Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Nov 1.
Published in final edited form as: J Invest Dermatol. 2010 Jun 17;130(11):2654–2663. doi: 10.1038/jid.2010.166

Effective narrow-band ultraviolet B radiation therapy suppresses the IL-23/IL-17 axis in normalized psoriasis plaques

Leanne M Johnson-Huang 1, Mayte Suárez-Fariñas 1,2, Mary Sullivan-Whalen 1, Patricia Gilleaudeau 1, James G Krueger 1, Michelle A Lowes 1
PMCID: PMC2955161  NIHMSID: NIHMS214887  PMID: 20555351

Abstract

Narrow-band ultraviolet B radiation (NB-UVB) therapy offers a well-established treatment modality for psoriasis. However, despite the common use of this form of treatment, the mechanism of action of NB-UVB is not well understood. We studied a group of 14 patients with moderate-to-severe psoriasis treated with carefully titrated and monitored NB-UVB for 6 weeks. Lesional plaques were classified as normalized (n=8) or non-responsive (n=6) based on their histological improvement and normalization. We characterized lesional myeloid dendritic cells (DCs) and T cells and their inflammatory mediators using immunohistochemistry and real-time PCR. NB-UVB suppressed multiple parameters of the IL-23/IL-17 pathway in normalized plaques, but not in non-responsive plaques. NB-UVB decreased numbers of CD11c+ DCs, specifically CD1c−CD11c+ “inflammatory” DCs, and their products, IL-20, iNOS, IL-12/23p40 and IL-23p19. Furthermore, effective NB-UVB suppressed IL-17 and IL-22 mRNA, which strongly correlated with lesion resolution. Therefore, in addition to its known role in suppressing IFN-γ production, NB-UVB radiation therapy can also target the IL-17 pathway to resolve psoriatic inflammation.

INTRODUCTION

Psoriasis, a chronic, debilitating skin disease affecting millions of Americans, is considered one of the most prevalent immune-mediated inflammatory diseases (Lebwohl, 2003). It has now become clear that infiltrating immune cells play very important roles in disease pathogenesis. T cells and DCs are significantly increased in lesional skin (LS) compared to uninvolved, “non-lesional” skin (NL) (Chamian et al., 2005; Lowes et al., 2005a). Along with the resident dermal myeloid DCs present in normal skin, an additional population of “inflammatory” DCs is detected in lesional psoriatic skin (Lowes et al., 2005a; Zaba et al., 2009a). DCs play an integral role in shaping the CD4+ T cell response, and the increased frequencies of IFN-γ and IL-17/IL-22-producing T cells observed in psoriatic lesional skin may be due to production of IL-12 and IL-23 by inflammatory DCs, respectively (Guttman-Yassky et al., 2008; Lowes et al., 2008; Pene et al., 2008; Zaba et al., 2007b). While, IFN-γ and IL-17 induce chemokine expression and cellular recruitment to the skin, IL-22 leads to aberrant keratinocyte proliferation and epidermal hyperplasia (Boniface et al., 2005; Nograles et al., 2008; Sa et al., 2007). Thus, the development of psoriatic lesions is likely due to the coordinated efforts of several inflammatory cytokine pathways and treatments targeting multiple facets of pathogenesis are of great value.

Ultraviolet radiation has been an effective treatment for psoriasis, as well as other skin diseases, for 85 years, since it was first used as a daily broadband source (310–320nm) combined with topical tar, known as the “Goeckerman” regimen (Goeckerman, 1925). Over the last 30 years, the introduction of fluorescent bulbs with a limited spectrum of 311–312nm (NB-UVB) has marked an advance in phototherapy, as this represents the wavelength with the optimal “phototherapy index.” When compared to conventional broad-band UVB therapy, treatment with NB-UVB, has been found to have greater bioactivity (Walters et al., 1999). However, we still do not fully understand the mechanism of action of this remarkably effective therapy.

At the cellular level, NB-UVB therapy has many immunosuppressive effects. It leads to a reduction of T cells (Carrascosa et al., 2007; Erkin et al., 2007) by inducing apoptosis (Krueger et al., 1995; Ozawa et al., 1999). Keratinocyte apoptosis also occurs as a result of in vitro NB-UVB (Aufiero et al., 2006), but it is probably not contributing to disease resolution in vivo as it is not detected after treatment (Krueger et al., 1995). Additionally, some studies have found a decrease Langerhans cell numbers after therapy (Murphy et al., 1993; Tjioe et al., 2003), although others have not found a significant decrease (Carrascosa et al., 2007; Erkin et al., 2007). Nevertheless, it is clear that NB-UVB impairs in vitro antigen presentation by both murine DCs and human Langerhans cells, rendering them tolerogenic rather than stimulatory (Baadsgaard et al., 1990; Goettsch et al., 1998; Murphy et al., 1993; Noonan et al., 1988; Simon et al., 1991). Thus, NB-UVB can suppress a broad range of immune cells.

More recently, there have been efforts to understand the impact of NB-UVB on inflammatory cytokine production. NB-UVB specifically targets IFN-γ-producing Th1 cells as well as upstream cytokines, IL-12 and IL-23 (Piskin et al., 2003; Piskin et al., 2004b; Walters et al., 2003). NB-UVB also has suppressive effects on additional inflammatory mediators, including IL-18, IL-8, IL-1β, and IL-6 (Sigmundsdottir et al., 2005; Walters et al., 2003). However, while it is clear that NB-UVB can suppress inflammatory cytokines, it is still unknown if NB-UVB can similarly target pathogenic IL-17 production.

