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CD4+ T cell-mediated neuroprotection is independent of T cell-derived BDNF in a mouse facial nerve axotomy model
Brain, Behavior, and Immunity 26 (2012) 886–890
Contents lists available at SciVerse ScienceDirect
Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi
Short Communication
CD4+ T cell-mediated neuroprotection is independent of T cell-derived BDNF in a mouse facial nerve axotomy model Junping Xin a,b,⇑, Nichole A. Mesnard a,b, Taylor Beahrs a,b, Derek A. Wainwright c, Craig J. Serpe b, Thomas D. Alexander b, Virginia M. Sanders d,1, Kathryn J. Jones e,1 a
Neuroscience Institute, Loyola University Medical Center, IL 60153, United States Research and Development Service, Hines VA Hospital, IL 60141, United States Department of Surgery, The Brain Tumor Center, University of Chicago, IL 60637, United States d Department of Molecular Virology, Immunology, & Medical Genetics, College of Medicine, The Ohio State University, OH 43210, United States e Department of Anatomy and Cell Biology, School of Medicine, Indiana University, IN 46202, United States b c
a r t i c l e
i n f o
Article history: Received 27 October 2011 Received in revised form 23 February 2012 Accepted 28 February 2012 Available online 7 March 2012 Keywords: CD4 T cell Brain-derived neurotrophic factor Facial nerve axotomy Motoneuron survival Conditional gene knockout
a b s t r a c t Background: The production of neurotrophic factors, such as BDNF, has generally been considered an important mechanism of immune-mediated neuroprotection. However, the ability of T cells to produce BDNF remains controversial. Methods: In the present study, we examined mRNA and protein of BDNF using RT-PCR and western blot, respectively, in purified and reactivated CD4+ T cells. In addition, to determine the role of BDNF derived from CD4+ T cells, the BDNF gene was specifically deleted in T cells using the Cre-lox mouse model system. Results: Our results indicate that while both mRNA expression and protein secretion of BDNF in reactivated T cells were detected at 24 h, only protein could be detected at 72 h after reactivation. The results suggest a transient up-regulation of BDNF mRNA in reactivated T cells. Furthermore, in contrast to our hypothesis that the BDNF expression is necessary for CD4+ T cells to mediate neuroprotection, mice with CD4+ T cells lacking BDNF expression demonstrated a similar level of facial motoneuron survival compared to their littermates that expressed BDNF, and both levels were comparable to wild-type. The results suggest that the deletion of BDNF did not impair CD4+ T cell-mediated neuroprotection. Conclusion: Collectively, while CD4+ T cells are a potential source of BDNF after nerve injury, production of BDNF is not necessary for CD4+ T cells to mediate their neuroprotective effects. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Our laboratory has previously shown that compared to wild type (WT) mice, immunodeficient mice lacking T and B cells have a significant decrease in motoneuron survival, which can be rescued by adoptive transfer of CD4+ T cells (Serpe et al., 1999, 2003). While multiple effector subsets of CD4+ T cells developed after facial nerve axotomy (Xin et al., 2008), mice with an impaired Th2 response exhibit a decrease in motoneuron survival after axotomy (Deboy et al., 2006). More recently, we determined that IL-10, a Th2-secreted anti-inflammatory cytokine, plays a critical role in supporting facial motoneuron survival after nerve injury, but that CD4+ T cells were not the primary source of IL-10 (Xin et al., ⇑ Corresponding author at: Neuroscience Institute, Loyola University Medical Center, 2160 S First Avenue, Maywood, IL 60153, United States. Tel.: +1 708 202 5723; fax: +1 708 202 2327. E-mail addresses: [email protected], [email protected] (J. Xin). 1 These authors share senior authorship. 0889-1591/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbi.2012.02.011
2011). Thus, the molecular mechanism responsible for CD4+ T cell-mediated neuroprotection remains to be elucidated. Brain-derived neurotrophic factor (BDNF) and its receptors are expressed in the thymus and may play an important role in T cell survival (Maroder et al., 1996; De Santi et al., 2009; Azoulay et al., 2008). It has also been demonstrated that mature resting CD4+ T cells express a low level of BDNF (Kerschensteiner et al., 1999; Ziemssen et al., 2002). Previously, our laboratory determined that lymph node cells, isolated from mice 9 days post-axotomy and reactivated in vitro for 24 h, are capable of secreting BDNF, and we proposed that the release of BDNF may underlie the mechanism of immune-mediated neuroprotection by the CD4+ T cells following nerve injury (Serpe et al., 2003, 2005). In the current study, using mice with T cells depleted of BDNF, we examined the ability of those cells to support facial motoneuron (FMN) survival after a facial nerve axotomy. Our results indicate that CD4+ T cells are capable of producing BDNF, however, to our surprise, that production is not required for T cellmediated neuroprotection of motoneurons from axotomy-induced cell death.
