting cells in the cultures, the age-related differences disappeared. There was no apparent age-related difference in secretion by cells isolated from the MLN.
Figure 1.
Anti-CT IgA levels in culture medium supernatants of cells isolated from spleen (SP), mesenteric lymph nodes (MLN), Peyer's patches (PP) and small intestinal lamina propria (LP) of young and old rats, killed 21 days after immunization. Results are expressed: (a) as ng IgA/106 cells in culture and (b) as ng IgA/anti-CT IgA-secreting cells (mean ± SEM). *Significantly different compared to young values (P < 0·05, n = 5/group).
IgA- and anti-CT IgA-secreting cells
Table 2 illustrates the numbers of SP and GALT IgA-secreting cells from young and old animals during the course of immunization, i.e. days 5, 7, 14 and 21. Cell numbers were independent of the immunization kinetics, i.e. the variation in the number of anti-CT IgA-secreting cells was not reflected in the total number of IgA-secreting cells. There were no obvious age-related differences in the numbers of IgA-secreting cells in SP and MLN. However, the numbers of IgA-secreting cells in the PP and the LP of old rats were significantly higher than the values in young animals at any postimmunization interval.
Table 2.
IgA-secreting cells/106 cells
| Spleen | Mesenteric lymph nodes | Peyer's patches | Lamina propria | ||||
|---|---|---|---|---|---|---|---|
| Young | Old | Young | Old | Young | Old | Young | Old |
| Day 5 | |||||||
| 427 ± 70 | 441 ± 111 | 12461 ± 2173 | 7568 ± 1364 | 1297 ± 277 | 3462 ± 785* | 2179 ± 905 | 4560 ± 1526* |
| Day 7 | |||||||
| 480 ± 39 | 463 ± 47 | 8748 ± 2226 | 7725 ± 1075 | 903 ± 155 | 2684 ± 516* | 1750 ± 460 | 4043 ± 788* |
| Day 14 | |||||||
| 674 ± 161 | 623 ± 191 | 12189 ± 2331 | 13561 ± 1611 | 962 ± 76 | 1904 ± 166* | 1934 ± 519 | 4466 ± 826* |
| Day 21 | |||||||
| 674 ± 131 | 790 ± 132 | 13485 ± 2811 | 16491 ± 3478 | 1033 ± 160 | 2258 ± 252* | 1296 ± 272 | 5123 ± 1695* |
Values represent the mean ± SEM for five animals.
Significantly different compared to values from young animals (P < 0·05).
Figure 2 presents the numbers of SP and GALT anti-CT IgA-secreting cells/106 cells isolated from young and old rats during the course of immunization. The temporal pattern of anti-CT IgA-secreting cell appearance was very similar in spleen and GALT and was not influenced by donor age. The number of antibody-secreting cells increased by day 7, subsequently decreased and increased again after boosting (days 14–21). However, there were fewer anti-CT IgA-secreting cells at the effector site, i.e. the intestinal mucosa, of old animals at all intervals after primary immunization. Furthermore, between days 5 and 14, the numbers of anti-CT IgA-secreting cells were lower at every site in the old age groups in comparison to the young animals. This pattern was most apparent at day 7; the number of anti-CT IgA-secreting cells in old rats was 50% lower in the PP and MLN, 75% lower in the SP and 90% lower in the LP in comparison to the young cohorts. The differences between the two age groups decreased with time after primary immunization, becoming insignificant in the PP, MLN and LP by day 14. This age difference reappeared 1 week after boosting (day 21) and the number of anti-CT IgA-secreting cells was four- to five-fold higher in the LP of young animals in comparison to old rats.
Figure 2.
Anti-CT IgA-secreting cells/106 cells in Peyer's patches (PP), mesenteric lymph nodes (MLN), spleen (SP) and intestinal lamina propria (LP) of 4 month-old or 24-month-old rats. Values represent the mean ± SEM for 10 animals. *Significantly different compared to values from young animals at the same interval after immunization (P < 0·05, n = 5/group).
