Effects of IL-2 on MMP expression in freshly isolated human NK cells and the IL-2-independent NK cell line YT

Abstract

Interleukin-2 is an important activation factor for natural killer (NK) cells but its effect on NK cell matrix metalloproteinases (MMP) production and matrix degradation is less well investigated. We have used freshly isolated human NK cells and the IL-2-independent NK cell line, YT, to investigate the effects of IL-2 stimulation on NK cell invasion of Matrigel and on MMP expression and production. In YT cells, we found opposing early and late effects of IL-2 stimulation with an early (2 h) increase in MMP-9 protein level and enhanced migration in the Matrigel invasion assay and by 30 hours a decreased mRNA expression of MMP-2, MMP-9, MMP-13, MT3-MMP, and MT6-MMP. We also found a preculture period of 48 hours with IL-2 to negatively affect YT cell migration. We furthermore found that freshly isolated human NK cells Matrigel invasion was MMP-dependent and it increased in response to IL-2. Importantly, in freshly isolated human NK cells we did not see a downregulation of MMPs after 24 hours IL-2 stimulation, but instead a significant upregulation of MT6-MMP mRNA. Because of the cellular localisation of MT6-MMP, which ensures a focalized proteolytic activity, and its high expression compared with the other MMPs in freshly isolated human NK cells makes it of interest to study further.

Fig. 1

IFN-γ produced in response to IL-2 stimulation. YT cells (5×10⁵ cells/mL) were stimulated with 0, 1, 10, 100 and 1000 U IL-2 for 24h and levels of IFN-γ were measured in supernatants. Results are described as mean values of at least three separate experiments using IFN-γ ELISA kit (OptEIA, BD Biosciences). A dose-response relationship was shown with an increase in IFN-γ production with increasing levels of IL-2.

Fig. 2

Interleukin 2 stimulation of YT cells migration across Matrigel membranes. YT cells (5×10⁵) were placed in the top well of Matrigel Invasion chambers and subjected to invasion assay for 48h with either no IL-2 or 100 U IL-2. Results are expressed as the number of invaded cells and each bar represents mean values (± SEM) from at least three separate experiments. Statistical significance was assessed using Student’s t-test, P < 0.05 (*two-tailed; #one-tailed). A) IL-2 stimulation significantly increased the amount of invaded cells by 52% (P < 0.05). B) YT cells pre-cultured for 48h with 100 U IL-2 prior to migration through Matrigel inserts invaded to a lesser degree than YT cells not pre-cultured with IL-2.

Fig. 3

The effect of IL-2 treatment on the expression of MMP-2, -9, -13, MT1-, MT3- and MT6-MMP in YT cells. Results were normalized to GAPDH expression and compared to unstimulated control cells. Significance of differences was tested with Welch Modified Two-Sample t-test. Stimulation with IL-2 significantly down regulated the expression of MMP-2, -9, -13, MT3- and MT6-MMP.

Fig. 4

Production of MMP-9 in response to IL-2 stimulation. Each bar represents mean values (± SEM) from at least three independent experiments. Statistical significance was assessed by two-tailed Student's t-test (*P < 0.05). A) YT cells (4×10⁵) were stimulated with 100 U IL-2 for 1, 2, 4, 10 and 24h. Stimulation with IL-2 resulted in an increase in levels of MMP-9 in supernatants with a peak at 2h (P < 0.05), followed by a decrease to 10h (P < 0.05), and returning back to baseline by 24h. B) Levels of MMP-9 in supernatant from YT cells cultured with or without 100 U IL-2 for 24h were also compared. Lower levels (50 %) of MMP-9 were found in supernatants from YT cells cultured for 24h with 100 U IL-2 compared to YT cells without IL-2.

Fig. 5

Interleukin 2 stimulation of human NK cell migration. Freshly isolated human NK cells (0.5×10⁶) were placed in the top well of Matrigel Invasion chambers and subjected to invasion assay for 48h with either 10 U IL-2 (as a control) or 100 U IL-2 with and without 10 µM GM6001. Results are expressed as number of invaded cells (± SEM). Statistical significance was assessed using two-tailed Student’s t-test (**P < 0.01; ***P < 0.0001). A concentration of 100 U IL-2 significantly increased the amount of invaded cells by 68% (P < 0.0001). Addition of GM6001 inhibited the migratory ability with 31% (P < 0.01).

Fig. 6

MMP expression analysis. Expression of MMP-2, -9, -13, MT1-, MT3- and MT6-MMP was analysed in freshly isolated as well as IL-2 stimulated human NK cells. Results, normalized to GAPDH expression, and compared to unstimulated control cells are expressed as fold change values. Statistical significance was assessed using two-tailed Student’s t-test (*P < 0.05; **P < 0.01). A) MMP-9, -13, MT1-, MT3- and MT6-MMP were found to be expressed in low levels. B) Stimulation with IL-2 (100 U and 1000 U) for both 2 and 24h led to a significant increase in the expression of MT6-MMP.