Impaired humoral immune responses to mycobacterial antigen in aged murine gut-associated lymphoid tissues

Mechanisms o f Ageing and Development, 54 (1990) 1431 161

143

Elsevier Scientific Publishers Ireland Ltd.

IMPAIRED HUMORAL IMMUNE RESPONSES TO MYCOBACTERIAL ANTIGEN IN AGED MURINE GUT-ASSOCIATED LYMPHOID TISSUES

HINDENORI KAWANISHI*, SHIN AJITSU and SCOTT MIRABELLA Gut Mucosal Immunity Laboratory, State University o f New York A t Stony Brook and Research Service, Northport Veterans Administration Medical Center, Northport, N Y 11768 (Received September 13th, 1989) SUMMARY

Senescence-related alterations of local gut mucosal immune responses to enteric mycobacterial antigen (Ag) were examined. Both aged (> 24 months old) and young adult (4--5 months old) BALB/c mice were enterically immunized with crude Mycobacterium paratuberculosis (M. paratbc) protoplasmic Ag, and in vitro Ag- and class-specific immunoglobulin (g) production by lymphocytes from gut-associated lymphoid tissues (GALT) (Peyer's patches, PP; mesenteric lymph nodes, MLN) and non-GALT (spleen, SPN) were determined against semipurified M. paratbc Ag. Ag-specific spontaneous immunoglobulin production by aged B cells from both GALT and non-GALT was enhanced only to a minor extent. Similarly, the functional activity of the Ag-specific T (Th) (CD3 + , CD4 + ) cell in both GALT and non-GALT was not profoundly affected by senescence (qualitative preservation). However, that of the suppressor T (Ts) (CD3 + , CD8 + ) cell was considerably diminished (qualitative defect). Thus, oral tolerance (systemic immunologic hyporesponsiveness) to M. paratbc Ag in aged mice is impaired. These age-related changes, manifested as hyperreactive humoral responses to the enteric microbial Ag, are due, at least in part, to hyporeactivity of the Ts cell, resulting in relative hyperfunction of the Ag-specific Th cell, despite the quantitative defect of the latter cell.

Key words: Aged gut-associated lymphoid tissues; Aging and immune responses of GALT; Mycobacterial Ag and GALT INTRODUCTION

A number of studies have demonstrated significant effects of aging on systemic *To whom all correspondence should be addressed at: Gut Mucosal Molecular Immunity Laboratory, Department of Medicine, UMDNJ I Robert Wood Johnson Medical School, One Robert Wood Johnson Place i CN 19, New Brunswick, NJ 08903-0019, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland

© 1990 Elsevier Scientific Publishers Ireland Ltd.

144

immune responses [1--4]. Animal model studies reveal an association between the age-realted decline in T cell-dependent immunologic responses and the decline in resistance against microbial infections [5--10]. In contrast, studies seeking Ag-specific gut mucosal immune responses have found less effects of age [11,12]; however, our previous results in aged BALB/c mice do not support their results [13--16]. Immunoregulatory T cell defects likely reside in aged gut-associated lymphoid tissues (GALT) as well. This discrepancy could be due to types of stimulants, different strains of mice, a n d / o r different assay systems used for determination of immunoregulatory functions. Resistance against tolerance induction in some strains of mice has been attributed to loss of specific suppressor cell activity [4,17,18], whereas the unresponsive state of helper T cells and B cells are also involved in the development of an animal's unresponsiveness. The real mechanism of these losses of tolerance in aging, however, is presently unclear. In this study, an enteric microbial Ag, Mycobacterium paratuberculosis (M. paratbc) protoplasmic macromolecules, was chosen as an antigenic stimulant. Live M. paratbc is the infectious agent in paratuberculosis, also known as Johne's disease, which is an insidious, orally transmitted mycobacterial disease of the intestinal mucosa found in herbivorous animals. Mice can be infected rather extensively by the routes of intraperitoneum and vein [19]. Thus, in the present study, we investigated whether the aging process impairs gutmucosal humoral immune responses of BALB/c mice to this enteric antigen, and if so, we sought to determine the immunoregulatory defects that are involved. MATERIALS AND M E T H O D S

Animals Specific pathogenic bacteria-free inbred normal mice (male), BALB/c NNia (H2), were obtained from the National Instituteon Aging (NIA) contract-supported colonies. Two age groups, the young adult (4--6 months old) and the aged (over 24 months old), were studied. These mice have a median survival of about 28 months. Animals with gross pathologic abnormalities were excluded. All mice were maintained under specific pathogen-free conditions at the Northport VA Medical Center Animal Facility. Bacterial Ag used for enteric immunization in vivo and stimulation of unseparated and separated lymphocytes in vitro Protoplasmic extracts prepared from M. paratbc bacilli were obtained from Dr. R.S Merkal, National Animal Disease Center, Ames, IA. The organisms were grown as a pellicle on Dorset-Henley medium, harvested by filtration on coarse filter paper, and suspended in approximately 3 vol. of distilled water. The suspension was passed through the Ribi press at 40 000 psi. It was centri-

