Immunological consequences of trauma and shock.

The immune system is a powerful, complex entity composed of numerous cell types and regulated by autocrine, paracrine, and hormonal mechanisms. Trauma and haemorrhagic shock induce numerous changes within this system which are ultimately deleterious and contribute to the high incidence of organ dysfunction and infectious complications seen following injury. Regional hypoxia and depletion of intracellular energy stores occur in response to diminished microcirculatory blood flow, and these changes alter cellular signalling and result in the release of pro-inflammatory cytokines and prostanoids which mediate further suppression of immune function. Neutrophil priming serves to induce tissue damage in critical organ systems such as the lungs, heart, liver, and gut, further insulting the injured organism. Depression of antigen presentation and cytokine elaboration by macrophages and other antigen presenting cells effectively prevents a normal response from the acquired immune system, and lymphocyte-monocyte interactions are squelched. The resulting depression in cell mediated and humoral immunity renders the organism susceptible to microbial infection and contributes to the morbidity and mortality associated with nosocomial infections. Hormonal modulation of the immune response is highly evident following trauma and haemorrhage, and the preponderance of male morbidity associated with sepsis can be explained by the depression in immune function seen in males, but not females in the pro-oestrous state. Despite the multitude of changes induced by trauma and haemorrhage, experimental studies have revealed several promising pharmacologic interventions which may serve to blunt the effect of injury on the immune system, and render the host competent to withstand the bacterial and viral challenges responsible for so much of the late mortality following severe injury.

Dehydroepiandrosterone restores immune function following trauma-haemorrhage by a direct effect on T lymphocytes.

Although a profound depression in immune function occurs following injury, the mechanism responsible for this is not fully understood. Furthermore, steroid hormones are known to be important mediators in the regulation of immune function. Although dehydroepiandrosterone (DHEA), the most plentiful steroid hormone, has been shown to stimulate immune function in normal animals, it is unknown whether DHEA has any salutary or detrimental effects on immune responses after trauma and haemorrhage. To study this, male mice were subjected to trauma, haemorrhage and resuscitation, following which they received either DHEA or vehicle subcutaneously. DHEA administration restored the normally depressed splenocyte proliferation as well as interleukin 2, interleukin 3, and interferon gamma elaboration following trauma and haemorrhage. In an attempt to determine the mechanisms mediating this effect, T cells were stimulated in vitro in the presence of DHEA and a variety of hormone antagonists. The stimulatory effect of DHEA on splenocyte proliferation was unaltered by the testosterone receptor antagonist flutamide, while the oestrogen antagonist tamoxifen completely abrogated its effect. In addition, DHEA administration normalized the elevated serum corticosterone level typically seen following injury. These results indicate, therefore, that DHEA improves splenocyte function after trauma and haemorrhage by directly stimulating T cells and also by preventing a rise in serum corticosterone.

Does uninjured skin release proinflammatory cytokines following trauma and hemorrhage?

HYPOTHESIS: Uninjured skin contributes to the elevation in circulating levels of proinflammatory cytokines seen following severe injury. DESIGN: Male C3H/HeN mice underwent trauma, trauma-hemorrhage and resuscitation, or closed long-bone fracture. Serum, skin, and liver samples were harvested at designated times after experimental treatment. MAIN OUTCOME MEASURES: Levels of interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were determined in serum and skin cultures at 1, 8, and 24 hours after trauma-hemorrhage. The RNA was isolated from liver and skin samples at 1, 2, 4, 8, and 24 hours from all 3 experimental groups, and gene expression of the cytokines was determined. RESULTS: Remote (nontraumatized) skin from trauma-hemorrhage animals released significantly more IL-6 and TNF-alpha into culture supernatants at 1 and 24 hours and significantly more IL-1beta at 1, 8, and 24 hours than did skin from sham animals. Serum levels of all 3 cytokines were significantly elevated at 1 and 24 hours after trauma-hemorrhage relative to sham animals. Gene expression of all 3 cytokines was detected in skin and liver following trauma-hemorrhage. Furthermore, gene expression of all 3 cytokines was detected in uninjured skin after soft tissue trauma and closed long-bone fracture. CONCLUSIONS: Proinflammatory cytokine gene expression is up-regulated in uninjured skin following trauma, trauma-hemorrhage, and long-bone fracture. This increase in gene expression correlates with increased cytokine production by cultured skin as well as increased circulating cytokine levels. These results suggest that uninjured skin may also contribute to the rise in circulating cytokine levels seen after injury.

Hypoxemia in the absence of blood loss upregulates iNOS expression and activity in macrophages.

