Toll-like receptors 3 ligation directly and indirectly affects mast cell cysteinyl leukotriene generation

Introduction

Mast cells are long-lived resident tissue cells numerously distributed throughout the body. These cells are a significant source of diverse mediators such as granule-associated preformed mediators (e.g. histamine, proteases, proteoglycans, metalloproteinases), de novo generated and secreted arachidonic acid metabolites [i.e. leukotrienes (LTs), prostaglandins (PGs) and thromboxanes (TXs)] as well as cytokines and chemokines [1, 2]. These mediators elicit pro-inflammatory, anti-inflammatory and immunoregulatory effects and act directly on various cell populations. Therefore, mast cells are important players in homeostasis maintenance via involvement in angiogenesis, tissue remodeling and repair, and regulation of vascular permeability [2-4]. These cells are also known to participate in the inflammatory processes [4-6]. Moreover, mast cells also take part in different pathological processes [3, 7, 8], including FceRI-mediated allergic reactions [6, 8-10].

Mast cells are particularly abundant beneath subepithelial layers of the skin, in the airways, gastrointestinal and genitourinary tracts, thus close to the external environment, virtually at the portals of infection [1, 2]. Furthermore, these cells express pathogen recognition receptors (PRRs), including Toll-like receptors (TLRs) [11], that detect bacteria-associated specific molecules [12]. Therefore, mast cells are considered to be engaged in both innate and adaptive immune response against bacteria [13-15]. More recently the question arose as to whether mast cells have similar structures to detect virus-derived components. Among the TLR family members, TLR3, TLR7/8 and TLR9 are the receptors specific to virus-associated molecular patterns [11, 16]. Although previously these receptors have been reported in mast cells, the data are scarce and mainly refer to mRNA transcript expression in cultured cell lines [17-22]. Thus, it could be assumed that mast cells are capable of being activated by viral ligands, and thereby involved in antiviral immune response and/or development of virus-induced diseases. Toll-like receptor 3 is regarded as the predominant receptor of cellular response to viral infections, because it detects a frequent by-product of viral replication, namely double-stranded (ds)RNA [23]. Therefore, the aim of our study was to determine whether polyinosinic-polycytidylic acid (poly(I:C)), TLR3-specific synthetic analogue of viral dsRNA, can directly activate mature mast cells to degranulation as well as generation of cysteinyl LTs (cysLTs), the key mediator of inflammation [23, 24]. Due to mast cells’ essential role in allergic reactions [6, 8-10], we also examined the effect of TLR3 ligation on FceRI-mediated mast cell response.

Material and methods

Isolation of mast cells



Mast cells were collected from peritoneal cavities by lavage with 50 ml of 1% Hank’s Balanced Salt Solution (HBSS) supplemented with 0.015% NaHCO3 (Life Technologies, Gaithersburg, MD, USA). Next, the abdominal massage (90 s) was performed and the cell suspension was removed from the peritoneal cavity, centrifuged (150 g, 5 min, 20°C), and washed twice in complete Dulbecco’s

Modified Eagle Medium (cDMEM) containing DMEM supplemented with 10% fetal calf serum (FCS), 10 µg/ml gentamicin and 2 mM glutamine (150 g, 5 min, 20°C) (Life Technologies). In order to prepare purified mast cells, the isotonic 72.5% Percoll (Sigma-Aldrich Co. St. Louis, MO, USA) density gradient centrifugation (190 g, 15 min, 20°C) was used and the cells were washed twice in cDMEM by centrifugation (150 g, 5 min, 20°C). Subsequently, the cells were counted and resuspended in the appropriate volume of medium for rat mast cells containing 1 mM MgCl2, 2.7 mM KCL, 137 mM NaCl, 1 mM CaCl2, 10 mM N-2-hydroxyethylpiperazine-N’-ethanesulphonic acid (HEPES) buffer, 5.6 mM glucose and 1 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich) (the pH of the medium was adjusted to 6.9) to obtain mast cell concentration of 1.5 × 106 cells/ml. Mast cells were prepared with purity over 98%, as determined by metachromatic staining with toluidine blue (Sigma-Aldrich).