J. Xin et al. / Brain, Behavior, and Immunity 26 (2012) 886–890
2. Materials and methods 2.1. Animals and surgical procedures Seven-week-old female C57Bl/6 wild-type and transgenic mice were obtained from Jackson Laboratory (Sacramento, CA, USA). Two transgenic groups of mice were used to create conditional BDNF gene knockout mice. One group, Lck-Cre, bears the Cre-recombinase gene driven by the distal promoter of the lymphocyte protein tyrosine kinase (Lck), which is a T cell receptor signaling component and only observed in T cells after T cell receptor a (Tcra) locus rearrangement. The second group possesses loxP sites on either side of exon 5 of the BDNF gene. Upon breeding these two groups, the litters contained two genotypes of mice, one Cre+/ genotype, expressing the Cre gene in T cells and leads to the deletion of the BDNF gene in T cells, and the other Cre/ genotype, which does not express the Cre and the BDNF gene in T cells remains intact. These mice were bred and prepared by Jackson laboratory. All mice were housed and surgery was performed as previously published (Serpe et al., 2003). All surgical procedures were completed in accordance with National Institutes of Health guidelines on the care and use of laboratory animals for research purposes. 2.2. Preparation of CD4+ T cells and reactivation Right (draining) cervical lymph nodes were collected from axotomized mice (n = 4/group) at 9 day post operative, and then CD4+ T cells were isolated via autoMACS using anti-CD4 magnetic beads as previously published (Xin et al., 2008). CD4+ T cells were plated in two sets of culture cambers with or without anti-CD3 coating. The cells that received anti-CD3 stimulation were defined as reactivated cells, because these cells were first activated by axotomy in vivo. Cells and supernatants were harvested at two time points, 24 and 72 h. Cells were used for RNA extraction and RT-PCR (Invitrogen, Carlsbad, CA). The supernatants were subjected to Western blot analysis. 2.3. RNA preparation, RT-PCR, and electrophoresis Complementary DNA was used in RT-PCR reactions in an iCycler (Applied Biosystems, Foster City, CA). Twenty-five microliter PCR reactions contained 1 SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 1 lL cDNA, and 200 nM forward and reverse primers. RT-PCR cycle parameters included an initial 95 °C for 10 min, followed by 45 cycles of 95 °C 30 s, 54 °C 30 s and 65 °C 30 s. BDNF PCR primers were designed from published mouse sequences, forward: 50 -CCATAAGGACGCGGACTTG-30 ; reverse: 50 -GACATGTTTGCGGCATCCA-30 . PCR products were separated on Criterion precast 10% nondenaturing polyacrylamide TBE gels (BioRad, Hercules, CA) for 90 min at 100 V. Gels were imaged on a STORM 860 Phosphoimager using Storm Scanner and ImageQuant programs. The PCR for detection of BDNF was performed using DNA samples from cervical lymph node cells with the method provided by Jackson Lab.
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following manufacture’s manual. An anti-human BDNF polyclonal antibody (1:1000) (Promega, San Luis Obispo CA) was used as primary antibody, and HRP-labeled anti-chicken IgG secondary antibody (1:500) (KPL, Gaithersburg, Maryland). Positive identification of BDNF was determined by a 27 kDa band (High-Range Rainbow Molecular Weight Marker; Amersham Biosciences). 2.5. Surface and intracellular staining, and flow cytometric analysis Single cell suspension of draining cervical lymph node cells were first incubated with phorbol myristate acetate (PMA, 50 ng/ ml) and ionomycin (500 ng/ml) for 6 h in the presence of brefeldin A (10 lg/ml) during the final 2 h. The T cells were permeabilized with Saponin and doubly stained with anti-CD4-APC (BD Pharmingen, San Diego, CA) and chicken anti-human BDNF (Promega, San Luis Obispo, CA). The stained cells were subjected to multi-color FACS analysis. 2.6. Tissue sectioning and cell counts, statistical analysis Coronal sections of the brainstem containing the facial nuclei were thaw-mounted onto SuperFrost Plus slides (Fisher) and facial motoneurons were counted under blind conditions as previously described (Serpe et al., 1999). Facial motoneuron survival was expressed as a percentage by comparing the number of cells on the right (injured) side to left (uninjured) side. The counting correction factor and section alignment procedures have been described in previous reports (Jones and LaVelle, 1985). Data are expressed as mean ± SEM. One-way ANOVA was performed to determine statistical differences among experimental groups at p < 0.05. 3. Results 3.1. Expression of BDNF mRNA and protein by CD4+ T cells CD4+ T cells from axotomized mice were reactivated in vitro with anti-CD3 or non-reactivated, without anti-CD3, for 24 or 72 h. As shown in Fig. 1A, following anti-CD3 reactivation, BDNF mRNA expression in CD4+ T cells was detected at 24 h, but not 72 h after anti-CD3 reactivation. Without reactivation, BDNF mRNA expression in CD4+ T cells was undetectable. These results suggest that CD4+ T cells express detectable levels of BDNF mRNA after being activated via injury and reactivated in vitro. In contrast, BDNF protein expression was detectable at 24 and 72 h in culture with and without anti-CD3 reactivation (Fig. 1B). However, the pattern of BDNF expression in the cell culture supernatant differs from the mRNA expression. First, BDNF protein was present in the culture supernatants regardless of whether or not the cells were activated with anti-CD3. Second, BDNF protein was secreted by CD4+ T cells in cervical lymph node from both axotomized and uninjured mice, suggesting that facial axotomy is not a requisite for CD4+ T cells to acquire BDNF-producing capability.
