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The Journal of Immunology, 2005, 175: 3273-3281.
Copyright © 2005 by The American Association of Immunologists

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

Jasim Uddin*, Hector H. Garcia{dagger},{ddagger}, Robert H. Gilman{dagger},§, Armando E. Gonzalez and Jon S. Friedland2,*

* Department of Infectious Diseases, Faculty of Medicine and the Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College (Hammersmith Campus), London, United Kingdom; {dagger} Departments of Microbiology and Pathology, Universidad Peruana Cayetano Heredia, Lima, Peru; {ddagger} Cysticercosis Unit, Instituto Nacional de Ciencias Neurológicas, Lima, Peru; § Department of International Health, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205; and School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 (161.5 ± 16 ng/ml), CXCL8/IL-8 (416 ± 6.2 ng/ml), and CXCL10/IFN-{gamma}-inducible protein (9.07 ± 0.6 ng/ml) secretion after 24 h, whereas direct astrocyte or neutrophil stimulation with TsAg had no effect. There was rapid accumulation of CCL2 and CXCL8 mRNA within 1 h, with somewhat delayed expression of CXCL10 mRNA initially detected 8 h poststimulation. Neutralizing anti-TNF-{alpha} 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-{kappa}B p65 and p50 by 1 h, with I{kappa}B{alpha} and I{kappa}B{beta} decay within 15 min. In addition, CoMTsAg induced transient nuclear binding of AP-1, which peaked 4 h poststimulation. In NF-{kappa}B blocking experiments using pyrrolidine dithiocarbamate, CoMTsAg-induced CCL2 secretion was reduced by up to 80% (p = 0.0006), whereas CXCL8 was inhibited by up to 75% (p = 0.0003). In summary, the data show that astrocytes are an important source of chemokines following larval Ag stimulation. Such chemokine secretion is NF-{kappa}B dependent, likely to involve AP-1, and is regulated in a paracrine loop by monocyte-derived TNF-{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neurocysticercosis (NCC),3 caused by CNS infection with larvae of the parasite Taenia solium, affects 50 million people worldwide (1, 2). Infection is endemic in South America, Asia, and sub-Saharan Africa, with increasing prevalence in countries such as the United States as a result of emigration from endemic areas (3, 4, 5). Infection is acquired via the fecal-oral route following ingestion of microscopic Taenia eggs (6). People with no direct contact with infected pigs are at risk of infection due to contamination of food or water by human tapeworm carriers (7, 8). In the human gut, Taenia eggs differentiate to invasive oncospheres, which penetrate the mucosal lining and enter the general circulation from where they disseminate and accumulate preferentially in the CNS forming fluid-filled cysticerci (4).

Clinical symptoms usually present after prolonged asymptomatic periods and depend on the size, location, and burden of infection (3). Epilepsy is the most common manifestation; in disease endemic regions, 25% of epilepsy may be due to NCC (9, 10, 11, 12). The transition from asymptomatic to symptomatic disease is thought to depend on cyst degeneration, a process that may be accelerated by antiparasite therapy (13). Studies in animal models suggest that the intact parasite maintains a Th2-permissive environment. The inflammatory response that leads to larval degeneration is characterized by a switch to a Th1-type cytokine profile, overexaggeration of which results in a chronic inflammatory response involving granuloma formation and tissue destruction (14, 15). Brain granulomas from NCC patients consist of mononuclear cells, granulocytes, and CNS microglial cells (16, 17). A prerequisite for granuloma formation is cell influx to sites of larval degeneration, and this will involve chemokines, which are fundamental in controlling cell trafficking (18). The source of such chemokines in NCC has not been investigated, although astrocytes that surround the perimeter of granulomas (17) are able to secrete a range of cytokines and may be important in orchestrating granuloma formation.

Astrocytes are the most abundant cell type in the CNS, constituting 50–75% of the total cell number (19, 20). Astrocytes and microglia function as primary immune effector cells of the CNS (21, 22). In addition, astrocytes have a central function in the maintenance of the blood-brain barrier (BBB), thereby indirectly contributing to control of CNS leukocyte trafficking (23). Astrocytes directly regulate CNS leukocyte invasion, secreting and expressing a number of chemokines and their associated receptors (24, 25, 26). TNF-{alpha} is an important stimulus for CNS chemokine expression (27). The role of chemokines in CNS infection has been most widely studied in viral infections, particularly HIV, where astrocyte chemokine secretion and chemokine receptor expression is important in mediating initial cellular entry and subsequent CNS pathology (25, 28, 29). Astrocyte-derived chemokines also contribute to pathology after CNS infection with the intracellular parasites Trypanosoma brucei and Toxoplasma gondii (30, 31).

