Isorhamnetin inhibits Prevotella intermedia lipopolysaccharide-induced production of interleukin-6 in murine macrophages via anti-inflammatory heme oxygenase-1 induction and inhibition of nuclear factor-κB and signal transducer and activator of transcription 1 activation.

BACKGROUND AND OBJECTIVE: Interleukin-6 (IL-6) is a key proinflammatory cytokine that has been considered to be important in the pathogenesis of periodontal disease. Therefore, host-modulatory agents directed at inhibiting IL-6 appear to be beneficial in terms of attenuating periodontal disease progression and potentially improving disease susceptibility. In the current study, we investigated the effect of the flavonoid isorhamnetin on the production of IL-6 in murine macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. MATERIAL AND METHODS: Lipopolysaccharide from P. intermedia ATCC 25611 was isolated using the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time PCR to quantify IL-6 and heme oxygenase-1 (HO-1) mRNA expression. The expression of HO-1 protein and the levels of signaling proteins were monitored using immunoblot analyses. The DNA-binding activity of nuclear factor-κB (NF-κB) was analyzed using ELISA-based assay kits. RESULTS: Isorhamnetin significantly down-regulated P. intermedia LPS-induced production of IL-6 as well as its mRNA expression in RAW264.7 cells. Isorhamnetin up-regulated the expression of HO-1 at both gene transcription and translation levels in cells stimulated with P. intermedia LPS. In addition, inhibition of HO-1 activity by tin protoporphyrin IX blocked the inhibitory effect of isorhamnetin on IL-6 production. Isorhamnetin failed to prevent LPS from activating either c-Jun N-terminal kinase or p38 pathways. Isorhamnetin did not inhibit NF-κB transcriptional activity at the level of inhibitory κB-α degradation. Isorhamnetin suppressed NF-κB signaling through inhibition of nuclear translocation and DNA binding activity of NF-κB p50 subunit and attenuated signal transducer and activator of transcription 1 signaling. CONCLUSION: Although further research is required to clarify the detailed mechanism of action, we propose that isorhamnetin may contribute to blockade of the host-destructive processes mediated by IL-6 and could be a highly efficient modulator of the host response in the treatment of inflammatory periodontal disease. Further research in animal models of periodontitis is required to better evaluate, the potential of isorhamnetin as a novel agent for treating periodontal disease.

Nonenzymatic glycation of high-density lipoprotein impairs its anti-inflammatory effects in innate immunity.

AIMS/HYPOTHESIS: In type 2 diabetes mellitus (T2DM), the abnormal protein and lipid composition of diabetic high-density lipoprotein (HDL) could impair its anti-inflammatory functions. Whether nonenzymatic glycation directly impaired the anti-inflammatory effects of HDL in innate immunity remained unclear. METHODS: Human acute monocytic leukemia cell line (THP-1) cells, mouse RAW 264.7 macrophages and primary human monocytes derived macrophages were pre-incubated with native HDL, diabetic HDL isolated from T2DM patients or HDL glycated with different doses of d-glucose in vitro and then challenged with lipopolysaccharide (LPS). The release of tumor necrosis factor (TNF)-α and IL-1ß was assayed by enzyme-linked immunosorbent assay (ELISA). Phosphorylation of Iκ-Bα in cytoplasm and nuclear translocation of NF-κB were detected by western blot. Glycation levels of native HDL, glycated HDL and diabetic HDL were determined using LC-MS/MS. RESULTS: The potency of diabetic HDL to inhibit the release of TNF-α (p < 0.05) and IL-1ß (p < 0.001) was dramatically attenuated compared with that of native HDL. Similarly, glycation of HDL in vitro impaired its ability to inhibit TNF-α and IL-1ß release in a glucose dose-dependent manner. Moreover, apoHDL still effectively inhibited the release of TNF-α and IL-1ß induced by LPS, but glycated apoHDL partly lost such abilities. Nonenzymatic glycation levels of glycated HDL and diabetic HDL increased 28 fold (p < 0.001) and 4 fold (p < 0.001), respectively compared with that of native HDL. CONCLUSIONS: In this study, we observed that diabetic HDL and HDL glycated in vitro both partly lose their protective effects to inhibit cytokines release induced by LPS in macrophages, and nonenzymatic glycation of the protein components of HDL plays key roles in these impairments.

