Neuropeptide regulation of proinflammatory cytokine responses.

Severe traumatic injuries and infections are frequently accompanied by life-threatening shock and are associated with increases in the proinflammatory cytokines, particularly tumor necrosis factor alpha (TNF-alpha). The body's first perception of injury is the nociceptive or pain response. This response is induced at the site of injury and is transmitted systemically by sensory neuropeptides, the tachykinins, released from sensory afferent c-fiber neurons. We studied the role of tachykinins in regulating the production of proinflammatory cytokines induced by the administration of bacterial lipopolysaccharide. Destruction of terminal sensory nerve endings before lipopolysaccharide administration abrogates tachykinin synthesis and down-regulates TNF-alpha transcription and secretion. In contrast, the responses of interleukins-1 and -6 are unaffected. Pretreating animals with an antagonist for the substance P-specific NK-1 receptor also down-regulated the TNF-alpha response, whereas blockade of the NK-2 receptor had no effect. These findings indicate that substance P contributes to the induction of those cytokines that are involved in precipitating the shock response.

HIV infection and aging: mechanisms to explain the accelerated rate of progression in the older patient.

Age is an important predictor of progression in HIV infections. Not only do older individuals' develop AIDS more rapidly than younger persons, they die more quickly after developing an AIDS-defining illness. While the elderly have higher morbidity and mortality rates from viral and bacterial infections, the mechanism(s) responsible for the more rapid progression of HIV infection in older individuals has not been described. Our results demonstrate that the destruction of T cells in both young and old HIV infected patients progresses at the same rate. HIV 1-infected cells from older individuals do not appear more susceptible to immune mediated destruction. The more rapid progression appears due to an inability of older persons to replace functional T cells that are being destroyed. These findings suggest that improved survival in older HIV infected individuals will require more aggressive antiretroviral therapies as well as continued research to identify and preserve immune system elements that control the virus.

Effect of hemocarboperfusion on organ blood flow and survival in porcine endotoxic shock.

OBJECTIVE: To evaluate the effects of hemocarboperfusion on hemodynamics, organ blood flow, and survival in endotoxin shock. DESIGN: Prospective, placebo-controlled, animal trial. SETTING: Research laboratory in a major university teaching hospital. SUBJECTS: Pentobarbital-anesthetized pigs. INTERVENTIONS: Twenty-eight pentobarbital-anesthetized pigs (18.5 to 22.3 kg) received 100 micrograms/kg of Escherichia coli endotoxin (lipopolysaccharide 0127) over 30 mins. Group 1 animals (n = 14) were controls and had blood diverted through an extracorporeal circuit without activated charcoal for 60 mins after lipopolysaccharide infusion. Group 2 animals (n = 14) underwent nonpulsatile hemocarboperfusion (activated charcoal SCN-1K). MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure, cardiac output, systemic vascular resistance, mean pulmonary arterial pressure, pulmonary vascular resistance, oxygen delivery, and regional blood flow (radiolabeled microsphere technique) were determined at baseline and every 30 mins for 150 mins. Results are presented as mean +/- SD. Parameters in the two groups were compared by two-way analysis of variance. A p < .05 was considered significant. The survival rate was ten (71%) of 14 animals in group 1 compared with 14 (100%) of 14 animals in group 2 (p < .05, Fisher's exact test). The mean cardiac output at the end of hemocarboperfusion was 1.6 +/- 0.6 L/min in group 1 compared with 3.0 +/- 0.9 L/min in group 2, and remained lower in group 1 animals throughout the experiment. Pulmonary arterial pressure and pulmonary vascular resistance were lower in the hemocarboperfusion-treated animals during and after hemocarboperfusion. Systemic vascular resistance increased by 70% after lipopolysaccharide infusion and returned to baseline values in the hemocarboperfusion group but remained increased in controls. Oxygen delivery was lower in group 1 at 90 and 150 mins (287 +/- 34 vs. 478 +/- 48 mL/min and 251 +/- 24 vs. 356 +/- 21 mL/min, respectively). Blood flow rates to the brain (38.5 +/- 7.5 vs. 27.1 +/- 5.4 mL/min/100 g), large intestine (26.6 +/- 1.1 vs. 17.7 +/- 2.5 mL/ min/100 g), and adrenal cortex (200 +/- 45 vs. 139 +/- 41 mL/min/100 g) were higher in the hemocarboperfusion group at the completion of carboperfusion but not at later time points. CONCLUSION: These data suggest that hemocarboperfusion may be of value in the treatment of septic shock.