We have investigated astrocyte chemokine secretion in NCC. Our data demonstrate for the first time that astrocytes but not neutrophils are a major source of the chemokines CCL2, CXCL8, and CXCL10. Chemokine secretion does not follow direct exposure to neurocysticercal (T. solium) Ags (TsAg) but is dependent on TNF-{alpha} derived from monocytes stimulated with TsAg and is regulated at the transcriptional level by an NF-{kappa}B-dependent mechanism. Such CNS networks may be important in increasing inflammatory cell influx and contributing to the pathology of NCC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

RPMI 1640, HBSS, Eagle’s MEM, L-glutamine, and sodium pyruvate were obtained from Invitrogen Life Technologies. FCS (endotoxin level <0.06 ng/ml) was obtained from Labtech International. Ficoll-Paque, nitrocellulose membrane (Hybond-C), nylon membrane (Hybond-N), ECL Hyperfilm, and [{gamma}-32P]ATP were obtained from Amersham Biosciences. Recombinant human TNF-{alpha}, IL-1 receptor antagonist, and neutralizing polyclonal rabbit anti-human TNF-{alpha} were obtained from PeproTech. The RNase protection assay system was from BD Pharmingen, and the DIG Wash and Blot set and CDP-Star were from Roche. The avidin-phosphatase was obtained from Tropix. The NF-{kappa}B, AP-1, and SP-1 consensus oligos and the T4 polynucleotide kinase were purchased from Promega. Premade 30% bisacrylamide stock used for PAGE was obtained from Anachem. Rabbit anti-human p65, p50, p52, c-Rel, rel-B, c-Fos, and I{kappa}B{alpha}{beta} Abs were obtained from Santa Cruz Biotechnology. HRP for chemokine ELISAs was purchased from BioSource International. All other materials and reagents were purchased from Sigma-Aldrich.

Preparation of TsAg

T. solium metacestodes (cysticerci) were dissected at postmortem from naturally infected pigs obtained from endemic areas in the Peruvian highlands. Metacestodes were homogenized in cold-buffered PBS using a glass homogenizer. Ag suspensions were subsequently prepared by sonication at 70 Hz for 3 min before storage at –70°C. Concentration of protein in the Ag preparation, which was used as a suspension, was quantified using a Bradford assay (Bio-Rad). Endotoxin contamination, measured using the Limulus amebocyte lysate assay, was minimal (between 0.11 and 5.46 pg/ml) and did not induce chemokine secretion in vitro.

Cell culture of U373MG cells, monocytes, and neutrophils

The human astrocytic U373MG cell line (no. 89081403; European Collection of Cell Cultures) was maintained in Eagle’s MEM supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, and 100 µg/ml ampicillin, in a humidified 5% CO2 atmosphere at 37°C. Confluent cultures were passaged with 0.25% trypsin-EDTA, seeding at a density of 3 x 10,000 cells/cm2. Primary human peripheral blood monocytes and polymorphonuclear leukocytes (PMNLs) were prepared from pooled buffy-coat residues from healthy donors (North London Blood Transfusion Service). Briefly, mononuclear cells and PMNLs were isolated by density gradient centrifugation over Ficoll-Paque. Monocytes were adhesion purified on tissue culture plastic for 1 h before being washed three times with sterile HBSS to remove nonadherent lymphocytes. PMNLs were separated from RBCs by hypertonic lysis followed by two washes using HBSS. Purified cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, and 100 µg/ml ampicillin, at 37°C in a humidified 5% CO2 atmosphere. Giemsa staining confirmed that >90% of the PMNLs were neutrophils.

Experimental protocol

Monocytes, U373MG cells, and PMNLs were seeded at a density of 1 x 105, 1 x 105, or 2 x 105 cells/cm2, respectively, in 6-well tissue culture plates. Cells were stimulated in triplicate with 100 µg/ml TsAg or various doses of LPS (from Escherichia coli serotype 0127:B8) for 24 h (monocytes and U373MG cells) or 14 h (neutrophils). Cell-free supernatants were subsequently collected and stored at –20°C before assay.

