The gene encoded for tryptophan decarboxylase (TDC), which is the key enzyme in terpenoil indole alkaloids pathway, was targeted to different subcellular compartments and stably expressed in transgenic tobacco (Nicotiana tabacum L.) plants at the levels detected by Western blot and tryptamine accumulation analysis. It was shown that the TDC was located in subcellular compartments, the chloroplasts and cytosol. The recombinant TDC targeted to chloroplasts and cytosol in tobacco plants was effectively expressed as soluble protein by Western blot analysis and enzymatic assay. The level of tryptamine accumulation in chloroplast was higher than that in cytosol and very low in vacuole and endoplasmic reticulum (ER) to be hardly detected by Western blot analysis. It was indicated that the highest amount of tryptamine was in chloroplasts, lower in endoplasmic reticula and the lowest in vacuoles as compared to those in wild type plants. The TDC targeted to different subcellular compartments of tobacco plants and its expression level were studied by different nucleotide sequences coding signal peptides at 5'-end of tdc gene in order to know the effects of the TDC in compartmentation on its functionality.
Poster: ECR 2018 / C-1169 / Gadoxetic acid magnetic resonance of the liver in cirrhotic patients with suspicious HCC after MDCT: preliminary results of a prospective monocentric study by: S. Fiore , E. Caramia, M. Gatti, L. J. Pavan, S. Molinaro, A. Depaoli, R. Faletti, P. Fonio; Turin/IT
Lipoxin A(4) (LXA(4)) is a potent negative modulator of the inflammatory response. The antiinflammatory activities of LXA(4), such as inhibition of agonist-induced polymorphonuclear cell (PMN) chemotaxis and upregulation of beta-2 integrins, require the expression of a G-protein-coupled, high-affinity LXA(4) receptor (LXA(4)R). We now report that stimulation of PMN with proinflammatory agonist N-formyl peptides (FMLP), calcium ionophore A(23187), or phorbol mirystate acetate (PMA) is followed by marked downregulation of LXA(4) binding (B(max) decrease of approximately 45%) and decreased activation of phospholipases A(2) (PLA(2)) and D (PLD). Elucidation of the mechanisms underlying these effects was addressed by structure-function analyses of the intracellular domains of LXA(4)R. Mutant molecule, S236/S237 --> A/G (LXA(4)R(pk)) and Y302 --> F (LXA(4)R(tk)) were obtained by site-directed mutagenesis to yield receptors lacking the putative targets for serine/threonine kinase- or tyrosine kinase-dependent phosphorylation. Expression of wild-type and mutated LXA(4)R sequences in CHO and HL-60 cells was used to examine LXA(4) ligand-receptor interactions and signal transduction events. Results indicated that cells expressing LXA(4)R(pk) or LXA(4)R(tk) displayed sustained activation of PLA(2) and PLD in contrast to the transient ones obtained with LXA(4)R(wt) (peak activation at 2-3 min). Moreover, inhibition of LXA(4)-dependent PLA(2) activity by PMA in LXA(4)R(wt) transfected CHO cells was not observed in cells expressing LXA(4)R(pk). Phosphopeptide immunoblotting revealed that the functional differences between wild-type and mutant LXA(4) receptors are accompanied by distinct changes in the receptor protein phosphorylation pattern. Further characterization of these and related LXA(4)R intracellular domains will help to better understand specific events that regulate the antiinflammatory activities of LXA(4).
