Tentative mechanism of lymphocyte chalone action

Primed in Sweden Copyright @ 1977 by Academic Press, Inc. . All rights of reproducrion in any form reserved ISSN 00144827

Experimental

TENTATIVE

MECHANISM

Cell Research 105 (1977) 137-141

OF LYMPHOCYTE

A. M. ATTALLAH’

CHALONE

ACTION

and J. C. HOUCK*

Biochemical Research Laboratory, Research Foundation of Children’s Hospital. Washington, DC 20009, USA

SUMMARY The cell-specific inhibition of PHA-stimulated lymphocyte transformation by splenic ultrafiltrates (50000-30000 D) differed in its kinetics from either cyclohexamide or cytosine arabinoside inhibition. Parallel inhibition of acid-soluble precursor [3H]TdR to acid-insoluble [?I]TdR was found for both ultrafiltered and exogenous cyclic AMP (CAMP), but not for cytosine arabinoside (cyt-a). While CAMP did not sinnificantlv increase the inhibition nroduced bv solenic ultrafiltrate. theophylline did. We suggest tentatively that lymphocyte chalone containing~ultrafiltrates might function via the production of increased intracellular CAMP which, in turn, could inhibit nucleoside kinases and hence the synthesis of macromolecular nucleic acid and mitosis.

The mitotic activity of lymphocytes has been shown to be under the control of a specific and endogenous negative feedback inhibitor [l], or lymphocyte chalone [2-4]. We have shown that this lymphocyte chalone can be extracted from lymphoid tissue (i.e., lymph node, spleen, and thymus) and can be concentrated by molecular ultrafiltration through Amicon Diaflo filters in a range of 30000 to 50000 D [2, 31. This lymphocyte chalone concentrate is capable of inhibiting the transformation of human lymphocytes in vitro whether stimulated by lectin or antigen [l-4] or in vivo as expressed by an inability of skin-grafted mice to reject their allograft [5]. Recently, Hauschka et al. [6] have indicated that CAMP is capable of significantly inhibiting the uptake of [3H]TdR into the Present address: ‘Clinical and Exnerimental Immunology, Naval Medical Research institute, Bethesda, MD 20014, USA. 2Virginia Mason Research Center. 1000Seneca Street, Seattle, WA 98101, USA.

acid-insoluble DNA fraction of Chang hamster ovary (CHO) fibroblast-like cells. They have found that this CAMP inhibition of thymidine uptake by these cells was mediated through the inhibition of nucleoside kinase activity intracellularly. This paper explores the apparent relationship between CAMP and chalone inhibition of lymphocyte transformation.

MATERIALS

AND METHODS

Lymphocyte chalone was prepared by extracting desiccated, defatted calf spleen with distilled water (20 ml/g) in the cold overnight. This splenic powder was prepared for us by the Viobin Corp. (Monticello, Ill.) by extraction of fresh tissue, using 1,2dichloroethanol. After aqueous extraction of the preparation of calf spleen, the supematant obtained from centrifugation at 4°C was subjected to molecular ultrafiltration, as has been described previously [2, 31. This spleen-derived lymphocyte &alone concentrate was made up in Medium 199, containing 20% fetal calf serum and the annromiate amounts of ah&mine and penicillin-streptomycm, as has been &scribed previously I2, 31. This medium was used to dilute the buffy coat of-normal human blood to a concentration of lo6 mononuclear cells/2 ml of medium, as judged Exp

Cell Res

IOS (1977)

138

Attallah

and Houck

Table 1. Inhibition of lymphocyte macromolecular synthesis at I2 and 27 h after PHA stimulation ‘ii inhibition

of “H uptake (6 h pulse)”

I2 h Inhibitor Splenic ultrafiltrate” (50 &ml) Cyclohexamide” (IO-’ M) Cyt-a” (IO-” M)

