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J Appl Physiol 100: 826-833, 2006. First published November 10, 2005; doi:10.1152/japplphysiol.00898.2005
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Influence of gender and age on T-cell responses in a murine model of trauma-hemorrhage: differences between circulating and tissue-fixed cells

Christian P. Schneider, Martin G. Schwacha, and Irshad H. Chaudry

Center for Surgical Research, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 25 July 2005 ; accepted in final form 8 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Clinical studies indicate that peripheral blood lymphocyte functions are depressed following trauma; however, it is unclear whether tissue-fixed lymphocyte functions are also altered under those conditions. Moreover, the impact of gender and age on peripheral T-cell responses following trauma-hemorrhage (TH) are unknown. To study this, immature (~3 wk of age), mature (~7 wk of age), and aged (~23 mo of age) male and proestrus female C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30 ± 5 mmHg for 90 min). Twenty-four hours after resuscitation, blood and splenocytes were harvested and T-cell functions assessed. In immature animals, TH induced an enhanced immune response in the splenic compartment and a suppressed response in the peripheral blood mononuclear cells (PBMC) that was independent of gender. Differential responses were observed in cells from mature mice. Splenic responses were enhanced following TH, independent of gender, whereas PBMC displayed gender dimorphism with suppressed proliferation and T-cell helper 1 responses in males but not in females. A similar pattern was observed in cells from aged mice. Splenic T cells from male mice displayed a suppressed CD4-to-CD8 ratio after TH, whereas no such change was observed in cells from proestrus females. In contrast, only PBMC from mature males displayed a suppressed CD4-to-CD8 ratio after TH. Thus gender differences exist in PBMC responses after TH that do not necessarily correlate with changes in the tissue-fixed compartment. Age is also an important factor in the immune responses after TH. In view of this, both gender and age should be taken into consideration in evaluating the immune status and in treatment of TH shock.

CD4; CD8; immunosuppression; peripheral blood mononuclear cells; spleen; T-cell helper 1/2

SEPSIS AND SUBSEQUENT multiple organ dysfunction syndrome (MODS) are the most common causes of death in surgical intensive-care units (4, 5, 29). Injury-induced anergy of the immune system has been postulated to be the major factor in the increased susceptibility of the trauma patient to serious infection and MODS (13, 25, 34, 44). Previous studies in patients (15, 32, 33, 49) and animal models (1) demonstrated that gender and age are crucial determinants in host immune responses following traumatic injury, shock, and/or infection. Although several clinical studies demonstrated that aging and male gender are associated with a higher risk of mortality after injury, an important limitation of these studies is that peripheral blood mononuclear cells (PBMC) were the only immune cell population analyzed (32, 33, 36, 49, 53, 54). It is unclear whether assessment of PBMC function under such conditions adequately reflected the overall immune status. In contrast, most animal studies of injury have primarily focused on tissue-fixed immune cells from various tissue compartments (i.e., spleen, liver, gut, etc.). In general, these studies have shown a suppression of T-cell function in the secondary lymphoid organs, such as the spleen (25, 46, 47), activation of hepatic and alveolar macrophages (3, 22, 37), and gut injury and cytokine generation (9, 28).

Aging causes a decrease in the number of naive T cells and production of T-cell cytokines with predominance toward a T-cell helper (Th) 2 phenotype (i.e., IL-4, IL-10) as opposed to a Th1 phenotype (i.e., IL-2, IFN-{gamma}) (27, 33). Moreover, Plackett et al. (38) suggested that this lack of an adequate amount of Th1 cytokines early after injury might correlate with the increased incidence of sepsis in aged animals. In addition to the effects of age, male rodents exhibit depressed immune responses and increased susceptibility to sepsis following trauma-hemorrhage, whereas proestrus females have maintained or enhanced immune responses under such conditions (1, 43). However, sexual dimorphic immune response of splenic T cells after traumatic injury appears to be reversed with aging (23). Other investigators have also proposed that a sexual dimorphism in the immune response in humans may exist before puberty (40).

