Eric S. Hungness

Mucosal and enterocyte IL-6 production during sepsis and endotoxemia--role of transcription factors and regulation by the stress response.

BACKGROUNDnSepsis and endotoxemia are associated with increased production of interleukin-6 (IL-6) in gut mucosa. Mucosal IL-6 may regulate enterocyte acute phase protein synthesis and intestinal IgA production. In addition, increased IL-6 has been proposed to be a mechanism of loss of mucosal integrity in critical illness. The purpose of this review is to describe current knowledge of the regulation of IL-6 production in the enterocyte/mucosa during inflammation caused by sepsis and endotoxemia.nnnDATA SOURCESnRecent publications describing the influence of sepsis, endotoxemia, and proinflammatory cytokines on mucosal/enterocyte IL-6 production.nnnCONCLUSIONSnIL-6 production is increased in gut mucosa during sepsis and endotoxemia and in cultured enterocytes after treatment with endotoxin or proinflammatory cytokines. The IL-6 gene is regulated by multiple transcription factors, including NF-kappaB, AP-1, and C/EBP. Because of the multiple important biological roles of IL-6, understanding the cellular and molecular mechanisms of mucosal/enterocyte IL-6 production as well as methods to modulate IL-6 production is of clinical importance in the setting of sepsis and other critical illness.

Early debridement for necrotizing pancreatitis: is it worthwhile?

BACKGROUNDnThe timing for debridement of necrotizing pancreatitis is controversial. We reviewed our experience with early and delayed surgical debridement in patients with necrotizing pancreatitis.nnnSTUDY DESIGNnThe records of patients diagnosed with acute necrotizing pancreatitis from January 1993 through June 2000 were reviewed retrospectively. Data were analyzed with respect to Ransons, APACHE II, and multiple organ failure scores, etiology, presence of infection, overall and ICU length of stay, time to first debridement, number of debridements, fluid requirements, days to enteral feeding, transfusion requirements, complications, and mortality.nnnRESULTSnTwenty-six patients (18 males, 8 females, mean age 51 years) were diagnosed with acute necrotizing pancreatitis. The admission Ransons score was 4.8, the APACHE II score was 11.7, and multiple organ failure score was 4.2. All but one patient underwent pancreatic debridement (4.3 debridements per patient). Eighteen patients (69%) had infected pancreatic necrosis. The timing of debridement was based on patients condition and surgeons preference. The presentation and demographics of patients who underwent early (<2 weeks) or late (>2 weeks) debridement did not differ significantly. Patients debrided early had a trend toward higher mortality (29% versus 18%) and experienced a higher number of major complications (p < 0.05). The six patients (23%) who died were older, had multiple organ failure scores, and more often had Candida in the infected necrosis (p < .05).nnnCONCLUSIONSnEarly debridement for acute necrotizing pancreatitis might not improve survival and might even be associated with increased number of complications. Most patients diagnosed with necrotizing pancreatitis eventually need debridement, but it might be beneficial to delay debridement if the patients condition allows for it.

Transcription factors C/EBP-β and -δ regulate IL-6 production in IL-1β-stimulated human enterocytes

In recent studies, treatment with IL‐1β of cultured Caco‐2 cells, a human intestinal epithelial cell line, resulted in transcriptional upregulation of IL‐6 production. The role of C/EBP‐β and ‐δ in enterocyte IL‐6 production is not known. Stimulation with IL‐1β of Caco‐2 cells transiently transfected with a luciferase reporter plasmid containing a wild‐type IL‐6 promoter resulted in an approximately 3.5‐fold increase in luciferase activity. This effect of IL‐1β was reduced by approximately 30% when the C/EBP binding site in the IL‐6 promoter was mutated, supporting a role of C/EBP in the regulation of IL‐6 production. When Caco‐2 cells were treated with IL‐1β in the presence of the MAPK inhibitor, PD‐98059, IL‐6 mRNA and protein levels were reduced by the same concentrations of PD‐98059 that inhibited C/EBP DNA binding activity in previous studies. Finally, overexpression of C/EBP‐β and ‐δ in IL‐1β‐treated Caco‐2 cells resulted in a 10–12‐fold increase in IL‐6 production. The results suggest that the β and δ isoforms of the C/EBP family of transcription factors at least in part regulate IL‐6 production in human intestinal epithelial cells.

