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Immunohistochemical examination of the role of Fas ligand and lymphocytes in the pathogenesis of human liver yellow fever

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Immunohistochemical examination of the role of Fas ligand and lymphocytes in the pathogenesis of human liver yellow fever
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  Virus Research 116 (2006) 91–97 Immunohistochemical examination of the role of Fas ligand andlymphocytes in the pathogenesis of human liver yellow fever Juarez Antonio Sim˜oes Quaresma a , ∗ , Vera Lucia Reis Souza Barros b , Elaine Raniero Fernandes c ,Carla Pagliari c , Fernanda Guedes c , Pedro Fernando da Costa Vasconcelos d ,Heitor Franco de Andrade Junior c , Maria Irma Seixas Duarte c a  N´ ucleo de Medicina Tropical, Universidade Federal do Par´ a, Av. Generalissimo Deodoro 92, 66055-240, Bel´ em, Par´ a, Brazil b  Departamento de Patologia, Instituto Evandro Chagas, Minist´ erio da Sa´ ude, Ananindeua, Brazil c  Departamento de Patologia, Faculdade de Medicina, Universidade de S˜ ao Paulo, S˜ ao Paulo, Brazil d  Departamento de Arbovirologia e Febres Hemorr´ agicas, Instituto Evandro Chagas, Minist´ erio da Sa´ ude, Bel´ em, Brazil Received 2 May 2005; received in revised form 24 August 2005; accepted 24 August 2005Available online 10 October 2005 Abstract Yellowfeverisaninfectious,non-contagiousdiseasecausedbyanRNAvirusofthefamily Flaviviridae ,whichistransmittedtomanbythebiteof hematophagousmosquitoes.Infectionwiththeyellowfeverviruscanprogresswithlesionsintheheart,kidneys,centralnervoussystem,andliver.Intheliver,thehistopathologicalpictureischaracterizedbynecrosis,steatosisandhepatocyteapoptosis,withapreferentialmidzonedistribution.Inthepresent study, liver samples from fatal patients with yellow fever were analyzed. The histopathological pattern was characterized by steatosis, lyticnecrosisandhepatocyteapoptosisassociatedwithamoderatemononuclearinflammatoryinfiltrate.Theinflammatorycomponentmainlyconsistedof CD4+ T lymphocytes, followed by CD8+ T lymphocytes, which showed a preferential portal and midzone distribution. Immunoreactivity toFas ligand was mainly observed in hepatocytes of the midzone region. Based on these findings, we conclude that lymphocytes play an importantrole in the genesis of hepatic lesions in severe yellow fever, inducing hepatocyte apoptosis through the binding to Fas receptors. However, furtherstudies are necessary to investigate the participation of other immune factors and to quantify the role of the cytotoxic cellular response in the lesionevolution during the course of disease in the liver.© 2005 Elsevier B.V. All rights reserved. Keywords:  Yellow fever; Immunopathology; Arbovirus; Necrosis; Apoptosis; Lymphocytes 1. Introduction Yellow fever is an acute infection disease whose symptomsvary within a broad spectrum of manifestations ranging frommild or subclinical cases to severe forms with intense vascu-lopathy and characterized by a clinical triad of jaundice, hem-orrhage and acute renal failure (Ishak et al., 1982; Monath andBarrett, 2003; Peters and Zaki, 2002). Yellow fever is caused byan arbovirus of the family  Flaviviridae , genus  Flavivirus , andits case-fatality rate has ranged from 20 to 50% (Vasconceloset al., 2004). Epidemiologically, this disease can occur in twoforms, an urban and a jungle form. The urban form is transmit- ∗ Corresponding author. Tel.: +55 91 3241 4681; fax: +55 91 3241 4681.  