The Mast Cell-restricted Tryptase mMCP-6 Has a Critical Immunoprotective Role in Bacterial Infections

Although it has been shown that mast cell-deficient mice have diminished innate immune responses against bacteria, the most important immunoprotective factors secreted from activated mast cells have not been identified. Mouse mast cell protease 6 is a tetramer-forming tryptase. This serine protease is abundant in the secretory granules and is exocytosed upon bacterial challenge. Here we have described the generation of a mast cell protease-6-null mouse. Our discovery that mice lacking this neutral protease cannot efficiently clear Klebsiella pneumoniae from their peritoneal cavities reveals an essential role for this serine protease, and presumably its human ortholog, in innate immunity. Although it has been shown that mast cell-deficient mice have diminished innate immune responses against bacteria, the most important immunoprotective factors secreted from activated mast cells have not been identified. Mouse mast cell protease 6 is a tetramer-forming tryptase. This serine protease is abundant in the secretory granules and is exocytosed upon bacterial challenge. Here we have described the generation of a mast cell protease-6-null mouse. Our discovery that mice lacking this neutral protease cannot efficiently clear Klebsiella pneumoniae from their peritoneal cavities reveals an essential role for this serine protease, and presumably its human ortholog, in innate immunity. Approximately 50% of the weight of a mature tissue mast cell (MC) 2The abbreviations and trivial names used are: MC, mast cell; CFU, colony-forming unit; DNP, 2,4-dinitrophenol; HSA, human serum albumin; ES, embryonic stem; hTryptase, human tryptase; MITF, microphthalmia transcription factor; mMCP, mouse MC protease; PCA, passive cutaneous anaphylaxis; tg/tg, WBB6F1-tg/tg mouse; TNF-α, tumor necrosis factor-α; Wsh/Wsh, B6.Cg-KitW-sh/KitW-sh mouse; W/Wv, WBB6F1-KitW/KitW-v mouse. consists of protease-serglycin proteoglycan complexes stored in the secretory granules. In humans, β tryptases are the most abundant MC-restricted neutral proteases (1Schwartz L.B. Lewis R.A. Austen K.F. J. Biol. Chem. 1981; 256: 11939-11943Abstract Full Text PDF PubMed Google Scholar, 2Miller J.S. Moxley G. Schwartz L.B. J. Clin. Investig. 1990; 86: 864-870Crossref PubMed Scopus (153) Google Scholar, 3Vanderslice P. Ballinger S.M. Tam E.K. Goldstein S.M. Craik C.S. Caughey G.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3811-3815Crossref PubMed Scopus (202) Google Scholar). The corresponding tryptases in mice are mouse MC protease (mMCP)-6 (4Reynolds D.S. Stevens R.L. Lane W.S. Carr M.H. Austen K.F. Serafin W.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3230-3234Crossref PubMed Scopus (172) Google Scholar, 5Reynolds D.S. Gurley D.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1991; 266: 3847-3853Abstract Full Text PDF PubMed Google Scholar) and mMCP-7 (6McNeil H.P. Reynolds D.S. Schiller V. Ghildyal N. Gurley D.S. Austen K.F. Stevens R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11174-11178Crossref PubMed Scopus (127) Google Scholar), with mMCP-6 being the most similar in amino acid sequence and substrate specificity to human tryptase (hTryptase) β1 (7Huang C. Friend D.S. Qiu W.T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar, 8Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 9Harris J.L. Niles A. Burdick K. Maffitt M. Backes B.J. Ellman J.A. Kuntz I. Haak-Frendscho M. Craik C.S. J. Biol. Chem. 2001; 276: 34941-34947Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). MCs are the only cells that express mMCP-6, and this serine protease is particularly abundant in those MCs that reside in the peritoneal cavity, skin, and lung (4Reynolds D.S. Stevens R.L. Lane W.S. Carr M.H. Austen K.F. Serafin W.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3230-3234Crossref PubMed Scopus (172) Google Scholar, 5Reynolds D.S. Gurley D.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1991; 266: 3847-3853Abstract Full Text PDF PubMed Google Scholar, 10Stevens R.L. Friend D.S. McNeil H.P. Schiller V. Ghildyal N. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 128-132Crossref PubMed Scopus (117) Google Scholar). Numerous biochemical studies have been carried out to understand the biosynthesis and substrate preference of mMCP-6. This tryptase is initially translated as a zymogen with a 245-mer mature domain. When the signal and propeptides are proteolytically removed, the mature protease spontaneously forms tetramers with the active site of each monomer facing the central core of the tetramer unit, as first described for its human ortholog (11Pereira P.J. Bergner A. Macedo-Ribeiro S. Huber R. Matschiner G. Fritz H. Sommerhoff C.P. Bode W. Nature. 