Therapeutic application of T cell receptor mimic peptides or cDNA in the treatment of T cell-mediated skin diseases.

An 8-amino acid peptide encoding a sequence of the transmembrane region of the T cell receptor alpha chain (TCR-alpha) was shown to inhibit T cell function by preventing functional assembly of the T cell receptor (mimic peptide). To avoid systemic immunosuppression by peptide application in vivo, we used a topical application of the peptide. In the system of murine contact sensitivity, topical application of the peptide inhibited the elicitation of contact sensitivity following application of a contact allergen in sensitized animals. Alternatively, when naked DNA encoding the peptide sequence was injected into skin before application of a contact allergen to sensitized animals, local immunosuppression was also observed. To investigate the effects of this peptide in humans, patients with psoriasis, atopic eczema, lichen planus, or contact dermatitis were treated topically with mimic peptide or control peptide. All patients except for one reported a marked improvement or cure of their skin disease following application of the TCR-alpha peptide, but not controls. These data indicate that TCR-alpha peptide or cDNA treatment might be a proper treatment for human T cell-mediated dermatoses substituting for corticosteroids.

Regulatory elements of the leukaemia inhibitory factor (LIF) promoter in murine bone marrow stromal cells.

Leukaemia inhibitory factor (LIF) plays an important role as a haematopoietically active cytokine. As described earlier in a murine model, interleukin 1 (IL-1) induced LIF mRNA and protein expression. We utilized the murine cell line +/+-1.LDA11 to further define regulatory mechanisms of LIF expression in bone marrow stromal cells. The production of LIF mRNA is stimulated by IL-1beta, TNF-alpha, and the cAMP analogue 8-bromoadenosine 3':5'-monophosphate (8BrcAMP). LIF mRNA expression is controlled at the transcriptional level. Different fragments from -542 to -45 bp 5' upstream of the transcriptional start site of the murine LIF gene were fused to the luciferase gene. All LIF-promoter luciferase constructs exhibited constitutive luciferase activity under serum free conditions. The level of luciferase activity decreased with LIF-promoter constructs of less than 249 bp (pLIF249) in size. When tested with the 314 bp LIF-promoter construct, incubation of stromal cells with IL-1beta (500 U/ml) resulted in a 1.57-fold stimulation, with TNF-alpha (500 U/ml) in 2.06-fold stimulation, and with 8BrcAMP (0.5 mM) in a 3. 42-fold stimulation of luciferase activity. By testing different deletion mutants we could narrow the IL-1 and TNF-alpha responsive promoter areas to the region -249 to -145 bp and the 8BrcAMP responsive area from -145 to -82 bp. Mobility shift experiments revealed that nuclear proteins from stromal cells form a DNA-protein complex by binding to the region from -249 to -145 bp of the LIF promoter.

Interferon-alpha (IFN-alpha) inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF) expression at the post-transcriptional level in murine bone marrow stromal cells.

Recently it has been shown that IFN-alpha inhibits expression of GM-CSF in adherent cells of human long-term bone marrow cultures (LTBMC) stimulated with interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) or endotoxin. The murine bone marrow stromal cell line +/+(-1).LDA11 was used to further define regulatory mechanisms of IFN-alpha inhibition on GM-CSF expression. This cell line originated from a murine Dexter type culture and exhibits a preadipocytic phenotype. As in human LTBMC, we could demonstrate a inhibitory effect of IFN-alpha co-incubation on GM-CSF activity in serum-free supernatants of +/+(-1).LDA11 stromal cell cultures stimulated with IL-1 or TNF-alpha or the combination of IL-1 plus TNF-alpha. IFN-alpha inhibitory effect on GM-CSF expression was shown to be dose dependent with minimal response at 10 U/ml and maximal inhibition at a dose of 500 U/ml. Northern blot analysis confirmed these data at the mRNA level. Reprobing of Northern blots for interleukin-6 (IL-6) mRNA showed increased expression after IFN-alpha incubation, demonstrating specific and differential regulatory effects of IFN-alpha on cytokine production in bone marrow stromal cells. Inhibition of GM-CSF mRNA by IFN-alpha was time dependent, starting at about 90-120 min post-treatment. Cycloheximide (CHX) incubation abolished the inhibitory effect of IFN-alpha on GM-CSF expression, suggesting the requirement of a labile protein. Reporter gene studies were used in order to evaluate the effect of IFN-alpha incubation on GM-CSF mRNA transcription in stromal cells. For this purpose, GM-CSF promoter fragments were subcloned into a luciferase expression vector. Neither constitutive nor TNF-alpha stimulated GM-CSF transcription was inhibited by IFN-alpha coincubation. On the other hand, actinomycin-D chase experiments revealed a reduced GM-CSF mRNA stability after IFN-alpha incubation. The induction of a RNAase, possibly a 2-5A-dependent RNAase, by IFN-alpha may be a possible cause for the increased GM-CSF mRNA decay. These results show a regulatory role for IFN-alpha in the bone marrow microenvironment possibly involved in the myelosuppressive effect of IFN-alpha therapy or viral infections.