[Frontiers in Bioscience 3, d125-135, January 15, 1998]

	[Frontiers in Bioscience 3, d125-135, January 15, 1998] Reprints PubMed CAVEAT LECTOR
	DOES CALORIC RESTRICTION ALTER IL-2 TRANSCRIPTION? Mohammad A. Pahlavani Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System and Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78284 Received 1/5/98 Accepted 1/9/98 3. INFLUENCE OF CALORIC RESTRICTION ON IL-2 EXPRESSION Interleukin-2 (IL-2) is a growth promoting cytokine that has received a great deal of attention over the past decade with respect to aging and cancer. It is produced primarily by helper T cells and regulates the growth and function of various cells that are involved in cellular and humoral immunity (19,20). The expression of IL-2 has been found to decrease with age in humans and rodents (reviewed in 21). The decline in IL-2 production has been shown to parallel the age-related decrease in immunologic function. Because caloric restriction enhances longevity and reduces pathology, there has been a great deal of interest on whether caloric restriction decreases pathology through action on the immune system, i.e., by altering IL-2 expression. The initial observation in this area was reported by Fernandes et al. (22); they showed that short-lived autoimmune prone (NZB X NZW) F1 mice fed normal calories (20 calories/day) were deficient in the production of IL-2 after 5 months of age. However, 50% caloric restriction preserved the production of IL-2 by the spleen cells of these animals; at 11 months of age, IL-2 production was approximately 2- to 3-fold higher for the caloric restricted mice. Furthermore, the caloric restricted mice responded vigorously to exogenous IL-2 in the thymocyte proliferation assay, while thymocytes from mice fed a normal diet lost much of their ability to respond to IL-2. In a later study, they showed that IL-2 activity and IL-2 receptor (IL-2R) expression (number of IL-2R sites per cell) were increased significantly in the concanavalin A (Con A)-stimulated spleen lymphocytes isolated from 19-month-old F344 rats fed a caloric restricted diet compared to the rats fed ad libitum (23). Since 1982, a number of different laboratories (24-30), including our laboratory (31-33), have shown that induction of IL-2 production by mitogens was greater for animals fed a caloric restricted diet (40% reduction in calories). For example, Hishinuma et al. (26) reported that Con A induction of IL-2 production in spleen cells isolated from 4-month-old male C3H/He mice fed a caloric restricted diet for 9 weeks was significantly increased compared to spleen cells from mice fed ad libitum. Using a limiting dilution assay, it was found that the proportion of IL-2 producing cells decreased with age. However, this decline was lower in mice fed a caloric restricted diet. For example, 32-month-old mice fed ad libitum retained only 15% of their helper T cell function (measured as IL-2 producing cells) compared to 7-month-old control mice (30). In old mice fed a caloric restricted diet, 53% of the helper T cell function was retained. Recently, it was demonstrated that caloric restriction prevents the rise in memory helper T cells (CD4+Pgp-1^high) with age in mice and maintains both a higher number of virgin/naive helper T cells (CD4+Pgp-1^lo) and a higher level of IL-2 production (34). Research from our laboratory has also shown that caloric restriction retards/reduces the age-related decrease in IL-2 expression. In an early study, we showed that caloric restriction significantly increased mitogen-induced lymphocyte proliferation and IL-2 production in F344 rats (31). In this study, rats (at 6 weeks of age) were subjected to a caloric restricted regimen. After 5, 12, 21, and 28 months of age, Con A induction of proliferation and IL-2 production (activity) by spleen lymphocytes were measured. We found that the proliferative response of lymphocytes to Con A in both caloric restricted rats and rats fed ad libitum declined significantly with increasing age. No differences were observed in mitogenesis and IL-2 production between caloric restricted rats and rats fed ad libitum at 5 and 12 months of age. However, the induction of proliferation and IL-2 expression was significantly higher for 21 and 22 month old caloric restricted rats compared to the rats fed ad libitum. In addition, we found that the increase in IL-2 activity was paralleled by an increase in the levels of the IL-2 mRNA transcript. Thus, caloric restriction altered the expression of IL-2 by increasing the transcription of the IL-2 gene. This was the first evidence that caloric restriction could alter the expression of a gene that plays a central role in cellular and humoral immune responses. Because aging is generally characterized by a reduced ability of an organism to maintain homeostasis in response to stress (35), we were interested in studying the effect of caloric restriction on the expression of the most predominant member of the heat shock protein 70 (HSP70) family, hsp70. Our laboratory had observed previously that the induction of hsp70 synthesis by heat shock was lower in lymphocytes isolated from old rats than from young rats (36). The age-related decrease in the induction of hsp70 synthesis was paralleled by a similar decrease in the induction of hsp70 mRNA. In a subsequent study, the induction of hsp70 expression in response to a mild hyperthermia and IL-2 expression in response to Con A were measured in T cells isolated from 24-month-old caloric restricted rats and ad libitum fed control rats. Figure 1 shows the effect of caloric restriction on hsp70 expression by hyperthermia and the induction of IL-2 expression by Con A. The induction of IL-2 mRNA levels was significantly higher in T cells isolated from old rats fed a caloric restricted diet than in old rats fed ad libitum. However, no differences were observed in the induction of hsp70 mRNA levels in the caloric restricted old rats and the old rats fed ad libitum (32). Thus, our study indicated that the influence of caloric restriction on the levels of mRNA transcripts appears to vary from gene to gene. Figure 1. Effect of caloric restriction on the induction of heat shock protein 70 (hsp70) mRNA levels by hyperthermia and IL-2 mRNA levels by Con A in spleen lymphocytes from F344 rats. The splenic T cells were isolated from 24-month-old rats fed ad libitum (AL) or 24-month-old rats fed a caloric restricted (CR) diet and were stimulated with Con A for 24 h or exposed to heat shock (42.5° C for 1 h) followed by incubation at 37° C for 1 h. The IL-2 and hsp70 mRNA levels were determined by Northern blot hybridization. The blots were quantified by Molecular Dynamic PhosphorImager, and the data are presented in the graph. Data were taken from Pahlavani et al. (32). The values () for the caloric restricted rats are significantly different from the values for the rats fed ad libitum* at p<0.05.