A single clinical study, examining PBMC from pre- and postmenopausal women, indicated that cells from premenopausal women (which had threefold higher estrogen plasma levels) produced significantly greater amounts of TNF-{alpha} and IFN-{gamma} (50). Although it is known that tissue immune cells from animals demonstrate an age-dependent gender dimorphic immune response following trauma-hemorrhage, it is unclear whether or not PBMC responses under such conditions are similarly affected by age and gender. It is our hypothesis that gender and age differentially influence tissue-fixed and peripheral T-cell immune function following trauma-hemorrhage. The present study used a murine model of soft tissue trauma and hemorrhagic shock to test this hypothesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Inbred male and female C3H/HeN mice (Charles River Laboratories, Wilmington, MA), 21–27 days (8–14 g body wt), 6–8 wk (23–26 g body wt), and 22–24 mo (28–37 g body wt; retired breeder) old were used for this study. All mice were specific pathogen free, as determined by the breeder. The animals were allowed to acclimate to the animal facility at University of Alabama at Birmingham. The group-housed mature (6–8 wk) and aged (22–24 mo) females were able to synchronize their estrus cycle. The stage of the estrus cycle was determined by daily examination of the vaginal smear at the same time each day and classified as described elsewhere (2). In this study, mature and aged female mice in the proestrus stage and premature females without estrus cycle were included. All procedures were carried out in accordance with the guidelines set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. The Institutional Animal Care and Use Committee of University of Alabama at Birmingham approved this study.

Experimental groups.   Female and male mice 21–27 days (immature), 6–8 wk (mature), or 22–24 mo (aged) were randomized into one or two groups containing six to eight animals each. Group 1 consisted of sham-operated mice, and group 2 animals underwent the trauma-hemorrhage procedure. Twenty-four hours after the end of resuscitation or sham operation, the animals were killed by methoxyflurane overdose, and blood and spleen were harvested aseptically. Cells isolated from each animal were kept separate and not pooled for analysis. To exclude the influence of such chronic diseases as hepatoma, focal leukocytic nodules of the liver, lymphoid hyperplasia of the spleen, cancer of the liver and lung, and splenomegaly, animals were examined after blood and spleens were harvested. None of the mice used had any of the above abnormalities.

Trauma-hemorrhage procedure.   Male and female mice of all ages in the trauma-hemorrhage groups were lightly anesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL) and restrained in a supine position. A 2.5-cm midline laparotomy (i.e., soft-tissue trauma) was performed, and the abdominal incision was then closed aseptically in two layers using 6-0 sutures (Ethilon, Ethicon, Somerville, NJ). Both femoral arteries were then aseptically cannulated with polyethylene 10 tubing (Clay-Adams, Parsippany, NJ) using a minimal dissection technique. Heparin (porcine intestines heparin, Elkins-Sinn, Cherry Hill, NJ; 2 U/25 g of body wt) was then administered and the animals were allowed to awaken. Blood pressure was monitored continuously by attaching one of the catheters to a blood pressure analyzer (Digi-Med BPA-190, Micro-Med, Louisville, KY). On awakening, the animals were bled through the other catheter to a mean arterial blood pressure of 30 ± 5 mmHg (blood pressure prehemorrhage was 90 ± 5 mmHg), which was maintained for 90 min. At the end of that period, animals were resuscitated with lactated Ringer solution (4 times the shed blood volume for 30 min). Lidocaine hydrochloride was applied to the groin incision sites, the catheters were removed, the vessels ligated, and the groin incision closed. Sham-operated animals underwent the same groin dissection, which included ligation of both femoral arteries; however, the mice did not receive low-dose heparin, hemorrhage, or fluid resuscitation.

