Advertisement

Aberrant Mucosal Mast Cell Protease Expression in the Enteric Epithelium of Nematode-Infected Mice Lacking the Integrin αvβ6, a Transforming Growth Factor-β1 Activator

      Infection of mice with the nematode Trichinella spiralis triggers recruitment and differentiation of intraepithelial intestinal mucosal mast cells expressing mouse mast cell protease 1 (Mcpt-1), which contributes to expulsion of the parasite. Expression of Mcpt-1 is transforming growth factor (TGF)-β1-dependent in vitro. TGF-β1, which is secreted within tissues as a biologically inactive complex with latency-associated peptide, requires extracellular modification to become functionally active. The integrin-ανβ6 mediates local activation of TGF-β1 in association with epithelia. Using T. spiralis-infected β6−/− mice, we show accumulation of mucosal mast cells in the lamina propria of the small intestine with minimal recruitment into the epithelial compartment. This was accompanied by a coordinate reduction in expression of both Mcpt-1 and -2 in the jejunum and increased tryptase expression, whereas Mcpt-9 became completely undetectable. In contrast, the cytokine stem cell factor, a regulator of mast cell differentiation and survival, was significantly up-regulated in T. spiralis-infected β6−/− mice compared with infected β6+/+ controls. Despite these changes, β6−/− mice still appeared to expel the worms normally. We postulate that compromised TGF-β1 activation within the gastrointestinal epithelial compartment is a major, but not the only, contributing factor to the observed changes in mucosal mast cell protease and epithelial cytokine expression in β6−/− mice.
      As a stereotypic TH2-regulated response to almost all gastrointestinal nematode infections of mammals, the population of mucosal mast cells (MMCs) expands rapidly both in the mucosa and within the epithelium.
      • Artis D
      • Humphreys NE
      • Bancroft AJ
      • Rothwell NJ
      • Potten CS
      • Grencis RK
      Tumor necrosis factor alpha is a critical component of interleukin 13-mediated protective T helper cell type 2 responses during helminth infection.
      • Ferguson A
      • Cummins AG
      • Munro GH
      • Gibson S
      • Miller HRP
      Roles of mucosal mast cells in intestinal cell mediated immunity.
      • Miller HRP
      Mucosal mast cells and the allergic response against nematode parasites.
      Intraepithelial MMCs are morphologically distinct from mast cells in nonmucosal sites and also differ in their content of granule mediators.
      • Ferguson A
      • Cummins AG
      • Munro GH
      • Gibson S
      • Miller HRP
      Roles of mucosal mast cells in intestinal cell mediated immunity.
      • Miller HRP
      Mucosal mast cells and the allergic response against nematode parasites.
      The intraepithelial accumulation of a high proportion of MMCs is most obvious in the mouse but occurs in many other species as well, including parasitized rats, ruminants, and equids. Studies using mast cell-deficient mice have established the importance of MMC in resistance against some, but not all, nematode parasites.
      • Ha TY
      • Reed ND
      • Crowle PK
      Delayed expulsion of adult Trichinella spiralis by mast cell-deficient W/Wv mice.
      • Faulkner H
      • Humphreys N
      • Renauld JC
      • Van Snick J
      • Grencis R
      Interleukin-9 is involved in host protective immunity to intestinal nematode infection.
      • Mitchell LA
      • Wescott RB
      • Perryman LE
      Kinetics of expulsion of the nematode, Nippostrongylus brasiliensis, in mast-cell-deficient W/WV mice.
      • Nawa Y
      • Ishikawa N
      • Tsuchiya K
      • Horii Y
      • Abe T
      • Khan AI
      • Bing S
      • Itoh H
      • Ide H
      • Uchiyama F
      Selective effector mechanisms for the expulsion of intestinal helminths.
      In mice, the highly soluble MMC granule-derived β-chymase mouse mast cell protease-1 (Mcpt-1) is abundantly expressed in the parasitized gut and is released both systemically and into the gut lumen during worm expulsion. BALB/c mice lacking Mcpt-1 (Mcpt-1−/− BALB/c F10) show significantly impaired expulsion of the intraepithelial nematode Trichinella spiralis in comparison to Mcpt-1+/+ BALB/c F10 controls.
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      Several studies suggest that a major role of MMC β-chymases is to promote epithelial permeability,
      • McDermott JR
      • Bartram RE
      • Knight PA
      • Miller HRP
      • Garrod DR
      • Grencis RK
      Mast cells disrupt epithelial barrier function during enteric nematode infection.
      • Scudamore CL
      • Thornton EM
      • McMillan L
      • Newlands GF
      • Miller HR
      Release of the mucosal mast cell granule chymase, rat mast cell protease-II, during anaphylaxis is associated with the rapid development of paracellular permeability to macromolecules in rat jejunum.
      and this may contribute to intestinal enteropathy.
      • Lawrence CE
      • Paterson YYW
      • Wright SH
      • Knight PA
      • Miller HRP
      Mouse mast cell protease-1 is required for the enteropathy induced by gastrointestinal helminth infection in the mouse.
      The multifunctional cytokine transforming growth factor (TGF)-β1 is a key regulator of Mcpt-1 expression and secretion in vitro,
      • Miller HR
      • Wright SH
      • Knight PA
      • Thornton EM
      A novel function for transforming growth factor-β1: upregulation of the expression and the IgE-independent extracellular release of a mucosal mast cell granule-specific β-chymase, mouse mast cell protease-1.
      • Brown JK
      • Knight PA
      • Wright SH
      • Thornton EM
      • Miller HR
      Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues.
      • Wright SH
      • Brown J
      • Knight PA
      • Thornton EM
      • Kilshaw PJ
      • Miller HR
      Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues.
      • Pemberton AD
      • Brown JK
      • Wright SH
      • Knight PA
      • Miller HR
      The proteome of mouse mucosal mast cell homologues: the role of transforming growth factor β1.
      as well as having chemotactic properties for mast cells.
      • Olsson N
      • Piek E
      • ten Dijke P
      • Nilsson G
      Human mast cell migration in response to members of the transforming growth factor-β family.
      TGF-β1 is secreted by many cell types, and the nascent protein is usually biologically inactive because it is complexed with latency-associated peptide (LAP). TGF-β1-LAP binds latent TGF-β-binding protein 1 (LT-BP1) and may also bind to LT-BP3/LT-BP4 in the extracellular matrix, described as the large latent complex. In the intestine, TGF-β1 is produced by enterocytes, promoting epithelial repair
      • Beck PL
      • Rosenberg IM
      • Xavier RJ
      • Koh T
      • Wong JF
      • Podolsky DK
      Transforming growth factor-β mediates intestinal healing and susceptibility to injury in vitro and in vivo through epithelial cells.
      • Podolsky DK
      Healing the epithelium: solving the problem from two sides.
      and reducing microbial-
      • Buzoni-Gatel D
      • Debbabi H
      • Mennechet FJ
      • Martin V
      • Lepage AC
      • Schwartzman JD
      • Kasper LH
      Murine ileitis after intracellular parasite infection is controlled by TGF-β-producing intraepithelial lymphocytes.
      • Roche JK
      • Martins CA
      • Cosme R
      • Fayer R
      • Guerrant RL
      Transforming growth factor beta1 ameliorates intestinal epithelial barrier disruption by Cryptosporidium parvum in vitro in the absence of mucosal T lymphocytes.
      • Yang X
      • Letterio JJ
      • Lechleider RJ
      • Chen L
      • Hayman R
      • Gu H
      • Roberts AB
      • Deng C
      Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β.
      and inflammatory bowel disease-associated enteropathy.
      • Yang X
      • Letterio JJ
      • Lechleider RJ
      • Chen L
      • Hayman R
      • Gu H
      • Roberts AB
      • Deng C
      Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β.
      • Hahm KB
      • Im YH
      • Parks TW
      • Park SH
      • Markowitz S
      • Jung HY
      • Green J
      • Kim SJ
      Loss of transforming growth factor β signalling in the intestine contributes to tissue injury in inflammatory bowel disease.
      • Hahm KB
      • Lee KM
      • Kim YB
      • Hong WS
      • Lee WH
      • Han SU
      • Kim MW
      • Ahn BO
      • Oh TY
      • Lee MH
      • Green J
      • Kim SJ
      Conditional loss of TGF-β signalling leads to increased susceptibility to gastrointestinal carcinogenesis in mice.
      • MacDonald TT
      Effector and regulatory lymphoid cells and cytokines in mucosal sites.
      Latent TGF-β1-LAP complex can be activated by a diverse range of factors. These include proteolysis by plasmin, metalloproteases, and cathepsins, and activation via TGF-β1-LAP-binding molecules including thrombospondin-1 and the αv integrin family (αvβ1, αvβ3, αvβ6, and αvβ8).
      • Annes JP
      • Munger JS
      • Rifkin DB
      Making sense of latent TGFβ activation.
      • Mu DZ
      • Cambier S
      • Fjellbirkeland L
      • Baron JL
      • Munger JS
      • Kawakatsu H
      • Sheppard D
      • Broaddus VC
      • Nishimura SL
      The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.
      Studies of null mice support roles for both integrin αvβ6 and TSP-1 as potential activators of TGF-β1in vivo
      • Crawford SE
      • Stellmach V
      • Murphy-Ullrich JE
      • Ribeiro SM
      • Lawler J
      • Hynes RO
      • Boivin GP
      • Bouck N
      Thrombospondin-1 is a major activator of TGF-β1 in vivo.
      • Huang XZ
      • Wu JF
      • Cass D
      • Erle DJ
      • Corry D
      • Young SG
      • Farese RV
      • Sheppard D
      Inactivation of the integrin β6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lungs and skin.
      • Ludlow A
      • Yee KO
      • Lipman R
      • Bronson R
      • Weinreb P
      • Huang XZ
      • Sheppard D
      • Lawler J
      Characterization of integrin β6 and thrombospondin-1 double-null mice.
      and integrin αvβ6 is expressed almost exclusively by epithelial cells.
      • Ludlow A
      • Yee KO
      • Lipman R
      • Bronson R
      • Weinreb P
      • Huang XZ
      • Sheppard D
      • Lawler J
      Characterization of integrin β6 and thrombospondin-1 double-null mice.
      • Breuss JM
      • Gillett N
      • Lu L
      • Sheppard D
      • Pytela R
      Restricted distribution of integrin beta-6 messenger-RNA in primate epithelial tissues.
      • Brown JK
      • McAleese SM
      • Thornton EM
      • Pate JA
      • Schock A
      • Macrae A
      • Scott PR
      • Miller HR
      • Collie DS
      Integrin-alpha(v) beta(6), a putative receptor for foot-and-mouth disease virus, is constitutively expressed in ruminant airways.
      We have confirmed the presence of β6 integrin transcripts in enterocytes of the jejunum in normal and T. spiralis-infected S129 mice, where it is coexpressed with TGF-β1.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      Integrin αvβ6 binds LAP and, in the presence of both intracellular β-actin and LT-BP1, activates TGF-β1.
      • Annes JP
      • Munger JS
      • Rifkin DB
      Making sense of latent TGFβ activation.
      • Munger JS
      • Huang X
      • Kawakatsu H
      • Griffiths MJ
      • Dalton SL
      • Wu J
      • Pittet JF
      • Kaminski N
      • Garat C
      • Matthay MA
      • Rifkin DB
      • Sheppard D
      The integrin αvβ6 binds and activates latent TGF β1: a mechanism for regulating pulmonary inflammation and fibrosis.
      Furthermore, integrin β6−/− mice show exaggerated lung inflammation,
      • Huang XZ
      • Wu JF
      • Cass D
      • Erle DJ
      • Corry D
      • Young SG
      • Farese RV
      • Sheppard D
      Inactivation of the integrin β6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lungs and skin.
      • Griffiths M
      • Huang XZ
      • Wu JF
      • Sheppard D
      Inactivation of the β6 integrin subunit gene protects against bleomycin-induced pulmonary fibrosis.
      which is reversed by epithelial expression of the human β6 transgene.
      • Huang X
      • Wu J
      • Zhu W
      • Pytela R
      • Sheppard D
      Expression of the human integrin β6 subunit in alveolar type II cells and bronchiolar epithelial cells reverses lung inflammation in β6 knockout mice.
      Integrin β6−/−/TSP-1−/− double-null mice exhibited a significantly higher incidence of tissue inflammation in the lung, glandular stomach epithelium, and other tissues than wild-type or single-null mice.
      • Ludlow A
      • Yee KO
      • Lipman R
      • Bronson R
      • Weinreb P
      • Huang XZ
      • Sheppard D
      • Lawler J
      Characterization of integrin β6 and thrombospondin-1 double-null mice.
      In β6−/− mice infected with the lumen-dwelling nematode Nippostrongylus brasiliensis, both MMC recruitment and Mcpt-1 expression were virtually ablated.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      In contrast, infection of β6−/− mice with T. spiralis, a nematode that parasitizes jejunal epithelium, resulted in greatly enhanced MMC recruitment into the lamina propria with significantly reduced recruitment to the epithelial compartment and greatly reduced expression of the integrin αEβ7 by the aberrantly located MMC.
      • Brown JK
      • Knight PA
      • Pemberton AD
      • Wright SH
      • Pate JA
      • Thornton EM
      • Miller HRP
      Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).
      Here, we demonstrate that there is a coordinate reduction in expression of both Mcpt-1 and -2 by MMC in β6−/− mice and that Mcpt-9 transcripts become completely undetectable. Interestingly, the cytokine stem cell factor (SCF), a key regulator of gastrointestinal nematode-induced MMC hyperplasia,
      • Donaldson LE
      • Schmitt E
      • Huntley JF
      • Newlands GFJ
      • Grencis RK
      A critical role for stem cell factor and c-kit in host protective immunity to an intestinal helminth.
      • Grencis RK
      • Else KJ
      • Huntley JF
      • Nishikawa SI
      The in vivo role of stem-cell factor (c-Kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice.
      is highly up-regulated in the epithelial compartment of β6−/− mice compared with β6+/+ controls. We postulate that the aberrant responses to T. spiralis infection in the β6−/− gastrointestinal mucosa are, in part, a result of compromised local TGF-β1 activation within the epithelium rather than any systemic effects.

