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Intestinal mucosal immunosenescence in rats
Experimental Gerontology 37 (2002) 197±203
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Intestinal mucosal immunosenescence in rats Douglas L. Schmucker* Department of Anatomy, Veterans Affairs Medical Center, Cell Biology and Aging (151E), University of California, 4150 Clement Street, San Francisco, CA 94121, USA Received 1 June 2001; accepted 1 September 2001
Abstract The elderly are characterized by systemic immunosenescence and high rates of morbidity and mortality associated with infectious diseases of the intestinal tract. Despite the consensus that the mucosal immune compartment is largely unaffected by aging, there are marked de®cits in the intestinal mucosal immune responses of old animals and elderly humans. However, little is known about the mechanism(s) whereby aging disrupts intestinal immunity. Events in the generation of an intestinal immune response may be susceptible to the insults of aging. The ®rst step involves the uptake of antigens by specialized follicular epithelial cells (M cells). There have been no studies on the ef®cacy of antigen uptake by M cells as a function of age. Little is known about the next step, i.e. antigen presentation by dendritic cells and subsequent isotype switching. The third event is the differentiation of putative immunolobulin A (IgA) plasma cells and their homing from the Peyer's patches (inductive site) to the lamina propria of the small intestine (effector site). Quantitative immunohistochemical and ¯ow cytometry analyses suggest that the homing of IgA immunoblasts may be compromised in old rats and monkeys. Local antibody production/secretion by mature IgA plasma cells in the intestinal wall constitutes the fourth step. In vitro anti-cholera toxin IgA antibody secretion by intestinal lamina propria lymphocytes is equivalent in cells isolated from young adult and senescent rats. The ®nal event in the mucosal immune response is the transport of IgA antibodies across the mucosal epithelial cells and their secretion onto the mucosal surface, i.e. receptor-mediated vesicular translocation of IgA by the intestinal epithelial cells. Binding assays did not detect age-associated declines in either the number or binding af®nity of the polymeric immunoglobulin receptor expressed on rodent and monkey intestinal epithelial cells. Published by Elsevier Science Inc. Keywords: Aging; Intestinal immunity; Immunoglobulin A; IgA secretion; Immunoblast homing
1. Intestinal mucosal immunity Mucosal surfaces constitute a discrete compartment of the immune system that is autonomous from the systemic arm by virtue of a different immunoglobulin isotype, immunoglobulin A (IgA), a unique process for initiating an immune response and an independent lymphocyte subpopulation. The intestine is the largest single immune organ; contains .70% of the plasma * Tel.: 11-415-221-4810, ext. 3450; fax: 11-415-750-6927. E-mail address: [email protected] (D.L. Schmucker). 0531-5565/02/$ - see front matter Published by Elsevier Science Inc. PII: S 0531-556 5(01)00184-X
cells and produces more IgA than the total production of immunoglobulin G (IgG). An effective immune response in the intestine involves (a) binding, uptake and transport of antigen at the mucosal surface, (b) antigen presentation by dendritic cells within the Peyer's patches and switching of lymphocytes to express the IgA isotype, (c) maturation and homing of Peyer's patch IgA immunoblasts to the effctor site, i.e. the intestinal lamina propria, (d) local antibody production by mature IgA plasma cells in the intestine and (e) transport of IgA antibodies across the intestinal epithelium to the mucosal surface by
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Fig. 1. (A) Anti-cholera toxin IgA titers in bile of young, mature and senescent rats ®ve days after intraduodenal boosting with cholera toxin. The titer is signi®cantly higher in young rats in comparison to old animals P , 0:01: Each values is the mean ^ SEM for ®ve rats and the data are expressed as reciprocal titers (titer 21). (B) Anti-cholera toxin concentrations in the intestinal lavage of young (2±6 year) and old (20±25 year) rhesus macaques at intervals during intraduondenal immunization with cholera toxin delivered by endoscope. The animals were immunized on days 0, 14 and 21 and each value represents the mean ^ SEM for ®ve macaques.
