Inter-relationship among macrophages, natural killer cells and neutrophils in early stages of Listeria resistance

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Inter-relationship among macrophages, natural killer cells and neutrophils in early stages of Listeria resistance Emil R Unanue Reports in the past few years have shown the involvement of different cells and cytokines in controlling the infection with the intracellular facultative pathogen Listeria monocytogenes. A synergistic interaction of T-cell-independent and -dependent processes takes place but the nature of these interactions and of the relevant cells and cytokines depends on both the stage of the infection and the tissue that is involved.

Addresses Department of Pathology, Center for Immunology, Washington University School of Medicine, Campus Box 8118, 660 South Euclid Avenue, St Louis, MO 63110, USA; e-mail: [email protected] Current Opinion in Immunology 1997, 9:35-43 Electronic identifier: 0952-7915-009-00035 © Current Biology Ltd ISSN 0959-7915 Abbreviations APC antigen-presenting cell IFN interferon IL interleukin L ligand NK natural killer R receptor SClD severe combined immunodeficient STAT signal tranducer and activator of transcription TNF tumor necrosis factor

Introduction

ListeHa monocytogenes infection in the mouse has long served as a basic experimental model to identify and study the mechanisms involved in host defense. It has been intellectually challenging and perhaps a bit surprising to consider the many components of the host immune and inflammatory responses involved in defense against Listeria. T h e process of host resistance is clearly multistaged in which the time of the infection, the nature of the infected tissue and organ, the innate and specific immune reactions influence it. While the initial ListeHa infective process was taken as a model to study cellular immunity at the macrophage level [1,2], it has become apparent that the process of control and elimination of Listeria involves a number of other cells and cytokines. Listeria is a Gram-positive organism that binds to a range of cells, not only macrophages but also epithelial and mesenchymal. Along these lines, a very recent study from Cossart's group at the Pasteur Institute [3°°] identified the Listeria receptor on epithelial cells [3°°]. T h e protein in Listeria that is required for entry is internalin [4]. T h e cell receptor on the Caco-2 epithelial cell is E-cadherin,

a protein involved in mediating homotypic adhesions in epithelial cells such as those of the intestine. T h e dynamics of entry into Caco-2 cells had been the subject of various studies [5-7]. Presumably, the internalin molecule of Listeria binding to E-cadherin forms a complex linked to the cytoskeleton of the epithelial celll [5] which then leads to the mechanical process that internalizes the microbe. This study of Mengaud et al. [3 °°] discusses how the process of enteric infection, the natural route, may take place, keeping in mind that the E-cadherin molecules are expressed on the basolateral surface of epithelial cells. T h e authors state that their results 'favor a mechanism of translocation through M cells as a primary step in infection. Entry into enterocytes would represent a secondary step taking place at the basolateral pole'. Regardless of the exact nature of the entry processs, this important study represents one major advance in our understanding of Listeria entry into cells. E-cadherin-internalin, of course, does not explain the mechanisms of entry into other cells, fibroblasts or macrophages, which lack E-cadherin. T h e molecular basis of interaction of ListeHa with t h e macrophage is not entirely clear and perhaps more than one component is involved. T h e macrophage scavenger receptor type I was identified as one molecule that binds to Gram-positive bacteria, including ListeHa, by way of their content of lipotechoic acid [8]. There are also reports of enhancement of binding to macrophages of Listeria coated with Clq (i.e. via the Clq receptor) [9] or C3 (via CR3 receptor) [10-12]. Once it is taken into the cell by phagocytosis, the organism can leave the vacuole as a result of its release of listeriolysin O, a pore-forming sulfhydryl activated cytolysin. ListeHa penetrates the cytoplasm where actin condensation takes place around the microbe. Listeria then moves in the cytoplasm and, presumably, cell to cell spread may take place without an extracellular phase of growth (reviewed in [13]). This ability of Listeria to cross into the cytoplasm favors the induction of CD8 + T cells [14] which bring in protective immunity (reviewed in [15]; see below). In fact, Listeria has now been molecularly engineered to produce a recombinant strain that expresses immunogenic epitopes. These Listeria serve as vehicles to transport different proteins into the cytosol and are processed into peptides that bind to M H C class I molecules to generate CD8 ÷ T cell immune responses. Such Listeria effectively induce specific cellular immunity [16,17,18°,19°]. T h e same effect has been accomplished using purified listeriolysin in liposomes, as a vehicle to transport antigens into the cytosol [20"].