Dexamethasone enhances 1alpha,25-dihydroxyvitamin D3 effects by increasing vitamin D receptor transcription.

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner.

Role of dexamethasone and oncostatin M on the formation of vacuoles in human fetal liver cells.

Combined treatment with dexamethasone and oncostatin M (DEX/OSM) or interleukin-6 (DEX/IL-6) resulted in the appearance of numerous large vacuoles in human fetal liver (HFL) cells and showed synergistic effects on the formation of vacuoles. The number of vacuoles formed by DEX, DEX/OSM, or DEX/IL-6 was significantly suppressed by RU-486, a glucocorticoid receptor antagonist. On the other hand, the size of vacuoles formed by OSM, IL-6, DEX/OSM, or DEX/IL-6 was significantly decreased to about 65% by madindoline A (MDL-A), which is a non-peptide antagonist of gp130 and an inhibitor of cytokines, such as IL-6, mediated by gp130 homodimerization, while RU-486 did not affect the size of vacuoles. Expression of IL-6 mRNA in HFL cells was markedly induced by OSM. Expression of IL-6R mRNA was induced by DEX. These results indicate that DEX contributes to the formation of vacuoles through glucocorticoid receptors and that OSM and IL-6 contribute to enlargement of these vacuoles.

The beta-adrenoceptor antagonist propranolol counteracts anti-inflammatory effects of isoflurane in rat endotoxemia.

BACKGROUND: Recent studies suggest that volatile anaesthetics have anti-inflammatory and preconditioning properties and that beta-adrenoceptors are involved in the signalling pathways for these effects. Concurrently, the blockade of beta-adrenoceptors has been shown to augment the release of inflammatory mediators in response to pro-inflammatory stimuli. We therefore aimed to investigate whether the beta-adrenoceptor antagonist propranolol might modulate the anti-inflammatory effects of isoflurane on the systemic and pulmonary release of pro-inflammatory cytokines in endotoxemic rats. METHODS: Forty anaesthetized and ventilated Sprague-Dawley rats were randomly treated as follows. Lipopolysaccharide (LPS) only (n = 8), endotoxemia with LPS [5 mg/kg, intravenously (i.v.)]. LPS-isoflurane (n = 8): endotoxemia and continuous inhalation of 1 minimum alveolar concentration (MAC) of isoflurane. LPS-isoflurane-propranolol (n = 8): administration of propranolol (3 mg/kg) before continuous inhalation of isoflurane and induction of endotoxemia. LPS-propranolol (n = 8): administration of propranolol (3 mg/kg) before endotoxemia without inhalation of isoflurane. Sham (n = 8): control-group only with surgical preparation. After 4 h of endotoxemia, levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-10 (IL-10) in plasma and bronchoalveolar fluid (BALF) were analysed. Release of nitric oxide (NO) and amount of inducible nitric oxide synthase (iNOS) protein in alveolar macrophages was measured by Griess assay or determined by Western Blotting, respectively. RESULTS: Inhalation of isoflurane reduced the release of TNF-alpha (P < 0.05) and IL-1beta (P < 0.05) in plasma and IL-1beta (P < 0.05) in BALF. Co-administration of propranolol significantly inhibited these effects. During inhalation of isoflurane, the increased release of NO and iNOS protein from alveolar macrophages was also completely inhibited by propranolol. CONCLUSION: Our results indicate for the first time, that blockade of beta-adrenoceptors counteracts the anti-inflammatory effects of isoflurane in endotoxemic rats.

Inhibitor kappaB and nuclear factor kappaB in granulocyte-macrophage colony-stimulating factor antagonism of dexamethasone suppression of the macrophage response to Aspergillus fumigatus conidia.

