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Effector-target cell interaction of lymph node cells from galactocerebroside-sensitized rats with oligodendrocytes of brain cell cultures
Journal ofNeuroimmunology, ] (1981) 191-194
|~!
Elsevier/North.Holland Biomedic~Press
Effector-target cell interaction of lymph node cell~ from l~alactocerebroside-sensitized rats with oligodendrocytes of brain cell cultures B. Niedieck and U. Lo.hmann Heinrich-Pette-~n~titutffir Experimentelle Virologie und Immunologic an tier Univer~i~it Hamburg, Martinistrasse 52, 2000 Hamburg 20 ( F.R. G.)
(l~¢ceived7 November, I~'80) (Accepted29 ~anuary,19:~1)
Summary Inbred Lewis rats :vere sensitized with galactocerebrosides (GC) and hemocy~J~ After 10 days lymph node cells (LNC) depleted of phagocytic cells were o b ~ These cells were tested on mixed brain cell cultures enriched with CK:-posi~ive oligodendrocytes. Within 3-48 h of incubation, conjugate formation betweea LNC and oligodendrocytes was observed. Interaction of effector cells with ~ tarot resulted in defective oligodendrocytes. Anti-GC-LNC did not adhere to o~ bfl© @strocytes or fibroblasts. Control LNC from rats sensitized only with the carri~ protein did not interact with oligodendrocytes.
Intredueflun Galactocerebrosides (GC) are able to induce a humora~ immune response if inoculated together with a carrier protein and Freund's adjuvant (Niedieck et ~1. 1%5). Anti-GC sera proved to be myelinotoxic on cerebellum tissue cultures (Dubois-Dalcq et al. 1970). The GC h~,pten is found not only in myelin but also o~ the cell surface of the myelin producing oligodendrocytes (Raft et al. 1975). These facts raised the following questions: (1) Is it possible to detect a ~ immune response to GC in addition to the humoral one and (2) do lymph node cells (LNC) from animals sensitized against GC interact with oligo~,~'ndrocytes in ~i~ro?
This work was suppofledby Gemeinut~zise Henic-Sfiftun8,Frankfi,~/Main. Federal P.~mb~¢ c~ Germany. Addrcs:.for reprint requests: Dr. Y).~iedieck, Heinrich-P~t~e-lns~mt,Maninisu'asse52, ~000 ".~m~bur8 20, F.R.C~.
1~2 l~ethods and Results Five experiments were done. In each, 2 inbred Lewis rats were injected into the hind footpads with an emulsion containing G C and keyh{-~te limpet hemocyanin in complete Freund's adjuvant. Every animal received 0.3 w.g GC and 0.3 mg hemocyanin (Niedieck 1975). Two control animals were inoculated with the same emu|sion without GC. Ten days later. LNC susp,msions, depicted of phagocytic cells, were aseptically prepared from tl:e draining lymph nodes. Fhe LNC of 2 rats were pooled to obtain enough cells for the examination of 10 cover slips. Mixed brain cell ~.ultures from syngeneic 4-day-old rats were obtained without the use of enzymes. Oligodendrocytes could be identified with anti-CC sera by immunofluorescence techniques (Raft et al. 1978). Cultures especially enriched with oligodendroc)tes (Lohmann and Niedieck, in preparation) were used as target for the effector LNC. The brain cells grown on cover slips in Petri dishes were ~,verlaid with an LNC suspension containing 2 × 106 L N C / c m 2 of the brain cell monolayer. After different intervals of incubation at 37°C, the cultures were washed and examined, either alive in lhe l:hase contrast microscope IM 35 (Zeiss) or after staining.
Fig. I. Neonatal rat brain cultures, A: Oligotlendrocyteafter 20 hours of incubationwith anIi-GC-LNC, Phaco. livingculture: × 450. B: Effcctor-targetcell conjugateafter 20 h of incub-'.tionwith anti-GC-LNC. May-(iriinw~M-(iicmsa: ~ 14(70.C: Olig~lcn6rtvcyteshowinga cytotoxiceffect after 20 h of incubz~tion with anti-GC..LNC.May-Crf~nwald.-Giemsa: × 17(70.D: Oligodendrocyteof a culture inct~batcdwith anti-hcmocyanin-LNCfor 2)h. Mas-(iriinv, ald-(iicmsa: ~',1400.