145 fuged at 40 000 g, and the supernatant was dialyzed overnight against water. It was again centrifuged at 40 g, but this time the midportion was harvested, leaving both the upper lipid layer and ribosomal pellet. The midportion was filtered through an 0.45 tam-Millipore filter and lyophilized. This antigenic material, referred to below as the crude M. paratbc Ribi Ag, was used for enteric immunization. Chromatographic separation of semipurified M. paratbc Ag was carried out as described below. Crude M. paratbc Ag was applied first to a G-100 gel filtration column, which was equilibrated with phosphate-buffered saline (PBS, pH 7.0). The Ag was eluted from the column with this buffer. Two peaks at 280-nm wavelength were obtained; the first peak had more antigenicity, as determined by an enzymelinked immunosorbent assay (ELISA), using anti-M, paratbc mouse serum. (The antiserum [positive reference] was prepared from mice enterically infected with live M. paratbc bacilli (wild strain 21) during their suckling period.) Next, the pooled first-peak samples were concentrated and dialyzed in 0.1 M Tris buffer (pH 8.0). The samples were then applied to a column packed with DEAE-Sephacel. Stepwise elution was carried out with increasing decimolar concentrations (0.1--0.6 M NaC1 in 0.1 M Tris buffer). This chromatography gave 7 peaks (p, Nos. 1--7). The fractions of each peak were pooled and concentrated. Optimal concentrations of the fractionated Ag (1, 25, 100 and 500/ag/ml) were tested against the positive reference serum, diluted at 1:2500 and 1 : 10 000. Results were recorded as the optimal density (OD) value at 492-nm wavelength. A fraction eluting with 0.3 M NaCI (i.e., peak 3) yielded the highest OD value at each Ag concentration. This fraction was used as the semipurified protoplasmic Ag in the present study. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (10070) analysis of crude M. paratbc Ag gave multiple bands with wide ranges of molecular weight, whereas that of the semipurified M. paratbc Ag gave one major band of mol. wt 25.8K, in addition to a few other much less prominent bands (Fig. 1). The semipurified M. paratbc protoplasmic Ag was employed in determining M. paratbc- and isotype-specific antibodies by ELISA. There was no significant Ig production by unprimed young and aged GALT and non-GALT cells in vitro against the semipurified Ag, regardless of the presence or absence of the Ag in cultures. In the ELISA, any of three Ag- M. avium, M. phlei and M. fortuitum did not crossreact with semipurified M. paratbc Ag when M. paratbc antisera were tested, and the sera were screened at 1:40 dilution. A relevant Ag, purified protein derivative (PPD) (Connaught Lab., Ontario, Canada) and a nonrelevant Ag, ovalbumin (Sigma Chemical Co., St. Louis, MO), were also tested.

Preparations of Cell Suspension of Peyer's Patches (PP), Mesenteric Lymph Nodes (MLN) and Spleen (SPN) Single cell suspensions from PP were prepared by a combined enzymatic and mechanical method [20]. MLN and SPN single cells suspensions were made as described previously [211.

146

Kd 92.5-) 66.2"-+ 45.0-) 31.0-~

21.5-) 14.4-)

1

2

r" 0

"!"

4

5

6

7

8

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10

Z

®~_.m

0 O "O

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Fig. 1. Sodium dodecyl sulfate polyacrylamide gel (SDS - - PAGE) (10%) analysis of crude Mycobacterium paratuberculosis protoplasmic Ag (lane 4), and semipurified Mycobacterium paratuberculosis protoplasmic Ag DEAE-Sephacel column - - separated fraction 3 (lane 5), and PPD (lane 6) under reducing conditions, according to the method of Laemmli (34).

147

Detection o f surface markers on cells separated The surface phenotypes of the isolated fresh and cultured cells were determined by direct immunofluorescence [21]. The Ab used included affinity-purified monoclonal anti-mouse CD3 + (Thy 1.2), CD4 + (L3T4) and CD8 + (Lyt 2); antimouse polyclonal Ig against murine IgF(ab')2; and affinity-purified class-specific anti-mouse IgM, IgG, and IgA (Zymed Laboratories, South San Francisco, CA). Culture medium Cells were suspended in RPMI 1640 supplemented with 5°70 heat-inactivated FCS (Hyclone, Sterile Systems, Inc., Logan, UT), penicillin at 100 units/ml, streptomycin at 100/~g/ml, gentamicin at 50/~g/ml and 5 x 10-5 M 2-mercaptoethanol (2-ME), 25 mM Hepes buffer, 25 mM L-glutamine, 1 mM sodium pyruvate and 0.1 mM nonessential amino acids (complete culture medium [CCM]). Further addition or depletion of supplements depended on the type of culture or assay used, and is indicated below. Development of MLN- and SPN-derived B cell hybridoma cell lines, producing class- and Ag (M. paratbc)-specific monoclonai antibodies (mAb) and purification of these mAb Two BALB/c mice, which were infected orally with live M. paratbc bacilli during their suckling period (5 × 10~ CFU/dose, 3 doses/week) beginning on 4th or 5th day after birth, were killed at 8 months. Single cell suspensions of MLN and SPN were prepared, and were stimulated with crude M. paratbc Ag (5/~g/ml) in the presence of B cell stimulatory factors (BSF) (20W0v/v) from our autoreactive PP-derived BSF-producing cloned hybridoma cell lines [22] for 5 days, followed by B cell separation, using anti-Fab panning. Viable B cells thus obtained were further stimulated with the Ag and BSF for the next 4m5 days in order to enrich the Agspecific B cells. The primed MLN or SPN B cells were hybridized with the Sp2/0Agl4 B cell fusion partner at a 1:1 ratio, and 1 × 104 mitomycin C (50 mg/ml)pretreated BALB/c SPN cells/well were used as filler cells. In addition, thymus conditioned medium (10 v/v) and endothelial growth factor (CR-ECGS) (100 units/ ml) (Collaborative Research, Inc., Lexington, MA) were added. Selective hypoxanthine-aminopterin-thymidine (HAT) medium was used throughout. Two or three of each isotype-specific anti-M, paratbc mAb-producing hybridomas were subsequently subcloned by limiting dilution, plating cells at 0.5 cells/well. Limiting dilution conditions were confirmed by Poisson analysis. Culture supernatants harvested from each expanded isotype-specific subclone culture were obtained, and each isotype- and M. paratbc Ag-specific mAb (IgM, IgG, and IgA) were purified by each isotype-specific affinity column chromatography. Microculture system for in vitro Ig production Appropriate numbers of unseparated or fractionated cells at a density of 2 x 105

