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a Center for Occupational and Environmental Health, Department of Community and Environmental Medicine, University of California, Irvine
Stephen C. Bondy, Department of Community & Environmental Medicine, University of California, Irvine, Irvine, CA 92697-1825 E-mail: scbondy{at}uci.edu.
Decision Editor: John A. Faulkner, PhD
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Abstract |
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INTERLEUKIN-6 (IL-6) is a multifunctional cytokine important in homeostasis and as an early marker of acute inflammatory response (1). Its level has been shown to increase in the serum during acute traumatic injury, and this level is positively correlated with the severity of tissue damage (2).
During the aging process, a compromise of the defense mechanism of cells may lead to the accumulation of exogenous and endogenous stress factors that may underlie an increased basal inflammatory response. Indeed, a substantial increase in IL-6 levels in peripheral blood mononuclear cells in elderly subjects has been documented. This is strongly correlated (3) with a parallel increase in levels of the systemic inflammatory marker C-reactive protein. Chronic production of cytokines such as IL-6 can result in cytotoxicity because they recruit and activate macrophages that produce high concentrations of reactive oxygen species (4). Thus, a chronically elevated basal level of inflammatory factors may be deleterious to an organism. This may account for the finding that, despite elevated monocyte function and greater production of cytokines such as IL-6, the older population is nevertheless more susceptible than the young population to viral and bacterial infections (5). An age-related increase in IL-6 has also been correlated with an enhanced potential for future disability in the older population (6).
Not only is the level of IL-6 increased in the aged population, but there is also an alteration in the level of regulatory proteins for this cytokine. It has been demonstrated that the level of plasma IL-6 soluble receptor is significantly lower in aged adults than it is in young adults (7). In contrast to previous studies that demonstrated an increase in IL-6 level in older subjects, Delpedro and his colleagues (8) found a decrease in IL-6 production by lipopolysaccharide-activated monocytes derived from subjects 65 years and older compared with the monocytes purified from 25-year-old donors. There is then some inconsistency as to whether mononuclear cells derived from elderly subjects are hyperactive or rather show a diminished response compared with those derived from the young. However, it is clear that the aging process does alter the responsiveness of blood-derived monocytes, and that changes in the production of cytokines and their receptors may reflect an altered immune response associated with senescence.
Whereas most studies have focused on assessing the level of IL-6 in plasma or the secretion of the cytokine by blood-derived leukocytes, the main purpose of the present study was to determine basal levels and effects of lipopolysaccharide (LPS) stimulation on levels of IL-6 mRNA within several organ tissues. The expression of this mRNA in the lung, liver, spleen, and kidney was quantitated in younger (7 months) and old (23 months) mice with and without stimulation by LPS. There was a major increase in all of the tissues of old animals upon activation. However, the younger animals displayed more variable responses to LPS, generally of a lower magnitude. It is possible that although organ-derived IL-6 may play a fundamental immune-related role in aged animals, in younger animals there is a greater range of functions involving this cytokine.
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Methods |
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Animal Treatment
Male B/6C3F1 mice, a hybrid between C57BL/6 mice and C3H mice from Harlan Labs (Indianapolis, IN), aged 7 or 23 months, were housed four per cage and were maintained on a 12 hour light–12 hour dark cycle in a temperature controlled (20 ± 1°C) room. Food and water were provided ad lib. Half of the mice were injected intravenously (infraorbitally, as recommended by Waynforth and Flecknell; (9)) with 100 µl of LPS (0.1 mg/ml dissolved in sterilized normal saline) 2 hours before sacrifice by cervical dislocation; naïve mice served as controls. The basis of selecting the 2-hour time point was to ensure that the acute early-phase response to LPS was monitored. Most groups consisted of three animals; results reported with fewer animals in a group were due to animal mortality prior to the conclusion of the experiment.
RNA Extraction
Tissues were excised quickly, blotted on absorbant paper to remove blood, and immediately placed in liquid nitrogen and stored at -70°C before processing. Total RNA was extracted by using the TRI REAGENT Kit obtained from Molecular Research Center, Inc. (Cincinnati, OH). RNA concentrations were determined by measuring absorption at a 260-nm wavelength.
Preparation of cDNA Probe
A rat IL-6 cDNA-containing plasmid (gift from Drs. Wolfgang Northemann and Georg Fey, Scripps Research Foundation, La Jolla, CA) was transformed into JM101 cells and then digested with PstI and BamHI restriction enzymes to obtain the insert for hybridization.
Dot Blot Analysis
Five-microgram aliquots of total RNA were denatured with Northern gel loading buffer (2 mM ethylenediamine tetra-acetic acid, 60% glycerol, 0.5% Bromphenol Blue, and 0.5% xylene cyanol). Dot blotting was performed with the Bio-Dot SF apparatus (Bio-Rad Laboratories, Hercules, CA). Zeta-Probe membranes (Bio-Rad Laboratories) were then hybridized with one of the cDNA probes labeled with [32P]dCTP to a specific activity of approximately 109 cpm/µg by using the RTS Radprime System (Life Technologies, Gaithersburg, MD). The membranes were autoradiographed at -70°C for periods varying from 8 hours to 7 days, using x-ray film (X-OMAT AR, Kodak, Rochester, NY); blots are displayed in Fig. 1. A densitometer (Eagle Eye, Stratagene, San Diego, CA) was used to quantify the bands in arbitrary units that correspond to the relative area-log pixel-density product under each blot. Values were normalized by using mRNA levels of a housekeeper gene (GAPDH) as the baseline.
