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CD4+CD25+ regulatory T cells and CD1-restricted NKT cells do not mediate facial motoneuron survival after axotomy
Journal of Neuroimmunology 176 (2006) 34 – 38 www.elsevier.com/locate/jneuroim
CD4+CD25+ regulatory T cells and CD1-restricted NKT cells do not mediate facial motoneuron survival after axotomy Cynthia A. DeBoy a,b,*, Susanna C. Byram a,b, Craig J. Serpe b, Danielle Wisuri b, Virginia M. Sanders c,1, Kathryn J. Jones a,b,1 a
c
Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Chicago, 2160 S. 1st Avenue, Maywood, IL 60153, United States b Research and Development Service, Hines VA Hospital, Hines, IL 60141, United States Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Graves Hall, 333 W. 10th Ave, Columbus, OH 43210, United States Received 4 January 2006; received in revised form 5 April 2006; accepted 5 April 2006
Abstract CD4+ T cells rescue facial motoneurons (FMN) from axotomy-induced cell death. The objective of this study is to determine if the CD4+ T regulatory subsets, CD4+CD25+ T or CD1d-restricted NKT cells are critical for FMN survival after facial nerve axotomy. Surviving FMN within facial motor nuclei from axotomized and control sides 4 weeks after axotomy were counted to determine percent FMN survival. Data generated by applying this paradigm to recombination activating gene-2-deficient mice reconstituted with CD4+ T cells depleted of CD4+CD25+ T cells and to CD1 / mice, deficient in CD1d-restricted NKT cells, suggest that neither regulatory CD4+ T subset is critical for FMN survival. D 2006 Elsevier B.V. All rights reserved. Keywords: CD4+ regulatory T cell; CD1-restricted NKT cell; Facial nerve axotomy; Motoneuron survival
1. Introduction Immune cells are capable of promoting neuro-protective effects after neuronal injury (Jones et al., 2005; Serpe et al., 1999). Specifically, the CD4+ T cell has been determined to be critical to mediate facial motoneuron (FMN) survival after facial nerve axotomy (Serpe et al., 2003). However, CD4+ T cell-mediated effects ranging from neuro-repair/protection to neuro-destruction have been described for different neuroinjury models (Fee et al., 2003; Hammarberg et al., 2000; Hauben et al., 2000; Hofstetter et al., 2003; Jones et al., 2002; Kipnis et al., 2002b; Moalem et al., 1999, 2000), necessitating further investigation. It is therefore important to identify * Corresponding author. Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Chicago, 2160 S. 1st Avenue, Maywood, IL 60153, United States. Tel.: +1 410 955 1118; fax: +1 410 502 6367. E-mail address: [email protected] (C.A. DeBoy). 1 Drs. Jones and Sanders share senior authorship. 0165-5728/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.04.006
the specific subsets of CD4+ T cells with neuro-protective potential. In addition to effector cells, CD4+ T cell subsets include regulatory T cells, including CD4+CD25+ regulatory T and CD4+CD1d-restricted natural-killer (NK) T cells, both of which have garnered considerable interest, as each can protect against autoimmune diseases (Bendelac et al., 1997; Gavin and Rudensky, 2003; Gumperz, 2004). CD4+CD25+ regulatory T cells suppress CD8+ T and CD4+ T cell proliferation and promote T cell tolerance (McHugh and Shevach, 2002; Wood and Sakaguchi, 2003). Although CD4+CD25+ regulatory T cells may promote FMN survival after facial nerve axotomy by regulating the ensuing immune response, the regulatory cell may alternatively suppress the CD4+ T cell-mediated neuro-protective effect, as CD4+CD25+ regulatory T cells have been found to increase neuro-damage after optic nerve injury (Kipnis et al., 2002a). CD1d-restricted NKT cell development and activation involves antigen presentation of lipid antigen presented in
C.A. DeBoy et al. / Journal of Neuroimmunology 176 (2006) 34 – 38
CD1d surface molecules on antigen presenting cells (Brigl and Brenner, 2004) and are therefore depleted in CD1deficient mice (Mendiratta et al., 1997; Smiley et al., 1997). Because of the capacity of CD1d-restricted NKT cells to suppress CNS inflammation (Teige et al., 2004) it is of interest to determine if the CD4+CD1d-restricted NKT cell plays a critical in mediating FMN survival. The objective of this study is to determine if the CD4+ regulatory subsets, CD4+CD25+ regulatory T or the CD4+CD1d-restricted NKT cells, are critical to FMN survival after facial nerve axotomy.
