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Cerebral Endothelial Cells Release TNF- After Stimulation with Cell Walls of Streptococcus pneumoniae and Regulate Inducible Nitric Oxide Synthase and ICAM-1 Expression Via Autocrine Loops¹

Dorette Freyer^*, Rahel Manz^*, Andreas Ziegenhorn^*, Markus Weih^*, Klemens Angstwurm^*, Wolf-Dietrich Döcke, Andreas Meisel^*, Ralf R. Schumann, Gilbert Schönfelder^§, Ulrich Dirnagl^* and Joerg R. Weber^2,*

^* Department of Neurology, Institute of Medical Immunology, and Institut für Mikrobiologie und Hygiene, Universitaetsklinikum Charité, Humboldt University Berlin, Germany; and ^§ Institut für Klinische Pharmakologie und Toxikologie, Free University Berlin, Berlin, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
TNF-, inducible NO synthase (iNOS), and ICAM-1 are considered to be key proteins in the inflammatory response of most tissues. We tested the hypothesis that cell walls of Streptococcus pneumoniae (PCW), the most common cause of adult bacterial meningitis, induce TNF-, iNOS, and ICAM-1 expression in rat primary brain microvascular endothelial cell cultures. We detected TNF- mRNA by RT-PCR already 1 h after stimulation with PCW, while TNF- protein peaked at 4 h (9.4 ± 3.6 vs 0.1 ± 0.1 pg/µg protein). PCW induced iNOS mRNA 2 h after stimulation, followed by an increase of the NO degradation product nitrite (18.1 ± 4 vs 5.8 ± 1.8 at 12 h; 18.1 ± 4 vs 5.8 ± 1.8 pmol/µg protein at 72 h). The addition of TNF- Ab significantly reduced nitrite production to 62.2 ± 14.4% compared with PCW-stimulated brain microvascular endothelial cells (100%). PCW induced the expression of ICAM-1 (measured by FACS), which was completely blocked by TNF- Ab (142 ± 18.6 vs 97.5 ± 12.4%; 100% unstimulated brain microvascular endothelial cells). Cerebral endothelial cells express TNF- mRNA as well as iNOS mRNA and release the bioactive proteins in response to PCW. PCW-induced NO production is mediated in part by an autocrine pathway involving TNF-, whereas ICAM-1 expression is completely mediated by this autocrine loop. By these mechanisms, cerebral endothelial cells may regulate critical steps in inflammatory blood-brain-barrier disruption of bacterial meningitis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Meningitis caused by Streptococcus pneumoniae, the most common agent in adults, is still associated with a surprisingly high mortality of 28% (1), and 50% of the survivors suffer from neurological sequelae (2).

Although separated from the peripheral immune system by the blood-brain barrier (BBB),³ various cell types of the brain generate a specific inflammatory response to bacteria and bacterial components, which contribute to neuronal damage. Of particular interest in this regard are the capillary endothelial cells because they 1) are the major cellular substrate of the BBB, 2) may present the site of entry for the bacteria into the brain (3), 3) are involved in the inflammatory recruitment of leukocytes (4), and 4) possess the biochemical ability to produce inflammatory mediators.

Astrocytes, microglia, and neuronal cells express and release TNF- in response to LPS (5), pneumococcal cell wall components (PCW) (6), hypoxia, and other stress factors (7, 8).

Although vascular injury and the break down of the BBB is a critical event in bacterial meningitis, it remains unclear whether brain microvascular endothelial cells (BMEC) express TNF- mRNA and release TNF- protein. TNF- production in response to cytokines and LPS is still controversial in endothelial cells. Intraerythrocytic Plasmodium falciparum induces the up-regulation of ICAM-1 but it does not induce TNF- release in HUVEC (9). Quantitative TNF- expression in BMEC has not yet been demonstrated, and the evidence for TNF- production by cerebral endothelial cells is based on occasionally positive TNF- immunoreactivity of these cells in multiple sclerosis lesions (10, 11).

