Histochemical and Ultrastructural Modification of Mucosal Mast Cell Granules in Parasitized Mice Lacking the β-Chymase, Mouse Mast Cell Protease-1
1998; Elsevier BV; Volume: 153; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65592-7
ISSN1525-2191
AutoresJonathan M. Wastling, Pamela A. Knight, Jan Ure, Steven H. Wright, Elisabeth M. Thornton, Cheryl L. Scudamore, John O. Mason, Austin Smith, H. R. P. Miller,
Tópico(s)Allergic Rhinitis and Sensitization
ResumoThe soluble β-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (−/−) mice, nor was mature mMCP-1 protein detected in (−/−) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 μg mMCP-1/g wet weight in uninfected mice to 129 and 229 μg/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in −/− mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected −/− mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from −/− IMMCs. These data show that disruption of themMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules. The soluble β-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (−/−) mice, nor was mature mMCP-1 protein detected in (−/−) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 μg mMCP-1/g wet weight in uninfected mice to 129 and 229 μg/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in −/− mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected −/− mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from −/− IMMCs. These data show that disruption of themMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules. Intestinal mucosal mast cells (IMMCs) in the mouse are distinguished from connective tissue mast cells (CTMCs) by their predominantly intraepithelial localization and unique fixation, by their histochemical and morphological properties,1Miller HRP Huntley JF Newlands GFJ Mackellar A Lammas D Wakelin D Granule proteinases define mast cell heterogeneity in the serosa and the gastrointestinal mucosa of the mouse.Immunology. 1988; 65: 559-566PubMed Google Scholar, 2Crowle PK Phillips DE Characteristics of mast cells in Chediak-Higashi mice: light and electron microscopic studies of connective tissue and mucosal mast cells.Exp Cell Biol. 1983; 51: 130-139PubMed Google Scholar and by their content of granule chymases.1Miller HRP Huntley JF Newlands GFJ Mackellar A Lammas D Wakelin D Granule proteinases define mast cell heterogeneity in the serosa and the gastrointestinal mucosa of the mouse.Immunology. 1988; 65: 559-566PubMed Google Scholar, 3Friend DS Ghildyal N Austen KF Gurish MF Matsumoto R Stevens RL Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype.J Cell Biol. 1996; 135: 279-290Crossref PubMed Scopus (195) Google Scholar Compared with other mammalian species, including rat, sheep, and human, in which IMMCs are relatively abundant and located in the lamina propria,4Miller HRP Mucosal mast cells and the allergic response against nematode parasites.Vet Immunol Immunopathol. 1996; 54: 331-336Crossref PubMed Scopus (112) Google Scholar, 5Irani A-MA Bradford TR Kepley CL Schechter NM Schwartz LB Deficiency of the tryptase-positive chymase-negative mast cell type in the gastrointestinal mucosa of patients with defective lymphocyte function.J Immunol. 1987; 138: 4381-4386PubMed Google Scholar, 6Enerback L Mucosal mast cells in the rat and man.Int Arch Allergy Appl Immunol. 1987; 82: 249-253Crossref PubMed Scopus (72) Google Scholar IMMCs in mice are rare unless the animal has recently harbored an intestinal nematode infection.1Miller HRP Huntley JF Newlands GFJ Mackellar A Lammas D Wakelin D Granule proteinases define mast cell heterogeneity in the serosa and the gastrointestinal mucosa of the mouse.Immunology. 