IL-1F6, IL-1F8, and IL-1F9 and the IL-1R6(RP2) receptor antagonist IL-1F5 constitute a novel IL-1 signaling system that is poorly characterized in skin. To further characterize these cytokines in healthy and inflamed skin, we studied their expression in healthy control, uninvolved psoriasis, and psoriasis plaque skin using quantitative RT-PCR and immunohistochemistry. Expression of IL-1F5, -1F6, -1F8, and -1F9 were increased 2 to 3 orders of magnitude in psoriasis plaque versus uninvolved psoriasis skin, which was supported immunohistologically. Moreover, treatment of psoriasis with etanercept led to significantly decreased IL-1F5, -1F6, -1F8, and -1F9 mRNAs, concomitant with clinical improvement. Similarly increased expression of IL-1F5, -1F6, -1F8, and -1F9 was seen in the involved skin of two mouse models of psoriasis. Suggestive of their importance in inflamed epithelia, IL-1α and TNF-α induced IL-1F5, -1F6, -1F8, and -1F9 transcript expression by normal human keratinocytes. Microarray analysis revealed that these cytokines induce the expression of antimicrobial peptides and matrix metalloproteinases by reconstituted human epidermis. In particular, IL-1F8 increased mRNA expression of human β-defensin (HBD)-2, HBD-3, and CAMP and protein secretion of HBD-2 and HBD-3. Collectively, our data suggest important roles for these novel cytokines in inflammatory skin diseases and identify these peptides as potential targets for antipsoriatic therapies.
The IL-1 family of cytokines is a set of structurally related molecules that are primary mediators of inflammation (1). This family now consists of 11 cytokines and 9 cell-surface receptors (reviewed in Refs. 2, 3), the best characterized of which are IL-1α, IL-1β, IL-1 receptor antagonist (IL-1ra), and IL-18. Seven novel members were recently identified from DNA databases almost simultaneously by several different groups (4–7), and to reduce confusion, a systematic nomenclature was proposed (8). IL-1F6 (9), IL-1F8 (10, 11), and IL-1F9 (10, 12) have all reported proinflammatory properties. IL-1F5 shares 52% amino acid similarity with IL-1ra (1) and as such antagonizes IL-1F6 (9) and IL-1F9 (12) at the IL-1R3(RAcP)–IL-1R6(Rrp2) receptor complex (10). IL-1F7 (6) acts as an attenuator of IL-18 activity (13) and may act as an inhibitor of innate immune signaling. IL-1F10 (14) has been reported to bind soluble IL-1R1 to unknown effect. Finally, IL-1F11 (IL-33) has been reported to be a Th2-inducing cytokine (15), having important roles in the stimulation of mast cells, eosinophils, basophils, and Th2 T cells, but also has a role in boosting IFN-γ production by NK cells (16).
Many of these cytokines are overexpressed in inflammatory disorders such as psoriasis (9, 12, 17, 18), which is a T cell-mediated skin disease characterized by red, scaly, and well-demarcated plaques on the skin, nail abnormalities, and, in some patients, a destructive arthritis (19). Importantly, overexpression of IL-1F6 in mouse basal keratinocytes leads to skin inflammation (9) that is exacerbated on an IL-1F5–deficient background, resulting in psoriasiform skin lesions. IL-1F5 (9), -1F6 (9, 12), and -1F9 (12) mRNA have been shown to be elevated in psoriasis plaques, and keratinocytes were identified as the predominant source (12). Taken together, these data indicate that IL-1F5, -1F6, -1F8, -1F9, and IL-1R6 constitute an distinct IL-1 signaling system analogous to IL-1α, -1β, -1ra, and IL-1R1 that is present and active in epithelia.
The regulation and function of these new IL-1 family members in the skin is poorly understood. To better define the role of this family in skin inflammation, we acquired biopsies of healthy control, uninvolved, and lesional psoriasis skin and assessed the expression of IL-1F5, -1F6, -1F8, -1F9, and their receptors by real-time quantitative RT-PCR (qRT-PCR), which we then confirmed by immunohistochemical evaluation. Next, we examined their mRNA expression levels in lesional skin during treatment with the anti–TNF-α biologic etanercept (Enbrel; Immunex). To gain further insight into the role of these cytokines in T cell-mediated skin inflammation, we assessed how their expression was modulated by the inflammatory cytokines IL-1α, TNF-α, IL-17A, and IL-22. IL-1F5 and -1F9 were efficiently induced by IL-1α, IL-17A, and TNF-α. However, although IL-1F9 mRNA and intracellular protein could be induced by these cytokines, secretion of IL-1F9 by keratinocytes required application of extracellular ATP. In terms of effector functions, these newly described cytokines induced the expression of a wide swath of epithelial defense proteins, with IL-1F8 being a particularly efficient inducer of human β-defensin (HBD) secretion. Taken together, our data suggest important roles for these novel cytokines in the pathogenesis of psoriasis and identify these peptides as potential targets for antipsoriatic therapies.
