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• W2052842832 abstract "Chronic pulmonary inflammation and infection are the leading causes of morbidity and mortality in cystic fibrosis (CF). While the effect of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on airways remains controversial, some groups have demonstrated increases in Na+ and Cl− in CF airway surface liquid compared to normal airways. We investigated the consequences of NaCl on pro-inflammatory chemokine and cytokine production by macrophages. Stimulation of mouse macrophages with increasing amounts of NaCl induced macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) production. Further, co-incubation of macrophages with NaCl in the presence of either lipopolysaccharide (LPS) or TNF-α synergistically increased MIP-2 production. Both the NaCl and NaCl plus LPS responses were partially dependent on endogenous production and autocrine signaling by TNF-α. To investigate the role of CFTR in MIP-2 production, we compared the responses of wild-type and ΔF508 CF mouse macrophages to NaCl and LPS. The responses of macrophages from both strains were indistinguishable. In addition, CFTR mRNA was not expressed in macrophages. Taken together, these findings suggest that NaCl stimulates MIP-2 production by macrophages through a mechanism that is partially dependent on TNF-α but independent of macrophage CFTR expression. Chronic pulmonary inflammation and infection are the leading causes of morbidity and mortality in cystic fibrosis (CF). While the effect of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on airways remains controversial, some groups have demonstrated increases in Na+ and Cl− in CF airway surface liquid compared to normal airways. We investigated the consequences of NaCl on pro-inflammatory chemokine and cytokine production by macrophages. Stimulation of mouse macrophages with increasing amounts of NaCl induced macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) production. Further, co-incubation of macrophages with NaCl in the presence of either lipopolysaccharide (LPS) or TNF-α synergistically increased MIP-2 production. Both the NaCl and NaCl plus LPS responses were partially dependent on endogenous production and autocrine signaling by TNF-α. To investigate the role of CFTR in MIP-2 production, we compared the responses of wild-type and ΔF508 CF mouse macrophages to NaCl and LPS. The responses of macrophages from both strains were indistinguishable. In addition, CFTR mRNA was not expressed in macrophages. Taken together, these findings suggest that NaCl stimulates MIP-2 production by macrophages through a mechanism that is partially dependent on TNF-α but independent of macrophage CFTR expression. Cystic fibrosis (CF) is the most common fatal hereditary disorder of the Caucasian population and is the result of the inheritance of mutations in the gene encoding CF transmembrane conductance regulator (CFTR). While many organs are affected in CF, the exuberant inflammatory response and chronic bacterial infections of the airways are major causes of morbidity and mortality.1Rosenstein BJ What is a cystic fibrosis diagnosis?.Clin Chest Med. 1998; 19: 423-441Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar In seeking to investigate the mechanism of CFTR involvement in the pathogenesis of lung disease in CF, we and others have shown that pulmonary inflammation is present in infants as early as 4 weeks after birth.2Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DW Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar Furthermore, in more than half of the infants studied, pulmonary inflammation was present even in the absence of detectable infection with common CF pathogens.2Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DW Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar These findings led us to propose that inactivation of CFTR function contributes to the initiation of early pulmonary inflammation in CF.2Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DW Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar, 3Riches DWH Lenfant C Pulmonary Inflammation in Cystic Fibrosis: Mechanisms and Consequences. Marcel Dekker, Inc., New York2003: 113-144Google Scholar Several hypotheses have been proposed to explain how mutations in the CFTR, a transmembrane protein that maintains airway surface liquid (ASL) function by regulating electrolyte transport across pulmonary epithelial cells,4Rich DP Anderson MP Gregory RJ Cheng SH Paul S Jefferson DM McCann JD Klinger KW Smith AE Welsh MJ Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.Nature. 