Release Of Β-glucuronidase Research Articles

Normal neutrophil functions in sphingosine kinase type 1 and 2 knockout mice

Sphingosine kinase (SPHK) has been implicated as an important element in neutrophil responses to diverse stimulatory agents. To get more insight into the role of the type 1 and 2 isoforms of SPHK in neutrophil functions, we made use of the respective SPHK knockout mice. Neutrophils isolated from the bone marrow of these mice showed normal increase of intracellular Ca 2+ when stimulated in vitro by fMLP, platelet-activating factor, the anaphylatoxin C5a, or ATP, and normal migration towards fMLP and C5a. Also, recruitment of neutrophils into the peritoneum towards the chemokines KC and MIP-2 or to LPS, and into the peripheral blood after fMLP injection was similar in SPHK knockout strains and wild-type animals. An in vivo model of bacterial lung infection revealed an accelerated progression of disease in SPHK2 (but not SPHK1) knockout mice as compared to wild-type controls. However, effector functions of SPHK-deficient neutrophils, such as superoxide production, β-glucuronidase release and their capacity to kill bacteria were unchanged as compared to wild-type cells. To conclude, the data derived from SPHK knockout mice do not support the hypothesis that any of the two lipid kinases plays a crucial role in signalling downstream of various neutrophil stimuli; SPHKs appear not to be essential for neutrophil recruitment and effector functions.

Synthesis, cytotoxicity, and anti-inflammatory evaluation of 2-(furan-2-yl)-4-(phenoxy)quinoline derivatives. Part 4

A number of 2-(furan-2-yl)-4-phenoxyquinoline derivatives have been synthesized and evaluated for anti-inflammatory evaluation. 4-[(2-Furan-2-yl)quinolin-4-yloxy]benzaldehyde ( 8), with an IC 50 value of 5.0 μM against β-glucuronidase release, was more potent than its tricyclic furo[2,3- b]quinoline isomer 3a (>30 μM), its 4′-COMe counterpart 7 (7.5 μM), and its oxime derivative 13a (11.4 μM) and methyloxime derivative 13b (>30 μM). For the inhibition of lysozyme release, however, oxime derivative 12a (8.9 μM) and methyloxime derivative 12b (10.4 μM) are more potent than their ketone precursor 7 and their respective tricyclic furo[2,3- b]quinoline counterparts 4a and 4b. Among them, 4-{4-[(2-furan-2-yl)-quinolin-4-yloxy]phenyl}but-3-en-2-one ( 10) is the most active against lysozyme release with an IC 50 value of 4.6 μM, while 8 is the most active against β-glucuronidase release with an IC 50 value of 5.0 μM. ( E)-1-{3-[(2-Furan-2-yl)quinolin-4-yloxy]phenyl}ethanone oxime ( 11a) is capable of inhibiting both lysozyme and β-glucuronidase release with IC 50 values of 7.1 and 9.5 μM, respectively. For the inhibition of TNF-α formation, 1-{3-[(2-furan-2-yl)quinolin-4-yloxy]phenyl}ethanone ( 6) is the most potent with an IC 50 value of 2.3 μM which is more potent than genistein (9.1 μM). For the inhibitory activity of fMLP-induced superoxide anion generation, 11a (2.7 μM), 11b (2.8 μM), and 13b (2.2 μM) are three of the most active. None of above compounds exhibited significant cytotoxicity.

Five New Diels-Alder Type Adducts from the Stem and Root Bark ofMorus mongolica

Five new Diels-Alder type adducts, mongolicins A - E, and nine known compounds, mongolicin F, chalcomoracin, mulberrofuran T, mulberrofuran G, mulberrofuran F, albanol B, kuwanon O, mulberrofuran H and kuwanon H, were isolated from the stem and root bark of Morus mongolica. Their structures were determined by spectroscopic analysis and chiroptical methods. Among them, compounds and showed potent anti-inflammatory activities (inhibition of release of beta-glucuronidase from rat PMNs induced by PAF) with inhibitory ratios of 80.4% (P<0.05) and 77.0% (P<0.001) at a concentration of 10(-5) mol L-1. Compounds and were active as antioxidatives (inhibition of liver microsomal lipid peroxidation induced by Fe2+-Cys system) with inhibitory ratios of 83.6% (P<0.05), 86.0% (P<0.05), and 63.0% (P<0.05) at a concentration of 10(-5) mol L-1.