      Materials and Methods

      Transgenic Mice, Parasite Infections, and Sample Preparation

      All experimental procedures involving laboratory animals were approved by The University of Edinburgh's Biological Services ethical review committee and were performed under license, as required by the United Kingdom's Animals (Scientific Procedures) Act 1986. Integrin-β6-null (S129 strain background: β6−/−) mice (Huang et al
      • Huang XZ
      • Wu JF
      • Cass D
      • Erle DJ
      • Corry D
      • Young SG
      • Farese RV
      • Sheppard D
      Inactivation of the integrin β6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lungs and skin.
      ) were originally obtained from Dr. Kairbaan Hodivala-Dilke (Cell Adhesion and Disease Laboratory, GKT School of Medicine, St. Thomas' Hospital, London, UK) and backcrossed with S129 β6+/+ controls (B&K Universal, Hull, UK) as previously described.
      • Brown JK
      • Knight PA
      • Pemberton AD
      • Wright SH
      • Pate JA
      • Thornton EM
      • Miller HRP
      Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).
      S129 β6+/+ and β6−/− mice, 8- to 15-week-old and age- and sex-matched, were infected by gavage with 250 muscle larvae, and groups (n = 4 to 6) were sacrificed at various time points after infection for sample preparation and assessment of parasite burden. Maintenance of and infection with Trichinella spiralis larvae, sample collection for histology, total RNA and protein, recovery of muscle larvae and/or adult worms, were performed as described by us previously.
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      For preparation of RNA from intestinal epithelium, mice were sacrificed on day 0 (uninfected) and day 12 after infection (worm expulsion phase) and epithelium purified and assessed for cellular composition as described previously.
      • Rosbottom A
      • Knight PA
      • McLachlan G
      • Thornton EM
      • Wright SW
      • Miller HR
      • Scudamore CL
      Chemokine and cytokine expression in murine intestinal epithelium following Nippostrongylus brasiliensis infection.
      • Knight PA
      • Pemberton AD
      • Robertson KA
      • Roy DJ
      • Wright SH
      • Miller HRP
      Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis.

      Histology and Immunocytochemistry

      Toluidine blue and esterase staining were performed as described previously.
      • Wastling JM
      • Knight P
      • Ure J
      • Wright S
      • Thornton EM
      • Scudamore CL
      • Mason J
      • Smith A
      • Miller HRP
      Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the β-chymase, mouse mast cell protease-1.
      Immunohistochemical staining of paraffin-embedded paraformaldehyde-fixed sections for Mcpt1, Mcpt2, and tryptase were performed according to published protocols.
      • Brown JK
      • Knight PA
      • Pemberton AD
      • Wright SH
      • Pate JA
      • Thornton EM
      • Miller HRP
      Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).
      • Pemberton AD
      • Brown JK
      • Wright SH
      • Knight PA
      • McPhee ML
      • McEuen AR
      • Forse PA
      • Miller HRP
      Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo.

      Mast Cell Cultures

      Bone marrow-derived mast cells (BMMCs) from β6+/+ and β6−/− mice were generated in the presence of recombinant mouse interleukin (IL)-3 (1 ng/ml; R&D Systems, Abingdon, UK), recombinant mouse IL-9 (5 ng/ml; R&D Systems Europe Ltd., Abingdon, UK), recombinant mouse SCF (50 ng/ml; PeproTech EC Ltd., London, UK), and recombinant human TGF-β1 (1 ng/ml; Sigma-Aldrich, Poole, UK) as described previously.
      • Pemberton AD
      • Brown JK
      • Wright SH
      • Knight PA
      • Miller HR
      The proteome of mouse mucosal mast cell homologues: the role of transforming growth factor β1.
      Viability was assessed using 0.2% nigrosin (Sigma-Aldrich) exclusion, and Mcpt-1 and -2 expression was assayed by enzyme-linked immunosorbent assay (ELISA) for up to 14 days ex vivo. Cytospins were prepared at 2- to 3-day intervals and stained with anti-Mcpt-1 antibody as described previously.
      • Pemberton AD
      • Brown JK
      • Wright SH
      • Knight PA
      • McPhee ML
      • McEuen AR
      • Forse PA
      • Miller HRP
      Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo.
      To assay the effects of TGF-β1 on BMMC protease transcription, BALB/c BMMCs were generated in the presence of recombinant mouse IL-3, IL-9, and SCF as described above with the addition of either recombinant human TGF-β1 (1 ng/ml; Sigma-Aldrich) or 1 μg/ml mouse anti-TGF-β1 IgG1 (clone 1D11; R&D Systems).
      • Pemberton AD
      • Brown JK
      • Wright SH
      • Knight PA
      • Miller HR
      The proteome of mouse mucosal mast cell homologues: the role of transforming growth factor β1.
      Samples for RNA extraction were collected from both sets of experiments as described previously.
      • Brown JK
      • Knight PA
      • Pemberton AD
      • Wright SH
      • Pate JA
      • Thornton EM
      • Miller HRP
      Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).

      Frequencies of Mast Cell Progenitors (MCPs) in β6−/− and β6+/+ Bone Marrow

      The MCP frequencies were determined by limiting dilution following a modification of a published method.
      • Brown JK
      • Donaldson DS
      • Wright SH
      • Miller HRP
      Mucosal mast cells and nematode infection: strain-specific differences in mast cell precursor frequency revisited.
      Briefly, bone marrow cells from β6+/+ and β6−/− mice were resuspended at 107 cells/ml in TI3S (1 ng/ml of recombinant human TGF-β1, 5 ng/ml of recombinant mouse IL-9, 1 ng/ml of recombinant mouse IL-3, and 50 ng/ml of recombinant mouse SCF). Serial dilutions, equivalent to 105, 104, 103, and 102 cells/well, were prepared in TI3S and plated out in replicates of 24 in 96-well plates. After 7 days wells were fed with 50 μl of TI3S and incubated for a further 7 days, after which cultures were harvested, freeze-thawed, and assayed for the presence of mature MMCs by Mcpt-2 ELISA.

      Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis

      Extraction of total RNA from jejunum, BMMCs, or isolated epithelium, and purification and removal of contaminating DNA using DNA-free DNase (Ambion Inc., Austin, TX) has been described previously.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      • Brown JK
      • Knight PA
      • Pemberton AD
      • Wright SH
      • Pate JA
      • Thornton EM
      • Miller HRP
      Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).
      For RT-PCR analysis, 1 μg of total RNA was reverse-transcribed as previously described, and 1/20th volume was amplified by PCR using gene-specific primers, with equivalent quantities of nonreverse-transcribed RNA as negative controls.
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      All reaction conditions were optimized to ensure the number of thermocycles used correlated with the amplification stage of the PCR. Primer sequences and product sizes are given in Table 1. Unless previously published, primers were designed from sequences in GenBank using the Primer 3 program available on http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi, checking specificity by searching the NCBI GenBank nucleotide database (http://www.ncbi.nlm.nih.gov). Amplifications for Mcpt-1, Mcpt-2, Mcpt-5, Mcpt-6, Mcpt-7, and carboxypeptidase A were performed for 40 seconds at 94°C, 40 seconds at 63°C, and 120 seconds at 72°C for 26 (Mcpt1, -2, -5), 30 (Mcpt-6, carboxypeptidase A), or 32 thermocycles (Mcpt-7) as appropriate. Amplifications for ATP5a, Mcpt-9, and SCF (KL-1/KL-2) were performed for 40 seconds at 94°C, 40 seconds at 55°C, and 120 seconds at 72°C for 28 thermocycles (ATP5a), 34 thermocycles (Mcpt-9), or 40 thermocycles (SCF KL-1/KL-2). Amplifications for αV and β6 integrin subunits were performed for 40 seconds at 94°C, 40 seconds at 52°C, and 120 seconds at 72°C for 32 thermocycles (αV) or 36 thermocycles (β6). PCR products were visualized on ethidium bromide-stained 1.6% agarose gels and images recorded and analyzed using a Kodak Digital Science Image Station 440CF and 1D Image Analysis software (Eastman-Kodak, Rochester, NY). To confirm the identity of the PCR products, some gels were subjected to Southern blotting following standard protocols and hybridization using digoxigenin-labeled internal gene-specific probes as described previously.
      • Wastling JM
      • Knight P
      • Ure J
      • Wright S
      • Thornton EM
      • Scudamore CL
      • Mason J
      • Smith A
      • Miller HRP
      Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the β-chymase, mouse mast cell protease-1.
      Table 1Primer Sequences Used in Standard RT-PCR for Identifying Protease Gene Transcripts
      GenePrimersPCR product size
      Mcpt-1
      Primer sequences from Wastling et al.41
      Forward: 5′-GGAAAACTGGAGAGAAAGAACCTAC-3′460 bp
      Reverse: 5′-GACAGCTGGGACAGAATGGGG-3′
      Mcpt-2
      Primer sequences from Wastling et al.41
      Forward: 5′-ATTTCATTGCCTAGTTCCTCTGAC-3′525 bp
      Reverse: 5′-CAGGATGAGAACAGGCTGGGAT-3′
      Mcpt-5
      Primer sequences from Wastling et al.41
      Forward: 5′-GGCAGAACAAACGTGAATGAGCC-3′418 bp
      Reverse: 5′-AAGAACCTTCTGGAAGCTCAGGG-3′
      Mcpt-6Forward: 5′-GCTCCTCTCTTTGAACCGGATC-3′212 bp
      Reverse: 5′-GGTGGGAGAGGCTCGTCATTA-3′
      Mcpt-7Forward: 5′-GGCTGGGGTAACATCGACAAT-3′317 bp
      Reverse: 5′-CAAGTAATAGGTGACCCGGGTGTA-3′
      Mcpt-9Forward: 5′-TTCCAAGTTCAATGACATCGTATTAC-3′159 bp
      Reverse: 5′-GATACTTTTTTTCACTCCAGTTCGC-3′
      CPA
      Primer sequences from Wastling et al.41
      Forward: 5′-ACACAGGATCGAATGTGGAG-3′689 bp
      Reverse: 5′-TAATGCAGGACTTCATGAGC-3′
      SCF (KL-1 and KL-2)
      Primers designed to distinguish between the two splice variants of SCF.
      Forward: 5′-TCCGAAGAGGCCAGAAACTA-3′368 bp (KL-1)
      Reverse: 5′-CAACTGCCCTTGTAAGACTTGA-3′284 bp (KL-2)
      αVForward: 5′-GAAGTTACTTCGGATTCGCCG-3′203 bp
      Reverse: 5′-CATCTTTGGCATAATCTCGATTGC-3′
      β6
      Primer sequences from Huang et al.28
      Forward: 5′-CAGTTCTGACATTGTTCAGA-3′356 bp
      Reverse: 5′-AGTTAATGGCAAAATGTGCT-3′
      ATP5a (ATP synthase H: ATP5a1)Forward: 5′-CTGTACTGCATCTACGTCGCGA-3′230 bp
      Reverse: 5′-AGCCTGCTTGGATAGATCGTCATA-3′
      * Primer sequences from Wastling et al.
      • Wastling JM
      • Knight P
      • Ure J
      • Wright S
      • Thornton EM
      • Scudamore CL
      • Mason J
      • Smith A
      • Miller HRP
      Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the β-chymase, mouse mast cell protease-1.
      Primers designed to distinguish between the two splice variants of SCF.
      Primer sequences from Huang et al.
      • Huang XZ
      • Wu JF
      • Cass D
      • Erle DJ
      • Corry D
      • Young SG
      • Farese RV
      • Sheppard D
      Inactivation of the integrin β6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lungs and skin.

      Real-Time RT-PCR Analysis of Transcripts in Epithelial RNA

      Standard housekeeping genes (GAPDH, creatine kinase, β-actin, ATP5a) were found to vary significantly in isolated epithelium (P.A.K., unpublished observations),
      • Knight PA
      • Pemberton AD
      • Robertson KA
      • Roy DJ
      • Wright SH
      • Miller HRP
      Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis.
      precluding the use of the formula described above for analyzing transcripts in isolated epithelium. We therefore set up quantitative real-time RT-PCR assays for the cytokines SCF, IL-4, IL-7, IL-13, and TGF-β1, generating PCR products to act as standards. All primer sequences and product sizes are given in Table 2. PCR products to act as standards for each transcript were generated from reverse-transcribed β6+/+-isolated epithelium (day 12 after infection), using the external primer sets, carrying out amplifications for 40 seconds at 94°C, 40 seconds at 55°C, and 120 seconds at 72°C for 40 thermocycles. PCR products were purified using a Roche High Pure PCR product purification kit (Roche, Lewes, UK) according to the manufacturer's instructions. PCR products were quantified and 10-fold dilutions set up to generate a standard curve, checking efficiency in the real-time PCR assays with the internal primers.
      Table 2Primer Sequences Used in Real-Time PCR for Identifying Cytokine Transcripts
      GeneInternal primersPCR product sizeExternal primersPCR product size
      SCFForward: 5′-GAAGTCAGTCTTTTCCTTGACAGT-3′
      Primer sequences from Sun et al.69
      70 bp
      Identical from both splice variants.
      Forward: 5′-ACGTGGACCAGTGGAAGAAC-3′181 bp
      Identical from both splice variants.
      Reverse: 5′-GCATGTCACATTATACTATTGCAAACA-3′
      Primer sequences from Sun et al.69
      Reverse: 5′-TTGCACATTCAGCATTCCTC-3′
      IL-4Forward: 5′-ACAGGAGAAGGGACGCCAT-3′
      Primer sequences from Overbergh et al.70
      95 bpForward: 5′-CCCCAGCTAGTTGTCATCCT-3′250 bp
      Reverse: 5′- GAAGCCCTACAGACGAGCTCA-3′
      Primer sequences from Overbergh et al.70
      Reverse: 5′-GCATGGAGTTTTCCCATGTT-3′
      IL-13Forward: 5′-AGACCAGACTCCCCTGTGCA-3′
      Primer sequences from Overbergh et al.70
      123 bpForward: 5′-TTGAGGAGCTGAGCAACATC-3′173 bp
      Reverse: 5′-TGGGTCCTGTAGATGGCATTG-3′
      Primer sequences from Overbergh et al.70
      Reverse: 5′-GGTTACAGAGGCCATGCAAT-3′
      IL-7Forward: 5′-ATTATGGGTGGTGAGAGCCG-3′
      Primer sequences from Overbergh et al.70
      231 bpForward: 5′-GGAGTCAGCTGCCTGAACTG-3′334 bp
      Reverse: 5′-GTTCCTGTCATTTTGTCCAATTCA-3′
      Primer sequences from Overbergh et al.70
      Reverse: 5′-TGGTTCATTATTCGGGCAAT-3′
      TGF-β1Forward: 5′-TGACGTCACTGGAGTTGTACGG-3′
      Primer sequences from Overbergh et al.70
      140 bpForward: 5′-ATACGCCTGAGTGGCTGTCT-3′214 bp
      Reverse: 5′-GGTTCATGTCATGGATGGTGC-3′
      Primer sequences from Overbergh et al.70
      Reverse: 5′-GCCATGAGGAGCAGGAAG-3′
      For production of PCR products as standards, external primers were used to generate RT-PCR products slightly larger than that generated by the internal primers used in the assay.
      * Primer sequences from Sun et al.
      • Sun LX
      • Lee JW
      • Fine HA
      Neuronally expressed stem cell factor induces neural stem cell migration to areas of brain injury.
      Identical from both splice variants.
      Primer sequences from Overbergh et al.
      • Overbergh L
      • Valckx D
      • Waer M
      • Mathieu C
      Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.
      For real-time RT-PCR analysis, 1 μg of total RNA from isolated epithelium from β6+/+ and β6−/− mice collected on day 0 and day 12 after infection was reverse-transcribed as previously described.
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      One hundred ng of cDNA or appropriate concentration of standard was amplified using 0.3 μmol/L of primers in 20 μl of SYBR Green master mix (2.5 mmol/L MgCl2) (Qiagen, Valencia, CA). PCR conditions were as follows: 95°C for 15 minutes, then 50 thermocycles of 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Reactions were performed in triplicate. Melting curves were calculated for each reaction to confirm identity of the PCR product and to ensure primer-dimer formation did not occur. Master mix only controls were included in all reactions as blanks, along with RNA only controls to check for DNA contamination. Cycle thresholds (Ct) and copy number were calculated using Opticon 2 software, correcting fluorescence values against background and blanks, with subsequent analysis performed in Microsoft Excel and using the InStat statistics package (GraphPad Software, Inc., San Diego, CA).

      Results

      MMC Distribution and Granule Protease Expression in Normal and Parasitized Jejunum of β6−/− and β6+/+ Mice