the polymeric immunoglobulin receptor (pIgR) (see Schmucker et al., 1996; Schmucker and Owen, 1997 for reviews). 2. Age and intestinal mucosal immunity The intestinal mucosal immune response is compromised in old animals and elderly humans (Haq and Szewczuk, 1991; Fujihashi et al., 2000; see Schmucker et al., 1996; Schmucker and Owen, 1997 for reviews). Several studies reported increases or no change in the serum IgA levels in old animals and humans in comparison to young subjects (Buckley et al., 1974; Amman et al., 1980; Finkelstein et al., 1984; Ebersole et al., 1985). Increased serum IgA levels in the elderly may re¯ect diminished epithelial cell transport of antibodies to the mucosal surfaces. Furthermore, serum IgA titers re¯ect the level of the monomeric, non-J chain-containing form of IgA that neither binds to the pIgR nor is transported to the mucosal surface as secretory IgA. Others have reported the absence of agerelated differences in the amounts of nonspeci®c immuno-globulins secreted into the intestinal lumen in vivo or into the medium by cultured duodenal biopsies (Penn et al., 1991; see Arranz and Ferguson, 1992 for a review). However, most investigators concur that the levels of nonspeci®c immunoglobulins in intestinal secretions are poor indices and that speci®c antibody titers are a more critical measure of the magnitude of the mucosal immune response (Smith et al., 1983;
Ebersole et al., 1985; Schmucker et al., 1988; Taylor et al., 1992; Vajdy and Lycke, 1992). Following intraduodenal immunization with cholera holotoxin, hepatobiliary and intestinal anti-cholera toxin IgA antibody titers decline with increasing age in rodents and monkeys, respectively (Schmucker et al., 1988; Taylor et al., 1992; Fig. 1). In addition, clinical studies have documented age-related declines in speci®c mucosal IgA antibody responses (Amman et al., 1980; Ganguly et al., 1986; Waldmann et al., 1987; Beyer et al., 1989; Fujihashi et al., 2000). 3. Effect of aging on binding and uptake of antigens in the intestine There is no evidence for age-related differences in the binding or uptake of antigens by M cells or follicle-associated epithelium overlying Peyer's patches and other lymphoid nodules in the intestine. Follicle weight and the yield of Peyer's patch lymphocytes decline with increasing age in mice (Kawanishi and Kiely, 1989). However, neither the number of Peyer's patches (i.e. 15.5, 15.7 and 14.4 patches per intestine in young adult, mature and senescent rats, respectively) nor the yield of lymphocytes from these aggregates varies signi®cantly with age in rats (Schmucker et al., 1988; Schmucker et al., 2001). Whether or not this critical initial step in the intestinal mucosal immune response is compromised in old animals remains unresolved.
D.L. Schmucker / Experimental Gerontology 37 (2002) 197±203
Fig. 2. The number of IgA 1 cells per microscopic ®eld in the Peyer's patches of young, mature and senescent immunologically naõÈve or cholera toxin immunized rats. The Peyer's patches in old rats contain twice the number of cells as those of young animals regardless of their immune status. Each value represents the mean ^ SEM of ®ve rats.
4. Effect of aging on antigen presentation and isotype switching After uptake by M cells or other follicle-associated epithelial cells, the antigens are presented to immunocompetent cells by dendritic cells in the Peyer's patches and, in the absence of tolerance, a mucosal immune response is initiated. There is con¯icting evidence concerning the effect of aging on the number, distribution and function of the epidermal analog of dendritic cells, i.e. Langerhans cells (Fagiolo et al., 1993; Sprecher et al., 1990; Haruna et al., 1995; Steger et al., 1996). However, other studies have reported that the antigen-presenting capacity, as well as other functions, of dendritic
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cells derived from peripheral blood monocytes remain unchanged as a function of donor age (Steger et al., 1997; Saurwein-Teissl et al., 1998; Castle et al., 1999; Lung et al., 2000). These studies did not use dendritic cells isolated from intestinal Peyer's patches, thereby precluding extrapolation of these data to the intestinal immune response. Recent studies have isolated and characterized rat intestinal dendritic cells and devised an in vitro system to assess antigen-presenting capacity (Liu and MacPherson, 1995a,b; MacPherson et al., 1995; Maric et al., 1996; Szabo et al., 1997). Age-related losses in intestinal B and/or T lymphocyte subpopulations or changes in the distributions of lymphocyte subsets, may contribute to or re¯ect mucosal immunosenescence (Haaijman et al., 1977; Rivier et al., 1983; Bienenstock et al., 1984; Wade et al., 1988; Fleming et al., 1993; Daniels et al., 1993). For example, we reported a 2±3 fold increase in the number of IgA 1 cells in the Peyer's patches in old versus young adult rats (Schmucker et al., 1988; Taylor et al., 1992; Fig. 2). The increase, coupled with a concomitant decline in the size of this cell subpopulation in the intestinal lamina propria, provided the ®rst evidence that aging compromises the homing of IgA 1 immunoblasts from the Peyer's patches (inductive site) to the intestinal mucosa (effector site) (Haaijman et al., 1977; Ebersole et al., 1988). There have been no studies on IgA 1 isotype switching in the Peyer's patches as a function of age. Information concerning age-related shifts in gut-associated lymphoid tissue (GALT) lymphocyte subpopulations that may in¯uence isotype switching is limited.
