Immunofluorescence localization of calmodulin in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNs) were purified (approximately equal to 99%) from peripheral blood of normal, adult volunteers. The indirect immunofluorescence technique was used to investigate the presence and the localization of calmodulin in human PMNs. The cellular distribution of calmodulin has been evaluated using an affinity chromatography-purified sheep IgG anti-calmodulin and fluorescein-conjugated rabbit anti-sheep IgG. The anti-calmodulin immunofluorescence pattern suggests that calmodulin is evenly distributed throughout the cytoplasm of human PMNs.

Studies of the intracellular Ca2+ levels in human adult skin mast cells activated by the ligand for the human c-kit receptor and anti-IgE.

The human c-kit receptor ligand, rhSCF, is the only cytokine known to be active on human mast cells, but its intracellular signal transduction pathway is still unknown. We compared the effect of rhSCF on intracellular Ca2+ levels in purified (> 70% pure) adult skin mast cells with two other immunologic stimuli, namely, anti-IgE and substance P. Both rhSCF (1 microgram/mL) and anti-IgE (3 micrograms/mL) induced a rapid (< 20 sec) and sustained (T1/2 for decay > 10 min) increase in free cytosolic Ca2+ concentration. In contrast, substance P (5 microM) elicited a very rapid (< 1 sec) and transient (T1/2 for decay congruent to 5 sec) rise in intracellular Ca2+ levels. Intracellular cAMP levels were then increased by pharmacologic means to examine the role of the cyclic nucleotide in controlling the Ca2+ response in skin mast cells. A combination of the general phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (200 microM) and the adenylate cyclase activator, forskolin (30 microM) was effective in inhibiting the Ca2+ response induced by rhSCF or anti-IgE (82 and 68% inhibition, respectively), while IBMX and forskolin alone were much less effective. The phosphodiesterase isozyme IV inhibitor, rolipram (10 microM), variably affected the increase in Ca2+ levels induced by anti-IgE, but it exerted a significant inhibitory activity on anti-IgE- or rhSCF-induced response in the presence of forskolin (30 micrograms/mL) (33 and 67%, respectively). Two different protein kinase C (PKC) activators TPA (200 nM) and bryostatin 1 (200 nM) similarly inhibited rhSCF- (22 and 32%, respectively) and anti-IgE-induced (24 and 32%) Ca2+ response. Finally, the kinase inhibitor genistein (30 micrograms/mL) was a somewhat more effective inhibitor of the rise in intracellular Ca2+ induced by rhSCF (100%) than that activated by anti-IgE (54%) (P < 0.05). These data indicate that rhSCF and anti-IgE may act on human mast cells through a common pathway to increase free cytosolic Ca2+ levels and this effect is similarly modulated by various drugs.

Inhibition of IgE-mediated release of histamine and peptide leukotriene from human basophils and mast cells by forskolin.

Forskolin, a diterpene compound isolated from the roots of Coleus forskohlii, activates adenylate cyclase in membranes from a variety of mammalian tissues. We found that forskolin (10(-7) to 3 X 10(-5) M) caused a concentration-related inhibition of IgE-mediated release of histamine and peptide leukotriene C4 (LTC4) from human basophils and lung mast cells. There was a significant linear correlation between the per cent inhibition of histamine and LTC4 release from both cell types. However, in both systems forskolin exerted a significantly greater inhibitory effect on LTC4 release than on histamine release. The concentration-response inhibition curve was paralleled by a forskolin-induced rise in cAMP levels in human leukocyte and mast cell preparations. The relationship between the effect of forskolin and the cAMP concentration was supported by the finding that forskolin inhibited the "first stage" of antigen-induced histamine release, but not the release caused by the Ca2+ ionophore, A23187. Propranolol, a competitive beta-receptor antagonist, did not block the inhibition of mediator release or the cAMP accumulation caused by forskolin. These data suggest that forskolin modulates the release of mediators of immediate hypersensitivity reactions via the activation of adenylate cyclase in human basophils and mast cells.

Modulation of mediator release from human basophils and pulmonary mast cells and macrophages by auranofin.

