Predominant expression of invariant V{alpha}14+ TCR {alpha} chain in NK1.1+ T cell populations

A novel T cell subset characterized by cell surface NK1.1⁺ TCRß⁺expression was investigated for its TCR usage, particularlythat of invariant V14 TCR, which was found to be preferentiallyused in peripheral CD4^–CD8^–T cells developed atextrathymic sites. We found that NK₊ ß T cell subsetsaccount for 0.4% in thymocytes, 5% in the splenic T cells and40.5% in the bone marrow T cells. Among these NK⁺ ßT cells, two distinct subsets were detected; cell surface TCRV14⁺and V14^– subpopulations. Almost all of NK⁺ ßthymocytes express V14 mRNA; however, only<20% were positive,while >80% were negative or undetectable for V14 TCR expressionon the cell surface in the thymus. Similarly,50% of NK⁺ ßT cells in spleen and bone marrow are V14⁺; as revealed by FACS.TCR repertoire analysis by nucleotide sequences on inverse PCRproducts demonstrated that most NK⁺ ß T cells expressan invariant TCR encoded by the V14J281 gene with a 1 base N-regionin all tissues. Thus, invariant V14 TCR is uniquely expressedon NK T cells, and can be a marker to distinguish NK, NK T andT cells.     

CD38 expression on mouse T cells: CD38 defines functionally distinct subsets of {alpha}{beta} TCR+CD4 CD8 thymocytes

We have examined CD38 expression on mouse lymphocytes usingthe rat mAb NIM-R5 and demonstrate that CD38 expression is restrictedto {small tilde}8% of thymocytes. Although CD38 is absent fromthe majority of CD4+ CD8^– and CD4^–CD8⁺ T cells,we detected a strong correlation between CD36 expression andß⁺CD4^–CD8^– T cells in the thymus, withnearly 80% of ß TCR⁺CD4^–CD8^– thymocytesbeing CD38⁺. Using heat stable antigen (HSA) and CD38, we dividedß⁺CD4⁺CD8^– thymocytes into four subsets: HSA⁺CD38^–,HSA^– CD38^hi, HSA^–CD3810^low and HSA^– CD38^–.Two established characteristics of ß TCR⁺CD4CD8^–cells, bias towards Vß 8.2 TCR expression and highlevels of IL-4 production, were used to establish a possiblerelationship between the above thymocyte subsets. Our presentdata show that the HSA⁺CD38^– subset is not biased towardsVß8.2 TCR expression whereas the HSA^– CD38^–subset does show this bias (–47%). Neither of these subsetsmake IL-4 upon CD3 mediated stimulation. In contrast, the CD38⁺subsets are heavily biased toward Vß8.2 expressionand produce large amounts of IL-4 upon stimulation, particularlythe CD38^low cells. Taken together, these data suggest that thesefour subsets represent various stages of a possible differentiationpathway for ß TCR⁺ CD4^–CD8^– cells, withthe HSA⁺CD38^– subset being the most Immature while theHSA^–CD38^low subset is the most functionally mature. Thesecharacteristics support the view that ap TCR⁺CD4^–CD8^–T cells represent an independent lineage with a distinct, butas yet obscure, role in immunity     

Brucella abortus induces a novel cytokine gene expression pattern characterized by elevated IL-10 and IFN-{gamma} in CD4+ T cells

Immunization of BALB/c mice with killed Brucella abortus (BA)has previously been shown to Increase serum lgG2a levels andlong-term T cell clones from these mice secrete T_h1-associatedcytokines: IFN- and IL-2 but not IL-4 or IL-5. We analyzed cytokinegene expression following primary immunization with BA to determinewhen CD4⁺ T cells first express cytokine genes and whether specifichypothesized cytokine patterns (e.g. T_h precursor, T_h0) couldbe identified prior to a T_h1-like pattern. Our results demonstrateda highly consistent and novel pattern of T_h 1/T_h2 cytokine geneexpression characterized by elevated IL-10 and IFN- in CD4⁺T cells which rapidly manifests itself and is sustained forat least 10 days after immunization. No elevation in IL-2 cytokinegene expression was observed and treatment of BA-immunlzed micewith blocking anti- IL-2 antibodies had no effect on the cytokinegene expression pattern, although treatment with anti-IFN antibodiesresulted in increased IL-4, IL-5, and IL-9 cytoklne gene expression,In the absence of any change In IFN- or IL-10 as early as 4days after immunization. These results suggest that a wholepathogen may trigger sufficient costimulatory signals to rapidlyinduce effector T cells in the absence of elevated IL-2 andthat IL-10 Is specifically elevated in certain T_h1-like responses.     

