The role of natural killer cells in host-parasite interactions

Phillip Scott and Giorgio Trinchieri

Universi ty of Pennsylvania Veterinary School of Med i c i ne and Wistar Institute of Anatomy and Biology, Phi ladelphia, USA

Natural killer cells contribute to resistance to infectious organisms, and may also influence the nature of the adaptive immune response associated with infection. During the past year, their role in these events has been more clearly defined. In addition, the results of several recent studies that have begun to define the mechanisms by which natural killer cells recognize their targets

will be important in further elucidating their role in infectious disease.

Current Opinion in Immunology 1995, 7:34-40

Introduction

Natural killer (NK) cells were originally identified in the peripheral blood and lymphoid organs of humans and experimental animals as cells capable of lysing a variety of cell types (including tumor-derived cell lines, virus- infected cells and, in some instances, normal cells) in vitro, in the absence of previous deliberate or known sensitization [1]. NK cells are currently defined as cy- totoxic cells that have the predominant morphology of large granular lymphocytes but that do not express on their surface the CD3 complex or any of the known T- cell receptor chains (TCK 0t, 13, y, 6). The majority of NK cells express the CD16 (FqRIIIA) and CD56 (N- CAM) antigens in humans, the NKI.1 antigen in mice, and the NKK-P1 antigen in rats. These antigens me- diate cytolytic reactions even when M H C class I or class II antigen expression is absent on the target cells. The cytotoxicity mediated by NK cells is clearly dis- tinct from that mediated by cytotoxic T lymphocytes (CTLs), which recognize specific antigenic peptides in association with M H C class I or class II molecules. Thus, cytotoxicity mediated by NK cells is often de- fined as non-MHC-requiring to distinguish it from the MHC-restricted cytotoxicity mediated by CTLs.

Many of the physiologic functions of NK cells are medi- ated at least in part by their ability to secrete cytokines. NK cells are powerful producers of interferon (IFN)- 3, and granulocyte-macrophage colony-stimulating factors (GM-CSFs), and have also been shown to be able to produce tumor necrosis factor (TNF)-et, macrophage colony-stimulating factor (M-CSF), interleukin (IL)-3, IL-8, and other cytokines [1-4]. NK cells are induced to produce cytokines by stimuli that act individually or often synergistically including cytokines such as IL-2,

IL-12, TNF-et and IL-1, triggering of surface receptors such as CD16, interaction with immune complexes and the interaction of still undefined surface receptors with ligands on NK-sensitive target cells [1,5].

The activities of NK cells (cytotoxicity, proliferation, production of cytokines) are regulated by various cy- tokines, both in vitro and in vivo. Although IFN-0t [6] and IL-2 [2] have been known for a long time to be among the most powerful enhancers of NK cell activity, a recently described cytokine, natural killer stimulatory factor (IL-12) has been shown to play a central role in the regulation of NK cell activity. IL-12 is a heterodimeric cytokine formed by two covalently-linked glycosylated chains of 35 kDa and 40 kDa. IL-12 was originally pu- rified from the supernatant fluids of Epstein-Barr virus transformed human B lymphoblastoid cell lines [7]. In addition to B cells, phagocytic cells i.e. monocytes, macrophages, and neutrophils, have been identified a s

the major physiological producers of IL-12 [8]. Phago- cytic cells produce IL-12 in response to whole bacteria, bacterial products such as lipopolysaccharide (LPS) and intracellular parasites. The cell types on which IL-12 ex- erts biological activity are T and NK cells and the major direct biological functions of IL-12 on these cell types are the induction of cytokine production, primarily IFN-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antigen-activated lym- phocytes [7].

On the basis of their ability to lyse tumor cells, virus- infected cells, and certain parasites, NK cells have been identified as an effector cell type involved in innate resistance that is not antigen specific. However, many recent studies have widened the interpretation of the role of NK cells in immunity to show that NK cells

Abbreviations CTL~ytotoxic T lymphocyte; IFN--intederon; IL--interleukin; LPS~lipopolysaccharide;

TCR--T cell receptor; Th-- T helper; TNF--tumor necrosis factor.

