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Mitogenic activity and immunological properties of bolesatine, a lectin isolated from the mushroom Boletus satanas Lenz
Int. J. Bloehem. Vol. 25, No. 5, pp. 789-792, 1993
0020-711X/93 $6.00 + 0.00 Per~tmon Press Ltd
Printed in Great Britain
MITOGENIC ACTIVITY AND IMMUNOLOGICAL PROPERTIES OF BOLESATINE, A LECTIN ISOLATED FROM THE MUSHROOM BOLETUS SATANAS L E N Z FIU~I~ICO LICASTEO,I * M ~ C ' m S ~ A Motto-I, x OLivn~ K a z r z , 2 Gu~ Dntm~Im~n, 2 EDMOI~D EKUE CREPPY3 and FIOp.EI~ZOSTIR~ I ~Dipartimento di Patologia Sperimentale, Universit/t di Bologna, Via San Giacomo 14, 1-40126 Bologna, Italy [Tel. (051) 242084; Fax (051) 251315], 2Institut de Biulogie Mul/sculalre et Cellulalre du CNRS and Universit~ Louis Pasteur, 15 rue Descartes, F-67084 Strasbourg, France and 3Laboratoire de Toxicolosie et d'Hygi~ne Appliqu~e, 3 tar Place de la Victoire, F-33076 Bordeaux Cedex, France (Received 23 April 1992)
Al~raetml. A lectin has been purified from the mushroom Boletus satanas Lenz. 2. The protein, called bolesatine, is mitogenic for human T lymphocytes in a dose- and time-dependent msnner.
3. Optimal mitogenic doses induce the release of interleukin-l~ and interleukin-2 from mononudenr cell cultures.
INTitODUCTION
Lectins are sngar-binding proteins which agglutinate cells or precipitate glycoconjugates (Goldstein et al., 1980; Sharon, 1983). These substances are found in plants and animal tissues, as well as in microorganisms (Sharon, 1983). Some lectins induce lymphocyte proliferation (Licastro et al., 1983a) and some of them are toxic and inhibit protein synthesis (Olsncs and Pihl, 1977; Stirpe et al., 1978, 1980; Barbieri et
a/., 1984).
Bolesatinc is a lectin purified from Boletus satanas Lenz (Kretz et al., 1989), a mushroom causing.serious gastroenteritis in humans (Maeder, 1975). Bolesatine is toxic and results in animal's death with an unspecific symptomatology (Kretz et al., 1991a). Furthermore, it inhibits D N A and protein synthesis in kidney cell cultures (Kretz et al., 1991b). Thus, this lectin might have a role in the toxicity of Boletus satanas Lenz. Here we present results regarding the effect of the lectin on human lymphocytes. Bolesatine is mitogenic for T cells and induces the release of interleukin-lu (IL-I~) and interleukin-2 (IL-2) in supernatants from mononuclear cell cultures.
MATERIALS AND MgrHO[~ Bolesatine isolation Bolesatine was isolated and purified from the mushroom Boletas satanas Lenz (Boletazeate)by two fast protein liquid chromatography (FPLC) methods on anion exchangers
*Addre~ correspondence to: Prof. F. ~ , Dipertimento di Patologia Sperimentale, Via San Giacomo 14,1-40126 Boingna, Italy. 789
after ammonium sulphate precipitation ~ et al., 1989). The protein concentration was determined according to the method of Bradford (1976). Separation of peripheral blood mononuclear cells Peripheral blood mononuclear ceJls were obtained from venous blood of healthy adult volunteers by.density gradient centrifugation (Bzyum, 1968) on lymphoprep (Immuno, Italy). Cells were 95% lymphocytes and 5% monocytes and cell viabifity ranged from 90 to 98%. Mononuclenr ~ were suspended in a serum-free medium which con~ted of Iseove's modification of Dullxx~o's medium (Gibco, U.S.A.) supplemented with bovine serum albumin (800/~8/ml; Cohn Fraction V, Sigma, U.S.A.), iron-free human tran~'errin (2/~g/ml; Sigma, U.S.A.), 100 U/m1 penicillin and 100 t*sJml streptomycin (Flow Labs, U.K.). Purification of T and B lymphocytes T and B lymphocytes were separated by rosette formation with sheep red blood cells and subsequent gradient centrifugnfion according to the method o f Roms$nani et al. (1977) with slight modifications (Francesehi et ai., 1981). Lymphocyte proliferative assay Cell cultures, with and without lectins, were