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.     

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.     

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.     

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.     

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.     

Expression of the IL-2 receptor {gamma} chain on various populations in human peripheral blood

We have established two rat mAbs, TUGH4 and TUGh5, specificfor the human chain of the IL-2 receptor (IL-2R), which isknown to be shared among receptors for IL-2, IL-4 and IL-7.The antibodies bound to cell lines transfected with the human chain gene but not to their parental cell lines, and precipitated65–70 and 80–90 kDa cell surface molecules fromlysates of human T cells surface-labeled with Na¹²⁵I and chemicallycross-linked with ^[125]IL-2 respectively. Flow cytometry withTUGh4 and TUGh5 detected the chain in a wide variety of peripheralblood cell populations including CD4⁺ T cells, CD20⁺ T cells,CD20⁺ B cells, CD56⁺ natural killer cells, CD4⁺ monocytes andgranulocytes, contrasting with expression of the and ßchains of IL-2R.     

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.     

Induction of human hsp60 expression in monocytic cell lines.

Both bacterial and mammalian heat shock proteins (HSP) are recognized by some T cells, and hsp60 recognition has been implicated in rheumatoid arthritis. We have developed a model to study the induction of hsp60 in human monocytic cell lines. An anti-mycobacterial hsp65 mAb (ML30), cross-reacting with human hsp60 was used to screen 21 human tumor cell lines in Western blot analysis. All T cell and B cell lymphomas constitutively expressed hsp60 protein at moderate to high levels, while little or no hsp60 protein was detected in two monocytic leukemia lines. Moderate to high levels of hsp60 mRNA and protein could be induced in the THP-I monocytic leukemia cell line by heat shock, retinoic acid, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha treatment, the highest levels obtained with a combination of IFN-gamma/TNF-alpha. This was also seen using two rabbit anti-hsp60 antisera directed against the N-terminal or C-terminal part of the human hsp60 protein. The determinants detected by the ML30 mAb or the two rabbit anti-hsp60 antisera were not cell surface expressed, as measured with immunofluorescence (FACS) analysis on control cultured or cytokine treated cell lines. This could be a useful model for studies related to the induction of hsp60 in human cells.     

Implication of the common {gamma} chain of the IL-7 receptor in intrathymic development of pro-T cells

The effects of IL-7 on the growth and differentiation of thymocyteswere analyzed using murine fetal thymua organ cultures (FTOC)in the presence of mAbs specific for the conventional IL-7 receptor(1L-7R) and for the common (c) chain. In FTOC, the developmentof CD4^–CD8^– double-negative thymocytes to CD4+CD8+double-positive (DP) and CD4+ or CD8+ single-positive (SP) cellswas not completely blocked by adding these mAbs, although cellgrowth was reduced by the treatment. To define a developingstage sensitive to the mAbs, most immature thymocytes, Pgp-1⁺c-kit cells, were cultured in the 2-deoxyguartosine treatedfetal thymus. In the presence of both mAbs in the culture, neitherDP nor SP thymocytes developed whereas either of the mAbs partiallyblocked their development. These results indicate that the Cchain is involved in early T cell development as an indispensablesubunlt of the functional IL-7 receptor complex.     

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.     

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.     

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.     

Monoclonal antibody against murine T cell receptor V alpha 14 cross-reacts with human CD3 epsilon and detects disulfide-linked dimeric form.

A monoclonal antibody against V14+ alpha-chain of murine T cell receptor (TCR) was established by fusing spleen cells from a rat immunized with a soluble chimeric TCR/IgG3 protein containing murine TCR V alpha 14J alpha 281 in place of the VHDHJH of an IgG3. lambda 1, and subjected to screening on a human transfectant (Jurkat variant) expressing the murine V14J281 alpha-chain. The anti-mouse V alpha 14 antibody precipitated TCR alpha beta molecules from Triton X-100-solubilized extracts of 125I-labeled murine thymocytes and spleen cells. Unexpectedly, the antibody showed cross-reactivity to the human CD3 epsilon molecule and detected a disulfide-linked 20 kDa dimeric form of human CD3 epsilon, which is a novel family component of the CD3 complex and is associated closely with the CD3 zeta-zeta homodimer as well as TCR alpha beta or TCR gamma delta.     

Maternal serum levels of interferon-gamma and interleukin-2 soluble receptor-alpha predict the outcome of early IVF pregnancies

