Clusterin synergizes with IL-2 for the expansion and IFN-γ production of natural killer cells

Abstract

CLU is a secreted, multifunctional protein implicated in several immunologic and pathologic conditions. As the level of serum CLU was shown to be elevated during inflammatory responses, we questioned if CLU might interact with circulating lymphocytes leading to functional consequences. To assess this possibility directly, mouse splenocytes and purified NK cells were cultured with varying dose of CLU, and its effect on cell proliferation was examined. Our data showed that CLU up-regulated DNA synthesis and expansion of NK cells significantly in response to a suboptimal, but not maximal, dose of IL-2, and CLU alone did not exhibit such effects. This CLU-mediated synergy required the co-presence of CLU at the onset of IL-2 stimulation and needed a continuous presence during the rest of the culture. Importantly, NK cells stimulated with CLU showed increased formation of cell clusters and a CD69 activation receptor, representing a higher cellular activation status compared with those from the control group. Furthermore, these NK cells displayed elevated IFN-γ production upon RMA/S tumor target exposures, implying that CLU regulates not only NK cell expansion but also effector function of NK cells. Collectively, our data present a previously unrecognized function of CLU as a novel regulator of NK cells via providing costimulation required for cell proliferation and IFN-γ secretion. Therefore, the role of CLU on NK cells should be taken into consideration for the previously observed, diverse functions of CLU in chronic inflammatory and autoimmune conditions.

Figure 1

Effect of CLU on the proliferation of whole splenocytes and purified NK cells. (A) Endogenous expression of CLU in total splenocytes and purified NK cells was monitored using Western blots (WB), as described in Materials and Methods. Cell lysates (20 μg), prepared from whole splenocytes (Spl) or purified NK cells, were subjected to SDS‐PAGE and immunoblotting with anti‐CLU Ab (C‐18 or M‐18). Diluted (1:10) C57BL/6 mouse serum (10 μl) was loaded as a positive control (CLU). (B and C) Total splenocytes (2×10⁵; B) or 1 × 10⁵ purified NK cells (C) were cultured for 3 days with 0, 0.5, 5, or 50 μg/ml CLU in the presence or absence of 100 U/ml IL‐2. ³H‐Thymidine (1 μCi) was added for an additional 12 h to count radioactivity incorporated into the DNA. The lower bar graphs are re‐plots of the upper graphs to compare ³H‐thymidine incorporation at 5 μg/ml CLU in the presence or absence of 100 U/ml of IL‐2. The data are representatives from 5 independent experiments. Error bars represent sd (**P<0.01, ***P<0.001).

Figure 2

CLU synergizes with IL‐2 for the proliferation of NK cells. (A) Purified NK cells were plated at 1 × 10⁵ cells/well and incubated for 3 days with various concentrations of IL‐2 (10, 100, 500, and 1000 U/ml) in the presence or absence of 5 μg/ml CLU. ³H‐Thymidine (1 μCi) was added for an additional 12 h to count radioactivity incorporated into the DNA. (B) Purified NK cells (5×10⁵) were labeled with 5 μM CFSE for 10 min, and cell division was analyzed by FACS, as described in Materials and Methods. Numbers in each graph indicate the percentage of cells undergoing cell division. FL1‐H, Fluorescence 1‐height. (C) Purified NK cells (1×10⁶) were cultured in the presence or absence of 5 μg/ml CLU and/or 100 U/ml IL‐2 for 6 days. Live cells were counted using a hemocytometer. The data shown are representatives of a minimum of 3 independent experiments. Error bars represent sd (*P<0.05).

Figure 3

CLU stimulates NK cell proliferation at the early onset. (A) Purified NK cells (1×10⁵; >95% purity) were cultured for a total 3.5 days with 100 U/ml IL‐2 in the absence or presence of 5 μg/ml CLU, which was added at the same time (0 h), 24 h, or 48 h following IL‐2 stimulation, as depicted in the left panel. Total incubation time with IL‐2 was 84 h in all conditions. ³H‐Thymidine incorporation into the DNA of NK cells was plotted as a bar graph (right panel). (B) Purified NK cells (1×10⁵; >95% purity) were cultured with 100 U/ml IL‐2 in the absence or presence of 5 μg/ml CLU. Twenty‐four hours later, media were removed and replaced with new ones containing 100 U/ml IL‐2 but without CLU (left panel). As controls, cells were cultured in the continuous presence of IL‐2, with or without 5 μg/ml CLU (left two bars). ³H‐Thymidine (1 μCi) was added at 2 days following washout and cultured for an additional 12 h, and radioactivity was compared. The level of ³H‐thymidine incorporated into NK cells was plotted as a bar graph (right panel). The data shown are representatives of 3 independent experiments. Error bars represent sd (*P<0.05).

Figure 4

Addition of prestimulated culture supernatants to the freshly isolated NK cells did not facilitate proliferation. (A) Purified NK cells (1×10⁵; with 80% purity) were stimulated with 100 U/ml IL‐2 in the absence or presence of 5 μg/ml CLU. Twenty‐four hours later, culture supernatants (sup) were collected and added to the freshly isolated NK cells (>95% purity) labeled with 5 μM CFSE. (B) Cells were cultured for 2 days in the presence of IL‐2, with or without 5 μg/ml CLU, and their divisions were analyzed by FACS, as described in Materials and Methods.

Figure 5

CLU causes aggregation of NK cells without significantly altering surface NK receptors. (A) Purified NK cells (7.5×10⁵) were incubated with 5 μg/ml CLU in the presence or absence of IL‐2 (100 U/ml) for 5 days prior to taking photos using an inverted microscope (40× and 100× original magnification). (B) Surface expression of CD27, CD44, CD11c, LFA‐1, I‐Ab, NKG2D, CD69, or ICAM‐1 was monitored by FACS using NK cells cultured with 100 U/ml IL‐2 in the presence or absence of 5 μg/ml CLU. The filled line represents isotype control, thin line IL‐2 alone, and thick line IL‐2 + CLU‐cultured NK cells. The results of this particular experiment represent those from 3 other independent experiments.

Figure 6

CLU promotes IFN‐γ secretion without affecting NK cytotoxicity. (A) Purified NK cells were incubated with 100 U/ml IL‐2 and/or 5 μg/ml CLU for 5 days. At the end of culture, cells were coincubated with RMA/S target cells in a 1:1 ratio for 6 h in the presence of Golgi stop, and intracellular staining for IFN‐γ was performed (n=3). Cells were gated on CD3^–NK1.1⁺ NK cells. (B) ⁵¹Cr‐labeled RMA/S cells were mixed with NK cells cultured for 5 days with IL‐2 alone or IL‐2 + CLU at indicated E:T ratios (n=6). Radioactivity released into the supernatant after 4 h was counted by using a γ‐counter. Percent of specific lysis was calculated as follows: 100 × (experimental release–spontaneous release)/(maximum release–spontaneous release).