Thomas Brzoska

Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures.

Abstract:  The vanilloid receptor subtype 1 (VR1)/(TRPV1), binding capsaicin, is a non‐selective cation channel that recently has been shown in human keratinocytes in vitro and in vivo. However, a description of VR1 localization in other cutaneous compartments in particular cutaneous nerve fibers is still lacking. We therefore investigated VR1 immunoreactivity as well as mRNA and protein expression in a series (n = 26) of normal (n = 7), diseased (n = 13) [prurigo nodularis (PN) (n = 10), generalized pruritus (n = 1), and mastocytosis (n = 2)], and capsaicin‐treated human skin (n = 6). VR1 immunoreactivity could be observed in cutaneous sensory nerve fibers, mast cells, epidermal keratinocytes, dermal blood vessels, the inner root sheet and the infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands. Upon reverse transcriptase‐polymerase chain reaction and Western blot analysis, VR1 was detected in mast cells and keratinocytes from human skin. In pruritic skin of PN, VR1 expression was highly increased in epidermal keratinocytes and nerve fibers, which was normalized after capsaicin application. During capsaicin therapy, a reduction of neuropeptides (substance P, calcitonin gene‐related peptide) was observed. After cessation of capsaicin therapy, neuropeptides re‐accumulated in skin nerves. In conclusion, VR1 is widely distributed in the skin, suggesting a major role for this receptor, e.g. in nociception and neurogenic inflammation.

α-Melanocyte-Stimulating Hormone and Related Tripeptides: Biochemistry, Antiinflammatory and Protective Effects in Vitro and in Vivo, and Future Perspectives for the Treatment of Immune-Mediated Inflammatory Diseases

Alpha-MSH is a tridecapeptide derived from proopiomelanocortin. Many studies over the last few years have provided evidence that alpha-MSH has potent protective and antiinflammatory effects. These effects can be elicited via centrally expressed melanocortin receptors that orchestrate descending neurogenic antiinflammatory pathways. alpha-MSH can also exert antiinflammatory and protective effects on cells of the immune system and on peripheral nonimmune cell types expressing melanocortin receptors. At the molecular level, alpha-MSH affects various pathways implicated in regulation of inflammation and protection, i.e., nuclear factor-kappaB activation, expression of adhesion molecules and chemokine receptors, production of proinflammatory cytokines and mediators, IL-10 synthesis, T cell proliferation and activity, inflammatory cell migration, expression of antioxidative enzymes, and apoptosis. The antiinflammatory effects of alpha-MSH have been validated in animal models of experimentally induced fever; irritant and allergic contact dermatitis, vasculitis, and fibrosis; ocular, gastrointestinal, brain, and allergic airway inflammation; and arthritis, but also in models of organ injury. One obstacle limiting the use of alpha-MSH in inflammatory disorders is its pigmentary effect. Due to its preserved antiinflammatory effect but lack of pigmentary action, the C-terminal tripeptide of alpha-MSH, KPV, has been delineated as an alternative for antiinflammatory therapy. KdPT, a derivative of KPV corresponding to amino acids 193-195 of IL-1beta, is also emerging as a tripeptide with antiinflammatory effects. The physiochemical properties and expected low costs of production render both agents suitable for the future treatment of immune-mediated inflammatory skin and bowel disease, fibrosis, allergic and inflammatory lung disease, ocular inflammation, and arthritis.

New Insights into the Functions of α-MSH and Related Peptides in the Immune System

Abstract: There is a substantial body of evidence that the tridecapeptide α‐melanocyte‐stimulating hormone (α‐MSH) functions as a mediator of immunity and inflammation. The immunomodulating capacity of α‐MSH is primarily because of its effects on melanocortin receptor (MC‐1R)‐expressing monocytes, macrophages, and dendritic cells (DCs). α‐MSH down‐regulates the production of proinflammatory and immunomodulating cytokines (IL‐1, IL‐6, TNF‐α, IL‐2, IFN‐γ, IL‐4, IL‐13) as well as the expression of costimulatory molecules (CD86, CD40, ICAM‐1) on antigen‐presenting DCs. In contrast, the production of the cytokine synthesis inhibitor IL‐10 is up‐regulated by α‐MSH. At the molecular level, these effects of α‐MSH are mediated via the inhibition of the activation of transcription factors such as NFκB. Not only α‐MSH but also its C‐terminal tripeptide (α‐MSH 11–13, KPV) was able to bind to MC‐1R and to modulate the function of APCs. In vivo, using a mouse model of contact hypersensitivity (CHS) systemic and topical application of α‐MSH or KPV inhibited the sensitization and the elicitation phase of CHS and was able to induce hapten‐specific tolerance. To investigate the underlying mechanisms of tolerance induction, we have performed in vivo transfer experiments. Treatment of naive mice with bone marrow‐derived immature haptenized and α‐MSH‐pulsed DCs resulted in a significant inhibition of CHS. Furthermore, tolerance induction was found to be mediated by the generation of CTLA4+ and IL‐10‐producing T lymphocytes. The potent capacity of α‐MSH to modulate the function of antigen‐presenting cells (APCs) has been further supported in another experimental approach. In vitro, by activating APCs, α‐MSH has been shown to modulate IgE production by IL‐4 and anti‐CD40 stimulated B lymphocytes. Moreover, in a murine model of allergic airway inflammation, systemic treatment with α‐MSH resulted in a significant reduction of allergen‐specific IgE production, eosinophil influx, and IL‐4 production. These effects were mediated via IL‐10 production, because IL‐10 knockout mice were resistant to α‐MSH treatment. Therefore, therapeutic application of α‐MSH or related peptides (KPVs) as well as α‐MSH/KPV‐pulsed DCs may be a useful approach for the treatment of inflammatory, autoimmune, and allergic diseases in the future.

