Lawrence B. Schwartz

Tryptase Levels as an Indicator of Mast-Cell Activation in Systemic Anaphylaxis and Mastocytosis

Better methods are needed to assess mast-cell activation in vivo and to distinguish the activation of mast cells from that of basophils. Tryptase, a neutral protease selectively concentrated in the secretory granules of human mast cells (but not basophils), is released by mast cells together with histamine and serves as a marker of mast-cell activation. In 17 patients with systemic mastocytosis, concentrations of tryptase in plasma were linearly related to those of histamine (P less than 0.01). Eleven of the 17 patients had tryptase levels of 4 to 88 ng per milliliter, indicating ongoing mast-cell activation. In each of six patients who experienced corresponding anaphylactic reactions after penicillin, aspirin, or melon ingestion, a wasp sting, exercise, or antilymphocyte globulin injection, tryptase levels in serum ranged from 9 to 75 ng per milliliter, indicating mast-cell activation during each of these events. In contrast, serum tryptase levels were less than 5 ng per milliliter in all patients presenting with myocardial disease (n = 8, 6 with hypotension) or sepsis (n = 6, 3 with hypotension) and in the controls (n = 20). One patient had a myocardial infarction after anaphylaxis in response to a wasp sting and an elevated tryptase level of 25 ng per milliliter. Thus, the plasma or serum tryptase level is a diagnostic correlate of mast-cell-related events.

Standards and standardization in mastocytosis: Consensus Statements on Diagnostics, Treatment Recommendations and Response Criteria

Although a classification for mastocytosis and diagnostic criteria are available, there remains a need to define standards for the application of diagnostic tests, clinical evaluations, and treatment responses. To address these demands, leading experts discussed current issues and standards in mastocytosis in a Working Conference. The present article provides the resulting outcome with consensus statements, which focus on the appropriate application of clinical and laboratory tests, patient selection for interventional therapy, and the selection of appropriate drugs. In addition, treatment response criteria for the various clinical conditions, disease‐specific symptoms, and specific pathologies are provided. Resulting recommendations and algorithms should greatly facilitate the management of patients with mastocytosis in clinical practice, selection of patients for therapies, and the conduct of clinical trials.

Treatment of Patients with the Hypereosinophilic Syndrome with Mepolizumab

BACKGROUND The hypereosinophilic syndrome is a group of diseases characterized by persistent blood eosinophilia, defined as more than 1500 cells per microliter with end-organ involvement and no recognized secondary cause. Although most patients have a response to corticosteroids, side effects are common and can lead to considerable morbidity. METHODS We conducted an international, randomized, double-blind, placebo-controlled trial evaluating the safety and efficacy of an anti-interleukin-5 monoclonal antibody, mepolizumab, in patients with the hypereosinophilic syndrome. Patients were negative for the FIP1L1-PDGFRA fusion gene and required prednisone monotherapy, 20 to 60 mg per day, to maintain a stable clinical status and a blood eosinophil count of less than 1000 per microliter. Patients received either intravenous mepolizumab or placebo while the prednisone dose was tapered. The primary end point was the reduction of the prednisone dose to 10 mg or less per day for 8 or more consecutive weeks. RESULTS The primary end point was reached in 84% of patients in the mepolizumab group, as compared with 43% of patients in the placebo group (hazard ratio, 2.90; 95% confidence interval [CI], 1.59 to 5.26; P<0.001) with no increase in clinical activity of the hypereosinophilic syndrome. A blood eosinophil count of less than 600 per microliter for 8 or more consecutive weeks was achieved in 95% of patients receiving mepolizumab, as compared with 45% of patients receiving placebo (hazard ratio, 3.53; 95% CI, 1.94 to 6.45; P<0.001). Serious adverse events occurred in seven patients receiving mepolizumab (14 events, including one death; mean [+/-SD] duration of exposure, 6.7+/-1.9 months) and in five patients receiving placebo (7 events; mean duration of exposure, 4.3+/-2.6 months). CONCLUSIONS Our study shows that treatment with mepolizumab, an agent designed to target eosinophils, can result in corticosteroid-sparing for patients negative for FIP1L1-PDGFRA who have the hypereosinophilic syndrome. (ClinicalTrials.gov number, NCT00086658 [ClinicalTrials.gov].).

