Received for publication March 21, on the macrophage system. In 2 and possibly 3 ofthe processes where

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1 Br. J. exp. Path. (1978) 59, 454 MODIFICATION OF THE TUBERCULIN REACTION BY LEVAN E. SHEZEN, J. LEIBOVICI AND M. WOLMAN* From the Department of Pathology, Sackler Medical School, Tel-Aviv Univer8ity, Tel-Aviv, I&rael Received for publication March 21, 1978 Summary.-High molecular levan (polyfructoside) inhibits the skin tuberculin reaction in guinea pigs as judged by the degree of induration and erythema. The effect is dose-dependent. No effect on cellular infiltration was observed in histological studies. The lymph nodes of levan-treated animals were smaller and exhibited a much milder granulomatous reaction than those of non-treated animals. HIGH MOLECULAR LEVAN (polyfructoside, which will be referred to as "levan") has been shown to possess several biological activities. It inhibits acute inflammatory response and wound healing (Wolmani, 1956; Wolman and Wolman, 1956), delays rejection of homologous skin grafts (Leibovici, Bleiberg and Wolman, 1975) and inhibits experimental allergic encephalomyelitis (Berman, Leibovici and Wolman, 1976). Levan has also been shown by us to have an antitumour effect (Leibovici, Sinai et at., 1975; Borit et al., 1977). Some of these effects may be partly explained by the coating of endothelial cells by levan which prevents passage of cells and macromolecules from blood vessels to the tissues. Evidence for such coating has been adduced by different techniques (Davies, Shilo and Hestrin, 1955; Behar and Shilo, 1969; Walters et al., 1978). Coating of the endothelial surfaces cannot, however, explain the effects of levan on wound healing and on destruction of neoplastic growths. Levan is known to affect immunological reactions (Miranda, 1972; Henig, Kazap and Leibovici, 1978). It acts as a T-independent B-cell mitogen (Coutinho and Moller, 1973) and also influences T cell function (Ben Sasson and Henkart, 1977). The work of our group and collaborative studies (Borit et al., 1977; Robertson et al., 1977; Kazap, Leibovici and Wolman, 1978; Aronson, Eldar and Wolman, 1978) have shown that levan has a marked effect on the macrophage system. In 2 and possibly 3 ofthe processes where levan exerted a marked influence, immunological phenomena play an important role. They are: homograft rejection, experimental allergic encephalomyelitis and defence against malignant tumours. In all these processes, although humoral immunity might be implicated, cellular immunity is generally believed to play a major role. (Brent et al., 1962; David and Paterson, 1965; Burnet, 1971). It is therefore possible that levan might exert an effect on cellular immunity. The present report deals with a study of the effects of levan on the classical form of cellular immunity, the delayed hypersensitivity reaction to tuberculin. MATERIALS AND METHODS Immunization.-Male and female Hartley guinea pigs weighing g were immunized by injections in the footpad and s.c. in the back with a total of 0 5 ml of complete Freund's adjuvant (CFA) containing 2 mg/ml of killed Mycobacterrium tuberculosis H37Ra (Difco Laboratories, Inc., Detroit, Mich.). CFA was used in the form of emulsion in phosphate-buffered saline (Biolab. Ltd., Jerusalem) 1:1. Tuberculin * Established investigator of the Chief Scientist's Bureau, Ministry of Health, Israel.

