Results

Absence of phosphorylated p65/RelA in normal skin

Although multiple studies endeavored to examine the expression pattern of NF-B family members in the epidermis and dermis, none have characterized the expression of the active phosphorylated form of RelA/p65 in the epidermis. Because of the critical role that NF-B plays in inflammatory responses, cellular proliferation, the cell cycle, and apoptosis and the contradictory reports of its specific function in keratinocytes, we wanted to study the expression of NF-B in both normal and psoriatic skin to establish, not only if differences existed but if those differences would correlate with disease status and response to therapy. Toward this end, we utilized a phospho-specific antibody targeted to serine 536 of RelA/p65, which is essential for transcriptional activation by NF-B. Skin samples were obtained from 4 healthy volunteer controls. Although it has been reported that a basal level of NF-B exists in the epidermis (^{Takao et al, 2003}), we found almost no active nuclear phosphorylated RelA in the nucleus of epidermal cells (Figure 1a). Activation of NF-B was confirmed utilizing the phospho-RelA antibody on samples of normal skin that were irradiated with UVB, a known inducer of NF-B activity. This UVB treatment led to a substantial (p<0.02) 25-fold increase in the level of phosphorylated RelA (Figure 1b and c).

Figure 1.

Absence of active phosphorylated RelA/p65 in the normal epidermis. Punch biopsies from four control subjects were analyzed by immunohistochemistry for expression of nuclear phosphorylated nuclear factor kappa B (NF-B)/RelA as described in Materials and methods. A representative sample shows strong expression of phosphorylated p65 was only detected in UVB-treated samples (compare A to B). The arrows denote examples of positive cells. No staining was observed with a negative control rabbit IgG antibody. (C) Chart demonstrating the average number of keratinocytes in epidermis expressing phosphorylated NF-B/RelA. ^*Denotes a significance of p<0.02.

Full figure and legend (194K)

Phosphorylated p65/Rel is present at basal constitutive levels in keratinocytes from non-lesional psoriatic skin

In light of the overwhelming evidence of the role of NF-B family members in immune regulation, apoptosis, keratinocyte regulation, and psoriasis, we wanted to examine whether the expression pattern of active nuclear phosphorylated NF-B differed between normal and psoriatic skin. There is established precedence for abnormal expression and regulation of proteins and cytokines in disease processes, including psoriasis (^{McKenzie and Sabin, 2003;McKenzie et al, 2003}). Therefore, we compared expression of active phosphorylated RelA/p65 in the normal epidermis to the non-lesional skin in our psoriasis patients. The skin samples were obtained from biopsies in ten patients who were subjects in a study of the efficacy and safety of the TNF-targeting pharmaceutical etanercept. Interestingly, and in contrast to the absence of active RelA in the epidermis from normal patients, those biopsies taken from the uninvolved skin of psoriatic patients demonstrated substantial basal levels of phospho-RelA/p65 in the epidermis (p<0.02). Specifically, there was an average 12-fold increase of active nuclear phosphorylated RelA/p65 in the nonlesional psoriatic epidermis compared to normal donor skin (Figure 2). These data demonstrate a significant difference in the expression of NF-B in the epidermis of psoriatic patients as compared with normal controls.

Figure 2.

Increased expression of phosphorylated nuclear factor kappa B (NF-B)/RelA in uninvolved psoriatic skin. The expression of nuclear phosphorylated RelA was examined via immunohistochemistry in four control subjects and ten psoriasis patients as explained in the Materials and methods utilizing a specific anti-phospho NF-B antibody. (A) Chart depicting the average number of keratinocytes in the epidermis expressing nuclear phosphorylated RelA. ^*Denotes significance of p<0.02. (B) Immunohistochemical analysis demonstrated the dramatic difference in expression levels of nuclear phosphorylated p65 between normal epidermis and the non-involved epidermis from psoriasis patients. Slide is a representative sample from the study population and the arrows denote examples of positive nuclear staining cells.

Full figure and legend (184K)

Phosphorylated p65/Rel is significantly upregulated in keratinocytes within psoriatic plaques

The differential expression of phosphorylated RelA between normal skin and non-lesional skin from psoriasis patients prompted examination of the epidermis from lesional psoriatic plaques for active nuclear phosphorylated NF-B expression. As shown in Figure 3, on average, there was a 5-fold increase (p<0.001) in phospho-RelA in the epidermis of plaques as compared with uninvolved skin from the same patient. In additional, there was a substantial increase in epidermal thickness, as well as expression of abnormal keratinocyte differentiation marker K16 (as has been previously reported).