TNF- mediates the up-regulation of adhesion molecules like selectins and ICAM-1 (12, 13). These adhesion molecules play a central role in the invasion of activated leukocytes into the CNS (4). Leukocytes essentially contribute to brain tissue damage in experimentally induced meningitis (14, 15, 16), and the number in the cerebrospinal fluid is correlated to the clinical outcome of patients with bacterial meningitis (2). Elevated levels of the soluble form of ICAM-1 were detected in the cerebrospinal fluid of patients with bacterial meningitis (17) and correlate well with the breakdown of the BBB (18). The inhibition of leukocyte adhesion has proved effective in reducing brain edema, increasing intracranial pressure and regional cerebral blood flow, leukocyte count, and lactate in the subarachnoideal space in experimental meningitis (14, 16, 19).

Beside firm adhesion of activated leukocytes, vasodilatation of pial venoles is an important step in early inflammation. There is increasing evidence that NO may be important not only as a potent vasodilator (20), but also by its cytotoxic properties (21). NO is a mediator of hemodynamic changes in in vivo models of septic shock (22, 23). NO possibly causes the blood flow increase in the early phase of bacterial meningitis (24, 25). This hyperemia contributes to vasogenic brain edema and to the increase of intracranial pressure. Vasodilatation may also augment leukocyte adhesion in early inflammation (26). However, NO has also a modulatory effect on leukocyte-endothelium interaction. NO synthase inhibition augments leukocyte adhesion (27, 28). In addition, NO produced by endothelial cells, macrophages, glia cells, and leukocytes may react with superoxide anions to peroxynitrite, a potential cytotoxic agent, resulting in damage of the BBB.

In this study, we investigated whether PCW can induce TNF-, inducible NO synthase (iNOS), and ICAM-1 in BMEC. Further, it was our hypothesis that PCW induced iNOS and that ICAM expression involves autocrine TNF--dependent pathways.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cerebral microvascular endothelial cells

Primary cultures of BMEC were prepared from 3-wk-old Wistar rats using modified methods as described earlier (29, 30). After removing the meninges and superficial vessels from the cortical tissue, the minced hemispheres were suspended in DMEM containing 1.1 U/ml collagenase (Serva, Heidelberg, Germany) and incubated for 1 h at 37°C. The suspension was cooled at 4°C and centrifuged with 25% BSA (Serva) for 20 min at 800 x g. The pellet was resuspended in DMEM with 0.1% collagenase/dispase (Boehringer Mannheim, Mannheim, Germany) and incubated for 30 min at 37°C. After centrifugation at 400 x g, the pellet was resuspended in DMEM with 0.05% DNase (Boehringer Mannheim) for 1 min. After washing in DMEM, the resulting crude cell fraction was seeded in collagen-coated culture plates and was grown in DMEM containing 20% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1.2 mM N-acetyl-L-alanyl-L-glutamine, 100 U/L heparin (Biochrom, Berlin, Germany), 20 mg/L endothelial cell growth factor (Boehringer Mannheim), 110 mg/L sodium pyruvate (Sigma, St. Louis, MO), and D-valine instead of L-valine to suppress the growth of astrocytes (30). Media were changed every day. The endothelial cells formed confluent monolayers after 6–8 days and were subsequently used in our experiments. Purity of the endothelial cell cultures were proven by using Abs directed against glial fibrillary acid protein (Dako, Hamburg, Germany), OX-42 (Genzyme, Cambridge, MA), and CD14 (Santa Cruz Biotechnology, Santa Cruz, CA). HRP-conjugated secondary Abs were used (Dako and Santa Cruz Biotechnology). Then the monolayers were screened by two experienced researches with light microscopy (magnification, x200) and the positive-stained cells were quantified and correlated to the number of v. Willebrandt-factor VIII-positive endothelial cells. These cells were counted in a Fuchs-Rosenthal chamber after trypsination. A total of 0.05% cells stained positive for glial fibrillary acid protein, and 1% of the cells stained positive for OX-42 and CD14. About 99% of the cultivated cells stained positive for v. Willebrandt-factor VIII (Dako).