The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of "self" versus "other" considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity. The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of "self" versus "other" considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity. The process of physiological cell death assures the elimination of functionally inappropriate cells in a manner that does not elicit inflammation (1.Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-256Crossref PubMed Scopus (12819) Google Scholar, 2.Savill J. Dransfield I. Gregory C. Haslett C. Nat. Rev. Immunol. 2002; 2: 965-975Crossref PubMed Scopus (1325) Google Scholar). Professional phagocytes, including resident macrophages and dendritic cells, participate in the efficient clearance of apoptotic corpses in vivo (3.Savill J.S. Wyllie A.H. 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Studies of neutrophil death and resulting phagocytosis by macrophages provided the first experimental evidence that a variety of cytokines and chemokines associated with inflammation, including interleukin (IL) 4The abbreviations used are: IL, interleukin; EGF, epidermal growth factor; PI, propidium iodide; PS, phosphatidylserine; LPS, lipopolysaccharide; RLU, relative light units; CFDA, 5,6-carboxyfluorescein diacetate succinimidyl ester; PBS, phosphate-buffered saline; CMTMR, 5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethyl rhodamine; TNFα, tumor necrosis factor α; PMA, phorbol 12-myristate 13-acetate; NF-κB, nuclear factor κB; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; TGRβ, transforming growth factor β; TLR, toll-like receptor.4The abbreviations used are: IL, interleukin; EGF, epidermal growth factor; PI, propidium iodide; PS, phosphatidylserine; LPS, lipopolysaccharide; RLU, relative light units; CFDA, 5,6-carboxyfluorescein diacetate succinimidyl ester; PBS, phosphate-buffered saline; CMTMR, 5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethyl rhodamine; TNFα, tumor necrosis factor α; PMA, phorbol 12-myristate 13-acetate; NF-κB, nuclear factor κB; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; TGRβ, transforming growth factor β; TLR, toll-like receptor.-6 and IL-8, are not secreted from phagocytes that engulf apoptotic targets (3.Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Scopus (1347) Google Scholar, 7.Meagher L.C. Savill J.S. Baker A. Fuller R.W. Haslett C. J. Leukocyte Biol. 1992; 52: 269-272Crossref PubMed Scopus (247) Google Scholar, 8.Hughes J. Liu Y. Damme J.V. Savill J. J. Immunol. 1997; 158: 4389-4397PubMed Google Scholar, 9.Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2552) Google Scholar, 10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). More significantly, the lack of inflammatory cytokine release reflects an affirmative inhibitory response. For example, whereas stimulation of macrophages via the Toll-like receptor (TLR) 4 signaling complex (11.Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar) upon engagement with bacterial lipopolysaccharide (LPS) triggers significant cytokine secretion, the additional ingestion of apoptotic cells attenuates this response potently (9.Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2552) Google Scholar, 10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 12.Voll R.E. Herrmann M. Roth E.A. Stach C. Kalden J.R. Girkontaite I. Nature. 1997; 390: 350-351Crossref PubMed Scopus (1505) Google Scholar). The ability of apoptotic cells to be cleared in a noninflammatory manner by professional phagocytes, such as macrophages, is a consequence of their specific expression of determinants for recognition and modulation of proinflammatory responses. The acquisition of these apoptotic determinants represents a gain of function and is common to all physiological cell deaths, without regard to suicidal stimulus (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Cells that die necrotically also are recognized by professional phagocytes; in contrast, however, necrotic corpses do not down-regulate inflammatory responses. Discrimination between apoptotic and necrotic corpses occurs on the level of binding and not engulfment and involves distinct and noncompeting mechanisms of recognition (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). The modulatory activity of the apoptotic corpse is manifest as an immediate-early inhibition of macrophage proinflammatory cytokine gene transcription and is exerted directly upon binding to the