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The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.

The records of 35 adult male patients with suspected Pneumocystis carinii pneumonia and the acquired immune deficiency syndrome were reviewed to determine the diagnostic utility of fiberoptic bronchoscopy. Twenty-two of the 24 patients (92%) shown to have Pneumocystis pneumonia were diagnosed by bronchoscopy; 20 patients were diagnosed after 1 bronchoscopy and 2 patients after 2 bronchoscopies. The sensitivity of bronchoscopy specimens for diagnosing Pneumocystis pneumonia was 79% for transbronchial biopsies (19 of 24), 78% for transbronchial biopsy touch preparations (7 of 9), 55% for bronchial washings (11 of 20), and 39% for bronchial brushings (7 of 18). The overall sensitivity of fiberoptic bronchoscopy was 85% (22 positive of 26 total bronchoscopies). Complications, occurring during 6 of the 37 bronchoscopies, were slight hemoptysis (1 patient), fever (2 patients), septic shock (1 patient), and pneumothorax (2 patients). Fiberoptic bronchoscopy is a safe and effective procedure for making the early diagnosis of Pneumocystis carinii pneumonia in adult male patients with the acquired immune deficiency syndrome.

Prostaglandins are known to affect ion transport and mucus secretion in the trachea, and at least part of their effect is thought to be mediated by cyclic AMP. Because no prostaglandin receptor assay is currently available, we have used an immunocytochemical probe for the intracellular localization of cyclic AMP to identify those specific cell types in dog and cat trachea that respond to prostaglandins. Using tracheal explants similar to those used by other investigators for in vitro studies of ion transport and glycoprotein secretion, we examined the effect of endogenous and exogenous prostaglandins on immunoreactive cyclic AMP. Endogenous prostaglandins, which may be secreted in response to minor distortion of tissue membranes, stimulated immunoreactive cyclic AMP in ciliated epithelial cells and in both serous and mucous submucosal gland cells. This was not observed in tissues assayed 60 min after dissection, or dissected in the presence of indomethacin or of BW755C. Exogenous prostaglandin E1 increased cyclic AMP in these same cell types. We conclude that prostaglandins stimulate cyclic AMP in specific cell types in the trachea, and that endogenous prostaglandins play a major role in many in vitro preparations.

In 4 unsedated, exercising dogs, we studied the effects of inhaled histamine aerosol on minute volume of ventilation, respiratory frequency, tidal volume, total pulmonary resistance, and dynamic pulmonary compliance. Inhalation (5 breaths) of 1 to 2 per cent histamine aerosols increased minute ventilation (mean, 50 per cent; p less than 0.001) by increasing respiratory frequency (mean, 166 percent; P less than 0.001), despite decreasing tidal volume (mean, 42 percent; P less than 0.0001). Total pulmonary resistance increased (mean, 200 per cent; P less than 0.001.) Breathing supplemental O2 did not affect the ventilatory response to histamine. Adding external resistive loads to a dog's airway did not simulate the pattern of rapid, shallow breathing produced by histamine. Inhalation of terbutaline prevented the changes in total pulmonary resistance and dynamic pulmonary compliance but did not alter the ventilatory response to histamine. When conduction in the cervical vagus nerves (which were implanted chronically in skin loops) was blocked by cooling, the ventilatory response to histamine was abolished. We concluded that histamine stimulates breathing by stimulation of receptors whose afferent pathways are in the vagus nerves; the effective stimulus is not bronchoconstriction but is presumably due to direct stimulation of airway receptors.

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.

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 less than 0.01), without altering monocyte chemotaxis, the production of platelet-activating 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 less than 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.

