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Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies.

Bonanno G, Procoli A, Mariotti A, Corallo M, Perillo A, Danese S, De Cristofaro R, Scambia G, Rutella S - J Transl Med (2010)

Bottom Line: In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline.Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF.Pegfilgrastim induced significant changes in immune cell number and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology, Catholic University Med, School, Rome, Italy.

ABSTRACT

Background: Pegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the in vivo and ex vivo release of immune modulating cytokines remain unexplored.

Methods: Twelve patients with gynecological cancers received carboplatin/paclitaxel chemotherapy and single-dose pegfilgrastim as prophylaxis of febrile neutropenia. Peripheral blood was collected prior to pegfilgrastim administration (day 0) and on days +7, +11 and +21, to quantify immunoregulatory cytokines and to assess type 1 DC (DC1), type 2 DC (DC2) and Treg cell mobilization. In vitro-differentiated, monocyte-derived DC were used to investigate endocytic activity, expression of DC maturation antigens and ability to activate allogeneic T-cell proliferation.

Results: Pegfilgrastim increased the frequency of circulating DC1 and DC2 precursors. In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline. Serum levels of hepatocyte growth factor and interleukin (IL)-12p40, but not transforming growth factor-β1 or immune suppressive kynurenines, significantly increased after pegfilgrastim administration. Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF. Finally, DC populations differentiated in vitro after clinical provision of pegfilgrastim were phenotypically mature, possessed low endocytic activity, and incited a robust T-cell proliferative response.

Conclusions: Pegfilgrastim induced significant changes in immune cell number and function. The enhancement of monocytic IL-12 secretion portends favorable implications for pegfilgrastim administration to patients with cancer, a clinical context where the induction of immune deviation would be highly undesirable.

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Ex vivo cytokine measurements and in vitro monocytic release of IL-10 and IL-12p40. Panel A: Patient serum was collected at the indicated time-points and used to evaluate IL-12p40, TGF-β1 and HGF levels by ELISA. Bars depict median values and interquartile ranges recorded in 12 independent experiments performed in duplicate. °p < 0.01 when comparing IL-12p40 levels on day +7 vs. day +21. °°p = 0.0036 when comparing IL-12p40 levels on day +11 vs. baseline and vs. day +21. *p = 0.0023 when comparing HGF levels on day +7 and day +11 vs. baseline. §p = 0.0062 when comparing HGF levels on day +7 and day +11 vs. baseline and vs. day +21. Panel B: Monocytes were purified on day +11 from the commencement of cytokine treatment, coincident with maximal mobilization into the peripheral blood. Cells (1 × 106) were stimulated with 1 μg/ml LPS in complete culture medium for up to 96 hours. Supernatants were harvested daily and used to measure IL-10 and IL-12p40 by ELISA. IL-10 and IL-12p40 levels were also estimated in 7 patients with gynecological cancers treated with daily G-CSF. Median values and interquartile range are shown. *p < 0.01 compared with IL-12p40 levels in supernatants of post-filgrastim monocytes.
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Figure 3: Ex vivo cytokine measurements and in vitro monocytic release of IL-10 and IL-12p40. Panel A: Patient serum was collected at the indicated time-points and used to evaluate IL-12p40, TGF-β1 and HGF levels by ELISA. Bars depict median values and interquartile ranges recorded in 12 independent experiments performed in duplicate. °p < 0.01 when comparing IL-12p40 levels on day +7 vs. day +21. °°p = 0.0036 when comparing IL-12p40 levels on day +11 vs. baseline and vs. day +21. *p = 0.0023 when comparing HGF levels on day +7 and day +11 vs. baseline. §p = 0.0062 when comparing HGF levels on day +7 and day +11 vs. baseline and vs. day +21. Panel B: Monocytes were purified on day +11 from the commencement of cytokine treatment, coincident with maximal mobilization into the peripheral blood. Cells (1 × 106) were stimulated with 1 μg/ml LPS in complete culture medium for up to 96 hours. Supernatants were harvested daily and used to measure IL-10 and IL-12p40 by ELISA. IL-10 and IL-12p40 levels were also estimated in 7 patients with gynecological cancers treated with daily G-CSF. Median values and interquartile range are shown. *p < 0.01 compared with IL-12p40 levels in supernatants of post-filgrastim monocytes.

Mentions: It is now recognized that the balance between IL-12 and IL-10 produced by the antigen presenting cell compartment dictates the outcome of an immune response, with IL-12 release leading to robust T-cell priming and IL-10 secretion primarily mediating the induction of T-cell unresponsiveness [23]. As shown in Figure 3A, serum IL-12p40 levels significantly increased after pegfilgrastim administration and returned to baseline on day +21. Conversely, IL-12p40 slightly declined in cancer patients given daily G-CSF, and returned to pre-treatment values by day +11. IL-10 serum levels were consistently below the ELISA lowest standard (7.8 pg/ml), either in patients treated with pegfilgrastim or in those given unconjugated G-CSF (data not shown). TGF-β and HGF play significant roles as immune modulating growth factors both physiologically and in pathological states such as cancer. In order to gain further insights into the immune modulation exerted by G-CSF, we also measured TGF-β and HGF levels before and after cytokine treatment. TGF-β levels displayed minor fluctuations in the peripheral blood of patients given either unconjugated G-CSF or pegylated G-CSF (Figure 3A). In contrast, the administration of pegfilgrastim was associated with an increase of serum HGF compared with baseline (Figure 3A). Importantly, serum HGF levels on day +11 were significantly higher in patients receiving filgrastim than in those given pegfilgrastim (p = 0.043). In both cohorts of patients, HGF returned to pre-treatment values on day +21 from the commencement of cytokine administration.


Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies.

