Download Virus Infection in Patients With Histiocytic Necrotizing Lymphadenitis

Microbiology and Infectious Disease / EBV, HTLV-I, AND PARVOVIRUS B19 IN HISTIOCYTIC NECROTIZING LYMPHADENITIS
Virus Infection in Patients With Histiocytic Necrotizing
Lymphadenitis in Taiwan
Detection of Epstein-Barr Virus, Type I Human T-Cell Lymphotropic
Virus, and Parvovirus B19
Chang-Fang Chiu, MD, PhD,1 Kuan-Chih Chow, PhD,2 Tze-Yi Lin, MD, PhD,3
Ming-Hsui Tsai, MD,4 Chi-Min Shih, MD,5 and Li-Mien Chen, MD6
Key Words: Histiocytic necrotizing lymphadenitis; EBV; Epstein-Barr virus; HTLV-I; Human T-cell lymphotropic virus type I; Parvovirus B19;
Fas ligand
Abstract
The relationship of Epstein-Barr virus (EBV), type I
human T-cell lymphotropic virus (HTLV-I), and
parvovirus B19 to histiocytic necrotizing lymphadenitis
was studied prospectively in 10 Taiwanese patients
using materials obtained by fine-needle aspiration and
lymph node biopsy. The presence of EBV was detected
by in situ hybridization for EBV-encoded RNA
expression. Immunocytochemistry was used to detect
virus-encoded protein for EBV and parvovirus B19.
DNA in situ hybridization and polymerase chain
reaction were performed to determine the existence of
HTLV-I provirus. Expressions of EBV-encoded RNA
and Fas ligand were detected in all cases. Expression of
EBV-encoded protein was identified in only 1 case.
Neither HTLV-I nor parvovirus B19 was detected in
any case.
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Histiocytic necrotizing lymphadenitis (HNL) is a selflimiting clinicopathologic disorder that is identified mainly in
the cervical lymph node of young adults.1,2 Malaise, fever,
cervical lymphadenopathy with severe pain, mild granulocytopenia, and lymphocytosis3,4 are common symptoms of the
disease. Histologically, 3 types of pathologic characteristics
have been identified in the lymph node: proliferative, necrotizing, and xanthomatous.5 Although these 3 types have
different clinical features, they may represent different phases
of disease progression. It is worth noting that these 3 types of
pathologic features can appear in the same lymph node at the
same time. The degree to which the pathologic types are
mixed could represent different stages of the disease. In
particular, the presence of reactive histiocytes and a high
percentage of atypical lymphocytes have been thought to
suggest viral infection.4-9
Epstein-Barr virus (EBV) and type I human T-cell
lymphotropic virus (HTLV-I) have been associated with malignant lymphomas, such as adult T-cell lymphoma/leukemia,10
African Burkitt lymphoma,11 Hodgkin lymphoma 12 and
peripheral T-cell lymphoma.13 Both viruses also have been
associated with several benign disorders, such as infectious
mononucleosis, oral hairy leukoplakia, and immature T-cell
lymphocytosis. 14,15 Because of the presence of atypical
lymphocytes and the reactive histiocytes, EBV and HTLV-I
were regarded as potential causative agents for HNL.4,6,8,9
Because virus-associated hemocytophagic syndrome resembled HNL in certain aspects, parvovirus B19 also was
proposed as an agent.9
By using polymerase chain reaction (PCR) and in situ
hybridization (ISH), EBV DNA was identified in a fraction
of HNL with a low number of positive cells.6,8,16 Since
HNL is a self-limiting benign disorder and can resolve
© American Society of Clinical Pathologists
Microbiology and Infectious Disease / ORIGINAL ARTICLE
spontaneously within a few weeks, characterizing the
mixture of pathologic types in the same affected lymph
node at the same time would indicate whether the disease
was in progression or resolution. The essence of identifying
a specific pathologic type therefore would depend on the
time the disease was diagnosed. We conducted a prospective study by using multiple sequential fine-needle aspirations (FNAs) followed by an excisional lymph node biopsy
in 10 Taiwanese patients to examine the relationship of
EBV, HTLV-I, and parvovirus B19 to HNL.
