Bacillus Calmette-Guérin potentiates monocyte responses to lipopolysaccharide-induced tumor necrosis factor and interleukin-1, but not interleukin-6 in bladder cancer patients

During the past decade, particular attention has been focused on treatment of bladder cancer patients with the bacterial agent bacillus Calmette-Guérin (BCG). In these studies, bladder cancer patients were instilled with BCG (75 mg/50 ml) once per week for 6 weeks, 1-2 weeks following trans-urethral resection of the bladder. Cystoscopy was performed after 6 weeks and, unless tumor progression was present, monthly treatments were given for 1 year. Blood was drawn 2 h after the last instillation, and monocytes were isolated (5 x 10(6) cells/ml) and treated, or not, with lipopolysaccharide (LPS) (20 microgram/ml) for tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) release. The levels of monokines were determined by a monokine-specific enzyme-linked immunosorbent assay. Our results clearly show that, after 18 h incubation, macrophages from BCG-treated bladder cancer patients produced from 2.8- to 1.9-fold and from 2.0- to 1.3-fold greater amounts of TNF alpha and IL-1 alpha respectively, compared to macrophages from healthy controls, 5-fold higher than bladder cancer patients not treated with BCG. IL-6 was not affected. In another set of experiments macrophages (5 x 10(6) cells/ml) from healthy subjects were pretreated, or not, with BCG (100 micrograms/ml) overnight and treated, or not, with LPS 20 microgram/ml alone and in combination with interleukin-1 receptor antagonist (IL-1ra) 250 ng/ml. Macrophages treated with BCG had a strong stimulatory effect on IL-1 alpha release (9.45 ng/ml) while LPS was less effective (3.59 ng/ml). The combination of BCG plus LPS produced an additive effect on IL-1 alpha release (13.71 ng/ml) compared to the effect of the compound alone. The addition of IL-1ra (250 ng/ml) to BCG was not effective, while when IL-1ra was added to BCG plus LPS only a partial inhibition of IL-1 alpha release was found (9.83 ng/ml), compared to BCG plus LPS without IL-1ra (13.71 ng/ml). These effects seem to be related to the inhibition of IL-1 alpha stimulated with LPS, but not BCG. The priming effect of BCG exerted on LPS-stimulated monocyte production of TNF alpha and IL-1 alpha from bladder cancer patients led us to study the possible modulation of fibrinogen and C-reactive protein in the serum of BCG-treated cancer patients. The plasma levels of fibrinogen and C-reactive protein were higher (approximately twice) in BCG-treated patients compared to values obtained in untreated patients or healthy controls. We conclude that the beneficial immunotherapeutic effects of BCG in bladder cancer patients are related to its capacity to prime macrophages to enhance the release of TNF alpha and IL-1 alpha, but not IL-6 in response to physiological secondary stimuli, or through the direct stimulation of BCG on IL-1 alpha or TNF alpha, which are directly involved in the killing of cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of xanthine oxidase and reactive oxygen intermediates in LPS- and TNF-induced pulmonary edema

We studied the role of reactive oxygen intermediates (ROI) in lipopolysaccharide (LPS)-induced pulmonary edema. LPS treatment (600 micrograms/mouse, IP) was associated with a marked induction of the superoxide-generating enzyme xanthine oxidase (XO) in serum and lung. Pretreatment with the antioxidant N-acetylcysteine (NAC)--1 gm/kg orally, 45 minutes before LPS--or with the XO inhibitor allopurinol (AP)--50 mg/kg orally at -1 hour and +3 hours--was protective. On the other hand nonsteroidal antiinflammatory drugs (ibuprofen, indomethacin, and nordihydroguaiaretic acid) were ineffective. These data suggested that XO might be involved in the induction of pulmonary damage by LPS. However, treatment with the interferon inducer polyriboinosylic-polyribocytidylic acid, although inducing XO to the same extent as LPS, did not cause any pulmonary edema, indicating that XO is not sufficient for this toxicity of LPS. To define the possible role of cytokines, we studied the effect of direct administration of LPS (600 micrograms/mouse, IP), tumor necrosis factor (TNF, 2.5 or 50 micrograms/mouse, IV), interleukin-1 (IL-1 beta, 2.5 micrograms/mouse, IV), interferon-gamma (IFN-gamma, 2.5 micrograms/mouse, IV), or their combination at 2.5 micrograms each. In addition to LPS, only TNF at the highest dose induced pulmonary edema 24 hours later. LPS-induced pulmonary edema was partially inhibited by anti-IFN-gamma antibodies but not by anti-TNF antibodies, anti-IL-1 beta antibodies, or IL-1 receptor antagonist (IL-1Ra).     

Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma

Endothelins (ETs) are potent bronchoconstrictor agents postulated to contribute to the pathophysiology of asthma and other respiratory disorders. An increase in both the expression and release of immunoreactive (ir) ETs was reported in bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic patients. We investigated whether dexamethasone (DEX), a potent anti-inflammatory and anti-asthmatic drug, regulates the basal and stimulated release of ETs from guinea-pig cultured tracheal epithelial cells. These airway epithelial cells spontaneously release ET-1 over 24 h. When incubated in the presence of 10(-7) and 10(-6) M DEX for 24 h, basal production of ET-1 decreased by 32 and 29%. Lipopolysaccharide (LPS; 1, 5, 10 micrograms/mL), tumor necrosis factor-alpha (TNF alpha; 5, 10 ng/mL), and interleukin-1 beta (IL-1 beta; 1, 5 ng/mL) significantly increased the basal release of ET-1 after 24 h. When these cells were pretreated with DEX (10(-7) M) for a 24-h period, then incubated in the presence of LPS (10 micrograms/mL), TNF alpha (10 ng/mL), or IL-1 beta (1 ng/mL) for another 24 h, the stimulated release of ET-1 was inhibited by 48, 31, and 38%, respectively. At 10(-6) M, DEX decreased the stimulated release by 45, 37, and 46%, respectively. The present results show that DEX can regulate the basal release and inhibit the pro-inflammatory cytokine-stimulated production of ET-1 from guinea-pig cultured tracheal epithelial cells. They suggest that the beneficial effect of glucocorticoids in asthma may be related to the inhibition of ET synthesis.     

Some experiences with hospital-based psychiatric aftercare over ten years

Smooth muscle cells represent a significant percentage of the total cells in the airway but their contribution to the inflammatory response seen in airway disease has not been studied. Hence, we have looked at the release of the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in response to bacterial lipopolysaccharide (LPS) and the pro-inflammatory cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). Human airway smooth muscle (HASM) cells released GM-CSF under basal conditions (45.4 +/- 13.1 pg ml-1) that was significantly enhanced by IL-1 beta and TNF alpha with a maximal effect seen at 10 ng ml-1 (1.31 +/- 0.07 ng ml-1 and 0.72 +/- 0.16 ng ml-1, respectively). In contrast, neither LPS nor IFN gamma produced a significant increase in GM-CSF release. However, HASM cells exposed to IL-1 beta, TNF alpha and IFN gamma generated more GM-CSF than that evoked by any cytokine alone (2.2 +/- 0.15 ng ml-1). The release of GM-CSF elicited by the cytokine mixture was inhibited by cycloheximide and dexamethasone. These data suggest that HASM cells might play an active part in initiating and/or perpetuating airway inflammation in addition to controlling airway calibre.     

Involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist in LPS-induced rabbit uveitis

The objective of the study was to investigate involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist (IL-1Ra) in lipopolysaccharide (LPS)-induced uveitis. Intravitreal injection of LPS (100 ng) to rabbits induced a massive leukocyte infiltration and protein leakage into the aqueous humor. Aqueous leukocyte counts and protein levels reached a peak 24 hr after this injection. The peak concentrations of aqueous TNF alpha (230 +/- 37 pg ml-1, at 9 hr) and IL-1 beta (185 +/- 80 pg ml-1, at 18 hr) preceded peak levels of aqueous leukocyte counts and protein levels. In contrast, the levels of aqueous IL-1Ra peaked at 48 hr (12,239 +/- 1964 pg ml-1) and a fairly high concentration of IL-1Ra remained when the inflammatory reactions subsided. Immunohistochemistry and leukocyte-depletion studies showed that infiltrating leukocytes were the major cellular sources of aqueous TNF alpha, IL-1 beta and IL-1Ra. Intravitreal injection of homologous TNF alpha (0.1-1.5 micrograms) or IL-1 beta (0.5-5 ng) reproduced a rapid leukocyte infiltration and protein leakage. Administration of anti-TNF alpha mAb (10 micrograms) suppressed the number of LPS-induced infiltrating neutrophils by 50%, mononuclear cells by 58%, and protein leakage by 42%. Administration of rabbit IL-1Ra (10 micrograms) also suppressed neutrophil influx by 78%, however, neither mononuclear cell influx nor protein leakage was inhibited by rabbit IL-1Ra. Co-administration of the two inhibitors enhanced inhibition of neutrophil infiltration to 88%, and protein leakage to 64%. We conclude that TNF alpha and IL-1 beta are the principal mediators of LPS-induced uveitis. Our observations also suggest that endogenous IL-1Ra may down-regulate inflammatory reactions.     

