Greg Noel

The effect of burn injury on suppressors of cytokine signalling.

The newly identified suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway, and they act as a negative feedback loop by subsequently inhibiting the Jak/STAT pathway either by direct interaction with activated Jaks or with the receptors. In this study we investigated the expression and translation of SOCS proteins after burn injury. Thermal injury increased the expression of SOCS3 compared with sham at 4 h, 24 h, and 10 days after thermal injury in the liver. SOCS3 protein was increased at 4 and 24 h after thermal injury in the liver. Expression of SOCS1 mRNA was not detected in sham or burn liver. SOCS2 mRNA and cytokine-inducible SH2-containing protein (CIS) mRNA were detected at the same levels for both sham and burn at all time points in the liver. In the spleen there was a trend towards an increase in SOCS1 mRNA at all time points; thermal injury significantly decreased SOCS2 mRNA compared with sham at 4 h, SOCS3 mRNA was significantly increased at 24 h compared with 10 days, and CIS mRNA was detected at the same levels for both sham and burn at all time points. In conclusion, thermal injury causes elevations in SOCS3 within 4 h after a burn, reaching a maximum at 24 h post injury. Levels continue to be elevated for up to 10 days post injury. SOCS3 may be very important in regulating the balance between immunosuppression and inflammation after thermal injury.

Postburn monocytes are the major producers of TNF-alpha in the heterogeneous splenic macrophage population.

Increased tumor necrosis factor (TNF)-&agr; production by postburn splenic macrophages is well documented. Splenic macrophages are a heterogeneous population, and the effect of thermal injury on these subpopulations has not been documented. We examined the effects of scald injury on myeloid cells with the phenotype of red pulp, white pulp, and marginal zone monocyte/macrophages. We found that thermal injury greatly increased the number of splenocytes with the phenotype of white pulp monocytes. These cells were the major producers of TNF-&agr; in the postburn spleen. Cells with the red pulp macrophage phenotype had an increased ability to make TNF-&agr; after burn injury, but had only half the capacity to make TNF-&agr; as did postburn monocytes. The postburn changes in TNF-&agr; production correlated with an increased in vivo susceptibility to endotoxin. The increase in monocytes in the spleen from postburn days 1 to 10 correlated with an increasing ability of splenocytes to produce granulocyte colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 2, and macrophage inflammatory protein 1-&agr;. These data suggest that the monocyte is a major source of inflammatory cytokines in the postburn spleen.

Neutrophils, not monocyte/macrophages, are the major splenic source of postburn IL-10.

Burn induces myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature polymorphonuclear neutrophils (PMNs) and monocytes, which protect against infection. Previous work from our laboratory demonstrated that inflammatory monocytes (iMos) were the major MDSC source of TNF-&agr; in the postburn spleen, and we hypothesized that they were also the major source of postburn IL-10. To test this hypothesis, we examined cytokine production by postburn CCR2 knockout (KO) mice, which have fewer iMos than burn wild-type (WT) splenocytes, but equal numbers of PMNs and F4/80 macrophages. Using cell sorting and/or intracellular cytokine techniques, we examined IL-10 production by postburn PMNs and iMos. Finally, we compared IL-10 production by postburn PMNs and iMos with culture-derived MDSCs. Splenocytes from postburn CCR2 KO mice produced less IL-6 and TNF-&agr; than WT burn splenocytes in response to LPS, but KO and WT burn splenocytes produced equal amounts of IL-10 in response to peptidoglycan. Depletion of PMNs from postburn splenocytes led to reductions in IL-10 and increases in IL-6 and TNF-&agr; in response to peptidoglycan, but not in response to LPS. Sorting or intracellular cytokine techniques gave consistent results: Burn PMNs made more IL-10 than sham PMNs and also more IL-10 than burn or sham iMos. Polymorphonuclear neutrophil and iMos subpopulations from culture-derived MDSCs produced the same cytokine profiles in response to LPS and peptidoglycan as did the PMNs and iMos from postburn spleens: PMNs made IL-10, whereas iMos made IL-6. Finally, LPS-induced mortality of burn mice was made worse by anti-Gr-1 depletion of all PMNs and 66% of iMos from burn mice. This suggests that PMNs play a primarily anti-inflammatory role in vitro and in vivo.

Differential immunological phenotypes are exhibited after scald and flame burns.