Migration of PKH26-labelled MLN cells after adoptive transfer
The effect of age on lymphocyte migration in vivo was assessed by monitoring the fate of fluorescent labelled MLN mononuclear cells from young or old donors following their intravenous injection into young or old host animals. The relative populations of T and B lymphocytes in the MLN cell suspensions isolated from both age groups were similar, i.e. 40% T and 45–50% B cells based on flow cytometric analysis (data not shown). Furthermore, the relative numbers of IgA- and anti-CT IgA-secreting cells were similar.
Cells were transferred into naı¨ve or immunized recipient rats, since the appearance of PKH26-labelled cells in the intestinal mucosa 20 hr after transfer was independent of the immunization state of the host animal.18,19 The data from both naı¨ve and immunized animals were pooled. Our results demonstrated that PKH26-labelled MLN cells from old donors did not migrate to the intestinal mucosa in young syngeneic recipients as efficiently as did similar cells from young donors (Table 3). The number of transferred cells recovered in the LP of old recipient animals was significantly lower in comparison to the young rats, regardless of the age of the donor cells. Moreover, the number of labelled cells observed in the intestinal mucosa was lowest when old donor cells were transferred into an old recipient.
Table 3.
Number of PKH26-labelled cells/field in the small intestinal lamina propria
| Recipient animals | Donor cells | No. PKH26-labelled cells/field | Mean ± SEM |
|---|---|---|---|
| Young | Young immunized | 1·56 ± 0·06I | 1·18 ± 0·14 |
| 1·35 ± 0·15I | |||
| 0·87 ± 0·27I | |||
| 1·20 ± 0·01N | |||
| 1·80 ± 0·80N | |||
| 0·86 ± 0·23I | |||
| 0·68 ± 0·14I | |||
| Old immunized | 0·80 ± 0·10I | 0·89 ± 0·17 | |
| 1·76 ± 0·95I | |||
| 0·65 ± 0·15N | |||
| 0·40 ± 0·20N | |||
| 1·05 ± 0·35I | |||
| 0·68 ± 0·18I | |||
| Old | Young immunized | 0·80 ± 0·10I | 0·58 ± 0·11* |
| 0·80 ± 0·20N | |||
| 0·30 ± 0·30N | |||
| 0·43 ± 0·10I | |||
| Old immunized | 0·80 ± 0·20N | 0·41 ± 0·11*† | |
| 0·15 ± 0·06N | |||
| 0·38 ± 0·09I | |||
| 0·33 ± 0·10I |
Values represent mean ± SD from immunized (I) and naı¨ve (N) recipient animals 20 hr after adoptive transfer of MLN cells from either young or old immunized (day 7) donor animals. The variance among recipient rats with the same immunization status was the same as among rats with different immunization status. Significantly different from
young recipient transferred young cells
young recipient transferred old cells (P < 0·05).
Discussion
The age-related increase in the serum IgA titre was accompanied by a concomitant decline in the serum titre of specifically induced anti-CT IgA. It has already been shown in humans, as well as in animals, that the serum IgA level increases, whereas specific IgA antibody titres to mucosal antigens decline with age.5–9,11 The present data support our contention that the serum IgA titre is not a valid measure of the magnitude of the mucosal immune response and that specific antibody titres are a more critical index. The age-related increase in serum IgA may reflect a shift in the relative distribution of IgA subclasses. For example, serum and saliva samples from elderly humans contain reduced levels of IgA2, an immunoglobulin of mucosal origin, in comparison to young subjects, despite normal IgA concentrations.20 An age-related shift in the ratio of monomeric to polymeric IgA in the serum may contribute to the increase in total IgA since the monomeric form cannot be transported by epithelial cells and is destined to remain in the serum.6 Ultimately, the reduced serum anti-CT IgA titre in old animals probably reflects a compromised immune response to a mucosal antigen since Kantele et al. demonstrated that 99% of the B cells secreting antibodies against mucosal antigens in the peripheral blood migrate to the mucosal effector site.21
The reduced serum anti-CT IgA antibody titres in old rats correlate with reduced in vitro IgA antibody secretion by GALT lymphocytes isolated from old animals. However, our study clearly demonstrates that the decline in antibody secretion at the effector site reflects fewer secreting cells, rather than diminished antibody secretion. Our results are in good agreement with previous studies demonstrating 40–70% declines in antibody secretion by PP and LP lymphocytes.22–24 However, these results do not account for alterations in antibody affinity or idiotypic shifts that may diminish the immune response. In fact, studies have shown shifts in the idiotypic repertoire and restricted heterogeneity in the affinity of antibodies secreted by SP, but not by MLN, lymphocytes from old mice.25
The number of IgA-secreting cells in the GALT and SP is independent of immunization status and is significantly higher in the PP and slightly higher in the LP of old rats in comparison to young animals. The large numbers of IgA-secreting cells in the PP and intestinal mucosa of old rats may reflect greater exposure to dietary antigens during their longer lives in comparison to younger cohorts, i.e. the accumulation of memory B cells at the inductive and effective sites.26 Previous quantitative immunohistochemical and flow cytometric studies in our laboratory have demonstrated an increase in the IgA cell population of the PP of old rats.27 These data suggest that IgA immunoblasts may not emigrate from the PP in response to antigenic stimulation as efficiently in old rats in comparison to young animals.
