CD2 expression on murine intestinal intraepithelial lymphocytes is bimodal and defines proliferative capacity

The CD2 molecule is normally expressed on nearly all murinelymphocytes, and is co-stimulatory in T cell activation viathe antigen receptor (TCR). A naturally occurring T lymphocytepopulation that is bimodal for CD2 expression was found in theintestinal intraepithelial lymphocytes (IEL). TCRß⁺IEL contain CD2^– and CD2⁺ cells of approximately equalproportion, while TCR⁺ IEL are predominantly CD2^–. Theproliferative response of IEL to stimulation with an anti-CD3mAb or with PMA plus ionomycin co-segregated with CD2 expression;the CD2⁺ subset proliferated vigorously under these conditionswhile the CD2^– subset was much less responsive. The respondingCD2⁺ IEL contained both TCRß⁺ and TCR⁺ cells. However,activation of the CD2^– IEL with anti-CD3 mAb resultedin only the expansion of TCR⁺ IEL, while activation with PMAplus ionomycin did not promote expansion of either the TCRß⁺or the TCR⁺ IEL. These findings parallel observations in theautoimmune lpr mouse, where massive numbers of peripheral TCRß⁺CD4^–CD8^–T cells that lack CD2 expression are also hyporesponsive tomltogenic stimulation. The apparent energy of CD2^–TCRß⁺IEL, as well as CD2^– T cells from lpr mice, demonstratesthat the absence of CD2 on TCRß⁺ T lymphocytes co-segregateswith nonresponsiveness.     

Phenotypic and functional assessment of intraepithelial lymphocytes bearing a 'forbidden' {alpha}{beta} TCR

Differences in the surface antigen phenotype, such as the expressionCD8 as an homodimer or the lack of Thy-1, on Intestinal Intraepitheliallymphocytes (IEL) are related, In part, to alternative differentiationpathways. The relationship of IEL lacking the pan-T cell markerCD5 to these IEL, their TCR repertoire and function has notbeen examined directly. We explored the TCR repertoire and functionof the CD5^– IEL subset In relation to the expression ofthe ‘autospecific’ V_ß6 TCR in Mls-1^a miceand to TCR. The results indicate that CD5 expression was absenton the majority of TCR IEL (96.9%) and on a significant proportionof TCR ß IEL (25.0%). Virtually all IEL In DBA/2 (Mls-1^a)mice that expressed the ‘autospecific’ V_ß6TCR were CD5^–, and this correlated with the expressionof CD8 . To assess the functional capacity of this subset ofIEL, we examined proliferation and IL-2 production in responseto TCR activation. Although CD5^– IEL proliferated in responseto anti-CD3, IEL bearing TCR V_ß6, In Mls-1^a mice,were not responsive to TCR-mediated activation. Similarly, TCR IEL were not responsive to stimulation by anti-TCR antibodies.The addition of exogenous IL-2, however, reconstituted the prollferativeresponse of both TCR IEL and the TCR V_ß6 expressingIEL. We conclude that the lack of CD5 defines a unique subsetof intraepithelial T cells expressing either TCR or ßthat Include potentially autoreactive cells that remain anergicin the absence of IL-2.     

IL-4 producing CD4+ TCR{alpha} {beta}int liver lymphocytes: influence of thymus, {beta}2-microglobulin and NK1.1 expression