Inhibition of phospholipase D activation by CYL-26z in formyl peptide-stimulated neutrophils involves the blockade of RhoA activation

5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity, which was assessed by the production of phosphatidylethanol (PEt) in the presence of ethanol, in rat neutrophils (IC 50 1.2 ± 0.2 μM). CYL-26z caused a slight but significant attenuation of the global protein tyrosine phosphorylation stimulated by fMLP only at concentrations of CYL-26z up to 30 μM. CYL-26z blocked the membrane recruitment of protein kinase C-α (PKC-α) at concentrations of CYL-26z ≥3 μM, but failed to affect the membrane association of PKC-βI and -βII. The translocation of RhoA to the membrane was attenuated by CYL-26z (IC 50 3.8 ± 0.8 μM) in fMLP-stimulated neutrophils, whereas CYL-26z caused no significant inhibition of the membrane recruitment of ADP-ribosylation factor (Arf). CYL-26z inhibited the activation of RhoA and dissociation of the RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex in fMLP-stimulated neutrophils (IC 50 1.8 ± 1.0 μM and 1.8 ± 0.9 μM, respectively). In a cell-free system, CYL-26z effectively attenuated the membrane association of RhoA in response to GTPγS (IC 50 1.3 ± 0.5 μM). In contrast, the GTPγS-stimulated translocation of Arf to membrane was suppressed only at concentrations of CYL-26z up to 30 μM. CYL-26z inhibited the fMLP-stimulated membrane expression of CD11b, CD45 and CD63, and the release of lysozyme and β-glucuronidase. These results indicate that CYL-26z inhibited the fMLP-stimulated PLD activity, mainly through the blockade of RhoA activation, and degranulation in rat neutrophils.

New Phenylpropane and Anti-inflammatory Diterpene Derivatives fromAmentotaxus formosana

One new diterpene, 8(14),15-sandaracopimaradiene-2alpha,3beta,18-triol (1), two new phenylpropane derivatives, i.e., (E)-methyl 2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoate (2) and (E)-2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoic acid (3), and two known diterpenes, ent-8(14),15-sandaracopimaradiene-2alpha,18-diol (4) and 8(14),15-sandaracopimaradiene-2alpha,18,19-triol (5), were isolated from the heartwoods and barks of Amentotaxus formosana, respectively. The anti-inflammatory activity of the diterpenes 1, 4, and 5 was assessed in vitro by determining their inhibitory effects on the chemical mediators released from mast cells, neutrophils, macrophages, and microglial cells. Compounds 1, 4, and 5 showed significant concentration-dependent inhibitory effects on the release of beta-glucuronidase from rat neutrophils in response to formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB) with IC50 values of 5.5 +/- 1.8, 8.4 +/- 2.9 and 19.2 +/- 3.3 microM, respectively. Compounds 1 and 5 also showed significant concentration-dependent inhibitory effects on superoxide anion generation in rat neutrophils stimulated with fMLP/CB and phorbol 12-myristate 13-acetate (PMA) with IC50 values of 12.6 +/- 1.2 and 9.4 +/- 1.7, and 10.7 +/- 3.3 and 12.9 +/- 0.9 microM, respectively.

Synthetic chalcones as potential anti-inflammatory and cancer chemopreventive agents

In an effort to develop potent anti-inflammatory and cancer chemopreventive agents, a series of chalcones were prepared by Claisen–Schmidt condensation of appropriate acetophenones with suitable aromatic aldehyde or prepared with appropriate dihydrochalcone reacted with appropriate alkyl bromide or prepared in one-pot procedure involving acetophenone and convenient aromatic aldehyde using ultrasonic agitation on basic alumina. The synthesized products were tested for their inhibitory effects on the activation of mast cells, neutrophils, macrophages, and microglial cells. The potent inhibitors of NO production in macrophages and microglial cells were further evaluated for their in vitro cytotoxic effects against several human cancer cell lines. 2′-Hydroxychalcones 1– 3, and 2′,5′-dihydroxychalcone 7 exhibited potent inhibitory effects on the release of β-glucuronidase or lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Two 2′-hydroxychalcones ( 1 and 3) showed potent inhibitory effects on superoxide anion generation in rat neutrophils in response to fMLP/CB. The previously reported chalcone, 5, 6, and 12, exhibited potent inhibitory effect on NO production in lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-activated N9 microglial cells or in LPS-activated RAW 264.7 macrophage-like cells. The potent inhibitors 5, 6, and 12 of NO production in macrophages or microglial cells revealed significant or marginal cytotoxic effects against several human cancer lines. Compound 12 manifested potent selective cytotoxicity against human MCF-7 cells and caused cell death by apoptosis. The present results demonstrated that 1– 3, and 7 have anti-inflammatory effects and 5, 6, and 12 are potential anti-inflammatory and cancer chemopreventive agents.

