- Email: [email protected]
Inhibition of DNA synthesis in lymphocytes by dialyzable components of human leukocyte extracts
CELLULAR
IMMUNOLOGY
Inhibition
K. SAITO, Department
31, 311-320
(1977)
of DNA Synthesis in Lymphocytes by Dialyzable Components of Human Leukocyte Extracts1
N. TAMAKI, L. A. FOSTER,~ B. BRENNESSEL, AND A. A. GOTTLIEB of Microbiology
awzdIm~nmology, Tulane University New Orleans, Louisiana 70112 Received
School
of Medicine,
May 25,1976
Dialysates prepared from human leukocytes contain molecular species which can suppress the uptake of [3H]thymidine by a continuous B cell line and by peripheral lymphocytes. These suppressors are capable of dialyzing through an exclusion limit of 3500 MW and can be separated from each major polypeptide-containing fraction of the lysate by gel filtration
a membrane having other and from the on Sephadex G-10.
INTRODUCTION Extracts of peripheral leukocytes obtained from donors sensitive to tuberculin or a variety of bacterial and/or fungal antigens contain dialyzable materials which are capable of transferring these sensitivities to nonsensitive recipients. This phenomenon was originally described by Lawrence and has been confirmed by others (1, 2) ; we shall refer to it here as the “transfer factor” phenomenon. While the bulk preparation of dialyzable material from such leukocyte extracts contains a variety of molecular species, it is likely that only some of these molecules are responsible for the immunological transfer activity of the bulk preparation. A major difficulty in the analysis of the transfer factor phenomenon is the lack of a readily reproducible in vitro assay. While the incorporation of [3H] thymidine into DNA by peripheral leukocytes has been used as an investigational parameter for the study of the effects of transfer factor preparations on lymphocyte proliferation (3), it appears that this assay is not antigen specific for the donor’s reactivities (14, 15) and may not measure the activity of those components of the dialysate responsible for the specific transfer of delayed hypersensitivity in man. filoreover, since this assay depends on the ability of preparations of transfer factor to augment [ 3H] thymidine uptake and concomitant DNA synthesis, molecules in transfer factor preparations which are capable of suppressing thymidine uptake and/or DNA synthesis would tend to modify the stimulatory effect(s) of other molecules in the leukocyte extract. Such inhibitory or suppressive molecules have 1 Supported by a grant from the National Institutes of Health, AI-13386 (previously AI-12090). 2 Present address : Department of Pathology, Wakayama Medical College, Wakayama, Japan. 3 Present address : Division of Immunology, Department of Medicine, New York University Medical Center, New York, New York. 311 Copyright @ 1977 by AcademicPress,Inc. All rights of reproductionin any form reserved.
ISSN OOOS- 8749
312
SAITO
ET
AL.
been described by our group (4, 5) and our observations have been confirmed by others (6-S). The present study was undertaken to determine whether similar inhibitory molecules are present in dialysates prepared from human leukocyte extracts. METHODS Source of cells and preparation of “crude” transfer factor. Buffy coat leukocytes, were obtained from normal donors by conventional methods. In general, a dialysate was prepared by repeated freeze-thawing of 1 to 5 x lo* leukocytes followed by treatment of the cell extract with DNase (100 pg/ml) according to the original method of Lawrence (1, 2) except that dialysis of the leukocyte extract was carried out against 20 vol of 5 mM ammonium bicarbonate using a dialysis membrane having a cutoff of 12,000 molecular weight. The dialysate was lyophilized and stored at -20°C. For studies involving human macrophages, these cells were collected from healthy donors by Dr. Isaac Djerassi, Fitzgerald-Mercy Medical Center, Philadelphia, according to techniques previously described by Dr. Djerassi and his colleagues (9, 10). Of cells obtained by this technique, 91% had the morphologic properties of macrophages ; 8% were neutrophils and 1% were small lymphocytes. Assays for polypefitide in leukocyte dialysates. Aliquots of the dialysate to be tested were adjusted to a volume of 3.0 ml by addition of 0.005 M phosphate buffer (pH 8.0). One milliliter of fluorescamine reagent (Fluram, Roche) containing 30 mg of Fluram/lOO ml of acetone was added rapidly to the sample and mixed thoroughly using a Vortex mixer. Fluorescence was measured using an Aminco fluorocolorimeter with a primary filter of 508 nm and a second filter having a cutoff of 4.55 nm. A reference curve was constructed using bovine serum albumin as a standard. Efect of dialyzable components of leukocyte extracts on the incorporation of [ 3H] thymidine by continuons lymphoid line cultures. SWB-16A lymphocytes were obtained through the courtesy of Dr. Philip Glade, and Molt 3 cells were the gift of Dr. Maria Hornung. These lines were maintained in RPMI-1640 medium containing 20% fetal calf serum, 0.002 M glutamine, and 40 pg/ml of gentamicin. For the experimental studies, 1.0 ml of the cell suspension containing 1.0 to 2.0 x lo6 cells/ml (cell viability 93 to 96%) was added to Falcon 3033 tissue culture tubes. The leukocyte dialysates were subjected to gel filtration on Sephadex G-10 as previously described (11). The effects of these fractions were evaluated by adding 0.1 ml of each fraction to individual tubes containing the SWB-16A cells, as described above. The cells were maintained for 3 hr at 37°C in a 5% COZ atmosphere, at which time 1 ,&i of [methyZ-3H]thymidine diluted in culture medium (specific activity = 19.1 C’/1 mmol) was added to each tube and incubation was continued for an additional 16 to 17 hr. At that time, the cell suspension was washed three times with normal saline ; 1 ml of 10% trichloroacetic acid was added to each tube and the resulting precipitate was allowed to remain at 4°C for 1 hr. The precipitate was then pelleted by centrifugation and taken up in 0.25 ml of a 1: 1 mixture of NCS solubilizer (Amersham/Searle) in toluene and counted in a scintillation mixture containing 0.5% PPO (2,5-diphenyloxazole) and 0.02% bisMSB [p-bis( 0-methylstyryl) benzene] in a 2: 1 mixture of toluene and Triton.