Figure 3.

Upregulation of active phosphorylated nuclear factor kappa B (NF-B) in lesional plaques. The expression of nuclear phosphorylated p65 was examined by immunohistochemistry in uninvolved and lesional epidermis from ten psoriasis patients. (A) Chart depicting the average number of phosphorylated NF-B positive keratinocytes in the epidermis. ^*Denotes significance of p<0.001. (B) Representative slide showing enhanced expression of phosphorylated NF-B detected in lesional plaques as compared to uninvolved skin. Arrows highlight examples of positive nuclear staining cells.

Full figure and legend (187K)

Etanercept reduces phosphorylated NF-B/RelA levels and reverses abnormal epidermal proliferation and differentiation

The high levels of TNF and NF-B found in the skin of psoriatic patients coupled with the success of recent immunologic therapies targeting TNF and immune receptors in the treatment of psoriasis prompted us to examine what effect etanercept therapy would have on phospho-RelA/p65 levels. We also wanted to determine if treatment with etanercept would lead to histological changes in keratinocyte differentiation and plaque morphology. To see if TNF's beneficial effects could be mediated via a decrease in NF-B activation we examined biopsy samples from etanercept patients at 0, 1, 3, and 6 mo. Consistent with the fact that NF-B is a downstream effector of TNF, we observed that the high levels of nuclear phosphorylated RelA/p65 shown previously were dramatically reduced (p<0.001) at each successive treatment interval (Figure 4a and 4b). Interestingly, this decrease in NF-B was complemented by a significant reduction in epidermal thickness (p<0.01) at each time period (Figure 4a). There was a significant correlation between these two outcome measures (Table I). Moreover, examination of keratinocyte differentiation also demonstrated a return of K16 levels to normal in 70%–80% of patients at 3 and 6 mo and this too correlated with the observed reduction in phosphorylated RelA/p65 (Table I).

Figure 4.

Etanercept downregulates expression of phosphorylated nuclear factor kappa B (NF-B). Punch biopsies from ten psoriasis patients were examined via immunohistochemistry utilizing an anti-phospho-NF-B specific antibody as described in Materials and methods. (A) Charts depicting the average number of keratinocytes expressing phosphorylated RelA and the average epidermal thickness from baseline plaques or at subsequent etanercept treatment points. ^*Denotes significance of p<0.01. (B) Immunohistochemical analysis of a representative sample demonstrating the reduction of phosphorylated RelA after treatment with etanercept at 1, 3, and 6 mo. Arrows highlight representative positively nuclear staining cells.

Full figure and legend (171K)

Table I - Correlation between reduction in phosphorylated NF-B, decrease in epidermal thickness and improvement of keratinocyte K-16 expression.

Full table

Discussion

This is the first report of an in vivo study to demonstrate a relative absence of nuclear phosphorylated RelA in normal skin, that non-lesional psoriatic skin contains a basal level of phosphorylated RelA, and significant overexpression of phospho-RelA in the epidermis of psoriatic plaques. The treatment of psoriatic patients with etanercept over a 6-mo period led to a dramatic downregulation of phospho NF-B/RelA in keratinocytes and this correlated with a dramatic reduction in epidermal hyperplasia/thickness and a return of normal keratinocyte markers of differentiation. Importantly, these patients also showed dramatic clinical improvement.

In the absence of stressful stimuli, NF-B is present in the cytoplasm in a resting state within normal keratinocytes (^{Takao et al, 2003}). Under normal conditions, NF-B has also been noted to play an important role in keratinocyte regulation and proliferation (^{Takao et al, 2003;Bernard et al, 2004}). But the function of NF-B in keratinocytes has been proposed to be quite paradoxical to its effect in other cell types, such as T or B cells (^{Dajee et al, 2003}). Skin defects are present in a number of mouse models in which Rel/NF-B function is perturbed (^{Gerondakis and Strasser, 2003}). For instance, the epidermis restricted expression of a dominant negative form of IB produced by mice with profoundly hyperplastic epithelia whereas mice that overexpressed RelA/p65 showed epidermal hypoplasia and growth inhibition (^{Takeda et al, 1999;Seitz et al, 2000a, b}). This is in contrast to studies on mice null for IB which demonstrate marked alterations in epidermal morphology, including hyperplastic epidermal keratinocytes and dermal infiltration of lymphocytes (^{Wilson et al, 1990;Chen et al, 2000a,2000b;Lee et al, 2000;Bell et al, 2003}).

