International Journal for Parasitology 34 (2004) 15171528 www.parasitology-online.

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Invited review

Natural killer cells and innate immunity to protozoan pathogens

Daniel S. Korbela, Olivia C. Finneyb, Eleanor M. Rileya,*
a

Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK b Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK Received 29 April 2004; received in revised form 16 September 2004; accepted 6 October 2004

Abstract Natural killer (NK) cells are lymphoid cells that mediate signicant cytotoxic activity and produce high levels of pro-inammatory cytokines in response to infection. During viral infection, NK cell cytotoxicity and cytokine production is induced principally by monocyte macrophage- and dendritic cell-derived cytokines but virally encoded ligands for NK cells are also beginning to be described. NK derived interferon-g (IFN-g) production is also essential for control of several protozoal infections including toxoplasmosis, trypanosomiasis, leishmaniasis and malaria. The activation of NK cells by protozoan pathogens is also believed to be cytokine-mediated although some recent studies suggest that direct recognition of parasites by NK cells also occurs. Both indirect signalling via accessory cell-derived cytokines and direct signalling, presumably through NK receptors, are needed in order for human malaria parasites (Plasmodium falciparum) to optimally stimulate NK activity. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Keywords: Natural killer cell; Innate immunity; Protozoa; Malaria; Plasmodium

1. Introduction Host resistance against viral, bacterial and protozoan pathogens depends on a complex interplay of innate and adaptive immune mechanisms. Acting as an early line of defence, the innate immune system contributes to the control of acute infection by mounting protective responses against invading pathogens before the onset of T and B cell-mediated immunity. Innate responses also stimulate and modulate adaptive immune responses. Natural killer (NK) cells are now known to be key players in these early innate responses (Carayannopoulos and Yokoyama, 2004). In this article, we briey review the biology of NK cells and their receptors and discuss our current understanding of NK cell function in innate immunity to protozoal infections, focussing on malaria infection in particular.

2. Natural killer cell biology The identication of a lymphocyte subset with the ability to exert cytolytic activity against certain tumour cells without prior stimulation led to the term natural killer cell (Herberman et al., 1975; Kiessling et al., 1975). It is now known that, in addition to killing of transformed cells (reviewed by Wu and Lanier, 2003), NK cells also play a role in allograft rejection (reviewed by Ruggeri et al., 2002) and in the control of a variety of pathogens, especially those that directly infect host cells (reviewed by Robbins and Brossay, 2002). Natural killer cells are large granular, bone marrow-derived lymphocytes which, in humans, are classically dened by surface expression of CD56 (an isoform of the neural adhesion molecule N-CAM) and the absence of expression of the thymocyte marker CD3. The expression of other surface molecules such as leukocyte marker 7 (Leu-7 or CD57), interleukin 2 receptor b chain (IL-2Rb or CD122) and killer cell lectin-like receptor B1 (KLRB1 or CD161) has also been used as a marker of the NK

* Corresponding author. Tel.: C44 207 927 2706; fax: C44 207 927 2807. E-mail address: eleanor.riley@lshtm.ac.uk (E.M. Riley).

0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.10.006

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cell subset but these molecules are also expressed on some T cell and NK-T subpopulations. The phenotypic characterisation of NK cells in mice is more complex. The most commonly used markers of murine NK cells are integrin subunit a2 (DX5 or CD49b), asialoganglioside M1 (ASGM1) and natural killer receptor P1C (NKR-P1C or NK1.1). However, murine CD4 and CD8 T lymphocytes can also express these markers (Slifka et al., 2000; Assarsson et al., 2000); ASGM1, for instance, is expressed by 2030% of nave and 90% of activated T cells (Slifka et al., 2000). Antibodies to ASGM1 and NK1.1 are typically used for NK cell depletion in mice, which inadvertently results in the simultaneous depletion of T cells expressing these markers; data from such studies thus need to be interpreted with caution. NK cells represent approximately 10% of peripheral blood mononuclear cells (PBMC) and 0.45% of mononuclear cells in secondary lymphoid organs (Robertson and Ritz, 1990). Natural cytotoxic mechanisms are similar to the mechanisms employed by cytotoxic T lymphocytes, i.e. secretion of the pore-forming protein perforin and of apoptosis-inducing granzymes and/or engagement of target-cell death receptors (Lieberman, 2003). NK-mediated lysis of infected host cells has been demonstrated for several rodent and human viral infections (French and Yokoyama, 2003), however, the physiological signicance of natural cytotoxicity in bacterial and protozoal infections is still a matter of debate (Mohan et al., 1997; Scharton-Kersten and Sher, 1997). The second major effector function of NK cells is the secretion of pro-inammatory cytokines and chemokines, for example the macrophage-activating factor interferon-g (IFN-g), tumour-necrosis factor a (TNF-a), lymphotoxin a (LT-a), granulocytemonocyte colony-stimulating factor (GM-CSF) and the macrophage inammatory protein CCL3 (Trinchieri, 1989; Robertson, 2002). IFN-g is central to host resistance to many infections, regulating several hundred genes associated with immune system functions and recruiting other effector cells to the site of infection (Boehm et al., 1997). IFN-g activates macrophages and neutrophils, stimulates the differentiation of Th1 CD4C lymphocytes, enhances antigen-presentation by the upregulation of MHC and MHC-associated molecules, and induces IgG subclass switching in B cells, thereby bridging the innate and adaptive arms of the immune system (Boehm et al., 1997). Hence, IFN-g secretion by NK cells has been demonstrated to play an important role in triggering potent immune responses to various classes of pathogens (Scharton-Kersten and Sher, 1997; Shtrichman and Samuel, 2001). Invading pathogens, or antigens of viral, bacterial and protozoan origin, are captured by DCs or monocyte macrophages which then release NK cell-activating cytokines (Biron et al., 1999; Ferlazzo et al., 2003; Lande et al., 2003; Sher et al., 2003b; Cooper et al., 2004). IL-2, IL-12, IL-15, IL-18, TNF-a and IFN-a/b all contribute to activation of NK cells whereas IL-4, IL-10 and TGF-b