Time course of appearance and disappearance of human mast cell tryptase in the circulation after anaphylaxis.

Tryptase, a neutral protease of human mast cells, is a potentially important indicator of mast cell involvement in various clinical conditions. The current study examined the time course of appearance and disappearance of tryptase in the circulation after an anaphylactic event and the stability of both endogenous and exogenous tryptase in terms of freeze-thawing and temperature. The peak level of tryptase after an experimentally induced systemic anaphylactic reaction occurred 1-2 h after the initiating bee sting in each of three subjects, in contrast to histamine levels which peaked at 5-10 min. In some cases elevated levels of tryptase may not be detected during the initial 15-30 min. Tryptase levels then declined under apparent first order kinetics with a t1/2 of approximately 2h. Similar disappearance kinetics were observed for two subjects presenting in the emergency room with immediate type reactions, one with severe asthma after indomethacin ingestion, the other with systemic anaphylaxis after a bee sting. Histamine levels declined rapidly and were back to baseline by 15-60 min. Measured levels of tryptase in serum or plasma were not diminished by up to four freeze-thaw cycles. Incubation of serum samples taken from subjects with elevated levels of tryptase at 22 and 37 degrees C indicated that greater than 50% of endogenous tryptase was still detected after 4 d. Purified tryptase added to serum or plasma and incubated as above was less stable: approximately 50% of exogenous tryptase in serum and approximately 15% in plasma was detected after 2d of incubation. Therefore, optimally samples should be stored frozen, but even those stored at room temperature for up to 4 d should be satisfactory. The best time to obtain samples for tryptase determinations is 1-2 h after the precipitating event, but depending on the magnitude of the initial response elevated levels of tryptase may be present in the circulation for several hours.

Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies

We developed an improved immunohistochemical technique for distinguishing human mast cells of the MCT (tryptase-positive, chymase-negative) and MCTC (tryptase-positive, chymase-positive) types utilizing a biotinylated murine anti-chymase monoclonal antibody (MAb), termed B7, and an alkaline phosphatase-conjugated murine anti-tryptase MAb, termed G3. The B7 MAb also was used to show the selective presence of chymase in mast cells. The distribution of MCT and MCTC cells in Carnoys fluid-fixed tissue sections of human lung, skin, small intestine, and tonsils was analyzed by the new technique and the results compared to those obtained with the older method using a rabbit polyclonal antichymase antibody and a mouse anti-tryptase MAb in indirect immunoperoxidase and indirect immunoalkaline phosphatase protocols, respectively. In tissues known to contain predominantly mature mast cells, there were no quantitative differences between the two techniques, although the staining intensity achieved with the anti-chymase MAb was greater and without development of high background, compared to results achieved with the polyclonal antibody. MCT cells were the predominant type seen in the alveoli of the lung (93%) and in the small intestinal mucosa (81%). MCTC cells predominanted in the skin (99%) and in the small intestinal submucosa (77%) and, to a lesser degree, in tonsils (60%). However, in newborn foreskin tissue which contains predominantly immature forms of mast cells, 75% of all mast cells were stained uniformly and intensely with B7, whereas only 43% were stained with the polyclonal anti-chymase antibody. Therefore, the use of MAb provides for better standardization of reagents and more accurate assessment of the distribution of human MCT and MCTC cells in tissues than previously available methods.

Human mast cells stimulate vascular tube formation. Tryptase is a novel, potent angiogenic factor.

The presence of mast cells near capillary sprouting sites suggests an association between mast cells and angiogenesis. However, the role of mast cells in blood vessel development remains to be defined. In an attempt to elucidate this relationship, we investigated the effect of human mast cells (HMC-1) and their products on human dermal microvascular endothelial cell (HDMEC) tube formation. Coculture of HMC-1 with HDMEC led to a dose-response increase in the network area of vascular tube growth. Moreover, the extent of neovascularization was enhanced greatly when HMC-1 were degranulated in the presence of HDMEC. Further examination using antagonists to various mast cell products revealed a blunted response (73-88% decrease) in the area of vascular tube formation if specific inhibitors of tryptase were present. Tryptase (3 microg/ml) directly added to HDMEC caused a significant augmentation of capillary growth, which was suppressed by specific tryptase inhibitors. Tryptase also directly induced cell proliferation of HDMEC in a dose-dependent fashion (2 pM-2 nM). Our results suggest that mast cells act at sites of new vessel formation by secreting tryptase, which then functions as a potent and previously unrecognized angiogenic factor.

