RESUMEN
BACKGROUND: Although 17α-ethinyl estradiol-3-sulfate (EES) reduces mortality in animal models of controlled hemorrhage, its role in a clinically relevant injury model is unknown. We assessed the impact of EES in a swine model of multiple injuries and hemorrhage. METHODS: The study was performed under Good Laboratory Practice, with 30 male uncastrated swine (25-50 kg) subjected to tibial fracture, pulmonary contusion, and 30% controlled hemorrhage for an hour. Animals were randomized to one of five EES doses: 0 (control), 0.3, 1, 3, and 5 mg/kg, administered postinjury. Subjects received no resuscitation and were observed for 6 hours or until death. Survival data were analyzed using Cox-proportional hazard regression. Left ventricular pressure-volume loops were used to derive preload recruitable stroke work as a measure of cardiac inotropy. Immediate postinjury preload recruitable stroke work values were compared with values at 1 hour post-drug administration. RESULTS: Six-hour survival for the 0, 0.3, 1, 3, and 5 mg/kg groups was 0%, 50%, 33.3%, 16.7%, and 0%, respectively. Following Cox regression, the hazard (95% confidence interval) of death was significantly reduced in the 0.3 (0.22 [0.05-0.93]) and 1 (0.24 [0.06-0.89]) mg/kg groups but not the 3 (0.49 [0.15-1.64]) and 5 (0.46 [0.14-1.47]) mg/kg groups. Mean survival time was significantly extended in the 1 mg/kg group (246 minutes) versus the 0 mg/kg group (96 minutes) (p = 0.04, t test). At 1 hour post-drug administration, inotropy was significantly higher than postinjury values in the 0.3 and 1 mg/kg groups (p = 0.003 and p < 0.001, respectively). Inotropy was unchanged in the 3 and 5 mg/kg groups but significantly depressed in the control (p = 0.022). CONCLUSION: Administration of EES even in the absence of fluid resuscitation reduces mortality and improves cardiac inotropy in a clinically relevant swine model of multiple injuries and hemorrhage. These findings support the need for a clinical trial in human trauma patients.
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Etinilestradiol/análogos & derivados , Traumatismo Múltiple/complicaciones , Choque Hemorrágico , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas/métodos , Estrógenos/análogos & derivados , Estrógenos/farmacología , Etinilestradiol/farmacología , Masculino , Contracción Miocárdica/efectos de los fármacos , Choque Hemorrágico/tratamiento farmacológico , Choque Hemorrágico/etiología , Choque Hemorrágico/fisiopatología , Análisis de Supervivencia , Porcinos , Resultado del TratamientoRESUMEN
BACKGROUND: Our in vivo rodent and pig model evidenced that estrogen and its derivative, ethinyl estradiol sulfate (EES), promote survival following hemorrhagic shock. To determine its mechanism, we first confirmed EES binding to estrogen receptor (ER) and improving/restoring cellular signaling, countering the assumption that EES, an ethinyl estradiol metabolite, is inactive. In addition, we examined if EES acts rapidly, consistent with nongenomic signaling. We selected the biomarkers of cardiovascular performance, reduction of apoptosis and proinflammatory responses, and elaboration of nitric oxide (NO) to validate efficacy. METHODS: A rat trauma-hemorrhage model, consisting of a midline laparotomy and controlled bleeding (60% blood loss) without fluid resuscitation, was used. At 30 minutes after hemorrhage, heart performance was monitored, and Western blots were used to quantify biochemical analytes. The specificity of EES for ER was profiled with ER antagonists. Binding studies by Sekisui XenoTech (Kansas City, KS) determined an LD50 value for EES binding the rat ER. RESULTS: The EES IC50 value was 1.52 × 10-8 Mol/L, consistent with pharmacologic efficacy. Ethinyl estradiol sulfate raised mean arterial pressure and ±derivative of pressure over time (dP/dT) significantly (but did not fully restore) within a 30-minute window. Levels of apoptosis and activation of NF-κB were dramatically reduced, as was elaboration of nitric oxide (NO) by inducible nitric oxide synthase. Phospho-endothelial nitric oxide synthase (eNOS) was restored to physiological levels. The restoration of cellular signaling occurs before restoration of cardiac contractility. CONCLUSION: Ethinyl estradiol sulfate is a potent drug for improving heart performance, which also dramatically reduces damage by apoptosis, proinflammatory activity, and NO production, validating that EES can blunt multiple harmful outcomes arising from hypoxia and hypovolemia. The actions are dependent on receptor engagement, where specificity is confirmed by ER antagonists. The constraint of a 30-minute sampling window affirms that the responses are nongenomic and very likely restricted to cell-surface receptor engagement. The rapidity of these responses makes EES promising for intervention in the "golden hour."
