Marco A. Martins

Cyclooxygenase-2-Derived Prostaglandin E2 and Lipoxin A4 Accelerate Resolution of Allergic Edema in Angiostrongylus costaricensis-Infected Rats: Relationship with Concurrent Eosinophilia

In noninfected rats, challenge with allergen following local IgE sensitization induced a pleurisy marked by intense protein exudation that plateaued from 30 min to 4 h after challenge, reducing thereafter. Infection of rats with Angiostrongylus costaricensis induced a 5-fold increase in blood eosinophil numbers by 25 days postinfection, whereas the numbers of eosinophils in the pleural cavity ranged from normal to a weak increase. In infected rats, identically sensitized, challenge with Ag induced a much shorter duration of pleural edema with complete resolution by 4 h, but no change in the early edema response. In parallel, infection increased the number of eosinophils recovered from the pleural cavity at 4 h, but not at 30 min, following allergen challenge. Pretreatment with IL-5 (100 IU/kg, i.v.) also increased eosinophil numbers in blood and, after allergen challenge, shortened the duration of the pleural edema and increased pleural eosinophil numbers. There were increases in the levels of both PGE2 and lipoxin A4 (LXA4) in pleural exudate. Selective cyclooxygenase (COX)-2 inhibitors, NS-398, meloxicam, and SC-236, did not alter pleural eosinophilia, but reversed the curtailment of the edema in either infected or IL-5-pretreated rats. Pretreatment of noninfected animals with the PGE analogue, misoprostol, or two stable LXA4 analogues did not alter the magnitude of pleural exudation response, but clearly shortened its duration. These results indicate that the early resolution of allergic pleural edema observed during A. costaricensis infection coincided with a selective local eosinophilia and seemed to be mediated by COX-2-derived PGE2 and LXA4.

Cutting Edge: Lipoxin (LX) A4 and Aspirin-Triggered 15-Epi-LXA4 Block Allergen-Induced Eosinophil Trafficking

Tissue eosinophilia prevention represents one of the primary targets to new anti-allergic therapies. As lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous “stop signals” produced in distinct pathologies including some eosinophil-related pulmonary disorders, we evaluated the impact of in situ LXA4/ATL metabolically stable analogues on allergen-induced eosinophilic pleurisy in sensitized rats. LXA4/ATL analogues dramatically blocked allergic pleural eosinophil influx, while concurrently increasing circulating eosinophilia, inhibiting the earlier edema and neutrophilia associated with allergic reaction. The mechanisms underlying this LXA4/ATL-driven allergic eosinophilia blockade was independent of mast cell degranulation and involved LXA4/ATL inhibition of both IL-5 and eotaxin generation, as well as platelet activating factor action. These findings reveal LXA4/ATL as a novel class of endogenous anti-allergic mediators, capable of preventing local eosinophilia.

The cellular prion protein modulates phagocytosis and inflammatory response

The cellular prion protein (PrPc) is a glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is abundantly expressed in the central nervous system. It is also expressed in a variety of cell types of the immune system. We investigated the role of PrPc in the phagocytosis of apoptotic cells and other particles. Macrophages from mice with deletion of the Prnp gene showed higher rates of phagocytosis than wild‐type macrophages in in vitro assays. The elimination of GPI‐anchored proteins from the cell surface of macrophages from wild‐type mice rendered these cells as efficient as macrophages derived from knockout mice. In situ detection of phagocytosis of apoptotic bodies within the retina indicated augmented phagocytotic activity in knockout mice. In an in vivo assay of acute peritonitis, knockout mice showed more efficient phagocytosis of zymosan particles than wild‐type mice. In addition, leukocyte recruitment was altered in knockout mice, as compared with wild type. The data show that PrPc modulates phagocytosis in vitro and in vivo. This activity is described for the first time and may be important for normal macrophage functions as well as for the pathogenesis of prion diseases.

Differential inhibition by two hetrazepine PAF antagonists of acute inflammation in the mouse

1 The injection of 100 or 300 μg of carrageenin into the mouse paw or pleural cavity produced a delayed inflammatory reaction at 48 h while platelet activating factor (PAF)‐induced paw oedema and pleurisy were maximal 30 min after its injection. 2 The PAF antagonist, WEB 2086, failed to inhibit mouse paw oedema and pleurisy induced by PAF, but reduced the first phase of oedema (1–4 h) induced by carrageenin without interfering with the second one (48–72 h). In contrast, another structurally‐related PAF antagonist, WEB 2170, inhibited dose‐dependently both oedema and pleurisy induced by PAF and by carrageenin (48 h). 3 Repeated injections of PAF into the mouse paw or pleural cavity led to significant auto‐desensitization. The animals desensitized to PAF and injected with carrageenin also displayed a significantly reduced oedema. 4 Our results suggest that PAF may be involved in the inflammatory response to carrageenin in mice. Furthermore, because the different receptor antagonists displayed distinct effects against PAF itself, different sites for in vivo interaction of PAF are available and are species‐ and drug‐dependent.

