11.1. INTRODUCTION

A critical role of various proteases in skin homeostasis as well as pathobiology, including itch, was described decades ago (Arthur and Shelley 1955; Rajka 1967, 1969; Shelley and Arthur 1955). Later, after the cloning of protease-activated receptors-1 (PAR-1) in 1991 (Vu et al. 1991) and the characterization of PAR-2 in the skin (D’Andrea et al. 1998; Derian et al. 1997; Hou et al. 1998; Santulli et al. 1995; Schechter et al. 1998; Steinhoff et al. 1999), some of the effects of endogenous or exogenous proteases could be attributed—at least in part—to the activation of those G-protein-coupled receptors. In 2000, a role of PAR-2 in skin neurogenic inflammation and later in human pruritus was established (Steinhoff et al. 2000, 2003). This review highlights our current understanding of proteases as histamine-independent pruritogens that are mainly—but not exclusively—mediated via protease-activated receptors. For more detailed information about the cellular mechanisms of PAR function, the reader is referred to exquisitely detailed reviews (Cottrell et al. 2002; Ossovskaya and Bunnett 2004; Steinhoff et al. 2005).

Various proteases such as mucunain, trypsin, kallikreins (KLK), or tryptase have been demonstrated to exert capacities as pruritogens in rodents or humans in vivo (Cormia and Dougherty 1960; Costa et al. 2008; Hägermark 1974; Hagermark et al. 1972; Hansson et al. 2002; Ny and Egelrud 2003; Reddy et al. 2008; Stefansson et al. 2008; Steinhoff et al. 2003; Ui et al. 2006). Accordingly, increased tryptase serum levels have been described in hemodialysis patients, interestingly correlating with itch severity, as well as in patients with atopic dermatitis (AD) (Dugas-Breit et al. 2005; Kawakami et al. 2006).

On the basis of these findings, it is important to understand the mechanisms how protease activated receptors, in particular, PAR-2 but probably also PAR-4, regulate protease-dependent, but histamine-independent itch. This is also of major medical interest since serine proteases like KLK, matriptase, prostasin, or tryptase have been implicated in various pruritic diseases including atopic dermatitis, Netherton syndrome, psoriasis, anaphylaxis-associated itch, dry skin itch, or renal insufficiency-associated itch, for example (Zhu et al. 2009a; Akiyama et al. 2009, 2010a, 2010b, 2010c).

However, the itch symptom as observed in patients with AD, other eczematous diseases, neuropathic or systemic disease-associated itch, or tumor-associated itch is mostly resistant to therapy with oral antihistamines. In this context, it is important to note that the itch sensation can be triggered by endogenous (KLK, matriptase, trypsins, and prostasin) or exogenous (e.g., house dust mite allergens Dpt. 1, 3, 9 or certain Staphylococcus aureus toxins) factors that are partly proteases (Jeong et al. 2008; Kato et al. 2009; Lee et al. 2010; Lutfi et al. 2012; Frateschi et al. 2011). Unfortunately, the mechanisms by which itch is controlled in antihistamine unresponsive pruritic diseases are still poorly understood in humans, and effective therapeutic options represent a significant unmet need (Hong et al. 2011; Buddenkotte and Steinhoff 2010).

As will be pointed out later in this book chapter in greater detail, PAR-2 is widely expressed by different cell types including suprabasal keratinocytes, endothelial cells, mast cells, neutrophils, macrophages, dendritic cells, and sensory nerve fibers, for example (Böhm et al. 1996). Recent findings indicate that keratinocytes can also be seen as a “sensory forefront” of neuronal activation and signaling (Elias and Steinhoff 2008). Upon activation by trigger factors that are potentially deleterious for the body system, such as microbes, noxious heat/cold, UV radiation, chemicals, allergens, or proteases, keratinocytes are capable of releasing factors that subsequently activate peripheral nerve endings to induce “neurogenic” inflammation, increased skin sensitivity, and pain or itch (Roosterman et al. 2006; Ma 2010). The factors that mediate this epidermal–neuronal communication during chronic inflammation and itch are, however, currently poorly understood and include at least ATP, endorphin, and endothelin-1 (ET-1) (Caterina et al. 2000; Imamachi et al. 2009; Kido et al. 2011).

Several studies indicate that proteases like KLK, trypsin, or tryptase are important activators of neuronal and/or keratinocyte-derived PAR-2, which subsequently induces itch directly (direct activation of nerve endings) or indirectly via activation of keratinocyte PAR-2 by endogenous or exogenous proteases. Activation of keratinocyte-derived PAR-2 leads to the release of as yet unidentified mediators, which activate sensory nerves and lead to itch and neuroinflammation, as well as nerve sprouting and/or skin hypersensitivity (Figure 11.1) (Frateschi et al. 2011; Stefansson et al. 2008; Moormann et al. 2006; Steinhoff et al. 1999, 2000, 2003, 2005). This chapter gives a comprehensive overview how proteases act via PAR-2 to trigger itch and skin inflammation.

