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Microbes and Infection 7 (2005) 1461–1468 www.elsevier.com/locate/micinf
Original article
TNF but not Fas ligand provides protective anti-L. major immunity in C57BL/6 mice Patricia Wilhelm a,b,1, Florian Wiede a,e,1, Anja Meissner a,2, Norbert Donhauser c,3, Christian Bogdan c,d, Heinrich Körner a,e,* a
Interdisziplinäres Zentrum für Klinische Forschung der Universität Erlangen-Nürnberg, Nachwuchsgruppe 1, Nikolaus-Fiebiger-Zentrum, Erlangen, Germany b Institut für Säugetiergenetik, Versuchstierzucht und -haltung, GSF, Forschungszentrum für Umwelt und Gesundheit GmbH, Neuherberg, Germany c Institut für Klinische Mikrobiologie, Immunologie und Hygiene der Universität Erlangen-Nürnberg, Erlangen, Germany d Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Freiburg, Germany e Comparative Genomics Center, School of Veterinary and Biomedical Sciences/School of Pharmacy and Molecular Sciences, Molecular Sciences Building 21, James Cook University, Townsville, Qld. 4811, Australia Received 15 March 2005; accepted 5 May 2005 Available online 05 July 2005
Abstract The pro-inflammatory cytokine TNF is essential for a protective immune response to some but not all strains of Leishmania major. TNFdeficient mice of a resistant genetic background succumbed rapidly to an infection with L. major BNI. Another member of the TNF superfamily, Fas ligand (FasL), has also been reported to be critical for the immune response to L. major. To test the relative importance of TNF versus FasL for the control of L. major BNI, we infected wildtype C57BL/6 (B6.WT), B6.TNF–/–, B6.gld and C57BL/6.gld x TNF–/– (B6.gld.TNF–/–) doublenegative mice. Visceral, fatal disease was only observed in B6.TNF–/– mice, but not in B6 gld mice. The course of infection and the immune response of B6.gld.TNF–/– mice were similar to those of B6.TNF–/– mice. B6.gld.TNF–/– mice had a high tissue parasite burden and expressed prominent amounts of inducible nitric oxide synthase (iNOS) in the skin, the lymph nodes (LN) and the spleen as previously reported for B6.TNF–/– mice, whereas the tissue parasite load and the iNOS expression of B6.gld mice resembled that of B6.WT controls. Neither the TNF- nor the FasL-deficiency exerted a detectable intrinsic effect on the proliferation of T cells. Thus, TNF, but not FasL is essential for the control of L. major BNI. The discrepancy between these and other published data are most likely due to the use of different strains of the pathogen. © 2005 Elsevier SAS. All rights reserved. Keywords: Protozoan parasite; Fas ligand; Tumor necrosis factor; Host defense
1. Introduction The genus Leishmania comprises different species of intracellular protozoan parasites that are able to infect a variety of Abbreviations: B6.gld, C57BL/6.gld; B6.gld.TNF–/–, C57BL/6.gld x TNF–/–; B6.TNF–/–, C57BL/6.TNF–/–; B6.WT, C57BL/6; CFSE, carboxyfluorescein diacetate succinimidylester; DN, double-negative; FasL, fas ligand; gld, generalized lymphoproliferative disorder; iNOS, inducible nitric oxide synthase; LN, lymph node. * Corresponding author. Tel.: +61 7 4781 4563; fax: +61 7 4781 6078. E-mail address: [email protected] (H. Körner). 1 P. Wilhelm and F. Wiede contributed equally to this manuscript. 2 Present address: Lehrstuhl für Immunologie, Universitätsklinikum Regensburg, Regensburg, Germany. 3 Present address: Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Erlangen-Nürnberg, Germany. 1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.05.005
mammalian hosts including humans and mice [1,2]. In experimental cutaneous leishmaniasis, the mouse model of Leishmania major infection, the control of parasite replication depends strongly on the genetically determined ability of the infected mouse strain to coordinate the expression of the cytokines IL-12, IFN-c and IL-4 [3]. The resistant mouse strain C57BL/6 (B6.WT) controls the pathogen predominantly at the site of infection and resolves the lesion. These mice show an early induction of IFN-ab [4] and IL-12, and an expansion of parasite-specific IFN-c- and IL-2-producing CD4+ Th1 cells [5] which, in concert with TNF, activate macrophages to exert inducible nitric oxide synthase (iNOS)dependent leishmanicidal activity [4,6–8]. In contrast, susceptible BALB/c mice develop a progressive, visceralizing and ultimately fatal disease. This alternative course of dis-
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ease is associated with an expansion of IL-4-, IL-5-, and IL-10-producing CD4+ Th2 cells propagated by the transient early presence of large amounts of IL-4 [3,9]. Recent infection experiments with TNF-negative mice, which were established in the resistant strain of C57BL/6 mice (B6.TNF–/–), have revealed an important role for TNF in the development of protective immunity to the BNI strain of L. major [10,11]. After infection with L. major BNI, B6.TNF–/– mice showed a delay of 1 week in the development of specific humoral and cellular immunity and a more disperse tissue expression of iNOS compared to wildtype mice. Despite these rather subtle changes the parasites disseminated throughout the visceral organs and the mice died within 6–8 weeks of infection [10]. In a second set of experiments we demonstrated that this rapidly fatal course of disease depended on the L. major strain used for the infection. B6.TNF–/– mice infected with L. major BNI strain succumbed to the infection, whereas an infection with L. major FRIEDLIN was confined to the draining lymph node (LN) [11]. The TNFdeficient mice survived infection with this strain for the duration of the observation period (14 weeks). Similarly, L. major LV39 caused local necrotizing skin lesions in B6.TNF–/– mice, but the parasite did not visceralize and the mice survived for the observation period of 12 weeks [12]. Another member of the TNF superfamily, Fas ligand (FasL), has also been implicated in the generation of protective immunity against L. major. The infection of a natural “loss of function” mutation of the Fas ligand (FasL), the “generalized lymphoproliferative” (gld) mouse strain, with L. major LV39 resulted in an aggravation of the disease compared to the respective wildtype controls [13,14]. Recent infection experiments with C57BL/6.gld mice and C57BL/6.gld x TNF–/– mice (B6.gld.TNF–/–) suggested that FasL rather than TNF played a dominant role in immunity to L. major LV39 [12]. To evaluate the role of FasL for protective immunity in our model, we infected wildtype, B6.gld, B6.TNF–/– and B6.gld.TNF–/– mice with L. major BNI, studied the immune response of the gene-deficient strains to L. major, and analyzed the intrinsic capability of T cells to proliferate and to produce IL-12 and IFN-c. Our data conclusively demonstrate that in contrast to the TNF-deficiency a loss-of function of FasL does not significantly influence the protective immune response to L. major BNI.
