Source Of Tumor Necrosis Factor Research Articles

Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4+ Tissue-resident Memory T cells in Crohn’s Disease

Background and aimsAs the most abundant memory T cells and major source of tumor necrosis factor α (TNF-α) in the intestinal mucosa of Crohn’s disease (CD) patients, CD4+ tissue-resident memory T (TRM) cells play a critical role in CD pathogenesis. We investigated the role of metabolic reprogramming in the regulation of proinflammatory and apoptosis-resistant phenotype for CD4+ TRM cells. MethodsCD4+ TRM cells were collected from intestinal resection tissues from control and CD patients. Transcriptomic and metabolomic analysis were performed to identify metabolic characteristics of CD4+ TRM cells. ELISA and qPCR experiments were utilized to assess cytokines level in CD4+ TRM cells, activation-induced cell apoptosis (AICD) rate was evaluated by flow cytometry. Transwell assay and wound healing assay were performed to detect the effect of CD4+ TRM cells on the migration of normal intestinal epithelial cells. ResultsTranscriptomic data combined with unbiased metabolomic analysis revealed an increased fatty acid oxidation (FAO) phenotype existed in CD4+ TRM cells from CD patients. The lipidomic data and stable isotope tracer experiments demonstrated that CD4+ TRM cells upregulated their lipid lipolysis and fatty acid uptake to fuel FAO in CD patients. Mechanistically, the activated NF-κB signaling increased transcription of genes involved in lipid lipolysis, fatty acid uptake and oxidation in CD4+ TRM cells from CD patients. Targeting FAO of CD4+ TRM cells reversed their apoptosis-resistant and proinflammatory phenotype in CD Patients. ConclusionCD4+ TRM cells process an accelerated FAO mediated by activated NF-κB signaling in CD patients, targeting FAO could reverse their apoptosis-resistant and proinflammatory phenotype. These findings shed a new light on the pathogenic mechanism investigation and novel therapy development in CD patients.

Priming astrocytes with TNF enhances their susceptibility to Trypanosoma cruzi infection and creates a self-sustaining inflammatory milieu

BackgroundIn conditions of immunosuppression, the central nervous sty 5ystem (CNS) is the main target tissue for the reactivation of infection by Trypanosoma cruzi, the causative agent of Chagas disease. In experimental T. cruzi infection, interferon gamma (IFNγ)+ microglial cells surround astrocytes harboring amastigote parasites. In vitro, IFNγ fuels astrocyte infection by T. cruzi, and IFNγ-stimulated infected astrocytes are implicated as potential sources of tumor necrosis factor (TNF). Pro-inflammatory cytokines trigger behavioral alterations. In T. cruzi-infected mice, administration of anti-TNF antibody hampers depressive-like behavior. Herein, we investigated the effects of TNF on astrocyte susceptibility to T. cruzi infection and the regulation of cytokine production.MethodsPrimary astrocyte cultures of neonatal C57BL/6 and C3H/He mice and the human U-87 MG astrocyte lineage were infected with the Colombian T. cruzi strain. Cytokine production, particularly TNF, and TNF receptor 1 (TNFR1/p55) expression were analyzed. Recombinant cytokines (rIFNγ and rTNF), the anti-TNF antibody infliximab, and the TNFR1 modulator pentoxifylline were used to assess the in vitro effects of TNF on astrocyte susceptibility to T. cruzi infection. To investigate the role of TNF on CNS colonization by T. cruzi, infected mice were submitted to anti-TNF therapy.ResultsrTNF priming of mouse and human astrocytes enhanced parasite/astrocyte interaction (i.e., the percentage of astrocytes invaded by trypomastigote parasites and the number of intracellular parasite forms/astrocyte). Furthermore, T. cruzi infection drove astrocytes to a pro-inflammatory profile with TNF and interleukin-6 production, which was amplified by rTNF treatment. Adding rTNF prior to infection fueled parasite growth and trypomastigote egression, in parallel with increased TNFR1 expression. Importantly, pentoxifylline inhibited the TNF-induced increase in astrocyte susceptibility to T. cruzi invasion. In T. cruzi-infected mice, anti-TNF therapy reduced the number of amastigote nests in the brain.ConclusionsOur data implicate TNF as a promoter of T. cruzi invasion of mouse and human astrocytes. Moreover, the TNF-enriched inflammatory milieu and enhanced TNFR1 expression may favor TNF signaling, astrocyte colonization by T. cruzi and egression of trypomastigotes. Therefore, in T. cruzi infection, a self-sustaining TNF-induced inflammatory circuit may perpetuate the parasite cycle in the CNS and ultimately promote cytokine-driven behavioral alterations.