Crude aqueous extracts of Ascaris suum (CE) have been used widely to study IgE-mediated reactions in various experimental preparations. Because some CE may contain a polypeptide, a mast cell degranulating peptide (MCDP), that degranulates mast cells by nonimmunologic mechanisms, various protocols have been used to ensure that the Ascaris preparation used did not contain MCPD. In general, these protocols have assumed MCDP had been without providing proof. Even protocols designed to isolate the major antigenic determinants from CE have usually been designed to evaluate immunogenic characteristics of the purified Ascaris; thus, few systematic comparisons of CE with purified Ascaris exist concerning mast cell degranulation, and few studies have demonstrated that MCDP has been removed during purification. Since Ascaris has proved to be useful in a variety of studies of IgE-mediated reactions, particularly in large animals (dog and sheep), we have developed a protocol to purify CE and MCDP and characterize their physiochemical and immunologic properties. We compared the allergenic activity of our purified Ascaris to that of CE and MCDP in skin and lung of natively sensitized dogs and in unsensitized rat peritoneal mast cells. Our results indicate that MCDP probably contaminates CE by less than 1.0%. However, the biologic activity of MCDP in dog lung appears insignificant and probably contributes little to CE-induced reactions in doses of CE commonly used (less than or equal to 100 mg injected). If a purified Ascaris preparation is essential, our protocol will yield an Ascaris preparation that has potent IgE-mediated effects in dog preparations with insignificant contamination by MCDP.

To determine the role of thromboxane A2 in the airway hyperresponsiveness induced by antigen challenge, we studied the effect of a thromboxane synthetase inhibitor, OKY-046, i.e., sodium (E)-3-[4-(1-imidazolylmethyl)-phenyl]-2-propanoate, in 6 ragweed-sensitized dogs. Airway responsiveness was assessed with dose-response curves of acetylcholine aerosol versus total pulmonary resistance before and 6 and 24 h after inhalation with ragweed antigen. This procedure was repeated in each dog during intravenous infusion of OKY-046 (100 micrograms/kg/min). OKY-946 did not alter the acute increase in total pulmonary resistance after antigen. At 6 h, there was a 7-fold increase in airway responsiveness, an effect that was prevented by OKY-046 (p less than 0.001). At 24 h, 18 h after OKY-046 was stopped, hyperresponsiveness was still significantly inhibited. OKY-046 did not alter the influx of neutrophils recovered by bronchoalveolar lavage performed at 6 h after antigen challenge. Antigen-induced airway hyperresponsiveness in dogs may depend upon the thromboxane A2 generation from inflammatory cells (e.g., neutrophils).

Because mast cells (MCs) in skin of atopic dogs are heterogeneous with respect to tissue fixation and staining properties, we determined the effect of antigen on each type of MC in vivo. Skin biopsies were done in anesthetized, ragweed-sensitized dogs before and at 1, 3, 6, and 24 hours after intradermal injections of ragweed antigen (n = 5) or glycerin diluent (n = 4). In each case, one biopsy specimen was fixed with formalin, and a second specimen from an adjacent abdominal site was fixed with basic lead acetate. In sections stained with Alcian blue, 49.7% more MCs (p less than 0.05) were detected in tissue fixed with basic lead acetate ("typical" plus "atypical" MCs: 2916 +/- 581/mm3; mean +/- SEM) than in tissue fixed with formalin ("typical" MCs: 1955 +/- 537/mm3). After antigen, the number of "typical" MCs detectable in tissue sections progressively decreased during the 24-hour period, whereas the number of "atypical" MCs was lowest at 1 hour and had increased at 24 hours. After diluent, MC numbers did not change significantly over time. A late-phase response (LPR), detected clinically as induration and edema, was present 6 hours after antigen in four of five dogs, but LPR was not detected after diluent. The size of LPR was correlated (r = 0.85; p less than 0.05) with the decrease in the number of "typical" MCs at 6 hours. We conclude that the response of the "typical" and "atypical" MC to antigen in vivo differs markedly. The "atypical" MCs participate in the early, acute response to antigen, and the "typical" MCs may be associated with the development of the LPR.

A heat-stable, glycoprotein-like material, osteopoietin, produced during bone marrow regeneration, has been shown to induce bone formation when implanted in the rat eye. The material was separated by ultrasonic treatment or by acid buffer (pH 3-5) from sponges implanted in the marrow. The extracted material free of bone or cell solids, induced bone formation in the anterior eye chamber of the rat, whereas the cell solids and control sponges similarly implanted did not.

