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Departments of
*
Molecular Cell Biology and
Pediatrics, Vrije Universiteit Medical Centre; and
Laboratory of Experimental Internal Medicine, Academic Medical Centre, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
, and IL-6, which lead to an increased permeability of
the blood-brain barrier
(BBB)2
(1). As a result of this enhanced permeability state,
leukocytes, predominantly granulocytes, are recruited to the CNS.
Activated granulocytes play a major role in bacterial clearance and
also secrete a variety of inflammatory mediators (e.g., reactive oxygen
species) that may not only kill bacteria, but also cause damage
to the CNS (4, 5, 6, 7, 8). These mediators play a key role in the
pathophysiology of bacterial meningitis and contribute to the
unfavorable outcome of the disease due to intracranial complications
such as decreased cerebral blood flow, raised intracranial pressure,
and brain edema (6, 9).
The cerebrospinal fluid (CSF) lacks the presence of cell populations
capable of initiating an effective immune response against invading
pathogens. Therefore, important questions are which (and how) resident
cell populations in the CNS initiate and regulate the inflammatory
response during meningitis. Resident CNS macrophages, and in
particular, the meningeal (MM) and perivascular (PVM) macrophages that
line the meninges and surround local blood vessels (10),
are prime candidates. The MM and PVM can be identified in the rat by
the selective expression of the ED2 Ag (11). They express
scavenger receptors and are highly phagocytic (12).
Furthermore, under various inflammatory conditions the MM and PVM can
express inflammatory mediators such as TNF-, IL-1, and
cyclooxygenase-2 (13, 14, 15). Until now only one study has
examined the role of the MM during bacterial meningitis
(16). It was observed that reduction of the MM by
intracisternal injection of clodronate liposomes did not affect
inflammation in the subarachnoidal space. Unfortunately, the liposome
treatment only resulted in a partial depletion of the MM, with no
further information about: 1) the effect on the PVM and other CNS cell
populations (e.g., microglial cells), 2) the effect on other parts of
the CNS like the spinal cord and cerebrum, and 3) possible side effects
on the periphery (e.g., Kupffer cells, splenic macrophages, and
monocytes).
We have recently described a depletion method that results in a complete and selective depletion of the MM and PVM (17). Here we use this method to study the role of the MM and PVM in an experimental bacterial meningitis model. Our results provide the first direct evidence for the role of MM and PVM in bacterial meningitis in particular, and in inflammation in the CNS in general.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Wistar rats were obtained from CPB-Harlan (Zeist, The Netherlands) and weighed 300–350 g at the time of the experiment. Animals were kept under routine laboratory conditions and were allowed free access to food and water. Microbiological status of the animals was according to Federation of European Laboratory Animal Science Associations recommendations. Approval of the Animal Ethics Committee of Vrije Universiteit was obtained for all animal experiments.
Preparation of liposomes
Multilamellar mannosylated liposomes were prepared as described
before (18). Briefly, 178 mg of phosphatidylcholine and 27
mg of cholesterol were dissolved in 8 ml of chloroform, which was added
to 9.25 mg of
p-amino-phenyl--D-mannopyranoside
(Sigma, St. Louis, MO) dissolved in 5 ml of methanol in a 500-ml
round-bottom flask. This was dried in vacuo on a rotary evaporator to
form a film. The molar ratio of
phosphatidylcholine/cholesterol/mannoside was 7:2:1 (according to Ref.
19). The lipid film was dispersed in 10 ml of PBS (0.15 M
NaCl in 10 mM phosphate buffer, pH 7.4) for the preparation of
PBS-containing mannosylated liposomes. To enclose the clodronate,
2.5 g (a gift of Roche Diagnostics, Mannheim, Germany) of
clodronate was dissolved in 10 ml of deionized water (adjusted to pH
7.3 with NaOH) in which the lipid film was dispersed, and the
preparations were kept for 2 h at room temperature, sonicated for
3 min, washed, and resuspended in 10 ml of PBS. PBS-containing control
liposomes and clodronate-containing liposomes were labeled with the
fluorescent dye DiI
(1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate;
D282; Molecular Probes, Eugene, OR) as described by Claassen
(20), with minor alterations. DiI was dissolved in
absolute ethanol at a concentration of 2.5 mg/ml, sonicated for 5 min,
and stored at 4°C. DiI was added to 1 ml of liposome suspension in a
final concentration of 62.5 µg/ml and incubated for 30 min at 37°C.
