RESEARCH doc

6BBYN305 Literature Based Project in Neurosciences

Cellular and Molecular Mechanisms of Itch

Camilla Siig

1207140

School of Bioscience Education

King’s College London

Guy’s Campus

Dissertation submitted in part fulfilment of a BSc (Hons)

in Biomedical Science

Abstract

The distinction between itch and pain has been a matter of debate for many years. Recent advancements in itch

research has begun to expose neurophysiological, cellular and molecular mechanisms of itch through human clinical

studies and animal models. Current knowledge affirms that itch is mediated by both unmyelinated C-fibres and Aδ

fibres, providing support for the labelled-line theory which advocates itch-specific neurocircuitry. Recent research

has implied the involvement of a myriad of mediators including histamine receptors, TRP channels, TLRs, Mrgprs,

inflammatory molecules and neuropeptides. This diversity of itch mediators and their complex interactions with pain

and itch suggests a more integrative approach is needed: population coding theory has been proposed in this

respect. This review will cover the development of an itch transmission theory and provide evidence for its

modulation in light of the current literature. The focus will be itch receptors and mediators, and their role in itch

signalling.

Table of Contents

1.0 Background 1

2.0 Itch Transmission 2

2.1 Peripheral Itch Coding and Receptors 2

2.2 Central Processing 4

3.0 Peripheral Mechanisms: Receptors and Mediators 4

3.1 Histamine 4

3.2 Mas-Related G Protein-Couple Receptors 6

3.3 Transient Receptor Potential Channels 6

3.4 Protease-activated receptors 8

3.5 Toll-like Receptors 9

3.6 Cytokines 9

3.7 Substance P 10

4.0 Modulation of the Itch Pathway 10

4.1 Peripheral Sensitisation 10

4.2 Central Sensitisation 11

5.0 Conclusion 11

6.0 References 14

Background

Itch, also referred to as pruritus, can be defined as an “unpleasant sensation that elicits the desire or reflex to

scratch” (Patel & Dong, 2011). Its clinical classification is complicated, though itch is broadly regarded as either

peripheral (pruritoceptive) or central (neurogenic or neuropathic). Twycross et al. (2003) propose four categories:

pruritoceptive, neuropathic, neurogenic and psychogenic. Pruritoceptive itch is the most well-understood,

encompassing symptoms such as inflammation, dryness and skin damage. Common conditions include xerosis,

atopic dermatitis and scabies (Cohen et al., 2012). Neuropathic itch on the other hand, is an area very much

unexplored. It is known to arise from impaired nerves of the central nervous system (CNS), causing disease anywhere

along the itch pathway (Yosipovitch G et al., 2003). Illnesses associated with neuropathic itch tend to emerge from

infections already causing neuropathic pain such as shingles, lesions of the trigeminal nerve, stroke and multiple

sclerosis (Oaklander, 2011). Neurogenic itch is characterised by a lack of neural pathology, such as in cholestasis;

and psychogenic itch evolves from psychiatric or psychological disorders including obsessive compulsive disorder and

delusional parasitophobia (Yosipovitch G et al., 2003). Concerning the alarming prevalence of pruritus among

patients, it is essential that the pathophysiology of abnormal itch can be diagnosed effectively. Most importantly,

understanding the complexity of itch mechanisms and mediators is the key to treating clinical conditions

successfully.

Regarding the development of an extensive itch theory, early studies noted behaviours indicating abolished pruritus

following ventral lateral cordotomies (surgical lesion of the ventral lateral funiculus (VLF)). Given that the

spinothalamic tract (STT) supports ascending axons of the VLF, it was concluded that itch sensation must be relayed

via the STT (Davidson et al., 2014). In 1997, Schmelz et al. first identified C fibres as “afferent nerve fibres with

particularly thin axons” necessary for mediating itch sensation. It is now understood that three types of primary

afferent nerve exist: Aβ (fast conducting and heavily myelinated), Aδ (slow conducting and myelinated) and C fibres

(slow conducting and unmyelinated). Those perceiving pain and itch involve Aδ and C fibres, which respond to

noxious and thermal stimuli; while those perceiving tactile sensations require Aβ conduction and respond to non-

noxious mechanical stimuli. Sharp pain corresponds to the fast conduction of Aδ fibres, and slower C fibres are

known to relay prolonged aching pain or itch. Overlapping neural circuitries associated with either of these

modalities initially led to the proposal of the “intensity theory”, which was accepted by the scientific community for

a long time (Potenzieri & Undem, 2012). Itch was considered to be a submodality of pain, differentiated only by the

intensity or pattern of neuronal firing. Contrary to this, recent development suggests a “labelled-line theory”

whereby itch pathways are distinctly marked by modality specific molecules, sensory neurons and neural pathways

(Han & Dong, 2014). The literature surrounding labelled-line theory is encouraging and consensus is gravitating

towards its explanation. Although not all itch phenomena can be fully interpreted yet, a new and promising biology

of itch is unfolding (Handwerker, 2014).

The current first line of treatment for itch relies on antihistamines. Though effective for minor allergies and insect

bites, antihistamines are largely inadequate at alleviating clinical itch, especially when the condition is chronic. The

limited knowledge of itch pathophysiology combined with the unavailability of itch animal models has hampered the

development of effective treatments for human itch. Currently, pruritogen injections and skin dehydration in vivo

models allow cutaneous symptoms associated with itch to be analysed. While models for systemically derived itch

mechanisms (such as cholestasis) have only recently become a possibility (Han & Dong, 2014); another issue

concerns the measurement of animal response behaviours versus direct sensation. For example, certain injection

sites induce a response where there is no differentiation between pain and itch behaviours (LaMotte et al., 2011). In

order to improve the prospects for itch treatment, it is necessary that current understanding of itch mechanisms be

further investigated. This review will provide a comprehensive and critical account on cellular and molecular

mechanisms of itch; delving into the neurophysiology underlying sensory processing as well as the plethora of

relevant receptors and molecular mediators. Abnormalities of the itch pathway will also be discussed, addressing the

histological structures of neuronal mechanisms in chronic itch conditions.

Itch Transmission

Peripheral Itch Coding and Receptors

The interaction between itch and pain has caused great confusion over the years. The intensity theory, originally

proposed in the early 20th century, was derived from the observation of overlapping pain and itch response spots in

human skin (Lewis et al., 1927). However, contradictions appeared. Tuckett (1982) demonstrated that pain did not

arise from itch with increasing frequency of electrical stimulation. Neither was pain found to dull into itch at lower

stimulation frequencies (Ochoa & Torejbork, 1989; Handwerker et al., 1991). Ensuing Schmelz’s 1997 discovery of

histamine-responsive C fibres, Andrew & Craig (2001) identified the lamina I region of the spinal cord as a “unique

subset of STT neurons” acquiring itch selectivity in response to histamine. Schmelz et al. (2003) supported this

conclusion in a more thorough investigation. Various pruritogen (itch producing) and algogen (pain producing)

substances were tested on three sub-classes of C-nociceptors. Their major finding determined that histamine-

positive C fibres are “selective” but not “specific” for pruritic stimuli. More recent studies reveal that chemically

silenced neurons expressing transient receptor potential vanilloid-1 (TRPV1) nociceptors exhibit severely impaired

itch response (Imamachi et al., 2009), as well as reduced thermal and pain sensitivity (Cavanaugh, 2009), suggesting

that TRPV1 is necessary for itch perception. However, the question remains: are pain and itch facilitated by separate

neuronal populations? Two major break-through papers from Sun & Chen (2007) and Sun et al. (2009) present

strong evidence for itch-specific neural pathways. Using genetically modified mice knockouts, they found that

gastrin-releasing peptide receptor (GRPR)-expressing neurons are exclusive to and necessary for itch perception (Sun

et al., 2009). Similarly, Liu et al. (2009) conclude that mas-related g-protein coupled receptors (Mrgprs) play a role in

itch-specific neural transmission. They discovered that ablation of an Mrgpr gene cluster significantly attenuated itch

response following chloroquine (CQ) stimulation. In addition, the deficit could be rescued by mouse MrgprA3 and

human MrgprX1. Interestingly, in 93% of cases, MrgprA3 was found to be co-expressed with GRP in a subpopulation

of dorsal root ganglion (DRG) neurons: a positive finding in line with Sun & Chen’s paper (2009). Another Liu et al.

