Independent Protective Effects for Tumor Necrosis Factor and Lymphotoxin Alpha in the Host Response to Listeria monocytogenes Infection

Although the essential role of tumor necrosis factor (TNF) inresistance to Listeria monocytogenes infection is well established,the roles of the related cytokines lymphotoxin alpha (LT ${alpha}$ ) andlymphotoxin beta (LTß) are unknown. Using C57BL/6mice in which the genes for these cytokines were disrupted,we examined the contributions of TNF, LT ${alpha}$ , and LTßin the host response to Listeria. To overcome the lack of peripherallymph nodes in LT ${alpha}$ ^–/– and LTß^–/–mice, bone marrow chimeras were constructed. TNF^–/–and LT ${alpha}$ ^–/– chimeras that lacked both secreted LT ${alpha}$ ₃and membrane-bound LT ${alpha}$ ₁ß₂ and LT ${alpha}$ ₂ß₁ werehighly susceptible and succumbed 4.5 and 6 days, respectively,after a low-dose infection (200 CFU). LTß^–/–chimeras, which lacked only membrane-bound LT, controlled theinfection in a manner comparable to wild-type (WT) chimeras.The Listeria-specific proliferative and gamma interferon T-cellresponses were equivalent in all five groups of infected mice(LT ${alpha}$ ^–/– and LTß^–/– chimeras,WT chimeras, and TNF^–/– and WT mice). TNF^–/–mice and LT ${alpha}$ ^–/– chimeras, however, failed to generatethe discrete foci of lymphocytes and macrophages that are essentialfor bacterial elimination. Rather, aberrant necrotic lesionscomprised predominantly of neutrophils with relatively few lymphocytesand macrophages were observed in the livers and spleens of TNF^–/–and LT ${alpha}$ ^–/– chimeras. Therefore, in addition to TNF,soluble LT ${alpha}$ ₃ plays a separate essential role in control of listerialinfection through control of leukocyte accumulation and organizationin infected organs.

INTRODUCTION

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Introduction

Tumor necrosis factor (TNF) and lymphotoxin alpha (LT ${alpha}$ ) are twostructurally related cytokines that mediate proinflammatoryactivities. The functions of TNF have been extensively characterized,and this factor is required for host control of both mycobacteriaand other intracellular pathogens, including Listeria monocytogenes(2, 12, 29). The activities of LT ${alpha}$ are less well understood,but recently an essential role for LT ${alpha}$ in the host response topulmonary tuberculosis has been demonstrated (28).

L. monocytogenes is a gram-positive facultative intracellularbacterium that infects macrophages, as well as epithelial cellsand hepatocytes. Although initially contained in the phagosome,Listeria is able to penetrate the vacuolar membrane and enterthe host cell cytosol through the action of the pore-formingtoxin listeriolysin O (LLO) (24). As a result, L. monocytogenesis a potent inducer of CD8 T-cell responses (3, 5). Listeriainfection also induces strong activation of antigen-specificCD4⁺ T cells. Although the role of CD4 T cells remains lesswell defined, it has recently been demonstrated that CD4 T-cellhelp is required for generation of functional CD8 T-cell memory(31, 32). While the effector mechanisms used by each cell typehave not been elucidated fully, studies in which CD8⁺ gammainterferon (IFN- ${gamma}$ )-deficient T cells were transferred indicatedthat IFN- ${gamma}$ is not required to effect the protection providedby these cells (14). Further studies revealed that transferof wild-type (WT) and TNF-deficient CD8⁺ T cells mediated antilisterialimmunity in wild-type but not TNF-deficient host mice, demonstratingthat TNF plays an essential role in the expression of antilisterialresistance (14, 34, 35). The function of lymphotoxin in protectiveimmunity to Listeria has not been reported.