Splenocyte and PBMC isolation.   The animals were killed with methoxyflurane overdose 24 h after the completion of the resuscitation. The spleens were removed, and blood was obtained aseptically by cardiac puncture. Splenocytes were isolated as previously described (45) and resuspended in complete media (RPMI 1640, 10% fetal bovine serum, penicillin-G 50 U/ml and streptomycin 50 µg/ml) or PBS containing 0.1% sodium azide at a final concentration of 1 x 106 cells/ml. PBMC were prepared from heparinized whole blood using a standard Ficoll-Hypaque (Ficoll-Paque Plus, Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient. Briefly, the blood was layered on top of the Ficoll Hypaque at a 2:1 ratio in 15-ml tubes and centrifuged for 20 min at 500 g. The PBMC were collected from the interface, washed two times in HBSS, and resuspended in either complete media or PBS containing 0.1% sodium azide at a final concentration of 1 x 106 cells/ml. Viability of splenocytes and PBMC was consistently >95%, as determined by trypan blue exclusion.

Preparation of splenocyte and PBMC cultures for cytokine production.   Cells were stimulated with surface-bound anti-CD3 (1 µg/ml; Pharmingen, San Diego, CA) and incubated for 48 h at 37°C, 5% CO2, and 90% humidity. Cell-free supernatants were collected and stored at –80°C until assayed for cytokine production. IL-2, IFN-{gamma}, IL-10, and IL-4 levels in cell-free splenocyte and PBMC supernatants were determined by ELISA (Pharmingen).

Splenocyte proliferation assay.   Splenocyte or PBMC suspensions in complete media were incubated in a 96-well microtiter plate containing 2 x 105 cells per well. The ability of cells to proliferate in response to anti-CD3 (Pharmingen) with 0 (negative control) or 1 µg/ml anti-CD3 was determined by incubation for 48 h. The extent of proliferation was measured using the [3H]thymidine incorporation technique as described by Stephan et al. (45).

Preparation of splenocytes and PBMC for flow cytometric analysis.   All antibodies and isotype controls conjugated to FITC, phycoerythrin (PE), streptavidin-Cy-chrome (Cy-Chrome) or biotin were purchased from Pharmingen. The following antibodies and isotype-specific controls were used: 17A2 (rat IgG2b, {kappa}; anti-CD3-Cy-Chrome), GK1.5 (rat IgG2b, {kappa}; anti-CD4-PE), 53-6.7 (rat IgG2b, {kappa}; anti-CD8{alpha}-FITC). PBMC and splenocytes were resuspended in PBS-0.1% sodium azide and were triple labeled with 1 µg/1 million cells of anti-CD3-Cy-Chrome, anti-CD4-PE, and anti-CD8{alpha}-FITC. Samples were incubated for 30 min on ice in the dark and thereafter washed once with PBS-0.1% sodium azide. Samples were kept on ice in the dark, and all measurements were analyzed within 30 min after completing the staining procedure. FITC, PE, and Cy-Chrome were analyzed with a Becton-Dickinson FACSort flow cytometer (San Jose, CA) fitted with a 488-nm argon laser and filter settings for FITC (530-nm-wide band-pass filter), PE (575-nm dichronic filter), and Cy-Chrome (650-nm-long band-pass filter). After appropriate instrument settings and spectral compensations, the instrument settings were not changed and stability was regularly checked. A minimum of 10,000 events were assessed using log-amplified fluorescence signals and linearly amplified side- and forward-scatter signals. PC-lysis version 1.0 software (Becton Dickinson) was used to analyze the data.

Statistics.   Data are presented as means ± SE of 6–8 animals in each group. One-way ANOVA for multiple comparison followed by Tukey's or Dunn's test were employed to determine the significance of the differences between experimental means. P < 0.05 was considered significant for all statistical analysis.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Effect of age and gender on splenic and PBMC T-cell proliferative responses following trauma-hemorrhage.   The extent of incorporation of [3H]thymidine by splenocytes and PBMCs after stimulation with anti-CD3 was related to both gender and age following trauma-hemorrhage (Fig. 1). In immature animals, splenocyte proliferation was increased compared with a decreased proliferative capacity by PBMC after trauma-hemorrhage (Fig. 1, A and B). Those changes were similar in both males and proestrus females. Similar changes were evident for splenocytes from mature animals of both genders, whereas proliferation of peripheral T cells from males was unaffected by trauma-hemorrhage but increased in females following trauma-hemorrhage (Fig. 1, C and D). In aged animals of both genders, the overall proliferative capacity of splenocytes was less compared with the younger groups and unaffected by gender and traumatic injury. Similar results were demonstrated for PBMC from aged females (Fig. 1, E and F). In contrast, PBMC from aged male animals displayed depressed splenocyte proliferation following trauma-hemorrhage.


