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• W2051363313 abstract "Alveolar macrophages and newly recruited monocytes are targets of infection by Mycobacterium tuberculosis. Therefore, we examined the expression of interferon regulatory factor 1 (IRF-1), which plays an important role in host defense against M. tuberculosis, in undifferentiated and differentiated cells. Infection induced IRF-1 in both. IRF-1 from undifferentiated, uninfected monocytic cell lines was modified during extraction to produce specific species that were apparently smaller than intact IRF-1. After infection by M. tuberculosis or differentiation, intact IRF-1 was recovered. Subcellular fractions were assayed for the ability to modify IRF-1 or inhibit its modification. A serine protease on the cytoplasmic surface of an organelle or vesicle in the “lysosomal/mitochondrial” fraction from undifferentiated cells was responsible for the modification of IRF-1. Thus, the simplest explanation of the modification is cleavage of IRF-1 by the serine protease. Recovery of intact IRF-1 correlated with induction of a serine protease inhibitor that was able to significantly reduce the modification of IRF-1. The inhibitor was present in the cytoplasm ofM. tuberculosis-infected or -differentiated cells. It is likely that induction of both IRF-1 and the serine protease inhibitor in response to infection by M. tuberculosis represent host defense mechanisms. Alveolar macrophages and newly recruited monocytes are targets of infection by Mycobacterium tuberculosis. Therefore, we examined the expression of interferon regulatory factor 1 (IRF-1), which plays an important role in host defense against M. tuberculosis, in undifferentiated and differentiated cells. Infection induced IRF-1 in both. IRF-1 from undifferentiated, uninfected monocytic cell lines was modified during extraction to produce specific species that were apparently smaller than intact IRF-1. After infection by M. tuberculosis or differentiation, intact IRF-1 was recovered. Subcellular fractions were assayed for the ability to modify IRF-1 or inhibit its modification. A serine protease on the cytoplasmic surface of an organelle or vesicle in the “lysosomal/mitochondrial” fraction from undifferentiated cells was responsible for the modification of IRF-1. Thus, the simplest explanation of the modification is cleavage of IRF-1 by the serine protease. Recovery of intact IRF-1 correlated with induction of a serine protease inhibitor that was able to significantly reduce the modification of IRF-1. The inhibitor was present in the cytoplasm ofM. tuberculosis-infected or -differentiated cells. It is likely that induction of both IRF-1 and the serine protease inhibitor in response to infection by M. tuberculosis represent host defense mechanisms. 12-O-tetradecanoylphorbol 13-acetate conditioned media electrophoretic mobility shift assay interferon interferon regulatory factor 1 M. bovis Bacille de Calmette-Guérin multiplicity of infection plasminogen activator inhibitor 2 phosphate-buffered saline phenylmethylsulfonyl fluoride secretory leukocyte protease inhibitor Tuberculosis begins with inhalation of Mycobacterium tuberculosis and infection of resident alveolar macrophages. Inflammation induced by M. tuberculosis then recruits monocytes (1Schmitt E. Meuret G. Stix L. Br. J. Haematol. 1977; 35: 11-17Crossref PubMed Scopus (36) Google Scholar), which also face infection in alveoli. The response of monocytes to M. tuberculosis is likely to differ from that of macrophages due to changes that occur during monocyte to macrophage differentiation. To date, few studies have compared infection of monocytes and macrophages. The human THP-1 cell line is a well established model system for such studies (2Tsuchiya S. Kobayashi Y. Goto Y. Okumura H. Nakae S. Konno T. Tada K. Cancer Res. 1982; 42: 1530-1536PubMed Google Scholar, 3Tsuchiya S. Yamabe M. Yamaguchi Y. Kobayashi Y. Konno T. Tada K. Int. J. Cancer. 