E-mail address:  juarez@ufpa.br (J.A.S. Quaresma). ted from a sick individual to a non-immunized person throughthe bite of infected  Aedes aegypti . In the jungle form in theSouth America, the virus is mainly transmitted accidentally toman by mosquitoes of the genus  Haemagogus , whose naturalhabitat are the forests and which become infected through con-tact with viremic animals, especially monkeys (Monath, 2001;Vasconcelos et al., 2001a,b).Clinically, after an asymptomatic period the patient maypresent fever, headache, generalized muscle pain, photophobia,shivering and jaundice, and can progress to hemorrhagic man-ifestations and acute renal failure (Elton and Romero, 1955;Monath, 2001).Inhepatictissue,thevirusmayinducelesionssuchasmacro-and microvesicular steatosis, eosinophilic degeneration andhepatocyte necrosis, which are characteristically more intensein the midzone region and are associated with a portal and 0168-1702/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.virusres.2005.08.019  92  J.A.S. Quaresma et al. / Virus Research 116 (2006) 91–97  acinar mononuclear infiltrate of mild intensity (Klotz and Belt,1930a,b; Kerr, 1973; Branquet, 1996). Quantitative analysis observation had shown that no substantial alterations in thereticular network were found in liver and the hepatic damageresulted mainly from massive apoptotic death of hepatocyteand lesser extent due to lytic necrosis. This histophatologicpicture was associated by an inflammatory infiltrate consistedof mononuclear cells with intensity disproportionate to intensedeath of hepatocytes, probably due to the apoptotic componentthat predominates in these cases with not activation of inflam-matory cascade (Vieira et al., 1983; Quaresma et al., 2005). Little is known about the role of the virus-host interaction,the cellular immune response and its role in the genesis of thehistopathological alterations observed in yellow fever liver asdone for hepatitis B and C (Ganem and Prince, 2004; Gremion and Cerny, 2005). We believe that such study would provide abetter understanding of the physiopathological aspects involvedin the genesis of hepatic lesions and the interaction with thehost immune response in an attempt to fill the gaps in theknowledge of the pathogenesis and clinical evolution of yellowfever. 2. Materials and methods 2.1. Diagnostic procedures and histological examination Samples of liver from Department of Pathology of EvandroChagas Institute (Belem, Brazil) were obtained by post-mortembiopsy specimen of 53 fatal patients from Brazil, with agebetween 03 and 74 years, 13.20% female and 86.79% male.The diagnosis was made by serology, viral isolation, andimmunohistochemistry. Samples were fixed in 10% neutral-buffered formalin, followed by paraffin embedding, micron-thick sectioned and stained by hematoxylin and eosin method.Immunohistochemical method for the detection of specific YFantigens using policlonal antibodies and light microscopy wascarried out as described elsewhere (Hall et al., 1991). Sections were also evaluated qualitatively according to the histologicalcharacteristics previously described (Quaresma et al., 2005). 2.2. Immunologic markers An immunohistochemical technique to characterize the phe-notypeoftheinflammatorycellsfollowedtheprotocolsrcinallydescribed by Hsu et al. (1981). The immunologic staining tech- niques for the detection of apoptosis were carried out as per themanufacturer’s instructions, as previously described by Gold etal. (1994). The following antibodies were used: CD45R0, CD4,CD8, CD95 and anti-apoptosis (APOPTAG plus peroxidase kit,Chemicon ® , USA).Forquantitativeanalysisofthephenotypeofthecells,cellularexpression of Fas ligand (FasL) and ApopTag positive cells weused in a grid-scale, with 10 × 10 subdivisions in an area of 0.0625mm 2 , to count fields under high magnification ( × 400)in all three areas of the hepatic lobule (I: peri-portal area; II:midzonal area, and III: central vein area). 