HLA Mismatching Favoring Host-Versus-Graft NK Cell Activity Via KIR3DL1 Is Associated With Improved Outcomes Following Lung Transplantation.

Chronic lung allograft dysfunction (CLAD) is linked to rejection and limits survival following lung transplantation. HLA-Bw4 recipients of HLA-Bw6 grafts have enhanced host-versus-graft (HVG) natural killer (NK) cell activity mediated by killer cell immunoglobulin-like receptor (KIR)3DL1 ligand. Because NK cells may promote tolerance by depleting antigen-presenting cells, we hypothesized improved outcomes for HLA-Bw4 recipients of HLA-Bw6 grafts. We evaluated differences in acute cellular rejection and CLAD-free survival across 252 KIR3DL1+ recipients from University of California, San Francisco (UCSF). For validation, we assessed survival and freedom from bronchiolitis obliterans syndrome (BOS), retransplantation, or death in 12 845 non-KIR typed recipients from the United Network for Organ Sharing (UNOS) registry. Cox proportional hazards models were adjusted for age, gender, ethnicity, transplant type, and HLA mismatching. HVG-capable subjects in the UCSF cohort had a decreased risk of CLAD or death (hazard ratio [HR] 0.57, 95% confidence interval [CI] 0.36-0.88) and decreased early lymphocytic bronchitis. The HVG effect was not significant in subjects with genotypes predicting low KIR3DL1 expression. In the UNOS cohort, HVG-capable subjects had a decreased risk of BOS, retransplant, or death (HR 0.95, 95% CI 0.91-0.99). Survival improved with the higher-affinity Bw4-80I ligand and in Bw4 homozygotes. Improved outcomes in HVG-capable recipients are consistent with a protective NK cell role. Augmentation of NK activity could supplement current immunosuppression techniques.

Bronchoalveolar lavage cell immunophenotyping facilitates diagnosis of lung allograft rejection.

Supplementary methods to identify acute rejection and to distinguish rejection from infection may improve clinical outcomes for lung allograft recipients. We hypothesized that distinct bronchoalveolar lavage (BAL) cell profiles are associated with rejection and infection. We retrospectively compared 2939 BAL cell counts and immunophenotypes against concomitantly obtained transbronchial biopsies and microbiologic studies. We randomly assigned 317 subjects to a derivation or validation cohort. BAL samples were classified into four groups: infection, rejection grade ≥A1, both or neither. We employed generalized estimating equation and survival modeling to identify clinical predictors of rejection and infection. We found that CD25(+) and natural killer cell percentages identified a twofold increased odds of rejection compared to either the infection or the neither infection nor rejection groups. Also, monocytes, lymphocytes and eosinophil percentages were independently associated with rejection. A four-predictor scoring system had high negative predictive value (96-98%) for grade ≥A2 rejection, predicted future rejection in the validation cohort and predicted increased risk of bronchiolitis obliterans syndrome in otherwise benign samples. In conclusion, BAL cell immunophenotyping discriminates between infection and acute rejection and predicts future outcomes in lung transplant recipients. Although it cannot replace histopathology, immunophenotyping may be a clinically useful adjunct.

Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient Kit(W-sh)/Kit(W-sh) sash mice.

BACKGROUND: Mast cell-deficient Kit(W)/Kit(W-v) mice are an important resource for studying mast cell functions in vivo. However, because they are compound heterozygotes in a mixed genetic background and are infertile, they cannot be crossed easily with other mice. OBJECTIVE: To overcome this limitation, we explored the use of Kit(W-sh)/Kit(W-sh) mice for studying mast cell biology in vivo. RESULTS: These mice are in a C57BL/6 background, are fertile and can be bred directly with other genetically modified mice. Ten-week-old Kit(W-sh)/Kit(W-sh) are profoundly mast cell-deficient. No mast cells are detected in any major organ, including the lung. Gene microarrays detect differential expression of just seven of 16,463 genes in lungs of Kit(W-sh)/Kit(W-sh) mice compared with wild-type mice, indicating that resting mast cells regulate expression of a small set of genes in the normal lung. Injecting 10(7) bone marrow-derived mast cells (BMMC) into tail veins of Kit(W-sh)/Kit(W-sh) mice reconstitutes mast cell populations in lung, stomach, liver, inguinal lymph nodes, and spleen, but not in the tongue, trachea or skin. Injection of BMMC into ear dermis or peritoneum reconstitutes mast cells locally in these tissues. When splenectomized Kit(W-sh)/Kit(W-sh) mice are intravenously injected with BMMC, mast cells circulate longer and are found more often in the liver and inguinal lymph nodes, indicating that the spleen acts as a reservoir for mast cells following injection and limits migration to some tissues. CONCLUSION: In summary, these findings show that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice possess unique attributes that favour their use for studying mast cell functions in vivo.

