Anti-inflammatory lipoxin A4 is an endogenous allosteric enhancer of CB1 cannabinoid receptor.

Allosteric modulation of G-protein-coupled receptors represents a key goal of current pharmacology. In particular, endogenous allosteric modulators might represent important targets of interventions aimed at maximizing therapeutic efficacy and reducing side effects of drugs. Here we show that the anti-inflammatory lipid lipoxin A(4) is an endogenous allosteric enhancer of the CB(1) cannabinoid receptor. Lipoxin A(4) was detected in brain tissues, did not compete for the orthosteric binding site of the CB(1) receptor (vs. (3)H-SR141716A), and did not alter endocannabinoid metabolism (as opposed to URB597 and MAFP), but it enhanced affinity of anandamide at the CB1 receptor, thereby potentiating the effects of this endocannabinoid both in vitro and in vivo. In addition, lipoxin A(4) displayed a CB(1) receptor-dependent protective effect against ß-amyloid (1-40)-induced spatial memory impairment in mice. The discovery of lipoxins as a class of endogenous allosteric modulators of CB(1) receptors may foster the therapeutic exploitation of the endocannabinoid system, in particular for the treatment of neurodegenerative disorders.

Pneumonia-induced sepsis in mice: temporal study of inflammatory and cardiovascular parameters.

The aim of the present work is to provide a better comprehension of the pneumonia-induced sepsis model through temporal evaluation of several parameters, and thus identify the main factors that determine mortality in this model. Klebsiella pneumoniae was inoculated intratracheally in anesthetized Swiss male mice. Inflammatory and cardiovascular parameters were evaluated 6, 24 and 48 h after the insult. The results show that severity of infection and the mortality correlated with the amount of bacteria. Six, 24 and 48 h after inoculation, animals presented pathological changes in lungs, increase in cell number in the bronchoalveolar lavage, leukopenia, increase in TNF-α and IL-1ß levels, hypotension and hyporesponsiveness to vasoconstrictors, the two latter characteristics of severe sepsis and septic shock. Significant numbers of bacteria in spleen and heart homogenates indicated infection spreading. Interestingly, NOS-2 expression appeared late after bacteria inoculation, whereas levels of NOS-1 and NOS-3 were unchanged. The high NOS-2 expression coincided with an exacerbated NO production in the infection focus and in plasma, as judging by nitrate + nitrite levels. This study shows that K. pneumoniae inoculation induces a systemic inflammatory response and cardiovascular alterations, which endures at least until 48 h. K. pneumoniae-induced lung infection is a clinically relevant animal model of sepsis and a better understanding of this model may help to increase the knowledge about sepsis pathophysiology.

Molecular mechanisms of topical anti-inflammatory effects of lipoxin A(4) in endotoxin-induced uveitis.

Lipoxin A(4) (LXA(4)) is a lipid mediator that plays an important role in inflammation resolution. We assessed the anti-inflammatory effect of LXA(4) on endotoxin-induced uveitis (EIU) in rats. The inflammatory cell number and levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)), and protein, as well as expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), in the anterior chamber of the eye were determined 24 h after lipopolysaccharide (LPS; 200 mug/paw) intradermal injection. The immunohistochemical reactivities of nuclear factor-kappaB (NF-kappaB) and c-Jun were also examined. Topical LXA(4) (1-10 ng/eye) pretreatment decreased the number of inflammatory cells and the protein leakage into the aqueous humor (AqH). In addition, topical LXA(4) (10 ng/eye) inhibited the LPS-induced production of IL-1beta, TNF-alpha, and PGE(2), and expression of COX-2 and VEGF. A decreased activation of NF-kappaB and c-Jun was also found in LXA(4)-treated eyes. It is very interesting that an anti-inflammatory effect was achieved even when LXA(4) (10 ng/eye) was applied topically after LPS challenge, as indicated by the reduction in the cellular and protein extravasations into the AqH. Moreover, topical treatment of corticosteroid prednisolone (200 mug/eye) beginning before or after LPS injection reduced all of the molecular and biochemical alterations promoted on EIU rats in an efficacy similar to that of LXA(4). Together, the present results provide clear evidence that pharmacological activation of LXA(4) signaling pathway potently reduces the EIU in rats. Therefore, LXA(4) stable analogs could represent promising agents for the management of ocular inflammatory diseases.

