The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schwartz, L. B.
Right arrow Articles by Fukuoka, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schwartz, L. B.
Right arrow Articles by Fukuoka, Y.
The Journal of Immunology, 2003, 170: 5667-5673.
Copyright © 2003 by The American Association of Immunologists

Tryptase Precursors Are Preferentially and Spontaneously Released, Whereas Mature Tryptase Is Retained by HMC-1 Cells, Mono-Mac-6 Cells, and Human Skin-Derived Mast Cells 1

Lawrence B. Schwartz2, Hae-Ki Min, Shunlin Ren, Han-Zhang Xia, Jiang Hu, Wei Zhao, George Moxley and Yoshihiro Fukuoka

Division of Rheumatology, Allergy and Immunology, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tryptase ({alpha} and {beta}) levels in serum are used to assess mast cell involvement in human disease. Using cultured cells, the current study examines the hypothesis that protryptase(s) are spontaneously secreted by mast cells at rest, whereas mature tryptase(s) are stored in secretory granules until their release by activated cells. HMC-1 cells have only {beta}-tryptase genes and the corresponding mRNA. Mono-Mac-6 cells have both {alpha}- and {beta}-tryptase genes but preferentially express {alpha}-tryptase. Mono-Mac-6 cells spontaneously secrete most of their tryptase, which consists of {alpha}-protryptase, whereas mature tryptase is retained inside these cells. HMC-1 cells also spontaneously secrete most of their tryptase, identified as {beta}-protryptase, and retain mature tryptase. Skin-derived mast cells retain most of their tryptase, which is mature, and spontaneously secrete protryptase(s). Total tryptase levels in plasma are detectable but no different in healthy subjects with and without the gene for {alpha}-tryptase, consistent with pro forms of both {alpha}- and {beta}-tryptase being spontaneously secreted. Thus, protryptase(s) are spontaneously secreted by resting mast cells, whereas mature tryptase is retained by mast cells until they are activated to degranulate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tryptase (EC 3.4.21.59) is the most abundant protein product produced by human mast cells and is derived principally from two genes on chromosome 16p13.3, {alpha}-tryptase and {beta}-tryptase (1). There are two major forms of {alpha}-tryptase ({alpha}I and {alpha}II) and three major forms of {beta}-tryptase ({beta}I, {beta}II, and {beta}III) (2). Also on chromosome 16p13.3 are the {delta}-tryptases, initially named mouse mast cell protease-7-like tryptases (3). The product of this gene has close homology to {alpha}{beta}-tryptases over exons 1–4, but exon 5 is more closely related to mouse mast cell protease-7. Although it is expressed by mast cells and possibly other cell types, a seemingly premature stop codon terminates translation 40 aa earlier than both {alpha}- and {beta}-tryptases (4). The product of the {beta}-tryptase gene(s) is autoprocessed in vitro from {beta}-protryptase to {beta}-pro’tryptase at acidic pH, optimally in the presence of heparin proteoglycan, and then to {beta}-tryptase by a dipeptidase, thought to be dipeptidyl peptidase I in humans (5). Tryptase in murine mast cells uses a different dipeptidase (6). Mature {beta}-tryptase is stored in secretory granules as an enzymatically active tetramer in a complex with heparin proteoglycan until the cells are activated to degranulate and release the protease:proteoglycan complex. In contrast, {alpha}-protryptase (and {delta}-tryptases) may not undergo autoprocessing from protryptase to pro’tryptase, because a tryptase-resistant Q-3 rather than a tryptase-sensitive R-3 is present in the -3 position of the propeptide (3, 5). Indeed, when mature {alpha}-tryptase is produced in vitro, although it forms a tetramer, the protein appears to be enzymatically inactive (7, 8, 9).

The major form of tryptase found in normal blood fails to bind to the G5 mAb, which recognizes recombinant human (rh)3 {beta}-tryptase but not rh{alpha}-protryptase (5, 10). Thus, an immunoassay using this mAb measures mature {beta}-tryptase. Although {beta}-tryptase levels are undetectable in normal serum (<1 ng/ml), they are elevated in the blood of most cases of systemic anaphylaxis with hemodynamic compromise, particularly when the precipitating agent is administered parenterally. In such cases, the magnitude of mast cell degranulation appears to be the primary determinant of clinical severity (11, 12, 13). In contrast, based on mAbs that recognize both {alpha}-protryptase and {beta}-tryptase, a total tryptase immunoassay was developed that detected levels of tryptase in baseline serum from essentially all individuals (mean ± SD, 4.9 ± 2.3 ng/ml) (14). So-called total tryptase levels are elevated in subjects with systemic mastocytosis and reflect the total body burden of mast cells (10).

Several issues relating to these mature and total tryptase assays must be clarified. Approximately 25% of individuals lack a gene for {alpha}-tryptase (15, 16). Whether such a genetic difference affects the level of tryptase in the blood has not been determined. Also, the possibility that {alpha}-protryptase might be processed to mature {alpha}-tryptase in vivo has not been excluded; thus, the ability of tryptase immunoassays to detect this putative form of the protein also should be determined. Finally, the hypothesis that mature forms of tryptase are preferentially stored in secretory granules, whereas immature forms of tryptase are preferentially selected for spontaneous secretion, has not been directly examined.

