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The effect of ingestion of heat-killed Histoplasma capsulatum yeast cells on the metabolism of arachidonic acid (20:4) to prostenoids and leukotrienes was examined in murine peritoneal macrophages (M phi s). H. capsulatum-containing M phi s exhibited a metabolite profile similar to that of zymosan-challenged phagocytes; however, there were differences with respect to the relative and total amounts of products produced. While proteose peptone-elicited M phi s exposed to H. capsulatum released quantitatively less prostaglandin E2 (PGE2) and leukotriene C4 than zymosan-treated M phi s, they metabolized a greater percentage of total product to prostenoids. In addition, whereas in vitro priming with gamma interferon increased both the PGE2 and leukotriene C4 contents of zymosan-stimulated M phi supernatants, similarly primed M phi s challenged with H. capsulatum selectively increased only PGE2 production. The immunosuppressive effect of a relative excess of prostenoids in H. capsulatum-containing M phi s may contribute to the overall disturbance in cell-mediated immunity characteristic of disseminated histoplasmosis.

Cytocentrifuge preparations of enzymatically dispersed human lung parenchymal mast cells were examined by light microscopy after fixation in either Mota's basic lead acetate or 10% neutral buffered formalin followed by toluidine blue staining at pH 0.5. Fixation in Mota's basic lead acetate allowed detection of all mast cells. However, after formalin fixation only 10.8 +/- 1.3%, range 4.7 to 17%, n = 8 remained detectable (i.e., formalin "resistant"). Therefore, the vast majority of human lung mast cells lose their metachromatic staining after formalin fixation (i.e., are formalin "sensitive"). Mast cells were then separated on the basis of diameter by countercurrent elutriation and on the basis of density by discontinuous Percoll gradients. Histochemically distinct populations of mast cell types emerged in all lungs studied. The proportion of formalin-resistant mast cells increased as a function of diameter: less than 5% at diameters of less than or equal to 11 mu and densities less than or equal to 1.063 g/ml, to 30 to 40% in cells of diameters greater than or equal to 16 mu and densities greater than or equal to 1.100 g/ml. Maximum anti-IgE challenge of nearly homogeneous formalin-sensitive mast cells (94.3 +/- 2.1% purity, n = 6) caused the generation of both leukotriene C4 (64.6 +/- 26.4 pg/mast cell) and PGD2 (114.8 +/- 37.5 pg/mast cell). Six- to eight-fold enrichment of formalin-resistant mast cells did not significantly alter the histamine release response or profiles of arachidonate metabolites. Similar results were obtained for the nonimmunologic stimulus ionophore A23187. We conclude that two histochemically distinct subpopulations, of mast cells are present in human lung suspensions. Although formalin-sensitive cells account for almost 90% of lung mast cells, formalin-resistant cells are separable by their large diameters and higher densities. Both subtypes show similar histamine release responses and arachidonate oxidation profiles.

Inhaled long-acting beta(2)-agonists improve asthma control when added to inhaled corticosteroid (ICS) therapy.To determine whether ICS therapy can be reduced or eliminated in patients with persistent asthma after adding a long-acting beta(2)-agonist to their treatment regimen.A 24-week randomized, controlled, blinded, double-dummy, parallel-group trial conducted at 6 National Institutes of Health-sponsored, university-based ambulatory care centers from February 1997 through January 1999.One hundred seventy-five patients aged 12 through 65 years with persistent asthma that was suboptimally controlled during a 6-week run-in period of treatment with inhaled triamcinolone acetonide (400 microg twice per day).Patients continued triamcinolone therapy and were randomly assigned to receive add-on therapy with either placebo (placebo-minus group, n = 21) or salmeterol xinafoate, 42 microg twice per day (n = 154) for 2 weeks. The entire placebo-minus group was assigned and half of the salmeterol group (salmeterol-minus group) was randomly assigned to reduce by 50% (for 8 weeks) then eliminate (for 8 weeks) triamcinolone treatment. The other half of the salmeterol group (salmeterol-plus group) was randomly assigned to continue both salmeterol and triamcinolone for the remaining 16 weeks (active control group).Time to asthma treatment failure in patients receiving salmeterol.Treatment failure occurred in 8.3% (95% confidence interval [CI], 2%-15%) of the salmeterol-minus group 8 weeks after triamcinolone treatment was reduced compared with 2.8% (95% CI, 0%-7%) of the salmeterol-plus group during the same period. Treatment failure occurred in 46.3% (95% CI, 34%-59%) of the salmeterol-minus group 8 weeks after triamcinolone therapy was eliminated compared with 13.7% (95% CI, 5%-22%) of the salmeterol-plus group. The relative risk (95% CI) of treatment failure at the end of the triamcinolone elimination phase in the salmeterol-minus group was 4.3 (2.0-9.2) compared with the salmeterol-plus group (P.001).Our results indicate that in patients with persistent asthma suboptimally controlled by triamcinolone therapy alone but whose asthma symptoms improve after addition of salmeterol, a substantial reduction (50%) in triamcinolone dose can occur without a significant loss of asthma control. However, total elimination of triamcinolone therapy results in a significant deterioration in asthma control and, therefore, cannot be recommended.