1990; 347: 358-363Crossref PubMed Scopus (487) Google Scholar contribute to airway disease in CF. Based on measurements of the elemental composition of ASL obtained from CF patients and healthy subjects, together with in vitro and in vivo models using bronchial epithelial cells, one hypothesis proposes that mutations in CFTR cause abnormalities in electrolyte transport across airway epithelia leading to elevations in NaCl concentration in ASL.5Gilljam H Ellin A Strandvik B Increased bronchial chloride concentration in cystic fibrosis.Scand J Clin Lab Invest. 1989; 49: 121-124Crossref PubMed Scopus (76) Google Scholar, 6Smith JJ Travis SM Greenberg EP Welsh MJ Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid.Cell. 1996; 85: 229-236Abstract Full Text Full Text PDF PubMed Scopus (899) Google Scholar, 7Goldman MJ Anderson GM Stolzenberg ED Kari UP Zasloff M Wilson JM Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis.Cell. 1997; 88: 553-560Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar, 8Zabner J Smith JJ Karp PH Widdicombe JH Welsh MJ Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro.Mol Cell. 1998; 2: 397-403Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar Other studies, however, have failed to detect differences in NaCl concentrations in ASL.9Hull J Skinner W Robertson C Phelan P Elemental content of airway surface liquid from infants with cystic fibrosis.Am J Respir Crit Care Med. 1998; 157: 10-14Crossref PubMed Scopus (77) Google Scholar, 10Jayaraman S Song Y Vetrivel L Shankar L Verkman AS Noninvasive in vivo fluorescence measurement of airway-surface liquid depth, salt concentration, and pH.J Clin Invest. 2001; 107: 317-324Crossref PubMed Scopus (168) Google Scholar, 11Matsui H Grubb BR Tarran R Randell SH Gatzy JT Davis CW Boucher RC Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease.Cell. 1998; 95: 1005-1015Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar, 12Knowles MR Robinson JM Wood RE Pue CA Mentz WM Wager GC Gatzy JT Boucher RC Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects.J Clin Invest. 1997; 100: 2588-2595Crossref PubMed Scopus (240) Google Scholar These studies have led to a second hypothesis in which mutations in CFTR promote increased absorption of isotonic ASL leading to dehydration and volume reduction of the ASL and impaired mucociliary clearance.13Guggino WB Cystic fibrosis and the salt controversy.Cell. 1999; 96: 607-610Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar Both hypotheses support the view that the ASL is abnormal in CF and arises as a consequence of the loss of CFTR activity. Both hypotheses have also focused attention on the mechanism by which abnormalities in ASL composition affect the function of airway epithelial cells and macrophages in inflammation and innate immunity in CF. In addition to expression by epithelial cells, sporadic reports have suggested that CFTR is expressed by macrophages and negatively regulates macrophage tumor necrosis factor-α (TNF-α) production. Using nonquantitative polymerase chain reaction (PCR), Yoshimura et al14Yoshimura K Nakamura H Trapnell BC Chu CS Dalemans W Pavirani A Lecocq JP Crystal RG Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin.Nucleic Acids Res. 1991; 19: 5417-5423Crossref PubMed Scopus (134) Google Scholar first reported that CFTR mRNA is expressed by human alveolar macrophages. Pfeffer et al15Pfeffer KD Huecksteadt TP Hoidal JR Expression and regulation of tumor necrosis factor in macrophages from cystic fibrosis patients.Am J Respir Cell Mol Biol. 1993; 9: 511-519Crossref PubMed Scopus (54) Google Scholar showed that monocyte-derived macrophages from CF patients express higher levels of TNF-α than macrophages from normal subjects following lipopolysaccharide (LPS) stimulation. Similar findings were reported by Thomas et al16Thomas GR Costelloe EA Lunn DP Stacey KJ Delaney SJ Passey R McGlinn EC McMorran BJ Ahadizadeh A Geczy CL Wainwright BJ Hume DA G551D cystic fibrosis mice exhibit abnormal regulation of inflammation in lungs and macrophages.J Immunol. 