Interference of antihistamines and anti-allergic drugs with antigen-induced paw edema in boosted and unboosted mice.

The protective effects of two antihistamines and two anti-allergic drugs against anaphylactic paw edema were studied in immunized animals that had or had not received a booster injection of antigen. The injection of 1 or 10 micrograms/paw ovalbumin induced acute paw edema of similar intensity in both groups. The antihistamine meclizine and the mixed anti histamine/anti-5-HT antagonist cyproheptadine reduced the anaphylactic reaction by 55 and 84% respectively, in non-boosted animals and were less effective against edema induced by 1 microgram antigen in boosted animals. The effectiveness of these drugs was also reduced when boosted mice were challenged with 10 micrograms antigen, where meclizine and cyproheptadine inhibited edema by 31 and 59%, respectively. The anti-allergic compounds ketotifen and azelastine, although effective against allergic inflammation in non-boosted mice, had a reduced or no effect in boosted mice. Our results suggest that allergic edema is less sensitive to antihistamine and anti-allergic drugs in boosted mice, which may be accounted for by an increased role of other mediators.     

Formation of LTB4 by fMLP-stimulated alveolar macrophages accounts for eosinophil migration in vitro.

Guinea pig alveolar macrophages obtained by bronchoalveolar lavage were isolated by adherence for 2 h and stimulated with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) for different time intervals. The supernatants then were tested for their chemotactic effect on guinea pig peritoneal normodense eosinophils and for release of thromboxane B2, leukotriene B4 (LTB4), and platelet activating factor (PAF). The supernatant from fMLP-stimulated alveolar macrophages induced a significant eosinophil attraction (96.0 +/- 11.9, number of migrating eosinophils [mean +/- SEM], n = 17) as compared to unstimulated macrophages (4.8 +/- 1.4, n = 15). This effect was not accounted for by fMLP carry-over to the macrophages because, in contrast to human eosinophils, fMLP has no chemotactic effect on guinea pig eosinophils. Pretreatment of eosinophils with BN 52021 (100 microM), a specific PAF antagonist, and with indomethacin (10 microM), a cyclooxygenase inhibitor, failed to inhibit migration of eosinophils induced by supernatants from either stimulated or unstimulated alveolar macrophages. In contrast, inhibition of the 5-lipoxygenase enzyme with N-(3-phenoxycinamyl)-acetohydroxamic acid (1 microM) suppressed eosinophil migration by alveolar macrophage supernatants (94.1 +/- 2.6% of inhibition, n = 6). Desensitization of eosinophils by and to LTB4 (10 nM) inhibited migration induced by supernatants from stimulated alveolar macrophages (87.5 +/- 5.4% of desensitization toward LTB4 and 83.1 +/- 5.4% of desensitization toward supernatants, n = 5). Under the present experimental conditions, LTB4 is the only agent implicated in eosinophil migration induced by supernatants from fMLP-stimulated alveolar macrophages.     

Lung responsiveness to antigen in sensitised mice of different strains

A new model of the isolated perfused lung from different strains of mice was developed. Lungs from Swiss, Balb/C and CBA mice actively sensitised to ovalbumin were challenged intratracheally (i.t.) by antigen on day 14. In Swiss mice instillation of ovalbumin led to the release of leukotriene (LT) C₄ significantly above basal values. Conversely, lungs from Balb/C and CBA mice were unresponsive to ovalbumin in terms of production of LTC₄. All strains failed to release histamine when challenged with antigen. Intratracheal instillation of platelet-activating factor (PAF), to lungs from non-sensitised animals, induced the release of comparable amounts of LTC₄, irrespective of the strain. In contrast, i.t. administration of fMLP to lungs from Swiss mice elicited release of significantly higher amounts of LTC₄ as compared to Balb/C and CBA mice. In separate experiments, ovalbumin was injected into the paws and anaphylactic oedema was evaluated. Balb/C and CBA required 1 μg to show an oedema formation, whereas the dose of ovalbumin for Swiss mice to develop a similar response was at least 30-fold lower. In conclusion, antigen provocation induced release of LTC₄ from lungs from Swiss mice but not from Balb/C or CBA. This difference may be accounted for by strain-dependent factors, such as antibody production and requires further investigation.

