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Background: Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood.Methods: We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique.Results: When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals.Conclusions: The novel microfluidic approach achieves leukocyte isolation in

Restoring oxygen delivery to ischemic organs after hemorrhagic shock is a fundamental goal of postinjury resuscitation. Currently, crystalloid resuscitation followed by transfusion of packed red blood cells (PRBCs) remains the standard of care; however, stored PRBC transfusion is not without adverse consequences. Allogenic blood transfusion for hemorrhagic shock has been linked to increased infection rates,1 and recent clinical studies indicate that the volume and timing of blood transfusion can increase the recipient's risk for developing postinjury multiple organ failure (MOF): specifically, transfusion of greater than 6 units of PRBC in the first 12 hours postinjury has been shown to be an independent risk factor for MOF, irrespective of the severity.2,3 The precise mechanisms in the pathogenesis of MOF remain unclear, and identification of these risk factors suggests that blood transfusion delivers an incremental insult contributing to a dysregulated systemic inflammatory response.4,5 Although it is known that stored red blood cells progressively change in size and shape with age,6 in vitro and clinical studies have offered greater insight into the proinflammatory effects of blood transfusion. Several bioactive substances accumulate with blood storage, including cytokines and proinflammatory lipids.7–9 Others have shown that there are extractable agents found in PRBC that can directly cause increased lung leak.10 Although removing leukocytes from stored PRBCs can reduce cytokine accumulation, proinflammatory lipids remain unfiltered. Furthermore, clinical studies demonstrate that despite leukoreduction, patients continue to experience adverse outcomes associated with transfusion.11–16 Conversely, avoiding PRBCs with the use of acellular hemoglobin-based oxygen carriers (HBOCs) to resuscitate severely injured patients is associated with a significantly lower systemic inflammatory response.17 In sum, it would seem that the predominant mechanism for the proinflammatory effect of PRBC transfusion lies in the interplay between the bioactive substances found in stored blood and the body's immune cells. Therefore, we think it is important to better understand how PRBCs affect the recipient's immune system, particularly in trauma where large-volume blood transfusions are common. In this study, we simulate major postinjury transfusion ex vivo and investigate the effect of PRBCs on circulating leukocyte gene expression. We hypothesize that PRBC transfusion induces inflammatory gene expression.

Technologies that enable the isolation of cell subtypes from small samples of complex populations will greatly facilitate the implementation of proteomics and genomics to human diseases. Transcriptome analysis of blood requires the depletion of contaminating erythrocytes. We report an automated microfluidic device to rapidly deplete erythrocytes from whole blood via deionized water lysis and to collect enriched leukocytes for phenotype and genomic analyses. Starting with blood from healthy subjects, we demonstrate the utility of this microfluidic cassette and lysis protocol to prepare unstimulated leukocytes, and leukocytes stimulated ex vivo with Staphylococcal enterotoxin B, which mimics some of the cellular effects seen in patients with severe bacterial infections. Microarrays are used to assess the global gene expression response to enterotoxin B. The results demonstrate that this system can isolate unactivated leukocytes from small blood samples without any significant loss, which permits more information to be obtained from subsequent analysis, and will be readily applicable to clinical settings.

Targeted expression of interleukin-10 (IL-10) has been proposed as a means to suppress acute and chronic inflammation. We explored the capacity of targeted adenoviral expression of human or viral IL-10 to improve outcome in a zymosan-induced model of acute lung injury and multisystem organ failure. Intratracheal administration of adenovirus expressing either human or viral IL-10 prior to zymosan administration significantly improved survival at a dose of 10(7) particles (P0.01), whereas the same recombinant vectors were ineffective at 10(8) particles and increased mortality at 10(9) particles. Improved survival after administration of 10(7) particles of adenovirus expressing viral or human IL-10 was associated with local tissue expression of IL-10 (100-300 pg/g wet wt). In contrast, mortality after administration of 10(9) particles was associated with markedly elevated IL-10 expression, both in the lung (10000-70000 pg/g wet wt) and systemically (1000-3000 pg/ml plasma), with evidence of an exaggerated systemic inflammatory response (plasma IL-6 and TNFalpha). Targeted gene expression of IL-10 can be used to treat acute inflammatory processes, but increased doses resulting in its systemic release are not associated with improvements in outcome, and may actually exacerbate acute inflammatory processes.