148

or 1 x 105/well were co-cultured in duplicate in wells of flat-bottomed 96-well microtiter plates in a final volume of 0.25 ml culture medium at 37°C for 10 days [21,23].

Quantitative measurement o f antibody secreted in microcultures An ELISA was used for the determination of Ag- and class-specific Ig secreted, as described below. Semipurified M. paratbc Ag (optimal, 10/ag/ml)-precoated wells of a polystyrene immunoplate II (Dynatech, Rockville, MD) were incubated with unknown test samples or each standard affinity-purified, class-specific anti-M. paratbc mAb prepared from our M. paratbc-specific B cell hybridomas, as described above, class-specific rabbit anti-mouse Ig conjugated to alkaline phosphatase, and finally p-nitrophenylphosphate substrate. The plates were read in the MR600 MicroELISA AutoReader (Dynatech, Rockville, MD). A standard curve was constructed for each assay to determine unknown samples. In preliminary experiments, the specificity of each isotype-specific, affinity purified, Ag-specific anti-Ig was determined by demonstrating that these anti-Ig were solid-phase absorbed against the other affinity-purified murine Ig and M. paratbc Ag. Cell fractionations o f B cells, CD4 + and CD8 + -bearing T cells from single cell suspensions o f GAL T (PP and M L N ) and S P N T and B cell populations were separated by panning on goat anti-mouse IgF(ab')2-coated petri dishes [21]. Further purification procedures are described in detail [15]. To purified CD4 + T cells, anti-CD4 mAb was added for panning after separation of Lyt2- T cells in some experiments. Functional ability to Ag-activated Th/si and Ts cell subpopulations Two Ag-reactive T cell-enriched subsets (CD4 + Th and CD8 + Ts cells) separated on anti-CD4 and anti-CD8 plates [13,15], were tested for their effect on classspecific Ig production by PP-derived B cells in the presence or absence of semipurified M. paratbc protoplasmic Ag. In the preliminary experiments, cocultures were done at different ratios of specified combinations to determine an optimal ratio of coculturing B cells to each immunoregulatory T effector cell, for evoking each appropriate immunoregulatory activity demonstrated in vitro Ag-specific enteric immunization Using a per oral intragastric tube, mice were immunized enterically with crude M. paratbc protoplasmic Ag (1 mg/0.2 ml per time) on days 1, 2, and 14. On day 21, these treated mice were killed. In addition, control mice were also treated i.g. with saline (0.2 ml/dose) as above. Statistical analysis Individual experiments were repeated at least three times. Results are expressed as

149

the mean of duplicate or triplicate cultures. The arithmetic means and standard errors were calculated. Group comparisons were made by employing the MannWhitney Two-Sample Rank Test. RESULTS

Ag-specific, class-specific immunoglobulin (Ig) production by G A L T and nonGAL T whole cells We determined Ag- and class-specific Ig production in vitro by young adult and aged GALT (PP and MLN) and non-GALT (SPN) cells derived from mice unprimed or primed enterically with crude M. paratbc protoplasmic Ag. Various in vitro concentrations of semipurified M. paratbc protoplasmic Ag, PPD, and ovalbumin were used as stimulants. Spontaneous Ag-specific IgM and IgA production were augmented in aged PP (Fig. 2a). IgM, IgG, and IgA production were increased in aged MLN (Fig. 2b) and non-GALT (SPN) (Fig. 2c). The production of Ag-specific IgA by the aged cells in the presence of the optimal concentration (1 /ag/ml) of semipurified M. paratbc protoplasmic Ag was augmented as compared to that by the young adult cells (P < 0.001), and was highest in PP, followed by MLN and SPN cells in that order. Similar results were also observed for IgM production, and IgG production differed only in the rank order of increase. Unprimed Ag, PPD, and ovalbumin evoked only marginal amounts of each corresponding Ag-specific, class-specific Ig by aged GALT and non-GALT cells (data not shown). Ag- and class-specific Ig production by G A L T and non-GALT B cells in the presence or absence o f the corresponding lymphoid tissue T cells or their subsets Effects of T cells were investigated in vitro on Ag-specific, class-specific Ig production in vitro by GALT and non-GALT B cells derived from young adult and aged mice unprimed or primed enterically with crude M. paratbc protoplasmic Ag (Table Ia--c). The semipurified M. paratbc protoplasmic Ag was employed as an in vitro Ag-specific stimulus at an optimal concentration of 1/ag/ml. In primed aged mice, GALT and non-GALT B ceils produced Ag-specific IgM and IgA, and IgM and IgG spontaneously only to a small extent over those of young adult mice, respectively. Ig production was enhanced only to a minor extent in the presence of semipurified M. paratbc protoplasmic Ag. As the ratio of T to B ceils was increased, each Ag- and isotype-specific Ig production was greatly augmented in aged primed mice under stimulation of the semipurified Ag; enhanced IgA production was observed in the rank order PP, MLN, and SPN cells at a T/B ratio of 2" 1. In contrast, IgM production was enhanced in the rank order MLN, PP and SPN, whereas IgG production was in the rank order SPN, MLN, and PP at the same T/B ratio.