1998; 392: 306-311Crossref PubMed Scopus (283) Google Scholar). A positively charged face forms on the surface of each monomer, thereby allowing mature mMCP-6 to interact with negatively charged serglycin proteoglycans in the Golgi complex. The resulting tryptase-serglycin macromolecular complexes are then targeted and packaged in the cell secretory granules. When exocytosed, these complexes are retained in connective tissues for hours because of their large sizes (12Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J. F.,Šali, A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar). Protease inhibitors are abundant in blood. Nevertheless, no circulating protease inhibitor has been identified that rapidly inactivates mMCP-6 or hTryptase β1. Substrate specificity studies carried out using varied peptide combinatorial libraries revealed that recombinant mMCP-6 (7Huang C. Friend D.S. Qiu W.T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar) and hTryptase β1 (8Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 9Harris J.L. Niles A. Burdick K. Maffitt M. Backes B.J. Ellman J.A. Kuntz I. Haak-Frendscho M. Craik C.S. J. Biol. Chem. 2001; 276: 34941-34947Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) prefer to cleave peptides having a Pro at residues P2 to P5 and a Lys or Arg at residue P1. However, due to the unique structural constraints of the tetramer unit, the abilities of mMCP-6 and hTryptase β1 to cleave large-sized proteins are very limited. Thus, the importance of these evolutionally conserved enzymes in MC-dependent reactions remains to be determined. MC development in vivo is highly dependent on the cytokine kit ligand/stem cell factor on the surface of mesenchymal cells and its tyrosine kinase receptor c-Kit/CD117 on the surface of MC-committed progenitors. Signaling through c-Kit results in the translocation of microphthalmia transcription factor (MITF) into the nucleus. WBB6F1 -KitW/KitW-v (W/Wv) mice are MC-deficient secondary to a point mutation in the intracellular domain of c-kit, which makes their MCs and progenitors less responsive to kit ligand. WBB6F1-tg/tg (tg/tg) mice, on the other hand, have reduced numbers of MCs, because they express a mutated isoform of MITF. Thus, transcription of the mMCP-6 gene and certain other MC-restricted genes are greatly diminished in MITF-deficient tg/tg mice (13Morii E. Oboki K. Ishihara K. Jippo T. Hirano T. Kitamura Y. Blood. 2004; 104: 1656-1661Crossref PubMed Scopus (32) Google Scholar). In 1996, Echtenacher et al. (14Echtenacher B. Maaönnel D.N. Huöltner L. Nature. 1996; 381: 75-77Crossref PubMed Scopus (812) Google Scholar) noted that W/Wv mice quickly die from septic peritonitis after their caecum is ligated and punctured. Malaviya et al. (15Malaviya R. Ikeda T. Ross E. Abraham S.N. Nature. 1996; 381: 77-80Crossref PubMed Scopus (984) Google Scholar) reported at the same time that W/Wv mice cannot efficiently combat a Klebsiella pneumoniae infection of their peritoneal cavities or lungs. The same phenomenon was observed in tg/tg mice (16Jippo T. Morii E. Ito A. Kitamura Y. J. Exp. Med. 2003; 197: 1417-1425Crossref PubMed Scopus (38) Google Scholar), confirming the importance of MCs in innate immunity. The latter data suggest that a MC-restricted gene whose transcription is highly dependent on MITF plays an essential immunoprotective role in bacterial infections. Although it has been concluded that MC-derived tumor necrosis factor-α (TNF-α) is needed to control bacterial infections (14Echtenacher B. Maaönnel D.N. Huöltner L. Nature. 1996; 381: 75-77Crossref PubMed Scopus (812) Google Scholar, 15Malaviya R. Ikeda T. Ross E. Abraham S.N. Nature. 