2.3. Negative controls and statistical analysis For negative and positive controls we included 10 liver sam-ples from patients with negative serology for the main hepa-totropic viruses (viruses of A, B, C and D hepatitis) and whichshowed no morphological alterations in the liver architeture,and 10 liver specimens from cases diagnosed as leptospirosisby clinical presentation, specific serology, histopathology andimmunohistochemical analysis. Statistical analysis was madeusing analysis of variance (ANOVA one-way) followed by theBonferroni test. The level of significance for these analyses wasestablished when  p ≤ 0.05. The analysis was performed usingthe GraphPad Prism 3.0 software for Windows (GraphPad Soft-ware, San Diego, CA). 3. Results Thepatternofhistopathologicalalterationswasmoreintensein the midzone region and was mainly characterized by hepato-cytic lesions such as macro- and microvesicular steatosis, focallytic necrosis and frequent eosinophilic bodies correspondingto apoptotic hepatocytes. A lymphomononuclear inflammatoryinfiltrate was observed which was minimal or moderate, and Fig. 1. Yellow fever liver: (A) histopathologic aspects of Councilman bodies ( ← ), macro and microvesicular steatosis (  ) (400 × ); (B) portal tract shownig minimalinflammatory infiltrate of lymphocytes (200 × ).   J.A.S. Quaresma et al. / Virus Research 116 (2006) 91–97   93     T   a    b    l   e    1    Y   e    l    l   o   w    f   e   v   e   r  —   m   e   a   n   a   n    d   s   t   a   n    d   a   r    d    d   e   v    i   a   t    i   o   n   o    f   a   p   o   p   t   o   s    i   s ,    l   y   m   p    h   o   c   y   t   e   s   a   n    d    F   a   s    L   e   x   p   r   e   s   s    i   o   n    i   n   a   c    i   n    i   a   n    d   p   o   r   t   a    l   t   r   a   c   t     A   p   o   p   t   o   s    i   s    C    D    4    5    R    O    C    D    4    C    D    8    F   a   s    L    Y    F    L    E    N    C    Y    F    L    E    N    C    Y    F    L    E    N    C    Y    F    L    E    N    C    Y    F    L    E    N    C    I    2 .    5    1       ±     0 .    6    4    0 .    5    0       ±     0 .    4    5    0 .    0    2       ±     0 .    0    0    2 .    2    9       ±     1 .    0    9    0 .    6    8       ±     0 .    0    7    0 .    0    6       ±     0 .    0    5    1 .    7    0       ±     0 .    5    5    0 .    2    2       ±     0 .    0    7    0 .    1    1       ±     0 .    0    5    1 .    2    0       ±     0 .    4    1    0 .    2    0       ±     0 .    0    5    0 .    1    2       ±     0 .    0    9    0 .    0    6       ±     0 .    0    0    0 .    0    1       ±     0 .    0    0    0 .    0    0       ±     0 .    0    0    I    I    1    6 .    4    1       ±     3 .    0    7    0 .    1    0       ±     0 .    6    2    0 .    0    3       ±     0 .    0    0    4 .    9    7       ±     1 .    8    5    1 .    1    2       ±     0 .    1    0    0 .    2    5       ±     0 .    0    8    3 .    0    4       ±     0 .    5    9    0 .    5    7       ±     0 .    0    8    0 .    1    3       ±     0 .    0    5    2 .    6    3       ±     0 .    8    5    0 .    5    7       ±     0 .    0    8    0 .    1    2       ±     0 .    0    9    0 .    1    0       ±     0 .    0    4    0 .    0    2       ±     0 .    0    0    0 .    0    1       ±     0 .    0    0    I    I    I    1 .    4    8       ±     0 .    7    3    0 .    3    2       ±     0 .    0    9    0 .    0    0       ±     0 .    0    0    0 .    6    1       ±     0 .    2    0    0 .    2    5       ±     0 .    0    5    0 .    0    0       ±     0 .    1    0    0 .    8    1       ±     0 .    2    9    0 .    2    1       ±     0 .    0    8    0 .    0    7       ±     0 .    0    6    0 .    5    5       ±     0 .    1    7    0 .    1    7       ±     0 .    1    8    0 .    0    5       ±     0 .    0    8    0 .    0    6       ±     0 .    0    0    0 .    0    1       ±     0 .    0    0    0 .    0    0       ±     0 .    0    0    P    T  –  –  –    1    0 .    2    6       ±     2 .    9    5    6 .    8    7       ±     0 .    4    0    6 .    8    7       ±     0 .    4    0    1    1 .    2       ±     2 .    