FIGURE 11.1

Role of exogenous, keratinocyte-, and immune-cellderived proteases on itch and inflammation in the skin. Upon stimulation by exogenous or endogenous proteases, PAR-2 becomes activated on nerve endings of primary afferent nerve fibers. Simultaneously, (more...)

Our review highlights the mechanisms by which proteases act via PAR receptors to exert important biological and pathobiological functions in the skin with a focus on itch and neurogenic inflammation. Understanding these mechanisms may lead to novel therapies against acute as well as chronic, recalcitrant itch.

11.2. PROTEASE-ACTIVATED RECEPTORS AND PROTEASES

Proteases and their inhibitors have diverse functional roles to maintain body homeostasis. Proteolysis does not only occur during stress, injury, or infection but is also necessary for normal tissue function, e.g., thrombin or factor VII are involved in blood coagulation, trypsin is an important protease for food digestion, and—in the skin—members of the KLK protease family are critical for epidermal differentiation, cornification and/or pigmentation (Babiarz-Magee et al. 2004; Derian et al. 1997; Lee et al. 2010; Lin et al. 2008; Santulli et al. 1995; Seiberg et al. 2000; Seiberg 2001). Extracellular proteolysis activity is also directly sensed by cells through the unique class of G-protein-coupled receptors (GPCRs), known as PARs, which consist of four receptors with unique cellular functions. These receptors become activated by proteolytic cleavage of the N-terminal end. Thereby, a tethered ligand sequence (TLS) is unmasked that binds to the second extracellular loop of the same receptor and subsequently leads to receptor activation, signaling, and receptor internalization. It is important to understand that, in contrast to other ligand binding GPCRs, such as neurokinin 1 receptor (NK1R) or endothelin A receptor (ETAR), activation and internalization of PARs results in endosomal trafficking, ubiquitination, and degradation of the receptors in lysosomes. Thus, replenishing of the cell surface with PARs depends on transport of stored receptors in intracellular vesicular structures and de novo synthesis of the receptor (Böhm et al. 1996a, 1996b; Déry et al. 1999; Roosterman et al. 2003). Thus, while binding of substance P (SP) or ET-1 to NK1R or ETAR, respectively, leads to internalization and recycling of the receptors, activation of PARs by proteases results in degradation of the internalized receptors (Böhm et al. 1996a; Roosterman et al. 2003, 2004; Zhang et al. 1999).

PARs are widely expressed in different cell types and activation of PARs on these cells induces different responses, respectively. PAR-1 was the first PAR to be cloned and was originally identified and termed as the thrombin receptor (Coughlin et al. 1992; Vu et al. 1991). PAR-1 is also activated by other coagulation proteases, matrix metalloproteinase 1, and microbial proteases (Goerge et al. 2006; Schuepbach and Riewald 2010). Besides PAR-1, PAR-3 and PAR-4 were also classified as thrombin-sensitive receptors (Gandhi et al. 2011; Oikonomopoulou et al. 2006b). PAR-2 was identified as a trypsin-activated, thrombin-insensitive receptor that is upregulated by inflammatory mediators, e.g., in endothelial cells (Santulli et al. 1995). Interestingly, PAR-2 can become transactivated by thrombin-cleaved PAR-1 (Lin and Trejo 2013; O’Brien et al. 2000). However, this is the only report of PAR-induced receptor transactivation so far. Proteolysis of PAR-2 is mediated by a broad array of extracellular proteases including members of serine proteases (e.g., KLK5, KLK14, trypsin, tryptase, prostasin, and matriptase) as well as cysteine proteases (cathepsin S, house dust mite (HDM) antigen Der p1) (Bocheva et al. 2009; Cattaruzza et al. 2011; Frateschi et al. 2011; Kauffman et al. 2006; Santulli et al. 1995; Stefansson et al. 2008). PAR-2 is also known to be activated by proteases produced by microbial agents such as house dust mites, cockroaches, certain bacteria, and probably also certain parasites (Shpacovitch et al. 2007). Several reports indicate that PAR-2 activation plays an important role in inflammation (e.g., neurogenic inflammation or rheumatic arthritis), tumor progression, allergic reaction, and pain (Kempkes et al. 2012; Lam et al. 2012; Lohman et al. 2012; Nichols et al. 2012; Poole et al. 2013; Seeliger et al. 2003; Steinhoff et al. 2000). Finally, several studies suggest that activation of PAR-2 can also elicit itch, either directly by activation of the receptor on sensory nerve fibers innervating the skin, or indirectly by activating keratinocytes or immune cells (e.g., mast cells), and thereby induce a cascade leading to release of pruritogens that in turn activate sensory nerve fibers in the skin (Akiyama et al. 2009, 2010a, 2010b, 2010c, 2012, 2013; Steinhoff et al. 2003).