Long-acting beta(2)-agonists are prescribed for patients with persistent asthma and are sometimes used without inhaled corticosteroids (ICSs). No evidence exists, however, to support their use as monotherapy in adults with persistent asthma.To examine the effectiveness of salmeterol xinafoate, a long-acting beta(2)-agonist, as replacement therapy in patients whose asthma is well controlled by low-dose triamcinolone acetonide, an ICS.A 28-week, randomized, blinded, placebo-controlled, parallel group trial conducted at 6 National Institutes of Health-sponsored, university-based ambulatory care centers from February 1997 to January 1999.One hundred sixty-four patients aged 12 through 65 years with persistent asthma that was well controlled during a 6-week run-in period of treatment with inhaled triamcinolone (400 microg twice per day).Patients were randomly assigned to continue triamcinolone therapy (400 microg twice per day; n = 54) or switch to salmeterol (42 microg twice per day; n = 54) or to placebo (n = 56) for 16 weeks, after which all patients received placebo for an additional 6-week run-out period.Change in morning and evening peak expiratory flow (PEF), forced expiratory volume in 1 second (FEV(1)), self-assessed asthma symptom scores, rescue albuterol use, asthma-specific quality-of-life scores, treatment failure, asthma exacerbation, bronchial reactivity, and markers of airway inflammation, compared among the 3 treatment groups.During the 16-week randomized treatment period, no significant differences between the salmeterol and triamcinolone groups were observed for conventional outcomes of clinical studies of asthma therapy-morning PEF, evening PEF, asthma symptom scores, rescue albuterol sulfate use, or quality of life. Both active treatments were superior to placebo. However, the salmeterol group had more treatment failures than the triamcinolone group (13/54 [24%] vs 3/54 [6%]; P =.004), as well as more asthma exacerbations (11/54 [20%] vs 4/54 [7%]; P =.04), greater increases in median (interquartile range) sputum eosinophils (2.4% [0.0% to 10.6%] vs -0.1% [-0.7% to 0.3%]; P.001), eosinophil cationic protein (71 [-2 to 430] U/L vs -4 [-31 to 56] U/L; P =.005), and tryptase (3.1 [2.1 to 7.6] ng/mL vs 0.0 [0.0 to 0.7] ng/mL; P.001). The duration of benefit when patients were switched from active treatment to placebo after 22 weeks of randomized treatment was not significantly longer in the triamcinolone group than in the salmeterol group.Patients with persistent asthma well controlled by low doses of triamcinolone cannot be switched to salmeterol monotherapy without risk of clinically significant loss of asthma control.

Regular use of inhaled beta-adrenergic agonists may have adverse effects in some asthma patients. Polymorphisms of the beta(2)-adrenergic receptor (beta(2)-AR) can affect its regulation; however, results of smaller studies of the effects of such polymorphisms on response to beta-agonist therapy have been inconsistent.We examined the possible effects of polymorphisms at codons 16 (beta(2)-AR-16) and 27 (beta(2)-AR-27) on response to albuterol by genotyping 190 asthmatics who had participated in a trial of regular versus as-needed albuterol use.During the 16-week treatment period, patients homozygous for arginine (Arg/Arg) at beta(2)-AR-16 who used albuterol regularly had a small decline in morning peak expiratory flow (AM PEF). This effect was magnified during a 4-week run-out period, when all patients returned to as-needed albuterol only. By the end of the study, Arg/Arg subjects who had used albuterol regularly had an AM PEF 30.5 +/- 12.1 liters/min lower (p = 0.012) than Arg/Arg patients who had used albuterol as needed only. Subjects homozygous for glycine at beta(2)-AR-16 showed no such decline. Evening PEF also declined in the Arg/Arg regular but not in as-need albuterol users. No significant differences between regular and as-needed treatment were associated with polymorphisms at beta(2)-AR-27.Polymorphisms of the beta(2)-AR may influence airway responses to regular inhaled beta-agonist treatment.

CD4+ "helper" T-lymphocytes in murine and human models have been divided into Th1 and Th2 subclasses, characterized by the profile of cytokines they secrete: INF-gamma (and perhaps IL-2 and TNF-beta) by Th1 cells, and IL-4 (and perhaps IL-5, IL-6, IL-10, and IL-13) by Th2 cells. Although a strict division into Th1 and Th2 phenotypes in humans (unlike murine systems) may not be possible, the asthmatic diathesis in humans appears to be one largely characterized by inflammatory responses associated with Th2 cells and their cytokines, particularly IL-4, IL-13, and IL-5. Other pulmonary disorders, such as those associated with infectious diseases including tuberculosis, appear to favor an immunologic response characteristic of Th1-cells, and its defining cytokine IFN-gamma. This apparent Th1/Th2 immune dysregulation in asthma is an area of active investigation and forms the basis for ongoing attempts to change this phenotype through a variety of approaches. These include immunotherapy with conventional antigens, designer peptides, oligonucleotides, and anti-IgE, and pharmacotherapy with immune modulating drugs, cytokines, cytokine agonists and cytokine antagonists, and antibodies. This field of investigation promises to usher in a whole new approach to our understanding of asthma and ways to approach its treatment.

SUMMARY IL-5 is a potent eosinophil viability-enhancing factor that has been strongly implicated in the pathogenesis of IgE-mediated inflammation in vivo. Recently published data have suggested that IL-5 (and related cytokines) may act by altering the expression of the anti-apoptotic regulator Bcl-2 or its homologues, but this is controversial. The behaviour of the recently described pro-apoptotic cysteine proteases (caspases) in eosinophils after IL-5 treatment has not been explored. We examined the effect of IL-5 on the expression of four major Bcl-2 homologues, as well as on the expression/activation of key members of the caspase cell death cascade in cultured circulating human eosinophils. The effect of relevant inducers of eosinophil apoptosis (glucocorticoid and Fas ligation) on these regulatory proteins was also examined. We observed baseline expression of the anti-apoptotic Mcl-1 and pro-apoptotic Bax proteins in immunoblots of eosinophil lysates, but not Bcl-x, Bcl-2. IL-5 treatment had the effect of maintaining this basal level of expression over time without altering the balance of Bcl-2 homologues. The (upstream) caspase 8 and (downstream) caspase 3 proenzymes were detected in eosinophils at baseline, and were processed during spontaneous and stimulated eosinophil death. IL-5 completely blocked caspase processing in spontaneous and dexamethasone-induced cell death, and significantly slowed processing during Fas ligation. Our data do not support the theory that IL-5 acts by altering the balance of anti-apoptotic and pro-apoptotic Bcl-2 homologues, but suggest that it may act by regulating activation of the caspase cell death cascade.

