Chronological changes in Epstein-Barr virus genome and subsets of peripheral mononuclear cells in a case of HLH

doi:10.4236/ojped.2011.13008

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Open Journal of Pediatrics, 2011, 1, 30-33

doi:10.4236/ojped.2011.13008 Published Online September 2011 (http://www.SciRP.org/journal/ojped/ OJPed

Published Online September 2011 in SciR es. http://www.scirp.org/journal/OJPed

Chronological changes in Epstein-Barr virus genome and

subsets of peripheral mononuclear cells in a case of HLH

Yukayo Ukeba-Terashita1, Yoshinori Saita1, Yoshinori Ito2, Hirokazu Kanegane3, Hiroshi Kimura4,

Ichiro Kobayashi1,5*

1Department of Pediatrics, Kitami Red Cross Hospital, Kitami, Japan;

2Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan;

3Department of Pediatrics, Graduate School of Medicine, University of T o yama, To yama, Japan;

4Department of Virology, Nagoya University Graduate School of Medicine, Nagoya, Japan;

5Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Email: *[email protected]

Received 30 April 2011; revised 1 June 2011; accepted 15 July 2011.

ABSTRACT

Hemophagocytic lymphohistiocytosis (HLH) is a rare

disorder characterized by high grade fever, heap-

tosplenomegaly, lymphadenopathy and cytope- nia in

association with hypercytokinemia and mac- rophage

activation. Epstein-Barr virus (EBV) is a common

cause of HLH particularly in Asian countries. Al-

though EBV usually infects B cells and causes infec-

tious mononucleosis, it is detected predominantly in

CD8+ T cells in EBV-associated HLH. In the present

study we found the EBV genome in various lineages

of the lymphoid cells including T, B, and natural kil-

ler cells in a 14-months-old boy with mild EBV-asso-

ciated HLH. Furthermore, chronological changes in

the copy number of EBV-DNA in each lineage are

reported. Profound decrease in the number of pe-

ripheral natural killer cells at the early stage could be

involved in the development of HLH.

Keywords: Epstein-Barr Virus; Hemophagocytic Lympho-

histio Cytosis; Natural Killer Cell

1. INTRODUCTION

Hemophagocytic lymphohistiocytosis (HLH) is a rare

disorder characterized by high grade fever, hepatosple-

nomegaly, lymphadenopathy and cytopenia in associa-

tion with hypercytokinemia and macrophage activation

[1]. HLH is classified into two clinical entities; primary

HLH which develops in inherited diseases such as fa-

milial hemophagocytic lymphohistiocytosis (FHL) or pr-

imary immunodeficiency, and secondary HLH which

develops associated with other acquired disorders such

as infection, collagen vascular diseases and malignancy

[1]. Sixty percent of the secondary HLH in children is

associated with Epstein-Barr virus (EBV) in Japan [2].

Although most cases of EBV-HLH have been reported

from Asian countries, reports from other regions have

been increasing [3,4]. EBV is a ubiquitous human h erpes

virus which usually infects B cells and causes self-lim-

iting infectious mononucleosis (IM). On the other hand,

persistent or recurrent IM-like disease associated with

abnormally high levels of both anti-EBV antibodies and

EBV genome is known as chronic active EBV infection

(CAEBV) [5]. EBV-associated HLH (EBV-HLH) occurs

in association with either CAEBV or a primary infection

of the virus [1-5]. Recent reports have demonstrated that

EBV infects NK or CD4+ T cells in CAEBV, whereas it

dominantly infects CD8+ T cells in EBV-HLH [5,6]. We

report here a case of HLH associated with a primary

infection of EBV and the chronological changes in

EBV-DNA of each lymphoid cell subset.

2. CASE REPORT

A previously healthy 14-month-old boy was admitted to

our hospital because of fever up to 41˚C persisting for

four days. He was born to non-consanguineous healthy

parents and had no history suggesting immunodeficiency

or hypersensitivity to mosquito bite. On the third hospi-

tal day, cervical lymphadenopathy and hepatospleno-

megaly were noted. Laboratory examination showed:

white blood cell count 7.89 × 109/l (neutrophils 17%,

lymphocytes 55%), hemoglobin 98 g/l, platelet count 45 ×

109/l, aspartate transaminase 543 IU/l, alanine transami-

nase 204 IU/l, total cho lesterol 0.81 g/l, triglyceride 2.24

g/l, C-reactive protein 115.3 mg/l, ferritin 2090 g/ l. Coa gu -

lation study showed: prothrombin time 17.8 seconds,

activated partial thromboplastin time 51.8 seconds, fi-

brinogen 2.66 g/l (reference range 2.00 g/l - 4.00 g/l) and

fibrin degradation products 93.9 g/ml (< 10 g/ml), sug-

Y. Ukeba-Terashita et al. / Open Journal of Pediatrics, 2011, 1, 30-33 31

gesting disseminated intravascular coagulation (DIC).

