Interleukin-1 Receptor Type 1 Is Essential for Control of Cerebral but Not Systemic Listeriosis
2007; Elsevier BV; Volume: 170; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.060507
ISSN1525-2191
AutoresMartina Deckert, Simona Virna, Monika Sakowicz‐Burkiewicz, Sonja Lütjen, Sabine Soltek, Horst Bluethmann, Dirk Schlüter,
Tópico(s)Immune Response and Inflammation
ResumoListeria monocytogenes may infect the central nervous system and several peripheral organs. To explore the function of IL-1 receptor type 1 (IL-1R1) in cerebral versus systemic listeriosis, IL-1R1−/− and wild-type mice were infected either intracerebrally or intraperitoneally with L. monocytogenes. After intracerebral infection with various numbers of attenuated Listeria, IL-1R1−/− mice succumbed due to an insufficient control of intracerebral Listeria, whereas all wild-type mice survived, efficiently restricting growth of Listeria. IL-1R1−/− mice recruited increased numbers of leukocytes, especially granulocytes, to the brain compared with wild-type mice. In contrast, both IL-1R1−/− and wild-type mice survived a primary and secondary intraperitoneal infection with Listeria without differences in the hepatic bacterial load. In addition, both strains developed similar frequencies of Listeria-specific CD4 and CD8 T cells after primary and secondary intraperitoneal infection. However, an intraperitoneal immunization before intracerebral challenge infection neither protected IL-1R1−/− mice from death nor reduced the intracerebral bacterial load, although numbers of intracerebral Listeria-specific CD4 and CD8 T cells and levels of inducible nitric oxide synthase, tumor necrosis factor, and interferon-γ mRNA were identical in IL-1R1−/− and wild-type mice. Collectively, these findings illustrate a crucial role of IL-1R1 in cerebral but not systemic listeriosis. Listeria monocytogenes may infect the central nervous system and several peripheral organs. To explore the function of IL-1 receptor type 1 (IL-1R1) in cerebral versus systemic listeriosis, IL-1R1−/− and wild-type mice were infected either intracerebrally or intraperitoneally with L. monocytogenes. After intracerebral infection with various numbers of attenuated Listeria, IL-1R1−/− mice succumbed due to an insufficient control of intracerebral Listeria, whereas all wild-type mice survived, efficiently restricting growth of Listeria. IL-1R1−/− mice recruited increased numbers of leukocytes, especially granulocytes, to the brain compared with wild-type mice. In contrast, both IL-1R1−/− and wild-type mice survived a primary and secondary intraperitoneal infection with Listeria without differences in the hepatic bacterial load. In addition, both strains developed similar frequencies of Listeria-specific CD4 and CD8 T cells after primary and secondary intraperitoneal infection. However, an intraperitoneal immunization before intracerebral challenge infection neither protected IL-1R1−/− mice from death nor reduced the intracerebral bacterial load, although numbers of intracerebral Listeria-specific CD4 and CD8 T cells and levels of inducible nitric oxide synthase, tumor necrosis factor, and interferon-γ mRNA were identical in IL-1R1−/− and wild-type mice. Collectively, these findings illustrate a crucial role of IL-1R1 in cerebral but not systemic listeriosis. Infection of the central nervous system (CNS) by Listeria monocytogenes (LM), a gram-positive facultative intracellular rod, may cause severe meningitis, encephalitis, and brain abscess. The risk for development and outcome of cerebral listeriosis is dependent on the immune status of the individual; immunocompromised, elderly, and very young individuals are at increased risk for the