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G Woerly

Publications and source records attributed to G Woerly.

31 records · Page 2Linked to original sources
PubMed1993

Evidence for shared receptor proteins for human interleukin-3 and granulocyte-macrophage colony-stimulating factor in the human M-07 cell line.

The biologic response of the human leukemia cell line M-07 to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 4 (IL-4) is mediated by a low number of high affinity receptors. Cross-competition studies revealed that IL-3 and GM-CSF partially inhibited the specific binding of the heterologous radiolabeled ligand, whereas IL-4 binding was not affected by these cytokines. The molecular mechanism of cross-competition was investigated by chemical crosslinking and immunoprecipitation. Trimolecular receptor complexes consisting of a major 73kDa and two minor 120 and 128kDa membrane proteins for IL-3, and a major 84kDa and two minor 120 and 130 kDa proteins for GM-CSF were found on M-07 cells. The 73 and 84kDa proteins represent distinct and non-linked membrane proteins and are identical with the cloned, low affinity IL-3 and GM-CSF receptor proteins (Gearing et al, 1989, Hayashida et al, 1990). The higher molecular weight proteins share common binding sites as evidenced by immunoprecipitation of double-crosslinked membranes. The 120/128kDa proteins are most likely identical with the recently cloned and shared beta-subunit of the IL-3 and GM-CSF receptor (Kitamura et al, 1991) containing a single or two IL-3 and/or GM-CSF molecules.

Binding, Competitive↗
PubMed1993

Pathology induced by interleukin-6.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in the immune response, hemopoiesis, and host defense. Recombinant human IL-6 (rhIL-6) was administered at high doses to mice, rats, and nonhuman primates. In all species, IL-6 had an immunostimulatory and hemopoietic (especially on megakaryocytes) effect. An acute-phase response was most pronounced in nonhuman primates, which was, however, not associated with any significant histopathological liver change. Finally, no evidence of glomerular pathology was found. Neutralizing antibodies were detected within 10 days of rhIL-6 administration in all species.

Acute-Phase Reaction↗
PubMed1992

Covalent binding of cyclosporine inhibits irreversibly T-lymphocyte activation.

A diazirine derivative of cyclosporine (PL-CS) was used to photolabel recombinant human cyclophilin (rhCyp), the cytosolic receptor for the immunosuppressant cyclosporine. The affinity of PL-CS for rhCyp and the immunosuppressive activity were 10-fold reduced as compared to cyclosporine A. Whereas cyclosporine immunosuppression was fully reversible, UV cross-linking of PL-CS resulted in permanent inhibition of lymphocyte activation as shown by proliferation of anti-CD3 stimulated human peripheral lymphocyte, interleukin (IL)-2 gene transcription and IL-2 synthesis in the human T-leukemia cell line Jurkat. In vivo photolabeling of viable Jurkat cells revealed that a 21-kDa complex was the major radiolabeled product which was identified as a cyclophilin-cyclosporine complex. In addition, cyclophilin B (25 kDa) and proteins of an unidentified nature at 40, 46 and 60 kDa were observed in Jurkat cells. The cyclosporine-resistant human fibroblast cell line MRC5 displayed a different labeling pattern: cyclophilin B (25 kDa) and a 65-kDa protein were the major labeled products, while the 46- and 60-kDa components were not detectable and cyclophilin was only faintly labeled. In summary, covalent cyclosporine binding caused irreversible lymphocyte inactivation and revealed in addition to cyclophilin other specifically labeled proteins in lymphoid cells. The role and identity of these proteins is presently unknown.

Affinity Labels↗
PubMed1992

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo.

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.

Amino Acid Isomerases↗
PubMed1991

Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for cyclosporine.

The immunosuppressive agent cyclosporine A (CSA) has been shown to reverse multidrug resistance (MDR) in malignant cells. In the present study, a 3H-cyclosporine diazirine analogue (3H-PL-CS) was used to photolabel viable MDR cells. The 170 kDa membrane P-glycoprotein, which functions as a drug efflux pump, was strongly labeled. The binding of 3H-cyclosporine diazirine analogue to P-glycoprotein was competable by excess cyclosporine A and by the nonimmunosuppressive cyclosporine H. These results suggest that cyclosporine reverses the MDR phenotype by binding directly to P-glycoprotein and that this binding is not dependent on the immunosuppressive potential of the cyclosporine derivative. The identification of P-glycoprotein as a cyclosporine binding protein has obvious implications for cancer chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Distribution of the cyclosporine binding protein cyclophilin in human tissues.

