FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1988018889> ?p ?o ?g. }

• W1988018889 endingPage "10858" @default.
• W1988018889 startingPage "10853" @default.
• W1988018889 abstract "GRO proteins are α-chemokine cytokines that attract neutrophils and stimulate the growth of a variety of cells. Previously, we observed that rabbit alveolar macrophages transcribe the genes for at least two GRO homologues. In order to study the role of GRO cytokines in lung inflammation, we cloned the predominant rabbit GRO cDNA (RabGRO) from alveolar macrophages, expressed bioactive recombinant protein (rRabGRO) in Escherichia coli, and developed a sensitive and specific enzyme-linked immunosorbent assay for RabGRO protein. We found that rabbit AM express and secrete GRO in vitro in response to both exogenous (e.g. lipopolysaccharide, heat-killed Staphylococcus aureus, and crystalline silica) and endogenous inflammatory stimuli (e.g. tumor necrosis factor-α) as determined by both radioimmunoprecipitation and enzyme-linked immunosorbent assay. Biologically significant amounts of GRO are present in vivo in the bronchoalveolar lavage fluid of rabbits with E. coli pneumonia; by in situ hybridization, GRO mRNA is detectable in infiltrating pulmonary leukocytes and bronchial epithelial cells. These results indicate that GRO chemokines are likely to be important mediators of the inflammatory response that accompanies acute infectious processes in the lungs. GRO proteins are α-chemokine cytokines that attract neutrophils and stimulate the growth of a variety of cells. Previously, we observed that rabbit alveolar macrophages transcribe the genes for at least two GRO homologues. In order to study the role of GRO cytokines in lung inflammation, we cloned the predominant rabbit GRO cDNA (RabGRO) from alveolar macrophages, expressed bioactive recombinant protein (rRabGRO) in Escherichia coli, and developed a sensitive and specific enzyme-linked immunosorbent assay for RabGRO protein. We found that rabbit AM express and secrete GRO in vitro in response to both exogenous (e.g. lipopolysaccharide, heat-killed Staphylococcus aureus, and crystalline silica) and endogenous inflammatory stimuli (e.g. tumor necrosis factor-α) as determined by both radioimmunoprecipitation and enzyme-linked immunosorbent assay. Biologically significant amounts of GRO are present in vivo in the bronchoalveolar lavage fluid of rabbits with E. coli pneumonia; by in situ hybridization, GRO mRNA is detectable in infiltrating pulmonary leukocytes and bronchial epithelial cells. These results indicate that GRO chemokines are likely to be important mediators of the inflammatory response that accompanies acute infectious processes in the lungs. Human GRO-α, -β, and -γ are members of the α-chemokine family of proteins, which also includes interleukin-8 (IL-8) 1The abbreviations used are: ILinterleukinAMalveolar macrophagesLPSlipopolysaccharideTNF-αtumor necrosis factor-αRPF2rabbit permeability factor-2rRabGROrecombinant RabGROBALFbronchoalveolar lavage fluidPBSphosphate-buffered salinerRabGRO-fprRabGRO fusion proteinPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatographyZASzymosan-activated serumMCP-1monocyte chemotactic protein-1Con Aconcanavalin AHKSAheat-killed Staphylococcus aureusRIPradioimmunoprecipitationELISAenzyme-linked immunosorbent assay. (1.Miller M.D. Krangel M.S. Crit. Rev. Immunol. 1992; 12: 17-46PubMed Google Scholar). Like most α-chemokines, GRO proteins are potent neutrophil chemoattractants and activators(2.Geiser T. Dewald B. Ehrengruber M.U. Clark-Lewis I. Baggiolini M. J. Biol. Chem. 1993; 268: 15419-15424Abstract Full Text PDF PubMed Google Scholar). In addition to their effects on neutrophils, these cytokines have many other biological activities, including regulatory effects on melanoma cell growth(3.Richmond A. Lawson D.H. Nixon D.W. Chawla R.K. Cancer. Res. 1985; 45: 6390-6394PubMed Google Scholar), fibroblast collagen production(4.Unemori E.N. Amento E.P. Bauer E.A. Horuk R. J. Biol. Chem. 1993; 268: 1338-1342Abstract Full Text PDF PubMed Google Scholar), monocyte activation and adhesion to endothelial cells(5.Walz A. Meloni F. Clark L.I. von T.V. Baggiolini M. J. Leukocyte Biol. 1991; 50: 279-286Crossref PubMed Scopus (96) Google Scholar, 6.Schwartz D. Andalibi A. Chaverri-Almada L. Berliner J.A. Kirchgessner T. Fang Z.T. Tekamp-Olson P. Lusis A.J. Gallegos C. Fogelman A.M. Territo M.C. J. Clin. Invest. 