Rapid acceleration of neutrophil apoptosis by tumor necrosis factor-{alpha}

We demonstrate here that human necrosis factor-, a potent neutrophilactivator, induces rapid (within 3 h) apoptosis of these cells,i.e. neutrophils treated with this cytokine exhibit (I) lightand electron microscopic changes characteristic to apoptoticcells, (II) reduced propidium iodide binding to DNA, and (III)the ladder form of DNA, as shown by agarose gel electrophoresis.These results suggest that apoptosis acceleration may be involvedin processes by which neutrophils are prevented from damagingtissues.     

The production of tumour necrosis factor a (TNF-{alpha}) by human endometrial cells in culture

The production of tumour necrosis factor a (TNF-) by culturedhuman endometrial epithelial and stromal cells prepared fromendometrium obtained at different stages in the menstrual cyclehas been investigated. TNF- was not detectable in the supernatantsof stromal cell cultures prepared from endometrial tissue obtainedat any time in the menstrual cycle. TNF- production by endometrialepithelial cells in culture varied depending on the time inthe cycle at which the endometrial tissue was taken. Cells preparedfrom tissue obtained during the late proliferative phase ofthe cycle produced more TNF- than those prepared from tissueobtained at other times in the cycle. In addition, a small increasein TNF- production was seen by cells prepared from tissue obtainedduring the mid-secretory phase of the cycle. Interieukin 1 (IL-1)(1.4–140 pmol/1) caused a dose-dependent increase in TNF-production by cells prepared from both proliferative and secretoryendometrium. Maximum LL-1-stimulated increases in TNF- productionwere similar in cells from both proUferative and secretory endometriumand typically reached from four to 10 times basal values. Highdoses of progesterone, either alone or in the presence of oestradiol,also affected TNF-a production by epithelial cells. TNF- productionby cells prepared from proliferative endometrium was increasedby progesterone. In contrast, TNF- production by cells preparedfrom secretory endometrium was decreased in the presence ofprogesterone. The effects of steroids on TNF- production wereless marked than that of IL-1, with values increasing or decreasingto a maximum of three times the basal value. Placental protein14 (PP14) (0.18 and 1.8 nmol/1) also increased TNF- productionby cells prepared from proliferative tissue, but had no effecton its production by cells prepared from secretory endometrium.PP14-stimulated TNF- levels typically only reached a maximumof two times basal values.     

Endocrinology: Ovarian hyperstimulation syndrome: pre-ovulatory serum concentrations of interleukin-6, interleukin-1 receptor antagonist and tumour necrosis factor-{alpha} cannot predict its occurrence

The pathogenesis of the ovarian hyperstimulation syndrome (OHSS)is poorly understood. Since significant elevations in cytokinesare found in 01155, our objective was to conduct a prospectivecase-controlled study to assess if preovulatory cytokine serumconcentrations can predict its occurrence. The study group wasselected from in-vitro fertilization patients who subsequentlydeveloped severe OHSS, along with a matched group who did notdevelop this complication (n = 20), and a healthy normal controlgroup (n = 10). Interleukin-6 (IL-6), interleukin-1 receptorantagonist (IL-1RA) and tumour necrosis factor- (TNF) measurementswere performed with sensitive immune-assays and confirmed withbioassays. Serum IL-6 (mean concentration ± SEM: 4.38± 0.36 pg/ml), IL-1RA (829 ± 292 pg/ml) and TNF(15.5 ± 132 pg/ml) concentrations did not show differencesthroughout the normal menstrual cycle group. Cytokine variabilityand pre-ovulatory values were similar in OHSS compared to controlledovarian hyperstiinulation (COH) patients. However, average follicularphase serum 1L-6 concentrations were higher in OHSS (8.71 ±0.41 pg/ml) and COH (7.66 ± 0.38 pg/ml) patients thanin normally menstruating women (4.34 ± 0.99 pg/ml) (P< 0.0001). Pre-ovulatory serum 1L-6 concentrations were alsohigher in OHSS (9 ± 0.94 pg/ml) and COH (73 ±0.97 pg/ml) patients than in controls (4.57 ± 1.1 pg/ml)(P < 0.01 and P < 0.04 respectively). IL-1RA and TNF concentrationswere comparable in all the groups. This study suggests thatcytokine measurements cannot be used to predict the occurrenceof OHSS prior to the administration of human chorionic gonadotrophin.     