Cyclophilin (CYP) is the major intracellular binding protein for the immunosuppressive drug cyclosporine (CS). CYP distribution was investigated in human tissues by solid-phase immunoassay, Western and Northern blot analysis as well as immunohistochemistry. CYP was found in all tissues examined at concentrations in the range of 1 microgram/mg protein. Furthermore, mRNA specific for CYP was found in every tissue, indicating local production of the protein. Immunohistochemical investigations revealed preferential parenchymal and only little stromal localization. Within certain organs, e.g. kidneys, regional differences of immunoreactive CYP was evident. The presence of CYP was also investigated in several lymphoid and non-lymphoid cell lines and was found at comparable concentrations. Immunogold staining confirmed cytosolic, but revealed also nuclear localization of CYP. The possible role of this abundant CS binding protein is discussed.

Amino Acid Isomerases↗
PubMed1990

Inhibition of interleukin 4 receptor expression on human lymphoid cells by cyclosporin.

The effect of the immunosuppressant cyclosporin (CsA) on the expression of interleukin (IL) 4 membrane receptors on human peripheral blood mononuclear cells (PBMC) was investigated after cell activation by anti-CD3 antibody, IL 2 or IL 4. Previous studies with 125I-labeled IL 4 identified on resting lymphocytes a trimolecular complex consisting of a 65/70-kDa doublet and a 110-kDa protein with approximately 300 high-affinity binding sites (Kd 100 pM) and approximately 9000 low-affinity binding sites (Kd 30 nM). Upon cell activation by anti-CD3 antibody both low- and high-affinity binding sites increased about threefold concomitant with up-regulation of all the cross-linked proteins. CsA inhibited anti-CD3 antibody-induced up-regulation of IL 4 receptor (IL 4R)-associated proteins as well as the expression of high-affinity binding sites. However, up-regulation of IL 4R by its own ligand or IL 2 and the growth-promoting effect of IL 4 on activated, IL 4R+ T cells were CsA resistant. Since CsA inhibits the synthesis of IL 4, exogenous IL 4 was added to the cultures and it partially reversed the inhibitory effect of CsA on cell proliferation as well as on IL 4R expression. It is concluded that the inhibitory effect of CsA on IL 4R expression may contribute to the immunosuppressive effect of the drug.

Affinity Labels↗
PubMed1990

Anti-CD3 antibody-induced expression of both p55 and p75 chains of the high affinity interleukin-2 receptor on human T lymphocytes is inhibited by cyclosporin A.

The inhibitory effect of cyclosporin (CsA) was investigated on human lymphocytes stimulated by anti-T-cell antibodies (anti-CD3 and -CD2) or mitogenic lectins. Whereas inhibition of cell proliferation (50%) occurred at 10 ng/ml CsA after cell activation via CD3 or CD2, higher CsA concentrations (300 ng/ml) were necessary to inhibit lectin-mediated cell activation (PHA, Con A). Exogenous recombinant interleukin-2 (rIL-2) partially reversed the inhibitory effect on antibody-stimulated cells only; however, at higher CsA concentrations (300 ng/ml) proliferation was again inhibited. Thus, CsA affected IL-2R expression and/or function at higher concentrations (300 ng/ml). CsA had no effect on receptor function as measured on IL-2-dependent cell growth of CTLL cells or preactivated lymphocytes. However, CsA inhibited both high and low affinity receptor expression as shown by [125I]IL-2 equilibrium binding studies on anti-CD3-stimulated cells. Cross-linking studies revealed that both p55 (TAC) and p75 chains of the IL-2R were not induced at low CsA concentrations (10 ng/ml). However, addition of rIL-2 reversed CsA inhibition of IL-2R expression. It is concluded that CsA, at least in anti-CD3-stimulated cells, inhibits IL-2R expression and cell proliferation with similar potency. Exogenous rIL-2 reverses CsA inhibition of IL-2R expression. This might be due to binding of rIL-2 to receptors which escape CsA inhibition, thereby up-regulating receptor expression which is drug resistant.

Antibodies, Monoclonal↗
PubMed1989

Identification of interleukin 4 receptor-associated proteins and expression of both high- and low-affinity binding on human lymphoid cells.

Interleukin 4 (IL4) produced by activated T cells expresses its biological effects on T and B lymphocytes by binding to specific membrane receptors. Cross-linking of human recombinant 125I-IL4 to peripheral blood mononuclear cells identifies a trimolecular complex consisting of a 65/70-kDa doublet and a 110-kDa protein. Scatchard analysis reveals about 300 IL4 binding sites/cell on resting cells with an equilibrium binding constant (Kd) of approximately 100 pM. Stimulation by anti-CD3 antibodies causes an up-regulation of IL4 receptors by a factor of 2 to 3 without any change in binding affinity. In addition to this high-affinity binding site a second class of a previously unidentified, low-affinity receptor (Kd approximately 30 nM, approximately 9000 sites/cell) is expressed on resting lymphocytes. The number of low-affinity binding sites for IL 4 also increases twofold upon cell activation. Exogenous IL 4 enhances the expression of its receptor on resting lymphocytes and this effect is further increased by anti-CD3 activation. Binding of IL4 to its receptor is specific, being only inhibited by IL 4 and not by IL2. By contrast, the gibbon leukemia cell line MLA 144 expresses only high-affinity receptors for IL4. Cross-linking studies reveal a 45/50-kDa IL 4 receptor-associated doublet in addition to the three proteins identified in human peripheral blood mononuclear cells. The functional significance of the different proteins composing the receptor for IL4 is discussed.