performed as previously described (Franceschi et al., 1978) with slight modifications (Licastro et al., 1983b). Briefly, mononuciear cells or purified T and B lymphocytes (2 × l0s viable cells in 0.1 rul of serum-free medium) were distributed in quadruplicate in microplate wells. Test cultures were stimulated with phytohemasglutinin-P (PHA; Difco Labs, U.S.A.) or bolesatine diluted in a serum-free medium. Cells were then cultured in a CO2 incubator. Cell proliferation was asse~d by pulsing the cultures with 0.5/~Ci/well of methyl-[Sl-l]thymidine (sp. act. 5Ci/ mmul; the Radiochemical Center, U.K.) during the last 6 hr. Cell cultures were harvested on glass fiber filters with the aid of a multiple cell-harvester (Skatron, Norway) and [~H]TdR incorporation was measured by liquid scintillation counting
790
FBDERICOLICASTROet al. 500 - mPHA20/~g/ml o Bolesotine0.01/~g/rnl 250 /~Bolesotine0.1/~g/ml 200
"T
|
0 Bo~otin~ 10/~g/ml
900
~IINo mitogen
800
r~IPHA 20 ~g/ml P-~Bolesotine 0.1/.tg/ml
7O0
T~ ~
600
~ 1~
g lo0 50
O--
O-,
O
O
O
72
96
120
°° 1O0
48
144
o
Hours
Fig. I. Time-dependent lymphocyte proliferation induced by three doses of bolesatine and one concentration of PHA. Results are expressed as mean + SE.
as previously described (Franceschi etal., 1978) with slight modifications (Licastro etal., 1982). IL-1= and IL-2 determination Mononuclear cells were cultured with or without PHA or bolesatine lectin for 24, 48 and 72 hr, then supernatants were collected by centrifugation and stored at -20°C. The amount of IL-I= and IL-2 in supernatants was determined with ELISA kits from Oncomembrane (U.S.A.) and British Biotechnology (U.K.), respectively. RESULTS Bolesatine had a strong mitogenic activity on human peripheral blood lymphocytes at 0.01 pg/ ml. Lymphocyte stimulation was dose- and timedependent, as shown in Fig. 1. Maximum mitogeaic activity was observed when a dose of 0.1 pg/ml of bolesatine was used and at this concentration lymphocyte proliferation was higher than that stimulated by an optimal concentration of PHA (20 #g/ml). At higher doses (10pg/ml), the lectin showed a toxic effect. When tested on purified T and B lymphocytes after 3 days of culture, the lectin induced the proliferation of T cells only (Fig. 2). Mononuclear cell cultures were activated for 2472 hr by optimal concentrations of bolesatine or P H A
m N o mitogen r--IPHA ~0 ~g/ml ~Bolosatinl 0.01/~g/ml FT"JBole~otine 0.1/~g/rnl
70 60-
~~
~
24
1
In 48
i
tn
,
72
Hours
Fig. 3. Time-dependent IL-I= release in supernatants of bolesatine- and PHA-activated mononuclear cells. Results are expressed as mean + SE. and supernatants were collected to detect the release of free cytokines. Bolesatine induced a very high and time-dependent secretion of free IL-I= from monocytes, whereas P H A did not stimulate a significant release of this cytokine (Fig. 3). Both bolesatine and P H A induced IL-2 secretion from activated human mononuclear cell cultures. The levels of free IL-2 in the supernatants from bolesatine-activated T lymphocyte~ were lower than those from cultures activated by PHA after 48 and 72 hr of incubation (Fig. 4). D~CUSSION
The proteinaceous toxin purified form Boletus satanas Lenz (Kretz etal., 1989) is toxic when injected in mice, being the LD~0 3.3 mg/kg of body weight (Kretz e t a l . , 1991a). Bolesatine inhibits D N A synthesis of cultured kidney cells in a dose-dependent manner, with an IC~oo f 0.32 p M (Kretz et al., 1991b). Protein synthesis is also inhibited "in the same culture conditions (IC~0 = 0.14 pM). The molecule binds to galactose residues of cell membrane receptors and behaves like a lectin, since the addition of D-galactose to the cell culture medium prevents the cytotoxicity of bolesatine (Kretz et al., 1991b). This notion has been confirmed by studies 1600
80 - 72 hour=
I1,.