Implantation: Tumour necrosis factor {alpha} inhibits in-vitro decidualization of human endometrial stromal cells

Interleukin-1 (IL-1) has been reported previously to inhibitthe in-vitro decidualization of human endometrial stromal cellsas assessed by progesterone-induced prolactin production andmorphological transformation. In this study we examined whetherother cytokines, such as tumour necrosis factor-(TNF), interferon-(IFN), IFN or granulocyte-macrophage colony-stimulating factor(GM-CSF), could affect the decidualization of human endometrialstromal cells in vitro. Of these cytokines, TNF significantlysuppressed prolactin production in a dose-dependent manner,with no apparent effect on cell number. The morphological transformationof endometrial stromal cells was also inhibited by TNF. TNFand IL-1 significantly suppressed cAMP-stimulated prolactinproduction by endometrial stromal cells. Neither the progesteroneconcentration in the supernatant of the endometrial stromalcell culture system nor intracellular calcium concentrationof the endometrial stromal cells were affected by the additionof TNF or IL-1. These results indicated that TNF and IL-1 suppressboth progesterone-induced and cAMP-mediated prolactin productionin endometrial stromal cells, and that this inhibition was notattributable to direct effects on progesterone metabolism orrelated to Ca2+-mediated signal transduction. These experimentssuggested that a local increase of TNF and IL-1 under certainpathological conditions in vivo may disturb blastocyst implantationand/or the maintenance of pregnancy by inhibiting the decidualizationof endometrial stromal cells.     

Concentration of interleukin-1{beta} correlates with prostaglandin E2 and F2{alpha} in human pre-ovulatory follicular fluid

To investigate the role(s) of interleukin-1 (IL-1) in humanovarian function, we measured the concentrations of IL–1,prostaglandins (PGs) and steroids in follicular fluid of 90stimulated ovaries, with reference to oocyte maturation. Concentrationsof IL-1 were significantly higher in the follicles from whichmature oocytes were recovered than in follicles from which oocytescould not be recovered (P < 0.05). IL-1 concentrations alsoincreased in association with oocyte maturation. Positive significantcorrelations were seen between IL-1 and prostaglandin E2 (PGE2)(r = 0.47, P < 0.001), and between IL-1 and prostaglandinF₂ (PGF₂) (r = 0.22, P < 0.05) in pre-ovulatory follicularfluid, but not between IL–1 and oestradiol, or betweenIL-1 and oestradiol, or between IL-1 and progesterone. 0Follicularfluid IL-1 might contribute to prostaglandin-induced oocytematuration and ovulation.     

Involvement of {alpha}1 and {alpha}4 integrins in gut mucosal injury of graft-versus-host disease

We have investigated the involvement of adhesion molecules inthe lymphocyte infiltration associated with acute intestinalgraft-versus-host disease (GVHD) induced by injection of C3Hlymph node cells into irradiated (C3H x DBA/2)F₁ mice. Firstwe analyzed the expression profile of adhesion molecules including1, 2, 4, 5, 6, L and ß7 integrins, CD44 and L-selectinof lymphocytes from lymph nodes and gut mucosa in normal mice.In normal mice, intraepithelial lymphocytes (IEL) and laminapropria lymphocytes (LPL) uniquely showed increased expressionof 1, 2 and ß7 integrins, and decreased expressionof L-selectin compared with that of lymphocytes of the lymphnodes and Peyer's patches. In mice with GVHD, IEL and LPL ofdonor lymph node cells origin underwent phenotyplc changes characterizedby the increased expression of 1, L and ß7 integrins,and the loss of L-selectin. The expression profile of adhesionmolecules on IEL and LPL of GVHD mice resembled that of normalmice except for the lack of 2 integrin. Treatment of GVHD micewith anti-1,-4 or-ß7 integrin antibody alone partiallyprevented the mucosal pathology of intestinal GVHD, whereasonly mice treated with anti-1 showed reduced donor lymphocyticinfiltration into the intestinal mucosa. In contrast, treatmentwith anti-L or anti-CD44 antibody did not affect the intestinalGVHD. Furthermore, dual blockade of both 1 and 4 integrins completelyinhibited the mucosal pathology and donor lymphocyte infiltrationof intestinal GVHD. These results indicate that 1 and 4 integrinsplay an important role in the pathology of intestinal GVHD.     