Proliferation and apoptosis of B220+ CD4 CD8 TCR{alpha}{beta}intermediate T cells in the liver of normal adult mice: implication for Ipr pathogenesis

Small numbers of T cells have been isolated from the normalmouse liver and many of these are of the CD4^–CD8^–TCRß⁺phenotype. Larger numbers of such cells are present in the liversof mice homozygous for the Ipr mutation and the liver has beenproposed to be the site of an extrathymlc T cell developmentpathway that is expanded in Ipr/lpr mice. Using a modified separationprocedure that increases the liver T cell yield, we have beenable to characterize a subset of CD4^–CD8^–TCRß^intermediateT cells that express the B220 epltope of the CD45 molecule,and resemble in this and many other ways the accumulating Tcells in Ipr lymph nodes. These cells are an actively dividingpopulation and even in healthy, unmanipulated mice a large proportionof them are undergoing apoptosis. We propose the model thatthe normal liver is a major site for T cell destruction andthat the Ipr defect results in failure of this process withleakage of B220⁺CD4^–CD8^–TCRß⁺ cells fromthe liver to peripheral lymphoid tissues, particularly lymphnodes.     

Migration pathways of CD4 T cell subsets in vivo: the CD45RC- subset enters the thymus via {alpha}4 integrin-VCAM-1 interaction

The present investigation examines the localization and migrationof purified T cell subsets in comparison with B cells, CD8 Tcells and CD4⁺CD8^– single-positive thymocytes. CD4 T cellsubsets in the rat are defined by mAb MRC OX22 ( anti-CD45RC),which distinguishes resting CD4 T cells (CD45RC⁺) from those(CD45RC^–) which have encountered antigen in the recentpast– subpopulations often referred to as ‘naive’and ‘memory’. Purified, ⁵¹Cr-labelled CD45RC⁺ CD4T cells broadly reflected the migration pattern of CD8 T cellsand B cells. Early localization to the spleen was followed bya redistribution to mesenteric lymph nodes (MLN) and cervicallymph nodes ( CLN) , B cells migrating at a slightly slowertempo. There was almost no localization of these subpopulationsto the small or large intestine [Peyer's patches (PP) excluded].In contrast, CD45RC^– CD4 T cells (indistinguishable insize from the CD45RC⁺ subset) localized in large numbers tothe intestine; they were present here at the earliest time point(0.5 h) , persisted for at least 48 h but did not accumulate,indicating a rapid exit. Numerically, localization of CD45RC^–CD4 T cells in the MLN could be accounted for entirely by afferentdrainage from the intestine. Unexpectedly, CD45RC^– CD4T cells (but not other subsets) localized and accumulated inthe thymus. In vivo treatment with mAb HP2/1 against the integrin₄ subunit inhibited almost entirely CD45RCT^– CD4 T cellmigration into the PP (98.1%), intestine (87.1%) , MLN (89.1%)and thymus (93.5%) migration into the CLN was only reduced byhalf. To distinguish between recognition of MAdCAM-1 and VCAM-1by ^4–containing integrins, recipients were treated withmAb 5F10 against rat VCAM-1. Except for the thymus and a smallreduction in CLN, localization of CD45RC^– CD4 T cellswas unaffected; entry to the thymus was almost completely blocked(92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC^–CD4 T cells alone showed enhanced localization to the gut andPP, probably via ₄ß₇-MAdCAM-1 interaction; ( II) thatmany CD45RC^– cells entered nonmucosal LN independentlyof ₄ integrin or VCAM-1; and (III) that entry of mature recirculatlngCD45RC^– CD4 T cells into the thymus across thymic endothellumwas apparently regulated by ₄ integrln-VCAM-1 interaction.     

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.     

Intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 are involved in protection mediated by CD3+TCR{alpha}{beta} T cells at the early stage after infection with Listeria monocytogenes in rats

To Investigate the significance of Intercellular adhesion molecule-1(ICAM-1) and leukocyte function-associated antlgen-1 (LFA-1)In host defense against infection with Intracellular parasites,we examined the effects of In vivo pretreatment with mAbs toICAM-1 (1A29) and LFA-1 (WT-1) on the protection against Infectionwith Listeria monocytogenesIn Fisher F344/N rats. Expressionof ICAM-1 and LFA-1 molecules on T cells In spleen, liver andperitoneal cavity of rats was down-regulated after i.p. administrationwith daily doses of 300 µg of either 1A29 or WT-1 for10 days. The survival rate of rats inoculated with viable Listeriawas significantly reduced byIn vivo pretreatment with 1A29 togetherwith WT-1 for 10 days but not by In vivo pretreatment with controlmAb. The numbers of bacteria In the spleen In rats pretreatedwith both 1A29 and WT-1 were significantly increased on day3 and day 6 after Infection with 1 x 10⁷ of viable Listeriacorresponding to 1/30 of LD₅₀ to normal rats. Thus, the resistanceagainst llsterial Infection was severely Impaired by combinationalpretreatment with mAbs In ICAM-1 and LFA-1. As shown In ourprevious report, the early appearance of CD3⁺TCRß^–T cells, presumably TCR cells, was evident In the peritonealcavity and liver of control rats at the early stage after llsterialInfection, while this was suppressed at this stage in rats pretreatedwith both 1A29 and WT1. These results suggest that the ICAM-1and LFA-1 adhesion pathway may be critically involved in protectlveroles of CD3⁺TCR_ß– T cells at the early stageof rat listeriosis.     

Ligand-induced autoregulation of IL-2 receptor {alpha} chain expression in murine T cell lines

The IL-2 receptor (IL-2R) is composed of three chains a, ßand . In mice, contrary to the human system, we have previouslydemonstrated that the IL-2Rß complex does not bindIL-2. Therefore, mouse IL-2 response is completely dependenton the expression of the IL-2R gene product. T cell clones expressingmouse IL-2Rß and the human IL-2R transgene have beenstudied. When cells are grown in IL-4, mouse IL-2R is not expressed.However, exposure to IL-2 leads to the expression of the endogenousmurine IL-2R subunit. The T cell line expressing mouse IL-2Rand human IL-2Rß can grow in IL-2 but does not expressendogenous murine IL-2 R. Transfection of these cells with thehuman IL-2R gene restores the capacity to induce murine IL-2R.This result demonstrates that IL-2-IL-2R interactions are requiredfor induction of IL-2R. The kinetics of induction and deinductionof murine IL-2R have been studied using clone 18.III. From negativecells, expression of murine IL-2R is a very slow phenomenon.From cells fully expressing IL-2R, deinduction is a two-stepprocess: after a rapid decrease of IL-2R the cells continueto express, for a long period of time, basal levels of murineIL-2R. When cells expressing basal levels of IL-2R are exposedto IL-2, induction of IL-2R is a very rapid phenomenon. Theautoregulatory loop formed by IL-2-IL-2R therefore displaysdifferent levels of functioning.     

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V{alpha}1 gene

Previous studies of the TCR chain gene have located promoterelements 5' to the start of the various V genes. The only fullycharacterized enhancer for the entire chain gene (V, J andC genes) has been located {small tilde}3 kb from the 3' endof C. We now report the existence of additional regulatory elementslocated in the introns of several murine V genes (V1, V3 andVB6.2.16). In the case of V1, this element appears to be a promoterwith bidirectional activity that is not T cell specific. Interestingly,upstream of the promoter in the antisense strand, an open readingframe has been found that codes for a small molecular weightprotein ({small tilde}60 amino acids) that contains a prollne-richregion and a tyrosine-isoleucine motif that has homology toIgß (the B29 gene product). A rabbit antiserum madeagainst this sequence has confirmed its existence by Westernblot and immunoprecipitation. Thus this V1 intronic promoterhas the potential not only to induce the formation of a truncatedV1 gene product, but also regulates the expression of a smallmolecular weight protein that may be involved in lymphocyteantigen receptor signaling. The activity of this promoter isregulated by changes in intracellular calcium. In the presenceof ionomycin the promoter is down-regulated in the sense directionand its activity is enhanced in the antisense direction. Thisresult suggests that this promoter can act differentially toproduce two very different gene products. The bidirectionalV1 promoter appears to be the first in the Ig superfamily toinduce potentially functional proteins in both directions.     

