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Article| October 30 2006
IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2–dependent mechanisms with implications for psoriasis pathogenesis
Jason R. Chan,
Jason R. Chan
1Discovery Research
Wendy Blumenschein,
Wendy Blumenschein
2Experimental Pathology and Pharmacology, Schering-Plough Biopharma (formerly DNAX Research, Inc.), Palo Alto, CA 94304
Erin Murphy,
Erin Murphy
Caroline Diveu,
Caroline Diveu
Maria Wiekowski,
Maria Wiekowski
3Immunology, Schering-Plough Research Institute, Kenilworth, NJ 07033
Susan Abbondanzo,
Susan Abbondanzo
Linda Lucian,
Linda Lucian
Richard Geissler,
Richard Geissler
4Drug Safety and Metabolism, Schering-Plough Research Institute, Lafayette, NJ 07848
Scott Brodie,
Scott Brodie
Alexa B. Kimball,
Alexa B. Kimball
5Department of Dermatology, Stanford University School of Medicine, Stanford, CA 94305
Daniel M. Gorman,
Daniel M. Gorman
Kathleen Smith,
Kathleen Smith
Rene de Waal Malefyt,
Rene de Waal Malefyt
Robert A. Kastelein,
Robert A. Kastelein
Terrill K. McClanahan,
Terrill K. McClanahan
Edward P. Bowman
CORRESPONDENCE Edward P. Bowman: eddie.bowman@spcorp.com
A.B. Kimball's present address is Massachusetts General and Brigham and Women's Hospitals, Harvard Medical School, Boston, MA 02114.
Abbreviations used: EM, electron micrograph; K16, keratin 16; KGF, keratinocyte growth factor 1.
Received: January 31 2006
Accepted: October 04 2006
Jason R. Chan, Wendy Blumenschein, Erin Murphy, Caroline Diveu, Maria Wiekowski, Susan Abbondanzo, Linda Lucian, Richard Geissler, Scott Brodie, Alexa B. Kimball, Daniel M. Gorman, Kathleen Smith, Rene de Waal Malefyt, Robert A. Kastelein, Terrill K. McClanahan, Edward P. Bowman; IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2–dependent mechanisms with implications for psoriasis pathogenesis . J Exp Med 27 November 2006; 203 (12): 2577–2587. doi: https://doi.org/10.1084/jem.20060244
Aberrant cytokine expression has been proposed as an underlying cause of psoriasis, although it is unclear which cytokines play critical roles. Interleukin (IL)-23 is expressed in human psoriasis and may be a master regulator cytokine. Direct intradermal administration of IL-23 in mouse skin, but not IL-12, initiates a tumor necrosis factor–dependent, but IL-17A–independent, cascade of events resulting in erythema, mixed dermal infiltrate, and epidermal hyperplasia associated with parakeratosis. IL-23 induced IL-19 and IL-24 expression in mouse skin, and both genes were also elevated in human psoriasis. IL-23–dependent epidermal hyperplasia was observed in IL-19−/− and IL-24−/− mice, but was inhibited in IL-20R2−/− mice. These data implicate IL-23 in the pathogenesis of psoriasis and support IL-20R2 as a novel therapeutic target.
Psoriasis is a common skin disorder that affects ∼2% of the worldwide population. Lesions are typically well-demarcated erythematous, scaly plaques. Histologically, there is marked epidermal hyperplasia (acanthosis) accompanied by parakeratosis (retention of keratinocyte nuclei in the stratum corneum) and a mixed dermal infiltrate, including CD4+ T cells, dendritic cells, macrophages, and mast cells. Neutrophilic exudates are often seen (Munro microabscesses) and CD8+ T cells are present in the epidermis. Dermal papillary blood vessels are dilated and tortuous, and there is increased expression of angiogenesis-associated genes (1).
The pathogenesis of psoriasis is unclear, but the effectiveness of cyclosporine and other newer agents implicates an important role for T cells. No clear-cut autoantigens, however, have been described, and it is likely that the innate immune system is also involved (2). Bacterial infections and physical trauma (Köebner's phenomenon) often precede lesion formation and neutrophil accumulation in the dermis, and an influx into the epidermis is observed in initial pre-pinpoint lesions (3). Plasmacytoid pre–dendritic cells initiate psoriasis in a xenograft animal model (4), and other cells, including dermal dendritic cells and NK T cells, have also been implicated (5, 6). Given their ability to link the innate and acquired immune systems, dysregulated cytokine production (elevated levels of TNF, IFN-γ, IL-1α, IL-1β, TGF-α, IL-6, and other cytokines in lesional skin) is postulated to establish chronic lesions by providing persistent proinflammatory signals in the skin (7). Immune deviation from a Th1 to Th2 cytokine milieu can ameliorate disease, which also supports the cytokine hypothesis (8). Additionally, a novel family of IL-10–related cytokines consisting of IL-19, IL-20, and IL-24 has recently been associated with psoriasis, but their roles in disease pathogenesis are unclear (9).
IL-23 is a recently described, IL-12–related heterodimeric cytokine (10). IL-23 and IL-12 both contain the IL-12p40 subunit, which is paired with IL-23p19 and IL-12p35, respectively. The heterodimeric receptors for IL-23 and IL-12 consist of IL-23R paired with IL-12Rβ1 and IL-12Rβ2 paired with IL-12Rβ1, respectively (11, 12). IL-23 has a dominant role over IL-12 in driving animal models of chronic autoimmune disease (13, 14), and IL-23 transgenic mice have systemic inflammation involving multiple organs, including the skin (15, 16). IL-23 is produced by dendritic cells and macrophages in response to bacteria, including Bordetella pertussis (17), Mycobacterium tuberculosis (18), Streptococcus pyogenes (19), and Klebsiella pneumoniae (20). IL-23 gene expression is increased in psoriatic lesions compared with uninvolved skin (21), but downstream consequences of IL-23 dysregulation are unclear. We demonstrate that intradermal delivery of IL-23 protein in mice results in acute skin histopathology that shares many features of psoriasis. We also show a role for the IL-19 family of cytokines, via the shared receptor IL-20R2, in IL-23–dependent epidermal hyperplasia, supporting IL-20R2 as a novel therapeutic target.
Elevated IL-23 is associated with human psoriasis
IL-23p19 mRNA expression was increased in lesional psoriatic skin compared with nonlesional skin from psoriatic and normal donors (Fig. 1 A, left), in agreement with a previous report (21). Similar results were observed for IL-12p40, including increased expression in nonlesional psoriatic skin compared with normal controls (Fig. 1 A, middle). In contrast, IL-12p35 expression was not increased but actually decreased in lesional psoriatic skin compared with nonlesional and normal skin (Fig. 1 A, right).
IL-23 drives inflammation and epidermal hyperplasia in mouse skin
Because IL-23 is elevated in human psoriasis, we injected IL-23 protein intradermally into mice to explore the downstream consequences of aberrant cutaneous IL-23 exposure. Wild-type mice treated daily with IL-23, but not saline, developed visually apparent erythema and induration (Fig. 1 B) and associated prominent dermal papillary blood vessels (Fig. 1 C). Induration was apparent by day 2 and persisted to day 7. Erythema was also apparent at day 2 but began to recede at day 5. Histological evaluation of IL-23–treated skin showed epidermal (and follicular) hyperplasia accompanied by parakeratosis at day 4 (Fig. 1 D, middle, red arrows). No rete ridges were observed, although hair follicles cut in the appropriate plane may falsely suggest otherwise. Focal neutrophilic exudates, reminiscent of Munro microabscesses, were often observed (Fig. 1 D, middle, green arrows). Markedly less parakeratosis and fewer focal neutrophilic exudates were observed by day 7 (unpublished data).
The specificity of this observation was confirmed using IL-23R–deficient mice (Fig. S1), which did not respond to intradermal IL-23 treatment (Fig. 1 D, right). IL-23–induced epidermal thickening was maximal by day 4 and began to decrease by day 7 (Fig. 1 E). IL-12 treatment, in comparison, did not induce significant changes in epidermal thickness. No changes in epidermal thickness were observed with TNF treatment either (Fig. 1 F), in agreement with a previous report (22). Bioactivity of these cytokines was confirmed by examining expression of IL-12–dependent (IFN-γ, CXCL9, and CXCL10) and TNF-dependent (VCAM-1) genes in skin samples (Fig. S2). Immunohistochemical analysis demonstrated a mixed dermal infiltrate consisting of CD4+ T cells, CD11c+ dendritic cells, F4/80+ macrophages, and neutrophils that began 1 d after IL-23 treatment and was markedly increased by day 4 (Fig. S3). No CD8+ T cells or increased numbers of mast cells were detected at either time point, in contrast to psoriasis (unpublished data). Aside from the obvious local changes in skin appearance, the IL-23 treatment regimen was well tolerated because the mice appeared healthy and did not lose weight during the course of the experiment. Serum cytokine levels, including GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, TNF, and IFN-γ, remained unchanged compared with saline-treated controls (unpublished data).
Epidermal thickening associated with IL-23 exposure was examined in detail using transmission electron microscopy. Hyperplastic skin was selected from animals without histopathologic evidence of suppurative dermatitis (day 4) for the purpose of defining ultrastructural features of the model in the absence of cellular infiltrates. Acanthosis (increased thickness of the stratum spinosum) was a predominant feature and accounted for the majority of the epidermal thickening and was characterized by spinous cell hyperplasia, hypertrophy, and spongiosis (intercellular edema) (Fig. 2 A). Spinous cells, although enlarged, maintained normal cytoplasmic contents and membrane structure. The granular cell layer (stratum granulosum) was more prominent in IL-23–treated treated skin when compared with untreated control skin. However, it was still very sparse, even in severely thickened skin, and often showed retained "end-stage" nuclei (i.e., nuclei with condensed and marginated chromatin). Nuclear fragmentation was not observed within this layer. The stratum corneum was characterized by altering areas of hyperparakeratosis (cells with retained epithelioid nuclei, keratohyalin granules, and compacted keratin filaments) and orthohyperkeratosis (Fig. 2 B). These results are discussed in the context of psoriasis keratinocyte biology in the Discussion. Additional transmission electron microscopy pictures are available.
