Cytokine response and polymerase chain reaction study of peripheral blood mononuclear cells in infants with human cytomegalovirus infection

ELSEVIER

FEMS Immunology

and Medical Microbiology

12 ( 1995) 153-

IMMUNOLOGY AND MEDICAL MICROBIOLOGY

I.58

Cytokine response and polymerase chain reaction study of peripheral blood mononuclear cells in infants with human cytomegalovirus infection Hideomi Asanuma Department

of Pediatrics.

School

Received

*, Kei ofMedicine.

Numazaki, Nobuo Nagata, Shunzo Chiba Sapporo

Medical

Uniwrsi/y.

S- 1. W-16

Chumku.

Sqymw

060.

Jtrptr17

IO May 1995: revised 24 July 1995: accepted 25 July 1995

Abstract We tried to detect human cytomegalovirus (HCMV) DNA in CD4 + and CD8 + T lymphocytes from fourteen infants with HCMV hepatitis using polymerase chain reaction (PCR) assay. HCMV was isolated from their urine and anti-HCMV IgM antibody was detected in their sera. One set of primers were designed from a region - a major immediate early (IE) gene. We detected HCMV IE DNA in the specimens obtained from six infants. HCMV IE DNA was detected from CD4 + cells in two cases and from CD8 + cells in one. In three cases, HCMV IE DNA was detected from both CD4 + and CD8 + cells. We also studied the relationship between HCMV infection and serum levels of cytokines. We determined serum levels of interleukin-4 (IL-4). tumor necrosis factor alpha (TNF-a) and soluble interleukin 2 receptor &IL-2R) which were associated with the activation of T lymphocytes by enzyme immunoassay. In the acute phase of HCMV infection. titers of sIL-2R were correlated with serum levels of liver enzymes in some cases. IL-4 and TNF-LU activities were not detected in sera. It is likely that expression of viral genome on T lymphocytes as well as activities of some cytokines are associated with active HCMV infection. Keyvord.s:

Human cytomegalovirus:

Polymerase

chain reaction;

T cell subsets: Cytokine:

1. Introduction Human cytomegalovirus (HCMV) is one of the causative agents of infantile hepatic dysfunction or hepatitis [ 1,2]. Acquired systemic HCMV infection is also a major complication in patients with immunosuppression associated with organ transplantation and

* Corresponding author. Tel: +81 Fax: +81 (01 I) 61 I-0352. 0928-8244/9S/$O9.50 SSDI

0928-8244(95)0006

0

1995

(01 I) 61 l-21 11 ext. 3413:

Federation I-5

of European

Microbiological

Soluble interleukin

2 receptor

AIDS. HCMV causes a variety of clinical manifestations, such as hepatitis. pneumonia, retinitis and gastroenteritis [3]. It is considered that blood transfusion from seropositive healthy donors to seronegative recipients transmits HCMV infection [4]. This fact is supported by the findings that use of leukocyte-deleted blood products can reduce the frequency of transfusion-associated HCMV infection [S]. Some investigators reported that HCMV DNA was detected in peripheral blood mononuclear cells (PBMCS) obtained from healthy seropositive donors Societies. All rights reserved

[6-81. However, others argued that none of these samples proved to be positive for HCMV DNA [9]. Schrier et al. [ 101 reported the presence of HCMV RNA in PBMC by in situ hybridization. It has not been sufficiently clarified whether PBMCs are the principal site of latency or not, but PBMCs are obviously related to the states of acute, chronic latent or reactivated HCMV infection. In this study. we concentrated on the subject of infants with liver dysfunction from primary acute HCMV infection. The final object of this study was to clarify the mechanisms of immunological reaction in the acute phase of primary perinatal HCMV infection and the roles of PBMC in the individuals with HCMV infection. We analyzed the localization of HCMV genome in peripheral blood T lymphocytes and determined serum levels of some cytokines in infants with HCMV infection.

