Immunology 1977 33 439
Further purification and chemical characterization of the lymphocyte-inhibitingfactor extracted from thymus (LIFT) NICOLE KIGER, IRINE FLORENTIN, G. LORANS & G. MATHE Institut de Cance'rologie et d'Immunogenetique (INSERM et Association Claude Bernard) HMpital Paul Brousse 14-16, Avenue Paul Vaillant Couturier 94800 Villejuif, France
Received 2 August 1976; acceptedfor publication 25 February 1977
specific extract from calf thymus, which inhibited, without toxicity, DNA synthesis in thymocytes (Kiger, Florentin & MatMe, 1972), in T lymphocytes stimulated in vivo by phytohaemagglutinin (PHA), and was in contrast less effective upon B lymphocytes stimulated by lipopolysaccharide from E. coli (LPS) (Florentin et al., 1976). Furthermore, this extract showed the ability to depress immune reactions involving T lymphocytes alone or in collaboration with B lymphocytes, but was unable to impair immune responses which only require B lymphocytes (Florentin et al., 1973). It was therefore used as a means of depressing cell mediated immunity, namely the graft-versus-host reaction (Kiger et al., 1973a,b). Initially, the crude thymic fraction, called T4, isolated from calf thymus by extraction in distilled water followed by ethanol fractionation was used. The active moiety was then partially purified by Sephadex G75 filtration and the molecular weight of the active molecule was estimated to 10,000-20,000 (Kiger et al., 1972). In the present paper, the extraction of the lymphocyte-inhibitory-factor (LIFT) is performed using lamb thymus as raw material, and two advances in the purification and characterization of this factor are described. Firstly, the tissue specificity of this extract is investigated by two approaches: by testing the effect of various lymphoid tissue extracts on the spontaneous DNA synthesis in thymocytes and the effect of the thymic extract on DNA synthesis in non-lymphoid cells.
Summary. A thymic extract which was previously demonstrated to contain a lymphocyte-inhibitingfactor (LIFT) based on its immunosuppressive activity in vivo and its antiproliferative properties on lymphocytes in vitro, has been purified using ultrafiltration procedures. Most of the activity measured by the ability to inhibit DNA synthesis in short term cultures of mouse thymocytes, was recovered in the 10,000-50,000 m. wt fraction (I fraction). In contrast, similar extracts from non-lymphoid organs were always ineffective in decreasing DNA synthesis in thymocytes. The I fraction was also inhibitory of DNA synthesis in mouse spleen cells stimulated by PHA whatever the time at which the fraction was added after the mitogen stimulation. This fraction, as well as the crude thymic extract, was ineffective in decreasing DNA synthesis in non-lymphoid target cells. The I fraction was further purified by Sephadex G50 filtration and DEAE Sephadex chromatography. The active molecule seemed to be a heat resistant basic peptide probably bound to a ribonucleotide moiety. INTRODUCTION We have previously described a species of nonCorrespondence: Dr Nicole Kiger, Institut de Canc~rologie et d'Immunogenetique, H6pital Paul Brousse, 14-16 Avenue Paul Vaillant Couturier, 94800 Villejuif, France.
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DC2 Amicon apparatus, Iyophilized and stored at -20°.
Preparation of crude extracts Mouse liver, lung, kidney and thymus and 6-weekold lamb liver, kidney and thymus were minced, ground up at 00 in distilled water and the mixture was then centrifuged for 1 h at 20,000 g. The supernatants from lamb organs were submitted to a fractional precipitation with ethanol. The fractions soluble in a final concentration of 42% ethanol, called T4 fraction in the case of the thymic extract, were dialyzed against distilled water on a
Filtration through Amicon systems The T4 fraction was submitted to molecular sieving through an Amicon System DC2 Amicon apparatus, (Lexington, Mass.) fitted out with Diafiber membrane cartridges HDX1 which separate mixed solutes into size-graded classes with a 50,000 molecular weight cut off. The retained fraction containing high molecular weight substances was called fraction S, the passing material with a presumed molecular weight lower than 50,000 was called fraction I. The S fraction was then submitted to Amicon Diaflo ultrafiltration to subdivide it into fractions, Si, S2, S3 with respective molecular weights in excess of 300,000, between 100,000 and 300,000 and between 50,000 and 100,000. All experimental procedures are recorded in Table 1. The crude extracts from mouse organs were also partially purified by Amicon Diaflo ultrafiltration, and
Secondly, a method is presented for a faster and simple isolation procedure which, having a better yield, allows further purification of the active principle, which is then partially characterized in terms of its physico-chemical, chemical and physiological properties. In the light of these results, the relation of this substance with other lymphocyte inhibitors is discussed. METHODS
Table 1. Extraction and purification of the LIFT: the underlined fractions are those which exhibit an inhibitory activity on the spontaneous or on the PHA induced DNA synthesis in lymphoid cell suspension Thymus
I
Suspension in distilled water: centrifugation 20,000 g,
1
h
Supernatant+ ethanol (42%4 final concentration)
Supernatant
Pellet discarded
Filtration through
an
=
T4 fraction
Amicon DC 2 apparatus
I fraction mol. wt < 50,000
S fraction mol. wt > 50,000
Filtration through Sephadex G 50 column
Filtration through Amicon membranes S fraction mol. wt > 300,000
Peak I S2 fraction 30,000< mol. wt < 100,000 S3 fraction 100,000< mol. wt < 50,000
I
I
Peak II
a~~~~ Peak III
Chromatography on DEAE Sephadex A 50 First
1
peak
I
(Amino acid analysis)
Purification and characterization of LIFT fractions with molecular weights higher or lower than 300,000 or between 50,000 and 10,000 were obtained. Each fraction was tested for protein concentration according to the Lowry, Rosebrough, Farr & Randall procedure, using bovine serum albumin as standard, as previously described (Kiger, 1972).
Sephadex filtration A further isolation of the active substance from the T4 fraction was obtained by gel filtration on Sephadex G50 :15 or 30 mg in protein of the fraction, dissolved in 1 ml distilled water, were passed through a 1 x 75 cm column, which was eluted with distilled water with a constant flow of 20 ml/h at 40, 2 ml fractions were collected and monitored with a spectrophotometer at 280 nm. Peaks distinguishable by optical density at 280 nm were collected, Iyophilized and used for activity testing. Anion exchange chromatography Further purification was carried out on a DEAESephadex A50 column (1 x 30 cm), equilibrated with a 0 01 M tris-HCI buffer pH 7-0. The column was developed with increasing steps of molarity (0 1, 0 2, 0 4, 1 M) of sodium chloride added to the buffer.
Polyacrylamide electrophoresis Analytical disc electrophoresis was performed as described by Davis using a 7% concentration of monomer in the lower gel at pH 9-5. Electrophoresis was carried out in a Tris-glycine buffer pH 8 at 5 mA per tube for 40 min. Bromophenol blue was added as a marker; at the end of the migration, gels were stained for 30 min with 055% amidoblack in 7 acetic acid and then destained. Amino acid analysis The fraction purified on DEAE-Sephadex was Iyophilized; 0 5 mg of this product was hydrolyzed with 6 N HCO for 24 h at 1100 in tubes sealed under vacuum. After hydrolysis, the content of the tube was evaporated the residue redissolved in 5 ml of 0-01 N HCO and chromatographed on an amino acid-analyser.