The NLRP3/ASC inflammasome promotes T-cell-dependent immune complex glomerulonephritis by canonical and noncanonical mechanisms

Interleukin (IL)-1β contributes to renal injury in immune complex glomerulonephritis. However, production of mature IL-1β depends on activation of the inflammasome that cleaves pro-IL-1β into its secretable form. A functional role of the NLRP3-containing inflammasome, which responds to various endogenous danger signals, was found in tubulointerstitial nephropathies, but its function in glomerular disease has not been established. To determine whether NLRP3 and its adapter molecule ASC contribute to glomerulonephritis, we induced T-cell-dependent autologous nephrotoxic serum nephritis in Nlrp3- and Asc-deficient mice. Renal expression of NLRP3/ASC inflammasome components and pro-IL-1β increased during nephrotoxic serum nephritis and was abundant in renal dendritic cells. This was associated with renal production of mature IL-1β, indicating inflammasome activation. Nlrp3 and Asc deficiency significantly attenuated glomerular injury, renal leukocyte infiltration, and T-cell activation. Production of mature IL-1β was abrogated in Asc-deficient mice, consistent with a loss of inflammasome-dependent IL-1β activation. Surprisingly, renal IL-1β secretion remained intact in Nlrp3-deficient mice, indicating noncanonical pro-inflammatory effects of NLRP3 in autologous nephrotoxic serum nephritis. This may include NLRP3-mediated glomerular release of pro-inflammatory high-mobility group box 1 protein as a noncanonical function of NLRP3/ASC in glomerulonephritis. Thus, therapeutic blockade of the NLRP3/ASC/IL-1β axis may be beneficial in glomerulonephritis.

TNF/TNF-R1 pathway is involved in doxorubicin-induced acute sterile inflammation

Dear Editor, Doxorubicin is an anthracycline antibiotic, which is used in treatment of cancer and it works by induction of apoptosis in cancerous cells.1 In vivo, apoptotic cells are rapidly cleared preferentially by monocytes and neutrophil migration is inhibited2 to limit tissue injury and inflammation. More recently, few anti-cancer drugs, including anthracyclines (doxorubicin) were shown to evoke apoptosis in cancerous cells, which is associated with immune system activation (i.e. immunogenic apoptosis).3, 4 In our previous study we reported that doxorubicin can induce acute inflammation in peritoneum, which is associated with apoptosis.5 Doxorubicin-killed cells could be also a source of damage-associated molecular patterns (DAMPs) leading to inflammation and tumor necrosis factor (TNF) production, which would amplify the inflammatory response triggered by DAMPs. TNF is a pleiotropic cytokine that binds to receptors TNF-R1 and TNF-R2 and, depending on cell type, triggers different signaling pathways, including cell death and inflammation.6 This study aimed to examine whether TNF contributes to the doxorubicin-induced acute sterile inflammation. Intraperitoneal injection of doxorubicin provoked an acute inflammatory response accompanied by the influx of neutrophils.5 We observed an increased level of LDH, a marker of tissue damage and secondary necrotic cells, already after 6 h with further increase after 16 h post injection of doxorubicin (Supplementary Figure 1a). Lavage fluid collected 16 h after doxorubicin injection contained increased levels of TNF as compared to vehicle-injected group (0.09±0.14 versus 2.11±0.84 pg/ml, P<0.0001). We next tested the involvement of TNF-R1 and TNF-R2 in the sterile inflammation in response to doxorubicin. Wild-type mice had abundant neutrophils in their abdominal cavities, but this response was markedly decreased in TNF-R1/2 double knockout mice (by 2.4-fold, Supplementary Figure 1b). The number of neutrophils attracted in response to doxorubicin-induced acute inflammation was 3.4-fold lower in TNF-R1-deficient mice than in controls (Figure 1a), but in TNF-R2-deficient mice, there was no difference as compared to wild-type mice (Figure 1b). These results show that TNF-R1 was involved in mediating the inflammatory response but TNF-R2 was not. Importantly, cell death in TNF-R1/2 double knockout mice was not affected in comparison to wild-type mice (data not shown) indicating that TNF does not contribute to the cell death process itself. Figure 1 Role of TNF-R1 and TNF-R2 and the effect of inhibiting soluble TNF on doxorubicin-induced acute inflammatory response. The number of neutrophils in peritoneal lavage was decreased in TNF-R1−/− (a) but not in TNF-R2−/− ( ... Further, we investigated whether acute inflammation can be reduced in wild-type mice by the administration of Etanercept, a recombinant dimeric soluble form of TNF-R2 that blocks the interaction of TNF with its cell surface receptors.7 Wild-type mice were injected intraperitoneally with Etanercept together with doxorubicin and 16 h later the recruited cells were phenotyped. Etanercept significantly reduced the recruitment of neutrophils (Figure 1c). This further confirms the importance of TNF in doxorubicin-induced acute inflammation. In conclusion, we report that intraperitoneal injection of doxorubicin in mice leads to increase in TNF levels in the lavage fluid. It has been also reported that patients undergoing chemotherapy with doxorubicin had elevated levels of TNF in their plasma.8 Similarly, mouse in vitro and in vivo studies demonstrated up-regulation of TNF after doxorubicin administration both on mRNA and protein levels.9, 10 Previously, we found that the majority of cells that died apoptotically due to doxorubicin treatment were monocytes/macrophages with some minor neutrophils.5 Therefore, it is possible that either dying macrophages or attracted neutrophils might be a source of TNF, and that by binding to TNF-R1 it could amplify the inflammation. We demonstrated that the acute inflammatory response to doxorubicin was reduced in TNF-R1- but not TNF-R2-deficient mice. In addition, Etanercept decreases the attraction of neutrophils after doxorubicin administration. These studies show that TNF and the TNF-R1 signaling pathway are key elements in the acute sterile inflammatory response to doxorubicin.

