key: cord-355413-ls2nud43
authors: Shi, Fu-Dong; Ransohoff, Richard M.
title: Nature killer cells in the central nervous system
date: 2010-01-29
journal: Natural Killer Cells
DOI: 10.1016/b978-0-12-370454-2.00028-4
sha: 
doc_id: 355413
cord_uid: ls2nud43

Natural killer (NK) cells, a prominent component of the innate immune system, are large granular lymphocytes that respond rapidly to a variety of insults via cytokine secretion and cytolytic activity. Recently, there has been growing insight into the biological functions of NK cells, in particular into their roles in infection, tumorurveillance and autoimmunity. Under these pathophysiological circumstances, NK cells readily home to the central nervous system (CNS) tissues to combat infection and presumably to curb progression of tumor. Bystander neuronal and/or glial cell damage can occur in this setting. Paradoxically, NK cells appear to have an inhibitory role for autoimmune responses within the CNS. As in the periphery, NK cells act in concert with T cells and other lymphocytes responsible for CNS pathology and immune regulation. Insights into the molecular signals and pathways governing the diverse biological effects of NK cells are keys for designing NK cell-based therapy for CNS infections, tumor and autoimmune diseases.NK cells readily accumulate in homing to CNS tissues under the pathophysiological situations. This process is tightly controlled by a number of chemokines and chemokine receptors. There is ample of evidence that NK cells within the CNS contribute to the control of infections and might limit progression of certain tumor. Bystander neuronal and/or glial cell damage can occur. In certain autoimmune conditions of the CNS, NK cells appear to have an inhibitory role. Disassociation of disease-inhibiting versus disease-promoting effects of NK cells is a key to harnessing NK cells for therapeutic purposes. To achieve this goal, a generation of genetic models with selective NK cell deficiency, and development of reagents (antibodies) for visualizing subsets of NK cells in situ will be necessary.

cells in human CNS tissues is still lacking. This is partly because of the lack of suitable antibodies for staining human NK cells in situ (see Chapter 31). Visualization of NK cells in mouse brains during experimental autoimmune encephalomyelitis (EAE) was achieved using anti-NK1.1 mAb (PK136) (Hammarberg et al., 2000) . Antibodies such as Ly49 have been successfully used for staining NK cells in lymphoid organs (O'Leary et al., 2006) . Suitability of these antibodies for staining CNS NK cells needs to be verified in additional studies.

Although direct visualization of NK cells in CNS tissues is technically challenging, there is little doubt that NK cells, as with other types of lymphocytes, enter the CNS during inflammatory processes. In fact, it has been reported that NK cells are among the earliest recruited cells during adoptive transfer EAE (Kerfoot and Kubes, 2002; Wekerle and Fierz, 1985) . Chemokine receptors such as CCR2, CCR5, CXCR3, CX3CR1 as well as lysophospholipid sphingosine 1-phosphate (S1P) are involved in the rapid NK-cell mobilization that occurs in inflammatory conditions (Ajuebor et al., 2007; Hokeness et al., 2005; Huang et al., 2006; Inngjerdingen et al., 2001; Jiang et al., 2004; Khan et al., 2006; Kveberg et al., 2002; Lavergne et al., 2003; Martin-Fontecha et al., 2004; Thapa et al., 2007; Wald et al., 2006; Walzer et al., 2007; Yu et al., 2007) , and several of these chemokine receptors (CCR5, CX3CR1) are directly involved in NK cell homing to the CNS (Huang et al., 2006; Martin-Fontecha et al., 2004; Thapa et al., 2007) . The biological implication of chemokine-guided homing of NK cells during CNS inflammation is discussed in greater detail in the following section.

The ability of NK cells to kill various transformed and virus-infected cells raises an important question whether direct NK cell cytolytic effects contribute to the pathogenesis of inflammatory, degenerative and autoimmune disorders of the nervous system. Neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease are characterized by the death of neurons in distinct functional neuron-anatomic systems. Multiple sclerosis (MS), on the other hand, is characterized by inflammation and demyelination within the spinal cord and brain, and axonal damage and brain atrophy also occur during the course of disease. The peripheral form of MS is Guillain-Barré syndrome, which is also characterized by demyelination and cellular infiltrates of the peripheral nervous system. Some of these diseases involve immunologic components or reactions, and some have been characterized extensively in the different systems. Autoreactive T cells and adaptive immune system components in the pathogenesis in some of these disorders are well characterized. As discussed here, evidence for direct NK cell cytolytic effects is emerging. Although the in vivo relevance of a great proportion of these studies needs to be validated, the current data emphasize the importance of NK cells either in direct cytotoxic effects or in collaboration with cells from both innate and adaptive immune systems in the initiation of these neurodegenerative and inflammatory diseases.

