Metin Artuc

Human skin: source of and target organ for angiotensin II

Abstract:u2002 The present study examined the expression of angiotensin receptors in human skin, the potential synthesis of angiotensin II (Ang II) in this location and looked for a first insight into physiological functions. AT1 and AT2 receptors were found within the epidermis and in dermal vessel walls. The same expression pattern was found for angiotensinogen, renin and angiotensin‐converting enzyme (ACE). All components could additionally be demonstrated at mRNA level in cultured primary keratinocytes, melanocytes, dermal fibroblasts and dermal microvascular endothelial cells, except for AT2 receptors in melanocytes. The ability of cutaneous cells to synthesize Ang II was proved by identifying the molecule in cultured keratinocytes. Furthermore, in artificially wounded keratinocyte monolayers, ACE‐mRNA expression was rapidly increased, and enhanced ACE expression was still found in cutaneous human scars 3 months after wounding. These findings suggest that the complete renin–angiotensin system is present in human skin and plays a role in normal cutaneous homeostasis as well as in human cutaneous wound healing.

Differential expression of angiotensin receptors in human cutaneous wound healing

Backgroundu2002 Angiotensin AT1 and AT2 receptors are expressed in human skin. Furthermore, AT2 receptors have been reported to be upregulated during tissue repair and remodelling in various noncutaneous human tissues.

Transition from atherosclerosis to aortic aneurysm in humans coincides with an increased expression of RAS components

While the renin-angiotensin system (RAS) is widely recognized to be involved in atherosclerosis, its potential role in the progression from atherosclerotic lesions to abdominal aortic aneurysm (AAA) is poorly understood. The present study aimed to investigate which components of the RAS may render the atherosclerotic aorta aneurysmatic. The expression of renin, prorenin/renin receptor, angiotensinogen, AT1- and AT2 receptors, cathepsin D, cathepsin G and chymase was examined by immunoblotting and immunohistochemistry in human atherosclerotic, aneurysmatic and healthy aortic tissues obtained from patients undergoing elective repair or at autopsy. AT1- and AT2 receptor mRNA expression was determined using quantitative real-time RT-PCR. All investigated local RAS components were up-regulated in atherosclerotic as compared to healthy tissues. AAA compared to atherosclerosis was characterized by a further increase in the expression of all RAS components except for the AT2 receptor. Cathepsin D was exclusively up-regulated in AAA. Most RAS components co-localized with infiltrating leukocytes or mast cells pointing to their contribution to inflammatory processes. Due to their proteolytic features, some RAS components (cathepsin D and cathepsin G and chymase) may contribute to AAA formation by accessory mechanisms. Taken together, our data suggest that in humans, RAS activation is not just a key-player in the pathogenesis of atherosclerosis, but that a further increasing activation may be involved in the transition from atherosclerosis to AAA.

Expression of prothrombin, thrombin and its receptors in human scars

Abstract:u2002 The coagulation system is thought to play a pivotal role during the initial phase of wound healing, but mechanisms and cells involved are only partly understood. We have therefore examined human scars for the expression of thrombin, its precursor prothrombin and the thrombin receptors, thrombomodulin (TM) and protease‐activated receptor‐1 (PAR‐1), compared with normal skin. Biopsies of scars were obtained from primary excision sites of melanoma patients (nu2003=u200320) and were compared with normal skin distant from the scar (nu2003=u200310), using immunohistochemistry. In addition, polymerase chain reaction analyses were performed on scar versus normal tissue and on cultured keratinocytes, fibroblasts and endothelial cells before and after stimulation with selected cytokines known to be active in wound healing. Normal epidermis was stained for prothrombin, thrombin, TM and PAR‐1, and dermal tissue was stained only for TM and PAR‐1. In scar tissue, thrombin and TM were upregulated in the epidermis and all four molecules in the dermis, independent of the age of the scars. In tissue extracts, mRNA expression of PAR‐1 and prothrombin expression were, however, unchanged and TM even slightly decreased in scars, compared with normal skin. On analysis of cultured cells, keratinocytes expressed mRNA for PAR‐1, TM and prothrombin, endothelial cells for PAR‐1 and TM, and fibroblasts for PAR‐1. An upregulation of PAR‐1 mRNA was induced in fibroblasts on exposure to tumor necrosis factor‐α (TNF‐α), while it remained unchanged in endothelial cells in response to TNF‐α. A downregulation of TM was induced in endothelial cells on exposure to TNF‐α. These findings, showing a marked modulation of thrombin, PAR‐1 and TM even in older human scar tissue, suggest that the coagulation system is not only involved during clotting, but also during the inflammatory and tissue remodelling phases of wound healing.

Phenotypic variability in human skin mast cells

Mast cells (MCs) are unique constituents of the human body. While inter‐individual differences may influence the ways by which MCs operate in their skin habitat, they have not been surveyed in a comprehensive manner so far. We therefore set out to quantify skin MC variability in a large cohort of subjects. Pathophysiologically relevant key features were quantified and correlated: transcripts of c‐kit, FcεRIα, FcεRIβ, FcεRIγ, histidine decarboxylase, tryptase, and chymase; surface expression of c‐Kit, FcεRIα; activity of tryptase, and chymase; histamine content and release triggered by FcεRI and Ca2+ ionophore. While there was substantial variability among subjects, it strongly depended on the feature under study (coefficient of variation 33‐386%). Surface expression of FcεRI was positively associated with FcεRIα mRNA content, histamine content with HDC mRNA, and chymase activity with chymase mRNA. Also, MC signature genes were co‐regulated in distinct patterns. Intriguingly, histamine levels were positively linked to tryptase and chymase activity, whereas tryptase and chymase activity appeared to be uncorrelated. FcεRI triggered histamine release was highly variable and was unrelated to FcεRI expression but unexpectedly tightly correlated with histamine release elicited by Ca2+ ionophore. This most comprehensive and systematic work of its kind provides not only detailed insights into inter‐individual variability in MCs, but also uncovers unexpected patterns of co‐regulation among signature attributes of the lineage. Differences in MCs among humans may well underlie clinical responses in settings of allergic reactions and complex skin disorders alike.