In this study we examined the roles of TNF, LT ${alpha}$ , and lymphotoxinbeta (LTß) in the host response to L. monocytogenesinfection by using mice with targeted disruption of the LT ${alpha}$ ,LTß, and TNF genes. Gene knockout mice were generateddirectly in C57BL/6 animals (18), and LT ${alpha}$ ^–/–, LTß^–/–,and WT donors were used to generate bone marrow radiation chimerasin syngeneic RAG^–/– recipients. This strategy overcameproblems related to the lack of peripheral lymphoid tissue inmice with an LT ${alpha}$ or LTß gene disruption (18, 23). TNF^–/–and LT ${alpha}$ ^–/– chimeric mice exhibited severe susceptibilityto low-dose intravenous infection with L. monocytogenes, despiteevidence of T-cell activation. LTß^–/–and WT chimeric mice both controlled and resolved the infectionnormally, indicating that LT ${alpha}$ , but not LTß, is essentialin the host response to L. monocytogenes.

MATERIALS AND METHODS

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Materials and Methods

Mice. WT C57BL/6 and C57BL/6.RAG-1^–/– mice (RAG is therecombinase activation gene) were obtained from the Animal ResourcesCentre (Perth, Australia). TNF, LT ${alpha}$ , and LTß gene knockoutmice were generated in the C57BL/6 genetic background as previouslydescribed (22, 26). Adult (>6-week-old) mice were used inall experiments. The mice were housed under specific-pathogen-freeconditions at the Centenary Institute animal facility untilinfection, when they were transferred and maintained in a level2 physical containment facility. Experiments were conductedby using protocols approved by the University of Sydney AnimalCare and Ethics Committee.

Generation of radiation bone marrow chimeras. Bone marrow cells were harvested from the long bones of donormice by flushing with phosphate-buffered saline, washed, andcounted. Recipient RAG^–/– mice were preconditionedwith 5.5 Gy gamma radiation on day –2 and again on day0. Bone marrow cells (1 x 10⁷ cells/recipient) from WT, LT ${alpha}$ ^–/–,and LTß^–/– mice were injected intravenouslyon day 0. The cage water was supplemented with trimethoprim(50 µg/ml) and sulfamethoxazole (0.25 mg/ml) (DBL, SydneyAustralia) from weeks 2 to 4 after transplantation. Chimeraswere used at least 3 months after preparation, when completecellular reconstitution had occurred. Cellular reconstitutionwas monitored by analysis of peripheral blood by flow cytometry.

Bacteria and experimental infections. L. monocytogenes was grown in tryptic soy broth (Difco, Detroit,Mich.) from a sample kindly provided by C. Cheers (Universityof Melbourne, Melbourne, Australia). The culture was initiatedfrom a single colony and cultured for 16 h at 37°C. Bacterialstocks were frozen in 30% (vol/vol) glycerol and stored at –70°C.LT ${alpha}$ , LTß, and WT chimeric mice, as well as TNF^–/–and WT mice, were infected with 200 CFU of L. monocytogenesintravenously via a lateral tail vein. The numbers of viablebacteria in target organs were monitored over time by platingserial dilutions of whole-organ homogenates on supplementedtryptic soy agar (Difco) and counting the bacterial coloniesthat had formed after 24 h of culture. Heat-killed L. monocytogenes(HKL) was prepared by incubating bacteria at 80°C for 1h.

Cytokine production from purified T cells. Single-cell suspensions from the spleens of L. monocytogenes-infectedmice 7 days after infection were stained with either rat anti-CD4(clone GK1.5) or anti-CD8 (clone 53.6-7) monoclonal antibodyfor 30 min at 4°C. Cells were then washed in 2% (vol/vol)fetal calf serum in phosphate-buffered saline and incubatedwith anti-rat immunoglobulin Dynabeads (Dynal, Australia) for30 min at 4°C. CD4⁺ and CD8⁺ T cells were then isolatedby two rounds of magnetic separation. The purities of the cellpopulations were confirmed by fluorescence-activated cell sortingstaining and analysis of an aliquot of cells from each isolation.Purities of more than 95% were obtained following separation(data not shown). For analysis of expression of TNF and LT ${alpha}$ mRNAtranscripts, total RNA was prepared from cell homogenates usingRNAzol B (Tel-Test, Friendswood, Tex.). cDNA was synthesizedusing SUPERSCRIPT II RNase H reverse transcriptase (GIBCO BRL)and was analyzed by reverse transcription-PCR using primersand methods described previously (26).

