Douglas E. Faunce

Aging and innate immune cells

The innate immune system serves an important role in preventing microbial invasion. However, it experiences significant changes with advancing age. Among the age‐associated changes are: Aged macrophages and neutrophils have impaired respiratory burst and reactive nitrogen intermediates as a result of altered intracellular signaling, rendering them less able to destroy bacteria. Aged neutrophils are also less able to respond to rescue from apoptosis. Aged dendritic cells (DC) are less able to stimulate T and B cells. The altered T cell stimulation is a result of changes in human leukocyte antigen expression and cytokine production, and lower B cell stimulation is a result of changes in DC immune complex binding. Natural killer (NK) cells from the elderly are less capable of destroying tumor cells. NK T cells increase in number and have greater interleukin‐4 production with age. Levels of various complement components are also altered with advancing age.

Innate immunity and aging

Advanced age is associated with defects in all of the cells of the innate immune system, including numbers, function, and early stages of activation. This review, presents the current state of the field on the impact of age on the innate immune system. The analysis of the literature suggests that a dysfunctional innate immune system is a contributing factor to aberrant outcomes after injury or infection and to the development of many of the diseases observed in the elderly. Gaining an understanding of the nature of the defects in innate immune cells may allow the development of therapeutic strategies aimed to restore innate immune function in aged individuals.

Age‐dependent decrease in Toll‐like receptor 4‐mediated proinflammatory cytokine production and mitogen‐activated protein kinase expression

Age‐related changes in immunity render elderly individuals more susceptible to infections than the young. Previous work by our laboratory and others showed that macrophages from aged mice are functionally impaired. Macrophages produce proinflammatory cytokines, tumor necrosis factor α (TNF‐α) and interleukin (IL)‐6, when stimulated with lipopolysaccharide (LPS), which signals through Toll‐like receptor‐4 (TLR4) and requires activation of mitogen‐activated protein kinases (MAPKs). We investigated whether aging is associated with alterations in TNF‐α and IL‐6 production and MAPK expression and activation in thioglycollate‐elicited peritoneal macrophages from mice. Kinetics and LPS dose‐responsiveness of macrophage TNF‐α production did not differ by age. Unstimulated macrophages did not differ by age in their cytokine production. However, LPS‐stimulated (100 ng/mL) cultures from aged mice produced 100 ± 30 pg/mL TNF‐α and 6000 ± 2000 pg/mL IL‐6, and those from young mice produced 280 ± 50 pg/mL and 10,650 ± 10 pg/mL, respectively (P<0.05). Likewise, levels of activated MAPKs did not differ by age in unstimulated macrophages, and LPS‐stimulated macrophages from aged mice had <70% activated p38 and c‐jun NH2‐terminal kinase (JNK) than those of young controls. Of particular interest, we observed >25% reduction of total p38 and JNK in macrophages from aged mice relative to young. In addition, surface TLR4 levels did not vary with age. We conclude that macrophages from aged mice exhibited suppressed proinflammatory cytokine production, which correlated with diminished total levels and LPS‐stimulated activation of p38 and JNK. These observations suggest that decreased MAPK expression could be a mechanism responsible for age‐related deterioration of the immune system.

Alcohol, injury, and cellular immunity

It is widely accepted that alcohol exposure is a causative factor in the occurrence of burn or other traumatic injury. It is less well known that individuals who have consumed alcohol before sustaining an injury suffer from increased morbidity and mortality compared with the morbidity and mortality of non-alcohol-consuming subjects with similar injuries. Complications due to bacterial infection are the most common burn sequelae in injured patients and are frequently associated with depressed immunity. Independently, alcohol exposure and injury have been shown to influence cellular immunity negatively. These changes in immunity are closely linked to injury- or alcohol-induced alterations in the cytokine milieu in both clinical studies and animal models. Not surprisingly, the combination of insult of alcohol exposure and burn injury results in immune suppression that is greater in magnitude and duration compared with either insult alone. The combined effects of alcohol and injury on immunity have been examined in a limited number of studies. However, results of these studies support the suggestion that altered cytokine production is an integral part of the immune dysregulation and increased mortality that is observed. In particular, the increased presence of macrophage-derived mediators observed after burn or alcohol exposure alone seems to be synergistically increased in a combined injury model. Although more research is needed, it is likely that therapeutic modalities that include manipulation of cytokine networks to boost cellular immunity may improve outcome for patients who sustain injuries subsequent to consuming alcohol.