2000; 164: 3870-3877Crossref PubMed Scopus (47) Google Scholar using macrophages from G551D CF mice. Taken together, these findings suggest that macrophages express CFTR mRNA and that loss of CFTR functional activity is associated with increased sensitivity to LPS. Given that mutations in CFTR may result in increased NaCl concentrations in the ASL of CF patients and that macrophages are an important source of pro-inflammatory chemokines and cytokines, we investigated the consequences of elevated NaCl on the production of pro-inflammatory cytokines by macrophages and the role of CFTR in this response. Here we show that NaCl stimulates the production of macrophage inflammatory protein-2 (MIP-2) by macrophages and amplifies MIP-2 production in response to LPS via a mechanism that is partially dependent on endogenous TNF-α production but independent of CFTR expression. These findings suggest that CFTR function is not required in the response of macrophages to either NaCl or LPS and favor a model in which macrophages respond to the abnormal environment of the lung created by loss of CFTR activity in other cell types. Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum were purchased from BioWhittaker (Walkersville, MD) and Atlanta Biologicals (Norcross, GA), respectively. Recombinant mouse TNF-α was purchased from R&D Systems, Inc. (Minneapolis, MN). All enzyme-linked immunosorbent assay (ELISA) kits were purchased from ELISA Tech (Aurora, CO). NaCl, mannitol, sodium gluconate, choline chloride, and Salmonella typhimurium-derived LPS were all obtained from Sigma-Aldrich Co. (St. Louis, MO). Sorbitol was obtained from EM Science (Gibbstown, NJ). C57Bl/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). tnf-α−/− mice were generously donated by Dr. Jack Routes (National Jewish Medical and Research Center, Denver, CO). tnf-r1/r2−/− mice and their C57Bl/6 × 129 controls were generously donated by Dr. Peter Henson (National Jewish Medical and Research Center). All mice were housed at the accredited National Jewish Medical and Research Center Biological Resource Center. ΔF508 mice, originally generated by Dr. William Colledge (University of Cambridge, Cambridge, UK),17Colledge WH Abella BS Southern KW Ratcliff R Jiang C Cheng SH MacVinish LJ Anderson JR Cuthbert AW Evans MJ Generation and characterization of a delta F508 cystic fibrosis mouse model.Nat Genet. 1995; 10: 445-452Crossref PubMed Scopus (193) Google Scholar and their C57Bl/6 wild-type controls were housed at the Health Science Research Facility at the University of Vermont (Burlington, VT). Monolayers of mouse bone marrow-derived macrophages were prepared as previously described.18Riches DW Underwood GA Expression of interferon-beta during the triggering phase of macrophage cytocidal activation. Evidence for an autocrine/paracrine role in the regulation of this state.J Biol Chem. 1991; 266: 24785-24792Abstract Full Text PDF PubMed Google Scholar Briefly, bone marrow cells from the tibias, femurs, and pelvises of mice were flushed with, and grown in, DMEM supplemented with 2 mmol/L l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% (v/v) fetal bovine serum, and 10% (v/v) L929 cell-conditioned medium, as a source of macrophage-colony stimulating factor. The bone marrow cells were seeded in 12-well culture dishes at 105 cells/well with 2 ml of medium/well. Macrophages were then cultured at 37°C under a 10% (v/v) CO2 atmosphere for 5 to 7 days with feeding after 5 days. ELISAs were used to determine the concentration of MIP-2 and TNF-α in cell culture supernatants of bone marrow-derived macrophages. The ELISA assays were performed according to the manufacturer's instructions. The sensitivity of both assays was <10 pg/ml. All ELISA data were normalized to cell count obtained using a Coulter Counter Model ZM from Coulter Corporate Communications (Hialeah, FL). Total RNA was isolated from bone marrow-derived macrophages, whole kidney, whole lung, and bronchoalveolar lavage cells using the TRIzol reagent (Life Technologies, Grand Island, NY). RT was performed on 1 μg of total RNA with random hexamers in a 50-μl reaction using the TaqMan RT reagents (Applied Biosystems, Foster City, CA). Real-time PCR was performed on the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). The 50-μl PCR reactions for CFTR contained either 60 or 100 ng of cDNA, 200 nmol/L fluorogenic probe, 500 nmol/L of each primer, and the other components within the TaqMan Universal PCR Master Mix. These probe and primer concentrations were determined following a rigorous optimization protocol. Three divergent regions of the cftr gene were examined by PCR to detect potential splice variants or differences in RT and/or PCR efficiency. The probes, labeled on the 5′-end with the reporter dye 6-carboxyfluorescein and on the 3′-end with the quencher dye 6-carboxytetramethylrhodamine, all spanned an exon-exon junction. The corresponding primers were thus in separate but adjacent exons. Table 1 shows the sequence (accession number M60493) and location of the probes and primers used to detect CFTR mRNA. The specificity of PCR was verified by no signal being present in the no-template and no-RT controls. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was evaluated using a PCR protocol similar to CFTR with the exceptions being that the probe concentration was 200 nmol/L, the primer concentration was 100 nmol/L, and the 5′ label was 6-carboxyrhodamine. The GAPDH probe and primer set were the TaqMan Rodent GAPDH Control Reagents part number 4308313 (Applied Biosystems). The threshold cycle (CT) was recorded for each sample. The relative CFTR mRNA expression levels were determined using the comparative CT method. The ΔCT (CFTR average CT − GAPDH average CT) was calculated. Next, the comparative difference in CFTR expression between the kidney, bronchoalveolar lavage cells, or lung and macrophages, ΔΔCT (ΔCT sample − ΔCT macrophage) was calculated. Finally, the relative-fold difference was determined by using the term, 2−ΔΔCT.Table 1Primers and Probes for Detection of CFTR by Real-Time PCRPrimers Exon 55′-TGGACAACTTGTTAGTCTTCTTTCCA-3′ Exon 65′-CCATCAGAAGAGTCACTTGTAAAGGA-3′ Exon 115′-GGATTATGCCGGGTACTATCAAAG-3′ Exon 125′-CTCCAAGAACTGTGTTGTCTTGTTC-3′ Exon 195′-TACTGAGGGCCTACTTCCTTCATAC-3′ Exon 205′-GTCCAGAGTCCTTTTAAGCTTGTCA-3′Probes Exon 5/65′-FAM-TGTGCCAAGGCAAGTCCTTCATCAAATTT-TAMRA-3′ Exon 11/125′-FAM-TGCCAACTACAGCAGGACATCACCAAGTT-TAMRA-3′ Exon 19/205′-FAM-TGAAAATTGGACTCCTGCCTTCAGATTCC-TAMRA-3′ Open table in a new tab Results are expressed as mean ± SE. Statistical analyses were performed using InStat version 3.0b for Macintosh (GraphPad, San Diego, CA). In the CF and non-CF mouse studies, statistical analyses were done using SAS version 8 (SAS Institute, Cary, NC). We used a two-way analysis of variance with preplanned contrasts to analyze MIP-2 (normalized to cell count) between CF and control. Before the actual analysis, we transformed MIP-2 normalized to cell count using log base 10 to stabilize the variance as described.19Curran-Everett D Taylor S Kafadar K Fundamental concepts in statistics: elucidation and illustration.J Appl Physiol. 1998; 85: 775-786PubMed Google Scholar To examine the effect of NaCl on MIP-2 production by macrophages, we incubated monolayers of C57Bl/6 bone marrow-derived macrophages with increasing amounts of NaCl (0 to 100 mmol/L; 315 to 490 mOsm/kg H2O) for 24 hours and quantified MIP-2 levels in the culture supernatants by ELISA. Addition of NaCl to the culture media yielded osmolalities as shown in Table 2. The range of NaCl concentrations was chosen to span the range of osmolalities and Na+ and Cl− concentrations reported in ASL from normal subjects and CF patients.6Smith JJ Travis SM Greenberg EP Welsh MJ Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid.Cell. 1996; 85: 229-236Abstract Full Text Full Text PDF PubMed Scopus (899) Google Scholar, 7Goldman MJ Anderson GM Stolzenberg ED Kari UP Zasloff M Wilson JM Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis.Cell. 