Microbicidal protein psoriasin is a multifunctional modulator of neutrophil activation

Abstract

As effector cells in host defence, neutrophils actively destroy invading microorganisms via a potent antimicrobial arsenal composed of oxidants and antimicrobial peptides. Psoriasin, an Escherichia coli-cidal antimicrobial protein, has been found to be overexpressed in psoriasis, a skin disease characterized by infiltration of neutrophils. In addition to its microbicidal activities and chemotaxis of neutrophils reported previously, we hypothesized that psoriasin might regulate other neutrophil functions such as cytokine and chemokine production, reactive oxygen species generation, and release of antimicrobial peptides. In the current study, we demonstrate that psoriasin activates neutrophils to produce a range of cytokines and chemokines including interleukin-6 (IL-6), IL-8/CXCL8, tumour necrosis factor-α, macrophage inflammatory protein-1α (MIP-1α)/CCL3, MIP-1β/CCL4 and MIP-3α/CCL20. Furthermore, psoriasin induces phosphorylation of mitogen-activated protein kinase p38 and extracellular signal-regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK), both of which are required for the production of cytokines and chemokines as evidenced by the inhibitory effects of p38 and ERK inhibitors on psoriasin-mediated neutrophil activation. Moreover, psoriasin stimulates the generation of reactive oxygen species from neutrophils, most likely via nicotinamide adenine dinucleotide phosphate oxidase activation. Finally, we demonstrate that psoriasin enhances messenger RNA expression of α-defensins, termed human neutrophil peptides (HNP) 1 to 3, and induces their extracellular release. Besides its antimicrobial properties, therefore, psoriasin may contribute to innate immunity through enhancing neutrophil host defence functions at sites of inflammation or infection.

Introduction

The innate immune system forms the first line of defence against invading microorganisms. In addition to the infiltrating neutrophils, macrophages and natural killer cells that are known to contribute to the cellular innate immunity in the skin, several reports have suggested that the epithelium participates in cutaneous innate immunity through the production of a vast arsenal of antimicrobial agents (reviewed in reference¹). These agents include defensins, cathelicidins and psoriasin, which are constitutively produced or locally induced in keratinocytes or phagocytes during infection or inflammation.¹

The antimicrobial protein psoriasin belongs to the S100 family of calcium-binding proteins and has been also termed S100A7.^2,3 Psoriasin has been shown to bind calcium, and its basal expression is influenced by extracellular calcium levels.^2,3 This microbicidal agent was initially identified as one of the proteins with highly up-regulated expression in psoriatic keratinocytes.⁴ Later studies have shown that the expression of psoriasin is not limited to psoriasis. Indeed, psoriasin has also been detected in a number of hyperproliferative and inflammatory skin diseases including atopic dermatitis.⁵ In addition, psoriasin has been reported to be overexpressed in carcinomatous tissues from breast and bladder, suggesting that it may be a candidate diagnostic marker for these disorders,^6,7 and may play a role in the regulation of cell growth, survival, or differentiation.^8,9 The expression of psoriasin in normal adult tissues is very low, but high expression levels have been detected in the fetal skin, implying its protective role in innate immunity of the newborn surface.¹⁰ Supporting this possibility, Gläser et al. investigated a skin antimicrobial agent that could preferentially and very effectively control the growth of Escherichia coli.¹¹ They found that psoriasin was a principal E. coli-cidal agent in healthy skin, which explained why skin regions that are often exposed to high concentrations of E. coli (such as anogenital and neonatal skin) are usually not infected with this gut bacterium.¹¹ Another report has also recently documented the antimicrobial properties of psoriasin against E. coli in skin wounds, where it is produced by wound-associated keratinocytes.¹² As for its killing mechanism, psoriasin directly interacts with the bacteria, and reduced interaction is associated with a decrease of its antibacterial function.¹² Furthermore, inactivation of the zinc-binding motif, but not of the calcium-binding motif, reduces the antibacterial function of psoriasin.¹² This is supported by the finding that incubation of psoriasin with zinc inhibits its microbicidal activity, whereas incubation with calcium neither inhibits nor increases this killing ability.¹¹

There is evidence that S100 members are involved in a proinflammatory axis associated with various inflammatory conditions. In fact, S100A8, S100A9 and S100A12 have been proven to be useful as diagnostic markers of inflammation, especially in non-infectious inflammatory diseases such as arthritis and chronic inflammatory lung and bowel disease.¹³ The observation that psoriasin is expressed in inflammatory diseases raised the question on its biological function in these conditions. To this end, Jingquan et al. have demonstrated the selective chemotactic activities of psoriasin towards CD4⁺ T lymphocytes and neutrophils.¹⁴

Based on these observations, and the fact that psoriasin is highly overexpressed in active psoriatic lesions where high amounts of infiltrating neutrophils have been observed, we speculated that in addition to its chemotactic activities, psoriasin may also have additional modulating effects on human neutrophil functions.

