Rapamycin inhibits airway leukocyte infiltration and hyperreactivity in guinea pigs

The effect of rapamycin on cell infiltration to the lung and on bronchial hyperreactivity induced by an intravenous injection of sephadex beads to guinea pigs was investigated. One day following the injection of sephadex the total cell number in bronchoalveolar lavage (BAL) fluid was significantly increased from 24.77 to 83.45×10⁶ cells. This was reflected in an increase in eosinophils, neutrophils, macrophages and lymphocytes. In addition, there was an increase in the reactivity of isolated bronchial strips to histamine. Rapamycin (5 mg/kg), administered two hours before the injection of sephadex, reduced the eosinophil, neutrophil, lymphocyte and macrophage number by 64%, 55%, 50% and 19%, respectively, and also inhibited the increased reactivity of isolated bronchial strips to histamine. These results suggest that rapamycin may reduce bronchial reactivity by the inhibition of leukocyte migration into the airways.

     

Effects of theophylline compared with prednisolone on late phase airway leukocyte infiltration in guinea pigs

The effects of prednisolone, theophylline or salbutamol treatment were studied on leukocyte numbers in bronchoalveolar lavage (BAL) fluid taken 72 h after ovalbumin challenge in sensitized guinea pigs. Ovalbumin challenge resulted in an approximate 3-fold increase in the number of eosinophils in BAL fluid. This increase was significantly reduced by oral administration of prednisolone (59% inhibition with 10 mg/kg x 2) theophylline (56% with 50 mg/kg x 2) but not by salbutamol (10 mg/kg x 2). A comparison with the bronchodilator potency of the above drugs indicated that in guinea pigs salbutamol appears relatively selective as a bronchodilator, prednisolone is selective as an inhibitor of eosinophilia whilst theophylline displays a balance of both activities.     

Effect of San-Ao-Tang on immediate and late airway response and leukocyte infiltration in asthmatic guinea pigs

San-Ao-Tang (SAT), a traditional Chinese medicines, has been used to treat patients with the bronchial asthma for several centuries. However, the therapeutic mechanisms of this Chinese medicine are still far from clear. To understand the mechanism of antiasthmatic property of SAT, a guinea pig model of allergic asthma was used to investigate the effects of SAT on Dermatophagoides pteronyssinus-induced. immediate and late asthmatic responses and airway inflammation. Our results showed that administration of SAT (10g/kg) extracts singificantly inhibited the antigen induced immediate asthmatic responses (IAR) in actively sensitized guinea pig. Examination of bronchoalveolar lavage fluid (BALF) revealed that SAT significantly inhibited the increase in neutrophil in the airway at 1, 2, 4, 6, 8 hr after antigen challenge. Histopathologic examination showed SAT suppressed the neutrophil infiltration into lung tissue. These results suggest that the antiasthmatic effect of SAT be mainly due to its bronchodilator effect and its ability to inhibit the neutrophil into the airway. The precise mechanism of action of SAT in asthma remains to be elucidated.     

Relationship between intracellular calcium and airway reactivity in guinea pigs.

The present study was carried out to examine the relationship between intracellular free calcium ion concentrations and its regulatory enzymes, sodium potassium adenosine triphosphatase (Na(+),K(+)-ATPase) and calcium adenosine triphosphatase (Ca(2+)-ATPase), with airway reactivity to inhaled histamine in guinea pigs. Forty-nine guinea pigs were included in this study. Of these, 34 animals responded to histamine bronchoprovocation challenge in vivo with a greater than 35% fall in specific airways conductance and were labeled as "reactive," and the remaining 15 were "nonreactive." The dose of histamine producing a 35% fall in specific airways conductance was labeled as ED(35) SGaw. The animals were then sacrificed, and the following biochemical measurements were carried out: intracellular free calcium ion concentrations [Ca(2+)](i) in leukocytes and isolated tracheal smooth muscle cells, activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase in tracheal homogenate, and plasma levels of lysophosphatidylcholine (LPC). Reactive guinea pigs showed significantly higher [Ca(2+)](i) and Na(+),K(+)-ATPase and Ca(2+)-ATPase activities. Airway reactivity (ED(35) SGaw) had significant negative correlation with [Ca(2+)](i), with activities of each of the ATPases and with plasma lysophosphatidylcholine. It is concluded that the level of [Ca(2+)](i) is an important determinant of airway reactivity. Intracellular calcium levels modulate airway response to histamine with higher levels being associated with greater reactivity.     

