Published studies indicate that peripheral injury activates the neural cellular circuitry in peripheral and central nervous tissue (Cenac et al., 2007; Trang et al., 2009). The resulted neuron-glia interactions elicit pain hypersensitivity (Cenac et al., 2007; Trang et al., 2009). The brain-derived neurotrophic factor (BDNF) plays a key role in eliciting pain hypersensitivity. Microglial cells are the major cell type to release BDNF upon excitation by activating the P2X4 receptor (P2X4R) (Tsuda et al., 2003). A hallmark of pain hypersensitivity is the over expression of P2X4R in microglial cells; however, its causative mechanism remains to be further elucidated (Tsuda et al., 2008). 
         It has been noted that mast cell activation is associated with pain hypersensitivity (Anaf et al., 2006; Zhang and Levy, 2008), such as in patients with abdominal hyperalgesia in irritable bowel syndrome and inflammatory bowel disease (Tache´ et al., 2002). The chemical mediators of mast cells (e.g., histamine and tryptase) (Smith et al., 2007; Dale and Vergnolle, 2008) are proposed playing roles in pain hypersensitivity. Yet, the underlying mechanism is unclear.
         The discovery of proteinase-activated receptor 2 (PAR2) has drawn great attention by PAR20 s critical role in eliciting pain hypersensitivity (Cenac et al., 2007; Zhang and Levy, 2008). Mast cells have the capacity to activate PAR2 by releasing mediator tryptase; the latter cleaves PAR2 and activates PAR2-bearing cells (Zhang and Levy, 2008). Activating PAR2 evokes a series of biochemical reactions in cells such as speeding up the mitogen-activated protein kinase (MAPK) phosphoryla- tion and caused the target gene transcription (Pan et al., 2008). It is likely but undetermined whether mast cell- induced PAR2 activation is involved in P2X4R expres- sion in microglial cells. Therefore, we hypothesized that mast cell activation promotes the expression of P2X4R or/and BDNF in microglial cells. The present study aimed to elucidate whether mast cell activation promotes the expression of P2X4R or/and BDNF in microglial cells via releasing tryptase; the latter cleaves PAR2 to activate microglial cells. The results demonstrate that mast cell activation plays a critical role in promoting P2X4R and BDNF expression in microglial cells.

                        MATERIALS AND METHODS

Reagents and Animals.
      BDNF ELISA kit (Millipore Bioscience Research Reagents, Temecula, CA). RNeasy Mini kit (Qiagen, Missis- sauga, Canada). iScriptTMcDNA Synthesis Kit (Bio-Rad, Mississauga, Canada). SuperScript III Platinum SYBR Green Two-Step qPCR Kit (Invitrogen, Burlington, ON, Canada). siRNAs, anti-b-actin, anti-phosphor MAPK, anti-PAR2, anti-ionized calcium binding adaptor molecule 1 (Iba-1) (Santa Cruz Biotechnology, Santa Cruz, CA). ATP, SB203580, SB202474, Corticotropin-releasing hormone, Pyr- ilamine, Cimetidine, Ketanserin, Azasetron, RS 39604 (Sigma Aldrich, St. Louis, MO). Anti-P2X4R (Everest Bioteck, Oxfordshire, UK). Pyrilamine, Cimetidine, Ketanserin, Azase- tron, RS 39604. Bis(5-amidino-2-benzimidazolyl)methane (BABIM) was a kind gift from Dr. Anna Diez (Institut de Recerca Biome`dica de Barcelona, Spain). Mouse PAR2 active peptide (SLIGRL) and the reverse peptide (LRGILS) were syn- thesized by the Department of Biochemistry at the Second Military Medical University. BALB/c mice (Shanghai Wan- Xiang Laboratory Animal Technology Co., Ltd). Male PAR2- deficient (PAR2-/-) and wild-type (PAR21/1) mice (genetic background: C57BL/6 strain; 8–10 weeks of age; Charles River Laboratories, Toulouse, France). The animal experimen- tal procedures were approved by the Experimental Animal Ethic Committee at the Second Military Medical University.