Preparation of PCW

An unencapsulated strain (PnR-527; National Reference Center, Jena, Germany) of S. pneumoniae was cultured overnight on Columbia agar plates, suspended in pyrogen-free saline, and heat-inactivated by boiling at 95°C for 30 min. The inactivated bacteria were disintegrated by ultrasound for 2 x 7 min/150 W, purified by centrifugation and washing, and again suspended in pyrogen-free saline. The optical density of the final preparation was 0.66 at 620 nm, correlating to an equivalent of 10⁷ CFU/ml. In Gram staining, there were <1% intact cells and the pH of the suspension was 7.4. DNA/RNA content of the preparation was <0.1 µg/ml (DNA Dip Stick Test; Invitrogen, Groningen, Netherlands), and the LPS concentration was below the detection limit of the Limulus amebocyte lysate test (BioWhittaker, Walkersville, MD).

Experimental protocol

At the day of the experiment, media were replaced by 400 µl/well serum- and endotoxin-free TNB-100 medium supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. For stimulation, 20 µl of PCW suspension was added at a concentration correlating to 500 x 10³ CFU/ml bacteria and incubated at 37°C and 5% CO₂. Dexamethasone (10^-6 M; Sigma-Aldrich, Deisenhofen, Germany), N^G-nitro-L-arginine (L-NA 10^-4 M; Research Biochemicals, Natick, MA), aminoguanidine (3 x 10^-4 M; Sigma-Aldrich), and a neutralizing polyclonal sheep anti-TNF- Ab (31) were added at the same time. One microliter of a 1:1000 dilution of this Ab neutralized 2 pg of TNF- standard. At times indicated, either supernatants were measured for nitrite and TNF- or cells were harvested for mRNA isolation and semiquantitative RT-PCR.

Semiquantitative RT-PCR of TNF- mRNA expression

For cell lysis, harvesting, and RNA isolation, we used the RNAid Kit by Bio 101 (La Jolla, CA). Competitive template RT-PCR from total RNA was performed as described (32). As control DNA fragment, a synthetic gene containing nonhomologous DNA with binding sites for 5' and 3' cytokine-specific primers were used. The sense and antisense primer sequences derived from rat TNF- and ß-actin have been described (32).

For quantification, cDNA samples were adjusted to equal input concentrations based on their ß-actin cDNA content. After electrophoresis of the PCR products on a 1.5% agarose gel, the proportions were estimated by measuring the intensity of ethidium bromide fluorescence. The level of cytokine gene expression was expressed in arbitrary units/µl cDNA. One arbitrary unit was defined as the highest dilution of the control fragment that yielded a detectable amplification product at the conditions used.

PCR products were purified (Qiaquick PCR Purification Kit; Qiagen, Hilden Germany), sequenced by cycle sequencing (Cycle Sequencing Kit; Pharmacia, Freiburg, Germany), and confirmed by BLAST search (data not shown). The following primer was used: 5'-CCCGTAGCCCACGTCGTAGC.

TNF- bioassay

TNF- bioactivity was measured by using an modified L 929 cytotoxicity assay (33). L929 cells were incubated with cell-culture supernatant in the presence of 1 µg/ml actinomycine D at 37°C for 20 h. Cell viability was quantified by the uptake of crystal violet in living cells, which was determined spectrophotometrically (595 nm) using an ELISA reader (Dynatech, Denkendorf, Germany). Equivalent concentrations of rat TNF- (a generous gift from Dr. P. Scholz, Schering AG, Berlin, Germany) were used as a standard.

TNF- ELISA

TNF- protein levels were measured with a rat ELISA kit (Cytoscreen KRC3012; Biosource, Ratingen, Germany).

