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(Journal of Leukocyte Biology. 2001;70:887-895.)
© 2001 by Society for Leukocyte Biology

Estrogen restores cellular immunity in injured male mice via suppression of interleukin-6 production

Kelly A. N. Messingham*,{dagger},{ddagger}, Scott A. Heinrich*,{dagger} and Elizabeth J. Kovacs*,{dagger},{ddagger},§

* Department of Cell Biology, Neurobiology, and Anatomy,
{dagger} The Burn and Shock Trauma Institute,
{ddagger} Alcohol Research Program,
§ Department of Surgery, and
|| Immunology and Aging Program, Loyola University Medical Center, Maywood, Illinois

Correspondence: Elizabeth J. Kovacs, Ph.D., Building 110, Room 4221, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153. E-mail: ekovacs{at}lumc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study examined whether estrogen treatment can improve immunity in male mice after combined ethanol and burn injuries. 17ß-Estradiol [estrogen, given subcutaneously (s.c.)] or oil (control) was administered at 30 min and 24 h postinjury. At 48 h postinjury, ethanol/burn-injured mice demonstrated significant suppression of cellular immunity. Estrogen treatment restored the delayed-type hypersensitivity (P<0.01) and splenocyte-proliferative (P<0.05) responses, reduced macrophage interleukin-6 (IL-6) (P<0.05), and increased survival after bacterial challenge (P<0.01). In vitro neutralization of IL-6, combined with macrophage supernatant experiments, confirmed that the beneficial effects of estrogen treatment were mediated through modulation of macrophage IL-6 production. Moreover, estrogen treatment resulted in a decrease in splenic nuclear factor-{kappa}B (NF-{kappa}B) activation in injured mice. There were no changes in cellular NF-{kappa}B or I{kappa}B{alpha} protein expression or I{kappa}B{alpha} phosphorylation at serine 32. Taken together, these studies suggest that estrogen treatment of injured male mice improves cellular immunity through direct modulation of NF-{kappa}B activation.

Key Words: cytokine • NF-{kappa}B • monocyte/macrophage • hormone • ethanol • burn


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been firmly established that a naturally occurring gender-based dimorphism of the immune response exists. This phenomenon has been largely attributed to the stimulatory effects of estrogen and inhibitory effects of testosterone on immunity [1 , 2 ]. Females exhibit more vigorous and better-sustained humoral and cell-mediated immune responses than those observed in males [3 4 5 6 7 ]. More recently, it has been established that this immunological dimorphism also influences outcomes for patients suffering from burns or other traumatic injuries. Not only are males predominately more affected by these types of injuries [8 ], but they are also at increased risk for morbidity and mortality due to septic complications, in comparison with females matched for age, severity of injury, and clinical course [8 , 9 ]. Experimental trauma models reveal a similar gender difference in immunity and subsequent survival after injury and septic challenge [10 11 12 13 14 15 ]. Furthermore, either chemical or surgical castration of male mice results in improved immune function and increased survival after injury, whereas administration of testosterone to female mice is detrimental to immunity and survival [11 12 13 14 15 ]. Collectively, these studies suggest that females are protected from the immune suppression seen in males by the presence of estrogen and/or the absence of testosterone.

Increased susceptibility to infection due to suppression of the cell-mediated immune response is the primary complication for patients who survive an initial burn or other traumatic injury [16 ]. After burn injury, macrophages have been shown to play a central role in this immune dysfunction [17 18 19 20 ] through altered production of inflammatory mediators such as interleukin-6 (IL-6) [17 18 19 , 21 22 23 24 ]. Although IL-6 normally plays a key role in maintaining immunity [25 , 26 ], abnormally high levels of this cytokine in sera and tissues of burn patients correlate with the severity of injury and subsequent morbidity and mortality, even in the absence of sepsis [18 , 21 , 27 28 29 ]. Furthermore, increased concentrations of circulating IL-6 coincide with impaired T cell responses after trauma [18 , 21 ]. Therefore, it has been suggested that increased inflammatory cytokine production is not only a marker of macrophage dysfunction but a means by which this type of cell can suppress immunity after injury [29 ].

Estrogen is known to regulate a variety of proinflammatory cytokines including IL-6 [30 31 32 ]. Both in vivo and in vitro exposure of macrophages to estrogen concentrations similar to those seen in cycling females decreases IL-6 production [32 , 33 ]. Although the IL-6 promoter lacks an estrogen-responsive element [33 34 35 ], on binding to estrogen, the receptor-ligand dimers interact directly with NF-{kappa}B, thereby preventing DNA binding and subsequent transcription [35 , 36 ].

To further examine the effects of burn injury on cellular immunity and its relation to the increased morbidity and mortality in these patients, our laboratory has developed a murine model of thermal injury with previous ethanol exposure [24 , 37 38 39 ]. This model was chosen because nearly 50% of the patients suffering from burn injuries have detectable levels of ethanol in their circulatory systems on admission [4 ]. Additionally, these patients suffer from increased mortality and infectious and/or surgical complications in comparison with patients exposed to burn injury alone [40 , 41 ]. In agreement with clinical observations, previous studies in our laboratory have shown that mice exposed to 100 mg/dL of circulating ethanol (equal to the consumption of 1–2 hard-liquor drinks) at the time of burn injury suffer from decreased delayed-type hypersensitivity (DTH) and splenocyte-proliferative responses, which are accompanied by increased circulating and macrophage-derived IL-6, in comparison with mice treated with either type of insult alone [24 , 37 , 39 ].