The Role of α‐MSH as a Modulator of Cutaneous Inflammation

Abstract: Among various neuropeptides such as substance P, calcitonin gene‐related peptide and others, α‐melanocyte‐stimulating hormone (α‐MSH) was found to be produced in the skin. Moreover, melanocortin receptor 1 (MC‐1R), which is specific for α‐MSH and ACTH, is expressed in the skin on keratinocytes, dendritic cells, macrophages and endothelial cells. In monocytes, macrophages and dendritic cells α‐MSH inhibits the production and activity of immunoregulatory and proinflammatory cytokines such as IL‐2, IFN‐γ, TNF‐α, and IL‐1. It downregulates the expression of costimulatory molecules such as CD86 and CD40 and induces the production of suppressor factors such as the cytokine synthesis inhibitory factor IL‐10. On endothelial cells α‐MSH is capable of downregulating the LPS‐induced expression of adhesion molecules such as vascular cell adhesion molecule (VCAM) and E‐selectin. Moreover, the LPS‐induced activation of transcription factors such as NFκB is downregulated by α‐MSH. In a mouse model i.v. or topical application of α‐MSH was found to inhibit the induction phase as well as the effector phase of contact hypersensitivity (CHS) reactions and to induce hapten‐specific tolerance. These findings indicate that the production of immunosuppressing neuropeptides such as α‐MSH by epidermal cells may play an essential role during the pathogenesis of immune and inflammatory reactions in the skin.

Mechanisms of the Antiinflammatory Effects of α-MSH: Role of Transcription Factor NF-κB and Adhesion Molecule Expression

ABSTRACT: The recruitment of leukocytes from the circulation to inflamed tissue is regulated by the expression of adhesion molecules on both leukocytes and endothelial cells. The proopiomelanocortin‐derived peptide α‐melanocyte stimulating hormone (α‐MSH) is known to modulate inflammation. Thus, we investigated the influence of α‐MSH on the LPS‐induced expression of the adhesion molecules E‐selectin and VCAM‐1 on endothelial cells. Human microvascular endothelial cells (HMEC‐1) were treated with LPS (100 ng/ml) alone or in the presence of α‐MSH (10−8 to 10−16 M). RT‐PCR analysis showed that α‐MSH significantly reduced LPS‐induced expression of VCAM‐1 and E‐selectin. Since many adhesion molecules contain regulatory NF‐κB sites in their promoter region, the role of α‐MSH in the activation of the transcription factor NF‐αB was also investigated. α‐MSH significantly downregulated the LPS‐mediated activation of NF‐κB, in a dose‐dependent manner. These findings indicate that modulation of the transcription factor NF‐κB is a crucial molecular event, one that seems to be responsible for the antiinflammatory effects of α‐MSH.

Human Peripheral Blood‐Derived Dendritic Cells Express Functional Melanocortin Receptor MC‐1R

ABSTRACT: The neuropeptide, α‐melanocyte‐stimulating hormone (α‐MSH) is well known for its immunomodulating capabilities. α‐MSH antagonizes the activity of numerous proinflammatory mediators; for example, Interleukin‐1 (IL‐1), IL‐6, tumor necrosis factor α (TNFα), and bacterial endotoxin. In vivoα‐MSH has been shown to suppress a contact hypersensitivity reaction in mice, and to induce hapten‐specific tolerance. Since antigen presenting cells (APC) represent key elements for tolerance induction, the effect of α‐MSH, and the expression of its receptor‐melanocortin receptor‐1 (MC‐1R), on human peripheral blood‐derived monocytes and dendritic cells (DC), was investigated. Semiquantitative RT‐PCR demonstrated that monocytes and DC express MC‐1R, but none of the other members of the MC‐receptor family. Moreover, the extent of MC‐1R expression correlated with the state of activation of these cells. Since the major ligand of MC‐1R is α‐MSH the question of whether α‐MSH affects the function of monocyte derived DC was further investigated. We found that the expression of the costimulatory molecules CD 86 and CD 40 was downregulated on DC in the presence of α‐MSH. Thus, α‐MSH may exert its immunosuppressive effects by altering the function of APC.