In experiments investigating monocyte-astrocyte networks, conditioned medium was prepared by stimulating monocytes with 100 µg/ml TsAg (CoMTsAg) for 8 h, after which sterile, cell-free supernatants were removed, aliquoted, and stored. Conditioned medium from unstimulated human monocytes cultured for 8 h (control conditioned medium; CoMCon) was the negative control. U373MG cells (1 x 105 cells/cm2) were seeded in 6-well tissue culture plates, 100-mm- or 150-mm-diameter dishes as appropriate for preparation of cell supernatants, RNA, or nuclear proteins. Cells were stimulated in triplicate for each experimental condition with either CoMTsAg or CoMCon at dilutions of 1/10, 1/50, or 1/100 for defined time points. Cell-free supernatants, cellular RNA, whole cell lysates, or nuclear extracts were subsequently collected and stored at either –20°C or –80°C (as appropriate) before being assayed.

In cytokine-neutralizing experiments, U373MG cells were preincubated with various doses of IL-1 receptor antagonist for 2 h before stimulation with CoMTsAg. The role of TNF-{alpha} was investigated by preincubating CoMTsAg for 2 h with various concentrations of neutralizing anti-TNF-{alpha} Ab before cellular stimulation. In NF-{kappa}B blocking experiments, U373MG cells were preincubated with 1, 10, or 100 µM pyrrolidine dithiocarbamate (PDTC; a broad spectrum NF-{kappa}B inhibitor) for 2 h before being stimulated with either CoMTsAg or TNF-{alpha} (10 ng/ml) for 24 h.

Chemokine ELISAs

CXCL8/IL-8 and CCL2/MCP-1 protein concentrations were measured using ELISAs based on matched Ab pairs (R&D Systems). The lower limit of sensitivity of the CCL2 and CXCL8 assays was 15 pg/ml. The level of CXCL10 (IFN-{gamma}-inducible protein) was measured using cytosets, according to manufacturer’s protocols (BioSource International). The lower limit of detection was 30 pg/ml.

RNA isolation and RNase protection assay

Total cellular RNA was extracted from U373MG cells using a guanidine thiocyanate and phenol mixture (Tri-reagent; Sigma-Aldrich). RNA was precipitated with isopropanol and washed with 70% ethanol before being dissolved in RNase-free water. mRNA analysis was conducted using a modified version of the RiboQuant multiprobe RNase protection assay protocol (BD Pharmingen). RNA (15 µg) was hybridized overnight with a multiprobe set containing cDNA templates for human XCL1, CCL5, CXCL10, CCL4, CCL3, CCL2, CXCL8, CCL1, and housekeeping genes L32 and GAPDH (hCK-5; BD Pharmingen), which had been biotinylated and in vitro transcribed using T7 RNA polymerase. After hybridization, RNA mixtures were treated with an RNase mixture at 30°C for 45 min, and protected mRNA species were then precipitated and separated on a 8 M urea-5% polyacrylamide gel before being transferred to Hybond-N by electroblotting and fixed by UV exposure (UV Stratalinker 1800; Stratagene). Blots were blocked overnight before incubation with alkaline phosphatase. After three washes, the substrate CDP-Star was added, and blots were exposed to enhanced chemiluminescence Hyperfilm for up to 1 h. Images were scanned and analyzed using Scion Image vBeta 4.0.2 (Scion). Data were normalized using housekeeping genes L32 and GAPDH.

EMSA

Nuclear extracts were prepared from U373MG cells grown in 150-mm-diameter tissue culture dishes, according to Clarke et al. (32). Briefly, cells were washed with ice-cold HBSS before extraction with a hypotonic buffer (5 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 containing protease inhibitors leupeptin, aprotinin, pepstatin, bestatin, and PMSF, all at 1 µg/ml). After addition of 0.25% Nonidet P-40, nuclei were pelleted, supernatant was removed, and nuclei were resuspended in a hypertonic buffer (5 mM HEPES, pH 7.9, 0.5 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol). After equilibration for 2 h at 4°C, nuclei were pelleted and soluble nuclear extract was aspirated and stored at –80°C. After protein concentrations were quantified by the Bradford assay, EMSAs were performed as follows. Double-stranded NF-{kappa}B or AP-1 consensus oligonucleotides were end labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase. Nuclear extracts (5–7 µg) and labeled oligo probes (specific activity >1 x 108 cpm) were mixed in binding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25 mg/ml poly(dI:dC)-poly(dI:dC)) for 20 min at room temperature before being subjected to 5% nondenaturing PAGE and autoradiography. Probe-binding specificity was confirmed in competition assays using 50-fold molar excess of cold, unlabeled NF-{kappa}B, AP-1, or SP-1 oligos. Supershift analysis to determine NF-{kappa}B subunit binding was performed by adding 1 µg of specific Abs raised against human p65, p50, p52, c-Rel, rel-B, or c-Fos (a subunit of AP-1) to the binding mixture 40 min before addition of radiolabeled oligo.

