Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy.

Autoimmunity to antigens of the central nervous system is usually considered detrimental. T cells specific to a central nervous system self antigen, such as myelin basic protein, can indeed induce experimental autoimmune encephalomyelitis, but such T cells may nevertheless appear in the blood of healthy individuals. We show here that autoimmune T cells specific to myelin basic protein can protect injured central nervous system neurons from secondary degeneration. After a partial crush injury of the optic nerve, rats injected with activated anti-myelin basic protein T cells retained approximately 300% more retinal ganglion cells with functionally intact axons than did rats injected with activated T cells specific for other antigens. Electrophysiological analysis confirmed this finding and suggested that the neuroprotection could result from a transient reduction in energy requirements owing to a transient reduction in nerve activity. These findings indicate that T-cell autoimmunity in the central nervous system, under certain circumstances, can exert a beneficial effect by protecting injured neurons from the spread of damage.

Innate and adaptive immune responses can be beneficial for CNS repair.

The limitation of immune responsiveness in the mammalian CNS has been attributed to the intricate nature of neuronal networks, which would appear to be more susceptible than other tissues to the threat of permanent disorganization when exposed to massive inflammation. This line of logic led to the conclusion that all forms of CNS inflammation would do more harm than good and, hence, the less immune intervention the better. However, mounting evidence indicates that some forms of immune-system intervention can help to protect or restore CNS integrity. We have shown that the innate immune system, represented by activated macrophages, can facilitate the processes of regeneration in the severed spinal cord. More recently, we found that autoimmune T cells that are specific for a component of myelin can protect CNS neurons from the catastrophic secondary degeneration, which extends traumatic lesions to adjacent CNS areas that did not suffer direct damage. The challenge, therefore, is to learn how to modify immune interactions in the traumatized CNS in order to promote its post-injury maintenance and repair.

Chemical mediators enhance the excitability of unmyelinated sensory axons in normal and injured peripheral nerve of the rat.

Ectopic excitation of nociceptive axons by chemical mediators may contribute to symptoms in neuropathic pain. In this study, we have measured the excitability of unmyelinated rat C-fiber axons in isolated segments of sural nerves under different experimental conditions. (1) We demonstrate in normal rats that several mediators including ATP, serotonin (5-HT), 1-(3-chlorophenyl)biguanide (5-HT3 receptor agonist), norepinephrine, acetylcholine and capsaicin alter electrophysiological parameters of C-fibers which indicate an increase of axonal excitability. Other mediators such as histamine, glutamate, prostaglandin E(2) and the cytokines tumor necrosis factor alpha, interleukin-1beta and interleukin-6 did not produce such effects. (2) The effects of several mediators were tested after peripheral nerve injury (partial ligation or spared nerve injury). Sural nerves from such animals did not show significant changes when compared with controls. (3) We tested whether the effects of chemical mediators on axonal excitability are due to actions on the sensory C-fiber afferents or the postganglionic sympathetic efferents. In order to distinguish these effects, we performed surgical sympathectomy of the lumbar sympathetic chain, including the L3, L4 and L5 ganglia. Sympathectomy did not markedly influence the effects of mediators on axonal excitability (except that the norepinephrine effect was significantly diminished). In conclusion, our data suggest a constitutive rather than inducible expression of axonal receptors for some chemical mediators on the axonal membrane of unmyelinated fibers. Most of the changes in axonal excitability take place in sensory C-fiber afferents rather than in postganglionic sympathetic efferents. Thus, it is possible that certain immune and glial cell mediators released in or around the nerve following injury or inflammation influence the excitability of intact nociceptive fibers. This mechanism could contribute to ectopic excitation of axons in neuropathic pain.

The remedy may lie in ourselves: prospects for immune cell therapy in central nervous system protection and repair.

The irreversible loss of function after axonal injury in the central nervous system (CNS) is a result of the lack of neurogenesis, poor regeneration, and the spread of damage caused by toxicity emanating from the degenerating axons to uninjured neurons in the vicinity. Now, 100 years after Ramon y Cajal's discovery that CNS neurons--unlike neurons of the peripheral nervous system--fail to regenerate, it has become evident that (a) CNS tissue is indeed capable of regenerating, at.least in part, provided that it acquires the appropriate conditions for growth support, and (b) that the spread of damage can be stopped and the postinjury rescue of neurons thus achieved, if ways are found to neutralize the mediators of toxicity, either by inhibiting their action or by increasing tissue resistance to them. In most physiological systems the processes of tissue maintenance and repair depend on the active assistance of immune cells. In the CNS, however, communication with the immune system is restricted. The accumulated evidence from our previous studies suggests that the poor posttraumatic repair and maintenance in the CNS is due at least in part to this restriction. Key factors in the recovery of injured tissues, but missing or deficient in the CNS, are the processes of recruitment and activation of immune cells. We therefore propose the development of immune cell therapies in which the injured CNS is exogenously provided with an adequate number of appropriately activated immune cells (macrophages for regrowth and autoimmune T cells for maintenance), controlled in such a way as to derive maximal benefit with minimal risk of disease. It is expected that these self-adjusting cells will communicate with the damaged tissue, monitor tissue needs, and control the dynamic course of CNS healing.

