ClGn. exp. Immunol. (1979) 37, 50-57.
Human transfer factor in vitro II. AUGMENTATION OF THE SECRETION OF LEUCOCYTE MIGRATION INHIBITORY FACTOR (LIF) BY LEUCOCYTE DIALYSATE AND BY ITS COMPONENTS L-SERINE AND GLYCINE R. G. I. ASHORN, LIISA RASANEN, KIRSI-MARJA MARNELA & K. J. E. KROHN Institute of Biomedical Sciences, University of Tampere, Box 607, SF-33 101 Tampere 10, Finland
(Accepted for publication 28 November 1978) SUMMARY
The effect of human transfer factor (TF) or its components L-serine and/or glycine on tuberculin (PPD), or leucoagglutinin (LA) induced leucocyte migration inhibitory factor (LIF) secretion was studied. Augmentation of LIF secretion was seen with low concentration (= 0-078 g/l) of TF when lymphocytes were cultured in minimum essential medium for suspension cultures (MEM-S), a culture medium lacking L-serine and glycine. High concentrations (03125-5'0 g/l dry weight) of TF were inhibitory in MEM-S. In RPMI 1640, a culture medium containing L-serine and glycine, TF was either inhibitory or had no effect. The combination of L-serine and glycine, at concentrations equivalent or lower than the optimum of TF, had an augmenting effect on LIF secretion identical to that of TF, but no inhibition at higher concentrations was seen. The results indicate that human TF contains components which have suppressive or augmenting effects on LIF secretion in vitro. The augmenting effect may be mainly due to L-serine and glycine and thus not related to TF's activity in vivo.
INTRODUCTION Human transfer factor (TF), a dialysate of leucocyte lysate, is known to convey delayed hypersensitivity from skin test positive donors to skin test negative recipients. This activity of transfer factor has been thought to be at least partly due to the transfer of immunological information (Fudenberg et al., 1974; Lawrence, 1969; 1974; Spitler, Levin & Fudenberg, 1973). Recipients of TF may, however, develop immune reactivities unrelated to the immune status of the donor (Arala-Chaves, Proenca & de Sousa, 1974; Burger et al., 1976a; Littman, Hirschman & David, 1977; Valdimarsson et al., 1974), and these observations suggest that in addition to immunologically specific activity, TF may also have an immunologically non-specific, adjuvant-like effect on the expression of recipients immune reactivities. Little is known of the mode of action and the chemical nature of the active component(s) of TF. A major problem in acquiring this basic information of TF has been the lack of a reliable in vitro assay. We have shown recently that the augmenting effect of TF on antigen- or mitogen-induced lymphocyte transformation may be mainly due to its content of L-serine and glycine, amino acids which are essential for lymphocyte proliferation and protein synthesis (Uotila et al., 1978). It is thus obvious that most of TF's in vitro effect on lymphocyte transformation is unrelated to its in vivo activity. This view gains further support from the finding that the in vivo activity of TF on skin reactivity is often not correlated to a simultaneous increase in the recipient's blast transformation index (Arala-Chaves et al., 1974; Fudenberg etal., 1974; Kirkpatrick, Rich & Smith, 1972; Krohn et al., 1976). Similar results have been obtained with the guinea-pig model for TF (Vanderbarck, Burger & Vetto, 1977; Welch et al., 1976). Correspondence: Dr K. Krohn, Institute of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland.
0099-9104/79/0070-0050$02.00 0 1979 Blackwell Scientific Publications
50
Transfer factor and LIF secretion
51
On the other hand, methods which measure the capacity of antigen-activated lymphocytes to secrete mediators of cellular immunity, such as the leucocyte migration inhibitory factor (LIF), seem to correlate better with skin test reactions in man and in the guinea-pig (Astor et al., 1973; Clausen, 1971; Hoffman, Spitler & Hsu, 1976). It is therefore important to know whether the observed in vitro inducing or potentiating activity of human TF on LIF secretion (Goust et al., 1976; Morison, 1976; Pizza et al., 1976; Read, Espinoza & Zabriskie, 1976; Wilson et al., 1977) is also connected to the contents of L-serine and glycine in TF, or whether this activity could be more closely related to TF's in vivo effect. In this paper we describe the effect of human TF on LIF secretion induced with an antigen, tuberculin (PPD), or a non-specific mitogen, leucoagglutinin (LA), and compare this effect of TF to that caused by Lserine and glycine. MATERIALS AND METHODS Transfer factor preparation. Human leucocyte dialysate was derived from a pool of 401 citrated buffy coats obtained from the Finnish Red Cross Blood Transfusion Service. For the preparation of dialysate, dextran sedimentation, freezing and thawing of the washed leucocytes, ultrafiltration of the cell lysate, and lyophilization of the ultrafiltrate were done as described previously (Krohn et al., 1977). Although the immune reactivities of the donors were unknown, the TF preparation can be considered PPD-positive, since all children in Finland have been vaccinated with BCG since the early 1940's. The preparation has also been shown to be active in the guinea-pig model (Krohn et al., 1979), as described by Vanderbarck et al. (1977) and Welch et al. (1976). Similarly prepared TF preparations from human buffy coat cells have also been shown to have a therapeutic effect in various clinical conditions (Krohn et al., 1977). Amino acid analysis. Analysis of the free amino acids in the horse serum was performed with a Beckman Multichrom M Amino Acid Analyzer as described previously (Uotila et al., 1978). The free amino acid content of the leucocyte dialysate used in the present study has been published previously (Uotila et al., 1978). Cell cultures. Mononuclear cells, containing an average of 85% lymphocytes, were isolated from citrated buffy coat cells (for mitogen-stimulated cultures), or from a PPD-positive donor (for antigen-stimulated cultures) on a Ficoll-Isopaque gradient (Boyum, 1968), and were suspended at a concentration of 106 cells/ml (antigen-stimulated cultures) or 2 x 106 cells/ml (mitogen-stimulated cultures) in MEM for suspension cultures (MEM-S) or RPMI 1640 (Flow Laboratories, Irvine, Scotland). The medium was supplemented with either 10% horse serum (Flow) dialysed for 36 hr with three changes, against MEM-S, or with 10% non-dialysed horse serum. Duplicate cultures containing 0-2 ml (antigen-stimulated cultures) or 0-1 ml (mitogen-stimulated cultures) of cell suspension per well were set up in U-bottomed microplates (Sterilin Ltd, Middlesex, UK). The cells were incubated with the following test substances: transfer factor in serial four-fold dilutions ranging from5OOOmg/l(4X*) to 49 mg/l (X/256) dry weight, and equivalent concentrations of amino acids found to be present in the trasfer factor preparation but not in MEM-S. The antigen, tuberculin (PPD), State Serum Institute, Denmark) and the mitogen, leucoagglutinin (LA, Pharmacia Fine Chemicals, Uppsala, Sweden), were used as stimulants. The antigen-stimulated cultures were incubated at 37°C in a 5% CO2 atmosphere for 6 days, in the presence of 12-5 mg/l of PPD. Immediately before collecting the supernatants, the control cultures were reconstituted with PPD. The mitogen-stimulated cultures were given a 30 min LA-pulse (30 mg/l), after which the cells were washed five times with Hanks's balanced salt solution (HBSS, Flow) to remove the unbound LA (Rasanen, Karhumaki & Krohn, 1978). The cultures were then reconstituted with the previous medium, serum and test substances, and were incubated at 37°C in a 5% CO2 atmosphere for 3 days before collecting the supernatants. Appropriate controls contained no addition, LA alone, or the corresponding amounts of test substances alone. All the supernatants were stored at - 80°C if not immediately tested. Leucocyte migration inhibitory factor assay. LIF activity in the lymphocyte culture supernatants was tested by the agarose migration method (Clausen, 1972) with minor modifications. Briefly, citrated or heparinized blood was sedimentated with dextran and the leucocyte-rich plasma was centrifuged on Ficoll-Isopaque. Leucocytes at the bottom of the tube, containing an average of 98% granulocytes and 2% mononuclear cells, were washed three times with HBSS before being used as indicator cells in the assay. The amount of erythrocytes varied, but as this did not seem to affect the test system, they were not lysed. The agarose plates prepared in RPMI 1640 with 10% horse serum were buffered with 5% sodium bicarbonate. Wells of 2-3 mm diameter were punched in the gel. The non-dialysed supernatants, supernatants dialysed for 36 hr, with three changes, against MEM-S and serial two-fold dilutions in MEM-S of the non-dialysed supernatants from the lymphocyte cultures were pre-incubated at 37°C for 30 min with the isolated granulocytes, after which 5,u1 of the suspension, containing 5 x 105 granulocytes, was pipetted into each well. The supernatants were tested with four replicate determinations. The plates were incubated at 37°C in a 5% CO2 atmosphere for 18 hr. The area of the well was excluded from the final area of migration. Means and standard deviations were calculated. The experimental approach. In the LIF assay two types of experiments were designed. In the first set, augmentation of LIF secretion, produced by various concentrations of the test substances, was determined by testing the effect of undiluted lymphocyte culture supernatants on granulocyte migration. The control migration inhibition percentage (MI%), test migration inhibition percentage (MIt%) and the augmenting effect of the test substance (AMI%) were calculated as follows: * X is the starting solution which is equivalent to 1-25 g/l.