INHIBITORS
OF LYMI’HOID
HUMAN
LEUKOCYTE
DNA
OIALYSATE
313
SYNTHESIS
0.442,000)
- 200
-
400 I a ”
- 600
0
35
40
45 FRACTION
50 55 NUMBER
60
65
70
800
FIG. 1. Gel filtration of human leukocyte dialysate (MW < 12,000). A dialysate of a leukocyte extract was prepared by a modification of the procedure of Lawrence (1, 2). The preparation was subjected to gel filtration on Sephadex G-10 and 0.1 ml of each fraction was added in to a l-ml culture containing 2 X lo0 cells/ml of the continuous lymphoid line SWB-16A RPMI-1640 medium containing 10% fetal calf serum. After 3 hr, 1 pCi of [?nethyl-3H]thymidine (specific activity = 19.1 ci/mmol) was added and incubation was continued for another 16-17 hr. After washing the cells three times with saline, 1 ml of 10% trichloroacetic acid was added. At that time, the precipitate was recovered and dissolved in 0.25 ml of a 1: 1 mixture of solubilizer (Amersham) in toluene and counted in a 2: 1 mixture of toluene and Triton X-100 containing 0.5% PPO and 0.02% bis-MSB. Viability of the cells was evaluated by the trypan blue technique and was over 93% at the conclusion of the experiment. The polypeptide content of each fraction was measured by the fluorescamine procedure and the results obtained are shown. The level of [3H]thymidine incorporation in four control cultures of SWB-16A cells to which only 5 mM ammonium bicarbonate had been added was 634 2 43 cpm.
For studies employing the Molt line of lymphoid cells, the washed cell pellet was suspended in 1.0 ml of physiological saline solution and 0.2 ml of the suspension was transferred to 5.0 ml of 10% cold trichloroacetic held at 4°C. After remaining in the cold for 60 min, the precipitates were filtered on Schleicher and Schuell B-6 nitrocellulose filters, washed five times with 5% trichloroacetic acid, dried, and counted in a liquid scintillator containing 0.5% PPO and 0.04% POPOP. Two control cultures were run in parallel: One set of cells was preincubated with 5 m&I ammonium bicarbonate solution and carried through without the addition of [3H] thymidine in order to measure absolute background ; the other was preincubated with 5 mM ammonium bicarbonate, [3H] thymidine was added, and the cells were incubated as above, in order to measure the baseline level of [3H] thymidine incorporation by these cultures. The viability of the cells in culture was evaluated by the trypan blue technique. Effect of dialyzebIe compomnts of leulzocytc cstmcts on the incovporatiort of Lymphocytes were recovered from [ 3H] thymidine by peripheral lymphocytes.
peripheral
blood of healthy individuals
by the Ficoll-Hypaque
method (2) and
314
SAITO
HUMAN
MACROPHAGE
ET
AL.
DIALYSATE
~M~l2,OOO)
I
12.0 II.0 _
zoo
10.0 -
400 9 SO0
4.0. 31)300 20-
I.::
FRACTION
NUMBER
of human macrophage dialysate (MW < 12,000). A dialysate of a macrophage extract was prepared as in Fig. 1. The preparation was subjected to gel filtration on Sephadex G-10 and O.l-ml aliquots of fractions 30-74 were assayed for their effects on the uptake of [‘Hlthymidine by SWB-16A lymphoid cells by the same procedure used in the experiment shown in Fig. 1. The polypeptide profile as determined by Fluram and the results of the [aH]thymidine incorporation assay are displayed. The level of [“HI thymidine incorporation in four control cultures of SWB-16A cells to which only 5 mM ammonium bicarbonate had been added was 710 2 38 cpm. FIG. 2. Gel filtration
suspended to a final concentration of lo6 cells/ml in a culture medium containing 80% RPMI-1640 (with L-glutamine) and 20% autologous plasma containing penicillin and streptomycin. One-fifth milliliter of cell suspension (2 x lo5 cells) was transferred to the wells of a sterile Falcon 3040 microtiter plate. The crude leukocyte dialysate was subjected to gel filtration, and the effects of the eluted fractions were tested by the addition of an aliquot of each fraction (in 0.02 ml) to each well of the microtiter plate. The cells were incubated at 37°C in a COz incubator for 3 to 144 hr, at which point 0.2 &i of [3H] thymidine (in 0.04 ml of culture medium) was added to each well. Incubation was carried out for an additional 18 hr, at which point the entire volume of the well was transferred to a test tube and washed three times in normal saline. Viability was then determined using trypan blue. The cell pellet was mixed with 5 ml of Aquasol (New England Nuclear Corp.) and transferred to a scintillation vial ; the test tube was rinsed with another 5 ml of Aquasol and the washed liquid was transferred to the same scintillation vial prior to counting in a Packard scintillation spectrometer. RESULTS In the course of fractionating crude leukocyte dialysates by gel filtration on Sephalex G-10, we had occasion to examine the effects of these fractions on the
INHIBITORS
OF
LYMI’HOID
DNA
SYNTHESIS
315
ability of lymphoid cells to incorporate [“HI thymidine into DNA. The eluted fractions were tested for their effects on the incorporation of [“H]thymidine by SWB16A lymphoid cells and the results obtained are shown in Fig. 1. The major polypeptide peak detected by reaction with Fluram is displayed. As can be seen, there are two regions of suppression in the elution pattern; a weak suppressor activity is associated with the major Fluram-reactive polypeptide peak, while a fraction exerting substantial suppression on these cells is found to elute behind the polypeptide peak. The two suppressors’ fractions are of nearly equal activity when tested against peripheral lymphocytes (Fig. 3) or against Molt cells (Fig. 5). In view of the functional similarity of these inhibitory components to the dialyzable substance from rat peritoneal macrophages which we have described previously (4), we proceeded to examine human macrophages to determine if a similar substance was present in these cells. A lysate of 8 x lo8 human macrophages was prepared and subjected to gel filtration in Sephadex G-10. The Fluramreactive polypeptide peak was identified, and the indicated fractions were tested for their effect on the incorporation of [sH]thymidine by continuously cultured SWB16A lymphocytes. The result is shown in Fig. 2. It is clear that a suppressor substance is present in the macrophage lysate and is not associated with the major Fluram-reactive fraction of these cells, but resembles the second suppressor found in the dialysate prepared from leukocyte lysates. In this study, the fractions preceding the Fluram peak were not examined for suppressive substances. As these results were originally observed using cells of the SWB-16A continuous lymphoid cell line, which are B-cell derivatives, it was of interest to determine whether these inhibitory fractions had similar effects on normal peripheral lymphocytes. Accordingly, studies were carried out to determine the effect of these fractions on the incorporation of [ 3H] thymidine by these cells. As shown in Fig. 3A, three inhibitory fractions were identified as a result of addition of the respective fractions from Sephadex G-10 to cultures of peripheral lymphocytes for 3 hr prior to addition of [3H]thymidine. An interesting observation, in this regard, is the effect of time on the ability of the fractions containing inhibitory activity to affect thymidine uptake by peripheral lymphocytes. Figures 3A and B illustrate the effect of addition of the same fractions to peripheral lymphocytes with incubation for 3 hr and 3 days, respectively, before addition of [3H] thymidine to cultures. In Fig. 3A, two inhibitory fractions, I1 and I2 corresponding to S1 and SII of prior studies, and a third fraction, I3 were observed. I3 appears to correspond to thymidine as noted in a separate study (Fig. 5). In contrast, after 7 days of incubation (Fig. 3C), the effects of the three inhibitory fractions are far less marked, but there is a significant augmentation of [3H] thymidine uptake displayed by material close to the maximum Fluram-reactive peak. Such augmentation is not observed after 3 hr or 3 days of incubation. These studies of the effects of these fractions on the uptake of [3H] thymidine by peripheral lymphocytes indicate that the two suppressor activities which we have identified are capable of suppressing the uptake of [3H] thymidine by normal peripheral lymphocytes and that the effects of these suppressors on the SWB-16A line are not attributable to some unusual property of these continuously cultured cells, but may be seen using normal peripheral cells as well. In prior work, we have found that components of leukocyte dialysates prepared by dialysis against a membrane having a 12,000-MW cutoff could be conveniently subfractionated by redialysis against a membrane having a cutoff of 3500 daltons.