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (105) yields only local expression, while transduction with higher particle numbers (107 and 1010) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (105) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.

Adenovirus (Ad) gene therapy has been proposed as a drug-delivery system for the targeted administration of protein-based therapies, including growth factors and biological response modifiers. However, inflammation associated with Ad transduction has raised concern about its safety and efficacy in acute inflammatory diseases. In the present report, intratracheal and i.v. administration of a first-generation adenoviral recombinant (E1,E3 deleted) either containing an empty cassette or expressing the anti-inflammatory cytokines viral or human IL-10 (IL-10) was administered to mice subjected to zymosan-induced multisystem organ failure or to acute necrotizing pancreatitis. Pretreatment of mice with the intratracheal instillation of Ad expressing human IL-10 or viral IL-10 reduced weight loss, attenuated the proinflammatory cytokine response, and reduced mortality in the zymosan-induced model, whereas pretreatment with a control adenoviral recombinant did not significantly exacerbate the response. Pretreatment of mice with pancreatitis using adenoviral vectors expressing IL-10 significantly reduced the degree of pancreatic and liver injury and liver inflammation when administered systemically, but not intratracheally. We conclude that adenoviral vectors can be administered prophylactically in acute inflammatory syndromes, and expression of the anti-inflammatory protein IL-10 can be used to suppress the underlying inflammatory process.

Tumor necrosis factor (TNF)-α and Fas ligand (FasL) are trimeric proteins that induce apoptosis through similar caspase-dependent pathways. Hepatocytes are particularly sensitive to inflammation-induced programmed cell death, although the contribution of TNF-α and/or FasL to this injury response is still unclear. Here, we report that d-galactosamine and lipopolysaccharide-induced liver injury in C57BL/6 mice is associated with increased hepatic expression of both TNF-α and FasL mRNA. Pretreatment of mice with a TNF-binding protein improved survival, reduced plasma aspartate aminotransferase concentrations, and attenuated the apoptotic liver injury, as determined histologically and by in situ 3′ OH end labeling of fragmented nuclear DNA. In contrast, pretreatment of mice with a murine-soluble Fas fusion protein (Fasfp) had only minimal effect on survival, and apoptotic liver injury was either unaffected or exacerbated depending on the dose of Fasfp employed. Similarly, mice with a spontaneous mutation in FasL (B6Smn.C3H- Faslgldderived from C57BL/6) were equally sensitive tod-galactosamine/lipopolysaccharide-induced shock. We conclude that the shock and apoptotic liver injury afterd-galactosamine/lipopolysaccharide treatment are due primarily to TNF-α release, whereas increased FasL expression appears to contribute little to the mortality and hepatic injury.

Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-α and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. β-Galactosidase expression in mice receiving intratracheal instillation of Adv/β-gal (adenovirus construct expressing β-galactosidase) was transient (less than 14 days), but a significant early increase of β-galactosidase expression was seen in mice lacking either or both TNF-α receptors. Absence of TNF-α or the p55 receptor significantly attenuated the Ab response to both adenovirus and β-galactosidase. Human IL-10 expression in the lung suppressed local TNF-α production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with β-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-α signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with β-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with β-galactosidase, and is nonimmunogenic in the lung.