1994; 94: 1968-1973Crossref PubMed Scopus (101) Google Scholar), myelopoiesis(7.Broxmeyer H.E. Sherry B. Lu L. Cooper S. Carow C. Wolpe S.D. Cerami A. J. Exp. Med. 1989; 170: 1583-1594Crossref PubMed Scopus (86) Google Scholar), and angiogenesis(8.Strieter R.M. Polverini P.J. Arenberg D.A. Walz A. Opdenakker G. Van Damme J. Kunkel S.L. J. Leukocyte Biol. 1995; 57: 752-762Crossref PubMed Scopus (248) Google Scholar, 9.Cao Y. Chen C. Weatherbee J.A. Tsang M. Folkman J. J. Exp. Med. 1995; 182: 2069-2077Crossref PubMed Scopus (123) Google Scholar). interleukin alveolar macrophages lipopolysaccharide tumor necrosis factor-α rabbit permeability factor-2 recombinant RabGRO bronchoalveolar lavage fluid phosphate-buffered saline rRabGRO fusion protein polyacrylamide gel electrophoresis high performance liquid chromatography zymosan-activated serum monocyte chemotactic protein-1 concanavalin A heat-killed Staphylococcus aureus radioimmunoprecipitation enzyme-linked immunosorbent assay. It is likely that GRO proteins are relevant in lung inflammation, as human alveolar macrophages (AM) express GRO mRNA and protein in response to stimulation with lipopolysaccharide (LPS)(10.Goodman R.B. Martin T.R. Chest. 1994; 105: 99SAbstract Full Text Full Text PDF PubMed Google Scholar), toxic shock syndrome toxin-1(11.Kurdowska A. Cohen A.B. Carr F.K. Stevens M.D. Miller E.J. Mullenbach G. Tekamp-Olsen P. Cytokine. 1994; 6: 124-134Crossref PubMed Scopus (15) Google Scholar), and tumor necrosis factor-α (TNF-α)(12.Becker S. Quay J. Koren H.S. Haskill J.S. Am. J. Physiol. 1994; 266: L278-L286PubMed Google Scholar). Although these in vitro studies suggest that GRO chemokines are mediators of the lung response to both exogenous and endogenous inflammatory stimuli, demonstration of their biological relevance in vivo is lacking. Rabbits, like humans and in contrast to rodents(1.Miller M.D. Krangel M.S. Crit. Rev. Immunol. 1992; 12: 17-46PubMed Google Scholar), produce a spectrum of α-chemokines that includes IL-8 (13.Yoshimura T. Yuhki N. J. Immunol. 1991; 146: 3-3488PubMed Google Scholar) and GRO(6.Schwartz D. Andalibi A. Chaverri-Almada L. Berliner J.A. Kirchgessner T. Fang Z.T. Tekamp-Olson P. Lusis A.J. Gallegos C. Fogelman A.M. Territo M.C. J. Clin. Invest. 1994; 94: 1968-1973Crossref PubMed Scopus (101) Google Scholar, 14.Jose P.J. Collins P.D. Perkins J.A. Beaubien B.C. Totty N.F. Waterfield M.D. Hsuan J. Williams T.J. Biochem. J. 1991; 278: 493-497Crossref PubMed Scopus (29) Google Scholar, 15.Johnson II, M.C. Goodman R.B. Kajikawa O. Wong V.A. Mongovin S.M. Martin T.R. Gene (Amst.). 1994; 151: 337-338Crossref PubMed Scopus (11) Google Scholar). We reported previously the molecular cloning of two distinct rabbit GRO cDNA homologues from LPS-stimulated AM (RabGRO and rabbit permeability factor-2 or RPF2)(15.Johnson II, M.C. Goodman R.B. Kajikawa O. Wong V.A. Mongovin S.M. Martin T.R. Gene (Amst.). 1994; 151: 337-338Crossref PubMed Scopus (11) Google Scholar). Of the two transcripts, RabGRO was the predominant AM-derived GRO homologue. We now report the expression in Escherichia coli of bioactive recombinant RabGRO protein (rRabGRO) and the development of a sensitive and specific RabGRO immunoassay. Using this assay, we have found that rabbit AM secrete native GRO protein in vitro in response to stimulation with a spectrum of biologically relevant agonists. We also report that biologically significant quantities of GRO protein are present in the bronchoalveolar lavage fluid (BALF) of rabbits with acute bacterial pneumonia and identify the sites of GRO mRNA expression in the lungs. These data suggest that GRO chemokines make important contributions to acute inflammatory responses in the lungs. The coding sequence for mature RabGRO protein (219 base pairs, 72 amino acids) (15.Johnson II, M.C. Goodman R.B. Kajikawa O. Wong V.A. Mongovin S.M. Martin T.R. Gene (Amst.). 