Cyclosporin G and metabolite binding to cyclophilin and a 50-kDa binding protein related to in vitro immunosuppression.

Seven purified metabolites of cyclosporin G (CsG) were studied for binding to cyclophilin and a 50-kDa binding protein (50-kDa BP). The ratios of the metabolite dissociation constants with respect to CsG were compared with in vitro immunosuppression by using the primary mixed lymphocyte suppression assay. The immunosuppressive potency ratio of the parent compounds, both cyclosporin A (CsA) and CsG, compared favorably with the drug dissociation constants for cyclophilin and the 50-kDa BP. Three of the seven metabolites had comparable binding and potency ratios for the 50-kDa BP. In contrast, none of the seven metabolites appreciably bound to cyclophilin in the concentration range tested.

Development of a radioreceptor assay to measure glucocorticoids.

We have developed a radioreceptor assay to measure glucocorticoids. The assay employs the partially purified 95-kDa receptor isolated from human liver and purified by size fractionation on high-performance liquid chromatography (HPLC). In the assay [3H]prednisolone competes with steroids (endogenous and exogenous) for binding to the receptor. Bound and free are separated by treatment with charcoal. The between-day precision [% coefficient of variation (CV)] at concentrations of 9.4, 18.7, and 69.9 micrograms/L prednisolone is 16.6, 9.3 and 4.5%, respectively. Specificity studies revealed that hydrocortisone, deoxycorticosterone, 4-pregnene-17 alpha,21-diol-3,20-dione, 17 alpha-hydroxyprogesterone, corticosterone and beta-hydroxyprogesterone all compete with [3H]prednisolone for binding to the receptor. Prednisone and 6 alpha-methyl prednisolone displace [3H]prednisolone to only a minor degree. The assay has been used to assess "glucocorticoid activity" in children with rheumatic diseases treated with prednisolone.

cis-urocanic acid down-regulates the induction of adenosine 3',5'-cyclic monophosphate by either trans-urocanic acid or histamine in human dermal fibroblasts in vitro.

It has been demonstrated that UVB radiation (290-320 nm) suppresses mammalian cell-mediated immunity by effecting the trans to cis isomerization of urocanic acid (UCA) in the stratum corneum, the uppermost layer of the skin. Trans-urocanic acid has been shown to be the photoreceptor for UVB-induced immune suppression and the cis-isomer has been demonstrated to be immunosuppressive. Little is known, however, about how the isomerization of UCA may affect the proximal or distal cells of the skin or the immune system. We report here that trans-UCA is biologically active in vitro in human dermal fibroblasts, inducing adenyl cyclase as measured by cAMP (adenosine 3',5'-cyclic monophosphate) formation in a dose-dependent manner similar to the action of histamine. Trans-UCA and histamine stimulate 50% of maximum activity at concentrations of 3.3 microM and 13.8 microM respectively. Cis-UCA does not increase cAMP in these human fibroblasts but actively down regulates the increase of cAMP induced by either histamine or trans-UCA. Cis-UCA down regulated the histamine response by 75% and the trans-UCA response by 60% at a concentration range of 1 mM to 1 nM. The trans-UCA induction of cAMP can also be downregulated with an H2 histamine receptor antagonist cimetidine. These results support the hypothesis that a cellular target for cis-UCA is the dermal fibroblast and the effects reported here may represent the initial biochemical and cellular event for UVB-induced immune suppression i.e. the immediate step following the isomerization of trans to cis-UCA is the down regulation of cAMP by cis-UCA. Regulation of such an important second messenger such as cAMP could then allow cascading signals to occur, leading to immune suppression.

Correlation of cyclosporine and metabolite binding to cyclophilin and a 50 kDa binding protein with in vitro immunosuppression: a preliminary report.

Seven purified cyclosporine (CsA) metabolites were analyzed for binding to cyclophilin and to a 50 kDa protein purified from a JURKAT cell line. In addition, the potency of the seven metabolites, relative to CsA, was obtained using a primary mixed lymphocyte culture (MLC) suppression assay. CsA, M1, 17, and 21 were found to be immunosuppressive in the concentration range used (0-500 ng/mL). These results were then compared to protein binding. CsA and metabolite 17 (M17) bound to both proteins. Conversely, M1, 13, 21, and 26 bound only to cyclophilin, while M8 and M18 bound only to the 50 kDa protein.