Colchicine demonstrates an array of anti-inflammatory properties of potential relevance to asthma. However, the efficacy of colchicine as an alternative to inhaled corticosteroid therapy for asthma is unknown. Five centers participated in a controlled trial testing the hypothesis that in patients with moderate asthma needing inhaled corticosteroids for control, colchicine provides therapeutic benefit as measured by maintenance of control when inhaled steroids are discontinued. Subjects were stabilized on triamcinolane acetonide (800 microg daily) and then enrolled in a 2-wk run-in during which all subjects took both colchicine (0.6 mg/twice a day) and triamcinolone. At the end of the run-in, all subjects discontinued triamcinolone and were randomized to continued colchicine (n = 35) or placebo (n = 36) for a 6-wk double-blind treatment period. The treatment groups were similar in terms of disease severity. After corticosteroid withdrawal, 60% of colchicine-treated and 56% of placebo-treated subjects were considered treatment failures as defined by preset criteria. No significant difference in survival curves was found between treatment groups (log rank = 0.38). Other measures, including changes in FEV1, peak expiratory flow, symptoms, rescue albuterol use, and quality of life scores, also did not differ between groups. Of note, subjects failing treatment had significantly greater methacholine responsiveness at baseline than did survivors (PC20, 0.81+/-1.38 versus 2.11+/-2.74 mg/ml; p = 0.01). An analysis of treatment failures suggested that the criteria selected for failure reflected a clinically meaningful but safe level of deterioration. We conclude that colchicine is no better than placebo as an alternative to inhaled corticosteroids in patients with moderate asthma. Additionally, we conclude that the use of treatment failure as the primary outcome variable in an asthma clinical trial where treatment is withdrawn is feasible and safe under carefully monitored conditions.

To determine whether hypoxic pulmonary vasoconstriction was associated with release of sulfidopeptide leukotrienes (SPLTs) from the lung, we measured SPLT activity by bioassay (guinea pig ileum) and radioimmunoassay in lymph, perfusate, and bronchoalveolar lavage (BAL) fluid from sheep lungs (n = 20) isolated and perfused in situ with a constant flow of autologous blood (100 ml.kg-1.min-1) containing indomethacin (60 micrograms/ml). The protocol consisted of three periods, each at least 1 h in duration. In experimental lungs, inspired O2 concentration (FIO2) was 28.2% in periods 1 and 3 and 4.2% in period 2. In control lungs, FIO2 was 28.2% throughout. Hypoxia increased pulmonary arterial pressure but did not alter peak tracheal pressure, lung lymph flow, or weight gain measured during the last 30 min of each period. SPLT activity was greatest in lung lymph and least in BAL fluid. Hypoxia did not alter SPLT activity in any fluid. Similar results were obtained in lungs not treated with indomethacin (n = 15). These data do not support the hypothesis that hypoxic pulmonary vasoconstriction is mediated by SPLTs.

Study of the role of mast cells in the human gastrointestinal tract has suffered from the inability to examine these cells in vitro. In addition, work in rodent systems suggests that there are substantial differences between intestinal mast cells and those from other tissues, making extrapolation of data from other sources difficult. We report a method for producing mast cell-containing single cell suspensions from human intestinal tissue by mechanical and enzymatic dispersion. This method yields 4.5 X 10(5) mast cells per gram of tissue in purity of 3.1 +/- 2.1%. These mast cells were functionally intact as assessed by survival in short-term culture, low spontaneous release, and appropriate IgE-mediated histamine release. They were morphologically intact on electron microscopy and conformed to published descriptions of human lung mast cells. The intestinal mucosal mast cells were also indistinguishable from human lung mast cells in histamine content, goat anti-human IgE dose-response curves, kinetics of histamine release, unresponsiveness to f-met peptide, and production of arachidonic acid metabolites, prostaglandin D2, and leukotriene C4. This procedure produces human intestinal mast cell suspensions in sufficient numbers to make pharmacologic characterization and further purification of this cell feasible.

