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Am J Physiol Cell Physiol 293: C978-C984, 2007. First published June 6, 2007; doi:10.1152/ajpcell.00092.2007
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RECEPTORS AND SIGNAL TRANSDUCTION

Estrogen receptor-{alpha} predominantly mediates the salutary effects of 17beta-estradiol on splenic macrophages following trauma-hemorrhage

Takao Suzuki, Tomoharu Shimizu, Huang-Ping Yu, Ya-Ching Hsieh, Mashkoor A. Choudhry, Kirby I. Bland, and Irshad H. Chaudry

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

Submitted 7 March 2007 ; accepted in final form 3 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although 17beta-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)-{alpha} or ER-beta and which signaling pathways are involved in such 17beta-estradiol effects. Utilizing ER-{alpha}- or ER-beta-specific agonists, this study examined the role of ER-{alpha} and ER-beta in 17beta-estradiol-mediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-{kappa}B are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent trauma-hemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER-{alpha} agonist propyl pyrazole triol (PPT; 5 µg/kg), the ER-beta agonist diarylpropionitrile (DPN; 5 µg/kg), 17beta-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF-{alpha} production and activation of MAPK and NF-{kappa}B were measured. Macrophage IL-6 and TNF-{alpha} production and MAPK activation were decreased, whereas NF-{kappa}B activity was increased, following trauma-hemorrhage. PPT or 17beta-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17beta-estradiol in preventing the suppression in macrophage cytokine production, it appears that ER-{alpha} plays the predominant role in mediating the salutary effects of 17beta-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-{kappa}B signaling pathways.

shock; mitogen-activated protein kinase; nuclear factor-{kappa}B; propyl pyrazole triol; diarylpropionitrile

A PREVIOUS STUDY (3) has shown that the capacity of splenic macrophages to produce proinflammatory cytokines decreased in male animals following trauma-hemorrhage. This was associated with depressed immune functions and increased susceptibility to subsequent sepsis (15, 31). However, such suppression in the cytokine production capacity of splenic macrophages was not observed in proestrus female animals following trauma-hemorrhage (3, 22). Additional studies (2, 3) have shown that male sex steroids suppress but female sex steroids maintain cytokine production by splenic macrophages following trauma-hemorrhages. Studies (17, 18) have also indicated that the capacity of splenic macrophages to produce cytokines is normalized by the administration of a single dose of 17beta-estradiol following trauma-hemorrhage. Although the effects of 17beta-estradiol on splenic macrophages appear to be mediated via estrogen receptors (ERs) (16), there are two major subtypes of ER, namely, ER-{alpha} and ER-beta (19), and it remains unknown which subtype of ER predominantly conveys the salutary effects of 17beta-estradiol on splenic macrophages following trauma-hemorrhage. Moreover, the mechanism by which 17beta-estradiol mediates its effect remains to be determined.

MAPKs are signaling molecules that play an important role in the regulation of immune responses including macrophage activation and cytokine production (4, 6, 11, 21). There are three major MAPK-dependent pathways: p38, ERK1/2, and JNK. All three MAPK families are activated by phosphorylation on both adjacent threonine and tyrosine residues. In addition to MAPKs, NF-{kappa}B has also been implicated in macrophage cytokine production (7, 9, 38). Furthermore, recent studies (5, 14) have revealed that NF-{kappa}B is activated by MAPKs. Thus, NF-{kappa}B is likely a downstream factor of MAPK signaling pathways for macrophage cytokine production. However, it is not known whether MAPKs and NF-{kappa}B are involved in the mechanism of the salutary effects of 17beta-estradiol on splenic macrophages following trauma-hemorrhage. In this study, we examined whether the salutary effects of 17beta-estradiol on cytokine production by splenic macrophages are mediated via ER-{alpha} or ER-beta and whether MAPKs or NF-{kappa}B play a role in mediating such effects of 17beta-estradiol.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.

Adult male (275–325 g) Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were used in this study. All experiments were performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Alabama (Birmingham, AL).

Trauma-hemorrhage procedure.

