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After Stimulation with Cell Walls of Streptococcus pneumoniae and Regulate Inducible Nitric Oxide Synthase and ICAM-1 Expression Via Autocrine Loops1
*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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),
NG-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 (Ads) and one of the amplified target cDNA (At), 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) (At/Ads) was plotted as a function of Ln (Nin). Nin is the known initial amount of deletion standard added to the PCR mixture. Interpolation of this linear plot to Ln (Ln At/Ads) = 0 (where At = Ads) 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
A 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-.
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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).
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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).
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, B and
C). A 4-fold higher PCW dose doubled iNOS transcription and
induced nitrite levels of 320.2 ± 60.4 pmol/µg protein (Fig. 5, B and C). We could not detect a further increase
of nitrite accumulation with higher doses of PCW than 500 x
103 CFU/ml (2000 x
103, 344.2 ± 23.6 pmol/µg).
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.
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA and release TNF- in response to PCW; 2)
PCW induce the expression of iNOS mRNA in BMEC; 3) BMEC up-regulate the
expression of the adhesion molecule ICAM-1 upon stimulation with PCW;
4) this ICAM-1 up-regulation is mediated by an autocrine TNF-
dependent pathway, whereas the nitrite release depends only in part on
this mechanism. 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.
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Acknowledgments |
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Footnotes |
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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, NG-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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C. J. Orihuela, S. Fillon, S. H. Smith-Sielicki, K. C. El Kasmi, G. Gao, K. Soulis, A. Patil, P. J. Murray, and E. I. Tuomanen Cell Wall-Mediated Neuronal Damage in Early Sepsis Infect. Immun., July 1, 2006; 74(7): 3783 - 3789. [Abstract] [Full Text] [PDF]
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 J. J. Alexander, A. Jacob, L. Bao, R. L. Macdonald, and R. J. Quigg Complement-Dependent Apoptosis and Inflammatory Gene Changes in Murine Lupus Cerebritis J. Immunol., December 15, 2005; 175(12): 8312 - 8319. [Abstract] [Full Text] [PDF]
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J. Thornton and L. S. McDaniel THP-1 Monocytes Up-Regulate Intercellular Adhesion Molecule 1 in Response to Pneumolysin from Streptococcus pneumoniae Infect. Immun., October 1, 2005; 73(10): 6493 - 6498. [Abstract] [Full Text] [PDF]
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K. Angstwurm, U.-K. Hanisch, T. Gassemi, M. B. Bille, M. Prinz, U. Dirnagl, H. Kettenmann, and J. R. Weber Tyrosine Kinase Inhibition Reduces Inflammation in the Acute Stage of Experimental Pneumococcal Meningitis Infect. Immun., June 1, 2004; 72(6): 3294 - 3298. [Abstract] [Full Text] [PDF]
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 C. Fiuza, M. Bustin, S. Talwar, M. Tropea, E. Gerstenberger, J. H. Shelhamer, and A. F. Suffredini Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells Blood, April 1, 2003; 101(7): 2652 - 2660. [Abstract] [Full Text] [PDF]
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 N. N. Khodarev, J. Yu, E. Labay, T. Darga, C. K. Brown, H. J. Mauceri, R. Yassari, N. Gupta, and R. R. Weichselbaum Tumour-endothelium interactions in co-culture: coordinated changes of gene expression profiles and phenotypic properties of endothelial cells J. Cell Sci., March 15, 2003; 116(6): 1013 - 1022. [Abstract] [Full Text] [PDF]
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E. A. Eugenin, M. C. Branes, J. W. Berman, and J. C. Saez TNF-{alpha} Plus IFN-{gamma} Induce Connexin43 Expression and Formation of Gap Junctions Between Human Monocytes/Macrophages That Enhance Physiological Responses J. Immunol., February 1, 2003; 170(3): 1320 - 1328. [Abstract] [Full Text] [PDF]
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 R. Nau and H. Eiffert Modulation of Release of Proinflammatory Bacterial Compounds by Antibacterials: Potential Impact on Course of Inflammation and Outcome in Sepsis and Meningitis Clin. Microbiol. Rev., January 1, 2002; 15(1): 95 - 110. [Abstract] [Full Text] [PDF]
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M. M. J. Polfliet, P. J. G. Zwijnenburg, A. M. van Furth, T. van der Poll, E. A. Dopp, C. Renardel de Lavalette, E. M. L. van Kesteren-Hendrikx, N. van Rooijen, C. D. Dijkstra, and T. K. van den Berg Meningeal and Perivascular Macrophages of the Central Nervous System Play a Protective Role During Bacterial Meningitis J. Immunol., October 15, 2001; 167(8): 4644 - 4650. [Abstract] [Full Text] [PDF]
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A. Willuweit, G. Sass, A. Schoneberg, U. Eisel, G. Tiegs, and M. Clauss Chronic Inflammation and Protection from Acute Hepatitis in Transgenic Mice Expressing TNF in Endothelial Cells J. Immunol., October 1, 2001; 167(7): 3944 - 3952. [Abstract] [Full Text] [PDF]
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 N. TSAO, H.P. HSU, C.M. WU, C.C. LIU, and H.Y. LEI Tumour necrosis factor-{alpha} causes an increase in blood-brain barrier permeability during sepsis J. Med. Microbiol., September 1, 2001; 50(9): 812 - 821. [Abstract] [Full Text] [PDF]
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M. Clauss, C. Sunderkotter, B. Sveinbjornsson, S. Hippenstiel, A. Willuweit, M. Marino, E. Haas, R. Seljelid, P. Scheurich, N. Suttorp, et al. A permissive role for tumor necrosis factor in vascular endothelial growth factor-induced vascular permeability Blood, March 1, 2001; 97(5): 1321 - 1329. [Abstract] [Full Text] [PDF]
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K. R. Munana, S. M. Vitek, B. C. Hegarty, D. L. Kordick, and E. B. Breitschwerdt Infection of Fetal Feline Brain Cells in Culture with Bartonella henselae Infect. Immun., January 1, 2001; 69(1): 564 - 569. [Abstract] [Full Text] [PDF]
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 Z. Radisavljevic, H. Avraham, and S. Avraham Vascular Endothelial Growth Factor Up-regulates ICAM-1 Expression via the Phosphatidylinositol 3 OH-kinase/AKT/Nitric Oxide Pathway and Modulates Migration of Brain Microvascular Endothelial Cells J. Biol. Chem., June 30, 2000; 275(27): 20770 - 20774. [Abstract] [Full Text] [PDF]
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