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, 286 (14), 12049-55

Overexpression of IL-32alpha Increases Natural Killer Cell-Mediated Killing Through Up-Regulation of Fas and UL16-binding Protein 2 (ULBP2) Expression in Human Chronic Myeloid Leukemia Cells

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Overexpression of IL-32alpha Increases Natural Killer Cell-Mediated Killing Through Up-Regulation of Fas and UL16-binding Protein 2 (ULBP2) Expression in Human Chronic Myeloid Leukemia Cells

Soyoung Cheon et al. J Biol Chem.

Abstract

IL-32 was recently identified as a proinflammatory cytokine that is induced by IL-18 in natural killer (NK) cells and is highly correlated with inflammatory disorders. However, the relationship between IL-32 and tumor progression is still unknown. In this study, we investigated whether overexpression of IL-32 affects susceptibility of chronic myeloid leukemia (CML) cells to NK cells. Interestingly, IL-32α-overexpressing CML cell lines, K562, Kcl22, and BV173, showed higher NK cell-mediated killing. Flow cytometry analysis revealed that overexpression of IL-32α induced increased expression of Fas and UL16-binding protein 2 (ULBP2) in CML cells. The direct relationship between overexpression of surface molecules by IL-32α and increased NK cell-mediated killing was confirmed by Fas or ULBP2 siRNA transfection. IL-32α-induced Fas and ULBP2 expression are regulated p38 MAPK. In addition, the transcription factor Ets1 plays a key role in ULBP2 specific expression by IL-32α overexpression in ULBP family members. Taken together, these data show that IL-32α stimulates Fas and ULBP2 expression via activation of p38 MAPK, which increases NK susceptibility of CML cells. Enhanced NK cell susceptibility of CML cells by IL-32α overexpression may improve the efficiency of NK cell-based immunotherapy.

Figures

FIGURE 1.
FIGURE 1.
Elevated NK susceptibility by IL-32α in CML cells. A, IL-32α expression in IL-32α-overexpressing K562 cells. K562 transfectants (vector or IL-32α) were lysed in cell lysis buffer as described under “Experimental Procedures,” the cell lysates were resolved on SDS-PAGE, and IL-32 expression was assessed by Western blotting with KU32–52 (mouse anti-human IL-32). The blot is representative of three independent experiments. B, shown is the NK susceptibility of K562 stable transfectants (vector and IL-32α). K562-stable transfectants (vector or IL-32α) were used as target cells, and IL-2-transfected human NK cells, NK-92MI, were used as effector cells. Each group of target cells was stained using CFSE for distinction from effector cells during flow cytometry analysis. CFSE-stained target cells were incubated with effector cells at a E:T ratio (0.25:1, 0.5:1, and 1:1) for 1 h. After incubation, co-incubated effector cells and target cells were stained with 7-AAD. The killing effect was analyzed by measurement of percentage of the CFSE and 7-AAD double-positive region. C, shown is the NK susceptibility of IL-32α-overexpressing CML Kcl22 and BV173 cell lines. Each transfectant was assayed for NK cytotoxicity. NK-92MI cells were used as effector cells at an E:T ratio of 2:1. Data presented are representative of three independent data sets for each condition (**, p < 0.05; ***, p < 0.001 versus vector control).
FIGURE 2.
FIGURE 2.
Induction of Fas and ULBP2 protein expression by IL-32α in CML cells. Surface expression of Fas and ULBP2 after IL-32α overexpression was measured in CML cell lines. CML transfectants were stained with PE-conjugated anti-human Fas and PE-conjugated anti-human ULBP2. FAS and ULBP2 expression in K562 (A and B), Kcl22 (C and D), and BV173 (E and F) was analyzed using flow cytometry. Data presented are representative of three independent data sets for each condition.
FIGURE 3.
FIGURE 3.
Induction of Fas and ULBP2 expression by IL-32α at mRNA level in K562 cells. Total RNAs were purified from K562 stable transfectants (vector or IL-32α) using TRIzol, and cDNAs were made using 2 μg of total RNA. cDNAs were used as templates for PCR amplification with Fas, ULBP1, ULBP2, ULBP3, and β-actin primers. A, Fas and ULBP2 expression by RT-PCR. B, Fas and ULBP2 expression by densitometry analysis using the TotalLabTM program. C, ULBP1 and ULBP3 expression by RT-PCR. Data presented are representative of three independent data sets for each condition.
FIGURE 4.
FIGURE 4.
Blocking effect of siRNA against Fas and ULBP2 in IL-32α-induced NK susceptibility in K562 cells. IL-32α-induced Fas and ULBP2 were blocked with specific siRNA. IL-32α-overexpressing K562 cells were transiently transfected with nonrelated control siRNA (GL3 siRNA), Fas siRNA, and ULBP2 siRNA using MicoPorator electroporation. After 48 h, K562 vector, K562-IL-32α, and siRNA-transfected K562-IL-32α (siGL3, siFas, and siULBP2) cells were analyzed. A, Fas expression in K562-vector, K562-IL-32α, and siRNA-transfected K562-IL-32α (siGL3 and siFas) cells. B, ULBP2 expression in K562 vector, K562-IL-32α, and siRNA-transfected K562-IL-32α (siGL3 and siULBP2) cells. C, NK susceptibility test. K562 vector, K562-IL-32α, and siRNA-transfected K562-IL-32α (siGL3, siFas, and siULBP2) cells were used as target cells, and NK-92MI cells were used as effector cells. Target cells were stained with CFSE as described under “Experimental Procedures,” and then the cells were incubated with NK-92MI cells at an optimal E:T ratio (E:T ratio: 0.25:1, 0.5:1, and 1:1). After a 1-h incubation, co-incubated effector cells and target cells were stained with 7-AAD. The killing effect was analyzed by measurement of percentage of CFSE and 7-AAD double-positive region. Data presented are representative of three independent data sets for each condition (*, p < 0.01; **, p < 0.05 versus K562-IL-32α).
FIGURE 5.
FIGURE 5.
p38 MAPK activation in IL-32α-induced Fas and ULBP2 expression. IL-32α-overexpressing K562 cells were incubated with 40 μm SB203580 (SB) for 48 h. After incubation time, the cells were stained with PE-conjugated anti-human Fas (CD95) Ab and PE-conjugated anti human ULBP2 Ab. A, Fas and ULBP2 expression by histogram analysis. B, K562 cells were transiently transfected with vector (pcDNA3.1+) or IL-32α cDNA (IL-32α/pcDNA3.1+). Αfter 24 h, K562 vector transfectants and IL-32α transfectants were lysed with cell lysis buffer as described under “Experimental Procedures.” Whole protein lysates were assessed with KU32–52 (mouse anti-human IL-32), rabbit anti-phosphor-p38 MAPK, and rabbit anti-total p38 MAPK. Data presented are representative of three independent data sets for each condition.
FIGURE 6.
FIGURE 6.
Blocking effect of Ets-1 in IL-32α-induced ULBP2 expression. K562 stable transfectants (vector or IL-32α) were transfected with Ets-1 siRNA. After incubation for 24 h, the cells were used for mRNA isolation, and the expression level of Ets-1 and ULBP family members was assessed. A, Ets-1 and ULBP family member expression by RT-PCR. B, ULBP2 expression by densitometry analysis using TotalLabTM program. C, protein level of ULBP2 on the Ets-1 down-regulated K562 stable transfectants (vector or IL-32α) was analyzed using flow cytometry. Data presented are representative of three independent data sets for each condition.

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