1994; 151: 337-338Crossref PubMed Scopus (11) Google Scholar) was amplified from LPS-stimulated rabbit AM mRNA by reverse transcriptase-polymerase chain reaction techniques using a 5′-primer with a BamHI restriction enzyme site (5′-CGGGATCCCGCGCTCACCGAGC-3′) and a 3′-primer with a BglII restriction enzyme site (5′-GAAGATCTTCCTCCTTTCCCAGG-3′). The primer sequences written in boldface correspond to base pairs coding for RabGRO. The RabGRO polymerase chain reaction product was confirmed by sequencing and directionally cloned into the expression vector pRSET (Invitrogen Co., San Diego, CA). The pRSET vector encodes a fusion protein that contains an N-terminal fusion peptide sequence with polyhistidine residues for Ni2+ affinity column purification of the fusion protein and an enterokinase cleavage site for removal of the fusion peptide sequence to generate the mature recombinant protein. The transformants were cultured in the presence of isopropyl-β-D-thiogalactopyranoside and M13/T7 bacteriophage to provide the T7 RNA polymerase required for transcription of the rRabGRO fusion protein (rRabGRO-fp) mRNA. After induction for 10 h, the bacterial pellet was resuspended in 20 mM PBS, pH 7.8, with lysozyme (100 μg/ml, Sigma) and then incubated on ice for 15 min with 10 mM EDTA and 1% Triton X-100, sonicated, flash-frozen in liquid nitrogen, and thawed at 37°C three times in succession. The supernatant was dialyzed overnight against PBS, pH 7.8 (Mr 1000 limit membrane, Spectrum Medical Industries, Inc., Los Angeles, CA). Following dialysis, the supernatant was mixed with Ni2+-activated resin and incubated at room temperature for 1 h. The resin was then washed with PBS at pH 7.8, 6.0, and 5.0 and then packed into a column. The rRabGRO-fp was eluted from the column in 1.0-ml fractions by rinsing with PBS at pH 2.0. Each fraction was analyzed by SDS-PAGE, and the fractions containing rRabGRO-fp were pooled and dialyzed against 100 mM NaCl overnight. The rRabGRO-fp was incubated overnight with enterokinase (Biozyme, South Wales, United Kingdom) at a concentration of 1 unit/μg fusion protein in 10 mM Tris-HCl, pH 8.0 and 10 mM CaCl2 to cleave the fusion peptide sequence. The reaction mixture was then loaded onto a C4 column (Dynamax-300A, Rainin Co., Emeryville, CA) and reverse-phase high performance liquid chromatography (HPLC) was performed using a 5-95% acetonitrile gradient. The resulting HPLC peaks were analyzed by SDS-PAGE, and the N-terminal amino acid sequences were determined by Edman degradation with a 475 Å pulse liquid protein sequencer (Applied Biosystems, Foster City, CA). Specific pathogen free female New Zealand White rabbits weighing approximately 3.0-3.5 kg (Western Oregon Rabbit Co., Philomath, OR) and 6-8-week-old female BALB/c mice (Simonsen Laboratories, Gilroy, CA) were housed in the Seattle Veterans Affair Medical Center vivarium prior to all experiments. A female goat was purchased locally and housed at the University of Washington vivarium. Rabbit AM were recovered by whole lung lavage as described previously(16.Martin T.R. Mathison J.C. Tobias P.S. Let'urcq D.J. Moriarty A.M. Maunder R.J. Ulevitch R.J. J. Clin. Invest. 1992; 90: 2209-2219Crossref PubMed Scopus (202) Google Scholar). Rabbit and human neutrophils were recovered from heparinized venous blood using Ficoll-Hypaque density gradient centrifugation (Mono-Poly resolving medium, ICN Biomedicals, Aurora, OH). The cells were washed twice in PBS and resuspended at the indicated concentrations for chemotaxis or immunoprecipitation studies. Rabbit and human neutrophil chemotaxis to rRabGRO was measured by the modified Boyden method using 48-well microchemotaxis chambers and nitrocellulose membranes with 3.0-μm pores for neutrophils as described(17.Falk W. Goodwin R.H.J. Leonard E.J. J. Immunol. Methods. 1980; 33: 239-247Crossref PubMed Scopus (251) Google Scholar). The samples were diluted with PBS, and 25 μl of each sample were added to the bottom well. Controls included 10% zymosan-activated human or rabbit serum (ZAS, positive control) or PBS alone (negative control). Neutrophils were added to the top wells, and the chambers were incubated for 2 h at 37°C in 5% CO2 and humidified air. Experiments were performed in quadruplicate, and chemotaxis was measured as the total number of leukocytes migrating through the filters in 10 consecutive high power microscopic fields. Results were expressed as percent maximal chemotaxis, in which the average number of leukocytes migrating toward PBS for each chamber was subtracted from each measurement (including ZAS) and then each measurement was divided by the adjusted value for ZAS. The value for ZAS was set as 100% maximal chemotaxis. Results are reported as mean ± S.E. To test the bioactivity of rRabGRO in the lung, 1.0 μg of LPS-free protein in 