Materials and Methods
Ten consecutive cases with biopsy-confirmed HNL were
collected in China Medical College Hospital, Taichung,
Taiwan, from October 1997 through June 1998. FNA was
performed during the first visit, and again on days 4, 7, and
14 after admission. Excision lymph node biopsy was scheduled for the day after the last FNA. All patients were
Taiwanese. None of the patients had symptoms of systemic
lupus erythematosus. The clinical record was reviewed, and
sections of each biopsy specimen were stained with H&E for
routine histopathologic examination. Imprints made from
FNA and excised lymph nodes were examined by Riu or
Wright-Giemsa staining.17 By using labeled streptavidinbiotin complex (DAKO, LSAB2 System, Carpinteria, CA),
the immunophenotype was studied with a panel of monoclonal antibodies, such as CD3, CD4, CD8, CD20, CD30,
CD43, CD45RO, CD56, CD68, lysozyme (DAKO, Kyoto,
Japan), and Fas ligand (Transduction Laboratories,
Lexington, KY). The specimen was fine-needle aspirate or
paraffin section. The chromogen was aminoethyl carbazole.
Each slide was counterstained with hematoxylin or methyl
green. Positive staining was recognized under a microscope
as crimson granules. Immunocytochemistry was used to
detect the gene product of virus by using antibodies to latent
membrane protein 1, early antigen diffused, EBV BZLF-1
replication activator (ZEBRA), and EBV-associated nuclear
protein (EBNA) 2 of EBV (DAKO, Kyoto, Japan) or to the
VP2 capsid protein of parvovirus B19 (DAKO, Kyoto,
Japan). Antibodies to ZEBRA and early antigen diffused
were used to determine whether EBV infection was lytic,
and antibodies to latent membrane protein 1 and EBNA-2
were used to determine whether EBV infection was latent.
The PCR procedure was described previously.14,18,19
Briefly, tissue sections positive for EBV-encoded RNA
(EBER) signal were marked on the glass slide under a microscope. A surgical blade was used to scrape the positive area
into a 0.5-mL microcentrifuge tube containing the PCR
mixture. The reaction mixture was then subjected to 35 cycles
of amplification using a standard procedure: denaturing DNA
© American Society of Clinical Pathologists
at 94°C for 1 minute, annealing at 56°C for 40 seconds, and
elongating at 72°C for 1.2 minutes. Primers for the EBV
BamH1 W region (a 10-12 tandem repeat of the EBV
genome) were listed as follows:
AA: 5´GCCAGAGGTAAGTGGACTTT-3´
(GenBank/M15973, sense nt 1400 to nt 1419);
AZ: 5´-TGGAGAGGTCAGGTTACTTA-3´
(GenBank/M15973, antisense nt 1640 to nt 1621).
The amplified product was resolved in 2% agarose gel.
The specificity of the 241-base-pair (bp) amplified product
was confirmed by Southern hybridization with biotinylated
BamH1 W fragment.
For HTLV-I detection, the following primers were
designed to match the pX region of HTLV-I.19
pX1: 5´-CGGATACCCAGTCTACGTGT-3´
(GenBank/L03562, sense nt 7338 to nt 7356);
pX2: 5´-GGTTCCATGTATCCATTTCG-3´
(GenBank/L03562, antisense nt 7663 to nt 7644).
The amplified product was resolved in 2% agarose gel.
The specificity of the 326-bp amplified product was
confirmed by Southern hybridization with biotinylated pX
region of HTLV-I. Positive control DNA was prepared from
a cell line (MT-2 cell) containing HTLV-I. The beta-globin
gene was used to determine the integrity of DNA in the
paraffin material.
The RNA ISH procedure was described previously.12,14
Briefly, a 4-µm section taken from paraffin-embedded tissue
was deparaffinized in xylene, dehydrated, and predigested
with 0.1 mg/mL of nuclease-free proteinase K (BoehringerMannheim, Mannheim, Germany) at room temperature for
15 minutes. The slide was washed with distilled water, rinsed
with 70% ethanol, and air-dried. One drop of fluorescein
isothiocyanate (FITC)-conjugated antisense probe (250
ng/mL in 50% formamide, 6× standard saline citrate, and
0.25% dry milk) to EBER that was expressed at 106 copies
in the affected cell was placed over each tissue section. The
probes with the following sequences were synthesized by
BRL (Bethesda, MD):
5´ FITC-ATGCTCTAGGCGGGAAGCCTCTCTTCTCCT3´ (GenBank/J02078, antisense nt 612 to nt 583);
5´ FITC-CGGTAGCACCGCACTGAGCGTTGGCGGTGT3´ (GenBank/J02078, antisense nt 560 to nt 531);
5´ FITC-CTGGTACTTGACCGAAGACGGCAGAAAGCAGA-3’ (GenBank/J02078, antisense nt 321 to
nt 290);
5´ FITC-CACGTCTCCTCCCTAGCAAAA-CCTCTAGGG3´ (GenBank/J02078, antisense nt 216 to nt 187).
