Effect of age and dietary restriction on expression of heat shock protein 70 in rat alveolar macrophages

Mechanisms of Ageing and Development 104 (1998) 59 – 73

Effect of age and dietary restriction on expression of heat shock protein 70 in rat alveolar macrophages Susan A. Moore a,b, Arturo Lopez a,b, Arlan Richardson a,b, Mohammad A. Pahlavani a,b,* a

Geriatric Research, Education, and Clinical Center (182), South Texas Veterans Health Care System, Audie Murphy VA Hospital, 7400 Merton Minter Bl6d., San Antonio, TX 78284, USA b Department of Physiology, Uni6ersity of Texas Health Science Center, San Antonio, TX 78284, USA Received 22 January 1998; received in revised form 15 April 1998; accepted 17 April 1998

Abstract Dietary restriction (DR) is the only effective experimental manipulation known to retard aging in rodents, and this manipulation has been shown to alter a variety of processes that change with age. However, there is no information on the effect of DR on macrophage function. In the present study, the effect of aging and DR on the ability of alveolar macrophages (AMs) to express the heat shock gene, hsp70 was studied. AMs were isolated by lavage from the lungs of young (4 – 6 months) and old (24 – 26 months) rats fed either ad libitum (AL) or a restricted diet (60% of AL). There was no age-related change in the number of cells recovered from young and old rats fed AL. However, the number of cells recovered from the lungs of the DR rats was reduced, and this decrease was statistically significant in young rats. The expression of heat shock protein 70 (hsp70) was measured by the level of the hsp70 mRNA transcript in total RNA isolated from AMs cultured under two conditions: in suspension and after adherence to plastic. When AMs were incubated at 37°C in suspension, no detectable hsp70 expression was observed; however, hsp70 expression was induced at 37°C when the AMs adhered to the plastic culture dishes. Hsp70 mRNA levels were rapidly induced by heat shock (43°C, 1 h) in AMs cultured both in suspension and on plastic. The induction of hsp70 expression did not change significantly with either age or DR in AMs cultured in suspension. In contrast, the induction of hsp70 mRNA levels by AMs * Corresponding author. Tel.: + 1 210 6175197; fax: + 1 210 6175312; e-mail: [email protected] 0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00052-9

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adherent to plastic culture plates decreased approximately 70% with age, and hsp70 induction was greater in AMs isolated from DR rats; this difference was statistically significant in young rats. The induction of hsp70 by heat shock (43°C, 1 h) also decreased with age in the adherent AMs, and DR increased the induction of hsp70 expression three- to fourfold in adherent AMs from both young and old rats. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Dietary restriction; Macrophage; Heat shock protein; Rat

1. Introduction Nutrition has been shown to have a significant impact on aging. Research in the 1950s and 1960s demonstrated that dietary restriction (DR), i.e. undernutrition not malnutrition, significantly increased the survival of rodents. This increase in survival has been observed with a variety of different nutritional techniques/regimens, and all these techniques/regimens have in common a reduction in the amount of diet that an animal consumes (reviewed in Richardson, 1985). Over the past decade, it has become apparent that total calories is the nutritional component of food restriction regimens that is responsible for the increase in survival (Masoro, 1990, 1992a,b). Because DR retards and reduces the incidence of almost all pathological lesions and because age-related changes in most physiological processes are reversed by DR, the general consensus of the research community is that DR increases the survival of rodents by retarding the aging process (Masoro, 1990, 1992a,b). Therefore, DR has become a powerful technique for studying aging. Although it is well established that DR increases the survival of rodents, it is currently unclear how DR alters aging and prolongs survival at the biochemical/ molecular level. The view that DR alters the aging process at the level of gene expression is attractive for the following reasons. First, gene expression is a critical site of cellular regulation in all cells/tissues of all living organisms. Second, changes in the expression of even one gene can have a dramatic effect on an organism. Third, alterations in various steps of gene expression have been observed with increasing age (Van Remmen et al., 1995). Over the past decade, the expression of heat shock genes has been used as a model for studying the regulation of gene expression at the transcriptional level. Various physical and chemical stimuli (e.g. heat shock, ethanol, amino acid analogs, heavy metals, free radicals, etc.) induce the expression of heat shock proteins or stress proteins, and this phenomenon is one of the most evolutionarily conserved processes in nature (Lindquist, 1986; Lindquist and Craig, 1988). Heat shock protein 70 (hsp70), which belongs to the HSP70 family, is the most inducible of the heat shock proteins by stress, and this protein appears to play a critical role in protecting cells against the adverse effects of heat shock and other stresses. For example, reduction in hsp70 levels is associated with increased thermosensitivity (Johnston and Kucey, 1988; Riabowol et al., 1988), and overexpression of hsp70 is associated with increased thermotolerance (Angelidis et al., 1991; Li et al., 1991).