The records of 9 adult male patients with the acquired immune deficiency syndrome (AIDS) and biopsy-proved Pneumocystis carinii pneumonia were reviewed to determine the correlation between serial pulmonary function tests and the presence or absence of Pneumocystis organisms in subsequent bronchoscopy specimens. At diagnosis, total lung capacity (TLC) or vital capacity (VC) was abnormally low in 4 patients (44%) and diffusing capacity (DLCO) was abnormally low in 8 patients (89%). The ratio of forced expiratory volume in one second (FEV1) to forced vital capacity (FVC) was elevated in all patients. After 21 to 47 days of specific therapy for Pneumocystis pneumonia, changes in DLCO, TLC, VC, and FEV1/FVC did not correlate with the presence or absence of Pneumocystis organisms in bronchoscopy specimens from 7 patients. However, changes in DLCO 105 to 258 days after diagnosis seemed to correlate with the late response to treatment in 6 patients. These results suggest that decisions to terminate specific therapy for Pneumocystis pneumonia in patients with AIDS should not be based on short-term changes in pulmonary function.

The relationship between airway responsiveness to inhaled antigen and histamine, immunologic release of lung histamine, immunologic responsiveness of skin, and specific immunoglobulin E (IgE) antibodies were examined in 11 inbred allergic dogs immunized with extracts of ragweed and grass and 5 nonimmunized control dogs from the same colony. Airway responsiveness to antigen and histamine was characterized by the doses that increased the airflow resistance of the total respiratory system to twice the control values (ED200). Highly significant correlations were found between airway responsiveness and cutaneous responsiveness to antigen and other immunologic characteristics (e.g., IgE and histamine released from lung by inhaled antigen) in all dogs. In ragweed-sensitized dogs, there was an inverse correlation between immunologic responsiveness (reflected by the cutaneous response to antigen and histamine released from lung by inhaled antigen) and nonimmunologic responsiveness of airways (histamine ED200: r = 0.73, P less than 0.05 and r = 0.75, P less than 0.01, respectively). Antigen ED200 was also correlated with histamine release from lung after antigen inhalation (r = 0.74; P less than 0.01). We conclude that airway reactions to inhaled antigen in allergic dogs are dependent not only on immunologic factors but also on the degree of nonimmunologic airway responsiveness to histamine and that these factors are correlated inversely.

Adenosine 3',5'-cyclic monophosphate (cAMP) is believed to mediate the transport of ions, water, and mucous glycoproteins in the respiratory tract. Because chemical measurements of total tissue levels of cAMP may not always reflect changes in specific cell types, we adapted standard immunocytochemical methods to examine the cellular localization of cAMP in dog and cat tracheae. The beta-adrenergic agonists terbutaline and isoproterenol increased immunoreactive cAMP in ciliated epithelial cells of dog and cat tracheae and in both serous and mucous gland cells of cat tracheae. Epithelial goblet cells did not respond to beta-adrenergic agonists in either species. This study provides information about the location of beta-receptors on individual cells in the trachea that is not available from chemical assays of either cAMP or beta-receptors in these tissues. Our results support the hypothesis that secretory functions in both serous and mucous submucosal gland cells and ciliated epithelial cells, but not goblet cells, may involve cyclic AMP-dependent mechanisms.

In allergic and nonallergic lung diseases, if intraluminal mast cells adhere to airway epithelium, inflammatory mediators released from activated mast cells may reach high local concentrations and thus greatly affect airway function. To determine whether mast cells adhere to airway epithelial cells, radiolabeled or unlabeled dog mastocytoma cells were incubated with cultured dog tracheal epithelial cells, with extracellular matrix substrates, and with cryostat-cut sections of dog trachea. Mast cells adhered well to cultured epithelial cells (35 +/- 13% adhesion, mean +/- 1 SD, n = 23) but adhered poorly to types I and IV collagen or to fibronectin (less than 7.5% mean adhesion in all cases). Similarly, in tracheal tissue sections, mast cells adhered preferentially to epithelial cells in surface epithelium or in submucosal glands but not to basal membrane or connective tissue. Adhesion to cultured epithelial cells was a characteristics of a subpopulation of mast cells, could persist for more than 48 h, did not require energy or the presence of divalent cations, and was not mediated by a known family of leukocyte-associated adhesion glycoproteins. Adhesion was completely abolished by pretreatment of mast cells with pronase E or proteinase K but not with trypsin (up to 10 micrograms/ml at 37 degrees C for 20 min each). In contrast, pretreatment of cultured epithelial cells with any of these proteinases had no effect on adhesion. It is concluded that dog mastocytoma mast cells adhere to dog tracheal epithelial cells and do so selectively. It is suggested that mast cell adhesion to airway epithelium may play a role in the effectiveness of mast cell-epithelial cell interactions, and thus, in certain lung diseases, airway function may be affected by intraluminal mast cells more than is currently appreciated.

