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Coagulation factors induce human skin mast cell and basophil degranulation via activation of complement 5 and the C5a receptor

2020; Elsevier BV; Volume: 147; Issue: 3 Linguagem: Inglês

10.1016/j.jaci.2020.08.018

1097-6825

Yuhki Yanase, Yoshimi Matsuo, Shunsuke Takahagi, Tomoko Kawaguchi, Kazue Uchida, Kaori Ishii, Akio Tanaka, Daiki Matsubara, Koichiro Ozawa, Michihiro Hide,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Chronic spontaneous urticaria (CSU), also called chronic idiopathic urticaria, is a common skin disorder characterized by daily or almost daily recurring wheal and flare, with itch occurring anywhere on the body for more than 6 weeks in association with degranulation of skin mast cells and basophils.1Min T.K. Saini S.S. Emerging therapies in chronic spontaneous urticaria.Allergy Asthma Immunol Res. 2019; 11: 470-481Crossref PubMed Scopus (7) Google Scholar,2Kolkhir P. Altrichter S. Munoz M. Hawro T. Maurer M. New treatments for chronic urticaria.Ann Allergy Asthma Immunol. 2020; 124: 2-12Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar The involvement of both exogenous factors (such as infections and psychological stress) and endogenous factors has been suggested. The latter includes the presence of autoantibodies against IgE or the high-affinity IgE receptor and autoantigens for IgE, such as thyroid peroxidase, double-stranded DNA, and IL-24 in sera of the patients.3Bansal C.J. Bansal A.S. Stress, pseudoallergens, autoimmunity, infection and inflammation in chronic spontaneous urticaria.Allergy Asthma Clin Immunol. 2019; 15: 56Crossref PubMed Scopus (15) Google Scholar,4Maurer M. Altrichter S. Schmetzer O. Scheffel J. Church M.K. Metz M. Immunoglobulin E-mediated autoimmunity.Front Immunol. 2018; 9: 689Crossref PubMed Scopus (52) Google Scholar Moreover, the increase in blood-coagulating and fibrinolytic activities has been shown in patients with moderate-to-severe CSU.5Takahagi S. Mihara S. Iwamoto K. Morioke S. Okabe T. Kameyoshi Y. et al.Coagulation/fibrinolysis and inflammation markers are associated with disease activity in patients with chronic urticaria.Allergy. 2010; 65: 649-656Crossref PubMed Scopus (104) Google Scholar,6Yanase Y. Takahagi S. Hide M. Chronic spontaneous urticaria and the extrinsic coagulation system.Allergol Int. 2018; 67: 191-194Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar However, the precise mechanisms of mast cell and basophil activation in CSU has remained unclear. We previously demonstrated that human umbilical vein endothelial cells (HUVECs) synergistically expressed tissue factor (TF) on their surface subsequent to treatment with histamine and LPS in vitro.7Yanase Y. Morioke S. Iwamoto K. Takahagi S. Uchida K. Kawaguchi T. et al.Histamine and TLR ligands synergistically induce endothelial-cell gap-formation by the extrinsic coagulating pathway.J Allergy Clin Immunol. 2018; 141: 1115-1118Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar We also showed that TF expression of peripheral monocytes in patients with CSU is significantly enhanced compared with that in healthy donors.8Saito R. Yanase Y. Kamegashira A. Takahagi S. Tanaka A. Uchida K. et al.Increase of tissue factor expression on the surface of peripheral monocytes of patients with chronic spontaneous urticaria.Allergy. 2020; 75: 971-974Crossref PubMed Scopus (3) Google Scholar Both TF expressed on HUVECs and human peripheral monocytes activated the extrinsic coagulation pathway and produced active forms of coagulation factors with serine protease activity, such as factor Xa and thrombin (IIa), resulting in the gap formation between HUVECs via the protease-activated receptor (PAR) PAR1.7Yanase Y. Morioke S. Iwamoto K. Takahagi S. Uchida K. Kawaguchi T. et al.Histamine and TLR ligands synergistically induce endothelial-cell gap-formation by the extrinsic coagulating pathway.J Allergy Clin Immunol. 