Notes: Long-term treatment with interferon alpha (IFN-α) has recently been shown to reduce the bone marrow infiltrate and cutaneous lesions in systemic mast cell disease. We therefore administered this cytokine to six patients with urticaria pigmentosa for up to 12 months, using subcutaneous injections of 5×106U, initially five times, and subsequently three times a week. The generally well-tolerated therapy resulted in marked improvement of the cutaneous symptoms, especially in three of the patients who suffered from very severe pruritus. Two of the patients with bone marrow infiltration showed normal findings after treatment. However, in none of the patients was there any change in the skin lesions, or decrease in the degree of cutaneous mast cell infiltration, as evidenced by light and electron microscopic examination. These findings indicate that IFN-α is highly effective in the control of symptoms, but otherwise does not influence the cutaneous lesions of urticaria pigmentosa.

Notes: The epidermis has been identified as an important site for the initiation of immunological events. In addition to the macrophage-like Langerhans cells, keratinocytes within the epidermal cells have been shown to act as immunoregulatory cells through the secretion of cytokines such as epidermal cell-derived thymocyte-activating factor (ETAF) and interleukin 3. Epidermal cell-derived interleukin 3 (EC IL-3), like lymphocyte-derived IL-3, induced the proliferation of IL-3-dependent mast cell-like cell lines. Biochemically, EC IL-3 was a heat-stable protein with a molecular weight of approximately 30 kD. Upon chromatofocusing, EC IL-3 exhibited three isoelectric points, at pI 7.8, 7.4, and 7.1. Furthermore, an antiserum against IL-3 neutralized EC IL-3 activity, suggesting that the molecules are closely related and share similar epitopes. ETAF-like macrophage-derived interleukin 1 (IL-1) is a low molecular weight protein with a multiplicity of amplifying effects on immunological and inflammatory reactions. Thus BALB/c mice were immunized with partially purified IL-1, and immune spleen cells were hybridized with plasmocytoma cells. Supernatants of the hybridoma cultures were screened for their capacity to inhibit IL-1-induced thymocyte proliferation. After expansion and cloning, one clone was selected for ascitic antibody production. The monoclonal anti-IL-1 antibody inhibited both the IL-1-dependent thymocyte and the fibroblast proliferation. Furthermore, the antibody blocked murine and human ETAF activity, suggesting that ETAF and IL-1 share antigenically similar domains. Moreover, by using the monoclonal antibody bound to Staphylococcus aureus cells, it was possible to immunoprecipitate IL-1. In contrast, anti-IL-1 antibody did not inhibit IL-2 or IL-3 activity. These findings demonstrate that the production of immunoregulatory cytokines is not confined to cells of the immune system and that keratinocytes through the production of ETAF and EC IL-3 may mediate inflammatory and hypersensitivity reactions. Furthermore, the monoclonal anti-IL-1 antibody may provide a useful tool for the development of new immunoassays to detect IL-1/ETAF and thereby facilitate the investigation of the role of these cytokines during the pathogenesis of inflammatory diseases.

Notes: The proopiomelanocortin (POMC) products α-melanocyte stimulating hormone (α-MSH) and adrenocorticotropin (ACTH) bind to specific receptors known as the melanocortin (MC) receptors. There is increasing evidence that the MC receptor subtype 1 (MC-1R) is expressed in vitro by several other cutaneous cell types besides melanocytes and keratinocytes. Our knowledge on the MC-1R expression in skin, however, remains fragmentary. In order to examine the expression of MC-1R in human skin cells in vitro and in situ, we made use of a recently described antibody directed against the amino acids 2-18 of the human MC-1R. Flow cytometry analysis revealed the highest MC-1R antigenicity in normal melanocytes and keratinocytes, followed by dermal fibroblasts, microvacualar endothelial cells and WM 35 melanoma cells. Little or no expression was detected in KB carcinoma cells and Fs4 fibroblasts. In normal human skin, immunoreactivity for the anti-MC-1R antibody was detected in hair follicle epithelia, sebocytes, secretory and ductal epithelia of weat glands, and periadnexal mesenchymal cells. Interfollicular epidermis was largely unreactive in adult skin as opposed to undifferentiated kertinocytes of fetal skin. Our findings form a framework within which MC-1 receptor expression can be studied in various skin diseases.