2.4. Western blot analysis
3.2. Facial motoneuron survival in the presence or absence of BDNF expression by T cells
The protein concentration of each sample was determined by Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Samples were separated on an 12% Ready Gel Tris–HCl Gel (Bio-Rad Laboratories, Hercules, CA) at 150 V for 45 min, and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) at 100 V for 60 min. The remainder of the Western blot analysis was accomplished by using a Protein Detector LumiGLO Reserve Western Blotting Kit (KPL, Gaithersburg, Maryland)
As detailed in the methods, two groups of transgenic mice, Lck-Cre and Loxp-BDNF, were used to create offspring with a conditional knockout of the BDNF gene (Fig. 2A). To confirm the successful deletion of BDNF gene expression in the Cre+/ mice, lymph node cells from Cre/ and Cre+/ mice underwent PCR detection of BDNF gene and anti-BDNF staining for protein expression. The deletion of BDNF gene was detected, as shown by bands with a larger size in Cre+/ mice (Fig. 2B). Of note, the intact BDNF
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J. Xin et al. / Brain, Behavior, and Immunity 26 (2012) 886–890
A
B
Fig. 1. BDNF production by wild-type CD4+ T cells. (A) Representative result for BDNF mRNA expression in CD4+ T cells, which was detected by RT-PCR at 24 h but not 72 h after anti-CD3 stimulation. (B) Western blot results of BDNF protein in the supernatant of CD4+ T cells of cervical lymph nodes from uninjured and axotomized, collection time of samples and anti-CD3 stimulation are as specified (M = molecular weight marker; PC = positive control).
A
B
Lck-Cre Cre Lck promoter
Loxp-BDNF
loxp
BDNF gene
loxp
Staining control
C
Cre+/-
Cre-/-
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2nd Ab only Con
D
BDNF Axo
E
100 90
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80 % FMN survival
WT
70 60 50 40 30 20
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10 0 WT
Cre-/-
Cre+/-
Fig. 2. FMN survival after axotomy in mice with or without BDNF gene specifically deleted in T cells. (A) A schematic drawing shows the parental mouse strains that were crossed to produce mice that have a specific deletion of the BDNF gene in T cells. (B) Representative PCR result for BDNF gene amplification in the lymph node cells (each lane represents one sample, the arrow indicates the amplicon after BDNF gene is deleted). (C) Intracellular staining of BDNF in lymph node cells, confirming the absence of BDNF expression in CD4+ T cells of mice with Cre+/ genotype. (D) Representative photomicrographs of thionin-stained facial motoneurons in the uninjured (Con) and axotomized (Axo) facial nuclei of wild-type (WT), BDNF conditionally knockout (Cre+/), and littermates with intact BDNF gene (Cre/) mice (100 original magnification). (E) The mean percentage of facial motoneuron (FMN) survival ± SEM at 4 weeks after axotomy. No significant differences were detected between groups (n = 6 for WT and 10 for Cre groups).
gene was presumably amplified from non-T cells. Consistently, the BDNF-positive CD4+ cells were noticeably higher in Cre/ mice
(0.21%) compared to Cre+/ mice, which was comparable to the background staining (0.05%, Fig. 2C).
J. Xin et al. / Brain, Behavior, and Immunity 26 (2012) 886–890
To determine whether expression of BDNF is necessary for CD4+ T cells to mediate their neuroprotective effects after axotomy, WT (n = 6), Cre/ (n = 10), and Cre+/ (n = 10) mice received a right facial nerve axotomy and FMN survival was assessed 28 days post-operative (Fig 2D). As shown in Fig 2E, no significant differences in FMN survival were observed in either Cre/ or Cre+/ group compared to WT. These results suggest the production of BDNF is not a requisite for T cell-mediated neuroprotection after axotomy.
4. Discussion In the past, BDNF production has been considered an important factor released from CD4+ T cells and responsible for immunemediated neuroprotection (Kerschensteiner et al., 1999; Ziv et al., 2006; Wolf et al., 2009; Linker et al., 2009; De Santi et al., 2011). Previous research (Serpe et al., 2005; Ziemssen et al., 2002; Kerschensteiner et al., 1999) has shown that BDNF is expressed by both resting and in vitro activated T cells. However, Edling et al. (2004) failed to detect BDNF mRNA expression in T cells following 72 h of in vitro activation, and concluded that BDNF are produced only in B cells. After comparison of these studies, we discerned that the discrepancy may have been caused by the difference of detection time. In the current study, we measured mRNA and protein expression at 24 and 72 h following anti-CD3 reactivation, and found that the detectable pattern of BDNF mRNA and protein were different. While BDNF protein expression was detected at both 24- and 72-h culture, BDNF mRNA expression was only detected at 24 h, not 72 h. These data suggest a rapid up- and down-regulation of BDNF mRNA in activated T cells, which is below detectable levels 72 h after reactivation. Interestingly, while BDNF mRNA expression in CD4+ T cell was detectable only after reactivation, its protein expression was detected regardless of axotomy and reactivation. This discrepancy may be caused by the different sensitivity of detection method, and/or by the accumulation of BDNF protein from the T cell pool, which makes BDNF protein detection easier. Further study is needed to elucidate the regulation of BDNF gene and protein expression. In addition, the implications of the CD4+ T cell’s intrinsic capability of BDNF production appear different in this facial nerve axotomy model when compared to other neuroinflammatory disease models (Linker et al., 2009; De Santi et al., 2011). Previously, based on the observation that application of BDNF to the proximal nerve stump supported FMN survival in immunodeficient mice (Serpe et al., 2005), we proposed that BDNF production by CD4+ T cells was critical to protect FMN from axotomy-induced cell death. The current study further showed in vitro that CD4+ T cells express BDNF. Next, a