Riccardo Bertini

Crucial pathophysiological role of CXCR2 in experimental ulcerative colitis in mice.

Polymorphonuclear leukocyte infiltration and activation into colonic mucosa are believed to play a pivotal role in mediating tissue damage in human ulcerative colitis (UC). Ligands of human CXC chemokine receptor 1 and 2 (CXCR1/R2) are chemoattractants of PMN, and high levels were found in the mucosa of UC patients. To investigate the pathophysiological role played by CXCR2 in experimental UC, we induced chronic experimental colitis in WT and CXCR2−/− mice by two consecutive cycles of 4% dextran sulfate sodium administration in drinking water. In wild‐type (WT) mice, the chronic relapsing of DSS‐induced colitis was characterized by clinical signs and histopathological findings that closely resemble human disease. CXCR2−/− mice failed to show PMN infiltration into the mucosa and, consistently with a key role of PMN in mediating tissue damage in UC, showed limited signs of mucosal damage and reduced clinical symptoms. Our data demonstrate that CXCR2 plays a key pathophysiological role in experimental UC, suggesting that CXCR2 activation may represent a relevant pharmacological target for the design of novel pharmacological treatments in human UC.

Targeting C5a: Recent Advances in Drug Discovery

Activation of complement via the innate and adaptive immune system is vital to the bodys defences in fighting infections. Unregulated complement activation is likely to play a crucial role in the pathogenesis of several diseases including psoriasis, adult (acute) respiratory distress syndrome (ARDS), bullous pemphigoid (BP), rheumatoid arthritis (RA) and ischemia-reperfusion (I/R) injury. The 74 amino acid peptide C5a is released after complement activation at sites of inflammation and is a potent chemoattractant for neutrophils, basophils, eosinophils, leukocytes, monocytes and macrophages. Recombinant proteins and humanized anti-C5 antibodies have been recently developed for blocking specific proteins of the complement system bringing renewed attention towards complement inhibition. Pharmacological studies have been highlighting the complement fragment C5a as an interesting target for the management of complement mediated diseases. Specific inhibition of C5a biological activity could gain therapeutic benefit without affecting the protective immune response. In the last few years several peptide and non-peptide antagonists of C5a have been discovered and tested in relevant pharmacological models; the availability of orally active compounds is rapidly helping to delineate the precise role of C5a in immunoinflammatory disorders. Moreover, mutagenesis data for the C5a/C5a receptor (C5aR) couple make C5aR a valuable model for mechanistic studies of peptidergic G-protein coupled receptors (GPCRs). The aim of this review is to outline the recent advances in C5a inhibition, especially highlighting the value of a multidisciplinary integrated approach in drug discovery.

Treatment with DF 2162, a non-competitive allosteric inhibitor of CXCR1/2, diminishes neutrophil influx and inflammatory hypernociception in mice.

Neutrophil migration into tissues is involved in the genesis of inflammatory pain. Here, we addressed the hypothesis that the effect of CXC chemokines on CXCR1/2 is important to induce neutrophil migration and inflammatory hypernociception.

Blockade of the chemokine receptor CXCR2 ameliorates adjuvant-induced arthritis in rats

Chemokine receptors CXCR1 and CXCR2 may mediate influx of neutrophils in models of acute and chronic inflammation. The potential benefits of oral administration of a CXCR1/2 inhibitor, DF 2162, in adjuvant‐induced polyarthritis (AIA) were investigated.

Corticosteroid-independent inhibition of tumor necrosis factor production by the neuropeptide urocortin

Urocortin (UCN) is a neuropeptide homologous with corticotropin-releasing factor (CRF), which has anti-inflammatory activities not all mediated by corticosteroids. In mice, UCN (1 microg/mouse sc) significantly reduced lipopolysaccharide (LPS)-induced serum tumor necrosis factor (TNF) and interleukin (IL)-1beta levels in vivo but did not affect serum IL-6. These effects were paralleled by a rise in corticosterone (CS) levels. Blockade of the CS increase by cyanoketone did not prevent TNF inhibition by UCN, suggesting the neuropeptide has anti-inflammatory mechanisms independent of the hypothalamus-pituitary-adrenal axis. In fact UCN had a direct inhibitory effect on LPS-induced TNF in rat Kupffer cells at concentrations between 10(-10) and 10(-16) M, and this effect was related to increased cAMP levels. However, the in vivo inhibition of LPS-induced IL-1beta by UCN was reversed by cyanoketone, indicating that the increase of endogenous glucocorticoids might be more important in IL-1beta inhibition than in TNF inhibition by UCN.Urocortin (UCN) is a neuropeptide homologous with corticotropin-releasing factor (CRF), which has anti-inflammatory activities not all mediated by corticosteroids. In mice, UCN (1 μg/mouse sc) significantly reduced lipopolysaccharide (LPS)-induced serum tumor necrosis factor (TNF) and interleukin (IL)-1β levels in vivo but did not affect serum IL-6. These effects were paralleled by a rise in corticosterone (CS) levels. Blockade of the CS increase by cyanoketone did not prevent TNF inhibition by UCN, suggesting the neuropeptide has anti-inflammatory mechanisms independent of the hypothalamus-pituitary-adrenal axis. In fact UCN had a direct inhibitory effect on LPS-induced TNF in rat Kupffer cells at concentrations between 10-10 and 10-16 M, and this effect was related to increased cAMP levels. However, the in vivo inhibition of LPS-induced IL-1β by UCN was reversed by cyanoketone, indicating that the increase of endogenous glucocorticoids might be more important in IL-1β inhibition than in TNF inhibition by UCN.