Figure 1
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  		Fig. 1. Proliferative capacity of splenocytes and peripheral blood mononuclear cells (PBMC) 24 h after sham procedure or trauma-hemorrhage (T-H). Immature splenocytes (A), immature PBMC (B), mature splenocytes (C), mature PBMC (D), aged splenocytes (E), and aged PBMC (F) were stimulated with anti-CD3, and proliferative capacity was measured by [3H]thymidine incorporation as described in MATERIALS AND METHODS. Data are means ± SE; n = 6–8/group. ANOVA: *P < 0.05 vs. sham/male.

 
Effect age and gender on splenic and PBMC T-cell cytokine production following trauma-hemorrhage.   The release of IL-2 and IFN-{gamma} by splenocytes from young and mature animals was increased in both genders following trauma-hemorrhage (Figs. 2, A and C, and 3, A and C), whereas the release of both these Th1 cytokines was unchanged in aged animals under such conditions (Figs. 2E and 3E). In PBMC from young mice, the overall cytokine productive capacity for IL-2 and IFN-{gamma} was lower than that seen in the other groups (Figs. 2B and 3B). In contrast to splenocytes, PBMC production of IFN-{gamma} was significantly decreased in young mice after trauma-hemorrhage (Fig. 3B). IL-2 and IFN-{gamma} release by PBMC from mature and aged animals was depressed in males but unchanged in proestrus females under such conditions (Figs. 2, D and F, and 3, D and F).


Figure 2
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  		Fig. 2. Production of IL-2 by splenocytes and PBMC 24 h after sham procedure or T-H. Immature splenocytes (A), immature PBMC (B), mature splenocytes (C), mature PBMC (D), aged splenocytes (E), and aged PBMC (F) were stimulated with anti-CD3, and IL-2 levels were measured by ELISA as described in MATERIALS AND METHODS. Data are means ± SE; n = 6–8/group. ANOVA: *P < 0.05 vs. sham/male.

 

Figure 3
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  		Fig. 3. Production of IFN-{gamma} by splenocytes and PBMC 24 h after sham procedure or T-H. Immature splenocytes (A), immature PBMC (B), mature splenocytes (C), mature PBMC (D), aged splenocytes (E), and aged PBMC (F) were stimulated with anti-CD3, and IFN-{gamma} levels were measured by ELISA as described in MATERIALS AND METHODS. Data are means ± SE; n = 6–8/group. ANOVA: *P < 0.05 vs. sham/male.

 
Trauma-hemorrhage led to significantly increased production of IL-4 by splenocytes and PBMC from young animals compared with sham animals (Fig. 4, A and B). A trend toward similar alterations for IL-10 was observed; however, it did not reach statistical significance (Fig. 5, A and B). Th2 cytokine release by splenocytes from mature animals was not altered following trauma-hemorrhage (Figs. 4B and 5B); however, its release was significantly increased in PBMC isolated from mature males compared with mature proestrus females under such conditions (Figs. 4D and 5D). In contrast, release of Th2 cytokines by tissue fixed cells and PBMC from aged animals following trauma-hemorrhage was only altered in females with increased IL-4 release, as opposed to decreased IL-10 release under such conditions (Figs. 4, E and F, and 5, E and F).


Figure 4
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  		Fig. 4. Production of IL-4 by splenocytes and PBMC 24 h after sham procedure or T-H. Immature splenocytes (A), immature PBMC (B), mature splenocytes (C), mature PBMC (D), aged splenocytes (E), and aged PBMC (F) were stimulated with anti-CD3, and IL-4 levels were measured by ELISA as described in MATERIALS AND METHODS. Data are means ± SE; n = 6–8/group. ANOVA: *P < 0.05 vs. sham/male.