1400
m No mitogen ~ P H A 20/~g/rnl 12~~oleaotine 0.1/~g/ml
1200 ... E 1000
$~ ,~
40
-~ ~ 0 50
-
~
-
10
-
41~
_
TcelI=
--~--[~ B cells
Fig. 2. Proliferation of purified T and B lymphocytes induced by bolesatine and PHA after 72 hr of culture. Results are expressed as mean + SE.
0
48 Hours
72
FiB. 4. Time-dependent IL-2 release in supernatants of bolesatine- and PHA-activated mononudear cells. Results are expressed as mean + SE.
Immunomodulation by bolesatine focused on the agglutinating activity of bolesatine
(Wretz, 19~2). Bolesatine is a potent mitogen for human T lymphocytes. A strong proliferation is induced by as little as 0.01/~g/ml of the compound and the maximum effect is observed with a concentration of 0.1 #g]ml. However, higher concentrations of the lectin inhibit cell proliferation, indicating a cytotoxic activity, possibly ascribable to the inhibition of protein synthesis. The high release of IL-I~, induced by bolesatine indicates that the lectin is a potent activator of human monocytes. Two different molecular forms of IL-1, called IL-I~ and IL-1/~, have been identified (Auron et al.., 1984; Lomedico et al., 1984). These 'molecules are synthesized in monocytes as 31 kDa precursors, which appear to be processed into lysosomes and transformed into the mature forms of 17kDa (Bakouche et al., 1987). After 18-24 hr of activation by LPS, IL-I~, precursor mostly remains cell associated (Lonnemann et al., 1989). The phosphorylation of the intracellular precursor of IL-lu facilitates the processing and the secretion of the 17 kDa mature form (Kohayashi et aL, 1988). The IL-I~ precursor appears to be also associated to the cell membrane (Beuscher et al., 1987) and the further processing of the molecule seems to be induced by extracellular serine proteases (Auron et al., 1987). Bolesatine is a very potent stimulus for the release of IL-I~ by human monocytes. It is possible that bolesatine, at variance with PHA, affects one of the above-mentioned metabolic steps; for instance, bolesatine may induce an increased level of phosphorylation or an augmented production of proteases able to cleave the membrane IL-I~ precursor. This notion may explain the differential release of IL-I~ induced by bolesatine in comparison to PHA. It is important to stress that we cultured human cells in a serum-free medium, thus exogenous proteases were not added to the cell cultures. The kinetics of IL- 1 secretion are peculiar, since the release of both IL-I~ and /~ is delayed following the synthesis and the precursor form accumulates in the cytoplasm (Hazuda et al., 1988). This observation has been recently confirmed and it has been suggested that most IL-I~, could be released after cell injury (Hogquist et al., 1991). It is important to keep in mind that bolesatine is toxic for cultured kidney cells. Thus, an alternative mechanism for the elevated timedependent release of free IL-I~, may be an injury to monocytes induced by bolesatine. The high level of IL-I~, may contribute to the elevated mitogenic activity of T lymphocytes, since this cytokine regulates the proliferation of activated T lymphocytes (Dinarello, 1989). Preliminary results show that bolesatine induces a high release of tumor necrosis factor-~ (TNF-~) from human mononuclear cell cultures after 24 hr of incubation (data not shown). Both IL-I~ and TNF-~ have been shown to enhance the expression of the ~, subunit of the IL-2 receptor and to synergize with IL-2 for the mito-
791
genesis of T lymphocytes (Smith et al., 1980; Lee et aL, 1987). The mitogenic activity of bolesatine is also mediated by the release of IL-2 by T helper cells. The level of this lymphokine in supernatants of proliferating lymphocytes depends upon the balance between its production by activated T helper cells and its absorption by high affinity receptors expressed on proliferating T lymphocytes (Smith, 1988). The reduced level of free IL-2 detected after 3 days in cultures activated by bolesatine may be secondary to an elevated expression of high affmity IL-2 receptors, with a consequent stimulation of mitogenic activity of T lymphocytes. The Boletus satanas mushroom is toxic and muses serious gastroenteritis in human beings. The toxicity of the mushroom may be partially ascribed to compounds, such as bolesatine, which are capable of inhibiting protein synthesis of epithelial cells and inducing a strong release of cytokines, such as IL-l~, and TNF-~ from gastroenteric macrophages. SUMMARY
Here we show that the lectin bolesatine isolated from the mushroom Boletus satanas Lenz has a strong immunomodulatory potential. It is able to stimulate the proliferation of human T lymphocytes and to promote the secretion of monokines and lymphokines, such as IL-lu and IL-2. Thus, the gastroenteritis induced by the ingestion of the mushroom may be partially ascribed to this lectin through mechanisms which activate immune responses. research was supported by M.U.R.S.T., CNR "Progetto Finalizzato Biotecnologie • Biostrumentazione", Pallotti's Legacy for Cancer Research, Ligue Nationale Fran~aise contre le Cancer, Comit~ D~partemental de la Gironde and INSERM Contract Recherche externe No. 89/2007.
