Distinct Roles for Lymphotoxin-α and Tumor Necrosis Factor in the Control of Leishmania donovani Infection
2004; Elsevier BV; Volume: 165; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63262-2
ISSN1525-2191
AutoresChristian Engwerda, Manabu Ato, Simona Stäger, Clare Alexander, Amanda C. Stanley, Paul M. Kaye,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoTumor necrosis factor (TNF) is critical for the control of visceral leishmaniasis caused by Leishmania donovani. However, the role of the related cytokine lymphotoxin (LT) α in this infection is unknown. Here we report that C57BL/6 mice deficient in TNF (B6.TNF−/−) or LTα (B6.LTα−/−) have increased susceptibility to hepatic L. donovani infection. Furthermore, the outcome of infection in bone marrow chimeric mice is dependent on donor hematopoietic cells, indicating that developmental defects in lymphoid organs were not responsible for increased susceptibility to L. donovani. Although both LTα and TNF regulated the migration of leukocytes into the sinusoidal area of the infected liver, their roles were distinct. LTα was essential for migration of leukocytes from periportal areas, an event consistent with LTα-dependent up-regulation of VCAM-1 on liver sinusoid lining cells, whereas TNF was essential for leukocyte recruitment to the liver. During visceral leishmaniasis, both cytokines were produced by radio-resistant cells and by CD4+ T cells. LTα and TNF production by the former was required for granuloma assembly, while production of these cytokines by CD4+ T cells was necessary to control parasite growth. The production of inducible nitric oxide synthase was also found to be deficient in TNF- and LTα-deficient infected mice. These results demonstrate that both LTα and TNF are required for control of L. donovani infection in noncompensatory ways. Tumor necrosis factor (TNF) is critical for the control of visceral leishmaniasis caused by Leishmania donovani. However, the role of the related cytokine lymphotoxin (LT) α in this infection is unknown. Here we report that C57BL/6 mice deficient in TNF (B6.TNF−/−) or LTα (B6.LTα−/−) have increased susceptibility to hepatic L. donovani infection. Furthermore, the outcome of infection in bone marrow chimeric mice is dependent on donor hematopoietic cells, indicating that developmental defects in lymphoid organs were not responsible for increased susceptibility to L. donovani. Although both LTα and TNF regulated the migration of leukocytes into the sinusoidal area of the infected liver, their roles were distinct. LTα was essential for migration of leukocytes from periportal areas, an event consistent with LTα-dependent up-regulation of VCAM-1 on liver sinusoid lining cells, whereas TNF was essential for leukocyte recruitment to the liver. During visceral leishmaniasis, both cytokines were produced by radio-resistant cells and by CD4+ T cells. LTα and TNF production by the former was required for granuloma assembly, while production of these cytokines by CD4+ T cells was necessary to control parasite growth. The production of inducible nitric oxide synthase was also found to be deficient in TNF- and LTα-deficient infected mice. These results demonstrate that both LTα and TNF are required for control of L. donovani infection in noncompensatory ways. 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To define the role of LTα in murine VL, and distinguish this role from that of TNF, we have analyzed the response to L. donovani infection in LTα- and TNF-deficient mice, as well as in chimeras generated using LTα−/− and TNF−/− BM cells. Data indicate that TNF and LTα are both important for host resistance to L. donovani in the liver in the first 14 days of infection, whereas later in infection, TNF but not LTα is critical for host survival. LTα and TNF production by radio-resistant cells was found to be required for efficient granuloma formation, although each cytokine appeared to be necessary for different stages in the recruitment of leukocytes. Furthermore, TNF and LTα produced by CD4+ T cells had nonredundant roles in host resistance and ultimately host survival. C57BL/6 mice were purchased from Tuck and Co. (Essex, UK) and were housed under conventional conditions. Mice deficient in TNF (B6.TNF−/−)12Korner H Cook M Riminton DS Lemckert FA Hoek RM Ledermann B Kontgen F Fazekas de St Groth B Sedgwick JD Distinct roles for lymphotoxin-alpha and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue.Eur J Immunol. 