A nonheparinized rat model of trauma-hemorrhage, as described previously (36), was used in this study. Briefly, male Sprague-Dawley rats (275–325 g) were fasted overnight before the experiment but allowed water ad libitum. Rats were anesthetized by isoflurane (Attane, Minrad, Bethlehem, PA) inhalation before the induction of soft tissue trauma (i.e., 5-cm midline laparotomy). The abdominal incision was then closed in two layers, and polyethylene catheters (PE-50, Becton-Dickinson, Sparks, MD) were placed in both femoral arteries and the right femoral vein. Rats were then placed into a Plexiglas box (21 x 9 x 5 cm) in a prone position and allowed to awaken, following which they were bled rapidly within 10 min to a mean arterial pressure (MAP) of 35–40 mmHg. This level of MAP was then kept by additional bleeding. The time after which the animals could no longer maintain a MAP of 35–40 mmHg without the infusion of some fluid is referred to as maximum bleedout (MBO). MAP was maintained at 40 mmHg until 40% of the shed blood was returned in the form of Ringer lactate solution. Animals were resuscitated with four times the shed blood volume with Ringer lactate solution over 60 min. Thirty minutes before the end of the resuscitation, rats received the ER-{alpha} agonist propyl pyrazole triol (PPT; 5 µg/kg body wt), the ER-beta agonist diarylpropionitrile (DPN; 5 µg/kg body wt), 17beta-estradiol (50 µg/kg body wt), or an equal volume of vehicle (~0.2 ml, 10% DMSO) subcutaneously. Doses of agonists were selected from an unpublished pilot study in which animals were treated with different doses and it was found that treatment of animals with PPT at 5 µg/kg body wt, DPN at 5 µg/kg body wt, or17beta-estradiol at 50 µg/kg body wt following trauma-hemorrhage normalized macrophage responses, similar to those observed in sham-operated animals. Following resuscitation, the catheters were removed, the vessels were ligated, and skin incisions were closed with sutures. Sham-operated animals underwent laparotomy and the same groin dissection, which included the ligation of the femoral artery and vein, but neither hemorrhage nor resuscitation was carried out. At 24 h after trauma-hemorrhage or sham operation, rats were anesthetized with isoflurane and exsanguinated to collect samples.

Isolation of splenic macrophages.

Spleens were removed aseptically and placed into 50-ml conical tubes with cold PBS (34). Spleens were then gently ground between frosted microscope slides to produce a single cell suspension and centrifuged at 400 g at 4°C for 15 min. Erythrocytes were lysed with buffer EL (Qiagen, Valencia, CA). The remaining cells were then washed, counted, suspended (1 x 107 cells/ml) in RPMI-1640, and incubated in 24-well plates. After 2 h of incubation (37°C at 5% CO2), nonadherent cells were removed, with the adherent cells being splenic macrophages. The procedure of adherence for macrophage isolation has been used for many years in several previous studies (1, 2). We routinely confirmed our preparations by spot checking the samples from time to time by 1) using cell morphology and 2) labeling cells with F4/80. We found that >95% cells were positive for macrophages (1, 2, 15).

Measurement of cytokine production.

Splenic macrophages were resuspended in 1 ml of fresh RPMI-1640 containing 10% FBS and antibiotics and cultured at 37°C and 5% CO2 for 24 h with 1 µg/ml LPS (from Escherichia coli 055:B5, Sigma). Supernatants were then harvested and analyzed for the presence of IL-6 and TNF-{alpha} using a DuoSet ELISA system (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Measurement of p38, ERK1/2, and JNK protein and phosphorylation levels.

As described previously (33), splenic macrophages were incubated with 1 µg/ml LPS for 30 min and lysed in lysis buffer. For the analysis of p38, ERK1/2, and JNK protein and phosphorylation, lysates were analyzed using SDS-PAGE and transferred to Immobilon P membranes (polyvinylidene difluoride, Millipore, Bedford, MA) using a semidry Trans-Blot system (Bio-Rad, Richmond, CA). Membranes were saturated with blocking buffer (10 mM Tris, 150 mM NaCl, and 0.05% Tween 20 supplemented with 5% dry milk) for 1 h at room temperature and incubated with antibodies to p38 protein, phospho-p38, ERK1/2 protein, phospho-ERK1/2, JNK protein, and phospho-JNK (Cell Signaling, Beverly, MA) at 4°C overnight. Membranes were then washed five times with Tris-buffered saline supplemented with 0.05% Tween 20 followed by an incubation with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Membranes were washed five times with Tris-buffered saline supplemented with 0.05% Tween 20 and probed using enhanced chemiluminescence dye, and phosphoproteins were autoradiographed.

