Patricia Bacon

Innate Immune-Related Receptors in Normal and Psoriatic Skin

CONTEXT A precise role for the innate immune system in psoriasis remains to be determined. Surface receptors, including Toll-like receptors (TLRs) that recognize bacterial ligands and CD91, which recognizes heat shock proteins (HSPs), are implicated in both innate and adaptive immunity. OBJECTIVE Since skin is exposed to various exogenous stimuli, which can provoke or exacerbate psoriasis, we characterized expression and function of TLRs, CD91, and HSPs in normal and psoriatic skin. DESIGN A variety of skin-derived cells and blood-derived cells were analyzed both in vivo and in vitro; samples were obtained from 24 different individuals for innate immune-related receptor expression and function. By comparing and contrasting individuals with healthy skin and psoriatic patients, several specific differences were identified. RESULTS Immunohistochemistry-based expression profiling revealed TLR1 expression in epidermal dendritic cells (DCs) and dermal dendritic cells (DDCs) in normal skin, as well as in pre-psoriatic skin and psoriatic plaques, with enhanced basal layer keratinocyte (KC) expression in pre-psoriatic and psoriatic plaques compared with normal skin; TLR2 expression primarily by DDCs; and TLR4 expression by epidermal DCs and DDCs, with mid-epidermal-layer KCs displaying cell surface staining. No TLR9 or CD14 was detected on DCs or KCs, although psoriatic plaques contained CD14-positive macrophages. Analysis of psoriatic epidermis revealed HSPs 27, 60, and 70. Keratinocytes were CD91 negative, but CD91 was expressed by fibroblasts and DDCs in normal and pre-psoriatic skin, with prominent accumulation of CD91-positive DDCs in psoriatic plaques. Cultured KCs revealed no surface expression of TLR2, TLR4, TLR9, or CD91. Exposure of fibroblasts, but not KCs, to lipopolysaccharide or HSPs triggered nuclear factor (NF)-kappaB activation. Heat shock proteins did induce maturation of blood-derived DCs accompanied by increased interleukin-12 production and enhanced antigen-presenting function. CONCLUSIONS These data demonstrate distinctive patterns of innate immune-related receptors by specific subsets of cells in normal and psoriatic skin, suggesting functional roles for HSPs and DCs in psoriasis.

The Notch Ligand Jagged-1 Is Able to Induce Maturation of Monocyte-Derived Human Dendritic Cells

Notch receptors play a key role in several cellular processes including differentiation, proliferation, and apoptosis. This study investigated whether the activation of Notch signaling would affect the maturation of dendritic cells (DCs). Direct stimulation of Notch signaling in DCs with a peptide ligand induced DC maturation, similar to LPS: DCs up-regulated maturation markers, produced IL-12, lost endocytosis capacity, and became able to activate allogeneic T cells. Furthermore, coculture of DCs with cells expressing Notch ligand Jagged-1 induced up-regulation of maturation markers, IL-12 production, T cell proliferative responses, and IFN-γ production. Our data suggest that activation of Notch by Jagged-1 plays an important role in maturation of human DCs. Additionally, they reveal a novel role for Notch signaling in cell maturation events distal to the cell fate decision fork. These data may have important medical implications, since they provide new reagents to induce DC activity, which may be beneficial as adjuvants in situations where an immune response needs to be elicited, such as tumor immunotherapy.

Regulation of apoptosis by p53 in UV-irradiated human epidermis, psoriatic plaques and senescent keratinocytes.