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Discussion |
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The aging process is associated with a higher responsivity of IL-6 mRNA to exogenous inflammatory stimuli. The intracellular level of T-cell IL-6, stimulated with phorbol 12-myristate 13-acetate, is significantly higher in aged donors than in younger blood donors (12). In the present study, injection of LPS in the 23-month-old mice produced a significant increase in IL-6 mRNA expression in all of the tissues analyzed. In contrast, the LPS-stimulated level of IL-6 mRNA expression varied in the 7-month-old mice with the specific tissue analyzed. To ensure that the changes in IL-6 levels are an acute early-phase response, the animals were sacrificed after 2 hours of exposure. It is possible that if the animals are exposed for longer periods of time, the modulation of IL-6 mRNA may change. Thus, over time, the expression of the cytokine may continue to increase in the younger animals whereas in the older mice it may start to decline.
In an interpretation of whole-organ cytokine mRNA expression, the possible contribution from residual blood cells should be minimized. However, prevention of mRNA degradation is paramount. Thus we did not attempt to perfuse tissue to remove blood cells; rather, we excised the tissue quickly and froze it immediately, blotting the tissue prior to freezing to minimize residual blood. Consequently, contributions to the measured IL-6 expression levels that are due to blood cells' remaining in tissue vasculature should be small.
The present study has focused on the genetic regulation of IL-6 mRNA expression and does not measure tissue levels of the cytokine directly. A future study will augment the results described here by a parallel assay of levels of IL-6 protein.
Although IL-6 may play a role in normal physiological maintenance of different organ systems, this cytokine is also an early marker of an acute inflammatory response (2). Whereas organ system-derived IL-6 may play a fundamentally immune-related role in the aged animals, it is possible that this is not the case in the younger animals and LPS activation might actually in some cases disrupt the basal expression of the cytokine necessary for homeostasis. Alternatively, younger animals may be able to protect themselves against excessive inflammatory responses by restricting the levels of the cytokine mRNA expression, and this control may be missing in the older animals. Thus, although the level of IL-6 mRNA expression in the 7-month-old animals modestly increases in the lung and actually declines in the kidney, the tissues of aged animals are all equally hyperresponsive, and, upon LPS stimulation, all of the tissues analyzed in the 23-month-old mice expressed a manyfold increase in levels of IL-6 mRNA.
The tissue cytokine mRNA responses found here resemble those of cytokines in plasma. Following intracerebroventricular injection of LPS, 2-year-old rats respond with a greater elevation of circulating levels of IL-6 than do 3-month-old rats (13). Similarly, the response of IL-6 and other cytokines to intraperitoneally injected LPS is greater in aged than in young mice (14). An augmented reactivity of periodontal cells, aged by repeated cycles of division, to LPS induction of IL-6 and other cytokines has also been reported (15). This increased reactivity of older cells to immunogenic stimuli may account for the greater susceptibility of old animals to LPS-induced lethality (16). However, such enhanced tissue susceptibility may be concurrent with an age-related depression of effective reactivity in tissues specifically responsible for immune activation, such as the spleen (17). This raises the possibility that the augmented changes found in aged animals are in fact inappropriately targeted and do not represent a physiologically effective response. Recent reports indicate that Toll-like receptor 4 (Tlr4) and the molecule CD14 are important mediators of LPS-invoked innate immunity (18)(19)(20). The increased response of the older animals to LPS may be the result of upregulation of CD14 or Tlr4 on the cell surface.
Although the exact mechanisms by which such age-dependent changes in inflammatory responses occur are unknown, the acceleration of processes leading to these age-associated alterations in cytokine profiles may play a role in the onset and progression of chronic age-related inflammatory diseases such as rheumatoid arthritis. Understanding the mechanisms underlying these age-related changes can allow the development of dietary regimens and pharmaceutical interventions that may ameliorate the dysregulation of the immune response and decrease the rate of progression of age-related degenerative diseases.
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Acknowledgments |
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Received May 21, 2001
Accepted August 6, 2001
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This article has been cited by other articles:
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  D. Harding, S. Dhamrait, A. Millar, S. Humphries, N. Marlow, A. Whitelaw, and H. Montgomery Is Interleukin-6 -174 Genotype Associated With the Development of Septicemia in Preterm Infants? Pediatrics, October 1, 2003; 112(4): 800 - 803. [Abstract] [Full Text] [PDF]
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value differs from that of corresponding younger mice (p < .02). GAPDH = glyceraldehyde phosphate dehydrogenase.