1996; 381: 77-80Crossref PubMed Scopus (984) Google Scholar), the number of TNF-α-expressing lipopolysaccharide-responsive macrophages in the lungs and peritoneal cavities of bacteria-infected mice greatly exceeds the number of MCs. There is also no evidence that MITF regulates the transcription of the TNF-α gene in any cell. Thus, additional factors more restricted to MCs must be required for an optimal antibacterial response in vivo to explain why MC-deficient mice are so susceptible to bacterial challenge. In support of this conclusion, Maurer et al. (17Maurer M. Echtenacher B. Huöltner L. Kollias G. Maaönnel D.N. Langley K.E. Galli S.J. J. Exp. Med. 1998; 188: 2343-2348Crossref PubMed Scopus (134) Google Scholar) note that MC activation significantly improves host defenses in TNF-α–/– mice, and Jippo et al. (16Jippo T. Morii E. Ito A. Kitamura Y. J. Exp. Med. 2003; 197: 1417-1425Crossref PubMed Scopus (38) Google Scholar) have found that the injection of TNF-α protein into the peritoneal cavities of tg/tg mice fail to completely restore their MC-dependent antibacterial responses. We have here described the generation of an mMCP-6–/– mouse strain. Using these mice, we have shown that mMCP-6 is required to efficiently combat K. pneumoniae infections. Targeting of the mMCP-6 Locus in Mouse Embryonic Stem Cells and Generation of the mMCP-6–/– Mouse Strain—The mMCP-6 gene (GenBankTM gene identification number 17229) resides in the tryptase locus on mouse chromosome 17A3.3 (18Wong G.W. Yasuda S. Morokawa N. Li L. Stevens R.L. J. Biol. Chem. 2004; 279: 2438-2452Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Using the bacterial artificial chromosome clone RP24–263I14 (Children’s Hospital Oakland Research Institute, CA) and oligonucleotides based on the genomic sequence of the chromosomal locus at GenBankTM accession number NT_039649, a PCR approach was carried out to obtain the mMCP-6-specific nucleotide sequences for the targeting vector shown in Fig. 1A. Employing the translation initiation site of the mMCP-6 transcript as a reference point (defined here as residue +1), a 2970-bp 5′ homology arm (nucleotides –2966 to +3) was cloned into the BglII-HindIII sites of the pKO Scrambler NTK-1907 plasmid (Stratagene) downstream of the phosphoglucokinase promoter-driven neomycin resistance gene (PGK-Neo). A 3196-bp 3′ homology arm (nucleotides +897 to +4092) was then cloned into the SalI-NotI sites upstream of PGK-Neo. The herpes simplex virus thymidine kinase cDNA driven by a mutant polyomavirus enhancer (MC1-TK) is present in this plasmid for negative selection. A mouse expressing Cre recombinase (Cre) under the control of an MC-specific promoter has not yet been created to selectively diminish the expression of ubiquitously expressed genes using a Cre/loxP approach. Because mMCP-6 is highly restricted to MCs, a knock-in approach was carried out by placing Cre with a SV40 poly(A) signal at the 3′ end of the 5′ homology arm using HindIII and XhoI restriction sites. The linearized targeting vector was electroporated into 129svJ × C57BL/6J F1 embryonic stem (ES) cells. ES cell clones that underwent homologous recombination were identified by blot analysis of BglI-digested ES cell DNA hybridized with a radiolabeled probe with using as a 3′ (nucleotides to clones were by PCR with a upstream of the 5′ homology arm and in the Cre ES cell clones were into which were then into mice were with and from this were for of the mutant mMCP-6 was carried out on by PCR using oligonucleotides the 5′ homology arm the of the mMCP-6 gene and the Cre mMCP-6 were to mice to any mutated gene in mMCP-6–/– Using a a mouse expressing of at chromosomal was identified in the This mouse was as the of a that was more the and mMCP-6–/– used in this mice were in a of The of M. to their and of In MCs were from the peritoneal with of was into the peritoneal to of the was in each and the resulting were at × for The cells were in of and a of in at × for peritoneal MCs were at the of the was by or by with and MCs were by in with a protease inhibitor were by the acid protein kit were by with The resulting were with for and mMCP-6 (12Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J. F.,Šali, A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar, H.P. Austen K.F. Friend D.S. Stevens R.L. J. Immunol. 1992; Google Scholar, N. Friend D.S. R. Austen K.F. McNeil H.P. Schiller V. Stevens R.L. J. Immunol. 