Averdunk, R.

Günther, T.

Clinical Chemistry and Laboratory Medicine (CCLM)

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T. Günther

R. Averdunk

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Reduced Lectin Stimulation of Lymphocytes from Magnesium-Deficient Rats

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Günther and Averdunk: Reduced lectin stimulation of lymphocytes in Mg deficiency 51J. Clin. Chem. Clin. Biochem.Vol. 17,1979, pp. 51-55Reduced Lectin Stimulation of Lymphocytes from Magnesium-Deficient RatsBy T. GüntherInstitut für Molekularbiologie und Biochemie andR. AverdunkInstitut für Klinische Chemie und Klinische Biochemie Freie Universität Berlin(Received July 25/September 26, 1978)Summary: Spleen cells and thymocytes from Mg-deficient rats, fed a Mg-deficient diet for 10-13 weeks, incorporate[3H]thymidine at half the rate of normal cells.The cells from Mg-deficient rats are less stimulated by lectins than those from normal animals.These effects correspond to the reduced content of -globulins in serum in Mg deficiency. They are caused by thealtered electrolyte content of the Mg-deficient cells.Verminderte Lectin-Stimulierung der Lymphocyten von Magnesium-Mangel-RattenZusammenfassung: Lymphocyten aus Milz und Thymus von Ratten, die 10-13 Wochen Mg-arm ernährt wurden,bauen [3H]Thymidin nur mit halb so hoher Geschwindigkeit ein wie die Zellen von normal ernährten Ratten.Die Lymphocyten von Mg-arm ernährten Ratten werden durch Lectine weniger stimuliert als normale Zellen.Dieses Verhalten erklärt den verminderten Gehalt des Serums an -Globulinen im Mg-Mangel. Es wird durch denveränderten Elektrolytgehalt der Lymphocyten im Mg-Mangel ausgelöst.IntroductionMg-deficient Yoshida ascites tumor cells (1) or fibro-blasts (2) (as well as Mg-deficient animals) exhibit areduced rate of growth and biosynthesis of DNA, RNAand protein. At the same time, there is an alteration inthe intracellular concentrations of Na+, K+, Ca2+, Mg2+and cAMP (3). Mg deficiency results in increased cellpermeability, which leads to a decrease in intracellular[K*] and [Mg2+] and to an increase in intracellular[Na+]and[Ca?+](l,4).The increased [Na+] releases stored Ca2+ frommitochocidria and produces a further increase incytösolic [Ca2^]. The elevated cytosolic [Ca2+] leadsto a further enhancement of permeability and to astimulation of adenyl cyclase; this, in turn, causes anincrease in intracellular [cAMP], which also increasespermeability. The altered concentrations of Na+, Ca2+,and especially K+ may cause the inhibition of DNA-,RNA- and protein biosynthesis (4).In agreement with the reduced protein biosynthesis, areduction in immunoglobulins and antibody formationis found during Mg deficiency (5—8).A transformation of lymphocytes to lymphoblastspreceeds antibody formation. In this process themembrane permeability is altered, resulting in anincreased leakiness of the cell membrane and increasedturnover of K+ and an enhanced uptake of Ca2+ (forlit. see 1. c. (9, 10)). These alterations correspond tothose in Mg deficiency. We therefore studied intra-cellular concentrations of Na+, K+, the rate of DNAsynthesis, and the stimulation of thymus and spleencells from normal and chronical Mg-deficient rats bylectins.Methods30 male Wistai rats with a body weight of 100 g were givenlow-magnesium feed (Altromin C 1035, Mg content1.6 mmol/kg) and distilled water ad libitum for 10-13 weeks.0340-076X/79/0017-0051 $02.00© by Walter de Gruyter & Co. · Berlin - New York52 G nther and Averdunk: Reduced lectin stimulation of lymphocytes in Mg deficiencyThe control animals were 10 Wistai rats of the same bodyweight which were fed Altromin (Mg content, 83 mmol/kg)and tap water (Mg content of the water, 0.4 mmol/1) ad libi-tum for the same time period.The animals were killed under ether anaesthesia.The number of