1980; 26: 171-176Crossref PubMed Scopus (1677) Google Scholar, 4Auwerx J. Experientia. 1991; 47: 22-31Crossref PubMed Scopus (658) Google Scholar). Growing THP-1 cells are monocyte-like, but they stop proliferating and differentiate to a macrophage-like state when treated with 12-O-tetradecanoylphorbol 13-acetate (TPA).1 One useful way to compare the two cell types is by examining the expression of the transcription factor interferon regulatory factor 1 (IRF-1), since it plays an important role in host defense against mycobacteria. For example, mice having a null mutation in the IRF-1 gene are susceptible to the normally non-pathogenic Mycobacterium bovis Bacille de Calmette-Guérin (M. bovis BCG) (5Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D., Le, J. Koh S.I. Kimura T. Green S.J. Mak T.W. Taniguchi T. Vilcek J. Science. 1994; 263: 1612-1615Crossref PubMed Scopus (785) Google Scholar) and are more susceptible than wild-type mice to infection by M. tuberculosis (6Cooper A.M. Pearl J.E. Brooks J.V. Ehlers S. Orme I.M. Infect. Immun. 2000; 68: 6879-6882Crossref PubMed Scopus (118) Google Scholar). IRF-1 is induced by many cytokines. It was purified, and its gene was cloned in the course of studies on induction of type I interferons (IFNs) (IFNα and IFNβ) by virus infection (7Miyamoto M. Fujita T. Kimura Y. Maruyama M. Harada H. Sudo Y. Miyata T. Taniguchi T. Cell. 1988; 54: 903-913Abstract Full Text PDF PubMed Scopus (794) Google Scholar) and on induction of gene expression in response to IFNα (8Pine R. Decker T. Kessler D.S. Levy D.E. Darnell J.E., Jr. Mol. Cell. Biol. 1990; 10: 2448-2457Crossref PubMed Scopus (277) Google Scholar). However, type II IFN (IFNγ) is far more potent than IFNα as an inducer of IRF-1, and virus infection is a poor inducer (8Pine R. Decker T. Kessler D.S. Levy D.E. Darnell J.E., Jr. Mol. Cell. Biol. 1990; 10: 2448-2457Crossref PubMed Scopus (277) Google Scholar). Thus, it is not surprising that disruption of the IRF-1 gene prevents induction of some IFNγ-regulated genes, including inducible nitric-oxide synthase (5Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D., Le, J. Koh S.I. Kimura T. Green S.J. Mak T.W. Taniguchi T. Vilcek J. Science. 1994; 263: 1612-1615Crossref PubMed Scopus (785) Google Scholar), but has little effect on viral induction of type I IFN genes or induction of gene expression by IFNα (9Kimura T. Nakayama K. Penninger J. Kitagawa M. Harada H. Matsuyama T. Tanaka N. Kamijo R. Vilcek J. Mak T.W. Taniguchi T. Science. 1994; 264: 1921-1924Crossref PubMed Scopus (266) Google Scholar, 10Reis L.F.L. Ruffner H. Stark G. Aguet M. Weissmann C. EMBO J. 1994; 13: 4798-4806Crossref PubMed Scopus (167) Google Scholar, 11Matsuyama T. Kimura T. Kitagawa M. Pfeffer K. Kawakami T. Watanabe N. Kündig T.M. Amakawa R. Kishihara K. Wakeham A. Potter J. Furlonger C.L. Narendran A. Suzuki H. Ohashi P.S. Paige C.J. Taniguchi T. Mak T.W. Cell. 1993; 75: 83-97Abstract Full Text PDF PubMed Scopus (558) Google Scholar). The susceptibility of IRF-1 null mutant mice to mycobacterial infection may be due to the resultant disruption of the normal pathway of response to IFNγ, since null mutations in IFNγ, its receptor, or inducible nitric-oxide synthase also increase susceptibility to mycobacteria (5Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D., Le, J. Koh S.I. Kimura T. Green S.J. Mak T.W. Taniguchi T. Vilcek J. Science. 