1994; Google Scholar, Wong G.W. Friend D.S. Gurish M.F. Qiu W.T. Huang C. Stevens R.L. Nature. PubMed Scopus Google Scholar). secondary and substrate were used for protein were at for in and they were then with an to as a protein peritoneal MCs from mice and mMCP-6–/– mice × were in for by for were in at with an with and and then with a from each were using an cell from from each were The of cell identified as MCs by the of their and surface was by J.P. 1991; PubMed Scopus Google Scholar). The the of the of the cell by and the surface is to the surface of the granules of were with the using a The of the cell was using a point and from mice and mMCP-6–/– mice were not to MC The cells in each were with a and the cell was on of of each with from mice and mMCP-6–/– mice were at in and in to the serglycin proteoglycans in the MC secretory granules. Thus, were with and for at and then with were in a with the and of the of the and observed in the we the of the as tissue and in Pro cells with a were were expressed as the number of of and from peritoneal MCs from mice and mMCP-6–/– mice were and In each MCs were in for at to cells were activated with human serum at an of cells was with and was in each and after TNF-α of the were at and and TNF-α were by and B6.Cg-KitW-sh/KitW-sh mice were with of and in of were then into the and the mice were with of in of The mice were and their were and in at for of in each was at using a pneumoniae were to on at in The were and in to × colony-forming and × and mMCP-6–/– mice were by injection of of the bacterial were to cell and bacterial and cytokine mice were and after The peritoneal of each was and was by of were in and cells were with a and the cell was by of or with were and The bacterial were The of the peritoneal were and the of TNF-α and in the were by were expressed as The of the was with the the by the Generation and of a mMCP-6–/– Mouse mouse not express mMCP-7 Stevens R.L. Austen K.F. J. Ghildyal N. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In mouse to express mMCP-6. Thus, to the role of mMCP-6 and its human ortholog in MC development and we used a homologous recombination approach to a on a mouse The targeting used in these is shown in Fig. 1A. We the downstream of the translation initiation site of the mMCP-6 gene with the cDNA that Cre The resulting mutated mMCP-6 gene amino the that the essential residue Thus, no mMCP-6 protein be expressed a transcript is of the resulting cloned ES cells and mutant the targeted of the mMCP-6 gene and In with the the of mMCP-6 protein were in the MCs that in the peritoneal cavities of these mice The we used was against a unique peptide that to residues in the mature domain of the protease (12Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J. F.,Šali, A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar). This amino acid sequence is by the to the of the mMCP-6 which was not by targeting that no protein were with this in MC from and mMCP-6–/– mice not we concluded that a protein was not Our mMCP-6–/– mice no and a in a analysis revealed numbers of MCs in the and peritoneal The peritoneal MCs from these mice also no in their or or in the and of their secretory granules and Thus, mMCP-6 is expressed in MC development D.S. Gurley D.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1991; 266: 3847-3853Abstract Full Text PDF PubMed Google Scholar), this neutral protease is not essential for the and of MC-committed progenitors in connective and of cutaneous and peritoneal cells with a of in of and of of peritoneal analysis of from peritoneal MCs. surface A cell cell cells with a of in of and of of peritoneal analysis of from peritoneal MCs. surface A cell cell in a MCs of the proteases and in their secretory granules in to the tryptase mMCP-6 (4Reynolds D.S. Stevens R.L. Lane W.S. Carr M.H. Austen K.F. Serafin W.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3230-3234Crossref PubMed Scopus (172) Google Scholar). by the of and stored in the peritoneal MCs in mMCP-6–/– mice were not the of protein in the peritoneal MCs of these mice, presumably due to the of more sites in the secretory MCs also of not Thus, the of mMCP-6 in a connective MC not the of other in the cell secretory granules. MCs from mMCP-6–/– of TNF-α and and a peritoneal MCs from and mMCP-6–/– mice were with and with in shown in Fig. no in the of TNF-α the was noted the of no in the of at was observed MCs a role in the in vivo and it has been concluded that the most important secreted from activated MCs in this response are and N. S. M. H. A. Immunol. PubMed Scopus Google Scholar, J. 1991; PubMed Scopus Google Scholar). We as have that the is reduced in MC-deficient mice to mice Because mMCP-6–/– mice a with that of their control we concluded that mMCP-6 not a role in the activation of MCs and their of and other in mMCP-6–/– having MC and secretory and mice the of a reduced to combat K. pneumoniae infections of their peritoneal of mMCP-6–/– mice after of K. pneumoniae with of and of these no mMCP-6–/– mice a with of