RESUMEN
Human α- and ß-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. ß-Protryptase is sequentially processed in vitro by autocatalysis at R(-3) followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R(-3)Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. ß-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q(-3)) and mutated ß-protryptase (R(-3)Q) as well as wild-type ß-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and ß-protryptases from human mast cells to their mature enzymatically active products.
Asunto(s)
Catepsinas/metabolismo , Precursores Enzimáticos/metabolismo , Mastocitos/enzimología , Procesamiento Proteico-Postraduccional , Triptasas/metabolismo , Catepsina B/metabolismo , Catepsina C/metabolismo , Catepsina L/metabolismo , Catepsinas/análisis , Línea Celular , Heparina/farmacología , Humanos , Espectrometría de Masas , Mastocitos/metabolismoRESUMEN
Human ß-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized tetramer. ß-Protryptase in solution can be directly processed to the mature enzyme by cathepsin (CTS) L and CTSB, and sequentially processed by autocatalysis at R(-3), followed by CTSC proteolysis. However, it is uncertain which CTS is involved in protryptase processing inside human mast cells, because murine bone marrow-derived mast cells from CTSC-deficient mice convert protryptase (pro-mouse mast cell protease-6) to mature mouse mast cell protease-6. This finding suggests that other proteases are important for processing human ß-protryptase. In the current study, reduction of either CTSB or CTSL activity inside HMC-1 cells by short hairpin RNA silencing or CTS-specific pharmacologic inhibitors substantially reduced mature ß-tryptase formation. Similar reductions of tryptase levels in primary skin-derived mast cells were observed with these pharmacologic inhibitors. In contrast, protryptase processing was minimally reduced by short hairpin RNA silencing of CTSC. A putative pharmacologic inhibitor of CTSC markedly reduced tryptase levels, suggesting an off-target effect. Skin mast cells contain substantially greater amounts of CTSL and CTSB than do HMC-1 cells, the opposite being found for CTSC. Both CTSL and CTSB colocalize to the secretory granule compartment of skin mast cells. Thus, CTSL and CTSB are central to the processing of protryptase(s) in human mast cells and are potential targets for attenuating production of mature tryptase in vivo.
Asunto(s)
Catepsina B/metabolismo , Catepsina C/metabolismo , Catepsina L/metabolismo , Precursores Enzimáticos/metabolismo , Mastocitos/enzimología , Triptasas/metabolismo , Animales , Catepsina B/genética , Catepsina B/inmunología , Catepsina C/genética , Catepsina C/inmunología , Catepsina L/genética , Catepsina L/inmunología , Línea Celular Tumoral , Precursores Enzimáticos/genética , Precursores Enzimáticos/inmunología , Humanos , Mastocitos/inmunología , Ratones , Ratones Mutantes , Vesículas Secretoras/enzimología , Vesículas Secretoras/genética , Vesículas Secretoras/inmunología , Piel/enzimología , Piel/inmunología , Triptasas/genética , Triptasas/inmunologíaRESUMEN
Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.
Asunto(s)
Anafilatoxinas/biosíntesis , Complemento C3/metabolismo , Complemento C4/metabolismo , Complemento C5/metabolismo , Mastocitos/metabolismo , Triptasas/metabolismo , Anafilatoxinas/química , Anticuerpos Monoclonales/química , Asma/enzimología , Complemento C3/química , Complemento C4/química , Complemento C5/química , Sulfato de Dextran/química , Sulfato de Dextran/metabolismo , Heparina/química , Heparina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Inmunidad Innata , Mastocitos/química , Estructura Cuaternaria de Proteína , Piel/química , Piel/metabolismo , Triptasas/químicaRESUMEN
BACKGROUND: MC(T) and MC(TC) types of human mast cells (MCs) are distinguished from one another on the basis of the protease compositions of their secretory granules, but their functional and developmental relationships have been uncertain. OBJECTIVE: These studies better define the functional properties and developmental relationship of MC(T) and MC(TC) cells. METHODS: Mast cells were dispersed from human skin and lung, purified with anti-Kit antibody, and separated into CD88+ and CD88- populations by cell sorting. These cells were evaluated by immunocytochemistry with antitryptase and antichymase mAbs; for chymase and tryptase mRNA by real-time RT-PCR; for conversion of MC(T) to MC(TC) cells during cell culture with recombinant human stem cell factor and recombinant human IL-6; and for degranulation and leukotriene C 4 (LTC 4 ) secretion when stimulated with anti-FcepsilonRI, substance P, C5a, and compound 48/80. RESULTS: Mature MC(T) and MC(TC) cells were separated from one another on the basis of selective expression of CD88, the C5aR, on MC(TC) cells. Lung MC(T) cells had negligible levels of chymase mRNA and retained their MC(T) phenotype in culture. Mature MC(TC) cells from skin and lung degranulated in response to FcepsilonRI cross-linking, C5a, compound 48/80, and substance P. Lung MC(TC) cells released LTC 4 on activation, but no LTC 4 was detected when skin-derived MC(TC) cells were activated. MC(T) cells from lung degranulated and released LTC 4 in response to anti-FcepsilonRI and substance P, but not to C5a and compound 48/80. CONCLUSION: These observations functionally distinguish MC(T) from MC(TC) types of human mast cells and suggest important differences that may affect their participation in diseases such as asthma and urticaria.