Auranofin, a new orally absorbable gold compound, inhibits IgE-(anti-IgE) and non-IgE-mediated (f-met-peptide and the Ca2+ ionophore A23187) histamine release from human basophils. Auranofin inhibits the release of histamine induced by phorbol myristate (TPA) and bryostatin 1 both in the presence and absence of extracellular Ca2+. Increasing the Ca2+ concentrations in the extracellular medium does not reduce the inhibitory effect of auranofin on anti-IgE- or A23187-induced secretion. Auranofin inhibits the de novo synthesis of sulfidopeptide leukotriene C4 (LTC4) induced by anti-IgE from basophils and mast cells purified from human lung. However, in both systems auranofin has a significantly greater inhibitory effect on LTC4 release than on histamine secretion. Finally, auranofin induces a concentration-dependent inhibition of A23187-induced leukotrine B4 (LTB4) release from purified human lung macrophages. These data suggest that auranofin modulates the release of preformed (histamine) and de novo synthesized (LTC4 and LTB4) chemical mediators from human inflammatory cells isolated from peripheral blood and human lung tissues.

Possible role of calmodulin in the control of histamine release from human basophil leukocytes.

We investigated the possible role of calmodulin (CaM) in the control of histamine release from human basophil leukocytes using several CaM antagonists. Trifluoperazine (TFP) (10(-6)-2 X 10(-5) M), pimozide (10(-6)-1.5 X 10(-5) M), chlorpromazine (CPZ) (10(-5)-10(-4) M) and promethazine (PMZ) (2 X 10(-5)-10(-4) M) inhibited in vitro histamine secretion from human basophils induced by several immunological (antigen, anti-IgE, and formyl-L-methionyl-L-leucyl-L-phenylalanine: f-met peptide) and nonimmunological (Ca2+ ionophore A23187 and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate: TPA) stimuli. Trifluoperazine sulfoxide (TFP-S) and chlorpromazine sulfoxide (CPZ-S), which have very low affinity to CaM, had practically no inhibitory effect on histamine release from human basophils. The inhibitory effect of TFP could be made irreversible by irradiating the cells with UV light. A sulfonamide derivative, the compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (2.5 X 10(-5)-2 X 10(-4) M), which selectively binds to CaM, inhibited the release of histamine from basophils. In contrast, the chloride deficient analogue, W-5, which interacts only weakly with CaM, had practically no inhibiting effect. The IC50 for enzyme release by a series of eight CaM antagonists was closely correlated (r = 0.91; p less than 0.001) with the CaM specific binding, supporting the concept that these agents act by binding to CaM and thereby inhibiting histamine release. TFP and W-7 inhibited histamine release in the absence and in the presence of increasing concentrations of extracellular Ca2+. These results emphasize the possible role of CaM in the control of histamine secretion from human basophils.

The releasability of lysosomal enzymes from neutrophil leukocytes in patients with rheumatoid arthritis.

OBJECTIVE: To study the enzyme content and the "releasability" of lysosomal enzymes (lysozyme and beta-glucuronidase) in neutrophils purified from peripheral blood of patients with rheumatoid arthritis (RA) or normal subjects. METHODS: Neutrophils were obtained from 13 patients (10 women and 3 men) with rheumatoid arthritis and from 11 healthy subjects (8 women and 3 men). We measured: (1) lysosomal enzyme (lysozyme and beta-glucuronidase) content; (2) spontaneous enzyme release; (3) lysosomal enzyme release after cell challenge with different segretagogues (FMLP, C5a, aggregated IgG, zymosan and Ca2+ ionophore A23187). RESULTS: The lysosomal enzyme content was not statistically different in control subjects and in patients with RA (7.4 +/- 1.9 vs 6.3 +/- 0.8 micrograms/10(6) neutrophils for lysozyme; 102.9 +/- 16.4 vs 78.9 +/- 11.2 micrograms/10(6) neutrophils for beta-glucuronidase in control and RA subjects, respectively, p = NS). Unstimulated release of lysozyme was significantly lower in RA patients (3.8 +/- 1.1%) when compared to control subjects (9.5 +/- 2.1%) (p < 0.05). In contrast, spontaneous release of beta-glucuronidase did not differ in the two groups (5.5 +/- 0.9% and 3.8 +/- 1.1% in control and RA subjects, respectively). Enzyme release induced by FMLP (3 x 10(-9)-3 x 10(-7) M), C5a (10(-8)-10(-7) M), aggregated IgG (0.1-0.6 mg/ml), or Ca2+ ionophore A23187 (0.1-1 microgram/ml) did not differ statistically in the two groups of subjects. Neutrophil stimulation by serum-treated zymosan, at the concentration of 0.3 mg/ml, induced a release of lysozyme that was significantly higher in patients with RA when compared to control subjects (p < 0.05), whereas zymosan-activated beta-glucuronidase secretion was similar in the two donor populations. CONCLUSIONS: This study suggests that the contribution of leukocytes to the inflammatory processes typical of RA does not depend on an altered "releasability" of preformed mediators from peripheral blood neutrophils.