R. G. I. Ashorn et al.
52 MI~%
=
area of migration+ s.d. with LA alone ) (1IIXmean 100 mean area of migration with no addition
( mean area of migration+ s.d. with LA+test substanceX -Ix 100 MI,% = I1 mean area of migration with test substance alone
AMI% = MIt%-MI'% The significance of the augmenting effect was determined by Student's t-test. With this test system, migration inhibitions close to maximum were often observed when testing supernatants of mitogenstimulated cultures, and so the amount of LIF in the culture supernatants was quantified more accurately by titration in the second set of experiments. Serial two-fold dilutions up to 1/128 were prepared from the culture supernatants containing an optimal concentration of TF with the highest AMI% values obtained in the first set of experiments. The culture supernatants containing L-serine, glycine, or L-serine+glycine at concentrations corresponding to that found in TF, or containing no test substance, were similarly diluted. Migration inhibitions were calculated, and the dilutions corresponding to 40% inhibition were determined by linear regression analysis of the migration inhibition values ranging from 15 to 65%.
RESULTS Preliminary experiments were carried out to determine the concentration of LA, where the effect of TF would be maximal. Lymphocytes were cultured in MEM-S, supplemented with 10% non-dialysed horse serum with or without different concentrations of TF or L-serine and/or glycine. The cultures were pulsed with various concentrations of LA. In these experiments, TF in concentrations ranging from 4X to X/256 augmented LIF secretion and the strongest augmentation was seen at an LA concentration of 30 mg/l. The optimal dose for TF varied between X and X/64, being most often X/16 (0.078 g/l). Dialysing the culture supernatants against MEM-S had no effect on their LIF activities (data not shown). Similar augmentations of LA responses were seen when the cultures in MEM-S were supplemented with L-serine or glycine at concentrations present in RPMI 1640 (Table 1). On the other hand, when the lymphocytes were cultured in RPMI 1640, TF had no augmenting effect on LIF production (Table 1). TABLE 1. The effect of transfer factor, L-serine and glycine on LA-induced LIF secretion in different culture media
Culture medium MEM-S+ 10% nondialysed horse serum RPMI 1640+10% nondialysed horse serum
LA alone
LA with TF (X/16*)
LA with serine (286 pmol/lt)
55+4+
71+3
76+3
72+1
75+2
80+ 2
74+ 1
n.t.§
n.t.§
A.5
LA with glycine LA with serine+glycine (286+133 ,umol/l¶) (133 ,pmol/lt)
* X = 1250 mg/l (dry weight). t Concentration present in RPMI 1640. + Migration inhibition percentage+ s.d. § Not tested.
Calculation of the concentrations of L-serine and glycine from the amino acid analysis of the leucocyte dialysate preparation used in this study showed that the amount of these two amino acids in TF at X/16 concentration was considerably lower than in RPMI 1640 (Table 2). Experiments were therefore devised where the effect of TF on LIF secretion was compared to that obtained with equivalent concentrations of the two amino acids, either alone or in combination. The results in Fig. 1 indicate that X/16 and higher dilutions of TF augmented LIF secretion as effectively as equivalent amounts of Lserine and glycine in combination. L-serine alone gave a somewhat lower response. With higher concentrations (X/8-4X) TF inhibited LIF secretion, the suppression effect being most pronounced with the highest concentration used (4X). Both L-serine alone and the combination of L-serine and glycine augmented LIF secretion, and with concentrations equivalent to X/4-4X of TF, the combination of the
Transfer factor and LIF secretion
53
20 ./
E-50
i
-60
-70
-80 _ X/4 X X/256 X/64 X/16 Dilution of test substances
4X
FIG. 1. Augmentation (AMI% > 0) or suppression (AMI%< 0) of leucocyte inhibition factor (LIF) mediated o) and ), L-serine (o granulocyte migration inhibition by serial dilutions of transfer factor (a . A). X 1-25 g/l ofleucocyte dialysate or equivalent amounts of the test amino acids L-serine+glycine (A (304 pmol/l for L-serine and 336 pmol/l for glycine). Vertical bars represent standard deviations of four replicate migration tests. - --
- --
=
amino acids gave significantly stronger augmentation than L-serine alone (P< 0001). The effect of glycine alone paralleled that of L-serine, but was usually somewhat weaker. In this experiment, as in most similar experiments, LA alone gave a rather strong migration inhibition (MI,,% > 60%) and thus the effect of the test substances could result only in a relatively small AMI%. Therefore, experiments were constructed where the amount of LIF in various lymphocyte culture supernatants was assessed by titration. An example of such an experiment is shown in Fig. 2, where the supernatants from lymphocyte cultures with TF concentration X/16 or equivalent amounts of L-serine,
two
TABLE 2. The concentrations of amino acids present in transfer factor and the complete culture, media, but not in MEM-S RPMI 1640+ MEM-S+ Transfer factor* 10% horse serum 10% horse serum
L-Serine Glycine L-Alanine L-Cysteine L-Proline L-Glutamic acid L-Aspartic acid Taurine o-Phospho-i.-serine
19t 21 26 2 14 20 9 21
16 31 15
302 164 15
5 16 3 4
180 152 153 4
1
Corresponds to a concentration of X/16 (X = 1250 mg/i). t Expressed as umol/l. *
54
R. G. I. Ashorn et al.
0
e40
-
-
-
=60
80
;.!:.
1/1 1/2 1/4 1/8 1/16 1/32 1/641/128 Dilution of the culture supernatonts
FIG. 2. Migration inhibition percentage caused by serial dilutions of supernatants from lymphocyte cultures in U) or together with X/16 MEM-S and 10% undialysed horse serum, containing LA alone (30 mg/l; concentration (0-078 g/l) of transfer factor ( . 0) or equivalent amounts of L-serine (19 pmol/l; o c) or L-serine+glycine (19+21 umol/l; A - - - A). The dilutions giving 40% migration inhibition, calculated by linear regression analysis of inhibition values ranging from 15 to 65% (*not included) were 1/6 for the control (r = 0 98), 1/10 for L-serine (r = 0 93), 1/20 for L-serine+glycine (r = 0.98) and 1/24 for transfer factor (r = 093). Vertical bars represent standard deviations of four replicate migration tests.