Western blotting for I{kappa}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 µg/ml PMSF, and protease inhibitors leupeptin, pepstatin, bestatin, and aprotinin all at 1 µg/ml) to 5 x 106 U373MG cells, followed by centrifugation at 800 x g for 5 min at 4°C. Protein concentration was determined by the Bradford assay; equal volumes of loading buffer (containing 50 mM HEPES, 10% glycerol, 5% DTT, 2% SDS, and bromphenol blue) were added to 50 µg of protein, and samples were boiled for 5 min before being frozen at –80°C. Samples were resolved on a 10% SDS-PAGE gel, transferred by electroblotting to a nitrocellulose membrane, and probed with 0.5 µg of rabbit anti-human I{kappa}B{alpha} or 0.8 µg of rabbit anti-human I{kappa}B{beta}. After incubation with peroxidase-conjugated goat anti-rabbit IgG, protein bands were visualized by chemiluminescence.

Data presentation and statistics

Data from ELISAs are means ± SEM of a triplicate experiment performed on at least two independent occasions. Data were analyzed using an unpaired Student’s t test, where p < 0.05 was taken as significant. RNase protection assays (together with densitometric analysis) and EMSAs shown are representative of at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TsAg stimulates cell-specific secretion of CCL2 and CXCL8 in monocytes but not in PMNLs or astrocytes

We first determined which cell types secreted chemokines in response to direct stimulation with TsAg. Human neutrophils are capable of responding to parasitic and proinflammatory microbial 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 ± 0.002 ng/ml CCL2 and 45.3 ± 6.4 ng/ml CXCL8, 100 µg/ml TsAg caused a 212-fold induction in the secretion of CCL2 (390 ± 13.5 ng/ml) and a 11-fold induction in CXCL8 (513 ± 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 µg/ml), induced a relatively high level of CXCL8 secretion from PMNLs (17.1 ± 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.



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  		FIGURE 1. Human astrocytic U373MG cells do not secrete CXCL8 or CCL2 in response to direct stimulation with TsAg. a, Monolayers of U373MG cells (astrocytes; 1 x 105/cm2), primary human neutrophils (PMNLs; 2 x 105/cm2), or primary human monocytes (1 x 105/cm2) were stimulated with medium (control) or 100 µg/ml TsAg (+) 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 µg/ml LPS for 24 h, and CCL2 and CXCL8 protein concentrations in culture supernatants were measured by specific ELISA. Data are means + SEM of a representative triplicate experiment performed on at least three independent occasions.

 
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. U373MG cells stimulated with CoMTsAg at a 1/10 dilution secreted 28.7 ± 3.3 ng/ml CCL2 within 4 h (Fig. 2a). This increase reached maximal amounts of 161.53 ± 16.02 ng/ml 24 h poststimulation. CoMTsAg was a potent stimulus for CCL2 secretion, as dilutions as low as 1/100 caused 27 ± 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 CoMCon-stimulated cells (Fig. 2b). CoMTsAg at a 1/10 dilution resulted in 276.5 ± 36.7 ng/ml CXCL8 secretion at 8 h with concentrations increasing over 48 h (452.40 ± 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.



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  		FIGURE 2. Kinetics of CoMTsAg-induced CCL2, CXCL8, and CXCL10 secretion from human astrocytic U373MG cells. CoMTsAg was used to stimulate monolayers of U373MG cells (1 x 105/cm2) over 48 h at dilutions of 1/10, 1/50, and 1/100. CoMCon was used at a dilution of 1/10. CCL2 (a), CXCL8 (b), and CXCL10 (c) concentrations (indicated by 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 represent basal monocyte-derived chemokine present in CoMTsAg. Results are mean values + SEM of a triplicate experiment representative of three independent experiments.

 
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 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 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).



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  		FIGURE 3. Kinetics of CoMTsAg-induced CCL2, CXCL8, and CXCL10 mRNA accumulation in astrocytic U373MG cells. Total cellular RNA was extracted at 0, 1, 2, 4, 8, and 24 h from 1 x 107 U373MG cells stimulated with either a 1/10 dilution of CoMTsAg or CoMCon (Con; for 24 h). Chemokine mRNA was assessed using a biotinylated multiprobe RNase protection assay. After computerized analysis, chemokine mRNA densitometry was normalized for loading using L32 and GAPDH densitometry and expressed as relative units. Shown are a representative autoradiograph (a) together with densitometric analysis of CCL2 (b), CXCL8 (c), and CXCL10 (d) mRNA. Data are representative of three independent experiments.

