FMLP-induced Histamine Release Research Articles

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+or-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.

Effects of protein kinase C and phospholipase C inhibitors on IgE-dependent and IgE-independent basophil histamine release.

This study was aimed to evaluate the effects of two protein kinase C (PKC) inhibitors (staurosporine and chelerythrine) and one phospholipase C (PLC) inhibitor (U73122) on basophil histamine release induced by anti-IgE, N-formyl-methionyl-leucyl-phenylalanine (FMLP), IL-3 and GM-CSF. Leukocytes were suspended in solutions containing physiological or low Na+ concentrations, since IL-3 and GM-CSF were shown to induce histamine release only when the inhibitory effect of extracellular Na+ has been removed. After incubation with PKC and PLC inhibitors, the stimuli were added and histamine release was measured by an automated fluorometric method. Staurosporine and chelerythrine exerted a significant inhibitory effect on histamine release induced by anti-IgE, IL-3 and GM-CSF at concentrations much higher than those required to inhibit PKC. FMLP-induced histamine release in a physiological Na+-containing medium was not significantly modified by staurosporine, although it was reduced by high concentrations of chelerythrine. A slight inhibition by high concentrations of staurosporine was found when basophils were suspended in a low Na+ medium. U73122 exerted a significant and dose-dependent inhibitory effect on basophil histamine release induced by anti-IgE, FMLP, IL-3 and GM-CSF. These results suggest that a prodegranulatory role of PKC in basophil histamine release induced by anti-IgE, FMLP, IL-3 and GM-CSF is unlikely; conversely, it is conceivable that PLC has a role in signal transduction and histamine release induced by the above stimuli.

Priming and inducing effects of interleukin‐3 on histamine release from cord‐blood basophils

The effect of interleukin-3 (IL-3) on histamine release from cord and adult blood basophils were evaluated. Leukocyte suspensions, obtained from adult patients with respiratory allergy (n = 15), normal adult subjects (n = 15), and neonates with (n = 15) and without (n = 19) atopic disposition, were stimulated with anti-IgE, fMLP, and IL-3. IgE-mediated histamine release was significantly higher in adult patients, either allergic or normal, than in neonates with or without atopic disposition. A trend toward higher fMLP-induced histamine release was found in allergic adult subjects. IL-3 had a weak direct histamine-releasing activity in allergic adult subjects and in neonates, but not in normal adult donors. A significant enhancing effect of IL-3 on histamine release induced by anti-IgE was observed in neonates with and without atopic disposition and in normal adult subjects, but not in atopic adult patients. IL-3 exerted a priming effect also when basophils were stimulated with fMLP, without any significant difference between neonates and adult subjects. Passive sensitization with IgE-rich serum resulted in a significant increase in anti-IgE-induced, but not in IL-3-induced, histamine release from cord-blood basophils. In conclusion, IL-3 primes cord-blood as well as adult blood basophils for a consecutive anti-IgE- or fMLP-induced histamine release and its activity is not limited by the low density of membrane IgE in cord-blood basophils.

Defective inhibition of sodium on basophil histamine release in patients with allergic rhinitis and bronchial asthma.

The aim of this study was to evaluate whether Na+ exerts its inhibitory effect on basophil histamine release induced by immunoglobulin E (IgE)-dependent (anti-IgE) and IgE-independent (N-formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin-3 (IL-3)) stimuli in patients with allergic rhinitis (n = 24) and allergic bronchial asthma (n = 10). Peripheral blood leucocytes were stimulated with anti-IgE, FMLP and IL-3 in the presence of high and low Na+ concentrations, and histamine release was measured using a fluorometric method. In standard Na(+)-containing medium, spontaneous and stimulated histamine release was higher in allergic patients (n = 34) (both rhinitic and asthmatic) than in healthy subjects (n = 41). Na+ removal from extracellular medium and its isosmotic substitution with choline chloride or with N-methyl-D-glucamine led to a significant increase of anti-IgE-, FMLP- and IL-3-induced histamine release in normal subjects, but not in allergic patients. The increase in Na+ concentration in the extra-cellular medium was accompanied by a dose-dependent decrease of anti-IgE- and FMLP-induced histamine release in normal subjects, but not in allergic patients. The behaviour of atopics and healthy subjects was different and not related to the basophil responsiveness to activating signals. The incubation of basophils from healthy subjects with sera from allergic patients did not have a significant influence on the inhibitory effect of Na+. Basophils from healthy subjects and atopic patients respond differently when stimulated in a low Na+ medium. The reduced sensitivity to the inhibitory effect of Na+ may contribute to basophil dysfunction in patients with respiratory allergy.

