Guinea Pig Eosinophils Research Articles

Lysophosphatidic Acid Enhances In Vivo Infiltration and Activation of Guinea Pig Eosinophils and Neutrophils via a Rho/Rho-Associated Protein Kinase-Mediated Pathway

Lysophosphatidic acid (LPA) has been shown to be a chemoattractant in in vitro studies. The present study was carried out to determine whether LPA enhances infiltration of inflammatory cells in in vivo studies with guinea pigs. LPA (1 – 10 μg/ml), when by guinea pigs for 5 min, substantially increased the numbers of eosinophils and neutrophils in the bronchoalveolar lavege fluid (BALF), which was recovered at over 4 h after the inhalation of LPA. Infiltration in BALF was significantly inhibited by inhalation of Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). LPA also increased superoxide production of eosinophils and neutrophils. In contrast, Y-27632 inhibited superoxide production. These findings suggest that LPA may contribute to infiltration and activation of inflammatory cells in bronchial asthma; furthermore, the Rho/ROCK-mediated pathway may be involved.

An estimation of β2‐adrenoceptor reserve on human bronchial smooth muscle for some sympathomimetic bronchodilators

The beta(2)-adrenoceptor on human pro-inflammatory cells is exquisitely sensitive to desensitization, whereas beta(2)-adrenoceptor-mediated relaxation of human airways smooth muscle (HASM) is relatively resistant to this phenomenon. An explanation for this discrepancy is that a large beta(2)-adrenoceptor 'reserve' exists on HASM cells for sympathomimetic bronchodilators, which protects against desensitization. The operational model of agonism was used to estimate the affinity of salbutamol, terbutaline, formoterol and procaterol for the beta(2)-adrenoceptors in methacholine (MCh)-contracted HASM from which the relationship between fractional receptor occupancy and relaxation was determined. This analysis was performed under conditions of fractional, irreversible, beta(2)-adrenoceptor inactivation and, for salbutamol and terbutaline only, by the comparative method of Barlow et al. The affinity of salbutamol for the beta(2)-adrenoceptor guinea-pig eosinophils and the receptor/occupancy-response relationship for the suppression of the respiratory burst (an index of pro-inflammatory cell function) was also determined. For salbutamol and terbutaline, both pharmacological approaches yielded in HASM discrepant affinity estimates (values differed, maximally, by 0.67 log(10) unit). However, affinity values more closely agreed (difference <0.47 log(10) unit), when operational analysis was performed on data corrected for 'fade' of the MCh-induced contraction. Plots of fractional beta(2)-adrenoceptor occupancy versus relaxation indicated a receptor 'reserve' for all agonists tested at all levels of response. In contrast, minimal receptor reserve was detected for the ability of salbutamol to suppress respiratory burst activity in eosinophils. These data may help explain the relative inability of sympathomimetic bronchodilators to render HASM tolerant to beta(2)-adrenoceptor-mediated relaxation.

Pharmacological characterization of guinea pig chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)

Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a G protein-coupled receptor activated by prostaglandin D 2 (PGD 2), has been identified as a receptor expressed on cell types critical to the pathogenesis of asthma. The cDNA encoding guinea pig CRTH2 was cloned and mRNA expression examined in selected tissues. Transcript profiling of guinea pig CRTH2 indicated relatively high levels of expression in bone marrow, intermediate levels in brain and relatively low levels in lung, spleen, thymus, lymph node, etc. Characterization of the molecular pharmacology of guinea pig CRTH2 revealed that guinea pig CRTH2 exhibited a greater affinity for Δ 12-PGJ 2, a stable PGD 2 metabolite relative to human CRTH2. The CRTH2 selective agonists 13,14-dihydro-15-keto PGD 2 and Δ 12-PGJ 2 induced the recruitment of eosinophils following intradermal administration of these ligands in guinea pigs. Chemotaxis of guinea pig eosinophils was elicited by either PGD 2 or Δ 12-PGJ 2, and was abolished by a CRTH2-specific antagonist. These results indicate that PGD 2 and the stable metabolite, Δ 12-PGJ 2, play important roles in CRTH2 activation in the guinea pig.

Rapid accumulation of eosinophils in lung lesions in guinea pigs infected with Mycobacterium tuberculosis.

