Craig J. Serpe

CD4-Positive T Cell-Mediated Neuroprotection Requires Dual Compartment Antigen Presentation

Our laboratory discovered that CD4-positive (CD4+) T cells of the immune system convey transitory neuroprotection to injured mouse facial motoneurons (FMNs) (Serpe et al., 1999, 2000, 2003). A fundamental question in the mechanisms responsible for neuroprotection concerns the identity of the cell(s) that serves as the antigen-presenting cell (APC) to activate the CD4+ T cells. Here, we first establish that CD4+ T cells reactive to non-CNS antigen fail to support FMN survival and, second, demonstrate a two-compartment model of CD4+ T cell activation. Mouse bone marrow (BM) chimeras were developed that discriminate between resident antigen-presenting host cell and BM-derived antigen-presenting donor cell expression of major histocompatibility complex II within central and peripheral compartments, respectively. After facial nerve transection, neither compartment alone is sufficient to result in activated CD4+ T cell-mediated FMN survival. Rather, CD4+ T cell-mediated neuroprotection appears to depend on both resident microglial cells in the central compartment and a BM-derived APC in the peripheral compartment. This is the first in vivo report demonstrating a neuroprotective mechanism requiring APC functions by resident (i.e., parenchymal) microglial cells.

CD4+ T, but not CD8+ or B, lymphocytes mediate facial motoneuron survival after facial nerve transection

The capacity of facial motor neurons (FMN) to survive injury and successfully regenerate is substantially compromised in immunodeficient mice, which lack T and B lymphocytes (). The goal of the present study was to determine which T cell subset (CD4+ and/or CD8+), and whether the B lymphocyte, is involved in FMN survival after nerve injury. All mice were subjected to a right facial nerve axotomy, with the left (uncut) side serving as an internal control. FMN survival, of the right (cut) side, was measured 4 weeks post-operative, and expressed as a percentage of the left (uncut) control side. FMN survival in wild-type mice was 86%+/-1.5. In contrast, FMN survival in CD4 KO mice was 60%+/-2.0. Reconstitution of either CD4 KO mice, or recombinase activating gene-2 knockout (RAG-2 KO) mice (which lack functional T and B cells) with CD4+ T cells alone restored FMN survival to wild-type levels (85%+/-1.2 and 84%+/-2.5, respectively). There was no difference in FMN survival between wild-type, CD8 KO and MmuMT (B cell deficient) mice. Reconstitution of RAG-2 KO mice with CD8+ T cells alone, or B cells alone, failed to restore FMN survival levels (65%+/-1.5 and 63%+/-1.0, respectively). It is concluded that, of the population of FMN that do not survive injury, CD4+ T lymphocytes, but not CD8+ T lymphocytes or B cells, mediate FMN survival after peripheral nerve injury.

Kinetics of facial motoneuron loss following facial nerve transection in severe combined immunodeficient mice

We have recently shown that cells of the acquired immune system are crucial components of motoneuron survival after injury (Serpe et al. [1999] J. Neurosci. 19:RC7). The goal of the present study was to determine the kinetics of facial motoneuron (FMN) loss in wild‐type, scid, and reconstituted scid mice after a right facial nerve axotomy at the stylomastoid foramen. Scid mice showed a significant decrease in FMN survival at all weekly postoperative (wpo) times. One, two, four, and ten wpo, ipsilateral FMN survival in scid mice was 90% ± 1.8%, 84% ± 1.3%, 52% ± 3.7%, and 45% ± 2.5%, respectively, of the contralateral, unoperated side. In contrast, FMN loss after axotomy in wild‐type and reconstituted scid mice was not observed until 4 wpo (86% ± 2.5% and 83% ± 3.5%, respectively) relative to the contralateral, unoperated side. However, the levels of FMN in both wild‐type and reconstituted scid mice were significantly higher than those in the nonreconstituted scid at 4 wpo. By 10 wpo, FMN survival in both wild‐type and reconstituted scid mice had continued to decline significantly (60% ± 2.1% and 58% ± 3.1%, respectively) relative to the contralateral, unoperated side but were still significantly higher than that of the nonreconstituted scid at 10 wpo. Several important controls were also added to this study. Because the scid mutation is present in all cells (although it specifically results in a loss of V(D)J recombination mechanisms, we wanted to rule out the actual DNA mutation as causal in FMN loss). To accomplish this, we used the recombinase‐activating gene‐2 knockout (RAG‐2 KO) mouse model, in which the RAG‐2 has been disrupted and prevents maturation of T and B cells. As with the scid model, there was a significant loss of FMN at 4 wpo in the RAG‐2 KO that could be reversed with whole splenocyte reconstitution. We also compared FMN numbers in nonaxotomized facial nuclei from both scid and RAG‐2 KO mice relative to wild‐type controls. No differences in normal numbers of FMN were found in either the mutation or the gene knockout model. The ability of T and B lymphocytes to rescue FMN from cell death after peripheral nerve injury supports the hypothesis that cells of the acquired immune system produce neurotrophic factors or neurocytokines to support neuronal survival until target reconnection occurs. J. Neurosci. Res. 62:273–278, 2000.

