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• W1993151719 abstract "Recent studies support the concept that pulmonary granulomatous inflammation directed by interferon (IFN)-γ, interleukin (IL)-12, and nitric oxide usually resolves in the absence of fibrosis. To determine whether nitric oxide participates in modulating the fibrotic response during the development of pulmonary granulomas in response to purified protein derivative (PPD), mice presensitized to PPD received daily intraperitoneal injections ofNG-nitro-d-arginine-methyl ester (d-NAME),NG-nitro-l-arginine-methyl ester (l-NAME), or aminoguanidine after delivery of PPD-coated beads to the lungs. Eight days later, morphometric analysis of lung granulomas revealed that l-NAME-treated mice when challenged with PPD in vitro for 36 hours had the largest pulmonary granulomas and the greatest collagen deposition among the treated groups. In addition, equivalent numbers of dispersed lung cells from l-NAME- and aminoguanidine-treated mice produced significantly higher levels of IL-4, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1α and significantly lower levels of eotaxin compared with d-NAME-treated mice. Cultures of dispersed lung cells from l-NAME-treated mice also produced significantly more IL-10 and less IL-12 compared with similar numbers of dispersed lung cells from d-NAME-treated mice. Cultures of isolated lung fibroblasts from l-NAME-treated mice expressed higher levels of C-C chemokine receptor 2 (CCR2) and CCR3 mRNA and contained less MCP-1 and eotaxin protein than a similar number of fibroblasts from d-NAME-treated mice. Thus, nitric oxide appears to regulate the deposition of extracellular matrix in lung granulomas through the modulation of the cytokine and chemokine profile of these lesions. Alterations in the cytokine, chemokine, and procollagen profile of this lesion may be a direct effect of nitric oxide on the pulmonary fibroblast and provide an important signal for regulating fibroblast activity during the evolution of chronic lung disease. Recent studies support the concept that pulmonary granulomatous inflammation directed by interferon (IFN)-γ, interleukin (IL)-12, and nitric oxide usually resolves in the absence of fibrosis. To determine whether nitric oxide participates in modulating the fibrotic response during the development of pulmonary granulomas in response to purified protein derivative (PPD), mice presensitized to PPD received daily intraperitoneal injections ofNG-nitro-d-arginine-methyl ester (d-NAME),NG-nitro-l-arginine-methyl ester (l-NAME), or aminoguanidine after delivery of PPD-coated beads to the lungs. Eight days later, morphometric analysis of lung granulomas revealed that l-NAME-treated mice when challenged with PPD in vitro for 36 hours had the largest pulmonary granulomas and the greatest collagen deposition among the treated groups. In addition, equivalent numbers of dispersed lung cells from l-NAME- and aminoguanidine-treated mice produced significantly higher levels of IL-4, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1α and significantly lower levels of eotaxin compared with d-NAME-treated mice. Cultures of dispersed lung cells from l-NAME-treated mice also produced significantly more IL-10 and less IL-12 compared with similar numbers of dispersed lung cells from d-NAME-treated mice. Cultures of isolated lung fibroblasts from l-NAME-treated mice expressed higher levels of C-C chemokine receptor 2 (CCR2) and CCR3 mRNA and contained less MCP-1 and eotaxin protein than a similar number of fibroblasts from d-NAME-treated mice. Thus, nitric oxide appears to regulate the deposition of extracellular matrix in lung granulomas through the modulation of the cytokine and chemokine profile of these lesions. Alterations in the cytokine, chemokine, and procollagen profile of this lesion may be a direct effect of nitric oxide on the pulmonary fibroblast and provide an important signal for regulating fibroblast activity during the evolution of chronic lung disease. It is not uncommon for chronic pulmonary granulomatous inflammation to result in irreversible tissue injury and end-stage fibrosis.1Kunkel SL Th1- and Th2-type cytokines regulate chemokine expression.Biol Signals. 