 

Figure 5
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  		Fig. 5. Production of IL-10 by splenocytes and PBMC 24 h after sham procedure or T-H. Immature splenocytes (A), immature PBMC (B), mature splenocytes (C), mature PBMC (D), aged splenocytes (E), and aged PBMC (F) were stimulated with anti-CD3 and IL-10 levels were measured by ELISA as described in MATERIALS AND METHODS. Data are means ± SE; n = 6–8/group. ANOVA: *P < 0.05 vs. sham/male.

 
Effect of age and gender on CD4-to-CD8 ratios following trauma-hemorrhage.   Changes in CD4-to-CD8 ratio within the spleen and PBMC were evaluated by positive staining for CD3 and for CD4 and CD8 following trauma-hemorrhage (Table 1). CD3+ lymphocytes from sham-operated animals of both sexes did not differ with regard to CD4 or CD8 expression, alone or in combination, in all age-related groups (data not shown). Trauma-hemorrhage decreased the CD4-to-CD8 ratio in CD3+ splenocytes from young male and female mice. No such changes were seen in PBMC in trauma-hemorrhage animals from this age group. In mature mice, the trauma-hemorrhage-induced changes in CD4-to-CD8 ratios in males were similar to those seen in splenocytes from young animals. CD4-to-CD8 ratios in mature proestrus females were unaffected by trauma-hemorrhage. Splenic T cells from aged mice also demonstrated a gender-dependent decrease in the CD4-to-CD8 ratio after trauma-hemorrhage similar to that observed in tissue-fixed T cells from the other groups. The CD4-to-CD8 ratio of peripheral T cells from aged animals of both genders remained unchanged following trauma-hemorrhage.


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  		Table 1. Effect of age and gender on T-cell CD4-to-CD8 ratio after trauma-hemorrhage

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The data presented here suggest that differences exist between tissue-fixed and peripheral T-lymphocyte responses following trauma-hemorrhage. Moreover, these responses were significantly influenced by both the gender and age of the animal. Splenic T cells demonstrated increased proliferation and a shift toward a Th1 response (i.e., IL-2, IFN-{gamma}) following injury that was independent of gender in immature and mature animals. In aged animals, however, splenocyte proliferation and Th1 cytokine production were not altered following trauma-hemorrhage. In contrast, PBMC from mature and aged animals showed gender-dependent alterations following trauma-hemorrhage, although PBMC responses were not different in immature animals under such conditions.

Traumatic injury and blood loss cause impairment of the cellular immune system, leading to increased susceptibility to secondary infections in animal models (1, 42, 44) and patients (29, 54). These studies have shown that trauma-hemorrhage led to a significant decrease in the total lymphocyte counts and T-cell proliferation that was associated with diminished Th1 cytokine production and increased production of Th2 cytokines (i.e., IL-4, IL-10). Moreover, previous studies have also indicated that trauma-induced changes in tissue-fixed T cells were different in males and females. Enhanced immune responses in proestrus female mice (which have elevated estrogen levels) and depressed immune responses in males were observed following trauma-hemorrhage (1, 24). In contrast, the present study found neither gender-specific alterations nor trauma-induced depression of immune responses in tissue-fixed splenic T cells. An important difference in the earlier studies is that splenocyte immune responses were assessed after stimulation with a mitogen [i.e., concanavalin A, phytohemagglutinin (PHA)], which is antigen presenting cell dependent. The present study used anti-CD3, which directly binds to the T-cell receptor. Preliminary experiments of the present study showed that PBMC from mice, compared with splenocytes from the same animals, did not respond to mitogenic stimuli such as concanavalin A or PHA (data not shown). Because the aim of the present study was to compare trauma-hemorrhage-induced alterations in tissue-fixed and peripheral T-cell immune responses, we assessed T-cell immune function after stimulation with anti-CD3. These findings suggest that the trauma-induced and gender-dependent changes in cytokine productive capacity by tissue-fixed T cells may be caused by antigen presenting cell dysfunction or alterations in coaccessory molecules on T cells (i.e., CD28, CD69) rather than a specific loss of T-cell receptor signaling.

