Portal de Periódicos da CAPES

Emergence of Anti-Red Blood Cell Antibodies Triggers Red Cell Phagocytosis by Activated Macrophages in a Rabbit Model of Epstein-Barr Virus-Associated Hemophagocytic Syndrome

2007; Elsevier BV; Volume: 170; Issue: 5 Linguagem: Inglês

10.2353/ajpath.2007.060772

1525-2191

Wen‐Chuan Hsieh, Yao Chang, Mei‐Chi Hsu, Bau-Shin Lan, Guan-Chung Hsiao, Huai‐Chia Chuang, Ih‐Jen Su,

Immune Cell Function and Interaction

Hemophagocytic syndrome (HPS) is a fatal complication frequently associated with viral infections. In childhood HPS, Epstein-Barr virus (EBV) is the major causative agent, and red blood cells (RBCs) are predominantly phagocytosed by macrophages. To investigate the mechanism of RBC phagocytosis triggered by EBV infection, we adopted a rabbit model of EBV-associated HPS previously established by using Herpesvirus papio (HVP). The kinetics of virus-host interaction was studied. Using flow cytometry, we detected the emergence of antibody-coated RBCs, as well as anti-platelet antibodies, at peak virus load period at weeks 3 to 4 after HVP injection, and the titers increased thereafter. The presence of anti-RBCs preceded RBC phagocytosis in tissues and predicted the full-blown development of HPS. The anti-RBC antibodies showed cross-reactivity with Paul-Bunnell heterophile antibodies. Preabsorption of the HVP-infected serum with control RBCs removed the majority of anti-RBC activities and remarkably reduced RBC phagocytosis. The RBC phagocytosis was specifically mediated via an Fc fragment of antibodies in the presence of macrophage activation. Therefore, the emergence of anti-RBC antibodies and the presence of macrophage activation are both essential in the development of HPS. Our observations in this animal model provide a potential mechanism for hemophagocytosis in EBV infection. Hemophagocytic syndrome (HPS) is a fatal complication frequently associated with viral infections. In childhood HPS, Epstein-Barr virus (EBV) is the major causative agent, and red blood cells (RBCs) are predominantly phagocytosed by macrophages. To investigate the mechanism of RBC phagocytosis triggered by EBV infection, we adopted a rabbit model of EBV-associated HPS previously established by using Herpesvirus papio (HVP). The kinetics of virus-host interaction was studied. Using flow cytometry, we detected the emergence of antibody-coated RBCs, as well as anti-platelet antibodies, at peak virus load period at weeks 3 to 4 after HVP injection, and the titers increased thereafter. The presence of anti-RBCs preceded RBC phagocytosis in tissues and predicted the full-blown development of HPS. The anti-RBC antibodies showed cross-reactivity with Paul-Bunnell heterophile antibodies. Preabsorption of the HVP-infected serum with control RBCs removed the majority of anti-RBC activities and remarkably reduced RBC phagocytosis. The RBC phagocytosis was specifically mediated via an Fc fragment of antibodies in the presence of macrophage activation. Therefore, the emergence of anti-RBC antibodies and the presence of macrophage activation are both essential in the development of HPS. Our observations in this animal model provide a potential mechanism for hemophagocytosis in EBV infection. Hemophagocytic syndrome (HPS) is a fatal disorder frequently associated with microbial infections such as Epstein-Barr virus (EBV),1Reisman RP Greco MA Virus-associated hemophagocytic syndrome due to Epstein-Barr virus.Hum Pathol. 1984; 15: 290-293Abstract Full Text PDF PubMed Scopus (69) Google Scholar cytomegalovirus,2Aouad A Dan ME Alangaden GJ Photo quiz. I. Reactive hemo-phagocytic syndrome caused by CMV infection.Clin Infect Dis. 1998; 26: 1295-1439Crossref PubMed Google Scholar and recently, H5N1 influenza virus.3To KF Chan PK Chan KF Lee WK Lam WY Wong KF Tang NL Tsang DN Sung RY Buckley TA Tam JS Cheng AF Pathology of fatal human infection associated with avian influenza A H5N1 virus.J Med Virol. 2001; 63: 242-246Crossref PubMed Scopus (390) Google Scholar Although this syndrome is diverse in etiology and genetic association, HPS shares common clinical and laboratory features and is characterized by fever, hepatosplenomegaly, hypercytokinemia, cytopenia, coagulopathy, and a systemic proliferation of macrophages with phagocytosis of blood cells.4Ost A Nilsson-Ardnor S Henter JI Autopsy findings in 27 children with haemophagocytic lymphohistiocytosis.Histopathology. 