(1) After 20 h of incubation, LNC from rats sensitized with GC accumulated ,,.| and adhered to the perikaryon and to the prc,cesses of oligodendrocytes. The tar~-t cells were covered with 10-50 LNC (Figs. IA and B). Frequently, p r o c e s ~ ef oligodendrocytes were marked by strings of adherent effet"or cells. In the 5 experiments, only t0-20% of oligodendrocytes were found with less than 10 LNC/celI. Most of these oligodendrocytes were more or less damage:l, as sho~n L~ Fig. IC. On protoplasmatic astrocytes no or only vet'y few LNC were seen. Ttms~ cells proved to be GC-negative but positive for the intracellular marker gl~al fibrillary acidic protein and therefore served as target comroi. (2) In the 5 control e:periments,0which were done with anti-hemocyanin LNC, we did not observe conjugate formation on oligodendrocytes after 20 h of incubation (Fig. ID). If on 20-30% of the oligodendrocytes up to 6 LNC were found in the entire process field, this was regarded as accidental, because no~ all unbound LNC were eliminated by the washing l~rocess. According to these criteria, the 5 control experiments were negative. The specificity of the LNC reaction was confirmed in 2 inhibition experiments. Ten per cent of heat-inactivated anti-GC serum or, for control, anti-henmc~anin serum were added to the anti-GC-LNC suspension. On cover slips incubated ,~ith anti-GC-LNC in the 0resence of anti-GC antibodies, no effector-target cell conjugation was observed, ~vhereas anti-hemocyanin antibodies did not inhibit conjugate formation according to the criteria mentioaed above.
Discussion While in experiments done with anti-cerebroside LNC most of the oligodeadrocytes were loaded with conjugates consisting of high numbers of effector cells, in the contro~ experiments with anti-hemocyanin LNC the majority of oligodendrcojtes were free and only a small percentage of cells was covered with a few LNC. S~ace it i~ known ~hat effector cells, after ~.. pcr;.od of conjugate for,:nation, &ssociate from their larger to adhere to and lyse other target cells, "~: is not -,urprising that oligod~ndrocytes have been found without anti-GC-LNC (Berke 19,':0). Although it is likely that damaged cells are lost during the washing process, defective digodendrocyt~,s are seen, being greater in number after 48 h of incubatiou ~h~a after 20h. 3"he interaction of L N C from rats sensitized again~,;tG C was specific: {I) Astrocytes and fibroblasts of the mixed brain cell cultures were not affected by anti-cerebroside LNC; (2) L N C from control rats that received only the carrier protein did not aggregate at and were not cytotoxic for oligodendrocytes. {3) In inhibition experiments, anti-GC antibodies prevented the production of effectortarget cellconjugates. Further experiments are planned to elucidate whether the observed reactions of anti-GC-LNC are caused by cytotoxic T cells or whether mechanisms of the so-called antibody-dependent cell-mediated cytotoxicity are involved (Lustig and Bianco 1976). The interaction of anti-cerebroside LNC with myelin-prodt~ng ofigodendrocytes seems of some interest for investigations c,~ncerning the etiotog~ and pathogenesis oi' demyelinating diseases.
194 Acknowledgments T h e a u t h o r s t h a n k Dr, L.F. Eng, S t a n f o r d Universiey, for p r o v i d i n g t h e anti-glial fibrillary acid p r o t e i n serum.
References Berke, G., Interaction of cytotoxic T lymphocytes and target ceilz, P~'ogr. Allergy, 27 0980) 69-133. Dubois-Dalcq, M., 3. Nicdieck and M. Buyse, A~tion of an~i-cerebro~ide sera on myelinated nervou~ tissue cultures, Path. Europ., 5 (19"~0) 331-3~,7. Lustig, H.J. and C. Bianco, Antibody-mediated c~ll cyt~,~oxicity in a defined system--Regulation by antigen, antibody, and complement, Jo Immmlology, ! 16 (1976) 253-260. Niedicck, B., E. Ku'~ert, O. Palacios and O. Drees, Immunocl~mical ,and serolo?,ical studies on tl~: lipid hapten of myefin with relationshii: to experimental allergic ,.'nceplialomyelitis. Ann. N.Y. Acad. Sci., 122 (1965) 266-276. Niedieck. B., On a glycolipid hapten of n~y¢lin, Progr. Allergy, 18 (1975) 353-422. Raff, M.~., R. Mirsky, K.L. Fields, F P. Lisak, S.H. Dorfman, D.H. Silberb~g, N.A. Gregson, S. /.eibo~vitz and M.C. Kennedy, Gala~ocerebroside is a specific cell-surface antigenic marker for oli~odendrocytes in ~ulturc, Nature (Load.), 274 (1978) 813-816.
|~!