Microglial Cell Preparation
      Following previous reports (Tsuda et al., 2008), mouse cortex microglial cells were isolated and cultured in DMEM medium supplemented with 10% fetal bovine serum, gluta- mine (1 lMol) and penicillin-streptomycin (1:1000) at 378C with 5% CO2 and 95% O2. On day 14, microglial cells were isolated from the culture with reported procedures (Tsuda et al., 2008). The purity of microglial cells was over 95% as analyzed by flow cytometry (with anti-Iba-1 antibody; Iba-1 is the specific marker of microglia).

HMC-1 Cell Culture
      Human mast cell line, HMC-1 cells, was a gift from Dr. Joseph H. Butterfield at Mayo Clinic. HMC-1 cells were cultured in RPMI1640 culture media supplemented with 10% fetal bovine serum, glutamine (2 lMol) and penicillin-strepto- mycin (1:1000).

ELISA
      Levels of BDNF in culture supernatant were determined by ELISA with BDNF-specific reagent kit following the man- ufacturer’s instruction.

Quantitative Real Time RT-PCR (qRT-PCR)
      Total RNA was extracted from the microglial cells using an RNeasy Mini kit. cDNA was synthesized using iScriptTMcDNA Synthesis Kit. The resulting cDNA was sub- jected to qPCR that was performed with a LightCycler using a SuperScript III Platinum SYBR Green Two-Step qPCR Kit. The amplified product was detected by the presence of an SYBR green fluorescent signal. The standard curve was designed with b-actin cDNA. The resulted amplicon was quantified with the standard curve. The primers and qPCR conditions included: P2X4R, forward: 50 -gcgtctgtgaagacctgtga-30 ; reverse: 50 - gatttggccaagacggaata-30 (246 bp; NCBI, NM_011026). b-actin: forward, 50 - ggacttcgagcaagagatgg-30 ; reverse, 50 -agcactgtgttggcg- tacag-30 ; 234 bp. NCBI, NM_001101). Annealing temperature:
608C, for 30 sec, 39 cycles.

RNA Interference (RNAi)
      Small interference RNA (siRNA) transfection was per- formed following reported procedures (Feng et al., 2008) as well as manufacturer’s instruction. The transfection was car- ried out when the cell confluence was about 30%-50%. One day before the transfection, cells were cultured in antibiotic- and serum-free media. A complex of siRNA oligonucletide and oligofectamine-mix was prepared; the complex was added to culture of microglial cells or HMC-1 cells to a final concen- tration of 60 pMol. The cells were incubated at 37℃ for 4 hr. Fifty micro milliliter growth media were then added to each well. The cells were cultured at 37℃; the expression of target genes was detected by Western blotting. The peak inhibitory effect was reached 24 hr post-transfection that sustained for another 72 hr and declined thereafter. The scramble siRNAs did not affect the target molecule expression. The transcription efficiency was over 90% and the inhibition was also over 90% and reproducible in all experiments. Cell viability was examined after the transfection. The viable cells were 95.6% in transfected cells while the viable cells were 115.3% in native controls.

Western Blotting
       Protein extracts (80 lg/well) were loaded onto 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with primary antibody (1lg/ml) in 5% nonfat milk in TBS at 48C overnight. This was followed by incubation with HRP-conjugated second antibody for 1 hr at room temperature with shaking. Reactions were developed using the Pierce ECL chemiluminescence substrate kit. Results were recorded with X ray film (Feng et al., 2007, 2008).

Flow Cytometry
        Microglial cells were collected and incubated with pri- mary antibodies (or isotype IgG) on ice for 30 min. For the intracellular staining, cells were fixed with 1% paraformalde- hyde on ice for 30 min and incubated with permealization reagents (BD FACS permeabilizing solution 2; catalogue num- ber: 340973) for 30 min on ice. The stained cells were ana- lyzed using a FACSarray (BD Bioscience, San Jose, CA). Data were analyzed with software FlowJo.