A recent study with a hemorrhagic-shock model found that administration of estrogen to male mice immediately after injury restored lymphocyte functions [15 ]. However, no studies to date have examined the effect of estrogen administration to male mice in a burn injury model. Therefore, the goal of the current study was to determine whether estrogen administration to male mice could improve cellular immunity and survival of bacterial challenge in burned mice with ethanol in their circulatory systems. Furthermore, these studies aimed to determine whether the beneficial effects of estrogen result from hormone-mediated reduction in IL-6 production through modulation of NF-{kappa}B activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Injury paradigm
Ten-week-old male C57BL/6 mice (Charles River Laboratories, Portage, MI) (body weight, ~24 g) were randomly assigned to one of four groups: saline/sham, ethanol/sham, saline/burn, or ethanol/burn. Thirty minutes before burn (or sham) injury, animals received an intraperitoneal (i.p.) injection of saline or of a 20% (v/v) ethanol solution in saline to achieve circulating ethanol levels of 100 mg/dL. The ethanol injections were based on previous studies in which an i.p. injection of 0.15 mL of a 20% ethanol solution resulted in 100 mg/dL of circulating ethanol at 30 min postinjection [37 , 38 ]. Animals with blood ethanol levels of 100 mg/dL demonstrate moderate impairment in balance and coordination, are quite active, and do not lose consciousness.

A dorsal-scald (burn) injury was administered as previously described [10 ]. Briefly, mice were anesthetized [40 mg/kg of sodium pentobarbital (Nembutal) in 0.9% normal saline i.p. (Abbott Laboratories, Abbott Park, IL)] and shaved with animal clippers to expose their dorsal surfaces. Animals were then placed into plastic templates designed to expose 15% of their total body surface area. The mouse and template were then immersed into a room temperature (sham) or 100°C (burn) water bath for 8 s. All animals were thoroughly dried with a towel to prevent further scalding and received i.p. resuscitation with 1.5 mL of 0.9% normal saline. The mice were then placed under warming lamps until recovery from anesthesia, after which they were returned to their cages. To avoid complications due to daily corticosterone fluctuations, animals were maintained on a 12-h light/dark cycle, and all procedures were performed between 8:30 and 10:30 a.m., when circulating corticosterone was at its lowest. It should be noted that the combination injury of ethanol and burn was much more severe than either injury alone; therefore, a relatively small (15%) total-body-surface-area burn was used to prevent high mortality rates. Thus, animals subjected to burns or ethanol alone received very moderate injury, which was not immune suppressive, in comparison with other burn models [20 , 22 ]. All animal studies described herein were performed in strict accordance with the guidelines set forth by the Loyola University Chicago institutional animal care and use committee.

Estrogen administration
Thirty minutes after injury, mice from each of the injury groups described above (saline/sham, ethanol/sham, saline/burn, and ethanol/burn) were subdivided and designated "+ oil" or "+ estrogen," resulting in a total of eight experimental groups. Each of these mice was treated with a 0.1-mL s.c. injection of sesame oil or 80 ng/mouse of 17ß-estradiol (estrogen) in sesame oil at 30 min and 24 h postinjury. This injection regimen was based on dose-response pilot studies examining injections of 0.08–800 ng/mouse/injection, which were conducted to determine the most beneficial dose of estrogen in regard to the DTH and splenocyte-proliferative responses (data not shown). Evaluation of serum estradiol revealed a gradual increase in circulating levels, which peaked at a concentration of 28.0 ± 3.8 pg/mL at 6 h after injection (data not shown). No differences in estradiol concentrations were observed between + oil and + estrogen groups of mice at the time of sacrifice (24 h after the last injection). Additionally, no differences in mortality (in the absence of bacterial challenge) were observed between groups of mice treated with oil versus estrogen.

Determination of DTH responses
DTH responses were induced as previously described [37 , 38 ]. Briefly, 5 days prior to thermal injury, all groups of experimental mice were sensitized to the hapten 2,4-dinitrofluorobenzene (DNFB) (ACROS Organics, Fairlawn, NJ) by applying 20 µL of a 0.5% solution in acetone/olive oil (4:1) directly to the shaved skin of the abdomen. Twenty-four hours after thermal injury, ear thickness measurements were made with a micrometer and then an eliciting dose (20 µL of 0.2% DNFB) was applied to the pinna of the left ear. At 24 h postelicitation (48 h postburn), both the unelicited (right) and the elicited (left) ears were measured. The magnitude of ear swelling was expressed as percent change in ear thickness using the following formula: (difference in thickness/preelicitation thickness) x 100%, where difference in thickness equals postelicitation minus preelicitation ear thickness. Right-ear (completely unmanipulated) measurements were obtained and served as internal controls for each animal. A group of naive (not sensitized or injured) animals received only the elicitation dose of DNFB for determination of nonspecific ear swelling caused by application of the hapten in oil. The naive mice showed a 3–9% increase in ear thickness in response to the eliciting dose of DNFB but did not demonstrate any differences in splenocyte proliferation or cytokine production in comparison with the sensitized saline/sham mice (data not shown).

