Calorie restriction modulates lymphocyte subset phenotype and increases apoptosis in MRLlpr mice

&nmunology Letters. 47 ( 1995) Elsevier Science B.V.

181-I86

IMLET 2428

Calorie restriction modulates lymphocyte subset phenotype and increases apoptosis in MRL/Zpr mice ’ Xianghong Luan, Weiguo Zhao, Bysani Chandrasekar, and Gabriel Femandes * Department

of Medicine, University of Texas Health Science Center, San Antonio, TX 78284, USA (Received

2 March 1995; revised 21 April 1995; accepted

Key words: Apoptosis;

Fas; Calorie restriction;

1. Summary Defective expression of the Fus apoptotic gene may account for overproduction of CD4-CD8-B220+ cells (double-negative) in MRL/MpJ-lpr/lpr (lpr) mice. Previous studies have shown that calorie restriction (CR) inhibits the development of autoimmune disease and extends life span in these animals. The present studies describe the effects of CR on the distribution of lymphocyte phenotypes, lymphocyte proliferative response, and cytokine release. The effects of CR on dexamethasone (DEX)-induced apoptosis were also studied using propidium iodide (PI) uptake and DNA fragmentation in splenocytes and lymph node (LN) cells. Weanling female mice were fed a nutritionally adequate semipurified diet either ad libitum (AL) or with 40% fewer calories than AL (CR), and killed at 5 months of age. CR mice had fewer palpable lymph nodes, and decreased serum anti-dsDNA antibodies. Mitogen (ConA, anti-CD3, and LPS) and superantigen (SEB)-induced proliferative response was significantly lower in lymphoid cells from AL fed animals. FACS analysis of cells from CR animals showed decreased CD4- CD8- cells in spleen (1.7-fold, P < 0.025) and LN (1.6-fold, P < 0.01) and significantly higher CD4+ (spleen, 1.7-fold, P < 0.0001; LN, 2.6-fold, P < 0.025) and CD8+ (spleen, 1.6-fold, P < 0.001; LN, 5.2-fold, P < 0.005) cells. ConA-stimulated IL-2 release was in-

Corresponding author: Gabriel Femandes, Ph.D., Professor, Department of Medicine/Clinical Immunology. University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 782847874, USA. Tel.: (210) 567-4663; Fax: (210) 567-4592. l

’ Data from parts of this paper were presented at the 58th Annual Meeting of the American College of Rheumatology at Minneapolis, Minnesota, during 23-27 October 1994. SSDlOl652478(95)00091-7

Autoimmunity;

6 June 1995)

Lupus; Lymphoproliferation

creased in CR animals (splenocytes, 7.5fold, P< 0.001; LN cells, 6.1-fold, P < 0.01). Finally, apoptosis in response to Dex was increased in CR animals as indicated by the presence of more PI-positive cells (spleen, 15.8%; LN, 10.7%; P < 0.01) and increased DNA fragmentation. In summary, the amelioration of autoimmune disease in MRL/lpr mice by CR is accompanied by prevention of the rise in ‘double-negative’ T cells and by maintenance of lymphocyte responsiveness to mitogens and DEX-induced apoptosis at higher levels.

2. Introduction MRL/lpr mice develop a disease characterized by lymphoid organ enlargement, hyper-gammaglobulinemia and the production of diverse autoantibodies typical of an autoimmune disease [1.2]. These pathological changes are accompanied by accumulation of an unusual subset of CD3+CD4_CDK 8220’ (doublenegative) T cells which may constitute up to 90% of the total T cells in enlarged LN. These cells respond weakly to stimulation with mitogens, antigens or anti-T-cell receptor antibody. Proliferation, IL-2 production and IL-2 receptor acquisition are markedly deficient in these cells after mitogen stimulation [3-61, indicating that both phenotypic and functional abnormalities are present in lymphocytes from MRL/lpr mice. Defects in the gene encoding the Fas antigen are also linked to the above alterations [7-91. The Fas antigen is a cell surface protein with structural homology to tumor necrosis factor receptors and the low-affinity nerve growth factor receptor [lo,1 11. Upon contact with the anti-Fas antibody, some lymphocytes that express Fas antigen undergo apoptosis [12- 141. Decreased Fas expression in lpr mice permits the development of T cells in which rearrangement/expression of the TCR/CD3 181