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Because hsp70 plays an important role in protecting cells against heat shock and other forms of stress, several laboratories have studied the effect of aging on hsp70 expression. These studies show that the induction of hsp70 expression by stress, such as hyperthermia, decreases significantly with increasing age in a variety of tissues, e.g. skin and lung (Blake et al., 1990), brain (Blake et al., 1991; Pardue et al., 1992), and liver (Heydari et al., 1993), from rodents and from peripheral blood lymphocytes (Deguchi and Kishimoto, 1989; Faassen et al., 1989) from humans. More recently, our laboratory showed that the ability of spleen lymphocytes from rats and peripheral blood lymphocytes from rhesus monkeys to express hsp70 in response to heat shock decreased with age, and that this decline occurred at the level of hsp70 transcription (Pahlavani et al., 1995). Heat shock proteins appear to play an important role in the function of cells in the immune system, including macrophages (reviewed in Young, 1992; Sribvastava et al., 1994). Macrophages play a critical role in initiation, maintenance, and resolution of the pulmonary immune response, and these cells are constantly exposed to environmental stresses. Various stimuli, such as bacterial lipopolysaccharide, inflammatory cytokines, phagocytosis and the respiratory burst, are known to induce expression of heat shock proteins in macrophage (Sribvastava et al., 1994), suggesting that these stress proteins may play an important role in the function and protection of these cells. Current evidence indicates that alteration in the ability of macrophages to respond to environmental stimuli results in increased vulnerability of the individual to disease (Brody and Brock, 1985). Therefore, it was of interest to study the effect of age and DR on the induction of hsp70 gene expression in alveolar macrophages (AMs) from rats. Our study shows that the induction of hsp70 by heat shock in AMs cultured in suspension was not altered with either age or DR. However, the induction of hsp70 mRNA was significantly decreased with age when AMs were allowed to adhere to plastic plates and when the adherent AMs were subjected to heat shock treatment. More importantly, our results show that the hsp70 mRNA levels were significantly higher in adherent AMs from young and old DR rats compared to AMs from the ad libitum (AL) fed rats.

2. Materials and methods

2.1. Animals and diets Male Fischer 344 rats (specific pathogen-free) were obtained at 3 weeks of age from Harlan Sprague – Dawley (Indianapolis, IN). Sentinel rats were sacrificed on receipt, and 6 weeks after the shipment for monitoring of viral antibodies (Sendai, Reo-3, GD-VII, PVM, KRU, H-I, SDA, LCM, and Adeno). The serum samples was sent to Microbiological Associates (Bethesda, MD) for analysis, and all tests were negative. This monitoring of sentinel rats was repeated every 3 months. All procedures for handling the rats were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and the sub-committee for Animal Studies at Audie L. Murphy Memorial

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Veterans Hospital. The rats were caged individually in a barrier facility on a 12-h light/dark cycle. Rats were fed a semi-synthetic soy protein diet (5770M-S Vitamin Fortified RP 101 Purified Diet; Purina Mills, St. Louis, MO), which consisted of the following ingredients: 21% RP101 soy protein isolate, 15% sucrose, 45.99% dextran, 6% corn oil, 5% mineral mix with reduced sodium, 3% solka floc, 0.35% DL-methionine, 0.33% choline chloride and 3.33% RP vitamin mix. At 6 weeks of age, the rats were randomly assigned to two groups. The control, AL group was given free access to the diet and the DR group received 60% of the diet consumed by the rats fed AL as previously described (Yu et al., 1985; Pahlavani et al., 1996). The soy protein diet has been shown to prevent the progression of chronic nephropathy, which has been reported in this strain of rats on other protein diets (Shimokawa et al., 1993). The median survival of the restricted rats was 938 days compared to a median survival of 813 days for the control rats that had free access to the diet. Both young and old rats were carefully examined for the presence of disease and major pathological lesions. Any rat with a tumor, an enlarged spleen, or signs of infection or pneumonia (e.g. increased number of cells from bronchoalveolar macrophages or abnormal differential cell counts) was excluded from the study.

2.2. Isolation and culturing of al6eolar macrophages Rats were killed by decapitation and the lungs were removed aseptically. Pyrogen-free saline (Baxter, Grant Perairie, TX) was instilled in an 8-ml aliquot into the lungs and then aspirated with a syringe. This procedure, known as lavage, removes cells from the airspace with the predominant cell type being AMs (Green and Kas, 1964; Fels and Cohn, 1996). Other cell types that are recovered include lymphocytes and polymorphonuclear leukocytes. The number and viability (trypan blue exclusion) of the cells obtained from the lungs of each rat were determined, and differential cell type (percentage of macrophages, neutrophils and lymphocytes) was measured by the Wright Giemsa stain method. After washing with pyrogen-free saline, AMs were resuspended in RPMI 1640 media (Gibco/BRL, Gaithersburg, MD) supplemented with L-glutamine, penicillin and streptomycin. The AMs were cultured under two conditions: they were incubated with shaking in polypropylene round-bottom culture tubes so that the AMs remained in suspension, or the AMs were incubated in 60-mm diameter tissue culture dishes and allowed to attach to the plastic surface of the culture dish. AMs maintained in suspension cultures are essentially inactive, e.g. they do not express proinflammatory cytokines, while AMs that adhere to plastic are primed to become activated and release various cytokines (e.g. tumor necrosis factor-a, interleukin-1, etc.) that are involved in macrophage-mediated functions. The AMs in suspension or adherent to plastic were cultured at 37°C under air and 5% carbon dioxide and heat shocked by incubating the cells at 43°C for 60 min.