Acknowledgements--Tl~s
Auron P. E., Webb A. C., Rosenwasser L. J., Mucci S. F., Rich A., Wolff S. M. and Dinarello C. 0984) Nucleotide sequence of human monocyte interleukin I precursor cDNA. Proc. hath. Acad. Sci. U.S.A. 81, 79007-7911. Auron P. E., Warner S. J. C., Webb A. C.. Cannon J. G., Bernheira H. A., McAdam K. J. P. W., Rosenwasser L. J., LoPreste G., Mucci S. F. and Dinarello C. A. 0987) Studies on the molecular nature of human interlenkin 1. J. Immun. 138, 1447-1456. Bakouche O., Brown D. C. and Lachman L. B. (1987) Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release. J. Immun. 138, 4249-4255. Barbieri L., Falasca A. I. and Stirpe F. (1984) Volkensin, the toxin of Adenia volkensii (kilyambiti plant). FEBS Left. 171, 277-279. Beuscher H. U., Fallon R. J. and Colten H. R. (1987) Macrophage membrane interleukin 1 regulates the
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expression of acute phase proteins in human hepatoma Hep 3B cells. J. Immun. 139, 1896-1901. Beynm A. (1968). Separation of leukocytes from blood and bone marrow. Scand. J. clin. lab. Invest. 21, Suppl. 97, 77--89. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Dinarello C. A. (1989) Interleukin-I and its biologically related cytokines. Adv. Immun. 44, 153-205. Franceschi C., Licnstro F., Paolucci P., Masi M., Cavicchi S. and Zannotti M. (1978) T and B lymphocyte subpopulations in Down's syndrome. A study of non-institutionalized subjects. J. merit. Defic. Res. 2:1, 179-191. Franceschi C., Licastro F., Chiricolo M., Bonetti F., Zannotti M., Fabris N., Mocchegiani E., Fantini M. P., Paolucci P. and Masi M. (1981) Deficiency of autologons mixed lymphocyte reactions and serum thymic factor level in Down's syndrome. J. Immun. 126, 2161-2164. Goldstein I. J., Hughes R. C., Monsigny M., Osawa T. and Sharon N. (1980) What should be called a lectin? Nature 285, 66. Hazuda D. J., Lee J. C. and Young P. R. (1988) The kinetics of interleukin 1 secretion from activated monocytes. Differences between interieukin 1~ and interleukin l/1. J. biol. Chem. 263, 8473-8479. Hogqulst K. A., Unanue E. R. and Chaplin D. (1991) Release of IL-1 from mononuclear phagocytes. J. Immun. 147, 2181-2186. Kohayashi Y., Appella E., Yamada M., Copeland T. D., Oppenheim J. J. and Matsushima K. (1988) Phosphorylation of intracellular precursor of human IL-1. J. Immun. 140, 2279-2287. Kretz O. (1992) Isolement et ~tude des propri~t~ physicochimiques, biologiques et toxicologiques de la bol&~atine, toxine prot~que glycoprot£,iqueextraite de Boletus satanas Lenz. PhD thesis, Louis Pasteur University, Strasbourg, France. Kretz O., Creppy E. E. and Dirbeimer G. (1991a) Disposition of the toxic protein, bolesatine, in rats: its resistance to proteolytic enzymes. Xenobiotica 21, 65-73. Kretz O., Creppy E. E. and Dirheimer G. (1991b) Characterization of bolesatine, a toxic protein from the mushroom Boletus satanas Lenz and its effects on kidney cells. Toxicology 66, 213-224. Kretz O., Creppy E. E., Bouianger Y. and Dirheimer G. (1989) Purification and some properties of bolesatine, a protein inhibiting/nvitro protein synthesis, from the mushroom Boletus satanas Lenz (Boletaceae). Arch. Toxic. Suppl. 13, 422-427.
et al.
