6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives as a new class of potent inhibitors of Interleukin-8-induced neutrophil chemotaxis.

A series of 6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives was synthesized. The compounds demonstrated to be novel, potent and selective inhibitors of Interleukin-8-induced human neutrophil chemotaxis. A SAR study was performed by varying the carbonyl function at position 5 and the chain linked to the amino group at position 6 of the scaffold. All the compounds of the series displayed inhibitory activity at nano- or picomolar concentrations against Interleukin-8-driven migration and no activity against fMLP- and C5a-induced chemotaxis. The binding tests of selected compounds on CXCR1 and CXCR2 receptors were negative. The most potent derivative showed in vivo efficacy in a mouse model of Zymosan-induced peritonitis.

Synthesis and biological evaluation of N-pyrazolyl-N'-alkyl/benzyl/phenylureas: a new class of potent inhibitors of interleukin 8-induced neutrophil chemotaxis.

Neutrophils chemotaxis is a complex multistep process that, if upregulated, causes acute inflammation and a number of autoimmune diseases. We report here the synthesis of a new N-(4-substituted)pyrazolyl-N'-alkyl/benzyl/phenylureas that are potent inhibitors of interleukin-8 (IL8)-induced neutrophil chemotaxis. The first series of compounds, obtained by functionalization with a urea moiety of the 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylic acid ethyl ester 3, blocked the IL8-induced neutrophil chemotaxis, while they did not block N-formylmethionylleucylphenylalanine-mediated chemotaxis. The most active compounds, 3-benzyl- (4d), 3-(4-benzylpiperazinyl)- (4i), 3-phenyl- (4k) and 3-isopropylureido (4a) derivatives, showed an IC50 of 10, 14, 45, and 55 nM, respectively. Several different molecules were then synthesized to obtain more information for SAR study. Compounds 4a, 4d, and 4k were inactive in the binding assays on CXCR1 and CXCR2 (IL8 receptors), whereas they inhibited the phosphorylation of PTKs (protein tyrosine kinases) in the 50-70 kDa region. Moreover, in the presence of the same derivatives, we observed a complete block of F-actin rise and pseudopod formation.

Chemokine receptor expression and function in childhood acute lymphoblastic leukemia of B-lineage.

Scanty information is available on chemokine receptor expression and function in childhood B-lineage acute lymphoblastic leukemia (ALL). Thirteen pro-B, 17 early pre-B, 12 pre-B, and 9 B-ALL/Burkitt lymphoma (BL) pediatric cases were tested for CXCR1 to CXCR5 and CCR1 to CCR7 expression. CXCR2, CXCR3, and CXCR4 were expressed in the majority of cases, while the other receptors were variably expressed or absent. CXCR4 mediated chemotaxis of all leukemic cell subtypes. Freshly isolated CCR7(+) early pre-B-ALL cells migrated to CCL19, whereas CCR7(+) pro-B- and pre-B-ALL cells were attracted by CCL19 only following culture with soluble recombinant CD40 ligand.

CCL3 (MIP-1alpha) induces in vitro migration of GM-CSF-primed human neutrophils via CCR5-dependent activation of ERK 1/2.

CCL3 (MIP-1alpha), a prototype of CC chemokines, is a potent chemoattractant toward human neutrophils pre-treated with GM-CSF for 15 min. GM-CSF-treated neutrophils migrate also to the selective CCR5 agonist CCL4 (MIP-1beta). CCL3- and CCL4-triggered migration of GM-CSF-primed neutrophils was inhibited by the CCR5 antagonist TAK-779. Accordingly, freshly isolated neutrophils express CCR5. Extracellular signal-regulated kinases (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) inhibitors blocked CCL3-induced migration of GM-CSF-primed neutrophils. When the activation of ERK-1/2 and p38 MAPK by CCL3 and the classical neutrophilic chemokine CXCL8 (IL-8) were compared, both the chemokines were capable of activating p38 MAPK. On the contrary, whereas both ERK-1 and ERK-2 were activated by CXCL8, no ERK-1 band was detectable after CCL3 triggering. Finally, neutrophil pre-treatment with GM-CSF activated both ERK-1 and ERK-2. This suggests that by activating ERK-1, GM-CSF renders neutrophils rapidly responsive to CCL3 stimulation throughout CCR5 which is constitutively expressed on the cell surface.

