Production of cytotoxic factor by peripheral blood mononuclear cells (PBMC) in patients with dengue haemorrhagic fever

Abstract

A unique cytokine, human cytotoxic factor (hCF), has been shown to occur in the sera of patients with dengue fever (DF) and dengue haemorrhagic fever (DHF). The present study was undertaken to investigate the ability of fresh PBMC of such patients to produce hCF. The PBMC were cultured for 24 h and the culture supernatants (CS) were analysed for the presence of hCF by cytotoxicity assay, competitive ELISA and dot blot tests. In 90% of 246 cases CS were positive for hCF by the three tests. CS were positive for hCF in PBMC collected from days 1-20 of illness but not at later periods. Higher cytotoxic activity was observed in CS of days 1-4 of illness and was highest in cases of DHF grade IV and lowest in cases of DF. Dot blot hybridization of RNA extracted from the PBMC of the patients showed the presence of mRNA for hCF in 94% of cases. A similar number of patients showed the presence of hCF in situ in the PBMC smears by fluorescent antibody technique. hCF was found only in CD4+ T cells. The findings thus present direct evidence of the production of hCF by CD4 T cells of cases of DF/DHF.

Fig. 1

A typical dot blot test for detection of human cytotoxic factor (hCF) in the culture supernatants (CS) of PBMC of the cases of various grades of dengue illness and normal healthy controls. Cultures were prepared (1 × 10⁶ cells/ml) in 4-cm Nunc Petri dishes and incubated at 37°C in the presence of 5% CO₂ and harvested after 24 h. CS were blotted on nitrocellulose membrane and were blocked. Blots were treated with anti-hCF antibody followed by anti-mouse IgG + horseradish peroxidase and then developed with substrate diaminobenzidine as described in Patients and Methods. The controls included CS from normal healthy individuals' PBMC (lane 9, A–H; lane 11, F–H); and purified hCF (1 μg/ml and its doubling dilutions) as positive control (lane 10, A–D).

Fig. 2

Presence of human cytotoxic factor (hCF) in the culture supernatants (CS) of PBMC collected on different days after the onset of illness. ▪, ELISA; □, dot blot; ♦, cytotoxicity assay. The ‘cut off’ value was obtained from the culture supernatants of PBMC of 50 normal healthy individuals as mean value + 2 s.d. and was 25% for ELISA and 10% for the cytotoxicity assay. The figures in parentheses represent the total number of patients in each group.

Fig. 3

Mean cytotoxic activity in the culture supernatants from controls and cases of various grades of illness. The bars over the column represent the size of the s.d. and the figures in parentheses represent total number of patients in each group.

Fig. 4

Dot blot hybridization analysis of total RNA isolated from PBMC of the cases of dengue fever (DF) (lane 1, A–D), DHF I (lane 1, E–H), DHF II (lane 2, A–D), DHF III (lane 2, E–H), DHF IV (lane 3, A–H), normal healthy controls (lane 4, A–H; lane 5, A–H), and dengue type 2 virus-infected mouse spleens (lane 6, A–H) as positive control. Each spot represents 5 μg of RNA from each patient/control or from mouse spleen blotted on nitrocellulose membrane. Hybridization was done as described in Patients and Methods.