Enhancement of basophil histamine release by interleukinl‐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 microM), and IL-3 (0.1-10 ng/ml), and histamine concentration was measured by an automated fluorometric method. A trend toward higher 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.

Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by N-formyl-methionyl-leucyl-phenylalanine

Background:Cyclosporin A (CsA) binds with high affinity to cyclophilin, a critical step in the molecular mechanism of action of cyclosporins, whereas cyclosporin H (CsH) has extremely low affinity for cyclophilin. CsH differs from CsA by the substitution of the L-methyl valine at position 11 with its D-isomer. Methods: We compared the effects of CsA and CsH on the release of preformed (histamine) and de novo synthesized inflammatory mediators (peptide leukotriene C 4) from peripheral blood basophils activated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). Results: CsH (8 to 800 nmol/L) concentration-dependently inhibited histamine and leukotriene C 4 release from purified and unpurified basophils activated by FMLP, whereas CsA (8 to 800 nmol/L) had little inhibitory effect on histamine release from basophils challenged with FMLP. Inhibition of histamine release from basophils challenged with FMLP was extremely rapid and was abolished by washing the cells (three times) before challenge. CsH (8 to 800 nmol/L) had no effect on the release of histamine caused by C5a, platelet activating factor, monocyte chemotactic activating factor, RANTES, IL-8, bryostatin 1, and phorbol myristate. Preincubation of basophils with granulocyte-macrophage colony-stimulating factor (30 and 100 pmol/L), but not IL-1β (30 and 100 ng/ml), concentration-dependently reversed the inhibitory effect of CsH on FMLP-induced histamine release. CsH competitively inhibited the effect of FMLP on histamine release from basophils. The dissociation constant (K d) for the CsH-FMLP receptor complex was approximately 9 × 10 -8 mol/L, more than 10-fold lower than that (≅ 1.3 × 10 -6 mol/L) of N-t-BOC-methionyl-L-leucyl-phenylalanine (BocMLP), a known formyl peptide receptor antagonist. CsH inhibited tritiated FMLP binding to human polymorphonuclear leukocytes with a concentration required to inhibit binding by 50% of approximately 5.4 × 10 -7 mol/L, whereas BocMLP was less potent with a concentration required to inhibit binding by 50% of approximately 9.1 × 10 -5 mol/L. Scatchard analysis revealed that the decreased tritiated FMLP binding caused by CsH was due to a decrease in the B max (0.22 ± 0.04 nmol/L/5 × 10 6 cells vs 0.09 ± 0.01 nmol/L/5 × 10 6 cells; p < 0.05), without a significant difference in the K d (5.16 ± 1.22 nmol/L vs 6.32 ± 2.42 nmol/L; p = NS). Conclusions: CsH is a potent and selective inhibitor of mediator release from basophils induced by activation of the formyl peptide receptor; it acts by interfering with agonist binding to FMLP receptors. (J A LLERGY C LIN I MMUNOL 1996;98:152-64.)