Background. Tumor necrosis factor-α (TNF-α) is a member of a large family of predominantly homotrimeric type II membrane-associated proteins with both proinflammatory and apoptosis-inducing properties. Although TNF-α expression has been studied extensively, little is known about the expression of other members of the TNF-α superfamily during acute inflammatory processes. Methods. TNF-α, Fas ligand (FasL), and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) messenger RNA (mRNA) expression were examined in liver, lung, spleen, and kidney after either a cecal ligation and puncture or endotoxemic shock with use of semiquantitative reverse transcriptase-polymerase chain reaction. Results. Cecal ligation and puncture increased TNF-α mRNA in lung and liver (both P

Apoptosis is a physiologic process that serves to eliminate cells during development or in response to immunologic regulation. In acute inflammation, however, apoptosis triggered by the overproduction of “death factors” such as TNF-α or Fas ligand (FasL) may contribute to tissue injury. Both TNF-α and FasL are presumed to convey an apoptotic signal by activating a cascade of cysteine-aspartate proteases, which includes IL-1β-converting enzyme or caspase-1. In the present study, we evaluated the contribution of TNF-α and FasL, as well as the role of caspase-1, in Con A-induced hepatitis. We report here that TNF-α and FasL mRNA and protein levels are both increased in the livers of Con A-challenged mice. Using a novel inhibitor of TNF-α, we can confirm that Con A-induced hepatitis is primarily TNF-α dependent. Blockade of FasL with a soluble Fas immunoadhesin does not prevent liver injury in animals treated with Con A alone. However, administration of a matrix metalloproteinase inhibitor exacerbates liver injury, in part through a FasL-dependent process, since pretreatment with the soluble Fas immunoadhesin reduces liver injury in this model. In addition, mice lacking functional caspase-1 are resistant to Con A-induced hepatitis, even after pretreatment with a matrix metalloproteinase inhibitor. We conclude that TNF-α plays a predominant role in Con A-induced liver injury, although concomitant activation of FasL can also lead to apoptotic injury. Furthermore, Con A-induced hepatitis is caspase-1 dependent.

Excessive tumor necrosis factor alpha (TNF alpha) production in response to Gram-negative bacteremia or endotoxemia can often lead to hypotension, shock, and increased mortality. Current approaches used to block the deleterious effects of exaggerated TNF alpha production rely on monoclonal antibodies or immunoadhesins that bind TNF alpha and thus prevent the interaction with its cellular receptors. This report examines whether a previously described inhibitor of matrix metalloproteinases, GM-6001, can inhibit TNF alpha processing and release and attenuate endotoxin-induced mortality. In human peripheral blood mononuclear cells stimulated in vitro with 1 microgram/mL endotoxin, GM-6001 at concentrations > 5 micrograms/mL blocked release of TNF alpha, but did not affect the release of either IL-1 beta or IL-6. GM-6001 also inhibited the release of soluble TNF receptor (p75) from peripheral blood mononuclear cells stimulated with endotoxin and/or TNF alpha. To confirm the role of secreted TNF alpha in endotoxic shock-induced mortality, C57BL/6 mice were challenged with either endotoxin alone (500 micrograms/mouse) or endotoxin (100 ng/mouse) plus D-galactosamine (8 mg/mouse). GM-6001 pretreatment (100 mg/kg) significantly attenuated the 90-minute plasma TNF alpha response in both models and improved survival in mice treated with low-dose endotoxin plus D-galactosamine. However, plasma IL-1 beta and IL-6 concentrations at 90 min after endotoxin treatment were unaffected by GM-6001 following lethal endotoxin challenge, confirming the in vivo specificity of this matrix metalloproteinase inhibitor for TNF alpha processing. These findings demonstrate that a novel inhibitor of matrix metalloproteinases can prevent the release of TNF alpha both in vitro and in vivo, and can abrogate the harmful sequelae of endotoxemic shock.

Background & Aims: Oxygen radicals and reactive oxygen species play an important role in inflammatory episodes in the bowel. Nonetheless, little is known about the regulation of colonic superoxide dismutases and key antioxidant enzymes with cytoprotective and radical detoxifying properties. The aim of this study was to examine the regulation of manganese superoxide dismutase (SOD) in acute acetic acid-induced colitis. Methods: Colitis was induced in adult rats by the rectal administration of 5% acetic acid. Total RNA and protein were isolated from the inflamed colon from 1 to 24 hours after the induction of colitis. MnSOD messenger RNA and protein levels were evaluated by Northern and Western analyses, respectively. MnSOD protein was localized in cross sections of the colon by immunocytochemistry. Results: MnSOD messenger RNA levels showed a rapid 14–96-fold induction in response to acetic acid administration. Western analysis showed a 22–49-fold induction in MnSOD protein levels. Immunocytochemistry showed induction of MnSOD protein, specifically in smooth muscle cells, epithelial cells at the base of the glands, and myenteric plexus neurons. Conclusions: MnSOD messenger RNA and protein levels are rapidly induced following the inflammatory insult, implicating a role for MnSOD in the acute phase of colonic inflammation. We suggest that induction of MnSOD in specific cell types may have a cytoprotective function.