9    3    1    0 .    1    9       ±     5 .    3    4    1 .    0    2       ±     0 .    6    4    7 .    5    2       ±     2 .    0    9    1    1 .    5       ±     1 .    3    9    0 .    7    1       ±     0 .    3    9  –  –  –     Y    F   :   y   e    l    l   o   w    f   e   v   e   r ,    L    E   :    l   e   p   t   o   s   p    i   r   o   s    i   s ,    N    C   :   n   o   r   m   a    l   c   o   n   t   r   o    l . disproportional to the intense degree of involvement of thehepatic parenchyma (Fig. 1A and B). In addition, hemorrhagicfoci and small neutrophil agglomerates were noted around theareas of lytic necrosis.Immunohistochemical examination revealed apoptotic(ApopTag-labeled) cells in all regions of the lobule, mainlyhepatocytes but also in Kupffer cells and in few mononucleatedinflammatory cells (Table 1). Immunolabeling for apoptosis was more intense in hepatocytes of the midzone areas (Fig. 2Aand B). The generally frequent immunolabeled apoptotic hep-atocytes correspond to the morphological aspect characteristicof the Councilman bodies.ActivatedTcellsimmunolabeledwiththeanti-CD45ROanti-bodywereobservedinboththeaciniandportaltract.Intheacini,the immunolabeling intensity was higher in the midzone region,followed in decreasing order by zones 1 and 3. ImmunolabeledT lymphocyte aggregates were observed around areas of lytichepatocyte necrosis. In the portal tract, the immunohistochem-ical pattern demonstrated a higher density of immunolabeled Tlymphocytes compared to the hepatic acini (Fig. 3A and B).CD4+ and CD8+ T cells were observed throughout the hep-atic parenchyma both in the portal tract and in the acini, witha higher concentration in zone 2, followed by zones 1 and 3.CD4+ T cells were more frequent than CD8+ T cells both in theacini and portal tract (Fig. 4A and B) (Fig. 5A and B). Expression of the FasL protein was observed in all hepaticlobulesandtendedtobehigherinthemidzoneregion.Ingeneral,immunoexpressionwasnotedespeciallyinhepatocytesandwasrare in cells lining the biliar ducts (Fig. 6A and B). 4. Discussion Thestudyofthelocalimmuneresponseisofgreatimportancefor the understanding and determination of yellow fever evolu-tion, especially in the liver, the target organ of disease (Maini etal., 2000; Ryo et al., 2000; Chisari and Ferrari, 1995). Monath(2001)mentionedtheoccurrenceofapoptosisduringsevereyellowfever.Moreover,inanexperimentalstudyinham-sters it was demonstrated by Xiao et al. (2001) that yellow fevervirus induced apoptosis in the infected animals. The histopatho-logical analysis of the present tissue specimens, as describedin the classical studies of  Councilman (1890) and Vieira et al. (1983), had demonstrated which Councilman/apoptotic bodieswereoneofthemarkedfindingsinoursample.Indeed,theapop-toticcellsshowedaclearpredominanceinthemidzoneareaandwere widely predominating over hepatocyte lytic necrosis.The relationship between apoptosis and infectious agentshas been the subject of numerous studies (Thompson, 1995;Harcwick, 1998). In some cases, probably it would represent atype of immune escape since the viral particles persist insideblebs lined with an intact membrane. However, it should beemphasized that the molecular mechanisms of virus-cell inter-action involved in the induction of cell death are extremelycomplex and varies according to the specific molecular inter-actions between the etiological agent and host cell (Roulston etal., 1999).  