1997; 88: 553-560Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar, 20Joris L Dab I Quinton PM Elemental composition of human airway surface fluid in healthy and diseased airways.Am Rev Respir Dis. 1993; 148: 1633-1637Crossref PubMed Google Scholar, 21Shapiro L Dinarello CA Hyperosmotic stress as a stimulant for proinflammatory cytokine production.Exp Cell Res. 1997; 231: 354-362Crossref PubMed Scopus (121) Google Scholar As shown in Figure 1A, unstimulated macrophages did not produce detectable amounts of MIP-2. Incubation with NaCl resulted in a dose-dependent significant increase (P < 0.05) in MIP-2 production beginning at 385 mOsm. The increase in MIP-2 production by NaCl was not due to contaminating LPS as stimulation of mouse macrophages from LPS-resistant C3H/HeJ mice yielded similar results (data not shown). To determine whether the increased production of MIP-2 was due to a general effect of hyperosmolarity, macrophages were exposed to either sorbitol or mannitol at the same osmolalities achieved with NaCl (Table 2). In contrast to NaCl, sorbitol and mannitol had a negligible effect on MIP-2 production (Figure 1A). These data indicate that the ability of NaCl to increase MIP-2 production by mouse macrophages is selective to NaCl and is not due to a general response to hyperosmolarity.Table 2Osmolalities and Corresponding Concentrations of Added OsmolitesmOsm/kgH2ONaCl (mmol/L)Sorbitol (mmol/L)Mannitol (mmol/L)Na− gluconate (mmol/L)Choline Cl− (mmol/L)3150 (100)000035020 (120)4040202038540 (140)8080404042060 (160)120120606045580 (180)1601608080490100 (200)200200100100Stimuli were prepared by adding the osmolites, at the concentrations shown, to DMEM. In the case of NaCl, the figures in parentheses represent the total concentration of NaCl in DMEM plus the added NaCl. The osmolality of each solution, including untreated DMEM, was measured by osmometry. Open table in a new tab Stimuli were prepared by adding the osmolites, at the concentrations shown, to DMEM. In the case of NaCl, the figures in parentheses represent the total concentration of NaCl in DMEM plus the added NaCl. The osmolality of each solution, including untreated DMEM, was measured by osmometry. Next, we determined whether Na+, Cl−, or both ions were necessary for maximal NaCl-induced MIP-2 production. Macrophages were incubated with increasing concentrations of Na+ gluconate or choline Cl− for 24 hours, and MIP-2 levels were measured in the culture supernatants. As can be seen in Figure 1B, at osmolalities below 450 mOsm, Na+ gluconate and choline Cl− significantly increased (P < 0.05) MIP-2 production to a similar extent as NaCl. At osmolalities above 450 mOsm, the level of MIP-2 produced by each ion was approximately half that seen from when both ions were present. Thus, although Na+ or Cl− alone can increase MIP-2, both are required for maximal MIP-2 production. While CF patients exhibit basal airway inflammation, infection with common gram-negative bacteria greatly augments the inflammatory response of the airways.2Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DW Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar To model the effects of gram-negative bacteria, we determined the effect of NaCl on macrophage MIP-2 production in the presence of LPS. Macrophage monolayers were incubated for 24 hours with increasing amounts of NaCl (0 to 100 mmol/L; 315 to 490 mOsm/kg H2O) in the presence or absence of LPS (1 ng/ml). Additionally, we determined the effect of NaCl on MIP-2 production in response to TNF-α (10 ng/ml). As shown in Figure 2A, while treatment with either LPS or TNF-α in medium alone (315 mOsm/kg H2O) induced significant (P < 0.05) MIP-2 production by macrophages (LPS: 17.13 ± 2.91, TNF-α: 2.07 ± 0.25 compared to unstimulated: 0.30 ± 0.11 ng MIP-2/106 cells), NaCl synergistically increased MIP-2 production in response to both stimuli. To determine whether the synergistic increase in MIP-2 production by NaCl was representative of a more general response to hyperosmolarity, macrophages were exposed to sorbitol at identical osmolalities to that of NaCl either alone or with LPS (1 ng/ml) or TNF-α (10 ng/ml) for 24 hours