Th2 bias of CD4+ NKT cells derived from multiple sclerosis in remission

Although CD1d-restricted NKT cells have been implicated as a participant in the regulatory mechanism of autoimmune diseases, it remains unclear how they would regulate human autoimmune diseases such as multiple sclerosis (MS). Furthermore, although the NKT cells comprise CD4(+) and CD4(-) populations, prior studies have often represented them as simply a CD4(-) population. Given that CD4(+) and CD4(-) NKT cells may represent functionally distinct populations, it appears crucial to examine the individual NKT subset in autoimmune diseases. Here we studied the frequency and cytokine phenotypes of the CD4(+) and CD4(-) NKT cells in fresh peripheral blood mononuclear cells, and of alpha-galactosylceramide-stimulated short-term cell lines obtained during the remission or relapse phase of MS as compared with from healthy subjects (HS). Here we report that CD4(+) NKT line cells expanded from MS in remission (MS-rem) would produce a larger amount of IL-4 than those from HS or from MS in relapse (MS-rel). They were significantly biased for T(h)2 as judged by the IL-4/IFN-gamma balance. However, there was no functional bias toward T(h)1 or T(h)2 in CD4(-) NKT line cells from MS-rem due to the defects in both IFN-gamma and IL-4 production, compared with HS. Of note, although double-negative NKT cells in the periphery were greatly reduced, the reduction of CD4(+) NKT cells was only marginal, if any, in MS-rem compared with HS. The T(h)2 bias of CD4(+) NKT line cells from MS-rem may support an immunoregulatory role for the CD4(+) NKT cells in vivo.     

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.     

Induction of human TCR gamma delta + and TCR gamma delta-CD2+CD3- double negative lymphocytes by bacterial stimulation

When human blood mononuclear cells (MNC) were incubated with heat-killed bacteria, proliferation of MNC was observed 5 days after stimulation, showing a peak on day 7. Interestingly, the bioassay of the culture supernatant and Northern blot analysis of mRNA demonstrated that no IL-2 production was associated with these proliferative responses. The induced lymphoblasts consisted predominantly of TCR gamma delta + (22.4 +/- 9.3%) and TCR gamma delta-CD2+CD3-(33.2 +/- 11.8%) double negative lymphocytes (n = 10), which were initially minor populations (less than 10%) in freshly isolated MNC. The prominent induction of TCR gamma delta + cells was confirmed by Northern blot analysis. TCR gamma delta + cells induced by bacterial stimulation seemed to generate from lymphocytes lacking the apparent expression of gamma delta TCR. The inducing capability for double negative cells is present in a large number of species of bacteria, especially Gram-positive bacteria. Gel filtration analysis of ultrasonicated filtrates of Staphylococcus aureus and Streptococcus pyogenes revealed that a substance with an Mr of 25-26 kd could be substituted for whole bacterial particles in the cell proliferative responses. In contrast to the purified protein derivative (PPD)-induced response, the response described here was inducible in the cord blood of neonates who had not yet been exposed to the corresponding bacterial infection. The physicochemical properties of the sonicated filtrates were different from those of PPD. These results suggested that the present phenomenon may be nonspecific, polyclonal (or oligoclonal) activation of TCR gamma delta + and TCR gamma delta -CD2+CD3- cells by bacterial stimulation.     

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.     

