Glutamine depletion potentiates leucocyte-dependent inflammatory events induced by carrageenan or Clostridium difficile toxin A in rats

This research investigated the effect of glutamine (Gln) depletion on leucocyte-dependent inflammatory events. Rats were treated intraperitoneally, 16 hr prior to the peak of every parameter evaluated, with either 0.9% NaCl, methionine-sulphoximine (MSO, an inhibitor of endogenous Gln synthesis, 25 mg/kg) or with MSO + Gln (MSO as above plus Gln 3 g/kg in three doses). MSO-induced Gln depletion increased paw oedema induced both by carrageenan (Cg) and by Clostridium difficile toxin A (TxA) (66.2% and 45.5%, respectively; P < 0.05). In dextran-injected animals, oedema and myeloperoxidase (MPO) activity were not modified by Gln depletion. In Cg-treated paws, Gln depletion increased MPO activity by 44% (P < 0.05), interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) concentrations by 47% and 52%, respectively (P < 0.05), and immunostaining for TNF-alpha in paw tissue. In TxA-injected paws, Gln depletion increased MPO activity (46%; P < 0.05). Gln depletion increased Cg- and TxA-induced neutrophil migration to subcutaneous air pouches by 56% and 77% (P < 0.05), respectively, but did not affect migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP). Gln infusions reversed all the effects of MSO. Leucocyte counts did not differ between groups. Gln depletion potentiates acute inflammation, possibly by increasing neutrophil migration through resident cell activation and production of IL-1beta and TNF-alpha. Gln supplementation reverses these effects and may be useful during inflammatory catabolic stress.     

Symptom pathogenesis during acute influenza: interleukin-6 and other cytokine responses.

In experimental human influenza infection initiated by nasal inoculation, the magnitude of viral replication, fever, and symptoms correlate with nasopharyngeal lavage fluid levels of various cytokines. Our aim was to assess these relationships in patients with naturally occurring acute influenza. Patients with culture-positive influenza illness of less than 36 hr of duration were studied. Nasopharyngeal washing were collected at enrollment and on Day 2, 4, 6 and 8 for quantitative virus isolation and IL-6, TNF-alpha, INF-alpha, INF-gamma and IL-10 determinations. Blood samples collected at entry and on Day 2 and 6 were processed to assess plasma cytokines and circulating influenza RNA. Patients received either oseltamivir or placebo for 5 days. We assessed the correlation between nasopharyngeal lavage fluid or blood levels of cytokines before treatment and viral titers, symptom severity and fever. Sixteen adult subjects (median age of 22 years) were studied. In this small group of patients no significant differences between placebo and oseltamivir patients were found in viral replication or measures of cytokines. Thus the data for all 16 subjects were pooled for analysis. At entry, influenza A viruses were cultured from nasopharyngeal washes at a median titer of 4.8 log(10)TCID(50)/ml of wash. Viral titers correlated positively with symptom score (P = 0.006) and temperature values (P < 0.001). Viral titers, fever and symptoms were highest at enrollment and fell in parallel during the subsequent days. RT-PCR assays failed to detect influenza RNA in the white blood cells from any patient. We observed a significant release, in both nasopharyngeal lavage fluid and in plasma, of IL-6, TNF-alpha, INF-alpha, INF-gamma and IL-10. At entry high IL-6 levels were detected in the nasopharyngeal lavage fluid (median 10.3 pg/ml) and plasma (median 5.1 pg/ml) of all patients. We found a positive correlation between plasma IL-6 levels and both symptom scores and temperature values (P < 0.05), as well as a positive correlation between nasopharyngeal lavage fluid levels of IL-6 and TNF-alpha and temperature (P < 0.05). We did not find significant associations between symptoms, fever and levels of INF-alpha, INF-gamma or IL-10. The magnitude of early decrease in viral titers correlated with initial levels of INF-gamma in nasopharyngeal lavage fluid (P < 0.05). Significant production of IL-6, TNF-alpha, INF-alpha, INF-gamma and IL-10 occurs in response to community acquired influenza A illness. As in experimental influenza, symptoms and fever in natural acute influenza correlate with the release of IL-6.     