Our results indicate that psoriasin induces the production of several cytokines and chemokines from neutrophils via activation of mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK). Furthermore, psoriasin mediates the generation of reactive oxygen species (ROS), most likely via a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and induces the messenger RNA (mRNA) expression, as well as the extracellular release of α-defensins known as human neutrophil peptide 1 to 3 (HNP1 to HNP3).

Materials and methods

Reagents

The recombinant human psoriasin (amino acid sequence: MSNTQAERSIIGMIDMFHKYTRRDDKIDKPSLLTMMKENFPNFLSACDKKGTNYLADVFEKKDKNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPCSGGSQ) with a GST tag at the N terminus was purchased from Abnova Corporation (Taipei, Taiwan). The recombinant psoriasin exhibited antimicrobial activity against E. coli (unpublished observation), and was not contaminated with traces of lipopolysaccharide. Rabbit polyclonal anti-phosphorylated p38, ERK and JNK antibodies, and p38, ERK and JNK antibodies were obtained from Cell Signaling Technology (Beverly, MA). The inhibitors SB203580 (Sigma-Aldrich, St Louis, MO), PD98059 (Cell Signaling Technology) and SP600125 (Calbiochem, La Jolla, CA) were used to study the MAPK pathway involved in the activation of neutrophils. Pertussis toxin and o-dianisidine dihydrochloride were purchased from Sigma-Aldrich. The 5-(and-6)chloromethyl-2′,7′-dichlorohydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) was from Molecular Probes (Eugene, OR), whereas both diphenylene iodonium chloride (DPI) and 1, 2-bis(o-amino phenoxyl) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) were obtained from Sigma-Aldrich.

Purification and stimulation of peripheral blood neutrophils

Informed consent according to Juntendo University Ethical Committee approval was obtained from healthy volunteers, and blood was drawn from the cubital vein. Neutrophils were isolated from heparinized blood by polymorphprep (Axis-Shield, Oslo, Norway) according to the manufacturer's instructions. To obtain a higher purity of neutrophils, cells were centrifuged twice using polymorphprep, giving a purity > 98%. The contaminating cells were mainly eosinophils (< 1·5%) and monocytes (< 0·5%). Cells at a final concentration of 2 × 10⁶ cells/ml were treated with various doses of psoriasin at 37° for 2–12 hr in RPMI-1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS) in a 5% CO₂ atmosphere. Following incubation, cells were centrifuged, and the supernatants were used for enzyme-linked immunosorbent assay (ELISA), while the cell pellet was used for total RNA extraction.

ELISA

Interleukin-6 (IL-6), IL-8/CXCL8, tumour necrosis factor-α (TNF-α), macrophage-inflammatory protein 1α (MIP-1α)/CCL3, MIP-1β/CCL4, MIP-3α/CCL20 and HNP1-3 released in the cell-free supernatants from non-stimulated or stimulated cultures with various doses of psoriasin added for the indicated time periods were measured using the appropriate ELISA kits. These were purchased from R&D Systems (Minneapolis, MN) for all cytokines and chemokines and from HyCult Biotechnology (Uden, the Netherlands) for HNP1–3. Supernatants were stored at −20° until use for ELISA according to the manufacturers’ instructions. In some experiments, where inhibitors were used, these inhibitors were added 2 hr before stimulation with psoriasin, and ELISA was performed as above.

Neutrophil apoptosis

Neutrophils were incubated at 37° in 5% CO₂, for 12 or 24 hr at 2 × 10⁵ cells in 100 μl of RPMI-1640/10% FCS in the presence or absence of psoriasin. Apoptosis was assessed by flow cytometry using fluorescein isothiocyanate (FITC)–annexin V and propidium iodide (Sigma-Aldrich), according to the manufacturer's instructions. After a 10-min incubation in the dark, cells were analysed using a FACScan (Becton-Dickinson, Bedford, UK). Apoptotic neutrophils were defined as annexin V-positive but propidium iodide-negative cells, necrotic neutrophils were defined as annexin V-positive and propidium iodide-positive cells, and viable neutrophils were defined as annexin V-negative and propidium iodide-negative cells. Results were expressed as the percentage of apoptotic, necrotic or viable cells.

Neutrophil degranulation: myeloperoxidase release assay

Myeloperoxidase (MPO) release was selected as a marker of degranulation of neutrophil azurophil granules, and was determined by the oxidation of o-dianisidine dihydrochloride by H₂O₂.¹⁵ Briefly, neutrophils (10⁶ cells) were incubated at 37° for the indicated periods with 10 μm psoriasin, 10 ng/ml phorbol 12-myristate 13-acetate (PMA) as a positive control, or medium alone. Cells were then centrifuged at 80 g for 6 min, and 100 μl of the cell-free supernatant was transferred to a new 96-well culture plate. An equivalent volume of a mixture containing 1·25 mg/ml o-dianisidine dihydrochloride and 0·004% H₂O₂ in Hanks’ balanced salt solution (HBSS) was added, followed by a 15-min incubation at room temperature. The reaction was stopped by the addition of 10 μl 1% NaN₃, and MPO release was assessed spectrophotometrically by measuring the changes in absorbance at 450 nm using SoftMax Pro (version 3) software (Molecular Devices, Sunnyvale, CA). The total release of MPO was measured from neutrophils lysed with 0·2% Triton X-100 detergent, and the per cent MPO release was calculated as the ratio of MPO released by psoriasin or PMA to the total amount released.