NK cell activity and a serum soluble CD25 was 14%

(normal; 18% - 45%) and 10,300 U/ml (normal < 520

U/ml), respectively. Although hemophagocytosis was

not evident in his bone marrow, seven of the eight items

of HLH criteria by Histiocyte Society were fulfilled [1].

Nafamostat mesilate was administered for DIC. Because

mild dilatation of coronary artery with a high echog enic-

ity of the arterial wall similar to that observed in Kawa-

saki disease was detected by echocardiography on the

7th day of hospitalization despite slight improvement of

both clinical and laboratory findings, high-dose intrave-

nous immunoglobulin (HD-IVIG) therapy (2 g/kg) was

started. Serological test for EBV before HD-IVIG ther-

apy showed IgG antibody to viral capsid antigen (VCA)

1:160, IgG antibody to early antigen 1:20, but negative

for IgG antibody to EBV nuclear antigen (EBNA) sug-

gesting a primary infection of EBV. Because his clinical

and laboratory findings rapidly improved, no further

therapy was performed.

Flow cytometric analysis of his peripheral blood at the

onset of HLH demonstrated a high level of CD8+HLA-

DR+ cells, wher eas the number of CD19+, CD20+, CD5 6+,

CD16+ , and CD57+ cells was low (Ta b l e 1 ). The num-

ber of CD8+HLA-DR+ cells declined in 20 days which

was associated with recovery of CD16+ or CD57+ cells.

The ratio of each subset has returned to normal ranges 3

months after the onset.

The copy number of EBV-DNA was analyzed by a

real-time polymerase chain reaction after separation of

his peripheral blood mononuclear cells (PBMC) into

CD19+, CD3, CD4+, CD8+, CD16+ and 56+ cells by an

immunobeads method (DynaBeads; Dynal A/S, Oslo,

Norway) as previously reported [7]. DNA was extracted

Table 1. Flow cytometric analyses of the peripheral mononu-

clear cells .

Date Apr 5 Apr 25 Jul 11 Nov 6

Absolute number

of PBMC (/mm3) 3666 3846 3872 5728

CD4 19.2% 41.6% 44.1% 36.4%

CD8 70.7% 40.6% 38.7% 34.0%

CD8×HLA-DR 56.7% 17.7% 11.2% 8.9%

CD16 2.3% 17.2% 12.2% 20.0%

CD56 1.4% ND 6.5% 14.9%

CD57 4.4% 7.6% 6.6% 12.2%

CD19 0.9% 0.7% 12.7% 18.2%

CD20 1.2% 0.6% 9.2% 12.9%

Abbreviation: PBMC, peripheral mononuclear cells; ND, not done.

from 2 × 106 PBMC using a QIAmp Blood Kit (Qiagen,

Hilden, Germany). A real-time quantitative PCR assay

with a fluorogenic probe was performed using TaqMan

PCR kit (PE Applied Biosystems, Foster City, CA). The

amount of EBV-DNA was calculated as the number of

virus copies per microgram PBMC DNA. The copy

number of EBV-DNA was extremely high at the onset in

all of CD19+, CD3, CD4+, CD8+, CD16+ and 56+ cells. A

high level infection with CD16+ or 56+ cells persisted at

least for five months, although the copy number of the

viral DNA declined with the clinical improvement (Ta-

ble 2). One year after the onset EBV-DNA was detect-

able only in CD19+ cells at a latent infection level com-

parable to normal individuals with a past infection of the

virus.

After the recovery from HLH, he has not shown any

IM-like symptoms or abnormal laboratory findings dur-

ing 2 years of follow up. IgG antibody to EBV early

antigen disappeared soon, whereas serum levels of IgG

antibodies to VCA and EBNA gradually elevated to

1:1280 and 1:40 in 5 months, respectively. NK activity

restored to 45%. No mutation was detected in either

SH2D1A or XIAP gene.

3. DISCUSSION

In the present study, we analyzed chronological changes

of EBV-DNA in each subset of PBMC in a case of

EBV-HLH. From his clinical course and the profile of

anti-EBV antibodies, HLH was likely associated with a

primary infection of EBV rather than CAEBV. Although

coronary lesion was transiently observed, the diagnostic

criteria for Kawasaki disease was not fulfilled [8]. In

addition to vasculitis such as Kawasaki disease, coronary

lesions develop in association with infection of the virus

to the endothelial cells in CAEBV or with systemic in-

flammation as observed in systemic-onset juvenile idio-

pathic arthritis [9,10].

Our case showed increased number of peripheral

CD8+HLA-DR+ cells and a decrease in both the number

and the activity of NK cells at the diagnosis, which is

consistent with the previous reports of EBV-HLH [11].