Measurement of NF-{kappa}B DNA binding activity.

NF-{kappa}B DNA binding activity was measured using Trans-AM ELISA-based kits (Active Motif, Carlsbad, CA) according to the manufacturer's instructions (30). Briefly, whole cell lysates of splenic macrophages stimulated with or without 1 µg/ml LPS for 1 h were incubated in a 96-well plate coated with an oligonucleotide containing the NF-{kappa}B consensus binding site. Activated transcription factors from the lysates specifically bound to the respective immobilized oligonucleotide were detected using antibodies to NF-{kappa}B p65 followed by a secondary antibody conjugated to horseradish peroxidase in an ELISA-like assay.

Statistical analysis.

Data are presented as means ± SE. Statistical differences between groups were determined by one-way ANOVA followed by Tukey's test as a post hoc test. Differences were considered significant if P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cytokine production.

IL-6 and TNF-{alpha} production by splenic macrophages decreased following trauma-hemorrhage (Fig. 1). However, PPT or 17beta-estradiol administration following trauma-hemorrhage normalized the production of these cytokines by splenic macrophages. Although the suppression of IL-6 and TNF-{alpha} production by splenic macrophages was also attenuated by DPN treatment following trauma-hemorrhage, the splenic macrophage capacity to produce these cytokines still remained significantly lower than shams.


Figure 1
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  		Fig. 1. IL-6 and TNF-{alpha} production by splenic macrophages from sham-operated animals treated with vehicle (sham) and trauma-hemorrhage animals treated with vehicle, propyl pyrazole triol (PPT), diarylpropionitrile (DPN), or 17beta-estradiol (E2). Splenic macrophages were isolated 24 h after trauma-hemorrhage and cultured with LPS (1 µg/ml) for 24 h. Cytokine levels in culture supernatants were determined by ELISA. Data are means ± SE of 5–6 animals/group. *P < 0.05 compared with sham-operated animals by ANOVA; #P < 0.05 compared with sham-operated animals and trauma-hemorrhage animals treated with vehicle by ANOVA.

 
p38, ERK1/2, and JNK phosphorylation.

To determine MAPK activation, the phosphorylation of p38 (Fig. 2), ERK1/2 (Fig. 3), and JNK (Fig. 4) in splenic macrophages with or without LPS stimulation was evaluated. The phosphorylation of p38, ERK1/2, and JNK in unstimulated splenic macrophages was decreased following trauma-hemorrhage (Figs. 2A, 3A, and 4A). Stimulation of these cells with LPS resulted in an increase in the phosphorylation of all three MAPKs in splenic macrophages from animals in all groups (Figs. 2A, 3A, and 4A); however, LPS-induced phosphorylation of p38, ERK1/2, and JNK was significantly lower in splenic macrophages from rats subjected to trauma-hemorrhage compared with those from sham-operated rats (Figs. 2B, 3B, and 4B). The administration of PPT or 17beta-estradiol following trauma-hemorrhage prevented the decrease in p38, ERK1/2, and JNK phosphorylation in splenic macrophages. However, the administration of DPN following trauma-hemorrhage had no significant effect on the phosphorylation of p38, ERK1/2, and JNK in splenic macrophages. There were no significant differences in p38, ERK1/2, and JNK protein expression in splenic macrophages from rats subjected to trauma-hemorrhage compared with rats subjected to sham operation (Figs. 2C, 3C, and 4C).


Figure 2
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  		Fig. 2. p38 phosphorylation and protein expression in splenic macrophages with (LPS+) or without LPS stimulation (LPS–) from sham-operated animals treated with vehicle and trauma-hemorrhage animals treated with vehicle, PPT, DPN, or E2. Splenic macrophages were isolated 24 h after traumahemorrhage, stimulated with 1 µg/ml LPS for 30 min, and lysed. Lysates were then analyzed for p38 phosphorylation (p-p38) and protein expression (A). Blots were reprobed for beta-actin for equal protein loading in various lanes. p38 blots obtained from 5 animals were analyzed using densitometry, and densitometric values for phosphorylation (LPS+; B) and total protein (C) were normalized to beta-actin and are shown as means ± SE. *P < 0.05 compared with sham-operated animals.

