Role of various carrageenans in autologous and allogeneic mixed lymphocyte reaction

CELLULAR

IMMUNOLOGY

68,402-409

(1982)

SHORT COMMUNICATION Role of Various Carrageenans in Autologous Mixed Lymphocyte Reaction

and Allogeneic

ISAMU SUGAWARAAND SHIGEAKI ISHIZAKA Department of Immunobiology, Karolinska Institute, Wallenberg Laboratory, Lilla Frescati, S-104 05, Stockholm, Sweden Received October 27. 1981: accepted February 11. 1982 Carrageenans (CGNs) are extracted from cell walls of certain algae of the Rhodophyta and are gel-forming polysaccharides. There are three kinds of purified carrageenans availablekappa, lambda, and iota CGN. All of them stimulated responding T lymphocytes in both autologous and allogeneic mixed lymphocyte reaction, lambda CGN being the most effective. Silica particles abrogated proliferation of responding cells in autologous and allogeneic mixed lymphocyte reaction by killing human monocytes. However, when 1 mg/ml of CGN was added to silica-added assay system, CGN could induce proliferation of responding T cells even in the absence of monocytes. It was concluded that CGN was a monocyte-independent Tlymphocyte mitogen that was also toxic for human monocytes.

INTRODUCTION Carrageenans, a mixture of sulfated D-galactose and 3,6-dehydro-D-galactose, have various biological and immunological properties (Fig. 1) ( 1). They are used as food additives and contribute to the stickiness of food (2). They have also been considered to be macrophage-toxic agents, but they are not toxic to lymphocytes (3). We have previously reported that kappa CGN’ was a mitogen for human lymphocytes and activated T lymphocytes. Lambda and iota CGN also activated human T cells and caused an increased thymidine uptake in B cells, but did not induce polyclonal and antibody synthesis.* It has been shown that CGNs are toxic to human monocytes.3 We have now investigated the effect of CGNs in autologous and allogeneic mixed lymphocyte reaction (MLR). We found that CGNs stimulated responding T cells in both autologous and allogeneic mixed lymphocyte reactions and that CGNs did so without monocyte help. ’ Abbreviations used: CGN, carrageenan; MNC, mononuclear cells; FCS, fetal calf serum; PHA, phytohemagglutinin; Con A, concanavalin A, MLR, mixed lymphocyte reaction. * Sugawara I., Ishizaka, S., and Moller, G., Carrageenans-powerful human lymphocyte mitogens, submitted for publication. 3Sugawara, I., and Ishizaka, S., Chemically modified carrageenans and cytotoxicity of human monocytes, submitted for publication. 402 OOOS-8749/82/060402-08$02.00/0 Copyright 0 1982 by Academic Press, Inc. Au right8 of repmdwtion in any foml mervcd.

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carrageenan

~~!F"?g&l 3 Lambda

FIG. 1. Structural

n

carrageenan

formulas of various CGNs (one repeating unit).

MATERIALS

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Isolation of mononuclear cells (MNC). Human peripheral blood from healthy adult donors was collected in heparin. It was then diluted l/2 in phsyiological saline and layered on Lymphoprep (Nyegaard, Oslo) (4). After centrifugation at 1500 rpm for 30 min, the cells at the interface were collected, pooled, and washed twice in balanced salt solution (BSS). Separation of T and non-T lymphocytes. In lo-ml plastic tubes (Falcon 2057), 4 X lo6 MNC/ml of MNC were mixed with 200 X lo6 fresh sheep erythrocytes in a final volume of 5 ml of RPM1 1640 (GIBCO Europe, Glasgow) containing 10% fetal calf serum (FCS) (previously absorbed with sheep and human erythrocytes and heat inactived). The cell mixture was incubated for 30 min at 37°C centrifuged for 5 min at 1000 rpm, and then stored overnight at 4°C before being carefully resuspended and layered on Lymphoprep. After centrifugation at 1500 rpm for 30 min at room temperature, the depleted cells at the interface were collected, pooled, and washed in BSS. This population is referred to as T lymphocytes after removal of sheep erythrocytes by osmotic shock. The purity of the T cells was about 95% and the T cells contained 1% phagocytic cells, as judged by latex particle ingestion test. Isolation