1.0 ml sterile, pyrogen-free 0.9% NaCl (approximately 10-7M solution) was instilled into the right lung of an anesthetized rabbit through an intratracheal catheter directed toward the right mainstem bronchus. For comparison, another rabbit was treated with 0.9% NaCl alone. The catheters were removed immediately following the instillations, and the animals were closely monitored to ensure adequate recovery from the procedure. The rabbits were euthanized 4 h later with pentobarbital, and selective right lung lavage was performed to obtain BALF for total and differential cell counts using methods as described(16.Martin T.R. Mathison J.C. Tobias P.S. Let'urcq D.J. Moriarty A.M. Maunder R.J. Ulevitch R.J. J. Clin. Invest. 1992; 90: 2209-2219Crossref PubMed Scopus (202) Google Scholar). A female goat was immunized with successive doses of rRabGRO-fp. Polyclonal goat IgG was purified from serum using protein G columns (Pierce), and a portion was biotinylated (Pierce). The specificity of the goat antisera was tested by Western blotting using biotinylated goat anti-RabGRO IgG or biotinylated nonimmune IgG and recombinant rabbit IL-8, rabbit monocyte chemotactic protein-1 (MCP-1)(18.Kajikawa O. Goodman R.B. Mongovin S.M. Wong V.A. Martin T.R. Am. Rev. Respir. Dis. 1993; 147: A751Google Scholar), recombinant human GRO-α (R & D Systems, Inc., Minneapolis, MN), and rRabGRO protein. Rabbit AM (2 × 106 cells/ml) were incubated for 1 h at 37°C in 24-well plates containing 1 ml of labeling medium containing 250 μCi of [35S]Cys (DuPont NEN). The cells were then stimulated by adding one of the following agents: LPS (E. coli, serotype 0111:B4, 10 μg/ml), intact or heat-inactivated human TNF-α (500 units/ml, R & D Systems), concanavalin A (ConA, 5 μg/ml), heat-killed Staphylococcus aureus (HKSA, 1 × 108 bacteria/ml), crystalline silica (α-quartz, 100 μg/ml SiO2), aluminum oxide (Al2O3, μg/ml), or media alone for an additional 19 h at 37°C. The supernatant from each well was collected and diluted 1:2 with radioimmunoprecipitation (RIP) buffer (100 mM Tris, pH 8.6, 150 mM NaCl, 0.5% Tween 20, and 0.1% bovine serum albumin) and then incubated with goat IgG coupled to protein G-agarose resin (Boehringer Mannheim, GmbH, Germany) for 24 h at 4°C. The protein-IgG-protein G-agarose resin complexes were recovered by centrifugation, washed, treated with 2-mercaptoethanol, and boiled for 5 min. The resulting protein mixture was then analyzed by SDS-PAGE using Coomassie Blue staining and autoradiography with quantitative phosphorimaging(19.Johnston R.F. Pickett S.C. Barker D.L. Electrophoresis. 1990; 11: 355-360Crossref PubMed Scopus (436) Google Scholar). A sandwich enzyme-linked immunosorbent assay (ELISA) was constructed using goat polyclonal anti-rRabGRO IgG as both the capturing and detecting antibody. After coating and overnight incubation at 4°C with nonbiotinylated immune goat IgG (1:350 dilution), microtiter plates were blocked with 10% non-fat milk for 1 h at 37°C. The blocking buffer was removed, samples and standards (serial dilutions of rRabGRO protein) were added, and the plates were incubated for 1 h at 37°C. After washing four times, biotinylated immune goat IgG (1:800 dilution) was added to the plates and they were again incubated for 1 h at 37°C. After washing, peroxidase-labeled streptavidin (Zymed Laboratories, Inc., South San Francisco, CA) was added, followed by incubation for 1 h at 37°C. The chromogen 3,3′,5,5′-tetramethylbenzidine (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) was added, and the optical density (A450) in each well was measured using a microtiter plate reader (Dynatech Co., Chantilly, VA). Based on a standard curve generated with rRabGRO protein, the lower limit of detection for the ELISA was 22 pg/ml. There was no cross-reactivity with either recombinant rabbit IL-8 or recombinant rabbit MCP-1 at concentrations varying from 10 pg/ml to 1 mg/ml in spiked rabbit BALF samples. For statistical analysis, undetectable GRO levels were assigned a value of 11 pg/ml (one-half the limit of detection of the assay). Rabbit AM (1 × 106 cells/ml in RPMI 1640 medium with 10% heat-inactivated fetal calf serum) were incubated with either LPS, intact or heat-inactivated human TNF-α (500 units/ml), ConA (5 μg/ml), HKSA (1 × 108 bacteria/ml), SiO2 (100 μg/ml), Al2O3 (100 μg/ml), or rabbit interferon-γ (INF-γ, 500 units/ml, Genentech, San