Claudio Canetti

Prostaglandin E2 Inhibits Alveolar Macrophage Phagocytosis through an E-Prostanoid 2 Receptor-Mediated Increase in Intracellular Cyclic AMP

Prostaglandin E2 is a potent lipid mediator of inflammation that effects changes in cell functions through ligation of four distinct G protein-coupled receptors (E-prostanoid (EP)1, EP2, EP3, and EP4). During pneumonia, PGE2 production is enhanced. In the present study, we sought to assess the effect of endogenously produced and exogenously added PGE2 on FcRγ-mediated phagocytosis of bacterial pathogens by alveolar macrophages (AMs), which are critical participants in lung innate immunity. We also sought to characterize the EP receptor signaling pathways responsible for these effects. PGE2 (1–1000 nM) dose-dependently suppressed the phagocytosis by rat AMs of IgG-opsonized erythrocytes, immune serum-opsonized Klebsiella pneumoniae, and IgG-opsonized Escherichia coli. Conversely, phagocytosis was stimulated by pretreatment with the cyclooxygenase inhibitor indomethacin. PGE2 suppression of phagocytosis was associated with enhanced intracellular cAMP production. Experiments using both forskolin (adenylate cyclase activator) and rolipram (phosphodiesterase IV inhibitor) confirmed the inhibitory effect of cAMP stimulation. Immunoblot analysis of rat AMs identified expression of only EP2 and EP3 receptors. The selective EP2 agonist butaprost, but neither the EP1/EP3 agonist sulprostone nor the EP4-selective agonist ONO-AE1-329, mimicked the effects of PGE2 on phagocytosis and cAMP stimulation. Additionally, the EP2 antagonist AH-6809 abrogated the inhibitory effects of both PGE2 and butaprost. We confirmed the specificity of our results by showing that AMs from EP2-deficient mice were resistant to the inhibitory effects of PGE2. Our data support a negative regulatory role for PGE2 on the antimicrobial activity of AMs, which has important implications for future efforts to prevent and treat bacterial pneumonia.

A Role for IL-18 in Neutrophil Activation

IL-18 expression and functional activity has been identified in several autoimmune and infectious diseases. To clarify the potential role of IL-18 during early innate immune responses, we have explored the capacity of IL-18 to activate neutrophils. Human peripheral blood-derived neutrophils constitutively expressed IL-18R (α and β) commensurate with the capacity to rapidly respond to IL-18. IL-18 induced cytokine and chemokine release from neutrophils that was protein synthesis dependent, up-regulated CD11b expression, induced granule release, and enhanced the respiratory burst following exposure to fMLP, but had no effect upon the rate of neutrophil apoptosis. The capacity to release cytokine and chemokine was significantly enhanced in neutrophils derived from rheumatoid arthritis synovial fluid, indicating differential responsiveness to IL-18 dependent upon prior neutrophil activation in vivo. Finally, IL-18 administration promoted neutrophil accumulation in vivo, whereas IL-18 neutralization suppressed the severity of footpad inflammation following carrageenan injection. The latter was accompanied by reduction in tissue myeloperoxidase expression and suppressed local TNF-α production. Together, these data define a novel role for IL-18 in activating neutrophils and thereby promoting early innate immune responses.

Leukotrienes: underappreciated mediators of innate immune responses.

Leukotrienes are bronchoconstrictor and vasoactive lipid mediators that are targets in the treatment of asthma. Although they are increasingly recognized to exert broad proinflammatory effects, their role in innate immune responses is less well appreciated. These molecules are indeed synthesized by resident and recruited leukocytes during infection. Acting via cell surface G protein-coupled receptors and subsequent intracellular signaling events, they enhance leukocyte accumulation, phagocyte capacity for microbial ingestion and killing, and generation of other proinflammatory mediators. Interestingly, a variety of acquired states of immunodeficiency, such as HIV infection and malnutrition, are characterized by a relative deficiency of leukotriene synthesis. The data reviewed herein point to leukotrienes as underappreciated yet highly relevant mediators of innate immunity.