The present report describes developmental, phenotypic and functionalfeatures of unconventional CD4⁺ TCRß lymphocytes.In C57BL/6 mice, the majority of liver lymphocytes expressingintermediate intensity of TCRß (TCRß^int)are CD4⁺NK1.1⁺ and express a highly restricted TCR V_ßrepertoire, dominated by V_ß8 with some contributionby V^ß7 and V^ß2. Although these cells expressthe CD4 co-receptor, they are present in H2-l Aß (Aß)^+/–gene disruption mutants but are markedly reduced in ß₂-microglobulin(ß₂m)^–/– mutant mice and hence are ß₂mdependent. Thymocytes expressing the CD4⁺NK1.1⁺ TCR ßphenotype are also (ß₂m) contingent, suggesting thatthese two T lymphocyte populations are related. The CD4⁺NK1.1⁺TCRßlymphocytes in liver and thymus share several markers such asLFA-1⁺, CD44⁺, CD5⁺, LECAM-1^– and IL-2Rßa. TheCD4⁺NK1.1 ⁺ TCRß^int liver lymphocytes were not detectedin athymic nuinu mice. We conclude that ß₂m expressionis crucial for development of the CD4⁺NK1.1⁺ TCRß^intliver lymphocytes and that thymus plays a major role. CD4⁺ TCRß^intliver lymphocytes were also identified in NK1.1⁺ mouse strains,there lacking the NK1.1 marker. We assume that the NK1.1 moleculeis a characteristic marker of the CD4⁺TCR"^int liver lymphocytesin NK1.1⁺ mouse strains, although its expression is not obligatoryfor their development. The liver lymphocytes from +₂m^+/–,but not from +₂m^–/–mice are potent IL-4 producersin response to CD3 or TCRß engagement and the IL-4production by liver lymphocytes was markedly reduced by treatmentwith anti-NK1.1 mAb. We conclude that the CD4+NK1.1⁺ TCRß^intliver lymphocytes are capable of producing IL-4 in responseto TCR stimulation.     

Alteration of a polyclonal to an oligoclonal immune response to cecal aerobic bacterial antigens in TCR{alpha} mutant mice with inflammatory bowel disease

Since lumenal bacteria have been postulated to play an Importantrole in the pathogenesis of inflammatory bowel disease (IBD),we investigated the humoral response to cecal aerobic bacterialantigens by Western blot analysis in TCR⁺ mice which spontaneouslydevelop IBD. The sera from TCR⁺ mice revealed an alterationof the recognition pattern against aerobic bacterial antigensfrom polyclonal to oligoclonal with age. This alteration wasnot observed in TCR⁺ and TCR⁺ mice. The alteration of the recognitionpattern in TCR⁺ mice was associated with production of autoantibodiesagainst tropomyosin and the development of IBD. The unique populationof CD4⁺ TCR^–ß⁺ cells in TCR⁺ mice may be involvedin the recongnition of these bacterial antigens and the absenceof the chain may result in the alteration of immune response.     

Maturation of medullary thymic epithelium requires thymocytes expressing fully assembled CD3-TCR complexes

Unlike meduilary thymic epithelial cells (TEC) of normal mice,meduilary TEC of TCR^– SCID mice are immature and disorganized.In order to assess directly the role of TCR⁺ cells in the developmentof medullary TEC, we bred mice which co-expressed the SCID geneticdefect and transgenes encoding clonotypic TCR chains. Immunohistologicexamination revealed that meduilary thymic epithelial cellsfrom TCRß transgenic SCID mice, whose thymocytes onlyexpress TCRß chains that inefficiently associate withCD3 and , components, remained immature and disorganized. Incontrast, meduilary TEC from TCRß transgenic SCIDmice, whose thymocytes express fully assembled CD3--TCRßcomplexes were mature and organized. Interestingly, the abilityof TCRß⁺-⁺-CD3³ thymocytes to induce maturation ofmeduilary TEC appeared not to be related to the antigen specificityof the TCR as thyml from positively selecting, negatively selectingand non-selecting TCRß transgenic SCID mice all possessedinduced meduilary thymic epithelial cells. In addition, we foundthat induction of meduilary TEC cells was associated with thepresence of meduilary thymocytes, including those of the CD4-CD8-TCRß⁺phenotype. The present findings demonstrate that fully assembledCD3--TCR complexes are required to induce maturation of meduilarythymic epithelial cells and indicate that thymocyte inductionof meduilary thymic epithelial cells may result from signalingindependently of their clonotyplc chains.     

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.     

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.     