IL-17A is not required for IL-23–dependent epidermal hyperplasia
IL-17A is a proinflammatory cytokine that lies downstream of IL-23 in the pathogenesis of experimental autoimmune encephalomyelitis and whose presence is correlated with the progression of collagen-induced arthritis (13, 23). Human psoriatic skin has elevated IL-17A gene expression in lesions relative to nonlesional skin and normal controls (Fig. 3 A). IL-17A mRNA expression was increased in IL-23–treated mouse skin at day 1 and remained elevated at day 4 (Fig. 3 C). We used a blocking monoclonal antibody to IL-17A to determine whether IL-17A is required for IL-23–dependent epidermal thickening. Anti–IL-17A did not inhibit IL-23– stimulated acanthosis (Fig. 3 E), nor did it affect IL-23–dependent erythema, induration, and parakeratosis (unpublished data). Anti–IL-17A treatment inhibited IL-23–stimulated G-CSF (Fig. 3 G) and MMP-13 expression (unpublished data), genes previously shown to be IL-17A regulated (24, 25). This anti–IL-17A antibody is efficacious at inhibiting experimental autoimmune encephalomyelitis, where IL-17A expressed in the central nervous system is a key pathogenic factor that contributes to paralysis (26), and also in inhibiting collagen-induced joint inflammation models, where IL-17A is a key pathogenic factor driving cartilage and bone destruction (unpublished data). Intradermal IL-17A delivery caused significant keratinocyte hypertrophy, but minimal if any hyperplasia by day 4 (unpublished data).
Role of TNF in IL-23–dependent epidermal hyperplasia
TNF is a validated target for various inflammatory diseases, including psoriasis (27, 28). TNF mRNA was elevated in human psoriatic skin relative to normal controls (Fig. 3 B) and was also induced in mouse skin by intradermal IL-23 treatment at day 1, but not at day 4 (Fig. 3 D). TNF protein was also detected by ELISA in IL-23–treated skin (unpublished data). We used a blocking monoclonal antibody to TNF to determine whether TNF is involved in IL-23–dependent epidermal thickening. In contrast to anti–IL-17A, anti-TNF treatment partially inhibited IL-23–dependent acanthosis (Fig. 3 F), and the degree of erythema and induration was decreased.
IL-23 indirectly stimulates keratinocyte proliferation
Immunohistochemical analysis of IL-23–treated skin at day 1 showed increased Ki67+ cells in the epidermis, suggesting that keratinocytes had received an IL-23–stimulated proliferative signal by day 1 (Fig. 4 A). This observation was confirmed by increased keratin 16 (K16) gene expression, which is associated with keratinocyte hyperplasia in psoriasis (29). Elevated K16 was evident at day 1, before changes in epidermal thickness, and was further increased at day 4 (Fig. 4 B). Increased expression of K16 was induced by IL-23, but not IL-12 or TNF (Fig. 4 C). Gene expression analysis of skin samples at day 1 suggested that the effects of IL-23 treatment on epidermal hyperproliferation were not due to increased gene expression of a panel of keratinocyte growth factors (Fig. 4 D). Furthermore, in vitro assays indicated that IL-23 did not have a direct effect on keratinocyte proliferation (Fig. 4 E).
IL-19 family members are stimulated by IL-23 and expressed in human psoriatic skin
Because IL-23 did not stimulate the expression of any growth factors tested at day 1 (a time point before visual epidermal thickening, but after a keratinocyte proliferative signal had been received based on Ki67 staining and K16 expression), we examined the novel IL-19 family of cytokines, which consists of IL-19, IL-20, and IL-24. These cytokines all bind the heterodimeric receptor IL-20R1/IL-20R2. IL-20 and IL-24 can also bind IL-22R1/IL-20R2 (30). These receptors are highly expressed in epithelial cells, including keratinocytes (31, 32), and are all elevated in human psoriatic skin (9), suggesting a possible role in disease pathology. IL-23 delivery to mouse skin elevated IL-19 and IL-24, but not IL-20, gene expression (Fig. 5, left). IL-23 did not increase IL-20R1, IL-20R2, or IL-22R1 gene expression in mouse skin (unpublished data). IL-19, IL-20, and IL-24 mRNA was also increased in human psoriatic skin relative to normal controls (Fig. 5, right). IL-20R1 and IL-22R1 were elevated in psoriatic skin compared with normal controls, whereas no significant changes were observed for IL-20R2 (Fig. S4).
Role of IL-20R2 in IL-23–dependent epidermal hyperplasia
IL-23 was delivered into the skin of IL-20R2−/− mice (Fig. S5) to simultaneously examine the role of all three IL-19 family cytokines. Epidermal thickening was significantly inhibited in IL-23–treated IL-20R2−/− mice compared with IL-23–treated wild-type controls (Fig. 6 A), despite the induction of IL-19 and IL-24 gene expression (Fig. 6 B). IL-23 was delivered into the skin of IL-19−/− and IL-24−/− mice (Fig. S6 and S7, respectively) to determine if IL-19 or IL-24 was required for IL-23–dependent epidermal hyperplasia. IL-23–stimulated epidermal thickening was observed in both IL-19−/− and IL-24−/− mice (Fig. 6 A). These data suggest that these cytokines are redundant in function because IL-24 and IL-19 gene expression was induced in IL-19−/− and IL-24−/− mice, respectively (Fig. 6, C and D). IL-20 gene expression was not significantly altered by IL-23 in skin from either IL-19−/− or IL-24−/− mice compared with wild-type controls (unpublished data). Importantly, in vitro stimulation of keratinocytes with increasing quantities of IL-19, IL-20, or IL-24 failed to stimulate proliferation, suggesting that IL-20R2 ligands are required but not sufficient for IL-23–stimulated epidermal hyperplasia (unpublished data).
Impaired leukocyte recruitment in IL-23–treated IL-20R2−/− mice
We performed immunohistochemical analysis on IL-23–treated IL-20R2−/− skin to determine whether impaired signaling through IL-20R2 affected the cellular composition of the dermal infiltrate. Although epidermal thickening was not apparent in IL-23–treated IL-20R2−/− mice, there were elevated numbers of CD4+ T cells and CD11c+ dendritic cells compared with saline-treated control mice, which was similar to IL-23–treated wild-type mice (Fig. 6, E and F, respectively). In contrast, there were reduced numbers of dermal F4/80+ macrophages and neutrophils in IL-20R2−/− mice (Fig. 6, G and H). Table I summarizes the differences between human psoriasis and wild-type or IL-20R2−/− mice in their responses to intradermal IL-23 treatment.
Because IL-23p19 and IL-12p40 gene expression was elevated in lesional psoriatic skin compared with nonlesional psoriatic skin and normal controls, we tested the hypothesis that IL-23 was involved in disease pathogenesis by injecting IL-23 intradermally into mice. IL-23 induced changes in mouse skin that share many features with human psoriasis, as summarized in Table I. The most striking similarity to psoriasis microscopically was the acanthosis (epithelial hyperplasia) and parakeratosis (keratinocyte nuclei in the stratum corneum).
Histological analysis of psoriatic skin reveals the absence of the normal granular layer where normal keratinocyte differentiation starts to give rise to a highly cross-linked, lipid-rich anuclear structure that provides the first barrier to the environment (i.e., stratum corneum). In normal skin, the granular layer is defined by numerous keratinocyte nuclei undergoing condensation and disintegration. Psoriatic skin, however, has little to no keratinocyte nuclei disintegration, which ultimately results in a modified stratum corneum with retained keratinocyte nuclei (parakeratosis). One difference between the acute IL-23–induced skin hyperplasia response and the chronic psoriatic lesional skin, however, is that a modified granular layer is present. IL-23–stimulated granular layer keratinocytes have nuclei condensation but no evidence of nuclei disintegration. This gives rise to parakeratosis, similar to psoriasis, by a slightly different mechanism than observed in psoriasis. Whether this is due to the IL-23–stimulated hyperplasia being an acute self-limiting condition versus the chronic nature of psoriatic lesions is under investigation. Reversible edema in the epidermis (spongiosis) was also observed in the acute model, which is not commonly seen in chronic psoriatic skin lesions. A rare disease, granular parakeratosis, is characterized by parakeratosis but in combination with a granular layer. IL-23 may also play a role in this disease due to the similarities between it and our observations in this mouse model.
An IL-12p40 antagonist that blocks IL-23 and IL-12p70 simultaneously was successful in a phase I psoriasis clinical trial (33), and IL-23p19, but not IL-12p35, gene expression was associated with human psoriasis (21). These data plus the observation that intradermal injections of IL-12 did not cause psoriasiform lesions in mice are consistent with IL-23 being the relevant target of anti–IL-12p40 therapy in psoriasis. It is noteworthy that IL-12 treatment did not induce psoriasiform lesions despite enhanced IFN-γ expression because human psoriatic lesions have a distinct signature of IFN-γ–regulated genes (34). This raises the possibility that IL-23 (dys)regulation may trigger psoriatic lesion formation and that IFN-γ–dependent processes may contribute to the chronicity of disease. This notion is consistent with IL-23 production early in an innate immune response to bacteria (17–20) that may initiate psoriatic lesions and with the multigenic nature of this disease (35).
IL-23 is a potent regulator of memory T cell development and/or expansion that is skewed to IL-17A secretion (36), and IL-23 has been implicated as a key disease driver in autoimmune disease models (13, 23). The human psoriatic data demonstrate that IL-17A gene expression is also associated with disease. Intradermal IL-23 injections stimulated elevated IL-17A expression; however, pretreatment with an antagonistic antibody did not ameliorate psoriasiform lesion formation. These data and the observation that direct IL-17A delivery has minimal effect on epidermal hyperplasia suggest that IL-17A, although downstream of IL-23 and associated with human psoriasis, is not required for disease pathogenesis. It is possible that IL-17F, whose expression is also skewed by IL-23 during memory T cell development (36) and is driven by IL-23 in this model (unpublished data), may compensate in some way for IL-17A because they both can bind IL-17RC (37). Interestingly, psoriatic patients are relatively resistant to cutaneous infections (38). IL-17A and IL-17F may play key roles in host defense to cutaneous infections because they stimulate neutrophil recruitment (39, 40), and IL-17A directly stimulates the anti-microbial gene β-defensin 2 in epithelial cells (41).
