Novel application of high-dose rate brachytherapy for severe, recalcitrant palmoplantar pustulosis.

Palmoplantar pustulosis (PPP) is a chronic pustular dermatitis of the palms and soles, which is frequently associated with significant pruritus and pain, often limiting daily activities. We present the case of a 36-year-old man with severe PPP who had treatment failure with multiple medical therapies but showed marked improvement with high-dose rate brachytherapy. Brachytherapy has the advantage of providing a conformal dose distribution over complex curved surfaces, such as the foot and ankle. Our observations suggest that brachytherapy may be a well-tolerated treatment option for patients with severe, refractory PPP.

Rapid and specific conversion of precursor interleukin 1 beta (IL-1 beta) to an active IL-1 species by human mast cell chymase.

Secretory granules of human dermal mast cells contain a chymotrypsin-like serine proteinase called chymase. In this study, we demonstrate that the inactive cytokine, 31 kD interleukin 1 beta (IL-1 beta), can be converted rapidly to an 18 kD biologically active species by human mast cell chymase. The product formed is three amino acids longer at the amino terminus than the mature IL-1 beta produced by peripheral blood mononuclear cells and has comparable biological activity. Because chymase is a secretory granule constituent, it is likely to be released into the surrounding tissue when mast cells degranulate. It is also known that non-bone marrow derived cells resident in skin (keratinocytes, fibroblasts) produce but do not process 31 kD IL-1 beta. In this context, chymase may be a potent activator of locally produced 31 kD IL-1 beta. Mast cells lie in close apposition to blood vessels in dermis; therefore, chymase mediated conversion of 31 kD IL-1 beta might be expected to have a critical role in the initiation of the inflammatory response in skin.

Human keratinocytes contain mRNA indistinguishable from monocyte interleukin 1 alpha and beta mRNA. Keratinocyte epidermal cell-derived thymocyte-activating factor is identical to interleukin 1.

Keratinocytes produce an IL-1 like factor termed epidermal cell-derived thymocyte-activating factor (ETAF). In this study, we show that ETAF and IL-1 are identical by the following criteria: Both normal and malignant human keratinocytes contain mRNAs identical to monocytic IL-1 alpha and IL-1 beta mRNA, as determined by an S1 nuclease protection assay; and IL-1 activity in medium conditioned by these cells can be neutralized by antibodies specific for human IL-1. The IL-1 alpha and IL-1 beta mRNAs can be identified in cultured human keratinocytes in the absence of identifiable stimulation; this basal level of mRNA can be further induced to accumulate with certain defined stimuli. Cultured normal human keratinocytes (HFKs) contain 2-4 times more IL-1 alpha than IL-1 beta mRNA; in contrast, human peripheral blood monocytes contain 10-20 times more IL-1 beta than IL-1 alpha mRNA. The IL-1 activity released by these HFK can be neutralized by an antibody that neutralizes both alpha and beta IL-1, but not by an antibody that neutralizes only IL-1 beta. While human monocytes produce a large excess of IL-1 beta after appropriate stimulation, these data suggest that IL-1 alpha is a major (and may be the predominant) form of IL-1 produced by human keratinocytes.

Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice.

Intercellular adhesion molecule 1 (ICAM-1) is one of three immunoglobulin superfamily members that bind to the integrins lymphocyte function associated 1 (LFA-1) and Mac-1 on leukocytes. We have generated mice that are genetically and functionally deficient in ICAM-1. These mice have elevated numbers of circulating neutrophils and lymphocytes, as well as diminished allogeneic T cell responses and delayed type hypersensitivity. Mutant mice are resistant to lethal effects of high doses of endotoxin (lipopolysaccharide [LPS]), and this correlates with a significant decrease in neutrophil infiltration in the liver. Production of inflammatory cytokines such as tumor necrosis factor alpha or interleukin 1 is normal in ICAM-1-deficient mice, and thus protection appears to be related to a diminution in critical leukocyte-endothelial interactions. After sensitization with D-galactosamine (D-Gal), ICAM-1-deficient mice are resistant to the lethal effect of low doses of exotoxin (Staphylococcus aureus enterotoxin B [SEB]), which has been shown to mediate its toxic effects via the activation of specific T cells. In this model, ICAM-1-mediated protection against SEB lethality correlates with a decrease in the systemic release of inflammatory cytokines, as well as with prevention of extensive hepatocyte necrosis and hemorrhage. ICAM-1-deficient mice sensitized with D-Gal, however, are not protected from lethality when challenged with low doses of endotoxin (LPS). These studies show that the different contribution of ICAM-1 in the activation of either T cells or macrophages is decisive for the fatal outcome of the shock in these two models. This work suggests that anti-ICAM-1 therapy may be beneficial in both gram-positive and -negative septic shock, either by reducing T cell activation or by diminishing neutrophil infiltration.

