Androgen-mediated modulation of macrophage function after trauma-hemorrhage: central role of 5alpha-dihydrotestosterone.

Androgens have been implicated as the causative factor for the postinjury immune dysfunction in males; however, it remains unknown whether androgens directly affect macrophages. To study this, male mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (mean arterial pressure, 30 +/- 5 mmHg for 90 min and then resuscitated). The mice received the 5alpha-reductase inhibitor 4-hydroxyandrostenedione (4-OHA) before resuscitation. Plasma TNF-alpha, IL-6, and IL-10 levels were elevated after trauma-hemorrhage and normalized by 4-OHA. TNF-alpha and IL-6 production by splenic macrophages was decreased after injury, whereas Kupffer cell production of these mediators was enhanced. 4-OHA normalized cytokine production. Androgens suppressed cytokine production by splenic macrophages from hemorrhaged mice, whereas it enhanced TNF-alpha and IL-6 production by Kupffer cells. The addition of 4-OHA in vitro normalized cytokine production by cells treated with testosterone, but it had no effect on dihydrotestosterone-treated cells. These results indicate that androgens directly affect macrophage function in males after trauma and hemorrhagic shock and that the intracellular conversion of testosterone to dihydrotestosterone is of particular importance in mediating the androgen-induced effects.     

Mechanism of cardiac depression after trauma-hemorrhage: increased cardiomyocyte IL-6 and effect of sex steroids on IL-6 regulation and cardiac function

A prolonged depression of cardiovascular function occurs in males after trauma-hemorrhagic shock (T-H). Although a correlation between increased circulatory IL-6 levels and poor outcome has been reported after T-H, it remains unknown whether T-H increases IL-6 levels locally in cardiomyocytes and whether there is a correlation between altered cardiac function and local IL-6 production after T-H. T-H was induced in normal, castrated (2 wk before T-H), and 17beta-estradiol (E2)-treated (0.5 mg sc, 1 wk before T-H) adult male rats. At 2 h after T-H or sham operation, cardiac output, heart rate, mean arterial pressure, positive and negative first derivative of pressure (+/-dP/dt), stroke volume, and total peripheral resistance were determined. Cardiomyocytes were isolated and divided into two parts: one was used for measurements of intracellular IL-6 levels using fluorescein-activated cell sorting, and the other was used to isolate RNA to determine IL-6 gene expression by quantitative real-time PCR. In addition, cardiac IL-6 protein levels were measured in freshly isolated hearts by Western blotting. Cardiac output, stroke volume, +dP/dt, -dP/dt, and total peripheral resistance were markedly altered after T-H. These parameters, except -dP/dt, improved significantly in the castrated group; however, all these parameters were restored in E2-treated males. Cardiomyocyte IL-6 mRNA expression and intracellular IL-6 production increased after T-H. Cardiac IL-6 protein levels increased after T-H in freshly isolated heart. Castration and E2 treatment attenuated cardiomyocyte intracellular IL-6 levels and cardiac IL-6 protein levels after T-H; however, only E2 treatment attenuated cardiomyocyte IL-6 gene expression. Thus there is an inverse correlation between cardiomyocyte IL-6 levels and cardiac function after T-H. The salutary effects of E2 on cardiac function after T-H may be due in part to decreased IL-6 synthesis in cardiomyocytes.     

Sex steroids regulate pro- and anti-inflammatory cytokine release by macrophages after trauma-hemorrhage

