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Lymphocyte enzyme activities in East African blacks: decrease in 5′nucleotidase and possible relation to immunosuppression
840
TRANSACTIONS OF THE ROYAL
SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 77, No. 6, 840-844 (1983)
Lymphocyte enzyme activities in East African blacks: decrease 5’nucleotidase and possible relation to immunosuppression A. G. LEVIN’, M. JONES’, D. M. KIRKHAM’,
in
T. SHAH’, T. J. PETERS*, I. D. HILLY, A. WASUNNA~ AND G. BRUBAKER’
‘Divisions of Clinical Sciences, ‘Clinical Cell Biology and 3Computing and Statistics, MRC Clinical Research Centre, Harrow, Middlesex HA1 3UJ, UK; 4Dept. of Surgery, University of Nairobi, Kenya; 5Shirati Hospital, Tanzania Summary
Microanalysis of subcellular organelle marker enzymes was applied to cryopreserved lymphocytes (obtained and orocessedin the field) from East African blacks with moderate to severemalnutrition and subject to‘locally endemic parasitic and infectious diseases.An initial study demonstrated that activities of these enzymes, with the partial exception of catalase, were stable to cryopreservation. Crvooreserved and thawed lvmuhocvte soecimens(1 to 3 x lo6 viable cells) from 26 Africans and 20 ‘Ca&&an controls were stu&ei. There was a highly significant decreasein S’nucleotidase activity in these African subjects. Activity of another plasma membrane enzyme, y-glutamyl transferase, and of marker enzymes for other intracellular organelles, was not significantly different between the two groups, indicating that the nucleotidase alteration is highly specific. S’Nucleotidase activity in a group of 17 East African blacks of high socio-economic status lay between the values obtained in the &he; two groups and was not signifi&tly different from eithe;. Further studies on S’nucleotidase showed no evidence that the enzvme is functionallv different in Africans. The differences in activitv of this enzyme in Africans may refiect the known ir&nuno-suppressive effects of infectious disease&d malnutrition or may have a genetic basis which may in turn be associatedwith the pathogenesis of secondary irnmunodeficiency.
Introduction
Although there is considerable animal and laboratory data suggesting that infectious disease and malnutrition depress immunity, evidence from human studies is limited (WHO, 1978). For logistic reasons, biological material is difficult to obtain and there is often a lack of adequate clinical information to correlate with the results of laboratory experiments. This is especially true in developing countries where these clinical problems are most acute. The availability of cryopreserved lymphocytes allows a wider range of laboratory investigations directed toward these problems, as is illustrated in this report. There has been relatively little study of biochemical mechanismsunderlying the behaviour of lymphocytes when immune reactivity is suppressed. Recent evidence suggests that assays of lymphocyte enzymes may be useful in investigating immune deficiency disorders (WEBSTER et al., 1979; SHAH et al., 1981). We here report results on the stability of lymphocyte enzymic activities to cryopreservation and enzymic analysis of cryopreserved lymphocytes from East African Blacks and a group of Caucasians. Subjects and Methods
Comparison of Fresh and Cyopreserued Lymphocytes
Initial experiments were designed to determine whether lymphocyte enzymic activities in cryopreserved cells could be detected and how such activity was affected by cryopreservation. 50 ml venous blood specimens were obtained from five subjects (Caucasian laboratory personnel--three males and two females ranging in age from 26 to 53 years). Lymphocytes were separated by standard density gradient methods (HARRIS& UKAEJIOFO,1969). Numbers of viable lymphocytes obtained varied from 10 x lo6 to 28 x 106.
Each specimen was divided; half was cryopreserved in liquid nitrogen with the technique employed in East Africa (STEEL
et al., 1974)and then thawed.The frozen and thawedand “fresh” lymphocytes from each subject were then assayed for six organellemarkerenzymesaswell asfor protein and DNA content. Subjects
Details of the 46 subjects studied are given in Table I. The Africans were in almost all caseshospital in-patients with a variety of minor medical and surgical conditions, either at the Kenyatta National Hospital, Nairobi or the Shirati Hospital in the North Mara Region of Tanzania. It is stressed,however, that all these individuals were representative of the general population of Kenya and Tanzania, in most cases subsisten& farmers, in so&e instances casually employed urban dwellers. They were subject to the moderate to severe orotein-calorie malnutrition and the infectious
and parasitic*diseaseswhich are endemic in Kenya and
Tanzania (VOGEL er al., 1974). Caucasian subjects were either working in Africa or were laboratory personnel at the Clinical Research Centre, Harrow, and were well nourished and in good health. There were.cells from approximately
equal numbersof Africans and Caucasiansin eachexperiment. To determine whether genetic factors were responsible for differences in S’nucleotidase activity, this enzyme was assayedin cryopreserved lymphocytes from 17 East African blacks of high socio-economic status (eight males, nine females, age range 22 to 40). These comprised 13 medical students, three nurses and a public health officer. This group is referred to as “healthy Africans”. Technique of Cryopreservatim
For each cell sample, 20 ml of venous blood was defibrinated. Thereafter the specimens taken in Africa were processed either in the Nairobi laboratory of the International Agency for Research on Cancer or in the laboratory
A.