77 h

[3H]Leu

[“H]UdR

[“H]Leu

[“H]TdR

9

2

21

42

83

76

6X

96

39

26

0

97

o Spleen ultrafiltrate, cyclohexamide. cyt-a and radioactive precursors were added 6 h prior to cell harvest.

by hemocytometry [2, 31. To this cell suspension were added the appropriate amounts of PHA-p (Difco). and these cultures were incubated in triplicate for 66 h at 37°C. or as otherwise indicated. At this time, each culture received I PCi of [3H]TdR [2, 31, and the incubation was continued for 6 h. After this further incubation, the cells were collected by centrifugation and rinsed twice in large volumes of 0. I5 N NaCl solution at 4°C. The cellular pellet resulting from the final rinse and centrifugation was then mixed with 2 ml of 5% trichloracetic acid (TCA) to precipitate the macromolecules and, after standing overnight in the cold, the supematant TCA was collected by centrifugation. This supematant (I ml) was mixed with IS ml of Aquasol liquid cocktail (New England Nuclear Corp.) and the radioactivity contained in this fraction was determined, using liquid scintillation counting in the usual manner[2,3]. The acid-insoluble pellet was solubilized with NCS solubilizer and mixed with the NCS scintillation cocktail and similarly counted in a liquid scintillation counter.

RESULTS Normal human lymphocytes were stimulated for 72 h and during this time splenic ultrafiltrate (50 pg/ml) was added at different intervals after stimulation. The incubation of these cultures was continued for a total of 72 h. One &i of [3H]TdR was added during the last 6 h of incubation, the cells were harvested, and the [3H]TdR uptake into TCA-insoluble DNA was determined. Results, presented in fig. 1, show

that there was a progressive decline in the inhibition of the [SH]TdR uptake. This inhibition was 50% if spleen extract was added concomitantly with the PHA addition. However, if after stimulation with PHA the splenic extract was added at a later time, the inhibition of [‘H]TdR incorporation was progressively decreased. A series of human lymphocyte cultures were stimulated with PHA for 12 h or alternatively for 27 h. Portions of the first group (12 h stimulation) were incubated for the last 6 h in the presence of 50 pg/ml of splenic extract or lo-” M cyclohexamide or IO-” M cytosine arabinoside (cyt-a). Also during this time, cultures were pulsed with I PCi of either [“Hlleucine or [“Hluridine. It is evident from the data in table I that the rate of uridine and leucine incorporation was not affected by splenic extracts in lymphocytes stimulated by PHA for 12 h, but the other Table 2. The effect of spleen u :f, db-CAMP and cyt-a on PHA stimulated lymphocytes comparing acid-soluble and acid-insoluble component % decrease in cpm/lOB cells Inhibitor

Acidsoluble

Splenic ultrafiltrate” IO0 Ccg/ml 7.5 pglml 50 pglml Dibutyryl cyclic AMP* 2.0~ IO-’ M 1.0~10 ‘M O.Sx IO-’ M 0.2~ IO-’ M Cytosine arabinoside” 5.0x We M 2.0X IO-” M I .0x IO-B M 0. Ix IO fi M

Acidinsoluble

100 98 70 47 26 15” 6”

60 34 4” 0”

4” 0 0 0

47 27 25 IO”

