Lymphocyte LIISA Institute
This paper describes experiments to determine whether human lymphocyte subpopulations stimulated with a variety of mitogens, leucoagglutinin (LA), concanavalin A (con A), pokeweed mitogen (PWM), protein A (prot A), and [email protected]
(anti-&m), synthesize lymphokines. T and B lymphocytes as well as unseparated mononuclear cells were stimulated with the mitogens, and the presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by an agarose migration method. Culture supernatants stimulated with LA or prot A were also fractionated on Sephadex G-100 columns, and LIF-containing fractions were tested for heat stability and the effect of monosaccharides. The results indicated that LA and con A caused LIF synthesis only in T-cell populations, while PWM stimulated both T and B lymphocytes and prot A and anti-&m were B-cell stimulants. Furthermore, LIF from LA-and prot-A-stimulated cultures behaved similarly upon physicochemical characterization.
INTRODUCTION Since the discovery that phytohemagglutinin (PHA) can induce blast transformation in lymphocytes (l), knowledge of mitogens has greatly increased. At present a wide variety of mitogenic substances are known, and their target cells and the mechanism of their action are under extensive study. In this investigation the following mitogens were used: leucoagglutinin (LA), concanavalin A (con A), pokeweed mitogen (PWM), protein A (prot A), and anti-pz-microglobulin (anti-&m). LA is obtained from the same plant as PHA (Phaseolus vulgaris) but is a pure product without erythroagglutinating properties (2, 3). Con A and PWM are well-known lymphocyte stimulants (4). Prot A from Staphylococcus aUrevS (5, 6) and anti-&m (7, 8) have recently been reported to be B-cell mitogens and polyclonal activators of antibody synthesis. As far as we know no report exists on lymphokine synthesis induced by LA, prot A, or anti&m. As lymphokine synthesis and other phenomena of lymphocyte activation are independent processes (9, lo), it was of interest to study whether these mitogens together with con A and PWM stimulated lymphocytes to synthesize lymphokines and if so whether they acted on T or B lymphocytes. In order to study whether leukocyte inhibitory factors (LIF) produced by cells stimulated with a T- or a B-cell mitogen were similar, lymphocyte culture supernatants were fractionated and LIF-containing fractions were characterized. 221
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The present paper will demonstrate that the mitogens, except for PWM, activate either T or B cells to produce LIF and that LIFs from LA- or prot-Astimulated cultures behave similarly upon physicochemical characterization. MATERIALS
Sepamtion of T and B lymphocytes. Mononuclear cells from healthy human individuals were obtained by Ficoll-Isopaque centrifugation of heparinized or &rated peripheral blood (11). T and B cells were purified as follows. The cells were incubated with 200 mg of carbonyl iron (Fluka AG, Buchs, Switzerland) in Hanks’ balanced salt solution (HBSS) supplemented with 10% fetal bovine serum (Flow Laboratories, Irvine, Scotland) for 45 min at 37°C after which free iron and the phagocytic cells were removed with a magnet. SRBC were treated with 50 U/ml of neuraminidase (Vibrio comma neuraminidase, Behringwerke, Marburg, Germany) by incubating 20% SRBC in HBSS at 37°C for 30 min, after which the SRBC were washed three times before use in rosette sedimentation. Solutions of 2 x lo7 lymphocytes, 0.5 ml of fetal bovine serum (absorbed twice with SRBC), 0.5 ml of HBSS, and 1.0 ml of 16% SRBC were mixed in polystyrene tubes (Falcon, Oxnard, Calif., U.S.A.) and centrifuged for 5 min at 200g and then incubated on ice for 30 min. Rosetted cells were gently resuspended and layered on Ficoll-Isopaque. Ficoll (14%) was used in preparing these FicollIsopaque solutions (12). The tubes were centrifuged for 20 min at lOOOg, and B-rich cells were collected from the interface and subjected to another rosetting Ficoll-Isopaque cycle. The T-cell-SRBC pellet was suspended in 1 ml of HBSS, and lysis of the SRBC was effected by hypotonic shock with 3 ml of distilled water for 10 sec. Finally the nonseparated mononuclear cells and T and B lymphocytes were washed three times with HBSS before culture. Cell cultures. The cells were suspended at a concentration of 2 X lo6 cells/ml in MEM for suspension cultures (MEM-S, Flow Laboratories) containing 10% horse serum (Flow Laboratories). Duplicate cultures containing 0.1 ml of cell suspension/well were set up in U-bottomed microplates (Sterilin Ltd., Middlesex, England). The cells were incubated with various concentrations of LA (Pharmacia Fine Chemicals, Uppsala, Sweden), con A (Pharmacia Fine Chemicals), PWM (Grand Island Biological Co., Grand Island, N.Y., U.S.A.), prot A (Pharmacia