Enzymatic digestion I fraction was submitted to enzymatic treatment: deoxyribonuclease (DNase from beef pancreas 1600 units/mg Sigma chemical Co.), insoluble trypsin
441
(Sigma) bound to polyacrylamide (250 units/g), insoluble protease (Sigma) bound to carboxymethyl cellulose (100 units/g), ribonuclease (Sigma) bound to agarose (650 units/g), as follows: 2 5 mg in protein of the fraction dissolved in 1 ml phosphate buffer saline (PBS) was digested with 200 ug/ml of DNase, 1 mg/ml of insolubilized trypsin 2 5 mg of insolubilized protease and 500 ug of insolubilized RNase. All digestions were performed at 370 for 16 h with continuous stirring. At this time, insoluble enzymes were removed by centriinactivate enzymes. Each digest was then submitted fugation, the supernatants were collected and submitted, as well as DNase digest, and I fraction alone, to heat treatment (15 mn at 1000) in order to to a diafiltration with a membrane which retained substances with molecular weight above 500 (UM 50 Amicon Corp. Lexington, Mass.) to remove small products of enzymatic digestion and immediately used for DNA synthesis inhibition assay. Influence of pH The I fraction was dissolved at a concentration of 2 5 mg in protein per ml of RPMI 1640 culture medium (Gibco) brought to the desired pH value and allowed to stand at 40 for then 1 h. Each fraction was adjusted at pH 7 and tested for DNA synthesis inhibition. DNA synthesis inhibition tests
Inhibition of the spontaneous DNA synthesis in thymocytes. Thymuses from 6-week-old mice were minced in RPMI 1640 culture medium. The resulting cell suspension was adjusted to 3 3 x 106 cells/ml. Three ml aliquots were incubated in glass culture tubes for 3 1/2 h at 370 in a humidified atmosphere of air-CO2 (95:5) after the addition of 250 ug of protein in 01 ml of the fraction to be tested (final concentration 20 pg/ml). A pulse of 3 pCi of tritiated thymidine (3H-TdR: specific activity 20 Ci/mmole, CEA France) was given during the last 30 min of incubation, at this time cell counts were made using a haemocytometer and viability was assayed by the trypan blue exclusion test. 3H-TdR uptake was determined by liquid scintillation spectrometry (Packard Tricarb Scintillation counter) as previously described (Kiger et al., 1972). Inhibition of spontaneous DNA synthesis in nonlymphoid cells. Established cell lines of mouse
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Nicole Kiger et al.
fibroblasts and of cells from a human breast cancer, maintained in continuous monolayer cultures, were used as target cells. 1 x 105 cells in 3 ml of McCoy's culture medium (Gibco Laboratories) were plated in plastic culture flasks and incubated at 370 for 24 h. At this time, the medium was removed and replaced by fresh medium, with or without 250 4ug protein of the fraction to be tested. Cultures were incubated for 5 h and pulsed with 10 ,uCi of 3H-TdR for the final 2 h of culture. Cells were harvested after trypsinization and prepared for radioactivity count-
expressed as the mean counts per minute (c.p.m.) of three or six cultures ± standard error. Inhibition of 3H-TdR incorporation was determined by the formula: % inhibition
=
in cultures with the fraction l 100 in cultures without the fractionJ
c.p.m.
c.p.m.
RESULTS
ing.
Tissue specificity of the thymic extract and its subfractions In a preliminary study, the influence of various mouse or lamb tissue extracts from thymus and nonlymphoid organs, on the spontaneous DNA synthesis in mouse thymocytes, was investigated. An inhibitory effect on 3H-TdR incorporation in thymocytes was observed when the cells were incubated for 3 h with a thymic fraction of molecular weight lower than 300,000 and more precisely with a fraction of molecular weight between 50,000 and 10,000 (Table 2). Similar fractions from liver, lung and kidney as well as crude extracts or fractions with a MW superior to 300,000 daltons from these organs and from thymus, were ineffective in decreasing DNA synthesis in thymocytes and even exerted a stimulatory effect. When the crude extracts were fractionated by alcohol precipitation, only the thymic fraction soluble in 42% ethanol exhibited inhibitory activity
Inhibition of DNA synthesis in PHA stimulated spleen cells. Spleen cells from 3-month-old C57B1/6 mice were cultured in RPMI 1640 culture medium supplemented with 5% mule serum (Gibco Laboratories) and antibiotics, at a concentration of 1 x 105 cells/0 2 ml in microculture plates (Falcon Microtest II) for 54 h in an air-CO2 atmosphere, along with 20 ul/well of 1/50 dilution of the stock solution of PHA (PHA MR68, Burroughs Wellcome). The fraction to be tested was added at time 0, 24, or 48 h at a final concentration of pg/ml. The cultures were pulsed with 1 pCi of 3H-TdR/well after 48 h of culture, harvested 4 h later using a multiple automated sample harvester (Mash II, Microbiological Associates). Expression of the results In all DNA synthesis inhibition
assays, results were
Table 2. Effect of various extracts from mouse or lamb organs on spontaneous DNA synthesis in mouse thymocytes
Organ Fraction
Liver
Lung
Kidney
Thymus
Crude organ extract 0 Mouse 0 Mouse (-) 38* Mouse (-) 35 Mouse (20,000 g supernatant) Crude extract filtered through Amicon membranes: Mouse (-) 60 Mouse (-) 30 Mouse (-) 30 Mouse 0 fraction of: mol. wt 300,000 0 Mouse (-) 30 Mouse (-) 60 Mouse 54 mol. wt 300,000 Mouse Mouse (-) 30 Mouse 67 mol. wt 10,000 Lamb (-) 50 Lamb 65 50,000 Crude extract submitted to ethanol
precipitation fraction soluble in 42%y ethanol
Lamb
5
Lamb (-)
10 Lamb
70
* % Inhibition of 3H-TdR incorporation in short term cultures of thymocytes in presence of the tissue fraction.
Purification and characterization of LIFT Table 3. Effect of T4 subfractions
3H-TdR uptake in thymocytes
on
74,472+ 1757 29,792+ 1730 51,145+ 1470 84,807+ 1680 55,108+ 1962 40,201 +42
50,000-10,000 100,000-50,000 300,000-100,000 > 300,000
*
%Y Inhibition
c.p.m. + s.e.*
Fraction added Molecular weight none 1 S3 S2 Si T4
443
60 31 - 14 26 46
Cultures were done in triplicate.
Table 4. Effect of the T4 and I fractions on spontaneous DNA synthesis in nonlymphoid cell lines
Cell lines Mouse fibroblasts Cells from a human breast cancer
Fraction added Mean c.p.m.+s.e. None T4 I None T4 I
(Table 2). This thymic fraction, usually referred to T4 fraction, was further purified by ultrafiltration through a DC2 Amicon apparatus. Results presented in Table 3 showed that the inhibitory activity on DNA synthesis in thymocytes was concentrated in the fraction with a mol. wt of between 50,000 and 10,000 (I fraction). A slight inhibitory effect was also observed with fractions of mol. wt. greater than 300,000 and with a fraction of molecular weight between 100,000 and 50,000. In order to further investigate the tissue specificity of the thymic extract, T4 and I fractions were tested for their inhibitory effect on DNA synthesis in non-lymphoid cells. Neither mouse fibroblasts nor human mammary carcinoma cells, growing as monolayer cell lines, were inhibited during the exponential phase of culture growth when incubated with T4 or I fractions (Table 4). Effect of the T4 subfractions on PHA stimulated spleen cells We have also investigated the activity of the different T4 subfractions on the PHA-induced DNA synthesis of spleen cells coming from normal mice. Table 5 shows that I and SI subfractions had marked inhibitory effect upon 3H-TdR uptake in PHA
18,235+ 1632 19,308+ 976 15,892+ 1038 79,830+ 3292 75,838+2374 115,095+ 1883
% Inhibition 0 13 8 0
Table 5. Effect of thymic fractions on 3H-TdR uptake in spleen cells from normal mice stimulated by PHA Fraction
Time
c.p.m. (mean+ s.e.)t
(h)* T4 I
Si S2
S3
0 24 48 0 24 48 0 24 48 0 24 48 0
30,435+ 3918 24,004+ 2546
7,399+616 11,919+ 134 7,089+ 1431 7,277+ 875 4,447+ 196
10,628± 1973 4,879+ 374 8,792± 1315
13,311+ 1315 30,818+ 2583 29,979+ 3063 20,923+ 1950
Y. Inhibition 22 76 61 77 76 85 65 84 71 56 0 0 31
* Time (after start of culture) the designated fraction was added. t Cultures were done in triplicate.