      Age- and sex-matched β6−/− and β6+/+ S129 mice (n = 4 to 6 mice/group/time point) were each infected with 250 T. spiralis L3 in four separate experiments to i) investigate infection kinetics and Mcpt-1 quantification by ELISA (experiments 1 and 2), ii) provide material for histological examination of tissues (experiment 3), and iii) isolate jejunal epithelium (experiment 4, day 0 and day 12 after infection only).
      Recruitment of esterase+ MMCs to the jejunal epithelium increased significantly during infection in S129 β6+/+ mice, whereas recruitment to this site was significantly compromised in infected β6−/− mice (Table 3 and Figure 2a). In β6−/− mice, MMCs accumulated in the lamina propria during infection as described previously.
      • Brown JK
      • Knight PA
      • Pemberton AD
      • Wright SH
      • Pate JA
      • Thornton EM
      • Miller HRP
      Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).
      In the current study, the numbers of toluidine blue+ and esterase+ MMCs were very similar (data not shown). Reduced recruitment of MMCs into the epithelium of β6−/− mice was associated with significantly (P ≤ 0.01) reduced numbers of Mcpt-1+ and Mcpt-2+ MMCs and with enhanced numbers of tryptase+ cells in the lamina propria (P ≤ 0.05) (Figure 1, Figure 2). This was concomitant with significantly (P ≤ 0.01) reduced expression of Mcpt-1 and Mcpt-2 in jejunal extracts (Figure 2, e and f). Transcripts for Mcpt-1 and Mcpt-2 were reduced, and Mcpt-5 and Mcpt-6 were enhanced in β6−/− jejunum, whereas there were no obvious differences in Mcpt-7 and carboxypeptidase A (Figure 3a). Interestingly, transcripts for Mcpt-9, which, like those for Mcpt-1 and Mcpt-2, are up-regulated during nematode infection by intraepithelial MMCs,
      • Hunt JE
      • Friend DS
      • Gurish MF
      • Feyfant E
      • Sali A
      • Huang C
      • Ghildyal N
      • Stechschulte S
      • Austen KF
      • Stevens RL
      Mouse mast cell protease 9, a novel member of the chromosome 14 family of serine proteases that is selectively expressed in uterine mast cells.
      • Friend DS
      • Ghildyal N
      • Gurish MF
      • Hunt J
      • Hu XZ
      • Austen KF
      • Stevens RL
      Reversible expression of tryptases and chymases in the jejunal mast cells of mice infected with Trichinella spiralis.
      were completely undetectable in β6−/− jejunum (Figure 3a) and in jejunal epithelium (Figure 3b), whereas Mcpt-9 was up-regulated in β6+/+ jejunal epithelium during T. spiralis infection, concomitantly with Mcpt-1 (Figure 3, a and b).
      Table 3Esterase+ MMC per Villus Crypt Unit (vcu) (±SE) in the Jejunum from β6+/+ and β6−/− Mice on Days 0 (Uninfected), 12, 16, and 28 after Infection with T. spiralis (n = 4 to 6)
      EpitheliumLamina propria
      β6+/+β6−/−β6+/+β6−/−
      Day 00.11 (±0.11)0.05 (±0.06)0.04 (±0.06)0.08 (±0.06)
      Day 129.44 (±3.2)1.45 (±0.30)
      P < 0.01; β6+/+ versus β6−/− Mann-Whitney nonparametric test.
      1.92 (±0.91)3.56 (±3.1)
      P < 0.01; β6+/+ versus β6−/− Mann-Whitney nonparametric test.
      Day 168.05 (±1.03)1.22 (±0.29)
      P < 0.01; β6+/+ versus β6−/− Mann-Whitney nonparametric test.
      2.46 (±0.65)8.7 (±1.61)
      P < 0.05,
      Day 285.22 (±0.51)1.7 (±0.47)
      P < 0.01; β6+/+ versus β6−/− Mann-Whitney nonparametric test.
      2.45 (±0.77)8.29 (±1.36)
      P < 0.01; β6+/+ versus β6−/− Mann-Whitney nonparametric test.
      * P < 0.05,
      P < 0.01; β6+/+ versus β6−/− Mann-Whitney nonparametric test.
      Figure thumbnail gr2
      Figure 2Mast cell counts in the jejunum from uninfected (day 0) and T. spiralis-infected (day 12 after infection) β6+/+ (filled bars) and β6−/− (open bars) mice (+SE) stained for esterase (a), tryptase (b), Mcpt-1 (c), and Mcpt-2 (d), and levels of Mcpt-1 (e) and Mcpt-2 (f) in the jejunum measured by ELISA, as described in Materials and Methods. *P < 0.05, **P < 0.01; Mann-Whitney nonparametric test. Note that reduced recruitment of MMCs into the epithelium of β6−/− mice is associated with significantly reduced Mcpt-1 and Mcpt-2 expression and enhanced tryptase expression in the lamina propria.
      Figure thumbnail gr1
      Figure 1Sections of jejunum from T. spiralis-infected (day 12 after infection) β6+/+ (A, C, and E) and β6−/− (B, D, and F) mice stained for Mcpt-1 (A and B), Mcpt-2 (C and D), and tryptase (E and F). Staining techniques are described in Materials and Methods. Positively stained MMCs are indicated by arrows. Note the decreased numbers of intraepithelial Mcpt-1+ and Mcpt-2+ MMCs, and increased numbers of tryptase+ MMCs, in β6−/− mice compared with controls. Scale bars = 25 μm.
      Figure thumbnail gr3
      Figure 3a: RT-PCR products for Mcpt-1(M1), Mcpt-2 (M2), Mcpt-5 (M5), Mcpt-6 (M6), Mcpt-7 (M7), Mcpt-9 (M9), carboxypeptidase A (CPA), and housekeeping gene (ATP5a) (HG) from RNA isolated from β6−/− and β6+/+ jejunum on day 12 of infection. Each lane represents a sample from a single mouse (n = 4 per group). Note transcripts for Mcpt-1 (M1) and Mcpt-2 (M2) are reduced, and Mcpt-5 (M5) and Mcpt-6 (M6) are enhanced in β6−/− jejunum, whereas there were no obvious differences in Mcpt-7 (M7) and carboxypeptidase A (CPA). Interestingly, transcripts for Mcpt-9 (M9) were completely undetectable in β6−/− jejunum. b: RT-PCR products for Mcpt-9 (M9) and housekeeping gene (ATP5a) (HG) from RNA isolated from epithelium from β6−/− and β6+/+ jejunum of uninfected controls (day 0) and from day 12 of infection. Each lane represents a sample from a single mouse (n = 4 per group). Note that transcripts for Mcpt-9 (M9) were up-regulated in the jejunal epithelium of β6+/+ mice but were completely undetectable in β6−/− epithelium. c: RT-PCR products for Mcpt-1(M1), Mcpt-2 (M2), Mcpt-5 (M5), Mcpt-6 (M6), Mcpt-7 (M7), Mcpt-9 (M9), and housekeeping gene (ATP5a) (HG) from RNA isolated from BMMCs cultured for 14 days in IL-3, IL-9, SCF, and TGF-β1 or in the absence of TGF-β1 and the addition of anti-TGF-β antibody (n = 4 per group; see Materials and Methods for details). Note that transcripts for Mcpt-1(M1) and Mcpt-2 (M2) are almost undetectable in the absence of TGF-β1, whereas there was no obvious difference in levels of transcripts for Mcpt-5 (M5), Mcpt-6 (M6), Mcpt-7 (M7), and Mcpt-9 (M9).

      In Vitro Expression of Granule Proteases by BMMCs—the Role of TGF-β1

      Previous studies have established a major role for TGF-β1 in the differentiation of bone marrow cells into MMC homologues expressing Mcpt-1 and -2.
      • Miller HR
      • Wright SH
      • Knight PA
      • Thornton EM
      A novel function for transforming growth factor-β1: upregulation of the expression and the IgE-independent extracellular release of a mucosal mast cell granule-specific β-chymase, mouse mast cell protease-1.
      • Brown JK
      • Knight PA
      • Wright SH
      • Thornton EM
      • Miller HR
      Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues.
      • Wright SH
      • Brown J
      • Knight PA
      • Thornton EM
      • Kilshaw PJ
      • Miller HR
      Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues.
      • Pemberton AD
      • Brown JK
      • Wright SH
      • Knight PA
      • Miller HR
      The proteome of mouse mucosal mast cell homologues: the role of transforming growth factor β1.
      To determine whether the aberrant protease phenotype of the MMCs recruited into the lamina propria might be attributed entirely to lack of active TGF-β1, or whether there are additional factors operating within the mucosa, we compared protease profiles in BMMCs cultured for 14 days in IL-3, IL-9, and SCF in the presence (TGF-β+) or absence (TGF-β) of exogenous TGF-β1 and with the addition of anti-TGF-β1 antibody to block endogenous TGF-β1. Although transcripts for Mcpt-1 and Mcpt-2 were almost undetectable in TGF-β) cultures, there were no obvious differences in levels of transcription of Mcpt-5, -6, -7, and -9 (Figure 3c). Real-time RT-PCR confirmed that transcription of Mcpt-9 was independent of TGF-β1in vitro (data not shown), and therefore its down-regulation in the β6−/− jejunum seems to be unrelated to activation of TGF-β1.

      Effect of the β6 Integrin Deletion on Mast Cells in Stomach, Colon, and Ear Pinnae

      To determine whether the absence of β6 integrin affected mast cell distribution throughout the gastrointestinal tract, the distribution of MMCs and the expression of their granule proteases were compared in the glandular stomach and in the colon of normal and β6−/− mice. Uninfected β6−/− mice have significantly (P ≤ 0.05) higher baseline numbers of esterase+ MMCs in the glandular stomach than β6+/+ controls (Table 4), but there was no difference in the number of gastric MMCs in T. spiralis-infected β6+/+ and β6−/− mice (day 12 after infection) (Table 4). This is in accordance with our previous observations with N. brasiliensis-infected β6−/− mice.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      However, infected β6−/− mice had significantly (P ≤ 0.05) reduced numbers of Mcpt-1+ MMCs, with a trend toward fewer Mcpt-2+ and more tryptase+ MMCs (Table 4). β6+/+ mice showed a significant (P ≤ 0.05) increase in numbers of esterase+, predominantly Mcpt-1+ MMCs in the colon after T. spiralis infection (Table 4). The population of MMCs in the colons of infected β6−/− mice was significantly reduced compare to that of infected β6+/+ controls, and this was associated with reduced Mcpt-1 expression. To determine whether mast cells elsewhere in the body were affected, the numbers of esterase+ connective tissue mast cells in the ear pinnae were compared in T. spiralis-infected and uninfected β6−/− and β6+/+ mice. The absence of β6 had no effect on this population of connective tissue mast cells in the ear (Table 4).
      Table 4Numbers of Mast Cells in the Stomach, Colon, and Ear Pinna from Uninfected (Day 0) and T. spiralis-Infected (Day 12 after Infection) β6+/+ and β6−/− Mice (±SE)
      Day 0Day 12
      β6+/+β6−/−β6+/+β6−/−
      Stomach
       Esterase+5.63 (±0.97)38.5 (±7.8)
      P < 0.05,
      51.6 (±7.7)65.7 (±7.3)
       Mcp-1+0.1 (±0.1)0.0 (±0.0)25.7 (±5.9)2.7 (±0.7)
      P < 0.01; Mann-Whitney nonparametric test.
       Mcpt-2+3.9 (±1.2)3.8 (±0.6)37.5 (±10.3)22.5 (±5.4)
       Tryptase+29.4 (±5.5)31.7 (±6.9)25.3 (±6.3)57.9 (±13.0)
      Colon
       Esterase+2.3 (±0.9)2.7 (±0.4)65.8 (±27.2)19.3 (±2.2)
      P < 0.05,
       Mcp-1+0.42 (±0.2)0 (±0)59.6 (±25.5)0.67 (±0.3)
      P < 0.05,
       Mcpt-2+0.2 (±0.2)0.3 (±0.2)20.8 (±19.6)6.2 (±4.0)
       Tryptase+29.4 (±5.5)31.8 (±6.9)25.3 (±6.3)57.9 (±13)
      Ear pinna
       Esterase+104.8 (±14.9)98.67 (±7.1)84.8 (±6.7)91.25 (±8.9)
       Mcp-1+0.0 (±0.0)0.0 (±0.0)0.0 (±0.0)0.0 (±0.0)
       Mcpt-2+0.0 (±0.0)0.0 (±0.0)0.0 (±0.0)0.0 (±0.0)
       Tryptase+29.4 (±5.5)47.7 (±8.6)41.3 (±2.8)39.2 (±9.3)
      β6+/+ versus β6−/−;
      * P < 0.05,
      P < 0.01; Mann-Whitney nonparametric test.

      Comparison of Cultured MMC Homologues Derived from Bone Marrow of β6−/− and β6+/+ Mice

      The possibility that systemic mechanisms contribute to the aberrant location and protease expression of MMCs in β6−/− mice was investigated. One possibility is that MMCs in β6−/− mice are inherently unable to differentiate and/or there is a difference in mast cell precursor (MCP) frequencies. To test this, we compared the development of MMC homologues from bone marrow cultures derived from β6+/+ and β6−/− mice. The frequencies of bone marrow-derived MCPs were assessed using a modification of a published technique,
      • Brown JK
      • Donaldson DS
      • Wright SH
      • Miller HRP
      Mucosal mast cells and nematode infection: strain-specific differences in mast cell precursor frequency revisited.
      • Weller CL
      • Collington SJ
      • Brown JK
      • Miller HRP
      • Al-Kashi A
      • Clark P
      • Jose PJ
      • Hartnell A
      • Williams TJ
      Leukotriene B-4, an activation product of mast cells, is a chemoattractant for their progenitors.
      and bone marrow from β6+/+ and β6−/− mice had similar MCP frequencies (Figure 4a). BMMCs from both strains were equivalent in their viability (Figure 4b) and their capacity to secrete Mcpt-1 and -2 into the culture supernatant (Figure 4c). There was equivalent expression of Mcpt-1 and Mcpt-2 in cell pellets harvested at day 14 (data not shown). BMMCs from β6+/+ and β6−/− mice cultured in the presence of TGF-β1 showed equivalent expression of the proteases Mcpt-1, -2, -5, -6, and -7 (Figure 4d), as well as the integrin αV(Figure 4e), in accordance with previous data showing that integrin αV is expressed by murine BMMCs.
      • Castells MC
      • Klickstein LB
      • Hassani K
      • Cumplido JA
      • Lacouture ME
      • Austen KF
      • Katz HR
      gp49BI-αvβ3 interaction inhibits antigen-induced mast cell activation.
      β6+/+, but not β6−/−, BMMCs showed barely detectable expression of the integrin β6. Overall these results suggest that there is no deficiency at the level of the bone marrow that would explain the aberrant protease expression and distribution of MMCs in the parasitized jejunum of β6−/− mice.
      Figure thumbnail gr4
      Figure 4a: Limiting dilution analysis of MCP frequency in bone marrow from β6+/+ (filled bars) and β6−/− (open bars) mice cultured in the cytokines described above, as assessed by Mcpt-2 ELISA (see Materials and Methods for details). NS, not significant. b: Viability of BMMCs from β6+/+ (filled bars) and β6−/− (open bars) mice (+SE) cultured for 14 days in the presence of TGF-β1, IL-3, IL-9, and SCF (see Materials and Methods for details) measured using an Improved Neubauer counting chamber and nigrosin exclusion. c: ELISA analysis of Mcpt-2 secretion into BMMC culture supernatant. d: RT-PCR products for Mcpt-1 (M1), Mcpt-2 (M2), Mcpt-5 (M5), Mcpt-6 (M6), Mcpt-7 (M7), Mcpt-9 (M9), and housekeeping gene ATP5a (HG) from β6+/+ and β6−/− BMMC RNA as indicated. e: RT-PCR products for αV, β6, and ATP5a (HG) from β6+/+ and β6−/− BMMC RNA as indicated, together with negative (no RT) and positive (β6+/+ epithelium) controls.