 

Figure 3
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  		Fig. 3. ERK phosphorylation and protein expression in splenic macrophages with or without LPS stimulation from sham-operated animals treated with vehicle and trauma-hemorrhage animals treated with vehicle, PPT, DPN, or E2. Splenic macrophages were isolated 24 h after trauma-hemorrhage, stimulated with 1 µg/ml LPS for 30 min, and lysed. Lysates were then analyzed for ERK phosphorylation (p-ERK) and protein expression (A). Blots were reprobed for beta-actin for equal protein loading in various lanes. ERK blots obtained from 5 animals were analyzed using densitometry, and densitometric values for phosphorylation (LPS+; B) and total protein (C) were normalized to beta-actin and are shown as means ± SE. *P < 0.05 compared with sham-operated animals.

 

Figure 4
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  		Fig. 4. JNK phosphorylation and protein expression in splenic macrophages with or without LPS stimulation from sham-opearted animals treated with vehicle and trauma-hemorrhage animals treated with vehicle, PPT, DPN, or E2. Splenic macrophages were isolated 24 h after trauma-hemorrhage, stimulated with 1 µg/ml LPS for 30 min, and lysed. Lysates were then analyzed for JNK phosphorylation (p-JNK) and protein expression (A). Blots were reprobed for beta-actin for equal protein loading in various lanes. JNK blots obtained from 5 animals were analyzed using densitometry, and densitometric values for phosphorylation (LPS+; B) and total protein (C) were normalized to beta-actin and are shown as means ± SE. *P < 0.05 compared with sham-operated animals.

 
NF-{kappa}B DNA binding activity.

To determine the activation of NF-{kappa}B, the DNA binding activity of the p65 subunit in splenic macrophages with or without LPS stimulation was evaluated. The DNA binding activity of NF-{kappa}B (Fig. 5) in unstimulated splenic macrophages was increased following trauma-hemorrhage. Although LPS stimulation increased the NF-{kappa}B DNA binding activity in cells from animals in all groups, the LPS-induced activation of NF-{kappa}B was higher following trauma-hemorrhage compared with shams. However, PPT or 17beta-estradiol administration following trauma-hemorrhage normalized the DNA binding activity of NF-{kappa}B. DPN administration, by contrast, did not significantly influence the NF-{kappa}B DNA binding activity in splenic macrophages following trauma-hemorrhage.


Figure 5
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  		Fig. 5. NF-{kappa}B DNA binding activity in splenic macrophages with or without LPS stimulation from sham-operated animals treated with vehicle and trauma-hemorrhage animals treated with vehicle, PPT, DPN, or E2. Splenic macrophages were isolated 24 h after trauma-hemorrhage, stimulated with 1 µg/ml LPS for 1 h, and lysed. Lysates were then analyzed for DNA binding activity of p65 for NF-{kappa}B using an ELISA-based assay. OD, optical density. Data are shown as means ± SE. *P < 0.05 compared with sham-operated animals.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our results indicated that IL-6 and TNF-{alpha} production by splenic macrophages decreased following trauma-hemorrhage. This was accompanied by the suppression of the phosphorylation of p38, ERK1/2, and JNK. In contrast to cytokine production and MAPK activation, the DNA binding activity of NF-{kappa}B was increased following trauma-hemorrhage. The administration of the ER-{alpha} agonist PPT or 17beta-estradiol following trauma-hemorrhage restored the production of IL-6 and TNF-{alpha} by splenic macrophages to the levels observed in sham-operated animals. Moreover, PPT or 17beta-estradiol administration also prevented the suppression of MAPK activity and the activation of NF-{kappa}B in macrophages. Although the administration of the ER-beta agonist DPN attenuated the decrease in macrophage cytokine production, the levels were not restored to the levels observed in sham-operated animals. In addition, DPN administration had no effect on the activation of MAPKs and NF-{kappa}B. These findings suggest that the effects of 17beta-estradiol on macrophages are mediated predominantly via ER-{alpha}.