(2010) study investigated toll-like receptor 7 (TLR7) in light of pain hypersensitivity; however, upon finding no

evidence for abnormal pain perception in TLR7 knockout mice, they considered its effects on itch. “A marked

reduction in scratching behaviour in response to nonhistaminergic pruritogens” was observed. TLR7, like MrgprA3,

was predominantly expressed in C fibres alongside TRPV1 and GRP, showing further support for itch-specific

neurophysiology. Han et al. (2013) reported similar results: mice possessing genetically ablated MrgrA3-positive

neurons display reduced itch sensation alongside unaffected pain sensitivity. In spite of capsaicin (an algogen)

application, MrgprA3-positive neurons solely expressing TRPV1, provoked itch behaviour and not pain. Taken

together, these data lean towards a “labelled-line” theory and provide consistent results advocating that itch specific

neurons exist.

Despite such positive findings, the labelled-line theory cannot explain everything. Firstly, pain and itch are both

prompted by multiple stimuli including mechanical, chemical, thermal and electrical methods. Not to mention

substantial integration of their functional mechanisms (Table 1) (Stander & Schmelz, 2006). Secondly, their

antagonistic relationship is puzzling: while the stimulation of pain may either inhibit or enhance itching sensation

(Stander & Schmelz, 2006), its suppression may also inhibit itch but not pain sensation (Heyer et al., 1997).

Moreover, the observation that nonhistaminergic pruritogens may involve multiple afferent pathways complicates

matters further. Ringkamp et al. (2011) used a selective conduction block of myelinated fibres on human subjects,

finding that a sub-group with A fibre-dominated itch had significantly reduced itch, pricking and burning sensation

elicited by cowhage (a plant known to induce itch). When comparing A fibre- with C fibre-dominated itch subjects,

their time courses were distinctly individual and each subgroup displayed matching peak responses respective to

their myelinated or unmyelinated characteristics. Similar findings demonstrate cowhage as a stimulus for C fibres

that are not related to histaminergic itch (Namer et al., 2008); and another primate study observed separate STT

neurons as responsive to both cowhage and histamine (Davidson et al., 2007). It seems that a population-coding

hypothesis explained by Campero et al. (2009) may resolve the inconsistencies between intensity and labelled-line

theory. It suggests that pain, itch and thermal sensations are transmitted by labelled afferents which proceed to

integrate and crosstalk (sometimes antagonistically) in the CNS. The response, therefore, does not necessarily

correspond to the stimulus: it may be an emergent sensation relative to the activation of multiple labelled lines (Ma,

2010).

Table 1. Common mediators, receptors and their contribution to pain and itch. Taken from Stander & Schmelz,

2006.

Mediator Receptors present in skin Pruritus PainHistamine H1, (H2), H4 (?) receptors Induction of itch via receptor

stimulationInduction of pain at high concentrations

Tryptase Proteinase-activated receptor-2

Induction of itch via receptor stimulation

In animal experiments

Endothelin Endothelin A-receptors Induction of itch via receptor stimulation

Induction of pain via receptor stimulation

Interleukins (IL-2, IL-4, and IL-6)

Receptor on nerve fibers: IL-2R, IL-6R

Delayed itch (secondary?) Sensitization

Substance P Neurokinin receptors (NKR 1–3) on mast cells and nerve endings

Induction of itch via mast cell degranulation, histamine, and tryptase release

Neurogenic inflammation, central sensitization

Capsaicin, heat, Transient receptor Induction of burning pruritus Induction of burning pain via

low pH potential (TRP): TRPV1 via receptor stimulation receptor stimulationBradykinin Bradykinin receptors (B1,

B2)Sensitization of nerve fibers for other chemical stimuli

Sensitization/activation of nerve fibers

Prostaglandins Prostaglandin receptors Sensitization of nerve fibers, potentiation of histamine-induced itch

Sensitization of nerve fibers

Nerve growth factor

TRK-A receptor Sensitization of nerve fibers speculated

Peripheral and central sensitization

Opioid peptides μ-, δ-receptors Inhibition of pruritus via peripheral receptors-induction of pruritus spinally

Inhibition of pain via central and peripheral receptors

Cannabinoids Cannabinoid receptors (CB1, CB2)

Suppression of histamine-induced itch

Peripheral and central analgesic effects

Central Processing

Although there is good indication for a population coding theory in terms of peripheral processing of itch, few

explicit details can be specified pertaining to the central component of the theory. Nonetheless, a combination of

neurophysiological, genetic and brain imaging studies begin to build a picture. It is common knowledge that pain (i.e.

scratching) may temporarily inhibit itch sensation. Additionally, heat and cold have similar actions (Ross, 2011). A

2010 paper by Zheng et al. presents an example whereby “inhibitory lamina II connections appear arranged to

modulate activity from different sets of peripheral unmyelinated fibres”, enabling reciprocal inhibition between the

two. Evidence for pain as a repressive mechanism of itch was discovered in experiments investigating the effects of

vesicular glutamate transporter (VGLUT) 2 deletion in TRIPV1-expressing mouse afferents (Lagerstrom et al., 2010).

They found substantially increased itch behaviour alongside reduced thermal pain response. Itch was alleviated upon

antihistamergic drug administration and genetic deletion of GRPR. Collectively, it can be deduced that VGLUT2 is

necessary for TRPV1 thermal nociception and regulation of normal itch perception. fMRI studies reveal that active

areas usually associated with itch include the prefrontal cortex (PFC), anterior cingulate cortex (ACC), insula,

somatosensory cortex (S2), and the cerebellum (Mochizuki et al., 2014). Importantly, the majority of subjects

showed regional activity correlating with itch intensity (Darsow et al., 2000). In particular, areas of the

periaqueductal grey matter (PAG) were especially dynamic, suggesting the region has a more primary role in itch

processing (Mochizuki et al., 2003). They also found that correlations between PAG activity and itch intensity were

resultant of pain stimuli, suggesting there may be descending modulatory pathways for itch. Though the data is

limited and discrepancies are dotted throughout the literature, recent genetic approaches combined with molecular

analysis and brain imaging provide a solid grounding for rapid development in this field.

Peripheral Mechanisms: Receptors and Mediators

Histamine

Histamine holds a historic place in the development of itch understanding, becoming the most studied pruritogen in

the literature to date (Han & Dong, 2014). It is now known to be an endogenous ligand of histamine receptors

(HisRs), synthesised from histidine and predominantly released by mast cells. It intimately links with the immune

system, triggering the familiarised “triple response of local vasodilation, local edema, and flare” and has functional

associations with allergy, anaphylaxis, gastric acid secretion and neural transmission (Thurmond et al., 2014). Today,

four HisRs are recognised: H1 receptor (H1R), H2 receptor (H2R), H3 receptor (H3R) and H4 receptor (H4R). They are all

G-protein coupled receptors (GPCRS), however, are expressed in different cells and activate separate signalling

pathways (Table 2) (Bongers et al., 2011). This overview of HisRs is somewhat simplified. Most have been discovered

via pharmacological observation and detailed signalling cascades have yet to be fully documented. Promiscuity in

binding affinities, multiple functions of ligands (i.e. agonist and antagonist) and complex transduction pathways

make it particularly challenging to characterise individual receptors (Thurmond et al., 2014).

Table 2. Histamine receptors: signal transduction. Based on Bongers et al., 2011.

HisR subtype

Cell expression G-protein coupling

cAMP production

Transduction cascade

H1R CNS neurons, smooth muscle cells, endothelial cells

Gq Increased PLC activation, IP3 & DAG production

H2R CNS neurons, gastric parietal cells, cardiac muscle cells

Gs Increased PKA activation, CREB phosphorylation

H3R Found mainly in CNS neurons as a presynaptic autoreceptor

Gi/o Decreased PKA inhibition, MAPK phosphorylation

H4R Immune cells i.e. peripheral blood leukocytes and mast cells

Gi/o Decreased PKA inhibition, MAPK phosphorylation, AP-1 activation

Pertaining to itch, there is robust evidence for histamine as a mediator. Of the four subtypes, H1R and H4R are the

most relevant (Thurmond et al., 2008). Although it has been suggested that H3R plays a role in H1R and H4R

activation (Rossbach et al., 2011), the literature surrounding this receptor is unresolved (Jeffry et al., 2011).