Genetic deficiency of human mast cell alpha-tryptase.

BACKGROUND: Human alpha- and beta-tryptases are proteases secreted by mast cells. Beta (but not alpha) tryptases are implicated in asthma. Genes encoding both types of tryptases cluster on chromosome 16p13.3. OBJECTIVE: This study examines the hypothesis, generated from mapping data, that alpha-alleles compete with some beta-alleles at one locus and that an adjacent locus contains beta-alleles exclusively. This hypothesis predicts that beta-alleles outnumber alpha and that some genomes lack alpha genes altogether. METHODS: To test this hypothesis, we developed PCR-based techniques to distinguish alpha from beta genes. We then genotyped genomic DNA from individuals and tryptase-expressing cell lines. RESULTS: In support of our hypothesis, we find that alpha-tryptase deficiency affects 80/274 (29%) of individuals surveyed. The genotype of the alpha-deficient individuals is betabetabetabeta, due to inheritance of four beta genes. The percentage of the population with the mixed genotypes alphaalphabetabeta and alphabetabetabeta is 21% and 50%, respectively. Accounting for all alpha- and beta-alleles at the tandem loci on 16p13.3, overall alpha-allele frequency is only 0.23, with beta-alleles considerably outnumbering alpha as hypothesized. In samples of defined ethnicity, alpha deficiency affects 45% of Caucasians, but a much lower percentage of other backgrounds, including African-Americans and Asians. Examination of cell lines reveals that HMC-1 and U-937 lack alpha-genes; thus, lack of alpha transcripts in these cells is due to absence of alpha-genes rather than beta-selective transcription. By contrast, alpha-transcribing Mono Mac 6 and KU812 cells contain alpha- and beta-genes. CONCLUSIONS: Genetic alpha-tryptase deficiency is common and varies strikingly between ethnic groups. Because beta-tryptases are implicated in allergic disorders, inherited differences in alpha/beta-genotype may affect disease susceptibility, severity and response to tryptase inhibitor therapy.

Dipeptidyl peptidase I is essential for activation of mast cell chymases, but not tryptases, in mice.

Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI +/+ and DPPI -/- mice. We find that DPPI -/- mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI -/- mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI -/- BMMCs is 25% that of DPPI +/- BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.

Mast cell tryptase activates extracellular-regulated kinases (p44/p42) in airway smooth-muscle cells: importance of proteolytic events, time course, and role in mediating mitogenesis.

We previously reported that mast cell tryptase is a potent mitogen for cultured airway smooth-muscle cells, but the early intracellular signals mediating this response are not known. In many cells, proliferative effects are mediated by a mitogen-activated protein kinase signaling pathway involving Raf-1, MAP kinase kinases (MEKs), and extracellular signal-regulated protein kinases (ERKs) 1 and 2. Therefore, we tested for tryptase-induced activation of ERK1 and 2 in cultured dog tracheal smooth-muscle cells. Tryptase, in nanomolar concentrations which potently stimulated DNA synthesis, increased dual phosphorylation of ERKs in cellular lysates as well as ERK2 kinase activity in immunoprecipitates. Pretreatment of cells with the MEK inhibitor PD098059 abolished tryptase-induced increases in DNA synthesis and attenuated increases in ERK2 activity. Irreversible inhibition of tryptase's proteolytic activity, using p-amidino phenylmethanesulfonyl fluoride, attenuated tryptase-induced increases in DNA synthesis and dual phosphorylation of ERKs by 76% and 40 to 60%, respectively. Tryptase also increased c-fos transcription as quantified in polymerase chain reactions. In concentrations that caused similar increases in DNA synthesis, tryptase and platelet-derived growth factor (PDGF-BB) increased ERK activity (and c-fos transcription) with markedly different kinetics, the tryptase-induced responses being slower in onset and more sustained. We conclude that tryptase-induced mitogenesis in airway smooth-muscle cells requires activation of ERK1 and 2; that these responses depend partially, but not completely, upon tryptase's properties as a protease; and that they are slower in onset and more sustained than those induced by PDGF-BB.