2. Material and methods 2.1. Mice Inbred C57BL/6 (B6.WT) and BALB/c mice were purchased from Charles River (Sulzfeld, Germany). C57BL/6.gld (B6.gld; FasL mutant) were obtained from The Jackson Laboratories (BarHarbor, USA). The generation and characteristics of our B6.TNF–/– were published previously [15,16]. B6.gld.TNF–/– mice were established by crossing B6.gld and
B6.TNF–/– mice and subsequently interbreeding the F1 generation [17]. All mouse lines were derived on a genetically pure C57BL/6 background and housed under standard conditions in the animal facilities of the Institute of Clinical Microbiology, Immunology and Hygiene and the NikolausFiebiger-Zentrum, University of Erlangen, Germany. 2.2. L. major parasites and the preparation of L. major antigen L. major promastigotes (MHOM/IL/81/FE/BNI) were propagated in vitro in blood agar cultures as described [18]. The virulence of the isolate was maintained by monthly passage in BALB/c mice. Stationary-phase promastigotes were harvested, washed four times and resuspended in PBS. The parasites were used for infection or subjected to four cycles of rapid freezing and thawing to prepare L. major antigen as described [10]. 2.3. L. major infection and evaluation of the systemic course of disease Mice were infected subcutaneously with 3 × 106 stationaryphase promastigotes of the third to seventh in vitro passage in a final volume of 50 µl in one hind footpad. The increase in lesion size was monitored weekly by measuring the footpad thickness with a metric caliper (Kroeplin Schnelltaster, Schlüchtern, Germany). The increase in footpad thickness (%) was determined as described [10]. The number of viable parasites in spleen and bone marrow was estimated using limiting dilution analysis by applying Poisson statistics and the v2 minimization method as described previously [19]. 2.4. Serum isotype detection, proliferation assay and detection of cytokines IgM, IgG1 and IgG2a serum antibodies reactive with L. major were detected by sandwich-ELISA and specific antibody titres were depicted as ELISA units relative to a L. major-positive serum pool as described [10]. The alkalinephosphatase labeled secondary detection antibody was added for 1 h at room temperature [10]. 2.5. Proliferation assay Mice of 6–8 weeks of age were sacrificed and draining popliteal LN were removed. Single cell suspensions were prepared by grinding the tissue between two sterile slides. Carboxyfluorescein diacetate succinimidylester (CFSE) proliferation assays were performed as described [20]. In order to determine the intrinsic capacity of the cells to proliferate in response to a stimulus, 5 × 105 CFSE-labeled T cells were stimulated with rat anti-CD3 (clone 17A2; BD Biosciences, Heidelberg, Germany) in combination with hamster antiCD28 (clone 31.51; BD Biosciences) coated to the surface of a microtiter plate at 10 or 5 µg/ml, respectively. After 48 h of
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incubation, the proportion of dividing cells was determined with a FACS-Calibur® flow cytometer (BD Biosciences) using the CellQuest® software (BD Biosciences). The assay was performed in duplicates. 2.6. Quantitative real-time PCR RNA was extracted from cells using the ″Perfect RNA Mini Kit″ (Eppendorf, Hamburg, Germany) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 to 2 µg of total RNA. The iCycler IQ-PCR System (Bio-Rad Laboratories GmbH, München, Germany) was used to quantify gene expression with SYBR-Green (Roche Applied Biosciences, Mannheim, Germany) following the manufacturer’s instructions. The following primers were used at a concentration of 200 nM: b-actin sense: 5′-AGA GGG AAA TCG TGC GTG AC-3′, b-actin antisense: 5′-CAA TAG TGA TGA CCT GGC CGT-3′; IL-4 sense: 5′-ACA GGA GAA GGG ACG CCA T-3′, IL-4 antisense: 5′-GAA GCC CTA CAG ACG AGC TCA-3′; IFN-c sense: 5′-TCA AGT GGC ATA GAT GTG GAA GAA-3′, IFN-c antisense: 5′TGG CTC TGC AGG ATT TTC ATG-3′. The specifically amplified cDNA was quantified in relation to b-actin transcripts from the same cDNA preparation and is shown as x-fold induction of the gene transcripts of activated cells compared to non-activated cells.
Fig. 1. Clinical course of L. major BNI infection. BALB/c, B6.wildtype, B6.TNF–/–, B6.gld and double-negative B6.gld.TNF–/– mice were s.c. infected with 3 × 106 L. major promastigotes into the right hind footpad and the percentage increase of thickness compared to the non-infected footpad was monitored. The data are given as the mean (± S.E.M.) of six (B6/WT, B6.TNF–/–) animals. One representative experiment of four is shown.
Tissue sections (7 µm) from embedded skin lesions, LN and spleens were prepared with a cryostat microtome (model HM 500 OM; Fa. Microm, Walldorf, Germany), thawed onto slides coated with Fro-Marker® (Science Services, Munich, Germany), surrounded with PAP PEN® (Science Services), air-dried, fixed in acetone (for 10 min at –20 °C) and briefly washed in PBS/0.05% Tween 20. Non-specific binding sites were blocked for 30 min with PBS/0.1% saponin/1% BSA/20% FCS. The detection of iNOS and L. major was performed by immunoperoxidase staining using 3-amino-9ethyl-carbazole as a substrate and hematoxylin counterstaining as described [21].
negative B6.gld.TNF–/– mice and the B6.TNF–/– mice showed a parallel course of lesion development and succumbed within 6 weeks to the infection as already described for B6.TNF–/– mice [10]. Neither B6.TNF–/– mice nor B6.gld.TNF–/– mice exhibited large or ulcerated skin lesions at the site of parasite inoculation. In contrast, both genotypes showed scaring after around 6 weeks and the local lesion of infected B6.gld.TNF–/– mice started to resolve [10]. LN cells and splenocytes were isolated and the parasite burden was analyzed at different time points of infection in a limiting dilution assay (Fig. 2). In the early phase of the infection (day 14) the number of viable parasites in the LN of B6.gld mice was only slightly elevated compared to B6.WT mice. The strikingly higher mean number of parasites in the LN of B6.gld mice at 15 weeks after infection could be due to the enlargement of LN in the gld background. After 42 days of infection B6.gld mice had a substantially lower number of L. major in LN and spleen than B6.gld.TNF–/– mice (Fig. 2). The parasite burden of B6.gld.TNF–/– mice was comparable to that of B6.TNF–/– mice [10].
3. Results
3.2. Anti-L. major immune response in B6.gld and double-negative B6.gld.TNF–/– mice
2.7. Antibodies and immunohistology
3.1. Course of leishmaniasis and parasite burden in LN and spleen of infected B6.gld and B6.gld.TNF–/– mice We infected B6.TNF–/–, B6.gld and the double-deficient B6.gld.TNF–/– mice together with B6.WT and BALB/c controls with L. major BNI and monitored the course of the infection for 14 weeks (Fig. 1). Within 6 weeks, the infected BALB/c mice developed large ulcerated lesions and became moribund [10]. The B6.WT control mice and the B6.gld mice controlled the infection and resolved the lesion. The double-
Whereas B6.WT T cells from draining LN of mice infected for 1 week already proliferated in response to L. major antigen, in double-negative B6.gld.TNF–/– mice an antigenspecific cellular response became detectable only after 2 weeks of infection (data not shown). This finding is in accordance with the results published for the B6.TNF–/– mice [10]. Humoral immunity does not contribute to the protective immune response to L. major. In contrast, a strong production of IgG antibodies can lead to the absence of delayed typehypersensitivity reaction and a more severe course of disease
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Fig. 2. Parasite burden in draining LN and spleen of BALB/c, B6.wildtype, B6.TNF–/–, B6.gld and double-negative B6.gld.TNF–/– mice. The number of viable parasites in the draining LN and the spleen was determined at day 14, day 42 and day 105 p.i. by limiting dilution analysis. The solid line represents the mean tissue parasite burden of four to six mice. One triangle represents one animal. One of three experiments is shown.