Studies indicate that macrophage immune responses in males aredepressed after trauma-hemorrhage, whereas they are enhanced in femalesunder such conditions. Nonetheless, the involvement of male and femalesex steroids in this gender-dependent dimorphic immune response aftertrauma-hemorrhage remains unclear. To study this, male C3H/HeN micewere castrated and treated with pellets containing either vehicle,5-dihydrotestosterone (DHT), 17-estradiol, or a combination ofboth steroid hormones for 14 days before soft tissue trauma (i.e.,laparotomy) and hemorrhagic shock (35 ± 5 mmHg for 90 min followed byadequate fluid resuscitation) or a sham operation. Twenty-four hourslater the animals were killed, plasma was obtained, and Kupffer celland splenic and peritoneal macrophage cultures were established. ForDHT-treated mice, we observed significantly decreased releases of theproinflammatory cytokines interleukin 1 (IL-1) and IL-6 bysplenic macrophage (50 and 57%, respectively) andperitoneal macrophage (51 and 52%, respectively)cultures after trauma-hemorrhage compared with releases by cultures ofcells from mice subjected to a sham operation; in contrast, responsesof splenic and peritoneal macrophage cultures from other groupssubjected to trauma-hemorrhage did not changesignificantly. In addition, only DHT-treated animals exhibited increased Kupffer cell IL-6 release (+634%). The release ofIL-10 in DHT-treated hemorrhaged animals was increased compared withthat in sham-operated animals but was decreased in estrogen-treated mice under such conditions. These results suggest that male and femalesex steroids exhibit divergent immunomodulatory properties with respectto cell-mediated immune responses after trauma-hemorrhage.

     

17 beta-Estradiol normalizes immune responses in ovariectomized females after trauma-hemorrhage

Recent studies indicate that immuneresponses in proestrus females are maintained after trauma-hemorrhagebut markedly depressed in ovariectomized females under such conditions.The current study tested the hypothesis that the decreased estrogenlevels after ovariectomy are responsible for this immune depression. Tostudy this hypothesis, ovariectomized female CBA/J mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35 ± 5 mmHg for 90 min, then resuscitated) or sham operation. The micereceived either 17-estradiol (E2; 100 µg/25 g body wt) or vehiclesubcutaneously during resuscitation. Immune cells were isolated 24 h thereafter. Splenocyte proliferation and interferon-, interleukin(IL)-2, and IL-3 release were significantly depressed aftertrauma-hemorrhage in vehicle-treated mice, whereas these functions weremaintained in E2-treated mice. Peritoneal macrophage IL-1 and IL-6release and splenic macrophage IL-6 and IL-12 release were alsosignificantly depressed in vehicle-treated mice after trauma-hemorrhage, and release of these cytokines was restored by E2treatment. In summary our findings indicate that the depressed splenicand peritoneal immune responses after trauma-hemorrhage can benormalized by a single dose of E2. Thus estrogen appears to be thecausative factor in the maintenance of immunocompetence in femalesafter trauma-hemorrhage, and its administration to ovariectomized orpostmenopausal females should be helpful in preventing immunedepression under such conditions.

     

Effects of gender and sex steroids on the immune response

Elevated immune responses and the higher incidence of autoimmune diseases in female (compared to male) humans and animals have been known for a long time. However, the scientific interest in this interrelationship has been limited both amongst immunologists and endocrinologists. It is mainly in the last ten years that investigations in this area have been intensifying. A number of fairly recent review articles confirm the increased interest in various aspects of this "interdiscipline" [1-4]. In the present paper we should like to make a new assessment of the state of knowledge. We shall firstly discuss heteroimmune response differences between males and females in humans, rodents and birds and then the roles of gender and sex hormones in autoimmune disease in various species. The general conclusions are the following. Gender and sex hormones have a clear effect on various hetero- and auto-immune responses but the mechanisms of action are still unknown; starting from sex hormones, steroids can be devised which have favourable effects on immune processes but lack undesirable hormonal effects; such hormonomimetics should be, in principle, applicable for the treatment of autoimmune disease.     

