N. Milazzo

Inhibitory Effect of the H1 Antagonist Loratadine on Histamine Release from Human Basophils

Loratadine is a powerful H1 antagonist commonly employed in the treatment of allergic disorders. The present study was performed to investigate whether loratadine, in addition to anti-H1 activity, is able to modulate histamine release from human basophils. Leukocyte suspensions were prepared by dextran sedimentation of peripheral venous blood drawn from 10 normal subjects. Leukocytes were stimulated with anti-IgE (1/5,000), N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10 microM) and the Ca2+ ionophore A23187 (1 microM), and histamine release in the cell supernatants was measured by an automated fluorometric method. Loratadine, at concentrations ranging from 1 to 50 microM, exerted a dose-dependent inhibitory effect on IgE-mediated and IgE-independent histamine release. The concentrations inhibiting 50% of histamine release were 30 microM (anti-IgE), 29 microM (FMLP) and 24 microM (Ca2+ ionophore A23187). The inhibitory activity of loratadine was optimal after incubation for 2 h at 37 degrees C and the effect of the drug was no longer evident when leukocytes were stimulated 2 h after cell washing. Increased extracellular Ca2+ concentrations reduced the inhibitory activity of loratadine, indicating that external Ca2+ and loratadine have antagonistic effects on basophil histamine release. These results indicate that loratadine, in addition to H1 receptor blocking activity, has the capacity to inhibit histamine release from human basophils.

Nasal neutrophilia and eosinophilia induced by challenge with platelet activating factor.

BACKGROUND It has been demonstrated that in vitro platelet activating factor-acether (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF) has the capacity to attract eosinophils and neutrophils. We investigated whether the same applies when human nasal airways are stimulated with PAF. METHODS Symptom scores and cytologic changes in nasal lavage fluids were evaluated in 10 atopic and 10 nonatopic subjects after nasal challenge with PAF, its precursor and metabolite, 1-O-hexadecyl-sn-glycero-3-phosphorylcholine (lyso-PAF), or saline solution. RESULTS Nasal obstruction was reported by all the atopic subjects and seven of the 10 nonatopic subjects after nasal challenge with PAF; other symptoms such as rhinorrhea, itching, and sneezing were generally mild. PAF induced neutrophilia, which appeared after 30 minutes in atopic subjects and after 1 hour in nonatopic subjects, and peaked at 3 hours in both. Less neutrophilia was found 3 hours after stimulation with lyso-PAF in both groups of subjects. PAF also induced eosinophilia, which appeared after 30 minutes in atopic subjects and only after 3 hours in nonatopic subjects. An increase in eosinophil counts was observed 3 hours after lyso-PAF stimulation in atopic but not in nonatopic subjects. CONCLUSION PAF can attract neutrophils and eosinophils into human nasal airways; however, the recruitment of inflammatory cells is more rapid in atopic than in nonatopic subjects, suggesting a different degree of responsiveness to PAF challenge in the two groups of subjects.

Nasal neutrophilia and release of myeloperoxidase induced by nasal challenge with platelet activating factor : Different degrees of responsiveness in atopic and nonatopic subjects

BACKGROUND Nasal challenge with platelet activating factor (PAF) is able to induce local neutrophilia, with a different degree of responsiveness in atopic subjects and in nonatopic subjects. We investigated whether nasal accumulation of neutrophils induced by PAF is accompanied by the release of neutrophil-derived mediators. METHODS Nasal lavages were performed before and after challenge with PAF (500 nmol), lyso-PAF (500 nmol), and saline solution in 10 patients with allergic rhinitis and 10 normal subjects to evaluate changes in neutrophil counts and the release of myeloperoxidase (MPO) and immunoreactive leukotriene B4. RESULTS PAF caused neutrophilia, which appeared after 30 minutes in atopic subjects and after 3 hours in nonatopic subjects. Furthermore, when compared with saline insufflation, PAF caused a significant release of MPO in the nasal lavage fluids collected 30 minutes, 3 hours, and 24 hours after challenge in atopic subjects and 3 hours after challenge in nonatopic subjects, with higher values in the former than in the latter. Neutrophil counts correlated with MPO levels in the nasal lavages collected after PAF challenge. A lower degree of neutrophilia was found 3 hours after stimulation with lyso-PAF in both groups of subjects, with a marginal release of MPO in atopic subjects only. No significant increase of immunoreactive leukotriene B4 levels in nasal lavages was found after challenge with either PAF or lyso-PAF. CONCLUSION These results indicate that PAF-induced neutrophilia in the nose is accompanied by the release of MPO, which appears earlier and is more marked in atopic subjects than in nonatopic subjects.

