TRANSACTIONS OF TWEROYAI.SOCIETYOF TROPICALMEDICINEAND HYGIENE(1991)
Changes in populations of immune effector haemorrhagic fever with renal syndrome
Richard M. Lewisl*, Ho Wang Lee’, Anthony F. See’, David B. Parrish’, Jung Sik Moon3, Dai Jung ‘Medical Division, United States Army Medical Research Institute of Kim3 and Thomas M. Cosgriff” Infectious Diseases. Frederick. Marvland. USA: ‘Institute for Viral Diseases, Korea University, ’ 3Capital Army Hospital, Seoul, Korea ‘ *
To characterize the immune response in haemorrhagic fever with renal syndrome, serial changes in immune effector cells were measured in 14 patients. Significant findings included initial elevations of all major leucocyte populations, increases in suppressor T cells and B cells, decreases in helper/suppressor cell ratios, and a dramatic increase in activated T cells. These changes were most marked in severely ill patients. Changes reverted to normal over approximately one week.
Introduction The characterization of the immune response to viral infections is important in understanhing the pathouhvsiologv of disease (OLDSTONE, 1979). Measurement 6f changes in the number and propoition of distinct populations of immune effector cells helps to define this response and can have prognostic significance (QUINNAN & ENNIS, 1980; DAHN et al., 1988). The association of specific immune cell functions with cell surface antigens has allowed the use of fluorescence-labelled monoclonal antibodies to the membrane proteins as significant tools in the characterization of the immune response to many viral diseases (BOYD, 1987). Haemorrhagic fever with renal syndrome (HFRS) is a viral illness which has been documented throughout the world (WHO, 1983) and is caused by viruses belonging to the genus Hantavirus (SCHMALJOHN et al., 1985). The severity of the syndrome varies with geographical area and is related to the particular strain of Hantavirus virus causing infection (LEDuc, 1987). Hantaan virus causes particularly severe disease (LEE, 1989) and Puumala virus relatively mild disease (LAHDEVIRTA, 1989). Seoul virus typically produces disease of intermediate severity (BYUN et al., 1986; LEE, 1989). Changes in populations of immune effector cells have been studied in experimental infection of newborn mice with Hantaan virus (ASADA et al., 1987), but similar studies have not been conducted to characterize the cellular immune response during the ‘Present addresses: Richard M. Lewis, Hemostasis and Thrombosis Laboratory, Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, USA; Thomas M. Cosgriff, Hematology-Oncology Division, Department of Medicine, Fitzsimmons Army Medical Center, Aurora, Colorado, USA. Address for correspondence: Richard M. Lewis, Building 29, HFB-470, 8800 Rockville Pike, Bethesda, MD 20892, USA.
course of human disease. To define the immune response in HFRS, granulocytes, monocytes, lymphocytes and lymphocyte sub-populations were measured in acutely ill HFRS patients.
Materials and Methods Patients Patients were enrolled in the study on admission to Capital Army Hospital, Seoul, Korea. Of the 14 patients studied, all were young, male soldiers, 21 to 25 years old; they were grouped according to disease severity. Severe disease was defined by the presence of shock or anuria (24 h urine volume
283 Blood cell counts White blood cell counts were performed on a Danam haematology analyser in the clinical laboratory of the Korea University Hospital. The white blood cell differential count was determined by calculating the percentage of lymphocytes, monocytes and granulocytes which were identified by their forward scatter/side scatter characteristics (FSC/SSC) patterns on the flow cytometer and staining characteristics using anti-Leu-M3 and anti-HLe-1. Flow cytometry Fluorescent antibody-labelled cells were analysed on a Becton Dickinson FACSCA~, with excitation at 488 nm using an argon laser. Lymphocytes in each patient’s sample were initially gated based on the FSC/SSC and staining characteristics using anti-leuM3 and anti-HLe- 1. Percentagesof positive cells were calculated as that portion with a fluorescence >95% of that of all unlabeled cells. The absolute number of lymphocytes was calculated from the differential and the white blood cell counts. The number of cells in each lymphocyte subset was determined from the number of lymphocytes and the percentage of cells with positive fluorescence. Analysis of data The data were analysed using a computer graphics program (Sigma-Plot@, Jandel Scientific, Corte Madera, California, USA) which calculated regression lines for changes in cell populations over time. Regression analysis was not performed for individual patients but calculations used all points on a particular graph. Regression lines were graphed, along with individual values and lines representing normal means and standard deviations. Results