Interleukin-1 beta modulates prostaglandin and progesterone production by primate luteal cells in vitro

Increasing evidence suggests that cytokine products of the immune system may play a regulatory role in corpus luteum regulation in several species. The role of cytokines in primate luteal function, however, remains unclear. In the present study we examined the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon-gamma (IFN-gamma) on progesterone and prostaglandin (PGE2, PGF2 alpha) production by primate luteal cells in vitro. Specifically, corpora lutea were removed from normally cycling cynomolgus monkeys (n = 30 corpora lutea) during either the early (Days 3-5 after the estimated LH surge), mid (Days 8-10), or late (Days 12-14) luteal phase of the menstrual cycle. The corpora lutea were dispersed into individual cells using collagenase, DNase, and hyaluronidase. Approximately 50,000 viable luteal cells per tube were incubated in Ham's F-10 medium with increasing concentrations of IL-1 beta (0.1-10 ng/ml), TNF alpha (1-100 ng/ml), or IFN-gamma (10-1000 U/ml) in the presence and absence of hCG for 8 h at 37 degrees C. TNF alpha and IFN-gamma had no effect on progesterone PGE2, or PGF2 alpha production during any phase of the cycle at the doses tested. In contrast, IL-1 beta significantly stimulated PGF2 alpha production in a dose-dependent manner during the mid and late luteal phases (p < 0.05). Human CG alone had no effect on PGE2 or PGF2 alpha production by dispersed luteal cells in vitro but inhibited IL-1 beta-stimulated PGF2 alpha production. As expected, hCG stimulated progesterone production by primate luteal cells in vitro. Interestingly, IL-1 beta inhibited this hCG stimulation of progesterone production. In summary, these date suggest that IL-1 beta is a potentially important modulator of prostaglandin production by the primate corpus luteum. In view of this, cytokine-mediated changes in prostaglandin production by the primate corpus luteum may participate in the physiological regulation of luteal function.     

Modulation of lipopolysaccharide-induced macrophage gene expression by Rhodobacter sphaeroides lipid A and SDZ 880.431

Rhodobacter sphaeroides lipid A (RsDPLA) and SDZ 880.431 (3-aza-lipid X-4-phosphate) are prototypic lipopolysaccharide (LPS) antagonists. Herein, we examined the ability of these structures to regulate murine macrophage tumor necrosis factor (TNF) secretion and LPS-inducible gene expression (tumor necrosis factor alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], IP-10, type 2 TNF receptor [TNFR-2], D3, and D8 genes). We report that RsDPLA alone (> 1 microgram/ml) induced low levels of TNF-alpha secretion and a selective pattern of gene expression in peritoneal exudate macrophages; SDZ 880.431 alone was completely inactive. When LPS was present at a low concentration (1 ng/ml), RsDPLA and SDZ 880.431 blocked TNF secretion and gene induction in a concentration-dependent fashion. In general, gene induction was measurably reduced by 10 to 30 ng of RsDPLA per ml or 300 ng of SDZ 880.431 per ml, but inhibition could be uniformly overridden by increasing the concentration of LPS. Although induction of all six genes by LPS was suppressed by either inhibitor, effective inhibitor concentrations depended on the gene of interest. Induction of TNFR-2 by LPS was relatively resistant to inhibition by RsDPLA, and induction of TNFR-2 and D3 was relatively resistant to inhibition by SDZ 880.431. When LPS was present at > or = 100 ng/ml, correspondingly high concentrations (> or = 20 micrograms/ml) of either inhibitor influenced gene expression in a bidirectional manner. Under these conditions, LPS-induced expression of IP-10, D3, and D8 was suppressed regardless of the LPS concentration used (concentrations tested up to 50 micrograms/ml), while expression of TNF-alpha mRNA was enhanced about fourfold. In toto, RsDPLA and SDZ 880.431, when present at low concentrations, act in a manner consistent with competitive inhibition of LPS, while at higher concentrations, these structures inhibit certain LPS responses noncompetitively and synergize with LPS for other responses.     