Retinal ganglion cell death is induced by microglia derived pro-inflammatory cytokines in the hypoxic neonatal retina

Hypoxic injury, including that resulting in the retinopathy of prematurity, may induce retinal ganglion cell (RGC) death in the neonatal retina. We hypothesized that this may be mediated by excess production of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by microglia. One-day-old Wistar rats were subjected to hypoxia for 2 h and the expression of TNF-α and IL-1β and their receptors was determined in the retina. The mRNA and protein expression of TNF-α, IL-1β, TNF-receptor 1 (TNF-R(1)), and IL-1 receptor 1 (IL-1R(1)) and the tissue concentration of TNF-α and IL-1β were up-regulated significantly after the hypoxic exposure. TNF-α and IL-1β immunoreactivity was localized in microglial cells, whereas that of TNF-R(1) and IL-1R(1) was restricted to RGCs, as confirmed by double immunofluorescence labelling. Along with this, increased expression of monocyte chemoattractant protein-1 and its receptor CCR2 was detected in the microglia. Primary cultured microglia subjected to hypoxia showed enhanced release of TNF-α and IL-1β. Primary cultured retinal ganglion cells (RGCs) treated with conditioned medium derived from hypoxic microglia showed enhanced apoptosis, which was significantly reduced when the cells were treated with microglia conditioned medium neutralized with TNF-α/IL-1β antibody. Our results suggest that activated microglial cells in hypoxic neonatal retina produce increased amounts of TNF-α and IL-1β that could induce RGC death.     

Actions of tumor necrosis factor-alpha on oocyte maturation and embryonic development in cattle

PROBLEM: Infertility can accompany mastitis in cattle. Involvement of tumor necrosis factor-alpha (TNF-alpha) in this phenomenon is suggested by observations that circulating concentrations of TNF-alpha are elevated after intramammary infection or infusion of endotoxin. It was hypothesized that (1) TNF-alpha acts on the oocyte during maturation to decrease the percent of oocytes that cleave and develop following fertilization; (2) exposure of embryos to TNF-alpha after fertilization reduces development to the blastocyst stage; and (3) TNF-alpha increases the proportion of blastomeres that undergo apoptosis in a stage-of-development dependent manner. METHOD OF STUDY: In one experiment, oocytes were matured with various concentrations of TNF-alpha and then fertilized and cultured without TNF-alpha. In another study, embryos were cultured with TNF-alpha for 8 days beginning after fertilization. Finally, embryos were collected at the two or four-cell stage (at 28-30 hr after insemination) or when > or = 9-cells (at day 4 after insemination) and cultured +/- TNF-alpha for 24 hr. The proportion of blastomeres undergoing apoptosis was then determined by the TUNEL procedure. RESULTS: Addition of TNF-alpha to maturation medium did not affect the proportion of oocytes that cleaved. However, the percent of oocytes that developed to the blastocyst stage at day 8 after insemination was reduced (P = 0.05) at all TNF-alpha concentrations tested (0.1-100 ng/mL). When added during embryo culture, there was no significant effect of TNF-alpha on the proportion of oocytes that became blastocysts. In addition, TNF-alpha did not induce apoptosis in two and four-cell embryos. For embryos > or = 9-cells, however, 10 and 100 ng/mL TNF-alpha increased (P < 0.05) the percent of blastomeres labeling as TUNEL-positive. CONCLUSION: TNF-alpha can have deleterious actions on oocyte maturation that compromise development of the resultant embryo. While exposure of fertilized embryos to TNF-alpha did not inhibit development to the blastocyst stage, TNF-alpha increased the percentage of blastomeres undergoing apoptosis when exposure occurred for embryos > or = 9-cells. Increased blastomere apoptosis could conceivably compromise subsequent embryo survival.     

