Lymphocyte lactate dehydrogenase isoenzymes in association with depressed mitogen responsiveness

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LACTATE DEiIYDXGE1;ASE ISOENZYMES IN 13I Tt; DEPRESSED r.'ITOCEN RESPONSIVENESS

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ABSTRACT Purified lymphocyte preparations from cancer patients were less responsive to the mitogen phytowere lymphocytes from healthy haemagglutinin (PHA) th SH -thymidine uptake over periods donors as measured by 83 The uptake of radiolabel in culture up to 96 hours. was paralleled by total cellular lactate production. The isoenzymic composition of lactate dehydrogenase (LD) in lymphocytes from healthy individuals was altered following PHA stimulation with increasing proportions of This the culture period. LD-1 and LD-2 throughout phenomenon was markedly reduced in lymphocytes from cancer patients. This defect in lymphocytes from cancer patients is thought to reflect an impaired capacity to accomplish an early mitogen-induced enhancement of glucose metabolism, which is a prerequisite for lymphocyte proliferation. INTRODUCTION The enzyme lactate dehydrogenase ((LD)L-lactate/ NAD+ oxidoreductase E.C.1.1.1.24) exists in five major The isoenzymes are isoenzyme forms in human tissue. tetramers composed of varying ratios of two protein the M (muscle) and H (heart) subunits and are subunits, designated LD-1 (H ), LD-2 (H,M) The di~tribk~r~n(~~"'~~~i~~~4 (HIV: > and LD-5 (M 4. enzgmes possibly deflects the importance of aerobic or anaerobic metabolism within a particular tissue or cell Net lactate production is (Everse and Kaplan, 1973). therefore thought to be influenced by the predominating LD isoenzyme type. 0309-1651la4/100857-09/$03.00/0

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stimulation is known to induce changes in Uitogen lymphocyte carbohydrate metabolism (800s and Loos, 1970; Actively proliferating lymphoLester et al., 1977). cytes accumulate lactate (Roes and Loos, 1970) and the LD isoenzyme pattern has been shown to differ between functional lymphocyte subpopulations (Plum and Ringoir, This pattern may also be used as a measure of 1975). cellular differentiation (Plum and Ringoir, 1977). The proliferative response of peripheral blood lymphocytes to mitogeq3stimulation may bg assessed by the incorporation of 1 iIl-thymidine ( i3ril-TdR) into newly synthesised DiJA.‘ . This technique has demonstrated a pronounced impairment in the responsiveness of cells from patients with malignancy to the mitogen phytohaemagglutinin (P;-IA) (Collins et al., 1986). It is possible that cellular events which precede the replicative phase are defective in these patients and the aims of this study were to measure glycolytic activities of X&-stimulated lymphocytes from cancer patients and from healthy controls, by estimation of both lactate accumulation in cell cultures and also by determination of the relative proportions of LD isoenzymes. MATERIALS AND XETtODS Lymphocyte

donors:

Following informed consent, heparinised blood samples were obtained from equal numbers of male and female individuals in the following categories:Group A:

Realthy individuais of mean age = n=8 67.2 f 1.0 years - S.E.M.,

Group B:

Patients with sc.uamous cell carcinoma the head and neck of mean age = n=8 63.6 5 0.8 years k S.E.X.,

All patients were presenting Dublin St. Luke's tiospital, treatment known to influence Lymphocyte

of

for the first time at and none were undergoing lymphocyte function.

isolation:

iliononuclear leukocytes were obtained from all blood samples within 2 hours of collection, using the method with further purification of lymphoof Boyum (1968), cytes by isopycnic centrifugation on Percoll using the The resultant procedure of Ulmer and Flad (1979).

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lymphocyte suspensions contained <4X contaminating monocytes as estimated morphologically by acridine orange/ethidium bromide staining under fluorescence There were no statistically significant microscopy. differences between the percentage of total T-cells,nor with the relative proportions of T--helper and Tsuppressor cell subsets in the two groups (Table 1) as determined by the attachment of the mouse monoclonal antibodies Anti-Leu 4, Anti-Leu 3a and Anti-Leu 2a Belgium) respectively and (Becton-Dickinson, Mechelen, visualised with FITC-conjugated rabbit anti-mouse IgG (North East Labs., Middlesex, England). Table

1:

T-lymphocyte

sub-populations Groups

*

% T-lymphocytes '73 TS cells '-70 TF cells I * Mean percentage 'Mean percentage

75.55 47.73 56.10

A 3 4.30 f 2.73 t 1.93

63.51 43.25 56.5

B 3 4.16 + 4.6 + 3.48

of lymphocytes + S.E.M. of total T-cells 2 S.E.M.

Lymphocyte

culture:

Purified cells/ml in plicate in presence of (Collins et established plates were 5% CO2 (in

lymphocytes were suspended at 5 x IO5 RPM1 1640 medium and cultured in trimicrotitre plates in 200~1 aliquots in the P;IA (200ng) as previously described Control cultures were also al., 1980). Culture in the absence of mitogen. incubated for up to 96 hours at 37OC in air).

