Dysregulation of IL-10 production with aging: Possible linkage to the age-associated decline in DHEA and its sulfated derivative

Experimental Gerontology,Vol. 31, No. 3, pp. 393--408, 1996 Copyright © 1996 Elsevier ScienceInc. Printed in the USA. All rights reserved 0531-5565/96 $15.00 + .00 ELSEVIER

SSDI: 0531-5565(95)02033-0

D Y S R E G U L A T I O N OF IL-10 P R O D U C T I O N W I T H AGING: POSSIBLE L I N K A G E TO THE A G E - A S S O C I A T E D DECLINE IN D H E A AND ITS S U L F A T E D D E R I V A T I V E

NINA F. L. SPENCER,1 STEVEN D. NORTON,2 LISA LYN HARRISON,2 GANG-ZHou LI t and RAYMOND A. DAYNES1'3 1Department of Pathology, University of Utah Medical School, Salt Lake City, Utah 84132 2paradigm Biosciences, Inc., Salt Lake City, Utah 84109 3Geriatric Research, Education and Clinical Center, Veterans Affairs Medical Center, Salt Lake City, Utah 84112

A b s t r a c t - - P e r i p h e r a l lymphoid ceils isolated from the spleens and peritoneal cavities of aged mice were found to constitutively secrete the multifunctional cytokine interleukin (IL)10 when cultured in vitro. B-Lymphocytes were implicated as the cell type responsible. Abnormal expression of this cytokine was also detected in vivo because high levels of mRNA for IL-10 were present in splenocytes freshly isolated from aged animals. In addition to the spontaneous secretion of IL-10, lymphoid cells from aged donors were hyperresponsive to exogenous stimulation with endotoxin, producing exaggerated quantities of both IL-10 and IL-6 in culture. Treatment of aged animals with dehydroepiandrosterone sulfate (DHEAS), a natural steroid, reversed the age-associated alterations in cytokine production, rendering the treated mice quite similar to mature adult controls. DHEAS treatment of aged mice also resulted in a lowering in the number of B 1 cells present in the peritoneal cavity and also reduced the titers of circulating autoantibodies specific for phosphatidylcholine (PtC). Based on its wide range of biologic activities, a dysregulation in the mechanisms that control IL-10 production could be a major contributor to immunosenescence. The ability of DHEAS treatment to restore normal control over the expression of IL-10 may explain how this steroid enhances immunocompetence in aged animals.

Key Words: aging, autoantibodies, B1 cells, cytokines, DHEA(S), immunosenescence, interleukin10, interleukin-6

Abbreviations: DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; ELISA, enzyme-linked immunosorbent assay; F1TC, fluorescein isothiocyanate; GM-CSF, granulocyte macrophage colony stimulating factor; IFN-~, interferon-~/; IL-, interleukin-; LPS, lipopolysaccharide; MHC, major histocompatability complex; NO, nitric oxide; TNF, tumor necrosis factor; PE, phycoerythrin; PtC, phosphatidylcholine; RT-PCR, reverse transcriptasepolymerase chain reaction. Correspondence to: Raymond A. Daynes, Department of Pathology, University of Utah Medical School, 50 N. Medical Drive, Salt Lake City, UT 84132 (Received 22 June 1995; Accepted 5 September 1995) 393

394

N.F. SPENCER et al.

INTRODUCTION THE IMMUNEsystem of mammals undergoes many alterations during the aging process (Miller, 1991b). In most individuals these changes lead to a progressive decline of appropriate immune responsiveness to exogenous antigens and a parallel increase in immune recognition and reactivity to self-constituents (reviewed in Thoman and Weigle, 1989; Miller, 1991b). The observed age-associated changes in immunocompetence could be responsible for the increased incidence of autoimmune diseases found in the elderly as well as their enhanced susceptibility to infectious agents, disease severity, and cancer development (Miller, 1991b). Fidelity of the immune system is known to depend upon the proper functioning of a subtle and well-balanced network of cytokines that control the survival, proliferation, and differentiation of lymphocytes and other lymphoid ceils. Therefore, there is a good possibility that the alterations in immune competence that accompany aging represent a product of the complex remodeling of cytokine production that characterizes the immunosenescent condition. T cells, B cells, and macrophage cell populations, the major cell types of the immune system, undergo well-defined phenotypic and functional changes with aging (reviewed in Thoman and Weigle, 1989; Miller, 1991b). These involve depressions in early signal transduction events, reduced calcium fluxes, and alterations in the inducibility and production of certain cytokine species by activated lymphoid cells (Vie and Miller, 1986; Buckler et al., 1988; Ernst et al., 1989; Thoman and Weigle, 1989; Philosophe and Miller, 1990; Miller, 1991a; Daynes and Araneo, 1992). Age-associated alterations in cytokines, especially the reduced potential to produce interleukin (IL)-2, leads to a diminished capacity of activated T and B cells to proliferate and differentiate into immune effector cells (Weksler and Hutteroth, 1974; Gillis et al., 1981; Nagel et al., 1988). Changes in inducible IL-4, IL-5, IL-6, and interferon gamma (IFNg), as well as macrophage production of tumor necrosis factor and IL-1, have all been reported to associate with advancing age (Heine and Adler, 1977; Inamizu et al., 1985; Ernst et al., 1990; Nagelkerken et aL, 1991; Cillari et al., 1992; Daynes and Araneo, 1992; Zhou et al., 1995). Dysregulations in the mechanisms that control cytokine production can also extend beyond conditions of hypo- or hyperresponsiveness to exogenous stimulation. Some cytokine species, like IL-6 for example, are produced constitutively in aged animals, creating a condition where all cytokine-responsive cell types are relegated to undertaking physiological processes under its continual influence (Daynes et al., 1993). Aberrant outcomes, like an ongoing acute phase response, nonspecific B cell stimulation, or osteoclast hyperactivity would logically result. These conditions do represent changes observed commonly in aged individuals. The numbers of CD5 + B cells increase in the elderly (Brohee et al., 1991; Booker and Haughton, 1992; Gottesman et al., 1993), and the reasons for this phenomenon are not fully understood. This subset of lymphocytes may represent a second B cell lineage that arises early in embryonic development from committed stem cell precursors. B cells differentiating down this developmental pathway have been designated B 1 cells and have been further characterized into one of two subpopulations (Hayakawa et al., 1985; Brohee et aL, 1991). Bla cells are characterized by the expression of CD5, Mac- l, and the B cell surface molecules IgM and B220, while the "sister" B lb cell population does not express the CD5 molecule (Hayakawa et al., 1985; Brohee et al., 1991). No functional distinction between Bla and B l b cells has yet been identified, and both populations of lymphocytes have been implicated in the production of autoreactive or self-reactive antibodies (Stall et al., 1992). Additionally, activated B 1 cells have been reported to be major producers of IL-10, a multifunctional cytokine with strong immunomodulatory properties (O'Garra et al., 1992). IL-10 is capable of being produced by activated