” Insignificant @
Lymphocyte

chalone

139

was not associated with a reduced amount of radioactivity in the intracellular acidsoluble DNA precursor pool, however. When both db-CAMP and spleen extract were given together to PHA-stimulated lymphocytes, the inhibitory effects of chaSplenic ultrafiltrateb lone and nucleotide were not additive, but, CAMP* % Inhibition’ b4dml) rather, the inhibition of stimulation was de0 lO-4 M 35 pendent solely upon the concentration of 50 lO-4 M 65 chalone present rather than cyclic nucleolO-4 M 100 97 0 50 63 tide, as shown in table 3. 50 0.5x 10-S 74 Theophylline is known to inhibit lympho50 1.0x 10-S 59 50 10.0x 10-S 65 cyte transformation [7]. Several dose comn % inhibition= binations of lymphocyte chalone and theomean cpm of cultures containing inhibitors lx 100. phylline were added to PHA-stimulated mean cpm of control cultures b Both inhibitors were added at the start of the 72 h lymphocytes for 72 h. [3H]TdR uptake into culture. acid-insoluble macromolecule was counted. The results in table 4 showed that the intwo compounds depressed the uptake of hibitory effects of these two materials were both uridine and leucine. additive. The results were somewhat different at DISCUSSION 27 h following stimulation. Both splenic extract and cyclohexamide did inhibit the syn- It can be seen from fig. 1 that there is a thesis of protein and DNA synthesis in linear decrease with time of the inhibition stimulated normal human lymphocytes. In of PHA-induced lymphocyte stimulation. contrast, cyt-a did not inhibit protein syn- As DNA synthesis requires the continuous thesis while profoundly inhibiting DNA synthesis. These results are representative Table 4. Inhibitory effects of splenic ultraof a typical experiment. filtrate and theophylline upon [3H]TdR upThe effects of various concentrations of take into the acid-insoluble components of spleen-derived lymphocyte chalone conPHA-stimulated lymphocytes centrate, dibutyryl cyclic AMP (db-CAMP), and various non-cytotoxic doses of cyt-a Splenic ultratItrate* upon the percent reduction of both acid- CMlW Theophylline” % Inhibition” soluble and acid-insoluble r3H]TdR cpm per 0 50 48 million lymphocytes in culture is shown in 50 1~10-~M 49 table 2. These results demonstrate an es- 50 1x 1O-3M 73 50 5x lO-3 M 99 sentially parallel inhibition of both acid0 lO-3 M 60 soluble and acid-insoluble [3H]TdR in prolO-3 M 25 58 50 10m3M 73 portion to the concentration of either lO-3 M 100 95 spleen-derived chalone or db-CAMP. Similar inhibition of the incorporation of [3H]- a % inhibition= mean cpm of cultures containing inhibitors IXl00 TdR into acid-insoluble DNA or of lectinmean cpm of control cultures stimulated lymphocytes in vitro by cyt-a * As table 3.

Table 3. Inhibitory

effects of mixtures of splenic ultrajiltrate and db-CAMP upon [3H]TdZ? uptake into the acid-insoluble components of 72 h PHA-stimulated lymphocytes

E.vp Crll

Rr,

/OS (19771

140

Attallah

and Houck

20 -

10 -

1 0

10

20

30

40

50

60

I 10

I 80

Fig. 1. Abscissa: (top) time of splenic UF addition (hours); (bottom) time after PHA 1ymph;cyte stimulatatt~ (hours); ordinate: % inhibition of [ H]TdR upTriplicate cultures of 5x105/ml normal human lymphocytes were incubated for 72 h with PHA with [3H]TdR present during the final 6 h. Fifty &ml sieved spleen extract (30000-50000 D) was added at various times after stimulation by PHA. The percent inhibition of f3H1TdR untake was determined with the cells growing in-media without spleen extract serving as controls.

recruitment of nucleoside precursors, it is tempting to speculate that the chalone shuts off the supply of precursors for macromolecular synthesis at some stage before the incorporation of deoxynucleoside triphosphates into DNA, but this would still allow the completion of DNA synthesis from the triphosphate pool formed prior to chalone addition. Baserga [8] has indicated the possibility that the initiation of DNA biosynthesis may be preceded by the synthesis of specific RNA and proteins. The concentration of such enzymes as nucleoside kinases and DNA polymerase would increase only at the time DNA synthesis begins [8]. In table 1, we have compared the effect of lymphocyte chalone on protein, RNA, and DNA synthesis with cyclohexamide (protein inhibitor) and cyt-a (DNA inhibitor). These results indicate that cyclohexamide was inhibiting protein synthesis in Exp Cd RPS IO5 (1977)

