Etanercept suppresses regenerative hyperplasia in psoriasis by acutely downregulating epidermal expression of interleukin (IL)-19, IL-20 and IL-24.

BACKGROUND: Psoriasis is a Th17/Th1-mediated skin disease that often responds to antitumour necrosis factor (TNF)-α therapies, such as etanercept. OBJECTIVES: To better define mechanisms by which etanercept improves psoriasis and to gain insight into disease pathogenesis. METHODS: We investigated the early biochemical and cellular effects of etanercept on skin lesions in responder patients prior to substantial clinical improvement (≤ 4 weeks). RESULTS: By 1 week, etanercept acutely suppressed gene expression of the interleukin (IL)-20 subfamily of cytokines (IL-19, IL-20, IL-24), which were found to be predominantly epidermis-derived and which are implicated in stimulating epidermal hyperplasia. Additionally, by 1 week of therapy, suppression of other keratinocyte-derived products (chemokines, antimicrobial proteins) occurred, while suppression of epidermal regenerative hyperplasia occurred within 1-3 weeks. Th17 elements (IL-23p19, IL-12p40, IL-17A, IL-22) were suppressed by 3-4 weeks. In vitro, TNF-α and IL-17A coordinately stimulated the expression of the IL-20 subfamily in normal keratinocytes. CONCLUSIONS: Based on the rapid suppression of regenerative hyperplasia, chemokines and other keratinocyte-derived products, including the IL-20 subfamily, we propose that epidermal activation is a very early target of etanercept. As many of these keratinocyte markers are stimulated by TNF-α, their rapid downregulation is likely to reflect etanercept's antagonism of TNF-α. Additionally, decreased epidermal hyperplasia might result specifically from acute suppression of the IL-20 subfamily, which is also a likely consequence of etanercept's antagonism of TNF-α. Thus, the IL-20 subfamily has potential importance in the pathogenesis of psoriasis and therapeutic response to etanercept.

Monocyte induction of IL-10 and down-regulation of IL-12 by iC3b deposited in ultraviolet-exposed human skin.

CD11b+ monocytic/macrophagic cells (Mo/Mph), which infiltrate into skin after UV irradiation, play an important role in UV-induced immunosuppression. Because in mice, blockade of CD11b (iC3b receptor) on monocytes and depletion of its ligand, iC3b, reverses UV-induced immunosuppression, we asked whether iC3b is deposited in human skin after UV, and whether iC3b can modulate the cytokine profile of Mo/Mph. Immunofluorescence studies revealed that iC3b was newly deposited in UV-exposed skin and was localized in apposition to infiltrating CD11b+ Mo/Mph. In addition, in situ hybridization studies showed that TNF-alpha mRNA was also induced in a similar microanatomic localization. To model the effects of these complex signals on infiltrating Mo/Mph following UV exposure, we then tested the effects of immobilized iC3b and TNF-alpha on resting blood monocytes. Both IL-10 mRNA synthesis and protein secretion were significantly induced by binding of iC3b in vitro and were synergistically increased by the presence of TNF-alpha. The effect was abrogated by a blocking Ab to CD11b, indicating CD11b-iC3b interaction. In contrast, iC3b binding resulted in suppression of IL-12 p40 mRNA and significantly inhibited the production of IL-12 p70 protein. Our studies thus define a novel mechanism for induction of tissue Mo/Mph into an IL-10high/IL-12low state via iC3b in combination with TNF-alpha.

Activated complement component 3 (C3) is required for ultraviolet induction of immunosuppression and antigenic tolerance.

Complement component 3 (C3), a critical regulator of innate immunity, may also play a role in the regulation of cognate immunity, such as contact sensitivity responses. Because ultraviolet (UV) radiation also activates C3 in the skin, we determined whether the immunosuppressed state that results when a contact sensitizer is applied through UVB-exposed skin requires the presence and activation of C3. This question was addressed through the use of C3-deficient mice, blockade of C3 cleavage to C3b, and accelerated degradation of iC3b by soluble complement receptor 1 (sCR1). Both C3-modulated systems totally reversed the failure to induce a contact sensitivity response to dinitrofluorobenzene (DNFB) upon primary sensitization at the UV-exposed site, as well as immunologic tolerance to a second DNFB immunization through normal skin. Treatment with sCR1 reduced the infiltration of CD11b+ leukocytes into the epidermis and dermis of UV-irradiated skin but did not reverse the UV-induced depletion of epidermal class II MHC+CD11blo Langerhans cells. These data, taken together with previous results showing abrogation of locally induced UV immunosuppression by in vivo anti-CD11b treatment, suggest a novel mechanism by which ligation of the leukocyte beta2 integrin, CD11b, by iC3b molecules formed from C3 activation in UV-exposed skin, modifies cutaneous CD11b+ cells such that skin antigen-presenting cells are unable to sensitize in a primary immune response, but actively induce antigenic tolerance.