STEROID REGULATION OF 1L-10 PRODUCTION

395

T cells, B cells and macrophages and possesses the capacity to affect physiological functions of many different cell types (reviewed in Moore et al., 1993). Increases in B1 cell numbers and activity have been observed to occur in several clinical conditions such as rheumatoid arthritis (Plater-Zyberk et al., 1992; Xu et al., 1992), systemic lupus erythematosus (SLE) (Reap et al., 1992), cancer (Stein et al., 1991), and B cell lymphocytic leukemia (Dighiero et al., 1991). Increased B 1 cell numbers and activity also closely correlate with increased levels of autoreactive antibodies (Dighiero et al., 1991; Stein et al., 1991; Reap et al., 1992; Xu et al., 1992). Species of antibodies found in the elderly include antithyroglobulin, antidouble-stranded DNA, and antibodies specific for a number of cell membrane components (Lyngbye and Kroll, 1971; Goidl et al., 1988; Kato and Hirokawa, 1993). Augmented activities by the autoreactive B1 cells, present in increased numbers with autoimmunity and aging, could represent a major contributing factor to these clinical conditions. There is a progressive reduction in the endogenous production of the adrenal steroid hormone dehydroepiandrosterone (DHEA) and its sulfated derivative, DHEAS (Yamaji and Ibayashi, 1969; Orentreich et al., 1984) with advancing age in humans. We have previously reported that this steroid can alter the behavior of activated murine T lymphocytes, both in vitro and in vivo (Araneo et al., 1991), and have demonstrated that lymphocytes from aged mice treated with DHEAS produced normal levels of IL-2, IL-3, IL-4, IL-5, IFN-~, and GM-CSF following activation (Daynes and Araneo, 1992). DHEAS treatment of aged animals also corrected the deficiencies in antibody responses following immunization and was able to eliminate the abnormal production of IL-6 found in aged animals (Daynes et al., 1993). In this report, we present evidence of a loss in normal control over IL-10 gene expression in old animals. Spontaneous production of this cytokine was observed in aged animals, as was an increase in the inducible production of IL-10 following endotoxin activation. Furthermore, it was determined that B cells from aged animals represent be the primary source of spontaneously produced IL-10. DHEAS treatment of aged animals was found to significantly correct the age-associated abnormalities in IL-10 production and eliminate the observed cellular hyperresponsiveness to activation-induced cytokine production. The DHEAS-mediated correction of control over IL-10 production, along with the previously described regulation of IL-6 expression, correlated with a decrease in the autoantibody titers observed in aged animals. MATERIALS AND METHODS Mice

Aged female CBA/CAHNNIA strain mice and mature adult female CBA/JCR strain mice were purchased from the National Institute on Aging and the National Cancer Institute, respectively. All animals were housed in the University of Utah Animal Resource Center. Animals used in these studies were 1.5-3 months (mature adult) or 18-24 months old (aged) at the onset of the experiments described. The Animal Resource Center at the University of Utah routinely monitors for the most prevalent murine pathogens and employs sentinel animals as a means for early detection of murine hepatitis virus. These monitoring procedures were negative throughout the study. All animals used in the present study exhibited no signs of illness or infection. Any mice with evidence of gross internal or external pathology were excluded from the study. In addition, any mice with apparent B cell lymphomas, as determined by flow cytometry, were also excluded from the study. The Internal Animal Care And Use Committee and the Animal Resource Center at the University of Utah guarantee strict compliance with regulations established by the Animal Welfare Act.

396

N.F. SPENCER et al.

DHEAS supplemental therapy DHEAS was purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. For in vivo administration, DHEAS was dissolved directly in the drinking water at a concentration of 100 txg/mL. Steroid supplemented drinking water was prepared fresh on a weekly basis.

Culture of murine peritoneal exudate cells and splenocvtes Cells obtained from peritoneal cavities and spleens of mature adult and aged animals were carefully cultured in the absence of any exogenous stimulants to determine spontaneous cytokine secretion. Additionally, experiments were carried out in which cells were cultured in the presence of varying concentrations of endotoxin (E. coli lipopolysaccharide serotype 0111 :B4, Sigma Chemical Co.) to evaluate cytokine production in response to an inductive stimulus. Briefly, mice were anesthetized with metofane and sacrificed by cervical dislocation. Peritoneal exudate cells were obtained by injecting 8 mL of Hanks/Heparin solution ( l x Hank's BSS with 10 mM HEPES, 2% FCS (v/v) and 20 U/mL Heparin) into the peritoneal cavity and withdrawing the fluid (usually resulting in a 6-7 mL return). Single cell suspensions were also prepared from the spleens of these animals. The collected peritoneal cells and splenocytes were washed three times in Dulbecco's phosphate-buffered saline and cultured at 4 x l06 cells/mL in freshly prepared serum-free medium consisting of RPMI 1640, lx Nutridoma-SR (BoehringerMannheim, Indianapolis, IN), 200 mM L-glutamine, antibiotics, and 5 x 10 -5 M 2-mercaptoethanol. In experiments where endotoxin was used for cell activation, unstimulated and stimulated cell cultures were incubated for 24 h at 37°C in an atmosphere of 5% CO 2 in air. Cell culture supernatants were then collected for quantitative evaluation of immunoactive IL-6 and IL-10 by ELISA.

Capture ELISA for quantitation of lL-lO and IL-6 The protocol used to quantify immunoreactive murine IL-10 and IL-6 represented a slight modification of the protocol reported by Schumacher et al. (1988) and has been reported in detail previously (Daynes et al., 199l). Monoclonal rat antimurine cytokine antibodies and murine recombinant IL-10 and IL-6 cytokine standards were purchased from PharMingen (San Diego, CA). Detection limits for IL-10 and IL-6 ELISAs are approximately 25 pg/mL and 10 pg/mL, respectively.

Reverse transcriptase (RT)-PCR for the identification of lL-lO mRNA RNA was prepared by the CsC1/guanidine method as previously described (Chirgwin et al., 1979). Reverse transcription was performed using 5 txg/mL total RNA in a 50 IxL reaction mixture containing 10 mM DTT (Gibco-BRL), I x RT buffer (Gibco-BRL, Gaithersburg, MD), 0.125 mM each of the four deoxyribonucleotide triphosphates, 0.5 Ixg of random primers (New England Biolabs, Beverly, MA), and 400 U M-MLV Reverse Transcriptase (Gibco-BRL). After a 60-rain incubation at 37°C, 2 p~g of DNase-free RNase was added and incubated for five minutes at 37°C. Water and NaC1 were then added to the reaction mixture to bring the total volume to 270 ~L and 0.4 M NaC1. A phenol/chloroform, chloroform extraction, and overnight precipitation with ethanol at -20°C followed. PCR was carried out with adaptations for rapid cycling with the 1605 air thermocycler (Idaho Technology, Idaho Falls, ID). Each 10 ~zL reaction contained 200 ng cDNA, 70 pmol of each