Effects of acute ethanol exposure on cellular immune responses in a murine model of thermal injury.

To test the effects of acute ethanol exposure on immune function after thermal injury, mice with blood alcohol levels of 100 mg/dL were given a 15% total body surface area dorsal scald or sham injury. Bacterial challenge resulted in 100 and 40% mortality in burn + ethanol‐ and burn + vehicle‐treated mice, respectively. Delayed‐type hypersensitivity responses were also significantly suppressed in burn + ethanol‐treated mice. At 1 and 4 days post‐burn, concanavalin A (ConA) ‐induced total splenocyte proliferation in burn + ethanol‐treated groups was significantly decreased (P < 0.01) compared with burn + vehicle‐ or sham‐treated animals. This decrease was not observed in total splenocytes cultured with anti‐CD3ε or among adherence‐depleted splenocytes given ConA or anti‐CD3ε. FACS analyses revealed no changes in splenocyte sub‐type ratios in burn + ethanol mice. The data herein demonstrate that acute ethanol exposure before thermal injury results in enhanced susceptibility to bacterial infection and markedly suppressed cellular immunity, which appears to be macrophage dependent. J. Leukoc. Biol. 62: 733–740; 1997.

Acute Ethanol Exposure Prior To Thermal Injury Results In Decreased T-cell Responses Mediated In Part By Increased Production Of Il-6

Previous studies by our laboratory have demonstrated that acute ethanol exposure prior to thermal injury results in suppression of cellular immune responses when compared with thermal injury alone. Ethanol exposure and burn injury are independently known to result in elevated IL-6, a cytokine with potent immunosuppressive properties. Therefore, we examined the role of IL-6 in the immune dysfunction in mice following a 15% body surface area scald (or sham) injury combined with acute ethanol (or vehicle) treatment. At 24 h post-injury, we observed slightly suppressed splenocyte proliferative responses and elevated circulating IL-6 (149 ± 15 pg/mL) in mice receiving burn alone compared with those receiving sham injury (31 ± 7 pg/mL). In contrast, burn + ethanol treated mice showed a profound suppression of splenocyte proliferation (20% of control) and significantly elevated circulating IL-6 levels (738 ± 218 pg/mL). The suppressed splenocyte proliferative response was found to be macrophage dependent. Furthermore, IL-6 production was significantly elevated (p < .05) in splenic macrophage cultures from burn + ethanol mice (159 ± 6 pg/mL) when compared with burn alone (109 ± 10 pg/mL). Treatment of the splenocyte cultures from burn + ethanol mice with an anti-IL6 monoclonal antibody resulted in partial restoration of splenocyte proliferation. Taken together, these data strongly suggest that the immune dysfunction observed in ethanol-exposed, thermally injured mice is mediated in part by elevated levels of IL-6.

Neutrophil chemokine production in the skin following scald injury

The present study was conducted to determine whether local production of neutrophil chemoattractant cytokines preceded the influx of neutrophils following dermal scald injury. To accomplish this, dermal tissue was examined for inflammatory infiltrate and the level of KC, a murine homolog of human interleukin-8, at various time points after scald injury. The studies reveal that there was a largely neutrophilic infiltrate at 1 day post-injury which persisted for 4 days. Dermal KC levels increased significantly at 4 h, returned to baseline at 8 h and were elevated again from 1 to 3 days post-burn (P < 0.01). At 3 days post-burn, KC was elevated 15-fold above the level in sham treated mice (P < 0.01). These observations demonstrate that the influx of neutrophils into the skin follows the expression of KC in the skin. This suggests that it should be possible to alter neutrophil accumulation at the wound site by manipulating the local chemokine signal.