development of cerebral listeriosis, which has a lethality of 30% despite antibiotic treatment.1Hof H Nichterlein T Kretschmar M Management of listeriosis.Clin Microbiol Rev. 1997; 10: 345-357PubMed Google Scholar The virulence of LM is dependent on several factors, including bacterial actin polymerase (ActA). ActA enables intracellular LM to move through the cytoplasm of the host cell by polymerization of host cell actin and to induce the formation of pseudopods, which extend from the infected cell to neighboring cells, triggering the uptake into uninfected cells.2Domann E Wehland J Rohde M Pistor S Hartl M Goebel W Leimeister-Wachter M Wuenscher M Chakraborty T A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin.EMBO J. 1992; 11: 1981-1990Crossref PubMed Scopus (326) Google Scholar, 3Kocks C Gouin E Tabouret M Berche P Ohayon H Cossart P L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein.Cell. 1992; 68: 521-531Abstract Full Text PDF PubMed Scopus (658) Google Scholar, 4Dussurget O Pizarro-Cerda J Cossart P Molecular determinants of Listeria monocytogenes virulence.Annu Rev Microbiol. 2004; 58: 587-610Crossref PubMed Scopus (230) Google Scholar LM deficient in ActA-dependent direct cell-to-cell spread are highly attenuated in murine systemic listeriosis.5Goossens PL Milon G Induction of protective CD8+ T lymphocytes by an attenuated Listeria monocytogenes actA mutant.Int Immunol. 1992; 4: 1413-1418Crossref PubMed Scopus (63) Google Scholar Furthermore, intracerebral (i.c.) infection with actA-deficient LM (ΔactA LM) induces nonlethal cerebral listeriosis, whereas i.c. inoculation of wild-type (WT) LM inevitably causes death of mice within 4 to 5 days before the onset of a LM-specific T cell response.6Schlüter D Oprisiu SB Chahoud S Weiner D Wiestler OD Hof H Deckert-Schlüter M Systemic immunization induces protective CD4+ and CD8+ T cell-mediated immune responses in murine Listeria monocytogenes meningoencephalitis.Eur J Immunol. 1995; 25: 2384-2391Crossref PubMed Scopus (43) Google Scholar, 7Virna S Deckert M Lütjen S Soltek S Foulds KE Shen H Körner H Sedgwick JD Schlüter D TNF is important for pathogen control and limits brain damage in murine cerebral listeriosis.J Immunol. 2006; 177: 3972-3982PubMed Google Scholar Murine cerebral listeriosis caused by infection with WT LM is characterized by a strong multiplication of LM in choroid plexus epithelial cells, ependymal cells, macrophages, microglia, and some neurons resulting in a prominent meningitis, ventriculitis, and lethal necrotizing brain stem encephalitis.8Schlüter D Chahoud S Lassmann H Schumann A Hof H Deckert-Schlüter M Intracerebral targets and immunomodulation of murine Listeria monocytogenes meningoencephalitis.J Neuropathol Exp Neurol. 1996; 55: 14-24Crossref PubMed Scopus (44) Google Scholar In addition, massive brain edema and neuronal apoptosis develop, which are reduced by intrathecally produced interleukin (IL)-10.9Deckert M Soltek S Geginat G Lütjen S Montesinos-Rongen M Hof H Schlüter D Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis.Infect Immun. 2001; 69: 4561-4571Crossref PubMed Scopus (68) Google Scholar An active systemic immunization before i.c. challenge infection induces LM-specific CD4 and CD8 T cells and reduces spread of LM from the ventricular system to the periventricular brain tissue and the brain stem, thereby preventing death in 60% of mice.6Schlüter D Oprisiu SB Chahoud S Weiner D Wiestler OD Hof H Deckert-Schlüter M Systemic immunization induces protective CD4+ and CD8+ T cell-mediated immune responses in murine Listeria monocytogenes meningoencephalitis.Eur J Immunol. 