A well-differentiated dog mastocytoma was characterized ultrastructurally using morphometric, histochemical, and biochemical methods. The ultrastructure of cells in the intact tumor was compared to the morphology of cells disaggregated from the tumor and cultured for periods as long as 4 weeks and to normal dog connective tissue mast cells. Most of the tumor cells contained histamine (mean = 5.81 pg/cell), demonstrated chloroacetate esterase activity histochemically, stained metachromatically with toluidine blue, and were similar in ultrastructure to normal dog mast cells. The proportion of mast cells in this tumor averaged 67%; eosinophils, fibroblasts, plasma cells, and macrophages also were present. The mean diameter of mast cells (12.79 micron) and the mean diameter of their cytoplasmic granules (473 nm) were similar to those reported for mast cells and mastocytoma cells from various species. The heterogeneity in appearance of the mastocytoma granules is consistent with a variable degree of granule maturation. After disaggregation or periods of culture ranging from 2 days to 4 weeks, the mean granule diameters were 15% larger than those measured in the intact mastocytoma cells, though other morphological features remained unchanged. Although the cells retained their distinct morphological features for at least 4 weeks, some of their physiological responses were lost after 1 week in culture. Our study showed that dog mastocytomas can be a source of a large, relatively homogeneous population of cells that are useful for elucidating some of the structural and functional properties of mast cells.

Inhalation of histamine diphosphate aerosol (1.6 per cent, 10 breaths) produced a 218 +/- 54.6 per cent (mean +/- SE) increase in airway resistance in 16 normal subjects with colds compared with a 30.5 +/- 5.5 per cent increase in 11 healthy control subjects (P less than 0.01). There was no significant difference in mean baseline airway resistance between the two groups. Inhalation of saline produced no significant change in airway resistance in either group. Isoproterenol hydrochloride (0.5 per cent, 1 breath) or atropine sulfate aerosol (0.2 per cent, 20 breaths) each reversed and prevented the increase in airway resistance by histamine, indicating that the bronchoconstriction was caused by smooth muscle contraction and that post-ganglionic, cholinergic pathways were involved in the mechanism. In 6 subjects with colds, citric acid aerosol (10 per cent, 5 breaths) caused bronchoconstriction that lasted up to 30 sec after inhalation, a significantly greater effect than that observed in control subjects or in the same subjects after recovery (P less than 0.05). Prior inhalation of atropine aerosol (0.2 per cent, 20 breaths) prevented the bronchoconstriction after citric acid aerosol in all 6 subjects. The threshold concentration of citric acid that produced cough in 7 subjects with colds was significantly lower than that in control subjects or in the 7 subjects after recovery (P less than 0.05), suggesting that the exaggerated cholinergic response was due to a decreased threshold for stimulation of the rapidly adapting sensory receptors in the airways. We have provided evidence that respiratory viral infections that produce airway epithelial damage temporarily cause these subjects to develop more bronchoconstriction after inhaling smaller doses of histamine than do healthy subjects. The fact that atropine prevents this response and that the threshold to cough is temporarily decreased is compatible with our hypothesis that airway epithelial damage by infection exposes and, thus, "sensitizes" the rapidly adapting airway receptors to inhaled irritants, causing increased bronchoconstriction via a vagal reflex. Damage to the airway epithelium may occur as a result of mechanical factors, inhaled chemicals, and pollutants, such as ozone, infections, or perhaps as a result of the action of materials released endogenously (e.g., from mast cells, white blood cells, or platelets). "Sensitization" of rapidly adapting sensory receptors in the airways may be an important factor in asthma and in other diseases of airways.

