Macrophages, NK cells and neutrophils in the cytokine loop of Listeria resistance

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Malo, D., Vogan, K., Vidal, S. et al. (1994), Haplotype mappingand sequenceanalysisof the mouseNramp genepredictssusceptibilityto infection with intracellular parasites.Genomics,23, 51-61. Medina E. & North, R.J. (1996), Evidence inconsistent with a role for the Beg genein resistanceto infection with virulent M. tuberculosis. .I. Exp. Med., 183, 1045-1051. Medina, E., Rogerson,B.J. & North, R.J. ( 1996). The Nrampl antimicrobial resistancegene segregates independentlyof resistanceto virulent M. tuberculosis. Immunology, 88, 479-481. Mock, B.A., Holiday, D.L., Cerretti, D.P. et al. (1994), Construction of a series of congenic mice with recombinantchromosome1 regionssurroundingthe genetic loci for resistanceto intracellular parasites (Ity, Lsh, Beg), DNA repair responses (Rep-l), and cytoskeletal protein villin (Vil). Infect. Immun., 62, 325328. Nikonenko,A.S. eral. (1996), Influenceof the mouseBeg, Tbc-I and xid genes on resistance and immune

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North, R.J. (1995). Mycobacterium tuberculosis is strikingly morevirulent for mice when given via the respiratory than by the intravenousroute.J. infect. Dis., 173, 1550-1553. Plant, J. & Glynn, A.A. (1979), Locating salmonellaresistance gene on mouse chromosome 1. Clin. Exp. Immunol., 37, 1-6.

Skamene,E., Gros, P., Gorget, A. et al. (1982), Genetic regulation of resistanceto intracellular pathogens. Nature (Lond.), 297, 506-509. Vidal, S.M., Malo, D., Vogan, K. et al. (1993), Natural resistanceto infection with intracellular parasites: identification of a candidategenefor Beg. Cell, 73, 469-485. Vidal, S., Tremblay, M.L., Govoni, G. et al. (1995), The /ty/LWBcg locus:naturalresistance to infection with intracellular parasitesis obrogatedby disruption of the Nrampl gene.J. Exp. Med., 182,655-666.

NK cells and neutrophils in the cytokine loop of Listeriu resistance E.R. Unanue

Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110 (USA)

This paper represents a brief comment on our research on the cellular events in Listeria infection. Our present interest is cent& on the early stageof the infection where the T-independent or cellular innate systemsplays a major role in resistance.Our coverage of referencesis not comprehensive.Our recent reviews have included a detailed analysis of them (Rogers et al, 1995; Tripp and Unanue, 1995; Unanue, 1996). Initially, in studying how the antigen-presenting function of macrophages was regulated, we were impressedby the very rapid and dramatic activation of macrophages during Listeria infection in the mouse (Belier et al., 1980). By 3 days after infection, the phenotype of tissue macrophages rapidly

ReceivedDecember13, 1996.

changed. These changes were particularly evident in the peritoneal macrophages, where its expression of class II MHC molecules shifted from a baseline of about lo-20% to close to 100% of positive macrophages. These macrophages were excellent presenting cells. The increase in class II-bearing macrophages was mostly accounted for by fresh new cells derived from blood (Scher et al., 1982). These early studies eventually identified interferon-y (IFNy) as the key cytokine responsible for this parameter of macrophage activation (for example, Sztein et al., 1984, reviewed in Unanue 1984), and also pointed to the T cell as a major producer of IFNy. Indeed, in transfer experiments, T cells from mice primed to