IL-23 stimulates macrophage TNF production (14) and IL-23p19 transgenic mice have elevated serum TNF levels (16). Neutralizing TNF partially inhibited epidermal hyperplasia induced by IL-23 in our study. This observation is consistent with the efficacy of anti-TNF–directed therapies in psoriasis (27, 28). TNF inhibition breaks the self-sustaining nature of psoriatic lesions by rapidly down-regulating several proinflammatory genes, including IL-23p19 and IL-19 (42). TNF alone, however, was not sufficient to induce epidermal hyperplasia, indicating that IL-23 stimulates other signaling pathways that synergize with TNF for disease pathogenesis.
The physiologic functions of the structurally related cytokines IL-19, IL-20, and IL-24 appear to be quite diverse despite their shared receptor profile. IL-19 may be involved in the induction of Th2 responses (43). IL-20 appears to play a key role in skin biology because IL-20 transgenic mice have aberrant epidermal differentiation with no immune cell infiltrates (32). Adenoviral delivery of IL-24 (MDA-7/mob-5/c49a) induces apoptosis in tumor cells (44), and IL-24 gene expression is increased during wound healing in the rat (45). The common link between IL-19, IL-20, and IL-24 is their use of heterodimeric receptors that share the IL-20R2 subunit: IL-20R1/IL-20R2 heterodimer and IL-22R1/IL-20R2 heterodimer (30). These receptors are primarily expressed in epithelial cells such as keratinocytes (31, 32) and are elevated in human psoriatic lesions (9, 32). Our human psoriatic data confirm that IL-20R1 and IL-22R2 are increased in psoriatic skin, although we did not detect a statistically significant increase in IL-20R2 mRNA compared with normal skin. Administration of IL-23 to IL-20R2−/− mice, but not IL-19−/− or IL-24−/− mice, resulted in significantly decreased acanthosis and parakeratosis. This suggests that either IL-19 or IL-24 may be sufficient to mediate epidermal hyperplasia via IL-20R2–containing receptors, although it is unclear whether these cytokines directly stimulate keratinocyte proliferation or alter the keratinocyte differentiation program.
Interestingly, cutaneous IL-23 exposure also drives high-level expression of IL-22, a cytokine structurally related to IL-19, IL-20, and IL-24 (unpublished data). IL-22 binds the heterodimeric receptor IL-22R1/IL-10RB, but it does not use IL-20R2. IL-22 activation of keratinocytes stimulates a variety of antimicrobial activities, such as psoriasin (S100A7), calgranulin A (S100A8), calgranulin B (S100A9), β-defensin 2, and β-defensin 3 (46–48). Therefore, cutaneous IL-23 expression in psoriatic skin drives both IL-22–dependent and IL-17A/IL-17F–dependent antimicrobial responses that protect the host.
The cellular mechanisms by which IL-23 drives IL-20R2–dependent epidermal hyperplasia and psoriasiform lesions remain to be investigated. Analysis of the inflammatory cell infiltrate in IL-23–treated IL-20R2−/− mice revealed decreased numbers of neutrophils and F4/80+ macrophages in the dermis, suggesting that these recruited cells may contribute to the lesion development in our model. In support of this possibility, neutrophil depletion inhibits acanthosis in the fsn/fsn mouse, which spontaneously develops flaky skin (49). Infiltrating myeloid cells can produce several factors associated with psoriasis, including iNOS, IL-19, MMP-12, and IL-23 (42).
T cells, monocytes, and NK cells express IL-23R (11), which raises the possibility that IL-23 may act directly on these cells to produce IL-19 and IL-24. Published data show that the major hematopoietic sources of IL-19 and IL-24 are monocytes and monocytes and T cells, respectively (50). IL-23–treated bone marrow macrophages do not express IL-19, IL-20, or IL-24 (unpublished data), suggesting that IL-19 and IL-24 expression may be indirectly stimulated by IL-23. This notion is supported by our data that keratinocytes do not respond to IL-23 and that IL-19 is expressed by keratinocytes in psoriatic lesions (9). TNF may be important here as IL-23 stimulates TNF expression in macrophages (14) and TNF directly stimulates IL-19 expression in normal human epidermal keratinocytes (unpublished data).
In summary, our data propose a molecular mechanism by which IL-23 dysregulation is one of the causative factors in psoriasis pathogenesis. Dysregulated cutaneous IL-23 production sets into motion several independent pathways. IL-23 contributes to the antimicrobial nature of psoriatic lesions by stimulating IL-17A (and IL-17F) and neutrophil recruitment. In parallel, IL-23 stimulates IL-19 and IL-24, which may directly act on keratinocytes in a TNF-regulated manner resulting in epidermal hyperplasia and/or altered keratinocyte differentiation. These functionally different arms suggest that IL-23 may have evolved as a "response to danger" cytokine invoking the body to protect itself by rapidly mobilizing antimicrobial components and instructing the epidermis to proliferate to provide additional protection from the environment. Given that IL-23–dependent epidermal hyperplasia was inhibited in IL-20R2−/− mice, targeting IL-20R2 and/or its associated receptors may be a novel therapeutic strategy for the treatment of this disease.
Human skin specimens.
Psoriasis patients (n = 45) and normal volunteers (n = 30) consented under a protocol approved by the Stanford Panel on Human Subjects. Psoriasis patients needed to have a Psoriasis Area Severity Index of at least 8 and a typical lesion at least 1 cm in size suitable for biopsy. The target lesion and the surrounding 5-cm area could not have been treated with any medicated topical formulation for at least 2 wk before obtaining the biopsy. Patients treated with systemic immunosuppressives including corticosteroids were excluded. One 4-mm biopsy from a lesional and an adjacent nonlesional site was collected from psoriatic patients. One 4-mm biopsy was obtained from each normal volunteer. Specimens were flash frozen in liquid nitrogen.
Reagents and antibodies.
Generation of IL-23R−/−, IL-20R2−/−, IL-19−/−, and IL-24−/− mice are described in Online supplemented material. Recombinant human and murine IL-23 have been described (10). Recombinant murine IL-12 was generated using approaches described previously (10). Murine TNF and human keratinocyte growth factor 1 (KGF) were purchased (PeproTech). Rat anti–IL-17A (JL7.1D10) (26), rat anti-TNF (MP6-XT22) (51), and isotype control antibody (25D2) were purified from hybridoma culture supernatants. Rat anti-Ki67 (TEC-3; DakoCytomation), rat anti-neutrophils (7/4; Serotec), rat anti-CD4 (L3T4; BD Biosciences), rat anti-F4/80 (A3-1; Serotec), hamster anti-CD11c (HL3; BD Biosciences), and rat anti-CD31 (MEC 13.3; BD Biosciences) were purchased.
IL-23–dependent skin inflammation model.
All animal protocols were approved by DNAX/Schering-Plough Biopharma's Institutional Animal Care and Use Committee. Hair was removed from the back of mice with electric clippers and a cream depilatory (Nair). 3 d later, mice were injected intradermally with IL-23, vehicle control, or other cytokines (IL-12 or TNF) in two locations on either side of the back for a total of 1 μg protein per mouse using a 29.5-gauge needle. Sterile saline was used as a vehicle control. Injections were performed daily until mice were killed as per the objective of the experiment. For antibody blocking studies, mice were treated subcutaneously with 0.2–1.0 mg anti–IL-17A, anti-TNF, or isotype control 2 d before the first cytokine injection. Mice were killed using carbon dioxide and blood was collected by cardiac puncture. Skin samples were removed from the prepared area, keeping at least 5 mm away from the hair boundary. Skin samples were frozen directly in liquid nitrogen for mRNA extraction, fixed in 10% neutral buffered formalin for histology, or embedded and frozen in OCT for immunohistochemistry.
Electron microscopy.
Skin samples were fixed in a solution containing 2.5% glutaraldehyde and 2% formaldehyde with 0.12 M sodium cacodylate followed by secondary fixation in buffered 1% OsO4. The samples were then dehydrated and embedded in epoxy resin by standard methods. Thin sections were stained with 5% uranyl acetate in 50% methanol and 1% aqueous lead citrate, and images were captured using an electron microscope (CM10; Philips).
RNA extraction and real-time quantitative PCR.
RNA isolation was performed by standard techniques, and gene expression was calculated using the Δ-ΔCt method (using the mean cycle threshold value for ubiquitin and the gene of interest for each sample. The equation 1.8e (Ct ubiquitin − Ct gene of interest) × 104 was used to obtain the normalized values.
Histology and immunohistochemistry.
Skin sections were stained with hematoxylin and eosin, and epidermal thickness was determined by measuring the average interfollicular distance from the basal lamina to the bottom of the stratum corneum in a blinded manner. Similar results were obtained for wild-type mice on 129SvEv, C57BL/6, or mixed B6 × 129 genetic backgrounds. For immunohistochemistry, 8-μm sections were fixed with 75% acetone/25% ethanol. Endogenous peroxidase was quenched with Peroxidase Blocking Reagent (DakoCytomation) and endogenous biotin was blocked with SP-2001 (Vector Laboratories). Primary antibodies were incubated for 30 min at room temperature and visualized using VECTASTAIN elite ABC kit (Vector Laboratories). Positive staining developed as a brown reaction precipitate.
In vitro keratinocyte proliferation assay.
In vitro keratinocyte proliferation assays were performed as described previously (52), with minor modification. Normal human epidermal keratinocytes (Cambrex) were seeded at 2,000/well in a 24-well plate in keratinocyte basal medium (Cambrex) supplemented with hydrocortisone (Cambrex) and 5 μg/ml human recombinant insulin (Roche Diagnostics). During seeding, cells were treated with vehicle control, 1–100 ng/ml human IL-23, or 10 ng/ml KGF as a control. After 6 d, cells were washed and incubated with 0.2% crystal violet (Sigma-Aldrich). Cells were lysed with 1% SDS and absorbance was read at 565 nm. The total number of cells per well was determined by reference to a standard curve. All conditions were performed in triplicate.
