Neurocysticercosis Regulation of Chemokine Secretion in Monocyte

Monocyte-Astrocyte Networks and the Regulation of Chemokine Secretion in Neurocysticercosis

This information is current as Jasim Uddin, Hector H. Garcia, Robert H. Gilman, Armando of September 24, 2021. E. Gonzalez and Jon S. Friedland J Immunol 2005; 175:3273-3281; ; doi: 10.4049/jimmunol.175.5.3273 http://www.jimmunol.org/content/175/5/3273 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Monocyte-Astrocyte Networks and the Regulation of Chemokine Secretion in Neurocysticercosis1

Jasim Uddin,* Hector H. Garcia,†‡ Robert H. Gilman,†§ Armando E. Gonzalez,¶ and Jon S. Friedland2*

Neurocysticercosis, caused by infection with larval Taenia solium, is a major cause of epilepsy worldwide. Larval degeneration, which is symptomatic, results in inflammatory cell influx. Astrocytes, the most abundant cell type and major cytokine-producing cell within the CNS, may be important in orchestrating inflammatory responses after larval degeneration. We investigated the effects of direct stimulation and of conditioned medium from T. solium larval Ag (TsAg)-stimulated monocytes (CoMTsAg) on neutrophil and astrocyte chemokine release. CoMTsAg, but not control conditioned medium, stimulated astrocyte CCL2/MCP-1 ,ng/ml), CXCL8/IL-8 (416 ؎ 6.2 ng/ml), and CXCL10/IFN-␥-inducible protein (9.07 ؎ 0.6 ng/ml) secretion after 24 h 16 ؎ 161.5) whereas direct astrocyte or neutrophil stimulation with TsAg had no effect. There was rapid accumulation of CCL2 and CXCL8 Downloaded from mRNA within 1 h, with somewhat delayed expression of CXCL10 mRNA initially detected 8 h poststimulation. Neutralizing anti-TNF-␣ inhibited CoMTsAg-induced CCL2 mRNA accumulation by up to 99%, causing total abolition of CXCL10 and up to 77% reduction in CXCL8 mRNA. CoMTsAg induced maximal nuclear binding of NF-␬B p65 and p50 by 1 h, with I␬B␣ and I␬B␤ decay within 15 min. In addition, CoMTsAg induced transient nuclear binding of AP-1, which peaked 4 h poststimulation. In NF-␬B blocking experiments using pyrrolidine dithiocarbamate, CoMTsAg-induced CCL2 secretion was reduced /In summary, the data show that http://www.jimmunol.org .(0.0003 ؍ whereas CXCL8 was inhibited by up to 75% (p ,(0.0006 ؍ by up to 80% (p astrocytes are an important source of chemokines following larval Ag stimulation. Such chemokine secretion is NF-␬B dependent, likely to involve AP-1, and is regulated in a paracrine loop by monocyte-derived TNF-␣. The Journal of Immunology, 2005, 175: 3273–3281.

eurocysticercosis (NCC),3 caused by CNS infection with Clinical symptoms usually present after prolonged asymptom- larvae of the parasite Taenia solium, affects 50 million atic periods and depend on the size, location, and burden of infec- N people worldwide (1, 2). Infection is endemic in South tion (3). Epilepsy is the most common manifestation; in disease America, Asia, and sub-Saharan Africa, with increasing preva- endemic regions, 25% of epilepsy may be due to NCC (9–12). The by guest on September 24, 2021 lence in countries such as the United States as a result of emigra- transition from asymptomatic to symptomatic disease is thought to tion from endemic areas (3–5). Infection is acquired via the fecal- depend on cyst degeneration, a process that may be accelerated by oral route following ingestion of microscopic Taenia eggs (6). antiparasite therapy (13). Studies in animal models suggest that the People with no direct contact with infected pigs are at risk of intact parasite maintains a Th2-permissive environment. The in- infection due to contamination of food or water by human tape- flammatory response that leads to larval degeneration is charac- worm carriers (7, 8). In the human gut, Taenia eggs differentiate to terized by a switch to a Th1-type cytokine profile, overexaggera- invasive oncospheres, which penetrate the mucosal lining and en- tion of which results in a chronic inflammatory response involving ter the general circulation from where they disseminate and accu- granuloma formation and tissue destruction (14, 15). Brain gran- mulate preferentially in the CNS forming fluid-filled cysticerci (4). ulomas from NCC patients consist of mononuclear cells, granulo- cytes, and CNS microglial cells (16, 17). A prerequisite for gran- *Department of Infectious Diseases, Faculty of Medicine and the Wellcome Trust uloma formation is cell influx to sites of larval degeneration, and Centre for Clinical Tropical Medicine, Imperial College (Hammersmith Campus), this will involve chemokines, which are fundamental in controlling † London, United Kingdom; Departments of Microbiology and Pathology, Univer- cell trafficking (18). The source of such chemokines in NCC has sidad Peruana Cayetano Heredia, Lima, Peru; ‡Cysticercosis Unit, Instituto Nacional de Ciencias Neurolo´gicas, Lima, Peru; §Department of International Health, Johns not been investigated, although astrocytes that surround the perim- Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205; and eter of granulomas (17) are able to secrete a range of cytokines and ¶School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru may be important in orchestrating granuloma formation. Received for publication January 19, 2005. Accepted for publication June 13, 2005. Astrocytes are the most abundant cell type in the CNS, consti- tuting 50–75% of the total cell number (19, 20). Astrocytes and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance microglia function as primary immune effector cells of the CNS with 18 U.S.C. Section 1734 solely to indicate this fact. (21, 22). In addition, astrocytes have a central function in the main- 1 This study was funded by the National Institutes of Health Grants AI-42037-01 and tenance of the blood-brain barrier (BBB), thereby indirectly con- AI-35894, International Training in Research in Emerging Diseases Training Grant TW00910, and The Wellcome Trust of Great Britain. tributing to control of CNS leukocyte trafficking (23). Astrocytes directly regulate CNS leukocyte invasion, secreting and expressing 2 Address correspondence and reprint requests to Prof. Jon S. Friedland, Department of Infectious Diseases, Imperial College London, Hammersmith Campus, Du Cane a number of chemokines and their associated receptors (24–26). Road, London W12 ONN, U.K. E-mail address: [email protected] TNF-␣ is an important stimulus for CNS chemokine expression 3 Abbreviations used in this paper: NCC, neurocysticercosis; BBB, blood-brain bar- (27). The role of chemokines in CNS infection has been most rier; CoM, conditioned medium; CoMCon, control conditioned medium; PDTC, pyrrolidine dithiocarbamate; PMNL, polymorphonuclear leukocyte; TsAg, Taenia so- widely studied in viral infections, particularly HIV, where astro- lium larval Ag. cyte chemokine secretion and chemokine receptor expression is

