High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells.

The cAMP-protein kinase A (PKA) pathway is the major signal transduction pathway involved in melanocyte-stimulating hormone receptor-mediated signaling and melanin production, whereas its role in the control of melanocyte proliferation is still controversial. In this study, we evaluated the effects of selective activation of the different PKA regulatory subunits type 1A (R1A) and type 2B (R2B) on melanocyte proliferation. Immunohistochemistry demonstrated that normal melanocytes lacked R1A protein whereas this subunit was highly expressed in all human melanomas studied (N=20) and in six human melanoma cell lines. Pharmacological activation of the R2 subunits by the cAMP analogue 8-Cl-cAMP inhibited proliferation and increased caspase-3 activity by 68.77+/-10.5 and 72+/-9% respectively, in all cell lines with the exception of the only p53-mutated one. Similar effects were obtained by activating R2 subunits with other analogues and by silencing R1A expression. The antiproliferative and proapoptotic effects of 8-Cl-cAMP were comparable to those observed with commonly used antitumoral drugs. Moreover, 8-Cl-cAMP potentiated the effects of these drugs on both cell proliferation and caspase-3 activity. In conclusion, this study first reports that human melanomas are characterized by a high R1/R2 ratio and that pharmacological and genetic manipulations able to revert this unbalanced expression cause significant antiproliferative and proapoptotic effects in melanoma cells.

Evaluation of proopiomelanocortin mRNA in the peripheral blood from patients with Cushing's syndrome of different origin.

ACTH-dependent Cushing's syndrome is due to ACTH overproduction originating from a pituitary corticotroph adenoma (Cushing's disease) or from ectopic tumors (ectopic ACTH syndrome). Due to difficulties in the differential diagnosis between these two forms of hypercortisolism it would be important to have molecular tools able to discriminate the two conditions. It is known that proopiomelanocortin (POMC) gene transcription can originate messengers of different length. ACTHomas show the normal 1072 nucleotides (nt) transcript, whereas ectopic tumors seem to be associated with a longer mRNA form (1450 nt). In order to analyse the presence of different POMC transcripts, we extracted total RNA from peripheral lymphocytes of 10 patients with Cushing's disease, 10 with ectopic Cushing syndrome, and 20 controls as well as from pituitary tissues (2 ACTH-omas and a normal pituitary polyA+ sample). Northern blot analysis correctly revealed a 1072 nt mRNA molecule in pituitary ACTH-oma and in the normal pituitary polyA+ RNA samples, whereas neither this molecule nor other alternative transcripts were detected in blood samples from patients and controls. These data were confirmed by the more sensitive RT-PCR technique. This study further underlines the need for alternative approaches in the diagnosis of ACTH-dependent Cushing's syndrome.

Toll-like receptor family and signalling pathway.

Toll is a Drosophila gene essential for ontogenesis and anti-microbial resistance. Several orthologues of Toll have been identified and cloned in vertebrates, namely Toll-like receptors (TLRs). Human TLRs are a growing family of molecules involved in innate immunity. TLRs are characterized structurally by a cytoplasmic Toll/interleukin-1 receptor (TIR) domain and by extracellular leucine-rich repeats. TLRs characterized so far activate the MyD88/interleukin-1 receptor-associated kinase (IRAK) signalling pathway. Genetic, gene-transfer and dominant-negative approaches have involved TLR family members (TLR2 and TLR4) in Gram-positive and Gram-negative bacteria recognition and signalling. Accumulating evidence suggests that TLR2 is also involved in signalling-receptor complexes that recognize components of yeast and mycobacteria. However, the definitive roles of other TLRs are still lacking. A systematic approach has been used to determine whether different human leucocyte populations selectively or specifically express TLR mRNA. Based on expression pattern, TLR can be classified as ubiquitous (TLR1), restricted (TLR2, TLR4 and TLR5) and specific (TLR3). Expression and regulation of distinct but overlapping ligand-recognition patterns may underlie the existence of a large, seemingly redundant TLR family. Alternatively, the expression of a TLR in a single cell type may indicate a specific role for this molecule in a restricted setting.