 
Monocyte-derived TNF-{alpha} 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-{alpha} and IL-1, which are secreted by human monocytes exposed to a diverse range of pathogens (36, 37, 38) and may regulate chemokine secretion in human astrocytic cells (39, 40). Preincubation of CoMTsAg with anti-TNF-{alpha} caused dose-dependent inhibition of CCL2, CXCL8, and CXCL10 secretion from U373MG cells. CCL2 secretion was most sensitive to anti-TNF-{alpha}, and preincubation with 0.1, 1, and 10 µg/ml caused 36, 57, and 73% decreases in CoMTsAg-induced CCL2 secretion, respectively (p = 0.04, 0.005, and 0.002, respectively; Fig. 4). In contrast, CXCL8 and CXCL10 secretion was less sensitive to anti-TNF-{alpha} treatment, which at maximal concentrations caused 46 and 69% decreases in CXCL8 and CXCL10 (p = 0.005 and 0.001, respectively; data not shown). Anti-TNF-{alpha} also inhibited CoMTsAg-induced chemokine mRNA accumulation (Fig. 5). On densitometric analysis, anti-TNF-{alpha} at a concentration of 10 µg/ml caused an ~84% reduction in CCL2 gene expression with almost total abolition of mRNA accumulation after preincubation with 100 µg/ml. Anti-TNF-{alpha} caused ~77% inhibition in CXCL8 mRNA accumulation. CXCL10 gene expression is the most sensitive to anti-TNF-{alpha} as preincubation of CoMTsAg with 10 µg/ml caused a 100% inhibition. Because anti-TNF-{alpha} inhibited chemokine secretion, we first investigated whether TNF-{alpha} could induce astrocyte chemokine secretion. U373MG cells stimulated with 10 ng/ml TNF-{alpha} for 24 h secreted 185.3 ± 24.4 ng/ml CCL2, 910.5 ± 52.3 ng/ml CXCL8, and 1132 ± 383 pg/ml CXCL10 (data not shown). Neutralization of this TNF-{alpha} bioactivity using 50 µg/ml anti-TNF-{alpha} caused levels of CCL2, CXCL8, and CXCL10 to return to control (data not shown). In addition, we confirmed that TsAg-stimulated monocytes secrete active TNF-{alpha} into CoMTsAg using the WEHI 164 bioassay. TNF-{alpha} secretion occurs in the first 24 h, with concentrations peaking at 130.1 ± 44.9 pg/ml at 8 h. Finally, we showed that TNF-{alpha} at this concentration drove CCL2 and CXCL8 secretion to a similar order of magnitude to that described above (data not shown). In contrast, no detectable TNF-{alpha} was secreted by TsAg-stimulated astrocytes. Preincubation of U373MG cells with IL-1 receptor antagonist caused no significant inhibition of CoMTsAg-induced chemokine secretion (data not shown).



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  		FIGURE 4. TNF-{alpha} mediates CoMTsAg-induced CCL2 secretion from U373MG cells. Aliquots of 1/10 diluted CoMTsAg were preincubated for 2 h at 37°C in the absence or presence of 0.01, 0.1, 1, or 10 µg/ml rabbit anti-human neutralizing anti-TNF-{alpha}, before stimulation of U373MG cell monolayers (1 x 105/cm2). Cell-free culture supernatants were collected after 24 h and analyzed for CCL2 concentrations by ELISA. Differences between groups were analyzed for statistical significance using an unpaired, two-tailed Student’s t test. Results are means + SEM of a triplicate experiment conducted on at least two separate occasions. Unstim, unstimulated.

 


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  		FIGURE 5. TNF-{alpha} mediates CoMTsAg-induced CCL2, CXCL8, and CXCL10 mRNA accumulation in U373MG cells. Aliquots of 1/10 diluted CoMTsAg were preincubated for 2 h at 37°C in the absence or presence of 1, 10, or 100 µg/ml rabbit anti-human neutralizing anti-TNF-{alpha}, before stimulation of U373MG cell monolayers (1 x 105/cm2). Control cells were stimulated with a 1/10 dilution of CoMCon. After 24 h culture, total cellular RNA was extracted and purified, and chemokine mRNA was assessed by biotinylated multiprobe RNase protection assay. A representative autoradiograph is shown from three independent experiments, and the densitometric analysis normalized using L32 and GAPDH expression is presented in the text.