Guinea pig eosinophils were positively identified in bronchoalveolar lavage populations and in the lung granulomas of Mycobacterium tuberculosis-infected guinea pigs. It is possible that the rapid influx of these cells, and their subsequent degranulation during acute pulmonary tuberculosis, may play a key role in the susceptibility of this animal model.

Role of Src in hypersensitization to phosphodiesterase inhibitors in beta2-adrenoceptor-desensitized eosinophils.

When guinea pig eosinophils were incubated with fenoterol, a beta2-agonist, for 120 min, not only desensitization of beta2-adrenoceptors but also hyperresponsiveness to phosphodiesterase (PDE) inhibitors, such as theophylline and rolipram, was observed. The fenoterol-induced beta2-adrenoceptor desensitization was not affected by pretreatment with either genistein, a broad-spectrum tyrosine kinase inhibitor, or PP2, a specific Src family tyrosine kinase inhibitor. On the other hand, both genistein and PP2 abolished the hyperresponsiveness to PDE inhibitors in beta2-adrenoceptor-desensitized eosinophils. These findings suggested that Src family tyrosine kinases play important roles in the hypersensitization of PDE to PDE inhibitors in beta2-adrenoceptor-desensitized eosinophils.

Lysophosphatidic acid enhances in vivo infiltration and activation of guinea pig eosinophils and neutrophils via a Rho/Rho-associated protein kinase-mediated pathway.

Lysophosphatidic acid (LPA) has been shown to be a chemoattractant in in vitro studies. The present study was carried out to determine whether LPA enhances infiltration of inflammatory cells in in vivo studies with guinea pigs. LPA (1 - 10 microg/ml), when by guinea pigs for 5 min, substantially increased the numbers of eosinophils and neutrophils in the bronchoalveolar lavege fluid (BALF), which was recovered at over 4 h after the inhalation of LPA. Infiltration in BALF was significantly inhibited by inhalation of Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). LPA also increased superoxide production of eosinophils and neutrophils. In contrast, Y-27632 inhibited superoxide production. These findings suggest that LPA may contribute to infiltration and activation of inflammatory cells in bronchial asthma; furthermore, the Rho/ROCK-mediated pathway may be involved.

The iodocyanopindolol and SM-11044 binding protein belongs to the TM9SF multispanning membrane protein superfamily

SM-11044 is the only β-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus. We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J. Biol. Chem. 272 (1997) 21244). The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA. This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains. The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF). Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF. The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion. Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined. Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide. Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of [ 125I]iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner. Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

Desensitization of β2-adrenoceptor and hypersensitization to phosphodiesterase inhibitors elicited by β2-agonists in guinea pig eosinophils

Background: Although the existence of functional β2-adrenoceptor on eosinophils has been reported, the effects of desensitization of β2-adrenoceptors on eosinophils have not been well documented. Objective: The effects of desensitization of β2-adrenoceptors on the degranulation of guinea pig eosinophils were investigated. Methods: Guinea pig eosinophils were stimulated with the calcium ionophore A23187, and eosinophil peroxidase (EPO) release was determined. Changes in intracellular cyclic adenosine monophosphate (cAMP) levels were also measured. Results: A23187-induced EPO release from guinea pig eosinophils was inhibited in a concentration-dependent manner by pretreatment for 5 minutes with fenoterol, clenbuterol, and salbutamol. Such effects of β2-agonists were abolished by pretreatment with KT5720, an inhibitor of protein kinase A. Desensitization of the inhibitory effects of β2-agonists was observed when the incubation time was prolonged. Fenoterol (10–6 mol/L) induced almost complete desensitization after 120 minutes of incubation, whereas clenbuterol did not bring about significant desensitization. The inhibitory effects of fenoterol and clenbuterol on A23187-induced EPO release were correlated with increases in the intracellular cAMP levels evoked by either compound. After incubation of eosinophils with 10–6 mol/L fenoterol for 120 minutes to induce complete desensitization of β2-adrenoceptors, the inhibitory effects of theophylline and rolipram were increased by about 100-fold in the desensitized cells, although the effects of forskolin and dibutyryl cAMP were not affected by β2-adrenoceptor desensitization. Conclusions: Prolonged incubation with β2-agonists induced desensitization of β2-adrenoceptors. Also, we postulated that hypersensitization of phosphodiesterase to its inhibitors occurs in β2-adrenoceptor–desensitized guinea pig eosinophils. (J Allergy Clin Immunol 2000;106:896-903.)