1996; 5: 197-202Crossref PubMed Scopus (27) Google Scholar For reasons that are presently unclear, the reparative process associated with interstitial pulmonary inflammation can progress uncontrollably, as evidenced by increased lung fibroblast proliferation and the deposition of collagenous material.2Antoniades HN Neville-Golden J Galanopoulos T Kradin RL Valente AJ Graves DT Expression of monocyte chemoattractant protein-1 mRNA in human idiopathic pulmonary fibrosis.Proc Natl Acad Sci USA. 1992; 89: 5371-5375Crossref PubMed Scopus (226) Google Scholar Clinical and laboratory evidence suggest that the progressive and unregulated reparative process in the lung is potentially related to the persistence of a variety of inflammatory signals.3Meduri GU Headley S Kohler G Stentz F Tolley E Umberger R Leeper K Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Plasma IL-1 and IL-6 levels are consistent and efficient predictors of outcome over time.Chest. 1995; 107: 1062-1073Crossref PubMed Scopus (628) Google Scholar Unfortunately, clinical strategies directed at preventing deleterious fibrotic responses through the nonselective inhibition of the inflammatory process with corticosteroid, cyclophosphamide, and azathioprine treatments have been both largely unsuccessful and often associated with severe side effects.4Schwartz MI Interstitial pulmonary fibrosis.in: Kelley WN Textbook of Internal Medicine. JP Lippincott, Philadelphia1989: 1902-1905Google Scholar Previous studies of experimental, antigen-driven granulomatous pulmonary inflammatory responses has demonstrated that inflammatory cytokine profiles have a major role in the degree of extracellular matrix deposition around these lesions.5Chensue SW Warmington K Ruth J Lincoln P Kuo M-C Kunkel SL Cytokine responses during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation: production of Th1 and Th2 cytokines and relative contribution of tumor necrosis factor.Am J Pathol. 1994; 145: 1105-1113PubMed Google Scholar For example, the pulmonary cytokine response to purified peptide derivative (PPD) from Mycobacteria is dominated initially by interferon (IFN)-γ and interleukin (IL)-12, and little deposition of extracellular matrix results around Sephadex beads coated with PPD that are embolized in the lungs of PPD-sensitized mice. In contrast, the cytokine response to Schistosoma mansoni egg antigen (SEA)-coated beads also embolized to the lungs of SEA-sensitized mice are dominated by IL-4 and accompanied by pulmonary fibroblast activation and excessive extracellular matrix deposition within the interstitium.5Chensue SW Warmington K Ruth J Lincoln P Kuo M-C Kunkel SL Cytokine responses during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation: production of Th1 and Th2 cytokines and relative contribution of tumor necrosis factor.Am J Pathol. 1994; 145: 1105-1113PubMed Google Scholar Thus, the evolution of the fibrotic process within the lung appears to be inherently dependent on the counter-regulatory actions of specific cytokine profiles.6Mosmann TR Coffman RL Th1 and Th2 cells. different patterns of lymphokine secretion lead to different functional properties.Annu Rev Immunol. 1989; 7: 145-173Crossref PubMed Scopus (6815) Google ScholarFurther examination of the mechanisms through which cytokine profiles dictate the pulmonary fibrotic response has revealed that the chemotactic cytokines or chemokines also play unique roles in this process.7Kunkel SL Lukacs NW Strieter RM Chensue SW Th1 and Th2 responses regulate experimental lung granuloma development.Sarcoidosis Vasc Diffuse Lung Dis. 1996; 13: 120-128PubMed Google Scholar, 8Smith RE Strieter RM Phan SH Kunkel SL C-C chemokines: novel mediators of the profibrotic inflammatory response to bleomycin challenge.Am J Respir Cell Mol Biol. 1996; 15: 693-702Crossref PubMed Scopus (87) Google Scholar Monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α are two such C-C chemokines that have garnered attention because of their demonstrable roles in experimental interstitial fibrosis. MCP-1 has been shown to be necessary for the development of fibrosis in the kidney.9Lloyd CM Minto