Anti-inflammatory Phloroglucinols and Terpenoids from Garcinia subelliptica

Three new phloroglucinols, garcinielliptones K (1), L (2), and M (3), and two new terpenoids, garcinielliptones N (4) and O (5), have been isolated from the seeds of Garcinia subelliptica. The structures of 1-5 including their relative configurations were elucidated by spectroscopic methods and supported by computer-generated molecular modeling. Compounds 2 and 3 showed potent inhibitory effects on the release of beta-glucuronidase, and on beta-glucuronidase and histamine, respectively, from peritoneal mast cells stimulated with p-methoxy-N-methylphenethylamine (compound 48/80) in a concentration-dependent manner. Compounds 2 and 3 showed potent effects on NO production in culture media of RAW 264.7 cells in response to lipopolysaccharide (LPS). Compound 2 also showed a potent effect on NO production in culture media of N9 cells in response to LPS/interferon-gamma (IFN-gamma).

Linear furanocoumarin protects rat myocardium against lipidperoxidation and membrane damage during experimental myocardial injury

The antioxidant activity and the membrane effects of linear furanocoumarin marmesinin isolated from Aegle marmelose was evaluated during experimental myocardial injury. Isoproterenol (150 mg kg –1 intraperitonially twice at an interval of 24 h) caused increase in the levels of serum marker enzymes via creatinekinase (CK), creatinekinase-MB (CK-MB) isoenzyme, lactatedehydrogenase (LDH) and lactatedehydrogenase isoenzyme (LDH 1). It also produced electrocardiographic changes such as increased heart rate, reduced R amplitude and ST elevation. Marmesinin at a dose of 200 mg kg –1, when administered orally, demonstrated a decrease in serum enzyme levels and restored the electrocardiographic changes towards normalcy. Myocardial injury was accompanied by the disintegration of lipidperoxides and the impairment of natural scavengers. Marmesinin oral treatment for 2 days before and during isoproterenol administration decreased the effect of lipidperoxidation. It was also shown to have a membrane stabilizing action by inhibiting the release of β-glucuronidase from the subcellular fractions. Thus, linear furanocoumarin marmesinin could have the protective effect against the damage caused by experimental myocardial injury.

Anti-inflammatory flavonoids and pterocarpanoid from Crotalaria pallida and C. assamica

One new isoflavone, 5,7,4′-trihydroxy-2′-methoxyisoflavone (3) and seven, and four known compounds were isolated from the barks of Crotalaria pallida and the seeds of C. assamica, respectively. The known compounds, apigenin (1) and 2′-hydroxygenistein (2), isolated from C. pallida, showed significant concentration-dependent inhibitory effects on the release of β-glucuronidase and lysozyme from rat neutrophils in response to formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB) with IC50 values of 2.8±0.1 and 17.7±1.9, and 5.9±1.4 and 9.7±3.5 μM, respectively. The known compounds, daidzein (4) and 2′-hydroxydaidzein (6), isolated from C. pallida, inhibited of the release of lysozyme and β-glucuronidase from rat neutrophils in response to fMLP/CB with IC50 values of 26.3±5.5 and 13.7±2.6 μM, respectively. Compounds 1 and 4 also showed significant concentration-dependent inhibitory effects on superoxide anion generation in rat neutrophils stimulated with fMLP/CB with IC50 values of 3.4±0.3 and 25.1±5.0 μM, respectively. Compounds 1 and 5, previously isolated from C. pallida, showed the inhibition of NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and LPS/interferon-γ (IFN-γ)-stimulated N9 microglial cells with IC50 values of 10.7±0.1 and 13.9±1.1 μM, respectively. Flavonoids, suppressed chemical mediators in inflammatory cells, may have value in treatment and prevention of central and peripheral inflammatory diseases associated with excess production of chemical mediators.

