Pharmacologic heterogeneity of human lung and colon cells: effect of terfenadine and cetirizine

H₁-blockers may have antiallergic properties which cause the blocking of eicosanoid release, and the effect of these drugs may differ according to the phenotype of mast cells. This study examined the ability of terfenadine and cetirizine to inhibit the release of arachidonic acid-derived mediators from human lung and colon cells. Dispersed cells were challenged with anti-IgE in the presence or absence of 10 μM of terfenadine or cetirizine, and the release of prostaglandin (PG)D₂ and leukotriene (LT)C₄/D₄ was assessed by enzyme immunoassay (EIA). Terfenadine caused significant inhibition of both PGD₂ and LTC₄/D₄ (49 ±9 and 29 ± 19%, respectively) from human lung cells but had a less marked effect on PGD₂ release from human colon cells (21 ± 9% for PGD² and 18 ± 9% for LTC₄/D₄). In contrast, although cetirizine caused significant inhibition of both mediators measured in lung cells (38 ± 16% for PGD₂ and 34 ± 19% for LTC₄), it did not cause any significant inhibition of either mediator from human colon cells. These findings suggest that H₁antagonists may have additional properties, and the differential effects of cetirizine on lung and colon tissue may indicate differences in mast cell phenotype.     

Modulation of eicosanoid and histamine release from human dispersed lung cells by terfenadine

Terfenadine is an H₁-blocker that may have antiallergic properties. A study was carried out to examine the ability of terfenadine to inhibit the release of histamine and arachidonic-acid-derived mediators from human lung cells. Cells were dispersed from fresh human lung tissue obtained from tour accident victims whose hearts were donated for transplantation and four lung cancer resections. Cells were dispersed by enzymatic digestion with type XIV protease and chymopapain, and this resulted in a cell population containing approximately 5% mast cells. The remaining cells were mainly macrophages. The cells were challenged with anti-IgE at a 1/1000 dilution. Cells were challenged without terfenadine and after a preincubation of 0.1, 1, and 10 umol terfenadine. The release of PGD₂ and LTC₄/D₄ was assessed with an EIA. Histamine was assayed by RIA with a monoclonal antibody against acylated histamine.
A release of both eicosanoids and histamine was observed m all experiments. An inhibition of eicosanoids was observed at both 1 and 10 μmol terfenadine (median percentage of inhibition of PGD₂: 38.00 ± 15.65 and 56,00 ± 13,12; median percentage of inhibition of LTC₄/D₄: 37.5 ± 19,80 and 52.5 ± 26.8). On the other hand, histamine release was not blocked by terfenadine.
Terfenadine inhibits, in a dose-dependent manner, the release of eicosanoids after challenge of dispersed lung cells by anti-IgE, and this effect may have some clinical relevance.     

SRS-A leukotrienes decrease the activity of human respiratory cilia

We have studied the effects of the slow reacting substance of anaphylaxis (SRS-A) constituents leukotrienes (LT) C₄ and D₄ on the ciliary activity of human respiratory cells. The ciliary beat frequency on human nasal cells harvested by cell scraping from the inferior turbinate was measured in a blind design by a microphoto-oscillographic technique. A total of 740 ciliated cells from seventy-four cell scrapings were studied. Mean baseline of ciliary beat frequency was 10.2 Hz. The ciliary beat frequency exhibited a pronounced variability in the spontaneous changes between the cell scrapings, yet less so within cell samples from the cell scrapings. We, therefore, evaluated the effect of the test solutions relative to the spontaneous decrease found during simultaneous perfusion with control solution of samples from the same cell scrapings. LTC₄, 3–300 nmol/l, caused a highly significantly dose-related decrease in the ciliary beat frequency by up to approximately 20% as compared to the corresponding control solution. The effect of LTC₄ was significantly inhibited by the SRS-A receptor antagonist FPL 55712 (10μmol/l), but not by indomethacin (10 μmol/l). LTD₄, 300 nmol/l, also decreased the ciliary beat frequency. LTB₄, which is a leukotriene, although without the sulphidopeptide side chain of the SRS-A leukotrienes, did not affect the ciliary beat frequency in a concentration of 100 nmol/l. This would seem to confirm the structure specificity of the elucidated effect of the SRS-A leukotrienes. Histamine (100 μmol/l) did not affect the ciliary beat frequency. The present study demonstrates that the SRS-A leukotrienes have a specific cilio-depressive effect, which may contribute to mucus obstruction in the lower airways in asthma and other chronic airway diseases.     