G. LEVIN
et al.
841
of Shirati Hospital, by a cryopreservation technique similar to that previously reported (STEEL et al., 1974). Cells were frozen at approximately l”C/min by placing ampoules of cells in a BF6 biological freezer plug set in the neck of an LR33 transportable liquid nitrogen refrigerator (Union Carbide). Specimens obtained in the UK were frozen by a two-stage technique (FARRANT et al., 1974). Usually four aliquots, of 2 to 4 x 10” cells, were obtained from each subject. Cells from African subjects were collected between 1972 and 1978 and from Caucasians between 1974 and 1981. Cryopreserved cells were rapidly thawed in a 37°C water bath after which tissue culture media (RPM1 with Hepes buffer? containing 20% foetal calf serum) was slowly added, with constant mixing. The cells were then centrifuged and prepared for assay of enzymic activities. The volume of the cell suspension was adjusted to equate the number of viable cells from each subject in each experiment. Serum removed before lymphocyte separation was tested for the presence of Hepatitis B-surface antigen by radioimmunoassay (Professor D. Dane, Middlesex Hospital Medical School) and only antigen-negative specimens were studied. Cell Preparations and Viability Cell viability was determined by the trypan blue (0.2%) exclusion test, and by more strict morphological criteria including refractility of the cell membrane and clear visualization of the nucleus. Enzymic Activiry Activity of the following enzymes representing various cell organelles was measured (PETERS, 1981): 5’nucleotidase (plasma membrane); y-glutamyl transferase (plasma membrane); lactate dehydrogenase (cytosol):, malate dehydrogenase (mitochondria); neutral o-glucosidase (endoplasmic reticulum); acid phosphatase (lysosomes); N-acetyl-fl-glucosaminidase (lysosomes); catalase (peroxisomes); o-naphthy1 esterases (specific vesicles). , Protein (SCHACTERLE & POLLACK, 1973) and DNA (KAPUSCINSKI & SKOCZYLAS, 1977) content of the cell homogenates were also measured. Comparison of the activities between fresh and cryopreserved cells were expressed as milliunits of enzyme activity per lo9 cells, based on the DNA content of the homogenate. This method was adopted because the 50% foetal calf serum in the freezing mixture could have affected a comparison between the fresh and the cryopreserved portions based on protein content. In all other experiments with cryopreserved cells, results were expressed as milliunits of enzyme activity/ mg protein, as this estimate was considered more reliable with the small numbers of cells involved. For statistical analysis the data was logarithmically transformed to reduce its skewness and p values were calculated with a technique designed to take into account bias which might result from differing numbers in various groups of subjects (BAKER & NELDER,
1978).
S’Nucleotidase Enzyme-substrate Reaction in Aficans’ and Caucasians’ Lymphocytes 5’Nucleotidase activity was assayed in the presence of varying concentrations of substrate and of varying concentrations of the specific enzyme inhibitor, o, S methylene adenosine diphosphate (Sigma Chemicals, Poole, Dorset, UK). Pooled cell homogenates from 24 African and 21 Caucasian subjects were used in these studies. Other studies compared pooled homogenates from three African and three Caucasian subjects with respect to pH optimum and Mg’ + requirements of the enzymic reaction. Immunoglobulin Determination Serum samples from 21 of the African assayed for immunoglobulin levels with techniques used in the Immunopathology Northwick Park Hospital (Dr. I. Chanarin Tidmarsh).
subjects were nephelometric Laboratory of and Miss E.
LYMPHOCYTE
842 Table
II-Lymphocyte
emymic
ENZYME
activities
ACTIVITIES
IN
EAST
in fresh and cryopreserved
Mean enzyme
Mean enzyme activity (Range) p value
_.
Crvooreserved (cytosol) - 36,300 (31,400-40,400)
p = 0.96
p = 0.51
Malate Dehydrogenase (mitochondria)
Neutral wglucosidase (endoplasmic reticulum)
34,900 (32,800-38,900)
35,700 (30,900-48,200)
Mean enzyme activity Ww) p value
cells
38,800 (33,700-45,500)
114.0 (89.7-172.0)
116.0 (68.7-185.0)
BLACKS
Fresh cells Lactate Dehydrogenase
Fresh Cells Cryopreserved SNucleotidase (plasma membrane) activity (Ran& ‘p value*
AFRICAN
14.3
13.1
(9.2-24.7)
(9.7-18.7)
p = 0.96
p = 0.42
N-Acetyl-fi-Glucosaminidase (lysosomes)
Catalase (peroxisomes)
60.0 (42.7-75.4)
62.8 (36.5-109.0)
658 (410-909)
p = 0.39
368 (105-707)
p = 0.056
Results expressed as milliunits enzyme activity/lO’ cells for 5 subjects *2 tailed paired t test employed
ndoplasmicl IEeticulum
Plasma membrane _ 4.0.E 2 Lo g3.0E 2 ;.2.0z '5 .G !ih9 '.Oz oN P
24
In 18 o.M)8
I
I
I I
Lvsosomes
16
I:
15
0.887
21
x
12 0.085
18
Specific vesicle
litochondri d Peroxisome MO-
225-
I
I
I I
12
25
0.927
19
0.329
1501 75 I
I
11 I
o6
7 0.058
25
20
0.078
22
20
0.644
3i 26
18
1.00
Fig. 1. Comparison of lymphocyte enzyme activities of African black (0) and Caucasian(0) subjects. Mean activities and 95% confidence limits are shown for each group (the confidence limits are asymmetrical becauselogarithmic transformations were used); p values have been calculated as described in the test.