Macrophages play a critical role in the innate immune system (15, 27). A previous study (32) from our laboratory has shown that the depressed macrophage functions following trauma-hemorrhage were associated with an increased susceptibility to subsequent sepsis. Therefore, it appears important to prevent the suppression of macrophage functions following trauma-hemorrhage to maintain the immune system and avoid subsequent infection. Previous studies (3, 37) have demonstrated that proestrus female animals, with high circulating levels of estrogen, did not show decreased cytokine production by macrophages, and they had normal immune functions compared with male mice following trauma-hemorrhage. Furthermore, proestrus females had significantly lower mortality following trauma-hemorrhage and the induction of subsequent sepsis than male mice (3, 10). Our previous studies (17, 18) showed that the administration of a single dose of 17beta-estradiol following trauma-hemorrhage improved macrophage functions. However, blockade of ER by the administration of EM-800 abolished the salutary effects of 17beta-estradiol (16), suggesting that the salutary effects of 17beta-estradiol are mediated via ER.

ER-{alpha} and ER-beta are the two major ER subtypes. Since the two subtypes of ER have different tissue distribution (19, 39), it appears important to determine which subtype of ER contributes to the effects of 17beta-estradiol in the target cells. In this study, we used selective agonists for ER subtypes. PPT is a selective ER-{alpha} agonist that has 410-fold binding selectivity over ER-beta (31). On the other hand, DPN, which is a selective ER-beta agonist, has 70-fold higher relative binding affinity and 170-fold higher relative estrogenic potency in transcription assays with ER-beta than ER-{alpha} (23). Although we did not determine the expression of ERs on macrophages in this study, it has been reported that both ER-{alpha} and ER-beta are expressed in the spleen and that ER-{alpha} is important for the function of splenic macrophages (20, 28, 29), which is consistent with our present finding of the predominance of the ER-{alpha} response in splenic macrophages following trauma-hemorrhage. Since in our study we did not determine the ER expression, the possibility that trauma-hemorrhage may influence the distribution of these receptors is not ruled out. It is also possible that the administration of PPT or DPN alters the level of the expression of ER-{alpha} or ER-beta. However, it remains to be determined whether PPT or DPN administration influences the expression of ER-{alpha} or ER-beta following trauma-hemorrhage.

A previous study (19) has indicated that there is no mRNA expression of ERs in the rat spleen. Furthermore, our preliminary analysis also did not reveal mRNA transcripts for ERs in spleen tissue (data not shown). However, whether the rat spleen expresses ER protein remains to be established, and, therefore, more studies are needed to determine the expression of ERs in the rat spleen. These studies should utilize both whole spleen tissue and isolated cells, and only then can a final conclusion can be drawn as to whether or not rat splenocytes express ERs. We (2, 25) have previously have shown that splenic T cells from female mice have relatively more ER-{alpha} mRNA expression compared with ER-beta, which is associated with the maintenance of T lymphocyte cytokine release. This study also investigated the mechanism by which 17beta-estradiol mediates the beneficial effect on macrophage cytokine production following trauma-hemorrhage. MAPKs play an important role in the regulation of macrophage activation (11, 24). Moreover, the production of various cytokines by macrophages involves MAPK signal transduction pathways (1, 11). The present results demonstrated that the activation of p38, ERK1/2, and JNK was suppressed in macrophages following trauma-hemorrhage. This suppression of MAPK activity was even seen in cells without additional stimulation in vitro, which suggests that the stress of trauma-hemorrhage per se suppresses some signaling pathways leading to MAPKs. The administration of PPT or 17beta-estradiol following trauma-hemorrhage prevented such suppression of MAPK activation in macrophages. Hence, it appears that PPT or 17beta-estradiol administration following trauma-hemorrhage normalizes macrophage cytokine production by preventing the suppression of p38, ERK, and JNK pathways. However, it still remains to be determined whether all three MAPKs or any one of them has a predominant role in producing the salutary effects of 17beta-estradiol or PPT under those conditions. In contrast to PPT and 17beta-estradiol, DPN administration had no effect on the activation of MAPKs in splenic macrophages despite the attenuation of the suppression in cytokine production in those cells. This finding suggests that signaling pathways other than those of MAPKs contribute to the effect of DPN on cytokine production under those conditions.