Much has been learned about the biochemical nature and pharmacologic activity of the products of arachidonic acid (AA) oxidation, but relatively little is known about the structures in nucleated cells into which AA is incorporated and from which it is initially mobilized. To address this question, we administered 3H-AA or other 3H-fatty acids in vitro to human lung mast cells and alveolar macrophages as well as to mouse and guinea pig peritoneal macrophages. The subcellular distribution of 3H label was assessed by electron microscopic autoradiography, and the nature of cell-associated 3H-lipids was determined by thin layer chromatography. Autoradiographic analysis of human lung mast cells localized virtually all of the 3H-AA to cytoplasmic lipid bodies. Lipid bodies are roughly spherical, variable osmiophilic, nonmembrane-bound structures that appear in the cytoplasm of a wide variety of cells, but we have found that these lipid bodies occur with increased frequency in granulocytes, macrophages, and mast cells at sites of inflammatory, immunologic, or neoplastic processes. Macrophages also incorporated 3H-AA predominantly into cytoplasmic lipid bodies. In contrast to mast cells, however, macrophages incorporated 3H-AA into the plasma membrane as well. Stimulation of macrophage phagocytosis resulted in striking alterations of the relationships of lipid bodies to intracellular membranes, so that many lipid bodies appeared adjacent to phagolysosomes. In addition, some phagolysosomes contained 3H label, which along with other morphologic evidence suggested that lipid bodies may discharge their contents into these structures. Mast cell and macrophage cytoplasmic lipid bodies appear to represent a major site of intracellular storage and metabolism of products of AA and perhaps other fatty acids taken up from the external milieu. These heretofore neglected organelles may thus influence cellular function in a wide variety of adaptive or pathologic processes.

Gaucher's disease is a lysosomal storage disease caused by a deficiency in the enzyme glucocerebrosidase. A small, heat-stable glycoprotein first obtained from Gaucher spleen (Ho, M. W., and O'Brien, J. S. (1971) Proc. Natl. Acad. Sci. U. S.A. 68, 2810-2813) has been observed to stimulate the activity of glucocerebrosidase isolated from normal tissue. It has been suggested that this material might be important in the physiological catabolism of glucocerebroside in normal individuals (Ho, M. W. (1974) in Enzyme Therapy in Lysosomal Storage Diseases (Tager, J. M., Hooghwinkel, G. J. M., and Daems, W. Th., eds) pp.239-246, North-Holland Publishing Co., Amsterdam). In order to investigate this suggestion, glucocerebrosidase activating factors were isolated and purified from control and Gaucher spleen and characterized. Although approximately the same mass of activator was isolated from both spleens, the two activators differ from one another in a number of important respects: (a) the activator from the control spleen is only 6 per cent as active (on a protein basis) as the activator from Gaucher spleen; (b) the amino acid compositions of the purified activators are significantly different; and (c) carbohydrate analysis of the purified activators indicates that the activator from Gaucher spleen is a glycoprotein, while that from control spleen is not. Comparative kinetic studies demonstrate that the anionic detergent, sodium taurocholate, and the acidic phospholipid, phosphatidylinositol, both stimulate glucocerebrosidase activity to a larger extent than the activator substance from Gaucher spleen. The activator from Gaucher spleen and human liver glucocerebrosidase both appear to contain significant hydrophobic character. We conclude that the activator is probably not physiologically important in the catabolism of glucocerebroside in normal tissues. The significance of the occurrence of this apparently unique glycoprotein activator in Gaucher spleen remains obscure; however, its presence represents another interesting aspect of Gaucher's disease that warrants further investigation.

We examined the role of cyclooxygenase-derived metabolites and epithelial cells in airflow-induced bronchospasm. Male dogs were anesthetized and collateral system resistance (Rcs) was measured with the wedged-bronchoscope technique. A 2-min high flow challenge with dry air in nine animals produced a mean increase in Rcs of 69 +/- 13% (SE). After treatment with indomethacin (5 mg/kg), the response was significantly attenuated; Rcs increased only 40 +/- 8%. Bronchoalveolar lavage performed 5 min after a dry air challenge yielded fluid with greater concentrations of prostaglandin D2 (PGD2) and thromboxane B2 than samples from unchallenged segments. Challenge with humidified air produced a smaller physiological response than did challenge with dry air. Lavage samples obtained after dry challenge had greater concentrations of PGD2 than samples taken after challenge with humidified air. After dry air challenge, epithelial cells in lavage fluid were increased by 454 and 515% when compared with control and humidified air challenge, respectively. Significant correlations were found between epithelial cell number and PGD2 recovered in lavage fluid after dry air challenges. We conclude that both epithelial cells and prostaglandins play an important role in peripheral lung responses to dry air.

Much has been learned about the biochemical nature and pharmacologic activity of the products of arachidonic acid (AA) oxidation, but relatively little is known about the structures in nucleated cells into which AA is incorporated and from which it is initially mobilized. To address this question, we administered 3H-AA or other 3H-fatty acids in vitro to human lung mast cells and alveolar macrophages as well as to mouse and guinea pig peritoneal macrophages. The subcellular distribution of 3H label was assessed by electron microscopic autoradiography, and the nature of cell-associated 3H-lipids was determined by thin layer chromatography. Autoradiographic analysis of human lung mast cells localized virtually all of the 3H-AA to cytoplasmic lipid bodies. Lipid bodies are roughly spherical, variable osmiophilic, nonmembrane-bound structures that appear in the cytoplasm of a wide variety of cells, but we have found that these lipid bodies occur with increased frequency in granulocytes, macrophages, and mast cells at sites of inflammatory, immunologic, or neoplastic processes. Macrophages also incorporated 3H-AA predominantly into cytoplasmic lipid bodies. In contrast to mast cells, however, macrophages incorporated 3H-AA into the plasma membrane as well. Stimulation of macrophage phagocytosis resulted in striking alterations of the relationships of lipid bodies to intracellular membranes, so that many lipid bodies appeared adjacent to phagolysosomes. In addition, some phagolysosomes contained 3H label, which along with other morphologic evidence suggested that lipid bodies may discharge their contents into these structures. Mast cell and macrophage cytoplasmic lipid bodies appear to represent a major site of intracellular storage and metabolism of products of AA and perhaps other fatty acids taken up from the external milieu. These heretofore neglected organelles may thus influence cellular function in a wide variety of adaptive or pathologic processes.