 
CoMTsAg induces nuclear binding of NF-{kappa}B and AP-1 in U373MG cells

Next, mechanisms important in regulating CoMTsAg-induced chemokine secretion were investigated, focusing on the role of NF-{kappa}B and AP-1. As shown in Fig. 6a, there was rapid NF-{kappa}B activation in U373MG cells 30 min after stimulation with CoMTsAg, which reached maximal levels within 1 h before returning to undetectable levels by 8 h. CoMTsAg-stimulated NF-{kappa}B binding was similar to that induced by TNF-{alpha} (data not shown). This was specific and competed out with a 50-fold molar excess of cold unlabeled NF-{kappa}B probe but not by the unrelated AP-1 oligonucleotide (Fig. 6b, right panel). Supershift analysis showed that p65 and p50 NF-{kappa}B subunits were specifically involved in CoMTsAg-stimulated NF-{kappa}B activation (Fig. 6b). Supershift data after stimulation with 10 ng/ml TNF-{alpha} were almost identical (data not shown). NF-{kappa}B activation is regulated in the cytoplasm by inhibitory I{kappa}B proteins released upon stimulus-specific, phosphorylation-dependent proteolysis (41). CoMTsAg induced rapid degradation of I{kappa}B{alpha} within 15 min, with maximal degradation in 2 h, before complete resynthesis at 4 h (Fig. 7a, middle panel). Degradation of I{kappa}B{beta}, usually associated with more prolonged NF-{kappa}B activation (42), was transient, occurring in 15 min, with maximal decay by 30 min, before resynthesis within 1 h (Fig. 7b, middle panel). TNF-{alpha}-induced I{kappa}B{alpha} degradation was more short-lived and biphasic, whereas I{kappa}B{beta} degradation was prolonged relative to that induced by CoMTsAg (Fig. 7). CoMCon did not alter I{kappa}B{beta} expression over 24 h (Fig. 7b, top panel), although it did cause limited I{kappa}B{alpha} decay at 4–24 h, which was consistent with weak late NF-{kappa}B activation (Fig. 7a, top panel, and data not shown).



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  		FIGURE 6. CoMTsAg stimulates nuclear translocation of NF-{kappa}B and AP-1 in astrocytic U373MG cells. Nuclear extracts were prepared from 2 x 107 U373MG cells stimulated with 1/10 diluted CoMTsAg for 0, 0.5, 1, 2, 4, 8, and 24 h. Equal amounts of nuclear protein (7 µg for NF-{kappa}B and 5 µg for AP-1) were incubated with 32P-end labeled NF-{kappa}B (a) or AP-1 consensus oligonucleotides (c), and protein-DNA complexes were separated by PAGE and visualized by autoradiography. Specificity of NF-{kappa}B and AP-1 binding was demonstrated by incubating extracts obtained either 1 h (NF-{kappa}B) or 4 h (AP-1) poststimulation with a 50-fold molar excess of cold, unlabeled specific or nonspecific probe. b, To determine involvement of specific NF-{kappa}B-family members, supershift analysis was performed on extracts obtained from 2-h CoMTsAg-stimulated cells. Extracts were incubated with 1 µg of rabbit anti-human Ab specific to NF-{kappa}B subunits p65, p50, p52, c-Rel, and rel-B (or c-Fos, an unrelated Ab to the AP-1 subunit) before addition of 32P-labeled NF-{kappa}B probe. Shown are data from a representative experiment repeated on at least three separate occasions.

 


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  		FIGURE 7. Kinetics of I{kappa}B{alpha} and I{kappa}B{beta} degradation and resynthesis in CoMTsAg-stimulated U373MG cells. Whole cell lysates were prepared from CoMCon (1/10 dilution)-, CoMTsAg (1/10 dilution)-, and TNF-{alpha} (10 ng/ml)-stimulated U373MG cells. After Bradford analysis, 50 µg of protein (with appropriate molecular mass markers) were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed for I{kappa}B{alpha} (a) and I{kappa}B{beta} (b) protein by immunoblotting using specific Abs. Blots shown are representative of experiments performed on at least three separate occasions.

 
CoMTsAg resulted in a delayed AP-1 activation in U373MG cells first detected at 1h, with maximal nuclear binding at 4 h (Fig. 6c), whereas CoMCon induced no detectable AP-1 nuclear binding (data not shown). Specificity of AP-1 binding was confirmed in competition experiments (Fig. 6c). Kinetics of AP-1 nuclear binding after TNF-{alpha} stimulation was similar to that induced by CoMTsAg although the magnitude of signal was greater.

