FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023951259> ?p ?o ?g. }

• W2023951259 endingPage "230" @default.
• W2023951259 startingPage "225" @default.
• W2023951259 abstract "It is generally recognized that two signals delivered to the T cell by ligation of the T-cell receptor complex (TCR) and a costimulatory receptor are necessary to generate a T-cell-derived immune response. However, evidence suggests that TCR-mediated signals alone induce T cells to become subsequently antigen-unresponsive (anergic) 1, 2 or even apoptotic 3-5 in the absence of antigen-independent, costimulatory signals. In contrast costimulation of resting T cells by the CD28 receptor promotes the upregulation of cytokine gene expression and secretion, T-cell proliferation and survival. 6-11 Attempts to delineate the signalling pathway by which CD28 can costimulate T cells have identified a number of intracellular effectors that are activated following CD28 ligation. For example, both the role of phosphatidylinositol 3-kinase (PI3K) 12-14 and protein tyrosine kinases (PTKs) 15-17 have been investigated as possible effectors of a CD28 mediated costimulatory transduction pathway. Data on the role of PI3K are somewhat conflicting, and although PI3K appears involved in CD28 costimulation of resting T cells, it did not appear involved in T-cell proliferation or interleukin-2 (IL-2) secretion from T cells in some systems. 18, 19 Interestingly, it has also been demonstrated in murine splenic T cells, that sphingomyelinase (SMase) can partially replace the ligation of CD28 as a costimulus of T-cell proliferation. 20 SMase hydrolyses sphingomyelin, a ubiquitous membrane sphingolipid to generate phosphocholine and ceramide. Significantly, ceramide has potent second messenger properties and has been reported to activate protein kinase Cζ (PKCζ), 21 c-Jun N terminal kinase (JNK) 22 and nuclear factor-κB (NF-κB). 20, 23 Furthermore, ceramide has been reported to mimic the effects of SMase in costimulating the proliferation of murine splenocytes as well as increasing IL-2 expression. 24 Therefore, SMase may represent an effector capable of transducing CD28 costimulatory signals. In order to address whether human resting T cells utilize SMase as a costimulatory effector, we attempted to substitute CD28-derived costimulation by addition of exogenous SMase or a cell-permeable ceramide. Accordingly we found that neither SMase nor C2 ceramide were capable of costimulating proliferation in human T cells stimulated with anti-CD3 antibodies. Surprisingly, we did observe that in T cells stimulated to proliferate by anti-CD3 and CD28 ligation, both sphingomyelinase and C2 ceramide were inhibitory to this process. However, the inhibition of T-cell proliferation did not prevent the expression of T-cell activation markers and could not be accounted for by T-cell apoptosis. Reagents were purchased from Sigma (Poole, UK) unless indicated otherwise. Chinese hamster ovary (CHO) K1 CD80 transfected cells, as previously described, were used. 9 Antibodies including OKT3 (αCD3), HB8784 (αCD25), L243 (αHLA-DR) were obtained from ATCC (Rockville, MD). UCHM1 (αCD14), BB-1 (αCD80) and BU12 (αCD19) were kind gifts, respectively, from Professor P. Beverley (Jenner Institute), Dr P. Linsley (Bristol-Myers Squibb, Seattle, WA) and Dr I. McLennan (University of Birmingham, UK) and αCD69 mAb was purchased from Serotec, Oxford, UK. Immunomagnetic sheep anti-mouse immunoglobulin (IgG) beads were purchased from Dynal (Dynal UK Ltd, Bromborough, UK) and [3H]thymidine was obtained from ICN Biomedicals Ltd (Basingstoke, Hants, UK). Resting T cells were prepared from whole blood of healthy volunteers. Mononuclear cells were recovered from a Ficoll 1·077 g/ml density gradient (Nycomed). T cells were isolated by negative selection using immunomagnetic beads as follows. After plastic adherence for 1 hr at 37OC in 10% v/v fetal calf serum (FCS) : RPMI, non-adherent mononuclear cells were subject to magnetic bead separation (Dynal 450) using anti-DR (L243), anti-B cell (CD19) and anti-monocyte (UCHM1) antibodies at 10 µg/ml to remove activated T cells, B cells and antigen-presenting cells (APCs). Purified T cells were cultured in RPMI (with 10% FCS, penicillin, streptomycin) in 96-well flat-bottomed plates at 37°, in an atmosphere of 5% CO2. 