Inducing and Enhancing Effects of IL-3, -5, and -6 and GM-CSF on Histamine Release from Human Basophils

The effects of interleukin (IL)-2, -3, -4, -5, -6, and -7 and granulocyte-macrophage colony-stimulating factor (GMCSF) on histamine release from human basophils were evaluated. IL-3 was the only cytokine with histamine-releasing activity. This activity was observed predominantly on basophils from allergic patients (mean release ± SEM, 33.9 ± 9.5%; n = 12), whereas basophils from normal subjects responded less frequently to stimulation with IL-3 (mean release ± SEM, 2.8 ± 1.0%; n = 22). The effect of IL-3 was time and temperature dependent, since release was optimal after incubation for 120 rain at 37°C. When cell-bound IgE were eluted at acid pH, basophils became unresponsive to IL-3; however, IL-3-induced histamine release correlated with anti-IgE-induced histamine release in allergics, but not in normals. IL-3, IL-5, IL-6, and GM-CSF enhanced significantly anti-IgE- and FMLP-induced histamine release. In contrast, IL-2, IL-4, and IL-7 were devoid of any significant histamine-releasing or -potentiating activity. These results indicate that IL-3 can induce and IL-3, -5, and -6 and GMCSF can enhance histamine release from human basophils, suggesting a possible role of these cytokines in the expression of allergic reactions.

Pharmacological investigations with different protein kinase C inhibitors on IgE-dependent and IgE-independent activation of human basophils.

In the present study different selective inhibitors of the multifunctional serine/threonine kinase protein kinase C (PKC) were investigated on classical activation pathways of basophils in comparison to the nonselective protein kinase inhibitor staurosporine. The potent inhibitors Ro 31-7549, Ro 31-8220, calphostin C and ilmofosine (BM 41.440), which show selectivity for PKC in vitro, significantly potentiated Fc epsilon RI-mediated histamine release up to 50% vs. controls at concentrations > 10(-7) mumol/l but were without any intrinsic histamine releasing activity. Direct activation of cellular PKC by phorbol ester was suppressed by all compounds apart from ilmofosine at the same concentrations. We did not observe statistically significant effects of selective PKC inhibitors on exocytosis induced by the peptide formylmeth-leu-phe (FMLP) or the ionophore A23187, whereas staurosporine potentiated the FMLP-induced histamine release in a dose-dependent fashion: maximum potentiation was 63.5 +/- 8.9% vs. control at 1 mumol/l (n = 4). The findings suggest that PKC exhibits differential functions during biochemical events of stimulus-secretion coupling in human basophils supposedly by its distinct subtypes. With respect to the present data, TPA-induced and IgE-mediated signals are not closely correlated.

Differential activation of human basophils by anti‐IgE and formyl‐methionyl‐leucylphenylalanine. Indications for protein kinase C‐dependent and ‐independent activation pathways

Upon activation, basophilic granulocytes release inflammatory mediators such as histamine. We studied histamine release of human basophils (64.1 +/- 9.6% pure) after cross-linking of membrane-bound IgE via anti-IgE, or after binding of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). A variability in the extent of histamine release upon stimulation by either anti-IgE or fMLP was found between donors. Kinetic studies revealed that the histamine release induced by anti-IgE (t1/2 greater than 240 s) was more than 20-fold slower than the almost instantaneous release upon stimulation with fMLP (t1/2 less than 10 s). Differences in the cell activation pathways triggered by these stimuli were further analyzed with staurosporine, an inhibitor of protein kinase C (PKC) and with wortmannin, an inhibitor of a PKC-independent pathway. Inhibition of PKC resulted in a partial inhibition of the anti-IgE-induced release, whereas the fMLP-induced release was slightly potentiated. The anti-IgE-induced release was completely inhibited in the presence of wortmannin. This inhibitor too, had no effect on the fMLP-induced release. We conclude that major differences exist in the signal-response coupling between the anti-IgE and fMLP-induced histamine release in human basophils. The so-called releasability of human basophils may be due to the availability of different cell activation pathways.