[Frontiers in Bioscience 3, d125-135, January 15, 1998]
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DOES CALORIC RESTRICTION ALTER IL-2 TRANSCRIPTION?

Mohammad A. Pahlavani

Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System and Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78284

Received 1/5/98 Accepted 1/9/98

3. INFLUENCE OF CALORIC RESTRICTION ON IL-2 EXPRESSION

Interleukin-2 (IL-2) is a growth promoting cytokine that has received a great deal of attention over the past decade with respect to aging and cancer. It is produced primarily by helper T cells and regulates the growth and function of various cells that are involved in cellular and humoral immunity (19,20). The expression of IL-2 has been found to decrease with age in humans and rodents (reviewed in 21). The decline in IL-2 production has been shown to parallel the age-related decrease in immunologic function. Because caloric restriction enhances longevity and reduces pathology, there has been a great deal of interest on whether caloric restriction decreases pathology through action on the immune system, i.e., by altering IL-2 expression. The initial observation in this area was reported by Fernandes et al. (22); they showed that short-lived autoimmune prone (NZB X NZW) F1 mice fed normal calories (20 calories/day) were deficient in the production of IL-2 after 5 months of age. However, 50% caloric restriction preserved the production of IL-2 by the spleen cells of these animals; at 11 months of age, IL-2 production was approximately 2- to 3-fold higher for the caloric restricted mice. Furthermore, the caloric restricted mice responded vigorously to exogenous IL-2 in the thymocyte proliferation assay, while thymocytes from mice fed a normal diet lost much of their ability to respond to IL-2. In a later study, they showed that IL-2 activity and IL-2 receptor (IL-2R) expression (number of IL-2R sites per cell) were increased significantly in the concanavalin A (Con A)-stimulated spleen lymphocytes isolated from 19-month-old F344 rats fed a caloric restricted diet compared to the rats fed ad libitum (23). Since 1982, a number of different laboratories (24-30), including our laboratory (31-33), have shown that induction of IL-2 production by mitogens was greater for animals fed a caloric restricted diet (40% reduction in calories). For example, Hishinuma et al. (26) reported that Con A induction of IL-2 production in spleen cells isolated from 4-month-old male C3H/He mice fed a caloric restricted diet for 9 weeks was significantly increased compared to spleen cells from mice fed ad libitum. Using a limiting dilution assay, it was found that the proportion of IL-2 producing cells decreased with age. However, this decline was lower in mice fed a caloric restricted diet. For example, 32-month-old mice fed ad libitum retained only 15% of their helper T cell function (measured as IL-2 producing cells) compared to 7-month-old control mice (30). In old mice fed a caloric restricted diet, 53% of the helper T cell function was retained. Recently, it was demonstrated that caloric restriction prevents the rise in memory helper T cells (CD4+Pgp-1^high) with age in mice and maintains both a higher number of virgin/naive helper T cells (CD4+Pgp-1^lo) and a higher level of IL-2 production (34).