Western blot analysis



Mast cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl, pH 8.0, with 150 mM NaCl, 1% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) (InvivoGen, San Diego, CA, USA)] containing protease inhibitor cocktail [1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 800 nM aprotinin, 50 µM bestatin, 15 µM E64, 20 µM leupeptin, 10 µM pepstatin A, 5 mM ethylenediaminetetraacetic acid (EDTA)] (Thermo Scientific, Rockford, IL, USA) for 30 min on ice and detergent-insoluble residues were removed. The protein concentration in the lysates was determined by Bradford Assay (Bio-Rad Laboratories, Inc., United States). The cell lysates (50 µg) were separated on NuPAGE 10% Bis-Tris Gel (Life Technologies) before transfer to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The expression of TLR3 was assessed using anti-TLR3 antibodies (1 : 200 dilution), followed by application of horseradish peroxidase-conjugated bovine anti-goat IgG (1 : 10 000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The specific protein was visualized by the enhanced chemiluminescence (ECL) system using ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) according to manufacturer’s instructions.



Histamine release assay



The purified mast cells were suspended in the medium and divided into 90 µl aliquots. Next, 10 µl of poly(I:C) (InvivoGen, San Diego, CA, USA) at final concentrations of 0.1 µg/ml or 100 µg/ml, compound 48/80 (Sigma-Aldrich) at final concentration of 5 µg/ml (positive control) or medium alone (negative control) was added and incubation in water bath with constant stirring was performed for 1 h or 1.5 h at 37°C. Then, 1.9 ml of cold medium was added to stop the reaction. The cell suspensions were centrifuged (150 g, 5 min, 4°C) and the supernatants were decanted into separate tubes and acidified with 3 N HCl. Subsequently, distilled water at a total volume of 2 ml was added to each tube containing cell pellet. The histamine content was assessed in cell pellets (residual histamine) as well as supernatants (released histamine) by a spectrofluorometric method using o-phthaldialdehyde (OPT) (Sigma-Aldrich). Histamine release was expressed as a percentage of the total cellular content of the amine.

In another series of experiments mast cells were preincubated with poly(I:C) at final concentrations of 0.1 µg/ml or 100 µg/ml, or medium alone for 1 h at 37°C. After washing, mast cells were challenged with anti-IgE (Serotec, Oxford, UK) at final concentration of 5 µg/ml or medium alone (negative control) for 30 min at 37°C. The reaction was stopped with the cold medium and histamine content was assessed, as described above.



CysLT release assay



The purified mast cells suspended in the medium were incubated with poly(I:C) at final concentrations of 0.1 µg/ml or 100 µg/ml, calcium ionophore A23187 (Sigma-Aldrich) at final concentration of 5 µg/ml (positive control) or medium alone (negative control) for 2 h or 3 h at 37°C. The supernatants were collected by centrifugation (150 g, 5 min, 20°C) and analyzed by an ELISA commercial kit (Cayman Chemical, Ann Arbor, MI, USA) detecting LTC4 and its degradation products LTD4 and LTE4. The sensitivity of this assay was < 13 pg/ml.

To determine the specificity of poly(I:C) effect on cysLT generation in some experiments mast cells were preincubated with goat polyclonal anti-TLR3 IgG antibodies or goat IgG isotype control (Santa Cruz Biotechnology) at final concentration of 40 µg/ml or medium alone (negative control) for 1 h at 37°C. Next, the cells were rinsed twice by centrifugation at 150 γ for 5 min and resuspended in the medium before poly(I:C) stimulation, as described.

In separate experiments, mast cells were incubated with anti-IgE at final concentration of 5 µg/ml, poly(I:C) at final concentrations of 0.1 µg/ml or 100 µg/ml, both poly(I:C) and anti-IgE, or medium alone (negative control) for 2 h at 37°C. The supernatants were collected by centrifugation and analysed for cysLTs, as described.



Statistical analysis



Statistical analysis included mean value, standard error of the mean (SEM) and Student’s t-test for “small groups”. Values of P < 0.05 were considered statistically significant.

Results

Mast cell TLR3 expression



To our knowledge, TLR3 protein has not been yet determined in mature tissue rat mast cells. Therefore, we examined these cells for TLR3 molecule expression. The western blot analysis revealed distinct 120 kDa band corresponding to TLR3 protein (Fig. 1). Thereby, we demonstrated that freshly isolated rat peritoneal mast cells constitutively express TLR3 protein.



Direct effect of poly(I:C) on mast cell histamine release and cysLT synthesis



First, the effect of TLR3 ligand, i.e. poly(I:C), on mast cells degranulation and preformed mediator release was investigated. We observed that poly(I:C), used at two different concentrations, did not induce mast cell degranulation and histamine secretion (Fig. 2A). In comparison, upon stimulation with compound 48/80, a potent trigger of mast cell degranulation, histamine release was up to 60.1 ±2.1% (mean ± SEM) after 1.5 h of incubation.