1997; 27: 2600-2609Crossref PubMed Scopus (276) Google Scholar and LTα−/− (LTα−/−)16De Togni P, Goellner J, Ruddle NH, Streeter PR, Fick A, Mariathasan S, Smith SC, Carlson R, Shornick LP, Strauss-Schoenberger J, Russell JH, Karr R, Chaplin DD: Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science, 264:703-707Google Scholar, 40Riminton SD Korner H Strickland DH Lemckert FA Pollard JD Sedgwick JD Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice.J Exp Med. 1998; 187: 1517-1528Crossref PubMed Scopus (145) Google Scholar were obtained from Bantin & Kingman (Hull, UK). B6.CD45.1 mice were obtained from Charles River (IFFA Credo, Saint Germain Sur L'Arbresle, France). B6.RAG-1−/− mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mouse strains were bred at the London School of Hygiene and Tropical Medicine under barrier conditions. Mice used in all experiments were sex-matched and used at 6 weeks of age. Chimeric mice were prepared by irradiating animals twice (48 hours apart) with 5.5 Gy and then engrafting with 107 fresh BM cells intravenously via the lateral tail vein within 2 hours of the second radiation exposure. Mice were maintained on antibiotics for 4 weeks after engraftment and infected with parasites 8 weeks after receiving BM. L. donovani (LV9) was maintained by passage in Syrian hamsters and amastigotes were isolated from infected spleens, as previously described.34Smelt SC Engwerda CR McCrossen M Kaye PM Destruction of follicular dendritic cells during chronic visceral leishmaniasis.J Immunol. 1997; 158: 3813-3821PubMed Google Scholar Mice were infected at 6 weeks of age by injecting 2 × 107 amastigotes intravenously via the lateral tail vein. Mice were killed at times indicated in the text by cervical dislocation and bled by severing the aorta. Livers and spleens were removed and parasite burden was determined from Giemsa-stained impression smears.41Engwerda CR Murphy ML Cotterell SE Smelt SC Kaye PM Neutralization of IL-12 demonstrates the existence of discrete organ-specific phases in the control of Leishmania donovani.Eur J Immunol. 1998; 28: 669-680Crossref PubMed Scopus (150) Google Scholar Parasite burden was expressed in Leishman-Donovan units, in which Leishman-Donovan unit is the number of amastigotes per 1000 host nuclei, multiplied by the organ weight.34Smelt SC Engwerda CR McCrossen M Kaye PM Destruction of follicular dendritic cells during chronic visceral leishmaniasis.J Immunol. 1997; 158: 3813-3821PubMed Google Scholar Liver sections from infected mice were stained with hematoxylin and eosin and the granulomatous response was assessed in two ways. First, granuloma density was determined from 25 fields of view per mouse liver (×40 magnification; n = 3 mice/group) and second, the maturation of granulomas was scored around infected Kupffer cells, as described elsewhere.42Murphy ML Cotterell SE Gorak PM Engwerda CR Kaye PM Blockade of CTLA-4 enhances host resistance to the intracellular pathogen, Leishmania donovani.J Immunol. 1998; 161: 4153-4160PubMed Google Scholar Hepatic mononuclear cells were isolated by passing livers through a 200-μm sieve and washing twice with phosphate-buffered saline supplemented with 2% (v/v) fetal calf serum (wash buffer). The cell pellet was resuspended in 33% (v/v) Percoll and centrifuged at 693 × g for 12 minutes at room temperature. Supernatant containing hepatocytes was removed and the leukocyte pellet was washed once in wash buffer, depleted of red blood cells using Red Cell Lysis Buffer (Sigma, Poole, UK), according to the manufacturer's instructions, underlayed with fetal calf serum, and centrifuged at 443 × g for 5 minutes. Cell pellets were washed once more with wash buffer and cells counted. Cells were labeled with antibody and analyzed by FACS as previously described.43Alexander CE Kaye PM Engwerda CR CD95 is required for the early control of parasite burden in the liver of Leishmania donovani-infected mice.Eur J Immunol. 2001; 31: 1199-1210Crossref PubMed Scopus (43) Google Scholar Antibodies used for FACS included rat anti-mouse CR3 (5C6), rat anti-mouse GR-1 (RB6 8C5), and PECy5-conjugated anti-mouse CD4 and CD8 (BioLegend, CA). Nonconjugated rat antibodies were detected with Alexa 488-conjugated goat anti-rat IgG (Molecular Probes, Eugene, OR). Antibodies used for histology included rat anti-mouse ICAM-1 (KAT-1), rat anti-mouse VCAM-1 (M/K-2) (both from Serotec, Kidlington, UK), and rabbit anti-mouse inducible nitric oxide synthase (NOS-2) (Calbiochem, La Jolla, CA). ICAM-1 and VCAM-1 staining was conducted on 6-μm acetone-fixed liver sections, and primary antibodies were detected with appropriate secondary detection reagents according to the manufacturer's instructions (Vector Laboratories, Peterborough, UK), and as previously described.34Smelt SC Engwerda CR McCrossen M Kaye PM Destruction of follicular dendritic cells during chronic visceral leishmaniasis.J Immunol. 