Bioconjugate Chem. 1991, 2, 16- 18
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ARTICLES Production of Mhcrophage-Derived Cytotoxic Factor by N-[3-[(Carbamoylmethyl)t hi01propionylated] Neoglycoproteins Pascal Breton, Patrick Midoux, Claire Petit, Marie-ThBrBse Bousser, Annie Claude Roche, Roger Mayer, and Michel Monsigny' Laboratoire de Biochimie des Glycoconjugues et Lectines EndogBnes, Centre de Biophysique Moleculaire CNRS, INSERM and Universite d'orleans, 1 Rue Haute, F-45071 Orleans Cedex 2, France. Received December 28, 1990
6-Phosphomannosylated bovine serum albumin (Man6P-BSA), a neoglycoprotein endocytosed by macrophages, bearing either 3-(2-pyridyldithio)propionylor 3- [ (carbamoylmethyl)thio]propionyl residues coming from alkylation of thiol residues by iodoacetamide were prepared and tested for their immunomodulator properties. The supernatants of mouse peritoneal macrophages incubated with Man6PBSA bearing 3- [ (carbamoylmethyl)thio]propionylgroups, and by a lesser extent 3-(2-pyridyldithio)propionyl groups, were cytotoxic to L929 cells, suggesting the presence of a tumor necrosis factor like compound. This macrophage-activation process is linked to the capacity of Man6P-BSA to be endocytosed via membrane lectins of macrophages, because the supernatants of macrophages incubated with unglycosylated conjugates were not cytotoxic. The cytotoxic activity induced by 3-[(carbamoylmethyl)thiolpropionyl groups bound onto Man6P-BSA was similar to that induced by Man6P-BSA bearing muramyl dipeptide, indicating that endocytosed neoglycoproteins bearing 3-[(carbamoylmethy1)thiolpropionyl residues are potent macrophage activators.
INTRODUCTION Muramyl dipeptide' (MDP) is the smallest fragment of the bacterial cell wall known to render macrophages cytostatic and cytotoxic against tumorigenic target cells (I). Recently we synthesized a series of acyl pseudopeptide analogues of muramyl dipeptide ( 2 , 3 ) . We have studied their capacity to activate macrophages inducing the release of cytotoxic factors and we found that some derivatives without the muramyl moiety were more active than MDP (2). Because macrophages are devoid of cell surface receptors for MDP ( 4 , 5 ) and knowing that MDP is at least 100 times more efficient when it is coupled to a macromolecular carrier (mannosylated serum albumin and polymer) recognized by membrane lectins of macrophages (6-8) than when it is free, the selected pseudopeptides were targeted to macrophages after coupling them onto 6-phosphomannosylated bovine serum albumin (Man6P-BSA). The pseudopeptides which possess an amino group instead of a carboxylic group were coupled onto glycosylated carriers via a disulfide bridge by using the heterobifunctional reagent SPDP (9). Surprisingly, an inactive pseudopeptide bound to Man6P-BSA by using this method was found as efficient as the active one bound to the same ligand. Man6P-BSA modified by PDP groups (introduced to allow conjugation with the pseudopeptide) was also found to induce the release of cytotoxic factors by macrophages. These surprising results prompt us to examine the effect of the carrier modifications introduced
* To whom reprint request should be addressed.