1988; 65: 559-566PubMed Google Scholar, 3Friend DS Ghildyal N Austen KF Gurish MF Matsumoto R Stevens RL Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype.J Cell Biol. 1996; 135: 279-290Crossref PubMed Scopus (195) Google Scholar At about the time of the immunological expulsion of intestinal nematodes, there is substantial IMMC hyperplasia in murine rodents and ruminants, as well as in other species, including guinea pigs and gerbils.7Rothwell TLW Immune expulsion of parasite nematodes from the alimentary tract.Int J Parasitol. 1989; 19: 139-168Crossref PubMed Scopus (149) Google Scholar, 8Morii Y Nawa Y Heparin containing mast cells in the jejunal mucosa of normal and parasitised Mongolian gerbils, Meriones unguiculatus.Int Arch Allergy Appl Immunol. 1992; 98: 415-419Crossref Scopus (21) Google Scholar Hyperplasia of IMMCs has also been described in parasitized humans,9Gutowska L Ruitenburg EJ Elgersma A Kociecka W Increase of mucosal mast cells in the jejunum of patients infected with Trichinella spiralis.Int Arch Allergy Appl Immunol. 1983; 71: 304-308Crossref PubMed Scopus (35) Google Scholar and this type of response may be common to most mammalian species infested with nematodes, suggesting that mast cells play a role in the extensive immunopathology associated with intestinal nematodiasis.4Miller HRP Mucosal mast cells and the allergic response against nematode parasites.Vet Immunol Immunopathol. 1996; 54: 331-336Crossref PubMed Scopus (112) Google Scholar, 6Enerback L Mucosal mast cells in the rat and man.Int Arch Allergy Appl Immunol. 1987; 82: 249-253Crossref PubMed Scopus (72) Google Scholar The hyperplasia and differentiation of IMMCs in response to nematode infection is highly T-cell dependent, and in murine rodents the ligand for c-kit, stem cell factor (SCF), plays a key role in nematode-induced IMMC hyperplasia.10Newlands GFJ Miller HRP Mackellar A Galli SJ Stem cell factor contributes to intestinal mucosal mast cell hyperplasia with Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor treatment decreases parasite egg production during N. brasiliensis infection.Blood. 1995; 86: 1968-1976PubMed Google Scholar, 11Donaldson 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: 559-567Crossref PubMed Scopus (99) Google Scholar Studies of parasitized rodents and sheep have shown that IMMCs express soluble chymases that, at about the time of the immunological expulsion of the parasites, are secreted systemically into the bloodstream and across the epithelium into the gut lumen.4Miller HRP Mucosal mast cells and the allergic response against nematode parasites.Vet Immunol Immunopathol. 1996; 54: 331-336Crossref PubMed Scopus (112) Google Scholar It is likely, therefore, that granule chymases serve a functional role in the protective response against gastrointestinal helminth parasites. It is probably significant that >95% of IMMCs in mice, and 40 to 50% of IMMCs in rats and sheep, are located intraepithelially at the time of worm elimination, because this would facilitate the access of secreted chymases to the epithelial tight junctions4Miller HRP Mucosal mast cells and the allergic response against nematode parasites.Vet Immunol Immunopathol. 1996; 54: 331-336Crossref PubMed Scopus (112) Google Scholar, 12Scudamore CL Thornton EM McMillan L Newlands GFJ Miller HRP 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: 1871-1881Crossref PubMed Scopus (130) Google Scholar and, possibly, to protease-activated receptors on the enterocytes.13Bohm SK Khitin LM Grady EF Aponte G Payan DG Bunnett NW Mechanisms of desensitization and resensitization of protease-activated receptor 2.J Biol Chem. 1996; 271: 22003-22016Crossref PubMed Scopus (209) Google Scholar There are, therefore, several mechanisms by which IMMCs could alter epithelial function or integrity. In rodents, the soluble β-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II (rMCP-II) are abundantly expressed by IMMCs; are 74% homologous at the amino acid level with net charges of +3 and +4, respectively14Huang R Blom T Hellman L Cloning and structural analysis of mMCP-1, mMCP-4 and mMCP-5, three mouse mast cell-specific proteases.Eur J Immunol. 