Western blot analysis

Neutrophils (2 × 10⁶ cells/ml) were incubated with 10 μm psoriasin for the indicated time periods. After stimulation, the lysates were obtained by lysing cells in lysis buffer [50 mm Tris–HCl (pH 8), 150 mm NaCl, 0·02% NaN₃, 0·1% sodium dodecyl sulphate (SDS), 1% nonidet P-40, containing 1 μm phenylmethylsulphonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin-A, 50 μg/ml aprotinin and 2 mm sodium orthovanadate]. Equal amounts of total protein were subjected to 12·5% SDS–polyacrylamide gel electrophoresis. Non-specific binding sites were blocked and the blots were incubated with polyclonal antibodies against phosphorylated p38, ERK and JNK or unphosphorylated p38, ERK and JNK overnight, according to the manufacturer's instructions. The membrane was developed with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). To quantify the intensity of the bands, densitometry using the software program image gauge (LAS-1000 plus, Fujifilm, Tokyo, Japan) was performed to provide correction for protein loading.

Measurement of intracellular ROS production

Flow cytometry was used to determine intracellular levels of ROS. After incorporation into cells, a non-polar compound, DCFH-DA, is converted into a membrane-impermeable, non-fluorescent polar derivative (DCFH) by cellular esterase. The trapped DCFH is rapidly oxidized to fluorescent 2′,7′-dichlorofluorescein (DCF) by intracellular peroxides such as H₂O₂.^16,17 Neutrophils at a density of 10⁶ cells/ml were suspended in 150 μl RPMI-1640/10% FCS, and incubated at 37° for the time indicated, with or without 50 μl titrated concentrations of psoriasin. The cells were washed, resuspended in Opti-MEM containing 1 μm DCFDA, and then incubated for 30 min at 37°. Following one wash in ice-cold phosphate-buffered saline, cells were analysed by flow cytometry. In inhibition experiments, the agents tested were added to cells 30 min before the addition of psoriasin.

Measurements of intracellular Ca²⁺ mobilization

Intracellular Ca²⁺ mobilization from neutrophils was measured by a no-washing method using a FLIPR Calcium Assay Kit (Molecular Devices). Neutrophils (100 μl) were seeded at a density of 2 × 10⁵ cells per well into poly-d-lysine coated 96-well black-walled clear bottom microtitre plates (Becton-Dicknson). Cells were then loaded for 1 hr at 37° in an equivalent volume of HBSS containing 20 mm HEPES, 2·5 mm probenecid (Sigma-Aldrich), and Calcium 3 Reagent (Molecular Devices) at pH 7·4 prepared according to the manufacturers’ instructions. To form a uniform monolayer of cells on the bottom of the wells, the microplate was gently centrifuged for 5 min with low acceleration and without break. The cell-containing plate was placed into a FlexStation II (Molecular Devices), with excitation, emission, and cut-off filter set to 485, 525 and 515 nm, respectively. A volume of 50 μl/well of agonist diluted in assay buffer was added to achieve the final indicated concentrations. Maximum change in fluorescence over baseline was used to determine agonist response, as quantified using SoftMax Pro (version 5) software.

Total RNA extraction and real-time quantitative polymerase chain reaction

Total RNA was extracted from neutrophils using TRIzol reagent (BRL, Life Technologies, Rockville, MD), according to the manufacturer's instructions. First-strand complementary DNA was synthesized from 3 μg total RNA with oligo-dT (12–18) primers using Superscript II RNase H^- reverse transcriptase (Life Technologies), as described previously.¹⁸ Real-time polymerase chain reaction (PCR) was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, NJ). Amplification and detection of HNP1-3 mRNA were analysed using a 7500 Real-Time PCR System (Applied Biosystems), according to the manufacturer's specifications. The HNP1-3 primer/probe set was obtained from Applied Biosystems Assays-on-Demand. To standardize mRNA concentrations, transcript levels of the housekeeping gene GAPDH were determined in parallel for each sample, and relative transcript levels were corrected by normalization based on the GAPDH transcript levels.

Statistical analysis

The statistical analysis was performed using Student's t-test or one-way analysis of variance (anova), and P < 0·05 was considered to be significant. The results are shown as mean ± SD.