T-cell responses to Listeria antigens. Spleens were removed, and single-cell suspensions were prepared.Erythrocytes were lysed in a hypotonic ammonium chloride lysisbuffer, and the remaining cells were washed, counted, and suspendedin complete RPMI medium (RPMI 1640 [Cytosystem, Sydney, Australia]with 10% fetal bovine serum [Trace, Sydney, Australia], 2 mML-glutamine [Sigma], 10 mM HEPES [Sigma], 10 mM Na₂CO₃, 0.5µM 2-mercaptoethanol [Sigma], 100 U/ml penicillin [Trace],and 100 µg/ml streptomycin [CSL, Melbourne, Australia]).To measure antigen-specific T-cell responses, splenocytes werecultured with HKL (2 x10⁷ CFU/ml) or medium (control) for 72h. To assess proliferative responses, cells were pulsed with1 µCi of [³H]thymidine (NEN Life Sciences, Boston, Mass.)for the final 6 h of culture and then harvested onto glass fiberfilters. The amount of incorporated [³H]thymidine was determinedby liquid scintillation spectroscopy (Pharmacia/Wallace Oy,Turku, Finland). Specific [³H]thymidine incorporation was calculatedby subtracting the mean counts per minute in unstimulated wellsfrom the mean counts per minute of test samples.

The concentration of IFN- ${gamma}$ in culture supernatants was determinedusing a capture enzyme-linked immunosorbent assay and a monoclonalantibody capture assay with the antibodies R4-6A2 and XMG1.2-biotin(Endogen, Woburn, Mass.) as previously described (17). No significantproliferation or IFN- ${gamma}$ release was detected in splenocytes fromuninfected chimeric and unmanipulated mice. To determine thefrequency of IFN- ${gamma}$ -producing cells, splenocytes were culturedfor 16 h in Multiscreen 96-well filtration plates (Millipore,Bedford, Mass.) precoated with anti-IFN- ${gamma}$ antibody R4-6A2 inthe presence of HKL, Listeria lysin O peptide_296-304 (LLO_296-304)(13), or medium alone. The enzyme-linked immunospot assay preparationswere then developed with anti-IFN- ${gamma}$ antibody XMG1.2-biotin andavidin-alkaline phosphatase as previously described (17).

Histology. Liver tissue samples were fixed in 10% neutral buffered formalin,processed in paraffin blocks, and sectioned (thickness, 5 µm).The sections were stained with hematoxylin and eosin and examinedto assess the histopathological responses of infiltrating leukocytesin the livers of L. monocytogenes-infected mice.

Statistical analysis. Statistical analyses of the results of immunological assaysand log-transformed bacterial counts were conducted using analysisof variance (ANOVA). Fisher's protected least-significant differenceANOVA post hoc test was used for pairwise comparisons of multigroupdata sets. P values of <0.05 were considered significant.Survival was calculated on a Kaplan-Meier nonparametric survivalplot, and significance was assessed by the Mantel-Cox log ranktest.

RESULTS

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Results

CD8⁺ and CD4⁺ T cells express LT ${alpha}$ and TNF following infection with L. monocytogenes. The expression of transcripts for LT ${alpha}$ and/or TNF was examinedin CD4⁺ and CD8⁺ T-cell populations to determine if both CD4⁺and CD8⁺ T cells produce LT ${alpha}$ and/or TNF during infection withL. monocytogenes. At 7 days postinfection CD4⁺ and CD8⁺ cellswere isolated separately from the spleen using antibody-coatedmagnetic beads, total RNA was extracted, and cDNA was reversetranscribed. Analysis of cDNA by reverse transcription-PCR indicatedthat following infection both CD4⁺ and CD8⁺ T cells up-regulatedthe expression of LT ${alpha}$ and TNF (Fig. 1).

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FIG. 1. Expression of LT ${alpha}$ and TNF is up-regulated in both CD4⁺ and CD8⁺ T cells during infection with L. monocytogenes. WT mice were infected with 200 CFU of L. monocytogenes intravenously, and at 7 days postinfection highly purified (>95%) CD4⁺ and CD8⁺ T cells were isolated from pooled spleen samples. RNA was extracted and cDNA was synthesized as outlined in Materials and Methods. The expression of TNF and LT ${alpha}$ was examined by reverse transcription-PCR. Lane 1, naive CD4⁺ spleen cells; lane 2, naive CD8⁺ spleen cells; lane 3, CD4⁺ spleen cells; lane 4, CD8⁺ spleen cells; lane 5, total spleen cells from infected mouse.