2 MODIFICATION OF THE TUBERCULIN REACTION BY LEVAN 455 PPD from M. tuberculosis (Central Veterinary Laboratory, Min. of Agriculture, Fisheries and Food, New Haw, Weybridge, Surrey, England), was used as antigen for obtaining the local tuberculin reaction. Native Aerobacter levanicum levan, prepared according to the method of Hestrin, Shilo and Feingold ( 1954) was purchased at the Dept. of Biological Chemistry, Technical Unit, The Hebrew University, Jerusalem. Its mol. wt was about 2 x 7. A 5% solution in saline was prepared according to the method of Shilo, Wolman and Wolman (1956). Levan was administered by daily i.p. injections beginning 1 day before immunization and continued throughout the experiment until the 25th day. Groups of 2-12 animals received daily doses of levan ranging from 15 to 150 mg per animal. The levanized and nonlevanized animals were skin-tested on the 24th day following sensitization by intradermal injection of PPD. Each guinea pig was injected with jug PPD, 1 lug PPD, both in 0-1 ml saline, and 0-1 ml saline on both sides of the abdomen. The reactions were examined 18 h following injection of antigen by determining the thickness of the lesion with a caliper, the diameter of the area of induration (both in mm) and by evaluating the intensity of hyperaemia in arbitrary units. All the guinea pigs were killed 25 days after the injection of CFA. Popliteal, inguinal and axillary lymph nodes of the animals as well as their spleens were excised and weighed. Pieces of the PPD-injected skin areas and of the lymph nodes and spleens were fixed in % formalin, embedded in paraffin and stained with H. and E. RESULTS Figure 1 shows the effect of different doses of levan, ranging from 15 to 150 mg/ day on the tuberculin reaction. With both 1 mg and mg PPD, a lesser increase in thickness of skin was observed in the levantreated than in the non-levanized animals. This inhibitory effect of levan was proportional to the levan dose, reaching 60% reduction in the thickness of the skin with 150 mg levan at 1,ug PPD. E =1.5- c. C.C\ 1.04X L u Levan dose (mg/day) FIG. 1.-Effect of levan on the intensity of local tuberculin reaction. Groups of (a) 12, (b) 2, (c) 16 and (d) 3 guinea-pigs were given (a) no levan, (b) 15, (c) 50 and (d) 150 mg levan daily. Levan treatment was begun one day before CFA administration. The increase in thickness of the skin was calculated by subtracting from the values found in the PPD injected sites, the values of the saline-injected sites. 1 jig PPD; *.,g PPD; A. Table I shows the influence of different doses of levan on the tuberculin reaction as judged by the diameter of the lesion and its degree of erythema. With both criteria, a reduced reaction was obtained in the presence of levan, proportional to the dose of the polysaccharide. Table II presents the effect of levan treatment on the weight of lymph nodes and spleens of CFA-treated guinea pigs 25 days after CFA administration. A 40% reduction of the average weight of lymph nodes was observed in the levan-treated animals. The spleens of the levan-treated TABLE I.-Effect of Levan on the Intensity of Local Tuberculin Reaction Diameter of lesion (mm) Degree of erythema (arbitrary units) Same conditions as for Fig. 1. PPD (pg) 1 Levan dose mg/day ± ± ± ±5 15-0± ± ±

3 456 E. SHEZEN, J. LEIBOVICI AND M. WOLMAN.", t+;\*^*r; - v--'t1 rr FiG. 2.-Lymph node of a non-levanized guinea-pig 27 days after injection of CFA. The architecture is destroyed and the lymphatic tissue is almost completely replaced by epithelioid cell granulomata. H. and E. x l. FIG. 3. Lymph node of a levan-treated guinea-pig 27 days after injection of CFA. The architecture and the lymphatic tissue are mostly preserved with scattered granulomata. H. and E. x 1.