stimulated spleen cells, independently of the time at which they were added to the culture. The T4 fraction had a slight effect in this experiment, when added at the same time as PHA and was strongly
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Table 6. Effect of a 24 h pre-incubation with the T4 and I fractions on subsequent 3H-TdR uptake of PHA stimulated spleen cells Fraction Experimental c.p.m. (mean+ s.e.)* % Inhibition added procedure
None T4 I
A B A B
30,259+ 966 17,221+ 3396 24,707+ 2714 18,564+ 578 28,033+ 1205
25
43 18 40 7
5 E c)
r'J
A, experimental cultures were incubated for 24 h with 80
,pg/ml of the different fractions and then stimulated in fresh medium. B, control cultures were incubated for 24 h with 80 pg/ml of the different fractions and then stimulated in fresh medium containing 80,pg/ml of the same fraction. * Cultures were done in triplicate.
effective in decreasing DNA synthesis when added 24 and 48 h after the beginning of the culture. The S2 fraction exerted an inhibitory effect when added at time 0 but was without any effect when added at 24 and 48 h. The S3 fraction slightly depressed mitogen induced DNA synthesis when added at time 0. Table 6 showed that the ability of mouse spleen cells to respond to PHA was depressed by about 40Y% by the presence of fraction T4 and I at a dose of 80 ,ug/ml during the whole course of cultures. When the spleen cells were preincubated for 24 h with these fractions and then washed before mitogen stimulation, their PHA responsiveness were not affected suggesting the T4 and I fractions did not act by damaging the cells. In all DNA synthesis inhibition tests, the I fraction proved to be the most strongly inhibiting fraction, so that this fraction was used for further purification work. Purification of the I fraction The I fraction, which represented 044% of the organ dry weight and about 10% of the T4 fraction, was fractionated into three parts by filtration through a column of Sephadex G50 (Fig. 1). The inhibitory activity was only recovered in the second peak (Peak II). In polyacrylamide gel electrophoresis this peak did not appear to be homogeneous and showed at least four bands (Fig. 2). Peak II was then submitted to chromatography
600 6 1.0
0-5 tg
10
_
0
20 Tube no.
*
V
50
h
30
Figure 1. Elution profile of the I fraction from a Sephadex G50 column.
on DEAE Sephadex A50 (Fig. 3). The active moiety, exhibiting a tenfold increase of the inhibitory activity when compared to the T4 fraction, was eluted in the first buffer (Tris-HCl 0 01 M without NaCl). All these purification procedures are outlined in Table 1. The maximum of absorbance in the ultraviolet of the active substance purified by DEAE Sephadex chromatography, occurred at 260 nm. The amino acid analysis of this peak is reported in Table 7. This analysis indicates a high-proportion of basic amino acids.
Heat sensitivity of the T4 and I fractions Table 8 shows that after heating 15 min at 1000 the T4 and I fractions remained capable of decreasing in vitro the spontaneous DNA synthesis in thymocytes.
pH sensitivity Fig. 4 shows that the inhibitory effect of the I fraction was maximal at pH 6, 7, 8 and reduced significantly at low pH (4 and 5).
Purification and characterization of LIFT
445
_
t.
--.*.. ..
..
... :.
.;: ...... .,: *;: :.: :.
Figure 2. Acrylamide gel electrophoresis of the three peaks eluted from the I fraction after filtration through a Sephadex G50 column. Table 7. Amino acid composition of the most purified thymic factor Amino acid Lys His NH3
Residue number for 100 10 residues
2
-
Arg
Asp
Thr
Ser
Glu
Pro
Ala
Val
Met
lie
Leu
Tyr
Phe
Gly
6
7
5
6
10
9
8
5
1
3
5
3
2
18
Enzymatic digestion of the I fraction Fig. 5 shows that after digestion with trypsin pronase and RNase, the inhibitory capacity of the I fraction towards the in vitro spontaneous DNA synthesis of the thymocytes disappeared, whereas DNase treatment did not reduce the inhibitory activity of the fraction.
DISCUSSION From a thymic extract which exhibits both an antiproliferative property when tested in vitro on lymphoid cells, and an immunosuppressive activity when injected into mice, we have isolated, by ultrafiltration procedures, four subfractions I, S3,
446
Nicole Kiger et al. 100 r
G z I c
0
80 F
._
100
2.0
0-
N
cS
c
0
40
50So-C
1.0
4
5
6
7
8
9
pH
Figure 4. Percentage of the inhibition of the in vitro spontaneous DNA synthesis in thymocytes is plotted as a function of the pH at which the I fraction was exposed. Control (100 %o) is the mean 3H-TdR uptake of culture incubated without any extract. Maximal deviation from the mean values are less than + 15%Y.
20 Tube no.
Figure3. Chromatography of the 2nd peak from Sephadex G50 on a DEAE-Sephadex A50 column. Table 8. Heat sensitivity of thymic fractions tested by their inhibitory effect upon spontaneous DNA synthesis in thymocytes
Fraction added c.p.m. (mean+ s.e.)* % Inhibition
2100 Q. I--
I -r 2
None Unheated T4 fraction Heated t T4 fraction Unheated I fraction Heatedt I fraction *
0
42,910+ 3030
0
11,156+429
74
10,512+ 1321 8452+ 1557
76 80
9639+ 2151
78
0
Fraction added I - I Enzyme treatment D Nase
- I Trypsin
- I Protease
- I R Nasp
Figure 5. Effect of enzymic digestion on the inhibitory capacity of I fraction.
Cultures were done in triplicate.
t The fractions were heated at 1000 for 15 min.
S2, S1 of increasing molecular weights. Testing the effects of these different subfractions upon the in vitro spontaneous 3H-TdR uptake in thymocytes, we have found that the I fraction, with molecular weight ranging between 10,000 and 50,000, was more active per unit protein than the T4 fraction starting material and that the activity of the residual proteins was clearly reduced after removal of the I fraction. Thus we have tentatively concluded that the inhibitoryactivity of the fractionT4 can be accounted for by the presence of the active low molecular weight moiety. Furthermore, the I fraction had the ability to inhibit PHA induced 3H-TdR incorporation in mouse spleen cells. In this case, the effect was
demonstrated to be independent of the time at which the I fraction was added after mitogen stimulation. This argues against the hypothesis of a competitive action of the inhibitor and PHA on the cell membrane, as well as against the possibility of an inhibition of the lectin effect by its binding to the thymic extract. In the same experiment, SI fraction (molecular weight greater than 300,000) exhibited the same inhibitory property as the I fraction. We can consider at least two likely explanations for these observations: either the lymphocyte inhibitor could be a low molecular weight entity contained in the I fraction, which is found in the SI fraction bound to a large carrier, or there could be two independent molecules with similar but unrelated physiological properties. These possibilities are under
investigation.