Fig. 3. The numbers of IgA 1 (A) and cholera toxin 1 (B) cells in the small intestinal lamina propria of young, mature and old naive and cholera toxin immunized rats. The number of IgA 1 cells declines markedly between young adult and mature animals, whereas the decline in the number of cholera toxin 1 cells occurs between maturity and senescence. Each value represents the mean ^ SEM for ®ve rats.
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Fig. 4. The number of ¯uorescent-labeled (PKH26) mesenteric lymph node cells isolated from young and senescent donor rats, transferred intravenously into young or senescent recipient animals and localized to the small intestinal lamina propria by ¯uorescence microscopy 20 h after transfer. The values in the senescent recipients receiving cells from either young ( p) or old ( pp) donors are signi®cantly lower than those in young recipients. Each value represents the mean ^ SEM for ®ve animals.
Kawanishi and Kiely reported a loss of CD8 1 cells in the Peyer's patches of old mice, whereas our ¯ow cytometry studies did not detect a shift in the size of this lymphocyte subpopulation in rats (Kawanishi and Kiely, 1987; Daniels et al., 1993). However, our quantitative immunohistochemical analysis did reveal a marked age-related shift in the distribution of CD8 1 lymphocytes within rat Peyer's patches. 5. Effect of aging on IgA immunoblast homing to the intestine The numbers of IgA 1 and cholera toxin 1 cells in
the rat intestinal lamina propria exhibit .60 and .50% age-related declines, respectively, at similar intervals following intraduodenal immunization (Schmucker et al., 1988; Fig. 3). Similar cell populations decline 3±4 fold in the peripheral blood of cholera toxin-immunized old rhesus macaques (Taylor et al., 1992; Fig. 3). Using lymphocyte adoptive transfer, we demonstrated that the homing of mesenteric lymph node lymphocytes to the intestinal lamina propria is diminished in senescent rats in comparison to young animals (Thoreux et al., 2000; Fig. 4). Homing of labeled cells was lowest when old recipients were injected with cells from old donors and greatest when young cells were injected into young hosts. The migration of lymphocytes isolated from young animals was also lower in old recipients in comparison to young hosts, suggesting that the agerelated decline in homing re¯ects de®cits in both cells isolated from old rats and in old recipient animals. 6. Effect of aging on local IgA antibody secretion Aging is accompanied by 40±70% declines in antibody secretion by Peyer's patch and intestinal lamina propria lymphocytes isolated from mice and rats (Kawanishi and Kiely, 1989; Rivier et al., 1983; Daniels et al., 1993). Daniels et al. measured antibody secretion by GALT lymphocytes isolated from young and old rats following intraduodenal immunization with cholera holotoxin. Five days after immunization, IgA antitoxin secretion by mesenteric lymph node
Fig. 5. Anti-cholera toxin IgA antibody secretion into the culture medium of cells isolated from the Peyer's patches and intestinal lamina propria of young adult and senescent rats seven days after intraduodenal boosting with cholera toxin. The data are expressed as ng of IgA antibody per 10 6 total cells (A) and ng of IgA antibody per 10 3 antibody-secreting cells (B). The antibody levels in cultures of old cells in (A) are signi®cantly lower than those measured in cultures from young rats P , 0:05: However, this apparent age-related difference disappears when the data are expressed per population of antibody-secreting cells (B). The values represent the means ^ SEM for ®ve animals per age group.