In this study, we sought to determine whether NB-UVB influences the IL-17/IL23 axis, which is considered a critical pathogenic pathway in psoriasis (Blauvelt, 2008; Di Cesare et al., 2009; Nestle et al., 2009). Psoriatic skin tissue was analyzed in index plaques demonstrating histological improvement after NB-UVB therapy (normalized) and those that did not respond (non-responsive) by real-time PCR and immunohistochemistry. We found that NB-UVB suppressed multiple parameters of the IL-23/IL-17 pathway in normalized plaques, but not in non-responsive plaques. Therefore, in addition to its known role in reducing IFN-γ production, NB-UVB therapy can also resolve psoriatic inflammation by targeting the IL-23/IL-17 axis.

RESULTS

Classification of response

In this study, 14 adult patients with moderate-to-severe psoriasis received a 6-week treatment regimen of regular, monitored NB-UVB radiation therapy. Psoriasis area and severity index (PASI) was decreased over 50% (PASI 50) in the vast majority of patients (93%), while 28% of patients achieved over 75% improvement (PASI 75) by week six of treatment (Table S1). Response of an index plaque to NB-UVB was further evaluated histologically by measuring epidermal thickness and keratinocyte differentiation by keratin 16 (K16) staining. Histological normalization in an index plaque does not always correlate with global clinical improvement as assessed by PASI. Plaques with epidermal thinning and normalization of K16 staining and mRNA expression by week 6 post-treatment were classified as normalized (Figure 1a, c–d), while non-responsive plaques retained a thickened epidermis and strong K16 staining and mRNA (Figure 1b, c–d). Of the 14 plaques, 8 were histologically classified as normalized, while 6 were considered non-responsive. Subsequent experiments presented here compare the outcome of NB-UVB therapy in both normalized and non-responsive plaques.

Figure 1. Classification of normalized and non-responsive plaques.

Figure 1

Open in a new tab

(a–b) Histological response was measured at baseline in non-lesional (NL) and lesional (LS) skin and skin 6 weeks post-treatment (week 6). Representative histology and immunohistochemistry showing hematoxylin and eosin (H&E) and K16 expression in (a) normalized plaques and (b) non-responsive plaques. Scale bar = 100μm. (c) Epidermal thickness of normalized (norm, black bars, n=7) and non-responsive plaques (non-resp, white bars, n=6) before and after NB-UVB therapy. Error bars represent the mean ± SEM. NL and wk6 values are compared to LS. (*) P<0.05, (***) P<0.001. (d) K16 mRNA expression normalized to hARP in both normalized (black bars) and non-responsive plaques (white bars). Error bars represent the mean ± SEM.

We also used multivariate μ scores (Wittkowski et al., 2004) to generate a “response score” for each plaque, taking into account both percent change in epidermal thickness and K16 mRNA. Plaques were ranked according to their response scores; the lower the response score, the better the improvement of the plaque. The 6 plaques classified as non-responsive had the highest response scores, while the remaining normalized plaques were ranked with lower response scores (Table S1). This analysis provided a non-biased, quantitative method to correlate response with the percent changes in gene expression or cell counts over the treatment period.

Inflammatory dendritic cells were decreased in normalized plaques

To begin to dissect the immune mechanism of disease control by NB-UVB therapy, immunohistochemistry for DC markers was performed on skin sections pre- and post-treatment. “Resident” myeloid DCs in normal dermis are identified by CD11c+ and CD1c (Zaba et al., 2009b). CD1c+ cells were not present in the epidermis. Dermal CD1c+ cells did not change in number during treatment with etanercept (Zaba et al., 2007a), and similarly did not change during the course of NB-UVB in normalized plaques (Figure 2a, c). LS skin of non-responsive plaques had a slight increase in CD1c+ cells over NL skin, although this result was not statistically significant.

Figure 2. Inflammatory myeloid DCs are reduced in normalized plaques.

Figure 2

Open in a new tab

(a, b) Representative immunohistochemistry of CD1c+ and CD11c+ cells. Bar, 100μm (c,d) Quantification of dermal CD1c+ or CD11c+ cells in normalized (black) and non-responsive plaques (white). Each circle represents a plaque. NL and wk6 are compared to LS. (**) P<0.01, (***) P<0.001. (e) CD11c+CD1c− cell numbers were calculated by subtracting CD1c+ counts from CD11c+ counts. (*) P<0.05. (f) Two-color immunofluorescence of CD11c (green) and CD1c (red) of normalized plaque. Cells co-expressing both markers appear yellow due to the superimposition of both green and red signals. Inset, high power magnification of double positive cells. The white line delineates the dermo-epidermal junction. Scale bar = 100μm.

In psoriasis lesions, we have previously found a dramatic increase in a second population of myeloid “inflammatory” DCs (Lowes et al., 2005a; Zaba et al., 2009a), that express CD11c, but not CD1c. Treatment of psoriasis with efalizumab, alefacept, cyclosporine or etanercept decreased these inflammatory myeloid DCs (Chamian et al., 2005; Haider et al., 2008; Lowes et al., 2005b; Zaba et al., 2007a). The effect of NB-UVB on inflammatory DCs was assessed by immunohistochemistry. While fewer dermal CD11c+ DCs were seen in NL skin compared to LS (Figure 2b, d), by week 6 of NB-UVB, dermal CD11c+ DCs were significantly reduced in normalized plaques, but not non-responsive plaques. Similar results were seen in the epidermis (Figure S1a).

