Emigration of selected subsets of {gamma}{delta}+ T cells from the adult murine thymus

Cells bearing the form of the TCR make up only 1–3% ofT cells in the adult murine thymus and peripheral lymphold organs.Evidence from studies of nude mice suggests that the developmentof at least some T cells is thymus dependent; however, untilnow it has not been directly demonstrated that cells are exportedfrom the thymus. In this paper we have used the technique oflabelling thymocytes in vivo with FITC, followed by flow cytometrlcanalysis to trace cells emigrating from the thymus to the spleen.Using this approach we have been able to demonstrate for thefirst time that T cells are exported from the adult murinethymus to the spleen. We also demonstrate that the cells emigratingto the spleen are a selected subset of thymocytes being heatstable antigen positive, Thy-1⁺, and expressing low levels ofCD44 (Pgp-1). In addition, investigation of TCR V; gene usageamong adult ⁺ thymocytes, recent emigrants, and spleen cells,indicated a selective emigration of cells expressing certainVgenes.     

Phenotypic and functional properties of murine {gamma}{delta} T cell clones derived from malaria immunized, {alpha}{beta} T cell-deficient mice

Six murine T cell clones expressing TCR were generated frommalaria immunized, ß T celldeficient mice. Phenotypiccharacterization of these clones has revealed that, in contrastto conventional ß T cells, there is a considerabledegree of heterogeneity among these clones with regard to theirsurface markers and their lymphokine profile. One clone wasfound to display significant anti-parasite activity in vivoupon adoptive transfer. We attempted to determine whether theprotective clone differs in one or more key characteristicsfrom the non-protective clones. Although no obvious patternpeculiar to the protective clone was observed, it appears thatmore than one parameter may, in combination, define a distinctprotective phenotype, and thus explain the functional differencebetween the protective and non-protective clones.     

The development of dermatitis infiltrated by {gamma}{delta} T cells in IL-7 transgenic mice

Transgenic mice constitutively expressing IL-7 developed severedermatitis with erythroderma and alopecia. The skin lesionswere characterized by massive infiltration of mononuclear cells.Immunofluorescence staining showed that most of the infiltratingcells were T cells with the majority bearing the TCR otherthan the V5 moiety. Furthermore, the number of T cells hadincreased in the lymphold organs of the dermatitis animals.These findings idicate the strong relationship between the expressionof IL-7 and the development of T cells in vivo and the pathologicalinvolvement of proliferated and/or activated T cells in skindisease.     

Thymic stroma is required for the development of human T cell lineages in vitro

Development of the T cell lineage is characterized by the homingof hematopoietic precursors to thymus, followed by their acquisitionof receptors for antigen. T cell receptors are ß or heterodimers associated with CD3 (TCR-CD3). Very early T cellprecursors in humans have been characterized as CD7+45+ cellswhich lack the T cell differentiation antigens CD1, CD2, CD3,CD4, and CD8. A phenotypically equivalent early thymocyte populationalso occurs in postnatal life, and we have previously shownthat interleukin 2 (IL2) promotes the development in vitro ofboth the ß and the T cells from these early thymocytes.Here we have analyzed the requirements of the induction of theIL2 pathway in early thymocytes, and their developmental potential.We show that: (I) thymic stromal cells, which are present inthymocyte suspensions, are necessary to induce the IL2 pathwayand the development of ß or T cell lineages fromearly thymocytes in vitro; and (II) when removed from the invivo environment, early thymocytes can develop in vitro intoTCR-CD3^– cells of the natural killer (NK) lineage. Weconclude that CD7+45+, CD1–2–3–4–8–early thymocytes are multipotential progenitors that, at least,have the capacity to develop into ß or T cell andNK lineages. The analysis of the mechanisms of generation andselection of human T and NK cell diversity, not feasible inbone marrow cultures, is now possible.     

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.     

Soluble human high-affinity receptor for IgE abrogates the IgE-mediated allergic reaction

The high-affinity receptor for IgE (FcRI) has a tetrameric structurecomposed of one, one ß, and two disulfide-linked subunits, of which the subunit binds IgE with high affinity.A recombinant soluble form of the ectodomain of the human FcRIsubunit (rsFcRI) was recently generated by gene engineeringand was verified to bind IgE with an affinity as high as thatof native FcRI on the cell surface. rsFcRI was prepared on alarge scale in order to analyze its biological function. rsFcRIcompletely inhibited IgE binding to the cell surface, resultingin abrogation of the chemical mediator release from RBL-2H3cells. Furthermore it completely abolished the passive cutaneousanaphylaxis (PCA) response by trapping IgE specifically whenitwas administered into rats prior to IgE sensltizatlon. Evenafter IgE sensitizatlon, treatment of rsFcRI substantially reducedthe PCA response. It was finally shown that rsFcRI inhibitedIgE binding to human peripheral blood basophils and the histaminerelease from them. In this paper we address the ability of rsFcRIto specifically prevent the IgE-mediated allergic reaction.     