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conventional non-microbial protein antigens resulted in exudates rich in class II-positive macrophages (Beller ef al., 1980). Consequently, we were greatly surprised upon examining SCID mice infected with Listeria ; this strain of mice had been identified by the laboratory of Mel Bosma as being defective in the production of lymphocytes (Bosma et al., 1983). SCID mice developed a carrier state for Listeria infection, maintaining infective organisms in spleen and liver for many weeks (Bancroft et al., 1986). An occasional mouse would succumb, with time, to the infection. The macrophages from the infected SCID mice were highly activated, expressing high levels of class II MHC molecules and producing nitric oxide as early as 3-4 days after infection (Beckerman et al., 1993). Clearly, such mice had a non-T-cell-dependent process that led to macrophage activation and the partial restriction of the infection (Rogers et al., 1995). A similar process of T-cell-independent resistance was noted in other infections with parameters very similar to listeriosis in SCID mice (for examples, see Gazinelli et al., 1993 ; Hunter et al., 1994 ; Orange et al., 1995 ; Anguita et al., 1996; Derrico and Goodrum, 1996; reviewed in Unanue, 1996). Three sets of cells and several molecules were involved in the early process of T-independent or “innate” or “natural” immunity that activated the macrophages and allowed the SCID mouse to show partial protection. The cells included the mononuclear phagocytes, the NK cells and the neutrophils. Central to the whole process was the macrophage. The macrophage was the main cell that took up Listeria and released the major early cytokines that set up the cascade of inflammatory events. Macrophages released IL12, TNF, IL1 and IL6 ; these cells also released the negative regulatory cytokines, like IL10 and TGFP. To note is that macrophages also released eicosanoids. All these, except for TGFP, have been shown to be involved in Listeriu resistance (reviewed in Unanue, 1996). All together, the role of the various cytokines is to activate and/or promote the recruitment of leukocytes and to prime the tissue for inflammation. One lesson from the study of listeriosis is that each cytokine has primary modes of action where its functional relevance becomes prominent. Concerning the other two major cells, NK cells were involved in the production of IFNy, upon reaction with macrophage-derived IL12 and TNF (Bancroft et al., 1989). Neutrophils were essential in the early stages of resistance, particularly in the liver phase of the infection (see below). The role of IFNy The main cytokine that controls IFNy, which in the SCID mouse,

the process is is exclusively

IN IMMUNOLOGY secreted by NK cells. In the thymic-sufficient mouse, it is secreted by CD4 and CD8 T cells. The initial studies from Robert Schreiber’s laboratory were to the point: resistance to infection with Listeriu by normal mice was impaired by administration of a powerful neutralizing antibody to IFNy (Buchmeir and Schreiber, 1985). We obtained similar results in the SCID mice using the same H22 antibody. These studies on the role of IFNy were amply confirmed in mice lacking the genes for IFNy or for the IFNy receptor (Huang et al., 1993 ; Dalton et al., 1993). Thus, the essential control mechanisms that produced resistance and maintained the carrier state depended on IFNy and the IFNy receptor. In the SCID mouse, we carried out parallel studies in culture and in vivo, in the latter by mainly using the neutralizing monoclonal antibodies. A conditioned medium from macrophages exposed to dead or live Listeria resulted in production of IFNy by NK cells (Bancroft et al., 1986). To note is that in vivo reduction of NK cell numbers by administration of antibodies to asialo-GM1 resulted in uncontrolled infection (Bancroft et al., 1989). The macrophage-conditioned media contained two cytokines that were required for the response of the NK cells. Initially, we showed that anti-TNF antibodies stopped the response (Wherry et al., 1991). However, purified recombinant TNFa, by itself, was not effective in driving IFNy production by NK cells. We then showed cooperativity between TNFcx and a secreted Listeria product; this result initially made us think that microbial products were the relevant molecules that synergized with TNF to activate the NK cells. However, the recognition of IL12 by Trinchieri (reviewed in Trinchieri and Gerosa, 1996) made us evaluate this new cytokine. Clearly, Catie Tripp demonstrated that IL12 was the missing second component in our reaction (Tripp et al., 1993). In our experience, no other cytokine has synergized to activate the NK cells. Contrary to the reports of others (Hunter et al., 1995), the IL1 family of proteins was ineffective. Our view is that IL1 has a role in listeriosis, but not by way of the NK cells + IFNy axis. In agreement with the culture studies, neutralizing both IL12 and TNF with monoclonal antibodies had a profound negative effect on the infection. The effect was shown both in SCID and in lymphocytesufficient mice (Tripp et al., 1994). The effects of anti-IL12 antibodies correlated with inhibition of macrophage expression of class II MHC molecules. Interestingly, these changes were corrected by the administration of recombinant IFNy: class II expression in macrophages returned and so did resistance to listeriosis, and the development of sterilizing immunity in normal mice, or the carrier state in SCID mice.