macrophage, independent of subsequent engulfment and soluble factor involvement (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Apoptotic cells target the proinflammatory transcriptional machinery of macrophages, with which they interact through a novel regulatory pathway. Inhibition appears to involve sequestration of critical transcriptional co-activator molecules without effect on proximal signaling events induced by inflammatory receptors, including innate immune receptors of the TLR family (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The ubiquity of apoptotic cells in all tissues throughout organismal life prompted us to ask whether this innate discrimination of apoptotic cells is limited to professional phagocytes. Certainly, normal homeostatic cell turnover in vivo, and especially in solid tissues and intact cellular strata, results in apoptotic cells that are in immediate contact with their neighbors independent of (and before the arrival of) mobile phagocytes (6.Wood W. Turmaine M. Weber R. Camp V. Maki R.A. McKercher S.R. Martin P. Development. 2000; 127: 5245-5252Crossref PubMed Google Scholar, 14.Saunders Jr., J.W. Science. 1966; 154: 604-612Crossref PubMed Scopus (720) Google Scholar, 15.Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-305Crossref PubMed Scopus (6720) Google Scholar, 16.Parnaik R. Raff M.C. Scholes J. Curr. Biol. 2000; 10: 857-860Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17.Monks J. Rosner D. Geske F.J. Lehman L. Hanson L. Neville M.C. Fadok V.A. Cell Death Differ. 2005; 12: 107-114Crossref PubMed Scopus (178) Google Scholar). We asked whether nonprofessional phagocytes discriminate and respond specifically to apoptotic cells in an anti-inflammatory manner. Here we describe studies that reveal that they do. Remarkably, this profound innate immune function is manifest fully and ubiquitously among professional and nonprofessional phagocytes and even nonphagocytic cell types. Cells and Death Induction—RAW 264.7 murine macrophages, DO11.10 murine T hybridoma cells, Jurkat human acute T leukemia cells, Ramos (RA-1) human Burkitt's B lymphoma cells, and PLB-985 human myelomonoblastic leukemia cells (generously provided by Dr. Peter Henson, National Jewish Medical and Research Center) were cultured at 37 °C in a humidified, 5% (v/v) CO2 atmosphere in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with heat-inactivated fetal bovine serum (10% v/v), 2 mm l-glutamine, and 50 μm 2-mercaptoethanol. HeLa human cervical carcinoma cells and 293T human transformed kidney epithelial cells were grown in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose (Mediatech) supplemented with fetal bovine serum (10% (v/v); HyClone Laboratories, Logan, UT) and 2 mm l-glutamine. Chinese hamster ovary cells were grown in α-minimal essential medium (Invitrogen) supplemented only with fetal bovine serum (10%, v/v). Human umbilical vein endothelial cells were grown in supplemented endothelial growth medium (Cambrex Bio Science, East Rutherford, NJ) on gelatin-coated plates. Immortalized murine 3T3 fibroblast cell lines were derived from mouse embryo fibroblasts following the 3T3 protocol of Todaro and Green (18.Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (2003) Google Scholar). Briefly, the embryo fibroblasts were cultured at 37 °C in a humidified, 5% (v/v) CO2 atmosphere in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose (Mediatech) supplemented with fetal bovine serum (10%, v/v; HyClone Laboratories), 2 mml-glutamine, and 50 μm 2-mercaptoethanol, replating at 3 × 105/60-mm diameter dish every 3 days. Immortalized cell lines were established from cells that grew from cultures that had become senescent. Physiological cell death (apoptosis) was induced by treatment of target cells with the macromolecular synthesis inhibitors actinomycin D (200 ng/ml, 12 h) or cycloheximide (1 μg/ml, 12 h) (19.Chang S.H. Cvetanovic M. Harvey K.J. Komoriya A. Packard B.Z. Ucker D.S. Exp. Cell Res. 2002; 277: 15-30Crossref PubMed Scopus (18) Google Scholar). Cells were killed pathologically (necrotic death) by incubation in phosphate-buffered saline (PBS) at 55 °C for 20 min (until trypan blue uptake indicated compromise of membrane integrity) (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). In all cases, target cells (viable, apoptotic, and necrotic) were washed twice and resuspended in the medium of the responder cells to be tested. In some experiments, apoptotic and viable target