Lee J. C., Truneh A., Smith M. F. and Tsang K. Y. (1987) Induction of interleuidn 2 receptor (TAC) by tumor necrosis factor in YT cells. J. Immun. 139, 19351938. Licastro F., Chiricolo M., Barbieri L. and Stirpe F. (1983a) Effect of 32 purified animal and plant lectins on human T lymphocytes. Lectins, Vol. 3, pp. 293-302. Walter de Gruyter, Berlin. Licastro F., Tabacchi P. L., Chiricolo M., Parente R., Cenci M., Barboni F. and Franceschi C. (1983b) Defective selfrecognition in subjects of far advanced age. Gerontology ~ , 64-72. Licastro F., Franceschi C., Chiricolo M., Battelli M. G., Tahacchi P. L., Cenci M., Barboni F. and Pallenzona D. (1982) DNA repair after gamma radiation and superoxide dismutase activity in lymphocytes from subjects of far advanced age. Carcinogenesis 3, 45-48. Lomedico P. T., Gubler U., Hellman C. P., Dukovich M., Giri J. G., Pan Y.-C. E., Collier K., Semionow R., Chua A. O. and Mizel S. B. (1984) Cloning and expression of murine interleuldn-1 cDNA in Facherichia coll. Nature 312, 458-462. Lonnemann G., Endres S., Van der Meer J. W. M., Cannon J. G., Koch K. M. and Dinarello C. A. (1989) Differences in the synthesis and kinetics of release of interlcukin 1~, interleuidn Ifl and tumor necrosis factor from human mononu¢lear cells. Eur. J. Immun. 19, 1531-1536. Maeder A. (1975) Toxicologic report 1974: Report of poisoning of humans by mushrooms in Switzerland. Bull. Swisse Mycol. 53, 151-153. Olsnes S. and Pih] A. (1977) Receptors and Recognition, Vol. 1, pp. 129-173. Chapman and Hall, London. Romagnani S., Maggi E., Amadori A., Giudizi G. and Ricei M. (1977) Co-operation between T and B lymphocytes from human tonsils in the response to mito~ns and antigens. Ciin. exp. Immun. 7,8, 332-340. Sharon N. (1983) Lectin receptors as lymphocyte surface markers. Adv. Immun. 34, 213-298. Smith K. A. (1988) Interieukin-2: Inception, impact, and implications. Science 240, 1169-I 176. Smith K. A., Lachman L. B., Oppenheim J. J. and Favata M. F. (1980) The functional relationship of the interleukins. J. lmmun. 151, 1551-1556. Stirpe F., Legg R. F., Onyon L. J., Ziska P. and Franz H. (1980) Inhibition of protein synthesis by a toxic lectin from Viscum album L. (mistletoe). Biochem. J. 190, 843-845. Stirpe F., Gasperi-Campani A., Barbieri L., Lorenzoni E., Montanaro L., Sperti S. and Bonetti E. (1978) Inhibition of protein synthesis by modeccin, the toxin of Modecca digitata. FEBS Lett. 85, 65-67.
0020-711X/93 $6.00 + 0.00 Per~tmon Press Ltd
Printed in Great Britain
MITOGENIC ACTIVITY AND IMMUNOLOGICAL PROPERTIES OF BOLESATINE, A LECTIN ISOLATED FROM THE MUSHROOM BOLETUS SATANAS L E N Z FIU~I~ICO LICASTEO,I * M ~ C ' m S ~ A Motto-I, x OLivn~ K a z r z , 2 Gu~ Dntm~Im~n, 2 EDMOI~D EKUE CREPPY3 and FIOp.EI~ZOSTIR~ I ~Dipartimento di Patologia Sperimentale, Universit/t di Bologna, Via San Giacomo 14, 1-40126 Bologna, Italy [Tel. (051) 242084; Fax (051) 251315], 2Institut de Biulogie Mul/sculalre et Cellulalre du CNRS and Universit~ Louis Pasteur, 15 rue Descartes, F-67084 Strasbourg, France and 3Laboratoire de Toxicolosie et d'Hygi~ne Appliqu~e, 3 tar Place de la Victoire, F-33076 Bordeaux Cedex, France (Received 23 April 1992)
Al~raetml. A lectin has been purified from the mushroom Boletus satanas Lenz. 2. The protein, called bolesatine, is mitogenic for human T lymphocytes in a dose- and time-dependent msnner.