Increased peripheral benzodiazepine binding sites and pentraxin 3 expression in the spinal cord during EAE: relation to inflammatory cytokines and modulation by dexamethasone and rolipram.

We have studied the mRNA expression of pentraxin 3 (PTX3) and the binding of the peripheral-type benzodiazepine receptor (PBR) ligand, [3H]-PK11195, in the spinal cord of Lewis rats where EAE was actively induced. PTX3 was induced during the active phase of EAE (day 10-14), it remained high up to 30 days and disappeared only 60 days later. Similarly, PK11195 binding peaked at day 14-17 during the recovery and it disappeared by day 60. On the other hand, the levels of TNF and IL-6 in the spinal cord were elevated at the peak and at the onset of clinical signs and returned to non-detectable by day 14-17. Dexamethasone abolished all these changes, while treatment with rolipram, delayed the appearance of the disease and then decreased its severity. However the peaks of TNF, IL-6, PBR and PTX3 levels in spinal cord were only delayed, but not reduced, by rolipram treatment. In conclusion, we show two types of inflammatory changes in EAE: acute, short term changes (TNF and IL-6), that correlate with the disease; and effects such as PTX3 expression and PK11195 binding that last longer after recovery from the disease.

Inhibition of monocyte chemotactic protein-1 synthesis by statins.

The beneficial effects of statins on the reduction of cardiovascular events has been partly attributed to their anti-inflammatory properties. In the complex of the different pathogenetic events leading to atherosclerosis, recent data suggest a central role of monocyte chemotactic protein-1 (MCP-1), because mice knock-out for MCP-1 or its receptor CC-chemokine receptor 2 were considerably resistant to plaque formation. In this study we investigated the effect of different statins on in vitro and in vivo production of MCP-1. Lovastatin and simvastatin caused a dose-dependent inhibition of MCP-1 production in peripheral blood mononuclear cells exposed to lipopolysaccharide or inactivated Streptococcus hemoliticus and in human endothelial cells exposed to interleukin-1beta. The addition of mevalonate overrode the inhibitory effect of statins indicating that mevalonate-derived products are important for chemokine production. The in vivo anti-inflammatory effect of statins was investigated using the mouse air-pouch model of local inflammation. Lovastatin and pravastatin were orally administered to mice according to a treatment schedule that significantly inhibited the hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity without affecting total blood cholesterol. At the dose of 10 mg/kg, lovastatin and pravastatin reduced by approximately 50% the lipopolysaccharide-induced leukocytes recruitment and the exudate MCP-1 production. In conclusion, statins, by inhibiting mevalonate-derived products, reduced both in vitro and in vivo the production of chemokines involved in leukocyte migration, and this effect is unrelated to their cholesterol-lowering action.

Selectivity release of the type II decoy IL-1 receptor.

The type II IL-1 receptor (IL-1RII) is a non-signalling molecule which acts as a decoy target for IL-1. Various signals (e.g. chemoattractants and phorbol ester) induce metalloprotease-mediated rapid shedding of the IL-1RII. The present study was designed to compare the susceptibility to shedding of the decoy IL-1RII vs the signalling IL-1RI. HEK 293 cells and COS cells were transfected with cDNAs encoding the human IL-1RI, IL-1RII or both. Slow spontaneous release and rapid phorbol ester-induced shedding were only observed for the decoy IL-1RII. Similarly, OVCAR-3 cells, which express substantial amounts of both IL-1RI and IL-1RII, only released the IL-1RII. These results indicate that the IL-RII, but not the IL-1RI, is susceptible to proteolytic shedding, a finding consistent with the decoy function of this molecule.

Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells.