Regional hypoxia, associated with hemorrhage, is thought to induce a variety of alterations in immune cell function, including upregulation of macrophage-inducible nitric oxide synthase (iNOS) expression and activity (NO production). Furthermore, NO may cause immune cell dysfunction similar to that associated with hemorrhagic shock. However, it remains unknown whether hypoxia per se in the absence of any blood loss is a sufficient stimulus to cause iNOS expression and NO production by macrophages. To study this, male Sprague-Dawley rats (275-325 g) were placed in a plastic box flushed with a gas mixture containing 5% O2-95% N2 for 60 min. Peritoneal and splenic macrophages were isolated 0-5.5 h thereafter, and blood samples were obtained. Nitrite and nitrate (stable degradation products of NO) production by splenic and peritoneal macrophages cultured for 48 h was significantly increased 3 and 5.5 h after hypoxemia. The increase in NO production by macrophages was preceded by elevated expression of iNOS mRNA at 1.5 h after hypoxia. Additionally, interferon-gamma (IFN-gamma) levels in plasma from rats subjected to hypoxemia were significantly elevated soon after the insult (0-1.5 h posthypoxemia), suggesting a causal relationship between IFN-gamma production and upregulation of iNOS activity. We propose that a hypoxemia-induced increase in macrophage iNOS activity following hemorrhage may in part be responsible for the observed immune dysfunction. Thus attempts to suppress macrophage iNOS activity after this form of trauma may be helpful in improving immune function under those conditions.

Dehydroepiandrosterone: an inexpensive steroid hormone that decreases the mortality due to sepsis following trauma-induced hemorrhage.

BACKGROUND: Recent studies suggest that male sex steroids play a role in producing immunodepression following trauma-hemorrhage. This notion is supported by studies showing that castration of male mice before trauma-hemorrhage or the administration of the androgen receptor blocker flutamide following trauma-hemorrhage in noncastrated animals prevents immunodepression and improves the survival rate of animals subjected to subsequent sepsis. However, it remains unknown whether the most abundant steroid hormone, dehydroepiandrosterone (DHEA), protects or depresses immune functions following trauma-hemorrhage. In this regard, DHEA has been reported to have estrogenic and androgenic properties, depending on the hormonal milieu. OBJECTIVE: To determine whether administration of DHEA after trauma-hemorrhage has any salutary or deleterious effects on immune responses, and whether it improves the survival of animals subjected to subsequent sepsis. DESIGN: Male C3H/HeN mice underwent laparotomy (ie, trauma-induced) and hemorrhagic shock (blood pressure, 35+/-5 mm Hg for 90 minutes) followed by fluid resuscitation, or sham operation. The animals then received 100 mg of DHEA (4 mg/kg) or propylene glycol (hereafter referred to as vehicle). At 24 hours after trauma-hemorrhage and resuscitation, the animals were killed and blood, spleens, and peritoneal macrophages were harvested. Splenocyte proliferation and interleukin (IL) 2 release and splenic and peritoneal macrophage IL-1 and IL-6 release were determined. In a separate set of experiments, sepsis was induced by cecal ligation and puncture at 48 hours after trauma-hemorrhage and resuscitation. For those studies, the animals received vehicle, a single 100-microg dose of DHEA, or 100 microg/d DHEA for 3 days following hemorrhage and resuscitation. Survival was monitored for 10 days after the induction of sepsis. RESULTS: Administration of DHEA restored the depressed splenocyte and macrophage functions at 24 hours after trauma-hemorrhage. Moreover, daily administration of DHEA for 3 days significantly increased the survival of animals subjected to subsequent sepsis (P=.01). CONCLUSION: The finding that DHEA markedly improves the depressed immune functions and survival of animals subjected to subsequent sepsis suggests that short-term treatment with DHEA after trauma-hemorrhage is a safe and novel approach for preventing immunodepression and for decreasing the mortality rate due to subsequent sepsis.

Thermal injury alters macrophage responses to prostaglandin E2: contribution to the enhancement of inducible nitric oxide synthase activity.

Prostaglandin E2 (PGE2) and macrophage (Mphi)-derived reactive nitrogen intermediates (RNI) have been implicated in T cell dysfunction after thermal injury. Normally, Mphi inducible nitric oxide synthase (iNOS) activity can be regulated by PGE2, however, it is unknown whether PGE2 modulates Mphi iNOS activity after thermal injury. Splenic Mphi isolated from mice 7 days after thermal injury produced higher levels of RNI than Mphi from sham mice when stimulated with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in combination. PGE2, when added concurrently with LPS, suppressed RNI production by Mphi from sham mice, whereas Mphi from injured mice were unaffected. When Mphi were pretreated with PGE2 before LPS, RNI production was suppressed in both populations. RNI production in response to IFN-gamma or IFN-gamma and TNF-alpha in combination was enhanced by PGE2 in both populations, however, the effect was markedly greater in Mphi from injured mice. The PGE2-mediated changes in RNI production were paralleled by similar changes in iNOS protein expression, suggesting that the effect of PGE2 was at the level of enzyme expression rather than activity. Dibutryl cAMP induced similar effects as PGE2, suggesting the response to PGE2 after thermal injury is independent of potential changes in PGE2-induced adenylate cyclase activity and is cAMP-mediated. The results indicate that Mphi from burned mice display an altered sensitivity to PGE2, resulting in enhanced iNOS activity. Thus, PGE2, which is elevated after thermal injury and can directly suppress T cell function, may also contribute to immune dysfunction through the enhancement of Mphi iNOS activity.