Requirement of IL-5 for induction of autoimmune hemolytic anemia in anti-red blood cell autoantibody transgenic mice

IL-5, IL-10 and lipopolysaccharide (LPS) are known to activateB-1 cells in vivo in normal mice and anti-red blood cell autoantibodytransgenic mice (HL mice). To assess the exact role of IL-5in proliferation and activation of peritoneal B-1 cells, weanalyzed IL-5 receptor chain-deficient HL (IL-5R^–/–x HL) mice generated by the cross between IL-5R^–/–and HL mice. In IL-5R^–/– x HL mice, Ig-producingB-1 cells in the peritoneal cavity were negligible, althoughthe total number of B-1 cells in the peritoneal cavity wereas many as 30% of that in HL mice. Moreover, LPS- or IL-10-induceddifferentiation of B-1 cells into antibody-producing cells wasseverely impaired in IL-5R^–/– x HL mice. We alsoused in vivo 5-bromo-2'-deoxyuridine labeling to estimate theproliferation of B-1 cells in IL-5R^–/– mice. Theabsence of IL-5R did not affect spontaneous proliferation ofperitoneal B-1 cells. However, induced proliferation of peritorealB-1 cells by oral administration of LPS was markedly impairedin IL-5R^–/– mice. These results suggest that IL-5is required for activation-associated proliferation of B-1 cellsbut not for their spontaneous proliferation and support theidea that IL-5 plays an important role on the induction of autoantibodyproduction from B-1 cells.     

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.     

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.     

Resident CD4+ {alpha}{beta} T cells of the murine female genital tract: a phenotypically distinct T cell lineage that rapidly proliferates in response to systemic T cell activation stimuli

A population of CD4⁺ cells has been identified in the murinefemale genital tract (FGT). Phenotypic studies of FGT CD4⁺ cellsdemonstrate that they express CD3 and that the majority of thesecells are ßTCR⁺Thy-1⁺. Most of the Thy-1⁺CD4⁺ßTCR⁺ cells resemble memory T cells based on their expressionof CD44, L-selectin and CD45RB antigens. The vast majority ofThy-1⁺CD4⁺ßTCR⁺ FGT cells are CD5⁺ and all of themare B220^–. Systemic stimuli including infection with Trypanosomabrucel brucel, injection with anti-CD3, or bacterial superantigensstaphylococcal enterotoxin A or B cause a rapid accumulationof CD4+cells in the FGT exceeding that observed for CD4⁺ cellsin spleen and lymph nodes (LN). Expansion of the FGT CD4⁺ cells,which are phenotypically distinct from the splenic and LN CD4⁺T cells, is due to local proliferation rather than an influxof cells from the circulation. The CD4⁺ population in the FGTof adult nu/nu mice is dramatically reduced, indicating itsthymic dependency. In lpr/lpr mice, FGT CD4 cells do not displaychanges characteristic of splenic or LN CD4 cells in the sameanimals. These findings demonstrate that the CD4⁺ cells of themurine FGT are thymic dependent, but that they constitute aT cell lineage that phenotypically and, probably functionally,is distinct from other peripheral CD4⁺ T cell populations.     

Cytokine dependence of V{gamma}3 thymocytes: mature but not immature V{gamma}3 cells require endogenous IL-2 and IL-7 to survive evidence for cytokine redundancy

It has previously been described that V3 cells can proliferateextensively in vitro in the presence of different cytokines.Here, the role of cytokines in the maintenance of V3 cells inthe thymus has been determined. Culture of fetal thymocytesin cell suspension for 24 h showed that, whereas immature TCR^lowHSA^highV3cells remained viable, all mature TCR^highHSA_lowV cells died.These cells died by apoptosis since protein synthesis was requiredand flow cytometric analysis as well as DNA gel electrophoresisshowed that the DNA was degraded to oligonucleosomal bands.Addition of IL-2, IL-4 or IL-7 to suspension cultures of fetalthymocytes rescued V3 cells from dying. Addition of IL-1, IL-3,IL-5, IL-6, IL-9, TNE- or IFN- was without effect. Phenotypicanalysis showed that the -chain of the IL-2 receptor (IL-2R)was expressed by part of the immature V3 thymocytes, all matureV3 cells expressed the p-chain of the IL-2 receptor (IL-2RP).Addition of anti-IL-2R mAb to fetal thymic organ culture (FTOC)resulted in a moderate reduction of the cell number of matureV3 thymocytes. Addition of anti-IL-2Ra, anti-IL-4 or anti-IL-7mAb had no effect. The cell number of mature V3 cells was highlyreduced when both anti-IL-2Rp and anti-IL-7 mAb were added toFTOC. These results show that IL-2 and IL-7 are actively involvedin the maintenance of mature V3 cells in the thymus. This cytokinedependence of mature Vthymocytes may explain their selectivelocalization in skin epithelium.     