Cutting Edge: Macrophage Inhibition by Cyclic AMP (cAMP): Differential Roles of Protein Kinase A and Exchange Protein Directly Activated by cAMP-1

cAMP has largely inhibitory effects on components of macrophage activation, yet downstream mechanisms involved in these effects remain incompletely defined. Elevation of cAMP in alveolar macrophages (AMs) suppresses FcγR-mediated phagocytosis. We now report that protein kinase A (PKA) inhibitors (H-89, KT-5720, and myristoylated PKA inhibitory peptide 14–22) failed to prevent this suppression in rat AMs. We identified the expression of the alternative cAMP target, exchange protein directly activated by cAMP-1 (Epac-1), in human and rat AMs. Using cAMP analogs that are highly specific for PKA (N6-benzoyladenosine-3′,5′-cAMP) or Epac-1 (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cAMP), we found that activation of Epac-1, but not PKA, dose-dependently suppressed phagocytosis. By contrast, activation of PKA, but not Epac-1, suppressed AM production of leukotriene B4 and TNF-α, whereas stimulation of either PKA or Epac-1 inhibited AM bactericidal activity and H2O2 production. These experiments now identify Epac-1 in primary macrophages, and define differential roles of Epac-1 vs PKA in the inhibitory effects of cAMP.

MIP‐1α[CCL3] acting on the CCR1 receptor mediates neutrophil migration in immune inflammation via sequential release of TNF‐α and LTB4

In the present study, we investigated the involvement of macrophage‐inflammatory protein‐1α (MIP‐1α)[CC chemokine ligand 3 (CCL3)], MIP‐1β[CCL4], regulated on activation, normal T expressed and secreted (RANTES)[CCL5], and CC chemokine receptors (CCRs) on neutrophil migration in murine immune inflammation. Previously, we showed that ovalbumin (OVA)‐triggered neutrophil migration in immunized mice depends on the sequential release of tumor necrosis factor α (TNF‐α) and leukotriene B4(LTB4). Herein, we show increased mRNA expression for MIP‐1α[CCL3], MIP‐1β[CCL4], RANTES[CCL5], and CCR1 in peritoneal cells harvested from OVA‐challenged, immunized mice, as well as MIP‐1α[CCL3] and RANTES[CCL5] but not MIP‐1β[CCL4] proteins in the peritoneal exudates. OVA‐induced neutrophil migration response was muted in immunized MIP‐1α[CCL3]−/− mice, but it was not inhibited by treatment with antibodies against RANTES[CCL5] or MIP‐1β[CCL4]. MIP‐1α[CCL3] mediated neutrophil migration in immunized mice through induction of TNF‐α and LTB4 synthesis, as these mediators were detected in the exudates harvested from OVA‐challenged immunized wild‐type but not MIP‐1α[CCL3]−/− mice; administration of MIP‐1α[CCL3] induced a dose‐dependent neutrophil migration, which was inhibited by treatment with an anti‐TNF‐α antibody in TNF receptor 1 (p55−/−)‐deficient mice or by MK 886 (a 5‐lipoxygenase inhibitor); and MIP‐1α[CCL3] failed to induce LTB4 production in p55−/− mice. MIP‐1α[CCL3] used CCR1 to promote neutrophil recruitment, as OVA or MIP‐1α[CCL3] failed to induce neutrophil migration in CCR1−/− mice, in contrast to CCR5−/− mice. In summary, we have demonstrated that neutrophil migration observed in this model of immune inflammation is mediated by MIP‐1α[CCL3], which via CCR1, induces the sequential release of TNF‐α and LTB4. Therefore, whether a similar pathway mediates neutrophil migration in human immune‐inflammatory diseases, the development of specific CCR1 antagonists might have a therapeutic potential.