Regarding H2R, its effects are minimal at best: both H2R agonists and antagonists fail to activate or inhibit itch

symptoms respectively (Bell et al., 2004). Having reached somewhat of a dead end in antihistamine treatment for

chronic itch, hope has been rekindled in view of recent data. Research is now pushing to re-evaluate the role of H1Rs

and consider H4R as a potential target for novel therapeutics.

Focusing first on H1Rs, it has become increasingly evident that TRPV1 is an important histamine sensor in the signal

transduction of itch. A 2006 paper by Han et al. presents the phospholipase C (PLC) β isoenzyme as a critical

mediator found in a subpopulation of C fibre nociceptors. Using Ca2+ imaging techniques, they noted high levels of

PLCβ3 expression in DRG neurons induced by histamine stimuli. Furthermore, subject to PLCβ3 deletion, mice

showed significantly impaired scratching behaviour, a result consistent with their primary findings. Woo et al. (2008)

later link PLC and TRPV1, asserting that diacylglycerol (DAG), a product of PLC hydrolysis, activates TRPV1 directly.

Alternatively, phospholipase A2 (PLA2) may also activate TRPV1 in histaminergic itch (Kim et al., 2004; Shim et al.,

2007). Several lines of evidence indicate H4R is involved in itch signalling and is independent from the H1R pathway

(Davidson & Giesler, 2010). In an assessment of the effects of H4R antagonist, JNJ 7777120, two models of dermatitis

were used in Rossbach et al.’s 2008 publication. Hapten-induced scratching was significantly reduced upon JNJ

7777120 administration; however, the presence of inflammation remained unaffected in both models. Combined

administration of an H1R and H4R antagonist proved more effective than individual application, suggesting H1R and

H4R are attributable to separate aspects of itch. Intracellular pathways relevant to H4R are still unclear. Nonetheless,

mitogen-activated protein kinase (MAPK) (Morse et al. 2001) and AP-1 activation (Gutzmer et al., 2009) have been

implied.

Mas-Related G Protein-Coupled Receptors

Mrgprs were first discovered in 2001 by Dong et al., being almost exclusively observed in tropomyosin receptor

kinase (Trk) A- expressing neurons in mice. Their classification has been split into three main families: MrgprA,

MrgprB and MrgprC, though a more distantly related group also exist (MrgprD-H) (McNeil & Dong, 2014). Only after

the generation of Mrgpr knockout mice did their significance in itch sensation become clear (Sun & Chen, 2007). The

closest human ortholog to MrgprA3 was revealed whilst experimenting with its agonist, CQ: Liu et al. (2009) found

MrgprX1 (receptors restricted to DRG neurons) in HEK293 cells also responded to CQ. Bovine adrenal medulla

peptide (BAM8-22), a potent pruritogen, showed specificity for MrgprC11 while MrgprA3 responded only to CQ (Liu

et al., 2009). Though these receptors are activated by distinct ligands, Wilson et al. (2011) demonstrates that

transient receptor potentional ankyrin-1 (TRPA1) is a common downstream target of Mrgpr-mediated

nonhistaminergic itch. Moreover, BAM8-22 has been shown to produce itch and pain sensations in humans, making

it attractive as a possible endogenous itch mediator for nonhistaminergic itch (Sikland et al., 2011). Based on the

knowledge that PLC couples to MrgprC11, Wilson et al. (2011) also investigated if gallein (a Gβγ inhibitor) affects itch

behaviour via MrgprA3. Significantly attenuated responses in Ca2+ signals and CQ-induced itch suggest that MrgprA3

requires Gβγ for TRPA1 coupling. The same experiment was carried out regarding MrgprC11, however no significant

change in BAM-evoked responses occurred. The data implies that PLC signalling must, by elimination, occur via Gq.

Unfortunately, no supporting observations have been documented yet. Finally, a recent paper by Liu et al. (2012)

shows that the amino acid β-alanine elicits itch mediated by MrgprD. MrgprD knockout mice were not responsive to

β-alanine; however, histamine-induced itch response was unchanged compared to wild type mice. These results

propose MrgprD is specific to β-alanine. Interestingly, Zylka et al. (2005) reported that MrgprD-expressing neurons

projected solely to the stratum granulosum layer of the epidermis. Though this seems reminiscent of labelled-line

theory, there is still confusion in that multiple stimuli (noxious, thermal and mechanical) and sensations (pain and

itch) are associated with these neurons (Jeffry et al., 2011). Nonetheless, the field is immature and the literature on

Mrgprs is incomplete. The future looks toward exposing the intracellular pathways linked with itch, perhaps

beginning with a specific PLC isoform.

Transient Receptor Potential Channels

The role of TRP channels is becoming increasingly recognised as a key component to itch transduction in the

periphery. They are part of the ion channel family, maintaining a tetrameric structure formed of six transmembrane

helices. There are as many as 27 members to date; however, those thought to participate in itch perception include:

TRPV1, TRPA1, and TRPV3 (Wilson & Bautista, 2014).

TRPV1, initially discovered using capsaicin (the active ingredient in hot chilli peppers that causes a burning

sensation), has now been established as a thermoreceptor activated at temperatures above 40°C. The relationship

between itch and pain has long been known; especially in consideration of the relieving effects pain has on itch.

Modern treatment using topical capsaicin induces “localised loss of nociceptive nerve fibre terminals in the

epidermis and dermis”, which implies the presence of TRPV1 or TRPV1-expressing primary afferents necessary for

pruriception (Wilson & Bautista, 2014). Imamachi et al. confirmed this in 2009 upon finding that TRPV1 knockouts

had significantly reduced scratch response to histamine injection. Other locations of TRPV1 expression include the

trigeminal ganglia, brain and DRG neurons. Pertaining to an intracellular signalling mechanism, Imamachi et al.

(2009) replicated Han et al.’s (2006) results, demonstrating that PLCβ is activated in a Gq dependent manner.

Although this evidence advocates that TRPV1 is a specific labelled line for histaminergic itch, recent papers suggest

otherwise. Patel et al. (2011) used Pirt (a molecule essential in TRPV1 modulation for pain sensation) knockout mice

to investigate whether it has a role in itch sensation. They found that both histamine-induced itch and CQ-induced

itch is substantially attenuated in comparison to wild type mice counterparts, indicating that Pirt is a novel mediator

of itch acting crucially via TRPV1 in both histaminergic and nonhistaminergic itch. Likewise, Than et al. (2013) found

that CQ activates TRPV1-expressing DRG neurons, accounting for “43.3% of the total CQ-excited neurons”; however,

TRPV1 was not a direct mediator in this case. In summary, TRPV1 is undeniably important for itch transduction and

may be more broadly involved than previously thought. Further experimentation should clarify mediators in

signalling pathways in order to build a better picture of itch mechanisms.

TRPA1 is localised in a subset of TRPV1-expressing neurons and is activated by a diverse array of endogenous and

exogenous irritants. Such endogenous inflammatory substances include 15dGJ2, PGA2 and Δ12-PGJ2, which may be

regulated by PLC-coupled receptors. Exogenous irritants include allyl isothiocyanate (AITC), cinnamaldehyde and

allicin – compounds of mustard, cinnamon and garlic extracts respectively (Wilson & Bautista, 2014). Wilson et al.

(2011) was first to identify that TRPA1 is required for histamine-independent itch downstream of MrgprA3 and

MrgprC11 in mice. Following this, Liu and Ji (2012) determined that both genetic and pharmacological blockage of

TRPA1 profoundly reduces oxidative itch. Furthermore, scratching could be attenuated in wild type mice with

systemic administration of antioxidants. In 2013, Wilson et al. elaborated on their 2011 findings, experimenting with

a mouse model of chronic itch. They confirmed that TRPA1 is required for transducing histamine-independent itch

elicited by CQ and BAM8-22. Remarkably, TRPA1 was found to induce pathophysiological markers in chronic itch,

including increases in epidermal thickness, epidermal hyperplasia, and upregulated gene expression associated with

MgpA3 receptors, protease activated receptor-2 (PAR2) and inflammatory bradykinin receptor (Bdkr2) (Wilson et al.,

2013). Another 2013 paper by Oh et al. used an interleukin-13 (IL-13- a critical cytokine for allergic inflammation)

mouse model to inhibit TRPA1 and genetically delete mast cells. In both cases itch was significantly diminished,

reaffirming TRPA1’s role in itch transduction. Interestingly, high expression of TRPA1 was observed in the “dermal

afferent nerves, mast cells, and the epidermis in the lesional skin biopsies from patients with atopic dermatitis”

when compared with skin from healthy subjects. Taking into account that IL-13 is produced by mast cells, it is in

accordance with the biopsies that IL-13 was found to robustly stimulate TRPA1 expression. A novel finding from Lieu

et al. (2014) indicates that TRPA1 is necessarily activated by the G protein-coupled bile acid receptor, TGR5, via a

Gβγ and PKC-mediated mechanism. Table 3 summarises the interactions between pruritogens, receptors and their

relationship with TRPV1 and/or TRPA1.