Purification and characterization of LIFT We have previously demonstrated that the inhibitory effect of the T4 fraction starting material upon thymocyte spontaneous DNA synthesis was reversible (Florentin et al. 1976). In the present paper, we show that a 24 h pre-incubation of spleen cells with either T4 or I fraction does not impair their ability to transform with PHA if cultures are performed in fraction free medium. This further argues against a cytotoxic effect of the fractions. Inhibition of DNA synthesis could simply be due to the decrease of tritiated thymidine penetration under the influence of the thymic extract. The observation that this extract was unable to decrease thymidine incorporation into non-lymphoid cells argues against this hypothesis. Furthermore, recent experiments have shown that it probably acts in a different way; indeed cytophotometric and autoradiographic studies have shown that the thymic factor inhibits the progression through the S phase but also on various positions of the mitotic cell cycle (Olsson, Kiger & Florentin, in press). We have previously demonstrated that the factor rapidly inhibits DNA and RNA synthesis in short term thymocyte cultures (Florentin, 1976). Experiments are in progress to further investigate the effect of this substance on protein synthesis as well as the mechanism of action of inhibition of DNA and RNA synthesis. We have previously demonstrated by Sephadex G75 filtration that all the activity of the I fraction was present in a molecular range between about 10,000 and 20,000, excluding the presence of cold thymidine as a cause of reduced incorporation of labelled thymidine into the cells. Furthermore, it is unlikely that the inhibitory activity of the I fraction is simply due to thymidine bound to a carrier protein as we have found that the spontaneous mitotic rate of thymocytes was also significantly impaired by incubation of cells with the thymic extract (Olsson et al., in press). LIFT, which is species non specific, exhibits a specificity of action for lymphoid cells since it does not inhibit DNA synthesis in non-lymphoid cells, i.e. mouse fibroblasts or human tumour cells. Conversely, similar extracts from non-lymphoid tissue, i.e. kidney, liver, lung are ineffective upon thymocytes. The I fraction has been further purified by filtration on Sephadex G50 followed by chromatography on DEAE-Sephadex A50. The conditions of elution of the inhibitory factor on anion-exchange chromato-
447
graphy indicated the basicity of the substance; this was confirmed by the amino acid analysis of the more purified fraction exhibiting a large amount of the basic amino acids. By digestion studies with enzymes, the active factor was found to be undigestible by DNase, destroyed by trypsin, pronase and RNase indicating that the active moiety may be a peptide bound to ribonucleosides, this latter conclusion seems to be confirmed by the maximal absorbance at 260 nm of the most purified fraction. This substance is heat insensitive as the activity remains intact even after heating 15 min at 100°. A number of inhibitors of lymphocyte proliferation have been described, namely, a globulins, factors secreted by established human lymphoid cell lines, and lymphoid chalones. Substances identified as a globulins, have been isolated from human sera and inhibit both immune responses in vivo and lymphocyte proliferation in vitro (Menzoian, Glasgow, Nimberg, Cooperband, Schmid, Saporoschetz & Mannick 1974, Riggio, Schwartz, Bull, Stenzel & Rubin, 1969, Miller, 1976). A similar factor inhibiting lymphocyte proliferation has been recovered in a thymic extract (Phillips, Carpenter & Lane, 1975). Few of these factors have been sufficiently purified, and when they have been characterized (Occhino, Glasgow, Coo-erband, Mannick & Schmid, 1973) they exhibit physico-chemical properties (molecular weight, sensitivity to pH, behaviour on anion exchange chromatography) which are different from those described for LIFT. Factors released by human lymphocytes in cultures have also been described which possess anti-proliferative capacities on lymphocytes when tested on mitogen-induced blastogenesis and which have been identified as glycoprotein. They are different from LIFT by their molecular weights (Chase, 1976) or by their conditions of inhibition of lymphocyte proliferation which are dependent on the time at which the fraction is added to the culture after stimulation by PHA (Milton, 1971). Other factors extracted from lymphocytes or released by human lymphocytes in cultures present important differences in the physico-chemical properties with LIFT such as different thermosensitivity and molecular weights (Papageorgiou, Tibbetts, Sorokin & Glade, 1974; Tibbetts, Glade & Papageorgiou, 1975), or important differences in their specificity, namely species specificity (Hersh, McCredie & Freireich, 1974).
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Nicole Kiger et al.
Several authors refer to the inhibitors of lymphoid DNA synthesis as lymphocyte chalones, which are tissue-specific but not species-specific inhibitors of lymphocyte proliferation. These inhibitors are generally considered as proteins (with molecular weights above 40,000) or as small peptides (with molecular weights lower than 5000), they are sensitive to proteolytic enzymes and to heat. Such inhibitors have beenextracted from spleen and lymph nodes (Moorhead, Paraskova-Tchnerno-zenska, Pirrie & Hayes, 1969, Lasalvia, Garcia-Giralt & Maciera-Coelho, 1970) and from thymus (Attallah, Sunshine, Hunt & Houck, 1975) these inhibitors are heat sensitive and have slightly larger molecular weights than LIFT but, at the present time, progress on their purification and characterization has been insufficient to allow further comparisons. In conclusion, at the present time, we cannot identify LIFT with other inhibitors of lymphocyte proliferation; it could be suggested that the active peptide we have isolated could be bound to one or more proteins giving rise to the substances described by different authors, but a number of physicochemical and even physiological properties seem to signify that they are unrelated substances.
ACKNOWLEDGMENTS The authors thank Miss Nicole Saou and Miss Martine Davigny for their excellent technical assistance and CERM Laboratories, 63 RIOM, France, who kindly provided the lyophilized lamb thymuses. This research was supported by Contrat libre INSERM no. 75-5-108-2 and Contrat DGRST nos 74-7-0620 and 75-7-0732. REFERENCES ATTALLAH A.M., SUNSHINE G.H., HUNT C.V. & HOUCK J.C. (1975) The specific and endogenous mitotic inhibitor of lymphocytes (chalone). Exp. Cell. Res. 93, 283. CHASE P.S. (1976) The effects of human serum fractions on phytohemagglutinin- and concanavalin A-stimulated human lymphocyte cultures. Cell. Immunol. 5, 544. FLORENTIN I., KIGER N. & MATHE G. (1976) The lymphocyte inhibiting factor extracted from the thymus (LIFT): inhibition of the DNA synthesis in vitro. Cell. Immunol. 23, 1. FLORENTIN I., KIGER N. & MATHE G. (1973) T lymphocyte specificity of a lymphocyte inhibiting factor extracted from the thymus. Europ. J. Immunol. 3, 624.
HERSH E.M., MCCEADIE K.B. & FREIREICH E.J. (1974) Inhibition of in vitro lymphocyte blastogenesis by inhibitor produced by cultured human lymphoblasts. Clin. exp. Immunol. 17, 463. KIGER N., FLORENTIN I. & MATHE G. (1972) Some effects of a partially purified lymphocyte inhibiting factor from calf thymus. Transplantation, 14, 448. KIGER N., FLORENTIN I. & MATHE G. (1973a) A lymphocyte inhibitory factor (chalone?) extracted from thymus immuno-suppressive effects. In: 'Chalones' concepts and current researches. Natl. Cancer Inst. Monograph, 38, 135. KIGER N., FLORENTIN I. & MATHE G. (1973b) Inhibition of graft-verses host reaction by pre-incubation of the graft with a thymic extract (lymphocyte chalone). Transplantation, 16, 393. LASALVIA E., GARCIA-GIRALT E. & MACEIRA-COELHO A. (1970) Extraction of an inhibitor of DNA synthesis from human peripheral blood lymphocyte and bovine spleen. Rev. Europ. Etudes Clin. et Biol., 15, 789. MENZOIAN J.O., GLASCOW A.H., NIMBERG R.D., COOPERBAND S.R., SCHMID K., SAPOROSCHETZ I. & MANNICK J.A. (1974) Regulation of T-lymphocyte function by immunoregulatory alphaglobulin (IRA). J. Immunol. 113, 266. MILLER F. (1976) Serum-derived immunosuppressive substances. I. Partial purification and range of action. Transplantation, 21, 179. MILTON J.D. (1971) Effect of an immunosuppressive serum a-2-glycoprotein with ribonuclease activity on the proliferation of human lymphocytes in culture. Immunology, 20, 205. MOORHEAD J.F., PARASKOVA-TCHERNOZENSKA E., PIRRIE A.J. & HAYES C. (1969) Lymphoid inhibitor of human lymphocyte DNA synthesis and mitosis in vitro. Nature, 224, 1207. OLSSON L., KIGER N. & FLORENTIN I. (1977) Effect of the lymphocyte-inhibiting factor extracted from thymus (LIFT) on thymocyte proliferation in vitro: a cytokinetic analysis. Cell. Tissue Kinet. (In presss). OCCHINO J.C., GLASGOW A.H., COOPERBAND S.R., MANNICK J.A. & SCHMID K. (1973) Isolation of an immunosuppresive peptide fraction from human plasma. J. Immunol. 110, 685. PAPAGEORGIOU P.S., TIBBETrS L., SOROKIN C.F. & GLADE P.R. (1974) Presence of a reversible inhibitor (s) for human lymphoid cell RNA, protein, and DNA synthesis in the extracts of established human lymphoid cell lines. Cell. Immunol. 11, 354. PHILLIPS S.M., CARPENTER C.B. & LANE P. (1975) Immunosuppressive a globulin from bovine thymus and serum: mode of action upon afferent and efferent arcs of the mouse immune response. N. Y. Acad. Sci. 249, 236. RIGGIo R.R., SCHWARTZ G.H., BULL F.G., STENZEL K.H. & RUBIN A.I. (1969) a2-globulins in renal graft refection. Effects on in vitro lymphocyte function. Transplantation. 8, 689. TIBBETTS L.M., GLADE P.R. & PAPAGEORGIOU P.S. (1975) Presence of a reversible inhibitor for human lymphoid cell DNA synthesis in the extracts of human peripheral lymphocytes. Cell. Immunol. 18, 384.