Currently, there is no positive marker to identify the inflammatory DCs. However, an approximation of the number of CD11c+CD1c− inflammatory DCs can be calculated by subtracting the number of CD1c+ cells from CD11c+ cells. Normalized plaques had decreased dermal CD11c+CD1c− inflammatory DCs, while non-responsive plaques retained a population of these cells (Figure 2e). To assess the relationship between response and the down-regulation of these cell subsets, we used multivariate μ scores as described above. There was a strong correlation between epidermal CD11c+ cells and response score (r=0.863, p =0.002; Figure S1b). Similarly, dermal CD11c+ DCs correlated significantly with response (r=0.675, p=0.015), while dermal CD1c+ cells did not (r=0.255, p=0.362; Figure S2). Moreover, inflammatory DCs also had a significant correlation with response score (r=0.664, p=0.017; Figure S2). To further characterize these DCs, two-color immunofluorescence was performed using antibodies against CD11c and CD1c. The majority of cells in NL skin co-stained for these two markers (Figure S3), while in LS skin, there was a higher proportion of cells staining for CD11c, but not CD1c (Figure 2f). After 6wk of NB-UVB, most cells were CD11c+CD1c+ in normalized plaques, suggesting a decrease in inflammatory DCs with effective treatment. Non-responsive plaques maintain a population of CD11c+CD1c− DCs after treatment (data not shown). These data support our concept that inflammatory DCs may be an important pathogenic cell population in psoriasis.

Decreased dendritic cell cytokines in normalized plaques

We have previously demonstrated that inflammatory myeloid DCs produce TNF, iNOS, IL-20, and IL-23 (Guttman-Yassky et al., 2008; Lowes et al., 2005a; Zaba et al., 2007a). In order to evaluate the expression of these cytokines during NB-UVB treatment, we performed quantitative real-time PCR on pre- and post-treatment skin RNA. There was significantly increased iNOS, IL-20, IL-12/23p40 and IL-23p19 in LS tissue compared to NL baseline skin (p<0.05 for most comparisons; Figure 3a). However, significant decreases were only observed in plaques that normalized after NB-UVB treatment (p<0.001 for all groups). In contrast, expression of these cytokines remained high in non-responsive plaques. Response scores significantly correlated with IL-23p19 mRNA expression (r=0.743, p=0.008) and IL-20 (r=0.88, p=0.002). We further confirmed the expression of IL-23 subunits at the protein level by performing two-color immunofluorescence with CD11c versus p40 or p19. Little p40 and p19 protein was seen in NL skin (Figure S4). LS skin demonstrated CD11c+ cells that co-stained for both IL-23 subunits, and by wk6 there was a reduction in both CD11c+ cells and the IL-23 subunits in normalized plaques (Figure 3b–c). Some cells expressed p40 or p19, but did not co-stain with CD11c, and are most likely macrophages (unpublished data). Furthermore, in non-responsive plaques, p19 and p40 protein expression was not reduced by NB-UVB at wk6 (Figure S5). These findings indicate that after NB-UVB therapy, normalization of psoriasis lesions is associated with a reduction in these inflammatory mediators, further implicating their potential role in psoriasis (summarized in Figure S6).

Figure 3. Decreased DC products in normalized plaques.

Figure 3

Open in a new tab

(a) mRNA expression levels normalized to hARP for the inflammatory DC products, iNOS, IL-20, IL-12/23p40, and IL-23p19 in both normalized (black bars, n=8) and non-responsive plaques (white bars, n=6). Error bars represent the mean ± SEM. NL and wk6 levels are compared to LS. (*) P<0.05, (**) P<0.01, (***) P<0.001. (b–c) Two-color immunofluorescence of CD11c+ myeloid DCs (red) with IL-23 subunits in a normalized plaque, (b) p40 and (c) p19 (green), demonstrating coexpression in baseline LS skin (yellow cells), which is diminished by week 6 post-therapy. Antibodies conjugated with a fluorochrome gave background epidermal fluorescence. The white line delineates the dermal epidermal junction. Inset, high power magnification of double positive cells. Scale bar = 100μm.

Decreased T cells in normalized plaques

We have previously shown that NB-UVB therapy effectively depletes T cells by inducing apoptosis (Ozawa et al., 1999). We confirmed this finding showing a significant increase in LS CD3+ T cells compared to NL skin in both dermis (Figure 4a–b) and epidermis (Figure S1c) that was reduced following NB-UVB (p<0.01). However, we extend this observation to show that a significant reduction in CD3+ T cells only occurs in plaques responding to NB-UVB (p<0.001; Figure 4b). To access the biological significance of the varying degrees of CD3+ T cell depletion with treatment, we correlated CD3+ T cells with response scores. There was a strong, statistically significant correlation between dermal CD3+ T cells and response scores (r=0.791, p=0.004; Figure 4c), as well as epidermal CD3+ T cells versus response scores (r=0.708, p=0.011; Figure S1d). These results indicate that a positive response to NB-UVB is associated with a profound decrease in T cells, in addition to the myeloid inflammatory DCs discussed above.

Figure 4. T cells are reduced in normalized plaques.

Figure 4

Open in a new tab

(a) Representative immunohistochemistry of CD3+ cells in NL, LS, and wk6 skin of a normalized plaque. Scale bar = 100μm. (b) Quantification of CD3+ T cell counts per mm of skin in both normalized (black circles) and non-responsive plaques (white circles). NL and wk6 counts are compared to LS. (**) P<0.01, (***) P<0.001. (c) Response scores were correlated with μ-scores for CD3+ cell counts. Spearman correlation coefficients (r) and p values are shown.