The human hair follicle: a reservoir of CD40+ B7-deficient Langerhans cells that repopulate epidermis after UVB exposure.

The ability of skin to maintain its protective structural and functional integrity depends on both resident and circulating cells. Until now, it was thought that dendritic antigen presenting cells of epidermis (Langerhans cells) were replaced by circulating bone marrow derived precursors. Here we show by immunostaining studies of timed biopsies taken from human skin after ultraviolet exposure, that hair follicle is a critical reservoir of Langerhans cells that repopulate epidermis depleted of Langerhans cells by a single four minimal erythema dose of ultraviolet B. Immunostaining with antibodies to thymidine dimers showed that ultraviolet B only penetrated the superficial hair follicle opening, whereas deeper follicle was relatively protected. Langerhans cells migrating from hair follicle into epidermis 72 h after ultraviolet exposure have a partial deficiency of molecules important to T cell costimulation. We used four color flow cytometry to show that Langerhans cells isolated from epidermis 72 h after ultraviolet B can upregulate CD40 but not B7-1 or B7-2 expression in culture, suggesting a different phenotype of hair follicle Langerhans cells. Therefore, the hair follicle is a specialized immune compartment of the skin that serves as an intermediate reservoir of Langerhans cells between bone marrow and epidermis, and that may play a critical role in immune surveillance.

Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.

In addition to being T lymphocyte-driven, psoriasis may be due in part to abnormal integrin expression. Normal-appearing (uninvolved) skin from psoriatic patients was examined to determine whether altered fibronectin or its receptor expression is detectable before development of psoriatic lesions. In contrast to skin from normal subjects, we detect by immunofluorescence the abnormal presence of plasma fibronectin in the basal cell layer of the epidermis of psoriatic uninvolved skin. Furthermore, increased fibronectin exposure superinduces the in vitro cell cycle induction and expansion of psoriatic nonlesional keratinocytes in response to a cocktail of T cell lymphokines. Fibronectin alone also appeared to increase cell cycle entry among uninvolved but not normal keratinocytes. Concordantly, the alpha5 integrin fibronectin receptor, but not alpha2 or alpha3, is overexpressed in the in vivo nonlesional psoriatic epidermis. The involvement of alpha5beta1 in the early outgrowth of clonogenic keratinocytes in the ex vivo culture was demonstrated by the ability of anti-alpha5 mAb to inhibit keratinocyte growth on fibronectin. Thus, the fibronectin receptor appears to be one of the components required for the development of the hyperresponsiveness of psoriatic keratinocytes to signals for proliferation provided by lymphokines produced by intralesional T lymphocytes in psoriasis.

Reversal of immunosuppression inducible through ultraviolet-exposed skin by in vivo anti-CD11b treatment.

In both human in vitro models and murine in vivo adoptive transfer studies, UV-induced class II MHC+ CD11b+ leukocytes that infiltrate the epidermis appear to mediate UV-induced immunosuppression. In the present study, their role is further probed using an anti-CD11b mAb (clone 5C6), which is effective in vivo in blocking CD11b+ monocyte/macrophage diapedesis into inflammatory lesions. A single exposure, low dose UV protocol (72 mJ/cm2) that resulted in tolerance only when dinitroflurobenzene was applied 48 h later through the UV-irradiated skin, but not through a distant non-UV-irradiated site, was used. In vivo anti-CD11b treatment in non-UV-irradiated mice did not block contact sensitivity responses. However, the ability to induce a primary contact sensitivity response was completely restored in UV-irradiated mice receiving anti-CD11b. This restoration was associated with partial restoration of papillary dermal class II MHC+ NLDC-145- cells. In vivo anti-CD11b treatment also blocked tolerance induction, which was associated with a 50% reduction in the infiltration of class II MHC+ CD11b+ Gr-1+ monocyte/macrophages into UV-irradiated skin. In addition, anti-CD11b treatment partially protected against epidermal UV injury, in that the epidermal structure was better preserved and the keratinocytes were less severely damaged. CD11b+ leukocytes may thus affect UV-irradiated skin through at least two mechanisms: 1) a class II MHC+ CD11b+ Gr-1+ monocyte/macrophage population inducing a state of tolerance to Ag(s) acquired in UV-irradiated skin, and 2) CD11b+ leukocytes capable of inflicting additional injury to both keratinocytes and constitutive APC damaged by UV photons.