[22]. Nevertheless, the quantification of the humoral immune response to a complex antigen can be used to determine the efficacy of the immune system. Furthermore, it has been shown that Th1 cells produce the Th1 cytokine IFN-c, which stimulates the production of IgG2a and inhibits IgG1, whereas the expansion of Th2 cells promotes B cell class switching towards IgG1 and IgE [23]. Thus, the type of T helper cell response determines which isotype is prominent in the humoral immune response [24]. Therefore, we analyzed the amount of L. major-specific IgM (days 7 and 14) and IgG1 and IgG2a antibodies (days 28 and 42) during the course of infection. The T cell-independent L. major-specific IgM response early after infection was similar in all genotypes with the exception of BALB/c (Fig. 3). The IgG production
of infected B6.gld mice was in the same order of magnitude (IgG1) or twofold higher (IgG2a) compared to B6.WT mice. In contrast, in the B6.gld.TNF–/– and the B6.TNF–/– mice antibodies of the IgG1 isotype could not be detected at days 28 and 42 after infection and IgG2a antibodies were expressed only at very low levels. The slightly increased expression of L. major-specific IgG2a antibodies points to a Th1-type response in B6.gld mice, which confirms earlier findings [10]. The almost complete absence of IgG production in TNFdeficient mice is in accordance with our earlier published data [10]. The absence of IgG antibodies in the gene-deficient B6.gld.TNF–/– and the B6.TNF–/– mice did not correlate with an improved course of disease like in immunoglobulindeficient BALB/c mice [22].
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respectively. In B6.gld T cells the level of IFN-c and IL4 mRNA was elevated 27- and 10-fold, respectively (Fig. 4B). Both IFN-c and IL-4 are upregulated strongly in B6.gld T cells and marginally in B6.gld.TNF–/– T cells. The fact that the expression of both cytokines is induced in a similar way in the two mouse strains points to a general phenomenon of the gld mutation. The balanced up-regulation of IFN-c and IL-4 mRNA probably prevents a deviation from the Th1 response. 3.4. Histological analysis of the site of infection and the draining LN in B6.gld mice
Fig. 3. Serum concentration of L. major-specific antibodies. The serum concentration of L. major-specific IgM (A), IgG1 (B) and IgG2a (C) was analyzed in B6.WT, BALB/c, B6.gld, B6.TNF–/–, B6.gld.TNF–/– mice after L. major infection. The concentration is shown in relative ELISA units.
3.3. Intrinsic capacity of B6.gld and double-negative B6.gld.TNF–/– T cells to proliferate and to produce IFN-c and IL-4 The proliferation of naive B6.WT, B6.gld and B6.gld.TNF–/– T cells was analyzed to rule out intrinsic deficiencies. T cells were isolated from non-infected mice, labeled with CFSE and stimulated in vitro with anti-CD3 and antiCD28 for 48 h B6.WT T cells divided three times within the period of observation (Fig. 4A) with 22% of cells in the third cycle of cell division. In contrast, both B6.gld and B6.gld.TNF–/– T cells demonstrated an increased cell proliferation with a substantial number of cells already in the fourth cycle of division (Fig. 4A). Almost half of the B6.gld.TNF–/– and B6.gld T cells had divided a third and fourth time (46% and 43%, respectively) within 48 h. A Th1-type response is characterized by IFN-c-expression, a Th2-type response by IL-4 expression. A L. major infection of B6.gld mice results in strong IFN-c-presence [13]. Therefore, the expression of IFN-c and IL-4 mRNA was tested by quantitative Real-Time RT-PCR to investigate the possibility that a deviation from a Th1 response was involved in the susceptibility of B6.gld.TNF–/– mice in our model. Relative to B6.WT T cells, B6.gld.TNF–/– T cells exhibited a three and twofold increased amount of IFN-c and IL-4 mRNA,
The expression of iNOS and the presence of parasites were analyzed in footpads, draining LN and spleen of B6.WT, B6.TNF–/– and B6.gld mice at days 7, 27 and 40 of infection. The expression of iNOS protein in the skin and LN of B6.WT and B6.gld mice was indifferent, both in terms of the overall amount of iNOS protein detected and the distribution and size of the iNOS clusters (Fig. 5A–F; and data not shown). In the LN of B6.TNF–/– mice, the iNOS foci were small, numerous and disperse. In both B6.WT and B6.gld mice they were considerably larger and confluent and lower in number (Fig. 5, panel D–F). In accordance with the limiting dilution analysis the popliteal LN of B6.TNF–/– mice (day 40 of infection) contained large numbers of parasites, whereas in the LN of B6.WT and B6.gld mice fewer parasites were detectable by immunohistology (Fig. 5, panel G–I). In spleen sections of B6.gld mice, which contained a limited number of parasite foci, iNOS protein was expressed (data not shown). This was not the case however, in B6.WT mice, while in the spleens of B6.TNF–/– mice both parasites and iNOS were detectable as reported before [10].
4. Discussion The L. major infection of B6.gld and double-negative B6.gld.TNF–/– mice resulted in a clinical course, which either resembled that of infected B6.WT mice (resolution of the lesion) or of B6.TNF–/– mice (viseralization of the parasite and death), respectively. We did not observe a specific role of FasL for the generation of a protective immune response in our model. In contrast, the almost identical course of infection in B6.TNF–/– and B6.gld.TNF–/– mice clearly demonstrated the central importance of TNF in the anti-leishmanial defense. Data on the role of TNF in the model of murine cutaneous leishmaniasis have been inconsistent and controversial. The pro-inflammatory cytokine TNF was examined extensively because of its potential effector function. Treatment of L. major LV39-infected CBA mice with TNF resulted in an attenuation of the course of disease and the reduction of lesion size and parasite burden [8,25,26]. Application of neutralizing anti-TNF antibodies only led to a transient aggravation of the disease in CBA or BALB/c mice infected with L. major
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Fig. 4. Intrinsic capacity of B6.WT, B6.gld and B6.gld.TNF–/– T cells to proliferate and to produce cytokines in response to CD3/CD28 mAbs. T cells were isolated from LN of naive mice and labeled with CFSE. The proliferation was triggered with CD3/CD28 mAbs coated on the surface of 96-well plates. (A) The single peak (thin line) represents the non-stimulated control cells. The proliferation-dependent decrease of CFSE labeling is shown in dark. (B) The proliferating cells were tested for expression of IL-4 and IFN-c by Real-Time RT-PCR. First, the specifically amplified cytokine cDNA was quantified in relation to a specifically amplified house keeping gene product (b-actin). Second, the relative up-regulation of the gene transcripts of activated B6.WT, B6.gld and B6.gld.TNF–/– T cells was compared to the relative expression of non-activated B6.WT, B6.gld and B6.gld.TNF–/– T cells. Third, the resulting values for activated/nonactivated B6.WT T cells were set at one and the mean of activated/non-activated B6.gld and B6.gld.TNF–/– T cells was depicted as x-fold induction. Every bar represents results generated from T cells taken independently from five mice (n = 5). The activated B6.gld T cells expressed a substantially increased amount of both IL-4 and IFN-c mRNA compared to B6.WT or B6.gld.TNF–/– cells (* P < 0.05).