The current study examines retained and spontaneously secreted tryptase in two human cell lines, HMC-1 cells derived from a mast cell leukemia (17) and Mono-Mac-6 (Mac-6) cells derived from a monoblastic leukemia (18). Only tryptase mRNA encoding {beta}-tryptase was previously found in HMC-1 cells (19, 20), whereas only tryptase mRNA encoding {alpha}-tryptase was found in Mac-6 cells (21). In contrast, most human lung-derived mast cells, skin-derived mast cells, and mast cells derived from fetal liver progenitors in vitro contain both {alpha}-tryptase and {beta}-tryptase mRNA (20, 22). HMC-1 cells contain ~50 ng of tryptase/106 cells (23), whereas human lung-derived (12 µg/106 cells) and skin-derived (35 µg/106 cells) mast cells contain substantially greater amounts (24). The current study concludes that precursor forms of both {alpha}- and {beta}-tryptases are preferentially secreted spontaneously. Thus, serum levels of precursor forms of tryptase in health subjects reflect the total body burden of mast cells, regardless of whether the subject is {alpha}-tryptase deficient, whereas serum levels of mature tryptase reflect mast cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Tosyl-Gly-Pro-Lys-p-nitroanilide, HEPES, MES, porcine heparin glycosaminoglycan (1,200–20,000 Da), dextran sulfate (500 kDa), 3-(cyclohexylamino)-1-propanesulfonic acid (Sigma-Aldrich, St. Louis, MO); HindIII, XhoI, Sac II, EcoRI, 100-bp DNA ladder, dNTP, genomic DNA purification kit, PCR size markers, DNase, oligodeoxythymidylate primers, and Taq DNA polymerase with the PCR reagents (Promega, Madison, WI); SYBR Green I (FMC Bioproducts, Rockland, ME); Moloney murine leukemia virus-derived reverse transcriptase (Life Technologies, Gaithersburg, MD); XL-1-Blue-competent cells, Pfu DNA polymerase, and the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA); and RNA isolation kit (Qiagen, Valencia, CA) were obtained as indicated. Tryptase from human lung and rh{beta}II-tryptase (both tetramers with subunits of 29 to 35 kDa by SDS-PAGE) were prepared and purified to homogeneity by immunoaffinity chromatography using the B2 mAb, and heparin-agarose chromatography as described (5, 25). rh{alpha}I-protryptase (a monomer of ~30 kDa by SDS-PAGE) also was prepared and purified as described (26). Purified preparations of tryptase were stored at -70°C in 20% (v/v) glycerol, 0.01 M MES (pH 6.5), 0.8 M NaCl. IgG1{kappa} mAb B12, which recognizes a conformational epitope present on tryptase, was prepared as before (14, 25). mAb B12 Affi-Gel was prepared by coupling B12 with activated Affi-Gel (Bio-Rad Laboratories, Hercules, CA). mAbs G4 (IgG1{kappa}) and G5 (IgG2b{kappa}), which recognize linear epitopes on all forms of tryptase and on {beta}-tryptase, respectively, were prepared as described (27, 28, 29). An HMC-1 mast cell leukemia cell line was provided by Dr. G. Gleich and Dr. J. Butterfield (Mayo Clinic, Rochester, MN) (17). The Mac-6 myelomonocytic leukemia cell line was provided by Dr. H. W. Ziegler-Heitbrock (University of Munich, Munich, Germany) (18). Primer synthesis and DNA sequencing reactions were performed by the Virginia Commonwealth University Nucleic Acids Core Laboratory (Richmond, VA). All studies using human serum, cells, and tissues were approved by the human studies Institutional Review Board of Virginia Commonwealth University (Richmond, VA). Tryptase ELISAs with the B12 mAb used for capture, and either biotin-G5 (mature tryptase) or biotin-G4 (total tryptase) used with streptavidin-alkaline phosphatase for detection was performed as described (10).

Preparation of mature {alpha}-tryptase

{alpha}I-Tryptase cDNA (26) in the pBluescript II SK vector (Stratagene) was mutated as recommended by the manufacturer (QuickChange Site-Directed Mutagenesis Kit, QCM) with slight modifications (30) to yield Q-3R by changing the -3 codon from CAA to CGA. First, two independent extension reactions were performed with Pfu DNA polymerase, one containing the sense primer, 5'- AGCGAGTGGGCATCGTTGGGGGTCAGG-3' and the other containing the antisense primer, 5'-CCTGACCCCCAACGATGCCCACTCGCT-3'. The underlined nucleotides show the mutation. Extension reactions were initiated by preheating the reaction mixtures to 95°C for 5 min, followed by five cycles at 95°C for 30 s, 58°C for 1 min, and 68°C for 8 min. Second, equal portions of the two reactions were mixed together along with an additional 1 U of Pfu DNA polymerase and the same sense and antisense primers and heated at 95°C for 5 min, followed by 22 cycles at 95°C for 30 s, 58°C for 1 min, and 72°C for 12 min and a final extension reaction at 72°C for 15 min. 10 U of DpnI were mixed into the final PCR mixture and incubated at 37°C for 2 h to destroy parental plasmid before transformation of XL-1-Blue cells was performed, which in turn were selected with ampicillin. After confirmation of the mutated sequence, Q-3R{alpha}-tryptase was excised by NotI and HindIII double digestion and ligated into the pFastBac1 transfer vector (Invitrogen, Carlsbad, CA). pFastBac1, which contained Q-3R{beta}-tryptase cDNA, was used to transform DH10Bac Escherichia coli-competent cells, which contained helper plasmid and bacmid DNA. Transformed cells with the transfer plasmid integrated into the bacmid by homologous recombination were selected on LB agar plates containing kanamycin (50 µg/ml), gentamicin (7 µg/ml),tetracycline (10 µg/ml), Bluo-gal (100 µg/ml; Invitrogen), and isopropyl {beta}-D-thiogalactoside (40 µg/ml). The inserted Q-3R{alpha}-tryptase cDNA sequence was confirmed using both vector- and tryptase-specific primers. Sf9 insect cells were then transfected with recombinant bacmid DNA mixed with CellFECTIN reagent (Invitrogen). Recombinant baculovirus was collected from the cell culture medium, produced in high titer, and used to express recombinant Q-3R {beta}-tryptase. Sf9 cells were infected at a multiplicity of infection of 0.1 and cultured at 2 x 106 cells/ml of Sf-900II serum-free medium. trans-Epoxysuccinylleucylamido(4-guanidino)butane (1 µM; Sigma-Aldrich) was added 2 days after transfection, and medium was typically harvested on day 4. Q-3R{alpha}-protryptase was purified from the medium (1–1.5 mg/L) by B2-agarose chromatography (26). Before immunoaffinity chromatography, the medium was adjusted to 1 M NaCl and 10 mM MES, pH 6.5. The B2 column, once loaded, was washed with several column volumes of 10 mM MES buffer, pH 6.5, containing 1 M NaCl and then 10 mM MES buffer, pH 6.5, containing 0.2 M NaCl. Elution occurred with 10 mM diethanolamine buffer, pH 10, containing 0.2 M NaCl, 50% ethylene glycol in 2-ml fractions. Eluted fractions were immediately neutralized with 1 M MES buffer, pH 6.5 (0.1 fraction volume). Tryptase was measured by the total tryptase ELISA.

To generate mature {alpha}-tryptase, the eluted Q-3R{alpha}-protryptase (10–30 µg/ml) was combined with dextran sulfate (100 µg/ml) and BSA (0.5 mg/ml) and dialyzed at 4°C overnight against 1 liter of 0.01 M MES buffer, pH 6.0, containing 0.15 M NaCl. Dialyzed Q-3R{alpha}-protryptase was incubated for 3 h at 37°C with purified human lung-derived tryptase (1–3% of Q-3R{alpha}-protryptase concentration), dipeptidylpeptidase I (10 µg/ml), and DTT (0.1 mM) in 10 mM MES buffer, pH 6.0, containing 0.15 M NaCl. Controls included omitting purified human lung-derived tryptase, omitting purified human lung-derived tryptase and dipeptidylpeptidase I, or omitting Q-3R{alpha}-protryptase. With all incubation components included, nearly all of the Q-3R{alpha}-protryptase was converted from a monomer to a tetramer by Superose 12 gel filtration performed in 10 mM MES buffer, pH 6.5, containing 1 M NaCl. In the absence of either lung-derived tryptase or dipeptidylpeptidase I, only monomer was detected. The N-terminal amino acid sequence of the processed tryptase was determined as described below.