Statistics
         All values were expressed as the means ±SD of at least three independent experiments. The values were analyzed using the two-tailed unpaired Student’s t-test when data con- sisted of two groups or by ANOVA when three or more groups were compared. The correlation between variables was analyzed using Pearso’s correlation coefficient. P < 0.05 was accepted as statistically significant.

                                        RESULTS
          Activation of Mast Cells Increases the Production of BDNF by Microglial Cells 
          Microglial cell-derived BDNF plays a critical role in eliciting pain hypersensitivity after neural injury (Coull et al., 2005); the causative factors inducing BDNF release are not fully understood. Mast cells are involved in pain hypersensitivity (Ohashi et al., 2008); the mechanism remains unclear. We thus speculated that there might be a connection between mast cell activa- tion and the release of BDNF from the microglia. In the first attempt, we primed HMC-1 cells with corticotro- phin releasing hormone (CRH; 100 nMol. CRH has the capacity to activate mast cells) (Cao et al., 2005; Wallon et al., 2008) and cocultured the primed HMC-1 cells with microglial cells for 72 hr to see if activated mast cells were capable of eliciting the BDNF release from microglial cells. Unexpectedly, in a dose range of  10-8–10-6 Mol of CRH, HMC-1 cells did not induce the release of BDNF from microglial cells (Fig. 1A,B). Considering that mast cells might only promote the expression (but not the release) of BDNF from micro- glia, in a coculture system, after coculture with CRH- activated HMC-1 cells for 24, or 48, or 72 hr, microglial cells were stimulated by adenosine triphosphate (ATP, 50 lMol. Microglial cells can respond to ATP to release BDNF) (Ulmann et al., 2008; Trang et al., 2009). As expected, ATP-elicited release of BDNF from microglial cells was increased significantly in culture, which was featured as two release peaks at 5 min and 60 min respectively after the addition of ATP that was similar to previous reports (Trang et al., 2009) (Fig. 1C,D). The results were further confirmed by Western blotting that showed the marked increase in BDNF protein in cellular extracts (Fig. 1F). Using as a control, some microglial  cells were treated with CRH (100 nMol) alone for 72 hr. Those microglial cells did not show any increase in
BDNF release upon ATP stimulation (Fig. 1E-con).

     Mast Cell Activation Increases P2X4R Expression on Microglial Cells
      Since ATP-increased BDNF release from micro- glial cells is via activating the P2X4R (Trang et al.,
2009), we inferred that, as a part of the mechanism, mast cell-promoted BDNF release was via increasing the expression of P2X4R in microglial cells. To test the hy- pothesis, we cocultured HMC-1 cells and microglial cells in the presence of CRH for 24–72 hr to see if the expression of P2X4R could be enhanced in microglial cells. Quantitative real time RT-PCR (qRT-PCR) results showed that activation of HMC-1 cells did increase the gene transcription of P2X4R in microglial cells (Fig. 2A). Furthermore, P2X4R protein was also increased in microglial cells as shown by Western blot- ting (Fig. 2B).

Mast Cell-Derived Tryptase Plays a Role in Promoting the Expression of P2X4R in Microglial Cells
       Mast cells contain an array of chemical mediators that can be released to the micro milieu upon activation. To elucidate which of the mediators were involved in promoting the expression of P2X4R in microglial cells, we selectively blocked several mediators (Brandt, et al., 2003; Feng et al., 2008) of mast cells (Table I) that might play roles in promoting the expression of P2X4R. Thus, under the same experimental conditions as described in Figure 1, antagonists of histamine, or sero- tonin, or tryptase were added to the culture. The results showed that blocking histamine and serotonin had little effect on P2X4R expression in microglial cells whereas anti-tryptase treatment abolished the increase in P2X4R expression (Fig. 3). To confirm the role of tryptase in the promoting P2X4R expression, some HMC-1 cells were pretreated with anti-tryptase (Fig. 1E) or pre-trans- fected with small interference RNA (siRNA) of tryptase, and then subjected to the coculture with microglial cells. Indeed, the promotion of P2X4R expression in micro- glial cells was abrogated (Fig. 2).