Insight into the mechanism by which metoclopramide improves immune functions after trauma-hemorrhage

Although studies have shownthat prolactin (Prl) and metoclopramide (Mcp) administration restoresthe depressed cell-mediated immune functions after hemorrhage, theunderlying mechanism responsible for the immunostimulatory effects ofMcp remains unknown. We hypothesized that Mcp improves immune responsesby upregulating the secretion of Prl. To test this hypothesis, maleC3H/HeN mice were subjected to sham operation or laparotomy (i.e., softtissue trauma) and hemorrhagic shock (Hem; 35 ± 5 mmHg for 90 min) and then resuscitated. Plasma Prl levels were determined 30 minafter Mcp (1 µg/g body wt sc at end of Hem) or vehicle (Veh)treatment in sham and Hem mice. The results indicate that plasma Prllevels increased significantly in Mcp-treated mice (sham-Veh 249.9 ± 5.3, Hem-Veh 229.9 ± 7.6, Hem-Mcp 596.9 ± 73.1 ng/ml,one-way ANOVA, P < 0.05 vs. Veh). To determine whetherMcp produces its salutary effects directly or indirectly via increasedPrl secretion, splenocyte proliferation and splenocyte interleukin(IL)-2 and IL-3 release from untreated sham or Hem mice were determinedin the presence of increasing concentrations of mouse Prl or Mcp. Theaddition of Mcp had no effect on splenocyte immune functions in vitro.However, the addition of Prl restored the hemorrhage-induced depressedsplenocyte proliferation as well as splenocyte IL-2 and IL-3 release invitro in a dose-dependent manner. Thus the beneficial effects of Mcp onimmune functions after Hem appear to be mediated by Prl. Because Mcpincreases plasma levels of the immunoenhancing hormone Prl, this agentshould be considered a useful adjunct for the treatment ofimmunodepression in trauma victims.

     

Sex steroid-mediated regulation of macrophage/monocyte function in a two-hit model of trauma-hemorrhage and sepsis

Studies have shown that 17beta-estradiol has salutary effects on immune functions after trauma-hemorrhage (TH). It remains unknown, however, whether 17beta-estradiol has a similar effect in a double-hit model of TH and subsequent sepsis. It is also unknown if under those conditions the circulating immune cells accurately represent immunological responses occurring in fixed tissues, such as the spleen. To study this, pre-castrated mice were hormonally treated and then subjected to soft-tissue trauma (i.e. midline laporatomy), hemorrhagic shock (MAP 35+/-5mmHg for 90 min followed by resuscitation) and 24 h later sepsis was induced by cecal ligation and puncture (CLP). Splenic macrophages (SMphi) and peripheral blood mononuclear cells (PBMC) were isolated and cultured with LPS. 5alpha-Dihydrotestosterone-treated mice showed a depressed pro-inflammatory cytokine production after TH-sepsis in both SMphi and PBMC. In contrast, the 17beta-estradiol treated groups showed suppressed pro-inflammatory cytokine production in the PBMC population under those conditions. In summary, 17beta-estradiol was able to prevent immune dysfunction after TH and subsequent sepsis. However, the beneficial effects of 17beta-estradiol were limited to tissue-fixed Mphi, suggesting compartmentalization of the response. Thus, events occurring in the tissue-fixed cells are not necessarily reflected in the circulating PBMC population.     

Salutary effects of androstenediol on cardiac function and splanchnic perfusion after trauma-hemorrhage

Recent studies have shown that dehydroepiandrosterone (DHEA) administration after trauma-hemorrhage (T-H) improves cardiovascular function and decreases cytokine production in male animals. Although androstenediol, one of the metabolites of DHEA, is reported to have estrogen-like activity, it remains unknown whether androstenediol per se has any salutary effects on cytokines and cardiovascular function after T-H. To examine this effect, male Sprague-Dawley rats underwent laparotomy and were bled to and maintained at a mean arterial blood pressure of 35-40 mmHg for approximately 90 min. The animals were resuscitated with four times the volume of maximal bleedout volume in the form of Ringer lactate. Androstenediol (1 mg/kg body wt i.v.) or vehicle was administered at the end of resuscitation. Twenty-four hours after resuscitation, cardiac function and organ blood flow were measured by using (85)Sr-microspheres. Circulating levels of nitrate/nitrite and IL-6 were also determined. Cardiovascular function and organ blood flow were significantly depressed after T-H. However, these parameters were restored by androstenediol treatment. The elevated plasma IL-6 levels after T-H were also lowered by androstenediol treatment. In contrast, plasma levels of nitrate/nitrite were the highest in the androstenediol-treated T-H animals. Because androstenediol administration after T-H decreases cytokine production and improves cardiovascular function, this agent appears to be a novel and useful adjunct for restoring the depressed cardiovascular function and for cytokine production in males after adverse circulatory conditions.     