Members of the Toll-like receptor (TLR) family probably play a fundamental role in pathogen recognition and activation of innate immunity. The present study used a systematic approach to analyze how different human leukocyte populations express specific transcripts for the first five characterized TLR family members. TLR1 was expressed in all leukocytes examined, including monocytes, polymorphonuclear leukocytes, T and B cells, and NK cells. In contrast TLR2, TLR4, and TLR5 were expressed in myelomonocytic elements. Exposure to bacterial products, such as LPS or lipoarabinomannan, or to proinflammatory cytokines increased TLR4 expression in monocytes and polymorphonuclear leukocytes, whereas IL-10 blocked this effect. TLR3 was only expressed in human dendritic cells (DC) wherein maturation induced by bacterial products or cytokines was associated with reduced expression. TLR3 mRNA expression was detected by in situ hybridization in DC and lymph nodes. These results demonstrate that TLR1 through TLR5 mRNAs are differentially expressed and regulated in human leukocytes. In particular, expression of TLR3 transcripts is restricted to DC that are the only elements which express the full TLR repertoire. These data suggest that TLR can be classified based on expression pattern as ubiquitous (TLR1), restricted (TLR2, TLR4, and TLR5 in myelomonocytic cells), and specific (TLR3 in DC) molecules.

Inducible expression of the long pentraxin PTX3 in the central nervous system.

PTX3 is a prototypic long pentraxin consisting of a C terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. PTX3 is induced by primary proinflammatory signals in various cell types, most prominently macrophages and endothelial cells. Other long pentraxins, such as murine or rat neuronal pentraxin 1 (NP1) and human neuronal pentraxin 2 (NPTX2), are expressed in the central nervous system (CNS). The present study was designed to investigate whether PTX3 is expressed in the brain and to define the structures and cells involved. Intracerebroventricular (i.c.v.), but not i.v., injection of LPS induced high levels of PTX3 mRNA in the mouse brain. In contrast NP1 is constitutively expressed in the murine CNS and is not modulated by LPS administration. I.c.v. IL-1beta was also a potent inducer of PTX3 expression in the CNS, whereas TNFalpha was substantially less effective and IL-6 induced a barely detectable signal. Central administration of LPS and IL-1 induced PTX3 also in the periphery (heart), whereas the reverse did not occur. Expression of PTX3 was also observed in the brain of mice infected with Candida albicans (C. albicans) or Cryptococcus neoformans. (C. neoformans). The kinetics of PTX3 gene induction were consistently different between C. albicans- and C. neoformans-infected mice, according to the diverse outcome of the CNS immune reaction. In situ hybridization revealed that i.c.v. injection of LPS induced a strong PTX3 expression in presumptive glial cells, in the white matter (corpus callosum, fimbria) and meningeal pia mater as well as in dentate gyrus hilus and granule cells. No constitutive expression of PTX3 was detected. Central expression of PTX3 may amplify mechanisms of innate resistance and damage in the CNS. The possibility of a direct interaction of PTX3 with neuronal cells, as suggested for NPTX2, remains to be explored.

Toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes.

Toll is a Drosophila gene essential for ontogenesis and antimicrobial resistance. Several hortologues of Toll have been identified and cloned in vertebrates, namely Toll-like receptors (TLR). Human TLR are a growing family of molecules involved in innate immunity. TLR are structurally characterized by a cytoplasmic Toll/interleukin-1R (TIR) domain and by extracellular leucine-rich repeats. TLR characterized so far activate the MyD88/IRAK signaling cascade, which bifurcates and leads to NF-kappaB and c-Jun/ATF2/TCF activation. Genetic, gene transfer, and dominant-negative approaches have involved TLR family members (TLR2 and TLR4) in lipopolysaccharide recognition and signaling. Accumulating evidence suggests that some TLR molecules are also involved in signaling receptor complexes that recognize components of gram-positive bacteria and mycobacteria. However, the definitive role of other TLR is still lacking. A systematic approach has been used to determine whether different human leukocyte populations selectively or specifically expressed TLR mRNA. Based on expression pattern, TLR can be classified as ubiquitous (TLR1), restricted (TLR2, TLR4, and TLR5), and specific (TLR3). Expression and regulation of distinct though overlapping ligand recognition patterns may underlie the existence of a numerous, seemingly redundant, TLR family. Alternately, the expression of a TLR in a single cell type may indicate a specific role for this molecule in a restricted setting.