Determination of spontaneous release of tryptase

HMC-1 cells were maintained in Iscove’s medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin D, 10% (v/v) heat-inactivated FCS, and 0.01% {alpha}-thioglycerol. Mac-6 cells were maintained in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin D, and 10% (v/v) heat-inactivated FCS. Both cell types were cultured at 37°C in an incubator maintained at 5% CO2. To examine the time course of spontaneous tryptase secretion, cells were washed three times and suspended in fresh medium at 0.5 x 106 cells/ml at time 0. Cells and medium were harvested at different times as indicated. Cells were separated from medium by centrifugation at 2000 rpm for 10 min at 4°C in a model PR6 centrifuge (IEC, Needham Heights, MA) using a swinging bucket rotor. Solubilized tryptase was obtained from the cell pellets by sonicating cells suspended at 107 cells/ml 10 mM MES, pH 6.0, containing 1 M NaCl at 4°C and centrifugation at 10,000 x g for 30 min at 4°C. Cell lysate supernatants and culture media were then analyzed as described.

Skin-derived mast cells were obtained as described (31). Briefly, cells were dispersed from fresh surgical skin using collagenase and hyaluronidase, partially purified by Percoll density-dependent sedimentation, and placed into culture in serum-free AIM-V medium containing 100 ng/ml rh stem cell factor (a gift from Amgen, Thousand Oaks, CA). Mast cells were studied after 6 wk of culture, by which time they were >99% pure and >97% viable. Media and cells were collected 6 days after addition of fresh medium. Tryptase levels in cells, after extraction of the protein by sonication in 10 mM MES, pH 6.5, containing 1 M NaCl as above, and in the medium were measured by the mature and total tryptase ELISAs.

Purification of tryptase from cell media

Cells and cell media were harvested 4 days after seeding the cells at 0.5 x 106 cells/ml. Tryptase in cell lysates (~109 cells) was solubilized as above and incubated with B12-Affi-Gel beads overnight at 4°C. The beads were washed with 10 volumes of 10 mM MES, pH 6.0, containing 1.0 M NaCl, 10 volumes of 10 mM diethanolamine, pH 10, containing 0.2 M NaCl and 50% ethylene glycol, and 10 volume of 10 mM Tris, pH 8.5, containing 0.2 M NaCl. Tryptase was then eluted with 8 M urea. Cell medium (5 liters) was adjusted to contain 1 M NaCl and incubated with B12-Affi-Gel beads overnight at 4°C. Washing and elution were as for the cell lysate tryptase. Each sample of eluted tryptase was dialyzed against 1.0 M NaCl and then against 0.12 M NaCl in 10 mM MES, pH 6.0. Dialyzed tryptase was subjected to heparin-agarose chromatography, being loaded in dialysis buffer and eluted with a salt gradient in 10 mM MES, pH 6.0, from 0.1 to 2.0 M NaCl. Protein was measured by the bicinchoninic acid-modified Lowry assay (32); tryptase was measured by total and mature tryptase ELISAs.

Amino acid sequencing

Tryptase preparations from HMC-1 and Mac-6 cell lysates and culture medium, purified as above, were further subjected to SDS-PAGE in 12% polyacrylamide gels under reducing conditions, blotted onto PVDF membranes (0.2 mm) in 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer, pH 11, containing 10% methanol at 50 V for 0.5–1 h, stained with 0.1% Coomassie Brilliant Blue R250 in 40% methanol, 1% acetic acid, destained with 50% methanol, 10% acetic acid, washed with water, and dried. The tryptase band was excised and subjected to N-terminal amino acid sequencing by the Protein Structure Laboratory (University of California, Davis, CA) using an Applied Biosystems 477A sequencer.

RT-PCR and genomic DNA analysis of tryptases in the HMC-1 and Mac-6 cells

Genomic DNA from each cell line was purified using a commercial genomic DNA purification kit, whereas total RNA from each cell line was extracted using a commercial RNA isolation kit, in each case according to the manufacturer’s instructions. Also, to remove genomic DNA from preparations of total RNA the samples were treated with RNase-free DNase. To assess the presence of tryptase in genomic DNA, PCR were performed in a total volume of 50 µl containing 1x reaction buffer with 50 ng of genomic DNA, 200 mM dNTP, and 20 pmol of each primer, including {alpha}-tryptase-specific and {beta}-tryptase-specific pairs (see below). To directly assess the presence of {alpha}-tryptase and {beta}-tryptase mRNA, cDNA was synthesized from 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase and oligodeoxythymidylate primers and subjected to PCR as above. {alpha}-Tryptase-specific primers (sense: 5'-TGCAGCAAGCGGGTATCGT-3'; antisense: 5'-AGTCTGGATGATGTAGAACTGT-3') and {beta}-tryptase-specific primers (sense: 5'-TGCAGCGAGTGGGCATCGT-3'; and antisense: 5'-GATCTGGGCGGTGTAGAACTGT-3') were used for PCR. In each case, the sense and antisense primers were located in exons 3 and 4, respectively, and amplified 386-bp regions from genomic DNA and 278-bp regions from cDNA. The optimal MgCl2 concentration for PCR was 0.5 mM. Hot start reactions (95°C for 5 min) were initiated with 1 U Taq DNA polymerase followed by 35 cycles of amplification in a volume of 50 µl. Each cycle consisted of 1 min at 94°C, 1 min at 62°C and 1 min at 72°C; the final extension was performed at 72°C for 10 min. Reaction conditions were as above except that a portion (2 of 20 µl) of the cDNA was used as template. PCR products were analyzed by electrophoresis in a 6% polyacrylamide gel and visualized by staining with SYBR Green I (1/10,000 dilution).