Analysis of splenocyte proliferation
When the mice were killed, spleens were aseptically removed, and single cell suspensions of splenocytes were plated into 96-well microtiter plates at a density of 400,000 cells per well in RPMI 1640 (Gibco-BRL, Grand Island, NY) supplemented with L-glutamine (2 mM), penicillin G (100 U/mL), streptomycin (100 µg/mL), 50 mM HEPES, 5 x 10-5 M 2-mercaptoethanol, and 10% heat-inactivated fetal bovine serum. The viability of the cells was confirmed to be >96% by trypan blue exclusion. Triplicate splenocyte cultures were incubated for 72 h at 37°C and 5% CO2 in air, in the presence of medium alone or concanavalin A [Con A (1 or 2 µg/mL)]. In some cases IL-6-neutralizing ({alpha}IL-6) or isotype control [immunoglobulin G (IgG)] antibodies were added at the time of culture initiation (0.1 µg/mL). A colorimetric assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)] was used to measure proliferation as previously described [10 ]. Briefly, after 68 h of incubation, plates were centrifuged, the supernatant was removed, and 50 µL of a 1-mg/mL solution of MTT (Sigma Chemical Co., St. Louis, MO) in phenol red-free RPMI 1640 were added to each well. Plates were then incubated for the remaining 4 h at 37°C in 5% CO2 in air. After the incubation, the untransformed MTT was removed after centrifuging plates by carefully inverting and blotting. Next, 50 µL of isopropanol were added, and the optical density of each well was measured using an automatic microplate reader at a wavelength of 540 nm. Triplicate cultures were averaged to generate an average for each animal, which was then used to determine the mean absorbance ± SE for each experimental group.

Preparation of macrophage supernatants
Purified splenic macrophages were obtained from total splenocyte suspensions by adherence depletion. Briefly, 250 µL of the total splenocyte suspension (containing 1.0 x 107 cells/mL) were plated in each well of a 96-well microtiter plate [24 ]. The cells were cultured for 1.5 h at 37°C in 5% CO2 in air. After adherence, the nonadherent cells were removed by washing twice with 37°C phosphate-buffered saline (PBS). This method yielded approximately 200,000 macrophages per well, which were >98% positive for Mac-3 and Di-I-acetylated low-density lipoprotein uptake (data not shown). The purified splenic macrophages were cultured in RPMI medium containing 10% fetal bovine serum for 18 h in the presence or absence of lipopolysaccharide [LPS (1 µg/mL)]. The supernatants were filtered through 0.22-µm-pore-size filters and stored at -80°C prior to evaluation of IL-6 content or effect on lymphocyte proliferation. To determine the inhibitory effect of macrophage-derived mediators, some of these supernatants were added at 10% of the final volume to proliferation cultures containing splenic lymphocytes from unmanipulated mice.

Preparation of splenic lymphocytes
Splenic lymphocytes from unmanipulated mice were obtained by incubating splenocyte cell suspensions for 1.5 h on tissue culture plastic. The nonadherent cells (T and B cells) were collected by washing with 37°C PBS and resuspended in medium and counted using trypan blue to ensure >96% viability. These lymphocytes were plated at 4.0 x 105 cells/well in the presence of Con A (2 gmg/mL) ± LPS-stimulated macrophage supernatants in the presence or absence of IL-6-neutralizing or isotype control antibodies as described above.

Evaluation of macrophage-derived IL-6
Splenic macrophage supernatants were assayed for the presence of IL-6 by enzyme-linked immunosorbent assay (ELISA). Cytokine concentrations were determined using a commercially available sandwich ELISA kit and matched capture and detection antibodies specific for murine IL-6 (Endogen Inc., Cambridge, MA). IL-6 was not detectable in unstimulated or Con A-stimulated macrophage cultures. Cytokine concentrations were interpolated from the linear range of a recombinant murine IL-6 standard curve, and the minimum detection level of this assay was 25 pg/mL.

Nuclear protein extraction and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from approximately 5 x 106 splenocytes stimulated with 1 µg of LPS for 15 min. All cells were washed in cold PBS and resuspended in 400 µL of hypotonic buffer [10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5 M dithiothreitol, 2 mM leupeptin, 1 µg/mL of aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM EGTA]. Cells were lysed by the addition of Nonidet-P40 (25 µL of a 10% solution), and nuclei were collected via centrifugation (10,000 g, 30 s). The nuclear proteins were prepared by incubation of the nuclei in 40 µL of lysis buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF) on an orbital shaker (4°C, 15 min) [42 ]. Protein concentrations were determined using a Bradford analysis kit (Bio-Rad Laboratories, Richmond, CA).

For EMSA, 4 µg of the extract were incubated at room temperature for 15 min with 0.2–0.5 ng of NF-{kappa}B probe [5'-AGT TGA GGG GAC TTT CCC AGG-3' (Santa Cruz Biotechnology, Santa Cruz, CA)] prelabeled with digoxygenin-11-UTP (Boehringer-Mannheim, Indianapolis, IN) and 3 µg of polyadenylic acid-polythymidylic acid to block nonspecific binding (Boehringer-Mannheim). The resulting complexes were subjected to electrophoresis at 4°C on a 6% polyacrylamide gel under nonreducing conditions (0.25x Tris-borate-EDTA buffer). Control samples contained 150-fold excess unlabeled oligonucleotide added to the nuclear extracts before the addition of the labeled probe. The proteins were then transferred to a positively charged nylon membrane (Boehringer-Mannheim) and detected using an antidigoxigenin antibody and chemiluminescent-film developing (Amersham Pharmacia, Piscataway, NJ).