Statistical analyses.
Human data was analyzed by Kruskal-Wallis with Dunn's Multiple Comparison after test. One-way or two-way ANOVA with Bonferroni after test were used where appropriate. P < 0.05 was considered to be statistically significant.
Online supplemented material.
Fig. S1 is the gene deletion strategy for the IL-23R knockout mouse. Fig. S2 shows the in vivo bioactivity of intradermally injected IL-12 and TNF. Fig. S3 shows the IL-23–dependent inflammatory cell infiltrate into mouse skin. Fig. S4 shows the IL-19 family receptor subunits in human psoriasis. Figs. S5–S7 show the gene deletion strategy for IL-20R2, IL-19, and IL-24 knockout mice, respectively. Fig. S8 is a high magnification hematoxylin and eosin–stained section showing general features of an IL-23–mediated model. Fig. S9 shows a high magnification hematoxylin and eosin–stained section illustrating an epidermal microabscess. Fig. S10 is a high magnification electron micrograph (EM) of spinous (bottom) and granular (top) layers (note presence of intercellular edema). Fig. S11 is a medium-high EM of junction between spinous and granular layers (note the presence of nuclear degeneration [condensed chromatin] in a granular cell [red arrow] vs. a more normal nucleus in a spinous cell [blue arrow]). Fig. S12 is a low magnification EM from a control animal illustrating the "normal" appearance of the epidermis (note that the granular layer is present, but not prominent, and there is little keratin). Fig. S13 is a high magnification EM from a control animal illustrating the "normal" appearance of the corneum (note the absence of nucleated keratinocytes and sparse keratohyalin granules). Table S1 is genotyping primers for analyzing knockout mice.
The authors would like to acknowledge Kevin Keane for reviewing histology; Don Cook and Sergio Lira for expertise in generating IL-19−/− mice; and Gil Asio and Sharon Osborn for histology assistance.
Schering-Plough Biopharma (formerly DNAX Research, Inc.) is a division of Schering-Plough. All authors, except A.B. Kimball, were employees of Schering-Plough at the time of these studies, which could be considered to pose a financial conflict of interest regarding the submitted manuscript.
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. A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits.
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. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain.
Kopp, T., P. Lenz, C. Bello-Fernandez, R.A. Kastelein, T.S. Kupper, and G. Stingl.
. IL-23 production by cosecretion of endogenous p19 and transgenic p40 in keratin 14/p40 transgenic mice: evidence for enhanced cutaneous immunity.
Wiekowski, M.T., M.W. Leach, E.W. Evans, L. Sullivan, S.C. Chen, G. Vassileva, J.F. Bazan, D.M. Gorman, R.A. Kastelein, S. Narula, and S.A. Lira.
. Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility, and premature death.
Fedele, G., P. Stefanelli, F. Spensieri, C. Fazio, P. Mastrantonio, and C.M. Ausiello.
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Verreck, F.A., T. de Boer, D.M. Langenberg, M.A. Hoeve, M. Kramer, E. Vaisberg, R. Kastelein, A. Kolk, R. de Waal-Malefyt, and T.H. Ottenhoff.
. Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria.
Veckman, V., M. Miettinen, J. Pirhonen, J. Siren, S. Matikainen, and I. Julkunen.
. Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells.
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Happel, K.I., M. Zheng, E. Young, L.J. Quinton, E. Lockhart, A.J. Ramsay, J.E. Shellito, J.R. Schurr, G.J. Bagby, S. Nelson, and J.K. Kolls.
. Cutting edge: roles of Toll-like receptor 4 and IL-23 in IL-17 expression in response to Klebsiella pneumoniae infection.
Lee, E., W.L. Trepicchio, J.L. Oestreicher, D. Pittman, F. Wang, F. Chamian, M. Dhodapkar, and J.G. Krueger.
. Increased expression of interleukin 23 p19 and p40 in lesional skin of patients with psoriasis vulgaris.
Jamieson, T., D.N. Cook, R.J. Nibbs, A. Rot, C. Nixon, P. McLean, A. Alcami, S.A. Lira, M. Wiekowski, and G.J. Graham.
. The chemokine receptor D6 limits the inflammatory response in vivo.
Langrish, C.L., Y. Chen, W.M. Blumenschein, J. Mattson, B. Basham, J.D. Sedgwick, T. McClanahan, R.A. Kastelein, and D.J. Cua.
. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.
Koenders, M.I., J.K. Kolls, B. Oppers-Walgreen, L. van den Bersselaar, L.A. Joosten, J.R. Schurr, P. Schwarzenberger, W.B. van den Berg, and E. Lubberts.
. Interleukin-17 receptor deficiency results in impaired synovial expression of interleukin-1 and matrix metalloproteinases 3, 9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall-induced arthritis.
Arthritis Rheum.
Fossiez, F., O. Djossou, P. Chomarat, L. Flores-Romo, S. Ait-Yahia, C. Maat, J.J. Pin, P. Garrone, E. Garcia, S. Saeland, et al.
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. Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis.
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. Efficacy and safety of infliximab monotherapy for plaque-type psoriasis: a randomised trial.
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. Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro.
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. IL-17 markedly up-regulates beta-defensin-2 expression in human airway epithelium via JAK and NF-kappaB signaling pathways.
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. TNF inhibition rapidly down-regulates multiple proinflammatory pathways in psoriasis plaques.
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. The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice.
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. Cutaneous rat wounds express c49a, a novel gene with homology to the human melanoma differentiation associated gene, mda-7.
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. IL-22 increases the innate immunity of tissues.
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. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes.
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. IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis.
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. Critical role of neutrophils for the generation of psoriasiform skin lesions in flaky skin mice.
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. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples.
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. Synergistic effects of epidermal growth factor (EGF) and insulin-like growth factor I/somatomedin C (IGF-I) on keratinocyte proliferation may be mediated by IGF-I transmodulation of the EGF receptor.
Figure S1
Targeting strategy for IL-23R-/- mice. The IL-23R targeting vector contains a 3.2-kb 5′ region and a 4.4-kb 3′ region of homology. The targeted locus has a 6.0-kb region containing exons 1, 2, and 3 replaced with a FRT-flanked Neo cassette. Arrows indicate the location of PCR primers (Table S1) used to discriminate the WT and properly targeted locus.
B6-IL23Rtm1Dnax (IL-23R-/-) mice were generated through a research agreement with Xenogen Biosciences. Using the mIL-23R sequence, a database search identified a BAC clone containing the IL-23R locus. The targeting vector was constructed with a 5′ homologous arm (3.2 kb) and 3′ homologous arm (4.4 kb) using PCR. The targeted locus has exons 1-3 replaced with a FRT-flanked Neo cassette. The targeting vector was electroporated into C57BL/6 ES cells, and G418-resistant colonies were screened for homologous recombination by Southern blotting with a probe flanking the 3′ arm. The 5′ arm integration was confirmed via PCR. IL-23R-/- mice were generated and a real-time PCR genotyping strategy to distinguish +/+ versus +/- versus -/- was established (Table S1). IL-23R-/- and WT controls were maintained on a B6 background.
- jpeg file
Bioactivity of intradermally injected IL-12 and TNF. Expression of IFN-γ (a), CXCL9/MIG (b), and CXCL10/IP-10 (c) mRNA at day 4 in skin samples from 129SvEv mice treated daily with saline, 1 mg IL-23, or 1 mg IL-12. Expression of VCAM-1 (d) mRNA in the skin from 129SvEv mice treated with saline (n = 3) or TNF (n = 4) for 24 h. Data are mean ± STD, n = 4. *, P < 0.05.
IL-23-dependent inflammatory cell infiltrate in mouse skin. Representative immunohistochemical staining for CD4+ T cells (a), CD11c+ dendritic cells (b), F4/80+ macrophages (c), and neutrophils (d) in WT B6x129 mice treated with saline at day 1 (left), 1 mg IL-23 at day 1 (middle), or at day 4 after daily treatments of 1 mg IL-23 (right).
Expression of IL-19 family receptor subunits in human psoriasis. IL-20R1 (top), IL-20R2 (middle), and IL-22R1 (bottom) mRNA expression was measured in human psoriatic skin. Bar represents median.
Targeting strategy for IL-20R2-/- mice. The IL-20R2 targeting vector contains a 6.4-kb 5′ region and a 5.3-kb 3′ region of homology. The targeted locus deletes the start codon and leader sequence in exon 1 and a portion of intron 1, inserting a LacZ-Neo cassette. Arrows indicate the location of PCR primers (Table S1) used to discriminate the WT and properly targeted locus.
B6;129-IL20R2tm1Dnax (IL-20R2-/-) mice were licensed from Deltagen. The targeting vector was constructed with a 5′ homologous arm (6.4 kb) and a 3′ homologous arm (5.3 kb) replacing the ATG and leader sequence from exon 1 and a portion of intron 1 with LacZ-Neo. IL-20R2-/- mice were generated at Deltagen, and a PCR genotyping strategy to distinguish +/+ versus +/- versus -/- was established (Table S1). IL-20R2-/- and WT controls were maintained on a mixed B6x129 background.
Targeting strategy for IL-19-/- mice. The IL-19 targeting vector contains a 1.8-kb 5′ region and a 4.0-kb 3′ region of homology. The targeted locus has a 2.4-kb region containing exons 3 and 4 replaced with a loxP-flanked Neo cassette. Arrows indicate the location of PCR primers (Table S1) used to discriminate the WT and properly targeted locus.