Activation of PAR2 is Involved in the Expression of P2X4R in Microglial Cells
        Tryptase has the capability to cleave PAR2 to acti- vate PAR2-bearing cells (Jacob et al., 2005). Therefore, we reasoned that activated HMC-1 cells released tryptase (Albrecht et al., 2007); the latter cleaved PAR2 on microglial cells that further promoted the expression of P2X4R. To prove the hypothesis, we detected the expression of PAR2 on microglial cells. As shown by flow cytometry, PAR2 expression was identified on the surface of microglial cells (Fig. 4). To confirm the role of PAR2 in the promotion of P2X4R in microglial cells, a batch of microglial cells was treated with active PAR2 peptide or control peptide; the treatment with active peptide, but not the control peptide, markedly increased the expression of P2X4R in microglial cells. In a further attempt, microglial cells were transfected with siRNA of PAR2 or microglial cells were prepared from PAR2-/- mice and then cocultured with activated HMC-1 cells. As expected, the PAR2-/- microglial cells did not show increase in P2X4R expression; the scram- ble siRNA did not affect the expression of P2X4R in microglial cells (Fig. 2).

MAPK Mediates PAR2-Induced BDNF Release from Microglial Cells
        It is reported that MAPK is involved in BDNF release from microglial cells (Ulmann et al., 2008; Trang et al., 2009). We then wondered if MAPK played any roles in the processes of PAR2-promoted P2X4R expression. With the same procedures aforementioned, microglial cells were cocultured with CRH-activated HMC-1 cells for 24–72 hr. As shown by Western blot- ting, phosphorylated MAPK p38 was significantly increased in microglial cell extracts (Fig. 5). Further- more, treatment with MAPK inhibitor (SB203580; 10 lMol; 10003 DMSO), but not the negative control MAPK inhibitor (SB202474), abolished the elevation of P2X4R expression in microglial cells induced by cocul- turing with CRH-activated HMC-1 cells. In an alterna- tive approach, microglial cells were treated with active PAR2 peptide in the presence or absence of SB203580. As expected, active PAR2 peptide effectively increased the expression of P2X4R that was abolished by the treatment with SB203580 (Fig. 2). The results indicate that MAPK is involved in P2X4R expression induced by mast cell activation.

                              DISCUSSION

        The present paper reports a set of novel data that mast cell activation was involved in the expression of BDNF and P2X4R, key molecules in the pathogenesis of pain hypersensitivity, from microglial cells in vitro. The critical mediator, tryptase, of mast cells cleaved PAR2 on microglial cells. The activation of PAR2 increased the expression of BDNF and P2X4R on microglial cells, which maintained the capacity to release high levels of BDNF upon appropriate stimuli (Ulmann et al., 2008; Trang et al., 2009).

          It has been recognized that mast cell activation is associated with the induction of pain hypersensitivity (Ohashi et al., 2008). The mediators, such as histamine and tryptase, are implicated involving the induction of pain hypersensitivity in the body (Cenac et al., 2007; Medhurst et al., 2008). The data show that mast cell activation markedly increases the expression of BDNF in microglial cells, but do not directly induce the release of BDNF, the key mediator of pain hypersensitivity (Ulmann et al., 2008; Trang et al., 2009). The fact reveals an important phenomenon that mast cell activa- tion keeps microglial cells at a status of hypersensitivity  by synthesizing and storing a quantity of BDNF. Since BDNF is the key effector molecule in pain hypersensi- tivity, the deposit of BDNF in the cytoplasm confers microglial cells the capacity to release BDNF upon ex- posure to appropriate stimuli. The inference was proved by subsequent experimental results that after coculture with activated HMC-1 cells, microglial cells release a high volume of BDNF in response to exposure to ATP in culture media.