Differential mechanisms in the stimulus-secretion coupling in human basophils: Evidence for a protein-kinase-C-dependent and a protein-kinase-C-independent route

Upon activation, basophilic granulocytes release inflammatory mediators, such as histamine. We studied histamine release (HR) of purified (64 +/- 10%) human basophils after cross-linking of membrane-bound IgE via anti-IgE or after binding of the chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). A variability in the extent of histamine release upon stimulation by either anti-IgE or FMLP was found between donors. Non-responders for FMLP showed high histamine release for anti-IgE, and vice versa. Inhibition of protein kinase C (PKC) by staurosporine (STSP) resulted in partial inhibition of the anti-IgE-induced HR, whereas inhibition of a PKC-independent pathway by wortmannin (WTM) totally blocked the anti-IgE induced histamine release. The HR induced by FMLP was not affected by either of these inhibitors. We conclude that major differences exist in the signal-response coupling between the anti-IgE and FMLP-induced HR in human basophils. The so-called releasability of human basophils may be due to the availability of different cell activation pathways.

Differential Effect of Hypertonicity on FMLP-, Anti-IgE- and Ca2+ Ionophore A23187-Induced Histamine Release from Human Basophil Leukocytes

Effects of different extracellular Na+ and K+ concentrations (respectively, 135, 155, 220, 260 mM NaCl, and 2.7, 20, 50, 100 mM KCl) on IgE-dependent and IgE-independent histamine release from human basophils were examined. High extracellular Na+ and K+ concentrations were shown to reduce N-formyl-methionyl-leucyl-phenyl-alanine- (FMLP), but not anti-IgE- or Ca2+ ionophore A23187-induced histamine release. A high extracellular Ca2+ (7.2 mM CaCl2) concentration increased basophil response to anti-IgE and FMLP. The enhancement of FMLP- but not of anti-IgE-induced histamine release was antagonized by high extracellular Na+ and K+ concentrations. When leukocytes were suspended in isotonic choline chloride solutions (choline is a nonpermeant monovalent cation), an enhancement of anti-IgE- and FMLP-induced histamine release was observed. This suggests that monovalent cations, namely Na+ ions, at physiological concentrations, downregulate histamine release from human basophils. At high choline chloride concentrations, FMLP-, but not anti-IgE-induced histamine release was inhibited. Thus, the reduction of FMLP-evoked histamine secretion from human basophils seems to be due to hypertonicity and not to the type of monovalent cation, either permeant or nonpermeant, contained in extracellular milieu. The different effects of a hypertonic solution on anti-IgE and FMLP-induced histamine release are probably related to the different cell activation pathways triggered by the two stimuli.

Differential Inhibition of Histamine Release from Human Basophils Induced by Antigen, Anti-IgE or N-formyl-&lt;i&gt;L&lt;/i&gt;-methionyl-&lt;i&gt;L&lt;/i&gt;-leucyl-&lt;i&gt;L&lt;/i&gt;-phenylalanine

We report differential effects of various compounds on inhibition of histamine release from washed leukocytes stimulated through IgE (using antigen or anti-IgE antibody) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors. Inhibition of IgE-induced release was seen for several compounds listed in order of potency (IC50 in microM): CI-922 (3,7-dimethoxy-4-phenyl-N-(1-H-tetrazol-5-yl)-4-H-furo-[3, 2-b]-indole-2-carboxamide, 1-arginate) less than NDGA less than BW755c less than proxicromil less than isamoxole less than phenidone. Other compounds including FPL 55712, meclofenamate, and indomethacin were inactive or enhanced IgE-mediated release. When FMLP was used to stimulate histamine release the order of potency (IC50) changed: meclofenamate = FPL 55712 = proxicromil less than CI-922 less than NDGA = BW755c less than indomethacin less than isamoxole = phenidone. The inhibition of FMLP-induced histamine release by FPL 55712, meclofenamate and indomethacin in the absence of inhibition (or even enhancement) of histamine release by the IgE mechanism suggests that different pathways of activation and/or control are critical in the release process when different stimuli are used to activate basophils.