Synthesis and biological evaluation of neutrophilic inflammation inhibitors.

In several non-infectious human diseases, such as ulcerous colitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), the extravasal recruitment of neutrophils plays a crucial role in the development of tissue damage, which, when persistent, can lead to the irreversible organ dysfunction. The neutrophil activation is controlled by a number of intracellular pathways, particularly by a cAMP-dependent protein kinase A (PKA) which also acts on phosphodiesterase IV (PDE4) gene stimulating the synthesis of this enzyme, able to transform cAMP to inactive AMP. PDE4 inhibitors enhance intracellular cAMP and decrease inflammatory cell activation. Several 3-cyclopentyloxy-4-methoxybenzaldehyde and 3-cyclopentyloxy-4-methoxybenzoic acid derivatives were synthesized and studied by us to evaluate their ability to inhibit the superoxide anion production in human neutrophils. These compounds were found able to inhibit the neutrophil activation and some of them increased the cAMP level on tumor necrosis factor-alpha-stimulated neutrophils. Moreover, they also inhibited selectively the human PDE4 enzyme, although they are less potent than the reference compound Rolipram. We report here synthesis, biological studies and some SAR considerations concerning the above mentioned compounds.

CCL19 and CXCL12 trigger in vitro chemotaxis of human mantle cell lymphoma B cells.

PURPOSE: Few data are available in the literature on chemokine receptor expression and migratory capability of mantle cell lymphoma (MCL) B cells. Information on these issues may allow us to identify novel mechanisms of chemokine-driven tumor cell migration. EXPERIMENTAL DESIGN: The research was designed to investigate: (a) expression of CCR1 to CCR7 and CXCR1 to CXCR5 chemokine receptors; and (b) chemotaxis to the respective ligands in MCL B cells and in their normal counterparts, i.e., CD5+ B cells. RESULTS: Malignant B cells from MCL patients and normal counterparts displayed similar chemokine receptor profiles. MCL B cells were induced to migrate by CXCL12 and CCL19, whereas normal CD5+ B cells migrated to the former, but not the latter chemokine. Overnight culture of MCL B cells and their normal counterparts with CXCL12 cross-sensitized other chemokine receptors to their ligands in some tumor samples but not in CD5+ B cells. CONCLUSIONS: CCR7 and CXCR4 ligands may play a key role in tumor cell migration and spreading in vivo. CXCL12 may additionally contribute by sensitizing MCL B cells to respond to the ligands of other chemokine receptors.

In vitro inhibition of human neutrophil histotoxicity by ambroxol: evidence for a multistep mechanism.

Neutrophils are major culprits for the protease/antiprotease imbalance during various lung diseases, that is, chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis and adult respiratory distress syndrome. Thus, these cells are presently considered an ideal target for the pharmacologic control of tissue injury during these diseases. This study was planned in order to investigate if ambroxol and its precursor bromhexine are actually capable of preventing alpha-1-antitrypsin (A1AT) inactivation by stimulated neutrophils and possibly to look into the mechanisms underlying this event. Ambroxol inhibited the production of superoxide anion by activated neutrophils, whereas bromhexine had no inhibitory effect. Ambroxol decreased the production of hypochlorous acid (HOCl) from activated neutrophils with high efficiency, whereas bromhexine had a modest activity. Ambroxol and bromhexine were capable of limiting the chlorination of monochlorodimedon by HOCl, displaying the capacity of directly scavenging the oxidant. Ambroxol decreased the release of elastase and myeloperoxidase from activated neutrophils, whereas bromhexine was ineffective. Ambroxol prevented the A1AT inactivation by neutrophils, whereas bromhexine was completely ineffective. Among drugs currently available for in vivo use in humans, ambroxol is unique by virtue of its ability to prevent neutrophil-mediated A1AT inactivation via inhibition of HOCl production as well as HOCl scavenging. Also taking into account its capacity for curbing elastase release, the drug displays the potential to lessen the burden of oxidants/proteases and to increase the antiprotease shield at the site of inflammation. Thus, ambroxol appears to be a good candidate for raising attempts to develop new therapeutic histoprotective approaches to inflammatory bronchopulmonary diseases.