strain LV39 [25–28]. Gene-targeted mice negative for TNFR 1 or both TNFR 1 and 2 developed a Th1 response, expressed IFN-c and iNOS, and were able to clear L. major strain FRIEDLIN [29]. Taken together, these findings suggested that TNF acts as a co-factor in the development of protective, antileishmanial immunity, but is dispensable for the ultimate control of the infection. Recently, however, infection of TNFnegative mice that had been established directly on a C57BL/6 background with the L. major BNI strain showed a different outcome. The mice were not able to establish protective immunity resulting in visceralization of the parasite and death of the mice [10]. In contrast to TNF the related FasL/Fas pathway has repeatedly been reported to be important for protection against L. major strain LV39. It was found that Fas-deficient MRL/lpr or B6/lpr mice did not resolve their skin lesions after infection although visceralization of the parasites did not occur [14]. An infection of doublenegative B6.gld.TNF–/– mice with L. major LV39 resulted in a more dramatic phenotype including necrosis at the site of infection and spreading of the parasite [12–14]. Nevertheless, the mice did not succumb to the infection. The studies mentioned above used L. major strains FRIEDLIN, LV39 and BNI as well as gene-deficient mice with variable levels of genetic backcrossing. In order to minimize the experimental problems associated with different genetic back-
grounds we developed gene-targeted inbred mouse strains that allowed us to study the individual and cooperative activities of TNF and FasL in leishmaniasis. The TNF gene was targeted in C57BL/6 ES cells directly [15], and the gld mice had been backcrossed to near-homogeneity thus minimizing the introduction of unrelated genes in the double-negative B6.gld.TNF–/– mice [17]. Moreover, in another study we addressed the discrepancy between the published results of L. major infection of TNF R1/2–/– [30,31] and B6.TNF–/– mice [10] and used two strains of L. major, FRIEDLIN and BNI in parallel infection experiments [11]. Our results clearly demonstrated that in some cases the L. major strain used for infection affected the outcome of the disease to a greater extent than the gene-deficiency investigated or the genetic background. The potential mechanism, that lead to this L. major strain-dependent immunity in TNF-negative mice could have been yet undefined structural differences in the lipophosphoglycan surface molecules of the two strains. These differences would trigger different tissue-specific innate effector mechanisms that would result in differential survival [11,32]. In earlier studies consistent results have been described in experimental leishmaniasis of IL-4-deficient mice [33]. In the present study we show that in L. major BNIinfected mice the loss-of-function mutation of FasL, gld, is not part of the protective immune response, whereas TNF is
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Fig. 5. Immunohistological analysis of iNOS expression and distribution of L. major promastigotes in skin lesions and draining LN of L. major infected B6.WT, B6.gld and B6.TNF–/– mice. Sections from foot pads (A–C) and draining LN (D–I) were stained for the presence of iNOS (A–F) or L. major promastigotes (G–I) using the immunoperoxidase technique and aminoethylcarbazole as a substrate (brown). The arrows point to single L. major amastigotes. The magnification is 400 times.
essential to survive an infection with the same strain of L. major. In contrast, infections with the L. major LV39 have provided evidence that FasL rather than TNF is decisive for the complete control of parasite replication [12–14]. Taken together, our present results and the previously published data provide another example for the strong impact of the genetic origin of L. major on the course of infection in immunodeficient mice.
Acknowledgments The study was supported by the Deutsche Forschungsgemeinschaft (Bo 996/3-1 and Bo 996/3-2 to C.B.; Ko 1315/33 to H.K.), a Competitive Research Incentive Grant (JCU to H.K.), the Federal Ministry of Education and Research (BMBF) and by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nürnberg (IZKF NW1 to H.K.).
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[13] F. Conceicao-Silva, M. Hahne, M. Schröter, J. Louis, J. Tschopp, The resolution of lesions induced by Leishmania major in mice requires a functional Fas (APO-1, CD95) pathway of cytotoxicity, Eur. J. Immunol. 28 (1998) 237–245. [14] F.-P. Huang, D. Xu, E.-O. Esfandiari, W. Sands, X. Wei, F.Y. Liew, Mice defective in Fas are highly susceptible to Leishmania major infection despite elevated IL-12 synthesis, strong Th1 responses, and enhanced nitric oxide production, J. Immunol. 160 (1998) 4143– 4147. [15] H. Körner, M. Cook, D.S. Riminton, F.A. Lemckert, R. Hoek, B. Ledermann, F. Köntgen, B. Fazekas de St Groth, J.D. Sedgwick, Distinct roles for lymphotoxin-a and tumour necrosis factor in lymphoid tissue organogenesis and spatial organisation defined in gene targeted C57BL/6 Mice, Eur. J. Immunol. 27 (1997) 2600–2609. [16] P. Wilhelm, D.S. Riminton, U. Ritter, F.A. Lemckert, C. Scheidig, R. Hoek, J.D. Sedgwick, H. Korner, Membrane lymphotoxin contributes to anti-leishmanial immunity by controlling structural integrity of lymphoid organs, Eur. J. Immunol. 32 (2002) 1993–2003. [17] H. Körner, E. Cretney, P. Wilhelm, J.M. Kelly, M. Röllinghoff, J.D. Sedgwick, M.J. Smyth, Tumor necrosis factor sustains the generalized lymphoproliferative disorder (gld) phenotype, J. Exp. Med. 191 (2000) 89–96. [18] W. Solbach, K. Forberg, E. Kammerer, C. Bogdan, M. Röllinghoff, Suppressive effect of cyclosporin A on the development of Leishmania tropica-induced lesions in genetically susceptible BALB/c mice, J. Immunol. 137 (1986) 702–711. [19] S. Stenger, H. Thüring, M. Röllinghoff, C. Bogdan, Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major, J. Exp. Med. 180 (1994) 783–793. [20] U. Ritter, A. Meissner, C. Scheidig, H. Körner, CD8alpha- and Langerin-negative dendritic cells, but not Langerhans cells, act as principal antigen-presenting cells in leishmaniasis, Eur. J. Immunol. 34 (2004) 1542–1550. [21] S. Stenger, N. Donhauser, H. Thüring, M. Röllinghoff, C. Bogdan, Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase, J. Exp. Med. 183 (1996) 1501–1514. [22] S.A. Miles, S.M. Conrad, R.G. Alves, S.M. Jeronimo, D.M. Mosser, A role for IgG immune complexes during infection with the intracellular pathogen Leishmania, J. Exp. Med. 201 (2005) 747–754.