The carcinogenic effects of sunlight in human epidermis may be thwarted by either: transient growth arrest and repair of DNA photodamage in keratinocytes (KCs); elimination of KCs with damaged DNA via apoptosis; or by stimulating a senescence switch whereby KCs become irreversibly growth arrested. Using normal human skin organ cultures and living epidermal equivalents, we demonstrate that in the proliferative basal layer, removal of KCs via apoptosis had a rapid onset (beginning within 2 h) following UV-light exposure generating progressively greater numbers of KCs with thymine dimers as the dose of UV-light was increased; involved induction of Apaf-1, activation of caspase-3, and was dependent on p53 activation as addition of a p53 chemical inhibitor blocked the apoptotic response. Suprabasal layer KCs underwent apoptosis at much later time points (>8 h). KCs in the basal layer repaired DNA damage more rapidly than KCs in suprabasal layers. Steady state levels of p53 increased in irradiated cells, and the increase was accompanied by phosphorylation of serine 9 and serine 15, but not serine 6 residues. By contrast, cultured KCs undergoing spontaneous replicative senescence were resistant to UV-induced apoptosis. Senescent KCs constitutively contained low levels of p53, which were neither increased nor phosphorylated or acetylated after UV-exposure and possessed minimal DNA binding activity, indicative of functional inactivation. Furthermore, treatment of senescent KCs with DNA damaging agent adriamycin did not result in activation of latent p53 or apoptosis. When KCs within psoriatic plaques were examined, they resembled senescent KCs in that they expressed p53, which was not phosphorylated or acetylated. Thus, UV-light induces DNA damage in human epidermal KCs triggering p53 activation, and subsequent apoptosis involving distinct cell layers and kinetics. However, the lack of p53 activation as seen in senescent KCs and psoriatic plaques, is associated with a relative resistance of KCs to UV-induced apoptosis. In conclusion, the sensitivity and resistance of KCs to apoptosis depends not only on the location within various layers of epidermis and levels of p53, but may also involve p53 activation via post-translational modifications.

In situ polymerase chain reaction-based localization studies support role of human herpesvirus-8 as the cause of two AIDS-related neoplasms: Kaposi's sarcoma and body cavity lymphoma

Several lines of investigation point to a new herpesvirus, human herpesvirus-8 (HHV-8), as the cause of two different neoplasms seen in AIDS patients-Kaposis sarcoma (KS) and body cavity B cell lymphoma. If this virus is the etiological agent, rather than another opportunistic infectious agent, it should be present in the earliest detectable clinical lesions on a temporal basis, and localize to specific target cells in a spatial pattern consistent with tumorigenic pathways. In this study, we take advantage of the clinical accessibility to biopsy early (patch stage) skin lesions of KS to address the temporal issue, combined with in situ PCR and dual immunostaining using a marker identifying malignant cells, to address the spatial localization issue. 21 different tissue samples were subjected to PCR analysis and in situ PCR with and without simultaneous immunostaining. In normal skin from healthy individuals, no HHV-8 DNA was detected by PCR or in situ PCR. However, in all PCR-positive tissues, distinct and specific in situ PCR staining was observed. In four different patch stage KS lesions, in situ PCR staining localized to nuclei of endothelial cells and perivascular spindle-shaped tumor cells. Later stage KS lesions (plaques and nodules) revealed additional positive cells, including epidermal keratinocytes (four of five), and eccrine epithelia (two of four). These patterns were nonrestricted to skin, as pulmonary KS also revealed HHV-8-specific infection of endothelial cells and KS tumor cells, as well as epithelioid pneumocytes (two of two). In body cavity B cell lymphoma by dual staining, HHV-8 was present in malignant tumor cells (EMA immunostained positive) and not in reactive lymphocytes. These results reveal an early temporal onset and nonrandom tissue and cellular distribution pattern for HHV-8 infection that is consistent with a causal link between this DNA virus and two AIDS-related neoplasms.

Tumor Suppressor Maspin Is Up-Regulated during Keratinocyte Senescence, Exerting a Paracrine Antiangiogenic Activity

Cell senescence is a physiological program of terminal growth arrest, which is believed to play an important role in cancer prevention. Senescent cells secrete multiple growth-regulatory proteins, some of which can affect tumor growth, survival, invasion, or angiogenesis. Changes in expression of different senescence-associated genes were analyzed in cultured human skin keratinocytes (KCs) that underwent replicative senescence or confluence-induced accelerated senescence. Senescent KC cultures showed a strong increase in mRNA and protein expression of maspin, a member of serine protease inhibitor family and an epithelial cell tumor suppressor with anti-invasive and antiangiogenic activities. Immunohistochemical analysis of 14 normal human skin samples (age range from 3 months to 84 years) showed that maspin is expressed by KCs in vivo and that the extent and intensity of maspin expression in the skin is significantly (P = 0.01) correlated with chronological age. Antiangiogenic activity of maspin secreted by senescent KCs was investigated in vitro by testing the effect of conditioned media from different KC cultures on endothelial cell migration in the presence or absence of several angiogenic factors. Media conditioned by senescent cultures (undergoing replicative or accelerated senescence), but not by proliferating KCs, strongly inhibited the stimulation of endothelial cell migration by all of the tested angiogenic factors. Neutralizing antibody against maspin abrogated this effect of conditioned media. These findings indicate that senescent KCs exert a paracrine antiangiogenic activity, and maspin is the principal contributor to this potentially tumor-suppressive effect of cellular senescence.