1994; 263: 1612-1615Crossref PubMed Scopus (785) Google Scholar, 12Cooper A.M. Dalton D.K. Stewart T.A. Griffin J.P. Russell D.G. Orme I.M. J. Exp. Med. 1993; 178: 2243-2247Crossref PubMed Scopus (1697) Google Scholar, 13Flynn J.L. Chan J. Triebold K.J. Dalton D.K. Stewart T.A. Bloom B.R. J. Exp. Med. 1993; 178: 2249-2254Crossref PubMed Scopus (2038) Google Scholar). However, IRF-1 might also play a role independent of the IFNγ system. In the present study, we examined changes in IRF-1 DNA binding activity and protein after mycobacterial infection. M. tuberculosisincreased both and induced a serine protease inhibitor activity that affected extraction of IRF-1. We suggest that induction of IRF-1 and the protease inhibitor may be functionally related as host defense responses to M. tuberculosis. All manipulations with viable M. tuberculosis were performed under biosafety level 3 containment. A clinical isolate of M. tuberculosis, TN913, from the Public Health Research Institute Tuberculosis Center was grown in Middlebrook 7H9 broth as previously described (14Zhao B.Y. Pine R. Domagala J. Drlica K. Antimicrob. Agents Chemother. 1999; 43: 661-666Crossref PubMed Google Scholar). THP-1 cells (3Tsuchiya S. Yamabe M. Yamaguchi Y. Kobayashi Y. Konno T. Tada K. Int. J. Cancer. 1980; 26: 171-176Crossref PubMed Scopus (1677) Google Scholar) obtained from the American Type Culture Collection were maintained between 0.6 and 6.0 × 105/ml in RPMI 1640 supplemented with penicillin/streptomycin (BioWhittaker) and 10% defined supplemented calf bovine serum (Hyclone). Before infection, as previously described (15Weiden M. Tanaka N. Qiao Y. Zhao B.Y. Honda Y. Nakata K. Canova A. Levy D.E. Rom W.N. Pine R. J. Immunol. 2000; 165: 2028-2039Crossref PubMed Scopus (97) Google Scholar), cells were untreated or treated with 20 nm TPA (Sigma) for 24 h. As indicated, cells were stimulated with recombinant human IFNγ (a gift from Amgen) at 1 ng/ml for the final 2 h before harvest for preparation of extracts. Conditioned media (CM) were collected 3 days post-infection from undifferentiated or TPA-treated THP-1 cells infected at the indicated multiplicity of infection (m.o.i.) or from parallel, uninfected cultures. The CM were sterilely filtered and used the same day or stored at 4 °C for use the next day. THP-1 cells maintained in complete media were collected by centrifugation, suspended as indicated in CM, then grown for 3 days before harvest for preparation of extracts. Experiments with primary cells were performed in accordance with all applicable laws and regulations. Cells obtained from healthy volunteers by bronchoalveolar lavage were suspended in RPMI 1640 plus 10% fetal bovine serum (Hyclone), placed in cell culture flasks, and infected with M. tuberculosis TN913 as previously described (15Weiden M. Tanaka N. Qiao Y. Zhao B.Y. Honda Y. Nakata K. Canova A. Levy D.E. Rom W.N. Pine R. J. Immunol. 2000; 165: 2028-2039Crossref PubMed Scopus (97) Google Scholar) for ∼16 h. Uninfected cells were cultured in parallel. Nonadherent cells then were removed with the media, and the adherent cells were washed gently with phosphate-buffered saline (PBS). The remaining alveolar macrophages were 90–95% pure based on microscopic examination of morphology. Peripheral blood monocytes were purified with anti-CD14 monoclonal antibody from buffy coats obtained from healthy volunteers, then cultured in RPMI 1640 plus 15% fetal bovine serum. Infection with a single cell suspension of M. tuberculosis H37Rv at a m.o.i. of ∼1 was begun after 4 or 5 days of adherence-induced differentiation and continued for ∼16 h. Uninfected cells were cultured in parallel. All steps were performed at 0–4 °C except as indicated. Media were removed from adherent cells. The cells were washed once with PBS and then were scraped into additional PBS. Undifferentiated, uninfected THP-1 cells growing in suspension were collected by centrifugation, suspended in PBS, collected again by