Interactions of ACTH and TGF beta on monocyte proliferation: implications for trauma and burn patients.

The purpose of this study was to try to elucidate a possible biobehavioral mechanism associated with decreased immune function in trauma patients by determining whether there is an interaction between the effects of ACTH, a stress hormone, and TGF beta, a cytokine, on peripheral blood lymphocyte proliferation. Peripheral mononuclear lymphocytes (PMLs) from healthy donors were preincubated with varying concentrations of ACTH for 24 hr, stimulated with concanavalin A and increasing concentrations of TGF beta, and incubated for 72 hr. Proliferation was assayed by tritiated thymidine incorporation. A parallel aliquot of PMLs were incubated in the presence of ACTH to determine the direct effect of ACTH on mononuclear cell TGF beta production. While harvested supernatant from cells incubated in the presence of ACTH did not contain any detectable TGF beta, ACTH as well as TGF beta were found to significantly decrease cellular proliferation independent of one another. An even greater decrease in cellular proliferation was found when both ACTH and TGF beta were used, compared to either ACTH or TGF beta alone. These results suggest a biobehavioral interaction between ACTH and TGF beta at the cellular level and that interactions to relieve stress may assist in improving function and recovery from trauma.

Urethan anesthesia protects rats against lethal endotoxemia and reduces TNF-alpha release.

Urethan is a commonly used animal anesthetic for nonrecovery laboratory surgery. However, urethan has diverse biological effects that may complicate the interpretation of experimental findings. This study examined the effect of urethan on the response to an intravenous bolus of lipopolysaccharide (LPS; 30 mg/kg) in rats. In instrumented rats, urethan (1.2 gm/kg i.p.) completely prevented the fall in arterial pressure immediately after LPS administration but did not prevent late cardiovascular collapse. In uninstrumented rats, urethan also attenuated indexes of organ injury measured 4 h after LPS administration, including mural bowel hemorrhage, hemoconcentration, hypoglycemia, metabolic acidosis, and lung myeloperoxidase activity, a measure of neutrophil sequestration. The peak increase in tumor necrosis factor-alpha (TNF-alpha) 90 min after LPS administration was reduced 88% by urethan (2,060 +/- 316 vs. 16,934 +/- 847 pg/ml; P < 0.001). In uninstrumented animals, urethan at 1.2 gm/kg reduced the 90% mortality rate of a lethal dose of LPS to 0-10% when given up to 24 h before LPS administration but did not reduce mortality when given 2 h after LPS. Urethan neither directly bound LPS by Limulus assay nor inhibited LPS-stimulated TNF-alpha mRNA expression in cultured mouse peritoneal macrophages, but TNF-alpha mRNA expression was suppressed by serum from a urethan-treated rat. Moreover, rauwolscine, which shares alpha 2-adrenoceptor-blocking activity with urethan, also prevented death from a subsequent 90% lethal dose LPS bolus. We conclude that urethan or its metabolites protect against LPS, in part, by reducing TNF-alpha release and speculate that this may be mediated by alpha 2-adrenoceptors. These actions of urethan make it an undesirable anesthetic agent for in vivo studies of sepsis or LPS.

Inhibition of apoptosis in polymorphonuclear neutrophils from burn patients.