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Innateimmunity

The early stages of the infection Much interest has developed in the analysis of the early stages of the infective process of Listeria. Particularly useful in delineating the various stages was the examination of infection in the severe combined immunodeficient (SCID) mouse [21,22"]. Lacking T and B cells, these mice nevertheless are able to mount partial resistance to listeriosis. T h e mice become chronic carriers of Listeria organisms and are unable to mount sterilizing immunity. T h e T-cell-independent process that controls Listefia resistance in SCID mice is part of the 'innate' or 'natural' immune process. Presumably this process is a component of resistance in normal mice, prior to the establishment of a T cell protective response. Clearly, sterilizing immunity for iisteriosis is a T-cell-dependent process (reviewed in [15]). Analysis of the early stages of Listeria infection, that is, the first 24-72 hours, allows the identification of those cells and cytokines involved. T h e cytokines that have been identified to play a role include IL-12, tumor necrosis factor (TNF)-ct, IL-1, IL-6 and IFN-T. These have been studied using either specific neutralizing monoclonal antibodies or gene ablated mice. T h e target site of action of the antibodies has not been made apparent in all studies. Regardless, the protective role of the cytokines is not only found in SCID mice but also in lymphocyte-sufficient (wild type) mice. Insofar as cell types involved, three cell types have been found to be essential for the T-cell-independent or innate component: the macrophage, the natural killer (NK) cell, and the neutrophil [21,22"]. T h e hypothesis that our laboratory has defended is that the macrophage first releases IL-12 and TNF-(~ following Listeria infection; and that both these cytokines, in synergy, drive the NK cells to produce IFN-T, which is then responsible for priming macrophages for cytocidal activity, and in curbing the growth of the organism [23,24]. This hypothesis was formulated when we found macrophage activation, both in terms of production of cytocidal molecules, such as nitric oxide, and expression of class II M H C molecules, following Listeria infection in SCID mice. In culture, the spleens from these mice elaborated IFN- T upon the addition of dead or live Listeria organisms: a macrophage-conditioned media added to NK cells resulted in IFN-T production. While the system was analyzed it became apparent that macrophages were elaborating at least two c y t o k i n e s - - I L - 1 2 and T N F c t - - that were essential for the NK cells to produce IFN-T. T h e two cytokines were identified using recombinant cytokines and also by the use of monoclonal antibodies to neutralize them in macrophage-conditioned media [25,26]. T h e production of IL-12 has been documented in vivo by its presence in blood and by Northern analysis of mRNA [27]. In culture, Listeria induces production of IL-12 by macrophages [24,28,29]. A provocative finding was made