Role of INK4a/Arf locus-encoded senescent checkpoints activated in normal and psoriatic keratinocytes

During malignant transformation in skin, epidermal keratinocytes (KCs) frequently acquire the capacity to by-pass cellular senescence, a response that normally limits their unrestricted proliferation. Despite growing interest in the role for senescence during aging of skin and cutaneous carcinogenesis, little is known regarding regulation of three proteins encoded by the INK4a/ARF locus (p12, p14(ARF), p16) in KCs. In this study, several molecular pathways are explored using cultured KCs and KCs freshly isolated from psoriatic plaques. p16 and p14(ARF) are predominantly expressed spontaneously when foreskin-derived early-passage KCs undergo confluency-induced premature senescence. Induction of p14(ARF) on confluency occurred with low E2F-1 levels. Suspension of KCs in methylcellulose induced p12 expression. Addition of various cytokines (interferon-gamma, tumor necrosis factor-alpha) or a phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] only induced p16, but not p14(ARF). Confluent KCs up-regulated Ras activity and the downstream signaling involving ERK. Addition of MAPK inhibitor blocked cytokine and TPA-induced p16 expression. Confluency and interferon-gamma induced premature senescence and p16 expression was linked to induction of the transcription factor Egr-1. KCs derived from chronic psoriatic plaques were characterized by enhanced p16, p14(ARF), and p12 expression accompanied by elevated Egr-1 levels. These results demonstrate that multiple and highly divergent stimuli can trigger the senescent checkpoint in human KCs with differential regulation of p16, p14(ARF), and p12. Although abnormal mitogenic signaling by oncogenic Ras is generally cited as being responsible for induction of premature senescence, our findings indicate that a broader perspective is warranted, to include confluency and cytokine-/TPA-induced pathways for KCs.

Low‐dose UV‐radiation sensitizes keratinocytes to TRAIL‐induced apoptosis

The impact of low‐dose ultraviolet light (UV‐light) on apoptotic susceptibility of keratinocytes (KCs) induced by TRAIL is unclear. Skin expresses a functional form of TRAIL, and while sun exposure influences TRAIL death receptors, a role for decoy receptors has not been evaluated. Unraveling mechanisms involving apoptotic sensitivity of KCs is important because skin is the first target of UV‐light, and a site for commonly occurring cancers. Since apoptosis is a homeostatic process eliminating UV‐light induced DNA damaged cells, elucidating molecular events regulating apoptosis enhances understanding of cutaneous photocarcinogenesis. Here we demonstrate low‐dose UV‐light enhances susceptibility of KCs to TRAIL‐induced apoptosis. Low‐dose UV‐light selectively reduces decoy receptors, without influencing death receptor levels. UV‐induced enhanced apoptotic susceptibility was reduced by over‐expression of decoy receptor TRAIL‐R4, but not TRAIL‐R3; or treatment with thiol compound pyrrolidine dithiocarbamate (PDTC), which also enhanced TRAIL‐R4 levels. Besides influencing decoy receptors, low‐dose UV‐light plus TRAIL also synergistically promoted cytochrome c and Smac release from mitochondria. Inhibitors directed against caspases 2, 3, 8, and 9 reduced the synergistic apoptotic response following low‐dose UV‐light plus TRAIL exposure; as did forced over‐expression of Bcl‐x and dominant negative (DN) constructs of FADD and caspase 9. Thus, relative levels of decoy receptors significantly influence susceptibility of KCs to TRAIL‐induced apoptosis with concomitant low‐dose UV‐light exposure; in addition to the apoptotic pathway mediated by mitochondrial permeabilization.

