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Involvement of the Chemokine RANTES (CCL5) in Resistance to Experimental Infection with Leishmania major

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Involvement of the Chemokine RANTES (CCL5) in Resistance to Experimental Infection with Leishmania major
    10.1128/IAI.72.8.4918-4923.2004. 2004, 72(8):4918. DOI: Infect. Immun. Tostes Gazzinelli and Leda Quercia VieiraCarlos Crocco Afonso, Mauro Martins Teixeira, Ricardo Glória de Souza, Luiz Antônio Rodrigues De-Freitas, LuísLuciana Santiago, Fernanda Oliveira Ferraz, Daniele da Helton da Costa Santiago, Carolina Ferreira Oliveira,  Leishmania major  Infection with (CCL5) in Resistance to Experimental Involvement of the Chemokine RANTES information and services can be found at: These include:  REFERENCES This article cites 24 articles, 11 of which can be accessed free CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new Information about commercial reprint orders: To subscribe to to another ASM Journal go to:  onN  ov  em b  er 1  8  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om  onN  ov  em b  er 1  8  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   I NFECTION AND  I MMUNITY , Aug. 2004, p. 4918–4923 Vol. 72, No. 80019-9567/04/$08.00  0 DOI: 10.1128/IAI.72.8.4918–4923.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved. Involvement of the Chemokine RANTES (CCL5) in Resistance toExperimental Infection with  Leishmania major  Helton da Costa Santiago, 1 Carolina Ferreira Oliveira, 1 Luciana Santiago, 1 Fernanda Oliveira Ferraz, 1 Daniele da Glo´ria de Souza, 1 Luiz Antoˆnio Rodrigues De-Freitas, 2 Luı´s Carlos Crocco Afonso, 3 Mauro Martins Teixeira, 1 Ricardo Tostes Gazzinelli, 1,4 and Leda Quercia Vieira 1 *  Departamento de Bioquı´mica e Imunologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, 1  andCentro de Pesquisas Rene´ Rachou, FioCruz, 4  Belo Horizonte MG, Centro de Pesquisas Gonc¸alo Muniz, FioCruz,Salvador, Bahia, 2  and Departamento de Cieˆncias Biolo´gicas and Nu´cleo de Pesquisa em Cieˆncias Biolo´gicas, Instituto de Cieˆncias Biolo´gicas e Exatas, Universidade Federal de Ouro Preto,Ouro Preto MG, 3  Brazil Received 20 August 2003/Returned for modification 17 December 2003/Accepted 3 April 2004 The expression and putative role of chemokines during infection with  Leishmania major  in mice wereinvestigated. CCL5 expression correlates with resistance, and blockade of CCL5 rendered mice more suscep-tible to infection. CCL5 is part of the cascade of events leading to efficient parasite control in  L. major  infection. Chemokines are cytokines with important roles in cell mi-gration and activation.  Leishmania  sp. infection induces theexpression of various chemokine genes (2, 13, 14).  Leishmania major   induces expression of CCL2 (MCP-1), CCL3 (MIP-1  ),CCL4 (MIP-1  ), CCL5 (RANTES), CXCL2 (MIP-2  ), andCXCL10 (  IP-10), along with the receptors CCR5, CCR2, andCCR1, in a time-dependent manner in mice (5, 9). CCR2 genedisruption was associated with increased susceptibility to  L. major   (20); however, CCL2 is important to resistance in hu-mans (15, 16) and mice (22, 25). Here we analyze the kineticsof chemokine expression in resistant and susceptible miceupon infection with  L. major  .C57BL/6 and BALB/c mice were infected with 10 6 stationaryforms of   L. major   (11). The mice were sacrificed 1, 2, 14, and42 days after infection, and RNA was extracted from lesionsfor reverse transcription (RT)-PCR analysis (3). The expres-sion of chemokines at the site of infection in resistant(C57BL/6) and susceptible (BALB/c) mice is shown in Fig. 1A and B. Expression levels of CXCL9 (Mig) and CCL5 increasedinitially in both strains, but expression was further increasedafter 2 weeks of infection in C57BL/6 mice. BALB/c, but notC57BL/6, mice expressed large amounts of mRNA for CCL2,CCL12 (MCP-5), and CXCL8 (KC). Expression levels of CXCL10 were similar in both strains. As the expression levelsof CCL2 and CCL5 diverged between the two mouse strains,these chemokines were investigated further.The differential expression of CCL2 and CCL5 was con-firmed by enzyme-linked immunosorbent assay (ELISA) (Fig.1C) (21). While BALB/c mice