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Sistema Cardiovascular , Etinilestradiol/análogos & derivados , Receptores de Estrógenos , Choque Hemorrágico , Animales , Apoptosis/efectos de los fármacos , Sistema Cardiovascular/efectos de los fármacos , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estrógenos/farmacología , Etinilestradiol/farmacología , Inflamación/tratamiento farmacológico , FN-kappa B/metabolismo , Óxido Nítrico/análisis , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Choque Hemorrágico/tratamiento farmacológico , Choque Hemorrágico/metabolismo , Choque Hemorrágico/fisiopatología , Transducción de Señal/efectos de los fármacos , Porcinos , Resultado del TratamientoRESUMEN
OBJECTIVE 17α-ethynylestradiol-3-sulfate (EE-3-SO4) is a highly water-soluble synthetic estrogen that has an extended half-life (â¼ 10 hours) over that of naturally occurring estrogen (â¼ 10 minutes). In this study, EE-3-SO4 was evaluated in a lateral fluid percussion-induced traumatic brain injury (TBI) model in rats. METHODS A total of 9 groups of Sprague-Dawley rats underwent craniectomy. Twenty-four hours later, lateral fluid percussion was applied to 6 groups of animals to induce TBI; the remaining 3 groups served as sham control groups. EE-3-SO4 (1 mg/kg body weight in 0.4 ml/kg body weight) or saline (vehicle control) was injected intravenously 1 hour after TBI; saline was injected in all sham animals. One day after EE-3-SO4/saline injection, intracranial pressure (ICP), cerebral perfusion pressure (CPP), and partial brain oxygen pressure (PbtO2) were measured in Groups 1-3 (2 TBI groups and 1 sham group), and brain edema, diffusion axonal injury, and cerebral glycolysis were assessed in Groups 4-6 using MRI T2 mapping, diffusion tensor imaging (DTI), and FDG-PET imaging, respectively. Four days after dosing, the open-field anxiety of animals was assessed in Groups 7-9 by measuring the duration that each animal spent in the center area of an open chamber during 4 minutes of monitoring. RESULTS EE-3-SO4 significantly lowered ICP while raising CPP and PbtO2, compared with vehicle treatment in TBI-induced animals (p < 0.05). The mean size of cerebral edema of TBI animals treated with EE-3-SO4 was 25 ± 3 mm3 (mean ± SE), which was significantly smaller than that of vehicle-treated animals (67 ± 6 mm3, p < 0.001). Also, EE-3-SO4 treatment significantly increased the fractional anisotropy of the white matter in the ipsilateral side (p = 0.003) and cerebral glycolysis (p = 0.014). The mean duration that EE-3-SO4-treated animals spent in the center area was 12 ± 2 seconds, which was significantly longer than that of vehicle-treated animals (4 ± 1 seconds; p = 0.008) but not different from that of sham animals (11 ± 3 seconds; p > 0.05). CONCLUSIONS These data support the clinical use of EE-3-SO4 for early TBI treatment.