B6;129-IL19tm1Dnax(N6) (IL-19-/-) mice were generated by the SPRI Mouse Genetics Group. A cDNA probe for mouse IL-19 was used to identify a BAC clone containing the entire IL-19 locus. A 12-kb subclone was identified containing exons 1-5. Using this plasmid, a targeting vector was constructed with a 1.8-kb BamHI fragment (5′ region of homology) and a 4.0-kb XmnI fragment (3′ region of homology). The targeted locus has exons 3 and 4 replaced with a loxP-flanked Neo cassette driven by an HSV TK promoter in the opposite direction of IL-19. The targeting vector was electroporated into 129 ES cells, and G418-resistant colonies were screened for homologous recombination by nested-set PCR analysis. Positive colonies were expanded, and verification of the 5′ and 3′ arm targeted to the locus was confirmed by Southern blotting with probes flanking the targeting arms. IL-19-/- mice were generated using approaches described previously (Chen, S.C., B. Mehrad, J.C. Deng, G. Vassileva, D.J. Manfra, D.N. Cook, M.T. Wiekowski, A. Zlotnik, T.J. Standiford, and S.A. Lira. 2001. J. Immunol. 166:3362-3368), and a PCR genotyping strategy to distinguish +/+ versus +/- versus -/- was established (Table S1). IL-19-/- and WT controls were maintained on a mixed B6x129 background.
B6-IL24tm1Dnax (IL-24-/-) mice were generated by the DNAX Gene Targeting Group. A cDNA probe for mouse IL-24 was used to identify a BAC clone containing the entire IL-24 locus. An IL-24 locus subclone of 12.7 kb was sequenced and found to contain exons 1-5. A targeting vector containing a 1.7-kb StuI and HpaI fragment (5′ region of homology) and a 5.3-kb XbaI and PacI fragment (3′ region of homology) was constructed. The targeted locus has exons 1 and 2 replaced with a loxP-flanked Neo cassette being driven by an HSV TK promoter in the opposite direction of IL-24. The targeting vector was electroporated into C57BL/6 ES cells, and G418-resistant colonies were screened for homologous recombination by nested-set PCR analysis. Positive colonies were expanded, and verification of the 5′ and 3′ arm targeted to the locus was confirmed by Southern blotting with probes flanking the targeting arms. Transient transfection with a plasmid expressing Cre recombinase resulted in deletion of the Neo cassette. IL-24-/- mice were generated using approaches as described previously (Ruuls, S.R., R.M. Hoek, V.N. Ngo, T. McNeil, L.A. Lucian, M.J. Janatpour, H. Korner, H. Scheerens, E.M. Hessel, J.G. Cyster, et al. 2001. Immunity. 15:533-543), and a PCR genotyping strategy to distinguish +/+ versus +/- versus -/- was established (Table S1). IL-24-/- and WT controls were maintained on a B6 background.
Hematoxylin and eosin-stained section showing general features of IL-23-mediated model.
Hematoxylin and eosin-stained section illustrating an epidermal microabscess.
Figure S10
EM of spinous (bottom) and granular (top) layers. Note presence of intercellular edema. Bar, 1.8 µM.
Medium-high EM of junction between spinous and granular layers. Note the presence of nuclear degeneration (condensed chromatin) in a granular cell (red arrow) versus a more normal nucleus in a spinous cell (blue arrow). Bar, 1.0 µM.
Low magnification EM from a control animal illustrating the "normal" appearance of the epidermis. Note that the granular layer is present, but not prominent and there is little keratin. Bar, 1.0 µM.
High magnification EM from a control animal illustrating the "normal" appearance of the corneum. Note the absence of nucleated keratinocytes and sparse keratohyalin granules. Bar, 0.3 µM.
Table S1- pdf file
IL-23 is associated with human psoriasis and drives epidermal hyperplasia in mice. Expression of IL-23p19, IL-12p40, and IL-12p35 mRNA in normal, nonlesional (NL), and lesional (L) human psoriatic skin. Bar represents median (A). Representative photographs (B) of 129SvEv mice at day 4 treated with daily injections of saline (left) or 1 μg IL-23 (right). Representative frozen sections stained for CD31. Samples were taken at day 4 from 129SvEv mice treated daily with saline (top) or 1 μg IL-23 (bottom). Red arrows indicate CD31 staining in the dermal papillary. Bar, 100 μm (C). Representative histological sections at day 4 of littermate wild-type C57BL/6 mice treated daily with saline (left) or 1 μg IL-23 (middle), and IL-23R−/− mice treated daily with 1 μg IL-23 (right). Red arrows indicate parakeratosis. Green arrows indicate neutrophilic exudates. Bar, 100 μm (D). Epidermal thickness was measured in 129SvEv mice treated daily with saline (◯) or 1 μg IL-23 (•). Data was pooled together from six independent experiments performed in quadruplicate. Mean ± SD is shown. *, P < 0.001 (E). Epidermal thickness was measured at day 4 in 129SvEv mice treated daily with saline, 1 μg IL-23, 1 μg IL-12, or 1 μg TNF. Data was pooled together from two independent experiments (F).
IL-23–stimulated epidermal thickening is due to keratinocyte hyperplasia in the spinous layer and altered granular layer differentiation. Low magnification (bar, 8.4 μm) of skin illustrating the major ultrastructural features of IL-23–induced epidermal hyperplasia, including hypertrophy and hyperplasia of the spinous layer, with spongiosis and retained nuclei as well as compacted keratin filaments in the keratin layer (A). Transmission electron photomicrograph of skin from an IL-23–treated mouse showing retained nuclei (single arrows) within the stratum corneum. Note the division between the cornified and granular layers (double arrow). Bar, 9.7 μm (B).
IL-17A is not required for IL-23–dependent epidermal hyperplasia. Expression of IL-17A (A) or TNF (B) mRNA in human psoriatic skin. Expression of IL-17A (C) or TNF (D) mRNA in mice treated daily with saline (open bars) or 1 μg IL-23 (filled bars). Data are mean ± SEM of three independent experiments using 129SvEv, C57BL/6, or B6 × 129 mice, where mRNA was pooled together from four mice per group. *, P < 0.05. Epidermal thickness at day 4 in 129SvEv mice treated daily with saline or 1 μg IL-23 in the presence of anti–IL-17A (E) or anti-TNF (F) blocking monoclonal antibody or isotype control. Sum of two independent experiments for each antibody is shown. Expression of G-CSF (G) at day 4 in 129SvEv mice (n = 4, mRNA was pooled together) treated daily with saline or IL-23 in the presence of blocking anti–IL-17A monoclonal antibody (filled bars) or isotype control (open bars). One of two representative experiments is shown.
IL-23 stimulates keratinocyte proliferation in mice. Representative immunohistochemical staining for Ki67 in mice at day 1 (A) after treatment with saline (top) or 1 μg IL-23 (bottom). Bar, 20 μm. Expression of K16 mRNA (B) in mice treated daily with saline (open bars) or 1 μg IL-23 (filled bars). Data are mean ± SEM of three independent experiments using 129SvEv, C57BL/6, or B6 × 129 mice, where mRNA was pooled together from four mice per group. *, P < 0.05. K16 mRNA expression in mice at day 4 treated daily with saline, 1 μg IL-23, 1 μg IL-12, or 1 μg TNF. Data are mean ± STD, n = 4. One of two representative experiments is shown (C). mRNA expression at day 1 for a panel of keratinocyte growth factors (D) in mice treated with saline (open bars) or IL-23 (filled bars). Data are mean ± SEM of three independent experiments using 129SvEv, C57BL/6, or B6 × 129 mice, where mRNA was pooled together from four mice per group. Keratinocyte proliferation assay (E) in which normal human keratinocytes were treated in triplicate with vehicle or IL-23 (1, 10, or 100 ng/ml). 10 ng/ml KGF was used as a positive control. One of three representative experiments is shown.
IL-19 family members are expressed in human psoriatic skin and stimulated by IL-23 in mouse skin. IL-19 (A), IL-20 (B), and IL-24 (C) mRNA expression was measured in mice (left) treated daily with saline (open bars) or 1 μg IL-23 (filled bars), and in human psoriatic skin (right). Mouse data are mean ± SEM of three independent experiments using 129SvEv, C57BL/6, or B6 × 129 mice, where mRNA was pooled together from four mice per group. *, P < 0.05. Bar represents median for human samples.
Role of IL-20R2 in IL-23–dependent epidermal hyperplasia. Epidermal thickness was measured at day 4 in wild-type, IL-20R2−/−, IL-19−/−, or IL-24−/− mice treated daily with saline (open symbols) or 1 μg IL-23 (filled symbols). Data from different wild-type strains were pooled together because no differences in IL-23–stimulated epidermal thickening were observed. Data are the aggregate of seven independent experiments (each strain of mouse was used in at least two separate experiments) (A). IL-19 and IL-24 gene expression in IL-20R2−/− mice at day 4 (B). IL-24 gene expression in IL-19−/− mice at day 4 (C). IL-19 gene expression in IL-24−/− mice at day 4 (D). Data are representative of two independent experiments where mRNA (n = 4) was pooled together. Representative immunohistochemical staining for CD4+ T cells (E), CD11c+ dendritic cells (F), F4/80+ macrophages (G), and neutrophils (H) at day 4 in IL-20R2−/− mice treated daily with saline (left) or 1 μg IL-23 (middle), or littermate wild-type mice at day 4 treated daily with 1 μg IL-23 (right). Bar, 100 μm.
Comparison of clinical and histological features of human psoriasis with IL-23–treated wild-type and IL-20R2−/− mouse skin
Human psoriasis
IL-23–treated wild-type skin
IL-23–treated IL-20R2−/− skin
Erythematous yes yes minimal
Well-demarcated from symptomless skin yes no n/a
White-silvery scale yes no, although small areas of flaky skin no, no evidence of flaky skin
Histological
Acanthosis yes yes significantly reduced
Granular layer no yes, but no evidence of nuclei disintegration no
Parakeratosis yes yes no
Elongation of rete ridges yes no n/a
Follicular hyperplasia no yes no
Epidermal spongiosis no yes no
Intracorneal pustules (Munro microabscesses) yes yes no
Recruitment to dermis of:
CD4+ lymphocytes yes yes yes
Dendritic cells yes yes yes
Macrophages yes yes reduced
Neutrophils yes yes no
Mast cells yes no n/a
CD8+ cells in epidermis yes no n/a
Prominent dermal papillary blood vessels yes yes no
Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses
Successful ultraviolet B treatment of psoriasis is accompanied by a reversal of keratinocyte pathology and by selective depletion of intraepidermal T cells.
Interleukin-17 cytokines: Effectors and targets in psoriasis—A breakthrough in understanding and treatment