Further purification and chemical characterization of the lymphocyte-inhibitingfactor extracted from thymus (LIFT) NICOLE KIGER, IRINE FLORENTIN, G. LORANS & G. MATHE Institut de Cance'rologie et d'Immunogenetique (INSERM et Association Claude Bernard) HMpital Paul Brousse 14-16, Avenue Paul Vaillant Couturier 94800 Villejuif, France
Received 2 August 1976; acceptedfor publication 25 February 1977
specific extract from calf thymus, which inhibited, without toxicity, DNA synthesis in thymocytes (Kiger, Florentin & MatMe, 1972), in T lymphocytes stimulated in vivo by phytohaemagglutinin (PHA), and was in contrast less effective upon B lymphocytes stimulated by lipopolysaccharide from E. coli (LPS) (Florentin et al., 1976). Furthermore, this extract showed the ability to depress immune reactions involving T lymphocytes alone or in collaboration with B lymphocytes, but was unable to impair immune responses which only require B lymphocytes (Florentin et al., 1973). It was therefore used as a means of depressing cell mediated immunity, namely the graft-versus-host reaction (Kiger et al., 1973a,b). Initially, the crude thymic fraction, called T4, isolated from calf thymus by extraction in distilled water followed by ethanol fractionation was used. The active moiety was then partially purified by Sephadex G75 filtration and the molecular weight of the active molecule was estimated to 10,000-20,000 (Kiger et al., 1972). In the present paper, the extraction of the lymphocyte-inhibitory-factor (LIFT) is performed using lamb thymus as raw material, and two advances in the purification and characterization of this factor are described. Firstly, the tissue specificity of this extract is investigated by two approaches: by testing the effect of various lymphoid tissue extracts on the spontaneous DNA synthesis in thymocytes and the effect of the thymic extract on DNA synthesis in non-lymphoid cells.
Summary. A thymic extract which was previously demonstrated to contain a lymphocyte-inhibitingfactor (LIFT) based on its immunosuppressive activity in vivo and its antiproliferative properties on lymphocytes in vitro, has been purified using ultrafiltration procedures. Most of the activity measured by the ability to inhibit DNA synthesis in short term cultures of mouse thymocytes, was recovered in the 10,000-50,000 m. wt fraction (I fraction). In contrast, similar extracts from non-lymphoid organs were always ineffective in decreasing DNA synthesis in thymocytes. The I fraction was also inhibitory of DNA synthesis in mouse spleen cells stimulated by PHA whatever the time at which the fraction was added after the mitogen stimulation. This fraction, as well as the crude thymic extract, was ineffective in decreasing DNA synthesis in non-lymphoid target cells. The I fraction was further purified by Sephadex G50 filtration and DEAE Sephadex chromatography. The active molecule seemed to be a heat resistant basic peptide probably bound to a ribonucleotide moiety. INTRODUCTION We have previously described a species of nonCorrespondence: Dr Nicole Kiger, Institut de Canc~rologie et d'Immunogenetique, H6pital Paul Brousse, 14-16 Avenue Paul Vaillant Couturier, 94800 Villejuif, France.
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Nicole Kiger et al.
440 more
DC2 Amicon apparatus, Iyophilized and stored at -20°.
Preparation of crude extracts Mouse liver, lung, kidney and thymus and 6-weekold lamb liver, kidney and thymus were minced, ground up at 00 in distilled water and the mixture was then centrifuged for 1 h at 20,000 g. The supernatants from lamb organs were submitted to a fractional precipitation with ethanol. The fractions soluble in a final concentration of 42% ethanol, called T4 fraction in the case of the thymic extract, were dialyzed against distilled water on a
Filtration through Amicon systems The T4 fraction was submitted to molecular sieving through an Amicon System DC2 Amicon apparatus, (Lexington, Mass.) fitted out with Diafiber membrane cartridges HDX1 which separate mixed solutes into size-graded classes with a 50,000 molecular weight cut off. The retained fraction containing high molecular weight substances was called fraction S, the passing material with a presumed molecular weight lower than 50,000 was called fraction I. The S fraction was then submitted to Amicon Diaflo ultrafiltration to subdivide it into fractions, Si, S2, S3 with respective molecular weights in excess of 300,000, between 100,000 and 300,000 and between 50,000 and 100,000. All experimental procedures are recorded in Table 1. The crude extracts from mouse organs were also partially purified by Amicon Diaflo ultrafiltration, and
Secondly, a method is presented for a faster and simple isolation procedure which, having a better yield, allows further purification of the active principle, which is then partially characterized in terms of its physico-chemical, chemical and physiological properties. In the light of these results, the relation of this substance with other lymphocyte inhibitors is discussed. METHODS
Table 1. Extraction and purification of the LIFT: the underlined fractions are those which exhibit an inhibitory activity on the spontaneous or on the PHA induced DNA synthesis in lymphoid cell suspension Thymus
I
Suspension in distilled water: centrifugation 20,000 g,
1
h
Supernatant+ ethanol (42%4 final concentration)
Supernatant
Pellet discarded
Filtration through
an
=
T4 fraction
Amicon DC 2 apparatus
I fraction mol. wt < 50,000
S fraction mol. wt > 50,000
Filtration through Sephadex G 50 column
Filtration through Amicon membranes S fraction mol. wt > 300,000
Peak I S2 fraction 30,000< mol. wt < 100,000 S3 fraction 100,000< mol. wt < 50,000
I
I
Peak II
a~~~~ Peak III
Chromatography on DEAE Sephadex A 50 First
1
peak
I
(Amino acid analysis)
Purification and characterization of LIFT fractions with molecular weights higher or lower than 300,000 or between 50,000 and 10,000 were obtained. Each fraction was tested for protein concentration according to the Lowry, Rosebrough, Farr & Randall procedure, using bovine serum albumin as standard, as previously described (Kiger, 1972).