Normal human polymorphonuclear neutrophils (PMN) undergo rapid apoptosis during in vitro culture. In contrast, apoptosis is inhibited in PMN from patients with severe burns. This inhibition is not an inherent property of the cells but is caused by thermolabile factors present in the plasma. Endotoxin and the proinflammatory cytokines interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) do not appear to be directly responsible. The ability of burn plasma to inhibit apoptosis was reduced by neutralizing antibodies to human granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF levels could not be detected in the burn plasma. However, the incubation of burn-derived or normal leukocyte populations consisting primarily of PMN in burn plasma induced the production of GM-CSF. The results suggest that activation of GM-CSF synthesis by factor(s) in burn plasma may play a role in regulating inflammation by the inhibition of apoptosis.

Regulation of acute phase gene expression following surgery and endotoxin administration in the anesthetized pig.

BACKGROUND: The hepatic acute phase response (APR) reflects an organism's integrated response to stress. This APR results in augmented synthesis and secretion of specific procoagulants and antiproteases and a complementary decrease in the synthesis and secretion of several constitutive proteins, such as albumin. The cytokines tumor necrosis factor (TNF) or interleukin-6 (IL-6) have been identified as proximal mediators of the APR in response to endotoxin stress. The authors hypothesized that TNF, IL-6, or both would be the proximal mediators of the APR in response to anesthesia and surgical stress. METHODS: The effects of a standardized surgical stress on the APR in pigs under general anesthesia with sodium pentobarbital and ketamine hydrochloride was investigated. Acute phase gene transcription was assayed in nuclei from serial liver biopsies obtained before and after 2.5 h of surgical stress, and after endotoxin administration. Tumor necrosis factor and IL-6 mRNA levels in this liver tissue were examined by Northern blot hybridization, and simultaneous plasma levels of these cytokines were measured using bioassays. RESULTS: The transcription rates of three positive acute phase genes--chymotrypsin inhibitor, inter-alpha-trypsin inhibitor and beta-fibrinogen--increased seven-, six-, and twofold, respectively (P < 0.05), and the transcription rate of albumin, a negative acute phase gene, decreased to 34% of baseline (P < 0.01) during the 2.5 h of anesthesia and surgical stress. During this initial 2.5 h, plasma concentrations of TNF and IL-6 did not change. Hepatic IL-6 mRNA expression was never observed, and TNF mRNA expression was undetectable in six of seven pigs. Subsequent 10-micrograms/kg endotoxin administration caused 20- and 100-fold increases in plasma concentrations of TNF and IL-6, respectively (P < 0.01), and were associated with substantial hepatic expression of the TNF and IL-6 mRNAs. These increments in cytokines were not associated with any further increase in the acute phase gene transcription rates. Thus, the APR was initially regulated at the transcriptional level during surgical stress independent of, and not augmentable by, an endotoxin-provoked increase in either plasma levels or hepatic mRNA expression of TNF or IL-6. CONCLUSIONS: Surgical stress induced hepatic acute phase gene transcription within 2.5 h in the absence of either systemic or local (hepatic) increases in TNF or IL-6. Subsequent endotoxin-induced increases in TNF or IL-6 did not alter this surgical stress-induced acute phase gene transcription.

Translocation. Incidental phenomenon or true pathology?

OBJECTIVE: This study was conducted to determine if reduction of early postburn endotoxemia influences the cytokine cascade, clinical manifestations of sepsis, and mortality rate. SUMMARY BACKGROUND DATA: Translocational endotoxemia has been demonstrated postburn in animals and humans. Endotoxin is known to induce the cytokine cascade, which leads to the clinical manifestations of sepsis. Whether reduction of postburn endotoxemia could influence the induction of cytokines has not been demonstrated. METHODS: In a prospective, randomized study, 76 burn patients were given polymyxin intravenously or served as control subjects. Polymyxin B was given intravenously for 1 week postburn in doses designed to neutralize circulating endotoxemia. RESULTS: In the polymyxin group, there was a statistically significant reduction in the plasma endotoxin concentration. There was, however, no reduction in the sepsis score or the interleukin-6 levels, and no differences in mortality rates were seen between the two groups. CONCLUSIONS: Early postburn translocational endotoxemia can be treated with anti-endotoxin agents such as polymyxin B. This, however, does not influence the cytokine cascade or the mortality rate. The systemic inflammatory response syndrome is caused by cytokine induction from the injury and is unaffected by a reduction in the plasma endotoxin concentration.