Enzymatically dispersed human lung parenchymal cells were fractionated according to size by countercurrent centrifugation elutriation. Human lung mast cells eluted throughout the procedure indicating heterogeneity of mast cell diameters. In seven individual lung elutriations, the mean histamine content ranged from 2.5 +/- 0.5 pg/mast cell for the smallest diameter mast cells (8-10 microns) to 10 +/- 2.5 pg/mast cell for the largest (16-20 microns). Intermediate sized mast cells had correspondingly intermediate histamine contents. The maximum release of histamine after anti-IgE stimulation varied with mast cell size. Small mast cells consistently released less histamine (10 +/- 3.6% net) than the largest diameter mast cells (38 +/- 6% net). This differential histamine release could not be explained by cell surface IgE content which was similar in mast cells of all sizes. The concentration of anti-IgE for maximum histamine release was the same (2 micrograms/ml) for mast cells of all sizes. The generation of PGD2, the predominant cyclooxygenase metabolite of the human lung mast cell, also was correlated positively with mast cell size and to the quantity of histamine released. Studies to date indicate no clear pattern in agonist receptor activities as judged by the inhibition of histamine release by PgE2, the beta-adrenergic agonist, fenoterol, and adenosine. We conclude that human lung mast cells are heterogeneous with regard to size and function.

Detailed studies of the biochemistry and pharmacology of mast cell-mediated inflammatory disorders have been hampered by the inability to purify human mast cells. We now report techniques to purify human lung mast cells to apparent homogeneity. The major purification steps are: 1) dispersion of lung fragments into a single-cell suspension with enzyme combinations (pronase-chymopapain, collagenase-elastase); 2) partial purification by countercurrent centrifugation elutriation (CCE); and 3) affinity column chromatography. Enzymatic dispersion yielded suspensions with congruent to 10(6) mast cells per gram of lung parenchyma in purities of 1.2 to 9.7%. Dispersed mast cells responded comparably to those in parent lung fragments to challenge with anti-human IgG and pharmacologic agonists. Elutriation of lung cell suspensions yielded mast cell-enriched fractions with purities up to 70%. High purity mast cell fractions were combined, passively sensitized with purified human penicillin (BPO)-specific IgE, and purified by a BPO-affinity column chromatography procedure. Post elutriation mast cell purities of 29 +/- 3.5% were increased to 84 +/- 3% (range 65 to 98%) by the affinity column. Short-term (24 hr) culture of column-purified mast cells allowed adherence of non-mast cell contaminants to tissue culture plates, further increasing purity (up to 100%). Purified mast cells were intact and functional as assessed by dye exclusion, survival in short-term culture, IgE-mediated histamine release, and modulation of release by the pharmacologic agonists adenosine, IBMX, prostaglandin E2, and fenoterol.

Human basophils release approximately 90 pmol of LTC4/micrograms histamine when challenged with anti-IgE antibody, but donor to donor variation produces a 1000-fold range of response. There is little conversion to LTC4 to LTE4 in purified preparations of basophils, but conversion to LTE4 does occur if cell densities are high during incubation. Like histamine release, leukotriene release is calcium and temperature dependent and is complete in 20 min, with a t1/2 of approximately 8 min. The process of desensitization also ablates leukotriene release, but there is a distinct two phase process where leukotriene release is enhanced after 5 min of desensitization, whereas histamine release is inhibited and total ablation of leukotriene release occurs only after 45 min of desensitization. Human basophils respond well to stimulation with covalently cross-linked trimeric IgE myeloma but respond poorly to dimeric IgE. This differential sensitivity to the two forms of cross-linked IgE is most exaggerated in the context of leukotriene release, where dimer is 30-fold less efficacious and 100- to 1000-fold less potent than trimer on some donors' basophils. This dichotomy of response is also observed in antigen-challenged cells, where the bivalent hapten, BPO2, also poorly induces leukotriene release in accord with the fact that it predominantly induces dimeric cross-links of penicillin-specific IgE. Anti-IgE dose-response curves reveal a region of dimeric cross-link dominance that may explain the peculiar differences observed in pharmacologic studies of basophil release induced with antigen vs anti-IgE. In addition, there is a continuum of "releasability," where some donors' basophils display no response (histamine or leukotriene release) to dimeric IgE, and others' basophils are essentially equally responsive to both dimeric and trimeric IgE. This releasability difference manifests itself by conferring increased sensitivity to antigenic challenge in those donors' basophils capable of responding to dimeric cross-links such that these donors' basophils are capable of releasing histamine upon antigen challenge while possessing only 50 molecules of cell surface antigen-specific IgE; other dimer-insensitive donors' basophils require 6 to 10-fold greater IgE densities for equal histamine release.