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2.3. Analysis of hsp70 mRNA Total RNA was isolated from AMs using the method previously described (Chomczynski and Sacchi, 1987), and the hsp70 mRNA levels in the RNA preparations were determined by Northern blot hybridization as described previously (Pahlavani et al., 1995). A total of 5–10 mg of total RNA was fractionated on a 1.2% agarose-formaldehyde gel and transferred to a nylon membrane using 10× SSC (0.15 M sodium chloride, 0.015 M sodium citrate). The relative levels of hsp70 mRNA in the RNA preparations were determined using a full length cDNA to rat hsp70, which was a gift from Dr F. Sharp, (Veterans Administration Hospital, San Francisco), as was described previously (Melton et al., 1984). After analyzing the membranes with the hsp70 probe, the membranes were subsequently hybridized to an oligomer directed against 18S rRNA (sequence:5%- GCCGTGCGTACTTAGACATGCATG-3%, synthesized by Genosys Woodlands, TX). The intensity of hybridization of the radioactive probes to hsp70 mRNA and 18S rRNA was measured using a Molecular Dynamics PhosphorImager, and hsp70 mRNA levels were expressed as the ratio of hsp70 mRNA to 18S rRNA.

2.4. Statistical analysis Data were analyzed by a Macintosh II computer using the Statview II statistical software package. One-way analysis of variance (ANOVA) was used to compare the mean response between groups. Data which appeared statistically significant by ANOVA were subsequently analyzed by Scheffe´ t-test to compare the means of multiple groups and were considered significant if PB 0.05.

3. Results The data on the effect of age and DR on the total cell number and percent macrophages recovered from bronchoalveolar lavage are presented in Table 1. Table 1 Effect of age and dietary restriction on the number and percentage of macrophages obtained from bronchoalveolar lavage Group

n

Total cells (×106) (x 9S.E.M.)

Percentage macrophages (x 9S.E.M.)

Young AL DR

17 11

8.429 0.60 6.15 9 0.46*

95.6 9 0.55 97.4 90.36

Old AL DR

17 15

7.72 90.70 6.27 90.41

94.9 90.62 95.1 9 1.05

* Significantly different from young AL rats, PB0.05.

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Fig. 1. Expression of hsp70 mRNA in AMs from F344 rats. AMs isolated from three young rats (4 – 6 months) were pooled and maintained in either suspension or adherent cultures. AMs were either incubated at 37°C (non-heat shock) for 2 h or heat shocked (43°C for 1 h) followed by incubation at 37°C for 1 h. The hsp70 mRNA levels were determined by Northern blot hybridization as described in Section 2, and a representative Northern blot is shown.

Although the total number of cells recovered from rats fed AL decreased slightly with age, this difference was not statistically significant. We also observed that the total number of cells obtained from the lungs of DR rats was lower compared to rats fed AL; this difference was statistically significant for young rats. There was no difference with age or DR in the percentage of macrophages, neutrophils and lymphocytes recovered (data not shown). Macrophages represented the majority (\ 94%) of the cells recovered from the lungs of the rats, and viability of the cells was greater than 95% in all groups studied (data not shown). The expression of hsp70 in AMs was measured under two culture conditions: in suspension and after adherence to plastic. In suspension, the AMs are in a resting, inactive state. In contrast, the AMs are primed to become activated when they adhere to the plastic surface of the culture plates (Friedman and Beller, 1987; Sporn et al., 1990). Fig. 1 shows that hsp70 mRNA was not detectable in AMs maintained in suspension at 37°C. In contrast, AMs that adhere to culture plates showed substantial levels of hsp70 mRNA when incubated at 37°C for 2 h (Fig. 1). Fig. 2A shows the time course for the induction of hsp70 mRNA in AMs after adherence to plastic. The levels of hsp70 mRNA were rapidly increased and were maximal 2 h after the cells were plated on the plastic culture plates. Therefore, hsp70 expression was measured in adherent AMs 2 h after the cells had been plated onto plastic culture plates in all subsequent studies. Clerget and Polla (1989) previously showed that the activation of human monocytes/macrophages induced hsp70 expression. Exposure of AMs to a brief heat shock (43°C for 1 h) resulted in a rapid induction of hsp70 mRNA levels in AMs in both suspension and adherent cultures as shown in Figs. 1 and 2B. The induction of hsp70 was maximum 60 min after heat shock for both adherent cells and cells maintained in suspension; however, the magnitude of induction of hsp70 was consistently higher for the adherent cells.