Inhibition of histamine release from human basophils in vitro by calmodulin antagonists.

Calmodulin is a ubiquitous and versatile Ca2+-binding protein that plays a pivoting role in cellular metabolism. We have investigated the possibility that calmodulin plays a role in immediate hypersensitivity reactions by evaluating the effects of two agents, trifluoperazine dihydrochloride (TFP) and the sulfonamide derivative N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) which selectively bind to calmodulin. TFP and W-7 cause a dose-dependent inhibition of histamine secretion from human basophils in vitro induced by several immunological (i.e., antigen and anti-IgE) and nonimmunological (i.e., formyl-methionine-containing peptide and the Ca2+ ionophore A23187) stimuli. These results indicating that two specific calmodulin antagonists are potent inhibitors of the secretory response of human basophils support the hypothesis that calmodulin may play a role in the control of the release of preformed mediators from human inflammatory cells.

Human skin mast cells express functional beta 1 integrins that mediate adhesion to extracellular matrix proteins.

We have evaluated the adhesion of human cutaneous mast cells to several components of the extracellular matrix (plasma fibronectin, laminin, collagen type I and IV) and studied whether these cells express the beta 1 integrins potentially involved in the adhesion to these proteins. Human skin mast cells (5.1 +/- 1.5% pure) spontaneously adhered to fibronectin and laminin (0.1 to 10 micrograms/ml) immobilized on plastic surfaces (e.g., 14 +/- 7.2% and 14 +/- 4.4% adhesion at 10 micrograms/ml, respectively). Similar results were obtained with a 90% pure mast cell preparation. In contrast, cutaneous mast cells did not adhere to collagen type I (1.6 +/- 0.5% adhesion) or type IV (1.2 +/- 0.8% adhesion). Control adhesion in BSA-coated wells was < 5%. Mast cell adhesion to fibronectin was optimal after an incubation period of 60 to 90 min (t1/2 = 28.2 +/- 6.2 min), whereas adhesion to laminin was faster (t1/2 = 10.1 +/- 1.2 min), being nearly optimal after a 15-min incubation period. Human skin mast cell adhesion to fibronectin and laminin was found to be dependent on the presence of divalent cations in the extracellular medium. Dual-color immunofluorescence and flow cytometry were used to evaluate whether human skin mast cells (51.3 +/- 3.9% pure) express beta 1 integrins that may mediate cell adhesion to extracellular matrix proteins. These mast cells were found to express VLA (very late Ag)-3 (75.3 +/- 35.6 specific fluorescence intensity) and, to lesser degree, VLA-4 and VLA-5 receptors (8.0 +/- 2.5 and 6.9 +/- 3.2 specific fluorescence intensity, respectively). In contrast, VLA-1, VLA-2, and VLA-6 integrins were not expressed significantly. mAb to VLA-3, VLA-4, and VLA-5 each inhibited by 70% skin mast cell adhesion to fibronectin. mAb to VLA-3 nearly abolished mast cells adhesion to laminin, whereas anti-VLA-4 and anti-VLA-5 were ineffective. Finally, immunosuppressant cyclosporin A (100 nM) and FK-506 (10 nM) significantly inhibited mast cell adhesion to both fibronectin and laminin (p < 0.05). Our data demonstrate that human skin mast cells spontaneously adhere to fibronectin and laminin, and that this adhesion is mediated by VLA-3, VLA-4, and/or VLA-5 integrins on these cells. Interactions between these beta 1 integrins and extracellular matrix proteins may be involved in perivascular tissue localization of human mast cells in vivo.

Activation of human basophils by bacterial products.

Staphylococcus aureus Cowan I (Cowan Staph A+), which synthesizes protein A (Staph A), induced histamine release from human basophils. In contrast, S. aureus Wood 46 (Wood Staph A-), which does not synthetize Staph A, did not induce histamine secretion. Soluble Staph A and Cowan Staph A+ induced histamine release by interacting with the alternative F(ab')2-binding site of IgE and/or IgG present on human basophils. Pepstatin A, a pentapeptide synthetized by various actinomycetes, induced histamine secretion by activating a specific membrane receptor, which is also activated by synthetic formylmethionine-containing peptides.

The mechanism of basophil histamine release induced by pepstatin A.