Pleiotropic role of lyn kinase in leukotriene B4–induced eosinophil activation

The authors have examined the role of the src-family of protein tyrosine kinases in leukotriene B4(LTB4)–induced activation of guinea-pig eosinophils. Western blot analysis identified the src-like protein tyrosine kinases p53lyn, p56lyn, p56/59hck, p55fgr, and p56lck whereas p60src, p62yes, p55blk, and p59fyn were not detected. LTB4 promoted a rapid increase in p53/56lyn activity in eosinophils, which peaked at 5 seconds and remained elevated at 60 seconds; hck, fgr, and lck were not activated. A role for p53/56lyn in eosinophil activation was investigated with the use of the src-selective inhibitor PP1 (1 μmol/L to 10 μmol/L), which attenuated LTB4-stimulated p53/56lyn activity and the phosphorylation of extracellular signal-regulated kinase–2 in intact cells. At comparable concentrations, PP1 was also shown to attenuate LTB4-induced nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase activation, chemotaxis, and Ca++-dependent [3H]arachidonic acid (AA) release. Moreover, an inhibitor of mitogen-activated protein kinase kinase-1, PD 098059, significantly inhibited LTB4-induced chemotaxis but had no effect on oxidant production or [3H]AA release. Collectively, these results implicate lyn kinase in LTB4-induced eosinophil activation through the recruitment of divergent cell-signaling pathways.

Pleiotropic role of lyn kinase in leukotriene B4–induced eosinophil activation

AbstractThe authors have examined the role of the src-family of protein tyrosine kinases in leukotriene B4(LTB4)–induced activation of guinea-pig eosinophils. Western blot analysis identified the src-like protein tyrosine kinases p53lyn, p56lyn, p56/59hck, p55fgr, and p56lck whereas p60src, p62yes, p55blk, and p59fyn were not detected. LTB4 promoted a rapid increase in p53/56lyn activity in eosinophils, which peaked at 5 seconds and remained elevated at 60 seconds; hck, fgr, and lck were not activated. A role for p53/56lyn in eosinophil activation was investigated with the use of the src-selective inhibitor PP1 (1 μmol/L to 10 μmol/L), which attenuated LTB4-stimulated p53/56lyn activity and the phosphorylation of extracellular signal-regulated kinase–2 in intact cells. At comparable concentrations, PP1 was also shown to attenuate LTB4-induced nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase activation, chemotaxis, and Ca++-dependent [3H]arachidonic acid (AA) release. Moreover, an inhibitor of mitogen-activated protein kinase kinase-1, PD 098059, significantly inhibited LTB4-induced chemotaxis but had no effect on oxidant production or [3H]AA release. Collectively, these results implicate lyn kinase in LTB4-induced eosinophil activation through the recruitment of divergent cell-signaling pathways.

Regulatory Mechanism of Eosinophil Peroxidase Release From Guinea Pig Eosinophils

The regulatory mechanism of degranulation of guinea pig peritoneal eosinophils was studied by determination of eosinophil peroxidase (EPO) release. β-Agonists, such as isoproterenol, salbutamol and fenoterol, effectively inhibited A23187-induced EPO release from guinea pig eosinophils. The inhibitory effects of β-agonists were attenuated by pretreatment with either propranolol, a non-selective β-antagonist, or ICI 118, 551, a selective β2-antagonist. Both theophylline and dibutyryl-cAMP (db-cAMP) also significantly inhibited A23187-induced EPO release. The inhibition of EPO release induced by db-cAMP was attenuated by pretreatment with KT5720, a protein kinase A inhibitor. In addition, calphostin C as well as cytochalasin D effectively inhibited A23187-induced EPO release. From the results of the present study, it was concluded that an increase in intracellular Ca2+ concentration may lead to exocytosis of eosinophil granules through activation of protein kinase C and microfilaments. β-Agonists and theophylline were effective in inhibiting degranulation of eosinophils by increasing intracellular cAMP level coupled with the activation of protein kinase A.