TNF^–/– and LT ${alpha}$ ^–/– chimeric mice, but not LTß^–/– chimeric mice, succumb to low-dose L. monocytogenes infection. To study functions of LT ${alpha}$ and LTß independent of secondarylymphoid organogenesis, LT ${alpha}$ ^–/–, LTß^–/–,and WT bone marrow chimeras were generated in irradiated RAG^–/–recipients. Full reconstitution of peripheral lymphoid tissuewith normal lymphoid architecture was observed 3 months aftertransplantation, and the numbers of peripheral blood leukocytesand percentages of CD4⁺ and CD8⁺ T lymphocytes were comparable(26, 28). To assess the contribution of secreted LT ${alpha}$ and TNFin the host response to L. monocytogenes infection, LT ${alpha}$ ^–/–,LTß^–/–, and WT chimeric mice, as wellas TNF^–/– and normal WT mice, were infected with200 CFU of L. monocytogenes via a lateral tail vein. The survivalof the infected mice and the bacterial load were monitored for2 weeks. All of the normal WT mice and WT chimeric mice survivedand had similar bacterial loads in their spleens and liversfollowing infection with L. monocytogenes (data not shown).TNF^–/– mice were extremely susceptible to infectionand succumbed 4 to 5 days postinfection (Fig. 2). These micehad significantly increased bacterial loads in their spleens(Fig. 3A) and livers (Fig. 3B) as early as 3 days postinfectioncompared to WT chimeras (Fig. 3) and normal WT controls (datanot shown). LT ${alpha}$ ^–/– chimeric mice were highly susceptibleto Listeria infection and succumbed 5 to 6 days postinfection(Fig. 2). Although similar numbers of bacteria were recoveredfrom both the spleens (Fig. 3A) and livers (Fig. 3B) of LT ${alpha}$ ^–/–and WT chimeras at 3 days postinfection, by day 5 there weresignificantly increased bacterial loads in LT ${alpha}$ ^–/–chimeric mice. In contrast, the LTß^–/–chimeras controlled the infection in a manner comparable toWT chimeric mice (Fig. 3). Therefore, both TNF and secretedLT ${alpha}$ independently mediate essential actions in the host responseto L. monocytogenes infection.

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FIG. 2. Survival of TNF^–/– mice and LT ${alpha}$ ^–/–, LTß^–/–, or WT chimeric mice following infection with L. monocytogenes. WT chimeric ( ${square}$ ), LT ${alpha}$ chimeric (•), LTß chimeric ( ${circ}$ ), and TNF^–/– ( ${blacksquare}$ ) mice were infected intravenously with 200 CFU of L. monocytogenes. The infected mice were monitored daily and euthanized when they showed signs of ill health. The data are the times to euthanasia for eight mice/group and are representative of one of three separate experiments. The P value was <0.001 for TNF^–/– versus WT chimeric mice and for LT ${alpha}$ chimeric versus WT chimeric mice as determined by a Mantel-Cox log rank test.

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FIG. 3. TNF and LT ${alpha}$ ^–/– chimeric mice fail to control infection with L. monocytogenes. WT chimeric ( ${square}$ ), TNF^–/– ( ${blacksquare}$ ), LT ${alpha}$ chimeric (•), and LTß chimeric ( ${circ}$ ) mice were infected intravenously with 200 CFU of L. monocytogenes, and the bacterial loads in the spleens (A) and livers (B) were monitored over time. The data are the means and standard errors for four mice per group and are representative of one of three experiments. The significance of differences between WT chimeric and TNF^–/– mice, as well as between LT ${alpha}$ ^–/– and WT chimeric mice, was determined by ANOVA (one asterisk, P < 0.01; two asterisks, P < 0.001).