Results

Psoriasin induces the production of cytokines and chemokines by neutrophils

To determine the stimulatory activities of psoriasin on human neutrophils, we first evaluated its ability to induce the production of various inflammatory cytokines and chemokines from neutrophils, as determined by ELISA. As shown in Fig. 1, the activation of neutrophils with 10 or 20 μm psoriasin resulted in a dose-dependent production of IL-6, IL-8/CXCL8, MIP-1α/CCL3 and MIP-3α/CCL20, whereas the production of TNF-α and MIP-1β/CCL4 was only significantly increased in cells stimulated with 20 μm psoriasin. We observed that the dosages higher that 20 μm did not further increase the production of cytokines and chemokines (data not shown). Furthermore, the stimulatory effect of psoriasin was time-dependent, reaching a maximum after 12 hr of incubation. Longer incubation (18 or 24 hr) also resulted in enhanced release of cytokines and chemokines; however, this production was accompanied by a dramatic decrease of cell viability, as evaluated by trypan blue exclusion and flow cytometry (data not shown).

Figure 1.

Psoriasin induces the production of cytokines and chemokines by neutrophils. Human neutrophils were stimulated with psoriasin at the concentrations of 10 μm (dots) or 20 μm (horizontal stripes) for 2–12 hr, and the amounts of various cytokines and chemokines released into the culture supernatants were determined by enzyme-linked immunosorbent assay. Values are compared between stimulated and non-stimulated cells (open bars). *P < 0·05, **P < 0·01. Each bar represents the mean ± SD of five independent experiments. IL-6, interleukin-6; TNF-α, tumour necrosis factor-α, MIP-1α, macrophage inflammatory protein-1α.

With the aim of determining whether psoriasin activates neutrophils via a receptor-mediated process, we evaluated the effects of pertussis toxin, an inhibitor for G protein, on neutrophil activation. As observed in Fig. 2(a,b), 200 ng/ml pertussis toxin failed to suppress psoriasin-induced IL-6 and IL-8/CXCL8 production. The same dose could significantly inhibit LL-37-induced IL-8 production (data not shown). Similar results were obtained for TNF-α, MIP-1α/CCL3, MIP-3α/CCL20 and MIP-1β/CCL4 (data not shown). Psoriasin is unlikely to stimulate neutrophils through G protein-coupled receptor(s).

Figure 2.

Pertussis toxin does not inhibit psoriasin-mediated neutrophil activation. Neutrophils were pretreated with 200 ng/ml pertussis toxin or medium alone for 2 hr. Cells were then challenged for 12 hr with 10 μm psoriasin, and the concentrations of interleukin-6 (IL-6) (a) and IL-8 (b) released into the culture supernatants were measured by enzyme-linked immunosorbent assay. Values are the mean ± SD of four separate experiments, and compared between the presence (PTx, pertussis toxin) and absence (Ctrl, control) of pertussis toxin. Med (medium alone: non-stimulated cells).

Psoriasin does not influence neutrophil apoptosis

Neutrophils undergo spontaneous apoptosis, and the antimicrobial peptide LL-37 is known to inhibit this apoptosis.^19,20 Therefore, we tested whether psoriasin may also influence neutrophil apoptosis. In untreated neutrophils, spontaneous apoptosis was observed in 20% of neutrophils at 12 hr, and markedly increased to approximately 40% at 24 hr, at 37° (Fig. 3a). Incubation for 24 hr also increased the number of necrotic cells (Fig. 3b). We found that the addition of psoriasin neither inhibited nor augmented neutrophil apoptosis, necrosis or viability, as compared with non-stimulated cells (Fig. 3a–c). This suggests that psoriasin and LL-37 activate human neutrophils by different methods, and confirms that the dosages of psoriasin used in the current study were not toxic.

Figure 3.

Psoriasin does not affect neutrophil apoptosis. Neutrophils were incubated for 12 or 24 hr with 10 μm psoriasin or medium alone in RPMI-1640/10% fetal calf serum at 37°. Cells were also incubated for 12 hr at 4° in the absence of psoriasin (0). Fluorescence-activated cell sorter analysis was used to determine the percentage of neutrophils, which were (a) apoptotic [fluorescein isothiocyanate (FITC) –annexin V-positive but propidium iodide-negative], (b) necrotic (FITC–annexin V-positive and propidium iodide-positive) and (c) viable (FITC–annexin V-negative and propidium iodide-negative). Values are compared between stimulated (Ps, psoriasin) and non-stimulated (Md, medium alone) cells, or between 12-hr and 24-hr incubation. Each bar represents the mean ± SD of four independent experiments.