Enhancement of basophil histamine release by interleukin-3 : reduced effect in atopic subjects

The inducing and enhancing effects of interleukin‐3 (IL‐3) on basophil histamine release in patients with respiratory allergy (n= 28) and in normal subjects (n= 22) were compared. Leukocyte suspensions. prepared by dextran sedimentation, were stimulated with anti‐IgE (1/5000), N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP, 1 μM), and IL‐3 (0.1–10 ngiml), and histamine concentration was measured by an automated fluorometric method. A trend toward highef histamine release after challenge with anti‐IgE, FMLP, and IL‐3 was found in atopic subjects. Preincubation of basophils with IL‐3 resulted in a dose‐dependent increase of anti‐IgE‐ and FMLP‐induced histamine release, with a more marked effect in nonatopic than in atopic subjects. Mean net enhancement of anti‐IgE‐induced histamine release by 10 ng/ml IL‐3 was 2.5±5% in atopic subjects and 29.6±4.2% in nonatopic subjects (P<0.001). The enhancement of FMLP‐induced histamine release by IL‐3 was 10.3 ± 3.9% in atopic patients and 29±2.4% in nonatopic subjects (P<0.01). In atopic subjects, a negative correlation was found between anti‐IgE‐ or FMLP‐induced histamine release and net enhancement by IL‐3 (r= ‐0.45, P<0.02; r= ‐0.48, P<0.01, respectively). The results of this study indicate that in atopic subjects IgE‐mediated histamine release can scarcely be enhanced by a basophil response modifier such as IL‐3. It is conceivable that the frequent basophil stimulation in atopic patients leads to a reduced sensitivity to the enhancing effect of IL‐3.

Antiallergic activity of loratadine: inhibition of leukotriene C4 release from human leucocytes

The H1 antagonist loratadine has the capacity to inhibit histamine release from human basophils. The aim of this study was to investigate whether loratadine can also inhibit leukotriene C4 (LTC4) release from human leucocytes. Basophil‐enriched mononuclear cell suspensions were prepared by centrifugation of peripheral venous blood (n= 10) on discontinuous Percoll gradients. Leucocytes were stimulated with anti‐IgE, N‐formylmethionyl‐leucyl‐phenylalanine (FMLP) and Ca2+ ionophore A23187; immunoreactive (i) LTC4 release in the cell supernatant was measured by a competitive radioimmunoassay and histamine release was evaluated by an automated fluorometric technique. Loratadine, in the concentration range of 1–50μM, exerted a dose‐dependent inhibitory effect on IgE‐mediated and IgE‐independent histamine and iLTC4 release. The concentrations inhibiting 50% of histamine release were 30 μM (anti‐IgE), 27μM (FMLP) and 19μM (Ca2+ ionophore A23187). The concentrations inhibiting 50% of iLTC4 release were 2–3 μM (anti‐IgE). 11 μM (FMLP) and 1.7μM (Ca2+ ionophore A23187). The inhibitory activity on iLTC4 release was optimal after preincubation for 2h at 37°C, and was no longer evident when leucocytes were stimulated 2h after cell washing. Increased extracellular Ca2+ concentrations reduced the inhibitory activity of loratadine. These results indicate that loratadine has the capacity to inhibit the release of preformed and newly generated mediators from human basophil‐enriched mononuclear cell suspensions.