We found, however, that poly(I:C) activated rat mast cells to cysLT generation and release (Fig. 2B). Poly(I:C) at concentration of 0.1 µg/ml stimulated mast cells to release up to 150.6 ±18.5 and up to 344.2 ±35.4 pg cysLT/1.5 × 106 mast cells after 2 h and 3 h of incubation, respectively. CysLT release from mast cells in response to activation with poly(I:C) used at 100 µg/ml was 202.0 ±19.7 pg/1.5 × 106 mast cells (time of stimulation – 2 h) and 392.5 ±27.7 pg/1.5 × 106 mast cells (time of stimulation – 3 h). In comparison, when challenged with calcium ionophore A23187 under the same experimental conditions, mast cells released up to 921.1 ±20.1 pg/1.5 × 106 mast cells (2 h-incubation) and 949.8 ±17.0 pg/1.5 × 106 mast cells (3 h-incubation) of cysLTs.

To determine the specificity of poly(I:C) action on mast cell cysLT generation and release, blocking experiments using anti-TLR3 antibodies were performed. We noticed that pretreatment of mast cells with these antibodies almost completely inhibited cysLT synthesis in response to poly(I:C) exposure. The isotype control antibodies did not affect poly(I:C)-induced cysLT generation by mast cells (Fig. 2B).



Effect of poly(I:C) on mast cell anti-IgE-mediated histamine release and cysLT synthesis



Next, the influence of poly(I:C) on mast cell anti-IgE-mediated degranulation and preformed mediator release was evaluated. As shown in Fig. 3A, pretreatment of mast cells with poly(I:C), used at concentrations of 0.1 µg/ml and

100 µg/ml for 1 h, did not affect histamine release induced by anti-IgE. On the contrary, we found that co-stimulation of mast cells with TLR3 ligand at concentration of 100 µg/ml and anti-IgE for 2 h resulted in a significant amplification of cysLT secretion (473.5 ±18.4 pg/1.5 × 106 mast cells, compared to 265.2 ±11.9 pg/1.5 × 106 mast cells for TLR3 ligand untreated cells) (Fig. 3B). However, simultaneous treatment of mast cells with poly(I:C) at concentration of 0.1 µg/ml and anti-IgE had no significant co-stimulatory effect on cysLT synthesis.

Discussion

So far, TLR3 mRNA has been found in human and murine immature mast cells [17, 19, 21, 26] as well as in mature human mast cells isolated from skin and lungs [27] and mouse mast cells obtained from fetal skin (FSMCs) [18]. Toll-like receptor 3 protein has been shown to be present in human and murine mast cell lines [17, 19, 21]. To our knowledge, only Kulka et al. [17] documented TLR3 at the protein level in mature native murine mast cells. In this study, we proved that mature connective tissue mast cells from rat peritoneal cavity constitutively express TLR3 protein. All these data indicate that mast cells could sense and respond to dsRNA viruses.

We also found that poly(I:C), a synthetic mimic of viral dsRNA, does not induce rat mature mast cells to degranulation and preformed mediator release. However, mast cells released cysLTs upon exposure to TLR3 ligand, though the amount of secreted mediators was relatively small. Nevertheless, it should be underlined that cysLTs are extremely potent proinflammatory mediators which produce their effects even at nanomolar concentrations [24, 25, 28]. Earlier studies showed that poly(I:C) does not activate immature bone marrow-derived mast cells (BMMCs) as well as mature FSMCs, human cutaneous and pulmonary mast cells and murine peritoneal mast cells to degranulation, as assessed by levels of b-hexosaminidase release [18, 19, 27]. Saluja et al. [26] observed no degranulation and cysLT generation by mouse in vitro-differentiated connective tissue-like mast cells (CTLMCs) and mucosal-like mast cells (MLMCs) in response to poly(I:C) stimulation. Burke et al. [29] stated that poly(I:C)-activated human cord blood-derived mast cells (CBMCs) do not produce cysLTs. To date, only Kulka et al. [17] demonstrated selective, i.e. without degranulation, poly(I:C)-induced cysLT production by human cultured mast cells (HCMCs).