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 3274 CHEMOKINE SECRETION IN NCC important in mediating initial cellular entry and subsequent CNS moved, aliquoted, and stored. Conditioned medium from unstimulated hu- pathology (25, 28, 29). Astrocyte-derived chemokines also con- man monocytes cultured for 8 h (control conditioned medium; CoMCon) ϫ 5 2 tribute to pathology after CNS infection with the intracellular par- was the negative control. U373MG cells (1 10 cells/cm ) were seeded in 6-well tissue culture plates, 100-mm- or 150-mm-diameter dishes as asites Trypanosoma brucei and Toxoplasma gondii (30, 31). appropriate for preparation of cell supernatants, RNA, or nuclear proteins. We have investigated astrocyte chemokine secretion in NCC. Cells were stimulated in triplicate for each experimental condition with Our data demonstrate for the first time that astrocytes but not neu- either CoMTsAg or CoMCon at dilutions of 1/10, 1/50, or 1/100 for de- trophils are a major source of the chemokines CCL2, CXCL8, and fined time points. Cell-free supernatants, cellular RNA, whole cell lysates, or nuclear extracts were subsequently collected and stored at either Ϫ20°C CXCL10. Chemokine secretion does not follow direct exposure to or Ϫ80°C (as appropriate) before being assayed. neurocysticercal (T. solium) Ags (TsAg) but is dependent on In cytokine-neutralizing experiments, U373MG cells were preincubated TNF-␣ derived from monocytes stimulated with TsAg and is reg- with various doses of IL-1 receptor antagonist for 2 h before stimulation ulated at the transcriptional level by an NF-␬B-dependent mech- with CoMTsAg. The role of TNF-␣ was investigated by preincubating ␣ anism. Such CNS networks may be important in increasing in- CoMTsAg for 2 h with various concentrations of neutralizing anti-TNF- Ab before cellular stimulation. In NF-␬B blocking experiments, U373MG flammatory cell influx and contributing to the pathology of NCC. cells were preincubated with 1, 10, or 100 ␮M pyrrolidine dithiocarbamate (PDTC; a broad spectrum NF-␬B inhibitor) for 2 h before being stimulated Materials and Methods with either CoMTsAg or TNF-␣ (10 ng/ml) for 24 h. Reagents