34 © Current Biology Ltd ISSN 0952-7915

Natural killer cells in host-parasite interactions Scott and Trinchieri 35

have a dual role as effector and regulatory cells in in- nate resistances and regulatory cells in antigen-specific immunity [9]. These regulatory functions of NK cells are probably more dependent on their ability to pro- duce lymphokines, particularly IFN-y, than on their cytotoxic activity [2,10]. Early in infection, NK cells are important for the antigen-independent activation of phagocytic cells and also for favoring the development of antigen-specific T helper (Th) cell type 1 producing IFN-T and IL-2 [11,12]. It must be noted that the par- ticipation of NK cells in induction of septic shock may represent a pathologic exaggeration of the physiologic functions of NK cells in innate resistance [13°,14°].

Here we report some of the advances in the last year defining the role of NK cells as effector cells, elucidating mechanisms of NK cell recognition, and describing the influence of NK cells on the development of adaptive immunity.

Natural killer cells are effector cellls of innate resistance against infectious agents

Because of their ability to respond to external stimuli without previous sensitization, NK cells are able to re- spond rapidly, although non-specifically, to the presence of infectious microorganisms, or in some cases, neoplas- tic cells. Together with phagocytic cells (e.g. monocytes, macrophages and neutrophils) NK cells are effector cells of the innate or natural resistance, which represents the first line of defense against infection. This defense can take the form of direct lysis of the pathogen, as shown with Cryptococcus neoformans [15,16], or can involve lysis of infected cells, as has been shown for Mycobacterium tuberculosis infected human monocytes [17°]. In many cases, however, it is the production of IFN-~/and the macrophage microbicidal activity induced by IFN-7 that is probably of central importance in resistance. This con- clusion has been made in part on the basis of the finding that the treatment of experimentally infected mice with anti-IFN-~' often abolishes the early resistance to infec- tion associated with NK cells.

The importance of NK cells in infectious disease has been studied in both humans and in mice. Although studies in mice models permit experimental manipu- lations, several problems are associated with studying NK cells in these models. Paramount among these is that the markers ot~en used to identify murine NK cells, NKI.1 and asialo GM1, are also expressed on other cell types [17°,18,19]. An additional handicap is that no mouse model exists that is deficient in NK cells; beige mice lack NK cytotoxic function, but have NK cells that produce IFN- T [20]. Recently, it was found that mice overexpressing the CD3 epsilon chain lack both NK and T cells, although these mice are not yet generally available for experimental use [21°]. For these reasons, scid mice, in which the only known source of

IFN-~t is the NK cell, have frequently been used to de- fine the factors involved in early NK cell activation. The most studied infection in scid mice has been with Liste- ria monocytogenes, for which macrophages were found to be required for NK-cell activation (reviewed in [22,23]). It is now known that Listeria induces the production of both TNF-Ct and lL-12 by macrophages, which subse- quently activate NK cells [22,24]. This pathway is not unique to Listeria and operates following infection with several bacteria and protozoa [22,25°]. Whether other cytokines and/or cells participate in the early activation of NK cells is unknown, but recent studies by Hunter et al. [26 °] suggest that macrophages may contribute an- other component to induce optimal IFN-y production by scid spleen cells taken from Toxoplasma infected mice.

Although studies in scid mice have predominated in this field, NK cells have also been shown to contribute to the innate immune response in conventional animals. For example, during the acute stage of Toxoplasma infec- tion in C57BL/6 nfice, IFN- 7 is produced by NKI.1 +, CD3 epsilon- cells [27°]. Furthermore, treatment of mice with anti-asialo-GM1 antisera enhanced suscepti- bility to infection in C57BL/6 mice, suggesting a critical role for NK cells in this infection [28]. In C3H mice, NK cells are induced within 24 hours of infection with Leish- maina, and contribute to both resistance to disease and to the early development of Th l cells [11]. The iden- tity of these cells has now been clearly established as a CD3- NK cell population [29°]. The initiation of this response requires IL-12, the production of which is stim- ulated soon after infection in vivo [29 °, 30°]. Interestingly, it was recently reported that Leishmania parasites also ac- tivate human NK cells present within cultures of normal human peripheral blood cells [31°].