BACKGROUND: The dexamethasone (DEX) immunosuppressive effect on macrophage killing activity and cytokine production in response to Aspergillus fumigatus conidia is antagonized by granulocyte-macrophage colony-stimulating factor (GM-CSF). The molecular mechanism is unknown. We postulated that this antagonism is mediated by inhibitor kappaB (I kappaB) induction by DEX and is opposed by acceleration of I kappaB degradation by GM-CSF with or without conidia stimulation, with corresponding effects on translocation and activation of nuclear factor kappa B (NF-kappaB). METHODS: We studied 2 types of cells, resident peritoneal macrophages from CD-1 mice and the murine macrophage RAW264.7 cell line. Cells were unstimulated or stimulated with conidia and simultaneously treated with DEX, GM-CSF, or DEX plus GM-CSF, for 2-4 hours. I kappaB degradation and NF-kappaB activation were assessed by Western blot. RESULTS: Macrophages stimulated with conidia alone increased NF-kappaB translocation. DEX increased I kappaB levels in cytoplasm and blocked translocation of NF-kappaB to the nucleus in unstimulated and conidia-stimulated macrophages. Conversely, GM-CSF decreased I kappaB levels. GM-CSF reversed the effect of DEX on I kappaB levels. NF-kappaB levels were minimal in DEX-treated macrophage nuclear extracts, compared with those from GM-CSF-treated and GM-CSF plus DEX-treated macrophages. CONCLUSION: GM-CSF can reverse the DEX immunosuppressive effect by enhancing I kappaB degradation and promoting NF-kappaB translocation. This would allow macrophage production of proinflammatory cytokines, facilitating resistance to aspergillosis.

Cortisol antiinflammatory effects are maximal at postoperative plasma concentrations.

OBJECTIVE: To determine the plasma concentration of cortisol that is needed for maximal suppression of the systemic inflammatory response to cardiac surgery with cardiopulmonary bypass. DESIGN: Prospective, randomized, double-blind clinical study of cardiac surgical patients. SETTING: Operating room and inpatient care facility of a university medical center. SUBJECTS: Sixty elective cardiac surgical patients scheduled for coronary artery bypass graft, cardiac valve replacement, or both. INTERVENTIONS: Patients were randomized to receive one of three different hydrocortisone doses, by intravenous infusion, for 6 hrs before, during, and immediately after surgery while also receiving etomidate to suppress endogenous cortisol production. MEASUREMENTS AND MAIN RESULTS: Serial determinations of plasma interleukin-6 were studied as a marker of systemic inflammation. Measurements of interleukin-10 were used as a marker of the compensatory antiinflammatory response. Plasma cortisol concentrations in an untreated control group rose from 17 microg/dL before surgery to a mean of 43 microg/dL by 4 hrs after surgery. A dose of hydrocortisone (4 microg/kg/min for 6 hrs) that maintained plasma cortisol between 40 and 50 microg/dL, starting 60-90 mins before surgery, significantly suppressed plasma interleukin-6 after surgery compared with control while significantly increasing plasma interleukin-10 during surgery. Plasma interleukin-6 after surgery was not suppressed further by increasing the dose of hydrocortisone to 8 microg/kg/min, although the mean peak plasma interleukin-10 concentration increased further compared with the group that received the 4 microg/kg/min hydrocortisone dose. CONCLUSIONS: At the doses studied, cortisol-induced suppression of plasma interleukin-6 during and after cardiac surgery appears to be a saturable phenomenon at the concentration of plasma cortisol that is normally achieved after surgery in untreated patients.

Raloxifene concurrently stimulates osteoprotegerin and inhibits interleukin-6 production by human trabecular osteoblasts.