We found that in contrast to cytokine production and MAPK activation, NF-{kappa}B activity was increased in macrophages following trauma-hemorrhage. Although NF-{kappa}B has been shown to contribute to various gene expressions including IL-6 and TNF-{alpha} in macrophages (7, 9, 37), our present results suggest that NF-{kappa}B appears not to mediate these cytokines production by splenic macrophages following trauma-hemorrhage. However, the administration of PPT or 17beta-estradiol averted the elevation of NF-{kappa}B activity, whereas DPN treatment had no effect. Therefore, it is likely that NF-{kappa}B mediates some effects of 17beta-estradiol or PPT rather than the effects on MAPK pathways and cytokine production capacity in macrophages. In addition, we recently found that NF-{kappa}B activity as well as cytokine productive capacity was decreased in splenic macrophages 2 h after trauma-hemorrhage. This finding and the present results suggest that NF-{kappa}B activity is suppressed at an early time point after trauma-hemorrhage and then is increased later. Thus, it is possible that the suppression in cytokine production by splenic macrophages following trauma-hemorrhage is mediated by NF-{kappa}B at an early time point but not at a late time point.

It has been suggested that MAPKs regulate cytokine expression not only at the transcriptional level but also at the posttranscriptional level. p38 regulates IL-6 and TNF-{alpha} expression by both stabilizing their mRNAs and promoting their translation. This role of p38 is mediated by its substrate, MAPMAPK-2 (8). ERK and JNK also regulate TNF-{alpha} induction by activating mRNA translation (12, 35). In view of this, according to our findings in this study, we can speculate that MAPKs posttranscriptionally regulate cytokine production whether NF-{kappa}B regulates the mRNA expression of cytokines or not. However, further investigations need to be performed to elucidate the detailed mechanism.

It can be argued that the present study utilized measurement at a single time point, i.e., at 24 h after treatment, and thus it remains unclear whether the salutary effects of 17beta-estradiol or PPT on macrophage signaling and cytokine production are sustained for longer than 24 h after treatment. Our previous studies (3, 10, 26), however, have shown that if the improvement in cell and organ function by any pharmacological agent is evident at 2, 5, or 24 h after treatment, those salutary effects are sustained for prolonged intervals and also improve the survival of animals. Thus, although a time point other than 24 h was not examined in this study, based on our previous work it would appear that the effects of 17beta-estradiol or PPT on macrophage cytokine release and MAPK and NF-{kappa}B activation would be evident even if one measured those effects at another time point following trauma-hemorrhage and resuscitation.

It could also be suggested that we should have administered 17beta-estradiol, PPT, or DPN alone in sham-operated groups in these experiments to determine if each of these treatments per se has any adverse or salutary effects. However, our recent study (13) has shown that the administration of PPT or DPN alone in sham groups did not produce any deleterious or salutary effects. Therefore, PPT or DPN administration in sham-operated animals was not carried out in this study.

In summary, our findings indicated that cytokine productive capacity and MAPK activation are suppressed, whereas NF-{kappa}B activity is increased, following trauma-hemorrhage. The administration of the ER-{alpha} agonist PPT or 17beta-estradiol was equally effective following trauma-hemorrhage in normalizing macrophage cytokine production and the activation of MAPK and NF-{kappa}B. The administration of the ER-beta agonist DPN, on the other hand, had less or no effect on those parameters. Thus, it can be concluded that the salutary effects of 17beta-estradiol on macrophage cytokine production following trauma-hemorrhage are mediated predominantly via ER-{alpha} and that these beneficial effects are likely mediated via MAPK pathways but not NF-{kappa}B at 24 h after trauma-hemorrhage.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of General Medical Sciences Grants R37-GM-39519 and R01-GM-37127.


   ACKNOWLEDGMENTS
 
Present address of H.-P. Yu: Department of Anesthesiology, College of Medicine Chang Gung University and Chang Gung Memorial Hospital, Taoyuan, Taiwan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Chaudry, Center for Surgical Research and Dept. of Surgery, Univ. of Alabama, at Birmingham, 1670 Univ. Blvd., Volker Hall, Rm. G094, 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.


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