0 _
20 -1
o
60
-
80~~~~~I ~
1/1 1/2 1/4 1/8 1/16 1/32 1/641/128 Dilution of the culture supernotants
FIG. 3. Migration inhibition percentage caused by serial dilutions of supernatant from lymphocytes cultures in MEM-S and 10% dialysed horse serum, containing LA alone (30 mg/l; * *) or together with X/16 concentration (0-078 g/l) of transfer factor ( -.) or equivalent amounts of L-serine (19 pmol/l; o - - - o) *) or L-serine+glycine (19+21 pmol/l; A - - - A). The dilutions giving 40% glycine (21 jumol/l; * migration inhibition, calculated by linear regression analysis of inhibition values ranging from 15 to 65% (*not included) were 1/2 for L-serine (r = 0-96) 1/1 for glycine (r = 0 98), 1/42 for L-serine4-glycine (r = 0 96) and 1/39 for transfer factor (r = 0.95). Vertical bars represent standard deviations of four replicate migration tests.
Transfer factor and LIF secretion
55
glycine or their combination were tested. The results indicate that the combination of L-serime and glycine once again augments LIF secretion as much as TF, and that the augmentation is about twice as strong as that caused by L-serine or glycine alone. All these experiments were performed with 10% non-dialysed serum which already contained L-serine and glycine. In experiments where only one of these two amino acids was added to the lymphocyte cultures, both amino acids were in fact present, and the effect on LIF synthesis was thus always a combination effect. The experiments shown in Fig. 2 were therefore repeated using dialysed horse serum, where the absence of glycine and L-serine was checked by amino acid analysis. The results of the experiments are shown in Fig. 3. Without addition of the test substances and in the complete absence of Lserine or glycine, LA caused hardly any detectable LIF secretion. The addition ofTF or the combination of L-serine and glycine caused similar LIF secretion, which was twenty and forty times higher than that caused by L-serine or glycine alone, as calculated from the 4000 migration inhibition values. These results show, therefore, that a synergistic action of L-serine and glycine was necessary for maximal LIF secretion, and that the effect of TF was as strong, but not stronger, than that of the two amino acids. The other amino acids which were found in the dialysate, but not in MEM-S (Table 2), had no effect on LIF synthesis. Finally, experiments were designed where the effects of TF, L-serine and glycine were tested on PPDinduced LIF secretion, using mononuclear cells derived from a PPD-positive donor, or from human cord blood. Stimulation of the cord blood cells with PPD caused no LIF secretion in the absence or presence of the test substances (data not shown). The results obtained with PPD-positive mononuclear cells, shown in Table 3, indicate that the effect of the test substances on antigen-induced LIF secretion is similar to that on mitogen-induced LIF secretion. TABLE 3. The effect of transfer factor, L-serine, and glycine on PPD-induced LIF secretion in different culture media PPD alone
Culture medium
PPD with TF
PPD with glycine PPD with serine+glycine (21 pmol/lt) (19+21 pmol/lt)
(X/16*)
PPD with serine (19 ,pmol/lt)
42+14§
34+16
12+4
37+8§
33 + 4§
28+ 3
12+11
30+ 0§
34+4
33+1
40+3
40+5
MEM-S+ 10% dialysed horse serum 9+7+ MEM-S+ 10% nondialysed horse serum 18+10 RPMI 1640+10% nondialysed horse serum 41+8 *
X
=
1250 mg/i (dry weight).
t Concentration equivalent to X/16 of TF. Migration inhibition percentage+ s.d. § Differs significantly (Student's t-test, P< 0 01) from the corresponding value of PPD alone.
+
DISCUSSION The results obtained in this study indicate that TF has a non-specific augmenting effect on antigen- and mitogen-stimulated LIF secretion, and that this effect is mainly due to the L-serine and glycine content of the leucocyte dialysate. The conclusion that TF, L-serine and glycine augment migration inhibition mainly by stimulating LIF secretion and not by increasing granulocyte responsiveness to LIF, can be drawn from the fact that depleting the culture supernatants of these test substances by dialysation had no effect on the LIF activity. We can deduce that this augmenting activity was due to the presence of Lserine and glycine from the following facts: (1) hardly any LIF secretion occurred when dialysed serum without TF or amino acid addition was used (Fig. 3, Table 3); (2) TF or L-serine, glycine and their combination augmented LIF secretion when experiments were carried out in medium which did not
56
R. G. I Ashorn et al.
itself contain these amino acids (MEM-S); and (3) the addition of TF in the test system had no effect when the redium used already contained a nearly optimal concentration of L-serine and glycine (RPMI 1640). In fact, in RPMI 1640, the addition of TF had an inhibitory rather than a stimulatory effect on LIF secretion. This finding may be due to the presence of substances in the leucocyte dialysate that suppress lymphocyte function-such a substance has previously been identified as nicotinamide (Burger et al., 1976b), but additional inhibitory substances may exist. The inhibitory effect of TF was especially strong when it was used in high concentrations (4X = 5 g/l); the optimum dose for augmentation of LIF synthesis was X/16, whereas L-serine and glycine concentrations corresponding to 4X of TF gave the highest augmenting effect (Fig. 1). Dilution of the lymphocyte culture supernatants cultured with either X/16 concentration of TE or with the equivalent L-serine+ glycine concentrations showed, however, that an equal amount of LIF was secreted in the presence of TF or these two amino acids (Fig. 2). Experiments carried out in dialysed serum, presented in Fig. 3, indicate that the effect of glycine and Lserine on LIF secretion is synergistic and not purely additive. This synergistic effect of the two amino acids may explain why with non-dialysed serum even the addition of one of the amino acids, at a low concentration, seemed to cause an effect equal to that obtained with TF or the combination of the two amino acids. Our results are thus in agreement with previous findings that lymphocytes are defective in synthesizing L-serine, and further suggest that the hydroxymethyltransferase reaction is insufficient for the interconversion of the two amino acids. It has been shown previously that L-serine and glycine are necessary for optimal lymphocyte DNA, RNA and protein synthesis (Dubrow, Pizer & Brody, 1973; Pizer & Regan, 1972; Regan et al., 1969; Uotila et al., 1978). Our present finding is the first to demonstrate a defect in the synthesis of a specific protein, LIF, in the absence of L-serine and/or glycine. According to some workers, LIF might exert its effect by virtue of being a serine protease or esterase (Bendtzen, 1977; Rocklin & Rosenthal, 1977). Thus, the necessity of L-serine in the catalytic site of the putative LIF enzyme would further contribute to the requirement of this amino acid. The present findings support the view that the non-specific augmenting in vitro effect of transfer factor on LIF secretion is mainly due to its L-serine and glycine content. It is obvious that L-serine and glycine cannot be responsible for TF's in vivo activity as these amino acids are normally present in human blood and their presence in TF is too slight to cause any significant increase in the plasma amino acid level. However, since a reliable in vitro test for TF, correlating with its in vivo activity, would be of great importance in the chemical characterization of TF, it is necessary to analyse carefully those in vitro activities of TF which are not related to its in vivo effect, as these non-specific activities may effectively conceal the more important specific ones. This work has been supported with grants from the Sigrid Juselius Foundation, Finnish Cultural Foundation, Cancer Foundation in Finland and Orion Pharmaceuticals. The skilled assistance of Mrs Eliisa Karhumaki, M.Sc., Mrs Eila Pohiola, Mrs Tuula Myllymaki, Mrs Raija Keskivali, Miss Eija Kyrola and Mr P. Virtanen is gratefully acknowledged. REFERENCES & VETTO, R.M. (1976a) Human transfer factor: effects on lymphocyte transformation. 5. Immunol. 117, 782. BURGER, D.R., VANDERBARCK, A.A., DAVEY, D., ANDERSON, W.A., VETTO, R.M. & FiNKE, P. (1976b) Human transfer factor: fractionation and biologic activity. 5. Immunol. 117, 789. CLAUSEN, J.E. (1971) Tuberculin induced migration inhibition of human peripheral leucocytes in agarose medium. Acta Allergol. 26, 56. CLAUSEN, J.E. (1972) Migration inhibitory effect of cell-free supernatants from mixed human lymphocyte cultures. ]. Immunol. 108, 453. DUBROW, R., PIZER, L.I. & BRODY, J.I. (1973) Serineglycine requirement of phytohaemagglutinin stimulated lymphocytes. ]. Nat. Cancer Inst. 51, 307.
