KdPT, a Tripeptide Derivative of α-Melanocyte–Stimulating Hormone, Suppresses IL-1β–Mediated Cytokine Expression and Signaling in Human Sebocytes

Acne vulgaris is the most common inflammatory disease of the pilosebaceous unit, affecting ~93% of adolescents with different degrees of severity (1). The pathogenesis of this disease is complex, involving sebaceous gland hypertrophy, increased seborrhea with the presence of oxidized lipids, pilosebaceous duct hyperkeratinization, and colonization with Propionibacterium acnes, resulting in perifollicular inflammation (2, 3). Among various mediators orchestrating the inflammatory response of the pilosebaceous unit in acne, several cytokines, including IL-1, IL-6, IL-8, and TNF-α, have been implicated (4–8). In particular, IL-1 appears to play a key role in the pathogenesis of acne. IL-1α was shown to be present in open comedones, the very early lesions of this disease (4). IL-1α induces follicular hyperkeratinization in an ex vivo model (9), whereas sebocytes respond to IL-1β with induction of proinflammatory cytokines. In light of these findings and in consideration of the role of IL-1 as a potent inducer of both IL-6 and IL-8 (10, 11), blocking of IL-1–mediated responses could offer a promising new immunomodulatory approach to combating acne.

To date, there is compelling evidence that α-melanocyte–stimulating hormone (α-MSH), a tridecapeptide originally described as a neurohormone, has pleiotropic anti-inflammatory effects (12), which have been demonstrated in various in vitro cell culture models. Moreover, these effects have also been shown in several animal models of, for example, contact dermatitis (13), vasculitis (14), allergic asthma (15), and fibrosis (16), to mention a few. The biologic actions of α-MSH are mediated via melanocortin receptors (MC-Rs), which belong to the superfamily of G protein-coupled receptors with seven transmembrane domains.

Interestingly, receptors for α-MSH have not only been detected in various cells of the immune system (17). They have also been identified in sebocytes under in vitro conditions (18, 19). In fact, α-MSH was shown to have weak inhibitory effects on IL-1β–induced secretion of IL-8 in human sebocytes in vitro (19). However, therapeutic exploitation of α-MSH as an anti-inflammatory agent in patients with acne is problematic for several reasons because natural and synthetic melanocortin peptides such as [Nle⁴, D-Phe⁷]-α–MSH (NDP-α–MSH) or adrenocorticotropic hormone directly increase lipogenesis in primary human sebocytes (20). Moreover, the melanotropic (pigment-inducing) activity of α-MSH analogs containing the central pharmacophor may be a limiting factor for their use, especially in Caucasian white-skinned patients.

KdPT (Lys-d-Pro-Thr) is a derivative of the C-terminal sequence of α-MSH in which the second amino acid, Pro, is replaced by its d-enantiomer and the third amino acid, Val, is exchanged for Thr. It is of interest that KPT, the l-enantiomer, is collinear with human IL-1β_193–195. Originally, KdPT was reported to attenuate the IL-1β–mediated hyperalgesic response in a rat-paw pressure test (21, 22). KdPT, however, does not appear to act as a simple or uniform IL-1β antagonist because the peptide did not have antipyretic activity against IL-1β in the rabbit pyrogen test (21). It was also inactive in the IL-1β–evoked relaxation of rabbit isolated mesenteric artery (23). So far, little research on KdPT has been conducted; consequently, the anti-inflammatory potential of this substance remains largely elusive.

In this study, we investigated the anti-inflammatory potential of KdPT on SZ95 sebocytes, which show the morphologic, phenotypic, and functional characteristics of primary human sebocytes (24). Because acne does not exist outside the human system and no animal models for this disease are available, this immortalized human sebocyte cell line has evolved as an important research tool in acne research to study the influence of various immune mediators and neuropeptides (25).

Our data provide compelling evidence for potent anti-inflammatory effects of KdPT on IL-1β–mediated IL-6 and IL-8 expression. These effects of KdPT were not mediated by binding to the MC-1R. We demonstrate that KdPT suppresses IL-1β–mediated NF-κB activation, possibly via interaction with the third Ig-like C2 domain of IL-1R type I (IL-1RI) and via reduced formation of oxidative stress. Because these anti-inflammatory effects of KdPT are not based on MC-1R binding, they cannot enhance melanogenesis. Hence, it is tempting to propose major potential for the tripeptide in the treatment of inflammatory diseases, including acne.