text:
United Nations Security Council resolution 858, adopted unanimously on 24 August 1993, after recalling resolutions 849 (1993) and 854 (1993) and noting a ceasefire between Abkhazia and Georgia and commitments to withdraw forces, the council established the United Nations Observer Mission in Georgia (UNOMIG) for an initial period of 90 days pending further extension.

In accordance with a report by the Secretary-General Boutros Boutros-Ghali, the peacekeeping force would consist of 88 military observers plus staff necessary to support UNOMIG. It would observe the ceasefire and report violations of it to the secretary-general, paying close attention to the city of Sukhumi and attempt to resolve any violations with the parties involved.

UNOMIG would be established for an initial period of 90 days which would be extended to six months based on whether progress had been made in implementing peace measures. Meanwhile, the secretary-general was requested to report back within three months on developments. The proposed deployment of mixed interim monitoring groups of Georgian/Abkhaz/Russian units was welcomed, calling on all concerned to pursue negotiations to ensure a comprehensive political settlement of the conflict.

See also
 Georgian–Abkhazian conflict
 List of United Nations Security Council Resolutions 801 to 900 (1993–1994)
 Sukhumi massacre
 United Nations resolutions on Abkhazia
 War in Abkhazia (1992–1993)

References

External links

Text of the Resolution at undocs.org

 0858
Abkhaz–Georgian conflict
1993 in Georgia (country)
1993 in Abkhazia
 0858
August 1993 events

redpajama_set_name: RedPajamaWikipedia


I'm so glad Mr. Obama has informed me I have nothing to worry about since its my neighbors and friends who are spying on me. I'm going to sleep so much better tonight.
Are "you" joking? That is suppose to make American's more understanding?
Everyone! You have nothing to worry about. Obama has Eric Holder reviewing everything with a secret court. A secret court. The same secret court where the Judge explained he has no authority to independently verify anything. Remember? Here, I'll help you refresh your memory here.
That is correct. Calm down. Relax. We'll take care of everything.
IRS targeting law abiding citizens . . .
Fast & Furious program where the feds ordered law abiding dealers to deliver guns to known drug cartel members which were found at the scenes of murdered Americans.
The guys who moved to dismiss the lawsuit after verdict, but before sentencing, of jackbooted Black Panthers with nightsticks who intimidated white people who attempted to vote.
Read the National Journal's article here.
Really, it's OK, Obama was a Constitutional Scholar, knows what is in the Constitution, and he's protecting all of us from it.
Anyone who continues to be a Democrat, and your neighbor. . . ? . . . Well, if you can't figure it out . . .
January 17, 2014 in Corruption, Government Surveillance, Liberty, NSA, Obama, TREASON, Tyranny.
How Many Bread Crumbs Are Needed To Make A Trail?
← 4th AMENDMENT: . . . shall not be violated . . .
I do believe it is coming down to the people (thanks in part to social media) being the watchdogs against an unruly government. What worries me is that the true media is in the government's pocket, and the majority of our elected officials are, as well. So when you have a majority, who does not pay attention; how in the hello do you wake them up? In essence; it is the people v. the government.
IMO….Snowden will go down as a 21st Century whitsleblower and Hero for Liberty …. and his reputation will be international.
I'm sure the Back-stabbing RINO Orrin Hatch is mighty Proud indeed !!!
Levin is very remorseful about endorsing this POS for Nov 2012.
Up until they went bad, the brown shirt and gestapo were neighbors and friends, too.
What Obama does not understand is that most evil sincerely believes what it does is for the best. Evil does not realize it is evil. The darkness does not understand the light, but the light DOES understand darkness. This is the problem with people like Obama and those who follow them. It always has been and it always will be — at least as long as man is runnign things it will be.
Thank you for explaining to everyone why the examples I gave of other "police forces" is relevant.
I have had many interactions with individuals from other countries during my career. No one else "trusts" law enforcement to take the proper actions against non authorities. This true because the military, police, etc. are used as political tools for authoritarianism's evil acts and deeds for the dictator/oligarchy.
Until today…. With the current NSA, IRS, FBI and Justice Dept.
We are in dark times. We the people must demand justice, not fairness, and return to the rule if law from the federal govt.
Otherwise the rule of man will destroy this nation and the rest of the world.
Americans have always been too trusting of authority — precisely for the reasons you mention. Up until just recently, we've had no reason to distrust our military/police. But this is because the government — or people in them — was still American in nature. Now, things have changed and we are ruled by people with a decidedly European mentality (i.e. materialistic).
I'll keep saying this — not because I am a pessimist, but because we need to understand what we are facing — but I fear that, where the spirit of tyranny within our government is concerned, we have already crossed the Rubicon.
I'm putting this up on all Posts about the NSA-FISC-Gov't Control and Anti-4th Amendment Posts. I think people should Re-Post this story far and wide ……………. THIS is what happens to Gov't DATA and Lists…… Gun Registration anyone ? The Question is WHAT DATABASE was the State of Maryland using for a person from another State .
Here is an example of "Information" used to harrass Innocent people. Gun-ownership info from another State was used by the State of Maryland to harass a law-abiding Citizen.
We live in a De Facto Police State Folks.


