Effects of SF on phagocytosis activity of RAW 264.7 cells
Our two initial observations were: 1) the uptake of 2-μm polystyrene beads by RAW264.7 cells was inversely related to the concentration of FBS in the culture medium (); and 2) SF addition stimulated this phagocytosis activity, but the degree of enhancement decreased as the FBS concentration was raised, and was not observed in cells incubated in complete medium containing 10% FBS (PI = 29.2 ± 8.5 and 29.5 ± 13.2, for 0 and 0.5 μM SF, respectively, which are not significantly different). Prior treatment of the cells for 24 h with 0.5 μM SF resulted in a highly significant increase in phagocytosis activity at low FBS concentrations (F(1, 115) = 13.98, p = 0.0003) and there was also an inverse correlation between the PI and FBS concentration. The magnitude of this stimulation was also dependent on SF concentration ().
Fig. 1 Effect of sulforaphane and fetal bovine serum (FBS) on phagocytosis activity of RAW 264.7 cells. (a) Each circle represents the phagocytosis index (PI) of each photo-field treated with 0.1% (v/v) acetonitrile as a control (●) and 0.5 μM (more ...)
Lowering of serum in the culture medium affects cell cycle dynamics and induces apoptosis in RAW 264.7 cells [19
]. In the absence of FBS, RAW 264.7 cell proliferation was depressed, and the cells showed significant changes in their morphology, including increased size and greatly elongated spindle shapes (). How these morphological changes relate to phagocytosis is unclear.
Participation of the Keap1/Nrf2 pathway in stimulation of phagocytosis by SF
SF is a potent inducer of the Keap1/Nrf2 pathway [21
]. Nrf2 has been also reported to interact with other cell-signaling pathways involved in cell proliferation and cell death such as the aryl hydrocarbon receptor (AhR) [24
], nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) [25
], p53 [26
], and Notch [27
]. We assessed the involvement of the Keap1/Nrf2 pathway in the enhancement of phagocytosis activity by SF treatment in two ways: 1) by examining the ability of Nrf2 inducers other than SF to stimulate phagocytosis, and 2) by determining whether stimulation of phagocytosis occurred in nrf2−/−
The inducer potencies of SF, benzyl isothiocyanate (BITC) and the triterpenoid TP-225 on the NQO1 activity of RAW 264.7 cells were measured in the presence of FBS (). The CD (Concentration required to Double) values for NQO1 induction for SF, BITC and TP-225 in DMEM containing 1% FBS were 0.57, 0.86, and 0.0014 μM, respectively. In contrast, no induction was observed with the same concentrations of these agents in the absence of FBS. On the other hand, treatment with both 0.5 and 2.0 μM SF induced NQO1 mRNA expression 2.8- and 5.8-fold even in the absence of FBS (). This suggests that lack of serum may disrupt posttranscriptional synthesis of Nrf2-dependent proteins. Treatment with SF or BITC (1.0 μM, 24 h) significantly stimulated the phagocytosis activity of RAW 264.7 cells in serum-free DMEM, but TP-225 (a powerful inducer of Nrf2) [13
] showed no effect on phagocytosis in the range of 0.005 to 0.02 μM (). These results suggest that stimulation of phagocytosis is not a common property of all inducers of the phase 2 response, but that it may be related to the specific chemistry of isothiocyanates, and be independent of the Keap1/Nrf2 pathway.
Fig. 2 Induction of NQO1 in RAW 264.7 cells by three phase 2 response inducers: sulforaphane (SF), benzyl isothiocyanate (BITC), and triterpenoid TP-225. (a) NQO1 enzyme specific activity (treated/control) after 24 h induction in presence of 1% FBS (●, (more ...)
Effects of phase 2 inducers on phagocytosis activity in RAW 264.7 cells in absence of Fetal Bovine Serum
This possibility was strengthened by the finding that the effects of SF on phagocytosis were also observed after only a very short (4 h) treatment. The phagocytosis index for untreated (number of fields counted n=29), 0.3 μM SF (n=30), and 1.0 μM SF (n=27) treated cells were 70.8 ± 35.0, 123.2 ± 43.7, and 132.7 ± 39.3, respectively; (p < 0.001 for increases compared to control).