IL-17 and IL-22 were reduced in normalized plaques

Recent studies have implicated IL-17 and IL-22 in psoriatic inflammation (Harper et al., 2009; Lowes et al., 2008). We performed real-time PCR of skin tissue RNA in order to evaluate pathogenic cytokine expression. We confirmed that IL-17, IFN-γ and IL-22 were increased in LS tissue compared to NL tissue, and there was a significant reduction in these cytokines in normalized plaques (p<0.001 for all; Figure 5a). In contrast, there was no reduction in these three cytokines in non-responsive plaques at wk6. We also found increased LS expression of β-defensin-4 (p<0.01) and myxovirus resistance-1 (p<0.01), down-stream target genes of IL-17 and IFN-γ respectively, which decreased with NB-UVB treatment only in normalized plaques (data not shown). When cytokine mRNA was correlated with response scores, a significant correlation was found with IL-22 (r=0.932, p=0.001) and IL-17 (r=0.868; p=0.002; Figure 5b). On the other hand, there was no correlation between IFNγ mRNA expression and response score (r=0.214, p=0.445), which may indicate that expression of IFN-γ mRNA is not necessarily associated with lesion resolution in response to NB-UVB. These cytokine data support earlier studies showing the importance of IL-17 and IL-22 in psoriatic inflammation, but to our knowledge, it is previously unreported that suppressing this axis is associated with histological normalization following NB-UVB.

Figure 5. Decreased IL-17 and IL-22 following therapy.

Figure 5

Open in a new tab

(a) mRNA expression levels normalized to hARP for the T cell products, IL-17, IFN-γ, and IL-22 in both normalized (black bars, n=8) and non-responsive plaues (white bars, n=6) in NL, LS, and wk6 skin. Error bars represent the mean ± SEM. NL and wk6 levels are compared to LS. (**) P<0.01, (***) P<0.001. (b) Response scores were correlated with μ-scores for mRNA expression. Spearman correlation coefficients (r) and p values are shown.

It is difficult to ascertain whether cytokine-related changes seen in the skin at the end of therapy are a direct result of NB-UVB radiation or simply a reflection of the improvement of psoriasis in general. Therefore, using intracellular cytokine staining techniques, we examined the ability of a single irradiation of 312nm NB-UVB to modulate cytokine expression in vitro. As serial shave biopsies are difficult to obtain for isolation of lesional T cells, we used peripheral blood mononuclear cells (PBMCs) as a surrogate source of T cells. PBMCs of normal volunteers were irradiated with different doses (25 or 50mJ) of NB-UVB, and cytokine synthesis was measured after a 4hr stimulation. As previously seen, IFN-γ was decreased by an average of 85% (Figure S7a). Furthermore, CD3+ T cell production of IL-22 and IL-17 was significantly decreased after NB-UVB by an average of 45% and 89%, respectively (Figure S7b). These data suggest that NB-UVB directly modulates production of these pathogenic cytokines. Taken together, our results indicate that NB-UVB radiation can target the IL-23/IL-17 pathway to resolve inflammation.

DISCUSSION

In this paper, we have focused on the effects of NB-UVB at the cellular and cytokine/inflammatory mediator levels to define a set of outcomes that were necessary for histological normalization after NB-UVB. Specifically, for lesion resolution there must be a reduction in myeloid inflammatory DCs and T cells and inflammatory products (iNOS, IL-23, IL-20, IFN-γ, IL-17, IL-22). Prior studies characterizing the effects of NB-UVB in psoriasis have shown a down-regulation of IFN-γ (Piskin et al., 2003; Piskin et al., 2004a; Piskin et al., 2004b), but we now show there is a greater correlation between decreased IL-22/IL-17 expression and histological improvement. We have previously shown that this general set of cell and cytokine alterations is also required for response to other therapies for psoriasis, including alefacept, efalizumab, etanercept and cyclosporine (Chamian et al., 2005; Haider et al., 2008; Lowes et al., 2005b; Zaba et al., 2007a). Despite the fact that all of these agents work in such different ways—targeting CD2, CD11a, TNF, and T cells respectively—their common feature is that they are all immunosuppressive. However, it is still unclear whether suppressing the IL-23/IL-17 axis is simply due to the depletion of immune cells by a particular therapy, or if the therapy itself can specifically inhibit cytokine production. Previous studies using flow cytometry have demonstrated that production of IFN-γ by dermal T cells is decreased after UVB irradiation, indicating that UVB can directly inhibit cytokine production by T cells (Piskin et al., 2003). Here, we also report that, in vitro, NB-UVB can directly suppress T cell production of IL-17 and IL-22.

Some studies have suggested that the clearing of the inflammatory cells is specifically related to how far the ultraviolet radiation can penetrate (Bruls et al., 1984; Krueger et al., 1995). For example, PUVA therapy, which uses psoralen and UVA radiation, penetrates much deeper than UVB radiation and is an even more effective therapeutic agent for psoriasis (Mahmoud et al., 2008; Yones et al., 2006), although it is no longer a preferred method of treatment as it appears to increase the risk of skin cancer (Patel et al., 2009). At the cellular level, sufficient penetration of NB-UVB into the upper dermis results in the clearing of myeloid DCs and T cells, which appears necessary for reduction of inflammatory cytokine production. Furthermore, previous studies have suggested that the dermal T cells that remain after NB-UVB produce less inflammatory cytokines, such as IFN-γ (Piskin et al., 2004a). Although we did not directly assess IL-17 and IL-22 production by lesional T cells, our in vitro results suggest that NB-UVB can also alter production of pro-inflammatory IL-17 and IL-22.

