Role Of Bradykinin B1 Receptor On Chronic Itch Sensation

Background and objectsAtopic dermatitis (AD) is accompanied by a disturbance of sensitization, which indicates the impact of chronic inflammation on the peripheral sensory neurons. AD patients and the animal model show increased and persistent pruritus or itching like scratching behaviors.Recently, it was suggested that bradykinin (BK) is involved in the pathological aggravation of AD, since activation of plasma kallikrein and the secretion of skin tissue kallikrein increase in human AD patients. Two kallikreins (plasma and tissue) cleave kininogens to release kinins, including BK and kallidin (Lys-BK), both of which are converted to des-Arg derivatives by carboxypeptidase N or M and become BK receptor 1 (B1R) agonists. BK is a mediator of inflammation; is produced from the plasma precursor kininogen by tissue injury, anoxia, or inflammation; and is responsible for nociceptor sensitization and pain, vasodilation, and capillary permeability. Furthermore, it is reported that BK participates in the release of some europeptides such as substance P indirectly. Robust calcium transients are observed in the presence of BK in primary dorsal root ganglia (DRG) culture.Whether BK participates in the chronic itch progress directly on the peripheral nerve endings or indirectly is not known. It is known, however, that B1R and BK receptor 2 (B2R) are G protein-coupled receptors that mediate kinin effects. In normal skin, B2R signaling predominates, however, and cutaneous inflammation results in enhanced B1R responses.Our previous studies have shown that itching is induced by BK, and the activation of kinin B1 receptor mediates the alloknesis response in complete Freund’s adjuvant (CFA)-inflamed mice. At the same time, in the epithelium of patients with asthma, BIR is up regulated and associated with airborne allergens that may play a role in the pathophysiology of atopic and allergic diseases. Stadnicki has suggested that B1R mRNA and protein increase in specimens from inflammatory bowel disease (IBD) patients and the enhanced BIR expression is related to the pathology of IBD. Nevertheless, a litter study on the itching sensation reported high BIR expression, focused on chronic itching in AD. Whether the expression of BIR changes and whether BIR expression is related to the etiology and development of the itching sensation in a chronic inflammatory state remains unclear.A recent report has indicated that treatment with diphenylcyclopropenone (DCP) often results in contact dermatitis, as well as intense itching, in both humans and mice. If this is the case, DCP-treated mice can be considered as a chronic inflammatory in vitro model. We focus on B1R function on itching behaviors in DCP-treated mice.In the present study, we evaluated scratching behaviors in a mouse model of AD, especially focusing on whether B1R affects itching behaviors and its possible regulatory mechanism in up regulating B1R.MethodsMale C57BL/6J mice (20-22 g) were used in this study. The dorsal hair of the mice was removed and then stimulated for the first day and the seventh day with DCP repeatedly under conventional conditions. Animal samples comprising skin tissues were obtained from experimental models of chronic inflammation induced by DCP and vehicle-treated mice. The experimental procedures and animal use and care protocols were approved by the Committee on Ethical Use of Animals at the Guangdong Academy of Medical Sciences (Guangzhou, China), following the National Institutes of Health’s animal use and care guidelines.Pruritus symptoms produced following repeated application of 1% DCP was characterized by a pronounced and long-lasting increase in spontaneous scratching behavior by the hind paw. And then we observed the effect of preventive treatment with BIR antagonist R892, kallikrein inhibitor NFM and B2R antagonist HOE-140 during the same phase.We examined skin tissues from experimental models of chronic inflammation induced by DCP. Immunohistochemical (IHC) staining of those tissues was analyzed after paraffin-embedded and formalin-fixed. The skin tissues were treated with RNAlater, followed by liquid nitrogen refrigeration were collected for Q-PCR, or obtained the tissues and stored with liquid nitrogen refrigeration immediately for western blot.The human HaCaT keratinocytes were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies). Cell lines were incubated at 37℃ with 5% CO2 and 95% humidity. All cells were obtained from Cell Bank, Shanghai Institutes for Biological Sciences (Shanghai, China). To study the effects of the PAR2 agonist, HaCaT keratinocytes were cultured with SLIGKV-NH2 (100 μM) or NF-κB inhibitor (PDTC:25 μM). Furthermore, to understand B1R function on keratinocytes, we assessed whether the B1R antagonist could exert a suppressing action on differentiation or proliferation of HaCaT cell lines by using Q-PCR and CCK8.Results1. Pruritus symptoms produced following repeated application of 1% DCP was characterized by a pronounced and long-lasting increase in spontaneous scratching behavior by the hind paw. This process was observed starting on day 3, reaching a maximum on day 10 after repeated application of 1% DCP on the seventh day.2. Antagonism of BIR significantly inhibited scratching in DCP-treated animalsPreventive treatment with B1R antagonist R892 (500 μg/kg, IP,30 min before observation), kallikrein inhibitor NFM (10mg/kg, IP,30 min before observation) significantly suppressed spontaneous scratching compared with the sham-treated group. However, preventive treatment with B2R antagonist HOE-140 (300 nmol/kg, IP,30 min before observation) during the same phase, did not significantly inhibit the scratching. Generally speaking, antagonism of BIR significantly inhibited scratching in DCP-treated animals.3. Kinin B1 receptors exert an important role in the neuropathic hypersensitivity induced by DCP treatment.To evaluate the effects of DCP on the expression of kinin B1 receptors, the mRNA and protein level from the skin of control and DCP-treated mice (on day 10 post application) were evaluated by means of real-time RT-PCR and Western blot assays, respectively. Basal expression of B1R was detected in the control group, while 10 days after DCP application, protein and mRNA levels of kinin BIR transcript were markedly increased in the skin tissue.4. Does the neural ending express B1R during the development of AD? To answer this question, we measured the colocalization of BIR expression with a well-known neural marker, PGP9.5, in the skin after DCP treatment. Little signal of BIR was detected in the PGP9.5 positive areas from treated mouse skin; nonetheless, B1R staining signal were markedly increased in keratinocytes at 10 days following DCP-treated compared to a sham treated group. There were also other cells showing BIR signals in the dermis. These results support the concept that keratinocytes expressed and up regulated B1R expression after DCP treatment.5. To study the effects of the PAR2 agonist, HaCaT keratinocytes were cultured with SLIGKV-NH2 (100μM) for approximately 24 h. By comparing B1R mRNA and protein levels with the control group, we confirmed that B1R expression was significantly augmented. A positive immunocytochemistry reaction for B1R was observed, located on the cell surface in HaCaT keratinocytes cells. In addition, SLIGKV-NH2 activated the NF-κB signaling pathway, while the NF-κB inhibitor (PDTC:25μM) intervened in the regulatory effects of PAR2 introducing BIR expression in HaCaT cells, following B1R down regulation.To understand B1R function on keratinocytes, we assessed whether the B1R antagonist could exert a suppressing action on differentiation or proliferation of HaCaT cell lines. However, these outcomes appeared to occur independently of the epidermal barrier protein filaggrin, since its levels were not affected. What is more, analysis of cell proliferation of HaCaT cells show that B1R antagonist R892 does not inhibit HaCaT cell growth by pretreatment of PAR2 agonist.Conclusions1. B1R facilitates the chronic itching sensation in a DCP-treated chronic inflammation experimental model.2. B1R is up regulated in the skin of DCP-treated mice.3. DCP treatment induced up regulation of B1R expression on keratinocytes, while delocalized with known neuronal maker Protein gene product 9.5 (PGP9.5).4. B1R expression was induced when HaCaT cells were exposed to PAR2 agonist, and NF-κB signal pathway activity has an important role in this process.