Results
Clinical Details and Lymphocyte Viability (Table I)
Although the African subjects were, on average, younger than the Caucasiansthere were no significant differences between the two groups. The data in this table also indicates that comparable numbers of cells of similar viability, assessedby two distinct techniques, were isolated from each subject group.
Comparison of Fresh and Cryopreserved Lymphocytes
(Table II) In fresh and cryopreserved lymphocytes from the
sameindividual there was no significant difference in the activity of five of the six enzymes tested. Catalase activity was reduced in the cryopreserved specimens tu,t tte;” quite reach statistical significance at the 00 Comparison of Lymphocyte Enzymic Activity in Africans and Caucasians (Fig. 1) S’Nucleotidase activities in Africans were signi-
ficantly lower than in Caucasians whereas there was no significant difference in y-glutamyl transferase activity, also a plasma membrane enzyme, between
A.
e.
the groups. There was no statistically significant difference between the subject groups with respect to other enzymes. For some of the enzymes, S’nucleotidase, lactate and malate dehydrogenases, neutral a-glucosidase, N-acetyl-/3-glucosaminidase, activities in both African and Caucasian females were higher than in males. Calculations of statistical significance due to race took into account anv sex differences. The S’nucleotidase activity of individuals in the “healthy African” group was 0.94 mU/mg protein (Caucasians, 1.72 and Africans renresentative of the general pomtlation, 0.69). Differences between the “healthy African” group and the Caucasians and other African subjects were not significant (p = 0.11 and 0.37, respectively). Statistical calculations again took into account any sex differences. Serum Immunoglobulin Levels of African Subjects No subject showed marked reduction and there was no correlation between immunoglobulin level and lymphocyte S’nucleotidase activity. The mean IgG was 13.6 g/l with a range of 7.2 to 26.0 (normal range in the Northwick Park Hospital laboratory 6.5 to 15.0 g/l). The mean IgA level was 2.46 g/l with a range of 0.5 to 4.1 (normal range 0.8 to 3.4 g/l) and the mean IgM 1.56 g/l, range 0.6 to 3.1 (normal range 0.6 to 3.1 g/l). Comparison of S’Nucleotidase Activity in Pooled Homogenates from Africans and Caucasians There was no difference in the kinetics of the enzyme-substrate reaction (Km for adenosine monophosphate) or in the effect of the 5’nucleotidase inhibitor (Ki for o, 13methylene adenosine diphosphate), or with respect to pH optimum and MgZf requirements of the enzymic reaction.
Discussion Decreased S’nucleotidase activity in Africans was selective (in that it was not due to gross impairment of the lymphocyte plasma membrane) and this is reflected by the finding that activity of another plasma membrane enzyme, y-glutamyl transferase, was not significantly different from Caucasians. Although studies on Caucasians with inununodeficiency syndromes suggest that decreased S’nucleotidase activitv is not exphcable in terms of altered proportions of B and T lvmnhocvtes (WEBSTER et al.. 1979). it is possible Thai the’results here reported may be’hue to differing proportions of B and T cells or of T cell subsets in Africans. Further studies combining assays of enzymic activity with the use of monoclonal antibodies now available against lymphocyte subpopulations, will help to answer this question. There is no evidence from our studies that S’nucleotidase is structurally or functionally different in African subjects compared with Caucasians. It is most likely that the Africans have less of the enzyme available and that this may be associated with clinical consequences. As a control for genetic differences in S’nucleotidase activitv. results from the “healthv African” group were ‘inconclusive, lying between results from the other two groups. It may be that both factors, environmental and genetic, are responsible for lower levels of S’nucleotidase in Africans. It is
L~wt-4
et al.