Analysis of the relationship between the presence and absence of the gene for {alpha}-tryptase and serum tryptase levels

Plasma and blood cells were derived from EDTA-anticoagulated venous blood collected from 109 healthy subjects (56 male and 53 female). Plasma was stored at -70°C, and genomic DNA was purified from peripheral blood leukocytes using a salting-out method (PureGene; Gentra, Minneapolis, MN) (33). Samples of DNA were assessed for the presence of the {alpha}-tryptase gene by PCR with {alpha}-tryptase-specific primers as described above. Purified genomic DNA (50 ng) was subjected to PCR as above except that 1.5 U of Taq DNA polymerase were included and 35 cycles were used. The PCR product was subjected to electrophoresis in 1.5% agarose gels and visualized with ethidium bromide. Plasma was subjected to the total tryptase ELISA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of recombinant forms of {alpha}- and {beta}-tryptases by ELISA

Mature and precursor forms of recombinant {alpha}- and {beta}-tryptases were prepared and analyzed by two ELISAs. Both mature recombinant {alpha}-tryptase and {beta}-tryptase were tetrameric by gel filtration and had the predicted N-terminal amino acid sequences of IVGG. Whereas recombinant {beta}-tryptase exhibited a specific activity with 0.1 mM tosyl-Gly-Pro-Lys-p-nitroanilide of ~25 U/mg, that of recombinant {alpha}-tryptase was <= 0.2 U/mg. Each ELISA used the B12 mAb for capture. One used biotin-conjugated G5 mAb for detection; the other used biotin-conjugated G4 mAb. As shown in Fig. 1A, the dose-response curves with the biotin-G4/B12 combination were no different among the recombinant and lung-derived forms of tryptase. In contrast, the biotin-G5/B12 combination failed to detect {alpha}-protryptase, {beta}-protryptase, and {beta}-pro’tryptase but showed similar dose-response curves for mature recombinant {beta}- and {alpha}-tryptases and lung-derived tryptase. Thus, in the context of mast cell {alpha}- and {beta}-tryptases, the biotin-G4/B12 sandwich serves as a total tryptase assay; the biotin-G5/B12 sandwich serves as an assay for mature tryptases.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 1. Detection of mature and precursor forms of {alpha}-tryptase and {beta}-tryptase by two ELISAs. Purified human lung-derived tryptase, rh{alpha}-protryptase (native sequence), rh{alpha}-tryptase (derived from Q-3R{alpha}-protryptase), rh{beta}-protryptase, rh{beta}-pro’tryptase, and rh{beta}-tryptase molecules, at concentrations from 0.01 to 0.6 ng/well, were measured as described in Materials and Methods using biotin-G4 (A) or biotin-G5 (B) for detection. The absolute amounts of each form of tryptase were determined by direct measurement of purified protein (bicinchoninic assay) and confirmed by comparing the optical density of the Coomassie blue-stained band after SDS-PAGE of each form of tryptase to that of human lung-derived tryptase.

 
Spontaneously secreted and retained levels of tryptase in cultures of HMC-1 and Mac-6 cell lines and of skin-derived mast cells

Total and mature tryptase levels were measured in the medium and in cell lysates collected at different times from cultures of HMC-1 and Mac-6 cells. As shown in Fig. 2, total tryptase levels in the medium progressively increased during the 3-day experiment for both HMC-1 and Mac-6 cells, whereas mature tryptase levels remained low throughout the culture (Table I). For HMC-1 cells, the total tryptase level detected in the medium after 3 days of culture (172 ± 27 ng/106 cell equivalents) was higher than the tryptase level detected inside the cells (36 ± 5 ng/106 cells). However, 42% of the tryptase that was retained in the cell was mature by ELISA, compared with only 3% of the spontaneously secreted tryptase. For Mac-6 cells the total tryptase level detected in the medium after 3 days of culture (37 ± 3 ng/106 cell equivalents) was considerably higher than that detected inside the cells (1.3 ± 0.3 ng/106 cells). Again, the portion of retained tryptase that by ELISA was mature (23%) was higher than for the spontaneously secreted tryptase (4%).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 2. Spontaneous secretion of tryptase by HMC-1 and Mac-6 cells. Cells in culture were washed and suspended in fresh medium at 0.5 x 106 cells/ml. Samples were harvested from three wells at each time point, separated into cells and medium, and assayed for tryptase with the two immunoassays as described. The data were normalized by the amount of tryptase divided by cell number (nanograms per 106 cells). A, HMC-1 cell line; B, Mac-6 cell line. Tryptases in medium and in the cell lysates are expressed as nanograms or micrograms per 106 cells or cell equivalents. Mean ± SD values are shown at each time point (n = 3).

 

View this table:
[in this window]
[in a new window]
  		Table I. Retained and secreted mature and total tryptase levels in HMC-1 and Mac-6 cellsa

 
Cultured skin-derived mast cells were examined 6 days after addition of fresh medium (Table II). For these primary cultures of nontransformed mast cells, the total tryptase level measured in the medium (5.2 ± 1.3 µg/106 cell equivalents) was lower than that retained by the cells (23 ± 3 µg/106 cells). Levels of tryptase associated with these skin-derived mast cells were substantially higher than for both leukemic cell types, being more comparable with freshly isolated skin-derived mast cells (35 µg/106 cells) (24). Like the leukemic cells, only a small portion of spontaneously secreted tryptase was mature (6%). In contrast, nearly all of the tryptase retained by the skin-derived mast cells was mature (94%).


View this table:
[in this window]
[in a new window]
  		Table II. Retained and secreted mature and total tryptase levels in skin-derived mast cellsa

 
Identification of mature and precursor forms of tryptase

To obtain more precise molecular data as to the types of tryptase either retained or secreted by HMC-1 and Mac-6 cells, tryptase from these sources was purified by sequential immunoaffinity and heparin-agarose chromatography and subjected to N-terminal amino acid sequencing. Whereas medium-derived tryptase eluted from heparin-agarose near 0.2 M NaCl, cell lysate-derived tryptase eluted at ~0.8 M NaCl, suggesting different physicochemical forms of this protein. Thus, cell lysate-derived tryptase elutes from heparin at an ionic strength comparable with that of enzymatically active, human lung-derived tryptase, whereas tryptase in the medium elutes at a salt concentration comparable with those of precursor forms of the protein (26).

After purification, both media and cell lysate tryptase preparations produced a single diffuse Coomassie blue-stained band after SDS-PAGE (data not shown). The protein in each of these bands was subjected to up to 12 cycles of N-terminal amino acid sequencing. Fig. 3 summarizes the results. The single sequence obtained for HMC-1 medium tryptase was consistent with {beta}-protryptase, indicating this was the predominant form that was spontaneously released. In contrast, the two N-terminal sequences for tryptase in the Mac-6 culture medium indicated that {alpha}-protryptase was the predominant form that was spontaneously secreted and that partial digestion of the propeptide had occurred, perhaps by an N-terminal peptidase. In contrast, tryptase in the cell lysates of both cell types yielded the mature sequence, consistent with the ELISA and chromatography data indicating that mature forms of tryptase predominate inside the cells.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3. N-Terminal amino acid sequence of retained and spontaneously secreted tryptase from HMC-1 and Mac-6 cells. In each case, the first N-terminal 12-aa positions were assigned. {alpha}-Tryptase and {beta}-tryptase can be distinguished from one another by the propeptide amino acids at positions -8, -3, and -2, namely, Val, Gln, and Ala for {alpha}-tryptase and Gly, Arg, and Val for {beta}-tryptase. Mature tryptase could not be subclassified by the obtainable sequence.