receptor is inefficient. The result is an overproduction of self-reactive T cells in the thymus since they fail to undergo apoptosis. They subsequently migrate in increased numbers to the periphery [15,16]. The double-negative cells in these animals can be reduced by treatment with phorbol ester (PMA) and IL-2 to cause differentiation to a more mature T-cell phenotype [17]. Also, treatment with IL-2/vaccine recombinant virus or a polyamine biosynthesis inhibitor [ 18,191 can prevent the rise in double-negative T-cell population. Insertion of a transgene for Fas also normalizes the number of double-negative cells [20]. Our previous studies have shown that calorie restriction (CR) extended the life span of MRL/lpr mice by inhibiting the development and expression of the lymphoproliferative syndrome [21]. We now propose that these effects of CR are accompanied by increasing apoptosis of T cells in peripheral organs such as LN and spleen by switching the phenotype more towards single-positive cells (CD4 or CD8).

3. Materials

3.1. Animals and diets Weanling (4 week old) female MRL/MpJ-lpr/lpr (lpr) and MRL/MpJ- + / + ( + / + ) mice obtained from the Jackson Laboratories (Bar Harbor, ME), were fed a nutritionally adequate semiputified diet containing 5% fat either ad libitum (AL) or 40% restricted calories (CR) as described earlier [22]. Animals were housed in a climate and light-controlled environment at the Laboratory Animal Care Facility at the University of Texas Health Science Center, San Antonio. A minimum of 20 mice in each group were studied. At 5 months of age, mice were killed by cervical dislocation, and spleens and LN were collected aseptically, quickly weighed, and used for further analyses. of serum anti-dsDNA

antibodies

Serum anti-dsDNA antibody concentrations were determined by direct ELISA as described earlier 1231. 3.3. Cell isolation, culture and proliferative mitogens

response to

Single-cell suspensions from aseptically collected spleens and LN were made, and enriched by centrifugation over Lympholyte-M (Cedarlane Laboratories, Ontario, Canada) as described earlier 1241. The proliferative response [ 3H]TdR incorporation) of lymphocytes to individual mitogens (ConA (2.5 pg/ml), LPS (40 182

3.4. Flow cytometric

analysis for lymphoid cell subsets

Single-cell suspensions diluted to 1 X lo6 cells/ sample were surface-labeled for one- or two-color immunofluorescence staining [24] and analyzed using a FACStar Plus flow cytometer (Becton Dickinson). 3.5. Cytokine levels Cytokine levels were measured in cell-free supernatants essentially as described earlier 1241 and the levels were quantitated by comparison to standard curves generated for each cytokine analyzed.

and methods

3.2. Measurement

pg/ml)), superantigen (staphylococcal enteroxin B (SEB; 2 pg/ml)), and anti-CD3 (IO pg/mI) was measured as described earlier [25]. For cytokine production, 5 X IO6 cells/ml were cultured in 24-well plates for 48 h in the presence of anti-CD3 antibodies (10 pg/ml), and cell-free supematants were collected and saved at - 70°C until assayed. For the induction of apoptotis, cells were cultured in 24-well plates at lo6 cells/ml for an indicated period of time in RPMI164O- 10% FCS with or without dexamethasone (DEX, IO-’ M).

3.6. DNA fragmentation 1 X lo6 LN cells were pelleted by centrifugation at 800 X g for 2 min at 23°C [26]. Cell pellets were resuspended in 50 ~1 of cell lysis buffer (10 mM EDTA, 50 mM Tris, pH 8.0, containing 0.5% (wt/v> N-lauroylsarcosine and 0.5 mg/ml proteinase K) and then incubated for 1 h in a water bath at 50°C. This was followed by the addition of 50 ~1 of RNase A (0.5 mg/ml) to each sample and incubated at 50°C for 1 h more. Cell lysates were then brought to 250 ~1 by the addition of TE (10 mM Tris Cl, pH 8.0, 1 mM EDTA) buffer, and extracted (twice) with phenol/ chloroform/ isoamyl alcohol (24: 24: 1). DNA was precipitated overnight at - 70°C by the addition of two volumes of ethanol. The precipitates were recovered by centrifugation at 13,000 X g for 10 min, air-dried, resuspended in TE buffer and stored at 4°C. The DNA was quantified spectrophotometrically (OD at 260 nm), and 8 pg of DNA/well was electrophoresed in TBE (89 mM Trisborate, 2 mM EDTA) buffer. A 123 bp DNA ladder was run simultaneously. DNA was visualized under UV light and photographed. 3.7. Propidium

iodide staining

One million cells were washed twice with PBS, then resuspended in 1 ml of PBS. PI was added at a concentration of 10 Fg/ml. The cells were incubated in the

dark for 1 h at 4°C washed with PBS, and then resuspended in 1 ml of PBS. The fluorescence intensity of individual nuclei was measured using a FACStar + flow cytometer [27]. 3.8. Statistical

analysis

Values are expressed as mean * SEM andP < 0.05 was considered significant when analyzed by Student’st test.