The accuracy of serum interleukin-6 and tumour necrosis factor as markers for ovarian torsion

BACKGROUND: The aim of this study was to investigate a possible role for interleukin-6 (IL-6) and tumour necrosis factor (TNF-alpha) as pre-operative markers for the diagnosis of ovarian torsion. METHODS: Twenty consecutive patients admitted to the gynaecological emergency room with suspected clinical diagnosis of ovarian torsion were prospectively assigned to the study. Blood samples were drawn pre-operatively and examined for serum concentrations of IL-6 and TNF-alpha. Surgeons were blinded to laboratory results prior to laparoscopy. RESULTS: The pre-operative diagnosis of ovarian torsion was confirmed during an urgent diagnostic laparoscopy in 8 (40%) patients. The surgical diagnosis among the remaining 12 patients was a large ovarian cyst not in torsion. In six out of eight (75.0%) patients with ovarian torsion serum IL-6 concentrations were elevated. None of the 12 patients without torsion had elevated serum IL-6 concentrations. This difference was statistically significant (P < 0.001). There was no significant difference in the proportion of women with elevated serum TNF-alpha concentrations, two of eight (25.0%) patients with torsion and four of 12 (33.3%) control cases. CONCLUSIONS: Elevated serum IL-6 concentrations, but not serum TNF-alpha concentrations, were significantly associated with the occurrence of ovarian torsion. In patients with vague clinical signs of ovarian torsion, serum IL-6 might help to distinguish which patients should undergo diagnostic laparoscopy.     

TNF-{alpha} regulates IL-4-induced Fc{varepsilon}RII/CD23 gene expression and soluble Fc{varepsilon}RII release by human monocytes

We examined the regulatory effects of TNF- on IL-4-induced geneexpression of the low-affinity receptor for IgE (FcRII/CD23)in human monocytes and IL-4-induced soluble FcRII (sFcRII) releasefrom monocytes. IL-4-induced FcRII expression on the surfaceof monocytes was reduced by TNF- as early as 1 day after cultureand the effect of TNF- increased with prolonged culture. Thepresent analysis was designed to examine whether or not TNF-could suppress IL-4-induced FcRII mRNA expression and enhancedIL-4-induced sFcRII release. The addition of TNF- to monocytecultures with IL-4 significantly reduced FcRII expression onthe surface of monocytes and significantly increased sFcRIIrelease from monocytes. Over time, there was an inverse relationshipbetween the disappearance of cell surface FcRII and the appearanceof sFcRII in culture supernatants. FcRII mRNA expression inmonocytes cultured with IL-4 was not affected by TNF- when examinedat 6 h after cultivation. When the cells were cultured withTNF- for more than 24 h, however, TNF- down-regulated IL-4-inducedFcRII mRNA levels. This correlated with the kinetics of down-regulationof IL-4-induced FCRII expression on the surface of monocytesby TNF-. These results suggest that TNF-dependent reductionof IL-4-;induced FcRII expression on the surface of monocytesresulted, at least in part, from the suppression of FcRII mRNAexpression and the enhancement of sFcRII release.     

Pregnancy: Cervical ripening with the cytokines interleukin 8, interleukin 1{beta} and tumour necrosis factor {alpha} in guinea-pigs