of human

T-cell subsets by complement-mediated

lysis utilizing

the

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monoclonal antibodies OKT4 and OKT8. The OKT4 antibody reacts with 50 to 60% of peripheral T cells, whereas the OKT8 antibody reacts with 30-40s human T cells. In order to isolate a T-cell population highly enriched in either OKT4reactive or OKT8-reactive cells, 50 X IO6 unfractionated T cells were resuspended in 1 ml of OKT4 or OKT8 antibody (Ortho Diagnostics, Raritan, N.J.) diluted l/250 in RPM1 1640 containing 5% FCS and incubated for 45 min at room temperature. After incubation, fresh rabbit serum was added at a final dilution of 1:10 and incubation was further carried out for 1 h at 37°C in a humid atmosphere. Complement-mediated cell lysis showed that the OK8-treated cells contained more than 90% OKTCpositive cells and that the OKCtreated cells contained more than 88% OKT8-positive cells. OKT4+ signifies a T-cell population remaining after treatment with OKT8 and complement and OKT8+ signifies a T-cell population remaining after treatment with OKT4 and complement (16). Mitogenic responses of 2 X I OSOKT4+ or OKT8+ cells to various CGNs. The 2 X lo5 OKT4+ cells or 2 X 1O5OKT8+ cells suspended in 0.1 ml of RPM1 1640 containing 10% human AB serum, glutamine, and gentamicin were placed in each well of microculture plates and the CGNs were added at a final concentration of 1 mg/ml. As control mitogens PHA (1 pg/ml) and Con A (2 @g/ml) were used. Each well contained 0.2 ml of cell suspension, the mitogen, and culture medium. The microculture plates were incubated at 37°C in a humidified atmosphere of 5% COz in air from Day 1 to Day 7. DNA synthesis was measured by incorporation of 0.8 &i [3H]thymidine during the last 8 hr at the culture period. The cells were harvested with a Titertek harvester (Flow Labs). The radioactivity remaining on the filter was determined in a liquid scintillation counter (Packard). One-way autologous and allogeneic mixed lymphocyte reactions. Both the autologous and allogeneic mixed lymphocyte reactions were conducted in round-bottomed microplates (Intermed, Copenhagen, Denmark) by mixing 1 X 1O5responder cells with an equal number of irradiated (2000 rad) stimulating cells in a total volume of 0.2 ml of RPM1 1640 supplemented with 2 mM glutamine, 1% penicillinstreptomycin (Flow Labs), and 10% heat-inactivated human AB serum. Each determination was performed in triplicate and consisted of T lymphocytes as responder cells stimulated by either autologous non-T lymphocytes in the autologous MLR or non-T cells from a nonrelated donor in the allogeneic MLR. After the cells were incubated for 7 days at 37°C in humidified 5% CO* in air, the cultures were pulsed daily with 0.8 &i of [3H]thymidine (Radiochemical Centre, Amersham) and incubated for an additional 8 hr at 37°C. The cells were then harvested using a multiple semiautomated sample harvester and the radioactivity incorporated in the cells was determined in a liquid scintillation counter (Packard). The results were expressed as counts per minute (cpm). In some experiments, silica suspension (KAC-2, Nippon Kotai Kenkyujo, JIMRO, Gunma, Japan) was added to non-T lymphocytes to deplete monocytes and was preincubated for 2 hr at 37°C. The CGNs used in our experiments were purchased from Sigma Chemical Company, St. Louis, Missouri. RESULTS (1) CGNs Can Induce Proliferation of OKT4+ and OKT8-k Cells Figure 2 shows the kinetic study of CGNs to 2 X 10’ OKT4+ or OKT8+ cells. We made three independent experiments. As shown in Fig. 2, CGNs could induce

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FIG. 2. Kinetics of CGN-induced DNA synthesis of 2 X lo5 OKT4+ or 2 X 10’ OKT8+ cells. Blastogenesis was determined by [3H]thymidine uptake. All the values show mean f SD of three independent experiments, each in triplicate. Left: OKT4+ (o), OKT4+ kappa CGN (1 mg/ml) (B), OKT4+ lambda CGN (I mg/ml) (X), OKT4+ iota CGN (1 mg/ml) (A). Right: OKT8+ (Cl), OKT8+ kappa CGN (1 mg/ml) (w), OKT8 + lambda CGN (1 mg/ml) (X), OKT8 + iota CGN (1 mg/ml). (A).

proliferation of OKT4+ and OKT8+ cells. There was no difference between OKT4+ and OKTS+ cells in the degree of the proliferation with various CGNs. Their peak responses were usually on Day 5. PHA and Con A induced DNA synthesis of both OKT4+ and OKT8+ cells significantly (data not shown). These findings are consistent with the report by Van Vauwe and Goossens (13). (2) CGNs Can Activate Responding T Cells in Autologous MLR CGNs are toxic to human monocyte? (6) and they have been shown to suppress antigen-induced lymphocyte transformation in vitro (5). If so, it is to be expected that CGNs would depress the proliferative response in autologous MLR. However, as shown in Fig. 3, CGNs induced proliferative changes in the autologous MLR. The response was significantly higher than that of responder cells and the CGN. We used two different doses-500 pg/ml and 1 mg/ml and the optimal dose for activating an autologous MLR was found to be 1 mg/ml. Lambda CGN was most potent in inducing proliferation of responding T cells, which usually peaked on Day 6. Kappa CGN was less potent compared with lambda and iota CGN (Fig. 3). (3) CGNs Can Also Induce Proliferative Allogeneic MLR

Change of Responder Cells in

AS shown in Fig. 4, CGNs could activate responding T cells in allogeneic MLR. The peak of the proliferation in allogeneic MLR was usually on Day 5 or 6. The proliferative response was always higher than that of responder T cells and the CGN. Lambda CGN was most effective in causing proliferation of responder cells.