Cellular protein extraction and Western blotting
Expression of NF-{kappa}B, I{kappa}B{alpha}, and phospho-serine 32 I{kappa}B{alpha} [p-ser-32 (New England Biolabs, Beverly, MA)] was examined by Western blot analysis. Protein extracts were prepared from 20 x 106 splenocytes stimulated with 1 µg/mL of LPS for 15 min. All cells were washed in cold PBS and resuspended in a triple-detergent lysis buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 0.2% NaN3, 1% Nonidet P-40, 1 mM PMSF, 100 mM Na3VO4). Cells were agitated on ice for 30 min, lysates were centrifuged at 14,000 g, and the supernatant was analyzed for total protein content using a Bradford analysis kit (Bio-Rad Laboratories).

For Western blot analysis,15–20 µg of protein were electrophoresed on 4–12% Tris/glycine acrylamide gels (Novex, San Diego, CA) and transferred to a polyvinylidene fluoride membrane (Novex). After protein transfer, nonspecific antibody-binding sites on the membrane were blocked by incubation with either 5% milkfat (I{kappa}B{alpha} or p-ser-32) or 5% normal donkey serum (NF-{kappa}B) in PBS. Membranes were then incubated with I{kappa}B{alpha}, NF-{kappa}B (1:500; Santa Cruz), or p-ser-32-specific antibodies overnight at 4°C. Specific protein bands were detected using a mouse anti-rabbit IgG (I{kappa}B{alpha} and p-ser-32, 1:5,000) (Amersham Pharmacia) or donkey anti-goat IgG (1:250,000) (Jackson Immunoresearch, West Grove, PA) conjugated to horseradish peroxidase for 1 h at room temperature followed by chemiluminescent detection on autoradiographic film (Amersham Pharmacia). The density of specific bands was analyzed using a densitometer. In some instances, tumor necrosis factor {alpha} (TNF-{alpha})-stimulated HeLa cells (New England Biolabs) were used as a positive control to confirm the position of the I{kappa}B{alpha} protein when using the p-ser-32 antibody.

Preparation of bacteria and infectious challenge after injury
Bacterial infectious challenge to thermally injured mice was performed as previously described [43 ]. Briefly, Pseudomonas aeruginosa (ATCC 19660) bacteria were cultured in 3% tryptic soy broth for 16–18 h at 37°C on an orbital shaker. At the end of the culture phase, the bacteria were centrifuged, and the pellet was washed three times and resuspended in PBS. Bacteria were quantified by spectrometry at 660 nm, and the suspension was adjusted to approximately 4,000 colony-forming units (CFUs)/mL. Immediately after thermal injury, mice were inoculated with 1,000 CFUs via topical application of the bacterial suspension directly to the burn wounds. The actual number of CFUs was confirmed by plating serial dilutions of the bacterial suspension on tryptic soy agar plates. After 18 h of culture at 37°C, the number of colonies per plate was counted. Data were expressed as percent survival after inoculation.

Statistical analysis
Data were expressed as means ± SE of each group unless otherwise indicated. When used, n is the number of animals in each group. In some cases, representative graphs of two to three identical experiments with similar results were produced. An analysis of variance was performed to assess differences among all experimental groups or to assess differences between in vitro effects of macrophage supernatants (medium, LPS, LPS+IgG, or LPS+anti-IL-6) generated from one group of mice. If the analysis of variance indicated a significant main or interactive effect, a Fisher’s protected least-significant-difference test was used to make post-hoc comparisons. A Mann-Whitney U test was used for a comparison of survival in bacterial-challenge experiments. A P of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DTH response
The DTH response was used as an in vivo measure of cellular immunity. As shown in Figure 1 , saline/sham, ethanol/sham, and saline/burn groups could generate robust increases (35–52%) in ear thickness, regardless of oil or estrogen treatment. Mice in the ethanol/burn + oil group demonstrated a dramatic decrease (P<0.01) in the amount of ear swelling (13%) in comparison with all other groups. Of particular interest to this study was the finding that estrogen administration to ethanol/burn-injured mice resulted in a complete restoration of the DTH response to the levels observed in saline/sham mice (given oil or estrogen injections).



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  		Figure 1. Effect of in vivo E2 treatment on the DTH response in mice given ethanol prior to burn injury. Mice were sensitized to DNFB 5 days before ethanol (or saline) injection and exposure to a burn (or sham) injury. Thirty minutes and 24 h postinjury, mice were given an s.c. injection of either 0.1 mL of sesame oil or 17ß-estradiol (E2, 80 ng) in oil. Ear thickness was recorded, and the DTH responses were elicited at 24 h postinjury. Final ear thickness measurements were made at 48 h postinjury, and the DTH responses were calculated. Data are expressed as the mean percent changes in ear thickness ± .SE from three combined experiments. n = 8 for saline/ethanol + E2; n = 9–12 for all other groups. *, P < 0.01 from all other groups.

 
Splenocyte proliferation
To evaluate the effect of estrogen administration to ethanol/burn-injured mice on lymphocyte function, splenic lymphocyte-proliferative responses were evaluated in response to the T cell mitogen Con A (2 µg/mL) (Fig. 2 ). In agreement with the DTH responses shown in Figure 1 , there were no differences in the ability of splenic lymphocytes isolated from saline/sham, ethanol/sham, and saline/burn-injured mice to proliferate in response to mitogen. However, splenocytes isolated from ethanol/burn-injured mice treated with oil demonstrated a significant (P<0.01) decrease in splenocyte proliferation. When ethanol/burn-injured mice were treated with estrogen after injury, the Con A-induced proliferation was restored to levels observed in control groups of mice.