The magnitude and time–course of the intestinal immune response in rats is different for each lymphoid site examined. Our finding that the number of anti-CT IgA-secreting cells in the intestinal mucosa is lower at any post-immunization interval in old versus young rats agrees with earlier studies.11,15,22 Furthermore, the time required to achieve the peak response is longer in old compared to young animals and occurs for the most part after boosting on day 14. For example, the primary and secondary peaks of mucosal secreting cells in young rats were equivalent and significantly greater than those observed in old animals, whose highest values occurred at day 21. Our ELISPOT data support the hypothesis that an age-related delay in the migration of IgA immunoblasts to the effector site, i.e. the intestinal mucosa, contributes to mucosal immunosenescence.
The adoptive transfer experiments demonstrated that the migration of MLN lymphocytes to the intestinal mucosa is dependent on the ages of both the recipient and the donor animals. The number of labelled cells migrating to the intestine is lowest when old recipient animals are injected with cells from old donors, suggesting that the migration of activated immunoblasts from the blood to the intestinal effector site is impaired with increasing age. Although this impairment may reflect the altered expressions of homing receptors or ligands, we have no evidence of potential mechanisms whereby ageing compromises such migration. On the one hand, there is no evidence that ageing diminishes the expression of either the integrin α4β7 or the mucosal addressin cell adhesion molecule-1 (MAdCAM-1). On the other hand, there are data suggesting that the expression of l-selectin, an integrin implicated in the homing of naı¨ve and memory lymphocytes to peripheral lymph nodes, is impaired by ageing and that this interferes with the migration of lymphocytes to secondary lymphoid tissues.28 Old l-selectin-deficient mice exhibit changes in lymphocyte distribution patterns similar to those observed in the wild phenotype, but the rate of change is accelerated in the former mice. Furthermore, Toichi et al. demonstrated that the age-related decline in delayed-type hypersensitivity (DTH) responses in short-lived senescence-accelerated mice is due to the impaired migration of inflammatory cells into the local site rather than a loss of DTH T cells.29
The distribution of lymphocytes in the donor MLN cell suspensions was identical to those used to measure in vitro antibody secretion. Both cell suspensions contained mucosally activated anti-CT immunoblasts, as well as other B- and T-cell populations, destined for the intestinal effector site. The absence of any age-related differences in the relative distributions of B and T lymphocytes, and IgA- and anti-CT IgA-secreting cells, in the donor MLN cell suspensions diminishes the possibility that shifts in the composition of GALT lymphocyte populations contribute to reduced migration to the mucosal site. In addition, both we and Fló et al. have demonstrated that there is no significant age-related change in the number or distribution of GALT CD4+ lymphocytes in rats.26–28 Moreover, the selective recruitment of MLN IgA immunoblasts from the peripheral blood into the intestinal mucosa is independent of the presence of specific antigen despite the fact that the subsequent retention, survival and proliferation of antigen-specific cells are dramatically influenced by cognate antigen.18,19
This study suggests that the response to a new antigen is altered with age in the intestinal mucosal immune system. The diminished antibody titre in the intestinal lumen reflects, in part, the fact that fewer IgA plasma cells reach the effective site, i.e. the intestinal lamina propria.
Acknowledgments
A Merit Review from the Department of Veterans Affairs and grants from the Danone Group and the University of California, San Francisco, supported this research. The authors thank Rose Wang for technical support.