PHA-induced

lymphocyte

transformation:

0.5uCi L3Hl-TdR (ICi/2mmol) were added to each well 24 hours prior-to harvesting on glass-fibre filters PH] -TdR incorporation into DNA was (Whatman GF/C). measured by standard liquid scintillation counting techniques. Estimation

of lactate

production:

Culture medium ana cells were removed from wells and were deproteinised by 0.6M perchloric acid. After centrifugation for 1Omin at 3,000 x g, lactate was

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measured in the su>ernatant dnd Wahlefeld, 1374). LD-isoenzyme

Reports,

using

Vol. 8, No. 70, October

1984

LD and NAD+ (Gutmann

fracZioilatlon .- -.-

Lymphocytes, either freshly isolated or followinS selected periods in culture were washed with PBS6 (Glbco Bio-cult) and lysed by resuspension at 10 cells/ml in 20mM Tris-HCl pH 7.4 followed by -ihres cycles of rapid freezing (-2O'C) and thawing (37OC). The 5 LD isoenzymes were immediately separated by electrophoresis on polyacrylamide gels (5.5% w/v) as described by Dietz and Lubrano (1967), the bands being visualised by LDH-Agaro staining reagent (Sigma Chemical Company). Quantitation of the 5 isoenzyme fractions was made following scanning of the gels at 560nm and this data allows the calculation of the ratio of H:M subunits. Statistical

analysis

of results:

One uay analysis of variance (Campbell, 1974) and the Student's t test were used tc. test the significance of experimental results. Probability values less than 0.05 were taken to indicate statistically significant differences between population means. Correlation coefficients (r) between different parameters were measured by regression analysis. RESULTS DNA-synthesis

and lactate

production:

Correlation between L3Hl-TdR incorporation into lymphocyte DNA and net lactate production are shown in In normal subjects both parameters were Fig. 1. maximal after 72 hours. (The time courses and magnitude of these events were altered in the patient population with both DNA synthesis and lactate production being at lower levels than normal, Decreased indicating decreased metabolic activity. cell viability at 120 hours prevented further In the absence investigation of this phenomenon). lactate production and DNA synthesis were of mitogen, stimulated in both groups minimally. Lymphocyte

LD isoenzyme

profiles:

The LD isoenzyme profiles together with H:M ratios in freshly isolated lymphocytes are shown in Fig. 2a. The major isoenzymes in controls and patients are

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PHA-induced DNA-synthesis ( a PHA, @ no mitogen) and lactate production (0-O PHA, C--Q no mitogen)

LD-2 and LD-3. The patients proportion of LD-1 and higher LD-5 relative to the controls, their overall H:M ratios.

however had a lower proportions of LD-4 and which is reflected in

The effects of exposure of lymphocytes to PHA on LD isoenzymes during culture was dramatic, as shown in Fig. 2b. In cells from healthy donors, levels of LD-3, -4 and -5 diminished and this resulted in an increasing ki:M ratio up to 72 hours in culture. In lymphocytes from the cancer patients, the increase in H:M subunit ratio was much less pronounced. The isoenzyme profiles at 72 hours (Fig. 2c) highlight these differences. In cells from Group A, the H:S ratio had increased from 1.63 to 5.33 and 91% of the isoenzyme forms were represented by LD-1 and LD-2. In the patients' cells, the Ii:hl ratio had increased from 1.32 to 2.23 and while there was an increased proportion of LD-1 and decreased proportions of LD-4 and LD-5, LD-2 and LD-3 still predominated. Analyses to directly correlate individual LD H:M subunit ratios and r3ki'i-TdR incorporation in patients and controls gave-r values of 0.60 and 0.75 respectively and neither values attained statistical significance.

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Fig.

2a

LD isoenzymes lymphocytes

Fig.

2b

LD subunit stimulation

Fig.

2c:

Vol. 8, No. 10, October

in fresh ly

ratios with

isola ted

fol lowing PHA

LD-1

u-2

Ix-3

G-4

m-5

(Ii41

W.p)

(HZMZ)

( IiM3)

(‘f4)