In spite of the strong evidence that NK cells are a critical component of the early innate immune response to sev- eral intracellular pathogens, several studies over the past year have suggested that these cells are not always as- sociated with resistance of these pathogens following infection [32°,33]. With Listeria infections, two stud- ies indicate that NKI.1 + cells may not be required for bacterial control in C57BL/6 mice [32°,34°]. Fur- thermore, mice treated with anti-NKl.1 monoclonal antibodies exhibited enhanced resistance to D'steria at day 5 of infection, although by day 7 there was no dif- ference between treated and untreated mice. As IFN-y is required for resistance to Listeria, and the anti-NKl.1 treated mice exhibited a significant decrease in the num- ber of IFN-y producing cells, these results are surprising. As mentioned above, however, one difficulty with inter- preting these results is the non-specificity of the NK1.1 marker. What may particularly confuse the situation is that NKI.1 has recently been shown to be present on a small population of CD4 + T cells that produce IL-4 [17°]. Thus, the in vivo outcome following adminis- tration of anti-NKl.1 may depend upon the relative balance between the IFN-y producing NK cells and the IL-4 producing CD4 + T cells that is achieved by the treatment, which may be difficult to predict.

36 Innate immunity

The ability of NK cells to participate in the resistance against infection by certain viruses is well documented in experimental animals [35] and is strongly suggested by the recurrent viral infections that occur in the few patients described to have a selective deficiency of NK cells (see, for example, [36]). In vitro, NK cells selectively lyse virus-infected cells, via a mechanism that is at least in part dependent on the production of IFN-tX, a potent stimulator of NK-cell activity [37,38]. In vivo, virus in- fection and IFN production are usually accompanied by a rapid activation of and increase in the number of NK cells, both systematically and localized in the infected area [39]. The NK response to virus infection usu- ally peaks at three days post-infection and is followed by an antigen-specific Th and CTL response, which peaks at seven to nine days post-infection [39]. The early N K response induces a significant reduction in the titer of certain viruses, as has been found during infec- tion with murine cytomegalovirus [35]. Other viruses, such as lymphocytic choriomeningitis virus (LCMV), are not only resistant to the antiviral effects of NK cells, but the IFN production and NK-cell activation induced by these viruses can even have pathogenic effects [35].

Mechanisms of natural killer cell recognition

Although the activity of NK cells has been considered to be non-specific and, unlike CD8 + CTLs, to be inde- pendent of the expression of MHC antigens on target cells, several lines of evidence in the past few years suggest that NK cells have clonally distributed recep- tors that recognize and bind to alleles of class I MHC antigens. Binding results in a negative signal being de- livered to the NK cell, thereby inhibiting cell-mediated cytotoxicity [40,41]. These receptors, which appear to be molecularly heterogenous, are expressed on subsets of NK cells in a clonally non-exclusive manner and recog- nize polymorphic differences on class I molecules. Un- like CD8 + T cells, NK cells recognize only supertypic or public specificities common to several HLA alleles. Resistance of target cells to killing by NK cells is con- ferred by the target cell by the 0tl/ct2 domains of class I HLA, which form the peptide binding groove [42]. Amino acid residue 74 (positioned in a side pocket of the groove of HLA-A molecules) has also been shown to be important in conferring resistance [43]. Furthermore, the relevant amino acid residues recognized by charac- terized class I receptors on human NK cells have been identified in positions 77 and 80 (Asn77, Lys80 or Ser77, Asn80) on HLA-C molecules [44,45] and in position 80 (Asn80 in the Bw4 supertypic epitope, or Thr80 in the Bw6 epitope) on HLA-B molecules [46"--48°]. All of these amino acid positions are in the peptide-binding groove of the class I molecules, raising the possibil- ity that peptide binding to class I molecules might be part of the structure recognized by NK cells or might

play a role in inducing conformational modifications of class I molecules recognized by NK cells.