Mast cell tissue inhibitor of metalloproteinase-1 is cleaved and inactivated extracellularly by alpha-chymase.

We previously reported that mast cell alpha-chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. Alpha-chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1's alpha-chymase-mediated cleavage products reveals hydrolysis at Phe(12)-Cys(13) and Phe(23)-Val(24) in loop 1 and Phe(101)-Val(102) and Trp(105)-Asn(106) in loop 3 of the NH(2)-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to alpha-chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, alpha-chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with alpha-chymase increases gel B activity 2- to 5-fold, suggesting that alpha-chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of alpha-chymase blocks degranulation-induced TIMP-1 processing (absent in alpha-chymase-deficient HMC-1 cells). Purified alpha-chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell alpha-chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.

Angiotensin II generation by mast cell alpha- and beta-chymases.

Mast cells secrete alpha- and beta-chymases. Primate alpha-chymases generate angiotensin (AT) II by selectively hydrolyzing AT I's Phe(8)-His(9) bond. This is distinct from the AT converting enzyme (ACE) pathway. In humans, alpha-chymase is the major non-ACE AT II-generator. In rats, beta-chymases destroy AT II by cleaving at Tyr(4)-Ile(5). Past studies predicted that AT II production versus destruction discriminates alpha- from beta-chymases and that Lys(40) in the substrate-binding pocket determines alpha-chymase Phe(8) specificity. This study examines these hypotheses by comparing AT II generation by human alpha-chymase (containing Lys(40)), dog alpha-chymase (lacking Lys(40)), and mouse mMCP-4 (a beta-chymase lacking Lys(40); orthologous to AT II-destroying rat chymase rMCP-1). The results suggest that human and dog alpha-chymase generate AT II exclusively and with comparable efficiency, although dog chymase contains Ala(40) rather than Lys(40). Furthermore, AT II is the major product generated by degranulation supernatants from cultured dog mast cells, which release tryptases and dipeptidylpeptidase as well as alpha-chymase. In contrast to rMCP-1, mMCP-4 beta-chymase readily generates AT II. Although there is competing AT I hydrolysis at Tyr(4), mMCP-4 does not destroy AT II quickly once it is formed. We conclude (1) that chymases are the dominant AT I-hydrolyzing mast cell peptidases, (2) that residues other than Lys(40) are key determinants of alpha-chymase AT I Phe(8) specificity, (3) that beta-chymases can generate AT II, and (4) that alpha- and beta-chymases are not strictly dichotomous regarding AT I cleavage specificity.

Characterization of human gamma-tryptases, novel members of the chromosome 16p mast cell tryptase and prostasin gene families.

Previously, this laboratory identified clusters of alpha-, beta-, and mast cell protease-7-like tryptase genes on human chromosome 16p13.3. The present work characterizes adjacent genes encoding novel serine proteases, termed gamma-tryptases, and generates a refined map of the multitryptase locus. Each gamma gene lies between an alpha1H Ca2+ channel gene (CACNA1H) and a betaII- or betaIII-tryptase gene and is approximately 30 kb from polymorphic minisatellite MS205. The tryptase locus also contains at least four tryptase-like pseudogenes, including mastin, a gene expressed in dogs but not in humans. Genomic DNA blotting results suggest that gammaI- and gammaII-tryptases are alleles at the same site. betaII- and betaIII-tryptases appear to be alleles at a neighboring site, and alphaII- and betaI-tryptases appear to be alleles at a third site. gamma-Tryptases are transcribed in lung, intestine, and in several other tissues and in a mast cell line (HMC-1) that also expresses gamma-tryptase protein. Immunohistochemical analysis suggests that gamma-tryptase is expressed by airway mast cells. gamma-Tryptase catalytic domains are approximately 48% identical with those of known mast cell tryptases and possess mouse homologues. We predict that gamma-tryptases are glycosylated oligomers with tryptic substrate specificity and a distinct mode of activation. A feature not found in described tryptases is a C-terminal hydrophobic domain, which may be a membrane anchor. Although the catalytic domains contain tryptase-like features, the hydrophobic segment and intron-exon organization are more closely related to another recently described protease, prostasin. In summary, this work describes gamma-tryptases, which are novel members of chromosome 16p tryptase/prostasin gene families. Their unique features suggest possibly novel functions.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Dipeptidyl peptidase I cleaves matrix-associated proteins and is expressed mainly by mast cells in normal dog airways.

