- Email: [email protected]
A radioreceptor assay for platelet-derived growth factor (PDGF): Application to PDGF release from platelets and monocytes
Suppl. XIII, 1991
ABSTRACTS
85
169 A RADfORECEF’TOR ASSAY FOR PLATELET-DERIVED Gm FACTOR (FQGF): APPUCATION TO PDGF RELEASE FROM PLATELETS AND MONOCYTES. D. An Yan, R. Sbatbati. G. Lazzerini, W. Bernini, R. Sic& MC. Cenni. M. Scarlattini, R. Ds Caterina. CNR Institute of Clinical Physiology. Pisa, My Platelet-derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF-like proteins are blood platelets, megakaryooytes. several transformed cell lines, monocytes and cultured endothelial cells. We have developed a radioreceptor assay to measure PDGF-like protein released by stfmulated platelets and monocytes. Subcultured confluent Swiss 3T3 cells, placed in serum-free medium (Eagle’s MEM) containing Pmg/ml bovine serum albumin 24 hrs before the assay, and layered in 6-well polystyrene plates, were used as a source of high-affinity, specific receptors for PDGF. 1251-labelled PDGF B-chain homodymer, specific activity >lOOO Cilmmol, was diluted with the medium to a final concentration of 12.5 pM. selected for optimal binding out of a concentration range from 3.1 to 600 pM. Recombinant PDGF B. O-600 pM final concentration (O-24 ng/tube), was used as standard for the calibration curve; 100-200 pl aliquots of unknown samples wsre used in an incubation volume of 1 ml. A preincubation (1 hr at 4°C with agitation) without tracer, followed by a 4 hr incubation with tracer in the same conditions, improved the assay sensitivity. After 3 washings, cells were solubilized with Triton X-100 and radioactivity counted. Binding capacity was (meanfSD, n=ll)lg.2f3.3%; detection limit was 0.56M.26 ng; nonspecific binding, evaluated by adding a large excess of standard PDGF (3000 PM) to the incubation mixture, was 0.7M.3%. Parallelism and recovery tests, together with the evaluation of the extent of cross-reaction with other growth factors (FGF, EGF, TGF-6, somatomedin C). were also performed to check the accuracy of the assay system. Time-course of PDGF accumulation in serum from whole blood incubated at 37°C up to 1 hour, showed a progressive increase of PDGF concentration up to 10.6f2.5 nglml, parallel to the accumulation of other alpha-granule proteins like beta-thromboglobulin an platelet factor 4. A plateau of production occurs earlier (30 min.) than production of thromboxane B2 (for which plateau is reached at 1 hour, and are not inhibited by aspirin. Monocytes from peripheral blood, obtained by sedimentation on Ficoll-Hypaque. rendered free of resetting pfatelek by incubation with autologous serum plus EDTA, are separated from lymphocytes by adherence to plastic. Measured level of PDGF-like protein was 1.6K).46 nglml in basal conditions. PDGF measurements have been also performed on monocytes stfmulated. by E. Coli extract at 37°C for several times up to 24 hours. The proposed method is a sensitive and reliable assay for PDGF-like material produced by human platelets and monocytes.
170
B (LTBq) PRODUCTION BY F'OLYHORPHONCJCLEAR RWAIRED LEUKOTRIENE CELIS (PHNs) IN CIRRBOSih (C): POSSIBLE ROLE OF'DRFECTIVE CR&LCELL gNTERACTION#. P. GFe,s+e, E. Ouero, V. Carloni, G. Laffi, F. MarXa, P. Gentlllnl, G.G. Nenci. Semeiotica Medica, Univ Perugia, Clin Medica II, Univ Florence, Italy. An impaired phagocytosis and chemotaxis of PMNs of C has been reported. We studied the capacity of PMNs of C patients and of healthy controls (HC), isolated or in whole blood, to produce LTB4 (measured by RIA) upon stimulation with A23187 or with STZ; PMNS. results (mean+SEM) are expressed as ng/lO Isolated PMNs A23(uM) Whole Blood A23(uM) HS(n=8) c(n=9) C(n=9) HS(n=8) zoo5 2.1f0.4 0.2 4.8kO.4 .0: 3.OkO.5 5.5k1.2 5 6.2+0.8 n.s 7.7kl.O 2 .025 18.9k3.6 27.8+1.8 10 8.6kO.7 n.s 9.3fl.O .0005 10 46.5k5.9 92.3+11 STZ(mg/ml) ZZ(mg/ml) 0.08f0.01 n.s 0.8kO.4 2.5 5.1k0.5 n.s 6.7k1.3 0.25 0.4f0.04 n.s 1.2f0.4 5 16.8k2.3 .Ol 1.25 26.1k2.6 0.5kO.02 .025 1.9kO.5 10 .05 22.2k2.9 31.7k3.4 2.5 C PMNs produce less LTB4; with A23187, which activates both PMN and platelet metabolism, the defect is greater with whole blood than with isolated PMNs while this is not the case with STZ, a PMN-specific stimulus. An intrinsic PMN defect and an altered cell-cell interaction, possibly involving defective platelets, can contribute to the impaired synthesis of LTB4 in C.