5 × 104 T cells/well were left unstimulated, or stimulated with combinations of anti-CD3 antibody (10 µg/ml OKT3) in the presence or absence of CD80 transfected CHO cells (at a ratio of 3 : 1 T cells to transfectants). CHO cells, transfected with the CD80 gene, were routinely monitored by fluorescence-activated cell sorting (FACS) for CD80 expression at the CHO cell surface. Transfectants were fixed in a solution of 0·025% (v/v) glutaraldehyde for 2 min in order to prevent proliferation. Additions of SMase (1 U hydrolyses 1 µmol of sphingomyelin min−1 at 37°, pH 7·4) or C2 ceramide were added to cultures as detailed in figure legends. Cellular proliferation was assayed by [3H]thymidine incorporation into cellular DNA (1 µCi/well, for the last 18 hr) at 72 hr and viability (exclusion of propidium iodide, 0·1 µg/ml), was measured by FACS. Cellular activation was measured by analysis of surface marker expression for CD69 and CD25 using FACS. Costimulated T cells were treated with or without C2 ceramide or SMase as detailed in figure legends. For staining, 2 × 105 cells were incubated with anti-CD25 or anti-CD69 antibodies at 10 µg/ml for 40 mins at 4°, washed in phosphate-buffered saline (PBS), followed by a secondary goat anti-mouse polyvalent fluorescein conjugated antibody for a further 40 min at 4°. Subsequently, these cells were analysed for levels of surface markers at 520 nm using a BD FACStar plus flow cytometer (BD Biosciences, San Jose, CA). Previous reports have suggested that in murine splenocytes, SMase or ceramide are capable of substituting for CD28 mediated T-cell costimulation. 20, 24 We therefore attempted to establish whether there was a similar role for SMase/C2 ceramide in human T-cell costimulation. A costimulatory assay was established ( Fig. 1) which showed that CHO cells transfected with B7 (CD80), a natural ligand of CD28, could costimulate resting T-cell proliferation, in the presence of anti-CD3 (αCD3) monoclonal antibody (mAb) but that neither stimuli alone could. This observation was in entirely in agreement with a large number of studies demonstrating a ‘two signal’ requirement for resting T-cell proliferation. However, when the ability of SMase and C2 ceramide were tested for costimulatory capacity we observed that neither SMase ( Fig. 1a) nor C2 ceramide ( Fig. 1b) were capable of synergizing with αCD3 mAb to promote T-cell proliferation. Thus, while our costimulation assays appeared to be intact using CD28 stimuli, neither the signals from SMase, nor one of the products of sphingomyelin hydrolysis (ceramide), costimulated proliferation. CD80 but not sphingomyelinase nor C2 ceramide costimulate resting human T-cell proliferation. Human resting T cells (5 × 104) were left unstimulated, or incubated with αCD3 mAb (10 µg/ml OKT3) alone and/or in the presence of 1·7 × 104 fixed CHO-CD80 cells, CHO control cells or increasing concentrations of (a) SMase (SM) or (b) C2 ceramide for 72 hr. Proliferation was measured by [3H]thymidine incorporation (c.p.m.). Data are the triplicate mean of a single representative experiment of n = 3 (SD indicated by horizontal bars). Given the lack of costimulatory effect of both C2 ceramide and SMase on costimulation, we sought evidence that these compounds were indeed active in other assays. Surprisingly, when we tested their effects on T cells receiving costimulation (αCD3 mAb plus CD80 stimulated cultures) we found that concentrations of SMase in excess of 5 × 10−5 U/ml inhibited the proliferation of costimulated resting T cells ( Fig. 2a). This was clearly a dose-dependent effect and was lost at concentrations of 1 × 10−5 U/ml or lower. Consistent with this effect of SMase, C2 ceramide was also observed to decrease the proliferation of costimulated T cells ( Fig. 2b). Given that the two compounds are related only by virtue of operating within the same signalling cascade, the two results strongly suggested that activation of the sphingomyelin cascade in this way, in costimulated T cells, actively suppressed proliferation. Sphingomyelinase activity or C2 ceramide decrease T cell proliferation. Resting T cells (5 × 104/well) were left unstimulated or incubated with a combination of αCD3 mAb (10 µg/ml), 1·7 × 104 fixed CD80 cells and various concentrations of (a) SMase (SM) or (b) C2 ceramide for 72 hr. Proliferation was measured by [3H]thymidine incorporation (c.p.m.). Data are the triplicate mean of a single representative experiment of n = 3 (SD indicated by horizontal bars). Because of a widely reported ability of ceramide to induce apoptosis, 23, 25-28 we next examined whether or not the decrease in proliferation we had observed correlated with a decrease in T-cell viability. Costimulated T cells were incubated with C2 ceramide and after 3 days assessed for viability, by FACS, of propidium iodide exclusion. At concentrations of C2 ceramide that had been observed to give substantial reductions in T-cell proliferation, there appeared to be no increase in T-cell death ( Fig. 3a). However, the ability of C2 ceramide to induce cell death in the Jurkat T-cell line was clearly seen in a dose-dependent manner over the same range ( Fig. 3b). Therefore, while C2 ceramide was indeed effective in causing apoptosis in Jurkats, the decrease in proliferation of costimulated normal T cells was not caused by cell death. C2 ceramide differentially affects Jurkat and