In five treadmill-exercising, unsedated dogs, we studied the effect of inhaled Ascaris suum antigen aerosols on minute volume of ventilation (VE), respiratory frequency (f), tidal volume (VT), total pulmonary resistance (RL), and dynamic pulmonary compliance (CLdyn), before and during cooling of the vagus nerves. With the vagi warm, inhaled antigen increased VE (mean + 62%; P less than 0.01)by increasing f (mean + 180%; P less than 0.01), despite a decrease in VT (mean - 42%; P less than 0.01). RL increased (mean + 170%; P less than 0.001) and CLdyn decreased (mean - 43%; P less than 0.005). With the vagi cool, inhaled antigen no longer affected VE, f, or VT (P greater than 0.5), although RL still increased and CLdyn still decreased. Inhalation of a bronchodilator, terbutaline, prevented the broncho-constriction induced by antigen but did not prevent the ventilatory response. We conclude that vagal afferent pathways mediate the ventilatory response to inhaled antigen and suggest that the primary stimulus for this response is not airway narrowing.

In 12 anesthetized dogs, we tested the effects of pharmacologic beta-adrenergic stimulation on the severity of tracheal mast cell reactions in vivo (Am Rev Respir Dis 1979; 119:62). In control dogs, tracheal arterial injections of compound 48/80 produced arteriovenous differences in histamine concentrations across the trachea (35 +/- 15 ng/ml), but not the lung; this finding suggested selective degranulation of tracheal mast cells. Tracheal arteriovenous histamine differences correlated (r = 0.70) with tracheal contractions (12 +/- 4 g/cm) induced by compound 48/80 in these animals. In other dogs, injections of isoproterenol (10 micrograms/kg) before compound 48/80 prevented the development of both tracheal arteriovenous differences and contractions. We concluded that pharmacologic beta-adrenergic stimulation inhibited degranulation of tracheal mast cells. The preparation appears suitable for examining the effects of endogenous stimuli on the severity of mast cell reactions in the central airways of anesthetized dogs.

We studied the effect of inhaled specific antigen on airflow resistance, histamine concentration, and mast cell numbers in airway tissues in allergic dogs. In each of six allergic dogs with open chests, inhalation of specific antigen aerosol (Ascaris suum) increased airflow resistance from 0.30+/-0.40 (mean+/-SE) to 2.8+/-0.41 cmH2O/1 per s (P less than 0.05); decreased mast cell number from 38.6+/-3.2 to 24.3+/-4.5 mast cells/mm2 (P less than 0.05); decreased histamine (per mg airway tissue) from 5.3+/-0.4 to 3.5+/-0.2 ng (P less than 0.05) in lobar bronchi 5-7 mm in diam; and released histamine into the blood perfusing the lung, control arterial plasma histamine; 2.8+/-0.64 ng/ml; antigen: 154+/-1.7 ng/ml (P less than 0.005). Specific antigen caused no significant changes in mast cells or histamine in bronchi 1-2 mm in diam. Control aerosols of nonspecific antigen or methacholine did not change levels of mast cells or histamine in airway tissues. These results suggest that experimental canine asthma involves local airway reactions with degranulation of mast cells and histamine release, as well as vagally mediated reflex bronchoconstriction.