The molecular basis for the accumulation of a substance which displays the immunological reactivity of alpha-1-antitrypsin within vesicles of liver parenchymal cells of individuals with hepatic cirrhosis and serum alpha-1-antitrypsin deficiency remains unclear. We recently reported that serum from a patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis was substantially deficient in sialyltransferease (EC 2.4.99.1) an enzyme which transfers sialic acid from cytidine 5'-monophosphate-N-acetylneuraminic acid to a variety of asialoglycoprotein acceptors. In the present report we have extended these studies to include serum from five additional patients with alpha-1-antitrypsin deficiency and juvenile hepatic cirrhosis as well as a liver specimen obtained at autopsy of one of these patients. We find the sialytransferase activity in serum from six patients with alpha-1-antitrypsin deficiency and hepatic cirrhosis to be 50% of healthy pediatric control values and 30% of pediatric patients with liver disease. However, serum from family members homozygous for alpha-1-antitrypsin deficiency but without hepatic cirrhosis, and serum from patients with a variety of other kinds of liver disease, failed to exhibit the marked sialytransferase deficiency. Similar assays carried out on a homogenate of a liver sample from one patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis indicated that the deficiency of sialyltransferase activity was not demonstrable in liver. Furthermore, a comparative kinetic analysis of serum and liver sialytransferase in normal and afflicted individuals failed to detect differences in substrate affinities which might account for a decrease in functional sialyltransferase capacity in individuals with alpha-1-antitrypsin deficiency and hepatic cirrhosis. These observations suggest that the serum sialyltransferase deficiency in such patients probably arises after chronic and extensive liver disease involving hepatic accumulation of alpha-1-antitrypsin rather than the enzyme deficiency being the primary cause of the hepatic cirrhosis and alpha-1-antitrypsin deficiency.

Atopic disease in humans results primarily from the activity of tissue mast cells and circulating basophils. These two very similar cell types have recently been purified to near homogeneity and studies have begun to identify the biochemical mechanisms of mediator release, to explore the effects of pharmacologic manipulation of the response, and to determine unambiguously which mediators are derived from mast cells and basophils. This report will review studies examining various parameters of histamine release, the role of cyclic AMP in histamine release, and the production of arachidonic acid metabolites and their pharmacologic modulation.

Prostaglandin (PG) D2 is the major cyclooxygenase metabolite of arachidonic acid released after immunologic stimulation of mast cells. In this report, we demonstrate that this PG is unlike other PGs previously investigated in that it enhances human basophil histamine release at concentrations of 1 to 100 nM. Enhancement is seen when antigen, the phorbol diester, 12-O-tetradecanoylphorbol-13-acetate and ionophore A23187 are used to initiate release and, in preparations of basophils, purified to 51 to 66% of total cell number as well as in preparations of mixed leukocytes. This enhancement is a late or "second stage" phenomenon as defined by the two stages of histamine release and is additive with the enhancement produced by D2O, but not with the enhancement produced by indomethacin or 5-hydroperoxyeicosatetraenoic acid. PGD2 also reverses the inhibition of release produced by drugs and hormones such as dimaprit and PGE2 that activate adenylate cyclase to increase cellular cyclic AMP levels. These data suggest that PGD2 may play an important role in allergic and immunologic reactions and suggest a mechanism by which mast cells and basophils can interact during these reactions.

Although the mechanism underlying hypoxic pulmonary vasoconstriction remains undefined, various reports have suggested that mast cells and cell-derived mediators may be important in the production of this phenomenon. We investigated the effect of reducing oxygen tension on the release from human lung fragments of the mast cell-derived mediators histamine, prostaglandin (PG) D2 and peptide leukotrienes, as well as the release of the largely non-mast cell-derived mediators PGE2, PGF2 alpha, prostacyclin metabolite (6-keto-PGF1 alpha) and the thromboxane A2 metabolite (thromboxane B2). The effect of reducing oxygen tension on both basal mediator release and release triggered by goat antihuman immunoglobulin E was studied. Reducing pO2 of buffer in which lung fragments were placed from 161 to 54 mm Hg resulted in no spontaneous release of histamine, PGD2 or peptide leukotrienes. However, hypoxia had a marked effect on mediator release triggered by goat antihuman immunoglobulin E. Although net histamine release was relatively unaffected (control 13.9 +/- 2.7%, hypoxic 12.7 +/- 2.1%), hypoxic treatment resulted in an 89% inhibition of PGD2 release (control 47.7 +/- 17.4 ng/g of lung, hypoxic 5.26 +/- 1.91 ng/g of lung) and an 81% inhibition of peptide leukotriene release (control 22.5 +/- 7.6 ng/g of lung, hypoxic 4.37 +/- 2.4 ng/g of lung). Similar inhibition was seen for non-mast cell-derived mediators, including PGF2 alpha, prostacyclin metabolite and thromboxane B2, and probably for PGE2. We conclude that hypoxic treatment of human lung fragments in vitro results in no spontaneous release of preformed or newly formed mediators but that it markedly alters mediator release after goat antihuman immunoglobulin E triggering.(ABSTRACT TRUNCATED AT 250 WORDS)

Gaucher's disease is a lysosomal storage disease caused by a deficiency in the enzyme glucocerebrosidase. A small, heat-stable glycoprotein first obtained from Gaucher spleen (Ho, M. W., and O'Brien, J. S. (1971) Proc. Natl. Acad. Sci. U. S.A. 68, 2810-2813) has been observed to stimulate the activity of glucocerebrosidase isolated from normal tissue. It has been suggested that this material might be important in the physiological catabolism of glucocerebroside in normal individuals (Ho, M. W. (1974) in Enzyme Therapy in Lysosomal Storage Diseases (Tager, J. M., Hooghwinkel, G. J. M., and Daems, W. Th., eds) pp.239-246, North-Holland Publishing Co., Amsterdam). In order to investigate this suggestion, glucocerebrosidase activating factors were isolated and purified from control and Gaucher spleen and characterized. Although approximately the same mass of activator was isolated from both spleens, the two activators differ from one another in a number of important respects: (a) the activator from the control spleen is only 6 per cent as active (on a protein basis) as the activator from Gaucher spleen; (b) the amino acid compositions of the purified activators are significantly different; and (c) carbohydrate analysis of the purified activators indicates that the activator from Gaucher spleen is a glycoprotein, while that from control spleen is not. Comparative kinetic studies demonstrate that the anionic detergent, sodium taurocholate, and the acidic phospholipid, phosphatidylinositol, both stimulate glucocerebrosidase activity to a larger extent than the activator substance from Gaucher spleen. The activator from Gaucher spleen and human liver glucocerebrosidase both appear to contain significant hydrophobic character. We conclude that the activator is probably not physiologically important in the catabolism of glucocerebroside in normal tissues. The significance of the occurrence of this apparently unique glycoprotein activator in Gaucher spleen remains obscure; however, its presence represents another interesting aspect of Gaucher's disease that warrants further investigation.