LD isoenzymes 72 hours stimulation with PEIA

after

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The observed similarities between the time courses and magnitudes of lactate production and DNA synthesis in mitogen stimulated lymphocytes from normal donors are in agreement with previous reports (Roos and Loos, 1970; Wang et al., 1976) and it is likely that increased glycolysis, as evidenced by lactate accumulation, is a necessary prelude to DNA synthesis. The depressed mitogen responsiveness of lymphocytes from cancer patients may be due, at least in part, to an inability of the cells to manifest an early and essential increase in aerobic glycolysis commensurate It is proposed (Hume with normal blastogenesis. 1978) that the major function of the enhanced et al., glycolytic rate in PRA-stimulated lymphocytes is the raising of the steady-state levels of glycolytic intermediates which can act as precursors for macromolecule synthesis. The earlier findings of Sagone indicating enhanced hexosemonophosphate et al. (1974), shunt activity in mitogen-stimulated cells, is consistent with this view. The relative proportions of LD isoenzymes in resting lymphocytes from healthy individuals are broadly comparable with those reported by Plum and Ringoir (1975). Our data suggests that in lymphocytes from cancer patients there is slight increase in the amounts of LD-4 and LD-5 relative to controls. Following PHA stimulation, major differences in LD isoenzymic composition were found in lymphocytes The H:M ratio in cells from the two donor groups. from healthy donors increased from 1.63 to 5.33 after 72 hours in culture, paralleling closely the increase in lactate output by mitogen stimulated cells. The lymphocytes from cancer patients did not manifest this alteration in the R:M ratio to any appreciable extent over the same period (1.32 to 2.23) and had diminished lactate production. Although the correlation between LD H:M ratio and F3R]-TdR incorporation did not attain statistical signific'ance in either group, nonetheless, both these parameters and total lactate production Kester et al. manifested very similar time courses. (1977) have demonstrated a qualitative alteration in the h:M ratio of LD isoenzymes in 72 hour mitogen-stimulated lymphocytes as one of a number of changes in the isoenzymic expression of many glycolytic sequence enzymes and have attributed this effect to altered gene expression.

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An early CAMP-mediated activation of LD subunit gene expression in cultured rat glioma cells has been A transient CAMP reported (Jungmann et al., 1983). surge is required for entry of mitogen-stimulated lymphocytes into the S-phase (Wang et al., 1978), allowing maturation of T-lymphocytes which Plum and Ringoir (1977) have shown to be associated with increased LD-1. Failure to manifest such an increase as observed in lymphocytes from cancer patients may thereby indicate a decreased capacity of such cells to function as normal mature T-lymphocytes. ACKNOWLEDGEMENTS This work was supported by a grant from the B. Hannigan Royal College of Surgeons in Ireland. was the recipient of a training grant from the Medical Research Council of Ireland. REFERENCES Separation of leucocytes from blood Boyum, A. (1968). and bone marrow. Scandanavian Journal of Clinical and Laboratory Investigation supplements, 1, l-20. Statistics for Biologists, Campbel1,R.C. (1974). 2nd ed. Cambridge University Press. Collins, P.B., Johnson, A.H. and Moriarty, Ivi. (1980). T-lymphocytes in human cancer: mitogen-responsiveness Irish Journal of lymphocytes in cancer patients. of i‘(;edical Science, 149, 301-306. Separation and Dietz, A.A. and Lubrano, T. (1967). quantitation of LDfi isoenzymes by disc electroAnalytical Biochemistry 20, 246-257. phoresis. Lactate dehyJ. and Kaplan, N.O. (1973). Everse, structure and function. In: drogenase: Advances in S.P. Colowick and N.O. Kaplan (eds). Academic Press, New York. Enzymology, 37, 61-134. L-(+)-lactate A.W. (1974). Gutmann, I. anddahlefeld, determination with lactate dehydrogenase and NAD+. lviethods of Enzymatic In: H.U. Bergmeyer (ed.) Academic Press, New York. 1464-1468. Analysis, Iiume, D.A., Radik, J.L., Berber, E. and Weidemann, K.J. Aerobic glycolysis and lymphocyte trans(1978).: Biochemical Journal, 174, 703-709. formation. Jungmann, R.A., Kelley, D.C., IvXles, M.F. and Milkowski, Cyclic AbiP regulation of lactate D.M. (1983). Journal of Biological Chemistry, 258, dehydrogenase. 5312-5318. Kester, M.V., Phillips, T.L. and Gracy, R.W. (1977). Changes in glycolytic enzyme levels and isoenzyme expression in human lymphocytes during blast formation.

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i;rchives of Biochemistry and Bioy;hysics, 182, 700-709. Plum, J. and Ringoir, s. (1975). A characterization of human B and T lymphocytes by their lactate dtthydrogenase isoenzyme Fattern. European Journal of immunology, 2, ;i71-274. Plum, J. and Ringoir, S. (1977). Lactate dehydrogenase isoenzyme patterns as a measure of cellular differentiation in lymphocytic cells. Journal of the Reticuloendothelial Society, 21.: 225-530. Roes: D. and Loos J.A. (1973). Changes in the carbohydrate meiabolism of mitogenically stimulated human peripheral lymphocytes. Experimental Cell Research, 77, 127-135. A.L. Jnr., LoBuglio, A.F. and Balceraak, S.P. Sagone, Alterations in hexose monophosphate shunt (1974) Cellular during lymphoblastic transformation. Immunology, 14, 443-452. Ulmer, A.J. and Flad, 3. (1379). Discontinuous density gradient separation of human mononuclear leucocytes using Percoll as gradient medium. Journal of-‘ Immunological I'icthods, 30, l-10. Wang, J.L., Sheppard, J.R. and Foker, J.E. (1978). ;lise and fall of cyclic AMP required for onset of lymphocyte DNA synthesis. Aerobic C. and Faker, J. (1976). Vlang, T., iliarquardt, Nature, glycolysis during lymphocyte proliferation. 702-705. 261,

Received:

25th June 1984.

Revised version accepted: 8th August 1984.

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