title: Selective down-regulation of T cell- and non-T cell-derived tumour necrosis factor alpha by thalidomide: comparisons with dexamethasone.
abstract: Both thalidomide and dexamethasone have been shown to inhibit the production of tumour necrosis factor alpha (TNF-alpha), but little is known of their cellular selectivity. Inhibition of monocyte TNF-alpha expression has been implicated in the clinical efficacy of thalidomide, and it has been suggested that the drug modulates only monocyte-derived cytokines. Given the importance of T lymphocyte responses in immunological disorders in which treatment with thalidomide has been successful, it is pertinent to study the effects of this drug on T cell-derived TNF-alpha. In the present investigations we have examined the influence of both thalidomide and dexamethasone on mitogen-induced elaboration of TNF-alpha by CD3+ peripheral blood mononuclear cells (PBMC) and the T cell line MOLT-4. PBMC from healthy human volunteers were stimulated optimally with phytohaemagglutinin (PHA) in the presence of varying concentrations of thalidomide or dexamethasone, and supernatants assayed for TNF-alpha and interleukin 2 (IL-2). Concurrently, PHA-stimulated PBMC were treated with 1 x 10(-1) mM thalidomide or dexamethasone and the cells fixed, permeabilised, stained with anti-CD3 and anti-TNF-alpha fluorescently labelled antibodies and analysed by flow cytometry. MOLT-4 cells were cultured in the presence or absence of the drugs following activation with phorbol myristate acetate (PMA)/ionophore, and supernatants analysed by enzyme-linked immunosorbent assay (ELISA) for cytokine expression. Thalidomide was found to inhibit PBMC-derived TNF-alpha, but not IL-2. In contrast, dexamethasone down-regulated both TNF-alpha and IL-2 in a dose-dependent manner. Thalidomide and dexamethasone both suppressed intracellular levels of TNF-alpha in CD3+ PBMC, reducing percentages of double positive staining cells by 28 and 52%, respectively, compared with controls. In addition, TNF-alpha production by CD3- PBMC was inhibited by 31% by thalidomide and by 47% by dexamethasone. In order to determine whether thalidomide was acting directly on T cells, or indirectly through effects on accessory cells, TNF-alpha production in the T cell line MOLT-4 was investigated. TNF-alpha secretion by PMA/ionophore activated MOLT-4 cells was reduced by 80% following thalidomide treatment and close to background levels following dexamethasone treatment. To verify that thalidomide was acting selectively to down-regulate TNF-alpha, IL-2 production by MOLT-4 cells was also measured and found to be unaffected by the drug. In contrast, dexamethasone reduced MOLT-4-derived IL-2 levels by 20%. These observations suggest that thalidomide, in addition to its known inhibitory effect on monocyte-derived TNF-alpha, is capable also of down-regulating T cell-derived TNF-alpha in a direct and selective manner. In addition, the inhibition of intracellular levels of TNF-alpha strengthens the evidence that the inhibitory effect of thalidomide is at the level of transcription and/or translation and does not reduce cellular TNF-alpha secretion. Such effects could explain the efficacy of thalidomide treatment in various immunological disorders where T cell activation plays an important role in the pathogenesis of the disease.
authors.affiliation: Medical Research Council Centre, Cambridge, UK.
authors.forename: T L
authors.initials: TL
authors.lastname: Rowland
authors.forename: S M
authors.initials: SM
authors.lastname: McHugh
authors.forename: J
authors.initials: J
authors.lastname: Deighton
authors.forename: P W
authors.initials: PW
authors.lastname: Ewan
authors.forename: R J
authors.initials: RJ
authors.lastname: Dearman
authors.forename: I
authors.initials: I
authors.lastname: Kimber
mesh_terms: D017252:CD3 Complex / Q000032:analysis; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007376:Interleukin-2 / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000187:drug effects; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects*; D013792:Thalidomide / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D017252:CD3 Complex; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D014409:Tumor Necrosis Factor-alpha; D013792:Thalidomide; D003907:Dexamethasone