     

Platelet involvement in rat paw edema induced by 2-methoxy-PAF

PAF-acether (PAF) or 2-methoxy-PAF (2-MX) caused a dose-dependent paw edema showing a 1: 25 ratio between their inflammatory activities. 2-MX caused a thrombocytopenia, whereas PAF did not alter the number of these cells. Both phospholipids induced reductions in total leukocyte count. Rat antiplatelet serum produced platelet depletion by PAF-induced paw edema was unaffected. The edema of 2-MX was significantly reduced by antiplatelet serum, under conditions where normal serum was inactive against the edema induced by PAF or 2-MX. Histopathological analysis of PAF and 2-MX-induced edema showed, in the first case, a small infiltrate of neutrophils, some lymphocytes, and several mastocytes around the vessels and, in the second, a neutrophilic infiltrate. These results suggest that PAF and 2-MX may produce edema through different mechanisms and that 2-MX causes edema in part through platelet activation.     

Release of platelet-activating factor (PAF-acether) and arachidonic acid metabolites from alveolar macrophages

Human, monkey and rat alveolar macrophages (AM) release PAF-acether in a dose-dependent fashion in the presence of 1 to 5 g/ml ionophore A 23187 (2.5 pmol of PAF-acether from 2.5×10⁵ cells) but not in the presence of zymosan. Arachidonic acid (AA) metabolites released from AM from these species were studied. Thromboxane A₂ TxA₂)—detected by its action on rabbit arteries—was released from human, monkey and rat AM upon addition of 0.5 mM AA. This release was inhibited by aspirin and indomethacin. Lipoxygenase and cyclooxygenase AA metabolites from rat AM were identified using high efficiency glass capillary column gas chromatography coupled to mass spectrometry. The cyclooxygenase metabolites PGF₂, E₂ and D₂ and TxB₂ were identified. The lipoxygenase-dependent AA metabolites were explored using aspirin-pretreated AM. Only 12 HETE was found.These data indicate that AM secrete several substances with bronchoconstrictive activity: PGF₂, D₂, TxA₂ and PAF-acether. Therefore an active role of AM in human and experimental bronchoconstriction must be considered.     

Platelet-activating factor (PAF-acether): Thromboxane-independent synergism with adrenaline on human platelets and recent insights into its mode of action

The mode of action of PAF-acether on human and rabbit plasma-free platelets is reviewed. PAF-acether and adrenaline synergize to trigger aggregation of human platelets, and this synergism is refractory to aspirin. When degranulated rabbit platelets are stimulated with PAF-acether, with thrombin or with the snake venom component convulxin, aggregation is obtained in the absence of detectable secretion. Collagen-induced aggregation is reduced, and is suppressed when aspirin is applied to the degranulated platelets. The formation of PAF-acether by platelets, and their stimulation by PAF-acether itself, should be added to the newly recognized pathways for platelet stimulation.     