erythrocytes and leucocytes, the proportionsof granulecytes and lymphocytes, the hemoglobin content,the hematocrit values, the Hb content per erythrocyte and themean cell volume were determined in a clinical chemical centrallaboratory by the usual routine methods.The concentrations of Na* and K* in the serum were measuredby flame photometry, and the concentrations of Ca2"1" andMg2* by atomic absorption spectrometry.For the determination of the Na+ and K"1" contents of the thymusand spleen, the organs were weighed immediately after theirexcision and ashed at about 700 °C. The ashes were taken upin 1.0 ml 0.1 mol/1 HC1 and the contents of Na+ and K+ weredetermined in a flame photometer (Eppendorf).For the determination of the rate of DNA synthesis and lectinstimulation, one thymus or spleen was taken from a controlanimal, or 2-3 from Mg-deficient rats, and homogenized insterile RPMI medium with 10% fetal calf serum (Seromed,M nchen). The homogenate was filtered under sterile condi-tions, and the filtrate was washed 3 X in the same medium bycentrifugation at 200 g. The cell concentration was determinedby counting in a Neubauer counting chamber.Bacterial growth in the medium was inhibited by 100 Mg/1streptomycin and 10s U/l penicillin.The incubation mixtures in stoppered incubation vessels (Fal-con) contained1.8 ml RPMI - 10% calf serum0.1 ml cells in RPMI - 10% calf serum0.1 ml lectin in RPMI - 10% calf serum10 μΐ [3H] thymidine, radioactivity 37 kBq = 1.0 μΟί (ΝΕΝChemicals, spec. act. 740 TBq/mol = 20 Ci/mmol).The cell concentration in the incubation mixtures was 2 X106/mLThe lectin concentrations werePhytohemagglutinin (Difco) (mg/1): 0,4, 8, 16, 32, 64Concanavalin A (Serva, Heidelberg) (mg/1): 0, 4, 8, 16Lipopolysaccharide (Difco) (mg/1): 0, 30, 40, 50, 60.The mixtures were gassed with 5 % CO2 containing air andincubated at 37 °C for 72 h in an incubation chamber underslow rotation.To save cells, most experiments were carried out on a microscale in a microtiter system (Falcon). The incubation mixturesfor this system contained0.1 ml cells in RPMI - 10% calf serum0.1 ml lectin in RPMI - 10% calf serum10 μΐ (3HJthymidine with 3.7 kBq = 0.1 μ€ί (spec. act.740 TBq/mol = 20 Ci/mmol).The cell and lectin concentrations were the same as in themacro-mixtures. The micro-mixtures were incubated in anincubation chamber gassed with 5 % CO2 containing air.For experiments with low-Mg RPMI medium, 10% calf serumand the same substances as in normal RPMI, except formagnesium, were mixed and sterilized by filtration. (Mgcontent of the low-Mg RPMI medium = 0.1 mmol/1).At the end of the experiment, the cells were centrifuged offfor 10 min at 800 £ and washed 3 X in cooled 0.15 mol/1NaCl. The cell sediment from the 2 ml incubation mixtureswas taken up in 2 ml 0.1 mol/1 NaOH, and the sediment fromthe 0.2 ml mixtures in 0.2 ml Q.I mol/1 NaOH. The radio-activity of a 0.1-ml aliquot was measured in 10 ml dioxanscintillation mixture in a Tricarb (Packard) counter.The DNA was determined according to Burton (11), and theprotein according to Lowry et al. (12).ResultsElectrolyte content in serum, spleen and thymusDuring the period of Mg deprivation, the changestypical for Mg deficiency occured. The Mg concentra-tion in the serum decreased to 0.32 ± 0.05 mmol/1(x ± SEM, n - 6), compared to a control value of0.98 ± 0.14 rnmol/1 (x ± SEM, n = 6). The Na+ and K*concentrations in the serum did not change, whereasthe Ca2+ concentration was increased by 4%. In thethymus and spleen of the magnesium-deficient rats, theNa+ content rose about 45%, and the K"1" contentdecreased about 67%.Blood countThe ν3Γίομ8 parameters of the blood count were deter-mined in order to facilitate comparison with other exper-iments (tab. 1). The erythrpcyte count, and thus thehematocrit values, decreased during the experiment. Thiscorresponds to the previously