Pepstatin A, a natural pentapeptide isolated from cultures of actinomycetes, induced histamine secretion from human basophils in the concentration range of 3 X 10(-7) to 10(-4) M. The characteristics of this reaction were similar to those of f-met-peptide-induced histamine release: pepstatin A-induced release required Ca2+, and the release reaction was complete within 2 min at 22 or 37 degrees C but did not occur at 4 degrees C. There was excellent correlation (r = 0.93; p less than 0.001) between the maximal histamine release induced by pepstatin A and f-met-peptide, but there was no relationship to the capacity of basophils to release with anti-IgE (r = -0.03) or the Ca2+ ionophore A23187 (r = -0.22). Release by pepstatin A was reversibly inhibited by two nonreleasing analogs of f-met-peptide, CBZ-Phe-Met and BOC-Met-Leu-Phe. BOC-Met-Leu-Phe competitively inhibited the effect of both f-met-peptide and pepstatin A on histamine release from basophils. The dissociation constant (Kd) for the BOC-Met-Leu-Phe-receptor complex in both conditions was approximately 10(-6) M. Furthermore, there was complete cross-desensitization between pepstatin A and f-met-peptide, whereas cells desensitized to pepstatin A released normally with anti-IgE and vice versa. A variety of pharmacologic agents had similar effects on both pepstatin A- and f-met-peptide-induced release (e.g., slight inhibition with cyclic AMP-active agents, no enhancement with D2O, and marked enhancement with cytochalasin B). We suggest that the natural pentapeptide pepstatin A induces histamine release from human basophils by activating a cell surface receptor(s) also activated by the synthetic tripeptide f-met-peptide.

Histamine release from human basophils by pepstatin A.

Pepstatin A, a pentapeptide isolated from cultures of actinomycetes, induced histamine secretion from human basophils in the concentration range of 3 X 10(-7) to 10(-4) M. The characteristics of this reaction were similar to those of f-met-peptide-induced histamine release: pepstatin A-induced release required Ca2+ and the release reaction was complete within 2 min at 22 or 37 degrees C, but did not occur at 4 degrees C. Release by both pepstatin A and f-met-peptide was reversibly inhibited by two non-releasing analogs of f-met-peptide, CBZ-Phe-Met and BOC-Met-Leu-Phe. Further, there was complete cross-desensitization between pepstatin A and f-met-peptide, while cells desensitized to pepstatin A released normally with anti-IgE and vice versa. A variety of pharmacological agents had similar effects on both pepstatin A and f-met-peptide-induced release (e.g., no enhancement with D2O; marked enhancement with cytochalasin B). We suggest that pepstatin A induces histamine release from human basophils by activating a cell surface receptor(s), also activated by the synthetic tripeptide f-met-peptide.

Studies of cell adhesion and flow cytometric analyses of degranulation, surface phenotype, and viability using human eosinophils, basophils, and mast cells.

Products derived from eosinophils, basophils, and mast cells are considered critical to the development of allergic diseases. Studies of the selective recruitment, accumulation, and/or activation of these cells during human allergic inflammatory reactions in vitro and in vivo have been facilitated by a wide variety of methods. Some have been developed to identify and isolate these cells from a variety of sites, including blood, airway secretions, and surgical or autopsy tissues. Once enriched in purity, assays of cell adhesion to endothelium, epithelium, matrix proteins, and purified, immobilized counterligands for integrins, selectins, or immunoglobulin gene superfamily structures can be performed in vitro under both static and flow conditions. Techniques involving flow cytometry, utilizing characteristics of cellular light scatter and immunofluorescence, have permitted the elucidation of cell surface phenotype and have aided in quantification of cellular degranulation and viability. These approaches have yielded new information on the function of human eosinophils, basophils, and mast cells and have suggested unique cell-specific pathways of cell recruitment, activation, and survival that may contribute to the pathogenesis of allergic diseases.

Modulation of the release of histamine and arachidonic acid metabolites from human basophils and mast cells by auranofin.

In these experiments the effects of pharmacological concentrations of auranofin, a new absorbable gold compound, were assessed on the release of histamine and peptide leukotriene C4 (LTC4) from human basophils and lung mast cells. Auranofin, at pharmacological concentrations, inhibited in vitro histamine and LTC4 release from human basophils induced by anti-IgE. Inhibition began at about 3 X 10(-7) M and was maximum at 10(-5) M. We also evaluated the effect of auranofin on the release of histamine and LTC4 induced by anti-IgE from mast cells purified from human lung. Auranofin (3 X 10(-7) to 10(-5) M) dose-dependently inhibited the release of histamine and LTC4 from human lung mast cells. Thus pharmacological concentrations of auranofin cause dose-related inhibition of histamine release and de novo synthesis of LTC4 by human basophils and lung mast cells.