IL-1β primes IL-8-activated human neutrophils for elastase release, phospholipase D activity, and calcium flux

Interleukin‐8 (IL‐8), the prototype of the α (i.e., C‐X‐C branch) chemokine family, induced elastase release in a concentration‐dependent manner (50‐1000 ng/mL) in cytochalasin B‐treated human polymorphonuclear leukocytes (PMNs). This response was potentiated about twofold if PMNs were preexposed to interleukin‐1β (IL‐1β) at concentrations that were by themselves inactive. The effect of IL‐1β was clearly observed after 5 min and was maximal after a 30‐min preincubation of the cells. The effect was present over the whole active concentration range of IL‐8 and was completely blocked by the presence of IL‐1 receptor antagonist. Priming of elastase release by IL‐1β was not associated with a change in receptor number or affinity for IL‐8. On the contrary, it was correlated with priming of phospholipase D activity and calcium flux activated by IL‐8. Preincubation of the cells with ethanol and/or La3+ inhibited IL‐8‐induced degranulation, suggesting that activation of phospholipase D and increase of [Ca2+]i were important for this response. In contrast, ethanol and La3+ did not decrease the priming effect of IL‐1β. IL‐8 and IL‐1β have been shown to be released by the same cell types and may be concomitantly present at sites of inflammation, giving rise to an amplification of the inflammatory response.

Dexamethasone modulation of in vivo effects of endotoxin, tumor necrosis factor, and interleukin-1 on liver cytochrome P-450, plasma fibrinogen, and serum iron.

Treatment of mice with endotoxin (lipopolysaccharide, LPS) and the two LPS‐induced monokines, tumor necrosis factor (TNF) and interleukin‐1 (IL‐1), caused a depression of liver cytochrome P‐450 and related drug‐metabolizing enzymes, as well as other acute‐phase changes including increase in plasma fibrinogen levels and hypoferremia. However, only IL‐1, not TNF or LPS, depressed cytochrome P‐450 in cultured hepatocytes, suggesting that the effect of TNF in vivo might be mediated by a second mediator. TNF‐or LPS‐stimulated monocytes released a factor capable of depressing cytochrome P‐450 in cultured hepatocytes. This factor was inhibited by anti‐IL‐1 antiserum, and its synthesis, like that of IL‐1, was inhibited by dexamethasone (DEX). Pretreatment of mice with DEX protected against the depression of liver cytochrome P‐450 by LPS or TNF but not by IL‐1, suggesting that IL‐1 directly depresses cytochrome P‐450 and that DEX acts by inhibiting IL‐1 synthesis in vivo induced by LPS or TNF. However, DEX did not inhibit two other effects of LPS and TNF in vivo: increase of plasma fibrinogen levels and decrease of plasma iron, suggesting that these might not be mediated by IL‐1. Therefore, the effect of DEX in vivo, although supporting the hypothesis that depression of liver cytochrome P‐450 by LPS and TNF is mediated by IL‐1, indicates the existence of IL‐1‐independent pathways in the acute‐phase response.

Reparixin, an Inhibitor of CXCR2 Function, Attenuates Inflammatory Responses and Promotes Recovery of Function after Traumatic Lesion to the Spinal Cord

It has been shown that the blockade of CXCR1 and CXCR2 receptors prevents ischemia/reperfusion damage in several types of vascular beds. Reparixin is a recently described inhibitor of human CXCR1/R2 and rat CXCR2 receptor activation. We applied reparixin in rats following traumatic spinal cord injury and determined therapeutic temporal and dosages windows. Treatment with reparixin significantly counteracts secondary degeneration by reducing oligodendrocyte apoptosis, migration to the injury site of neutrophils and ED-1-positive cells. The observed preservation of the white matter might also be secondary to the enhanced proliferation of NG2-positive cells. The expression of macrophage-inflammatory protein-2, tumor necrosis factor-α, interleukin (IL)-6, and IL-1β was also counteracted, and the proliferation of glial fibrillary acidic protein-positive cells was markedly reduced. These effects resulted in a smaller post-traumatic cavity and in a significantly improved recovery of hind limb function. The best beneficial outcome of reparixin treatment required 7-day administration either by i.p. route (15 mg/kg) or subcutaneous infusion via osmotic pumps (10 mg/kg), reaching a steady blood level of 8 μg/ml. Methylprednisolone was used as a reference drug; such treatment reduced cytokine production but failed to affect the rate of hind limb recovery.

Depression of liver drug metabolism and increase in plasma fibrinogen by interleukin 1 and tumor necrosis factor: a comparison with lymphotoxin and interferon

Different recombinant cytokines were studied for their effects on mouse liver in vivo: interleukin 1-alpha and -beta, tumor necrosis factor, lymphotoxin, interferon-alpha A/D and gamma depressed liver cytochrome P450-dependent drug metabolism (measured by ethoxycoumarin deethylase activity) 24 h after treatment, at doses in the microgram range, while IL-2 had no effect on this enzymatic system. Interleukin 1 (both alpha and beta), tumor necrosis factor and lymphotoxin also increased plasma fibrinogen, a marker of liver acute phase inflammatory response. Interferon-gamma and tumor necrosis factor had an additive effect in depressing liver drug metabolism. When tested in vitro on isolated hepatocytes, only interleukin 1 depressed P450-dependent drug metabolism, while all the other cytokines were inactive, thus suggesting that their effect on the liver in vivo is not a direct effect but is mediated by other mediators.

Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxis

The signalling pathways leading to CXCL8/IL‐8‐induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline‐rich tyrosine kinase 2 (Pyk2), a non‐receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3‐kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8‐mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1‐mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase‐dead mutant of Pyk2, blocks CXCL8‐induced chemotaxis of HL‐60‐derived PMN‐like cells, thus pinpointing the key role of Pyk2 in CXCL8‐induced chemotaxis.