Dehydroepiandrosterone Sulfate Directly Activates Protein Kinase C-β to Increase Human Neutrophil Superoxide Generation

Abstract

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in the human circulation and is secreted by the adrenals in an age-dependent fashion, with maximum levels during the third decade and very low levels in old age. DHEAS is considered an inactive metabolite, whereas cleavage of the sulfate group generates dehydroepiandrosterone (DHEA), a crucial sex steroid precursor. However, here we show that DHEAS, but not DHEA, increases superoxide generation in primed human neutrophils in a dose-dependent fashion, thereby impacting on a key bactericidal mechanism. This effect was not prevented by coincubation with androgen and estrogen receptor antagonists but was reversed by the protein kinase C inhibitor Bisindolylmaleimide 1. Moreover, we found that neutrophils are unique among leukocytes in expressing an organic anion-transporting polypeptide D, able to mediate active DHEAS influx transport whereas they did not express steroid sulfatase that activates DHEAS to DHEA. A specific receptor for DHEAS has not yet been identified, but we show that DHEAS directly activated recombinant protein kinase C-β (PKC-β) in a cell-free assay. Enhanced PKC-β activation by DHEAS resulted in increased phosphorylation of p47^phox, a crucial component of the active reduced nicotinamide adenine dinucleotide phosphate complex responsible for neutrophil superoxide generation. Our results demonstrate that PKC-β acts as an intracellular receptor for DHEAS in human neutrophils, a signaling mechanism entirely distinct from the role of DHEA as sex steroid precursor and with important implications for immunesenescence, which includes reduced neutrophil superoxide generation in response to pathogens.

Dehydroepiandrosterone sulphate stimulates superoxide generation by neutrophils in a PKC-dependent manner. Neutrophils express the steroid transporter OATP-D and DHEAS is shown to activate PKC-beta directly.

Dehydroepiandrosterone (DHEA) and its sulfate ester DHEA sulfate (DHEAS) are the most abundant steroids in the human circulation, representing the major products of the adrenal zona reticularis. In humans and higher primates DHEAS secretion shows a characteristic, age-associated pattern with very high levels in the neonatal period, a drop to very low levels during the first few months of life, and a continuous increase starting between the sixth and tenth year of age, also termed “adrenarche” (1). Intraindividual maximal levels are achieved during the third decade of life followed by a steady decline starting in the fifth decade (adrenopause) with levels decreasing to 10–20% of maximal levels around 70 yr of age (1). This age-related decline in DHEAS does not reflect a general loss of adrenocortical output because levels of cortisol are maintained and are even slightly raised with age (2). Surprisingly, the consequences of adrenopause for human physiology are poorly understood.

DHEA serves as a key intermediate in human sex steroid synthesis (3) and beneficial, androgenic effects have been observed during DHEA replacement in individuals with adrenal insufficiency and thus pronounced adrenal androgen deficiency (4, 5, 6). In contrast, DHEAS is assumed to have no independent biological function other than representing a precursor for DHEA regeneration and a metabolite for eventual DHEA excretion. Previous reports have suggested immune modulatory effects of DHEA (7, 8, 9, 10). However, the majority of these studies have been carried out in rodents, which are not capable of adrenal DHEA synthesis and, consequently, have very low circulating DHEAS levels, and many of those studies employed DHEA at concentrations several orders of magnitude above physiological levels.