Bonanno G, Procoli A, Mariotti A, Corallo M, Perillo A, Danese S, De Cristofaro R, Scambia G, Rutella S - J Transl Med (2010)

Ex vivo cytokine measurements and in vitro monocytic release of IL-10 and IL-12p40. Panel A: Patient serum was collected at the indicated time-points and used to evaluate IL-12p40, TGF-β1 and HGF levels by ELISA. Bars depict median values and interquartile ranges recorded in 12 independent experiments performed in duplicate. °p < 0.01 when comparing IL-12p40 levels on day +7 vs. day +21. °°p = 0.0036 when comparing IL-12p40 levels on day +11 vs. baseline and vs. day +21. *p = 0.0023 when comparing HGF levels on day +7 and day +11 vs. baseline. §p = 0.0062 when comparing HGF levels on day +7 and day +11 vs. baseline and vs. day +21. Panel B: Monocytes were purified on day +11 from the commencement of cytokine treatment, coincident with maximal mobilization into the peripheral blood. Cells (1 × 106) were stimulated with 1 μg/ml LPS in complete culture medium for up to 96 hours. Supernatants were harvested daily and used to measure IL-10 and IL-12p40 by ELISA. IL-10 and IL-12p40 levels were also estimated in 7 patients with gynecological cancers treated with daily G-CSF. Median values and interquartile range are shown. *p < 0.01 compared with IL-12p40 levels in supernatants of post-filgrastim monocytes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2992497&req=5

Figure 3: Ex vivo cytokine measurements and in vitro monocytic release of IL-10 and IL-12p40. Panel A: Patient serum was collected at the indicated time-points and used to evaluate IL-12p40, TGF-β1 and HGF levels by ELISA. Bars depict median values and interquartile ranges recorded in 12 independent experiments performed in duplicate. °p < 0.01 when comparing IL-12p40 levels on day +7 vs. day +21. °°p = 0.0036 when comparing IL-12p40 levels on day +11 vs. baseline and vs. day +21. *p = 0.0023 when comparing HGF levels on day +7 and day +11 vs. baseline. §p = 0.0062 when comparing HGF levels on day +7 and day +11 vs. baseline and vs. day +21. Panel B: Monocytes were purified on day +11 from the commencement of cytokine treatment, coincident with maximal mobilization into the peripheral blood. Cells (1 × 106) were stimulated with 1 μg/ml LPS in complete culture medium for up to 96 hours. Supernatants were harvested daily and used to measure IL-10 and IL-12p40 by ELISA. IL-10 and IL-12p40 levels were also estimated in 7 patients with gynecological cancers treated with daily G-CSF. Median values and interquartile range are shown. *p < 0.01 compared with IL-12p40 levels in supernatants of post-filgrastim monocytes.
Mentions: It is now recognized that the balance between IL-12 and IL-10 produced by the antigen presenting cell compartment dictates the outcome of an immune response, with IL-12 release leading to robust T-cell priming and IL-10 secretion primarily mediating the induction of T-cell unresponsiveness [23]. As shown in Figure 3A, serum IL-12p40 levels significantly increased after pegfilgrastim administration and returned to baseline on day +21. Conversely, IL-12p40 slightly declined in cancer patients given daily G-CSF, and returned to pre-treatment values by day +11. IL-10 serum levels were consistently below the ELISA lowest standard (7.8 pg/ml), either in patients treated with pegfilgrastim or in those given unconjugated G-CSF (data not shown). TGF-β and HGF play significant roles as immune modulating growth factors both physiologically and in pathological states such as cancer. In order to gain further insights into the immune modulation exerted by G-CSF, we also measured TGF-β and HGF levels before and after cytokine treatment. TGF-β levels displayed minor fluctuations in the peripheral blood of patients given either unconjugated G-CSF or pegylated G-CSF (Figure 3A). In contrast, the administration of pegfilgrastim was associated with an increase of serum HGF compared with baseline (Figure 3A). Importantly, serum HGF levels on day +11 were significantly higher in patients receiving filgrastim than in those given pegfilgrastim (p = 0.043). In both cohorts of patients, HGF returned to pre-treatment values on day +21 from the commencement of cytokine administration.

Bottom Line: In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline.Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF.Pegfilgrastim induced significant changes in immune cell number and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology, Catholic University Med, School, Rome, Italy.

ABSTRACT

Background: Pegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the in vivo and ex vivo release of immune modulating cytokines remain unexplored.

Methods: Twelve patients with gynecological cancers received carboplatin/paclitaxel chemotherapy and single-dose pegfilgrastim as prophylaxis of febrile neutropenia. Peripheral blood was collected prior to pegfilgrastim administration (day 0) and on days +7, +11 and +21, to quantify immunoregulatory cytokines and to assess type 1 DC (DC1), type 2 DC (DC2) and Treg cell mobilization. In vitro-differentiated, monocyte-derived DC were used to investigate endocytic activity, expression of DC maturation antigens and ability to activate allogeneic T-cell proliferation.

Results: Pegfilgrastim increased the frequency of circulating DC1 and DC2 precursors. In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline. Serum levels of hepatocyte growth factor and interleukin (IL)-12p40, but not transforming growth factor-β1 or immune suppressive kynurenines, significantly increased after pegfilgrastim administration. Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF. Finally, DC populations differentiated in vitro after clinical provision of pegfilgrastim were phenotypically mature, possessed low endocytic activity, and incited a robust T-cell proliferative response.

Conclusions: Pegfilgrastim induced significant changes in immune cell number and function. The enhancement of monocytic IL-12 secretion portends favorable implications for pegfilgrastim administration to patients with cancer, a clinical context where the induction of immune deviation would be highly undesirable.

Show MeSH
Related in: MedlinePlus