Trauma-hemorrhage activates signal transduction pathways in mouse splenic T cells.

Severe impairment in the functions of immune-competent cells has been observed following trauma and hemorrhage. Inappropriate release of cytokines during trauma and hemorrhagic shock disrupt T lymphocyte functions and enable cells to activate genes whose products are detrimental for maintaining a much-needed humoral and cell-mediated immunity. The intracellular events for gene activation are mediated by cytoplasmic transcription factors present as nascent (signal transducer and activator of transcription 1 (STAT 1)) or as a complex (nuclear factor kappaB (NF-kappaB)). Receptor-initiated phosphorylation activates these transcription factors prior to their nuclear translocation and binding to cognate DNA sequences. Because T cell functions are critical to efficient functioning of the immune system, we investigated whether expression of transcription factors, STAT1 and NF-kappaB, is perturbed in splenic T cells following trauma and hemorrhage. To study this, enriched T cells harvested from spleens (pooled from three or four mice per group) of sham, trauma (consisting of midline laparotomy), sham+trauma, hemorrhage (blood pressure maintained at approximately 30 mmHg for 90 min followed by adequate fluid resuscitation), and trauma+hemorrhage groups at 16-18 h after surgical procedure were probed for signal expressions in the presence and absence of interferon-gamma using electrophoretic mobility shift and Western immunoblot assay procedures. Hemorrhage with or without trauma induced activation of Janus kinase 1, STAT1, and NF-kappaB in T cells. Stimulation of T cells with interferon-gamma led to activation of all these signals in all groups including experimental controls. STAT1 activation was accompanied by Janus kinase 1 phosphorylation, whereas NF-kappaB activation was mediated by phosphorylation and rapid degradation of IkappaBalpha. These studies demonstrate that hemorrhagic shock, with or without laparotomy, is sufficient to induce activation of transcription factors in splenic T cells. Thus, attempts to prevent the activation of transcription factors following hemorrhage by pharmacologic means might be helpful for maintaining cell-mediated immunity under these conditions.

Gut and liver: the organs responsible for increased nitric oxide production after trauma-hemorrhage and resuscitation.

OBJECTIVE: To determine which organs produce the increased levels of nitric oxide (NO) seen after hemorrhage and resuscitation. ANIMALS AND INTERVENTIONS: Adult male rats underwent laparotomy (ie, trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the maximum bleedout volume was returned in the form of Ringer lactate. The rats were then resuscitated with Ringer lactate, 4 times the maximum bleedout volume for 1 hour. Sham-operated animals underwent only the surgical procedure. MAIN OUTCOME MEASURES: Plasma levels of nitrate/nitrite (NO3-/NO2-, stable products of NO) were measured by colorimetric assay at the maximum bleedout volume; at the end of hemorrhage; at the end of resuscitation; and 1.5, 4, 8, and 24 hours after resuscitation. In additional rats, the heart, liver, small intestine, kidneys, and spleen were harvested 4 hours after resuscitation for the measurement of NO3-/NO2- levels. Moreover, tissue perfusion was determined in the above-mentioned organs by radioactive microspheres 4 hours after resuscitation in other groups of animals. RESULTS: Plasma levels of NO3-/NO2- were similar to those of sham-operated animals during hemorrhage and at the end of resuscitation. One and a half hours after the end of resuscitation, however, NO production increased significantly. The peak levels of plasma NO3-/NO2- occurred at 4 hours, and the levels remained elevated even 24 hours after resuscitation. Tissue NO3-/NO2- levels were significantly increased in the liver, small intestine, and spleen 4 hours after resuscitation. In contrast, the levels of NO3-/NO2- were similar to those of sham-operated animals in the heart and kidneys at all times. Blood flow in the heart was maintained after hemorrhage, whereas hepatic, intestinal, splenic, and renal perfusion decreased significantly. CONCLUSIONS: The gut and liver seem to be the sites responsible for the increased NO production seen after trauma and hemorrhage. The overproduction of NO is most likely caused by up-regulation of inducible NO synthase. Thus, attempts to reduce NO production using specific inhibitors for inducible NO synthase might be helpful for improving hepatic and intestinal functions after trauma and hemorrhagic shock.