1995; 25: 2384-2391Crossref PubMed Scopus (43) Google Scholar, 10Kwok LY Miletic H Lütjen S Soltek S Deckert M Schlüter D Protective immunosurveillance of the central nervous system by Listeria-specific CD4 and CD8 T cells in systemic listeriosis in the absence of intracerebral Listeria.J Immunol. 2002; 169: 2010-2019PubMed Google Scholar In addition to T cells, a large number of macrophages and granulocytes are recruited to the LM-infected brain accompanied by an up-regulation of several cytokines including IL-1. The biological activity of IL-1α and IL-1β is exerted via the IL-1 receptor type 1 (IL-1R1).11Glaccum MB Stocking KL Charrier K Smith JL Willis CR Maliszewski C Livingston DJ Peschon JJ Morrissey PJ Phenotypic and functional characterization of mice that lack the type I receptor for IL-1.J Immunol. 1997; 159: 3364-3371PubMed Google Scholar, 12Labow M Shuster D Zetterstrom M Nunes P Terry R Cullinan EB Bartfai T Solorzano C Moldawer LL Chizzonite R McIntyre KW Absence of IL-1 signaling and reduced inflammatory response in IL-1 type I receptor-deficient mice.J Immunol. 1997; 159: 2452-2461PubMed Google Scholar The IL-1 receptor type 2 is not involved in signal transduction but acts as a decoy receptor, which can be shed from the cell surface and inhibits binding of IL-1 to the IL-1R1. In addition, binding and signaling of IL-1 is regulated by an endogenous IL-1R antagonist (IL-1Ra), which inhibits binding of IL-1 to IL-1R1.13Sims JE IL-1 and IL-18 receptors, and their extended family.Curr Opin Immunol. 2002; 14: 117-122Crossref PubMed Scopus (181) Google Scholar In murine systemic listeriosis, IL-1R1−/− on a mixed 129/Sv × C57BL/6 background11Glaccum MB Stocking KL Charrier K Smith JL Willis CR Maliszewski C Livingston DJ Peschon JJ Morrissey PJ Phenotypic and functional characterization of mice that lack the type I receptor for IL-1.J Immunol. 1997; 159: 3364-3371PubMed Google Scholar, 12Labow M Shuster D Zetterstrom M Nunes P Terry R Cullinan EB Bartfai T Solorzano C Moldawer LL Chizzonite R McIntyre KW Absence of IL-1 signaling and reduced inflammatory response in IL-1 type I receptor-deficient mice.J Immunol. 1997; 159: 2452-2461PubMed Google Scholar as well as female B6CBAF1/J overexpressing IL-1Ra14Hirsch E Irikura VM Paul SM Hirsh D Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice.Proc Natl Acad Sci USA. 1996; 93: 11008-11013Crossref PubMed Scopus (248) Google Scholar had an increased susceptibility with increased numbers of LM in liver and spleen and decreased survival rates, respectively, as compared with respective WT animals. However, after backcrossing to the C57BL/6 background, IL-1R1−/− mice were as resistant as WT mice to systemic listeriosis,11Glaccum MB Stocking KL Charrier K Smith JL Willis CR Maliszewski C Livingston DJ Peschon JJ Morrissey PJ Phenotypic and functional characterization of mice that lack the type I receptor for IL-1.J Immunol. 1997; 159: 3364-3371PubMed Google Scholar further illustrating that C57BL/6 mice are more resistant to listeriosis than 129/Sv and CBA/J mice.15Cheers C McKenzie IF Resistance and susceptibility of mice to bacterial infection: genetics of listeriosis.Infect Immun. 1978; 19: 755-762PubMed Google Scholar In addition, in LM-susceptible mice a blockade of IL-1R1 resulted in an increased bacterial load on systemic infection,16Havell EA Moldawer LL Helfgott D Kilian PL Sehgal PB Type I IL-1 receptor blockade exacerbates murine listeriosis.J Immunol. 1992; 148: 1486-1492PubMed Google Scholar and a simultaneous inhibition of IL-1α, IL-1β, and IL-1R1 prevented the recruitment of neutrophils to LM-associated inflammatory foci.17Rogers HW Tripp CS Schreiber RD Unanue ER Endogenous IL-1 is required for neutrophil recruitment and macrophage activation during murine listeriosis.J Immunol. 