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'Inside Llewyn Davis' sad, folky, wonderful
Kevin Noonan | Tuesday, February 11, 2014
Emily Hoffmann
Someone in this section today, Scene Writer John Darr is (hopefully) reviewing "Benji," the latest album from San Francisco-based folk act Sun Kil Moon. As is standard practice in this section, I'd never heard of the band. But another Scene writer quickly informed me of its sound and style.
"They're folky. And they sing about death a lot," the Scene writer said. "Yeah, all of their songs are pretty much about death."
Usually, this kind of description would only serve to remind me why I've never heard of most any of the bands we review in Scene, and why I let the more knowledgeable and talented writers handle music reviews.
On this particular day however, I was struck by another thought ¾ that sounded a lot like "Inside Llewyn Davis," the latest film from the Coen Brothers. The movie depicts the world of Llewyn Davis, a semi-fictional singer-songwriter in 1961 who specializes in acoustic folk songs, almost all of which are about death.
Granted, based on my taste in music, I wouldn't normally be drawn to a movie that boasts of following a week in the life of a folk singer in 1961, but as a fan of Coen Brothers movies ("The Big Lebowski," "No Country for Old Men," "Fargo"), I decided to give it a shot.
As it turns out, it instantly became one of my favorite movies of the last year, and yet even now as I attempt to relate my reaction to the film, I'm not sure why I enjoyed it or even really watched it.
First of all, it's not really a movie. There isn't a real plot or a storyline. Llewyn Davis is sort of a bum, clearly a talented folk singer but in a time that didn't have much love for folk music, and he isn't really a nice guy.
In fact, there are no real likeable characters in the movie, and not in the contemporary anti-hero, bad-but-you-still-root-for-them kind of unlikeable. It's more of, "These people are actually kind of awful and I don't really care if they succeed or fail but I kind of hope they get punched in the face" kind of unlikeable. Oscar Isaac as Llewyn Davis is one part tormented artist, battling demons through his music and four parts irascible and irritating jerk.
Carey Mulligan's Jean, as Davis' friend's wife and one-time adulterer, is a woman in a difficult position in a difficult time for women, but it's not hard to imagine her character being generally awful to be around in the best of times and places.
The only two redeemable characters in the film are Justin Timberlake's Jim, a fellow folk-singer, and an orange tabby cat that Davis accidentally loses throughout the film. Jim is the really only artist in the film that hits it big, but he's kind of a dolt and Llewyn makes fun of him throughout the film. The cat, which the Coen Brothers said they only included because they were worried there wasn't enough plot in their script, is adorable and yet one of the saddest elements in an all around sad movie.
But even with all that, I loved it. The movie is less a story or an exploration of character as it is an illustration of time, place and environment. The film feels tangible and real, as if you can smell the nastiness of the dingy dive bars that Llewyn performs and get dirt under your fingernails from Greenwich Village side streets where Davis takes his beatings.
The Coen brothers based Llewyn Davis off a few folk-singers from the 60s, but began with the idea of wondering what might happen if real-life folk singer Dave Van Ronk outside of a bar in the Village. That's the sort of feel this movie embodies ¾ it rambles a bit, punches a bit and explores the world of folk music, leading to depressing losses and failures for its main characters, but with a hopeful heart.
In the opening scene and again in one of the last scenes of the film, Llewyn performs a song called, "Hang Me, Oh Hang Me," which is, as you might imagine, not quite in the same spirit of Pharrell's "Happy." The song's lyrics are sad, but he performs with an intriguing acceptance of fate mixed with a morbid hopefulness that makes you think things might not always be this bad.
Davis never gets anywhere in his week long journey, but as the film ends, a young man with wild hair, a guitar on his lap and a harmonica strapped to his chest takes the stage to begin his first set at Davis' regular spot. Maybe the sad, cynical world of Llewyn Davis, which really is the sad, cynical world of folk music, has some hope after all. Maybe not, of course, since the film's final moments are less than hopeful for Davis himself, but there's a sense of hope hidden in there among all the death and sadness that makes for a fascinating film.
4.5/5 Shamrocks
Tags: Coen, Film, Folk, Inside Llewyn Davis, Movies
About Kevin Noonan
I'm a senior from Kansas City studying Marketing with a minor in Journalism, Ethics and Democracy. I've written for The Observer since I was a freshman, and now serve as editor for Scene.
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redpajama_set_name: RedPajamaCommonCrawl


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Los cazas droides o droides buitre eran unos droides utilizados por el poderoso monopolio de la Federación de comercio y luego usados por los aliados separatistas en la saga de la Guerra de las Galaxias.

La Federación de Comercio le compró a la compañía Xi-Char Cathedral Factories una enorme cantidad de estos androides, que se alojaban en los cruceros Lucrehulk. Estos robots tenían dos formas en las cuales podían transformarse dependiendo de la situación de combate en la que estuviesen:

 Podían ser unos cazas de pequeño tamaño, con la capacidad de ir a grandes velocidades pro el espacio y derribar así otros cazas enemigos.

También podían descender a tierra y transformarse en unos robots de enorme tamaño, que desintegraban con sus cañones gemelos a todo los que se le cruzasen en su camino o cumplir funciones de reconocimiento.

En la batalla de Naboo miles de cazas droides de la Federación lucharon en contra de los cazas estelares Naboo N-1, logrando derribar muchos. Pero los que más bajas causaron en los cazas de los Naboo fueron en realidad los cuadrilásers de la nave de control principal de la Federación de comercio.

Durante las Guerras Clon los cazas droides siguieron en uso y lucharon junto con los cazas estelares geonoosianos en contra de la República Galáctica y a favor de los ideales separatistas. Avanzada la guerra, Xi-Char fue comprada por la Tecno Unión, y está comenzó a fabricarlos de una manera más eficaz.

Eran apodados "droides buitre" porque se asemejaban a estos animales. Según se fueron produciendo por diversas industrias su color fue variando, del marrón clásico utilizado por la Federación, pasaron al azul y negro debido a que estos eran los colores de la CSI.

Durante el asedio al planeta Coruscant, cientos de estos androides protegieron a La Mano Invisible, una nave insignia del Ejército Separatista. Ciertas modificaciones permitieron que los cazas droides llevasen cuatro misiles cargados con droides abeja. Uno de estos disparó su carga, la cual se vació encima del caza del General Obi-Wan Kenobi. De inmediato, los pequeños droides dañaron todos los sistemas de esta, aunque Kenobi logró sobrevivir.

Los cazas droides también lucharon en el planeta Utapau para repeler las fuerzas de la República Galáctica y permitir así el escape del Consejo Separatista y del General Grievous, aunque este último fue asesinado más tarde por Obi-Wan.

Una vez finalizada la guerra, estos androides fueron desactivados y utilizados como chatarra.