AW Dorf ME Proudfoot A Wells TN Salant DJ Gutierrez-Ramos JC RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis.J Exp Med. 1997; 185: 1371-1380Crossref PubMed Scopus (439) Google Scholar Interestingly, studies by Gharaee-Kermani et al10Gharaee-Kermani M Denholm EM Phan SH Costimulation of fibroblast collagen and transforming growth factor β1 gene expression by monocyte chemoattractant protein-1 via specific receptors.J Biol Chem. 1996; 271: 17779-17784Crossref PubMed Scopus (396) Google Scholar have revealed that MCP-1 has direct stimulatory effects on de novo synthesis of transforming growth factor (TGF)-β, which in turn augments collagen generation by cultured rodent pulmonary fibroblasts. Other experimental studies have shown that MIP-1α clearly contributes to the pulmonary fibrotic response to bleomycin.11Smith RE Strieter RM Zhang K Phan SH Standiford TJ Lukacs NW Kunkel SL A role for C-C chemokines in fibrotic lung disease.J Leukocyte Biol. 1995; 57: 782-787Crossref PubMed Scopus (168) Google Scholar Clinical observations also support a role for MCP-1 and MIP-1α in pulmonary fibrosis as both are elevated in the bronchoalveolar lavage and open-lung biopsies from patients with interstitial lung fibrosis.2Antoniades HN Neville-Golden J Galanopoulos T Kradin RL Valente AJ Graves DT Expression of monocyte chemoattractant protein-1 mRNA in human idiopathic pulmonary fibrosis.Proc Natl Acad Sci USA. 1992; 89: 5371-5375Crossref PubMed Scopus (226) Google Scholar, 12Iyonaga K Takeya M Saita N Sakamoto O Yoshimura T Ando M Takahashi K Monocyte chemoattractant protein-1 in idiopathic pulmonary fibrosis and other interstitial lung diseases.Hum Pathol. 1994; 25: 455-463Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 13Standiford TJ Rolfe MR Kunkel SL Lynch JP Becker FS Orringer MB Phan S Strieter RM Altered production and regulation of monocyte chemoattractant protein-1 from pulmonary fibroblasts isolated from patients with idiopathic pulmonary fibrosis.Chest. 1993; 103: 121SPubMed Google Scholar, 14Standiford TJ Rolfe MW Kunkel SL Lynch JP Burdick M Gilbert AR Orringer MB Whyte RI Strieter RM Macrophage inflammatory protein-1α expression in interstitial lung disease.J Immunol. 1993; 151: 2852-2863PubMed Google Scholar Eotaxin represents another C-C chemokine that may also exert a prominent role in pulmonary inflammation because of its ability to recruit eosinophils.15Jose PJ Griffiths-Johnson DA Walsh DT Moqbel R Totty NF Truong O Hsuan JJ Williams TJ Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea-pig model of allergic airways inflammation.J Exp Med. 1994; 179: 881-887Crossref PubMed Scopus (763) Google Scholar Although a causal link between eosinophils and pulmonary fibrosis has yet to be established, eosinophils are found in abundance in fibrotic tissue,16Peterson MW Monick M Hunninghake GW Prognostic role of eosinophils in pulmonary fibrosis.Chest. 1987; 92: 51-56Crossref PubMed Scopus (124) Google Scholar and activated eosinophils are a rich source of many inflammatory cytokines.17Zhang K Gharaee-Kermani M Jones ML Warren JS Phan SH Lung monocyte chemoattractant protein-1 gene expression in bleomycin-induced pulmonary fibrosis.J Immunol. 1994; 153: 4733-4741PubMed Google Scholar Thus, the cumulative balance of pro- and antifibrotic cytokines and chemokines during a pulmonary inflammatory response may be critical in regulating the tissue reparative process.Endogenous nitric oxide synthesis is a key regulator of interstitial fibrotic responses in numerous organs, including the kidney,18Morrissey JJ Ishidoya S McCracken R Klahr S Nitric oxide generation ameliorates the tubulointerstitial fibrosis of obstructive nephropathy.J Am Soc Nephrol. 1996; 7: 2202-2212PubMed Google Scholar heart,19Numaguchi K Egashira K Takemoto M Kadokami T Shimokawa H Sueishi K Takeshita A Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats.Hypertension. 1995; 26: 957-962Crossref PubMed Scopus (202) Google Scholar and vasculature.20Babal P Pechanova O Bernatova I Stvrtina S Chronic inhibition of NO synthesis produces myocardial fibrosis and arterial media hyperplasia.Histol Histopathol. 