Table 3. TRPV1 and TRPA1 in itch transduction. Adapted from Zhang, 2014.

Pruritogens Pruritic receptor Excited ion channelsHistaminergic Histamine H1R, H3R, H4R TRPV1, others?

Nonhistaminergic

CQ MrgprA3 TRPA1, TRPC3SLIGRL, BAM8-22 Mrgpr C11 TRPA1Cowhage PAR2, PAR4 TRPA1?IL-13 ? TRPA1IL-31 IL-31RA TRPV1, TRPA1TSLP TSLPR TRPA1LTB4 BLT1 TRPV1, TRPA1

TRPV3 is widely expressed in skin keratinocytes and is activated by both innocuous and noxious temperatures above

33° (Steinhoff & Biro, 2009). Yoshioka et al. (2009) suspected a role for TRPV3 in pruritus, however, no evidence

could sufficiently support this idea. A 2012 paper by Yamamoto-Kasai et al. discovered that TRPV3 knockout mice

exhibited significantly less scratching behaviour after acetone ether water treatment (AEW- a treatment that causes

skin dehydration, used to replicated chronic itch in mice), in comparison to TRPV3-positive counterparts. This

suggests TRPV3 does indeed contribute to itch perception. In addition to this, atopic dermatitis patients showed

increased levels of TRPV3 mRNA expression, better linking the findings in murine pruritus to human pruritus.

Although there is the indication that prostaglandin E2 (a pruritogen) is released from keratinocytes displaying TRPV3

overexpression, the data needs to be reproduced in light of itch rather than pain (Huang et al., 2008). TRPV3 may

hold promise for itch therapeutics, however, future efforts should be made to elucidate itch-promoting mechanisms

within keratinocytes.

Protease-activated receptors

Protease-activated receptors (PARs) are part of the GPCR family, featuring four subtypes altogether: PAR1-4. Of

those four, PAR2 is believed to be itch related, specifically as a mediator of nonhistaminergic itch. While PAR4 may

also regulate itch, the field is very much premature and what limited data there is, is generally inconclusive. PARs are

unusually activated by proteolytic cleavage of their N terminus. Once the N terminus is freed, a tethered ligand

sequence (TLS) becomes exposed, causing activation of the receptor upon binding (Kempkes et al., 2014).

Endogenous itch-inducing proteases include serine proteases such as trypsin, tryptase, matriptase or prostasin; while

exogenous itch-inducing proteases include the likes of mucunain (a component of cowhage), cathepsin S, kallikreins

(KLK) and tryptase (Han & Dong, 2014; Kempkes et al., 2014).

PAR2 receptors, in particular, are essential for histamine-independent itch. Steinhoff et al. first confirmed their

identity in 2003, where they appeared to be restricted to cutaneous sensory nerves. The same study saw that

patients with atopic dermatitis expressed upregulated levels of PAR2 which were correlated with itch severity,

suggesting a strong link between the two. Dugas-Breit et al. (2005) noticed a similar relationship in dialysis patients

who exhibited raised serum levels of mast cell tryptase, and asserted the possibility that immune cells may be

directly involved in the mediation of pruritus. Several recent papers have reinforced this notion upon finding

contribution of other proteases to itch response (Tsujii et al., 2009; Andon et al., 2012). Although much remains

unknown about PAR signalling pathways, the relevance of TRP channels has been recognised. The most robust

findings associate PAR2 with TRPA1: firstly, TRPA1 and PAR2 were found to be frequently colocalised in DRG neurons

(Dai et al., 2007). Secondly, TRPA1 currents were increased by activation of PAR2; and thirdly, application of PLC

inhibitors, but not PKC inhibitors, suppressed said TRPA1 currents (Dai et al., 2007). Grant et al. (2007) suggests a

pathway involving PLCβ, acting via the TRPV4 channel, however, conflicting results see that both “PLC and PKC

mediate PAR2-induced sensitisation of TRPV1” (Amadesi et al., 2004). It may be that PAR2 is mediated by multiple

signalling pathways and/or that PAR2 activation is limited to sensory nerves of the skin. At present, it is difficult to

reason which is the case. Still, it is certain that PARs are clinically relevant in human pruritic skin diseases and thus,

are worthy of further study.

Toll-like Receptors

Toll-like receptors (TLRs) are widespread throughout the CNS and peripheral nervous system (PNS), being expressed

in cells such as “microglia, astrocytes, oligodendrocytes, Schwann cells and neurons” (Li & Ji, 2014). They are known

to participate in autoimmune function, recognising endogenous ligands called danger-associated molecular patterns

(DAMPS); and to regulate adaptive immunity, detecting characteristic molecular structures of pathogens named

pathogen-associated molecular patterns (PAMPS) (Liu et al., 2012). Classed as single transmembrane proteins, nine

TLRs exist in total. With respect to itch sensation, TLR3 and TLR 7 are most pertinent.

In 2010, Liu et al. recorded TLR7 as a novel mediator of itch sensation. Their expression was limited to TRPV1-

positive nociceptors along with coexpression of GRP and MrgprA3. Activation of TLR7 by imiquimod indicated the

itch response was induced directly by a nonhistaminergic pathway; a conclusion that was reproduced by Kim et al. in

2011. Interestingly, Liu et al. (2010) also saw significant reduction in itch behaviour in CQ, endothelin-1 and SLIGRL-

HN2, suggesting that TLR7 has a broader impact on itch signalling than initially perceived. Moreover, they found that

using histaminergic pruritogens had no effect on scratching behaviour in TLR7 knockouts, compared with wild type

mice, reinforcing the idea that TLR7 may be quite general in its action. A later study by Liu et al. (2012) finds that

TLR3 evokes itch in a similar way to TLR7, however, both histaminergic and nonhistaminergic pruritis were markedly

decreased in TLR3 deficient mice. Unlike TLR7, TLR3 may be indirectly regulating itch transmission through the CNS.

This could explain the differences between Liu et al.’s 2010 and 2012 studies. Considering these data, TLRs may play

a vital role in itch sensation, both directly and indirectly. The challenge remains to identify what signalling

specificities are distinct to TLRs compared with other immune cells, and to uncover the role of molecular

mechanisms underlying TLR transduction.

Cytokines

Cytokines are secreted proteins that are released from activated immune and skin cells such as mast cells and

keratinocytes respectively (Cevikbas et al., 2014). Although they make up a large family, only a few have been

identified as itch mediators thus far. Interleukin-31 (IL-31), a member of the IL-6 family, is the most studied itch

cytokine. It is preferentially secreted from T helper type 2 (TH2) cells and signals through a heterodimeric receptor

complex formed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR) (Dillon et al., 2004).

Consequentially, JAK tyrosine kinases are recruited causing stimulation of STAT, phosphatidylinositol 3-kinase and

various MAPK signalling pathways (Cornelissen et al., 2012). In 2006, Bando et al. located IL-31 mRNA alongside

OSMRβ in a small subset of DRGs and trigeminal ganglia, however fails to specify whether sensitisation of DRGs

occurs directly or indirectly (2006). Transgenic mice acquiring IL-31 overexpression “developed severe pruritus,

alopecia and skin lesions”, showing solid evidence for a role in itch sensation (Dillon et al., 2004). Not to mention,

these results have been replicated in both humans and NC/Nga mice (Takaoka et al., 2005; Szegedi et al., 2012).