[23] T.R. Mosmann, H. Cherwinski, M.W. Bond, M.A. Giedlin, R.L. Coffman, Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins, J. Immunol. 136 (1986) 2348–2357. [24] F.D. Finkelman, I.M. Katona, T.R. Mosmann, R.L. Coffman, IFNgamma regulates the isotypes of Ig secreted during in vivo humoral immune responses, J. Immunol. 140 (1988) 1022–1027. [25] F.Y. Liew, C. Parkinson, S. Millott, A. Severn, M. Carrier, Tumour necrosis factor (TNF alpha) in leishmaniasis. I. TNFa mediates host protection against cutaneous leishmaniasis, Immunol. 69 (1990) 570– 573. [26] R.G. Titus, B. Sherry, A. Cerami, Tumor necrosis factor plays a protective role in experimental murine cutaneous leishmaniasis, J. Exp. Med. 170 (1989) 2097–2104. [27] C.M. Theodos, L. Povinelli, R. Molina, B. Sherry, R.G. Titus, Role of tumor necrosis factor in macrophage leishmanicidal activity in vitro and resistance to cutaneous leishmaniasis in vivo, Infect. Immunol. 59 (1991) 2839–2842. [28] S. de Kossodo, G.E. Grau, J.A. Louis, I. Müller, Tumor necrosis factor alpha (TNF-alpha) and TNF-beta and their receptors in experimental cutaneous leishmaniasis, Infect. Immun. 62 (1994) 1414–1420. [29] S.T. Kanaly, M. Nashleanas, B. Hondowicz, P. Scott, TNF receptor p55 is required for elimination of inflammatory cells following control of intracellular pathogens, J. Immunol. 163 (1999) 3883–3889. [30] L.Q. Vieira, M. Goldschmidt, M. Nashleanas, K. Pfeffer, T. Mak, P. Scott, Mice lacking the TNF receptor p55 fail to resolve lesions caused by infection with Leishmania major, but control parasite replication, J. Immunol. 157 (1996) 827–835. [31] M. Nashleanas, S. Kanaly, P. Scott, Control of Leishmania major infection in mice lacking TNF-receptors, J. Immunol. 160 (1998) 5506–5513. [32] G.F. Spath, L.F. Lye, H. Segawa, D.L. Sacks, S.J. Turco, S.M. Beverley, Persistence without pathology in phosphoglycan-deficient Leishmania major, Science 301 (2003) 1241–1243. [33] N. Noben-Trauth, W.E. Paul, D.L. Sacks, IL-4- and IL-4 receptordeficient BALB/c mice reveal differences in susceptibility to Leishmania major parasite substrains, J. Immunol. 162 (1999) 6132–6140.
Original article
TNF but not Fas ligand provides protective anti-L. major immunity in C57BL/6 mice Patricia Wilhelm a,b,1, Florian Wiede a,e,1, Anja Meissner a,2, Norbert Donhauser c,3, Christian Bogdan c,d, Heinrich Körner a,e,* a
Interdisziplinäres Zentrum für Klinische Forschung der Universität Erlangen-Nürnberg, Nachwuchsgruppe 1, Nikolaus-Fiebiger-Zentrum, Erlangen, Germany b Institut für Säugetiergenetik, Versuchstierzucht und -haltung, GSF, Forschungszentrum für Umwelt und Gesundheit GmbH, Neuherberg, Germany c Institut für Klinische Mikrobiologie, Immunologie und Hygiene der Universität Erlangen-Nürnberg, Erlangen, Germany d Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Freiburg, Germany e Comparative Genomics Center, School of Veterinary and Biomedical Sciences/School of Pharmacy and Molecular Sciences, Molecular Sciences Building 21, James Cook University, Townsville, Qld. 4811, Australia Received 15 March 2005; accepted 5 May 2005 Available online 05 July 2005
Abstract The pro-inflammatory cytokine TNF is essential for a protective immune response to some but not all strains of Leishmania major. TNFdeficient mice of a resistant genetic background succumbed rapidly to an infection with L. major BNI. Another member of the TNF superfamily, Fas ligand (FasL), has also been reported to be critical for the immune response to L. major. To test the relative importance of TNF versus FasL for the control of L. major BNI, we infected wildtype C57BL/6 (B6.WT), B6.TNF–/–, B6.gld and C57BL/6.gld x TNF–/– (B6.gld.TNF–/–) doublenegative mice. Visceral, fatal disease was only observed in B6.TNF–/– mice, but not in B6 gld mice. The course of infection and the immune response of B6.gld.TNF–/– mice were similar to those of B6.TNF–/– mice. B6.gld.TNF–/– mice had a high tissue parasite burden and expressed prominent amounts of inducible nitric oxide synthase (iNOS) in the skin, the lymph nodes (LN) and the spleen as previously reported for B6.TNF–/– mice, whereas the tissue parasite load and the iNOS expression of B6.gld mice resembled that of B6.WT controls. Neither the TNF- nor the FasL-deficiency exerted a detectable intrinsic effect on the proliferation of T cells. Thus, TNF, but not FasL is essential for the control of L. major BNI. The discrepancy between these and other published data are most likely due to the use of different strains of the pathogen. © 2005 Elsevier SAS. All rights reserved. Keywords: Protozoan parasite; Fas ligand; Tumor necrosis factor; Host defense
1. Introduction The genus Leishmania comprises different species of intracellular protozoan parasites that are able to infect a variety of Abbreviations: B6.gld, C57BL/6.gld; B6.gld.TNF–/–, C57BL/6.gld x TNF–/–; B6.TNF–/–, C57BL/6.TNF–/–; B6.WT, C57BL/6; CFSE, carboxyfluorescein diacetate succinimidylester; DN, double-negative; FasL, fas ligand; gld, generalized lymphoproliferative disorder; iNOS, inducible nitric oxide synthase; LN, lymph node. * Corresponding author. Tel.: +61 7 4781 4563; fax: +61 7 4781 6078. E-mail address: [email protected] (H. Körner). 1 P. Wilhelm and F. Wiede contributed equally to this manuscript. 2 Present address: Lehrstuhl für Immunologie, Universitätsklinikum Regensburg, Regensburg, Germany. 3 Present address: Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Erlangen-Nürnberg, Germany. 1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.05.005
mammalian hosts including humans and mice [1,2]. In experimental cutaneous leishmaniasis, the mouse model of Leishmania major infection, the control of parasite replication depends strongly on the genetically determined ability of the infected mouse strain to coordinate the expression of the cytokines IL-12, IFN-c and IL-4 [3]. The resistant mouse strain C57BL/6 (B6.WT) controls the pathogen predominantly at the site of infection and resolves the lesion. These mice show an early induction of IFN-ab [4] and IL-12, and an expansion of parasite-specific IFN-c- and IL-2-producing CD4+ Th1 cells [5] which, in concert with TNF, activate macrophages to exert inducible nitric oxide synthase (iNOS)dependent leishmanicidal activity [4,6–8]. In contrast, susceptible BALB/c mice develop a progressive, visceralizing and ultimately fatal disease. This alternative course of dis-