Fine Chemicals), or with medium alone (control cultures). This incubation was a 30-min pulse, after which the cells were washed five times with HBSS to remove the unbound mitogen. In order to check whether residual mitogen was present in the cultures, the protein synthesis inhibitor puromycin (Sigma, St. Louis, MO., U.S.A.) was used as follows. Cells were pulse-treated with the largest concentration of mitogens used and cultured in the presence of 5 pg/ml of puromycin. Previous experiments demonstrated that lymphocyte pulse treatment with anti-/&m [dialyzed overnight against phosphate-buffered saline (PBS), Dakopatts A/S, Copenhagen, Denmark] gave very poor production of LIF. Therefore anti-&m was present during the whole culture period. In order to eliminate the direct migration-inhibitory action of anti-&m, the control cultures were reconstituted with corresponding amounts of this mitogen before being assayed, All cultures were incubated at 37°C in a 5% CO, atmosphere for 3 days unless otherwise stated. Cell markers. Monocytes were identified by nonspecific esterase activity according to Yam et al. (13). Identification of T cells was by SRBC rosette formation.
Briefly, lo6 mononuclear cells or purified T or B lymphocytes were mixed with 0.5 ml of HBSS, 0.1 ml of 470 SRBC, and 0.6 ml of 12% Ficoll (14), and the mixture was centrifuged for 5 min at 200g and then incubated on ice for 30 min. B lymphocytes were identified by readily detectable surface membrane immunoglobulin. A total of lo8 cells were spun down, and 50 ~1 of a 1: 8 dilution of FITCconjugated anti-immunoglobulin (IgG + IgA + IgM, Behringwerke) was added to the cell pellet. After incubation on ice for 30 min, the cells were washed three times with PBS containing 0.1% sodium azide. One drop of 50% glycerol in PBS was added to the cell pellet, and the suspension was transferred to a microscope slide and the number of fluorescent cells counted with Ploem illumination. Characterization of leukocyte inhibitory factor. Supernatants for gel filtration chromatography were prepared as follows. Mononuclear cells were pulse treated with medium alone (control cultures), 40 pg/ml of LA, or 80 pg/ml of prot A, after which they were washed three times with HBSS. The cells were cultured in serum-free MEM-S in polystyrene tubes at a concentration of 4 X lo6 cells/ml for 2 days. Supernatants (30-80 ml) were dialyzed overnight against distilled water and lyophilized. The powder was dissolved in 2 ml of volatile buffer (0.05 N ammonium bicarbonate ; acetic acid, pH 7.2), clarified by centrifugation for 10 min at 10009, and applied to a 1.6 X 85-~111 column of Sephadex G-100. The column had been precalibrated with rabbit serum albumin (Sigma ; MW, 68,000) and cu-chymotrypsinogen A (Sigma ; MW, 23,000) by measuring the optical density of the eluate fractions at 280 nm. All elutions were done with a volatile buffer, and the eluate was pooled into four fractions. Fraction I contained the void volume and material eluting before the albumin marker, and fraction II contained molecules eluting in the same volume as albumin. The fraction between the albumin and the chymotrypsinogen markers was referred to as fraction III, and fraction IV contained material eluting in the same volume as the chymotrypsinogen marker. The fractions were lyophilized and dissolved in l/20 of the original volume of MEM-S supplemented with 10% horse serum. In order to study the thermostability of fractions II from control and stimulated cultures, the fractions were incubated for 30 min in 56 or 80°C water baths and then assayed. The effect of N-acetyl-D-glucosamine and L-fucose was studied as described by Rocklin (15) after dilution (1: 2-l : S) of the fractions. Leukocyte migration inhibitory factor assay. LIF activity was tested in the culture supernatants by the agarose migration method (16) with minor modifications. After sedimentation of blood, the leukocyte-rich plasma was centrifuged on Ficoll-Isopaque. Cells at the bottom of the tube were 9S% pure granulocytes and were washed three times before use as indicator cells in the assay. The agarose plates were buffered with 5% CO%-bicarbonate. Wells of 2.3-mm diameter were punched in the gel, and 5 ~1 of culture supernatant containing lo6 granulocytes was pipetted into each well. The supernatants were tested with four to six replicate determinations. The area of the well was excluded from the final area of migration. The migration index (MI) was calculated as follows : area of migration in the presence of test supernatant n#u= area of migration in the presence of control supernatant The method worked with standard deviations ranging from 0 to 18.0% (mean, 7.2%). Generally migration indices smaller than 0.85 represented significant inhibition of migration as calculated by Student’s t test.