Sephadex filtration A further isolation of the active substance from the T4 fraction was obtained by gel filtration on Sephadex G50 :15 or 30 mg in protein of the fraction, dissolved in 1 ml distilled water, were passed through a 1 x 75 cm column, which was eluted with distilled water with a constant flow of 20 ml/h at 40, 2 ml fractions were collected and monitored with a spectrophotometer at 280 nm. Peaks distinguishable by optical density at 280 nm were collected, Iyophilized and used for activity testing. Anion exchange chromatography Further purification was carried out on a DEAESephadex A50 column (1 x 30 cm), equilibrated with a 0 01 M tris-HCI buffer pH 7-0. The column was developed with increasing steps of molarity (0 1, 0 2, 0 4, 1 M) of sodium chloride added to the buffer.
Polyacrylamide electrophoresis Analytical disc electrophoresis was performed as described by Davis using a 7% concentration of monomer in the lower gel at pH 9-5. Electrophoresis was carried out in a Tris-glycine buffer pH 8 at 5 mA per tube for 40 min. Bromophenol blue was added as a marker; at the end of the migration, gels were stained for 30 min with 055% amidoblack in 7 acetic acid and then destained. Amino acid analysis The fraction purified on DEAE-Sephadex was Iyophilized; 0 5 mg of this product was hydrolyzed with 6 N HCO for 24 h at 1100 in tubes sealed under vacuum. After hydrolysis, the content of the tube was evaporated the residue redissolved in 5 ml of 0-01 N HCO and chromatographed on an amino acid-analyser.
Enzymatic digestion I fraction was submitted to enzymatic treatment: deoxyribonuclease (DNase from beef pancreas 1600 units/mg Sigma chemical Co.), insoluble trypsin
441
(Sigma) bound to polyacrylamide (250 units/g), insoluble protease (Sigma) bound to carboxymethyl cellulose (100 units/g), ribonuclease (Sigma) bound to agarose (650 units/g), as follows: 2 5 mg in protein of the fraction dissolved in 1 ml phosphate buffer saline (PBS) was digested with 200 ug/ml of DNase, 1 mg/ml of insolubilized trypsin 2 5 mg of insolubilized protease and 500 ug of insolubilized RNase. All digestions were performed at 370 for 16 h with continuous stirring. At this time, insoluble enzymes were removed by centriinactivate enzymes. Each digest was then submitted fugation, the supernatants were collected and submitted, as well as DNase digest, and I fraction alone, to heat treatment (15 mn at 1000) in order to to a diafiltration with a membrane which retained substances with molecular weight above 500 (UM 50 Amicon Corp. Lexington, Mass.) to remove small products of enzymatic digestion and immediately used for DNA synthesis inhibition assay. Influence of pH The I fraction was dissolved at a concentration of 2 5 mg in protein per ml of RPMI 1640 culture medium (Gibco) brought to the desired pH value and allowed to stand at 40 for then 1 h. Each fraction was adjusted at pH 7 and tested for DNA synthesis inhibition. DNA synthesis inhibition tests
Inhibition of the spontaneous DNA synthesis in thymocytes. Thymuses from 6-week-old mice were minced in RPMI 1640 culture medium. The resulting cell suspension was adjusted to 3 3 x 106 cells/ml. Three ml aliquots were incubated in glass culture tubes for 3 1/2 h at 370 in a humidified atmosphere of air-CO2 (95:5) after the addition of 250 ug of protein in 01 ml of the fraction to be tested (final concentration 20 pg/ml). A pulse of 3 pCi of tritiated thymidine (3H-TdR: specific activity 20 Ci/mmole, CEA France) was given during the last 30 min of incubation, at this time cell counts were made using a haemocytometer and viability was assayed by the trypan blue exclusion test. 3H-TdR uptake was determined by liquid scintillation spectrometry (Packard Tricarb Scintillation counter) as previously described (Kiger et al., 1972). Inhibition of spontaneous DNA synthesis in nonlymphoid cells. Established cell lines of mouse
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Nicole Kiger et al.
fibroblasts and of cells from a human breast cancer, maintained in continuous monolayer cultures, were used as target cells. 1 x 105 cells in 3 ml of McCoy's culture medium (Gibco Laboratories) were plated in plastic culture flasks and incubated at 370 for 24 h. At this time, the medium was removed and replaced by fresh medium, with or without 250 4ug protein of the fraction to be tested. Cultures were incubated for 5 h and pulsed with 10 ,uCi of 3H-TdR for the final 2 h of culture. Cells were harvested after trypsinization and prepared for radioactivity count-
expressed as the mean counts per minute (c.p.m.) of three or six cultures ± standard error. Inhibition of 3H-TdR incorporation was determined by the formula: % inhibition
=
in cultures with the fraction l 100 in cultures without the fractionJ
c.p.m.
c.p.m.
RESULTS
ing.
Tissue specificity of the thymic extract and its subfractions In a preliminary study, the influence of various mouse or lamb tissue extracts from thymus and nonlymphoid organs, on the spontaneous DNA synthesis in mouse thymocytes, was investigated. An inhibitory effect on 3H-TdR incorporation in thymocytes was observed when the cells were incubated for 3 h with a thymic fraction of molecular weight lower than 300,000 and more precisely with a fraction of molecular weight between 50,000 and 10,000 (Table 2). Similar fractions from liver, lung and kidney as well as crude extracts or fractions with a MW superior to 300,000 daltons from these organs and from thymus, were ineffective in decreasing DNA synthesis in thymocytes and even exerted a stimulatory effect. When the crude extracts were fractionated by alcohol precipitation, only the thymic fraction soluble in 42% ethanol exhibited inhibitory activity
Inhibition of DNA synthesis in PHA stimulated spleen cells. Spleen cells from 3-month-old C57B1/6 mice were cultured in RPMI 1640 culture medium supplemented with 5% mule serum (Gibco Laboratories) and antibiotics, at a concentration of 1 x 105 cells/0 2 ml in microculture plates (Falcon Microtest II) for 54 h in an air-CO2 atmosphere, along with 20 ul/well of 1/50 dilution of the stock solution of PHA (PHA MR68, Burroughs Wellcome). The fraction to be tested was added at time 0, 24, or 48 h at a final concentration of pg/ml. The cultures were pulsed with 1 pCi of 3H-TdR/well after 48 h of culture, harvested 4 h later using a multiple automated sample harvester (Mash II, Microbiological Associates). Expression of the results In all DNA synthesis inhibition
assays, results were
Table 2. Effect of various extracts from mouse or lamb organs on spontaneous DNA synthesis in mouse thymocytes
Organ Fraction
Liver
Lung
Kidney
Thymus
Crude organ extract 0 Mouse 0 Mouse (-) 38* Mouse (-) 35 Mouse (20,000 g supernatant) Crude extract filtered through Amicon membranes: Mouse (-) 60 Mouse (-) 30 Mouse (-) 30 Mouse 0 fraction of: mol. wt 300,000 0 Mouse (-) 30 Mouse (-) 60 Mouse 54 mol. wt 300,000 Mouse Mouse (-) 30 Mouse 67 mol. wt 10,000 Lamb (-) 50 Lamb 65 50,000 Crude extract submitted to ethanol
precipitation fraction soluble in 42%y ethanol
Lamb
5
Lamb (-)
10 Lamb
70
* % Inhibition of 3H-TdR incorporation in short term cultures of thymocytes in presence of the tissue fraction.
Purification and characterization of LIFT Table 3. Effect of T4 subfractions
3H-TdR uptake in thymocytes
on
74,472+ 1757 29,792+ 1730 51,145+ 1470 84,807+ 1680 55,108+ 1962 40,201 +42
50,000-10,000 100,000-50,000 300,000-100,000 > 300,000
*
%Y Inhibition
c.p.m. + s.e.*
Fraction added Molecular weight none 1 S3 S2 Si T4
443
60 31 - 14 26 46
Cultures were done in triplicate.