produced CCL2 early at the siteof infection, CCL2 was detectable only at week 2 postinfectionin C57BL/6 mice. The two strains of mice showed similar levelsof CCL2 from week 4 of infection. Similar levels of CCL5 weredetected in the early and late stages of infection in bothC57BL/6 and BALB/c mice. However, C57BL/6 mice had sig-nificantly greater quantities of CCL5 than BALB/c mice at weeks 4 and 6 of infection. The decrease in CCL5 levels ob-served in C57BL/6 mice coincided with the resolution of in-fection and inflammation at the site of infection (data notshown).Further studies were performed to verify whether CCL2 andCCL5 were markers of susceptibility and resistance, as sug-gested for BALB/c and C57BL/6 mice. Hence, we infectedsusceptible interleukin-12 knockout (IL-12   /   ) and gammainterferon knockout (IFN-    /   ) C57BL/6 mice and resistantIL-4   /   BALB/c mice (6, 10, 23) with  L. major   (Fig. 2A).CCL5 and CCL2 expression was determined by real-time RT-PCR and ELISA. CCL5 protein and mRNA expression levelscorrelated well. As shown in Fig. 2, CCL5 expression at week6 of infection was higher in the resistant (IL-4   /   ) and lower inthe susceptible (IL-12   /   and IFN-    /   ) mouse strains. Con- versely, there was no correlation between expression of CCL2protein and susceptibility or resistance to infection (Fig. 2Band C). Moreover, there was no equivalence between CCL2mRNA and protein expression levels.The results described above suggest that CCL5 may be rel-evant to resistance against  L. major   infection. To verify thispossibility, C57BL/6 mice were treated daily subcutaneously with Met-RANTES (10   g/mouse; kindly provided by A. E.Proudfoot, Serono Pharmaceuticals, Geneva, Switzerland), afunctional antagonist of CCR1 and CCR5 (1, 7, 12). Treatmentstarted at week 2 of infection, when no significant increase inCCL5 mRNA expression was observed. Treatment with Met-RANTES led to a transitory increase in lesion size (Fig. 3A).Mice were sacrificed at weeks 3 and 5 after treatment. At 3 weeks, there was an increase in IL-4 mRNA but no change inIFN-  expression in lesions (Fig. 3B). Met-RANTES also pro-moted an impressive down-regulation of the production of IFN-  in draining lymph nodes, whereas IL-4 production wasunchanged (Table 1). Tissue parasitism in these lesions wasevaluated by PCR (19) and was higher in the Met-RANTES-treated group at week 3 of treatment (Fig. 3C), which wasfurther confirmed in another experiment by serial dilution * Corresponding author. Mailing address: Departamento de Bio-quı´mica e Imunologia, ICB-Universidade Federal de Minas Gerais, CP486, 30161-970, Belo Horizonte MG, Brazil. Phone: 55-31-34992656.Fax: 55-31-3499-2614. E-mail:   onN  ov  em b  er 1  8  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   FIG. 1. Kinetics of CXC and CC chemokine mRNA expression in the footpads of C57BL/6 and BALB/c mice infected with  L. major  . (A) Mice were infected with  L. major   and sacri fi ced at 1 day, 2 days, 2 weeks, and 6 weeks postinfection, and the hind infected footpad was used in assaysof mRNA expression by RT-PCR and ELISA. Representative gels are shown. (B) Densitometric analysis was performed, and quanti fi cation wasnormalized to the levels of hypoxanthine phosphoribosyltransferase (HPRT) expression. Results are expressed as  n -fold increases over results withnoninfected (NI) control animals. (C) ELISA for detection of chemokines at the site of infection was performed at 1 and 2 days and at 2, 4, 6,and 8 weeks postinfection. Brie fl  y, footpad proteins were extracted at 50 mg of tissue/100  l of phosphate-buffered saline to which 0.4 M NaCl,0.05% Tween 20, and protease inhibitors (0.1 mM phenylmethylsulfonyl  fl uoride, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KIaprotinin A/100 ml) were added. The samples were centrifuged for 10 min at 3,000   g  , and the supernatant was immediately used for ELISAs.ELISA plates were coated overnight with sheep anti-mouse CCL2 (Pharmingen, San Diego, Calif.) or CCL5 (RD Systems, Minneapolis, Minn.),and ELISAs were performed as recommended by the manufacturer. The anti-CCL2 assay had a sensitivity of 16 pg/ml, and the anti-CCL5 assayhad a sensitivity of 32 pg/ml. * and **, statistical differences (  P   0.05) between results for C57BL/6 and BALB/c for each point, determined byStudent ’ s  t  test. In panel B, each point represents the mean (  standard error [SE]) for at least three mice per time point of one experiment of three performed.V OL  . 