Immunostaining of intracellular TNF-

Endothelial cells were incubated with PCW for 90 min, then fixed in 4% paraformaldehyde with 4% sucrose for 20 min. After washing with PBS, cells were incubated with 0.1 mM glycine for 20 min and then permeabilized with 0.2% Triton X-100 for 90 s followed by incubation with an Ab against TNF- (IP-400; Genzyme) or a normal rabbit IgG at the same concentration (AB-10S-C; R&D Systems, Wiesbaden, Germany) for 1 h. All further steps were conducted according to the instructions of the LSAB (labeled streptvidin-biotin) kit (Dako).

Semiquantitative RT-PCR for quantification of iNOS mRNA expression

Total cellular RNA was isolated from about 10⁶ cells according to a modified method described by Chomczynski and Sacchi (34) using trizol reagent (Life Technology, Karlsruhe, Germany). Contaminating DNA was removed by incubating the RNA with DNase (Promega, Madison, WI) as recommended by the supplier. Integrity of the RNA was verified by gel electrophoresis, and RNA quantity was determined by optical density.

Reverse transcription of extracted RNA was performed by standard procedures (35) using Moloney murine leukemia virus reverse transcriptase (Life Technology).

Semiquantitative RT-PCR was performed as described by (36) using GAPDH mRNA as an internal standard for RNA preparation and reverse transcriptase reaction. Deletion mutants of rat iNOS and rat GAPDH, which were used as external standards for quantification of PCR products, were constructed as described (37) and cloned in pGEM-T (Promega) and pCRII (Invitrogen) vectors. The resulting plasmids were linearized by BamHI (GAPDH) and SacII (iNOS) under appropriate conditions.

We amplified rat iNOS using synthesized 20-mer oligonucleotide primers encoding rat iNOS, 5'-GCA GAA TGT GAC CAT CAT GG-3' (bases 1295 through 1314) as sense primer and 5'-ACA ACC TTG GTG TTG AAG GC-3' (bases 1701 through 1720) as antisense primer spanning an intron. These primers yielded a PCR product 426 bp long. The length of the deletion mutant was 357 bp. Rat-specific GAPDH was amplified using 23- and 24-mer oligonucleotides, 5'-ATC CTG CAC CAC CAA CTG CTT AG3-' (bases 515 through 537) as sense primer and 5'-CAG GAA ATG AGC TTC ACA ATG TTG-3' (bases 980 through 1003), yielding a PCR product of 489 bp. The respective deletion mutant was 440 bp long.

In addition, each forward primer was synthesized with a CY5 5'-labeling and added to 1/10 of the final primer concentration allowing the direct quantification of PCR products with a laser fluorescence DNA sequencer (ALFexpress; Pharmacia Biotech, Freiburg, Germany).

The competitive PCR was performed by amplifying a constant amount of iNOS or GAPDH cDNA together with a serial diluted DNA of the respective deletion standard.

PCR reaction mixture contained 10 mM Tris-HCl, 2 mM and 1.5 mM MgCl₂, for GAPDH and iNOS, respectively, 0.1 mM dNTPs (Pharmacia, Uppsala, Sweden), 2 µM of each primer, 0.2 µM of CY5-labeled primer, and 2.5 U of Taq DNA-polymerase (Perkin-Elmer, Weiterstadt, Germany) in a final volume of 50 µl. Amplification for iNOS and GAPDH was run under the following conditions: denaturation at 94°C for 3 min, annealing at 61°C for 60 s, and extension at 72°C for 30 s. iNOS-PCR was performed with 29 cycles and GAPDH-PCR with 30 cycles.

For iNOS and GAPDH, the resulting two PCR products, one of the amplified deletion standard (A_ds) and one of the amplified target cDNA (A_t), were separated by a nondenaturating 5% polyacrylamide gel, and CY5-labeled bands were quantified by fluorescence detection using the ALFexpress DNA sequencer.