Circulating DHEAS is very low in patients with acute systemic inflammation, such as sepsis (11), and in patients with chronic autoimmune disease, e.g. rheumatoid arthritis and systemic lupus erythematosus (12, 13); in the latter, chronic exogenous glucocorticoid treatment decreases residual endogenous DHEAS production even further. However, to what extent these endocrine changes impact on the immune response in affected individuals remains to be established. Physiological aging is also associated with a decline in immune function, termed “immunesenescence” (14), which includes decreased neutrophil activity (15). This leaves elderly individuals more susceptible to bacterial infections because neutrophils represent the major cellular defense against rapidly dividing bacteria. The essential elements of this defense mechanism are phagocytosis of the microbe, release of toxic enzymes from cytoplasmic granules, and the generation of superoxide free radical (O₂·⁻) via assembly and activation of the NADPH oxidase system. Cortisol is well known to inhibit human neutrophil superoxide generation (16). We have previously shown that neutrophil superoxide generation was suppressed in older adults after hip fracture, and this was associated with a raised cortisol-DHEAS ratio, which was highest in the those patients developing early postoperative infections (17). Crucially we could demonstrate in vitro that physiological concentrations of DHEAS were able to overcome the suppressive effects of cortisol on neutrophil superoxide generation (17).

This study has therefore investigated whether DHEAS is able to regulate neutrophil function directly and by which mechanism, a question of major interest because to date no specific receptor for either DHEA or DHEAS has been conclusively identified.

Results

DHEAS increases human neutrophil superoxide generation

We have previously demonstrated in vitro that incubation of human neutrophils with DHEAS prevents the glucocorticoid-mediated inhibition of bacterial formylated peptide formyl methionyl leucyl phenylalanine (fMLP)-stimulated superoxide generation (17), raising the possibility that the sulfate ester of DHEA may have distinct biological activity and may be able to directly influence innate immune cell function in vivo. Figure 1A shows that neutrophils treated with DHEAS before stimulation with fMLP showed a concentration-dependent increase in superoxide generation that was significant at concentrations within the physiological range (10 μm; P < 0.05). The unconjugated steroid DHEA was without effect, even at the supraphysiological dose of 1 μm (Fig. 1B).

Fig. 1.

Superoxide generation by human neutrophils incubated with DHEA and DHEAS, respectively. Neutrophils were isolated from human peripheral blood and incubated with medium alone or containing DHEAS (0.1–10 μm) (A) or, DHEA (10–1000 nm) (B). C, Neutrophils were treated with GM-CSF, in the absence or presence of 10 μm DHEAS. In all experiments superoxide generation in response to fMLP was then measured using a lucigenin-based assay. Data are mean ± sd of five separate experiments. In panel A, asterisk indicates P < 0.05 compared with vehicle. In panel C, asterisk indicates P < 0.05 compared with GM-CSF alone; **, P < 0.04 compared with fMLP alone.

To determine whether neutrophil function could also be enhanced by DHEAS in conditions encountered at sites of inflammation, we treated human neutrophils with the proinflammatory cytokine, granulocyte macrophage colony-stimulating factor (GM-CSF), which enhances neutrophil responsiveness to bacterial components, before incubation with 10 μm DHEAS and stimulation with fMLP. Under these conditions, human neutrophil superoxide generation was increased 5-fold by the priming cytokine GM-CSF, and this effect was significantly enhanced by DHEAS at physiological concentrations (Fig. 1C).

Human neutrophils show specific expression of an organic anion transporter polypeptide that mediates cross-membrane influx of DHEAS

In contrast to lipophilic DHEA, DHEAS is very hydrophilic and therefore requires active influx transport into cells, which can be mediated by several members of the organic anion-transporting polypeptide (OATP) family (18). OATP mRNA expression analysis revealed the specific expression of OATP-D in human neutrophils (Fig. 2A). Interestingly, the peripheral mononuclear blood cell (PBMC) fraction containing the remaining blood leukocytes did not express any OATPs (Fig. 2A), suggesting that responsiveness to DHEAS by immune cells is a phenomenon unique to neutrophils.

Fig. 2.

Specific expression of an active influx transporter for DHEAS in human neutrophils. Total mRNA was extracted from peripheral blood neutrophils (N) and human PBMCs and analyzed by RT-PCR. A, Specific mRNA expression of OATP A-F, OTAP-8, and OAT4, with RNA from kidney as a positive control (+ve). B, Expression of the enzymes involved in the interconversion of DHEA and DHEAS, steroid sulfatase, STS, and SULT2A1. RNA from placenta and adrenal served as positive controls (+). C, Representative chromatograms illustrating the detection of DHEAS in cell lysate from neutrophils incubated with DHEAS (10 μm) for 30 min. DHEA is the internal standard at a concentration of 5 ng/ml. All images shown are representative of three separate analyses performed. D, Intracellular DHEAS concentration in human neutrophils (N) and PBMCs incubated for 30 and 60 min, respectively, with DHEAS (10 μm). DHEAS concentrations were measured using liquid chromatography/tandem mass spectrometry represented as means ± sem based on three independent triplicate experiments.

Human neutrophils did not express steroid sulfatase (STS) and DHEA sulfotransferase (SULT2A1) (Fig. 2B), which catalyze the interconversion of DHEA and DHEAS, thereby excluding cleavage of DHEAS to DHEA or sulfation of DHEA within human neutrophils.

Because OATP-D has only been reported previously to transport prostaglandins, thyroid hormones, benzylpenicillin, and vasopressin (18) it was important to demonstrate the functionality of the transporter. To determine the effective and specific uptake of DHEAS by human neutrophils, we devised uptake experiments employing freshly isolated human neutrophils and PBMC as control. Cells were incubated with DHEAS (10 μm) for 30 or 60 min. Supernatant was removed, followed by washing of the cells and subsequent cell lysis. This was followed by solid phase extraction of DHEAS and subsequent quantification using ultra high pressure liquid chromatography-tandem mass spectrometry. Figure 2C shows representative total ion chromatograms illustrating the detection of DHEAS in cell lysate from neutrophils incubated with DHEAS. DHEA was used as the internal standard. Figure 2D shows that DHEAS was taken up rapidly by neutrophils and that maximal uptake appeared to have occurred by 30 min because no further increase was seen by 60 min. In contrast, there was no significant uptake by PBMC.

The effect of DHEAS on neutrophil superoxide generation is independent of sex steroid receptor activation but is blocked by protein kinase C inhibition

To further determine whether the effects of DHEAS might be due to intracellular conversion of DHEAS to DHEA and sex steroids, we performed several studies. First, the effects of DHEAS were not blocked by coincubation with the androgen receptor antagonist flutamide, or the estrogen receptor antagonist ICI 182,780 (Fig. 3A), excluding the mediation of the observed effects of DHEAS via downstream conversion to sex steroids. Interestingly, some of the actions of DHEAS have been attributed previously to conversion to sex steroids and interactions with the estrogen receptor, suggested by reversal of some observed effects by the estrogen receptor antagonist tamoxifen (19). However, tamoxifen also inhibits the protein kinase C (PKC) signaling pathway (20), and this led us to investigate whether effects of DHEAS on neutrophil superoxide generation might be mediated by activation of PKC. Figure 3B shows that incubation of neutrophils with the broad range PKC inhibitor bisindolylmaleimide 1 (BIM) before addition of DHEAS reduced superoxide generation to control levels. Neutrophils treated with GM-CSF, which mediates its effect on superoxide generation through activation of the MAPK ERK, produced a robust respiratory burst, which was not inhibited significantly by BIM (Fig. 3B). None of the inhibitors alone had any effect on superoxide generation.

Fig. 3.

DHEAS effects on neutrophil superoxide generation are PKC dependent. A, Human neutrophils were preincubated with the estrogen receptor antagonist ICI 182,780 (ICI) or the androgen receptor antagonist flutamide (fluta) in the presence or absence of 10 μm DHEAS. B, Human neutrophils were treated with 10 μm DHEAS or GM-CSF after preincubation with the PKC inhibitor (BIM). Superoxide generation in response to fMLP was measured using a lucigenin-based assay and compared with an untreated control. Data are expressed as percentage of control and are mean ± sd of four separate experiments. *, P < 0.04 compared with fMLP alone. C, DHEAS induces PKC-mediated phosphorylation of the NADPH oxidase component p47^phox. Neutrophils were radiolabeled with ³²PO₄ before addition of medium alone (Ctrl), GM-CSF, or 10 μm DHEAS in the absence or presence of BIM. p47^phox was then immunoprecipitated and isolated using protein G-coated magnetic beads. The ³²P-labeled p47^phox was separated by SDS-PAGE and visualized using a phosphor imager (Typhoon 9200; Amersham Biosciences, Piscataway, NJ). Coincubation with DHEAS increased p47^phox phosphorylation, which could be reversed by coincubation with the PKC inhibitor BIM. The image shown is representative of two separate experiments performed. D, Densitometric analysis as mean data for the two experiments. AU, Arbitrary units.

DHEAS induces phosphorylation of the NADPH oxidase component p47^phox

Superoxide generation in neutrophils occurs via activation of the NADPH oxidase complex, a multicomponent enzyme composed of membrane-bound flavocytochrome b₅₅₈, a heterodimer consisting of gp91^phox and p22^phox, and the cytosolic factors p47^phox, p67^phox, and guanosine triphosphatase Rac1 or Rac2 (21). PKC is one of the protein kinase regulators of the NADPH oxidase in leukocytes, inducing the phosphorylation-dependent activation of the cytosolic components resulting in their translocation to the membrane. As a marker of NADPH oxidase modulation, we determined whether p47^phox was phosphorylated after DHEAS treatment and whether this was also PKC dependent. Figure 3, C and D, shows that p47^phox phosphorylation was induced by DHEAS and blocked by the PKC inhibitor BIM, providing further, direct evidence for PKC-mediated activation of the NADPH oxidase complex by DHEAS. GM-CSF also induced p47^phox phosphorylation and acted as a positive control. Taken together, these data indicate that DHEAS enhanced neutrophil superoxide generation via activation of the NADPH complex mediated by activation of the PKC-signaling pathway.

DHEAS specifically activates PKC-β

PKC is a family of at least 10 isoenzymes, five of which are expressed at a significant level in human neutrophils, namely PKC-α, -βI, -βII, -δ, and -ζ (22). To investigate which PKC isoenzymes were activated by DHEAS, the subcellular location of these isoenzymes was determined, because PKC translocates from the soluble cytosolic fraction to the particulate membrane fraction upon activation (23). Western blots of the cytosolic and membrane fractions of control and DHEAS-treated human neutrophils showed that PKC-βI and -βII were activated by DHEAS (Fig. 4, A and B). No consistent translocations after DHEAS treatment were seen for the other PKC isoenzymes present in neutrophils. The positive control phorbol myristate acetate (PMA) translocated PKC-α, -βI, -βII, and -δ, but not atypical PKC-ζ, which is phorbol insensitive. Furthermore, sc-204199, a small molecule inhibitor that shows a high degree of selectivity toward PKC-β, was able to completely inhibit DHEAS-induced superoxide generation (Fig. 4C).

Fig. 4.

PKC is activated in response to DHEAS treatment leading to NADPH oxidase activation. A and B, DHEAS specifically activates PKC-β. Neutrophils were cultured with medium alone, 10 μm DHEAS, or 20 nm PMA, and cells were lysed and the cell membrane (M) and cytosol (C) fractions were isolated by differential centrifugation. Fractions were assessed for PKC-α, β (Ι and ΙΙ), -δ, and -ζ by Western blotting. A, DHEAS incubation results in an increase in translocation of PKC-βI and -II from the cytosol to the membrane, indicative of activation. The subcellular distribution of the other PKC isoforms was not consistently altered by DHEAS. B, Densitometric analysis of the PKC-β blots with data shown as mean ± sem. C, Human neutrophils were treated with 10 μm DHEAS after preincubation with the PKC-β-selective inhibitor sc-204199. Superoxide generation in response to fMLP was measured using a lucigenin-based assay and compared with an untreated control. The image shown is representative of three separate experiments. D, Rat brain PKC was combined with a range of concentrations of DHEAS or PMA and a biotinylated PKC peptide substrate in the presence of γ³²P-ATP. At the end of the assay, the substrate was collected on to a streptavidin-coated membrane, and radioactive content was determined by scintillation counting. The data shown are mean ± sem of three independent experiments; *, P < 0.05. RLU, Relative light units.