Dipeptidyl peptidase I (DPPI) is a cysteine protease found in many tissues, including the lung. Major cell types expressing DPPI in vitro include myelomonocytic cells, cytotoxic T cells, and mast cells. After activation and degranulation, cytotoxic T cells and mast cells secrete DPPI. With a goal of clarifying possible roles for DPPI in lung diseases, we sought to identify cells expressing DPPI in lung tissue, hypothesizing that lung mast cells are major producers of DPPI and that secreted DPPI cleaves extracellular matrix proteins. To address these hypotheses, we used immunohistochemical techniques to localize DPPI in normal dog airways, lung, and cultured mast cells, and we used purified DPPI to examine cleavage of matrix-associated proteins in vitro. We found that mast cells are the major identifiable source of DPPI in airways and that macrophages are the major source in alveoli. Within mast cells, DPPI localizes to cytoplasmic granules. We also found that DPPI endoproteolytically cleaves the extracellular matrix proteins fibronectin and collagen types I, III, and IV. The finding of DPPI in airway mast cells and its cleavage of matrix proteins suggest the possibility that DPPI plays a role in mast cell-mediated turnover of matrix proteins and in airway remodeling of chronic airway diseases such as asthma.

Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase.

Proteinase-activated receptor-2 (PAR-2) is a G-protein coupled receptor. Tryptic proteases cleave PAR-2 exposing a tethered ligand (SLIGKV), which binds and activates the receptor. Although PAR-2 is highly expressed by cultured keratinocytes and is an inflammatory mediator, its precise localization in the normal and inflamed human skin is unknown, and the proteases that activate PAR-2 in the skin have not been identified. We localized PAR-2 in human skin by immunohistochemistry, examined PAR-2 expression by RT-PCR and RNA blotting, and investigated PAR-2 activation by mast cell tryptase. PAR-2 was localized to keratinocytes, especially in the granular layer, to endothelial cells, hair follicles, myoepithelial cells of sweat glands, and dermal dendritic-like cells. PAR-2 was also highly expressed in keratinocytes and endothelial cells of inflamed skin. PAR-2 mRNA was detected in normal human skin by RT-PCR, and in cultured human keratinocytes and dermal microvascular endothelial cells by Northern hybridization. Trypsin, tryptase and a peptide corresponding to the tethered ligand (SLIGKVNH2) increased [Ca2+]i in keratinocytes, measured using Fura-2/AM. Although tryptase-containing mast cells were sparsely scattered in the normal dermis, they were numerous in the dermis in atopic dermatitis, and in the dermis, dermal-epidermal border, and occasionally within the lower epidermis in psoriasis. Tryptase may activate PAR-2 on keratinocytes and endothelial cells during inflammation.

Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2.

1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.

Expression of HPV16 early region genes in basal keratinocytes of transgenic mice elicits a multistage pathway to squamous carcinoma. We report that infiltration by mast cells and activation of the matrix metalloproteinase MMP-9/gelatinase B coincides with the angiogenic switch in premalignant lesions. Mast cells infiltrate hyperplasias, dysplasias, and invasive fronts of carcinomas, but not the core of solid tumors, where they degranulate in close apposition to capillaries and epithelial basement membranes, releasing mast-cell-specific serine proteases MCP-4 (chymase) and MCP-6 (tryptase). MCP-6 is shown to be a mitogen for dermal fibroblasts that proliferate in the reactive stroma, whereas MCP-4 can activate progelatinase B and induce hyperplastic skin to become angiogenic in an in vitro bioassay. Notably, premalignant angiogenesis is abated in a mast-cell-deficient (KITW/KITWWv) HPV16 transgenic mouse. The data indicate that neoplastic progression in this model involves exploitation of an inflammatory response to tissue abnormality. Thus, regulation of angiogenesis during squamous carcinogenesis is biphasic: In hyperplasias, dysplasias, and invading cancer fronts, inflammatory mast cells are conscripted to reorganize stromal architecture and hyperactivate angiogenesis; within the cancer core, upregulation of angiogenesis factors in tumor cells apparently renders them self-sufficient at sustaining neovascularization.