A novel itch mediator is Thymic Stromal Lymphopoietin (TSLP). In 2013, Wilson et al. found that TSLP directly

activates cutaneous sensory neurons that coexpress TSLP receptors (TSLPRs) and TRPA1 and communicate with

epithelial cells. This produced robust scratching in a mouse model of atopic dermatitis. Additionally, epithelial cells

utilised “ORAI1-mediated Ca2+ influx to regulate cytokine expression and release” downstream of nonhistaminergic

signalling via PAR2 activation in keratinocytes. It is not clear yet whether lymphocytes are necessary for TSLP-evoked

itch. Future studies should investigate tissue specific TSLPR knockout mice to determine its contributions to itch

through sensory neurons and immune cells respectively. Overall, novel therapeutics may target the immune system

for itch relief, however, it is essential to understand how the immune and nervous systems communicate in order to

implement such treatments.

Substance P

Substance P (SP) is a neuropeptide, dominantly expressed in cutaneous nociceptive nerve endings and causes

neurogenic inflammation once released. Otherwise, SP can be found in skin mast cells, making it particularly relevant

to allergic reactions. Early records show intradermal injection of SP produces flare, wheal and itching, and that

antihistamines inhibit such responses (Hagermark et al., 1978). Thus, SP regulates histaminergic itch and does so via

neurokinin one (NK1) receptors (Andoh et al., 1998). In 1998, Andoh et al. found that mast cell-deficient mice

exhibit scratching behaviour, implying that mast cells may in fact, be negligible in SP-induced itching. Conversely

Tatemoto et al. (2006) suggested that MrgX2 receptors contribute to mast cell degranulation by activating G

proteins. Regardless, studies have shown aprepitant, a selective NK1 receptor antagonist, to be effective in chronic

itch patients (Stander et al., 2010), as well as murine models of atopic dermatitis (Ohmura et al., 2004). Very little is

known about mechanisms underlying SP-induced itch; although, nitric oxide (Andoh & Kuraishi, 2003) and

leukotriene B4 (Andoh et al., 2001) have been implied.

Modulation of the Itch Pathway

Peripheral Sensitisation

In conditions causing pathological itch, the neural pathways are functionally abnormal. This leads to sensitisation of

the peripheral and/or central neurocircuitries, thus subsequent excitation of spontaneous or hypersensitive itch

response occurs (Schmelz, 2014). Such examples have been observed in chronic pruritus patients whereby electrical

and pruritic stimuli induce an enhanced itch response in comparison to normal skin of healthy individuals (Ozawa et

al., 2009; can Laarhoven et al., 2010). Examples of hyperinnervation are evident in both experimental and clinical

studies whereby epidermal nerve fibre densities are significantly increased (Kinkelin et al., 2000; Tominaga et al.,

2007). These results may be explained by remarkable increases in nerve growth factor (NGF) expression seen in the

epidermis and immune cells (Kinkelin et al., 2000; Groneberg et al., 2005; Tominaga et al., 2007; Suga et al., 2013).

In line with this hypothesis, Rukwied et al. (2013) demonstrated that pre-treatment of NGF causes sensitisation of

cowhage-but not histamine-induced itch. This also suggests that histaminergic- and nonhistaminergic-itch

sensitisation is mediated by separate mechanisms: cowhage-induced itch may be specific to peripheral sensitisation

mechanisms while histamine-induced itch may be modulated by central sensitisation mechanisms. It has also been

implied that tumour necrosis factor (TNF)-α acts as an upregulator of epidermal NGF via MAPK signalling pathways

(Takaoka et al., 2009), and is ultimately derived from mast cells (Kakurai et al., 2006). Additionally, class 3

semaphorins (Sema3A), a nerve growth inhibitor, were shown to be downregulated in lesional skill of atopic

dermatitis (Tominaga et a., 2008). Interestingly, while Sema3A can inhibit NGF-induced nerve sprouting, NGF causes

collapsing of nerve growth cones (Tominaga & Takamori, 2014). This implies a delicate balance between Sema 3A

and NGF levels is needed for normal epidermal innervation. Understanding and targeting appropriate regulation of

these growth factors may be a promising target for future therapeutics, especially concerning chronic itch.

Central Sensitisation

The distinctions between pain and itch become all the more unclear as research delves into central sensitisation

mechanisms. Enhanced itch processing occurs similarly to hyperalgesia (an exaggerated pain response to noxious

stimuli due to hyperexcitability of nociceptors). Pruriceptors localised in CNS neurons acquire a lower threshold for

action potential firing, thus itch transmission occurs in spite of non-pruritic stimulation (Schmelz, 2014). This

phenomenon has been termed alloknesis. Having developed a reliable animal model of alloknesis, Akiyama et al.

(2012), found that upon injection of histamine in the rostral back, light mechanical stimulation away from the

application site, elicits scratching behaviour. This is in agreement with Ikoma et al.’s 2004 study in which noxious

stimuli primarily evoked itch in atopic dermatitis patients. With regard to specific targets participating in central

sensitisation, recent development is only beginning to expose such contributors. In 2012, Liu et al. demonstrated

that spinal cord slices of TLR3 knockout mice revealed significantly attenuated pruritus. Thus, TLR3 may be critically

involved in the regulation of neuronal excitability, itch transmission and central sensitisation. A later 2013 paper by

Zhao et al. developed a mouse model expressing constitutively active serine/threonine kinase BRAF in sodium

channel Nav1.8-gated neurons. These mice were found to have enhanced and persistent expression of itch-sensing

genes (GRP & MrgprA3 in TRPV1-positive nociceptors) in the spinal cord, which was reflected in amplified itch

response behaviours. Inhibition of BRAF or GRP signalling relieved some of the itch, thus, it can be deduced that

BRAF and GRP are key regulators in central sensitisation (Zhang et al., 2013). Especially concerning chronic patients,

investigating sensitisation of the itch pathway may be extremely beneficial. The difficultly will be in differentiating

between pain and itch processing, however, existing knowledge regarding pain sensitisation may assist in this

process.

Conclusion

Chronic itch is a serious condition that can detrimentally affect quality of life. Despite the development of

antihistamines, the medical needs of such patients are severely lacking. Current understanding concerning the way

itch is perceived has set aside the intensity theory and taken up a new, labelled-line theory. Recent research

suggests that major mediators involved in itch include HisRs, TRP channels, TLRs, Mrgprs, inflammatory molecules

and neuropeptides. Itch may be signalled by either endogenous or exogenous pruriceptive stimuli and can be

categorised into histaminergic and nonhistaminergic itch. While evidence implies the activation of itch specific

primary afferents, contradiction arises on deeper contemplation. It seems that several stimuli and mediators are

shared between itch and pain, leading to excitation of multiple afferent pathways in certain cases. Moreover, a

confusing antagonistic relationship exists between the two. Taking into account the evidence for peripheral and

central sensitisation, population coding may emerge as an all-encompassing itch theory; nonetheless, further

research is needed. Hopefully, combining methods of genetic manipulation, in culture research, brain imaging and

clinical observation will improve research impact and successfully reveal the mysteries of itch.

References

Akiyama T, Carstens E. (2014) Spinal coding of itch and pain. In E. Carstens & T. Akiyama (Eds.), Itch: Mechanisms and

Treatment (pp. 319-39). Boca Raton, FL: CRC Press.

Akiyama R, Carsten MI, Ikoma A, Cevikbas F, Steinhoff M, Carstens E. Mouse model of touch-evoked itch (alloknesis).

J Invest Dermatol. 2012; 132(7): 1886-91.

Amadesi S, Nie J, Vergnolle N, Cottrell GS, Grady EF, Trevisani M, Manni C, Geppetti P, McRoberts JA, Ennes H, Davis

JB, Mayer EA, Bunnett NW. Protease-activated receptor 2 sensitises the capsaicin receptor transient receptor

potential vanilloid receptor 1 to induce hyperalgesia. J Neurosci. 2004; 24(18): 4300-12.

Andoh T, Katsube N, Maruyama M, Kuraishi Y. Involvement of leukotriene B(4) in substance P-induced itch-

associated response in mice. J Invest Dermatol. 2001; 117(6): 1621-6.

Andoh T, Kuraishi Y. Nitric oxide enhances substance P-induced itch-associated responses in mice. Br J Pharmacol.

2003; 138(1): 202-8.

Andoh T, Nagasawa T, Satoh M, Kuraishi Y. Substance P induction of itch-associated response mediated by cutaneous

NK1 tachykinin receptors in mice. J Pharmacol Exp Ther. 1998; 286(3): 1140-5.

Andoh T, Takayama Y, Yamakoshi T, Lee JB, Sano A, Shimizu T, Kuraishi Y. Involvement of serine protease and

proteinase-activated receptor 2 in dermatophyte-associated itch in mice. J Pharmacol Exp Ther. 2012; 343(1): 91-6.

