Susan C. Seatter

PRE-EXPOSURE TO HYPOXIA OR SEPTIC STIMULI DIFFERENTIALLY REGULATES ENDOTOXIN RELEASE OF TUMOR NECROSIS FACTOR, INTERLEUKIN-6, INTERLEUKIN-1, PROSTAGLANDIN E2, NITRIC OXIDE, AND SUPEROXIDE BY MACROPHAGES

UNLABELLED Shock states with resulting inadequate cellular oxygen delivery may contribute to macrophage (M phi) activation or dysregulation. In this study we compared the effects of transient anoxia and endotoxin pretreatment (LPS1) on M phi mediator release with a second endotoxin stimulus (LPS2). METHODS In vitro cultures of murine peritoneal exudate M phi were exposed to 2 hours of hypoxic or normoxic conditions, then incubated 22 hours under identical normoxic conditions +/- 10 ng/mL of LPS1 pretreatment. During the final 24 hours all M phis were exposed to a range of LPS2 concentrations. The M phi supernatants were assayed for tumor necrosis factor (TNF), interleukin 1 (IL-1), interleukin 6 (IL-6), prostaglandin E2 (PGE2), nitric oxide (NO), and superoxide release. RESULTS LPS1 markedly inhibited M phi TNF release by LPS2, but hypoxia had no effect on LPS2-triggered TNF release. Hypoxia increased M phi IL-6 production in the absence of LPS1, but inhibited the LPS1 augmentation seen under normoxic conditions. Pretreatment with LPS1 increased NO production from LPS2 under normoxic conditions, but hypoxia inhibited this effect. Superoxide production increased by LPS1 under normoxic conditions, but hypoxia significantly inhibited superoxide release. CONCLUSIONS The effects of transient anoxic exposure on LPS2-triggered M phi function are markedly different from the effects of pretreatment with septic stimuli (LPS1).

Endotoxin Pretreatment Of Human Monocytes Alters Subsequent Endotoxin-triggered Release Of Inflammatory Mediators

In trauma or sepsis, monocytes and macrophages release mediators such as tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), and prostaglandin E2 (PGE2). Although patients may be exposed to more than one stimulus, the effect of repetitive endotoxin (LPS) stimulation on human monocytes is poorly characterized. Human peripheral blood monocytes obtained from healthy volunteers were pretreated with endotoxin (LPS1) for 24 h. Cultures were then restimulated for 24 h with a second, activating LPS stimulus (LPS2) at various concentrations and supernatant mediators (TNF, IL-1, IL-6, and PGE2) measured. Serum cytokine levels of normal monocyte donors were compared to basal and LPS-stimulated cytokine release of their monocytes in vitro. LPS2 increased all mediators in a dose-dependent manner in the absence of LPS1 pretreatment. LPS1 significantly increased LPS2-triggered monocyte secretion of IL-1, IL-6, and PGE2, but inhibited TNF release. Cell-associated TNF and IL-1 were also inhibited and enhanced in parallel with supernatant levels of the respective cytokines. Serum cytokine levels were low, showed wide variation, and correlated poorly with in vitro LPS-triggered cytokine production. Human monocyte mediator production is differentially regulated by endotoxin pretreatment. Provocative in vitro testing of monocytes could identify prior LPS exposure and may be more useful than serum cytokine measurements.

Mechanisms of reprogrammed macrophage endotoxin signal transduction after lipopolysaccharide pretreatment