Dendritic cells (DCs) play a key role in the interface between the innate and acquired immune systems. In response to both exogenous as well as endogenous signals, DCs undergo a programmed maturation to become an efficient, antigen-presenting cell. Yet little is known regarding the differential responses by endogenous versus exogenous stimuli on DC maturation. In the present report, we have compared the phenotypic, functional, and genome-wide expression responses associated with maturation by bone marrow derived DCs to either an endogenous danger signal, tumor necrosis factor-alpha (TNF-alpha), or a microbial product, bacterial lipopolysaccharide (LPS). Examination of the cell surface expression of DCs as well as cytokine production demonstrated that patterns of DC maturation varied dramatically depending upon the stimulus. Whereas LPS was highly effective in terms of inducing phenotypic and functional maturation, TNF-alpha exposure produced a phenotypically distinct DC. Gene expression patterns in DCs 6 and 24 h after LPS and TNF-alpha exposure revealed that these activation signals produce fundamentally different genomic responses. Supervised analysis revealed that the expression of 929 probe sets discriminated among the treatment groups, and the patterns of gene expression in TNF-alpha stimulated DCs were more similar to unstimulated cells at both 6 and 24 h post-stimulation than to LPS-stimulated cells at the same time points. These findings reveal that DCs are capable of a varying phenotypic response to different antigens and endogenous signals.

Control of dendritic cell (DC) function is critical for strategies to modulate innate and acquired immune responses. We examined whether transduction of murine DCs with adenoviral vectors (Adv) expressing interleukin (IL)-10 could alter their phenotype and T cell stimulatory function. Murine bone marrow-derived DCs were transduced with AdV encoding human IL-10 or green fluorescent protein (GFP). Whereas transduction of immature DCs with AdV/GFP resulted in dose-dependent maturation, DCs transduced with Adv/IL-10 maintained an immature state with low major histocompatibility complex (MHC) class II, CD86, and IL-12 expression. The Adv/IL-10 transduced DCs were phenotypically unique, characterized by suppression of IL-12 expression, failure to stimulate Th1 or Th2 cytokine responses, and retained capacity to endocytose antigen. Importantly, Adv/IL-10-transduced DCs were biologically active in vivo, in that administration of these DCs into mice before a generalized peritonitis significantly improved survival. We conclude that Adv/IL-10 transduction of DCs provides an efficient means to modulate DC function. The capacity to modify DCs by adenoviral expression of IL-10 may provide a novel ex vivo or in vivo approach to mitigate acute and chronic inflammatory diseases like sepsis.

The analysis of gene expression data in clinical medicine has been plagued by the lack of a critical evaluation of accepted methodologies for the collection, processing, and labeling of RNA. In the present report, the reliability of two commonly used techniques to isolate RNA from whole blood or its leukocyte compartment was compared by examining their reproducibility, variance, and signal-to-noise ratios. Whole blood was obtained from healthy subjects and was either untreated or stimulated ex vivo with Staphylococcus enterotoxin B (SEB). Blood samples were also obtained from trauma patients but were not stimulated with SEB ex vivo. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system or from isolated leukocytes. Biotin-labeled cRNA was hybridized to Affymetrix GeneChips. The Pearson correlation coefficient for gene expression measurements in replicates from healthy subjects with both techniques was excellent, exceeding 0.985. Unsupervised analyses, including hierarchical cluster analysis, however, revealed that the RNA isolation method resulted in greater differences in gene expression than stimulation with SEB or among different trauma patients. The intraclass correlation, a measure of signal-to-noise ratio, of the difference between SEB-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples than in whole blood: 0.75 vs. 0.46 ( P = 0.002). At the P < 0.001 level of significance, twice as many probe sets discriminated between SEB-stimulated and unstimulated blood with leukocyte isolation than with PAXgene. The findings suggest that the method of RNA isolation from whole blood is a critical variable in the design of clinical studies using microarray analyses.