843
possible however, that the “healthy Africans” we studied were also affected to a certain degree by endemic infectious disease and by less than optimal nutrition. It is well recognized that the purine nucleotides which are hydrolysed by S’nucleotidase play an important immunoregulatory function. Specific immunodeficiency syndromes are associated with congenital absence of lymphocyte adenosine deaminase and purine nucleoside phosphorylase. More recently, deficiencv of lvmnhocvte S’nucleotidase has been associated with both sex-linked and common variable hypogammaglobulinaemia (JOHNSON et al., 1977; EDWARDS et al., 1978). Recent studies with the highly specific assay used in the present study have indicated that hypogammaglobulinaemia patients have lymphocyte S’nucleotidase activities of less than 5% of control values (SMITH et al., 1982). This deficiency also seems to be highly selective in that the plasma membrane enzymes y-glutamyl transferase, leucine aminopeptidase and adenosine diphosphatase were unaffected. In the immunodeficiency syndromes genetic factors are of prime importance and immunoglobulin levels are often very low. While the decreased S’nucleotidase activity in Africans may be partly or wholly genetically determined, there is no evidence of hypogammaglobulinaemia in the African subjects. The S’nucleotidase deficiency in African patients may be a biochemical manifestation of the relative imrnunodepression associated with malnutrition and infectious disease. Alternatively these factors may induce secondary immunodeficiency in a setting when there is a genetically determined lower level of S’nucleotidase. This report also indicates that field studies of lymphocyte enzyme activity are possible. The volume of blood required for these experiments is small enough to be collected on a routine basis. Lymphocytes can be separated and cryopreserved with basic laboratory facilities. The specialized equipment required is both easily available and durable. Sources of liquid nitrogen in many developing countries (usually connected with artificial insemination programmes) are relatively accessible. Activities of the enzymes tested were stable to cryopreservation, with the possible exception of catalase which is known to be particularly affected by freezing and thawing (SHIKAMA & YANAZAKI, 1961). The cells used in this investigation were collected from three to eight years before being tested. We have no evidence that any biochemical or immunological property of cryopreserved lymphocytes is affected by prolonged storage in liquid nitrogen; indeed there is good evidence that such biological properties are unaffected by the duration of storage (FARRANT, 1980). Acknowledgements This work was supported in part by Grant G973/753/T from the UK Medical Research Council, by Grant SP1559 from the Cancer Research Campaign of the-UK, by a grant from the United Cancer Council, Rochester, New York and by Contract No. NOICP 43296 between the National Institutes of Health, USANCINCP and the International Agency for Research on Cancer. We are grateful to Mrs. J. Safari and Mr. Z. Siso for their assistance in field studies, to Drs. A. D. B. Webster and M. E. Balis for their helpful comments and Ms. Rosamund Greensted for secretarial assistance.
844
LYMPHOCYTE
ENZYME
ACTIVITIES
References Baker, R. J. & Nelder, J. A. (1978). Generalized Linear Interactive Modelling (GLZM) Release 3, Oxford: Numerical Algorithms Group. Edwards. N. L., Magilva. D. B., Cassidv, J. T. & Fox, I. H. (1978). Lymphocyte ecto-5’nucleotidase in agammaglobuhnaernia. Science, 201, 628630. Farrant, J. (1980). Practical Aspects. In: Lao Temperature Preservation in Medicine and Biology: Ashwood-Smith, M. J. & Farrant, J. (Editors). Tunbndge Wells: Pitman Medical, pp. 285-310. Farrant, J., Knight, S. C., McGann, L. L. & O’Brien, J. (1974). Optimal recovery of lymphocytes and tissue culture cells following rapid cooling. Nuture, 249, 452-453. Harris, R. & Ukaejioto, E. 0. (1969). Tissue typing usinga one-step lymphocyte separation procedure. Lancer, II, Joh%h S. M North M. E Asherson, G. L., Allsop, J.? Wat&, R.“W. El & Webster, A. D. B. (i977). Lymphocyte purine S’nucleotidase deficiency in primary hypogammaglobulinaemia. Lancet, i, 168-170. Kapuscinski, J. & Skoczylas, B. (1977). A simple and rapid fluorimetric method for DNA microassay. Analytical Biochemistry, 83, 252-257.
Peters, T. J. (1981). Investigation of tissue organelles by a combination of analytical subcellular fractionation and enzymic microanalysis: a new approach to pathology. Jcnmal of Clinical Patholoa, 34, 1-12. Schacterle, G. R. & Pollack, R. L. (1973). A simplified method ,for ,the quantitauve assay of small amounts of ~;oz$i;~rn biologic material. Analytical Baochemwy, 51,
IN
EAST
AFRICAN
BLACKS
Shah, T., Webster, A. D. B. & Peters, T. J. (1981). Analytical subcellular fractionation with enryrmc ,m~croassayof human peripheral blood lymphocytes. Clmrc~l Science. 60. 3-4P. Shikama, K. &Yanazaki, I. (1961). Denaturation of catalase by freezing and thawing. Nature, 190, 83-84. Smith, G. P., Shah, T., Webster, A. D. B. & Peters, T. J. (1982). Studies on the kinetic properties and subcellular localization of adenine nucleotide phosphatases in peripheral blood lymphocytes from control subjects and patients with conu-nonvariable primary hypoganunaglols&~~;6rua. Clmscal and Experimental Immunology, 46, Steel, C. M:,Levin, A G.,Tsu, T. & Gross, R. L. (1974).A bank of frozen peripheral-blood lymphocytes for in vitro immunological studies on East African cancer patients. International Journal of Cancer, 13, 489-493. Vogel, L. C., Muller, A. S., Odingo, R. S., Onyango, Z. & De Geus, A. (Editors) (1974). Health and Disease in Kenya, Nairobi: East African Literature Bureau, pp. 181-374 and 409-428. Webster, A. D. B., Rowe, M., Johnson, S. M., Asherson, G. L. & Harkness, A. (1979). Ecto-S’nucleotidase deficiency in primary hypogammaglobulinaemia. CZBA Foundation Svmnosium. 68. 135-144. World Health Orga&ation. (1978). Immunodeficietq. Technical Report 630. Geneva: World Health Organization, pp. 52-i2.
