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.     

Involvement of protein kinase C-{varepsilon} in glucocorticoid-induced apoptosis in thymocytes

Apoptosis is induced in immature thymocytes by physiologicalpeak levels of glucocortlcold hormones, especially in murineand rat cells. Endogenous glucocortlcolds may have some rolein thymic selection. Glucocorticold-lnduced thymocyte apoptosisappears to be dependent on protein kinase C (PKC), since itis inhibited by PKC inhibitors. PKC Is a family of closely relatedenzymes, consisting of Ca²⁺-dependent (PKC-, -ßI,-ßII, and ) and Ca²⁺-Independent (PKC-, -ß,(L), -, -, and -) isozymes. In the present study, we analyzedthe role of PKC in glucocorticold-lnduced apoptosis in murinethymocytes and found that glucocorticold selectively inducesan increase in Ca²⁺ -Independent PKC activity in the paniculatefraction of Immature thymocytes but not in that of mature Tcells. The increase as well as the apoptosis was inhibited byactlnomycln D, cyclohexlmkte, or the glucocortteoid receptorantagonist, RU 38486. Immunoblottlng studies revealed the selectivetranslocatlon of PKC-from the cytosollc fraction to the paniculatefraction upon glucocortlcold treatment. These results suggestthat glucocorticold-lnduced apoptosis in immature thymocytesinvolves glucocorticold receptor-mediated and selective activationof PKC-through de novo synthesis of macromolecules.     

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.     

Nickel and skin irritants up-regulate tumor necrosis factor-{alpha} mRNA in keratinocytes by different but potentially synergistic mechanisms

A critical role of tumor necrosis factor (TNF)- in irritantcontact dermatitis and in the challenge phase of allergic contactdermatitis has recently been demonstrated in vivo. As in situhybridization studies have indicated that keratinocytes werethe cellular source of TNF- in these reactions, we studied themechanisms of TNF- mRNA induction in keratinocytes by agentsthat induce contact dermatitis. Murine Ia^–;/CD3^–epidermal cells were stimulated with phorbol myristate acetate(PMA), dimethylsulfoxide (DMSO), sodium dodecyl sulfate (SDS)and NiSO₄, all of which up-regulated epidermal cell TNF- mRNAproduction. In contrast, trinitrobenzenesulfonic acid and trinitrochlorobenzenedid not significantly up-regulate TNF- mRNA. These results wereconfirmed with murine keratinocyte cell lines. In keratinocytestransfected with a chloramphenicol acetyltransferase constructcontaining the –1059 to +138 base pair TNF- promoter,increased promoter activity was observed upon stimulation withPMA and DMSO. In addition, PMA stimulation did not affect thestability of TNF- mRNA. The PMA- but also the DMSO- and SDSinducedup-regulation of TNF- mRNA was abolished by an inhibitor ofprotein kinase C (PKC). In contrast, NISO4 up-regulated TNF-mRNA by a PKC-independent mechanism, did not increase TNF- promoteractivity, but markedly increased the stability of the TNF- mRNA.Co-stimulation with PMA and NISO₄ induced a marked increasein TNF-a mRNA over that obtained with each agent alone. Thus,whereas PKC-dependent irritants act by up-regulating TNF- promoteractivity, nickel acts via post-transcrlptional regulation. Ourresults, also establish that some irritants and irritant sensitizersdirectly induce TNF- in keratinocytes without intermediate Langerhanscell derived signals.     

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.     

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.     

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.     