Mast cells are believed to play an important role in the pathogenesis of asthma, and several investigators have suggested that increased numbers of mast cells in the airway lumen or increased releasability of histamine from these mast cells are responsible for chronic airway hyperreactivity. To determine whether mast cells in the lumen of the airways of hyperreactive Basenji greyhound (BG) dogs differ from those of mongrel dogs with normal airway reactivity, we investigated the morphologic and functional characteristics of mast cells recovered by bronchoalveolar lavage (BAL). BAL was performed in five BG and five mongrel dogs with 900 cc of a buffered salt solution. The recovered lavage fluid contained 115 +/- 19 X 10(6) and 116 +/- 14 X 10(6) (mean +/- SEM) cells in BG and mongrel dogs, respectively. The proportion of all mast cells within the recovered cell population as enumerated after fixation with basic lead acetate and staining with alcian blue was not different in BG and mongrel dogs and averaged 0.80 +/- 0.07% and 1.1 +/- 0.3%, respectively. Typical mast cells as identified after fixation with paraformaldehyde were rare; however, significantly more mast cells were found in mongrel (0.03 +/- 0.009%) than in BG dogs (0.004 +/- 0.002%; p less than 0.02). Mast cells recovered from BG and mongrel dogs were not different in their low spontaneous histamine release (2.0 +/- 0.5% and 2.9 +/- 0.8%), their histamine release on stimulation with the calcium ionophore A23187 (maximum release 44.8 +/- 5.7% and 41.5 +/- 3.9%), and their lack of response to compound 48/80 (maximum release 5.8 +/- 1.8% and 6.1 +/- 6.0%).(ABSTRACT TRUNCATED AT 250 WORDS)

Because adherence of histamine to glass is well-known, we tested for its adherence to a mechanical homogenizer commonly used in the extraction of histamine from tissue samples. During 60 sec of homogenization, 15% to 17% of the histamine originally present in the samples "disappeared," and the reason for the disappearance was reversible binding of histamine to the homogenizer. Adding trace amounts of [14C]histamine to each sample before homogenization and measuring the disappearance of radioactivity during homogenization permitted correction for binding to the homogenizer. This technique for correction was validated by the measurement of endogenous concentrations of histamine in the tracheal posterior membranes of six dogs (range of mean concentrations: 0.63 to 1.51 ng/mg wet weight) followed by the measurement of known amounts of exogenous histamine added before homogenization to tracheal tissue samples from the same dogs. In the latter samples, 96 +/- 13% (mean +/- SEM) of the histamine added was measured by our technique. We conclude that binding of histamine to mechanical homogenizers may be an important cause of inaccuracy of the enzymatic assay for the measurement of histamine concentrations in tissue but that such binding may but that such binding may be easily corrected for.

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators.

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.

Histamine can be recovered from the blood of ragweed-sensitized dogs after aerosol antigen challenge, although its source is unknown. Neutrophils and eosinophils have been recovered from bronchoalveolar lavage fluid (BALF) obtained under identical conditions. We investigated the time course of changes in histamine levels in plasma and BALF taken from ragweed-sensitized dogs after aerosol challenge. Changes in the numbers of circulating neutrophils, eosinophils, lymphocytes, monocytes, and platelets were also studied. After 3 min, total pulmonary resistance (RL) was maximally increased and systolic blood pressure was maximally decreased. Histamine levels in plasma and BALF were increased and circulating eosinophils and neutrophils were decreased. After 15 min, platelet numbers were reduced. By 90 min, changes in RL, blood pressure, plasma and BALF histamine concentrations, and circulating neutrophils and eosinophils had returned to base-line values, but platelet numbers remained significantly decreased. Sham challenge caused no significant changes in any of these variables. Intravenous administration of histamine in doses large enough to attain plasma levels comparable with those achieved after aerosol antigen challenge resulted in no concomitant rise in BALF histamine levels. We conclude that antigen challenge in sensitized dogs causes increases in BALF and plasma histamine levels and is associated with a reduction in circulating neutrophils, eosinophils, and platelets. It is likely that antigen causes airway mast cells to release mediators that move down a concentration gradient from the airways to the pulmonary circulation.

We studied whether antigen-induced airway hyperresponsiveness was associated with pulmonary inflammation in 11 anesthetized ragweed-sensitized dogs. Airway responsiveness to acetylcholine aerosol was determined before and at 2, 6, and 24 h after ragweed or sham aerosol challenge. Pulmonary inflammation was assessed by bronchoalveolar lavage (BAL) performed at either 2 or 6 h. Total pulmonary resistance increased 11-fold at 5 min after ragweed. Airway responsiveness was unchanged at 2 h but was increased 6.6-fold at 6 h in 8 of 11 dogs (P less than 0.001); hyperresponsiveness persisted from 4 days to 4 mo. Airway responsiveness was unchanged by aerosols of diluent. Neutrophils in BAL fluid increased approximately sixfold at 2 h (P less than 0.02) and at 6 h (P less than 0.02) after antigen challenge. There were fewer eosinophils in fluid recovered at 6 h after antigen compared with 2 h lavages (P less than 0.05). In three nonresponders, BAL showed no significant changes in neutrophils and eosinophils after antigen. Thus antigen-induced hyperresponsiveness is associated with the presence of pulmonary inflammation, presumably arising from the airways and involving both neutrophils and eosinophils.