Francisco, CA), or 0.9% NaCl (10 μl/ml) for 2, 6, or 20 h at 37°C and 5% CO2. To minimize adherence, the incubations were performed in gently agitated polypropylene tubes. After incubation, the cell supernatants were removed, clarified by centrifugation, and the levels of GRO protein were measured by ELISA. Anesthetized rabbits were treated with either a 1.0 ml bolus of 2 × 1010 colony-stimulating factor E. coli (serotype K1) in sterile, pyrogen-free 0.9% NaCl (n = 8) or NaCl alone (n = 8) through an intratracheal catheter directed toward the right mainstem bronchus. The animals were euthanized 4 or 24 h later, and selective right lung lavage was performed. Bronchoalveolar lavage fluid cell counts and differentials were calculated and GRO protein levels were measured by ELISA. In situ hybridization was performed on right lung tissue from rabbits treated with endobronchial E. coli (1 × 109 colony-stimulating factor) and sacrificed after 4 h as described above. Lung tissue samples were frozen in liquid Freon® and placed in O.C.T. Compound (Miles, Inc., Elkhart, IN). The embedded samples were then sectioned (9-μm slices) and mounted onto gelatin-coated slides (Fisher). After treatment with a 4% paraformaldehyde solution, the slides were dehydrated with graded ethanol rinses. Antisense and sense (base pairs 111-140(15.Johnson II, M.C. Goodman R.B. Kajikawa O. Wong V.A. Mongovin S.M. Martin T.R. Gene (Amst.). 1994; 151: 337-338Crossref PubMed Scopus (11) Google Scholar)) RabGRO cDNA probes were made by end labeling synthesized oligonucleotides with 35S-dATP using terminal deoxynucleotidyltransferase (DuPont). The specificity of the probe for rabbit GRO cDNA was confirmed by the absence of hybridization signals with rabbit IL-8, IL-1, and TNF-α cDNA on Southern blots (data not shown). Hybridization was performed at 48°C overnight by incubating each slide in a buffer containing 50% formamide and 1 × 106 cpm antisense or sense (negative control) RabGRO cDNA. Post-hybridization washings were performed twice in 1 × standard saline citrate solution at 66°C for 30 min and once at room temperature for 1 h. The slides were then dehydrated with successive ethanol rinses and dipped into warmed Kodak NTB2 emulsion solution (Eastman Kodak Co.) for autoradiographic analysis. After exposure for 9 days at 4°C, the slides were developed, fixed, counterstained with hematoxylin-eosin, and visualized by light microscopy. The coding sequence for mature RabGRO was amplified by polymerase chain reaction and cloned into the pRSET expression vector. After confirming the sequence for the RabGRO cDNA, the recombinant protein was expressed in JM109 E. coli and purified from bacterial lysates by Ni2+ affinity chromatography. Analysis by SDS-PAGE confirmed that the purified protein was of the expected size for rRabGRO-fp (approximately 14,000 kDa) (Fig. 1A). The rRabGRO-fp was then treated with enterokinase, and the products were purified by reverse-phase HPLC (Fig. 1B). The HPLC peak labeled “rRabGRO” was found to contain mature rRabGRO of the expected sequence as determined by N-terminal amino acid sequencing. The chemotactic activity of rRabGRO was tested in vitro using rabbit and human neutrophils. For comparison, recombinant human GRO-α (R & D Systems) was also tested. Recombinant RabGRO protein stimulated chemotaxis of rabbit neutrophils at concentrations as low as 3 × 10-10M, with maximal activity at approximately 10-7M and a decline in activity at 10-6M (Fig. 2A). In contrast, rRabGRO had little chemotactic activity for human neutrophils over the same concentration range. Similarly, human GRO-α showed no significant chemotactic activity for rabbit neutrophils, despite being chemotactically active for human neutrophils (Fig. 2B). To assess in vivo bioactivity, rRabGRO was instilled endobronchially into a rabbit. This produced a brisk influx of neutrophils into the lung (45% neutrophils in the BALF cell count from the treated lung at 4 h). For comparison, the BALF from the untreated lung in the same rabbit and from the lungs of a rabbit treated with sterile 0.9% NaCl alone contained only 6 and 5% neutrophils, respectively. After isolating total IgG from immune goat serum, the specificity of the anti-rRabGRO IgG was tested by Western analysis using rRabGRO, recombinant rabbit MCP-1, rabbit IL-8 (each produced using similar methods(18.Kajikawa