Purification and characterization of cyclosporine and FK-506 binding proteins from a human T-helper cell line.

Cytosolic proteins that specifically bind cyclosporine A and FK-506 were isolated and purified from the JURKAT human T-helper cell line. These binding proteins were purified by affinity, molecular weight exclusion and weak cation exchange column chromatography. Radiolabeled cyclosporine A specifically bound to a approximately 17 kDa molecule which is cyclophilin and also bound to a approximately 50 kDa protein(s). Radiolabeled FK-506 did not bind to the approximately 17 kDa molecular weight protein, but specifically bound to soluble approximately 10 kDa and approximately 50 kDa proteins.

A preliminary study to evaluate an in vitro assay for determining patient whole blood immunosuppressive cyclosporine A and metabolite activity: comparison with cytosolic binding assays using cyclophilin or a 50-kilodalton binding protein, and the Abbott TDx cyclosporine A parent, and parent and metabolites assays.

Thirty-five allograft recipients undergoing cyclosporine A (CsA) therapy were randomly selected to evaluate a "novel" in vitro assay that determines CsA and metabolite immunosuppressive activity in whole blood. The assay uses a third party mixed lymphocyte culture (MLC) system to which patient whole blood extracts containing CsA and metabolites are added. The ability of the extracted CsA and metabolites to inhibit proliferation in this system is proportional to the immune suppressive activity in whole blood. Comparison of the MLC suppression assay against Abbott TDx parent, TDx parent and metabolites, and radioreceptor assays utilizing cyclophilin or a 50-kDa binding protein isolated from JURKAT cytosol gave the following correlation coefficients: r = 0.612, r = 0.672, r = 0.362, and r = 0.775, respectively.

Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action.

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1-induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1.

Synthetic C-terminal peptide of IL-1 functions as a binding domain as well as an antagonist for the IL-1 receptor.

Fragments of IL-1 beta were chemically synthesized and tested for biological activity as well as binding of radiolabelled peptides to the IL-1 receptor. A peptide from the extreme C-terminal region of IL-1 beta was found to antagonize intact, native IL-1 beta in the thymocyte bioassay. In addition, this C-terminal region peptide, when radiolabelled, can function as a ligand for the IL-1 receptor on murine cell lines and effectively compete with intact radiolabelled recombinant IL-1 beta.

Mast cells induced in vitro by interleukin 3 from native murine thymus cells.

To shed further light on the induction and characterization of thymus-derived mast cells, we cultured a variety of cell populations from murine thymus tissues (Balb/c) in the presence or absence of interleukin 3 (IL-3). The whole cell population and the non-adherent T cell-depleted population developed mast cells. The morphological studies revealed granulated cells; the granules were stained with toluidine blue, alcian blue (pH 3.0), and metachromatic dyes. Electron microscopy revealed altered mast cell granules. These cells contained relatively low amounts of histamine (approximately 1700 ng/10(6) cells), were IL-3 (but not IL-2)-dependent, and did not possess T-cell, B-cell, or macrophage markers. No phagocytosis was observed. The cells also had 20 alpha-hydroxysteroid dehydrogenase, and both IL-3 and IgE (145,600/cell) high affinity receptors. The frequency analysis showed 17 precursor cells per 10(6) thymic cells. The results indicate that the thymus indeed contains progenitors of mast cells responsive to IL-3, and that the mast cells are derived from non-T, non-phagocytic, and non-adherent cells of the thymus. Their T-lymphocyte product (IL-3) dependency, ultrastructural appearance, granular stainability, and low content of histamine may support the view that the mast cells originating from the thymus probably belong to a mucosal mast cell lineage.

Biologic effects of prolonged melphalan treatment of murine long-term bone marrow cultures and interleukin 3-dependent hematopoietic progenitor cell lines.