2. Materials and methods 2.1. Animals and surgical procedures Female mice, 8– 12 weeks of age including C57BL/6J, BALB/c, and CD1-deficient( / ) (BALB/c background) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and recombination activating gene-2-deficient (RAG-2 / ) (C57BL/6J background) mice were purchased from Taconic Laboratories (Germantown, NY). Pathogenfree conditions were maintained under 12 h light/dark cycles. No manipulations were made until mice acclimated for at least 1 week. Procedures were performed in accordance with NIH guidelines. Facial nerve axotomy was performed as described (Byram et al., 2003; Jones and LaVelle, 1985; Serpe et al., 2003, 1999). Using aseptic techniques and 3% halothane for anesthesia, the right facial nerve of each mouse was completely transected at its exit from the stylomastoid foramen. At the time of sacrifice, behavioral observations were made to assure that no recovery from facial paralysis occurred. 2.2. Preparation of CD4+CD25neg lymphocytes and cell reconstitutions Lymph nodes were aseptically removed from C57BL/6 mice, prepared as a single cell suspension in Hank’s balanced salt solution (HBSS) (Gibco Invitrogen Corp., Carlsbad, CA) + 5% fetal calf serum (FCS) (Gibco Invitrogen Corp.). Supernatant was removed after cells were centrifuged at 300g for 10 min. Cells were resuspended in DNASE buffer (pH = 7.4) for 25 min at 25 -C. DNASE buffer consists of: PBS (Gibco Invitrogen Corp.), 0.5 mM MgCl2 (Sigma-Aldrich), 1 mM CaCl2 (Sigma-Aldrich), 0.5% BSA (Sigma-Aldrich), and 100 U/ml Dnase (Amersham, Piscataway, NJ). Supernatant was removed and cells were resuspended in running buffer which consists of: PBS (Gibco Invitrogen Corp.) + 0.5% bovine serum albumin (Sigma-Aldrich, St. Louis MO) + 2 mM EDTA (Ambion, Austin, TX). A CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec) was used to remove CD4+CD25+ regulatory T cells from a lymphocyte population. Manufacturer’s instructions
35
were followed. Briefly, per 107 lymphocytes, 40 Al running buffer and 10 Al Biotin-Antibody Cocktail were incubated for 10 min at 4 -C, after which 30 Al of running buffer, 20 Al of anti-biotin MicroBeads, and 10 Al of anti-CD25-PE antibody (clone 7D4) were mixed and incubated in the dark for 15 min at 4 -C. Cells were washed and resuspended in running buffer and passed through a 30 AM filter (Miltenyi Biotec) prior to magnetic cell separation with autoMACS automated cell sorter (Miltenyi Biotec) to remove nonCD4+ T cells. The unlabeled CD4+ T cell population was centrifuged and resuspended in 90 Al running buffer + 10 Al Anti-PE MicroBeads per 107 lymphocytes. After a 15min incubation at 4 -C and washing, CD4+CD25+ cells were removed after separation was performed with an autoMACS automated cell sorter. The unlabeled fraction of cells containing CD4+CD25neg cells was centrifuged, and resuspended in running buffer. Remaining magnetically labeled cells were removed with the autoMACS cell sorter. Unlabeled cells were labeled with anti-CD4 MicroBeads and positively selected as described previously (Byram et al., 2004; Serpe et al., 2003). CD4+ T cells were resuspended in PBS to 0.5 107 cells/ ml and 0.2 ml of cell suspension or vehicle alone was injected into the lateral tail vein of recipient mice 1 week prior to axotomy. 2.3. Immunofluorescence staining and flow cytometric analysis Antibody labeling and analysis of isolated CD4+ CD25neg cells were performed as previously described (Byram et al., 2003). Antibodies, purchased from Pharmingen, included FITC-labeled anti-CD4 (clone GK1.5), PE-labeled anti-CD25 (Clone, 3C7), and the isotypematched controls, FITC-labeled rat IgG2b n and PElabeled Rat IgG2b n, respectively. CD4+CD25neg cells population contained greater than 99% of CD4+ T cells and less than 0.2% of contaminating CD25+ cells (data not shown). The percent purity of CD4+CD25neg cells from lymph nodes removed from reconstituted mice 5 weeks after reconstitution contained less than 0.2% of CD4+CD25+ T cells (data not shown). 2.4. Cell-counting procedures Mice were sacrificed by CO2 asphyxiation 4 weeks after facial nerve axotomy. A 3-mm region of brain, containing the facial nucleus, was fixed in methacarn for 24 h. Methacarn fixative consists of: 60% methanol (FisherScientific), 30% 1,1,1-trichloroethane (TCE) (SigmaAldrich), and 10% glacial acetic acid (J.T. Baker, Phillipsburgh, NJ). Tissue was dehydrated in serial washes with isopropyl alcohol and placed in TCE for 16 h. Vacuum infiltration was performed at 60 -C, 23 psi. Tissue was embedded in paraffin. Microtome sections (12 Am) were
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C.A. DeBoy et al. / Journal of Neuroimmunology 176 (2006) 34 – 38
3. Results 3.1. CD4+CD25neg T cells promote WT levels of FMN survival after facial nerve axotomy when reconstituted into RAG-2 / mice To establish if the CD4+CD25+ regulatory T cell is critical for FMN survival after facial nerve axotomy, RAG2 / mice, deficient in functional T and B cells (Shinkai et al., 1992), were reconstituted with CD4+ T cells depleted of CD4+CD25+ regulatory T cells (CD4+CD25neg). FMN survival 4 weeks after injury compared to C57BL/6J WT mice, at 65 T 2.1% (n = 8), was significantly decreased ( p < 0.05) in RAG-2 / mice to 54 T 4.0% (n = 5) of the uninjured control side (Fig. 1). FMN survival 4 weeks after facial nerve axotomy was significantly increased ( p < 0.05) in RAG-2 / mice reconstituted with CD4+CD25neg cells to 69% T 3.8% (n = 7) of the uninjured control side ( p < 0.05), compared to RAG-2 / mice, which was not statistically increased above WT levels. These data suggest
Fig. 1. Effects of reconstitution with a population of CD4+CD25+ regulatory T cell-depleted CD4+T cells (CD4+CD25aneg) on facial motoneuron (FMN) survival 4 weeks after facial nerve axotomy in RAG2 deficient (RAG-2 / ) mice. (a) Cresyl violet-stained FMN in control and axotomized facial motor nuclei of wild-type (WT) and RAG-2 / mice with or without reconstitution (original magnification, 4). (b) Average percent facial motoneuron (FMN) survival (T S.E.M.) 4 weeks after facial nerve axotomy in WT and RAG-2 / mice with and without reconstitution. *denotes significant differences from WT and reconstituted RAG-2 / mice at p <0.05.