Immune-mediated neuroprotection of axotomized mouse facial motoneurons is dependent on the IL-4/STAT6 signaling pathway in CD4+ T cells

The CD4(+) T lymphocyte has recently been found to promote facial motoneuron (FMN) survival after nerve injury. Signal Transducer and Activator of Transcription (STAT)4 and STAT6 are key proteins involved in the CD4(+) T cell differentiation pathways leading to T helper type (Th)1 and Th2 cell development, respectively. To determine which CD4(+) T cell subset mediates FMN survival, the facial nerve axotomy paradigm was applied to STAT4-deficient (-/-) and STAT6-/- mice. A significant decrease in FMN survival 4 weeks after axotomy was observed in STAT6-/- mice compared to wild-type (WT) or STAT4-/- mice. Reconstituting STAT6-/- mice with CD4(+) T cells obtained from WT mice promoted WT levels of FMN survival after injury. Furthermore, rescue of FMN from axotomy-induced cell death in recombination activating gene (RAG)-2-/- mice (lacking T and B cells) could be achieved only by reconstitution with CD4(+) T cells expressing functional STAT6 protein. To determine if either the Th1 cytokine, interferon-gamma (IFN-gamma) or the Th2 cytokine IL-4 is involved in mediating FMN survival, facial nerve axotomy was applied to IFN-gamma-/- and IL-4-/- mice. A significant decrease in FMN survival after axotomy occurred in IL-4-/- but not in IFN-gamma-/- mice compared to WT mice, indicating that IL-4 but not IFN-gamma is important for FMN survival after nerve injury. In WT mice, intracellular IFN-gamma vs. IL-4 expression was examined in CD4(+) T cells from draining cervical lymph nodes 14 days after axotomy, and substantial increase in the production of both CD4(+) effector T cell subsets was found. Collectively, these data suggest that STAT6-mediated CD4(+) T cell differentiation into the Th2 subset is necessary for FMN survival. A hypothesis relevant to motoneuron disease progression is presented.

IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection

We have previously shown that immunodeficient mice exhibit significant facial motoneuron (FMN) loss compared to wild-type (WT) mice after a facial nerve axotomy. Interleukin-10 (IL-10) is known as a regulatory cytokine that plays an important role in maintaining the anti-inflammatory environment within the central nervous system (CNS). IL-10 is produced by a number of different cells, including Th2 cells, and may exert an anti-apoptotic action on neurons directly. In the present study, the role of IL-10 in mediating neuroprotection following facial nerve axotomy in Rag-2- and IL-10-deficient mice was investigated. Results indicate that IL-10 is neuroprotective, but CD4+ T cells are not the requisite source of IL-10. In addition, using real-time PCR analysis of laser microdissected brainstem sections, results show that IL-10 mRNA is constitutively expressed in the facial nucleus and that a transient, significant reduction of IL-10 mRNA occurs following axotomy under immunodeficient conditions. Dual labeling immunofluorescence data show, unexpectedly, that the IL-10 receptor (IL-10R) is constitutively expressed by facial motoneurons, but is selectively induced in astrocytes within the facial nucleus after axotomy. Thus, a non-CD4+ T cell source of IL-10 is necessary for modulating both glial and neuronal events that mediate neuroprotection of injured motoneurons, but only with the cooperation of CD4+ T cells, providing an avenue of novel investigation into therapeutic approaches to prevent or reverse motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

Brain-derived neurotrophic factor supports facial motoneuron survival after facial nerve transection in immunodeficient mice