316
SAITO
ET
AL.
1 w !OO 300 400 ; 500 600 100 800
3oo
.._..................
..,,
lAtlCmWD
0
ii
loo
z 2 zoo-
1 9 ‘2
200
1 \ ii , j,; 8 :, ‘3 . .,i... ..;.. J( /Y+ . ..-....... $.-.-I
I t F d a loo-
ml ..,.... h” IHIHCO, 400 0t 1 CWROL NO
-..............
600 700 0 50
-“n J.
.J\ ,,..
@.I
.,......
150
200
WI
BActcRwNI)
FIG. 3. Gel filtration of human leukocyte dialysate (MW < 12,000) : Effects of recovered fractions on peripheral lymphocytes. Cultures of peripheral lymphocytes purified by FicollHypaque sedimentation were established in wells of microtiter plates at levels of 2 X IO” cells/O.2 ml. The respective fractions were each tested separately by addition to each well. Incubation was carried out for 3 (A), 72 (B), and 144 hr (C), at which times 0.2 $Zi of [SHJthymidine was added to each well, and incubation was continued for another 18 hr. The cells were recovered, washed, and counted by the procedures described under Methods. The term background refers to the result obtained by carrying cells through the procedure without the addition of thymidine. Control cultures to which only 5 mM ammonium bicarbonate was added were run in parallel, and the range of [‘Hlthymidine incorporation manifested by these cultures is displayed.
INHTRTTORS
OF
LYMPHOTD
DNA
317
SYNTHESIS
600 2 u 800
I
TF (3500
< M < 12000)
E BOO
FRACTION
NUMBER
FIG. 4. Differential dialysis of human leukocyte dialysate. Effects of low molecular weight (< 3500, A) and high molecular weight (> 3500, B) subfractions of leukocyte extract on uptake of [“Hlthymidine by SWB-16A lymphoid cells: a leukocyte extract was dialyzed against a membrane having a cutoff of 3500 MW. The small (< 3500) and large (> 3500) fractions were were passed separately through a Sephadex G-10 column and the indicated fractions assayed by Fluram for polypeptide content and their effect on [3H]thymidine uptake by SWB16A lymphoid cells. The level of [“Hlthymidine incorporation in four control cultures of SWB16A cells to which only 5 mM ammonium bicarbonate had been added was 844 k 58 cpm.
the same leukocyte dialysate which was studied in the experiment shown in Fig. 1 was redialyzed in this way, and the dialyzable as well as the retained fraction were each subjected to gel filtration on Sephadex G-10. The eluates were tested for their ability to suppress [ 3H] thymidine uptake. The results are depicted in Figs. 4A and B. The material passing through the 3500-MW dialysis membrane appeared to contain suppressive activity, whereas the retentate displayed no inhibitory properties. In view of the finding by Opitz et a/. (13) that suppression of thymidine incorporation in lymphocytes by macrophages might be due to the release of endogenous thymidine from the macrophages, it was important to assess whether or not either of the suppressive materials in the leukocyte dialysates was thymidine. unlabeled thymidine was passed through the same column used to Accordingly, Accordingly,
318
SAITO HUMAN
LEUKOCYTE
ET AL. DIALYZATE
( M<3500
)
05
80 IO
NO70 60 3 c ~zoo- 50
; pz &
20: I
* 40
?
25:
30 30
IOO20
35
1.0
IO
20
30
40
60
50 FRACTION
70
60
90
4.0 IOU
NO.
FIG. 5. Gel filtration of human leukocyte dialysate (MW < 3500) : Differential elution of the suppressors and unlabeled thymidine. A leukocyte extract was dialyzed using a dialysis membrane having a cutoff of 3500 MW. The dialysate was recovered, concentrated, and applied to a column of Sephadex G-10 in 5 mM ammonium bicarbonate and elution was carried out with this solvent. The fractions were assayed by Fluram and for their effects on the ability of Molt cells to incorporate [‘Hlthymidine. The polypeptide profile is shown and the presence of two suppressor species ( SI and Sn) is indicated. After washing the column with 5 mM ammonium bicarbonate, 2 mg of unlabeled thymidine was applied and elution was carried out in the same way as for the dialysate. The absorbance profile at 260 nm is shown, and it can be seen that thymidine elutes at a point distinct from the Fluram-reactive peak or suppressors Sr and Sn. The level of [‘Hlthymidine incorporation in four control cultures of Molt cells to which only 5 mM ammonium bicarbonate had been added was 1614 _’ 38 cpm.
fractionate the dialysate. As shown in Fig. 5, the Sr and Srr suppressor fractions eluted ahead of unlabeled thymidine from the Sephadex G-10 column under these conditions. In these experiments as in others (Fig. 3), significant inhibition was also observed in the region corresponding to thymidine. To determine whether these inhibitors were present in all lymphoid cells, a lysate was prepared from SWB-16A cells, which are a B-cell lymphoid line sustained in continuous culture. The lysate was sequentially dialyzed against the 12,000- and 3500-MW membranes, respectively. The final dialysate contained relatively little Fluram-reactive material and displayed no significant suppression of [ 3H] thymidine uptake by other SWB-16A cells (Fig. 6). DISCUSSION These studies provide evidence for the existence of at least three substances, in lysates of buffy coat leukocytes, which are capable of suppressing DNA synthesis in peripheral lymphocytes as well as continuously cultured B lymphocytes. We have identified these as Sr and S n, with respect to their action on the SWB-
present
INHIBITORS DIALYZATE
20
30
OF LYMPHOID (Mc3500)
FROM
DNA SWB-16A
40 FRACTION
SYNTHESIS LYMPHOID
50
319
CELLS
60
NO.
