Functional regulation and proteomic characterization of human natural killer cells through recombinant human granulocyte‐colony stimulating factor treatment

Abstract

Recombinant human granulocyte‐colony stimulating factor (rhG‐CSF) is the most common hematopoietic growth factor used for chemotherapy‐induced neutropenia or mobilization of stem cells. Natural killer (NK) cells are critical for host defense against infected cells and tumors. However, the cellular and molecular events of NK cells responding to rhG‐CSF remain unclear. Toward this end, we treated human NK cells with rhG‐CSF and assessed their cytotoxic function as well as proteomic characteristics. Unlike the other tested hematopoietic cytokines, rhG‐CSF decreased NK cell‐mediated cytotoxicity without affecting the viability of NK cells. The rhG‐CSF also reduced the production of nitric oxide and expression of inducible nitric oxide synthase in recombinant human interleukin (rhIL)‐2‐activated NK cells. By using cytokine array, rhG‐CSF reduced secretion of growth‐related oncogene‐α from NK cells. Intriguingly, rhG‐CSF did not affect production of various inflammatory cytokines [MCP‐1, IL‐6, tumor necrosis factor‐α (TNF‐α), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and IL‐8], which are markedly stimulated by rhGM‐CSF. Proteomic analysis of rhG‐CSF‐ and rhGM‐CSF‐treated NK cells uncovered unique proteins possibly involving rhG‐CSF effect. These proteins were classified into six groups as follows: glycolysis, apoptosis, cytotoxicity, inflammation, antigen processing and presentation, and the others. We conclude that rhG‐CSF may impair NK‐mediated cytotoxicity accompanied by alterations in protein expression profile distinct from that of rhGM‐CSF.

G-CSF downregulates natural killer cell-mediated cytotoxicity in donors for hematopoietic SCT

“…Although the level of TRAIL did not change significantly, the cytotoxicity of NK cells was decreased by G-CSF in this study and in our previous study. 7 One possibility is TRAIL production in other immune cells, such as CTLs, provokes cytotoxicity to the target. One study indicated that multipeg-G-CSF enhanced CTL function and maximized GVL effects; 21 therefore, G-CSF effect on other cells may influence the plasma levels of TRAIL in this study.…”

“…13 G-CSF increases the alloAg-presenting capability of immune accessory cells. 7,14 However, in one study it diminished the cytotoxic ability of NK cells, 15 and in another (with 104 healthy donors), it decreased immune function in G-CSF-mobilized PBSCs. 16 Our previous study isolated CD56 þ CD3 À NK cells from peripheral blood and demonstrated that G-CSF was effective at inhibiting NK cell activity through the regulation of several cytotoxicityrelated proteins.…”

“…5,6 Our previous study demonstrated that the cytotoxicity of CD56 þ CD3 À NK cells isolated from human peripheral blood was markedly increased after treatment with SCF, IL-3 and granulocyte/macrophage-CSF; in contrast, G-CSF attenuated NK cell-mediated cytotoxicity. 7 Moreover, G-CSF-mediated downregulation of NK cell activity may be associated with several cytotoxicity-related protein expressions, such as triosephosphate isomerase (TPI), calreticulin and ubiquitin-conjugating enzyme E2H. 7 NK cell activity is also involved in a variety of pathologic conditions, such as autoimmune diseases and transplant rejection.…”

“…7 Moreover, G-CSF-mediated downregulation of NK cell activity may be associated with several cytotoxicity-related protein expressions, such as triosephosphate isomerase (TPI), calreticulin and ubiquitin-conjugating enzyme E2H. 7 NK cell activity is also involved in a variety of pathologic conditions, such as autoimmune diseases and transplant rejection. 8 In particular, for hematopoietic SCT (HSCT) of leukemia patients, NK cells have a critical role in regulating immune responses, including GVHD and GVL effects.…”

“…16 Our previous study isolated CD56 þ CD3 À NK cells from peripheral blood and demonstrated that G-CSF was effective at inhibiting NK cell activity through the regulation of several cytotoxicityrelated proteins. 7 Because of the controversy regarding NK cell activity in the setting of G-CSF-mobilized allo-HSCT, clarification of the role of G-CSF in regulating NK cell activity is necessary. The present study assayed NK cell activity in both G-CSF-treated NK-92MI cell lines and in NK cells isolated from the peripheral blood of donors treated with G-CSF before allo-HSCT.…”