Arachidonate 5-lipoxygenase and cyclooxygenase metabolites from guinea pig eosinophils and macrophages.

To investigate the cellular sources of arachidonate metabolites during asthma, in vitro productions of thromboxane (TX) B2, prostaglandin (PG) D2, leukotriene (LT) B4 and cysteinyl (Cys) LTs from guinea pig eosinophils and macrophages simultaneously stimulated with the Ca ionophore A23187 were investigated. Eosinophils produced high levels of LTB4 and TXB2 and relatively low levels of CysLTs and PGD2. Although macrophages released abundant TXB2 and a little PGD2, 5-lipoxygenase metabolites were below detectable levels. In conclusion, eosinophils produced both cyclooxygenase and 5-lipoxygenase metabolites, while arachidonate metabolism in the macrophages was almost completely inclined toward the cyclooxygenase pathway.

Regulatory mechanism of eosinophil peroxidase release from guinea pig eosinophils.

The regulatory mechanism of degranulation of guinea pig peritoneal eosinophils was studied by determination of eosinophil peroxidase (EPO) release. Beta-agonists, such as isoproterenol, salbutamol and fenoterol, effectively inhibited A23187-induced EPO release from guinea pig eosinophils. The inhibitory effects of beta-agonists were attenuated by pretreatment with either propranolol, a non-selective beta-antagonist, or ICI 118,551, a selective beta2-antagonist. Both theophylline and dibutyryl-cAMP (db-cAMP) also significantly inhibited A23187-induced EPO release. The inhibition of EPO release induced by db-cAMP was attenuated by pretreatment with KT5720, a protein kinase A inhibitor. In addition, calphostin C as well as cytochalasin D effectively inhibited A23187-induced EPO release. From the results of the present study, it was concluded that an increase in intracellular Ca2+ concentration may lead to exocytosis of eosinophil granules through activation of protein kinase C and microfilaments. Beta-agonists and theophylline were effective in inhibiting degranulation of eosinophils by increasing intracellular cAMP level coupled with the activation of protein kinase A.

Arachidonate 5-Lipoxygenase and Cyclooxygenase Metabolites From Guinea Pig Eosinophils and Macrophages

Arachidonate 5-Lipoxygenase and Cyclooxygenase Metabolites From Guinea Pig Eosinophils and Macrophages

Role of arachidonic acid in leukotriene B 4-induced guinea-pig eosinophil homotypic aggregation

The activation of eosinophils with the lipid mediator, leukotriene B 4, induces their homotypic aggregation. Upon activation with leukotriene B 4, eosinophils release a significant amount of arachidonic acid, a process dependent on the activation of phospholipase A 2. Here, we have evaluated whether arachidonic acid could induce aggregation of eosinophils and whether the release of arachidonic acid mediated the aggregation induced by leukotriene B 4. The exogenous administration of arachidonic acid induced a concentration-dependent eosinophil homotypic aggregation. Pretreatment of eosinophils with a 5-lipoxygenase inhibitor or a leukotriene B 4 receptor antagonist abrogated arachidonic-acid-induced aggregation. Arachidonic acid induced a significant increase in leukotriene B 4 levels and desensitised leukotriene B 4-induced aggregation in a dose-dependent manner. Moreover, this desensitisation was effectively reversed by a 5-lipoxygenase inhibitor. However, arachidonic acid failed to induce a rise in intracellular Ca 2+ in eosinophils and failed to desensitise these cells to rises in intracellular Ca 2+ induced by leukotriene B 4. Pretreatment of eosinophils with the phospholipase A 2 inhibitor, mepacrine, inhibited the aggregation responses induced by 1 nM leukotriene B 4 by approximately 50% but had no significant effect on the other concentrations of leukotriene B 4 tested (0.1 to 100 nM). In conclusion, arachidonic acid stimulates eosinophil aggregation indirectly via the release of leukotriene B 4. Although a significant amount of arachidonic acid is released in response to activation of eosinophils with leukotriene B 4, the arachidonic acid released does appear to play a major role in mediating leukotriene B 4-induced eosinophil aggregation.

Expression and translocation of Rac2 in eosinophils during superoxide generation.