STEROIDREGULATIONOFIL-IOPRODUCTION

397

primer (primers were 21 nucleotides), 0.8 mM dNTP, 2.5 ~Ci [32p]dCTP (25 Ci/mmol; ICN), l x reaction buffer (10x stock consisted of 500 mM Tris pH 8.3, 30 mM MgCl 2, 200 mM KC1, and 5 mg/mL BSA), and 0.72 U Taq DNA Polymerase (5 U/I~L, Gibco-BRL diluted 1:7). For consistency, master mixes of primer, 1× reaction buffer, dNTP, [32p]dCTP, and Taq DNA polymerase were prepared and aliquoted to the cDNA samples. Each 10 txL reaction was added to glass microcapillary tubes (#1705, Idaho Technology), the ends sealed, and the tubes inserted into the air thermocycler. PCR conditions were: denaturation, 94°C for one second, annealing, 59°C for one second; and elongation, 72°C for eight seconds. Sixteen cycles and 28 cycles were performed for [3-actin and IL-10, respectively. Following PCR, the ends of the microcapillary tubes were scored and samples were removed with a Captrol microaspirator. The entire 10 ~L sample was added to an equal volume of stop solution, heated to 95°C for five minutes, and electrophoresed in a 6% acrylamide gel. Bands were detected by autoradiographic exposure for 15 h at -70°C. Sizes of bands were estimated by the migration of 32p-end-labeled Msp 1 digest of pBR322. Gene-specific sequences were derived from GenBank submissions. Oligonucleotides used for these analyses are as follows:~ -actin: 5'GGG TCA GAA GGA CTC CTA TG3' and 5'GTA ACA ATG CCA TGT TCA AT3'. IL-10: 5'CTG GCA TGA GGA TCA GCA GG3' and 5'CAC CTG CTC CAC TGC CTT GC3' Dynabead cell enrichments T Cell Enrichment Freshly isolated splenocytes (1 × 106 cells/50 ~L 5% FCS-DPBS) were incubated at a 1:1 bead/cell ratio two times for 20 rain each with agitation at 4°C with sheep antirat IgG Dynabeads M-450 (Dynal, New York, NY). Following incubation, the beads were removed using a Dynal MPC (Magnetic Particle Concentrator). Following depletion, the cells were separated for use in culture, mRNA analysis, and FACS analysis. B Cell Enrichment Freshly isolated splenocytes (1 x 106 cells/50 p,L 5% FCS-DPBS) were incubated with biotinylated ct-CD3, ot-CD4, and o~-CD8 antibodies (PharMingen, San Diego, CA) at 0.1 ~g Ab/50 IxL 5% FCS-DPBS for 20 min on ice. Cells were then subjected to the bead depletion with streptavidin Dynabeads M-280 (Dynal) and analysis as described for the T cell enrichment. Phosphatidylcholine (PtC) autoantibody ELISA assay ELISA plates were coated with 45 ~g/mL PtC (L-a-phosphatidylcholine, distearoyl [c18:0], Sigma Chemical) diluted in ethanol from a 2.6 mg/mL stock (dissolved in ethanol). The plates were incubated overnight in the dark at room temperature allowing the ethanol to evaporate. Plates were blocked with 20% fetal calf serum in PBS for two hours in the dark and then washed with 1× PBS. Standard and samples, diluted in blocking buffer (10% fetal calf serum in PBS), were incubated on the blocked plates for one hour in the dark. Plates were washed in 1× PBS, coated with anti-IgM-HRP (Southern Biotechnology Associates, Birmingham, AL) at a l:1000 dilution and incubated for one hour in the dark. Plates were then washed in 1× PBS prior to adding chromagen/substrate. Supernatant from a CD5 + B cell lymphoma (CH12.LX), which spontaneously produces anti-PtC antibody of the IgM isotype (Bishop, 1992), was used as the standard.

398

N.F. SPENCER et al.

Flow cytometry

Splenocytes and peritoneal exudate cells (3 × 105-106) were preincubated on ice for five minutes in 50 IxL of staining buffer (1× PBS supplemented with 2% FCS and 0.2% sodium azide) with 0.5-1.0 Ixg Fc-block (PharMingen) and, in the case of peritoneal cells, 100 txg of rat gamma globulin (Jackson ImmunoResearch, West Grove, PA) to reduce background staining. Cells were then stained on ice for 20 min with antibodies, 1.0 txg, directly labeled with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) (PharMingen). Directly labeled isotype-matched control antibodies were used as negative controls. Cells were washed three times in cold staining buffer and resuspended in 0.5 mL of staining buffer containing 1.0 Ixg/mL propidium iodide (Sigma Chemical Co.). For each sample, 5 × 105 cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Dead cells were excluded from analysis by propidium iodide staining. Background staining with control antibodies was usually < 1%. Statistical analysis

Data are represented as the mean of all the experiments performed (the number of experiments is indicated in figure legends) _++ SEM. Analysis of variance was performed, followed by appropriate post hoc tests with the statistical analysis program SPSS. The data from aged and aged + DHEAS groups are compared to the data from the mature adult groups. RESULTS Aging appears to result in a dysregulation in the mechanisms that control normal production

of lL-lO Splenocytes, peripheral lymph node cells (inguinal, brachial, and axillary), mesenteric lymph node cells, and peritoneal exudate cells isolated from mature adult (three months) and aged (24 months) CBA strain mice were cultured overnight in serum-free medium without additional stimulation. Supernatants were collected after 24 h and quantitatively analyzed for the presence of IL-10 by capture ELISA. The data demonstrates that splenocytes and peritoneal exudate cells from aged animals constitutively produce IL-10 at levels far exceeding that found from similar cell populations isolated from younger syngeneic animals (Fig. 1A). Lymphoid cells isolated from peripheral lymph nodes, or the mesenteric lymph nodes of aged animals, did not spontaneously produce sufficient quantities of IL-10 for detection (data not shown). All cell supernatants tested were devoid of IL-2 and IL-4, reducing the likelihood that the observed presence of IL-10 was the result of artifactual lymphocyte stimulation due to the culture conditions employed (data not shown). Therefore, in addition to an abnormality in the control of IL-6 production in aged animals (Daynes et al., 1993), aging also results in a dysregulation in control over production of the pleiotropic cytokine IL-10 by cells residing in some, but not all, secondary lymphoid organs. Unlike the age-associated dysregulation in IL-6 production, where the serum from aged animals possesses detectable IL-6, no IL-10 could be detected in the serum of aged animals (data not shown). To provide additional support that IL-10 is, indeed, being actively produced in vivo, experiments were conducted to establish whether the murine IL-10 gene was being transcribed by freshly isolated splenocytes from aged donors. Spleens were obtained from young and aged

399

STEROIDREGULATIONOFIL-10PRODUCTION [] mature adult

A. 125010O0. IL-10

pg/rra

B. mature adult

[] aged

T

q¢

750-

T

500. ~

aged

IL-10

~-actin

250IL-10/[~-actin

0

0.006 0.001

1.8

2.2

i

spleen

peritonealexudate cells

FIG, 1. The mechanisms that control interleukin-10 production are dysregulated as a consequence of aging. Splenocytes and peritoneal exudate cells (A) were collected from mature adult (three months old) and aged (24 months old) CBA mice and cultured overnight in serum-free medium. The 24-h culture supernatants were quantitatively analyzed for IL-10 by capture ELISA. Data is represented as the mean of ten experimental points ~-+_SEM. *p < 0.05 as compared to mature adult. Spleens were also removed from mature adult (three months old) and aged (24 months old) CBA mice and immediately frozen in liquid nitrogen. RT-PCR (B) was performed on mRNA isolated from homogenized spleens. PCR data is representative of four experiments, Ratios of IL-10/[3-actindensitometric values are included below the gel picture.