D.L. Schmucker / Experimental Gerontology 37 (2002) 197±203
cells isolated from young rats was greater than that by cells from old animals. Despite the fact that Peyer's patches of old rats contained two-fold fewer anticholera toxin IgA-secreting cells in comparison to those of young animals, Peyer's patch cells isolated from old rats secreted signi®cantly more antibodies than similar cells from young animals. A 2.5-fold age-related increase in the number of CD8 1 lymphocytes in the intestinal lamina propria may re¯ect increased immunosuppression of IgA immunoblast maturation and/or antibody secretion at the effector site. This may contribute to more frequent tolerogenic responses in old animals (see Mayer, 1997 for a review). We recently reported that in vitro secretion of anticholera toxin IgA antibodies by lymphocytes isolated from the intestinal lamina propria of senescent rats was signi®cantly lower (.60%) than that of cells obtained from young animals (Thoreux et al., 2000; Fig. 5). Nevertheless, when antibody secretion was expressed relative to the number of anti-cholera toxin IgA-secreting cells in each culture, the agerelated difference was no longer evident. Therefore, the age-related decline in antibody titers at the effector site re¯ects fewer secreting cells rather than diminished antibody secretion per cell. 7. Effect of aging on IgA secretion by the mucosal epithelium The intestinal immune response culminates in the transport of IgA antibodies to the apical surface of enterocytes and their secretion onto the mucosal surface. This requires receptor-mediated endocytosis of the antibodies at the basal plasma membrane and their translocation to the mucosal surface in endocytic vesicles (see Underdown and Schiff, 1986 for a review). Nearly all mucosal epithelial cells express pIgR on their basolateral surfaces. Fifteen years ago, we reported a 4±6 fold age-related decline in the transport of polymeric IgA from blood to bile in rats (Schmucker et al., 1985). We also showed a concomitant 3±4 fold decline in the number of hepatic polymeric IgA receptors across this same age span, i.e. 3 and 25 months (Daniels et al., 1985). We and others subsequently showed that (a) the rat hepatic pIgR mRNA steady state level declines 20% between
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these ages and (b) the incorporation of [ S]-cysteine into pIgR lags in cultured hepatocytes isolated from old rats in comparison to cells from young animals (Gregoire et al., 1992; van Bezooijen et al., 1994). These data suggest that the expression of hepatic pIgR is diminished with increasing age and contributes to reduced IgA secretion into the bile and, ultimately, to a diminished intestinal immune response. These studies were performed using rat hepatocytes and there are no data concerning the effect of age on polymeric IgA transport across the small intestinal mucosa. Although crypt enterocytes exhibit a 3-fold greater binding of polymeric IgA than do villus tip cells, the expression of pIgR on the basolateral plasma membranes of rat small intestinal mucosal epithelial cells remains unchanged during aging (Daniels and Schmucker, 1987). The crypt-to-villus tip gradient in pIgR expression is identical in young and old rats and a similar pattern is seen in the rhesus macaque (Taylor et al., 1992; Daniels et al., 1988). The absence of an age-related change in the expression of pIgR on enterocytes suggests that antibody binding by intestinal epithelial cells remains unscathed by aging. 8. Conclusions The intestinal mucosal immune response to a variety of antigens is diminished in old animals and elderly humans. However, information concerning the effect(s) of aging on the individual events in the etiology of the intestinal immune response is limited. On the one hand, there are few data concerning the effect of aging on the initial steps in this process, e.g. those occurring at the inductive site (antigen uptake and presentation, isotype switching). On the other hand, we recently demonstrated an age-related de®cit in the homing of IgA immunoblasts to the effector site. Therefore, diminished antibody titers in the intestinal lavage of senescent animals most likely re¯ect fewer plasma cells rather than a decline in local antibody production. Acknowledgements This research was supported by the Department of Veterans Affairs, The Research Evaluation and
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D.L. Schmucker / Experimental Gerontology 37 (2002) 197±203 Mucosal immune response to cholera toxin in aging rats. I. Antibody and antibody-containing cell response. Immunology 64, 691±695. Schmucker, D.L., Heyworth, M., Owen, R.L., 1996. Impact of aging on gastrointestinal mucosal immunity. Dig. Dis. Sci. 41, 1183± 1193. Schmucker, D.L., Thoreux, K., Owen, R.L., 2001. Aging impairs intestinal immunity. Mech. Aging Develop. in press. Smith, D., Ebersole, J., Taubman, M., 1983. Local and systemic immune response in aged hamsters. Immunology 50, 407±413. Sprecher, E., Becker, Y., Kraal, G., 1990. Effect of aging on epidermal dendritic cell populations in C57BL/6J mice. J. Invest. Dermatol. 94, 247±253. Steger, M., Maczek, C., Grubeck-Loebenstein, B., 1996. Lymphocyte migration in l-selectin-de®cient mice. Altered subset migration and aging of the immune system. J. Immunol. 157, 1096±1106. Steger, M., Maczek, C., Grubeck-Loebenstein, B., 1997. Peripheral blood dendritic cells reinduce proliferation in in vitro aged T cell populations. Mech. Aging Develop. 93, 125±130. Szabo, M., Butcher, E., McEvoy, L., 1997. Specialization of mucosal follicular dendritic cells revealed by mucosal addressin-cell adhesion molecule-1 display. J. Immunol. 158, 5584±5588.