Effects of Ding-Chuan-Tang on bronchoconstriction and airway leucocyte infiltration in sensitized guinea pigs

Ding-Chuan-Tang (DCT), a traditional Chinese medicine, has been used in treatment of the bronchial asthma for several centuries. However, the therapeutic mechanism of these Chinese medicine are still far from clear. To understand the mechanism of antiasthmatic property of DCT. A guinea pig model of allergic asthma was used to investigate the effects of DCT on ovalbumin-induced early and late asthmatic responses and airway inflammation, particularly the extent of eosinophil infiltration, and examine it direct beta2-adrenoceptor agonist activity in guinea-pig isolated trachea. We had used three different protocals in ovalbumin sensitized guinea pigs by administrating 10 g/kg of DCT extracts to sensitized guinea pigs 30 min before antigen challenge (group I), 5 hr after antigen challenge (group II) and 2.5 g/kg once daily from the day of sensitization to the day of challenge. Our result showed that administration of DCT singificantly inhibited the antigen induced immediate asthmatic responses (IAR) in group I and inhibited both IRA and late asthmatic responses (LAR) in actively sensitized guinea pig in group III. DCT caused concentration-dependent relaxations in strips of guinea pig trachea contracted with carbachol, however ICI-118551, a selective beta2-adrenoceptor antagonist, didn't significantly competitively inhibit the relaxations caused by DCT. Furthermore, examination of bronchoalveolar lavage fluid (BALF) revealed that DCT significantly inhibited the increase in percent of eosinophils in the airway after antigen challenge in three group. Histopathologic examination showed DCT suppressed the eosinophil infiltration into lung tissue. These results suggest that the antiasthmatic effect of DCT is mainly due to its bronchodilatation effect and its ability to inhibit the eosinophil into the airway and there is prophylactic effect of DCT on allergen-induced airway inflammation.     

Eosinophil chemotactic factor (ECF). III. Generation in human peripheral leukocytes.

Eosinophil chemotactic factor (ECF) can be released from human peripheral leukocytes by an IgE-anti-IgE reaction, by the calcium ionophore A 23187 and during phagocytosis. Supernatants and sonicates of unstimulated cells contain little or no ECF. On stimulation, however, ECF activity increases in the cells and even more so in the supernatants. This holds for purified neutrophils (PMNs) as well as for basophil-containing mononuclear cell preparations. These findings contrast with those in lung homogenates, where ECF-A is present in mast cells in a preformed state. On chromatographic analysis, the ECF present within cells does not differ from that released into the supernatant. For the generation of ECF, calcium and an intact cell are necessary. Inhibition of deoxyribonucleic acid (DNA) and protein synthesis do not affect ECF generation or release. The metabolic inhibitor, 2-deoxyglucose (2-DG), suppresses ECF activity in leukocyte supernatants to a greater extent than in the cell pellets while the reserve is true for colchicine. This further confirms that ECF is not preformed within cells and that its generation and release are two active, distinct metabolic processes.     

Antagonism of airway reactivity induced by ovalbumin antigen in guinea pigs by 5-amino-4-imidazolecarboxamide riboside

The effect of 5-amino-4-imidazolecarboxamide riboside (AICA riboside), a modulator of purine metabolism, was studied on antigen-induced bronchospasm in ovalbumin (OA)-sensitized guinea pigs. In separate experiments, sodium cromoglycate (SCG) and terbutaline were used to compare their effectiveness with that of AICA riboside (wt/vol). AICA riboside and SCG were administered as an aerosol daily for a minimum of 2 weeks before OA aerosol challenge. Terbutaline, as an aerosol, was administered once 5 minutes before OA challenge. Airway reactivity was determined through the use of a whole-body plethysmography by monitoring specific airway resistance (SRaw). OA aerosol challenge of 0.05%, 0.1%, and 0.25% (wt/vol), administered for a period of 1 minute, increased SRaw. Each of the three agents attenuated the effect of OA on SRaw, although terbutaline demonstrated more consistency and potency as compared to either AICA riboside or SCG. However, at moderate degrees of OA challenge, AICA riboside appeared to be as effective as either agent. Although the mechanism of action of AICA riboside remains uncertain, it may have therapeutic benefit in the treatment of asthma or allergic diseases.     