Elsevier/North.Holland Biomedic~Press
Effector-target cell interaction of lymph node cell~ from l~alactocerebroside-sensitized rats with oligodendrocytes of brain cell cultures B. Niedieck and U. Lo.hmann Heinrich-Pette-~n~titutffir Experimentelle Virologie und Immunologic an tier Univer~i~it Hamburg, Martinistrasse 52, 2000 Hamburg 20 ( F.R. G.)
(l~¢ceived7 November, I~'80) (Accepted29 ~anuary,19:~1)
Summary Inbred Lewis rats :vere sensitized with galactocerebrosides (GC) and hemocy~J~ After 10 days lymph node cells (LNC) depleted of phagocytic cells were o b ~ These cells were tested on mixed brain cell cultures enriched with CK:-posi~ive oligodendrocytes. Within 3-48 h of incubation, conjugate formation betweea LNC and oligodendrocytes was observed. Interaction of effector cells with ~ tarot resulted in defective oligodendrocytes. Anti-GC-LNC did not adhere to o~ bfl© @strocytes or fibroblasts. Control LNC from rats sensitized only with the carri~ protein did not interact with oligodendrocytes.
Intredueflun Galactocerebrosides (GC) are able to induce a humora~ immune response if inoculated together with a carrier protein and Freund's adjuvant (Niedieck et ~1. 1%5). Anti-GC sera proved to be myelinotoxic on cerebellum tissue cultures (Dubois-Dalcq et al. 1970). The GC h~,pten is found not only in myelin but also o~ the cell surface of the myelin producing oligodendrocytes (Raft et al. 1975). These facts raised the following questions: (1) Is it possible to detect a ~ immune response to GC in addition to the humoral one and (2) do lymph node cells (LNC) from animals sensitized against GC interact with oligo~,~'ndrocytes in ~i~ro?
This work was suppofledby Gemeinut~zise Henic-Sfiftun8,Frankfi,~/Main. Federal P.~mb~¢ c~ Germany. Addrcs:.for reprint requests: Dr. Y).~iedieck, Heinrich-P~t~e-lns~mt,Maninisu'asse52, ~000 ".~m~bur8 20, F.R.C~.
1~2 l~ethods and Results Five experiments were done. In each, 2 inbred Lewis rats were injected into the hind footpads with an emulsion containing G C and keyh{-~te limpet hemocyanin in complete Freund's adjuvant. Every animal received 0.3 w.g GC and 0.3 mg hemocyanin (Niedieck 1975). Two control animals were inoculated with the same emu|sion without GC. Ten days later. LNC susp,msions, depicted of phagocytic cells, were aseptically prepared from tl:e draining lymph nodes. Fhe LNC of 2 rats were pooled to obtain enough cells for the examination of 10 cover slips. Mixed brain cell ~.ultures from syngeneic 4-day-old rats were obtained without the use of enzymes. Oligodendrocytes could be identified with anti-CC sera by immunofluorescence techniques (Raft et al. 1978). Cultures especially enriched with oligodendroc)tes (Lohmann and Niedieck, in preparation) were used as target for the effector LNC. The brain cells grown on cover slips in Petri dishes were ~,verlaid with an LNC suspension containing 2 × 106 L N C / c m 2 of the brain cell monolayer. After different intervals of incubation at 37°C, the cultures were washed and examined, either alive in lhe l:hase contrast microscope IM 35 (Zeiss) or after staining.
Fig. I. Neonatal rat brain cultures, A: Oligotlendrocyteafter 20 hours of incubationwith anIi-GC-LNC, Phaco. livingculture: × 450. B: Effcctor-targetcell conjugateafter 20 h of incub-'.tionwith anti-GC-LNC. May-(iriinw~M-(iicmsa: ~ 14(70.C: Olig~lcn6rtvcyteshowinga cytotoxiceffect after 20 h of incubz~tion with anti-GC..LNC.May-Crf~nwald.-Giemsa: × 17(70.D: Oligodendrocyteof a culture inct~batcdwith anti-hcmocyanin-LNCfor 2)h. Mas-(iriinv, ald-(iicmsa: ~',1400.