4. Results 4.1. Body and organ weights, and serum anti-dsDNA

OOL-= = -d Ii32 1163 1,128 1,256 1,512 111024 l~O48ll4C'h Dilution of serum

Fig. 2. Effect of CR on serum anti-dsDNA antibody titers (ELISA) in MRL/pr and MRL/ + / + mice. CR was initiated from weanling (1 month), and the mice were killed at 5 months of age. Data are the mean * SEM of observations from 4 to 8 mice/group. CR significantly (P < 0.01) inhibited serum anti-dsDNA antibody levels as compared to ad-libitum AL fed group. In contrast congenic + / + mice had very low to undetectable levels of the antibody.

antibodies

Before the dietary regimen was initiated, the weanling mice had similar (N 27 g) body weights (Fig. 1). Between 1 and 3 months of age, mice on the AL diet gained weight while the CR animals lost a small amount of weight. Both AL and CR animals were relatively stable between 3 and 5 months when the animals were killed. A similar pattern in body weights was observed in + / + mice (Fig. 1). Both spleen and LN weights were significantly lower in CR fed mice as compared to the organs from AL fed mice (spleen; AL, 0.73 -t 0.09 g; CR, 0.12 f 0.02 g, P < 0.01; LN, AL, 3.23 t_ 0.49 g, CR, 0.09 + 0.02 g, P < 0.01). A similar pattern in spleen weights was observed in + / + mice (AL, 0.13 f 0.02 g, CR, 0.18 &-0.02 g, P < 0.01). As disease progression in lpr mice correlates with increased anti-dsDNA antibodies in serum, we compared the effects of AL and CR on antibody levels at 5 months of age. As shown in Fig. 1, the levels of serum anti-dsDNA antibodies in lpr mice were significantly lower (P < 0.01) in CR mice as compared to AL mice. MRL/ + / + mice, which are resistant to the development of autoimmune disease, had very low to undetectable levels of the antibodies (Fig. 2).

4.2. Proliferative

responses

to mitogens

Cell proliferation in response to mitogens is a charactristic of ‘mature’ T cells, so we compared the effects of AL and CR on the proliferative response of lymphocytes from lpr (AL and CR) and + / + mice. Both spleen and LN cells from CR mice had significantly higher proliferative response to SEB and anti-CD3 (Fig. 3). Also a similar response was found to ConA and LPS in both spleen and LN (data not shown). Proliferative response to all the 4 stimulators was found higher for splenocytes rather than for LN cells of CR mice. 4.3. Phenotype

of T cells from lymph nodes

Lymphadenopathy in lpr mice is due to proliferation and accumulation of large numbers of non-malignant CD3+CD4_CD8T lymphocytes in the LN. To explore the effects of AL and CR on accumulation of double-negative T cells in the peripheral organs, lym-

0

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SEB

Spleen

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antl-CD3

Medlnm

SEB anti-CD3

Lymph

node

5

Age (mon.) Fig. 1. Effect of CR on body weights in MRL/ Zpr and MRL / + / + mice. CR was initiated from weanling (1 month), and the mice were killed at 5 months of age. Values are meani-SEM. Both MRL/lpr and MRL/+/+ mice on CR diet had a significantly lower body weight as compared to respective AL fed groups.

Fig. 3. Effect of CR on proliferative response ([‘H]TdR incorporation) of splenocytes and LN cells from MRL/ lpr and MRL/ + / + mice at 5 months af age. Data are the mean * SEM of observations from 4 to 8 mice/group. CR significantly (P < 0.01) increased proliferative response to the indicated mitogens. ’ P < 0.05. . P < 0.01 (fpr, AL vs. CR), +P < 0.005, ‘+P < 0.025 (&-AL vs. +/+l

AL).