Effect of anesthesia and surgery on plasma cytokine levels.

Cytokines released in response to stress may have a profound impact on circulatory stability. There is no information on the effect of general anesthesia alone on plasma cytokine levels and little information on cytokine release following surgery. Plasma cytokine levels and hemodynamic parameters were measured during anesthesia and abdominal surgery under sterile and nonpyrogenic conditions in seven pigs anesthetized with ketamine and pentobarbital. Tumor necrosis factor (TNF) was measured by bioassay. Bioassays of low and high sensitivity were used to measure interleukin 6 (IL-6). Measurements were made sequentially during: (1) 4 hours observation with anesthesia alone; (2) 2 hours following laparotomy and traumatic intestinal manipulation (IM) sufficient to produce shock; and (3) after an intravenous bolus of 1 microgram/kg endotoxin as a positive control. Arterial blood pressure decreased following IM from 91.5 +/- 5.8 to 48.6 +/- 3.2 mm Hg, (mean +/- SE, P < .05), with no further change following endotoxin. Heart rate was unchanged during the experiment, and central venous pressure decreased after endotoxin (P < .05). There were no increases in TNF or IL-6 (using a low sensitivity assay) with anesthesia alone or following IM with shock, but both increased after endotoxin administration (P < .05); using a high sensitivity assay, IL-6 did not change during anesthesia alone but did increase fivefold following IM with shock (P < .05) and 50-fold following endotoxin administration (P < .05). We conclude that in a porcine model under sterile and nonpyrogenic conditions, prolonged anesthesia does not increase plasma cytokine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased production of TGF-beta and Il-6 by aged spleen cells.

Aging is accompanied by a progressive decline in immunity in every species that has been studied. Despite its ubiquity, the causes of immunosenescence are unknown. Transforming growth factor beta (TGF-beta) is a cytokine with potent immunosuppressive properties. Cells from aged mice produce increased levels of TGF-beta in vitro along with similar increases in interleukin 6 (Il-6), a cytokine which is immunosuppressive at elevated concentrations. Il-6 does not upregulate TGF-beta production, but high concentrations of Il-6 increase the percentage of cells expressing the TGF-beta receptor. Increased TGF-beta production and Il-6-induced upregulation of the TGF-beta receptor may be factors contributing to age-associated immunosuppression.

Inhibitory effects of interleukin 6 on immunity. Possible implications in burn patients.

Certain disease states are associated with abnormal increases in the monokine interleukin 6. Increased levels of interleukin 6 have been demonstrated in serum from patients with burns and are associated with systemic increases in endotoxin levels. Using a murine in vitro experimental model, we have studied the effects of interleukin 6 on various measures of immunity. Our data indicate that levels equivalent to the concentrations found in serum of burn victims inhibit T-cell proliferation. The inhibitory effect is dose and time dependent, is specific for T cells, is not due to impairment of interleukin 2 production or of interleukin 2 receptor expression, and is dependent on macrophages. These data suggest that extraordinary increases in interleukin 6 levels may be related to impaired T-cell responses and to an increased susceptibility to infection in the patient with burns.

The Baltimore Sepsis Scale: measurement of sepsis in patients with burns using a new scoring system.

