Human Cord Blood-Derived Mast Cells Are Activated by the Nod1 Agonist M-TriDAP to Release Pro-Inflammatory Cytokines and Chemokines

Mast cells are among the first cells of our immune system to encounter exogenous danger. Intracellular receptors such as nucleotide-binding oligomerization domain (Nod) play an important role in responding to invading pathogens. Here, we have investigated the response of human mast cells to the Nod1 ligand M-TriDAP. Human cord blood-derived mast cells (CBMCs) were activated with M-TriDAP alone, or in combination with the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and zymosan. Release of pro-inflammatory chemokines and cytokines was measured by ELISA, cytometric bead array and LUMINEX, and degranulation was evaluated by analysis of histamine release. M-TriDAP induced a dose-dependent release of IL-8, MIP-1 , MIP-1 and TNF. In contrast, degranulation could not be observed. When cells were treated with M-TriDAP in combination with the TLR4 agonist LPS, but not with TLR2 agonist zymosan, the secretion of cytokines was augmented. We here present results demonstrating that human CBMCs are stimulated by the Received: April 29, 2010 Accepted after revision: October 12, 2010 Published online: November 24, 2010 Journal of Innate Immunity Dr. Carolina Lunderius Andersson Department of Medicine, Clinical Immunology and Allergy Unit Karolinska Institutet, KS L2:04 SE–171 76 Stockholm (Sweden) Tel. +46 8 517 766 97, Fax +46 8 33 57 24, E-Mail carolina.lunderius @ ki.se © 2010 S. Karger AG, Basel 1662–811X/11/0032–0142$38.00/0 Accessible online at: www.karger.com/jin Activation of Human Mast Cells by the Nod1 Agonist M-TriDAP J Innate Immun 2011;3:142–149 143 and best illustrations of the role of mast cells in the defence against bacterial infections was demonstrated using mast cell-deficient mice [6, 7] . These mice are not protected against pathogens unless they are reconstituted with mast cells. A combination of different mechanisms are used by mast cells to sense and respond to pathogens [8] , either directly through receptors such as Toll-like receptors (TLRs) [9] , dectin-1 [10] and the mannose receptor CD48 [11] or indirectly, for instance through cross-linking of Fc receptors by superantigens [12] . These events initiate signalling pathways that lead to activation of nuclear factor B (NF B), which in turn activate pro-inflammatory genes, including genes for cytokines and chemokines. In recent years, a number of pattern-recognition receptors (PRRs) of the host cells have been described as binding different pathogen-associated molecular patterns (PAMPs) [13, 14] . TLRs represent prominent examples of PRRs. A selection of PAMPs includes bacterial components such as lipopolysaccharide (LPS), nucleic acids, zymosan and peptidoglycan (PGN); thus, PAMPs are composed of a great variety of structures [15] . The effectiveness of the innate sensing network relies on the numerous receptors, their unique specificities, and how they cooperate in the recognition of different structures of pathogens. In addition to cell surface receptors, intracellular PRRs such as the nucleotide-binding oligomerization domain proteins Nod1 and Nod2, are also important players in responding to pathogens [16] . Nod proteins sense inflammatory stimuli through their leucine-rich repeat domains [17] . As Nod proteins are intracellular, they are primarily thought to sense intracellular bacteria. However, recent studies have shown that the Nod ligands may also be translocated into the cytoplasm by various mechanisms [18, 19] . Whereas Nod1 recognizes peptidoglycans from mainly Gram-negative bacteria, such as Helicobacter pylori, Nod2 can sense both Gram-negative and -positive bacteria through motifs such as muramyl dipeptide (MDP) found in all bacterial peptidoglycans [20] . GM-TriDAP (GlcNAc-MurNAcL -AlaD -Glumeso-DAP) is a naturally occurring peptidoglycan degradation product specifically recognized by Nod1 [20] . The aim of the present study was to investigate the response of human mast cells to M-TriDAP, a diaminopimelate-containing MurNAc tripeptide, which is specifically recognized by Nod1 [20–22] . Here, human cord blood-derived mast cells (CBMCs) were activated with M-TriDAP alone or in combination with the TLR ligands LPS and zymosan, and the release of pro-inflammatory mediators was measured. Our results demonstrate that activation of the intracellular Nod1 protein leads to a degranulation-independent secretion of cytokines, thus providing further evidence for the role of mast cells as important contributors to host defence. Materials and Methods Reagents The calcium ionophore A23187, LPS from Escherichia coli O55:B5, zymosan A from Saccharomyces cerevisiae and SP600125 were all purchased from Sigma Aldrich, St. Louis, Mo., USA. Ultra-pure M-TriDAP was synthesized as previously described [20] , and kindly provided by Drs. Mireille Hervé and Dominique Mengin-Lecreulx, Université Paris-Sud, France. Cell signalling inhibitors SB203580, LY294002, PD98059 and wortmannin were from Calbiochem, Merck KgaA, Darmstadt, Germany. Mast Cell Cultures CBMCs were obtained as previously described [23, 24] . Briefly, CD34+ cells were purified with CD34 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) from umbilical cord blood and maintained in complete StemPro medium (Invitrogen, Carlsbad, Calif., USA) supplemented with 100 ng/ml SCF, 10 ng/ml IL-6 (Amgen, Thousand Oaks, Calif., USA), 2 m M L -glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin for the first 4 weeks. In addition, during the first week of culture, 10 ng/ml IL-3 was included in the media. After 4 weeks, the cells were cultured in RPMI 1640 medium (Sigma Aldrich, St. Louis, Mo., USA) supplemented with 10% FBS (Invitrogen, Carlsbad, Calif., USA), 100 ng/ml SCF and 10 ng/ml IL-6, 0.01 M HEPES, 0.5 ! non-essential amino acids, 2 m M L -glutamine, 100 units/ml penicillin, 0.1 mg/ ml streptomycin and 48 M  -mercaptoethanol (Sigma Aldrich, St. Louis, Mo., USA). Mast cell maturity and purity was evaluated by staining for the mast cell-specific protease tryptase [25] . The purity of mast cells used in experiments exceeded 95%. The use of CBMCs in this study was approved by the ethical committee at Uppsala University Hospital, Sweden. All donors gave informed consent. Analysis of Nod1 and Nod2 Protein Expression in CBMCs Nod1 and Nod2 protein expression in CBMCs was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Stockholm, Sweden). Briefly, CBMCs were fixed with Biolegends fixation buffer and Fc receptors were blocked using Human TruStain Fc Receptor Blocking Solution, after which cells were permeabilized using a permeabilization buffer (all from Biolegend, San Diego, Calif., USA). To detect intracellular expression of Nod1, the following antibodies were utilized: -CARD4 (Abcam, Cambridge, UK), followed by a fluorescein (FITC)-labelled secondary antibody (swine-rabbit IgG; Dako, Glostrup, Denmark). Rabbit IgG was used as isotype control (Dako). To detect Nod2 expression, an -CARD15 antibody (clone 2D9; Abcam) was used followed by a FITC-labelled secondary antibody (rabbit-mouse IgG; Dako). Mouse IgG1 was used as isotype control (Dako).