1998; 32: 310-316Crossref PubMed Scopus (120) Google Scholar, 5Su IJ Hsieh HJ Lee CY Histiocytic medullary reticulosis: a lethal form of primary EBV infection in young children in Taiwan.Lancet. 1989; 1: 389Abstract PubMed Scopus (19) Google Scholar, 6Chen RL Su IJ Lin KH Lee SH Lin DT Chuu WM Lin KS Huang LM Lee CY Fulminant childhood hemophagocytic syndrome mimicking histiocytic medullary reticulosis. An atypical form of Epstein-Barr virus infection.Am J Clin Pathol. 1991; 96: 171-176PubMed Google Scholar, 7Janka GE Familial hemophagocytic lymphohistiocytosis.Eur J Pediatr. 1983; 140: 221-230Crossref PubMed Scopus (434) Google Scholar Among the viral pathogens responsible for HPS, EBV accounts for more than 60% of HPS cases in young children.5Su IJ Hsieh HJ Lee CY Histiocytic medullary reticulosis: a lethal form of primary EBV infection in young children in Taiwan.Lancet. 1989; 1: 389Abstract PubMed Scopus (19) Google Scholar, 6Chen RL Su IJ Lin KH Lee SH Lin DT Chuu WM Lin KS Huang LM Lee CY Fulminant childhood hemophagocytic syndrome mimicking histiocytic medullary reticulosis. An atypical form of Epstein-Barr virus infection.Am J Clin Pathol. 1991; 96: 171-176PubMed Google Scholar The pathogenesis of EBV-associated HPS has been proposed to result from a dysregulated cytotoxic T-cell response with macrophage activation in response to EBV infection in clinical conditions such as X-linked lymphoproliferative disorders and sporadic hemophagocytic lymphohistiocytosis (HLH).7Janka GE Familial hemophagocytic lymphohistiocytosis.Eur J Pediatr. 1983; 140: 221-230Crossref PubMed Scopus (434) Google Scholar, 8Sumegi J Huang D Lanyi A Davis JD Seemayer TA Maeda A Klein G Seri M Wakiguchi H Purtilo DT Gross TG Correlation of mutations of the SH2D1A gene and Epstein-Barr virus infection with clinical phenotype and outcome in X-linked lymphoproliferative disease.Blood. 2000; 96: 3118-3125PubMed Google Scholar, 9Su IJ Chen RL Lin DT Lin KS Chen CC Epstein-Barr virus (EBV) infects T lymphocytes in childhood EBV-associated hemophagocytic syndrome in Taiwan.Am J Pathol. 1994; 144: 1219-1225PubMed Google Scholar, 10Su IJ Wang CH Cheng AL Chen RL Hemophagocytic syndrome in Epstein-Barr virus-associated T-lymphoproliferative disorders: disease spectrum, pathogenesis, and management.Leuk Lymphoma. 1995; 19: 401-406Crossref PubMed Scopus (123) Google Scholar The phagocytic process by macrophages is by no means a random event but involves a meticulous interaction between ligands on the surface of phagocytosed cells and the receptors on the activated macrophages.11Aderem A Underhill DM Mechanisms of phagocytosis in macrophages.Annu Rev Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2122) Google Scholar, 12Larroche C Mouthon L Pathogenesis of hemophagocytic syndrome (HPS).Autoimmun Rev. 2004; 3: 69-75Crossref PubMed Scopus (199) Google Scholar Because macrophage activation is a common phenomenon in infectious diseases, the relative rarity of HPS and the frequent association of HPS with EBV raise such a possibility that EBV may play a specific role in triggering HPS. Furthermore, the major blood cells engulfed by macrophages in EBV-associated HPS are red blood cells (RBCs) or platelets, distinct from the predominant lymphocytes in H5N1 influenza infection and other conditions such as Rosai-Dorfman disease.3To KF Chan PK Chan KF Lee WK Lam WY Wong KF Tang NL Tsang DN Sung RY Buckley TA Tam JS Cheng AF Pathology of fatal human infection associated with avian influenza A H5N1 virus.J Med Virol. 2001; 63: 242-246Crossref PubMed Scopus (390) Google Scholar, 13Kara IO Ergin M Sahin B Inal S Tasova Y Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman's disease) previously misdiagnosed as Toxoplasma lymphadenitis.Leuk Lymphoma. 2004; 45: 1037-1041Crossref PubMed Scopus (7) Google Scholar To investigate why specific blood cells are selectively phagocytosed by macrophages in different conditions should help to clarify the pathogenesis of virus-associated HPS. One clue to resolve this issue comes from the observations that virus infection may induce a wide spectrum of polyclonal B-cell and antibody responses against RBCs, platelets, lymphocytes, and endothelial cells.14Papesch M Watkins R Epstein-Barr virus infectious mononucleosis.Clin Otolaryngol Allied Sci. 2001; 26: 3-8Crossref PubMed Scopus (59) Google Scholar, 15Haukenes G Viggen B Boye B Kalvenes MB Flo R Kalland KH Viral antibodies in infectious mononucleosis.FEMS Immunol Med Microbiol. 