BACKGROUND: Elevated maternal serum levels of interleukin-2 soluble receptor-alpha (IL-2 sRalpha), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been associated with pregnancy loss. The aim of our study was to evaluate the predictive value of these cytokines in the outcome of early IVF pregnancies. METHODS: One hundred and fifty-nine consecutive IVF patients who were subsequently diagnosed to have a biochemical pregnancy (n = 23), a first-trimester miscarriage (n = 19) or a normal term delivery (n = 117) were included in this study. Serum was collected from the initial pregnancy test, 11 days after a day 3 embryo transfer, and all samples were analysed for IL-2 sRalpha, TNF-alpha and IFN-gamma by commercially available enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IL-2 sRalpha levels were significantly higher in patients with an early pregnancy loss compared with patients with a normal term delivery (849.5 +/- 69.6 versus 693.5 +/- 31.2 pg/ml, P = 0.02), and a cut-off point of IL-2 sRalpha >1000 pg/ml predicted a poor pregnancy outcome (44.4 versus 22.7% pregnancy loss, IL-2 sRalpha >or=1000 versus IL-2 sRalpha <1000 pg/ml; P = 0.02). IFN-gamma-positive patients had twice the risk for poor IVF pregnancy outcome compared with IFN-gamma-negative subjects (40.8 versus 20.0%, respectively; P < 0.02), including a significantly lower implantation rate (37.6 +/- 0.05 versus 50.0 +/- 0.03%, respectively; P = 0.02). There was no difference in pregnancy outcome based upon serum levels, or the ability to detect the presence of TNF-alpha. No differences in levels of these cytokines were found based on the aetiology of the patients' infertility. CONCLUSIONS: Elevated maternal serum levels of IL-2 sRalpha and IFN-gamma as early as 11 days after embryo transfer are associated with poor IVF pregnancy outcome.     

Critical proline residues of the cytoplasmic domain of the IL-5 receptor {alpha} chain and its function in IL-5-mediated activation of JAK kinase and STAT5

The high-affinity receptor (R) for IL-5 consists of a uniquea chain (IL-5Rc) and a ß chain (ßc) thatisshared with the receptors for IL-3 and granulocyte macrophagecolony stimulating factor (GM-CSF). We defined two regions ofIL-5R for the IL-5-induced proliferative response, the expressionof nuclear proto-oncogenes, and the tyrosine phosphorylationof cellular proteins including ßc, SH2/SH3-containingproteins and JAK2 kinase. In the studies described here, wedemonstrate that IL-5, IL-3 or GM-CSF stimulation induces thetyrosine phosphorylation of JAK2, and to a lesser extent JAK1,and of STAT5. Mutational analysis revealed that one of the prolineresidues, particularly Pro352and Pro355, in the membrane-proximalproline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmicdomain of IL-5R is required for cell proliferation, and forboth JAK1 and JAK2 activation. In addition, transfectants expressingchimeric receptors which consist of the extracellular domainof IL-5R and the cytoplasmic domain of ßc respondedtoIL-5 for proliferation and tyrosine phosphorylation of JAK1.Intriguingly, electrophoretic mobility shift assay analysisrevealed that STAT5 was activated in cells showing either JAK1or JAK2 tyrosine phosphorylation. These results indicate thatactivation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-inducedtyrosine phosphorylation and ultimately mitogenesis, and thatPro352 and Pro355 in the proline-rich sequence appear to playmore essential roles in cell growth andin both JAK1/STAT5 andJAK2/STAT5 activation than Pro353 does.     

Polymorphism of the human T cell receptor alpha chain variable genes: identification of a highly polymorphic V gene probe

In this study, we report the RFLP of the human T cell receptor (TCR) alpha chain variable gene segments. Using DNA samples from 20 individuals and three restriction endonucleases (BamHI, EcoRI and HindIII), the degree of RFLP of a number of different V gene segments was defined. Half of the V alpha subfamilies (6/12) were characterized by a predominant hybridization pattern, with only a few individuals displaying a second pattern. However, one particular V gene family, V alpha 6, has at least five allelic forms that segregated consistently in familial studies. The V alpha polymorphisms revealed in this study, together with those exhibited by V beta gene subfamilies, should prove useful in studying possible associations between TCR gene usage and disorders of the immune system.     

Activation of Vgamma9Vdelta2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens. Vgamma9Vdelta2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-gamma revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate gammadelta T cells, were shown to induce the inhibition of HCV replication mediated by Vgamma9Vdelta2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vgamma9Vdelta2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.     

IL-2 receptor {alpha} chain (CD25JAC) expression defines a crucial stage in pre-B cell development

The analysis of the expression of the a chain of the IL-2 receptor(CD25.TAC) on the surface of B lineage cells In mouse bone marrowreveals that it is a useful marker to distinguish pre-B-I frompre-B-II cells. CD25 Is not expressed on CD45R(B220)⁺ c-kit⁺CD43⁺ TdT⁺ ₅⁺ C_µ^– slg^– lgH chain locus DJ_H-rearrangedpre-B-I cells of mouse bone marrow. It is expressed on largecycling CD45R(B220)⁺ c-kit⁺ CD43⁺ TdT⁺ ₅⁺ C_µ^– sig^–and on small resting CD45R(B220)⁺ c-kit⁺ CD43^– TdT⁺ ₅⁺C_µ^– sig^– sig- IgH chain locus VHDJH-rearrangedpre-B-II cells. Therefore, the transition from pre-B-I to largepre-B-II cells is marked by the downregulation of c-kit andterminal deoxynucleotldyl transferase (TdT), and by the upregulattonof CD25. SCID, RAG-2T, _µMT and ₆T mutant mice do havenormal, If not elevated numbers of pre-B-I cells but lack allCD25⁺ pre-B-II cells in their bone marrow. The expression ofa transgenic H chain under control of the _µH chain enhancerin RAG-2T bone marrow B lineage precursors allows the developmentof large and small CD25⁺ pre-B-II cells. The results suggestthat the differentiation of pre-B-I to pre-B-II cells in mousebone marrow requires the expression of _µH chains and surrogateL chains in membranes, probably on the surface of precursorB cells.