150

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Semipurified M. paratbc Protoplasmic Ag Concentrations Employed in Vitro Fig. 2a. Fig. 2. Secreted Ag- and class-specific immunoglobulins (Ig) (ng/ml) in vitro by Peyer's patch (PP) cells (a), mesenteric lymph node (MLN) cells (h) and spleen (SPN) cells (c) from young adult and aged BALB/ c mice. These lymphoid cells were stimulated with various amounts of semipurified M. paratuberculosis protoplasmic Ag. All values are expressed as mean ng/ml _+ S.E., in three experiments, in each of which three mice were used.

In young adult mice, an increase in the ratio of T cells to B cells from primed GALT (in particular, PP), but not from SPN, in the coculture significantly enhanced Ag- and isotype-specific IgA production. The production of other Ig isotypes was only slightly or not at all augmented; the actual amounts produced were

151

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much lower than those produced by aged GALT and non-GALT T and B cell cocultures (P < 0.001). The unprimed Ag, PPD/ovalbumin, did not induce significant levels of Agspecific Ig by B ceils alone or by B cells plus T cells in mixed cultures regardless of in vivo prepriming with crude M. paratbc Ag (data not shown).

152

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Semipurified M. paratbc Protoplasmic Ag Concentrations Employed in Vitro Fig. 2c.

Effects o f CD4 + T cells and CD8 + T cells on Ag- and isotype-specific Ig production by B cells derived from young adult and aged mice primed enterically As shown in Table IIa,b, aged GALT B cells produced predominantly IgM and IgA in the presence of aged CD4 + T cells, whereas aged non-GALT B cells produced predominantly IgM and IgG in the presence of aged CD4 + T cells (P < 0.001). IgA production in the coculture of aged GALT B and CD4 + T cells at a ratio of 1(B) to 2(T) was highest exceeding even IgG production in coculture of aged non-GALT B and CD4 + T cells at the same ratio.

3 4 ± 6b(<5) c 5 9 ± 8(14 ± 7) 78 ± 12(20 ± 6) 95± 8(38±9)

59±11(18±4) 148 ± 13 (33 ± 6) 225 +- 21 ( 5 6 ± 5) 351±23(83±8)

<5(<5) 1 0 ± 2(<5) 2 6 ± 4(<5) 30±5(<5)

Young adu~

YoungaduR

Aged

IgG

lgM

AntbM. paratbclgProduced(ng/mO(1 × lO~Bcells/well) ~

<5(<5) 14 ± 4 (<5) 44 ± 5 ( 9 ± 3) 63±8(15±5)

Aged

5 3 ± 9 ( 2 1 ± 4) 2 4 0 ± 18 ( 6 9 ± 9) 325 + 24 (98 ± 8) 519±38(115±12)

Youngadu~

lgA

'Cultured for 10 days. bMean ng/ml ± S.E.M. in three experiments with in vitro Ag stimulation. ¢Mean ng/ml ± S.E.M. in three experimetns without in vitro Ag stimulation. Three or four mice were employed in each experiment.

BceUsalone I (B): 0.5 (T) I(B):I(T) I(B):2(T)

B: T cell ratio co-cultured with~without semipurified M. paratbc protoplasmic Ag (1 ~glmO

118 379 603 776

± 12 (52 ± 7) ±26(103 ± 11) ± 23 (158 ± 20) ± 4 2 ( 1 8 4 ± 18)

Aged

ANTIGEN- AND CLASS-SPECIFIC IMMUNOGLOBULIN (Ig) PRODUCTION IN VITRO BY PEYER'S PATCH (PP)-DERIVED B CELLS 1N THE PRESENCE/ABSENCE OF PP-DERIVED T CELLS FROM YOUNG ADULT (4--6 MONTHS OLD) AND AGED (OVER 24 MONTHS OLD) MICE PREPRIMED ENTERICALLY WITH M. P A R A TUBERCULOSIS (PARA TBC) ANTIGEN (Ag).

TABLE I (a)

t~

41 ± 7 ( < 5 ) 68±7(18±3) 85±5(26±2) 72 ± 6 ( 3 0 __+ 4)

68 ± 8(25 + 3) 2 1 0 ± 1 6 ( 4 8 ± 5) 306±23(60___7) 415 ± 27 (88 ± 6) 44 _ 4 (< 5)

20 ± 3 ( < 5 ) 30±4(<5) 38±5(<5)

Young adult

Young adult

Aged

IgG

IgM

A nti-M, paratbc Ig Produced (ng/ml) (1 x l i t B cells~well) °

36 ± 8 ( < 5 ) 101 ± 9 ( 2 2 ± 4 ) 158± 12(32±3) 193 ± 15 (40 ± 5)

Aged

139 ± 1 0 ( 3 4 ± 4)

55 ± 5(10 ± 3) 84± 8(21±4) 110± 12(30±3)

Young adult

lgA

aCultured for 10 days. bMean n g / m l - S.E.M. in three experiments with in vitro Ag stimulation. ~Mean n g / m l __. S.E.M. in three experimetns without in vitro Ag stimulation. Three or four mice were employed in each experiment.