TCR isoform containing the Fc receptor {gamma} chain exhibits structural and functional differences from isoform containing CD3{xi}

The structure and function of the TCR-CD3 complex containinga homodimer of the gamma chain of the high affinity receptorfor IgE (FcR) (FcR⁺ TCR) was investigated by transfecting theFcR gene into a CD3^–, CD3^–, FcR^– T cell line.Introduction of FcR, as well as CD3, induced a high expressionof the TCR-CD3 complex on the cell surface. Transfected FCRformed a homodimer and associated firmly with the TCRßdimer but only weakly with the CD3. Stimulation of both FcRand CD3 transfectants by antibodies against TCR or CD3 inducedaccumulation of inositol phosphates, the Ca²⁺ response, IL-2production, and growth inhibition. On the other hand, antigenstimulation of transfectants expressing FcR as well as CD3 inducedIL-2 production, but only the latter exhibited the antigen-inducedgrowth inhibition. In vitro kinase assay suggested that theCD3 dimer but not the FcR dimer associates with the Fyn kinase.These results indicate that the FcR homodlmer Is able to forma functional TCR complex but that the mode of assembly and thesignaling function of FcR⁺ TCR, including its association withtyrosine klnase(s), may differ from the TCR-CD3 complex containingCD3 homodimers (⁺ TCR). This provides an example which illustratesthat different TCR isoforms mediate distinct signals and functions.     

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.     

Double-stranded RNA and bacterial lipopolysaccharide enhance sensitivit to TNF-{alpha}-mediated cell death

The effect of double-stranded RNA (dsRNA) and bacterial lipopolysaccharideon the sensitivity to tumor necrosis factor (TNF)--medlatedcell death was studied In an In vitro system. Since secretionof TNF- Is a part of the early host response to viral and bacterialinfection, we examined whether mimicking the Infection withviral and bacterial products could affect the response of cellsto TNF-. Incubation of WEHI 164 fibrosarcoma cells with dsRNAor lipopolysaccharide (LPS) significantly increased their sensitivityto TNF--mediated lysis and to TNF-secreting inflammatory T cell-mediatedlysis. Thus, these products could induce Increased sensitivityto TNF- In cells In an inflammatory focus, possibly contributingto selective elimination of Infected but not healthy cells bythis non-specific cytokine. Additionally, our data show thatboth dsRNA and LPS, as well as TNF- Itself, rapidly Induce nuclearfactor-xB (NF-*B), a DNA-bindlng protein Implicated In regulationof gene expression. We suggest that NF-xB could regulate genescrucial for the induction of cell death by TNF-.     

Cytokine production by CD16 CD56bright natural killer cells in the human early pregnancy decidua

Using a cell sorter, CD16^–CD56^bright natural killer (NK)cells were sorted from decidual mononuclear cells at an earlystage of pregnancy. These cells were examined by the reversetranscrlptase-polymerase chain reaction (RT-PCR) method fortheir expression of mRNA coding for the following 12 cytokines:IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), macrophage colonystimulating factor (M-CSF), tumornecrosis factor- (TNF-), interferond- (IFN-), and leukemia inhibitoryfactor (LIF). Although mRNA coding for every cytokine was detectedin decidual mononuclear cells, mRNAs coding for only G-CSF,GM-CSF, M-CSF, TNF-, IFN-, and LIF were detected In CD16^–CD56^brightNK cells. Also, the supernatant of CD16^–CD56^bright NKcell cultures was found to contain G-CSF, GM-CSF, M-CSF, TNF-,IFN-, and LIF. These findings indicate that CD16^–CD56^brightNK cells produce many different cytokines and that these cytokinesmay play an important role in a successful pregnancy.     

Monoclonal anti-{lambda}5 antibody FS1 identifies a 130 kDa protein associated with {lambda}5 and Vpre-B on the surface of early pre-B cell lines