Temporal correlation between UV radiation locally-inducible tolerance and the sequential appearance of dermal, then epidermal, class II MHC+CD11b+ monocytic/macrophagic cells.

We performed a time course study in order to define the in vivo relationship between the induction of active suppression of contact sensitization and the presence of various cells in ultraviolet-exposed dermis and epidermis implicated in locally inducible immune tolerance: class II major histocompatibility complex (MHC)+CD11b(lo)Gr-1- Langerhans cells (LC), class II MHC-CD45+CD3+ dendritic epidermal T cells, class II MHC+CD11b+Gr-1- monocytes or class II MHC+CD11b+Gr-1+ monocytic/macrophagic cells. Partial tolerance (50%) was first detectable 6 h after a single 72 mJ/cm2 ultraviolet B exposure and maximum tolerance at 48 h post-ultraviolet exposure. By flow cytometry, a low granularity LC subset had disappeared from the epidermis within 6 h after ultraviolet exposure, followed by a slower decrease in the high granularity Langerhans cells subset. Within the dermis at the 6-h time point, small numbers of infiltrating monocytic/macrophagic cells are already apparent. By 24 h post-ultraviolet exposure, at which time tolerance has increased to 70%, the infiltrating monocytic/macrophagic population had risen to 1.2% of the total dermal cell population and was observed for the first time in the epidermis along with other infiltrating leukocytes (i.e., polymorphonuclear leukocytes). By 48 h post-ultraviolet exposure, when a state of maximum tolerance is obtained, both constitutive epidermal and dermal antigen-presenting cell populations were at or near their nadir of depletion. The infiltrating monocyte/macrophage population, however, exhibited a dramatic increase in the epidermis at 48 and 72 h. Thus, the ability to locally induce a state of in vivo tolerance is closely associated with the expansion of class II MHC+CD11b+Gr-1+ and -monocytic/macrophagic cells in the dermis and epidermis.

Differential regulation of IL-1 and IL-1 receptor antagonist in HaCaT keratinocytes by tumor necrosis factor-alpha and transforming growth factor-beta 1.

Cytokines such as TNF alpha and TGF beta 1 have potent effects on keratinocyte differentiation and have been implicated in cutaneous injury, immunologic reactions, and wound healing. To determine whether such conditions might alter the balance of epidermal keratinocyte IL-1 and the IL-1 receptor antagonist (IL-1ra), TNF alpha and TGF beta 1 were added to HaCaT cells, a human adult keratinocyte cell line. mRNA levels of IL-1 alpha, IL-1 beta, and IL-1Ra were detected by polymerase chain reaction (PCR) on reverse transcribed RNA extracts, followed by Southern blot of the PCR products, 35S-labeled probe hybridization, and quantification against standard curves. TNF alpha (100 ng/ml) at the 3-h time point significantly induced increases in mRNA expression of II-1 alpha (9.2 +/- 2.9 fold increase) and IL-1 beta (2.5 +/- 0.7 fold increase) (n=7) which were concordant with increases in IL-1 alpha protein (7.1 +/- 1.3 fold increase) and Il-beta protein (4.4 +/- 1.0 fold increase) measured by ELISA 24 h after stimulation. By contrast, icIL-1Ra mRNA and protein levels were not affected by TNF alpha. TGF beta 1 induced a mild increase in IL-1 alpha mRNA (3.8 +/- 1.8 fold) and protein (3.5 +/- 1.2 fold). TGF beta 1 did not affect IL-1 beta mRNA levels but caused variable increases in IL-1 beta protein levels. TGF beta 1 did not alter icIL-1Ra mRNA or protein levels. Inhibition of RNA synthesis with actinomycin D demonstrated that the rate of degradation of IL-1 beta mRNA was reduced by treatment with TNF alpha. This stabilization of IL-1 beta mRNA was specific, because TGF beta 1 did not stabilize IL-1 beta mRNA, and TGF beta 1 and TNF alpha did not increase the stability of Il-1 alpha mRNA. icIL-1Ra mRNA was fairly stable over a 20 hour period and its slow degradation was not affected by treatment with either TNF alpha or TGF beta 1, indicating a higher steady state stability of icIL-1ra mRNA relative to IL-1 mRNA's. Given the high rate of degradation of IL-1 alpha and IL-1 beta mRNA, levels of these mRNAs may rapidly decrease while the icIL-1ra mRNA levels remain constant, thus allowing for rapid dampening of IL-1 activity soon after the stimuli provoking an inflammatory or reparative response have abated. In conclusion, TNF alpha and TGF beta 1, cytokines with potent effects on inflammation and differentiation, both induce keratinocyte IL-1 alpha mRNA and protein levels, but differentially regulate IL-1 beta mRNA. They both exert little effect on IL-1 Ra levels, which were constitutively highly stable. Such differential regulation provides mechanisms for separately controlling the relative activity of these cytokines under normal and disordered conditions.

