Host's innate immune response to fungal and bacterial agents in vitro: up-regulation of interleukin-15 gene expression resulting in enhanced natural killer cell activity

Abstract

Natural killer (NK) cells play an important role in the first line of defence against viral infections. We have shown earlier that exposure of human peripheral blood mononuclear cells (PBMC) to viruses results in rapid up-regulation of NK cell activity via interleukin-15 (IL-15) induction, and that this mechanism curtails viral infection in vitro. By using Candida albicans, Escherichia coli and Staphylococcus aureus, we now show here that exposure of PBMC to fungi and bacteria also results in an immediate increase of NK cytotoxicity. Reverse transcriptase-polymerase chain reaction and Western blot analyses as well as the use of antibodies against different cytokines revealed that IL-15 induction played a predominant role in this NK activation. These results indicate that IL-15 is also involved in the innate immune response against fungal and bacterial agents.

Figure 1

Effect of different pathogens on enhancement of NK cytocidal activity in the presence or absence of anti-IL-15 mAb. PBMC, pre-exposed to each of the indicated pathogens, were incubated with or without IL-15 neutralizing mAb (10 µg/ml). After 24 hr incubation at 37°, PBMC were incubated with ⁵¹Cr-labelled K562 cells at an E : T ratio of 20 : 1 for 16 hr (see Materials and methods). The data shown are expressed as percentage of NK cytotoxicity and represent mean ± SEM of triplicate determination using PBMC from three donors. The isotype-matched control antibody (for the IL-15 neutralizing antibodies) was also tested, no decrease of NK activity was observed (data not shown)

Figure 2

Western blot analysis of IL-15 protein expression in PBMC treated with different pathogens. PBMC exposed to the indicated pathogens for 24 hr were lysed and run on 15% SDS–PAGE. After electroblotting onto nylon membrane, IL-15 mAb (M112) (1 µg/ml) was incubated with membrane to allow the specific binding of mAb to IL-15 protein on the membrane. The blots were developed using AP-conjugated anti-mouse, BCIP and NBT. The experiment was repeated with three donors and similar results were obtained each time (not shown). Uninfected PBMC (band 1), PBMC treated with HHV-6 (band 2), with C. albicans (band 3), with E. coli (band 4), and with S. aureus (band 5).

Figure 3

Kinetics of IL-15 mRNA expression in PBMC exposed to different pathogens. PBMC (5 × 10⁶ cells) were exposed to different pathogens for various time periods (0, 6, 12, 18 and 24 hr). Cells were then lysed, and total RNA was extracted and measured. Total RNA was reverse-transcribed (RT) with oligo d(T), and IL-15 mRNA was amplified using PCR. Bands were detected following blotting on a nylon membrane as described in Materials and Methods. (a) IL-15 and β-actin bands obtained from PBMC treated and untreated with different pathogens at different time points. (b) Curves showing the increase (fold) of IL-15 mRNA levels in PBMC treated with different pathogens at different time points. Quantification of IL-15 mRNA was based on its expression relative to the amplified amount of β-actin mRNA.

Figure 4

Effect of different anti-cytokine antibodies on enhancement of NK activity by different pathogens. PBMC were treated with C. albicans (a), E. coli (b), S. aureus (c) and HHV-6 (d). followed by the addition of mAb against IL-15 (10µ/ml), IL-12 (10 µg/ml), IL-2 (10 µg/ml), IFN-α and TNF-α (10 µg/ml) separately. After 24 hr incubation at 37°, PBMC were harvested and their NK activity was determined by chromium release assay. Data are expressed as percentage of cytotoxicity, representing mean ± SEM. The experiment was repeated three times and similar results were obtained. *, P < 0·05; **, P < 0·01; ***, P < 0·001 compared with PBMC stimulated with respective pathogen in the absence of mAb.

Figure 5

Kinetic analyses of NK activity of PBMC exposed to different pathogens. PBMC treated with C. albicans, E. coli, S. aureus, and HHV-6 were collected at different time points (shown are the results for samples taken at 12, 24, 36 and 48 hr), and then resuspended at a concentration of 4 × 10⁶ cells/µl with complete medium. The NK cytotoxic activity was determined by measuring the chromium release by K562 labelled cells incubated with stimulated PBMC for 16 hr at 37°. The experiment was repeated three times and similar results were obtained. For all samples taken at 6 hr (not shown), the cytotoxic activity was below that observed for 12-hr samples.