FIG. 6. Effect of the low molecular weight subfraction of SWB-16A cells on the uptake of [‘Hlthymidine by SWB-16A cells. An extract was prepared from 5.2 X 10’ SWB-16A cells and subjected to dialysis against a membrane having a cutoff of 3500 MW. The dialysis was subjected to gel filtration on a column of Sephadex G-10 using 5 mM ammonium bicarbonate as eluant. The fractions were assayed for polypeptide content by the Fluram technique and for their effects on [“Hlthymidine uptake by SWB-16A cells. The level of [3H]thymidine incorporation in four control cultures of SWB-16A cells to which only 5 nh4 ammonium bicarbonate had been added was 631 * 39 cpm.
16A line, and I1 and 12, with respect to their action on peripheral lymphocytes. A third inhibitor, corresponding to Is, was observed to elute in the region of thymidine (Fig. 5) and probably represents endogenous thymidine recovered from the leukocyte lysate. None of these suppressive activities appears to be present in the continuously cultured B-cell line (SWB-16A), and this would suggest their absence from B cells in V&JO. Sn appears to be present in human macrophages (Fig. 2), but may also be derived from other cell types, as it appears to be comparable to the suppressive factor reported by Burger et nl., who have identified this component as nicotinamide (S). This material may also be similar if not identical to the dialyzable suppressor which we have previously identified in peritoneal macrophages (4). A similar substance appears to have been found in rabbit macrophages by Ulrich (7) and in mouse macrophages by Unanue and co-workers (6). The presence of these suppressive substances in leukocyte dialysates may have an important bearing on the effects of these dialysates on lymphocyte DNA synthesis both in viva and in vitro. Studies of the effects of transfer factor preparations on antigen-triggered proliferation of lymphocytes in vitro have suggested that this assay does not measure specific transfer factor activity, since it is not antigen specific for the reactivities of the donor. Interpretation of these results may be affected by the presence in these preparations of inhibitory substances such as the suppressor molecules identified in these studies. Removal of one suppressor (Sn) by gel filtration on Sephadex G-10 and further fractionation of the major Fluram-
320
SAITO
ET
AL.
reactive polypeptide peak to remove the other (S1) may give rise to leukocyte extract preparations containing biologically active transfer factor which could be usefully employed for further study of this phenomenon. In this regard it is of interest to note that the suppressors appear to be most active when incubated with peripheral lymphocytes from 3 to 72 hr, while augmentation of [ 3H] thymidine uptake is induced by substances present close to the major polypeptide peak, if these fractions are permitted to stay in contact with the lymphocytes for 144 hr. This would suggest that by proper choice of time of exposure of lymphocytes to the particular fraction under study, one can selectively study either suppression or stimulation. We also noted that, in contrast to the effects observed using peripheral lymphocytes, significant augmentation as well as suppression were noted after only 3 hr of contact of the respective fractions with the Molt lymphoid cell line, which is derived from a human leukemia cell having the character of a T cell. It is possible that the target cells for the factor in the dialysates responsible for augmentation may be analogous to the type of T lymphocyte represented in the Molt line. Such a cell might arise during culture of peripheral lymphocytes over a 6-day period. Both suppressor fractions (S1 and S,,) appear to be capable of acting on peripheral lymphocytes. The relative effects of augmentation and suppression of [3H]thymidine uptake by different types of lymphoid cells may offer an opportunity to study these respective functions and to ascertain whether modulation and/or balance of these effects occurs in viva. REFERENCES 1. Lawrence, H. S., Harvey Lect. 68, 239, 1973. 2. Lawrence, H. S., J. Clin. Invest. 34, 219, 1955. 3. Ascher, M. S., Schneider, W. J., Valentine, F. T., and Lawrence, H. S., Proc. Nat. Acad. Sci. USA 71, 1178,1974. 4. Waldman, S. R., and Gottlieb, A. A., Cell. Imwwnol. 9, 142, 1973. 5. Saito, K., Foster, L. G., and Gottlieb, A. A., J. Int. Res. Commult. 3, 323, 1975. 6. Calderon, J., Williams, R. T., and Unanue, E. R., Proc. Nat. Acad. Sci. USA 71, 4273, 1974. 7. Ulrich, F., Biochem. Biophys, Res. Cownu~z. 60, 1453, 1974. 8. Burger, D. R., Vandenbark, A. A., Daves, D., Anderson, W. A., Jr., Vetto, M. R., and Finke, P., J. Iwzwaunol. 117, 797, 1976. 9. Djerassi, I., Kim, T. S., Ciesielka, W., Chaimongkol, B., and Suvansri, U., Transfusion. 15, 353, 1973.
10. Djerassi, I., Kim, T. S., Mitrakul, C., Suvansri, U., and Ciesielka, W., J. Med. (Basel) 1, 358, 1970.
11. Gottlieb, A. A., Foster, L. G., and Waldman, S. R., Lancet 2, 822, 1973. 12. Boyum, A., Stand. J. Clin. Lab. Znvcst. 21, 31, 1968. 13. Opitz, H. G., Niethammer, D., Lemke, H., Flad, H. D., and Huget, R., Cell Immunol. 16, 379, 1975. 14. Cohen, L., Holzman, R. S., Valentine, F. T., and Lawrence, H. S., J. Exp. Med. 143, 791, 1976.
15. Hamblin, A., Maini, R. N., and Dumonde, D. C. In. “Transfer Factor. Basic Properties and Clinical Applications” (M. S. Ascher, A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 49-57. Academic Press, New York, 1976.