centrifugation, and suspended again in PBS. Cells that had been adherent or in suspension were then collected by centrifugation. Lysates and extracts were prepared with non-ionic detergent or without detergent. Lysates and extracts from cells infected by M. tuberculosis were filter-sterilized before removal from bio-safety level 3 containment. For preparations with non-ionic detergent, cell pellets were suspended in 4 volumes of buffer I (0.5% Nonidet P-40, 0.1 mm EDTA, 20 mm Hepes, pH 7.9, 10% glycerol, 1 mmdithiothreitol, 0.4 mm phenylmethylsulfonyl fluoride (PMSF), 3 μg/ml aprotinin, 1 μg/ml leupeptin, and 2 μg/ml pepstatin) and incubated on ice for 10 min. As indicated, cells were lysed, and extracts were prepared without protease inhibitors. Nuclei were sedimented by centrifuging the lysates at 1,000 ×g for 10 min. The supernatants were recovered and adjusted to 0.3 m NaCl to produce the cytoplasmic extracts. The nuclear pellets were suspended with buffer I, sedimented again by centrifuging, and suspended with 4 volumes of buffer I plus 0.4m NaCl. The suspended nuclei were incubated for 30 min with occasional mixing. The suspensions were clarified by centrifuging at 15,000 × g for 10 min. The supernatants were recovered as the nuclear extracts. Extracts were frozen rapidly on crushed dry ice and stored at −80 °C. For preparations without detergent, cells were suspended in 9 volumes of buffer II (10 mm Hepes, pH 7.9, 10 mm KCl, 3 mm MgCl2, 1 mm dithiothreitol) and allowed to swell for ∼20 min. A lysate was prepared by disruption in a Dounce homogenizer with 25–40 strokes of a loose-fitting pestle to achieve 80–90% lysis, as determined by microscopic examination. Nuclei were then sedimented as described above. The supernatant was recovered and adjusted by the addition of 1/3 volume of 40% glycerol, 0.4 m NaCl in buffer II to produce a cytoplasmic extract. Nuclear pellets were resuspended in 3 volumes of buffer II containing 0.4 m NaCl and 10% glycerol. Suspended nuclei were extracted as described above. Alternatively, the lysate was adjusted by the addition of 1/3 volume of 40% glycerol, 0.4 m NaCl in buffer II and used for subcellular fractionation. Subcellular fractions prepared by differential centrifugation are designated based on the well established distribution of the predominant organelle(s) and vesicles (16de Duve C. J. Cell Biol. 1971; 50: 20-55Crossref PubMed Scopus (277) Google Scholar). Vesicles derived from plasma membrane trafficking compartments including early or late endosomes, Golgi apparatus, and endoplasmic reticulum are primarily in the “microsomal” fraction, whereas a small portion of plasma membrane often sediments with nuclei (16de Duve C. J. Cell Biol. 1971; 50: 20-55Crossref PubMed Scopus (277) Google Scholar, 17Marino M.W. Dunn A. Grail D. Inglese M. Noguchi Y. Richards E. Jungbluth A. Wada H. Moore M. Williamson B. Basu S. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8093-8098Crossref PubMed Scopus (693) Google Scholar, 18Chang K.-J. Bennett V. Cuatrecasas P. J. Biol. Chem. 1975; 250: 488-500Abstract Full Text PDF PubMed Google Scholar, 19Dunn W.A. Hubbard A.L. Aronson N.N., Jr. J. Biol. Chem. 1980; 255: 5971-5978Abstract Full Text PDF PubMed Google Scholar). All steps were performed at 0–4 °C. Adjusted lysate from Dounce homogenization was centrifuged at 1,000 × g for 10 min to sediment nuclei. The post-nuclear supernatant was removed thoroughly, and a portion was set aside. The remainder was centrifuged at 13,000 × g for 10 min to sediment lysosomes and mitochondria. The post-lysosomal/mitochondrial supernatant was removed thoroughly, and a portion was set aside. The remainder was centrifuged at 130,000 × g for 1 h to sediment microsomes and ribosomes. The post-microsomal/ribosomal supernatant, also