Impact of sex and age on bone marrow immune responses in a murine model of trauma-hemorrhage.

Although studies have demonstrated that trauma markedly alters the bone marrow immune responses, sex and age are crucial determinants under such conditions and have not been extensively examined. To study this, 21- to 27-day-old (premature), 6- to 8-wk-old (mature), and 20- to 24-mo-old (aged) male and female (proestrus) C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30 +/- 5 mmHg for 90 min) followed by fluid resuscitation. Twenty-four hours after resuscitation, bone marrow cells were harvested. Trauma-hemorrhage induced an increased number of the early pluripotent stem cell-associated bone marrow cell subsets (Sca1(+)CD34(-)CD117(+/-)lin(+/-)) in young mice. The CD117(+) proportion of these cell subsets increased in mature proestrus females, but not in males. Aged males displayed significant lower numbers of Sca1(+)CD34(-)CD117(+/-)lin(+/-) cells compared with young male mice. Trauma-hemorrhage also increased development of granulocyte/macrophage progenitor cells (CD11b(+)Gr-1(+)). Proliferative responses to granulocyte macrophage colony-stimulating factor were maintained in mature and aged proestrus females, but decreased in young mice and mature males. Augmented differentiation into monocyte/macrophage lineage in mature and aged proestrus females was observed and associated with the maintained release of TNF-alpha and IL-6. Conversely, increased IL-10 and PGE(2) production was observed in the male trauma-hemorrhage groups. Thus, sex- and age-specific effects in bone marrow differentiation and immune responses after trauma-hemorrhage occur, which are likely to contribute to the sex- and age-related differences in the systemic immune responses under such conditions.     

Differences in the expression of LPS-receptors are not responsible for the sex-specific immune response after trauma and hemorrhagic shock

Several studies demonstrated a sex-specific cytokine secretion by macrophages following trauma-hemorrhage (T-H) and incubation with lipopolysaccharide A (LPS). Although LPS is known to act via the receptors CD14 and TLR4 on macrophages, it remains unknown whether differences in LPS receptor expression in males and females may be responsible for the gender-specific LPS induced cytokine response following (T-H). To study this, male and proestrus female mice (C3H/HeN) were subjected to trauma (laparotomy) followed by hemorrhage or sham operation. At 2 h thereafter, SMphi and PMphi were harvested and cultured for 2 h. The expression of CD14 and TLR4 was measured by flow cytometry on unstimulated SMphi and PMphi as well as after LPS stimulation. The results indicate that the expression of CD14 and TLR4 on SMphi and PMphi from female and male mice was similar in sham-operated animals and after (T-H). Incubation of macrophages with LPS did not alter CD14 and TLR4 expression in the study groups. Thus, the sex specific LPS induced cytokine secretion after (T-H) is not caused by differences in LPS receptor expression on Mphi of male and female mice.     