Abbreviations: CMTP,3-[(carbamoylmethyl)thio]propanoyl; BSA, bovine serum albumin; DTT, dithiothreitol; LDL, lowdensity lipoprotein; LPS, lipopolysaccharide;ManGP, N-[[ p - ( 0 6-phospho-a-~-mannopyranosyl)phenyl] thioamidyl]; MDP,muramyl dipeptide; PBS, phosphate-buffered saline; PDP, 3-(2p y r i d y 1 t h i o ) p r o p i o n y l ; SPDP, N-succinimidyl 3 - ( 2 pyridy1dithio)propionate; TNF, tumor necrosis factor. 1043-1802/91/2902-0016$02.50/0
for the coupling of the pseudopeptide onto its carrier on the macrophage activation. We showed that Man6P-BSA bearing N - [3-[ (carbamoylmethyl)thio]propionyl] (CMTP, COCHzCHzSCHr CONH2) groups generated after alkylation of thiol functions by iodoacetamide was a potent macrophage activator when the conjugates are efficiently internalized by the membrane lectins of macrophages. EXPERIMENTAL PROCEDURES Chemicals. Bovine serum albumin (BSA) and Trisacryl GF05 were purchased from IBF-Biotechnics (Villeneuvela-Garenne, France); N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) and iodoacetamide were from Pierce (Rockford,IL, USA); dithiothreitol (DTT) was from Serva (Heidelberg, FRG); all buffers were extemporaneously prepared and abundantly outgassed under reduced pressure. BSA bearing 16N-[[p-(0-6-phospho-a-~-mannopyranosyl)phenyl]thioamidyl] residues (ManGP16-BSA) was prepared as previously described (10). Muramyl dipeptide (MDP) was synthesized according to ref 11 and (ManGP)16-BSA bearing eight MDP residues [ (Man6P)16-,(MDP)s-BSA] was prepared as previously described (6). Preparation of N-[3-(2-Pyridyldithio)propionylated] Protein a n d Neoglycoprotein [ (Man6P)16-,(PDP)15-BSA and of (PDP)~B-BSA]. To 20 mg of (Man6P)le-BSA or BSA in 1 mL of 0.1 M borate buffer (pH 8.6) was added SPDP (4 mg; 12.8 bmol) in 130 p L of ethanol and the mixture was stirred for 20 h at 20 "C. ( M ~ ~ G P ) ~ G - , ( P D P ) ~ ~was - B Spurified A by gel filtration on Trisacryl GF05 eluted by PBS followed by gel purificat,ion on Trisacryl GF05 in HzO/l-butanol (95:5; v/v) and then freeze-dried. (PDP),,-BSA was purified by gel filtration on Trisacryl GF05 in HzO/l-butanol(955;v/v) and then freeze-dried. Fifteen PDP groups were coupled to (Man6P)l6-BSA and BSA as determined from absorbance at 343 nm in the presence of DTT (9). 0 1991 American Chemical Society
Production of Macrophage-Derived Cytotoxic Factor
Preparation of N-[ 3- [ (Carbamoylmethyl) t hiolpropionylated] Protein and Neoglycoprotein [ ( M ~ ~ G P ) I ~ - , ( C M T P ) ~ ~and - B S(CMTP)~S-BSA)]. A Ten milligrams of ( M ~ ~ G P ) ~ ~ - , ( P D P ) I ~or- B(PDP)15SA BSAin 0.1M citrate-phosphate buffer (pH 5.3) was treated with DTT (25 mM final concentration) at 20 "C for 30 min. DTT and other small molecules were eliminated by gel filtration on Trisacryl GF05 eluted by the same citratephosphate buffer outgassed by bubbling nitrogen. Free thiol groups of proteins reacted with iodoacetamide (1% final concentration) for 1h a t 20 "C, the pH being adjusted to 7.5 with a 0.2 M sodium carbonate solution. The solution volume was reduced by freeze-drying. (Man6P)16-,(CMTP)IS-BSA and (CMTP)ls-BSA were finally purified on Trisacryl GF05 columns as described above. Macrophage Supernatants. (C57BL/6 X Balb/c) F1 mice (6-7 weeks old) purchased from CSEAL (Orleans, France) were injected ip with 2 mL of thioglycolate medium (Institut Pasteur, Paris, France). Four days later, peritoneal macrophages were harvested by lavages of the peritoneal cavity and identified by neutral red uptake, as previously described (6). Thioglycolate-elicited peritoneal macrophages (2.5 X lo5) were plated in 16 mm diameter well of multiwell plates (Falcon, Becton-Dickinson, Sunnyvale, CA) in serum-free RPMI medium. Upon 2-h incubation at 37 "C in a humidified atmosphere containing 5 90 C02, nonadherent cells were discarded; adherent cells were cultured in 0.5 mL of serum-free RPMI medium supplemented with antibiotics and L-glutamine and in the presence or absence of 100 pg/mL stimulating agents; incubations were conducted