According to Bouaboula et al. (38), for estimation of the initial amount of target cDNA in the original test samples, natural log (Ln) (A_t/A_ds) was plotted as a function of Ln (N_in). N_in is the known initial amount of deletion standard added to the PCR mixture. Interpolation of this linear plot to Ln (Ln A_t/A_ds) = 0 (where A_t = A_ds) leads to the initial amount of iNOS or GAPDH cDNA, which was present in the test sample. PCR products were sequenced as described above using the following primers: iNOS, 5'-CAGCCTCAGAGTCCTTCATG; GAPDH, 5'-TGCTTAGCCCCCCTGGCCAAG.

iNOS Western blot

The method used was described in detail (39). Briefly, rat cerebral endothelial cells were boiled in a SDS sample buffer for 3 min. 5 µg protein per lane were loaded on 7.5% SDS-polyacrylamid mini gels followed by electrophoresis. Blotting was performed semidry onto polyvinyldifluoride membranes, blocked with 5% nonrat dry milk, and detected with the primary monoclonal iNOS-Ab (Transduction Laboraties, Lexington, KY), a secondary anti-mouse horseradish peroxidase-linked Ab (Sigma, St. Louis, MO) and enhanced chemoluminescence (Amersham, Freiburg, Germany) using Kodak x-ray films.

Nitrite assay

Nitrite, a primary reaction product of NO in aqueous solution, was determined by the Griess reaction: the supernatant of cell culture was centrifuged at 400 x g, aliquots of 100 µl were incubated with 100 µl Griess reagent (0.1% naphthylthylene, 1% sulfanilamide in 5% H₃PO₄), and the absorption at 550 nm was measured using a MR5000 ELISA reader (Dynatech, Denkendorf, Germany).

Determination of ICAM-1 (CD54) expression

After incubation with PCW for 10–12 h, cells were washed in TNB-100, followed by an incubation in 2% BSA for 10 min to block the unspecific binding of Ab. Afterward, the cells were incubated with FITC-labeled mAb against ICAM-1 (rat anti-ICAM-1, mouse IgG1, clone 1A29; PharMingen, San Diego, CA) in a dilution of 1:200 for 30 min at 37°C. After binding of the Ab, the cells were trypsinated, centrifuged, and resuspended in fixation solution containing 2% glucose, 1% formaldehyde, and 5 mM NaN₃. The mean fluorescence intensity was detected using a FACScan (Becton Dickinson, Heidelberg, Germany).

Protein determination

Cells were lysed with 0.5% SDS solution overnight. Then, 50 µl of cell lysate were incubated with 150 µl BCA reagent (Pierce, Rockford, IL) for 30 min at 37°C. Optical density was measured at 550 nm using an ELISA reader.

Data analysis

Data are expressed as means ± SD. For comparisons between groups, Kruskal-Wallis one-way ANOVA was used. To detect statistically significant differences between two groups, the nonparametric Mann-Whitney U test was performed using SPSS 6.1 statistical software (SPSS, Chicago, IL). Values of p < 0.05 were considered as statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Kinetics of PCW-induced TNF- release and TNF- RNA expression

After the cells were incubated with PCW (500 x 10³ CFU/ml), TNF- mRNA expression was detected as early as 1 h and reached a maximum 2 h after stimulation (Fig. 1A and Fig. 2). Bioactive TNF- and TNF- protein measured by ELISA reached a peak already after 4 h of incubation with PCW (Fig. 1, B and C). During the following 20 h, TNF- protein as well as the bioactivity dropped, but there was still a significant protein content in the supernatant compared with control (Fig. 1, B and C). Intracellular TNF- was demonstrated by the immunostaining 90 min after incubation of the BMEC with PCW (Fig. 3A). Isotype-matched control staining confirmed the specificity of intracellular TNF-.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. TNF- mRNA expression reached a maximum 2 h after stimulation with PCW (A; n = 6). The mRNA expression was followed by a rapid increase of TNF- (B shows bioactive TNF-, n = 4; C shows TNF- protein detected with ELISA, n = 7) *, Statistical significant increase compared with unstimulated BMEC (p < 0.05).