mAbs specific for mouse 5 protein were prepared by fusion ofspleen cells from a hamster Immunized with recomblnant 5 proteinsynthesized in bacteria and the mouse myeloma cell line SP2/0-Ag14.Here we report the characteristics of the antibodies producedby the FS1 hybridoma. FS1 antibody stains a variety of mousepre-B cell lines but not B cell lines or T cell lines. The stainingof the pre-B cell lines Awl and C-7 by phycoerythrin (PE)-con]ugatedFS1 (FS1 - PE) can be blocked by prelncubatlon of these cellswith unconjugated FS1 antibody or with affinity purified polyclonal5 specific Ig but not with normal hamster or mouse IgG or withaffinity purified polyclonal antl-Mb-1 Ig. From these experimentswe concluded that FS1 specifically recognizes 5 protein. Weused FS1 -PE to probe for surface (s) 5⁺ cells in normal BALB/cmouse bone marrow. Such cells were undetectable when total bonemarrow or FACS sorted subpopulatlons were analyzed. However,when B220^plus;, CD43⁺, s5⁺ bone marrow cells were cultured for4 days on the stromal cell line FLST2 in the presence of IL-7,s5 expression became apparent. Further expansion of these cellsin IL7 alone augmented the s5 expression to readily detectablelevels. This modulation may indicate that s5 expression on normalbone marrow cells in vivo is transient and that at any givenmoment only a small fraction of bone marrow cells expresseslow levels of 5 protein on the surface. Alternatively the bindingof our FS1 mAb to the s5 molecules on normal bone marrow cellsmay be blocked by other proteins binding to the 8X5 complexin vivo and directly ex vivo. Previous analysis of surface 5associated proteins on early mouse pre-B cell lines using apolyclonal anti-5 rabbit antlserum had suggested that s5 proteinwas associated with a high molecular weight protein. Analysisof 5 and Its associated proteins on early pre-B cell lines usingour FS1 mAb confirmed our previous finding and showed that theearly 5 receptor contains at least three proteins: 5, V_pre-B,and an as yet uncharacteiized protein with a molecular weightof 130,000 designated p130.     

Growth inhibition of Mycobacterium bovis by IFN-{gamma} stimulated macrophages: regulation by endogenous tumor necrosis factor-{alpha} and by IL-10

Murine bone marrow-derived macrophages (BMM) are able to inhibitthe intracellular growth of Mycobacterium bovis and Mycobacteriumtuberculosis H37Rv after activation with recomblnant (r) IFNand growth inhibition is mediated by reactive nitrogen intermediates(RNI) derived from L-arglnlne. We now demonstrate that tumornecrosis factor (TNF)- acts as an endogenous cofactor in theinduction of mycobacterial growth inhibition. TNF- was producedby BMM stimulated with rlFN- and infected with mycobacterla,and a specific antlserum to TNF- Inhibited rlFN--lnduced productionof RNI as well as growth inhibition of M. bovis. IL-10, a cytokinewhich suppresses antlmycobacterial macrophage functions, wasalso produced by BMM activated with hFN- and infected with M.bovis. IFN--induced production of TNF- and of reactive nitrogenintermediates as well as mycobacterial growth inhibition wereinhibited by exogenous IL-10, but only when given prior to IFN-stimulation. We conclude that the outcome of mycobacterial infectionis regulated by a coordinate interplay between IFN-, TNF- andIL-10.     

Composition of TCR-CD3 complex in human intestinal intraepithelial lymphocytes: lack of Fc{varepsilon}Rl{gamma} chain

Human intestinal intraepithelial lymphocytes (DEL) are a uniquepopulation of predominantly CD8ß⁺ TCRß⁺lymphocytes and, to a lesser extent, TCR⁺ lymphocytes that proliferatepoorly to anti-CD3 mitogenic signals but display significantcytolytic activity. Studies in mouse model systems have shownthat the chain of the high-CD3 affinity receptor for IgE (FcRl)may substitute for the chain in the TCR-CD3 complex of iIEL.This has suggested that the functional properties of these cellsmay be associated with an altered composition of the TCR-CD3complex. We therefore analyzed the TCR-CD3 complex of normalhuman iIEL. One-and two-dimensional non-reducing/reducing SDS-PAGEanalysis of CD3, CD3, CD3, and FcRr chain immunopreclpitatesof cell surface radiolabeled proteins with subunit-specificantibodies revealed a TCR-CD3 complex without associated FcRrchains. Thus, normal human NEL contain a TCR-CD3 complex thatconsists predominantly of , homodimers in association with theß TCR and CD3, and , similar to the majority of peripherallymphocytes. This indicates that the distinct properties ofhuman DEL are not associated with substitutions of the FcRlchain in the TCR-CD3 complex.