Sepsis remains a significant clinical conundrum, and recent clinical trials with anticytokine therapies have produced disappointing results. Animal studies have suggested that increased lymphocyte apoptosis may contribute to sepsis-induced mortality. We report here that inhibition of thymocyte apoptosis by targeted adenovirus-induced thymic expression of human IL-10 reduced blood bacteremia and prevented mortality in sepsis. In contrast, systemic administration of an adenovirus expressing IL-10 was without any protective effect. Improvements in survival were associated with increases in Bcl-2 expression and reductions in caspase-3 activity and thymocyte apoptosis. These studies demonstrate that thymic apoptosis plays a critical role in the pathogenesis of sepsis and identifies a gene therapy approach for its therapeutic intervention.

A hammerhead ribozyme directed against murine TNFalpha (mTNFalpha) mRNA has been constructed. In vitro studies showed that this ribozyme was released from the parent molecule by flanking cis-acting hammerhead and hairpin ribozymes. This same anti-mTNFalpha ribozyme specifically cleaved both synthetically derived substrate RNA and mTNFalpha mRNA within a pool of total cellular RNA. Endogenous delivery of this anti-mTNFalpha ribozyme via the self-cleaving cassette reduced mTNFalpha mRNA and protein levels in lipopolysaccharide (LPS)-stimulated, stably transfected murine macrophage RAW 264.7 cells. When complexed to liposomes and exogenously delivered to mouse peritoneal macrophages, the same ribozyme, with and without the cis-acting ribozymes, reduced mTNFalpha protein levels. However, an irrelevant ribozyme delivered in an identical fashion was also effective at reducing mTNFalpha protein levels. These data suggest that anti-mTNFalpha ribozymes can be constructed which efficiently cleave mTNFalpha mRNA, but irrelevant RNA/liposome complexes also effectively limit TNFalpha mRNA expression and can mimic functional ribozyme activity under in vitro conditions.

Inducible nitric oxide synthase (iNOS) is expressed during cutaneous wound repair. Mounting evidence suggests that wound nitric oxide (NO) augments collagen accumulation. We hypothesized that in vivo transfection of wound cells with the iNOS gene would increase physiological wound NO levels and thus augment collagen accumulation. Polyvinyl alcohol sponges were instilled with a mammalian expression plasmid (pMP6) containing either the chloramphenicol acetyl transferase (CAT) reporter or murine iNOS gene driven by a CMV immediate-early promoter. Plasmid DNA was injected alone or in complex with cationic liposomes, and the sponges were placed subcutaneously in male Sprague-Dawley rats which had received a longitudinal dorsal midline incision. Animals were sacrificed at different time points post-wounding and the sponges assayed for CAT activity, transfected iNOS mRNA, total nitrate and nitrite concentration (NOx) (as an index of wound NO synthesis), and hydroxyproline content (as an index of sponge collagen accumulation). The results demonstrate that wound cells were more efficiently transfected by naked DNA than by liposome mediated transfection and that maximal expression of both iNOS and CAT occurred at 48 hrs with a rapid decline after this time point. After 7 days, iNOS transfected sponges had accumulated significantly more collagen than those transfected with CAT. We conclude that cutaneous wounds can be successfully transfected by direct injection of naked DNA and that increased iNOS expression precedes an increase in collagen synthesis.