Selective induction of MCP-1 in human mesangial cells by the IL-6/sIL-6R complex.

Interleukin (IL) 6, an autocrine growth factor for mesangial cells, and chemokines, which are released from activated mesangial cells and induce leukocyte infiltration, play a critical role in the progression of immune system mediated renal diseases. Since the reciprocal relationship between IL-6 and chemokines in renal inflammation has been barely investigated, we have analyzed whether IL-6 (500 ng/ml), alone or in combination with the soluble form of its receptor (sIL-6R, 200 ng/ml), can induce normal human mesangial cells (NHMC) to release alpha and/or beta chemokines: MCP-1 (monocyte chemoattractant protein 1), IL-8, Rantes (regulated on activation, normal T cell expressed and secreted), and MIP-1alpha (macrophage inflammatory protein 1alpha). Whereas IL-6 or sIL-6R alone were ineffective in inducing significant chemokine release from NHMC, the simultaneous treatment with IL-6 and sIL-6R showed a significant interaction, leading to a strong synergic effect on MCP-1 synthesis and release without exerting any relevant activity on IL-8, Rantes, or MIP-1alpha. Consistently with the unresponsiveness to IL-6, mRNA and protein expression analysis of the two subunits which form the functional IL-6 receptor showed that NHMC express only the gp130 signal-transducing chain and not the subunit-specific IL-6R (gp80). These findings support an unexpected role of the IL-6 system in kidney inflammatory reactions through the selective regulation of monocyte recruitment.

Autocrine production of IL-10 mediates defective IL-12 production and NF-kappa B activation in tumor-associated macrophages.

IL-12 is a central cytokine in the activation of inflammation and immunity and in the generation of Th1-type responses. Tumor-associated macrophages (TAM) from mouse and human tumors showed defective production of IL-12. Defective IL-12 production was associated with lack of p50/p65 NF-kappa B activation. TAM produced increased amounts of the immunosuppressive cytokine IL-10. Abs against IL-10 restored the defective capacity of TAM to produce IL-12. Our data suggest that during tumor growth an IL-10-dependent pathway of diversion of macrophage function can be activated into the tumor microenvironment and results in the promotion of the IL-10+ IL-12- phenotype of TAM. Blocking IL-10, as well as other immunosuppressive cytokines present in the tumor microenvironment, such as TGF-beta, may complement therapeutic strategies aimed at activating type I antitumor immune responses.

Bacterial lipopolysaccharide activates NF-kappaB through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells. Differential expression of TLR-4 and TLR-2 in endothelial cells.

A missense mutation in the cytoplasmic domain of the Toll-like receptor-4 (TLR-4) has been identified as the defect responsible for lipopolysaccharide (LPS) hyporesponsiveness in C3H/HeJ mice. TLR-4 and TLR-2 have recently been implicated in LPS signaling in studies where these receptors were overexpressed in LPS non-responsive 293 human embryonic kidney cells. However, the signaling role of TLR-4 or TLR-2 in human cells with natural LPS response remains largely undefined. Here we show that human dermal microvessel endothelial cells (HMEC) and human umbilical vein endothelial cells express predominantly TLR-4 but very weak TLR-2 and respond vigorously to LPS but not to Mycobacterium tuberculosis 19-kDa lipoprotein. Transient transfection of non-signaling mutant forms of TLR-4 and anti-TLR-4 monoclonal antibody inhibited LPS-induced NF-kappaB activation in HMEC, while a monoclonal antibody against TLR-2 was ineffective. In contrast to LPS responsiveness, the ability of HMEC to respond to 19-kDa lipoprotein correlated with the expression of TLR-2. Transfection of TLR-2 into HMEC conferred responsiveness to 19-kDa lipoprotein. These data indicate that TLR-4 is the LPS signaling receptor in HMEC and that human endothelial cells (EC) express predominantly TLR-4 and weak TLR-2, which may explain why they do not respond to 19-kDa lipoprotein. The differential expression of TLRs on human EC may have important implications in the participation of vascular EC in innate immune defense mechanisms against various infectious pathogens, which may use different TLRs to signal.