Mast cells and eicosanoid mediators: a system of reciprocal paracrine and autocrine regulation

Summary:  When activated by specific antigen, complement, or other transmembrane stimuli, mast cells (MCs) generate three eicosanoids: prostaglandin (PG)D₂, leukotriene (LT)B₄, and LTC₄, the parent molecule of the cysteinyl leukotrienes (cysLTs). These diverse lipid mediators, which are generated from a single cell membrane-associated precursor, arachidonic acid, can initiate, amplify, or dampen inflammatory responses and influence the magnitude, duration, and nature of subsequent immune responses. PGD₂ and cysLTs, which were originally recognized for their bronchoconstricting and vasoactive properties, also serve diverse and pivotal functions in effector cell trafficking, antigen presentation, leukocyte activation, matrix deposition, and fibrosis. LTB₄ is a powerful chemoattractant for neutrophils and certain lymphocyte subsets. Thus, MCs can contribute to each of these processes through eicosanoid generation. Additionally, MCs express G-protein-coupled receptors specific for cysLTs, LTB₄, and another eicosanoid, PGE₂. Each of these receptors can regulate MC functions in vivo by autocrine and paracrine mechanisms. This review focuses on the biologic functions for MC-associated eicosanoids, the regulation of their production, and the mechanisms by which eicosanoids may regulate MC function in host defense and disease.     

In vitro effects of H1-antihistamines on histamine and PGD2 release from mast cells of human lung, tonsil, and skin

Mast cells from different anatomic sites differ in cytochemistry and response to various secretory stimuli. We have investigated whether responsiveness to the second-generation H₁-receptor antagonists, which are important first-line drugs for the relief of symptoms in patients with chronic urticaria and allergic rhinoconjunctivitis, also differs according to the site of origin of mast cells. The effects of terfenadine, ketotifen, and cetirizine were therefore examined in relation to the IgE-dependent release of histamine and prostaglandin D₂ (PGD₂) from dispersed human lung, tonsil, and skin mast cells. Terfenadine had a biphasic effect on lung and skin mast cells: at low concentrations, a concentration-dependent inhibition of histamine release from lung and skin mast cells was observed, whereas at higher concentrations the drug stimulated mediator release. Even at a high concentration, terfenadine inhibited mediator release from tonsil mast cells. Ketotifen had low potency as an inhibitor of mediator release from lung and tonsil mast cells. In skin mast cells, no inhibition of mediator release was observed below 1.0 μM, and above that concentration it induced mediator release. Cetirizine, a much less lipophilic drug than the others tested, did not induce mediator release from mast cells even at concentrations up to 100 μM. This drug showed concentration-dependent inhibition of IgE-dependent mediator release from lung and tonsil mast cells only. Our results show that human mast cells are heterogeneous with respect to modulation of mediator release by these H₁-antihistamines. In particular, differences were observed between skin mast cells and those dispersed from lung and tonsils.     

Dopamine selectively reduces GABAB transmission onto dopaminergic neurones by an unconventional presynaptic action

The functioning of midbrain dopaminergic neurones is closely involved in mental processes and movement. In particular the modulation of the inhibitory inputs on these cells might be crucial in controlling firing activity and dopamine (DA) release in the brain. Here, we report a concentration-dependent depressant action of dopamine on the GABA_B IPSPs intracellularly recorded from dopaminergic neurones. Such effect was observed in spite of the presence of D₁/D₂ dopamine receptor antagonists. A reduction of the GABA_B IPSPs was also caused by noradrenaline (norepinephrine) and by l -β-3,4-dihydroxyphenylalanine ( l -DOPA), which is metabolically transformed into DA. The DA-induced depression of the IPSPs was partially antagonised by the α2 antagonists yohimbine and phentolamine. DA did not change the postsynaptic effects of the GABA_B agonist baclofen, suggesting a presynaptic site of action. Furthermore, DA did not modulate the GABA_A-mediated IPSP. The DA-induced depression of the GABA_B IPSP occluded the depression produced by serotonin and was not antagonized by serotonin antagonists. The DA- and 5-HT-induced depression of the GABA_B IPSP persisted when calcium and potassium currents were reduced in to the presynaptic terminals. These results describe an unconventional presynaptic, D₁ and D₂ independent action of DA on the GABA_B IPSP. This might have a principal role in determining therapeutic/side effects of l -DOPA and antipsychotics and could be also involved in drug abuse.     