called the cytosol, was removed thoroughly. Each pellet was resuspended in the same volume of the same buffer as the fraction that was its source. Thus, equal volumes of each fraction are derived from equal numbers of the initial cells. An EMSA was carried out as previously described (20Pine R. Nucleic Acids Res. 1997; 21: 4346-4354Crossref Scopus (142) Google Scholar). The radiolabeled probe was an oligonucleotide from −117 to −89 of the IFNα/β-stimulated gene 15, which includes the IFN-stimulated response element (21Reich N. Evans B. Levy D. Fahey D. Knight E., Jr. Darnell J.E., Jr. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6394-6398Crossref PubMed Scopus (281) Google Scholar). Cell lysates, extracts, subcellular fractions, immunodepleted extracts (see below), partially purified IRF-1 (prepared by phosphocellulose (Whatman P11) chromatography of nuclear extracts from IFNγ-stimulated HeLa cells), proteases, protease inhibitors, and control buffers were included as indicated for individual assays. Unlabeled hepatocyte nuclear factor 4 distal element (22Sladek F.M. Zhong W. Lai E. Darnell J.E., Jr. Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (853) Google Scholar) or IFN-stimulated response element oligonucleotides were included at 100-fold molar excess to provide nonspecific or specific competition, respectively, as indicated for individual assays. All the components for each assay except reaction buffer, nonspecific DNA, and oligonucleotides were assembled on ice. Incubation was started by the addition of a mixture of these remaining components and then carried out for 30 min at room temperature before electrophoresis on 6% polyacrylamide gels at 4 °C with 20 mm Tris-borate, pH 8.3, 0.4 mm EDTA buffer. Images were obtained, and results were quantified with a PhosphorImager (Molecular Dynamics). All specific antibodies were directed against human antigens. Rabbit anti-neutrophil/monocyte elastase and anti-cathepsin G as well as purified human neutrophil/monocyte elastase and cathepsin G were obtained from Calbiochem. Goat anti-secretory leukocyte protease inhibitor (SLPI) and recombinant human SLPI were obtained from R&D Systems. Anti-plasminogen activator inhibitor 2 (PAI-2) and recombinant human PAI-2 were obtained from American Diagnostica. Normal rabbit or goat IgG obtained from Zymed Laboratories Inc. was used as a nonspecific, control antibody for the respective specific antibodies. Protein A- or protein G-conjugated-Sepharose 4B beads were obtained from Zymed Laboratories Inc. Immunodepletion was performed at 4 °C by mixing 5 μl (5 μg) of control or specific antibody with 50 μl of a cytoplasmic extract (∼150 μg of protein) prepared in buffer I and adjusted to 150 mm NaCl without or with added target protein as an external standard for 2 h, adding 50 μl of a 50% slurry of protein A or protein G beads (for reactions with rabbit or goat antibodies, respectively) and mixing for 4 h more and then recovering the beads by centrifugation at 12,000 × gfor 20 s. The immunodepleted supernatants were then removed. The recovered beads were washed 3 times with buffer I plus 300 mm NaCl, and then bound material was eluted by boiling in SDS-PAGE sample buffer for 3 min. Eluates were recovered after centrifugation at 12,000 × g for 20 s. The immunodepleted supernatants and the eluates were then frozen in crushed dry-ice and stored at –80 °C. To control for the efficiency of immunodepletion, elastase (1.5 μg), cathepsin G (1 μg), SLPI (50 ng), or PAI-2 (750 ng) was added to an extract, and immunodepletion was performed with the respective specific antibody or the control antibody (data not shown). As judged by immunoblot of the recovered supernatants and of the eluted immunoprecipitates, the amount of each added protein