Antigen-specific T-cell responses following L. monocytogenes infection. The T-cell response to Listeria infection peaked between days7 and 10 in WT mice (data not shown), but an antigen-specificT-cell response could be detected after 5 days. The abilityof the TNF^–/– mice or LT ${alpha}$ ^–/– chimericmice to mount T-cell responses to L. monocytogenes was assessedto determine if the failure to control infection was due toa deficiency in the activation of T cells. Splenocytes fromTNF^–/– mice and LT ${alpha}$ ^–/–, LTß^–/–,and WT chimeras infected for 5 days all produced comparableamounts of IFN- ${gamma}$ following 3 days of ex vivo culture in the presenceof HKL (Fig. 4A). The proliferative potential of these cellswas also assessed at this time by determining the incorporationof [³H]thymidine during the final 6 h of culture. Splenocytesfrom all groups displayed similar antigen-specific proliferativeresponses (Fig. 4B). There were also comparable numbers of Listeria-specificsplenic IFN- ${gamma}$ -producing cells at 5 days postinfection in allgroups in response to HKL stimulation (Fig. 4C). Further analysisto determine if the susceptibility of the LT ${alpha}$ chimeric mice wasdue to a lack of a CD8 T-cell response was then undertaken.WT and LT ${alpha}$ chimeric mice displayed a similar number of T cellsproducing IFN- ${gamma}$ in response to the CD8-specific LLO_296-304 epitope(Table 1). Furthermore, the percentages of IFN- ${gamma}$ -producing Tcells detected by intracellular staining were also similar inboth CD4⁺ and CD8⁺ T cells from WT and LT ${alpha}$ chimeric mice.

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FIG. 4. TNF^–/– and LT ${alpha}$ ^–/– chimeric mice have the potential to mount normal T-cell responses. WT chimeric (open bars), LT ${alpha}$ chimeric (bars with diagonal lines), LTß chimeric (bars with patterned lines), and TNF^–/– (solid bars) mice were infected intravenously with 200 CFU of L. monocytogenes. At 5 days postinfection splenocytes were prepared and cultured in the presence of HKL or medium alone (media) for 3 days. Total IFN- ${gamma}$ release was measured by an enzyme-linked immunosorbent assay (A), and proliferation was determined by measuring the incorporation of [³H]thymidine during the final 6 h of culture (B). (C) Cells were cultured in the presence of HKL overnight, and the frequency of IFN- ${gamma}$ -producing cells was determined by the enzyme-linked immunospot assay. The data are the means ± standard errors of the means for four mice per group and are representative of one of two experiments.

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TABLE 1. Equivalent CD4⁺ and CD8⁺ T-cell responses in WT and LT ${alpha}$ ^–/– chimeric mice at 5 days postinfection^a

LT ${alpha}$ ^–/– chimeric mice do not form normal foci of leukocytes in response to infection with L. monocytogenes. To determine if the increased susceptibility of TNF^–/–and LT ${alpha}$ ^–/– chimeric mice to infection was associatedwith different patterns of cellular recruitment, liver histologywas assessed at days 3 and 5 postinfection. A few small discretefoci (10 or more cells) were seen in the WT and LTßchimeric mice on day 3, with even fewer foci evident in theLT ${alpha}$ chimeric mice (WT chimeric mice, 19 ± 3.4 foci/liversection; LTß chimeric mice, 14 ± 3.2 foci/liversection; LT ${alpha}$ chimeric mice, 6 ± 3.4 foci/liver section).At this time TNF^–/– mice already displayed extensiveinflammation and necrosis in the liver. By day 5, compact, discretecollections of macrophages and lymphocytes were seen in WT chimeras(Fig. 5A) and LTß^–/– chimeras (Fig. 5B),and these collections were similar to those observed in normalWT mice (data not shown). In contrast, the foci that formedin LT ${alpha}$ ^–/– chimeric mice (Fig. 5C) and in TNF^–/–mice (Fig. 5D) were grossly abnormal. These aberrant foci containednumerous neutrophils and relatively few lymphocytes and macrophages,and extensive areas of necrosis were observed in all lesions.

