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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.
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