Elevation of plasma cytokines in disorders of excessive daytime sleepiness: role of sleep disturbance and obesity

Excessive daytime sleepiness (EDS) and fatigue are frequent symptoms in the general population and the chief complaint of the majority of patients at Sleep Disorders Centers. There is evidence that the inflammatory cytokines tumor necrosis factor-alpha (TNF alpha), interleukin-1beta (IL-1beta), and IL-6 are involved in physiological sleep regulation and that their administration to humans is associated with sleepiness and fatigue. To explore whether plasma levels of TNF alpha, IL-1beta, and IL-6 are elevated in patients with EDS, we measured morning plasma levels of TNF alpha, IL-1beta, and IL-6 in 12 sleep apneics, 11 narcoleptics, 8 idiopathic hypersomniacs, and 10 normal controls. TNF alpha was significantly elevated in sleep apneics and narcoleptics compared to that in normal controls (P < 0.001 and P = 0.001, respectively). Plasma IL-1beta concentrations were not different between sleep disorder patients and controls, whereas IL-6 was markedly and significantly elevated in sleep apneics compared to that in normal controls (P = 0.028). The primary factor influencing TNF alpha values was the degree of nocturnal sleep disturbance, whereas the primary determinant for IL-6 levels was the body mass index. Our findings suggest that TNF alpha and IL-6 might play a significant role in mediating sleepiness and fatigue in disorders of EDS in humans.     

Cytokine-mediated growth hormone release from cultured ovine pituitary cells

Previous studies have demonstrated that intravenous lipopolysaccharide (LPS) will increase concentrations of growth hormone (GH). One possible explanation for this may reside in the response of the pituitary to specific cytokines. This study sought to determine the effects of recombinant bovine tumor necrosis factor alpha (TNF), recombinant ovine (ro) interleukin-1alpha (IL-1alpha), roIL-1beta, ro interleukin-2 (IL-2), and ro gamma-interferon (INT) on GH release from cultured sheep pituitary cells. Sheep were sacrificed and pituitary cells cultured in DMEM with 10% fetal bovine serum for 3 days. On day 4, cells were washed and serum-free DMEM added to cells. IL-1alpha and IL-1beta were used at 0.2, 2 and 20 ng/ml and the remaining cytokines at 2, 20 and 200 ng/ml. Neither IL-2 nor INT had effects on basal or on GH-releasing hormone (GRH)-stimulated GH release. TNF inhibited GRH-stimulated GH release (p < 0.05). Both IL-1alpha and IL-1beta stimulated GH release from cultured pituitary cells at all doses tested (p < 0.01). Neither IL-1alpha nor IL-1beta had an effect on GRH-stimulated GH release. IL-1 effects were inhibited by H-89 (p < 0.05; a protein kinase A inhibitor) and by nifedipine (p < 0.05; a calcium channel blocker). Both of these mechanisms are central signal transduction mechanisms mediating GRH-stimulated GH release. IL-1-stimulated GH release is partially inhibited (p < 0.05) by lipoxygenase pathway blockers. Phorbol myristate acetate downregulation of protein kinase C did not alter IL-1-stimulated GH release. IL-1beta increased the content of both GH and GH mRNA in cultured sheep pituitary cells. We conclude that IL-1 produces a strong stimulus to GH release, which is mediated by calcium entry and protein kinase A activation. IL-1 also activates lipoxygenase pathways. This latter pathway as well as calcium entry were shown to mediate LPS stimulation of GH release from cultured pituitary cells. The similarity between IL-1 and LPS signal transduction suggests that LPS may activate pituitary production of IL-1 to produce the stimulus to GH. The lack of inhibitory effects of INT, TNF and IL-2 as opposed to what is seen in the rat may suggest a partial mechanism to explain the different effects of LPS on GH release between sheep and that seen in cattle and rats.     