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Fig. 3 shows the effect of age and dietary restriction on the induction of hsp70 mRNA levels by heat shock in AMs in suspension cultures. The induction of hsp70 mRNA in AMs from young rats was similar to that observed in AMs from old rats. Although the induction of hsp70 was increased slightly in cells from old DR rats compared to old rats fed AL, the increase was not statistically significant. Fig. 4A shows the effect of age and dietary restriction on the induction of hsp70 mRNA levels 2 h after AMs adhere to plastic. The induction of hsp70 mRNA levels by adherence was approximately 70% lower for AMs isolated from old rats fed AL compared to AMs isolated from young rats fed AL. Interestingly, AMs isolated from young DR rats showed significantly higher (\ 150%) induction of hsp70 mRNA than AMs isolated from young rats fed AL. We also observed greater induction of hsp70 mRNA in the old, DR rats; however, this increase was not statistically significant. No difference was observed to occur in either the amount of total RNA isolated or changes in the cell viability with age or DR that could account for the changes in hsp70 mRNA levels. Fig. 4B shows the effect of aging and dietary restriction on the induction of hsp70 mRNA levels by heat shock in AMs that had adhered to tissue culture dishes. An age related decrease in the induction of hsp70 mRNA levels of

Fig. 2. The time course of hsp70 induction in AMs by adherence (A) or by heat shock (B). The AMs were isolated from three young rats (4 – 6 months), pooled, plated on plastic culture dishes, and incubated at 37°C (graph A). At various times, cells were harvested, and the hsp70 mRNA was determined by Northern blot hybridization. The data in graph B show the time course for the induction of hsp70 expression by heat shock. AMs were pooled from five young rats (4 – 6 months) and cultured in suspension (open circles) or allowed to adhere to plastic culture plates (closed circles) for 2 h. Cells were heat shocked (43°C for 1 h) and incubated at 37°C for various times. Total RNA was extracted from the cells, and the hsp70 mRNA and 18S rRNA levels were determined by Northern blot hybridization as described in Section 2. The hsp70 mRNA levels are expressed as the ratio of hsp70 mRNA to 18S rRNA.

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Fig. 3. Effect of age and dietary restriction on the induction of hsp70 expression by heat shock in suspensions of AMs. AMs were isolated from young (4 – 6 months) and old (24 – 26 months) rats fed either AL or a restricted diet (DR) as described in Section 2. Cells were heat shocked (43°C, 1 h) and incubated at 37°C for 1 h. Total RNA was isolated from the cells and analyzed by Northern blot hybridization as described in Section 2. The hsp70 mRNA levels are expressed as the ratio of hsp70 mRNA to 18S rRNA. Mean 9S.E.; n =6.

approximately 60% was observed in AMs isolated from rats fed both AL and a restricted diet. Dietary restriction had a dramatic effect on hsp70 induction by heat shock in AMs that had adhered to plastic. Hsp70 mRNA levels were threeto four-fold higher in AMs obtained from either young or old rats fed a restricted diet compared to young and old rats fed AL. Interestingly, the level of hsp70 expression after heat shock was similar for the young rats fed AL and the old DR rats.

4. Discussion The immune system represents the major first-line of defense in protecting organisms against a variety of environmental insults, e.g. pathogenic microorganisms and neoplasia. The protective action of the immune response requires interaction between a variety of cell types, e.g. T cells, B cells, natural killer cells, and macrophages. Although macrophages are not antigen specific, they play a critical role in the regulation of the immune response. For example, they ingest and process antigenic particles and display the resulting fragments in a form that is stimulatory for T cells, and during the process of phagocytosis and activation, macrophages release a variety of cytokines that are necessary for the initial activation and proliferation of T cells. In addition, macrophages produce more than 75 substances that have anti-microbial and tumoricidal activities (Nathan and Cohn, 1985). Thus, alterations in macrophage function could have profound effects on the immune response (De la Fuente, 1985).

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One of the characteristic features of senescence in mammals is the age-related decline in the immune system (reviewed in Thoman and Weigle, 1989; Murasko and Goonewardene, 1990; Miller, 1991; Pahlavani and Richardson, 1996), and it has been argued that this decline plays a major role in the dramatic increase in morbidity and mortality that is observed with increasing age (Walford, 1974; Makinodan and Kay, 1980; Inamizu et al., 1985; Effros and Walford, 1987). For example, respiratory infections such as influenza and pneumonia increase dramatically with age and are major causes of death in the elderly (Inamizu et al., 1985). Because of the potential importance of the immune system in aging, a great deal of research has focused on the mechanisms responsible for the age-related decline in immune function. Research with both humans and rodents demonstrates that aging affects a variety of immune functions (Thoman and Weigle, 1989; Murasko and Goonewardene, 1990; Miller, 1991; Pahlavani and Richardson, 1996), and it appears that T cell functions are particularly sensitive to aging (Miller, 1991; Pahlavani and Richardson, 1996).

Fig. 4. Effect of age and dietary restriction on the induction of hsp70 expression by adherence (A) and by heat shock (B) in AMs. AMs were isolated from young (4 – 6 months) and old (24 – 26 months) rats fed either AL or a restricted diet (DR) as described in Section 2. Cells were incubated at 37°C for 2 h on plastic culture dishes, and total RNA was isolated from the cells and analyzed by Northern blot hybridization (graph A). Mean 9 S.E.; n =5 for Y-AL; n =4 for Y-DR; n = 5 for O-AL; n = 4 for O-DR. a Significantly different from young rats, PB 0.05. b Significantly different from young rats fed AL, PB 0.05. The data in graph B shows the effect of age and dietary restriction on the induction of hsp70 expression by heat shock in cultures of adherent AMs. Cells were incubated at 37°C for 2 h on plastic culture dishes. Cells were then heat shocked (43°C, 1 h) followed by incubation at 37°C for an additional hour. Total RNA was isolated from the cells and analyzed by Northern blot hybridization as described in Section 2. The hsp70 mRNA levels are expressed as the ratio of hsp70 mRNA to 18S rRNA. Mean 9 S.E.; n=4 for Y-AL; n = 3 for Y-DR; n =3 for O-AL; n =4 for O-DR. * Significantly different from young rats, P B0.05. ** Significantly different from rats fed AL, PB 0.001.