Eosinophils induce tissue injury by releasing granule-associated cytotoxic proteins, lipid mediators and superoxide anions in response to appropriate stimuli. Superoxide generation associated with respiratory burst is largely dependent on the assembly of the NADPH oxidase complex in the membrane, consisting of membrane-bound cytochrome b558 and translocated p47phox and p67phox. The activation of this complex is critically dependent on the translocation of GTP-bound Rac1, or its homologue Rac2, from the cytosol to the membrane in neutrophils. Rac expression has not yet been fully characterized in eosinophils. We proposed that eosinophils translate and express Rac2 and its GDP-dissociation inhibitor, RhoGDI. Furthermore, we hypothesized that Rac2 translocates along with p47phox and p67phox proteins from the cytosol to the plasma membrane during respiratory burst. By reverse transcription-polymerase chain reaction analysis and sequencing of the amplified product, guinea-pig eosinophils were found to express Rac2 mRNA, exhibiting 93% homology with the human Rac2 sequence. Rac1 mRNA was also detected in eosinophils but not its translated product. In contrast, Rac2 protein expression was detected using a specific antibody. In subcellular fractions, Rac2 was found to translocate, along with p47phox and p67phox, from cytosol to plasma membrane-associated fractions following phorbol myristate acetate stimulation, while RhoGDI remained within cytosolic fractions. These findings suggest that Rac2 is preferentially expressed and activated in eosinophils, and is likely to be a crucial regulator of the release of reactive oxygen species from these cells during inflammatory reactions.

Production of singlet oxygen by eosinophils activated in vitro by C5a and leukotriene B4

Using the trans-methoxyvinylpyrene analogues of benzo[ a]pyrene-7,8-dihydrodiol (MVP) as a singlet oxygen ( 1O 2) chemiluminescence probe, we have demonstrated that guinea pig eosinophils release 1O 2 when activated with the physiological agonists C5a and leukotriene B 4. This release, which occurs at agonist concentrations as low as 10 −7 M, occurs more rapidly than activation with phorbol ester (10 −6 M), is similar in level, but is more transitory. In addition, the release of 1O 2 occurs in the absence of added bromide ions and represents, we propose, an important feature of eosinophil-mediated inflammatory damage.

Oxidation Regulates the Inflammatory Properties of the Murine S100 Protein S100A8

The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites. S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.

O–210 - Regulatory mechanism of eosinophil peroxidase release from guinea pig eosinophils

O–210 - Regulatory mechanism of eosinophil peroxidase release from guinea pig eosinophils

Anti VLA-4 monoclonal antibody inhibits eosinophil infiltration in allergic conjunctivitis model of guinea pig

Purpose. To determine the contribution of very late activation antigen-4 (VLA-4) molecule to eosinophil infiltration into the conjunctiva in an actively sensitized allergic conjunctivitis model of guinea pig, effects of a monoclonal antibody against VLA-4 was examined in vivo. Methods. A rat anti-mouse VLA-4 mAb (PS 2. 3), which cross-reacts with guinea pig VLA-4, inhibited the adhesion and transmigration of guinea pig eosinophils to /through human umbilical vein endothelial cells (HUVEC) in vitro. Hartley guinea pigs were actively sensitized by intraperitoneal injection of ovalbumin with aluminum hydroxide. Two weeks later, 2.5% ovalbumin was dropped into the eye for the antigen challenge. In the treatment group, 4 mg/kg (body weight) of the anti VLA-4 mAb (PS2·3), and in the control group, the same amount of a control Ab was intraperitoneally injected respectively at 4 hours before the antigen challenge. From both groups, the eyelids and eyeballs were excised at 2, 4, 10, and 24 hours after the antigen challenge, fixed, stained with Hansel solution and the number of the infiltrating eosinophils in the conjunctiva was counted. Results. In the control group, infiltration of eosinophils to the conjunctiva increased with time, peaked at 12 hours, and then gradually decreased until 24 hours after the antigen challenge. In the anti VLA4-mAb treated group, eosinophil infiltration was almost completely inhibited at least until 24 hours after the antigen challenge. Conclusion. VLA-4 molecule was elucidated to play a critical roll in the eosinophil infiltration in experimentally-induced allergic conjunctivitis model of guinea pig.