animals, immediately frozen in liquid nitrogen, and m R N A was prepared as described in the Materials and Methods. R T - P C R was performed on the c D N A preparations from both sources. As shown in Fig. 1B, minimal IL-10 m R N A was detectable in splenocytes isolated directly from mature adult mice. In contrast, a large amount of IL-10 m R N A was found in the spleen cells obtained from the aged animals. The finding of small amounts of immunoreactive IL-10 after the 24-h culture of normal adult splenocytes and the absence of detectable IL-10 m R N A from freshly collected spleen tissue samples implies that the manipulations e m p l o y e d to prepare cells for tissue culture may be providing a stimulus that causes some cells to produce very low levels o f IL-10. The data is supportive, however, o f the argument that IL-10 is being actively transcribed and produced in vivo in selected lymphoid compartments from aged animals.

B cells appear to be the major contributor to the abnormality in IL-IO expression seen in aged animals Spleens were removed from mature adult (three months) and aged (24 months) C B A strain mice. The cells obtained from animals of both age groups were divided equally into three aliquots followed by preparation of T cell-enriched and B cell-enriched fractions using Dynabead depletion techniques as described in the Materials and Methods. The level o f B and T cell purity between experiments varied between 82-92%, as determined by flow cytometric analysis. The enriched cell populations were then cultured overnight under serum-free conditions. Figure 2A demonstrates that the spontaneous IL-10 produced by splenocytes from aged animals is primarily a product of the B cells since IL-10 product was detected in the B cell-enriched cultures but not in the T cell-enriched cultures, Interestingly, IL-10 m R N A (Fig. 2B) could be detected by R T - P C R in both the T and B-enriched cell populations, although greater amounts were present in extracts from the B cell-enriched fractions.

400

N.F. SPENCER et al.

IL-10 pg/ml

A.

[]

Pre-Bead

275 250225 2001751501257 10075-~ 5025 -~ 0

[]

T cell enriched

[]

B cell el

B. Total

T cell enriched

B cell enriched

1L-10

~-actin

i

mature adult

aged

IL- I0/~-actin

0.01

0.08

0.05

0.5

0.01

1.9

FIG. 2. B cells from aged animals appear to be the major contributors to dysregulated IL-10 production. Splenocyte suspensions from mature adult (three months old) and aged (24 months old) were enriched for T cells or B cells as described in the Materials and Methods. Twenty-four-hour culture supernatants were quantitatively analyzed for IL-10 by ELISA (A). Data is represented as the mean of three experiments ++ SEM. RT-PCR (B) was performed on mRNA isolated from the cell subsets immediately after enrichment. PCR data is representative of three experiments, Ratios of IL-10/[3actin densitometric values are included below the gel picture.

Treatment of aged animals with DHEAS corrects the IL-IO dysregulation It has been previously reported that treating aged animals with low doses o f D H E A S causes a reversal in many o f the immune cell functions used to define immunosenescence (Daynes and Araneo, 1992). Because a dysregulation in the mechanisms that control IL-10 production might represent a contributing factor in immunosenescence, aged animals were treated with D H E A S to establish its influence in the regulation o f IL-10 expression. Aged C B A strain mice (25 months) were treated with D H E A S in their drinking water (100 txg/mL) for three weeks prior to analysis. Splenocytes and peritoneal exudate cells were then obtained from groups o f mature adult, aged, and the DHEAS-treated group of aged animals. The isolated cell populations were then evaluated for constitutive production o f IL-10 by culturing them overnight in serum-free medium. As shown in Fig. 3A, minimal immunoreactive IL-10 was present in the supernatants of splenocytes and peritoneal exudate cells obtained from aged animals provided with DHEAS. Though the DHEAS-treated group was still significantly different from the young animals, the D H E A S group was also significantly different from the untreated aged group, indicating that D H E A S treatment does alter IL-10 levels. Similar results have been obtained with cell populations isolated from aged animals maintained on D H E A S treatment for as long as 20 weeks prior to analysis (data not shown). A n RT-PCR evaluation of m R N A prepared from freshly isolated splenocytes from mature adult, aged, and DHEAS-treated aged animals determined that IL-10 m R N A was still present following DHEAS-treatment o f aged animals (Fig. 3B). A method that provides a quantitative analysis o f m R N A is necessary, however, to establish whether, or how, D H E A S is influencing IL-10 gene expression. When splenocyte preparations from aged animals were stimulated with endotoxin in vitro, a significant amount o f IL-10 and IL-6 were produced over the subsequent 24-h period (Fig. 4A and B). Using endotoxin doses between 0.01 and 10 tzg/mL, activated splenocytes from aged animals consistently produced four to five times the quantity of these two cytokines when

401

STEROIDREGULATIONOF IL-10PRODUCTION

B.

[] mature adult

mature adult

[] aged A. 800-

[] aged + DHEAS 9¢ 9¢

T

70o-

aged

aged + DHEAS

IL-IO

6oo500-

IL-10 pg/ml

~-actin

400300-

~

IL-10/13-actin

0.02

0.005

2.1

2.8

1,3

1,5

2001000

i

spleen

peritoneal exudate cells

FIG. 3. Supplemental DHEAS treatment of aged mice controls unregulated production of IL-10. Splenocytes and peritoneal exudate ceils (A) were collected from CBA mature adult (three months old), aged (25 months old), and aged (25 months old) animals provided chronic DHEAS treatment (> three weeks). The 24-h culture supernatants were quantitatively analyzed for IL-10 by capture ELISA. Data is represented as the mean of eight experimental points ±+ SEM. *p < 0.05 as compared to mature adult. Spleens were removed and fragments were immediately frozen in liquid nitrogen from CBA mature adult (three months old), aged (25 months old), and aged (25 months old) animals provided chronic DHEAS treatment (> three weeks). RT-PCR (B) was performed on mRNA isolated from spleens from mature adult, aged, and aged animals treated with DHEAS. PCR data is representative of four experiments. Ratios of IL-10/13-actin densitometric values are included below the gel picture.

compared to similar splenocyte preparations from mature adult donors. Splenocytes from aged animals on DHEAS treatment for three weeks produced near mature-adult quantities of IL-10 and IL-6 in response to endotoxin exposure, indicating that the effects of this steroid extend beyond a correction of the processes that result in spontaneous cytokine production. mature adult

A.

2400 2200200018001600IL-10 1400pg/ml 12001000800600400-

B.