Glossary
Abbreviations
- CT
cholera toxin
- GALT
gut-associated lymphoid tissues
- LP
lamina propria
- MLN
mesenteric lymph nodes
- PP
Peyer's patches
- SP
spleen
References
- 1.Schmucker DL, Owen RL. Aging and gastrointestinal mucosal immune response. Cur Opinion Gastroenterol. 1997;13:534–41. [Google Scholar]
- 2.Schmucker DL, Gilbert R, Jones AL, Hradek GT, Bazin H. Effect of aging on the hepatobiliary transport of dimeric immunoglobulin A in the male Fischer rat. Gastroenterology. 1985;88:436–43. doi: 10.1016/0016-5085(85)90504-9. [DOI] [PubMed] [Google Scholar]
- 3.Jeandel C, Laurain MC, Decottignies F. Infectious diarrhea in the aged. Rev Prat. 1996;46:184–8. [PubMed] [Google Scholar]
- 4.Owen RL, Lew JF. Gastrointestinal infections in the elderly. In: Surawicz C, Owen RL, editors. Gastrointestinal and Hepatic Infections. Philadelphia: W.B. Saunders; 1995. pp. 551–64. [Google Scholar]
- 5.Arranz E, O'mahony S, Barton JR, Ferguson A. Immuno-senescence and mucosal immunity: significant effects of old age on secretory IgA concentrations and intraepithelial lymphocyte counts. Gut. 1992;33:882–6. doi: 10.1136/gut.33.7.882. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Ebersole JL, Smith DJ, Taubman MA. Secretory immune responses in ageing rats. I. Immunoglobulin levels. Immunology. 1985;56:345–50. [PMC free article] [PubMed] [Google Scholar]
- 7.Paganelli R, Scala E, Quinti I, Ansotegui IJ. Humoral immunity in aging. Aging. 1994;6:143–50. doi: 10.1007/BF03324229. [DOI] [PubMed] [Google Scholar]
- 8.Smith DJ, Ebersole JL, Taubman MA. Local and systemic immune response in aged hamsters. Immunology. 1983;50:407–13. [PMC free article] [PubMed] [Google Scholar]
- 9.Taylor LD, Daniels CK, Schmucker DL. Ageing compromises gastrointestinal mucosal immune response in the Rhesus monkey. Immunology. 1992;75:614–18. [PMC free article] [PubMed] [Google Scholar]
- 10.Lim TS, Messiha N, Watson RR. Immune components of the intestinal mucosae of ageing and protein deficient mice. Immunology. 1981;43:401–7. [PMC free article] [PubMed] [Google Scholar]
- 11.Schmucker DL, Daniels CK, Wang R, Smith K. Mucosal immune response to cholera toxin in ageing rats. I Antibody and antibody-containing cell response. Immunology. 1988;64:691–5. [PMC free article] [PubMed] [Google Scholar]
- 12.Schmucker DL, Daniels CK. Aging, gastrointestinal infections, and mucosal immunity. J Am Geriat Soc. 1986;34:377–84. doi: 10.1111/j.1532-5415.1986.tb04321.x. [DOI] [PubMed] [Google Scholar]
- 13.Daniels CK, Schmucker DL, Jones AL. Age-dependent loss of dimeric immunoglobulin A receptors in the liver of the Fischer 344 rat. J Immunol. 1985;134:3855–8. [PubMed] [Google Scholar]
- 14.Daniels CK, Schmucker DL, Bazin H, Jones AL. Immunoglobulin A receptor of rat small intestinal enterocytes is unaffected by aging. Gastroenterology. 1988;94:1432–40. doi: 10.1016/0016-5085(88)90683-x. [DOI] [PubMed] [Google Scholar]
- 15.Bianchi AT, Van der Heiden PJ, Stok W, Rozing J, Coolen C. Decrease of IgA-secreting cells in the lamina propria of mice during aging. Aging: Immunol Infect Dis. 1990;2:241–50. [Google Scholar]
- 16.Lycke N. A sensitive method for the detection of specific antibody production in different isotypes from single lamina propria plasma cells. Scand J Immunol. 1986;24:393–403. doi: 10.1111/j.1365-3083.1986.tb02127.x. [DOI] [PubMed] [Google Scholar]