2018; 141: 1115-1118Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Leakage of plasma containing the activated coagulation factors with serine protease activity is predicted to activate skin mast cells and basophils expressing PARs, and consequentially develop clinical edema formation. However, actual degranulation of human mast cells and basophils by activated coagulation factors has never been reported. Here, we have demonstrated that the activated extrinsic coagulation factors do not activate human skin mast cells (hsMCs) and basophils by themselves but induce degranulation of hsMCs and basophils via the production of complement C5a acting on the C5a receptor (C5aR). We first studied the effect of PAR1 and PAR2 agonists and various activated factors produced in the extrinsic coagulation pathway and consequent fibrinolytic pathways. As expected, hsMCs expressed mRNA of PAR1 and marginally expressed PAR2 (see Fig E1 in this article's Online Repository at www.jacionline.org). However, neither TRAP-6 (a PAR1 agonist) nor AC55541 (a PAR2 agonist) nor the activated extrinsic coagulation factors VIIa, Xa, and IIa nor the fibrinolytic factor plasmin induced degranulation of hsMCs by themselves (Fig 1, A). We then studied degranulation of hsMCs in response to the complement activation products C3a and C5a. As shown in Fig 1, B, C5a, but not C3a, induced degranulation of hsMCs. Because serine proteases, such as coagulation and/or fibrinolysis factors, may convert C5 into C5a and C5b, we investigated the effect of C5 in the presence of factor VIIa, factor Xa, factor IIa, or plasmin on the degranulation of hsMCs. Although neither C5 nor factor VIIa nor factor Xa nor factor IIa induced degranulation of hsMCs by themselves, C5 did induce degranulation of hsMCs in the presence of factor Xa, factor IIa, or plasmin, suggesting that C5a converted by factor Xa, factor IIa, or plasmin from C5 induces degranulation of hsMCs (Fig 1, C, D). C5a cleaved from C5 by factor Xa and factor IIa induced histamine release from hsMCs as well (see Fig E2 in this article's Online Repository at www.jacionline.org). However, C3 induced a slight degranulation of hsMCs even in the presence of factor Xa and factor IIa (Fig 1, C). Moreover, nafamostat mesylate, a serine protease inhibitor, reduced C5 plus IIa– and C5 plus plasmin–induced degranulation of hsMCs in a concentration-dependent manner. This suggests that serine protease activity of activated coagulation and/or fibrinolysis factors is essential and sufficient for the conversion of C5 to C5a and consequent activation of hsMCs (Fig 1, E). Furthermore, C5a-, C5 plus IIa–, and C5 plus plasmin–induced degranulation of hsMCs were clearly inhibited by pretreatment for 15 minutes with a C5a receptor (C5aR) antagonist, W-54011, in a concentration-dependent manner (Fig 1, F). These results suggest that activated coagulation factors cleave C5 into C5a, which then induces degranulation of hsMCs via C5aR on the surface of mast cells. Human basophils also express C3aR, C5aR, and a small level of PAR1 and PAR2 at mRNA levels (see Fig E3 in this article's Online Repository at www.jacionline.org). We then investigated the effect of C3a and C5a on histamine release from human peripheral basophils in PBMCs. As shown in Fig 2, A, C5a, but not C3a, induced histamine release from human peripheral basophils. Moreover, neither C5 nor the coagulation factors Xa or IIa induced histamine release by themselves, but the combination of C5 and factor IIa or factor Xa did (Fig 2, B). These results suggest that C5a converted by the activated coagulation factors, but not C5 or coagulation factors by themselves, induce basophil