1991; 21: 1611-1621Crossref PubMed Scopus (100) Google Scholar; and probably, therefore, serve similar functions in vivo. The introduction of rMCP-II into the perfused mesenteric artery of normal rats is associated with rapid development of intestinal mucosal permeability.12Scudamore CL Thornton EM McMillan L Newlands GFJ Miller HRP 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: 1871-1881Crossref PubMed Scopus (130) Google Scholar Similarly, systemic challenge with worm antigen in rats sensitized by previous infection is associated with immediate and substantial release of rMCP-II into the gut lumen and is accompanied by the translocation of plasma proteins across the intestinal epithelium.12Scudamore CL Thornton EM McMillan L Newlands GFJ Miller HRP 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: 1871-1881Crossref PubMed Scopus (130) Google Scholar One of the major functions of these soluble β-chymases is, therefore, to promote mucosal permeability in intestinal allergic hypersensitivity reactions. However, it is also likely that these abundant chymases serve other as yet undefined functions. To address the functions of these β-chymases more directly, we have used a targeting strategy to generate mice lacking a functional mMCP-1 gene. Our results suggest that mMCP-1 plays a role in the regulation of mast cell hyperplasia during nematode infection and that its absence is associated with altered size and internal structure of IMMC granules. Using a full-length cDNA clone for mMCP-1 as a template, a 100-bp probe was generated by polymerase chain reaction (PCR) using primers that amplified a 100-bp region of the 5′-untranslated region of mMCP-1 to minimize the possibility of inadvertently isolating clones from related protease genes.14Huang R Blom T Hellman L Cloning and structural analysis of mMCP-1, mMCP-4 and mMCP-5, three mouse mast cell-specific proteases.Eur J Immunol. 1991; 21: 1611-1621Crossref PubMed Scopus (100) Google Scholar The probe was labeled with digoxigenin (DIG) (Boehringer Mannheim, Lewes, UK) in the PCR and used to isolate a full-length genomic clone from a 129-strain mouse genomic λ-library. This clone was mapped and consisted of a 15-kb fragment containing the mMCP-1 gene with extensive 5′ and 3′ flanking regions. A combination of DNA sequencing and Southern blotting was used to confirm the identity of the mMCP-1 clone. A 4.35-kbPvuII-HindIII and 3.9-kbHindIII-SalI restriction fragment incorporating the 5′ and 3′ flanking regions of the mMCP-1 gene was cloned into pSP72 (Promega, Southampton, UK). A 4.8-kb fragment containing all five exons of the mMCP-1 gene itself was replaced in the above construct by a neo gene driven by a human β-actin promoter and including an SV40 polyadenylation signal,15Joyner AL Skarnes WC Rossant J Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells.Nature. 1989; 338: 153-156Crossref PubMed Scopus (129) Google Scholar to form the targeting vector. The targeting vector was linearized by HindIII digest and electroporated into 129 strain mouse embryonic stem (ES) cells. Linearized DNA was added to 9.0 × 107 E14 Tg2A ES cells and exposed to a 0.8-kV 3.0-μF pulse and plated out at 5 × 106 cells/100-cm Petri dish. Cells were subjected to selection by G418 (175 μg/ml) on days 1, 3, 5, 7, and 9. Resistant clones containing the correct targeting event were identified by Southern blotting using two external screening probes (Figure 1A). After homologous recombination an endogenous EcoRI fragment was converted from a 12-kb fragment to a 10.2-kb fragment detected by the first probe (A) because of the substitution of the five exons of mMCP-1 with theneo cassette. The replacement event was confirmed using a second probe (B) to detect generation of a 9.1-kb EcoRI fragment. All confirmed clones were analyzed with a third probe derived