-'--
. *

280026002400220020001800IL-6 1600pg/ml 14oo12001000800600400-

TT

aged + DHEAS

---{3--- mature adult

*~ T

-----O-- aged

I ~

aged + DHEAS ~ . L 1 3 r

~t

2oo1

o

o;1

o11

1'o

[LPS], p,g/ml

1'o

i

100

o

0

o/ol

o'1

t'o

1'o

[LPS], I,tg/ml

FIG. 4. DHEAS supplementation reduces the exaggerated response of aged lymphocytes to stimulation. Spleens were collected from CBA mature adult (three months old), aged (24 months old), and aged (24 months old) animals provided chronic DHEAS treatment (three weeks). Splenocytes were cultured in the absence and presence of varying concentrations of LPS (A and B). The 24-h culture supernatants were quantitatively analyzed for IL-10 (A) and IL-6 (B) by capture ELISA. Data is represented as the mean of two experiments. *p < 0.05 as compared to mature adult.

i

100

402

N.F. SPENCER et al.

D H E A S Treatment o f Aged Mice Reduces the Numbers o f B1 Cells and T Cells in Their Peritoneal Cavities and Simultaneously Lowers Circulating Titers o f Autoantibodies As previously reported (Hayakawa et al., 1985; Brohee et al., 1991; Gottesman et al., 1993), and as shown here (Table 1), the number of CD5 + ( Bl a) B cells is increased in mice as a consequence of aging. While total numbers o f splenocytes in aged animals were not significantly elevated compared to that observed in mature adults, the absolute numbers of CD5 + B ceils were increased in the spleens of aged animals. Total numbers o f cells in peritoneal cavities o f aged animals were also significantly elevated compared to mature adult numbers. This increase in total cell number was due to elevated numbers of B l a and B l b as well as T cells present in the peritoneal cavities of the aged mice. The elevation in total numbers of lymphocytes found in the peritoneal cavities of aged animals were not seen following a treatment with D H E A S for a three-week period (Table 1). D H E A S treatment of aged animals depressed both the levels of B 1 and T cells present in the peritoneal cavities o f aged mice but did not have an observed influence on splenic B 1 cell numbers or on the total numbers of splenocytes in aged animals. Thus, under the experimental conditions employed, any corrective effect of D H E A S treatment on lymphoid cell types and numbers appears to be restricted to the peritoneal cavity of aged animals. Another consequence of old age is characterized in both rodents and humans by increases in numerous species o f autoreactive IgM and lgG antibodies. B 1 cells are strongly implicated in the production of autoreactive antibodies and are the major B cell type capable of IL-10 production (O'Garra et al., 1992). Through autocrine pathways, therefore, B 1 cell I L - I 0 might promote nonspecific B 1 cell activation, causing enhanced levels of autoreactive antibodies to be produced. Experiments were conducted to question whether differences existed in the levels of serum anti-PtC antibodies between mature adult and old C B A female mice. As seen in Fig. 5,

T A B L E I.

DHEAS

TREATMENT IN AGED ANIMALS RESTORES PERITONEAL B

1

CELL NUMBERS T O NORMAL MATURE

ADULT LEVELS a

Group Mature adult Aged Aged + DHEAS

total splenocvte

total peritoneal peritoneal T

83_+8 (100%)b 108+13 (130%)b.c 121_+14 (146%)b,c

5.4_+0.4 (100%)b 13_+1.5 (241%)b,~ 6.5_+1.2 (120%)b

peritoneal Bla

0.3_+0.05

1.3±0.4

( 1 0 0 % ) t'

(100%) h

2.9_+0.3 (967%)b,'~ 2.2_+0.9 (733%)b,"

3.5_+0.3 (269%)bx ~.0_+ 0.7 (77%)b

peritoneal Blb 1.0_+0.2 (100%)8 2.8_+0.8 (280%)b," 1.9_+ 1.2 (190%)b

splenicBla 4.3_+1.l (100%)b 13.9-+3.1 (323%)b,c 17.2+2.3 (400%)b,c

~Splenocytes and peritoneal cells were collected from mature adult (6-12 weeks), aged (76-96 weeks), and aged (76-96) CBA female mice provided DHEAS treatment (three weeks). Using flow cytometry, the absolute numbers of various cell populations were calculated based on the percentage of a given cell type present. Peritoneal T cells were determined by positive staining with anti-CD5 PE and negative staining for anti-IgM FITC. Splenic and peritoneal B la cells were determined by positive staining with both anti-IgM FITC and anti-CD5 PE. Peritoneal Blb cells were determined by subtracting the percentage of Bla cells from cells staining positive with anti-IgM FITC and anti-Mac-1 PE. Data are presented as the mean -+_+S.E.M. (xxl0-% bpercent increase in cell numbers as compared to mature adult. Cp < 0.05, as compared to mature adult.

403

STEROID REGULATION OF IL-10 PRODUCTION q¢

11000-

11000-

100009000 -

T ~:.:.:.:.:.:.:.:.~:.:.:.:.:+:.:.:

8000-

i~i~i'i~ii~ii~i~i~i~

7000-

::::::::::::::::::::::::::::::::::::::::::::::

6000-

.................

-~

10000. 90O0 -

=

8000.

"k

.~

7000-

]-

.~

6000.

~

4000. 3000-

I

(p
IT1

(p
5000-

w

4000-

-==

3000-

:.:.:.:,:.:.:.:.:.:,:.:.:.:.:.:.:.:. T

!i!~!~!!!~i~i~!~!~!~!~!!i!~i~i~

:.:.:.:.:.:.:.:.:,:.:.:.:.:.:.:.:.i T

2000 -

10000

young

aged

aged + D H E A S

1 young

•...................,...........: .................. . ..,.................: +:c.:+>>>:+>>:+:+:t :-:-:.:.:.:.:.:.:,:-:-:-:.:.:.:,:,i ::::::::::::::::::::::::::::::::::: i aged

i aged + D H E A S

FIG. 5. DHEAS therapy reduces the serum titers of anti-PtC antibodies to near normal mature adult levels in old mice. Blood was collected from mature adult (three months old), aged (24 months old), and aged (24 months old) CBA mice provided chronic DHEAS treatment. The DHEAS-treated animals were provided with steroid for times between 3-20 weeks. Equivalently reduced serum titers of anti-PtC antibodies were found in all groups treated with steroid; therefore, the data was pooled from 28 mature adult, 23 aged, and 23 aged ++ DHEAS animals and presented ++ SEM. Sera was analyzed for anti-PtC antibodies by ELISA as described in Materials and Methods. anti-PtC antibody titers are elevated in aged animals as compared to serum titers in mature adult controls. This increase in titer correlates with the observed elevations in the anti-PtC-producing B 1 cell numbers (Allison and Nawata, 1992). DHEAS treatment was determined to affect the production of autoreactive antibodies known to be produced by B 1 cells. As presented in Fig. 5, the elevated levels of anti-PtC antibodies normally found in aged animals are reduced towards normal mature adult levels in aged animals following a short duration D H E A S treatment. Again, the antibody titers from DHEAS-treated group are significantly lower than those of the untreated aged group. It appears, therefore, that D H E A S treatment is s o m e h o w able to influence the state of activation of B 1 cells involved in autoantibody production.