While some studies have shown that NB-UVB therapy can deplete CD1a+ Langerhans cells (Murphy et al., 1993; Tjioe et al., 2003), to our knowledge, it is previously unreported that it can also deplete myeloid CD11c+ DCs, both in the epidermis and the dermis. Non-responsive plaques retained a population of CD11c+ CD1c− DCs and concurrent mRNA expression of inflammatory DC products, indicating that depletion of these cells is necessary for resolution of psoriatic lesions. Since DCs are the bridge between the innate and adaptive immune responses driving subsequent T cell responses, it seems logical that they need to be eliminated first, in order to prevent further T cell activation.

There have been some concerns stemming from data in animal models that targeting IL-12 and IL-23 with monoclonal antibodies will potentially increase overall cancer risk (Maeda et al., 2006). In humans, systemic immunosuppression clearly increases the risk of skin carcinomas and alters their aggressiveness (Berg and Otley, 2002). Hence, it is important to establish the potential relationship between immunosuppression in human skin and associated cancer risk. Suppression of p40 cytokine synthesis in the skin during NB-UVB raises the same potential concerns as blocking p40 cytokines using monoclonal antibodies. Recent studies have shown that careful, long-term administration of NB-UVB is not associated with increased risk in any skin cancers, including basal and squamous cell carcinomas and melanoma (Hearn et al., 2008; Patel et al., 2009; Weischer et al., 2004). Neither is there any reported risk of internal cancer from NB-UVB therapy. Thus, blocking the IL-23/IL-17 pathway in the skin with NB-UVB appears to be relatively safe and non-carcinogenic, suggesting that targeting this axis per se does not lead to cutaneous cancer. Another factor to consider is IL-22, as it has been shown to promote some cancers, probably due to its trophic effects on epithelia (Bard et al., 2008; Zhang et al., 2008). This pro-carcinogenic factor is also reduced by UVB therapy

In conclusion, this study clearly demonstrates the set of changes that are necessary for resolution of psoriasis lesions: a reduction in myeloid inflammatory DCs and their products (iNOS, IL-23, IL-20) associated with a decrease in T cells and additional pathogenic cytokines (IL-17, IFN-γ, IL-22). These changes were not seen in non-responsive plaques. These results establish a group of cells and products that must be effectively targeted for clearing of psoriasis, and confirm the IL-23/IL-17 pathway as an essential therapeutic target.

MATERIALS AND METHODS

Study design and skin biopsies

We conducted a therapeutic clinical trial under a Rockefeller University IRB-approved protocol in which 14 psoriasis patients received treatment with regular, monitored NB-UVB radiation therapy (Table S1). This trial is registered at clinicaltrials.gov under NCT00220025. Informed consent was obtained and the study was performed in adherence with the Declaration of Helsinki Principles. Patients were initially treated with 50% of their minimal erythemal dose, with increments of 5–10% 3–4 times per week, for up to 6 weeks. 6mm diameter skin punch biopsies from NL skin were taken prior to treatment as a baseline; pre- and post-treatment lesional biopsies were taken from an index plaque. Each biopsy was cut in two: half was stored in OCT for cryosections, and half snap-frozen in liquid nitrogen for RNA extractions. Skin tissue from one plaque (normalized) was only used for RNA.

Antibodies

All antibodies used for immunohistochemistry, immunofluorescence, and flow cytometry are listed in Tables S2, S3 and S4.

Immunohistochemistry

Skin sections were stained as previously described (Zaba et al., 2007b). Positive cells per millimeter were counted manually using computer-assisted image analysis software (ImageJ 1.42, National Institutes of Health).

Immunofluorescence

Skin sections (n=3 for normalized and non-responsive plaques) were stained as previously described (Zaba et al., 2007b). Images were acquired using appropriate filters of a Zeiss Axioplan 2I microscope with Plan Apochromat 20 × 0.7 numerical aperture lens and a Hagamatsu orca ER-cooled charge-coupled device camera, controlled by METAVUE software (MDS Analytical Technologies). Dermal collagen fibers gave green autofluorescence. Antibodies conjugated with a fluorochrome often gave background epidermal fluorescence. Cells co-expressing both markers appear yellow due to the superimposition of both green and red signals. The white line delineates the dermal epidermal junction.

mRNA extraction and real-time PCR

RNA extraction and real-time PCR using Taqman gene expression assays (Table S5) were performed as previously described (Chamian et al., 2005). Custom primers for IFN-γ, K16, and IL-12/23p40 were generated as previously described (Chamian et al., 2005). Data normalized to hARP housekeeping gene were quantified by software provided with Applied Biosystems PRISM 7700 (Sequence Detection Systems, version 1.7). The normalized PCR data were log2 transformed before statistical analysis.

In vitro NB-UVB irradiation and flow cytometry

PBMCs were prepared from heparinized venous blood of healthy volunteers by Ficoll sedimentation (n=3). Cells (106 cells/mL) were irradiated with NB-UVB (TL-01) at varying doses (25mJ/cm2 and 50mJ/cm2) in uncovered tissue culture plates in phosphate-buffered saline, then stimulated for 4 hours with phorbol myristate acetate (25 ng/mL), ionomycin (1 μg/mL), and brefeldin A (10 μg/mL) (all from Sigma-Aldrich Corp, St Louis, MO). Unactivated controls were treated with brefeldin A only. Cells were stained as previously described (Zaba et al., 2009a). Samples were acquired by an LSR-II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Treestar, Ashland, OR).

Statistical analysis

A repeated measures ANOVA with Tukey’s multiple comparison test was used to compare RT-PCR data and cell counts from LS skin versus respective NL skin or post-treatment skin pairs. A p-value less than 0.05 was considered significant.