Raloxifene reduces bone loss and prevents vertebral fractures in postmenopausal women. Its skeletal effects are mediated by estrogen receptors (ER) and their modulation of paracrine osteoblastic factors. Receptor activator of nuclear factor-kappa B ligand is essential for osteoclasts and enhances bone resorption, whereas osteoprotegerin (OPG) neutralizes receptor activator of nuclear factor-kappa B ligand. Here, we assessed the effects of raloxifene on OPG production in human osteoblasts (hOB). Raloxifene enhanced gene expression of ER-alpha and progesterone receptor. Moreover, raloxifene increased OPG mRNA levels and protein secretion by hOB in a dose- and time-dependent fashion by 2- to 4-fold with a maximum effect at 10(-7) M and after 72 h (P < 0.001). Treatment with the ER antagonist ICI 182,780 abrogated the effects of raloxifene on OPG production. Moreover, raloxifene enhanced osteoblastic differentiation markers, type 1 collagen secretion, and alkaline phosphatase activity by 3- and 2-fold, respectively (P < 0.001). In addition, raloxifene inhibited expression of the bone-resorbing cytokine IL-6 by 25-45% (P < 0.001). In conclusion, our data suggest that raloxifene stimulates OPG production and inhibits IL-6 production by hOB. Because OPG production increases with osteoblastic maturation, enhancement of OPG production by raloxifene could be related to its stimulatory effects on osteoblastic differentiation.

Anti-inflammatory activity of creatine supplementation in endothelial cells in vitro.

1 Creatine (CR) supplementation augments muscle strength in skeletal muscle cells by increasing intracellular energy pools. However, the effect of CR supplementation on endothelial cells remains to be clarified. 2 In this study, we investigated whether CR supplementation had any anti-inflammatory activity against human pulmonary endothelial cells in culture. 3 We confirmed that supplementation with 0.5 mM CR significantly increased both intracellular CR and phosphocreatine (PC) through a CR transporter while keeping intracellular ATP levels constant independent of CR supplementation and a CR transporter antagonist. 4 In the assay system of endothelial permeability, supplementation with 5 mM CR significantly suppressed the endothelial permeability induced by serotonin and H(2)O(2). 5 In cell adhesion experiments, supplementation with 5 mM CR significantly suppressed neutrophil adhesion to endothelial cells. 6 In the measurement of adhesion molecules, CR supplementation with more than 0.5 mM CR significantly inhibited the expressions of ICAM-1 and E-selectin on endothelial cells, and the inhibition was significantly suppressed by an adenosine A(2A) receptor antagonist. 7 The present study suggests that CR supplementation has anti-inflammatory activities against endothelial cells.

Dexamethasone regulates endothelin-1 and endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells.

Endothelin-1 (ET-1) lowers intraocular pressure (IOP) in animal models by regulating aqueous humour dynamics through both inflow and outflow mechanisms. Moreover, ET's concentration is elevated in glaucoma patients and in animal models of glaucoma. Glucocorticoid therapy often can lead to increase IOP in susceptible individuals including patients with primary open angle glaucoma (POAG). In this study, we examined the effects of dexamethasone (Dex), a frequently used anti-inflammatory glucocorticoid, on the synthesis and release of endothelin-1 and on the expression of endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells, an established source for ET-1 in the anterior chamber. As measured by ET-1 immunoreactivity, ET-1 was concentration-dependently increased following 24hr Dex treatment, with a maximum concentration (100 nM) causing a threefold increase of ET-1 release. Western blot analysis of HNPE cells showed the expression of endothelin receptor A (ET(A)) and endothelin receptor B (ET(B)) with approximate molecular weights of 40 kDa. Dex treatment decreased ET(A) receptor expression at all Dex doses, but up-regulated ET(B) receptors with 10nM Dex having the greatest effect. Quantitative PCR demonstrated that Dex also increased the mRNA of pre-pro-ET-1 (ppET-1) and ET(B) but decreased the mRNA of ET(A). RU486, a glucocorticoid receptor antagonist, was able to block Dex's actions on ET release and ET(B) receptor expression, but did not block its action on ET(A) receptor expression. Endothelin receptors were minimally expressed in HNPE cells as determined in binding experiments (B(max): ET(A) 17, ET(B) 25 fmolmg(-1) membrane protein). However Dex treatment stimulated a dramatic increase in ET(B) receptor density while decreasing ET(A) receptors (B(max): ET(A) 11, ET(B) 116 fmolmg(-1) membrane protein). The regulation of endothelin and its receptors could be a novel mechanism associated with glucocorticoid's effects on intraocular pressure. The increase in ET-1 and disproportionate regulation in ET receptor expression by Dex could promote dysregulation in ET's mechanism on both inflow and outflow, thus affecting aqueous humour dynamics in the anterior chamber of the eye.