DISCUSSION The immune system undergoes numerous cellular and molecular changes as a consequence of aging. S o m e of these alterations can directly translate into depressions in protective immune function, while others appear to associate with pathophysiological end points including autoimmunity (Thoman and Weigle, 1989; Miller, 1991b). The research reported herein has determined that a dysregulation occurs in the mechanisms that control gene expression of IL-10 in aged animals. Splenocytes and peritoneal exudate cells from aged mice were found to spontaneously secrete IL-10 in culture, and m R N A for IL-10 was confirmed to exist in vivo by RT-PCR analysis. B lymphocytes were established to be the major source of this abnormal cytokine secretion, even though m R N A for IL-10 could be detected in freshly isolated cell populations derived from aged mice enriched for either T cells or B cells. It has been previously reported that purified CD4 + T cells from aged murine donors produce significantly more IL-10 in response to anti-CD3 stimulation than CD4 + T cells from mature adult animals (Hobbs et al., 1994). No detectable nonspecific production of IL-10 was observed in this report, a finding consistent to our present results. From these new studies, it now appears that the dysregulation

404

N.F. SPENCER et al.

in IL-10 expression associated with old age extends beyond an activation-induced hyperactivity of T cells, and must include other cellular sources of IL-10, such as hyperactive B cells. Some of the age-associated changes in T cell, macrophage, and B cell functions that define immunosenescence (Thoman and Weigle, 1989; Miller, 1991b) may be linked to the present observation of dysregulated control over IL-10 production. For example, IL-10 has been reported to directly inhibit IL-2 synthesis by activated T cells (Fiorentino et al., 1989; Taga and Tasoto, 1992), reduce the expression of class II MHC molecules on monocytes/macrophages (de Waal Malefyt et al., 1991), and depresses B7 costimulatory molecule expression on activated macrophages (Ding et al., 1993). Previous studies have also demonstrated that IL-10 can reduce stimulated macrophage production of numerous cytokines (Fiorentino et al., 1991), as well as inhibit antibody production by B cells in response to either T-independent or T-dependent antigens (Pecanha et al., 1992). Many of these conditions are similar to those reported to occur as a consequence of aging. We have recently established that treatment of old animals with modest amounts DHEAS, a natural steroid whose endogenous production in humans declines with advancing age (Yamaji and Ibayashi, 1969; Orentreich et al., 1984), restores certain components of the senescent routine immune system (Daynes and Araneo, 1992). Our present study demonstrates that the age-associated dysregulated production of the pleiotropic cytokine IL-10 can be effectively corrected by treating aged animals with DHEAS. These findings add to those previously reported with IL-6, where the age-associated dysregulation in production of this cytokine was effectively corrected with steroid treatment (Daynes et al., 1993). DHEAS treatment, however, was unable to correct the abnormal transcription of the IL-10 gene, because we were still able to find IL-10 mRNA in the spleens of DHEAS-treated aged animals. The abnormal expression of IL-10 could be attributed to a number of factors including changes in the stability, translation, and DNA binding characteristics of mRNA. We are currently investigating the mechanism(s) responsible for the influence of DHEAS on the production of this cytokine by aged animals. DHEAS treatment of aged mice was also capable of reducing the hypersensitivity of their splenocytes to the stimulatory effects of endotoxin. The reestablishment of a normal control over inducible IL-10 production and the elimination of constitutive expression of this cytokine in vivo could be a major contributing factor to the normalization of both activated T-cell function and antibody production previously observed to occur in aged animals treated with DHEAS (Daynes and Araneo, 1992). We have previously reported that increases in IgM and IgG tissue-reactive autoantibodies associated with old age could be reduced to near mature adult levels following DHEAS treatment (Daynes et al., 1993). The age-associated increases in autoreactive antibody titers were originally ascribed to the dysregulation of IL-6 production observed to occur with aging (Daynes et al., 1993). Based on findings from the present study it is likely that IL-10 is also contributing to this potentially pathologic phenotype. The presence of autoantibodies in the serum of elderly individuals and aged animals is commonly observed (Mariotti et al., 1992; Franceschi et al., 1995). In healthy elderly centenarians, however, there is a unique absence of organ specific autoantibodies (Mariotti et al., 1992; Franceschi et al., 1995). This finding suggests that the age-associated changes that allow autoantibodies to be produced may be contributing to the pathophysiology of aging. Recently, Llorente et al. established that the production of autoantibodies by B lymphocytes from patients with the autoimmune condition SLE is largely dependent on the endogenous abnormal production of IL- 10 (Llorente et al., 1994). Because SLE and aging appear to both involve hyperactive B cells, it is possible that B-cell IL-10 is contributing

STEROID REGULATION OF IL-10 PRODUCTION

405

to the conditions that allow autoantibody production to occur in old age and could, therefore, be a crucial factor involved in the aging process. The underlying mechanism(s) responsible for the increased numbers of B 1 cells in old age and certain autoimmune disease states is presently unknown. One mechanism by which the absolute numbers of a particular lymphoid cell type can be controlled in vivo is related to their susceptibility or resistance to apoptosis. It is possible that B 1 cells in old animals are somehow rendered less sensitive to the apoptotic pathway. Indeed, it has been suggested from work in humans that the age-associated increase in the level of autoantibodies and B 1 cells might be due to an age-related depression in B-cell susceptibility to apoptosis (Franceschi et al., 1995). IL-10 has been reported to prevent spontaneous death of germinal center B cells by its capacity to induce expression of the bcl-2 protein (Levy and Brouet, 1994). It is, therefore, possible that the abnormal production of IL-10 in old age is a contributing factor to both the elevated B 1 cells and their subsequent hyperactivity through a mechanism involving bcl-2. Dysregulated production of IL-10 and other cytokines has been observed to occur in a number of autoimmune, neoplastic, and infections disease states. These include SLE (Llorente et al., 1994), HIV infection (Benjamin et al., 1992; Clerici et al., 1994), tuberculosis (Barnes et al., 1993), melanoma (Chen et al., 1994), and certain types of lymphomas (Finke et al., 1993). Furthermore, the constitutive expression of IL-10 has sometimes been directly implicated as a causative factor in the pathophysiology of these conditions (Benjamin et al., 1992; Barnes et al., 1993; Finke et al., 1993; Chen et al., 1994; Clerici et al., 1994; Levy and Brouet, 1994; Llorente et al., 1994). The results of the present study suggest that the "natural" process of aging represents yet another complex biological condition where the constitutive presence of IL-10 and the responses it mediates, may be providing some significant and consistent contributions to the undesirable consequences associated with aging. Once a more complete determination of the role(s) played by IL-10 in the immunosenescent process is established, the knowledge gained could lead to an increased understanding of the effects IL-10 may have in the many clinical conditions that are known to associate with a dysregulated expression of this cytokine. In clinical conditions where IL-10 is contributing to the pathophysiology of the disease state, therapeutic interventions with agents capable of reducing its production could prove to be clinically beneficial. Aging may represent a prime candidate for such intervention strategies. Acknowledgments--This work was funded by National Institutes of Health Grants CA25917, AG11475, Center of

Excellence in Molecular Hematology grant P50 DK49219, and by DVA Medical Research Funds. REFERENCES ALLISON,A.C. and NAWATA, Y. Cytokines mediating the proliferation and differentiation of B-1 lymphocytesand their role in ontogeny and phylogeny. Ann. NY Acad. Sci. 651, 200-219, 1992. ARANEO, B.A., DOWELL,T., TERUI, T., DEIGEL, M., and DAYNES, R.A. Dihydrotestosteroneexerts a depressive influence on the production of interleukin-4 (IL-4), IL-5, and gamma-interferon,but not IL-2 by activated murine T cells. Blood 78(3), 688-699, 1991. BARNES, P.F., LU, S, ABRAMS, J.S., WANG, E., YAMAMURA,M., and MODLIN, R.L. Cytokine production at the site of disease in human tuberculosis. Infect. Immunol. 61(8), 3482-3489, 1993. BENJAMIN, D., KNOBLOCH, T., and DAYTON, M.A. Human B-cell interleukin-10: B-Cell lines derived from patients with acquired immunodeficiencysyndromeand Burkitt's lymphomaconstitutively secrete large quantities of interleukin-10. Blood 80(5), 1289-1298, 1992.