11.3. PAR-2 EXPRESSION IN THE SKIN

The skin is composed of three layers: epidermis, dermis, and subcutis. Several studies showed that PAR-2 expression in different cell types (keratinocytes, endothelial cells, skin-innervating nerve fibers, mast cells, neutrophils, and dendritic cells) is important for a functional skin homeostasis. The epidermis consists of several keratinocyte layers (stratum corneum, stratum granulosum, stratum spinosum, and stratum basale) that create a barrier to protect the body from the environment. Besides being a protection barrier, the epidermis can also be understood as a sensor for a broad variety of stimuli (e.g., UV radiation, temperature, pH changes, touch, chemicals, allergens, and microbial proteases). The stratum basale is formed by highly proliferating keratinocyte-specific stem cells and immature keratinocytes. These cells do not express PAR-2, but during the maturation process of keratinocytes, the expression levels of PAR-2-activating proteases (e.g., matriptase, KLK and prostasin) and PAR-2 itself become significantly upregulated. Different studies showed that PAR-2 activation of keratinocytes influences cell function by decreasing keratinocyte proliferation and inducing maturation of keratinocytes. Recent reports demonstrate the importance of PAR-2 for epidermal barrier homeostasis (Demerjian et al. 2008; Hachem et al. 2006). Keratinocytes of the stratum granulosum produce specific lipids (lamellar bodies) that prevent epidermal water loss. After acute permeability barrier disruption, e.g., by tape stripping, the pH of the stratum corneum increases from acidic (pH ~5) toward a more neutral pH. This, in turn, induces the activity of serine proteases, such as KLK5 and 14 (Brattsand et al. 2005; Hachem et al. 2003), which can activate PAR-2 leading to impairment of epidermal barrier recovery by downregulation of lamellar body secretion and induction of cornification. Jeong and colleagues observed that proteolytic allergens from cockroach and house dust mites delayed barrier recovery and lamellar body secretion in vivo (Jeong et al. 2008). Barrier recovery was normalized after application of a PAR-2 antagonist or protease inhibitors prior to epidermal barrier disruption. PAR-2 and the proteolytic activity of its activating proteases are tightly regulated by protease inhibitors during keratinocyte differentiation.

Different members of the KLK family have a broad spectrum of activity in the skin, and the proteolytic activity of proteases are regulated by different members of the serine protease inhibitor/lymphoepithelial-Kazal-type 5 inhibitor (Spink/LEKTI) family during keratinocyte differentiation (Deraison et al. 2007; Schechter et al. 2005). KLK5 and 14 are capable of activating PAR-2, which inhibits keratinocyte proliferation and induces the maturation of keratinocytes (Oikonomopoulou et al. 2006a, 2006b; Stefansson et al. 2008). KLK5 and KLK7 are important to initiate shedding of dead keratinocytes from the stratum granulosum by degrading corneodesmosomes (Brattsand et al. 2005). Therefore, it is important that the skin maintains a regulated proteolytic activity of KLKs during skin homeostasis. Consequently, during keratinocyte differentiation the gene expression of KLK inhibitors such as Spink5/LEKTI1, Spink9/LEKTI2, and Spink6/LEKTI3 are downregulated while KLK5, 7, and 14 as well as PAR-2 expression are upregulated. This fine-tuned interaction of proteases and protease inhibitors probably initiates keratinocyte differentiation and finally skin shedding (Deraison et al. 2007; Fischer et al. 2013; Reiss et al. 2011; Schechter et al. 2005). Vice versa, dysregulation of the LEKTI/KLK system leads to uncontrolled increased activity of KLKs, which, in turn, induces inflammatory reactions, scaly skin, and pruritus (Borgoño et al. 2007; Ishida-Yamamoto et al. 2005; Meyer-Hoffert et al. 2010). The impact of KLK dysregulation is best described by one form of a severe genetic skin disease, Netherton syndrome. Patients with this life-limiting form of ichthyosis harbor mutations in the Spink5 gene that results in the disability of LEKTI1 to inhibit KLK5 and 7 activities (Briot et al. 2009, 2010; Fortugno et al. 2011). The patients have severe skin barrier disruption defects leading to dehydration, chronic skin inflammation, itch, and a high risk of severe skin infections. Similar to patients with AD, Netherton syndrome patients and ichthyosis patients suffer from universal itch and develop a scaly, reddish skin (Briot et al. 2009, 2010; Fortugno et al. 2011). Of note, not only KLKs and protease inhibitors are important regulators of keratinocyte differentiation and skin barrier, but also PAR-2 and its activating proteases. This is of interest because the hyperkeratosis in different pruritic and inflammatory skin diseases (e.g., AD or Netherton syndrome) is accompanied by increased expression levels of PAR-2 and PAR-2-activating proteases (Briot et al. 2009, 2010; Elias and Steinhoff 2008).

Beside keratinocytes, the epidermis also contains melanocytes that produce melanin after sun exposure to protect the skin against UV radiation. Interestingly, PAR-2 has an additional important role for the melanin transport from melanocytes into keratinocytes. Upon UVB exposure, melanocytes produce and store melanin in so-called melanosomes, located in dendritic-like cell structures. Nearby keratinocytes constrict and phagocyte the melanosomes (Okazaki et al. 1976; Yamamoto and Bhawan 1994). UVB radiation enhances secretion of proteases into the extracellular matrix of the epidermis and thereby triggers PAR-2 activity on keratinocytes (Seiberg et al. 2000; Seiberg 2001). This process however leads to increased phagocytosis of melanosomes and increased skin tanning (Lin et al. 2008). Inhibition of PAR-2 activity on the other hand reduces pigment transfer after UVB exposure and also induces depigmentation of the skin (Seiberg et al. 2000; Seiberg 2001).