O. Goodman R.B. Mongovin S.M. Wong V.A. Martin T.R. Am. Rev. Respir. Dis. 1993; 147: A751Google Scholar)), and recombinant human GRO-α. The anti-rRabGRO IgG reacted strongly with both the fusion protein and the cleaved mature rRabGRO protein (data not shown). Although there was very weak cross-reactivity with human GRO-α, there was no cross-reactivity with either rabbit IL-8 or rabbit MCP-1. The specificity of the goat anti-rRabGRO antibodies for native rabbit GRO protein was further confirmed by RIP (described below). We performed RIP and ELISA measurements of stimulated rabbit AM supernatants to measure the expression of newly synthesized and secreted GRO protein. The immunoprecipitation experiments were conducted using the supernatants of adherent rabbit AM metabolically labeled with [35S]cysteine and stimulated for 19 h with a variety of endogenous and exogenous proinflammatory agents. Immunoprecipitation of these supernatants with the goat anti-rRabGRO IgG resulted in a major band of the expected size (approximately 7 kDa) for each stimulus (Fig. 3). Further analysis of the gels by phosphorimaging allowed quantitation of GRO protein in the supernatants. SiO2, Al2O3, LPS, human TNF-α, and HKSA were each strong inducers of GRO secretion, while ConA and heat-inactivated human TNF-α were very weak stimuli. GRO protein was not detected when RIP was performed with nonimmune goat serum. Because adherence alone appeared to be a stimulus for GRO protein production in the RIP studies, we measured GRO levels by ELISA using the supernatants of rabbit AM cultured in polypropylene tubes which were periodically agitated. These results showed significant time- and stimulus-dependent differences in the accumulation of GRO protein in the supernatants of relatively nonadherent AM (Fig. 4). In response to stimulation by HKSA and LPS, significant amounts of extracellular GRO were detected after only 2 h of incubation. For HKSA, GRO increased steadily and was maximal by 20 h of incubation. For LPS, maximal amounts of GRO were detected at 4 h of incubation and the concentration failed to increase further at 20 h. Incubation with SiO2 caused the secretion of relatively small amounts of GRO protein during the first 6 h, but relatively high concentrations of GRO were present in the supernatants at 20 h. Stimulation with ConA and Al2O3 did not result in significant secretion of GRO when compared with incubation with 0.9% NaCl (negative control). Likewise, human TNF-α and rabbit IFN-γ (both intact and heat-inactivated) failed to stimulate increased amounts of GRO in the supernatants of nonadherent AM (data for heat-inactivated proteins not shown). Significantly increased amounts of GRO protein were detected in the BALF of rabbits treated with endobronchial E. coli when compared with rabbits treated with NaCl alone at both 4 and 24 h (p < 0.03 by unpaired t tests for both groups) (Table 1). There was also a significant increase in the number and percentage of neutrophils in the E. coli-treated animal BALF at both 4 and 24 h when compared with the NaCl-treated animals (p < 0.0001 for both groups) and a significant positive relationship between GRO protein levels and the number and percentage of BALF neutrophils (r = 0.82, p < 0.001 for BALF total neutrophils versus BALF GRO concentration).TABLE 1 To better define which lung cells in the lung express GRO mRNA in acute pulmonary inflammation, we used in situ hybridization techniques to localize GRO mRNA in lung tissue from rabbits with E. coli pneumonia. Hybridization signals with the RabGRO antisense probe were detected in bronchial epithelial cells and in inflammatory cells in the lung parenchyma around the airways (Fig. 5, A and C). The tissue inflammatory cells were strongly positive and appeared to be either macrophages or neutrophils infiltrating the peribronchial lung parenchyma. Hybridization with the negative control (sense) RabGRO probe produced virtually no signals (Fig. 5, B and D). Alveolar macrophages are important cellular mediators of pulmonary inflammation, in part due to their ability to elaborate a variety of leukocyte chemotactic factors(20.Nathan C.F. J. Clin. Invest. 