suppress NK cell function (Biron et al., 1999; Colucci et al., 2003). IL-12, a pro-inammatory cytokine mainly produced by professional antigen-presenting cells, i.e. macrophages and dendritic cells (DC), is the most potent inducer of NK cell cytotoxicity and IFN-g secretion and thus, together with IL-18, plays a crucial role in triggering NK-mediated immune responses (Trinchieri, 1998; Wei et al., 1999).

3. NK receptors Whilst stimulation by cytokines contributes signicantly to their activation, NK cells also have the ability to directly sense transformed or infected cells and immediately respond to this stimulus. Engagement of an elaborate repertoire of ligands via a similarly complex repertoire of surface receptors enables NK cells to discriminate between normal host cells and abnormal, potentially dangerous, cells without prior sensitisation. A delicate balance of inhibitory and stimulatory signals is maintained to ne-tune the activation state of NK cells (reviewed by Lanier, 2003). One aspect of this process is the recognition of missing self, rst proposed by Klaus Karre in the 1980s, whereby NK cells scan potential target cell surfaces and become activated if the target cells cannot provide sufciently strong inhibitory signals (Karre, 1985). This is now understood to be due to the need, under normal physiological conditions, for NK cells to receive inhibitory signals provided by engagement of MHC class I-specic inhibitory receptors in order to prevent their activation (Lanier, 2003; Moretta and Moretta, 2004). Down-regulation or loss of MHC class I molecules on target cells can skew the balance of inhibitory and activating signals towards the triggering of NK cell activation. More recently it has been appreciated that NK cells also recognise altered-self in that NK cells can also be activated by cells expressing a normal complement of MHC class I if there is sufciently strong engagement of activating NK receptors by neoantigens, induced by stress, tumour transformation or infection (Raulet, 2003). Activating and inhibitory NK cell receptors (Table 1) have now been shown to recognise a wide range of self and non-self molecules. In contrast to lymphocytes of the adaptive immune system, NK cells do not utilise somatic gene re-arrangements to generate clones with the capacity for diverse antigen recognition, rather they achieve versatility by simultaneous expression of multiple receptors, inhibitory and activating, with different specicities (reviewed by Moretta et al., 2001; McQueen and Parham, 2002). NK receptors include members of the immunoglobulin (Ig) superfamily (e.g. killer Ig-like receptors (KIR), natural cytotoxicity receptors (NCR), LIR/ILT, Siglec-7) and members of the C-type lectin family such as Ly49 and NKG2 receptors (reviewed by McQueen and Parham, 2002; Shilling et al., 2002; Yokoyama and Plougastel, 2003).