Zinc, but not calcium, inhibits psoriasin-mediated neutrophil activation

It has been suggested that the antibacterial action of psoriasin against E. coli is inhibited by zinc but not by calcium.¹¹ To test whether these divalent ions may influence psoriasin-mediated neutrophil activation, neutrophils were preincubated with ZnSO₄ or CaCl₂ before stimulation with psoriasin. The results revealed that 5 μm Zn²⁺ almost completely inhibited psoriasin-induced IL-6 and IL-8/CXCL8 production. Although psoriasin is a Ca²⁺ binding protein, Ca²⁺ at concentrations as high as 50 μm was unable to affect neutrophil activation (Fig. 4a,b). Similar results were obtained for other cytokines/chemokines tested (data not shown).

Figure 4.

Effects of zinc and calcium on psoriasin-induced cytokine/chemokine production. Neutrophils were pretreated with ZnSO₄ (5 μm) or CaCl₂ (50 μm) for 2 hr before the addition of 10 μm psoriasin for 12 hr. The concentrations of released interleukin-6 (IL-6) (a) and IL-8 (b) into the culture supernatants were measured by enzyme-linked immunosorbent assay. Values are the mean ± SD of five independent experiments, and compared between the presence and absence (Ctrl, control) of zinc or calcium. **P < 0·01. Med (medium alone: non-stimulated cells).

Phosphorylation of p38 and ERK is required for the production of cytokines/chemokines

Human antimicrobial peptides such β-defensins and LL-37 have been reported to induce phosphorylation of MAPK p38 and ERK in various immune and inflammatory cells.^21–24 We speculated that psoriasin may have similar properties to activate MAPKs in neutrophils. Cells were stimulated with 10 μm psoriasin for 10–30 min, and the phosphorylated and unphosphorylated p38, ERK or JNK levels were determined using Western blot analysis. Psoriasin induced phosphorylation of both p38 and ERK at 10 and 20 min compared with non-stimulated cells, and this phosphorylation was markedly enhanced at 30 min. In contrast to p38 and ERK, the phosphorylation of JNK by psoriasin was not observed (Fig. 5a right and left panels).

Figure 5.

Psoriasin induces the phosphorylation of mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase (ERK) that are further required for neutrophil activation. (a) Neutrophils (2 × 10⁶ cells/ml) were stimulated with 10 μm psoriasin for the indicated time periods, and phosphorylated p38 (p-p38), ERK (p-ERK) or c-Jun N-terminal kinase (p-JNK), and unphosphorylated p38 (p38), ERK (ERK) or JNK (JNK) levels in cellular lysates were determined by Western blot analysis. Left panel: one representative of six separate experiments with similar results is shown. Right panel: bands were quantified by densitometry using the software program Image Gauge (LAS-1000plus) to allow correction for protein loading. Data represent the ratio of the intensity of phosphorylated protein (p-p38, p-ERK or p-JNK) divided by respective unphosphorylated protein (p38, ERK or JNK). Values are the mean ± SD of six independent experiments. *P < 0·05, **P < 0·01 as compared between stimulated and non-stimulated (0) cells. (b) Neutrophils were preincubated with 10 μm of SB203580 (SB), 10 μm of PD98059 (PD), 20 μm SP600125 (SP) or medium alone for 2 hr, after which the cells were stimulated for 12 hr with 10 μm psoriasin. Interleukin-6 (IL-6; left panel) and IL-8 (right panel) released into supernatants were determined by enzyme-linked immunosorbent assay. Values are the mean ± SD of four separate experiments. **P < 0·01 as compared between the presence and absence (Ctl, control) of each MAPK inhibitor. Med (medium alone: non-stimulated cells).

To determine whether activation of MAPKs was required for psoriasin-mediated production of cytokines and chemokines, cells were incubated with specific inhibitors for p38, ERK or JNK for 2 hr before stimulation with psoriasin. The presence of SB203580 (p38 inhibitor) and PD98059 (ERK inhibitor), but not SP600125 (JNK inhibitor) markedly suppressed the production of IL-6 and IL-8/CXCL8 (Fig. 5b). Similar inhibitory effects of SB203580 and PD98059 on other cytokines and chemokines tested were also observed (data not shown). We confirmed that the treatment of neutrophils with MAPK inhibitors did not affect the cell viability as assessed by a fluorescence-activated cell sorting-based annexin V/propidium iodide staining (data not shown).

Psoriasin stimulates the generation of ROS by neutrophils

The kinetics of psoriasin-stimulated ROS generation in human neutrophils was measured using the cell permeant, oxidation-sensitive dye DCFDA. This dye is non-fluorescent until oxidized by ROS, and an increase of fluorescence of DCFDA indicates oxidation by peroxides, peroxynitrites or hydroxyl radicals,²⁵ while dihydroethidium (DHE) is selectively oxidized by superoxide anion.²⁶ In the preliminary experiments, we observed that psoriasin induced significant increases in DCFDA oxidation in a time-dependent fashion from 2·5 to 20 min, with a maximal oxidation at 20 min (data not shown). In dose–response experiments, a 20-min incubation with psoriasin induced DCFDA oxidation dose dependently, reaching a peak at 10 μm, before decreasing at 20 μm (Fig. 6a right and left panels). Using DHE as a selective probe for superoxide anion, psoriasin could only slightly induce DHE oxidation at various doses and time periods tested (data not shown).