4 MODIFICATION OF THE TUBERCULIN REACTION BY LEVAN 457 TABLE II.-Effect of Levan Treatment of Guinea Pigs Sensitized with CFA on the Weight of Lymph Nodes and Spleen. Average weight Average weight Levan treatment Number of lymph nodes Number of spleen (50 mg/day) of animals (in g) of animals (in g) ± *91±0-28 Inguinal, popliteal and axillary lymph nodes and the spleens of guinea pigs were weighed on the 25th day after CFA injection. Levan treatment was begun 1 day before CFA administration and continued throughout the experiment. guinea pigs, however, increased in weight by about 70%. Figures 1 and 2 show the histological changes in lymph nodes of levan-treated and untreated guinea pigs 25 days after CFA administration. In the non-levanized animals most of the lymph node parenchyma was replaced by epithelioid-cell granulomata. Nests of lymphocytes were scattered in between these structures (Fig. 1). In the levan-treated animals most of the normal constituents of the lymph node was preserved. A moderate number of similar granulomata was found in the lymphoid tissue (Fig. 2). Thus, lymphatic tissue was preserved in the levan-treated animals to a much greater extent than in the non-levanized guinea pigs. The histological picture of the spleens of levantreated animals (not presented here) showed numerous foci of foamy, levanladen macrophages. In the PPD-injected areas of the skin, the known reaction (Wiener, Spiro and Zunker, 1965) with infiltration of polymorphs and macrophages was observed in the dermis. The epidermis was also partly infiltrated. The intensity of cellular infiltration in the skin was found to be similar in the levanized and non-levanized animals. DISCUSSION The data presented show that levan inhibits the tuberculin reaction in guinea pigs. A clear reduction in the intensity of the reaction was obtained as judged by the thickness of the lesion, its diameter and the intensity of erythema. Sheper et al. (1977) stressed the necessity of determining all 30 3 parameters of the tuberculin test, especially as modificators may influence only one of them. The intensity of cellular infiltration appeared, however, not to be affected. Thus, while in acute inflammation levan inhibits both the passage of plasma and of polymorphs across the vessel walls, in the tuberculin reaction transfer of plasma is inhibited, while mononuclear cell infiltration is not. The fact that lymph nodes in the levantreated animals increased less in weight and had less granulomata than the nontreated ones suggests an effect of levan on the sensitization step of the immune response. This could be brought about by inhibition of antigen processing by levanladen macrophages. The effect of levan could also be due to its influence on the efferent pathway of the immune response. Levan has been shown in the past to have an antiinflammatory effect (Wolman and Wolman, 1956), and cortisone has been shown to inhibit the tuberculin reaction by such an effect (Gell and Hinde, 1951). The reduction in the intensity of edema and erythema by levan (Fig. 1 and Table I) might have been caused by a diminution in the amount of mediators (such as the lymph node permeability factor, LNPF, or lymphokine) released by lymphocytes or prevented from reaching their target organs. LNPF was shown, in fact, to affect erythema as well as cellular infiltration (Willoughby, Walters and Spector, 1965). Our observation might raise an interesting possibility. We have shown that levan markedly reduced oedema and erythema with no apparent effect on cellular infiltra-

