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Incorporation of fatty acids by concanavalin A-stimulated lymphocytes and the effect on fatty acid composition and membrane fluidity.

ABSTRACT: The fatty acid compositions of the neutral lipid and phospholipid fractions of rat lymph node lymphocytes were characterized. Stimulation of rat lymphocytes with the T-cell mitogen concanavalin A resulted in significant changes in the fatty acid composition of both neutral lipids and phospholipids (a decrease in the proportions of stearic, linoleic and arachidonic acids and an increase in the proportion of oleic acid). Membrane fluidity was measured using nitroxide spin-label e.s.r., and increased during culture with concanavalin A. Culturing the lymphocytes in the absence of mitogen did not affect fatty acid composition or membrane fluidity. The uptake and fate of palmitic, oleic, linoleic and arachidonic acids were studied in detail; there was a time-dependent incorporation of each fatty acid into all lipid classes but each fatty acid had a characteristic fate. Palmitic and arachidonic acids were incorporated principally into phospholipids whereas oleic and linoleic acids were incorporated in similar proportions into phospholipids and triacylglycerols. Oleic acid was incorporated mainly into phosphatidylcholine, palmitic and linoleic acids were incorporated equally into phosphatidylcholine and phosphatidylethanolamine, and arachidonic acid was incorporated mainly into phosphatidylethanolamine. Supplementation of the culture medium with particular fatty acids (myristic, palmitic, stearic, oleic, linoleic, alpha-linolenic, arachidonic, eicosapentaenoic or docosahexaenoic acid) led to enrichment of that fatty acid in both neutral lipids and phospholipids. This generated lymphocytes with phospholipids differing in saturated/unsaturated fatty acid ratio, degree of polyunsaturation, index of unsaturation and n - 6/n - 3 ratio. This method allowed the introduction into lymphocyte phospholipids of fatty acids not normally present (e.g. alpha-linolenic) or usually present in low proportions (eicosapentaenoic and docosahexaenoic). These three n - 3 polyunsaturated fatty acids replaced arachidonic acid in lymphocyte phospholipids. Fatty acid incorporation led to an alteration in lymphocyte membrane fluidity: palmitic and stearic acids decreased fluidity whereas the unsaturated fatty acids increased fluidity. It is proposed that the changes in lymphocyte phospholipid fatty acid composition and membrane fluidity brought about by culture in the presence of polyunsaturated fatty acids are responsible for the inhibition of lymphocyte functions caused by these fatty acids.

SUBMITTER: Calder PC

PROVIDER: S-EPMC1138191 | biostudies-other | 1994 Jun

REPOSITORIES: biostudies-other

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Project description:1. Rat lymph-node cells cultured in serum and medium 199 were activated to transform and proliferate by concanavalin A. Initial cell activation was assessed by measuring the enhanced radioactive labelling of cells with [(3)H]uridine produced by concanavalin A during the first 6h of culture. 2. In medium containing serum the degree of activation was dependent on the ratio of concanavalin A to non-diffusible serum macromolecules; however, cells could be activated to a normal extent in a medium containing only diffusible molecules. This indicates that certain serum macromolecules buffer cell receptor sites against reaction with concanavalin A. 3. At high concanavalin A concentrations labelling was depressed below that of control cultures without concanavalin A. This inhibition of labelling (i) occurred in calf serum, but not in homologous serum, (ii) was removed by pretreatment of serum with various complement inhibitors, and (iii) first appeared after 1h of culture following an initial phase of cell activation by concanavalin A. Cells pre-labelled with [(3)H]uridine slowly released the label into the culture medium; this rate of release was suddenly accelerated after 1h of culture with concanavalin A if complement was present. The results suggest that inhibition of labelling requires the sequential binding of concanavalin A and then complement to the cell surface. 4. Results from experiments in which calf serum was mixed in various proportions with calf serum which had been preheated to inactivate complement, suggest (i) a requirement for complement in stoicheiometric quantities dependent on the number of cells being inhibited, and (ii) that preheated serum can inactivate complement in unheated serum. 5. The proliferative response over 3 days of culture was assessed by measuring the enhanced labelling of cells with [(3)H]thymidine produced by concanavalin A. In preheated calf serum two types of inhibition were noted. (i) A progressive inhibition at high concanavalin A concentrations so that the optimum response was shifted to lower concanavalin A concentrations as the duration of culture was extended; it is suggested that this reflects the secretion of complement by cultured cells. (ii) An inhibition of the optimum response appearing late in the culture period at high cell concentrations; it is suggested that this is due to the exhaustion of medium nutrients in most actively growing cultures.

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Incorporation of fatty acids by concanavalin A-stimulated lymphocytes and the effect on fatty acid composition and membrane fluidity.

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