Skewed T cell receptor V{alpha} repertoire among superantigen reactive murine T cells

Reactivity of murine T cells with viral or bacterial superantigensis clearly correlated with the expression of TCR V_ßdomains. Thus, T cells responding to the minor lymphocyte stimulatorylocus (Mls-1^a) or staphylococcal enterotoxin B (SEB) expresspredominantly TCR V_ß6 or V_ß8.2 respectively.We have investigated the involvement of the other major variableelement of the TCR, the V domain, in these superantigen responses.Using a panel of anti-TCR V mAbs, It is demonstrated that theTCR V repertoire among superantigen stimulated V_ß6⁺or V_ß8.2⁺ blasts (responding to Mls-1^a or SEB respectivelyin vitro) is altered in comparison with anti-CD3 stimulatedcells expressing the same V domains. Furthermore, the TCR Vrepertoire is strongly skewed in TCR V_ß8.2 transgenicmice that have undergone extensive peripheral clonal deletionafter SEB injection. These data imply that the V domain influencessuperantigen recognition by sthe TCR.     

Dendritic cells and macrophages are required for Th1 development of CD4+ T cells from {alpha}{beta} TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-{gamma} production is IFN-{gamma}-dependent

We have examined the antigen presenting cell (APC) requirementsfor primary T cell activation and T helper (Th) cell phenotypedifferentiation using naive CD4⁺ T cells from ß TCRtransgenic mice. Purified dendritic cells were the principalcell required for induction of primary ovalbumln peptide specificT cell activation and clonal expansion. However, dendritic cellsdid not induce differentiation of T cells toward Th1 or Th2phenotype. Addition of IL-4 during primary dendritic cell stimulationsof T cells resulted in the development of a Th2 phenotype whichproduced high levels of IL-4 during secondary and tertiary stimulation.In contrast, development of Th1 cells producing high levelsof IFN- could not be induced with dendritic cells alone butrequired the addition of appropriately activated macrophages.Addition of splenic or peritoneal B cells did not induce Th1development. Activated splenic macrophages induced Th1 developmentvia a non-MHC restricted mechanism. Thus, requirements for inductionof proliferation of naive CD4⁺ T cells are distinct from thosedirecting Th1 phenotype development. IL-12 could replace therequirement for macrophages to induce Th1 development when Tcells were activated with dendritic cells. Furthermore, thisIL-12 mediated development of Th1 cells producing high levelsof IFN- was dependent on IFN-.     

Immunology: CD56+ lymphoid cells in human first trimester pregnancy decidua as a source of novel transforming growth factor-{beta}2-related immunosuppressive factors

The lymphomyeloid cells isolated from normal first trimesterpregnancy decidua may be separated into a CD56⁺ population ofnatural killer (NK)-lineage cells with the morphology of granulatedlymphocytes, and a CD56^– population which includes othercell types. Unlike CD56+ NK cells in peripheral blood, decidualCD56⁺ cells lack type III Fc receptors (CD16) and did not expresssignificant levels of either type I FcR (CD64) or type II FcR(CDw32). By contrast to the decidual CD56^– cells, CD56⁺cells could release biologically active transforming growthfactor (TGF)-in vitro, detectable using an normal rat kidneyfibroblast colony-forming assay. The CD56⁺ cells could be stainedusing an antibody specific for TGF-2, and similarly stainingcells could be detected in intact biopsies of normal pregnancydecidua. Bioactive TGF- is known to suppress the generationof cytotoxic cells in vitro, and high performance liquid chromatographyfractionation of supernatants conditioned by CD56⁺ but not CD56–cells contained reproducible peaks of immunosuppressive activityat 40–45 and 15–20 kDa, similar to the TGF-2 immunosuppressiveactivity in supernatants conditioned by unfractionated decidua.     

Stimulation of prostaglandin (PG) F2{alpha} and PGE2 release by tumour necrosis factor-{alpha} and interleukin-1{alpha} in cultured human luteal phase endometrial cells