Data from 14 patients were analysed; 4 had mild disease. 6 moderate diseaseand 4 severe HFRS. On initial testing, total white blood cell counts were
frequently elevated, as were granulocyte and monocvte counts (Fig. 1). The degree of increase in these counts appearedto correlate with diseaseseverity: the increaseswere highest in patients wtih severe illness. Total lymphocyte counts were elevated in patients with severe illness, although regression slopes suggested that all patient groups had elevations earlier in the disease course (Fig. 2). The increase in total lymphocyte counts was due to increased numbers of both T (CD3+) and B (CD19+) cells (Fig. 2). Analysis of T cell subsets showed an increase in cytotoxic-suppressor cells (CDS+) (Fig. 3). All of these changes appeared to be most pronounced in patients with severe disease. The number of helper cells (CD4+) remained within the normal range, with relatively higher values in patients with severeillness. The end-result of these changeswas an initial decrease in the helper-suppressor ratio with a gradual increase during the 2 weeks of study. The number of activated T cells (HLA-DR+) was increased dramatically in all patients (Fig. 4). As well as absolute numbers for activated T lymphocytes, Fig. 4 shows the percentagesof T lymphocytes which were activated. Becauseactivated lymphocytes often become larger and more granular, the data were re-analysed by measuring the number of HLA - DR + lymphocytes that exhibited FSC/SSC similar to the larger monocytes. Very few additional lymphocytes were identified by this method. All were cytotoxic/ suppressor T cells. There were no changes in the number of natural killer cells (CD57+) (data not shown). Over the course of approximately one week, values for the various cell populations returned to the normal range. Discussion
Immune effector cells are crucial in the defence of human pathogens, including viruses. Changes in populations of these cells over the course of illness can provide important information related to host defences and pathogenetic mechanisms. The pattern of
DAY OF ILLNESS
Fig. 1. Countsof A, B: total bloodcells;C, D: granulocytes;E, F: monocytes.Cell countsweredividedinto two groups;onegroupfrom patients with eithermild (A) or moderate(0) disease(A, C, E) andthe othergroupfrom patientswith severedisease(0) (B, D, F). The horizontallines representthe meansof normal individuals and 2 standarddeviationsaboveand below the notmsl mean value.
DAY OF ILLNESS
Fii. 2. Countsof A, B: total lymphocytes;C, D: T lymphocytes(CD3+); E, F: B lymphocytes(CD19+).The resultsfrommild (A) andmoderate (0) patients arc shown in A, C, and E, and thosefrom severepatients(Cl) in B, D and F.
DAY OF ILLNESS
Fig. 3. Countsof: A, B: helpercells(CD4+); C, D: suppressorcells(CD8+). The resultsfrommild (A or A) andmoderate(0) patientsareshown in A and C and those of severepatients (0) in B and D. the lymphocyte response, however, exbibia considerable diversity among viral diseases. As reviewed by BARANSKI & YOUNG (1987), lymphocytosis has been
associatedwith viral infection and can be the result of increased numbers of B or T cells. In other cases, lymphocytopenia has been associatedwith other viral infections. HSIA et al. (1989) reported increased levels of lymphocytes during rhinovirus infection which probably reflected an increase in T lymphocytes, in
particular natural killer (CD16) and cytotoxic suppressor (CD8) cells. In contrast, LEVANWWSKI et al. (1986) measured a decreasein total T lymphocytes (CD3) during rhinovirus infection, which was accounted for by decreasesin both cytotoxicihelper (CD4) and cytotoxickuppressor (CD8) cells. Similar patterns have been reported for measles (DAGAN et al., 1987). In the studies of patients with HFRS, the numbers
285 loo so so70- o 60-
0.6 0.6 A
0 0 0
0.4 0.2 O.O6
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Cl hlro, 1s
DAY OF ILLNE!SS Fig. 4. Counts of activated lymphocytes-T (HLA-DR+): A, B: counts per kc; C, D: percwmgcs of T lymphocytes. A and C show mild (A) and moderate (0) patients’ vehtcs, B and D show the valua for severe patients (Cl). Four high values src not shown on graph B.