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FIG. 5. Histopathological response to L. monocytogenes infection is abnormal in the livers of TNF^–/– and LT ${alpha}$ ^–/– chimeric mice. Livers from infected WT, LT ${alpha}$ ^–/–, and LTß^–/– chimeric mice and TNF^–/– mice were removed at day 5 postinfection and processed for histology. WT (A) and LTß^–/– (B) chimeric mice showed infiltrating macrophages and lymphocytes in small, compact, discrete collections of cells, whereas LT ${alpha}$ ^–/– chimeras (C) and TNF^–/– (D) mice exhibited large necrotic lesions with numerous neutrophils. Original magnification, x100. The sections show typical lesions from one of four livers per group from one of two representative experiments.

DISCUSSION

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Discussion

This study defined independent, essential, functions for TNFand LT ${alpha}$ in the murine response to the intracellular pathogenL. monocytogenes. The requirement for TNF was confirmed hereby progressive infection and profound susceptibility to L. monocytogenesin TNF^–/– mice. The median survival time after infectionwas only 4.5 days, and a significant increase in the bacterialload in the spleen and liver was evident by 3 days postinfection.This most likely reflects the role of TNF in facilitating earlyrecruitment of neutrophils, which are crucial for the containmentof early Listeria infection (6, 9). This is in addition to therole of TNF during the adaptive phase of the response, in whichTNF is essential for the recruitment of T cells and macrophagesinto lesions to effect bacterial clearance (2). These data areconsistent with the profound susceptibility of mice treatedwith anti-TNF antisera and TNF receptor I-deficient mice toL. monocytogenes (23, 29) and with the observation that therapywith recombinant TNF protected mice from a lethal L. monocytogeneschallenge (15).

The role of other TNF superfamily members in antilisterial immunityhas been less clear. This study revealed a separate and independentrole for the soluble form of LT, the homotrimer LT ${alpha}$ ₃, but notfor the membrane-bound heterotrimeric forms, LTß2 ${alpha}$ ₁and LTß₁ ${alpha}$ ₂, in host control of L. monocytogenes infections.The membrane-bound heterotrimers signal through the unique LTßreceptor, and the signal is essential for formation of peripherallymphoid tissue during embryogenesis (25). Use of LT ${alpha}$ ^–/–and LTß^–/– chimeras overcomes the problemof the lack of secondary lymphoid organs in LTß^–/–-deficientmice (26, 28), which limits the development of normal T-cellresponses.

LTß^–/– chimeras lack membrane-bound LTbut are sufficient in the LT ${alpha}$ secreted homotrimer LT ${alpha}$ ₃, whereasLT ${alpha}$ ^–/– chimeras are deficient in both soluble andmembrane-bound LT. The LT ${alpha}$ ^–/– chimeras, but not theLTß^–/– chimeras, were highly susceptibleand succumbed 6 days after low-dose infection. Similar susceptibilitywas observed in LT ${alpha}$ -deficient chimeras and TNF^–/–mice exposed to Mycobacterium tuberculosis by the aerosol route(2, 28). The LT ${alpha}$ ₃ defect was not due to an indirect effect ofa failure to produce TNF, as macrophages from LT ${alpha}$ -deficient mice(and LTß-deficient mice) exhibited normal TNF secretionin response to lipopolysaccharide and IFN- ${gamma}$ stimulation (28).LT ${alpha}$ was also not required to control early bacterial replication.Unlike TNF^–/– mice, LT ${alpha}$ ^–/– chimeras containedbacillary growth as effectively as WT chimeric mice for thefirst 72 h. However, LT ${alpha}$ was required for effective containmentand elimination of Listeria, as after 5 days of infection boththe spleens and livers of the LT ${alpha}$ ^–/– chimeric micedisplayed increased bacterial loads. This was not due to aninability to develop an antigen-specific T-cell response asboth LT ${alpha}$ ^–/– chimeras and TNF^–/– micedeveloped normal Th-1-like T-cell responses.