double staining for BDNF and T cells at the injury site was not performed in the present study due to technical difficulties, making this aspect of the research unclear as far as obtaining the in vivo evidence that a portion of BDNF was derived from T cells localizing to the injury site. Unexpectedly, however, the deletion of BDNF in T cells did not abolish the immune-mediated neuroprotection, suggesting that while T cells may have the capability of producing BDNF, other possibilities need to be explored as to what cell may be making BDNF after a nerve injury, including the possibility that CD4+ T cells may modulate other cells to produce BDNF, which is actually supported by our recent finding that IL-10 is essential for FMN survival after axotomy, but that CD4+ T cells were not the primary source of the IL-10 (Xin et al., 2011). Therefore, besides direct secretion of BDNF or IL-10, CD4+ T cell may have alternative roles in supporting FMN survival. In addition, we cannot exclude the possibility of compensation by change of T cell number and phenotype in the facial nucleus and by increased production of other neurotrophic
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factors, such as glial-derived neurotrophic factor, Neurotrophin-3, and ciliary neurotrophic factor, known to promote motoneuron survival after injury (Kondo et al., 2003; Zhao et al., 2004). The current study focused on the presence or absence of BDNF mRNA and protein expression by T cells and did not compare the level of BDNF among groups. In the future, we intend to utilize multiple approaches including immunocytochemistry staining and quantitative PCR to investigate the pattern and quantity of neurotrophic factor and cytokine gene and protein expression in central nervous system resident cells and infiltrating T cells, which may be of help to dissect the roles and contribution of each factor in supporting facial motoneuron survival after facial never injury. Disclosure We declare no conflict of interest or financial interests. Acknowledgements K.J.J. and V.M.S. were supported by National Institutes of Health Grant (NS40433), and J.X. was supported by a Grant from Muscular Dystrophy Association (MDA202906). We thank Richard Batka and Drs. Melissa Haulcomb, Keith Fargo, Eileen Foecking and Susan McGuire for support. References Azoulay, D., Urshansky, N., Karni, A., 2008. Low and dysregulated BDNF secretion from immune cells of MS patients is related to reduced neuroprotection. J. Neuroimmunol. 195, 186–193. De Santi, L., Cantalupo, L., Tassi, M., Raspadori, D., Cioni, C., Annunziata, P., 2009. Higher expression of BDNF receptor gp145trkB is associated with lower apoptosis intensity in T cell lines in multiple sclerosis. J. Neurol. Sci. 277, 65–70. De Santi, L., Polimeni, G., Cuzzocrea, S., Esposito, E., Sessa, E., Annunziata, P., Bramanti, P., 2011. Neuroinflammation and neuroprotection: an update on (future) neurotrophin-related strategies in multiple sclerosis treatment. Curr. Med. Chem. 18, 1775–1784. Deboy, C.A., Xin, J., Byram, S.C., Serpe, C.J., Sanders, V.M., Jones, K.J., 2006. Immunemediated neuroprotection of axotomized mouse facial motoneurons is dependent on the IL-4/STAT6 signaling pathway in CD4(+) T cells. Exp. Neurol. 201, 212–224. Edling, A.E., Nanavati, T., Johnson, J.M., Tuohy, V.K., 2004. Human and murine lymphocyte neurotrophin expression is confined to B cells. J. Neurosci. Res. 77, 709–717. Jones, K.J., LaVelle, A., 1985. Changes in nuclear envelope invaginations in axotomized immature and mature hamster facial motoneurons. Brain Res. 353, 241–249. Kerschensteiner, M., Gallmeier, E., Behrens, L., Leal, V.V., Misgeld, T., Klinkert, W.E., Kolbeck, R., Hoppe, E., Oropeza-Wekerle, R.L., Bartke, I., Stadelmann, C., Lassmann, H., Wekerle, H., Hohlfeld, R., 1999. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J. Exp. Med. 189, 865–870. Kondo, S., Kishi, H., Tokimitsu, Y., Muraguchi, A., 2003. Possible involvement of glial cell line-derived neurotrophic factor and its receptor, GFRalpha1, in survival and maturation of thymocytes. Eur. J. Immunol. 33, 2233–2240. Linker, R.A., Lee, D.H., Demir, S., Wiese, S., Kruse, N., Siglienti, I., Gerhardt, E., Neumann, H., Sendtner, M., Luhder, F., Gold, R., 2009. Functional role of brainderived neurotrophic factor in neuroprotective autoimmunity: therapeutic implications in a model of multiple sclerosis. Brain 133, 2248–2263. Maroder, M., Bellavia, D., Meco, D., Napolitano, M., Stigliano, A., Alesse, E., Vacca, A., Giannini, G., Frati, L., Gulino, A., Screpanti, I., 1996. Expression of trKB neurotrophin receptor during T cell development. Role of brain derived neurotrophic factor in immature thymocyte survival. J. Immunol. 157, 2864– 2872. Serpe, C.J., Byram, S.C., Sanders, V.M., Jones, K.J., 2005. Brain-derived neurotrophic factor supports facial motoneuron survival after facial nerve transection in immunodeficient mice. Brain Behav. Immun. 19, 173–180. 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. 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. Wolf, S.A., Steiner, B., Akpinarli, A., Kammertoens, T., Nassenstein, C., Braun, A., Blankenstein, T., Kempermann, G., 2009. CD4-positive T lymphocytes provide a neuroimmunological link in the control of adult hippocampal neurogenesis. J Immunol 182, 3979–3984.
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Xin, J., Wainwright, D.A., Mesnard, N.A., Serpe, C.J., Sanders, V.M., Jones, K.J., 2011. IL-10 within the CNS is necessary for CD4(+) T cells to mediate neuroprotection. Brain Behav. Immun. 25, 820–829. Xin, J., Wainwright, D.A., Serpe, C.J., Sanders, V.M., Jones, K.J., 2008. Phenotype of CD4+ T cell subsets that develop following mouse facial nerve axotomy. Brain Behav. Immun. 22, 528–537. Zhao, Z., Alam, S., Oppenheim, R.W., Prevette, D.M., Evenson, A., Parsadanian, A., 2004. Overexpression of glial cell line-derived neurotrophic factor in the CNS rescues motoneurons from programmed cell death and promotes their longterm survival following axotomy. Exp. Neurol. 190, 356–372.