 
Tryptase genes and their expression in HMC-1 and Mac-6 cells

Previous reports indicated that HMC-1 cells express predominantly {beta}I-tryptase mRNA (19, 20) and Mac-6 cells express predominantly {alpha}-tryptase mRNA (21). In the current study, PCR and RT-PCR were used to examine genomic DNA and mRNA for {alpha}-tryptase and {beta}-tryptase. Primers specific for {alpha}-tryptase and {beta}-tryptase were designed. As shown in Fig. 4, amplification by PCR with {alpha}-tryptase-specific primers yielded a 278-bp product with an {alpha}-tryptase cDNA template but no product with a {beta}-tryptase cDNA template (lanes 6 and 7, respectively). Likewise, {beta}-tryptase-specific primers produced a 278-bp product with a {beta}-tryptase cDNA template and no product with an {alpha}-tryptase cDNA template (lanes 11 and 10, respectively). Whereas genomic DNA from both HMC-1 and Mac-6 cells yielded a 386-bp product reflecting a {beta}-tryptase gene (lanes 3 and 4, respectively), only Mac-6 genomic DNA yielded an {alpha}-tryptase product (lane 2), indicating HMC-1 cells were deficient for the {alpha}-tryptase gene (lane 1). RT-PCR using {alpha}-tryptase primers for PCR showed that {alpha}-tryptase mRNA was present in Mac-6 cells (lane 9), but not in HMC-1 cells (lane 8). In contrast, RT-PCR using {beta}-specific primers for PCR showed that {beta}-tryptase mRNA was present in both HMC-1 cells (lane 12) and Mac-6 cells (lane 13). However, there appeared to be far less {beta}-tryptase product from Mac-6 cells than from HMC-1 cells, suggesting a corresponding paucity of {beta}-tryptase mRNA in Mac-6 cells.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 4. Analysis of {alpha}-tryptase and {beta}-tryptase in genomic DNA and mRNA of HMC-1 and Mac-6 cells. Samples of genomic DNA from HMC-1 (HD) and Mac-6 (MD) cells were amplified by PCR with {alpha}-tryptase-specific and {beta}-tryptase-specific primers. Samples of RNA (HR and MR) were subjected to RT-PCR using the same primer pairs. The size marker lane is noted by mw. The {beta}-tryptase RT-PCR product (50 µl) from Mac-6 cells was purified and concentrated before loading, whereas 5 µl of each of the other reaction mixtures were applied to the gel. DNA was visualized with SYBR Green I.

 
Plasma tryptase levels in subjects with and without the gene for {alpha}-tryptase

Samples of genomic DNA from the peripheral blood leukocytes of 109 healthy subjects were assessed for the presence or absence of the {alpha}-tryptase gene by PCR. Tryptase gene haplotypes may bear either one {alpha} and one {beta} gene, or, alternatively, two {beta} genes; with our method, a sample typing positive for the {alpha}-tryptase gene may represent either a heterozygote ({alpha}{beta}:{beta}{beta}) or a homozygote ({alpha}{beta}:{alpha}{beta}). Representative PCR results are shown for nine of these subjects in Fig. 5A, where the results reflect the presence of an {alpha}-tryptase gene in six of the nine samples. Overall, 28 of the 109 specimens (26%) lacked the {alpha}-tryptase gene. All 109 DNA samples contained {beta}-tryptase gene(s) (data not shown). Total tryptase levels also were measured in corresponding samples of plasma, as illustrated in Fig. 5B. The median (25th percentile, 75th percentile) plasma tryptase value among {alpha}-tryptase+ subjects, 5.2 (4.1, 7.5) ng/ml was slightly but not significantly (p = 0.11, Mann-Whitney rank sum test) higher than that of {alpha}-tryptase- subjects, 4.7 (3.4, 6.3) ng/ml. The range also was higher among {alpha}-tryptase+ (1.1–19 ng/ml) than {alpha}-tryptase- (1.8–8.4 ng/ml) subjects. Total tryptase levels in male subjects (4.7 (3.5, 6.8) ng/ml) were slightly lower than in female subjects (5.6 (4.3, 7.5) ng/ml) (p = 0.02). Regression analyses using log-transformed tryptase values and correcting for the sex difference confirmed no significant effect of the {alpha}-tryptase gene on total tryptase levels. Thus, the median total plasma tryptase level of healthy subjects lacking the {alpha}-tryptase gene was not significantly different from the median for those having at least one {alpha}-tryptase gene.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 5. Plasma tryptase levels in subjects with and without the gene for {alpha}-tryptase. A, Analysis of genomic DNA for {alpha}-tryptase. Genomic DNA was extracted from peripheral blood leukocytes and amplified by PCR using {alpha}-tryptase-specific primers as described. Lanes 1–9, Representative PCR results from 9 of the 109 subjects in the study group; H, HMC-1 cells; M, Mac-6 cells. A 100-bp DNA ladder was used to estimate molecular mass (mw). Arrows point to the 300- and 400-bp markers, and to the 386-bp PCR product. B, Plasma total tryptase levels. Box and whisker plots show median (horizontal bar within gray boxes), 25th and 75th percentiles (box ends), 10th and 90th percentiles (whiskers), and outliers (•).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A major finding of the current study is that precursors of both {alpha}-tryptase and {beta}-tryptase are spontaneously released, while fully processed {beta}-tryptase is retained by the cells, principally in secretory granules. This finding applies to both tryptase-expressing leukemic cell lines (HMC-1 and Mac-6) and primary cultures of skin-derived mast cells. The consensus 12-aa {alpha}-protryptase was dominant in tryptase purified from the medium of Mac-6 cultures, whereas the {beta}-protryptase sequence was dominant in tryptase purified from the culture medium of HMC-1 cells. This is the first time the {beta}-protryptase molecule has been directly observed in a mast cell system and supports this consensus pro sequence as being the actual pro sequence. In contrast to medium-derived tryptase, cell lysate-derived tryptase exhibited a sequence consistent with the mature portion of either {beta}-tryptase or {alpha}-tryptase. Because prior work showed that recombinant human {beta}-protryptase could be autoprocessed next to R-3 to {beta}-pro’tryptase, and then to {beta}-tryptase by dipeptidyl peptidase I, whereas {alpha}-protryptase did not undergo autoprocessing (5), we presume that most if not all of the mature tryptase detected in the HMC-1 cells, Mac-6, cells and skin-derived mast cells is mature {beta}-tryptase. This is certainly the case in HMC-1 cells, which lack the {alpha}-tryptase gene. However, for Mac-6 cells, which express small amounts of {beta}-tryptase mRNA along with larger amounts of {alpha}-tryptase mRNA, the gene of origin for the mature tryptase has not been directly determined. In the case of skin- and lung-derived mast cells, it is likely that most, if not all, of the mature tryptase is {beta}-tryptase. As reported previously (7, 8, 9, 34) and confirmed in the current study, mature {alpha}-tryptase, like mature {beta}-tryptase, forms a tetramer, but has little, if any, enzymatic activity. Because tryptase purified from skin- and lung-derived mast cells has a specific enzymatic activity nearly identical with that of mature recombinant {beta}-tryptase, little if any mature {alpha}-tryptase is likely present.