IMMUNOLOGY
Inhibition
K. SAITO, Department
31, 311-320
(1977)
of DNA Synthesis in Lymphocytes by Dialyzable Components of Human Leukocyte Extracts1
N. TAMAKI, L. A. FOSTER,~ B. BRENNESSEL, AND A. A. GOTTLIEB of Microbiology
awzdIm~nmology, Tulane University New Orleans, Louisiana 70112 Received
School
of Medicine,
May 25,1976
Dialysates prepared from human leukocytes contain molecular species which can suppress the uptake of [3H]thymidine by a continuous B cell line and by peripheral lymphocytes. These suppressors are capable of dialyzing through an exclusion limit of 3500 MW and can be separated from each major polypeptide-containing fraction of the lysate by gel filtration
a membrane having other and from the on Sephadex G-10.
INTRODUCTION Extracts of peripheral leukocytes obtained from donors sensitive to tuberculin or a variety of bacterial and/or fungal antigens contain dialyzable materials which are capable of transferring these sensitivities to nonsensitive recipients. This phenomenon was originally described by Lawrence and has been confirmed by others (1, 2) ; we shall refer to it here as the “transfer factor” phenomenon. While the bulk preparation of dialyzable material from such leukocyte extracts contains a variety of molecular species, it is likely that only some of these molecules are responsible for the immunological transfer activity of the bulk preparation. A major difficulty in the analysis of the transfer factor phenomenon is the lack of a readily reproducible in vitro assay. While the incorporation of [3H] thymidine into DNA by peripheral leukocytes has been used as an investigational parameter for the study of the effects of transfer factor preparations on lymphocyte proliferation (3), it appears that this assay is not antigen specific for the donor’s reactivities (14, 15) and may not measure the activity of those components of the dialysate responsible for the specific transfer of delayed hypersensitivity in man. filoreover, since this assay depends on the ability of preparations of transfer factor to augment [ 3H] thymidine uptake and concomitant DNA synthesis, molecules in transfer factor preparations which are capable of suppressing thymidine uptake and/or DNA synthesis would tend to modify the stimulatory effect(s) of other molecules in the leukocyte extract. Such inhibitory or suppressive molecules have 1 Supported by a grant from the National Institutes of Health, AI-13386 (previously AI-12090). 2 Present address : Department of Pathology, Wakayama Medical College, Wakayama, Japan. 3 Present address : Division of Immunology, Department of Medicine, New York University Medical Center, New York, New York. 311 Copyright @ 1977 by AcademicPress,Inc. All rights of reproductionin any form reserved.
ISSN OOOS- 8749
312
SAITO
ET
AL.
been described by our group (4, 5) and our observations have been confirmed by others (6-S). The present study was undertaken to determine whether similar inhibitory molecules are present in dialysates prepared from human leukocyte extracts. METHODS Source of cells and preparation of “crude” transfer factor. Buffy coat leukocytes, were obtained from normal donors by conventional methods. In general, a dialysate was prepared by repeated freeze-thawing of 1 to 5 x lo* leukocytes followed by treatment of the cell extract with DNase (100 pg/ml) according to the original method of Lawrence (1, 2) except that dialysis of the leukocyte extract was carried out against 20 vol of 5 mM ammonium bicarbonate using a dialysis membrane having a cutoff of 12,000 molecular weight. The dialysate was lyophilized and stored at -20°C. For studies involving human macrophages, these cells were collected from healthy donors by Dr. Isaac Djerassi, Fitzgerald-Mercy Medical Center, Philadelphia, according to techniques previously described by Dr. Djerassi and his colleagues (9, 10). Of cells obtained by this technique, 91% had the morphologic properties of macrophages ; 8% were neutrophils and 1% were small lymphocytes. Assays for polypefitide in leukocyte dialysates. Aliquots of the dialysate to be tested were adjusted to a volume of 3.0 ml by addition of 0.005 M phosphate buffer (pH 8.0). One milliliter of fluorescamine reagent (Fluram, Roche) containing 30 mg of Fluram/lOO ml of acetone was added rapidly to the sample and mixed thoroughly using a Vortex mixer. Fluorescence was measured using an Aminco fluorocolorimeter with a primary filter of 508 nm and a second filter having a cutoff of 4.55 nm. A reference curve was constructed using bovine serum albumin as a standard. Efect of dialyzable components of leukocyte extracts on the incorporation of [ 3H] thymidine by continuons lymphoid line cultures. SWB-16A lymphocytes were obtained through the courtesy of Dr. Philip Glade, and Molt 3 cells were the gift of Dr. Maria Hornung. These lines were maintained in RPMI-1640 medium containing 20% fetal calf serum, 0.002 M glutamine, and 40 pg/ml of gentamicin. For the experimental studies, 1.0 ml of the cell suspension containing 1.0 to 2.0 x lo6 cells/ml (cell viability 93 to 96%) was added to Falcon 3033 tissue culture tubes. The leukocyte dialysates were subjected to gel filtration on Sephadex G-10 as previously described (11). The effects of these fractions were evaluated by adding 0.1 ml of each fraction to individual tubes containing the SWB-16A cells, as described above. The cells were maintained for 3 hr at 37°C in a 5% COZ atmosphere, at which time 1 ,&i of [methyZ-3H]thymidine diluted in culture medium (specific activity = 19.1 C’/1 mmol) was added to each tube and incubation was continued for an additional 16 to 17 hr. At that time, the cell suspension was washed three times with normal saline ; 1 ml of 10% trichloroacetic acid was added to each tube and the resulting precipitate was allowed to remain at 4°C for 1 hr. The precipitate was then pelleted by centrifugation and taken up in 0.25 ml of a 1: 1 mixture of NCS solubilizer (Amersham/Searle) in toluene and counted in a scintillation mixture containing 0.5% PPO (2,5-diphenyloxazole) and 0.02% bisMSB [p-bis( 0-methylstyryl) benzene] in a 2: 1 mixture of toluene and Triton.