The NK cell-dependent death of sympathetic neurons resident in the superior cervical ganglia of rats, observed after the exposure to the drug guanethidine (Hickey et al., 1992; Hougen et al., 1992) , is the first in vivo disorder of the nervous system in which NK cells appear to be the dominant effector cells. The pathogenic mechanism observed appeared to represent a novel type of autoimmune reaction: an exogenously/chemically induced alteration in a specific subset of cells that was suggested to target them for direct NK cell-mediated killing.

Interestingly, neuronal cells from the peripheral system and the CNS appear to have different susceptibility to NK cell killing. Ljunggren and colleagues have carried out a series of well-designed studies addressing this puzzling phenomenon. Initially, it was demonstrated that NK cells could readily kill syngeneic dorsal root ganglia (DRG) neurons by a perforin-dependent mechanism (Backstrom et al., 2000 (Backstrom et al., , 2003 . Ventral spinal cord neurons and hippocampal neurons of the CNS were resistant to lysis. The resistance to NK cell-mediated lysis of the latter neurons was not related to protection by MHC class I molecules, since similar  2 -micro-globulin / neurons were equally resistant to lysis (Backstrom et al., 2003) .

NK cell function is tightly regulated by multiple signals transmitted via inhibitory and activating receptors. The prerequisite for NK cell killing is its activation via signalling from activating receptor ligand pathways. NK cell activation generally appears to be elicited by a distinct set of molecules that have weak homology with MHC class I molecules. The activating receptor NKG2D which differs dramatically from other NKG2 receptor proteins is of particular interest since it, in contrast to other NK cell-activating receptors, is constitutively expressed on NK cells. The endogenous ligand of NKG2D in the mouse was recently identified as retinoic acid early inducible gene-1 (RAE-1)-encoded proteins and minor histocompatibility antigen H60 (Cerwenka et al., 2000; Diefenbach et al., 2000; Malarkannan et al., 1998) . Differential expression of NKG2D and its ligand on neuronal cells from the peripheral system or the CNS appears a key mechanism underlying variable susceptibility to NK lyses. RAE-1, the product of which is a ligand for the NK cell-activating receptor NKG2D, was expressed at high levels in the DRG neurons. In contrast, RAE-1 was expressed only at very low levels in the resistant CNS-derived neurons. Blocking NK cells with anti-NKG2D antibodies inhibited NK cell-mediated killing of the DRG neurons.

These findings are important in revealing novel immunopathologic effects of several CNS diseases. Indeed, progressive motor and sensory neuropathy developed in a patient with chronic NK cell lymphocytosis (CNKL) (Noguchi et al., 2005) . A sural nerve biopsy revealed infiltration of NK cells into the nerve fascicles, and demyelinating changes with secondary axonal degeneration. The infiltrating NK cells were adjacent to myelinated fibres, showing damage of Schwann cell membranes. Treatment with oral prednisolone resulted in rapid improvement of sensory disturbance and weakness with a significant decrease of NK cells in the blood and disappearance of the conduction block. These observations suggest that the infiltrating NK cells may directly damage myelin and Schwann cells, thus causing demyelination.

Since expression of NKG2D ligands is likely regulated by viral infection or transformation, and the inhibitory MHC class I expression is low or absent in the nervous system, it is plausible that a viral infection or transformation could well break the balance of activating/inhibiting activities on NK cells, and NK cell-mediated immune pathology would occur in such circumstances.

Human oligodendrocytes do not express MHC class II molecules; thus, direct MHC-restricted injury mediated by myelin-reactive CD4  T cells is less likely to occur. The migration of NK cells to the CNS during inflammatory responses and lack of inhibitory signal MHC class I expression within the CNS invites prediction that direct cytolytic effects of NK cells contribute to oligodendrocyte damage and demyelination to CNS diseases such as MS. Antel and colleagues demonstrated using in vitro assays that human oligodendrocytes, as well as other glial elements (astrocytes, microglia), were susceptible to injury mediated by peripheral blood-derived mononuclear cell preparations (MNCs), which were enriched for NK cells by depleting CD3  , with or without CD19  cells through the use of either magnetic beads or cell sorting (Antel et al., 1998; Morse et al., 2001) . Cytotoxic effects of the NK cell-enriched effectors were dependant on pre-exposure of these cells to IL-2. Furthermore, it was found that autologous oligodendrocytes were as susceptible to injury mediated by IL-2-activated NK cells as were heterogonous oligodendrocytes.

In searching for receptor ligand pathways that control the NK cell and oligodendrocyte interactions, it was found that human adult oligodendrocytes and foetal astrocytes expressed ligands for NKG2D in vitro, whereas neurons, microglia, and adult astrocytes did not (Saikali et al., 2007) . Disruption of the NKG2D-NKG2D ligand interaction using blocking antibodies significantly inhibited killing of primary human oligodendrocytes mediated by activated human NK cells (Saikali et al., 2007) . These results imply that NKG2D-NKG2D ligand interactions can potentially contribute to cytotoxic responses mediated by activated immune effector cells in the inflamed CNS, as observed in MS.