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The sample was denatured at 75°C on a thermal plate
for 5 minutes and moved to a moist chamber at 37°C for 2
hours. After extensive wash with 2× standard saline citrate,
hybridization product was detected using anti-FITC antibodies conjugated with alkaline phosphatase (NEN,
Boston, MA). The chromogenic development was
processed in a mixture of 4-nitroblue tetrazolium and 5bromo-4-chloro-3-indolphosphate. The slide was counterstained with nuclear fast red or methyl green. Positive
staining was recognized under the microscope as brownish
purple granules. A specimen of nasopharyngeal carcinoma
with positive serology for EBV was used as a positive
control. Paraffin sections of normal tonsil and normal
appendix were used as negative controls.
For detecting proviral DNA of HTLV-I, the procedure
was similar to that described in the preceding paragraphs
except that (1) biotinylated HTLV-I pX probe was used; (2)
the denaturing temperature was set at 100°C; and (3) MT-2
cells containing HTLV-I were used as the positive control.
Results
❚Table 1❚ summarizes the clinicopathologic features
and the ISH, PCR, and immunostaining results. The
immunophenotyping study showed a reactive lymph node
by patchy positive areas for CD3, CD30, CD43, CD45RO,
CD68, and lysozyme. Cells positive for CD3, CD30, CD43,
and CD45RO were mainly lymphocytes. Cells positive for
CD68 and lysozyme were macrophages. Unlike normal
lymph node, the ratio of T-cell numbers was between 5%
and 25% of the total mononuclear cells in the areas
containing karyorrhectic bodies. Among T cells, only a few
(<1%) were CD56+.
The ages of the 10 patients ranged from 14 to 37 years
(mean ± SD, 22.0 ± 7.8 years). Three patients were male,
and 7 were female.
Expression of CD68 was detected by immunostaining
in all cases. The relative abundance of CD68+ cells,
however, varied from case to case. A representative
example is shown in ❚Image 1❚.
In the proliferative phase, CD68 expression was
detected mainly in the cytoplasm of histiocytes, whereas
lymphocytes and other cells were negative (Image 1A). By
ISH, expression of EBER was identified in all 10 cases.
The relative abundance of EBER-positive cells also varied
from case to case. In the proliferative phase, EBER expression essentially was detected in the nuclei of lymphocytes
within the affected lymph node, whereas histiocytes and the
other cells were negative (Image 1B).
In the necrotizing phase, the CD68 signal was mixed
with tingible bodies. The margins of germinal follicles and
the border between histiocytes were indistinguishable
(Image 1C). In this phase, EBV signal was identified in the
nuclei of lymphocytes and in the karyorrhectic bodies
within the cytoplasm of histiocyte (Image 1D).
In the xanthomatous phase, CD68 expression
decreased dramatically, and foamy histiocytes became
predominant (Image 1E). In this phase, the EBER signal
also became limited (Image 1F). In the lymph node
imprint, the EBER signal (in lymphocytes indicated by
arrows) and CD68+ histiocytes were detected simultaneously ❚Image 2A❚. The presence of EBV was confirmed by
PCR in all 10 cases (Table 1). However, the expression of
❚Table 1❚
Clinical Features and ISH, PCR, and Immunostaining Results for 10 Patients With HNL*
Case No./Sex/Age (y)
1/M/16
2/F/37
3/F/16
4/F/26
5/F/23
6/F/14
7/M/21
8/F/29
9/M/19
10/F/15
11/M/45
12/M/47
13/F/47
14/F/50
15/F/25†
16/M/15†
Diagnosis
HNL
HNL
HNL
HNL
HNL
HNL
HNL
HNL
HNL
HNL
NPC
NPC
ATLL
ATLL
AML
ALL
EBER/ISH
+
+
+
+
+
+
+
+
+
+
+
+
+
–
–
–
EBV/PCR
+
+
+
+
+
+
–
+
+
+
+
+
+
–
–
–
pX/PCR
–
–
–
–
–
–
–
–
–
–
–
–
+
+
–
–
Parvovirus B19/IM
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+
+
ALL, acute lymphocytic leukemia; AML, acute myelogenous leukemia; ATLL, adult T-cell lymphoma/leukemia; EBER, EBV-encoded RNA; EBV, Epstein-Barr virus; HNL,
histiocytic necrotizing lymphadenitis; HTLV-I, type I human T-cell lymphotropic virus; IM, immunostaining; ISH, in situ hybridization; NPC, nasopharyngeal carcinoma;
PCR, polymerase chain reaction; pX, pX region of HTLV-I.
* Cases 11 to 16 were positive controls for EBV, HTLV-I, and parvovirus B19, respectively.