α-Melanocyte-Stimulating Hormone Inhibits Allergic Airway Inflammation

α-Melanocyte-stimulating hormone (α-MSH) is a neuropeptide controlling melanogenesis in pigmentary cells. In addition, its potent immunomodulatory and immunosuppressive activity has been recently described in cutaneous inflammatory disorders. Whether α-MSH is also produced in the lung and might play a role in the pathogenesis of inflammatory lung conditions, including allergic bronchial asthma, is unknown. Production and functional role of α-MSH were investigated in a murine model of allergic airway inflammation. α-MSH production was detected in bronchoalveolar lavage fluids. Although aerosol challenges stimulate α-MSH production in nonsensitized mice, this rapid and marked stimulation was absent in allergic animals. Treatment of allergic mice with α-MSH resulted in suppression of airway inflammation. These effects were mediated via IL-10 production, because IL-10 knockout mice were resistant to α-MSH treatment. This study provides evidence for a novel function of α-MSH linking neuroimmune functions in allergic airway inflammation.

Modulation of Cutaneous Inflammation by Angiotensin-Converting Enzyme

Cutaneous neurogenic inflammation is a complex biological response of the host immune system to noxious stimuli. Present evidence suggests that zinc metalloproteases may play an important role in the regulation of neurogenic inflammation by controlling the local availability of neuropeptides, such as substance P (SP), that are capable of initiating or amplifying cutaneous inflammation after release from sensory nerves. To address the hypothesis that the dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) is capable of modulating skin inflammation, we have analyzed murine allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) using wild-type C57BL/6J (ACE+/+) or genetically engineered mice with a heterozygous deletion of somatic ACE (ACE+/−). In 2,4-dinitro-1-fluorobenzene-sensitized ACE+/− mice, ACD was significantly augmented in comparison to ACE+/+ controls as determined by the degree of ear swelling after exposure to hapten. Likewise, systemic treatment of ACE+/+ mice with the ACE inhibitor captopril before sensitization or elicitation of ACD significantly augmented the ACD response. In contrast, local damage and neuropeptide depletion of sensory nerves following capsaicin, injection of a bradykinin B2, or a SP receptor antagonist before sensitization significantly inhibited the augmented effector phase of ACD in mice with functionally absent ACE. However, in contrast to ACD, the response to the irritant croton oil was not significantly altered in ACE+/− compared with ACE+/+ mice. Thus, ACE by degrading bradykinin and SP significantly controls cutaneous inflammatory responses to allergens but not to irritants, which may explain the frequently observed exacerbation of inflammatory skin disease in patients under medication with ACE inhibitors.

Detection of melanocortin-1 receptor antigenicity on human skin cells in culture and in situ

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.

Molecular Basis of the α‐MSH/IL‐1 Antagonism

ABSTRACT: The neuropeptide α‐melanocyte stimulating hormone (α‐MSH) is recognized as a potent mediator of immune and inflammatory reactions. Accordingly, α‐MSH in vitro, as well as in vivo, antagonizes the proinflammatory activities of cytokines such as interleukin‐1 (IL‐1), IL‐6, and tumor necrosis factor a (TNFα). Since the molecular basis of these antiinflammatory effects is not well known, the influence of α‐MSH on IL‐1β‐induced chemokine production and transcription factor activation was investigated in human keratinocytes. α‐MSH, in a dose‐dependent manner, after 48 h, significantly reduced the IL‐1b mediated secretion of the C‐X‐C chemokines IL‐8 and Groα. This was confirmed by semiquantitative RT‐PCR, which revealed a marked downregulation in IL‐8 and Groα mRNA expression. Furthermore, we determined the effect of α‐MSH on the IL‐1β‐induced activation of the nuclear factor κB (NFκB)‐a major transcription factor for chemokine genes. Electrophoretic mobility‐shift‐assays showed that α‐MSH, in a dose range from 10−6 to 10−12 M, significantly downregulated the IL‐1b‐induced activation of NFκB 10 minutes after stimulation. Therefore, NFκB inactivation by α‐MSH appears to be a crucial event, one that is responsible for the downregulation of cytokine gene transcription.