D.S. Korbel et al. / International Journal for Parasitology 34 (2004) 15171528 Table 1 Properties of NK cell receptors Receptor Killer Iglike receptors (KIR) LIR/ILT Family Ig Species H, Hd, Ro Genes Multigene Known ligands 9 Activating = Broad MHC I specicity Others? and/or ; inhibitory 9 Activating = nBroad MHC I specicity gpUL18a, others? and/or ; Inhibitory Activating ? Activating Activating Activating Inhibitory Viral haemagglutininsa Viral haemagglutininsa ? Sialic acid Function Selected references

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Ig

Multigene

Winter et al. (1998), Vilches and Parham (2002) and Carrington and Norman (2003) Martin et al. (2002a) and Natarajan et al. (2002) Moretta et al. (2000) and Sato et al. (2001)) Moretta et al. (2000) Moretta et al. (2000) Moretta et al. (2000) and Vitale et al. (2001)) Crocker (2002) and Nicoll et al. (2003) Meyaard et al. (1997) Sivori et al. (2000) Raulet (2003) Matsumoto et al. (1998), George et al. (1999), Nakamura et al. (1999), Furukawa et al. (2002) and Yokoyama and Plougastel (2003) Braud et al. (1998) and Vance et al. (1998) Iizuka et al. (2003) and Yokoyama and Seaman (1993) Hornung et al. (2002) and Akira and Hemmi (2003)

NKp30 NKp44 NKp46 NKp80 Siglec-7 LAIR 2B4 NKG2D Ly49

Ig Ig Ig Ig Ig Ig Ig CLD CLD

H, Hd, Ro H H, Hd, Ro H H H H, Ro H, Hd, Ro H, Hd, Ro

Single Single Single Single Single Single Single Single Multigene

Inhibitory Ep-CAM Activating CD48 Activating MICA/B, ULBP1/2/3a, Rae-1, H60 9 Activating = HLA I, m157a, Hm1-C4a Others? and/or ; Inhibitory 9 Activating = HLA-E, Qa-1b and/or ; inhibitory 9& Activating = C-type lectin-related molecules, and/or others? ; inhibitory Activating Pathogen-associated moleculesa

CD94NKG2 KLRB1

CLD

H, Hd, Ro

Multigene

CLD

H, Hd, Ro

Multigene

TLR

Toll receptor

A, V

Multigene

Ig, Immunoglobulin superfamily; CLD, C-type lectin domain family; H, human; Hd, humanoids; Ro, rodents; A, arthropods; V, vertebrates. a Non-self ligands.

Whilst most of these receptors show little evidence of polymorphism, remarkable diversity is found among the KIR gene family in primates and among the functionally equivalent Ly49 receptor genes in rodents. The human KIR locus on chromosome 19q13.4 is spread over approx. 150 KB, coding for up to 15 KIR genes (Carrington and Norman, 2003). Haplotypic and allelic variability are responsible for the enormous heterogeneity found in the KIR genotype of a population with each individual expressing a characteristic set of inhibitory and activating KIR. Furthermore, stochastic variation in the KIR loci expressed by different NK clones leads to a polyclonal NK repertoire within an individual. The realisation that the diversity of individual KIR genotypes within a population is comparable to the diversity found in human leukocyte antigen (HLA) genotypes (reviewed by Hsu et al., 2002) suggests that similar selective pressures for diversication may be acting on both loci and it is thus not surprising that specic recognition of class I MHC molecules by inhibitory KIRs forms an essential part of the self/non-self discrimination mechanism of NK cells. By contrast, the identity of physiological ligands for activating KIR is still a matter of intense investigation. Although interactions of HLA molecules with activating KIR have been reported (Winter

et al., 1998) functional interactions with non-MHC molecules cannot be excluded. The polymorphic C-type lectin-like receptors of the Ly49 multigene family are the rodent analogue of human KIR, as they recognise MHC class I and MHC class I-like molecules and individual Ly49 haplotypes vary in the number of genes for activating and inhibitory receptors (reviewed by Yokoyama and Plougastel, 2003). The leukocyte immunoglobulin-like receptor (LIR/ILT) family of receptors shows structural similarities to certain KIR molecules but shows only limited allelic variation and gene content is conserved across haplotypes (Young et al., 2001; Martin et al., 2002a). For example, the minimally polymorphic inhibitory receptor LIR-1/ILT-2 (LILRB1) is expressed on human NK cells and binds to a wide range of MHC class I and MHC-I-like molecules (Young et al., 2001; Natarajan et al., 2002). The multigene, C-type lectin family of NKG2 molecules is present in rodents and humans. The inhibitory heterodimers of NKG2A, -B, -C, or -E with CD94 are known to recognise non-classical MHC-I molecules (HLA-E/Qa-1) and thus contribute to the discrimination between self and non-self (Braud et al., 1998; Vance et al., 1998). In humans, the related NKG2D homodimer is an activating receptor and recognises molecules which bind to cytomegalovirus