Dendritic cells facilitate accumulation of IL-17 T cells in the kidney following acute renal obstruction

Acute urinary obstruction causes interstitial inflammation with leukocyte accumulation and the secretion of soluble mediators. Here we show that unilateral ureteral ligation caused a progressive increase in renal F4/80(+) and F4/80(-) dendritic cells, monocytes, neutrophils and T-cells 24-72 h following obstruction. Depletion of dendritic cells by clodronate pretreatment showed these cells to be the most potent source of tumor necrosis factor and other pro-inflammatory mediators in the obstructed kidney. F4/80(+) dendritic cells and T-cells co-localized in the cortico-medullary junction and cortex of the obstructed kidney. Cytokine secretion patterns and surface phenotypes of T-cells from obstructed kidneys were found to include interferon-gamma-secreting CD4(+) and CD8(+) memory T-cells as well as interleukin 17 (IL-17)-secreting CD4(+) memory T-cells. Depletion of the intra-renal dendritic cells prior to ligation did not numerically reduce T-cells in obstructed kidneys but attenuated interferon-gamma and IL-17-competent T-cells. Our study shows that intra-renal dendritic cells are a previously unidentified early source of proinflammatory mediators after acute urinary obstruction and play a specific role in recruitment and activation of effector-memory T-cells including IL-17-secreting CD4(+) T-cells.

Estrogen-TNF interactions and vascular inflammation

cardiovascular disease (CVD), especially coronary heart disease (CHD), is by far the leading cause of death in women ([29][1], [40][2]). As the prevalence of these diseases increases after menopause, much effort has been devoted to determining whether endogenous estrogen confers a protective effect

Increase in CD30 ligand/CD153 and TNF-alpha expressing mast cells in basal cell carcinoma.

Mast cells are a significant source of tumor necrosis factor (TNF) superfamily members, such as TNF-alpha, CD30 ligand/CD153 (CD30L) and CD40L/CD154. Furthermore, the expression of some of these proteins in mast cells has been associated with tumorigenesis, and mast cells have been found to be increased in number in the basal cell carcinoma (BCC) lesion. In this study, we have examined the expression of TNF-alpha, CD30L and CD40L immunoreactivity in mast cells in the healthy-looking skin and lesional skin of ten patients with superficial spreading BCC. Also, the counterparts of these molecules, TNF receptor (TNFR) I and II as well as CD30 and CD40, were analysed immunohistochemically. We found that numbers of mast cells and Kit-positive cells were significantly increased in the dermal BCC lesion. The percentage of CD30L-positive mast cells and the number of CD30-positive cells were significantly increased in the upper dermis of the BCC lesion as well. In addition, the numbers of TNF-alpha-positive mast cells and cells with TNFRI and TNFRII were markedly increased in the upper lesional dermis. In contrast, no mast cells positive for CD40L could be detected, even though the lesional dermis contained increased numbers of CD40 positive cells. The BCC epithelium was positive for TNFRI, TNFRII and CD40, but not for CD30, though the larger basal buds appeared to be less intensely stained for TNFRI and CD40. In conclusion, mast cells positive for CD30L and TNF-alpha, but not CD40L, are increased in number in the lesional dermis in BCC. These data suggest plausible pathways whereby mast cells can be activated and to interact with other cells and thereby contribute to the tumorigenesis in BCC.