Ziemssen, T., Kumpfel, T., Klinkert, W.E., Neuhaus, O., Hohlfeld, R., 2002. Glatiramer acetate-specific T-helper 1- and 2-type cell lines produce BDNF: implications for multiple sclerosis therapy. Brain-derived neurotrophic factor. Brain 125, 2381–2391. Ziv, Y., Ron, N., Butovsky, O., Landa, G., Sudai, E., Greenberg, N., Cohen, H., Kipnis, J., Schwartz, M., 2006. Immune cells contribute to the maintenance of neurogenesis and spatial learning abilities in adulthood. Nat. Neurosci. 9, 268–275.
Contents lists available at SciVerse ScienceDirect
Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi
Short Communication
CD4+ T cell-mediated neuroprotection is independent of T cell-derived BDNF in a mouse facial nerve axotomy model Junping Xin a,b,⇑, Nichole A. Mesnard a,b, Taylor Beahrs a,b, Derek A. Wainwright c, Craig J. Serpe b, Thomas D. Alexander b, Virginia M. Sanders d,1, Kathryn J. Jones e,1 a
Neuroscience Institute, Loyola University Medical Center, IL 60153, United States Research and Development Service, Hines VA Hospital, IL 60141, United States Department of Surgery, The Brain Tumor Center, University of Chicago, IL 60637, United States d Department of Molecular Virology, Immunology, & Medical Genetics, College of Medicine, The Ohio State University, OH 43210, United States e Department of Anatomy and Cell Biology, School of Medicine, Indiana University, IN 46202, United States b c
a r t i c l e
i n f o
Article history: Received 27 October 2011 Received in revised form 23 February 2012 Accepted 28 February 2012 Available online 7 March 2012 Keywords: CD4 T cell Brain-derived neurotrophic factor Facial nerve axotomy Motoneuron survival Conditional gene knockout
a b s t r a c t Background: The production of neurotrophic factors, such as BDNF, has generally been considered an important mechanism of immune-mediated neuroprotection. However, the ability of T cells to produce BDNF remains controversial. Methods: In the present study, we examined mRNA and protein of BDNF using RT-PCR and western blot, respectively, in purified and reactivated CD4+ T cells. In addition, to determine the role of BDNF derived from CD4+ T cells, the BDNF gene was specifically deleted in T cells using the Cre-lox mouse model system. Results: Our results indicate that while both mRNA expression and protein secretion of BDNF in reactivated T cells were detected at 24 h, only protein could be detected at 72 h after reactivation. The results suggest a transient up-regulation of BDNF mRNA in reactivated T cells. Furthermore, in contrast to our hypothesis that the BDNF expression is necessary for CD4+ T cells to mediate neuroprotection, mice with CD4+ T cells lacking BDNF expression demonstrated a similar level of facial motoneuron survival compared to their littermates that expressed BDNF, and both levels were comparable to wild-type. The results suggest that the deletion of BDNF did not impair CD4+ T cell-mediated neuroprotection. Conclusion: Collectively, while CD4+ T cells are a potential source of BDNF after nerve injury, production of BDNF is not necessary for CD4+ T cells to mediate their neuroprotective effects. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Our laboratory has previously shown that compared to wild type (WT) mice, immunodeficient mice lacking T and B cells have a significant decrease in motoneuron survival, which can be rescued by adoptive transfer of CD4+ T cells (Serpe et al., 1999, 2003). While multiple effector subsets of CD4+ T cells developed after facial nerve axotomy (Xin et al., 2008), mice with an impaired Th2 response exhibit a decrease in motoneuron survival after axotomy (Deboy et al., 2006). More recently, we determined that IL-10, a Th2-secreted anti-inflammatory cytokine, plays a critical role in supporting facial motoneuron survival after nerve injury, but that CD4+ T cells were not the primary source of IL-10 (Xin et al., ⇑ Corresponding author at: Neuroscience Institute, Loyola University Medical Center, 2160 S First Avenue, Maywood, IL 60153, United States. Tel.: +1 708 202 5723; fax: +1 708 202 2327. E-mail addresses: [email protected], [email protected] (J. Xin). 1 These authors share senior authorship. 0889-1591/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbi.2012.02.011
2011). Thus, the molecular mechanism responsible for CD4+ T cell-mediated neuroprotection remains to be elucidated. Brain-derived neurotrophic factor (BDNF) and its receptors are expressed in the thymus and may play an important role in T cell survival (Maroder et al., 1996; De Santi et al., 2009; Azoulay et al., 2008). It has also been demonstrated that mature resting CD4+ T cells express a low level of BDNF (Kerschensteiner et al., 1999; Ziemssen et al., 2002). Previously, our laboratory determined that lymph node cells, isolated from mice 9 days post-axotomy and reactivated in vitro for 24 h, are capable of secreting BDNF, and we proposed that the release of BDNF may underlie the mechanism of immune-mediated neuroprotection by the CD4+ T cells following nerve injury (Serpe et al., 2003, 2005). In the current study, using mice with T cells depleted of BDNF, we examined the ability of those cells to support facial motoneuron (FMN) survival after a facial nerve axotomy. Our results indicate that CD4+ T cells are capable of producing BDNF, however, to our surprise, that production is not required for T cellmediated neuroprotection of motoneurons from axotomy-induced cell death.