Accepted
for
publication
31st March,
1983.
TRANSACTIONS OF THE ROYAL
SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 77, No. 6, 840-844 (1983)
Lymphocyte enzyme activities in East African blacks: decrease 5’nucleotidase and possible relation to immunosuppression A. G. LEVIN’, M. JONES’, D. M. KIRKHAM’,
in
T. SHAH’, T. J. PETERS*, I. D. HILLY, A. WASUNNA~ AND G. BRUBAKER’
‘Divisions of Clinical Sciences, ‘Clinical Cell Biology and 3Computing and Statistics, MRC Clinical Research Centre, Harrow, Middlesex HA1 3UJ, UK; 4Dept. of Surgery, University of Nairobi, Kenya; 5Shirati Hospital, Tanzania Summary
Microanalysis of subcellular organelle marker enzymes was applied to cryopreserved lymphocytes (obtained and orocessedin the field) from East African blacks with moderate to severemalnutrition and subject to‘locally endemic parasitic and infectious diseases.An initial study demonstrated that activities of these enzymes, with the partial exception of catalase, were stable to cryopreservation. Crvooreserved and thawed lvmuhocvte soecimens(1 to 3 x lo6 viable cells) from 26 Africans and 20 ‘Ca&&an controls were stu&ei. There was a highly significant decreasein S’nucleotidase activity in these African subjects. Activity of another plasma membrane enzyme, y-glutamyl transferase, and of marker enzymes for other intracellular organelles, was not significantly different between the two groups, indicating that the nucleotidase alteration is highly specific. S’Nucleotidase activity in a group of 17 East African blacks of high socio-economic status lay between the values obtained in the &he; two groups and was not signifi&tly different from eithe;. Further studies on S’nucleotidase showed no evidence that the enzvme is functionallv different in Africans. The differences in activitv of this enzyme in Africans may refiect the known ir&nuno-suppressive effects of infectious disease&d malnutrition or may have a genetic basis which may in turn be associatedwith the pathogenesis of secondary irnmunodeficiency.
Introduction
Although there is considerable animal and laboratory data suggesting that infectious disease and malnutrition depress immunity, evidence from human studies is limited (WHO, 1978). For logistic reasons, biological material is difficult to obtain and there is often a lack of adequate clinical information to correlate with the results of laboratory experiments. This is especially true in developing countries where these clinical problems are most acute. The availability of cryopreserved lymphocytes allows a wider range of laboratory investigations directed toward these problems, as is illustrated in this report. There has been relatively little study of biochemical mechanismsunderlying the behaviour of lymphocytes when immune reactivity is suppressed. Recent evidence suggests that assays of lymphocyte enzymes may be useful in investigating immune deficiency disorders (WEBSTER et al., 1979; SHAH et al., 1981). We here report results on the stability of lymphocyte enzymic activities to cryopreservation and enzymic analysis of cryopreserved lymphocytes from East African Blacks and a group of Caucasians. Subjects and Methods
Comparison of Fresh and Cyopreserued Lymphocytes
Initial experiments were designed to determine whether lymphocyte enzymic activities in cryopreserved cells could be detected and how such activity was affected by cryopreservation. 50 ml venous blood specimens were obtained from five subjects (Caucasian laboratory personnel--three males and two females ranging in age from 26 to 53 years). Lymphocytes were separated by standard density gradient methods (HARRIS& UKAEJIOFO,1969). Numbers of viable lymphocytes obtained varied from 10 x lo6 to 28 x 106.
Each specimen was divided; half was cryopreserved in liquid nitrogen with the technique employed in East Africa (STEEL
et al., 1974)and then thawed.The frozen and thawedand “fresh” lymphocytes from each subject were then assayed for six organellemarkerenzymesaswell asfor protein and DNA content. Subjects
Details of the 46 subjects studied are given in Table I. The Africans were in almost all caseshospital in-patients with a variety of minor medical and surgical conditions, either at the Kenyatta National Hospital, Nairobi or the Shirati Hospital in the North Mara Region of Tanzania. It is stressed,however, that all these individuals were representative of the general population of Kenya and Tanzania, in most cases subsisten& farmers, in so&e instances casually employed urban dwellers. They were subject to the moderate to severe orotein-calorie malnutrition and the infectious
and parasitic*diseaseswhich are endemic in Kenya and
Tanzania (VOGEL er al., 1974). Caucasian subjects were either working in Africa or were laboratory personnel at the Clinical Research Centre, Harrow, and were well nourished and in good health. There were.cells from approximately
equal numbersof Africans and Caucasiansin eachexperiment. To determine whether genetic factors were responsible for differences in S’nucleotidase activity, this enzyme was assayedin cryopreserved lymphocytes from 17 East African blacks of high socio-economic status (eight males, nine females, age range 22 to 40). These comprised 13 medical students, three nurses and a public health officer. This group is referred to as “healthy Africans”. Technique of Cryopreservatim
For each cell sample, 20 ml of venous blood was defibrinated. Thereafter the specimens taken in Africa were processed either in the Nairobi laboratory of the International Agency for Research on Cancer or in the laboratory
A.