It has been suggested that the collagenolytic enzymes releasedfrom white blood cells which infiltrate the pregnant human uterinecervix at term are responsible for connective tissue changeswhich take place during the ripening process. Similarly, aninfiltration of inflammatory cells occurs in pregnant guinea-pigseither spontaneously at term or at preterm after treatment withthe antiprogestin onapristone. The objective of this study wasto evaluate the effects of the inflammatory cytokines interleukin8 (IL-8), interleukin 1 (IL-1), tumour necrosis factor (TNF-)and a combination of IL-1 and TNF- on cervical ripening in guinea-pigsduring advanced pregnancy. The cytokines were applied locally(intracervically) in a gel for 2 days and the effects were assessedon the third day by both extensibility measurements and morphologicalevaluation. IL-8 treatment on days 42 and 43 post coitum (p.c)and on days 48 and 49 p.c. (term: day 67± 3 p.c.) significantly(P < 0.05) increased cervical extensibility at both stagesof pregnancy. Although IL-1 treatment (days 42 and 43 p.c.)led to a slight increase in cervical extensibility, this effectwas not statistically significant. An electron microscope studyperformed on days 48 and 49 p.c. revealed a pronounced cervicalripening accompanied by the dissolution of collagen fibres,stromal oedema and the infiltration of polymorphonuclear leukocytesin all cytokine-treated groups. The morphological effects ofIL-8 and IL-1 were indistinguishable from those observed duringnormal cervical ripening at term. In contrast, TNF-, both aloneand in combination with IL-1, brought about a severe inflammatoryreaction with a massive infiltration of lymphocytes, marcophagesand polymorphonuclear leukocytes at the investigated dose. Weconclude that the local application of the inflammatory cytokinesIL-8, IL-1 and TNF- produces cervical ripening without inducinglabour in pregnant guinea-pigs; the morphological effects ofIL-8 and IL-1 being similar to the physiological cervical ripening.Our data support the view that cytokines, particularly IL-8,may play an important role during physiological, pathologicaland induced cervical ripening and could be clinically usefulas an adjunct to labour and delivery.     

Elevated expression of tumour necrosis factor alpha in cultured granulosa cells from women with endometriosis

Fertilization and oocyte cleavage rates have previously been demonstrated to be lower for women with endometriosis undergoing IVF compared with controls. This might be related to impaired oocyte function, possibly due to an inflammatory milieu in the pelvis of these women, where an elevated concentration of many cytokines is documented. The aim of this study was to examine whether granulosa cells from women with endometriosis deviated with respect to production of the inflammatory cytokines interleukin-1beta, interleukin-6, interleukin-8 and tumour necrosis factor alpha (TNFalpha) compared with granulosa cells from healthy women, undergoing IVF for male infertility. The effect of human chorionic gonadotrophin on cytokine production was also investigated. Granulosa cells in follicular fluid were obtained at oocyte retrieval for IVF. Incubated cell culture media were analysed by enzyme-linked immunosorbent assay. The basal production of all four cytokines was higher in cells from women with endometriosis when compared to controls, although the increase was only significant for TNFalpha. Chorionic gonadotrophin had no significant effect, although it had a tendency to suppress cytokine release in both patient categories. Whether aberrant cytokine production in granulosa cells from women with endometriosis may disturb fertilizing capacity of oocytes requires study.     

Tumor necrosis factor-{alpha} up-regulates Bcl-2 expression and decreases calcium-dependent apoptosis in human B cell lines

Group I and Epstein–Barr virus-negative Burkitt's lymphomacell lines and the B104 lymphoma cell line which expresses aphenotype of immature B cells undergo apoptosis after cross-linkingof their surface Ig receptors or after exposure to a calciumionophore. We show here that tumor necrosis factor (TNF)- protectsthese B cell lines against Ca²⁺-dependent apoptosis. Protectionwas associated with up-regulatlon of bcl-2 mRNA and proteinexpression. The increase of Bcl-2 expression induced by TNF-was inhibited by chelerythrine, a specific inhibitor of proteinkinase C (PKC), suggesting that Bcl-2 expression was dependenton PKC activation. Furthermore, we show that phorbol estersand cyclosporin A (CsA), which prevent Ca²⁺-dependent apoptosis,up-regulated Bcl-2 expression. The effect of CsA on Bcl-2 expressionis controlled by calcineurin since we have shown that FK506but not rapamycin had the same effect on Bcl-2 expression, whereasokadaic acid, an inhibitor of phosphatases 1, 2A and 2C, wasineffective. These data provide direct evidence that TNF- preventsCa²⁺-dependent apoptosis by a Bcl-2-dependent mechanism mediatedby PKC.     