cells were fixed by incubation with formaldehyde (125 mm in PBS, 25 °C, 20 min; Polysciences, Inc., Warrington, PA) or were cycled through three rounds of freezing and thawing. Target preparations then were washed twice and resuspended in the medium of the responder cells to be tested. Plasma membrane vesicles were prepared from HeLa cells following the approach of Baumann et al. (20.Baumann N.A. Vidugiriene J. Machamer C.E. Menon A.K. J. Biol. Chem. 2000; 275: 7378-7389Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Monolayers of cells, either untreated or induced to die with actinomycin D (and still adherent), were stimulated to vesiculate by incubation at 37 °C in Vesiculation Buffer (10 mm Hepes (pH 7.4), 150 mm NaCl, 2 mm CaCl2, 2 mm dithiothreitol, and 25 mm formaldehyde). Supernatants were collected after ∼2.5 h (when abundant small membrane vesicles were apparent in the culture fluid). Nonadherent cells were removed (1,000 × g for 10 min), and vesicles were pelleted from the cleared supernatant by centrifugation (30,000 × g, 60 min, 4 °C). Cytofluorimetric analysis indicated that vesicles were ∼0.8 μm in diameter and free of contaminating intact cells. Phagocytosis Assay and Other Cytofluorimetric Analyses—Phagocytosis was assessed as previously described for macrophages (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Target cells were labeled green with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA; 0.2 μm; Molecular Probes, Inc., Eugene, OR) and were then induced to undergo apoptotic cell death, killed pathologically by heat treatment, or left untreated. Phagocytes (or cells being tested for phagocytic activity) were labeled red with 5-(and 6)-(((4-chloromethyl)benzoyl)-amino) tetramethyl rhodamine (CMTMR; 10 μm; Molecular Probes). In all cases, cells were labeled on the day preceding the experiment and cultured in serum-containing medium overnight to eliminate unbound label. Labeled phagocytes were co-cultured with the apoptotic, necrotic, or viable target cells for 30 min at 37 °C. Cells were harvested with PBS supplemented with 0.4 mm Na2EDTA and analyzed cytofluorimetrically on a FACSCaliber instrument (BD Biosciences). Cytofluorimetric data were processed with WinMDI software (Joe Trotter, Scripps Research Institute, La Jolla, CA). Cells that were both CMTMR-positive (Exλ = 488 nm, Emλ = 610 ± 15 nm) and CFDA-positive (Exλ = 488 nm; Emλ = 530 ± 15 nm) and that had scatter properties of the phagocyte population represented phagocytes that had engulfed targets. Engulfment is calculated as the fraction of double-positive phagocytes (all CMTMR-positive cells that also are CFDA-positive). Most targets that are bound but not engulfed are disrupted and do not remain adherent during the analysis, although they could be enumerated under static microscopic examination (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). The accessibility of phosphatidylserine was revealed by the binding of fluorescein isothiocyanate-conjugated annexin V (Pharmingen; San Diego, CA; Exλ = 488 nm, Emλ = 525 nm). Cells were harvested and washed twice with cold PBS. Cells were resuspended in 100 μl of binding buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2) and incubated with 5 μl of fluorescein isothiocyanate-conjugated annexin V for 15 min in the dark at 25 °C. After incubation, 400 μl of binding buffer was added per sample, and cells were analyzed cytofluorimetrically. Propidium iodide (PI) was employed to assess plasma membrane integrity. PI was added to cells at 1 μg/ml immediately before cytofluorimetric analysis (Exλ = 488 nm, Emλ = 610 nm). Transfections and Luciferase Assays—Apoptotic modulation of specific transcription (e.g. dependent on nuclear factor κB (NF-κB) or the IL-8 promoter) was assessed in various cell types following transfection of relevant transcriptional reporter constructs, using a dual luciferase strategy, as described previously (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The efficiencies of transfection were measured in parallel with farnesylated green fluorescent protein as a transfection marker (21.Harvey K.J. Lukovic D. Ucker D.S. Cytometry. 