A dysfunctional immune system is known to be part of the pathophysiology after burn trauma. However, reports that support this have used a variety of methods, with numerous variables, to induce thermal injury. We hypothesized that, all other parameters being equal, an injury infliction by a scald would yield different immunological responses than one inflicted by a flame. Here, we demonstrated that both burn methods produced a full-thickness burn, yet there was more of an increase in subdermal temperature, hematocrit, mortality, and serum IL-6 concentrations associated with the scald burn. On postinjury day 1, the scald-burned mice showed diminished lymphocyte numbers, interferon &ggr; production, and lymphocyte T-bet expression as compared with sham- and flame-burned mice. On postburn day 8, spleens from both sets of thermally injured animals showed an increase in proinflammatory myeloid cells as compared with sham-burned mice. Furthermore, the T-cell numbers, T-bet expression, and phenotype were changed such that interferon &ggr; production was higher in scald-burned mice than in sham- and flame-burned mice. Altogether, the data show that differential immunological phenotypes were observed depending on the thermal injury method used.

Concerns over interspecies transcriptional comparisons in mice and humans after trauma

We have read with interest the study by Seok et al. (1) describing transcriptional responses of the immune systems of humans and mice. The authors perform Affymetrix GeneChip-based microarray assays on blood samples collected from blunt trauma, burn, and endotoxemia patients and mouse models of these pathologies. They report little correlation between human and murine genomic responses. Furthermore, the authors assert that mouse models of human disease are of questionable value due to the low biological similarity they observed.

STRESS HORMONE SECRETION AND GUT SIGNAL TRANSDUCER (STAT) PROTEINS AFTER BURN INJURY IN RATS

A burn injury triggers traumatic reactions characteristics of a stress. Here we investigated the early responses of prolactin (PRL), corticosterone (CS), and signal transducer and activator of transcription 5 (STAT5) in male Sprague-Dawley rats after burn injury. PRL and CS levels were determined in blood serum. STAT5 and phospho-STAT5 levels were determined in jejunum total protein extracts. The results confirmed an expected increase of CS between 4 and 6 h after the burn injury. Unexpectedly, PRL secretion was suppressed during the same time frame. These hormone levels returned to normal 6 to 8 h after burn injury. STAT5 was increased in the jejunum after burn injury, and its phosphorylation was increased between 8 and 11 h after burn injury. These changes in STAT5 were not temporally correlated with either the hormone changes that we observed or with previously documented changes of the gut function after burns.

A RIBONUCLEOTIDE REDUCTASE INHIBITOR REVERSES BURN-INDUCED INFLAMMATORY DEFECTS

Immature myeloid cells have been implicated as a source of postburn inflammation, and the appearance of these cells correlates with enhanced upregulation of hematopoiesis. The role of proliferative cells in postburn immune changes has not been directly tested. Gemcitabine, a ribonucleotide reductase inhibitor, has been shown to deplete proliferative immature myeloid cells in tumor models while sparing mature cells, leading to restored lymphocyte function and tumor regression. We treated burn mice at postburn day 6 (PBD6) with 120 mg/kg gemcitabine. On PBD8, splenocytes were taken and stimulated with LPS, peptidoglycan, or concanavalin A. The blood and spleen cell populations were enumerated by flow cytometry or automated cell counter. In addition, mice treated with gemcitabine were given LPS or infected with Pseudomonas aeruginosa at PBD8, and mortality was monitored. Gemcitabine depleted burn-induced polymorphonuclear leukocytes and inflammatory monocytes without affecting mature F4/80 macrophages. This was accompanied by reduced TNF&agr;, IL-6, and IL-10 production by burn splenocytes. Burn splenocytes stimulated with mitogens exhibited increased nitric oxide production relative to sham mice. In vivo treatment of burn mice with gemcitabine blocked these burn-induced changes without damaging lymphocyte function. Treatment of burn mice with gemcitabine ameliorated burn-induced susceptibility to LPS and infiltration of polymorphonuclear leukocytes into the liver and lung. Finally, gemcitabine treatment blocked the protective effect of burn injury upon P. aeruginosa infection. Our report shows that proliferative cells are major drivers of postburn immune changes and provides evidence that implicates immature myeloid cells in these processes.

Mice treated with a benzodiazepine had an improved survival rate following Pseudomonas aeruginosa infection.

