Warren M. Gold

Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs.

Supernatants obtained by degranulation of dog mastocytoma cells greatly increased the sensitivity and the magnitude of the contractile response of isolated dog bronchial smooth muscle to histamine. The enhanced contractile response was reversed completely by H1-receptor antagonists and was prevented by an inhibitor of tryptase (a major protease released with histamine from secretory granules of mast cells). The potentiation of histamine-induced contractions was reproduced by active tryptase in pure form. The contractions due to the combination of histamine and purified tryptase were abolished by the Ca2+ channel blockers nifedipine and verapamil. The bronchoconstricting effects of KCl and serotonin, which, like histamine, contract airway smooth muscle by a mechanism predominantly involving membrane potential-dependent Ca2+ transport, were also potentiated by tryptase. However, the contractile effects of acetylcholine, which contracts dog airway smooth muscle by a mechanism independent of Ca2+ channels, were unaffected by tryptase. These findings show a striking promotion of agonist-induced bronchial smooth muscle contraction by mast cell tryptase, via direct or indirect effects on Ca2+ channels, and the findings therefore suggest a novel potential mechanism of hyperresponsiveness in dog bronchi.

Alterations in human leukocyte function induced by ingestion of eicosapentaenoic acid

Two groups of six adults with persistent asthma, who were identical clinically, received 0.1 or 4 g of purified eicosapentaenoic acid ethyl ester (EPA) daily for 8 weeks. Both doses increased significantly the generation of leukotriene B5 (LTB5) from EPA by polymorphonuclear (PMN) and mononuclear leukocytes, while only the high dose decreased leukocyte arachidonic acid (AA) and the generation of LTB4 and prostaglandin E2 from AA. Only the high dose led to inhibition of PMN leukocyte chemotaxis to multiple stimuli by a mean of 57–70% (P<0.01), without altering monocyte chemotaxis, the production of plateletactivating factor by mononuclear leukocytes, or the IgE-dependent release of histamine from basophils. Both doses of EPA increased the responses of T lymphocytes to phytohemagglutinin by a mean of 73% or more (P<0.01) without modifying the numbers of helper and suppressor T lymphocytes. EPA affects the functions of several types of leukocytes critical to inflammation and immunity.

In Vivo Demonstration of Nonadrenergic Inhibitory Innervation of the Guinea Pig Trachea

To determine if electrical stimulation of autonomic nerves could excite nonadrenergic inhibitory motor pathways in the guinea pig respiratory system in vivo, we studied the effects of electrical stimulation of the cervical vagi and sympathetic nerve trunks on pressure changes (P(p)) within an isolated, fluid-filled cervical tracheal segment which reflected changes in trachealis muscle tone. We preserved the innervation and circulation of the segment as evidenced by a rise in P(p) with vagus nerve stimulation and a fall in P(p) with intravenous isoproterenol. In five atropine-treated animals, stimulation of the cut vagi or sympathetic nerve trunks resulted in a mean fall in P(p) of 7.9 and 8.2 cm H(2)O, respectively. Treatment with propranolol attenuated the response to sympathetic stimulation but not vagal stimulation. To determine if these relaxation responses were mediated by an adrenergic or nonadrenergic mechanism, we studied an additional five animals that had been treated with 6-hydroxydopamine to destroy adrenergic nerve endings. In 6-hydroxydopamine, atropine, and propranolol-treated animals, sympathetic nerve stimulation decreased P(p) only 0.65 cm H(2)O, confirming the elimination of adrenergic nerve influences, whereas vagus nerve stimulation decreased P(p) 17.7 cm H(2)O. After sectioning the recurrent laryngeal nerves, the mean decrease in P(p) during vagus nerve stimulation was only 3.2 cm H(2)O. These findings demonstrate the presence of nonadrenergic inhibitory nerves in the guinea pig trachea in vivo. They further show that nonadrenergic inhibitory nerve effects are elicited during electrical stimulation of the vagus nerves and that interruption of the recurrent laryngeal nerves diminishes the magnitude of these effects.

Dog mastocytoma cells produce transforming growth factor beta 1.

Transforming growth factor-beta (TGF beta) promotes deposition of extracellular matrix and is associated with fibrotic conditions both in experimental animals and in humans. Although a role for mast cells has been suspected in the pathogenesis of fibrosis, no potent mediator capable of stimulating fibroblast growth or extracellular matrix deposition has been identified in mast cell supernatants. We report here the constitutive production of TGF beta 1 by four dog mastocytoma cell lines. TGF beta 1 was identified by characteristic biologic activity, blockade of biologic effect by specific neutralizing antibody, and by recognition of a band with the appropriate migration by western blot. TGF beta 1 mRNA, but not TGF beta 2 or TGF beta 3 mRNA, was also produced constitutively by all four cell lines. Quantitation by bioassay revealed baseline TGF beta secretion of approximately 1 ng/10(6) cells over 48 h. Stimulation of mastocytoma cells with phorbol ester increased the rate of release of TGF beta 1, most markedly in the first 30 min after stimulation, without increasing TGF beta 1 mRNA. Dog mastocytoma cells produced TGF beta 1 primarily in a latent form, inactive until treated with acid. Both pure TGF beta 1 and TGF beta-containing mastocytoma cell-conditioned media inhibited mitogenesis and proliferation in dog mastocytoma cell lines, suggesting that mast cell tumor lines would not grow preferentially based on their ability to produce TGF beta. These studies may make possible further investigation of the mechanism by which mast cells contribute to the induction of fibrosis.