observed changes inchronic Mg deficiency (13,14,15). The loss of Hb wassomewhat less severe than the decrease in erythrocytecount, so that the Hb content per erythrocyte roseslightly. This effect does not result from a smaller cellvolume of the Mg-deficient erythrocytes. They werefound to be somewhat swollen (tab. 1). The number ofleucocytes increased by 52%; granulocytes and lympho-cytes were equally affected.Tab. 1. Blood count from 5 normal and 7 Mg-deficient rats. Mean ± S. E. M.MCH = mean cell hemoglobin, MCV = mean cell volumeErythro-cytes[1012/1)Hb[g/1]Hematocrit MCH. [pg]MCVIfl]Leuko-cytes[109/1]Granulo-cytefractionLymphocytefractionnormal 7.15 ±0.19' 14.86 ± 0.28 0.376 ±0.011 20.5 ± 0.5 0.52 ± 0.01 6.46 ± 0.18 0.085 ± 0.006 0.882 ± 0.016rats(n = 5)Mg 5.80 ± 0.21 13.70 ± 0.21 0.340 ± 0.005 22.3 ± 0.5 0.57 ± Q.Ol' 9.82 ± 0.48 0.090 ± 0.006 0.870 ± 0.017deficientrats(n = 7) - · . ·J. Clin. Cherm Clin. Biochem. / Vol. 17,1979 / No. 1Günther and Averdunk: Reduced lectin Stimulation of lymphocytes in Mg deficiency 53ImmunoglobulinsIn the present experiments, we found a decrease in theserum proteins, particularly the -globulins (not shown),in agreement with earlier results (5-8).Lymphocyte stimulationIn the process of antibody formation, lymphocytesare transformed to lymphoblasts, and these multiplyclonally. The blast transformation can be experimentallyinduced by stimulation with lectins.Table 2 shows that the rate of incorporation of[3H]thymidine into spleen and thymus cells of chronic-ally Mg-deficient rats is reduced to half the controlvalue. The same difference between normal and Mg-deficient cells is found when the cells are incubated ina low-Mg RPMI-medium with 10% calf serum. In thismedium, the rates of incorporation were only 20%lower than in normal medium, in both Mg-deficientand normal cells.The stimulation of spleen and thymus cells from normalrats by phytohemagglutinin, lipopolysaccharide orconcanavalin A is 1.5 times higher than the stimulationof cells from Mg-deficient animals. In low-Mg RPMImedium, the stimulation by phytohemagglutinin,lipopolysaccharide or concanavalin A of normal andMg-deficient spleen and thymus cells is in each caseabout 20% lower than in normal RPMI medium. Theoptimal lectin concentrations for cells from normaland Mg-deficient animals were not significantly differ-ent.After the addition of lectin and subsequent incubation,some of the cells clump and die. After 2 days, all thesurviving lymphocytes proliferate (16).For a quantitative evaluation of the above results, it istherefore necessary to know whether the cells fromMg-deficient animals are destroyed at a higher rate bylectins, or whether they proliferate less vigorously. Insome of the experiments, therefore, we determinedthe DNA and protein contents of the cells after thethree-day incubation with lectins. We found that theprotein and DNA contents of the cultures of spleenand thymus cells from normal rats were not significantlychanged by the lectins and the incubation in low-MgRPMI medium. With the cell cultures from Mg-deficientrats we again found the same DNA and protein contents,with and without lectins, and in both media. From thisone can conclude that the cells from Mg-deprivedanimals are not significantly more sensitive to lectinsthan normal cells. However, the protein and DNAcontents of cultures of spleen and thymus cells fromMg-deficient animals were 25% lower than the controlsafter 3 days incubation, although the number of cellsat the beginning of the experiment was the same. Thisindicates that the cells from Mg-deficient rats have alower proliferation rate.Tab. 2. Effects of lectins on the incorporation of [3H]thymidine in spleen cells and thymocytes from normal and Mg-deficient rats innormal and low-Mg medium. The lectin concentrations at which maximal stimulation occurred were 16 mg/1 for