Effect of clotting factors concentrates on lymphocyte and neutrophil function in vitro.

Immunological abnormalities have been reported in haemophiliacs. Although infections with HIV, hepatitis and other viruses may contribute to these abnormalities, immune defects are detectable also in HIV seronegative haemophiliacs. It is likely that chronic exposure to extraneous proteins in clotting factor concentrates (CFCs) may play a role in immunomodulation, but the underlying mechanisms remain unclear. The results of the present paper show that: a) soluble HLA class I (sHLA-I), soluble Fas-ligand (sFas-L) and transforming growth factor beta 1 (TGF-beta1) are detectable in plasma derived but not in recombinant CFCs; b) the level of sHLA-I and sFas-L is proportional to the grade of CFCs purity whereas TGF-beta1 showed very variable levels; c) soluble molecules detected in CFCs exert immunomodulatory effects in vitro like apoptosis induction in Jurkat cells and inhibition of mixed lymphocyte reaction response, antigen-specific lymphocyte cytotoxic activity and neutrophil chemotaxis.

Differential regulation of spontaneous and immune complex-induced neutrophil apoptosis by proinflammatory cytokines. Role of oxidants, Bax and caspase-3.

Neutrophil apoptosis represents a crucial step in the mechanisms governing the resolution of neutrophilic inflammation. Several soluble mediators of inflammation modulate neutrophil survival, retarding their apoptosis, whereas neutrophil activation by immune complexes (IC) results in the acceleration of apoptosis. To investigate neutrophil fate at the site of inflammation, we studied the effects of interleukin (IL)-2, IL-6, IL-8, IL-15, GM-CSF, and fMLP on spontaneous and IC-induced neutrophil apoptosis and the mechanisms regulating the survival of these cells. Spontaneous apoptosis was inhibited by GM-CSF, IL-6, and IL-15, but only GM-CSF overturned IC-induced apoptosis. No role of oxidants on the modulation of IC-dependent apoptosis was found. Indeed, fMLP or GM-CSF augmented the IC-dependent oxidative response, whereas the other compounds were ineffective. CGD neutrophils showed low levels of spontaneous apoptosis, but when exposed to IC, underwent a sharp increment of the apoptotic rate in a GM-CSF-inhibitable manner. Conversely, the expression of the proapoptotic protein Bax in 18-h aged neutrophils was down-regulated by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a nearly threefold Bax up-regulation, which was completely reversed only by GM-CSF. Accordingly, the spontaneous activity of caspase-3 was inhibited by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a sharp increment of enzymatic activity, and only GM-CSF inhibited the IC-dependent acceleration. Our results show that apoptosis of resting and IC-activated neutrophils is regulated differently, GM-CSF being the most potent neutrophil antiapoptotic factor. The results also unveil the existence of an oxidant-independent, Bax- and caspase-3-dependent, intracellular pathway regulating neutrophil apoptosis.

Delayed neutrophil apoptosis induced by synovial fluid in rheumatoid arthritis: role of cytokines, estrogens, and adenosine.

The fate of neutrophils at sites of inflammation, where these cells are likely exposed to both anti- and proapoptotic influences, needs to be clarified. To investigate this issue, we studied the survival of neutrophils in the presence of articular fluids from RA joints before and after immune complex activation. Eight of eleven samples of RA synovial fluid studied were found to inhibit spontaneous and immune complex-stimulated neutrophil apoptosis. No relationships were found between GM-CSF and TNF-alpha concentrations measured on each sample of synovial fluid studied and the levels of neutrophil apoptosis detectable in the presence of the same synovial fluid. Furthermore, no activity on neutrophil survival was observed at either physiologic or pharmacologic concentrations of estradiol. On the contrary, the synovial fluid anti-apoptotic activity correlates (r(2) = 0.8818, p < 0.0001) with the adenosine detected at concentrations in each sample ranging from 18.7 to 52.4 microM. Finally, synovial fluids were incapable of interfering with neutrophil activation evaluated as superoxide anion production. Our results suggest that the microenvironment of rheumatoid synovial fluid is a proinflammatory milieu responsible for the in loco persistence of activated and long-surviving neutrophils.