RPMI 1640, HBSS, Eagle’s MEM, L-glutamine, and sodium pyruvate were Chemokine ELISAs obtained from Invitrogen Life Technologies. FCS (endotoxin level Ͻ0.06 ng/ml) was obtained from Labtech International. Ficoll-Paque, nitrocellu- CXCL8/IL-8 and CCL2/MCP-1 protein concentrations were measured us- Downloaded from lose membrane (Hybond-C), nylon membrane (Hybond-N), ECL Hyper- ing ELISAs based on matched Ab pairs (R&D Systems). The lower limit film, and [␥-32P]ATP were obtained from Amersham Biosciences. Recom- of sensitivity of the CCL2 and CXCL8 assays was 15 pg/ml. The level of binant human TNF-␣, IL-1 receptor antagonist, and neutralizing polyclonal CXCL10 (IFN-␥-inducible protein) was measured using cytosets, accord- rabbit anti-human TNF-␣ were obtained from PeproTech. The RNase pro- ing to manufacturer’s protocols (BioSource International). The lower limit tection assay system was from BD Pharmingen, and the DIG Wash and of detection was 30 pg/ml. Blot set and CDP-Star were from Roche. The avidin-phosphatase was obtained from Tropix. The NF-␬B, AP-1, and SP-1 consensus oligos and the T4 polynucleotide kinase were purchased from Promega. Premade 30% RNA isolation and RNase protection assay http://www.jimmunol.org/ bisacrylamide stock used for PAGE was obtained from Anachem. Rabbit Total cellular RNA was extracted from U373MG cells using a guanidine anti-human p65, p50, p52, c-Rel, rel-B, c-Fos, and I␬B␣␤ Abs were ob- thiocyanate and phenol mixture (Tri-reagent; Sigma-Aldrich). RNA was tained from Santa Cruz Biotechnology. HRP for chemokine ELISAs was precipitated with isopropanol and washed with 70% ethanol before being purchased from BioSource International. All other materials and reagents dissolved in RNase-free water. mRNA analysis was conducted using a were purchased from Sigma-Aldrich. modified version of the RiboQuant multiprobe RNase protection assay pro- Preparation of TsAg tocol (BD Pharmingen). RNA (15 ␮g) was hybridized overnight with a multiprobe set containing cDNA templates for human XCL1, CCL5, T. solium metacestodes (cysticerci) were dissected at postmortem from CXCL10, CCL4, CCL3, CCL2, CXCL8, CCL1, and housekeeping genes naturally infected pigs obtained from endemic areas in the Peruvian high- L32 and GAPDH (hCK-5; BD Pharmingen), which had been biotinylated lands. Metacestodes were homogenized in cold-buffered PBS using a glass and in vitro transcribed using T7 RNA polymerase. After hybridization, by guest on September 24, 2021 homogenizer. Ag suspensions were subsequently prepared by sonication at RNA mixtures were treated with an RNase mixture at 30°C for 45 min, and 70 Hz for 3 min before storage at Ϫ70°C. Concentration of protein in the protected mRNA species were then precipitated and separated ona8M Ag preparation, which was used as a suspension, was quantified using a urea-5% polyacrylamide gel before being transferred to Hybond-N by elec- Bradford assay (Bio-Rad). Endotoxin contamination, measured using the troblotting and fixed by UV exposure (UV Stratalinker 1800; Stratagene). Limulus amebocyte lysate assay, was minimal (between 0.11 and 5.46 pg/ Blots were blocked overnight before incubation with alkaline phosphatase. ml) and did not induce chemokine secretion in vitro. After three washes, the substrate CDP-Star was added, and blots were exposed to enhanced chemiluminescence Hyperfilm for up to 1 h. Images Cell culture of U373MG cells, monocytes, and neutrophils were scanned and analyzed using Scion Image vBeta 4.0.2 (Scion). Data The human astrocytic U373MG cell line (no. 89081403; European Collec- were normalized using housekeeping genes L32 and GAPDH. tion of Cell Cultures) was maintained in Eagle’s MEM supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential ␮ EMSA amino acids, and 100 g/ml ampicillin, in a humidified 5% CO2 atmosphere at 37°C. Confluent cultures were passaged with 0.25% trypsin- Nuclear extracts were prepared from U373MG cells grown in 150-mm- ϫ 2 EDTA, seeding at a density of 3 10,000 cells/cm . Primary human pe- diameter tissue culture dishes, according to Clarke et al. (32). Briefly, cells ripheral blood monocytes and polymorphonuclear leukocytes (PMNLs) were washed with ice-cold HBSS before extraction with a hypotonic buffer were prepared from pooled buffy-coat residues from healthy donors (North (5 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 containing protease London Blood Transfusion Service). Briefly, mononuclear cells and inhibitors leupeptin, aprotinin, pepstatin, bestatin, and PMSF, all at 1 ␮g/ PMNLs were isolated by density gradient centrifugation over Ficoll-Paque. ml). After addition of 0.25% Nonidet P-40, nuclei were pelleted, superna- Monocytes were adhesion purified on tissue culture plastic for 1 h before tant was removed, and nuclei were resuspended in a hypertonic buffer (5 being washed three times with sterile HBSS to remove nonadherent lym- mM HEPES, pH 7.9, 0.5 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% phocytes. PMNLs were separated from RBCs by hypertonic lysis followed glycerol). After equilibration for2hat4°C, nuclei were pelleted and sol- by two washes using HBSS. Purified cells were maintained in RPMI 1640 Ϫ ␮ uble nuclear extract was aspirated and stored at 80°C. After protein con- medium supplemented with 10% FCS, 2 mM glutamine, and 100 g/ml centrations were quantified by the Bradford assay, EMSAs were performed ampicillin, at 37°C in a humidified 5% CO2 atmosphere. Giemsa staining ␬ Ͼ as follows. Double-stranded NF- B or AP-1 consensus oligonucleotides confirmed that 90% of the PMNLs were neutrophils. were end labeled using [␥-32P]ATP and T4 polynucleotide kinase. Nuclear 8 Experimental protocol extracts (5–7 ␮g) and labeled oligo probes (specific activity Ͼ1 ϫ 10 cpm) were mixed in binding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM Monocytes, U373MG cells, and PMNLs were seeded at a density of 1 ϫ EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25 105,1ϫ 105,or2ϫ 105 cells/cm2, respectively, in 6-well tissue culture mg/ml poly(dI:dC)-poly(dI:dC)) for 20 min at room temperature before plates. Cells were stimulated in triplicate with 100 ␮g/ml TsAg or various being subjected to 5% nondenaturing PAGE and autoradiography. Probe- doses of LPS (from Escherichia coli serotype 0127:B8) for 24 h (mono- binding specificity was confirmed in competition assays using 50-fold mo- cytes and U373MG cells) or 14 h (neutrophils). Cell-free supernatants were lar excess of cold, unlabeled NF-␬B, AP-1, or SP-1 oligos. Supershift subsequently collected and stored at Ϫ20°C before assay. analysis to determine NF-␬B subunit binding was performed by adding 1 In experiments investigating monocyte-astrocyte networks, conditioned ␮g of specific Abs raised against human p65, p50, p52, c-Rel, rel-B, or medium was prepared by stimulating monocytes with 100 ␮g/ml TsAg c-Fos (a subunit of AP-1) to the binding mixture 40 min before addition of (CoMTsAg) for 8 h, after which sterile, cell-free supernatants were re- radiolabeled oligo. The Journal of Immunology 3275

stimuli by producing chemokines (34, 35). At 24 h, monocytes secreted signiﬁcant amounts of CCL2 and CXCL8 after direct stimulation with TsAg, whereas astrocytes and PMNLs did not (Fig. 1a). Compared with control, in unstimulated cells that secreted 1.84 Ϯ 0.002 ng/ml CCL2 and 45.3 Ϯ 6.4 ng/ml CXCL8, 100 ␮g/ml TsAg caused a 212-fold induction in the secretion of CCL2 (390 Ϯ 13.5 ng/ml) and a 11-fold induction in CXCL8 (513 Ϯ 43 ng/ml) in monocytes. There was a minimal induction in CXCL8 secretion following stimulation of PMNLs with TsAg, whereas the positive control, LPS (10 ␮g/ml), induced a relatively high level of CXCL8 secretion from PMNLs (17.1 Ϯ 1.7 ng/ml; data not shown). We did not analyze PMNL CXCL8 secretion beyond 14 h because of signiﬁcant decreases in cell viability (data not shown). TsAg-stimulated U373MG cells did not secrete more CCL2 or CXCL8 than control cells. CCL2 and CXCL8 increased in a dose-dependent manner after LPS stimulation, demonstrating that these cells can secrete chemokines in response to microbial stimuli (Fig. 1b). The possibility that these and subsequent data

were due to LPS contamination was excluded by the limulus assay. Downloaded from

CoMTsAg induces secretion of CCL2, CXCL8, and CXCL10 from astrocytic U373MG cells Because TsAg had no direct effect on astrocyte chemokine secretion, we examined whether a cytokine network might be active.

FIGURE 1. Human astrocytic U373MG cells do not secrete CXCL8 or U373MG cells stimulated with CoMTsAg at a 1/10 dilution se- http://www.jimmunol.org/ CCL2 in response to direct stimulation with TsAg. a, Monolayers of creted 28.7 Ϯ 3.3 ng/ml CCL2 within 4 h (Fig. 2a). This increase U373MG cells (astrocytes; 1 ϫ 105/cm2), primary human neutrophils (PMNLs; 2 ϫ 105/cm2), or primary human monocytes (1 ϫ 105/cm2) were stimulated with medium (control) or 100 ␮g/ml TsAg (ϩ) for 24 h. Cell- free supernatants were subsequently harvested and analyzed for CCL2 and CXCL8 protein by ELISA. b, U373MG cells were stimulated with medium (control; Con) or with 0.01, 0.1, 1, or 10 ␮g/ml LPS for 24 h, and CCL2 and CXCL8 protein concentrations in culture supernatants were measured by speciﬁc ELISA. Data are means ϩ SEM of a representative triplicate by guest on September 24, 2021 experiment performed on at least three independent occasions.

Western blotting for I␬B Western blot analysis was performed according to standard procedures (33). Briefly, cell lysates were prepared by adding ice-cold lysis buffer (PBS containing 0.1% SDS, 0.1% Nonidet P-40, 0.5% deoxycholate, 10 mM NaF, 1 mM sodium orthovanadate, 170 ␮g/ml PMSF, and protease inhibitors leupeptin, pepstatin, bestatin, and aprotinin all at 1 ␮g/ml) to 5 ϫ 106 U373MG cells, followed by centrifugation at 800 ϫ g for 5 min at 4°C. Protein concentration was determined by the Bradford assay; equal vol- umes of loading buffer (containing 50 mM HEPES, 10% glycerol, 5% DTT, 2% SDS, and bromphenol blue) were added to 50 ␮g of protein, and samples were boiled for 5 min before being frozen at Ϫ80°C. Samples were resolved on a 10% SDS-PAGE gel, transferred by electroblotting to a nitrocellulose membrane, and probed with 0.5 ␮g of rabbit anti-human I␬B␣ or 0.8 ␮g of rabbit anti-human I␬B␤. After incubation with perox- idase-conjugated goat anti-rabbit IgG, protein bands were visualized by chemiluminescence. Data presentation and statistics Data from ELISAs are means Ϯ SEM of a triplicate experiment performed on at least two independent occasions. Data were analyzed using an unpaired Student’s t test, where p Ͻ 0.05 was taken as significant. RNase protection assays (together with densitometric analysis) and EMSAs shown FIGURE 2. Kinetics of CoMTsAg-induced CCL2, CXCL8, and are representative of at least three independent experiments. CXCL10 secretion from human astrocytic U373MG cells. CoMTsAg was used to stimulate monolayers of U373MG cells (1 ϫ 105/cm2) over 48 h at Results dilutions of 1/10, 1/50, and 1/100. CoMCon was used at a dilution of 1/10. TsAg stimulates cell-specific secretion of CCL2 and CXCL8 in CCL2 (a), CXCL8 (b), and CXCL10 (c) concentrations (indicated by monocytes but not in PMNLs or astrocytes brackets) were measured by ELISA in cell-free culture supernatants at 0, 2, 4, 8, 24, and 48 h after stimulation. Chemokine concentrations at 0 h We first determined which cell types secreted chemokines in re- represent basal monocyte-derived chemokine present in CoMTsAg. Re- sponse to direct stimulation with TsAg. Human neutrophils are sults are mean values ϩ SEM of a triplicate experiment representative of capable of responding to parasitic and proinflammatory microbial three independent experiments. 3276 CHEMOKINE SECRETION IN NCC reached maximal amounts of 161.53 Ϯ 16.02 ng/ml 24 h poststimulation. CoMTsAg was a potent stimulus for CCL2 secretion, as dilutions as low as 1/100 caused 27 Ϯ 0.7 ng/ml CCL2 to be secreted within 8 h. Cells stimulated with CoMTsAg at a dilution of 1/100 did not drive CXCL8 secretion greater than that in CoM- Con-stimulated cells (Fig. 2b). CoMTsAg at a 1/10 dilution resulted in 276.5 Ϯ 36.7 ng/ml CXCL8 secretion at 8 h with concentrations increasing over 48 h (452.40 Ϯ 7.39 ng/ml; Fig. 2b). The higher concentration of CoMTsAg (1/10) was required to induce CXCL10 secretion, and the absolute amounts were low compared with CCL2 and CXCL8 (Fig. 2c). Chemokine values quoted were taken after accounting for monocyte-derived chemokine levels present in the CoMTsAg; this was represented by the baseline chemokine secretion at 0 h. CoMTsAg induces differential CCL2, CXCL8, and CXCL10 mRNA accumulation in astrocytic U373MG cells The kinetics of CCL2, CXCL8, and CXCL10 gene expression determined by RNase protection assay were consistent with protein Downloaded from secretion data. CCL2 mRNA appeared within 1-h stimulation with CoMTsAg and peaked after 4 h before it began to fall after 8 h (Fig. 3, a and b). Kinetics of CXCL8 mRNA accumulation followed a similar pattern, although there was constitutive expression of CXCL8 mRNA at baseline and in CoMCon-stimulated cells

(Fig. 3, a and c). In contrast to CCL2 and CXCL8, the overall http://www.jimmunol.org/ accumulation of CXCL10 mRNA was modest, with initial detection of gene expression occurring 8 h poststimulation and levels rising a further 33% by 24 h (Fig. 3, a and d). Monocyte-derived TNF-␣ is essential for driving CoMTsAg- induced chemokine secretion from U373MG cells To investigate mediators potentially important in CoMTsAg, we focused on the proinflammatory cytokines TNF-␣ and IL-1, which are secreted by human monocytes exposed to a diverse range of by guest on September 24, 2021 pathogens (36–38) and may regulate chemokine secretion in human astrocytic cells (39, 40). Preincubation of CoMTsAg with anti-TNF-␣ caused dose-dependent inhibition of CCL2, CXCL8, and CXCL10 secretion from U373MG cells. CCL2 secretion was most sensitive to anti-TNF-␣, and preincubation with 0.1, 1, and 10 ␮g/ml caused 36, 57, and 73% decreases in CoMTsAg-induced CCL2 secretion, respectively ( p ϭ 0.04, 0.005, and 0.002, respectively; Fig. 4). In contrast, CXCL8 and CXCL10 secretion was less sensitive to anti-TNF-␣ treatment, which at maximal concentrations caused 46 and 69% decreases in CXCL8 and CXCL10 ( p ϭ 0.005 and 0.001, respectively; data not shown). Anti-TNF-␣ also inhibited CoMTsAg-induced chemokine mRNA accumulation (Fig. 5). On densitometric analysis, anti-TNF-␣ at a concentration of 10 ␮g/ml caused an ϳ84% reduction in CCL2 gene expression with almost total abolition of mRNA accumulation after preincubation with 100 ␮g/ml. Anti-TNF-␣ caused ϳ77% inhibition in CXCL8 mRNA accumulation. CXCL10 gene expression is the most sensitive to anti-TNF-␣ as preincubation of CoMTsAg with 10 ␮g/ml caused a 100% inhibition. Because anti-TNF-␣ inhibited chemokine secretion, we first investigated whether TNF-␣ could induce astrocyte chemokine secretion. U373MG cells stimulated with 10 ng/ml TNF-␣ for 24 h secreted 185.3 Ϯ 24.4 ng/ml CCL2, FIGURE 3. Kinetics of CoMTsAg-induced CCL2, CXCL8, and 910.5 Ϯ 52.3 ng/ml CXCL8, and 1132 Ϯ 383 pg/ml CXCL10 CXCL10 mRNA accumulation in astrocytic U373MG cells. Total cellular ϫ 7 (data not shown). Neutralization of this TNF-␣ bioactivity using RNA was extracted at 0, 1, 2, 4, 8, and 24 h from 1 10 U373MG cells 50 ␮g/ml anti-TNF-␣ caused levels of CCL2, CXCL8, and stimulated with either a 1/10 dilution of CoMTsAg or CoMCon (Con; for 24 h). Chemokine mRNA was assessed using a biotinylated multiprobe RNase CXCL10 to return to control (data not shown). In addition, we ␣ protection assay. After computerized analysis, chemokine mRNA densitome- confirmed that TsAg-stimulated monocytes secrete active TNF- try was normalized for loading using L32 and GAPDH densitometry and ex- ␣ into CoMTsAg using the WEHI 164 bioassay. TNF- secretion pressed as relative units. Shown are a representative autoradiograph (a) to- occurs in the first 24 h, with concentrations peaking at 130.1 Ϯ gether with densitometric analysis of CCL2 (b), CXCL8 (c), and CXCL10 (d) 44.9 pg/ml at 8 h. Finally, we showed that TNF-␣ at this concen- mRNA. Data are representative of three independent experiments. The Journal of Immunology 3277

FIGURE 6. CoMTsAg stimulates nuclear translocation of NF-␬B and Downloaded from AP-1 in astrocytic U373MG cells. Nuclear extracts were prepared from FIGURE 4. TNF-␣ mediates CoMTsAg-induced CCL2 secretion from 2 ϫ 107 U373MG cells stimulated with 1/10 diluted CoMTsAg for 0, 0.5, U373MG cells. Aliquots of 1/10 diluted CoMTsAg were preincubated for 1, 2, 4, 8, and 24 h. Equal amounts of nuclear protein (7 ␮g for NF-␬B and 2 h at 37°C in the absence or presence of 0.01, 0.1, 1, or 10 ␮g/ml rabbit 5 ␮g for AP-1) were incubated with 32P-end labeled NF-␬B(a) or AP-1 ␣ anti-human neutralizing anti-TNF- , before stimulation of U373MG cell consensus oligonucleotides (c), and protein-DNA complexes were sepa- ϫ 5 2 monolayers (1 10 /cm ). Cell-free culture supernatants were collected rated by PAGE and visualized by autoradiography. Specificity of NF-␬B after 24 h and analyzed for CCL2 concentrations by ELISA. Differences http://www.jimmunol.org/ and AP-1 binding was demonstrated by incubating extracts obtained either between groups were analyzed for statistical significance using an un- 1 h (NF-␬B) or 4 h (AP-1) poststimulation with a 50-fold molar excess of paired, two-tailed Student’s t test. Results are means ϩ SEM of a triplicate cold, unlabeled specific or nonspecific probe. b, To determine involvement experiment conducted on at least two separate occasions. Unstim, of specific NF-␬B-family members, supershift analysis was performed on unstimulated. extracts obtained from 2-h CoMTsAg-stimulated cells. Extracts were incubated with 1 ␮g of rabbit anti-human Ab specific to NF-␬B subunits p65, tration drove CCL2 and CXCL8 secretion to a similar order of p50, p52, c-Rel, and rel-B (or c-Fos, an unrelated Ab to the AP-1 subunit) magnitude to that described above (data not shown). In contrast, no before addition of 32P-labeled NF-␬B probe. Shown are data from a rep- detectable TNF-␣ was secreted by TsAg-stimulated astrocytes. resentative experiment repeated on at least three separate occasions.

Preincubation of U373MG cells with IL-1 receptor antagonist by guest on September 24, 2021 caused no signiﬁcant inhibition of CoMTsAg-induced chemokine secretion (data not shown). NF-␬B and AP-1. As shown in Fig. 6a, there was rapid NF-␬B activation in U373MG cells 30 min after stimulation with CoMT- CoMTsAg induces nuclear binding of NF-␬B and AP-1 in sAg, which reached maximal levels within 1 h before returning to U373MG cells undetectable levels by 8 h. CoMTsAg-stimulated NF-␬B binding Next, mechanisms important in regulating CoMTsAg-induced chemokine secretion were investigated, focusing on the role of

FIGURE 5. TNF-␣ mediates CoMTsAg-induced CCL2, CXCL8, and CXCL10 mRNA accumulation in U373MG cells. Aliquots of 1/10 diluted CoMTsAg were preincubated for2hat37°C in the absence or presence of FIGURE 7. Kinetics of I␬B␣ and I␬B␤ degradation and resynthesis in 1, 10, or 100 ␮g/ml rabbit anti-human neutralizing anti-TNF-␣, before CoMTsAg-stimulated U373MG cells. Whole cell lysates were prepared stimulation of U373MG cell monolayers (1 ϫ 105/cm2). Control cells were from CoMCon (1/10 dilution)-, CoMTsAg (1/10 dilution)-, and TNF-␣ (10 stimulated with a 1/10 dilution of CoMCon. After 24 h culture, total cellular ng/ml)-stimulated U373MG cells. After Bradford analysis, 50 ␮g of pro- RNA was extracted and puriﬁed, and chemokine mRNA was assessed by tein (with appropriate molecular mass markers) were separated by SDS- biotinylated multiprobe RNase protection assay. A representative autoradio- PAGE, transferred to nitrocellulose membrane, and analyzed for I␬B␣ (a) and graph is shown from three independent experiments, and the densitometric I␬B␤ (b) protein by immunoblotting using speciﬁc Abs. Blots shown are rep- analysis normalized using L32 and GAPDH expression is presented in the text. resentative of experiments performed on at least three separate occasions. 3278 CHEMOKINE SECRETION IN NCC Downloaded from http://www.jimmunol.org/

FIGURE 8. PDTC inhibits CoMTsAg- and TNF-␣-induced CCL2 and CXCL8 secretion from U373MG cells. Monolayers of U373MG cells (1 ϫ 105/cm2) were preincubated with increasing concentrations of PDTC (1, 10, or 100 ␮M) for 2 h before stimulation with CoMTsAg (1/10 dilution; a and ␣

c) or TNF- (10 ng/ml; b and d) for 24 h, after which cell-free culture supernatants were collected and assayed for CCL2 (a and b) and CXCL8 (c and by guest on September 24, 2021 d) by speciﬁc ELISA. Results are expressed as means ϩ SEM of a triplicate experiment, which is representative of two separate experiments. Differences between groups were analyzed for statistical signiﬁcance using an unpaired, two-tailed Student’s t test.