Increased in vitro cysteinyl leukotriene release from blood leukocytes in patients with asthma, nasal polyps, and aspirin intolerance

In vitro cysteinyl leukotriene (cLT) release from blood leukocytes was measured in eight normal individuals (NI), nine patients with nasal polyps (NP) without aspirin intolerance, and eight patients with NP, asthma, and aspirin intolerance (AI). Blood leukocytes were prestimulated with interleukin-3 (IL-3) and incubated with acetylsalicylic acid (ASA) (10 and 100 μ/ml) together with C5a (10⁻⁸ mol/1) for 18 h. cLT release (LTC₄, LTD₄ and LTE₄) from blood leukocytes was measured with a competitive enzyme-linked immunoassay. Background cLT release was 259±66 pg/ml (mean±SEM) in the NI group, 185±33 pg/ml in the NP group, and 578±136 pg/ml in the AI group ( P = 0.002). After incubation with 10 μ/ml ASA, cLT concentration was lower in normal subjects (346±72 pg/ml) and in patients with NP (209±53) than in patients with AI (686±75 pg/ml, P = 0.002). After incubation with 100 μ/ml ASA, cLT concentrations were 285±72 pg/ml in the NI group, 313±77 pg/ml in the NP group, and 654±121 pg/ml in the AI group ( P = 0.04), respectively. Simultaneous incubation with ASA 10 μ/ml and C5a (10⁻⁸mol/1) resulted in a cLT concentration of 751±171 pg/ml in the NI group, 343±102 pg/ml in the NP group, and 2196±480 pg/ml in patients with AI ( P = 0.0006), whereas simultaneous incubation with ASA l00g/ml and C5a (10⁻⁸mol/1) resulted in 268±51 pg/ml in the NI group, 412±97 pg/ml in the NP group, and 1701±368 pg/ml in the AI group ( P = 0.005). In patients with AI, cLT release from blood leukocytes is altered when compared with normals and patients with NP. The presented cLT-release assay could be of potential use in the in vitro diagnosis of AI.     

Influence of Tumour Condition on the Macrophage Activity in Candida albicans Infection

To better understand the interactions between opportunistic fungi and their hosts, we investigated hydrogen peroxide (H₂O₂)_, nitric oxide and TNF-α production by peritoneal macrophages from Ehrlich tumour-bearing mice (TBM) during microbial infections. For this purpose, TBM at days 7, 14 and 21 of tumour progression were inoculated intraperitoneally with C. albicans and evaluated after 24 and 72 h. We observed that TBM showed significant increases in H₂O_2, TNF-α levels and fungal clearance at day 7 after C. albicans infection. However, as the tumour advanced, there was a progressive decline in the release of H₂O₂ and TNF-α that was paired with the dissemination of C. albicans . These results demonstrate that protective macrophage activities against Candida albicans are limited to the initial stages of tumour growth; continued solid tumour growth weakened the macrophage response and as a consequence, weakened the host's susceptibility to opportunistic infections.     

Increased Expression of Glutathione by Estradiol, Tumor Necrosis Factor-Alpha, and Interleukin 1-Beta in Endometrial Stromal Cells

Problem  The intracellular antioxidant system, based on glutathione (GSH), plays a key role in endometrial detoxification reactions and has been proposed to be involved in the pathogenesis endometriosis. This study was designed to evaluate whether estradiol (E₂) and proinflammatory cytokines have any effects on expression of glutathione in endometrial stromal cells (ESCs).
Method of study  Glutathione levels were measured utilizing high-performance liquid chromatography following in vitro culture and treatment of ESCs with estradiol, tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1β).
Results  The GSH level in E₂ (10⁻⁸  m ) treatment group was significantly higher than in the control group at 48 h ( P  < 0.05). In vitro treatment of ESCs with TNF-α 10 ng/mL as well as E₂ (10⁻⁸  m ) plus TNF-α 10 ng/mL for 48 hr also led to a significant increase in GSH level ( P  < 0.05; P  < 0.05, respectively). Both IL-1β 10 ng/mL and E₂ (10⁻⁸  m ) plus IL-1β 10 ng/mL for 48 hr increased GSH level significantly ( P  < 0.05; P  < 0.05, respectively) as well.
Conclusions  These findings might suggest that increased production of estradiol and proinflammatory cytokines in the peritoneal cavity possibly leads to the establishment of endometriosis through increased level of GSH.     