that was specifically removed was far in excess of the amounts of the endogenous proteins, which were undetectable when extracts were directly assayed by immunoblot. The control antibodies did not reduce the amount of added target protein in the supernatants or recover any in the immunoprecipitates. Cytoplasmic or nuclear extracts, immunodepleted extracts, eluted immunoprecipitates, partially purified IRF-1, and protein standards in cytoplasmic extract buffer all were adjusted to 1× SDS-PAGE loading buffer before analysis by immunoblotting. All samples were boiled for 3 min, electrophoresed on 10% SDS-polyacrylamide gels, and then transferred to nitrocellulose membranes (Bio-Rad) by electroblotting. Membranes were blocked with 0.5% nonfat dry milk in PBS for detection of IRF-1 or with 5% nonfat dry milk and 0.2% Tween 20 in PBS for detection of cathepsin G, elastase, PAI-2, or SLPI. Rabbit polyclonal antiserum against human IRF-1 (8Pine R. Decker T. Kessler D.S. Levy D.E. Darnell J.E., Jr. Mol. Cell. Biol. 1990; 10: 2448-2457Crossref PubMed Scopus (277) Google Scholar) or the antibodies against the other antigens (described above) were added to the respective solution. Membranes were washed with PBS, then incubated with horseradish peroxidase-conjugated secondary antibodies. Goat anti-rabbit immunoglobulin G and rabbit anti-goat immunoglobulin G (Zymed Laboratories Inc.) were used to detect rabbit and goat primary antibodies, respectively. Membranes were washed with PBS, then incubated with LumiGLO chemiluminescent substrate (Kirkegaard and Perry Laboratories). Signals were detected with x-ray film. We first investigated whether infection of macrophages by M. tuberculosis would alter IRF-1 DNA binding activity as measured by EMSA (Fig 1 A). Alveolar macrophages had clearly detectable constitutive IRF-1 DNA binding activity (lane 1). The activity was near or below the lower limit of detection in peripheral blood monocyte-derived macrophages and THP-1 macrophages (lanes 5 and 9), as is typical of many cells (8Pine R. Decker T. Kessler D.S. Levy D.E. Darnell J.E., Jr. Mol. Cell. Biol. 1990; 10: 2448-2457Crossref PubMed Scopus (277) Google Scholar, 20Pine R. Nucleic Acids Res. 1997; 21: 4346-4354Crossref Scopus (142) Google Scholar, 23Pine R. Darnell J.E., Jr. Mol. Cell. Biol. 1989; 9: 3533-3537Crossref PubMed Scopus (13) Google Scholar, 24Parrington J. Rogers N.C. Gewert D.R. Pine R. Veals S.A. Levy D.E. Stark G.R. Kerr I.A. Eur. J. Biochem. 1993; 214: 617-626Crossref PubMed Scopus (38) Google Scholar, 25Neish A.S. Read M.A. Thanos D. Pine R. Maniatis T. Collins T. Mol. Cell. Biol. 1995; 15: 2558-2569Crossref PubMed Google Scholar, 26Matikainen S. Ronni T. Hurme M. Pine R. Julkunen I. Blood. 1996; 88: 114-123Crossref PubMed Google Scholar, 27Improta T. Pine R. Cytokine. 1997; 9: 383-393Crossref PubMed Scopus (15) Google Scholar). In each case, infection induced IRF-1 DNA binding activity (lanes 3, 7, and10). The slower mobility induced complex comigrates within vitro translated or partially purified full-length IRF-1 (lane 11) bound to the oligonucleotide probe (8Pine R. Decker T. Kessler D.S. Levy D.E. Darnell J.E., Jr. Mol. Cell. Biol. 1990; 10: 2448-2457Crossref PubMed Scopus (277) Google Scholar). The identity of the complexes was confirmed by use of specific competitor oligonucleotide (for example, lanes 2, 4,6, and 8) and by reaction with anti-IRF-1 antibody (data not shown). The faster mobility-induced complex, labeled A, also contained a species of IRF-1. Altogether, total IRF-1 DNA binding activity clearly increased upon infection. Quantification of IRF-1 DNA binding activity relative to a nonspecific DNA-binding protein that served as an internal standard (Fig. 1 B), showed that infection with M. tuberculosis caused full-length