Tumor necrosis factor-alpha production in whole blood after cardiopulmonary bypass: downregulation caused by circulating cytokine-inhibitory activities.

OBJECTIVES: Cardiopulmonary bypass is associated with the release of proinflammatory cytokines (tumor necrosis factor alpha, interleukin 1beta, interleukin 6, and interleukin 8) and anti-inflammatory cytokines (interleukin 10 and transforming growth factor beta(1)). On the one hand this cytokine release is related to the postoperative systemic inflammatory response syndrome, and on the other hand it is related to deterioration of the immune system, for example in monocyte or polymorphonuclear neutrophil function, leading to an increased susceptibility to infections. To gain further insight into the alterations of immune cell reactivity and possible regulatory mechanisms, we studied lipopolysaccharide-induced tumor necrosis factor alpha synthesis in whole blood from cardiac surgical patients. METHODS: Fifteen patients undergoing elective heart surgery with cardiopulmonary bypass were included in the study. Ex vivo lipopolysaccharide-induced tumor necrosis factor alpha synthesis was measured in a whole blood assay before, during, and after bypass. Corresponding tumor necrosis factor alpha messenger RNA levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction. In addition, the influence of patient serum on whole blood responsiveness and its relationship to anti-inflammatory cytokines were evaluated in vitro. RESULTS: Tumor necrosis factor alpha synthesis was significantly reduced after 30 minutes of cardiopulmonary bypass and showed the lowest values at the end of bypass (mean +/- SD 0.109 +/- 0.105 ng/10(6) white blood cells after 30 minutes of bypass and 0.050 +/- 0.065 ng/10(6) white blood cells at the end of bypass, vs 0.450 +/- 0.159 ng/10(6) white blood cells preoperatively, P <.001). As a further indication of reduced cytokine biosynthesis, diminished messenger RNA levels for tumor necrosis factor alpha were detected. Serum withdrawn from patients at the end of cardiopulmonary bypass reduced tumor necrosis factor alpha synthesis in heterologous blood from healthy volunteers highly significantly to 39.93% +/- 23.18% relative to control serum (P =.005) and preoperatively drawn serum (P =.024). This effect was dose dependent and was not specific for lipopolysaccharide-induced tumor necrosis factor alpha synthesis. Anesthesia and heparin administration did not influence tumor necrosis factor alpha production significantly. Ex vivo tumor necrosis factor alpha synthesis was negatively related to interleukin 10 serum levels, positively but weakly related to interleukin 4, and was not related to transforming growth factor beta(1) (Spearman correlation coefficients -0.565, P <.001, 0.362, P <.001, and -0.062, P =.460, respectively). However, interleukin 10 levels in patient serum after cardiopulmonary bypass were 300-fold below the quantities needed for half-maximal inhibition of tumor necrosis factor alpha synthesis in vitro. Moreover, the inhibitory activity could not be removed by immune absorption of interleukin 10. CONCLUSIONS: These results suggest that during cardiac operations cytokine-inhibitory serum activities are released or newly formed. These activities could not be explained by the actions of interleukins 4 and 10 or transforming growth factor beta(1). Although their exact nature remains undetermined, these substances may contribute to the diminished immune cell functions after cardiopulmonary bypass and thus need further characterization.

Desipramine antagonized corticosterone-induced apoptosis in cultured PC12 cells.

AIM: To study possible action mechanism of a tricyclic antidepressant, desipramine (DIM). METHODS: Cultured PC12 cells were exposed to corticosterone in the absence or presence of DIM for 5 d. Agarose gel electrophoresis, flow cytometry, and electron microscopy were used to detect the apoptosis of PC12 cells. RESULTS: Corticosterone 10 micromol/L treatment for 5 d elicited typical apoptotic biochemical and morphological changes including condensed chromatin shaped like crescent moon, nuclear fragmentation, and DNA degradation. The highest percentage of apoptotic cells accumulated to 28 % +/- 9 %. Agarose gel electrophoresis showed typical DNA ladders pattern. While in the presence of DIM 1 or 5 micromol/L, apoptosis percentage was markedly decreased with lightened DNA ladder and ultrastructure of the cells was improved. CONCLUSION: DIM could antagonize the apoptosis in PC12 cells induced by corticosterone, which may be one of the cellular mechanisms of its antidepressant effect.