In order to assess whether sulfidopeptide leukotrienes are generated following nonimmunologic stimulation of inflammatory cells in vivo, 11 subjects complaining of symptoms of rhinitis when exposed to cold and dry environments were challenged by nasal breathing, first with warm, moist air and then with cold, dry air. Nasal lavages with normal saline were performed before and after each exposure. Immunoreactive leukotriene in the lavage fluids was significantly increased following cold, dry air exposure (2.6 ng/ml) compared with that after warm, moist air exposure (0.7 ng/ml) or at baseline (0.4 ng/ml) (p less than 0.01 in both instances). Six more subjects, denying cold-air sensitivity, were subjected to the same protocol and had no mediator and symptom score changes after cold, dry air challenge. Leukotriene changes after cold, dry air were highly concordant with increments in histamine, prostaglandin D2,N-alpha-tosyl-L-arginine methyl ester (TAME) esterase(s) activity and symptom scores (p less than 0.001). Separation of leukotrienes by high performance liquid chromatography in the nasal washes of 3 subjects showed variable amounts of LTC4, D4, and E4, suggesting metabolism of the former to the latter 2. To our knowledge, this is the first in vivo demonstration of leukotriene production in response to a physical stimulus, and it suggests a possible role of these and other inflammatory mediators in pathologic conditions, such as exercise-induced asthma, that involve physical causative factors.

Glucocorticoids exert their actions through a time-dependent, receptor-mediated, protein synthesis- and RNA synthesis-dependent mechanism. We have assessed the effects of 24-h culture of human neutrophils with dexamethasone on degranulation, chemotaxis, binding to vascular endothelium and formation of leukotriene B4. Purified neutrophils contained an average of 2896 [3H]dexamethasone binding sites per cell with a Kd of 4.1 X 10(-9) M for [3H]dexamethasone binding. Cells exposed to dexamethasone (10(-6) M) released equal or greater quantities of the lysosomal enzymes, lysozyme and beta-glucuronidase in response to formylmethionyl-leucyl-phenylalanine, serum activated zymosan, and the tumor promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate compared to controls. Culture with dexamethasone also did not inhibit neutrophil chemotaxis in response to a range of concentrations of formylmethionyl-leucyl-phenylalanine, or did it inhibit binding of neutrophils to cultured endothelial cells stimulated by either leukocyte activators (formylmethionyl-leucyl-phenylalanine and platelet-activating factor) or endothelial activators (interleukin-1, lipopolysaccharide or 12-O-tetradecanoylphorbol-13-acetate). Spontaneous adherence of neutrophils to endothelial cells was inhibited (82.9 +/- 6.8% of control, P less than .025, n = 18). Neither in vitro or in vivo glucocorticoids inhibited neutrophil leukotriene B4 formation induced by either the calcium ionophore A23187 or serum activated zymosan. We conclude that human neutrophils are not functionally inactivated by glucocorticoids and suggest that the mechanism by which glucocorticoids inhibit neutrophil accumulation at inflammatory sites may be by inhibition of the production of chemoattractants and endothelial activators rather than inhibition of their actions.

Acid phosphatase (EC 3.1.3.2) isoenzyme profiles of extracts of splenic tissue and serum from patients with Gaucher's disease were measured by a mini-column ion-exchange chromatographic method [Clin. Chem., 23, 000 (1977)]. Diagnosis of Gaucher's disease in the five patients studied was confirmed by demonstrating decreased (2.3 to 4.1% of normal) glucocerebrosidase activity in the spleen. With p-nitrophenyl phosphate as substrate, increased acid phosphatase activity (three-to eight-fold normal) was demonstrated in spleen tissue from Gaucher;s disease patients; isoenzyme profiles by the ion-exchange column technique showed acid phosphatase isoenzyme 5 to be the predominant isoenzyme. Comparison of acid phosphatase isoenzyme profiles from patients with Gaucher's disease and prostatic carcinoma revealed distinct differences in the activities of isoenzymes 2 and 5. The isoenzyme-5 measurement thus appears to provide a diagnostic test for Gaucher's disease that can be done reapidly and easily in the routine clinical chemistry laboratory.

The release of inflammatory mediators (histamine, PGD2, TxB2, and LTC4) from purified human lung mast cells was characterized by kinetic and anti-IgE dose-response parameters. The relative rate of mediator release was histamine greater than PGD2 = TxB2 greater than LTC4, with one half maximal release occurring at approximately 2, 5, and 10 min, respectively. In 2 experiments, stimulation with anti-IgE caused significant quantities of platelet-activating factor (PAF) to appear rapidly (2 min) in the cell pellet; cell-associated PAF declined to low levels by 45 min. The optimal concentration of anti-IgE for the release of the arachidonate cyclooxygenase metabolites PGD2 and TxB2 (0.3 microgram/ml) was 10- to 30-fold less than that required for the release of histamine and LTC4 (3 to 10 micrograms/ml), suggesting that these release processes may have differential IgE Fc receptor cross-linking requirements. At optimal histamine release, the magnitude of the release of each arachidonate metabolite was found to correspond to the magnitude of histamine release, however, suggesting that the 2 processes are linked either in series or in parallel.