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(CMV) proteins (UL16-binding proteins; ULBP) as well as the MHC-like molecules MICA and MICB (Bauer et al., 1999; Cosman et al., 2001). Among the NKG2D ligands in mice are members of the retinoic acid early transcript 1 family (Rae1), the minor histocompatibility molecule H-60 and the ULBP-like transcript Mult1 (Cerwenka et al., 2000; Diefenbach et al., 2000; Carayannopoulos et al., 2002). NKG2D ligands are not usually expressed by normal, healthy tissues but expression of MICA, MICB, Rae1, ULBP and ULBP-like proteins is upregulated in response to cellular stress in transformed (Groh et al., 1999; Cerwenka et al., 2000; Diefenbach et al., 2000; Girardi et al., 2001; Jinushi et al., 2003) and infected cells (Das et al., 2001; Groh et al., 2001; Tieng et al., 2002; Welte et al., 2003); accordingly, NKG2D has been implicated in immunity to tumours and in anti-viral defence (reviewed by Raulet, 2003). The inhibitory killer cell lectin-like receptor KLRG1 has recently been shown to be up-regulated upon NK cell activation in vivo and to suppress IFN-g secretion and NK cytotoxicity in vitro, thus acting as a terminator of NK activity (Robbins et al., 2002); however, the exact role KLRG1 and the identity of its ligands is not yet clear. So far, the non-polymorphic family of activating NCR consists of four members in mice and in humans, NKp30, NKp44, NKp46 and NKp80, all of which are still orphan receptors. However, Mandelboim and colleagues recently reported the engagement of NKp46 by viral haemagglutinin proteins (Mandelboim et al., 2001) and NKp30 appears to be involved in the activation of NK cells by DCs; the ligand, though, still remains to be identied (Ferlazzo et al., 2002). The importance of pattern recognition receptors (PRR) in the regulation of NK cell activity is still poorly understood. Toll-like receptors (TLR) bind to CpG motifs, lipopolysaccharides, and other, mostly pathogenassociated molecules (reviewed by Kopp and Medzhitov, 2003). They are principally involved in the activation of macrophages and dendritic cells and are known to play a part in early immune responses to viral, bacterial and protozoan infections (reviewed by Aderem and Ulevitch, 2000). Nevertheless, since TLRs are also expressed on NK cells and lipophosphoglycan puried from a protozoan parasite has been reported to engage TLR-2 on NK cells (Becker et al., 2003), we cannot rule out the possibility that engagement of NK-TLRs is relevant to innate immunity. Recently, the inhibitory a2,8-linked disialic acid-specic receptor Siglec-7 has been implicated in the recognition of non-MHC-I glycoconjugate molecules (Crocker, 2002; Nicoll et al., 2003). Protection of MHC-Ilow or MHC-Idecient host cells (e.g. dorsal root ganglion neurons and erythrocytes (Botto et al., 1990; Backstrom et al., 2003) from NK cytotoxicity by the binding of self molecules to Siglec-7 may be part of an intriguing new mechanism of self/non-self discrimination. It is possible that engagement of Siglec-7 by surface sialic acid-containing glycoproteins

may allow some extracellular parasites to evade NKmediated killing whereas pathogens that lack sialic acid expression (e.g. Trichophyton spp., Esquenazi et al., 2003) may be susceptible to NK-mediated attack.

4. Activation of NK cells by viral and protozoan pathogens In the last two decades, signicant advances have been made in our understanding of NK cell function in various infectious diseases (reviewed by Bancroft, 1993; SchartonKersten and Sher, 1997). Although the vast majority of data stem from in vitro and in vivo studies of viral infections (reviewed by Biron et al., 1999; Yokoyama and Scalzo, 2002), there is a rapidly accumulating body of evidence supporting a role for NK cells in control of protozoal diseases. 4.1. Viral infections It is now well established that NK cell cytotoxicity and IFN-g production play a crucial role in resolution of herpesvirus, papillomavirus and inuenza virus infections in mice and man (reviewed by Biron et al., 1999). In addition, NK cell interactions with HIV-1 and human T cell leukaemia virus have been demonstrated (Ruscetti et al., 1986; Tasca et al., 2003). Generally, NK cell activation in viral diseases appears to be mediated in an indirect manner by cytokines, mostly IL12 and IFN-a/b secreted by monocytemacrophages (reviewed by Biron et al., 1999). However, a number of recent studies have reported engagement of NK cell receptors by viral antigens in vitro. For example, the haemagglutinin protein of inuenza virus and the haemagglutininneuraminidase complex of parainuenza virus were shown to directly trigger NK-mediated cytotoxicity in vitro by specic interaction with the activating receptor NKp46 (Mandelboim et al., 2001) although the physiological relevance of direct NK cellvirus interactions in the control of infection has not been demonstrated convincingly in vivo. More recently, the groups of Vidal, Lanier and Yokoyama have provided convincing evidence that resistance to murine CMV depends on Ly49H stimulation by a viral, MHC-like glycoprotein (Lee et al., 2001; Arase et al., 2002; Voigt et al., 2003), which offers a very plausible explanation for the differential in vivo susceptibility of different mouse strains to CMV (reviewed by Lee et al., 2002). 4.2. Leishmaniasis NK cells also provide the basis for early resistance to leishmaniasis as suggested by the nding that the course of disease in Leishmania major infection is more severe in NKdepleted mice and that Leishmania amazonensis