taken throughout the entire caudal-rostral extent of the facial nucleus. Floating sections were mounted on slides, dried, and soaked in Xylene (Fischer-Scientific) for 5 min to remove paraffin. Re-hydration with alcohol dilutions was performed prior to staining with cresyl violet. The facial nerve from each side was used to align matching axotomized and uninjured control sides. Surviving neurons with clear nuclei and dark nuclei were counted on every other matched section and total percent survival of the uninjured control side was determined. Cell counting was done such that the identity of each group was not known. A one-way ANOVA and Neuman– Keul’s test was used to measure statistical significance.
Fig. 2. Facial motoneuron (FMN) survival levels 4 weeks after facial nerve axotomy in wild-type (WT), and CD1-deficient (CD1-/-) mice. (a) Cresyl violet-stained FMN in control and axotomized facial motor nuclei (original magnification, 4). (b) Average percent FMN survival levels (T S.E.M.).
C.A. DeBoy et al. / Journal of Neuroimmunology 176 (2006) 34 – 38
that CD4+CD25+ regulatory T cells are not critical for CD4+ T cell-mediated rescue of FMN from axotomyinduced cell death. 3.2. CD1 /
mice have WT levels of FMN survival
To determine if CD1d-restricted NKT cells are critical for WT levels of FMN survival after nerve injury, facial nerve axotomy was performed on WT and CD1 / mice. FMN survival 4 weeks after injury in WT and CD1 / mice were 63 T 1.3% (n = 5) and 66 T 4.4% (n = 7) of the uninjured control side, respectively (Fig. 2). These data indicate that CD1d-restricted NKT cells are not critical for WT levels of FMN survival after facial nerve axotomy.
4. Discussion Data from this study indicate that the CD4+ regulatory subsets, CD4+CD25+ regulatory T and CD1d-restricted NKT cells, are not critical to mediate FMN survival after facial nerve axotomy. A recent study has suggested that CD4+CD25+ regulatory T cells exacerbate neuronal damage after optic nerve injury (Kipnis et al., 2002a). In the present study however, there is no statistically significant difference in FMN survival after facial nerve axotomy in RAG-2 / mice, reconstituted with a CD4+ T cell population depleted of CD4+CD25+ regulatory T cells compared to WT mice, suggesting that CD4+CD25+ regulatory T cells, while not critical to promote WT levels of FMN survival, do not inhibit CD4+ T cell-mediated rescue of FMN from axotomy-induced cell death. Although it has been established that CD4+ T cells are, but NK cells are not critical to mediate FMN survival after facial nerve axotomy (Byram et al., 2003; Serpe et al., 2003), it remained unknown if the CD4+ T cell subset, NKT cells are critical for the FMN survival process. Mice deficient in major-histocompatibility complex II (MHC II), the surface molecule required to present antigen for effector CD4+ T cell development (Janeway, 1999), have been used to determine that MHC II is critical for CD4+ T cellmediated rescue of FMN from axotomy-induced cell death (Byram et al., 2004). It has been found that NKT cells, which require CD1d-mediated development, are present in MHC II / mice (Cardell et al., 1995). Therefore, in MHC II / mice, CD1d-restricted NKT cells, in the absence of functional effector CD4+ T cells are not sufficient to mediate FMN survival. Because the failure of NKT-cell mediated rescue of FMN in MHC II / mice could be due to other deficiencies, we have extended these observations to determine if CD1d-restricted NKT cells are critical for WT levels of FMN survival by using a mouse model in which effector CD4+ T cells are present and CD1drestricted NKT cells are deficient. The data presented in this report establish conclusively that CD1d-restricted NKT cells are not critical to mediate FMN survival. It is likely