Generation of response score

The percent change of each gene or leukocyte count was calculated using the following formula: ((wk6-LS)/(LS-NL)*100). Multivariate μ scores (Wittkowski et al., 2004) were then used to produce an overall histological response score (“response score”) for each plaque, combining the percent change in ET and K16 mRNA. We have used this classification method previously (Haider et al., 2008). The lower the response score, the better the improvement of the index plaque. μ scores were also calculated for the percent change in expression for each gene or cell count (e.g. IL-17 score) and were then correlated with response score by Spearman-type correlation coefficients (r).

Supplementary Material

Acknowledgments

Research was supported by a Clinical and Translational Science Award grant UL 1RR024143; the Doris Duke Foundation supported LMJ-H and MAL; MAL is also supported by NIH K23 AR052404. We thank Dr. A. North for her guidance with immunofluorescent images.

Abbreviations

DCs

dendritic cells

LS

lesional

NL

non-lesional

NB-UVB

narrow-band ultraviolet B

K16

keratin 16

BDCA-1

blood dendritic cell antigen-1

iNOS

inducible nitric oxide synthase

ET

epidermal thickness

PBMCs

peripheral blood mononuclear cells

Footnotes

Conflict of interest

The authors do not have any conflict of interest.

References

  1. Aufiero BM, Talwar H, Young C, Krishnan M, Hatfield JS, Lee HK, et al. Narrow-band UVB induces apoptosis in human keratinocytes. J Photochem Photobiol B. 2006;82:132–139. doi: 10.1016/j.jphotobiol.2005.08.011. [DOI] [PubMed] [Google Scholar]
  2. Baadsgaard O, Salvo B, Mannie A, Dass B, Fox DA, Cooper KD. In vivo ultraviolet-exposed human epidermal cells activate T suppressor cell pathways that involve CD4+CD45RA+ suppressor-inducer T cells. J Immunol. 1990;145:2854–2861. [PubMed] [Google Scholar]
  3. Bard JD, Gelebart P, Anand M, Amin HM, Lai R. Aberrant expression of IL-22 receptor 1 and autocrine IL-22 stimulation contribute to tumorigenicity in ALK+ anaplastic large cell lymphoma. Leukemia. 2008;22:1595–1603. doi: 10.1038/leu.2008.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berg D, Otley CC. Skin cancer in organ transplant recipients: Epidemiology, pathogenesis, and management. J Am Acad Dermatol. 2002;47:1–17. doi: 10.1067/mjd.2002.125579. quiz 18–20. [DOI] [PubMed] [Google Scholar]
  5. Blauvelt A. T-helper 17 cells in psoriatic plaques and additional genetic links between IL-23 and psoriasis. J Invest Dermatol. 2008;128:1064–1067. doi: 10.1038/jid.2008.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, Morel F. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. J Immunol. 2005;174:3695–3702. doi: 10.4049/jimmunol.174.6.3695. [DOI] [PubMed] [Google Scholar]
  7. Bruls WA, Slaper H, van der Leun JC, Berrens L. Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol. 1984;40:485–494. doi: 10.1111/j.1751-1097.1984.tb04622.x. [DOI] [PubMed] [Google Scholar]
  8. Carrascosa JM, Tapia G, Bielsa I, Fuente MJ, Ferrandiz C. Effects of narrowband UV-B on pharmacodynamic markers of response to therapy: an immunohistochemical study over sequential samples. J Cutan Pathol. 2007;34:769–776. doi: 10.1111/j.1600-0560.2006.00694.x. [DOI] [PubMed] [Google Scholar]
  9. Chamian F, Lowes MA, Lin SL, Lee E, Kikuchi T, Gilleaudeau P, et al. Alefacept reduces infiltrating T cells, activated dendritic cells, and inflammatory genes in psoriasis vulgaris. Proc Natl Acad Sci U S A. 2005;102:2075–2080. doi: 10.1073/pnas.0409569102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Di Cesare A, Di Meglio P, Nestle FO. The IL-23/Th17 axis in the immunopathogenesis of psoriasis. J Invest Dermatol. 2009;129:1339–1350. doi: 10.1038/jid.2009.59. [DOI] [PubMed] [Google Scholar]
  11. Erkin G, Ugur Y, Gurer CK, Asan E, Korkusuz P, Sahin S, et al. Effect of PUVA, narrow-band UVB and cyclosporin on inflammatory cells of the psoriatic plaque. J Cutan Pathol. 2007;34:213–219. doi: 10.1111/j.1600-0560.2006.00591.x. [DOI] [PubMed] [Google Scholar]
  12. Goeckerman WH. Treatment of psoriasis. North West Med. 1925;24:229–231. [Google Scholar]
  13. Goettsch W, Hurks HM, Garssen J, Mommaas AM, Slob W, Hoekman J, et al. Comparative immunotoxicology of ultraviolet B exposure I. Effects of in vitro and in situ ultraviolet B exposure on the functional activity and morphology of Langerhans cells in the skin of different species. Br J Dermatol. 1998;139:230–238. doi: 10.1046/j.1365-2133.1998.02359.x. [DOI] [PubMed] [Google Scholar]