Left portahepatic shunt: A novel technique to decrease excessive portal venous inflow during live donor liver transplantation

A critical shortage of cadaveric livers and an increasing population on the waiting list have encouraged the development of living donor liver transplantation. Living donor liver transplantation accounts for approximately 10% of all liver transplants being performed. One problem unique to lobar transplantation relates to hepatic congestion and associated functional impairment of the transplanted lobe. This congestion is multifactorial and includes low graft-weight ratio, middle hepatic vein drainage problems, and excessive portal venous inflow. Increasing disparities of graft-weight ratios exaggerate the importance of the other two factors. Finally, animal data from asplanchnic and anhepatic models implicate the intestine as the source of tumor necrosis factor released into the circulation following portal unclamping. Tumor necrosis factor, in turn, leads to adhesion molecule upregulation and endothelial cell disruption and potentiates allograft dysfunction. Although veno-venous bypass may alleviate some of these problems, it does not prevent excessive mesenteric venous flows, nor does it prevent the tumor necrosis factor production associated with portal clamping during the portal anastomotic phase. We have recently developed the technique of temporary left portahepatic shunt. This allows portasystemic decompression during implantation and diverts mesenteric blood from the liver during the crucial period after unclamping. Early lobar congestion can be avoided, and the shunt may be left in place for as long as 2 hours while volume homeostasis is restored. This technique allows the tailoring of the portal flow to the graft size to avoid any liver congestion or dysfunction.

Intrinsic renal cells are the major source of tumor necrosis factor contributing to renal injury in murine crescentic glomerulonephritis.

Macrophages are prominent participants in crescentic glomerulonephritis (GN) and have been suggested to be the major source of TNF in this cell-mediated form of glomerular inflammation. Intrinsic renal cells also have the capacity to produce TNF. For dissecting the contribution of local versus bone marrow (BM)-derived TNF in inflammatory renal injury, TNF chimeric mice were created by transplanting normal wild-type (WT) BM into irradiated TNF-deficient recipients (WT-->TNF-/- chimeras) and vice versa (TNF-/- -->WT chimeras). A model of crescentic GN induced by an intravenous injection of sheep anti-murine glomerular basement membrane antibody was studied in WT mice, mice with complete TNF deficiency (TNF-/-), and chimeric mice. Crescentic GN was attenuated in TNF-/- mice with fewer crescents (crescents, 13.7 +/- 1.7% of glomeruli) and reduced functional indices of renal injury (serum creatinine, 15.2 +/- 0.8 micromol/L). Similar protection (crescents, 14.3 +/- 1.9% of glomeruli; serum creatinine, 18.9 +/- 1.1 micromol/L) was observed in chimeric mice with intact BM but absent renal-derived TNF (WT-->TNF-/- chimeras), suggesting a minor contribution of infiltrating leukocytes to TNF-mediated renal injury. Chimeric mice with TNF-deficient leukocytes but intact intrinsic renal cell-derived TNF (crescents, 20.5 +/- 2.0% of glomeruli; serum creatinine, 21.6 +/- 1.4 micromol/L) developed similar crescentic GN to WT mice (crescents, 22.3 +/- 1.4% of glomeruli; serum creatinine, 24.8 +/- 1.9 micromol/L). Cutaneous delayed-type hypersensitivity after subdermal challenge with the nephritogenic antigen was attenuated in the absence of BM cell-derived TNF but unaffected in WT-->TNF-/- chimeric mice. These studies suggest that intrinsic renal cells are the major cellular source of TNF contributing to inflammatory injury in crescentic GN.

Tumor necrosis factor-alpha from bone marrow-derived cells is not essential for the expression of adhesion molecules in lipopolysaccharide-induced nasal inflammation.