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining.

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.

Cutaneous allergic response in atopic dogs: Relationship of cellular and histamine responses

We studied the cutaneous response to intradermal antigen using clinical, histologic, and physiologic criteria in ragweed-sensitized dogs. The clinical response was measured early (the wheal at 20 minutes) and late (induration at 6 hours). We assessed cutaneous responsiveness to histamine before and 6 hours after injection (intradermally) of ragweed (n = 5, antigen group) and diluent (n = 4, 10% glycerin in 0.9% NaCl, sham group); we measured the wheal in response to histamine (1.0 ng to 0.1 mg, intradermally), constructed a dose-response curve, and interpolated the provocative dose (milligrams) of histamine required to create a wheal 10 mm larger than the response to saline control. Skin biopsy specimens were obtained before and after injection of either ragweed or diluent. Consistent with the human late-phase response, neutrophils and eosinophils were present in the dermis at 1 hour, maximal at 6 hours, and decreased at 24 hours. Mononuclear cells increased significantly at 6 hours and were the predominant cells present at 24 hours after antigen. The late clinical response correlated only with influx of eosinophils (rs = 0.85; p less than 0.005). Histamine responsiveness increased markedly after antigen (p less than 0.0001), did not change after glycerin diluent (sham), and was correlated with the intensity of neutrophil influx at 6 hours (rs = 0.69; p less than 0.05), and to a much greater degree with mononuclear cell influx at 6 hours (rs = 0.85; p less than 0.005).

Immunologic and Physiologic Characterization of the Role of Reaginic Antibodies in Experimental Asthma in Dogs

Examination of the role of reaginic antibodies in experimental canine asthma indicates that the immunologic species involved in the airway response to inhaled antigens in allergic dogs is in the 0.030

Pulmonary function abnormalities in never-smoking flight attendants exposed to secondhand tobacco smoke in the aircraft cabin.

Objective: To determine whether the flight attendants who were exposed to secondhand tobacco smoke in the aircraft cabin have abnormal pulmonary function. Methods: We administered questionnaires and performed pulmonary function testing in 61 never-smoking female flight attendants who worked in active air crews before the smoking ban on commercial aircraft (preban). Results: Although the preban flight attendants had normal FVC, FEV1, and FEV1/FVC ratio, they had significantly decreased flow at mid- and low-lung volumes, curvilinear flow-volume curves, and evidence of air trapping. Furthermore, the flight attendants had significantly decreased diffusing capacity (77.5% ± 11.2% predicted normal) with 51% having a diffusing capacity below their 95% normal prediction limit. Conclusions: This cohort of healthy never-smoking flight attendants who were exposed to secondhand tobacco smoke in the aircraft cabin showed pulmonary function abnormalities suggestive of airway obstruction and impaired diffusion.

Chronic dyspnea and hyperventilation in an awake patient with small subcortical infarcts

A 79-year-old woman presented with chronic dyspnea and hyperventilation. There was no evidence of pulmonary disease. Hyperventilation persisted during sleep and after high-dose administration of a narcotic. A head MRI revealed bilateral medial thalamic infarctions. Central neurogenic hyperventilation was diagnosed in this alert patient. The case may illustrate a role for the thalamus in regulating ventilation, but another small infarct not visible on MRI also could be responsible.

Mast cell exocytosis : evidence that granule proteoglycan processing is not coupled to degranulation

It has been hypothesized that the dissolution of mast cell granules at the time of degranulation results from proteoglycan cleavage coupled to exocytosis. To address this hypothesis, we studied granule proteoglycan before and after exocytosis in dog mastocytoma cells, which solubilize granule contents during exocytosis. 35S-labeled proteoglycans were extracted from unstimulated whole cells and cell degranulation supernatant. Sequential anion-exchange and gel filtration chromatography, followed by specific glycosaminoglycan digestion, identified chondroitin sulfate and heparin glycosaminoglycan and proteoglycan in unstimulated cells and degranulated material alike. Glycosaminoglycan type and charge density in degranulation supernatant were unchanged compared with unstimulated cells. There was no decrease in proteoglycan size with cell activation and exocytosis. Thus, granule release and solubilization does not appear to require exocytosis-coupled degradation of granule proteoglycans. Release in association with high-m.w. proteoglycans may serve to limit rates of diffusion and activity of proteases and other mast cell mediators.