      Expression of Cytokines in Normal and Parasitized β6−/− and β6+/+ Jejunal Epithelium and Mucosa

      SCF is vital for the generation of intestinal mastocytosis observed during T. spiralis infection
      • Donaldson LE
      • Schmitt E
      • Huntley JF
      • Newlands GFJ
      • Grencis RK
      A critical role for stem cell factor and c-kit in host protective immunity to an intestinal helminth.
      • Grencis RK
      • Else KJ
      • Huntley JF
      • Nishikawa SI
      The in vivo role of stem-cell factor (c-Kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice.
      and may be chemotactic for intestinal MMCs.
      • Pennock JL
      • Grencis RK
      In vivo exit of c-kit+/CD49dhi/β7+ mucosal mast cell precursors from the bone marrow following infection with the intestinal nematode Trichinella spiralis.
      Because MMCs in wild-type mice predominantly localize in the epithelium and have access to SCF on the lateral membranes of enterocytes,
      • Klimpel GR
      • Langley KE
      • Wypych J
      • Abrams JS
      • Chopra AK
      • Niesel DW
      A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.
      we examined the expression of this cytokine within the jejunal epithelium. Real-time PCR was used to quantify the abundance of transcripts for SCF in jejunal epithelium isolated from β6+/+ and β6−/− mice on day 0 and on day 12 after infection with T. spiralis(Figure 5a). Transcripts encoding SCF were significantly up-regulated in the epithelium of T. spiralis-infected β6−/− mice in comparison to the constitutive expression seen in β6+/+ controls (P ≤ 0.05) (Figure 5a). There were no significant differences in the levels of SCF transcripts detected in whole jejunum from T. spiralis-infected β6+/+ and β6−/− mice using real-time PCR (data not shown). RT-PCR using primers that distinguish between the two splice variants of SCF, KL-1 and KL-2, demonstrated an increased transcription of KL-1 with respect to KL-2 in β6−/− epithelium (Figure 5, b and c).
      Figure thumbnail gr5
      Figure 5a: Abundance of SCF transcripts from RNA from β6+/+ (filled bars) and β6−/− (open bars) isolated jejunal epithelium taken on day 0 and day 12 after T. spiralis infection, as quantified by real-time PCR (infected versus uninfected, β6+/+ versus β6−/−; *P < 0.05, Mann-Whitney nonparametric test. NS, not significant). b: RT-PCR products for KL-1 and KL-2 splice variants of SCF from RNA isolated from β6−/− and β6+/+ epithelium on day 12 of T. spiralis infection, as indicated. Each lane represents a sample from a single mouse (n = 4 per group). c: Ratios of KL-1 RT-PCR products with respect to KL-2. See Materials and Methods for details.
      T. spiralis-induced MMC hyperplasia is dependent, among other factors, on the Th2 cytokines IL-4 and IL-13,
      • Helmby H
      • Grencis RK
      IL-18 regulates intestinal mastocytosis and Th2 cytokine production independently of IFN-gamma during Trichinella spiralis infection.
      • McDermott JR
      • Humphreys NE
      • Forman SP
      • Donaldson DD
      • Grencis RK
      Intraepithelial NK cell-derived IL-13 induces intestinal pathology associated with nematode infection.
      • Urban JF
      • Schopf L
      • Morris SC
      • Orekhova T
      • Madden KB
      • Betts CJ
      • Gamble HR
      • Byrd C
      • Donaldson D
      • Else K
      • Finkelman FD
      Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T cell-dependent mechanism.
      which are also products of mast cells.
      • Finkelman FD
      • Urban JF
      The other side of the coin: the protective role of the T(H)2 cytokines.
      • Grencis RK
      Cytokine regulation of resistance and susceptibility to intestinal nematode infection—from host to parasite.
      Therefore we quantified the epithelial expression of these cytokines in comparison with TGF-β1 and of IL-7, which is highly expressed by intestinal epithelial cells.
      • Jiang Y
      • McGee DW
      Regulation of human lymphocyte IL-4 secretion by intestinal epithelial cell-derived interleukin-7 and transforming growth factor-β.
      Transcripts for IL-4 and IL-13 were undetectable in uninfected β6+/+ and β6−/− epithelium but increased significantly (P ≤ 0.05) in both groups on day 12 of infection (Figure 6, a and b). Transcription of IL-4 and IL-13 was, however, comparable in the epithelium of β6+/+ and β6−/− mice. Levels of TGF-β1 remained constant in uninfected and T. spiralis-infected β6+/+ and β6−/− mice (Figure 6d), in accordance with our previous observations.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      Interestingly, the epithelial cytokine IL-7 was significantly up-regulated in infected β6−/− mice but remained at uninfected levels in β6+/+ controls (Figure 6c). This may relate to the fact that IL-7 mRNA can be down-regulated by TGF-β1 in some cell types.
      • Tang JH
      • Nuccie BL
      • Ritterman I
      • Liesveld JL
      • Abboud CN
      • Ryan DH
      TGF-β down-regulates stromal IL-7 secretion and inhibits proliferation of human B cell precursors.
      Figure thumbnail gr6
      Figure 6Abundance of transcripts for IL-4 (a), IL-13 (b), IL-7 (c), and TGF-β1 (d) as indicated, from RNA from β6+/+ (filled bars) and β6−/− (open bars) isolated jejunal epithelium taken on day 0 and day 12 after T. spiralis infection, as quantified by real-time PCR (infected versus uninfected, β6+/+ versus β6−/−; *P < 0.05, Mann-Whitney nonparametric test. NS, not significant).

      Comparison of TH2-Driven Inflammation and of CD3+ T-Cell Populations in the Parasitized Jejunum of β6−/− and β6+/+ Mice

      There is evidence to suggest that many components of a complex TH2-driven response, including goblet cell-derived mucins, locally derived T cells, and eosinophils play a role in elimination of worms in addition to MMCs and Mcpt-1.
      • Miller HRP
      Mucosal mast cells and the allergic response against nematode parasites.
      • Finkelman FD
      • Shea-Donohue T
      • Morris SC
      • Gildea L
      • Strait R
      • Madden KB
      • Schopf L
      • Urban JF
      Interleukin-4- and interleukin-13-mediated host protection against intestinal nematode parasites.
      • Khan WI
      • Blennerhasset P
      • Ma C
      • Matthaei KI
      • Collins SM
      Stat6 dependent goblet cell hyperplasia during intestinal nematode infection.
      • Miller HRP
      Gastrointestinal mucus, a medium for survival and for elimination of parasitic nematodes and protozoa.
      Therefore we assessed the numbers of goblet cells, eosinophils, and CD3+ T cells in the jejunum of T. spiralis-infected (day 12 after infection) and control β6+/+ and β6−/− mice (Table 5). Goblet cell and eosinophil numbers increased to similar levels in infected β6−/− jejunum and β6+/+ controls. This is in accordance with previous observations with N. brasiliensis-infected β6−/− mice.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      There was no overall increase in CD3+ T cells in either group, also in accordance with previous observations.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      However, β6−/− mice had reduced numbers of CD3+ T cells in the epithelium with slightly increased numbers in the lamina propria (β6+/+ versus β6−/−; *P ≤ 0.01 day 12 after infection Mann-Whitney nonparametric test).
      Table 5Numbers of Goblet Cells, Eosinophils, and CD3+ Lymphocytes in the Jejunum from Uninfected (Day 0) and T. spiralis-Infected (Day 12 after Infection) β6+/+ and β6−/− Mice (±SE)
      Day 0Day 12
      β6+/+β6−/−β6+/+β6−/−
      Goblet cells9.84 (±0.34)10.32 (±0.86)16.98 (±1.77)17.93 (±0.58)
      Eosinophils34.8 (±7.2)57.9 (±15.5)152.32 (±31.0)159 (±32.1)
      CD3+ lymphocytes
       Epithelium8.74 (±0.9)6.3 (±0.98)11.05 (±2.12)3.6 (±0.25)
      P < 0.01; Mann-Whitney nonparametric test.
       Lamina propria4.05 (±0.38)4.71 (±0.59)5.77 (±0.65)8.64 (±0.65)
      P < 0.05,
      β6+/+ versus β6−/−; NS, not significant. Note that goblet cell and eosinophil numbers increase normally in the β6−/− jejunum on infection, but β6−/− mice had reduced numbers of CD3+ T cells in the epithelium with slightly increased numbers in the lamina propria.
      * P < 0.05,
      P < 0.01; Mann-Whitney nonparametric test.

      Expulsion Kinetics of T. spiralis from β6−/− and β6+/+ Mice

      There were no significant differences in mean worm burdens at any stage of infection, between β6+/+ and β6−/− mice (Figure 7a), nor of total muscle larvae (β6+/+ 12,241 (±2181) β6−/− 16,628 ± 4602; day 35 after infection (NS), despite the significantly reduced concentrations of Mcpt-1 in the serum of β6−/− mice (Figure 7b) (days 13, 17, and 27; β6+/+ versus β6−/−; *P ≤ 0.05; Mann-Whitney nonparametric test). To check the expulsion kinetics of the S129 background and to verify the time points chosen were appropriate, SPF S129 control mice were simultaneously infected with the same dosage (Figure 7a). Although worm burdens were significantly reduced by day 13 in comparison to day 6 (P ≤ 0.05), in accordance with previous data from both S129 and BALB/c mice,
      • Lawrence CE
      • Paterson JCM
      • Higgins LM
      • MacDonald TT
      • Kennedy MW
      • Garside P
      IL-4-regulated enteropathy in an intestinal nematode infection.
      worms persisted until after day 20 after infection, indicating that T. spiralis worms are expelled more slowly in S129 mice than is generally observed in BALB/c mice at this dosage.
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      • Lawrence CE
      • Paterson JCM
      • Higgins LM
      • MacDonald TT
      • Kennedy MW
      • Garside P
      IL-4-regulated enteropathy in an intestinal nematode infection.
      Figure thumbnail gr7
      Figure 7a: Mean total worm burdens (+SE) from S129 β6+/+ (solid line) and β6−/− (dashed line) mice infected with 300 T. spiralis L3, typical of two experiments. Mean total worm burdens (+SE) are also shown from S129 mice infected with 300 T. spiralis L3 at the same time, to show expulsion kinetics (dotted line) (infected versus uninfected; *P < 0.05, **P < 0.01; Mann-Whitney nonparametric test). b: Concentrations of Mcpt-1 in the serum (μg/ml) from S129 β6+/+ (solid line) and β6−/− (dashed line) mice infected with T. spiralis (as shown in ) (β6+/+ versus β6−/−; *P < 0.05; Mann-Whitney nonparametric test).