costimulated T-cell viability. (a) αCD3 mAb, CD80 stimulated resting T cells (2 × 105 T cells, 0·7 × 105 CD80 transfectants/well) were incubated for 72 hr with or without C2 ceramide (these data are paired with data shown in Fig. 2b), or (b) Jurkat T cells (2 × 105 cells/well) were incubated for 24 hr with or without C2 ceramide, and analysed by FACS for viability. All wells were equalized for vehicle content at 1% v/v ethanol. Propidium iodide uptake was measured by FACS. A representative experiment of n = 3 is shown. Given that C2 ceramide appeared not to be exerting its effects on cell proliferation by inducing cell death, we next examined whether SMase or C2 ceramide were inhibiting all early activation events or whether a more specific effect on cell proliferation was occurring. We therefore assessed the expression of early activation markers CD69 and CD25 to establish whether their expression was inhibited by C2 ceramide or SMase. These experiments ( Fig. 4) clearly showed that CD69 was upregulated at the T-cell surface more rapidly than CD25 as expected. Slightly lower levels of CD25 or CD69 expression were observed in the presence, ceramide or sphingomyelinase, respectively. However the degree of modulation was limited and unlikely to explain the effects of SMase/C2 ceramide on proliferation of costimulated T cells. Thus, we concluded that the effects of C2 ceramide and SMase on T-cell proliferation were likely to be derived from their action on relatively specific targets and did not result from generalized inhibition of all T-cell activation pathways, nor were these molecules simply toxic. Neither sphingomyelinase nor C2 ceramide prevent costimulated T cells from upregulating CD25 and CD69 expression. Resting T cells (2 × 105 T cells and 0·7 × 105 CD80 transfectants/well) were stimulated with αCD3 mAb, CD80 in the presence/absence of 7·5 × 10−5 U/ml SMase or 30 µm C2 ceramide. CD25 and CD69 surface expression were measured by FACS over a 4-day period post-stimulation. Changes in expression are represented by the histograms depicted. Control is the level of CD25/CD69 observed on unstimulated resting T cells (day 0). A representative experiment of n = 2 is shown. In this study we have investigated the effects of cell-permeable C2 ceramide and SMase on the costimulation of resting human T cells. In standard costimulation assays we demonstrated that human T cells could proliferate in response to αCD3 mAb when costimulation was provided by CD80 expressing transfectants ( Fig. 1) but not to either stimulus alone. This once again confirms the observations of previous studies regarding the importance of CD28 costimulation to resting human T-cell proliferation. 6-8 Having established a clearly costimulation-dependent system we then tested the ability of two signalling intermediates (SMase and ceramide) activated by the CD28 pathway to substitute for CD28 costimulation. Similar experiments performed using murine T cells found that either SMase or ceramide could costimulate the proliferation of cells cultured with αCD3 mAb. 20, 24 In contrast to these studies, we were unable to show a costimulatory effect of either C2 ceramide or SMase. The reasons for these differences are unclear; however, one possibility is that there may be species differences between human and mouse T cells in terms of their sensitivity to these signalling intermediates. The data observed in this study clearly do not preclude a role for SMase in CD28 signalling but suggest that neither SMase nor C2 ceramide are sufficient for costimulating human resting T cells. An alternative possibility is that there are differences in the costimulatory requirements of human peripheral blood T cells and mouse splenocytes. While data concerning the differential response of human and murine T cells to SMase/C2 ceramide are limited, 20, 24 other activation differences in human and murine T cell responses have been previously reported. For example, wortmannin, an inhibitor of PI3K activity, can ablate activation of human resting T cells, 13, 18, 29 while murine T cells are reported to be resistant. 30 Nonetheless, it is difficult to be certain that such differences are not simply a result of different experimental conditions, and these comparisons should be interpreted with caution. Surprisingly, we also observed that while SMase/C2 ceramide were incapable of costimulating T-cell proliferation, both showed consistent effects in inhibiting the proliferation of costimulated T cells ( Fig. 2). This observation concurred with previous findings on the role of a ceramide analogue (1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP)), which decreased the proliferation of mitogenically stimulated murine splenocytes. 31 Accordingly, the authors suggested that PDMP interfered with T-cell proliferation by its conversion to dimethylsphingosine, an inhibitor of protein kinase C (PKC). 