A dialysis culture system for the study of the production and modulation of growth-regulatory molecules: studies using the P388D1 macrophage cell line

Abstract

P388D1 macrophage-like cells have previously been shown to produce both mitogenic and inhibitory regulators of porcine smooth muscle cell (pSMC) growth. The mitogenic activity was shown to have a molecular mass of > 10 kDa while the inhibitory activity was in the range of 2–6 kDa. In the present study, we present a novel dialysis culture system where P388D1 cells were grown in dialysis membranes with a 12 kDa cut-off which allowed continuous production of fractions of the culture medium. Using pSMC as target cells, mitogenic activity was found to be retained by the dialysis membrane while the low molecular mass inhibitory activity passed freely through the membrane. The effect of the macrophage-activators phorbol myristate acetate (PMA), concanavalin A (ConA) and interferon-gamma in combination with lipopolysaccharide (IFN gamma/LPS) were investigated in the dialysis culture system. PMA, ConA and IFN gamma/LPS were found to enhance the production of mitogenic activity by P388D1 cells. PMA also increased the production of growth-inhibitory activity, while ConA abolished inhibitor production and IFN gamma/LPS had no effect on the amount of inhibitory activity produced by P388D1 cells. The experiments show that the balance of production of mitogenic and inhibitory activities by macrophages can be modulated by agents that alter the state of activation of the cells. This could be of profound significance in the influence of macrophages on smooth muscle cell growth during the development of atherosclerosis.