1997; 12: 623-629PubMed Google Scholar This free radical is produced by many cells through the utilization of l-arginine by at least three distinct nitric oxide synthase (NOS) isoforms.21Moncada S Palmer RJR Higgs EA Nitric oxide: physiology, pathophysiology, and pharmacology.Pharmacol Rev. 1991; 43: 109-123PubMed Google Scholar The three major classifications of NOS are as follows: NOS I is a constitutive isoform found in neurons, NOS II is an inducible isoform found in activated macrophages and epithelial and smooth muscle cells, and NOS III is a constitutive isoform found in endothelial cells.22Nathan C Nitric oxide is a secretory product of mammalian cells.FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4132) Google Scholar NOS I and III are calcium-dependent enzymes that produce nanomolar pulses of nitric oxide continuously, whereas NOS II is expressed only after gene induction and can be involved in the generation of micromolar quantities of nitric oxide that may be cytotoxic.22Nathan C Nitric oxide is a secretory product of mammalian cells.FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4132) Google Scholar The major functions of NOS I through III include neurotransmission, microbicidal and tumoricidal effects, and vasoregulation, respectively.21Moncada S Palmer RJR Higgs EA Nitric oxide: physiology, pathophysiology, and pharmacology.Pharmacol Rev. 1991; 43: 109-123PubMed Google Scholar At present, the precise role of each NOS isoform in pulmonary fibrotic disease is unknown, but immunohistochemical studies have shown that NOS II expression is increased whereas NOS III is decreased in patients with early- and intermediate-stage interstitial fibrotic disease.23Saleh D Barnes PJ Giaid A Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 1997; 155: 1763-1769Crossref PubMed Scopus (229) Google Scholar Furthermore, the by-product of protein nitration by nitric oxide and superoxide, nitrotyrosine, has also been detected in interstitial fibrotic lung disease.23Saleh D Barnes PJ Giaid A Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 1997; 155: 1763-1769Crossref PubMed Scopus (229) Google Scholar However, other studies suggest that exogenous nitric oxide has a beneficial role in idiopathic pulmonary fibrosis,24Yoshida M Taguchi O Gabazza EC Yasui H Kobayashi T Kobayashi H Maruyama K Adachi Y The effect of low-dose inhalation of nitric oxide in patients with pulmonary fibrosis.Eur Respir J. 1997; 10: 2051-2054Crossref PubMed Scopus (28) Google Scholar but only when it is administered in quantities that are similar to those generated by NOS I and III.25Maruyama K Kobayasi H Taguchi O Chikusa H Muneyuki M Higher doses of inhaled nitric oxide might be less effective in improving oxygenation in a patient with interstitial pulmonary fibrosis.Anesth Analg. 1995; 81: 210-211PubMed Google Scholar, 26Channick RN Hoch RC Newhart JW Johnson FW Smith CM Improvement in pulmonary hypertension and hypoxemia during nitric oxide inhalation in a patient with end-stage pulmonary fibrosis.Am J Respir Crit Care Med. 1994; 149: 811-814Crossref PubMed Scopus (49) Google Scholar Thus, nitric oxide may have dichotomous effects on the pulmonary interstitial fibrotic process that are related to the enzymatic source of this mediator.In this study, we have addressed the role of nitric oxide in the regulation of collagen deposition around PPD-bead pulmonary granulomas. In a previous study, we showed that NOS II was prominent in many cells in the lung by day 4 in the PPD-bead granuloma model, and the granulomatous response on day 4 was markedly augmented after the inhibition of nitric oxide synthesis.27Hogaboam CM Chensue SW Steinhauser ML Huffnagle GB Lukacs NW Strieter RM Kunkel SL Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide.J Immunol. 1997; 159: 5585-5593PubMed Google Scholar Furthermore, the cytokine and chemokine profile of dissociated lung cells fromNG-nitro-l-arginine-methyl ester (l-NAME)-treated, PPD-challenged mice reflected a cytokine and chemokine profile more in line with that observed during a granulomatous response to SEA-beads in SEA-sensitized mice.5Chensue SW Warmington K Ruth J Lincoln P Kuo M-C Kunkel SL Cytokine responses during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation: production of Th1 and Th2 cytokines and relative contribution of tumor necrosis factor.Am J Pathol. 