View larger version (102K): [in this window] [in a new window]   FIGURE 2. Gel electrophoresis of the PCR product after TNF- RT-PCR. Lane 1 (left), bp ladder; lane 2, BMEC after incubation for 2 h with PCW (500 x 103 CFU/ml); lane 3, control in serum-free media; lane 4, control DNA-fragment alone; lane 5 (right), cDNA.

View larger version (134K): [in this window] [in a new window]   FIGURE 3. Immunostaining for intracellular TNF- of confluent monolayers of BMEC. After incubation with PCW, all cells stained positive, demonstrating BMEC as source of TNF- (A). Unstimulated BMEC remain unstained (B).

Incubation of BMEC with PCW (500 x 10³ CFU/ml) induced a significant expression of iNOS mRNA already at 2 h followed by a significant increase of nitrite in the supernatant at 12 h (18.1 ± 4.0 vs 5.8 ± 1.8 pmol/µg protein), which continued to increase until 72 h (415.6 ± 9.7 vs 177.9 ± 10.7 pmol/µg protein) (Fig. 4, A and B). Nitrite production was also observed in unstimulated BMEC of the control group. Accordingly, we found a minimal iNOS mRNA expression in these control cells. The nitrite accumulation was not detected in the absence of cells. iNOS expression as demonstrated by Western blot confirms the above-mentioned mRNA and nitrite results (Fig. 4, C and D).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. iNOS mRNA expression (A) was followed by nitrite accumulation in the supernatant of BMEC (B). *, Statistically significant difference compared with unstimulated BMEC (p < 0.05, n = 6). iNOS bands at 130 kDa in the Western blot had a maximum at 12 and 24 h (C). Minimal iNOS activation appeared in unstimulated BMEC (D).

Dose dependence of PCW-induced TNF- and NO release from BMEC

Already the lowest dose of PCW (7.8 x 10³ CFU/ml) led to a significant release of bioactive TNF- (0.82 ± 0.41 vs 0.28 ± 023 pg/µg protein). A stimulation with 500 x 10³ CFU/ml of PCW resulted in a further increase of TNF- (5.5 ± 3.18 pg/µg protein; Fig. 5A), whereas a higher concentration of PCW did not generate additional TNF- release (Fig. 5B; 500 x 10³ CFU/ml, 6.62 ± 1.01 pg/µg protein; 1000 x 10³ CFU/ml, 5.76 ± 1.84 pg/µg protein; 2000 x 10³ CFU/ml, 6.41 ± 2.41 pg/µg protein).

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Dose dependency of TNF- (A, bioactivity; B, protein measured by ELISA), iNOS mRNA (C), and nitrite (D) after stimulation with PCW. *, Statistically significant difference compared with resting BMEC (A and C) using the Wilcoxon Test (p < 0.05, n = 6).

Effect of dexamethasone, L-NA, and aminoguanidine on endothelial TNF- and NO production

BMEC were incubated with PCW (500 x 10³ CFU/ml) and the different inhibitors for 8 h and 48 h before supernatants were assayed for TNF- and NO content, respectively. The glucocorticoid dexamethasone completely inhibited TNF- as well as NO production (Fig. 6). Dexamethasone also decreased basal NO release (Fig. 6). Aminoguanidine and L-NA significantly reduced NO production to 18.2 ± 20.6% and 64.9 ± 28.4%, respectively (Fig. 6). As expected, TNF- release was not effected by aminoguanidine as well as by L-NA.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Effect of dexamethasone, L-NA, aminoguanidine (AG), and TNF- Ab on TNF- and nitrite concentration in the supernatant. *, Statistically significant difference compared with untreated PCW-stimulated BMEC (100%) (p < 0.05, n = 6).