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 A4 is an endogenous allosteric enhancer of the CB1 cannabinoid receptor. Lipoxin A4 was detected in brain tissues, did not compete for the orthosteric binding site of the CB1 receptor (vs. 3H-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 A4 displayed a CB1 receptor-dependent protective effect against β-amyloid (1–40)-induced spatial memory impairment in mice. The discovery of lipoxins as a class of endogenous allosteric modulators of CB1 receptors may foster the therapeutic exploitation of the endocannabinoid system, in particular for the treatment of neurodegenerative disorders.

Tumour necrosis factor‐alpha and leukotriene B4 mediate the neutrophil migration in immune inflammation

We investigated the mediators responsible for neutrophil migration induced by ovalbumin (OVA) in immunized mice and the mechanisms involved in their release. OVA administration promoted dose‐ and time‐dependent neutrophil migration in immunized, but not in non‐immunized mice, which was mediated by leukotriene B4 (LTB4) and tumour necrosis factor (TNF)α, since it was inhibited by LTB4 synthesis inhibitor (MK 886) or by LTB4 receptor antagonist (CP 105,696), by dexamethasone and by antiserum to TNFα (82, 85, 63 and 87%, respectively). Confirming TNFα involvement, OVA challenge in immunized p55 TNF receptor deficient mice (p55−/−) did not promote neutrophil migration (control: 2.90±0.68; p55−/−: 0.92±0.23×106 neutrophils cavity−1). OVA‐stimulated peritoneal cells from immunized mice released a neutrophil chemotactic factor which mimicked, in naive mice, neutrophil migration induced by OVA. Supernatant chemotactic activity is due to TNFα and LTB4, since its release was inhibited by MK 886 (93%) and dexamethasone (90%), and significant amounts of these mediators were detected. TNFα and LTB4 released by OVA challenge seem to act through a sequential mechanism, since MK 886 inhibited (88%) neutrophil migration induced by TNFα. Moreover, peritoneal cells stimulated with TNFα released LTB4. CD4+ T cells are responsible for TNFα release, because the depletion of this subset prevented the release of TNFα (control: 400±25; immunized: 670±40; CD4+ depleted: 435±18 pg ml−1). In conclusion, neutrophil migration induced by OVA depends on TNFα released by CD4+ cells, which acts through an LTB4‐dependent mechanism.

Involvement of LTB4 in zymosan-induced joint nociception in mice: participation of neutrophils and PGE2

Leukotriene B4 (LTB4) mediates different inflammatory events such as neutrophil migration and pain. The present study addressed the mechanisms of LTB4‐mediated joint inflammation‐induced hypernociception. It was observed that zymosan‐induced articular hypernociception and neutrophil migration were reduced dose‐dependently by the pretreatment with MK886 (1–9 mg/kg; LT synthesis inhibitor) as well as in 5‐lypoxygenase‐deficient mice (5LO−/−) or by the selective antagonist of the LTB4 receptor (CP105696; 3 mg/kg). Histological analysis showed reduced zymosan‐induced articular inflammatory damage in 5LO−/− mice. The hypernociceptive role of LTB4 was confirmed further by the demonstration that joint injection of LTB4 induces a dose (8.3, 25, and 75 ng)‐dependent articular hypernociception. Furthermore, zymosan induced an increase in joint LTB4 production. Investigating the mechanism underlying LTB4 mediation of zymosan‐induced hypernociception, LTB4‐induced hypernociception was reduced by indomethacin (5 mg/kg), MK886 (3 mg/kg), celecoxib (10 mg/kg), antineutrophil antibody (100 μg, two doses), and fucoidan (20 mg/kg) treatments as well as in 5LO−/− mice. The production of LTB4 induced by zymosan in the joint was reduced by the pretreatment with fucoidan or antineutrophil antibody as well as the production of PGE2 induced by LTB4. Therefore, besides reinforcing the role of endogenous LTB4 as an important mediator of inflamed joint hypernociception, these results also suggested that the mechanism of LTB4‐induced articular hypernociception depends on prostanoid and neutrophil recruitment. Furthermore, the results also demonstrated clearly that LTB4‐induced hypernociception depends on the additional release of endogenous LTs. Concluding, targeting LTB4 synthesis/action might constitute useful therapeutic approaches to inhibit articular inflammatory hypernociception.