Mast cell expression of gelatinases A and B is regulated by kit ligand and TGF-beta.

Our prior work shows that cultured BR cells derived from dog mastocytomas secrete the 92-kDa proenzyme form of gelatinase B. We provided a possible link between mast cell activation and metalloproteinase-mediated matrix degradation by demonstrating that alpha-chymase, a serine protease released from secretory granules by degranulating mast cells, converts progelatinase B to an enzymatically active form. The current work shows that these cells also secrete gelatinase A. Furthermore, gelatinases A and B both colocalize to alpha-chymase-expressing cells of canine airway, suggesting that normal mast cells are a source of gelatinases in the lung. In BR cells, gelatinase B and alpha-chymase expression are regulated, whereas gelatinase A expression is constitutive. Progelatinase B mRNA and enzyme expression are strongly induced by the critical mast cell growth factor, kit ligand, which is produced by fibroblasts and other stromal cells. Induction of progelatinase B is blocked by U-73122, Ro31-8220, and thapsigargin, implicating phospholipase C, protein kinase C, and Ca2+, respectively, in the kit ligand effect. The profibrotic cytokine TGF-beta virtually abolishes the gelatinase B mRNA signal and also attenuates kit ligand-mediated induction of gelatinase B expression, suggesting that an excess of TGF-beta in inflamed or injured tissues may alter mast cell expression of gelatinase B, which is implicated in extracellular matrix degradation, angiogenesis, and apoptosis. In summary, these data provide the first evidence that normal mast cells express gelatinases A and B and suggest pathways by which their regulated expression by mast cells can influence matrix remodeling and fibrosis.

Clustering of activating mutations in c-KIT's juxtamembrane coding region in canine mast cell neoplasms.

The proto-oncogene c-KIT encodes a growth factor receptor, KIT, with ligand-dependent tyrosine kinase activity that is expressed by several cell types including mast cells. c-KIT juxtamembrane coding region mutations causing constitutive activation of KIT are capable of transforming cell lines and have been identified in a human mast cell line and in situ in human gastrointestinal stromal tumors, but have not been demonstrated in situ in neoplastic mast cells from any species. To determine whether c-KIT juxtamembrane mutations occur in the development of mast cell neoplasms, we examined canine mastocytomas, which are among the most common tumors of dogs and which often behave in a malignant fashion, unlike human solitary mastocytomas. Sequencing of c-KIT cDNA generated from tumor tissues removed from seven dogs revealed that three of the tumors contained a total of four mutations in an intracellular juxtamembrane coding region that is completely conserved among vertebrates. In addition, two mutations were found in three mast cell lines derived from two additional dogs. One mutation from one line matched that found in situ in one of the tumors. The second was found in two lines derived from one dog at different times, indicating that the mutation was present in situ in the animal. All five mutations cause high spontaneous tyrosine phosphorylation of KIT. Our study provides in situ evidence that activating c-KIT juxtamembrane mutations are present in, and may therefore contribute to, the pathogenesis of mast cell neoplasia. Our data also suggest an inhibitory role for the KIT juxtamembrane region in controlling the receptor kinase activity.

Neutrophil elastase and elastase-rich cystic fibrosis sputum degranulate human eosinophils in vitro.