Identification of a human dermal macrophage population responsible for constitutive restraint of primary dermal fibroblast proliferation.

Primary human dermal cell suspensions prepared from the papillary dermis of keratomed skin strips were used to investigate the effect of indigenous dermal macrophages (HLA-DR+, CD11c+ CD11b+ CD1c- phagolysosome+) upon dermal fibroblast proliferation. Rapid dermal fibroblast expansion was induced upon immunomagnetic bead removal of CD11b+ or CD11c+ cells as well as by removal of more inclusive subsets contained within the DR+ population, but the removal of mast cells, endothelial cells, and CD1c+ dermal Langerhans cells from dermal cell suspensions failed to result in proliferation of the remaining cell subsets. Removal of 1B10+ fibroblasts from macrophage depleted (CD11b-) dermal cell suspensions essentially abrogated the unrestrained proliferation of the CD11b- dermal cells. Flow cytometric cell cycle analysis of cultured macrophage-depleted dermal cells confirmed that the unrestrained proliferating cells contain procollagen I+ as well as procollagen I- dermal fibroblasts. Inhibition of primary fibroblast expansion by adding a supernatant from unfractionated dermal cells suggested that a growth-inhibitory soluble activity of >30,000 kDa dominates the cytokine mixture released by unfractionated fresh dermal cells ex vivo. Inhibitory activity counterbalanced positive fibroblast growth- stimulatory cytokines released by dermal cells because neutralizing antibodies to insulin-like growth factor 1 and interleukin-1 beta resulted in decreased CD11b- dermal cell fibroblast proliferation. These data indicated an important role for dermal macrophages of the DR+ CD11b+ CD11c+ DC1c- phenotype in the normal homeostatic restraint of primary human dermal fibroblast proliferation.

Intralesional T-lymphocyte activation as a mediator of psoriatic epidermal hyperplasia.

An early cellular event in the development of psoriatic lesions is infiltration of target tissue by macrophages and activated T lymphocytes. Lesional psoriatic skin contains activated memory T lymphocytes with production of mRNA for lymphokines such as interleukin-2, interferon-gamma, and tumor necrosis factor-alpha that is elevated relative to normal or uninvolved psoriatic skin. That the T-cell activation and cellular lymphokine production have a crucial role in the maintenance of epidermal hyperplasia in the psoriatic lesion is indicated by the beneficial effect of immunosuppressive agents in the treatment of psoriasis (cyclosporin A, FK506, anti-CD3, anti-CD4). A link between immune activation and psoriasis is also indicated by immunogenetic associations in this disease. Also, psoriatic keratinocytes appear to have been modulated by T-cell lymphokines in vivo, because they abnormally express molecules uniquely induced on keratinocytes by the T-cell product interferon-gamma. Indeed, T cells producing interferon-gamma have been cloned from psoriatic lesions, and they are able to induce keratinocyte class II major histocompatibility complex and intercellular adhesion molecule expression. These lesion-derived T-cell clones can induce growth of keratinocytes, and specifically lesional psoriatic T cells produce factors that induce increased keratinocyte colony formation, as well as increased cell cycle entry of the normally quiescent stem cell population. Interferon-gamma, although a growth inhibitor on its own, acts cooperatively with other T-cell-produced growth factors to cause keratinocyte growth induction. Furthermore, relative to normal stem cells, keratinocyte stem cells (beta 1 integrin+ K1/K10-) in psoriatic uninvolved epidermis are significantly hyperresponsive to the growth-stimulatory lymphokine milieu created by lesional T lymphocytes. Whether such abnormalities in responsiveness are associated with new genetic linkages reported in families of psoriasis patients is unknown. As the epidermis of lesional psoriatic skin can be demonstrated to produce elevated levels of factors that can further potentiate T-cell activation, a self-sustaining cycle can be constructed of T-cell recruitment, intralesional activation, release of factors that preferentially stimulate psoriatic epidermal stem cells to proliferate, and further epidermal potentiation of the T-cell-mediated lesions.

Active induction of unresponsiveness (tolerance) to DNFB by in vivo ultraviolet-exposed epidermal cells is dependent upon infiltrating class II MHC+ CD11bbright monocytic/macrophagic cells.