ATLa, an Aspirin-Triggered Lipoxin A4 Synthetic Analog, Prevents the Inflammatory and Fibrotic Effects of Bleomycin-Induced Pulmonary Fibrosis

Despite an increase in the knowledge of mechanisms and mediators involved in pulmonary fibrosis, there are no successful therapeutics available. Lipoxins (LX) and their 15-epimers, aspirin-triggered LX (ATL), are endogenously produced eicosanoids with potent anti-inflammatory and proresolution effects. To date, few studies have been performed regarding their effect on pulmonary fibrosis. In the present study, using C57BL/6 mice, we report that bleomycin (BLM)-induced lung fibrosis was prevented by the concomitant treatment with an ATL synthetic analog, ATLa, which reduced inflammation and matrix deposition. ATLa inhibited BLM-induced leukocyte accumulation and alveolar collapse as evaluated by histology and morphometrical analysis. Moreover, Sirius red staining and lung hydroxyproline content showed an increased collagen deposition in mice receiving BLM alone that was decreased upon treatment with the analog. These effects resulted in benefits to pulmonary mechanics, as ATLa brought to normal levels both lung resistance and compliance. Furthermore, the analog improved mouse survival, suggesting an important role for the LX pathway in the control of disease establishment and progression. One possible mechanism by which ATLa restrained fibrosis was suggested by the finding that BLM-induced myofibroblast accumulation/differentiation in the lung parenchyma was also reduced by both simultaneous and posttreatment with the analog (α-actin immunohistochemistry). Interestingly, ATLa posttreatment (4 days after BLM) showed similar inhibitory effects on inflammation and matrix deposition, besides the TGF-β level reduction in the lung, reinforcing an antifibrotic effect. In conclusion, our findings show that LX and ATL can be considered as promising therapeutic approaches to lung fibrotic diseases.

The critical role of leukotriene B4 in antigen-induced mechanical hyperalgesia in immunised rats.

We investigated the mediators responsible for mechanical hypersensitivity induced by antigen challenge in rats immunised with ovalbumin (OVA). Challenge with OVA (12.5–100 μg, intraplantar) caused a dose‐ and time‐dependent mechanical hypersensitivity, which peaked 3 h after, decreased thereafter and reached control levels 24 h later. Levels of TNFα, IL‐1β and cytokine‐induced neutrophil chemoattractant 1 (CINC‐1) were increased in paw skin after antigen challenge. OVA‐evoked hypersensitivity was partially inhibited (about 51%) by pretreatment with anti‐TNFα, IL‐1β and IL‐8 sera or with IL‐1 receptor antagonist (IL‐1ra), but not anti‐NGF serum. Pretreatment with thalidomide (45 mg kg−1) or pentoxifylline (100 mg kg−1) also partially inhibited the hypersensitivity at 1–3 h after challenge. Pretreatment with indomethacin (5 mg kg−1) or atenolol (1 mg kg−1) reduced the OVA‐induced hypersensitivity at 1 and 3 h, but not at 5 h after challenge, while the combination of B1 and B2 bradykinin receptor antagonists was ineffective over the same times. Pretreatment with MK886 (5‐lipoxygenase‐activating protein inhibitor, 3 mg kg−1), CP 105696 (LTB4 receptor antagonist; 3 mg kg−1) or dexamethasone (0.5 mg kg−1) inhibited the hypersensitivity from 1 to 5 h. Furthermore, LTB4 levels were increased in the paw skin of challenged rats. In conclusion, our results suggest that the TNFα‐, IL‐1β‐ and CINC‐1‐driven release of prostaglandins, sympathetic amines and LTB4 mediates the first 3 h of mechanical hypersensitivity induced by antigen challenge in rats. At 5 h after OVA administration, although TNFα has some role, LTB4 is the critical nociceptive mediator.