recently by Dighe et al. [30°°]; macrophages from normal mice challenged with Listeria produced low levels of IL-12 p40 chain, but this amount was increased about 20-fold by the addition of I F N - T. Such an increment was not observed in macrophages of IFN-T receptor (R) null mice, nor macrophages engineered to contain a dominant negative mutant protein of the IFN- T receptor antagonist chain. Similar requirements for the presence of I F N - T for the elicitation of IL-12 effects were noted in murine tuberculosis infection [31] and in human monocytes [32-34]. Aside from macrophages, IL-12 has also been shown to be synthesized and secreted by dendritic cells [35] as well as by neutrophils [36]. (Dendritic cells have been shown to take up Listeria [37] and, for that matter, can be killed by apoptosis resulting from the release of listeriolysin O [38].) Results compatible with the protective role of IL-12 in listeriosis have been obtained in a number of other murine infections: Toxoplasma gondii [39-41], Lyme borreliosis [42], streptococci [43], Salmonella typhimurium [44], cytomegalovirus [45], Mycobacterium avium [46,47], M. tuberculosis [48], Bacillus Calmette-Gu~rin [31], malaria [49], Schistosoma mansori [50], for example. For a general review on IL-12 by its discoverer, see [51"]. IL-12 was shown to be essential for resistance to Listeria infection in both normal and SCID mice [52]. Neutralizing the activity of IL-12 using specific antibodies resulted in poor macrophage activation and a marked increase in Liste~ia growth in liver and spleen [52,53]. That the effects could be attributed to the lack of production of IFN-T was proven by the injection of I F N - T following administration of the antibody [52]. Administration of IFN- T corrected the defects and such mice resisted infection. T h e effects of anti-IL-12 administration were less pronounced in lymphocyte-sufficient mice during a secondary challenge (i.e. wild type mice first infected with an immunizing dose of Listeria and then challenged days later with a lethal dose [54]). This would suggest that there are other mechanisms of induction of I F N - T production operating in the immune mice. Along these lines, we and other groups such as those of Ken Murphy and Alan Sher have speculated that IL-12 is the cytokine that bridges the innate immune mechanisms to the specific adaptive response. In support of this statement, Murphy's laboratory [55], together with Ann O'Garra's laboratory [55], showed that the induction of IL12 by macrophages was a key step in the differentiation of CD4 ÷ T cells to a T h l phenotype (i.e. to cells that produce IFN- T and little IL-4). T h e inductive stimulus in their experimental system was Listetia organisms interacting with macrophages. Importantly, another manner by which to induce IL-12 is during the T-celI-APC interaction. This involves the molecules CD40-CD40 ligand (L) (in T cells and APCs, respectively; [56°,57"]). For a recent review

Macrophages, natural killer cells and neutrophils in Listeria resistance Unanue

on the role of "CD40 and its ligand in host defense" see [58]. This phenomenon of the T-celI-APC interaction activating the APC for cytokine production has been described previously for IL-1 [59]. Indeed, the release of I L - I was shown to be also mediated by the C D 4 0 - C D 4 0 L molecules [60]. T N E together with IL-12, was shown to be essential for NK cells to produce IFN-T [24,26]. In vivo administration of neutralizing anti-TNF to SCID mice resulted in the inhibition of macrophage activation, and with it increased Listeria growth in tissues [61]. T h e role of T N F is multiple: in contrast to the results with anti-IL-12 cited above, administration of IFN-y to Listeria-infected mice given anti-TNF corrected some parameters of macrophage activation but did not reduce the uncontrolled growth of Listeria [52]. For example, expression of M H C class II molecules by macrophages was restored. T h e effects of T N F were noted also in lymphocyte-sufficient mice [62-68]. Finally, a recent noteworthy study indicated that virulent listeriolysin ÷ Listeria are more effective than the avirulent Listeriolysin- strains in inducing T N F production by macrophages [69]. IL-1 has been shown to be required for resistance to listeriosis in vivo [70-72]. Our laboratory studies indicate that the protective action of IL-1 was not by way of the IL-12 +TNF---~NK= IFN-T axis [70,71]. Neither the addition of monoclonal antibodies to IL-l(x, IL-113 nor the type 1 IL-1R inhibited the in vitro response of NK cells to macrophage-conditioned media containing IL-12 and T N F ; nor did the addition of IL-I to the culture have an effect. Others have argued, however, for a role for IL-1 in the generation of IFN-T [73]. In normal mice IL-1 was shown to synergize with IL-12 in the production of IFN-T by y8 T cells [74]. In our laboratory, Howard Rogers found that the injection of monoclonal antibodies to both IL-lct and IL-~ and to the type I IL-1 receptor were required to show heightened susceptibility to Listeriosis in wild type and SCID mice [70,71]. Such mice did produce IFN-y, however. IL-1 neutralization resulted in two effects. First, there was a decreased number of neutrophils in infective foci (see below). Second, the macrophages were unresponsive to IFN-T. This latter effect needs to be examined further. Whether IL-1 has other effects in vivo such as participating in the activation of endothelial ceils, needs to be evaluated.