72, 2004 NOTES 4919   onN  ov  em b  er 1  8  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   analysis (data not shown). All changes had disappeared by week 5 of treatment. To evaluate why the effects of Met-RANTES were transient, we investigated the presence of anti-Met-RANTES antibodies in serum. Not only were there ele- vated titers of antibodies in serum (Fig. 3D), but there was alsoan increase in the expression of CCL5 mRNA following Met-RANTES treatment (data not shown). Hence, it is possiblethat the effect of Met-RANTES (a competitive antagonist of CCL5 binding to CCR1 and CCR5) is transient due either (i)to the appearance of antibodies (as shown here) which recog-nize and, possibly, prevent the action of Met-RANTES, or (ii)to the increase in the levels of CCL5 (as suggested by the FIG. 2. Course of infection, chemokine expression, and protein production at the site of infection in IL-12, IFN-  , and IL-4 knockout (   /   )mice and their wild-type control. (A) Mice were infected in both hind footpads with 10 6 stationary-phase  L. major   promastigotes, and lesions weremeasured weekly. (B) Chemokine expression was determined at 2 and 6 weeks postinfection by semiquantitative real-time RT-PCR and compared with the expression in noninfected controls (NI). Results are expressed in arbitrary units normalized to results for noninfected control animals.(C) ELISA for chemokine production was performed with lesions at 2 and 6 weeks after infection, as described in the legend to Fig. 1. Each pointrepresents the mean (  SE) for three mice per point. Different letters indicate a  P   value of   0.05 between results for different knockout mice atthe same time point, determined by Student ’ s  t  test. Experiments were repeated, with similar results.4920 NOTES I NFECT . I MMUN .   onN  ov  em b  er 1  8  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   FIG. 3. (A) Effect of Met-RANTES on the course of infection by  L. major  ; (B) cytokine mRNA expression by RT-PCR at the site of infection;(C) quanti fi cation of   L. major   gp63 mRNA expression at the lesion site; (D) production of anti-MetCCL5 antibody in sera from treated andnontreated animals. (A) C57BL/6 mice were infected with  L. major   in the hind footpads and treated daily from week 3 to 8 postinfection withintralesion injections of Met-RANTES (10  g/animal) or with phosphate-buffered saline (PBS; vehicle). Lesions were measured weekly, and eachpoint represents the mean result (  SE) for four to eight animals per time point of one experiment of two performed. (B) Animals were sacri fi cedat weeks 3 and 5 of treatment, and the infected footpads were used to assay mRNA expression by RT-PCR. Representative gels are shown.Densitometric analysis was performed, and the quanti fi cation was normalized to the levels of HPRT expression. To determine the Th1/Th2balance, the densities of the bands were compared by dividing values obtained for IFN-  by values obtained for IL-4. Each point represents means(  SE) of results for three mice per time point of one of two experiments performed. (C) Parasite load was determined by RT-PCR for  L. major  gp63 using various dilutions of the total lesion cDNA, at 3 weeks of treatment (6 weeks of infection). The densitometric analysis was performedand normalized by HPRT levels at the same dilution factor. Each point represents the mean (  SE) of results for three to four mice.(D) Anti-Met-RANTES antibodies were determined by ELISA of a 1:20 dilution of the serum at the end of the experiment (5 weeks of treatment).Bars represent means (  SE) of results for four to six mice from two different experiments for the absorbance of the serum. *, statistical differences(  P   0.05), determined by Student ’ s  t  test. NI, noninfected; O.D., optical density.V OL  . 72, 2004 NOTES 4921   onN  ov  em b  er 1  8  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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