1994; 8: 219-224Crossref PubMed Scopus (25) Google Scholar, 16Lin YS Lin CF Fang YT Kuo YM Liao PC Yeh TM Hwa KY Shieh CC Yen JH Wang HJ Su IJ Lei HY Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity.Clin Exp Immunol. 2005; 141: 500-508Crossref PubMed Scopus (47) Google Scholar The cell types that are opsonized, ie, prepared for phagocytosis by specific antibodies, may represent the selective targets of phagocytosis by activated macrophages mediated through Fc receptors. Of note, production of Paul-Bunnell (PB) heterophile antibodies that agglutinate RBCs is a prevailing serological marker for acute EBV infection or infectious mononucleosis.15Haukenes G Viggen B Boye B Kalvenes MB Flo R Kalland KH Viral antibodies in infectious mononucleosis.FEMS Immunol Med Microbiol. 1994; 8: 219-224Crossref PubMed Scopus (25) Google Scholar, 17Patarca R Fletcher MA Structure and pathophysiology of the erythrocyte membrane-associated Paul-Bunnell heterophile antibody determinant in Epstein-Barr virus-associated disease.Crit Rev Oncog. 1995; 6: 305-326Crossref PubMed Scopus (9) Google Scholar The prevalence of anti-RBCs or heterophile antibodies in EBV infection may explain the frequent association of HPS with EBV. Therefore, we hypothesize that anti-RBC antibodies may play a pivotal role in triggering the phagocytosis of red cells in EBV-associated HPS. To test this hypothesis, we adopted a rabbit model of EBV-associated HPS previously established by Hayashi and colleagues18Hayashi K Ohara N Teramoto N Onoda S Chen HL Oka T Kondo E Yoshino T Takahashi K Yates J Akagi T An animal model for human EBV-associated hemophagocytic syndrome: herpesvirus papio frequently induces fatal lymphoproliferative disorders with hemophagocytic syndrome in rabbits.Am J Pathol. 2001; 158: 1533-1542Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Hayashi K Teramoto N Akagi T Animal in vivo models of EBV-associated lymphoproliferative diseases: special references to rabbit models.Histol Histopathol. 2002; 17: 1293-1310PubMed Google Scholar, 20Hayashi K Jin Z Onoda S Joko H Teramoto N Ohara N Oda W Tanaka T Liu YX Koirala TR Oka T Kondo E Yoshino T Takahashi K Akagi T Rabbit model for human EBV-associated hemophagocytic syndrome (HPS): sequential autopsy analysis and characterization of IL-2-dependent cell lines established from herpesvirus papio-induced fatal rabbit lymphoproliferative diseases with HPS.Am J Pathol. 2003; 162: 1721-1736Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar using EBV-related Herpesvirus papio (HVP). In this rabbit model, HVP is previously found to infect T and B cells, distinct from the predominant or exclusive infection of T or natural killer (NK) cells in HLH cases.9Su IJ Chen RL Lin DT Lin KS Chen CC Epstein-Barr virus (EBV) infects T lymphocytes in childhood EBV-associated hemophagocytic syndrome in Taiwan.Am J Pathol. 1994; 144: 1219-1225PubMed Google Scholar, 21Ishii E Ohga S Imashuku S Kimura N Ueda I Morimoto A Yamamoto K Yasukawa M Review of hemophagocytic lymphohistiocytosis (HLH) in children with focus on Japanese experiences.Crit Rev Oncol Hematol. 2005; 53: 209-223Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Although not entirely similar to the disease entity of human HLH, this animal model still represents a valuable tool to investigate the pathogenesis of virus-associated HPS. In this study, we extended the study to the kinetics of virus-host interaction, and the development of anti-virus and anti-RBC antibodies was longitudinally followed, with correlation to the presence of hemophagocytosis in tissues. In vitro and ex vivo phagocytosis assay was further performed to clarify the role of anti-RBC antibodies in RBC phagocytosis by activated macrophages mediated via Fc receptor. The rabbit model of EBV-associated HPS was previously established by Hayashi and colleagues18Hayashi K Ohara N Teramoto N Onoda S Chen HL Oka T Kondo E Yoshino T Takahashi K Yates J Akagi T An animal model for human EBV-associated hemophagocytic syndrome: herpesvirus papio frequently induces fatal lymphoproliferative disorders with hemophagocytic syndrome in rabbits.Am J Pathol. 