In the dermis, PAR-2 is expressed by dermal fibroblasts, mast cells, endothelial cells, and dermis/epidermis-innervating peripheral nerve endings. Human mast cells produce proinflammatory molecules such as histamine, proteoglycans, and PAR-2-activating proteases (e.g., tryptase and chymase). These mediators are stored in intracellular secretory granules that are released upon mast cell activation. Mast cells play an important role in several skin disorders including inflammation, hypersensitivity, and wound healing (Benoist and Mathis 2002). Although tryptase is able to stimulate PAR-2, the enzyme is a less potent activator of PAR-2 compared to trypsin, because tryptase activity seems to depend on receptor glycosylation and on the presence of sialic acid on the cell surface (Compton et al. 2002; Seymour et al. 2005). Interestingly, dermal mast cells themselves express PAR-2, suggesting a possible autocrine activation mechanism of PAR-2 on these cells. Upon allergen contact, mast cells release histamine that has several effects on the organism. Regarding neurogenic inflammation, it activates cutaneous sensory nerves and is therefore responsible for smooth muscle contraction, vasodilation, plasma extravasation, and instant itch sensations (Gazerani et al. 2009; Harvima et al. 2010; Steinhoff et al. 2003). Usually, histamine and tryptase are cosecreted upon mast cell stimulation. However, PAR-2 stimulation of mast cells did not seem to be followed by corelease of tryptase in skin mast cells (He et al. 2005; Moormann et al. 2006). In contrast, mast cells isolated from tonsils were observed to secrete tryptase upon PAR-2 stimulation but not histamine (He et al. 2005). Further investigations are needed to fully elucidate these somewhat contradictory observations. However, the PAR-2-profiling of mast cells differs between tissues; whereas human mast cells from the colon also express PAR-2; it was observed that lung mast cells were PAR-2-negative and did not respond to PAR-2 agonists (He et al. 2004, 2005).

11.4. FUNCTIONAL ROLE OF PAR-2 IN ITCH

In 1955, Arthur and Shelley published that the protease mucunain is the active compound of cowhage (Mucuna pruriens) and induces itch but not pain in humans (Arthur and Shelley 1955; Shelley and Arthur 1955). In particular, they observed that mucunain was responsible for a long lasting, histamine-independent itch (>30 min without wheal or flare in contrast to histamine-induced itch). Although the mechanism how mucunain induces itch was unknown, they already hypothesized that the protease may activate a “protease receptor,” which is expressed by nerves or indirectly exerts its pruritic effects by inducing the release of a pruritogen from epidermal cells. Although this study was of significant importance, it took several years before Rajka and colleagues followed up on this idea and demonstrated that the proteases trypsin and chymotrypsin are capable of inducing itch in humans (Rajka et al. 1967, 1969). However, it was not before 1995 after the murine and in 1996 the human PAR-2 were cloned that the protease-mediated histamine-independent itch was on the cusp of being better understood (Boehm et al. 1996; Nystedt et al. 1995a, 1995b; Santulli et al. 1996).

In 2000, we were able to detect PAR-2 in terminal nerve endings of a subpopulation of polymodal peptidergic C nerve fibers in the skin, and we could demonstrate that rat dorsal root ganglion (DRG) neurons respond to nanomolar concentrations of trypsin or tryptase (Steinhoff et al. 2000). This was of interest because further studies confirmed the hypothesis that PAR-2 activation on DRGs induces not only neurogenic inflammation but also histamine-independent itch (Steinhoff et al. 1999, 2003). Our subsequent studies showed that PAR-2 expression in keratinocytes and skin nerve fibers were increased in patients with AD (Briot et al. 2010; Buddenkotte et al. 2005; Steinhoff et al. 2003). Interestingly, AD patients also display increased tryptase serum levels, which correlate with itch sensation (Steinhoff et al. 2003). Similarly, patients with renal insufficiency can suffer from severe pruritus because of insufficient clearance of urinary excreted and pruritogenic substances. Dugas-Breit and colleagues were able to show that the severity of itch in dialysis patients correlated well with the patients’ increased serum levels of mast cell tryptase, suggesting an important role of mast cell tryptase and PAR-2 in the pathogenesis of renal pruritus (Dugas-Breit et al. 2005).