Beneficial immune activity after CNS injury: prospects for vaccination.

A recent study in our laboratory showed, against all expectations, that macrophages and a particular type of T cell, by promoting regrowth and reducing the post-traumatic spread of damage in the injured rat optic nerve or spinal cord, have a beneficial effect on the injured CNS. Macrophages in the CNS have long been thought to have predominantly destructive effects. Autoimmunity in general, and in the CNS in particular, has never been documented as a purposeful physiological response of benign character. Our results suggest that after traumatic injury to the central nervous system (CNS), both of these immune cell types potentially have beneficial effects: macrophages can promote repair and T cells of a particular specificity can reduce the spread of damage. However, possibly because of the immune-privileged character of the CNS, the spontaneously evoked physiological activities of both macrophages and T cells in the CNS are restricted, and appear to need well-controlled boosting in order to be effective. It thus appears that (i) a stress signal transmitted from the traumatized tissue (in this case the CNS) for recruitment of the adaptive immune system does not have to be pathogen-related in order to evoke a response, (ii) a response to self is not necessarily a quirk of nature, and (iii) an autoimmune response, provided that it is well-regulated, helps the individual to cope with stress signals from the traumatized CNS, and thus plays a role in maintenance of the injured tissue without posing a threat to the organism.

Accumulation of passively transferred primed T cells independently of their antigen specificity following central nervous system trauma.

The central nervous system (CNS) enjoys a unique relationship with the immune system. Under non-pathological conditions, T cells move through the CNS but do not accumulate there. CNS trauma has been shown to trigger a response to CNS self-antigens such as myelin basic protein (MBP). Here, we examined whether the injured CNS tissue undergoes changes that permit T cell accumulation. We found that injury to CNS white matter, such as the optic nerve, led to a transiently increased accumulation of T cells (between days 3 and 21). In Lewis rats with unilaterally injured optic nerves, systemic administration of passively transferred T cells recognizing either self-antigen (MBP) or non-self-antigen (ovalbumin) resulted in accumulation of the T cells in injured optic nerve, irrespective of their antigenic specificity. The effect of the T cells on the damaged nerve, the lack of selectivity in T cell accumulation and the mechanism underlying non-selective accumulation are discussed.

Autoimmune T cells retard the loss of function in injured rat optic nerves.

We recently demonstrated that autoimmune T cells protect neurons from secondary degeneration after central nervous system (CNS) axotomy in rats. Here we show, using both morphological and electrophysiological analyses, that the neuroprotection is long-lasting and is manifested functionally. After partial crush injury of the rat optic nerve, systemic injection of autoimmune T cells specific to myelin basic protein significantly diminished the loss of retinal ganglion cells and conducting axons, and significantly retarded the loss of the visual response evoked by light stimulation. These results support our challenge to the traditional concept of autoimmunity as always harmful, and suggest that in certain situations T cell autoimmunity may actually be beneficial. It might be possible to employ T cell intervention to slow down functional loss in the injured CNS.

T lymphocytes play a role in neuropathic pain following peripheral nerve injury in rats.

A catastrophic consequence of peripheral nerve injury is the development of abnormal, chronic neuropathic pain. The inflammatory response at the injury site is believed to contribute to the generation and maintenance of such persistent pain. However, the physiological significance and potential contribution of T cells to neuropathic pain remains unclear. Here we show that T cells infiltrate injured sciatic nerves following chronic constriction injury (CCI), but not uninjured nerves. Congenitally athymic nude rats, which lack mature T cells, developed a significantly reduced mechanical allodynia and thermal hyperalgesia following CCI, compared with their heterozygous littermates. To understand further the role played by different T-cell subsets, we generated polarized populations of type 1 and type 2 T cells, with different cytokine secretion profiles, from spleens of sciatic nerve-injured heterozygous rats. Passive transfer of type 1 T cells, which produce proinflammatory cytokines, into nude rats enhanced the recipients' pain hypersensitivity to a level similar to that of heterozygous donor rats. In contrast, passive transfer of polarized type 2 T cells, which produce anti-inflammatory cytokines, into heterozygous rats modestly though significantly attenuated their pain hypersensitivity. Thus, injection of type 1 and type 2 T-cell subsets produces opposing effects on neuropathic pain. These findings suggest the modulation of the T-cell immune response as a potential target for the treatment of neuropathic pain.