both periods of incubation and subsequently inhibiting DNA synthesis. This contrasts with lymphocyte chalone inhibition in that protein synthesis was inhibited only at the time of DNA synthesis. The inhibition of DNA synthesis with a longer stimulation time (21-27 h) could be due to the inhibition of the synthesis of enzymes essential to DNA synthesis by lymphocyte chalone. With cyt-a, protein synthesis was not inhibited during this period of stimulation, while the synthesis of DNA was completely inhibited. It is not clear why cyt-a should effect protein synthesis initially and not at later culture interval. The data in table 2 demonstrate an essentially parallel inhibition of both acidsoluble and acid-insoluble [3H]TdR in proportion to the concentration of either lymphocyte chalone or db-CAMP. Similar inhibition of the incorporation of r3H]TdR into acid-insoluble DNA during the lectinstimulated lymphocyte in vitro by cyt-a was not associated with a reduced amount of radioactivity in the intracellular acid-soluble DNA precursor pool, however. Since CAMP has been shown to be capable of inhibiting the nucleoside kinase activity of the fibroblast in vitro and hence DNA synthesis, it is tempting to conclude that lymphocyte chalone exerts its inhibitory effect on DNA synthesis through CAMP. The data in table 3 demonstrate that the inhibitory effects on lymphocyte DNA synthesis by chalone and db-CAMP were not additive. Rather, the inhibition of DNA synthesis in this case was dependent solely on the concentration of lymphocyte chalone present. We may therefore speculate that once the chalone has acted on the cell, the cell surface and/or the phosphodiesterase system are changed in such a way that they will not respond further to exogenous CAMP.

.

Lymphocyte chalone

141

their proliferation would be profoundly inIn contrast with the results of lymphocyte chalone plus CAMP, the data in table 4 hibited. Clearly, a necessary proof of this proshowed that the inhibition of DNA synthesis by chalone and theophylline of PHA posed mechanism for lymphocyte chalone transformation of normal human lympho- activity would be the demonstration of an cytes was additive. This could be due to the increased adenyl cyclase activity in broken elevation of intracellular CAMP after the membrane preparations by addition of the stimulation of its synthesis by lymphocyte appropriate chalone. Such experiments are currently under way. chalone action and theophylline inhibition of subsequent CAMP degradation by phosphodiesterases. REFERENCES These observations lead us to tentatively 1. Moorhead, J F, Paraskova-Tchemozenska, E, Pirrie, A J &Hayes, C, Nature 224 (1969) 1207. suggest a possible mechanism of action for J, Irausquin, H & Leikin, S, Science 173 the lymphocyte chalone in vitro [9], namely 2. Houck, (1971) 1139. that the presence of chalone maintains a 3. Attallah, A M, Sunshine, G, Hunt, C V & Houck, C , Exp cell res 93 ( 1975)283. high level of activity of adenyl cyclase suf- 4. JLasalvia, E, Garcia-Giralt, E & Macieira-Coelho, A, Rev Eur etud clin biol 15 (1970) 789. ficient to insure a relatively high intracelluHouck, J C, Attallah, AM &Lilly, J R, Nature 245 lar concentration of CAMP. As a conse- 5. (1973) 148. quence of this CAMP concentration, a long- 6. Hauschka, P V, Everhart, L P & Rubin, R W, Proc natl acad sci US 69 (1972) 3542. term inhibition of nucleoside kinase activity 7. Smith, J W, Steiner, A L & Parker, C W, J clin invest 50 (1971) 442. may well be produced, as in the CHO fibroBaserga, R, Cancer res 25 (1%5) 581. blasts [5]. In the absence of nucleoside ki- 8. 9. Attallah, A M, Chalones (ed J C Houck) p. 141. ElsevierlNorth-Holland, Amsterdam (1976). nase activity, little DNA synthesis would be possible in these diploid human lympho- Received July 5, 1976 cytes in culture, and as a consequence, Accepted November 1, 1976

Exp Cell Res 105 (1977)