1997; 158: 3813-3821PubMed Google Scholar NOS-2 staining was performed on 6-μm paraformaldehyde-fixed liver sections, and the primary antibody detected with an alkaline phosphatase-conjugated goat anti-rabbit antibody (Jackson Laboratories, West Grove, PA). Alkaline phosphatase was visualized using appropriate detection reagents according to the manufacturer's instructions (Vector Laboratories). Sections were dehydrated and mounted before microscopic examination. In some cases, sections were counterstained with hematoxylin (Sigma) before dehydration and mounting. Spleens were passed through a 20-μm sieve and red blood cells were lysed in Gey's solution (130 mmol/L NH4Cl, 5 mmol/L KCl, 8.4 mmol/L Na2HPO4, 180 μmol/L KH2PO4, 5.6 mmol/L d-glucose, 0.001% (w/v) phenol red, 1 mmol/L MgCl2·6H2O, 280 μmol/L MgSO4.7H2O, 1.5 mmol/L CaCl2, 13 mmol/L NaHCO3) for 8 minutes at room temperature and washed twice in RPMI 1640. Splenic mononuclear cells were then enriched over Histopaque 1083 (1700 rpm, 15 minutes, room temperature; Sigma), washed, and resuspended in complete culture media. CD4+ T cells were positively selected from naïve splenocyte preparations using magnetic-activated cell sorting according to protocols recommended by the manufacturers of the metallo-conjugated anti-CD4 antibodies, and positive selection columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells isolated by this procedure were greater than 98% pure, as assessed by FACS. B6.RAG-1−/− mice were reconstituted with 1 × 106 CD4+ T cells in 200 μl of RPMI 1640 via the lateral tail vein 24 hours before infection with 2 × 107 L. donovani amastigotes. The efficiency of reconstitution was assessed by FACS at the termination of experiments (day 14 after infection) with quantum red-conjugated anti-CD4 (H129.19) monoclonal antibody (Sigma). RNA was isolated from liver tissue using Tri Reagent (Sigma), and an RNAeasy mini kit with on-column DNase digestion (Qiagen, Valencia, CA), according to the manufacturers' instructions. RNA was reverse-transcribed into cDNA as described previously.30Engwerda CR Mynott TL Sawhney S De Souza JB Bickle QD Kaye PM Locally up-regulated lymphotoxin alpha, not systemic tumor necrosis factor alpha, is the principle mediator of murine cerebral malaria.J Exp Med. 2002; 195: 1371-1377Crossref PubMed Scopus (216) Google Scholar The number of NOS-2, LTα, TNF, and HPRT (housekeeping gene) cDNA molecules in each sample was calculated by real-time reverse transcriptase-polymerase chain reaction using TaqMan probes in an Assay-on-Demand Gene Expression kit (Applied Biosystems, Foster City, CA) and a Corbett Research RG-3000 Rotor Gene, according to the manufacturers instructions. Standard curves were constructed with known amounts of NOS-2, LTα, TNF, and HPRT cDNA, and the number of cytokine molecules per 1000 HPRT molecules in each sample was calculated. The statistical significance of differences between different groups was tested using the unpaired Student's t-test with SigmaPlot software (SPSS Inc., Richmond, CA), except when the distribution of hepatic histological responses were compared using χ2 analysis with Microsoft Excel software (Microsoft Corp., Seattle, WA). All data are presented as the mean values ± standard errors unless otherwise stated. To distinguish the roles of LTα and TNF in the liver during VL, we infected mice deficient in these cytokines (B6.LTα−/− and B6.TNF−/−, respectively) with L. donovani and followed the course of infection throughout 56 days. In C57BL/6 mice, hepatic parasite burden peaked between days 14 to 28 after infection before resolving (Figure 1A). The liver parasite burdens in both LTα- and TNF-deficient mice were significantly increased early in infection (day 14 after infection), suggesting a critical role for both these cytokines in early control of L. donovani growth in the liver. However, after day 14 of infection, distinct patterns of parasite growth emerged in LTα- and TNF-deficient mice. B6.LTα−/− mice were able to control infection, whereas parasite growth continued in the livers of B6.TNF−/− mice, and these animals died between days 42 to 56 after infection, as previously reported.21Murray HW Jungbluth A Ritter E Montelibano C Marino MW Visceral leishmaniasis in mice devoid of tumor necrosis factor and response to treatment.Infect Immun. 2000; 68: 6289-6293Crossref PubMed Scopus (94) Google Scholar Whether the TNF-deficient mice died because of progressive infection because of the absence
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