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Aging enhances lymphocyte cytokine defects after injury¹

TIMOTHY P. PLACKETT^*,, ERIC M. SCHILLING^*,, DOUGLAS E. FAUNCE^,,, MASHKOOR A. CHOUDHRY^,,, PAMELA L. WITTE^*, and ELIZABETH J. KOVACS^*,,,,2

Departments of
^* Cell Biology, Neurobiology, and Anatomy and
Surgery,
Immunology and Aging Program, and
Burn and Shock Trauma Institute, Loyola University Chicago, Maywood, Illinois, USA

²Correspondence: Departments of Cell Biology, Neurobiology, and Anatomy and Surgery, Loyola University Chicago, Stritch School of Medicine, Building 110, Room 4221, 2160 South 1st Avenue, Maywood, IL 60513, USA. E-mail: ekovacs{at}lumc.edu

SPECIFIC AIMS

Mortality and sepsis after traumatic injury are greater in the elderly than in young individuals, and the altered lymphocyte response that occurs in healthy individuals is proposed to be a contributing factor. The specific aim of the current study was to compare the delayed-type hypersensitivity (DTH) response and splenocyte production of cytokines by young and aged mice subjected to sham and injury, given that we previously showed that aged, injured mice were less likely to survive after a 15% total body surface area burn and were more immunosuppressed.

PRINCIPAL FINDINGS

1. The DTH of aged mice is significantly lower following injury
To determine if there are differences in in vivo cell-mediated immune responses with advancing age and if the changes are more pronounced with injury, the DTH response to 2,4-dinitrofluorobenzene was measured. Although young mice were able to mount a normal DTH response regardless of injury, aged, sham-injured mice experienced a decrease in DTH (36%) relative to both groups of young animals. Following injury, aged mice had an 80% decrease in the DTH response compared with all other groups (P<0.01).

2. The production of T helper cell type 2 (TH2) cytokines is elevated with advancing age and is augmented by injury
To further examine the role of aging on the immune response after burn injury, we examined the ex vivo production of TH2 cytokines. Splenocytes were obtained from mice 24 h after injury and stimulated overnight with concanavalin A (ConA). The supernatants were then assayed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-4 and IL-10. With advancing age, IL-4 and IL-10 production increased dramatically. IL-4 was twice as high with age, and IL-10 rose from below the detectable limit in young mice to 3 times the limit in aged mice (P<0.01). Following injury, IL-10 continued to be undetectable in the supernatants of young, injured mice (Fig. 1 ). However, in aged mice, IL-10 levels decreased significantly after injury but continued to remain elevated in comparison with young mice (P<0.01). IL-4 levels decreased following injury, regardless of age (P<0.05), but there was no difference between young, injured and aged, injured animals.

View larger version (12K): [in this window] [in a new window]   Figure 1. Increased production of IL-10 with age. Splenocytes were harvested 24 h post-injury and cultured in the presence of ConA (2 µg/ml) for 18 h. IL-10 was assayed by ELISA. Data were combined from three independent experiments and are shown as mean IL-10 levels ± SE; n = 4–11 young and 11–14 aged mice per group. *, P < 0.01 from both young groups; #, P < 0.01 from aged, sham mice, determined by ANOVA with Newman-Keuls post-hoc analysis.

3. The production of TH1 cytokines is decreased after injury
As a corollary to the altered TH2 cytokine profile, the TH1 cytokine profile was also examined. Splenocyte supernatants were assayed for IL-2, IL-12, and interferon- (IFN-) following stimulation of the cells with ConA. IL-2 production was 25% lower in the aged, sham animals than in the young, sham animals (P<0.01). After injury, production of IL-2 was lower in the young and aged, injured mice (38% and 68%, respectively), but this decrease was more pronounced in the aged, injured mice (P<0.01). There was no difference between the young and aged, sham-injured animals with respect to splenocyte production of IFN- or IL-12. As with IL-2, IFN- was decreased after injury, regardless of age (P<0.01), and this decrease was greatest for the aged, burn-injured animals (11,584±1847 pg/ml vs. 4374±955 pg/ml) (Fig. 2 ). However, although after the burn, IL-12 was decreased by over 55% for the young and aged, injured animals in comparison with either sham animal (P<0.01), there were no differences in the production of IL-12 between young or aged, burn-injured animals.

View larger version (15K): [in this window] [in a new window]   Figure 2. Decreased IFN- production with injury. Splenocytes were harvested 24 h post-injury and cultured in the presence of ConA (2 µg/ml) for 18 h. IFN- was assayed by ELISA. Data were combined from five independent experiments and are shown as mean IFN- levels ± SE; n = 14–18 young and 13–16 aged mice per group. *, P < 0.01 from both sham groups; #, P < 0.01 from young, burn mice, determined by ANOVA with Newman-Keuls post-hoc analysis.

CONCLUSIONS

Our findings demonstrate that there is a suppression of the DTH response in aged mice and that this age-related immunosuppression is worse after a traumatic injury. The suppressed DTH response is accompanied by alterations in the delicate balance between the TH1 and TH2 phenotype. With advancing age, there is an increase in the production of TH2 cytokines, in particular IL-4 and IL-10, after stimulation of the splenic lymphocytes (Fig. 3 ). In addition, there is a decrease in the TH1 cytokine IL-2. After burn injury, lymphocytes from young and aged animals produce decreased levels of TH1 and TH2 cytokines. In aged, injured animals, the decrease in TH1 cytokines was much greater than what occurred to young, injured animals. This switch to an increase in the TH2 phenotype in aged, injured mice may be a cause of the decreased survival in this group relative to others. Further studies are needed to determine whether the alteration in the age- and injury-related TH phenotype is the direct result of the increased TH2 phenotype in aged, sham-injured animals relative to their younger counterparts. Studies are also needed to determine if fixing the TH1:TH2 balance restores immunity to the aged, injured mice.

View larger version (13K): [in this window] [in a new window]   Figure 3. Summary of the immune response following burn injury in young and aged mice. Young, injured mice experience an equal decrease in TH1 and TH2 cytokines. Aged, injured mice experience a disproportionate decrease in TH1 cytokines, which may contribute to an inadequate inflammatory response and subsequent sepsis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0452fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0452fje

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