Alloxan Diabetes Reduces Pleural Mast Cell Numbers and the Subsequent Eosinophil Influx Induced by Allergen in Sensitized Rats

Alloxan damages insulin-producing cells and has been used as an inducer of experimental diabetes in several animal species. In this study, administration of alloxan (40 mg/kg, i.v.) to rats was followed by a selective and time-dependent reduction in the number of pleural mast cells (50 +/- 2.2%, p < 0.01; mean +/- SEM), while mononuclear cell and eosinophil counts were not altered. As compared to naive rats, the reduction in mast cell numbers was first noted 48 h following alloxan administration and remained unaltered for at least 60 days. It is noteworthy, that the depletion in the mast cell population was not accompanied by alterations in the total amount of histamine stored per cell. Sensitized rats turned diabetic by alloxan treatment performed 72 h before challenge showed a less pronounced antigen-induced mast cell degranulation compared to nondiabetic rats. Moreover, rats injected with alloxan 72 and 48 but not 24 h before challenge, reacted to allergenic challenge with 50% reduction in the number of eosinophils recruited to the pleural cavity within 24 h. We found that the less pronounced eosinophil accumulation did not relate to an intrinsic cell locomotor abnormality since eosinophils from diabetic rats presented similar chemotactic responses to LTB4 and PAF in vitro as compared to matching controls. Insulin (3 IU/rat) restored basal levels of mast cells and reversed the subsequent inhibition of allergen-induced pleural eosinophilia, suggesting a causative relationship between these phenomena. Treatment with insulin also significantly increased the number of mast cells in the pleural cavity of naive rats (from 637 +/- 57 to 978 +/- 79 x 10(3) cells/cavity, p < 0.001). Consistently, previous depletion of mast cells by means of local treatment with compound 48/80 significantly reduced the antigen-induced eosinophil recruitment in sensitized animals. We conclude that the reduction in the pleural mast cell population noted in alloxan-treated rats could be directly implicated in the diminished pleural eosinophil influx following allergen challenge. This hyporesponsiveness is independent of an intrinsic abnormality of cell chemotaxis, but can be imitated by local mast cell depletion.

Pharmacological modulation of Paf‐induced rat pleurisy and its role in inflammation by zymosan

1 The intrapleural injection of Paf‐acether into rats caused, at 30 min, a marked exudation accompanied by a reduction in the pleural leucocyte count. At 6 h, the exudate volume had decreased and a significant increase in the total leucocyte count, particularly eosinophils was noted. 2 Two Paf‐acether antagonists, WEB 2086 and 48740 RP abrogated the pleural leucopenia observed 30 min after Paf‐acether administration, whereas the exudation was inhibited only by the former. Pleurisy was also reduced by about 60% with dexamethasone, by about 45% with BW 755C or LY 171883, a mixed cyclo‐oxygenase/lipoxygenase inhibitor and a peptido‐leukotriene antagonist respectively, and by about 30% with indomethacin, flurbiprofen or piroxicam. 3 Repeated daily intrapleural injections of Paf‐acether led to a state of progressive desensitization to Paf‐acether itself, whereas responsiveness to 5‐hydroxytryptamine was maintained. In addition, the Paf‐induced auto‐desensitization was largely inhibited by WEB 2086. 4 Pleurisy induced by zymosan, but not by carrageenin, was significantly reduced in Paf‐acether‐desensitized animals. These results were consistent with those obtained with WEB 2086 which supressed zymosan‐induced but not carrageenin‐induced pleurisy. 5 This study suggests that Paf‐acether‐induced pleurisy in the rat may be mediated by lipoxygenase arachidonic acid metabolites and that pleurisy induced by zymosan, but not by carrageenin, is largely dependent upon Paf‐acether.

Design, Synthesis, and Pharmacological Evaluation of N-Acylhydrazones and Novel Conformationally Constrained Compounds as Selective and Potent Orally Active Phosphodiesterase-4 Inhibitors

Among a small series of tested N-acylhydrazones (NAHs), the compound 8a was selected as a selective submicromolar phosphodiesterase-4 (PDE4) inhibitor associated with anti-TNF-α properties measured both in vitro and in vivo. The recognition pattern of compound 8a was elucidated through molecular modeling studies based on the knowledge of the 3D-structure of zardaverine, a PDE4 inhibitor resembling the structure of 8a, cocrystallized with the PDE4. Based on further conformational analysis dealing with N-methyl-NAHs, a quinazoline derivative (19) was designed as a conformationally constrained NAH analogue and showed similar in vitro pharmacological profile, compared with 8a. In addition 19 was found active when tested orally in LPS-evoked airway hyperreactivity and fully confirmed the working hypothesis supporting this work.