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc{gamma}R) encoded by an alternatively spliced transcript of the Fc{gamma}RII gene

Low affinity FcR are a heterogeneous group of glycoproteinswhich exist in transmembrane (TM) as well as in soluble forms.Two membrane isoforms of the murine type II FcR, FcRilb1 andFc;Rilb2, have been described. They result from the translationof alternatively spliced premRNA, FcRilb2 lacking sequencesof the first intracytoplasmic domain (IC1). Soluble forms ofFcR (sFcR) have previously been shown to result from proteolysisof membrane receptors. We report here the identification, inmacrophages, of a mRNA derived from the FCRll gene by splicingexons encoding the TM and IC1 domains, i.e. corresponding toa TM-deleted FcRllb2 mRNA. A soluble protein possibly encodedby this mRNA was identified in macrophage supernatants. In accordancewith FcR nomenclature, we propose to name this new FcRll IsoformFcRllb3. It is the most abundant 8FcR present in serum, as comparedwith 8FcR resulting from cleavage of membrane FcR.     

Homing and in situ differentiation of resident pulmonary lymphocytes

At birth, T lymphocytes which colonize the lung are mainly ofthe subset, while ß T cells predominate in the spleen.Thus, the lung is a preferred site for the homing of T cellsin the perinatal period. However, after birth, the pattern ofV gene usage among resident pulmonary lymphocytes (RPL) changeswith age, from a predominance of V6 at birth to a predominanceof V4 in older mice. The generation of the V6 fraction appearsto be thymus dependent, since in athymic nude mice, the V6 populationpresent at birth is replaced by V4 T cells. In the postnatalperiod, both RAG-1 and RAG-2 genes are expressed at high levelsin the RPL population. TCR bearing cells are among those thatexpress RAG genes, indicating that maturation of T cells takesplace in this organ. In addition, transfer experiments revealthat lymphoid precursors are present in the lung. The stageof differentiation of these precursors will be characterizedin future studies. The data presented here indicate that pulmonaryT lymphocytes are derived from both migrants of thymic originand from precursors which have undergone differentiation andselection in the lung. The population that is generated in situand that has not been selected in the thymus may include cellsthat are typical for the pulmonary environment.     

Ovary: Immunolocalization of transforming growth factor-{beta}1 and transforming growth factor-{beta}2 in the mouse ovary during gonadotrophin-induced follicular maturation

An immunohistochemical approach was utilized to evaluate thecellular distribution of transforming growth factor-₁ (TGF₁)and transforming growth factor ₂ (TGF₂) at different stagesof follicle development in the prepubertal mouse ovary underthe following conditions: (i) after pregnant mare's serum gonadotrophin(PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin(HCG) treatment; (iii) after PMSG and HCG treatment plus mating.In the immature ovary, TGFF₁ and TGF₂ immunoreactivities arelocalized in theca and granulosa cells and in oocytes. AfterPMSG treatment, TGF₁ and TGF₂ immunoreactivities are localizedin granulosa cells; in addition, TGF₂ staining is noted in thematrix surrounding antral cells. Staining for both TGF₁ andTGF₂ drops in the theca but persists in the oocyte. PMSG plusHCG treatment results in a significant increase in TGF₁ andTGF₂ immunoreactivity in the theca and in the maintenance ofTGF₁ staining in both basal granulosa cells and cumulus cellswhereas TGF₂ immunoreactivity is essentially localized in thematrix surrounding cumulus cells. Staining for TGF₁ and TGF₂persists in the oocyte. Following PMSG plus HCG treatment andmating, TGF₁ immunoreactivity is localized in the luteal cellsof corpora lutea and TGF₂ shows a similar localization pattern.This study provides evidence that TGF₁ and TGF₂ peptides areexpressed in specific cell types during induced follicular maturationin the mouse ovary.     

Sharing of the IL-2 receptor {gamma} chain with the functional IL-9 receptor complex

The third subunit, the so-called common (_c) chain, of the IL-2receptor is shared among the receptors for IL-2, IL-4, IL-7and IL-15, and dysfunction of the _c chain is thought to causeX-linked severe combined immunodeficiency (XSCID) ascribed toimpairment of early T cell development. However, cytokines linkedto XSCID are as yet unidentified. A mAb specific for the _c chain,TUGm2, profoundly inhibited cell proliferation in response toIL-9. Another mAb, TUGm3, immunoprecipltated [¹²⁵I]IL-9 cross-linkedwith either the IL-9 receptor or the _c chain. These resultsdemonstrate that the _c chain is included in the functional receptorcomplex for IL-9, which was initially characterized as a T cellgrowth factor and is essential for IL-9-dependent growth signaltransductlon.     