Airway reflexes are difficult to study in conscious animals because associated changes in ventilation alter intrathoracic airway dimensions. By studying an isolated segment of extrathoracic trachea, we have overcome this problem. In each of 2 dogs, we created surgically an isolated tracheal segment just below the larynx, sealed at one end and tapered at the other to a 3-mm opening via a skin fistula. A chronic tracheostomy was also created near the thoracic outlet. We monitored intraluminal pressure (Pseg) of the isolated segment to reflect changes in smooth muscle tone. During anesthesia, with pentobarbital, gentle mechanical stimulation of the carina, deflation of the lungs, and asphyxia for one min increased Pseg (+9 to +/- 16 cm H2O). Lung inflation and alveolar hyperventilation decreased Pseg (-9 to -16 cm H2O). Five breaths of 2 per cent histamine aerosol increased Pseg (+5 cm H2O) when resting tone was normal. We also coated lumen of the isolated segment with tantalum powder and documented roentgenologically changes in the size of the segment that reflected changes in smooth muscle tone; constriction and dilation in response to asphyxia and lung inflation, respectively, were demonstrated directly by this technique. In conscious dogs, lung inflation decreased Pseg, and carinal stimulation increased Pseg. Instillation of lidocaine hydrochloride (Xylocaine) into the isolated tracheal segment blocked cough caused by mechanical stimulation of the segment, but carinal stimulation still caused constriction of the segment under these conditions which indicated that afferent, but not effrent parasympathetic innervation of the segment had been blocked selectively. Conversely, instillation of atropine sulfate into the isolated tracheal segment blocked constriction of the segment caused by carinal stimulation, but mechanical stimulation of the segment still caused cough under these conditions, which indicated that efferent, but not afferent parasympathetic innervation of the segment had been blocked selectively. We conclude that an innervated extrathoracic tracheal segment constricts and dilates via cholinergic pathways and is suitable for the study of airway reflexes in conscious dogs.

Discrepancies exist between reported effects of histamine on the alpha-adrenergic responsiveness of the canine trachealis muscle. Therefore, using an in situ preparation described recently (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 49: 84-94, 1980), we tested the effects of histamine and other agents on alpha-adrenergic responses in the trachealis muscle of 34 anesthetized dogs. Under basal conditions, maximum alpha-adrenergic responses were small [3 +/- 1 (SE) g/cm] compared with those induced by a supramaximal parasympathetic stimulus (45 +/- 3 g/cm). After exposure of the muscle to histamine, responsiveness increased markedly to alpha-adrenergic stimuli, which included tracheal arterial injections of norepinephrine and phenylephrine or electrical stimulation of sympathetic nerves. Augmented alpha-adrenergic responsiveness persisted for 20 min after the end of contractions induced by histamine. Serotonin and long-acting, but not short-acting, cholinergic agonists also potentiated alpha-adrenergic responsiveness. We concluded that exposure of the trachealis muscle in living dogs to a variety of constrictor stimuli potently augmented its alpha-adrenergic responsiveness.