The reason for this diversity in outcome of NF-B activation may be a reflection of the cell type and differentatiation state of the cell (^{Ozes et al, 1999;Romashkova and Makarov, 1999;Ernfors, 2000}). An interesting model proposed by^{Kaufman and Fuchs (2000)} tries to take into account the peculiar findings of NF-B activity in the epidermis. They suggest that a balancing act is played between cell-cycle withdrawal, cell survival and differentiation. In addition to an apparent cell-cycle arrest effect, NF-B in the epidermis also protects against apoptosis by enhancing the expression of anti-apoptotic factors (^{Wrone-Smith et al, 1997;Seitz et al, 2000b}). It is through a marriage of cell-cycle withdrawal and protection against apoptosis that NF-B is able to coordinate homeostasis within the normal epidermis.

The data and models discussed above are all in reference to normal skin, normal epidermis, and normal keratinocytes. Psoriasis is clearly an abnormal state, and one would anticipate changes in the epidermis and keratinocytes. Perhaps, this is why we observed, in contrast to our control subjects, a basal level of nuclear phosphorylated RelA/p65 in the non-lesional skin of our psoriatic patients and in turn this may be one factor, in the backdrop of an altered cellular milieu, which predisposes to psoriatic plaque formation. For example, when normal keratinocytes are co-cultured with psoriatic T cells (^{Bata-Csorgo et al, 1995}) they do not undergo increased growth or hyperproliferation. But keratinocytes established from non-lesional psoriatic skin are markedly abnormal even before they are cocultured with lymphocytes (^{Jackson et al, 1999}). In culture these abnormal keratinocytes release abnormal amounts of interferon gamma (IFN), and have subnormal transcription factor activation in response to cytokine stimulation (^{Werner and Smola, 2001}). Although IFN promotes growth arrest of normal keratinocytes (^{Nickoloff et al, 1984;Saunders and Jetten, 1994}), it induces proliferation of psoriatic keratinocytes (^{Baker et al, 1988,1993}). These findings have led to the hypothesis that the existence of abnormalities in the signaling pathways of psoriatic keratinocytes predisposes them to display an overactive wound healing response when exposed to stimulation from inflammatory mediators (^{McKenzie and Sabin, 2003}). A recent study published by^{Watanabe et al (2004)} identified a mutation in Crohn's disease patients that predisposes them to the development of the disease. Interestingly, a mutation in the gene encoding nucleotide-binding oligomerization domain 2 (NOD2) appeared to be the culprit. They reported that the function of NOD2 was to limit the proinflammatory effects mediated by Toll-like receptor 2 (TLR2) stimulation. The mutated form of NOD2 found in Crohn's patients was unable to inhibit TLR2 signaling, and this apparently skewed the system towards inflammation. Interestingly, this is an NF-B-dependent phenomenon because NF-B is downstream of TLR-2 and its activation leads to production of IL-12. Expression of both IL-12 and IL-2 has also been found to be upregulated in psoriasis (^{Lee et al, 1988;Yawalkar et al, 1998}).