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ease is associated with an expansion of IL-4-, IL-5-, and IL-10-producing CD4+ Th2 cells propagated by the transient early presence of large amounts of IL-4 [3,9]. Recent infection experiments with TNF-negative mice, which were established in the resistant strain of C57BL/6 mice (B6.TNF–/–), have revealed an important role for TNF in the development of protective immunity to the BNI strain of L. major [10,11]. After infection with L. major BNI, B6.TNF–/– mice showed a delay of 1 week in the development of specific humoral and cellular immunity and a more disperse tissue expression of iNOS compared to wildtype mice. Despite these rather subtle changes the parasites disseminated throughout the visceral organs and the mice died within 6–8 weeks of infection [10]. In a second set of experiments we demonstrated that this rapidly fatal course of disease depended on the L. major strain used for the infection. B6.TNF–/– mice infected with L. major BNI strain succumbed to the infection, whereas an infection with L. major FRIEDLIN was confined to the draining lymph node (LN) [11]. The TNFdeficient mice survived infection with this strain for the duration of the observation period (14 weeks). Similarly, L. major LV39 caused local necrotizing skin lesions in B6.TNF–/– mice, but the parasite did not visceralize and the mice survived for the observation period of 12 weeks [12]. Another member of the TNF superfamily, Fas ligand (FasL), has also been implicated in the generation of protective immunity against L. major. The infection of a natural “loss of function” mutation of the Fas ligand (FasL), the “generalized lymphoproliferative” (gld) mouse strain, with L. major LV39 resulted in an aggravation of the disease compared to the respective wildtype controls [13,14]. Recent infection experiments with C57BL/6.gld mice and C57BL/6.gld x TNF–/– mice (B6.gld.TNF–/–) suggested that FasL rather than TNF played a dominant role in immunity to L. major LV39 [12]. To evaluate the role of FasL for protective immunity in our model, we infected wildtype, B6.gld, B6.TNF–/– and B6.gld.TNF–/– mice with L. major BNI, studied the immune response of the gene-deficient strains to L. major, and analyzed the intrinsic capability of T cells to proliferate and to produce IL-12 and IFN-c. Our data conclusively demonstrate that in contrast to the TNF-deficiency a loss-of function of FasL does not significantly influence the protective immune response to L. major BNI.
2. Material and methods 2.1. Mice Inbred C57BL/6 (B6.WT) and BALB/c mice were purchased from Charles River (Sulzfeld, Germany). C57BL/6.gld (B6.gld; FasL mutant) were obtained from The Jackson Laboratories (BarHarbor, USA). The generation and characteristics of our B6.TNF–/– were published previously [15,16]. B6.gld.TNF–/– mice were established by crossing B6.gld and
B6.TNF–/– mice and subsequently interbreeding the F1 generation [17]. All mouse lines were derived on a genetically pure C57BL/6 background and housed under standard conditions in the animal facilities of the Institute of Clinical Microbiology, Immunology and Hygiene and the NikolausFiebiger-Zentrum, University of Erlangen, Germany. 2.2. L. major parasites and the preparation of L. major antigen L. major promastigotes (MHOM/IL/81/FE/BNI) were propagated in vitro in blood agar cultures as described [18]. The virulence of the isolate was maintained by monthly passage in BALB/c mice. Stationary-phase promastigotes were harvested, washed four times and resuspended in PBS. The parasites were used for infection or subjected to four cycles of rapid freezing and thawing to prepare L. major antigen as described [10]. 2.3. L. major infection and evaluation of the systemic course of disease Mice were infected subcutaneously with 3 × 106 stationaryphase promastigotes of the third to seventh in vitro passage in a final volume of 50 µl in one hind footpad. The increase in lesion size was monitored weekly by measuring the footpad thickness with a metric caliper (Kroeplin Schnelltaster, Schlüchtern, Germany). The increase in footpad thickness (%) was determined as described [10]. The number of viable parasites in spleen and bone marrow was estimated using limiting dilution analysis by applying Poisson statistics and the v2 minimization method as described previously [19]. 2.4. Serum isotype detection, proliferation assay and detection of cytokines IgM, IgG1 and IgG2a serum antibodies reactive with L. major were detected by sandwich-ELISA and specific antibody titres were depicted as ELISA units relative to a L. major-positive serum pool as described [10]. The alkalinephosphatase labeled secondary detection antibody was added for 1 h at room temperature [10]. 2.5. Proliferation assay Mice of 6–8 weeks of age were sacrificed and draining popliteal LN were removed. Single cell suspensions were prepared by grinding the tissue between two sterile slides. Carboxyfluorescein diacetate succinimidylester (CFSE) proliferation assays were performed as described [20]. In order to determine the intrinsic capacity of the cells to proliferate in response to a stimulus, 5 × 105 CFSE-labeled T cells were stimulated with rat anti-CD3 (clone 17A2; BD Biosciences, Heidelberg, Germany) in combination with hamster antiCD28 (clone 31.51; BD Biosciences) coated to the surface of a microtiter plate at 10 or 5 µg/ml, respectively. After 48 h of
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incubation, the proportion of dividing cells was determined with a FACS-Calibur® flow cytometer (BD Biosciences) using the CellQuest® software (BD Biosciences). The assay was performed in duplicates. 2.6. Quantitative real-time PCR RNA was extracted from cells using the ″Perfect RNA Mini Kit″ (Eppendorf, Hamburg, Germany) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 to 2 µg of total RNA. The iCycler IQ-PCR System (Bio-Rad Laboratories GmbH, München, Germany) was used to quantify gene expression with SYBR-Green (Roche Applied Biosciences, Mannheim, Germany) following the manufacturer’s instructions. The following primers were used at a concentration of 200 nM: b-actin sense: 5′-AGA GGG AAA TCG TGC GTG AC-3′, b-actin antisense: 5′-CAA TAG TGA TGA CCT GGC CGT-3′; IL-4 sense: 5′-ACA GGA GAA GGG ACG CCA T-3′, IL-4 antisense: 5′-GAA GCC CTA CAG ACG AGC TCA-3′; IFN-c sense: 5′-TCA AGT GGC ATA GAT GTG GAA GAA-3′, IFN-c antisense: 5′TGG CTC TGC AGG ATT TTC ATG-3′. The specifically amplified cDNA was quantified in relation to b-actin transcripts from the same cDNA preparation and is shown as x-fold induction of the gene transcripts of activated cells compared to non-activated cells.
Fig. 1. Clinical course of L. major BNI infection. BALB/c, B6.wildtype, B6.TNF–/–, B6.gld and double-negative B6.gld.TNF–/– mice were s.c. infected with 3 × 106 L. major promastigotes into the right hind footpad and the percentage increase of thickness compared to the non-infected footpad was monitored. The data are given as the mean (± S.E.M.) of six (B6/WT, B6.TNF–/–) animals. One representative experiment of four is shown.