These observations can be understood by recent studies, showing that PAR-2 is expressed in a population of TRPV1-positive peptidergic nerve fibers (Amadesi et al. 2004, 2006). PAR-2-induced activation of PAR-2/TRPV1 nerve fibers induces the release of SP and calcitonin gene-related product (CGRP), which, in turn, induce secretion and activation of inflammatory mediators (as described below) and can increase itch sensitivity (Akiyama et al. 2013; Grant et al. 2007; Ständer et al. 2010; Steinhoff et al. 2000). The expression of PAR-2 in keratinocytes seems to correlate with severity of pruritus in chronic itch diseases. Mouse models treated to develop chronic dry skin phenotype also developed an itch phenotype (Yosipovitch 2004). Similar to patients with AD or chronic eczema, these mice also developed hyperkeratosis and skin barrier dysfunction (Akiyama et al. 2010a, 2010b, 2010c, 2012; Yosipovitch 2004). Furthermore, the expression of PAR-2 and PAR-2-activating proteases was increased in the skin, and the skin was highly innervated by nerve fibers. In a skin barrier disruption mouse model, proteases from cockroach and mite allergens induced activation of PAR-2, and the mice presented a delayed epidermal barrier recovery (Jeong et al. 2008; Yosipovitch 2004). Another study used NC mice that develop an atopic-like dermatitis when housed in a nonpathogen free/conventional environment (Takahashi et al. 2005). In the lesional skin of these mice, tryptase activity was significantly increased (Tsujii et al. 2009). The intense scratching behavior of the NC mice was widely reduced by intravenous injection of the protease inhibitor nafamostat mesilate (Tsujii et al. 2009). In correlation to this study, other groups established that PAR-2 knockout mice display a reduced response in oxazolone and picryl chloride induced allergic dermatitis models (Kawagoe et al. 2002). These results have a highly translational value because patients with chronic eczema and AD suffer from allergen-induced itch that increases epidermal barrier disruption. This can be partly explained by increased protease-activity on PAR-2 that is expressed by keratinocytes and skin nerve fibers (Ikoma et al. 2011; Lee et al. 2010; Steinhoff et al. 1999).

Subsequent studies in various skin mouse models helped to understand the molecular mechanisms behind PAR-2-induced itch and the role of PAR-2 in chronic pruritic diseases. In particular, intradermal injections of trypsin or tryptase were able to induce histamine-independent scratching behavior in mice (Costa et al. 2008; Tsujii et al. 2009; Ui et al. 2006). Using different protease inhibitors as well as antagonist or anti-PAR-2 antibodies, PAR-2 evoked itch sensation was significantly suppressed (Costa et al. 2008; Tsujii et al. 2009; Ui et al. 2006).

Different studies demonstrate that uncontrolled protease activity in the skin of mice induces atopic-dermatitis like phenotypes (Briot et al. 2009, 2010; Frateschi et al. 2011; Kim et al. 2012). It was shown in Spink5 knockout mice that increased and uncontrolled proteolytic activity of the serine proteases KLK5 and KLK7 induces a skin phenotype, which had similarity to the Netherton syndrome in humans (Briot et al. 2009). In addition, cathepsin S overexpressing mice and keratinocyte-specific prostasin transgenic mice developed atopic-like dermatitis (Kim et al. 2012). These data show that proteases are important for skin homeostasis and are tightly regulated to maintain a normal skin homeostasis. Dysregulation results in severe skin diseases accompanied with severe pruritus. However, a more recent study proved that the severe skin phenotype is driven by PAR-2 activity, as an epidermal PAR-2 overexpressing mouse model also results in an atopic-like dermatitis phenotype, including hyperkeratosis and skin barrier disruption (Frateschi et al. 2011). The mice display increased scratching behavior and develop skin lesion during adolescence. The tail and paw skin appears to be dry and scaly (Frateschi et al. 2011). We observed in these mice that the skin is highly innervated with nerve fibers and the mice develop hyperkinesis and allokinesis (yet unpublished observation and part of upcoming manuscript).

In understanding PAR-2-induced itch, humans are maybe the preferred research object compared to animals as the intensity of itch sensation can be described by the object itself. Studies using cowhage showed that the protease mucunain induces PAR-2-related itch most intensely when the spicules were inserted down to the level of the basal membrane (Arthur and Shelley 1955; Shelley and Arthur 1955). Interestingly, itch was never induced when spicules were inserted into the skin area after the epidermis and upper dermis had been removed. These results are similar to histamine-evoked itch or pain. Intradermal injections of histamine induce itch sensation, but subcutaneous injection was rather painful then itchy (Rosenthal et al. 1977). Our own studies revealed that intradermal injection of a human PAR-2 agonist as well as tryptase induce pruritus in humans and that intralesional injection of tryptase enhances and prolongs itch in AD patients (Steinhoff et al. 2003). We used codeine to investigate the release of histamine and tryptase in healthy volunteers and AD patients, and thereby we were able to show that tryptase release was increased in AD patients and that it induced a histamine-independent itch in humans (Steinhoff et al. 2003). Therefore, tryptase could induce antihistamine treatment resistant itch in AD patients (Steinhoff et al. 2003).