1987; 79: 319-326Crossref PubMed Scopus (2146) Google Scholar). Among these factors, IL-8 is thought by many investigators to be the major neutrophil chemoattractant in the lung(21.Kunkel S.L. Standiford T.J. Kasahara K. Strieter R.M. Exp. Lung Res. 1991; 17: 17-23Crossref PubMed Scopus (478) Google Scholar). However, studies using specific antisera to neutralize IL-8 in the supernatants of activated human AM (22.Sylvester I. Rankin J.A. Yoshimura T. Tanaka S. Leonard E.J. Am. Rev. Respir. Dis. 1990; 141: 683-688Crossref PubMed Scopus (61) Google Scholar) and BALF from patients with adult respiratory distress syndrome (23.Miller E.J. Cohen A.B. Nagao S. Griffith D. Maunder R.J. Martin T.R. Weiner-Kronish J.P. Sticherling M. Christophers E. Matthay M.A. Am. Rev. Respir. Dis. 1992; 146: 427-432Crossref PubMed Scopus (432) Google Scholar) suggest that a significant proportion of AM-derived neutrophil chemotactic activity is due to other non-IL-8 chemoattractants. Possible candidates include the GRO subgroup of α-chemokines, which are closely related to IL-8 and are also potent neutrophil chemoattractants and activators(2.Geiser T. Dewald B. Ehrengruber M.U. Clark-Lewis I. Baggiolini M. J. Biol. Chem. 1993; 268: 15419-15424Abstract Full Text PDF PubMed Google Scholar). In this study, our goals were: 1) to express recombinant rabbit GRO protein and develop a specific and sensitive ELISA; 2) to investigate GRO protein expression by rabbit AM in response to stimulation with both endogenous and exogenous inflammatory agents; and 3) to determine whether GRO protein and mRNA can be detected in the lung in vivo during the acute inflammatory response that accompanies bacterial pneumonia. In order to generate the species-specific reagents necessary to measure rabbit GRO protein, we first produced rRabGRO as a fusion protein in E. coli. We used a prokaryotic expression system for producing the recombinant protein because GRO proteins have been shown not to undergo post-translational sulfation, glycosylation, or phosphorylation(24.Derynck R. Balentien E. Han J.H. Thomas H.G. Wen D.Z. Samantha A.K. Zachariae C.O. Griffin P.R. Brachmann R. Wong W.L. Matsushima K. Richmond A. Biochemistry. 1990; 29: 10225-10233Crossref PubMed Scopus (65) Google Scholar). RabGRO amino acid sequence alignment with all reported full-length GRO homologues demonstrated identities ranging from 41% for 9E3 (chicken GRO homologue) to 78% for human GRO-β (Table 2). Given the high degree of identity with human GRO proteins, it is perhaps surprising that rRabGRO had little chemotactic activity for human neutrophils. However, this confirms previous observations by other investigators that 125I-labeled human GRO-α does not bind to rabbit neutrophils (25.Thomas K.M. Taylor L. Prado G. Romero J. Moser B. Car B. Walz A. Baggiolini M. Navarro J. J. Immunol. 1994; 152: 2496-2500PubMed Google Scholar) and further underscores the necessity of using species-specific reagents when studying α-chemokines(26.Rot A. Cytokine. 1991; 3: 21-27Crossref PubMed Scopus (77) Google Scholar).TABLE 2 The rRabGRO-fp proved to be a good immunogen for raising specific goat anti-rabbit GRO polyclonal antibodies, as Western analysis confirmed that the goat antibodies did not cross-react with either rabbit IL-8 or MCP-1. The antibodies did show a small amount of cross-reactivity for recombinant human GRO-α, which is not surprising given the high degree of homology between these proteins (Table 2). As RabGRO and RPF2 are nearly identical at the mature protein level (93% identity), the anti-rRabGRO antibodies are likely to cross-react with RPF2 as well. We have also shown that rabbit AM express and secrete GRO protein in response to stimulation by LPS, TNF-α, SiO2, Al2O3, and HKSA. Adherence and/or culture conditions alone also appear to stimulate GRO expression. Under nonadherent conditions, LPS, HKSA, and SiO2 were the most potent inducers of GRO protein" @default.
• W1988018889 created "2016-06-24" @default.
• W1988018889 creator A5020975579 @default.
• W1988018889 creator A5026870299 @default.
• W1988018889 creator A5031392392 @default.
• W1988018889 creator A5031423575 @default.
• W1988018889 creator A5048753086 @default.
• W1988018889 creator A5057087120 @default.
• W1988018889 creator A5066325637 @default.
• W1988018889 creator A5089312224 @default.
• W1988018889 date "1996-05-01" @default.
• W1988018889 modified "2023-10-14" @default.
• W1988018889 title "Molecular Expression of the α-Chemokine Rabbit GRO in Escherichia coli and Characterization of Its Production by Lung Cells in Vitro and in Vivo" @default.