Materials and Methods
Twenty individuals with chronic plaque psoriasis and 20 normal controls were enrolled (age range 18–75 y). Entry criteria were the manifestation of one or more well-demarcated, scaly, erythematous psoriatic plaques that were not limited to the scalp and no systemic antipsoriatic treatments for 2 wk before biopsy. Biopsies sites varied between patients depending on site of active plaques, whereas biopsies of uninvolved and control skin were from the buttocks.
Biopsies were also obtained from uninvolved and lesional skin of individuals responding to treatment for chronic plaque psoriasis with the biologic agent etanercept (Enbrel; Immunex). Thirty subjects were enrolled based on criteria previously published (20). Inclusion criteria included age >18 y and stable plaque-type psoriasis involving at least 10% body surface area. Exclusion criteria included use of systemic psoriasis therapy within 4 wk, topical therapy within 2 wk, or severe comorbid diseases. For 12 wk, subjects received etanercept 50 mg twice a week s.c. (open label). At baseline, 6-mm punch biopsies were obtained under local anesthesia (lidocaine) from uninvolved skin and a target plaque. Subsequent biopsies were taken on days 1, 6, 14, 21, and 28 of therapy from the same target plaque.
Informed consent was obtained from all subjects under protocols approved by the Institutional Review Board of the University of Michigan. This study was conducted in compliance with good clinical practice and according to the Declaration of Helsinki Principles.
RPLP0. Mouse skin mRNA was extracted, reverse transcribed, and transcripts quantified as above using primers for murine IL-1 family genes (IL-1A Mm99999060_m1, IL-1F5 Mm00497802_m1, IL-1F6 Mm00457645_m1, IL-1F8 Mm01337545_m1, and IL-1F9 Mm00463327_m1) and 18S rRNA housekeeping gene (Hs99999901_s1). Data were tested for normality and statistical significance calculated using Student t test or Mann–Whitney U test as appropriate using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA).
n = 6.
Normal human keratinocyte (NHK) cultures were established from sun-protected adult human skin as described (212
Reconstituted human epidermal (RHE) cultures were obtained from MatTek (EPI-200; MatTek, Ashland, MA). After overnight equilibration in 5 ml normal maintenance medium (MatTek), medium was refreshed and supplemented with recombinant human cytokines as indicated. Medium was sampled at 24 and 48 h. In addition, 4-mm-diameter discs were punched from RHE cultures and formalin fixed, paraffin embedded, sectioned at 5 μm, and stained for HBD-2 expression (1:200 dilution, goat anti-BD-2; PeproTech, Rocky Hill, NJ) or IL-1F9 expression (10 μg/ml; Santa Cruz Biotechnology) and visualized with 3,3′-DAB (BD Pharmingen).
RNA processing and microarray hybridization
22) was used to process the raw data. Raw microarray data has been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and is accessible through Gene Expression Omnibus Series accession number GSE25400.
Estimation of HBD-2 was carried out as previously described (23
Expression of IL-1F5, -1F6, -1F8, and -1F9 is increased in plaque psoriasis skin
Given the proinflammatory nature of IL-1α, the identification of new members of the IL-1 family raised intriguing possibilities about their involvement in epithelial inflammation. Thus, we examined the expression of these IL-1 family cytokines and their receptors using real-time qRT-PCR on snap-frozen biopsies of healthy, nonlesional psoriasis, and PP skin from 20 volunteers.
The expression of several IL-1 family members was significantly increased in PP skin (Fig. 1). IL-1β (p < 0.001), IL-1F5 (p < 0.001), IL-1F6 (p < 0.001), IL-1F8 (p < 0.001), and IL-1F9 (p < 0.001) mRNA were all significantly increased compared with symptomless psoriasis skin and healthy control skin. As assessed in terms of fold change relative to its negligible expression in normal skin, IL-1F6 mRNA was upregulated to a much greater degree than its antagonist cytokine IL-1F5 (Fig. 1D, 1E). This upregulation in PP was also evident in terms of protein expression, in which IL-1F5 (Fig. 1H), -1F6 (Fig. 1I), -1F8 (Fig. 1J), and -1F9 (Fig. 1K) proteins were all upregulated in the keratinocytes of lesional skin compared with healthy control skin as assessed immunohistochemically. In addition, IL-1F5, -1F8, and -1F9 were also detected in the mononuclear cell infiltrate in lesional skin. We also assessed the expression of all IL-1 receptor transcripts in healthy, nonlesional, and plaque skin. Of note, IL-1R2 (the IL-1α/β decoy receptor) was significantly downregulated (p < 0.001) in plaque skin, as was IL-1R4 (p < 0.001), the receptor for IL-1F11 (IL-33), IL-1R8, and IL-1R9, for which ligands and functions in skin are currently unknown. However, neither IL-1R3 (IL-1RAcP) nor IL-1R6 (IL-1Rrp2) was differentially expressed in these tissues (Supplemental Fig. 1).