In the context of tissue injury that occurs in MS, the inflammatory milieu in MS lesions may provide conditions required for NK cell activation, raising the possibility that such effector cells would play a role in the pathogenesis. In addition to direct cytotoxicity, cytokine release by NK cells may also participate in tissue damage as well as in regulating T cell immune responses. Interferon-gamma (IFN-) is a pleiotropic cytokine produced by T cells and NK cells that has been implicated as a deleterious factor in MS, the immune-mediated demyelinating disorder. In vitro, purified developing and mature oligodendrocytes die in the presence of IFN- by apoptosis and necrosis, respectively. Transgenic expression of PLP/SOCS1 (proteolipid protein regulating suppressor of cytokine signalling 1), brings about diminished oligodendrocyte responsiveness to IFN- attributable to the targeted expression of SOCS1 in these cells (Balabanov et al., 2006) . Consequently, oligodendrocytes in the PLP/SOCS1 transgenic mice are protected against the injurious effect of IFN-. Although both NK cells and T cells produce IFN-, NK cells are the principal sources of early IFN- production prior to T cell activation. This time kinetic might be particularly relevant for early oligodendrocyte damage during inflammation.

Efficient early control of viral infections is determined by viral tissue tropism and rate of replication as well as the host's ability to mount an effective immune response. Cellular cytotoxicity, in particular, that of NK cells and cytotoxic T cells, is central to the early antiviral immune response. Table 28 .1 illustrates several immunedeficient conditions in humans, which stem from mutations affecting NK cells (Biron et al., 1989; Gilmour et al., 2001; Markel et al., 2004; Moins-Teisserenc et al., 1999) .

A number of studies have demonstrated the recruitment and activation of NK cells following infection with a wide range of viruses. However, not all viral infections P A R T I I I are susceptible to NK-mediated clearance, and susceptibility depends upon the effector mechanisms induced. For example, the induction of both cytotoxicity and IFN- production by NK cells following murine cytomegalovirus (CMV) and influenza virus infection results in reduced virally induced disease and enhanced survival. Along the same time, deficient IFN- production by NK cells correlates with the absence of an effective innate response to lymphocytic choriomeningitis virus infection. Moreover, the organs targeted by viral infection can also influence the participation of NK cells. Indeed, it has been shown that the NK response to murine CMV is perforin-dependent within the spleen, whereas production of IFN- is required for viral clearance from the liver. These results indicate that the importance of the NK cell response to viral infection can depend upon multiple factors, including the tissue infected, as well as the effector mechanisms induced.

Although a number of studies have documented the possible role for NK cells in controlling CNS infection with CMV, influenza and other viruses, the following studies provide relatively direct evidence for the importance of NK cells during CNS viral infection:

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus. Infection of susceptible mice (SJL) with TMEV causes a biphasic disease characterized by grey matter inflammation followed by late chronic demyelination (Roos and Wollmann, 1984; Rosenthal et al., 1986) . After inoculation, CNS TMEV titres were higher in SJL mice compared with C57BL/10 mice, correlating with a 50% lower NK cell activity in the SJL mice than in the C57BL/10 mice (Paya et al., 1989) . Clinically, SJL mice are much more susceptible than C57BL/10 mice to TMEV. When resistant (C57BL/10) mice were depleted of NK cells using either mAb NK1.1 or polyclonal anti-asialo-GM1, TMEV induced the development of diffuse encephalitis and meningitis early in the post-infection period. However, the second phase of TMEV-induced CNS disease (demyelination) was observed only in resistant C57BL/10 mice treated with anti-asialo-GM1. Experiments with beige/beige mice of C57BL/10 background showed a mild degree of grey matter inflammation but no demyelination (Paya et al., 1989) . NK cells are critical effectors in protecting against TMEV-induced grey matter disease, whereas a different population of either NK1.1-NK cells, or other activated lymphocytes, may be critical in resistance or susceptibility to demyelination.

In support of the involvement of NK cells during TMEV of the CNS, another study demonstrated that stressed mice developed clinical signs of encephalitis, thymic atrophy, and adrenal hypertrophy after infection with Theiler's virus (Welsh et al., 2004) . This syndrome was associated with significantly reduced virus-induced NK cell cytotoxic activity in restrained mice at 1 day post-infection, which may account for the reduced viral clearance from the CNS.

Mouse hepatitis virus (MHV) is a coronavirus that causes infection of the CNS (Marten et al., 2001; Wang et al., 1990) . Intracerebral infection of susceptible strains of mice with MHV results in an acute encephalomyelitis followed by a chronic immune-mediated demyelinating disease that is similar in pathology to the human demyelinating disease MS (Walsh et al., 2007; Zuo et al., 2006) . Intracerebral infection of RAG1 / mice with a recombinant CXCL10-expressing murine coronavirus (MHV) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of NK cells within the CNS (Walsh et al., 2007) . Accumulation of NK cells resulted in a reduction in viral titres that was dependant on IFN- secretion (Walsh et al., 2007) . These results indicate that the CXCL10-guided NK cell homing to the CNS might play a pivotal role in defence following coronavirus infection of the CNS.

Semliki Forest virus (SFV) is a positive-stranded RNA virus (Atkins et al., 1999; Smithburn and Haddow, 1944) . Infection of C57BL/6 mice with SFV leads to pronounced CNS cellular infiltration and apoptosis of microglial and neuronal cells (Alsharifi et al., 2006) . In this model, NK cells and, to a lesser degree, cytotoxic T cells are major contributors in combating SFV infection.

Mice lacking the Tc cell compartment ( 2 -microglobulin-deficient mice, and thus CD8  T cells) exhibit susceptibility similar to wild-type mice. Depletion of NK cells significantly delayed the mean time to death but did not prevent mortality in SFV-infected B6 mice suggesting that cytolytic activity of NK cells is detrimental, while IFN- production is beneficial for recovery from SFV infection (Alsharifi et al., 2006) .

With greater than 1.6 million Americans infected annually (Armstrong et al., 2001) , herpes simplex virus type 1 and 2 (HSV-1, HSV-2) are pathogens with a significant impact on public health. Typically, infection results in a life-long latent infection of the host (Halioua and Malkin, 1999; Whitley, 2002) . The transmission of HSV-2 in the human population includes asymptomatic shedding of the virus even in the presence of CD8  cytotoxic T lymphocytes and the production of a viral glycoprotein that indirectly elicits NK cell death (Bellner et al., 2005; Posavad et al., 2000; Wald et al., 2000) . In a mouse model of HSV-2 infection, it was shown that mice deficient in CCR5 (CCR5 / ) displayed a significant reduction in cumulative survival following infection in comparison with wild-type HSV-2-infected controls. Associated with decreased resistance to viral infection, CCR5 / mice yielded significantly more virus and expressed higher levels of tumour necrosis factor alpha (TNF-), CXCL1, CCL2, CCL3 and CCL5 in the vagina, spinal cord, and/or brain stem than did wild-type mice. In addition, when comparing wildtype HSV-2-infected mice with CCR5 / mice prior to or after infection, there were significantly more NK cells (NK1.1  CD3  ) residing in the brain stem and spleen of infected wild-type mice. Functionally, NK activity from cells isolated from the brain stem of HSV-2-infected wild-type mice was greater than that from HSV-2-infected CCR5 / mice. Further, antibody-mediated depletion of NK cells resulted in an increase in HSV-2 levels in the vaginal, spinal cord and brain stem tissue of wild-type mice but not CCR5 / mice (Kveberg et al., 2002) . Collectively, the absence of CCR5 expression significantly impacts the ability of the host to control genital HSV-2 infection, inflammation and spread associated with a specific reduction in NK cell expansion, infiltration and activity in the nervous system.

Congenital toxoplasmosis poses a public health problem, being capable of causing foetal death and ocular and neurological sequelae in congenitally infected children.

Congenital infection occurs only when mothers first encounter Toxoplasma gondii (T. gondii) during pregnancy (Remington et al., 1994; Roberts and Alexander, 1992) . Resistance to T. gondii is mainly mediated by type 1 cytokines, such as IFN- and interleukin 2 (IL-2), whereas type 2 cytokines, such as IL-4 and IL-10, are associated with increased susceptibility to infection (Hunter et al., 1996; Khan et al., 1994) . Susceptibility of the pregnant host to toxoplasmosis may be attributable to a type 2-cytokine bias that is maintained during gestation (Shirahata et al., 1992) . Cell-mediated immune responses involving CD4 and CD8T cells and NK cells play a protective role in T. gondii primary infection (Goldszmid et al., 2007; Scharton-Kersten et al., 1998; Scorza et al., 2003; Scott and Trinchieri, 1995; Subauste et al., 1992) . To clarify the roles of NK cells and IFN- in protection against primary congenital toxoplasmosis, Abou-Bacar and colleagues (2004) used recombination activating gene 2 knockout (KO) (RAG-2 / ) mice, which lack T and B lymphocytes, in comparison with the wild-type BALB/c model. RAG-2 / mice had a significantly lower risk of foetal toxoplasmosis than BALB/c mice. This protection was associated with an increased number of maternal NK cells, IFN- secretion by spleen cells, and decreased parasitemia. In the RAG-2 / mice, NK cell depletion increased the rate of foetal infection. These data suggest that a partially protective immunity against congenital toxoplasmosis is achieved owing to the increased number of NK cells in RAG-2 / mice (Abou-Bacar et al., 2004) . Protective effect of NK cells was confirmed in another study using the SCID model (Kang and Suzuki, 2001) .