RESULTS Stirnc~lation of Mononl~cleav Cells and Lymphocyte
Mononuclear cells obtained after Ficoll-Isopaque centrifugation contained an average of 69.7% (range S&76%) T lymphocytes, 19.370 (16-23s) B lymphocytes, and 11.1% (S-25%) monocytes. The separation method used yielded Tcell populations contaminated with an average of 2.1% (< 0.54%) B lymphocytes and less than 0.5% monocytes, and B-cell populations contaminated with 2.6% (l-5%) T cells and 3.0% (l-4%) monocytes. Figure 1 shows the mean results of mitogen-induced LIF synthesis in five to nine experiments. Mononuclear cells produced LIF with all five mitogens tested. Purified T cells were stimulated by LA and con A, whereas B lymphocytes responded to prot A and anti-&m. PWM caused elaboration of LIF in both Tand B-cell populations. Greater inhibition of migration was obtained with LA, prot A, and PWM than with con A and anti-&m. No inhibition of migration was .
PWM f f .
CONCENTRATION OF STIMULANTS activity in culture supernatants of unseparatedmononuclear cells (A-A), T lymphocytes (w-w), and B lymphocytes (O-O). The concentration of LA, con A, and prot A is expressed as micrograms per milliliter. The concentration of PWM and anti-&m FIG.
is expressed as final dilutions of the stock solution 10 ml of MEM-S) and the anti-serum, respectively. meanf SE.
(the contents of one vial were dissolved in pm = puromycin. The values represent the
1 I 34567
DAY OF CULTURE FIG. 2. Kinetics of LIF elaboration by mononuclear (A---A), T (a---H), and B 0) cells. The concentration of LA was 10 pg/ml for mononuclear cells, 0.6 fig/ml for (*T lymphocytes, and 40 Fg/ml for B lymphocytes. Con A was used at a concentration of 320 pg/ml. The dilution of PWM was 1 : 1280 for mononuclear cells, 1 : 320 for T cells, and 1 : 20 for B lymphocytes. Mononuclear and B cells were pulse-treated with 20 pg/ml of prot A, and T lymphocytes were treated with 80 pg/ml. Anti+m was used at a dilution of 1 : 4. The values represent the mean f SE.
seen in cultures pulse-treated puromycin (Fig. 1). Kinetics
mitogens and cultured
in the presence of
Figure 2 presents the mean results on kinetics in four to seven experiments. It was shown that the greatest LIF activity was induced with highest mitogen doses and that such strong stimulus would obscure changes in the kinetic response curve. Therefore in kinetic experiments suboptimal concentrations of mitogens shown in previous experiments to give a good response were used, otherwise the largest concentration tested was chosen. It is possible that even if T- or B-cell-rich populations had not produced measurable amounts of LIF during the 3-day culture, the activity of remaining contaminating lymphocytes might have been revealed after longer periods of culture. However, LA and con A caused no LIF synthesis in B-lymphocyte populations, nor did prot A and anti-&m activate T-lymphocyte cultures. The
Fractionation of LA- and Prot-A-Stimulated Culture Supernatants by Sephadex G-100 Chromatography Stimulant
Expt. No. I
0.97a 0.96 1.06
0.69 0.56 0.47
1.03 0.98 1.00
0.97 1.02 0.99
0.40 0.45 0.52
1.01 0.91 0.90
I 2 3
a Migration index.