183

Calorie restriction

Ad libitum

400

0

*

Ipr-AL

z

8

Y2 6

Y2 a

1

IL-2

1

IL-4

Spleen 0

0 0

12

3

4

0

12

a-CD4

3

4

a-CD4

IL-2

Lymph

IL-4

node

Fig. 6. Effect of AL and CR diets on cytokine levels (pg/ml) in cell-free supematants. Data are the mean f SEM of observations from 4 to 6 mice/group. P < 0.01. l

4

4

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MRL/ + / + mice, in which CD4+ cells were 51% (data not shown). Similar phenotypic differences were observed in splenocytes from Zpr (AL and CR) mice (Fig. 5).

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Fig. 4. A representative contour plot (out of three) showing the effects of CR on double-negative and single-positive cells in spleen and LN isolated from MRL/lpr mice. The LN cells were subjected to two-color immunofluorescence staining utilizing FITC-conjugated CD4 and PE-conjugated CD8 mAbs, and subsequently 5X 10’ cells were analyzed. A reversal in T-cell phenotype from ‘double-negative’ (CD4- CD8- ) to single-positive (CD4+ CDfi- or CD4- CD8+ ) cells was noted.

phocytes were subjected to two-color immunofluorescence staining with FITC-conjugated anti-CD4 and PEanti-CD8 mAbs. In AL MRL/lpr mice, 83% of LN cells were CD4_CD8-, but this dropped to 52% in CR mice (Fig. 4). This effect of CR was accompanied by higher levels of mature cells (both CD4+CD8and CD4_CD8+ cells) (Fig. 5). Approximately 30% of LN cells were CD4+ and 15% were CD8+. While CR did improve the phenotypic distribution of T cells, the levels did not reach those of (presumably normal)

Spleen

CD4Ch-

CD4+

CDS+

It has been repeatedly observed that ConA-stimulated IL-2 production is markedly reduced in LN cells from MRL/lpr mice. This profound defect is partly restored by CR. IL-2 levels were 6-fold higher in LN cells and 7.5-fold higher in splenocytes from CR mice compared to AL (Fig. 6). Also in CR fed mice, a 3-fold increase in IL-4 secretion by splenocytes was observed, while other cytokines such as IL-6, IL-IO, and IFN-7 were minimally altered (data not shown). 4.5. Apoptosis Fresh LN cells from MRL/lpr or MRL/ + / + mice were stimulated with and without DEX, harvested at 18 h, and analyzed for apoptosis by FACS analysis

Lymph node

Ig+

CD4-

CD4+

CDS+

Ig+

Ch-

Fig. 5. Effect of ad-libitum AL and CR diets on lymphocyte subsets in spleen and LN isolated from hfRL/lpr mice. One-color immunofluorescence analysis was performed utilizing FITC-conjugated mAb. Data are the mean +SEM of observations from 4 to 6 mice/group. ‘P < 0.01.

184

4.4. Cytokine release

Media

Dexamethasone

Fig. 7. Effects of CR on DEX-induced PI staining of lymphocytes isolated from LN and spleens of MRL/ lpr mice. CR, as compared to AL, significantly increased DEX-induced PI-positive cells in LN from AL lpr mice. Values are mean*SEM of observations from 4 to 5 mice/group. * P < 0.01, ’ ‘P < 0005 (lpr, CR vs. AL), ‘P < 0.05, “P < 0.005 (Ipr-AL vs. + / +-AL).

Fig. 8. Effects of CR on apoptosis of lymph node cells. LN cells (1 X IO61 from MRL/lpr or MRL/ + / + mice were incubated with or without DEX (lo-’ M) for 18 h. DNA was extracted, electrophoresed and visualized in UV. CR induced accelerated apoptosis in the LN cells from lppr mice similar to that observed in + / + mice.

and DNA ladder formation. By FACS analysis cells cultured in media alone showed 29.6% of LN cells from MRL/ + / + mice were apoptotic versus only 23.5% of the cells from AL MRL/lpr mice (as assessed by PI staining). CR restored the percentage of apoptotic cells to 29% (Fig. 7). Similar pattern in PI staining was observed in splenocytes from lpr and + / + mice (Fig. 7). These results were confirmed by agarose gel electrophoresis of cellular DNA showing a characteristic DNA ladder formation (Fig. 8). DEX-induced apoptosis was the highest in LN of + / + mice (80%), followed by CR (69%) and AL (59%) fed lpr mice.