Table 4. Effect of the T4 and I fractions on spontaneous DNA synthesis in nonlymphoid cell lines
Cell lines Mouse fibroblasts Cells from a human breast cancer
Fraction added Mean c.p.m.+s.e. None T4 I None T4 I
(Table 2). This thymic fraction, usually referred to T4 fraction, was further purified by ultrafiltration through a DC2 Amicon apparatus. Results presented in Table 3 showed that the inhibitory activity on DNA synthesis in thymocytes was concentrated in the fraction with a mol. wt of between 50,000 and 10,000 (I fraction). A slight inhibitory effect was also observed with fractions of mol. wt. greater than 300,000 and with a fraction of molecular weight between 100,000 and 50,000. In order to further investigate the tissue specificity of the thymic extract, T4 and I fractions were tested for their inhibitory effect on DNA synthesis in non-lymphoid cells. Neither mouse fibroblasts nor human mammary carcinoma cells, growing as monolayer cell lines, were inhibited during the exponential phase of culture growth when incubated with T4 or I fractions (Table 4). Effect of the T4 subfractions on PHA stimulated spleen cells We have also investigated the activity of the different T4 subfractions on the PHA-induced DNA synthesis of spleen cells coming from normal mice. Table 5 shows that I and SI subfractions had marked inhibitory effect upon 3H-TdR uptake in PHA
18,235+ 1632 19,308+ 976 15,892+ 1038 79,830+ 3292 75,838+2374 115,095+ 1883
% Inhibition 0 13 8 0
Table 5. Effect of thymic fractions on 3H-TdR uptake in spleen cells from normal mice stimulated by PHA Fraction
Time
c.p.m. (mean+ s.e.)t
(h)* T4 I
Si S2
S3
0 24 48 0 24 48 0 24 48 0 24 48 0
30,435+ 3918 24,004+ 2546
7,399+616 11,919+ 134 7,089+ 1431 7,277+ 875 4,447+ 196
10,628± 1973 4,879+ 374 8,792± 1315
13,311+ 1315 30,818+ 2583 29,979+ 3063 20,923+ 1950
Y. Inhibition 22 76 61 77 76 85 65 84 71 56 0 0 31
* Time (after start of culture) the designated fraction was added. t Cultures were done in triplicate.
stimulated spleen cells, independently of the time at which they were added to the culture. The T4 fraction had a slight effect in this experiment, when added at the same time as PHA and was strongly
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Nicole Kiger et al.
Table 6. Effect of a 24 h pre-incubation with the T4 and I fractions on subsequent 3H-TdR uptake of PHA stimulated spleen cells Fraction Experimental c.p.m. (mean+ s.e.)* % Inhibition added procedure
None T4 I
A B A B
30,259+ 966 17,221+ 3396 24,707+ 2714 18,564+ 578 28,033+ 1205
25
43 18 40 7
5 E c)
r'J
A, experimental cultures were incubated for 24 h with 80
,pg/ml of the different fractions and then stimulated in fresh medium. B, control cultures were incubated for 24 h with 80 pg/ml of the different fractions and then stimulated in fresh medium containing 80,pg/ml of the same fraction. * Cultures were done in triplicate.
effective in decreasing DNA synthesis when added 24 and 48 h after the beginning of the culture. The S2 fraction exerted an inhibitory effect when added at time 0 but was without any effect when added at 24 and 48 h. The S3 fraction slightly depressed mitogen induced DNA synthesis when added at time 0. Table 6 showed that the ability of mouse spleen cells to respond to PHA was depressed by about 40Y% by the presence of fraction T4 and I at a dose of 80 ,ug/ml during the whole course of cultures. When the spleen cells were preincubated for 24 h with these fractions and then washed before mitogen stimulation, their PHA responsiveness were not affected suggesting the T4 and I fractions did not act by damaging the cells. In all DNA synthesis inhibition tests, the I fraction proved to be the most strongly inhibiting fraction, so that this fraction was used for further purification work. Purification of the I fraction The I fraction, which represented 044% of the organ dry weight and about 10% of the T4 fraction, was fractionated into three parts by filtration through a column of Sephadex G50 (Fig. 1). The inhibitory activity was only recovered in the second peak (Peak II). In polyacrylamide gel electrophoresis this peak did not appear to be homogeneous and showed at least four bands (Fig. 2). Peak II was then submitted to chromatography
600 6 1.0
0-5 tg
10
_
0
20 Tube no.
*
V
50
h
30
Figure 1. Elution profile of the I fraction from a Sephadex G50 column.
on DEAE Sephadex A50 (Fig. 3). The active moiety, exhibiting a tenfold increase of the inhibitory activity when compared to the T4 fraction, was eluted in the first buffer (Tris-HCl 0 01 M without NaCl). All these purification procedures are outlined in Table 1. The maximum of absorbance in the ultraviolet of the active substance purified by DEAE Sephadex chromatography, occurred at 260 nm. The amino acid analysis of this peak is reported in Table 7. This analysis indicates a high-proportion of basic amino acids.
Heat sensitivity of the T4 and I fractions Table 8 shows that after heating 15 min at 1000 the T4 and I fractions remained capable of decreasing in vitro the spontaneous DNA synthesis in thymocytes.
pH sensitivity Fig. 4 shows that the inhibitory effect of the I fraction was maximal at pH 6, 7, 8 and reduced significantly at low pH (4 and 5).
Purification and characterization of LIFT
445
_
t.
--.*.. ..
..
... :.
.;: ...... .,: *;: :.: :.
Figure 2. Acrylamide gel electrophoresis of the three peaks eluted from the I fraction after filtration through a Sephadex G50 column. Table 7. Amino acid composition of the most purified thymic factor Amino acid Lys His NH3
Residue number for 100 10 residues
2
-
Arg
Asp
Thr
Ser
Glu
Pro
Ala
Val
Met
lie
Leu
Tyr
Phe
Gly
6
7
5
6
10
9
8
5
1
3
5
3
2
18
Enzymatic digestion of the I fraction Fig. 5 shows that after digestion with trypsin pronase and RNase, the inhibitory capacity of the I fraction towards the in vitro spontaneous DNA synthesis of the thymocytes disappeared, whereas DNase treatment did not reduce the inhibitory activity of the fraction.
DISCUSSION From a thymic extract which exhibits both an antiproliferative property when tested in vitro on lymphoid cells, and an immunosuppressive activity when injected into mice, we have isolated, by ultrafiltration procedures, four subfractions I, S3,
446
Nicole Kiger et al. 100 r
G z I c
0
80 F
._
100
2.0
0-
N
cS
c
0
40
50So-C
1.0
4
5
6
7
8
9
pH
Figure 4. Percentage of the inhibition of the in vitro spontaneous DNA synthesis in thymocytes is plotted as a function of the pH at which the I fraction was exposed. Control (100 %o) is the mean 3H-TdR uptake of culture incubated without any extract. Maximal deviation from the mean values are less than + 15%Y.
20 Tube no.
Figure3. Chromatography of the 2nd peak from Sephadex G50 on a DEAE-Sephadex A50 column. Table 8. Heat sensitivity of thymic fractions tested by their inhibitory effect upon spontaneous DNA synthesis in thymocytes
Fraction added c.p.m. (mean+ s.e.)* % Inhibition
2100 Q. I--
I -r 2
None Unheated T4 fraction Heated t T4 fraction Unheated I fraction Heatedt I fraction *
0
42,910+ 3030
0
11,156+429
74
10,512+ 1321 8452+ 1557
76 80
9639+ 2151
78
0
Fraction added I - I Enzyme treatment D Nase
- I Trypsin
- I Protease
- I R Nasp
Figure 5. Effect of enzymic digestion on the inhibitory capacity of I fraction.
Cultures were done in triplicate.
t The fractions were heated at 1000 for 15 min.
