The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2

Cytokines interact with hematopoietin superfamily receptors and stimulate receptor dimerization. We demonstrate that chemoattractant cytokines (chemokines) also trigger biological responses through receptor dimerization. Functional responses are induced after pairwise crosslinking of chemokine receptors by bivalent agonistic antichemokine receptor mAb, but not by their Fab fragments. Monocyte chemoattractant protein (MCP)-1-triggered receptor dimerization was studied in human embryonic kidney (HEK)-293 cells cotransfected with genes coding for the CCR2b receptor tagged with YSK or Myc sequences. After MCP-1 stimulation, immunoprecipitation with Myc-specific antibodies revealed YSK-tagged receptors in immunoblotting. Receptor dimerization also was validated by chemical crosslinking in both HEK-293 cells and the human monocytic cell line Mono Mac 1. Finally, we constructed a loss-of-function CCR2bY139F mutant that acted as a dominant negative, blocking signaling through the CCR2 wild-type receptor. This study provides functional support for a model in which the MCP-1 receptor is activated by ligand-induced homodimerization, allowing discussion of the similarities between bacterial and leukocyte chemotaxis.

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (Eδ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by Eδ and the TCR α enhancer, and that it prevents Eδ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.

Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.

Upregulation of urotensin II receptor in preeclampsia causes in vitro placental release of soluble vascular endothelial growth factor receptor 1 in hypoxia

Preeclampsia is a hypertensive disorder of pregnancy caused by abnormal placental function, partly because of chronic hypoxia at the utero-placental junction. The increase in levels of soluble vascular endothelial growth factor receptor 1, an antiangiogenic agent known to inhibit placental vascularization, is an important cellular factor implicated in the onset of preeclampsia. We investigated the ligand urotensin II (U-II), a potent endogenous vasoconstrictor and proangiogenic agent, for which levels have been reported to increase in patients with preeclampsia. We hypothesized that an increased sensitivity to U-II in preeclampsia might be achieved by upregulation of placental U-II receptors. We further investigated the role of U-II receptor stimulation on soluble vascular endothelial growth factor receptor 1 release in placental explants from diseased and normal patients. Immunohistochemistry, real-time PCR, and Western blotting analysis revealed that U-II receptor expression was significantly upregulated in preeclampsia placentas compared with controls (P<0.01). Cellular models of syncytiotrophoblast and vascular endothelial cells subjected to hypoxic conditions revealed an increase in U-II receptor levels in the syncytiotrophoblast model. This induction is regulated by the transcriptional activator hypoxia-inducible factor 1a. U-II treatment is associated with increased secretion of soluble vascular endothelial growth factor receptor 1 only in preeclamptic placental explants under hypoxia but not in control conditions. Interestingly, normal placental explants did not respond to U-II stimulation.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1 beta maturation

Background: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1 beta and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results: Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE(2) and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1 beta and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)alpha and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1 beta and PGE(2) did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-alpha and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1 beta was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE(2) production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1 beta maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE(2) production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphoryl

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (h beta c). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of h beta c. We report here a comprehensive screen of the entire h beta c molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of h beta c that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most h beta c mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together; these results highlight key regions involved in h beta c activation, dissociate h beta c tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by h beta c. (C) 1998 by The American Society of Hematology.

A new small molecule C5a receptor antagonist inhibits the reverse-passive arthus reaction and endotoxic shock in rats

C5a is implicated as a pathogenic factor in a wide range of immunoinflammatory diseases, including sepsis and immune complex disease, Agents that antagonize the effects of C5a could be useful in these diseases. We have developed some novel C5a antagonists and have determined the acute anti-inflammatory properties of a new small molecule C5a receptor antagonist against C5a- and LPS-induced neutrophil adhesion and cytokine expression, as well as against some hallmarks of the reverse Arthus reaction in rats. We found that a single i.v. dose (1 mg/kg) of this antagonist inhibited both C5a- and LPS-induced neutropenia and elevated levels of circulating TNF-alpha, as well as polymorphonuclear leukocyte migration, increased TNF-alpha levels and vascular leakage at the site of immune complex deposition. These results indicate potent anti-inflammatory activities of a new C5a receptor antagonist and provide more evidence for a key early role for C5a in sepsis and the reverse Arthus reaction. The results support a role for antagonists of C5a receptors in the therapeutic intervention of immunoinflammatory disease states such as sepsis and immune complex disease.