Peripheral T cells from mature animals showed gender-dependent response to trauma-hemorrhage. Cells from proestrus females exhibited an increased proliferative response and maintained cytokine release capacity, whereas cells from male animals exhibited decreased proliferation and a Th2 cytokine profile after trauma-hemorrhage. These results are comparable with those seen in mitogen-stimulated tissue-fixed T cells following trauma-hemorrhage (1). A single clinical study, in which PBMC were studied, indicated that cells from premenopausal women (which had threefold higher estrogen plasma levels) produced significantly greater amounts of TNF-{alpha} and IFN-{gamma} (50). It remains unknown, however, whether sex steroids modulate peripheral T-cell responses directly or act indirectly via antigen presenting cells in the circulation following trauma-hemorrhage. Nonetheless, a direct action on T cells seems plausible, since steroid receptors are present on this immune cell population in rodents (41, 51) and humans (55).

Aging is a complex process that affects a wide variety of body functions, including those of the immune system. In particular, T cells undergo dramatic changes during the aging process (27). In an aged population, it has been postulated that the loss of immune competence was related to alterations in T-cell function, which might represent the leading cause for the increased morbidity and mortality after traumatic injury and/or infection (27, 32, 49). In contrast, studies have shown that immature individuals are vulnerable to trauma and infection due to immaturity of the immune system (13, 19). The present results demonstrate that PBMC from immature animals showed depressed proliferation and shift toward a Th2 response after trauma-hemorrhage. This trauma-hemorrhage-induced change in PBMC response was independent of gender, in accordance with previous findings (19). Similar observations have also been made in children after head injury (31, 48). With regard to gender, girls did not have a better outcome after pediatric traumatic brain injury than boys (35).

The differential effects of estrogens and androgens on immune functions are indicated by the observation that thymus involution in young mice starts earlier in males than in females (10). Angele et al. (2) have shown that thymic apoptosis is increased in male mice but not in proestrus females following trauma-hemorrhage. Moreover, in vitro treatment of thymocytes with 5{alpha}-dihydrotestosterone induces apoptosis, suggesting that the gender-dependent dimorphic immune response following trauma-hemorrhage is in part due to an androgen-induced increase in thymic apoptosis. However, it remains to be determined whether aging influences this effect of trauma-hemorrhage on thymic apoptosis. Moreover, experimental studies have shown that androgens caused an impairment of the immune response following trauma-hemorrhage and female sex hormones are immunoprotective under those conditions (1). The present data show similar alterations in the immune responses by peripheral T cells from proestrus females and males following trauma-hemorrhage. With aging, it has generally been demonstrated that lymphocyte proliferation decreased, and this decrease was associated with diminished IL-2 receptor expression (18, 23, 52). The data presented herein confirm those results showing diminished proliferation in aged sham animals compared with their mature groups. Among these changes, a shift toward a Th2 phenotype in the aged group was observed (16, 29, 38). Although splenocytes from aged mice did not demonstrate trauma-hemorrhage or gender-dependent immune alterations, tissue-fixed T cells from aged females produced larger amounts of IL-4 in response to anti-CD3 following trauma-hemorrhage. Furthermore, the overall production of IL-10 was greater in splenocytes from aged animals compared with younger mice. Likewise, it has been shown that aged humans (13, 16, 33) and animals (23, 30, 44) have increased IL-10 production, which suppresses proinflammatory cytokine production.