SZ95 sebocytes (25) were maintained in Sebomed basal medium supplemented with 5 ng/ml human epidermal growth factor, 10% FCS (both from Biochrom, Berlin, Germany), 1% l-glutamine, 1% penicillin/streptomycin, and 1 mM CaCl₂ in a humidified atmosphere containing 5% CO₂ at 37°C. Human melanocytes obtained from Tebu-bio (Portland, OR) were routinely grown in MGM-M2 plus all supplements (Cascade Biologics, Portland, OR). Mouse B16-F1 melanoma cells (26) were cultured in MEM supplemented with 10% heat-inactivated FCS, 2 mM l-glutamine, 1% nonessential amino acids, 1% vitamin solution, 50 IU/ml penicillin, and 50 μg/ml streptomycin (conditions as described for sebocytes).

SZ95 sebocytes were seeded into 6-cm Ø tissue culture dishes at a density of 5 × 10⁵ cells per dish. On the next day, cells were switched to Sebomed basal medium containing 0.1% FCS. After 24 h of serum deprivation, cells were treated with IL-1β in the doses indicated (ImmunoTools, Friesoythe, Germany) in combination with various doses of KdPT (Bachem, Weil, Switzerland). In some experiments, cells were preincubated for 16 h with the Mn complex of 7-hydroxy-flavone (Calbiochem-Novabiochem, Schwalbach, Germany) prior to IL-1β treatment. For MC-1R competition experiments, a peptide corresponding to aa 87–132 of human Agouti signaling peptide (Phoenix Pharmaceuticals, Belmont, CA) was coincubated at 10⁻⁶ M simultaneously with KdPT and IL-1β. After treatment, cells were washed with PBS and harvested for real-time RT-PCR analysis. Total RNA was then isolated by the RNeasy kit (Qiagen, Hilden, Germany). Following DNAse I treatment, cDNA was synthesized using oligo-dT primers and RevertAid M-MuLV reverse transcriptase (Fermentas Life Sciences, Hanover, MD), according to the manufacturer’s instructions. Real-time RT-PCR was performed in a total volume of 20 μl with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), and a 200 nM concentration of each primer. The primer sequences for IL-6 were sense: 5′-AGCCACTCACCTCTTCAGAACG-3′; and antisense: 5′-GGTTCAGGTTGTTTTCTGCCAG-3′; and for IL-8 were sense: 5′-CTTGGCAGCCTTCCTGATTTC-3′; and antisense: 5′-TTCTGTGTTGGCGCAGTGTG-3′. Reactions were carried out in duplicates in an ABI Prism 7000 sequence detector supplied with SDS 2.2 software (Applied Biosystems), using the following conditions: an initial activation step (2 min at 50°C), a single denaturation step (15 min at 95°C) followed by 40 cycles of 15 s at 95°C and 60 s at 60°C, and a final cycle of 15 s at 95°C, 30 s at 60°C, and 15 s at 95°C. Levels of gene expression in each sample were quantified applying the 2^-ΔΔCT method with glyceraldehyde-3-phosphate dehydrogenase as an endogenous control. Glyceraldehyde-3-phosphate dehydrogenase primers were identical with those reported before (27). For the detection of IL-1RI mRNA, cDNA from SZ95 sebocytes was amplified by established primers (28), using a single denaturation step of 5 min at 94°C followed by 35 cycles of 1 min at 94°C, 1 min of 60°C, 1 min of 72°C, and a final cycle of 5 min at 72°C, yielding a 340-bp product.

SZ95 sebocytes seeded into 3.5 cm Ø tissue culture dishes were serum deprived and treated with stimuli, as indicated above. After incubation for 24 h, culture supernatants were collected and centrifuged for removal of cell detritus. Aliquots were stored at −80°C until use. IL-6 and IL-8 protein levels were then determined using commercially available ELISAs (ImmunoTools and DPC Biermann, Bad Nauheim, Germany). Triplicate wells were used for each individual sample.

SZ95 sebocytes were seeded into 3.5-cm Ø tissue culture dishes at a density of 2 × 10⁵ cells per dish. After serum deprivation as outlined above, cells were washed three times with PBS, followed by simultaneous treatment with 5 μM 5-(and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) (Molecular Probes, Karlsruhe, Germany), IL-1β (1 ng/ml), or IL-1β plus KdPT (10⁻⁶ M or 10⁻⁸ M) for 20 min in PBS with glucose (5 mM) at 37°C in the dark. After treatment, cells were trypsinized, centrifuged, and resuspended in PBS with glucose (5 mM). Oxidative conversion of CM-H₂DCFDA to the fluorescent product in living cells was assessed by flow cytometry using a FACSCalibur (BD Biosciences, San Jose, CA), equipped with a 488-nm argon laser. A total of 1 × 10⁴ cells from each sample was acquired. CellQuest Pro software (BD Biosciences) was used for analyzing the data. The median of FL-1 channel of fluorescence was used as the parameter to evaluate the intracellular oxidative stress because it matches the maximal number of cells with the highest fluorescence.

Serum-deprived SZ95 sebocytes were stimulated with IL-1β (1 ng/ml) or IL-1β plus KdPT (10⁻⁶ M) for 15 min. Cells were then trypsinized and washed with ice-cold PBS. The resulting pellets were resuspended into ice-cold lysis buffer (10 mM HEPES, pH 7.3; 0.1 mM EDTA; 10 mM NaCl; 1 mM DTT; 5% glycerol; 50 mM NaF; 10 mM sodium molybdate) containing the Complete Protease Inhibitor Set (Roche, Mannheim, Germany), followed by spectrophotometric protein measurement. Identical amounts of protein (50 μg/lane) were resolved on 10% SDS-PAGE. The proteins were electrotransferred to nitrocellulose membranes. After blocking with 2% Blocking Reagent (Roche), membranes were incubated with a mAb against IκBα (1:500; IMGENEX, San Diego, CA) in 1% Blocking Reagent overnight at 4°C. After washing, membranes were incubated with an anti-mouse HRP-conjugated Ab (1:10,000 dilution) (Amersham Pharmacia, Piscataway, NJ) in 1% Blocking Reagent for 1 h at room temperature. Bound Abs were detected using the ECL Plus chemiluminescent reagent (Amersham-Pharmacia). Identical protein loading was confirmed by stripping the blot and reprobing it with an anti–α-tubulin Ab (Oncogene, San Diego, CA).

SZ95 sebocytes were seeded into eight-well chamber slides and were fixed with methanol for 30 min at −20°C. Nonspecific binding was blocked with 5% donkey serum for 1 h at room temperature. Cells were then incubated for 1 h with the rabbit polyclonal Ab against anti–NF-κB p65 (Santa Cruz Biotechnology, San Diego, CA) (1 μg/ml). Bound Abs were visualized with a donkey anti-rabbit (1:1500) Ab coupled to Texas Red (Dianova, Hamburg, Germany). For nuclear staining, 4′,6-diamidino-2-phenylindole (DAPI) (Fluka, Taufkirchen, Germany) was used. Cells were imaged and examined with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Red fluorescence was excited using the TX3 filter (530–585 nm); for DAPI staining an excitation wavelength of 365 nm was used and emission was measured at 615 nm.

SZ95 sebocytes were seeded into 10-cm Ø tissue culture dishes at a density of 5 × 10⁶ cells. Following deprivation, cells were stimulated for 15, 30, or 60 min with IL-1β (1 ng/ml), KdPT (10⁻⁶ M), or both agents. Cells were then washed with ice-cold PBS, scraped in low-salt buffer A consisting of 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and the Complete Protease Inhibitor Set, and then incubated on ice for 20 min. The lysates were subsequently passed 10 times through a 26G1/2-gauge needle. After centrifugation, nuclear pellets were resuspended in high-salt buffer C (20 mM HEPES, pH 7.9; 400 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1mM DTT; and protease inhibitors, as mentioned above), followed by intense shaking at 4°C for 30 min. Nuclear lysates were cleared by centrifugation and the protein content determined by the DC Protein Assay Kit (Bio-Rad, Hercules, CA). The NF-κB consensus oligonucleotide (Santa Cruz Biotechnology) was end labeled using [γ³²P]ATP and T4 polynucleotide kinase (Fermentas), followed by column purification (QIAquick Nucleotide Removal Kit, Qiagen). Binding reactions were carried out in a 20-μl volume of 4 mM HEPES (pH 7.5), 10 mM KCl, 0.5 mM MgCl₂, 4% (w/v) Ficoll, 0.2 mM DTT containing 14 μg protein extract, 2 μg poly [dI-dC], 2 μg BSA, and 60,000 cpm of ³²P-labeled NF-κB consensus oligonucleotide. Reaction samples were incubated for 20 min at room temperature. For supershift assays, EMSA samples were incubated with 2 μg of the respective supershift Ab recognizing p65 or p50 (Santa Cruz Biotechnology) for 20 min at room temperature prior to addition of ³²P-labeled NF-κB oligonucleotide. Reaction samples were separated on a 4.5% native PAGE at 150 V for 2.5 h and detected by autoradiography.

Competition binding experiments were performed in 96-well U-bottom microplates (BD Biosciences, Heidelberg, Germany), each well containing 100 μl murine B16-F1 or human SZ95 sebocyte cell suspensions adjusted to 4 × 10⁶ cells/ml. The binding medium consisted of MEM with Earle’s salts, 0.2% BSA, and 0.3 mM 1,10-phenanthroline. Triplicates of competitor peptide solution (50 μl), yielding a final concentration ranging from 1 × 10⁻⁴ to 1 × 10⁻¹² M, were added, followed by the addition of 50,000 cpm [¹²⁵I]NDP-α–MSH in 50 μl to each well (29). The incubation conditions were 15°C for 3 h for B16-F1 cells and 15°C for 3 h or 37°C for 2 h for SZ95 cells. The reaction was stopped by placing the plates on ice for 10 min. The cell-bound radioactivity was collected on filters (Unifilter-96 GF/B; Packard, Meriden, CT) by use of a cell harvester, and the radioactivity was counted on a TopCount scintillation counter (Packard). The IC₅₀ values were calculated with Prism software (GraphPad Software, San Diego).

The potential melanotropic in vitro effect of KdPT at concentrations ranging from 10⁻⁶ to 10⁻¹⁰ M versus NDP–α-MSH (10⁻⁸ M) as a positive control was assayed on B16-F1 melanoma cells seeded at a density of 2500 cells per well in 96-well tissue culture plates. The melanin content in the supernatants of the cells after 72 h was determined photometrically at 405 nm, as described previously (30).

Human rIL-1β (eBioscience, Frankfurt, Germany), 25 μg, was labeled with Alexa Fluor 488, using a commercially available protein labeling kit (Invitrogen, Darmstadt, Germany). SZ95 sebocytes were then seeded into 3.5-cm Ø tissue culture dishes at a density of 2 × 10⁵ cells per dish. After serum deprivation, as described above, cells were washed with PBS, trypsinized, centrifuged, and transferred to FACS tubes. For the labeling reaction, cells were incubated on ice for 15 min with Alexa Fluor 488-labeled IL-1β. KdPT (10⁻⁷ M) or unlabeled IL-1β (1 ng/ml) was preincubated for 15 min. Cells were finally washed twice with ice-cold PBS to remove unbound molecules. Cell-bound fluorochrome-labeled IL-1β was visualized by flow cytometry, using the FACSCalibur, as outlined above.

The crystal structures of the complex between human IL-1β and IL-1RI were obtained from the Protein Data Bank (accession code 11TB) (31). The resulting structures were analyzed by Deep View (Swiss Institute of Bioinformatics, Lausanne, Switzerland). Modeling and docking of the tripeptide KdPT to the receptor was followed by analysis of the resulting complex using HyperChem (Hypercube, Gainesville, FL). Figures are presented using both HyperChem and DeepView.

Real-time RT-PCR analysis, ELISAs, and melanin assays were performed in at least three independent experiments. Data were then calculated as means ± SD, followed by statistical analysis using the Student t test for unpaired experiments; p values < 0.05 were regarded as significant.

We first investigated the effect of KdPT on IL-1β–induced IL-6 and IL-8 mRNA expression by real-time RT-PCR. IL-1 via activating receptor subtype I (IL-1–RI) is a well-known inducer of both cytokines (10, 11). However, the impact of IL-1β on IL-6 and IL-8 expression in SZ95 sebocytes has not been examined in detail. IL-1β treatment for 8 h led to a dose-dependent increase in the mRNA amounts of both cytokines (Fig. 1A, 1B). For all subsequent functional studies with KdPT, a dose of IL-1β at 1 ng/ml was used.

Incubation of SZ95 sebocytes with various doses of KdPT (10⁻⁶ M–10⁻¹⁰ M) per se did not lead to any detectable changes in the relative mRNA amounts of both proinflammatory cytokines (Supplemental Fig. 1). In contrast, KdPT dose-dependently and significantly reduced IL-6 mRNA and IL-8 mRNA expression when coincubated with IL-1β (Fig. 1C, 1D). In fact, the tripeptide at 10⁻⁴, 10⁻⁶, and 10⁻⁸ M suppressed IL-1β–induced IL-6 and IL-8 mRNA expression almost to basal levels (calculated IC₅₀ for IL-6 mRNA suppression: ~10⁻⁹ M; for IL-8 mRNA suppression: <10⁻¹⁰ M). Dose-kinetic studies employing different IL-1β doses further revealed that the attenuating effect of KdPT depends on the concentration of coincubated IL-1β. Although, at IL-1β doses of 0.1 and 1 ng/ml KdPT at all tested concentrations significantly suppressed IL-6 and IL-8 mRNA expression, a suppressive effect in the presence of 10 ng/ml IL-1β could be achieved only at 10⁻⁴ or 10⁻⁶ M of the tripeptide (Table I).

The suppressive effect of KdPT on IL-6 and IL-8 mRNA expression in SZ95 sebocytes was subsequently confirmed at the protein level, using ELISA. As expected, stimulation with IL-1β resulted in a marked increase in the secreted amounts of IL-6 and IL-8. In accordance with the mRNA expression data, KdPT significantly and dose-dependently attenuated the inductive effect of IL-1β on the protein secretion of both cytokines (Fig. 1E, 1F).

To determine the molecular mechanism of the suppressive effect of KdPT on IL-1β–mediated cytokine expression in SZ95 sebocytes, we next analyzed the NF-κB pathway, which is well established to be activated by IL-1β.

First, SZ95 sebocytes were treated with IL-1β alone or in the presence of KdPT at different time points. Nuclear extracts were isolated, and NF-κB-DNA binding activity was assessed by EMSA. In control cells, low NF-κB binding activity was detected, which was strongly enhanced after treatment with IL-1β (1 ng/ml) for 15, 30, and 60 min (Fig. 2A). Supershift experiments employing anti-p65 and anti-p50 Abs disclosed that the IL-1β–induced NF-κB–DNA binding complexes consist of p65/p65 homodimers and p65/p50 heterodimers. In accordance with the lack of any effect on IL-6 and IL-8 expression, KdPT (10⁻⁶ M) alone did not elicit any detectable effect on NF-κB DNA binding. Cotreatment of cells with IL-1β plus KdPT, however, attenuated DNA binding of NF-κB, presumably owing to reduced generation of p65/p65 homodimers (Fig. 2A).

The KdPT-mediated attenuation of the NF-κB pathway induced by IL-1β was confirmed by immunofluorescence analysis of p65 in SZ95 sebocytes. In nontreated cells, p65 immunostaining excluded the nuclei in virtually all cells. Instead, a diffuse cytoplasmic and perinuclear staining was detectable (Fig. 2B, left panel). Stimulation with IL-1β (1 ng/ml) for 30 min resulted in a clear-cut change of p65 immunostaining from the cytoplasm to the nucleus in the majority of cells, as shown by double staining with an anti-p65 Ab and the nuclear tracker DAPI (Fig. 2B, middle panel). Costimulation with IL-1β plus KdPT (10⁻⁶ M), in contrast, reduced the number of SZ95 sebocytes with nuclear p65 staining, compared with cells exposed to IL-1β alone (Fig. 2B, right panel).

To further elucidate the molecular mechanism of KdPT-mediated attenuation of NF-κB activation by IL-1β, we finally performed Western immunoblotting of IκBα in cytoplasmic extracts of SZ95 sebocytes. NF-κB activation is triggered by phosphorylation of its inhibitor protein IκBα on Ser-32 and Ser-36 (32). As a result, IκBα disassociates from NF-κB in the cytoplasm and is degraded by the proteasome. As illustrated in Fig. 2C, treatment of SZ95 with IL-1β (1 ng/ml) for 15 min resulted in reduced protein amounts of IκBα, compared with unstimulated cells, indicating enhanced protein degradation. Although KdPT alone did not have any effect, cotreatment with IL-1β plus KdPT partially abrogated the cytokine-induced effect on IκBα degradation (Fig. 2C).

Because intracellular generation of reactive oxygen species (ROS) plays an important role in IL-1β–induced NF-κB activation (33), we hypothesized that KdPT may suppress IL-1β–mediated signaling by suppressing intracellular ROS generation. SZ95 sebocytes were thus loaded with the ROS-detecting fluoroprobe CM-H₂DCFDA and stimulated with IL-1β (1 ng/ml) alone or together with KdPT (10⁻⁶ M). As shown by FACS analysis, IL-1β stimulation led to a rightward shift in the mean fluorescence intensity, indicating increased amounts of intracellular ROS, compared with amounts in nontreated cells (Fig. 3A). KdPT alone did not have any effects (data not shown). In contrast, KdPT reduced IL-1β–induced ROS production, as demonstrated by a marked leftward shift in the mean fluorescence intensity, compared with IL-1β alone (Fig. 3A). In a series of three independent experiments, this KdPT-mediated decrease in IL-1β–induced intracellular ROS production was −30 ± 1.27% (p < 0.001).

To confirm the role of oxidative stress as an intracellular signaling event during IL-1β–mediated proinflammatory cytokine expression in SZ95 sebocytes, we pretreated the cells with the Mn complex of 7-hydroxy-flavone (MnCpx3). MnCpx3 was previously shown to exhibit potent superoxide dismutase activity and protection against lipid peroxidation in vitro (34). MnCpx3 significantly suppressed IL-1β–mediated mRNA expression of both IL-6 and IL-8 in SZ95 sebocytes (Fig. 3B, 3C).

In the next set of experiments, we determined whether KdPT mediates its biological effects via binding to MC-1Rs expressed by SZ95 sebocytes. In accordance with previous data (19), MC-1R mRNA was detectable by RT-PCR in these cells (data not shown). However, blocking experiments with a peptide analog of Agouti signaling protein (ASIP) acting as an MC1R/MC-4R antagonist (35, 36) did not result in abrogation of the suppressive effect of KdPT on IL-1β–induced IL-6 or IL-8 mRNA expression (Fig. 4A, 4B). To clearly exclude the involvement of an MC-1R–mediated action of KdPT in SZ95 sebocytes, radioligand binding assays with radio-labeled ¹²⁵I-NDP–α-MSH were then performed. SZ95 sebocytes failed to show any specific NDP-α–MSH radioligand binding, whereas B16-F1 melanoma cells used as positive control readily revealed specific binding of the tracer (Fig. 4C). These data indicate that in SZ95 sebocytes, binding sites for α-MSH are below the detection limit of classical radioligand binding assays and, more importantly, exclude any MC-R–mediated signaling of KdPT as an essential component of its biological effect. In accordance with the latter finding, KdPT—in contrast to α-MSH—failed to bind to B16-F1 murine melanoma cells, too (Fig. 4C). To finally complement the data of these binding assays with a more functional test, melanin production was determined in B16-F1 melanoma cells in response to KdPT and NDP-α–MSH. NDP-α–MSH (10⁻⁸ M) significantly increased the secreted amount of melanin into the culture supernatants of B16-F1 melanoma cells after 72 h of incubation, whereas KdPT at doses from 10⁻⁶ to 10⁻¹⁰ M did not elicit any melanotropic effect (Fig. 4D).

These findings demonstrate that KdPT acts independently of MC-Rs. Moreover, lack of a pigment-inducing effect represents important preclinical information for the therapeutic exploitation of this anti-inflammatory tripeptide.

Because the amino acid sequence of KPT is contained within the IL-1β (fragment 193–195), we next considered the possibility of KdPT binding to the signaling-sufficient IL-1RI. RT-PCR analysis disclosed the expression of IL-1RI in SZ95 sebocytes (Fig. 5A). However, subsequent radioligand binding studies using iodinated IL-1β did not yield specific binding in SZ95 sebocytes, possibly owing to changes in the binding behavior of the radiotracer by the incorporated [¹²⁵I] (data not shown). Thus, we performed an alternative approach in which human IL-1β was fluorochrome labeled in vitro with Alexa Fluor 488. SZ95 sebocytes were then incubated with the IL-1β–fluorochrome. Treatment of cells with the fluorochrome-tagged IL-1β resulted in surface labeling, which was reduced in the presence of non–fluorochrome-labeled IL-1β (1 ng/ml; reduction of fluorescence intensity versus control: −32.0 ± 14.4%). KdPT (10⁻⁷ M) similarly reduced cell surface binding of the fluorochrome-tagged IL-1β (reduction of fluorescence intensity versus control: −45.3 ± 13.3%) (Fig. 5B).

To gain a better understanding, we used computer modeling for a closer look into a possible binding of KdPT to IL-1RI. The crystal structures of the IL-1β/IL-1RI complex revealed that the amino acids in positions 193–195 of IL-1β do not interact with the receptor (Fig. 6A). We thus turned to previously reported site-directed mutation studies to investigate if any Lys, Pro, or Thr residues on IL-1β had been shown to be important or critical for receptor binding. Interestingly, Lys²⁰⁹ of IL-1β was found to be absolutely essential for activity, with mutants showing <1% of activity (37, 38). Examination of the crystal structure of the IL-1β/IL-1RI complex showed that the residue Lys²⁰⁹ interacted with Glu²⁵², which is found on a loop between two β strands (fragment 240–264) with the sequence ²⁴⁰IAYWKWNGSVIDE²⁵²DDPVLGEDYYSV²⁶⁴, which is itself located on the third Ig-like C2 domain of IL-1RI. This loop on the receptor is rich in acidic residues; in particular, there are 3 Asp and 1 Glu residues in a continuous sequence (residues 251–254). Therefore, models were constructed on the basis that Lys¹ in KdPT might enable strong binding via interaction with one or more of these acidic residues. In addition, the possibility was investigated that Thr³ in KdPT might also interact with residues on the above loop. In the final model, a complex between KdPT and IL-1RI was predicted in which the ε-amino group of Lys¹ side chain H-bonds to Glu²⁵² and Asp²⁵³, whereas the N-terminal amino group of Lys¹ binds also Glu²⁵² and to Asp²⁵¹. The C-terminal carboxylic acid group of Thr³ binds to Asn²⁴⁶ and Ser²⁴⁸. d-Pro² may form hydrophobic interactions with Val²⁴⁹ (Fig. 6B).

In this report, we have investigated anti-inflammatory potential and its underlying molecular mechanism in SZ95 sebocytes of KdPT, a tripeptide derivative of the C-terminal end of α-MSH. Our findings provide evidence for a significant suppressive effect by this small molecule in a wide range of concentrations (micromolar to subnanomolar) on IL-1β–induced IL-6 and IL-8 expression in the above in vitro cell culture model. Because there is no established animal model for acne, it is of some interest that KdPT, in fact, possesses anti-inflammatory effects in vivo [e.g., in mouse models of experimentally induced colitis (39)]. The latter finding may indicate that KdPT may possess anti-inflammatory effector mechanisms additional to antagonism of IL-1β at the IL-1R level. Interestingly, the KdPT-related α-MSH tripeptide homolog KPV was reported to exert its anti-inflammatory effects via PepT1, an H⁺-coupled oligopeptide transporter expressed in the gut (40). Thus, we cannot rule out that peptide transporters such as PepT1 may also be involved in the anti-inflammatory effect of KdPT.