Tissue sections (7 µm) from embedded skin lesions, LN and spleens were prepared with a cryostat microtome (model HM 500 OM; Fa. Microm, Walldorf, Germany), thawed onto slides coated with Fro-Marker® (Science Services, Munich, Germany), surrounded with PAP PEN® (Science Services), air-dried, fixed in acetone (for 10 min at –20 °C) and briefly washed in PBS/0.05% Tween 20. Non-specific binding sites were blocked for 30 min with PBS/0.1% saponin/1% BSA/20% FCS. The detection of iNOS and L. major was performed by immunoperoxidase staining using 3-amino-9ethyl-carbazole as a substrate and hematoxylin counterstaining as described [21].
negative B6.gld.TNF–/– mice and the B6.TNF–/– mice showed a parallel course of lesion development and succumbed within 6 weeks to the infection as already described for B6.TNF–/– mice [10]. Neither B6.TNF–/– mice nor B6.gld.TNF–/– mice exhibited large or ulcerated skin lesions at the site of parasite inoculation. In contrast, both genotypes showed scaring after around 6 weeks and the local lesion of infected B6.gld.TNF–/– mice started to resolve [10]. LN cells and splenocytes were isolated and the parasite burden was analyzed at different time points of infection in a limiting dilution assay (Fig. 2). In the early phase of the infection (day 14) the number of viable parasites in the LN of B6.gld mice was only slightly elevated compared to B6.WT mice. The strikingly higher mean number of parasites in the LN of B6.gld mice at 15 weeks after infection could be due to the enlargement of LN in the gld background. After 42 days of infection B6.gld mice had a substantially lower number of L. major in LN and spleen than B6.gld.TNF–/– mice (Fig. 2). The parasite burden of B6.gld.TNF–/– mice was comparable to that of B6.TNF–/– mice [10].
3. Results
3.2. Anti-L. major immune response in B6.gld and double-negative B6.gld.TNF–/– mice
2.7. Antibodies and immunohistology
3.1. Course of leishmaniasis and parasite burden in LN and spleen of infected B6.gld and B6.gld.TNF–/– mice We infected B6.TNF–/–, B6.gld and the double-deficient B6.gld.TNF–/– mice together with B6.WT and BALB/c controls with L. major BNI and monitored the course of the infection for 14 weeks (Fig. 1). Within 6 weeks, the infected BALB/c mice developed large ulcerated lesions and became moribund [10]. The B6.WT control mice and the B6.gld mice controlled the infection and resolved the lesion. The double-
Whereas B6.WT T cells from draining LN of mice infected for 1 week already proliferated in response to L. major antigen, in double-negative B6.gld.TNF–/– mice an antigenspecific cellular response became detectable only after 2 weeks of infection (data not shown). This finding is in accordance with the results published for the B6.TNF–/– mice [10]. Humoral immunity does not contribute to the protective immune response to L. major. In contrast, a strong production of IgG antibodies can lead to the absence of delayed typehypersensitivity reaction and a more severe course of disease
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Fig. 2. Parasite burden in draining LN and spleen of BALB/c, B6.wildtype, B6.TNF–/–, B6.gld and double-negative B6.gld.TNF–/– mice. The number of viable parasites in the draining LN and the spleen was determined at day 14, day 42 and day 105 p.i. by limiting dilution analysis. The solid line represents the mean tissue parasite burden of four to six mice. One triangle represents one animal. One of three experiments is shown.
[22]. Nevertheless, the quantification of the humoral immune response to a complex antigen can be used to determine the efficacy of the immune system. Furthermore, it has been shown that Th1 cells produce the Th1 cytokine IFN-c, which stimulates the production of IgG2a and inhibits IgG1, whereas the expansion of Th2 cells promotes B cell class switching towards IgG1 and IgE [23]. Thus, the type of T helper cell response determines which isotype is prominent in the humoral immune response [24]. Therefore, we analyzed the amount of L. major-specific IgM (days 7 and 14) and IgG1 and IgG2a antibodies (days 28 and 42) during the course of infection. The T cell-independent L. major-specific IgM response early after infection was similar in all genotypes with the exception of BALB/c (Fig. 3). The IgG production
of infected B6.gld mice was in the same order of magnitude (IgG1) or twofold higher (IgG2a) compared to B6.WT mice. In contrast, in the B6.gld.TNF–/– and the B6.TNF–/– mice antibodies of the IgG1 isotype could not be detected at days 28 and 42 after infection and IgG2a antibodies were expressed only at very low levels. The slightly increased expression of L. major-specific IgG2a antibodies points to a Th1-type response in B6.gld mice, which confirms earlier findings [10]. The almost complete absence of IgG production in TNFdeficient mice is in accordance with our earlier published data [10]. The absence of IgG antibodies in the gene-deficient B6.gld.TNF–/– and the B6.TNF–/– mice did not correlate with an improved course of disease like in immunoglobulindeficient BALB/c mice [22].
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respectively. In B6.gld T cells the level of IFN-c and IL4 mRNA was elevated 27- and 10-fold, respectively (Fig. 4B). Both IFN-c and IL-4 are upregulated strongly in B6.gld T cells and marginally in B6.gld.TNF–/– T cells. The fact that the expression of both cytokines is induced in a similar way in the two mouse strains points to a general phenomenon of the gld mutation. The balanced up-regulation of IFN-c and IL-4 mRNA probably prevents a deviation from the Th1 response. 3.4. Histological analysis of the site of infection and the draining LN in B6.gld mice
Fig. 3. Serum concentration of L. major-specific antibodies. The serum concentration of L. major-specific IgM (A), IgG1 (B) and IgG2a (C) was analyzed in B6.WT, BALB/c, B6.gld, B6.TNF–/–, B6.gld.TNF–/– mice after L. major infection. The concentration is shown in relative ELISA units.
3.3. Intrinsic capacity of B6.gld and double-negative B6.gld.TNF–/– T cells to proliferate and to produce IFN-c and IL-4 The proliferation of naive B6.WT, B6.gld and B6.gld.TNF–/– T cells was analyzed to rule out intrinsic deficiencies. T cells were isolated from non-infected mice, labeled with CFSE and stimulated in vitro with anti-CD3 and antiCD28 for 48 h B6.WT T cells divided three times within the period of observation (Fig. 4A) with 22% of cells in the third cycle of cell division. In contrast, both B6.gld and B6.gld.TNF–/– T cells demonstrated an increased cell proliferation with a substantial number of cells already in the fourth cycle of division (Fig. 4A). Almost half of the B6.gld.TNF–/– and B6.gld T cells had divided a third and fourth time (46% and 43%, respectively) within 48 h. A Th1-type response is characterized by IFN-c-expression, a Th2-type response by IL-4 expression. A L. major infection of B6.gld mice results in strong IFN-c-presence [13]. Therefore, the expression of IFN-c and IL-4 mRNA was tested by quantitative Real-Time RT-PCR to investigate the possibility that a deviation from a Th1 response was involved in the susceptibility of B6.gld.TNF–/– mice in our model. Relative to B6.WT T cells, B6.gld.TNF–/– T cells exhibited a three and twofold increased amount of IFN-c and IL-4 mRNA,
The expression of iNOS and the presence of parasites were analyzed in footpads, draining LN and spleen of B6.WT, B6.TNF–/– and B6.gld mice at days 7, 27 and 40 of infection. The expression of iNOS protein in the skin and LN of B6.WT and B6.gld mice was indifferent, both in terms of the overall amount of iNOS protein detected and the distribution and size of the iNOS clusters (Fig. 5A–F; and data not shown). In the LN of B6.TNF–/– mice, the iNOS foci were small, numerous and disperse. In both B6.WT and B6.gld mice they were considerably larger and confluent and lower in number (Fig. 5, panel D–F). In accordance with the limiting dilution analysis the popliteal LN of B6.TNF–/– mice (day 40 of infection) contained large numbers of parasites, whereas in the LN of B6.WT and B6.gld mice fewer parasites were detectable by immunohistology (Fig. 5, panel G–I). In spleen sections of B6.gld mice, which contained a limited number of parasite foci, iNOS protein was expressed (data not shown). This was not the case however, in B6.WT mice, while in the spleens of B6.TNF–/– mice both parasites and iNOS were detectable as reported before [10].
