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From the Division of Clinical Research,* National Health Research Institutes, Tainan; and the Graduate Institutes of Microbiology and Basic Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan
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Abstract |
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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.11,12 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.3,13 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.14-16 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.15,17 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 colleagues18-20 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.9,21 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.
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Materials and Methods |
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The rabbit model of EBV-associated HPS was previously established by Hayashi and colleagues18
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 x g for 30 minutes to remove cell debris, filtered with a 0.45-µm filter (Millipore, Billerica, MA), and then centrifuged at 100,000 x 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 x 107 copies of HVP as quantified by real-time polymerase chain reaction (PCR)]. Control rabbits were inoculated with phosphate-buffered saline (PBS).
Histopathological and Immunohistochemical Examinations
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 CD79
(B-cell marker, cross-reaction with rabbit CD79) (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.
Detection of Antibody Responses to HVP Viral Capsid Antigen (VCA) in Rabbits
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.
Immunomagnetic Purification of T Cells, B Cells, and Monocytes/Macrophages in Peripheral Blood Mononuclear Cells (PBMCs) and Spleen
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).
PCR Detection of HVP DNA in PBMCs or Component Fractions of T Cells, B Cells, and Monocytes/Macrophages
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.18 ß-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).
Quantification of HVP Virus Load by Real-Time PCR
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 1x 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.
In Situ Hybridization to Detect Virus-Encoded Small RNA, EBER1
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).
Flow Cytometric Detection of Antibody-Coated Red Cells and Anti-Platelets in HVP-Infected Rabbits
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 x 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.
Detection of PB Heterophile Antibodies and Preabsorption Test
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 x 108 normal rabbit red cells for 30 minutes at 37°C twice before performing the agglutination assay.
In Vitro Phagocytosis 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.
Antibody Coating and ex Vivo Phagocytosis of Rabbit Red Cells
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.
Purification of Fab and Fc Fragments for Phagocytosis Assay
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.
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Results |
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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).
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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)
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(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. Detection of Anti-RBC Antibodies, PB Heterophile Antibodies, and Anti-Platelet Antibodies in Serum of HVP-Infected Rabbits
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)
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, 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.
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) cells to observe the antibody-coated platelets. As shown in Figure 3D
, the antibody-coated platelets could be observed at day 9 after HVP inoculation and, similar to anti-RBC antibodies, the titers of anti-platelet antibodies increased with correlation to the severity of diseases.
Figure 4, A and B
, summarizes the kinetics of virus-host interaction in this HPS animal model. The first event detectable after virus inoculation was the appearance of HVP DNA in rabbit sera at day 12, followed by the production of anti-VCA antibodies in sera and the presence of viral genomes in PBMCs and tissues at day 19. The production of anti-RBC/heterophile antibodies developed at a relatively late stage, usually occurring when the virus load peaked during weeks 3 to 4 after HVP inoculation, and increased rapidly thereafter. Notably, the red cell phagocytosis in rabbit tissues could only be observed after the emergence of anti-RBC antibodies, suggesting that the coating of red cells with antibodies is a prerequisite for phagocytosis of red cells. The increasing titers of anti-RBC antibodies represented a reliable indicator to predict the full-blown diseases and impending mortality in HVP-infected rabbits.
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To clarify whether both the presence of antibodies against red cells and macrophage activation are essential for the hemophagocytic process, we performed serial in vitro phagocytic studies. First, the human promonocytic cell line U937 was treated with or without TPA and tested for the activity to engulf FITC-labeled latex beads. U937 cells could phagocytose the fluorescent beads only when the U937 cells were preactivated by TPA (data not shown). Furthermore, the TPA-treated U937 cells could only engulf Coombs-treated cells (Figure 5A
, right; Coombs cell) but not control red cells (Figure 5A
, control), indicating that the coating of a red cell with anti-RBC antibodies was required for phagocytosis by activated macrophages. For clarification of the relationship between the titers of antibodies present on red cell surfaces and the degree of RBC phagocytosis, different ratios of antibody-coated RBCs were used to measure the percentages of phagocytosis by flow cytometry. The fraction of macrophages was gated (Figure 5B
, top left), and the fluorescence intensity of the gated macrophages was measured. The Coombs cell study showed a higher nonspecific background attributable to the adherence of RBC lysate to the surface of activated macrophages. The fluorescence intensity of phagocytosed Coombs cells was measured over the M1-defined region (Figure 5B
, bottom left). As shown in Figure 5B
(right), the activities of RBC phagocytosis by macrophages increased with the ratio of Coombs cells (human IgG-coated RBCs). Ex vivo phagocytosis assay further showed that the TPA-activated rabbit monocytes could engulf rabbit erythrocytes only when pretreated with HVP-infected rabbit sera (Figure 5C
, right), but not the uninfected sera (Figure 5C
, left). To define clearly the specificity of the immunoglobulin fragments that reacted with macrophages, excess Fab fragments, Fc fragments, or the whole IgG molecules were used to compete with the antibodies of Coombs cells. Figure 5D
demonstrates that Fc fragments as well as whole IgG molecules, but not Fab fragments, could completely block the engulfment of Coombs-treated cells by TPA-activated U937 cells, indicating that phagocytosis is mediated through Fc receptor. Taken together, the data shown above indicate that both the presence of anti-red cell antibodies and macrophage activation are essential for RBC phagocytosis, which is mediated via Fc fragments.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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, the emergence of anti-RBC antibodies always preceded the development of RBC phagocytosis in tissues and the increasing titers of anti-RBC antibodies predicted the full-blown development of HPS and the impending mortality of HVP-infected rabbits. Given that this rabbit model of EBV-associated HPS is not entirely similar to human HLH cases, the findings we observed in this study can still provide valuable information on the mechanism of hemophagocytosis in virus-associated HPS. Although the presence of anti-RBC antibodies or PB heterophile antibodies in this animal model can explain the frequent association of childhood HPS with EBV, the ubiquitous EBV infection and the relative rarity of EBV-associated HPS, however, challenge the role of anti-RBC antibodies in the development of EBV-HPS. As shown in this study, we demonstrated that both the phagocytic process and the specific types of blood cells phagocytosed by macrophages represent nonrandom biological events. In the absence of anti-RBC antibodies, red cell phagocytosis did not occur even in the presence of activated macrophages and vice versa. This observation may therefore explain the relative infrequency of HPS in most viral infections other than those observed with EBV and in virus-associated HLH patients.