These findings suggest a possible explanation for data in- dicating that infection of target cells by herpes viruses only enhances their susceptibility to lysis if the cells express class I MHC [49] or that NK cell clones are heterogenous in lysing these infected target cells and that their activity is controlled by polymorphic tar- get elements [50"]. Because self class I molecules have been shown to protect normal cells from lysis medi- ated by autologous NK cells [51 °] and only because minimal variation in class I expression is observed in herpes virus infected target cells [49,50"], the results above suggest that occupancy of class I molecules by virus-derived peptides may result in the suppression of their ability to prevent NK cell mediated lysis and, con- sequently, in an enhanced susceptibility to lysis of the tar- get cells. Similarly, it has previously been demonstrated that the addition in culture of large quantities ofpeptides able to bind to class I alleles of target cells, enhances the cells' susceptibility to lysis [52,53].

The interaction of NK cells with class I molecules on target cells induces a global inactivation of multiple NK cell activating signals that usually lead to cell-mediated cytotoxicity. This indicates that NK cells must be trig- gered by any one of a set of distinct target-cell ligands, but that all these signals can be overruled by class I me- diated inhibition [54"]. Interestingly, the recognition of class I by NK cell clones also prevents their production of cytokines such as IFN-y [55] but it does not inhibit their proliferation [56°], suggesting that class I molecules may play a role in regulating both NK cell cytotoxicity and cytokine production.

Many of the receptors identified on NK cells are type II integral membrane proteins with C-type lectin homol- ogy. It is noteworthy that many members of the C-type receptor family on NK cells (e.g. NKI-1, NKR-P1, and CD69) are triggering molecules that induce cytotoxicity and cytokine production [57], whereas other receptors (e.g. the murine Ly49 and the human kp43 [CD94]), are inhibitory, able to recognize class I polymorphic molecules [41]. Although these lectin-like receptors can bind carbohydrates, they may also bind to polypeptide or conformational sequences. This has been shown for CD23, a member of the C-type lectin family. Although CD23 binds to IgE independently of carbohydrates, it binds the carbohydrate structure of CD21 [58]. The hy- pothesis of a role for carbohydrate recognition activity has recently been supported by the data of Bezouska et al. [59]. The data suggests that oligosaccharides that bind to NKR-P1 with high affinity are able to inhibit binding of NKR-P1 protein to target cells, thereby preventing target cell killing, and the activation of signal trans- duction in NK cells, and when incorporated into the membrane of NK-resistant target cells, making them sensitive to NK cell-mediated cytotoxicity. The ability of NK cells to recognize carbohydrates and be activated by them, offers another possible explanation for the abil-

Natural killer cells in host-parasite interactions Scott and Trinchieri 37

ity of NK cells to recognize infected target cells or for the direct recognition of bacteria and parasites.

Role of natural killer cells in septic shock

In mice, a lethal shock syndrome known as generalized Shwartzman reaction can be elicited by two consecutive injections of lipopolysaccharide LPS, the first intrader- mally and the second, 24 hours later, intravenously. The priming effect of the first injection is due to LPS-in- duced IL-12, which stimulates IFN-~' production; IL-12 and IFN-~', together with TNF and IL-1, also play a role in the lethal response to the LPS challenge [60°]. In this model NK cells are probably the most important source of endogenous IFN-3', since in vivo depletion of N K cells prevents LPS-induced lethal reactions in nfice [13°]. IL-12 is also produced in response to high doses of LPS, or alternatively to low doses of LPS in nfice that had been previously primed by BCG infection. In both cases, IL-12 is responsible for IFN-y production [14°,61°]. Thus, antibodies against IL-12 prevent IFN- T produc- tion and lethality in these endotoxin shock models [14°]. SCID mice produce equivalent or higher levels of lL-12 and IFN- compared with wild-type mice, suggesting that NK cells are also present in this endotoxic shock model and are the main cell type responsible for IFN-~' produc- tion and therefore have a major role in the LPS-induced morbidity and lethality [14°]. The pathogenic dysregu- lation of cytokine production, involving IL-12 and NK cells, observed in endotoxic shock or in the generalized Shwartzman reaction most likely represents an exagger- ation of the physiological cytokine response (discussed above) in response to infection.