2001; 43: 273-278Crossref PubMed Scopus (27) Google Scholar). Cells were co-transfected with pNF-κB-Luc, a plasmid containing the firefly (Photinus pyralis) luciferase gene, the expression of which is driven by a basal transcriptional promoter linked to four copies of the κB motif (Clontech), together with pRL-SV40, a Renilla (sea pansy; R. reniformis) luciferase control vector, the constant expression of which is dependent on the SV40 early enhancer/promoter region (Promega, Madison, WI). RAW 264.7 macrophages (5.0 × 106 cells/60-mm diameter dish) and HeLa, 293T, and Chinese hamster ovary cells, at ∼75% confluence, were transfected using Effectene Transfection Reagent (Qiagen, Valencia, CA). 3T3 cells were transfected using the MEF1 Nucleofector Kit (AMAXA Biosystems; Gaithersburg, MD), with MEF Nucleofector Solution 1 and a machine setting of A-23. Jurkat, Ramos RA-1, and human umbilical vein endothelial cells were transfected using GenePORTER2 Transfection Reagent (Gene Therapy Systems, San Diego, CA). The next day, the cells were replated in 24-well plates (1.0 × 105 cells/well) and incubated without or with the indicated target cells (at a target cell/macrophage ratio of 10:1) and/or a proinflammatory stimulus in a final volume of 2 ml. The proinflammatory stimuli used included LPS (100 ng/ml; Escherichia coli O111:B4; Sigma), tumor necrosis factor-α (TNFα; 10 ng/ml; R&D Systems; Minneapolis, MN), IL-1β (5 ng/ml; R&D Systems), and phorbol 12-myristate 13-acetate (PMA; 1.3 ng/ml; EMD Biosciences, San Diego, CA) alone or with ionomycin (200 ng/ml; Molecular Probes). Cell extracts were prepared after further incubation as indicated, and luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega) in an FB12 luminometer (Zylux; Oak Ridge, TN). Each condition was repeated in triplicate wells, and the luciferase activities in cells from each well were determined independently. Within any experiment, Renilla luciferase activities among samples varied less than 6%. The firefly luciferase activity in each sample was normalized with respect to the internal Renilla luciferase activity, and the relative level of normalized firefly luciferase activity compared with the activity in an untreated population was taken as a measure of specific (e.g. NF-κB-dependent) transcriptional activity. Stably transfected reporter cells were generated by transfection of 293T cells with another NF-κB-Luc reporter construct, 4×NF-κB(HIV)tkluc (22.Wissink S. van de Stolpe A. Caldenhoven E. Koenderman L. van der Saag P.T. Immunobiology. 1997; 198: 50-64Crossref PubMed Scopus (40) Google Scholar), and an unlinked vector conferring hygromycin resistance. Cells resistant to hygromycin (50 μg/ml) were selected, cloned at limiting dilution, and tested for NF-κB-dependent responsiveness. Extracts were prepared and analyzed as above, except that the luciferase assay system (Promega) was used. Data with one clone, B2, are described here. Quantification of Cytokine Release—Cytokine production was assessed following incubation of responder cells with target cells. Where indicated, proinflammatory stimuli were added simultaneously with the addition of targets. Culture supernatants were withdrawn from wells at the indicated times and frozen at –20 °C until analysis. Secreted cytokines were quantified by ELISA, using matched pair cytokine-specific capture and biotinylated reporter antibodies for murine IL-6 (eBiosciences; San Diego, CA) or human IL-8 (BIOSOURCE, Camarillo, CA). The reporter reactions were developed with horseradish peroxidase-conjugated streptavidin (R&D Systems) and measured spectrophotometrically at 450 nm (corrected for turbidity at 550 nm; Microplate Autoreader model EL311; Bio-Tek Instruments, Winooski, VT). Cellular Extract Preparation and Immunoblot Analysis—Activation of Akt and inhibition of ERK1/2 were assessed in 3T3 cells cultured overnight in serum-free medium and left unstimulated or stimulated for 15 min with a 5-fold excess of apoptotic DO11.10 cells (the apoptotic cells, which had been cultured under serum-free conditions, were centrifuged briefly onto the adherent 3T3 cells to initiate the interaction) and/or subsequent stimulation with epidermal growth factor (EGF; 10 nm; Calbiochem). After washing, cell extracts were prepared from the adherent 3T3 cells. Cells were lysed in lysis buffer (150 mm NaCl, 50 mm HEPES (pH 7.5), 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 200 μm orthovanadate). Lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatants were stored at –70 °C. Protein samples (20 μg each, determined by the bicinchoninic acid protein assay; Pierce) were boiled in 5× sample buffer, run on 12% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA). Blots were blocked with 5% dry milk in 150 mm PBS, 20 mm Tris HCl, pH 7.5, before probing