maximum inhibition of migration occurred on Days 3, 4, or 5 with LA, con A, PWM and prot A. In contrast, anti-pzm gave the greatest inhibition of migration on Day 1. Physicochemical
Table 1 shows the results of fractionation of LA- and prot-A-stimulated culture supernatants by Sephadex G-100 chromatography. Leukocyte-inhibitory activity from both LA- and prot-A-stimulated cultures eluted mainly in fraction II, i.e., in the albumin region. Table 2 shows that heating the fractions for 30 min at 56 or 80°C resulted in loss of leukocyte-inhibitory activity. In three experiments heating at 56°C produced a slight loss of the inhibitory activity, while heating at 80°C totally abolished it. Table 2 also shows the effect of monosaccharides on TABLE
Effect of Heat and Monosaccharides on Leukocyte Inhibitory Stimulant
1 2 3
0.69. 0.56 0.47
0.79 0.59 0.59
1.01 0.94 0.99
n.d.* 0.63 0.66
n.d. 1.00 0.90
n.d. 0.62 0.71
0.40 0.45 0.52
0.52 0.62 0.67
1.00 0.98 1.01
0.60 0.73 0.74
0.93 0.99 0.94
0.63 0.72 0.70
Mean Prot A
Mean a Migration index. b Not done.
1 2 3
leukocyte-inhibitory activity. N-Acetyl-D-glucosamine the activity, while L-fucose had no effect.
DISCUSSION We have shown that mitogens LA, con A, PWM, prot A, and anti-&m activate lymphocytes to synthesize a lymphokine. LIF derived from LA- and prot-A-stimulated lymphocytes was also physicochemically characterized and no difference was found by the methods used. Migration inhibition phenomena should be treated with care because many factors, e.g., toxicity or an agglutinating effect of the stimulants or medium exhaustion, may cause inhibition of migration ( 17). To exclude the direct inhibitory action of the mitogens on indicator cell migration, we have pulse-treated the cells with mitogens (except for anti-&m). However, the possibility exists that during culture some of the cell-bound mitogen is released and on testing directly inhibits migration. The demonstration of complete lack of inhibition of migration in cultures containing puromycin, which in itself does not significantly affect granulocyte migration (18)) indicates that the inhibition of migration in pulse-treated cultures was not even in part due to residual mitogen. In fact, stimulation of migration was seen in mononuclear and B-cell populations treated with puromycin. This is due to spontaneous elaboration of LIF by B lymphocytes, which takes place in unstimulated control cultures (18) but is blocked together with the mitogeninduced LIF synthesis in puromycin cultures. The problem of residual mitogen becomes important in gel filtration experiments where large volumes of supernatants were concentrated and thus even small amounts of residual mitogens may have affected the results. However, as the molecular weight of LA has been reported to be 126,000-140,000 (2, 3) and that of prot A, 42,000 (19), these mitogens would have eluted mainly in fractions I and III, respectively. It is therefore evident that mitogens present in the concentrates would elute either before or after fraction II, which contained leukocyte inhibitory activity. Our finding that the molecular weight of LIF produced by LA- and prot-Astimulated cells lies close to that of albumin is compatible with Rocklin’s finding of the molecular weight of LIF produced by con A-stimulated cells (20). LIF activity has been reported to be blocked by N-acetyl-n-glucosamine ( 15) and to be abolished at 80°C (21). Our observations are compatible with these findings. Thus LIF from cells stimulated with a T- or a B-cell mitogen seems to behave similarly. Except for PWM, the mitogens used in this investigation activated either purified T of B lymphocytes to produce lymphokines. This cell specifity is compatible with earlier reports (4-7). The issue as to the target cells of PWM is controversial. Some investigators claim PWM to be both a T- and a B-cell stimulant (22, 23), while others favor the opinion that activation of B cells by PWM is T-cell dependent (24-27). Our B-cell populations contained an average of 2.6% contaminating T lymphocytes. On the basis of our results it cannot be deduced whether LIF production in B-cell populations was dependent on T-cell help. There seem to be differences between the stimulating capacity of various mitogens. Thus LA, PWM, and prot A are considerably stronger stimulants than con A and anti-&m. The stimulating patterns of the mitogens also differ from each other. Of the two T-cell stimulants, LA causes more LIF synthesis in T-cell populations than in unseparated mononuclear cells, while con A does the opposite.
