Cytolytic mechanisms involved in non-MHC-restricted cytotoxicity in Chediak-Higashi syndrome

Abstract

To determine the mechanisms responsible for the impaired lymphocyte-mediated cytotoxicity in Chediak-Higashi syndrome (CHS), we investigated the killing ability of peripheral blood lymphocytes (PBL) from three patients with CHS using several kinds of target cells that were sensitive to perforin, Fas ligand (FasL), and/or tumour necrosis factor-alpha (TNF-alpha). Freshly isolated CHS PBL did not kill K562 target cells, killing of which by normal PBL was perforin-dependent, as demonstrated by complete inhibition by concanamycin A (CMA), an inhibitor of perforin-based cytotoxicity. In contrast, the CHS PBL exhibited substantial cytotoxicity against Jurkat cells, which was only partially inhibited by CMA treatment but not by the addition of neutralizing anti-FasL or anti-TNF-alpha antibodies. IL-2-activated CHS PBL exhibited substantial levels of cytotoxicity against K562 and Jurkat cells, the levels being 74% and 83% of the respective normal control values, respectively. CMA treatment showed that while the cytotoxicity of IL-2-activated CHS PBL against K562 was largely dependent on perforin, that against Jurkat was largely not. IL-2-activated CHS PBL expressed FasL mRNA, and killed Fas transfectants. These findings indicate that CHS PBL have an ability to kill some target cells via a perforin-mediated pathway, especially when they are activated by IL-2. It was also demonstrated that CHS PBL can exert cytotoxicity against certain target cells by utilizing FasL and an undefined effector molecule other than perforin, FasL, or TNF-alpha.

Fig. 1

Induction of apoptosis by anti-Fas MoAb and tumour necrosis factor-alpha (TNF-α). K562, Jurkat, Fas/WR19L, and WR19L cells (1 × 10⁶/ml) were cultured in medium alone or in the presence of anti-Fas MoAb (7C11, 5 μg/ml) or TNF-α (10 ng/ml) for 4 h. Then the cells were fixed and permeabilized with ethanol as described in Patients and Methods. Percentages of apoptotic cells (less than 2 N) were determined by flow cytometry after staining with propidium iodide. Representative data from three independent experiments are shown.

Fig. 2

Cytotoxicity of peripheral blood lymphocytes (PBL) from Chediak-Higashi syndrome (CHS) patients (CHS 1, 2, 3) and age-matched controls (control 1, 2) against K562 target cells, and the blocking effect of concanamycin A (CMA). Freshly isolated (a) and IL-2-activated (b) PBL were preincubated with or without CMA (100 nm) for 2 h. The pretreated cells were then tested for cytotoxicity against K562 cells at E:T ratios of 40:1 (▪) and 20:1 (□) in a standard 4-h ⁵¹Cr-release assay. Results are expressed as the mean ± s.d., and error bars are sometimes smaller than the plot symbol. Representative data from two independent experiments are shown.

Fig. 3

Cytotoxicity of peripheral blood lymphocytes (PBL) from Chediak-Higashi syndrome (CHS) patients (CHS 1, 2, 3) and age-matched controls (control 1, 2) against Jurkat target cells, and the blocking effect of concanamycin A (CMA), anti-FasL MoAb, or anti-tumour necrosis factor-alpha (TNF-α) pAb. Freshly isolated (a) and IL-2-activated (b) PBL were preincubated with or without CMA (100 nm) for 2 h, anti-FasL MoAb (NOK2, 10 μg/ml) for 30 min, and/or anti-TNF-α pAb (× 20 dilution) for 30 min as indicated. The pretreated cells were then tested for cytotoxicity against Jurkat cells at E:T ratios of 40:1 (▪) and 20:1 (□) in a standard 4-h ⁵¹Cr-release assay. Results are expressed as the mean ± s.d., and error bars are sometimes smaller than the plot symbol. Representative data from two independent experiments are shown.

Fig. 4

FasL-mediated cytotoxicity of IL-2-activated peripheral blood lymphocytes (PBL) from Chediak-Higashi syndrome (CHS) patients (CHS 1, 2, 3) and age-matched controls (control 1, 2) against Fas transfectants. PBL were cultured with IL-2 (100 U/ml) for 72 h and then tested for cytotoxicity against Fas/WR19L in the presence or absence of anti-FasL MoAb (NOK2, 10 μg/ml) or wild-type WR19L target cells at E:T ratios of 40:1 (▪) and 20:1 (□) in a 12-h ⁵¹Cr-release assay. Results are expressed as the mean ± s.d., and error bars are sometimes smaller than the plot symbol. Representative data from two independent experiments are shown.

Fig. 5

Expression of FasL mRNA in IL-2-activated peripheral blood lymphocytes (PBL) from Chediak-Higashi syndrome (CHS) patients and normal controls. PBL from CHS patients (CHS 1, lane 1; CHS 2, lane 2; CHS 3, lane 4) and age-matched controls (control 1, lane 3; control 2, lane 5) were activated with IL-2 (100 U/ml) for 24 h and then FasL and β₂-microglobulin mRNAs were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) as described in Patients and Methods. Representative data from two independent experiments are shown.