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FIG. 3. The effect of various CGNs on autologous MLR. Autologous MLR was performed in roundbottomed microplates by mixing IO5 responding T cells with an equal number of irradiated (2000 rad) stimulating non-T cells and various CGNs. Each value shows mean + SD of four independent experiments, each in triplicate. NT(X), irradiated non-T cells. Left: T cells (o), T + kappa CGN (1 mg/ml) (W), T + lambda CGN (1 mg/ml) (X), T + iota CGN (1 mg/ml) (A). Right: T + NT(X) (a), T + NT(X) + kappa CGN (1 mg/ml) (m), T + NT(X) + lambda CGN (I mg/ml) (X), T + NT(X) + iota CGN (1 mg/ml) (A).

Kappa CGN was less effective. The optimal dose was 1 mg/ml. The peak of proliferation of responding cells usually appeared earlier than that of allogeneic MLR alone. We performed four independent experiments. (4) Silica Particles Abrogate Proliferation of Responding T Cells in Both Autologous and Allogeneic MLR, but CGNs Still Activate Responding T Cells in Autologous and Allogeneic MLR The silica suspension we used for our three independent experiments was toxic to monocytes but not to lymphocytes. Silica could inhibit the proliferative response in autologous and allogeneic MLR by killing the monocytes directly. In spite of a decreased response in MLR, CGNs could reverse the response. Therefore, CGNs may activate responding T cells directly without help of monocytes. The dose of silica used for monocyte killing was 25 /*g/ml (Figs. 5 and 6). DISCUSSION Carrageenans are known to be macrophage-toxic agents (3). From this point of view, CGNs have been considered to be immunosuppressive sulfated polygalactans. They are also. used as food additives to promote stickiness of food (2). Carrageenans are also toxic to human monocytes3 (5). Monocytes are necessary for autologous and allogeneic MLR (7,8). Thus, it is expected that the proliferative change in autologous and allogeneic MLR would be abrogated by the addition of

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FIG. 4. The effect of CGNs on aliogeneic MLR. IO5 T cells were mixed with IO5 irradiated non-T cells (2000 rad) from a nonrelated donor and various CGNs in round-bottomed microplates. After a certain period of incubation, the cultures were pulsed with 0.8 &i of [‘Hlthymidine and incubated for an additional 8 hr at 37°C. Results are expressed as mean -CSD of four independent experiments, each in triplicate. NT(X), irradiated allogeneic non-T cells. Left: T cells ( * * * ), T -t NT(X) (X * * . X), T 0). Right: T-t NT(X) + lambda CGN (I mg/ml) -t NT(X) 4 kappa CGN (I mg/ml) (O(I 1), T + NT(X) + iota CGN (1 mg/ml) (X X ).

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FIG. 5. The effect of various CGNs on silica-added autologous MLR. Two hours after preiacubation with silica pan&s (25 pg/ml), various CGNs were added to autologous MLR assay system containing silica particles. NT(X), irradiated non-T cells (2000 rad). Results are expressed as mean k SD of three independent experiments, each in triplicate. Left: T + NT(X) (X * * * X), T -t NT(X) + silica + kappa CGN (1 mg/ml) (0 IJ). Right: T + NT(X) C silica (. * a), T + NT(X) f silica + lambda CGN T + NT(X) + silica -I- iota CGN (1 mg/mI) (X __ X). (1 mg/ml) (I -•),

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FIG. 6. The effect of various CGNs on silica-added allogeneic MLR. Two hours after preincubation with silica particles (25 pg/ml), various CGNs were added to allogeneic MLR assay system containing silica particles. NT(X), irradiated allogeneic non-T cells (2000 rad). All the values show mean f SD of three independent experiments, each in triplicate. Left: T + NT(X) (X * * . X), T + NT(X) + silica + kappa (CGN (1 mg/mI) (0 0). Right: T + NT(X) + silica (a * . ), T + NT(X) + silica + iota CGN (1 mg/ml) (X-X), T + NT(X) + silica t lambda CGN (1 mg/ml) (m-m).