Interleukin-1beta induces in vivo tolerance to lipopolysaccharide in mice

Endotoxin or lipopolysaccharide (LPS) tolerance may be partially due to the secretion of potent anti-inflammatory cytokines following severe Gram-negative infections, or by low doses of LPS. In this work, we describe the effects of interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF-), two early cytokines secreted after LPS exposure, in the induction of LPS tolerance. Our results demonstrate that mice treated with three daily doses of 100 ng of IL-1 were tolerant to LPS-induced shock. However, TNF- was unable to induce an LPS refractory state. Given the fact that 100 ng of IL-1 increase the plasma levels of glucocorticoids, we evaluated whether a daily injection of dexamethasone (DEX) alone was able to reproduce the LPS-like tolerant state. However, no signs of LPS refractoriness were detected, except when DEX was administered concomitantly with a dose of IL-1 that does not induce corticosterone secretion (12 ng/mouse). This dose was found to induce in vitro up-regulation of the glucocorticoid receptors (GcR) of peritoneal macrophages following 24 h of treatment. In addition, we demonstrate that IL-1 is capable of inducing the down-regulation of Toll-like receptor 4 (TLR4), a crucial molecule in the signal transduction of LPS. Taken together, our results indicate that IL-1 can generate tolerance to LPS in vivo, and suggest that the regulation of mechanisms of the down-regulation of TLR4, as well as those involved in the expression of GcR and/or in the secretion of glucocorticoids, would be crucial for these effects.     

Ginseng modulates the immune response by induction of interleukin-12 production

In infections with intracellular microorganisms such as mycobacteria and Leishmania parasites as well as certain extracellular chronic infections such as Pseudomonas aeruginosa a Th1 response with activation of macrophages is desirable. Several studies indicate that such a response is associated with better recovery from infection, improved course of the chronic infection, and higher survival rate. In Th1 responses there is increased interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) production, whereas that of interleukin-10 (IL-10) is decreased. The present study indicated that Ginseng modulation of stimulated peripheral blood mononuclear cells (PBMC) results in a higher IL-12 production. The enhanced IL-12 production could induce a stronger Th1 response, resulting in better protection against infection with a variety of pathogens.     

Interleukin-6 modifies mRNA expression in mouse skeletal muscle

Aim: The aim of this study was to test the hypothesis that interleukin (IL)‐6 plays a role in exercise‐induced peroxisome proliferator‐activated receptor γ co‐activator (PGC)‐1α and tumor necrosis factor (TNF)‐α mRNA responses in skeletal muscle and to examine the potential IL‐6‐mediated AMP‐activated protein kinase (AMPK) regulation in these responses. Methods: Whole body IL‐6 knockout (KO) and wildtype (WT) male mice (4 months of age) performed 1 h treadmill exercise. White gastrocnemius (WG) and quadriceps (Quad) muscles were removed immediately (0′) or 4 h after exercise and from mice not run acutely. Results: Acute exercise reduced only in WT muscle glycogen concentration to 55 and 35% (P < 0.05) of resting level in Quad and WG respectively. While AMPK and Acetyl CoA carboxylase (ACC) phosphorylation increased 1.3‐fold (P < 0.05) in WG and twofold in Quad immediately after exercise in WT mice, no change was detected in WG in IL‐6 KO mice. The PGC‐1α mRNA content was in resting WG 1.8‐fold higher (P < 0.05) in WT mice than in IL‐6 KO mice. Exercise induced a delayed PGC‐1α mRNA increase in Quad in IL‐6 KO mice (12‐fold at 4 h) relative to WT mice (fivefold at 0′). The TNF‐α mRNA content was in resting Quad twofold higher (P < 0.05) in IL‐6 KO than in WT, and WG TNF‐α mRNA increased twofold (P < 0.05) immediately after exercise only in IL‐6 KO. Conclusion: In conclusion, IL‐6 affects exercise‐induced glycogen use, AMPK signalling and TNF‐α mRNA responses in mouse skeletal muscle.     