OBJECTIVE: To determine if transfection of SW48 colon cancer cells with the type II transforming growth factor-beta (TGF-beta) receptor restores growth inhibition and reverses the in vitro and in vivo malignant phenotype. SUMMARY BACKGROUND DATA: The authors have previously shown that SW48 colon cancer cells that are replication error positive in both alleles lack functional cell surface TGF-beta type I (RI) and type II (RII) receptors and are insensitive to TGF-beta1-induced growth inhibition. METHODS: SW48 cells were stably transfected with the cDNA for the normal type II TGF-beta receptor (RII). Once transfected, the cells were evaluated for in vitro phenotypic changes and in vivo changes in tumor growth. RESULTS: Denaturing sequencing gel electrophoresis of the reverse transcriptase-polymerase chain reaction product from SW48 cells revealed that the RII coding sequence contained a single base deletion mutation. When these cells were transfected with normal RII cDNA, Northern and Western blot analyses revealed increased levels of RII mRNA and protein. Affinity labeling techniques revealed that RII-transfected SW48 cells produced functional RI and RII protein. Transfection of SW48 cells also led to changes in cell phenotype, as shown by inhibition of both in vitro growth rate and incorporation of [3H]-thymidine. SW48 cells expressing normal RII also exhibited reduced cloning efficiency in semisolid medium and reduced growth as a xenograft in NOD/LtSz-scid/J mice. CONCLUSIONS: The results confirm that RII is a tumor-suppressor protein that is required for TGF-beta-induced growth inhibition in SW48 colon cancer cells.

We have observed a rapid induction of manganese superoxide dismutase (MnSOD) in epithelial, neuronal, and smooth muscle cells (SMC) after acetic acid-induced colitis. To examine the regulation of MnSOD in the SMC more specifically, primary cultures of colonic SMC were developed by enzymatic digestion of the circular muscle layer from an adult rat. SMC were treated for 2-72 h with 0.5 microgram/ml Escherichia coli endotoxin [lipopolysaccharide (LPS)], 10 ng/ml tumor necrosis factor (TNF)-alpha, or 2 ng/ml interleukin-1 beta (IL-1 beta). Cotreatments were performed with IL-1 beta and 4 microM actinomycin or 50 microM cycloheximide. Northern analysis demonstrated 23-fold, 8-fold, and 6-fold inductions of MnSOD mRNA by IL-1 beta, LPS, and TNF-alpha, respectively. Induction of MnSOD by IL-1 beta was eliminated by actinomycin but not by cycloheximide, implicating a requirement for de novo transcription. Western analysis resulted in a 23.7-fold induction of MnSOD protein after 48-h treatment with IL-1 beta. Induction of MnSOD by IL-1 beta and other inflammatory mediators may serve as a protective mechanism to reduce oxygen free radical- and nitric oxide-mediated cell damage during inflammation.

BACKGROUND & AIMS: Manganese superoxide dismutase (MnSOD) is rapidly induced in myenteric plexus neurons (MPNs) in acute colitis and may protect cells from nitric oxide toxicity. Inducible nitric oxide synthase (iNOS) regulation was examined in acute colitis, and MnSOD and iNOS were examined in primary cultures of MPNs. METHODS: Acute colitis in rats was induced with 5% acetic acid. iNOS messenger RNA (mRNA) was analyzed by Northern analysis, and reduced nicotinamide adenine dinucleotide phosphate diaphorase was used to identify potential NO synthase activity. MnSOD and iNOS mRNA levels were evaluated in cultured MPNs after treatment with tumor necrosis factor alpha, interleukin (IL) 1beta, or IL-6. iNOS and MnSOD protein expression in control and IL-1beta-treated neurons was evaluated by immunofluorescence microscopy. RESULTS: iNOS mRNA was detected in the mucosal and muscularis layers after the initiation of colitis. Reduced nicotinamide adenine dinucleotide phosphate diaphorase localized NO synthase activity to MPNs in controls and in epithelial cells and MPNs in the inflamed colon. In MPN cultures, tumor necrosis factor alpha and IL-1beta treatment resulted in induction of MnSOD, but only IL-1beta induced iNOS. Immunolocalization confirmed that the neurons were the primary source of iNOS and MnSOD. CONCLUSIONS: Induction of MnSOD and iNOS are coordinated and may limit NO cytotoxicity. The function of iNOS in gut neurons remains to be delineated. (Gastroenterology 1996 Jul;111(1):56-64)