ARALA-CHAVES, M.P. PROENCA, R. & DE SOUSA, M. (1974) Transfer factor therapy in a case of complex immunodeficiency. Cell. Immunol. 10, 371. ASTOR S.H. SPITLER L.E. FRICK O.L. & FUDENBERG, H.H. (1973) Human leucocyte migration inhibition in agarose using four antigens: Correlation with skin test reactivity. ]. Immunol. 110, 1174. BENDT2EN. K. (1977) Human leucocyte inhibitory factor (LIF). III. Further investigations on the serine protease nature of this lymphocine and its preference for arginine amides. Scand. J. Immunol. 6, 1055. BO6UM, A. (1968) Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, Suppl. 97, 77. BURGER, D.R., VANDERBARCK, A.A., FiNKE, P., NOLTE, J.E.
Transferfactor and LIF secretion FUDENBERG, H.H., LEVIN, A.S., SpiTuR, L.E., WYBRAN, J. & BYrERs, V. (1974) The therapeutic uses of transfer factor. Hosp. Pract. 9, 95. GOUST, J.M., MoULIAS, R., PHILIPE, R. & FUDENBERG, H.H. (1976) In vitro assay for transfer factor by means of leucocyte migration inhibition test (LMT). Transfer factor, basic properties and clinical applications (ed. by M.S. Archer, A.A. Gottlieb & C.H. Kirkpatrick), p. 137. Academic Press, New York. HOFFMAN, P.M., SPITLER, L.E. & Hsu, M. (1976) Leucocyte-migration inhibition in guinea-pigs. I. Correlation with skin test reactivity and macrophage-migration inhibition. Cell. Immunol. 21, 358. KIRKPATRICK, C.H., RICH, R.R. & SMITH, T.K. (1972) Effect of transfer factor on lymphocyte function in anergic patients. ]. cdin. Invest. 51, 2948. KROHN, K., GROHN, P., HoRsmAN-EIMO, M. & VIROLAINEN, M. (1976) Fractionation studies on human leucocyte dialysates. Demonstration of three components with transfer factor activity. Med. Biol. 54, 384. KROHN, K., UOTILA, A., VAISANEN, J. & GROHN, P. (1977) Studies on the chemical composition and biological properties of transfer factor. Z. Immun.-Forsch. 153, 395. KROHN, K., VOTILA, A. ASHORN, R. & KARHUMAmI, E. (1979) Augmentation of skin reactivities in antigen primed guinea pigs by transfer factor and other cellular dialysates. Immune regulators in transfer factor (ed. by A. Khan, C.H. Kirkpatrick and N.O. Hill). Academic Press, New York. LAWRENCE, H.S. (1969) Transfer factor. Adv. Immunol. 11, 195. LAWRENCE, H.S. (1974) Transfer factor in cellular immunity. Harvey Lec . 68, 239. LITTMAN, B.H., HIRSCHMAN, E.M. & DAVID, J.R. (1977) Augmentation of 3H-thymidine incorporation by lymphocytes in the presence of antigen and fractions of dialyzable transfer factor: A non-specific phenomenon. Cell. Immunol. 28, 158. MORISON, W.L. (1976) Phytohaemagglutinin and transfer factor in the leucocyte migration inhibition test in patients with sarcoidosis. Thorax, 31, 87. PIZER, L.I. & REGAN, J.D. (1972) Basis for the serine requirements in leucemic and normal human lymphocytes. Reduced levels of the enzymes in the phosphorylated pathway. ]. Nat. Cancer Ins. 48, 1897. PIZZA, G., VIZA, D., BOUCcHEIX, C.I. & CORRADO, F. (1976) Studies with in vitro produced transfer factor. Transfer factor, basic properties and clinical applications (ed. by M.S.
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Ascher, A.A. Gottlieb & C.H. Kirkpatrick), p. 173. Academic Press, New York. RASANEN, L., KARHUMAKI, E. & KROHN, K. (1978) Elaboration of leucocyte migration inhibitory factor by human lymphocyte subpopulations stimulated with mitogens. Cell. Immunol. 37, 221. READ, S.E., EsPINozA, L.R. & ZABRISKIE, J.B. (1976) In vitro assay for transfer factor using direct migration inhibition. Transfer factor, basic properties and clinical applications (ed. by M.S. Ascher, A.A. Gottlieb & C.H. Kirkpatrick), p. 129. Academic Press, New York. REGAN, J.D., VoDopIicK, H., TAKEDA, S., LEE, W.H. & FAULCON, F.M. (1969) Serine requirement in leucemic and normal blood cells. Science, 163, 1452. ROCKLIN, R.E. & ROSENTHAL, A.S. (1977) Evidence that human leucocyte inhibitory factor (LIF) is an esterase. J. Immunol. 119, 249. SPITLER, L.E., LEVIN, A.S. & FUDENBERG, H.H. (1973) Human lymphocyte transfer factor. Methods Cancer Res. 8, 59. UOTILA, A., MARNELA, K.-M., HAMBLIN, A. & KROHN, K. (1978) Activity of human transfer factor in vitro. Identification of L-serine and glycine as components with augmenting effect in the lymphocyte transformation assay. Scand. J. Immunol. 7, 495. VALDIMAaSSON, H., HAMBLETON, G., HENRY, K. & MCCONNEL, J. (1974) Restoration of T-lymphocyte deficiency with dialyzable leucocycte extract. Clin. exp. Immunol. 16, 141. VANDERBARCK, A.A., BURGER, D.R. & VETTO, R.M. (1977) Human transfer factor activity in the guinea-pig: Absence of antigen specificity. Clin. Immunol. Immunopathol. 8, 7. WAITHE, W.I., DAUPHINAIS, C., HATHAWAY, P. & HIRSCHORN, K. (1975) Protein synthesis in stimulated lymphocytes. II. Amino acid requirements. Cell. Immunol. 17, 323. WELCH, T.M., TRIGLIA, R., SPITLER, L.E. & FUDENBERG, H.H. (1976) Preliminary studies on human 'Transfer factor' activity in guinea-pigs. Systemic transfer of cutaneous delayed type hypersensitivity to PPD and SK-SD. Clin. Immunol. Immunopath. 5, 407. WILSON, G.B., WELCH, T.M., KNAPP, D.R., HORSMANHEIMO, A. & FUDENBERG, H.H. (1977) Characterization of Tx, an active subfraction of human dialyzable transfer factor. I. Identification of the major component in TFg, a precursor of Tx, as hypoxanthine. Clin. Immunol. Immunopathol. 8, 55 1.
