Recent Observations Regarding Interferon, Keratinocytes and Lymphocytes In vitro and In vivo

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111185/1/jde03658.pd

Specifically targeting ERK1 or ERK2 kills Melanoma cells

<p>Abstract</p> <p>Background</p> <p>Overcoming the notorious apoptotic resistance of melanoma cells remains a therapeutic challenge given dismal survival of patients with metastatic melanoma. However, recent clinical trials using a BRAF inhibitor revealed encouraging results for patients with advanced BRAF mutant bearing melanoma, but drug resistance accompanied by recovery of phospho-ERK (pERK) activity present challenges for this approach. While ERK1 and ERK2 are similar in amino acid composition and are frequently not distinguished in clinical reports, the possibility they regulate distinct biological functions in melanoma is largely unexplored.</p> <p>Methods</p> <p>Rather than indirectly inhibiting pERK by targeting upstream kinases such as BRAF or MEK, we directly (and near completely) reduced ERK1 and ERK2 using short hairpin RNAs (shRNAs) to achieve sustained inhibition of pERK1 and/or pERK2.</p> <p>Results and discussion</p> <p>Using A375 melanoma cells containing activating BRAF^V600Emutation, silencing ERK1 or ERK2 revealed some differences in their biological roles, but also shared roles by reduced cell proliferation, colony formation in soft agar and induced apoptosis. By contrast, chemical mediated inhibition of mutant BRAF (PLX4032) or MEK (PD0325901) triggered less killing of melanoma cells, although they did inhibit proliferation. Death of melanoma cells by silencing ERK1 and/or ERK2 was caspase dependent and accompanied by increased levels of Bak, Bad and Bim, with reduction in p-Bad and detection of activated Bax levels and loss of mitochondrial membrane permeability. Rare treatment resistant clones accompanied silencing of either ERK1 and/or ERK2. Unexpectedly, directly targeting ERK levels also led to reduction in upstream levels of BRAF, CRAF and pMEK, thereby reinforcing the importance of silencing ERK as regards killing and bypassing drug resistance.</p> <p>Conclusions</p> <p>Selectively knocking down ERK1 and/or ERK2 killed A375 melanoma cells and also increased the ability of PLX4032 to kill A375 cells. Thus, a new therapeutic window is open for future clinical trials in which agents targeting ERK1 and ERK2 should be considered in patients with melanoma.</p

Merkel Cell Tumor of the Thigh

This case of a Merkel cell carcinoma is unusual due to the occurrence of the tumor on the thigh; most Merkel cell tumors have been found on the sun-exposed region of the head and neck. Histologically, the nodule was composed of sheets of uniform, poorly differentiated cells with a high nuclear to cytoplasmic ratio. Electron miscroscopy revealed perinuclear filaments, scattered dense core granules, and complex, interdigitating processes within cytoplasmic membranes. Treatment consisted of surgical excision of the tumor with a wide margin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72478/1/j.1524-4725.1988.tb03374.x.pd

Histamine and cis-urocanic acid augment tumor necrosis factor-alpha mediated induction of keratinocyte intercellular adhesion molecule-1 expression

Early cellular and molecular events in inflamed skin include the active participation of epidermal keratinocytes (KCs) and dermal mast cells which can produce diffusible mediators such as tumor necrosis factor-alpha (TNF-Α), histamine, and urocanic acid (UCA). Rapid induction of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) by KCs is observed following a highly diverse array of stimuli which can provoke both irritant, inflammatory, as well as allergic and immune reactions. To determine if the aforementioned mediators could interact in either an additive or synergistic fashion with each other, cultured KCs were exposed to these mediators alone and in combination, and the degree of ICAM-1 mRNA and protein quantitated. Whereas histamine or cis-UCA alone only weakly induced KC ICAM-1, when they were combined with TNF-Α, significant augmentation was observed by Northern blot hybridization studies, immunostaining, and FACS analysis. Other histamine derivatives such as L-histidine, 1-methylhistidine, 3-methylhistidine, or all-trans-UCA had no effect. Histamine pretreatment did not affect cell surface high affinity TNF-Α receptors, as determined by ligand binding and immunodetection, and did not induce KC TNF-Α production. The KC histamine receptor was also characterized and found not to be influenced by TNF-Α, cis-UCA, all-trans-UCA, or diphenyhydramine (an H 1 antagonist), but it was inhibited by cimetidine (an H 2 antagonist). These results demonstrate that 1) KCs can be induced to express ICAM-1 by exposure to histamine and cis-UCA, 2) histamine and cis-UCA can also augment TNF-Α inducible ICAM-1 mRNA and cell surface protein expression, 3) this augmentation does not directly involve changes in KC TNF-Α receptor number, affinity, or TNF-Α production and, 4) KCs possess a type 2 histamine receptor which is not the photoreceptor for UCA. These findings highlight the potential for cross-talk between molecules produced by resident cutaneous cell types above (i.e., KCs) and below (i.e., mast cells) the epidermal basement membrane zone. These cells and their mediators can cooperate to respond to either exogenous or endogenous stimuli leading to rapid and strong KC ICAM-1 expression. Such induction of this important adhesion molecule by KCs ensures the retention of T lymphocytes necessary to participate in the maintenance of cutaneous immunohomeostasis. © 1993 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49885/1/1041560218_ftp.pd

Induction of the Synthesis of Triton-Soluble Proteins in Human Keratinocytes by Gamma Interferon