Long-Term Cultured Human Skin Mast Cells Are Suitable for Pharmacological Studies of Anti-Allergic Drugs Due to High Responsiveness to FcεRI Cross-Linking

Human skin mast cells proliferated in the presence of interleukin (IL)-4+SCF (expanding 18-fold in 8 weeks) and acquired profound responsiveness towards high affinity IgE receptor (FcεRI) cross-linking, liberating about 75% of their histamine. In a proof-of-concept, we found that these cells are useful for pharmacological testing. Even a subtle inhibition of degranulation can be visualized. This model might prove valuable in tests of novel anti-allergic drugs.

Skin mast cells develop non-synchronized changes in typical lineage characteristics upon culture.

Despite their hematopoietic origin, mast cells (MCs) develop exclusively in tissues, hampering their ample use in research. To circumvent this problem, tissue‐derived MCs are typically first expanded in culture, but the changes MCs may undergo in the novel micromilieu are poorly defined. Here, we monitor skin MCs from a number of donors over time, revealing profound yet non‐synchronized modulations in culture. While tryptase and chymase, the most specific markers, strongly decline, FcεRI surface expression, and FcεRI‐mediated histamine release steeply increase (from ≈15.5% to ≈60%), replicated by similar increments in TNF‐α secretion. Interestingly, the modulations are independent of cell cycle progression, as they are comparable in the growth and postgrowth phase, implying they primarily result from microenvironmental conditioning. The data highlight a high degree of MC versatility, but also advise that results based on cultured MCs should be viewed with some caution, as they may not accurately reflect their counterparts in situ.

Testosterone exerts selective anti-inflammatory effects on human skin mast cells in a cell subset dependent manner.

Androgens are known to exert anti‐inflammatory effects but their impact on mast cells (MCs) remains to be determined. Here, we show that MCs isolated from human foreskin samples (male) and those from breast skin (female) express the androgen receptor, albeit with a 10‐fold difference between the subsets. While fundamental MC properties (FcεRI, c‐Kit, tryptase; histamine release upon FcεRI cross‐linking) were unaffected or slightly reduced (chymase) by testosterone, the hormone had a more profound impact on the production of cytokines, with IL‐6 being a target (reduction by 53%). Interestingly, this effect was limited to breast skin MCs (15 of 16 donors displayed this phenomenon), but was not reproduced by foreskin MCs. Collectively, effector functions of human skin MCs are modulated by androgens in a gene‐selective and MC subset‐specific fashion. Possibly, MCs from women are more susceptible to testosterone. We also demonstrate that MC IL‐6 production is highly variable among individuals.

Mast cell-derived TNF-α and histamine modify IL-6 and IL-8 expression and release from cutaneous tumor cells.

Abstract:u2002 The coincidence of skin tumors and elevated mast cell (MC) numbers has been known for many years. However, it has remained controversial whether, in this context, MCs promote or inhibit tumor growth. Addressing this problem, different melanoma and squamous cell carcinoma cell lines were co‐cultivated with primary, dermal MC for 24u2003h and gene or protein expression of cytokines tumor necrosis factor (TNF‐α), interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) estimated. Co‐culture with MCs led to an increase in IL‐8 gene expression and IL‐8 protein release from melanoma cells and IL‐6 and IL‐8 gene expression and protein release from squamous cell carcinoma cells, respectively. Moreover induction of IL‐6 and IL‐8 was primarily regulated by MC‐derived TNF‐α. Our data suggest an interplay between MCs and tumor cells, which results in altered cytokine release and may, thus, have an impact on tumor growth, invasion and neovascularisation.

IL-4 and human skin mast cells revisited: reinforcement of a pro-allergic phenotype upon prolonged exposure

Mast cells (MCs), unique cellular elements of the body, are commonly associated with IgE-mediated reactions and manifestations of Th2-type immunity. A key characteristic of the lineage is its heterogeneity, with subsets displaying significant variation depending on maturation stage, species, tissue, microenvironment and other. Heterogeneity also affects MC responses to extracellular cues. Indeed, IL-4, the signature cytokine of Th2-immunity, can affect MCs in opposing ways ranging from the induction of apoptosis to positive regulation of lineage characteristics. It is unknown, however, whether IL-4 alters the phenotype of terminally differentiated human cutaneous MCs. Using our well-established technique for homogeneous purification of human skin MCs, we now report that prolonged contact with IL-4 not only increases MC expansion, but also phenotypically and functionally re-shapes the cells. FcεRI cell surface expression, FcεRIα-specific mRNA and FcεRI-mediated histamine release are all augmented by IL-4, while histamine release elicited by the non-immunological stimulus, substance P, remains unaffected. IL-4’s potential to mold MCs is broad and similarly detectable across donors. Intriguingly, IL-4 impacts granule-associated mediators, especially histamine whose synthesis is boosted in the presence of IL-4. To our knowledge, an increase in histamine production by IL-4 has not been described yet for any type of MCs, but may well contribute to its pro-allergic effect given the significance of this biogenic amine to allergic symptoms. Collectively, IL-4 alters human skin MCs after long-term exposure mimicking chronic disorders by strengthening MC numbers and intensifying processes associated with allergic inflammation.