with a phospho-Akt(Thr308)-specific rabbit anti-serum (Cell Signaling, Beverly, MA) or an affinity-purified phospho-ERK1/2 (Thr183/Tyr185)-specific polyclonal rabbit IgG (Promega, Madison, WI). Following incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase, immunoreactive bands were visualized by the luminol reaction (ECLplus; Amersham Biosciences). Equivalent loading of protein samples was monitored by Ponceau S staining (0.25% (w/v) (Sigma) in 0.1% acetic acid; 5 min) of blotted proteins. Specific Recognition and Response to Apoptotic Cells Is Not Limited to Macrophages—The anti-inflammatory response triggered in macrophages by their specific recognition of apoptotic cells is exerted on the level of cytokine gene transcription (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Although key transcriptional activators of cytokine gene expression, such as NF-κB (23.Shakhov A.N. Collart M.A. Vassalli P. Nedospasov S.A. Jongeneel C.V. J. Exp. Med. 1990; 171: 35-47Crossref PubMed Scopus (733) Google Scholar, 24.Collart M.A. Baeuerle P. Vassalli P. Mol. Cell. Biol. 1990; 10: 1498-1506Crossref PubMed Google Scholar), are not the molecular targets of apoptotic modulation, modulation is evident on the level of NF-κB-dependent transcription, and an NF-κB-dependent transcriptional reporter serves as a sensitive, reliable, and convenient readout for the modulatory effect exerted by apoptotic targets (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The experiment in Fig. 1A exemplifies this analysis. We transiently transfected RAW 264.7 macrophages with pNF-κB-Luc, a plasmid containing the firefly luciferase gene, the expression of which is driven by a basal transcriptional promoter linked to four copies of the κB motif. Macrophages were co-transfected with a constitutive (NF-κB-independent) Renilla luciferase control vector, which served as an internal normalization control for transfection efficiency and cell viability. Following transfection, macrophages were incubated with different target cell populations and/or bacterial LPS, a potent proinflammatory agonist. Firefly and Renilla luciferase activities then were measured. As indicated by these luciferase reporters, LPS-activated NF-κB-dependent transcription (but not global transcription) in macrophages is inhibited specifically following their interaction with apoptotic cells; necrotic and viable cells do not exert this effect (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). This response is elicited by apoptotic cells generally, regardless of species, cell type, or suicidal stimulus. Here, murine DO11.10 T cells and human HeLa epithelial carcinoma cells, triggered to die with different suicidal stimuli (inhibitors of translation and transcription, respectively), were equally effective at triggering modulation in these murine macrophages. As a first test of the ability of nonprofessional phagocytes to recognize and respond to apoptotic cells, we examined the responsiveness of HeLa cells, the same cells used as targets in Fig. 1A, utilizing the identical transcriptional reporter strategy. The transfected HeLa cells were incubated with apoptotic, necrotic, or viable populations of target cells and/or the inflammatory cytokine TNFα as a stimulus of an NF-κB-dependent transcriptional response. Significantly, HeLa cells do not die in response to TNFα unless the NF-κB-dependent response is attenuated (e.g. by an inhibitor of macromolecular synthesis) (25.Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2933) Google Scholar, 26.Wang C.-Y. Mayo M.W. Baldwin A.S.J. Science. 1996; 274: 784-787Crossref PubMed Scopus (2509) Google Scholar, 27.Harvey K.J. Lukovic D. Ucker D.S. J. Cell Biol. 2000; 148: 59-72Crossref PubMed Scopus (83) Google Scholar). Just as with LPS-stimulated macrophage responsiveness, robust TNFα-activated NF-κB-dependent transcription in HeLa cells was inhibited specifically and profoundly following the interaction of those cells with apoptotic, but not necrotic or viable, targets (Fig. 1B). It is notable that the ranges of absolute values of NF-κB-dependent luciferase activities were quite different in HeLa cells and macrophages at comparably early times (as much as 300-fold; see Fig. 1), reflecting differences in the efficiencies of transfection and transgene expression. Still, expressed relative to basal luciferase levels, these data present a consistent pattern of modulation and reveal an identical response to apoptotic targets in different responder cell populations. Again, this selective response to apoptotic cells occurred without species restriction. Most dramatically, HeLa cells even were able to recognize and respond specifically to homotypic apoptotic cells. The Characteristic Repertoire of Anti-inflammatory Responses Elicited Specifically upon Apoptotic Cell Recognition Is Evident in Murine Fibroblasts—To begin a more comprehensive exploration of the recognition and response to apoptotic targets by nonprofessional phagocytes, we examined nontransformed murine fibroblasts. Immortalized murine embryo fibroblast cells, derived by the 3T3 protocol of Todaro and Green (18.Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (2003) Google Scholar), were highly phagocytic for dead cells (Fig. 2A; for consistency, we used DO11.10 cells as targets in this and the following experiments). Apoptotic and necrotic cell targets were engulfed rapidly and to equal extents by 3T3 fibroblasts, whereas viable cells were not ingested (Fig. 2A; we take the low level of engulfment of "viable" cells to reflect the small fraction of dead and dying apoptotic cells present in any cell culture). These 3T3 fibroblasts secrete the inflammatory cytokine IL-6 in response to a variety of proinflammatory stimuli, including IL-1β, TNFα, and, to a lesser degree, bacterial LPS (Fig. 2B and data not shown) (28.Kurt-Jones E.A. Sandor F. Ortiz Y. Bowen G.N. Counter S.L. Wang T.C. Finberg R.W. J. Endotoxin Res. 2004; 10: 419-424Crossref PubMed Google Scholar). We characterized the release of IL-6 from fibroblasts following their interaction with target cells as one indication of inflammatory responsiveness; IL-6 secretion in macrophages reflects inflammatory responsiveness generally (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). IL-1β-stimulated IL-6 secretion was potently attenuated when fibroblasts interacted with apoptotic targets, but not with necrotic or viable targets (Fig. 2B). Apoptotic target cell contact was necessary for this response, since supernatants from apoptotic cell cultures could not substitute for the target cells themselves to modulate IL-6 secretion (data not shown). The ability of apoptotic cells to block IL-6 secretion by IL-1β-stimulated fibroblasts parallels their ability to abrogate the secretion of IL-6 and other inflammatory cytokines by LPS-stimulated macrophages (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163
In this work, we characterize nor-β-lapachone-loaded (NβL-loaded) microcapsules prepared using an emulsification/solvent extraction technique. Features such as surface morphology, particle size distribution, zeta potential, optical absorption, Raman and Fourier transform infrared spectra, thermal analysis data, drug encapsulation efficiency, drug release kinetics and
To evaluate safety and efficacy of weekly (qw) and every other week (q2w) dosing of sarilumab, a fully human anti-interleukin 6 receptor α (anti-IL-6Rα) monoclonal antibody, for moderate-to-severe rheumatoid arthritis (RA).
Methods
In this dose-ranging study, patients (n=306) with active RA, despite methotrexate, were randomly assigned to placebo or one of five subcutaneous doses/regimens of sarilumab: 100 mg q2w, 150 mg q2w, 100 mg qw, 200 mg q2w, 150 mg qw for 12 weeks, plus methotrexate. The primary end point was ACR20 at Week 12. Secondary endpoints included ACR50, ACR70, Disease Activity Score in 28 joints (C reactive protein). Safety, pharmacokinetics, pharmacodynamics and efficacy in population subgroups were assessed.
Results
The proportion of patients achieving an ACR20 response compared with placebo was significantly higher for sarilumab 150 mg qw (72.0% vs 46.2%, multiplicity adjusted p=0.0203). Higher ACR20 responses were also attained with 150 mg q2w (67%; unadjusted (nominal) p=0.0363) and 200 mg q2w (65%; unadjusted p=0.0426) versus placebo. Sarilumab ≥150 mg q2w reduced C reactive protein, which did not return to baseline between dosing intervals. Infections were the most common adverse event; none were serious. Changes in laboratory values (neutropenia, transaminases and lipids) were consistent with reports with other IL-6Rα inhibitors.
Conclusions
Sarilumab improved signs and symptoms of RA over 12 weeks in patients with moderate-to-severe RA with a safety profile similar to reports with other IL-6 inhibitors. Sarilumab 150 mg and sarilumab 200 mg q2w had the most favourable efficacy, safety and dosing convenience and are being further evaluated in Phase III.