phytohemag-glutinin, 50 mg/1 for lipopolysaccharide and 8 mg/1 for concanavalin A. Mean ± S. E. M. from 7 different experiments each.Rate of [3H)thymidine incorporationNormal mediumSpleenNormal ratsunstimulated[counts/min ·106 cells]"536 ± 86Phyto-hemagglutininstimulatedunstimulated3.74 ± 0.28Lipo-polysaccharidestimulatedunstimulated1.87 ± 0.24Mg-deficient mediumunstimulated[counts/min ·106 ceilsj435 ± 75Phyto-hemagglutininstimulatedunstimulated3.08 ± 0.28Lipo-polysaccharidestimulatedunstimulated1.68 ± 0.23Mg-deficient 294 ± 36rats (n = 7)2.63 ± 0.26 1.22 ± 0.17 237 ±50 2.00 ±0.18ThymusNormal rats 505 ± 125 2.23 ± 0.18 19.6 ± 2.0Nfe-deficient 220 ± 61 1.96 ± 0.32 12.0 ± 0.5rats (n = 7)360 ±72177 ± 302.00 ±0.131.62 ± 0.291.18 ±0.10unstimulated[counts/min ·106 cells]Phyto^hemagglutininstimulatedunstimulatedConcanavalin Astimulatedunstimulatedunstimulated[counts/min ·106 cells)Phyto-hemagglutininstimulatedunstimulatedConcanavalin Astimulatedunstimulated16.0 ± 1.09.5 ± 0.6J. Clin. Chem. Clin. Biochem. / Vol. 17,1979 / No. 154 Günther and Averdunk: Reduced lectin stimulation of lymphocytes in Mg deficiencyDiscussionTypical symptoms of chronic Mg deficiency appearedin our experimental animals during the 10 to 13 weeksof Mg-deficient diet. These included a decrease in theMg concentration in the serum, mild anemia, leuco-cytosis and a decrease in the serum proteins, especiallythe 7-globulins. The leucocytosis was less pronouncedthan that found by other workers (15), who reporteda 2.5-fold increase.In one animal, an infiltratively growing lymphocyticthymoma had formed after 90 days. This correspondsto the results of others (14), who found a rate of suchtumors between 5 and 20%, depending on the strain.The most significant result was the reduced incorpora-tion of thymidine in the spleen and thymus cells ofMg-deficient animals and the reduced responsivenessof these cells to lectins.The rates of stimulation in the control cells, four-foldfor phytohemagglutinin and 19-fold for concanavalin A,are relatively low compared to those in peripherallymphocytes. One reason for this is the high celldensity of 2 · 106/ml used in these experiments; with105/ml the stimulation rates are ten times higher (16).Rirthermore, in the thymus 10-20% of the cells aremitotically active, in contrast to the peripheral lympho-cytes (18).The spleen and thymus cells from Mg-deficient ratsincorporated thymidine at half the control rates, buttheir DNA and protein contents were only about 25%lower. It follows that the spleen and thymus cells ofMg-deficient animals have a lower rate of DNA synthesisor proliferation than normal cells. A related phenome-non is the lower responsiveness of the Mg-deficientspleen and thymus cells to lectins.After incubation in low-Mg RPMI medium, the DNAsynthesis rates and the responsiveness of both controland Mg-deficient cells are about 20% lower. It isunlikely that this effect results from a reduced bindingof lectins to their receptors at the cell membrane in thelow-Mg medium, because there was no alteration in theoptimal lectin concentrations in the low-Mg medium.The reduced responsiveness in cells from Mg-deprivedanimals and in Mg-poor RPMI medium explains thedecrease in the -globulin fraction in the serum andthe reduced immunizability of Mg-deprived rats (15).The cause of this behaviour must lie in changes in theelectrolyte and cell metabolism of Mg-deficient cells.In these cells, an increased permeability causes a dropin the K+ concentration and a rise in the intracellularconcentrations of Na+, Ca2+ and cAMP, and an inhibi-tion of the synthesis of protein and DNA (1, 3, 4). Wealso observed these changes in the Na+ and K+ concentra-tions in the spleen and thymus cells of the Mg-deficientrats.The permeability of the cell membrane is also increasedduring lectin stimulation (9, 10). The effects of Mgdeficiency