Alveolar macrophage activation after trauma-hemorrhage and sepsis is dependent on NF-kappaB and MAPK/ERK mechanisms

The acute respiratory distress syndrome (ARDS) is a major cause of morbidity after injury. We hypothesized that alveolar macrophage (AMPhi) chemokine and cytokine release after hemorrhage and sepsis is regulated by NF-kappaB and MAPK. Adult male rats underwent soft tissue trauma and hemorrhagic shock (~90 min) followed by crystalloid resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) 20 h after resuscitation. AMPhi were harvested, and TNF-alpha, IL-6, and macrophage inflammatory protein (MIP)-2 release and serum IL-6 and TNF-alpha levels were measured at 5 h after HCLP. Lung tissues were analyzed for activation of NF-kappaB, myeloperoxidase activity, and wet/dry weight ratio. In control animals, AMPhi were stimulated with LPS with or without inhibitors of NF-kappaB and MAPK. Serum TNF-alpha and IL-6 levels and spontaneous AMPhi TNF-alpha and MIP-2 release were elevated (P < 0.05) after HCLP, concomitantly with the development of lung edema and leukocyte activation. Activation of NF-kappaB increased in lungs from the hemorrhage and CLP group compared with shams. Inhibition of NF-kappaB or the upstream MAPK significantly decreased LPS-stimulated AMPhi activation. Because enhanced release of inflammatory mediators by AMPhi may contribute to ARDS after severe trauma, inhibition of intracellular signaling pathways represents a target to attenuate organ injury under those conditions.     

Flutamide restores cardiac function after trauma-hemorrhage via an estrogen-dependent pathway through upregulation of PGC-1

Although previous studies have shown that flutamide improves cardiovascular function after trauma-hemorrhage, the mechanisms responsible for the salutary effect remain unknown. We hypothesized that flutamide mediates its beneficial effects via an estrogen-dependent pathway through upregulation of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC-1). PGC-1, a key regulator of cardiac mitochondrial ATP production, induces mitochondrial DNA (mtDNA)-encoded genes such as cytochrome-c oxidase (COX) subunit I, II, and III (COX I, COX II, and COX III), which regulates mitochondrial oxidative phosphorylation. To test this hypothesis, male rats underwent trauma-hemorrhage (mean arterial pressure of 35-40 mmHg for approximately 90 min) followed by resuscitation. At the onset of resuscitation, rats received vehicle, flutamide (25 mg/kg body wt), flutamide in combination with estrogen receptor (ER) antagonist ICI-182,780 (3 mg/kg body wt), or ICI-182,780 alone. Flutamide administration after trauma-hemorrhage restored the depressed cardiac function and increased cardiac testosterone, estrogen levels, and aromatase activity. These increases were accompanied by normalized cardiac ER-alpha and ER-beta protein levels, PGC-1, and COX I mRNA expression, mitochondrial COX activity, and ATP contents. However, cardiac dihydrotestosterone, 5alpha-reductase II, androgen receptor protein levels, and mtDNA-encoded genes COX II and COX III were unaffected by flutamide treatment. The flutamide-mediated restoration of cardiac function, the increases in aromatase activity and estrogen levels, ER-alpha, ER-beta, PGC-1, COX I, COX activity, and ATP contents were, however, abolished when ER antagonist ICI-182,780 was administrated along with flutamide. These findings suggest that the salutary effect of flutamide on cardiac function after trauma-hemorrhage is mediated via an estrogen-dependent pathway through upregulation of PGC-1.     

Estradiol improves cardiac and hepatic function after trauma-hemorrhage: role of enhanced heat shock protein expression