The suspension was washed three times with 2 ml of PBS (24,000 x
g, 15 min) to remove free DiI, and the pellet was
resuspended in 400 µl of PBS.
Stereotaxical injection of liposomes
The rats were deeply anesthetized with an i.m. injection of 1.0 ml/kg body weight of a mixture of 4 parts ketamine (1% solution of Ket; Aesco, Boxtel, The Netherlands) and 3 parts xylazine (2% solution of Rompun; Bayer, Brussels, Belgium) and mounted in a stereotaxic frame. Control or clodronate liposomes (50 µl) were slowly (over a total time period of 25 min) injected stereotaxically with a blunt Hamilton syringe (Hamilton Bonaduz, Bonaduz, Switzerland) into the 4th ventricle. Coordinates (Bregma +11.6 and 5.5 mm dorsoventral) were derived from the atlas of Paxinos and Watson (21).
Bacterial meningitis model
Streptococcus pneumoniae type 6A (isolated from the
CSF of a meningitis patient) were incubated in brain heart infusion
broth at 37°C. After 16–17 h, 5 ml of this preincubation culture was
mixed with 20 ml of fresh brain heart infusion medium and further
incubated for 3–5 h at 37°C until OD620 =
1 was reached. Rats were mounted in a stereotaxic frame and injected
intracisternally with 
5 x 106 CFU of
S. pneumoniae, 7 days post clodronate liposome
(n = 11) or PBS liposome (n = 7)
injection. Twelve hours post meningitis induction rats were clinically
scored (0 = no clinical symptoms, 1 = red nose and eyes,
2 = reduced activity/apathy/piloerection, 3 =
convulsions/respiratory problems) and sacrificed with an i.p. injection
of 1 ml of Nembutal (Sanofi Sante Animale, Benelux B.V., Maassluis, The
Netherlands). The inoculum (S. pneumoniae) was plated
(1 x 100–1 x
10-7) on sheep blood agar-hemin menadione
(BA-hm) plates and cultured overnight at 37°C to determine the amount
of CFU injected.
Sample collection and processing
Blood was collected via a heart puncture for blood smears, and
1 x 100–1 x
10-4 eg of EDTA blood was plated on sheep BA-hm
plates for identification and quantification of the bacteria. Serum was
collected for measurement of TNF-, IL-1, IL-6 (National Institute
for Biological Standards and Control, South Mimms, Potter Bar,
Hertfordshire, U.K.), and macrophage-inflammatory protein
(MIP)-2 (BioSource International, Camarillo, CA) by ELISA. CSF was
aspirated via a cisterna magna puncture, and 1 x
100–1 x 10-7 was
plated on BA-hm plates, for the quantification of the bacteria. The
leukocytes were counted using a hemocytometer, and cytocentrifuge
preparations were made and stained with May-Grünwald Giemsa dye
(Merck, Darmstadt, Germany), according to standard histological
staining methods. CSF samples were then centrifuged, cells were stored
in 10% DMSO/90% NBCS at -80°C, and the supernatant was
stored at -80°C for determination of MIP-2 by ELISA. The cerebellum,
cerebrum, spinal cord (cranial-caudal), liver, cervical lymph nodes,
and spleen were collected, snap-frozen, and stored at -80°C until
histological evaluation.
Bacterial peritonitis model
Seven days post clodronate liposome (n = 6) and PBS liposome (n = 5) injection into the 4th ventricle as described above, the rats received an i.p. injection of 3 x 108 CFU/0.5 ml S. pneumoniae type 6A (culture conditions as described above). Four hours after this bacteria injection into the peritoneum, the rats were sacrificed by O2/CO2 exposure. The peritoneal cavity was washed with 20 ml of RPMI 1640 (4°C) medium (Life Technologies, Paisley, Scotland), cells were counted, and cytocentrifuge preparations were stained with May-Grünwald Giemsa dye (Merck), to determine the percentages of leukocytes.