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Taylor, L., Daniels, C.K., Schmucker, D.L., 1992. Aging compromises gastrointestinal mucosal immune response in the rhesus monkey. Immunology 75, 614±618. Thoreux, K., Owen, L., Schmucker, D.L., 2000. Intestinal lymphocyte number, migration and antibody secretion in young and old rats. Immunology 101, 161±167. Underdown, B., Schiff, J., 1986. Immunoglobulin A: strategic defense initiative at the mucosal surface. Ann. Rev. Immunol. 4, 389±417. Vajdy, M., Lycke, N., 1992. Cholera toxin adjuvant promotes longterm immunological memory in the gut mucosa to unrelated immunogens after oral immunization. Immunology 75, 488± 492. Van Bezooijen, R., Wang, R.K., Lechner, M., Schmucker, D.L., 1994. Aging affects the regulation of preferentially expressed liver proteins in male Fischer 344 rats. Exp. Gerontol. 29, 186± 196. Wade, A., Green-Johnson, J., Szewczuk, M., 1988. Functional changes in systemic and mucosal lymphocyte repertoires with age: an update review. Aging: Immunol. Infect. Dis. 1, 65±97. Waldmann, R., Bergmann, M., Stone, J., 1987. Age-dependent antibody response in mice and humans following oral in¯uenza immunization. J. Clin. Immunol. 7, 327±332.
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Intestinal mucosal immunosenescence in rats Douglas L. Schmucker* Department of Anatomy, Veterans Affairs Medical Center, Cell Biology and Aging (151E), University of California, 4150 Clement Street, San Francisco, CA 94121, USA Received 1 June 2001; accepted 1 September 2001
Abstract The elderly are characterized by systemic immunosenescence and high rates of morbidity and mortality associated with infectious diseases of the intestinal tract. Despite the consensus that the mucosal immune compartment is largely unaffected by aging, there are marked de®cits in the intestinal mucosal immune responses of old animals and elderly humans. However, little is known about the mechanism(s) whereby aging disrupts intestinal immunity. Events in the generation of an intestinal immune response may be susceptible to the insults of aging. The ®rst step involves the uptake of antigens by specialized follicular epithelial cells (M cells). There have been no studies on the ef®cacy of antigen uptake by M cells as a function of age. Little is known about the next step, i.e. antigen presentation by dendritic cells and subsequent isotype switching. The third event is the differentiation of putative immunolobulin A (IgA) plasma cells and their homing from the Peyer's patches (inductive site) to the lamina propria of the small intestine (effector site). Quantitative immunohistochemical and ¯ow cytometry analyses suggest that the homing of IgA immunoblasts may be compromised in old rats and monkeys. Local antibody production/secretion by mature IgA plasma cells in the intestinal wall constitutes the fourth step. In vitro anti-cholera toxin IgA antibody secretion by intestinal lamina propria lymphocytes is equivalent in cells isolated from young adult and senescent rats. The ®nal event in the mucosal immune response is the transport of IgA antibodies across the mucosal epithelial cells and their secretion onto the mucosal surface, i.e. receptor-mediated vesicular translocation of IgA by the intestinal epithelial cells. Binding assays did not detect age-associated declines in either the number or binding af®nity of the polymeric immunoglobulin receptor expressed on rodent and monkey intestinal epithelial cells. Published by Elsevier Science Inc. Keywords: Aging; Intestinal immunity; Immunoglobulin A; IgA secretion; Immunoblast homing
1. Intestinal mucosal immunity Mucosal surfaces constitute a discrete compartment of the immune system that is autonomous from the systemic arm by virtue of a different immunoglobulin isotype, immunoglobulin A (IgA), a unique process for initiating an immune response and an independent lymphocyte subpopulation. The intestine is the largest single immune organ; contains .70% of the plasma * Tel.: 11-415-221-4810, ext. 3450; fax: 11-415-750-6927. E-mail address: [email protected] (D.L. Schmucker). 0531-5565/02/$ - see front matter Published by Elsevier Science Inc. PII: S 0531-556 5(01)00184-X
cells and produces more IgA than the total production of immunoglobulin G (IgG). An effective immune response in the intestine involves (a) binding, uptake and transport of antigen at the mucosal surface, (b) antigen presentation by dendritic cells within the Peyer's patches and switching of lymphocytes to express the IgA isotype, (c) maturation and homing of Peyer's patch IgA immunoblasts to the effctor site, i.e. the intestinal lamina propria, (d) local antibody production by mature IgA plasma cells in the intestine and (e) transport of IgA antibodies across the intestinal epithelium to the mucosal surface by
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Fig. 1. (A) Anti-cholera toxin IgA titers in bile of young, mature and senescent rats ®ve days after intraduodenal boosting with cholera toxin. The titer is signi®cantly higher in young rats in comparison to old animals P , 0:01: Each values is the mean ^ SEM for ®ve rats and the data are expressed as reciprocal titers (titer 21). (B) Anti-cholera toxin concentrations in the intestinal lavage of young (2±6 year) and old (20±25 year) rhesus macaques at intervals during intraduondenal immunization with cholera toxin delivered by endoscope. The animals were immunized on days 0, 14 and 21 and each value represents the mean ^ SEM for ®ve macaques.