Pathways involved in proliferating, senescent and immortalized keratinocyte cell death mediated by two different TRAIL preparations

Abstract: Properly regulated keratinocyte cell death is fundamentally important to maintain structural integrity and homeostatic function of epidermis. Moreover, from an oncological perspective, therapeutic approaches selectively targeting apoptosis of malignant cell types while sparing normal keratinocytes in surrounding skin is desirable. Apo2Ligand/tumor necrosis factor‐related apoptosis‐inducing ligand (Apo2L/TRAIL) has been observed to preferentially induce cytopathic effects on transformed/malignant cell types compared with their non‐neoplastic counterparts. In this report, two different biologically active preparations of Apo2L/TRAIL, a non‐tagged version, NT‐Apo2L/TRAIL, and a leucine zipper fusion protein, LZ‐Apo2L/TRAIL, were examined for their ability to trigger apoptosis in normal human keratinocytes, and in an immortalized cell line (HaCaT cells). Differences between these preparations were observed, including: NT‐Apo2L/TRAIL induced less keratinocyte apoptosis compared with LZ‐Apo2L/TRAIL; NT‐Apo2L/TRAIL also induced less apoptosis of HaCaT cells compared with LZ‐Apo2L/TRAIL; LZ‐Apo2L/TRAIL but not NT‐Apo2L/TRAIL induced cytotoxic effects when keratinocytes became growth arrested due to undergoing spontaneous replicative senescence – a biological state previously observed to be resistant to UV‐light‐induced apoptosis. Similarities between preparations included: an enhanced ability for both Apo2L/TRAIL preparations to kill a greater relative percentage of HaCaT cells compared with keratinocytes; enhanced cytotoxicity towards keratinocytes that had their NF‐B activity inhibited; a dependence of both Apo2L/TRAIL preparations on FADD and caspase activation; triggering of the same caspase cascades including caspase 8 and 3; and an ability to induce apoptosis even when HaCaT cells and keratinocytes were transduced to overexpress either Bcl‐2 or Bcl‐xL (survival factors that reduce susceptibility to UV‐light‐induced apoptosis). These results indicate that while both preparations of Apo2L/TRAIL possess biological activity, there are important differences as regards their ability to induce apoptosis in normal and immortalized keratinocytes. Moreover, the death receptor pathway triggered by LZ‐Apo2L/TRAIL can overcome the apoptotic resistance normally observed in response to UV‐light mediated by Bcl‐2/Bcl‐xL, as well as by the state of cellular senescence. Unraveling the molecular basis for these differential biological effects may reveal a new strategic role for these death receptor/ligands linked to apoptosis in maintaining the dynamic balance of keratinocyte proliferation, differentiation, and cell death necessary to achieve a homeostatic thickness and function of normal skin. In addition, it may be possible to utilize these Apo2L/TRAIL preparations for the treatment of various sun‐induced skin cancers as they can differentially trigger apoptosis of transformed keratinocytes, or keratinocytes with abnormal NF‐κB signaling, while sparing adjacent normal keratinocytes.

Role for Id‐1 in immunobiology of normal keratinocytes and in basal cell carcinoma

Abstract:  It has been established that Id proteins can block the basic helix–loop–helix (HLH) transcription factors, thereby impacting the onset of senescence in keratinocytes, as well as influencing tumorigenesis involving squamous cell carcinomas. However, the ability of Id‐1 to influence the immunologic response of epithelial cells to cytokines implicated in cutaneous oncology such as gamma interferon (IFN‐γ) has not been determined. Using a whole population of human keratinocytes infected with a retrovirus to induce over‐expression of Id‐1, the influence on early differentiation of rapidly proliferating keratinocytes was assessed, as was the response to IFN‐γ. While induction of involucrin, a marker of early differentiation, was not altered in Id‐1 overexpressing keratinocytes, the IFN‐γ mediated increase in intercellular adhesion molecule‐1 (ICAM‐1) and HLA‐DR was reduced. No change in constitutive or inducible levels of MHC class I antigen, CD95 (Fas antigen) or LFA‐3 (CD58) was observed in this system. Immunostaining and Western blot analysis revealed over‐expression of Id‐1 in basal cell carcinomas (BCCs). These tumors not only strongly and diffusely expressed Id‐1, but were also characterized by reduced ICAM‐1 and HLA‐DR expression. Thus, dysregulated Id‐1 may not only contribute to delaying the senescence program in keratinocytes, it may also contribute to the escape of the relatively undifferentiated tumor cells in BCC from immune surveillance.