Contact sensitizers, tumor Ags, and microbial pathogens presented through UV-exposed skin result in T cell-mediated immune tolerance (inhibition of acquisition of responsiveness) to these normally potent immunogens. The APC in UV-exposed skin that delivers the signals inducing tolerance remains highly controversial and is the subject of this study. Application of the contact sensitizer, 2,4-dinitro-1-fluorobenzene (DNFB), to C3H/HeN mice immediately after a single dose of 72 mJ/cm2 UVB (138 mJ/cm2 total UVB) resulted in unresponsiveness to an initial DNFB ear challenge, but failed to block the development of responsiveness after a second sensitization on previously unexposed skin (no tolerance). A state of tolerance could only be achieved if a delay of 72 h was allowed to elapse between the UV exposure and the initial sensitization. Epidermal cell suspensions (EC) were prepared from the skin of normal controls and from skin exposed to the same UV dose 3 days before (UV-EC). Three days after in vivo UV exposure, Langerhans cells (CD11blow Ia+) were depleted, and CD11bbright Ia+ macrophages had appeared in the epidermis along with GR-1+ neutrophils. Intracutaneous injection of 2,4 dinitrobenzenesulfonic acid (DNBSO3)-haptenated UV-EC, but not normal controls, resulted in the induction of locally inducible Ag-specific tolerance to DNFB, indicating the presence and dominance of tolerogenic signal within in vivo-irradiated epidermis. Removal of CD11b+ and class II MHC+ cells within UV-EC showed that a CD11b+ class II MHC+ population was indeed critical for tolerance induction. In addition, tolerance induction by UV-EC was not a result of surviving, UV-exposed Langerhans cells, because haptenated 3-day cultured EC from epidermis removed 5 h after UV exposure (before leukocytic infiltration) failed to induce a tolerogenic state. In conclusion, the ability of UV-exposed skin to induce peripheral adult tolerance to a normally potent immunogen is critically dependent on inflammatory class II MHC+, CD11bbright monocytic/macrophagic cells that infiltrate UV-irradiated skin at the same time the ability to tolerize is acquired.

CD11b+ macrophages that infiltrate human epidermis after in vivo ultraviolet exposure potently produce IL-10 and represent the major secretory source of epidermal IL-10 protein.

Because activated human macrophages can be potent sources of IL-10, and because immunosuppressive tolerance-inducing macrophages populate the skin after UV exposure, we determined whether IL-10 is induced after UV exposure of human skin and whether it is related to the immigrating macrophages. Keratomes were obtained from control skin or from skin obtained 72 h after a single exposure to four minimal erythemal doses of UVB. Quantitative reverse-transcriptase PCR on total RNA extracted immediately from skin keratomes showed that IL-10 mRNA was elevated in UV-exposed skin. Epidermal cell suspensions from non-UV-exposed keratomes (C-EC) and UV-exposed keratomes (UV-EC) were fractionated by sequential immunobead selection. IL-10 mRNA was reproducibly 200- to 400-fold higher in CD11b+ UV-EC (macrophages) relative to CD11b- UV-EC (keratinocytes). IL-10 mRNA was not detected in C-EC that contained the CD1a+ population (Langerhans cells) nor in CD1a- C-EC keratinocytes from normal skin. As determined by ELISA, CD11b+ UV-EC IL-10 cell-associated protein was fivefold higher than that of CD11b- UV-EC; this was confirmed by flow cytometric visualization of IL-10 protein in permeabilized cells. CD11b+ UV-EC macrophages secreted IL-10 protein into the supernatant at a level of 333 +/- 51 pg/10(6) cells, whereas UV-EC keratinocytes did not secrete detectable levels of IL-10 (n = 3), although UV did induce low levels of IL-10 mRNA and cell-associated protein in keratinocytes. Therefore, although human keratinocytes accumulate intracellular IL-10 after in vivo UV exposure, the most potent production and secretion of IL-10 in the epidermis seems to be that of UV-induced macrophages. Skin-infiltrating macrophage secretion of such a potent immunoregulatory cytokine may account for the delayed immunosuppressive environment of sunburned skin and the altered APC activity of the infiltrating macrophages.

Distinction of class II MHC+ Langerhans cell-like interstitial dendritic antigen-presenting cells in murine dermis from dermal macrophages.