- 17.Czerkinsky C, Nilsson LA, Nygren H, Ouchterlony Ö, Tarkowski A. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Meth. 1983;65:109–21. doi: 10.1016/0022-1759(83)90308-3. [DOI] [PubMed] [Google Scholar]
- 18.Husband AJ. Kinetics of extravasation and redistribution of IgA-specific antibody-containing cells in the intestine. J Immunol. 1982;128:1355–9. [PubMed] [Google Scholar]
- 19.Butcher E. Lymphocyte homing and intestinal immunity. In: Ogra P, Mestecky J, Lamm M, Strober W, Bienenstock J, McGhee J, editors. Mucosal Immunology. 2. San Diego: Academic Press; 1999. pp. 507–22. [Google Scholar]
- 20.Ventura MT. Evaluation of IgA1-IgA2 levels in serum and saliva of young and elderly people. Allergologia Immunopathologia. 1991;19:183–5. [PubMed] [Google Scholar]
- 21.Kantele A, Kantele JM, Savilahti E, Westerholm M, Arvilommi H, Lazarovits A, Butcher EC, Mäkelä PH. Homing potentials of circulating lymphocytes in humans depend on the site of activation: oral, but not parenteral, typhoid vaccination induces circulating antibody-secreting cells that all bear homing receptors directing them to the gut. J Immunol. 1997;158:574–9. [PubMed] [Google Scholar]
- 22.Kawanishi H, Kiely J. Immune-related alterations in aged gut-associated lymphoid tissues in mice. Dig Dis Sci. 1989;34:175–84. doi: 10.1007/BF01536048. [DOI] [PubMed] [Google Scholar]
- 23.Kawanishi H, Senda S, Ajitsu S. Aging-associated intrinsic defects in IgA production by murine Peyer's patch B cells stimulated by autoreactive Peyer's patch T cell hybridoma-derived B cell stimulatory factors (BSF) Mech Ageing Dev. 1989;49:61–78. doi: 10.1016/0047-6374(89)90068-7. [DOI] [PubMed] [Google Scholar]
- 24.Daniels CK, Schmucker DL, Irvin BJ. Differential IgA response in the aging Fischer rat following mucosal stimulation with cholera toxin or B subunit. Aging: Immunol Infect Dis. 1993;4:95–107. [Google Scholar]
- 25.Szewczuk MR, Wade AW. Aging and the mucosal-associated lymphoid system. Ann N Y Acad Sci. 1983;409:333–44. doi: 10.1111/j.1749-6632.1983.tb26881.x. [DOI] [PubMed] [Google Scholar]
- 26.Fló J, Massouh E. Age-related changes of naïve and memory CD4 rat lymphocyte subsets in mucosal and systemic lymphoid organs. Dev Comp Immunol. 1997;21:443–53. doi: 10.1016/s0145-305x(97)00023-2. 10.1016/s0145-305x(97)00023-2. [DOI] [PubMed] [Google Scholar]
- 27.Daniels CK, Perez P, Schmucker DL. Alterations in CD8+ cell distribution in gut-associated lymphoid tissues (GALT) of the aging Fischer 344 rat: a correlated immunohistochemical and flow cytometric analysis. Exp Gerontol. 1993;28:549–55. doi: 10.1016/0531-5565(93)90043-d. [DOI] [PubMed] [Google Scholar]
- 28.Steeber DA, Green NE, Sato S, Tedder TF. Lymphocyte migration in l-selectin-deficient mice. Altered subset migration and aging of the immune system. J Immunol. 1996;157:1096–106. [PubMed] [Google Scholar]
- 29.Toichi E, Hanada K, Hosokawa K, Higuchi K, Hosokawa M, Imamura S, Hosono M. Age-related decline in humoral immunity caused by the selective loss of Th cells and decline in cellular immunity caused by the impaired migration of inflammatory cells without a loss of TDTH cells in SAMP1 mice. Mech Ageing Dev. 1997;99:199–217. doi: 10.1016/s0047-6374(97)00100-0. 10.1016/s0047-6374(97)00100-0. [DOI] [PubMed] [Google Scholar]