2001; 158: 1533-1542Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar using the EBV homologue virus HVP. The HVP-producing baboon lymphoblastoid cell line 594S was cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (ICN, Aurora, OH) and 100 U/ml penicillin-streptomycin (Life Technologies, Inc.). Culture supernatants of 594S cells were centrifuged at 8000 × g for 30 minutes to remove cell debris, filtered with a 0.45-μm filter (Millipore, Billerica, MA), and then centrifuged at 100,000 × g (L-100XP; Beckman Coulter, Hialeah, FL) for 60 minutes to obtain concentrated virus stocks. New Zealand White rabbits (each weighing ∼2 kg) were obtained from Taiwan Livestock Research Institute (Tainan, Taiwan). Each rabbit was inoculated intravenously with virus stocks concentrated from 200 ml of culture supernatants of 594S cells [containing ∼5 × 107 copies of HVP as quantified by real-time polymerase chain reaction (PCR)]. Control rabbits were inoculated with phosphate-buffered saline (PBS). After inoculation with virus pellets, rabbits were sacrificed weekly under euthanasia with excess pentobarbital sodium (MTC Pharmaceuticals, Hamilton, ON, Canada). Control rabbits were sacrificed until all of the HVP-infected rabbits died of illness. The organs, including spleen, lymph nodes, livers, kidneys, thymus, lungs, and spinal cord, were examined macroscopically and then fixed with 3.7% formalin. The formalin-fixed, paraffin-embedded tissue blocks were sectioned at 3- to 5-μm thickness and stained for conventional histopathology with hematoxylin and eosin. Bone marrow and blood smears of rabbits were stained with a modified Wright or Liu A/Liu B stain. For immunohistochemical studies of the distribution of T cells and B cells in tissues, freshly frozen specimens were sectioned at 5-μm thickness and immunostained with monoclonal mouse anti-human CD79α (B-cell marker, cross-reaction with rabbit CD79α) (DAKO, Glostrup, Denmark) and anti-CD5 (T cell) (Serotec Ltd., Oxford, UK) for 60 minutes at room temperature. The clone of CD5 monoclonal antibody used in this study is specific for T cells and does not cross-react with B cells. Cells were then washed twice with PBS and immunoreacted with goat anti-mouse horseradish peroxidase-conjugated IgG (horseradish peroxidase) (Amersham Bioscience, London, UK) for 60 minutes at room temperature. After washing, the sections were reacted with AEC (3-amino-9-ethylcarbazole) substrate (DAKO) and then examined under a microscope. Anti-VCA IgG in sera was determined by an indirect immunofluorescence assay using EBV/indirect immunofluorescence assay slides (Meridian Bioscience, Cincinnati, OH). Different dilutions of rabbit sera (1:10 ∼ 1:640) were added to each well of EBV/indirect immunofluorescence assay slides and incubated for 30 minutes at room temperature. The slides were washed with PBS and then reacted with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). After further incubation at room temperature for 30 minutes, slides were washed and observed under a fluorescence microscope. For controls, secondary antibody alone (FITC-labeled anti-rabbit IgG) and another irrelevant secondary antibody, FITC-labeled goat anti-mouse IgG (Jackson ImmunoResearch), were simultaneously run on the same test. Mononuclear cells of rabbits were isolated from whole blood and spleen by Ficoll gradient separation and labeled by incubating with mouse anti-rabbit IgM (B cells), anti-rabbit CD5 (T cells), or anti-human CD14 (cross-reaction with rabbit CD14) (Serotec Ltd.) for 15 minutes at room temperature. Cells were then washed twice with PBS and magnetically labeled with goat anti-mouse IgG MicroBeads (Miltenyi Biotec, Auburn, CA) for 15 minutes at 4°C. After washing, the CD5+ T cells, IgM+ B cells, or CD14+ monocytes/macrophages were separated by an immunomagnetic procedure incorporating the MACS system (Miltenyi Biotec). DNA extraction was performed according to the manufacturer's description of QIAamp DNA blood kit (Qiagen GmbH, Hilden, Germany). For detection of HVP virus genome, we used one primer pair for HVP EBNA1 (HPNA-1S: 5′-CTGGGTTGTTGCGTTCCATG-3′, HPNA-1A: 5′-TTGGGGGCGTCTCCTAACAA-3′) and two primer pairs for HVP EBNA2 (HPNA2-231S: 5′-ACCACTGGGACCAGTTTGGT-3′, HPNA2-1612A: 5′-AGAGGACTGAGGTTCTTGC-3′; PNA2-1485S: 5′-AGCCTAGGCCCAATAGCTCA-3′ and HPNA2-1691A: 5′-CCTCCCATTGGTTGTCAGGG-3′) as previously described.18Hayashi K Ohara N Teramoto N Onoda S Chen HL Oka T Kondo E Yoshino T Takahashi K Yates J Akagi T An animal model for human EBV-associated hemophagocytic syndrome: herpesvirus papio frequently induces fatal lymphoproliferative disorders with hemophagocytic syndrome in rabbits.Am J Pathol. 