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that the critical CD4+ T cells involved in mediating FMN survival are the effector cells. These data therefore suggest that CD1d-restrictred NKT cells are not necessary to regulate the CD4+ T cell-mediated rescue of FMN from axotomy-induced cell death. This study also indicates that the CD1d surface molecule, involved in antigen presentation of lipid antigen (Brigl and Brenner, 2004), is also not critical for the FMN survival process after facial nerve axotomy, and further emphasizes the importance of the MHC II molecule. Furthermore, while the antigens involved in initiating the immune response following facial nerve axotomy are not known, the findings presented here suggest that lipid antigen may not be critical to induce the CD4+ T cell-mediated rescue of FMN from axotomy-induced cell death. In conclusion, the CD4+ T cell regulatory subsets, CD4+CD25+ regulatory T, and CD1drestricted NKT cells are not critical to mediate FMN survival. References Bendelac, A., Rivera, M.N., Park, S.H., Roark, J.H., 1997. Mouse CD1specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15, 535 – 562. Brigl, M., Brenner, M.B., 2004. CD1: antigen presentation and T cell function. Annu. Rev. Immunol. 22, 817 – 890. Byram, S.C., Serpe, C.J., Pruett, S.B., Sanders, V.M., Jones, K.J., 2003. Natural killer cells do not mediate facial motoneuron survival after facial nerve transection. Brain Behav. Immun. 17, 417 – 425. Byram, S.C., Carson, M.J., DeBoy, C.A., Serpe, C.J., Sanders, V.M., Jones, K.J., 2004. CD4-positive T cell-mediated neuroprotection requires dual compartment antigen presentation. J. Neurosci. 24, 4333 – 4339. Cardell, S., Tangri, S., Chan, S., Kronenberg, M., Benoist, C., Mathis, D., 1995. CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice. J. Exp. Med. 182, 993 – 1004. Fee, D., Crumbaugh, A., Jacques, T., Herdrich, B., Sewell, D., Auerbach, D., Piaskowski, S., Hart, M.N., Sandor, M., Fabry, Z., 2003. Activated/effector CD4+ T cells exacerbate acute damage in the central nervous system following traumatic injury. J. Neuroimmunol. 136, 54 – 66. Gavin, M., Rudensky, A., 2003. Control of immune homeostasis by naturally arising regulatory CD4+ T cells. Curr. Opin. Immunol. 15, 690 – 696. Gumperz, J.E., 2004. CD1d-restricted ‘‘NKT’’ cells and myeloid IL-12 production: an immunological crossroads leading to promotion or suppression of effective anti-tumor immune responses? J. Leukoc. Biol. 76, 307 – 313. Hammarberg, H., Lidman, O., Lundberg, C., Eltayeb, S.Y., Gielen, A.W., Muhallab, S., Svenningsson, A., Linda, H., van Der Meide, P.H., Cullheim, S., Olsson, T., Piehl, F., 2000. Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. J. Neurosci. 20, 5283 – 5291. Hauben, E., Butovsky, O., Nevo, U., Yoles, E., Moalem, G., Agranov, E., Mor, F., Leibowitz-Amit, R., Pevsner, E., Akselrod, S., Neeman, M., Cohen, I.R., Schwartz, M., 2000. Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion. J. Neurosci. 20, 6421 – 6430. Hofstetter, H.H., Sewell, D.L., Liu, F., Sandor, M., Forsthuber, T., Lehmann, P.V., Fabry, Z., 2003. Autoreactive T cells promote posttraumatic healing in the central nervous system. J. Neuroimmunol. 134, 25 – 34.
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Janeway Jr., C.A., 1999. The role of self-recognition in receptor repertoire development. Members of the Janeway Laboratory. Immunol. Res. 19, 107 – 118. Jones, K.J., LaVelle, A., 1985. Changes in nuclear envelope invaginations in axotomized immature and mature hamster facial motoneurons. Brain Res. 353, 241 – 249. Jones, T.B., Basso, D.M., Sodhi, A., Pan, J.Z., Hart, R.P., MacCallum, R.C., Lee, S., Whitacre, C.C., Popovich, P.G., 2002. Pathological CNS autoimmune disease triggered by traumatic spinal cord injury: implications for autoimmune vaccine therapy. J. Neurosci. 22, 2690 – 2700. Jones, K.J., Serpe, C.J., Byram, S.C., Deboy, C.A., Sanders, V.M., 2005. Role of the immune system in the maintenance of mouse facial motoneuron viability after nerve injury. Brain Behav. Immun. 19, 12 – 19. Kipnis, J., Mizrahi, T., Hauben, E., Shaked, I., Shevach, E., Schwartz, M., 2002. Neuroprotective autoimmunity: naturally occurring CD4+CD25+ regulatory T cells suppress the ability to withstand injury to the central nervous system. Proc. Natl. Acad. Sci. U. S. A. 99, 15620 – 15625. Kipnis, J., Mizrahi, T., Yoles, E., Ben-Nun, A., Schwartz, M., Ben-Nur, A., 2002. Myelin specific Th1 cells are necessary for post-traumatic protective autoimmunity. J. Neuroimmunol. 130, 78 – 85. McHugh, R.S., Shevach, E.M., 2002. The role of suppressor T cells in regulation of immune responses. J. Allergy Clin. Immunol. 110, 693 – 702. Mendiratta, S.K., Martin, W.D., Hong, S., Boesteanu, A., Joyce, S., Van Kaer, L., 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6, 469 – 477.