94  J.A.S. Quaresma et al. / Virus Research 116 (2006) 91–97  Fig. 2. Yellow fever liver: (A) immunohistochemistry for apoptosis showing hepatocytes in the acini (200 × ); (B) quantitative analysis of apoptotic hepatocytes inthe areas I, II and III of the hepatic acini (*  p ≤ 0.05, ANOVA/Bonferroni).Fig. 3. Yellow fever liver: (A) immunohistochemistry of T lymphocytes (400 × ); (B) quantitative analysis of T lymphocytes with predominance of cells in themidzonal region and portal tract. (*  p ≤ 0.05, ANOVA/Bonferroni). Many factors are involved in the induction of programmedcell death during the course of C hepatitis, a  Flaviviridae  agent,with emphasis on the role of cytotoxic T lymphocytes throughtheir FasL, TNF-  , and IFN-  , as well as a direct viral cyto-pathic effect. The direct viral cytopathic effect and its potentialin inducing apoptosis have been described for other mosquito-borned flaviviruses such as dengue, yellow fever, and West Nile(Marinneauetal.,1998;Monath,2001).Intheyellowfeverliver, Fig.4. Yellowfeverliver:(A)immunohistochemistryofCD4lymphocytesintheacini(400 × );(B)quantitativeanalysisofdistributionofCD4positivecellsshowingpredominance in the midzonal region. (*  p ≤ 0.05, ANOVA/Bonferroni).   J.A.S. Quaresma et al. / Virus Research 116 (2006) 91–97   95Fig. 5. Yellow fever liver: (A) immunohistochemistry of CD8 lymphocytes in the portal tract (200 × ); (B) quantitative analysis of distribution of CD8 positive cellsshowing predominance in the midzonal region. (*  p ≤ 0.05, ANOVA/Bonferroni). contrary of is observed in the B and C hepatitis, the presence of an inflammatory infiltrate consisting mainly of helper and cyto-toxic T lymphocytes in a preferential midzone distribution, inaddition to the characteristic immunolabeling for FasL in zone2 hepatocytes, is an indication of the role of T lymphocytes inthe genesis of yellow fever lesions, since previous studies hadshown preferential distribution of yellow fever antigens in mid-zonal region (Monath et al., 1989; De La Monte et al., 1983;De Brito et al., 1992; Deubel et al., 1997). Probably, viral inter-action with MHC-related molecules leads to the activation of cytolyticCD8+TlymphocytesthatbindtoTlymphocyterecep-tors or through FasL, inducing apoptosis through the interactionwith specific death domains (FADD or TRADD) or throughothermechanismsinvolvingthereleaseofgranzymes/perforins.Thesemechanismshavebeencharacterizedasbeinginvolvedinthe pathogenesis of other chronic, acute, and fulminant forms of hepatitis(Galleetal.,1995;Ryoetal.,2000).Inthosestudies,the authorscalledattentiontotheroleofcytotoxicCD8+Tlympho-cytesasoneofthemechanismsunderlyingthegenesisofhepaticparenchymallesionsinpatientswithBandChepatitis(BertolettiandMaini,2000).Ryoetal.(2000),studyingpatientswithsigns and symptoms of acute hepatitis of viral etiology, detected anincrease of FasL immunoexpression in the hepatic tissue of thepatients, which was accompanied by an increase in the expres-sion of specific ligands on tissue and circulating lymphocytesassociated with an intense apoptotic component. In addition,experimental studies using murine models of fulminant liverfailure, have demonstrated that the administration of anti-Fasantibodies induces intense hepatocyte destruction (Ogasawaraet al., 1993). In the present study, despite the observation of anincrease in FasL expression compared to normal controls, themassive apoptosis detected during this process was not accom-panied by the intense lymphocytic infiltration observed by otherin acute and fulminant viral hepatitis (Makinen, 2004). The causes of this discrete inflammatory component accom-panied by an intense degree of apoptosis which has been quan-tified and interpreted as the main causal factor of hepatocytedeath, remain unknown but it is known that apoptosis does notinduce an important inflammatory response, and the apoptoticbodies are phagocytosed by neighboring macrophages, and thusdonotactivatelargeregionalinflammatoryresponse(Kerretal.,1972;MajnoandJoris,1995;KaufmannandHengartner,2001). Fig. 6. Yellow fever liver: (A) immunohistochemistry of CD95 (FasL–Fas-ligant) in the acini (400 × ); (B) quantitative analysis of distribution of FasL positive cellsshowing predominance in the midzonal region. (*  p ≤ 0.05, ANOVA/Bonferroni).
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