Selectivity of Thy-1 monoclonal antibodies in enhancing neurite outgrowth

Neuroscience Letters', 137 (1992) 75 77 :c 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Selectivity of Thy-1 monoclonal antibodies in enhancing neurite outgrowth Stuart A. L i p t o n a-e, D a n a Leifer ad'e a n d Colin J. Barnstable f Departments o[Neurology, "Children's Howital, ~'Beth Israel Hospital, '~Brigham & Women's Hospital, aMassaehusetts General Hospital and "Program in Neuroscienee. Harvard Medical School, Boston, MA 02115 (USA) and t Department o['Ophthabnology and Vis'ual Science, Yale University School of Medicine. New Haven. CT 06510 ( USA ) (Received 2 October 1991 ; Accepted 3 December 1991 ) Key words'." Immunoglobulin-like domain; Rodent retinal ganglion cell; Thy-I glycoprotein; Active site: Central nervous system: Cell culture; Axonal/dendritic regeneration Thy-1 monoclonal antibodies (MAbs) have previously been shown to promote neurite outgrowth from retinal ganglion cells and a variety of other neurons. We have studied the effect on neurite outgrowth of several Thy-1 MAbs with quantitatively similar binding properties and found that only certain Thy-1 MAbs promote neurite outgrowth. This finding suggests that the antibody effects depend on specific interactions with one or more active sites on the Thy-I glycoprotein

Thy-1 is arguably the most abundant glycoprotein on the neuronal surface in mammals [17, 22], yet its function has remained elusive. Because it bears structural homology to immunoglobulins, it has been suggested that Thy1 may be a recognition molecule in the nervous system [17, 22]. Several other neuronal glycoproteins that function as adhesion molecules are in the immunoglobulin superfamily, such as N-CAM, NILE, MAG and contactin [2, 5, 7, 18, 19]. Thus, it is possible that Thy-I is part of a system forming an adhesive hierarchy for pathway recognition and outgrowth. Several lines of evidence suggest that Thy-I is specifically involved in neurite outgrowth, maintenance, or regeneration. (i) We have demonstrated and others have confirmed that immobilized or cross-linked Thy-1 monoclonal antibodies (MAbs) can promote neurite outgrowth in primary neuronal cultures [8, 14, 16]. (ii) Mahanthappa and Patterson [14] have shown that Thy-I MAbs also enhance neurite outgrowth from PCI2 cells and that mutant PC12 cells that are deficient in Thy-I have more abundant neurites than normal PC12 cells in culture. They therefore suggested that Thy-1 stabilized existing neurites or inhibited new outgrowth and that Thy-1 antibodies block these effects. Morris and colleagues have proposed a similar role for Thy-I based on Correspondence: D. Leifer, Children's Hospital, Enders Bldg, Room 350, 300 Longwood Avenue, Boston, MA 02115, USA. Fax: (1) (617) 730-0636.

in vivo observations that it appears on neuronal processes as they reach their targets and form stable connections [23, 24]. (iii) In lymphocytes and neurons, Thy-1 MAbs induce an increase in [Ca>]~ or in Ca > current, phenomena known to be correlated with neurite outgrowth [1, 6, 9, 11, 20]. (iv) Using anti-idiotype MAbs against Thy-1 MAbs, we have found a putative Thy-1 receptor on astrocytes in the nervous system that appears to mimic the effects ofThy-1 MAbs in enhancing neurite outgrowth [4]. In this paper, using a panel of Thy-I MAbs in rodents, we report that only certain of these MAbs promote process outgrowth despite similar concentration and affinity for Thy-1. Neuronal outgrowth was quantified on 1 2-week-old postnatal rat and mouse retinal ganglion cells, isolated and cultured as described previously [8]. The quantification of neuronal outgrowth was performed using a computer-aided interactive graphics package to trace each neurite [10, 13]. These neurons were identified by retrograde transport of the fluorescent dye granular blue injected in vivo into the superior colliculus 2 days prior to dissociation of the retina. When plated on plain glass or on conventional tissue culture substrates the majority of retinal ganglion cells did not elaborate processes after a day in culture [8]. Fig. 1 illustrates that certain Thy-1 MAbs were effective in promoting neurite outgrowth from retinal ganglion cells while others were not. For example, as previously shown, MRC OX7 and 2G12 enhance outgrowth from

