British Journal of Haematofogy, 1976, 34, 631.

Two Biochemical Markers in Lymphocyte Subpopulations ANDREW T. HUANG, GERALD L. LOGUE AND HELEN L. ENGELBRECHT

Department of Medicine, Duke University Medical Center, and VA Hospital, Durham, North Carolina (Received 2 April 1976; acceptedfor publication 9 June 1976) SUMMARY.Human blood lymphocytes can be separated into two populations according to the presence of surface complement receptors. Lymphocytes containing complement receptors (CR+) were found to have a high rate of RNA synthesis or turnover accompanied by increased protein synthesis. Lymphocytes not containing complement receptors (CR- ) while maintaining a low profile in RNA synthesis, had a 10-12-fold greater activity in adenosine deaminase enzyme which is believed to be related to lymphocyte-immune responses and cell-mediated immunity. These two biochemical characteristics can be useful tools for future studies in lymphocyte functions. By using these two biochemical markers, we found that CLL lymphocytes were predominantly of the CR+ type, had high active RNA synthesis, and very low adenosine deaminase level. Lymphocytes from two patients with X-linked agammaglobulinaemia showed a picture opposite to that of CLL. Human peripheral blood lymphocytes may be classified on the basis of proposed immunologic function. Surface characteristics of these cells have been used in this classification (Jondal et al, 1972). Lymphocytes which function in the regulation of so-called cellular immunity form rosettes with sheep red cells and lack surface-bound immunoglobulins as well as receptors for the third component of complement (C3) (Abrahamsohn et al, 1974). Lymphocytes which are involved in humoral immune recognition generally have surfacebound immunoglobulins, receptors for Fc fragments of immunoglobulins, and receptors for lymphocytes. C3. The former cells have been termed ‘T’ lymphocytes, and the latter, ‘By Investigation of various functions of these circulating lymphocytes has been hampered because of the difficulty of obtaining reasonably pure subpopulations. Lymphocytes can, however, be reproducibly separated into two distinct subpopulations by virtue of the presence or absence of complement receptors. Incubation of lymphocytes with autologous human red cells coated with complement in the absence of antibody and their subsequent centrifugation on Ficoll-Hypaque gradients allows this separation. Lymphocytes which have formed rosettes with complement coated red cells will be carried with the red cells to the bottom fraction of the gradient. Studies of the surface characteristics of these lymphocyte subpopulations have been amply reported. However, insufficient attention has been given to the biochemical differences in these subpopulations. We have been interested in enzymology and biosynthesis of nucleic Correspondence:Dr Andrew T. Huang, Division of Hematology and Oncology, Department of Medicine, Duke University Medical Center, Box 3942, Durham, North Carolina 27710, U.S.A.

631

632

A. T. Huang, G. L. Logue and H. L. Engelbrecht

acids and their macromolecular structures. While attempting to study the biochemical variations during their rcsponse to mitogens we observed two useful markers of these subpopulations related to the purine metabolic pathway and the synthesis of ribonucleic acids. Lymphocytes which did not possess complement receptors (CR- lymphocytes) had a 10-12-fold higher adenosine deaminase activity than cells with complement receptors (CR+ lymphocytes). On the other hand, CR+ lymphocytes were found to have a strikingly active synthesis of RNA while the CR- lymphocytes maintained a low baseline synthesis. The purpose of our report is to demonstrate these two distinct biochemical differences in normal CR+ and CR- lymphocytes and to show that these markers are useful in studying lymphocytes of patients with various diseases. MATERIALS AND METHODS Soiirce of Lymphocytes Lymphocytes were obtained from venous blood from normal volunteers, untreated patients with chronic lymphocytic leukaemia (lcucocyte counts ranged from 15.0 to 88.0 x rog/l.; Table 11), and X-linked aganiniaglobulinaemia.*Whole blood was allowed to scttle, leaving leucocyte-rich plasma in the upper fraction. This fraction was then incubated with carbonyl iron at I g/Io ml of plasma (purchased from GAF Corporation, New York, N.Y.) at 37°C for 30 min. The cell suspension in 5 nil volume was placed on a 15 ml Ficoll-Hypaque gradient (50% Hypaquc:g% Ficoll = 1/3 vlv) and centrifuged at 10ooo g for 20 min. Erythrocytes, neutrophile leucocytcs and monocytes were found in thc bottom fraction. Thc lymphocytes were removed from the interface, washed in Minimal Essential Medium (CIBCO, New York), counted, and were ready for further studies.