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Although the effect of aging on T and B cell function has been studied extensively, the information on how aging affects macrophage function is limited and inconsistent. For example, the production of interleukin-1, which is required for the initial stimulation of T cells, has been reported to decrease (Effros and Walford, 1987) or not change (Kuffman, 1986) with age in peritoneal macrophages from rodents. De la Fuente (1985) has reported that phagocytosis (as measured by the ingestion of iron particles) decreased significantly with age in peritoneal macrophages from mice. However, Gottesman et al. (1985) found no difference in the ability of monocytes/macrophages from old and young human donors to support the mixed lymphocytic response or in presenting the soluble antigen to mouse T cells (Perkins et al., 1982). Similarly, Goodwin and Messner (1979) found no age-related difference in the ability of monocytes/macrophages from human donors to produce prostaglandins, e.g. PGE2. In contrast to peripheral monocyte/ macrophage function that does not seem to change with age, several functions of the AMs have been reported to decrease with age. For example, AMs from aged mice have been shown to have a decreased capacity to kill microorganisms and to produce the respiratory burst (Berton and Gordon, 1983; Ganguly et al., 1984; Hayakawa et al., 1985; Yokota et al., 1988; Davila et al., 1990; Lavie et al., 1992). In addition, the production of tumor necrosis factor-a and interleukin-1 by AMs has been reported to decrease with age (Effros et al., 1991). Although it has been argued that peripheral monocyte/macrophage function in general is preserved with age (Yehuda and Weksler, 1992), studies suggest that alveolar macrophage functions might be altered with age (Higashimoto et al., 1993). Because macrophages play a critical role in the immune defense system and because it is unclear if aging alters macrophage function, we studied the effect of age and life-long DR on the ability of AMs to express the heat shock protein, hsp70. We chose to study hsp70 expression because it appears that an age-related decline in the ability of cells to express hsp70 in response to stress is a universal phenomenon. For example, investigators over the past decade have consistently shown that the induction of hsp70 by heat shock is reduced with age and that this occurs in a variety of tissues and cell types (reviewed in Heydari et al., 1994). In addition, research from our laboratory has demonstrated that the age-related defect in the induction of hsp70 expression occurs at the level of transcription (Heydari et al., 1993; Pahlavani et al., 1995). Thus, we felt that it was highly probable that the induction of hsp70 expression by stress would be affected if macrophage function were compromised with age. When AMs were incubated at 37°C in suspension, no detectable hsp70 expression was observed. However, hsp70 mRNA levels were rapidly induced in response to heat shock (Fig. 1). The expression of heat shock genes, including hsp70, is regulated at the transcriptional and post-transcriptional levels. In our previous studies in hepatocytes (Heydari et al., 1993) and lymphocytes (Pahlavani et al., 1995) we observed an excellent correlation between the induction of hsp70 mRNA levels and the protein levels or hsp70 synthesis. This has also been observed by other investigators (Richardson and Holbrook, 1996). In this study, we found that the induction of hsp70 expression was similar in AMs isolated from young and old