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Fig. I. Various Thy-I antibodies differentially enhance outgrowth by rodent retinal ganglion cells. The ordinate represents the percentage of retinal ganglion cells elaborating one or more processes longer than the diameter of the cell body after one day in culture. On the abscissa is the panel of Thy-1 MAbs or control glass substrate. The MAbs were immobilized on glass cover slips, as described previously [8]. OX7 and 2G12 were incubated with Long Evans rat cultures grown as detailed in Leifer et al. [8], while M5/49. M5/54, and M5/56 were used on C57BL/6 mouse cultures prepared as described by Tauck et al. [2 t]. The degree of outgrowth in the control glass cover slips was determined in both rat and mouse cultures and was essentially identical for each, so the data have been combined. The error bars represent S.D. Statistical analysis was performed using an extension of Fisher's exact test for matrices, as described in Leifer et al. [8]. The outgrowth of cultures plated on MAbs OX7, 2G12 and M5/49 was significantly increased compared to outgrowth on MAbs M5/54, M5/56. or on control glass cover slips (P<0.001 after Bonferroni's correction). Control results on plain glass cover slips using either rat or mouse cells were indistinguishable. Saturating concentrations of each antibody (approximately 10 /~g/ml) were incubated on the cover slips 3 h prior to the addition of the retinal cell cultures [8]. This concentration of MAbs was known to saturate the glass cover slips from parallel incubations with various concentrations of radiolabeled MAbs. For each treatment at least 127 retinal ganglion cells were counted in triplicate experiments.

rat retinal g a n g l i o n cells; 70-85% o f the cells grew at least one process l o n g e r t h a n the d i a m e t e r o f the cell b o d y c o m p a r e d to only 30% in c o n t r o l cultures p l a t e d on plain glass. Several o t h e r Thy-1 M A b s were used t h a t react with m o u s e tissues b u t n o t rat [3]. Therefore, these M A b s were tested in retinal cultures f r o m mice. Thy-1 M A b M5/49 p r o m o t e d o u t g r o w t h . In contrast, M A b s M5/54 a n d M 5 / 56 d i d n o t increase the p e r c e n t a g e o f cells with processes over the c o n t r o l s p l a t e d on plain glass. S i m i l a r c o n c e n trations o f M A b s were used in each case. In addition, the M A b s t h a t p r o m o t e o u t g r o w t h represent v a r i o u s I g G subclasses (IgG~ in the case o f O X 7 a n d IgG2, for 2G12 a n d M5/49). Therefore, i s o t y p e does n o t a p p e a r to be the critical f a c t o r in o b t a i n i n g an effect.

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Fig. 2.'On-rate' of different Thy-I MAbs as measured by their binding to neural tissue bearing Thy-1 glycoprotein on its surface. Assays of relative affinity were made using 3SS-methionine labeled antibodies. These assays were conducted by measuring the amount of radiolabeled antibody binding to cortical membranes immobilized in wells of microtiter plates. The 'on-rate' is shown here; the 'off-rate' was so slow (many hours) that it could not be measured within the time frame of these studies. Thus, the relative affinity of the antibodies of Thyq gtycoprotein was in Fact determined by the 'on-rate~. These studies show no significant difference in affinity between the Thy-I antibodies tested,