Prcpnratiori of Conrplearcnt-Coa~cedErythrocytcs Human erythrocytes were coated with the third component of complement in its immune adherent form (C3b) by incubating with anti-I antiscra and human complement in a biphasic temperature reaction mixture (Logue et nl, 1973). After completion of reaction, these red cclls (EC3b) were assayed for membrane bound C3 by the absorption of anti-C3 antibodies from fluid phase. The bound C3 were found to be between 0.8 and 1.5 x 1 0 - lg~ per red cell. To form human complement coated-human erythrocyte (HEC) rosettes, lymphocytes were mixed with C3-coated RBC in a ratio of I :IO in McCoy’s 5A tissue culture medium. The mixture of cells was incubated for 13 min or 180 min at 37°C. Association of one lymphocyte with a minimum of three erythrocytes was defined as a rosette. The mixture was then placed on top of a second Ficoll-Hypaquc gradient. After centrifugation, the top and bottom fractions were removed and washed in MEM for further studies. The top fraction contained isolated lymphocytes while the bottom fraction contained free erythrocytes, erythrocyte-erythrocyte, and lymphocyte-erythrocyte rosettes. Anti-iriimunoglobulin InzmunoJuorescence Test of Lymphocyte Fractions Fluorescent anti-IgG, IgM, and IgA reagents (purchased from Meloy Laboratories) were * Patient I : IgG 125,IgA 4.5 and IgM 3.1 mg/dl with normal range for his age group, IgG 535-1432, IgA 92-581, IgM 27-111 mg/dl. Patient 2 : IgG 180mg/dl, IgA and IgM were undetectablewith normal range for his age group, IgG 656-1755, IgA 89-559 and IgM 24-98 mg/dl.

Biochemical Markers in Lymphocytes

633

used to detect the presence of immunoglobulins on the lymphocyte surface (Abrahamsohn et al, 1974).This study confirmed that all lymphocytes sedimented in a rosette form to the bottom fraction had surface immunoglobulins while I-4% contamination of these cells were found in the top fraction. The bottom fraction appeared homogeneous and contained only lymphocytes with surface immunoglobulins.