There are some observations that murine and human mast cells are capable of responding to TLR3 stimulation by producing type I interferons (IFNs) [17], key factors for the induction of antiviral defense. What is more, poly(I:C)-activated mast cells produce and secrete proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin (IL)-6 [18, 21]. Furthermore, mast cell activation via TLR3 ligation results in selective synthesis and release of several chemokines, including CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, CXCL8, and CXCL10 [18, 19, 21, 26, 29]. Mast cell-derived proinflammatory cytokines as well as chemokines strongly influence the development of antiviral immune response and act as NK and cytotoxic CD8+ T cell chemoattractants. It might be suggested that cysLTs generated by mast cells in response to TLR3 ligation intensify inflammatory processes by enhancing vascular permeability thereby affecting cellular migration during virus infection.

It is well documented that viral infections induce exacerbations of bronchial asthma [30-32], but the underlying mechanisms remain unclear. Thus, we studied the influence of TLR3 ligation on FceRI-mediated mature rat mast cell degranulation and cysLT generation. We documented that mast cell priming with TLR3 ligand for 1 h had no effect on anti-IgE-induced degranulation. However, mast cell co-stimulation with poly(I:C), but only at the higher concentration used, and anti-IgE for 2 h caused significant augmentation in cysLT generation. It might be speculated that high concentrations of poly(I:C) may up-regulate FceRI expression and thereby increase IgE-dependent cysLT release. Previously, it was observed that poly(I:C) pretreatment for 5-12 h had no effect on IgE-mediated degranulation of HCMCs and LAD cells [17] and priming with TLR3 ligand, up to 24 h, did not affect FceRI-dependent b-hexosaminidase release as well as cysLT and LTB4 generation by CTLMCs and MLMCs derived from cultured mouse bone marrow cells [26]. However, the prolonged, up to 96 h, exposure to TLR3 ligand caused a significant increase in CTLMs and MLMCs degranulation and induced an amplified secretion of cysLTs from CTLMCs [26]. The observations that mast cell activation by dsRNA can induce augmentation of cysLT generation in response to FceRI-mediated stimulation seem to be of great importance. It is a commonly held belief that cysLTs play an important role in the pathogenesis of acute and chronic asthma. These mediators display a variety of disorder-provoking physiological effects such as increased microvascular permeability leading to pulmonary edema, impaired ciliary activity responsible for enhanced mucus secretion and decreased respiratory self-clearance, as well as respiratory tract smooth muscle cell hyperplasia contributing to airway remodeling [33]. CysLTs are also known to be essential in the course of allergic rhinitis [34].

In conclusion, we clearly demonstrated that native mature tissue mast cells constitutively express TLR3 molecule and can be activated via poly(I:C), a synthetic dsRNA construct, to cysLT generation and release. These observations might suggest that mast cells can be directly activated to proinflammatory mediator release during dsRNA virus infections. Furthermore, data showing that priming/co-stimulation with TLR3 ligand may affect FceRI-dependent mast cell activation indicate that dsRNA virus infections could influence allergic reaction severity. However, to clarify the impact of TLR3 ligation on mast cell reactivity, further studies are necessary.



The authors declare no conflict of interests.



This work was supported by the National Science Centre in Poland (Grant No. 2011/03/N/NZ6/03528) and by the Medical University of Łódź (Grant No. 502-03/6-164-01/502-64-005).

References

 1. Gilfillan AM, Austin SJ, Metcalfe DD (2001): Mast cell biology: introduction and overview. Adv Exp Med Biol 716: 2-12.

 2. Rao KN, Brown MA (2008): Mast cells: multifaceted immune cells with diverse roles in health and disease. Ann N Y Acad Sci 1143: 83-104.

 3. Heib V, Becker M, Taube C, Stassen M (2008): Advances in the understanding of mast cell function. Br J Haematol 142: 683-694.

 4. Galli SJ, Tsai M (2008): Mast cells: versatile regulators of inflammation, tissue remodeling, host defense and homeostasis. J Dermatol Sci 49: 7-19.

 5. Crivellato E, Ribatti D (2005): Involvement of mast cells in angiogenesis and chronic inflammation. Curr Drug Targets Inflamm Allergy 4: 9-11.

 6. Theoharides TC, Kalogeromitros D (2006): The critical role of mast cells in allergy and inflammation. Ann N Y Acad Sci 1088: 78-99.

 7. Weller CL, Collington SJ, Williams T, Lamb JR (2011): Mast cells in health and disease. Clin Sci 120: 473-484.

 8. Anand P, Singh B, Jaggi AS, Singh N (2012): Mast cells: an expanding pathophysiological role from allergy to other disorders. Naunyn Schmiedebergs Arch Pharmacol 385: 657-670.