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parasitaemia cannot be efciently limited in the absence of NK cells (Laskay et al., 1993; Scharton and Scott, 1993; Laurenti et al., 1999). Although NK cytotoxicity against tumour cell lines is increased in resistant compared to susceptible animals early in infection, and NK cells destroy L. major- and L. amazonensis-infected macrophages in vitro, NK-mediated killing appears not to be essential to resistance, as the ability to control infection is only slightly impaired in Leishmania tropica-infected beige mice (Kirkpatrick and Farrell, 1982; Resnick et al., 1988; Scharton and Scott, 1993; Brodskyn et al., 1997). The beige mutation selectively disrupts the lytic pathway of NK cells but does not affect the cytokine secretion pathway (Roder and Duwe, 1979). Taken together, these data point to NK cell effector function in leishmaniasis being cytokinemediated rather than cytotoxicity-mediated. Consistent with this idea, it was shown that rapid IFN-g production during the rst hours and days of L. major infection is crucial for survival and that NK cells are the initial source of this cytokine (Scharton and Scott, 1993; Scharton-Kersten and Scott, 1995). Furthermore, severe combined immunodecient (SCID) mice, which lack T cells but have normal NK function, are able to contain L. major parasites in the draining lymph nodes, arguing for the existence of a T cell-independent mechanism to limit parasite spread; neutralisation of IFN-g or depletion of NK cells prior to infection of SCID mice abrogated their ability to control parasite spread (Laskay et al., 1995). Likewise, spontaneous healing of, and protection from, leishmaniasis in humans appears to be associated with the ability to rapidly respond to Leishmania aethiopica infection by NK cell proliferation and cytokine secretion (Maasho et al., 1998). A wealth of data indicates that the activation of NK cells in leishmaniasis is generally an indirect, cytokine/chemokine-mediated rather than a direct, NK receptor-mediated phenomenon. IL-12 and IL-18 are major regulators of innate and adaptive immune responses to L. major infection (Scharton-Kersten et al., 1995; Wei et al., 1999). Leishmania major- and Leishmania donovani-susceptible mice are effectively cured by treatment with exogenous IL-12 (Heinzel et al., 1993; Murray and Hariprashad, 1995) and the lack of NK cell-activating chemokines results in suboptimal NK cell-mediated defence (Vester et al., 1999). Further studies in rodents suggest that dendritic cells are important for NK-mediated protection and constitute the principal source of IL-12 early in leishmaniasis, as transient production of this cytokine by DCs in the rst 24 h of Leishmania infection appears to be the initial event that is required to trigger NK cell activation (Gorak et al., 1998; Berberich et al., 2003; Sher et al., 2003b). Intriguingly, it has recently been demonstrated that live promastigotes of L. donovani and L. aethiopica activate puried human NK cells to secrete IFN-g in the absence of other antigen-presenting cells (Nylen et al., 2003) and that direct stimulation of the TLR-2 on NK cells by a L. major