Selective potentiation of lymphocyte-derived macrophage chemotactic factor release in complete Freund's adjuvant-treated guinea pigs

Dinitrophenol (DNP)-ovalbumin(OA)-induced tissue macrophage reaction in sensitized guinea pigs is enhanced by treatment with complete Freund's adjuvant (CFA). The enhancement of the reaction may be due to the increased production of a T-lymphocyte-derived macrophage chemotactic factor (LDMCF) because treatment of animals with CFA potentiates antigen- and concanavalin A(ConA)-induced release of LDMCF activity from spleen cells of the CFA-treated animals in vitro. This potentiating effect by CFA seems to be ascribed to the release of an adherent-cell-derived soluble factor from the CFA-treated animals. The adherent cell-derived factor, LDMCF-potentiating factor (LDMCF-PF), preferentially potentiates the release of LDMCF activity but not of eosinophil chemotactic activity from antigen- or Con-A-stimulated T lymphocytes. Protein synthesis is required for release of LDMCF-PF. Molecular weight of LDMCF-PF activity is assumed to be about 10,000-20,000. LDMCF-PF activity is sensitive to trypsin, to neuraminidase, and also to alkalinity at pH 11, suggesting that LDMCF-PF is a glycoprotein. The present study provides one explanation for the enhanced macrophage reaction in delayed-type hypersensitivity reactions.     

Effects of leukotriene B4 receptor antagonist, LY293111Na, on antigen-induced bronchial hyperresponsiveness and leukocyte infiltration in sensitized guinea pigs

OBJECTIVE AND DESIGN: LY29311 Na, 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy] propoxy] -phenoxy]-benzoic acid sodium salt, is a novel leukotriene B4 (LTB4) receptor antagonist. Its effects on guinea pig models of asthma were compared with those of dexamethasone. METHODS: Effects of LY293111Na were tested in antigen (ovalbumin, OA)-induced bronchial hyperresponsiveness (BHR) and leukocyte accumulation in actively sensitized guinea pigs. Its effects on antigen-induced acute bronchoconstriction in passively sensitized guinea pigs were also studied. RESULTS: LY293111 Na (10 to 30 mg/kg p.o., 1 h before and 6 h after OA challenge) inhibited BHR to acetylcholine. LY293111 Na (3 mg/kg p. o.) significantly inhibited accumulation of neutrophils in bronchoalveolar lavage (BAL) fluid 24 h after antigen challenge but it did not inhibit accumulation of eosinophils and macrophages at any doses used. In contrast, dexamethasone (30 mg/kg p.o., 4 h before OA challenge) not only inhibited BHR but also reduced the infiltration of all three types of leukocytes. A significant increase of LTB4 levels in BAL fluid was noted at 3 and 15 min after the antigen challenge. LY293111Na did not inhibit antigen-induced acute bronchoconstriction in passively sensitized guinea pigs. CONCLUSION: These results indicate that LTB4 may participate in antigen-induced BHR but not in eosinophil infiltration and acute bronchoconstriction in guinea pigs.     

Effects of xiao-qing-long-tang (XQLT) on bronchoconstriction and airway eosinophil infiltration in ovalbumin-sensitized guinea pigs: in vivo and in vitro studies