1 (B): 2 ( T )

B cells alone I (B): 0.5 (T) I(B):I(T)

B: T cell ratio co-cultured with~without semipurified M. paratbc protoplasmic Ag (1 l~g/mO

99 231 368 543

± 7(20 ± ± 21 (35 ± ± 31 (49 ± ± 38 (60 _

Aged 3) 5) 2) 4)

ANTIGEN- A N D CLASS-SPECIFIC 1 M M U N O G L O B U L I N (lg) P R O D U C T I O N I N VITRO BY MESENTER1C L Y M P H NODE (MLN)-DERIVED B CELLS IN T H E P R E S E N C E / A B S E N C E OF M L N - D E R I V E D T C E L L S F R O M Y O U N G A D U L T ( 4 - - 6 M O N T H S OLD) A N D A G E D (OVER 24 M O N T H S OLD) MICE P R E P R I M E D E N T E R I C A L L Y W I T H C R U D E M. PARA TUBERCULOSIS (PARA TBC) P R O T O P L A S M I C A N T I G E N (Ag).

T A B L E I(b)

4 0 _ 5 ( 2 0 - 4) 58__.6(35_+3) 70-+9(40-+5) 41 _+ 3(38 -+ 6)

83 _+ 6 ( 5 0 - 5) 156_+ 14 (36_+6) 213_+21(110-+4) 207 -+ 25032 - 9)

Aged

44_+ 4(26-+ 3) 94_+ 6 (48 -+ 5) 109_+ 15(32-+2) 338_+25(121 -+ 12) 116_+ 1300-+ 5) 4 0 4 _ + 3 6 ( 1 5 6 -+ 8) 39 _+ 2(>5) 533 -+ 39(183 _+ 13)

Young adult

Young adult

Aged

IgG

IgM

Anti-M. paratbc Ig Produced (ng/ml) (1 x 10J B cells~well)~

19_+ 3(<5) 33-+4(<5) 48_+7(<5) 21 _+ 5(<5)

Young adult

IgA

•Cultured for 10 days. bMean ng/ml -+ S.E.M. in three experiments with in vitro Ag stimulation. cMean ng/ml _+ S.E.M. in three experimetns without in vitro Ag stimulation. Three or four mice were employed in each experiment.

B cells alone I (B): 0.5 (T) I(B):I(T) 1 (B):2(T)

B: Tcell ratio co-cultured with/without semipurified M. paratbc protoplasmic A g (I ~g/ml)

49-+ 8(10-+ 4) 85-+11(21_+2) 138___ 15(36-+5) 171 _+ 17(48 _+ 4)

Aged

ANTIGEN- AND CLASS-SPECIFIC IMMUNOGLOBULIN (Ig) PRODUCTION I N VITRO BY SPLEEN (SPN)-DERIVED B CELLS IN THE PRESENCE/ABSENCE OF SPN-DERIVED T CELLS FROM YOUNG ADULT (4--6 MONTHS OLD) AND AGED (OVER 24 MONTHS OLD) MICE PREPRIMED ENTERICALLY WITH M. P A R A TUBERCULOSIS (PARA TBC) ANTIGEN (Ag).

TABLE l(c)

U,

156 TABLE II(a) A N T I G E N - A N D CLASS-SPECIFIC I M M U N O G L O B U L I N (Ig) P R O D U C T I O N I N VITRO BY GUTASSOCIATED L Y M P H O I D TISSUE (GALT)-DERIVED B CELLS IN THE PRESENCE OF GALTDERIVED CD4 + T CELLS/CD8 + T CELLS FROM YOUNG A D U L T (4--6 MONTHS OLD) AND AGED (OVER 24 MONTHS OLD) MICE P R E P R I M E D ENTERICALLY W I T H CRUDE M. PARATUBERCULOSIS (PARA TBC) P R O T O P L A S M I C A N T I G E N (Ag).

Co-culture condition in presence of semipurified

Anti-M. paratbc lg Produced (ng/ml) (1 x 10~B cells~well)a

M. paratbc Ag (] laglmO

IgM

Young

IgG

Aged

adult B cells alone B cells plus CD4 + T cells (TI) I(B):0.5(T1) I(B):I(TI) I(B):2(TI) B cells plus CD4 + T cells (TI) plus CD8 + T cells (T2) I(B):I(TI):0.5(T2)

I(B):1(I"1):I(T2) I(B):I(T1):2(T2)

6 6 ± l0 b

IgA

Young

Aged

Young

adult 98 ± 18

10±

Aged

adult 5

27 ±

4

65_

11

102 ± 16

1 0 4 ± 20 235±24 437 ± 38

263 ± 31 502±34 728 ± 73

44 ± 5 76± 7 124 ± 1 0

65 ± 5 93±11 141 ± 17

196 ± 10 283+11 496 ± 14

233 ± 23 357±30 650 ± 41

191 ± 16 103 ± 30 48 ± I0

516 ± 34 453 ± 31 414 ± 45

54 ± 7 27 ± 11 <5

104 ± 10 101 ± 6 79 ± 7

105 ± 13 32 ± 5 <5

323 ± 27 281 ± 29 253 ± 22

aCultured for 10 days. bMean ng/ml ± S.E.M. in three experiments. Five or seven mice were employed in each experiment.