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Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Etinilestradiol/análogos & derivados , Animales , Modelos Animales de Enfermedad , Etinilestradiol/farmacología , Etinilestradiol/uso terapéutico , Conducta Exploratoria/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
As with sharks and horseshoe crabs, some designs of nature need only minor evolutionary adjustments during the millennia to remain superbly adapted. Such is the case at the molecular level for the nuclear receptors (NRs), which seem to have originated concomitantly with the earliest metazoan lineage of animals. A wide array of NRs persists today throughout all animal phyla with many different functions, yet they share a highly conserved protein structure, a testament to their having evolved through numerous gene duplications. Of particular interest for this readership are the estrogen-related receptors (ERRs), which have significant supportive roles in energy creation and regulation, mitochondrial function and biogenesis, development, tissue repair, hypoxia, and cancer. Thus, placed at the nexus of energetics and homeostasis, ERR (in association with the coregulatory molecules peroxisome proliferator-activated receptor-γ coactivator-1α and -ß) can facilitate repair from injury and adaptations to stressful environments. Whereas it is curious that ERRs and some other NRs exist as "orphans" by virtue of having no known cognate ligand, increasing interest in the estrogen receptor has led to the development of synthetic ligands and screening for naturally occurring molecules, either capable of modulating ERR activity. Thus, what is needed now is a nomenclature update for the ERR to focus the mind on energetics and metabolism, the most compromised and crucial systems after trauma and shock.
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Metabolismo Energético , Evolución Molecular , Mitocondrias , Receptores de Estrógenos , Choque , Heridas y Lesiones , Animales , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Choque/genética , Choque/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Heridas y Lesiones/genética , Heridas y Lesiones/metabolismoRESUMEN
Estrogen plays an important role as a neuroprotector in the central nervous system (CNS), directly interacting with neurons and regulating physiological properties of non-neuronal cells. Here we evaluated estrogen sulfate (E2-SO4) for traumatic brain injury (TBI) using a Sprague-Dawley rat model. TBI was induced via lateral fluid percussion (LFP) at 24 h after craniectomy. E2-SO4 (1 mg/kg BW in 1 mL/kg BW) or saline (served as control) was intravenously administered at 1 h after TBI (n=5/group). Intracranial pressure (ICP), cerebral perfusion pressure (CPP), and partial brain oxygen pressure (pbtO2) were measured for 2 h (from 23 to 25 h after E2-SO4 injection). Brain edema and diffuse axonal injury (DAI) were assessed by diffusion tensor imaging (DTI), and cerebral glycolysis was measured by (18)F-labeled fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging, at 1 and 7 days after E2-SO4 injection. E2-SO4 significantly decreased ICP, while increasing CPP and pbtO2 (p<0.05) as compared with vehicle-treated TBI rats. The edema size in the brains of the E2-SO4 treated group was also significantly smaller than that of vehicle-treated group at 1 day after E2-SO4 injection (p=0.04), and cerebral glycolysis of injured region was also increased significantly during the same time period (p=0.04). However, E2-SO4 treatment did not affect DAI (p>0.05). These findings demonstrated the potential benefits of E2-SO4 in TBI.
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Edema Encefálico/tratamiento farmacológico , Lesiones Encefálicas/tratamiento farmacológico , Estrona/análogos & derivados , Glucólisis/efectos de los fármacos , Animales , Edema Encefálico/diagnóstico , Edema Encefálico/etiología , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/diagnóstico , Lesión Axonal Difusa/diagnóstico , Lesión Axonal Difusa/tratamiento farmacológico , Imagen de Difusión Tensora , Modelos Animales de Enfermedad , Estrona/administración & dosificación , Estrona/farmacología , Fluorodesoxiglucosa F18 , Masculino , Tomografía de Emisión de Positrones , Radiofármacos , Ratas , Ratas Sprague-DawleyRESUMEN
During sepsis, a complex network of cytokine, immune, and endothelial cell interactions occur and disturbances in the microcirculation cause organ dysfunction or even failure leading to high mortality in those patients. In this respect, numerous experimental and clinical studies indicate sex-specific differences in infectious diseases and sepsis. Female gender has been demonstrated to be protective under such conditions, whereas male gender may be deleterious due to a diminished cell-mediated immune response and cardiovascular functions. Male sex hormones, i.e., androgens, have been shown to be suppressive on cell-mediated immune responses. In contrast, female sex hormones exhibit protective effects which may contribute to the natural advantages of females under septic conditions. Thus, the hormonal status has to be considered when treating septic patients. Therefore, potential therapies could be derived from this knowledge. In this respect, administration of female sex hormones (estrogens and their precursors) may exert beneficial effects. Alternatively, blockade of male sex hormone receptors could result in maintained immune responses under adverse circulatory conditions. Finally, administration of agents that influence enzymes synthesizing female sex hormones which attenuate the levels of pro-inflammatory agents might exert salutary effects in septic patients. Prospective patient studies are required for transferring those important experimental findings into the clinical arena.