THE TRANSCRIPTION FACTOR ACTIVATOR PROTEIN-1 IS ACTIVATED AND INTERLEUKIN-6 PRODUCTION IS INCREASED IN INTERLEUKIN-1β-STIMULATED HUMAN ENTEROCYTES

The intestinal mucosa is an active participant in the inflammatory and metabolic response to sepsis, endotoxemia, and other critical illness. The genes for various cytokines, e.g., interleukin 6 (IL-6), are regulated by multiple transcription factors, including nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1). In recent studies, treatment with IL-1beta of cultured Caco-2 cells, a human intestinal epithelial cell line, resulted in increased NF-kappaB DNA binding. The effect of IL-1beta on AP-1 activity in the enterocyte and the potential role of AP-1 in enterocyte IL-6 production are not known. We treated Caco-2 cells with IL-1beta and determined AP-1 activity by electrophoretic mobility shift assay (EMSA) and IL-6 production by enzyme-linked immunosorbent assay (ELISA). Treatment of Caco-2 cells with IL-1beta resulted in a dose- and time-dependent increase in AP-1 DNA binding. Supershift analysis suggests that activated AP-1 contained c-Jun, JunD, c-Fos, FosB, and Fra1 subunits. When Caco-2 cells were transiently transfected with an AP-1 luciferase reporter plasmid, stimulation with IL-1beta resulted in increased luciferase activity, suggesting that AP-1 DNA binding increased gene activation. Additional luciferase assays were performed with a plasmid containing a wild-type or AP-1-mutated IL-6 promoter. Stimulation of these cells with IL-1beta gave rise to results supporting the role of AP-1 in the regulation of IL-6 production. Geldanamycin, which has been shown in studies to inhibit AP-1 activation, blocked IL-1beta-induced AP-1 luciferase gene activation and IL-6 production. These results suggest that the AP-1 family of transcription factors is activated by IL-1beta in human enterocytes and that AP-1 may at least in part regulate IL-6 production in these cells.

The stress response decreases NF-κB activation in liver of endotoxemic mice

Recent studies suggest that the stress (heat shock) response protects cells and tissues from inflammatory and other noxious insults. The transcription factor nuclear factor-kappa B (NF-&kgr;B), normally sequestered in the cytoplasm by its inhibitory protein I&kgr;B, regulates many genes involved in the inflammatory response to critical illness. Endotoxemia is associated with increased NF-&kgr;B activity in liver but the effect of the stress response on endotoxin-induced NF-&kgr;B activation in the liver is not known. We hypothesized that the stress response inhibits NF-&kgr;B DNA binding activity in liver during endotoxemia. The stress response was induced in mice by hyperthermia (42°C for 3 min) or sodium arsenite (10 mg/kg) and resulted in increased hepatic heat shock protein-72 levels. After induction of the stress response, mice were injected subcutaneously with endotoxin (12.5 mg/kg) or a corresponding volume of sterile saline. NF-&kgr;B DNA binding activity in the nuclear fraction of liver tissue increased and cytoplasmic I&kgr;B-&agr; levels decreased after endotoxin injection, with a maximal effect seen at 60 min. The endotoxin-induced increase in NF-&kgr;B DNA binding activity and decrease in I&kgr;B-&agr; levels were inhibited by prior induction of the stress response. In additional experiments, treatment of mice with sodium arsenite after induction of endotoxemia blunted the increase in NF-&kgr;B activity, indicating a therapeutic potential of sodium arsenite, in addition to its preventive effect. The present results suggest that the protective effects of the stress response in vivo may, at least in part, be due to inhibited NF-&kgr;B activation.

IL-1β activates C/EBP-β and δ in human enterocytes through a mitogen-activated protein kinase signaling pathway

The enterocyte is an active participant in the inflammatory and metabolic response to sepsis, endotoxemia and other critical illnesses and is the site for cytokine and acute phase protein production in these conditions. The role of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors in the response to inflammatory stimuli in the enterocyte is not well understood. In the present study, we treated Caco-2 cells with IL-1beta and determined C/EBP DNA binding activity by electrophoretic mobility shift assay. The involvement of the alpha, beta, and delta isoforms was determined by supershift analysis and Western blot analysis of proteins from the nuclear fraction. The role of the mitogen-activated protein kinase (MAPK) signaling pathway was assessed by treating cells with the MAPK inhibitor PD-98059. Treatment of the Caco-2 cells with IL-1beta resulted in increased CCAAT/enhancer binding protein DNA binding activity. Supershift analysis and Western blotting indicated that this response to IL-1beta mainly reflected the delta isoform, and to a lesser degree the beta isoform. Treatment of the cells with PD-98059 inhibited the IL-1beta-induced increase in beta and delta activity. The results suggest that members of the CCAAT/enhancer binding protein family of transcription factors are activated in enterocytes during inflammatory conditions characterized by high levels of IL-1beta.

Home Oxygen Therapy: Adjunct or Risk Factor?