The degree of help by aged GALT and SPN CD4 + T cells did not differ (P > 0.05), irrespective of whether aged or young adult corresponding B cells were used as targets. Amounts of IgM produced were 409 ± 41, of IgG 110 __. 69, and of IgA 469 ± 32 ng/ml (1 x 105 B cells/well) at a ratio of young adult GALT B cells (1): aged CD4 + T cells (2). Those of IgM produced 399 ± 35; of IgG 327 ± 37, and of IgA 123 ± 13 ng/ml (1 × 105 B cells/well) at a ratio of young adult SPN B cells (1): aged CD4 + T cells (2). Next, in the presence of B cells and C D 4 + T cells at a ratio of I(B) to I(T), we determined the effect of CD8 + T cell on B cell production. Preliminary studies had showed CD8 + T cells alone to have no significant helper or suppressor activity toward B cell Ig production in vitro.. However, increasing concentrations of young adult CD8 + GALT or SPN T cells evoked considerable suppression in the mixed culture of B and CD4 + T cells from both lymphoid tissues of young adult mice, whereas similar concentrations of aged CD8 + T cells evoked only marginal suppression ( P < 0.001) (Table 2a,b). Finally, to determine further whether the functional (suppressor) activity of aged

157 TABLE II (b) ANTIGEN-AND CLASS-SPECIFIC IMMUNOGLOBULIN (lg) PRODUCTION IN VITRO BY SPLEEN (SPN)-DERIVED B CELLS IN THE PRESENCE OF SPN-DERIVED CD4+ T CELLS/ CD8 + T CELLS FROM YOUNG ADULT (4--6 MONTHS OLD) AND AGED (OVER 24 MONTHS OLD) MICE PREPRIMED ENTERICALLY WITH CRUDE M. PARATUBERCULOSIS (PARA TBC) PROTOPLASMIC (Ag). Co-culture condition in presence of semipurified M. paratbc Ag (1 ~g/mO

Anti-M. paratbc lg Produced (ng/mO (1 x lO~B cells/well)~ IgM Young adult

B cells alone B cells plus CD4 + T cells (TI) I(B):0.5(T1) I(B):I(T1) l(B):2(T1) B cells plus CD4 + T cells (TI) plus CD8 + T cells (T2) l(B):l(T1):0.5(T2) I(B):I(TI):I(T2) I(B):I(T1):2(T2)

53-+ 8b

lgG Aged

96_+ 7

Young adult

30_+ 8

IgA Aged

85_+ 9

188 _+ 10 201 -+ 21 299_+ 19 343 _+ 22 420_+ 24 513 -+ 32

99-+ 13 214_+ 20 180_+ 18 347 _+ 28 353 + 29 531 _+ 56

181 _ 14 313 _+ 20 102_ 5 265 _ 24 24 -+ 6 241 _+ 16

113_.+ 12 345 _+ 27 53 ± 9 299_+ 25 <5 267 _+ 12

Young adult

<5

22_+ 4 53 _+ 9 ll0_+ 7

<5 <5 <5

Aged

43_+ 7

83 _+ 5 141 _+ 6 213 _ 9

132 _+ 7 126_+ 8 93 _+ 5

~Cultured for l0 days. bMean ng/ml --+ S.E.M. in three experiments. Five or seven mice were employed in each experiment.

CD8 + T cells was selectively i m p a i r e d , y o u n g adult B cells, y o u n g adult C D 4 + T cells a n d aged CD8 + T ceils f r o m Ag p r i m e d G A L T a n d n o n - G A L T (SPN) were cocultured at a ratio o f 1 : 1 : 2, respectively. The following results revealed n o difference in the p r o d u c t i o n o f each Ag-specific Ig class c o m p a r e d to those o f the cocultures o f all aged cell types, i n d i c a t i n g rather selective defects o f the aged Ts (CD8 + ) cells. I n G A L T , 208 _+ 29 n g / m l o f IgM (l × 105 B cells/well), 84 _+ 12 n g / m l o f IgG a n d 236 _+ 30 n g / m l o f IgA, were p r o d u c e d , whereas in n o n - G A L T 264 _+ 21 n g / m l o f IgM (1 x 10 ~ B cells/well), 143 _+ 19 n g / m l o f IgG, a n d 60 _+ 12 n g / m l o f IgA were p r o d u c e d . DISCUSSION T h e results o f the work reported here have d e m o n s t r a t e d that local gut m u c o s a l m e m o r y specific to a p a r t i c u l a r enteric m i c r o b i a l Ag, M . p a r a t b c p r o t o p l a s m i c antigenic p r o t e i n , is in a h y p e r r e s p o n s i v e state in senescent mice. Those oral i n t o l e r a n c e to the Ag in aged mice is at least a t t r i b u t a b l e to hyporeactive effector f u n t i o n o f the