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Andrógenos/inmunología , Estrógenos/inmunología , Choque Séptico/epidemiología , Choque Séptico/inmunología , Antagonistas de Receptores Androgénicos/uso terapéutico , Animales , Sistema Cardiovascular/inmunología , Deshidroepiandrosterona/uso terapéutico , Femenino , Flutamida/uso terapéutico , Humanos , Inmunidad Celular , Masculino , Trasplante de Células Madre Mesenquimatosas , Ratones , Factores Sexuales , Choque Séptico/prevención & controlRESUMEN
Abstract Traumatic brain injury (TBI) is a significant public health problem in the United States. Despite preclinical success of various drugs, to date all clinical trials investigating potential therapeutics have failed. Recently, sex steroid hormones have sparked interest as possible neuroprotective agents after traumatic injury. One of these is 17ß-estradiol (E2), the most abundant and potent endogenous vertebrate estrogen. The goal of our study was to investigate the acute potential protective effects of E2 or the specific G protein-coupled estrogen receptor 1 (GPER) agonist G-1 when administered in an intravenous bolus dose 1 hour post-injury in the lateral fluid percussion (LFP) rodent model of TBI. The results of this study show that, when assessed at 24 hours post-injury, E2 or G-1 confers protection in adult male rats subjected to LFP brain injury. Specifically, we found that an acute bolus dose of E2 or G-1 administered intravenously 1 hour post-TBI significantly increases neuronal survival in the ipsilateral CA 2/3 region of the hippocampus and decreases neuronal degeneration and apoptotic cell death in both the ipsilateral cortex and CA 2/3 region of the hippocampus. We also report a significant reduction in astrogliosis in the ipsilateral cortex, hilus, and CA 2/3 region of the hippocampus. Finally, these effects were observed to be chiefly dose-dependent for E2, with the 5 mg/kg dose generating a more robust level of protection. Our findings further elucidate estrogenic compounds as a clinically relevant pharmacotherapeutic strategy for treatment of secondary injury following TBI, and intriguingly, reveal a novel potential therapeutic target in GPER.
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Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/prevención & control , Estradiol/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Animales , Lesiones Encefálicas/patología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Masculino , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The aged patient stands at the nexus of significant biomedical and bioethical issues in transplantation. This in itself can be seen as a microcosm of an imminent global tempest, stemming from expanding numbers and longer lives of the aged population. As a candidate for receiving organ and tissue transplants, the geriatric patient is challenging because they present unique physiology for medical management. As organ and tissue donors, the aged are perceived of as providing "marginal" organs, which drives the fear that the graft will fail before the recipient. Such difficulties lead inexorably to several unique bioethical considerations for transplantation with this population. The thorny conundrums for society hinge on fairness versus discrimination based on age, played out under the enormous and probably intractable problem of severe donor organ shortages. Fortunately, recent findings offer some rather unexpected new and favorable prospects. Notably, aged donors can provide organs with good, lifesaving function, even though there are nonetheless age-related compromises present. On the other side of the coin, there is less doubt that recipients can have their lives extended with high quality through transplantation. Here they benefit from some (counterintuitively) positive attributes for aging, such as reduced immune function, making immunosuppression less rigorous. Finally, the pressure of organ and tissue shortages plus the lifting of bans on embryonic stem cell research have portents for an explosive alternative to transplantation of adult organs. Stem cells also lend credibility to prospects for realizing regenerative medicine, assuming ethical and religious concerns can be satisfied.