† Prominent hemocytophagia and aplastic anemia were observed in bone marrow aspirates after high-dose induction chemotherapy.
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© American Society of Clinical Pathologists
Microbiology and Infectious Disease / ORIGINAL ARTICLE
A
B
C
D
E
F
❚Image 1❚ (Case 3) Paraffin-embedded tissue sections. Expression of CD68 and Epstein-Barr virus–encoded RNA (EBER) in the
proliferative, necrotizing, and xanthomatous phases of histiocytic necrotizing lymphadenitis. A, By immunohistochemistry, CD68
expression was detected mainly in the cytoplasm of histiocytes in the proliferative phase, whereas replicating lymphocytes and
the other cells were negative. B, By in situ hybridization, expression of EBER was identified mainly in the nuclei of lymphocytes
within the affected lymph node, whereas histiocytes and the other cells were negative. The specimen was counterstained with
nuclear fast red. C, The CD68 signal was spread out in the area containing tingible bodies. The margins of germinal follicles and the
border between histiocytes were indistinguishable. D, The Epstein-Barr virus signal was located in the nuclei of lymphocytes and in
the karyorrhectic bodies within the cytoplasm of histiocytes. The specimen was counterstained with methyl green. E, In the
xanthomatous phase, CD68 expression was dramatically less, and foamy histiocytes were predominant. F, In the xanthomatous
phase, the EBER signal was limited. The specimen was counterstained with nuclear fast red. (Original magnification, ×600.)
© American Society of Clinical Pathologists
Am J Clin Pathol 2000;113:774-781
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Chiu et al / EBV, HTLV-I, AND PARVOVIRUS B19 IN HISTIOCYTIC NECROTIZING LYMPHADENITIS
A
B
❚Image 2❚ (Case 7) Simultaneous detection of CD68+ and Epstein-Barr virus–encoded RNA (EBER)-positive cells in the lymph
node imprint of histiocytic necrotizing lymphadenitis. A, By combining in situ hybridization and immunostaining for the lymph
node imprint, the EBER signal (arrows) was located in lymphocyte and karyorrhectic bodies of CD68+ histiocytes. B, Lymph
node imprint stained by Riu staining. (Original magnification, ×600.)
ZEBRA and early antigen diffused was identified in only 1
case (case 3, day 4). Latent membrane protein 1, EBNA-2,
and VP2 were not detected (data not shown). In contrast
with 2 cases of adult T-cell lymphoma/leukemia, an HTLVI sequence was not detected by PCR or ISH in patients with
HNL (Table 1). Expression of Fas ligand ❚Image 3A❚ and
❚Image 3B❚ was identified by immunocytochemistry in the
CD68+ histiocytes ❚Image 3C❚ in a lymph node imprint.
Discussion
Viral infection has long been proposed in the disease
progression of HNL. In the present study, we used ISH,
PCR, and immunocytochemistry to detect HTLV-I,
parvovirus B19, and EBV in HNL. The results show that
lymphocytes in affected lymph nodes from these patients
were infected with EBV. This conclusion is based on the
detection of viral DNA by PCR in tissue sections, and localization of EBER expression in lymphocytes and karyorrhectic bodies within the cytoplasm of histiocytes. Previous
reports have described patterns of HNL with atypical
lymphocytes and suggested viral infection.3-9,20 In some
studies, many patients with HNL indeed had elevated antibody titers to EBV.3,8 Nevertheless, positive serologic data
can result from recent EBV exposure, and such data may not
indicate the presence of virus at the time of examination.
Hollingsworth et al6 attempted to document the potential
etiologic correlation between HNL and EBV or between
HNL and human herpesvirus type 6.6 Although the human
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herpesvirus type 6 signal was not detected in any of the
HNLs examined, EBV DNA was detected in 11 (55%) of 20
HNL cases by PCR. Moreover, the EBV signal was located
in 6 cases of PCR-positive HNL by ISH.6 Interestingly,
because of discrepancies in the positive results among their
collaborative laboratories, Hollingsworth et al6 suggested
that a positive result for EBV should be confirmed by
repeated examination or by other methods.
As noted, identifying a mixture of various pathologic
phases in an affected lymph node suggests that the disease
could be in progression. Therefore, diverse results obtained
from different laboratories may reflect the time the disease
was diagnosed and the time the tissue was examined.3,5,6,8,20
If this is so, it would explain the irreconcilable results in the
study by Hollingsworth et al.6 Different laboratories might
obtain different biopsy specimens or different regions of the
same specimen that contained the different phases of HNL.