Although studies indicate that 17beta-estradiol administration after trauma-hemorrhage (T-H) improves cardiac and hepatic functions, the underlying mechanisms remain unclear. Because the induction of heat shock proteins (HSPs) can protect cardiac and hepatic functions, we hypothesized that these proteins contribute to the salutary effects of estradiol after T-H. To test this hypothesis, male Sprague-Dawley rats ( approximately 300 g) underwent laparotomy and hemorrhagic shock (35-40 mmHg for approximately 90 min) followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. 17beta-estradiol (1 mg/kg body wt) was administered at the end of the resuscitation. Five hours after T-H and resuscitation there was a significant decrease in cardiac output, positive and negative maximal rate of left ventricular pressure. Liver function as determined by bile production and indocyanine green clearance was also compromised after T-H and resuscitation. This was accompanied by an increase in plasma alanine aminotransferase (ALT) levels and liver perfusate lactic dehydrogenase levels. Furthermore, circulating levels of TNF-alpha, IL-6, and IL-10 were also increased. In addition to decreased cardiac and hepatic function, there was an increase in cardiac HSP32 expression and a reduction in HSP60 expression after T-H. In the liver, HSP32 and HSP70 were increased after T-H. There was no change in heart HSP70 and liver HSP60 after T-H and resuscitation. Estradiol administration at the end of T-H and resuscitation increased heart/liver HSPs expression, ameliorated the impairment of heart/liver functions, and significantly prevented the increase in plasma levels of ALT, TNF-alpha, and IL-6. The ability of estradiol to induce HSPs expression in the heart and the liver suggests that HSPs, in part, mediate the salutary effects of 17beta-estradiol on organ functions after T-H.     

Mechanism of salutary effects of estradiol on organ function after trauma-hemorrhage: upregulation of heme oxygenase

A growing body of evidence indicates that heme degradation products may counteract the deleterious consequences of hypoxia and/or ischemia-reperfusion injury. Because heme oxygenase (HO)-1 induction after adverse circulatory conditions is known to be protective, and because females in the proestrus cycle (with high estrogen) have better hepatic function and less hepatic damage than males after trauma-hemorrhage, we hypothesized that estrogen administration in males after trauma-hemorrhage will upregulate HO activity and protect the organs against dysfunction and injury. To test this hypothesis, male Sprague-Dawley rats underwent 5-cm laparotomy and hemorrhagic shock (35-40 mmHg for 93 +/- 2 min), followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. 17beta-Estradiol and/or the specific HO enzyme inhibitor chromium mesoporphyrin (CrMP) were administered at the end of resuscitation, and the animals were killed 24 h thereafter. Trauma-hemorrhage reduced cardiac output, myocardial contractility, and serum albumin levels. Portal pressure and serum alanine aminotransferase levels were markedly increased under those conditions. These parameters were significantly improved in the 17beta-estradiol-treated rats. Estradiol treatment also induced increased HO-1 mRNA expression, HO-1 protein levels, and HO enzymatic activity in cardiac and hepatic tissue compared with vehicle-treated trauma-hemorrhage rats. Administration of the HO inhibitor CrMP prevented the estradiol-induced attenuation of shock-induced organ dysfunction and damage. Thus the salutary effects of estradiol administration on organ function after trauma-hemorrhage are mediated in part via upregulation of HO-1 expression and activity.     

Mast cells are critical for the production of leukotrienes responsible for neutrophil recruitment in immune complex-induced peritonitis in mice

Mast cells are secretory cells strategically located in the vicinity of blood vessels where they can readily initiate and modulate various inflammatory processes, including plasma exudation and leukocyte infiltration. We have previously shown that 50% of the neutrophil influx during immune complex peritonitis in mice is due to mast cells. Eicosanoids are important mediators of various inflammatory processes including neutrophil infiltration. The possibility that mast cells are essential for the production of leukotrienes (LT) involved in the elicitation of neutrophils in immune complex peritonitis was investigated in mast cell-deficient, WBB6F1-W/WV, and normal, WBB6F(1-)+/+, mice. The time course and amounts of immunoreactive PGE2, 6-keto-PGF1 alpha, and TX3B2 released into the peritoneal exudates were similar in both sets of mice. LTB4 and LTC4 levels, however, were twofold higher in +/+ than in W/WV mice 2 h after stimulation. HPLC analysis of the peritoneal exudate confirmed the presence of leukotrienes. The 5-lipoxygenase inhibitor A-63162 blocked leukotriene production in a dose-dependent manner in both sets of mice. However, this compound caused a significant reduction (60%) of neutrophil infiltration only in WBB6F(1-)+/+ but not in the mast cell-deficient mice. Mast cell reconstitution of WBB6F1-W/WV mice restored the effect of A-63162 on PMN recruitment. These data suggest that mast cells in the vicinity of blood vessels are important for the synthesis of leukotrienes responsible for PMN recruitment.     