Human transfer factor in vitro II. AUGMENTATION OF THE SECRETION OF LEUCOCYTE MIGRATION INHIBITORY FACTOR (LIF) BY LEUCOCYTE DIALYSATE AND BY ITS COMPONENTS L-SERINE AND GLYCINE R. G. I. ASHORN, LIISA RASANEN, KIRSI-MARJA MARNELA & K. J. E. KROHN Institute of Biomedical Sciences, University of Tampere, Box 607, SF-33 101 Tampere 10, Finland
(Accepted for publication 28 November 1978) SUMMARY
The effect of human transfer factor (TF) or its components L-serine and/or glycine on tuberculin (PPD), or leucoagglutinin (LA) induced leucocyte migration inhibitory factor (LIF) secretion was studied. Augmentation of LIF secretion was seen with low concentration (= 0-078 g/l) of TF when lymphocytes were cultured in minimum essential medium for suspension cultures (MEM-S), a culture medium lacking L-serine and glycine. High concentrations (03125-5'0 g/l dry weight) of TF were inhibitory in MEM-S. In RPMI 1640, a culture medium containing L-serine and glycine, TF was either inhibitory or had no effect. The combination of L-serine and glycine, at concentrations equivalent or lower than the optimum of TF, had an augmenting effect on LIF secretion identical to that of TF, but no inhibition at higher concentrations was seen. The results indicate that human TF contains components which have suppressive or augmenting effects on LIF secretion in vitro. The augmenting effect may be mainly due to L-serine and glycine and thus not related to TF's activity in vivo.
INTRODUCTION Human transfer factor (TF), a dialysate of leucocyte lysate, is known to convey delayed hypersensitivity from skin test positive donors to skin test negative recipients. This activity of transfer factor has been thought to be at least partly due to the transfer of immunological information (Fudenberg et al., 1974; Lawrence, 1969; 1974; Spitler, Levin & Fudenberg, 1973). Recipients of TF may, however, develop immune reactivities unrelated to the immune status of the donor (Arala-Chaves, Proenca & de Sousa, 1974; Burger et al., 1976a; Littman, Hirschman & David, 1977; Valdimarsson et al., 1974), and these observations suggest that in addition to immunologically specific activity, TF may also have an immunologically non-specific, adjuvant-like effect on the expression of recipients immune reactivities. Little is known of the mode of action and the chemical nature of the active component(s) of TF. A major problem in acquiring this basic information of TF has been the lack of a reliable in vitro assay. We have shown recently that the augmenting effect of TF on antigen- or mitogen-induced lymphocyte transformation may be mainly due to its content of L-serine and glycine, amino acids which are essential for lymphocyte proliferation and protein synthesis (Uotila et al., 1978). It is thus obvious that most of TF's in vitro effect on lymphocyte transformation is unrelated to its in vivo activity. This view gains further support from the finding that the in vivo activity of TF on skin reactivity is often not correlated to a simultaneous increase in the recipient's blast transformation index (Arala-Chaves et al., 1974; Fudenberg etal., 1974; Kirkpatrick, Rich & Smith, 1972; Krohn et al., 1976). Similar results have been obtained with the guinea-pig model for TF (Vanderbarck, Burger & Vetto, 1977; Welch et al., 1976). Correspondence: Dr K. Krohn, Institute of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland.
0099-9104/79/0070-0050$02.00 0 1979 Blackwell Scientific Publications
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Transfer factor and LIF secretion
51
On the other hand, methods which measure the capacity of antigen-activated lymphocytes to secrete mediators of cellular immunity, such as the leucocyte migration inhibitory factor (LIF), seem to correlate better with skin test reactions in man and in the guinea-pig (Astor et al., 1973; Clausen, 1971; Hoffman, Spitler & Hsu, 1976). It is therefore important to know whether the observed in vitro inducing or potentiating activity of human TF on LIF secretion (Goust et al., 1976; Morison, 1976; Pizza et al., 1976; Read, Espinoza & Zabriskie, 1976; Wilson et al., 1977) is also connected to the contents of L-serine and glycine in TF, or whether this activity could be more closely related to TF's in vivo effect. In this paper we describe the effect of human TF on LIF secretion induced with an antigen, tuberculin (PPD), or a non-specific mitogen, leucoagglutinin (LA), and compare this effect of TF to that caused by Lserine and glycine. MATERIALS AND METHODS Transfer factor preparation. Human leucocyte dialysate was derived from a pool of 401 citrated buffy coats obtained from the Finnish Red Cross Blood Transfusion Service. For the preparation of dialysate, dextran sedimentation, freezing and thawing of the washed leucocytes, ultrafiltration of the cell lysate, and lyophilization of the ultrafiltrate were done as described previously (Krohn et al., 1977). Although the immune reactivities of the donors were unknown, the TF preparation can be considered PPD-positive, since all children in Finland have been vaccinated with BCG since the early 1940's. The preparation has also been shown to be active in the guinea-pig model (Krohn et al., 1979), as described by Vanderbarck et al. (1977) and Welch et al. (1976). Similarly prepared TF preparations from human buffy coat cells have also been shown to have a therapeutic effect in various clinical conditions (Krohn et al., 1977). Amino acid analysis. Analysis of the free amino acids in the horse serum was performed with a Beckman Multichrom M Amino Acid Analyzer as described previously (Uotila et al., 1978). The free amino acid content of the leucocyte dialysate used in the present study has been published previously (Uotila et al., 1978). Cell cultures. Mononuclear cells, containing an average of 85% lymphocytes, were isolated from citrated buffy coat cells (for mitogen-stimulated cultures), or from a PPD-positive donor (for antigen-stimulated cultures) on a Ficoll-Isopaque gradient (Boyum, 1968), and were suspended at a concentration of 106 cells/ml (antigen-stimulated cultures) or 2 x 106 cells/ml (mitogen-stimulated cultures) in MEM for suspension cultures (MEM-S) or RPMI 1640 (Flow Laboratories, Irvine, Scotland). The medium was supplemented with either 10% horse serum (Flow) dialysed for 36 hr with three changes, against MEM-S, or with 10% non-dialysed horse serum. Duplicate cultures containing 0-2 ml (antigen-stimulated cultures) or 0-1 ml (mitogen-stimulated cultures) of cell suspension per well were set up in U-bottomed microplates (Sterilin Ltd, Middlesex, UK). The cells were incubated with the following test substances: transfer factor in serial four-fold dilutions ranging from5OOOmg/l(4X*) to 49 mg/l (X/256) dry weight, and equivalent concentrations of amino acids found to be present in the trasfer factor preparation but not in MEM-S. The antigen, tuberculin (PPD), State Serum Institute, Denmark) and the mitogen, leucoagglutinin (LA, Pharmacia Fine Chemicals, Uppsala, Sweden), were used as stimulants. The antigen-stimulated cultures were incubated at 37°C in a 5% CO2 atmosphere for 6 days, in the presence of 12-5 mg/l of PPD. Immediately before collecting the supernatants, the control cultures were reconstituted with PPD. The mitogen-stimulated cultures were given a 30 min LA-pulse (30 mg/l), after which the cells were washed five times with Hanks's balanced salt solution (HBSS, Flow) to remove the unbound LA (Rasanen, Karhumaki & Krohn, 1978). The cultures were then reconstituted with the previous medium, serum and test substances, and were incubated at 37°C in a 5% CO2 atmosphere for 3 days before collecting the supernatants. Appropriate controls contained no addition, LA alone, or the corresponding amounts of test substances alone. All the supernatants were stored at - 80°C if not immediately tested. Leucocyte migration inhibitory factor assay. LIF activity in the lymphocyte culture supernatants was tested by the agarose migration method (Clausen, 1972) with minor modifications. Briefly, citrated or heparinized blood was sedimentated with dextran and the leucocyte-rich plasma was centrifuged on Ficoll-Isopaque. Leucocytes at the bottom of the tube, containing an average of 98% granulocytes and 2% mononuclear cells, were washed three times with HBSS before being used as indicator cells in the assay. The amount of erythrocytes varied, but as this did not seem to affect the test system, they were not lysed. The agarose plates prepared in RPMI 1640 with 10% horse serum were buffered with 5% sodium bicarbonate. Wells of 2-3 mm diameter were punched in the gel. The non-dialysed supernatants, supernatants dialysed for 36 hr, with three changes, against MEM-S and serial two-fold dilutions in MEM-S of the non-dialysed supernatants from the lymphocyte cultures were pre-incubated at 37°C for 30 min with the isolated granulocytes, after which 5,u1 of the suspension, containing 5 x 105 granulocytes, was pipetted into each well. The supernatants were tested with four replicate determinations. The plates were incubated at 37°C in a 5% CO2 atmosphere for 18 hr. The area of the well was excluded from the final area of migration. Means and standard deviations were calculated. The experimental approach. In the LIF assay two types of experiments were designed. In the first set, augmentation of LIF secretion, produced by various concentrations of the test substances, was determined by testing the effect of undiluted lymphocyte culture supernatants on granulocyte migration. The control migration inhibition percentage (MI%), test migration inhibition percentage (MIt%) and the augmenting effect of the test substance (AMI%) were calculated as follows: * X is the starting solution which is equivalent to 1-25 g/l.