Rectal administration of tranilast ameliorated acute colitis in mice through increased expression of heme oxygenase‐1

Mast cells play a key role in the pathophysiology of inflammatory bowel disease (IBD). Tranilast, a mast cell stabilizer, has been empirically used for IBD in Japan, but its precise role in the treatment of IBD is largely unknown. To investigate the role of tranilast for the treatment of IBD, tranilast was administered intrarectally to mice with dextran sulfate sodium (DSS)‐induced colitis. Tranilast ameliorated DSS colitis clinically and pathologically, as demonstrated by decreased number and degranulation of mast cells in the colon. mRNA expression was increased for tumor necrosis factor‐α, interferon‐γ and interleukin (IL)‐6, and decreased for IL‐10 in the colon of DSS colitis mice. In contrast, tranilast markedly decreased expression of mRNAs for the pro‐inflammatory cytokines, and increased that of the anti‐inflammatory cytokines. Moreover, tranilast increased heme oxygenase (HO)‐1 expression on colonic epithelial cells as well as on colon‐infiltrating cells of DSS colitis. In conclusion, tranilast ameliorated DSS colitis by regulating mast cell degranulation, decreasing inflammatory cytokines and increasing anti‐inflammatory cytokines. Tranilast might exert these effects partly through enhanced HO‐1 expression in the colon, suggesting a potential adjunctive therapy for IBD.     

Frequency of human cytomegalovirus-specific T cells during pregnancy determined by intracellular cytokine staining

The characteristics of cytomegalovirus (CMV)-specific T-cell immunity was investigated in pregnant women with primary, latent, or reactivated CMV infection, and in a comparative group of non-pregnant women. Forty-six pregnant and 8 non-pregnant women were examined based on the presence of serum antibody activity against CMV and viral excretion in urine. The frequency of CMV-specific CD4(+) T cells in peripheral blood lymphocytes was determined by staining for intracellular cytokines, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha. There was no change in the frequencies of CMV-specific CD4(+) T cells in CMV-seropositive normal non-pregnant and pregnant women at any gestation. However, the frequency of CMV-specific CD4(+) T cells in pregnant women associated with CMV reactivation or reinfection was significantly higher than in CMV-seropositive normal pregnant and non-pregnant women. There were no CMV transmissions to the infants of all these women. These CMV-specific T cells responses in pregnant women may contribute some to block the intrauterine CMV infection in their infants.     

Altered claudin expression is a feature of chronic plaque psoriasis

Epithelial tight junctions play a central role in cell-cell adhesion and are necessary for the selective paracellular movement of ions. Claudins are key components of tight junctions and their expression is altered in gut epithelia in a variety of inflammatory enteropathies, including ulcerative colitis and Crohn's disease. Psoriasis is a chronic inflammatory skin disease affecting approximately 2% of the western population, with significantly increased occurrence in individuals with Crohn's disease. Initial studies investigated the expression of claudins in skin of healthy volunteers and patients with chronic plaque psoriasis. We report here that claudins-1 and -3 are the major protein species present in the epidermis of healthy skin; they are expressed on the surface of epidermal keratinocytes, consistent with their localization to tight junctions. In plaques of psoriasis, claudin-1 was not identifiable in the epidermis, although typical staining patterns were observed in clinically normal, uninvolved skin of patients with psoriasis. Claudin-3 was present in the epidermal granular cell layer in normal skin, but was only identified within the cytosol of epidermal keratinocytes in both involved and uninvolved skin of psoriasis patients. We examined further whether exposure of keratinocytes in vitro to pro-inflammatory cytokines mimicked the observed changes in claudin expression seen in chronic plaque psoriasis; lipopolysaccharide, interferon-gamma and tumour necrosis factor-alpha had no effect on claudin protein expression or distribution. Addition of interleukin-1beta, however, resulted in down-regulation of claudins-1 and -3. Tumour necrosis factor-alpha and interleukin-1beta were further used in an in vivo model of skin inflammation; interleukin-1beta alone modulated claudin protein expression in this system. These data demonstrate that epidermal claudin expression is altered in chronic plaque psoriasis and that expression is in part modulated by interleukin-1beta.     

Apoptosis in cultured rat hepatocytes: the effects of tumour necrosis factor alpha and interferon gamma.

