Micha Feld

A sensory neuron–expressed IL-31 receptor mediates T helper cell–dependent itch: Involvement of TRPV1 and TRPA1

BACKGROUND Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. OBJECTIVE We sought to determine whether immune cell-derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31-induced itch. METHODS We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects. RESULTS Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31-induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)-deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca(2+) release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo. CONCLUSION IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA(+)/TRPV1(+)/TRPA1(+) neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma.

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.

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.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of β-arrestin1 and PP2A with noninternalized NK(1)R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

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.

Intracellular degradation of somatostatin-14 following somatostatin-receptor 3-mediated endocytosis in rat insulinoma cells

Somatostatin receptor (SSTR) endocytosis influences cellular responsiveness to agonist stimulation and somatostatin receptor scintigraphy, a common diagnostic imaging technique. Recently, we have shown that SSTR1 is differentially regulated in the endocytic and recycling pathway of pancreatic cells after agonist stimulation. Additionally, SSTR1 accumulates and releases internalized somatostatin‐14 (SST‐14) as an intact and biologically active ligand. We also demonstrated that SSTR2A was sequestered into early endosomes, whereas internalized SST‐14 was degraded by endosomal peptidases and not routed into lysosomal degradation. Here, we examined the fate of peptide agonists in rat insulinoma cells expressing SSTR3 by biochemical methods and confocal laser scanning microscopy. We found that [125I]Tyr11‐SST‐14 rapidly accumulated in intracellular vesicles, where it was degraded in an ammonium chloride‐sensitive manner. In contrast, [125I]Tyr1‐octreotide accumulated and was released as an intact peptide. Rhodamine‐B‐labeled SST‐14, however, was rapidly internalized into endosome‐like vesicles, and fluorescence signals colocalized with the lysosomal marker protein cathepsin D. Our data show that SST‐14 was cointernalized with SSTR3, was uncoupled from the receptor, and was sorted into an endocytic degradation pathway, whereas octreotide was recycled as an intact peptide. Chronic stimulation of SSTR3 also induced time‐dependent downregulation of the receptor. Thus, the intracellular processing of internalized SST‐14 and the regulation of SSTR3 markedly differ from the events mediated by the other SSTR subtypes.

New mechanism underlying IL-31–induced atopic dermatitis

Background TH2 cell–released IL‐31 is a critical mediator in patients with atopic dermatitis (AD), a prevalent and debilitating chronic skin disorder. Brain‐derived natriuretic peptide (BNP) has been described as a central itch mediator. The importance of BNP in peripheral (skin‐derived) itch and its functional link to IL‐31 within the neuroimmune axis of the skin is unknown. Objective We sought to investigate the function of BNP in the peripheral sensory system and skin in IL‐31–induced itch and neuroepidermal communication in patients with AD. Methods Ca2+ imaging, immunohistochemistry, quantitative real‐time PCR, RNA sequencing, knockdown, cytokine/phosphokinase arrays, enzyme immune assay, and pharmacologic inhibition were performed to examine the cellular basis of the IL‐31–stimulated, BNP‐related itch signaling in dorsal root ganglionic neurons (DRGs) and skin cells, transgenic AD‐like mouse models, and human skin of patients with AD and healthy subjects. Results In human DRGs we confirmed expression and co‐occurrence of oncostatin M receptor &bgr; subunit and IL‐31 receptor A in a small subset of the neuronal population. Furthermore, IL‐31 activated approximately 50% of endothelin‐1–responsive neurons, and half of the latter also responded to histamine. In murine DRGs IL‐31 upregulated Nppb and induced soluble N‐ethylmaleimide–sensitive factor activating protein receptor–dependent BNP release. In Grhl3PAR2/+ mice house dust mite–induced severe AD‐like dermatitis was associated with Nppb upregulation. Lesional IL‐31 transgenic mice also exhibited increased Nppb transcripts in DRGs and the skin; accordingly, skin BNP receptor levels were increased. Importantly, expression of BNP and its receptor were increased in the skin of patients with AD. In human skin cells BNP stimulated a proinflammatory and itch‐promoting phenotype. Conclusion For the first time, our findings show that BNP is implicated in AD and that IL‐31 regulates BNP in both DRGs and the skin. IL‐31 enhances BNP release and synthesis and orchestrates cytokine and chemokine release from skin cells, thereby coordinating the signaling pathways involved in itch. Inhibiting peripheral BNP function might be a novel therapeutic strategy for AD and pruritic conditions.