There is conflicting evidence as to which autonomic receptors mast cells possess and whether the receptors are capable of modulating mediator release. We have studied dog mastocytoma cells because they are available in large numbers in a relatively pure form, unlike normal dog mast cells. Mastocytoma nodules from a dog were excised and disaggregated with collagenase to provide a cell suspension of mastocytoma cells of greater than 92% purity. The presence of autonomic receptors was assessed by both radioligand binding assays and by evaluating pharmacologic modulation of mediator release. In the radioligand binding assays, beta-adrenergic receptors were estimated by [3H]dihydroalprenolol binding, alpha-adrenergic receptors by [3H]prazosin binding and cholinergic receptors by [3H]quinuclidinyl benzilate binding. Nonspecific binding was determined in each case by incubation in the presence of the specific antagonists propranolol, phentolamine, and atropine, respectively. The effect of autonomic agonists on immunologic and nonimmunologic histamine release was examined, using the beta-adrenergic agonists isoproterenol and terbutaline, the alpha-adrenergic agonist phenylephrine with and without propranolol, and the cholinergic agonist acetylcholine. Dose-response curves were constructed both for the autonomic agonists and the histamine-releasing agents. Results from the radioligand binding and the pharmacologic studies were concordant. These dog mastocytoma cells had a high density of beta-receptors (21,500 +/- 3,300; mean +/- SE beta-receptors/cell, n=5) of which the predominant subtype appeared to be beta2. No evidence was found for the presence of alpha-adrenergic or cholinergic receptors either by direct receptor binding or by their actions on histamine release.

The role of arachidonic acid metabolites in the pathogenesis of airway inflammation and clinical asthma is currently unknown. The addition of eicosapentaenoic acid (EPA) to the diet of humans has been shown to generate metabolites that are less potent than their arachidonic acid counterparts. The substitution of EPA for arachidonic acid metabolites in patients might cause a decrease in airway inflammation and an improvement in clinical asthma. We studied the effect of addition of EPA to the diet of twelve asthmatic patients. Standard clinical evaluations and pulmonary function tests were done on weeks 0, 3, 6, 10, 12 and 14. Patients ingested either low-dose EPA (0.1 g/day) or high-dose EPA (4.0 g/day) from weeks 6-14 (total of 8 weeks). There was no difference in clinical status or pulmonary function between groups at the start of the study. There was no change in clinical status or pulmonary function between or within groups at the end of 8 weeks of EPA ingestion.

We studied the roentgenograms, pulmonary function tests, and physical findings of 294 shipyard workers to evaluate asbestos exposure-cigarette smoking interactions. Roentgenographic parenchymal opacities, decreased pulmonary diffusing capacity for carbon monoxide, decreased flow at low lung volume, rales, and clubbing were each significantly related to the number of years elapsed since first exposure to asbestos and cigarette smoking status when analyzed by logistic regression. A dose-dependent cigarette smoking response that was consistent with synergism was present only for parenchymal opacities and decreased flow at low lung volume. These findings suggest that decreased flow at low lung volume, possibly reflecting peribronchiolar fibrosis, may be a functional corollary to smoking-associated parenchymal roentgenographic opacities among some asbestos-exposed individuals.

The effect of short-term ozone (O3) exposure on pulmonary mast cell function was examined. Guinea pigs were continuously exposed to 1.0 ppm O3 for 2, 4, and 8 hr. O3 exposure produced a significant decrease in lung histamine concentration. Two-hour exposure to O3 caused a 22.4 +/- 7.0% decrease in lung histamine concentration compared with controls. Ozone exposures of 4 and 8 hr caused lung histamine concentrations to decrease by 43.7 +/- 7.7 and 49.0 +/- 7.5% (P less than 0.05), respectively, without significant changes in lung water or protein, or evidence of cytotoxicity. These results suggest that O3 or its metabolites affect pulmonary mast cell function by stimulating the release of histamine from the lung.

Three allergic dogs were trained to swallow an esophageal balloon, breathe through a tracheostomy tube, and stand quietly in a wooden stall. The mechanical properties of their lungs were measured reproducibly over many months. Average values were: pulmonary resistance 1.33 cmH2O/l per s, static lung compliance 104 ml/cmH2O, and functional residual capacity 982 ml. In two dogs inhalation of histamine aerosol caused a 374% increase in resistance; inhalation of Ascaris suum aerosol caused a 307% increase in resistance. The increased resistance was associated with mild-to-moderate respiratory distress and slightly increased functional residual capacity. Aerosols of isoproterenol or atropine sulfate inhibited the increased resistance. We conclude that the conscious allergic dog is a suitable subject for the long-term study of asthma. Our present results are consistent with our earlier findings in anesthetized dogs, that a vagal reflex is involved in acute bronchomotor responses to inhalation of histamine as well as specific antigen.