Differential T cell response in central and peripheral nerve injury: connection with immune privilege.

The central nervous system (CNS), unlike the peripheral nervous system (PNS), is an immune-privileged site in which local immune responses are restricted. Whereas immune privilege in the intact CNS has been studied intensively, little is known about its effects after trauma. In this study, we examined the influence of CNS immune privilege on T cell response to central nerve injury. Immunocytochemistry revealed a significantly greater accumulation of endogenous T cells in the injured rat sciatic nerve than in the injured rat optic nerve (representing PNS and CNS white matter trauma, respectively). Use of the in situ terminal deoxytransferase-catalyzed DNA nick end labeling (TUNEL) procedure revealed extensive death of accumulating T cells in injured CNS nerves as well as in CNS nerves of rats with acute experimental autoimmune encephalomyelitis, but not in injured PNS nerves. Although Fas ligand (FasL) protein was expressed in white matter tissue of both systems, it was more pronounced in the CNS. Expression of major histocompatibility complex (MHC) class II antigens was found to be constitutive in the PNS, but in the CNS was induced only after injury. Our findings suggest that the T cell response to central nerve injury is restricted by the reduced expression of MHC class II antigens, the pronounced FasL expression, and the elimination of infiltrating lymphocytes through cell death.

Factor XIIIa as a nerve-associated transglutaminase.

Recent findings have led to changes in the traditional concept of nerve recovery, including the realization that injured nerves, like any other injured tissue, need the assistance of blood-derived cells and factors in order to heal. We show that factor XIIIa (FXIIIa, the potentially active a2subunit of factor XIII), an enzyme that participates in blood coagulation by stabilizing the fibrin clot, is also active in the nervous system where it may play a key role in the healing of injured tissue. We demonstrate that the plasma, macrophages and nerves of fish contain a 55 kDa form of transglutaminase that cross-reacts immunologically with the a-subunit of FXIII in mammals (80 kDa). The fish enzyme in the plasma, unlike its mammalian counterpart, is active, pointing to a difference in control of the coagulation pathway in the two species. Analysis of FXIIIa expression in mammalian neural tissues and their response to injury revealed high levels of the enzyme in media conditioned by peripheral nerves as compared with medium conditioned by nerves of the central nervous system. Furthermore, similarity was observed in the postinjury behavior of FXIIIa in regenerating nerve tissues (peripheral nervous system of mammals and the central nervous system of fish). We suggest that the postinjury level of factor XIIIa in the nervous system may be related to the tissue's regenerative capacity, and that FXIIIa may therefore be a link underlying a possible association between the processes of blood coagulation and nerve healing.

Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity.

Neurotrophins (NTs) promote neuronal survival and maintenance during development and after injury. However, their role in the communication between the nervous system and the immune system is not yet clear. We observed recently that passively transferred activated T cells of various antigen specificities home to the injured central nervous system (CNS), yet only autoimmune T cells specific to a CNS antigen, myelin basic protein (MBP), protect neurons from secondary degeneration after crush injury of the rat optic nerve. Here we examined the involvement of NTs in T-cell-mediated neuroprotection, and the possible significance of the antigen specificity of the T cells in this activity. Analysis of cytokine and NT expression in various rat T cell lines showed that the T cells express mRNA for cytokines of Th1, Th2, and Th3 phenotypes. In addition, the T cells express mRNA and protein specific to nerve growth factor, brain-derived neurotrophic factor, NT-3, and NT-4/5. Antigen activation significantly increased NT secretion. Thus, reactivation of CNS autoimmune T cells by locally presented antigens to which they are specific can lead to enhanced secretion of NTs and possibly also of other factors in injured optic nerves. mRNA for TrkA, TrkB and p75 receptors was expressed in the injured nerve, suggesting that these specific receptors can mediate the effects of the T-cell-derived NTs. The neuroprotective effect of the passively transferred autoimmune anti-MBP T cells in injured optic nerves was significantly decreased after local applicaiton of a tyrosine kinase inhibitor known to be associated with NT-receptor activity. These results suggest that the neuroprotective effect of autoimmune T cells involves the secretion of factors such as NTs by the T cells reactivated by their specific antigen in the injured CNS. T cell intervention in the injured CNS might prove to be a useful means of promoting post-injury CNS maintenance and recovery, possibly via supply of NTs and other factors.