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rats (Fig. 3). To our knowledge, this is the first report showing that the induction of hsp70 expression by heat shock does not decrease with age in a mammalian cell/tissue. This is in contrast to what we observed in other cells in the immune system. For example, a 50 – 70% decrease in the induction of hsp70 expression by heat shock was observed with age in resting T and B lymphocytes from male Fischer 344 rats and from resting peripheral blood lymphocytes from rhesus monkeys (Pahlavani et al., 1995). Thus, the ability of resting, non-activated AMs in suspension to respond to hyperthermia and express hsp70 does not change with age. However, we did observe age-related changes in hsp70 expression in adherent AMs. The expression of hsp70 was induced when AMs adhered to plastic culture dishes, and we found that the hsp70 mRNA levels induced by adherence were approximately 70% lower in AMs isolated from old rats compared to AMs isolated from young rats (Fig. 4). We also observed that the induction of hsp70 expression by heat shock decreased with age in adherent AMs. Therefore, our results strongly suggest that the activation of AMs might be affected by aging because hsp70 expression is compromised with age in adherent AMs and not in AMs in suspension. For example, macrophage activation occurs in a two-step process: priming, which is induced by adherence, and triggering, which is induced by bacterial lipopolysaccharide, interleukin-1 and other agents (Clerget and Polla, 1989; Kantengwa and Polla, 1991). Our data suggest that the priming step of macrophage activation might be altered by age. Interestingly, Lavie et al. (1992) reported that heat stress resulted in a diminished capacity of peritoneal macrophages (PM) from old mice to generate superoxide anions compared to PM from young rats. For example, heat shock (42.5°C for 20 min) was found to be the threshold temperature for irreversible loss of activity of PM function in old mice, whereas at this temperature, PM from young mice recovered up to 70% of their superoxide generating activity. The major observation of our study is that DR has a profound effect on hsp70 expression in adherent AMs. Research over the past two decades has shown that DR reduces/delays the incidence of most age-related diseases and alters a wide variety of physiological processes (Richardson, 1985; Masoro, 1990, 1992a,b). In addition, studies from our laboratory have shown that DR can alter gene expression at the level of transcription (Pahlavani et al., 1994). Walford’s laboratory initially demonstrated that DR altered immune function when it was shown that mitogen-induced lymphocyte proliferation was enhanced in DR mice (Walford, 1974). Subsequently, a variety of immunological parameters have been shown to be altered by DR (Windruch et al., 1979; Weindruch et al., 1986). However, there is currently no information on the effect of life-long DR on macrophage function. We found that the levels of hsp70 mRNA induced by adherence were dramatically increased by DR in AMs isolated from young rats and that the induction of hsp70 expression by heat shock in adherent AMs from both young and old rats was increased three- to four-fold by DR. Dietary restriction had no effect on hsp70 expression in AMs in suspension (Fig. 3); therefore, DR appears to affect the priming function of AMs. We previously showed that the induction of hsp70 expression by heat shock in hepatocytes from Fischer 344 rats was enhanced by DR

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(Pahlavani et al., 1994); however, the increase in hsp70 expression observed in hepatocytes was modest (less than 60%) compared to the effect of DR on hsp70 expression in AMs. Interestingly, we recently observed that DR had no effect on the induction of hsp70 expression in spleen lymphocytes from Fischer 344 rats (Pahlavani et al., 1996). One surprising observation from our study was that DR had a dramatic effect on hsp70 expression in adherent AMs from young rats (Fig. 4). In other words, the effect of DR on AMs occurred within 3–4 months of the implementation of the DR. Thus, DR did not increase hsp70 expression by delaying the age-related decline in hsp70 expression. Rather, DR appears to alter the animal physiologically in such a way that the induction of hsp70 expression by AMs is enhanced soon after the implementation of DR. It would be of interest to determine if DR affects other functions of primed or activated AMs that would be associated with immune function, e.g. phagocytosis and antigen presentation. Based upon our observations with hsp70, we would predict that DR would enhance the priming or activation of AMs. These changes could be beneficial to an organism because of the important roles of macrophages in the immune defense system, especially in the lungs. However, activated macrophages also produce a variety of pro-inflammatory cytokines and reactive oxygen species that can be cytotoxic and damaging to cells (Friedman and Beller, 1987; Sporn et al., 1990). In summary, our study shows that the effect of aging on the induction of hsp70 by heat shock can vary depending upon the physiological status of the cell. In resting, non-activated AMs, the induction of hsp70 expression did not change with age; however, in adherent AMs, which are primed to be activated, hsp70 induction by heat shock decreased approximately 60% with age. Dietary restriction also had no effect on the induction of hsp70 expression in resting, non-activated AMs. However, the induction of hsp70 by heat shock in adherent AMs was dramatically increased (three- to four-fold) by DR in AMs from both young and old rats. Thus, our study shows for the first time that DR can alter the function of macrophages.

Acknowledgements This research was supported in part by grants from the National Institutes of Health (AG01548, AG00205, AG00677, and AG14088), the Office of Research and Development, Department of Veterans Affairs, and a grant from the Nathan Shock Center of Excellence in Basic Biology of Aging (PO3 AG13319). The excellent secretarial assistance of Erin K. Patterson is acknowledged.

References Angelidis, C.E., Lazaridis, I., Pagoulatos, G.N., 1991. Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance. Eur. J. Biochem. 199, 35 – 39.