Biologic treatment of psoriasis decreases IL-1F5, -1F6, -1F8, and -1F9 expression in skin
Therapies targeting TNF-α such as etanercept, a human TNFR fusion protein, have been shown to have high efficacy in psoriasis (24). To appreciate how expression of the IL-1 family of cytokines is modulated during disease remission, we obtained skin biopsies from patients responding to etanercept therapy for chronic plaque psoriasis. Biopsies were taken on days 1, 6, 14, 21, and 28 of treatment and processed for mRNA analysis, revealing that etanercept treatment lead to significantly decreased expression of IL-1α (p < 0.001, ANOVA), IL-1β (p < 0.001), IL-1F5 (p < 0.01), -1F6 (p < 0.001), -1F8 (p < 0.001), and -1F9 (p < 0.001) (Fig. 2).
IL-1F5, -1F6, -1F8, and -1F9 are overexpressed in two mouse models of psoriasis
Given the dramatic overexpression of IL-1F5, -1F6, -1F8, and -1F9 in involved psoriasis skin, we went on to examine whether these cytokines were overexpressed in two established mouse models of psoriasis. We analyzed mRNA from lesional and unaffected control skin from the KC-Tie2 [Tg(KRT5-tTA)1Dmt × Tg(TetOS-Tek)1Dmt] transgenic model (25, 26) and the imiquimod-induced mouse model (27) of psoriasis (Fig. 3). In both of these mouse models, the inflamed skin showed striking and significant overexpression of IL-1α, IL-1F5, -1F6, -1F8, and -1F9 transcripts compared with skin from monotransgenic control littermates (Fig. 3A) or nonimiquimod-treated skin (Fig. 3B), respectively. Moreover, immunohistochemistry revealed robust expression of IL-1F6 in the lesional skin of KC-Tie2 mice (Fig. 3C) compared with monotransgenic littermates (Fig. 3D).
Psoriasis-associated cytokines induce IL-1F5, -1F8, and -1F9 expression by keratinocytes
To begin to understand the induction of IL-1 cytokines in inflamed skin, we treated normal NHK with the psoriasis-associated cytokines TNF-α, IL-1α, IL-17A, and IL-22. Stimulation of NHK with TNF-α dose-dependently induced IL-1F5, -1F8, and -1F9 mRNAs (Fig. 4A). IL-1α treatment led to dose-dependent increased expression of IL-1F5, -1F8, and -1F9 (Fig. 4B). IL-17A induced IL-1F6 and -1F9 expression (Fig. 4C, 4D), and although IL-22 alone was ineffective, it potentiated the effects of IL-17A for the induction of IL-1F6 (p < 0.05) and IL-1F9 (p < 0.001) (Fig. 4C, 4D).
The induction of IL-1F9 protein was confirmed in IL-1α– and TNF-α–treated RHE cultures (Fig. 4E). Individually, both IL-1α (Fig. 4F) and TNF-α (Fig. 4F) could induce keratinocyte expression of IL-1F9; however, a combination of the two cytokines was most effective (Fig. 4E, 4F). Although this stimulation was particularly efficient at inducing IL-1F9 protein expression in tissue as detected immunohistochemically, little secreted IL-1F9 was detected in conditioned culture medium. Given that like IL-1α, IL-1F5–IL-1F9 do not have leader sequences that target their secretion (7), and IL-1α is likely secreted by an ATP-dependent alternative pathway (28), we stimulated NHK with exogenous ATP for 2 h following 24 h of cytokine stimulation (Fig. 4C, 4F). Application of ATP markedly induced secretion of IL-1F9 from the keratinocyte cultures, as measured by ELISA of conditioned medium at 2 h (Fig. 4F).