The present investigation was designed to study the histamine release and pharmacologic characteristics of dispersed human lung mast cells, particularly in comparison with parenchymal tissue fragments. Dispersed human lung mast cells were prepared by enzymatic treatment (yield, 0.5 to 2 x 10(6) mast cells/g tissue). Purity was 1 to 8% (mean, 3.6% +/- 0.7%), and histamine content varied from 2 to 6 pg/cell (mean, 3.6 +/- 0.5 pg/cell). Release, studied using anti-IgE as the stimulus, was relatively rapid, being essentially complete within 15 min when high concentrations of anti-IgE (greater than or equal to 0.3 microgram/ml) were used and was not enhanced by phosphatidyl serine. The concentration of drug required to inhibit histamine release by 50% in dispersed cells for a series of pharmacologic agents, including the beta-adrenergic agent fenoterol, the prostaglandin E2, and the phosphodiesterase inhibitor isobutylmethylxanthine, were 0.1 to 1 microM, 50 microM, and 0.5 mM, respectively; similar results were obtained in simultaneous experiments performed using tissue fragments. Adenosine enhanced release (19 +/- 3.4%) at low concentrations (10 microM) and inhibited release (61 +/- 5.1%) at high concentrations (1mM). The H2 agonist, dimaprit (at 10(-5) to 10(-7) M) and prostaglandin D2 (at 10(-4) to 10(-6) M) had no effect on histamine release, whereas deuterium oxide potentiated histamine release. This study serves to quantitate the pharmacologic effects of several agents on anti-IgE-mediated histamine release from dispersed human lung mast cells and has further suggested that the dispersed cell system is similar to the standard chopped lung system in dose-response relationships, kinetics, and pharmacologic modulation. It also indicates that the enzymatic treatment of the cells does not affect the release characteristics or functional capacity of several different receptors, and that this preparation, therefore, appears suitable as an in vitro human model of mediator release that can be used for the evaluation of pharmacologic agents and for further mast cell purification.

Arachidonic acid metabolism has been explored in preparations of purified human lung mast cells prelabeled with arachidonic acid (AA). Cells were of 83 to greater than 96% purity, and each experiment was performed with four to six different preparations of mast cells. After overnight culture of the purified cells in the presence of 3H-AA, followed by extensive washing in buffer, mast cell uptake of labeled AA was 61.4 +/- 14.8 pmol/10(6) cells with 21 +/- 2.4% of the label in phospholipids, 73 +/- 2.1% in neutral lipids, and 3.6 +/- 0.8% as free AA. Analysis of the distribution of radioactivity in phospholipid classes revealed 51.4 +/- 5.5% of the label in phosphatidylcholine, 14.5 +/- 1.6% in phosphatidylinositol, 12.0 +/- 3.0% in phosphatidylethanolamine, and 9.1 +/- 2.4% in sphingomyelin, with the rest in other phospholipid classes. Challenge of these cells with an optimal concentration of anti-IgE led to the release of 20 +/- 4.0% of cellular histamine and to a reduction in labeled phosphatidylcholine and phosphatidylinositol to 75.5 +/- 8.8% and 84.2 +/- 4.5% of the control levels, respectively, (p less than 0.05); anti-IgE challenge produced no statistically significant change in the quantities of other labeled phospholipids. Activation of human lung mast cells with anti-IgE led to the release of 3.4 +/- 1.3% of the cellular 3H as AA and AA metabolites (1.5 +/- 0.6% as unmetabolized AA) in conjunction with 16 +/- 4.3% of the cellular histamine. Although activation of human lung mast cells with ionophore A23187 caused 70 +/- 1.1% histamine release, a similar quantity of AA and AA metabolites was released (a total of 4.0 +/- 0.8% with 2.3 +/- 1.5% as unmetabolized AA). Analysis of the released metabolites by liquid scintillation spectrometry after high performance liquid chromatography separation showed that approximately equal amounts of metabolites were produced after mast cell activation with anti-IgE and ionophore A23187. In this series of experiments approximately equal amounts of cyclooxygenase and lipoxygenase products were generated.(ABSTRACT TRUNCATED AT 400 WORDS)

Mediators released from human basophils and mast cells in response to immunologic and other stimuli are felt to be important in the pathophysiology of several nasal and pulmonary disease processes. Recently, we have developed techniques to purify these cells, thus allowing precise ultrastructural, biochemical and pharmacological studies of mediator release. Previous literature has emphasized the similarities of the two cell types including their metachromatic staining and IgE-mediated release of mediators. However, we now appreciate that several differences exist between the two cell types. At the ultrastructural level, basophil release is characterized by individual granules emptying to the cell exterior, while mast cell granules fuse intracellularly and release their contents through cytoplasmic channels. Functionally, basophils are 10- to 30-fold more sensitive than mast cells to anti-IgE, but the kinetics of release are less rapid. Basophil, but not mast cell, release is (I) inhibited by histamine H2 agonists and glucocorticoids; (II) enhanced by PgD2 and (III) modulated by arachidonic acid lipoxygenase metabolites. Significant quantities of slow-reacting substance of anaphylaxis, PgD2 and platelet-activating factor are generated by mast cells. Basophils produce little or none of these mediators. Studies with these purified, relevant human cell types should provide important insights into the cellular basis of hypersensitivity states and their control.