IRF-1 to increase ∼4-fold in differentiated THP-1 cells and monocyte-derived macrophages. The increase was ∼2-fold in alveolar macrophages compared with their unusually high constitutive level of IRF-1. Thus, in primary macrophages and THP-1 macrophages, infection by M. tuberculosis clearly resulted in induction of IRF-1 DNA binding activity. The induced level of IRF-1 was similar in the alveolar and monocyte-derived macrophages and somewhat lower in the THP-1 macrophages. M. tuberculosis infection of THP-1 monocytes also induced IRF-1 DNA binding activity (Fig.2 A). Constitutive expression of IRF-1 was quite low (lane 1). The complexes that were detected (labeled A, B, and C) migrated more rapidly than the typical complex containing full-length IRF-1. Nuclear extracts from monocytes stimulated with IFNγ produced an increase in the rapidly migrating complexes, yet the typical complex was not induced (lane 2). Compared with extracts from uninfected, unstimulated cells, nuclear extracts from infected monocytes yielded a complex that had the mobility of full-length IRF-1 bound to the IFN-stimulated response element (lane 3). Furthermore, the abundance of complex A increased and that of complexes B and C decreased. As in differentiated THP-1 cells and primary macrophages, infection of undifferentiated THP-1 cells by M. tuberculosis led to an increase in total IRF-1 DNA binding activity. When nuclear extracts of IFNγ-stimulated infected monocytes were assayed (lane 4), the typical complex formed abundantly, whereas the complexes B and C were much less abundant than after IFNγ stimulation of uninfected cells. Recovery of full-length IRF-1 from THP-1 monocytes was a specific effect of infection byM. tuberculosis. After infection by M. bovis BCG at the same m.o.i. (lanes 5 and 6) or after phagocytosis of latex beads (data not shown), predominantly faster mobility IRF-1 complexes were detected. The complexes formed with extracts from untreated or IFNγ-treated infected monocytes had essentially the same mobility as the complexes formed with extracts from untreated or IFNγ-treated THP-1 macrophages (compare lanes 3 and 4 with lanes 7 and 8). Thus, infection of THP-1 monocytes by M. tuberculosis and differentiation increased formation of typical complexes and decreased formation of rapidly migrating complexes detected by EMSA of the respective nuclear extracts compared with extracts from uninfected, undifferentiated cells. Immunoblots were performed (Fig. 2 B) to identify IRF-1 species present in the extracts that had been analyzed by EMSA. Anti-IRF-1 antiserum detected a pattern of proteins that corresponded precisely with the protein-DNA complexes detected by EMSA. Constitutively expressed IRF-1 recovered from monocytes appeared as three species, labeled A, B, and C(lanes 1 and 1′). Each of those was induced by IFNγ (lane 2). Extracts of infected monocytes contained an additional, slower mobility IRF-1 species, and total recovery of IRF-1 increased (lane 3). IFNγ treatment of infected monocytes strongly induced the additional IRF-1 species, and to a lesser extent, higher mobility species were recovered. IRF-1 species A was constitutively present in macrophage extracts (lane 5). Recovery of this species increased slightly, and the slowest mobility IRF-1 species was abundant in extracts from IFNγ-treated macrophages (lane 6). Consistent with the EMSA results, the slower mobility-induced species comigrated with in vitro translated or partially purified full-length IRF-1 (lane 7) (8Pine R. Decker T. Kessler D.S. Levy D.E. Darnell J.E., Jr. Mol. Cell. Biol. 1990; 10: 2448-2457Crossref PubMed Scopus (277) Google Scholar). Comparison of the EMSA and immunoblot results indicated that the ratio of IRF-1 DNA binding activity and protein were