Whether either of the immunoassays used in the current study detects {delta}-tryptase was not directly addressed. However, like {alpha}-protryptase, {delta}-protryptase has a Q-3, possibly making autoprocessing inefficient. Further, in Western blots with extracts of various types of mast cells, a low mw band reflecting the 40 amino acid deficit of {delta}-tryptase was not apparent (our unpublished results). Thus, {delta}-tryptase may be present in relatively low amounts compared with {alpha}- and {beta}- tryptases or may not be recognized by the mAb(s) used for Western blotting. Nevertheless, {delta}-tryptase remains to be precisely quantified.

Why protryptase molecules are spontaneously secreted by mast cells is unclear. Failure to adopt a particular conformation needed for targeting the protein to secretory granules is one possibility. A region within the pro portion that actively targets the protein to a constitutive secretory pathway is another possibility. A third consideration is that the protein is targeted to the secretory granules through its high affinity interaction with heparin proteoglycan, which for {beta}-tryptase occurs after the protein has been processed to the mature, tetrameric enzyme. {beta}-Protryptase binds to heparin with lower affinity and is optimally processed to the mature tetramer when bound to heparin. If production of {beta}-protryptase in excess of cofactor heparin occurred, it would most likely lead to inefficient processing of the excess portion of the enzyme, and spontaneous secretion of the unprocessed protein. Whether Mac-6 cells can express small amounts of heparin proteoglycan or use distinct processing enzymes and stabilizing cofactors must be considered.

Although {alpha}-tryptase and {beta}-tryptase mRNA are found together in preparations of lung-derived and skin-derived mast cells (20) and increase in parallel during the development of mast cells from fetal liver progenitors in vitro (22), the current study supports previous observations in Mac-6 cells that indicate that differential expression of these genes is possible (19, 21). Mac-6 cells express predominantly {alpha}-tryptase and can now be said to also contain small amounts of {beta}-tryptase mRNA. Other cell lines, U-937 monocytic leukemia cells and the KU812 basophil leukemia cells, express predominantly {beta}-tryptase mRNA, whereas normal basophils from peripheral blood of two individuals expressed predominantly {alpha}-tryptase mRNA (20, 21, 35). Normal peripheral blood monocyte preparations of 85% purity that were T cell depleted reportedly contain trace amounts of tryptase mRNA (21), but basophils possibly present in this preparation should be considered as a potential source of the tryptase mRNA detected. Factors regulating the differential expression of tryptase genes remain to be determined.

A finding of practical importance to clinicians is that the ELISA using the G5 mAb for detection preferentially detects mature tryptase, regardless as to whether the product originates from the {alpha}-tryptase or {beta}-tryptase genes. This is likely even though all subtypes of these gene products were not examined. For example, the primary sequence of {beta}III-tryptase is entirely represented in {alpha}- and the other {beta}-tryptases. Thus, the total and mature tryptase assays should recognize the precursor and mature forms of this tryptase variant. Because mature tryptase is stored in secretory granules and released during degranulation, measurement of this form of tryptase in biological fluids should reflect the magnitude of mast cell activation, as has been postulated previously (10, 11, 36). In contrast, the ELISA using the G4 mAb for detection detects both pro and mature forms of {alpha}-tryptase and {beta}-tryptase, i.e., the total tryptase level. Because the tryptase in normal blood is measurable by the total but not mature tryptase assay, it is likely that protryptase(s) is the major form in normal blood. Although it was previously postulated that {alpha}-protryptase accounted for the protryptase in normal blood (10), this cannot be the case in subjects who lack a gene for {alpha}-tryptase. The current study shows that both {alpha}-protryptase and {beta}-protryptase are spontaneously secreted by tryptase-producing cells in culture. The total tryptase level, in a subject without symptoms or signs of anaphylaxis, is likely to reflect mast cell number. Indeed, the total tryptase level is elevated (>20 ng/ml) in people with systemic mastocytosis during nonacute periods (10, 37, 38). However, total tryptase levels also rise during systemic anaphylaxis (14). The current study shows the difference in median total tryptase levels in the plasma of subjects with (5.2 ng/ml) and without (4.7 ng/ml) the {alpha}-tryptase gene is small and is not statistically significant. If each haploid genome contains either one {alpha}-tryptase and one {beta}-tryptase gene, or two {beta}-tryptase genes, as postulated (16), then subjects with four {beta}-tryptase genes in their diploid genomes produce almost the same levels of total plasma tryptase as do the combined groups with either two {beta}-tryptase and two {alpha}-tryptase, or three {beta}-tryptase and one {alpha}-tryptase genes. However, whether a difference might have been appreciated if the latter two groups had been identified and separately analyzed was not directly assessed by the current study. Also, the subtypes of {alpha}- and {beta}-tryptases were not assessed, leaving open the possibility of variations in tryptase levels that correspond to specific subtypes.

Subjects with systemic mastocytosis have elevated levels of total tryptase (>20 ng/ml) and little if any mature tryptase in their plasma or serum during nonacute periods, presumably reflecting the increased burden of mast cells in tissues such as liver, spleen, and bone marrow. Some subjects with acute myelocytic leukemia and myelodysplastic syndromes also have markedly elevated levels of total tryptase (39, 40). Based on the immunoassay characteristics of the current study, most of the plasma tryptase detected in such patients is likely to be precursor forms of {alpha}- and {beta}-tryptases; however, direct N-terminal sequencing of tryptase purified from such sources would provide the best evidence for this inference. Unfortunately, plasma tryptase purified from two mastocytosis subjects had blocked N termini (our unpublished data). Whether the absence or presence of {alpha}-tryptase in the genomes of individuals with these disorders affects total tryptase levels to a greater extent than for healthy subjects remains to be studied. Also, it is possible that the coordinated expression of {beta}-protryptase and heparin proteoglycan in mastocytosis mast cells is awry and leads to the spontaneous secretion of a higher portion of the tryptase produced by such cells. The HMC-1 cells used in the current study secrete a far greater portion of their {beta}-tryptase compared with cultured skin-derived mast cells, even though the latter cells produce substantially more tryptase.