1994; 153: 2093-2101PubMed Google Scholar In this study, we report on the role of IL-1R1-mediated immune reactions in cerebral and systemic listeriosis of LM-resistant C57BL/6 mice and illustrate that IL-1R1 is critical for control of LM in the brain and survival of both primary and secondary cerebral listeriosis only, but not of systemic listeriosis. Age- and sex-matched C57BL/6 WT, obtained from Harlan-Winkelmann (Borchen, Germany), and IL-1R1−/− mice,12Labow M Shuster D Zetterstrom M Nunes P Terry R Cullinan EB Bartfai T Solorzano C Moldawer LL Chizzonite R McIntyre KW Absence of IL-1 signaling and reduced inflammatory response in IL-1 type I receptor-deficient mice.J Immunol. 1997; 159: 2452-2461PubMed Google Scholar backcrossed to C57BL/6 mice for 10 generations, were used. All animals were kept under conventional conditions in an isolation facility throughout the experiments. Experiments were approved and supervised by local governmental institutions. WT LM (serovar 1/2a, EGD, SLCC 5835), recombinant ovalbumin-expressing LM (LMova),18Shen H Miller JF Fan X Kolwyck D Ahmed R Harty JT Compartmentalization of bacterial antigens: differential effects on priming of CD8 T cells and protective immunity.Cell. 1998; 92: 535-545Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar ΔactA LM,19Hauf N Goebel W Fiedler F Sokolovic Z Kuhn M Listeria monocytogenes infection of P388D1 macrophages results in a biphasic NF-kappaB (RelA/p50) activation induced by lipoteichoic acid and bacterial phospholipases and mediated by IkappaBalpha and IkappaBbeta degradation.Proc Natl Acad Sci USA. 1997; 94: 9394-9399Crossref PubMed Scopus (88) Google Scholar and recombinant ovalbumin-expressing ΔactA LM (ΔactA LMova)7Virna S Deckert M Lütjen S Soltek S Foulds KE Shen H Körner H Sedgwick JD Schlüter D TNF is important for pathogen control and limits brain damage in murine cerebral listeriosis.J Immunol. 2006; 177: 3972-3982PubMed Google Scholar were grown in tryptose soy broth, and aliquots of log-phase cultures were stored at −80°C. For each experiment, the respective strain of LM was thawed from the stock solution and diluted appropriately in sterile pyrogen-free phosphate-buffered saline (PBS) (pH 7.4). Anesthetized mice were infected intracerebrally with 1 × 102 WT LM or 6 × 101, 6 × 102, and 6 × 103 ΔactA LM, respectively, into the right caudate nucleus as described previously.8Schlüter D Chahoud S Lassmann H Schumann A Hof H Deckert-Schlüter M Intracerebral targets and immunomodulation of murine Listeria monocytogenes meningoencephalitis.J Neuropathol Exp Neurol. 1996; 55: 14-24Crossref PubMed Scopus (44) Google Scholar For immunization, mice were infected intraperitoneally (i.p.) with 1 × 106 ΔactA LM or 5 × 104 LMova 28 days before i.c. challenge infection with 6 × 102 ΔactA LM or ΔactA LMova, respectively. For systemic listeriosis, mice were i.p. infected with 1 × 104 WT LM for primary and 1 × 106 WT LM for secondary infection. On the other hand, systemic listeriosis was induced by i.p. infection with 5 × 104 LMova and i.p. reinfection with 5 × 106 LMova. For each experiment, the bacterial dose used for infection was controlled by plating an inoculum on tryptose soy agar and counting colonies after incubation at 37°C for 48 hours. For immunohistochemistry on frozen sections, mice were perfused intracardially with 0.9% NaCl in methoxyflurane anesthesia. Brains of three to six animals per group were dissected, and blocks were mounted on thick filter paper with Tissue-Tek O.C.T. Compound (Miles Scientific, Naperville, IL), snap-frozen in isopentane precooled on dry ice, and stored at −80°C. In addition to H&E staining, immunohistochemistry was performed on frozen sections as described previously.20Schlüter D Lohler J Deckert M Hof H Schwendemann G Toxoplasma encephalitis of immunocompetent and nude mice: immunohistochemical characterisation of Toxoplasma antigen, infiltrates and major histocompatibility complex gene products.J Neuroimmunol. 