Animals↗
PubMed1988

Cyclosporine--relationship of side effects to mode of action.

Although cyclosporine has high specificity for the immune system, immunosuppressive therapy with CsA is often complicated by nephrotoxicity. The main morphologic targets of CsA nephrotoxicity include the tubular epithelial and endothelial cells. These cells were investigated in vitro. CsA caused a dose- and time-dependent inhibition of cell growth, vacuolization and fatty change in adherent cells, detachment, and cell death. Inhibition of 3H-TdR incorporation in cells of both tubular epithelial and endothelial origin occurred between 3 microM and 10 microM. Electron microscopy studies revealed cellular swelling, dilatation of the endoplasmic reticulum, and the presence of lipid droplets and giant mitochondria. The content of the main CsA-binding protein, cyclophilin, in these cell-lines was 5-10 micrograms/mg protein and did not differ in various cell lines, including T cells. Immunohistochemistry using rabbit anticyclophilin antibody revealed diffuse distribution of cyclophilin in the cytosol, nuclear membrane, and nucleolus. Whereas lymphoid cell functions are inhibited at 10-100 nM, CsA had no effect on tubular epithelial and endothelial cells at these concentrations. At concentrations of 3-10 microM, CsA caused growth inhibition and cytotoxicity on cells of lymphoid and nonlymphoid origin. Present evidence shows little, if any, relationship of side-effects to the mode of action of CsA.

Animals↗
PubMedDate not supplied

Expression of Th1 and Th2 immunoregulatory cytokines by human eosinophils.

Besides cytotoxic mediators, human eosinophils can produce proinflammatory and anti-inflammatory cytokines, as well as growth factors and chemokines. The demonstration that eosinophils from patients could produce IL-5, IL-4 and IL-2 suggested their participation in the regulation of immune response. In the present work, we have examined the presence of Th1 (IFN-gamma, IL-2) and Th2 (IL-4, IL-5, IL-10 and IL-13) cytokines in eosinophils from patients or donors by intracellular flow cytometry, and by immunocytochemistry. Whereas almost 100% eosinophils expressed intracellular IFN-gamma, IL-2 and IL-10, the expression of IL-4, IL-5 and IL-13 is more variable and increased in patients versus donors. The differential release by eosinophils of Th2 versus Th1 cytokines is suggested both by the decrease of the intracellular content after culture, restricted to IL-4, IL-5 and IL-13, associated with an accumulation of IL-4 and IL-5 in the presence of Brefeldin. These results indicate that, through the preferential release of Th2 cytokines, human eosinophils can participate in the polarization of the immune response.

Animals↗
PubMedDate not supplied

Differentiation of eosinophils from cord blood cell precursors: kinetics of Fc epsilon RI and Fc epsilon RII expression.

Expression of Fc epsilonRI and Fc epsilonRII/CD23 was examined by immunocytochemistry and flow cytometry on eosinophils differentiated from human cord blood cells in the presence of human interleukin-3 (rhIL-3), granulocyte/macrophage colony stimulating factor (rhGM-CSF) and interleukin-5 (rhIL-5) and on blood eosinophils purified from normal donors or patients with idiopathic hypereosinophilic syndrome (HES). On cord blood derived eosinophils, Fc epsilonRI expression started at 1 week of culture and increased to reach a plateau at 3 weeks of culture. Fc epsilonRII/CD23 appeared slightly later, after 2 weeks of culture, and the percentage of Fc epsilonRII/CD23-positive eosinophilic cells increased and stayed in plateau. Fc epsilonRI expression on cord blood derived eosinophils was downregulated after culture with interleukin-2 (rhIL-2), interleukin-4 (rhIL-4), rhIL-5, interferon-alpha (rhIFN-alpha), interferon-gamma (rhIFN-gamma). In contrast, the expression of Fc epsilonRII/CD23 on cord blood derived eosinophilic cells was upregulated after culture with rhIL-4, rhIL-5 and rhIFN-gamma, and downregulated with rhIL-2 and rhIFN-alpha. Fc epsilonRI was expressed on about 30% normal donor eosinophils as well as on normodense eosinophils from HES patients but significantly decreased on hypodense eosinophils. In contrast, Fc epsilonRII/CD23, expressed on a very small proportion of normal donor eosinophils, increased from normodense to hypodense eosinophils. These results suggest that Fc epsilonRI on eosinophils might represent one differentiation antigen expressed relatively early, with decreased expression through maturation or activation, whereas Fc epsilonRII/CD23 might rather be considered as a marker of eosinophil activation.

Cell Differentiation↗