S.A. Moore et al. / Mechanisms of Ageing and De6elopment 104 (1998) 59–73

71

Berton, G., Gordon, S., 1983. Superoxide release by peritoneal and bone marrow-derived macrophages. Modulation by adherence and cell activation. Immunology 49, 693 – 704. Blake, M.J., Tytell, M., Fargnoli, J., Holbrook, N.J., 1990. Discordant expression of heat shock protein mRNAs in tissues of heat-stressed rats. J. Biol. Chem. 265, 15275 – 15279. Blake, M.J., Fargnoli, J., Gershon, D., Holbrook, N.J., 1991. Concomitant decline in heat-induced hyperthermia and HSP70 mRNA expression in aged rats. Am. J. Physiol. 260, 663 – 667. Brody, J.A., Brock, D.B., 1985. Epidemiological and statistical characteristics of the United States elderly population. In: Finch, C.E., Schneider, E.L. (Eds.), Handbook of the Biology of Aging, Van Nostrand Reinhold, New York, pp. 546 – 574. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 157 – 161. Clerget, M., Polla, B.S., 1989. Erythrophagocytosis induces heat shock protein synthesis by human monocytes-macrophages. Proc. Natl. Acad. Sci. USA 87, 1081 – 1085. Davila, D.R., Edwards, C.K., Arkins, S., 1990. Interferon-g-induced priming for secretion of superoxide anion and tumor necrosis factor-a declines in macrophages from aged rats. FASEB J. 4, 2906 – 2911. Deguchi, Y., Kishimoto, S., 1989. Enhanced expression of the heat shock protein gene in peripheral blood mononuclear cells of patients with active systemic lupus erythematosus. FEBS Lett. 259, 158–160. De la Fuente, M., 1985. Changes in the macrophage function with aging. Comp. Biochem. Physiol. 81, 935–938. Effros, R.B., Walford, R.L., 1987. Infections and immunity. In: Goidl, E.A. (Ed.), Aging and the Immune Response. Dekker, New York, pp. 45 – 65. Effros, R.B., Svoboda, K., Walford, R.L., 1991. Influence of age and caloric restriction on macrophage IL-6 and TNF production. Lymphokine Cytokine Res. 10, 347 – 351. Faassen, A.E., O’Leary, J.J., Rodysill, K.J., Bergh, N., Hallgren, H.M., 1989. Diminished heat-shock protein synthesis following mitogen stimulation of lymphocytes from aged donors. Exp. Cell. Res. 183, 326–334. Fels, A.O., Cohn, A.Z., 1996. The alveolar macrophage. J. Appl. Physiol. 60, 353 – 369. Friedman, A., Beller, D.I., 1987. The effect of adherence on the in vitro induction of cytocidal activity by macrophages. Immunology 61, 469 – 474. Ganguly, R., Shamlin, P.B., Craig, C.P., 1984. Microbial activity and superoxide production by macrophages in aging rats. Allergy Immunol. 30, 225 – 229. Goodwin, J.S., Messner, R.P., 1979. Sensitivity of lymphocytes to prostaglandin E2 increases in subjects over age 70. J. Clin. Invest. 64, 434– 439. Gottesman, S.R., Walford, R.L., Thorbeche, G.J., 1985. Proliferative and cytotoxic immune functions in aging mice. III. Exogeneous interleukin-2 rich supernatant only partially restores allorectivity in vivo. Mech. Ageing Dev. 31, 103–113. Green, G.M., Kas, E.H., 1964. The role of the alveolar macrophage in the clearance of bacteria from the lung. J. Exp. Med. 119, 167–172. Hayakawa, H., Sato, A., Yagi, T., 1985. Superoxide generation by alveolar macrophages from aged rats: improvement by in vitro treatment with IFN-g. Mech. Ageing Dev. 80, 199 – 221. Heydari, A., Wu, B., Takahashi, R., Strong, R., Richardson, A., 1993. Expression of heat shock protein 70 is altered by age and diet at the level of transcription. Mol. Cell. Biol. 13, 2909 – 2918. Heydari, A.R., Takahashi, Y., Guttsman, A., You, S., Richardson, A., 1994. HSP70 and aging. Experientia 50, 1092–1098. Higashimoto, Y., Fukuchi, Y., Shimada, Y., Ishida, L., Ohata, M., Furuse, T., Shu, C., Teramoto, S., Matsuse, T., Sudo, E., Orimo, H., 1993. The effects of aging on the function of alveolar macrophages in mice. Mech. Ageing Dev. 69, 207 – 217. Inamizu, T., Chang, M., Makinodan, T., 1985. Influence of age on the production and regulation of interleukin-1 in mice. Immunology 55, 447 – 455. Johnston, R., Kucey, B.L., 1988. Competitive inhibition of hsp70 gene expression causes thermosensitivity. Science 242, 1551–1554. Kantengwa, S., Polla, B.S., 1991. Flavonoids, but not protein kinase C inhibitors, prevent stress protein synthesis during erythrophagocytosis. Biochem. Biophys. Res. Commun. 180, 308 – 314.