ABSTRACTS
85
169 A RADfORECEF’TOR ASSAY FOR PLATELET-DERIVED Gm FACTOR (FQGF): APPUCATION TO PDGF RELEASE FROM PLATELETS AND MONOCYTES. D. An Yan, R. Sbatbati. G. Lazzerini, W. Bernini, R. Sic& MC. Cenni. M. Scarlattini, R. Ds Caterina. CNR Institute of Clinical Physiology. Pisa, My Platelet-derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF-like proteins are blood platelets, megakaryooytes. several transformed cell lines, monocytes and cultured endothelial cells. We have developed a radioreceptor assay to measure PDGF-like protein released by stfmulated platelets and monocytes. Subcultured confluent Swiss 3T3 cells, placed in serum-free medium (Eagle’s MEM) containing Pmg/ml bovine serum albumin 24 hrs before the assay, and layered in 6-well polystyrene plates, were used as a source of high-affinity, specific receptors for PDGF. 1251-labelled PDGF B-chain homodymer, specific activity >lOOO Cilmmol, was diluted with the medium to a final concentration of 12.5 pM. selected for optimal binding out of a concentration range from 3.1 to 600 pM. Recombinant PDGF B. O-600 pM final concentration (O-24 ng/tube), was used as standard for the calibration curve; 100-200 pl aliquots of unknown samples wsre used in an incubation volume of 1 ml. A preincubation (1 hr at 4°C with agitation) without tracer, followed by a 4 hr incubation with tracer in the same conditions, improved the assay sensitivity. After 3 washings, cells were solubilized with Triton X-100 and radioactivity counted. Binding capacity was (meanfSD, n=ll)lg.2f3.3%; detection limit was 0.56M.26 ng; nonspecific binding, evaluated by adding a large excess of standard PDGF (3000 PM) to the incubation mixture, was 0.7M.3%. Parallelism and recovery tests, together with the evaluation of the extent of cross-reaction with other growth factors (FGF, EGF, TGF-6, somatomedin C). were also performed to check the accuracy of the assay system. Time-course of PDGF accumulation in serum from whole blood incubated at 37°C up to 1 hour, showed a progressive increase of PDGF concentration up to 10.6f2.5 nglml, parallel to the accumulation of other alpha-granule proteins like beta-thromboglobulin an platelet factor 4. A plateau of production occurs earlier (30 min.) than production of thromboxane B2 (for which plateau is reached at 1 hour, and are not inhibited by aspirin. Monocytes from peripheral blood, obtained by sedimentation on Ficoll-Hypaque. rendered free of resetting pfatelek by incubation with autologous serum plus EDTA, are separated from lymphocytes by adherence to plastic. Measured level of PDGF-like protein was 1.6K).46 nglml in basal conditions. PDGF measurements have been also performed on monocytes stfmulated. by E. Coli extract at 37°C for several times up to 24 hours. The proposed method is a sensitive and reliable assay for PDGF-like material produced by human platelets and monocytes.
170
B (LTBq) PRODUCTION BY F'OLYHORPHONCJCLEAR RWAIRED LEUKOTRIENE CELIS (PHNs) IN CIRRBOSih (C): POSSIBLE ROLE OF'DRFECTIVE CR&LCELL gNTERACTION#. P. GFe,s+e, E. Ouero, V. Carloni, G. Laffi, F. MarXa, P. Gentlllnl, G.G. Nenci. Semeiotica Medica, Univ Perugia, Clin Medica II, Univ Florence, Italy. An impaired phagocytosis and chemotaxis of PMNs of C has been reported. We studied the capacity of PMNs of C patients and of healthy controls (HC), isolated or in whole blood, to produce LTB4 (measured by RIA) upon stimulation with A23187 or with STZ; PMNS. results (mean+SEM) are expressed as ng/lO Isolated PMNs A23(uM) Whole Blood A23(uM) HS(n=8) c(n=9) C(n=9) HS(n=8) zoo5 2.1f0.4 0.2 4.8kO.4 .0: 3.OkO.5 5.5k1.2 5 6.2+0.8 n.s 7.7kl.O 2 .025 18.9k3.6 27.8+1.8 10 8.6kO.7 n.s 9.3fl.O .0005 10 46.5k5.9 92.3+11 STZ(mg/ml) ZZ(mg/ml) 0.08f0.01 n.s 0.8kO.4 2.5 5.1k0.5 n.s 6.7k1.3 0.25 0.4f0.04 n.s 1.2f0.4 5 16.8k2.3 .Ol 1.25 26.1k2.6 0.5kO.02 .025 1.9kO.5 10 .05 22.2k2.9 31.7k3.4 2.5 C PMNs produce less LTB4; with A23187, which activates both PMN and platelet metabolism, the defect is greater with whole blood than with isolated PMNs while this is not the case with STZ, a PMN-specific stimulus. An intrinsic PMN defect and an altered cell-cell interaction, possibly involving defective platelets, can contribute to the impaired synthesis of LTB4 in C.