The histamine-cytokine network in allergic inflammation

Histamine is synthesized and released by human basophils, mast cells, and neurons. Its pleiotropic effects are mediated by the activation of 4 receptors: H 1, H 2, H 3, and H 4. With the advent of selective antagonists (the antihistamines widely used to treat allergic disorders), the H 1-receptor was the first member of the receptor family to be pharmacologically defined. Increasing evidence indicates that, in addition to exerting immediate vascular and bronchial responses, histamine might modulate the immune reaction by interacting with T cells, macrophages, basophils, eosinophils, and monocytes. We have shown that, in vitro, histamine induces a concentration-dependent release of IL-6 and β-glucuronidase from macrophages isolated from the human lung parenchyma. These effects are inhibited by fexofenadine, an H 1-receptor antagonist, but not by ranitidine, an H 2-receptor antagonist. This observation raises the possibility that long-term treatment with fexofenadine might have beneficial effects on immune dysregulation and tissue damage/remodeling associated with histamine-mediated macrophage activation.

Secretory phospholipases A2 activate selective functions in human eosinophils.

Secretory phospholipases A(2) (sPLA(2)s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA(2). We have examined the effects of two structurally different sPLA(2)s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA(2) induced a concentration-dependent release of beta-glucuronidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition, sPLA(2)s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of the sPLA(2)s examined induced the production of IL-5, the de novo synthesis of leukotriene C(4) and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA(2)s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes' effects. In addition, inactivation of sPLA(2) enzymatic activity by bromophenacyl bromide did not influence the release of beta-glucuronidase or of cytokines. Stimulation of eosinophils by sPLA(2)s was associated with activation of extracellular signal-regulated kinases 1/2. These results indicate that sPLA(2)s selectively activate certain proinflammatory and immunoregulatory functions of human eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.

Structure–activity relationship studies on chalcone derivatives: the potent inhibition of chemical mediators release

Some chalcones exert potent anti-inflammatory activities. 2′,5′-Dialkoxychalcones and 2′,5′-dihydroxy-4-chloro-dihydrochalcone inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-activated N9 microglial cells and in LPS-activated RAW 264.7 macrophage-like cells have been demonstrated in our previous reports. These compounds also suppressed the inducible NO synthase (iNOS) expression and cyclooxygenase-2 (COX-2) activity in RAW 264.7 cells. In an effort to continually develop potent anti-inflammatory agent, a series of chalcones were prepared by Claisen–Schmidt condensation of appropriate acetophenones with appropriate aromatic aldehyde and then evaluated their inhibitory effects on the activation of mast cells, neutrophils, macrophages, and microglial cells. Most of the 2′,5′-dihydroxychaclone derivatives exhibited potent inhibitory effects on the release of β-glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Some chalcones showed potent inhibitory effects on superoxide anion generation in rat neutrophils in response to fMLP/CB. Compounds 1 and 5 exhibited potent inhibitory effects on NO production in macrophages and microglial cells. Compound 11 showed inhibitory effect on NO production and iNOS protein expression in RAW 264.7 cells. The present results demonstrated that most of the 2′,5′-dihydroxychaclones have anti-inflammatory effects. The potent inhibitory effect of 2′,5′-dihydroxy-dihydrochaclones on NO production in LPS-activated macrophage, probably through the suppression of iNOS protein expression, is proposed to be useful for the relief of septic shock.

The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway.

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1beta is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1beta and HMGB1 respond at different times to different stimuli: IL-1beta secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.

Two Novel and Anti-Inflammatory Constituents ofArtocarpus rigida

With the scope of our search for biologically active compounds, two new phenolic compounds, artocarpols G (1) and H (2), and two known compounds, rubraflavone C (3) and trans-stilbene-2,4,3′,5′-tetrol, were isolated from the root bark of Artocarpus rigida. Their structures were determined by spectroscopic methods and comparison with data reported in the literature. Compound 4, previously isolated from this plant, strongly inhibited in a concentration-dependent manner the release of β-glucuronidase and histamine from mast cell degranulation caused by compound 48/80, with IC50 values of 10.9±1.4 and 13.2±0.6 μM, respectively. Compound 4 also showed a concentration-dependent inhibitory effect on the formyl-peptide-stimulated superoxide anion formation in neutrophils with an IC50 value of 26.0±5.6 μM.