11.5. DIFFERENCES BETWEEN PAR-2- AND MrgprC11-INDUCED ITCH

Over recent years, several studies investigating the role of murine PAR-2 in inflammation and itch utilized a peptide analog that is synthesized according to the TLS sequence of the murine PAR-2. The use of synthetic peptide sequences is frequently deployed to investigate activation and mechanism of PAR-induced cellular responses. An interesting publication of Liu and colleagues demonstrated recently that the murine-amidated peptide sequence SLIGRL-NH2 is not only capable of inducing PAR-2 activation but additionally addresses the Mas-related G-protein-coupled receptor MrgprC11 (Liu et al. 2011). This receptor is highly potent to activate Bam8-22-induced itch responses in mice (Sikand et al. 2011; Wilson et al. 2011). Liu and colleagues demonstrated that MrgprC11 could be mainly responsible for SLIGRL-NH2-induced scratching behavior in mice (Liu et al. 2011). The authors used both PAR-2 and MrgprC11 knockout mice for their studies. SLIGRL-NH2-induced itch was only abolished in MrgprC11 knockout mice but not in PAR-2 knockout mice. As an explanation, the authors pointed out that the activation of MrgprC11 is mainly induced by the RL-NH2 feature of the peptide structure. Proteases, such as trypsin, tryptase, or KLK, activate PAR-2 by cleaving the receptor upstream of the TLS sequence (Adams et al. 2011; Hollenberg et al. 2008; Ramachandran et al. 2009). Thereby, the receptor structure itself will be shortened to , which induces activation of the PAR-2 receptor. This also means that the synthetic activating peptide sequence for MrgprC11 and PAR-2 does not occur naturally by proteolytic cleavage of the PAR-2 receptor in mice. Because there are three different synthetic PAR-2 peptide sequences commercially available (SLIGRL, SLIGRL-NH2, and fuoryl-SLIGRLO-NH2), future studies will need to furnish proof, if all three peptide sequences are potent PAR-2 and/or Mrgprc11 activators. Liu and colleagues presented evidence in their work that already the loss of the L amino acid (at position 6 after unmasking the TLS) inhibits the peptide’s potential to activate MrgprC11. Interestingly, although the authors quantified an intense itch response to intradermal trypsin injection in PAR-2 knockout mice, the concentration of the used trypsin was significantly higher (8000 pmol) compared to the study of Ui and colleagues applying 75 pmol of the less potent PAR-2-activating protease tryptase (Liu et al. 2011; Ui et al. 2006). However, trypsin and tryptase are not only exclusive PAR-2 activators but also capable of accomplishing proteolytic cleavage of PAR-4 or PAR-1 as well as of other preproteins, which may have a pruritic effect distant from PARs (Antalis et al. 2011; Brown et al. 2006; Kahn et al. 1998). Furthermore, the pruritic response to intradermal injection of trypsin, tryptase, or the human synthetic agonist SLIGKV-NH2 are, as far as we know, MRGPRX1-independent (the human homolog of MrgprC11) (Lee et al. 2010; Steinhoff et al. 2003). Although Liu and colleagues describe SLIGRL-NH2 as a potent itch activator mainly functioning via MrgprC11, it is wrong to conclude that PAR-2 expressed in DRGs would be nonfunctional and not important for itch in both human and mice (Liu et al. 2011). An armada of publications on this topic clearly favors the importance of proteases and neuronal PAR-2 function induced by proteases on itch, but not pain (Cormia and Dougherty 1960; Arthur and Shelley 1955; Shelley and Arthur 1955; Rajka 1967; Hägermark 1974; Steinhoff et al. 2003; Akiyama et al. 2012a, 2012b; Andoh et al. 2012a, 2012b; Kim et al. 2012; Frateschi et al. 2011; Olivry et al. 2013). Thus, the effect of a promiscuous synthetic agonist (SLIGRL-NH2) cannot replace the observations revealed using natural proteases, PAR-2 overexpression approaches, functional studies using proteases and human studies. We also have to consider species differences, because human studies clearly indicate PAR-2 as an itch receptor in humans. Although we cannot exclude a protease-dependent but PAR-2-independent itch pathway in neurons, increased protease and PAR-2 expression in the skin of atopic dermatitis-like mouse models and AD patients suffering from severe itch sensations indeed underline the importance of PAR-2 in itch (Briot et al. 2009, 2010; Frateschi et al. 2011; Kim et al. 2012; Lee et al. 2010; Steinhoff et al. 2003). Further studies will be necessary to understand the molecular mechanism of itch and the role of PAR-2 in pruritic skin diseases.