To determine whether activation of PKC by DHEAS was a direct effect of the steroid or was mediated via an intermediate signaling event, DHEAS was tested in an in vitro PKC assay using rat brain PKC, which contains high levels of classical PKCs including PKC-β. PMA was again used as a positive control. Figure 4D shows that DHEAS was able to activate rat brain PKC in the physiological range of 1–10 μm in this cell-free assay, providing evidence for direct activation of PKC by DHEAS.

DHEAS directly activates recombinant human PKC-β

To determine whether DHEAS activation of PKC shows any isoenzyme selectivity, DHEAS was also tested in a micellar-based assay using a range of recombinant PKC isoenzymes, including classical PKC-βI and -II, novel PKC-δ, and atypical PKC-ζ. Results showed that DHEAS directly activated PKC-βI and -βII in a concentration-dependent manner (Fig. 5A), while exerting no effect on PKC-δ (Fig. 5B) and PKC-ζ (data not shown). DHEA did not activate any of the PKC isoenzymes tested (data not shown).

Fig. 5.

DHEAS activates PKC-β directly in vitro. Glutathione-S-transferase-tagged recombinant PKC-βI and -βII (A) or PKC-δ (B) were incubated with DHEAS at the concentrations shown or 20 nm PMA, and activity was assessed in a micelle-based assay as incorporation of ³²P from [³²P]ATP into a synthetic peptide substrate. Data are expressed as fold stimulation over assay buffer alone and are mean ± sd (n = 3); *, P < 0.05 compared with value for solvent alone.

Discussion

Here we have shown that PKC-β acts as an intracellular receptor for DHEAS in human neutrophils, mediating its stimulatory effects on neutrophil superoxide generation via activation of the NADPH oxidase complex. To our knowledge, this is the first report of the direct activation of a major serine/threonine protein kinase by DHEAS and, as such, provides evidence for a novel signaling mechanism for this major steroid that is distinct from that of nonconjugated DHEA.

Interestingly, the ability to respond to circulating DHEAS in vivo appears to be a unique feature of neutrophils because only human neutrophils showed specific expression of the organic anion transporter polypeptide OATP-D. OATP-D has not previously been reported as a transporter of sulfated steroids (18), but our data showing rapid uptake of DHEAS by neutrophils suggest that it can mediate active influx transport of DHEAS. By contrast, none of the other OATP transporters previously implicated in the cross membrane transport of DHEAS or other steroids were expressed in neutrophils or any of the other blood leukocytes. Tamai et al. (24) had shown previously that OATP-D was the only OATP expressed by peripheral blood leukocytes, and our data suggest that, in fact, within the leukocyte pool OATP-D expression is restricted to neutrophils.

Neutrophils are the dominant leukocyte in blood and function as the major defense against rapidly dividing bacteria. The observed immune enhancing effects of DHEAS targeted at this cell population thus uncover an important novel aspect of the endocrine-immune axis. This may be particularly important in older humans because serum DHEAS declines with age while levels of immune-suppressing glucocorticoids remain constant (2), thus resulting in a relative glucocorticoid excess in old age. The loss of DHEAS is paralleled by a decline in immunity, including reduced neutrophil bactericidal function (25, 26). However, before our observations no causal relationship between these two events had been established. Our data demonstrate that DHEAS acts as a significant modulator of neutrophil superoxide generation, and thus a diminished modulatory role could have detrimental effects, in particular in relation to susceptibility to bacterial infections in older adults.