Both the role and source of tumor necrosis factor (TNF-alpha) in lipopolysaccharide (LPS)-induced nasal inflammation were investigated using TNF-alpha gene deficient (TNF-alpha -/-) mice and chimeric mice that are TNF-alpha gene deficient only in bone marrow-derived cells. In the present study, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression levels in the nasal mucosa were significantly decreased following intranasal instillation of LPS in TNF-alpha gene deficient mice compared to those in wild type mice. In contrast, ICAM-1 and VCAM-1 mRNA expressions were not significantly decreased although TNF-alpha mRNA expression was dramatically decreased in TNF-alpha gene deficient bone marrow-transplanted-chimeric (TNF-alpha -/--->+/+) mice compared to those in wild type bone marrow-transplanted-control (TNF-alpha +/+-->+/+) mice. These results indicate that the elevation of TNF-alpha mRNA in the nasal mucosa is mainly originated from bone marrow-derived cells. However, even low expression of TNF-alpha at local inflammation sites is sufficient to induce the expression of adhesion molecules in acute LPS-induced experimental rhinitis.

Microglia and Macrophages Are the Major Source of Tumor Necrosis Factor in Permanent Middle Cerebral Artery Occlusion in Mice

The proinflammatory cytokine tumor necrosis factor (TNF) is known to be expressed in brain ischemia; however, its cellular and temporal appearance is not fully settled. In this study, nonradioactive in situ hybridization for murine TNF mRNA was performed on brain sections from adult C57x129 mice at 6 hours, 12 hours, 24 hours, 2 days, 5 days, or 10 days (six to eight mice per group) after induction of permanent focal cerebral ischemia. Cortical infarct volumes were estimated, and TNF mRNA-expressing cells were counted within the infarct and infarct border using Cast-Grid analysis. At 12 hours, a peak of 19.2 +/- 5.1 TNF mRNA-expressing cells/mm2 was counted, contrasting two to three times lower values at 6 and 24 hours (6.4 +/- 4.6 and 9.2 +/- 3.4 cells/mm2, respectively) and <2 cells/mm2 at 48 hours and later stages. The TNF mRNA-expressing cells were distributed along the entire rostrocaudal axis of the cortical infarcts and occasionally within the caudate putamen. At all time points, TNF mRNA colocalized with Mac-1-positive microglia/macrophages but not with Ly-6G (Gr-1)-positive polymorphonuclear leukocytes. Similarly, combined in situ hybridization for TNF mRNA and immunohistochemistry for glial fibrillary acidic protein at 12 and 24 hours revealed no TNF mRNA-expressing astrocytes at these time points. Translation of TNF mRNA into bioactive protein was demonstrated in the neocortex of C57B1/6 mice subjected to permanent middle cerebral artery occlusion. In summary, this study points to a time-restricted microglial/macrophage production of TNF in focal cerebral ischemia in mice.

Macrophages are the major source of tumor necrosis factor alpha in the porcine corpus luteum.

This study was designed to determine the source of tumor necrosis factor (TNF) alpha within the porcine corpus luteum (CL). 1) Sections of frozen or paraffin-embedded CL from various stages of the estrous cycle were incubated with the following primary antibodies: anti-human recombinant TNFalpha, anti-porcine macrophage-specific antigen, or anti-alpha-actin (marker of pericyte and smooth muscle cells). Dolichos biflorus lectin-peroxidase was used as an endothelial cell label. Positive immunostaining for TNFalpha was apparent in porcine CL throughout the estrous cycle. TNFalpha immunoreactivity was primarily localized in cells along septal/vascular tracts, and exhibited spatial and temporal distribution similar to that of cells labeled with anti-macrophage antibodies. Large luteal cells exhibited weak staining for TNFalpha in paraffin sections, whereas microvascular endothelial cells were consistently negative in both frozen and paraffin sections. 2) Enriched subpopulations of macrophages, endothelial cells, and large and small luteal cells were isolated by density gradient and immunomagnetic bead separation techniques. TNFalpha secretion by each subpopulation was determined by measuring bioactive TNFalpha in incubation media using a specific in vitro bioassay. Macrophage subpopulations secreted up to 100-fold greater quantities of bioactive TNFalpha (up to 400 pg/10(6) cells) than did other subpopulations. In contrast, endothelial cell and small luteal cell subpopulations released very small amounts (< 8 pg/10(6) cells) of bioactive TNFalpha. Large luteal cells secreted slightly greater amounts of TNFalpha (10-15 pg/10(6) cells). Local macrophages appear to be the primary source of TNFalpha in the porcine CL.

Liver as a focus of impaired oxygenation and cytokine production in a porcine model of endotoxicosis.