in the presence of 10 pg/mL polymyxin B (Sigma, St. Louis, MO). Culture supernatants were collected after 24-h incubation at 37 "C. Before testing, supernatants were rendered cell-free by centrifugation at 2000g for 10 min. Cytotoxic Factor Assay. The cytotoxic activity of macrophage culture supernatants was determined according to ref 12. Briefly, L929 cells (kindly given by Dr. C. De Chastellier, Institut Pasteur, Paris, France) in exponential phase growth were pretreated with 2 pg/mL actinomycin D (Calbiochem, La Jolla, CA) for 2 h at 37 "C in a humidified atmosphere containing 5% COS (13). Actinomycin D pretreated L929 (actD-L929) cells were collected after trypsin treatment, and seeded in 96-well microplates (4 X lo4 cells suspended in 0.05 mL serumfree RPMI medium per well). After 3-4 h at 37 "C, diluted supernatants from stimulated macrophages were added (0.05 mL) and the actD-L929 cells were further incubated at 37 "C for 18-20 h in a humidified atmosphere with 5% CO2. Three wells containing cells treated under identical conditions were used. Microplates were washed with PBS and the percentage of cell lysis was determined after staining the cells with 0.05 mL of crystal violet (0.2% in 2 % ethanol). The plates were then washed with tap water and the dye was solubilized by adding 0.05 mL/well of 0.3% sodium dodecyl sulfate. Absorbance of each well was read at 570 nm on Dinatech Micro-Elisa autoreader (Artek System Corp., Farmingdale, NY). Percent cytotoxicity was calculated according to (ANs - &)/A,, X 100 where ANSand As were the absorbances ofwells containing target cells incubated with supernatant dilutions of nonstimulated and stimulated macrophages, respectively. All experiments were conducted four times under identical conditions. Results from single experiments were expressed as mean values of triplicate determinations. Results from different experiments were very similar. All cell lines were regularly checked under fluorescence
Bioconjugate Chem., Vol. 2, No. 1, 1991
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SUPERNATANT DILUTIONS
Figure 1. Thioglycolate-elicited peritoneal macrophages were incubated a t 37 "C for 24 h in the presence of polymyxin B with either 100 pg/mL (ManGP)16-,(MDP)*-BSA (W), (Man6P)ls(CMTP)l6-BSA (A),(ManGP)l6-,(PDP)15-BSA (O), (CMTP)lsBSA ( O ) , (PDP)lS-BSA (a),or (ManGP)le-BSA (A). Cytotoxic activity was then tested with the corresponding supernatants incubated 18-20 h with actD-L929 cells. Cytolysis was assessed by counting viable cells upon crystal violet coloration. Supernatants were collected from two wells and a triplicate cytolysis assay was performed (mean deviation < 10%).
microscope to detected putative mycoplasma contamination by using either bisbenzimidazole (14) or a benzoxazinone derivative (15). RESULTS
Effects of PDP and CMTP Conjugates on the Macrophage Cytotoxicity. (ManGP)16-BSA and BSA were substituted upon reaction with SPDP by 15 342pyridy1dithio)propionyl groups [ (Man6P)16-,(PDP)15BSA and (PDP)ls-BSA]. These conjugates, after treatment with DTT to generate thiol groups, were reacted at neutral pH with iodoacetamide to give (ManGP)16-BSA and BSA bearing 15 3- [ (carbamoylmethyl)thio]propionyl groups (Man6P)I~-,(CMTP)~~-BSA and (CMTP)IS-BSA, respectively. Mouse thioglycolate-elicitedperitoneal macrophages were incubated at 37 "C in the presence of 100 pg/mL of each conjugate. The incubations were performed in the presence of polymyxin B to avoid side effects due to LPS contamination in serum, medium, or sample preparations. The culture supernatants were collected after 24 h and their cytotoxicity activities were tested on a tumor necrosis factor (TNF) sensitive L929 cell line. Supernatants from macrophages incubated with ( M ~ ~ G P ) ~ G - , ( C M T P ) ~ ~were - B S found A as cytotoxic as (ManGP)ls-,(MDP)s-BSA on actD-L929 cells: 50% cytotoxicity dose was obtained at l / 2 supernatant ~ dilution (Figure 1). Supernatants from macrophages incubated with (ManGP)16-,(PDP)ls-BSA were found to