158 Ag-specific suppressor T (Ts) cell, whereas the in vitro T and B cell coculture experiments indicate that the effector activity of the Ag-specific helper T (Th) cell in both GALT and non-GALT (SPN) is much less affected by aging than is the Ts cell. When immunized enterically, the Th cells augmented Ag-specific IgM and IgA responses in GALT and the IgM and IgG responses in non-GALT. Suppressor cells have received considerable attention in studies of in vitro and in vivo host immune responses to Ag-specific and non-specific stimuli, because the Ts cell has been demonstrated to play an important role in regulating the immune response as well as inducing and maintaining tolerance to exogenous and self antigens [24]. An increase in Ts cells in aged animals has been reported [25], as have increases in suppressor activity [25--27]. On the other hand, identification of Ts cells by functional analyses has suggested a decrease in Ts cells in aging animals and humans [4,13,17,18,28]. The latter observation supports the present results, in particular those obtained by the cross coculture experiments, which demonstrate that semipurifled M paratbc protoplasmic Ag-specific Ts cells are induced in lower amounts in old than in young adult mice. As a result, aged mice show a considerable decrease in Ag-specific immunosuppression in GALT. Our previous work also demonstrated that non-specific T cell mitogen (Con A)activated lymphocytes from GALT of aging mice (BALB/c) manifested a loss in the ability to nonspecifically suppress the response to mitogens [15]. These inconsistent experimental observations appear to be attributable to different methodological approaches and the use of different organs and also different strains of mice, in addition to different assay systems in some experiments. Thus, the present observation may not be a generalized feature of gut mucosal immune responses in aged mice. The proportion of mature B cells responsive to a variety of Agis often decreased in aged mice, although only to a slight extent [1,13,29,30]. However, studies of bone marrow cells in aged mice have generally demonstrated that the ability to generate B cells from the stem pool is not diminished with age [30,31]. The unusual findings at the B cell level were also demonstrated by the finding that responses to an abundant environmental antigen, phosphorylcholine (PC), by splenic B cells were significantly increased with age in BALB/c mice [32]. This high proportion of PC-specific splenic B cells was assumed to be due to an increase in the frequency of newly generated PC-specific B cells. PC-specific precursor cell pools were also increased. The authors presumptively speculated that aging may be accompanied by progressive changes in the read-out of heavy- and/or light-chain variableregion genes. Although it is not yet clear whether such changes in variable region gene expression are extensive in aged animals, our findings of augmentation of all classspecific and Ag-specific Ig production in vitro under stimulation with primed Ag could be consistent with the results of the above studies of PC-specific B cell respon-

159

ses in aged mice. The age-associated Ag-specific hyperresponsiveness to certain enterically immunized Ag could be fundamentally due to an alteration in variableregion gene expression of Ag-responsive B cells concomitant with aging. In addition, there is a more selective decrease in mucosal and probably also non-mucosal Agspecific suppressor T cell activity, as shown here. In the present studies, spontaneous Ag-specific Ig production by preprimed B cells derived from aged GALT and non-GALT (SPN) is augmented. This suggests that the Ag-specific aged B cell in vivo is impaired, at least in the later stage of maturation induced by the primed Ag. In any event, the enhancement of Ag-specific humoral responses was more prominent in GALT than in SPN, indicating that the major site of immune reactivity is in GALT, when immunization occurs through the gastrointestinal route. Cinader and his associates [33] demonstrated that there is a decrease in function of one subset of Th cells and one subset of Ts cells in aging C57BL/6 mice. On the other hand, there is a concomitant increase in another type of Th cell, which is independent of the carrier-hapten bridge. The two types of Th cells appear to be independently operable during age-dependent changes. Based on our present findings, the two kinds of T helper cells could be the conventional Th cell subset and the suppressor-inducer Th cell subset. The regulatory balance of these independent effector Th cells affects the functional capacity of the Ts cell. Finally, the present results suggest that the diminished suppressor activity in aged GALT is due mainly to a marked senescence-associated decrease in Ag-specific Ts cell effector function, but whether or not another type of immunoregulatory T cell, such as the suppressor-inducer T cell, is defective, and/or whether or not the intrinsic defect lies, if any, in the aged Ts cell remains to be investigated. Furthermore, it has yet to be resolved whether functional impairment of the Ts cell in aged GALT contributes to favoring a host immune defense in vivo through the generation of more microbial-Ag-specific cytotoxic cells. ACKNOWLEDGEMENT

This work was supported by Public Health Service grant AG 07603 from the National Institute of Health and partly by a Veterans Administration Merit Review Fund. Dr. Kawanishi is a recipient of a Clinical Investigatorship Award, Research and Development Program, Veterans Administration. REFERENCES 1 2 3

T. Makinodan and M. Kay, Age influence on the immune system. Adv. Immunol., 29 (1980) 287-330. M.R. Szewczuk and R.J. Campbell, Loss of immune competence with age may be due to auto-antiidiotypic antibody regulation. Nature, 286 (1980) 164--166. U. Ponnappan, B. Cinader, V. Gerber and K. Blaser, Age-dependent changes in isotype expression and down-regulation of C57BL/6 mice. Scand. J. ImmunoL, 25 (1987) 45--54.