To determine whether the liver is a focus of insufficient oxygenation and whether liver is a source of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in a porcine model of endotoxicosis. In vivo, prospective, controlled, repeated-measures, experimental study. Experimental physiology laboratory in a university. Juvenile pigs, weighing 22 to 35 kg. Catheters for blood sampling were inserted into the carotid artery, portal vein, hepatic vein, and pulmonary artery of anesthetized animals. Ultrasonic flow probes were placed on the portal vein and the hepatic artery. During surgery, normal saline was infused intravenously at 25 mL/kg/hr. Following stabilization, animals were allocated randomly to one of two groups. The endotoxemic group (n = 6) received 50 mg/kg of purified Escherichia coli lipopolysaccharide infused into the external jugular vein over 1 hr. The control group (n = 6) received a sham saline infusion infused over 1 hr. Once the endotoxin or sham infusion was initiated, the rate of the intravenous saline infusion was increased to 48 mL/kg/hr for the remainder of the experiment. Measurements were obtained before the endotoxin or sham infusion, immediately after the infusion, and every 30 mins thereafter for 4 hrs. Blood gases, lactate, and bioactive TNF and IL-6 concentrations were measured from the carotid artery, portal vein, hepatic vein, and pulmonary artery. The porcine model is characterized by systemic hypotension, pulmonary hypertension, and maintenance of cardiac output. Despite decreased hepatic oxygen delivery in endotoxemic animals (p < .02), there was no change in hepatic oxygen consumption compared with controls. Throughout the experiment, there was net hepatic consumption of lactate in both groups. There was no significant hepatic production (or consumption) of TNF or IL-6 in either group. In this porcine model of endotoxicosis, there is a reduction of hepatic oxygen delivery but dysoxia is not present. The liver is not a source of TNF or IL-6 in this model of endotoxicosis.

Splanchnic origin of cytokines in a porcine model of mesenteric ischemia-reperfusion

<h2>Abstract</h2> <b>Background:</b> The purpose of this study was to determine whether the gut or the liver was the source of tumor necrosis factor (TNF), interleukin-6 (IL-6), and endogenous endotoxin in a porcine model of mesenteric ischemia-reperfusion. <b>Methods:</b> Endotoxin, TNF, and IL-6 levels were measured from the carotid artery (CA), portal vein (PV), and hepatic vein (HV) every 30 minutes for 330 minutes in anesthetized pigs after occlusion of the superior mesenteric artery (SMA; n=7) and after sham operation (n = 7). In animals subjected to mesenteric ischemia, the SMA clamp was released twice: once at 240 minutes (for 40 seconds) and once at 300 minutes (for the remainder of the experiment). <b>Results:</b> In control animals, TNF and IL-6 levels remained at baseline at all vascular sites for the duration of the experiment. In the SMA ligation group, TNF levels peaked before release of the SMA clamp. Compared with TNF levels in the CA (27 ± 3.7 pU/ml, unchanged from baseline), TNF levels were higher in the PV and in the HV (47 ± 1.7 and 44 ± 4.0 pU/ml, respectively; <i>p</i> < 0.05). In contrast, IL-6 appeared in the circulation immediately after first release of the SMA clamp. At this instant, compared with levels in the CA (1381 ± 305 pU/ml), IL-6 levels in the PV and HV were higher (1884 ± 276 and 1795 ± 213 pU/ml, respectively; <i>p</i> < 0.05). Endotoxin remained at baseline levels (1.0 ± 0.3 endotoxin unit/ml) throughout the experiment in both groups of animals, and gut efflux of endotoxin never exceeded gut influx. <b>Conclusions:</b> TNF is produced in a partially perfused splanchnic bed during SMA clamping (e.g., pancreas, duodenum, liver, left colon). IL-6 is produced in gut during SMA clamping and is released when the SMA is unclamped. There is no apparent splanchnic release of endotoxin during or after SMA clamping in this model. (Surgery 1998;123:79-88.)