the polymeric immunoglobulin receptor (pIgR) (see Schmucker et al., 1996; Schmucker and Owen, 1997 for reviews). 2. Age and intestinal mucosal immunity The intestinal mucosal immune response is compromised in old animals and elderly humans (Haq and Szewczuk, 1991; Fujihashi et al., 2000; see Schmucker et al., 1996; Schmucker and Owen, 1997 for reviews). Several studies reported increases or no change in the serum IgA levels in old animals and humans in comparison to young subjects (Buckley et al., 1974; Amman et al., 1980; Finkelstein et al., 1984; Ebersole et al., 1985). Increased serum IgA levels in the elderly may re¯ect diminished epithelial cell transport of antibodies to the mucosal surfaces. Furthermore, serum IgA titers re¯ect the level of the monomeric, non-J chain-containing form of IgA that neither binds to the pIgR nor is transported to the mucosal surface as secretory IgA. Others have reported the absence of agerelated differences in the amounts of nonspeci®c immuno-globulins secreted into the intestinal lumen in vivo or into the medium by cultured duodenal biopsies (Penn et al., 1991; see Arranz and Ferguson, 1992 for a review). However, most investigators concur that the levels of nonspeci®c immunoglobulins in intestinal secretions are poor indices and that speci®c antibody titers are a more critical measure of the magnitude of the mucosal immune response (Smith et al., 1983;
Ebersole et al., 1985; Schmucker et al., 1988; Taylor et al., 1992; Vajdy and Lycke, 1992). Following intraduodenal immunization with cholera holotoxin, hepatobiliary and intestinal anti-cholera toxin IgA antibody titers decline with increasing age in rodents and monkeys, respectively (Schmucker et al., 1988; Taylor et al., 1992; Fig. 1). In addition, clinical studies have documented age-related declines in speci®c mucosal IgA antibody responses (Amman et al., 1980; Ganguly et al., 1986; Waldmann et al., 1987; Beyer et al., 1989; Fujihashi et al., 2000). 3. Effect of aging on binding and uptake of antigens in the intestine There is no evidence for age-related differences in the binding or uptake of antigens by M cells or follicle-associated epithelium overlying Peyer's patches and other lymphoid nodules in the intestine. Follicle weight and the yield of Peyer's patch lymphocytes decline with increasing age in mice (Kawanishi and Kiely, 1989). However, neither the number of Peyer's patches (i.e. 15.5, 15.7 and 14.4 patches per intestine in young adult, mature and senescent rats, respectively) nor the yield of lymphocytes from these aggregates varies signi®cantly with age in rats (Schmucker et al., 1988; Schmucker et al., 2001). Whether or not this critical initial step in the intestinal mucosal immune response is compromised in old animals remains unresolved.
D.L. Schmucker / Experimental Gerontology 37 (2002) 197±203
Fig. 2. The number of IgA 1 cells per microscopic ®eld in the Peyer's patches of young, mature and senescent immunologically naõÈve or cholera toxin immunized rats. The Peyer's patches in old rats contain twice the number of cells as those of young animals regardless of their immune status. Each value represents the mean ^ SEM of ®ve rats.