It was i m p o r t a n t to ensure t h a t a difference in affinity o f the v a r i o u s M A b s for Thy-1 was n o t the r e a s o n f o r the s u p e r i o r i t y o f some M A b s over others in increasing outgrowth. To q u a n t i f y affinity a technique was used similar to t h a t o f M a s o n a n d W i l l i a m s [15] w h o m e a s u r e d the affinity o f O X 7 for Thy- 1: with r a d i o l a b e l e d Thy- 1 M A b they m e a s u r e d the ' o n - r a t e ' a n d 'off-rate, o f the a n t i b o d ies o n t o Thy-1 c o n t a i n i n g tissues. The relative ' o n - r a t e s ' o f r a d i o l a b e l e d (35S-methionine) M A b s a g a i n s t m o u s e n e u r o n s b e a r i n g Thy-1 were very similar as s h o w n in Fig. 2 which plots label b o u n d vs. time. In this case, the b i n d ing kinetics o f M5/49, a M A b t h a t p r o m o t e s o u t g r o w t h , was nearly identical to t h a t o f M5/54, an M A b not enh a n c i n g o u t g r o w t h ; similar b i n d i n g results were f o u n d for M5/56 a n d were p r e v i o u s l y r e p o r t e d for O X 7 [15]. F u r t h e r m o r e , as p r e v i o u s l y f o u n d by M a s o n a n d Williams, the 'off-rates' were extremely slow a n d therefore d i d n o t greatly influence the a p p a r e n t affinity. Thus, the affinities o f the v a r o u s Thy-1 M A b s were related to the ' o n - r a t e ' a n d were, therefore, quite similar. In s u m m a r y , certain Thy-1 M A b s p r o m o t e o u t g r o w t h o f r o d e n t retinal g a n g l i o n cells a n d others d o not. This v a r i a t i o n c a n n o t be explained by trivial causes such as differences in c o n c e n t r a t i o n or affinity. These results suggest that neurite o u t g r o w t h i n d u c e d by Thy-1 M A b s is not merely the non-specific consequence o f b i n d i n g antib o d y to a very a b u n d a n t cell-surface m o l e c u l e ( T h y - I ) . In fact, this study raises the possibility t h a t there is an

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active site or sites on Thy-1 and that binding to only particular region(s) induces conformational changes that are critical in mediating the effect on neurite outgrowth. We are grateful to Paul Harcourt and Michael Phillips for technical assistance and to Drs. Leonard Levin and Evan Dreyer for helpful discussions. A preliminary account of this work was presented to the Society for Neuroscience at their 1988 meeting [11]. This study was supported by NIH Grants EY06087, CHD00888, and EY05206, NSF Grant BNS-8606145, the American Paralysis Association, and the American Health Assistance Foundation, and Research to Prevent Blindness, Inc. I Connor, J.A., Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central neurons, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 6179 6183. 2 Cunningham, B.A., Hemperly, J.]., Murray, B.A., Prediger, E.A., Brackenbury, R. and Edelman, G.M., Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing, Science, 236 (1987) 799 806. 3 Davignon, D., Martz. E., Reynolds, T., K~rzinger, K. and Springer, T.A., Lymphocyte function-associated antigen 1 (LFA-1): a surface antigen distinct from Lyt-2, 3 that participates in T lymphocyte-mediated killing. Proc. Natl. Acad. Sci. U.S.A., 78 (19811 4535 4539. 4 Dreyer, E.B., Leifer, D., Levin, L.A., Barnstablc, C.J. and Lipton, S.A., A novel astrocyte receptor for neuronal Thy-1 affects neurite outgrowth, submitted. 5 Jessell, T.M., Adhesion molecules and the hierarchy of neural development, Neuron, I (1988)3 13. 6 Kroczek, R.A., Gunter, K.C., Germain, R.N. and Shevach, E.M., Thy-1 functions as a signal transduction molecule in T lymphocytes and transfected B lymphocytes, Nature, 322 (1986) 181 183. 7 Lagenaur. C. and Lemon, V., An LI-like molecule, the 8D9 antigen, is a potent substrate for neurite extension, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7753 7757. 8 Leifer, D., Lipton, S.A., Barnstable, C.J. and Masland, R.H., Monoclonal antibody to Thy-I enhances regeneration of processes by rat retinal ganglion cells in culture, Science, 224 (1984) 303 306. 9 Lipton, S.A., Bursting of calcium-activated cation-selective channels is associated with neurite regeneration in a mammalian central neuron, Ncurosci. Lett., 82 (1987) 21 28. 10 Lipton, S.A., Frosch. M.P., Phillips, M,D., Tauck, D.L. and Aizenman. E., Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture, Science, 239 (1988} 1293 1296.