In conclusion, the current study supports the previous hypothesis that levels of mature tryptase in biological fluids serve as an indicator for mast cell activation, because this form is selectively stored in mast cell secretory granules. In contrast, levels of the precursors of mature tryptase reflect mast cell numbers, because they are spontaneously secreted by mast cells. Further, whether or not the four putative tryptase genes in the normal diploid chromosome lack {alpha}-tryptase does not influence the circulating level of tryptase precursors in healthy subjects.


   Footnotes
 
1 This study was supported in part by Grants AI24087 and AI27517 from the National Institutes of Health (Bethesda, MD) and by a grant from Pharmacia Diagnostics (Uppsala, Sweden). Back

2 Address correspondence and reprint requests to Dr. Lawrence B. Schwartz, Division of Rheumatology, Allergy and Immunology, Department of Internal Medicine, Virginia Commonwealth University, P.O. Box 980263, Richmond, VA 23298. E-mail address: lbschwar{at}vcu.edu Back

3 Abbreviations used in this paper: rh, recombinant human; Mac-6, Mono-Mac-6. Back

Received for publication January 2, 2003. Accepted for publication March 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schwartz, L. B.. 1994. Tryptase: a mast cell serine protease. Methods Enzymol. 244:88.[Medline]
  2. Vanderslice, P., S. M. Ballinger, E. K. Tam, S. M. Goldstein, C. S. Craik, G. H. Caughey. 1990. Human mast cell tryptase: multiple cDNAs and genes reveal a multigene serine protease family. Proc. Natl. Acad. Sci. USA 87:3811.[Abstract/Free Full Text]
  3. Pallaoro, M., M. S. Fejzo, L. Shayesteh, J. L. Blount, G. H. Caughey. 1999. Characterization of genes encoding known and novel human mast cell tryptases on chromosome 16p13.3. J. Biol. Chem. 274:3355.[Abstract/Free Full Text]
  4. Wang, H. W., H. P. McNeil, A. Husain, K. Liu, N. Tedla, P. S. Thomas, M. Raftery, G. C. King, Z. Y. Cai, J. E. Hunt. 2002. {delta} Tryptase is expressed in multiple human tissues, and a recombinant form has proteolytic activity. J. Immunol. 169:5145.[Abstract/Free Full Text]
  5. Sakai, K., S. Ren, L. B. Schwartz. 1996. A novel heparin-dependent processing pathway for human tryptase: autocatalysis followed by activation with dipeptidyl peptidase I. J. Clin. Invest. 97:988.[Medline]
  6. Wolters, P. J., C. T. N. Pham, D. J. Muilenburg, T. J. Ley, G. H. Caughey. 2001. Dipeptidyl peptidase I is essential for activation of mast cell chymases, but not tryptases, in mice. J. Biol. Chem. 276:18551.[Abstract/Free Full Text]
  7. Huang, C., L. Li, S. A. Krilis, K. Chanasyk, Y. Tang, Z. Li, J. E. Hunt, R. L. Stevens. 1999. Human tryptases {alpha} and {beta}/II are functionally distinct due, in part, to a single amino acid difference in one of the surface loops that forms the substrate-binding cleft. J. Biol. Chem. 274:19670.[Abstract/Free Full Text]
  8. Marquardt, U., F. Zettl, R. Huber, W. Bode, C. Sommerhoff. 2002. The crystal structure of human {alpha}1-tryptase reveals a blocked substrate-binding region. J. Mol. Biol. 321:491.[Medline]
  9. Selwood, T., Z. M. Wang, D. R. McCaslin, N. M. Schechter. 2002. Diverse stability and catalytic properties of human tryptase {alpha} and {beta} isoforms are mediated by residue differences at the S1 pocket. Biochemistry 41:3329.[Medline]
  10. Schwartz, L. B., K. Sakai, T. R. Bradford, S. L. Ren, B. Zweiman, A. S. Worobec, D. D. Metcalfe. 1995. The {alpha} form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis. J. Clin. Invest. 96:2702.
  11. Schwartz, L. B., D. D. Metcalfe, J. S. Miller, H. Earl, T. Sullivan. 1987. Tryptase levels as an indicator of mast-cell activation in systemic anaphylaxis and mastocytosis. N. Engl. J. Med. 316:1622.[Abstract]
  12. Schwartz, L. B., J. W. Yunginger, J. S. Miller, R. Bokhari, D. Dull. 1989. The time course of appearance and disappearance of human mast cell tryptase in the circulation after anaphylaxis. J. Clin. Invest. 83:1551.
  13. Van der Linden, P.-W. G., C. E. Hack, J. Poortman, Y. C. Vivié-Kipp, A. Struyvenberg, J. K. Van der Zwan. 1992. Insect-sting challenge in 138 patients: relation between clinical severity of anaphylaxis and mast cell activation. J. Allergy Clin. Immunol. 90:110.[Medline]
  14. Schwartz, L. B., T. R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J. K. Van der Zwan, P.-W. G. Van Der Linden. 1994. Development of a new, more sensitive immunoassay for human tryptase: use in systemic anaphylaxis. J. Clin. Immunol. 14:190.[Medline]
  15. Guida, M., M. Riedy, D. Lee, J. Hall. 2000. Characterization of two highly polymorphic human tryptase loci and comparison with a newly discovered monkey tryptase ortholog. Pharmacogenetics 10:389.[Medline]
  16. Soto, D., C. Malmsten, J. L. Blount, D. J. Muilenburg, G. H. Caughey. 2002. Genetic deficiency of human mast cell {alpha}-tryptase. Clin. Exp. Allergy 32:1000.[Medline]
  17. Butterfield, J. H., D. Weiler, G. Dewald, G. J. Gleich. 1988. Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk. Res. 12:345.[Medline]
  18. Ziegler-Heitbrock, H. W., E. Thiel, A. Futterer, V. Herzog, A. Wirtz, G. Riethmuller. 1988. Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int. J. Cancer 41:456.[Medline]
  19. Nilsson, G., T. Blom, M. Kusche-Gullberg, L. Kjellen, J. H. Butterfield, C. Sundstrom, K. Nilsson, L. Hellman. 1994. Phenotypic characterization of the human mast-cell line HMC-1. Scand. J. Immunol. 39:489.[Medline]
  20. Xia, H.-Z., C. L. Kepley, K. Sakai, J. Chelliah, A.-M. A. Irani, L. B. Schwartz. 1995. Quantitation of tryptase, chymase, Fc{epsilon}RI{alpha}, and Fc{epsilon}RI{gamma} mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction. J. Immunol. 154:5472.[Abstract]