3. Optimal mitogenic doses induce the release of interleukin-l~ and interleukin-2 from mononudenr cell cultures.
INTitODUCTION
Lectins are sngar-binding proteins which agglutinate cells or precipitate glycoconjugates (Goldstein et al., 1980; Sharon, 1983). These substances are found in plants and animal tissues, as well as in microorganisms (Sharon, 1983). Some lectins induce lymphocyte proliferation (Licastro et al., 1983a) and some of them are toxic and inhibit protein synthesis (Olsncs and Pihl, 1977; Stirpe et al., 1978, 1980; Barbieri et
a/., 1984).
Bolesatinc is a lectin purified from Boletus satanas Lenz (Kretz et al., 1989), a mushroom causing.serious gastroenteritis in humans (Maeder, 1975). Bolesatine is toxic and results in animal's death with an unspecific symptomatology (Kretz et al., 1991a). Furthermore, it inhibits D N A and protein synthesis in kidney cell cultures (Kretz et al., 1991b). Thus, this lectin might have a role in the toxicity of Boletus satanas Lenz. Here we present results regarding the effect of the lectin on human lymphocytes. Bolesatine is mitogenic for T cells and induces the release of interleukin-lu (IL-I~) and interleukin-2 (IL-2) in supernatants from mononuclear cell cultures.
MATERIALS AND MgrHO[~ Bolesatine isolation Bolesatine was isolated and purified from the mushroom Boletas satanas Lenz (Boletazeate)by two fast protein liquid chromatography (FPLC) methods on anion exchangers
*Addre~ correspondence to: Prof. F. ~ , Dipertimento di Patologia Sperimentale, Via San Giacomo 14,1-40126 Boingna, Italy. 789
after ammonium sulphate precipitation ~ et al., 1989). The protein concentration was determined according to the method of Bradford (1976). Separation of peripheral blood mononuclear cells Peripheral blood mononuclear ceJls were obtained from venous blood of healthy adult volunteers by.density gradient centrifugation (Bzyum, 1968) on lymphoprep (Immuno, Italy). Cells were 95% lymphocytes and 5% monocytes and cell viabifity ranged from 90 to 98%. Mononuclenr ~ were suspended in a serum-free medium which con~ted of Iseove's modification of Dullxx~o's medium (Gibco, U.S.A.) supplemented with bovine serum albumin (800/~8/ml; Cohn Fraction V, Sigma, U.S.A.), iron-free human tran~'errin (2/~g/ml; Sigma, U.S.A.), 100 U/m1 penicillin and 100 t*sJml streptomycin (Flow Labs, U.K.). Purification of T and B lymphocytes T and B lymphocytes were separated by rosette formation with sheep red blood cells and subsequent gradient centrifugnfion according to the method o f Roms$nani et al. (1977) with slight modifications (Francesehi et ai., 1981). Lymphocyte proliferative assay Cell cultures, with and without lectins, were performed as previously described (Franceschi et al., 1978) with slight modifications (Licastro et al., 1983b). Briefly, mononuciear cells or purified T and B lymphocytes (2 × l0s viable cells in 0.1 rul of serum-free medium) were distributed in quadruplicate in microplate wells. Test cultures were stimulated with phytohemasglutinin-P (PHA; Difco Labs, U.S.A.) or bolesatine diluted in a serum-free medium. Cells were then cultured in a CO2 incubator. Cell proliferation was asse~d by pulsing the cultures with 0.5/~Ci/well of methyl-[Sl-l]thymidine (sp. act. 5Ci/ mmul; the Radiochemical Center, U.K.) during the last 6 hr. Cell cultures were harvested on glass fiber filters with the aid of a multiple cell-harvester (Skatron, Norway) and [~H]TdR incorporation was measured by liquid scintillation counting
790
FBDERICOLICASTROet al. 500 - mPHA20/~g/ml o Bolesotine0.01/~g/rnl 250 /~Bolesotine0.1/~g/ml 200
"T
|
0 Bo~otin~ 10/~g/ml
900
~IINo mitogen
800
r~IPHA 20 ~g/ml P-~Bolesotine 0.1/.tg/ml
7O0
T~ ~
600
~ 1~
g lo0 50
O--
O-,
O
O
O
72
96
120
°° 1O0
48
144
o
Hours
Fig. I. Time-dependent lymphocyte proliferation induced by three doses of bolesatine and one concentration of PHA. Results are expressed as mean + SE.