J. Xin et al. / Brain, Behavior, and Immunity 26 (2012) 886–890
2. Materials and methods 2.1. Animals and surgical procedures Seven-week-old female C57Bl/6 wild-type and transgenic mice were obtained from Jackson Laboratory (Sacramento, CA, USA). Two transgenic groups of mice were used to create conditional BDNF gene knockout mice. One group, Lck-Cre, bears the Cre-recombinase gene driven by the distal promoter of the lymphocyte protein tyrosine kinase (Lck), which is a T cell receptor signaling component and only observed in T cells after T cell receptor a (Tcra) locus rearrangement. The second group possesses loxP sites on either side of exon 5 of the BDNF gene. Upon breeding these two groups, the litters contained two genotypes of mice, one Cre+/ genotype, expressing the Cre gene in T cells and leads to the deletion of the BDNF gene in T cells, and the other Cre/ genotype, which does not express the Cre and the BDNF gene in T cells remains intact. These mice were bred and prepared by Jackson laboratory. All mice were housed and surgery was performed as previously published (Serpe et al., 2003). All surgical procedures were completed in accordance with National Institutes of Health guidelines on the care and use of laboratory animals for research purposes. 2.2. Preparation of CD4+ T cells and reactivation Right (draining) cervical lymph nodes were collected from axotomized mice (n = 4/group) at 9 day post operative, and then CD4+ T cells were isolated via autoMACS using anti-CD4 magnetic beads as previously published (Xin et al., 2008). CD4+ T cells were plated in two sets of culture cambers with or without anti-CD3 coating. The cells that received anti-CD3 stimulation were defined as reactivated cells, because these cells were first activated by axotomy in vivo. Cells and supernatants were harvested at two time points, 24 and 72 h. Cells were used for RNA extraction and RT-PCR (Invitrogen, Carlsbad, CA). The supernatants were subjected to Western blot analysis. 2.3. RNA preparation, RT-PCR, and electrophoresis Complementary DNA was used in RT-PCR reactions in an iCycler (Applied Biosystems, Foster City, CA). Twenty-five microliter PCR reactions contained 1 SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 1 lL cDNA, and 200 nM forward and reverse primers. RT-PCR cycle parameters included an initial 95 °C for 10 min, followed by 45 cycles of 95 °C 30 s, 54 °C 30 s and 65 °C 30 s. BDNF PCR primers were designed from published mouse sequences, forward: 50 -CCATAAGGACGCGGACTTG-30 ; reverse: 50 -GACATGTTTGCGGCATCCA-30 . PCR products were separated on Criterion precast 10% nondenaturing polyacrylamide TBE gels (BioRad, Hercules, CA) for 90 min at 100 V. Gels were imaged on a STORM 860 Phosphoimager using Storm Scanner and ImageQuant programs. The PCR for detection of BDNF was performed using DNA samples from cervical lymph node cells with the method provided by Jackson Lab.
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following manufacture’s manual. An anti-human BDNF polyclonal antibody (1:1000) (Promega, San Luis Obispo CA) was used as primary antibody, and HRP-labeled anti-chicken IgG secondary antibody (1:500) (KPL, Gaithersburg, Maryland). Positive identification of BDNF was determined by a 27 kDa band (High-Range Rainbow Molecular Weight Marker; Amersham Biosciences). 2.5. Surface and intracellular staining, and flow cytometric analysis Single cell suspension of draining cervical lymph node cells were first incubated with phorbol myristate acetate (PMA, 50 ng/ ml) and ionomycin (500 ng/ml) for 6 h in the presence of brefeldin A (10 lg/ml) during the final 2 h. The T cells were permeabilized with Saponin and doubly stained with anti-CD4-APC (BD Pharmingen, San Diego, CA) and chicken anti-human BDNF (Promega, San Luis Obispo, CA). The stained cells were subjected to multi-color FACS analysis. 2.6. Tissue sectioning and cell counts, statistical analysis Coronal sections of the brainstem containing the facial nuclei were thaw-mounted onto SuperFrost Plus slides (Fisher) and facial motoneurons were counted under blind conditions as previously described (Serpe et al., 1999). Facial motoneuron survival was expressed as a percentage by comparing the number of cells on the right (injured) side to left (uninjured) side. The counting correction factor and section alignment procedures have been described in previous reports (Jones and LaVelle, 1985). Data are expressed as mean ± SEM. One-way ANOVA was performed to determine statistical differences among experimental groups at p < 0.05. 3. Results 3.1. Expression of BDNF mRNA and protein by CD4+ T cells CD4+ T cells from axotomized mice were reactivated in vitro with anti-CD3 or non-reactivated, without anti-CD3, for 24 or 72 h. As shown in Fig. 1A, following anti-CD3 reactivation, BDNF mRNA expression in CD4+ T cells was detected at 24 h, but not 72 h after anti-CD3 reactivation. Without reactivation, BDNF mRNA expression in CD4+ T cells was undetectable. These results suggest that CD4+ T cells express detectable levels of BDNF mRNA after being activated via injury and reactivated in vitro. In contrast, BDNF protein expression was detectable at 24 and 72 h in culture with and without anti-CD3 reactivation (Fig. 1B). However, the pattern of BDNF expression in the cell culture supernatant differs from the mRNA expression. First, BDNF protein was present in the culture supernatants regardless of whether or not the cells were activated with anti-CD3. Second, BDNF protein was secreted by CD4+ T cells in cervical lymph node from both axotomized and uninjured mice, suggesting that facial axotomy is not a requisite for CD4+ T cells to acquire BDNF-producing capability.