Functional expression of ionotropic purinergic receptors on mouse taste bud cells

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 μ m ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca²⁺ imaging showed that similarly applied 1 μ m ATP, 30 μ m BzATP (a P2X₇ agonist), or 1 μ m 2MeSATP (a P2Y₁ and P2Y₁₁ agonist) increased intracellular Ca²⁺ concentration, but 100 μ m UTP (a P2Y₂ and P2Y₄ agonist) and α,β-meATP (a P2X agonist except for P2X₂, P2X₄ and P2X₇) did not. RT-PCR suggested the expression of P2X₂, P2X₄, P2X₇, P2Y₁, P2Y₁₃ and P2Y₁₄ among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X₂. The exposure of the basolateral membranes to 3 m m ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 μ m PPADS (a non-selective P2 blocker) and 1 μ m KN-62 (a P2X₇ blocker). These results showed for the first time the functional expression of P2X₂ and P2X₇ on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.     

Comparative vascular effects of histamine, prostaglandin (PG) D2 and its metabolite 9α, 11β-PGF2 in human skin

In this double-blind study we have investigated the vascular effects of prostaglandin, (PG) D₂, in normal skin and compared these effects with histamine and the initial PGD₂ metabolite 9α, 11β-PGF₂. In eight healthy subjects the vascular response to intradermal injections of histamine, PGD₂, a combination of histamine and PGD₂, and 9α, 11β-PGF₂, was assessed by measurement of the weal and flare area. Histamine caused dose-related increases in weal area ( P <0.01). The weal response due to PGD₂ was greater than saline control only at a dose of 71.0 and 710 nmol ( P <0.05). Because of the small size of the weal produced by PGD₂ when compared with histamine, it was not possible to determine their relative potencies. Histamine and PGD₂ caused dose-related increases in flare area ( P <0.05), and when compared at a response level of 10 cm² and 15 cm², histamine was 45 and 251 ( P <0.01) times more potent than PGD₂ in molar terms. Weal and flare responses due to 9α, 11β-PGF₂ were similar to those observed with the equimolar concentration of PGD₂. The weal and flare responses when PGD₂ and histamine when combined were not significantly different from that predicted by a purely additive effect. We conclude that histamine is likely to be an important mediator contributing towards increased vascular permeability and vasodilatation following immunological activation of skin mast cells in vivo , while PGD₂ and its metabolite 9α, 11β-PGF₂ play only a minor role.     

Acquisition of Interleukin-5 Secretion by Human Naive T-Helper Cells is Regulated by Distinct Signals from both the T-Cell Receptor/CD3 Complex and CD2

We developed a human naive T-helper (Th) cell differentiation model with anti-CD3 monoclonal antibody (MoAb), using a B-cell line as source of costimulation. In this system, we examined the contribution of the T-cell receptor (TCR)/CD3-derived signals and that of lymphocyte function-associated antigen-1 (LFA-1) and CD2 in regulating Th-cell subset differentiation. We found that lowering the level of anti-CD3 MoAb decreased tumour necrosis factor-α (TNF-α) production, while increasing secretion of the Th2 cytokines, interleukin-5 (IL-5) and interleukin-13 (IL-13). Secretion of interferon-γ (IFN-γ) was not influenced by the strength of the anti-CD3 signal. Under conditions where Th0 cells are generated, co-culture with anti-CD2 F(ab')₂ MoAb led to the generation of Th cells that secreted 30–35% less IL-5, while not affecting secretion of IFN-γ or granulocyte–macrophage colony-stimulating factor (GM-CSF). By contrast, anti-CD18 MoAb F(ab')₂ inhibited IFN-γ and GM-CSF levels only in the primary stimulation, but did not affect cytokine levels after restimulation. Neither anti-CD2 nor anti-CD18 F(ab')₂ MoAbs could alter cytokine secretion profiles of peripheral blood-derived memory/effector Th cells. Our results indicate that acquisition of IL-5 secretion capability by Th cells is regulated mainly by signals transduced by the TCR/CD3 complex and by the presence of interleukin-4 (IL-4), while the CD2/LFA-3 pathway plays an additional, but minor, role. These regulatory CD2-derived signals, however, are distinct from those generated by the TCR/CD3 complex.     