Splanchnic origin of cytokines in a porcine model of mesenteric ischemia-reperfusion

Background: The purpose of this study was to determine whether the gut or the liver was the source of tumor necrosis factor (TNF), interleukin-6 (IL-6), and endogenous endotoxin in a porcine model of mesenteric ischemia-reperfusion. Methods: Endotoxin, TNF, and IL-6 levels were measured from the carotid artery (CA), portal vein (PV), and hepatic vein (HV) every 30 minutes for 330 minutes in anesthetized pigs after occlusion of the superior mesenteric artery (SMA; n = 7) and after sham operation (n = 7). In animals subjected to mesenteric ischemia, the SMA clamp was released twice: once at 240 minutes (for 40 seconds) and once at 300 minutes (for the remainder of the experiment). Results: In control animals, TNF and IL-6 levels remained at baseline at all vascular sites for the duration of the experiment. In the SMA ligation group, TNF levels peaked before release of the SMA clamp. Compared with TNF levels in the CA (27 ± 3.7 pU/ml, unchanged from baseline), TNF levels were higher in the PV and in the HV (47 ± 1.7 and 44 ± 4.0 pU/ml, respectively; p < 0.05). In contrast, IL-6 appeared in the circulation immediately after first release of the SMA clamp. At this instant, compared with levels in the CA (1381 ± 305 pU/ml), IL-6 levels in the PV and HV were higher (1884 ± 276 and 1795 ± 213 pU/ml, respectively; p < 0.05). Endotoxin remained at baseline levels (1.0 ± 0.3 endotoxin unit/ml) throughout the experiment in both groups of animals, and gut efflux of endotoxin never exceeded gut influx. Conclusions: TNF is produced in a partially perfused splanchnic bed during SMA clamping (e.g., pancreas, duodenum, liver, left colon). IL-6 is produced in gut during SMA clamping and is released when the SMA is unclamped. There is no apparent splanchnic release of endotoxin during or after SMA clamping in this model. (Surgery 1998;123:79-88.)

Expression of TNF and the Necessity of TNF Receptors in Bleomycin-Induced Lung Injury in Mice

Bleomycin (BLM) induction of lung fibrosis in mice is an established model to study the mechanism of pulmonary fibrosis. Cytokine secretion has been implicated as a fundamental component of the lung fibrotic process observed in response to BLM. Among the cytokines implicated in lung fibrosis, Tumor necrosis factor (TNF) alpha has been considered to play a fundamental role. In the present study, we characterized the cellular sources of TNF during BLM-induced lung injury and examined the importance of TNF receptors in this process. To characterize the expression of TNF, we utilized two strains of mice, one sensitive (C57BL/6) and one resistant (BALB/c) to BLM-induced lung injury. Mice received BLM (120 mg/kg total) or saline, as control, by multiple subcutaneous injections. BLM induced the development of inflammation in subpleural areas only in the lungs of BLM-sensitive mice. These subpleural areas were characterized by infiltration of CD68-positive macrophages and increased collagen deposition. BLM enhanced the expression of TNF mRNA in BLM-sensitive, but not in BLM-resistant, mice. In situ hybridization studies localized the expression of TNF in the areas of BLM-induced inflammation in 6% and 27% of macrophages at 14 and 21 days post BLM treatment. In addition to TNF, BLM exposure resulted in the upregulated expression of transforming growth factor (TGF)-β1, but not interleukin (IL)-1, mRNA in the lungs of both murine strains at 14 and 21 days. This upregulated expression of TGF-β1 mRNA was greater in the lungs of BLM-sensitive mice. In separate experiments, double TNF receptor knockout mice were exposed to BLM. These animals demonstrated an increased expression of TNF, but not TGF-β1, mRNA in response to BLM and did not exhibit histologic evidence of lung injury following BLM exposure. In summary, the upregulation of TNF mRNA in macrophages correlated with the appearance of inflammation following BLM exposure and was limited to the BLM-sensitive strain. Furthermore, in addition to the release of the TNF ligand, it appears that the presence of TNF receptors is necessary for the development of BLM-induced lung injury, and signaling through these receptors may contribute to the regulation of the TGF-β1 mRNA expression observed in response to bleomycin. These results provide further support for a role of macrophages and TNF in the induction of lung inflammation

Myocardium is a major source of proinflammatory cytokines in patients undergoing cardiopulmonary bypass