Adenosine Deamiiiase Assay Samples from the top and bottom fractions of a second Ficoll-Hypaque gradient were treated identically before the enzyme study. Erythrocytes were lysed by the method of Fallon ct a1 (1962).Lymphocytes were then suspended in 0.1M potassium phosphate buffer, pH 6.5, sonicated, and centrifuged at 15 ooo rev/min for 20 min to remove the particulate matter. The supernatants were removed and used as the source of enzyme. Protein concentrations Enzyme assay (Pfrogner, 1967)was were determined by the method of Lowry et a1 (1951). performed by incubating the lymphocyte extracts with tritiated adenosine containing 5000 cpm per assay with the adenosine concentration ranging from 0.6 to 27.4nmoles. The enzyme activity was determined by a paper chromatographic separation of adenosine (Rf = 0.15) from inosine (Rf = 0.42) and hypoxanthine (Rf = 0.31)using ammonium sulphate-waterisopropanol (80:18:2)as solvent. An International Unit (U) was defined as that amount of enzyme which converted I pmole of adenosine per hour per mg protein. Study of RNA Synthesis Lymphocytes after the first Ficoll-Hypaque gradient separation from monocytes, polymorphonuclear leucocytes and red cells and before complement-mediated rosetting step, were incubated in McCoy's gA tissue culture medium with tritiated uridine (25 Ci/mmole, New England Nuclear) at 2 pCi/ml specific activity. After 3 h of incubation in radioactive medium, the cells were removed, washed with cold uridine in phosphate buffered saline twice, and were then subjected to HEC rosetting process. The mixture of cells were layered 011 top of another Ficoll-Hypaque gradient. The bottom fractions were examined under microscope for the confirmation of rosette formation. These two fractions were divided and processed for liquid scintillation counting and autoradiography study (Moses, 1964). For liquid scintillation counting of tritiated uridine, the top and bottom fractions were precipitated by 5% trichloroacetic acid a t 0°C for 15 min (Schneider, 1945).The precipitates which contained the macromolecular form of RNA were collected on top of glass-fibre filter disks before scintillation counting with toluene-phosphor as scintillator (Huang et al, 1972).For autoradiography study, each fraction was suspended in autologous plasma and was dispersed on clean glass slides. The slides were coated with Kodak NTB3 emulsion in complete darkness, incubated for 3 0 d at o"C, and developed with microdol-X. The slides were then stained with Giemsa, pH 6.6.Extensive scoring was then performed on both fractions. RESULTS

Di@rences in A DA Activity between CR+ and CR- Lymphocytes Incubation of Cg-coated red cells and lymphocytes yielded 16+ 3% HEC rosettes. These rosetted lymphocytes sedimented to the bottom fraction of the second Ficoll-Hypaque

634

A. T. Huang, G. L. Logue and H. L. Engelbrecht TABLE I. Adenosine deaniinase activity in normal lymphocytes

ADA activity (1.U.)

11.9 12.1

3 4

13.5

2.6

9.1 10.6 16.6

Below detection

5 6 7

MeanfSD ~

3.5 Below detection

I 2

0.2

0.4

8.2

0.9

11.7rt3.5

1.1 f 1.3

~

Seven studies of ADA activity in normal lymphocytes. CR+ and CR-- lymphocytes were separated after EC3-rosetting and subsequent density gradient centrifugation. Both fractions of cells were briefly treated to lyse the erythrocytes. The supernatant fractions of lymphocyte sonicates were assayed for ADA as described in Methods,

density gradient. The top fraction consisted of single lymphocytes without red cell association. Rosetted lymphocytes scdimenting to the bottom fraction maintained their rosette arrangements throughout the entire study. This method of lymphocyte separation yielded a rcproducibly homogeneous population of CR+ lymphocytes in the lower fraction of the gradient. Typing of the lymphocytes by fluorescent anti-immunoglobulin antibody technique confirmed that the CR lymphocytes also have surface immunoglobulins. Adenosine deaminase assay was performed on the extracts of those two fractions of lymphocytes. CR+ lymphocytes were found to have a mean activity of I.O+ 1.3 International Units. Extracts from the CR- lymphocytes had a mean activity of 11.6f 3.5 International Units (Table I). The study was extended to patients with chronic lymphocytic leukaemia and X-linked agammaglobulinaeniia. Table I1 showed that CLL CR+ lymphocytcs had ADA activity as low as their normal counterparts. Extracts from the CR- lymphocytes of chronic lymphocytic leukaemia have an activity approximately 50% of that of normal extracts. In two patients with X-linked agammaglobulinaemiawithout detectable circulating CR + lymphocytes, the lymphocyte ADA activities were within the normal variation of CR- lymphocytes, being 8.8 and 9.7 International Units respectively.