Altered dopaminergic modulation of sympathetic nervous system activity in idiopathic edema.

Hormonal and mean arterial pressure responses to posture and isometric handgrip exercise were examined in 6 women with idiopathic edema and 10 age- and weight-matched normal women before and after 5-7 days of administration of the dopamine agonist, bromocriptine (2.5 mg three times a day). Edema patients demonstrated greater orthostatic weight gain, greater upright epinephrine values, and greater supine and upright norepinephrine values than did the control group. However, supine and upright plasma dopamine levels were similar in the two groups. In edema patients there was a greater supine and posture related norepinephrine and epinephrine to dopamine ratio than in normal controls. These abnormalities were not corrected by treatment with bromocriptine. Supine and upright plasma norepinephrine and epinephrine levels were decreased following bromocriptine treatment in normal subjects but not in edema patients. These data are consistent with the concept that there is decreased dopaminergic regulation of sympathetic nervous activity in patients with idiopathic edema.

Role of the sympathetic nervous system in blood pressure maintenance in obesity.

A group of 10 borderline hypertensive obese subjects had higher (P less than 0.05) supine plasma norepinephrine (NE), epinephrine, and PRA levels as well as greater (P less than 0.05) NE responses to upright posture and isometric handgrip exercise than 12 nonobese controls. Plasma NE as well as mean arterial pressure (MAP) responses to posture and handgrip in the obese patients demonstrated a significant decrement at week 2 after the onset of a low calorie diet. Decrements in plasma NE and MAP responses to posture were correlated (r = 0.61; P less than 0.05) throughout an 8-week period of weight loss in these borderline hypertensive obese subjects. In 15 normotensive obese subjects as well as in the 10 borderline hypertensive obese subjects, weight reduction associated with a very low calorie intake was accompanied by a reduction in supine plasma NE, epinephrine, and MAP 2 weeks after the onset of dieting. PRA decreased after 8 weeks of dieting in both obese groups, and these PRA decrements were correlated with reductions in MAP and decrements in plasma NE. We conclude that enhanced sympathetic activity may play a role in the maintenance of elevated blood pressure in obese individuals. Decreases in PRA and blood pressure associated with weight loss in both normotensive and hypertensive obese individuals occurs, in part, secondary to reductions in plasma NE levels.

Evidence for dopaminergic control of circadian variations in thyrotropin secretion.

This study examines the influence of dopamine of recumbent circadian secretory patterns of plasma TSH. Seven euthyroid men were studied during a control (no medication) and a bromocriptine period (2.5 mg bromocriptine three times a day for 5 days). In the control period, a clear daily pattern in plasma TSH was evidenced, with highest levels occurring during the late evening presleep period and the fist few hours after the onset of nocturnal sleep. During the control period, recumbent 24-h plasma TSH was linearly correlated (gamma = 0.41; P less than 0.05) with clock time. Bromocriptine lowered the mean 24-h TSH from a control level of 2.1 +/- 0.2 to 1.1 microunits/ml. Further, bromocriptine eliminated the early nocturnal TSH surge and the correlation of plasma TSH with clock time. Thus, circadian variations in plasma TSH levels may be modulated, in part, by a dopaminergic mechanism. However, we cannot rule out a direct effect of bromocriptine on the anterior pituitary resulting in decreased late evening rises in plasma TSH.

Influence of sodium homeostasis on dopaminergic modulation of aldosterone, renin, and prolactin secretion in man.

This study examines the effect of sodium homeostasis on the plasma aldosterone, renin, and PRL responses to the dopamine antagonist metoclopramide in normal individuals. Responses to metoclopramide were evaluated after receiving a 10-meq sodium, 80-meq potassium diet for 5 days and after receiving a 200-meq sodium, 80-meq diet for 5 days. On both occasions, the subjects had reached sodium equilibrium states, as determined by urinary sodium measurements, at the time that they received metoclopramide. Maximum absolute incremental aldosterone responses to metoclopramide were considerably greater (P less than 0.001) in the subjects after 5 days on a 10-meq sodium intake (23.2 +/- 2.0 ng/dl) than after 5 days on a 200-meq sodium intake (6.6 +/-0.9 ng/dl). Greater PRL and PRA responses (P less than 0.05) were also noted with low (10 meq) daily sodium intake, although the influence of sodium intake was less than that observed on the aldosterone response to metoclopramide. PRA responses to metoclopramide occur late (45 min) compared to aldosterone and PRL responses, which were noted 5 min after drug administration, suggesting an indirect effect of dopamine in modulating renin secretion. The proportion of total renin which was inactive was greater with the higher sodium intake. These data suggest that sodium homeostasis may influence dopaminergic modulation of renin, aldosterone, and PRL secretion and may effect the interrelationship between active and inactive renin.