Notes: Abstract Cytokines released by mast cells as well as their effects on mast cell functions appear to be of major importance in the pathogenesis of mast cell mediated skin diseases. In addition, the identification of some key mediators which were found to play a crucial in vivo rôle in certain disease states may allow the development of new therapeutie strategies using cytokines or cytokine antagonists for the treatment of inflammatory skin diseases. So far, encouraging results have been obtained when diseases with mast cell involvement such as atopic eczema or mastocytosis have been treated with different IFNs. Future trials using IFNs and other newly detected cytokines or their antagonists are required to establish effective therapy regimens.

Notes: Abstract: To investigate the pathomechanisms of leukocytoclastic vasculitis (LcV) we compared mouse models of LcV with non-vasculitic irritant contact dermatitis (ICD). Criteria for LcV as met by the immune complex-mediated Arthus reaction (Art-r) were also fulfilled by the localized Shwartzman reaction (Shw-r) and by cutaneous Loxoscelism (Lox) (injection of venom from Loxosceles reclusa containing sphingomyelinase D). After depletion of PMN (by γ-irradiation) vessel damage could not be elicited in these models, distinguishing them from models of direct endothelial insult (necrotizing ICD). Depletion of complement could only delay, but not inhibit the Art-r, and did not change ICD, Lox or the Shw-r. The Shw-r exclusively revealed a sustained local expression of vascular adhesion molecules for 24 h in the preparatory phase (LPS s.c.), not observed in the Art-r, in Lox or ICD. Subsequent challenge with LPS i.p. was associated with upregulation of Mac-1 and ICAM-1 on PMN, but not of VLA-4 or LFA-1 (FACS analysis). Cytokines which were able to replace LPS in priming for LcV in the Shw-r (TNF-α and IL-1β) also induced sustained expression of adhesion molecules, whereas IL-12 and IFN-γ did neither. Neutralizing IL-12 or IFN-γ also inhibited neither LcV nor sustained expression of adhesion molecules, whereas anti-TNF-α inhibited both. Anti-TNF-α had no marked inhibitory effects in the Art-r, in Lox or ICD. Combined (but not separate) neutralization of both E-selectin and VCAM-1 by antibodies suppressed LcV independent from reducing influx of PMN, proving that their sustained expression is decisive for the Shw-r and interferes with normal diapedesis. Since Loxosceles venom is known to dysregulate diapedesis and degranulation of PMN in vitro, since adherent immune complexes activate PMN at the vessel wall, and since adhesion molecules are dysregulated in the Shw-r, we suggest that LcV develops when activation of PMN coincides with vascular alterations which interfere with normal diapedesis.

Notes: Abstract There is strong evidence for a complex network-like interaction between cytokines, growth factors and other mediators being responsible for cell growth and differentiation as well as for the outcome of an inflammatory reaction. Therefore, the regulation of the production of the ubiquitous proinflammatory cytokine interleukin 6 (IL-6) by transforming growth factor β (TGFP) was investigated. Human peripheral blood mononuclear cells (PBMC), human normal keralinocytes (HNK), and an epidermoid carcinoma cell line (KB) were treated with TGFβ or TGFβ2 and subsequently IL-6 secretion was evaluated. Addition of TGFβ1 as well as TGFβ2 to PBMC, HNK and KB cells resulted in a significantly increased release of IL-6 activity. The inducing effect of TGFβ was dose dependent and maximal when supernatants were harvested 48 h after stimulation. In addition, upon Western blot analysis using a monoclonal IL-6 antibody significantly increased amounts of IL-6 protein were detected in KB cell supernatants following stimulation with TGFβ 1. These results were further confirmed at the transcriptional level using a cDNA probe specific for IL-6 and Northern blot analysis. Accordingly, an increased IL-6 inRNA expression in PBMC or KB cells was detected following TGFβl treatment. These findings indicate that TGFβ in contrast to its antiinflammatory capacities also may stimulate IL-6 production in PBMC and keratinocytes. This further supports the possibly important immunoregulatory role of growth factors such as TGFβ.