before measurement of MIP-2 levels in culture supernatants. As shown in Figure 2B, the effect of sorbitol on either LPS or TNF-α-induced MIP-2 production was negligible. Collectively, these data indicate that NaCl greatly augments LPS- and TNF-α-induced MIP-2 production by macrophages and that this effect is not simply due to hyperosmolarity. Many pro-inflammatory cytokines, including MIP-2, have been shown to be induced by TNF-α.22Calkins CM Heimbach JK Bensard DD Song Y Raeburn CD Meng X McIntyre Jr, RC TNF receptor I mediates chemokine production and neutrophil accumulation in the lung following systemic lipopolysaccharide.J Surg Res. 2001; 101: 232-237Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 23Czermak BJ Sarma V Bless NM Schmal H Friedl HP Ward PA In vitro and in vivo dependency of chemokine generation on C5a and TNF-alpha.J Immunol. 1999; 162: 2321-2325PubMed Google Scholar For this reason, we investigated the hypothesis that NaCl increases MIP-2 production in a TNF-α-dependent fashion. First, we determined if NaCl increased TNF-α production. Monolayers of C57Bl/6 bone marrow-derived macrophages were incubated with increasing amounts of NaCl (0 to 100 mmol/L; 315 to 490 mOsm/kg H2O) for 24 hours, and TNF-α levels were measured in culture supernatants by ELISA. As shown in Figure 3A, NaCl significantly (P < 0.05) increased TNF-α production. Further, the increase in TNF-α production qualitatively paralleled that of MIP-2 (Figure 1A). Second, to determine the role of endogenously produced TNF-α on MIP-2 production, we investigated the effect of NaCl on macrophages from tnf-α−/− and tnf-r1/r2−/− mice. Macrophages from tnf-α−/− and tnf-r1/r2−/− mice and their wild-type controls (C57Bl/6 and C57Bl/6 × 129, respectively) were incubated with increasing amounts of NaCl (0 to 100 mmol/L; 315 to 490 mOsm/kg H2O) for 24 hours, and the amount of MIP-2 in the culture supernatants was determined. Both the inability of macrophages to produce TNF-α (Figure 3B) and the inability to respond to TNF-α (Figure 3C) resulted in a significant (P < 0.05), though incomplete, inhibition of MIP-2 production in response to NaCl. Similar experiments were performed on C57Bl/6 macrophages in the presence of an anti-TNF-α neutralizing antibody. Here, too, MIP-2 production was significantly (P < 0.05) but incompletely inhibited (data not shown). Collectively, these findings suggest that the production of MIP-2 in response to NaCl is partially dependent on endogenously produced TNF-α via signaling through TNF-R1 and/or TNF-R2. Next, we investigated the role of autocrine signaling by TNF-α on the augmentation of LPS-induced MIP-2 production by NaCl. To determine whether NaCl potentiated TNF-α production by LPS, we exposed C57Bl/6 macrophages to increasing amounts of NaCl (0 to 100 mmol/L; 315 to 490 mOsm/kg H2O) in the presence of LPS (1 ng/ml) for 24 hours and then measured the amount of TNF-α present in the culture supernatants. As shown in Figure 4A, macrophages produced ∼1 ng of TNF-α/106 cells following stimulation with LPS alone, whereas incubation with LPS in the presence of NaCl resulted in a significant (P < 0.05) potentiation of TNF-α production. To directly evaluate the role of autocrine-signaling by TNF-α in the synergistic increase of MIP-2 production by NaCl and LPS, macrophage monolayers from tnf-α−/− and tnf-r1/r2−/− mice and their wild-type controls (C57Bl/6 and C57Bl/6 × 129, respectively) were incubated with increasing concentrations of NaCl in the presence of LPS (1 ng/ml) for 24 hours before determination of MIP-2 levels. Compared to wild-type controls, macrophages from tnf-α−/− (Figure 4B) and tnf-r1/r2−/− (Figure 4C) produced less MIP-2 in response to LPS alone. Strikingly, the potentiating effect of NaCl on LPS-induced MIP-2 production was significantly (P < 0.05) though incompletely reduced in macrophages from both strains of mutant mice. Similar results were obtained with wild-type macrophages when endogenous TNF-α activity was blocked with a neutralizing antibody (data not shown). Thus, autocrine signaling by TNF-α also contributes to the NaCl-induced potentiation of MIP-2 production in response to LPS. Given our