160 4 5 6 7 8 9 10

11

12 13 14 15

16

17 18

19

20 12

22

23

24 25

M.L. Thomas and W.O. Weigle, Deficiency in suppressor T cell activity in aged animals: Reconstitution of this activity by interleukin 2. J. Exp. Med., 157(1983) 2184--2189. S. Ohar, S.R. Sharki and R.A. Lenora, Aging and the respiratory system. Med. Clin. N. Am., 50 (1976) ll21--1139. I.D. Gardner and J.S. Remington, Age-related decline in resistance of mice to infection with intracellular pathogens. Infect. Immunol., 16 (1977) 593--598. M.M.B. Kay, Parainfluenza infection of aged mice results in autoimmune disease. Clin. Immunol. Immunopathol., 12 (1979) 308--315. P.J. Patel, Aging and cellular defense mechanisms: Ag-related changes in resistance of mice to Listeria monocytogenes. Infect. Immunol., 32 (1981) 557--562. T. Makinodan, S.J. Jameo, T. Inamizu and M.-P. Change, Immunological basis for susceptibility to infection in the aged. Gerontology, 30 (1984) 279--289. Y. Yokota, Y. Wakai, M. Yasuhiro, S. Goto, M. Nichida and Kuwahara, S. Degradation of host defenses against respiratory tract infection by Klebsiella pneumoniae in aged mice. Infect. Immunol. 56(1988) 966--97 I. M.R. Szewczuk and R.J. Campbell, Differential effect of aging on the heterogeneity of the immune response to a T-dependent antigen in systemic and mucosal-associated lymphoid tissues. J. Immunol. 126 (1981) 472--477. D.N. Ernest, W.O. Weigle and M.L. Thoman, Retention of T cell reactivity in mitogens and alloantigens by Peyer's patches of aged mice. J. Immunok 138 (1987) 26--3 I. H. Kawanishi and J. Kiely, Immunoregulatory defects in murine aged Peyer's patches. Ear. J. Immunol. 17(1987) 1223--1228. H. Kawanishi and J. Kiely, Effect of aging on murine gut-associated lymphoid tissue. Dig. Dis. Sci. 34 (1989) 175--184. H. Kawanishi and J. Kiely, Impaired non-specific suppressor-inducer T cell activity in aged marine Peyer's patches, which can be restored partially by interleukin 2 in vitro. Immunology, 66 (1989) 61 --68. H. Kawanishi, S. Senda and S. Ajitsu, Aging-associated intrinsic defects in IgA production of murine Peyer's patch T cell hyhridoma-derived B cell stimulatory factors (BSF). Mech. Ageing Dev., 49 (1989) 61--78. D.R. Barthod, S. Kysela and A.D. Steinberg, Decline in suppressor T cell function with age in female NZB mice. J. Immunol., 112 (1974) 9--16. S.R.S. Gottesman, R.L. Walford and G.J. Thorbecke, Proliferative and cytotoxic immune functions in aging mice. II. Decreased generation of specific suppressor cells in alloreactive cultures. J. Immunol., 133 (1984) 1782--1790. R.J. Chiodini, H.J. Van Kruiningen, W.R. Thayer, R.S. Markal and J.A. Contu, Possible role of mycobacteria in inflammatory bowel disease. I. An unclassified mycobacterium species isolated from patients with Crohn's disease. Dig. Dis. Sci., 29 (1984) 1073--1079. H. Kawanishi, Immunocompetence of normal human appendiceal lymphoid cells: In vitro studies. Immunology, 60 (1987) 19--28. H. Kawanishi, L.E. Saltzman and W. Strober, Mechanisms regulating IgA class-specific immunoglobulin production in murine gut-associated lymphoid tissues. I. T cells derived from Peyer's patches that switch sIgM B cells to sIgA B cells in vitro. J. Exp. Med., 1570983) 433--450. H. Kawanishi, S. Senda, S. Ajitsu and S. Mirabella, Autoreactive T cell hybridoma-derived B cell stimulatory factor(s) governing IgA isotype immunoglobin production by marine Peyer's patch B cells. Immunol. Invest., 17(1988) 587--613. H. Kawanishi, L.E. Saltzman and W. Strober, Characteristics and regulatory function of marine Con A-induced, cloned T cells obtained from Peyer's patches and spleen: Mechanisms regulating isotype-specific immunoglobulin production by Peyer's patch B cells. J. Immunok, 129 (1982) 475 --483. G.L. Asherson, V. Colizzi and M. Zembola, An overview of T-suppressor cell circuits. Ann. Rev. Immunol., 2 (1986) 127--153. Callard, R.E. Aging of the immune system. In M.M.B. Kay and T. Makinodan (eds.), Handbook oJ Immunology in Aging, CRC Press, Inc., Boca Raton, Florida, 1981, pp. 103--122.

161 26

27

28

29

30 31 32 33

34

R. DeKruyff, Y. Kim, G. Siskind and M. Weksler, Age-related changes in the in vitro immune response. Increased suppressor activity in immune and aged mice. J. Immunol., 125 (1980) 142-147. G. Doria, C. Mancini and L. Adorino, Immunoregulation in senescence: Increased inducibility of antigen-specific suppressor T cells and loss of cell sensitivity to immunosuppression in aging mice. Proc. Natl. Acad. Sci. USA, 79 (1982) 3803--3807. R.M. Gorczynski, M. Kennedy and S. MacRae, Altered lymphoid recognition repertoire during aging. III. Changes in MHC restriction patterns in parental T lymphocytes and diminution in T suppressor function. Immunology, 52 (1984) 611--620. S. Kishimoto, T. Takahana and H. Mizumachi, In vitro immune response to the 2, 4, 6-trinitrophenyl determinant in aged C57BL/6J mice: Changes in the humoral immune response to avidity for the TNP determinant, and responsiveness to LPS effect with aging. J. Immunol., 116 (1976) 294 --300. D.E. Harrison, C.M. Antly and J.W. Doubleday, Stem cell lines from old immunodeficient donors give normal responses in young recipients. J. Immunol., 118 (1987) 1223--1227. D. Zharhary and N.R. Klinman, Antigen responsiveness of the mature and generative B cell populations of aged mice. J. Exp. Med., 157(1983) 1300--1308. D. Zharhary and N.R. Klinman, A selective increase in the generation of phosphorylcholine-specific B cells associated with aging. J. lmmunol., 136 (1986) 366--370. B. Cinader, T. Amagai, T. Matsuzawa and K. Nakano, Genetic programs for the changes in immune responsiveness in adult life. In T. Hraba and M. Hasek (eds.), Cellular and Molecular Mechanisms o f Immunologic Tolerance, Marcel Dekker, Inc., New York, 1980, pp. 17--198. J.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227 (1970) 680--685.