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The administration of anti-TNF antibodies likewise resulted in the same results as giving anti-1112 antibodies, but in contrast, the deleterious effects were not abolished by IFNy (Tripp ef al., 1994). While IFNy did correct some of the deficit in macrophage activation, like class II expression, it failed to control the infection. Our conclusion was that TNF exercised a number of effects not mediated by IFNy. This could include activation of the vascular bed, changes in blood flow, activation of other leukocytes, etc. We found that the effects of IFNy in activating the macrophages were to a great extent attributed to the production of NO’ (Beckerman et al., 1993). Karen Beckerman led our group to show that the Listeria-activated macrophages produced NO’ and that neutralization of NO’ either in vivo or in culture abrogated the listeticidal effect. These results were confirmed by other investigators when using NO null mice (MacMicking et af., 1995). Recent studies have further examined the biology of IFNy. The laboratory of Robert Schreiber, with whom we have had long-standing collaboration, went on to show that the early resident macrophages were crucial in the response to IFNy (Dighe et al., 1995). They did their studies by engineering a dominant-negative construct of the a chain of the JFNy receptor expressed on the lysozyme promoter. The mutant gene was expressed on resident macrophages and not in newly arrived monocytes. Such mice were susceptible to listeriosis. We suggest that the tissue macrophages initiates the process of resistance, after uptake of Listeria, by releasing IL12 and TNF, perhaps to a limiting degree ; rapidly, however, IFNy provides a positive feedback loop on the macrophages to produce more cytokines that then activates the system to full operation. Indeed, although Listeria or other microbes, or their products, trigger IL12 from macrophages, the release can be small, but it is markedly enhanced if the macrophage is first primed with IFNy (FIesch et al., 1995; Hayes et al., 1995; Ma et al., 1996). Finally, it is of note that mice lacking the STAT-l gene, by homologous recombination, do not respond to IFN signalling and are highly susceptible to Listeria infection (Meraz et al., 1996). Recently, we explored whether IFNy had a different effect in mice already immune to Listeria (Tripp et al., 1995b). Using the H22 neutralizing antibody, Listeria-primed mice were unable to mount an effective response upon a secondary challenge with a large dose of microbes. The dose of anti-IFNy antibodies required to see an effect on the infection was about 3 times higher than in the normal or SCID mice. This was an indication that the levels of IFNy were higher, an issue that can explain the failure of some to note an effect (Samson et al., 1995). In the

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same experiments, we found that monoclonal antibodies to IL12 had a lesser effect, implying that 1FN-y release is less dependent on it. We are aware of the recent studies from Harty and Bevan which indicate that some of the protective effects of CD8 T cells may not require IFNy (Harty and Bevan, 1995). Thus, there may be other, perhaps less dominant pathways, not using IL12 and/or IFNy for macrophage activation. In essence, certain features of the early protective pathway appear to stand out: the importance of an early release of IL12/TNF by the macrophage, the presence of responding cells that produce IFNy, the target of IFNy on the inflammatory response, particularly on the macrophage.

The role of other cytokines:

IL1

We found that neutralization of both ILla and IL1 p impaired resistanceto listeriosis (Rogers et al., 1992, 1994). Both cytokines had to be neutralized with specific monoclonal antibodies. In our studies, we also included antibodies to the type I IL1 receptor. Howard Rogers showed that such treated mice showed impairment of early neutrophil accumulation in the peritoneal cavity, the site of infection. The treated mice were also considerably impaired in macrophage activation. The number of class II-positive macrophages was low. The mice, however, expressed IFNy which could easily be titrated in blood. Moreover, culture of the macrophages of anti-IL1 treated mice with IFNy did not result in the expression of class II MHC molecules. In essence, IL1 is involved in the early recruitment of blood neutrophils to the site of infection. The molecular basis of this recruitment has not been identified. There was no apparent role for IL1 when cultures of peritoneal macrophages from normal mice were studied for their response to IFNy. Indeed, addition of anti IL1 antibodies did not impair the response. Thus, the effect of anti IL1 in inhibiting the macrophage response took place in viva We believe that IL1 may be involved in the early maturation of the monocyte to an IFNy responsive state. Finally, to prove that at least some of the deleterious effects of IL1 were caused by its effects on neutrophils, we had to prove that these cells were an active participant. This was the case, as discussednext.