(1) After 20 h of incubation, LNC from rats sensitized with GC accumulated ,,.| and adhered to the perikaryon and to the prc,cesses of oligodendrocytes. The tar~-t cells were covered with 10-50 LNC (Figs. IA and B). Frequently, p r o c e s ~ ef oligodendrocytes were marked by strings of adherent effet"or cells. In the 5 experiments, only t0-20% of oligodendrocytes were found with less than 10 LNC/celI. Most of these oligodendrocytes were more or less damage:l, as sho~n L~ Fig. IC. On protoplasmatic astrocytes no or only vet'y few LNC were seen. Ttms~ cells proved to be GC-negative but positive for the intracellular marker gl~al fibrillary acidic protein and therefore served as target comroi. (2) In the 5 control e:periments,0which were done with anti-hemocyanin LNC, we did not observe conjugate formation on oligodendrocytes after 20 h of incubation (Fig. ID). If on 20-30% of the oligodendrocytes up to 6 LNC were found in the entire process field, this was regarded as accidental, because no~ all unbound LNC were eliminated by the washing l~rocess. According to these criteria, the 5 control experiments were negative. The specificity of the LNC reaction was confirmed in 2 inhibition experiments. Ten per cent of heat-inactivated anti-GC serum or, for control, anti-henmc~anin serum were added to the anti-GC-LNC suspension. On cover slips incubated ,~ith anti-GC-LNC in the 0resence of anti-GC antibodies, no effector-target cell conjugation was observed, ~vhereas anti-hemocyanin antibodies did not inhibit conjugate formation according to the criteria mentioaed above.
Discussion While in experiments done with anti-cerebroside LNC most of the oligodeadrocytes were loaded with conjugates consisting of high numbers of effector cells, in the contro~ experiments with anti-hemocyanin LNC the majority of oligodendrcojtes were free and only a small percentage of cells was covered with a few LNC. S~ace it i~ known ~hat effector cells, after ~.. pcr;.od of conjugate for,:nation, &ssociate from their larger to adhere to and lyse other target cells, "~: is not -,urprising that oligod~ndrocytes have been found without anti-GC-LNC (Berke 19,':0). Although it is likely that damaged cells are lost during the washing process, defective digodendrocyt~,s are seen, being greater in number after 48 h of incubatiou ~h~a after 20h. 3"he interaction of L N C from rats sensitized again~,;tG C was specific: {I) Astrocytes and fibroblasts of the mixed brain cell cultures were not affected by anti-cerebroside LNC; (2) L N C from control rats that received only the carrier protein did not aggregate at and were not cytotoxic for oligodendrocytes. {3) In inhibition experiments, anti-GC antibodies prevented the production of effectortarget cellconjugates. Further experiments are planned to elucidate whether the observed reactions of anti-GC-LNC are caused by cytotoxic T cells or whether mechanisms of the so-called antibody-dependent cell-mediated cytotoxicity are involved (Lustig and Bianco 1976). The interaction of anti-cerebroside LNC with myelin-prodt~ng ofigodendrocytes seems of some interest for investigations c,~ncerning the etiotog~ and pathogenesis oi' demyelinating diseases.
194 Acknowledgments T h e a u t h o r s t h a n k Dr, L.F. Eng, S t a n f o r d Universiey, for p r o v i d i n g t h e anti-glial fibrillary acid p r o t e i n serum.
References Berke, G., Interaction of cytotoxic T lymphocytes and target ceilz, P~'ogr. Allergy, 27 0980) 69-133. Dubois-Dalcq, M., 3. Nicdieck and M. Buyse, A~tion of an~i-cerebro~ide sera on myelinated nervou~ tissue cultures, Path. Europ., 5 (19"~0) 331-3~,7. Lustig, H.J. and C. Bianco, Antibody-mediated c~ll cyt~,~oxicity in a defined system--Regulation by antigen, antibody, and complement, Jo Immmlology, ! 16 (1976) 253-260. Niedicck, B., E. Ku'~ert, O. Palacios and O. Drees, Immunocl~mical ,and serolo?,ical studies on tl~: lipid hapten of myefin with relationshii: to experimental allergic ,.'nceplialomyelitis. Ann. N.Y. Acad. Sci., 122 (1965) 266-276. Niedieck. B., On a glycolipid hapten of n~y¢lin, Progr. Allergy, 18 (1975) 353-422. Raff, M.~., R. Mirsky, K.L. Fields, F P. Lisak, S.H. Dorfman, D.H. Silberb~g, N.A. Gregson, S. /.eibo~vitz and M.C. Kennedy, Gala~ocerebroside is a specific cell-surface antigenic marker for oli~odendrocytes in ~ulturc, Nature (Load.), 274 (1978) 813-816.