Physiological concentrations of zinc inhibit the release of histamine from human basophils and lung mast cells.

We have previously shown that physiological concentrations of zinc (congruent to 7 X 10(-6) M) inhibit the release of histamine from human basophil leukocytes (Marone et al., J. Pharmacol. Exp. Ther. 217: 292, 1981). In these experiments we compared the effect of zinc chloride on the release of chemical mediators from human basophils and mast cells isolated from human lung. Preincubation (5 min, 37 degrees C) of human basophils and lung mast cells with zinc chloride (10(-6)-3 X 10(-5) M) caused dose-related inhibition of histamine and peptide leukotriene C4 (LTC4) release induced by anti-IgE. Increase Ca2+ concentrations (0.3 to 6 mM) in the extracellular medium completely reversed the inhibitory effect of zinc on anti-IgE-mediated histamine secretion. Zinc chloride was a competitive antagonist of the action of Ca2+ in histamine secretion induced by anti-IgE with a dissociation constant (Kd) of about 10(-5) M in both the basophil and mast cell systems. Thus physiological concentrations of zinc inhibit the release of histamine from human basophils and lung mast cells, presumably by blocking Ca2+ uptake induced by anti-IgE activation.

Forskolin inhibits the release of histamine from human basophils and mast cells.

We found that forskolin (10(-7) to 3 X 10(-5) M) caused dose-related inhibition of antigen-induced histamine release from human basophil leukocytes. The dose-response inhibition curve was paralleled by a forskolin-induced increase in cyclic AMP (cAMP) levels in human leukocyte preparations. The kinetics of inhibition of histamine release and of the increase in leukocyte cAMP were the same. In a second series of experiments we evaluated the effect of forskolin on antigen-induced histamine release from chopped human lung passively sensitized with serum from an allergic patient. Forskolin (10(-7) to 3 X 10(-5) M) dose-dependently inhibited the release of histamine from human lung mast cells. Thus forskolin appears to modulate the release of mediators of the immediate hypersensitivity reaction, presumably through activation of adenylate cyclase in human basophils and mast cells.

Histamine release from human basophils induced by platelet activating factor: the role of extracellular calcium, interleukin-3, and granulocyte-macrophage colony-stimulating factor.

Although we have demonstrated that platelet activating factor (PAF) directly induces histamine release from human basophils, other studies have failed to report similar effects. In an attempt to understand the variability of these results, we examined the effect of some factors that could influence the basophils' response to PAF such as, extracellular Ca2+ and cytokines (interleukin-3 and granulocyte-macrophage colony-stimulating factor [GM-CSF]). The secretion of histamine induced by PAF was optimal when the cells were incubated in Ca2+ for 2 to 5 minutes, whereas it declined at longer time intervals up to 15 minutes. If cytochalasin B (5 micrograms/ml) was coincubated with PAF (1 mumol/L) to enhance the secretory response, histamine release was maximal at time 0 and decreased in parallel with the time of the basophils' exposure to Ca2+, like 0.1 microgram/ml anti-IgE-induced histamine secretion but unlike 1 mumol/L formyl-methionyl-leucyl-phenylalanine-induced histamine secretion. We found that there is synergy between interleukin-3 (1 to 3 ng/ml) and PAF (1 mumol/L) for secretion of histamine from human basophils (p < 0.05) and that GM-CSF (10 ng/ml) significantly (p < 0.02) potentiates the secretion of histamine activated by PAF (1 mumol/L). Our results demonstrate that: (1) the kinetics of the interaction between Ca2+ and the activation pathway that leads to histamine secretion are central events in the release reaction elicited by PAF in human basophils, and (2) interleukin-3 and GM-CSF can potentiate the secretory response of human basophils stimulated by PAF.

The antineoplastic bryostatins affect human basophils and mast cells differently.