Immunohistochemistry for light microscopy
Cryostat sections (8 µm) were cut serially, picked up on gelatin coated slides, and air dried overnight in a container with silica gel. Immunohistochemistry was applied after a 10-min fixation in dehydrated acetone to examine the presence of macrophages, neutrophils, and the expression of cerebral endothelial adhesion molecules. The murine mAbs in this study include the anti-rat macrophage markers ED1 and ED2. ED1 recognizes a lysosomal membrane-related Ag on both monocytes and macrophages, and ED2 recognizes an Ag on a subset of mature rat macrophages including the MM and PVM in the CNS (22). HIS48 recognizes an Ag on all granulocytes (23). 1A29 (a gift of T. Tamatani, Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) directed against ICAM-1 (24), 3A12 (a gift of W. F. Hickey, Dartmouth Medical School, Lebanon, NH) for platelet endothelium cell adhesion molecule (PECAM; Ref. 25), and 5F10 (a gift of R. Lobb, Biogen, Cambridge, MA), which recognizes VCAM-1 (26) on rat cerebral endothelial cells. Abs and conjugates were diluted in 0.1 M Tris buffer, pH 7.6, with 0.1% BSA (Organon Technika, Oss, The Netherlands) and used at optimal final dilution of 2 µg/ml for ED1-biotin and ED2-biotin; 6 µg/ml for 5F10; 2.75 µg/ml for 3A12; 1/5 of the 1A29 supernatant, and 1 µg/ml for His48. Control slides included incubations in which the primary Ab was omitted. All incubations were conducted horizontally at room temperature. After incubation with the first Ab for 60 min, the slides were rinsed in Tris buffer, pH 7.6, with 0.1% BSA, incubated with conjugate/0.1% normal rat serum for 60 min, and washed again with Tris/BSA buffer. As conjugates we used streptavidin-alkaline phosphatase-conjugated rabbit anti-mouse IgG (DAKO, Glostrup, Denmark) and alkaline phosphatase-conjugated rabbit anti-mouse IgG + IgM (DAKO). Alkaline phosphatase activity was demonstrated by incubation in naphthol AS-BI phosphate substrate (27) in 0.1 M Tris, pH 8.7, buffer for 10 min. Sections were rinsed in 0.1 M Tris, pH 7.6, light counterstained with hematoxylin, and mounted in VectaMount (Vector Laboratories, Burlingame, CA). The sections were examined with a microscope (Nikon Eclipse E800; Melville, NY), and the recordings were made with a Nikon DXM1200 camera.
Quantification of leukocytes
The number of leukocytes were counted with a light microscope using x400 magnification. Two hundred total white blood cells were counted per CSF cytocentrifuge preparation and, subsequently, the percentage of neutrophils, eosinophils, macrophages, and lymphocytes of the total amount of white blood cells was counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Before investigating the role of the MM and PVM during meningitis,
we evaluated the kinetics of the S. pneumoniae meningitis
model in detail. We counted the number of bacteria and leukocytes in
the CSF and blood of the animals after 4, 12, 24, and 48 h. We
observed a decline in the number of bacteria in the CSF, 12 h post
meningitis induction, followed by an increase after 24 and 48 h
(Fig. 1
a). There were no
detectable bacteria in the blood between 12 and 24 h, and they
reappeared at 48 h post S. pneumoniae injection into
the cisterna magna (Fig. 1b). To see whether the decrease in
bacteria correlated with the presence of granulocytes, which play a
major role in bacterial clearance, we quantified the cells present in
the CSF. We observed a maximal influx of granulocytes, mainly
neutrophils (75%), at 12 and 24 h post S.
pneumoniae inoculation, followed by a decrease after 48 h
(Fig. 1c). The granulocytes were mainly situated within the
subarachnoidal and Virchow-Robin space, and only a low percentage
seemed to be located within the parenchyma of the brain (Fig. 1d). Based on these observations, we decided to investigate
the effect of MM and PVM depletion 12 h post meningitis
induction.