2001; 158: 1533-1542Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar β-Actin primers were used as an internal control: AC3: 5′-GGAGCCTTGAATACACCCAA-3′ and AC4: 5′-GAGGCGTACAGGGATAGCA C-3′. For each PCR reaction, 35 cycles were performed on 50 ng of DNA in 25 μl of PCR reaction mixture, which consisted of 50 nmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3, 1.5 mmol/L MgCl2, 200 nmol/L dNTP, 0.2 μmol/L primers, and 1.25 U of Taq polymerase (Takara, Shiga, Japan). For detection of HVP virus load in sera of infected rabbits, quantitative real-time PCR was performed using the LightCycler 1.5 (Roche Diagnostics GmbH, Mannheim, Germany) and LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH). Virus DNA in rabbit sera were extracted by QIAamp DNA mini kit (Qiagen GmbH) according to the manufacturer's protocol. A total of 4 μl of sample DNA was used in a reaction volume of 20 μl, and the assay contained 4 mmol/L magnesium chloride, 0.25 μmol/L of each primer, 2 μl of 1× LightCycler FastStart DNA Master SYBR Green I mixture. The reaction conditions for the assay were as follows: one segment of denaturation at 95°C for 10 minutes; 45 cycles of amplification at three segments of 95°C for 10 seconds, 57°C for 5 seconds, and 72°C for 10 seconds; three segments of melting at 95°C, 65°C for 15 minutes, and 95°C; and finally one segment of cooling at 40°C for 30 seconds. The primers used in this assay were HPNA2-1485S and HPNA2-1691A. Rabbit spleens, lymph nodes, thymuses, lungs, and livers were fixed with formalin and paraffin-embedded. Sections of 3-μm thickness were put onto glass slides and subjected to detection of EBER1. In situ hybridization was performed according to the manufacturer's protocol provided in the EBV probe in situ hybridization kit (Novocastra Laboratories Ltd., Newcastle on Tyne, UK). Rabbit blood samples were collected before and after the inoculation with HVP. Rabbit red cells were isolated from whole blood by Ficoll gradient separation, washed twice with PBS, and then incubated with FITC-labeled anti-rabbit immunoglobulin (DAKO) for 30 minutes. After setting forward scatter/side scatter detectors to log mode and gating the RBC region, surface fluorescence on the rabbit red cells was measured by FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ) and analyzed by CellQuest software. FITC-labeled mouse IgG (Becton, Dickinson and Company) was used as isotype control in this experiment. To clarify whether anti-platelet antibodies were also present in this animal model, assays for anti-platelet antibodies were also performed. Platelet-rich plasma was separated from whole blood by centrifugation (250 × g for 10 minutes). After washing and counting, 106 platelets were incubated with HVP-infected rabbit serum for 30 minutes. Platelets were then washed twice with PBS and double-labeled with anti-rabbit CD41/CD61 PE (Serotec Ltd.) and anti-rabbit Ig FITC (DAKO) for another 30 minutes. After washing with PBS, fluorescence on platelets was measured and analyzed by FACSCalibur. For cellular enzyme-linked immunosorbent assay, 107 platelets were seeded in each well of a 96-well plate and then incubated at 37°C for 2 hours. For fixation, 4% paraformaldehyde was added into each well after carefully washing each well and then incubated at room temperature for 15 minutes. After blocking with PBS/1% bovine serum albumin, different dilutions of rabbit plasma were added into wells and incubated at room temperature for 2 hours. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Amersham Biosciences) was then added after washing the plate and incubated for 1 hour. Finally, tetramethyl benzidine substrate was applied to elicit a chromogenic signal, and the plate was measured under an enzyme-linked immunosorbent assay reader. Determination of PB heterophile antibodies in rabbit sera was performed with Monospot latex kit (Meridian Bioscience), which contains the PB heterophile determinants. Briefly, 50 μl of sample was added on one well of the disposable slide, and then a drop of latex reagent was added next to the drop of sample. After mixing and rotating gently for 3 minutes, the mixture was examined for the presence or absence of agglutination. To investigate whether heterophile antibodies represent the predominant component of anti-red cell antibodies in the infected serum, a preabsorption test was performed. The infected rabbit sera were preincubated with 1 × 108 normal rabbit red cells for 30 minutes at 37°C twice before performing the agglutination assay. The promonocytic cell line U937 was differentiated by activation with 5 ng/ml phorbol ester (TPA) (Sigma, St. Louis, MO) for 48 hours. FITC-labeled latex beads (Sigma) were added into TPA-treated or untreated U937 cells and incubated for 2 hours at 37°C. After washing four times with PBS, phagocytosis could be observed under fluorescence microscopy. For investigating phagocytosis of red cells by macrophages, normal human red cells and Coombs-treated cells (Gamma Biologicals Inc., Houston, TX) were labeled with FITC using the Flurorotag FITC conjugation kit (Sigma). FITC-labeled normal human red cells and FITC-labeled Coombs-treated cells were used in this study. The FITC-labeled target cells were incubated with TPA-activated U937 cells at 37°C for 2 hours. After incubation, unengulfed red cells were lysed by RBC lysis buffer, and the remaining cells were examined by fluorescence microscopy or flow cytometry. Rabbit monocytes were isolated from PBMCs by using the immunomagnetic approach mentioned above. The rabbit monocytes were activated by TPA and tested for the engulfment of FITC-labeled normal rabbit red cells that were precoated or not coated with HVP-infected rabbit sera. After co-incubation of activated monocytes and red cells for 2 hours, phagocytosis of red cells was examined by fluorescence microscopy. To determine whether phagocytosis of red cells is mediated via Fc fragments, Fab and Fc fragments were separately prepared by papain digestion using the ImmunoPure Fab preparation kit (Pierce, Rockford, IL). In brief, 10 mg of purified human IgG (Sigma) was digested with immobilized papain for 5 hours at 37°C. Then chromatography was used to purify Fab and Fc fragments of IgG on a column with protein G (Pierce). The Fc, Fab fragments, or whole IgG were added as blockers (at a final concentration of 1 mg/ml) in the phagocytosis assay described above. A total of 11 rabbits were used for this study, eight receiving HVP inoculation and three as control. Five HVP-infected rabbits were serially sacrificed under euthanasia to observe the dynamic virus-host interaction and the sequential development of diseases. The remaining three HVP-infected rabbits developed fulminating HPS and died within 1 month (on days 23 to 30 after virus inoculation). They appeared physically healthy until the last week before they died. The rabbits first lost appetite and then became emaciated. A few of them had rhinorrhea admixed with blood and then developed dyspnea. All three control rabbits were free from symptoms and remained healthy at the end of the experiment. Necropsy of the infected rabbits revealed dark purple (congestion) and swollen lymph nodes, as well as hepatosplenomegaly. The kidneys and thymuses looked normal, but lungs showed congestion and edema. All control rabbits looked otherwise normal. Histopathological examinations of the infected rabbit tissues showed progressive lymphoid depletion in spleen and lymph nodes (Figure 1A) with lymphoid infiltration of predominantly CD5+ T cells around perivascular areas in many organs such as livers, kidneys, and lungs. The liver showed mild fatty changes with portal infiltration of predominant CD5+ T-lymphoid cells. The bone marrow showed mild hyperplasia at day 20 but progressed to marked hypoplasia at death. The histological pictures of hemophagocytosis first appeared at 3 weeks after HVP inoculation, with predominant engulfment of red cells by macrophages in