A small synthetic molecule capable of preferentially inhibiting the production of the CC chemokine monocyte chemotactic protein-1.

Blocking chemokine production or action is a major target for pharmacological intervention in different human diseases. Bindarit (2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propan oic acid) dose-dependently inhibited MCP-1 and TNF-alpha production induced in vitro in monocytes by LPS and Candida albicans. It did not affect the production of the cytokines IL-1, IL-6, or the chemokines IL-8, MIP-1alpha and RANTES. In the air pouch model in mice, oral treatment reduced monocyte recruitment and local MCP-1 production, induced by carrageenan or IL-1 injection. In NZB/W mice, a model of lupus nephritis, oral treatment prolonged survival and delayed the onset of proteinuria. The results presented here show that bindarit is a preferential inhibitor of the production of MCP-1 in vitro and in vivo and suggest that its beneficial effects in models of joint and kidney inflammation are related to its anti-MCP-1 action. It is therefore possible to selectively and differentially regulate chemokines by targeting their production with small synthetic molecules.

Up-regulation of CCR2 chemokine receptor expression and increased susceptibility to the multitropic HIV strain 89.6 in monocytes exposed to glucocorticoid hormones.

Glucocorticoid hormones (GC) are potent antiinflammatory agents widely used in the treatment of diverse human diseases. The present study was aimed at assessing the effect of GC on chemokine receptor expression in human monocytes. Dexamethasone (Dex) up-regulated mRNA expression of the monocyte chemotactic protein (MCP-1, CCL2) chemokine receptor CCR2. The effect was selective in that other chemokine receptors were not substantially affected. Stimulation by Dex was observed after 4 h of exposure at concentrations of 10(-7) to 10(-5) M. Steroids devoid of GC activity were inactive, and the GC receptor antagonist, RU486, inhibited stimulation. Dex did not affect the rate of nuclear transcription, but augmented the CCR2 mRNA half-life. Augmentation of CCR2 expression by Dex was associated with increased chemotaxis. Finally, Dex treatment induced productive replication of the HIV strain 89.6, which utilizes CCR2 as entry coreceptor, in freshly isolated monocytes. Together with previous findings, these results indicate that at least certain pro- and antiinflammatory molecules have reciprocal and divergent effects on expression of a major monocyte chemoattractant, MCP-1, and of its receptor (CCR2). Augmentation of monocyte CCR2 expression may underlie unexplained in vivo effects of GC as well as some of their actions on HIV infection.

Characterization of type II intracellular IL-1 receptor antagonist (IL-1ra3): a depot IL-1ra.

Three molecular forms of the IL-1 receptor antagonist (IL-1ra) have been identified and cloned. Secreted IL-1ra (sIL-1ra or IL-1ra1) contains a classical leader peptide giving a released mature protein. Two intracellular isoforms, icIL-1ra type I (IL-1ra2) and icIL-1ra type II (IL-1ra3), have no leader sequence, thus predicting that these proteins remain intracellular. In an effort to define its biological role, we structurally and functionally characterized IL-1ra3. Endogenous immunoreactive IL-1ra3 was detected in a variety of inflammatory cells and tissues. We used a gene transfer strategy to explore the possible intracellular functions of IL-1ra3 (and IL-1ra2) and the cell-associated agonist IL-1alpha. The intracellular IL-1ra3 isoform, as well as IL-1ra2, does not block the action of exogenous and endogenous IL-1 under these conditions. Intact IL-1ra3 was released from the cells killed by NK effectors. The intracellular isoforms may represent a reservoir of IL-1ra, released upon cell death, whose function is to limit the pro-inflammatory action of cell debris.

