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.

Basal keratinocytes from uninvolved psoriatic skin exhibit accelerated spreading and focal adhesion kinase responsiveness to fibronectin.

We previously proposed that the keratinocyte hyperproliferative state in psoriatic skin results from a combination of T cell cytokine interaction with basal keratinocytes that exist in a primed state. We now provide evidence that basal keratinocytes from psoriatic uninvolved skin are in a preactivated state with regard to their interaction with fibronectin. Freshly isolated basal keratinocytes (K(1)/K(10)(-)) from non-lesional psoriatic skin demonstrated a significantly higher percentage of spreading cells 1 h after plating on fibronectin-coated plates than keratinocytes isolated from normal skin (p =0.0002). No differences were observed on collagen-laminin-coated plates, however. The keratinocyte spreading on fibronectin-coated plates involved alpha 5 beta 1 and alpha V beta 1 integrins. To address the potential signaling cascades that may respond to integrin changes in psoriatic keratinocytes, focal adhesion kinase changes were assessed. The percentage of keratinocytes from psoriatic uninvolved skin that exhibit positive focal adhesion kinase staining was significantly greater than the percentage from healthy volunteers after 1 h incubation on fibronectin (p =0.006). Additionally, focal adhesion kinase isolated from uninvolved psoriatic keratinocytes had a greater degree of tyrosine phosphorylation. Thus, the proliferative effect of fibronectin in combination with T cell lymphokines on psoriatic uninvolved basal keratinocyte progenitors may be due to abnormal in vivo integrin-driven focal adhesion kinase activity and downstream signaling.

Overexpression of the oncofetal Fn variant containing the EDA splice-in segment in the dermal-epidermal junction of psoriatic uninvolved skin.

The extracellular matrix protein, Fn, has critical functions in cell attachment, migration, differentiation, and proliferation. We have previously shown that fibronectin (Fn) is abnormally expressed and potentiates entry into the cell cycle of basal keratinocytes in uninvolved psoriatic skin, in combination with T cell lymphokines. It is not known what type of Fn is present in psoriatic skin, however, and how this Fn may regulate signaling. Embryonic forms of cellular Fn containing extra domains, designated EDA and EDB, are generated by alternative splicing and are seen in proliferating, developing tissue and in wound healing. Because the EDA segment enhances the integrin binding sequence Arg, Gly, Asp (RGD), which, when present, has been shown to be critical in integrin-extracellular matrix signaling, we were particularly interested in determining whether or not EDA-containing Fn (EDA+Fn) represented the aberrantly expressed Fn in psoriasis. Increased EDA+ Fn protein was demonstrated by immunostaining at the dermal-epidermal junction in clinically uninvolved skin from six of six patients with psoriasis, but not in skin from control subjects. Using reverse transcription polymerase chain reaction an increased ratio of EDA+ Fn versus EDA- Fn mRNA was present in epidermal samples from psoriatic but not control individuals. Interestingly, the EDA+Fn in the psoriatic epidermis had the IIICS region spliced out (EDA+, FDB-, IIICS-, III9+), which was shared with normal epidermis (EDA-, EDB-, IIICS-, III9+). These results suggest a selective predominance of the EDA+ Fn isoform at the dermal-epidermal junction of psoriatic skin. The consistent aberrant localization of EDA+ Fn at the dermal-epidermal junction in uninvolved skin of psoriatics may confer the hyperresponsiveness of psoriatic uninvolved basal keratinocytes for rapid cellular proliferation in response to T cell signals. Key words: immunohistochemistry/integrin/keratinocyte/RT-PCR.

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.

Identification and quantitation of interferon-gamma producing T cells in psoriatic lesions: localization to both CD4+ and CD8+ subsets.

Interferon-gamma (IFN-gamma) produced by lesional T cell clones is critical for the induction into G1 of the cell cycle by psoriatic keratinocyte stem cells; however, direct data demonstrating psoriatic lesional T cell subset IFN-gamma expression, and quantitation at a single cell level to calculate in vivo proportions, are lacking. In this study, using flow cytometry of freshly isolated normal and psoriatic lesional T cells from keratome biopsies, we found elevated CD3+, CD4+, and CD8+ T cells in all compartments of psoriatic skin, compared with normals. Using Brefeldin A to induce short-term intracellular accumulation of IFN-gamma in T cells capable of IFN-gamma production, we found that 90% of psoriatic patients have IFN-gamma-producing T cells at a greater proportion of their CD3+ cells than normals, with a mean of 16%+/-3%, as compared with 4%+/-2% in normal epidermis (p = 0.01). Expressed as density in the tissue, the IFN-gamma+ CD3+ cell number in psoriatic epidermis was 97+/-22 per mm2 surface area, as compared with 4.4+/-1.8 per mm2 of normal epidermis (p = 0.002). Thus, the total number of IFN-gamma+CD3+ T cells in the skin of a patient with 20% involvement is estimated to be 3.9 x 10(8). CD4+ and CD8+ IFN-gamma+ T cells were both elevated in psoriatic epidermis (p = 0.04 and p = 0.008, respectively) relative to normal skin. In the dermis, only 44% of patients demonstrated a higher percentage of IFN-gamma-producing T cells than did normals (p = 0.1), possibly indicating dilution, in some patients, by fresh infiltrating T cells. Interleukin-4 was not found by a combination of flow cytometry, reverse transcriptase-polymerase chain reaction, western blot, and immunoprecipitation. In conclusion, a significant portion of lesional T cells in psoriasis are IFN-gamma producing, without interleukin-4. The increased numbers of both IFN-gamma+CD4+ and IFN-gamma+CD8+ T cells indicate that both CD4+ and CD8+ IFN-gamma+ T cells are present in appropriate anatomic locations to sustain the lesional pathology.