Asunto(s)
Pulmón/inmunología , Mastocitos/inmunología , Receptor de Anafilatoxina C5a/análisis , Degranulación de la Célula/efectos de los fármacos , Separación Celular , Células Cultivadas , Quimasas , Complemento C5a/farmacología , Humanos , Leucotrieno C4/biosíntesis , Mastocitos/clasificación , ARN Mensajero/análisis , Receptor de Anafilatoxina C5a/deficiencia , Receptores de IgE , Serina Endopeptidasas/análisis , Serina Endopeptidasas/genética , Piel/inmunología , Sustancia P/farmacología , Triptasas , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
BACKGROUND: Alpha and beta-tryptase levels in serum are clinical tools for the evaluation of systemic anaphylaxis and systemic mastocytosis. Basophils and mast cells are known to produce these proteins. OBJECTIVE: The current study examines the effect of the alpha,beta-tryptase genotype on basophil tryptase levels and the type of tryptase stored in these cells. METHODS: Tryptase extracted from purified peripheral blood basophils from 20 subjects was examined by using ELISAs measuring mature and total tryptase and by using an enzymatic assay with tosyl-Gly-Pro-Lys-p-nitroanilide. Tryptase genotypes (4:0, 3:1, and 2:2 beta/alpha ratios) were assessed by using a hot-stop PCR technique with alpha,beta-tryptase-specific primers. Total alpha,beta-tryptase mRNA was measured by means of competitive RT-PCR, and ratios of alpha to beta-tryptase mRNA were measured by means of hot-stop RT-PCR. RESULTS: Tryptase in all but one of the basophil preparations was mature and enzymatically active. Tryptase quantities in basophils were less than 1% of those in tissue mast cells. Tryptase genotypes (beta/alpha) among the 20 donors were 4:0 in 7, 3:1 in 7, and 2:2 in 6. Tryptase protein and mRNA levels per basophil were not affected by the tryptase genotype. CONCLUSION: Basophils from healthy subjects contain modest amounts of mature and enzymatically active tryptase unaffected by the tryptase genotype.
Asunto(s)
Basófilos/enzimología , Serina Endopeptidasas/genética , Línea Celular , Genotipo , Humanos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/análisis , TriptasasRESUMEN
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.
Asunto(s)
Precursores Enzimáticos/metabolismo , Mastocitos/enzimología , Serina Endopeptidasas/metabolismo , Piel/enzimología , Células Cultivadas , Precursores Enzimáticos/sangre , Precursores Enzimáticos/genética , Precursores Enzimáticos/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Humanos , Isoenzimas/sangre , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Mastocitos/metabolismo , ARN Mensajero/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Serina Endopeptidasas/sangre , Serina Endopeptidasas/genética , Serina Endopeptidasas/aislamiento & purificación , Piel/citología , Piel/metabolismo , Triptasas , Células Tumorales CultivadasRESUMEN
Fc receptors for IgG (FcgammaR) are widely expressed in the hematopoietic system and mediate a variety of inflammatory responses. There are two functional classes of FcgammaR, activation and inhibitory receptors. Since IgG immune complexes (IgG IC) bind each class with similar affinity, co-expression of these receptors leads to their co-ligation. Thus, expression levels of this antagonistic pair play a critical role in determining the cellular response. Murine mast cells co-express the activation receptor FcgammaRIII and the inhibitory receptor FcgammaRIIb and can be activated by IgG IC. Mast cell activation contributes to allergic and other inflammatory diseases-particularly those in which IgG IC may play important roles. Using mouse bone marrow-derived mast cells, we report that IL-4 selectively increases FcgammaRIII expression without altering FcgammaRIIb. This enhanced expression could be induced by Stat6 activation alone, and appeared to be mediated in part by increased FcgammaRIIIalpha protein synthesis without significant changes in transcription. The increase in FcgammaRIII expression was functionally significant, as it was matched by enhanced FcgammaR-mediated degranulation and cytokine production. Selective regulation of mast cell FcgammaR by interleukin-4 could alter inflammatory IgG responses and subsequently disease severity and progression.