Over-expression of CD3{varepsilon} transgenes blocks T lymphocyte development

We have reported previously that mice carrying >30 copiesof the human CD3 transgene completely lose their T lymphocytesand NK cells (36). Here we demonstrate by immunohistology thatin the most severely immunodeficient mouse, tg26, the thymusis very small, has sizeable vacuoles and does not contain recognizableT lymphocytes except for a small percentage of Thy- 1⁺ cellsand B cells. Cell surface phenotyping and TCR and -ßrearrangement studies confirm that the arrest in T lymphocytedevelopment precedes the arrest in rag-1^null, rag-2^null andTCRß^null mice. Since the T cell progenitors in whichthe arrest occurred were absent in the transgenic mice, indirectapproaches were taken to examine the causes of the block inT cell development. Analyses of 12 independently establishedmutant mouse lines, generated with five different transgenicconstructs, revealed that the severity of the abrogation inT cell development was dependent on the number of copies oftransgenes. Since the number of transgene copies generally correlatedwith the levels of expression of the transgenic CD3 proteins,we concluded that over-expression of the CD3 protein was thelikely cause of the block in T lymphocyte development. The Tcell immunodeficiency was caused by either the human or themurine CD3 protein. Since transgene coded mRNAs were found insignificantly higher quantities than endogenous CD3 mRNAs infetal thymi on days 13 and 14 of gestation, over-expressiontook place very early in development, probably prematurely.Over-expression of the CD3 transgene in thymocyte precursorsmay therefore affect T lymphocyte development in the absenceof TCR and possibly in the absence of the other CD3 proteins.More importantly, over-expression of the CD3 protein in thymocytesof mice with a low copy number of transgenes had a significanteffect on late thymic development Over-expression of the CD3protein in immature thymocytes mimicked the effects caused byexposure of CD4^–; CD8^– thymocytes to anti-CD3 treatment:apoptosis and lack of TCRß expression. We thereforespeculate that in the homozygous tg26 animals the arrest inT cell development was caused by excessive signal transductionevents rather than by a toxic effect of the transgenic protein.     

Herpesvirus saimiri immortalization of {alpha}{beta} and {gamma}{delta} human T-lineage cells derived from CD34+ intrathymic precursors in vitro

Herpesvirus saimiri (HVS), an agent that can infect many humancell types, has been shown to immortalize selectively TCR ß⁺CD3⁺T lymphocytes. Human T cell precursors defined as CD34⁺CD3^–CD4^–CD8^–were isolated from thymic samples and exposed to HVS in thepresence of either IL-2 or IL-7. Cultures lacking the viruswere non-viable by day 15. Test cultures, in contrast, showeda sustained proliferative activity lasting >5 months, allowingthe phenotypical and molecular analysis of the cellular progeny.In the presence of IL-7, TCR ß⁺ cells with three differentphenotypes (mainly CD4⁺CD8^–, but also CD4⁺CD8⁺ and CD4^–CD8⁺)were immortalized, whereas no TCR ⁺ cells were recovered. Kineticstudies showed that the expansion of immortalized TCR ß⁺cells was preceded by a gradual loss of CD34⁺ cells followedby a transient accumulation of two distinct cell subsets: firstCD1⁺CD4⁺CD3^– cells and then CD4⁺CD8⁺ thymocytes. Thisresembles early phenotypic changes occurring during normal intrathymicT cell development. In the presence of IL-2, in contrast, onlyTCR ⁺ cells were immortalized (mainly CD4^–CD8⁺, but alsoCD4^–CD8^–). The results show that HVS can be usedto read the CD3⁺ cellular outcome of T cell differentiationassays, including ⁺ CD4^–CD8⁺, ⁺CD4^–CD8^–, ß⁺CD4⁺CD8^–,ß⁺CD4^–CD8⁺ and ß⁺CD4⁺CD8⁺ T cells.A clear role for different cytokines (IL-2 for ⁺ cells, IL-7for ß⁺ cells) in early T cell commitment was alsoapparent.     

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.     

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.     

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.