Although several studies have reported beneficial effects of DHEA supplementation upon resistance to parasitic infections in mice (10, 27), very few have considered resistance to bacterial infection (9, 28) or investigated direct effects on human immune cells. Moreover these reports did not determine whether the improvement involved increased neutrophil function or whether the effect was due, in fact, to increased levels of DHEAS. Interestingly, Schuld et al. (29) reported that serum DHEAS was significantly increased in humans 6–12 h after a systemic challenge with bacterial endotoxin, suggesting an integration of the response to bacterial infections by the innate immune system and the hypothalamic-pituitary-adrenal axis. However, this effect was dependent upon the dose of endotoxin administered with only the lowest dose producing an increase in serum DHEAS, and a later publication from the same group showed no effect of endotoxin challenge on either serum DHEA or DHEAS levels, although cortisol was raised after 4 h (30).

Although neutrophils are known to express androgen receptors (31), our investigations into the mechanism of action of DHEAS on neutrophil superoxide generation indicate that this was not via conversion of DHEAS to DHEA and on to sex steroids, because neutrophils lacked expression of steroid sulfatase required for cleavage of DHEAS to DHEA, and coincubations with estrogen and androgen receptor antagonists were without effect, thereby precluding a sex steroid-mediated effect. A specific cellular receptor for DHEA or DHEAS has not previously been characterized (32), although recent studies have provided some evidence for direct binding of DHEA to a G protein-coupled membrane receptor in vascular endothelial cells (33) and subsequent activation of classic serine threonine kinase cascades, including the ERK MAPK and phosphatidylinositol 3-kinase pathways (34). Our data suggest, for the first time, that in neutrophils PKC-β serves as the intracellular receptor for DHEAS, leading to phosphorylation of the cytosolic components of NADPH oxidase and generation of superoxide (Fig. 6). Moreover, DHEAS did not induce superoxide generation in the absence of fMLP (data not shown), indicating that it acts as a priming agent and ensuring that its actions are only effected in the presence of pathogenic stimuli.

Fig. 6.

Proposed model of the mechanism by which DHEAS enhances human neutrophil superoxide generation. The schematic shows cross-membrane transport of DHEAS into neutrophils via OATP-D. DHEAS is then able to activate PKC-β leading to phosphorylation of p47^phox, one of the cytosolic components of the NADPH oxidase complex, resulting in association with the membrane-localized components and activation of the enzyme with generation of superoxide.

NADPH oxidase activity is the major source of superoxide production in phagocytic leukocytes such as neutrophils, and PKC is a key regulator of the NADPH oxidase, inducing the phosphorylation-dependent activation of the cytosolic components resulting in their translocation to the membrane (35). Moreover, in the study by Fontayne et al. (36), PKC-β was shown to be capable of phosphorylating p47^phox, and in the present study PKC-βΙ and PKC-βΙΙ were the only isoforms to translocate to the membrane in response to DHEAS. Moreover, DHEAS induced phosphorylation of p47^phox in neutrophils, and this was PKC dependent. Interestingly the literature contains reports of the activation of PKC isoenzymes by other sulfated steroids. Specifically, cholesterol sulfate has been shown to induce cell differentiation of keratinocyte and squamous cell lines and to activate multiple recombinant PKC isoforms in vitro (37, 38). Cholesterol sulfate was most effective for activation of the novel PKCs (38), whereas DHEAS had no effect on novel PKC-δ in vitro at physiological levels or in whole-cell assays. In addition, as seen for DHEA in our study, the nonsulfated cholesterol also had no effect on PKC activity (37).

In conclusion, our findings establish, for the first time, a specific biological activity for DHEAS as an important enhancer of human neutrophil activity with its actions mediated via direct activation of a major serine/threonine protein kinase. This function is distinct from its role as a precursor to DHEA and downstream sex steroid synthesis. Furthermore, we propose a broader influence for sulfated steroids, including DHEAS, in those cells expressing transporters for these abundant molecules.

Materials and Methods

Neutrophil isolation

Human neutrophils were isolated from the peripheral blood of healthy male donors as described previously (39) and were always greater than 97% pure, as judged by differential Giemsa staining, and the main contaminating cells were eosinophils. Neutrophils were resuspended in RPMI 1640 medium (Life Technologies, Paisley, Scotland, UK), supplemented with 10% human AB serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine (Sigma-Aldrich, Poole, UK).