INHIBITORS
OF LYMI’HOID
HUMAN
LEUKOCYTE
DNA
OIALYSATE
313
SYNTHESIS
0.442,000)
- 200
-
400 I a ”
- 600
0
35
40
45 FRACTION
50 55 NUMBER
60
65
70
800
FIG. 1. Gel filtration of human leukocyte dialysate (MW < 12,000). A dialysate of a leukocyte extract was prepared by a modification of the procedure of Lawrence (1, 2). The preparation was subjected to gel filtration on Sephadex G-10 and 0.1 ml of each fraction was added in to a l-ml culture containing 2 X lo0 cells/ml of the continuous lymphoid line SWB-16A RPMI-1640 medium containing 10% fetal calf serum. After 3 hr, 1 pCi of [?nethyl-3H]thymidine (specific activity = 19.1 ci/mmol) was added and incubation was continued for another 16-17 hr. After washing the cells three times with saline, 1 ml of 10% trichloroacetic acid was added. At that time, the precipitate was recovered and dissolved in 0.25 ml of a 1: 1 mixture of solubilizer (Amersham) in toluene and counted in a 2: 1 mixture of toluene and Triton X-100 containing 0.5% PPO and 0.02% bis-MSB. Viability of the cells was evaluated by the trypan blue technique and was over 93% at the conclusion of the experiment. The polypeptide content of each fraction was measured by the fluorescamine procedure and the results obtained are shown. The level of [3H]thymidine incorporation in four control cultures of SWB-16A cells to which only 5 mM ammonium bicarbonate had been added was 634 2 43 cpm.
For studies employing the Molt line of lymphoid cells, the washed cell pellet was suspended in 1.0 ml of physiological saline solution and 0.2 ml of the suspension was transferred to 5.0 ml of 10% cold trichloroacetic held at 4°C. After remaining in the cold for 60 min, the precipitates were filtered on Schleicher and Schuell B-6 nitrocellulose filters, washed five times with 5% trichloroacetic acid, dried, and counted in a liquid scintillator containing 0.5% PPO and 0.04% POPOP. Two control cultures were run in parallel: One set of cells was preincubated with 5 m&I ammonium bicarbonate solution and carried through without the addition of [3H] thymidine in order to measure absolute background ; the other was preincubated with 5 mM ammonium bicarbonate, [3H] thymidine was added, and the cells were incubated as above, in order to measure the baseline level of [3H] thymidine incorporation by these cultures. The viability of the cells in culture was evaluated by the trypan blue technique. Effect of dialyzebIe compomnts of leulzocytc cstmcts on the incovporatiort of Lymphocytes were recovered from [ 3H] thymidine by peripheral lymphocytes.
peripheral
blood of healthy individuals
by the Ficoll-Hypaque
method (2) and
314
SAITO
HUMAN
MACROPHAGE
ET
AL.
DIALYSATE
~M~l2,OOO)
I
12.0 II.0 _
zoo
10.0 -
400 9 SO0
4.0. 31)300 20-
I.::
FRACTION
NUMBER
of human macrophage dialysate (MW < 12,000). A dialysate of a macrophage extract was prepared as in Fig. 1. The preparation was subjected to gel filtration on Sephadex G-10 and O.l-ml aliquots of fractions 30-74 were assayed for their effects on the uptake of [‘Hlthymidine by SWB-16A lymphoid cells by the same procedure used in the experiment shown in Fig. 1. The polypeptide profile as determined by Fluram and the results of the [aH]thymidine incorporation assay are displayed. The level of [“HI thymidine incorporation in four control cultures of SWB-16A cells to which only 5 mM ammonium bicarbonate had been added was 710 2 38 cpm. FIG. 2. Gel filtration
suspended to a final concentration of lo6 cells/ml in a culture medium containing 80% RPMI-1640 (with L-glutamine) and 20% autologous plasma containing penicillin and streptomycin. One-fifth milliliter of cell suspension (2 x lo5 cells) was transferred to the wells of a sterile Falcon 3040 microtiter plate. The crude leukocyte dialysate was subjected to gel filtration, and the effects of the eluted fractions were tested by the addition of an aliquot of each fraction (in 0.02 ml) to each well of the microtiter plate. The cells were incubated at 37°C in a COz incubator for 3 to 144 hr, at which point 0.2 &i of [3H] thymidine (in 0.04 ml of culture medium) was added to each well. Incubation was carried out for an additional 18 hr, at which point the entire volume of the well was transferred to a test tube and washed three times in normal saline. Viability was then determined using trypan blue. The cell pellet was mixed with 5 ml of Aquasol (New England Nuclear Corp.) and transferred to a scintillation vial ; the test tube was rinsed with another 5 ml of Aquasol and the washed liquid was transferred to the same scintillation vial prior to counting in a Packard scintillation spectrometer. RESULTS In the course of fractionating crude leukocyte dialysates by gel filtration on Sephalex G-10, we had occasion to examine the effects of these fractions on the
INHIBITORS
OF
LYMI’HOID
DNA
SYNTHESIS
315
ability of lymphoid cells to incorporate [“HI thymidine into DNA. The eluted fractions were tested for their effects on the incorporation of [“H]thymidine by SWB16A lymphoid cells and the results obtained are shown in Fig. 1. The major polypeptide peak detected by reaction with Fluram is displayed. As can be seen, there are two regions of suppression in the elution pattern; a weak suppressor activity is associated with the major Fluram-reactive polypeptide peak, while a fraction exerting substantial suppression on these cells is found to elute behind the polypeptide peak. The two suppressors’ fractions are of nearly equal activity when tested against peripheral lymphocytes (Fig. 3) or against Molt cells (Fig. 5). In view of the functional similarity of these inhibitory components to the dialyzable substance from rat peritoneal macrophages which we have described previously (4), we proceeded to examine human macrophages to determine if a similar substance was present in these cells. A lysate of 8 x lo8 human macrophages was prepared and subjected to gel filtration in Sephadex G-10. The Fluramreactive polypeptide peak was identified, and the indicated fractions were tested for their effect on the incorporation of [sH]thymidine by continuously cultured SWB16A lymphocytes. The result is shown in Fig. 2. It is clear that a suppressor substance is present in the macrophage lysate and is not associated with the major Fluram-reactive fraction of these cells, but resembles the second suppressor found in the dialysate prepared from leukocyte lysates. In this study, the fractions preceding the Fluram peak were not examined for suppressive substances. As these results were originally observed using cells of the SWB-16A continuous lymphoid cell line, which are B-cell derivatives, it was of interest to determine whether these inhibitory fractions had similar effects on normal peripheral lymphocytes. Accordingly, studies were carried out to determine the effect of these fractions on the incorporation of [ 3H] thymidine by these cells. As shown in Fig. 3A, three inhibitory fractions were identified as a result of addition of the respective fractions from Sephadex G-10 to cultures of peripheral lymphocytes for 3 hr prior to addition of [3H]thymidine. An interesting observation, in this regard, is the effect of time on the ability of the fractions containing inhibitory activity to affect thymidine uptake by peripheral lymphocytes. Figures 3A and B illustrate the effect of addition of the same fractions to peripheral lymphocytes with incubation for 3 hr and 3 days, respectively, before addition of [3H] thymidine to cultures. In Fig. 3A, two inhibitory fractions, I1 and I2 corresponding to S1 and SII of prior studies, and a third fraction, I3 were observed. I3 appears to correspond to thymidine as noted in a separate study (Fig. 5). In contrast, after 7 days of incubation (Fig. 3C), the effects of the three inhibitory fractions are far less marked, but there is a significant augmentation of [3H] thymidine uptake displayed by material close to the maximum Fluram-reactive peak. Such augmentation is not observed after 3 hr or 3 days of incubation. These studies of the effects of these fractions on the uptake of [3H] thymidine by peripheral lymphocytes indicate that the two suppressor activities which we have identified are capable of suppressing the uptake of [3H] thymidine by normal peripheral lymphocytes and that the effects of these suppressors on the SWB-16A line are not attributable to some unusual property of these continuously cultured cells, but may be seen using normal peripheral cells as well. In prior work, we have found that components of leukocyte dialysates prepared by dialysis against a membrane having a 12,000-MW cutoff could be conveniently subfractionated by redialysis against a membrane having a cutoff of 3500 daltons.