1994; 145: 1105-1113PubMed Google Scholar As previous studies have shown that increased IL-4, MCP-1, and MIP-1α are associated with the development of experimental lung fibrosis,8Smith RE Strieter RM Phan SH Kunkel SL C-C chemokines: novel mediators of the profibrotic inflammatory response to bleomycin challenge.Am J Respir Cell Mol Biol. 1996; 15: 693-702Crossref PubMed Scopus (87) Google Scholar the specific aim of the present study was to determine whether the inhibition of nitric oxide would alter the resolution phase in the PPD-bead granuloma model and modify chemokine and chemokine receptor expression by isolated lung fibroblasts from mice with PPD-bead granulomas.Materials and MethodsPPD-Bead Pulmonary Granuloma ModelSpecific-pathogen free (SPF), female CBA/J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and were maintained under SPF conditions before and during experiments. All mice received a subcutaneous and intraperitoneal injection of complete Freund's adjuvant (CFA; Sigma Chemical Co., St. Louis, MO) diluted 1:1 with normal saline as previously described in detail.5Chensue SW Warmington K Ruth J Lincoln P Kuo M-C Kunkel SL Cytokine responses during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation: production of Th1 and Th2 cytokines and relative contribution of tumor necrosis factor.Am J Pathol. 1994; 145: 1105-1113PubMed Google Scholar Immediately after the injection of CFA, all mice were fed a chemically defined diet from Zeigler Bros. (Gardiners, PA) that was deficient in l-arginine. Previous investigators have used this solid pellet chow to effectively reduce baseline nitric oxide synthesis in mice.28Granger DL Hibbs JB Broadnax LM Urinary nitrate excretion in relation to murine macrophage activation: influence of dietary l-arginine and oral NG-monomethyl-l-arginine.J Immunol. 1991; 146: 1294-1302PubMed Google Scholar In the present study we observed that this diet did not alter the normal growth patterns of the mice over the entire course of the experiment, but it did markedly reduce detectable levels of urinary nitric oxide metabolites such as nitrite and nitrate (data not shown). Sixteen days after CFA sensitization, all mice received 3000 Sepharose 4B beads covalently coupled to purified protein derivative (PPD) from Mycobacterium sp. by intravenous injection. Introduction of PPD-coated beads in this manner ensures that the beads embolize to the lungs and elicit a granulomatous response. After the introduction of PPD-coated beads, the mice were divided into groups of five and received daily intraperitoneal injections of l-NAME, d-NAME, or aminoguanidine during the PPD-bead embolization period. All compounds were administered at a dose of 8 mg/kg as previously described.27Hogaboam CM Chensue SW Steinhauser ML Huffnagle GB Lukacs NW Strieter RM Kunkel SL Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide.J Immunol. 1997; 159: 5585-5593PubMed Google Scholar, 29Feder LS Stelts D Chapman RW Manfra D Crawley Y Jones H Minnicozzi M Fernandez X Paster T Egan RW Kreutner W Kung TT Role of nitric oxide on eosinophilic lung inflammation in allergic mice.Am J Respir Cell Mol Biol. 1997; 17: 436-442Crossref PubMed Scopus (100) Google Scholar l-NAME is a nondiscriminating inhibitor of all three isoforms of nitric oxide synthase, whereas d-NAME is a structural enantiomer of l-NAME that lacks nitric oxide synthase inhibitory actions.30Rees DD Palmer RMJ Shulz R Hodson HF Moncada S Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo.Br J Pharmacol. 1990; 101: 746-751Crossref PubMed Scopus (1841) Google Scholar Aminoguanidine has been used by a number of investigators to more selectively inhibit NOS II activity.29Feder LS Stelts D Chapman RW Manfra D Crawley Y Jones H Minnicozzi M Fernandez X Paster T Egan RW Kreutner W Kung TT Role of nitric oxide on eosinophilic lung inflammation in allergic mice.Am J Respir Cell Mol Biol. 1997; 17: 436-442Crossref PubMed Scopus (100) Google Scholar, 31Corbett JA McDaniel ML Selective inhibition of inducible nitric oxide synthase by aminoguanidine.Methods Enzymol. 