Notes: Abstract: The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. It has been observed that certain skin diseases such as psoriasis and atopic dermatitis have a neurogenic component. Neuropeptides released by sensory nerves that innervate the skin and often contact epidermal and dermal cells can directly modulate functions of keratinocytes, Langerhans cells (LC), mast cells, dermal microvascular endothelial cells and infiltrating immune cells. Among these neuropeptides the tachykinins substance P (SP) and neurokinin A (NKA), calcitonin gene related peptide (CGRP), vasoactive intestinal peptide (VIP) and somato statin (SOM) have been reported to effectively modulate skin and immune cell functions such as cell proliferation, cytokine production or antigen presentation under physiological or pathophysiological conditions. Expression and regulation of their corresponding receptors that are expressed on a variety of skin cells as well as the presence of neuropeptidespecific peptidases such as neutral endopeptidase (NEP) or angiotensinconverting enzyme (ACE) determine the final biological response mediated by these peptides on the target cell or tissue. Likewise, skin cells like keratinocytes or fibroblasts are a source for neurotrophins such as nerve growth factor that are required not only for survival and regeneration of sensory neurons but also to control responsiveness of these neurons to external stimuli. Therefore, neuropeptides, neuropeptide receptors, neuropeptidedegrading enzymes and neurotrophins participate in a complex, interdependent network of mediators that modulate skin inflammation, wound healing and the skin immune system. This review will focus on recent studies demonstrating the role of tachykinins, CGRP, SOM and VIP and their receptors and neuropeptide-degrading enzymes in mediating neurogenic inflammation in the skin.

Notes: Dermal microvascular endothelial cells (ECs) are both source and target of the pro-opiomelanocortin (POMC) peptides ACTH and α-melanocyte-stimulating hormone (α-MSH). The availability of neuropeptides as important modulators of innate and adaptive immune responses is controlled by neuropeptide-specific peptidases such as neutral endopeptidase (NEP) or angiotensin-converting enzyme (ACE). In this study, we have tested the possibility that NEP or ACE expressed by ECs may influence the local bioavailability of POMC peptides. Incubation of ACTH1−39 with cell membranes prepared from the high NEP-/low ACE-expressing microvascular EC line 1 (HMEC-1) or from low NEP-/high ACE-expressing primary human dermal ECs (HDMECs) for 30–480 min resulted in a decrease in ACTH immunoreactivity (IR) over time in membrane supernatants that could be partially blocked with NEP inhibitors as detected by radioimmunoassay. In parallel, α-MSH IR increased peaking after 60 min. Fragments generated by incubation of HMEC-1 or HDMEC membranes with ACTH1−39, ACHT1−24 or α-MSH for 1–120 min were further analysed by mass spectroscopy. HMEC-1 membranes generated peptide products which could be altered by inhibition of NEP, but not ACE. Likewise, HDMEC membranes fragmented ACTH similar to HMEC-1 membranes in the presence of NEP inhibitors. Some of the proteins can be assigned to regular proteolytic cleavage, while others seem to be modified. Importantly, HMEC-1 and HDMEC membranes also slowly degraded α-MSH, suggesting that EC proteolytic peptidases locally control ACTH/α-MSH bioavailability, which may be important in controlling cutaneous inflammation.

Notes: α-Melanocyte-stimulating hormone (α-MSH) exerts numerous immunomodulatory and anti-inflammatory activities, which at least partly are mediated through the melanocortin receptor-1 (MC-1R), expressed on monocytes, dermal fibroblasts, dendritic cells (DCs), endothelial, and epithelial cells. Accordingly, α-MSH downregulates the production of proinflammatory cytokines and the expression of costimulatory molecules on antigen-presenting cells (APCs) via inhibiting the activation of transcription factors such as NF-κB, while upregulating the production of suppressor factors such as IL-10. Besides α-MSH, its C-terminal-tripeptide KPV and the IL-1β-derived tripeptide KPT are capable of modulating APC functions. Using a mouse model of contact hypersensitivity (CHS), systemic and epicutaneous application of α-MSH, KPV, or KPT inhibited CHS induction and induced hapten-specific tolerance. However, using MC-1R-deficient mice (MC-1Re/e), tolerance induction was found to be independent of MC-1R expression. To further investigate the mechanisms responsible for tolerance induction, adoptive transfer experiments were performed. α-MSH-treated haptenized DCs inhibited CHS and induced hapten-specific tolerance, via induction of regulatory T lymphocytes (Treg). In contrast, using a murine model of intestinal inflammation [Dextransulfate (DSS)-induced colitis], the expression of a functional MC-1R was found to be crucial for α-MSH to exert its anti-inflammatory activity; in wt mice, weight loss was reduced and the survival rate significantly was improved upon treatment with α-MSH or KPV. However, DSS colitis was significantly aggravated in MC1-Re/e mice, resulting in the death of all animals. Bone marrow transplantation from wt mice did not alter the course of inflammation, indicating that MC-1R expression on non-hematopoietic cells is crucial for host defense. These findings further support the therapeutic potential of α-MSH-related peptides for the treatment of inflammatory, autoimmune, and allergic diseases.