Table 2. Chronological changes of EBV-DNA (copies/g DNA) in

each subset.

DateApr 5 Apr 25 Jun 7 Sep5 Jan 9 Apr 16, 2008

PBMC110 × 1044.77 × 104269 858 54 86

CD317.9 × 1043.46 × 10420 823 ND ND

CD41.22 × 1040.74 × 10417 249 ND ND

CD840.2 × 1043.55 × 1040 527 ND ND

CD16ND 5.14 × 1041483 311 99 ND

CD5697.8 × 1045.13 × 1041295 1769 ND ND

CD1913.9 × 1042.16 × 104119 932 173 508

Abbreviation: PBMC, peripheral mononuclear cell s ; ND, not done.

Y. Ukeba-Terashita et al. / Open Journal of Pediatrics, 2011, 1, 30-33

The dominant population of EBV-infected cells is re-

ported to be CD8+ T cells in EBV-HLH, whereas the

ma jor target s of EBV in CAEBV are CD4+ T cells or NK

cells [5-7]. EBV-infected CD8+Tcells are activated and

produce cytokines such as IFN-γ, TNF-α and inter-

leukin-6, which activate macrophages and endothelial

cells resulting in hemophagocytosis, DIC, and multi-

organ failure [1,5,12]. NK cells, as well as EBV-specific

cytotoxic T cells (CTL), play a critical role in the elimi-

nation of EBV-infected cells [1]. This is supported by the

fact that the functional defects in cytotoxic activity of

CTL and NK cells such as X-linked lymphoproliferative

syndrome and FHL predispose for EBV-HLH [1]. In

acute IM the number of NK cells, although it usually

increases, is inversely correlated with the severity of the

disease [13]. Thus, the numerical defect of NK cells as

observed in our case could cause the development of

EBV-HLH or severe IM. Our case showed subsequent

recovery of both NK activity and the number of CD16+ ,

56+ or 57+cells as well as decline in CD8+HLA-DR+

cells, which was associated with clinical improvement.

Furthermore, no mutation was detected in causative

genes of XLP such as SH2DA or XIAP gene. Thus, al-

though mutations of other FHL-related genes have not

been tested, the numerical and functional abnormality

was unlikely to be intrinsic to his NK cells. Of note was

the finding that high lev els of EBV-DNA was detected in

CD16+ and 56+ cells, in addition to T and B cells, despite

the decreased number of the subsets in our case. Al-

though other methods such as in situ hybridization and

immunohistochemistry were not performed, a high level

of infection in CD 16+ and 56+ cells was confirmed by

serial examination for several months. The copy number

of EBV-DNA in PBMC of our case was comparable to

that in previously reported cases of EBV-HLH, CAEBV

and post-transplantational lymphoproliferative disorder

[14]. Although EBV-HLH has a tendency to have larger

viral burdens than acute phase of IM, it is difficult to

differentiate between these two diseases simply by viral

load in whole PBMC [15]. The copy number of EBV-

DNA gradually declined in all of the subsets and was

finally detectable in B cells only at a latency level one

year after the onset of the disease. These findings are in

contrast to NK cell-type CAEBV or NK cell lymphoma

which shows a clonal expansion of EBV-infected NK

cells [5]. It is known that EBV is able to infect NK cells

at an early stage of IM, although the precise mechanisms

of infection remain unclear [16]. As well, EBV genome

is also detected in bo th NK and B cells under some con-

ditions such as EBV-HLH but usually at a lower level

than CD8+ T cells [6]. Isobe et al have reported that in

vitro infection of EBV induces apoptosis of NK cells

[17]. Thus, it is possible that the infection by EBV in-

duced apoptosis of NK cells in vivo and allowed un-

regulated activation of EBV-infected CD8+T cells. De-

creased number of peripheral B cells in association with

HLH as observed in our case has also been reported [18].

Imashuku et al have demonstrated depletion of B cells in

the spleen of the patients with HLH and suggested in-

volvement of cytokines produced by activated T or NK

cells and/or Fas-FasL-mediated apoptosis [18].

Although our case showed both clinical and labora-

tory improvement before the commencement of HD-

IVIG therapy suggesting self-limiting nature, it is possi-

ble that the therapy accelerated the improvement by its

anti-inflammatory mechanism [19]. Further studies are

required to clarify the mechanisms of the decrease in the

number of peripheral NK cells in EBV-HLH without un-

derlying primary immunodeficiencies or FHL.

In conclusion, EBV infected various subsets of PBMC

including NK cells in a case of HLH associated with a

primary infection of EBV. EBV genome gradually de-

clined in association with his clinical improvement and

was finally detectable in only B cells at a latency level.

Transient decrease in NK cells could be involved in the

development of EBV-HLH in our case.

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