The use of home oxygen therapy has become increasingly commonplace and is frequently prescribed by medical specialists. In this study, we have identified a generally unexpected risk of home oxygen therapy. We performed a retrospective review of 3673 consecutive patients treated at our adult burn center over a 10-year period from 1992 to 2001. We identified 27 patients with burns directly attributable to oxygen therapy and also noted an increased incidence of these injuries over the study period. The average age of the patients was 68.1 +/- 9.2 years (range, 40-82 years). Twenty-three were using oxygen at home, three in nursing homes, and one was an inpatient in an acute care facility. Twenty-five patients (93%) were receiving oxygen therapy for the diagnosis of chronic obstructive pulmonary disease. Twenty-four patients (89%) were smoking while using oxygen, two were lighting pilot lights, and one was lighting his wifes cigarette. Four patients (15%) sustained burns greater than 10% TBSA. Seventeen patients (63%) had only partial thickness burns. Thirteen patients (48%) required admission for treatment of their burn injuries. The average length of stay for those admitted was 4.4 days. The average hospital charge for admitted patients was US dollars 8055. There were four deaths (15%), all of which were correlated only with the extent of injury. Although intuitively obvious to most health care professionals, not all patients understand that oxygen therapy and cigarettes or open flame can result in a significant injury. Although some practitioners have advocated not prescribing home oxygen for those who continue to smoke, an alternative means of reducing the incidence of this preventable complication appears warranted. Prevention efforts should focus on the counseling of patients and their caregivers as well as educating primary care physicians, nurses, and home health providers as to the dangers of oxygen use.

Bilateral Synchronous Breast Cancer and HER-2/neu Overexpression

Bilateral synchronous breast cancer appears to have a worse prognosis than comparable unilateral breast cancer. HER-2/neu expression in bilateral breast cancer has not been reported. The purpose of this study was to review the characteristics of patients with bilateral synchronous breast cancer and to report the incidence of HER-2/neu overexpression. Between 1984 and 1998, 58 patients were diagnosed with bilateral synchronous breast cancer (defined as both cancers diagnosed within 3 months). The paraffin blocks from both breast specimens were available and immunostained in 21 patients. Of 42 breast specimens, there were 31 invasive carcinomas and 11 noninvasive carcinomas. Of the 21 paired specimens immunostained for HER-2/neu, 11 were invasive cancers in both breasts, nine were invasive cancers in one breast and noninvasive cancers in the other breast, and one was noninvasive cancers in both breasts. Of the 31 invasive carcinomas, HER-2/neu was overexpressed (2–3+) in 22 (71%) and negative (0–1+) in nine (29%). In contrast, 35 of 101 (34.7%) consecutive unilateral invasive breast cancer specimens from our institution overexpressed HER-2/neu. The difference in HER-2/neu overexpression between patients with bilateral synchronous breast cancer and unilateral breast cancer (22/31 v.s. 35/101) was statistically significant (chi squareu2009=u200911.3, p u2009 < u20090.001). In cases where both breasts had invasive carcinoma, HER-2/neu overexpression could be either in one (six patients) or both breasts (four patients). The increased mortality of patients with bilateral synchronous breast cancer may be due to the higher incidence of HER-2/neu overexpression.

Hyperthermia-induced heat shock activates the transcription factor C/EBP-β and augments IL-6 production in human intestinal epithelial cells

BACKGROUNDnInterleukin (IL)-6 production is increased in gut mucosa during sepsis and endotoxemia. The heat shock response augments IL-6 production under these conditions, but the mechanism is not known. We hypothesized that heat shock stimulates IL-6 production in enterocytes by increasing expression and activity of the transcription factor C/EBB.nnnSTUDY DESIGNnCultured Caco-2 cells, a human intestinal epithelial cell line, underwent induction of the heat shock response by hyperthermia (43 degrees C for 1 hour). Other cells were kept at 37 degrees C. Cells were then treated with 0.5 ng/mL human recombinant IL-1beta for 4 hours. C/EBP-beta and delta DNA binding activity was determined by electrophoretic mobility shift assay and supershift analysis. In additional experiments, Caco-2 cells were transfected with expression plasmids for C/EBP-beta and delta, after which cells were subjected to hyperthermia and treatment with IL-1beta.nnnRESULTSnC/EBP-beta, but not delta, protein levels and DNA binding activity were increased in Caco-2 cells expressing the heat shock response. Induction of the heat shock response augmented IL-6 production in IL-1beta-treated cells overexpressing C/EBP-beta, but not delta.nnnCONCLUSIONSnIncreased IL-6 production in IL-1beta-treated enterocytes expressing the heat shock response might be caused by upregulated expression and activity of CIEBP-beta. Because recent studies suggest that IL-6 might be an antiinflammatory cytokine and might exert protective effects in gut mucosa and enterocytes, understanding mechanisms by which the heat shock response augments IL-6 production might have important clinical implications.