+

Active Synthesis of RNA in CR+ Lyrnyhocytes CR+ lymphocytes appear to have a much greater rate of RNA synthcsis or turnover as evidenced by increased tritiated uridine incorporation into the acid precipitable fraction of the cells. When the entire circulating population of lymphocytes was cultured with tritiated uridinc, most of the radioactivity was found in the CR+ lymphocytes fraction. The diffcrences in specific activity are presented in Table 111. In 106 cells, CR- lymphocytes have a

Biochemical Markers in Lymphocytes TABLE 11. Comparison of ADA activity in normal and CLL lymphocytes ADA specifrc activity (I.U.) Source of lymphocytes

CR-

Normal (seven studies)

CR+

II.7k3.5

I.Ik1.3

4.3

0.8 0.3 0.3

CLL* I

* 3 4 Mean 5 SD

2.0

3.3 9.7 4.8k2.9

1.5

0.7k0.5

~

ADA activities of four studies of CLL lymphocytes.

CLL cells were handled as described in legend for Table I. * Leucocyte counts: Patient I , 15 x 10~11.;Patient 2, 88 x 109/l.; Patient 3 , 34 x 10~11.Patient ; 4, 40 x 10~11.

TABLE 111. RNA synthesis in normal lymphocytes [3H]Uridine incorporated (cpm per 106 lymphocytes) No. of study

CR -

CR+

3522

13900 12419 23463

3980 1078 49 5

Mean f SD

2269+1505

I0000

14946f5110

[3H]uridine uptake studies were performed on lymphocytes from four normal donors. Purified total lymphocytes were incubated in tissue culture media containing [3H]uridine at a specific activity of 2 pCi/ml for 3 h, at which time cells were washed to remove unincorporated radioactivity. CR+ and CRlymphocytes were separated by rosette formation. Separated fractions were precipitated with 5% TCA at 0°C. Precipitates were collected on glass-fibre filter disks, washed extensively with 5% TCA and alcohol, and dried. The radioactivity of the disks were counted in a Beckman liquid scintillation counter using toluetiephosphor as scintillator.

63 5

A. T. Huang, G. L. Logue and H. L. Engelbrecht 636 baseline RNA synthesis of 2269f 1505 (SD) counts per minute (cpni). The CR+ population incorporated tritiated uridine to 14 946+ 5110 cpm. This striking difference in the rate of incorporation could be visualized by autoradiography study of the same cclls (Fig I). As seen in the figure, the CR+ population had a large number of tritium grains on their nuclei while a few grains were seen in the CR- population. DISCUSSION Many surface characteristics and immunological functions have been attributed to human blood lymphocytes. HEC rosetting and subsequent density gradient separations offer a useful tool in the study of lymphocytes with and without complement receptors. The binding of complement-coated erythrocytes to lymphocytes occurs rapidly and is firm. Microscopic examination following sedimentation reveals that virtually all of the lymphocytes carried to the bottom fraction have formed rosettes with the red cells. Thus, this technique reproducibly givcs a homogeneous population of CR + lymphocytes in sufficient quantity for biochemical study. Normal CR+ and CR- lymphocytes separatcd by this procedure show striking differences in their biochemical behaviours. CR - cells have a 10-12-fold greater adenosine deaniinasc activity while their RNA synthesis appears to be at a low Icvcl. CR+ lymphocytcs, on the other hand, have low ADA activity while maintaining a very high rate of synthesis of RNA. The enzyme adenosinc deaminase (ADA, EC 3.5.4.4)is related to cell division. Tissue cells having a higher rate of multiplication have been found to be rich in ADA as compared to non-dividing cells (Grcen & Chan, 1973). This enzyme is present in high concentration in lymphoid tissue (Edwards et al, 1971)and many observations strongly suggest the importance of this enzyme in the normal inxnunologic response (Hall, 1963). A rise in lymphocyte adenosine deaminase level was found to correlate with a rise of specific antibodies in rcsponse to bacterial infection. Furthermore, suppression of this enzyme by a specific inhibitor abolishes lymphocyte-mediated cytolysis of pre-sensitized tumour cclls (Wolberg ct nl, 1975).The absence of this enzyme in childrcn with severe combined iiiimune dcficicncy disease brings this cnzyme to the forefront ofclinical investigation (Meuwissen ct ul, 1975).The presence of a high adenosine deaminase activity in CR- lymphocytes described in this report indicates that CR- lymphocytes may multiply more readily and arc more closely related to lymphocyte-mediated cytolysis. Synthesis of RNA in human lymphocytcs has been extensively investigatcd in the past using the entire circulating population of lymphocytes (e.g. Cooper & Rubin, 1966). Stimulation of early RNA synthesis has been vicwed as neccssary for the subsequent response to mitogen or antigen. Studies of this type have not becn conducted in different subpopulations of lymphocytes. Our studies of entire resting lymphocyte populations freshly obtained from normal donors revealed that 10% of the cells had a greater uptake of tritiated uridinc as evidence of RNA synthesis or turnover. When these cells are separated according to the technique described above, the cells active in RNA synthesis were the CR+ lymphocytes. Studies which are presented elsewhere (Logue & Huang, 1976) show that following a 3 h incubation of complement coated red cells with these lymphocytes, further increasc in RNA synthesis is noted. The time required (10-15min) to separate CR+ from CR- lym-