The role of neutrophils The involvement of neutrophils in listeriosis was suggested by several results. A correlation was found between neutrophilia in infected foci and resistance to infection among strains of mice (Ste-

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venson et al., 1981; Czuprynski et al., 1985). Pixie Campbell’s laboratory called attention to a neutrophil listericidal effect in culture experiments (Czuprynski et al., 1984; Alford et al, 1990). And Conlan and North made a detailed analysis of neutrophil-rich infective foci in the liver during the first few days after infection (Conlan et al., 1991, 1993). As mentioned, in our laboratory, Howard Rogers became interested in the early neutrophilia when he found an influx of peritoneal neutrophils a few hours after infection. Such an influx was markedly impaired in anti-ILl-treated mice (Rogers et al., 1992, 1994). In a different experimental approach, mice were depleted of neutrophils by the use of monoclonal antibodies and then injected with Listeria. These mice rapidly succumbed to infection (Rogers and Unanue, 1993). Results similar to his were obtained by others using similar experimental approaches (for examples Conlan and North, 1994; Czuprynski et al., 1994a, b; reviewed in Unanue, 1996). Overall, the experiments indicated that neutrophils curbed the early spread of Listeria from infected foci. In the case of the liver infection, the neutrophil reduced the dimensions of the infected foci. In their absence, the infection spread and the mouse died of acute liver injury (Rogers et al., 1996). The liver infection with Listeria is of great interest. A scenario sees the early uptake of blood-borne Listeria by the Kupffer cells, followed by an initial reduction in viable Listeria; and then the transfer of the microbe into the hepatocyte. In the hepatocyte, the Listeria infection causes its death by apoptosis, at about the same time that neutrophils accumulate (Rogers et al., 1996). We examined the apoptosis of infected hepatocytes both in vivo and in culture. In vivo, the lesions containing the microabscess were examined for apoptotic nuclei using TUNEL (for terminal deoxynucleotide transferase-mediated dUTP-biotin-dependent nick-end Iabelling). The centre of the abscess was rich in neutrophils and in dead cells and it was not possible to identify the nature of the TUNEL+ cells. However, at the edges of the lesion TUNEL+ hepatocytes were clearly identified, many of which contained Listeria. Depleting the mice of circulating neutrophils showed the lesions made up of TUNELpositive, heavily infected hepatocytes. Culture of primary hepatocytes infected with Listeria confirmed the in vivo findings and added new information. Listeria grew uncontrolled in the hepatocytes which underwent death by apoptosis. Culture of the cells with cytokines did not inhibit growth: nor did the addition of neutrophils. Infected hepatocytes, however, released chemotactic factors for neutrophils which were also more adhesive to them. In unpublished experiments using mice lacking the ICAMgene, we find no impairment of the

IN IMMUNOLOGY early liver-phase of Listeria resistance. (These mice were produced by Dr. J.C. Gutierrez-Ramos at the Center for Blood Research in Boston). Thus, the scenario that our studies support is that the infected hepatocyte cannot defend itself unless it does so by calling forth acute inflammation. Although there are claims that cytokine-treated hepatocytes curbed Listeria growth, we were unable to find such results. Our results suggest that apoptosis is part of the program of resistance of the liver cells. Others have found that apoptotic hepatocytes develop high levels of transglutaminase (Tarcsa et al., 1987). This activity may extensively cross-link cytosolic proteins and inhibit the dissemination of Listeria.

The negative

cytokines:

IL10

IL10 was produced when macrophages were infected with Listeria (Tripp et al., 1993). The conditioned media from Listeria-infected macrophages were rich in all four cytokines (i.e., ILl, ILlO, IL12 and TNF). Thus, the ratio of IL12 to IL10 becomes critical because it establishes whether macrophage activation predominates. Indeed, addition of antibodies to IL10 in culture markedly increased the response of NK cells. In our studies, we could identify the fact that IL10 reduced the production of IL12 by macrophages, but it also had an effect on the production of IFNy by the NK cells. Our most seminal results on the effects of IL10 in vivo were found in mice having circulating immune complexes. Indeed, mice that formed antigen-antibody complexes at the early stage of Listeria infection rapidly succumbed. This was a dramatic effect extensively studied by Skip Virgin in our laboratory (Virgin et al., 1985a, b, c). We infected mice primed to ovalbumin, with Listeria, at the same time challenging them with the soluble protein. Within a few days, the mice died of uncontrolled infection. Needless to say, mice infected without injection of ovalbumin resisted the process. The heightened susceptibility was transferred to naive mice with anti-ovalbumin antibodies, and not with T cells. Although the Virgin effects could be ascribed to several factors, one component is the production of ILlO. Indeed, addition of Listeria and ovalbumin anti-ovalbumin complexes to cultures of macrophages increased the production of IL10 (Tripp et al., 1995a). Along these lines, transfer of immune complexes to SCID mice resulted in the increased susceptibility to infection, but this was not found if anti-IL10 antibodies were also administered (Tripp et al., 1995a). These experiments are important in that immune complex formation is a frequent occurrence in chronic infectious processes. The immune complex,