4. Effect of aging on antigen presentation and isotype switching After uptake by M cells or other follicle-associated epithelial cells, the antigens are presented to immunocompetent cells by dendritic cells in the Peyer's patches and, in the absence of tolerance, a mucosal immune response is initiated. There is con¯icting evidence concerning the effect of aging on the number, distribution and function of the epidermal analog of dendritic cells, i.e. Langerhans cells (Fagiolo et al., 1993; Sprecher et al., 1990; Haruna et al., 1995; Steger et al., 1996). However, other studies have reported that the antigen-presenting capacity, as well as other functions, of dendritic
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cells derived from peripheral blood monocytes remain unchanged as a function of donor age (Steger et al., 1997; Saurwein-Teissl et al., 1998; Castle et al., 1999; Lung et al., 2000). These studies did not use dendritic cells isolated from intestinal Peyer's patches, thereby precluding extrapolation of these data to the intestinal immune response. Recent studies have isolated and characterized rat intestinal dendritic cells and devised an in vitro system to assess antigen-presenting capacity (Liu and MacPherson, 1995a,b; MacPherson et al., 1995; Maric et al., 1996; Szabo et al., 1997). Age-related losses in intestinal B and/or T lymphocyte subpopulations or changes in the distributions of lymphocyte subsets, may contribute to or re¯ect mucosal immunosenescence (Haaijman et al., 1977; Rivier et al., 1983; Bienenstock et al., 1984; Wade et al., 1988; Fleming et al., 1993; Daniels et al., 1993). For example, we reported a 2±3 fold increase in the number of IgA 1 cells in the Peyer's patches in old versus young adult rats (Schmucker et al., 1988; Taylor et al., 1992; Fig. 2). The increase, coupled with a concomitant decline in the size of this cell subpopulation in the intestinal lamina propria, provided the ®rst evidence that aging compromises the homing of IgA 1 immunoblasts from the Peyer's patches (inductive site) to the intestinal mucosa (effector site) (Haaijman et al., 1977; Ebersole et al., 1988). There have been no studies on IgA 1 isotype switching in the Peyer's patches as a function of age. Information concerning age-related shifts in gut-associated lymphoid tissue (GALT) lymphocyte subpopulations that may in¯uence isotype switching is limited.
Fig. 3. The numbers of IgA 1 (A) and cholera toxin 1 (B) cells in the small intestinal lamina propria of young, mature and old naive and cholera toxin immunized rats. The number of IgA 1 cells declines markedly between young adult and mature animals, whereas the decline in the number of cholera toxin 1 cells occurs between maturity and senescence. Each value represents the mean ^ SEM for ®ve rats.
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Fig. 4. The number of ¯uorescent-labeled (PKH26) mesenteric lymph node cells isolated from young and senescent donor rats, transferred intravenously into young or senescent recipient animals and localized to the small intestinal lamina propria by ¯uorescence microscopy 20 h after transfer. The values in the senescent recipients receiving cells from either young ( p) or old ( pp) donors are signi®cantly lower than those in young recipients. Each value represents the mean ^ SEM for ®ve animals.
Kawanishi and Kiely reported a loss of CD8 1 cells in the Peyer's patches of old mice, whereas our ¯ow cytometry studies did not detect a shift in the size of this lymphocyte subpopulation in rats (Kawanishi and Kiely, 1987; Daniels et al., 1993). However, our quantitative immunohistochemical analysis did reveal a marked age-related shift in the distribution of CD8 1 lymphocytes within rat Peyer's patches. 5. Effect of aging on IgA immunoblast homing to the intestine The numbers of IgA 1 and cholera toxin 1 cells in
the rat intestinal lamina propria exhibit .60 and .50% age-related declines, respectively, at similar intervals following intraduodenal immunization (Schmucker et al., 1988; Fig. 3). Similar cell populations decline 3±4 fold in the peripheral blood of cholera toxin-immunized old rhesus macaques (Taylor et al., 1992; Fig. 3). Using lymphocyte adoptive transfer, we demonstrated that the homing of mesenteric lymph node lymphocytes to the intestinal lamina propria is diminished in senescent rats in comparison to young animals (Thoreux et al., 2000; Fig. 4). Homing of labeled cells was lowest when old recipients were injected with cells from old donors and greatest when young cells were injected into young hosts. The migration of lymphocytes isolated from young animals was also lower in old recipients in comparison to young hosts, suggesting that the agerelated decline in homing re¯ects de®cits in both cells isolated from old rats and in old recipient animals. 6. Effect of aging on local IgA antibody secretion Aging is accompanied by 40±70% declines in antibody secretion by Peyer's patch and intestinal lamina propria lymphocytes isolated from mice and rats (Kawanishi and Kiely, 1989; Rivier et al., 1983; Daniels et al., 1993). Daniels et al. measured antibody secretion by GALT lymphocytes isolated from young and old rats following intraduodenal immunization with cholera holotoxin. Five days after immunization, IgA antitoxin secretion by mesenteric lymph node
Fig. 5. Anti-cholera toxin IgA antibody secretion into the culture medium of cells isolated from the Peyer's patches and intestinal lamina propria of young adult and senescent rats seven days after intraduodenal boosting with cholera toxin. The data are expressed as ng of IgA antibody per 10 6 total cells (A) and ng of IgA antibody per 10 3 antibody-secreting cells (B). The antibody levels in cultures of old cells in (A) are signi®cantly lower than those measured in cultures from young rats P , 0:05: However, this apparent age-related difference disappears when the data are expressed per population of antibody-secreting cells (B). The values represent the means ^ SEM for ®ve animals per age group.