Ionic regulation of human basophil releasability. I. Inhibitory effect of copper

The effects of copper (CuSO4 and CuCl2) onin vitro histamine release from human basophils stimulated by anti-IgE and Ca2+ ionophore A23187 were evaluated. Both CuSO4 and CuCl2 caused a dose-related inhibition of histamine release, which was more pronounced on anti-IgE-than on Ca2+ ionophore-induced histamine release. The concentration which produced 50% inhibition of anti-IgE-induced histamine release was 1.3 μM for CuSO4 and 1.5 μM for CuCl2; the maximal inhibition of Ca2+ ionophore-induced histamine release was 33% for CuCl2 (4 μM) and 51% for CuSO4 (16 μM). The inhibitory effect on anti-IgE-induced histamine release persisted also when extracellular Cu2+ was removed by cell washing before IgE-induced histamine release persisted also when extracellular Cu2+ was removed by cell washing before stimulation, whereas no inhibition of Ca2+ ionophore-induced histamine release was found when extracellular Cu2+ was removed. The activity of Cu2+ was independent of any effects of deuterium oxide and colchicine, two agents known to interact with microtubules. Increased extracellular Ca2+ concentrations reduced the inhibitory effect of CuCl2 on Ca2+ ionophore-induced histamine release, and Schild plot analysis demonstrated that Cu2+ ions are competitive antagonists of Ca2+ ions.These results indicate that Cu2+ ions in the micromolar range down-regulate anti-IgE- and Ca2+ ionophore-induced histamine release. Since Cu2+ concentration in human plasma is in the micromolar range (30 μM with 10–30% of free Cu2+), it is conceivable that Cu2+ ions contribute to thein vivo regulation of histamine release from human basophils.

In vivo and ex vivo inhibitory effects of loratadine on histamine release in patients with allergic rhinitis

Background The aim of this study was to evaluate the in vivo and ex vivo effects of the H.‐antagonist loratadine on histamine release.

Ionic regulation of human basophil releasability. III. Effects of Na+ and Ca2+ on histamine release induced by different stimuli

The effects of Na+ and Ca2+ ions on histamine release from human basophils stimulated by anti-IgE, N-formyl-methionyl-leucyl-phenylalanine (FMLP), 4 beta-phorbol 12-myristate 13-acetate (PMA) and Ca2+ ionophore A23187 were evaluated. Isosmotic replacement of Na+ in the extracellular medium with the nonpermeant Na+ analogue choline+ or with glucose led to a significant increase in anti-IgE- (1/5000: 43.7 +/- 7.3% in high Na+ vs 68.9 +/- 7.3% in low Na+, mean +/- SEM, n = 8, P < 0.001), FMLP- (1 microM: 37.9 +/- 2.3% vs 49.5 +/- 4.3%, n = 8, P < 0.01) and PMA-(160 nM: 12.7 +/- 0.9% vs 27.3 +/- 4.3%, n = 8, P < 0.05) induced histamine release, whereas A23187-induced histamine release was reduced (1 microM: 90.4 +/- 2.4% vs 45.4 +/- 3.4%, n = 8, P < 0.0001). The progressive increase in extracellular Na+ concentration was accompanied by a decrease of basophil response to anti-IgE, FMLP and PMA; in contrast, A23187-induced histamine release was up-regulated by Na+. The Na+/H+ exchanger monensin, in the concentration range of 10(-8)-10(-4) M, exerted a dose-dependent inhibitory effect on anti-IgE-, FMLP- and PMA-induced histamine release, but not on A23187-induced histamine release. Extracellular Ca2+ up-regulated the histamine release induced by all the above stimuli. Removal of extracellular Na+ lowered the requirement of extracellular Ca2+ for anti-IgE, FMLP- and PMA-induced histamine release. In contrast with previous observations showing that Na+ supports histamine release from rat peritoneal mast cells and rat basophilic leukaemia cells, these results indicate that Na+ strongly inhibits histamine release from human basophils stimulated by anti-IgE, FMLP and PMA, whereas it enhances Ca2+ ionophore A23187-induced histamine release. The effects of Na+, which are probably related to modulation of membrane potential and/or intracellular pH, vary depending on the cell type and the stimulus employed for cell activation.