316
SAITO
ET
AL.
1 w !OO 300 400 ; 500 600 100 800
3oo
.._..................
..,,
lAtlCmWD
0
ii
loo
z 2 zoo-
1 9 ‘2
200
1 \ ii , j,; 8 :, ‘3 . .,i... ..;.. J( /Y+ . ..-....... $.-.-I
I t F d a loo-
ml ..,.... h” IHIHCO, 400 0t 1 CWROL NO
-..............
600 700 0 50
-“n J.
.J\ ,,..
@.I
.,......
150
200
WI
BActcRwNI)
FIG. 3. Gel filtration of human leukocyte dialysate (MW < 12,000) : Effects of recovered fractions on peripheral lymphocytes. Cultures of peripheral lymphocytes purified by FicollHypaque sedimentation were established in wells of microtiter plates at levels of 2 X IO” cells/O.2 ml. The respective fractions were each tested separately by addition to each well. Incubation was carried out for 3 (A), 72 (B), and 144 hr (C), at which times 0.2 $Zi of [SHJthymidine was added to each well, and incubation was continued for another 18 hr. The cells were recovered, washed, and counted by the procedures described under Methods. The term background refers to the result obtained by carrying cells through the procedure without the addition of thymidine. Control cultures to which only 5 mM ammonium bicarbonate was added were run in parallel, and the range of [‘Hlthymidine incorporation manifested by these cultures is displayed.
INHTRTTORS
OF
LYMPHOTD
DNA
317
SYNTHESIS
600 2 u 800
I
TF (3500
< M < 12000)
E BOO
FRACTION
NUMBER
FIG. 4. Differential dialysis of human leukocyte dialysate. Effects of low molecular weight (< 3500, A) and high molecular weight (> 3500, B) subfractions of leukocyte extract on uptake of [“Hlthymidine by SWB-16A lymphoid cells: a leukocyte extract was dialyzed against a membrane having a cutoff of 3500 MW. The small (< 3500) and large (> 3500) fractions were were passed separately through a Sephadex G-10 column and the indicated fractions assayed by Fluram for polypeptide content and their effect on [3H]thymidine uptake by SWB16A lymphoid cells. The level of [“Hlthymidine incorporation in four control cultures of SWB16A cells to which only 5 mM ammonium bicarbonate had been added was 844 k 58 cpm.
the same leukocyte dialysate which was studied in the experiment shown in Fig. 1 was redialyzed in this way, and the dialyzable as well as the retained fraction were each subjected to gel filtration on Sephadex G-10. The eluates were tested for their ability to suppress [ 3H] thymidine uptake. The results are depicted in Figs. 4A and B. The material passing through the 3500-MW dialysis membrane appeared to contain suppressive activity, whereas the retentate displayed no inhibitory properties. In view of the finding by Opitz et a/. (13) that suppression of thymidine incorporation in lymphocytes by macrophages might be due to the release of endogenous thymidine from the macrophages, it was important to assess whether or not either of the suppressive materials in the leukocyte dialysates was thymidine. unlabeled thymidine was passed through the same column used to Accordingly, Accordingly,
318
SAITO HUMAN
LEUKOCYTE
ET AL. DIALYZATE
( M<3500
)
05
80 IO
NO70 60 3 c ~zoo- 50
; pz &
20: I
* 40
?
25:
30 30
IOO20
35
1.0
IO
20
30
40
60
50 FRACTION
70
60
90
4.0 IOU
NO.
FIG. 5. Gel filtration of human leukocyte dialysate (MW < 3500) : Differential elution of the suppressors and unlabeled thymidine. A leukocyte extract was dialyzed using a dialysis membrane having a cutoff of 3500 MW. The dialysate was recovered, concentrated, and applied to a column of Sephadex G-10 in 5 mM ammonium bicarbonate and elution was carried out with this solvent. The fractions were assayed by Fluram and for their effects on the ability of Molt cells to incorporate [‘Hlthymidine. The polypeptide profile is shown and the presence of two suppressor species ( SI and Sn) is indicated. After washing the column with 5 mM ammonium bicarbonate, 2 mg of unlabeled thymidine was applied and elution was carried out in the same way as for the dialysate. The absorbance profile at 260 nm is shown, and it can be seen that thymidine elutes at a point distinct from the Fluram-reactive peak or suppressors Sr and Sn. The level of [‘Hlthymidine incorporation in four control cultures of Molt cells to which only 5 mM ammonium bicarbonate had been added was 1614 _’ 38 cpm.
fractionate the dialysate. As shown in Fig. 5, the Sr and Srr suppressor fractions eluted ahead of unlabeled thymidine from the Sephadex G-10 column under these conditions. In these experiments as in others (Fig. 3), significant inhibition was also observed in the region corresponding to thymidine. To determine whether these inhibitors were present in all lymphoid cells, a lysate was prepared from SWB-16A cells, which are a B-cell lymphoid line sustained in continuous culture. The lysate was sequentially dialyzed against the 12,000- and 3500-MW membranes, respectively. The final dialysate contained relatively little Fluram-reactive material and displayed no significant suppression of [ 3H] thymidine uptake by other SWB-16A cells (Fig. 6). DISCUSSION These studies provide evidence for the existence of at least three substances, in lysates of buffy coat leukocytes, which are capable of suppressing DNA synthesis in peripheral lymphocytes as well as continuously cultured B lymphocytes. We have identified these as Sr and S n, with respect to their action on the SWB-
present
INHIBITORS DIALYZATE
20
30
OF LYMPHOID (Mc3500)
FROM
DNA SWB-16A
40 FRACTION
SYNTHESIS LYMPHOID
50
319
CELLS
60
NO.