406

N.F. SPENCERet al.

BISHOP, G. Signalling to a CD5 + B-cell clone through surface Ig and MHC class II molecules. Ann. NYAcad. Sci. 651, 228-240, 1992. BOOKER, J.K. and HAUGHTON, G. Characterization of expanded populations of peritoneal CD5 B cells specific for pbosphatidyl choline in old B10.H-2"H-4bp/Wts Mice. Ann. N Y Acad. Sci. 651, 494-497, 1992. BROHEE, D., VAN HAEUERBEEK, M., KENNES, B., and NEVE, P. Changing patterns of CD5 + CD20 + double positive lymphocytes with ageing and cytotoxic chemotherapy. Mech. Ageing Dev. 58(2-3), 127-138, 1991. BUCKLER, A.J., VIE, H., SONENSHEIN, G.E., and MILLER, R.A. Defective T lymphocytes in old mice. Diminished production of mature c-myc RNA after mitogen exposure not attributable to alterations in transcription or RNA stability. J. lmmunol. 140(7), 2442-2446, 1988. CHEN, Q., DANIEL, V., MAHER, D.W., and HERSEY, P, Production of lL-10 by melanoma cells: Examination of its role in immunosuppression mediated by melanoma, h~t. J. Cancer 56, 755-760, 1994. CHIRGWIN, J.M., PRYZBYLA, G., MCDONALD, P.J., and RUTTER, W.J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemisto, 18(24), 5294-5299, 1979. C1LLARI, E., MILANO, S., DIELI, M., ARCOLEO, F., PEREGO, R., LEONI, F., GROMO, G., and SEVERN, A. Thymopentin reduces the susceptibility of aged mice to cutaneous leishmaniasis by modulating CD4 T-cell subsets. hnmunology 76, 362-366, 1992. CLERICI, M., WYNN, T.A., BERZOFSKY, J.A., BLATT, S.P., HENDRIX, C.W., SHER, A., COFFMAN, R.L., and SHEARER, G.M. Role of interleukin-10 in T helper cell dysfunction in asymptomatic individuals infected with the human immuuodeficiency virus. J. Clin. Invest. 93, 768-775, 1994. DAYNES, R.A. and ARANEO, B.A. Prevention and reversal of some age-associated changes in immunologic responses by supplemental dehydroepiandrosterone sulfate therapy. Aging lmmunol. Inject. Dis. 3(3), 135-154, 1992. DAYNES, R.A., ARANEO, B.A., ERSHLER, W.B., MALONEY, C., LI, G.-Z., and RYU, S.-Y. Altered regulation of IL-6 production with normal aging: Possible linkage to the age-associated decline in dehydroepiandrosterone and its sulfated derivative. J. lmmunol. 150(12), 5219-5230, 1993. DAYNES, R.A., DOWELL, T., and ARANEO, B.A. Platelet derived growth factor is a potent biologic response modifier of T cells..L Exp. Med. 174, 1323-1334, 1991. DE WAAL MALEFYT, R., HAANEN, J., SPITS, H., ROCAROLO, M.G., TE VELDE, A., FIGDOR, C., JOHNSON, K., KASTELEIN, R., YSSEL, H., and DE VRIES, J.E. Human T cell proliferation by diminishing the antigenpresenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J. E,tp. Med. 174(4), 915-924, 1991. DIGHIERO, G., TRAVADE, P., CHEVRET, S., FENAUX, P., CHASTANG, C., BINET, J.L., and CLL, T.F.C.G.O. B-Cell chronic lymphocytic leukemia: Present status and future directions. Blood 78(8), 1901-1914, 1991. DING, L., LINSLEY, P.S., HUANG, L.Y., GERMAIN, R., and SHEVACH, E.M. IL-10 inhibits macrophages costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J. lmmunol. 151(3), 1224-1234, 1993. ERNST, D.N., HOBBS, M.V., TORBETT, B.E., GLASEBROOK, A.L., REHSE, M.A., BOTTOMLY, K., HAYAKAWA, K., HARDY, R.R., and WEIGLE, W.O. Differences in the expression profiles of CD45RB, Pgp-l, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4 + T cells from young and aged mice. J. lmmunol. 145(5), 1295-12302, 1990. ERNST, D.N., WEIGLE, W.O., MCQUITTY, D.N., ROTHERMEL, A.L., and HOBBS, M.V. Stimulation of routine T cell subsets with anti-CD3 antibody. Age-related defects in the expression of early activation molecules. J. hnmunol. 142(5), 1413-1421, 1989. FINKE, J., TERNES, P.. MERTELSMANN, R., and DOLKEN, G. Expression of interleukin 10 in B lymphocytes of different origin. Leukemia 7( 11 ), 1852-1857, 1993. FIORENTINO, D.F., BOND, M.W., and MOSMANN, T.R. Two types of mouse T helper cell. IV. TH2 clones secrete a factor that inhibits cytokine production by TH1 clones. J. E_~p. Med. 170, 2081 2095, 1989. FIORENTINO, D.F., ZLOTNIK, A., MOSMANN, T.R., HOWARD, M., and O'GARRA, A. 1L-10 inhibits cytokine production by activated macrophages. J. hnmunol. 147(1 l), 3815-3822, 1991. FRANCESCHI, C., MONTI, D., SANSONI, P., and CASSAR1ZZA, A. The immunology of exceptional individuals: The lesson of centenarians, hnmunol. Today 16, 12-16, 1995. GILLIS, S., KOZAK, R., DURANTE, M., and WEKSLER, M.E. Immunological studies of aging. J. Clin. hlvest. 67(4), 937-942, 1981. GOIDL, E.A., STASHAK, P.N., MARTIN-MCEVOY, S.J., and HIERNAUX, J.R. Age related changes in serum immunoglobulin isotypes and isotype subclass levels among standard long-lived and autoimmune and immunodeficient strains of mice, Aging hnmunol. Infect. Dis. 1, 227-236, 1988. GOTTESMAN, R.S., EDINGTON, J., TSIAGBE, V.K., and THORBECKE, G.J. Influence of cytokines and the effect of aging on colony formation by B-cell subsets. Aging, hnmunol, lnt~,ct. Dis. 4(3), 197-211, 1993.