A new sepsis scale has been developed, and it consists of 13 parameters, each of which is measured daily on a variable point scale. In 41 patients with major burns the Baltimore Sepsis scale correlated well with death or survival, with occurrence of septicemia, and with the level of serum interleukin-6. It did not correlate well with the level of plasma endotoxin or with the type of organism that was grown in blood cultures (gram-positive or gram-negative). We propose that the Baltimore Sepsis Scale would be an accurate and easy scale to use for the measurement of interventions that are aimed at improving the septic state.

Altered organ growth and zinc and copper distribution in endotoxin-treated neonatal mice.

Organ weights and the distribution of zinc and copper were compared in HLA/ICR mice that received intraperitoneal injections of 10 micrograms of Serratia marcescens lipopolysaccharide W or of sterile physiologic saline at 2 d of age. Between 5 and 28 d of age, body weight gains were similar in both groups. At 5 and 7 d of age, lipopolysaccharide W-treated mice had significantly lower thymus weights (p less than 0.01). At 7 d of age, liver weight was significantly increased (p less than 0.01) in lipopolysaccharide W-treated mice. Compared with tissue copper concentration in coeval saline-treated mice, lipopolysaccharide W treatment significantly increased copper concentration in thymus at 5 d of age (p less than 0.05) and significantly decreased concentration of this metal in liver at 7 d of age (p less than 0.05) and in spleen at 14 d of age (p less than 0.05). Liver zinc concentration was significantly lower (p less than 0.05) in 28-d-old mice that had received lipopolysaccharide W. When expressed on the basis of total organ burdens of zinc or copper, only the liver burden of zinc in 5-d-old lipopolysaccharide W-treated mice was significantly increased (p less than 0.05). Lipopolysaccharide W treatment consistently decreased copper concentration in liver cytosol and the amounts of zinc and copper bound to metallothionein, a transition metal-binding protein, in liver cytosol. These effects of lipopolysaccharide W on organ size and metal distribution may contribute to the adverse effects that persist after endotoxin exposure in early life.

Pathologic concentrations of interleukin 6 inhibit T cell responses via induction of activation of TGF-beta.

Interleukin 6 levels are increased in a variety of clinical conditions including bacterial and viral infections, HIV infection, autoimmune diseases, certain neoplasias, and traumatic injury. In general, all these conditions are characterized by suppression of one or more manifestations of the immune response. Concentrations of IL 6 comparable to those found in the sera of immunosuppressed, thermally injured patients selectively inhibit T cell proliferative responses. This suppression is independent of IL 2-mediated responses, is dependent on macrophage activity, and is reversed by antisera specific for transforming growth factor-beta (TGF-beta).

Increased levels of circulating interleukin 6 in burn patients.

The serum levels of interleukin 6 (IL-6) were determined in a population of burn patients. In all patients, IL-6 levels were increased over a 3-week interval with peak concentrations reached during the first week after injury. Patients receiving intravenous polymyxin B therapy according to a regimen designed to reduce endotoxemia manifested greatly reduced levels of both circulating endotoxins and IL-6. Certain patients not treated with polymyxin B showed extraordinarily large increases in IL-6 which were associated with lethal or life-threatening clinical complications. Increased IL-6 levels were also associated with decreased percentage of circulating T cells and corresponding increases in B cells. However, IL-6 did not produce any direct inhibitory effects in vitro on T cell representation or function.

Control of endotoxemia in burn patients by use of polymyxin B.

A group of patients with severe burns were entered into two sequential prospective randomized trials for reduction of endotoxemia by the use of intravenous polymyxin B. The first group underwent polymyxin administration during the first week after burn injury in a bell-shaped dosage form constructed to resemble the level of endotoxemia as previously documented. This group showed a statistically highly significant reduction in endotoxin levels and a suggestive, but not statistically significant, reduction in wound infection and mortality in the treated group compared with controls. The second group of patients underwent treatment with perioperative polymyxin B given in conjunction with an excisional procedure of the burn wound. In this group, polymyxin B also accomplished a reduction in endotoxemia from preoperative to postoperative cases, but there was no significant reduction in clinical complication rate or mortality. In the dosages used, polymyxin B is nontoxic and promises to be a useful part of the surgeon's armamentarium in dealing with severe complications of gram-negative sepsis.