K. pneumoniae This was with a in the of into the peritoneal cavities of mMCP-6–/– mice This was of the number of circulating which were and mMCP-6–/– mice the of the infection not with bacterial in the and of mice in mMCP-6 and data not and bacterial peritoneal K. pneumoniae and mMCP-6–/– mice of K. pneumoniae and were at time the number of in each peritoneal was from cell in a and in with mMCP-6–/– a in the of to the site of infection bacterial were by the of of each peritoneal There were significantly bacterial in the peritoneal cavities of mMCP-6–/– mice after infection We in studies that the of K. pneumoniae was not diminished by to active recombinant mMCP-6, thereby out a of this protease (8Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In support of we also noted that the of of recombinant mMCP-6 or its human ortholog hTryptase β1 into the peritoneal cavities or lungs of W/Wv mice results in a (7Huang C. Friend D.S. Qiu W.T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar, 8Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Because of human cells to recombinant mMCP-6 or hTryptase β1 not their results in a in the of and protein (8Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), the data suggest that active mMCP-6 plays a role in bacterial infections by cells in sites to of factors that the of into the site to control the Nevertheless, the by which mMCP-6 these cells remains to be determined. In to the that W/Wv mice cannot efficiently combat a K. pneumoniae infection of their peritoneal Malaviya et al. (15Malaviya R. Ikeda T. Ross E. Abraham S.N. Nature. 1996; 381: 77-80Crossref PubMed Scopus (984) Google Scholar) reported of TNF-α and in the peritoneal cavities of these mice after a in mice after and which was with of in the peritoneal cavities of these mice J. Caughey G.H. P.J. J. Clin. Investig. 2004; PubMed Scopus Google Scholar). Because TNF-α and in the peritoneal cavities of and mMCP-6–/– mice were the of the infection these not as for the we observed in In with this conclusion, MCs from mice of TNF-α When in vivo and in mouse MCs expressed and acid However, of these were MC-restricted as were the of the cell. Although MCs of mice in combat infections mice Y. Y. Y. A. Y. T. H. S. E. A. T. H. J. Immunol. PubMed Scopus Google Scholar). is that plays an role in this response because of its which the of and their from MCs also large of varied of serine proteases in their secretory granules. The MCs in the peritoneal cavities of mice mMCP-7 Stevens R.L. Austen K.F. J. Ghildyal N. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), and the corresponding MCs in mice G.W. Yasuda S. S. G. Li L. J.A. Stevens R.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), yet these mouse are not highly susceptible to The other serine proteases found in mouse MCs and are of the chromosome of mMCP-6, the latter of serine proteases for their activation P.J. Caughey G.H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). mice have in the and to mice J. Caughey G.H. P.J. J. Clin. Investig. 2004; PubMed Scopus Google Scholar). is that any chromosome protease plays a role in MC-dependent bacterial The data suggest that mMCP-6 is the that mice from bacterial infections. In support of this conclusion, transcription of the mMCP-6 gene in MCs is highly dependent on MITF E. T. Jippo T. K. K. K. S. M. Kitamura Y. Blood. 1996; PubMed Google Scholar), thereby why tg/tg mice are so susceptible to bacterial infections. The discovery that mMCP-6–/– mice have in an K. pneumoniae infection a role for this evolutionally conserved serine protease in innate immunity. are highly susceptible to and other infections. The that hTryptase MCs and their progenitors be with of Y. Li L. R. C. A. E. M. S. M. A. Wong G.W. Stevens R.L. Blood. 2001; PubMed Scopus (75) Google Scholar, N. M. Friend D.S. H. J. J.A. J. 2001; PubMed Scopus Google Scholar, J.A. G. C. Immunol. 2004; PubMed Scopus Google Scholar) with the that have reduced numbers of hTryptase MCs in their G. Schwartz L.B. J. Immunol. Google Scholar) that this protease plays a immunoprotective role in The that no human has been identified hTryptase MCs is evidence that these immune cells are essential to it to be out that the is hTryptase inhibitors in an to and connective tissue in and other MC-dependent Our data the that the of these hTryptase inhibitors in in a of bacterial infections. We B. M. and C. for and This used the Mouse Research Research and of The of M. by the of and of