5 458 E. SHEZEN, J. LEIBOVICI AND M. WOLMAN tion. Geczy, Geczy and de Weck (1976) have demonstrated that lymphokines affected more markedly cellular infiltration than erythema. Cyclophosphamide influenced erythema only. The findings considered together suggest that the 2 processes might be effected through different mediators. This work was supported by a research grant (M.W.) from the United States- Israel Binational Science Foundation (BSF), Jerusalem, Israel. REFERENCES ARONSON, M., ELDAR, T. & WOLMAN, M. (1978) Effects of High Molecular Levan on Macrophages. Submitted for publication. BEHAR, A. & SHILO, M. (1969) Effect of Native Levan on Membrane Permeability. I. Effect on the Vascular Membrane in Acute Nonspecific Inflammation. A Study with the Light Microscope. Am. J. Pathol., 57, 383. BEN-SASSON, S. A. & HENKART, P. A. (1977) Enhancement of Weak Mixed Lymphocyte-type Reactions by Hydrophilic Polymers. J. Immunol., 119, 227. BERMAN, Z., LEIBOVICI, J. & WOLMAN, M. (1976) Effect of Levan on the Stages of Development of Experimental Allergic Encephalomyelitis. Isr. J. Med. Sci., 12, BORIT, A., LEIBoVICI, J., SANDBANK, U., SINAI, Y. & WOLMAN, M. (1977) Inhibition of 3LL Carcinoma of Mice by Levan Treatment. Isr. J. Med. Sci., 13, 859. BRENT, L., B3ROWN, J. B. & MEDAWAR, P. B. (1962) Quantitative Studies on Tissue Transplantation Immunity. VI. Hypersensitivity Reactions Associated with the Rejection of Homografts. Proc. Roy. Soc., Ser. B, 156, 187. BURNET, F. M. (1971) Immunological Surveillance in Neoplasia. Transpl. Rev., 7, 3. COUITINHO, A. & MOLLER, G. B. (1973) Cell Mitogenic Properties of Thymus-independent Antigens. Nature, New Biol., 245, 12. DAVID, J. R. & PATERSON, P. Y. (1965) In Vitro Demonstration of Cellular Sensitivity in Allergic Encephalomyelitis. J. exp. Med., 122, DAVIES, A., SHILO, M. & HESTRIN, S. (1955) The Influence of Aerobacter Levan on the Permeability of the Blood Vessels of the Skin: Studies with Antibody Globulins and Trypan Blue. Br. J. exp. Path., 36, 500. GECZY, C. L., GECZY, A. F. & DE WECK, A. L. (1976) Antibodies to Guinea Pig Lymphokines. II. Suppression of Delayed Hypersensitivity Reactions by a "Second Generation" Goat Antibody Against Guinea Pig Lymphokines. J. Immunol., 117, 66. GELL, P. G. H. & HINDE, I. T. (1951) The Histology of the Tuberculin Reaction and its Modification by Cortisone. Brit. J. exp. Path., 32, 516. HENIG, S., KAZAP, I. & LEIBOVICI, J. (1978) Suppression of Humoral Immune Response in Mice by Administration of High Molecular Levan. Experimentia, in press. HESTRIN, S., SHILO, M. & FEINGOLD, D. S. (1954) Infection Promoting Activity of Levan and Dextran as a Function of Degree of Polymerisation. Br. J. exp. Path., 35, 7. KAZAP, I., LEIBOVICI, J. & WOLMAN, M. (1978) Blood Levels of High Molecular Levan in Mice as a Function of the Route and Duration of Administration. Arzneimittelf. Drug Res. in press. LEIBOVICI, J., BLEIBERC, I. & WOLMAN, M. (1975) Effect of Native Levan on Homograft Rejection in Mice. Proc. Soc. exp. Biol. Med., 149, 348. LEIBOVICI, J., SINAI, Y., WOLMAN, M. & DAVIDAI, G. (1975) Effect of High Molecular Levan on the Growth and Spread of AKR Lymphoma. Cancer Res., 35, MIRANDA, J. (1972) Studies on Immunological Paralysis. IX. The Immunogenicity and Tolerogenicity of Levan (Polyfructose) in Mice. Immunology, SHEPER, R. J., NOBLE, B., PARKER, D. & TURK, J. L. (1977) The Value of an Assessment of Erythema and Increase in Thickness of the Skin Reaction for a Full Appreciation of the Nature of Delayed Hypersensitivity in the Guinea Pig. Int. Archs. Allergy Appl. Immun., 54, 58. WALTERS, M. N. -I., WOLMAN, M., ROBERTSON, T. A., & PAPADIMITRIOU, J. M., (1977) The inhibitory Effects of High Molecular Levan on Transport Across the Vascular Wall Stimulated by Histamine. J. Path., 124, 195. SHILO, M., WOLMAN, M. & WOLMAN, B. (1956) Inhibition of Inflammatory Response of Skin to Staphylococcus Aureus by High Polymer Levan. Br. J. exp. Path., 37, 219. WIENER, J., SPIRO, D. & ZUNKER, H. 0. (1965) A Cellular Study of Tuberculin Sensitivity. Amer. J. Path., 47, 723. WILLOUGHBY, D. A., WALTERS, M. N. I. & SPECTOR, W. G. (1965) Lymph Node Permeability Factor in the Dinitrochlorobenzene Skin Hypersensitivity R-action in Guinea Pigs. Immunol., 8, 578. WOLMAN, M. (1956) Histological Changes Produced by Injection of Polysaccharid&s. A.M.A. Arch, Pathol., 62, 149. WOLMAN, M. & WOLMAN, B. (1956) Effect of Polysaccharides on the Formation of Granulation Tissue. A.MAT.A. Arch. Pathol., 62, 74.