5. Discussion CR maintains the vigor of immunological responses that otherwise rapidly wane during aging both in normal and in disease-prone mice [28,29]. Compared to AL fed mice, proportion of single-positive CD4 or CD8 cells in spleen and LN increased and the proportion of doublenegative cells decreased significantly. These singlepositive more mature T-cell subsets showed increased IL-2 production and proliferated 3- to 9-fold higher than AL fed mice when stimulated with various mitogens. Thus, the functional recovery of splenocytes and LN cells in CR lpr mice is related to alterations in the distribution of lymphocyte phenotypes. Similarly, in the context of aging-related changes in the immune system,

CR slowed age-related impairments in the immune system, including maintenance of a more youthful T-cell profile [ 301. Homozygous expression of the lpr gene leads to autoimmunity and lymphadenopathy [7-g]. The Zpr mutation involves a rearrangement of the gene for the Fas antigen, which is expressed in the thymus and mediates apoptosis. An insertion of an early transposable element (ETn) in intron 2 of the Fas gene results in diminished Fas expression [7,8]. It has been suggested that Fus plays a role in both thymic selection and T-cell survival in the periphery and that the accelerated autoimmunity in ipr mice results from a defect in both of these pathways. However, it is possible that activation via Fas is required at some point, but not in all steps of T-cell death [31]. The abnormal doublenegative T cells which accumulate have a defect in the Fas gene, but they still have the ability to undergo either spontaneous or DEX-induced apoptosis in vitro [31]. By binding to their receptors, glucocorticoids either induce or inhibit the transcription of the responsive genes, including apoptosis-related genes [32,33]. Role of CR on increasing apoptosis in other organs such as liver has been recently described [34.35]. Decreased carcinogenesis and tumor formation in CR fed mice was implicated to increased apoptosis [34,3.5]. To our knowledge, the role of CR on enhanced apoptosis in immune cells has not yet been described. Several earlier reports in rodents describing inhibition of autoimmunity and extension of life span by CR may possibly be due to presence of an efficient apoptotic mechanisms in immune cells. In some way, CR restores or normalizes the mechanism(s) involved in the glucocorticoid-induced apoptotic process, and accelerates apoptosis. It is quite possible that an increased serum corticosterone level which is known to be elevated in CR fed animals [36,37] might have also contributed in inhibition of autoimmune disease by accelerating apoptosis. This hypothesis needs to be further tested in AL and CR fed Zpr mice. Recent studies undertaken in CR fed animals have revealed an altered membrane composition and changes in membrane fluidity, which might also influence mitogen and antigen receptors and modulate transmembrane signaling responses of lymphocytes [38-401. In Ipr mice an abnormal development of T cells in the thymus is thought to be related to the expression of an unusual CD4-CDS-B220+ peripheral T-cell subset that results in lymphadenopathy. Landolfi et al. [ 151 have demonstrated demethylation of CD8 gene in CD4_CD8LN cell and in the CD4-CD8-B220+ thymocytes in lpr mice, indicating that these abnormal cells expressed CD8 marker earlier [15]. Also, a population of cells that did express CD2, CD4, and CD8 markers earlier had 185

lost their expression after differentiation [ 151, resulting in defective signal transduction mechanisms in CD4-CDS-T lymphocytes. Simultaneous treatment of these double-negative cells with the protein kinase C activator, PMA and IL-2, induced differentiation to a more mature T-cell phenotype, with loss of cell surface B220. The cells transiently expressed IL-2 receptors and grew in an IL-2-dependent manner. Furthermore, a portion of the cells acquired antigen receptor and Lyt-2. Thus, treatment with appropriate agonists can restore the differentiating capabilities of double-negative T cells, possibly circumventing the Fus gene defect. Although the present study demonstrates a significant decrease in the number of double-negative cells in CR MRL/lpr mice, the level of apoptosis was, however, not proportionately increased. This implies that perhaps other mechanisms beside apoptosis may be involved in preventing the rise in double-negative cells by CR. Further studies are needed to understand the precise mechansm(s) involved in delaying or inhibiting autoimmune disorders and malignancy by CR 1411. Also, studies are needed to determine whether CR alters thymic selection as well as expression of genes involved in inducing or opposing apoptosis, including restoration of defective signal transduction pathways as the mechanism(s) for ameliorating autoimmune disease.

Acknowledgements The work was supported by NIH Grants ROl AG03417, AG-10531, DE-10863 and AREC at UTHSC. We also thank Dr. Dean A. Troyer, Department of Pathology for critically reading the manuscript.

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