G. LEVIN
et al.
841
of Shirati Hospital, by a cryopreservation technique similar to that previously reported (STEEL et al., 1974). Cells were frozen at approximately l”C/min by placing ampoules of cells in a BF6 biological freezer plug set in the neck of an LR33 transportable liquid nitrogen refrigerator (Union Carbide). Specimens obtained in the UK were frozen by a two-stage technique (FARRANT et al., 1974). Usually four aliquots, of 2 to 4 x 10” cells, were obtained from each subject. Cells from African subjects were collected between 1972 and 1978 and from Caucasians between 1974 and 1981. Cryopreserved cells were rapidly thawed in a 37°C water bath after which tissue culture media (RPM1 with Hepes buffer? containing 20% foetal calf serum) was slowly added, with constant mixing. The cells were then centrifuged and prepared for assay of enzymic activities. The volume of the cell suspension was adjusted to equate the number of viable cells from each subject in each experiment. Serum removed before lymphocyte separation was tested for the presence of Hepatitis B-surface antigen by radioimmunoassay (Professor D. Dane, Middlesex Hospital Medical School) and only antigen-negative specimens were studied. Cell Preparations and Viability Cell viability was determined by the trypan blue (0.2%) exclusion test, and by more strict morphological criteria including refractility of the cell membrane and clear visualization of the nucleus. Enzymic Activiry Activity of the following enzymes representing various cell organelles was measured (PETERS, 1981): 5’nucleotidase (plasma membrane); y-glutamyl transferase (plasma membrane); lactate dehydrogenase (cytosol):, malate dehydrogenase (mitochondria); neutral o-glucosidase (endoplasmic reticulum); acid phosphatase (lysosomes); N-acetyl-fl-glucosaminidase (lysosomes); catalase (peroxisomes); o-naphthy1 esterases (specific vesicles). , Protein (SCHACTERLE & POLLACK, 1973) and DNA (KAPUSCINSKI & SKOCZYLAS, 1977) content of the cell homogenates were also measured. Comparison of the activities between fresh and cryopreserved cells were expressed as milliunits of enzyme activity per lo9 cells, based on the DNA content of the homogenate. This method was adopted because the 50% foetal calf serum in the freezing mixture could have affected a comparison between the fresh and the cryopreserved portions based on protein content. In all other experiments with cryopreserved cells, results were expressed as milliunits of enzyme activity/ mg protein, as this estimate was considered more reliable with the small numbers of cells involved. For statistical analysis the data was logarithmically transformed to reduce its skewness and p values were calculated with a technique designed to take into account bias which might result from differing numbers in various groups of subjects (BAKER & NELDER,
1978).
S’Nucleotidase Enzyme-substrate Reaction in Aficans’ and Caucasians’ Lymphocytes 5’Nucleotidase activity was assayed in the presence of varying concentrations of substrate and of varying concentrations of the specific enzyme inhibitor, o, S methylene adenosine diphosphate (Sigma Chemicals, Poole, Dorset, UK). Pooled cell homogenates from 24 African and 21 Caucasian subjects were used in these studies. Other studies compared pooled homogenates from three African and three Caucasian subjects with respect to pH optimum and Mg’ + requirements of the enzymic reaction. Immunoglobulin Determination Serum samples from 21 of the African assayed for immunoglobulin levels with techniques used in the Immunopathology Northwick Park Hospital (Dr. I. Chanarin Tidmarsh).
subjects were nephelometric Laboratory of and Miss E.
LYMPHOCYTE
842 Table
II-Lymphocyte
emymic
ENZYME
activities
ACTIVITIES
IN
EAST
in fresh and cryopreserved
Mean enzyme
Mean enzyme activity (Range) p value
_.
Crvooreserved (cytosol) - 36,300 (31,400-40,400)
p = 0.96
p = 0.51
Malate Dehydrogenase (mitochondria)
Neutral wglucosidase (endoplasmic reticulum)
34,900 (32,800-38,900)
35,700 (30,900-48,200)
Mean enzyme activity Ww) p value
cells
38,800 (33,700-45,500)
114.0 (89.7-172.0)
116.0 (68.7-185.0)
BLACKS
Fresh cells Lactate Dehydrogenase
Fresh Cells Cryopreserved SNucleotidase (plasma membrane) activity (Ran& ‘p value*
AFRICAN
14.3
13.1
(9.2-24.7)
(9.7-18.7)
p = 0.96
p = 0.42
N-Acetyl-fi-Glucosaminidase (lysosomes)
Catalase (peroxisomes)
60.0 (42.7-75.4)
62.8 (36.5-109.0)
658 (410-909)
p = 0.39
368 (105-707)
p = 0.056
Results expressed as milliunits enzyme activity/lO’ cells for 5 subjects *2 tailed paired t test employed
ndoplasmicl IEeticulum
Plasma membrane _ 4.0.E 2 Lo g3.0E 2 ;.2.0z '5 .G !ih9 '.Oz oN P
24
In 18 o.M)8
I
I
I I
Lvsosomes
16
I:
15
0.887
21
x
12 0.085
18
Specific vesicle
litochondri d Peroxisome MO-
225-
I
I
I I
12
25
0.927
19
0.329
1501 75 I
I
11 I
o6
7 0.058
25
20
0.078
22
20
0.644
3i 26
18
1.00
Fig. 1. Comparison of lymphocyte enzyme activities of African black (0) and Caucasian(0) subjects. Mean activities and 95% confidence limits are shown for each group (the confidence limits are asymmetrical becauselogarithmic transformations were used); p values have been calculated as described in the test.