CXCL8 (3–73)K11R/G31P antagonizes ligand binding to the neutrophil CXCR1 and CXCR2 receptors and cellular responses to CXCL8/IL-8

We recently reported that CXCL8 (3–73)K11R is a high affinity agonist of neutrophil activation and chemotactic responses. In this report we employed CXCL8 (3–73)K11R as a template to generate CXCL8/IL-8 analogues with antagonist activities, using site-directed mutagenesis to introduce conservative amino acid substitutions into the first turn within the molecule's β-pleated sheet region (G31P, P32G) and, in association with these, into the putative receptor-recognition site (T12S, H13F, F17S). We then examined their impact on the analogues' biological activities and found that a G31P substitution rendered CXCL8 (3–73)K11R a high affinity antagonist of CXCL8/IL-8. The ranking (in the order of decreasing CXCL8/IL-8 antagonist activities) of the CXCL8 (3–73)K11R analogues we generated was, G31P>T12S/G31P>H13F/G31P>T12S/H13F/G31P≫P32G≈T12S/P32G≈H13F/P32G>T12S/H13F/P32G; CXCL8 (3–73)K11R/F17S did not inhibit CXCL8/IL-8-dependent responses. CXCL8 (3–73)K11R/G31P had no discernible agonist (β-glucuronidase release, chemotactic) activity, but at 12.5 ng/ml it bound to purified neutrophils more avidly than did 1.25 μg/ml CXCL8/IL-8. Furthermore, CXCL8 (3–73)K11R/G31P competitively antagonized the binding of CXCR1- and CXCR2-specific antibodies to these receptors. Taken together, these data thus provide further impetus to the study of the potential efficacy of CXCL8 (3–73)K11R/G31P as a broad-spectrum antagonist of the ELR–CXC chemokines in experimental and clinical settings.

Differential Regulation of Neutrophil Phospholipase D Activity and Degranulation

One of the proposed functions of phosphatidic acid (PA) formation from phospholipase D (PLD) activation in neutrophils is to promote degranulation induced by receptor agonists. The present study shows that the time course and dose response of PA formation and degranulation induced by N-formyl–methionyl–leucyl-phenylalanine (fMLP) differed. PLD activation and degranulation also exhibited different dose response to genistein and epigallocatechin gallate (EGCG), inhibitors of protein tyrosine kinases. Genistein inhibited PLD activity with an IC50 value of 12.2 μM in fMLP- and 107 μM in phorbol myristate acetate (PMA)-stimulated cells. It required higher concentrations of genistein to inhibit degranulation than to inhibit PLD activity induced by fMLP. EGCG in the range of 40–400 μM had no effect on PLD activity but it inhibited the release of β-glucuronidase and elastase by fMLP-stimulated cells. These results demonstrate differential regulation of PLD activity and degranulation of primary granules by genistein and EGCG in fMLP-stimulated neutrophils.

Lack of fusion of azurophil granules with phagosomes during phagocytosis of Mycobacterium smegmatis by human neutrophils is not actively controlled by the bacterium.

Biogenesis of phagolysosomes is a very rapid event in neutrophils which takes place with nascent unclosed phagosomes, leading to the release of lysosomal enzymes such as beta-glucuronidase in the extracellular medium. We have previously shown that, under nonopsonic conditions, both pathogenic and nonpathogenic mycobacteria uncouple phagocytosis from fusion of azurophil granules (specialized secretory lysosomes) with phagosomes. In the present study we questioned whether they actively act on neutrophils to block this process or use phagocytic receptors that negatively control the biogenesis of phagolysosomes. As for live unicellular Mycobacterium smegmatis, we observed that nonopsonic phagocytosis of heat-killed mycobacteria did not induce the release of beta-glucuronidase, indicating that M. smegmatis does not actively act on the fusion process in neutrophils. In contrast, phagocytosis of unicellular M. smegmatis opsonized in immune serum or that of small nonopsonized mycobacterial aggregates restored the biogenesis of phagolysosomes. Aggregates were internalized in a CR3- and cholesterol-dependent manner as unicellular mycobacteria. However, aggregates but not unicellular bacteria triggered F-actin and Hck recruitment at the phagosomes, events that have been associated with lysosome fusion. Thus, we propose that M. smegmatis does not actively control the fusion of azurophil granules at early time points postinfection and that mycobacterial aggregates recruit large clusters of receptors at the neutrophil surface which could trap proteins implicated in the biogenesis of phagolysosomes.