In further support that the Keap1/Nrf2 pathway is not involved, the effect of SF was assessed on peritoneal macrophages from two strains of nrf2−/− mice which had either the genetic background of C57BL/6 mice or were 50% hybrids of C57BL/6 and SKH1 hairless mice. Peritoneal cells obtained from these mice were plated at 105 cells/well in 8-well chamber slides and incubated for 3 h (37°C, 5% CO2). After removal of nonadherent cells with PBS, SF (0.1, 0.3, and 1 μM) or 0.1% (v/v) acetonitrile were added to the medium (RPMI 1640) in the absence of FBS and incubated for 24 h. Exposure to SF of peritoneal macrophages obtained from SKH1 wild-type female mice resulted in a dose-dependent stimulation of phagocytosis. The PIs for these macrophages were increased 15-22% (p < 0.001) at SF concentrations of 0.3-1.0 μM (data not shown). However, macrophages from nrf2−/− mice, both from C57BL/6 and SKH1/C57BL/6 hybrids showed lower PI, but greater sensitivity to stimulation by SF (). This strongly suggests that induction of Nrf2-dependent genes is not required for stimulation of phagocytosis by SF.
Effects of sulforaphane on phagocytosis activity in primary peritoneal macrophages of nrf2−/− mice
MIF inactivation and phagocytosis
Synthesis and secretion of the cytokine MIF are early and prominent inflammatory responses of macrophages and other cells. MIF is a homotrimer (114 amino acids in humans) that promotes the tautomerization of dopachrome, an enzymatic activity that can be quantified spectroscopically [11
]. Recently three laboratories [28
] independently reported that a number of isothiocyanates, including SF, potently and irreversibly inactivated the enzymatic function of MIF by modifying its highly nucleophilic N
-terminal proline residues. Indeed, MIF was identified as the most prominent target of phenethyl isothiocyanate in Jurkat T lymphocytes [28
]. Furthermore, Onodera and colleagues [12
] reported earlier that treatment of RAW 267.4 cells with either 1 or 10 μg/ml MIF for 3 h increased their phagocytosis index for latex beads. These observations are in apparent conflict with the above findings that two isothiocyanates (SF and BITC) that inactivate MIF both enhance phagocytosis in these cells by mechanisms that may involve the participation of MIF. This paradox led us to undertake the following experiments.
RAW 264.7 cells were treated for 24 h with a range of concentrations of SF (0 - 4 μM) in the medium containing either 0 or 10% FBS, and the dopachrome tautomerase activity was measured in the lysates of washed cells and in the culture medium. These activities were both normalized to the proliferation ability of the cells under similar treatment (). The presence of 10% FBS reduced MIF tautomerase activity levels in both compartments. Furthermore, addition of SF also lowered the MIF tautomerase activity in a concentration-dependent manner in these compartments. Thus, the presence of either SF or FBS suppresses MIF enzymatic activity levels. In contrast, as shown above, FBS depresses and SF enhances phagocytosis in RAW 264.7 cells.
Fig. 3 Inactivation of MIF by sulforaphane treatment in RAW 264.7 cells. Relative MIF tautomerase activity of cell lysates (upper boxes) and medium (lower boxes) incubated with serial dilutions of SF for 24 h in the medium in the absence (○) and presence (more ...)
We therefore examined the effect on phagocytosis of the direct treatment of RAW 264.7 cells with recombinant mouse MIF (2, 10, 50 ng/ml) for 24 h in serum-free medium (). This treatment dramatically suppressed the phagocytosis index in proportion to MIF concentration. However, if recombinant MIF was first treated with SF to abolish its tautomerase activity completely, and the excess SF was removed, these preparations no longer stimulated phagocytosis (). This phenomenon was abolished in the presence of 10% FBS. We therefore conclude that at least part of the stimulating activity of SF on phagocytosis is mediated by its modification of MIF (abolition of tautomerase activity; conformational changes).
Fig. 4 Effect of addition of recombinant mouse MIF on phagocytosis activity of RAW 264.7 cells. PI values of cells treated with intact MIF (○) and sulforaphane-inactivated MIF (□) in serum-free medium and intact MIF in the medium containing 10% (more ...)
Although the same types of RAW 267.4 macrophage-like cells were used in our and Onodera’s experiments, contradictory effects of MIF on phagocytosis were observed in the two systems: inhibition in our, and stimulation in Onodera’s system. However, the experimental systems were quite different with respect to the FBS concentration of the medium (0% and 10%); the types and sizes of beads used for quantifying phagocytosis (2.0 μm polystyrene and 0.75 μm latex beads); antibody (polystyrene beads were coated with IgG, latex beads were uncoated); time of exposure to MIF (24 h and 48 h) and to beads (10 min and 120 min); and ratio of bead number to phagocytic cells.