as previously described (Franceschi etal., 1978) with slight modifications (Licastro etal., 1982). IL-1= and IL-2 determination Mononuclear cells were cultured with or without PHA or bolesatine lectin for 24, 48 and 72 hr, then supernatants were collected by centrifugation and stored at -20°C. The amount of IL-I= and IL-2 in supernatants was determined with ELISA kits from Oncomembrane (U.S.A.) and British Biotechnology (U.K.), respectively. RESULTS Bolesatine had a strong mitogenic activity on human peripheral blood lymphocytes at 0.01 pg/ ml. Lymphocyte stimulation was dose- and timedependent, as shown in Fig. 1. Maximum mitogeaic activity was observed when a dose of 0.1 pg/ml of bolesatine was used and at this concentration lymphocyte proliferation was higher than that stimulated by an optimal concentration of PHA (20 #g/ml). At higher doses (10pg/ml), the lectin showed a toxic effect. When tested on purified T and B lymphocytes after 3 days of culture, the lectin induced the proliferation of T cells only (Fig. 2). Mononuclear cell cultures were activated for 2472 hr by optimal concentrations of bolesatine or P H A
m N o mitogen r--IPHA ~0 ~g/ml ~Bolosatinl 0.01/~g/ml FT"JBole~otine 0.1/~g/rnl
70 60-
~~
~
24
1
In 48
i
tn
,
72
Hours
Fig. 3. Time-dependent IL-I= release in supernatants of bolesatine- and PHA-activated mononuclear cells. Results are expressed as mean + SE. and supernatants were collected to detect the release of free cytokines. Bolesatine induced a very high and time-dependent secretion of free IL-I= from monocytes, whereas P H A did not stimulate a significant release of this cytokine (Fig. 3). Both bolesatine and P H A induced IL-2 secretion from activated human mononuclear cell cultures. The levels of free IL-2 in the supernatants from bolesatine-activated T lymphocyte~ were lower than those from cultures activated by PHA after 48 and 72 hr of incubation (Fig. 4). D~CUSSION
The proteinaceous toxin purified form Boletus satanas Lenz (Kretz etal., 1989) is toxic when injected in mice, being the LD~0 3.3 mg/kg of body weight (Kretz e t a l . , 1991a). Bolesatine inhibits D N A synthesis of cultured kidney cells in a dose-dependent manner, with an IC~oo f 0.32 p M (Kretz et al., 1991b). Protein synthesis is also inhibited "in the same culture conditions (IC~0 = 0.14 pM). The molecule binds to galactose residues of cell membrane receptors and behaves like a lectin, since the addition of D-galactose to the cell culture medium prevents the cytotoxicity of bolesatine (Kretz et al., 1991b). This notion has been confirmed by studies 1600
80 - 72 hour=
I1,.
1400
m No mitogen ~ P H A 20/~g/rnl 12~~oleaotine 0.1/~g/ml
1200 ... E 1000
$~ ,~
40
-~ ~ 0 50
-
~
-
10
-
41~
_
TcelI=
--~--[~ B cells
Fig. 2. Proliferation of purified T and B lymphocytes induced by bolesatine and PHA after 72 hr of culture. Results are expressed as mean + SE.
0
48 Hours
72
FiB. 4. Time-dependent IL-2 release in supernatants of bolesatine- and PHA-activated mononudear cells. Results are expressed as mean + SE.