The increased epidermal thickness present in psoriatic plaques is the net sum of both rates of proliferation and cell death. Moreover, aside from high levels of TNF and NF-B, many other cytokines, such as IFN, and their downstream targets are upregulated in psoriasis (^{Bowcock et al, 2001}). It would be naïve to suggest that NF-B is the sole suspect in this process, but NF-B activation is also clearly associated with resistance to apoptosis (^{Beg and Baltimore, 1996;Wrone-Smith et al, 1997;Foo and Nolan, 1999;Wang et al, 1999a, b}). Thus, one would predict that blocking NF-B activation would make keratinocytes more susceptible to apoptosis, with fewer rather than more viable cells in the epidermis. Unfortunately, under normal conditions, the opposite is observed with the blockade of NF-B. How could such a paradox exist? First, in psoriasis it is clear that the keratinocytes are not normal; therefore, what is observed under normal conditions may not apply in this chronic inflammatory condition. Second, it would seem reasonable to consider the context in which we are looking at NF-B activity and to consider the role of other molecular regulators of apoptosis, the cell cycle and proliferation in the skin. For example, another major cellular pathway regulated by NF-B is the cell cycle (^{Chaturvedi et al, 1999}). It has been shown that NF-B activating stimuli such as IFN and tissue plasminogen activator (TPA) induce growth arrest in normal keratinocytes in vitro (^{Qin et al, 1999}). Therefore, the observation that constitutive activation of NF-B in transgenic mice models produced epidermal thinning may in fact represent a dominance of the cell cycle inhibitory effect over the anti-apoptotic effect. Thus, although the cells were not undergoing cell death, they were not proliferating either. So if we extrapolate to normal skin, it may be that the anti-proliferative effects of NF-B are superior to its anti-apoptotic effect in the absence of inflammation or apoptotic stimuli. In support of this view, transgenic mice expressing anti-apoptotic genes Bcl-x or Bcl-2 have no significant increase in epidermal thickness in the absence of cell-cycle changes. In fact, it is only when the homeostasis of the skin is altered by various proinflammatory or tumorigenic stimuli that a distinctive phenotype becomes apparent (^{Valle Blazquez et al, 1997;Rodriguez-Villanueva et al, 1998}). Such a scenario exists in psoriasis, therefore the disruption of balance between cell-cycle inhibitory and proliferative effects would tip the scale towards epidermal proliferation in the context of a self-sustaining cytokine-mediated feedback loop.

In agreement with the high levels of TNF and phosphorylated NF-B found in psoriatic patients, and the crucial link between TNF-mediated signaling and NF-B, we observed a substantial and significant reduction in the levels of phospho-NF-B after treatment with etanercept. This suggests that NF-B may not only play an important role in the pathogenesis of psoriasis, but that it may be a reasonable therapeutic target. Recent work by Zollner et al supports this hypothesis. They demonstrated that the inhibition of superantigen-mediated T cell activation by the selective proteasome inhibitor PS-519 led to significant resolution of psoriasis in a severe combined immunodeficiency (SCID-hu) model (^{Zollner et al, 2002}). PS-519 is known to affect the inhibitory regulator of NF-B and IB, and the work demonstrated a concentration-dependent suppression of NF-B DNA binding.

In light of the results from these experiments and the current literature, we propose a significant role for NF-B in the pathogenesis of psoriasis. Although there is ample evidence in the literature to substantiate our utilization of nuclear-phosphorylated NF-B as a proxy for NF-B activation, relying exclusively on immunostaining to detect nuclear phospho-RelA does not necessarily indicate a functional state of NF-B activation. Therefore, although confident in our results, we recognize the limitation of our in vivo studies, and have initiated follow-up experiments to reaffirm our results. Due to limited patient samples, we are utilizing the remainder of the 6mm biopsy specimens from our study population to perform RT-PCR-mediated gene expression studies that will reaffirm the etanercept-mediated repression of NF-B activity.

NF-B's role in psoriasis is multifactorial, with influence on T cells, dendritic cells and keratinocytes. In lymphocytes, downregulation of TNF leads to downregulation of NF-B, which not only abrogates additional TNF production but also eliminates the production of necessary inflammatory cytokines and cell adhesion markers. The role of NF-B in the keratinocyte is more complicated and controversial (^{Kaufman and Fuchs, 2000}). We believe that in the setting of a chronic inflammatory state such as psoriasis, there is an imbalance between the anti-apoptotic role and the cell-cycle inhibitory role of NF-B whereby the scale is tipped toward protection against cell death in the context of a constitutive cytokine-rich inflammatory milieu. This allows for the increased epidermal thickness and hyperproliferation seen in plaques. Alternatively, perhaps the proposed function of NF-B in normal keratinocytes is altered in the abnormal keratinocytes present in psoriatic patients and therefore proliferation and increased thickness is observed as opposed to cell-cycle arrest. Further work will be needed to expand on these ideas and confirm the potential benefit of NF-B targeted therapies in the treatment of psoriasis as well as other inflammatory and autoimmune disorders.