  21. Huang, R., M. Åbrink, A. E. Gobl, G. Nilsson, M. Aveskogh, L. G. Larsson, K. Nilsson, L. Hellman. 1993. Expression of a mast cell tryptase in the human monocytic cell lines U-937 and Mono Mac 6. Scand. J. Immunol. 38:359.[Medline]
  22. Xia, H. Z., Z. M. Du, S. Craig, G. Klisch, N. Noben-Trauth, J. P. Kochan, T. H. Huff, A. M. Irani, L. B. Schwartz. 1997. Effect of recombinant human IL-4 on tryptase, chymase, and Fc{epsilon} receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells. J. Immunol. 159:2911.[Abstract]
  23. Butterfield, J. H., D. A. Weiler, L. W. Hunt, S. R. Wynn, P. C. Roche. 1990. Purification of tryptase from a human mast cell line. J. Leukocyte Biol. 47:409.[Abstract]
  24. Schwartz, L. B., A. M. A. Irani, K. Roller, C. Castells, N. M. Schechter. 1987. Quantitation of histamine, tryptase and chymase in dispersed human T and TC mast cells. J. Immunol. 138:2611.[Abstract]
  25. Schwartz, L. B., T. R. Bradford, D. C. Lee, J. F. Chlebowski. 1990. Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer: production of monoclonal antibodies recognizing active tryptase. J. Immunol. 144:2304.[Abstract]
  26. Sakai, K., S. D. Long, D. A. D. Pettit, G. A. Cabral, L. B. Schwartz. 1996. Expression and purification of recombinant human tryptase in a baculovirus system. Prot. Exp. Purif. 7:67.[Medline]
  27. Schwartz, L. B., J. V. Foley, K. F. Austen, N. A. Soter, R. Shepard, G. F. Murphy. 1985. Localization of tryptase to human cutaneous mast cells and keratinocytes by immunofluorescence and immunoperoxidase cytochemistry with monoclonal antitryptase antibody. J. Allergy Clin. Immunol. 76:182.[Medline]
  28. Schwartz, L. B.. 1985. Monoclonal antibodies against human mast cell tryptase demonstrate shared antigenic sites on subunits of tryptase and selective localization of the enzyme to mast cells. J. Immunol. 134:526.[Abstract]
  29. Irani, A.-M. A., T. R. Bradford, C. L. Kepley, N. M. Schechter, L. B. Schwartz. 1989. Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies. J. Histochem. Cytochem. 37:1509.[Abstract/Free Full Text]
  30. Wang, W., B. A. Malcolm. 1999. Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. BioTechniques 26:680.[Medline]
  31. Kambe, N., M. Kambe, J. P. Kochan, L. B. Schwartz. 2001. Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes. Blood 97:2045.[Abstract/Free Full Text]
  32. Redinbaugh, M. G., R. B. Turley. 1986. Adaption of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions. Anal. Biochem. 153:267.[Medline]
  33. Miller, S. A., D. D. Dykes, H. F. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215.[Free Full Text]
  34. Pereira, P. J., A. Bergner, S. Macedo-Ribeiro, R. Huber, G. Matschiner, H. Fritz, C. P. Sommerhoff, W. Bode. 1998. Human {beta}-tryptase is a ring-like tetramer with active sites facing a central pore. Nature 392:306.[Medline]
  35. Blom, T., L. Hellman. 1993. Characterization of a tryptase mRNA expressed in the human basophil cell line KU812. Scand. J. Immunol. 37:203.[Medline]
  36. Schwartz, L. B.. 2001. Clinical utility of tryptase levels in systemic mastocytosis and associated hematologic disorders. Leuk. Res. 25:553.[Medline]
  37. Akin, C., L. B. Schwartz, T. Kitoh, H. Obayashi, A. S. Worobec, L. M. Scott, D. D. Metcalfe. 2000. Soluble stem cell factor receptor (CD117) and IL-2 receptor {alpha} chain (CD25) levels in the plasma of patients with mastocytosis: relationships to disease severity and bone marrow pathology. Blood 96:1267.[Abstract/Free Full Text]
  38. Valent, P., H.-P. Horny, L. Escribano, B. J. Longley, C. Y. Li, L. B. Schwartz, G. Marone, R. Nunez, C. Akin, K. Sotlar, et al 2001. Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leuk. Res. 25:603.[Medline]
  39. Sperr, W. R., B. Stehberger, F. Wimazal, M. Baghestanian, L. B. Schwartz, M. Kundi, H. Semper, J. H. Jordan, A. Chott, J. Drach, et al 2002. Serum tryptase measurements in patients with myelodysplastic syndromes. Leuk. Lymphoma 43:1097.[Medline]
  40. Sperr, W. R., J. H. Jordan, M. Baghestanian, H. P. Kiener, P. Samorapoompichit, H. Semper, A. Hauswirth, G. H. Schernthaner, A. Chott, S. Natter, et al 2001. Expression of mast cell tryptase by myeloblasts in a group of patients with acute myeloid leukemia. Blood 98:2200.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
W. W. Raymond, S. Su, A. Makarova, T. M. Wilson, M. C. Carter, D. D. Metcalfe, and G. H. Caughey
{alpha}2-Macroglobulin Capture Allows Detection of Mast Cell Chymase in Serum and Creates a Reservoir of Angiotensin II-Generating Activity
J. Immunol., May 1, 2009; 182(9): 5770 - 5777.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Fukuoka, H.-Z. Xia, L. B. Sanchez-Munoz, A. L. Dellinger, L. Escribano, and L. B. Schwartz
Generation of Anaphylatoxins by Human {beta}-Tryptase from C3, C4, and C5
J. Immunol., May 1, 2008; 180(9): 6307 - 6316.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. N. Trivedi, Q. Tong, K. Raman, V. J. Bhagwandin, and G. H. Caughey
Mast Cell {alpha} and beta Tryptases Changed Rapidly during Primate Speciation and Evolved from {gamma}-Like Transmembrane Peptidases in Ancestral Vertebrates
J. Immunol., November 1, 2007; 179(9): 6072 - 6079.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. C. M. Strik, A. Wolbink, D. Wouters, B. A. Bladergroen, A. R. Verlaan, I. S. van Houdt, S. Hijlkema, C. E. Hack, and J. A. Kummer
Intracellular serpin SERPINB6 (PI6) is abundantly expressed by human mast cells and forms complexes with {beta}-tryptase monomers
Blood, April 1, 2004; 103(7): 2710 - 2717.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schwartz, L. B.
Right arrow Articles by Fukuoka, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schwartz, L. B.
Right arrow Articles by Fukuoka, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS