Up-regulation of Microglial CD11b Expression by Nitric Oxide
2006; Elsevier BV; Volume: 281; Issue: 21 Linguagem: Inglês
10.1074/jbc.m600236200
ISSN1083-351X
AutoresAvik Roy, Yiu K. Fung, Xiaojuan Liu, Kalipada Pahan,
Tópico(s)Neuropeptides and Animal Physiology
ResumoIncreased expression of CD11b, the β-integrin marker of microglia, represents microglial activation during neurodegenerative inflammation. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) induced the production of NO and increased the expression of CD11b in mouse BV-2 microglial cells and primary microglia. Either a scavenger of NO (PTIO) or an inhibitor of inducible nitric-oxide synthase (L-NIL) blocked this increase in microglial CD11b expression. Furthermore, co-microinjection of PTIO with LPS was also able to suppress LPS-mediated expression of CD11b and loss of dopaminergic neuronal fibers and neurotransmitters in striatum in vivo. Similarly, other inducers of NO production such as interferon-γ, interleukin-1β, human immunodeficiency virus type-1 gp120, and double-stranded RNA (poly(IC)) also increased the expression of CD11b in microglia through NO. The role of NO in the expression of CD11b was corroborated further by the expression of microglial CD11b by GSNO, an NO donor. Because NO transduces many intracellular signals via guanylate cyclase (GC), we investigated the role of GC, cyclic GMP (cGMP), and cGMP-activated protein kinase (PKG) in microglial expression of CD11b. Inhibition of LPS- and GSNO-mediated up-regulation of CD11b either by NS2028 (a specific inhibitor of GC) or by KT5823 and Rp-8-bromo-cGMP (specific inhibitors of PKG), and increase in CD11b expression either by 8-bromo-cGMP or by MY-5445 (a specific inhibitor of cGMP phosphodiesterase) alone suggest that NO increases microglial expression of CD11b via GC-cGMP-PKG. In addition, GSNO induced the activation of cAMP response element-binding protein (CREB) via PKG that was involved in the up-regulation of CD11b. This study illustrates a novel biological role of NO in regulating the expression of CD11b in microglia through GC-cGMP-PKG-CREB pathway that may participate in the pathogenesis of devastating neurodegenerative disorders. Increased expression of CD11b, the β-integrin marker of microglia, represents microglial activation during neurodegenerative inflammation. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) induced the production of NO and increased the expression of CD11b in mouse BV-2 microglial cells and primary microglia. Either a scavenger of NO (PTIO) or an inhibitor of inducible nitric-oxide synthase (L-NIL) blocked this increase in microglial CD11b expression. Furthermore, co-microinjection of PTIO with LPS was also able to suppress LPS-mediated expression of CD11b and loss of dopaminergic neuronal fibers and neurotransmitters in striatum in vivo. Similarly, other inducers of NO production such as interferon-γ, interleukin-1β, human immunodeficiency virus type-1 gp120, and double-stranded RNA (poly(IC)) also increased the expression of CD11b in microglia through NO. The role of NO in the expression of CD11b was corroborated further by the expression of microglial CD11b by GSNO, an NO donor. Because NO transduces many intracellular signals via guanylate cyclase (GC), we investigated the role of GC, cyclic GMP (cGMP), and cGMP-activated protein kinase (PKG) in microglial expression of CD11b. Inhibition of LPS- and GSNO-mediated up-regulation of CD11b either by NS2028 (a specific inhibitor of GC) or by KT5823 and Rp-8-bromo-cGMP (specific inhibitors of PKG), and increase in CD11b expression either by 8-bromo-cGMP or by MY-5445 (a specific inhibitor of cGMP phosphodiesterase) alone suggest that NO increases microglial expression of CD11b via GC-cGMP-PKG. In addition, GSNO induced the activation of cAMP response element-binding protein (CREB) via PKG that was involved in the up-regulation of CD11b. This study illustrates a novel biological role of NO in regulating the expression of CD11b in microglia through GC-cGMP-PKG-CREB pathway that may participate in the pathogenesis of devastating neurodegenerative disorders. Microglia are considered as CNS 2The abbreviations used are: CNS, central nervous system; iNOS, inducible nitric-oxide synthase; LPS, lipopolysaccharide; IL, interleukin; HIV-1, human immunodeficiency virus type-1; PKG, cGMP-activated protein kinase; CREB, cAMP response element-binding protein; GC, guanylate cyclase; l-NIL, l-N6-(1-Iminoethyl)-lysine; 8-Br-cGMP, 8-bromo-cGMP; FACS, fluorescence-activated cell sorter; TH, tyrosine hydroxylase; RT-PCR, reverse transcription-PCR; ASO, antisense oligonucleotide; ScO, scrambled oligonucleotide. resident professional macrophages that function as the principal immune effector cells of the CNS responding to any pathological event. Activation of microglia has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer disease, Parkinson disease, Creutzfeld-Jacob disease, HIV-associated dementia, stroke, and multiple sclerosis (1Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar). It has been found that activated microglia accumulate at sites of injury or plaques in neurodegenerative CNS (1Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar, 2Barcia C. Sanchez Bahillo A. Fernandez-Villalba E. Bautista V. Poza Y Poza M Fernandez-Barreiro A. Hirsch E.C. Herrero M.T. Glia. 2004; 46: 402-409Crossref PubMed Scopus (169) Google Scholar, 3Dauer W. Przedborski S. Neuron. 2003; 39: 889-909Abstract Full Text Full Text PDF PubMed Scopus (4099) Google Scholar, 4Wu D.C. Jackson-Lewis V. Vila M. Tieu K. Teismann P. Vadseth C. Choi D.K. Ischiropoulos H. Przedborski S. J. Neurosci. 2002; 22: 1763-1771Crossref PubMed Google Scholar, 5Carson M.J. Glia. 2002; 40: 218-231Crossref PubMed Scopus (205) Google Scholar, 6Rock R.B. Gekker G. Hu S. Sheng W.S. Cheeran M. Lokensgard J.R. Peterson P.K. Clin. Microbiol. Rev. 2004; 17: 942-964Crossref PubMed Scopus (547) Google Scholar). Although activated microglia scavenge dead cells from the CNS and secrete different neurotrophic factors for neuronal survival (1Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar, 5Carson M.J. Glia. 2002; 40: 218-231Crossref PubMed Scopus (205) Google Scholar, 6Rock R.B. Gekker G. Hu S. Sheng W.S. Cheeran M. Lokensgard J.R. Peterson P.K. Clin. Microbiol. Rev. 2004; 17: 942-964Crossref PubMed Scopus (547) Google Scholar), it is believed that severe activation causes various autoimmune responses leading to neuronal death and brain injury (1Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar, 2Barcia C. Sanchez Bahillo A. Fernandez-Villalba E. Bautista V. Poza Y Poza M Fernandez-Barreiro A. Hirsch E.C. Herrero M.T. Glia. 2004; 46: 402-409Crossref PubMed Scopus (169) Google Scholar, 3Dauer W. Przedborski S. Neuron. 2003; 39: 889-909Abstract Full Text Full Text PDF PubMed Scopus (4099) Google Scholar, 4Wu D.C. Jackson-Lewis V. Vila M. Tieu K. Teismann P. Vadseth C. Choi D.K. Ischiropoulos H. Przedborski S. J. Neurosci. 2002; 22: 1763-1771Crossref PubMed Google Scholar, 5Carson M.J. Glia. 2002; 40: 218-231Crossref PubMed Scopus (205) Google Scholar, 6Rock R.B. Gekker G. Hu S. Sheng W.S. Cheeran M. Lokensgard J.R. Peterson P.K. Clin. Microbiol. Rev. 2004; 17: 942-964Crossref PubMed Scopus (547) Google Scholar). During severe activation microglia not only secrete various neurotoxic molecules but also express different proteins and surface markers. Among different surface markers, CD11b is the most potential one with immense biological significance (6Rock R.B. Gekker G. Hu S. Sheng W.S. Cheeran M. Lokensgard J.R. Peterson P.K. Clin. Microbiol. Rev. 2004; 17: 942-964Crossref PubMed Scopus (547) Google Scholar, 7Ling E.A. Wong W.C. Glia. 1993; 7: 9-18Crossref PubMed Scopus (590) Google Scholar). It acts as a binding protein for intracellular cell adhesion molecule-1 and complement C3bi (8Schwarz M. Nordt T. Bode C. Peter K. Thromb. Res. 2002; 107: 121-128Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). It is reported that in various neuroinflammatory diseases, the increased Cd11b expression corresponds to the severity of microglial activation (1Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar, 6Rock R.B. Gekker G. Hu S. Sheng W.S. Cheeran M. Lokensgard J.R. Peterson P.K. Clin. Microbiol. Rev. 2004; 17: 942-964Crossref PubMed Scopus (547) Google Scholar, 7Ling E.A. Wong W.C. Glia. 1993; 7: 9-18Crossref PubMed Scopus (590) Google Scholar). Morphologically, microglial activation is associated with intense ramification and cytoskeletal rearrangement in which changes in shape and motility correlate with increased expression of CD11b (1Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar, 6Rock R.B. Gekker G. Hu S. Sheng W.S. Cheeran M. Lokensgard J.R. Peterson P.K. Clin. Microbiol. Rev. 2004; 17: 942-964Crossref PubMed Scopus (547) Google Scholar, 7Ling E.A. Wong W.C. Glia. 1993; 7: 9-18Crossref PubMed Scopus (590) Google Scholar). During this activation process, the cytoplasmic domain of CD11b is believed to interact increasingly with cytoskeletal protein (1Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar). However, the mechanism by which microglial expression of CD11b is increased in neurodegenerative CNS remains unclear. Because activated microglia also express inducible nitric-oxide synthase (iNOS) to produce an excessive amount of NO, a molecule implicated virtually in all reported neurodegenerative and neuroinflammatory conditions (9Stewart V.C. Heales S.J. Free Radic. Biol. Med. 2003; 34: 287-303Crossref PubMed Scopus (165) Google Scholar, 10Ischiropoulos H. Beckman J.S. J. Clin. Investig. 2003; 111: 163-169Crossref PubMed Scopus (657) Google Scholar), we were prompted to investigate whether NO plays a role in the microglial expression of CD11b. Here we report that NO is instrumental in increasing the expression of CD11b in microglia. Different inducers of NO production such as LPS, IFN-γ, IL-1β, HIV-1 gp120, and poly(IC) stimulated microglial expression of CD11b via NO. Furthermore, we also demonstrate that NO employed the guanylate cyclase (GC)-cGMP-cGMP-activated protein kinase (PKG)-cAMP response element-binding protein (CREB) pathway to up-regulate the expression of CD11b in microglia. Reagents—Fetal bovine serum and Dulbecco's modified Eagle's medium/F-12 were from Mediatech Inc. LPS (Escherichia coli), actinomycin D, and poly(IC) were purchased from Sigma. HIV-1 coat protein gp120 was obtained from US Biological. l-N6-(1-Iminoethyl)-lysine (l-NIL), NS-2028 (an inhibitor of guanylate cyclase), 8-Br-cGMP, MY-5445 (an inhibitor of cGMP phosphodiesterase), and KT5823 (an inhibitor of PKG) were obtained from Biomol. Recombinant mouse IFN-γ and IL-1β were obtained from R&D. Isolation of Mouse Microglia—Microglial cells were isolated from mixed glial cultures according to the procedure of Giulian and Baker (11Giulian D. Baker T.J. J. Neurosci. 1986; 6: 2163-2178Crossref PubMed Google Scholar). Briefly, on day 7-9 the mixed glial cultures were washed three times with Dulbecco's modified Eagle's medium/F-12 and subjected to shaking at 240 rpm for 2 h at 37 °C on a rotary shaker. The floating cells were washed and seeded on to plastic tissue culture flasks and incubated at 37 °C for 2 h. The attached cells were removed by trypsinization and seeded onto new plates for further studies. Ninety to ninety-five percent of this preparation was found to be positive for Mac-1 surface antigen. Mouse BV-2 microglial cells (kind gift from Virginia Bocchini of University of Perugia) were also maintained and induced as indicated above. Assay for NO Synthesis—Synthesis of NO was determined by assay of culture supernatants for nitrite, a stable reaction product of NO with molecular oxygen, using Griess reagent as described (12Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 13Liu X. Jana M. Dasgupta S. Koka S. He J. Wood C. Pahan K. J. Biol. Chem. 2002; 277: 39312-39319Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Flow Cytometry—Surface expression of CD11b on BV-2 microglial cells was checked by flow cytometry as described earlier (14Dasgupta S. Jana M. Liu X. Pahan K. J. Biol. Chem. 2002; 277: 39327-39333Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Dasgupta S. Jana M. Liu X. Pahan K. J. Biol. Chem. 2003; 278: 22424-22431Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Briefly, 1 × 10 6 cells suspended in RPMI 1640-fetal bovine serum were incubated in the dark with appropriately diluted fluorescein isothiocyanate-labeled antibodies to CD11b (Mac-1 Integrin) (BD Pharmingen) at 4 °C for 30 min. Following incubation, cell suspension was centrifuged, washed thrice, and resuspended in 500 μl of RPMI-fetal bovine serum. The cells were then analyzed through FACS (Becton Dickinson). A minimum of 10,000 cells was accepted for FACS analysis. Cells were gated based on morphological characteristics. Apoptotic and necrotic cells were not accepted for FACS analysis. Immunofluorescence Analysis—It was performed as described earlier (16Jana A. Pahan K. J. Neurosci. 2004; 24: 9531-9540Crossref PubMed Scopus (86) Google Scholar). Briefly, cover slips containing 100-200 cells/mm2 were fixed with 4% paraformaldehyde for 20 min followed by treatment with cold ethanol (-20 °C) for 5 min and two rinses in phosphate-buffered saline. Samples were blocked with 3% bovine serum albumin in phosphate-buffered saline-Tween-20 (PBST) for 30 min and incubated in PBST containing 1% bovine serum albumin and rabbit anti-CD11b (1:50). After three washes in PBST (15 min each), slides were further incubated with Cy2 (Jackson ImmunoResearch Laboratories, Inc.). For negative controls, a set of culture slides was incubated under similar conditions without the primary antibodies. The samples were mounted and observed under a Bio-Rad MRC1024ES confocal laser-scanning microscope. Microinjection of LPS into the Striatum of C57BL/6 Mice—Male C57BL/6 mice (8-10-week old) were anesthetized with ketamine and xylazine and underwent cerebellar operations in a Kopf small animal stereotaxic instrument (David Kopf, CA). Briefly, the animal was mounted in a stereotaxic frame on a heating blanket. Body temperature was maintained at 37 ± 0.5 °C during the time of surgery. A midsagittal incision was made to expose the cranium and a hole <0.5 mm in diameter was drilled with a dental drill over the cerebrum according to the following coordinates: 5 mm anterior to lambda, lateral (L) 2.2 mm, ventral (V) 3.5 (as shown in Fig. 6A). Four micrograms of LPS in the presence or absence of l-NIL (10 μg) and PTIO (10 μg) dissolved in 3 μl of saline was injected using a 5-μl syringe (Hamilton, Reno, Nevada) over a period of 2 min, and the needle was held in place for another minute before withdrawing it from the skull to prevent reflux up the needle tract. Similarly control mice received 3 μl of saline. The incision was closed with surgical staples and covered with a mixture of bacitracin and Hurricane (20% benzocaine). Tyrosine Hydroxylase Immunostaining—Five days after microinjection, mice were perfused with 4% paraformaldehyde, and their brains were processed for immunohistochemical studies. Sections (10 μm) were incubated with a polyclonal anti-tyrosine hydroxylase (TH, 1,000 dilution, Calbiochem) for 24 h at 4 °C. Biotinylated secondary antibodies followed by avidin-biotin complex were used. Immunoreactivity was visualized by Vectastatin Elite ABC kit (Vector Laboratories, Inc.). Striatal optical density of TH immunostaining, measured by SpotDenso Analysis Tools in Fluorochem 8800 Imaging System, was used as an index of striatal density of TH innervation. Measurement of Dopamine and Its Metabolite Levels in Striatal Tissues—After 5 days of microinjection, mice were sacrificed, and their striata were dissected out and stored at -80 °C until analysis. On the day of the assay, striatal tissues were sonicated in 0.2 m perchloric acid (0.5 ml/100 mg of tissue) containing isoproterenol (100 ng/100 mg of tissue) as internal standard. After centrifugation at 20,000 × g for 15 min at 4 °C, the pH of supernatants was adjusted to pH 3.0 with 1 m sodium acetate. After filtration, 10 μl of supernatant was injected onto a Eicompak SC-3ODS column (Complete Stand-Alone HPLC-ECD System EiCOMHTEC-500 from JM Science Inc., Grand Island, NY) and analyzed for dopamine, dihydroxyphenylacetic acid, and homovanillic acid following manufacturer's protocol. Briefly, the mobile phase consisted of 20% methanol and 80% 0.1 m citrate-acetate buffer (pH 3.5) with 220 mg/l sodium octane sulfate and 5 mg/l disodium EDTA. The flow rate was maintained at 340-400 μl/min. Semiquantitative RT-PCR Analysis—Total RNA was isolated from BV-2 microglial cells, primary microglia, or striatal tissues surrounding the point of microinjection using Ultraspec-II RNA reagent (Biotecx Laboratories, Inc.) following manufacturer's protocol. To remove any contaminating genomic DNA, total RNA was digested with DNase. Semiquantitative RT-PCR was carried out as described earlier (17Dasgupta S. Jana M. Zhou Y. Fung Y.K. Ghosh S. Pahan K. J. Immunol. 2004; 173: 1344-1354Crossref PubMed Scopus (104) Google Scholar) using a RT-PCR kit from Clontech. Briefly, 1 μg of total RNA was reverse-transcribed using oligo(dT)12-18 as primer and MMLV reverse transcriptase (Clontech) in a 20-μl reaction mixture. The resulting cDNA was appropriately diluted, and diluted cDNA was amplified using titanium TaqDNA polymerase and the following primers. Amplified products were electrophoresed on a 1.8% agarose gel and visualized by ethidium bromide staining: CD11b, sense: 5′-CAGATCAACAATGTGACCGTATGGG-3′, antisense: 5′-CATCATGTCCTTGTACTGCCGCTTG-3′; IL-1β, sense: 5′-CTCCATGAGCTTTGTACAAGG-3′, antisense: 5′-TGCTGATGTACCAGTTGGGG-3′; glyceraldehyde-3-phosphate dehydrogenase, sense: 5′-GGTGAAGGTCGGTGTGAACG-3′, antisense: 5′-TTGGCTCCACCCTTCAAGTG-3′. The relative expression of either CD11b or IL-1β (CD11b or IL-1β/glyceraldehyde-3-phosphate dehydrogenase) was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech Corporation). Real Time PCR analysis for CD11b mRNA—It was performed using the ABI-Prism7700 sequence detection system (Applied Biosystems) as described earlier (17Dasgupta S. Jana M. Zhou Y. Fung Y.K. Ghosh S. Pahan K. J. Immunol. 2004; 173: 1344-1354Crossref PubMed Scopus (104) Google Scholar). Briefly, reactions were performed in 96-well optical reaction plates on cDNA equivalent to 50 ng of DNase-digested RNA in a volume of 25 μl, containing 12.5 μl of TaqMan Universal Master mix and optimized concentrations of carboxyfluorescein-labeled probe, forward and reverse primers, following manufacturer's protocol. All primers and FAM-labeled probes for mouse CD11b and glyceraldehyde-3-phosphate dehydrogenase were obtained from Applied Biosystems. The mRNA expression of CD11b was normalized to the level of glyceraldehyde-3-phosphate dehydrogenase mRNA. Data were processed by the ABI Sequence Detection System 1.6 software and analyzed by analysis of variance. Electrophoretic Mobility Shift Assay—Nuclear extract preparation and electrophoretic mobility shift assay was performed as described previously with some modifications (15Dasgupta S. Jana M. Liu X. Pahan K. J. Biol. Chem. 2003; 278: 22424-22431Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 18Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M.J. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, oligonucleotides containing the consensus binding sequence for CREB (5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′) (Promega) was radiolabeled with [γ-32P]ATP using polynucleotide T4 kinase. Labeled probe was purified with chroma spin column (BD Biosciences). Six-micrograms of nuclear extract was incubated with binding buffer and nonspecific oligonucleotides for 15 min in ice prior to incubation with labeled probe for another 15 min. Subsequently, samples were separated on a 6% polyacrylamide gel in 0.25× TBE buffer (Tris borate-EDTA), which were then dried and exposed to generate autoradiograms. Bacterial LPS Increases the Expression of CD11b in Mouse BV-2 Microglial Cells via NO—Microglia express CD11b, however, during microglial activation the expression of CD11b is markedly increased. Because microglial activation is also associated with the production of NO, we investigated the role of NO in microglial expression of CD11b. Mouse BV-2 microglial cells were stimulated with different concentrations of LPS, the prototype inducer of different immune cells including CNS microglia (18Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M.J. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Kim H.S. Whang S.Y. Woo M.S. Park J.S. Kim W.K. Han I.O. J. Neuroimmunol. 2004; 151: 85-93Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). It is clear from Fig. 1A that LPS dose dependently increased the expression of CD11b in BV-2 cells with maximum increase observed at 0.75 or 1.0 μg/ml of LPS. An assay of nitrite in culture supernatants shows that the increase in CD11b expression was associated with the production of NO (Fig. 1B). Next during a time-dependent analysis of LPS-mediated up-regulation of CD11b expression, we observed that LPS was unable to increase the mRNA expression of CD11b within 6 h of stimulation (Fig. 1C). However, the increase in CD11b expression was visible at 12 h with the maximum up-regulation observed at 24 h of stimulation (Fig. 1C). When we measured the time-dependent production of nitrite in response to LPS, we observed that the induction of NO production began at 12 h and reached the maximum at 24 h (Fig. 1D). In fact, the expression of CD11b paralleled to the production of NO suggesting a possible involvement of NO in the increase in CD11b expression. We then compared the expression pattern of another inducible gene in microglia. In contrast to the expression pattern of CD11b, marked induction of IL-1β mRNA was observed within 6 h of stimulation with LPS (Fig. 1C). Because the expression of IL-1β was observed before the increase in CD11b expression, we also investigated whether IL-1β was playing a role in LPS-induced expression of CD11b. The time course of IL-1β-induced CD11b expression shows that IL-1β was unable to stimulate the expression of CD11b within 12 h of stimulation (Fig. 1E). However, at 24 h of stimulation, the up-regulation of CD11b was clearly visible (Fig. 1E) suggesting that IL-1β may not be involved in LPS-induced expression of CD11b. Therefore, to investigate the role of NO in LPS-mediated up-regulation of CD11b, we examined the effect of l-NIL (an inhibitor of NOS) and carboxyl PTIO (a scavenger of NO) on the LPS-mediated increase in CD11b mRNA expression in BV-2 glial cells. It is clearly evident from semiquantitative RT-PCR analysis that both l-NIL and PTIO markedly inhibited LPS-mediated expression of CD11b (Fig. 1, F and G). Quantitative real time PCR analysis also reveals a marked inhibition of LPS-mediated expression of CD11b mRNA by l-NIL and PTIO (Fig. 1H). Next we investigated the effect of l-NIL and PTIO on the expression of CD11b protein in LPS-stimulated cells. Because CD11b is a surface protein, we analyzed its expression by FACS using FITC-labeled antibodies against CD11b. Fig. 2A represents auto-fluorescence, as this was observed in unconjugated normal BV-2 glial cells. As areas under M1 and M2 in Fig. 2, A-E represent auto-fluorescence and fluorescence, respectively, because of CD11b there was some expression of CD11b on the surface of normal BV-2 glial cells (Fig. 2B) in contrast to marked increase in CD11b expression on the surface of LPS-stimulated cells (Fig. 2C). Consistent to the inhibition of CD11b mRNA expression, both l-NIL and PTIO markedly inhibited LPS-mediated stimulation of CD11b protein expression (Fig. 2, D and E). Immunofluorescence analysis of CD11b in BV-2 microglial cells also shows that LPS stimulation increased the expression of CD11b and that l-NIL and PTIO attenuated LPS-mediated CD11b expression (Fig. 2F). Taken together, these studies suggest that LPS up-regulates the expression of CD11b in BV-2 microglial cells via NO. LPS Increases the Expression of CD11b in Mouse Primary Microglia via NO—To understand whether NO is required for the increase in CD11b expression by LPS in primary cells, we examined the effect of l-NIL and PTIO on LPS-mediated expression of CD11b in mouse primary microglia (Fig. 3). Consistent with the induction of NO production and increase in CD11b mRNA expression in BV-2 microglial cells, LPS induced the production of NO (Fig. 3A) and increased the expression of CD11b mRNA as revealed by semiquantitative RT-PCR (Fig. 3B) and quantitative real time PCR analyses (Fig. 3C)in mouse primary microglia. However, either blocking the production of NO by l-NIL or scavenging NO by PTIO (Fig. 3A) markedly suppressed LPS-mediated stimulation of CD11b mRNA expression (Fig. 3, B and C) in primary microglia. Apart from CD11b, microglia express many other surface markers such as, CD18, CD11a, CD11c, etc. In addition, CD11b pairs with CD18 to exhibit functional activity, therefore, we wondered whether NO was also regulating the expression of other surface markers including CD18. In mouse primary microglia, LPS increased the expression of CD18, CD11a, and CD11c compared with control (Fig. 3D). Interestingly, similar to the regulation of CD11b, both l-NIL and PTIO suppressed the LPS-mediated increase in CD18, CD11c, and CD11a expression in primary microglia (Fig. 3D) suggesting that NO is capable of regulating the expression of various surface markers of microglia associated with its activation. Involvement of NO in Proinflammatory Cytokine-, Double-stranded RNA- and HIV-1 gp120-mediated Increase in CD11b Expression in BV-2 Microglial Cells—Microglia are activated under various pathological conditions, such as inflammation, viral infection, etc. (1Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar, 20Saha R.N. Pahan K. J. Neurochem. 2003; 86: 1057-1071Crossref PubMed Scopus (90) Google Scholar, 21Gonzalez-Scarano F. Martin-Garcia J. Nat. Rev. Immunol. 2005; 5: 69-81Crossref PubMed Scopus (896) Google Scholar). Because LPS increased the expression of CD11b in microglia through NO, we investigated whether other inducers of microglial activation also increase CD11b expression via NO. Therefore, BV-2 microglial cells were stimulated with proinflammatory cytokines (IL-1β and IFN-γ), HIV-1 coat protein gp120 (22Dawson V.L. Dawson T.M. Uhl G.R. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3256-3259Crossref PubMed Scopus (334) Google Scholar), and double-stranded RNA in the form of polyinosinic-polycytidilic acid (poly(IC)) (23Auch C.J. Saha R.N. Sheikh F.G. Liu X. Pahan K. FEBS Lett. 2004; 563: 223-228Crossref PubMed Scopus (45) Google Scholar) in the presence or absence of l-NIL and PTIO. All four stimuli (IFN-γ, IL-1β, gp120, and poly(IC)) induced the production of NO and increased the expression of CD11b in BV-2 microglial cells (Fig. 4). Similar to the inhibition of LPS-mediated expression of CD11b, either blocking the production of NO by l-NIL or scavenging NO by PTIO knocked down IFN-γ-, IL-1β-, gp120-, and poly(IC)-mediated increase in CD11b mRNA expression (Fig. 4, A-D) suggesting that different neuroinflammatory and neurodegenerative stimuli also up-regulate the expression of CD11b in microglia via NO. LPS-mediated Increase in CD11b Expression in Vivo in the Striatum Depends on NO—Using various approaches, the studies presented above have shown that different neurotoxins increase the expression of CD11b in microglial cells or cultured primary microglia via NO. However, these studies do not indicate whether NO may have the capacity to influence the expression of CD11b in vivo in the CNS. It is increasingly becoming clear that microglial activation plays an important role in the loss of dopaminergic neurons in striatum and nigra of patients with Parkinson disease (3Dauer W. Przedborski S. Neuron. 2003; 39: 889-909Abstract Full Text Full Text PDF PubMed Scopus (4099) Google Scholar, 4Wu D.C. Jackson-Lewis V. Vila M. Tieu K. Teismann P. Vadseth C. Choi D.K. Ischiropoulos H. Przedborski S. J. Neurosci. 2002; 22: 1763-1771Crossref PubMed Google Scholar, 5Carson M.J. Glia. 2002; 40: 218-231Crossref PubMed Scopus (205) Google Scholar). Therefore, we were prompted to investigate whether NO is also involved in the up-regulation of CD11b expression in vivo in the striatum. As expected, the microinjection of LPS but not saline into the striatum of 8-10-week-old male C57BL/6 mice induced the expression of iNOS (Fig. 5B) and CD11b (C and D). Next to analyze the role of NO in LPS-mediated expression of CD11b, PTIO was microinjected together with LPS. As revealed by semiquantitative RT-PCR (Fig. 5C) and quantitative real time PCR (Fig. 5D), PTIO knocked down LPS-mediated increase in CD11b expression in vivo in the striatum (Fig. 5, C and D). This has been further suppo
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