optimal dosesof CGNs because CGNs are toxic to monocytes. As already depicted in Figs. 3 and 4, CGNs induced proliferation of the responder cells contrary to our expectation. In addition, when silica particles were added to autologous and allogeneic mixed cultures, there was no proliferation of the responding cells presumably because silica blocked the monocyte function (9). When CGNs were added to the silica-treated cultures, the responding lymphocytes proliferated. We concluded that CGNs could directly activate responding T cells, while they were toxic to monocytes. It is, however, worthwhile to mention that the effects of CGNs vary with dose (10). Although 1 mg/ml of CGNs can activate T cells and kill monocytes, 500 pg/ml of CGNs can hardly activate lymphocytes even though monocytes are killed. T lymphocytes respond to autologous non-T lymphocytes in coculture by proliferation. This phenomenon has been termed autologous mixed lymphocyte reaction (MLR). The responding T lymphocytes in autologous MLR and allogeneic MLR belong to a different subpopulation of T cells (12). Now antibodies derived from hybridomas are available to separate various T lymphocyte subpopulations to obtain highly purified monocytes and B cells ( 13- 15). Recently Van Vauwe and Goossens reported that OKT4+ cells respond well to autologous non-T cells but OKT8+ cells responded poorly to autologous non-T cells in autologous MLR, while both OKT4+ and OKT8+ cells responded well to allogeneic non-T cells in allogeneic MLR (13). From our data concerning DNA

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synthesis of OKT4+ and OKT8+ cells to various CGNs, it was assumed that in autologous MLR, CGNs could stimulate OKT8+ cells and OKT4+ cells unresponsive to autologous non-T cells and in allogeneic MLR, CGNs could induce proliferation of both unstimulated OKT4+ and OKT8+ cells. We may also add the possibility that CGNs stimulate responder T blasts responding to autologous or allogeneic non-T cells. Kappa CGN is composed of D-galactose-4-sulfate and 3,6-anhydro-o-galactose units. The molecular weight of one unit is about 402 and there is one sulfate radical per unit. Iota CGN is a kappa CGN-like structure and has two sulfate radicals per unit (about 453 MW of one unit). Lambda CGN consists of equal proportions of 1,3-linked and 1,4-linked D-galactose units. One unit is about 563 in molecular weight and has three sulfate radicals (Fig. 1) (1). In our assay system lambda CGN, which has the highest negative charge of the three CGNs, was most effective in inducing the proliferative change of the responder cells in autologous and allogeneic MLR. Kappa CGN was least effective in causing proliferation of the responding lymphocytes. Iota CGN was intermediate between kappa and lambda CGN. It seems that the higher negatively charged molecules cause the strongest proliferative change of the responder cells ( 11). ACKNOWLEDGMENT This work was made possible in part by support from Mr. Mitsuo Sugawara, who is a lawyer and director of Tokyo Marunouchi Law Center, Tokyo, Japan, and the Swedish Cancer Society and the Swedish Medical Research Council.

REFERENCES 1. Thomson, A. W., Fowler, E. F., and Pugh-Humphreys, R. G. P., Ini. J. Immunopharmacol. 1,247, 1919. 2. Ishizaka, S., Hasuma, T., Otani, S., and Morisawa, S., J. Immunol. 125, 2232, 1980. 3. Catanzaro, P. J., Schwartz, H. J., and Graham, R. C., Amer. J. Pathol. 64, 387, 1971. 4. Boyurn, A., Stand. J. Clin. Lab. Invest. Zl(Supp1. 97), 1968. 5. Lake, W. W., Bite, D., Schwartz, H. J., and Salvaggio, J., J. Immunol. 107, 1745, 1971. 6. Thomson, A. W., and Horne, C. H. W., Brir. J. Exp. Pathol. 57, 455, 1976. 7. Dock, N. L., and Davey, F. R., Clin. Immunol. Immunopathol. 17, 123, 1980. 8. Alter, B. J., and Bach, F. H., Cell. Immunol. 1, 207, 1970. 9. Allison, A. C., Harrington, J. S., and Birbech, M., J. Exp. Med. 124, 141, 1966. 10. Pawleac, G., and Brons, G., Clin. Exp. Immunol. 31, 426, 1978. 11. Diamanstein, T., Vogt, W., Rtihl, H., and Bochert, G., Eur. J. Immunol. 3, 488, 1973. 12. Palacios, R., Llorente, L., Alarcon-Segovia, D., Ruiz-Arguelles, A., and Diaz-Jouanen, E., J. Clin. Invest. 65, 1527, 1980. 13. Van Vauwe, J., and Goossens, J., Immunology 42, 157, 1981. 14. Reinherz, E. L., Kung, P. C., Pesando, J. M., Ritz, J., Goldstein, G., and Schlossman, S. F., J. Exp. Med. 150, 1472, 1979. 15. Breard, J., Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F., J. Immunol. 124(4), 1943, 1980.