Moalem, G., Leibowitz-Amit, R., Yoles, E., Mor, F., Cohen, I.R., Schwartz, M., 1999. Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat. Med. 5, 49 – 55. Moalem, G., Yoles, E., Leibowitz-Amit, R., Muller-Gilor, S., Mor, F., Cohen, I.R., Schwartz, M., 2000. Autoimmune T cells retard the loss of function in injured rat optic nerves. J. Neuroimmunol. 106, 189 – 197. Serpe, C.J., Kohm, A.P., Huppenbauer, C.B., Sanders, V.M., Jones, K.J., 1999. Exacerbation of facial motoneuron loss after facial nerve transection in severe combined immunodeficient (scid) mice. J. Neurosci. 19, RC7. Serpe, C.J., Coers, S., Sanders, V.M., Jones, K.J., 2003. CD4+ T, but not CD8+ or B, lymphocytes mediate facial motoneuron survival after facial nerve transection. Brain Behav. Immun. 17, 393 – 402. Shinkai, Y., Rathbun, G., Lam, K.P., Oltz, E.M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A.M., et al., 1992. RAG-2deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855 – 867. Smiley, S.T., Kaplan, M.H., Grusby, M.J., 1997. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science 275, 977 – 979. Teige, A., Teige, I., Lavasani, S., Bockermann, R., Mondoc, E., Holmdahl, R., Issazadeh-Navikas, S., 2004. CD1-dependent regulation of chronic central nervous system inflammation in experimental autoimmune encephalomyelitis. J. Immunol. 172, 186 – 194. Wood, K.J., Sakaguchi, S., 2003. Regulatory T cells in transplantation tolerance. Nat. Rev., Immunol. 3, 199 – 210.
CD4+CD25+ regulatory T cells and CD1-restricted NKT cells do not mediate facial motoneuron survival after axotomy Cynthia A. DeBoy a,b,*, Susanna C. Byram a,b, Craig J. Serpe b, Danielle Wisuri b, Virginia M. Sanders c,1, Kathryn J. Jones a,b,1 a
c
Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Chicago, 2160 S. 1st Avenue, Maywood, IL 60153, United States b Research and Development Service, Hines VA Hospital, Hines, IL 60141, United States Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Graves Hall, 333 W. 10th Ave, Columbus, OH 43210, United States Received 4 January 2006; received in revised form 5 April 2006; accepted 5 April 2006
Abstract CD4+ T cells rescue facial motoneurons (FMN) from axotomy-induced cell death. The objective of this study is to determine if the CD4+ T regulatory subsets, CD4+CD25+ T or CD1d-restricted NKT cells are critical for FMN survival after facial nerve axotomy. Surviving FMN within facial motor nuclei from axotomized and control sides 4 weeks after axotomy were counted to determine percent FMN survival. Data generated by applying this paradigm to recombination activating gene-2-deficient mice reconstituted with CD4+ T cells depleted of CD4+CD25+ T cells and to CD1 / mice, deficient in CD1d-restricted NKT cells, suggest that neither regulatory CD4+ T subset is critical for FMN survival. D 2006 Elsevier B.V. All rights reserved. Keywords: CD4+ regulatory T cell; CD1-restricted NKT cell; Facial nerve axotomy; Motoneuron survival
1. Introduction Immune cells are capable of promoting neuro-protective effects after neuronal injury (Jones et al., 2005; Serpe et al., 1999). Specifically, the CD4+ T cell has been determined to be critical to mediate facial motoneuron (FMN) survival after facial nerve axotomy (Serpe et al., 2003). However, CD4+ T cell-mediated effects ranging from neuro-repair/protection to neuro-destruction have been described for different neuroinjury models (Fee et al., 2003; Hammarberg et al., 2000; Hauben et al., 2000; Hofstetter et al., 2003; Jones et al., 2002; Kipnis et al., 2002b; Moalem et al., 1999, 2000), necessitating further investigation. It is therefore important to identify * Corresponding author. Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Chicago, 2160 S. 1st Avenue, Maywood, IL 60153, United States. Tel.: +1 410 955 1118; fax: +1 410 502 6367. E-mail address: [email protected] (C.A. DeBoy). 1 Drs. Jones and Sanders share senior authorship. 0165-5728/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.04.006
the specific subsets of CD4+ T cells with neuro-protective potential. In addition to effector cells, CD4+ T cell subsets include regulatory T cells, including CD4+CD25+ regulatory T and CD4+CD1d-restricted natural-killer (NK) T cells, both of which have garnered considerable interest, as each can protect against autoimmune diseases (Bendelac et al., 1997; Gavin and Rudensky, 2003; Gumperz, 2004). CD4+CD25+ regulatory T cells suppress CD8+ T and CD4+ T cell proliferation and promote T cell tolerance (McHugh and Shevach, 2002; Wood and Sakaguchi, 2003). Although CD4+CD25+ regulatory T cells may promote FMN survival after facial nerve axotomy by regulating the ensuing immune response, the regulatory cell may alternatively suppress the CD4+ T cell-mediated neuro-protective effect, as CD4+CD25+ regulatory T cells have been found to increase neuro-damage after optic nerve injury (Kipnis et al., 2002a). CD1d-restricted NKT cell development and activation involves antigen presentation of lipid antigen presented in
C.A. DeBoy et al. / Journal of Neuroimmunology 176 (2006) 34 – 38
CD1d surface molecules on antigen presenting cells (Brigl and Brenner, 2004) and are therefore depleted in CD1deficient mice (Mendiratta et al., 1997; Smiley et al., 1997). Because of the capacity of CD1d-restricted NKT cells to suppress CNS inflammation (Teige et al., 2004) it is of interest to determine if the CD4+CD1d-restricted NKT cell plays a critical in mediating FMN survival. The objective of this study is to determine if the CD4+ regulatory subsets, CD4+CD25+ regulatory T or the CD4+CD1d-restricted NKT cells, are critical to FMN survival after facial nerve axotomy.