Interaction of dendritic cells with skin endothelium: A new perspective on immunosurveillance.

The goal of this study was to determine the mechanisms by which dendritic cells (DCs) in blood could interact with endothelium, a prerequisite to extravasation into tissues. Our results indicate that DCs express both HECA-452-reactive and nonreactive isoforms of P-selectin glycoprotein ligand 1 (PSGL-1) and can tether and roll efficiently on E- and P-selectin under flow conditions in vitro. Freshly isolated blood DCs were further observed to roll continuously along noninflamed murine dermal endothelium in vivo. This interaction is strictly dependent on endothelial selectins, as shown by experiments with blocking antibodies and with E- and P-selectin-deficient mice. We hypothesize that DCs in blood are constitutively poised at the interface of blood and skin, ready to extravasate upon induction of inflammation, and we showed that cutaneous inflammation results in a rapid recruitment of DCs from the blood to tissues. We propose that this is an important and previously unappreciated element of immunosurveillance.

1,24-Dihydroxyvitamin D3 (tacalcitol) prevents skin T-cell infiltration.

BACKGROUND: 1,24-Dihydroxyvitamin D3 (tacalcitol), a vitamin D(3) compound, has been used to treat T cell-mediated inflammatory skin diseases such as psoriasis, prurigo and vitiligo. The best-known mechanism of action of this compound is inhibition of the abnormal proliferation of keratinocytes and subsequent maturation; however, its effects on skin T-cell recruitment have not yet been evaluated. Cutaneous lymphocyte-associated antigen (CLA), a surface glycoprotein expressed on T cells, plays a critical role in skin T-cell infiltration. We recently reported that 1,25-dihydroxyvitamin D3 inhibits skin infiltration of CD4+ T cells by suppressing CLA expression on T cells. OBJECTIVES: In this study, we investigated the effect of tacalcitol on CLA epitope decoration and on the levels of gut or lymph node homing receptor expression in human T cells. METHODS: We cultured human T cells with tacalcitol and analysed the effect on CLA expression and skin-homing ability, and evaluated glycosyltransferase mRNAs. We also performed an in vivo study using an antigen-dependent delayed-type hypersensitivity (DTH) mouse model and investigated the effect of tacalcitol on skin-infiltrating CD4+ T cells. RESULTS: Tacalcitol downregulated the expression of CLA and, in parallel, the E- and P-selectin ligand function; however, it exerted no effect on other homing receptors. Subcutaneously and intraperitoneally administered tacalcitol downregulated skin infiltration of effector CD4+ T cells in an in vivo DTH mouse model. CONCLUSIONS: These findings suggest that tacalcitol reduces skin inflammation by partially downregulating CLA expression levels.

Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P and E selectin under physiological flow.