11.6. FUNCTIONAL ROLE OF PAR-2 IN NEUROGENIC INFLAMMATION

Serine and cystein proteases, such as thrombin, kallikreins, matriptase, tryptase, trypsin, cathepsin G and S, have acute effects on the inflammatory response in the human body (Kim et al. 2012; Stefansson et al. 2008; Seitz et al. 2007; Steinhoff et al. 2005). Recruitment of downstream cellular signaling cascades of proteolytic-activated PAR-2 finally accelerates widespread inflammatory processes that are expressed by keratinocyte activation, vasodilation, extravasation of plasma proteins, and infiltration of neutrophils (Seeliger et al. 2003; Steinhoff et al. 2005; Briot et al. 2010). The precise underlying signaling and intercellular communication pathways induced by PAR-2 in keratinocytes, dermal endothelial cells and in skin sensory nerves are not fully understood as of yet. The NF-kB pathway appears to play an essential role in PAR-2 mediated inflammation of skin diseases (Shpacovitch et al. 2002; Budenkotte et al. 2005; Moormann et al. 2006; Macfarlane et al. 2005; Goon et al. 2008; Dejean et al. 2012). A key component of PAR-2 induced acute and chronic inflammation appears to be a secretion of Neuropeptides such as SP and CGRP from sensory nerves in the skin (Steinhoff et al. 2000; Vergnolle et al. 2001). Histological analysis of DRG coherently reveals a high amount of PAR-2⁺ neurons coexpressing CGRP or SP, respectively (Steinhoff et al. 2000). That serine proteases engage in neurogenic inflammation has been shown for example for tryptase. In cutaneous tissue, tryptase originated from mast cells activates PAR-2 on sensory afferents leading to secretion of CGRP and SP, both neuropeptides capable of initiating inflammatory processes through their corresponding target receptors, most likely CGRP1 receptor (CGRR2 also exists) and the NK1R, respectively (Steinhoff et al. 2000, 2003; Obreja et al. 2005; Costa et al. 2008). In a dual mechanistic mode, CGRP induces vasodilation in arteriolar vessels resulting in erythema (flare), while SP induces edema (wheal) by activating postcapillary venules, the prerequisite for immune cell infiltration into the inflammatory tissue. CGRP further boosts SP release from cutaneous nerve terminals and protects SP from degradation by neutral endopeptidases, thereby amplifying SP-induced neurogenic inflammation (Scholzen et al. 2001; Schlereth et al. 2013). Simultaneously, SP activates residing mast cells to release tryptase that not only can activate PAR-2 but inactivates CGRP to counteract the CGRP-sustained inflammatory spiral. These tryptase-releasing mast cells are not only found in close proximity to PAR-2 expressing cells such as C-fibers, dermal endothelial cells, or keratinocytes (D’Andrea et al. 1998, 2000; Steinhoff et al. 2003) during inflammation, but themselves express PAR-2 (also PAR-1) (Moormann et al. 2006). Therefore, mast cells may release tryptase and stimulate inflammatory or propruritic responses in mast cells in an autocrine or paracrine fashion via PAR-2. A role for the tryptase/PAR-2 ligand-receptor axis might be of importance in the pathophysiology of AD because tryptase levels in this skin disease are significantly upregulated (Steinhoff et al. 2003; Hallgren and Pejler 2006).

For decades, the preventing effect of repeatedly applied capsaicin to ameliorate neurogenic inflammation (as does denervation) is well established (Jancsó et al. 1967), but it took further decades to identify the main receptor target of that natural compound in spicy pepper, namely TRPV1 (Caterina et al. 1997). The concept of desensitization of neuronal receptors to chemical stimuli that parallels the failure of the stimuli to cause pain or an inflammatory response was later commonly applied to investigate agonist/receptor tandems that induce neurogenic inflammation. In the acute setting, capsaicin leads to nociceptive behavior as well as neurogenic inflammation implicating a close functional cross talk on receptor level between TRPV1 and PAR-2. Cotreatment experiments of PAR-2-agonists with capsaicin effectively support a partial dependency of PAR-2/TRPV1 in conducting their function. Several groups observe a PAR-2-dependent potentiation of TRPV1 activity in protease induced inflammatory pain (Dai et al. 2004), hyperalgesia (Amadesi et al. 2004), and neurogenic inflammation (Hoogerwerf et al. 2001). A recent study aiming to further solve the signaling mechanism by which PAR-2 mediates neurogenic inflammation identifies coupling of the PAR to TRPV4, PAR-2-induced generation of arachidonic acid-derived lipid mediators such as 5´-6´-EET and tyrosine phosphorylation (Tyr110) of TRPV4 as important steps in this process (Poole et al. 2013). Also, activation and translocation of PKD1 to 3 are described to engage in the process of PAR-2-initiated neurogenic inflammation, which in part appears to be further dependent on PKCɛ (Amadesi et al. 2009). A functional interaction of PAR-2 and TRPA1 has been described in DRG neurons where PAR-2 activation (possibly through trypsin or tryptase released in response to tissue inflammation) leads to phospholipase C (PLC) as well as phosphotidylinositol 4,5-bisphosphate (PIP2) activation. Subsequent TRPA1 sensitization then leads to PAR-2/TRPA1-mediated inflammatory pain (Dai et al. 2007). The interaction between PAR-2 and TRPV1 or TRPA1 channels has to await further investigation.