from the neo cassette to ensure single integration events. The frequency of correctly targeted clones was 5 out of 192 (2.6%). Targeted ES cell clones were separately microinjected into blastocysts collected 3.5 days postcoitum from C57BL/6 mice and implanted into C57BL/6 × CBA pseudopregnant foster females 2.5 days postcoitum. ES cell-derived progeny identified by coat color were screened by Southern blot analysis and long-template PCR (LT-PCR) to detect the correct targeting event, and targeted progeny were backcrossed with MF-1 strain mice. Genomic DNA was prepared by phenol/chloroform extraction16Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar and amplified using LT-PCR (Boehringer Mannheim). The positions of the primers used are indicated in Figure 1A. Two pairs of oligonucleotide primers were used: primers P1 and P2, which amplify the wild-type mMCP-1 allele to give a product of 5 kb, and primers P1 and P3, which amplify the correctly targeted allele to give a product of 7.3 kb. Primer sequences were as follows: 5′-TGAGCAGGTGGAGACTCCTGATAGT-3′ (P1), 5′-AGCTCTCTGGTACTCTTTGGTTCA-3′ (P2), and 5′-TGCATTAATGAATCGGCCAACGCGC-3′ (P3).14Huang R Blom T Hellman L Cloning and structural analysis of mMCP-1, mMCP-4 and mMCP-5, three mouse mast cell-specific proteases.Eur J Immunol. 1991; 21: 1611-1621Crossref PubMed Scopus (100) Google Scholar Reactions were carried out in 50-μl volumes in thin-walled 0.2-ml tubes (Perkin Elmer MicroAmp; Perkin Elmer Corp., Norwalk, CT) containing 20- to 100-ng template DNA, 350 μmol/L deoxynucleotide triphosphates, 300 nmol/L of each primer, 1× Boehringer Mannheim Expand PCR buffer (50 mmol/L Tris-HCl pH 9.2, 16 mmol/L (NH4)2SO4, 1.75 mmol/L MgCl2), and 3.5 U Taq/Pwo enxyme mix. After an initial denaturation step for 2 minutes at 94°C, the DNA was amplified for 10 seconds at 94°C, 30 seconds at 65°C, and 4 minutes at 68°C for 10 thermocycles, followed by 10 seconds at 94°C, 30 seconds at 65°C, and 4 minutes at 68°C, with the latter step extended by 20 seconds at each cycle for 20 thermocycles and a final elongation step of 7 minutes at 68°C. The PCR products were analyzed on 1% agarose gels. The level of detection was increased and the authenticity of the PCR products was confirmed by Southern hybridization with a DIG (Boehringer Mannheim)-labeled cDNA probe specific for a region within probe B, 3′ of the targeted construct and common to LT-PCR products from both alleles, without including LT-PCR primer sequences. The cDNA probe was amplified and DIG labeled by PCR of a 147-bp fragment using a probe B cDNA clone as a template and substitution of deoxynucleotide triphosphate with DIG-11-deoxyuridine triphosphate labeling mixture in the PCR (Boehringer Mannheim). Primers were 5′-ACATGCATAAGAATAAACACTGTGG-3′ and 5′-ACAGGTTTAATGGCTTCCAGAAAGG-3′. For Southern detection the DIG-labeled probe was denatured at 95°C (10 minutes), chilled on ice, and hybridized at 20 ng/ml in a total volume of 10 ml Rapidhyb (Amersham, Rainham, UK) hybridization buffer/membrane. After hybridization for 3 hours, membranes were washed at high stringency (65°C in 0.1× standard saline citrate/0.1% sodium dodecyl sulfate). Hybridized probe was detected with anti-DIG alkaline phosphatase antibody using colorimetric detection with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. The LT-PCR products detected by Southern hybridization from a typical wild-type (mMCP-1+/+) homozygote (mMCP-1−/−) and heterozygote (mMCP-1+/−) mouse are shown in Figure 1B. The mouse-adapted strain of Nippostrongylus brasiliensis (generously supplied by Dr. J. Urban) was maintained by alternate passage through BALB/c and Swiss White strains of mice and charcoal cultures as described previously.17Scudamore CL McMillan L Thornton EM Wright SH Newlands GFJ Miller HRP Mast cell heterogeneity in the gastrointestinal tract: variable expression of mouse mast cell protease-1 (mMCP-1) in intraepithelial mucosal mast cells in nematode infected and normal BALB/c mice.Am J Pathol. 