was similar to that induced by TNF-␣ (data not shown). This was binding after TNF-␣ stimulation was similar to that induced by specific and competed out with a 50-fold molar excess of cold CoMTsAg although the magnitude of signal was greater. unlabeled NF-␬B probe but not by the unrelated AP-1 oligonucle- otide (Fig. 6b, right panel). Supershift analysis showed that p65 ␬ and p50 NF-␬B subunits were specifically involved in CoMTsAg- CoMTsAg-induced chemokine secretion is NF- B dependent stimulated NF-␬B activation (Fig. 6b). Supershift data after stim- To further investigate the role of NF-␬B, U373MG cells were pre- ulation with 10 ng/ml TNF-␣ were almost identical (data not treated for 2 h with PDTC, a broad-spectrum NF-␬B inhibitor. shown). NF-␬B activation is regulated in the cytoplasm by inhib- CoMTsAg-induced CCL2 secretion was highly sensitive to PDTC itory I␬B proteins released upon stimulus-specific, phosphoryla- treatment, and 1 ␮M caused 41% reduction in secretion ( p ϭ tion-dependent proteolysis (41). CoMTsAg induced rapid degra- 0.007), with 100 ␮M PDTC causing CCL2 concentrations to fall dation of I␬B␣ within 15 min, with maximal degradation in 2 h, by 80% to levels seen in control cells ( p ϭ 0.0006; Fig. 8a). before complete resynthesis at 4 h (Fig. 7a, middle panel). Deg- CoMTsAg-induced CXCL8 secretion was similarly sensitive to radation of I␬B␤, usually associated with more prolonged NF-␬B PDTC. Pretreatment with 1 ␮M and 100 ␮M PDTC resulted in activation (42), was transient, occurring in 15 min, with maximal CXCL8 secretion decreasing 30% and 75%, respectively ( p ϭ decay by 30 min, before resynthesis within 1 h (Fig. 7b, middle 0.007 and 0.0003; Fig. 8c). In comparison, TNF-␣-induced CCL2 panel). TNF-␣-induced I␬B␣ degradation was more short-lived and CXCL8 secretion was somewhat less sensitive to PDTC (Fig. and biphasic, whereas I␬B␤ degradation was prolonged relative to 8, b and d). that induced by CoMTsAg (Fig. 7). CoMCon did not alter I␬B␤ expression over 24 h (Fig. 7b, top panel), although it did cause limited I␬B␣ decay at 4–24 h, which was consistent with weak late Discussion NF-␬B activation (Fig. 7a, top panel, and data not shown). CCL2, CXCL8, and CXCL10 are chemokines that play key roles CoMTsAg resulted in a delayed AP-1 activation in U373MG in CNS inflammation (43–45). This study demonstrates that para- cells first detected at 1h, with maximal nuclear binding at 4 h (Fig. crine networks between human monocytes and astrocytes after 6c), whereas CoMCon induced no detectable AP-1 nuclear bind- TsAg stimulation are important regulators of CCL2, CXCL8, and ing (data not shown). Specificity of AP-1 binding was confirmed CXCL10 gene expression and secretion. Such chemokine secretion in competition experiments (Fig. 6c). Kinetics of AP-1 nuclear is critically regulated at the transcriptional level by NF-␬B and The Journal of Immunology 3279 involves AP-1 binding to gene promoters. The network is depen- Delayed CXCL10 production by astrocytes may be important in dent on TNF-␣. The data suggest that astrocytes may have a cen- promoting the shift from a protective Th2 profile to the develop- tral role in mediating cell influx after larval degeneration in NCC. ment of Th1 responses associated with progression to symptomatic Astrocytes have a multiplicity of functions (46) and form an NCC (14, 57, 60). essential part of the BBB (47). Such anatomical positioning allows TNF-␣-mediated secretion of CCL2 and CXCL8 from astro- communication between astrocytes and circulating peripheral im- cytes was dependent on NF-␬B, as blockade with PDTC, which mune cells. Interactions between monocyte and astrocytic cells blocks the dissociation of I␬B from cytoplasmic NF-␬B (64), re- have been implicated in regulating CCL2 secretion in both cell sulted in a significant reduction in secretion of both chemokines. In types (48). Our data identify a TNF-␣-dependent monocyte-astro- addition, AP-1 nuclear activation, although not directly correlated cyte network that causes transcription-dependent secretion of to chemokine secretion, was also observed in CoMTsAg-stimu- CCL2, CXCL8, and CXCL10 from astrocytic cells. Astrocytes are lated astrocytes. The activation of this transcription factor was rel- thus activated in NCC, and astrogliosis, a reflection of astrocytic atively delayed when compared with NF-␬B. Such findings are cell activation, has been detected in patients with active NCC (17, consistent with the presence of binding sites for both transcription 49). In contrast, direct stimulation with TsAg did not cause CCL2 factors in the promoters of CCL2, CXCL8, and CXCL10 genes or CXCL8 secretion from astrocytic cells, whereas stimulation (65, 66) and with previous work indicating that TNF-␣ may induce with even low LPS concentrations (0.01 ␮g/ml) was able to induce activation of NF-␬B and AP-1 in astrocytes (67, 68). CoMTsAg high-level secretion of both chemokines, indicating that the bio- induced activation of NF-␬B and AP-1 with a kinetic profile chemical pathways activated by TsAg and LPS are distinct. Sim- roughly equivalent to that induced by 10 ng/ml TNF-␣, although ilarly, neutrophils did not respond to TsAg with chemokine secre- the magnitude of activation in particular for AP-1 was greater after Downloaded from tion. The exact mechanisms by which TsAg drives monocyte TNF-␣ stimulation. Such differences may simply reflect differ- TNF-␣ secretion are the subject of ongoing research, but prelim- ences in TNF-␣ concentrations because CoMTsAg was found to inary data indicate that signaling is not via either TLR 2 or 4. contain TNF-␣ levels of 130 Ϯ 26 pg/ml (data not shown). CoMT- CCL2, important in monocytic cell recruitment (50), was po- sAg induced activation of p65/p50 subunits as did TNF-␣ stimu- tently up-regulated in astrocytes after stimulation with CoMTsAg. lation, which is the most potent NF-␬B family gene transactivator