BACKGROUND: Xiao-qing-long-tang (XQLT sho-seiru-to), a traditional Chinese medicine, has been used to treat patients with bronchial asthma in Oriental countries for several centuries. However, the therapeutic mechanisms of this Chinese medicine remain a matter of considerable debate. Therefore, a series of experiments using ovalbumin-sensitized guinea pigs was performed to elucidate the possible antiasthmatic effect of XQLT. METHODS: The effect of XQLT on ovalbumin-induced airway inflammation in a guinea pig model of allergic asthma was examined, and early and late asthmatic responses were measured in terms of airway resistance and extent of eosinophil infiltration. Furthermore, the bronchorelaxing effect of XQLT was measured in isolated guinea pig trachea. RESULTS: XQLT significantly inhibited the antigen-induced immediate asthmatic response (IAR) and late asthmatic response (LAR) in actively sensitized guinea pigs. Cumulative administration of XQLT caused concentration-dependent relaxation of the carbachol-precontracted guinea pig trachea. The bronchorelaxing effect of XQLT was reversed by ICI-118551, a selective beta2-adrenoceptor antagonist. Furthermore, examination of bronchoalveolar lavage fluid (BALF) revealed that XQLT significantly suppressed the increase in eosinophils (24 h after antigen challenge) in the airway. In addition, XQLT significantly attenuated the increase in eosinophils at 1, 6, 24, 48, and 72 h after antigen challenge when it was administered once daily from the day of sensitization to the day of challenge. Histopathologic examination results showed that XQLT suppressed eosinophil infiltration into lung tissue. CONCLUSIONS: These results demonstrate that the antiasthmatic effects of XQLT appear to be partly mediated by stimulation of beta2-adrenoceptors, leading to bronchorelaxation, and that XQLT inhibits the infiltration of eosinophils into the airway. Thus, XQLT may be useful for the prevention or treatment of asthma.     

Synergistic effect of heparin and chemotactic factor on polymorphonuclear leukocyte aggregation and degranulation.

The in vitro effects of therapeutic amounts of polyanionic heparin on human polymorphonuclear leukocytes (PMN) aggregation and on the release of cationic lactoferrin from PMN-specific granules were investigated. Incubation of 1 X 10(7) human PMNs with 0.3 unit/ml of heparin followed by stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) 2 X 10(-7) M significantly increased PMN aggregation, compared with controls. Cytochalasin B potentiated aggregation, which was further increased by incubation of the PMNs with heparin. Similarly, heparin also increased PMN degranulation and lactoferrin release following stimulation with FMLP with or without cytochalasin B, compared with controls. In addition, human lactoferrin complexed with heparin on a sucrose density gradient and caused a significant shift in the migration of 3H-heparin. Finally, rabbits pretreated with intravenous heparin resulting in prolongation of their activated partial thromboplastin time (APTT) to 1.5 to 2.5 times baseline had more profound reduction in PMN counts following a challenge with the secretagogue phorbol myristate acetate (PMA). These studies demonstrate that heparin can interact synergistically with chemotactic stimuli known to evoke lactoferrin release, which in turn leads to enhancement of PMN aggregation. Our data further suggest that heparin may be contraindicated in the treatment of syndromes with increased PMN aggregation such as endotoxin-induced Schwartzman-type reactions.     

Characterization of three macrophage chemotactic factors from PPD-induced delayed hypersensitivity reaction sites in guinea pigs, with special reference to a chemotactic lymphokine.

Three types of macrophage chemotactic factors (MCFs) were separated from purified protein derivative (PPD)-induced delayed hypersensitivity (DHR) skin lesions in guinea pigs, and MCF-c was purified isotachophoretically. The macrophage chemotactic activity (MCA) of the skin extract was mostly associated with MCF-a and c. MCF-c (mol wt 110,000) seemed more significant for mediating the macrophage reaction than MCF-a (mol wt 150,000), which exhibited common antigenicity with serum IgG. MCF-b (mol wt 14,000), manifesting common antigenicity with the fifth component of complement (C5), was apparently less significant. MCF-c was thermostable and had an isoelectric point (pI) of 5.2 +/- 0.1. The protein exhibited marked macrophage chemotactic activities (MCA) but no neutrophil chemotactic, lymphocyte chemotactic, and MIF activity in excess of that in vitro. Selective accumulation of macrophages was observed in response to intradermal MCF-c injection. Our studies suggest that MCF-c at 24-hour skin sites of DHR may exist as specific lymphokine preparations with high potential for monocyte infiltration.     