Protection against Paracoccidioides brasiliensis infection conferred by the prophylactic administration of native and recombinant ArtinM

We determined the prophylactic effect of both the d-mannose-binding lectin ArtinM extracted from the seeds of Artocarpus integrifolia (jackfruit) and its recombinant counterpart during the course of experimental paracoccidioidomycosis induced in BALB/c mice. Four experimental protocols of prophylaxis were employed to evaluate the most protective regimen of ArtinM administration. It was demonstrated that the best effect was obtained by administration of two ArtinM doses on days 10 and 3 before the challenge with Paracoccidioides brasiliensis. By following this protocol, the lungs of mice that received native or recombinant ArtinM exhibited reduced fungal burden and granuloma incidence. In addition, the protocol augmented contents of IL-12, IFN-gamma, TNF-alpha and NO. On the other hand, the control group consisting of untreated infected mice had higher pulmonary levels of IL-4 and IL-10. In conclusion, prophylaxis with ArtinM significantly reproduces the effect of its therapeutic administration, i.e, it confers resistance to P. brasiliensis infection in mouse models by promoting IL-12 production and favours Th1-immunity.

Inhibitors of TACE and Caspase-1 as anti-inflammatory drugs

TNF-alpha neutralising agents such as Infliximab (Remicade(R)), Etanercept (Enbrel(R)) and the IL-1 receptor antagonist Anakinra (Kineret(R)), are currently used clinically for the treatment of many inflammatory diseases such as Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriatic arthritis and psoriasis. These protein preparations are expensive to manufacture and administer, need to be injected and can cause allergic reactions. An alternative approach to lowering the levels of TNF-alpha and IL-1 beta in inflammatory disease, is to inhibit the enzymes that generate these cytokines using cheaper small molecules. This paper is a broad overview of the progress that has been achieved so far, with respect to small molecule inhibitor design and pharmacological studies (in animals and humans), for the metalloprotease Tumour Necrosis Factor-alpha Converting Enzyme (TACE) and the cysteine protease Caspase-1 (Interieukin-1 beta Converting Enzyme, ICE). Inhibitors of these two enzymes are currently considered to be good therapeutic targets that have the potential to provide relatively inexpensive and orally bioavailable anti-inflammatory agents in the future.

IL-1 beta breaks tolerance through expansion of CD25(+) effector T cells

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn's disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1 beta drives proliferation and cytokine production by CD4(+)CD25(+)FoxP3(-) effector/memory T cells, attenuates CD4(+)CD25(+)FoxP3(+) regulatory T cell function, and allows escape of CD4(+)CD25(-) autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1 beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity

A conformationally biased decapeptide agonist of human C5a (C5a(65-74)Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a(12-20)-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

Purpose We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the antihypertensive drug losartan (LOS). Results We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumorassociated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. Conclusions Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

The generation of H-1-NMR-detectable mobile lipid in stimulated lymphocytes: Relationship ts cellular activation, the cell cycle, and phosphatidylcholine-specific phospholipase C

Mobile Lipids detected using H-1-NMR in stimulated lymphocytes were correlated with cell cycle phase, expression of the interleukin-2 receptor alpha and proliferation to assess the activation status of the lymphocytes. Mobile lipid levels, IL-2R alpha expression and proliferation increased after treatment with PMA and ionomycin. PMA or ionomycin stimulation alone induced increased IL-2R alpha expressiom but not proliferation, PMA- but not ionomycin-stimulation generated mobile lipid, Treatment with anti-CD3 antibody did not increase IL-2R alpha expression or proliferation but did generate increased amounts of mobile lipid, The cell cycle status of thymocytes treated with anti-CD3, PMA or ionomycin alone indicated an. accumulation of the cells in the G(1) phase of the cell cycle, The generation of mobile lipid was abrogated in anti-CD3 antibody-stimulated thymic lymphocytes but not in splenic lymphocytes, using a phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor which blocked cells in the G(1)/S phase of the cell cycle, This suggests that the H-1-NMR-detectable mobile Lipid may be generated in anti-CD3 antibody-stimulated thymic lymphocytes by the action of PC-PLC activity via the catabolism of PC, in the absence of classical signs of activation. (C) 1997 Academic Press.