1997; 150: 1661-1672PubMed Google Scholar Null (mMCP-1−/−) mice and MF-1 (mMCP-1+/+) controls were infected with 500N. brasiliensis L3, and infection was monitored by fecal egg counts. Mice were killed by exsanguination under terminal anesthesia, and small (<1 cm) samples of jejunum were immediately snap frozen in liquid nitrogen for RNA analysis, and on dry ice for immunoassay of mMCP-1, and stored at −70°C before extraction. Two separate but adjacent samples of jejunum approximately 6 to 8 cm long were taken 2 to 3 cm distal to the ligament of Trietz, and worms were counted in situ using a dissecting microscope after opening and flattening the intestine onto stiff blotting paper. After counting, the jejunum was gently lifted off the paper and rolled with villi outermost onto the tip of a plastic pipette (pastette) and immediately transferred to Carnoy's fluid or 4% paraformaldehyde dissolved in phosphate-buffered saline (PF/PBS) as described previously.17Scudamore CL McMillan L Thornton EM Wright SH Newlands GFJ Miller HRP Mast cell heterogeneity in the gastrointestinal tract: variable expression of mouse mast cell protease-1 (mMCP-1) in intraepithelial mucosal mast cells in nematode infected and normal BALB/c mice.Am J Pathol. 1997; 150: 1661-1672PubMed Google Scholar, 18Wastling JM Scudamore CL Thornton EM Newlands GFJ Miller HRP Constitutive expression of mouse mast cell protease-1 in normal BALB/c mice and its up-regulation during intestinal nematode infection.Immunology. 1997; 90: 308-313Crossref PubMed Scopus (52) Google Scholar Samples of ear pinnae from the same mice were also snap frozen or fixed in Carnoy's fluid or PF/PBS. After fixation for 6 hours in PF/PBS or overnight in Carnoy's fluid, the tissues were transferred to 70% ethanol and stored at 4°C for a minimum of 24 hours before trimming, processing, and embedding in paraffin wax. Similar procedures for collecting jejunum were followed for uninfected (control) mice. Total RNA was extracted from snap-frozen ear pinnae and jejunum by maceration in 1 to 2 ml of Tri-Reagent (Sigma, Poole, UK) with a mortar and pestle precooled to −70°C as described previously.18Wastling JM Scudamore CL Thornton EM Newlands GFJ Miller HRP Constitutive expression of mouse mast cell protease-1 in normal BALB/c mice and its up-regulation during intestinal nematode infection.Immunology. 1997; 90: 308-313Crossref PubMed Scopus (52) Google Scholar The quantity and quality of the total RNA was determined by measurement of the absorbance at 260/280 nm in a Beckman DU 650 spectrophotometer. Because mast cell-associated heparin can copurify with RNA and is known to inhibit the PCR reaction, RNA samples were treated with heparinase before reverse transcription (RT).19Tsai M Miyamoto M Tam S-Y Wang Z-s Galli SJ Detection of mouse mast cell-associated protease mRNA: heparinase treatment greatly improves RT-PCR of tissues containing mast cell heparin.Am J Pathol. 1995; 146: 335-343PubMed Google Scholar Aliquots of 4 μg RNA were incubated with 4 U heparinase I (Sigma) in 5 mmol/L Tris-HCl pH 7.5, 1 mmol/L CaCl2, and 40 U RNase inhibitor (Promega) in a total volume of 40 μl for 2 hours at 25°C. Serial dilutions of the heparinase-treated samples were reverse transcribed in 20-μl volumes containing 1, 0.1, or 0.01 μg RNA, 1 mmol/L deoxynucleotide triphosphates, 20 U RNase inhibitor, 2.5 μmol/L (dT) is oligonucleotide primers, 1× RT buffer, and 2.5 mmol/L MgCl2; 50 U of avian myeloblastosis virus reverse transcriptase reaction was diluted to 100 μl, and 10 μl were used for each PCR reaction. The cDNA was amplified for 1 minute at 94°C, 2 minutes at 63°C, and 3 minutes at 72°C for 30 thermocycles in 50-μl volumes in thin-walled 0.2-ml tubes (Perkin Elmer MicroAmp) containing 250 μmol/L deoxynucleotide triphosphates, 250 nmol/L of each primer, 1× Boehringer Mannheim PCR buffer (10 mmol/L Tris-HCl, 50 mmol/L KCl, and 1.5 mmol/L MgCl2, pH 8.3) and 2.5 UTaq DNA polymerase (Boehringer Mannheim). Oligonucleotide primers used are detailed in Table 1. Five pairs of oligonucleotide primers were used to identify transcription of chymase genes commonly expressed in mouse mast cells: mMCP-1, 2, 4, and 5.14Huang R Blom T Hellman L Cloning and structural analysis of mMCP-1, mMCP-4 and mMCP-5, three mouse mast cell-specific proteases.Eur J Immunol. 