Relationship between tumour necrosis factor {alpha} and sex steroid concentrations in the follicular fluid of women with immunological infertility

Experimental evidence suggests a tight relationship betweencytokines and the reproductive system. Tumour necrosis factor (TNF), a cytokine produced by activated macrophages and mesenchymalcells, seems to participate in the control of fertility. Therefore,the present study was undertaken to evaluate the concentrationsof TNF in the follicular fluid of female patients with immunologicalinfertility, as well as the possible role of this cytokine infollicular development. Concentrations of TNF, 17β-oestradiol,progesterone, androstenedione and testosterone were measuredin the follicular fluid of patients with immunological infertilityand patients with a tubal factor of infertility, who servedas a control group. Patients with immunological infertilityhad significantly higher concentrations of TNF in their follicularfluid compared to the control group. In contrast, oestradiolconcentrations were significantly lower in the former group.The intrafollicular concentrations of the other steroids measureddid not differ significantly between the two groups. The fertilizationrate of ova from follicles included in the study was significantlylower in patients with immunological infertility compared tocontrol subjects (19.1 and 57.1% respectively). In conclusion,this study shows that patients with infertility of immunologicalorigin have increased follicular fluid concentrations of TNFand lower oestradiol concentrations. We speculate that elevatedTNF concentrations in the human follicle may negatively influenceboth ovulation- and fertilization-related events.     

Reactive oxygen species stimulate prostaglandin F2 alpha production in human endometrial stromal cells in vitro

BACKGROUND: The present study was undertaken to investigatethe effect of reactive oxygen species on prostaglandin F2 (PGF2)production by human endometrial stromal cells (ESC). METHODSAND RESULTS: Isolated ESC were incubated with hydrogen peroxide,which induces lipid peroxidation. Hydrogen peroxide increasedboth intracellular and medium concentrations of PGF2 (P <0.01). A time course study showed that hydrogen peroxide significantlyincreased PGF2 concentrations in the medium after 6 h incubation(P < 0.01), after which no further increase was observed.To study whether the increase in PGF2 production caused by hydrogenperoxide was mediated by cyclooxygenase, ESC were incubatedwith indomethacin (0.5 µg/ml), an inhibitor of cyclooxygenase,in the presence of hydrogen peroxide. Indomethacin significantlyblocked the increases in PGF2 production caused by hydrogenperoxide (P < 0.01). Hydrogen peroxide also increased PGF2production by decidualized ESC (P < 0.01), induced by theincubation with medroxyprogesterone acetate (10^–6 mol/l)and oestradiol (10^–8 mol/l). CONCLUSIONS: Reactive oxygenspecies stimulate PGF2 production in ESC, suggesting that theymight influence endometrial function by regulating PGF2 production.     

Time-dependent effects of transforming growth factor {alpha} on aromatase activity in human granulosa cells

Transforming growth factor a (TGF) is implicated as a paracrinegrowth factor in the regulation of human granulosa cell function.To investigate this further, we have examined the actions ofTGF on the basal and folliclestimulating hormone (FSH)-stimulatedaromatase activity of human granulosa cells to determine howthis growth factor influences oestrogen biosynthesis in thefollicle. Granulosa cells from women having in-vitro fertilizationduring untreated or gonadotrophin-stimulated cycles were culturedfor 1–6 days in the presence or absence of FSH or TGFat a range of doses. Aromatase activity, expressed as oestradiolproduction, was determined after culture during a 3 h test period.After 2 days, TGF (1–300 ng/ml) decreased basal and FSH-stimulatedaromatase activity in a dose-dependent manner (ED50 = 3 ng/ml).In contrast, after 4 days, TGF enhanced both basal and FSH-stimulatedaromatase activity. Repeated experiments revealed a consistentpattern of inhibition on day 2, which was more marked in thepresence of FSH (reduction by 30.6 ± 9.1%, mean ±SEM; n = 14; P < 0.01), and stimulation on day 4 in boththe absence (increased by 61.4 ± 20.6%, mean ±SEM; n = 6; P < 0.05) and presence of FSH (increased by 36.0± 15.2%, mean ± SEM; n = 8; P < 0.05). Theresults provide further evidence that TGF is a paracrine factorin the control of oestrogen biosynthesis, but the actions canbe either inhibitory or stimulatory depending on the durationof exposure.     