Neutrophil superoxide production

Neutrophils were incubated in the absence or presence of DHEAS (Sigma-Aldrich), and/or GM-CSF (50 ng/ml; Serotec Ltd., Oxford, UK) for 1 h at 37 C, in a 5% CO₂ atmosphere. The generation of superoxide in response to 100 nm fMLP (Sigma-Aldrich) was measured over a 10-min period using a lucigenin-based assay as described previously (40) and superoxide generation calculated from the area under the curve. Neutrophils were also pretreated for 30 min with the broad-range PKC inhibitor Bisindolylmaleimide 1 (CN Biosciences, Nottingham, UK) at 1 μm, the PKCβ-selective inhibitor sc-204199 (Santa Cruz Biotechnology, Santa Cruz, CA) at 100 nm, the androgen receptor antagonist flutamide (Sigma-Aldrich) at 100 nm, or the estrogen receptor antagonist ICI 182,780 (Sigma-Aldrich) at 100 nm, before addition of 10 μm DHEAS for an additional 1 h and measurement of superoxide production.

OATP, STS, and SULT2A1 mRNA expression analysis

Total cellular RNA was isolated from human PBMCs and highly purified (>99%) neutrophils using the one-step TRI Reagent (Sigma-Aldrich) with contaminating DNA removed by precipitation with chloroform. Reverse transcription was performed using AMV reverse transcriptase (Promega, Southampton, UK) and random hexamer primers (Sigma Genosys). Qualitative PCR analysis for expression of the DHEA/DHEAS shuttle system was carried out using the following primers. DHEA sulfotransferase (hydroxysteroid sulfotransferase 2A1, SULT2A1): forward, 5′-CAGGAAGAACCATAGAGAAGA TCTG-3′; reverse, 5′-GTCTTACACAATGACCCCAGTC-3′; STS: forward, 5′-AGGACTTCCCACCGATGAGATTACCTTTG-3′; reverse, 5′-AAAAG GGTCAGGATTAGGGCTGCTAGGAA-3′; OATPs: as previously published (41), except for OATP-A (OATP1A2), forward, 5′ GGTGTGGGATTACATACATTTT-3′; reverse, 5′-GAATAGTGTAATTAAGGTCA GACAGGGC-3′; OATP-C (OATP1B1): forward, 5′-GTTCAACCTGAATT GAAATCAC-3′; reverse, 5′-GATGTGGAATTATATGTCCTAC-3′. PCR amplification was carried out using the following conditions: initial denaturation at 94 C (5 min), followed by 35 cycles of 30 sec at 94 C, 30 sec at 60 C, and 30 sec at 72 C, with a final extension of 5 min at 72 C. PCR products were resolved on 1% agarose gels and stained with ethidium bromide.

DHEAS uptake assays

To determine the uptake of DHEAS by immune cells, human neutrophils and peripheral blood mononuclear cells were incubated with DHEAS (10 μm) in PBS for 30 or 60 min, followed by washing of the cells and subsequent cell lysis. This was followed by solid-phase extraction of DHEAS and subsequent quantification using uPLC-tandem mass spectroscopy. For the extraction of DHEAS from cell lysates, 1 ml C18(EC) solid-phase extraction cartridges were used. The samples were passed through the column at a flow rate of 1 ml/min with the addition of DHEA (5 ng/ml) as an internal standard. The columns were subsequently washed with 5% methanol and steroids eluted with 1.2 ml of methanol. The samples were evaporated to dryness at 50 C under constant nitrogen flow and reconstituted in 1 ml of 50:50 methanol/water. The extraction was optimized, and validated extraction efficiency for DHEAS was 88.7% ± 0.005%. The samples were analyzed using a Waters Xevo triple-quad mass spectrometer with an electrospray ionization source in positive mode, linked to an acuity UPLC system (Waters, Manchester, UK). DHEAS and DHEA were separated using a two-solvent gradient system (solvent A = water 0.1% formic acid; solvent B = methanol 0.1% formic acid) on a 1.7-μm BEH C₁₈ column (2.1 × 50 mm) the column was heated to 40 C throughout these experiments. The flow rate was 0.6 ml/min; the starting gradient was 45% B, increasing to 47% B at 1 min, then 57% B at 2 min, and finally 98% B at 2.5 min, followed by a reequilibrium step at 3 min. DHEAS concentrations were calculated by reference to a calibration series from 1–500 ng/ml using linear regression with DHEA as an internal standard. The assay was linear from 2–500 ng/ml with an interassay reproducibility SD (RSD) of approximately 10%. In addition to neutrophils and PBMCs, cell-free controls were run with only PBS and DHEAS added. Supernatant was removed from the tube, and the reaction tube was washed and extracted as for cell-containing samples. DHEAS uptake assays were performed in three independent, triplicate experiments.