similar under all conditions and for all species of IRF-1. There was also a close correlation between the mobility of the protein-DNA complexes detected by EMSA and the mobility of the IRF-1 protein species. Furthermore, infection or differentiation led to decreased recovery of higher mobility IRF-1 species and increased recovery of full-length IRF-1. To unambiguously demonstrate which protein species was contained in which protein-DNA complexes, we next performed two-dimensional analyses. To increase sensitivity, the extracts were prepared from IFNγ-treated cells. Fig. 3 Ashows the results obtained with extracts from monocytes (left panel) and macrophages (right panel). The initial separation by EMSA (in a parallel lane not used for a SDS-PAGE sample) is shown aligned in the same position as the lane used for a SDS-PAGE sample. The immunoblot result from the SDS-PAGE separations of the initial protein samples and the EMSA complexes formed by those samples is shown beneath the EMSA separations. Each complex resolved by EMSA contained one species of IRF-1 that precisely comigrated with a species resolved by SDS-PAGE alone. Thus, each species of IRF-1 protein detected in extracts from monocytes was found in only one of the complexes detected by EMSA, and the slower mobility complex detected by EMSA of extracts from macrophages contained full-length IRF-1. The two-dimensional analyses of extracts from uninfected and infected monocytes (Fig. 3 B) also showed that one characteristic higher mobility IRF-1 species was present in each protein-DNA complex resolved by EMSA of monocyte extract (left panel) and that the slower mobility complex resolved by EMSA of the extract from infected monocytes contained only full-length IRF-1 (right panel). Thus, the complexes resolved by EMSA reflect the presence of the corresponding distinct species of IRF-1 and can be used as an assay for their abundance. We hypothesized that the presence of higher mobility IRF-1 species was the result of protease activity and found that full-length IRF-1 was recovered in nuclear extracts when THP-1 monocytes were lysed with non-ionic detergent and sufficient (2 mm) PMSF (Fig.4 A, lane 2). This result suggested that higher mobility IRF-1 species were produced during extraction, in which case they might also be produced in vitro. A cytoplasmic extract from untreated THP-1 cells was prepared after lysis with non-ionic detergent in the absence of protease inhibitors. The extract was mixed with partially purified IRF-1 in the absence or presence of protease inhibitors, and whether high mobility IRF-1 species were produced was determined in an EMSA (Fig. 4 B). IRF-1 assayed with no extract produced the typical protein-DNA complex expected for intact IRF-1 (lane 1). Addition of the extract (lane 2) produced the specific species previously observed for endogenous IRF-1 recovered from THP-1 monocytes. PMSF, which reversibly inhibits cysteine proteases and irreversibly inhibits serine proteases, substantially reduced production of higher mobility IRF-1 species (lanes 3and 4). In contrast, E64, a specific cysteine protease inhibitor, did not (lanes 5 and 6). To confirm that the E64 was active, its ability to inhibit papain was tested. Papain completely degraded IRF-1 (lane 7), but inclusion of E64 at 50 μg/ml completely protected the IRF-1 against proteolysis (lane 8). Thus, monocytes contain a serine protease that can lead to productio" @default.
• W2051363313 created "2016-06-24" @default.
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• W2051363313 date "2002-06-01" @default.
• W2051363313 modified "2023-10-06" @default.
• W2051363313 title "Host Defense Responses to Infection by Mycobacterium tuberculosis" @default.
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