2. Materials and methods

2.1. Patients We analyzed fourteen infants who had hepatic dysfunction associated with perinatal HCMV infection. Clinical specimens (blood and urine) in acute and chronic phase were collected from these infants. The mean age of patients was 7.57 + 4.33 (mean k S.D.) months old, ranging from 2 months to IS months of age with a male/female ratio of 6/8. Diagnosis of HCMV infection was obtained by HCMV isolation from urine and by presence of positive specific IgM antibodies to HCMV [ 1 l]. Serum IgG and IgM antibodies to HCMV were determined using available ELISA kits in the acute phase (Enzygnost, Behringwerke AG, Marburg, Germany). The fourteen infants were seronegative for hepatitis A, B, C and Epstein-Barr viruses. Informed consent was obtained from the parents of each patient prior to collection of samples. 2.2. Virus isolation from urine Urine of the acute phase was obtained from infants with hepatic dysfunction. HCMV from urinary specimens was isolated according to the standard tissue culture technique using MRC-5 cells [I I]. The

presence of HCMV was confirmed by observation of its characteristic cytopathic effect (CPE), and by immunofluorescent staining with monoclonal antibodies against HCMV immediate early (IE) and early (E) antigens (Ortho Diagnostics Systems. Carpinteria, CA) [ 11,121. 2.3. ~~~~~~n~~c~earcell preparations PBMCs were isolated from heparinized peripheral blood by Ficoll-Paque (Pharmacia LKB, Uppsala, Sweden) gradient centrifugation and resuspended in RPM1 1640 (Gibco, Paisley, UK) with 10% FCS (Gibco) to a concentration of lo-20 X 10h cells/ml. 2.4. Analysis ofji'ow cytometry for characterization of T lymphocytes To analyze the conditions of cellular immunity of infants with hepatic dysfunction associated with perinatal primary HCMV infection, we assessed the surface markers on PBMCs obtained from two infants by flow cytometry using the following fluorescein-conjugated monoclonal antibodies (mAb): Leu4 (CD3), Leu3a (CD4), Leu2a (CD8), Leu7 (CD57) and HLA-DR (Becton Dickinson, Mountain View, CA). Paired isotype-specific control antibodies were run with each sample. Fluorescence was compared with appropriate fluorescein-labeled isotype controls (IgG-FITC: B ec t on Dickinson) using logarithmic-tolinear conversion tables. 2.5. Fractionation

ofT lymphocytes

Dynabeads M-450 CD2, CD4, and CD8 (Dynal A.S., Oslo, Norway) were added to PBMC suspensions and incubated for 60 min at 4°C on a tilt rotation device. The rosetted cells were isolated by placing the test tube in the Dynal Magnetic Particle Concentrator (MPC) for 3 min and washed three times in PBS with FCS. The test tube was removed from the Dynal MPC. Then the rosetted cells were resuspended in PBS with FCS. To detach the Dynabeads, DETACHaBEAD was added to the resuspension. Then the mixture was incubated for 1 h at room temperature. To remove the detached beads the test tube was placed in the Dynal MPC for 3 min. The positive cell suspension was removed from the

H. A.sanurna et nl. / FEMS tmmunolog~

md Medical

Micmhiolo~y

test tube while the beads were attached to the wall of the tube by the Dynal MPC.

3. Results

2.6. Polymerase

acute phase of prima?

3. I. Characterization chain reaction (PCR) assay

DNA was extracted from cells after fractionation with Dynabeads, and we amplified a part of HCMV IE gene using PCR assay. Reaction mixture in a volume of 50 ml contained 10 mM Tris-HCI pH 8.3, 50 mM KCI. 0.1% Gelatin, 1.5 mM MgCl?, 1 mM of each dNTP, 0.05 FM with respect to each primer of PIE- 1 (5’~CAAGAGAAAGATGGACCCTGAT-3’ nucleotides 980- 100 1) and PIE-2 (5’-CAGGACATCTTTCTCGGGGTTC-3’ nucleotides 13041325) [ 131 based on the nucleotide sequences determined by Akrigg et al. [14], and 2.5 U of Taq polymerase (Promega, Madison, WI). Each cycle consisted of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 2 min. Cell lysate of MRC-5 cells and HCMV, a laboratory strain AD169 (from the American Type Culture Collection, Rockville, MD)-infected MRC-5 cells served as respectively negative and positive controls. The 30 cycles were performed with DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT). PCR products were electrophoresed through 4% agarose gels, stained with 1 Fg/ml ethidium bromide and visualized with an ultraviolet transilluminator as reported previously [ 131.