Interestingly, peripheral T cells from aged mice exhibited gender differences in the immune response following trauma-hemorrhage. PBMC from aged male mice showed depressed proliferation and decreased IL-2 and IFN-{gamma} release compared with aged females following trauma-hemorrhage. These changes contrast with an earlier study demonstrating reversed gender-dependent immune responses with aging (23). In the former study, however, only acyclic aged female mice were included, whereas the present study used aged proestrus females. We also observed a greater production of the Th2 cytokine IL-10 in aged proestrus females, which decreased following trauma-hemorrhage compared with unchanged IL-10 levels in males. This gender difference may also be explained by the estrogen action, since Islander et al. (21) demonstrated that estrogen receptor-{alpha} is required for the increased production of IL-10 in aged female mice.

Tissue trauma causes not only functional changes in T cells but also a significant decrease in total systemic lymphocyte counts, including CD4+ and CD8+ cells (13, 25, 34). It has been demonstrated that CD4+-to-CD8+ T-cell ratio decreases under such conditions, particularly due to a decline in CD4+ subpopulation (20). In the present study, the trauma-hemorrhage-induced decline in CD4-to-CD8 ratio was also evident in splenocytes from premature animals of both genders. Conversely, in mature and aged animals, this decrease in CD4-to-CD8 ratio occurred only in males but not in proestrus females. Additionally, the overall CD4-to-CD8 ratio decreased with aging in a gender-independent manner. However, the relative importance of the decline in CD4-to-CD8 ratio following injury remains to be elucidated. Foulds et al. (14) showed that stimulated CD8+ T cells proliferated more readily than CD4+ T cells. One explanation for this observation may be the fact that generation of appropriate helpers and rapid deployment of numerous cytotoxic effectors are essential for the development of an effective adaptive immune response. Despite the maintenance of normal T-cell numbers with age, there is a considerable decrease in CD4- and CD8-mediated responses (11, 17).

An interesting component of research in the area of hemorrhagic shock is resuscitation strategies. Such strategies are designed to restore organ perfusion and modulate postinjury inflammation; however, a clear optimal choice for the resuscitation fluid is unclear (39). Although in our studies we chose crystalloid as a resuscitant, some studies suggest that artificial colloids and hypertonic fluids have immunomodulatory properties (6, 7, 12, 39). Moreover, recent findings by Lee et al. (26) in a rat model of hemorrhage using male animals suggest that modified fluid gelatin promotes a proinflammatory response compared with lactated Ringer or 4% hydroxyethel starch. The impact of either gender or age on resuscitation strategies is unclear but should be an area of potential future investigation.

The present study demonstrates that differences exist in tissue-fixed and peripheral T-cell immune responses following trauma and hemorrhagic shock. Nonetheless, the study is somewhat limited since it only evaluated a single time point following trauma-hemorrhage and did not examine other tissue compartments, such as the mesenteric lymph nodes, which may be important to the overall immune status of the individual (8). Moreover, other important immune cell populations (i.e., macrophages, B cells, and neutrophils) were not studied. Future studies examining the influence of age and gender on post trauma-hemorrhage immune function are needed to evaluate these parameters. Nonetheless, it can be concluded that evaluation of peripheral immune function might not entirely reflect the overall immune status of the patient. The study further demonstrated that T-cell immune responses after injury were influenced by both gender and age. Although many of the changes that occur are subtle and may not be of much consequence to an individual in normal, steady-state conditions, they may become very relevant in conditions of stress, such as injury and/or infection. Therefore, both parameters should be taken into consideration in evaluation and treatment of trauma and shock. In particular, increased estrogen levels seem to be protective also in aged females. Nonetheless, age- and gender-dependent alterations of the immune response following trauma-hemorrhage are complex, and further studies are needed in this regard.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by National Institute of General Medical Sciences Grant R01 GM-37127. M. G. Schwacha is supported in part by National Institute of Allergy and Infectious Diseases Independent Scientist Award K02 AI-049960.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
Present address of Christian P. Schneider: Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Chaudry, Center for Surgical Research, Univ. of Alabama at Birmingham, G 094Volker Hall, 1670 Univ. Blvd., Birmingham, AL 35294-0019 (e-mail: Irshad.Chaudry{at}ccc.uab.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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