This may increase influx of blood-derived monocytes into the CNS complex (41). http://www.jimmunol.org/ and migration of resident microglial cells after exposure to cystic- I␬B degradation kinetics revealed distinct differences in re- ercal Ags. Consistent with these results, astrocytes have previously sponses to TNF-␣ and CoMTsAg stimulation. I␬B␣ degradation in been found to be the major source of CCL2 within the CNS (24). response to TNF-␣ was relatively short-lived and showed a bipha- Studies in CCL2Ϫ/Ϫ mice suggested that CCL2 secretion is im- sic profile compared with kinetics obtained after CoMTsAg stim- portant in mediating both entry of monocytic cells and develop- ulation. I␬B␤ degradation was relatively prolonged in response to ment of Th2 immune responses during CNS inflammation (51). TNF-␣. Such differences suggest that TNF-␣ is not solely respon- However, in NCC inflammation is associated with a switch to a sible for mediating the CoMTsAg effects. Th1 phenotype. CCL2 is likely have additional direct affects on In conclusion the data are consistent with a model in which brain endothelial cells (which express CCR2) and therefore on larval degeneration causes the release of previously masked im- by guest on September 24, 2021 BBB permeability (52). munogenic Ags that initially activate microglial cells and the small CXCL8, a potent neutrophil chemoattractant also able to attract number of resident macrophages (present as a result of low-level monocytes and lymphocytes (53–55), was up-regulated in re- transient transendothelial migration through the BBB), which are sponse to CoMTsAg, and the absolute concentrations were higher stimulated to produce and secrete CCL2, CXCL8, and CCL3. Se- when compared with CCL2. In the normal CNS, there are few cretion of these chemokines causes transendothelial migration of PMNLs. CNS neutrophilia is a relatively acute occurrence and is peripheral monocytes and neutrophils across the BBB via chemo- associated with clinically serious brain injury due to raised intra- tactic activity and direct effects on BBB permeability. Cellular cranial pressure, cerebral infarction, and encephalitis, all of which influx of monocytes and neutrophils would be further amplified by are potential complications of anti-helminthic therapy in patients TNF-␣-dependent monocyte-astrocyte networks that increase with NCC (4, 56). Neutrophils have been detected in brain lesions CCL2 and CXCL8 secretion as well as initiate secretion of from NCC patients (16) as well in brain parenchyma early during CXCL10 in a NF-␬B- and/or AP-1-dependent fashion, which the course of experimental NCC in a mouse model of disease (57). drives CNS lymphocyte influx. Such lymphocyte influx and che- CNS neutrophil recruitment mediated primarily by CXCL8 may mokine secretion likely contribute to shaping Th1-type cytokine explain the relatively acute affects of treatment-associated deteri- profiles, which may be important in augmenting larval degenera- oration observed in some NCC patients. tion and in mounting Ag-Ab immune responses. The net result is In contrast to CCL2 and CXCL8, CXCL10 gene expression and cell influx, chronic granulomatous inflammation, tissue damage, secretion by astrocytes in response to CoMTsAg stimulation was and clinical symptomatology. delayed and of a lower order of magnitude. The delayed production of CXCL10, a chemoattractant for activated T cells (58, 59), Disclosures is consistent with the relatively late recruitment of lymphocytes to The authors have no financial conflict of interest. sites of infection and inflammation. T cells are important for granuloma development and have been detected in large numbers in late-stage brain granulomas in NCC patients (6, 60). Evidence References from a murine model of NCC suggests that ␥␦ T cells are one of 1. White, A. C., Jr. 1997. Neurocysticercosis: a major cause of neurological disease the predominant cell types in NCC, and knock-out mice have re- worldwide. Clin. Infect. Dis. 24: 101–113. 2. Roman, G., J. Sotelo, O. Del Brutto, A. Flisser, M. Dumas, N. Wadia, D. Botero, duced neurological symptomatology (61). Although CXCL10 is M. Cruz, H. Garcia, P. R. de Bittencourt, et al. 2000. A proposal to declare able to recruit activated T cells in general, this chemokine prefer- neurocysticercosis an international reportable disease. Bull. World Health Organ. entially recruits T cells expressing the CXCR3 receptor, which is 78: 399–406. 3. Carpio, A. 2002. Neurocysticercosis: an update. Lancet Infect. Dis. 2: 751–762. predominantly expressed on Th1 cells (62). In addition, CXCL10 4. White, A. C., Jr. 2000. Neurocysticercosis: updates on epidemiology, pathogen- may block the recruitment of CCR3 expressing Th2 cells (63). esis, diagnosis, and management. Annu. Rev. Med. 51: 187–206. 3280 CHEMOKINE SECRETION IN NCC

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