Recombinant human gamma interferon (r-IFN-γ) induces the synthesis and expression of HLA-DR antigen on cultured, normal, human keratinocytes depleted of Langerhans cells. After removal of r-IFN-γ from the culture medium of keratinocytes that are expressing HLA-DR antigen, the cells continue to express this antigen for at least 2 days. r-IFN-γ induces, in a dose dependent fashion, the synthesis of several triton-soluble proteins with the most prominent having an apparent molecular weight of 53,000. Whereas normal keratinocytes do not express HLADR antigen in vivo, they do express HLA-DR in a variety of skin diseases such as lichen planus, graft-versushost disease, and mycosis fungoides. We propose that an understanding of lymphocyte-keratinocyte interactions in vivo may be achieved by further studies of the mechanism of action of r-IFN-γ on cultured keratinocytes and that the results may provide insight into the pathophysiology leading to a number of common inflammatory and neoplastic skin diseases

Role of NF-κB Activity in Apoptotic Response of Keratinocytes Mediated by Interferon-γ, Tumor Necrosis Factor-α, and Tumor-Necrosis-Factor-Related Apoptosis-Inducing Ligand

An important step in tumorigenesis involves loss of sensitivity to various apoptotic signals by malignant cells, imbuing them with an enhanced survival phenotype. NF-κB also regulates epidermal thickness, susceptibility to apoptosis, and tumor formation in skin. Keratinocytes were examined for their susceptibility to apoptosis using cytokines produced during an immunologic response to tumor antigens, i.e., interferon-γ and/or tumor necrosis factor-α (TNF-α). The role for NF-κB in this response was examined using a retroviral vector containing a degradation-resistant form of IκBα. Whereas interferon-γ and TNF-α either alone or in combination did not induce apoptosis in keratinocytes, after infection with the retrovirus to block NF-κB activation they became susceptible to TNF-α but not Fas-induced apoptosis. Moreover, when keratinocytes with repressed NF-κB activity were simultaneously treated with interferon-γ, there was a synergistic induction of apoptosis by TNF-α that was dependent on FADD, tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL), and caspase activation. Molecular abnormalities accompanying repressed NF-κB activity included failure to induce TNF-RII receptor together with enhanced levels of TRAIL death receptor 4. The ability of interferon-γ when combined with TNF-α to mediate keratinocyte apoptosis included induction of TRAIL coupled with diminished capacity of keratinocytes with repressed NF-κB activity to increase the TRAIL decoy receptor-1, as well as lower levels of several NF-κB-dependent antiapoptotic proteins accompanied by enhanced caspase 8 levels. These results indicate that interferon-γ and TNF-α synergistically induce keratinocyte apoptosis when concomitant induction of NF-κB is blocked. Participants in the apoptotic response mediated by NF-κB, besides cell-survival proteins, include modulation of TRAIL and both death and decoy receptors. Thus, not only does NF-κB signaling influence the intrinsic survival pathway for keratinocytes in normal skin, but it may also play a role in determining the apoptotic response to cytokines generated during an immune response via TRAIL produced by the keratinocytes themselves

Lymphocyte Trafficking in Psoriasis: A New Perspective Emphasizing the Dermal Dendrocyte with Active Dermal Recruitment Mediated Via Endothelial Cells Followed by Intra-Epidermal T-Cell Activation

Prominent within the inflammatory infiltrate of psoriasis are HLA-DR positive T lymphocytes and factor XIIIa positive dermal dendrocytes. Many investigators studying psoriasis have assumed that the HLA-DR positive T cells are activated, and thereby capable of producing lymphokines such as gamma interferon. However, by immunohistochemical analysis, greater than 95% of the dermal T cells in psoriatic lesions are Ki-67 negative, which suggests that they are in a resting or non-cycling (Go) state. In contrast to the darmal T-cell population, the epidermal T-cell population contains a greater population of Ki-67 positive lymphocytes. The entry of the T cells into the epidermis is, therefore, apparently associated with an important activation event, which in all likelihood involves interaction with the keratinocyte. The presence of activated intraepidermal T cells has been substantiated by the ability to detect gamma interferon mRNA by polymerase chain reaction in epidermal sheets of psoriatic lesions. The pathophysiologic implication in psoriasis for these distinctions and compartmentalization involving dermal and epidermal T cells are placed into the context of a cascade of cellular trafficking events, which are further dissected into a specific network of molecular mediators of inflammation. This report suggests that more attention should be placed on the microenvironment of the skin, with specific emphasis on the mechanism by which T cells accumulate in the dermis and epidermis, and elucidation of the selective inductive and recruitment capabilities of endothelial cells, perivascular dermal dendrocytes, and keratinocytes

Immunological functions of non-professional antigen-presenting cells: new insights from studies of T-cell interactions with keratinocytes

T-cell activation in the absence of co-stimulatory signals can lead to induction of anergy. Professional antigen-presenting cells (APCs) of bone marrow origin, such as macrophages and dendritic cells, can provide co-stimulation through molecules such as B7-1 and B7-2. In addition, cells of epithelial origin can function as `non-professional' APCs when activated. In these circumstances, the functional consequences of the T cell-APC interaction may differ, perhaps due to the nature of the co-stimulatory pathways utilized and/or the cytokines encountered by the T cell. Here, Brian Nickoloff and Laurence Turka suggest that these differences may be important in regulating immune responses to local antigens and also in maintaining self-tolerance.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31295/1/0000201.pd