This may reflect differences in helper cell requirements. LA-induced LIF synthesis may be more independent of monocyte help but with con A T lymphocytes may need the help of phagocytic cells or B lymphocytes. In this investigation we have confirmed earlier findings that lymphokine production is a common property of T and B lymphocytes (28-30). Lymphokines are generally connected with cell-mediated immunity. However, it is difficult to fit B-lymphocyte lymphokines, if present in V&O, into phenomena of cell-mediated immunity, and thus the concept of lymphokines as mediators of cellular immunity needs reevaluation. REFERENCES 1. Nowell, P. C., Cancer Res. 20, 462, 1960. 2. Weber, T. H., Aro, H., and Nordman, C. T., Biochim. Biophys. Acta 263, 94, 1972. 3. Risinen, V., Weber, T. H., and G&beck, R., Eur. J. Biochem. 38, 193, 1973. 4. Oppenheim, J. J., and Rosenstreich, D. L. (Eds.), “Mitogens in Immunobiology.” Academic Press, .New York, 1976. 5. Forsgren, A., Svedjelund, A., and Wigzell, H., Eur. J. Immunol. 6, 207, 1976. 6. Mijller, G., and Landwall, P., Stand. J. Immunol. 6, 357, 1977. 7. Ringden, O., and Mtiller, E., Stand. J. Zmmutzol. 4, 171, 1975. 8. Ringden, O., and Johansson, B. G., Stand. J. Immunol. 6, 281, 1977. 9. Wahl, S. M., Iverson, G. M., and Oppenheim, J. J., J. Exp. Med. 140, 1631, 1974. 10. Littman, B. H., David, J. R., and Rocklin, R. E., Cell. Zmmunol. 24, 241, 1976. 11. Biiyum, A., Stand. J. Cliw. Lab. Invest. 21, Suppl. 97, 77, 1968. 12. Parish, C. R., and Hayward, J. A., Proc. Rey. Sot. Ser. B. 187, 65, 1974. 13. Yam, L. T., Li, C. Y., and Crosby, W. H., Amer. J. Clis. Pathol. 55, 283, 1971. 14. Pattengale, P. K., and Reichelderfer, P. S, Zmmunol. Comnzun. 4, 179, 1975. 15. Rocklin, R. E., J. Immu~aol. 116, 816, 1976. 16. Clausen, J. E., J. Immunol. 108, 453, 1972. 17. Taylor, M. M., Burman, C. J., and Fantes, K. H., Cell. Immultol. 19, 41, 1975. 18. Arvilommi, H., and Risinen, L., Nature (Londofz) 257, 144, 1975. 19. BjGrk, I., Eur. J. Biochem. 29, 579, 1972. 20. Rocklin, R. E., J. Immunol. 114, 1161, 1975. 21. Bendtzen, K., Acta Pathol. Microbial. Scalzd. Sect. C 84, 471, 1976. 22. Greaves, &I., Janossy, G., and Doenhoff, M., J. Exp. Med. 140, 1, 1974. 23. Chess, L., MacDermott, R. P., and Schlossman, S. F., J. Ztnnmnol. 113, 1113, 1974. 24. Lohrmann, H.-P., Novikovs, L., and Graw, R. G., I. Exp. Med. 139, 1553, 1974. 25. Brochier, J., Samarut, C., Gueho, J. P., and Revillard, J. P., Znt. Arch. Allergy 51, 101, 1976. 26. Keightley, R. G., Cooper, M, D., and Lawton, A. R., J. Immmzol. 117, 1538, 1976. 27. Janossy, G., de la Concha, E. G., Luquetti, A., Snajdr, M. J., Waxdal, M. J., and PlattsMills, T. A. E., Stand. J. Zmmunol. 6, 109, 1977. 28. Yoshida, T., Sonozaki, H., and Cohen, S., J. Exp. Med. 138, 784, 1973. 29. Rocklin, R. E., MacDermott, R. P., Chess, L., Schlossman, S. F., and David, J. R., J. Exp. Med. 140, 1303, 1974. 30. Mackler, B. F., Altman, L. C., Rosenstreich, D. L., and Oppenheim, J. J., Nature (London) 249, 834, 1974.