1996; 268: 398-408Crossref PubMed Google Scholar, 32Ridger VC Pettipher ER Bryant CE Brain SD Effect of the inducible nitric oxide synthase inhibitors aminoguanidine and l-N6-(1-iminoethyl)lysine on zymosan-induced plasma extravasation in rat skin.J Immunol. 1997; 159: 383-390PubMed Google Scholar, 33Shiraishi T DeMeester SR Worrall NK Ritter JH Misko TP Ferguson Jr, TB Cooper JD Patterson GA Inhibition of inducible nitric oxide synthase ameliorates rat lung allograft rejection.J Thorac Cardiovasc Surg. 1995; 110: 1449-1460Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Aminoguanidine has also been shown to inhibit the induction of NOS II in the endotoxin-challenged lung.34Ruetten H Thiemermann C Prevention of the expression of inducible nitric oxide synthase by aminoguanidine or aminoethyl-isothiourea in macrophages and in the rat.Biochem Biophys Res Commun. 1996; 225: 525-530Crossref PubMed Scopus (80) Google Scholar l-NAME and aminoguanidine were used in the present study to determine the relative contribution of all of the isoforms nitric oxide synthase (ie, NOS I, II, and III) as compared with the inducible nitric oxide synthase (ie, NOS II) on the development of extracellular matrix around PPD-bead granulomas. Based on results obtained by monitoring urine nitrite/nitrate levels, it appeared that both compounds equally reduced systemic nitric oxide production during the 8-day treatment protocol (data not shown). All mice were maintained on the l-arginine-deficient diet throughout the 8 days of PPD-bead challenge, after which whole lungs were removed and fixed for morphometric analysis or cultured for assessment of cytokine and chemokine synthetic capacity.Morphometric Analysis and Masson Trichrome Staining of Collagen in Granulomatous LungsWhole lungs from d-NAME-, l-NAME-, or aminoguanidine-treated mice 8 days after PPD-bead administration were fully inflated by intratracheal administration of 4% paraformaldehyde. Lungs were then dissected out and fixed in fresh 4% paraformaldehyde for an additional 24 hours. Routine histological techniques were used to paraffin-embed this tissue, and 5-μm sections of whole lung were prepared for Masson trichrome staining as previously described.35Szapiel SV Elson NA Fulmer JD Hunninghake GW Crystal RG Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse.Am Rev Respir Dis. 1979; 120: 893-899PubMed Google Scholar At the conclusion of this staining protocol, lung sections were counterstained with Mayer's hemotoxylin (Mayer & Myles Laboratories, Coopersburg, PA) for the visualization and identification of nucleated cells composing the granulomas. Quantitative digital morphometric analysis of granulomas was performed using a protocol described in detail elsewhere.27Hogaboam CM Chensue SW Steinhauser ML Huffnagle GB Lukacs NW Strieter RM Kunkel SL Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide.J Immunol. 1997; 159: 5585-5593PubMed Google Scholar A minimum of 10 granulomas were analyzed in each whole-lung tissue section, and granulomas were considered in this analysis if a full cross section of the bead nidus was visible. Using the application program IP Lab Spectrum-R4, images of the granulomas were captured, and copies of the image were made. The color wavelengths of the copied image were transformed into digital readings, allowing for quantification of the various color wavelengths using pixels as the unit of measure. Using the original image for comparison, the color spectra of each copied image was then adjusted until the collagen (highlighted in the original image by Masson trichrome staining) was green while the remaining granuloma was black. Percent collagen was then calculated by dividing the total pixel area of the granuloma by the pixel area corresponding to collagen.Assessment of Cytokine and Chemokine Profiles from Dissociated Granulomatous LungsGranulomatous lungs from d-NAME-, l-NAME-, or aminoguanidine-treated mice were mechanically dissociated over steel mesh using a plunger from a 20-ml syringe. Red blood cells in the dissociated cell suspensions were lysed using a hypotonic lysing buffer (150 mmol/L NH4Cl, 10 mmol/L NaHCO3, 1 mmol/L EDTA) for 2 minutes at 4°C. The remaining cells were suspended in RPMI containing 10% fetal bovine serum and were added at a density of 4.0 × 105 cells/well in six-well tissue culture plates. The dispersed lung cells were then challenged with 3 μg/ml PPD for 36 hours at 37°C in a humidified CO2 incubator as previously described27Hogaboam CM Chensue SW Steinhauser ML Huffnagle GB Lukacs NW Strieter RM Kunkel SL Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide.J Immunol. 