IL-1 and IL-1 receptor antagonist regulation during keratinocyte cell cycle and differentiation in normal and psoriatic epidermis.

Changes in the levels of IL-1 (IL-1alpha, IL-1beta, and its receptor antagonist, IL-1RA) occur upon keratinocyte differentiation in vitro and are associated in vivo with abnormal differentiated and hyperproliferative states of psoriatic keratinocytes. A flow cytometric procedure, capable of detecting changes in the intracellular levels of IL-1, was used to determine whether intracellular IL-1/IL-1RA levels in psoriatic and normal keratinocytes alter during in vivo differentiation and the cell cycle. Increases in the IL-1RA levels and IL-1alpha levels were observed as both normal and psoriatic keratinocytes differentiated from basal stem cells (beta1 integrin+, small size) into transient amplifying cells (TAC; beta1 integrin+, large size). Upon further differentiation (beta1 integrin-, large size) both IL-1RA and IL-1alpha levels dropped. However, while psoriatic IL-1beta levels increased as cells differentiated into TACs, little change occurred in the IL-1beta levels of normal keratinocytes during differentiation. Changes in IL-1/IL-1RA levels were also detected as keratinocytes progressed through the cell cycle. Within the basal stem cell population of both normal and psoriatic keratinocytes, the IL-1alpha and IL-1RA levels increased between G0/G1 and S but not between S and G2/M. However, psoriatic basal keratinocyte IL-1beta levels differed from those of normal keratinocytes by showing no increase between S and G2/M. The IL-1/IL-1RA levels of normal TAC increased throughout the cell cycle. However, in psoriatic TAC, a slight decrease in IL-1alpha and IL-1RA levels was observed between G0/G1 and S followed by a delayed increase between S and G2/M. IL-1beta levels in psoriatic TAC varied little throughout the cell cycle. Thus, we were able to detect precisely the regulation of IL-1/IL-1RA intracellular levels during the keratinocyte cell cycle and differentiation, showing notably decreased IL-1beta upregulation in psoriatic keratinocytes progressing through the cell cycle.

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.

Significantly increased occurrence of HLA-DQB1*0301 allele in patients with ocular cicatricial pemphigoid.

To determine the immunogenetic characteristics of patients with immune-mediated subepithelial blistering diseases of mucous membranes, we performed HLA-typing for the class II MHC gene DQB1*0301 allele using a direct method. Genomic DNA extracted from Caucasian patients was amplified by polymerase chain reaction using primer pairs specific for the DQB loci followed by Southern blotting with a peroxidase-conjugated sequence-specific oligonucleotide probe. Seventy-six percent (16/21) of patients with ocular mucosal disease (with or without oral mucosal and skin diseases) carried the DQB1*0301 allele; by contrast, only 33% (14/42) of race-, age-, and geography-matched normal individuals carried the DQB1*0301 allele (p < 0.005). The relative risk for ocular disease if DQB1*0301 allele is present is 6.4, similar to the relative risk of 8 for patients with ocular but no oral disease (pure ocular cicatricial pemphigoid, p < 0.025). In patients with oral mucosal disease (with or without ocular mucosal and skin diseases), 68% (15/22) carried the DQB1*0301 allele (p < 0.025). When patients with ocular disease were excluded, however, the increased occurrence of the DQB1*0301 allele in patients with oral disease was not statistically significant (64%, 7/11, p < 0.25). In patients with subepidermal blistering skin disease but no oral or ocular disease, there was no increase in the occurrence of the DQB1*0301 allele (38%, 5/13, p > 0.5). The significantly increased occurrence of the DQB1*0301 allele in patients with ocular mucosal disease may point to a distinct immunogenetic factor that predisposes patients to develop an ocular scarring process.

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.

Defective in vivo expression and apparently normal in vitro expression of a newly identified 105-kDa lower lamina lucida protein in dystrophic epidermolysis bullosa.

We have previously identified a novel 105-kDa lower lamina lucida protein detected by the autoantibodies from a group of patients who developed a unique immune-mediated subepidermal bullous dermatosis. We sought to determine if this novel basement membrane zone (BMZ) protein is normally expressed in the skin of patients with various subsets of epidermolysis bullosa (EB). Indirect immunofluorescence microscopy performed on non-lesional skin sections from patients with three major EB subsets revealed absence or significantly reduced expression of this novel BMZ protein in 20 out of 23 skin sections from patients with generalized dominant and recessive dystrophic EB. However, immunoblot analyses with the autoantibodies on Western-blotted proteins revealed that a comigrating 105-kDa protein is present in both cytosol extracts (n = 6) and conditioned media (n = 3) of cultured dermal fibroblasts derived from patients with dystrophic EB, as well as those cultured from two healthy individuals. Although the reason for such disparate findings is not known, the defective in vivo expression of this novel 105-kDa protein in dystrophic EB is presumably not due to a failure of fibroblasts to synthesize or secrete the protein. It is possible, however, that the 105-kDa protein may be unable to incorporate into the BMZ because it is produced in a dysfunctional form, or its BMZ binding site is missing. It is also possible that other structural alterations in skin BMZ, which occur in dystrophic EB, result in masking of the antigenic binding by the autoantibody when intact BMZ is probed.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes.

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.