2. Materials and methods 2.1. Animals and surgical procedures Female mice, 8– 12 weeks of age including C57BL/6J, BALB/c, and CD1-deficient( / ) (BALB/c background) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and recombination activating gene-2-deficient (RAG-2 / ) (C57BL/6J background) mice were purchased from Taconic Laboratories (Germantown, NY). Pathogenfree conditions were maintained under 12 h light/dark cycles. No manipulations were made until mice acclimated for at least 1 week. Procedures were performed in accordance with NIH guidelines. Facial nerve axotomy was performed as described (Byram et al., 2003; Jones and LaVelle, 1985; Serpe et al., 2003, 1999). Using aseptic techniques and 3% halothane for anesthesia, the right facial nerve of each mouse was completely transected at its exit from the stylomastoid foramen. At the time of sacrifice, behavioral observations were made to assure that no recovery from facial paralysis occurred. 2.2. Preparation of CD4+CD25neg lymphocytes and cell reconstitutions Lymph nodes were aseptically removed from C57BL/6 mice, prepared as a single cell suspension in Hank’s balanced salt solution (HBSS) (Gibco Invitrogen Corp., Carlsbad, CA) + 5% fetal calf serum (FCS) (Gibco Invitrogen Corp.). Supernatant was removed after cells were centrifuged at 300g for 10 min. Cells were resuspended in DNASE buffer (pH = 7.4) for 25 min at 25 -C. DNASE buffer consists of: PBS (Gibco Invitrogen Corp.), 0.5 mM MgCl2 (Sigma-Aldrich), 1 mM CaCl2 (Sigma-Aldrich), 0.5% BSA (Sigma-Aldrich), and 100 U/ml Dnase (Amersham, Piscataway, NJ). Supernatant was removed and cells were resuspended in running buffer which consists of: PBS (Gibco Invitrogen Corp.) + 0.5% bovine serum albumin (Sigma-Aldrich, St. Louis MO) + 2 mM EDTA (Ambion, Austin, TX). A CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec) was used to remove CD4+CD25+ regulatory T cells from a lymphocyte population. Manufacturer’s instructions
35
were followed. Briefly, per 107 lymphocytes, 40 Al running buffer and 10 Al Biotin-Antibody Cocktail were incubated for 10 min at 4 -C, after which 30 Al of running buffer, 20 Al of anti-biotin MicroBeads, and 10 Al of anti-CD25-PE antibody (clone 7D4) were mixed and incubated in the dark for 15 min at 4 -C. Cells were washed and resuspended in running buffer and passed through a 30 AM filter (Miltenyi Biotec) prior to magnetic cell separation with autoMACS automated cell sorter (Miltenyi Biotec) to remove nonCD4+ T cells. The unlabeled CD4+ T cell population was centrifuged and resuspended in 90 Al running buffer + 10 Al Anti-PE MicroBeads per 107 lymphocytes. After a 15min incubation at 4 -C and washing, CD4+CD25+ cells were removed after separation was performed with an autoMACS automated cell sorter. The unlabeled fraction of cells containing CD4+CD25neg cells was centrifuged, and resuspended in running buffer. Remaining magnetically labeled cells were removed with the autoMACS cell sorter. Unlabeled cells were labeled with anti-CD4 MicroBeads and positively selected as described previously (Byram et al., 2004; Serpe et al., 2003). CD4+ T cells were resuspended in PBS to 0.5 107 cells/ ml and 0.2 ml of cell suspension or vehicle alone was injected into the lateral tail vein of recipient mice 1 week prior to axotomy. 2.3. Immunofluorescence staining and flow cytometric analysis Antibody labeling and analysis of isolated CD4+ CD25neg cells were performed as previously described (Byram et al., 2003). Antibodies, purchased from Pharmingen, included FITC-labeled anti-CD4 (clone GK1.5), PE-labeled anti-CD25 (Clone, 3C7), and the isotypematched controls, FITC-labeled rat IgG2b n and PElabeled Rat IgG2b n, respectively. CD4+CD25neg cells population contained greater than 99% of CD4+ T cells and less than 0.2% of contaminating CD25+ cells (data not shown). The percent purity of CD4+CD25neg cells from lymph nodes removed from reconstituted mice 5 weeks after reconstitution contained less than 0.2% of CD4+CD25+ T cells (data not shown). 