In analogy to IL-1, the action of IL-6 in early listeriosis also does not appear to work by way of the NK cell IFN-T pathway. Mice lacking the IL-6 gene by homologous recombination were found to be highly susceptible to early growth of Listeria in the liver and spleen [75"]. IL-6 reduced blood neutrophilia, although liver microabscesses were found. A similar effect was previously found by Cheer's laboratory [76] using anti-IL-6 neutralizing

37

antibodies. Mice lacking the transcription factor nuclear factor-IL-6 were highly sensitive to listeriosis [77].

IFN-T

T h e essential role of IFN-T in listeriosis, or in any infection with an intracellular infectious agent for that matter, was first shown by the Schreiber laboratory [78] by the use of a potent neutralizing antibody. I F N - y activates the macrophage which changes to the phenotype typical of activated macrophages [1,15]. Since the original study [78], other laboratories have amply confirmed the role of IFN-y. As mentioned, in the SCID mouse [23] the maintenance of homeostasis between host and Listeria and the maintenance of the carrier state is dependent on IFN-y. In the SCID mouse the induction of IFN-y production follows the release of IL-12 and T N F ; in the lymphocyte-sufficient mouse it depends on the activation of T h l CD4÷ T ceils. There have been many reports in recent years on the biology of IFN-T. In the context of this review, several recent studies are worth noting. T h e Schreiber laboratory in the paper of Dighe et al. [30 °°] offered convincing proof that the resident tissue macrophage is an important participant in early listeriosis, by its response to IFN-T. T h e y molecularly engineered a dominant negative IFN-y receptor c~ chain under the control of the human lysozyme promoter. Transgenic mice bearing this gene construct were found to express it in tissue macrophages but not in recently activated macrophages, and showed heightened susceptibility to Listeria infection. T h e data would imply, therefore, that the resident macrophages were an important element in the initiation of Listeria resistance. In accordance with this suggestion, mice that lacked IFN- T responsiveness by ablation of either the I F N - T receptor (x chain gene [79] or the IFN-Tstructural gene [80,81 °'] were highly susceptible to infection. T h e laboratory of Robert Schreiber just produced mice with ablation of the signal transducer and activator of transcription (STAT)-I gene [82 °° ] which is the transcription factor for activation of IFN-T induced genes [83]. T h e y found in a number of assays that macrophages from these mice did not respond to IFN-T. Such mice were not able to control Listeria infection. T h e mode of action of I F N - T may well be through the induction of reactive nitrogen or oxygen intermediates. Administration of aminoguanidine (an inhibitor of the inducible form of nitric oxide synthase) to mice, at frequent intervals, maintained an inhibition of nitric oxide production by tissue macrophages and increased sensitivity to Listeria infection [84]. T h e macrophages treated in vitro with the inhibitor also had reduced listericidal activity. Although some reports failed to find an effect of nitric oxide inhibitors in listeriosis, this is likely because of dose/time effects [85,86]. Indeed, it