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by triggering more ILlO, could contribute to downregulation of macrophage activation.

What have we learned by studying the T-independent activation induced by Lhteria ? Several lessons have come out of these studies. First, the resistance to an infectious process is made up of multiple stages or phases, all of which are coordinated in generating protective immunity and elimination of the microbe. The way in which each phaseis displayed may depend very much on the tissue site of infection. Second, as we mentioned before, each phase is influenced by a predominant cell and cytokines. Lastly, the immune system can deal with a microbe in the absence of a specific adaptive lymphocyte response. Several elements can be identified at each stage of the response, and these are worth a brief comment. First, the macrophage is a central cell that can be triggered to respond early in the infection. The production of cytokines by the macrophage sets the system to move forward. Also, in responseto Listeriu, the macrophagesexpress high levels of adhesive and costimulatory molecules. These favour an optimal antigen-presenting function. Second, effector cells vary in importance depending on the stage and tissue involved. The study of the neutrophil in protecting the liver infection is a clear demonstration of this. Finally, a point to addressis that of cell death as a component of the infection. We first became aware of this when studying the release of ILlj3 by macrophages (Hogquist et al., 1991). Macrophages that underwent apoptosisactivated the IL1 p converting enzyme that led to the release of biologically active ILl. Thus, cell death was a means of calling forth inflammation. This is a point that was clearly demonstrated in the early infection of the hepatocyte described above. It is interesting to note that the liver infection with Listeria takes place not only in the primary infection, but also in the previously immunized mouse having a protective memory response.In these cases,despite strong T-cell immunity, neutrophils are still required (Czuprynski et al., 1994b; Appelberg et al., 1994; Rakhmilevich, 1995). Listeriosis in SCID mice allowed us to examine functions of resistance that can develop independently of the lymphocyte. Clearly, the macrophage system rapidly primes the system through cytokines and the recruitment of other leukocytes, and now poises the cellular system to influence the lymphocyte response. Listeriosis triggers primarily a Thl response (Rogers et al., 1992), an issue that was extensively developed by Ken Murphy and Anne

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O’Garra when they and their associates examined the differentiation of T cells into Thl and Th2 subsets(Hsieh et al., 1993). Clearly, addition of Listeria organisms to the culture skewed the response to a ThI phenotype and led to their identification of IL12 asone key regulatory cytokine. In summary, in listeriosis, there is a blending of the early T-cell-independent phase with the lymphocyte response that eventually results in sterilizing immunity. Future studies should address the unique properties of T cells that are responsible for elimination of Listeria organisms from infective foci.

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Innate versus acquired immunity

in listeriosis

F. Brombacher (‘I (*I and M. Kopf (2) (‘)&lax-Planck-Institute for Immunobiology, Stiibeweg 51, 79108 Freiburg (Germany), and c2)BaselInstitute for Immunology, Base1(Switzerland)

Introduction Listeria monocytogenes are Gram-positive bacteria which induce their own internalization into phagocytes and non-phagocytic cells, where they eventually survive and replicate in the cytoplasm. Infection is characterized by an immediate innate immune responseof the host, where phagocytes kill Received December13, 1996. (*) Corresponding author.

90% of the inoculum during the first hours and where a proinflammatory cytokine response is induced which determines the further host defence. Surviving Listeria infiltrate tissuesand rapidly replicate. The recruitment of activated macrophages and neutrophils will limit their growth, until Listeriaspecific T cells arrive at day 4 to 5, which leads to granulomatous formation and consequent eliminaabout