Victoria Shpacovitch

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.

Role of protease-activated receptors in inflammatory responses, innate and adaptive immunity

Serine proteases are well known as enzymes involved in digestion of dietary proteins, blood coagulation, and homeostasis. Only recent groundbreaking studies revealed a novel role of serine proteases as signaling molecules acting via protease‐activated receptors (PARs). Important effects of PAR activation on leukocyte motility, cytokine production, adhesion molecule expression, and a variety of other physiological or pathophysiological functions have been described in vitro and in vivo. The crucial role of PAR activation during disease progression was revealed in animal models of different gastrointestinal pathologies, neuroinflammatory and neurodegenerative processes, skin, joint and airway inflammation, or allergic responses. This review focuses on the findings related to the impact of PAR deficiency in animal models of inflammatory and allergic diseases. Additionally, we observe the role of PAR activation in the regulation of functional responses of innate and adaptive immune cells in vitro. Understanding the mechanisms by which PARs exert the effects of serine proteases on immune cells may lead to new therapeutic strategies in inflammation, immune defense, and allergy.

Tumor-derived matrix metalloproteinase-1 targets endothelial proteinase-activated receptor 1 promoting endothelial cell activation.

In the vascular system, circulating tumor cells interact with endothelial cells. Tumor-endothelial cross-talk transforms the intravascular milieu to a prothrombotic, proinflammatory, and cell-adhesive state called endothelial cell activation (ECA). In the present study, we analyze the potential of metastatic tumor-derived soluble factors to transform the vascular endothelium into a prothrombotic and proinflammatory activated state. Supernatant from cultured melanoma and colon cancer cells (A375, WM9, A7, and HT-29) induced an acute activation of macrovascular and microvascular endothelial cells (human umbilical vein endothelial cells and human dermal microvascular endothelial cells) as shown by intracellular calcium flux and secretion of von Willebrand factor and interleukin-8, all markers of acute ECA. This process was inhibited using specific proteinase-activated receptor 1 (PAR1) inhibitors (RWJ-58259 and SCH-79797), indicating a mediating role for endothelial thrombin receptors. Immunofluorescence, Western blot analysis, and collagenase activity assay of tumor cells and culture supernatant revealed the presence of matrix metalloproteinase-1 (MMP-1), a recently described activator of PAR1. Inhibition of MMP-1 in supernatant from cultured tumor cells significantly attenuated ECA. Additional studies using isolated human MMP-1 (5 nmol/L) proved the presence of a functional MMP-1/PAR1 axis in tumor-endothelial communication. These findings show a new pathway of tumor-endothelial cross-talk via an intravascular MMP1/PAR1 axis in microvascular and macrovascular endothelium. Inhibition of this cross-talk may be a powerful means to prevent tumor-induced ECA and thus thrombotic and inflammatory cell adhesion.

Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices

Proteinase‐activated receptor‐2 (PAR2) belongs to a novel subfamily of G‐protein‐coupled receptors with seven‐transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist‐stimulated human neutrophils show significantly enhanced migration in 3‐D collagen lattices. PAR2 agonist stimulation also induced down‐regulation of L‐selectin display and up‐regulation of membrane‐activated complex‐1 very late antigen‐4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)‐1β, IL‐8, and IL‐6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up‐regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.

Pituitary adenylate cyclase activating polypeptide: an important vascular regulator in human skin in vivo.

Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.

Agonists of proteinase-activated receptor-2 affect transendothelial migration and apoptosis of human neutrophils

Abstract:  Skin is the first barrier preventing microorganism invasion in host. Wounds destroy this defense barrier and, without an appropriate care, may lead to sepsis. Neutrophil activation and immigration plays an important role at the inflammatory stage of wound healing. Neutrophils are known to express proteinase‐activated receptors (PARs), which can be activated by serine proteases, also by enzymes involved in wound healing. We previously reported that PAR2 agonists up‐regulate cell adhesion molecule expression and cytokine production by human neutrophils. Here, we demonstrate that PAR2 agonists (serine proteases as well as synthetic peptides) reduce transendothelial migration of neutrophils and prolong their life in vitro. Synthetic PAR2 agonist also enhanced protective interferon (IFN)γ‐induced FcγRI expression at neutrophil cell surface. Of note, IFNγ is a cytokine, which was used in clinical trials to reactivate human neutrophil functions during sepsis. Moreover, we observed a significant increase of PAR2 expression on cell surface of neutrophils from septic patients as compared with healthy volunteers. Together, our results indicate that PAR2 may be involved in the pathophysiology of neutrophil‐endothelial interactions during wound healing or later during sepsis in humans, potentially by affecting neutrophil apoptosis, transendothelial migration and Fcγ receptor‐mediated phagocytosis.