the sinuses of spleens, lymph nodes, and bone marrow of HVP-infected rabbits (Figure 1B). The severity of red cell engulfment by activated macrophages was associated with disease progression. Plasma cells, atypical lymphocytes, and nucleated red cells could also be observed in blood smears of HVP-infected rabbits with HPS (data not shown). All sera obtained from HVP-inoculated rabbits were anti-VCA IgG-positive, whereas sera from the preinoculated rabbits and from control rabbits showed negative reactivity. Although seroconversion varied in titers among the HVP-infected rabbits, antibodies to VCA became detectable on day 19 and persisted thereafter (Figure 2A). The presence of viral genomes in PBMCs from infected rabbits was assessed by PCR to detect EBNA1 and EBNA2 gene regions of HVP DNA. As shown in Figure 2B (top), HVP-EBNA2 genomes (the same for EBNA1, data not shown) were detectable in PBMCs from day 19 after virus inoculation. Quantitative PCR studies showed that HVP DNA in rabbit sera was detectable from day 12, and the viral load peaked during weeks 3 to 4 after HVP inoculation and declined thereafter. HVP DNA could also be detected by PCR in most other tissues, such as spleens, lymph nodes, livers, and lungs from HVP-infected rabbits (data not shown). By immunomagnetic purification, HVP DNA could be detected in the fractions of purified CD5+ T cells, IgM+ B cells, and CD14+ monocytes of PBMCs in HVP-infected rabbits from day 19 after virus inoculation (Figure 2B, bottom). In situ EBER1 hybridization was further performed on formalin-fixed tissues of HVP-infected rabbits. EBER1 RNA of HVP was detectable in lymphocytes of spleens (Figure 2C, top), lymph nodes, and thymuses. The EBER1 signals were first detected at the marginal zone of white pulp of spleens on day 14 and then extensively in spleens (Figure 2C, bottom) and lymph nodes in the following weeks. EBER1 could also be detected in most infiltrating lymphocytes of lungs and livers obtained in week 4 after HVP inoculation or at necropsy. Immunohistochemical studies revealed that the infiltrating lymphoid cells in liver and lungs were mostly CD5 T cells, and only rarely B cells (data not shown). The in situ EBER1 signals correlated to the white pulps of the spleen, which contained B- and T-immunoreactive cells (Figure 2C, top). To confirm the specificity of HVP-infected cells, immunomagnetic purification and fractionation of T and B cells was performed on spleen lymphocytes. HVP DNA could be detected in the fractions of T and B cells in spleen (Figure 2C, bottom right). Quantitative real-time PCR analysis revealed that B cells contained a mean of 414 copies of HVP genome per 100 ng of cell DNA, higher than the 225 copies of HVP genome in T-cell fraction. The CD14+ fraction contained too few cells to be available for DNA extraction and further analysis. Using FITC-labeled anti-rabbit immunoglobulin in flow cytometric analysis, we found that red cells from HVP-infected rabbits were coated with antibodies. The antibody-coated red cells appeared at around 2 weeks after virus inoculation, and the reactivities increased rapidly thereafter: 2% on day 13, 20% on day 22, and 60% on day 24. In the last week immediately before death, more than 90% of red cells were coated with antibodies (Figure 3A). Considering that patients with infectious mononucleosis produce heterophile antibodies that agglutinate red cells, we speculated that a link may exist between the heterophile antibodies and anti-red cell autoantibodies in our rabbit model. An agglutination test using a Monospot kit showed that all HVP-infected rabbits produced PB heterophile antibodies, whereas control rabbits did not (Figure 3B, top). The antibody-coated red cells closely correlated to the titers of heterophile antibodies during disease progression (Table 1). Preabsorption of HVP-infected sera with normal rabbit erythrocytes could significantly reduce the agglutination activity of heterophile antibodies. The titers of heterophile antibodies decreased from 1:250 to below 1:50 after preabsorption (Figure 3B, bottom). These results indicate that the anti-red cell antibodies in the serum of HVP-infected rabbits cross-reacted with PB heterophile antibodies in this rabbit model.Table 1Titers of Anti-RBC and Heterop