Neurogenic airway inflammation induced by repeated intra-esophageal instillation of HCl in guinea pigs

This study was conducted to investigate if repeated intra-esophageal acid administrations may induce neurogenic inflammation in the airways and nodose ganglion in a guinea pig model. Guinea pigs were sedated and perfused with 0.1 N HCl in the distal esophagus via a nasoesophageal catheter for 14 consecutive days. Substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and calcitonin gene-related peptide concentration were measured by ELISA or radioimmunoassay. Neuropeptide expression in the airways and nodose ganglion was detected by immunohistochemistry and assessed semi-quantitatively. Inflammation was found in the trachea and bronchi. There was a threefold increase in substance P concentration in the trachea, main bronchi, and lung homogenate and a twofold increase in NKA and NKB concentration in the main bronchi, lung homogenate, and bronchial alveolus lavage fluid, respectively. The SP and NKA expressions in the airways and nodose ganglion were also significantly increased. Chronic intra-esophageal acid instillation induces significant neurogenic inflammation in the airways and nodose ganglion in the vagus nerve in guinea pigs.     

Participation of collagenase and elastase in LPS-induced airway hyperresponsiveness in guinea pigs

Bacterial lipopolysaccharide (LPS) cause airway hyperreactivity in guinea pigs pretreated with metopirone. LPS inhalation resulted in an increase in airway muscarinic reactivity measured by intravenous acetylcholine injection 1–4 h after the inhalation of LPS. The increase of pulmonary capillary permeability was observed 1–24 h after the inhalation of LPS, whereas the increase of leukocytes in bronchoalveolar lavage fluid (BALF) was observed 2 and 24 h after the inhalation of LPS. Increased cells are mainly neutrophil, eosinophil, and macrophage. From the histopathological study, acute mucosal injury and loss of epithelial cilia were observed 1–24 h after the inhalation of LPS. In order to investigate the phlogistic substance in LPS-induced hypereactivity, the roles of collagenase and elastase were investigated. The activities of both enzymes were elevated 2 h after the inhalation of LPS. The inhalation of collagenase and elastase caused bronchial hyperreactivity and increased pulmonary permeability. The combined administration of prednisolone (10 mg/kg/day) and cyclephosphamide (10 mg/kg/day) for five days decreased LPS-induced hyperreactivity, pulmonary capillary increase, collagenase and elastase activities, and the number of nucleated cells in BALF 2 h after the inhalation of LPS. These results indicate the participation of collagenase and elastase in the onset of LPS-induced airway hyperreactivity in guinea pigs.     

Allergenic and blastogenic reactivity of three antigens from Mycobacterium tuberculosis in sensitized guinea pigs.

Three antigens from a culture filtrate of Mycobacterium tuberculosis H37Rv were purified by affinity chromatography, using monoclonal antibodies. The molecular weights of the purified antigens are 17,000 to 19,000, 32,000 to 33,000, and 39,000, respectively, and by their migration patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, they all appeared to be single-chain polypeptides. Western blot and enzyme-linked immunosorbent assay analyses indicated that the antigens are non-cross-reactive. All antigens generated an intermediate to strong skin reaction when tested in guinea pigs previously immunized with a live M. bovis BCG vaccine or with an oil emulsion preparation of phenol-or heat-killed M. tuberculosis. Lymphocytes isolated from peripheral blood or lymph nodes of similarly immunized guinea pigs could be stimulated by purified protein derivative and the purified antigens. Qualitative differences in stimulatory capacity between the preparations were demonstrated. The antigens may prove useful in further studies of the immunology and pathogenesis of tuberculosis.     

Comparison of the airway hyperreactivity produced by single and multiple antigen exposures in sensitized guinea pigs

Chronic airway hyperreactivity is a hallmark feature of asthma, but animal models of airway hyperreactivity often utilize a single antigen challenge. Therefore, we compared the airway hyperreactivity produced by single and multiple antigen challenges in ovalbumin-sensitized guinea pigs. Significant (2-fold) leftward shifts in dose-response curves for i. v. methacholine- or LTD₄-induced bronchoconstriction in anesthetized and ventilated animals occurred 24 h following a single ovalbumin challenge. This nonspecific airway hyperreactivity was prevented by pretreatment with ketotifen or dexamethasone. However, airway hyperreactivity was no greater 24h following the last of 3 daily antigen challenges than after 1 challenge and was absent 72h following one antigen challenge. These results raise concern over the similarity of antigen-induced airway hyperreactivity in guinea pigs to the chronic airway hyperreactivity in asthmatics.