The influence of natural killer cells on the development of the adaptive immune response

In addition to contributing to resistance during the early phases of infection, it has been postulated that NK cells may influence the nature of the adaptive im- mune response [12,62,63]. Recently, a direct role for these cells has been demonstrated in vitro with human cells [64], and in vivo in mice immunized with antigen and IL-12 [65°,66°]. In the human system, depletion o f CD16 + NK cells impaired the capacity o f IL-12 to promote the development of Th l clones. In mice, it was found that administration o f IL-12 with leish- manial antigens promoted Th l cell development and resistance [65°]. When NK cells were depleted at the time of immunization, CD4 + Th l cells failed to de- velop. In this system, NK cells appeared to function as a source o f IFN- T that was critical for CD4 + T h l cell development. Similarly, in studies using the antigen

KLH (keyhole limpet hemocyanin), the ability of IL-12 to promote IFN- T production was abrogated when NK cells were removed, although in contrast to the leishma- nial system, IL-12 still inhibited IL-4 production, and depletion of IFN- T had no effect on the efficiency of inmaunization [66°].

These results do not imply that NK cells are an abso- lute requirement for the development of Th l cells; in fact, it has been shown that these cells are not required for in vitro Thl development in T C R transgenic models [67,68]. Furthermore, Romano and colleagues [69] have found that NK cells are not required for the development of Thl cells following immunization with a Candida al- bicans vaccine. Similarly, C3H mice still develop a Th l response following Leishntania major infection when NK cells are depleted, but the response is delayed and the an- imals are correspondingly more susceptible [29°]. Where NK cells may be particularly important is in situations in which antigens or infections that tend to induce a strong IL-4 response in the absence of adjuvant, such as is the case with soluble leishmanial antigens in BALB/c mice [65 °] and keyhole limpet hemocyanin in C57BL/6 mice [66°].

Conclusion

NK cells play multiple roles in host-parasite interactions. These cells lyse pathogens and pathogen-infected cells, but their effector function is not limited to their lytic capacity as in many cases, the production of cytokines may be their primary contribution to resistance. We now know that NK cells, through the cytokines they produce, can also influence the development of the adaptive im- mune response. Furthermore, the recognition that cer- tain pathogens can stimulate the production of cytokines critical for NK cell activation, such as IFN-0t/~, TNF ot and IL-12, has led to the provision of a model for how NK cells can be induced soon after infection. We are also beginning to understand better the mechanism(s) by which NK cells recognize targets, although this field is still hampered by the incomplete characterization of the receptors on NK cells and also by the fact that each NK cell most likely expresses several different receptors, some able to transduce positive signals resulting in cytotoxicity and cytokine secretion and others prevalently delivering negative inhibitory signals. Nonetheless, rapid progress in the characterization of the structure and specificity of the class I recognizing receptor of NK cells, and the more recent advance in the characterization of the specificity of the lectin-like receptors, is beginning to enable us to speculate on some of the possible mechanisms involved in NK cell recognition of infectious agents. The exact role of the possible mechanism of recognition depends upon a more complete characterization of the various receptors involved in regulating NK cell activation.

38 Innate i m m u n i t y

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • o of outstanding interest

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The first direct demonstration of a role for natural killer cells in l ipopolysacc- haride-induced generalized Shwartzman-like reaction.

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This paper demonstrates that interleukin (IL)-I 2 is required, but not suffi- cient, for IFN-y production and lethality in (low dose) l ipopolysaccharide- induced shock in BCG-primed mice. The ability of T- and B-cell deficient scidmice to produce both IL-12 and interferon (IFN)-y suggests a role for natural killer cells in IFN-y production.

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The authors found that overexpression of the human CD3 epsilon chain ablates natural killer and T-cell development. The findings are important not only for understanding the relationship between T and NK cells, but also because they may provide a model to study immune responses in the absence of both T and NK cells.

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Natura l k i l le r cells in hos t -paras i te in te rac t ions Scott and Trinchier i 39

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P Scott, University of" Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104, USA. G Trinchieri, The Wistar Institute, 3601 Spruce Street, Philadel- phia, PA 19104, USA.