1991; 21: 1611-1621Crossref PubMed Scopus (100) Google Scholar, 20Ghildyal N McNeil HP Stechschulte S Austen KF Silberstein D Gurish MF Somerville LL Stevens RL IL-10 induces transcription of the gene for mouse mast cell protease-1, a serine protease preferentially expressed in mucosal mast cells of Trichinella spiralis-infected mice.J Immunol. 1992; 149: 2123-2129PubMed Google Scholar, 21Serafin WE Reynolds DS Rogelj S Lane WS Conder GA Johnson SS Austen F Stevens RL Identification and molecular cloning of a novel mouse mucosal mast cell serine protease.J Biol Chem. 1990; 265: 423-429Abstract Full Text PDF PubMed Google Scholar, 22Serafin WE Sullivan TP Conder GA Ebrahimi A Marcham P Johnson SS Austen KF Reynolds RS Cloning of the cDNA and the gene for mouse mast cell protease-4.J Biol Chem. 1991; 266: 1934-1941Abstract Full Text PDF PubMed Google Scholar, 23McNeil HP Austen KF Somerville LL Gurish MF Stevens RL Molecular cloning of the mouse mast cell protease-5 gene.J Biol Chem. 1991; 266: 20316-20322Abstract Full Text PDF PubMed Google Scholar, 24Zaheer A Zhong W Lim R Expression of mRNAs of multiple growth factors and receptors by neuronal cell lines: detection with RT-PCR.Neurochem Res. 1995; 20: 1457-1463Crossref PubMed Scopus (32) Google Scholar All primers were designed to span an intron to ensure that genomic DNA was distinguishable from cDNA PCR products. In addition, oligonucleotide primers for SCF, transcribed by jejunal epithelial cells25Anderson DM Lyman A Baird JM Wignall J Eisenman C Rauch CJ March HS Boswell SD Gimpel D Cosman D Williams DE Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms.Cell. 1990; 63: 235-243Abstract Full Text PDF PubMed Scopus (726) Google Scholar, 26Klimpel GR Chopra AK Langley KE Wypych J Annable CA Kaiserlian D Ernst PB Peterson JW A role for stem cell factor and c-kit in the murine intestinal tract secretory response to cholera toxin.J Exp Med. 1995; 182: 1931-1942Crossref PubMed Scopus (40) Google Scholar and mouse carboxypeptidase A27Jippo T Tsujino K Kim H Kim D Lee Y Nawa Y Kitamura Y Expression of mast-cell specific proteases in tissues of mice studied by in situ hybridization.Am J Pathol. 1997; 150: 1373-1382PubMed Google Scholar(Table 1), were used in some PCRs (infected jejunum only). Primers for a 514-bp fragment from mouse β-actin cDNA, 5′-TGTGATGGTGGGAATGGGTCAG (5′ primer) and 5′-TTTGATGTCACGCACGATTTCC (purchased from Stratagene, Cambridge, UK), were included in all PCR reactions as a control to eliminate variations in the heparinase/RT reactions that could affect the efficiency of subsequent PCR reactions. The RNA concentrations 1, 0.1, and 0.01 μg RNA/20 μl in the RT reaction (final amounts in PCR of 100, 10, and 1 ng) were determined in initial pilot studies. PCRs for carboxypeptidase A and SCF were carried out at a single RNA concentration of 10 ng/PCR reaction with the sole purpose of detecting the presence or absence of these transcripts. Controls included in the PCR reactions were RNA only (no cDNA) and primers only (no target).Table 1PCR Primers and Product SizesGenePrimerSequenceProduct size from cDNA (bp)mMCP-1*Ghildyal et al,105′5′-GGAAAACTGGAGAGAAAGAACCTAC4603′5′-GACAGCTGGGGACAGAATGGGGmMCP-2†Serafin et al,215′5′-ATTTCATTGCCTAGTTCCTCTGAC5253′5′-CAGGATGAGAACAGGCTGGGATmMCP-4‡Serafin et al,22 and5′5′-GTAATTCCTCTGCCTCGTCCTTC4543′5′-GGACAGGATGGACACATGCTTTmMCP-5§McNeil