         It is known that stimulation of P2X4R on micro- glial cells promotes the release of BDNF (Tsuda et al., 2003, 2008; Ulmann et al., 2008). Therefore, to under- stand the regulatory mechanism in expression of P2X4R on microglial cells can be important in the elucidation of the mechanism of pain hypersensitivity. Mechanical injury of peripheral nerve can be one of the etiologies of up regulation of expression of P2X4R (Ulmann et al., 2008). Our results add novel information to the mechanism of expression of P2X4R; under an environment of mast cell activation, the promotion of expression of P2X4R was observed in cocultured microglial cells. Furthermore, the longer stimulation with activated mast  cells, the higher expression of P2X4R in cocultured microglial cells. The fact implicates that mastocytosis in the central nervous system may facilitate the expression of P2X4R in microglial cells.

         It is noted that mast cell-derived tryptase is associated with pain hypersensitivity (Barbara et al., 2007). Our data further support the notion by providing evi- dence that mast cell activation promoted BDNF expres- sion in the microglia that could be abolished by blocking tryptase. This is in line with previous reports that irrita- ble bowel syndrome-dependent excitation of dorsal root ganglia can be inhibited by serine protease inactivation (Cenac et al., 2007; Barbara et al., 2007). Difference was noted between our results and previous data that treat- ment with histamine receptor 1 antagonists could block the excitation of dorsal root ganglia (Barbara et al., 2007) whereas our data show that blocking histamine re- ceptor do not block the effect of mast cell activation induced release of BDNF from microglial cells. That is because we observed the expression of P2X4R was promoted by activation of PAR2, but not by histamine receptors.

         PAR2 is proposed playing a role in the pathophysiology of several disorders, such as irritable bowel syndrome (Cenac et al., 2007), immune diseases in central  nervous system (Noorbakhsh et al., 2006) and other inflammatory conditions (Bunnett, 2006). In line with  these previous studies, the present study adds fresh data to the study of PAR2. Activation of PAR2 in microglial cells promotes the expression of P2X4R; the latter is the so called ‘‘pain receptor’’; activation of P2X4R triggers the release of BDNF to elicit pain (Ulmann et al., 2008; Trang et al., 2009). This notion is supported by further observation that the more P2X4R is expressed in microglial cells, the more BDNF release can be resulted in upon proper stimuli as observed by the present study. Others also reported similar results (Trang et al., 2009).

       CRH is one of the major mediators in stress response (Aguilera et al., 2008). The philosophy that we choose CRH to activate mast cells is based on the fact that chronic stress induces CRH release (Aguilera et al., 2008) and CRH has the capacity to activate mast cells (Wallon et al., 2008). On the other hand, CRH has a close relation with the enhancement of viscerosensitivity (Tache´ et al., 2002,2005) under unknown mechanism. The present study lines up these events that enhancement of release of CRH may activate mast cells to release tryptase; the latter cleaves PAR2 on microglial cells to increase the expression of P2X4R; under proper stimulation, P2X4R is activated to release the BDNF. However, the critical physiopathological significance of the present finding needs to be further understood with
an in vivo animal model of pain hypersensitivity. It is also reported that microglial cells have CRH receptor 1 (Wang et al., 2002). It is conceivable that microglial cells might respond to CRH. Our data demonstrate that CRH does not induce BDNF release from microglial cells. However, the results do not rule out the possibility that CRH may cause other activities to occur in microglial cells, such as release of tumor necrosis factor-a (Wang et al., 2003), or other cytokines (Yang et al., 2005).

        In summary, the present study revealed that mast cell activation promoted the expression of BDNF and P2X4R in microglial cells. These microglial cells deposited a quantity of BDNF that could be released to the micro milieu upon appropriate stimuli.

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