EBV-associated HPS usually develops in patients with abnormal immune disorders such as X-linked lymphoproliferative disorders22
or in rare conditions in which EBV infects T or NK cells such as HLH and NK/T cell lymphoma.9,10,23
In X-linked lymphoproliferative disorders, the mutation of a suppressor SAP protein leads to dysregulated CTL response in primary EBV infection.24,25
In sporadic HLH patients, EBV usually infects T or NK cells rather than B cells, and EBV latent membrane protein-1 has been demonstrated to initiate the TRAFs/NF-
B/SAP/ERK signaling pathway to activate T cells and up-regulate Th1 cytokine secretion.25-28
Alternatively, HLH may result from mutations of perforin genes, leading to an ineffective CTL response to EBV infection.21
Distinct from the predominant or exclusive infection of T or NK cells by EBV in HLH,9,21
HVP infects T cells, B cells, and possibly macrophages in this rabbit model. Virus infection of B cells may induce polyclonal activation of B lymphocytes and leads to the formation of a wide variety of autoantibodies, including those directed against smooth muscle, blood groups, lymphocytes, platelets, and erythrocytes.29-31
Summarizing the data, we presented in this study, we propose that the development of HPS requires two essential events, one involving dysregulated CTL immune response, which results in hypersecretion of Th1 cytokines and the subsequent activation of macrophages, and the other branch involving the autoreactive polyclonal B-cell response that produces autoantibodies against blood cells. Whether monocytes/macrophages can be infected by EBV is still controversial. Recent studies, however, have demonstrated the capability of EBV to infect and replicate in freshly isolated human monocytes.32
The infection of macrophages by EBV may further activate macrophages. In this animal model, the infectivity of tissue macrophages by HVP was inconclusive because of too few cells to be obtained for flow cytometric analysis. However, HVP DNA could be detected in the CD14+ monocyte fraction in PBMC. Therefore, the possibility of HVP infection of macrophages may exist. The infection of monocytes/macrophages by EBV/HVP may further activate the macrophages to secrete monokines and make the cytokine storm worse in HPS.
Among the wide variety of autoantibodies produced, PB heterophile antibodies are specifically associated with primary EBV infection.17 In past decades, extensive efforts have been given to study cross-species reactivity patterns and antigenic epitopes of PB heterophile antibodies.17,33 Epitopes of PB antibodies in erythrocyte membrane have been recognized to be sialoglycopeptides containing the O-linked disialyl Gal-GalNAc oligopeptide. This epitope forms the basis for commercially available kits currently used for antibody detection in the diagnosis of infectious mononucleosis and was adopted in this study. Several explanations have been proposed to describe the mechanism of developing heterophile antibodies in EBV infection. Molecular mimicry of host cell DNA or proteins to viral gene products is the most prevailing theory.31,34 By preabsorption of HVP-infected sera with rabbit red cells in this study, the titers of heterophile antibodies were reduced significantly, suggesting that the PB heterophile antibodies may cross-react with or even represent the major components of anti-RBC antibodies in HVP-infected rabbit serum. One critical question is whether the finding of anti-RBC antibodies in this animal model can be applied to human HPS or HLH patients. We retrospectively retrieved serum samples from a total of 20 EBV-associated HPS patients for indirect Coombs test, and eight of them were positive (W.-C.H. and I.-J.S, unpublished data), suggesting that anti-RBC antibodies do exist in some of the EBV-HPS patients. We are now performing a prospective multicenter study using direct Coombs test to clarify the positivity of anti-RBC antibodies in EBV-HPS patients.
In this study, we further clarified the kinetics of virus-host interaction in this animal model of EBV-associated HPS.18-20 The HVP viruses appear to first infect lymphoid cells in the marginal zone of spleens, followed by an extensive infection in spleen, lymph nodes, livers, and lungs before mortality. The rabbits developed symptoms and signs typical to that of human HPS such as coagulopathy, cytopenia, and depletion of lymphoid systems and bone marrow. Because of the limitation and commercial availability of antibodies against rabbits, especially those of cytokines and antigens of blood cells, a detailed description of cytokine changes is not available in this study. Although this HVP rabbit model of HPS is not entirely similar to human HLH cases, the findings we observed in this study can still provide valuable information to understand the pathogenesis of human EBV-HPS. Importantly, this animal model should also provide a valuable tool for designing the therapeutic trial to improve the management of virus-associated HPS patients.
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Footnotes |
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Supported by the National Science Council and the National Health Research Institutes, Taiwan.
Accepted for publication January 25, 2007.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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N, Sever-Prebilic M, Crnic-Martinovic M, Prebilic I: Severe autoimmune hemolitic anemia as a potentially fatal complication of EBV infectious mononucleosis. Int J Hematol 2001, 74:352-353[Medline]
aszewska E, Kurowska E, Duk M, Koscielak J: Paul-Bunnell antigen and a possible mechanism of formation of heterophile antibodies in patients with infectious mononucleosis. Acta Biochim Pol 2003, 50:1205-1211[Medline]
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