Human Brain Endothelial Cells and Astrocytes Produce IL-1β but not IL-10

The ability of human brain endothelial cells to produce mRNA for interleukin-10, and release IL-10 in culture supernatants after in vitro stimulation with LPS, TNF-α and γ-IFN was assessed and compared to that of astrocytes, peripheral blood mononuclear cells and human umbilical vein endothelial cells. IL-1β and β₂-microglobulin release were also analysed. IL-10 and TNF-α mRNA presence was investigated in normal brain as well as in three plaques from two multiple sclerosis patients. While increased IL-1β and β₂-microglobulin release in the supernatants of stimulated cells could be detected in all the studied cell lineages, IL-10 mRNA and protein release was only seen in LPS-stimulated PBMNCs. Similarly, mRNA for IL-10 was not detected in CNS tissues, while TNF-α was present in all plaques. The lack of production of significant amounts of IL-10 by astrocytes and human brain endothelial cells suggests that these cells may not be the primary source of in vivo IL-10-mediated down-regulation of immune reactions within the central nervous system.     

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following β2-adrenergic stimulation of ICa,L in frog ventricular myocytes

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the β₂-adrenergic stimulation of the L-type Ca²⁺ current ( I _Ca,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 μM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of I _Ca,L were used to determine the gradient of local vs. distant cAMP concentration (α). IBMX (100 μM), a non-selective PDE inhibitor, reduced α from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 μM) or PDE4 inhibition by Ro 20-1724 (3 μM) reduced α by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 μM), a non-selective phosphatase inhibitor, or H89 (1 μM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of I _Ca,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon β₂-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of β₂-adrenergic receptors with nearby Ca²⁺ channels via local elevations of cAMP.     

D1 and D2 receptor-mediated dopaminergic modulation of visual responses in cat dorsal lateral geniculate nucleus

The modulatory effects of dopamine (DA) on the visual responses of relay cells of the dorsal aspect of cat lateral geniculate nucleus (dLGN) were tested using local micro-iontophoretic application of DA and application of the receptor-specific agonists SKF38393 (SKF, D₁/D₅) and quinpirole (QUIN, D₂/D₃/D₄) in the anaesthetized alcuronium-treated cat. The effects of DA and QUIN were clearly dose-dependent: small amounts caused a weak and transient facilitation of visual activity (10–30 % increase) preferentially in Y-type relay cells, which changed to a moderate reduction of visual responses when the dose was increased (50 %, maximal 70 %). The effect of SKF was mainly suppressive and increased with the amount of drug applied (up to 90 % reduction). The selective antagonists SCH23390 (SCH, D₁) and sulpiride (SULP, D₂) reduced the effects of co-applied DA agonists. We found little evidence for a specific dopaminergic modulation of the surround inhibition (stimulus-driven lateral inhibition) although DA slightly facilitated the transmission of weak signals (small stimuli). Nevertheless, some dopaminergic effects seem to be mediated via inhibitory interneurons regulating the strength of sustained or recurrent inhibition. Application of DA agonists during blockade of GABA_A receptors indicates a direct suppression of relay cells via D₁ receptors, an excitation of relay cells via D₂ receptors and - with increasing amounts of D₂ agonist - probably also an excitation of inhibitory interneurons, which results in an indirect inhibition of dLGN relay cells (predominantly of the X-type). The results are discussed in relation to the impairment of visual functions in Parkinson's disease.     

Leukotriene pathway genetics and pharmacogenetics in allergy

Leukotrienes (LT) are biologically active lipid mediators known to be involved in allergic inflammation. Leukotrienes have been shown to mediate diverse features of allergic conditions including inflammatory cell chemotaxis/activation and smooth muscle contraction. Cysteinyl leukotrienes (LTC₄, LTD₄ and, LTE₄) and the dihydroxy leukotriene LTB₄ are generated by a series of enzymes/proteins constituting the LT synthetic pathway or 5-lipoxygenase (5-LO) pathway. Their function is mediated by interacting with multiple receptors. Leukotriene receptor antagonists (LTRA) and LT synthesis inhibitors (LTSI) have shown clinical efficacy in asthma and more recently in allergic rhinitis. Despite growing knowledge of leukotriene biology, the molecular regulation of these inflammatory mediators remains to be fully understood. Genes encoding enzymes of the 5-LO pathway (i.e. ALOX5 , LTC4S and LTA4H ) and encoding for LT receptors ( CYSLTR1/2 and LTB4R1/2 ) provide excellent candidates for disease susceptibility and severity; however, their role remains unclear. Preliminary data also suggest that 5-LO pathway/receptor gene polymorphism can predict patient responses to LTSI and LTRA; however, the exact mechanisms require elucidation. The aim of this review was to summarize the recent advances in the knowledge of these important mediators, focusing on genetic and pharmacogenetic aspects in the context of allergic phenotypes.     