Gender difference in cell-mediated immunity after thermal injury is mediated, in part, by elevated levels of interleukin-6.

The gender difference in normal immune function has been well documented, however, there is only limited information regarding whether such a difference occurs after injury. To investigate this, we examined cell‐mediated immune responses in male and female mice given a 15% total body surface area dorsal scald or sham injury. Both delayed‐type hypersensitivity (DTH) and splenocyte proliferative responses were significantly suppressed in males at 1 day and in females at 7 and 10 days post burn (P < 0.01). The decreased splenocyte proliferation was found to be macrophagedependent and suppression of both immune parameters corresponded with elevated interleukin‐6 (IL‐6) levels. Furthermore, post‐burn treatment with an anti‐IL‐6 antibody partially restored the DTH response in males at 1 day and females at 10 days post injury and completely restored splenocyte proliferation. These data demonstrate a possible mechanism for the gender difference in cell‐mediated immune responses after thermal injury. J. Leukoc. Biol. 67: 319–326; 2000.

CD1d-Restricted NKT Cells Contribute to the Age-Associated Decline of T Cell Immunity

NKT cells are known to regulate effector T cell immunity during tolerance, autoimmunity, and antitumor immunity. Whether age-related changes in NKT cell number or function occur remains unclear. Here, we investigated whether young vs aged (3 vs 22 mo old) mice had different numbers of CD1d-restricted NKT cells and whether activation of NKT cells by CD1d in vivo contributed to age-related suppression of T cell immunity. Flow cytometric analyses of spleen and LN cells revealed a 2- to 3-fold increase in the number of CD1d tetramer-positive NKT cells in aged mice. To determine whether NKT cells from aged mice differentially regulated T cell immunity, we first examined whether depletion of NK/NKT cells affected the proliferative capacity of splenic T cells. Compared with those from young mice, intact T cell preparations from aged mice had impaired proliferative responses whereas NK/NKT-depleted preparations did not. To examine the specific contribution of NKT cells to age-related T cell dysfunction, Ag-specific delayed-type hypersensitivity and T cell proliferation were examined in young vs aged mice given anti-CD1d mAb systemically. Compared with young mice, aged mice given control IgG exhibited impaired Ag-specific delayed-type hypersensitivity and T cell proliferation, which could be significantly prevented by systemic anti-CD1d mAb treatment. The age-related impairments in T cell immunity correlated with an increase in the production of the immunosuppressive cytokine IL-10 by splenocytes that was likewise prevented by anti-CD1d mAb treatment. Together, our results suggest that CD1d activation of NKT cells contributes to suppression of effector T cell immunity in aged mice.

An age‐associated increase in pulmonary inflammation after burn injury is abrogated by CXCR2 inhibition

Burn patients over the age of 60 are at a greater risk for developing pulmonary complications than younger patients. The mechanisms for this, however, have yet to be elucidated. The objective of this study was to determine whether increased chemoattraction plays a role in the age‐related differences in pulmonary inflammation after burn injury. At 6 or 24 h after receiving sham or 15% total body surface area scald injury, lungs from young and aged mice were analyzed for leukocyte content by histological examination and immunostaining. Lungs were then homogenized, and levels of neutrophil chemokines, MIP‐2 and KC, were measured. At 6 h after burn, the number of neutrophils was four times higher in the lungs of both burn groups compared with aged‐matched controls (P<0.05), but no age difference was evident. At 24 h, in contrast, neutrophils returned to sham levels in the lungs of young, burn‐injured mice (P<0.05) but did not change in the lungs of aged, burn‐injured mice. Pulmonary levels of the neutrophil chemokine KC but not MIP‐2 were consistently three times higher in aged, burn‐injured mice compared with young, burn‐injured mice at both time‐points analyzed. Administration with anti‐CXCR2 antibody completely abrogated the excessive pulmonary neutrophil content by 24 h (P<0.05), while not affecting the inflammatory response of the wounds. These studies show that CXCR2‐mediated chemoattraction is involved in the pulmonary inflammatory response after burn and suggest that aged individuals sustaining a burn injury may benefit from treatment strategies that target neutrophil chemokines.