The significant, overriding defect observed in both LT ${alpha}$ ^–/–chimeras and TNF^–/– mice was a profoundly inadequateinflammatory response. After 72 h of infection the number ofinflammatory foci identified in the LT ${alpha}$ ^–/– chimericmice was lower than the number seen in both the WT and LTßchimeric mice. These differences were most apparent by 5 dayspostinfection, when, rather than responding to L. monocytogenesby formation of tight foci of T cells and macrophages, as seenin the WT and LTß^–/–chimeras, the LT ${alpha}$ ^–/–chimeras and TNF^–/– mice responded with diffusecollections of cells, mainly neutrophils, that failed to containthe infection. Extensive tissue necrosis resulting from thesecretion of oxidative products from activated neutrophils andincreased protein exudation were observed. Similar aberrantlesions were observed in TNF- and LT ${alpha}$ -deficient mice infectedwith M. tuberculosis (28). Recombinant murine LT ${alpha}$ ₃ induces theleukocyte adhesion molecules vascular cell adhesion molecule1, intracellular adhesion molecule 1, and E-selectin on endothelialcells in vitro and stimulates the T-cell- and monocyte-attractingchemokines RANTES, IP-10, and MCP-1 (7, 8). Dysregulated chemokineexpression accompanied abnormal granuloma formation in TNF^–/–chimeras infected with M. tuberculosis (2, 27, 28). Since TNFand LT ${alpha}$ clearly display independent proinflammatory activitiesand LT ${alpha}$ ₃ can signal through the same receptors, TNFRI and TNFRII,it is possible that LT ${alpha}$ ₃ activity is also mediated via an additionalsignaling pathway, such as HVEM. Alternatively, LT ${alpha}$ ₃ may signalthrough the same receptors as TNF, but the timing and site ofproduction and therefore the effects of LT ${alpha}$ ₃ signaling may betemporally distinct.

Independent effector functions for TNF and LT ${alpha}$ have been describedin several other murine models of infection. Control of Toxoplasmagondii required both TNF and LT ${alpha}$ acting in synergy (30), andprotection against Mycobacterium bovis BCG infection was moreprofoundly impaired in TNF^–/– LT ${alpha}$ ^–/–double-knockout mice than in their LT ${alpha}$ ^–/– counterparts(4, 16). In a murine model of cerebral malaria, LT ${alpha}$ was essentialfor the development of the immunopathology, whereas the centralnervous system inflammation progressed independent of TNF (11).Interpretation of the outcome of infections in nonchimeric LT-deficientmice is complicated by their lack of peripheral lymphoid tissues.Impaired activation and expansion of virus-specific CD8⁺ T cellshave been observed in LT ${alpha}$ ^–/– mice infected with herpessimplex virus (19), lymphocytic choriomeningitis virus (33),Theiler’s murine encephalomyelitis virus (20), and influenzavirus (21). Impaired T-cell responses to lymphocytic choriomeningitisvirus (33) and also to Leishmania major (36) required intactperipheral lymphoid tissue; however, interestingly, LTß-deficientchimeras with sufficient LT ${alpha}$ ₃ resisted L. major infection (36).

Studies of M. tuberculosis and high-dose Listeria infectionsin LTß receptor knockout mice revealed susceptibilityto both infections which was not solely attributable to a lackof secondary lymphoid tissue and implicated membrane-bound LT ${alpha}$ ßheterotrimer signaling in protective responses (10). This contrastswith our observations of protective responses to Listeria andM. tuberculosis in LTß^–/– chimeras (28;this study). These discrepancies may reflect indirect effectsof LTß receptor deficiency, such as a failure in maturationof antigen-presenting dendritic cells (1, 10).

In conclusion, this study revealed independent functions ofTNF and LT ${alpha}$ , but not LTß, in the protective responseto L. monocytogenes. TNF- and LT ${alpha}$ -deficient T cells do not displayintrinsic defects in activation or effector function. Rather,both of the cytokines are required for the early recruitmentof macrophages and T cells to the site of infection and activatingmacrophages to eliminate the intracellular bacterial infection.

ACKNOWLEDGMENTS

This work was supported by the National Health and Medical ResearchCouncil of Australia and the New South Wales Department of Healththrough its research infrastructure grant to the Centenary Instituteof Cancer Medicine and Cell Biology. D.R. was supported by anAustralian postgraduate award.

We thank Jenny Kingham and staff for excellent animal care.

FOOTNOTES

* Corresponding author. Mailing address: Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW, 2042, Australia. Phone: 61-2-95155210. Fax: 61-2-93513968. E-mail: wbritton{at}medicine.usyd.edu.au.

Editor: J. L. Flynn

REFERENCES

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