STEROIDREGULATIONOF IL-10PRODUCTION

407

HAYAKAWA~ K., HARDY, R.R., HERZENBERG, L.A., and HERZENBERG, L.A. Progenitors for Ly-I B cells are distinct from progenitors for other B cells. J. Exp. Med. 161, 1554-1468, 1985. HEINE, J.W. and ADLER, W.H. The quantitative production of interferon by mitogen-stimulated mouse lymphocytes as a function of age and its effect on the lymphocytes proliferative response..L Immunol. 118(4), 1136-1139, 1977. HOBBS, M.V., WEIGLE, W.O., and ERNST, D.N. Interleukin-10 production by splenic CD4 + cells and cell subsets from young and old mice. Cell. Immunol. 154, 264-272, 1994. INAMIZU, T., CHANG, M.-P., and MAKINODAN, T. Influence of age on the production and regulation of interleukin-1 in mice. Immunology 55, 447-455, 1985. KATO, K. and HIROKAWA, K. Qualitative and quantitative analysis of autoantibody production in aging mice. Aging hnmunol. Infect. Dis. 1(3), 177-190, 1993. LEVY, Y. and BROUET, J.C. Interleukin-10 prevents spontaneous death of germinal center B cells by induction of the bcl-2 protein. J. Clin. Invest. 93, 424-428, 1994, LLORENTE, L., RICHAUD-PATIN, Y., FIOR, R., ALCOCER-VARELA, J., WIJDENES, J., FOURRIER, B.M., GALANAUD, P., and EMILIE, D. In vivo production of interleukin-10 by non-T cells in rheumatoid arthritis, Sjtigrens syndrome, and systemic lupus erythematosus. Arthritis Rheum. 37(1 l), 1647-1655, 1994. LYNGBYE, J. and KROLL, J. Quantitative immunoelectrophoresis of proteins in serum from a normal population: season-, age-, and sex-related variations. Clin. Chem. 17(6), 495-500, 1971. MARIOTTI, S., SANSONI, P., BARBESINO, G., CATUREGLI, P., MONTI, D., COSSARIZZA, A., GIACOMELLI, T., PASSERI, G., FAGIOLO, U., PINCHERA, A., and FRANCESCHI, C. Thyroid and other organ-specific autoantibodies in healthy centenarians. Lancet 339, 1506-1508, 1992. MILLER, R.A. Accumulation of byporesponsive, calcium extruding memory T cells as a key feature of age-dependent immune dysfunction. Clin. lmmunol, lmmunopathol. 58(3), 302-317, 1991a. MILLER, R.A. Aging and immune function. Int. Rev. Cytol. 124, 187-214, 1991b. MOORE, K.W., O'GARRA, A., DE WAAL MALEFYT, R., VIEIRA, P., and MOSMANN, T. Intedeukin-10. Annu. Rev. Immunol. 11, 165-190, 1993. NAGEL, J.E., CHOPRA, R.K., CHREST, F.J., MCCOY, M.T., SCHNEIDER, E.L., HOLBROOK, N.J., and ADLER, W.H. Decreased proliferation, interleukin 2 synthesis, and interleukin 2 receptor expression are accompanied by decreased mRNA expression in phytohemagglutinin-stimulated cells from elderly donors. J. Clin. Invest. 81(4), 1096-1102, 1988. NAGELKERKEN, L., HERTOGH-HUIJBREGTS, A., DOBBER, R., and DRAGER, A. Age-related changes in lymphokine production related to a decreased number of CD45RBhi T cells. Eur. J. Immunol. 21(2), 273-281, 1991. O'GARRA, A., CHANG, R., GO, N., HASTINGS, R., HAUGHTON, G., and HOWARD, M. Ly-1 B (B-l) cells are the main source of B cell derived interleukin 10. Fur. ,1. lmmunol. 22, 711-717, 1992. ORENTREICH, N., BRIND, J.L., RIZER, R.L., and VOGELMAN, J.H. Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulbood. J. Clin. Endocrinol. Metab. 59(3), 551-555, 1984. PECANHA, L.M., SNAPPER, C.M., LEES, A., and MOND, J.J. Lymphokine control of type 2 antigen response. IL- 10 inhibits IL-5- but not IL-2- induced Ig secretion by T cell-independent antigens. J. lmmunol. 148(11), 3427-3432, 1992. PHILOSOPHE, B. and MILLER, R.A. Diminished calcium signal generation in subsets of T lymphocytes that predominate in old mice. J. Gerontol. 45, B87-B93, 1990. PLATER-ZYBERK, C., BROWN, C.M,S., ANDREW, E.M., and MAINI, R.N. CDS+B in rheumatoid arthritis. Ann. NY Acad. Sci. 651, 540-550, 1992. REAP, E.A., SOBEL, E.S., COHEN, P.L., and EISENBERG, R.A. The Role of CD5 + B cells in the production of autoantibodies in murine systemic lupus erytbematosus. Ann. NY Acad. Sci. 651, 588-590, 1992. SCHUMACHER, J.H., O'GARRA, A., SHRADER, B., VAN KIMMENADE, A., BOND, M.W., MOSMANN, T.R., and COFFMAN, R.L. The characterization of four monoclonal antibodies specific for mouse IL-5 and development of mouse and human IL-5 enzyme-linked immunosorbent. J. Immunol. 141, 1576-1581, 1988. STALL, A.M., ADAMS, S., HERZENBERG, L.A., and KANTOR, A.B. Characteristics and Development of the murine B-lb (Ly-l B Sister) cell population. Ann. NYAcad. Sci. 651, 33-43, 1992. STEIN, R., WITZ, IP, OVADIA, J., GOLDENBERG, D.M., and YRON, I. CD5 + B cells and naturally occurring autoantibodies in cancer patients. Clin. Exp. lmmunol. 85, 418-423, 1991. TAGA, K. and TASOTO, G. 1L-10 inhibits human T cell proliferation and IL-2 production. J. Immunol. 148, 11431148, 1992. THOMAN, M.L. and WEIGLE, W.O. The cellular and subcellular bases of immunosenescence. Adv. lmmunol. 46, 221-261, 1989.

408

N.F. SPENCERet al.

VIE, H. and MILLER, R.A. Decline with age, in the proportion of mouse T cells that express IL-2 receptors after mitogen stimulation. Mech. Ageing Dev. 33(3), 313-322, 1986. WEKSLER, M. and HUTTEROTH, T.H. Impaired lymphocyte function in aged humans. J. Clin. Invest. 53(1), 99-104, 1974. XU, H.J., ROBERTS-THOMSON, P.J., AHERN, M.J., and AOLA, H. Comparative Evaluation of CD5 B cells in patients with rheumatoid arthritis and essential mixed cryoimmunoglobulinemia using FACS analyzer and FACSCAN flow cytometer. Ann. NY Acad. Sci. 651, 594-598, 1992. YAMAJI, J. and IBAYASHI, H. Plasma dehydroepiandrosterone sulfate in normal and pathologic conditions. J. Clin. Endocrinol. Metab. 29, 273-278, 1969. ZHOU, T., EDWARDS, C.K., III, and MOUNTZ, J.D. Prevention of age-related T cell apoptosis defect in CD2-fastransgenic mice. J. Exp. Med. 182, 129-137, 1995.