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

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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

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HUMAN

MACROPHAGE

ET

AL.

DIALYSATE

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I

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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.

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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

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 Leukocy...
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