Numerous studies have shown that motoneuron survival can be facilitated by neurotrophic factors (NTF) after injury. However, the ability of specific NTF to rescue facial motoneurons (FMN) from axotomy-induced death in immunodeficient mice has not been tested. Therefore, one goal of this study was to determine if brain-derived neurotrophic factor (BDNF), an NTF with a known ability to rescue FMN from axotomy-induced death, supports FMN from axotomy-induced death in recombinase activating gene-2 knockout (RAG-2 KO) mice that lack functional T and B lymphocytes. Nerve growth factor, which has been shown not to play a role in motoneuron survival, was used as a negative control. Brain derived neurotrophic factor treatment restored FMN survival to wild-type (WT) control levels 4 weeks post-operative (wpo) (80% +/- 1.9, 83% +/- 2.4, respectively). The second goal of this study was to begin to elucidate if CD4+ T cells produce NTF after facial nerve axotomy. Cervical lymph nodes were collected from WT mice 9 days post-operative, re-activated with anti-CD3 and supernatant collected 24 h later. Immediately after injury, the supernatant was administered to RAG-2 KO mice leading to an increase in FMN survival equivalent to WT controls (80% +/- 1.4, 84% +/- 2.1, respectively, 4 wpo). In addition, cervical lymph node supernatant treated with anti-BDNF attenuated FMN rescue in RAG-2 KO mice (62% +/- 3.3) 4 wpo. These data lend support to the hypothesis that CD4+ T cells produce NTF that support motoneuron survival before target reconnection occurs.

Functional recovery after facial nerve crush is delayed in severe combined immunodeficient mice

The goal of the current study was to determine if T and B lymphocytes play a role in functional recovery after peripheral nerve injury. The time course of behavioral recovery following facial nerve crush injury at the stylomastoid foramen was established in scid mice which lack functional T and B cells and reconstituted scid mice as compared to wild-type mice. The average time necessary for recovery of full eye blink reflex and vibrissae movements in wild-type mice was 10.3+/-0.2 and 9.9+/-0.34 days, respectively. In contrast, recovery of full eye blink reflex and vibrissae movements took 14.8+/-0.54 and 12.3+/-0.41 days, respectively, in scid mice. Reconstitution of scid mice with whole splenocytes resulted in functional recovery times similar to wild-type, with eye blink reflex recovery and vibrissae movement being 10.5+/-0.3 and 10.0+/-0.0 days, respectively. These results suggest that the delayed behavioral recovery time observed in scid mice may be due to the absence of T and B lymphocytes.

Natural killer cells do not mediate facial motoneuron survival after facial nerve transection.

The goal of the current study was to determine if natural killer (NK) cells mediate facial motoneuron (FMN) survival following injury. Wild-type (WT), perforin/recombinase activating gene-2 knockout (pfp/RAG-2 KO), and common gamma-chain (gammac)/RAG-2 KO mice received a right facial nerve axotomy. In WT mice, FMN survival was 86+/-1.0% relative to the contralateral control side. In contrast, pfp/RAG-2 and gammac/RAG-2 KO mice exhibited significant decreases in FMN survival ( approximately 20% and approximately 30%, respectively), relative to WT. Reconstitution of pfp/RAG-2 and gammac/RAG-2 KO mice with normal NK cells alone, failed to restore FMN survival levels to those of WT, but did restore functional lytic activity against YAC-1 cells. Reconstitution of pfp/RAG-2 and gammac/RAG-2 KO mice with splenocytes, and pfp/RAG-2 KO mice with CD4+ T-lymphocytes alone or in combination with NK cells, restored FMN survival levels to those of WT. Thus, NK cells appear to not be a component of immune cell-mediated rescue of motoneurons from axotomy induced cell death.

CD4+ T cell-mediated neuroprotection is independent of T cell-derived BDNF in a mouse facial nerve axotomy model

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.

Motoneurons and CD4+ effector T cell subsets: Neuroprotection and repair

Abstract Both neuroprotective and neurodestructive effects of the immune system have been described, although the regulatory nature of such contradictory actions have yet to be determined. We combined the facial nerve injury with immunodeficient mouse models and our findings provide the foundation for a working model of CD4+T cell-mediated motoneuron survival and axonal regeneration after injury. Key to this model is the new concept that CD4+ effector T cell subsets play distinctive roles in motoneuron reparative processes, with the Th2 cell mediating FMN survival and the Th1 cell mediating functional recovery. This concept places the motoneuron as the central regulator of the immune response that occurs after direct axonal trauma. The postulated Th2/Th1 paradigm creates a balance essential to the health of the motoneuron and to its ability to mount a graded response to injury that is geared toward survival and structural/functional recovery for the organism, respectively. Understanding the inherent capabilities of the CNS to direct the local immune reaction to injury is essential to elucidation of the pathophysiology inherent in neurodegenerative diseases, particularly ALS and other motoneuron disorders, in which compromised neurons may lose the ability to regulate a local immune response, such that an imbalance occurs and results in a pro-inflammatory, neurodestructive environment. In the case of motoneuron disease, such as ALS, we hypothesize that the diseased motoneuron cannot direct a neuroprotective immune response, resulting in a destructive pro-inflammatory environment near the diseased motoneuron.