2.7. Assays ,for detection

of

cell free

leukin 2 receptor (sIL-2R), interleukin tumor necrosis factor alpha (TNF-CY)

soluble

inter-

4 (IL-4) and

We obtained sera from I I out of 14 patients with HCMV hepatitis in the acute phase. We observed changes of serum levels of sIL-2R and liver enzymes in three cases. Serum sIL-2R, IL-4 and TNF-a activities were determined by commercially available ELISA test kits (sIL-2R from T Cell Diagnostic, Cambridge, MA, IL-4 and TNF-a from Research and Diagnostic Systems. Minneapolis, MN) according to the manufacturer’s instructions. As the comparative study, we also examined serum levels of sIL-2R from eleven age-matched infants with congenital heart disease (CHD). When their sera were obtained, they were free from any infectious diseases and from the state of heart failure.

15s

I2 C1995) 153- 15X

of lymphocyte perinatal

subsets during

HCMV infection

The percentages of each surface marker of PBMC from two infants with hepatic dysfunction associated with primary HCMV infection were showed as follows: CD3, respectively 49.6% and 56.2%; CD4, 3 1.2% and 33.9%; CD8, 22.0% and 23.9%; CD57. 4.6% and 18.1%; and HLA-DR, 48.7% and 33.9%. The CD4/CD8 ratios were 1.42 and 1.42. 3.2. Detection of HCMV DNA by PCR assay HCMV DNA was detected from fractionated T lymphocytes of acute phase in 6 of 14 cases by PCR assay. In three cases, HCMV IE DNA was detected from both CD4 + and CD8 + cells. HCMV IE DNA was detected from CD4 + cells in two cases and from CD8 + cells in one case (Table I). HCMV IE DNA was not detected from T lymphocytes of chronic phase. Fig. 1 summarizes the results obtained from case 4. The PCR products were analyzed on 4% agarose gel and stained with ethidium bromide. The fragment-size of aimed products was 346 bp. 3.3. Serum levels

of IL-4

and TNF-a

The elevation of serum IL-4 and TNF-LY titers was not observed in eleven samples obtained from infants with HCMV hepatitis. 3.4. Serum 1eLlels of sIL-2R Serum levels of sIL-2R were determined by ELISA in eleven patients. as shown in Fig. 2. In sera Table I Detection

of HCMV DNA by PCR assay

CWZ

CD2 +

CD4 +

CD8 +

I 2 3

_ _ _

+ + +

+ + _

4 5 6

+ + _

+ _

+ + _

+

H. Asanuma

156

et al. / FEMS Immunology

and Medical

Microbiology

12 (1995) 153-158

GOT slL-2R cpT ,li/rnLI ,I”/L,

~346

I

I

’

I

bp 0

o*Set isolation of HCMV PCR

Fig.

I. Erhidium

bromide-stained

P means positive control, representing amplified AD1 69 strain. Lanes from CD2+

I, 2

(+)

(+)

8

6 (+I

(+)

!JlOllthS

10 (+)

(+)

(+)

agarose gel of the amplified

PCR products obtained from case 4. N means negative control and

tion with HCMV

4

2

DNA

Fig. 3. The clinical course of a case.

of the

and 3 show the products after amplifica-

IE primer pair. Lane

I

shows amplified products

cells. Lanes 2 and 3 respectively

show amplified

products from CD4 + and CD8 + cells. The arrowhead indicates the amplified 346 bp DNA.

from the patients with HCMV infection, sIL-2R titers were significantly elevated (1928.8 + 747.3 U/ml, P < 0.05) than those of CHD infants (I 124.3 t 380.7 U/ml).

that serum sIL-2R indicated relatively high levels at the initial stage and thereafter decreased. The changes of serum levels of sIL-2R and GOT (equal to AST) and GPT (equal to ALT) of a case, representative of three, are shown in Fig. 3. Although HCMV had been isolated from urine during this time course, the serum levels of liver enzymes and sIL-2R decreased. The change of serum levels of sIL-2R was correlates with that of liver enzymes in these three cases.

3.5. The clinical course 4. Discussion The changes of serum levels of sIL-2R and liver enzymes in three cases were observed for several months. In these cases, we observed the tendency

slL-2R u rnI, 3500 .

. . .

3000 2500

-

1500r. 2000

# 1

(:lll)

1

I

hepatic dysfunctm assoaated wth HCMV

Fig. 2. Serum levels of soluble mterleukin 2 receptor &IL-2R) determined by ELISA. The serum titer of healthy children less than two years of age, is presented as the shaded area (706.6+ 286.0 U/ml).