As demonstrated by our mechanistic studies, KdPT attenuates the activation of NF-κB pathway, presumably by binding to IL-1RI and consequently by reduction of IL-1β–mediated intracellular ROS production. ROS formation is a well-known upstream signal mediator activating the redox-sensitive transcription factor NF-κB (33). The role of intracellular ROS as a mediator of IL-1β–mediated IL-6 and IL-8 expression could be confirmed by preincubating SZ95 sebocytes with MnCpx3. Likewise, the antioxidant N-acetylcysteine suppressed IL-1β–mediated IL-6 and IL-8 expression (M. Böhm and A. Kokot, unpublished observations).

Different enzymatic pathways may contribute to the upregulation of intracellular ROS production either directly or via side-chain reactions—for example, the expression of 5-lipoxygenase (5-LOX). 15-LOX-1 and cyclooxygenase-2 were detected in SZ95 sebocytes at the mRNA and protein level (41). Because 5-LOX has previously been shown to be the main source of ROS after IL-1β stimulation in lymphoid cells (42), it is likely that 5-LOX contributes to IL-1β–induced ROS generation in SZ95 sebocytes, too. Alternatively, IL-1β–activated ROS may be generated by distinct isoforms of NADPH oxidase. Only recently, IL-1β–mediated NF-κB activation in MCF7 epithelial cells was found to activate Nox2 (the phagocytic NADPH oxidase isoform that is also expressed by a number of nonphagocytic cell types), which, after recruitment to the endosomal compartment, generates ROS (43). However, as demonstrated by our NF-κB pathway studies, KdPT did only partially inhibit IL-1β–mediated NF-κB activation. The difference between the potent suppressive effect of KdPT on IL-1β–mediated IL-6 or IL-8 production and its less prominent impact on IL-1β–mediated-NF-κB activation may be related to the complex regulation of proinflammatory cytokine expression. NF-κB–independent mechanisms controlling the relative amounts of IL-8 transcripts do exist, such as via altered mRNA stability (44).

Regarding the potential adverse effects of KdPT, it was important to determine, whether KdPT binds MC-Rs, especially MC-1R. The data in this study provide clear evidence that KdPT does not bind MC-1R and does not require α-MSH binding sites for its anti-inflammatory action. KdPT lacked any melanotropic effect on B16-F1 melanoma cells, as shown by the blocking experiments with the MC-1R/4 antagonist ASIP and the results of the competitive NDP-α–MSH radioligand/KdPT binding assays. Moreover, KdPT failed to alter the amount of melanin in normal human melanocytes cultured under in vitro conditions (39). These findings are salutary with regard to the future exploitation of the peptide for treatment of patients with inflammatory diseases, including acne, because KdPT-induced cutaneous hyperpigmentation would be an unwanted adverse effect.

In summary, we have shown for the first time that KdPT has potent anti-inflammatory effects under in vitro conditions. The peptide acts antagonistically to IL-1β, presumably by binding to the signaling-sufficient IL-1RI. Consequently, IL-1β–mediated intracellular ROS formation, NF-κB activation, and IL-6 and IL-8 transcription are attenuated. The lack of melanotropic activity suggests that this peptide is a promising future candidate for the treatment of many inflammatory human diseases, not only those involving inflamed sebaceous glands.

We thank Britta Ringelkamp, Mara Apel, and Heidi Tanner for expert technical assistance and Claus Kerkhoff for critical reading of the manuscript.

Disclosures M.B. is an inventor of antioxidative tripeptides (EP070222401). C.C.Z. is an inventor of the SZ95 sebaceous gland cell line (AU770518B, CA2360762, CN100366735C, DE19903920, EP1151082, HU0200048, IL144683, JP2000-597413, KR689120, PL194865, US2002034820). All other authors have no financial conflicts of interest.