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Innateimmunity

is clear that mice with a disruption of the inducible nitric oxide synthase gene have decreased resistance to listeriosis [87"°]. Finally, I shall discuss whether IFN-y is absolutely required for all aspects of Listeria resistance. In our experience with SCID mice, the presence of IFN-y was absolutely required but didn't lead to sterilizing immunity. T h e effects of primary infection in normal mice have been consistent in showing a major essential role. In the secondary or memory response arguments exist. Tripp etal. [54] found such a requirement using the H-22 monoclonal antibody made in the Schreiber laboratory, but in this case Tripp needed to inject 2-3-fold higher amounts than for the primary response. This could explain the failure of others to see a deleterious effect in the memory response by using a regular dose of neutralizing antibodies [68,88]. A very important and provocative study is that of Harty and Bevan [81°°]. T h e y showed by adoptive T cell transfers that a protective response could be elicited by CD8 ÷ T cells from mice lacking the structural gene for IFN-y. This study is exciting and demands a careful look at non-IFN-y pathways of resistance.

The control of the macrophage activation pathway: IL-IO While the previously discussed cytokines exert a powerful positive effect, they are in fact balanced by other sets of molecules that exert a negative control in the infectious process. The molecules that have been mostly discussed include IL-10, IL-4, transforming growth factor (TGF)-I3 and the prostaglandins. In the context of listeriosis, it is IL-10 that has received the most attention. T r i p p e t al. [24] were the first to notice IL-10 production in cultures of spleen cells from SCID mice challenged with Lister~a. Their studies indicated that IL-10 exerted an inhibitory role on IL-12 release from macrophages as well as in the response of the NK cell to IL-12 and T N E In fact, addition of IL-10 to cultures containing IL-12 and T N F reduced IFN-y secretion by NK cells [89]. Particularly provocative are the recent experiments showing the effects of IL-10 in listerioris [90°°]. Previous studies had indicated that formation of antigen-antibody complexes in vivo resulted in profound susceptibility to Listeria infection [91-93]. The mechanism of inhibition is probably multifactorial and involves the inhibition of a number of macrophage functions by the engagement of their Fc receptors. (In fact, a recent study [94 °] documented that antigen-antibody complexes inhibited IFN-'/class II expression by preventing phosphorylation of STAT-1.) Addition of antigen-antibody complexes and Listetia to macrophages resulted in an alteration of the IL-12/IL-10 ratio with the latter being the predominant cytokine [90°°]. Consequently, the innate mechanism did not effectively activate the macrophage. Antigen-antibody complexes arc found in situations of chronic microbial infection such as malaria, Leishmaniasis and leprosy. Many of these infections show depression of cellular immunity,

an issue which is poignant from the studies of Virgin and Trippet al. [90",91-93]. Finally, the effects of IL-10 may depend on the stage of the infection. Indeed, an analysis using anti-IL-10 antibodies from Czuprynski's laboratory indicated an impairment of the T cell phase of clearance of Listeria [951.

The role of neutrophils Early indications that neutrophiis were playing a role in Listeria infection came from studies showing a correlation between early neutrophilia in infected foci and degree of infection [96,97]. But, it was Pixie Campbell in Denver who first called attention to a listericidal effect of neutrophils by her observations in cultures [98,991. In listeriosis, the infective foci during the first two days of infection are rich in neutrophils. Striking is the early liver infection, characterized by small microabscesses: hepatocytes are heavily infected with Listeria and are surrounded by neutrophils [100-103]. T h e usual time of appearance of neutrophils in Listeria infection is about 2 a. A,8 hrs. T h e liver lesion develops hours after the Listeria is taken up by Kuppfer cells. Invariably, a certain number of Listeria are transported into the hepatocyte through a process that needs to be evaluated insofar as its precise mechanism. Once in the hepatocyte the microbe grows exuberantly. Subsequently, the number of infective foci decreases as does the amount of ListeHa, and the number of small granulomas develops. In the SCID mouse, in contrast, the Listeria persists and the chronic infective foci are formed by accumulations of macrophages and neutrophils. Several groups have examined the functional role of neutrophils by using monoclonal antibodies that deplete them from blood and tissues [104-109] for several hours. T h e monoclonal antibody RB6-8C5 has been successfully used. In essence, depletion of neutrophils resulted in a pronounced acceleration of the infective process. We have interpreted the participation of the neutrophil to involve two processes [22°]: first, the control of local infection and the spread of the infection to other sites; and second, in the liver, to curb the dissemination of the microbe to the liver cells. Mice depleted of neutrophils died from acute liver failure [110°°1. By doing time-sequence histological analysis of neutrophil-depleted mice, Howard Rogers in my laboratory [110 -°] found that following infection, the hepatocyte died via apoptosis. Hepatocytes that contained ListeHa organisms were terminal deoxynucleotide transfer-mediated dUTP-biotin-dependent nick end labelling ( T U N E L ) positive. Listeria organisms also infected cultures of primary hepatocytes and induced their death by apoptosis, as was evident by DNA fragmentation. In vivo, in the absence of neutrophil, the infection spread and the mice succumbed to acute liver failure. We also examined the possibility that cytokines contributed to the liver death