IL-1F8 functions as an inducer of antimicrobial peptide and MMP expression by keratinocytes
Although induction of NF-κB activation by IL-1F6 (10), -1F8 (10), and -1F9 (10, 12Fig. 5. Subsequent qRT-PCR analysis confirmed that several antimicrobial peptides (AMP) are significantly induced by IL-1F8, including HBD-2 (DEFB4; Fig. 6A), lipocalin2 (Fig. 6C), HBD-3 (Fig. 6D), CAMP (Fig. 6F), elafin (Fig. 6G), serpinB1 (Fig. 6H), and IL-8 (Fig. 6I). IL-1F8–induced secretion of the defensins HBD-2 and HBD-3 into the conditioned cell-culture medium was detected by ELISA (Fig. 6B, 6E). The upregulation of HBD-2 protein expression by IL-1α– and IL-1F8–treated RHE cultures could also be detected immunohistochemically as shown in Fig. 6J–L. Collectively, these data illustrate the induction of AMP by IL-1F8.
Our microarray analysis also highlighted a number of MMP as targets of IL-1F8 and IL-1F9 treatment of RHE cultures (Fig. 5), which is of interest, as a number of MMPs are overexpressed in lesional psoriasis skin (29, 30). Using qRT-PCR, we demonstrate that IL-1F8 could significantly induce 3.6-fold and 2.6-fold increases in MMP9 and MMP19 mRNA, respectively (Fig. 7C, 7G), and IL-1F9 induced 2.8-fold and 2.9-fold increases in MMP10 and MMP19 transcript expression, respectively (Fig. 7E, 7G). Interestingly, MMP1 (18-fold, p < 0.05) and MMP9 (2.8-fold, p < 0.01) were significantly elevated in lesional psoriasis skin (Fig. 7).
Epidermal inflammation is driven by the coordinated action of a number of cytokines and growth factors, including the IL-1 family. With the discovery of IL-1F5 (4–7, 31), IL-1F6 (7), IL-1F8 (5, 7), and IL-1F9 (6), these cytokines, together with their receptors IL-1R6(RP2) and IL-1R3(RAcP), form a novel and distinct IL-1 signaling system. Because overexpression of IL-1F6 in the basal keratinocytes of an IL-1F5–deficient mouse led to psoriasiform hyperplasia (9), we proceeded to characterize this cytokine family in human psoriasis skin. Expression of IL-1F5, -1F6, -1F8, and -1F9 transcripts were increased 2 to 3 orders of magnitude in PP versus PN skin (Fig. 1), which was supported immunohistologically and is in accord with the earlier findings that IL-1β (32), IL-18 (33), IL-1F5 (17), IL-1F6 (9), and IL-1F9 (9, 17) are all markedly elevated in lesional psoriasis skin. IL-1α mRNA and protein have been previously shown to be decreased in psoriasis plaques (32), which is consistent with the data we present in this study (Fig. 1A). Interestingly, neither receptor for these cytokines was differentially expressed (IL-1R3 and IL-1R6; Supplemental Fig. 1). We also found significantly decreased expression of IL-1α, IL-1β, IL-1F5, IL-1F6, IL-1F8, and IL-1F9 mRNAs in skin during treatment of psoriasis with etanercept (Fig. 2), which is consistent the earlier findings that two other IL-1 family members, IL-1β (34) and IL-18 (35), are downregulated during effective treatment of psoriasis and with studies in a mouse-human psoriasis xenograft system that show markedly decreased IL-1F6 expression in the skin lesions during effective treatment with etanercept (36).
Given the marked expression of these cytokines in psoriasis lesions and that the overexpression of IL-1F6 leads to psoriasiform inflammation in the mouse (9), we examined two mouse models of psoriasis (Fig. 3) and determined that IL-1F5, -1F6, -1F8, and -1F9 were all dramatically elevated in the lesional skin of both KC-Tie2 and imiquimod-treated mice. We also demonstrate that IL-1F6 protein was robustly expressed in the lesional skin of KC-Tie2 mice (Fig. 3C). It is particularly striking that in the KC-Tie2 mice, IL-1F6 mRNA is expressed 25 times more abundantly than that of its receptor antagonist IL-1F5 (Fig. 3A).