Neutrophils, eosinophils, and their proinflammatory constituents are important mediators of airway disease, and high levels of neutrophil proteases and eosinophil cationic protein (ECP) are found in sputum from patients with cystic fibrosis (CF). To investigate whether neutrophil proteases or CF sputum causes eosinophil degranulation, purified eosinophils from atopic asthmatic subjects were incubated for 2 h with neutrophil elastase, cathepsin G, and CF sputum, and the release of ECP was measured. We found that the percent release of ECP was higher after incubation with neutrophil elastase (10(-5) M) than with a buffer control [6.1 +/- 0.8 (SE) vs. 1.7 +/- 0.1%; P < 0.003] and represented >50% of the release caused by positive controls [Ca2+ ionophore A-23187 (5 x 10(-6) M) or serum-coated Sephadex beads]. The release of ECP after incubation with cathepsin G (2.3 +/- 0.2%) and CF sputum (6.2 +/- 2.0%) was also significantly higher than that with a buffer control (P < 0.05). Neutralization of free elastase activity with alpha1-proteinase inhibitor reduced the mean percent degranulation of eosinophils by neutrophil elastase by 50% (P = 0.0004) and by CF sputum by 75% (P = 0.02). Preincubation of eosinophils with cytochalasin B (10 mg/ml) and depletion of the incubation medium of Ca2+ also significantly attenuated degranulation of eosinophils incubated with purified free neutrophil elastase or CF sputum (P < 0.05). We conclude that neutrophil proteases, especially neutrophil elastase, and elastase-rich CF sputum cause degranulation of eosinophils in a mechanism partially dependent on Ca2+ and actin filaments.

Characterization of genes encoding known and novel human mast cell tryptases on chromosome 16p13.3.

Tryptases are serine proteases implicated in asthma and are very highly expressed in human mast cells. They fall into two groups, alpha and beta. Although several related tryptase mRNAs are known, it is unclear which if any are transcripts of separate haploid genes. The studies described here investigated the nature and number of human tryptases and sought possibly novel members of the family. To this end, two human bacterial artificial chromosome (BAC) clones containing tryptase genes were identified and mapped to chromosome 16p13.3, of which approximately 2.2 megabases are syntenic with the part of mouse chromosome 17 containing tryptase genes mouse mast cell protease (mMCP)-6 and -7. Sequencing and restriction mapping suggest that the BACs may partially overlap. Sequenced BAC genes correspond to three known beta-tryptases (betaI, betaII, and betaIII), an alpha-like gene, and a pair of novel hybrid genes related partly to alpha/beta-tryptases and partly to orthologs of mMCP-7. betaII and betaIII, betaI and alphaII, as well as the two mMCP-7-like genes, may be alleles at single loci; in total, there are at least three nonallelic tryptase genes in the isolated BAC clones. DNA blotting and restriction analysis suggest that the BACs include most members of the immediate tryptase family. Thus, chromosome 16p13.3 harbors a cluster of known and previously undescribed members of the tryptase gene family.

Porcine origin of human sputum trypsin?

From purulent cystic fibrosis (CF) sputum, previous investigators partially purified a trypsinlike protease. A similar purified enzyme is available commercially as "human sputum trypsin." To explore the nature and origin of this preparation, we purified and NH2 terminally sequenced its major protein component. The resulting sequence, Ile-Val-Gly-Gly-Tyr-Thr-(Cys)-Ala-Ala-Asn-Ser-Val/Ile-Pro-Tyr-Gln-Val -Ser-Leu-Asn-Ser, differs from known human proteins but is identical to porcine trypsin, including the Val/Ile polymorphism at residue 12. Specific activity and electrophoretic and inhibition profiles and immunoreactivity of sputum and porcine pancreatic trypsin are nearly identical. Because porcine trypsin is a major ingredient of digestive enzyme supplements taken by CF patients with pancreatic dysfunction, we propose that one or more lots of human sputum trypsin derive from enzyme supplements and are of porcine origin. The path by which trypsin ends up in sputum is unknown. Because sputum trypsin is active but susceptible to inactivation by plasma alpha1-proteinase inhibitor, it is unlikely to derive from trypsin absorbed into the bloodstream. However, it may originate from tracheally aspirated stomach contents or from digestive supplement-contaminated saliva mixed with expectorated sputum. The imbalance between proteases and antiproteases in CF bronchial secretions allows trypsin to remain active despite sensitivity to serpins and secretory leukocyte proteinase inhibitor. Furthermore, because sputum trypsin activates human progelatinase B, it may be responsible in part for the reported presence of activated matrix metalloproteinases in CF sputum.