lipophosphoglycan (LPG) leads to up-regulation of TLR-2 and increased production of IFN-g and TNF-a (Becker et al., 2003), suggesting the existence of an additional, accessory cell-independent route of NK cell activation in leishmaniasis. However, levels of IFN-g production by pure NK cells were low (detectable by ELISPOT but not by ELISA or intracellular staining, Nylen et al., 2003) suggesting that accessory cell-derived cytokines may be required for amplication of the direct response. 4.3. Trypanosomiasis Early NK cell activity also inuences the course of disease in American and African trypanosomiases. Depletion of NK cells in Trypanosoma cruzi-resistant mice strains led to higher parasitaemia, an increased mortality very early in infection and a delayed onset of IFN-g production by T cells (Rottenberg et al., 1988; Sakai et al., 1999; Une et al., 2000). Ex vivo analysis of lymphocyte phenotypes in acute Chagas disease in humans supports the idea that NK cells are activated by T. cruzi before T cell immunity develops (Sathler-Avelar et al., 2003). The importance of cytotoxic mechanisms in resistance to Trypanosoma parasites is still under discussion. In the early 80s, ex vivo studies in mice revealed an enhanced cytolytic activity of NK cells as early as 24 h into T. cruzi infection in mice and direct, NK-mediated destruction of the extracellular epimastigote and trypomastigote forms of T. cruzi and Trypanosoma lewisi in vitro (Hatcher et al., 1981; Hatcher and Kuhn, 1982; Albright et al., 1984); intriguingly, Trypanosoma musculi parasites are not susceptible to NK lysis and do not enhance NK cell lysis of tumour cell lines (Albright et al., 1984). However, as observed for NK cell activity in vivo in Leishmania infection, the beige mutation does not have a severe effect on the outcome of T. cruzi infection (Hatcher et al., 1981). In line with this observation, another more recent report indicates that NK cell cytotoxicity is not crucial for control of T. cruzi infection and survival of IFN-a/b-decient mice (Une et al., 2003). Although mice decient in either the perforin/granzyme or the Fas/FasL cytolytic pathway succumb to T. cruzi infection very early, indicating a crucial role of cytotoxicity in the control of infection, it is not possible (from the experiments reported) to determine whether cytotoxicity is mediated by cytotoxic T cells or NK cells (Muller et al., 2003). Paralleling the data on resistance mechanisms in early leishmaniasis, several in vitro and in vivo studies have shown that IL-12-stimulated NK cells can control parasitaemia by IFN-g secretion in the rst days of infection with T. cruzi (Rottenberg et al., 1988; Aliberti et al., 1996; Cardillo et al., 1996; Hunter et al., 1996; Silva et al., 1998; Une et al., 2000). Again, NK cells appear to be activated mainly in an indirect manner by macrophage or dendritic cell derived cytokines. Glycosylphosphatidylinositol (GPI)

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from T. cruzi and Trypanosoma brucei activates murine macrophages in vitro (Tachado and Schoeld, 1994; Camargo et al., 1997a,b) and the Tc52 protein released by T. cruzi has been shown to activate human dendritic cells via TLR-2 (Ouaissi et al., 2002). On the other hand, T. cruzi glycolipids have been reported to directly stimulate murine NK cell activity (De Arruda Hinds et al., 1999). It remains to be established which of these recognition and activation pathways are physiologically relevant in vivo. 4.4. Toxoplasma gondii It is now well established that NK cells also participate in early resistance to Toxoplasma gondii infection (reviewed by Sher et al., 2003a). Toxoplasma gondii-induced NK cell cytotoxicity against YAC-1 tumour cells is macrophage dependent and NK cells show enhanced cytotoxic activity against T. gondii-infected macrophages as well as against the extracellular tachyzoite form (Hauser et al., 1982, 1983; Kamiyama and Hagiwara, 1982; Hauser and Tsai, 1986; Subauste et al., 1992). However, again, cytotoxicity seems to be of minor importance for resistance since the selective defect in NK cell cytotoxicity in beige mice has no apparent effect on their survival (Hughes et al., 1988; Johnson and Sayles, 1995). By contrast, the severe outcome of disease in NK-depleted T. gondii-infected mice implies an important protective function of NK cells other than cytotoxicity (Hunter et al., 1995). Accordingly, it was demonstrated that intact tachyzoites as well as parasite extracts stimulate IFNg production by NK cells and that survival of the animals is fully dependent on this cytokine (Denkers et al., 1993; Gazzinelli et al., 1993; Sher et al., 1993; Scharton-Kersten et al., 1996). In initial reports, the indirect in vitro activation of NK cells by extracellular and intracellular stages of T. gondii was found to be totally dependent on IL-12 producing macrophages (Gazzinelli et al., 1993). However, more recent reports also suggest a physiological role for dendritic cells in the recognition of T. gondii and the initial stimulation of NK cells (Reis e Sousa et al., 1999; Aliberti et al., 2000; Scanga et al., 2002; Sher et al., 2003a). Again, parasite-derived GPI proteins appear to be among the factors responsible for the induction of IL-12 as suggested by the activation of the NF-kB pathway in macrophages (Debierre-Grockiego et al., 2003). 4.5. Malaria Important insight into the role of NK cells in immunity to malaria was provided by studies on IL-12-related mechanisms in early stages of the disease (reviewed by Stevenson et al., 2001). Infection of normally susceptible A/J mice with Plasmodium chabaudi chabaudi AS (P. chabaudi AS) can be signicantly ameliorated by administration of exogenous IL12 very early in the course of infection leading to the development of IFN-g-, TNF-a- and nitric oxide-dependent

immunity (Stevenson et al., 1995). Furthermore, in vivo depletion of NK cells in P. chabaudi AS-resistant C57BL/6 mice results in a severely increased pathology, and IL-12treated but NK-depleted A/J mice fully fail to control the infection (Mohan et al., 1997). Similarly, self-resolving P. chabaudi AS and Plasmodium yoelii infections are characterised by the ability of the host to mount a strong IFN-g response as early as 24 h after challenge (De Souza et al., 1997) and NK cell-depleted animals show a signicantly reduced IFN-g response and are not able to control infection with either P. chabaudi AS or a non-lethal P. yoelii strain, suggesting a major contribution of NK-derived IFN-g to the resolution of infection in early stages (De Souza et al., 1997; Choudhury et al., 2000). Whilst interpretation of these studies might be complicated by inadvertent depletion of T cell subsets expressing NK-like markers (as described in Section 2), the observation that NK cell-depleted SCID mice succumb to non-lethal P. yoelii infection even earlier than SCID or anti-Thy 1.1-treated mice (Choudhury et al., 2000) conrms a role for NK cells that is independent of any effect of T cells. The in vivo role of NK cytotoxicityas opposed to IFN-g productionin resistance to murine malaria is still unclear but beige mice do show a signicantly higher parasitaemia in the early phase of infection with Plasmodium berghei, and NK-mediated lysis of P. berghei-infected erythrocytes renders parasites incapable of productively infecting malaria-nave mice (Solomon, 1986; Mohan et al., 1997; Stevenson et al., 2001). All of the studies cited above were carried out with blood stage malaria infections. However, g-irradiated P. berghei sporozoites can also induce IFN-g in vitro and in vivo, and activate splenic NK cells to lyse murine tumour cells, as early as 24 h after inoculation (Ojo-Amaize et al., 1984). More recently it has been shown that CD8C T cellmediated, nitric oxide- and IFN-g-dependent protective immunity induced by vaccination with irradiated P. yoelii sporozoites or DNA constructs requires both IL-12 and NK cells (Doolan and Hoffman, 1999). This study is an excellent example of the interplay between innate and adaptive immunity. In recent years the role of NK cells in control of human malaria infections has begun to be investigated. Work in our laboratory has revealed that, in non-immune donors, NK cells are among the rst cells in peripheral blood to produce IFN-g in response to Plasmodium falciparum-infected red blood cells (Artavanis-Tsakonas and Riley, 2002). In this in vitro setting we observed that NK cells are activated during the rst 18 h of exposure to infected erythrocytes, whereas gd T cells and NK T cells respond later, after 24 to 48 h. This activation was dependent on IL-12, and to a lesser extent IL-18, released from accessory cells (Artavanis-Tsakonas and Riley, 2002). The results of a study in children with acute P. falciparum infection suggested a positive correlation between the lytic activity of natural killer cells towards the human leukaemia line K562 ex vivo and the extent of parasitaemia (Ojo-Amaize et al., 1981)

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and Orago and Facer (1991) have provided evidence that puried NK cells from both healthy and P. falciparuminfected individuals directly lyse parasitised erythrocytes in vitro. This work has recently been conrmed (Mavoungou et al., 2003). In experimental P. falciparum infections of non-immune individuals, carried out in the course of vaccine evaluation, elevated plasma levels of IFN-g and soluble granzyme A were observed at the time of parasite release from the liver into the circulation, indicating a possible role for inammatory cytokines and cytotoxicity in the initial defence against blood stage infection in vivo (Hermsen et al., 2003). Parasitised red blood cells undergo massive structural alterations during trophozoite and schizont development (reviewed by Cooke et al., 2001), characterised by abnormal exposure on the cell surface of erythrocyte membrane components such as spectrin and Band 3 (reviewed by Crandall and Sherman, 1994) and surface expression of parasite-encoded neoantigens (reviewed by Craig and Scherf, 2001). Thus, it is of interest that lysis by NK cells of erythrocytes harbouring schizont-stage P. falciparum was found to be signicantly more efcient than killing of uninfected erythrocytes, suggesting a specic recognition of the altered erythrocyte surface (Orago and Facer, 1991). In support of this idea, we demonstrated that optimal activation of NK-derived IFN-g production in vitro requires contact between NK cells and P. falciparum-infected erythrocytes (Artavanis-Tsakonas and Riley, 2002; Artavanis-Tsakonas et al., 2003). Thus, the activation of human NK cells by blood stages of P. falciparum appears to depend on at least two signals,

i.e. cytokines released by bystander cells such as monocyte macrophages or dendritic cells and direct recognition of the infected red blood cell by NK cell receptors (Fig. 1). The ability for specic recognition of malaria-infected erythrocytes could be explained by the abnormal expression of ligands for stimulatory NK cell receptors, e.g. activating KIR, natural cytotoxicity receptors or Toll-like receptors, or alternatively by the down-regulation or complete loss of a ligand for inhibitory receptors such as Siglec-7. Interestingly, the ability of NK cells to respond to parasitised erythrocytes in vitro by IFN-g production is a stable phenotype of individual donors but varies signicantly between individuals (Artavanis-Tsakonas and Riley, 2002). The observed differences in NK reactivity are not a result of varying levels of IL-12 and IL-18 released by bystander cells since administration of exogenous cytokines does not augment the response to infected erythrocytes by NK cells of non-responding donors (Artavanis-Tsakonas and Riley, 2002). We therefore hypothesise that the heterogeneity in the response to P. falciparum-infected erythrocytes is due to differences in expressed repertoires of activating or inhibitory NK cell receptors. This might reect haplotypic variation in the KIR genotype among individuals and/or extensive functional allelic polymorphism in KIR and TLR (Uhrberg et al., 1997; Arbour et al., 2000; Rajalingam et al., 2001). Interestingly, in this context, genetic analysis of a small cohort of individuals has revealed a signicant association between NK responses to Plasmodium-infected erythrocytes and KIR genotype (Artavanis-Tsakonas et al., 2003) suggesting thatas has recently been suggested for susceptibility to HIV

Fig. 1. Model for the activation of natural killer cells by malaria-infected erythrocytes. We propose that, very early in blood-stage infection, dendritic cells and monocytemacrophages are activated by Plasmodium-infected erythrocytes. The activated cells mature and release cytokines including interleukin-12 (IL-12), interleukin-18 (IL-18) and possibly interleukin-15 (IL-15) which, in turn, activate natural killer (NK) cells to produce pro-inammatory cytokines such as interferon-g (IFN-g) and to release cytotoxic proteins including granzymes and perforin. In addition to this cytokine-mediated (bystander) activation, optimal activation of NK cells appears to require the direct recognition of the infected erythrocytes, presumably by NK receptors. The IFN-g released by NK cells stimulates macrophages to phagocytose parasitised erythrocytes. IFN-g furthermore links innate immunity to malaria to the adaptive immune system by inducing further DC maturation and the differentiation of T helper 1 cells. DC, dendritic cell; IFN-g, interferon-g; IL-12, interleukin-12; IL-15, interleukin-15; IL-18, interleukin-18; NK cell, natural killer cell.

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and hepatitis C virus (Martin et al., 2002b; Khakoo et al., 2004)there may be intrinsic, genetically-determined differences in malaria susceptibility that can be explained by variation in the innate immune response. Following stimulation of PBMC with infected erythrocytes we also observed signicantly increased expression of both CD94 and NKG2A on responding NK cells, suggesting a regulatory function for the inhibitory CD94:NKG2A heterodimeric receptor in limiting potential pathology caused by continual NK cell activation (Artavanis-Tsakonas et al., 2003). If conrmed, this implies that activation of NK cells by malaria-infected red blood cellsand likely other protozoan pathogens toois the result of integration by the cell of a complex array of signals emanating from both activating and inhibitory receptors. In this respect, the nal stages of pathogen-mediated NK activation may be very similar to those already described for transformed and MHC class I-decient cells. However, conrmation of the protective function of NK cells in human malaria, and determination of the role of KIR or other polymorphisms in inuencing susceptibility to malaria, will require extensive evaluation in Plasmodium-exposed populations.

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5. Concluding remarks NK cells appear to play an important role in the early immune response to a wide variety of pathogens, including a number of protozoal infections. In all cases, NK activation depends on cytokines released by accessory cells such as macrophages and dendritic cells but intriguing evidence is beginning to emerge of direct interactions between protozoa, or protozoan-infected cells, and NK cell receptors. It is likely that pathogen-derived ligands for activating NK receptors exist and we look forward to these ligands and their receptors being identied in the next few years. The possibility that polymorphismsat the level of the NK receptor and possibly also at the level of the parasiteencoded ligandmay inuence the outcome of NK pathogen interactions and thus the course of infection, remains an interesting possibility.

Acknowledgements We would like to thank Katerina Artavanis-Tsakonas, Michael Walther, Dan Davis, Karina McQueen and Peter Parham for data, advice and discussion. Work on NK cells in our laboratory is funded by the UK Medical Research Council, The Wellcome Trust and Boehringer Ingelheim Fonds. DSK is a Boehringer Ingelheim Fonds pre-doctoral fellow. The work of OCF has been supported by a PCC Garnham Fund Fellowship from the Royal Society of Tropical Medicine and Hygiene.

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