Although IL-1F6, -1F8, and -1F9 have been shown to activate NF-κB (10, 12), JNK, and/or ERK1/2-mediated pathways in cell lines (10), no function has yet been assigned to these molecules in skin. Thus, we treated reconstituted epidermal cultures with rIL-1F6, -1F8, and -1F9 and performed microarray analysis. Strikingly, these cytokines induced a broad pattern of increased AMP, MMP, and growth factor expression (Fig. 5). In particular, IL-1F8 was effective at inducing keratinocytes to express HBD-2 and HBD-3, the secretion of which was readily detectable in conditioned culture medium (Fig. 6). This is an interesting observation in light of the fact that the mononuclear cell infiltrate in psoriasis plaques is positive for IL-1F6, -1F8, and -1F9 immunoreactivity (Fig. 1H–K) and that the expression of these cytokines can be induced in blood monocytes in culture (A. Johnston, unpublished observation) and thus may contribute to AMP and MMP expression in inflamed skin. In addition, the mutual induction of IL-1α, IL-1β, and IL-1F9 may act as a positive-feedback loop driving AMP expression by the epidermis. It is interesting to note that the increases in MMP expression in PP versus PN or healthy control skin tend to be larger than those induced by cytokine treatment of RHE cultures (Fig. 7). These differences may relate to the fact that RHE cultures continue to express the regenerative maturation program of epidermal differentiation that is also characteristic of psoriasis (37), though they are capable of reverting to a more normal differentiation profile after grafting to immunocompromised mice (38).
Decorin and betacellulin (BTC) were both consistently downregulated by the IL-1 family cytokines in our microarray experiments (Fig. 5), and both of these transcripts are heavily downregulated in lesional psoriasis skin (23, 39). BTC is the only epidermal growth factor receptor ligand that is decreased in psoriasis skin (23, 40). Its role in skin biology is largely unknown, although transgenic overexpression of the extracellular domain of BTC in the mouse leads to alterations in hair follicle development and cycling and increased angiogenesis at experimental wounding sites (41). Decorin has been reported to interact with TGF-β (42), and via this interaction, it is thought to participate in cell cycle control (43).
Whereas others have shown that IL-1F9 is induced in NHK by treatment with IL-1α and TNF-α (5), in this study, we demonstrate that treatment with exogenous ATP is required for secretion of IL-1F9 into the medium (Fig. 4E, 4F). The same phenomenon was recently described for IL-1F6 release from transfected murine bone marrow-derived macrophages (44). IL-1F6, -1F8, and -1F9, like IL-1α and -1β, lack signal sequences for conventional secretion (7) via the endoplasmic reticulum/Golgi pathway and are secreted via a nonclassical pathway involving multivesicular bodies and exosomes (28) in a process triggered by extracellular ATP. Interestingly, like IL-1α (45), one role of IL-1F5, IL-1F6, IL-1F8, and/or IL-1F9 may involve acting as a transcription factor and as such would not require secretion. To that end, both IL-1F7 (46) and IL-1F11 (IL-33) (47) translocate to the nucleus, where they function as a transcription factors.
We typically required microgram per milliliter quantities of IL-1F5, -1F6, -1F8, and -1F9 for in vitro activity, which is consistent with earlier findings (10, 12). Thus, we must consider the possibility that these cytokines require some form of posttranslational modification that is not included in the commercial recombinant preparations we have used, such as glycosylation (48), and/or trimming of N-terminal amino acid residues (36). It will be important to focus on understanding how these molecules might function intracellularly as transcription factors and to further characterize how they are processed within the cell for secretion, as both modes of action may be important in tissue homeostasis and defense.
IL-1 was the first cytokine discovered in skin (49), and with the discovery that IL-1 is crucial for T cell differentiation (50, 51), IL-1 immunobiology is undergoing a renaissance. In light of the current study, we now have a better understanding of the broader IL-1 family in the pathobiology of psoriasis. Further, given that we have shown that these new IL-1 family members are able to drive AMP expression in skin and generate a psoriasis-like gene expression profile, it is possible that the use of an IL-1R6 antagonist, analogous to IL-1ra/anakinra in the cryopyrin-associated periodic syndromes (52, 53) and rheumatoid arthritis (54), may prove to be therapeutically beneficial in psoriasis.
The authors have no financial conflicts of interest.
This work was supported in part by a National Psoriasis Foundation/USA Discovery grant (to A.J.), a Dermatology Foundation Research grant (to A.J.), the National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health (R01 AR 052889 to J.T.E.), and the American Skin Association and Dermatology Foundation (to J.E.G.). J.T.E. is supported by the Ann Arbor Veterans Affairs Hospital.
The sequences presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE25400.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- antimicrobial peptide
- human β-defensin
- IL-1 receptor antagonist
- keratinocyte chemoattractant
- matrix metalloproteinase
- normal human keratinocyte
- uninvolved psoriasis
- psoriasis plaque
- quantitative RT-PCR
- reconstituted human epidermis
- room temperature.
- Received October 5, 2010.
- Accepted December 13, 2010.