Figure 6.

Psoriasin-stimulated generation of reactive oxygen species (ROS) in neutrophils. (a) Left panel: representative fluorescence-activated cell sorting profile for various doses of psoriasin-induced DCFDA oxidation at 20 min with non-stimulated cells (black lines, Med: medium alone). 2·5 μm psoriasin-induced ROS generation is represented by green lines, 5 μm (red lines), 10 μm (blue lines), and 20 μm (orange lines). Right panel: averages of mean fluorescence of four independent experiments. Values are compared between stimulated and non-stimulated (Med, medium alone) cells. *P < 0·05, **P < 0·01. Each bar represents the mean ± SD. (b) and (c) Effect of ROS scavengers. DCFDA oxidation was determined after 20 min of stimulation with 10 μm psoriasin in the presence or absence of titrated concentrations of (b) DPI or (c) BAPTA-AM. Data are corrected from spontaneous DCFDA oxidation, and are expressed as fluorescence mean. The data represent the average of six experiments (± SD). *P < 0·01 as compared between the presence and absence of inhibitor. (d) Cells were incubated for 1 hr at 37° in Hanks’ balanced salt solution containing HEPES, probenecid and Calcium 3 Reagent, and then stimulated with 10 or 20 μm psoriasin as described in the Materials and methods. LL-37 (10 μg/ml) was used as a positive control. Data are representative of four separate experiments, and shown as fluorescence change corrected from spontaneous fluorescence by non-stimulated cells. Arrow indicates the addition of stimulant.

Generation of ROS upon psoriasin stimulation is most likely mediated by NADPH oxidase

To further characterize the ROS generated upon psoriasin stimulation in neutrophils, antioxidants were used as scavengers of selective species of oxidants. The broad-spectrum inhibitor of flavoprotein-containing oxidoreductases DPI has been reported to inhibit the release of ROS by blocking NADPH oxidase activity.²⁷ Consistently, various dosages of DPI noticeably reduced psoriasin-mediated ROS production in a dose-dependent manner (Fig. 6b). DPI at concentrations as low as 2·5 μm was effective, and the inhibitory effect was maximal at 10 μm. Because DPI has also been reported to inhibit mitochondrial ROS generation,²⁸ it was possible that the effect of psoriasin resulted from the activation of mitochondrial respiratory chain enzymes. We tested this possibility by pretreating neutrophils with rotenone, a potent inhibitor of the mitochondrial electron transport chain. Rotenone could not suppress the ROS production induced by psoriasin (data not shown). Similarly, ROS generation was not via xanthine oxidase activation, as shown by the fact that allopurinol, an inhibitor for xanthine oxidase,²⁹ failed to suppress psoriasin-mediated ROS production (data not shown). Because the intracellular Ca²⁺ chelator BAPTA-AM has been shown to potently inhibit ROS generation in neutrophils,³⁰ its possible involvement was also examined. As seen in Fig. 6(c), BAPTA-AM did not significantly inhibit psoriasin-induced ROS generation. This finding was supported by the fact that psoriasin could not induce intracellular Ca²⁺ flux, whereas cathelicidin LL-37, used as a positive control, evoked significant increases in intracellular Ca²⁺ concentrations (Fig. 6d). Together, these results suggest that the psoriasin-mediated ROS generation is mediated by flavoenzyme, most likely an NADPH oxidase.

Psoriasin induces the gene expression and extracellular release of HNP1-3

It has been previously shown that stimulation of neutrophils from guinea pigs resulted in extracellular release of antimicrobial agents such as α-defensins and cathelicidin CAP11.³¹ We envisaged that psoriasin may also activate human neutrophils to release α-defensins. To this end, we first evaluated the effect of psoriasin on mRNA expression of HNP, a type of α-defensin located in neutrophil azurophil granules, with a predominance of HNP1, -2 and -3.^32–34 Incubation of neutrophils with 10 μm psoriasin resulted in increases of gene expression of HNP1-3. This effect was time-dependent, reaching a peak at 8 hr before decreasing after 12 hr of incubation (Fig. 7a). We observed that 20 μm psoriasin did not cause a significant further increase of mRNA expression of HNPs (data not shown).

Figure 7.

Psoriasin increases the gene expression and extracellular release of human neutrophil peptides 1 to 3 (HNP1-3). (a) Neutrophils (2 × 10⁶ cells) were incubated with 10 μm psoriasin for 2–12 hr. Following the incubation, total RNA was extracted, converted into complementary DNA, and real-time polymerase chain reaction was performed to analyse the changes in gene expression. Each bar shows the mean ± SD from five separate experiments. Values represent transcript numbers, and are compared between stimulated and non-stimulated (Med, medium alone) cells. *P < 0·05, **P < 0·01. (b) Supernatants from cells stimulated with 10 μm (dots) or 20 μm (horizontal stripes) of psoriasin for the indicated time periods were used for detection of released concentrations of HNP1-3 by enzyme-linked immunosorbent assay. Values are compared between stimulated and non-stimulated cells (open bars). **P < 0·01. Each bar represents the mean ± SD of five independent experiments.