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.     

Different cytoplasmic structure of the CD3{zeta} family dimer modulates the activation signal and function of T cells

The TCR complex transduces the antigen recognition signal throughcommon activation motifs present in both CD3 chains and , dimerswithin the complex. We have investigated functional roles ofthe cytoplasmic domain in and CD3 for T cell activation inearly and late responses by comparing the signaling capabilityof the TCR complexes containing mutant lacking some or allmotifs, or chain, another family molecule. The results withthe mutant , lacking all motifs indicated that CD3 can transducesignals to cause earty activation events and production of IL-2upon antigen stimulation in the absence of , motifs. However,any one of the ; motifs was required to respond to Thy-1 stimulationand this requirement cannot be replaced by other CD3 chains.Such , motif-dependent responses were also observed in tyrosinephosphoryiation of a 90 kDa protein upon TCR stimulation. Furthermore,we found that the C-terminal unique region of the chain exhibitsinhibitory function in phosphoryiation and Ca2⁺ response uponTCR stimulation as well as IL-2 production upon Thy-1 stimulation.Collectively, the present analyses suggest that two types ofsignals are induced through the TCR-CD3 complex: (I) the commonmotif-dependent signals which are mediated equally through ,dimers and CD3, and (II) , specific motif-dependent signals.Differences in the cytoplasmic domain of , family moleculesmay modulate the cooperation of these two signals, resultingin alteration of T cell functions.     

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.     

Molecular cloning and characterization of guinea pig Fc{gamma}RIII: expression but not function is independent of the {gamma} chain of Fc{gamma}FceRI

We have isolated two cDNA clones encoding the guinea pig receptorfor the Fc portion of lgG2 (Fc2R) from a guinea pig peritonealmacrophage cDNA library. Analysis of the predicted amino acidsequence of the one cDNA clone indicated that the guinea pigFc2R Is a type I transmembrane protein and has 72% DNA sequencehomology and 57% protein sequence homology with the human FcRIII.Therefore, we propose that the guinea pig Fc2R Is referred toas guinea pig FcRIII. The most important finding In this reportis that the obtained cDNA directed the cell surface expressionof the Fc2R on COS-7 cells without association with the chainof the high-affinity IgE receptor (FcRly) which is requiredfor human and mouse FcRIII to be expressed on the cell surface.Furthermore, we demonstrated that the endocytosis activity ofFcRIII is dependent upon the association with FcRl, suggestingthat FcRl is Involved in the functions of guinea pig FcRIII.The other clone was found to lack the sequence encoding transmembraneand cytoplasmic domains, suggesting the presence of a solubleform of guinea pig FcRIII. Northern blot analysis and RT-PCRshowed that a transmembrane form of guinea pig FcRIII was expressedin peritoneal macrophages, but not in neutrophils In spite ofthe fact that they express Fc2R, indicating that the Fc2R onneutrophils is a product of a distinct gene.     

In vitro and in vivo recovery of IFN-{gamma}, but not IL-2, production by IL-12 in mice with plasma ceIL tumors

We have previously found that T ceILs from mice bearing plasmaceIL tumors (PCT mice) demonstrate decreased proliferation asweIL as decreased production of the T^h 1-associated cytokinesIL-2 and IFN- in response to polyclonal stimulation. In thepresent study, we have examined soluble factors as possibleelements required to rescue this decreased proliferation andcytokine production by splenocytes from PCT mice. We find thatthe addition of supernatants from stimulated normal splenocyteshas no effect on proliferation or IL-2 production by splenocytesfrom PCT mice. In contrast, these supernatants completely restoreIFN- production by splenocytes from PCT mice. We have foundthat IL-12 is responsible for the observed increase in IFN-production because: (i) addition of anti-IL-12 antibody blocksthis recovery of IFN- production by these supernatants, (ii)the addition of recombinant IL-12 to cultures of splenocytesfrom PCT mice results in increased IFN- production and (iii)In vivo treatment of PCT mice in IL-12 also results in increasedIFN- production by the subsequently activated splenocytes, buthas little effect on proliferation or IL-2 production. Theseresults demonstrate that both in vitro and in vivo, IL-12 selectivelyrestores the decreased production of IFN- by splenocytes fromPCT mice.