Troglitazone prevents the inhibitory effects of inflammatory cytokines on insulin-induced adipocyte differentiation in 3T3-L1 cells

Tumor necrosis factor (TNF) is implicated in wasting syndromes and insulin resistance in chronic infection and obese-linked diabetes. TNF (10 ng/ml) inhibited adipocyte differentiation of 3T3-L1 cells, and in these TNF treated cells little insulin-stimulated glucose uptake was observed. Treatment of 3T3-L1 cells with troglitazone (1-10 microM) partially prevented this inhibitory effect of TNF on adipogenesis, and enhanced expression of C/EBP alpha and GLUT4, even in the presence of TNF. Troglitazone also prevented the inhibitory effects of interleukin-1, interleukin-6, and leukemia inhibitory factor, but not of transforming growth factor beta on adipocyte differentiation of 3T3-L1 cells. These effects might contribute to the antidiabetic effect of troglitazone in obese diabetic animals.     

CsA, FK506, corticosteroids and rapamycin inhibit TNF alpha production by cultured PTEC

In this study we investigated the effect of immunosuppressive drugs on the interleukin-1 alpha (IL-1 alpha) enhanced tumor necrosis factor alpha (TNF alpha) production by proximal tubular epithelial cells (PTEC). Under basal conditions cultured PTEC produce between 0 to 390 pg/ml/10(5) cells of TNF alpha. Upon stimulation with IL-1 alpha an enhancement of TNF alpha production was seen in each cell line tested, ranging from 230 to 2424 pg/ml/10(5) cells. The presence of cyclosporin A (CsA) during stimulation with IL-1 alpha inhibited the enhanced TNF alpha production in a dose dependent fashion, with a maximal inhibition of 90% at a concentration of 250 ng/ml. Inhibition was at the level of mRNA as could be demonstrated by Northern blot analysis. FK506, corticosteroids and rapamycin also inhibited TNF alpha production in a dose dependent fashion, although not as effectively as CsA. Two corticosteroids were tested for their inhibitory effect on TNF alpha production. It was found that dexamethasone at a concentration of 10 ng/ml inhibited TNF alpha production for almost 40%. A 100-fold higher concentration of hydrocortisone was necessary to yield similar inhibition. The effect of rapamycin on the IL-1 alpha enhanced TNF alpha production differed from the effect of CsA. While CsA induced a maximal inhibition of 90%, rapamycin only induced a maximal inhibition of 37%, and even less inhibition at higher concentrations of the drug. The presence of the various drugs was essential for their inhibitory effect, because removal of the drug from the PTEC by washing immediately resulted in loss of inhibition. Combinations of CsA and FK506 or rapamycin were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ricin toxin contains three lectin sites which contribute to its in vivo toxicity

Ricin intoxication of mammalian cells is initiated by B chain (RTB) binding to cell surface galactosides. Recombinant insect-derived RTB mutants with modifications in lectin-site subdomains 2 gamma, 1 alpha/2 gamma, and 1 alpha/1 beta/2 gamma were reassociated with plant RTA and tested for lethality in C57B1/6 6-8 weeks old mice. The LD50 of intraperitoneally injected castor bean ricin was 75 ng per 18 g mouse. The LD50 of single-site 2 gamma mutant heterodimer was 100 ng: the LD50 of the double-site 1 alpha/2 gamma mutant heterodimer was 500 ng, and the LD50 of the triple-site 1 alpha/1 beta,2 gamma mutant heterodimer was > 10 micrograms. Plant RTA alone had an LD50 of 300 micrograms. Animals died between 1 and 10 days post-injection. Histopathological examination of morbid animals receiving an LD50 dose of each toxin revealed only apoptosis in the thymus and spleen. The present data provide clear evidence for participation of three lectin sites in ricin in vivo toxicity. These results suggest an origin for some of the normal tissue toxicities observed with clinical trials of doubly blocked ricin conjugates and suggest modification of at least three RTB subdomains will be necessary in genetically engineered ricin fusion proteins.     