Next we evaluated whether psoriasin could also stimulate neutrophils to extracellularly release their respective proteins. After stimulation of cells with 10 or 20 μm psoriasin for 2–12 hr, the release of HNP1-3 in cell-free supernatants was determined by a specific ELISA kit. As depicted in Fig. 7(b), psoriasin dose-dependently enhanced the release of HNP1-3 after a 12-hr incubation. The treatment of neutrophils with psoriasin failed to trigger detectable HNP1-3 protein in the cell lysates, as assessed by Western blot analysis (data not shown), so it was concluded that psoriasin-induced HNP1-3 mRNA levels are very low to synthesize enough HNP protein.

Degranulation of neutrophils upon psoriasin stimulation

To confirm whether the ability of psoriasin to trigger exocytosis of HNP1-3 correlates with its capacity to degranulate neutrophils, we assessed the extracellular release of MPO, a marker of azurophil granules.¹⁵ The results depicted in Fig. 8 demonstrate that psoriasin significantly induced neutrophil degranulation as compared with non-stimulated cells. MPO release was not significantly detected until 12 hr after psoriasin stimulation, and then increased markedly. PMA-induced and psoriasin-induced MPO release was 37·13% and 25·69% of total release, respectively, whereas the spontaneous release was 14·82%. The PMA was used as a positive control. Higher dosages of psoriasin did not yield a different result (data not shown).

Figure 8.

Myeloperoxidase (MPO) release from psoriasin-exposed neutrophils. MPO release from neutrophils was determined from 0·5 to 12 hr after incubation with 10 μm psoriasin (dots), 10 ng/ml phorbol 12-myristate 13-acetate (PMA; horizontal stripes) used as a positive control, or medium alone (open bars), as described in Materials and methods. Values represent changes in absorbance at 450 nm, and are compared between stimulated and non-stimulated cells. *P < 0·05, **P < 0·01.

Discussion

Antimicrobial protein psoriasin is markedly overexpressed in psoriasis, a skin disease that is histopathologically characterized by a preferential epidermal infiltration of neutrophils and T lymphocytes.³⁵ Besides its bactericidal properties, psoriasin has also been shown to selectively chemoattract neutrophils and CD4⁺ T cells.¹⁴ Based on this finding, we anticipated that psoriasin might have additional stimulatory effects on neutrophils other than chemotaxis. Supporting our hypothesis, the current study revealed that psoriasin mediates the production of several inflammatory cytokines and chemokines from neutrophils through activation of MAPK p38 and ERK. Moreover, psoriasin induces ROS generation via an NADPH oxidase and increases mRNA expression as well as extracellular release of HNP1-3.

Comprising more than half of the circulating white blood cells in humans, neutrophils are the predominant inflammatory cells recruited to sites of infection or inflammation. After the onset of an inflammatory reaction, large numbers of neutrophils are still present at the inflamed site long after the cessation of neutrophil influx.³⁶ These cells have been shown to be activated by a wide variety of stimuli including antimicrobial peptide LL-37, which inhibits neutrophil apoptosis^19,20 and modulates cytokine production.²⁰ To our knowledge, the current study shows for the first time that psoriasin, another antimicrobial protein, induces the generation of cytokines and chemokines such as IL-6, IL-8/CXCL8, TNF-α, MIP-1α/CCL3, MIP-1β/CCL4 and MIP-3α/CCL20, which are produced by neutrophils,^37–41 and contribute to the pathogenesis of psoriasis.^42,43 Although the amounts of cytokines/chemokines induced by psoriasin appear to be very low, one could suppose that at an infection/inflammation site, psoriasin may work synergistically with other antimicrobial agents such as LL-37 or human β-defensins to reach bioactive production. Indeed, these latter peptides are known to activate neutrophils,^1,19,20 and have been shown to stimulate several cell types synergistically.²¹

To characterize the mechanism by which psoriasin activates neutrophils, a receptor-mediated process was first examined by investigating the effects of pertussis toxin. However, because pertussis toxin failed to suppress psoriasin-induced cytokine/chemokine production, a G-protein-coupled pathway was ruled out. We next focused on the pathways of MAPK cascades p38 and ERK, because these kinases have been shown to be involved in a large variety of cellular activities, ranging from cell survival and proliferation to expression of proinflammatory cytokines and chemokines.^44,45 Recently, we and other investigators have shown that human β-defensins and LL-37 activate keratinocytes, mast cells and monocytes through p38 and ERK.^21–24 In the present study, psoriasin induced phosphorylation of p38 and ERK, but not JNK. Both p38 and ERK, but not JNK, were required for psoriasin stimulatory ability, as evidenced by the inhibitory effects of p38-specific and ERK-specific inhibitors. Finally, we revealed that similar to its microbicidal ability,^11,12 psoriasin-mediated neutrophil activation was also dependent on zinc but not on calcium.