      Discussion

      Here, we show compromised transcription and expression of the MMC-specific proteases Mcpt-1 and Mcpt-2 in the jejunum of T. spiralis-infected β6−/− mice. In contrast, expression of the MMC proteases Mcpt-5, Mcpt-6, and Mcpt-7, as measured by RT-PCR or anti-tryptase antibody, are unaffected or enhanced (Figure 1, Figure 2, Figure 3). These observations tally with the reduced recruitment of MMC to the epithelial compartment and their accumulation in the lamina propria, which increased during the course of infection (Table 3).
      • Brown JK
      • Knight PA
      • Pemberton AD
      • Wright SH
      • Pate JA
      • Thornton EM
      • Miller HRP
      Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).
      They are also consistent with the hypothesis that intraepithelial MMCs express the highly TGF-β1-dependent granule proteases Mcpt-1 and Mcpt-2
      • Miller HR
      • Wright SH
      • Knight PA
      • Thornton EM
      A novel function for transforming growth factor-β1: upregulation of the expression and the IgE-independent extracellular release of a mucosal mast cell granule-specific β-chymase, mouse mast cell protease-1.
      • Brown JK
      • Knight PA
      • Wright SH
      • Thornton EM
      • Miller HR
      Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues.
      because of their proximity to activated TGF-β1 in association with integrin αvβ6 on the lateral membranes of enterocytes. The reduced expression of Mcpt-1 in the glandular stomach and colon (Table 4) suggests that integrin αvβ6 activation of TGF-β1 may be important for epithelial MMC differentiation throughout the gastrointestinal tract, consistent with the epithelial expression of integrin αvβ6 in these tissues,
      • Ludlow A
      • Yee KO
      • Lipman R
      • Bronson R
      • Weinreb P
      • Huang XZ
      • Sheppard D
      • Lawler J
      Characterization of integrin β6 and thrombospondin-1 double-null mice.
      • Breuss JM
      • Gillett N
      • Lu L
      • Sheppard D
      • Pytela R
      Restricted distribution of integrin beta-6 messenger-RNA in primate epithelial tissues.
      which we have confirmed by RT-PCR (P.A.K., unpublished observations). However, as will be discussed below, there are a number of observations that indicate that the events in the epithelium are more complex than might be expected if the only function of αVβ6 on enterocytes was to activate latent TGF-β1.
      With regard to the MMC population in the parasitized jejunum there were three striking findings associated with the absence of β6 integrin: reduced expression of Mcpt-1 and -2, absence of expression of Mcpt-9, and failure of MMCs to enter the epithelium. We also see higher baseline numbers of gastric MMCs in β6−/− mice than in β6+/+ controls, which may reflect altered MCP development or recruitment. We therefore considered the possibility that the targeting of β6 integrin might have unintended consequences for mast cell development, perhaps through effects of the mutation on an adjacent locus, which directly affected mast cell differentiation and gene expression. However, this was not supported by our in vitro observations on the growth and differentiation of MMC homologs. BMMCs from β6−/− mice cultured in the presence of TGF-β1 differentiate and express Mcpt-1 and Mcpt-2 and the integrin αV at comparable levels with BMMCs from β6+/+ controls. It is worth noting that MCP frequency in bone marrow is similar in both β6−/− and β6+/+ mice, and connective tissue mast cells in the ear pinna are unaffected by the deletion of β6 integrin (Table 4 and Figure 4). Furthermore, Mcpt-9 was equally expressed by both β6+/+ and β6−/− BMMCs cultured in the presence of TGF-β1. Together, these in vivo and in vitro observations strongly suggest that it is unlikely that targeting the β6 integrin has any direct effect on MMCs or their precursors.
      It is unlikely that the failure to activate TGF-β1 is the sole mechanism that drives the aberrant mast cell response. Mcpt-9 is reported to be regulated in vitro by IL-10 and SCF,
      • Hunt JE
      • Friend DS
      • Gurish MF
      • Feyfant E
      • Sali A
      • Huang C
      • Ghildyal N
      • Stechschulte S
      • Austen KF
      • Stevens RL
      Mouse mast cell protease 9, a novel member of the chromosome 14 family of serine proteases that is selectively expressed in uterine mast cells.
      but in our studies transcript levels for Mcpt-9 were similar in cultures of MMC homologs supplemented with TGF-β1 and in the cultures of BMMCs grown without TGF-β1(Figure 3c). Neither of these cultures had exogenously added IL-10, although both were supplemented with SCF. Mcpt-9 was originally identified in uterine mast cells,
      • Hunt JE
      • Friend DS
      • Gurish MF
      • Feyfant E
      • Sali A
      • Huang C
      • Ghildyal N
      • Stechschulte S
      • Austen KF
      • Stevens RL
      Mouse mast cell protease 9, a novel member of the chromosome 14 family of serine proteases that is selectively expressed in uterine mast cells.
      and because in wild-type mice this protease is expressed by intraepithelial mast cells during T. spiralis infection, the lack of expression of Mcpt-9 in β6−/− mice may reflect the fact that few of the cells are intraepithelial. The assumption here is that there is a lack of an appropriate environmental signal, although this signal is apparently present in our BMMC cultures. On the other hand, expression of this protease may be down-regulated by other environmental signals in the lamina propria.
      A second striking finding that is likely to have a bearing on the differentiation of MMC was the substantial up-regulation of transcription of epithelial SCF in T. spiralis-infected β6−/− mice. This may be an additional outcome of decreased epithelial levels of active TGF-β1; TGF-β1 has been shown to inhibit SCF production by ovarian epithelial cells.
      • Ismail RS
      • Cada M
      • Vanderhyden BC
      Transforming growth factor-β regulates Kit ligand expression in rat ovarian surface epithelial cells.
      In wild-type mice, levels of epithelial SCF transcripts did not change on infection, in agreement with previous findings in mice infected with Nippostrongylus brasiliensis.
      • Rosbottom A
      • Knight PA
      • McLachlan G
      • Thornton EM
      • Wright SW
      • Miller HR
      • Scudamore CL
      Chemokine and cytokine expression in murine intestinal epithelium following Nippostrongylus brasiliensis infection.
      SCF is a key regulator of T. spiralis-induced intestinal mastocytosis,
      • Donaldson LE
      • Schmitt E
      • Huntley JF
      • Newlands GFJ
      • Grencis RK
      A critical role for stem cell factor and c-kit in host protective immunity to an intestinal helminth.
      • Grencis RK
      • Else KJ
      • Huntley JF
      • Nishikawa SI
      The in vivo role of stem-cell factor (c-Kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice.
      SCF is produced by the small intestinal epithelium
      • Rosbottom A
      • Knight PA
      • McLachlan G
      • Thornton EM
      • Wright SW
      • Miller HR
      • Scudamore CL
      Chemokine and cytokine expression in murine intestinal epithelium following Nippostrongylus brasiliensis infection.
      and has both systemic and local effects on mast cell development. In vivo and in vitro studies have shown SCF to have multiple effects on mast cell biology, including directing migration
      • Pennock JL
      • Grencis RK
      In vivo exit of c-kit+/CD49dhi/β7+ mucosal mast cell precursors from the bone marrow following infection with the intestinal nematode Trichinella spiralis.
      • Tan BL
      • Yazicioglu MN
      • Ingram D
      • McCarthy J
      • Borneo J
      • Williams DA
      • Kapur R
      Genetic evidence for convergence of c-Kit- and alpha(4) integrin-mediated signals on class IAPI-3kinase and the Rac pathway in regulating integrin-directed migration in mast cells.
      and, in conjunction with IL-3 and IL-4, mast cell growth and proliferation.
      • Tsuji K
      • Zsebo KM
      • Ogawa M
      Murine mast-cell colony formation supported by IL-3, IL-4, and recombinant rat stem-cell factor, ligand for c-Kit.
      The soluble form of SCF (KL-S) is predominantly generated by proteolytic cleavage from the transmembrane precursor KL-1, but less efficiently from KL-2, in which the major proteolytic cleavage site is removed by splicing.
      • Huang EJ
      • Nocka KH
      • Buck J
      • Besmer P
      Differential expression and processing of 2 cell associated forms of the Kit-ligand—Kl-1 and Kl-2.
      Preferential expression of KL-1 rather than KL-2 can be associated with increased production of the soluble form of SCF in epithelial cells.
      • Ismail RS
      • Cada M
      • Vanderhyden BC
      Transforming growth factor-β regulates Kit ligand expression in rat ovarian surface epithelial cells.
      • Huang EJ
      • Nocka KH
      • Buck J
      • Besmer P
      Differential expression and processing of 2 cell associated forms of the Kit-ligand—Kl-1 and Kl-2.
      Here, we have found that the KL-1 splice variant of SCF is preferentially expressed in infected β6−/− mice in relation to KL-2, possibly favoring local production of SCF that can be more readily cleaved to its soluble form. Local up-regulation of SCF, diffusing from the epithelium, may contribute to increased proliferation of MMCs in the lamina propria of β6−/− mice. This may partially explain the unusual MMC distribution observed in β6−/− mice, in addition to reduced clearance of MMCs via the epithelium.
      • Brown JK
      • Knight PA
      • Pemberton AD
      • Wright SH
      • Pate JA
      • Thornton EM
      • Miller HRP
      Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).
      Previous observations have shown that expulsion of the intraepithelial nematode T. spiralis is partially dependent on the presence of MMCs
      • Ha TY
      • Reed ND
      • Crowle PK
      Delayed expulsion of adult Trichinella spiralis by mast cell-deficient W/Wv mice.
      • Faulkner H
      • Humphreys N
      • Renauld JC
      • Van Snick J
      • Grencis R
      Interleukin-9 is involved in host protective immunity to intestinal nematode infection.
      • Mitchell LA
      • Wescott RB
      • Perryman LE
      Kinetics of expulsion of the nematode, Nippostrongylus brasiliensis, in mast-cell-deficient W/WV mice.
      • Nawa Y
      • Ishikawa N
      • Tsuchiya K
      • Horii Y
      • Abe T
      • Khan AI
      • Bing S
      • Itoh H
      • Ide H
      • Uchiyama F
      Selective effector mechanisms for the expulsion of intestinal helminths.
      and, specifically, of Mcpt-1.
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      • McDermott JR
      • Bartram RE
      • Knight PA
      • Miller HRP
      • Garrod DR
      • Grencis RK
      Mast cells disrupt epithelial barrier function during enteric nematode infection.
      However, the role of this chymase in worm expulsion was demonstrated in a rapid responder BALB/c strain of mouse onto which the Mcpt-1−/− genotype was back-crossed through 10 generations.
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      • McDermott JR
      • Bartram RE
      • Knight PA
      • Miller HRP
      • Garrod DR
      • Grencis RK
      Mast cells disrupt epithelial barrier function during enteric nematode infection.
      Our detailed kinetic studies in the 129 strain mice (Figure 7a) indicate that they may be a slower responder phenotype than the BALB/c strain,
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      with worms persisting until days 23 to 27 after infection. Furthermore, there was still low-level expression of Mcpt-1 in the jejunum, and 4 to 5 μg/ml Mcpt-1 were present in sera of β6−/− mice on days 13 to 17. This is in contrast to Mcpt-1−/− mice in which there is a complete absence of the protease, even though MMCs migrate normally into the epithelium.
      • Knight PA
      • Wright SH
      • Lawrence CE
      • Paterson YY
      • Miller HR
      Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
      The lack of effect on worm expulsion of the reduced numbers of intraepithelial MMCs and decreased expression of Mcpt-1 in β6−/− mice may, therefore, reflect the presence in the mucosa of significant quantities of Mcpt-1, albeit with systemic levels reduced by ∼70% compared with controls, and the possible contribution of additional effectors, such as tryptase, from the expanded MMC population in the lamina propria (Figure 2a).
      Despite the reduced numbers of intraepithelial MMCs and CD3+ T cells in β6−/− mice, there was no significant reduction of levels of IL-4 or IL-13, although there was a trend toward lower levels of IL-13. This implies that the intestinal epithelium itself, or other infiltrating cells, are a source of these cytokines; intraepithelial NK cells in T. spiralis-infected mice have recently been identified as a source of IL-13.
      • McDermott JR
      • Humphreys NE
      • Forman SP
      • Donaldson DD
      • Grencis RK
      Intraepithelial NK cell-derived IL-13 induces intestinal pathology associated with nematode infection.
      The consistent expression of TGF-β1 transcripts in the epithelium of β6+/+ and β6−/− mice, as well as the fact that levels are unchanged during infection, is in accordance with our previous observations.
      • Knight PA
      • Wright SH
      • Brown JK
      • Huang X
      • Sheppard D
      • Miller HR
      Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
      It is of interest that the epithelial cytokine IL-7 is significantly increased on infection in β6−/− mice. TGF-β1 and IL-7 have counter-regulatory effects in a number of cell types.
      • Ebert EC
      Inhibitory effects of transforming growth factor-β (TGF-β) on certain functions of intraepithelial lymphocytes.
      • Huang M
      • Sharma S
      • Zhu LX
      • Keane MP
      • Luo J
      • Zhang L
      • Burdick MD
      • Lin YQ
      • Dohadwala M
      • Gardner B
      • Batra RK
      • Strieter RM
      • Dubinett SM
      IL-7 inhibits fibroblast TGF-β production and signaling in pulmonary fibrosis.
      • Tsujimoto T
      • Lisukov IA
      • Huang NH
      • Mahmoud MS
      • Kawano MM
      Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells.
      TGF-β1 is known to down-regulate IL-7 mRNA,
      • Tang JH
      • Nuccie BL
      • Ritterman I
      • Liesveld JL
      • Abboud CN
      • Ryan DH
      TGF-β down-regulates stromal IL-7 secretion and inhibits proliferation of human B cell precursors.
      whereas IL-7 can down-regulate TGF-β1 mRNA.
      • Dubinett SM
      • Huang M
      • Dhanani S
      • Wang JY
      • Beroiza T
      Down-regulation of macrophage transforming growth-factor-β messenger RNA expression by IL-7.
      The function of this cytokine in nematode infection is not known.
      Here, we demonstrate that expression of the integrin ανβ6 is vital for normal MMC differentiation and protease expression in the gastrointestinal tract and affects epithelial transcription of the cytokines IL-7 and SCF in the epithelium. Our observations strongly support our hypothesis that the integrin ανβ6 regulates MMC differentiation in the gastrointestinal epithelium through local activation of TGF-β1, which may be spatially or cell-type specific. However, our observations also suggest that αVβ6 on enterocytes has diverse and as yet unknown functions in the epithelium apart from activation of latent TGF-β1.