  14. Guttman-Yassky E, Lowes MA, Fuentes-Duculan J, Zaba LC, Cardinale I, Nograles KE, et al. Low expression of the IL-23/Th17 pathway in atopic dermatitis compared to psoriasis. J Immunol. 2008;181:7420–7427. doi: 10.4049/jimmunol.181.10.7420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haider AS, Lowes MA, Suarez-Farinas M, Zaba LC, Cardinale I, Khatcherian A, et al. Identification of Cellular Pathways of “Type 1,” Th17 T Cells, and TNF- and Inducible Nitric Oxide Synthase-Producing Dendritic Cells in Autoimmune Inflammation through Pharmacogenomic Study of Cyclosporine A in Psoriasis. J Immunol. 2008;180:1913–1920. doi: 10.4049/jimmunol.180.3.1913. [DOI] [PubMed] [Google Scholar]
  16. Harper EG, Guo C, Rizzo H, Lillis JV, Kurtz SE, Skorcheva I, et al. Th17 Cytokines Stimulate CCL20 Expression in Keratinocytes In Vitro and In Vivo: Implications for Psoriasis Pathogenesis. J Invest Dermatol. 2009 doi: 10.1038/jid.2009.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hearn RM, Kerr AC, Rahim KF, Ferguson J, Dawe RS. Incidence of skin cancers in 3867 patients treated with narrow-band ultraviolet B phototherapy. Br J Dermatol. 2008;159:931–935. doi: 10.1111/j.1365-2133.2008.08776.x. [DOI] [PubMed] [Google Scholar]
  18. Krueger JG, Wolfe JT, Nabeya RT, Vallat VP, Gilleaudeau P, Heftler NS, et al. Successful ultraviolet B treatment of psoriasis is accompanied by a reversal of keratinocyte pathology and by selective depletion of intraepidermal T cells. J Exp Med. 1995;182:2057–2068. doi: 10.1084/jem.182.6.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lebwohl M. Psoriasis. Lancet. 2003;361:1197–1204. doi: 10.1016/S0140-6736(03)12954-6. [DOI] [PubMed] [Google Scholar]
  20. Lowes M, Chamian F, Abello MV, Fuentes-Duculan J, Lin SL, Nussbaum R, et al. Increase in TNF-alpha and inducible nitric oxide synthase-expressing dendritic cells in psoriasis and reduction with efalizumab (anti-CD11a) Proc Natl Acad Sci USA. 2005a;102:19057–19062. doi: 10.1073/pnas.0509736102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lowes MA, Kikuchi T, Fuentes-Duculan J, Cardinale I, Zaba LC, Haider AS, et al. Psoriasis Vulgaris Lesions Contain Discrete Populations of Th1 and Th17 T Cells. J Invest Dermatol. 2008;128:1207–1211. doi: 10.1038/sj.jid.5701213. [DOI] [PubMed] [Google Scholar]
  22. Lowes MA, Turton JA, Krueger JG, Barnetson RS. Psoriasis vulgaris flare during efalizumab therapy does not preclude future use: a case series. BMC Dermatol. 2005b;5:9. doi: 10.1186/1471-5945-5-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maeda A, Schneider SW, Kojima M, Beissert S, Schwarz T, Schwarz A. Enhanced photocarcinogenesis in interleukin-12-deficient mice. Cancer Res. 2006;66:2962–2969. doi: 10.1158/0008-5472.CAN-05-3614. [DOI] [PubMed] [Google Scholar]
  24. Mahmoud BH, Hexsel CL, Hamzavi IH, Lim HW. Effects of visible light on the skin. Photochem Photobiol. 2008;84:450–462. doi: 10.1111/j.1751-1097.2007.00286.x. [DOI] [PubMed] [Google Scholar]
  25. Murphy GM, Norris PG, Young AR, Corbett MF, Hawk JL. Low-dose ultraviolet-B irradiation depletes human epidermal Langerhans cells. Br J Dermatol. 1993;129:674–677. doi: 10.1111/j.1365-2133.1993.tb03330.x. [DOI] [PubMed] [Google Scholar]
  26. Nestle FO, Kaplan DH, Barker J. Psoriasis. N Engl J Med. 2009;361:496–509. doi: 10.1056/NEJMra0804595. [DOI] [PubMed] [Google Scholar]
  27. Nograles KE, Zaba LC, Guttman-Yassky E, Fuentes-Duculan J, Suarez-Farinas M, Cardinale I, et al. Th17 cytokines interleukin (IL)-17 and IL-22 modulate distinct inflammatory and keratinocyte-response pathways. Br J Dermatol. 2008;159:1086–1091. doi: 10.1111/j.1365-2133.2008.08769.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Noonan FP, De Fabo EC, Morrison H. Cis-urocanic acid, a product formed by ultraviolet B irradiation of the skin, initiates an antigen presentation defect in splenic dendritic cells in vivo. J Invest Dermatol. 1988;90:92–99. doi: 10.1111/1523-1747.ep12462045. [DOI] [PubMed] [Google Scholar]
  29. Ozawa M, Ferenczi K, Kikuchi T, Cardinale I, Austin LM, Coven TR, et al. 312-nanometer ultraviolet B light (narrow-band UVB) induces apoptosis of T cells within psoriatic lesions. J Exp Med. 1999;189:711–718. doi: 10.1084/jem.189.4.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Patel RV, Clark LN, Lebwohl M, Weinberg JM. Treatments for psoriasis and the risk of malignancy. J Am Acad Dermatol. 2009;60:1001–1017. doi: 10.1016/j.jaad.2008.12.031. [DOI] [PubMed] [Google Scholar]
  31. Pene J, Chevalier S, Preisser L, Venereau E, Guilleux MH, Ghannam S, et al. Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes. J Immunol. 2008;180:7423–7430. doi: 10.4049/jimmunol.180.11.7423. [DOI] [PubMed] [Google Scholar]
  32. Piskin G, Koomen CW, Picavet D, Bos JD, Teunissen MB. Ultraviolet-B irradiation decreases IFN-gamma and increases IL-4 expression in psoriatic lesional skin in situ and in cultured dermal T cells derived from these lesions. Exp Dermatol. 2003;12:172–180. doi: 10.1034/j.1600-0625.2003.120208.x. [DOI] [PubMed] [Google Scholar]