of monocytes, granulocytes and lymphocytes, including the quantity of lymphocyte subsets, were measured. All leucocyte populations were elevated, including total white blood cell count. This is consistent with numerous reports of HFRS, including a very early clinical report that 91% of the cases had leucocyte counts above 10 OOO/mm3,and 33% above 20 ooo/mms (POWELL, 1954). Of the major white cell populations, the monocytes remained significantly above normal values longer than the other populations. The lymphocyte counts were not elevated and remained within the normal range in those patients with mild or moderate disease. Lymphocyte counts in a study of 58 patients with nephropathia epidemica were reported as decreased during the, leucocytosis phase, days 3-5 (LAHDEVIRTA, 1971); nephropathia epidemica follows a milder course than HFRS. In general, most cell counts were correlated with the increase in the white blood cell counts. The cell population which exhibited the most dramatic increase, as determined by percentage of total cells, were the activated lymphocytes (HLA-DR+). Generally, the populations of lymphocytes remained constant and the absolute numbers changed, basedon the values of the total white blood cell count. Although the importance of immune effector cells during the course of HFRS has not been previously defined, the contribution of lymphocytes in mouse Hantaan virus infection has been examined. Studies by ASDAand others (1987) in Yamanishi’s laboratory, using adoptive transfer techniques in nude mice, showed that the B cell and some T lymphocyte
sub-populations were important for survival. Using athymic BALB/c mice it was shown that adoptive transfer of cytotoxic/suppressor (Lytl.2) cells or immune serum was effective in protection from viral infection. If lymphocytes were transferred after viral inoculation, lymphocytes of all subsets or, secondarily, lymph tes depleted of only helper (L3T4+) cells?were“re fective in viral clearance. In other mouse studies, cytotoxic T lymphocytes could be generated from the spleens of Hantaan-infected mice (ASADAet al., 1988). Of the lymphocyte data in this sNdy, the most striking included that of the activated T cells and the helper/suppressor ratios. The helper/suppressor ratios, even though they were not significantly different from the normal range, showed a consistent increase over time, which may reflect a decreaseof suppressor cells rather than an increase of helper cells. YONGMINGet al. (1988a) noted changes in the proportion of atypical lymphocytes in HFRS in Chinese patients. The number of total lymphocytes in the present study of Korean patients did not change; in addition, gates on the flow cytometer were reset to evaluate cells which were larger and more complex (increased forward and side scatter) than would be expected for lymphocytes: cells which stained positively for lymphocyte markers were not observed in these areas. Blood smearswere not examined for the presence of atypical cells. HFRS replicates in adherent mononuclear cells isolated from human peripheral blood and can be isolated from macrophages and granulocytes of infected rats (NAGAI et al., 1985). In the studies of
286 HFRS patients, no attempts were made to isolate virus from specific leucocvte Dopulations. The increases in monocyte and- gr&uiocyte populations suggest that, if infected by Hantaan virus, there is no negative effect on cell number. In another report by YONGMING et al. (1988b), a gradual increase in suppressor cells with no concomitant change in helper cells has been reported. It was concluded that the smxxessors alone accounted for the change in the help&uppressor cell ratio. In this studv of Korean HFRS oatients. when initial observations began 6-8 d afte; onset~suppressorcells were increased and decreasedgradually over the following three weeks. The increase over time in the helper/ suppressor ratio could be accounted for by a decrease in suppressor cells and constant values of the helper subpopulation. Acknowledgements
Thanks to Dr Pyung-Woo Lee and Dr Youn Ho Chung for laboratory space and assistance. The help of Dr Ross Graham and Dr Kenneth Dixon at the United States Army Research Unit, Republic of Korea, was appreciated. References
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