findings that macrophages produce increased amounts of MIP-2 in response to NaCl and the reports suggesting that macrophages express CFTR, we next determined if CFTR activity was involved in the regulation of MIP-2 production.14Yoshimura K Nakamura H Trapnell BC Chu CS Dalemans W Pavirani A Lecocq JP Crystal RG Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin.Nucleic Acids Res. 1991; 19: 5417-5423Crossref PubMed Scopus (134) Google Scholar To this end we used ΔF508 CF mice as this is the most common mutation in CF, occurring in ∼70% of patients,24Kerem B Rommens JM Buchanan JA Markiewicz D Cox TK Chakravarti A Buchwald M Tsui LC Identification of the cystic fibrosis gene: genetic analysis.Science. 1989; 245: 1073-1080Crossref PubMed Scopus (3224) Google Scholar to investigate the production of MIP-2 by macrophages. Macrophage monolayers from ΔF508 and C57Bl/6 wild-type littermate control mice were incubated in medium alone, NaCl (450 mOsm), LPS (1 ng/ml), or both LPS and NaCl together for 24 hours before measurement of MIP-2 accumulation in the culture supernatants. Figure 5 shows that macrophages from wild-type and ΔF508 CF mice produced indistinguishable amounts of MIP-2 in response to all stimuli tested. Analysis of TNF-α production by ELISA assay also demonstrated no difference in the responses of ΔF508 and C57Bl/6 macrophages (data not shown). These findings indicate that the production of MIP-2 induced by NaCl and LPS, as well as the synergistic increase in chemokine production observed following costimulation with LPS and NaCl, occurs independently of CFTR activity in macrophages. Based on these findings, we next determined if CFTR mRNA was expressed by mouse macrophages. Kidney and lung mRNAs were used as positive controls for CFTR expression.25Morales MM Carroll TP Morita T Schwiebert EM Devuyst O Wilson PD Lopes AG Stanton BA Dietz HC Cutting GR Guggino WB Both the wild type and a functional isoform of CFTR are expressed in kidney.Am J Physiol. 1996; 270: F1038-F1048PubMed Google Scholar Real-time PCR probes were designed to overlap exon-exon boundaries to negate amplification of genomic DNA. No CFTR was detected in samples that did not receive reverse transcriptase as part of their preparation, indicating a lack of contamination with genomic DNA (data not shown). GAPDH was also amplified to verify the integrity of the RNA/cDNA and to provide a means of controlling for variations in total RNA between samples. The maximum number of amplification cycles used to detect mRNA expression was 40. Therefore, the CT of any samples in which the mRNA could not be detected was reported as >40. Table 3 shows that, although CFTR mRNA was readily detected in kidney and lung, it was not detected in macrophages using any of the three primer pairs and probe sets tested. Minimally, kidney cells express 1600- to 3100-fold more CFTR mRNA than macrophages. Lack of detection of CFTR mRNA in macrophages was not a result of degradation of the RNA as GAPDH expression was detected in samples of macrophage RNA and in equivalent levels to that of kidney. We also investigated CFTR mRNA expression in mouse alveolar macrophages. Although CFTR mRNA was detected in RNA isolated from the lungs of BALB/c mice, it was not detected in alveolar macrophages (Table 3).Table 3CFTR Expression by Real-Time PCRCFTR primer pair and probe setCFTR average CTGAPDH average CTExpression level of CFTR relative to macrophageMacrophageExon 5–6>4017.00ND*ND, not detected.Exon 11–12>4017.00NDExon 19–20>4017.00NDKidneyExon 5–629.1316.85>1686Exon 11–1228.2416.85>3114Exon 19–2028.9416.85>1917Alveolar macrophageExon 5–6>4023.03ND" @default.
• W2052842832 created "2016-06-24" @default.
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• W2052842832 date "2006-07-01" @default.
• W2052842832 modified "2023-10-17" @default.
• W2052842832 title "Regulation of Chemokine Expression by NaCl Occurs Independently of Cystic Fibrosis Transmembrane Conductance Regulator in Macrophages" @default.
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• W2052842832 doi "https://doi.org/10.2353/ajpath.2006.051042" @default.
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