In vivo GM-CSF prevents dexamethasone suppression of killing of Aspergillus fumigatus conidia by bronchoalveolar macrophages.

Dexamethasone (DEX) is a potent immunosuppressive agent used in the treatment of several disorders. However, despite its beneficial effects, DEX puts patients at risk for opportunistic infections, especially pulmonary aspergillosis. Previously we reported that in vitro granulocyte-macrophage colony-stimulating factor (GM-CSF) blocks the immunosuppressive action of DEX on bronchoalveolar macrophages (BAMs). Here we report that BAMs freshly isolated from mice treated intraperitoneally with DEX for 24 h had significantly (P<0.01) reduced killing of conidia, i.e., 15 +/- 5% conidia killed by BAMs from DEX-treated mice versus 35 +/- 3% by BAMs from mice given saline, 38 +/- 5% by BAMs from mice given GM-CSF, and 39 +/- 1% by BAMs from mice given both DEX and GM-CSF. On the other hand, in another compartment GM-CSF could not block the DEX reduction of spleen weight and spleen cellularity. Unlike GM-CSF, granulocyte colony-stimulating factor did not block DEX suppression of BAMs. GM-CSF given 24 h before DEX resulted in blocking of DEX suppression of BAM conidiacidal activity. However, when DEX was given 24 h before GM-CSF, DEX suppression of BAM was not reversed. These data show that GM-CSF in vivo blocks the in vivo immunosuppressive effects of DEX on BAM killing of conidia and suggest a potential use of GM-CSF in patients at risk for aspergillosis due to immunosuppressive DEX treatment.

Inhibition of glucocorticoid-mediated, caspase-independent dendritic cell death by CD40 activation.

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells, including T cells, monocytes/macrophages, osteoclasts, and dendritic cells (DC). However, the mechanism(s) by which GC exert anti-inflammatory effects is still largely unknown. It is already well known that GC treatment inhibits DC maturation and interleukin (IL)-12 production by DC. In this study, we investigated the apoptosis induction of DC by a synthetic GC, dexamethasone (Dex). The stimulation with Dex resulted in DC apoptosis in a dose- and time-dependent manner as it was measured by determining annexin V-positive cells and mitochondrial potential. In contrast, monocytes that are precursor cells of DC are resistant to Dex-mediated apoptosis. The Dex-induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z-VAD-fmk. It is interesting that agonistic CD40 antibody completely inhibited Dex-induced cell death, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC-mediated DC apoptosis. Taken together, our findings revealed an important role of GC and CD40 signaling in the regulation of immune responses in which DC play a key role in the inflammatory process of various immunomediated diseases.

Anti-inflammatory drugs block cytokine mRNA accumulation in the skin and improve the clinical condition of reactional leprosy patients.