Bacterial lipopolysaccharide causes rapid shedding, followed by inhibition of mRNA expression, of the IL-1 type II receptor, with concomitant up-regulation of the type I receptor and induction of incompletely spliced transcripts.

The IL-1 type I receptor (IL-1RI) is part of a signaling complex together with the IL-1R accessory protein, whereas available information is consistent with a "decoy" model of function for the IL-1 type II receptor (IL-1RII). The present study was designed to investigate the effect of bacterial LPS on IL-1R in human monocytes. LPS causes rapid release of the IL-1RII, an effect blocked by a metalloprotease inhibitor. Subsequently, LPS-treated monocytes showed a drastic reduction of IL-1RII mRNA. In contrast, LPS induced IL-1RI and, to a lesser extent, IL-1AcP expression. LPS-induced augmented expression of the canonical 5-kb IL-1RI mRNA was accompanied by the appearance of 2.4-kb IL-1RI transcripts. The use of probes representative of different regions of the IL-1RI mRNA, as well as cDNA cloning, revealed that the 2.4-kb inducible band includes incompletely spliced, polyadenylated transcripts potentially encoding truncated versions of the receptor. The observation that the prototypic proinflammatory molecule LPS has divergent effects on IL-1Rs, with inhibition of IL-1RII and stimulation of IL-1RI and IL-1R accessory protein, is consistent with the view that these molecules subserve opposite functions in the pathophysiology of the IL-1 system. The rapid shedding of IL-1RII by monocytes early in recruitment may serve to buffer the systemic action of IL-1 leaking from sites of inflammation. This early event, followed by prolonged inhibition of IL-1RII expression and up-regulation of IL-1RI, may render monocytes more responsive to IL-1 at sites of inflammation.

Up-regulation of CCR1 and CCR3 and induction of chemotaxis to CC chemokines by IFN-gamma in human neutrophils.

Human neutrophils (polymorphonuclear leukocytes; PMN) respond to some CXC chemokines but do not migrate to CC chemokines. Recent work has shown that chemokine receptors can be modulated by inflammatory cytokines. In this study, the effect of IFN-gamma, a prototypic Th1 cytokine, on chemokine receptor expression in PMN was investigated. IFN-gamma caused a rapid (approximately 1 h) and concentration-dependent increase of CCR1 and CCR3 mRNA. The expression of CCR2, CCR5, and CXCR1-4 was not augmented. IFN-gamma-treated PMN, but not control cells, expressed specific binding sites for labeled monocyte-chemotactic protein (MCP)-3 and migrated to macrophage-inflammatory protein (MIP)-1alpha, RANTES, MCP-3, MIP-5/HCC2, and eotaxin. 7B11, a mAb for CCR3, inhibited the chemotactic response of IFN-gamma-treated PMN to eotaxin, and aminoxypentane-RANTES blocked PMN migration to RANTES. These results suggest that the selectivity of certain chemokines for their target cells may be altered by cytokines produced within an inflammatory context. Since PMN may play a role in orienting immunity toward Th1 responses, it is possible to speculate that IFN-gamma not only promotes Th1 differentiation directly, but also reorients the functional significance of Th2 effector cytokines by broadening the spectrum of their action to include PMN.

Divergent effects of LPS on expression of IL-1 receptor family members in mononuclear phagocytes in vitro and in vivo.