After primary infection, HCMV becomes latent and is reactivated by several factors. Functions and effects of activated T lymphocytes or cellular immune systems in both primary and secondary HCMV infections have been investigated. Cytotoxic CD8 + T lymphocytes and natural killer (NK) cells have complementary roles in the recovery from HCMV infection [ 151. In asymptomatic healthy carriers, increased numbers of subsets of CD8 + , CD57 + cells have been associated with the states of persistent HCMV infection [ 161. HCMV mononucleosis patients have elevated levels of CD8 + cells and decreased levels of CD4 + ceils if 71. HCMV infection also influences the ratios of surface markers on T lymphocytes. Although HCMY infection frequently occurs in the context of pre-existing immunosuppression, as seen in patients with AIDS, HCMV infection itself frequently causes depressed cellular immunity [18]. Tim6n et al. [i9] reported that PBMCs obtained from children with acute

H. Asnnuma

rt al. / FEMS lmmunolog~

HCMV infection showed impairment in their proliferative response to some stimulations. We tried to study the roles of cells of immune system in infants with hepatic dysfunction associated with perinatal primary HCMV infection. The cellular immunological states of these infants was not severely damaged and the ratios of surface markers on T lymphocytes from infants with HCMV hepatitis was not different from age-matched healthy infants [20]. We were able to detect viral genomes in the fractionated peripheral blood T lymphocytes. These results support previous findings which referred to the correlation between T lymphocytes and HCMV infection. Concerning the detection of HCMV DNA, there is no predominance between CD4 + and CD8 + cells. It was speculated that both CD4 + and CD8 + cells had significant roles in immunologic reaction for HCMV infection. Nakano et al. [21] also reported the importance of the reaction of CD4 + and CD8 + cells using flow cytometry. There were two cases that HCMV DNA was detected in fractionated CD4 + and/or CD8 + cells but not in CD2 + cells (pan T cell). It was suspected that CD2 + /CD4 + or CD2 + /CD8 + cells with HCMV DNA were not collected in enough number after the fractionation by immunomagnetic beads (Dynabeads M450 CD2, Pan-T). This is considered to be due to the problem of cross-reactivity of the monoclonal antibodies against CD2 and CD4 or CD8 of the immunomagnetic beads. As it was speculated that HCMV infection occurred to a significant extent in CD4 + or CD8 + cells, we determined serum levels of cytokines that are associated with activation of T lymphocytes. IL-2 is secreted from CD4 + Thl cells and stimulates CD8 + cytotoxic T lymphocytes and NK cells. IL-2 has the critical role as a transmitter of commands in the cellular immunity. In general, the levels of serum sIL-2R are correlated with serum IL-2 levels. On the other hand, IL-4 is secreted from CD4 + Th2 cells and contributes to the secretion of immunoglobulins. Our study showed that the serum sIL-2R titers of HCMV hepatitis infants indicated significantly high level in the acute phase than that of CHD infants. Although there are some reports [22,23] on the relationship between serum levels of cytokines and the condition of heart failure, the reason for a slight elevation of sIL-2R in CHD infants is unknown (the

and Mrdicul

Microbiology

157

12 (lYY5J 153%IS8

reported serum titer of healthy children less than two years of age is 706.6 + 286.0 U/ml [24]). In some cases, the changes of serum sIL-2R titers of infants with HCMV hepatitis were speculated to be correlated with the condition of liver dysfunction. HCMV infection may also affect the activation of cellular immunity and the secretion of IL-2 that stimulates NK cells with cytotoxic activity. The elevation of IL-4 titer was not observed in this study. It was also speculated that the level of localized IL-4 was too low or the secretion of IL-4 from Th2 cells was inhibited by HCMV infection. We also examined the relationships between markers of liver dysfunction associated with HCMV infection and serum levels of TNF-(u. TNF-(r has the important role that modulates the inflammatory process during various types of infection. TNF-cx stimulates HCMV IE activity in the study of in vitro [25]. HCMV IE gene products also enhance expression of the TNF-a gene [26]. In the present study, TNF-(Y activities in sera were not observed. In septic patients, HCMV antigenemia and high levels of serum TNF-(r were reported [27]. However, these findings cannot be compared with ours because our subjects were not in septic or immunosuppressive conditions. We conclude that expression of viral genome on T lymphocytes as well as activities of some cytokines were associated with perinatal primary HCMV infection.

Acknowledgements We thank Michihide Mikami, Ogyu Laboratory, for expert technical assistance with flow cytometry. This work was supported by research grants from the Ministry of Education, Science and Culture of Japan, and the Clinical Pathology Research Foundation of Japan.

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