Dermal cells are capable of initiating contact-hypersensitivity responses but the precise identification of the antigen-presenting cell within murine dermis is lacking. Class II major histocompatibility complex (MHC)+ cells with dendritic shape and lacking endothelial factor VIII but expressing the dendritic antigen-presenting cell marker NLDC-145 were observed in the perivascular and interstitial dermis of BALB/c and C3H/HeN skin. The heterogeneous class II MHC+ cells could be divided into two subsets: each was class II MHC+ CD45+ (bone marrow derived) GR-1- (non-neutrophil/macrophage) CD3- (non T), but one subset was CD11b+ (beta 2 integrin) and the other was CD11b-. Ultrastructural examination of class II MHC+ cells revealed the presence of a Langerhans cell-like/indeterminant cell subset with indented nuclei, dendritic morphology, active cytoplasm, and dense intermediate filaments. Phagolysomes and Birbeck granules were not observed in such cells, indicating these were distinct from dermal macrophages and from classical epidermal Langerhans cells, respectively. Cells with a monocyte/macrophage ultrastructural appearance were also noted, likely representing the class II MHC subset expressing CD11b and Ly6c (monocyte/endothelial antigen). Dermal cells in suspension were capable of processing and presenting large protein antigens to antigen-specific T-cell hybridomas; dermal cells also induced the syngeneic mixed lymphocyte reaction. The dermal antigen-presentation activities were totally abrogated by removal of class II MHC+ cells, but not by removal of CD11b+ cells or Ly6c+ cells, indicating that potent antigen-presenting cell activity was restricted to the class II MHC+ CD11b- Ly6c- subset (Langerhans cell-like/indeterminant cells). In conclusion, within a complex array of dermal leukocytes a murine dermal class II MHC+ cell population expressing a Langerhans cell-like/dendritic antigen-presenting cell phenotype and exhibiting potent antigen processing and presenting activity can be identified. The positioning of potent interstitial dendritic antigen-presenting cells at the interface of the vasculature with the dermal interstitium provides rapid access to an antigen-presenting cell as T cells first egress into the skin.

Cyclosporin A rapidly inhibits epidermal cytokine expression in psoriasis lesions, but not in cytokine-stimulated cultured keratinocytes.

To better understand the cellular target(s) of cyclosporin action in psoriasis, we have studied the effects of systemic short-term (7 d), low-dose (3-7.5 mg/kg) cyclosporin A administration on the expression of the cytokines interleukin (IL)-8 and IL-1 beta in psoriatic lesions. RNA blot hybridization analysis of pretreatment keratome biopsies revealed that expression of both cytokine mRNAs was highly variable from patient to patient. Significant covariation of both cytokine mRNA levels was noted (r = 0.86, p < 0.0001). However, there was no significant correlation between expression of either cytokine and clinical severity, as measured by the pretreatment Psoriasis Area and Severity Index (PASI). IL-1 beta protein levels measured by enzyme-linked immunosorbent assay (ELISA) were highly correlated with IL-1 beta mRNA levels, indicating that the differences in transcript levels accurately reflect differences in epidermal cytokine protein. Significant reductions in both cytokine transcripts and in IL-1 beta immunoreactive protein were noted in the high expression subgroup after 1 week of cyclosporin A therapy, prior to detectable clinical improvement. In contrast to its pronounced effects on epidermal cytokine expression in vivo and the allogeneic mixed lymphocyte reaction in vitro, cyclosporine A did not inhibit the induction of intercellular adhesion molecule (ICAM)-1 or IL-8 mRNAs by cultured keratinocytes in response to IL-1 beta or the combination of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. These data suggest that epidermal keratinocytes respond to signals produced by activated T cells by coordinate expression of multiple cytokines, and that cyclosporin A acts primarily through blockade of T cells, rather than through keratinocyte activation.

Neutrophils, differentiated macrophages, and monocyte/macrophage antigen presenting cells infiltrate murine epidermis after UV injury.

We asked whether, as in humans, a population of antigen-presenting macrophages infiltrates the epidermis of ultraviolet (UV)-exposed BALB/c mice. Using three-color flow cytometry on cell suspensions plus in situ immunofluorescence microscopy, the phenotype of normal Langerhans cells was class II major histocompatibility complex (MHC+), CD11b+, NLDC-145+, BM8+ CD45+ and homogeneous. By contrast, in epidermal cells harvested 3 d following UV (UV-EC), there were two subsets of class II MHC+ cells: 1) class II MHChi CD11b+, and 2) class II MHClo CD11b-. Neither expressed the Langerhans cell markers BM8 and NLDC-145. In addition, there were two major populations of class II MHC- CD11b+ cells; half of these expressed the GR-1 neutrophil marker. Langerhans and dendritic epidermal T cells were markedly reduced after UV injury. By electron microscopy, immunomagnetic bead-purified CD11b+ cells in UV-EC were comprised of neutrophils, differentiated macrophages, and mononuclear cells with prominent lysosomes, but no Birbeck granules; the class II MHC+ subset resembled a monocytic cell in between differentiated macrophages and indeterminate dendritic cells. Functionally, immediately following in vivo UV exposure, the allogeneic antigen-presenting cell capacity of UV-EC was reduced to 21 +/- 6% of control epidermal cells (C-EC); by 3 d, antigen-presenting cell activity of UV-EC had recovered to 59 +/- 11% of C-EC, although at this time NLDC-145+ Langerhans cells had reached their lowest number. The recovered antigen-presenting cell activity was critically dependent upon the class II MHChiCD11b+ cells. Sensitization of BALB/c mice through skin that contained these antigen-presenting cells (3 d after UV) resulted in tolerance to dinitrofluorobenzene. By contrast, sensitization through UV-exposed skin immediately after the exposure resulted in unresponsiveness without tolerance, demonstrating temporal association of tolerance with leukocytic infiltration. In summary, murine epidermis responds to an acute UV injury in vivo with an initial abrogation of antigen-presenting activity followed by epidermal infiltration with neutrophils, differentiated macrophages, and monocytic antigen-presenting cells that are distinct from Langerhans cells with regard to expression of Langerhans cell markers and ultrastructure.