Research from our laboratory has also shown that caloric restriction retards/reduces the age-related decrease in IL-2 expression. In an early study, we showed that caloric restriction significantly increased mitogen-induced lymphocyte proliferation and IL-2 production in F344 rats (31). In this study, rats (at 6 weeks of age) were subjected to a caloric restricted regimen. After 5, 12, 21, and 28 months of age, Con A induction of proliferation and IL-2 production (activity) by spleen lymphocytes were measured. We found that the proliferative response of lymphocytes to Con A in both caloric restricted rats and rats fed ad libitum declined significantly with increasing age. No differences were observed in mitogenesis and IL-2 production between caloric restricted rats and rats fed ad libitum at 5 and 12 months of age. However, the induction of proliferation and IL-2 expression was significantly higher for 21 and 22 month old caloric restricted rats compared to the rats fed ad libitum. In addition, we found that the increase in IL-2 activity was paralleled by an increase in the levels of the IL-2 mRNA transcript. Thus, caloric restriction altered the expression of IL-2 by increasing the transcription of the IL-2 gene. This was the first evidence that caloric restriction could alter the expression of a gene that plays a central role in cellular and humoral immune responses.

Because aging is generally characterized by a reduced ability of an organism to maintain homeostasis in response to stress (35), we were interested in studying the effect of caloric restriction on the expression of the most predominant member of the heat shock protein 70 (HSP70) family, hsp70. Our laboratory had observed previously that the induction of hsp70 synthesis by heat shock was lower in lymphocytes isolated from old rats than from young rats (36). The age-related decrease in the induction of hsp70 synthesis was paralleled by a similar decrease in the induction of hsp70 mRNA. In a subsequent study, the induction of hsp70 expression in response to a mild hyperthermia and IL-2 expression in response to Con A were measured in T cells isolated from 24-month-old caloric restricted rats and ad libitum fed control rats. Figure 1 shows the effect of caloric restriction on hsp70 expression by hyperthermia and the induction of IL-2 expression by Con A. The induction of IL-2 mRNA levels was significantly higher in T cells isolated from old rats fed a caloric restricted diet than in old rats fed ad libitum. However, no differences were observed in the induction of hsp70 mRNA levels in the caloric restricted old rats and the old rats fed ad libitum (32). Thus, our study indicated that the influence of caloric restriction on the levels of mRNA transcripts appears to vary from gene to gene.

Figure 1. Effect of caloric restriction on the induction of heat shock protein 70 (hsp70) mRNA levels by hyperthermia and IL-2 mRNA levels by Con A in spleen lymphocytes from F344 rats. The splenic T cells were isolated from 24-month-old rats fed ad libitum (AL) or 24-month-old rats fed a caloric restricted (CR) diet and were stimulated with Con A for 24 h or exposed to heat shock (42.5° C for 1 h) followed by incubation at 37° C for 1 h. The IL-2 and hsp70 mRNA levels were determined by Northern blot hybridization. The blots were quantified by Molecular Dynamic PhosphorImager, and the data are presented in the graph. Data were taken from Pahlavani et al. (32). The values (*) for the caloric restricted rats are significantly different from the values for the rats fed ad libitum at p<0.05.