Agonists of Proteinase-Activated Receptor-2 Enhance IFN-γ-Inducible Effects on Human Monocytes: Role in Influenza A Infection

Proteinase-activated receptor-2 (PAR2) is expressed by different types of human leukocytes and involved in the development of inflammatory and infectious diseases. However, its precise role in the regulation of human monocyte and macrophage function during viral infection remains unclear. Also, the ability of PAR2 agonists to enhance the effects induced by immune mediators during infection or inflammation is still poorly investigated. Therefore, we investigated the ability of a PAR2 agonist to enhance IFN-γ-induced suppression of influenza A virus replication in human monocytes. We found that this effect correlates with an increased abundance of IκBα after costimulation of cells with PAR2 agonist and IFN-γ. Remarkably, coapplication of PAR2 agonist and IFN-γ also enhances the effects of IFN-γ on IFN-γ-inducible protein 10 kDa release, and CD64 and αVβ3 surface expression by human monocytes. Together, these findings indicate a potentially protective role of PAR2 activation during the progression of influenza A virus infection. This effect could be associated with the ability of PAR2 agonists to enhance IFN-γ-induced protective effects on human monocytes.

α-1-Antitrypsin and IFN-γ Reduce the Severity of IC-Mediated Vasculitis by Regulation of Leukocyte Recruitment In Vivo

IC-mediated vasculitis (ICV) can be life threatening. The cellular and immune mechanisms controlling ICV are poorly understood. Therefore, we investigated the role of α-1-antitrypsin (α1AT) and IFN-γ in reducing the severity of ICV in a mouse model in vivo. To induce ICV, mice were challenged with the reverse passive Arthus reaction (RPA), the prototypic in vivo model for leukocytoclastic vasculitis (LcV), and the modulation of vascular permeability, edema formation, and leukocyte recruitment was studied. To further analyze the dynamics of RPA, we applied intravital microscopy in the dorsal skinfold chamber. α1AT continuously led to reduced leukocyte recruitment. α1AT interfered with neutrophil recruitment through a KC-dependent mechanism and reduced KC-elicited neutrophil activation. In contrast to α1AT, IFN-γ-reduced leukocyte recruitment during RPA was clearly independent of KC. We also revealed that the recruitment of neutrophils during RPA was a prerequisite for full KC expression. Thus, therapeutic administration of α1AT and IFN-γ might be beneficial for limiting the duration and severity of ICV.

Role of proteinase-activated receptor-2 in anti-bacterial and immunomodulatory effects of interferon-γ on human neutrophils and monocytes

Recent studies show that proteinase‐activated receptor‐2 (PAR2) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR2 activation in the anti‐microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR2 to the anti‐microbial response of isolated human innate immune cells. We found that PAR2 agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon‐γ (IFN‐γ). However, co‐application of PAR2‐cAP and IFN‐γ did not enhance the phagocytic and bacteria‐killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN‐γ enhances PAR2 agonist‐induced monocyte chemoattractant protein 1 (MCP‐1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide‐3 kinase and janus kinase molecules are involved in the synergistic effect of PAR2 agonist and IFN‐γ on MCP‐1 secretion. Our findings suggest a potentially protective role of PAR2 agonists in the anti‐microbial defence established by human monocytes and neutrophils.

Interferon-γ induces upregulation and activation of the interleukin-31 receptor in human dermal microvascular endothelial cells.

Abstract:  Interleukin‐31 (IL‐31), a recently discovered cytokine derived from T helper cells, is involved in chronic dermatitis and pruritus. This study demonstrates for the first time that the IL‐31 receptor complex for IL‐31 is substantially upregulated in human dermal microvascular endothelial cells after stimulation with interferon‐γ (IFN‐γ). Activation of the IL‐31 receptor complex results in the induction of the intracellular ERK1/2 signaling pathway and downregulation of IFN‐γ‐induced monokine induced by IFN‐γ expression. Inhibitor studies revealed that the IFN‐γ‐induced IL‐31RA upregulation is processed via JNK and PI3 kinase activation. In sum, our study points toward an interaction between the TH1‐derived cytokine IFN‐γ and the TH2‐derived cytokine IL‐31 on endothelial cells.