Results
Clinical Details and Lymphocyte Viability (Table I)
Although the African subjects were, on average, younger than the Caucasiansthere were no significant differences between the two groups. The data in this table also indicates that comparable numbers of cells of similar viability, assessedby two distinct techniques, were isolated from each subject group.
Comparison of Fresh and Cryopreserved Lymphocytes
(Table II) In fresh and cryopreserved lymphocytes from the
sameindividual there was no significant difference in the activity of five of the six enzymes tested. Catalase activity was reduced in the cryopreserved specimens tu,t tte;” quite reach statistical significance at the 00 Comparison of Lymphocyte Enzymic Activity in Africans and Caucasians (Fig. 1) S’Nucleotidase activities in Africans were signi-
ficantly lower than in Caucasians whereas there was no significant difference in y-glutamyl transferase activity, also a plasma membrane enzyme, between
A.
e.
the groups. There was no statistically significant difference between the subject groups with respect to other enzymes. For some of the enzymes, S’nucleotidase, lactate and malate dehydrogenases, neutral a-glucosidase, N-acetyl-/3-glucosaminidase, activities in both African and Caucasian females were higher than in males. Calculations of statistical significance due to race took into account anv sex differences. The S’nucleotidase activity of individuals in the “healthy African” group was 0.94 mU/mg protein (Caucasians, 1.72 and Africans renresentative of the general pomtlation, 0.69). Differences between the “healthy African” group and the Caucasians and other African subjects were not significant (p = 0.11 and 0.37, respectively). Statistical calculations again took into account any sex differences. Serum Immunoglobulin Levels of African Subjects No subject showed marked reduction and there was no correlation between immunoglobulin level and lymphocyte S’nucleotidase activity. The mean IgG was 13.6 g/l with a range of 7.2 to 26.0 (normal range in the Northwick Park Hospital laboratory 6.5 to 15.0 g/l). The mean IgA level was 2.46 g/l with a range of 0.5 to 4.1 (normal range 0.8 to 3.4 g/l) and the mean IgM 1.56 g/l, range 0.6 to 3.1 (normal range 0.6 to 3.1 g/l). Comparison of S’Nucleotidase Activity in Pooled Homogenates from Africans and Caucasians There was no difference in the kinetics of the enzyme-substrate reaction (Km for adenosine monophosphate) or in the effect of the 5’nucleotidase inhibitor (Ki for o, 13methylene adenosine diphosphate), or with respect to pH optimum and MgZf requirements of the enzymic reaction.
Discussion Decreased S’nucleotidase activity in Africans was selective (in that it was not due to gross impairment of the lymphocyte plasma membrane) and this is reflected by the finding that activity of another plasma membrane enzyme, y-glutamyl transferase, was not significantly different from Caucasians. Although studies on Caucasians with inununodeficiency syndromes suggest that decreased S’nucleotidase activitv is not exphcable in terms of altered proportions of B and T lvmnhocvtes (WEBSTER et al.. 1979). it is possible Thai the’results here reported may be’hue to differing proportions of B and T cells or of T cell subsets in Africans. Further studies combining assays of enzymic activity with the use of monoclonal antibodies now available against lymphocyte subpopulations, will help to answer this question. There is no evidence from our studies that S’nucleotidase is structurally or functionally different in African subjects compared with Caucasians. It is most likely that the Africans have less of the enzyme available and that this may be associated with clinical consequences. As a control for genetic differences in S’nucleotidase activitv. results from the “healthv African” group were ‘inconclusive, lying between results from the other two groups. It may be that both factors, environmental and genetic, are responsible for lower levels of S’nucleotidase in Africans. It is
L~wt-4
et al.