 9. Brown JM, Wilson TM, Metcalfe DD (2008): The mast cell and allergic diseases: role in pathogenesis and implications for therapy. Clin Exp Allergy 1: 4-18.

10. Stelekati E, Orinska Z, Bulfone-Paus S (2007): Mast cells in allergy: innate instructors of adaptive responses. Immunobiol 6: 505-519.

11. Brzezińska-Błaszczyk E, Wierzbicki M (2010): Mast cell Toll-like receptors (TLRs). Postepy Hig Med Dosw 64: 11-21.

12. Takeda K, Akira S (2004): Microbial recognition by Toll-like receptors. J Dermatol Sci 34: 73-82.

13. Marshall JS (2004): Mast-cell responses to pathogens. Nat Rev Immunol 4: 787-799.

14. Abraham SN, St John AL (2010): Mast cell-orchestrated immunity to pathogens. Nat Rev Immunol 10: 440-452.

15. Tete S, Tripodi D, Rosati M, et al. (2012): Role of mast cells in innate and adaptive immunity. J Biol Regul Homeost Agents 26: 193-201.

16. Blasius AL, Beutler B (2010): Intracellular toll-like receptors. Immunity 32: 305-315.

17. Kulka M, Alexopoulou L, Flavell RA, Metcalfe DD (2004): Activation of mast cells by double-stranded RNA: evidence for activation through Toll-like receptor 3. J Allergy Clin Immunol 114: 174-182.

18. Matsushima H, Yamada N, Matsue H, Shimada S (2004): TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. J Immunol 173: 531-541.

19. Orinska Z, Bulanova E, Budagian V, et al. (2005): TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment. Blood 106: 978-987.

20. Sundstrom JB, Little DM, Villinger F, et al. (2004): Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells. J Immunol 172: 4391-4401.

21. Yang H, Wei J, Zhang H, et al. (2009): Upregulation of Toll-like receptor (TLR) expression and release of cytokines from P815 mast cells by GM-CSF. BMC Cell Biol 10: 37.

22. Yoshioka M, Fukuishi N, Iriguchi S, et al. (2007): Lipoteichoic acid downregulates Fc?RI expression on human mast cells through Toll-like receptor 2. J Allergy Clin Immunol 120: 452-461.

23. Sen GC, Sarkar SN (2005): Transcriptional signaling by double-stranded RNA: role of TLR3. Cytokine Growth Factor Rev 16: 1-14.

24. Singh RK, Gupta S, Dastidar S, Ray A (2010): Cysteinyl leukotrienes and their receptors: molecular and functional characteristics. Pharmacology 85: 336-349.

25. Peters-Golden M, Henderson WR Jr. (2007): Leukotrienes. N Engl J Med 357: 1841-1854.

26. Saluja R, Delin I, Nilsson GP, Adner M (2012): FceRI-mediated mast cell reactivity is amplified through prolonged Toll-like receptor-ligand treatment. PLoS One 7: e43547.

27. Kulka M, Metcalfe DD (2006): TLR3 activation inhibits human mast cell attachment to fibronectin and vitronectin. Mol Immunol 43: 1579-1586.

28. Arm JP (2004): Leukotriene generation and clinical implications. Allergy Asthma Proc 25: 37-42.

29. Burke SM, Issekutz TB, Mohan K, et al. (2008): Human mast cell activation with virus-associated stimuli leads to the selective chemotaxis of natural killer cells by a CXCL8-dependent mechanism. Blood 111: 5467-5476.

30. Busse WW, Lemanske RF Jr, Gern JE (2010): Role of viral respiratory infections in asthma and asthma exacerbations. Lancet 376: 826-834.

31. Dulek DE, Peebles RS Jr. (2011): Viruses and asthma. Biochim Biophys Acta 11: 1080-1090.

32. Kloepfer KM, Gern JE (2010): Virus/allergen interactions and exacerbations of asthma. Immunol Allergy Clin North Am 30: 553-563.

33. Holgate ST, Peters-Golden M, Panettieri RA, Henderson WR Jr. (2003): Roles of cysteinyl leukotrienes in airway inflammation, smooth muscle function, and remodeling. J Allergy Clin Immunol 111: S18-34.

34. Peters-Golden M, Henderson WR Jr. (2005): The role of leukotrienes in allergic rhinitis. Ann Allergy Asthma Immunol 94: 609-618.