PKC activation assays

Cellular in vivo activation of PKC was assessed by translocation of the enzyme from the cytosolic to membrane fraction of neutrophils treated with either 10 μm DHEAS or 20 nm PMA (Sigma) for 30 min at 37 C. After treatment, subcellular fractions were prepared as previously described (22), and the cytosolic and membrane fractions were assessed for PKC-α, -β (Ι, ΙΙ), -δ, and -ζ by Western blotting using isoenzyme-specific antibodies (Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence (GE Healthcare, Amersham, UK).

Direct in vitro activation of PKC was measured using two different methods: first, in a cell-free system using the SignaTect PKC activity assay and rat brain PKC (Promega Ltd., Southampton, UK) according to the manufacturer’s instructions. PKC activation was also assessed using a micellar-based assay. Recombinant glutathione-S-transferase-tagged PKC-βI, -βII, -δ or -ζ (150 ng) (kindly provided by Dr. M. Kubbutat, ProQinase Ltd, Freiburg, Germany) were incubated with PKC-α-19-31/Ser25 (NeoMPS, Strasbourg, France) as substrate, exactly as previously described (42). Activation of PKC by DHEA or DHEAS (500 nm to 50 μm) was assessed and compared with a solvent control.

Phosphorylation of p47^phox

Neutrophils (3 × 10⁷) were resuspended in phosphate-free buffer (20 mm HEPES, pH 7.4; 130 mm NaCl; 3 mm KCl; 1 mm MgCl₂; 1 mm CaCl_-2; and 5 mm glucose) and incubated with 0.5 mCi/ml ³²PO₄ (GE Healthcare) for 1 h at 30 C, before addition of 50 ng/ml GM-CSF (Serotec) or 10 μm DHEAS in the absence or presence of 1 μm BIM for 30 min at 37 C. Cells were then resuspended in 1 ml of lysis buffer (50 mm Tris-HCl, pH 8.0; 150 mm NaCl, 1% Triton X-100; 10% glycerol; 4 mm aminoethylbenzene sulfonyl fluoride; 10 μg/ml leupeptin; 10 μg/ml pepstatin-A; 10 μg/ml aprotinin; 2.5 mm NaF; and 5 mm NaVO_4; all from Sigma), homogenized, centrifuged at 14,000 × g for 5 min at 4 C and the supernatant was collected. The lysate was incubated with 5 μg of a monoclonal anti-p47^phox antibody (a kind gift from Dr. F. B. Wientjes, University College London) or irrelevant isotype control antibody (Dako Ltd., Cambridge, UK). Immunoprecipitates were harvested using the μMACS protein G microbead technology (Miltenyi Biotec Ltd., Bisley, UK) according to manufacturer’s instructions and resolved on a 10% SDS-PAGE gel, and autoradiography was performed using a phosphorimaging screen (Molecular Dynamics, Sunnyvale, CA).

Statistical analysis

Statistical significance was assessed by Student’s t test after ANOVA, and P < 0.05 was taken as a significant difference between means.