32 However, this would seem an unlikely mechanism for the inhibition of costimulated T-cell proliferation observed in our study as CD69 upregulation, a PKC-dependent event, 33 was not prevented by C2 ceramide. Others showed a role for C6 ceramide in MOLT-4 leukaemic cell cycle arrest (acute lymphoblastic leukaemia), 34 observing the inhibitory action of ceramide on proliferation was mediated by dephosphorylation of the retinoblastoma gene product leading to decreased levels of the proto-oncogene c-Myc. 34, 35 Thus, the retinoblastoma gene product may represent a substrate of C2 ceramide in human T cells. Our attempts to abrogate the activity of sphingomyelinase in costimulation assays with the inhibitor chloroquine were not possible to be interpreted meaningfully because of the cytotoxicity of the inhibitor. Despite inhibition of T-cell proliferation, we did not observe an increase in cell death in C2 ceramide or SMase treated cells. While Jurkat cells were sensitive to C2 ceramide-induced cell death ( Fig. 3), costimulated T cells were resistant. The sensitivity of Jurkat cells to ceramide-induced cell death correlates with a widely reported role for ceramide as a mediator of Fas- 25-27 or tumour necrosis factor receptor (TNFR)- 23, 28 induced apoptosis. Conversely the resistance of costimulated T cells to ceramide-induced cell death agrees with observations on the ability of CD28 to overcome insult and activation-induced cell death by upregulation of the survival factor Bcl-xL and the observed resistance of activated T cells to Fas-induced death. 3, 11, 36 Alternatively, ceramide-induced apoptosis has also been found to be inhibited by addition of exogenous diacylglycerol to MOLT-4 cells 34 and human myeloid leukaemic cells 37 and therefore the resistance of costimulated T cells to C2 ceramide-induced apoptosis, which we observed may be caused by the generation of diacylglycerol following CD3 stimulation. Finally, when the activation status of costimulated T cells was examined over a period of 4 days, it was found that αCD3 mAb, CD80 stimulation of resting human T cells induced peak expression of CD69 and CD25 at day 1 and day 4, respectively, post-stimulation ( Fig. 4). Neither SMase nor C2 ceramide markedly altered the expression of these markers ( Fig. 4), although there was a modest inhibition of CD25 expression as a result of C2 ceramide and of CD69 as a result of SMase or C2 ceramide at days 4 and 1, compared to controls, but not comparable to the inhibitory effect of these molecules in proliferation assays. These data concur with the selective role of the ceramide analogue PDMP in inhibiting proliferation, but not CD25 expression, as previously observed in murine splenocytes. 31 This suggests that the SMase pathway has a relatively specific effect on cell-cycle progression and not a generalized effect on all aspects of T-cell activation. However, a recent report indicated that ceramide, generated following ligation of Fas, decreased intracellular IL-2 content in Jurkat cells by inhibiting intracellular store-operated calcium release-activated calcium channels, thus preventing a sustained increase in Ca2+i and the binding of the transcription factor nuclear factor of activated T cells (NFAT) to the IL-2 promoter. 38 Such a mechanism would be generally consistent with our observations that ceramide is inhibitory to the early stages of T-cell proliferation, but does not ablate all signals resulting from TCR stimulation, such as those required for CD69 upregulation, which is known to be calcium independent. In summary, we found that while CD80 costimulated human resting T cells, neither SMase nor C2 ceramide could provide a sufficient signal to induce T-cell proliferation while both proved capable of inhibiting proliferation. However, in contrast to the reported pro-apoptotic role for ceramide, we found that C2 ceramide was unable to induce the death of costimulated T cells at concentrations that were capable of inhibiting T-cell proliferation. These data combined with those demonstrating the activation of SMase following CD28 ligation, suggest that SMase activation is insufficient for costimulation and may operate an inhibitory pathway for T-cell proliferation. This work was supported by the BBSRC. D.M.S. is an ARC senior research fellow." @default.
• W2023951259 created "2016-06-24" @default.
• W2023951259 creator A5042350027 @default.
• W2023951259 creator A5048447114 @default.
• W2023951259 date "2000-06-01" @default.
• W2023951259 modified "2023-09-27" @default.
• W2023951259 title "Lack of costimulation by both sphingomyelinase and C2 ceramide in resting human T cells" @default.
• W2023951259 cites W113197063 @default.
• W2023951259 cites W1488633968 @default.
• W2023951259 cites W1490870966 @default.
• W2023951259 cites W1531973880 @default.