2.4. Cell-counting procedures Mice were sacrificed by CO2 asphyxiation 4 weeks after facial nerve axotomy. A 3-mm region of brain, containing the facial nucleus, was fixed in methacarn for 24 h. Methacarn fixative consists of: 60% methanol (FisherScientific), 30% 1,1,1-trichloroethane (TCE) (SigmaAldrich), and 10% glacial acetic acid (J.T. Baker, Phillipsburgh, NJ). Tissue was dehydrated in serial washes with isopropyl alcohol and placed in TCE for 16 h. Vacuum infiltration was performed at 60 -C, 23 psi. Tissue was embedded in paraffin. Microtome sections (12 Am) were
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3. Results 3.1. CD4+CD25neg T cells promote WT levels of FMN survival after facial nerve axotomy when reconstituted into RAG-2 / mice To establish if the CD4+CD25+ regulatory T cell is critical for FMN survival after facial nerve axotomy, RAG2 / mice, deficient in functional T and B cells (Shinkai et al., 1992), were reconstituted with CD4+ T cells depleted of CD4+CD25+ regulatory T cells (CD4+CD25neg). FMN survival 4 weeks after injury compared to C57BL/6J WT mice, at 65 T 2.1% (n = 8), was significantly decreased ( p < 0.05) in RAG-2 / mice to 54 T 4.0% (n = 5) of the uninjured control side (Fig. 1). FMN survival 4 weeks after facial nerve axotomy was significantly increased ( p < 0.05) in RAG-2 / mice reconstituted with CD4+CD25neg cells to 69% T 3.8% (n = 7) of the uninjured control side ( p < 0.05), compared to RAG-2 / mice, which was not statistically increased above WT levels. These data suggest
Fig. 1. Effects of reconstitution with a population of CD4+CD25+ regulatory T cell-depleted CD4+T cells (CD4+CD25aneg) on facial motoneuron (FMN) survival 4 weeks after facial nerve axotomy in RAG2 deficient (RAG-2 / ) mice. (a) Cresyl violet-stained FMN in control and axotomized facial motor nuclei of wild-type (WT) and RAG-2 / mice with or without reconstitution (original magnification, 4). (b) Average percent facial motoneuron (FMN) survival (T S.E.M.) 4 weeks after facial nerve axotomy in WT and RAG-2 / mice with and without reconstitution. *denotes significant differences from WT and reconstituted RAG-2 / mice at p <0.05.
taken throughout the entire caudal-rostral extent of the facial nucleus. Floating sections were mounted on slides, dried, and soaked in Xylene (Fischer-Scientific) for 5 min to remove paraffin. Re-hydration with alcohol dilutions was performed prior to staining with cresyl violet. The facial nerve from each side was used to align matching axotomized and uninjured control sides. Surviving neurons with clear nuclei and dark nuclei were counted on every other matched section and total percent survival of the uninjured control side was determined. Cell counting was done such that the identity of each group was not known. A one-way ANOVA and Neuman– Keul’s test was used to measure statistical significance.
Fig. 2. Facial motoneuron (FMN) survival levels 4 weeks after facial nerve axotomy in wild-type (WT), and CD1-deficient (CD1-/-) mice. (a) Cresyl violet-stained FMN in control and axotomized facial motor nuclei (original magnification, 4). (b) Average percent FMN survival levels (T S.E.M.).
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that CD4+CD25+ regulatory T cells are not critical for CD4+ T cell-mediated rescue of FMN from axotomyinduced cell death. 3.2. CD1 /
mice have WT levels of FMN survival
To determine if CD1d-restricted NKT cells are critical for WT levels of FMN survival after nerve injury, facial nerve axotomy was performed on WT and CD1 / mice. FMN survival 4 weeks after injury in WT and CD1 / mice were 63 T 1.3% (n = 5) and 66 T 4.4% (n = 7) of the uninjured control side, respectively (Fig. 2). These data indicate that CD1d-restricted NKT cells are not critical for WT levels of FMN survival after facial nerve axotomy.