Neutralization of PCW induced TNF- activity

PCW-induced bioactive TNF- was neutralized by an Ab to TNF-. Ab concentrations sufficient to block all TNF- biological activity significantly reduced the release of NO to 62.2 ± 14.4% (Fig. 6). TNF- Ab added to unstimulated BMEC had no effect on NO release (29.5 ± 9.7% vs 30 ± 11.5% unstimulated BMEC).

Externally added TNF- resulted in a dose-dependent release of nitrite (0 pg/ml, 124 ± 64 pmol/µg protein; 312.5 pg/ml, 117 ± 81 pmol/µg protein; 1250 pg/ml, 121 ± 78 pmol/µg protein; 5000 pg/ml, 158 ± 103 pmol/µg protein; significantly different p < 0.05 compared with unstimulated BMEC; n = 7).

Effect of PCW on ICAM-1 up-regulation on BMEC

As previously shown, ICAM-1 was constitutively expressed on cultured BMEC. This expression was significantly increased after stimulation with PCW (at 10–12 h) to 142 ± 18.6% of control values, which was prevented by dexamethasone (109 ± 18.7%; Fig. 7). In the FACS analysis, an increase of the mean as well as the mean fluorescence intensity was measured (399.4 ± 69.3 mean fluorescence intensity = 100% in unstimulated BMEC). In contrast, the TNF--induced increase of ICAM-1 was not inhibited by dexamethasone (149.7 ± 15.9 vs 138.7 ± 9%). When TNF- activity was neutralized with a TNF- Ab, ICAM-1 expression was blocked (97.5 ± 12.4%; Fig. 7). TNF- Ab had no effect on unstimulated BMEC (365.2 ± 15.8 mean fluorescence intensity).

View larger version (17K): [in this window] [in a new window]   FIGURE 7. PCW and TNF- induced a significant up-regulation of ICAM-1 in BMEC (*, p < 0.05, n = 6), which was significantly inhibited by dexamethasone (**, p < 0.05, n = 6) and blocked by TNF- Ab (**, p < 0.05, n = 6) compared with untreated PCW-stimulated BMEC, whereas TNF--induced ICAM-1 expression was not altered by dexamethasone treatment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Thus, our data suggest that BMEC generate key mediators of the inflammatory response to cell wall components of S. pneumoniae.

Although microvascular cerebral endothelial cells are a major part of the BBB, surprisingly little is known about the role of these cells in acute inflammation. The activation of BMEC is a critical step in the inflammatory cascade. Several recent studies focused on the stimulatory properties of TNF- and other proinflammatory cytokines on endothelial cells and BMEC (12, 13, 40).

There is evidence for an important role of TNF- in bacterial meningitis as it has been detected in the cerebrospinal fluid of meningitis patients (41, 42) as well as in experimentally induced meningitis (41, 43). The definite cellular source of TNF- in inflammatory CNS disease remains unclear. Astrocytes and microglial cells release TNF- after stimulation with PCW (6), LPS, or cytokines (44). Neuron-derived TNF- has been demonstrated to play a role in posttraumatic regeneration of the brain (45).

In vivo evidence for TNF- release by endothelial cells is scarce. Cerebral endothelial cells showed occasional immunoreactivity for TNF- in multiple scleroses lesions (10, 11).

TNF- activity was detectable in the supernatant of confluent porcine pulmonary artery enthothelial cells incubated with Escherichia coli LPS. A maximum of TNF- release was found at 6 h, whereas IL-1 was not released by porcine pulmonary artery endothial cells (46). TNF- secretion measured by ELISA was found in HUVEC treated with IFN- and cross-linked by anti-E-selectin and anti-ICAM-1 Abs (47).

Our study demonstrates for the first time that BMEC can express TNF- mRNA and release the bioactive protein. These data suggest that BMEC participate in the early steps of bacterial inflammation. Endothelial inflammation may be an important factor for the damage of the BBB but may also contribute to the process of bacterial invasion.

NO is a central mediator in inflammation and bacterial meningitis. NO may be responsible, in part, for the blood flow increase in the early phase of bacterial meningitis (24, 25). This blood flow increase plays an important role in brain edema generation, which develops in parallel to the NO increase (48). NO also has a toxic effect on the BBB (49) and on neurons (21) in bacterial meningitis. TNF--induced cytotoxicity in bovine BMEC is significantly reduced by the NO synthase inhibitor N-iminoethyl-L-ornithine or by unspecific inhibition with dexamethasone (50), indicating NO as a possible mediator of cellular damage. In addition, TNF- enhances NO synthase activity and nitrite production in bovine BMEC (51) and may augment NO toxicity.

In our study, iNOS mRNA was expressed in response to PCW in a dose-dependent manner, followed by an increase in the release of NO, as measured from nitrite accumulation. iNOS expression and NO release were delayed in comparison to TNF- induction. The addition of a neutralizing TNF Ab resulted in a significant decrease of NO, suggesting an autocrine regulatory loop. In contrast to LPS-stimulated porcine pulmonary endothelial cells (46), the NO release was only inhibited in part (63%) by a neutralizing Ab, indicating that a TNF--independent intracellular pathway may be involved in PCW-induced NO production in BMEC. Dexamethasone inhibition of iNOS probably results from a down-regulation of NF- B activation (52).

In accordance with previous publications (53, 54), we found that resting, unstimulated BMEC release nitrite and express iNOS mRNA. The spontaneous release of low quantities of NO (51, 55) from BMEC may be the result of a minor activation from the cell culture conditions, as BMEC in brain slices under more physiological conditions do not express iNOS (53).

Like other inflammatory stimuli, PCW are capable to induce ICAM-1 up-regulation in BMEC. In human BMEC stimulated with LPS, ICAM-1 up-regulation can be inhibited by dexamethasone (12), which is in agreement with our findings in rat BMEC, whereas ICAM-1 up-regulation induced by TNF- is not altered by dexamethasone (12). Both PCW and TNF- induce ICAM-1 expression. In human BMEC, a maximum expression of ICAM-1 was found with a similar low concentration of TNF- as it was used in our experiments (13). Incubation with a low TNF- concentration, which was effective in BMEC, did not up-regulate ICAM-1 in HUVEC. Only a 10-fold higher concentration than the one used in our study resulted in ICAM-1 expression (56).

A neutralizing Ab directed against TNF- inhibited PCW-induced ICAM-1 expression. BMEC regulate ICAM-1 expression exclusively via an autocrine TNF--dependent pathway, which may be enhanced by TNF- produced by astrocytes, microglia, and other cells.

We conclude that cerebral endothelial cells produce TNF- and NO in response to pneumococcal cell walls. NO release is mediated in part by an autocrine pathway involving TNF-. PCW-induced ICAM-1 expression is completely mediated by this autocrine loop. A precise knowledge of these underlying mechanisms is necessary to understand early endothelial activation and to design therapeutic strategies in the treatment of CNS infection.

	Acknowledgments

 
We thank Dr. Wolf Bürger for preparation of PCW, Renate Gusinda for excellent technical assistance, Claudia Muselmann for help with PCR and sequencing, and Joachim G Schulz for performing the iNOS Western blots.

	Footnotes

 
¹ This study was supported by grants of the Deutsche Forschungsgemeinschaft, SFB 507 project B6 (J.R.W.) and Di 454/8-2 as well the Hermann & Lilly Schilling Foundation (U.D.).

² Address correspondence and reprint requests to Dr. Joerg R. Weber, Department of Neurology, Unversitaetsklinikum Charité, Humboldt University Berlin, D-10098 Berlin, Germany. E-mail address:

³ Abbreviations used in this paper: BBB, blood-brain barrier; BMEC, brain microvascular endothelial cells; L-NA, N^G-nitro-L-arginine; iNOS, inducible NO synthase; PCW, cell walls of Streptococcus pneumoniae.

Received for publication January 11, 1999. Accepted for publication August 2, 1999.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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