T-cell activation is potentiated by cytokines released by lesional psoriatic, but not normal, epidermis.

BACKGROUND AND DESIGN: T-cell activation appears to be critical for the maintenance of psoriatic lesions. In this study, we determined whether cytokines released by epidermal cells from psoriatic lesions are providing signals that result in propagation of intralesional T-cell activation. Supernatants were obtained from epidermal cell cultures derived from skin biopsy specimens of psoriatic patients and normal subjects. These supernatants were added to purified normal CD4+ T cells activated via T-cell receptor (immobilized anti-CD3 and fibronectin) or via other activating pathways (anti-CDw60 or UM4D4). RESULTS: Psoriatic supernatants (n = 9), but not normal supernatants (n = 7, P < .0006), potentiated T-cell stimulation with anti-CD3 and fibronectin to 172% +/- 41% over control stimulation levels. The degree of lesional psoriatic epidermal cell potentiation correlated with the clinical severity of the lesion (r = .82, P = .007). Psoriatic epidermal cytokine potentiation of T-cell activation was not limited to T-cell receptor mediated stimulation; potentiation of anti-CDw60-stimulated CD4+ T cells was also observed. Neutralizing antisera to interleukin 1 and interleukin 8, but not interleukin 6, were found to reduce only partly the observed potentiation of T-cell activation. To determine whether cyclosporine is down modulating T-cell-potentiating cytokine activity in psoriasis, we compared samples obtained during a double-blind clinical trial of intralesional cyclosporine. T-cell-potentiating activity from psoriatic lesional sites treated with cyclosporine was not significantly modulated relative to the activity derived from vehicle-treated or untreated sites. CONCLUSION: These data demonstrate that lesional psoriatic epidermal cells release a balance of cytokines that potentiate T-cell activation. Because normal epidermal cells do not potentiate T-cell activation in this system, these findings demonstrate a mechanism by which the epidermis may non-specifically potentiate and perpetuate T-cell activation in psoriatic lesions.

Retinoic acid and phorbol ester synergistically up-regulate IL-8 expression and specifically modulate protein kinase C-epsilon in human skin fibroblasts.

Phorbol ester (TPA) and retinoic acid (RA) are two potent immunomodulatory agents whose actions are mediated through distinct signal transduction pathways involving protein kinase C (PKC) and nuclear RA receptors, respectively. We have investigated the interactions between these two pathways in the regulation of expression of the inflammatory cytokine IL-8 in human skin fibroblasts. TPA (as previously reported) and RA both induced IL-8 mRNA and protein in a time- and dose-dependent manner. IL-8 mRNA induction by TPA (10 nM) was maximal (15-fold) within 6 h, and returned to baseline within 24 h of treatment, although maximal induction (10-fold) by RA (1 microM) did not occur until 24 h posttreatment. Induction of IL-8 by TPA was blocked by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, which inhibits PKC and cAMP-dependent protein kinases (PKA), but not by N-(2-ganidinoethyl)-5-isoquinoline sulfonamide, which preferentially inhibits PKA, consistent with the participation of PKC in the induction of IL-8 by TPA. In contrast, induction of IL-8 by RA was inhibited by both 1-(5-isoquinoline sulfonamide and N-(2-gamidinoethyl)-5-isoquinoline sulfonamide, suggesting the participation of PKA in the induction of IL-8 by RA. However, activation of PKA by addition of cAMP analogues was not sufficient to induce IL-8 expression. Induction of IL-8 by RA also did not appear to be mediated indirectly through induction of IL-1, because addition of IL-1R antagonist did not block IL-8 induction by RA. RA and TPA added in combination synergistically enhanced expression of IL-8 mRNA, measured at 6 (2-fold) and 24 h (10-fold) posttreatment. To investigate the mechanism of this synergy, the effect of TPA and RA on fibroblast PKC activation and PKC isozyme levels were determined. TPA, either alone or together with RA, but not RA alone, stimulated phosphorylation of an endogenous 80-kDa PKC substrate. Dermal fibroblasts expressed three PKC isozymes (alpha, (delta, and (epsilon). TPA, but not RA, down-regulated PKC-alpha, neither TPA or RA affected the level of PKC-delta, and both TPA and RA down-regulated PKC-epsilon. This latter effect was enhanced 2-fold by addition of RA and TPA together. These data suggest that modulation of PKC-epsilon may be a common participant in the regulation of IL-8 expression by TPA and RA.