FIG. 6. Effect of the low molecular weight subfraction of SWB-16A cells on the uptake of [‘Hlthymidine by SWB-16A cells. An extract was prepared from 5.2 X 10’ SWB-16A cells and subjected to dialysis against a membrane having a cutoff of 3500 MW. The dialysis was subjected to gel filtration on a column of Sephadex G-10 using 5 mM ammonium bicarbonate as eluant. The fractions were assayed for polypeptide content by the Fluram technique and for their effects on [“Hlthymidine uptake by SWB-16A cells. The level of [3H]thymidine incorporation in four control cultures of SWB-16A cells to which only 5 nh4 ammonium bicarbonate had been added was 631 * 39 cpm.
16A line, and I1 and 12, with respect to their action on peripheral lymphocytes. A third inhibitor, corresponding to Is, was observed to elute in the region of thymidine (Fig. 5) and probably represents endogenous thymidine recovered from the leukocyte lysate. None of these suppressive activities appears to be present in the continuously cultured B-cell line (SWB-16A), and this would suggest their absence from B cells in V&JO. Sn appears to be present in human macrophages (Fig. 2), but may also be derived from other cell types, as it appears to be comparable to the suppressive factor reported by Burger et nl., who have identified this component as nicotinamide (S). This material may also be similar if not identical to the dialyzable suppressor which we have previously identified in peritoneal macrophages (4). A similar substance appears to have been found in rabbit macrophages by Ulrich (7) and in mouse macrophages by Unanue and co-workers (6). The presence of these suppressive substances in leukocyte dialysates may have an important bearing on the effects of these dialysates on lymphocyte DNA synthesis both in viva and in vitro. Studies of the effects of transfer factor preparations on antigen-triggered proliferation of lymphocytes in vitro have suggested that this assay does not measure specific transfer factor activity, since it is not antigen specific for the reactivities of the donor. Interpretation of these results may be affected by the presence in these preparations of inhibitory substances such as the suppressor molecules identified in these studies. Removal of one suppressor (Sn) by gel filtration on Sephadex G-10 and further fractionation of the major Fluram-
320
SAITO
ET
AL.
reactive polypeptide peak to remove the other (S1) may give rise to leukocyte extract preparations containing biologically active transfer factor which could be usefully employed for further study of this phenomenon. In this regard it is of interest to note that the suppressors appear to be most active when incubated with peripheral lymphocytes from 3 to 72 hr, while augmentation of [ 3H] thymidine uptake is induced by substances present close to the major polypeptide peak, if these fractions are permitted to stay in contact with the lymphocytes for 144 hr. This would suggest that by proper choice of time of exposure of lymphocytes to the particular fraction under study, one can selectively study either suppression or stimulation. We also noted that, in contrast to the effects observed using peripheral lymphocytes, significant augmentation as well as suppression were noted after only 3 hr of contact of the respective fractions with the Molt lymphoid cell line, which is derived from a human leukemia cell having the character of a T cell. It is possible that the target cells for the factor in the dialysates responsible for augmentation may be analogous to the type of T lymphocyte represented in the Molt line. Such a cell might arise during culture of peripheral lymphocytes over a 6-day period. Both suppressor fractions (S1 and S,,) appear to be capable of acting on peripheral lymphocytes. The relative effects of augmentation and suppression of [3H]thymidine uptake by different types of lymphoid cells may offer an opportunity to study these respective functions and to ascertain whether modulation and/or balance of these effects occurs in viva. REFERENCES 1. Lawrence, H. S., Harvey Lect. 68, 239, 1973. 2. Lawrence, H. S., J. Clin. Invest. 34, 219, 1955. 3. Ascher, M. S., Schneider, W. J., Valentine, F. T., and Lawrence, H. S., Proc. Nat. Acad. Sci. USA 71, 1178,1974. 4. Waldman, S. R., and Gottlieb, A. A., Cell. Imwwnol. 9, 142, 1973. 5. Saito, K., Foster, L. G., and Gottlieb, A. A., J. Int. Res. Commult. 3, 323, 1975. 6. Calderon, J., Williams, R. T., and Unanue, E. R., Proc. Nat. Acad. Sci. USA 71, 4273, 1974. 7. Ulrich, F., Biochem. Biophys, Res. Cownu~z. 60, 1453, 1974. 8. Burger, D. R., Vandenbark, A. A., Daves, D., Anderson, W. A., Jr., Vetto, M. R., and Finke, P., J. Iwzwaunol. 117, 797, 1976. 9. Djerassi, I., Kim, T. S., Ciesielka, W., Chaimongkol, B., and Suvansri, U., Transfusion. 15, 353, 1973.
10. Djerassi, I., Kim, T. S., Mitrakul, C., Suvansri, U., and Ciesielka, W., J. Med. (Basel) 1, 358, 1970.
11. Gottlieb, A. A., Foster, L. G., and Waldman, S. R., Lancet 2, 822, 1973. 12. Boyum, A., Stand. J. Clin. Lab. Znvcst. 21, 31, 1968. 13. Opitz, H. G., Niethammer, D., Lemke, H., Flad, H. D., and Huget, R., Cell Immunol. 16, 379, 1975. 14. Cohen, L., Holzman, R. S., Valentine, F. T., and Lawrence, H. S., J. Exp. Med. 143, 791, 1976.
15. Hamblin, A., Maini, R. N., and Dumonde, D. C. In. “Transfer Factor. Basic Properties and Clinical Applications” (M. S. Ascher, A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 49-57. Academic Press, New York, 1976.