CoMTsAg-induced chemokine secretion is NF-{kappa}B dependent

To further investigate the role of NF-{kappa}B, U373MG cells were pretreated for 2 h with PDTC, a broad-spectrum NF-{kappa}B inhibitor. CoMTsAg-induced CCL2 secretion was highly sensitive to PDTC treatment, and 1 µM caused 41% reduction in secretion (p = 0.007), with 100 µM PDTC causing CCL2 concentrations to fall by 80% to levels seen in control cells (p = 0.0006; Fig. 8a). CoMTsAg-induced CXCL8 secretion was similarly sensitive to PDTC. Pretreatment with 1 µM and 100 µM PDTC resulted in CXCL8 secretion decreasing 30% and 75%, respectively (p = 0.007 and 0.0003; Fig. 8c). In comparison, TNF-{alpha}-induced CCL2 and CXCL8 secretion was somewhat less sensitive to PDTC (Fig. 8, b and d).



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  		FIGURE 8. PDTC inhibits CoMTsAg- and TNF-{alpha}-induced CCL2 and CXCL8 secretion from U373MG cells. Monolayers of U373MG cells (1 x 105/cm2) were preincubated with increasing concentrations of PDTC (1, 10, or 100 µM) for 2 h before stimulation with CoMTsAg (1/10 dilution; a and c) or TNF-{alpha} (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 d) by specific ELISA. Results are expressed as means + 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CCL2, CXCL8, and CXCL10 are chemokines that play key roles in CNS inflammation (43, 44, 45). This study demonstrates that paracrine networks between human monocytes and astrocytes after TsAg stimulation are important regulators of CCL2, CXCL8, and CXCL10 gene expression and secretion. Such chemokine secretion is critically regulated at the transcriptional level by NF-{kappa}B and involves AP-1 binding to gene promoters. The network is dependent on TNF-{alpha}. The data suggest that astrocytes may have a central role in mediating cell influx after larval degeneration in NCC.

Astrocytes have a multiplicity of functions (46) and form an essential part of the BBB (47). Such anatomical positioning allows communication between astrocytes and circulating peripheral immune cells. Interactions between monocyte and astrocytic cells have been implicated in regulating CCL2 secretion in both cell types (48). Our data identify a TNF-{alpha}-dependent monocyte-astrocyte network that causes transcription-dependent secretion of CCL2, CXCL8, and CXCL10 from astrocytic cells. Astrocytes are thus activated in NCC, and astrogliosis, a reflection of astrocytic cell activation, has been detected in patients with active NCC (17, 49). In contrast, direct stimulation with TsAg did not cause CCL2 or CXCL8 secretion from astrocytic cells, whereas stimulation with even low LPS concentrations (0.01 µg/ml) was able to induce high-level secretion of both chemokines, indicating that the biochemical pathways activated by TsAg and LPS are distinct. Similarly, neutrophils did not respond to TsAg with chemokine secretion. The exact mechanisms by which TsAg drives monocyte TNF-{alpha} secretion are the subject of ongoing research, but preliminary data indicate that signaling is not via either TLR 2 or 4.

CCL2, important in monocytic cell recruitment (50), was potently up-regulated in astrocytes after stimulation with CoMTsAg. This may increase influx of blood-derived monocytes into the CNS and migration of resident microglial cells after exposure to cysticercal Ags. Consistent with these results, astrocytes have previously been found to be the major source of CCL2 within the CNS (24). Studies in CCL2–/– mice suggested that CCL2 secretion is important in mediating both entry of monocytic cells and development of Th2 immune responses during CNS inflammation (51). However, in NCC inflammation is associated with a switch to a Th1 phenotype. CCL2 is likely have additional direct affects on brain endothelial cells (which express CCR2) and therefore on BBB permeability (52).

CXCL8, a potent neutrophil chemoattractant also able to attract monocytes and lymphocytes (53, 54, 55), was up-regulated in response to CoMTsAg, and the absolute concentrations were higher when compared with CCL2. In the normal CNS, there are few PMNLs. CNS neutrophilia is a relatively acute occurrence and is associated with clinically serious brain injury due to raised intracranial pressure, cerebral infarction, and encephalitis, all of which are potential complications of anti-helminthic therapy in patients with NCC (4, 56). Neutrophils have been detected in brain lesions from NCC patients (16) as well in brain parenchyma early during the course of experimental NCC in a mouse model of disease (57). CNS neutrophil recruitment mediated primarily by CXCL8 may explain the relatively acute affects of treatment-associated deterioration observed in some NCC patients.