Psoriatic skin reveals the in vivo presence of an epidermal IL-1 inhibitor.

Production of inhibitor(s) of IL-1 activity can be induced in keratinocytes by exposure to UVB. We describe in this study the characterization of an endogenous constitutively expressed IL-1 inhibitor which is present in extracts of human psoriatic epidermal keratome biopsies. Size-fractionated extracts of normal human epidermis did not reveal IL-1 inhibitory factor(s) activity in normal epidermis. Psoriatic epidermal extracts, however, contained virtually no IL-1 bioactivity and inhibited the activity of recombinant human IL-1 beta. This IL-1 inhibitor has a molecular weight of approximately 30 kDa and a pI of 5.3, as revealed by fast protein liquid chromatography size fractionation and chromatofocusing of psoriatic epidermal extracts. IL-1 inhibitory activity was not blocked by neutralizing anti-TGF beta monoclonal antibody. It did not have any inhibitory effect upon normal cellular proliferation but could block the IL-1 induction of IL-2 production by LBRM.33 cells as late as 4 h after exposure of LBRM.33 cells to IL-1. Thus, in vivo human psoriatic epidermis expresses an IL-1 inhibitor that specifically inhibits IL-1 activity but which appears distinct from previously described UV-induced epidermal IL-1 inhibitory activity or TGF beta.

Ocular cicatricial pemphigoid occurring as a sequela of Stevens-Johnson syndrome.

Ocular cicatricial pemphigoid is a chronic scarring inflammation of the ocular mucosae that can lead to blindness. Although cicatricial pemphigoid has been classified clinically, histopathologically, and immunopathologically, no definite initiating factor or precipitating factor has been identified. In this report, we describe five cases of ocular cicatricial pemphigoid that developed following an acute episode of severe ocular inflammatory injury secondary to Stevens-Johnson syndrome. The time lag between the onset of Stevens-Johnson syndrome and cicatricial pemphigoid ranged from a few months to 31 years. All five patients had linear immune deposits characteristic of cicatricial pemphigoid along the basement membrane zone of mucosal biopsy specimens as detected by either direct immunofluorescence microscopy or direct immunoperoxidase staining. In two patients whose serum was tested, a Western blot assay taken of keratinocyte antigens that had undergone electrophoresis reacted with the serum and identified a 120-kd epidermal antigen detected by the IgG class antibodies. All five patients were treated with systemic immunosuppressive therapy for cicatricial pemphigoid, and we obtained objective responses. Severe ocular mucosal injury such as that which occurs in Stevens-Johnson syndrome may be a precipitating factor in the development of ocular cicatricial pemphigoid.

Elevated thymidine phosphorylase activity in psoriatic lesions accounts for the apparent presence of an epidermal "growth inhibitor," but is not in itself growth inhibitory.

An apparent tissue-specific growth inhibitor, or chalone, obtained from psoriatic lesions was tentatively identified in the 100-kDa fraction based upon inhibition of DNA synthesis, as measured by [3H]-thymidine uptake by a squamous cell carcinoma cell line, SCC 38. This fraction, however, failed to inhibit SCC 38 cell growth when assessed directly in a neutral red uptake assay. Characterization of the inhibitor of [3H]-thymidine uptake revealed it to have biochemical properties identical to thymidine phosphorylase: 1) molecular weight close to 100 kDa, 2) isoelectric point of 4.2, and 3) thymidine phosphorylase enzyme activity. Thus, we conclude that its ability to inhibit [3H]-thymidine uptake was due to thymidine catabolism rather than inhibition of DNA synthesis or growth inhibition. Examination of thymidine phosphorylase activity in keratome biopsies from psoriatic and normal skin demonstrated a twentyfold increase in activity in psoriatic lesions relative to non-lesional or normal skin. This increase in metabolism of thymidine was due to thymidine phosphorylase rather than uridine phosphorylase activity. The correlation between increased thymidine phosphorylase activity and increased keratinocyte proliferation in vitro (cultured) and in vivo (psoriasis), suggests that this enzyme may play a critical role in providing the thymidine necessary for keratinocyte proliferation.