Properties of a specific interleukin 1 (IL 1) receptor on human Epstein Barr virus-transformed B lymphocytes: identity of the receptor for IL 1-alpha and IL 1-beta

The properties of specific human interleukin 1 (IL 1) receptors on human Epstein Barr virus-transformed B lymphocytes (EBV-B) were studied. Purified human IL 1-beta from a myelomonocytic cell line (THP-1) was labeled with 125I by the Bolton-Hunter method without detectable loss of biological activity. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the highest amount of 125I-IL 1-beta. Maximal binding was reached within 20 min at 4 degrees C. Scatchard plot analysis of the binding of 125I-IL 1-beta to VDS-O cells yielded a Kd (dissociation constant) of 2.4 to 5.9 X 10(-10) M with 110 to 220 binding (receptor) sites/cell. The binding of 125I-IL 1-beta to VDS-O cells was also inhibited by F(ab)'2 fragments of anti-human IL 1 and recombinant human IL 1-alpha, as well as by unlabeled human IL 1-beta but not by recombinant lymphotoxin, recombinant tumor necrosis factor, or phorbol myristic acid, suggesting that IL 1-alpha and IL 1-beta bind specifically to the same receptor. The m.w. of IL 1 receptor on human EBV-B cells was estimated to be 60,000 by both the chemical cross-linking method and high pressure liquid chromatography (HPLC) gel filtration analysis of receptor extracted from membrane enriched fraction by a non-ionic detergent (CHAPS). The isoelectric point of solubilized human IL 1 receptor was 7.3 on HPLC chromatofocusing. The evidence of existence of IL 1 receptor on human EBV-B cells additionally supports the hypothesis that IL 1 may be an autocrine signal for these cells.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

Analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.     

Determination of fragment D-dimer derivatives in serum reactive with antiserum against human fragment D-dimer

A previous study demonstrated the preparation of an antiserum having enough specificity and sensitivity for a radioimmunoassay to determine fragment D-dimer derivatives. Using the antiserum the contents of fragment D-dimer derivatives in the sera of normal subjects and patients were determined. The content in normal subjects was 0.260 +/- 0.07 micrograms/ml (mean +/- SD) an that in patients with elevated levels of FDP ranged from 0.30 to 28 micrograms/ml. The values of fragment D-dimer derivatives and FDP in sera of some patients did not necessarily change in parallel, although there seems to be generally a positive correlation between them.