and lectins on the membrane may be additive.If the changes in the concentrations of ions and sub-strates caused by increased permeability are not com-pensated by increased active transport rates, the intra-cellülar K* concentration decreases, among otherthings. If this decrease exceeds 10-20% of the normalK+ concentration, DNA and protein synthesis decreasein parallel to the K* concentration (19).Hie decreased responsiveness of Mg-deficient lympho-cytes to lectins corresponds to the reduction in thenumber of plaque-forming cells after i. v. injection ofsheep erythfocytes into Mg-deprived animals (8), andexplains the reduced antibody synthesis in Mg deficiency.The cause may be the reduced rate of protein synthesisdue to the decrease in intracellular K+ concentration.Due .to the complex mechanism of the immune response,the details of which are still not completely clear, othereffects may interfere in Mg deficiency. The Mg contentdecreases and the intracellular concentrations of cAMPand Ca, and probably also of Ca2+ ions, increase. Theincrease in the intracellular Ca2+ concentration plays adecisive role in the immune response. The Ca ipnpphoreA 23187, which increases the intracellular [Ca2+], isby itself mitogenic. An increase in the intracellularCa2+ concentration is also involved in the effects ofother lectins. It may represent a common member inthe reaction chain because the effects of süboptimalconcentrations of A 23187 and phytohemagglutininare additive (20). Nevertheless, the DNA synthesis andresponsiveness of Mg-deficient lymphocytes to lectinswere reduced.The reason for this behaviour may be the severe reduc-tion in the intracellular K+ concentration in Mgdeficiency which decreases the rate of protein synthesis.Apparently lymphocyte stimulation requires bothan increase in the Ca2+ concentration and a sufficientK+ concentration. This might also be an explanationfor the fact that the less specific Ca ionophore X 537 Ais not mitogenic (9). It apparently reduces the intra-cellular K+ concentration too far. The inhibition oflymphocyte stimulation by ouabain, the specificinhibitor of Na+-K+ transport, can be explained in thesame way (9).Since the development of leukocytosis and thymomasis associated with a higher synthesis rate in these cells,another, presently unknown mechanism must beresponsible for their formation.J. Clin. Chem. Clin. Biochem. / Vol. 17,1979 / No. 1Günther and Averdunk: Reduced lectin stimulation of lymphocytes in Mg deficiency 55References1. Günther, T. & Averdunk, R. (1970), Z. Klin. Chem. Klin.Biochem. 8, 621-625.2. Sanui, H. & Rubin, H. (1977), J. Cell. Physiol. 92, 23-32.3. Averdunk, R., Ostapovicz, B. & Günther, T. (1975), Z.Klin. Chem. Klin. Biochem. 13, 361-366.4. Günther, T., Averdunk, R. & Ising, H. in: M. Cantin (ed.)Proc. 2. Intern. Symposium on Magnesium, Spectrum Publ.Inc. 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Symposion über Mg-Mangel in der menschlichen Pathologie, (Durlach, J., ed.)Vittel, p. 501-516.16. Bernheim, J. L. & Mendelsohn, J. (1977), in Regulatorymechanisms in Lymphocyte activation (Lucas, D. O., ed.)Acad. Press. Inc., p. 479-505.17. Bois, P. (1964), Nature 204, 1316.18. Whitfield, J. F., Perris, A. D. & Rixon, R. H. (1969), J.Cell. Physiol. 74, 1-8.19. Ledbetter, M. L. S. & Lubin, M. (1977), Exp. Cell Res.105, 223-236.20. Crumpton, M. J., Perles, B. & Auger, J. (1977), Exp. CelL Res.tional Cell Biology (Brinkley, B. R. & Porter, R. K., eds.)Rockefeller Univers. Press, p. 119-127.Prof. Dr. Th. GüntherInstitut für Molekularbiologieund Biochemieder Freien Universität BerlinArnimallee 22D-1000 Berlin 33J. Clin. Chem. Clin. Biochem. / Vol. 17,1979 / No. l

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Reduced Lectin Stimulation of Lymphocytes from Magnesium-Deficient Rats

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