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.

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy.

Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation.

Mast cells have been implicated in the pathogenesis of the matrix degradation observed in the cartilaginous and osseous structures of the rheumatoid joint. We previously reported that human mast cell tryptase, a 134-kD granule-associated neutral protease, is present in rheumatoid synovium and can activate collagenase in crude culture medium in vitro. the present study attempts to depict the precise mechanism of this activation. To express full activation of latent collagenase, matrix metalloproteinase 3 (MMP-3) or stromelysin, can be activated by tryptase in a time and dose-dependent manner. Tryptase was not capable of generating active collagenase in the crude media from cultured rheumatoid synoviocytes depleted of proMMP-3 by immunoadsorption. In addition, the function of the tissue inhibitor of metalloproteinases (TIMP) was not altered by tryptase, and SDS-PAGE analysis revealed no degradation of TIMP by tryptase. The tryptase dependent activation of synoviocyte procollagenase thereby appears to be entirely dependent upon its ability to activate proMMP-3.

A common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects.

Many patients with asthma have increased wheezing with colds. We hypothesized that rhinovirus colds might increase asthma by augmenting airway allergic responses (histamine release and eosinophil influx) after antigen challenge. Seven allergic rhinitis patients and five normal volunteers were infected with rhinovirus type 16 (RV16) and evaluated by segmental bronchoprovocation and bronchoalveolar lavage. Segmental challenge with saline and antigen was performed 1 mo before infection, during the acute infection, and 1 mo after infection. Lavage was performed immediately and 48 h after antigen challenge. Data were analyzed by two-way analysis of variance, and a P value of < or = 0.05 was considered to be significant. All volunteers inoculated with RV16 developed an acute respiratory infection. BAL fluid obtained from allergic rhinitis subjects during the acute viral infection, and 1 mo after infection, showed the following significant RV16-associated changes after antigen challenge: (a) an enhanced release of histamine immediately after local antigen challenge; (b) persistent histamine leak 48 h afterwards; and (c) a greater recruitment of eosinophils to the airway 48 h after challenge. These changes were not seen in non-allergic volunteers infected with RV16 and challenged with antigen, nor in allergic volunteers repetitively challenged with antigen but not infected with RV16, nor in RV16 infected allergic volunteers sham challenged with saline. We conclude that rhinovirus upper respiratory infection significantly augments immediate and late allergic responses in the airways of allergic individuals after local antigen challenge. These data suggest that one mechanism of increased asthma during a cold is an accentuation of allergic responses in the airway which may then contribute to bronchial inflammation.

Definitions, Criteria and Global Classification of Mast Cell Disorders with Special Reference to Mast Cell Activation Syndromes: A Consensus Proposal

Activation of tissue mast cells (MCs) and their abnormal growth and accumulation in various organs are typically found in primary MC disorders also referred to as mastocytosis. However, increasing numbers of patients are now being informed that their clinical findings are due to MC activation (MCA) that is neither associated with mastocytosis nor with a defined allergic or inflammatory reaction. In other patients with MCA, MCs appear to be clonal cells, but criteria for diagnosing mastocytosis are not met. A working conference was organized in 2010 with the aim to define criteria for diagnosing MCA and related disorders, and to propose a global unifying classification of all MC disorders and pathologic MC reactions. This classification includes three types of ‘MCA syndromes’ (MCASs), namely primary MCAS, secondary MCAS and idiopathic MCAS. MCA is now defined by robust and generally applicable criteria, including (1) typical clinical symptoms, (2) a substantial transient increase in serum total tryptase level or an increase in other MC-derived mediators, such as histamine or prostaglandin D2, or their urinary metabolites, and (3) a response of clinical symptoms to agents that attenuate the production or activities of MC mediators. These criteria should assist in the identification and diagnosis of patients with MCAS, and in avoiding misdiagnoses or overinterpretation of clinical symptoms in daily practice. Moreover, the MCAS concept should stimulate research in order to identify and exploit new molecular mechanisms and therapeutic targets.