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  		Figure 2. Effect of in vivo E2 treatment on the splenocyte-proliferative response in mice given ethanol prior to burn injury. Thirty minutes and 24 h postinjury, mice were given s.c. injections of either 0.1 mL of sesame oil or 17ß-estradiol (E2, 80 ng) in oil. Mice were sacrificed 48 h postinjury, and 4 x 105 splenocytes/well were cultured for 72 h in the presence of Con A (2 µg/mL). Proliferation was evaluated using the MTT assay. Data are expressed as mean absorbance at 540 nm ± SE from two combined experiments. n = 5 for saline/ethanol + E2, n = 6–9 for all other groups. *, P < 0.05 from all other groups.

 
Splenic macrophage-derived and circulating IL-6
To determine whether the beneficial effects of estrogen administration on the DTH and splenocyte-proliferative responses could be the result of regulating IL-6 production, IL-6 secretion by splenic macrophages cultured in the presence or absence of LPS (1 µg/mL) was assayed (Fig. 3 ). Macrophages cultured in medium alone (no LPS) did not produce detectable levels of IL-6 (data not shown). Although there was a trend towards increasing IL-6 levels in the LPS-stimulated cultures generated from saline/sham, ethanol/sham, and saline/burn-injured mice, there was no significant difference in IL-6 production in these groups (regardless of estrogen or oil treatment). Macrophage production of IL-6 from ethanol/burn + oil mice was significantly increased (P<0.01) above all other groups. Estrogen treatment of the mice in the ethanol/burn group decreased LPS-induced splenic-macrophage IL-6 production in comparison with that observed from macrophages isolated from ethanol/burn-injured mice treated with oil; however, the level of IL-6 detected was still significantly greater (P<0.05) than those levels secreted by macrophages isolated from all other groups receiving estrogen injections. Moreover, estrogen treatment of ethanol/burn-injured mice also resulted in a twofold decrease in circulating IL-6 at 24 h postinjury (from 615 ± 286 to 277 ± 59 pg/mL) (n=4; P<0.05).



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  		Figure 3. Effect of in vivo E2 treatment on macrophage IL-6 in mice given ethanol prior to injury. Adherence-purified splenic macrophages were cultured at a density of 2 x 105/well with or without LPS (1 µg/mL) for 18 h. Macrophage supernatants were assayed for IL-6 by ELISA. Data are expressed as the mean IL-6 concentrations (pg/mL) ± SE. n = 3–6 per group, and data are representative of three identical experiments for supernatants. *, P > 0.01 from all other groups; #, P > 0.05 from all other groups treated with E2.

 
The role of IL-6 in splenocyte proliferation
To determine whether IL-6 mediated the suppression of splenocyte proliferation observed in cultures generated from ethanol/burn-injured mice, and to evaluate the role of IL-6 in the proliferation of splenocytes isolated from ethanol/burn + estrogen mice, splenocyte proliferation was evaluated in the presence of neutralizing antibodies (anti-IL-6) or isotype control antibodies (IgG). Cultures were stimulated with suboptimal mitogen concentrations (1 µg/mL of Con A) to allow upward or downward modulation of the proliferative response that might not be seen when a maximal response (2 µg/mL of Con A) is induced. As shown in Figure 4 , addition of isotype control antibodies did not affect proliferative responses. The addition of neutralizing antibodies (anti-IL-6) to the proliferation cultures had no affect on proliferation of splenocytes isolated from saline/sham, ethanol/sham, and saline/burn group mice but resulted in complete restoration (P<0.05) of the proliferative response in cultures generated from the ethanol/burn + oil mice (Fig. 4A) . In contrast, when mice were treated with estrogen, there were no differences in proliferation between any of the injury treatment groups (Fig. 4B) , nor did the addition of anti-IL-6 or IgG influence the magnitude of splenocyte proliferation.



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  		Figure 4. Effect of E2 treatment on IL-6-mediated suppression of splenocyte proliferation in mice given ethanol prior to burn injury. Thirty minutes and 24 h postinjury, each mouse was given an s.c. injection of either 0.1 mL of sesame oil (A) or 17ß-estradiol (E2, 80 ng) (B) in oil. Mice were sacrificed at 48 h postinjury, and 4 x 105 splenocytes/well were cultured for 72 h in the presence of Con A (1 µg/mL) alone or combined with IL-6-neutralizing (anti-IL-6) or IgG control (IgG) antibodies (0.1 µg/mL). Proliferation was evaluated using the MTT assay. Data are expressed as mean absorbance at 540 nm ± SE. n = 5 for saline/ethanol + E2; n = 6–9 for all other groups. *, P < 0.05 from all groups treated with IgG isotype control; #, P < 0.05 from all other groups.

 
The effects of macrophage supernatants on lymphocyte proliferation
To establish whether estrogen treatment restored the DTH and splenocyte-proliferative responses via alterations in macrophage-derived factors, specifically IL-6, nonadherent splenocytes (splenic lymphocytes) were cultured in the presence of supernatants from LPS-stimulated macrophages isolated from each of the eight treatment groups. These supernatants were then added to cultures of Con A-stimulated (1 µg/mL) splenic lymphocytes with or without IL-6-neutralizing antibodies (anti-IL-6) or isotype control (IgG) antibodies. As shown in Figure 5 A and B , supernatants from unstimulated (medium) macrophages had no effect on lymphocyte proliferation. In contrast, when supernatants from LPS-stimulated macrophages obtained from ethanol/burn + oil mice were added to lymphocyte proliferation cultures, a significant (P<0.01) decrease in proliferation was observed (Fig. 4A) . This suppression of lymphocyte proliferation was completely restored when anti-IL-6 antibodies were added but was unaffected by the addition of IgG control antibodies. It is of particular interest to this study that when mice from each of the treatment groups were given estrogen after injury, the macrophage supernatants generated from all groups of mice, including the ethanol/burn group, had no effect on the proliferation of splenic lymphocytes (Fig. 5B) .



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  		Figure 5. Estrogen modulation of macrophage-mediated suppression of lymphocyte proliferation. Macrophage supernatants generated from mice given oil (A) or E2 (B) after administration of ethanol (or saline) prior to a burn (or sham) injury. Splenic macrophages (2x105 cells/well) were cultured in the presence of LPS (1 µg/mL) or medium for 18 h. Supernatants (designated medium or LPS) were added at 10% of the total culture volume to Con A (2 µg/mL)-stimulated nonadherent splenic lymphocytes (5x105/well). IL-6-neutralizing antibodies (anti-IL-6) or isotype control antibodies (IgG) were added at culture initiation (0.1 µg/mL). After 72 h, proliferation was evaluated using the MTT assay. Data are expressed as mean absorbance at 540 nm ± SE. IgG control antibodies (LPS+IgG) were added to triplicate cultures containing supernatants from n = 3–4 animals per group (1 experiment). All other triplicate lymphocyte cultures contained supernatants from five ethanol/sham + E2 mice or six to nine mice from all other groups (two combined experiments). *, 7P < 0.01 from all other cultures containing LPS-stimulated supernatants.

 
NF-{kappa}B activation in splenocytes
Members of the NF-{kappa}B family of transcription factors are known to regulate IL-6 production in response to LPS stimulation, and estrogen is known to decrease NF-{kappa}B activation at doses similar to those found in estrogen-treated mice (~30 pg/mL). To begin to explore the mechanism(s) by which in vivo estrogen administration modulates IL-6 levels and thus restores immunity in these mice, nuclear extracts generated from splenocytes from all groups of mice were analyzed for NF-{kappa}B-binding activity by EMSA. Splenocytes from saline/burn + oil or ethanol/burn + oil groups of mice demonstrated increased NF-{kappa}B binding in comparison with splenocytes from saline/sham- or ethanol/sham-treated mice (Fig. 6 A ). When these same groups of mice were treated with estrogen, there was a reduction in NF-{kappa}B binding. Further examination of NF-{kappa}B activation in four individual ethanol/burn-injured mice treated with oil or estrogen revealed a similar reduction in DNA binding in extracts from splenocytes isolated from three of four individual estrogen-treated mice in comparison with those treated with oil (Fig. 6B) .



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  		Figure 6. NF-{kappa}B activation in splenocytes isolated from mice treated with oil or estrogen after injury. Mice were given an i.p. injection of ethanol (or saline) 30 min prior to burn (or sham) injury and treated with estrogen (E2, 80 ng/mouse) in oil or oil control at 30 min and 24 h after injury. Mice were sacrificed at 48 h postinjury, and splenocytes from individual mice were cultured with LPS (1 mg/mL) for 15 min. Each lane represents an extract made of splenocytes from an individual mouse. NF-{kappa}B-binding activity in 4 µg of nuclear protein was measured using the EMSA. (A) Splenocytes from mice in each group were assayed to evaluate the effects on NF-{kappa}B binding. Specific NF-{kappa}B binding was confirmed by adding a 150-fold excess of unlabeled NF-{kappa}B oligonucleotide ("cold compete") or a 150-fold excess of a nonspecific oligonucleotide ("non-specific") to the reaction mixture. (B) Nuclear extracts from four individual mice from the ethanol/burn + oil or ethanol/burn + E2 groups. Data are representative of at least three identical experiments.

 
Evaluation of NF-{kappa}B expression in splenocytes
To determine whether the decreased NF-{kappa}B activation found in the splenocytes isolated from ethanol/burn + estrogen mice was the result of a decrease in protein expression, NF-{kappa}B expression was evaluated by Western blot. Evaluation of total cellular NF-{kappa}B expression in all groups of mice revealed no differences in protein expression regardless of injury type or estrogen treatment (Fig. 7 A ). Further analysis of protein expression in splenocytes from four individual ethanol/burn + oil or ethanol/burn + estrogen mice revealed a slight but insignificant decrease in NF-{kappa}B expression in the ethanol/burn + estrogen group of mice (Fig. 7B) .



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  		Figure 7. NF-{kappa}B and I{kappa}B{alpha} protein expression and I{kappa}B{alpha} phosphorylation in splenocytes isolated from mice treated with oil or estrogen after injury. Mice were given an i.p. injection of ethanol (or saline) 30 min prior to burn (or sham) injury and treated with estrogen [E2 (80 ng/mouse)] in oil or oil control at 30 min and 24 h after injury. Mice were sacrificed at 48 h postinjury, and splenocytes from individual mice were cultured with LPS (1 mg/mL) for 15 min. For all Western blots, each lane represents an extract made of splenocytes from an individual mouse. Fifteen micrograms of protein were evaluated for NF-{kappa}B (A), I{kappa}B{alpha},and phospho-I{kappa}B{alpha} (p-ser-32) expression. To confirm these findings, extracts from four individual mice from the ethanol/burn + oil or ethanol/burn + E2 groups were also examined for NF-{kappa}B (B), I{kappa}B{alpha}, and phospho-I{kappa}B{alpha} (p-ser-32) expression. Densitometric analysis revealed no differences between groups (results not shown). Western blots are representative of three identical experiments.