1997; 159: 5585-5593PubMed Google Scholar, 36Lukacs NW Chensue SW Strieter TM Warmington K Kunkel SL Inflammatory granuloma formation is mediated by TNF-α-inducible intercellular adhesion molecule-1.J Immunol. 1994; 152: 5883-5889PubMed Google Scholar before 1-ml aliquots were removed and stored at −20°C before ELISA analysis.Granuloma Lung Fibroblast CulturePulmonary granuloma fibroblasts from d-NAME- and l-NAME-treated mice were subsequently grown out from mixed lung cell primary cultures (see above), as previously described.37Lukacs NW Chensue SW Smith RE Strieter RM Warmington K Wilke C Kunkel SL Production of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 by inflammatory granuloma fibroblasts.Am J Pathol. 1994; 144: 711-718PubMed Google Scholar Briefly, these cells were transferred to 175-ml tissue culture flasks and were fed Dulbeco's modified Eagle's medium (DMEM) containing 1% (v/v) antibiotic/antimycotic and 15% (v/v) fetal bovine serum (FBS). After a minimum of three passages, homogeneous populations of fibroblasts were transferred to six-well tissue culture plates for experiments. Before use in any experiment, lung fibroblasts were transferred to two-well Labtek chamber culture slides and stained for α-actin, desmin, and α-naphthyl acetate esterase. After the third passage, lung fibroblasts stained for α-actin, suggesting a myofibroblast-type phenotype, but cultures of these cells were found to be completely free of α-naphthyl acetate esterase-positive cells such as macrophages (data not shown). After ensuring the homogeneity of fibroblast cultures, each well in a six-well tissue culture plate was initially seeded with approximately 1.0 × 106 fibroblasts. When cell confluence was reached, the DMEM was removed and IL-4 (R&D Systems, Minneapolis, MN) or IFN-γ (Genzyme, Cambridge, MA) suspended at 10 ng/ml in RPMI containing 10% FBS was added. Twenty-four hours later, cell-free supernatants were removed for ELISA measurements, and the adherent fibroblasts were washed and subjected to RNA isolation or prepared for flow cytometry. Cultured lung fibroblasts were used in these experiments up to the sixth passage.Cytokine and Chemokine MeasurementMurine IL-4, IL-10, IL-12, MCP-1, MIP-1α, eotaxin, and TGF-β levels were determined in 50-μl supernatant samples from dispersed lung cells or purified lung fibroblasts using a standardized sandwich ELISA as previously described.27Hogaboam CM Chensue SW Steinhauser ML Huffnagle GB Lukacs NW Strieter RM Kunkel SL Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide.J Immunol. 1997; 159: 5585-5593PubMed Google Scholar Briefly, Nunc-immuno ELISA plates (MaxiSorp) were coated with the appropriate cytokine capture antibody at a dilution of 1 μg/ml coating buffer (0.6 mol/L NaCl, 0.26 mol/L H3BO4, 0.08 mol/L NaOH, pH 9.6) for 16 hours at 4°C. Excess capture antibody was washed away and each plate was blocked for 90 minutes with 2% bovine serum albumin (BSA)/PBS at 37°C. After the blocking period, each ELISA plate was washed with PBS/Tween 20 (0.05%, v/v), and 50-μl samples (no dilution or 1:10) were added to wells in duplicate for 1 hour at 37°C. Recombinant murine IL-4, IL-10, IL-12, MCP-1, MIP-1α, eotaxin, and TGF-β standard curves were used to calculate cytokine concentrations. The plates were then thoroughly washed, and the appropriate biotinylated polyclonal rabbit anti-cytokine antibody (3.5 μg/" @default.
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• W1993151719 date "1998-12-01" @default.
• W1993151719 modified "2023-10-17" @default.
• W1993151719 title "Collagen Deposition in a Non-Fibrotic Lung Granuloma Model after Nitric Oxide Inhibition" @default.
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