Biochemical Markers in Lymphocytes

I . Autoradiography of [ 3H]uridine labellcd normal lymphocytes. Lymphocytes were treatcd as described in legend for Table I11 but without acid precipitation. The radiolabclled lymphocytes were washed and dispersed in autologous plasiiia. A thin smear was prepared on a glass slide, fixed in methanol and dipped into Kodak NTB3 photosensitivc emulsion in the dark. The slides were developed with Microdol X after 4 weeks of incubation at 0°C in the dark. Photography was performed after 3 inin of staining in Gienisa solution at a magnification of x 1000.Left: CR-- ; right CR+.

FIG

(Facing p 636)

Biochemical Markers in Lymphocytes

63 7

phocytes for these studies is not sufficient to allow stimulation of RNA synthesis. That is, the baseline RNA synthesis of the CR+ cells is greater than that of CR- cells prior to their interaction with the complement coated red cells. The active incorporation of tritiated uridine into these CR+ lymphocytes reflects increased RNA synthesis or turnover. The radioactive precursor was incorporated into RNA rather than into DNA. The radiolabelled macromolecules were alkaline-sensitive and were extractable by the method used for RNA (Scherrer et al, 1963). Furthermore, when tritiated thymidine was added to these cells for 3 h of incubation, insignificant amount of the label was incorporated by them. Our preliminary studies aiso show that the RNA synthesized by these CR+ lymphocytes are of the polydisperse type, including 45S, 3 5 S , 28S, 16S, 5s and 4s RNA species. These tritiated RNA are mainly found around the nuclei by autoradiography. This indicates that the CR+ lymphocytes may have a constant and active transcriptional activity as compared to CR- lymphocytes. Using radioactive amino acids as precursors we find that these cells are also much more active in protein synthesis (Logue & Huang, 1976). The observation indicates that the active RNA transcription is followed by the production of proteins. Utilizing these types of biochemical studies we have undertaken the investigation of lymphocytes from patients with several immunologic diseases. Adenosine deaminase studies in the B-cell deficient X-linked agammaglobulinaemicpatients show that their lymphocytes have a very high enzyme activity comparable to that of normal CR- lymphocyte population. Alternatively, extracts of CLL lymphocytes consistently have decreased ADA activity prior to fractionation into lymphocyte subpopulations. This low ADA activity could be primarily attributed to a high number of CR+ lymphocytes seen in these patients studied. The subnormal ADA activity in CR- lymphocytes of CLL patients may be either the result of contamination with CR+ lymphocytes or that their CR- lymphocytes have lower enzyme activity. W e feel that such biochemical studies of lymphocyte subpopulations will yield considerable information concerning normal immunologic functions and give insight into the biochemical deficiencies in patients with various immunodeficiency syndromes. ACKNOWLEDGMENTS