Memory T lymphocytes extravasate at sites of inflammation, but the mechanisms employed by these cells to initiate contact and tethering with endothelium are incompletely understood. An important part of leukocyte extravasation is the initiation of rolling adhesions on endothelial selectins; such events have been studied in monocytes and neutrophils but not lymphocytes. In this study, the potential of T lymphocytes to adhere and roll on endothelial selectins in vitro was investigated. We demonstrate that T cells can form tethers and rolling adhesions on P selectin and E selectin under physiologic flow conditions. Tethering and rolling on P selectin was independent of cell-surface cutaneous lymphocyte antigen (CLA) expression, which correlated strictly with the capacity of T cells to form rolling adhesions under flow on E selectin. T cell tethering to P selectin was abolished by selective removal of cell surface sialomucins by a P. haemolytica O-glycoprotease, while cutaneous lymphocyte antigen expression was unaffected. A sialomucin molecule identical or closely related to P selectin glycoprotein ligand-1 (PSGL-1), the major P selectin ligand on neutrophils and HL-60 cells, appears to be a major T cell ligand for P selectin. P selectin glycoprotein ligand-1 does not appear to support T cell rolling on E selectin. In turn, E selectin ligands do not appear to be associated with sialomucins. These data demonstrate the presence of structurally distinct ligands for P or E selectins on T cells, provide evidence that both ligands can be coexpressed on a single T cell, and mediate tethering and rolling on the respective selectins in a mutually exclusive fashion.

Cytokines: IL-20 - a new effector in skin inflammation.

The newly discovered cytokine interleukin-20 (IL-20) is structurally related to IL-10, yet it appears to be an autocrine factor for keratinocytes that regulates their participation in inflammation.

Human keratinocytes produce but do not process pro-interleukin-1 (IL-1) beta. Different strategies of IL-1 production and processing in monocytes and keratinocytes.

Keratinocytes comprise the majority of cells in the epidermis, the interleukin-1 rich layer of tissue contiguous with the outside world. Keratinocytes produce IL-1 alpha and beta mRNA in vitro, but only IL-1 alpha biological activity has been identified in keratinocyte cultures. In contrast, monocytes secrete biological activities attributable to both species of IL-1. Using several monoclonal antibodies to IL-1 beta, significant amounts of IL-1 beta protein could be found in keratinocyte cultures; all of this immunoreactive IL-1 beta was in the 31-kD form. This latent cytokine has been shown to bind inefficiently to the IL-1 receptor and to be (in relative terms) biologically inactive. Chymotrypsin cleaves 31-kD IL-1 beta at Tyr 113-Val 114, generating an 18-kD IL-1 species with activity equivalent to the authentic mature IL-1 beta (NH2-terminal Ala 117). Treatment of 31-kD keratinocyte IL-1 beta with chymotrypsin also generated an 18-kD molecule and significant IL-1 activity. Monocytes contain an IL-1 convertase enzyme that cleaves the IL-1 beta promolecule at Ala 117. We demonstrate here that keratinocytes do not contain such an IL-1 convertase activity, nor do they contain any activity capable of productively processing 31-kD IL-1 beta into a biologically active form. These data suggest that keratinocytes (and other non-bone marrow-derived cells) produce IL-1 beta in an inactive form that can be processed only after leaving the cell.

Interleukin 1 binds to specific receptors on human keratinocytes and induces granulocyte macrophage colony-stimulating factor mRNA and protein. A potential autocrine role for interleukin 1 in epidermis.

Cultured human keratinocytes have been shown to produce IL-1 alpha and beta mRNA and protein. IL-1 biological activity has been identified in normal human epidermis; in vitro, most biologically active IL-1 resides in a cell-associated compartment. The potential for autocrine effects of IL-1 on human keratinocytes was assessed by measurement of keratinocyte IL-1 receptors. Both high- and low-affinity cell surface receptors that bound recombinant (r) IL-1 alpha and beta with comparable affinities could be identified on cultured human keratinocytes, using 125I-labeled rIL-1. Chemical crosslinking experiments identified a cell surface molecule of roughly 72,500 Mr that bound 125I-labeled IL-1, similar to the molecular weight of previously described IL-1 receptors on fibroblasts, B cells, and T cells. To assess the biological consequences of keratinocyte IL-1 binding, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression was measured. The addition of exogenous rIL-1 alpha led to a dose-dependent increase in the accumulation of GM-CSF mRNA, as measured by a sensitive and specific S1 nuclease assay. This increase in mRNA was reflected in a marked increase in GM-CSF biological activity as measured by proliferation of blast cells from chronic myelogenous leukemia patients. The biological activity was completely inhibitable by an antibody to human rGM-CSF. GM-CSF activates mature neutrophils and macrophages and appears to enhance the efficiency of Langerhans cell antigen presentation to T cells. Release of IL-1 from injured or activated keratinocytes may lead to enhanced epidermal GM-CSF gene expression via an autocrine mechanism, thus enhancing local host defense.

Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation.

Interleukin 1 (IL-1) is a family of polypeptides initially found to be produced by activated monocytes and macrophages that mediate a wide variety of cellular responses to injury and infection. Epidermal epithelial cells (keratinocytes) produce "epidermal cell-derived thymocyte activating factor" or ETAF, which has been recently shown to be identical to IL-1. Human epidermis is normally exposed to significant amounts of solar ultraviolet radiation. Certain ultraviolet wavelengths (UVB, 290-320 nm) are thought to be responsible for most of the immediate and long-term pathological consequences of excessive exposure to sunlight. In this study, we asked whether exposure to UVB irradiation induced IL-1 gene expression in cultured human keratinocytes. Cultured human keratinocytes contain detectable amounts of IL-1 alpha and beta mRNA and protein in the absence of apparent stimulation; these levels could be significantly enhanced 6 h after exposure to 10 ng/ml of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Exposure to UVB irradiation with an emission spectrum comparable to that of sunlight (as opposed to that of an unfiltered artificial UV light source) significantly increased the steady state levels IL-1 alpha and beta mRNA in identical populations of human keratinocytes. This was reflected in the production of increased IL-1 activity by these cultures in vitro. In the same cell population, exposures to UVB irradiation did not alter the level of actin mRNA; therefore, the effect of UV irradiation on IL-1 represents a specific enhancement of IL-1 gene expression. Local increases of IL-1 may mediate the inflammation and vasodilation characteristic of acute UVB-injured skin, and systemic release of this epidermal IL-1 may account for fever, leukocytosis, and the acute phase response seen after excessive sun exposure.

Parathyroid hormone and lipopolysaccharide induce murine osteoblast-like cells to secrete a cytokine indistinguishable from granulocyte-macrophage colony-stimulating factor.

Osteoblasts are the cells responsible for the secretion of collagen and ultimately the formation of new bone. These cells have also been shown to regulate osteoclast activity by the secretion of cytokines, which remain to be defined. In an attempt to identify these unknown cytokines, we have induced primary murine osteoblasts with two bone active agents, parathyroid hormone (PTH) and lipopolysaccharide (LPS) and analyzed the conditioned media (CM) for the presence of specific cytokines. Analysis of the CM was accomplished by functional, biochemical, and serological techniques. The data indicate that both PTH and LPS are capable of inducing the osteoblasts to secrete a cytokine, which by all of the techniques used, is indistinguishable from granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF is not constitutive and requires active induction. Production of the cytokine is dependent on the dose of PTH or LPS added. It has been demonstrated that the addition of GM-CSF to bone marrow cultures results in the formation of increased numbers of osteoclasts. Therefore, these data suggest that osteoblasts not only participate in bone remodeling by formation of new matrix but may regulate osteoclast activity indirectly by their ability to regulate hematopoiesis.

Inflammatory and hyperproliferative skin disease in mice that express elevated levels of the IL-1 receptor (type I) on epidermal keratinocytes. Evidence that IL-1-inducible secondary cytokines produced by keratinocytes in vivo can cause skin disease.