Therefore, their results would be different. Furthermore,
unlike malignant lymphomas that contain EBV or HTLV-I,
HNL is a self-limiting disorder and may harbor virus only
transiently. A case report by Yen et al8 described the presence of EBV in HNL and the skin manifestations in a 6-yearold boy. We confirmed their findings by detecting EBV in all
10 cases by 3 independent methods: ISH, PCR, and
immunocytochemistry. Nevertheless, the number of EBVpositive cells varied from one case to another. HTLV-I, on
the other hand, was identified in only 2 positive control cases
of adult T-cell lymphoma/leukemia, and parvovirus B19 was
detected in bone marrow aspirates from 2 patients with
severe hemocytophagia. These results suggest that HTLV-I
© American Society of Clinical Pathologists
Microbiology and Infectious Disease / ORIGINAL ARTICLE
A
B
C
❚Image 3❚ (Case 7) Imprint from an excised lymph node.
Expression of Fas ligand in the histiocytes of a lymph node
imprint from a patient with histiocytic necrotizing
lymphadenitis. By immunocytochemistry, the expression of
Fas ligand was detected in activated histiocytes (A) and at
the boundary between histiocytes and engulfed lymphocytes
(B). The histiocyte with engulfed lymphocytes is a CD68+
histiocyte (C). (Original magnification, ×600.)
and parvovirus B19 may not have a role in the pathogenesis
of HNL in most Taiwanese patients.4,9
Previous studies demonstrated the association of EBV
with an array of diseases, such as Hodgkin disease, primary
cutaneous amyloidosis, persistent polyclonal B-cell lymphocytosis, T-cell lymphoma, and nasopharyngeal carcinoma.12,14,18-21 Despite the presence of viral DNA, only a
limited number of viral genes were expressed.22 At present,
it is unclear what controls the viral gene expression and how
the host factor(s) might interact with the virus infection,
which in turn would determine the disease manifestation.20
The disease progression of HNL considered together with
infectious mononucleosis could provide a valuable insight to
the relationship between virus infection and host response.23
Takano et al24 pointed out that karyorrhectic debris
morphologically resembled apoptotic bodies. Elegant in
situ end-labeling studies clearly showed that nuclear DNA
© American Society of Clinical Pathologists
fragmentation, a sign of the early apoptosis, not only was
identified within the necrosis area, but also was located in
the germinal center.7,25,26 Furthermore, most of the apoptotic cells were T cells. Identification of perforin in the
apoptotic foci by immunohistochemistry also suggests that
apoptosis could be initiated by cell-mediated cytotoxicity.7
Interestingly, unlike normal lymph node, T cells were
estimated to be fewer than 30% of the total mononuclear
cells in the necrotic area of HNL.7,26 Among these T cells,
only a few were CD56+ or CD16+ natural killer cells.25,27
We obtained similar results.
Moreover, the present study sheds light on the issues of
EBV infection, apoptosis, and histiocytosis by showing that
EBER-positive lymphocytes can be engulfed by histiocytes
before apoptosis (Images 2A, 2B, 3B, and 3C). The presence
of Fas ligand in the affected area clearly indicates that Fas
ligand expression is a mechanism that can be associated with
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Chiu et al / EBV, HTLV-I, AND PARVOVIRUS B19 IN HISTIOCYTIC NECROTIZING LYMPHADENITIS
apoptosis in HNL.28 Such results are supported further by
the fact that apoptosis alone may not warrant immediate
phagocytosis 29,30 and that Fas ligand expression is not
restricted to T cells.31
At present, there is no clear explanation for the
increased activity of histiocytes during the proliferative and
necrotizing phases of HNL. Although expression of early
antigen diffused and ZEBRA in an affected area indicates a
correlation between acute EBV infection and pathogenesis of
HNL, the role of EBV in HNL remains to be determined.
Our observation provides a focus for future studies to elucidate the mechanism and the order by which viral infection,
apoptosis, and histiocytosis interact with each other.
From the 1Section of Hematology/Oncology, Department of
Internal Medicine, and the Departments of 2Medical Research,
3Pathology, and 4Otolaryngology, China Medical College
Hospital; and the 5Department of Pathology and 6Section of
Hematology, Department of Internal Medicine, Armed Forces
Taichung General Hospital, Taichung, Taiwan.
Supported by the grant DMR-88-043 from the China Medical
College Hospital, Taichung, Taiwan.
Address reprint requests to Dr Chow: Dept of Medical
Research, China Medical College Hospital, 2 Yuh-Der Road,
Taichung, Taiwan 404, Taiwan.
Acknowledgments: We thank Pao-Hsiang Wen and Wen-Jye
Lo for their technical assistance and S.H. Chiou, PhD, for useful
discussion and critically reading the manuscript.
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