be less cytotoxic: 50% cytotoxicity dose was obtained at l / 1 6 supernatant dilution (Figure 1). Under the same conditions, supernatants from macrophages incubated with (Man6P)le-BSA or (PDP)ls-BSA or (CMTP)15-BSA did not induce cytotoxicity against actD-L929 cells (Figure 1). The viability and the growth of L929 cells were not affected when they were cultured in medium containing 100 pg/mL of either ( M ~ ~ ~ P ) I ~ - , ( P D P ) ~ ~ or- B S A ( M ~ ~ G P ) ~ ~ - , ( C M T P ) ~ S -(data B S A not shown). As a further control, (Man6P)ls-BSA was incubated with iodoacetamide and purified by gel filtration under the same conditions described for t h e p r e p a r a t i o n of (M~~GP)~G-,(CMTP)~~-BSA. The culture supernatants of macrophages collected after incubation with this iodoacetamide-treated (ManGP)16-BSA were tested on actDL929 cells and the cytotoxicity was as low as that obtained
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Bioconjugafe Chem., Vol. 2, No. 1, 1991
with supernatants from macrophages incubated in the presence of (ManGP)16-BSA or (PDP)lb-BSA or (CMTP)lb-BSA (data not shown). This result indicated that the cytotoxic effect of (ManGP)ls-,(CMTP)15-BSA was not due to traces of iodoacetamide nor to modifications of ManGP16-BSA in the presence of iodoacetamide. DISCUSSION
These results demonstrated that supernatants from macrophages incubated with 6-phosphomannosylated BSA bearing 3-[(carbamoylmethyl)thio]propionyl (CMTP) and in a less extent 3-(2-pyridyldithio)propionyl(PDP) groups can provide a signal to render macrophages cytotoxic for actD-L929 cells. The cytotoxic effect was due to 3- [(carbamoylmethyl)thio]propionylor 3-(2-pyridyldithio) propionyl groups linked to lysine residues of BSA bearing Man6P residues, a neoglycoprotein endocytosed by macrophages via membrane lectins. Macrophages did not possess receptor for N-[3-[ (carbamoylmethyl)thio]propionyl] groups as evidenced by the inefficiency of BSA bearing N- [ 3- [ (carbamoylmethyl)thio] propionyl] groups to render macrophages cytotoxic for L929 cells. Modified proteins such as LDL in which lysine residues have been modified by acetylation, maleylation, or succinylation bind to the scavanger receptor of macrophages (16)and specially maleylated BSA which binds to this receptor were effective in rending macrophages cytotoxic (17). In this case a specific cell surface receptor was implicated. Like MDP, macrophage activation by 3-[ (carbamoylmethyl)thio]propionyl group required its internalization into macrophages which was performed with specific carrier such as BSA bearing mannose 6-phosphate residues specifically recognized by membrane lectins whereas unglycosylated BSA was totally ineffective. Recently, Petit et al. reported that MDP linked to Man6P-BSA or Man-BSA induced the release of tumor necrosis factor (TNF) by macrophages (18). The cytotoxic effect of macrophages incubated with (ManGP)16-,(CMTP)lb-BSA (50% cytotoxicity a t l/zm supernatant dilution) was close to that obtained with (Man6P)40-,(MDP)g-BSA (50% cytotoxicity a t '/256 supernatant dilution) (18). As in the case of MDP, the cytotoxic factor released by peritoneal macrophages stimulated by ( M ~ ~ G P ) I ~ - , ( C M T P ) ~ ~could - B S Abe TNF. In conclusion, proteins bearing 3-(2-pyridyldithio)propionyl or 3- [ (carbamoylmethyl)thio]propionylgroups can induced the release by macrophages of cytotoxic factors if they are internalized by macrophages. Thereby, the heterobifunctional reagent SPDP used to link active molecules or drugs onto macromolecular carriers must be taken with care. This activation process is linked to the capacity of the carrier to be endocytosed. A 3-[(carbamoylmethyl)thio]propionyl group linked on the e-amino group of lysine of BSA bearing mannose 6-phosphate residues is an immunoactivator much less expensive than but as efficient as MDP bound to the same carrier. ACKNOWLEDGEMENT
P.B. received a fellowship from MRT. P.M. and A.C.R. are Charg6 and Directeur de Recherche INSERM, respectively.