Interleukin (IL)-1 induces a cascade of secondary cytokines in a large number of cell types in vitro, including monocytes, fibroblasts, synovial cells, and keratinocytes. Although it has been proposed that autocrine or paracrine activation of such cells by IL- 1 in situ could orchestrate a local inflammatory response, formal proof for such an hypothesis has been lacking. In an attempt to lower the threshold for secondary cytokine production in these cells in response to IL-1, we have generated transgenic mice (designated IR10) which overexpress functional type 1 IL-1 receptor in basal layer of epidermis keratinocytes. As predicted, keratinocytes from these animals were substantially more responsive to exogenous IL-1 than nontransgenic keratinocytes when stimulated in vitro. When challenged with known inducers of keratinocyte IL-1 synthesis and release, skin of IR10 mice exhibited an exaggerated inflammatory response, characterized by epidermal hyperplasia and an acute dermal inflammatory cell infiltrate. In this setting, the secondary epidermal cytokines gro-alpha and GM-CSF were strongly induced in transgenic epidermis but not in control skin. To confirm that these changes were indeed related to IL-1 mediated activation pathways, IR10 mice were crossed to a distinct line of transgenic mice that overexpress 17-kD IL-l alpha in basal keratinocytes. Double transgenic mice derived from this cross breeding experiment developed spontaneous inflammation of the skin, similar in appearance to that induced by PMA, both histologically and macroscopically, and distinct from that seen in either parental strain spontaneously. Furthermore, secondary cytokines were more strongly induced in the double transgenic than in either parental strain. These findings conclusively demonstrate the potential for functional autocrine pathways of keratinocyte activation mediated by IL-1 alpha in vivo, and suggest that level of expression of type 1 IL-1 receptor may function as a significant control point in physiologic IL-1 mediated autocrine pathways.

Targeted expression of bcl-2 to murine basal epidermal keratinocytes results in paradoxical retardation of ultraviolet- and chemical-induced tumorigenesis.

The antiapoptotic protein bcl-2 is found up-regulated in a number of malignant and premalignant skin conditions of keratinocyte origin, but in normal skin, it is expressed at low levels only in interfollicular epidermis. To investigate whether unregulated bcl-2 expression could affect the incidence of epidermal tumors, we have generated a mouse line that over-expresses human bcl-2 in the basal layer of epidermis under the control of the human keratin 14 promoter. These mice were subjected to both UVB photocarcinogenesis and classical two-stage chemical carcinogenesis. Although transgenic bcl-2 in these mice reduces the formation of sunburn cells after short-term UVB irradiation, chronically UVB irradiated K14/bcl-2 mice were protected against tumor development, because transgenic mice developed tumors much later and at a significantly lower frequency than controls. Immunohistochemical analyses of the UVB-induced tumors revealed no significant differences in the degree of inflammatory cell infiltrates. When either K14/bcl-2 mice or F(1) progeny of matings with mice expressing an activated Ha-ras oncogene (K14/bcl-2/ras) were treated with 9,10-dimethyl-1,2-benzanthracene/phorbol 12-myristate 13-acetate, the latency of first papilloma appearance was the same in transgenic mice and controls, but further papillomas developed more slowly in the mutant mice. Moreover, the K14/bcl-2/ras mice developed far fewer albeit larger tumors/mouse than did the ras/+ controls. The rate of conversion to malignant carcinomas, the carcinoma grade, and the frequency of lymph node metastases were not significantly different between mutants and controls. We conclude that, despite its antiapoptotic function, bcl-2, overexpressed in basal epidermal keratinocytes, exerts a paradoxical retardation on the development of skin tumors induced by chemical carcinogens and particularly by UVB.

Outcome assessment in cellulitis clinical trials: is telephone follow up sufficient?

The US Food and Drug Administration has scrutinized clinical trial methodology in cellulitis, partly because the definition and timing of cure are debatable. We analysed the validity of telephone self-report as a proxy for in-person follow up in a cellulitis treatment trial comparing cephalexin alone with cephalexin-plus-trimethoprim/sulfamethoxazole. Our results demonstrate poor agreement between these two methods of outcome determination and have implications for future cellulitis clinical trial design and clinical management.

The activated keratinocyte: a model for inducible cytokine production by non-bone marrow-derived cells in cutaneous inflammatory and immune responses.