Mecrophages, natural killer cells end neutrophils in Listerie resistance Unanue

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Figure 1

Pathogen

Q

~

~

S

NK cell

Resident macrophage or

monocyte Thl

3

MHC class II, costimulators Activated macrophage

© A resident macrophage that takes up Listeria produces a number of cytokines, some of which induce the NK cell to produce IFN-7 (induction is represented by +). Other cytokines, like IL-10, inhibit this process (inhibition is represented by -). The IFN-7 drives the macrophage to an activated state. The activated macrophages express high levels of MHC class II molecules (represented by two parallel lines) and costimulators, like intercellular adhesion molecule (ICAM)-I and B7 molecules (represented by a wavy line). The activated macrophage is active in: reducing the growth of Listeria in vacuoles (closed circle) or in the cytosol following vesicle lysis (broken circle); in presenting peptides to Listeria-specific T cells (marked as Th0); and in inducing the differentiation of the ThO cell to Thl (by its release of IL-12). NO" represents nitric oxide. Reproduced from [114].

program. Using antibodies, inhibitors, or gene ablated mice we could find no role for TNF, IFN-T, nitric oxide or IL-1 [110°']. T h e issue remains unresolved whether the hepatocyte can develop listericidal activity as a result of exposure to local cytokines. Some have found effects in cultures of hepatocytes treated with cytokines [111,112], but others have failed [110°',113]. To note is that the secondary response to Listeria in lymphocyte-sufficient mice is still neutrophil-dependent despite strong T cell immunity [108,109]. At this time, we favor the scenario that the apoptosis program is part of the local response of the hepatocyte which involves the attraction of neutrophiis and their adherence to the infected cells. T h e liver adhesive molecule for neutrophils is not ICAM-1. Mice lacking the ICAM-1 gene develop neutrophil micro-abscesses after listeriosis (JC Gutievrez-Ramos, ER Unanue, unpublished data).

Conclusions

Figure 1 is a sketch of some of the events taking place during Listeria infection [114]. These events have been outlined as a result of intensive research by many laboratories during the past three decades, starting with the seminal studies of George Mackaness [1]. Despite these efforts, listeriosis will keep our interest alive as long as it remains a fertile model to examine regulatory events of both thymic-independent and -dependent processes. T h e figure, taken from a recent review [114], depicts a scenario for the cellular interactions taking place in listeriosis. T h e control of ListeHa infection requires the coordinated and integrated activity of both the cellular innate and specific adaptive systems. No other experimental model demonstrates these components as clearly as murine listeriosis. T h e SCID model allows us

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Innate immunity

to identify and examine T-cell-independent processes that are essential to control infection. Infection of the SCID mouse shows the immune system operating in its most primitive or elementary form. T h e Listeria model also allows us to probe the interdependence of both cellular systems. T h e macrophage lineage responds to Listeria by cytokines that influence the tissue response, the NK cell system and, at the same time, sets the system for the T cell response not only by presenting peptides but by the display of helper molecules, of which IL-12 is a major one.

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2.

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