histamine release. We further investigated whether TF-triggered activation of the coagulation cascade induces histamine release from human peripheral basophils via the production of C5a. In the extrinsic coagulation cascade, the complex of TF and factor VIIa converts factor X to factor Xa, after which then Xa converts factor II to factor IIa (see Fig E4 in this article's Online Repository at www.jacionline.org). As shown in Fig 2, C, the mixture of TF, factor VIIa, factor X, factor II, and C5—but not TF, activated coagulation factors, or C5 alone—induced histamine release from basophils. Moreover, the TF-induced activation of basophils was clearly blocked by a C5aR antagonist in a concentration-dependent manner (Fig 2, D). The C5aR antagonist did not inhibit anti-IgE–induced basophil activation (see Fig E5 in this article's Online Repository at www.jacionline.org). These results suggest that TF triggered the activation of the extrinsic coagulation cascade producing the activated coagulation factors, Xa and IIa, which convert C5 to C5a, and finally activates basophils via C5aR. To date, several articles have implied that activated coagulation factors, such as Xa and IIa, directly activate hsMCs via PAR1 or PAR2 in CSU.9Moormann C. Artuc M. Pohl E. Varga G. Buddenkotte J. Vergnolle N. et al.Functional characterization and expression analysis of the proteinase-activated receptor-2 in human cutaneous mast cells.J Invest Dermatol. 2006; 126: 746-755Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar Although we could not rule out the involvement of PARs for mast cell and basophil activation on account of the experiments having a small number of subjects, we demonstrated that C5a resulting from the extrinsic coagulation system induces degranulation of hsMCs and basophils via C5aR. Thus, we established an alternative hypothesis of mast cell and/or basophil activation through the coagulation pathway and complement (see Fig E6 in this article's Online Repository at www.jacionline.org). Further studies of the role of C5a in CSU pathogenesis would help clarify the mechanism of CSU in more detail. The combination of C5aR antagonists with conventional medications for CSU, such as H1 antihistamines, could be more effective in treating patients with refractory CSU than current approaches are. We wish to thank Dr Faiz Kermani for his manuscript review. Recombinant human TF, stem cell factor, C5a, and C3a were purchased from R&D Systems Inc (Minneapolis, Minn). X-vivo-15 medium was from Lonza (Walkersville, Md). The RNeasy Mini Kit and the Quanti Tect Reverse Transcription Kit were from Qiagen (Valencia, Calif). Reverse-phase HPLC was performed with equipment from Shimadzu (Kyoto, Japan). C5 and C3 from human serum, plasmin from human plasma and BSA were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Factors VIIa, X, II, and IIa were from Haematologic Technologies Inc (Essex, Vt). Factor Xa was from Abcam (Cambridge, United Kingdom). Plasmin from human plasma, TRAP-6, and AC55541 were from TOCRIS (Bristol, United Kingdom). Nafamostat mesylate was from Tokyo Chemical Industry Co, Ltd (Tokyo, Japan). W-54011 was from Cayman Chemical Company (Ann Arbor, Mich). Substance P was from the Peptide Institute, Inc (Osaka, Japan). The EasySep Human Basophil Enrichment Kit was from Stem Cell Technologies, Inc (British Columbia, Canada). hsMCs were isolated from the skin of patients with neurofibromatosis type I without CSU, as previously described.E1Matsuo Y. Yanase Y. et al.Neuromedin U directly induces degranulation of skin mast cells, presumably via MRGPRX2.Allergy. 