We investigated the cytotoxic effects of tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on rat hepatocytes in culture. Under phase contrast microscopy, we found a small number of dying hepatocytes in control cultures, each having been transformed into a cluster of small spheres. Under transmission electron microscopy, these cells showed the characteristics of apoptosis. TNF alpha and a combination of TNF alpha and IFN gamma exerted a cytotoxic effect, whereas IFN gamma showed no significant cytotoxicity when assessed by neutral red assay and by measuring LDH activity in culture medium. Under phase contrast microscopy, the number of apoptotic cells increased with the addition of either TNF alpha or IFN gamma, and markedly with the addition of both. DNA extracted from apoptotic cells cultured with TNF alpha and IFN gamma was fragmented, and a set of bands of the '200 bp ladder', which is characteristic of the DNA of apoptotic cells, was observed in agarose gel electrophoresis. These findings indicate that cultured hepatocytes die from apoptosis. TNF alpha killed cultured rat hepatocytes by increasing apoptosis, and this effect was potentiated by the addition of IFN gamma, which by itself was also weakly cytotoxic.     

Estradiol reduces basal and cytokine induced monocyte adhesion to endothelial cells

OBJECTIVE: To investigate the effect of 17beta-estradiol (E2) on binding of monocytes to human aortic endothelial cells (HAECs) with or without cytokine induction. METHODS: Confluent monolayers of HAECs were incubated with or without E2 for 48 h prior to the monocyte adhesion assay. In studies with cytokines, 1 ng/ml tumor necrosis factor-alpha (TNF-alpha), 20 U/ml interleukin-1beta (IL-1beta) or both were added to the culture medium for the final 24 or 4 h. For the measurement of monocyte adhesion, 3H-thymidine labeled human THP-1 monocytes (4 x 10(5) cells per well) were added to the confluent monolayer of HAECs and incubated at 37 degrees C for 90 min. The unbound THP-1 cells were removed by gentle washing, and bound cells were digested with NaOH and quantified by measuring radioactivity. RESULTS: When HAECs were pretreated for 48 h with E2 the basal adhesion of THP-1 cells was reduced by an average of 28%. Estrogen significantly reduced cytokine-induced adhesion by 30-35% when the cytokines were added for 4 h. When the cytokine treatment was prolonged to 24 h, pretreatment of HAECs with E2 had no effect on THP-1 cell adhesion. CONCLUSIONS: E2 reduces basal and short-term cytokine induced monocyte binding to HAECs. Since monocyte adhesion to vascular endothelial cells is one of the initial steps in the pathogenesis of atherosclerosis, E2 may mediate vascular protection by reducing monocyte-endothelial cell binding in the early stages of atherogenesis.     

Impaired production of proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation in elderly humans.

Ageing is associated with decreased resistance to bacterial infections and concomitant increased circulating levels of inflammatory cytokines. The purpose of the present study was to research age-related changes in levels of early mediators of the acute-phase response in whole blood supernatants following LPS stimulation, representing an ex vivo model of sepsis. Levels of tumour necrosis factor-alpha (TNF-alpha), IL-1beta and IL-6 in whole blood supernatants were measured after in vitro LPS stimulation for 24 h in 168 elderly humans aged 81 years from the 1914 cohort in Glostrup, Denmark and in 91 young controls aged 19-31 years. Levels of TNF-alpha and IL-1beta were significantly lower in elderly humans compared with young controls, whereas no difference was detected with regard to IL-6. Elderly humans with low body mass index had the lowest levels of IL-1beta. Young women had lower levels of proinflammatory cytokines compared with young men, but this difference was blurred by ageing. No relation was found between circulating plasma levels of TNF-alpha and levels after in vitro LPS stimulation. In conclusion, decreased production of TNF-alpha and IL-1beta after exposure to LPS may reflect impaired host defence against infections in the elderly and be of importance in elderly humans with underlying health disorders. However, the clinical relevance is questionable in healthy elderly people because decreased levels were found compared with young men but not compared with young women.