Andrew D, Craig AD. Spinothalamic lamina I neurons selectively sensitive to histamine: a central neural pathway for

itch. Nat Neurosci. 2001; 4(1): 72-7.

Bando T, Morikawa Y, Komori T, Senba E. Complete overlap of interleukin-31 receptor A and oncostatin M receptor

beta in adult dorsal root ganglia with distinct developmental expression patterns. Neuroscience. 2006; 142(4): 1263-

71.

Bell JK, McQueen DS, Rees JL. Involvement of histamine H4 and H1 receptors in scratching induced by histamine

receptor agonists in BalbC mice. Br J Pharmacol. 2004; 142(2): 374-80.

Bongers G, de Esch I, Leurs R. Molecular pharmacology of the four histamine receptors. Adv Exp Med Biol. 2011;

709:11-9.

Campero M, Baumann TK, Bostock H, Ocha JL. Human cutaneous C fibres activated by cooling, heating and menthol.

J Physiol. 2009; 578(Pt 23): 5633-52.

Cavanaugh DJ, Lee H, Lo L, Shield SD, Zylka MJ, B AI, Anderson DJ. Distinct subsets of unmyelinated primary sensory

fibres mediate behavioural responses to noxious thermal and mechanical stimuli. Proc Natl Acad Sci U S A. 2009;

106(22): 9075-80.

Cevikbas F, Kempkes C, Buhl T, Mess C, Buddenkotte J, Steinhoff M. (2014) Role of interleukin-31 and oncostatin M in

itch and neuroimmune communication. In E. Carstens & T. Akiyama (Eds.), Itch: Mechanisms and Treatment (pp.

237-57). Boca Raton, FL: CRC Press.

Clough GF, Boutsiouki P, Church MK. Comparison of the effects of levocetirizine and loratadine on histamine-induced

wheal, flare, and itch in human skin. Allergy. 2001; 56(1): 985-8.

Cohen KR, Frank J, Salbu RL, Israel I. Pruritus in the elderly. P&T. 2012; 27(4): 227-232.

Cornelissen C, Luscher-Firzlaff J, Baron JM, Luscher B. Signalling by IL-31 and functional consequences. Eur J Cell Bio.

2012; 91(6-7): 552-66.

Dai Y, Wang S, Tominaga M, Yamamoto S, Fukuoka T, Higashi T, Kobayashi K, Obata K, Tamanaka H, Noguchi K.

Sensitisation of TRPA1 by PAR2 contributes to the sensation of inflammatory pain. J Clin Invest. 2007; 117(7): 1979-

87.

Darsow U, Drzezga A, Frisch M, Munz F, Weilke F, Bartenstein P, Schwaiger M, Ring J. Processing of histamine-

induced itch in the human cerebral cortex: a correlation analysis with dermal reactions. J Invest Dermatol. 2000;

115(6): 1029-33.

Davidson S, Giesler G. The multiple pathways for itch and their interactions with pain. Trends Neurosci. 2010; 33(12):

550-558.

Davidson S, Moser H, Giesler G. (2014) Ascending pathways for itch. In E. Carstens & T. Akiyama (Eds.), Itch:

Mechanisms and Treatment (pp. 373-91). Boca Raton, FL: CRC Press.

Denham KJ, Boutsiouki P, Clough GF, Church MK. Comparison of the effects of desloratadine and levocetirizine on

histamine-induced wheal, flare and itch in human skin. Inflamm Res. 2003; 52(10): 424-7.

Dillon SR, Sprecher C, Hammond A, Bilsborough J, Rosenfeld-Franklin M, Presnell SR, Haugen S, Mauewe M, Harder

B, Johnston J, Bort S, Mudri S, Kuijper JL, Bukowski T, Shea P, Dong DL, Dasovich M, Grant FJ, Lockwood L, Levin SD,

LeCiel C, Waggie K, Day H, Topouzis S, Kramer J, Kuestner R, Chen Z, Foster D, Parrish-Novak J, Gross JA. Interleukin

31, a cytokine produced by activated T cells, induces dermatitis in mice. Nat Immunol. 2004; 5(7): 752-60.

Dugas-Breit S, Schopf P, Dugas M, Schiffl H, Rueff F, Przybilla B. Baseline serum levels of mast cell tryptase are raised

in hemodialysis patients and associated with severity of pruritus. J Dtsch Dermatol Ges. 2005; 3(5): 343-7.

Gutzmer R, Mommert S, Gschwandtner M, Zwingmann K, Stark H, Werfel T. The histamine H4 receptor is

functionally expressed on T(H)2 cells. J Allergy Clin Immunol. 2009; 123(3): 619-25.

Grant AD, Cottrell GS, Amadesi S, Trevisani M, Nicoletti P, Materazzi S, Altier C, Cenac N, Zamponi GW, Bautistia-Cruz

F, Lopez CB, Joseph EK, Levine JD, Liedtke W, Vanner S, Vergnolle N, Geppetti P, Bunnett NW. Protease-activated

receptor 2 sensitises the transient receptor potential vanilloid 4 ion channel to cause mechanicam hyperalgesia in

mice. J Physiol. 2007; 578(Pt 3): 715-33.

Groneberg DA, Serowka F, Peckenschneider N, Artuc M, Grutzkau A, Fischer A, Henz BM, Welker P. Gene expression

and regulation of nerve growth factor in atopic dermatitis mast cells and the human mast cell line-1. J

Neuroimmunol. 2005; 161(1-2): 87-92.

Hagermark O, Hokfelt T, Pernow B. Flare and itch induced by substance P in human skin. J Invest Dermatol. 1978;

71(4): 233-5.

Han L, Dong X. Itch mechanisms and circuits. Annu Rev Biophys. 2014; 43: 331-55.

Han L, Ma C, Liu Q, Weng HJ, Cui Y, Tang Z, Kim Y, Nie H, Qu L, Patel HL, Li Z, McNeil B, He S, Guan Y, Xiao B, LaMotte

RH, Dong X. A subpopulation of nociceptors specifically link to itch. Nat Neurosci. 2013; 16(2): 174-82.

Han SK, Mancino V, Simon MI. Phospholipase Cβ 3 mediates the scratching response activated by the histamine H1

receptor on C-fibre nociceptive neurons. Neuron. 2006; 52(4): 691-703.

Handwerker HO. (2014) Itch hypothesis: from pattern to specificity and to population coding. In E. Carstens & T.

Akiyama (Eds.), Itch: Mechanisms and Treatment (pp. 1-9). Boca Raton, FL: CRC Press.

Handwerker HO, Forster C, Kirchhoff C. Discharge patterns of human C-fibres induced by itching and burning stimuli.

J Neurophysiol. 1991; 66(1): 307-15.

Heyer G, Dotzer M, Diepgen TL, Handwerker HO. Opiate and H1 antagonist effects on histamine induced pruritus and

alloknesis. Pain. 1997; 73 (2):237-43.

Huang SM, Lee H, Chung MK, Park U, Yu YY, Bradshaw HB, Coulombe PA, Walker JM, Caterina MJ. Overexpressed

transient receptor potential vanilloid 3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin

E2. J Neurosci. 2008; 28(51): 13727-37.

Ikoma A, Cevikbas F, Kempkes C, Steinhoff M. Anatomy and neurophysiology of pruritus. Semin Cutan Med Surg.

2011; 30(2): 64-70.

Ikoma A, Fartasch M, Heyer G, Miyachi Y, Handwerker H, Schmelz M. Painful stimuli evoke itch in patients with

chronic pruritus: central sensitisation for itch. Neurology. 2004; 62(2): 212-7.

Imamachi N, Park GH, Lee H, Anderson DJ, Simon MI, Basbaum AI, Han SK. TRPV1-expressing primary afferents

generate behavioural responses to pruritogens via multiple mechanisms. Proc Natl ACad Sci U S A. 2009; 106(27):

11330-5.

Jeffry J, Kim S, Chen ZF. Itch signalling in the nervous system. Physiology (Bethesda). 2011; 26(4): 286-92.

Kakurai M, Montekforte R, Suto H, Tsai M, Nakae S, Galli SJ. Mast cell-derived tumour necrosis factor can promote

nerve fibre elongation in the skin during contact hypersensitivity in mice. Am J Pathol. 2006; 169(5): 1713-21.