Effect of novel selective non-peptide kinin B(1) receptor antagonists on mouse pleurisy induced by carrageenan.

Two novel selective non-peptide kinin B(1) receptor antagonists, the benzodiazepine antagonist and SSR240612, were evaluated in carrageenan-induced mouse pleurisy. The peptide R-715 (0.5 mg/kg, i.p.) and the non-peptide benzodiazepine (3 mg/kg, i.p.) antagonists significantly decreased cellular migration (predominantly neutrophils), without altering plasma exudation. SSR240612 (1 mg/kg, i.p.) diminished total cells and neutrophils, besides exudation. Oral administration of SSR240612 (10 mg/kg) also reduced total cell and neutrophil counts. Only the benzodiazepine antagonist inhibited the lung myeloperoxidase activity. No tested antagonist significantly altered the lung and pleural TNFalpha and IL-1beta production. We provide interesting evidence on the anti-inflammatory in vivo effects of non-peptide B(1) receptor antagonists.

Antiinflammatory and antiallodynic actions of the lignan niranthin isolated from Phyllanthus amarus. Evidence for interaction with platelet activating factor receptor.

Previous studies have shown that the extracts obtained from Phyllanthus amarus, and some of the lignans isolated from it, exhibit pronounced antiinflammatory properties. In the present study, we have assessed whether the antiinflammatory actions of these lignans can be mediated by interaction with platelet activating factor (PAF) receptor or interference with the action of this lipid. The local administration of nirtetralin, phyltetralin or niranthin (30 nmol/paw), similar to WEB2170 (a PAF receptor antagonist, 30 nmol/paw), significantly inhibited PAF-induced paw oedema formation in mice. The extracts of P. amarus (100 microg/ml) and niranthin (30 microM), but not nirtetralin or phyltetralin (30 microM), decreased the specific binding of [(3)H]-PAF in mouse cerebral cortex membranes. Furthermore, both niranthin and WEB2170 displaced, in a concentration-dependent manner, the [(3)H]-PAF binding sites. The mean IC(50) values from these effects were 6.5 microM and 0.3 microM, respectively. Additionally, both niranthin and WEB2170 (30 nmol/paw) inhibited the increase of myeloperoxidase activity induced by PAF injection in the mouse paw. When assessed the mouse model of pleurisy induced by PAF, pretreatment with niranthin (100 micromol/kg, p.o.) or WEB2170 (1.7 micromol/kg, i.p.) significantly inhibited PAF-induced protein extravasations. Moreover, in the rat model of PAF-induced allodynia, both niranthin (30 nmol/paw) and WEB2170 (30 nmol/paw) treatment significantly inhibited PAF-induced allodynia. In addition, niranthin had a rapid onset and long-lasting antiallodynic action when compared with WEB2170. Collectively, the present findings suggest that niranthin exhibits antiinflammatory and antiallodynic actions which are probably mediated through its direct antagonistic action on the PAF receptor binding sites.

Lipoxin A4 inhibits acute edema in mice: implications for the anti-edematogenic mechanism induced by aspirin.

