An early alteration in the phospholipid metabolism of lymphocytes by phytohemagglutinin.

Abstract

Phytohemagglutinin (PHA), a glycoprotein derived from the kidney bean Phaseolus vulgaris, initiates a sequential activation of RNA, protein, and DNA synthesis that leads to cell division in human lymphocytes cultured in vitro.1-3 In this response the lymphocytes present a synchronized cell system for studying the molecular steps in the transformation of a resting cell to the growing state. Definition of the interaction of PHA with the lymphocyte might thus identify key processes controlling cell replication. That PHA interacts with the surface of the lymphocyte is suggested by the striking ability of PHA to agglutinate lymphocytes. The induction of growth achieved by mixing lymphocytes with a different major histocompatibility locus4 or by the treatment of leukocytes with antileukocyte antibody5 implicates cell surface interactions in the regulation of lymphocyte growth.In an attempt to obtain biochemical evidence for action of PHA on the membranous system of the lymphocyte, the incorporation Of P3204= into the phospholipid constituents has been studied. We have found that PHA rapidly and selectively stimulates the incorporation of PI204= into phosphatidyl inositol. On the basis of this observation and the reported relationship of phosphatidyl inositol metabolism to protein transport, we propose a hypothesis concerning the role of membranes in growth induction in lymphocytes.Materials and Methods.-M1aterials: The dextran used was Dextran 500 (mol wt 518,000, Pharmacia, Uppsala, Sweden). The heparin was in the form of sodium heparin (1000 units/ml, Organon Inc., W. Orange, N.J.). P3204m was carrier-free and in the H3PO4 form (E. R. Squibb and Sons, N.Y.). To a 5-ml vial of Difco Phytohemagglutinin M, 5 ml of 0.9% NaCl was added and the solution was designated 1 unit/ml. One unit of PHA will optimally induce DNA synthesis in a 300-ml culture containing 50-100 X 101 lymphocytes.1 Chromatographically pure phosphatidyl inositol was obtained from Pierce Chemical Co., Rockford, Ill. 2-Phosphoinositol was a gift from L. E. Hokin. Silica gel H, according to Stahl (E. Merck AG, Darmstadt, Germany), was employed for thin-layer chromatography. The -lleucine-UL-H3 (New England Nuclear Corp., Boston) had a specific activity of 5 c/mM. The cycloheximide was obtained from Calbiochem, Los Angeles. Methods: Preparation of the lymphocytes: Sufficient amounts of dextran and heparin were added to human blood to give a final concentration of 0.5% dextran and 30 units heparin/ml. The blood was allowed to settle for 1 hr, after which the leukocyte-rich plasma layer was drawis off and passed over a column of glass beads to remove other leukocytes from the small lymphocyte suspension.6 Apl)proximately 80% of the lymphocytes were recovered in the eluate, which was 95-99% lymphocytes with respect to leukocytes, and contained 5-10 red blood cells/lymphocyte. The red cells were subsequently removed from the lymphocytes during the work-up of the samples and did not contribute to the results. The cells were suspended in culture tubes at 2-4 X 1...

Cyclic adenosine 3′,5′-monophosphate in human lymphocytes. Alterations after phytohemagglutinin stimulation

“…The early response occurred within 1 min, sufficiently fast to suggest that cyclic AMP could play a major role in the early metabolic effects of PHA. Other early changes in lymphocyte metabolism in PHAstimulated cells include increased incorporation of 'P orthophosphate into phosphatidylinositol (20), an increased rate of phosphorylation and acetylation of nuclear protein (21), and an increased rate of incorporation of radioactive precursors into RNA and protein (22). These changes appear to occur less rapidly than the early elevation in cyclic AMP concentration.…”

Effects of tumor necrosis factor on cell growth and expression of transferrin receptors in human fibroblasts.

“…Several observations suggested that the accumulation of free Ca2+ in cytosol might act as a signal for cell proliferation (18). Diacylglycerol (DG), generated by the breakdown of phosphatidylinositol 4,5-bisphosphate, was found to induce the activation of protein kinase C, and has been recognized as an important mediator for cell proliferation (3,15). Evidence has been obtained that TPA can act as a substitute for DO to activate protein kinase C (8,10), and that FGF induces DG formation, protein kinase C activation, and Ca2+ mobilization (26).…”

The rapid induction by phytohemagglutinin of increased α-aminoisobutyric acid uptake by lymphocytes

“…The preincubation period produced no alteration in the rate of AIB uptake either in the presence or absence of PHA indicating an absence of exchange diffusion. 2 Removal of PHA from lymphocytes. Having demonstrated that PHA can alter the membrane function of lymphocytes, we next investigated whether this effect would be sustained or reversed when the bound PHA was removed from the lymphocyte cell surface.…”

“…While numerous investigators have studied a variety of metabolic alterations which occur after lymphocytes are exposed to PHA (for a recent review see Reference 1), little attention has been focused on the effect that PHA may have on the lymphocyte plasma membrane. Recently, Fisher and Mueller (2) have reported that within minutes after PHA is added to lymphocytes there is an increase in the rate of 8P incorporation into membrane phospholipids. However, the consequences of PHA binding to lymphocytes on the functional characteristics of the plasma membrane need to be further explored.…”