Stem Cell Factor-Induced Leukotriene B4 Production Cooperates with Eotaxin to Mediate the Recruitment of Eosinophils During Allergic Pleurisy in Mice

The understanding of the mechanisms underlying eosinophil recruitment in vivo may aid in the development of novel strategies for the treatment of allergic disorders. In this study, we investigated the role of chemokines in the cascade of events leading to eosinophil recruitment in a stem cell factor (SCF)- and leukotriene B4 (LTB4)-dependent allergic pleurisy model in mice. The intrapleural administration of the eosinophil-active chemokines eotaxin, RANTES, and macrophage-inflammatory protein 1α (MIP-1α) induced a time- and dose-dependent eosinophil recruitment. Pretreatment with anti-eotaxin, but not anti-RANTES or anti-MIP-1α, blocked the recruitment of eosinophils following Ag challenge of sensitized animals, and significant eotaxin immunoreactivity was detected in the pleural cavity of these animals. Similarly, only the anti-eotaxin inhibited the eosinophil recruitment induced by injection of SCF in naive animals. However, blockade of SCF did not inhibit the release of eotaxin after Ag challenge of sensitized mice. Akin to its effects on SCF and in the allergic reaction, eotaxin-induced eosinophil recruitment was blocked by the LTB4 receptor antagonist CP105696. Nevertheless, SCF, but not eotaxin, appeared to regulate the endogenous release of LTB4 after Ag challenge. Finally, we show that low doses of eotaxin synergized with LTB4 to induce eosinophil recruitment in the pleural cavity. Overall, the present results show that eotaxin and SCF-induced LTB4 cooperate to induce eosinophil recruitment into sites of allergic inflammation. Cooperation between inflammatory mediators must be an important phenomenon in vivo, explaining both the ability of lower concentrations of mediators to induce a full-blown functional response and the effectiveness of different strategies at inhibiting these responses.

Neutrophil infiltration is implicated in the sustained thermal hyperalgesic response evoked by allergen provocation in actively sensitized rats

&NA; It has been proposed that allergen provocation induces hyperalgesia but the involvement of immunoglobulin E and leukocytes remains poorly understood. Here, we have compared the profile of allergen‐evoked thermal hyperalgesic response in both passively and actively sensitized rats, and investigated the role of leukocytes in allergen‐evoked nociception. Wistar rats were passively sensitized with an intraplantar injection of immunoglobulin E anti‐dinitrophenylated bovine serum albumin monoclonal antibody (0.5 &mgr;g/paw), and challenged with dinitrophenylated bovine serum albumin (0.5 &mgr;g/paw) 24 h later. Alternatively, the animals were actively sensitized with a mixture of Al(OH)3 and ovalbumin and challenged intraplantarly with ovalbumin (12 &mgr;g/paw) 14 days later. We found that the thermal hyperalgesic responses set in very rapidly and with comparable intensity in both passively and actively sensitized rats. However, while in the former group the response was shorter, peaking within 1 h and reducing thereafter, a marked plateau was observed from 1 to 6 h post‐challenge in the latter group. Actively sensitized rats also had higher neutrophil influx in the plantar tissue, as attested by both myeloperoxidase activity and histological analysis. Treatment of actively sensitized rats with either fucoidin (10 mg/kg, i.v) or anti‐rat neutrophil antiserum (i.p.) reduced neutrophil accumulation and the late hyperalgesic response noted from 3 to 6 h post‐challenge. Thus, we conclude that though immunoglobulin E‐mediated mechanisms can cause thermal hyperalgesia, components of the cellular immune reaction are crucial in order to amplify and sustain the immediate hyperalgesic response triggered by allergen, in a process dependent on neutrophil recruitment.

Warifteine, a bisbenzylisoquinoline alkaloid, decreases immediate allergic and thermal hyperalgesic reactions in sensitized animals.

Warifteine is a bisbenzylisoquinoline alkaloid isolated from the Cissampelos sympodialis Eichl (Menispermaceae). This plant is used in the folk medicine for the treatment of airway respiratory diseases. A murine model of immediate allergic reaction was used to evaluate warifteine treatment in the IgE production, leukocyte activation, thermal hyperalgesia, mast cell degranulation and scratching behavior. BALB/c mice treated with warifteine (0.4-10 mg/Kg) 1 h before OVA sensitization reduced OVA induced paw edema as well as the OVA-specific IgE serum titers as compared with non-treated and OVA-sensitized animals. Warifteine also reduced the mice death evoked by IgE-dependent anaphylactic shock reaction at 30 min after intravenous OVA challenge. To assess the effect of warifteine treatment on T cell proliferative response, spleen cells from warifteine treated or non-treated and OVA-sensitized animals were evaluated. Spleen cells from warifteine treated animals (2.0 mg/kg) did not proliferate following OVA stimulation as compared with spleen cell cultures from non-treated animals. This response may be related with the increase of NO production as observed in peritoneal macrophage cultures treated with warifteine. Thermal hyperalgesia evoked by IgE or histamine/5-hydroxytryptamine challenge was inhibited on rats at dose of 4.0 mg/kg. Warifteine treatment (0.6 or 6.0 microg/ml) also decreased the IgEalphaDNP-BSA sensitized mast degranulation after DNP-BSA challenge measured by histamine release. In addition, compound 48/80-induced scratching behavior was also sensitive to warifteine treatment. These results demonstrate for the first time that warifteine treatment reduced the allergy-associated responses.