Keratinocytes, fibroblasts, endothelial cells, and other sessile cells resident to human skin can be induced in vitro to synthesize and secrete cytokine molecules. Cytokines are small protein molecules produced upon injury or cellular activation which influence immune and inflammatory events; as such, they have been best understood previously as products of leukocytes. The appreciation that cultured non-bone marrow-derived cells from skin could produce cytokines capable of initiating an inflammatory response or facilitating an immune response has led to speculation that cells resident to skin may be less passive participants in such phenomena than previously thought. Using as a model the cultured keratinocyte, which produces both interleukin-1 alpha and beta and an interleukin-1 receptor, models of autocrine and paracrine activation of this cell have been constructed. Such "activated keratinocytes," or by analogy other activated resident skin cells, produce a spectrum of cytokines in vitro which could potentially influence leukocyte adhesion to endothelium, direction migration of leukocytes towards the activated cell (presumably an inflammatory nidus), and activation of leukocyte functions in situ. The putative role of regulation of cytokine and cytokine-receptor regulation in mediating the activation of such cells (and thus, presumably, local inflammation) is discussed. An important aspect of this hypothetical model is that in the absence of activation (which characterizes normal uninflamed skin), cytokine production and its consequences do not occur. The conclusion reached is that based on in vitro data it is plausible to guess that local inflammatory or immune responses can be both initiated and facilitated by locally produced cytokines.

Hydrocortisone reduces both constitutive and UV-elicited release of epidermal thymocyte activating factor (ETAF) by cultured keratinocytes.

Epidermal thymocyte activating factor (ETAF) is spontaneously released into the media by PAM 212 and A 431 cell lines and cultured normal human keratinocytes. ETAF from all 3 cell types can substitute for interleukin 1 (IL-1) in the augmentation of proliferation of a helper T-cell clone (D10.G4.1) induced by mitogen. Hydrocortisone (HC) substantially reduces the release of ETAF by these keratinocytes and, further, appears to induce the release of an inhibitor of lymphocyte activating factor activity of IL-1. Irradiation with UVC causes increased ETAF release into the media. Hydrocortisone abrogates this effect. Thus HC reduces both constitutive and elicited release of ETAF. ETAF plays a major role in inflammation; the ability of HC to block ETAF release by keratinocytes may account for the anti-inflammatory effect of glucocorticosteroids on the skin.

The interleukin-1 axis and cutaneous inflammation.

Since the discovery that epidermal cell-derived thymocyte-activating factor was identical to interleukin (IL)-1 alpha and -beta in 1986, these molecules have been implicated in the pathogenesis of skin diseases. In 1995, it has become clear that a group of gene products function to regulate the activity of IL-1. IL-1 alpha and mature 17-kD IL-1 beta (cleaved from precursor by IL-1 beta-converting enzyme) bind to the type 1 IL-1 receptor to transduce a signal. This process can be antagonized at the level of the receptor by two distinct forms of the IL-1 receptor antagonist, which bind to the type I receptor but do not transduce a signal. The process can also be antagonized at the level of the ligand by either cell-bound or soluble type 2 IL-1 receptor. This type 2 IL-1 receptor binds ligand but does not transduce a signal. Keratinocytes can make each of these variables in vitro, and the balance between agonists and antagonists dictates the biologic outcome of a putative IL-1-mediated event. Transgenic mice that overexpress each of these factors individually in epidermis will be useful for enhancing our understanding of the cutaneous biology of IL-1.

Keratinocyte membrane-associated epidermal cell-derived thymocyte-activating factor (ETAF).

This study describes the association between secreted keratinocyte interleukin 1 (IL-1) and its presence on the keratinocyte cell surface. These properties were studied in normal and transformed human keratinocytes as well as in transformed murine keratinocytes. We will present evidence that the secretion of IL-1 by human and murine keratinocytes is associated with the presence of IL-1 on the keratinocyte membrane. In addition, although transformed murine keratinocytes secrete other cytokines, namely keratinocyte T-cell growth factor (KTGF) and IL-3, no KTGF or IL-3 activity can be demonstrated on the cell surface.