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Muramylpeptides: chemical structure, biological activity and mechanism of action. Mol. Cell. Biochem. 41, 27-47. (2) Breton, P. (1989) Synthesis and characterization of putative macrophage activators: pseudopeptide analogues of muramyl dipeptide. Thesis, OrlBans, France. (3) Breton, P., Monsigny, M., and Mayer, R. (1990) Synthesis of new acylpseudopeptides analogous to N-acetylmuramyl dipeptide (MDP). Tetrahedron 46, 4265-4276. (4) Tenu, J. P., Roche, A. C., Yapo, A., Kieda, C., Monsigny, M., and Petit, J. F. (1982) Absence of cell surface receptors for muramylpeptides in mouse peritoneal macrophages. Biol. Cell 44, 157-164. ( 5 ) Fogler, W. E., and Figler, I. J. (1986) The activation of tumoricidal properties in human blood monocytes by muramyl dipeptide requires specific intracellular interaction. J. Zmmunol. 136, 2311-2317. (6) Monsigny, M., Roche, A. C., and Bailly, P. (1984) Tumoricidal activation of murine alveolar macrophages by muramyldipeptide substituted mannosylated serum albumin. Biochem. Biophys. Res. Commun. 121, 579-584. (7) Roche, A. C., Bailly, P., and Monsigny, M. (1985) Macrophage activation by MDP bound to neoglycoproteins: metastasis eradication in mice. Invasion Metastasis 5, 218-232. ( 8 ) Derrien, D., Midoux, P., Petit, C., NBgre, E., Mayer, R., Monsigny, M., and Roche, A. C. (1989) Muramyl Dipeptide Bound to Poly-L-Lysine Substituted with Mannose and Gluconoyl Residues as Macrophage Activators. Glycoconjugates J. 6, 241-255. (9) Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-conjugation. N-succinimidyl3-( 2pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. 173, 723-737. LO) Roche, A. C., Midoux, P., Bouchard, P., and Monsigny, M. (1985) Membrane lectins on human monocytes. Maturationdependent modulation of 6-phosphomannose and mannose receptors. FEBS Letters 193, 63-68. 11) Merser, C., Sinay, P., and Adam, A. (1975) Total synthesis and adjuvant activity of bacterial peptidoglycan derivatives. Biochem. Biophys. Res. Commun. 66, 1316-1322. 12) Fish, H., and Gifford, G. E. (1983) A photometric and plaque assay for macrophage mediated tumor cell cytotoxicity. J . Immunol. Methods 57, 311-325. 13) Ostrove, J. M., and Gifford, G. E. (1979) Stimulation of RNA synthesis in L929 cells by rabbit tumor necrosis factor. Proc. SOC.Exp. Biol. Med. 160, 354-358. 14) Chen, T. R. (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoescht 33258 stain. Exp. Cell Res. 104, 255-262. (15) Monsigny, M., Midoux, P., Depierreux, C., Le Bris, M. T., and Valeur, B. Benzoxazinone kanamycin A conjugate. A new fluorescent probe suitable to detect mycoplasma in cell culture. Biol. Cell, in press. (16) Brown, M. S., and Goldstein, J. L. (1983) Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu. Rev. Biochem. 52,223261. (17) Somers, S. D., Hamilton, T. A., and Adams, D. 0. (1987) Maleylated bovine serum albumin triggers cytolytic function in selected populations of primed murine macrophages. J. Immunol. 139, 1361-1368. (18) Petit, C., Monsigny, M., and Roche, A. C. (1990) Macrophages activation by muramyldipeptide bound to neoglycoproteins and glycosylated polymers: Cytotoxic factor production. J. Biol. Response Modif. 9, 33-43.