2.4. Western blot analysis
3.2. Facial motoneuron survival in the presence or absence of BDNF expression by T cells
The protein concentration of each sample was determined by Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Samples were separated on an 12% Ready Gel Tris–HCl Gel (Bio-Rad Laboratories, Hercules, CA) at 150 V for 45 min, and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) at 100 V for 60 min. The remainder of the Western blot analysis was accomplished by using a Protein Detector LumiGLO Reserve Western Blotting Kit (KPL, Gaithersburg, Maryland)
As detailed in the methods, two groups of transgenic mice, Lck-Cre and Loxp-BDNF, were used to create offspring with a conditional knockout of the BDNF gene (Fig. 2A). To confirm the successful deletion of BDNF gene expression in the Cre+/ mice, lymph node cells from Cre/ and Cre+/ mice underwent PCR detection of BDNF gene and anti-BDNF staining for protein expression. The deletion of BDNF gene was detected, as shown by bands with a larger size in Cre+/ mice (Fig. 2B). Of note, the intact BDNF
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A
B
Fig. 1. BDNF production by wild-type CD4+ T cells. (A) Representative result for BDNF mRNA expression in CD4+ T cells, which was detected by RT-PCR at 24 h but not 72 h after anti-CD3 stimulation. (B) Western blot results of BDNF protein in the supernatant of CD4+ T cells of cervical lymph nodes from uninjured and axotomized, collection time of samples and anti-CD3 stimulation are as specified (M = molecular weight marker; PC = positive control).
A
B
Lck-Cre Cre Lck promoter
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loxp
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BDNF Axo
E
100 90
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Fig. 2. FMN survival after axotomy in mice with or without BDNF gene specifically deleted in T cells. (A) A schematic drawing shows the parental mouse strains that were crossed to produce mice that have a specific deletion of the BDNF gene in T cells. (B) Representative PCR result for BDNF gene amplification in the lymph node cells (each lane represents one sample, the arrow indicates the amplicon after BDNF gene is deleted). (C) Intracellular staining of BDNF in lymph node cells, confirming the absence of BDNF expression in CD4+ T cells of mice with Cre+/ genotype. (D) Representative photomicrographs of thionin-stained facial motoneurons in the uninjured (Con) and axotomized (Axo) facial nuclei of wild-type (WT), BDNF conditionally knockout (Cre+/), and littermates with intact BDNF gene (Cre/) mice (100 original magnification). (E) The mean percentage of facial motoneuron (FMN) survival ± SEM at 4 weeks after axotomy. No significant differences were detected between groups (n = 6 for WT and 10 for Cre groups).
gene was presumably amplified from non-T cells. Consistently, the BDNF-positive CD4+ cells were noticeably higher in Cre/ mice
(0.21%) compared to Cre+/ mice, which was comparable to the background staining (0.05%, Fig. 2C).
J. Xin et al. / Brain, Behavior, and Immunity 26 (2012) 886–890
To determine whether expression of BDNF is necessary for CD4+ T cells to mediate their neuroprotective effects after axotomy, WT (n = 6), Cre/ (n = 10), and Cre+/ (n = 10) mice received a right facial nerve axotomy and FMN survival was assessed 28 days post-operative (Fig 2D). As shown in Fig 2E, no significant differences in FMN survival were observed in either Cre/ or Cre+/ group compared to WT. These results suggest the production of BDNF is not a requisite for T cell-mediated neuroprotection after axotomy.
4. Discussion In the past, BDNF production has been considered an important factor released from CD4+ T cells and responsible for immunemediated neuroprotection (Kerschensteiner et al., 1999; Ziv et al., 2006; Wolf et al., 2009; Linker et al., 2009; De Santi et al., 2011). Previous research (Serpe et al., 2005; Ziemssen et al., 2002; Kerschensteiner et al., 1999) has shown that BDNF is expressed by both resting and in vitro activated T cells. However, Edling et al. (2004) failed to detect BDNF mRNA expression in T cells following 72 h of in vitro activation, and concluded that BDNF are produced only in B cells. After comparison of these studies, we discerned that the discrepancy may have been caused by the difference of detection time. In the current study, we measured mRNA and protein expression at 24 and 72 h following anti-CD3 reactivation, and found that the detectable pattern of BDNF mRNA and protein were different. While BDNF protein expression was detected at both 24- and 72-h culture, BDNF mRNA expression was only detected at 24 h, not 72 h. These data suggest a rapid up- and down-regulation of BDNF mRNA in activated T cells, which is below detectable levels 72 h after reactivation. Interestingly, while BDNF mRNA expression in CD4+ T cell was detectable only after reactivation, its protein expression was detected regardless of axotomy and reactivation. This discrepancy may be caused by the different sensitivity of detection method, and/or by the accumulation of BDNF protein from the T cell pool, which makes BDNF protein detection easier. Further study is needed to elucidate the regulation of BDNF gene and protein expression. In addition, the implications of the CD4+ T cell’s intrinsic capability of BDNF production appear different in this facial nerve axotomy model when compared to other neuroinflammatory disease models (Linker et al., 2009; De Santi et al., 2011). Previously, based on the observation that application of BDNF to the proximal nerve stump supported FMN survival