Materials and Methods

Patient studies

The study was conducted according to the Declaration of Helsinki Principles, and participants gave their written informed consent. Adult patients with moderate to severe psoriasis were treated with etanercept monotherapy (25 mg subcutaneously twice weekly) for 24 wk under a protocol approved by the UMDNJ-Robert Wood Johnson Medical School Institutional Review Board. Systemic and photo-therapies were excluded for 1 mo prior to dosing; topical medications were excluded for 2 wk prior to dosing. Eucerin cream (Beiersdorf, Wilton, Connecticut) was the standard moisturizer used throughout the study. Patients were told not to apply moisturizer prior to study evaluations. Clinical efficacy was assessed using PASI. Skin biopsies were obtained at baseline, 1, 3, and 6 mo. Skin biopsies were snap frozen in optimal cutting temperature compound (OCT, Tissue Tek, Sakura, Torrance, California) and saved at -70°C. Laboratory-based evaluations were blinded to the clinical results until all data were collected.

UVB exposure of human subjects

Under a protocol approved by the UMDNJ-Robert Wood Johnson Medical School Institutional Review Board, four healthy volunteers between the ages of 18–60 were recruited and their minimum erythema dose (MED) was determined. MED of a subject is the minimum dose of UVB light that induces mild redness (sunburn) on the skin 24 h after the light exposure. The patients were irradiated with 2 MED of UVB on either the left or right hip and 6 mm punch biopsy was taken immediately after UVB exposure. Subsequently, two more punch biopsies were taken from each patient at 24 and 48 h after the UVB light exposure. The biopsies were frozen immediately in OCT and saved at -70°C.

Histology and immunohistochemistry

Skin punch biopsy specimens (6 mm) were obtained from target plaques at weeks 0, 4, 12, and 24. A non-lesional skin biopsy was also obtained at week 0. Biopsy specimens for UVB-treated skin were obtained on Day 1 immediately after UVB irradiation and at 72 h post-treatment. Serial cryostat-cut sections were stored at -70°C before use. Immunoperoxidase studies of the number of epidermal nuclear-staining phosphorylated NF-B positive cells and keratinocyte expression of k16 keratin were performed (Vectastain ABC kit, Vector Laboratories, Burlingame, California) as previously described (^{Gottlieb et al, 2003a}). Briefly, slides were thawed to room temperature, fixed in BFA for 15 min, and then endogenous peroxidase activity was quenched with 0.3% H₂O₂. After washing in PBS the sections were blocked for 30 min with goat serum in PBS. The primary antibody in PBS containing goat serum was applied for 30 min at room temperature. After rinsing in PBS, sections were incubated with biotinylated secondary antibody in PBS containing goat serum, washed in PBS, and incubated with Vectastain Elite ABC Peroxidase reagent (Vector Laboratories). Finally, after rinsing in PBS, the reaction product was visualized using diaminobenzidine. Primary antibodies used were anti-phospho RelA/p65 (Cell Signaling Technologies, Beverly, Massachusetts) 1:50, and anti K-16 (1:400). As negative controls, all experiments were performed in the presence of equal concentrations of normal rabbit IgG isotype control.

Three blinded investigators, including board certified dermatologists, examined immunostained sections with an image analyzer attached to a light microscope (Image pro-plus 3.1, Mediscybernectic, Silver Spring, Maryland). The average number of positively nuclear stained epidermal nuclear phospho-NF-B cells in five 200 fields was determined. Epidermal thickness was quantitated by averaging the epidermal thickness in three 100 fields in H&E-stained sections. For keratinocyte expression of K16, semiquantitative scores of 0 to 4+ were assigned for each section read. Normal expression was defined as 0. Abnormal expression was defined as 1+, 2+, 3+, or 4+. There was less than 5% discordance between observers in all instances.

Statistical methods

Statistical analysis was performed using the paired or unpaired t test unless otherwise specified. Correlation between variables was analyzed by the Pearson's correlation coefficient. Two-tailed p<0.05 was considered to indicate statistical significance.

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Acknowledgments

This is an investigator-initiated study, supported in part by grants from Amgen and a Clinical Immunology Center of Excellence grant from the Federation of Clinical Immunology Societies (FOCIS). Dr Gottlieb is an investigator, consultant, and speaker for Amgen.