R. G. I. Ashorn et al.
52 MI~%
=
area of migration+ s.d. with LA alone ) (1IIXmean 100 mean area of migration with no addition
( mean area of migration+ s.d. with LA+test substanceX -Ix 100 MI,% = I1 mean area of migration with test substance alone
AMI% = MIt%-MI'% The significance of the augmenting effect was determined by Student's t-test. With this test system, migration inhibitions close to maximum were often observed when testing supernatants of mitogenstimulated cultures, and so the amount of LIF in the culture supernatants was quantified more accurately by titration in the second set of experiments. Serial two-fold dilutions up to 1/128 were prepared from the culture supernatants containing an optimal concentration of TF with the highest AMI% values obtained in the first set of experiments. The culture supernatants containing L-serine, glycine, or L-serine+glycine at concentrations corresponding to that found in TF, or containing no test substance, were similarly diluted. Migration inhibitions were calculated, and the dilutions corresponding to 40% inhibition were determined by linear regression analysis of the migration inhibition values ranging from 15 to 65%.
RESULTS Preliminary experiments were carried out to determine the concentration of LA, where the effect of TF would be maximal. Lymphocytes were cultured in MEM-S, supplemented with 10% non-dialysed horse serum with or without different concentrations of TF or L-serine and/or glycine. The cultures were pulsed with various concentrations of LA. In these experiments, TF in concentrations ranging from 4X to X/256 augmented LIF secretion and the strongest augmentation was seen at an LA concentration of 30 mg/l. The optimal dose for TF varied between X and X/64, being most often X/16 (0.078 g/l). Dialysing the culture supernatants against MEM-S had no effect on their LIF activities (data not shown). Similar augmentations of LA responses were seen when the cultures in MEM-S were supplemented with L-serine or glycine at concentrations present in RPMI 1640 (Table 1). On the other hand, when the lymphocytes were cultured in RPMI 1640, TF had no augmenting effect on LIF production (Table 1). TABLE 1. The effect of transfer factor, L-serine and glycine on LA-induced LIF secretion in different culture media
Culture medium MEM-S+ 10% nondialysed horse serum RPMI 1640+10% nondialysed horse serum
LA alone
LA with TF (X/16*)
LA with serine (286 pmol/lt)
55+4+
71+3
76+3
72+1
75+2
80+ 2
74+ 1
n.t.§
n.t.§
A.5
LA with glycine LA with serine+glycine (286+133 ,umol/l¶) (133 ,pmol/lt)
* X = 1250 mg/l (dry weight). t Concentration present in RPMI 1640. + Migration inhibition percentage+ s.d. § Not tested.
Calculation of the concentrations of L-serine and glycine from the amino acid analysis of the leucocyte dialysate preparation used in this study showed that the amount of these two amino acids in TF at X/16 concentration was considerably lower than in RPMI 1640 (Table 2). Experiments were therefore devised where the effect of TF on LIF secretion was compared to that obtained with equivalent concentrations of the two amino acids, either alone or in combination. The results in Fig. 1 indicate that X/16 and higher dilutions of TF augmented LIF secretion as effectively as equivalent amounts of Lserine and glycine in combination. L-serine alone gave a somewhat lower response. With higher concentrations (X/8-4X) TF inhibited LIF secretion, the suppression effect being most pronounced with the highest concentration used (4X). Both L-serine alone and the combination of L-serine and glycine augmented LIF secretion, and with concentrations equivalent to X/4-4X of TF, the combination of the
Transfer factor and LIF secretion
53
20 ./
E-50
i
-60
-70
-80 _ X/4 X X/256 X/64 X/16 Dilution of test substances
4X
FIG. 1. Augmentation (AMI% > 0) or suppression (AMI%< 0) of leucocyte inhibition factor (LIF) mediated o) and ), L-serine (o granulocyte migration inhibition by serial dilutions of transfer factor (a . A). X 1-25 g/l ofleucocyte dialysate or equivalent amounts of the test amino acids L-serine+glycine (A (304 pmol/l for L-serine and 336 pmol/l for glycine). Vertical bars represent standard deviations of four replicate migration tests. - --
- --
=
amino acids gave significantly stronger augmentation than L-serine alone (P< 0001). The effect of glycine alone paralleled that of L-serine, but was usually somewhat weaker. In this experiment, as in most similar experiments, LA alone gave a rather strong migration inhibition (MI,,% > 60%) and thus the effect of the test substances could result only in a relatively small AMI%. Therefore, experiments were constructed where the amount of LIF in various lymphocyte culture supernatants was assessed by titration. An example of such an experiment is shown in Fig. 2, where the supernatants from lymphocyte cultures with TF concentration X/16 or equivalent amounts of L-serine,
two
TABLE 2. The concentrations of amino acids present in transfer factor and the complete culture, media, but not in MEM-S RPMI 1640+ MEM-S+ Transfer factor* 10% horse serum 10% horse serum
L-Serine Glycine L-Alanine L-Cysteine L-Proline L-Glutamic acid L-Aspartic acid Taurine o-Phospho-i.-serine
19t 21 26 2 14 20 9 21
16 31 15
302 164 15
5 16 3 4
180 152 153 4
1
Corresponds to a concentration of X/16 (X = 1250 mg/i). t Expressed as umol/l. *
54
R. G. I. Ashorn et al.
0
e40
-
-
-
=60
80
;.!:.
1/1 1/2 1/4 1/8 1/16 1/32 1/641/128 Dilution of the culture supernatonts
FIG. 2. Migration inhibition percentage caused by serial dilutions of supernatants from lymphocyte cultures in U) or together with X/16 MEM-S and 10% undialysed horse serum, containing LA alone (30 mg/l; concentration (0-078 g/l) of transfer factor ( . 0) or equivalent amounts of L-serine (19 pmol/l; o c) or L-serine+glycine (19+21 umol/l; A - - - A). The dilutions giving 40% migration inhibition, calculated by linear regression analysis of inhibition values ranging from 15 to 65% (*not included) were 1/6 for the control (r = 0 98), 1/10 for L-serine (r = 0 93), 1/20 for L-serine+glycine (r = 0.98) and 1/24 for transfer factor (r = 093). Vertical bars represent standard deviations of four replicate migration tests.