RESUMEN
BACKGROUND: This study evaluated the effect of 17ß-estradiol (E2) administration on cardiovascular parameters in male rats after trauma-hemorrhage and for an extended period (3 hours) of severe hypotension, based on blood-pool single photon emission computed tomography imaging. METHODS: After a 5-cm midline laparotomy, male Sprague-Dawley rats were injected intravenously with Tc-bovine serum albumin; the animals were then bled for >45 minutes to reach maximum bleedout (MBO; removal of 60% of the circulating blood volume). E2 (1 mg/kg body weight, 0.4 mL/kg) or vehicle (cyclodextrin [CD], 0.4 mL/kg) was injected intravenously at MBO; no additional fluid was administered for 3 hours. Imaging was performed continually for a maximum of 3 hour post-MBO. The percentages of injected dose in heart, brain, liver, and kidney were quantified from the imaging (n = 8/group) at 0 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes post-MBO. Mixed model analysis of variance was used to analyze the difference between the two groups over six imaging time points (30-180 minutes post-MBO). RESULTS: The percentages of injected dose of Tc-bovine serum albumin in heart, kidney, and liver after E2 administration were significantly higher than those after CD administration (p = 0.036, 0.025, and 0.028 for heart, kidney, and liver, respectively), whereas those in brain were not different between E2 and CD administration (p = 0.343). CONCLUSIONS: The significantly larger blood volume maintained in heart, kidney, and liver of rats after E2 therapy compared with control supports the notion that E2 produces salutary effects on the cardiovascular system after trauma-hemorrhage and even extended periods of severe hypotension.
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Hipotensión/tratamiento farmacológico , Choque Hemorrágico/tratamiento farmacológico , Tomografía Computarizada de Emisión de Fotón Único , Heridas y Lesiones/complicaciones , Animales , Presión Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Estradiol/uso terapéutico , Hipotensión/diagnóstico por imagen , Hipotensión/etiología , Masculino , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Choque Hemorrágico/diagnóstico por imagen , Choque Hemorrágico/etiología , Heridas y Lesiones/fisiopatologíaRESUMEN
Healing of the burn wound is a critical component of the burn patient's successful recovery. While inflammation is a critical component of the healing process, it is unknown whether the inflammatory response differs between non-burn and burn wounds. To study this, mice were subjected to major burn injury or sham procedure. Wound cells were collected by implantation of polyvinyl alcohol sponges beneath the burn site in injured mice or beneath uninjured skin in sham mice (i.e., non-burn wound). Three days thereafter, skin, wound fluid, and infiltrating cells were collected for analysis. Significant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL-6), monocyte chemoattractant protein (MCP)-1, and keratinocyte-derived chemokine (KC) were observed in burn wound tissue and the wound fluid from both non-burn and burn wounds. Burn injury induced 3-fold higher levels of KC and 50-fold higher levels of IL-6 in the wound fluid compared with non-burn injury. Significant numbers of the cells from both burn and non-burn wounds were CD11b(+), GR1(+), and F4/80(+), suggestive of a myeloid suppressor cell phenotype, whereas CD3(+) T-cells were negligible under both conditions. LPS induced TNF-alpha, IL-6, IL-10, MCP-1, KC, and nitric oxide production in both cell populations, however, IL-6, IL-10, MCP-1, and KC levels were suppressed in burn wound cell cultures. These findings indicate that significant differences in the wound inflammatory response exist between burn and non-burn cutaneous wounds and that the unique characteristics of the inflammatory response at the burn site may be an important contributing factor to post-burn wound healing complications.
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Quemaduras/inmunología , Quemaduras/patología , Citocinas/biosíntesis , Mediadores de Inflamación/análisis , Piel/inmunología , Animales , Antígeno CD11b/análisis , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Quimiocina/análisisRESUMEN
Reduced immune function is frequently a consequence of serious injury such as trauma-hemorrhage (T-H). Injury may lead to reduced T-cell activation, resulting in decreased engagement of costimulatory molecules after antigen recognition and in subsequent immunological compromise and anergy. We hypothesized that inhibition of CD28 expression is one possible mechanism by which immune functions are suppressed after T-H. Male C3H/HeN mice (with or without ovalbumin immunization) were subjected to sham operation or T-H and sacrificed after 24 hours. Splenic T cells were then stimulated with concanavalin A or ovalbumin in vivo or in vitro, and CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD69, and phospho-Akt expression was determined. T-cell proliferation/cytokine production was measured in vitro. Stimulation-induced CD69, CD28, and phospho-Akt up-regulation were significantly impaired after T-H compared with sham-operated animals; however, CTLA-4 expression was significantly higher in the T-H group. Over a 3-day span, stimulated T cells from sham-operated animals showed significantly higher proliferation compared with the T-H group. IL-2 and IFN-gamma were elevated in sham-operated animals, whereas IL-4 and IL-5 rose in the T-H group, revealing a shift from T(H)1 to T(H)2 type cytokine production after T-H. Dysregulation of the T-cell costimulatory pathway is therefore likely to be a significant contributor to post-traumatic immune suppression.