D.L. Schmucker / Experimental Gerontology 37 (2002) 197±203
cells isolated from young rats was greater than that by cells from old animals. Despite the fact that Peyer's patches of old rats contained two-fold fewer anticholera toxin IgA-secreting cells in comparison to those of young animals, Peyer's patch cells isolated from old rats secreted signi®cantly more antibodies than similar cells from young animals. A 2.5-fold age-related increase in the number of CD8 1 lymphocytes in the intestinal lamina propria may re¯ect increased immunosuppression of IgA immunoblast maturation and/or antibody secretion at the effector site. This may contribute to more frequent tolerogenic responses in old animals (see Mayer, 1997 for a review). We recently reported that in vitro secretion of anticholera toxin IgA antibodies by lymphocytes isolated from the intestinal lamina propria of senescent rats was signi®cantly lower (.60%) than that of cells obtained from young animals (Thoreux et al., 2000; Fig. 5). Nevertheless, when antibody secretion was expressed relative to the number of anti-cholera toxin IgA-secreting cells in each culture, the agerelated difference was no longer evident. Therefore, the age-related decline in antibody titers at the effector site re¯ects fewer secreting cells rather than diminished antibody secretion per cell. 7. Effect of aging on IgA secretion by the mucosal epithelium The intestinal immune response culminates in the transport of IgA antibodies to the apical surface of enterocytes and their secretion onto the mucosal surface. This requires receptor-mediated endocytosis of the antibodies at the basal plasma membrane and their translocation to the mucosal surface in endocytic vesicles (see Underdown and Schiff, 1986 for a review). Nearly all mucosal epithelial cells express pIgR on their basolateral surfaces. Fifteen years ago, we reported a 4±6 fold age-related decline in the transport of polymeric IgA from blood to bile in rats (Schmucker et al., 1985). We also showed a concomitant 3±4 fold decline in the number of hepatic polymeric IgA receptors across this same age span, i.e. 3 and 25 months (Daniels et al., 1985). We and others subsequently showed that (a) the rat hepatic pIgR mRNA steady state level declines 20% between
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these ages and (b) the incorporation of [ S]-cysteine into pIgR lags in cultured hepatocytes isolated from old rats in comparison to cells from young animals (Gregoire et al., 1992; van Bezooijen et al., 1994). These data suggest that the expression of hepatic pIgR is diminished with increasing age and contributes to reduced IgA secretion into the bile and, ultimately, to a diminished intestinal immune response. These studies were performed using rat hepatocytes and there are no data concerning the effect of age on polymeric IgA transport across the small intestinal mucosa. Although crypt enterocytes exhibit a 3-fold greater binding of polymeric IgA than do villus tip cells, the expression of pIgR on the basolateral plasma membranes of rat small intestinal mucosal epithelial cells remains unchanged during aging (Daniels and Schmucker, 1987). The crypt-to-villus tip gradient in pIgR expression is identical in young and old rats and a similar pattern is seen in the rhesus macaque (Taylor et al., 1992; Daniels et al., 1988). The absence of an age-related change in the expression of pIgR on enterocytes suggests that antibody binding by intestinal epithelial cells remains unscathed by aging. 8. Conclusions The intestinal mucosal immune response to a variety of antigens is diminished in old animals and elderly humans. However, information concerning the effect(s) of aging on the individual events in the etiology of the intestinal immune response is limited. On the one hand, there are few data concerning the effect of aging on the initial steps in this process, e.g. those occurring at the inductive site (antigen uptake and presentation, isotype switching). On the other hand, we recently demonstrated an age-related de®cit in the homing of IgA immunoblasts to the effector site. Therefore, diminished antibody titers in the intestinal lavage of senescent animals most likely re¯ect fewer plasma cells rather than a decline in local antibody production. Acknowledgements This research was supported by the Department of Veterans Affairs, The Research Evaluation and
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