As the cornerstone of the innate immune response, neutrophils are the archetypical phagocytic cells that actively destroy pathogenic microorganisms. To achieve this essential role in host defence, neutrophils deploy a potent antimicrobial arsenal that includes oxidants (oxidative mechanism) and antimicrobial proteins (oxygen-independent mechanism).⁴⁶ Neutrophils possess a membrane-bound multicomponent enzyme complex termed the NADPH oxidase that, when activated, generates large quantities of ROS.^47,48 The phagocyte NADPH oxidase is dormant in resting cells and can be rapidly activated by a variety of soluble mediators (e.g. chemoattractant peptides and chemokines) and particulate stimuli (e.g. bacteria and immune complexes).⁴⁹ To investigate whether psoriasin could contribute to neutrophil host defence functions, its effect on ROS generation was first examined. Psoriasin enhanced the ROS generation in neutrophils, most probably via NADPH oxidase activation, as demonstrated by the inhibitory effect of DPI, which is known to suppress NADPH oxidase. This was also supported by the finding that neither rotenone (mitochondrial inhibitor) nor allopurinol (inhibitor for xanthine oxidase) affected psoriasin-mediated ROS generation.

The second evidence that psoriasin contributes to the neutrophil host defence was confirmed by the ability of psoriasin to induce the exocytosis of the antimicrobial granule components, α-defensins. Four α-defensin molecules (HNP1, HNP2, HNP3 and HNP4) have been described.^32–34,50 HNP1, HNP2 and HNP3, which differ only in the first amino acid, account for 5–7% of total neutrophil proteins.³³ By contrast, HNP4, with an amino acid sequence distinct from other HNP sequences, comprises less than 2% of total defensins in neutrophils.⁵⁰ HNP are generally synthesized in neutrophil precursor cells (promyelocytes) and in the bone marrow, and the mature peptides are stored in the azurophil granules of neutrophils, where they are extracellularly released upon stimulation.¹ It is believed that mature neutrophils that circulate in the blood contain large levels of HNP but no longer synthesize either the mRNA or the proteins themselves.⁵¹ In contrast to this, the current report shows that mature neutrophils have the capacity to synthesize HNP1-3 mRNA. Our results are in accord with previous reports that have identified defensin precursors in mature human neutrophils, and have demonstrated that peripheral blood neutrophils retain a substantial ability to biosynthesize HNP1-3 mRNA.^52,53 This is not surprising because mature neutrophils have been also reported to synthesize a certain degree of RNA and protein.⁵⁴ However, it appears that the levels of HNP1-3 mRNA induced by psoriasin are not enough for protein production because an attempt to detect HNP1-3 in cell lysates failed (data not shown). Therefore, psoriasin probably induces HNP1-3 release from azurophil granules rather than protein production. This was also confirmed by the ability of psoriasin to increase the activity of MPO, a marker for azurophil granules.

Besides its chemotactic property, we provide novel evidence that psoriasin activates both the oxidative and non-oxidative killing mechanisms of neutrophils. The biological responses of leucocytes to chemotactic factors are divided into those related to cellular motility and those related to the cytotoxic or microbicidal ability of leucocytes. Interestingly, these responses are regulated differently. Low doses of chemoattractants stimulate chemotaxis-related functions, whereas the cytotoxic properties require higher concentrations.^55,56 Accordingly, while nanomolar concentrations of psoriasin stimulate neutrophil chemotaxis,¹⁴ micromolar concentrations were necessary for neutrophil activation in our experimental conditions. This is in full agreement with an early observation that psoriasin concentrations as high as 1–5 μm were needed for its killing activity against E. coli.¹² Although there is no description of the exact amounts of psoriasin in vivo, a rough estimate of antimicrobial proteins in skin extracts indicates that psoriasin represents the most abundant antimicrobial protein in healthy skin followed by LL-37 and human β-defensin-2,⁵⁷ which have been estimated at 300 μm and 20 μm, respectively, in psoriatic skin.⁵⁸ Therefore, assuming that psoriasin increases greatly at sites of inflammation or infection, it may reach optimal concentrations in vivo that are adequate for the stimulation of the biological activities of neutrophils.

Neutrophils are known to participate in innate immunity, so our finding that psoriasin activates human neutrophils to produce inflammatory cytokines/chemokines, generate ROS, and release antimicrobial peptides provides insight into the novel mechanism by which psoriasin contributes to the modulation of host defence, particularly at inflammation/infection sites. This control of immunity by psoriasin was to our knowledge previously unknown, and adds a new element to the understanding of antimicrobial agent-mediated host defence.