Combinations of CD45 isoforms are crucial for immune function and disease

Expression of the CD45 Ag in hemopoietic cells is essential for normal development and function of lymphocytes, and both mice and humans lacking expression exhibit SCID. Human genetic variants of CD45, the exon 4 C77G and exon 6 A138G alleles, which alter the pattern of CD45 isoform expression, are associated with autoimmune and infectious diseases. We constructed transgenic mice expressing either an altered level or combination of CD45 isoforms. We show that the total level of CD45 expressed is crucial for normal TCR signaling, lymphocyte proliferation, and cytokine production. Most importantly, transgenic lines with a normal level, but altered combinations of CD45 isoforms, CD45(RABC/+) and CD45(RO/+) mice, which mimic variant CD45 expression in C77G and A138G humans, show more rapid onset and increased severity of experimental autoimmune encephalomyelitis. CD45(RO/+) cells produce more TNF-alpha and IFN-gamma. Thus, for the first time, we have shown experimentally that it is the combination of CD45 isoforms that affects immune function and disease.     

Dickeya dadantii pectic enzymes necessary for virulence are also responsible for activation of the Arabidopsis thaliana innate immune system

Soft‐rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions. We focused on the production of AtrbohD‐dependent reactive oxygen species (ROS), callose deposition and cell death as indicators of these reactions. We observed a significant reduction in ROS and callose formation with a bacterial mutant in which genes encoding five pectate lyases (Pels) were disrupted. Treatment of plant leaves with bacterial culture filtrates containing Pels resulted in ROS and callose production, and both reactions were dependent on a functional AtrbohD gene. ROS and callose were produced in response to treatment with a cellular fraction of a T2SS‐negative mutant grown in a Pels‐inducing medium. Finally, ROS and callose were produced in leaves treated with purified Pels that had also been shown to induce the expression of jasmonic acid‐dependent defence genes. Pel catalytic activity is required for the induction of ROS accumulation. In contrast, cell death observed in leaves infected with the wild‐type strain appeared to be independent of a functional AtrbohD gene. It was also independent of the bacterial production of pectic enzymes and the type III secretion system (T3SS). In conclusion, the work presented here shows that D. dadantii is recognized by the A. thaliana innate immune system through the action of pectic enzymes secreted by bacteria at the site of infection. This recognition leads to AtrbohD‐dependent ROS and callose accumulation, but not cell death.     

Regional adipose tissue metabolism in postmenopausal women after treatment with exogenous sex steroids

In order to evaluate the effect of exogenous sex steroids on adipose tissue metabolism, two groups of postmenopausal women were studied. In one of the groups, the effect of 50 micrograms ethinyl estradiol (EE) was investigated given orally alone and in combination with 10 mg norethisterone acetate (NET). This combination is reminiscent of an old high dose oral contraceptive. In the other group, the effect of 3 mg 17 beta-estradiol was evaluated when administered percutaneously alone and in combination with 300 mg micronized progesterone given orally. These substances and doses were chosen to provide a "physiological" hormonal influence. In the femoral region 50 micrograms EE induced an increase in LPL activity. This elevated LPL value was reversed with the addition of 10 mg NET. Moreover, during treatment with 50 micrograms EE, a decrease in norepinephrine stimulated lipolysis was seen in the abdominal region. The percutaneous administration of 17 beta-estradiol with or without micronized progesterone, however, was inert as regards subcutaneous adipose tissue metabolism. Our findings indicate, therefore, that EE in doses used in oral contraception might promote lipid accumulation in the femoral adipose tissue depot.