Three molecules, interleukin 1 (IL-1) receptor I (IL-1RI), IL-1 receptor II (IL-1RII or decoy) and IL-1 receptor accessory protein (IL-1R AcP or IL-1RIII), are involved in IL-1 binding and signal transduction. In addition, three homologous genes (T1/ST2, MyD88 and rsc786) have been identified. Expression of the signal transducing type I R and of the decoy type II R in human monocytes is regulated by pro- and anti-inflammatory signals. The present study was designed to evaluate comprehensively how a prototypic pro-inflammatory signal, bacterial lipopolysaccharide (LPS), affects expression of IL-1R family members in mononuclear phagocytes in vitro and in vivo. Resting human monocytes expressed high levels of IL-1RII, IL-1R AcP, MyD88 and rsc786, whereas low levels of IL-1RI and T1/ST2 were present. In vitro exposure to LPS augmented expression of IL-1RI, T1/ST2 and MyD88, whereas it inhibited that of IL-1RII and rsc786. Expression of IL-1R AcP in monocytes was less substantially affected by LPS. The expression of IL-1R family members was also studied in organs of mice given LPS. As expected on the basis of in vitro results, organs (e.g. spleen, lungs and peritoneal exudate cells) from LPS-treated mice showed increased levels of IL-1RI, T1/ST2 and MyD88. Intriguingly, while expression of IL-1RII was inhibited in peritoneal macrophages after LPS, in accordance with in vitro results, increased IL-1RII mRNA was observed in organs such as liver, lungs and spleen. This unexpected effect of LPS was drastically reduced in mice rendered neutropenic by 5-fluorouracil. Therefore, we conclude that the apparent induction of IL-1RII in certain organs of LPS-treated mice is due to recruitment of myeloid cells which express high levels of decoy RII. Therefore, members of IL-1R family are independently and divergently regulated in mononuclear phagocytes exposed to the prototypic pro-inflammatory signal LPS in vitro and in vivo.

Interferon-gamma inhibits expression of the long pentraxin PTX3 in human monocytes.

PTX3 is a prototypic long pentraxin expressed by various cell types, most prominently monocytes and endothelial cells, in response to interleukin-1 (IL-1), tumor necrosis factor (TNF) and bacterial products. In the present report, we show that interferon-gamma (IFN-gamma) inhibits the expression of the PTX3 gene induced by exposure to IL-1, TNF or lipopolysaccharide in human monocytes. This effect is dose dependent and observable when IFN-gamma is added from 24 h before up to 3 h after the addition of IL-1. While the time course of the IL-1-induced PTX3 mRNA expression is not affected, IFN-gamma reduces the stability of the PTX3 mRNA as well as its transcription. The inhibition of PTX3 expression is restricted to monocytes in that no inhibition occurs in cytokine-stimulated fibroblasts and endothelial cells. Under the same conditions, as expected, IFN-gamma augmented monocyte chemotactic protein-1 expression in the same cell preparations. PTX3 protein secretion by activated monocytes is also suppressed by exposure to IFN-gamma. Altogether, these data identify a negative pathway of regulation mediated by IFN-gamma, which may occur under inflammatory conditions.

Selective inhibition of expression of the chemokine receptor CCR2 in human monocytes by IFN-gamma.

IFN-gamma is a potent activator of mononuclear phagocyte function and promotes the development of Th1 responses. Moreover, it induces and modulates chemokine production in a variety of cell types, including mononuclear phagocytes. In the present study, we examined the effect of IFN-gamma on the expression of CC chemokine receptors in human monocytes. IFN-gamma selectively and rapidly inhibited expression of the monocyte chemotactic protein (MCP) receptor CCR2 with an ED50 of approximately 50 U/ml. The effect was rapid (detectable after 1 h) and reversible. Other chemokine receptors (CCR1, CCR3, CCR4, and CCR5) were not substantially affected, and CXCR4 was reduced. IFN-gamma acted in concert with LPS, TNF-alpha, and IL-1beta in inhibiting CCR2 expression. IFN-gamma-treated monocytes showed a shorter half-life of CCR2 mRNA compared with untreated cells, whereas the rate of nuclear transcription was unaffected. The inhibition of CCR2 mRNA expression by IFN-gamma was associated with a lower number of surface receptors and lower chemotactic responsiveness. Thus, IFN-gamma, an inducer of MCP-1 and MCP-3 in mononuclear phagocytes, selectively inhibits expression of the MCP receptor CCR2 in monocytes. These results are consistent with an emerging paradigm of divergent regulation by several agents of chemokine production and receptor expression in monocytes. The inhibition of MCP-1R expression may serve as a means of retaining mononuclear phagocytes at sites of inflammation and as a feedback mechanism in the regulation of recruitment from the blood.