1991; 31: 185-198Abstract Full Text PDF PubMed Scopus (72) Google Scholar In brief, sections were stained by an indirect immunoperoxidase protocol using rat anti-mouse CD45 (clone M1/9.3.4.HL.2), CD4 (clone G.K.1.5.), CD8 (clone 2.43), and Ly6-G (clone RB6-8C5; all antibodies from the American Type Culture Collection, Manassas, VA) as primary antibodies and peroxidase-linked sheep anti-rat IgG F(ab′)2 (Amersham Pharmacia, Freiburg, Germany) as secondary antibody. In addition, the avidin-biotin complex technique using rat anti-mouse F4/80 (clone F4/80; American Type Culture Collection) as primary antibody, biotinylated mouse serum-preadsorbed mouse anti-rat IgG F(ab′)2 (Dianova, Hamburg, Germany) as secondary antibody, and streptavidin-biotin complex (Dako, Hamburg, Germany) was used. LM was demonstrated immunohistochemically by incubating sections with a polyclonal rabbit anti-LM antiserum (Difco, Freiburg, Germany) followed by peroxidase-labeled goat anti-rabbit IgG F(ab′)2 fragments (Dianova). Brain edema was histologically analyzed on immunostained frozen sections by visualization of immunoglobulin deposits in brain tissue. Sections were stained with goat anti-mouse IgG and goat anti-mouse IgM (Vector Laboratories, Burlingame, CA) followed by biotin-labeled anti-goat antibodies and the Vectastain Elite ABC kit (Vector Laboratories). Peroxidase reaction products were visualized using 3,3′-diaminobenzidine and H2O2 as co-substrate. Sections were lightly counterstained with hemalum. CSF (∼7 μl/mouse) was obtained from mice after intracardial perfusion with 0.9% NaCl by puncturing the cisterna cerebellomedullaris with a fine glass capillary as described previously.21Schlüter D Deckert-Schlüter M Schwendemann G Brunner H Hof H Expression of major histocompatibility complex class II antigens and levels of interferon-gamma, tumour necrosis factor, and interleukin-6 in cerebrospinal fluid and serum in Toxoplasma gondii-infected SCID and immunocompetent C.B-17 mice.Immunology. 1993; 78: 430-435PubMed Google Scholar The CSF of five mice per experimental group was pooled and mixed with an equal volume of sterile distilled 0.1 mol/L PBS to reduce losses. CSF was stored at −80°C before being analyzed in a commercially available mouse IL-1α and IL-1β-ELISA (Quantikine M kits; both from R&D Systems, Wiesbaden, Germany), which were used as recommended by the manufacturer. At the indicated time points postinfection (p.i.), brains and livers were dissected from sacrificed mice and homogenized separately with sterile tissue grinders. Tenfold serial dilutions of the homogenates were plated on tryptose-soy agar. Bacterial colonies were counted microscopically after incubation at 37°C for 48 hours. At the indicated days p.i., animals were anesthetized with Metofane (Janssen, Neuss, Germany) and intracardially perfused with 0.9% NaCl to remove contaminating intravascular leukocytes from the brain. Thereafter, brains were dissected, minced through a 100-μm cell strainer (Becton-Dickinson, Heidelberg, Germany), and leukocytes separated by Percoll gradient centrifugation (Amersham Pharmacia) as described previously.22Schlüter D Hein A Dörries R Deckert-Schlüter M Different subsets of T cells in conjunction with natural killer cells, macrophages, and activated microglia participate in the intracerebral immune response to Toxoplasma gondii in athymic nude and immunocompetent rats.Am J Pathol. 