4. Discussion Data from this study indicate that the CD4+ regulatory subsets, CD4+CD25+ regulatory T and CD1d-restricted NKT cells, are not critical to mediate FMN survival after facial nerve axotomy. A recent study has suggested that CD4+CD25+ regulatory T cells exacerbate neuronal damage after optic nerve injury (Kipnis et al., 2002a). In the present study however, there is no statistically significant difference in FMN survival after facial nerve axotomy in RAG-2 / mice, reconstituted with a CD4+ T cell population depleted of CD4+CD25+ regulatory T cells compared to WT mice, suggesting that CD4+CD25+ regulatory T cells, while not critical to promote WT levels of FMN survival, do not inhibit CD4+ T cell-mediated rescue of FMN from axotomy-induced cell death. Although it has been established that CD4+ T cells are, but NK cells are not critical to mediate FMN survival after facial nerve axotomy (Byram et al., 2003; Serpe et al., 2003), it remained unknown if the CD4+ T cell subset, NKT cells are critical for the FMN survival process. Mice deficient in major-histocompatibility complex II (MHC II), the surface molecule required to present antigen for effector CD4+ T cell development (Janeway, 1999), have been used to determine that MHC II is critical for CD4+ T cellmediated rescue of FMN from axotomy-induced cell death (Byram et al., 2004). It has been found that NKT cells, which require CD1d-mediated development, are present in MHC II / mice (Cardell et al., 1995). Therefore, in MHC II / mice, CD1d-restricted NKT cells, in the absence of functional effector CD4+ T cells are not sufficient to mediate FMN survival. Because the failure of NKT-cell mediated rescue of FMN in MHC II / mice could be due to other deficiencies, we have extended these observations to determine if CD1d-restricted NKT cells are critical for WT levels of FMN survival by using a mouse model in which effector CD4+ T cells are present and CD1drestricted NKT cells are deficient. The data presented in this report establish conclusively that CD1d-restricted NKT cells are not critical to mediate FMN survival. It is likely
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that the critical CD4+ T cells involved in mediating FMN survival are the effector cells. These data therefore suggest that CD1d-restrictred NKT cells are not necessary to regulate the CD4+ T cell-mediated rescue of FMN from axotomy-induced cell death. This study also indicates that the CD1d surface molecule, involved in antigen presentation of lipid antigen (Brigl and Brenner, 2004), is also not critical for the FMN survival process after facial nerve axotomy, and further emphasizes the importance of the MHC II molecule. Furthermore, while the antigens involved in initiating the immune response following facial nerve axotomy are not known, the findings presented here suggest that lipid antigen may not be critical to induce the CD4+ T cell-mediated rescue of FMN from axotomy-induced cell death. In conclusion, the CD4+ T cell regulatory subsets, CD4+CD25+ regulatory T, and CD1drestricted NKT cells are not critical to mediate FMN survival. References Bendelac, A., Rivera, M.N., Park, S.H., Roark, J.H., 1997. Mouse CD1specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15, 535 – 562. Brigl, M., Brenner, M.B., 2004. CD1: antigen presentation and T cell function. Annu. Rev. Immunol. 22, 817 – 890. Byram, S.C., Serpe, C.J., Pruett, S.B., Sanders, V.M., Jones, K.J., 2003. Natural killer cells do not mediate facial motoneuron survival after facial nerve transection. Brain Behav. Immun. 17, 417 – 425. Byram, S.C., Carson, M.J., DeBoy, C.A., Serpe, C.J., Sanders, V.M., Jones, K.J., 2004. CD4-positive T cell-mediated neuroprotection requires dual compartment antigen presentation. J. Neurosci. 24, 4333 – 4339. Cardell, S., Tangri, S., Chan, S., Kronenberg, M., Benoist, C., Mathis, D., 1995. CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice. J. Exp. Med. 182, 993 – 1004. Fee, D., Crumbaugh, A., Jacques, T., Herdrich, B., Sewell, D., Auerbach, D., Piaskowski, S., Hart, M.N., Sandor, M., Fabry, Z., 2003. Activated/effector CD4+ T cells exacerbate acute damage in the central nervous system following traumatic injury. J. Neuroimmunol. 136, 54 – 66. Gavin, M., Rudensky, A., 2003. Control of immune homeostasis by naturally arising regulatory CD4+ T cells. Curr. Opin. Immunol. 15, 690 – 696. Gumperz, J.E., 2004. CD1d-restricted ‘‘NKT’’ cells and myeloid IL-12 production: an immunological crossroads leading to promotion or suppression of effective anti-tumor immune responses? J. Leukoc. Biol. 76, 307 – 313. Hammarberg, H., Lidman, O., Lundberg, C., Eltayeb, S.Y., Gielen, A.W., Muhallab, S., Svenningsson, A., Linda, H., van Der Meide, P.H., Cullheim, S., Olsson, T., Piehl, F., 2000. Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. J. Neurosci. 20, 5283 – 5291. Hauben, E., Butovsky, O., Nevo, U., Yoles, E., Moalem, G., Agranov, E., Mor, F., Leibowitz-Amit, R., Pevsner, E., Akselrod, S., Neeman, M., Cohen, I.R., Schwartz, M., 2000. Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion. J. Neurosci. 20, 6421 – 6430. Hofstetter, H.H., Sewell, D.L., Liu, F., Sandor, M., Forsthuber, T., Lehmann, P.V., Fabry, Z., 2003. Autoreactive T cells promote posttraumatic healing in the central nervous system. J. Neuroimmunol. 134, 25 – 34.
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