      Acknowledgements

      We thank Kairbaan Hodivala-Dilke (Cell Adhesion and Disease Laboratory Guy's, King's, and St. Thomas' School of Medicine, St. Thomas' Hospital, London, UK) and Dean Sheppard (University of California, San Francisco, CA) for supplying the β6−/− breeding stock; and Eileen Duncan, Liz Moore, Neil McIntyre, and everyone at the Department of Pathology, Easter Bush Veterinary Centre, University of Edinburg, for technical support.

      References

        • Artis D
        • Humphreys NE
        • Bancroft AJ
        • Rothwell NJ
        • Potten CS
        • Grencis RK
        Tumor necrosis factor alpha is a critical component of interleukin 13-mediated protective T helper cell type 2 responses during helminth infection.
        J Exp Med. 1999; 190: 953-962
        • Ferguson A
        • Cummins AG
        • Munro GH
        • Gibson S
        • Miller HRP
        Roles of mucosal mast cells in intestinal cell mediated immunity.
        Ann Allergy. 1987; 59: 40-43
        • Miller HRP
        Mucosal mast cells and the allergic response against nematode parasites.
        Vet Immunol Immunopathol. 1996; 54: 331-336
        • Ha TY
        • Reed ND
        • Crowle PK
        Delayed expulsion of adult Trichinella spiralis by mast cell-deficient W/Wv mice.
        Infect Immun. 1983; 41: 445-447
        • Faulkner H
        • Humphreys N
        • Renauld JC
        • Van Snick J
        • Grencis R
        Interleukin-9 is involved in host protective immunity to intestinal nematode infection.
        Eur J Immunol. 1997; 27: 2536-2540
        • Mitchell LA
        • Wescott RB
        • Perryman LE
        Kinetics of expulsion of the nematode, Nippostrongylus brasiliensis, in mast-cell-deficient W/WV mice.
        Parasite Immunol. 1983; 5: 1-12
        • Nawa Y
        • Ishikawa N
        • Tsuchiya K
        • Horii Y
        • Abe T
        • Khan AI
        • Bing S
        • Itoh H
        • Ide H
        • Uchiyama F
        Selective effector mechanisms for the expulsion of intestinal helminths.
        Parasite Immunol. 1994; 16: 333-338
        • Knight PA
        • Wright SH
        • Lawrence CE
        • Paterson YY
        • Miller HR
        Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.
        J Exp Med. 2000; 192: 1849-1856
        • McDermott JR
        • Bartram RE
        • Knight PA
        • Miller HRP
        • Garrod DR
        • Grencis RK
        Mast cells disrupt epithelial barrier function during enteric nematode infection.
        Proc Natl Acad Sci USA. 2003; 100: 7761-7766
        • Scudamore CL
        • Thornton EM
        • McMillan L
        • Newlands GF
        • Miller HR
        Release of the mucosal mast cell granule chymase, rat mast cell protease-II, during anaphylaxis is associated with the rapid development of paracellular permeability to macromolecules in rat jejunum.
        J Exp Med. 1995; 182: 1871-1881
        • Lawrence CE
        • Paterson YYW
        • Wright SH
        • Knight PA
        • Miller HRP
        Mouse mast cell protease-1 is required for the enteropathy induced by gastrointestinal helminth infection in the mouse.
        Gastroenterology. 2004; 127: 155-165
        • Miller HR
        • Wright SH
        • Knight PA
        • Thornton EM
        A novel function for transforming growth factor-β1: upregulation of the expression and the IgE-independent extracellular release of a mucosal mast cell granule-specific β-chymase, mouse mast cell protease-1.
        Blood. 1999; 93: 3473-3486
        • Brown JK
        • Knight PA
        • Wright SH
        • Thornton EM
        • Miller HR
        Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues.
        Clin Exp Allergy. 2003; 33: 132-146
        • Wright SH
        • Brown J
        • Knight PA
        • Thornton EM
        • Kilshaw PJ
        • Miller HR
        Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues.
        Clin Exp Allergy. 2002; 32: 315-324
        • Pemberton AD
        • Brown JK
        • Wright SH
        • Knight PA
        • Miller HR
        The proteome of mouse mucosal mast cell homologues: the role of transforming growth factor β1.
        Proteomics. 2006; 6: 623-631
        • Olsson N
        • Piek E
        • ten Dijke P
        • Nilsson G
        Human mast cell migration in response to members of the transforming growth factor-β family.
        J Leukoc Biol. 2000; 67: 350-356
        • Beck PL
        • Rosenberg IM
        • Xavier RJ
        • Koh T
        • Wong JF
        • Podolsky DK
        Transforming growth factor-β mediates intestinal healing and susceptibility to injury in vitro and in vivo through epithelial cells.
        Am J Pathol. 2003; 162: 597-608
        • Podolsky DK
        Healing the epithelium: solving the problem from two sides.
        J Gastroenterol. 1997; 32: 122-126
        • Buzoni-Gatel D
        • Debbabi H
        • Mennechet FJ
        • Martin V
        • Lepage AC
        • Schwartzman JD
        • Kasper LH
        Murine ileitis after intracellular parasite infection is controlled by TGF-β-producing intraepithelial lymphocytes.
        Gastroenterology. 2001; 120: 914-924
        • Roche JK
        • Martins CA
        • Cosme R
        • Fayer R
        • Guerrant RL
        Transforming growth factor beta1 ameliorates intestinal epithelial barrier disruption by Cryptosporidium parvum in vitro in the absence of mucosal T lymphocytes.
        Infect Immun. 2000; 68: 5635-5644
        • Yang X
        • Letterio JJ
        • Lechleider RJ
        • Chen L
        • Hayman R
        • Gu H
        • Roberts AB
        • Deng C
        Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β.
        EMBO J. 1999; 18: 1280-1291
        • Hahm KB
        • Im YH
        • Parks TW
        • Park SH
        • Markowitz S
        • Jung HY
        • Green J
        • Kim SJ
        Loss of transforming growth factor β signalling in the intestine contributes to tissue injury in inflammatory bowel disease.
        Gut. 2001; 49: 190-198
        • Hahm KB
        • Lee KM
        • Kim YB
        • Hong WS
        • Lee WH
        • Han SU
        • Kim MW
        • Ahn BO
        • Oh TY
        • Lee MH
        • Green J
        • Kim SJ
        Conditional loss of TGF-β signalling leads to increased susceptibility to gastrointestinal carcinogenesis in mice.
        Aliment Pharmacol Ther. 2002; 16: 115-127
        • MacDonald TT
        Effector and regulatory lymphoid cells and cytokines in mucosal sites.
        in: Defense of Mucosal Surfaces: Pathogenesis, Immunity and Vaccines. Springer-Verlag Berlin, Berlin1999: 113-135
        • Annes JP
        • Munger JS
        • Rifkin DB
        Making sense of latent TGFβ activation.
        J Cell Sci. 2003; 116: 217-224
        • Mu DZ
        • Cambier S
        • Fjellbirkeland L
        • Baron JL
        • Munger JS
        • Kawakatsu H
        • Sheppard D
        • Broaddus VC
        • Nishimura SL
        The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.
        J Cell Biol. 2002; 157: 493-507
        • Crawford SE
        • Stellmach V
        • Murphy-Ullrich JE
        • Ribeiro SM
        • Lawler J
        • Hynes RO
        • Boivin GP
        • Bouck N
        Thrombospondin-1 is a major activator of TGF-β1 in vivo.
        Cell. 1998; 93: 1159-1170
        • Huang XZ
        • Wu JF
        • Cass D
        • Erle DJ
        • Corry D
        • Young SG
        • Farese RV
        • Sheppard D
        Inactivation of the integrin β6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lungs and skin.
        J Cell Biol. 1996; 133: 921-928
        • Ludlow A
        • Yee KO
        • Lipman R
        • Bronson R
        • Weinreb P
        • Huang XZ
        • Sheppard D
        • Lawler J
        Characterization of integrin β6 and thrombospondin-1 double-null mice.
        J Cell Mol Med. 2005; 9: 421-437
        • Breuss JM
        • Gillett N
        • Lu L
        • Sheppard D
        • Pytela R
        Restricted distribution of integrin beta-6 messenger-RNA in primate epithelial tissues.
        J Histochem Cytochem. 1993; 41: 1521-1527
        • Brown JK
        • McAleese SM
        • Thornton EM
        • Pate JA
        • Schock A
        • Macrae A
        • Scott PR
        • Miller HR
        • Collie DS
        Integrin-alpha(v) beta(6), a putative receptor for foot-and-mouth disease virus, is constitutively expressed in ruminant airways.
        J Histochem Cytochem. 2006; 54: 807-816
        • Knight PA
        • Wright SH
        • Brown JK
        • Huang X
        • Sheppard D
        • Miller HR
        Enteric expression of the integrin is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1.
        Am J Pathol. 2002; 161: 771-779
        • Munger JS
        • Huang X
        • Kawakatsu H
        • Griffiths MJ
        • Dalton SL
        • Wu J
        • Pittet JF
        • Kaminski N
        • Garat C
        • Matthay MA
        • Rifkin DB
        • Sheppard D
        The integrin αvβ6 binds and activates latent TGF β1: a mechanism for regulating pulmonary inflammation and fibrosis.
        Cell. 1999; 96: 319-328
        • Griffiths M
        • Huang XZ
        • Wu JF
        • Sheppard D
        Inactivation of the β6 integrin subunit gene protects against bleomycin-induced pulmonary fibrosis.