Lipoxin A4 (LXA4) is a lipid mediator that plays an important role in the resolution of inflammation. However, the role of LXA4 and aspirin (ASA)-triggered lipoxins (ATLs) in inflammatory edema formation remains unclear. Here, we investigated the inhibitory role played by LXA4 in the carrageenan-induced and other inflammatory mediator-induced edematogenic response in mice, and also assessed the role of ATLs in the anti-edematogenic action of aspirin. Our results showed that LXA4 (1-20 ng/paw or 5 microg/kg i.p.) was effective in inhibiting carrageenan-induced paw edema from 30 min to 2 h. LXA4 (10 ng/paw) was also able to acutely inhibit PAF-, histamine-, PGE2- or bradykinin-induced paw edema, as well as the PAF-induced myeloperoxidase activity increase in the paws. Likewise, LXA4 (10 ng/cavity) also inhibited the pleural edema triggered by histamine (1h), and this response was not followed by leukocyte accumulation. Of note, the lipoxin receptor (ALX-r) antagonist Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe, 200 ng/paw) significantly reverted the anti-edematogenic effect of ASA (300 mg/kg p.o.) against carrageenan, PAF, PGE2 and BK, without affecting the anti-edematogenic action caused by indomethacin (3 mg/kg i.p.) in the carrageenan-induced paw edema. Collectively, our results demonstrate for the first time that LXA4 displays an acute and rapid onset anti-edematogenic activity that does not discriminate among different pro-inflammatory stimuli, an effect that is most likely independent of its action on the leukocyte influx. Finally, the present study demonstrates that ATLs exert a very important role in the acute anti-edematogenic action of ASA.

Dual role of lipoxin A4 in pneumosepsis pathogenesis.

Lipoxin A4 (LXA4) is an endogenous lipid mediator with potent anti-inflammatory actions but its role in infectious processes is not well understood. We investigated the involvement of LXA4 and its receptor FPR2/ALX in the septic inflammatory dysregulation. Pneumosepsis was induced in mice by inoculation of Klebsiella pneumoniae. LXA4 levels and FPR2/ALX expression in the infectious focus as well as the effects of treatment with receptor agonists (LXA4 and BML-111) and antagonists (BOC-2 and WRW(4)) in early (1h) and late (24h) sepsis were studied. Sepsis induced an early increase in LXA4, FPR2/ALX lung expression, local and systemic infection and inflammation, and mortality. Treatment with BOC-2 in early sepsis increased leukocyte migration to the focus, and reduced bacterial load and dissemination. Inhibition of 5- and 15-lipoxygenase in early sepsis also increased leukocyte migration. Early treatment with WRW(4) and BOC-2 improved survival. Treatment with authentic LXA4 or BML-111 in early sepsis decreased cell migration and worsened the infection. In late sepsis, treatment with BOC-2 had no effect, but LXA4 improved the survival rate by reducing the excessive inflammatory response, this effect being abolished by pretreatment with BOC-2. Thus, the anti-inflammatory and pro-resolution mediator LXA4 and its receptor FPR2/ALX levels were increased in the early phase of sepsis, contributing to the septic inflammatory dysregulation. In addition, LXA4 has a dual role in sepsis and that its beneficial or harmful effects are critically dependent on the time. Therefore, a proper interference with LXA4 system may be a new therapeutic avenue to treat sepsis.

Aspirin-triggered lipoxin induces CB1-dependent catalepsy in mice.

Evidence are that inhibition of cyclooxygenase 2 (COX-2) enhances endocannabinoid signaling, indicating a crosstalk between these two eicosanoid pathways. Aspirin, a non-selective COX inhibitor, acetylates COX-2 with generation of a lipoxygenase (LOX) substrate, whose end product is the 15-epi-lipoxin A(4) (15-epi-LXA(4)), an aspirin-triggered lipoxin. Our objective was to investigate whether 15-epi-LXA(4) would potentiate in vivo effects of the endocannabinoid anandamide (AEA). Catalepsy was selected as a behavioral parameter and tested 5 min after AEA injection in all experiments. AEA induced dose-dependent (200 pmol/2 microl, i.c.v.) catalepsy. A sub-dose of AEA (10 pmol/2 microl, i.c.v.) was potentiated by aspirin (300 mg/kg, p.o.) via a 5-LOX-dependent step. The cataleptic effect induced by the interaction between sub-doses of 15-epi-LXA(4) (0.01 pmol/2 microl, i.c.v.) and AEA (10 pmol/2 microl, i.c.v.) was prevented by the cannabinoid CB(1) receptors antagonist SR141716A (1mg/kg, i.p.), but not by the antagonist of lipoxin ALX receptors Boc-2 (10 microg/kg, i.p.). While previous studies have shown that COX inhibition itself may enhance endocannabinoid effects, here we add another piece of evidence revealing that a LOX-derivative produced in consequence of COX-2 acetylation participates in this process.