Kim BM, Lee SH, Shim WS, Oh U. Histamine-induced Ca(2+) influx via the PLA(2)/lipoxygenase/TRPV1 pathway in rat

sensory neurons. Neurosci Lett. 2004; 361(1-3): 159-62.

Kim SJ, Park GH, Kim D, Lee J, Min H, Wall E, Lee CJ, Simon MI, Lee SJ, Han SK. Analysis of cellular and behaviour

responses to imiquimod reveals a unique itch pathway in transient receptor potential vanilloid 1 (TRPV1)-expressing

neurons. Proc Natl Acad Sci U S A. 2011; 108(8): 3371-6.

Kinkelin I, Motzing S, Koltenzenburg M, Brocker EB. Increase in NGF content and nerve fibre sprouting in human

allergic contact eczema. Cell Tissue Res. 2000; 302(1): 31-7.

Kempkes C, Buddenkotte, Cevikbas F, Buhl T, Steinhoff M. (2014) Role of PAR-2 in neuroimmune communication and

itch. In E. Carstens & T. Akiyama (Eds.), Itch: Mechanisms and Treatment (pp. 193-213). Boca Raton, FL: CRC Press.

Lagerstrom MC, Rogoz K, Abrahamsen B, Persson E, Reinius B, Nordenankar K, Olund C, Smith C, Mendez JA, Chen

ZF, Wood JN, Wallen-Mackenzie A, Kullander K. VGLUT2-dependent sensory neurons in the TRPV1 population

regulate pain and itch. Neuron. 2010; 68(3): 529-42.

LaMotte RH, Shimada SG, Sikand P. Mouse models of acute, chemical itch and pain in humans. Exp Dermatol. 2011;

20(1): 778-82.

Lewis T, Grant RT, Marvin HM. Vascular reactions of the skin to injury. X. The intervention of a chemical stimulus

illustrated especially by the flare: the response to faradism. Heart. 1927; 14: 139-60.

Lieu T, Jayawwera G, Zhao P, Poole DP, Jensen D, Grace M, McIntyre P, Bron R, Wilson YM, Krappitz M, Haerteis S,

Korbmacher C, Steinhoff MS, Nassini R, Materazzi S, Geppetti, Corvera CU, Bunnett NW. The bile acid receptor TGR5

activates the TRPA1 channel to induce itch in mice. Gastroenterology. 2014; 146(6): 1417-28.

Liu T, Berta T, Xu ZZ, Park CK, Zhang L, Lu N, Liu Q, Liu Y, Gao YJ, Liu YC, Ma Q, Dong X, Ji RR. TLR3 deficiency impairs

spinal cord synaptic transmission, central sensitisation, and pruritus in mice. J Clin Invest. 2012; 122(6): 2195-207.

Liu T, Gao YJ, Ji RR. Emerging role of Toll-like receptors in the control of pain and itch. Neurosci Bull. 2012; 28(2): 131-

44.

Liu T, Ji RR. (2014) Toll-like receptors and itch. In E. Carstens & T. Akiyama (Eds.), Itch: Mechanisms and Treatment

(pp. 257-71). Boca Raton, FL: CRC Press.

Liu T, Xu ZZ, Park CK, Berta T, Ji RR. Toll-like receptor 7 mediates pruritus. Nat neurosci. 2010; 13(12): 1460-2.

Liu Q, Sikand P, Ma C, Tang Z, Han L, Li Z, Sun S, LaMotte RH, Dong X. Mechanisms of itch evoked by β-alanine. J

Neurosci. 2012; 32(42): 14532-7.

Liu Q, Tang Z, Surdenikova L, Kim S, Patel KN, Kim A, Ru F, Guan Y, Weng HJ, Geng Y, Undem BJ, Kollarik M, Chen ZF,

Anderson DJ, Dong X. Sensory neuron-specific GPCR Mrgprs are itch receptors mediating choloroquine-induced

pruritus. Cell. 2009; 139(7): 1353-65.

Ma Q. Labelled lines meet and talk: population coding of somatic sensations. J Clin Invest. 2010; 120(11): 3773-8.

McNeil B, Dong X. (2014) Mrgprs as itch receptors. In E. Carstens & T. Akiyama (Eds.), Itch: Mechanisms and

Treatment (pp. 213-37). Boca Raton, FL: CRC Press.

Mochizuki H, Papoiu ADP, Yosipovitch G. (2014) Brain processing of itch and scratching. In E. Carstens & T. Akiyama

(Eds.), Itch: Mechanisms and Treatment (pp. 391-409). Boca Raton, FL: CRC Press.

Mochizuki H, Tashiro M, Kano M, Sakurada Y, Itoh M, Yanai K. Imaging of central itch modulation in the human brain

using positron emission tomography. Pain. 2003; 105(1-2): 339-46.

Morse KL, Behan J, Laz TM, West RE Jr, Greenfeder SA, Anthes JC, Umland S, Wan Y, Hipkin RW, Gonsiorek W, Shin N,

Gustafson EL, Qiao X, Wang S, Hedrick JA, Greene J, Bayne M, Monsma FJ Jr. Cloning and characterisation of a novel

human histamine receptors. J Pharmacol Exp Ther. 2001; 296(3): 1058-66.

Oaklander AL. Neuropathic Itch. Semin Cutan Med Surg. 2011; 30(2): 87092.

Ochoa J, Torebjork E. Sensations evoked by intraneural microstimulation of C nociceptor fibres in human skin nerves.

J Physiol. 1989; 415: 583-99.

Oh MH, Oh SY, Lu J, Lou H, Myers AC, Zhu Z, Zheng T. TRPA1-dependent pruritus in IL-13- induced chronic atopic

dermatitis. J Immunol. 2013; 191(11): 5371-82.

Ozawa M, Tsuchiyama K, Gomi R, Kurosaki F, Kawamoto Y, Aiba S. Neuroselective transcutaneous electrical

stimulation reveals neuronal sensitisation in atopic dermatitis. J Am Acad Dermatol. 2009; 60(4): 609-14.

Patel KN, Dong X. Itch: cells, molecules, and circuits. ACS Chem Neurosci. 2011; 2(1):17-25.

Patel KN, Liu Q, Meeker S, Undem BJ, Dong X. Pirt, a TRPV1 modulator, is require for histamine-dependent and –

independent itch. PLoS One. 2011; 6(5): e20559.

Potenzieri C, Undem BJ. Basic mechanisms of itch. Clin Exp Allergy. 2012; 42(1): 8-19.

Ringkamp M, Schepers RJ, Shimada SG, Johanek LM, Hartke TV, Borzan J, Shim B, LaMotte RH, Meyer RA. A role for

nociceptive, myelinated nerve fibres in itch sensation. J Neurosci. 2011; 31(42): 14831-9.

Ross SE. Pain and itch: insights into the neural circuits of aversive somatosensation in health and disease. Curr Opin

Neurobiol. 2011; 21(6): 880-7.

Rossbach K, Nassenstein C, Gschwandtner M, Schnell D, Sander K, Seifert R, Stark H, Kietzmann M, Baumer W.

Histamine H1, H3 and H4 receptors are involved in pruritus. Neuroscience. 2011; 190: 89-102.

Rossbach K, Wendorff S, Sander K, Stark H, Gutzmer R, Werfel T, Keitzmann M, Baumer W. Histamine H4 receptor

antagonism reduces hapten-induced scratching behaviour but not inflammation. Exp Dermatol. 2009; 18(1): 57-63.

Rukweid RR, Main M, Weinkauf B, Schmelz M. NGF sensitises nociceptors for cowhage- but not histamine-induced

itch in human skin. J Invest Dermatol. 2013; 133(1): 268-70.

Schmelz M. (2014) Sensitisation for itch. In E. Carstens & T. Akiyama (Eds.), Itch: Mechanisms and Treatment (pp.

431-41). Boca Raton, FL: CRC Press.

Schmelz M, Schmidt R, Bickel A, Handwerker HO, Torejbork HE. Specific C-receptors for itch in human skin. J

Neurosci. 1997; 17(20): 8003-8.

Schmelz M, Schmidt R, Weidner C, Hilliges M, Torebjork HE, Handwerker HO. Chemical response pattern of different

classes of C-nociceptors to pruritogens and algogens. J Neurophysiol. 2003; 89(5): 2441-8.