Psychological stress has a high incidence after burn injury, therefore, anxiolytic drugs are often prescribed. Unfortunately, to date, no burn study has investigated the effects of anxiolytic drugs on the ability to fight infection. This study was undertaken to determine if psychological stress, anxiety-modulating drugs, or both, alter survival following an infection. On day 0, 7-week-old male C57Bl/6 mice either received a 15% full-thickness flame burn or were sham treated (anesthesia and shaved), whereas controls received no treatment. Mice received midazolam (1 mg/kg intraperitoneally) or saline daily and were stressed by exposure to rat in a guinea pig cage or placed in an empty cage for 1 hour a day, beginning on postburn day 1. For the survival experiments, mice either received bacteria after 2 or 8 consecutive days of predator exposure and drug treatment, which continued daily for 7 days after inoculation. In a separate set of experiments, after eight daily injections of midazolam, mice were given lipopolysaccharide, bacteria, or saline and were killed 12 hours later. Mice that received midazolam had improved survival rates when compared with their saline-treated counterparts, and the protective effect was more significant the more days they received the drug. For most of the cytokines, the bacteria-induced increase was significantly attenuated by midazolam as was the amount of bacteria in the liver. The protective effect seems to be independent of the drugs anxiolytic activity as there were no significant differences in survival between the predator-stressed and the nonstressed mice. The mechanisms responsible for the protective effect remain to be elucidated.

Mouse embryonic stem cells undergo charontosis, a novel programmed cell death pathway dependent upon cathepsins, p53, and EndoG, in response to etoposide treatment

Embryonic stem cells (ESCs) are hypersensitive to many DNA damaging agents and can rapidly undergo cell death or cell differentiation following exposure. Treatment of mouse ESCs (mESCs) with etoposide (ETO), a topoisomerase II poison, followed by a recovery period resulted in massive cell death with characteristics of a programmed cell death pathway (PCD). While cell death was both caspase- and necroptosis-independent, it was partially dependent on the activity of lysosomal proteases. A role for autophagy in the cell death process was eliminated, suggesting that ETO induces a novel PCD pathway in mESCs. Inhibition of p53 either as a transcription factor by pifithrin α or in its mitochondrial role by pifithrin μ significantly reduced ESC death levels. Finally, EndoG was newly identified as a protease participating in the DNA fragmentation observed during ETO-induced PCD. We coined the term charontosis after Charon, the ferryman of the dead in Greek mythology, to refer to the PCD signaling events induced by ETO in mESCs.

Heat shock stress ameliorates cytokine mixture-induced permeability by downregulating the nitric oxide and signal transducer and activator of transcription pathways in Caco-2 cells.

Proinflammatory cytokines are known to impair intestinal barrier function and to activate signaling pathways, whereas heat shock responses prevent cytokine-induced mucosal damage. We hypothesized that heat shock response blocks the effects of proinflammatory cytokines by regulating nitric oxide (NO) production and the activities of the Janus kinase/signal transducer and activator of transcription (STAT) pathway. A monolayer of Caco-2 cells were pretreated with sodium arsenite (SA, 500 &mgr;mol/L) for 1 h, followed by a 1-h recovery, and then stimulated with a cytokine mixture (cytomix: tumor necrosis factor &agr; [10 ng/mL], interferon beta [1000 U/mL], and interleukin [IL] 1&bgr; [1 ng/mL]) for 24 h. The permeability of horseradish peroxidase and fluorescein isothiocyanate-conjugated Dextran and transepithelial resistance and potential difference were measured in Ussing chambers. Interleukin-6, IL-8, NO, inducible NO synthase mRNA, STAT activity, and suppressor of cytokine signaling (SOCS) expression were measured in medium or cell lysates. Cytomix resulted in increased epithelial permeability of both fluorescein isothiocyanate-conjugated Dextran and horseradish peroxidase; whereas treatment of Caco-2 cells with SA 500 &mgr;mol/L blocked the cytomix-induced permeability changes. In addition, SA treatment decreased cytomix-induced NO production and inducible NO synthase mRNA expression and decreased the levels of STAT1, STAT3, SOCS1, and SOCS3. The SA treatment also decreased cytomix-induced IL-6 and IL-8 production in a dose-dependent manner. In conclusion, cytomix increased epithelial permeability, which is associated with increased NO and STAT activities. The SA treatment ameliorated cytomix-induced permeability, possibly through the downregulation of the NO and Janus kinase/STAT pathways.