  33. Piskin G, Sylva-Steenland RM, Bos JD, Teunissen MB. T cells in psoriatic lesional skin that survive conventional therapy with NB-UVB radiation display reduced IFN-gamma expression. Arch Dermatol Res. 2004a;295:509–516. doi: 10.1007/s00403-004-0460-9. [DOI] [PubMed] [Google Scholar]
  34. Piskin G, Tursen U, Sylva-Steenland RM, Bos JD, Teunissen MB. Clinical improvement in chronic plaque-type psoriasis lesions after narrow-band UVB therapy is accompanied by a decrease in the expression of IFN-gamma inducers -- IL-12, IL-18 and IL-23. Exp Dermatol. 2004b;13:764–772. doi: 10.1111/j.0906-6705.2004.00246.x. [DOI] [PubMed] [Google Scholar]
  35. Sa SM, Valdez PA, Wu J, Jung K, Zhong F, Hall L, et al. The effects of IL-20 subfamily cytokines on reconstituted human epidermis suggest potential roles in cutaneous innate defense and pathogenic adaptive immunity in psoriasis. J Immunol. 2007;178:2229–2240. doi: 10.4049/jimmunol.178.4.2229. [DOI] [PubMed] [Google Scholar]
  36. Sigmundsdottir H, Johnston A, Gudjonsson JE, Valdimarsson H. Narrowband-UVB irradiation decreases the production of pro-inflammatory cytokines by stimulated T cells. Arch Dermatol Res. 2005;297:39–42. doi: 10.1007/s00403-005-0565-9. [DOI] [PubMed] [Google Scholar]
  37. Simon JC, Tigelaar RE, Bergstresser PR, Edelbaum D, Cruz PD., Jr Ultraviolet B radiation converts Langerhans cells from immunogenic to tolerogenic antigen-presenting cells. Induction of specific clonal anergy in CD4+ T helper 1 cells. J Immunol. 1991;146:485–491. [PubMed] [Google Scholar]
  38. Tjioe M, Smits T, van de Kerkhof PC, Gerritsen MJ. The differential effect of broad band vs narrow band UVB with respect to photodamage and cutaneous inflammation. Exp Dermatol. 2003;12:729–733. doi: 10.1111/j.0906-6705.2003.00057.x. [DOI] [PubMed] [Google Scholar]
  39. Walters IB, Burack LH, Coven TR, Gilleaudeau P, Krueger JG. Suberythemogenic narrow-band UVB is markedly more effective than conventional UVB in treatment of psoriasis vulgaris. J Am Acad Dermatol. 1999;40:893–900. doi: 10.1016/s0190-9622(99)70076-9. [DOI] [PubMed] [Google Scholar]
  40. Walters IB, Ozawa M, Cardinale I, Gilleaudeau P, Trepicchio WL, Bliss J, et al. Narrowband (312-nm) UV-B suppresses interferon gamma and interleukin (IL) 12 and increases IL-4 transcripts: differential regulation of cytokines at the single-cell level. Arch Dermatol. 2003;139:155–161. doi: 10.1001/archderm.139.2.155. [DOI] [PubMed] [Google Scholar]
  41. Weischer M, Blum A, Eberhard F, Rocken M, Berneburg M. No evidence for increased skin cancer risk in psoriasis patients treated with broadband or narrowband UVB phototherapy: a first retrospective study. Acta Derm Venereol. 2004;84:370–374. doi: 10.1080/00015550410026948. [DOI] [PubMed] [Google Scholar]
  42. Wittkowski KM, Lee E, Nussbaum R, Chamian FN, Krueger JG. Combining several ordinal measures in clinical studies. Stat Med. 2004;23:1579–1592. doi: 10.1002/sim.1778. [DOI] [PubMed] [Google Scholar]
  43. Yones SS, Palmer RA, Garibaldinos TT, Hawk JL. Randomized double-blind trial of the treatment of chronic plaque psoriasis: efficacy of psoralen-UV-A therapy vs narrowband UV-B therapy. Arch Dermatol. 2006;142:836–842. doi: 10.1001/archderm.142.7.836. [DOI] [PubMed] [Google Scholar]
  44. Zaba L, Cardinale I, Gilleaudeau P, Sullivan-Whalen M, Suárez-Fariñas M, Fuentes-Duculan J, et al. Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses. J Exp Med. 2007a;204:3183–3194. doi: 10.1084/jem.20071094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zaba LC, Fuentes-Duculan J, Eungdamrong NJ, Abello MV, Novitskaya I, Pierson KC, et al. Psoriasis is characterized by accumulation of immunostimulatory and Th1/Th17 cell-polarizing myeloid dendritic cells. J Invest Dermatol. 2009a;129:79–88. doi: 10.1038/jid.2008.194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zaba LC, Fuentes-Duculan J, Steinman RM, Krueger JG, Lowes MA. Normal human dermis contains distinct populations of CD11cBDCA-1 dendritic cells and CD163FXIIIA macrophages. J Clin Invest. 2007b;117:2517–2525. doi: 10.1172/JCI32282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zaba LC, Krueger JG, Lowes MA. Resident and “inflammatory” dendritic cells in human skin. J Invest Dermatol. 2009b;129:302–308. doi: 10.1038/jid.2008.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang W, Chen Y, Wei H, Zheng C, Sun R, Zhang J, et al. Antiapoptotic activity of autocrine interleukin-22 and therapeutic effects of interleukin-22-small interfering RNA on human lung cancer xenografts. Clin Cancer Res. 2008;14:6432–6439. doi: 10.1158/1078-0432.CCR-07-4401. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS214887-supplement-supplement_1.pdf (12.9MB, pdf)

RESOURCES