0 _
20 -1
o
60
-
80~~~~~I ~
1/1 1/2 1/4 1/8 1/16 1/32 1/641/128 Dilution of the culture supernotants
FIG. 3. Migration inhibition percentage caused by serial dilutions of supernatant from lymphocytes cultures in MEM-S and 10% dialysed horse serum, containing LA alone (30 mg/l; * *) or together with X/16 concentration (0-078 g/l) of transfer factor ( -.) or equivalent amounts of L-serine (19 pmol/l; o - - - o) *) or L-serine+glycine (19+21 pmol/l; A - - - A). The dilutions giving 40% glycine (21 jumol/l; * migration inhibition, calculated by linear regression analysis of inhibition values ranging from 15 to 65% (*not included) were 1/2 for L-serine (r = 0-96) 1/1 for glycine (r = 0 98), 1/42 for L-serine4-glycine (r = 0 96) and 1/39 for transfer factor (r = 0.95). Vertical bars represent standard deviations of four replicate migration tests.
Transfer factor and LIF secretion
55
glycine or their combination were tested. The results indicate that the combination of L-serime and glycine once again augments LIF secretion as much as TF, and that the augmentation is about twice as strong as that caused by L-serine or glycine alone. All these experiments were performed with 10% non-dialysed serum which already contained L-serine and glycine. In experiments where only one of these two amino acids was added to the lymphocyte cultures, both amino acids were in fact present, and the effect on LIF synthesis was thus always a combination effect. The experiments shown in Fig. 2 were therefore repeated using dialysed horse serum, where the absence of glycine and L-serine was checked by amino acid analysis. The results of the experiments are shown in Fig. 3. Without addition of the test substances and in the complete absence of Lserine or glycine, LA caused hardly any detectable LIF secretion. The addition ofTF or the combination of L-serine and glycine caused similar LIF secretion, which was twenty and forty times higher than that caused by L-serine or glycine alone, as calculated from the 4000 migration inhibition values. These results show, therefore, that a synergistic action of L-serine and glycine was necessary for maximal LIF secretion, and that the effect of TF was as strong, but not stronger, than that of the two amino acids. The other amino acids which were found in the dialysate, but not in MEM-S (Table 2), had no effect on LIF synthesis. Finally, experiments were designed where the effects of TF, L-serine and glycine were tested on PPDinduced LIF secretion, using mononuclear cells derived from a PPD-positive donor, or from human cord blood. Stimulation of the cord blood cells with PPD caused no LIF secretion in the absence or presence of the test substances (data not shown). The results obtained with PPD-positive mononuclear cells, shown in Table 3, indicate that the effect of the test substances on antigen-induced LIF secretion is similar to that on mitogen-induced LIF secretion. TABLE 3. The effect of transfer factor, L-serine, and glycine on PPD-induced LIF secretion in different culture media PPD alone
Culture medium
PPD with TF
PPD with glycine PPD with serine+glycine (21 pmol/lt) (19+21 pmol/lt)
(X/16*)
PPD with serine (19 ,pmol/lt)
42+14§
34+16
12+4
37+8§
33 + 4§
28+ 3
12+11
30+ 0§
34+4
33+1
40+3
40+5
MEM-S+ 10% dialysed horse serum 9+7+ MEM-S+ 10% nondialysed horse serum 18+10 RPMI 1640+10% nondialysed horse serum 41+8 *
X
=
1250 mg/i (dry weight).
t Concentration equivalent to X/16 of TF. Migration inhibition percentage+ s.d. § Differs significantly (Student's t-test, P< 0 01) from the corresponding value of PPD alone.
+
DISCUSSION The results obtained in this study indicate that TF has a non-specific augmenting effect on antigen- and mitogen-stimulated LIF secretion, and that this effect is mainly due to the L-serine and glycine content of the leucocyte dialysate. The conclusion that TF, L-serine and glycine augment migration inhibition mainly by stimulating LIF secretion and not by increasing granulocyte responsiveness to LIF, can be drawn from the fact that depleting the culture supernatants of these test substances by dialysation had no effect on the LIF activity. We can deduce that this augmenting activity was due to the presence of Lserine and glycine from the following facts: (1) hardly any LIF secretion occurred when dialysed serum without TF or amino acid addition was used (Fig. 3, Table 3); (2) TF or L-serine, glycine and their combination augmented LIF secretion when experiments were carried out in medium which did not
56
R. G. I Ashorn et al.
itself contain these amino acids (MEM-S); and (3) the addition of TF in the test system had no effect when the redium used already contained a nearly optimal concentration of L-serine and glycine (RPMI 1640). In fact, in RPMI 1640, the addition of TF had an inhibitory rather than a stimulatory effect on LIF secretion. This finding may be due to the presence of substances in the leucocyte dialysate that suppress lymphocyte function-such a substance has previously been identified as nicotinamide (Burger et al., 1976b), but additional inhibitory substances may exist. The inhibitory effect of TF was especially strong when it was used in high concentrations (4X = 5 g/l); the optimum dose for augmentation of LIF synthesis was X/16, whereas L-serine and glycine concentrations corresponding to 4X of TF gave the highest augmenting effect (Fig. 1). Dilution of the lymphocyte culture supernatants cultured with either X/16 concentration of TE or with the equivalent L-serine+ glycine concentrations showed, however, that an equal amount of LIF was secreted in the presence of TF or these two amino acids (Fig. 2). Experiments carried out in dialysed serum, presented in Fig. 3, indicate that the effect of glycine and Lserine on LIF secretion is synergistic and not purely additive. This synergistic effect of the two amino acids may explain why with non-dialysed serum even the addition of one of the amino acids, at a low concentration, seemed to cause an effect equal to that obtained with TF or the combination of the two amino acids. Our results are thus in agreement with previous findings that lymphocytes are defective in synthesizing L-serine, and further suggest that the hydroxymethyltransferase reaction is insufficient for the interconversion of the two amino acids. It has been shown previously that L-serine and glycine are necessary for optimal lymphocyte DNA, RNA and protein synthesis (Dubrow, Pizer & Brody, 1973; Pizer & Regan, 1972; Regan et al., 1969; Uotila et al., 1978). Our present finding is the first to demonstrate a defect in the synthesis of a specific protein, LIF, in the absence of L-serine and/or glycine. According to some workers, LIF might exert its effect by virtue of being a serine protease or esterase (Bendtzen, 1977; Rocklin & Rosenthal, 1977). Thus, the necessity of L-serine in the catalytic site of the putative LIF enzyme would further contribute to the requirement of this amino acid. The present findings support the view that the non-specific augmenting in vitro effect of transfer factor on LIF secretion is mainly due to its L-serine and glycine content. It is obvious that L-serine and glycine cannot be responsible for TF's in vivo activity as these amino acids are normally present in human blood and their presence in TF is too slight to cause any significant increase in the plasma amino acid level. However, since a reliable in vitro test for TF, correlating with its in vivo activity, would be of great importance in the chemical characterization of TF, it is necessary to analyse carefully those in vitro activities of TF which are not related to its in vivo effect, as these non-specific activities may effectively conceal the more important specific ones. This work has been supported with grants from the Sigrid Juselius Foundation, Finnish Cultural Foundation, Cancer Foundation in Finland and Orion Pharmaceuticals. The skilled assistance of Mrs Eliisa Karhumaki, M.Sc., Mrs Eila Pohiola, Mrs Tuula Myllymaki, Mrs Raija Keskivali, Miss Eija Kyrola and Mr P. Virtanen is gratefully acknowledged. REFERENCES & VETTO, R.M. (1976a) Human transfer factor: effects on lymphocyte transformation. 5. Immunol. 117, 782. BURGER, D.R., VANDERBARCK, A.A., DAVEY, D., ANDERSON, W.A., VETTO, R.M. & FiNKE, P. (1976b) Human transfer factor: fractionation and biologic activity. 5. Immunol. 117, 789. CLAUSEN, J.E. (1971) Tuberculin induced migration inhibition of human peripheral leucocytes in agarose medium. Acta Allergol. 26, 56. CLAUSEN, J.E. (1972) Migration inhibitory effect of cell-free supernatants from mixed human lymphocyte cultures. ]. Immunol. 108, 453. DUBROW, R., PIZER, L.I. & BRODY, J.I. (1973) Serineglycine requirement of phytohaemagglutinin stimulated lymphocytes. ]. Nat. Cancer Inst. 51, 307.