BACKGROUND Dysregulation of macrophage tumor necrosis factor (TNF) and interleukin-(IL-1) release results from repetitive lipopolysacharride (LPS) stimulation. In this study we investigated the mechanisms of LPS pretreatment (LPSp) signal transduction producing altered LPS-activated (LPSa) cytokine release. METHODS Murine macrophages were treated with medium alone, actinomycin D, cycloheximide, a protein kinase C inhibitor (H7), or the nitric oxide synthase inhibitor L-NMA. Macrophages were then pretreated with 100 ng/ml LPSp and cultured in medium alone, a nitric oxide donor (sodium nitroprusside), or a cyclic adenosine monophosphate donor (8 bromoadenosine) for 20 hours. Cultures were then washed, and fresh medium containing 1 microgram/ml LPSa was added. TNF and IL-1 release in 24-hour supernatant was measured by bioassays. RESULTS LPSp inhibited TNF and enhanced IL-1 release. The results with actinomycin D and cycloheximide suggested that LPSp effects did not require transcription, but IL-1 enhancement required protein synthesis. Addition of 8-bromo-cyclic adenosine monophosphate, H7, or nitroprusside prevented LPSp-induced augmentation of IL-1 but had no effect on inhibition of TNF release. Inhibition of LPSp-induced nitric oxide production with L-NMA had no effect on TNF or IL-1. CONCLUSIONS Complex, independent, but incompletely understood signal transduction pathways for LPSp-induced alterations in LPSa-stimulated macrophage TNF and IL-1 release were shown. Understanding altered signal transduction from prior LPS stimulation may suggest new therapies to control dysregulated macrophage cytokine release in sepsis.

LPS pretreatment reprograms macrophage LPS-stimulated TNF and IL-1 release without protein tyrosine kinase activation.

Pretreatment of macrophages with low‐dose endotoxin (LPSp) profoundly alters cytokine release in response to subsequent LPSa activation. These qualitative and quantitative alterations in cytokine release have been termed macrophage reprogramming. Macrophage activation by LPS is thought to occur via a mechanism involving an early protein tyrosine kinase (PTK) phosphorylation step. PTK inhibition with genistein or herbimycin A blocks LPSa‐stimulated secretion of tumor necrosis factor (TNF) and interleukin‐1 (IL‐1). In this study we investigated whether a PTK pathway participates in LPSp pretreatment reprogramming. We show that LPSp pretreatment inhibited TNF and augmented IL‐1 release in response to subsequent LPSa stimulation. Blockade of PTK activation pathways during the interval when macrophages were exposed to LPSp prevented mitogen‐activated protein kinase phosphorylation, as well as LPSp‐stimulated release of TNF and IL‐1, but did not block LPSp reprogramming effects. We conclude that LPSp pretreatment reprogramming of macrophage cytokine production does not require PTK activation. J. Leukoc. Biol. 61: 88–95; 1997.

Risk factors and patterns of injury in snowmobile crashes.

OBJECTIVE To evaluate risk factors for snowmobile injury and patterns of injury. METHODS We performed a retrospective analysis of patients with snowmobile injury at three trauma centers. Data were collected from trauma databases and patients charts from January 1988 through April 1996; we obtained statistics from the Minnesota Department of Natural Resources for comparison purposes. RESULTS There were 274 patients identified. The average age was 29 years (SD 12, range 1.6-77). The male:female ratio was 6.6:1. Helmets were used in 35%, not used in 10%, and not reported in 55%. Ethanol consumption was reported in 44% of patients. The average speed of the snowmobile at the time of the accident, when reported, was 47 mph/75 kph (n = 103, range 10-100 mph/16-166 kph). Of these patients, 26% (n = 27) reported a speed in excess of the legal limit (55 mph/88 kph). Accidents were more common in the afternoon and evening hours, and most accidents were caused by the snowmobile striking terrain or man-made objects. Mortality rate was 3.6% for this patient group (10 of 274). The average injury severity score (ISS) was 15 (SD 11). The average Glasgow Coma Score (GCS) was 14. The average number of patients who went to the intensive care unit and the total lengths of stay were 2 +/- 5 and 8 +/- 9 days, respectively. Neither GCS nor ISS correlated with reported speed. The frequencies of different types of injuries are as follows: fractures of upper and lower extremities (n = 184), serious head injury (n = 92), facial fractures or soft tissue injury to head or neck (n = 88), thoracic injury (n = 80), spine injuries (n = 50), intraabdominal injuries (n = 41), and pelvic fractures (n = 31). CONCLUSIONS Snowmobile injuries are related to ethanol use and the high speed attained by the newer generation of snowmobiles. Extremity fractures were a common component of snowmobile injury in this series, and rates of such injuries are similar to rates injuries in motorcycle accidents in states with helmet laws. Efforts at prevention of snowmobile injuries should be targeted at rider education and enforcement of alcohol restrictions.