The innate immune system plays an instrumental role in generating and directing the adaptive immune responses (Shi et al., 2001) . NK cells represent a critical first line of defence against malignant transformation. Earlier results by Karre and Ljunggren demonstrated that NK cells can preferentially kill and reject cells that fail to express 'self ' MHC class I molecules (Karre et al., 1986) . These findings led to the formation of the famous 'missing self hypothesis' (Ljunggren and Karre, 1990) . Over the years, the missing self hypothesis has been repeatedly demonstrated in a variety of experimental tumour systems by different groups of investigators, and a number of molecular pathways governing the interactions of NK cell-target cells have been revealed. Surveillance against 'missing self ' may thus be one, but not the only function of NK cells (Ljunggren and Malmberg, 2007) . Non-surgical resectable tumours within the CNS constitute significant challenges for physicians. Furthermore, studies have documented frequent immune system defects in intracranial tumour-bearing patients and an inability of certain lymphocyte subset to mediate antitumour effector functions in the CNS.

Malignant melanoma is notorious for metastasis to discrete locations such as testis and brain. Malignant melanoma is the third most common type of cancer that metastasizes to the brain (Prins et al., 2006) , which presents clinicians with few treatment options. Although nearly a dozen melanoma antigens specifically recognized by T cells have been identified, melanoma cells are still able to avoid immune destruction in most instances. Because the generation of an effective antitumour immune response requires both the presence of foreign antigen and a costimulatory molecule or signal, tumour cells displaying tumour antigens may avoid immune detection because of the absence of appropriate costimulation. Thus, anti-tumour immune responses might be achieved by more effective local delivery of costimulatory molecules. Activation and expansion of NK cells may independently lyse tumour cells, or provide T cells with costimulatory molecules including cytokines, and overall enhance antigen presentation to T cells.

Several attempts have been made in an effort to use NK cells to target CNS melanoma. The specific receptor for IL-2 on NK cells allows several approaches to deliver IL-2 intrathecally and activate NK cells. Ewend and associates carried out a study in C57BL/6 mice that were simultaneously given intracranial injections of tumour and of irradiated B16F10 melanoma cells transduced to secrete IL-2 (Prins et al., 2006) . IL-2 therapy generated antitumour responses capable of extending the survival of animals that received simultaneous intracranial tumour challenges either locally or at distant sites in the brain. Non-transduced melanoma cells had little effect. Elimination of T-cell and NK subsets using gene KO mice and antibody-depletion techniques demonstrated that NK cells were most important for the initial anti-tumour response, whereas CD4  T cells were not necessary. These studies demonstrate that local IL-2 therapy in the brain not only generates an immediate local antitumour immune response, but also establishes long-term immunologic memory capable of eliminating subsequent tumour challenges within and outside of the CNS. Furthermore, the antitumour response to paracrine IL-2 in the brain differed significantly from that in the flank, suggesting that the intrinsic CNS cells involved in initiating immunity within the brain have different cytokine requirements from their peripheral counterparts.

Using the same model, a recent study showed that DCs administration induced dramatic anti-tumour immune protection in CD8 KO mice that were challenged with B16 melanoma both subcutaneously and in the brain (Ewend et al., 2000) . The CNS anti-tumour immunity was dependant on both CD4  T cells and NK cells. Administration of non-Ag-loaded, immature DC resulted in significant CD4  T cell and NK cell expansion in the draining lymph nodes at 6 days post-vaccination, which persisted for 2 weeks. Finally, Ag-loaded DC administration in CD8 KO mice was associated with robust infiltration of CD4  T cells and NK cells into the brain tumour parenchyma (Ewend et al., 2000) .

Glioma cells interfere with anti-tumour immune responses by expressing immune inhibitory cell surface molecules, such as HLA-G, or by releasing soluble immunosuppressants such as transforming growth factor (TGF-). They rarely metastasize outside the brain, raising the possibility of immune-mediated control of these cells outside, but not inside, the brain.

IL-2, as well as growth hormone, is potent in enhancing NK cell activity against glioma both in human trials and in several experimental systems (Eisele et al., 2006; Hayes et al., 1995; Shimizu et al., 2005; Wischhusen et al., 2005) . As receptors governing NK cell action and effector functions are being elucidated, more sophisticated means of manipulating NK cells have been generated. As noted above, NKG2D is a powerful, activating NK cell receptor (Wischhusen et al., 2005) . Accordingly, activating the innate immune system by forcing glioma cells to express danger signals such as NKG2D ligands is a promising strategy of immunotherapy for these tumours. The remaining challenges would be to downregulate HLA-E expression on glioma cells and suppress production of TGF- by glioma. Both HLA-E and TGFbeta can down-regulate NKG2D expression on glioma, which enable these tumour cells to escape NK cell surveillance.

Various studies have documented the role of NK cells in surveillance and suppression of other type of CNS tumours including medulloblastoma (Castriconi et al., 2007) , astrocytoma and neuroblastoma (Kang et al., 2004) . On the other hand NK cells appear to have little, if any, role in suppressing CNS lymphomas (Yamasaki et al., 2003) .

Clearly, cumulative evidence suggests that NK cells play a role in curbing malignant transformation and progression of many primary and metastatic CNS tumours (Table 28 .2). Direct cytotoxic effects and collaboration with T cells and other immune cells are required to achieve these functions (Table 28 .2). Effective therapies harnessing NK cells will be facilitated through understanding of the molecular signalling pathways that will be governing NK cell activation, expansion and maintenance. Specific anatomical factions within the CNS should also be considered. Furthermore, effort must be taken in suppressing the capacity of certain tumours to down-regulate activating signals and production of inhibitory proteins against NK cells.