Bryostatins, macrocyclic lactones from the marine bryozoan Bugula neritina, are potent antineoplastic agents and multi-potential stimulators of immune cells. We have examined the effects of bryostatins on mediator release from human basophilic leukocytes and human tissue mast cells. Bryostatins 1, 2, and 5 (10 to 3,000 nmol/L) induced histamine secretion from purified and unpurified peripheral blood basophils, whereas they caused no release of peptide-leukotriene C4 from these cells. The rate of histamine release caused by bryostatin 1 was slower than that caused by anti-IgE (t1/2 +/- SEM = 38.2 +/- 4.7 minutes v 8.9 +/- 0.2 minutes; P < .01), whereas the temperature dependence was similar (optimum release at 37 degrees C, approximately 30% less at 30 degrees C, and no release at 22 degrees C or 4 degrees C). The addition of increasing concentrations of extracellular Ca2+ to the medium caused histamine release in the presence of bryostatins. Subeffective concentrations of bryostatins and anti-IgE produced a synergistic effect on histamine release from basophils. Staurosporine, chelerythrine, and calphostin C (0.1 to 10 nmol/L), which are protein kinase C inhibitors, inhibited the histamine secretion activated by bryostatin 1 and tetradecanoylphorbol-acetate (TPA). Preincubation with granulocyte-monocyte colony-stimulating factor (GM-CSF; 1 and 5 nmol/L) and interleukin-3 (IL-3; 10 ng/mL) potentiated the activation of human basophils induced by bryostatin 1. Neither bryostatin 1 nor bryostatin 2 induced the release of histamine from mast cells isolated from human lung or skin tissues. However, brief (10 minutes) preincubation with bryostatin 1 (3 to 300 nmol/L) potently inhibited the histamine secretion induced by anti-IgE from skin or lung mast cells. Bryostatin 1 was a more potent (by approximately 30 times) inhibitor of IgE-mediated histamine release than was TPA. The heterogeneous effects exerted by bryostatins on human basophils and mast cells can be of interest for those designing therapeutic trials using these agents.

Substance P activates the release of histamine from human skin mast cells through a pertussis toxin-sensitive and protein kinase C-dependent mechanism.

Using pharmacologic agents, we explored the mechanism by which a potent neuropeptide, substance P, induces the secretion of histamine from human skin mast cells and compared their effects on substance P-induced histamine release to the secretion activated by anti-IgE. Histamine release from human cutaneous mast cells induced by substance P was inhibited by the Ge-protein inhibitor pertussis toxin that, in turn, did not affect the IgE-mediated secretion. Similarly to anti-IgE, two activators of protein kinase C, tetradecanoylphorbol acetate (TPA) and bryostatin 1, significantly inhibited the substance P-induced response. In contrast, drugs that enhance intracellular levels of cAMP, an inhibitor of protein kinases, genistein, and a protease inhibitor, AEBSF, did not affect substance P-induced histamine secretion, whereas these compounds significantly reduced the response initiated by anti-IgE. Our data demonstrate that substance P activates human cutaneous mast cells by acting on G proteins and protein kinase C. Our results also suggest that the biochemical pathways underlying mast cell activation by substance P and anti-IgE are to a great extent unrelated.

Adenosine potentiates mediator release from human lung mast cells.

Micromolar concentrations of adenosine were found to potentiate the release of histamine and leukotriene C4 (LTC4) from immunologically activated human lung mast cells (HLMC). Structurally modified congeners of adenosine including 5'-N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyladenosine (R-PIA) also potentiated mediator release. A rank order of potency was established where NECA greater than R-PIA for the potentiation of both LTC4 production and histamine secretion. Mast cells isolated by either enzymatic or mechanical means from human lung parenchyma were both similarly responsive to the modulatory effects of adenosine and analogues, and the potency series of NECA greater than R-PIA also applied. Moreover, histamine release induced by the calcium ionophore A23187 was augmented by NECA, R-PIA, and adenosine and in that potency order. Dipyridamole, an agent thought to impede the intracellular uptake of adenosine, failed to reverse the nucleoside's enhancement of IgE-mediated secretion. The irreversible inhibitor of adenosine deaminase, deoxycoformycin, did not modify the adenosine enhancement of stimulated secretion. Low concentrations of methylxanthines, which antagonize responses mediated at cell surface adenosine receptors, were inconsistent in their effects. Theophylline modestly reversed the adenosine-induced potentiation of IgE-mediated LTC4 generation but not histamine release. Studies employing 8-phenyltheophylline were complicated by the methylxanthine possessing inhibitory properties of its own at concentrations expected to antagonize a nucleoside-mediated effect. In total, these results suggest that the response of HLMC to adenosine describes properties most consistent with an A2/Ra-like process, although an interaction via an, as yet, uncharacterized cell surface receptor cannot be excluded.