Role of nuclear factor-kappaB activation in cytokine- and sphingomyelinase-stimulated inducible nitric oxide synthase gene expression in vascular smooth muscle cells

Inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF alpha), are known to activate sphingomyelinase (SMase) and nuclear factor-kappaB (NF-kappaB) in certain cell types, which also stimulate inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains unknown whether the SMase pathway is involved in iNOS gene expression in VSMCs. Therefore, the present study was designed to examine whether SMase induces iNOS gene expression via the NF-kappaB activation pathway similar to that of IL-1beta and TNF alpha in cultured rat VSMCs. Neutral SMase, although less potently than IL-1beta and TNF alpha, stimulated nitrite/nitrate (NOx) production, and iNOS messenger RNA and protein expression, as assessed by Northern and Western blot analyses, respectively. Neutral SMase, IL-1beta, and TNF alpha activated NF-kappaB, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, iNOS expression, and NF-kappaB activation stimulated by TNF alpha, but not by IL-1beta. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-kappaB activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1beta and TNF alpha, but not neutral SMase, caused a transient decrease in IkappaB-alpha protein levels, whereas IkappaB-beta protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated IkappaB-alpha degradation. Several cell-permeable ceramide analogs (C2, C6, and C8), hydrolysis products of sphingomyelin, activated NF-kappaB less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-kappaB activation in mediation of neutral SMase-induced iNOS expression, but distinct from the proteasome-mediated IkappaB-alpha degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).     

Modulation of kinin B1 but not B2 receptors-mediated rat paw edema by IL-1beta and TNFalpha

The modulatory effects of IL-1beta and TNF alpha on the rat paw edema induced by B1 agonists have been analyzed. In naive rats, i.d. injection of B1 agonists, des-Arg9-bradykinin and des-Arg10-kallidin (up to 300 nmol), causes a minimal increase in paw volume, while the B2 agonist tyrosine8-bradykinin (0.3-10 nmol) induces graded paw edema. The injection of des-Arg9-bradykinin (10-100) nmol or des-Arg10-kallidin (1-100 nmol), in paws pre-treated with IL-1beta or TNF alpha (both 5 ng/paw; 60 and 30 min prior, respectively), caused a graded edema formation. The edemas induced by des-Arg9-bradykinin (100 nmol) were evident at 15 min, reaching the maximum 60 and 30 min after treatment with IL-1beta (0.64 +/- 0.06 ml) or TNF alpha (0.47 +/- 0.05 ml), respectively, being reduced at 360 min. The B1 antagonist des-Arg9-NPC 17731 (1-30 nmol), but not the B2 antagonist Hoe 140 (10 nmol), produced marked inhibition of des-Arg9-bradykinin-induced paw edema. Dexamethasone (0.5 mg/kg, s.c., 4 h) or cycloheximide (1.5 mg/kg, s.c., 6 h) significantly prevented the edema caused by des-Arg9-bradykinin (100 nmol) in rats treated with IL-1beta (81 +/- 5% and 59 +/- 3%) or TNF alpha (78 +/- 4% and 43 +/- 2%). Indomethacin (2 mg/kg, i.p.) or meloxicam (3 mg/kg, i.p.), 1 h prior, significantly reduced the edema induced by des-Arg9-bradykinin (100 nmol) in IL-1beta (40 +/- 6% and 69 +/- 8%) or TNF alpha (43 +/- 3% and 53 +/- 9%) treated rats. It is suggested that i.d. injection of the IL-1beta or TNF alpha, produced up-regulation of B1 receptor-mediated paw edema, being this effect sensitive to dexamethasone and cycloheximide and to cyclo-oxygenase pathway.     

Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE2 was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE2, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGF beta and tumor necrosis factor-alpha(TNF). The production of 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) and of PGF2 alpha were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGF beta and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE2 biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6-12 h, and levels had appreciably diminished by 24 h after treatment. Both TGF beta and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE2 resulting from TNF stimulation was blocked by the addition of an interleukin-1 beta (IL-1 beta) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1 beta production. TGF beta regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines.     

Modulation by endothelin-1 of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells: interaction of endothelin-1 with cytokines

The interaction of endothelin-1 (ET-1) with either interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on the release of tissue plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was investigated in a culture system of vascular endothelial cells derived from human umbilical vein. The t-PA:Ag release was significantly decreased by either IL-1 beta or TNF alpha; ET-1 intensified the suppressive effect of the cytokines. In contrast, PAI-1:Ag release was significantly increased by either IL-1 beta or TNF alpha; ET-1 significantly reduced the stimulatory effect of the cytokines. The data suggest that endothelial cell-mediated fibrinolysis may be modulated by ET-1.