1995; 146: 999-1007PubMed Google Scholar Leukocytes from spleen were isolated by mincing spleen tissue through a 70-μm cell strainer (Becton-Dickinson). Isolated cerebral leukocytes were analyzed by double or triple immunofluorescence staining followed by flow cytometry. All antibodies were rat anti-mouse antibodies from Becton-Dickinson. To block unspecific binding of antibodies to FC receptors, i.c. leukocytes were first incubated with anti-CD16/32 (clone 2.4G2) at 4°C for 10 minutes. Subsequently, the cells were stained with a cocktail of fluorochrome-labeled antibodies at 4°C for 20 minutes. CD4 and CD8 T cells were detected by staining with anti-CD4-PE (clone RM4-5) and anti-CD8-FITC (clone 53-6.7) in combination with anti-CD45-CyChrome (clone 30-F11). Macrophages and granulocytes were identified by staining with anti-Ly6G-PE (clone RB6-8C5), anti-CD11b-FITC (clone M1/70), and anti-CD45-CyChrome. Macrophages are CD11b+ CD45high; granulocytes are CD11b+ CD45high Ly6Ghigh.23Schlüter D Meyer T Strack A Reiter S Kretschmar M Wiestler OD Hof H Deckert M Regulation of microglia by CD4+ and CD8+ T cells: selective analysis in CD45-congenic normal and Toxoplasma gondii-infected bone marrow chimeras.Brain Pathol. 2001; 11: 44-55Crossref PubMed Scopus (33) Google Scholar Control staining was performed with isotype-matched control antibodies. Flow cytometry was performed on a FACScan (Becton-Dickinson), and the data were analyzed with WinMDI or Cell Quest software. The frequency of i.c. LM-specific CD4 and CD8 T cells was determined by an interferon (IFN)-γ-specific ELISPOT as described previously.9Deckert M Soltek S Geginat G Lütjen S Montesinos-Rongen M Hof H Schlüter D Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis.Infect Immun. 2001; 69: 4561-4571Crossref PubMed Scopus (68) Google Scholar In brief, isolated i.c. and splenic leukocytes (1 × 105/well, 1 × 104/well, and 1 × 103/well) were added in triplicate to 96-well ELISPOT plates coated with rat anti-mouse IFN-γ (Biosource, Camarillo, CA). Isolated leukocytes were co-incubated with spleen cells from noninfected WT C57BL/6 mice (2 × 105/well), which were either preloaded with listeriolysin (LLO)190–201- (10−6) or ovalbumin (OVA)257–264- (10−8 m/moL) peptide. Controls included co-incubation of isolated leukocytes with spleen cells without peptide loading. ELISPOT plates were incubated overnight and developed with biotin-labeled rat anti-mouse IFN-γ, peroxidase-conjugated streptavidin, and aminoethylcarbazole (Sigma-Aldrich, St. Louis, MO). The spots were counted microscopically, and the frequency of antigen-specific cells was calculated from triplicate wells as the number of spots per leukocytes seeded. IFN-γ, tumor necrosis factor (TNF), inducible nitric-oxide synthase (iNOS), granulocyte chemotactic protein-2 (GCP-2), RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-2 (MIP-2) mRNA transcripts, and hydroxyphosphoribosyltransferase (HPRT) mRNA expression were analyzed in brain tissue homogenates following a protocol described in detail previously.9Deckert M Soltek S Geginat G Lütjen S Montesinos-Rongen M Hof H Schlüter D Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis.Infect Immun. 2001; 69: 4561-4571Crossref PubMed Scopus (68) Google Scholar Primer and probe sequences for IFN-γ, TNF, iNOS, and HPRT were as described previously.24Deckert-Schlüter M Albrecht S Hof H Wiestler OD Schlüter D Dynamics of the intracerebral and splenic cytokine mRNA production in Toxoplasma gondii-resistant and -susceptible congenic strains of mice.Immunology. 1995; 85: 408-418PubMed Google Scholar, 25Schlüter D Kaefer N Hof H Wiestler OD Deckert-Schlüter M Expression pattern and cellular origin of cytokines in the normal and Toxoplasma gondii-infected murine brain.Am J Pathol. 