IL-8(3–73)K11R Is a High Affinity Agonist of the Neutrophil CXCR1 and CXCR2

In studies aimed at developing a high affinity IL-8 antagonist, our first objective was to generate a high-affinity IL-8 analogue. We targeted amino acids within the receptor-binding domain and found that IL-8(3–73)K11R induced significantly more neutrophil β-glucuronidase release than either IL-8 or the alternate analogues and, in chemotaxis assays, induced 2–3-fold greater neutrophil responses than IL-8. Furthermore, in competitive radio- or biotinylated-ligand binding assays, IL-8(3–73)K11R was more effective than IL-8, IL-8(3–73), or its T12S, H13F, and K11R/T12S/H13F analogues in blocking IL-8 binding to neutrophils; 1.8 pmol IL-8(3–73)K11R inhibited by 50% the binding of ≈20 pmol 125I-IL-8 to neutrophils. Both IL-8 (a CXCR1/CXCR2 ligand) and the CXCR2-specific ligand GROα differentially inhibited binding of 125I-IL-8(3–73)K11R to neutrophils, albeit weakly, suggesting that IL-8(3–73)K11R is a high affinity ligand for both the CXCR1 and CXCR2. Thus IL-8(3–73)K11R is an excellent candidate for further studies aimed at generating a high affinity IL-8 antagonist.

Histamine induces exocytosis and IL-6 production from human lung macrophages through interaction with H1 receptors.

Increasing evidence suggests that a continuous release of histamine from mast cells occurs in the airways of asthmatic patients and that histamine may modulate functions of other inflammatory cells such as macrophages. In the present study histamine (10(-9)-10(-6) M) increased in a concentration-dependent fashion the basal release of beta-glucuronidase (EC(50) = 8.2 +/- 3.5 x 10(-9) M) and IL-6 (EC(50) = 9.3 +/- 2.9 x 10(-8) M) from human lung macrophages. Enhancement of beta-glucuronidase release induced by histamine was evident after 30 min and peaked at 90 min, whereas that of IL-6 required 2-6 h of incubation. These effects were reproduced by the H(1) agonist (6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane carboxamide but not by the H(2) agonist dimaprit. Furthermore, histamine induced a concentration-dependent increase of intracellular Ca(2+) concentrations ([Ca(2+)](i)) that followed three types of response, one characterized by a rapid increase, a second in which [Ca(2+)](i) displays a slow but progressive increase, and a third characterized by an oscillatory pattern. Histamine-induced beta-glucuronidase and IL-6 release and [Ca(2+)](i) elevation were inhibited by the selective H(1) antagonist fexofenadine (10(-7)-10(-4) M), but not by the H(2) antagonist ranitidine. Inhibition of histamine-induced beta-glucuronidase and IL-6 release by fexofenadine was concentration dependent and displayed the characteristics of a competitive antagonism (K(d) = 89 nM). These data demonstrate that histamine induces exocytosis and IL-6 production from human macrophages by activating H(1) receptor and by increasing [Ca(2+)](i) and they suggest that histamine may play a relevant role in the long-term sustainment of allergic inflammation in the airways.

Histamine-Induced Activation of Human Lung Macrophages

Background: Histamine plays a central role in the pathogenesis of allergic and inflammatory diseases by modulating vascular and airway responses. Increasing evidence suggests that histamine also regulates the function of inflammatory and immune cells. Macrophages are primarily involved in inflammatory diseases of the lung. We explored the ability of low concentrations of histamine to induce the release of proinflammatory mediators from human lung macrophages. Methods: Macrophages purified (>95%) from lung parenchyma by Percoll density gradients and adherence to polystyrene dishes were incubated (37°C, 2–24 h) with histamine (10^–9–10^–6M). At the end of incubation, the release of β-glucuronidase and IL-6 was determined. Results: Histamine induced a concentration-dependent release of β-glucuronidase and IL-6 with a maximum release after 2 and 6 h of incubation, respectively. Exocytosis induced by histamine was noncytotoxic and was Ca²⁺- and temperature-dependent. The effect of histamine was inhibited by the H₁ receptor antagonist fexofenadine but not by the H₂ antagonist ranitidine. Conclusions: These data indicate that histamine is an effective stimulus for exocytosis and cytokine production from human lung macrophages. These effects are inhibited by pharmacological concentrations of fexofenadine. Our results suggest that histamine may contribute to the long-term evolution of lung inflammation and tissue remodelling in allergic diseases by modulating the production of proinflammatory and immunoregulatory mediators by macrophages.