The aim of this study was to investigate in what ways in vivo anti-inflammatory treatment affects cytokine mRNA expression in situ in both erythema nodosum leprosum and reversal reaction patients. Serial biopsies were collected from the patients undergoing leprosy reactions before and during pentoxifylline (n = 7) or thalidomide (n = 3) treatment for erythema nodosum leprosum and prednisone (n = 3) for reversal reaction. Clinical evolution of the skin lesion was assessed during the study and semiquantitative reverse transcription-polymerase chain reaction was used to investigate cytokine mRNA expression at the lesion site. Results showed expression of interferon-gamma, interleukin-6, interleukin-10, interleukin-12 p40, and tumor necrosis factor-alpha in all patients tested at the onset of reactional episodes, but interleukin-4 mRNA was rarely detected in the lesions (n = 4). Follow-up analysis showed that, irrespective of the drugs used, tumor necrosis factor-alpha mRNA was diminished in 10 of the 13 patients tested. A concomitant decrease of mRNA accumulation was also observed for interferon-gamma (nine of 11 patients), interleukin-6 (nine of 11), and interleukin-12 p40 (six of eight). An inhibitory effect on interleukin-10 mRNA was likewise seen after thalidomide and pentoxifylline, but not subsequent to prednisone treatment. The data also demonstrated that cytokine mRNA inhibition correlates to the resolution of the inflammatory response in situ (n = 10), whereas the persistence/enhancement of cytokine message expression after treatment was associated with worsening of the skin condition, as seen in three erythema nodosum leprosum patients whose maintenance of local inflammation was accompanied by the appearance/persistence of interleukin-4 gene expression in situ subsequent to anti-inflammatory treatment. In summary, the participation of cytokines in leprosy inflammatory episodes seems to be directly associated with the patients' clinical evolution following therapy for reaction.

Glycine: a new anti-inflammatory immunonutrient.

The mechanism of the immunosuppressive effects of glycine and its pathophysiological applications are discussed in this review. Glycine has been well characterized in spinal cord as an inhibitory neurotransmitter which activates a glycine-gated chloride channel (GlyR) expressed in postsynaptic membranes. Activation of the channel allows the influx of chloride, preventing depolarization of the plasma membrane and the potentiation of excitatory signals along the axon. Glycine has recently been shown to have similar inhibitory effects on several white blood cells, including hepatic and alveolar macrophages, neutrophils, and lymphocytes. Pharmacological analysis using a GlyR antagonist strychnine, chloride-free buffer, and radiolabeled chloride has provided convincing evidence to support the hypothesis that many white blood cells contain a glycine-gated chloride channel with properties similar to the spinal cord GlyR. Molecular analysis using reverse transcription-polymerase chain reaction and Western blotting has identified the mRNA and protein for the beta subunit of the GlyR in total RNA and purified membrane protein from rat Kupffer cells. Dietary glycine is protective in rat models against endotoxemia, liver ischemia-reperfusion, and liver transplantation, most likely by inactivating the Kupffer cell via this newly identified glycine-gated chloride channel. Glycine also prevents the growth of B 16 melanomas cell in vivo. Moreover, dietary glycine is protective in the kidney against cyclosporin A toxicity and ischemia-reperfusion injury. Glycine may be useful clinically for the treatment of sepsis, adult respiratory distress syndrome, arthritis, and other diseases with an inflammatory component.

Relative sensitivity of Tc-99m WBC versus In-111 WBC in a patient with Crohn disease and steroid use.

Numerous studies have shown that chemotaxis is affected by certain antibiotics and steroids. The authors present the case of a patient with Crohn disease relapse with multiple small-bowel fistulae and mesenteric abscesses. Whereas the Tc-99m WBC scan failed to show the intra-abdominal inflammatory foci, an In-111 WBC scan performed within a week delineated the abscesses very well, and these were later confirmed at surgery. This case is presented not only to illustrate the relative sensitivities of a Tc-99m WBC versus an In-111 WBC scan, but also to discuss the impediment to polymorphonuclear chemotaxis by steroids, which may be a contributory factor to the sensitivities of the different radiopharmaceuticals selected for detection of intra-abdominal septic foci.

The effects of glucocorticoids and a glucocorticoid antagonist (RU 38486) on experimental acute pancreatitis in rat.

The effects of glucocorticoids on acute pancreatitis are a matter of dispute. In animal experiments, dexamethasone and hydrocortisone significantly decreased the serum amylase activities 8 hours after the induction of pancreatitis. In the dexamethasone treated group, the serum IL-6 level was significantly decreased at 4 and 8 hours, while in the hydrocortisone treated group, all the IL-6 values were significantly diminished vs. the control group. As compared to the control, a glucocorticoid antagonist (RU 38486) did not influence the serum amylase activity, but significantly increased the serum IL-6 level. These results suggest that glucocorticoids may play a role in the control of pancreatitis caused by inhibition of cytokine production.