• W2023951259 cites W1583840873 @default.
• W2023951259 cites W1601124709 @default.
• W2023951259 cites W1607687470 @default.
• W2023951259 cites W1631147967 @default.
• W2023951259 cites W1634026430 @default.
• W2023951259 cites W1965631826 @default.
• W2023951259 cites W1969158591 @default.
• W2023951259 cites W1976942018 @default.
• W2023951259 cites W1982332591 @default.
• W2023951259 cites W1983481853 @default.
• W2023951259 cites W1985789743 @default.
• W2023951259 cites W1988903428 @default.
• W2023951259 cites W1998166016 @default.
• W2023951259 cites W2003474943 @default.
• W2023951259 cites W2008161863 @default.
• W2023951259 cites W2020316950 @default.
• W2023951259 cites W2027127436 @default.
• W2023951259 cites W2037626742 @default.
• W2023951259 cites W2040572245 @default.
• W2023951259 cites W2041525823 @default.
• W2023951259 cites W2041576624 @default.
• W2023951259 cites W2049525996 @default.
• W2023951259 cites W2053858732 @default.
• W2023951259 cites W2054565917 @default.
• W2023951259 cites W2065399078 @default.
• W2023951259 cites W2070673941 @default.
• W2023951259 cites W2074820885 @default.
• W2023951259 cites W2077888296 @default.
• W2023951259 cites W2091783976 @default.
• W2023951259 cites W2100117872 @default.
• W2023951259 cites W2110357545 @default.
• W2023951259 cites W2162688081 @default.
• W2023951259 cites W2170548924 @default.
• W2023951259 doi "https://doi.org/10.1046/j.1365-2567.2000.00030.x" @default.
• W2023951259 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2327007" @default.
• W2023951259 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10886399" @default.
• W2023951259 hasPublicationYear "2000" @default.
• W2023951259 type Work @default.
• W2023951259 sameAs 2023951259 @default.
• W2023951259 citedByCount "11" @default.
• W2023951259 countsByYear W20239512592014 @default.
• W2023951259 countsByYear W20239512592015 @default.
• W2023951259 countsByYear W20239512592018 @default.
• W2023951259 countsByYear W20239512592021 @default.
• W2023951259 crossrefType "journal-article" @default.
• W2023951259 hasAuthorship W2023951259A5042350027 @default.
• W2023951259 hasAuthorship W2023951259A5048447114 @default.
• W2023951259 hasBestOaLocation W20239512591 @default.
• W2023951259 hasConcept C185592680 @default.
• W2023951259 hasConcept C190283241 @default.
• W2023951259 hasConcept C2777851122 @default.
• W2023951259 hasConcept C41625074 @default.
• W2023951259 hasConcept C55493867 @default.
• W2023951259 hasConcept C61322309 @default.
• W2023951259 hasConcept C86803240 @default.
• W2023951259 hasConcept C95444343 @default.
• W2023951259 hasConceptScore W2023951259C185592680 @default.
• W2023951259 hasConceptScore W2023951259C190283241 @default.
• W2023951259 hasConceptScore W2023951259C2777851122 @default.
• W2023951259 hasConceptScore W2023951259C41625074 @default.
• W2023951259 hasConceptScore W2023951259C55493867 @default.
• W2023951259 hasConceptScore W2023951259C61322309 @default.
• W2023951259 hasConceptScore W2023951259C86803240 @default.
• W2023951259 hasConceptScore W2023951259C95444343 @default.
• W2023951259 hasIssue "2" @default.
• W2023951259 hasLocation W20239512591 @default.
• W2023951259 hasLocation W20239512592 @default.
• W2023951259 hasLocation W20239512593 @default.
• W2023951259 hasOpenAccess W2023951259 @default.
• W2023951259 hasPrimaryLocation W20239512591 @default.
• W2023951259 hasRelatedWork W1492356485 @default.
• W2023951259 hasRelatedWork W1969158591 @default.
• W2023951259 hasRelatedWork W1972090215 @default.
• W2023951259 hasRelatedWork W1977563244 @default.
• W2023951259 hasRelatedWork W1994004807 @default.
• W2023951259 hasRelatedWork W2049114455 @default.
• W2023951259 hasRelatedWork W2093676869 @default.
• W2023951259 hasRelatedWork W2118560029 @default.
• W2023951259 hasRelatedWork W2136238444 @default.
• W2023951259 hasRelatedWork W4252607908 @default.
• W2023951259 hasVolume "100" @default.
• W2023951259 isParatext "false" @default.
• W2023951259 isRetracted "false" @default.
• W2023951259 magId "2023951259" @default.
• W2023951259 workType "article" @default.