Proinflammatory cytokines, such as tumor necrosis factor–α, interleukin-6, and interleukin-8, and antiinflammatory cytokines, such as interleukin-10, may play an important role in patient responses to cardiopulmonary bypass. We sought to define whether the myocardium and the lungs serve as important sources of these cytokines under conditions of cardiopulmonary bypass. Ten patients (age 64 ± 3 years, mean ± standard error of the mean) undergoing elective coronary artery bypass grafting were monitored with an arterial catheter, a coronary sinus catheter, and a pulmonary artery catheter. Plasma levels of tumor necrosis factor–α, interleukin-6, interleukin-8, and interleukin-10 were measured simultaneously in peripheral arterial blood, coronary sinus blood, and mixed venous blood before heparin administration, 1 minute before aortic crossclamping, 5 minutes after aortic declamping, and at 0.5, 1, 1.5 and 2 hours after aortic declamping. The durations of cardiopulmonary bypass and aortic crossclamping were 114 ± 9 and 64 ± 5 minutes, respectively. Levels of tumor necrosis factor–α and interleukin-6 were significantly higher in coronary sinus blood than in arterial blood after aortic declamping. Tumor necrosis factor–α and interleukin-6 levels were also higher in mixed venous blood than in arterial blood within 1 hour after declamping. There were no significant differences among the three sampling sites with respect to interleukin-8 and interleukin-10 levels. In one patient who had postoperative myocardial infarction, however, interleukin-8 levels were three times as high as in coronary sinus blood than in arterial blood. These data indicate that the myocardium is a major source of tumor necrosis factor–α and interleukin-6 in patients undergoing cardiopulmonary bypass. The lungs may consume rather than release proinflammatory cytokines in the early phase of reperfusion. The source under these conditions of the antiinflammatory cytokine interleukin-10 remains to be determined. (J T HORAC C ARDIOVASC S URG 1996;112:806-11)

Human adrenal cells express tumor necrosis factor-alpha messenger ribonucleic acid: evidence for paracrine control of adrenal function.

Tumor necrosis factor (TNF) is gaining increasing importance in clinical medicine. It plays a role in the interaction of the immune system with the hypothalamic-pituitary-adrenal axis. In the present study various morphological methods, including immunohistochemistry, electron microscopy, and in situ hybridization were applied to characterize the localization and distribution of TNF in the human adrenal gland. Double immunostaining revealed an astonishing degree of intermingling of steroid-producing cells and chromaffin cells. Macrophages could be found in all regions of the adrenal gland, but particularly in the transition zone of cortex and medulla. The steroid-producing cells of the inner zone of the cortex express major histocompatibility complex class II molecules. On the ultrastructural level, immune cells, steroid cells, and catecholamine-producing cells were found in direct contact. The combination of immunohistochemistry and in situ hybridization was optimally suited to define the exact cellular source of TNF in the human adrenal. TNF is produced in macrophages, but above all in 17 alpha-hydroxylase-positive cells (steroid-producing cells) in the zona reticularis and medulla. No signal was found in chromaffin cells. TNF may induce major histocompatibility complex class II in human adrenal gland in a paracrine or autocrine manner. It is concluded that TNF may have an important role in normal human adrenal physiology.

Expression of the Tumor Necrosis Factor Gene by Dermal Fibroblasts in Response to Ultraviolet Irradiation or Lipopolysaccharide

To examine the effects of different wavelengths of ultraviolet (UV) radiation on tumor necrosis factor (TNF) production, we took advantage of mice carrying a chloramphenicol acetyl transferase (CAT) reporter transgene bearing the entire TNF promoter and 3'-untranslated region. Aside from constitutive expression in the thymus, CAT activity was detected only in locally UVB- or UVC-irradiated skin. After UVB irradiation, markedly greater amounts of CAT activity were traced to the dermis rather than the epidermis; by contrast, almost all CAT activity was localized to the epidermis after UVC irradiation. Fibroblasts have not been shown previously to express the TNF gene, i.e., the TNF gene is highly methylated and inaccessible to exogenous modulation in 3T3 fibroblasts. However, the present report reveals that cultured dermal fibroblasts are capable of producing both CAT and TNF in response to treatment in vitro with either UVB irradiation, UVC irradiation, or lipopolysaccharide. These findings indicate that dermal fibroblasts may serve not only as a target for but also as a source of TNF.

Mechanism for macrophage activation against Corynebacterium parvum--participation of T cells and its lymphokines.

It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T-cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c(-)+/+ mice was abrogated completely by the pre-treatment of spleen cells with anti-Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c-nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin-P (a PHA-induced lymphokine) made it possible for BALB/c-nu/nu mice to produce TNF, associated with an induction of Lyt-1+ cells and Lyt-2+ cells. However, treatment with the lymphokine did not augment the increases of Ia+ cells or liver and spleen weights. These results suggest that increasing the number of Ia+ cells is not sufficient to bring about TNF production; Ia+ cells must also be stimulated by T cells or T-cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia+ cells against C. parvum.