The mechanism of alkylating agent-induced leukemia is unknown. For the determination of whether chronic alkylating agent treatment of hematopoietic stem cells in vitro was detectably leukemogenic, murine long-term bone marrow cultures (LTBMC) and clonal interleukin 3 (IL-3)-dependent multipotential hematopoietic progenitor cell lines [B6SUtA clone (cl) 27 and Ro cl 3-1] derived from LTBMC were chronically pulse treated in vitro with the alkylating agent melphalan [L-phenylalanine mustard (L-PAM)]. Weekly treatment of C3H/HeJ or CD-1 Swiss mouse LTBMC with 3 X 10(-6)M L-PAM significantly decreased cumulative production of nonadherent granulocytes and granulocyte-macrophage progenitor cells responsive to L-cell or WEH1-3 cell colony-stimulating factor compared to the production seen in untreated control cultures; it also significantly reduced the hematopoietic longevity (13 wk compared to greater than 20 wk for untreated control cultures). Weekly, twice weekly, or daily (3 X 10(-6)M) L-PAM treatment of IL-3-dependent cell lines induced gradual L-PAM adaptation in the absence of a detectable change in the maximum binding capacity of 125I-labeled IL-3. No leukemogenic variants of line B6SUtA cl 27 were detectably induced. However, 3 stably expressed marker chromosomes were induced after 12 months of L-PAM treatment of line B6SUtA cl 27. Thus IL-3-dependent hematopoietic progenitor cells slowly adapt to L-PAM when in suspension culture in vitro. Physiologic expression of drug toxicity in LTBMC may prevent this hematopoietic cell gradual adaptation.

Evidence for specific receptors for interleukin 3 on lymphokine-dependent cell lines established from long-term bone marrow cultures.

Utilizing 125I-labelled IL 3, an assay for specific IL 3 receptors on continuous cell lines was developed. The 32D-cl 23 and FDC-P1 cell lines examined were originally derived from long-term cultures of murine bone marrow supplemented with WEHI-3-conditioned media. Both of these cell lines have been shown to be absolutely dependent on IL 3, a component of WEHI-3-conditioned media, for growth in vitro. The binding of radiolabeled IL 3 to these cells was found to be specific, saturable, reversible, and time and temperature dependent. The specificity of IL 3 binding was demonstrated by the failure of a number of different lymphokines, hormones, and proteins to compete for the binding of radiolabeled IL 3. The IL 3-dependent cell lines uniquely express detectable receptors, whereas other cell lines of various types and origin known to be IL 3 independent had no detectable specific binding of IL 3. A Kd of 5.4 X 10(-11) M and a maximum binding capacity of 6.25 fmol per 1 X 10(6) cells was calculated for 32D-cl 23 from equilibrium binding studies. A similar analysis for FDC-P1 yielded a Kd of 1.7 X 10(-11) and a maximum binding capacity of 1.2 fmol per 1 X 10(6) cells. The data are consistent with the presence of 4000 to 5000 specific high-affinity binding sites/cell on 32D-cl 23, whereas FDC-P1 has 1500 to 2500 specific sites/cell. The Kd calculated for the FDC-P1 cell line from kinetic binding data was 2.5 X 10(-11) M, which agreed well with the Kd of 1.7 X 10(-11) M calculated from equilibrium data.

Thymosin alpha 1-like peptides: localization and biochemical characterization in the rat brain and pituitary gland.

Using a radioimmunoassay for thymosin alpha 1, endogenous thymosin-like peptides were characterized in the rat brain and pituitary gland. Thymosin alpha 1-like peptides were present in high concentrations in hypothalamus and pituitary extracts. These peptides were characterized using gel filtration techniques and the main peak of immunoreactive thymosin had a molecular weight similar to that of thymosin alpha 1 (3108 daltons). Using HPLC techniques, one main peak of immunoreactivity was present in brain extracts, whereas two peaks were present in pituitary extracts, one of which coeluted with thymosin alpha 1. The discrete regional distribution of thymosin alpha 1-like peptides was investigated and the highest densities of immunoreactive thymosin were present in the median eminence and arcuate nucleus of the hypothalamus, as well as the neurointermediate lobe of the pituitary. Due to the anatomical proximity of immunoreactive thymosin to loci containing known releasing factors and hormones, thymosin alpha 1-like peptides may function as neuroendocrine regulatory agents.