• W1988018889 cites W1489136175 @default.
• W1988018889 cites W1510097734 @default.
• W1988018889 cites W1527149701 @default.
• W1988018889 cites W153681275 @default.
• W1988018889 cites W1565900869 @default.
• W1988018889 cites W1589729211 @default.
• W1988018889 cites W1596383605 @default.
• W1988018889 cites W1600880529 @default.
• W1988018889 cites W1607098455 @default.
• W1988018889 cites W1965061750 @default.
• W1988018889 cites W1965694122 @default.
• W1988018889 cites W1968235120 @default.
• W1988018889 cites W1973195685 @default.
• W1988018889 cites W1979694727 @default.
• W1988018889 cites W1983203261 @default.
• W1988018889 cites W1983239749 @default.
• W1988018889 cites W1983405778 @default.
• W1988018889 cites W1988742153 @default.
• W1988018889 cites W1989438935 @default.
• W1988018889 cites W1999193723 @default.
• W1988018889 cites W2004355153 @default.
• W1988018889 cites W2004446611 @default.
• W1988018889 cites W2017347652 @default.
• W1988018889 cites W2020979927 @default.
• W1988018889 cites W2037464100 @default.
• W1988018889 cites W2058757949 @default.
• W1988018889 cites W2065165441 @default.
• W1988018889 cites W2070193280 @default.
• W1988018889 cites W2110105332 @default.
• W1988018889 cites W2127844945 @default.
• W1988018889 cites W2147657681 @default.
• W1988018889 cites W2147819976 @default.
• W1988018889 cites W2148687063 @default.
• W1988018889 cites W2153131035 @default.
• W1988018889 cites W2154232005 @default.
• W1988018889 cites W2215231529 @default.
• W1988018889 cites W2339444913 @default.
• W1988018889 cites W2340351358 @default.
• W1988018889 cites W2613386474 @default.
• W1988018889 cites W4239357337 @default.
• W1988018889 cites W1892153687 @default.
• W1988018889 doi "https://doi.org/10.1074/jbc.271.18.10853" @default.
• W1988018889 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8631900" @default.
• W1988018889 hasPublicationYear "1996" @default.
• W1988018889 type Work @default.
• W1988018889 sameAs 1988018889 @default.
• W1988018889 citedByCount "24" @default.
• W1988018889 crossrefType "journal-article" @default.
• W1988018889 hasAuthorship W1988018889A5020975579 @default.
• W1988018889 hasAuthorship W1988018889A5026870299 @default.
• W1988018889 hasAuthorship W1988018889A5031392392 @default.
• W1988018889 hasAuthorship W1988018889A5031423575 @default.
• W1988018889 hasAuthorship W1988018889A5048753086 @default.
• W1988018889 hasAuthorship W1988018889A5057087120 @default.
• W1988018889 hasAuthorship W1988018889A5066325637 @default.
• W1988018889 hasAuthorship W1988018889A5089312224 @default.
• W1988018889 hasBestOaLocation W19880188891 @default.
• W1988018889 hasConcept C104317684 @default.
• W1988018889 hasConcept C105795698 @default.
• W1988018889 hasConcept C126322002 @default.
• W1988018889 hasConcept C13373296 @default.
• W1988018889 hasConcept C150903083 @default.
• W1988018889 hasConcept C153911025 @default.
• W1988018889 hasConcept C185592680 @default.
• W1988018889 hasConcept C202751555 @default.
• W1988018889 hasConcept C203014093 @default.
• W1988018889 hasConcept C207001950 @default.
• W1988018889 hasConcept C2776914184 @default.
• W1988018889 hasConcept C2777714996 @default.
• W1988018889 hasConcept C2779884254 @default.
• W1988018889 hasConcept C33923547 @default.
• W1988018889 hasConcept C547475151 @default.
• W1988018889 hasConcept C55493867 @default.
• W1988018889 hasConcept C71924100 @default.
• W1988018889 hasConcept C86803240 @default.
• W1988018889 hasConcept C89423630 @default.
• W1988018889 hasConceptScore W1988018889C104317684 @default.
• W1988018889 hasConceptScore W1988018889C105795698 @default.
• W1988018889 hasConceptScore W1988018889C126322002 @default.
• W1988018889 hasConceptScore W1988018889C13373296 @default.
• W1988018889 hasConceptScore W1988018889C150903083 @default.
• W1988018889 hasConceptScore W1988018889C153911025 @default.
• W1988018889 hasConceptScore W1988018889C185592680 @default.
• W1988018889 hasConceptScore W1988018889C202751555 @default.
• W1988018889 hasConceptScore W1988018889C203014093 @default.

• next