The PAR-2-triggered inflammatory response to serine proteases certainly comprises a strong neurogenic element, but a nonneurogenic component cannot be neglected because administration of CGRP and SP receptor antagonists fail to completely ameliorate symptoms of inflammation as, e.g., formation of edema. This is supported by findings in skin tissues where PAR-2 agonists activate intracellular signaling cascades leading to activation and nuclear translocation of the proinflammatory transcription factor NF-κB (Buddenkotte et al. 2005). PAR-2-induced activation of NF-κB is crucial for the upregulation of cell adhesion molecules (e.g., ICAM-1 [Shpacovitch et al. 2002; Buddenkotte et al. 2005] and E-selectin [Seeliger et al. 2003]). Of note, both PAR-2 as well as NF-kB appear to be critical for the pathophysiology of atopic dermatitis (Pastore et al. 2000; Steinhoff et al. 2003; Macfarlane et al. 2005; Goon et al. 2008). In addition, PAR-2 induces release of cytokines (IL-6, IL-8, GRO-α) and prostaglandins (LTB4, PGE2) from keratinocytes or dermal endothelium (Hou et al. 1998; Shpacovitch et al. 2002; Takei-Taniguchi et al. 2012; Zhu et al. 2009b). Especially, cytokines may also contribute to neurogenic inflammation, pain, and pruritus as receptors bound by the IL-6 cytokine family [e.g., oncostatin M receptor (OSMR), leukemia inhibitory factor receptor (LIFR)] are expressed by cutaneous sensory neurons. Recent studies utilizing PAR-2-deficient mice further demonstrate that PAR-2 affected cutaneous inflammation in vivo is probably mediated by release of the vasodilator nitric monoxide and involves SP and NF-kB (Seeliger et al. 2003).

A detailed picture of the regulation of the neurogenic inflammation elicited by PAR-2 agonists has not been fully painted yet, but the physiological control of cell responses to various inflammatory stimuli will require regulation at several levels. The first level of regulation affects the operational capability of PAR-2 agonist prior to receptor activation, which is commonly based on the balance between agonist (protease) inhibitors and agonist (protease). Dysregulation of such balance often leads to severe disease states, for example, overexpressed CAP1/Prss8 (channel-activating protease-1/protease serine S1 family member 8) causes ichthyosis and a dysbalance of the PAR-2 activating KLK5 serine protease and its inhibitor LEKTI contributes to atopic lesions in Netherton syndrome (Briot et al. 2009). A second level of regulating neurogenic inflammation processes is directed against the receptor by multiple strategies. In the way PAR-2 sensitizes TRPV4 to cause mechanical hyperalgesia in mice (Grant et al. 2007), and interacts with TRPA1 in mouse DRG neurons to sensitize for inflammatory pain (Dai et al. 2007), PAR-2 function itself on keratinocytes could be regulated through such sensitization or, in contrast, desensitization. Further possible ways to modify PAR-2 function/PAR-2 activation on the receptor level include receptor trafficking/recycling, ligand uncoupling from receptor binding site, modulation of ligand/receptor affinity or endocytosis by the extracellular microenvironment. On a third, the intracellular level, the modulation of PAR-2-induced signal transduction and their amplification/reduction mechanisms in nerves, endothelial cells and keratinocytes may significantly influence downstream signaling and regulation of transcription factors involved in PAR-2-mediated inflammation and itch. The impact of these molecular mechanisms in human disease, however, is very poorly understood.

11.7. ROLE OF PAR-4 IN ITCH

Recent studies also suggest a possible role of PAR-4 in inflammation and itch. In 2008, Tsujii and colleagues demonstrated that PAR-4 stimulation induced histamine-depending scratching behavior in mice (Tsujii et al. 2008). However, the same authors also published later, that a specific PAR-4 agonist failed to induce PAR-4-specific scratching behavior in mice (Tsujii et al. 2009). In the same year, Akiyama and colleagues presented a study that showed that PAR-4 and histamine-induced scratching behavior seem to be independent from each other, as the authors have not observed cross-tachyphylaxis between PAR-4 agonist and histamine stimulation (Akiyama et al. 2009, 2010c). We are only at the beginning of understanding the role of PAR-4 as a potential itch receptor, and further studies will need to show if PAR-4-induced itch depends on PAR-4 activation and if the expression of PAR-4 ligands and the receptor itself is modulated during inflammation and itch on sensory nerve endings or other cell types in the skin.

11.8. CONCLUSIONS AND FUTURE DIRECTIONS

Various proteases from animals, plants, and skin cells themselves are important itch inducers that mainly function via PAR-2 in the rodent and human skin. PAR-2 is upregulated in various acute and chronic pruritic diseases in mice and humans, and the increased generation and release of certain kallikreins, tryptase, or prostasin correlating with itch intensity supports the concept that proteases via PAR-2 are important histamine-independent inducers in acute and chronic itch. Although the exact molecular mechanisms of PAR-2-induced itch are still poorly understood, its clinical relevance in severe, pruritic human skin diseases is indisputable. However, we are still at the beginning of understanding PAR-2-induced itch and neurogenic inflammation in the skin and other organs including the lung and gastrointestinal tract or brain. Further studies will be necessary to demonstrate if PAR-2 activation on sensory nerves in the skin is sufficient to directly induce itch in humans, or if a complex cascade involving keratinocytes and/or immune cells in the skin is also involved, or even more important for the PAR-2-induced, histamine-independent itch.

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