Shim WS, Tak MH, Lee MH, Kim M, Koo JY, Lee CH, Kim M, Oh U. TRPV1 mediates histamine-induced itching via the

activation of phospholipase A2 and 12-lipoxygenase. J Neurosci. 2007; 27(9): 2331-7.

Sikland P, Dong X, LaMotte RH. BAM8-22 peptide produces itch and nociceptive sensations in humans independent

of histamine release. J Neurosci. 2011; 31(20): 7563-7.

Simone DA, Zhang X, Li J, Zhang JM, Honda CN, LaMotte RH, Giesler GJ Jr. Comparison of responses of primate

spinothalamic tract neurons to pruritic and algogenic stimuli. J Neurophysiol. 2004; 91(1): 213-22.

Suga H, Sugaya M, Miyagaki T, Ohmatsu H, Fujita H, Kagami S, Asano Y, Tada Y, Kadono T, Sato S. Association of

nerve growth factor, chemokine (C-C motif) ligands and immunoglobulin E with pruritus in cutaneous T-cell

lymphoma. Acta Derm Venereol. 2013; 92(2): 144-9.

Sun YG, Chen ZF. A gastrin-releasing peptide receptors mediates the itch sensation in the spinal cord. Nature. 2007;

448(7154): 700-3.

Sun YG, Zhao ZQ, Meng XL, Yin J, Liu XY, Chen ZF. Cellular basis of itch sensation. Science. 2009; 325(5947): 1531-4.

Stander S, Schmelz M. Chronic itch and pain—similarities and differences. Eur J Pain. 2006; 10(5): 473-8.

Stander S, Siepmann D, Herrgott I, Sunderkotter C, Luger TA. Targeting the neurokinin receptor 1 with aprepitant: a

novel antipruritic strategy. PLoS One. 2010; 5(6): e10968.

Steinhoff M, Biro T. A TR(I)P to pruritus research: role of TRPV3 in inflammation and itch. J Invest Dermatol. 2009;

129(3): 531-5.

Steinhoff M, Neisiu U, Ikoma A, Fartasch M, Heyer G, Skov PS, Luger TA, Schmelz M. Proteinase-activated receptor-2

mediates itch: a novel pathway for pruritus in human skin. J Neurosci. 2003; 23(15): 6176-80.

Szegedi K, Kremer AE, Kezic S, Teunissen MB, Bos JD, Luiten RM, Res PC, Middelkamp-Hup MA. Increased frequencies

of IL-31- producing T cells are found in chronic atopic dermatitis skin. Exp Dermatol. 2012; 21(6): 431-6.

Takaoka A, Arai I, Sugimoto M, Yamaguchi A, Tanaka M, Nakaike S. Expression of IL-31 gene transcripts in NC/Nga

mice with atopic dermatitis. Eur J Pharmacol. 2005; 516(2): 180-1.

Takaoka K, Shirai Y, Saito N. Inflammatory cytokine tumour necrosis factor-alpha enhances nerve growth factor

production in human keratinocytes, HaCaT cells. J Pharmacol Sci. 2009; 111(4): 381-91.

Tatemoto K, Nozaki Y, Tsuda R, Konno S, Tomura K, Furuno M, Ogasawata H, Edamura K, Takagi H, Iwamura H,

Noguchi M, Naito T. Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors. Biochem

Biophys Res Commun. 2006; 349(4): 1322-8.

Than JY, Li L, Hasan R, Zhang X. Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel-expressing sensory

neurons by the pruritogen choloroquine. J Biol Chem. 2013; 288(18): 12818-27.

Thurmond RL, Gelfand EW, Dunford PJ. The role of histamine H1 and H4 receptors in allergic inflammation: the

search for new antihistamines. Nat Rev Drug Discov. 2008; 7(1): 41-53.

Thurmond RL, Kazerouni K, Chaplan SR, Greenspan AJ. (2014) Peripheral neuronal mechanism of itch. In E. Carstens

& T. Akiyama (Eds.), Itch: Mechanisms and Treatment (pp. 143-93). Boca Raton, FL: CRC Press.

Tominaga M, Ogawa H, Takamori K. Decreased production of semaphoring 3A in the lesional skin of atopic

dermatitis. Br J Dermatol. 2008; 158(4): 842-4.

Tominaga M, Ozawa S, Ogawa H, Takamori K. A hypothetical mechanism of intraepidermal neurite formation in

NC/Nga mice with atopic dermatitis. J Dermatol Sci. 2007; 46(3): 199-210.

Tominaga M, Takamori K. (2014) Sensitisation of itch signalling. In E. Carstens & T. Akiyama (Eds.), Itch: Mechanisms

and Treatment (pp. 293-305). Boca Raton, FL: CRC Press.

Tsujii K, Andoh T, Ui H, Lee JB, Kuraishi Y. Involvement of trpytase and proteinase-activated receptor-2 in

spontaneous itch-associated response in mice with atopy-like dermatitis. J Pharmacol Sci. 2009; 109(3): 388-95.

Tuckett RP. Itch evoked by electrical stimulation of the skin. J Invest Dermatol. 1982; 79(6): 368-73.

Twycross R, Greaves MW, Handwerker H, Jones EA, Libretto SE, Szepietowski JC, Zylicz Z. Itch: scratching more than

the surface. QJM. 2003; 96(1):5-26.

van Laarhoven AI, Kraaimaat FW, Wilder-Smith OH, van Riel PL, van de Kerkhof PC, Evers AW. Sensitivity to itch and

pain in patients with psoriasis and rheumatoid arthritis. Exp Dermatol. 2013; 22(8): 530-4.

Wilson SR, Nelson AM, Batia L, Takeshi M, Estandian D, Owens DM, Lumpkin EA, Bautista DM. The ion channel TRPA1

is requires for chronic itch. J Neurosci. 2013; 33(22): 9283-94.

Wilson SR, Bautista DM. (2014) Role of transient receptor potential channels in acute and chronic itch. In E. Carstens

& T. Akiyama (Eds.), Itch: Mechanisms and Treatment (pp. 281-93). Boca Raton, FL: CRC Press.

Wilson SR, Gerhold KA, Bifolck-Fisher A, Liu Q, Patel KN, Dong X, Bautista DM. TRPA1 is require for histamine-

independent, Mas-related G protein-coupled receptor-mediated itch. Nat Neurosci. 2011; 14(5): 595-602.

Wilson SR, The L, Batia LM, Beattie K, Katibah GE, McClain SP, Peelegrino M, Estandian DM, Bautista DM. The

epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell. 2013; 155(2): 285-95.

Woo DH, Jung SJ, Zhu MH, Park CK, Kim YH, Oh SB, Lee CJ. Direct activation of transient receptor potential vanilloid

(TRPV1) by diaclyglycerol (DAG). Mol Pain. 2008; 4: 42.

Yoshioka T, Imura K, Asakawa M, Suzuki M, Oshima I, Hirasawa T, Sakata T, Horikawa T, Arimura A. Impact of the

Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice. J Invest Dermatol.

2009; 129(3): 714-22.

Yosipovitch G, Greaves MW, Schmelz M. Itch. Lancet. 2003; 361(9358): 609-4.

Zhang X. Targeting TRP ion channels for itch relief. Naunyn Schmiedebergs Arch Pharmacol. 2014. doi:

10.1007/s00210-014-1068-z

Zhang X, Mak S, Li L, Parra A, Denlinger B, Belmonte C, McNaughton PA. Direct inhibition of the cold-activated

TRPM8 ion channel Gαq. Nat Cell Biol. 2012; 14(8): 851-8.

Zheng J, Lu Y, Perl ER. Inhibitory neurones of the spinal substantia gelatinosa mediate interaction of signals from

primary afferents. J Physiol. 2010; 588(12): 2065-75.

Zhao ZQ, Huo FQ, Jeffry J, Hampton L, Demehri S, Kim S, Liu XY, Barry DM, Wan L, Liu ZC, Li H, Turkoz A, Ma K,

Cornelius LA, Kopan R, Battey JF Jr, Zhong J, Chen ZF. Chronic itch development in sensory neurons requires BRAF

signalling pathways. J Clin Invest. 2013; 123(11): 4769-80.

Zylka MJ, Rice FL, Anderson DJ. Topographically distinct epidermal nociceptive circuits revealed by axonal tracers

targeted to Mrgprd. Neuron. 2005; 45(1): 17-25.