1997; 150: 1021-1035PubMed Google Scholar For the detection of MIP-2, GCP-2, and RANTES mRNA the following primers and probes were used: MIP-2 (forward): 5′-GCTGTTGTGGCCAGTGAAC-3′, MIP-2 (reverse): 5′-TTCAGGTCAAGGCAAACTT-3′, MIP-2 (probe): 5′-TCCAGAGCTTGAGTGTGACG-3′; GCP2 (forward): 5′-GAAAGCTAAGCGGAATGCAC-3′, GCP2 (reverse): 5′-GGGACAATGGTTTCCCTTTT-3′, GCP2 (probe): 5′-CCACCCACGGATTTTCTTTA-3′; and RANTES (forward): 5′-GGTACCATGAAGATCTCTGC-3′, RANTES (reverse): 5′-GGGTCAGAATCAAGAAACCC-3′, RANTES (probe): 5′-CTCTCCCTAGAGCTGCCTCG-3′. RNA was extracted from tissue homogenates using an RNA extraction kit (Dianova). After reverse transcription of mRNA using the Superscript RT kit (Life Technologies, Eggenstein, Germany), PCR reactions were performed in a volume of 30 μl. PCR reaction conditions were optimized for each set of primers. PCR was performed at different cycle numbers to ensure that amplification occurred in the linear range. PCR products were electrophoresed through an agarose gel and the DNA was transferred to a nylon membrane (Amersham Pharmacia). Blots were hybridized using specific oligonucleotide probes, which were 3′-end-labeled with digoxigenin by use of a DIG oligonucleotide 3′-end labeling kit (Roche, Mannheim, Germany). A DIG luminescent kit (Roche) was used to visualize the hybridization products. Quantitation of RNA was performed with an imaging densitometer and Quantity One software (Bio-Rad, München, Germany) as described previously.9Deckert M Soltek S Geginat G Lütjen S Montesinos-Rongen M Hof H Schlüter D Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis.Infect Immun. 2001; 69: 4561-4571Crossref PubMed Scopus (68) Google Scholar The intensity of each band was determined and related to the intensity of the respective autoradiogram band obtained for the internal control, HPRT. The results were expressed as fold increase over the respective RNA levels in uninfected animals of the same strain. For statistical evaluation of the experimental data, the WINKS software (Texasoft, Cedar Hill, TX) was used. Survival analysis was performed with the Mantel-Haenszel log-rank test. Student's t-test and the Wilcoxon rank sum test were used to analyze differences in CFU and cell numbers between WT and TNF-deficient mice. P values <0.05 were accepted as significant. To determine the kinetics of IL-1α and IL-1β in cerebral listeriosis, both nonimmunized and immunized WT mice were infected i.c. with 6 × 102 ΔactA LM and cytokines were measured in CSF by ELISA (Figure 1, A and B). Both IL-1α and IL-1β rapidly increased and peaked as early as one day after i.c. infection, with higher levels in immunized than nonimmunized mice. Thereafter, both cytokines sharply declined in the CSF of both nonimmunized and immunized animals and were close or identical to baseline levels of uninfected mice at day 7 p.i. These findings provide the basis to explore further the function of IL-1R1 in murine cerebral listeriosis. After i.c. infection with 1 × 102 WT LM, all IL-1R1−/− and WT mice succumbed (Figure 2A). However, IL-1R1−/− mice died significantly earlier than WT animals and harbored significantly more LM in their brains at day 1 p.i. (Figure 2B). To analyze further whether IL-1R1−/− mice are more susceptible to cerebral listeriosis than WT mice, animals were infected with various amounts of attenuated ΔactA LM. After infection with 6 × 101 and 6 × 102 ΔactA LM, 90% of nonimmunized IL-1R1−/− mice succumbed up to days 7 and 6 p.i., respectively, whereas all WT mice survived (Figure 2, C and E). A further increase of the dose of infection to 6 × 103 ΔactA LM resulted in death of 100% of IL-1R1−/− mice until day 4 