 
Evaluation of I{kappa}B{alpha} expression and phosphorylation in splenocytes
The transcription factor NF-{kappa}B is maintained in an inactive form by its inhibitor protein, I{kappa}B{alpha}, thereby preventing NF-{kappa}B-induced transcription of various cytokine genes, including IL-6 [44 , 45 ]. A variety of steroid hormones are known to regulate the production of I{kappa}B{alpha}, resulting in alterations in transcription of NF-{kappa}B-dependent genes [45 , 46 ]. Evaluation of cellular I{kappa}B{alpha} content in all groups of mice revealed no differences in protein expression regardless of injury type or estrogen treatment (Fig. 7A) . Further analysis of protein expression in splenocytes from four individual ethanol/burn + oil or ethanol/burn + estrogen mice revealed no differences in I{kappa}B{alpha} expression in these groups of mice (Fig. 7B) .

The phosphorylation of I{kappa}B{alpha} on serine-32 and serine-36 results in polyubiquitination and subsequent degradation of the inhibitor, resulting in translocation of NF-{kappa}B to the nucleus and activation of target genes [44 , 46 ]. Phosphorylation of I{kappa}B{alpha} was evaluated by Western blot using a serine-32-specific antibody. Because multiple proteins contain phosphorylated serine residues, TNF-{alpha}-stimulated HeLa cells, which contain high levels of phosphorylated I{kappa}B{alpha}, were utilized as a positive control. Evaluation of I{kappa}B{alpha} phosphorylation in all groups of mice revealed no differences in any group of mice regardless of injury type or estrogen treatment (Fig. 7A) . Further analysis of splenocytes from four individual ethanol/burn + oil or ethanol/burn + estrogen mice revealed no differences in I{kappa}B{alpha} phosphorylation in these groups of mice (Fig. 7B) .

Survival of bacterial challenge
Previous studies that used an identical model of ethanol exposure followed by burn injury found a significant decrease in survival of bacterial challenge in ethanol/burn-injured mice in comparison with mice subjected to burn injury alone (saline/burn). The saline/burn-injured mice are not significantly immune suppressed by this type of injury and thus serve as a control for immune-dependent susceptibility to infection [39 ]. To determine the ability of estrogen treatment to improve survival of bacterial challenge in the ethanol/burn group of mice, mice from the saline/burn and ethanol/burn + oil or ethanol/burn + estrogen groups were inoculated immediately postinjury with 1,000 CFUs of P. aeruginosa applied directly to the burn wound. As shown in Figure 8 , bacterial challenge resulted in approximately 76% survival in the saline/burn group of mice at 14 days postinjury. In contrast, there was only 9% survival in the ethanol/burn + oil group of mice. Estrogen treatment of ethanol/burn-injured mice resulted in a significant reduction (P<0.01) in the number of deaths, with 66% survival at the end of the 14-day experiment.



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  		Figure 8. Influence of estrogen treatment on percent survival from injury and bacterial challenge. Thirty minutes and 24 h postinjury, ethanol/burn mice were given an s.c. injection of either 0.1 mL of sesame oil or 17ß-estradiol (E2, 80 ng) in oil. Mice in the saline/burn group did not receive additional treatments postinjury. Immediately after burn injury, all groups of mice had 1,000 CFUs of P. aeruginosa applied topically to the burn wound sites. Data are percents survival in each group based on the total number of animals in that group. n = 9–12 mice per group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human males are at a significantly increased risk for developing septic complications after burn or other traumatic injury [8 , 9 ] and suffer from increased mortality in comparison with females that are matched for age and both type and severity of injury. The improved outcome in females has been attributed to the immune stimulatory effects of estrogen [1 ]. In support of this, recent studies using a hemorrhagic-shock model of injury found that administration of estrogen improved lymphocyte functions after injury [15 ]. The studies described in this report are the first to examine the immunomodulatory effects of estrogen treatment in a burn injury model. Furthermore, these studies identify a reduction in NF-{kappa}B-mediated cytokine production as a possible mechanism for the improved immune function after trauma in the presence of proestrus concentrations of estrogen.

These results contrast with others obtained by Gregory and colleagues [10 , 23 ], in which estrogen was found to be responsible for the decreased DTH and splenocyte-proliferative responses observed in female mice. but not in males at 10 days postburn. This discrepancy is the result of several factors. First, in the female mice, a 10-day time point was chosen to demonstrate maximal suppression of immunity postburn, whereas male mice were shown to exhibit maximal immune suppression between 24 and 48 h postburn. Additionally, the 1,000-fold-higher baseline estrogen concentration in females, combined with a spike (10- to 15-fold increase) in circulating hormone after injury [10 , 47 ], results in pregnancy levels of estrogen in female mice after burn injury, which are known to be immune suppressive [10 , 48 , 49 ]. In contrast, normal cycling concentrations of estrogen at the time of traumatic injury correlate with increased cellular immunity [14 , 15 , 50 ]. Thus, the effects of estrogen administration on immune function are most likely dose dependent and gender specific. This concept is supported by the finding that estrogen administration to ovariectomized mice treated with ethanol/burn (as described in the current study) does not restore the DTH or splenocyte-proliferative responses [K. A. N. Messingham and E. J. Kovacs, unpublished results]. It is probable that treatment of intact female mice with estrogen would have no effect on immunity or, more likely, might actually serve to augment immune dysfunction.