S2, S1 of increasing molecular weights. Testing the effects of these different subfractions upon the in vitro spontaneous 3H-TdR uptake in thymocytes, we have found that the I fraction, with molecular weight ranging between 10,000 and 50,000, was more active per unit protein than the T4 fraction starting material and that the activity of the residual proteins was clearly reduced after removal of the I fraction. Thus we have tentatively concluded that the inhibitoryactivity of the fractionT4 can be accounted for by the presence of the active low molecular weight moiety. Furthermore, the I fraction had the ability to inhibit PHA induced 3H-TdR incorporation in mouse spleen cells. In this case, the effect was
demonstrated to be independent of the time at which the I fraction was added after mitogen stimulation. This argues against the hypothesis of a competitive action of the inhibitor and PHA on the cell membrane, as well as against the possibility of an inhibition of the lectin effect by its binding to the thymic extract. In the same experiment, SI fraction (molecular weight greater than 300,000) exhibited the same inhibitory property as the I fraction. We can consider at least two likely explanations for these observations: either the lymphocyte inhibitor could be a low molecular weight entity contained in the I fraction, which is found in the SI fraction bound to a large carrier, or there could be two independent molecules with similar but unrelated physiological properties. These possibilities are under
investigation.
Purification and characterization of LIFT We have previously demonstrated that the inhibitory effect of the T4 fraction starting material upon thymocyte spontaneous DNA synthesis was reversible (Florentin et al. 1976). In the present paper, we show that a 24 h pre-incubation of spleen cells with either T4 or I fraction does not impair their ability to transform with PHA if cultures are performed in fraction free medium. This further argues against a cytotoxic effect of the fractions. Inhibition of DNA synthesis could simply be due to the decrease of tritiated thymidine penetration under the influence of the thymic extract. The observation that this extract was unable to decrease thymidine incorporation into non-lymphoid cells argues against this hypothesis. Furthermore, recent experiments have shown that it probably acts in a different way; indeed cytophotometric and autoradiographic studies have shown that the thymic factor inhibits the progression through the S phase but also on various positions of the mitotic cell cycle (Olsson, Kiger & Florentin, in press). We have previously demonstrated that the factor rapidly inhibits DNA and RNA synthesis in short term thymocyte cultures (Florentin, 1976). Experiments are in progress to further investigate the effect of this substance on protein synthesis as well as the mechanism of action of inhibition of DNA and RNA synthesis. We have previously demonstrated by Sephadex G75 filtration that all the activity of the I fraction was present in a molecular range between about 10,000 and 20,000, excluding the presence of cold thymidine as a cause of reduced incorporation of labelled thymidine into the cells. Furthermore, it is unlikely that the inhibitory activity of the I fraction is simply due to thymidine bound to a carrier protein as we have found that the spontaneous mitotic rate of thymocytes was also significantly impaired by incubation of cells with the thymic extract (Olsson et al., in press). LIFT, which is species non specific, exhibits a specificity of action for lymphoid cells since it does not inhibit DNA synthesis in non-lymphoid cells, i.e. mouse fibroblasts or human tumour cells. Conversely, similar extracts from non-lymphoid tissue, i.e. kidney, liver, lung are ineffective upon thymocytes. The I fraction has been further purified by filtration on Sephadex G50 followed by chromatography on DEAE-Sephadex A50. The conditions of elution of the inhibitory factor on anion-exchange chromato-
447
graphy indicated the basicity of the substance; this was confirmed by the amino acid analysis of the more purified fraction exhibiting a large amount of the basic amino acids. By digestion studies with enzymes, the active factor was found to be undigestible by DNase, destroyed by trypsin, pronase and RNase indicating that the active moiety may be a peptide bound to ribonucleosides, this latter conclusion seems to be confirmed by the maximal absorbance at 260 nm of the most purified fraction. This substance is heat insensitive as the activity remains intact even after heating 15 min at 100°. A number of inhibitors of lymphocyte proliferation have been described, namely, a globulins, factors secreted by established human lymphoid cell lines, and lymphoid chalones. Substances identified as a globulins, have been isolated from human sera and inhibit both immune responses in vivo and lymphocyte proliferation in vitro (Menzoian, Glasgow, Nimberg, Cooperband, Schmid, Saporoschetz & Mannick 1974, Riggio, Schwartz, Bull, Stenzel & Rubin, 1969, Miller, 1976). A similar factor inhibiting lymphocyte proliferation has been recovered in a thymic extract (Phillips, Carpenter & Lane, 1975). Few of these factors have been sufficiently purified, and when they have been characterized (Occhino, Glasgow, Coo-erband, Mannick & Schmid, 1973) they exhibit physico-chemical properties (molecular weight, sensitivity to pH, behaviour on anion exchange chromatography) which are different from those described for LIFT. Factors released by human lymphocytes in cultures have also been described which possess anti-proliferative capacities on lymphocytes when tested on mitogen-induced blastogenesis and which have been identified as glycoprotein. They are different from LIFT by their molecular weights (Chase, 1976) or by their conditions of inhibition of lymphocyte proliferation which are dependent on the time at which the fraction is added to the culture after stimulation by PHA (Milton, 1971). Other factors extracted from lymphocytes or released by human lymphocytes in cultures present important differences in the physico-chemical properties with LIFT such as different thermosensitivity and molecular weights (Papageorgiou, Tibbetts, Sorokin & Glade, 1974; Tibbetts, Glade & Papageorgiou, 1975), or important differences in their specificity, namely species specificity (Hersh, McCredie & Freireich, 1974).
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Nicole Kiger et al.
Several authors refer to the inhibitors of lymphoid DNA synthesis as lymphocyte chalones, which are tissue-specific but not species-specific inhibitors of lymphocyte proliferation. These inhibitors are generally considered as proteins (with molecular weights above 40,000) or as small peptides (with molecular weights lower than 5000), they are sensitive to proteolytic enzymes and to heat. Such inhibitors have beenextracted from spleen and lymph nodes (Moorhead, Paraskova-Tchnerno-zenska, Pirrie & Hayes, 1969, Lasalvia, Garcia-Giralt & Maciera-Coelho, 1970) and from thymus (Attallah, Sunshine, Hunt & Houck, 1975) these inhibitors are heat sensitive and have slightly larger molecular weights than LIFT but, at the present time, progress on their purification and characterization has been insufficient to allow further comparisons. In conclusion, at the present time, we cannot identify LIFT with other inhibitors of lymphocyte proliferation; it could be suggested that the active peptide we have isolated could be bound to one or more proteins giving rise to the substances described by different authors, but a number of physicochemical and even physiological properties seem to signify that they are unrelated substances.
ACKNOWLEDGMENTS The authors thank Miss Nicole Saou and Miss Martine Davigny for their excellent technical assistance and CERM Laboratories, 63 RIOM, France, who kindly provided the lyophilized lamb thymuses. This research was supported by Contrat libre INSERM no. 75-5-108-2 and Contrat DGRST nos 74-7-0620 and 75-7-0732. REFERENCES ATTALLAH A.M., SUNSHINE G.H., HUNT C.V. & HOUCK J.C. (1975) The specific and endogenous mitotic inhibitor of lymphocytes (chalone). Exp. Cell. Res. 93, 283. CHASE P.S. (1976) The effects of human serum fractions on phytohemagglutinin- and concanavalin A-stimulated human lymphocyte cultures. Cell. Immunol. 5, 544. FLORENTIN I., KIGER N. & MATHE G. (1976) The lymphocyte inhibiting factor extracted from the thymus (LIFT): inhibition of the DNA synthesis in vitro. Cell. Immunol. 23, 1. FLORENTIN I., KIGER N. & MATHE G. (1973) T lymphocyte specificity of a lymphocyte inhibiting factor extracted from the thymus. Europ. J. Immunol. 3, 624.
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