4. Discussion The L. major infection of B6.gld and double-negative B6.gld.TNF–/– mice resulted in a clinical course, which either resembled that of infected B6.WT mice (resolution of the lesion) or of B6.TNF–/– mice (viseralization of the parasite and death), respectively. We did not observe a specific role of FasL for the generation of a protective immune response in our model. In contrast, the almost identical course of infection in B6.TNF–/– and B6.gld.TNF–/– mice clearly demonstrated the central importance of TNF in the anti-leishmanial defense. Data on the role of TNF in the model of murine cutaneous leishmaniasis have been inconsistent and controversial. The pro-inflammatory cytokine TNF was examined extensively because of its potential effector function. Treatment of L. major LV39-infected CBA mice with TNF resulted in an attenuation of the course of disease and the reduction of lesion size and parasite burden [8,25,26]. Application of neutralizing anti-TNF antibodies only led to a transient aggravation of the disease in CBA or BALB/c mice infected with L. major
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Fig. 4. Intrinsic capacity of B6.WT, B6.gld and B6.gld.TNF–/– T cells to proliferate and to produce cytokines in response to CD3/CD28 mAbs. T cells were isolated from LN of naive mice and labeled with CFSE. The proliferation was triggered with CD3/CD28 mAbs coated on the surface of 96-well plates. (A) The single peak (thin line) represents the non-stimulated control cells. The proliferation-dependent decrease of CFSE labeling is shown in dark. (B) The proliferating cells were tested for expression of IL-4 and IFN-c by Real-Time RT-PCR. First, the specifically amplified cytokine cDNA was quantified in relation to a specifically amplified house keeping gene product (b-actin). Second, the relative up-regulation of the gene transcripts of activated B6.WT, B6.gld and B6.gld.TNF–/– T cells was compared to the relative expression of non-activated B6.WT, B6.gld and B6.gld.TNF–/– T cells. Third, the resulting values for activated/nonactivated B6.WT T cells were set at one and the mean of activated/non-activated B6.gld and B6.gld.TNF–/– T cells was depicted as x-fold induction. Every bar represents results generated from T cells taken independently from five mice (n = 5). The activated B6.gld T cells expressed a substantially increased amount of both IL-4 and IFN-c mRNA compared to B6.WT or B6.gld.TNF–/– cells (* P < 0.05).
strain LV39 [25–28]. Gene-targeted mice negative for TNFR 1 or both TNFR 1 and 2 developed a Th1 response, expressed IFN-c and iNOS, and were able to clear L. major strain FRIEDLIN [29]. Taken together, these findings suggested that TNF acts as a co-factor in the development of protective, antileishmanial immunity, but is dispensable for the ultimate control of the infection. Recently, however, infection of TNFnegative mice that had been established directly on a C57BL/6 background with the L. major BNI strain showed a different outcome. The mice were not able to establish protective immunity resulting in visceralization of the parasite and death of the mice [10]. In contrast to TNF the related FasL/Fas pathway has repeatedly been reported to be important for protection against L. major strain LV39. It was found that Fas-deficient MRL/lpr or B6/lpr mice did not resolve their skin lesions after infection although visceralization of the parasites did not occur [14]. An infection of doublenegative B6.gld.TNF–/– mice with L. major LV39 resulted in a more dramatic phenotype including necrosis at the site of infection and spreading of the parasite [12–14]. Nevertheless, the mice did not succumb to the infection. The studies mentioned above used L. major strains FRIEDLIN, LV39 and BNI as well as gene-deficient mice with variable levels of genetic backcrossing. In order to minimize the experimental problems associated with different genetic back-
grounds we developed gene-targeted inbred mouse strains that allowed us to study the individual and cooperative activities of TNF and FasL in leishmaniasis. The TNF gene was targeted in C57BL/6 ES cells directly [15], and the gld mice had been backcrossed to near-homogeneity thus minimizing the introduction of unrelated genes in the double-negative B6.gld.TNF–/– mice [17]. Moreover, in another study we addressed the discrepancy between the published results of L. major infection of TNF R1/2–/– [30,31] and B6.TNF–/– mice [10] and used two strains of L. major, FRIEDLIN and BNI in parallel infection experiments [11]. Our results clearly demonstrated that in some cases the L. major strain used for infection affected the outcome of the disease to a greater extent than the gene-deficiency investigated or the genetic background. The potential mechanism, that lead to this L. major strain-dependent immunity in TNF-negative mice could have been yet undefined structural differences in the lipophosphoglycan surface molecules of the two strains. These differences would trigger different tissue-specific innate effector mechanisms that would result in differential survival [11,32]. In earlier studies consistent results have been described in experimental leishmaniasis of IL-4-deficient mice [33]. In the present study we show that in L. major BNIinfected mice the loss-of-function mutation of FasL, gld, is not part of the protective immune response, whereas TNF is
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Fig. 5. Immunohistological analysis of iNOS expression and distribution of L. major promastigotes in skin lesions and draining LN of L. major infected B6.WT, B6.gld and B6.TNF–/– mice. Sections from foot pads (A–C) and draining LN (D–I) were stained for the presence of iNOS (A–F) or L. major promastigotes (G–I) using the immunoperoxidase technique and aminoethylcarbazole as a substrate (brown). The arrows point to single L. major amastigotes. The magnification is 400 times.
essential to survive an infection with the same strain of L. major. In contrast, infections with the L. major LV39 have provided evidence that FasL rather than TNF is decisive for the complete control of parasite replication [12–14]. Taken together, our present results and the previously published data provide another example for the strong impact of the genetic origin of L. major on the course of infection in immunodeficient mice.
Acknowledgments The study was supported by the Deutsche Forschungsgemeinschaft (Bo 996/3-1 and Bo 996/3-2 to C.B.; Ko 1315/33 to H.K.), a Competitive Research Incentive Grant (JCU to H.K.), the Federal Ministry of Education and Research (BMBF) and by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nürnberg (IZKF NW1 to H.K.).
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