843
possible however, that the “healthy Africans” we studied were also affected to a certain degree by endemic infectious disease and by less than optimal nutrition. It is well recognized that the purine nucleotides which are hydrolysed by S’nucleotidase play an important immunoregulatory function. Specific immunodeficiency syndromes are associated with congenital absence of lymphocyte adenosine deaminase and purine nucleoside phosphorylase. More recently, deficiencv of lvmnhocvte S’nucleotidase has been associated with both sex-linked and common variable hypogammaglobulinaemia (JOHNSON et al., 1977; EDWARDS et al., 1978). Recent studies with the highly specific assay used in the present study have indicated that hypogammaglobulinaemia patients have lymphocyte S’nucleotidase activities of less than 5% of control values (SMITH et al., 1982). This deficiency also seems to be highly selective in that the plasma membrane enzymes y-glutamyl transferase, leucine aminopeptidase and adenosine diphosphatase were unaffected. In the immunodeficiency syndromes genetic factors are of prime importance and immunoglobulin levels are often very low. While the decreased S’nucleotidase activity in Africans may be partly or wholly genetically determined, there is no evidence of hypogammaglobulinaemia in the African subjects. The S’nucleotidase deficiency in African patients may be a biochemical manifestation of the relative imrnunodepression associated with malnutrition and infectious disease. Alternatively these factors may induce secondary immunodeficiency in a setting when there is a genetically determined lower level of S’nucleotidase. This report also indicates that field studies of lymphocyte enzyme activity are possible. The volume of blood required for these experiments is small enough to be collected on a routine basis. Lymphocytes can be separated and cryopreserved with basic laboratory facilities. The specialized equipment required is both easily available and durable. Sources of liquid nitrogen in many developing countries (usually connected with artificial insemination programmes) are relatively accessible. Activities of the enzymes tested were stable to cryopreservation, with the possible exception of catalase which is known to be particularly affected by freezing and thawing (SHIKAMA & YANAZAKI, 1961). The cells used in this investigation were collected from three to eight years before being tested. We have no evidence that any biochemical or immunological property of cryopreserved lymphocytes is affected by prolonged storage in liquid nitrogen; indeed there is good evidence that such biological properties are unaffected by the duration of storage (FARRANT, 1980). Acknowledgements This work was supported in part by Grant G973/753/T from the UK Medical Research Council, by Grant SP1559 from the Cancer Research Campaign of the-UK, by a grant from the United Cancer Council, Rochester, New York and by Contract No. NOICP 43296 between the National Institutes of Health, USANCINCP and the International Agency for Research on Cancer. We are grateful to Mrs. J. Safari and Mr. Z. Siso for their assistance in field studies, to Drs. A. D. B. Webster and M. E. Balis for their helpful comments and Ms. Rosamund Greensted for secretarial assistance.
844
LYMPHOCYTE
ENZYME
ACTIVITIES
References Baker, R. J. & Nelder, J. A. (1978). Generalized Linear Interactive Modelling (GLZM) Release 3, Oxford: Numerical Algorithms Group. Edwards. N. L., Magilva. D. B., Cassidv, J. T. & Fox, I. H. (1978). Lymphocyte ecto-5’nucleotidase in agammaglobuhnaernia. Science, 201, 628630. Farrant, J. (1980). Practical Aspects. In: Lao Temperature Preservation in Medicine and Biology: Ashwood-Smith, M. J. & Farrant, J. (Editors). Tunbndge Wells: Pitman Medical, pp. 285-310. Farrant, J., Knight, S. C., McGann, L. L. & O’Brien, J. (1974). Optimal recovery of lymphocytes and tissue culture cells following rapid cooling. Nuture, 249, 452-453. Harris, R. & Ukaejioto, E. 0. (1969). Tissue typing usinga one-step lymphocyte separation procedure. Lancer, II, Joh%h S. M North M. E Asherson, G. L., Allsop, J.? Wat&, R.“W. El & Webster, A. D. B. (i977). Lymphocyte purine S’nucleotidase deficiency in primary hypogammaglobulinaemia. Lancet, i, 168-170. Kapuscinski, J. & Skoczylas, B. (1977). A simple and rapid fluorimetric method for DNA microassay. Analytical Biochemistry, 83, 252-257.
Peters, T. J. (1981). Investigation of tissue organelles by a combination of analytical subcellular fractionation and enzymic microanalysis: a new approach to pathology. Jcnmal of Clinical Patholoa, 34, 1-12. Schacterle, G. R. & Pollack, R. L. (1973). A simplified method ,for ,the quantitauve assay of small amounts of ~;oz$i;~rn biologic material. Analytical Baochemwy, 51,
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Shah, T., Webster, A. D. B. & Peters, T. J. (1981). Analytical subcellular fractionation with enryrmc ,m~croassayof human peripheral blood lymphocytes. Clmrc~l Science. 60. 3-4P. Shikama, K. &Yanazaki, I. (1961). Denaturation of catalase by freezing and thawing. Nature, 190, 83-84. Smith, G. P., Shah, T., Webster, A. D. B. & Peters, T. J. (1982). Studies on the kinetic properties and subcellular localization of adenine nucleotide phosphatases in peripheral blood lymphocytes from control subjects and patients with conu-nonvariable primary hypoganunaglols&~~;6rua. Clmscal and Experimental Immunology, 46, Steel, C. M:,Levin, A G.,Tsu, T. & Gross, R. L. (1974).A bank of frozen peripheral-blood lymphocytes for in vitro immunological studies on East African cancer patients. International Journal of Cancer, 13, 489-493. Vogel, L. C., Muller, A. S., Odingo, R. S., Onyango, Z. & De Geus, A. (Editors) (1974). Health and Disease in Kenya, Nairobi: East African Literature Bureau, pp. 181-374 and 409-428. Webster, A. D. B., Rowe, M., Johnson, S. M., Asherson, G. L. & Harkness, A. (1979). Ecto-S’nucleotidase deficiency in primary hypogammaglobulinaemia. CZBA Foundation Svmnosium. 68. 135-144. World Health Orga&ation. (1978). Immunodeficietq. Technical Report 630. Geneva: World Health Organization, pp. 52-i2.
Accepted
for
publication
31st March,
1983.