During CNS infection, cytolytic activity of NK cells contributes to elimination of viral and bacterial infected cells and controls the magnitude of inflammation. Debris from neuronal and/or glial cell death is taken up by antigen-presenting cells (APCs) and presentation to T cells. Cytokine (IFN-) secretion by NK cells increases MHC class II expression by APC and, thus, favours generation of Th1 type of T helper cells. Thus, NK cells function not only as the initial line of host defence, but also as fuel to the generation of adaptive immune responses. Overall, NK cells are expected to boost immune response within the CNS. Paradoxically, emerging evidence suggests that NK cells can inhibit CNS inflammation and control the magnitude of autoimmunity (Table 28 .2).

MS is a classic autoimmune disease characterized by extensive CNS inflammation and immune-mediated destruction of myelin. Consequently, the function of myelin sheaths becomes compromised and neurological impairment occurs. The pathogenesis of MS is mirrored, in part, in EAE, which can be induced in susceptible strains of mice with neuron-antigens and complete Freund's adjuvant. The roles of NK cells in the pathogenesis of MS and EAE have been investigated. In patients with MS, NK cells (CD56 and CD57) are present in the peripheral blood with reduced numbers and cytolytic activity (Shibatomi et al., 2001; Trinchieri, 1989) . This finding is not unique to MS and similar phenotype of NK cells have been documented in many other types of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, etc (Shibatomi et al., 2001) . In parallel, patients with MS and other autoimmune diseases have defective functions of 'regulatory cells', including NKT cells and CD4  CD25  regulatory T cells (Treg) (La Cava et al., 2006) . A reduced number and/or compromised function of NK cells, NKT cells and Treg cells invite a hypothesis that autoimmunity is associated with a result of global defective regulatory cell functions in these patients. This hypothesis has been tested in several EAE models. Because gene encoding NK cells cannot be targeted by the current technology, several depleting antibodies have been used to study the function of NK cells in vivo. Several groups have utilized anti-NK1.1 mAb and observed that depletion of NK cells by injecting anti-NK1.1 mAb leads to exacerbation of EAE (Zhang et al., 1997) . Apparently, both peripheral and CNS NK cells are absent in this experimental system. It is, therefore, not possible to differentiate the role of NK cells in the periphery and in the CNS.

NK cell homing to CNS is controlled by a specific chemokine receptor ligand pathway involving CX3CR1 and fractalkine (CX3CL1). CX3CR1 is expressed almost exclusively by CNS glial cells (Boehme et al., 2000; Cardona et al., 2006) . Thus, germ-line deletion of CX3CR1 leads to impaired homing of NK cells to the CNS. This model would be ideal in addressing CNS inflammation/autoimmunity in relation to NK cells. Interestingly, upon immunization, CX3CR1-deficient mice with reduced NK cells in the CNS and intact NK cells in the periphery developed their wild-type controls. Thus, lack of CNS NK cells alone is sufficient to cause exacerbated CNS inflammation and autoimmunity (Huang et al., 2006) . It is also conceivable that chemokine guided NK cell homing to CNS might serve as pathway that can be therapeutically targeted (Figure 28.1) .

Infection is suggested to play a role triggering the initiation of MS in some patients (Bendelac and Medzhitov, 2002; Pulendran and Ahmed, 2006; Shirahata et al., 1992) . The use of complete Freund's adjuvant in the induction of EAE may mimic this process (Fearon and Locksley, 1996) . In these patients or in EAE animals, NK cells may contribute to the demyelination through bystander damage while controlling the infection. Once infection is controlled, NK cells may function to inhibit the excessive (auto) immune responses elicited by pathogens. The immune system may use NK cell as a versatile regulator to tune its capacity in combating infection and avoiding autoimmunity.

The mechanism underlying this unique role for NK cells within the CNS during EAE is still elusive. A close survey of the literature reveals multiple steps where NK cells can regulate inflammation and intervene in the loss of self-tolerance. Importantly, the findings also caution against inferring a similar role for NK cells in all types of autoimmune phenomena or during separate stages of the same disease (Flodstrom et al., 2002; Yokoyama and Plougastel, 2003) . NK cells can both promote and inhibit autoreactive T cells. These possibilities have been extensively reviewed recently (Shi and Van Kaer, 2006) . The specific CNS anatomical location, as reflected by diverse CNS APCs and multiple antigens may also influence the outcome of autoimmunity. As with EAE, it appears that NK cells control T cell proliferation in an antigen non-specific manner, both in the periphery and within the CNS (Shi, et al., unpublished) . Recently, it has been demonstrated that human NK cells kill resting but not activated microglia via NKG2D-and NKp46-mediated recognition (Lünemann et al., 2008) . This study emphasizes the potential importance of interactions between NK cells and CNS resident APCs. However, whether the regulatory effects of NK cells can be attributed to the action of NK cells on APCs, directly on T cells, or both, is not known and currently under investigation.