Autoimmune T cells as potential neuroprotective therapy for spinal cord injury.

Autoimmune T cells against central nervous system myelin associated peptide reduce the spread of damage and promote recovery in injured rat spinal cord, findings that might lead to neuroprotective cell therapy without risk of autoimmune disease.

THF-gamma 2-mediated reduction of pulmonary metastases and augmentation of immunocompetence in C57BL/6 mice bearing B16-melanoma.

Immunotherapy with the immunomodulating thymic humoral factor-gamma 2 (THF-gamma 2) octapeptide, combined with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy, will be used for enhancing host immune response to arrest pulmonary metastases of a B16-F10.9 melanoma tumor. In this experimental model of pulmonary metastasis, the highly metastatic B16-F10.9 melanoma tumor cells (2 x 10(5)) were inoculated into the footpad of mice to form a primary tumor. The tumor-bearing leg was surgically removed on reaching the size of 5.5 mm, which resulted in the appearance of metastases in the lungs of the animals. After tumor excision, mice were treated intraperitoneally with a single dose of BCNU (20 or 35 mg/kg) followed by a series of intraperitoneal THF-gamma 2 injections (1 microgram/0.5 ml/injection). Relative to untreated mice and those receiving chemotherapy alone, the antitumor action of the combined THF-gamma 2 chemoimmunotherapy protocol was significantly augmented according to the following in vivo parameters: (a) decreased postsurgical spontaneous metastatic burden; (b) prolonged survival time; (c) increased resistance to tumor cell challenge; and (d) massive infiltration of lymphocytes, polymorphonuclear cells, and macrophages in the lung tissue. The THF-gamma 2 immunotherapy also prevented a decrease in lymphocyte reactivity, otherwise induced by the tumor/BCNU chemotherapy. THF-gamma 2 immunotherapy resulted in restoration of the response to Lipopolysaccharide mitogenic stimulation and the allogeneic response. Our data suggest that postoperative THF-gamma 2 immunotherapy could be a valuable adjunct to anticancer chemotherapy as a treatment for metastatic arrest of melanoma tumor.

Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion.

Partial injury to the spinal cord can propagate itself, sometimes leading to paralysis attributable to degeneration of initially undamaged neurons. We demonstrated recently that autoimmune T cells directed against the CNS antigen myelin basic protein (MBP) reduce degeneration after optic nerve crush injury in rats. Here we show that not only transfer of T cells but also active immunization with MBP promotes recovery from spinal cord injury. Anesthetized adult Lewis rats subjected to spinal cord contusion at T7 or T9, using the New York University impactor, were injected systemically with anti-MBP T cells at the time of contusion or 1 week later. Another group of rats was immunized, 1 week before contusion, with MBP emulsified in incomplete Freund's adjuvant (IFA). Functional recovery was assessed in a randomized, double-blinded manner, using the open-field behavioral test of Basso, Beattie, and Bresnahan. The functional outcome of contusion at T7 differed from that at T9 (2.9+/-0.4, n = 25, compared with 8.3+/-0.4, n = 12; p<0.003). In both cases, a single T cell treatment resulted in significantly better recovery than that observed in control rats treated with T cells directed against the nonself antigen ovalbumin. Delayed treatment with T cells (1 week after contusion) resulted in significantly better recovery (7.0+/-1; n = 6) than that observed in control rats treated with PBS (2.0+/-0.8; n = 6; p<0.01; nonparametric ANOVA). Rats immunized with MBP obtained a recovery score of 6.1+/-0.8 (n = 6) compared with a score of 3.0+/-0.8 (n = 5; p<0.05) in control rats injected with PBS in IFA. Morphometric analysis, immunohistochemical staining, and diffusion anisotropy magnetic resonance imaging showed that the behavioral outcome was correlated with tissue preservation. The results suggest that T cell-mediated immune activity, achieved by either adoptive transfer or active immunization, enhances recovery from spinal cord injury by conferring effective neuroprotection. The autoimmune T cells, once reactivated at the lesion site through recognition of their specific antigen, are a potential source of various protective factors whose production is locally regulated.