2018; 73: 2256-2260Crossref PubMed Scopus (7) Google Scholar Briefly, skin samples were cut into fragments and incubated in a solution of HBSS containing type 2 collagenase (1.5 mg/mL), hyaluronidase (0.7 mg/mL), type I DNase (0.3 mg/mL), 1% FCS, and 1 mM CaCl2 for 2 hours at 37°C. The dispersed cells were separated from residual tissues by filtration through a 70-mesh sieve and suspended in X-vivo-15 media. The isolated mast cells were cultured in X-vivo-15 media with 100 ng/mL of recombinant human stem cell factor for 1 to 2 months.E1Matsuo Y. Yanase Y. et al.Neuromedin U directly induces degranulation of skin mast cells, presumably via MRGPRX2.Allergy. 2018; 73: 2256-2260Crossref PubMed Scopus (7) Google Scholar Human PBMCs were isolated from fresh blood of healthy donors without CSU by using Ficoll-Paque Plus density gradient separation. Basophils were isolated from PBMCs by magnetic depletion of nonbasophils by using the EasySep Human Basophil Enrichment Kit. Total RNA was extracted from mast cells, PBMCs, and basophils by using the RNeasy Mini kit (Qiagen, Venlo, The Netherlands), and cDNA was generated by using the QuantiTect Reverse transcription kit (Qiagen). Levels of mRNA expression of TF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were evaluated by using the ABI 7300 Real-Time PCR System (Applied Biosystems, Carlsbad, Calif) with Power SYBR Green PCR Master Mix (Applied Biosystems). The specific primer pairs were as follows: forward primer for GAPDH, 5′-GAAGGTGAAGGTCGGAGTCA-3′; reverse primer for GAPDH, 5′-GAAGATGGTGATGGGATTTCC-3′, forward primer for PAR1, 5-GTATCCCATGCAGTCCCTCTCC-3′; reverse primer for PAR1, 5′- GTAATGCGCAATCAGGAGGACG-3′, forward primer for PAR2, 5-TCTGCATCTGTCCTCACTGG-3′; and reverse primer for PAR2, 5′-TGAAGATGGTCTGCTTCACG-3′. The expression of GAPDH was measured as an internal control to calibrate gene expression. Levels of mRNA expression of C3aR and C5aR were evaluated by using ABI 7300 Real-time PCR System by Taqman gene expression assay (Hs 00269693_s1 for C3aR, Hs 00704891_s1 for C5aR, Hs02758991_g1 for GAPDH). Degranulation of hsMCs was evaluated by measuring the release of β-hexosaminidase, a granule marker that hydrolyzes p-nitropheny-N-acetyl-β-glucosamine to the chromophore p-nitorophenol, as descrived previously.E1Matsuo Y. Yanase Y. et al.Neuromedin U directly induces degranulation of skin mast cells, presumably via MRGPRX2.Allergy. 2018; 73: 2256-2260Crossref PubMed Scopus (7) Google Scholar Briefly, hsMCs were resuspended in piperazine-N,N'-bis(2-ethanesulfonic acid) buffer containing 119 mM NaCl, 5 mM KCl, 1.0 mM CaCl2, 0.4 mM MgCl2, 5.6 mM glucose, 25 mM piperazine-N,N'-bis(2-ethanesulfonic acid), and 1 mg/mL of BSA ( pH 7.2). The cells were allowed to settle onto a 96-well plate. hsMCs were pretreated with or without W-54011 or nafamostat mesylate and then stimulated with indicated concentrations of stimuli for 15 minutes at 37°C. Histamine release tests with human PBMCs or hsMCs were performed as described previously with a goat anti-IgE antibody as a positive control.E1Matsuo Y. Yanase Y. et al.Neuromedin U directly induces degranulation of skin mast cells, presumably via MRGPRX2.Allergy. 2018; 73: 2256-2260Crossref PubMed Scopus (7) Google Scholar Histamine was extracted and measured by reverse-phase HPLC. The activity of the TF-triggered extrinsic coagulation cascade was evaluated by using Spectrozyme FXa substrate. We first added 50 μL of assay buffer to each well of a 96-well plate. We then added 75 μL of assay buffer containing factors VIIa and X with or without human recombinant TF and incubated the plates for 30 minutes at 37°C. After incubation, 25 μL of Spectrozyme FXa substrate was added and incubated at 37°C for 30 minutes. We finally measured the absorbance of each well at 405 nm by using a microplate reader. The difference between each group was tested by using 1-way ANOVA followed by the Tukey test performed with GraphPad PRISM software, version 6 (GraphPad Software, San Diego, Calif).