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Linfocitos T CD4-Positivos/inmunología , Tolerancia Inmunológica/inmunología , Activación de Linfocitos/inmunología , Transducción de Señal/inmunología , Bazo/citología , Heridas y Lesiones/inmunología , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/enzimología , Antígeno CTLA-4 , Proliferación Celular , Células Cultivadas , Concanavalina A/inmunología , Citocinas/biosíntesis , Activación Enzimática , Hemorragia/inmunología , Lectinas Tipo C , Ratones , Ovalbúmina/inmunología , Proteínas Proto-Oncogénicas c-akt , Bazo/inmunologíaRESUMEN
Healing of the burn injury site is a critical component of the patient's successful recovery from this form of trauma. Previous studies from our laboratory have demonstrated that gammadelta T-cells via the production of growth factors are important in burn wound healing. Nonetheless, the role of these cells in burn wound inflammation remains unknown. To study this, wild-type (WT) and gammadelta T-cell receptor-deficient (delta TCR) C57BL/6 male mice were subjected to burn injury or sham procedure. Wound cells were collected by implantation of polyvinyl alcohol sponges beneath the burn site in injured mice or beneath uninjured skin in sham mice. At 3 days after injury, infiltrating cells, wound fluid, and skin were collected for analysis. Burn injury markedly increased skin tumor necrosis factor-alpha (TNF-alpha) and monocyte chemoattractant protein 1 levels. In WT mice, the numbers of infiltrating cells were similar between nonburn wounds and burn wounds. In contrast, deltaTCRmice displayed a 6-fold reduction in the cellular infiltrate. Burn injury in WT mice caused a marked increase in burn wound TNF-alpha, monocyte chemoattractant protein 1, and interleukin 6 content as compared with nonburn wounds, whereas in delta TCRmice, the burn-induced increase of TNF-alpha and interleukin 6 was not observed. The wound cell infiltrate at 3 days postinjury was devoid of gammadelta T-cells in WT mice. It was predominately of myeloid origin expressing high levels of CD11b and F4/80. In conclusion, these findings suggest that resident gammadelta T-cells are important in the recruitment of inflammatory cells and regulation of the inflammatory response at the wound site after thermal injury.
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Quemaduras/fisiopatología , Dermatitis/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Subgrupos de Linfocitos T/fisiología , Cicatrización de Heridas/fisiología , Animales , Antígenos de Diferenciación/biosíntesis , Quemaduras/patología , Antígeno CD11b/biosíntesis , Complejo CD3/biosíntesis , Quimiocina CCL2/biosíntesis , Citocinas/metabolismo , Interleucina-6/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Piel/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.
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Apoptosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Estradiol/farmacología , Peróxido de Hidrógeno/farmacología , Mitocondrias/fisiología , Animales , Aorta/citología , Caspasas/fisiología , Células Cultivadas , Masculino , Mitocondrias/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Sustancias Protectoras/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll-like receptors (TLR). Although mitogen-activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP approximately 35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF-alpha, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF-alpha, MCP-1, and KC production by Kupffer cells were significantly increased following trauma-hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG-stimulated TLR9 signaling increased TNF-alpha and IL-6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, -4, and -9 signaling. In contrast, TNF-alpha and IL-6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma-hemorrhage.