Influence of age upon the metabolism of zinc in livers of C57BL/6J mice.

The kinetics of accumulation and loss of zinc from the liver following subcutaneous administration of 10 mg of zinc per kg were examined in young adult (6 months old) and old (24 months old) male C57BL/6J mice. After zinc treatment, total liver zinc concentrations rose equally in both groups and returned to basal levels at 96 h post treatment. However, differences were found in the subcellular distribution of zinc in these two age groups. The concentration of zinc in the cytosolic fraction (104,000 g supernate) prepared from the livers of old mice attained its maximum at 24 h post treatment. In contrast, the concentration of zinc in the cytosolic fraction of liver from young adult mice peaked at 48 h post treatment. This difference in accumulation of zinc in the cytosol was reflected by differences in the binding of zinc to metallothionein, a cytosolic transition metal binding protein. In old mice the highest amounts of zinc bound to metallothionein were found at 24 h post treatment: in young adults the maximal zinc binding to this protein occurred at 48 h post treatment. Examination of the relationship between cytosolic zinc contents and the binding of zinc to metallothionein in young adult and old mice suggested similar regulatory processes in the two age groups. Thus, age-dependent differences in accumulation and loss of zinc from the cytosolic fraction of liver probably reflect factors other than differences in regulation of the synthesis of metallothionein by this essential metal.

Altered expression of lymphocyte Il-2 receptors in burned patients.

Impairment of T-cell function is a consistent observation in burned patients. Concomitant with this impairment is an increase in serum factors which inhibit interleukin-2-mediated T-cell functions. These factors are heat labile and do not behave like endotoxins. Nonetheless, treatment of burned patients with endotoxin-neutralizing regimens of polymyxin B reduces the levels of these factors, suggesting that they are generated in response to endotoxin exposure. In addition to factors which inhibit Il-2 responses burn serum contains increases of circulating soluble, cell-free Il-2 receptors. However, the level of Il-2R is not altered by polymyxin B treatment and does not appear to be a direct result of endotoxin exposure. These observations suggest that multiple causes contribute to T-cell impairment in burned patients.

Supplemental zinc restores antibody formation in cultures of aged spleen cells. III. Impairment of II-2-mediated responses.

The effects of zinc on interleukin-2(IL-2)-dependent T cell responses of lymphocytes from immunodepressed aged mice and from young adult animals were studied. Concentrations of zinc which have been shown to restore antibody formation in cells from aged mice and to increase the production of Il-1 and Il-4 inhibited the production of Il-2. Cells from both young adults and aged mice were inhibited similarly. Zinc also impaired the ability of aged T cells to proliferate in response to concanavalin A and exogenous Il-2, but enhanced the proliferation of similarly activated splenic cultures containing both T and B cells. Cultures of isolated B lymphocytes produced antibody to sheep red blood cells if the cells were provided with supplemental zinc and Il-1. In contrast, recombinant Il-2 with or without zinc did not activate antibody formation. The results support the premise that the restorative effects of zinc are independent of Il-2 and that Il-2 is not a necessary mediator for antibody production in the aged.

Activation of thymocyte responses to interleukin-1 by zinc.

In vitro proliferative responses of murine thymocytes to interleukin-1 are enhanced by supplementing the cultures with the trace nutrient zinc. Zine not only enhances the responses of cells suboptimally activated by PHA but can also prime the cells to respond to IL-1 in the absence of activation by PHA. Zinc affects the early stages of the proliferative response. The data suggest that zinc may enhance the cellular uptake of IL-1 or may facilitate enzymatic steps subsequent to IL-1 binding.