Role of immunoglobulins G1 and G2 in anaphylactic shock in the guinea pig

Heating serum from actively sensitised guinea pigs did not remove its ability to sensitise recipient animals in vivo and parenchymal lung strips in vitro to anaphylaxis. Thermoresistant antibodies should thus account for the transferable sensitising effect, which persists for at least 9 days. IgG1 and IgG2, contained in the serum, were separated by affinity chromatography to determine the importance and the participation of these subclasses in passive anaphylactic shock. IgG1, present in smaller amounts than IgG2, was more effective in sensitising isolated lung strips. The intravenous administration of ovalbumin to guinea pigs, which had been injected with 0.8 mg/kg of IgG1 or 2 mg/kg of IgG2 9 days beforehand, induced an intense bronchoconstriction with leucopenia and moderate thrombopenia, suggesting an as yet undescribed role for IgG2 in passive tissue sensitisation. The use of mepyramine, an antagonist of the histamine H1 receptor, WEB 2086, an antagonist of platelet-activating factor, and nordihydroguaiaretic acid, a dual inhibitor of cyclooxygenase and lipooxygenase, alone or associated, demonstrated that the anaphylactic contraction of lung strips from guinea pigs sensitised by IgG1 is mediated by histamine and arachidonate derivatives, whereas that of lung strips from guinea pigs sensitised with IgG2 is mostly mediated by histamine. In addition, the association of the three potential antagonists slightly reduced the anaphylactic contraction of lung strips provided by guinea pigs sensitised by serum. Our results, using a sensitisation procedure considered until now to involve exclusively IgE antibodies, indicate that IgG1 and IgG2 are in fact the essential antibodies for passive anaphylactic shock in the guinea pig.     

Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins.

Bordetella pertussis expresses factors such as filamentous hemagglutinin, agglutinogens, pertactin, and pertussis toxin, which participate in bacterial adhesion; pertussis toxin, dermonecrotic toxin, lipopolysaccharide, and tracheal cytotoxin, which are responsible for toxic effects; and adenylate cyclase-hemolysin, which is required to initiate infection. By using a murine respiratory model, we showed that the RGD sequences of filamentous hemagglutinin and pertactin are important for bacterial persistence. However, mutants deficient in filamentous hemagglutinin and agglutinogens or in pertactin and the RGD sequence of filamentous hemagglutinin behaved as did wild-type B. pertussis, i.e., induced bronchopneumonia, alveolitis, and an influx of macrophages, lymphocytes, and polymorphonuclear leukocytes into bronchoalveolar lavage fluids. These results suggest that these adhesins are not involved in the induction of pulmonary lesions following infection. The intensity of inflammation was markedly reduced after infection with mutants deficient in either hemolytic activity or pertussis toxin expression, whereas a mutant devoid of adenylate cyclase activity behaved as did the avirulent mutant. Pertussis toxin and adenylate cyclase-hemolysin may act indirectly by altering immune cell functions and thus allowing other factors, such as filamentous hemagglutinin, agglutinogens, and pertactin, to trigger adhesion and lipopolysaccharide, dermonecrotic toxin, and tracheal cytotoxin to induce their toxic effects. However, it is possible that pertussis toxin is also responsible for the induction of some pulmonary alterations.     

LTB4, a potent chemotactic factor for purified guinea-pig eosinophils: interference of PAF-acether antagonists.

Two populations of eosinophils were separated upon a discontinuous metrizamide gradient from peritoneal lavages of polymyxin B-treated guinea-pigs. One population was of low density (between 20 and 22% of metrizamide, purity: 63 +/- 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 +/- 2%, n = 26). Responses to chemotactic stimuli were studied using a micro-Boyden chamber, results being expressed as the chemotactic index (CI, mean +/- S.E.M.), i.e. the ratio between the number of eosinophils migrating at 40 microns through a cellulose nitrate filter in the presence of the agonist and the number of cells migrating in the presence of the solvent alone. The normal density eosinophils responded more to LTB4 (CI = 19.4 +/- 4.6 with LTB4 10(-8) M; P less than 0.05; n = 9) than to PAF-acether (CI = 6.2 +/- 1.4 with PAF-acether 10(-8) M; P less than 0.05; n = 20). By contrast, low density eosinophils responded less intensely to LTB4 (CI = 7.6 +/- 1.8 with LTB4 10(-8) M; P less than 0.01; n = 6) and to PAF-acether (CI = 2.4 +/- 0.4 with PAF-acether 10(-8) M; P less than 0.05; n = 14). Guinea-pig eosinophils failed to migrate in response to FMLP and lyso PAF-acether.(ABSTRACT TRUNCATED AT 250 WORDS)