11 Lipton, S.A. and Kater, S.B., Neurotransmitter regulation of neuronal outgrowth, plasticity and survival, Trends Neurosci., 12 (1989) 265 270. 12 Lipton, S.A., Levin, L.A. and Barnstable, C.]., Various Thy-I antibodies differentially enhance outgrowth by rodent retinal ganglion cells, Soc. Neurosci. Abstr., 24 (1988) 747. 13 Lipton. S.A., Wagner, J.A., Madison, R.D. and D'Amore, RA., Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture, Proc. Natl. Acad. Sci. U.S.A.. 85 (1988} 2388 2392. 14 Mahanthappa, N.K. and Patterson. PH., Antibodies to Thy-I promote neurite outgrowth from rat sympathetic neurons in vitro, and promote neurite initiation by rat adrenal chromaffin and P ( I 2 cells in the absence of NGF, Soc. Neurosci. Abstr., 13 (1987) 5. 15 Mason, D.W. and Williams, A.F., The kinetics of antibody binding to membrane antigens in solution and at the cell surface, Biochem. J., 187(1980) 1 20. 16 Messer, A., Snodgrass, G.L. and Maskin. A., Enhanced survival of cultured cerebellar Purkinje cells by plating on antibody to Th5-1, Cell. Mol. Neurobiol.. 4 (1984) 285 290. 17 Morris,R., Thy-I in developing nervous tissue, Dev, Neurosci., 7 (1985) 133 160. 18 Poltorak, M., Sadoul, R., Keilhauer, G., Landa. C., Fahrig, T. and Schachner, M., Myelin-associated glycoprotein, a member of the L2/HN K-1 family of neuronal adhesion molecules, is involved in neuron oligodendrocyte and oligodendrocyte oligodendrocytc interaction, J. CelI Biol..105 (1987) 1893 1899. 19 Ranscht, B., Sequence of contactin, a 130-kD glycoprotein concentrated in areas of interneuronal contact, defines a new member of the immunoglobulin supcrgene family in the nervous system, J. Cell Biol., 107(1988) 1561 1573. 20 Saleh, M.. Lang, R.J. and Bartlett, P.F., Thy-I-mediated regulation of a low-threshold transient calcium current in cultured sensory neurons, Proc. Natl. Acad. Sci. U.S,A. 85 (1988) 4543 4547. 21 Tauck, D.L., Frosch, M.P. and Lipton, S.A., Bursting of calciunlactivated cation-selective channels is associated with neuritc regeneration in a mammalian central neuron. Neurosci. Lett., 82 (1987) 21 28. 22 Williams, A.F. and Gagnon, J., Neuronal cell Thy-I glycoprolcin: homology with immunoglobulin, Science. 216 (1982} 696 703. 23 Xue, G.P., Calvert, R.A. and Morris, R.J., Expression of neuronal surface glycoprotein Thy-I is under post-transcriptional control, and is spatially regulated, in the developing olli~ctory system, Development, 109 (1990) 851 864. 24 Xue, G.E, Pliego Rivero, B. and Morris, R.J., The surface glycoprotein Thy-I is excluded from growing axons during development: a study of the expression of Thy-I during axonogenesis in hippocampus and lk~rebrain. Development. 112 ( 1991 ) 161 176.