Asunto(s)
Hemorragia/fisiopatología , Macrófagos del Hígado/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 4/fisiología , Receptor Toll-Like 9/fisiología , Animales , Células Cultivadas , Quimiocina CCL2/metabolismo , Hemorragia/patología , Interleucina-6/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Macrófagos del Hígado/patología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C3H , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 9/agonistas , Factor de Necrosis Tumoral alfa/metabolismo , Heridas y Lesiones/patología , Heridas y Lesiones/fisiopatología , Zimosan/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Although Kupffer cell, splenic, and peritoneal macrophage functions are markedly altered following trauma-hemorrhage (T-H), it remains unclear whether T-H also affects splenic dendritic cell (sDC) functions. We hypothesized that sDC functions will also be compromised following T-H. Male C3H/HeN (6- to 8-wk) mice were randomly assigned to sham operation or T-H. T-H was induced by midline laparotomy and approximately 90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (four times the shed blood volume in the form of Ringer's lactate). Two hours later, the mice were sacrificed; sDC were isolated; and the changes in their apoptosis, MHC class II expression, and ability to produce costimulatory cytokines and Ag presentation were measured. The results indicate that sDC Ag presentation capacity was significantly decreased and MHC class II expression was also significantly decreased following T-H. Moreover, LPS-induced IL-12 production and LPS- or IL-12-induced IFN-gamma production following T-H were significantly decreased. Thus, the markedly decreased MHC class II expression and cytokine (IL-12, IFN-gamma) production following T-H may be the cause for the depressed sDC Ag presentation under those conditions. This depression in Ag presentation could contribute to the host's enhanced susceptibility to sepsis following T-H.
Asunto(s)
Células Dendríticas/inmunología , Hemorragia/inmunología , Bazo/inmunología , Heridas y Lesiones/inmunología , Animales , Presentación de Antígeno/inmunología , Apoptosis/inmunología , Citocinas/sangre , Modelos Animales de Enfermedad , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II , Masculino , RatonesRESUMEN
Posttraumatic activation of macrophages enhances development of systemic inflammation/immunosuppression and organ dysfunction. We hypothesized that Kupffer cells are the main source of monocyte chemoattractant protein-1 (MCP-1) production after trauma-hemorrhage, that administration of 17beta-estradiol (E2) after trauma-hemorrhage modulates MCP-1 release and reduces remote organ damage, and that salutary effects of E2 are mediated via estrogen receptor (ER)-alpha. To test these hypotheses, female B57BL/J6 mice received E2 (50 microg/25 g) or vehicle after trauma-hemorrhage and female 129 Sve ER-beta-/- transgenic mice and ovariectomized wild-type mice received E2 or ER-alpha agonist propyl pyrazole triol (50 microg/25 g) after trauma-hemorrhage. Systemic MCP-1 and interleukin-6 and their release by liver, spleen, and lung macrophages were determined by flow cytometry 4 hours after trauma-hemorrhage. Prior Kupffer cell depletion with gadolinium chloride significantly decreased systemic MCP-1 and interleukin-6 after trauma-hemorrhage and was associated with decreased edema/neutrophil infiltration in lung and liver. Kupffer cells were the only macrophages showing significant MCP-1 release, which was markedly reduced by E2 or propyl pyrazole triol in wild-type and in ER-beta-/- mice. Pretreatment of mice with anti-MCP-1 antiserum prevented an increase in myeloperoxidase and edema in lung and liver. These findings suggest that Kupffer cell-derived MCP-1 plays a major role in remote organ dysfunction after trauma-hemorrhage.