Ionic regulation of human basophil releasability. II. Non‐releasing basophils are converted into releasing basophils in a low‐Na+ medium

The effects of different extracellular Na+ and CV2+ concentrations on histamine release from human basophils were investigated. Isosmotic replacement of extracellular Na + either with choline +, a non‐permeant Na + analogue, or glucose significantly increased spontaneous and anti‐IgE‐induced histamine release. Basophils from 12 of 49 normal subjects, which were found not to release histamine upon challenge with an optimal dose of anti‐IgE in a 135 mM NaCl buffer, were converted into releasing basophils when stimulation with anti‐IgE was performed in a low‐Na+ medium. The increase in Na + concentration in the extracellular medium was accompanied by a reduction in the magnitude of basophil response to anti‐IgE, which was significantly more pronounced in non‐releasers than in releasers (per cent inhibition by 70 mM NaCl 75.5 + 3.2 vs 43.5 + 9.0, P < 0.01). At higher Na+ concentrations a progressive and almost complete abrogation of histamine release was observed in non‐releasers, but not in releasers (maximal per cent inhibition at 140 mM NaCl 97.3+1.3 vs 50.4 + 8.6). The Na+/H+ exchanger monensin had a dose‐dependent inhibitory effect on anti‐IgE‐induced histamine release, and the concentration inhibiting 50% of histamine release was l.5 × 10−7M. When basophils were challenged in the presence of different Na+ and C2+ concentrations, it was shown that the two cations have antagonistic effects, which is to say that they down‐regulate and upregulate histamine release, respectively. Moreover, the requirement of extracellular Ca2+ was lowered in a low‐Na+ medium. These results suggest that Na+ and Ca+ ions contribute with opposite effects to the modulation of basophil response to anti‐IgE and that non‐releasing basophils are converted into releasing basophils in a low‐Na + medium.

Impairment of the inhibitory effect of sodium on basophil histamine release in patients with systemic sclerosis.

It has been demonstrated that Na+ down‐regulates IgE‐dependent and IgE‐independent histamine release from basophils of normal subjects. The aim of this study was to evaluate whether Na+ exerts its inhibitory effect on basophil histamine release in patients with systemic sclerosis (SSc). Peripheral blood leucocytes were stimulated with anti‐IgE, n‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) and IL‐3 in the presence of high and low Na+ concentrations, and histamine release was measured by a fluorometric method. The dose–response curves of histamine release induced by the above stimuli were similar in SSc patients (n = 15) and in normal subjects (n = 39). Na+ removal from the extracellular medium and its isosmotic replacement with choline chloride led to a significant increase of anti‐IgE‐and fMLP‐induced histamine release in normal subjects, but not in SSc patients. In the former population, histamine release induced by an optimal dose of anti‐IgE (1/5000) was 26.4 ± 3.1% in high Na+ and 59.3 ± 3.5% in low Na+ (mean ± s.e.m., P < 0.0001), whereas in the latter population mean histamine release was 20.4 ± 5.1% in high Na+ and 15.8 ± 2.9% in low Na+ (P NS). A similar trend was observed when basophils were stimulated with fMLP. Na+ exerted a dose‐dependent inhibitory effect on anti‐IgE‐ and fMLP‐induced histamine release in normal subjects, but not in SSc patients. IL‐3‐induced histamine release from basophils of SSc patients was increased in a low‐Na+ solution, but to a lesser extent when compared with normal controls. Therefore basophils from normal subjects and SSc patients behave in a different way when stimulated in a low‐Na+ medium. The inhibitory effect of Na+ on basophil histamine release is impaired in SSc patients, and this abnormality could contribute to basophil dysfunction.