We thank Mrs R. Bossen for her preparation of the manuscript, Mrs M. M. Riddle and Ms B. Conner for their excellent technical contributions. This research is supported by grants from USPHS (NCI) CA I 1265, North Carolina Health Funds, and VA Research No. 4700-02 and 52 88-01. REFERENCES

I., NILSSON, U.R. & ABDOU, N.I. ABRAHAMSOHN, (1974)Relationship of immunoglobulin to complement receptors of human B cells. Journal of Immunology, I I Z , 1931. COOPER, H.L. & RUBIN, A.D. (1966)Synthesis of nonribosomal RNA by lymphocytes: a response to phytohemagglutinin treatment. Science, 152, 516. EDWARDS,Y.H.,HOPKINSON, D.A. & HARRIS, H. (1971) Adenosine deaminase isozymes in human tissues. Annals ofHuman Genetics, 35, 207.

FALLON, H.J., FREI,E., 111, DAVIDSON, J.D.,TRIER, J.S. & Bum, D. (1962)Leukocyte preparations from human blood: evaluation of their morphologic and metabolic state. Journal of Laboratory and Clinical Medicine, 59, 779. GREEN,H. & CHAN,T. (1973)Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine deaminase. Science, 182, 837. HALL, J.G. (1963)Adenosine deaminase activity in lymphoid cells during antibody production.

63 8

A. T. Huang, G. L. Logue and H. L. Engelbrecht

Australian Journal of Experimental Biology and Medical Science, 41, 93. HLJANG, A.T., KREMBR, W.B., LASZLO, J. & SETLOW, R.B. (1972) DNA repair in human leukaemic lymphocytes. Nature: N e w Biology, 240, 114. JONDAL, M., HOLM, G. & WIGZELL, H. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. Journal of Experimental Medicine, 136,207. LOGUE, G.L. & HUANG,A.T. (1976) Human lymphocyte complement receptors. 11. Stimulation of lymphocyte RNA synthesis by complement coated human red cells. Clinical Immunology and Immunopathology (submitted). LOCUE, G.L., ROSSE,W.F. & ADAMS,J.P. (1973) Complement-dependent immune adherence rneasured with human granulocytes: changes in the antigenic nature of red cell bound C3 produced by incubation in human serum. Clinical Immunology and Immunopathology, I,398. LOWRY, O.H.,ROSEBROUGH, N.J., FARR,A.L. & RANDALL, R.J. (1951) Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265.

MBUWISSJN, H.J., POLLARA, B. & PICKBRING, R.J. (1975) Combined immunodeficiency disease associated with adenosine deaminase deficiency.Journal ofPediatrics, 86, 169. MOSES,M.J. (1964) Application of autoradiography to electron microscopy. Journal of Histochemistry and Cytochemistry, 12, I I 5. PFROGNER, N. (1967) Adenosine deaminase from calf spleen. I. Purification. Archives of Biochemistry and Biophysics, 119,141. SCHERRER, K., LATHAM, H. & DARNELL, J.C. (1963) Demonstratiou of an unstable RNA and of a precursor to ribosomal RNA in HeLa cells. Proceedings ofthe National Academy of Sciences ofthe United States of America, 49, 240. SCHNBIDER, W.C. (1945)Phosphorus compounds in animal tissues. I. Extraction and estimation of deoxypentose nucleic acid and of pentose nucleic acid. Journal ofBiologira1 Chemistry, 161, 293. WOLBERG, G., ZIMMERMAN, T.P., Hxmsm, K., WINSTON, M. & CHU, L.C. (1975) Adenosine inhibition of lymphocytemediated cytolysis: possible role of cyclic adenosine monophosphate. Science, 187,957.

Two biochemical markers in lymphocyte subpopulations.

British Journal of Haematofogy, 1976, 34, 631. Two Biochemical Markers in Lymphocyte Subpopulations ANDREW T. HUANG, GERALD L. LOGUE AND HELEN L. ENG...
567KB Sizes 0 Downloads 0 Views