        Mol Biol Cell. 1996; 7: 964
        • Huang X
        • Wu J
        • Zhu W
        • Pytela R
        • Sheppard D
        Expression of the human integrin β6 subunit in alveolar type II cells and bronchiolar epithelial cells reverses lung inflammation in β6 knockout mice.
        Am J Respir Cell Mol Biol. 1998; 19: 636-642
        • Brown JK
        • Knight PA
        • Pemberton AD
        • Wright SH
        • Pate JA
        • Thornton EM
        • Miller HRP
        Expression of integrin-alpha(E) by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth-factor-beta(1)-activating integrin alpha(v)beta(6).
        Am J Pathol. 2004; 165: 95-106
        • Donaldson LE
        • Schmitt E
        • Huntley JF
        • Newlands GFJ
        • Grencis RK
        A critical role for stem cell factor and c-kit in host protective immunity to an intestinal helminth.
        Int Immunol. 1996; 8: 559-567
        • Grencis RK
        • Else KJ
        • Huntley JF
        • Nishikawa SI
        The in vivo role of stem-cell factor (c-Kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice.
        Parasite Immunol. 1993; 15: 55-59
        • Rosbottom A
        • Knight PA
        • McLachlan G
        • Thornton EM
        • Wright SW
        • Miller HR
        • Scudamore CL
        Chemokine and cytokine expression in murine intestinal epithelium following Nippostrongylus brasiliensis infection.
        Parasite Immunol. 2002; 24: 67-75
        • Knight PA
        • Pemberton AD
        • Robertson KA
        • Roy DJ
        • Wright SH
        • Miller HRP
        Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis.
        Infect Immun. 2004; 72: 6076-6086
        • Wastling JM
        • Knight P
        • Ure J
        • Wright S
        • Thornton EM
        • Scudamore CL
        • Mason J
        • Smith A
        • Miller HRP
        Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the β-chymase, mouse mast cell protease-1.
        Am J Pathol. 1998; 153: 491-504
        • Pemberton AD
        • Brown JK
        • Wright SH
        • Knight PA
        • McPhee ML
        • McEuen AR
        • Forse PA
        • Miller HRP
        Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo.
        Clin Exp Allergy. 2003; 33: 1005-1012
        • Brown JK
        • Donaldson DS
        • Wright SH
        • Miller HRP
        Mucosal mast cells and nematode infection: strain-specific differences in mast cell precursor frequency revisited.
        J Helminthol. 2003; 77: 155-161
        • Hunt JE
        • Friend DS
        • Gurish MF
        • Feyfant E
        • Sali A
        • Huang C
        • Ghildyal N
        • Stechschulte S
        • Austen KF
        • Stevens RL
        Mouse mast cell protease 9, a novel member of the chromosome 14 family of serine proteases that is selectively expressed in uterine mast cells.
        J Biol Chem. 1997; 272: 29158-29166
        • Friend DS
        • Ghildyal N
        • Gurish MF
        • Hunt J
        • Hu XZ
        • Austen KF
        • Stevens RL
        Reversible expression of tryptases and chymases in the jejunal mast cells of mice infected with Trichinella spiralis.
        J Immunol. 1998; 160: 5537-5545
        • Weller CL
        • Collington SJ
        • Brown JK
        • Miller HRP
        • Al-Kashi A
        • Clark P
        • Jose PJ
        • Hartnell A
        • Williams TJ
        Leukotriene B-4, an activation product of mast cells, is a chemoattractant for their progenitors.
        J Exp Med. 2005; 201: 1961-1971
        • Castells MC
        • Klickstein LB
        • Hassani K
        • Cumplido JA
        • Lacouture ME
        • Austen KF
        • Katz HR
        gp49BI-αvβ3 interaction inhibits antigen-induced mast cell activation.
        Nat Immunol. 2001; 2: 436-442
        • Pennock JL
        • Grencis RK
        In vivo exit of c-kit+/CD49dhi/β7+ mucosal mast cell precursors from the bone marrow following infection with the intestinal nematode Trichinella spiralis.
        Blood. 2004; 103: 2655-2660
        • Klimpel GR
        • Langley KE
        • Wypych J
        • Abrams JS
        • Chopra AK
        • Niesel DW
        A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.
        J Exp Med. 1996; 184: 271-276
        • Helmby H
        • Grencis RK
        IL-18 regulates intestinal mastocytosis and Th2 cytokine production independently of IFN-gamma during Trichinella spiralis infection.
        J Immunol. 2002; 169: 2553-2560
        • McDermott JR
        • Humphreys NE
        • Forman SP
        • Donaldson DD
        • Grencis RK
        Intraepithelial NK cell-derived IL-13 induces intestinal pathology associated with nematode infection.
        J Immunol. 2005; 175: 3207-3213
        • Urban JF
        • Schopf L
        • Morris SC
        • Orekhova T
        • Madden KB
        • Betts CJ
        • Gamble HR
        • Byrd C
        • Donaldson D
        • Else K
        • Finkelman FD
        Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T cell-dependent mechanism.
        J Immunol. 2000; 164: 2046-2052
        • Finkelman FD
        • Urban JF
        The other side of the coin: the protective role of the T(H)2 cytokines.
        J Allergy Clin Immunol. 2001; 107: 772-780
        • Grencis RK
        Cytokine regulation of resistance and susceptibility to intestinal nematode infection—from host to parasite.
        Vet Parasitol. 2001; 100: 45-50
        • Jiang Y
        • McGee DW
        Regulation of human lymphocyte IL-4 secretion by intestinal epithelial cell-derived interleukin-7 and transforming growth factor-β.
        Clin Immunol Immunopathol. 1998; 88: 287-296
        • Tang JH
        • Nuccie BL
        • Ritterman I
        • Liesveld JL
        • Abboud CN
        • Ryan DH
        TGF-β down-regulates stromal IL-7 secretion and inhibits proliferation of human B cell precursors.
        J Immunol. 1997; 159: 117-125
        • Finkelman FD
        • Shea-Donohue T
        • Morris SC
        • Gildea L
        • Strait R
        • Madden KB
        • Schopf L
        • Urban JF
        Interleukin-4- and interleukin-13-mediated host protection against intestinal nematode parasites.
        Immunol Rev. 2004; 201: 139-155
        • Khan WI
        • Blennerhasset P
        • Ma C
        • Matthaei KI
        • Collins SM
        Stat6 dependent goblet cell hyperplasia during intestinal nematode infection.
        Parasite Immunol. 2001; 23: 39-42
        • Miller HRP
        Gastrointestinal mucus, a medium for survival and for elimination of parasitic nematodes and protozoa.
        Parasitology. 1987; 94: S77-S100
        • Lawrence CE
        • Paterson JCM
        • Higgins LM
        • MacDonald TT
        • Kennedy MW
        • Garside P
        IL-4-regulated enteropathy in an intestinal nematode infection.
        Eur J Immunol. 1998; 28: 2672-2684
        • Ismail RS
        • Cada M
        • Vanderhyden BC
        Transforming growth factor-β regulates Kit ligand expression in rat ovarian surface epithelial cells.
        Oncogene. 1999; 18: 4734-4741
        • Tan BL
        • Yazicioglu MN
        • Ingram D
        • McCarthy J
        • Borneo J
        • Williams DA
        • Kapur R
        Genetic evidence for convergence of c-Kit- and alpha(4) integrin-mediated signals on class IAPI-3kinase and the Rac pathway in regulating integrin-directed migration in mast cells.
        Blood. 2003; 101: 4725-4732
        • Tsuji K
        • Zsebo KM
        • Ogawa M
        Murine mast-cell colony formation supported by IL-3, IL-4, and recombinant rat stem-cell factor, ligand for c-Kit.
        J Cell Physiol. 1991; 148: 362-369
        • Huang EJ
        • Nocka KH
        • Buck J
        • Besmer P
        Differential expression and processing of 2 cell associated forms of the Kit-ligand—Kl-1 and Kl-2.
        Mol Biol Cell. 1992; 3: 349-362
        • Ebert EC
        Inhibitory effects of transforming growth factor-β (TGF-β) on certain functions of intraepithelial lymphocytes.
        Clin Exp Immunol. 1999; 115: 415-420
        • Huang M
        • Sharma S
        • Zhu LX
        • Keane MP
        • Luo J
        • Zhang L
        • Burdick MD
        • Lin YQ
        • Dohadwala M
        • Gardner B
        • Batra RK
        • Strieter RM
        • Dubinett SM
        IL-7 inhibits fibroblast TGF-β production and signaling in pulmonary fibrosis.
        J Clin Invest. 2002; 109: 931-937
        • Tsujimoto T
        • Lisukov IA
        • Huang NH
        • Mahmoud MS
        • Kawano MM
        Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells.
        Blood. 1996; 87: 3375-3383
        • Dubinett SM
        • Huang M
        • Dhanani S
        • Wang JY
        • Beroiza T
        Down-regulation of macrophage transforming growth-factor-β messenger RNA expression by IL-7.
        J Immunol. 1993; 151: 6670-6680
        • Sun LX
        • Lee JW
        • Fine HA
        Neuronally expressed stem cell factor induces neural stem cell migration to areas of brain injury.
        J Clin Invest. 2004; 113: 1364-1374
        • Overbergh L
        • Valckx D
        • Waer M
        • Mathieu C
        Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.
        Cytokine. 1999; 11: 305-312