Effect of in vitro aspirin stimulation on basophils in patients with aspirin-exacerbated respiratory disease

Background Basophil activation has been implicated in the pathogenesis of aspirin-exacerbated respiratory disease (AERD). However, a comprehensive analysis of basophil responses to aspirin in terms of mediator release, cytokine secretion and increased expression of surface activation markers has not been performed.
Objective To study the in vitro effects of aspirin on the concurrent release of histamine, leukotriene C₄ (LTC₄) and IL-4 from human basophils and to also evaluate changes in surface activation markers (CD63, CD69 and CD203c) expressed by these cells.
Methods Basophil-enriched cell suspensions from 10 patients with AERD and 10 healthy volunteers were incubated with lysine-aspirin for up to 3 h. Cells were analysed for expression of CD63, CD69 and CD203c using flow cytometry. Cell-free supernatants were evaluated for histamine, and LTC₄ release and for IL-4 secretion.
Results Aspirin-induced expression of CD63, CD69 and CD203c yielded 30%, 80% and 70% sensitivity, respectively, but with poor specificity. There was no significant difference in LTC₄ synthesis between groups. None of the patients with AERD (or controls) released IL-4 in response to aspirin. A higher dose of 5 mg/mL aspirin-mediated non-specific effects on basophils.
Conclusion Basophil responses to in vitro aspirin challenge are poor indicators of clinical sensitivity. Aspirin activates some basophils by means of mechanisms that differ from the classical IgE-mediated pathway. Our study also shows that the use of 27 m m of aspirin (5 mg/mL) by previous investigators causes non-specific basophil activation, thereby eliminating its usefulness in a cell-based diagnostic test for AERD. Evaluation of in vitro basophil activation has low clinical value in identifying aspirin-induced respiratory reactions.     

Histamine releasability of basophils and skin mast cells in chronic urticaria

In order to clarify the pathogenetic role of basophils and mast cells in chronic urticaria, histamine and leukotrienes (LT) C₄ release was examined in washed mixed leukocytes (n=8) and skin mast cells (n=5) from patients with chronic urticaria and compared with the same cells from normal controls (n=9). Anti-IgE-stimulated basophil histamine release was significantly reduced in urticaria patients (median 2.9% vs 15.1% in normal controls), whereas histamine release to A23187, FMLP, and PAF, as well as anti-IgE-induced LTC₄ release, showed no differences in both groups. In contrast, anti-IgE-stimulated skin mast cells from urticaria patients reacted similarly to those of controls (median histamine release 11.4% vs 14.2% in normal controls). Pretreatment of the cells with interleukin (IL)-3 upregulated responsiveness of basophil histamine release to anti-IgE in urticaria patients (median histamine release 14.3%), but pretreatment with the H₂-antagonist cimetidine showed no effect. These data show that reduced basophil histamine releasability in chronic urticaria is not H₂ mediated. It is a stimulus-, mediator-, and cell type-restricted phenomenon that can, at least partially, be reversed in the presence of the cytokine IL-3.     

Possible mechanisms of the concentration-dependent action of substance P to induce histamine release from rat peritoneal mast cells and the effect of extracellular calcium on mast-cell activation

Rat peritoneal mast cells purified on a Percoll gradient were loaded with the fluorescent Ca²⁺ indicator fura-2 and were challenged with different concentrations of substance P (SP), and intracellular calcium concentrations ([Ca²⁺]_i) were measured by a spectrofluorometric assay. SP at 5 × 10⁻⁶ mol/1 and 10⁻⁵ mol/1 caused a significant histamine release with a significant increase in [Ca²⁺]_i in a dose-dependent manner. However, SP at 10⁻⁸-10⁻⁶ mol/1 did not induce either histamine release or increase in [Ca²⁺]_i. Extracellular calcium at 0.9 mM inhibited the histamine release with a significant reduction of [Ca²⁺]ⁱ compared with that of the cells in a nominally calcium-free condition. These results indicate that the action of SP on rat mast cells relies upon [Ca²⁺]_i to induce histamine release.