Immunomodulation by bolesatine focused on the agglutinating activity of bolesatine
(Wretz, 19~2). Bolesatine is a potent mitogen for human T lymphocytes. A strong proliferation is induced by as little as 0.01/~g/ml of the compound and the maximum effect is observed with a concentration of 0.1 #g]ml. However, higher concentrations of the lectin inhibit cell proliferation, indicating a cytotoxic activity, possibly ascribable to the inhibition of protein synthesis. The high release of IL-I~, induced by bolesatine indicates that the lectin is a potent activator of human monocytes. Two different molecular forms of IL-1, called IL-I~ and IL-1/~, have been identified (Auron et al.., 1984; Lomedico et al., 1984). These 'molecules are synthesized in monocytes as 31 kDa precursors, which appear to be processed into lysosomes and transformed into the mature forms of 17kDa (Bakouche et al., 1987). After 18-24 hr of activation by LPS, IL-I~, precursor mostly remains cell associated (Lonnemann et al., 1989). The phosphorylation of the intracellular precursor of IL-lu facilitates the processing and the secretion of the 17 kDa mature form (Kohayashi et aL, 1988). The IL-I~ precursor appears to be also associated to the cell membrane (Beuscher et al., 1987) and the further processing of the molecule seems to be induced by extracellular serine proteases (Auron et al., 1987). Bolesatine is a very potent stimulus for the release of IL-I~ by human monocytes. It is possible that bolesatine, at variance with PHA, affects one of the above-mentioned metabolic steps; for instance, bolesatine may induce an increased level of phosphorylation or an augmented production of proteases able to cleave the membrane IL-I~ precursor. This notion may explain the differential release of IL-I~ induced by bolesatine in comparison to PHA. It is important to stress that we cultured human cells in a serum-free medium, thus exogenous proteases were not added to the cell cultures. The kinetics of IL- 1 secretion are peculiar, since the release of both IL-I~ and /~ is delayed following the synthesis and the precursor form accumulates in the cytoplasm (Hazuda et al., 1988). This observation has been recently confirmed and it has been suggested that most IL-I~, could be released after cell injury (Hogquist et al., 1991). It is important to keep in mind that bolesatine is toxic for cultured kidney cells. Thus, an alternative mechanism for the elevated timedependent release of free IL-I~, may be an injury to monocytes induced by bolesatine. The high level of IL-I~, may contribute to the elevated mitogenic activity of T lymphocytes, since this cytokine regulates the proliferation of activated T lymphocytes (Dinarello, 1989). Preliminary results show that bolesatine induces a high release of tumor necrosis factor-~ (TNF-~) from human mononuclear cell cultures after 24 hr of incubation (data not shown). Both IL-I~ and TNF-~ have been shown to enhance the expression of the ~, subunit of the IL-2 receptor and to synergize with IL-2 for the mito-
791
genesis of T lymphocytes (Smith et al., 1980; Lee et aL, 1987). The mitogenic activity of bolesatine is also mediated by the release of IL-2 by T helper cells. The level of this lymphokine in supernatants of proliferating lymphocytes depends upon the balance between its production by activated T helper cells and its absorption by high affinity receptors expressed on proliferating T lymphocytes (Smith, 1988). The reduced level of free IL-2 detected after 3 days in cultures activated by bolesatine may be secondary to an elevated expression of high affmity IL-2 receptors, with a consequent stimulation of mitogenic activity of T lymphocytes. The Boletus satanas mushroom is toxic and muses serious gastroenteritis in human beings. The toxicity of the mushroom may be partially ascribed to compounds, such as bolesatine, which are capable of inhibiting protein synthesis of epithelial cells and inducing a strong release of cytokines, such as IL-l~, and TNF-~ from gastroenteric macrophages. SUMMARY
Here we show that the lectin bolesatine isolated from the mushroom Boletus satanas Lenz has a strong immunomodulatory potential. It is able to stimulate the proliferation of human T lymphocytes and to promote the secretion of monokines and lymphokines, such as IL-lu and IL-2. Thus, the gastroenteritis induced by the ingestion of the mushroom may be partially ascribed to this lectin through mechanisms which activate immune responses. research was supported by M.U.R.S.T., CNR "Progetto Finalizzato Biotecnologie • Biostrumentazione", Pallotti's Legacy for Cancer Research, Ligue Nationale Fran~aise contre le Cancer, Comit~ D~partemental de la Gironde and INSERM Contract Recherche externe No. 89/2007.
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