ARALA-CHAVES, M.P. PROENCA, R. & DE SOUSA, M. (1974) Transfer factor therapy in a case of complex immunodeficiency. Cell. Immunol. 10, 371. ASTOR S.H. SPITLER L.E. FRICK O.L. & FUDENBERG, H.H. (1973) Human leucocyte migration inhibition in agarose using four antigens: Correlation with skin test reactivity. ]. Immunol. 110, 1174. BENDT2EN. K. (1977) Human leucocyte inhibitory factor (LIF). III. Further investigations on the serine protease nature of this lymphocine and its preference for arginine amides. Scand. J. Immunol. 6, 1055. BO6UM, A. (1968) Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, Suppl. 97, 77. BURGER, D.R., VANDERBARCK, A.A., FiNKE, P., NOLTE, J.E.
Transferfactor and LIF secretion FUDENBERG, H.H., LEVIN, A.S., SpiTuR, L.E., WYBRAN, J. & BYrERs, V. (1974) The therapeutic uses of transfer factor. Hosp. Pract. 9, 95. GOUST, J.M., MoULIAS, R., PHILIPE, R. & FUDENBERG, H.H. (1976) In vitro assay for transfer factor by means of leucocyte migration inhibition test (LMT). Transfer factor, basic properties and clinical applications (ed. by M.S. Archer, A.A. Gottlieb & C.H. Kirkpatrick), p. 137. Academic Press, New York. HOFFMAN, P.M., SPITLER, L.E. & Hsu, M. (1976) Leucocyte-migration inhibition in guinea-pigs. I. Correlation with skin test reactivity and macrophage-migration inhibition. Cell. Immunol. 21, 358. KIRKPATRICK, C.H., RICH, R.R. & SMITH, T.K. (1972) Effect of transfer factor on lymphocyte function in anergic patients. ]. cdin. Invest. 51, 2948. KROHN, K., GROHN, P., HoRsmAN-EIMO, M. & VIROLAINEN, M. (1976) Fractionation studies on human leucocyte dialysates. Demonstration of three components with transfer factor activity. Med. Biol. 54, 384. KROHN, K., UOTILA, A., VAISANEN, J. & GROHN, P. (1977) Studies on the chemical composition and biological properties of transfer factor. Z. Immun.-Forsch. 153, 395. KROHN, K., VOTILA, A. ASHORN, R. & KARHUMAmI, E. (1979) Augmentation of skin reactivities in antigen primed guinea pigs by transfer factor and other cellular dialysates. Immune regulators in transfer factor (ed. by A. Khan, C.H. Kirkpatrick and N.O. Hill). Academic Press, New York. LAWRENCE, H.S. (1969) Transfer factor. Adv. Immunol. 11, 195. LAWRENCE, H.S. (1974) Transfer factor in cellular immunity. Harvey Lec . 68, 239. LITTMAN, B.H., HIRSCHMAN, E.M. & DAVID, J.R. (1977) Augmentation of 3H-thymidine incorporation by lymphocytes in the presence of antigen and fractions of dialyzable transfer factor: A non-specific phenomenon. Cell. Immunol. 28, 158. MORISON, W.L. (1976) Phytohaemagglutinin and transfer factor in the leucocyte migration inhibition test in patients with sarcoidosis. Thorax, 31, 87. PIZER, L.I. & REGAN, J.D. (1972) Basis for the serine requirements in leucemic and normal human lymphocytes. Reduced levels of the enzymes in the phosphorylated pathway. ]. Nat. Cancer Ins. 48, 1897. PIZZA, G., VIZA, D., BOUCcHEIX, C.I. & CORRADO, F. (1976) Studies with in vitro produced transfer factor. Transfer factor, basic properties and clinical applications (ed. by M.S.
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Ascher, A.A. Gottlieb & C.H. Kirkpatrick), p. 173. Academic Press, New York. RASANEN, L., KARHUMAKI, E. & KROHN, K. (1978) Elaboration of leucocyte migration inhibitory factor by human lymphocyte subpopulations stimulated with mitogens. Cell. Immunol. 37, 221. READ, S.E., EsPINozA, L.R. & ZABRISKIE, J.B. (1976) In vitro assay for transfer factor using direct migration inhibition. Transfer factor, basic properties and clinical applications (ed. by M.S. Ascher, A.A. Gottlieb & C.H. Kirkpatrick), p. 129. Academic Press, New York. REGAN, J.D., VoDopIicK, H., TAKEDA, S., LEE, W.H. & FAULCON, F.M. (1969) Serine requirement in leucemic and normal blood cells. Science, 163, 1452. ROCKLIN, R.E. & ROSENTHAL, A.S. (1977) Evidence that human leucocyte inhibitory factor (LIF) is an esterase. J. Immunol. 119, 249. SPITLER, L.E., LEVIN, A.S. & FUDENBERG, H.H. (1973) Human lymphocyte transfer factor. Methods Cancer Res. 8, 59. UOTILA, A., MARNELA, K.-M., HAMBLIN, A. & KROHN, K. (1978) Activity of human transfer factor in vitro. Identification of L-serine and glycine as components with augmenting effect in the lymphocyte transformation assay. Scand. J. Immunol. 7, 495. VALDIMAaSSON, H., HAMBLETON, G., HENRY, K. & MCCONNEL, J. (1974) Restoration of T-lymphocyte deficiency with dialyzable leucocycte extract. Clin. exp. Immunol. 16, 141. VANDERBARCK, A.A., BURGER, D.R. & VETTO, R.M. (1977) Human transfer factor activity in the guinea-pig: Absence of antigen specificity. Clin. Immunol. Immunopathol. 8, 7. WAITHE, W.I., DAUPHINAIS, C., HATHAWAY, P. & HIRSCHORN, K. (1975) Protein synthesis in stimulated lymphocytes. II. Amino acid requirements. Cell. Immunol. 17, 323. WELCH, T.M., TRIGLIA, R., SPITLER, L.E. & FUDENBERG, H.H. (1976) Preliminary studies on human 'Transfer factor' activity in guinea-pigs. Systemic transfer of cutaneous delayed type hypersensitivity to PPD and SK-SD. Clin. Immunol. Immunopath. 5, 407. WILSON, G.B., WELCH, T.M., KNAPP, D.R., HORSMANHEIMO, A. & FUDENBERG, H.H. (1977) Characterization of Tx, an active subfraction of human dialyzable transfer factor. I. Identification of the major component in TFg, a precursor of Tx, as hypoxanthine. Clin. Immunol. Immunopathol. 8, 55 1.