et al.235′5′-GGCAGAACAAACGTGAATGAGCC4183′5′-AAGAACCTTCTGGAAGCTCAGGGCarboxypeptidase A¶Primer sequences from Tsai et al.195′5′-ACACAGGATCGAATGTGGAG6893′5′-TAATGCAGGACTTCATGAGCSCF∥Primer sequences from Zaheer et al.245′5′-GCCTTTCCTTATGAAGAAGA5753′5′-TGCAACAGGGGGTAACATAAATGGPrimers designed using cDNA sequence from* Ghildyal et al,10Newlands GFJ Miller HRP Mackellar A Galli SJ Stem cell factor contributes to intestinal mucosal mast cell hyperplasia with Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor treatment decreases parasite egg production during N. brasiliensis infection.Blood. 1995; 86: 1968-1976PubMed Google Scholar† Serafin et al,21Serafin WE Reynolds DS Rogelj S Lane WS Conder GA Johnson SS Austen F Stevens RL Identification and molecular cloning of a novel mouse mucosal mast cell serine protease.J Biol Chem. 1990; 265: 423-429Abstract Full Text PDF PubMed Google Scholar‡ Serafin et al,22Serafin WE Sullivan TP Conder GA Ebrahimi A Marcham P Johnson SS Austen KF Reynolds RS Cloning of the cDNA and the gene for mouse mast cell protease-4.J Biol Chem. 1991; 266: 1934-1941Abstract Full Text PDF PubMed Google Scholar and§ McNeil et al.23McNeil HP Austen KF Somerville LL Gurish MF Stevens RL Molecular cloning of the mouse mast cell protease-5 gene.J Biol Chem. 1991; 266: 20316-20322Abstract Full Text PDF PubMed Google Scholar¶ Primer sequences from Tsai et al.19Tsai M Miyamoto M Tam S-Y Wang Z-s Galli SJ Detection of mouse mast cell-associated protease mRNA: heparinase treatment greatly improves RT-PCR of tissues containing mast cell heparin.Am J Pathol. 1995; 146: 335-343PubMed Google Scholar∥ Primer sequences from Zaheer et al.24Zaheer A Zhong W Lim R Expression of mRNAs of multiple growth factors and receptors by neuronal cell lines: detection with RT-PCR.Neurochem Res. 1995; 20: 1457-1463Crossref PubMed Scopus (32) Google Scholar Open table in a new tab Primers designed using cDNA sequence from PCR products were separated on 1.2% agarose gels and recorded with a charge-coupled device camera linked to an image processor (Appligene, Warford, UK), and the band intensities were analyzed using a densitometry package (Quantiscan II for Windows 95; Biosoft, Cambridge, UK). The intensity of each band was measured relative to the intensity of its corresponding β-actin signal. The authenticity of the PCR products was confirmed by Southern hybridization using gene-specific oligonucleotide probes, the sequences of which are shown in Table 2. One hundred pmol of each oligonucleotide probe was 3′ end-labeled with DIG-dideoxyuridine triphosphate using terminal transferase (Boerhinger Mannheim) according to the manufacturer's instructions. Membranes were hybridized with DIG-labeled probes at 10 pmol/ml in a total volume of 10 ml of Rapidhyb (Amersham) hybridization buffer/membrane for 3 hours at 40°C. Washing steps at high stringency, antibody incubations, and visualization of bands with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium were carried out as described in the section on LT-PCR screening. All PCR products were of the expected sizes for cDNA, and Southern blotting with gene-specific oligonucleotide probes confirmed their identity. No bands were detected in the RNA-only and no-target controls. Contamination from genomic DNA (indicated by a higher-kb band of 800 to 1000 bp for the proteinase genes) was negligible (<5% of cDNA product in samples containing higher RNA concentrations).Table 2Oligonucleotide Probes for PCR ProductsPCR productProbe sequencemMCP-15′-AAGGCAGGCTGCACATAGmMCP-25′-CTCAGAGGTACCAGATGAmMCP-45′-CCCAAGGGTTATTAGAAGAGCTCmMCP-55′-TACAGACAGGCCAGATCACarboxypeptidase A5′-AACTCCACCTGCATTGGCACTSCF5′-CAAACTTGGATTATCACTTGCATOligonucleotide probes were designed using the cDNA sequences indicated in Table 1. Open table in a new tab Oligonucleotide pro
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