In contrast to CCL2 and CXCL8, CXCL10 gene expression and secretion by astrocytes in response to CoMTsAg stimulation was delayed and of a lower order of magnitude. The delayed production of CXCL10, a chemoattractant for activated T cells (58, 59), is consistent with the relatively late recruitment of lymphocytes to 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 from a murine model of NCC suggests that {gamma}{delta} T cells are one of the predominant cell types in NCC, and knock-out mice have reduced neurological symptomatology (61). Although CXCL10 is able to recruit activated T cells in general, this chemokine preferentially recruits T cells expressing the CXCR3 receptor, which is predominantly expressed on Th1 cells (62). In addition, CXCL10 may block the recruitment of CCR3 expressing Th2 cells (63). Delayed CXCL10 production by astrocytes may be important in promoting the shift from a protective Th2 profile to the development of Th1 responses associated with progression to symptomatic NCC (14, 57, 60).

TNF-{alpha}-mediated secretion of CCL2 and CXCL8 from astrocytes was dependent on NF-{kappa}B, as blockade with PDTC, which blocks the dissociation of I{kappa}B from cytoplasmic NF-{kappa}B (64), resulted in a significant reduction in secretion of both chemokines. In addition, AP-1 nuclear activation, although not directly correlated to chemokine secretion, was also observed in CoMTsAg-stimulated astrocytes. The activation of this transcription factor was relatively delayed when compared with NF-{kappa}B. Such findings are consistent with the presence of binding sites for both transcription factors in the promoters of CCL2, CXCL8, and CXCL10 genes (65, 66) and with previous work indicating that TNF-{alpha} may induce activation of NF-{kappa}B and AP-1 in astrocytes (67, 68). CoMTsAg induced activation of NF-{kappa}B and AP-1 with a kinetic profile roughly equivalent to that induced by 10 ng/ml TNF-{alpha}, although the magnitude of activation in particular for AP-1 was greater after TNF-{alpha} stimulation. Such differences may simply reflect differences in TNF-{alpha} concentrations because CoMTsAg was found to contain TNF-{alpha} levels of 130 ± 26 pg/ml (data not shown). CoMTsAg induced activation of p65/p50 subunits as did TNF-{alpha} stimulation, which is the most potent NF-{kappa}B family gene transactivator complex (41).

I{kappa}B degradation kinetics revealed distinct differences in responses to TNF-{alpha} and CoMTsAg stimulation. I{kappa}B{alpha} degradation in response to TNF-{alpha} was relatively short-lived and showed a biphasic profile compared with kinetics obtained after CoMTsAg stimulation. I{kappa}B{beta} degradation was relatively prolonged in response to TNF-{alpha}. Such differences suggest that TNF-{alpha} is not solely responsible for mediating the CoMTsAg effects.

In conclusion the data are consistent with a model in which larval degeneration causes the release of previously masked immunogenic Ags that initially activate microglial cells and the small number of resident macrophages (present as a result of low-level transient transendothelial migration through the BBB), which are stimulated to produce and secrete CCL2, CXCL8, and CCL3. Secretion of these chemokines causes transendothelial migration of peripheral monocytes and neutrophils across the BBB via chemotactic activity and direct effects on BBB permeability. Cellular influx of monocytes and neutrophils would be further amplified by TNF-{alpha}-dependent monocyte-astrocyte networks that increase CCL2 and CXCL8 secretion as well as initiate secretion of CXCL10 in a NF-{kappa}B- and/or AP-1-dependent fashion, which drives CNS lymphocyte influx. Such lymphocyte influx and chemokine secretion likely contribute to shaping Th1-type cytokine profiles, which may be important in augmenting larval degeneration and in mounting Ag-Ab immune responses. The net result is cell influx, chronic granulomatous inflammation, tissue damage, and clinical symptomatology.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was funded by the National Institutes of Health Grants AI-42037-01 and AI-35894, International Training in Research in Emerging Diseases Training Grant TW00910, and The Wellcome Trust of Great Britain. Back

2 Address correspondence and reprint requests to Prof. Jon S. Friedland, Department of Infectious Diseases, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, U.K. E-mail address: j.friedland{at}imperial.ac.uk Back

3 Abbreviations used in this paper: NCC, neurocysticercosis; BBB, blood-brain barrier; CoM, conditioned medium; CoMCon, control conditioned medium; PDTC, pyrrolidine dithiocarbamate; PMNL, polymorphonuclear leukocyte; TsAg, Taenia solium larval Ag. Back

Received for publication January 19, 2005. Accepted for publication June 13, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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