Asunto(s)
Receptor beta de Estrógeno/inmunología , Hemorragia/inmunología , Macrófagos del Hígado/inmunología , Activación de Macrófagos/inmunología , Edema Pulmonar/inmunología , Heridas y Lesiones/inmunología , Animales , Antiinflamatorios/farmacología , Anticuerpos/inmunología , Anticuerpos/farmacología , Quimiocina CCL2/inmunología , Estradiol/farmacología , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/deficiencia , Femenino , Gadolinio/farmacología , Hemorragia/patología , Interleucina-6/inmunología , Macrófagos del Hígado/patología , Hígado/inmunología , Hígado/patología , Pulmón/inmunología , Pulmón/patología , Activación de Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Infiltración Neutrófila/inmunología , Fenoles , Edema Pulmonar/patología , Pirazoles/farmacología , Bazo/inmunología , Bazo/patología , Heridas y Lesiones/patologíaRESUMEN
We hypothesized that administration of androgen receptors antagonist flutamide following trauma-hemorrhage (T-H) in metestrus females will maintain immune function and reduce remote organ damage under those conditions. Female B57BL/J6 mice (metestrus state, 8-12 weeks old) underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mmHg for 90 min) and then received 17beta-estradiol (E2; 50 microg/25 g), flutamide (625 microg/25 g), or E2 + flutamide. Four hours after resuscitation, plasma cytokine and chemokine (TNF-alpha, IL-6, IL-10, IFN-gamma, and MCP-1) concentrations and their release in vitro by hepatic and pulmonary tissue macrophages (M Phi) were determined by flow cytometry. Organ damage was assessed by edema formation (wet-to-dry weight ratio) and neutrophil infiltration [myeloperoxidase (MPO) activity]. Administration of E2, flutamide, or E2 + flutamide following T-H resulted in a significant decrease in systemic TNF-alpha, IL-6, and MCP-1 concentrations under those conditions. This was accompanied by significantly decreased in vitro TNF-alpha release by Kupffer cells after administration of E2, flutamide, or E2 + flutamide. The in vitro release of proinflammatory cytokines by alveolar M Phi, however, was reduced significantly only by the addition of E2 or E2 + flutamide but not by the addition of flutamide. A significant decrease in pulmonary and hepatic edema formation as well as neutrophil infiltration in the lung was observed after E2, flutamide and E2 + flutamide administration. In contrast, hepatic neutrophil infiltration was only significantly reduced following E2 and E2 + flutamide administration. Thus, although flutamide does not produce synergistic, salutary effects with E2, its administration in females following T-H also produces salutary effects on the immune and organ function, similar to E2 administration under those conditions.
Asunto(s)
Estradiol/administración & dosificación , Flutamida/administración & dosificación , Hemorragia/tratamiento farmacológico , Hígado/patología , Pulmón/patología , Metestro/efectos de los fármacos , Animales , Quimiocina CCL2/efectos de los fármacos , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Citocinas/efectos de los fármacos , Citocinas/inmunología , Citocinas/metabolismo , Activación Enzimática/inmunología , Femenino , Citometría de Flujo/métodos , Hemorragia/inmunología , Inflamación , Inyecciones Subcutáneas , Macrófagos del Hígado/citología , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/inmunología , Hígado/inmunología , Pulmón/inmunología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Metestro/inmunología , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Peroxidasa/inmunología , Sensibilidad y EspecificidadRESUMEN
Severe injury induces immune dysfunction resulting in increased susceptibility to opportunistic infections. Previous studies from our laboratory have demonstrated that post-burn immunosuppression is mediated by nitric oxide (NO) due to the increased expression of macrophage inducible nitric oxide synthase (iNOS). In contrast, others suggest that injury causes a phenotypic imbalance in the regulation of Th1- and Th2 immune responses. It is unclear whether or not these apparently divergent mediators of immunosuppression are interrelated. To study this, C57BL/6 mice were subjected to major burn injury and splenocytes were isolated 7 days later and stimulated with antiCD3. Burn injury induced NO-mediated suppression of proliferative responses that was reversed in the presence of the NOS inhibitor L-monomethyl-L-arginine and subsequently mimicked by the addition of the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). SNAP also dose-dependently suppressed IFN-gamma and IL-2 (Th1), but not IL-4 and IL-10 (Th2) production. Delaying the addition of SNAP to the cultures by 24 h prevented the suppression of IFN-gamma production. The Th2 shift in immune phenotype was independent of cGMP and apoptosis. The addition of SNAP to cell cultures also induced apoptosis, attenuated mitochondrial oxidative metabolism and induced mitochondrial membrane depolarization. However, these detrimental cellular effects of NO were observed only at supra-physiologic concentrations (>250 microM). In conclusion, these findings support the concept that NO induces suppression of cell-mediated immune responses by selective action on Th1 T cells, thereby promoting a Th2 response.