NK cells readily accumulate in homing to CNS tissues under the pathophysiological situations. This process is tightly controlled by a number of chemokines and chemokine receptors. There is ample of evidence that NK cells within the CNS contribute to the control of infections and might limit progression of certain tumours. Bystander neuronal and/or glial cell damage can occur. In certain autoimmune conditions of the CNS, NK cells appear to have an inhibitory role. Activation and expansion of NK cells through engaging IL-2 receptors on NK cells not only inhibit several CNS tumours, but also might slow the progression of MS and other autoimmune diseases (Bielekova et al., 2006; Li et al., 2005) . Furthermore, the ability of IFN- and IFN- to ameliorate MS in humans and IFN- to inhibit EAE in mice may reflect the ability of these cytokines to transiently activate NK-dependent regulatory responses. However, because IFN treatment also upregulates Qa-1 expression on T cells (Ota et al., 2005) , the short duration and usually modest nature of these therapeutic effects may reflect a Qa-1-dependent decrease in NK cell activation and associated immunoregulatory activity (Lu et al., 2007) .

Disassociation of disease-inhibiting versus diseasepromoting effects of NK cells is a key to harnessing NK cells for therapeutic purposes. To achieve this goal, a generation of genetic models with selective NK cell deficiency, and development of reagents (antibodies) for visualizing subsets of NK cells in situ will be necessary. Optimization of methods to produce NK cells in large quantities for therapeutic usage is also important. Clearly, understanding the molecular signals and pathways governing these differential biological effects of NK cells as well as their cross talk with T cells is key to designing NK cell-based therapy for CNS infections, tumours and autoimmune diseases.

Role of NK cells and gamma interferon in transplacental passage of Toxoplasma gondii in a mouse model of primary infection

CCR5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine T cell-mediated hepatitis

NK cell-mediated immunopathology during an acute viral infection of the CNS

Non-MHC-restricted cellmediated lysis of human oligodendrocytes in vitro: relation with CD56 expression

Incidence of herpes simplex virus type 2 infection in the United States

The molecular pathogenesis or Semliki Forest virus: a model virus made useful?

Natural killer cell-mediated lysis of dorsal root ganglia neurons via RAE1/ NKG2D interactions

Direct NK cellmediated lysis of syngenic dorsal root ganglia neurons in vitro

Suppressor of cytokine signaling 1 expression protects oligodendrocytes from the deleterious effects of interferongamma

A proinflammatory peptide from herpes simplex virus type 2 glycoprotein G affects neutrophil, monocyte, and NK cell functions

Adjuvants of immunity: harnessing innate immunity to promote adaptive immunity

Regulatory CD56(bright) natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis

Severe herpesvirus infections in an adolescent without natural killer cells

The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia

Expression of a killer cell receptor-like gene in plastic regions of the central nervous system

Control of microglial neurotoxicity by the fractalkine receptor

Both CD133 and CD133-medulloblastoma cell lines express ligands for triggering NK receptors and are susceptible to NK-mediated cytotoxicity

Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice

Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages

TGF-beta and metalloproteinases differentially suppress NKG2D ligand surface cells

Intracranial paracrine interleukin-2 therapy stimulates prolonged antitumor immunity that extends outside the central nervous system

The instructive role of innate immunity in the acquired immune response

The natural killer cell-friend or foe in autoimmune disease?

Defective expression of the interleukin-2/interleukin-15 receptor beta subunit leads to a natural killer cell-deficient form of severe combined immunodeficiency

TAP-1 indirectly regulates CD4T cell priming in Toxoplasma gondii infection by controlling NK cell IFN-gamma production

Epidemiology of genital herpesrecent advances

Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNSinfiltrating T and natural killer cells

Improved long term survival after intracavitary interleukin-2 and lymphokine-activated killer cells for adults with recurrent malignant glioma

Exogenously-induced, natural killer cell-mediated neuronal killing: a novel pathogenetic mechanism

Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-alpha/beta-induced inflammatory responses and antiviral defense in liver

Effect of immunosuppressive agents on the guanethidine-induced sympathectomy in athymic and euthymic rats

The neuronal chemokine CX3CL1/ fractalkine selectively recruits NK cells that modify experimental autoimmune encephalomyelitis within the central nervous system

Cells and cytokines in resistance to Toxoplasma gondii

Expression and regulation of chemokine receptors in human natural killer cells

Regulation of pulmonary fibrosis by chemokine receptor CXCR3

Requirement of non-T cells that produce gamma interferon for prevention of reactivation of Toxoplasma gondii infection in the brain

Lymphokine activated killer cells from umbilical cord blood show higher antitumor effect against anaplastic astrocytoma cell line (U87) and medulloblastoma cell line (TE671) than lymphokine activated killer cells from peripheral blood

Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy

Overlapping roles of P-selectin and alpha 4 integrin to recruit leukocytes to the central nervous system in experimental autoimmune encephalomyelitis

Interleukin-12 enhances murine survival against acute toxoplasmosis

CCR5 is essential for NK cell trafficking and host survival following Toxoplasma gondii infection

Sphingosine 1 phosphate induces the chemotaxis of human natural killer cells. Role for heterotrimeric G proteins and phosphoinositide 3 kinases

CD4 CD25 Tregs and NKT cells: regulators regulating regulators

Fractalkine mediates natural killer-dependent antitumor responses in vivo

Cutting edge: in vivo blockade of human IL-2 receptor induces expansion of CD56(bright) regulatory NK cells in patients with active uveitis

In search of the 'missing self ': MHC molecules and NK cell recognition

Prospects for the use of NK cells in immunotherapy of human cancer

Regulation of activated CD4 T cells by NK cells via the Qa-1-NKG2A inhibitory pathway

iTRAQ is a useful method to screen for membrane-bound proteins differentially expressed in human natural killer cell types

Human NK cells kill resting but not activated microglia via NKG2D-and NKp46-mediated recognition

The molecular and functional characterization of a dominant minor H antigen, H60

The mechanisms controlling NK cell autoreactivity in TAP2-deficient patients

MHV infection of the CNS: mechanisms of immunemediated control

Induced recruitment of NK cells to lymph nodes provides IFNgamma for T(H)1 priming

Association of a syndrome resembling Wegener's granulomatosis with low surface expression of HLA class-I molecules

NK cell-mediated lysis of autologous human oligodendrocytes

Peripheral neuropathy associated with chronic natural killer cell lymphocytosis

T cell-and B cell-independent adaptive immunity mediated by natural killer cells

IFN-gamma-mediated negative feedback regulation of NKT-cell function by CD94/NKG2

Role of natural killer cells as immune effectors in encephalitis and demyelination induced by Theiler's virus

Long term persistence of herpes simplex virus-specific CD8 CTL in persons with frequently recurring genital herpes

NK and CD4 cells collaborate to protect against melanoma tumor formation in the brain

Translating innate immunity into immunological memory: implications for vaccine development

Three or more routes for leukocyte migration into the central nervous system

Toxoplasmosis

Studies on a murine model of congenital toxoplasmosis: vertical disease transmission only occurs in BALB/c mice infected for the first time during pregnancy

DA strain of Theiler's murine encephalomyelitis virus induces demyelination in nude mice

Mechanism of Theiler's virus-induced demyelination in nude mice

NKG2D-mediated cytotoxicity toward oligodendrocytes suggests a mechanism for tissue injury in multiple sclerosis

Infection of mice lacking the common cytokine receptor gammachain (gamma(c)) reveals an unexpected role for CD4 T lymphocytes in early IFN-gamma-dependent resistance to Toxoplasma gondii

A GRA1 DNA vaccine primes cytolytic CD8() T cells to control acute Toxoplasma gondii infection

The role of natural killer cells in host-parasite interactions

Innate immunity and autoimmunity: from self-protection to selfdestruction

Reciprocal regulation between natural killer cells and autoreactive T cells

A novel role for interleukin-18 in human natural killer cell death: high serum levels and low natural killer cell numbers in patients with systemic autoimmune diseases

Growth hormone enhances natural killer cell activity against glioma

Correlation between increased susceptibility to primary Toxoplasma gondii infection and depressed production of gamma interferon in pregnant mice

Semliki Forest virus I isolation and pathogenic properties

Human lymphokineactivated killer cells are cytotoxic against cells infected with Toxoplasma gondii

Susceptibility of CCR5-deficient mice to genital herpes simplex virus type 2 is linked to NK cell mobilization

Biology of natural killer cells

Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons

IFN-gamma acts on T cells to induce NK cell mobilization and accumulation in target organs

Expression of CXC chemokine ligand 10 from the mouse hepatitis virus genome results in protection from viral-induced neurological and liver disease

Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor

Demyelination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated

T cell approach to demyelinating diseases

The effects of restraint stress on the neuropathogenesis of Theiler's virus infection II: NK cell function and cytokine levels in acute disease

Herpes simplex virus infection

HLA-E protects glioma cells from NKG2D-mediated immune responses in vitro: implications for immune escape in vivo

Experimental appraisal of the lack of antitumor natural killer cell-mediated immunosurveillance in response to lymphomas growing in the mouse brain

Immune functions encoded by the natural killer gene complex

Defective antitumor responses in CX3CR1-deficient mice

Regulation of Experimental Autoimmune Encephalomyelitis by Natural Killer (NK) cells

Mouse hepatitis virus pathogenesis in the central nervous system is independent of IL-15 and natural killer cells

We thank Dr R. Liu, Dr W. Piao, and Dr. Dayao and Ms. Linda Swanson for their wonderful assistance in preparation of this chapter. The Muscular Dystrophy Association, the Barrow Neurological Foundation, and the National Institutes of Health support the authors' work in the laboratories.