in immunodeficient mice (Serpe et al., 2005), we proposed that BDNF production by CD4+ T cells was critical to protect FMN from axotomy-induced cell death. The current study further showed in vitro that CD4+ T cells express BDNF. Next, a double staining for BDNF and T cells at the injury site was not performed in the present study due to technical difficulties, making this aspect of the research unclear as far as obtaining the in vivo evidence that a portion of BDNF was derived from T cells localizing to the injury site. Unexpectedly, however, the deletion of BDNF in T cells did not abolish the immune-mediated neuroprotection, suggesting that while T cells may have the capability of producing BDNF, other possibilities need to be explored as to what cell may be making BDNF after a nerve injury, including the possibility that CD4+ T cells may modulate other cells to produce BDNF, which is actually supported by our recent finding that IL-10 is essential for FMN survival after axotomy, but that CD4+ T cells were not the primary source of the IL-10 (Xin et al., 2011). Therefore, besides direct secretion of BDNF or IL-10, CD4+ T cell may have alternative roles in supporting FMN survival. In addition, we cannot exclude the possibility of compensation by change of T cell number and phenotype in the facial nucleus and by increased production of other neurotrophic
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factors, such as glial-derived neurotrophic factor, Neurotrophin-3, and ciliary neurotrophic factor, known to promote motoneuron survival after injury (Kondo et al., 2003; Zhao et al., 2004). The current study focused on the presence or absence of BDNF mRNA and protein expression by T cells and did not compare the level of BDNF among groups. In the future, we intend to utilize multiple approaches including immunocytochemistry staining and quantitative PCR to investigate the pattern and quantity of neurotrophic factor and cytokine gene and protein expression in central nervous system resident cells and infiltrating T cells, which may be of help to dissect the roles and contribution of each factor in supporting facial motoneuron survival after facial never injury. Disclosure We declare no conflict of interest or financial interests. Acknowledgements K.J.J. and V.M.S. were supported by National Institutes of Health Grant (NS40433), and J.X. was supported by a Grant from Muscular Dystrophy Association (MDA202906). We thank Richard Batka and Drs. Melissa Haulcomb, Keith Fargo, Eileen Foecking and Susan McGuire for support. References Azoulay, D., Urshansky, N., Karni, A., 2008. Low and dysregulated BDNF secretion from immune cells of MS patients is related to reduced neuroprotection. J. Neuroimmunol. 195, 186–193. De Santi, L., Cantalupo, L., Tassi, M., Raspadori, D., Cioni, C., Annunziata, P., 2009. Higher expression of BDNF receptor gp145trkB is associated with lower apoptosis intensity in T cell lines in multiple sclerosis. J. Neurol. Sci. 277, 65–70. De Santi, L., Polimeni, G., Cuzzocrea, S., Esposito, E., Sessa, E., Annunziata, P., Bramanti, P., 2011. Neuroinflammation and neuroprotection: an update on (future) neurotrophin-related strategies in multiple sclerosis treatment. Curr. Med. Chem. 18, 1775–1784. Deboy, C.A., Xin, J., Byram, S.C., Serpe, C.J., Sanders, V.M., Jones, K.J., 2006. Immunemediated neuroprotection of axotomized mouse facial motoneurons is dependent on the IL-4/STAT6 signaling pathway in CD4(+) T cells. Exp. Neurol. 201, 212–224. Edling, A.E., Nanavati, T., Johnson, J.M., Tuohy, V.K., 2004. Human and murine lymphocyte neurotrophin expression is confined to B cells. J. Neurosci. Res. 77, 709–717. Jones, K.J., LaVelle, A., 1985. Changes in nuclear envelope invaginations in axotomized immature and mature hamster facial motoneurons. Brain Res. 353, 241–249. Kerschensteiner, M., Gallmeier, E., Behrens, L., Leal, V.V., Misgeld, T., Klinkert, W.E., Kolbeck, R., Hoppe, E., Oropeza-Wekerle, R.L., Bartke, I., Stadelmann, C., Lassmann, H., Wekerle, H., Hohlfeld, R., 1999. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J. Exp. Med. 189, 865–870. Kondo, S., Kishi, H., Tokimitsu, Y., Muraguchi, A., 2003. Possible involvement of glial cell line-derived neurotrophic factor and its receptor, GFRalpha1, in survival and maturation of thymocytes. Eur. J. Immunol. 33, 2233–2240. Linker, R.A., Lee, D.H., Demir, S., Wiese, S., Kruse, N., Siglienti, I., Gerhardt, E., Neumann, H., Sendtner, M., Luhder, F., Gold, R., 2009. Functional role of brainderived neurotrophic factor in neuroprotective autoimmunity: therapeutic implications in a model of multiple sclerosis. Brain 133, 2248–2263. Maroder, M., Bellavia, D., Meco, D., Napolitano, M., Stigliano, A., Alesse, E., Vacca, A., Giannini, G., Frati, L., Gulino, A., Screpanti, I., 1996. Expression of trKB neurotrophin receptor during T cell development. Role of brain derived neurotrophic factor in immature thymocyte survival. J. Immunol. 157, 2864– 2872. Serpe, C.J., Byram, S.C., Sanders, V.M., Jones, K.J., 2005. Brain-derived neurotrophic factor supports facial motoneuron survival after facial nerve transection in immunodeficient mice. Brain Behav. Immun. 19, 173–180. 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. 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. Wolf, S.A., Steiner, B., Akpinarli, A., Kammertoens, T., Nassenstein, C., Braun, A., Blankenstein, T., Kempermann, G., 2009. CD4-positive T lymphocytes provide a neuroimmunological link in the control of adult hippocampal neurogenesis. J Immunol 182, 3979–3984.
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