Droides de Star Wars
Vehículos de Star Wars
Vehículos de la Confederación


The compact size of the Boston/Cambridge area makes bicycling a popular mode of transportation for students and employees. In addition to riding on main streets, there are a wealth of paths, parks and bicycle-friendly streets that can make bicycling one of your most efficient, healthy and enjoyable forms of transportation. Since bicycling in traffic can be a challenge, riders should be cautious and follow the rules of the road.
Current MIT employees and students can buy subsidized annual Blue Bikes memberships and ride at a discounted hourly rate. The subsidized annual membership for MIT employees and students is $35 (regularly $99). Follow these instructions to signup as a new member or renew your membership under MIT. An MIT certificate is required in order to register.
NOTE: Unpaid postdoctoral fellows or research fellows, see separate instructions.
Members of the MIT community can register their bicycles with the MIT Parking and Transportation Office. Register your bike online to deter theft and to help recover your bike if it is stolen.
The MIT Parking & Transportation Office administers bicycle compounds located in the rear of Building 13, under Building E53 in the Hermann Garage, and in Building E62. Cards to access to the compounds can be requested by sending an email to commuting@mit.edu. Your bicycle must be registered with the MIT Parking & Transportation Office in order to gain access to a secure bike compound.
To find a bicycle fix-it station, check out the MIT bike map.
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Return to IBI Corporate Website »
Letter to Stakeholders
Passive House Design
Efficient Energy
Smart design is good for everyone; our clients, the people in our communities, and ultimately, the environment. That's why we plan and deliver projects to meet or exceed the highest environmental standards and constantly seek to reduce energy consumption and emissions. Resilience is incorporated into our designs to better manage potential climate events, and aligning with this philosophy, we have implemented a Sustainability, Environmental and Climate Action Policy.
Sustainable design principles are integral to IBI's vision for smart cities, and for providing healthy, long-lasting communities for future generations. Careful consideration is given to all of the unique physical and digital aspects of a project – from the soil, water and bedrock geology, to its plants, people and future uses – all of which are balanced by consideration for the consequences of technologies being deployed and their carbon footprints, both locally and globally.
Approximately 25% of IBI's registered architects, engineers, landscape architects and planners are LEED-accredited* professionals
*Leadership in Energy and Environmental Design (LEED) is a green building certification program used worldwide.
Leaders in Passive House Design
Derived from the German "Passivhaus", a passive house is a voluntary standard for energy efficiency in a building which reduces its ecological footprint. Passive house buildings consume up to 90% less heating and cooling energy than conventional buildings1. The passive house high-performance building standard is the only internationally-recognized, proven, science-based energy standard in construction. Certification ensures that designers and consultants are expertly qualified to design buildings to meet the standard.
University of Toronto Scarborough Campus Student Residence
IBI Group is leading the consultant team as part of a design-build contract for a 750-bed passive house student residence that aims to become one of the largest passive house projects in North America. By incorporating advanced energy efficiency principles and construction techniques, the building will provide much needed student housing to accommodate both short- and long-term campus growth projections. Consulting work commenced in mid-2020, with an anticipated project completion of December 2022.
1075 Nelson
As co-designer of 1075 Nelson, a 178-metre-high skyscraper in Vancouver, IBI will demonstrate that contemporary, high-rise architecture can be more sustainable to conform to rigorous passive house standards. Set to become the tallest passive house building in the world, it will contain 480 residential units, and was approved for development in November 2020.
The Future of Business
'Net-Zero' Stakeholder Engagement
Through our Digital Engagement Venues (DEVs), IBI is delivering innovative virtual stakeholder engagement and internal programming on a global scale that significantly reduces carbon emissions. Our fully interactive platform incorporates individual and group chats, live-streamed broadcasts, and bespoke content strategies, while eliminating the need for travel and expensive food and beverage programs.
Supporting this initiative, our data analytics team produced a carbon footprint calculator, which compared IBI's physical Canada West Leadership Summit held in person in 2019 with the virtual event held in 2020.
The impact of going virtual with this single event roughly equates to eliminating the annual CO2 emissions of more than eight cars2.
Kilograms of CO2 Produced from Canada West Leadership Summit
(In-Person)
(Virtual)
IBI's Carbon Footprint Calculator
Parametric modelling uses the processing power of a computer to optimize design and planning. Data that would take us months to analyze manually can be assessed in a matter of seconds by our parametric design team, allowing us to efficiently interpret data to understand the carbon impact of our projects. IBI's dynamic carbon model enables designers to calculate the total energy required to produce a project, and can even show how the choice of material used can impact that project's carbon footprint. It also allows designers to model the potential for carbon sequestration (the process of removing or capturing carbon from the atmosphere over the long term), a critical factor in net-zero and net-positive design solutions. The model has been developed to integrate with open-source databases like EC3, a tool created through a not-for-profit alliance of architecture, engineering and construction organizations, including the American Association of Architects (AIA) and the Carbon Leadership Forum.
Driving Clean and Efficient Energy
In late 2019, IBI took an equity investment in SWTCH, a company providing turnkey solutions for electric vehicle (EV) charging and energy management, specifically in multi-tenant buildings. With growing EV adoption, SWTCH identified the potential for utilities to benefit from the integration of intelligent EV charging, optimized grid management and energy storage technologies. SWTCH is running an innovative pilot at our Toronto headquarters to demonstrate how an EV charging management platform with bi-directional charging capabilities (back and forth between the EV charging station and the power grid) can significantly reduce the costs of EV charging, while enhancing the overall efficiency of the grid.
Bolstering Our Environmental Capabilities
In December 2020, IBI acquired the assets of Cole Engineering, strengthening our capabilities and competitiveness in water and wastewater demand modelling and planning, water infrastructure and facilities design, and providing opportunities to enhance our BlueIQ™ water operations optimization solution. Additionally, Cole's training and accreditation program for water facilities staff directly aligns with our priorities in operations optimization and asset management.
In February 2021, IBI acquired Peters Energy Solutions Inc., augmenting our existing energy management business and work in sustainable community development. This acquisition bolsters our capabilities in renewable energy consulting, including solar, wind and energy storage, and the environmental, regulatory and economic considerations of planning, designing and delivering green energy solutions.
Taking a Stand to Combat Climate Change
IBI Group is proud to stand with the following organizations seeking to enable transformational climate solutions:
American Institute of Architects (AIA) 2030 Challenge
Royal Institute of British Architects Climate and Biodiversity Declaration
Royal Architectural Institute of Canada Climate Emergency Declaration
(1) Passive House Canada
(2) Natural Resources Canada – The average Canadian vehicle, which burns 2,000 L of gasoline every year, releases about 4,600 kg of CO2 into the atmosphere
Stephen Taylor, Senior VP & CFO
IBI Group Inc.
Tel +1 416 596 1930
Julia Harper, Manager, Global Communications
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Will salmon and steelhead once again spawn on the Mokelumne above Pardee?
Photo of Upper Mokelumne River (c) 2009, Katherine K. Evatt.
Record numbers of salmon and steelhead have returned to the Mokelumne River Fish Hatchery in recent years, but the outlook is even brighter with the release of a study that shows the possibility of the reintroduction of the iconic salmonid species in a 13.7 mile stretch of the Mokelumne above Lake Pardee.
The California Department of Fish and Wildlife reported a modern record of more than 19,900 fall-run Chinook salmon returned to spawn in the lower Mokelumne in the fall of 2017. The 2018 fall salmon returns were also impressive, with a total return of 17,474 fish.
These record runs have contributed greatly to the ocean recreational and commercial fishery over the past couple of years. The Mokelumne, a relatively small river, provided 33 percent of the Central Valley fall Chinooks caught in the recreational fishery and 43 percent of the commercial fishery in 2018, according to the Pacific Fishery Management Council (PFMC).
If a pilot study bears fruit, salmon and steelhead may be once again spawning in the Mokelumne above Pardee, making the magic taking place in CDFW and EBMUD's stewardship of the lower Mokelumne extend into some of the salmon's historic upstream habitat.
With that in mind, the Foothill Conservancy, East Bay Municipal Utility District, other nonprofit groups, businesses, state and local agencies, and tribal interests — working together as the Upper Mokelumne River Salmonid Restoration Team — have been exploring the potential to restore fall-run Chinook salmon to the upper Mokelumne River, according to a news release from the Conservancy and EBMUD.
The recently released study conducted by Cramer Fish Sciences shows that 13.7 miles of Mokelumne River above Pardee show the potential to support both spawning and rearing of chinook salmon.
"The next step in exploring this potential will be a pilot project, likely reintroducing a small number of salmon upstream of Pardee Reservoir," according to the Conservancy and EBMUD.
"Those 'excess' fish could be available for direct reintroduction into the upper watershed without reducing the hatchery production. This new study shows that we may be able to return some of the returning salmon to their historic spawning habitat in the upper Mokelumne for the first time since Woodbridge and Pardee dams were built early in the last century," said Bell.
Reintroducing salmon to the watershed could provide positive ecosystem-level impacts by restoring marine nutrients to the river and watershed and food for fish and wildlife, according to the scientists.
The Mokelumne's salmon are critical to California's recreational and commercial salmon fishery. The Mokelumne and other Central Valley salmon stocks generate millions of dollars every year from Morro Bay in the South to Shelter Cove in the north.
Michelle Workman, Supervising Fisheries Biologist for East Bay Municipal Utility District, said the Upper Mokelumne Salmon Restoration Team has been "working diligently" to develop the science and engage multiple stakeholders and agencies to help move this process from planning to implementation.
"We look forward to continuing this valuable collaboration for the benefit of the Mokelumne River fishery and for the enjoyment of a renewed public trust resource in the river above Pardee Reservoir," said Workman.
Grants from the Lower Mokelumne River Partnership and the Firedoll Foundation funded the salmon habitat study.