Normally, IL-6 is produced in response to a variety of stimuli, including LPS, IL-1, and TNF-{alpha}, and plays a key role in the regulation of the immune response, acute-phase response, and hematopoiesis [25 , 26 , 51 ]. However, after burn injury, abnormally elevated levels of IL-6 serve as predictive indices regarding the probability of a patient suffering not only from septic complications but also an unchecked inflammatory response (multiple-organ failure or systemic-inflammatory-response syndrome) ultimately resulting in death [27 28 29 , 52 ]. Experimental models of burn injury have demonstrated a robust elevation in both circulating and macrophage-derived IL-6 and have established a role for this aberrant cytokine production in the suppression of lymphocyte proliferation and cytokine production [23 , 24 ]. The data described in this report further demonstrate that male mice subjected to ethanol/burn injury displayed a suppression of splenocyte proliferation, which was mediated by macrophage-derived IL-6. Although the mechanism of this suppression of lymphocyte function by IL-6 is not fully understood, it might occur directly through the induction of G0/G1 cell cycle arrest [53 ] or indirectly by altering production of cytokines that decrease T cell functions, including transforming growth factor-ß [18 ] and prostaglandin E2 [54 ].

In our current studies, estrogen treatment of ethanol/burn-injured mice resulted in a significant but not complete ablation of macrophage IL-6 production. This finding, in light of the complete restoration of DTH and splenocyte-proliferative responses in estrogen-treated ethanol/burn-injured mice, might be due to the general stimulatory properties of estrogen on immunity [1 , 2 ]. However, it is also possible that a threshold concentration of IL-6 must be reached in the splenic microenvironment in order to exert suppressive effects on T cell function, which was not achieved in the estrogen-treated ethanol/burn mice, or that hormone treatment might render immune cells more resistant to the inhibitory effects of high IL-6 concentrations. Alternatively, estrogen treatment might also exert its beneficial effects through regulation of a variety of other cytokines (e.g., IL-2 or IL-4), which would influence lymphocyte function. In fact, previous studies suggest that IL-6 is not the only mediator of the decreased ear swelling response in the ethanol/burn-injured mice because in vivo neutralization of IL-6 results in only a partial restoration of the DTH response but complete restoration of splenocyte proliferation [37 ].

NF-{kappa}B activation is accepted as an integral component of a variety of inflammatory disorders; however, its role in immune suppression caused by burns, trauma, and sepsis has only recently been explored. Clinical studies have identified increased activation of NF-{kappa}B as a predictor of mortality in septic patients [55 , 56 ]. Additionally, Bohrer and colleagues [55 ] found that overexpression of I{kappa}B{alpha}, in a murine endotoxemia model increased survival, suggesting that NF-{kappa}B activation plays a direct role in the pathogenesis of septic shock. The current studies suggest that hormone modulation may improve outcome in injuries associated with a dysregulated inflammatory response. Conflicting reports exist as to whether estrogen modulates I{kappa}B{alpha}expression and/or phosphorylation [36 , 57 ]; however, in the current studies, administration of estrogen did not influence I{kappa}B{alpha} expression or phosphorylation in any experimental group. Thus, it is not likely that increased inhibitor binding to NF-{kappa}B is responsible for changes in DNA binding observed when ethanol/burn-injured mice are treated with estrogen.

The observation that nuclear extracts of splenocytes from saline/burn + oil mice had similar DNA binding to that of extracts from the ethanol/burn + oil mice was not expected because these mice are not immune suppressed and their macrophages do not produce significant amounts of IL-6. Because promoter or transcriptional activity was not evaluated per se, it is possible that either the NF-{kappa}B activation measured by gel shift does not translate directly to IL-6 gene transcription or other posttranscriptional changes are responsible for the differences in IL-6 production in these mice.

It has been suggested that the androgen-to-estrogen ratio might be more important to immune function than the concentration of either steroid alone [14 , 15 ]. However, pilot studies designed to examine the requirement of testosterone for estrogen to exert its beneficial effects on male mice suggest that the circulating level of estrogen might be more important in the current model of ethanol exposure followed by burn injury. In these studies, gonadectomized male mice given estrogen after injury had DTH and splenocyte-proliferative responses similar to those seen in intact males (Messingham and Kovacs, unpublished results).

In conclusion, these studies show that estrogen administration to male mice after the combined injuries of ethanol exposure followed by burns improved immunity, in part, by decreasing IL-6 production. These results support the necessity for gender-specific treatments for traumatic injury and suggest that manipulation of the hormonal milieu after injury might improve outcome. Moreover, these studies are the first to provide a potential mechanism through which the hormone-mediated restoration of immunity after injury could occur.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grants AA12034, AA11134, and AA11134-51. We sincerely thank Lisa A. Duffner, Elizabeth A. Durbin, M.S., and Christine V. Fontanilla, M.S., for their generous assistance with animal procedures and sample collection. Elisabeth Hahn, Ph.D., helped with infection studies. We also thank Lisa L. Shafer for her helpful comments regarding the manuscript.

Received January 31, 2001; revised July 30, 2001; accepted August 4, 2001.


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