The level of tumor necrosis factor-{alpha} producing cells in the spinal cord correlates with the degree of Theiler's murine encephalomyelitis virus-induced demyelinating disease

The levels of tumor necrosis factor (TNF)- producing cells wereanalyzed in mice with Theller's murine encephalomyelitis virus-induceddemyelinating disease (TMEV-IDD). Using an ELISPOT assay, wedemonstrate an increase in TNF- producing cells in the spinalcords of TMEV-infected SJL/J mice, especially at an active diseasestage. The numbers of TNF- producing cells were extremely highin susceptible SJL/J mice compared with the numbers in resistantBALB/c and C57BL/6 mice. TNF- producing cells were also immunohistochemicallyidentified in active lesions of TMEV-IDD at acute as well aschronic stages. The percentage of TNF- producing cells comparedwith the total number of cells isolated from spinal cords washigher in TMEV-infected SJL/J mice than resistant BALB/c andC57BL/6 mice. Correspondingly, the level of TNF- was much higherin the culture supernatants of both infiltrating cells in thespinal cords and spleen cells from clinically affected animalsthan that from similarly treated resistant mice. Treatment ofvirus-infected mice with a mAb specific for TNF- at the beginningof the onset of disease suppressed the development of the demyelinatingdisease. These findings suggest that TNF- may play an importantrole in the pathogenicity of TMEV-IDD.     

Endocrinology: Prostaglandin F2{alpha} stimulates release of insulin-like growth factor binding protein-3 from cultured human granulosa-luteal cells

Human ovarian follicular fluid contains a number of insulin-likegrowth factor binding proteins (IGFBP) of which IGFBP-3 is themost abundant. IGFBP-3 synthesis is growth hormone-regulated.We studied the effect of prostaglandin F₂ (PGF₂) on IGFBP-3secretion by cultured human granulosa-luteal cells from follicularaspirates of women participating in an in-vitro fertilizationprogramme. The IGFBP-3 concentration was measured using a specificmonoclonal immunofluorimetric assay. Contrary to a previousreport on unstimulated follicles, this study demonstrated apositive correlation between follicular fluid IGFBP-3 concentrationand follicular size. PGF2a was found to stimulate in a dose-dependentfashion the secretion of IGFBP-3. Significant (p < 0.05)effects were found at PGF₂ concentrations of 10^–8, 10^–7and 10^–6 M. Because IGFBP-3 inhibits progesterone productionstimulated by insulin-like growth factor (IGF)-I, the PGF₂-inducedstimulation of IGFBP-3 production may be one of the mechanismswhereby PGF₂ exerts its luteolytic effect via the IGF system     

Uterus and endometrium: Indomethacin reduces contraction of isolated non-pregnant human uterine artery induced by prostaglandin F2{alpha}

The purpose of this study was to explore whether cyclo oxygenaseproducts derived from endothelium or vascular smooth muscleparticipate In the response of human uterine artery to prostaglandinF² Experiments were performed using human uterine arterial rings.Prostaglandin F² (0.4 nM-1 µM) induced contraction ofhuman uterine arteries with both Intact and denuded endothellumwith similar potency and efficacy (pD² values: 7.93±0.01and 8.07±0.03 for vessels with and without endotheliuinrespectively; maximal response values: 89.1±4.7% and92.3±3.8% for vessels with and without endotheliuin respectively).Indomethacin (10 µM) significantly sup pressed the maximumeffects of prostaglandin F² and induced a shift towards theright of the prostaglandin F² concentration-response curves,regardless of the endo thelial condition. On the other hand,in both types of preparations, OKY-046 (10 µM), an inhibitorof throm boxane synthesis, did not affect prostaglandin F²-inducedcontraction of human uterine arteries. It is concluded thatin human uterine artery prostaglandin F²-induced contractionis mediated, at least in part, through constrictor prostanoid(s)of vascular smooth muscle origin that is not thromboxane A₂.