Tumour necrosis factor- (TNF-) and interleukin-1 (IL-1) areimportant mediators of cell signalling in the uterus. Prostaglandins(PG) have been implicated in the increase of endometrial vascularpermeability which occurs during the implantation process. Thisstudy evaluates the effect of these two pleiotropic cytokineson PGF₂ and PGE₂ release from human luteal phase endometrialglandular epithelial cells (GEC) and stromal cells (STC) inculture. Basal PGF and PGE release did not differ significantlyfrom each other or among cell types, and declined significantlywith increasing number of days in culture. On day 3, basal PGrelease had decreased to half of that on day 1 of culture. However,both cell types were still able to respond to the addition ofexogenous arachidonic acid (5 µM) on day 3 of culture,with PG release by GEC being elevated 7- to 10-fold and by STCmoderately, but still significantly, on day 4. The permissiveeffect of arachidonic acid on the stimulation of PG releasemay indicate the down-regulation of phospholipase A₂ with continuedtime in culture. However, the addition of arachidonic acid (5µM) on day 0 of culture, while able to cause significantlyincreased PG release from GEC, had no effect on STC. In contrast,the addition of a combination of arachidonic acid (5 µM),and either recombinant human TNF- (10 µg rhTNF-/I) or10 µg rhlL-1/I, had a synergistic action and caused thesignificantly increased release of PGF and PGE from both celltypes, compared with that achieved with either arachidonic acidor the cytokine alone (although GEC responded more than STC).During the first 24 h after the addition of rhTNF- or rhlL-1,both cytokines stimulated PG release from both cell types ina dose- and time-dependent fashion. Neither cycloheximide (10µM) nor actinomycin D (10 µM) affected basal PGrelease, but both blocked cytokine-induced PG release from bothcell types. These results suggest that there is a differentialcontrol of human endometrial cell PG biosynthesis, and thatPG release may be regulated through gene activation.     

Pregnancy: Effects of hyperosmolar glucose, prostaglandin-F2{alpha} and 15-methyl-prostaglandin-F2{alpha} on human placental cells in vitro

The objective of this study was to assess the ability of certaindrugs, used for local injection therapy of ectopic pregnancy,to suppress the activities of cultured human placental cells.Placental cells from legal first trimester abortions were preparedby collagenase treatment and density gradient centrifugation.The cells were exposed to hyperosmolar glucose (500 mg/ml),15-methyl-prostaglandin-F₂ (15-m-PGF₂; 10–7 to 10–3mol/l) and prostaglandin-F₂ (PGF₂; 10–5 to 5X10–3mol/l) for 30 min on days 2–4 after seeding. The effectson the secretion of human chorionic gonadotrophin (HCG) andprogesterone, as well as on the protein content per culturewell, were measured. Hyperosmolar glucose was the most effectivedrug and caused a marked decrease of the protein content inthe culture wells and a reduction of progesterone secretion.Of the two prostaglandins, only 15-m-PGF₂ affected the viabilityof the cells and reduced the protein content of the wells. Theclinical effectiveness of the two groups of drugs seems to besimilar but certain in-vitro effects are different. Thus invivo they may act on different target tissues. Against thisbackground, the combination of hyperosmolar glucose and prostaglandinsmight be an interesting approach for local injection therapyfor tubal pregnancy.     

IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T cells

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and T(h)1 response. IL-18 has a structural homology to IL-1, particularly IL-1beta. IL-18R, composed of IL-1R-related protein (IL-18Ralpha) and IL-1R accessory protein-like (IL-18Rbeta), belongs to the IL-1R family. Furthermore, IL-18R at least partly shares the signal transducing system with IL-1R. Thus, the IL-18-IL-18R system has a striking similarity to the IL-1-IL-1R system. For this reason, we regarded it important to investigate whether, like IL-18, IL-1beta synergizes with IL-12 in inducing IFN-gamma production from human T cells and plays an important role in the T(h)1 response. Here we show that IL-12 and IL-1beta synergistically induce T cells to proliferate and produce IFN-gamma without their TCR engagement. IL-12 stimulation induced an increase in the proportion of T cells positive for IL-18R. Then, IL-12-stimulated T cells responded to IL-18 or IL-1beta by their proliferation and IFN-gamma production, although levels of IL-1beta-induced responses were lower. CD4(+)CD45RA(+) T cells, although they constitutively expressed IL-18Rbeta mRNA, did not express IL-18Ralpha mRNA. Phytohemagglutinin (PHA) stimulation alone induced IL-18Ralpha mRNA without affecting the expression of IL-18Rbeta mRNA. T(h)1-inducing conditions (PHA, IL-12 and anti-IL-4) further increased this expression. We also show that T(h)1 cells but not T(h)2 cells have increased expression of IL-18R and IL-1R, and produce IFN-gamma in response to IL-18 and/or IL-1beta.