p.i., whereas all WT mice still survived (Figure 2G). In each of the experimental groups, the poor outcome of IL-1R1−/− mice was paralleled by a significantly increased i.c. bacterial load as compared with WT mice (Figure 2, D, F, and H). Collectively, these findings illustrate that the IL-1R1 is essential for survival and efficient control of LM in the CNS. To determine the impact of IL-1R1 on the spread and distribution of LM as well as the accompanying inflammatory leukocytes, a detailed histopathological analysis was performed after i.c. infection with 6 × 102 ΔactA LM. Remarkably, differences were conspicuous as early as day 1 p.i. Individual WT mice harbored a few meningeal CD45+ infiltrates, corresponding mainly to Ly6G+ granulocytes, F4/80+ macrophages, and single CD4+ and CD8+ T cells. In addition, single CD45+ leukocytes were present in the lateral ventricle of WT mice, whereas bacteria were only exceptionally visible in the meninges and the ventricular system. In marked contrast, IL-1R1−/− mice had already developed ventriculitis involving the lateral, third, and fourth ventricle in addition to meningitis with LM homing to these target sites. CD45+ leukocytes, consisting predominantly of Ly6G+ granulocytes and a few F4/80+ macrophages as well as single CD4+ and CD8+ T and B220+ B cells, were present in the meninges. Cerebral listeriosis progressed more rapidly in IL-1R1−/− mice as compared with WT animals. At day 3 p.i., IL-1R1−/− mice were unable to restrict bacteria to the ventricular system (Figure 3a), thereby allowing diffuse bacterial infiltration of the periventricular brain tissue, the basal ganglia, the hippocampus, the neocortex, and the cerebellum. This widespread distribution of LM was accompanied by a diffuse encephalitis affecting the same anatomical structures, strong inflammation of the massively enlarged lateral, third, and fourth ventricles, in addition to meningitis (Figure 3, c and e). In contrast, both bacteria and inflammatory CD45+ leukocytes were largely confined to the ventricular lumen and the subependymal brain tissue in WT mice (Figure 3, b, d, and f). At day 5 p.i., the brains of terminally ill IL-1R1−/− mice harbored huge numbers of bacteria covering the ventricular system as well as the periventricular brain tissue, and inflammation was widespread (Figure 3, g and k). CD45+ leukocytes were scattered throughout the brain, in particular infiltrating the basal ganglia, the hippocampus, and wide areas of the neocortex (Figure 3i). Infiltrates consisted of large numbers of Ly6G+ granulocytes, F4/80+ macrophages/microglial cells, and some CD4+ and CD8+ T cells. At this time point, ventriculitis was less severe in WT mice with the ependyma being partly intact (Figure 3, j and l), thereby restricting bacteria mainly to the lumen of the ventricular system and the ependyma (Figure 3h). Consequently, inflammatory infiltrates were mainly located in the ventricular system, only spreading to the basal ganglia immediately adjacent to the lateral ventricle (Figure 3, j and l). To quantitate the increased numbers of inflammatory leukocytes and to define the subtypes of inflammatory leukocytes, i.c. infiltrates were analyzed by flow cytometry after i.c. infection with 6 × 102 ΔactA LM. At days 1 and 3 p.i. with ΔactA LM, IL-1R1−/− mice harbored significantly more i.c. inflammatory leukocytes than WT animals (Figure 4A). This increase was caused by an enhanced recruitment of granulocytes into the CNS of IL-1R1−/− mice, whereas other subpopulations of leukocytes, ie, CD4 and CD8 T cells and macrophages, did not differ between the two strains of mice (Figure 4B). An increased recruitmen
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