72

S.A. Moore et al. / Mechanisms of Ageing and De6elopment 104 (1998) 59–73

Kuffman, C.A., 1986. Endogeneous pyrogen/interleukin-1 production in aged rats. Exp. Gerontol. 21, 75–81. Lavie, L., Weinreb, O., Gershon, D., 1992. Age-related alterations in superoxide anion generation in mouse peritoneal macrophages studied by repeated stimulations and heat shock treatment. J. Cell. Physiol. 152, 382–388. Li, G.G., Liu, Y.K., Mak, J.Y., Chen, L., Lee, W.M., 1991. Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene. Proc. Natl. Acad. Sci. USA 88, 1681–1685. Lindquist, S., 1986. The heat-shock response. Ann. Rev. Biochem. 55, 1151 – 1191. Lindquist, S., Craig, E.A., 1988. The heat-shock proteins. Ann. Rev. Genet. 22, 631 – 677. Makinodan, T., Kay, M.M.B., 1980. Age influence on the immune system. Adv. Immunol. 29, 287 – 298. Masoro, E.J., 1990. Assessment of nutritional components in prolongation of life and health by diet. Proc. Soc. Exp. Biol. Med. 193, 31–34. Masoro, E.J., 1992a. Retardation of aging processes by nutritional means. Ann. New York Acad. Sci. 673, 29–35. Masoro, E.J., 1992b. Potential role of the modulation of fuel use in the anti-aging action of dietary restriction. Ann. New York Acad. Sci. 663, 403 – 411. Melton, D.A., Kreig, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., Green, M.R., 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035 – 7056. Miller, R.A., 1991. Aging and immune function. Int. Rev. Cytol. 124, 187 – 215. Murasko, D.M., Goonewardene, I.M., 1990. T-cell function in aging: mechanisms of decline. Ann. Rev. Gerontol. 10, 71–96. Nathan, C.F., Cohn, Z.A., 1985. Cellular components of inflammation: monocytes and macrophages. In: Kelly, W., Harris, E., Reddy, S., Sledge, C.B. (Eds.), Textbook of Rheumatology, 2nd ed., vol. 1. Saunders, Philadelphia, pp. 144–169. Pahlavani, M.A., Richardson, A., 1996. Effect of age on the expression of interleukin-2. Mech. Ageing Dev. 89, 125–154. Pahlavani, M.A., Haley-Zitlin, V., Richardson, A., 1994. Influence of dietary restriction on gene expression: changes in the transcription of specific genes. In: Yu, B.P. (Ed.), Modulation of the Aging Process by Dietary Restriction. CRC Press, Boca Raton, FL, pp. 143 – 156. Pahlavani, M.A., Denny, M., Moore, S.A., Weindruch, R., Richardson, A., 1995. The expression of heat shock protein 70 decreases with age in lymphocytes from rats and rhesus monkeys. Exp. Cell Res. 218, 310–318. Pahlavani, M.A., Harris, M.D., Richardson, A., 1996. Expression of heat shock protein 70 in rat spleen lymphocytes is affected by age but not by food restriction. J. Nutr. 126, 2069 – 2075. Pardue, M.L., Ballinger, D.G., Hogan, N.C., 1992. The heat shock response. Cells coping with transient stress. Ann. New York Acad. Sci. 663, 125 – 138. Perkins, E.H., Massucci, J.M., Glover, P.L., 1982. Antigen presentation by peritoneal macrophages from young adult and old mice. Cell. Immunol. 70, 1 – 10. Riabowol, K.T., Mizzen, L.A., Welch, W.J., 1988. Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70. Science 242, 433 – 436. Richardson, A., 1985. The effect of age and nutrition on protein synthesis by cells and tissues from mammals. In: Watson R.R. (Ed.), Handbook of Nutrition and Aging. CRC Press, Boca Raton, FL, pp. 31–48. Richardson, A., Holbrook, N.J., 1996. Aging and the cellular response to stress: reduction in the heat shock response. In: Holbrook, N.J., Martin, G.R., Lockshine, R.A. (Eds.), Cellular Aging and Cell Death. Wiley-Liss, New York, pp. 67 – 80. Shimokawa, I., Higami, Y., Hubbard, G.B., McMahan, C.A., Masoro, E.J., Yu, B.P., 1993. Diet and the suitability of the male Fischer 344 rat as a model for aging research. J. Gerontol. 48, B27 – 32. Sporn, S.A., Eierman, D.F., Johnson, C.E., 1990. Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair. J. Immunol. 144, 4434–4443.

S.A. Moore et al. / Mechanisms of Ageing and De6elopment 104 (1998) 59–73

73

Sribvastava, P.K., Udono, H., Blachere, N.E., Li, Z., 1994. Heat shock proteins transfer peptides during antigen processing and CLT priming (review). Immunogenetics 39, 93 – 98. Thoman, M.L., Weigle, W.O., 1989. The cellular and subcellular bases of immunosenescence. Adv. Immunol. 46, 221–261. Van Remmen, H., Ward, W., Sabia, R.V., Richardson, A., 1995. Effect of age on gene expression and protein degradation. In: Masoro E.J., (Ed.), Handbook of Physiology, Volume on Aging. Oxford University Press, New York, pp. 171– 234. Walford, R.L., 1974. Immunologic theory of aging: current status. FASEB J. 33, 2020 – 2025. Weindruch, R., Walford, R.L., Figiel, S., Guthrie, D., 1986. The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. J. Nutr. 116, 641 – 648. Windruch, R., Kristie, J.A., Cheney, K.E., Walford, R.L., 1979. Influence of controlled dietary restriction on immunologic function and aging. Fed. Proc. 38, 2007 – 2016. Yehuda, A.B., Weksler, M.C., 1992. Immune senescence: mechanisms and clinical implications. Cancer Invest. 10, 535–541. Yokota, Y., Wakai, Y., Mine, Y., 1988. Degradation of host defenses against respiratory tract infection by Klebsiella pneumoniae in aged mice. Infect. Immun. 56, 966 – 971. Young, D.B., 1992. Heat shock proteins: immunity and autoimmunity (review). Curr. Opin. Immunol. 4, 396–400. Yu, B.P., Masoro, E.J., McMahan, C.A., 1985. Nutritional influences on aging of Fischer 344 rats: I. Physical, metabolic, and longevity characteristics. J. Gerontol. 40, 657 – 670.

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