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We have recently developed a liposome-based depletion method
(17) that allows us to investigate the role of the MM and
PVM during inflammation in the CNS. This method is based on a single
intraventricular injection of clodronate liposomes resulting in a
complete and selective depletion of the MM and PVM throughout the CNS.
We induced the bacterial meningitis model 7 days post depletion of the
MM and PVM. At this time point, the liver and draining lymph nodes,
which suffer from a mild and temporal macrophage depletion using this
regime, are completely repopulated with macrophages (17).
First, we tested whether this method could also be used to deplete the
MM and PVM during meningitis induction; both the MM and PVM were still
completely absent from all CNS areas tested (Fig. 2, a and b). We
evaluated the animals for clinical symptoms 12 h post S.
pneumoniae inoculation and observed that the depleted group was
clearly more ill than the control group (Fig. 3a). In correlation with the
more severe disease course (p = 0.04), we
observed profoundly higher bacterial counts in CSF (approximately
x600) and blood (approximately x20) of the MM- and PVM-depleted
animals (Fig. 3, b and c).
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Although the results described above were in agreement with a
possible role of the MM and PVM in bacterial clearance, the dominant
clearing cell is probably the granulocyte (2, 6).
Therefore, it was important to investigate whether MM and PVM depletion
was affecting the influx of granulocytes and other leukocytes into the
CSF. We observed that the number of leukocytes that had extravasated
was significantly decreased in comparison with the control group (Fig. 4a). Also no signs of
increased cell death were observed. The leukocytes were mainly composed
of granulocytes (90%), and no difference in composition was noted
between depleted animals and controls (Fig. 4b). To exclude
the possibility that the decreased influx of granulocytes in the
clodronate liposome-treated animals was caused by a direct effect of
the clodronate liposomes on circulating neutrophils, a control
experiment was performed. Seven days after the depletion of the MM and
PVM, S. pneumoniae peritonitis was induced, and the number
of immigrated cells in the peritoneal cavity was quantified 4 h
post peritonitis induction. There was no significant difference between
the control and depleted animals (Fig. 5a). Analysis of
May-Grünwald Giemsa-stained peritoneal cell cytocentrifuge
preparations showed that 4 h after i.p. bacteria injection the
major infiltrating population is formed by the granulocytes (90%);
this was the same for the control, MM-, and PVM-depleted animal groups
(Fig. 5b). In other words, the intrinsic migratory capacity
of leukocytes was not affected by the liposome treatment.
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It is known that high levels of proinflammatory cytokines
correlate with the pathophysiological symptoms and the clinical outcome
in meningitis patients (reviewed in Ref. 1). Therefore, it
was important to establish whether the enhanced numbers of bacteria in
the circulation of the depleted animals were accompanied by higher
levels of systemic proinflammatory cytokines. Significantly higher
levels of IL-6 in the serum of the depleted group in comparison with
the control group were observed (Fig. 6c). We could not observe
detectable levels of TNF- (<15 pg/ml) and IL-1 (<15 pg/ml) in
the serum of either group.
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To investigate whether the decrease in neutrophil influx after MM
and PVM depletion was the possible consequence of a lowered chemokine
expression in the CSF, we measured the expression levels of MIP-2.
MIP-2 is both essential (28, 29) as well as sufficient (P.
Zwijnenburg, unpublished observation) for the infiltration of
granulocytes into the CSF during rodent bacterial meningitis. Moreover,
MM and PVM appear to be the only endogenous CNS sources of MIP-2 in
meningitis (28). Surprisingly, CSF MIP-2 levels of the
depleted animals were much higher (530–2577 pg/ml) in comparison with
the control meningitis group (34–448 pg/ml) (Fig. 6
a). We
could even detect elevated MIP-2 levels (7–22 pg/ml) in the serum of
depleted animals as compared with controls, which displayed levels that
were below detection (<10 pg/ml) (Fig. 6b). Thus, the
decrease in leukocyte influx could not simply be explained by a
decrease in MIP-2 expression.
MM and PVM depletion increases the expression of vascular adhesion molecules on the BBB during experimental meningitis
An alternative explanation for the decreased influx of
granulocytes into the CSF in MM- and PVM-depleted animals was a
possible decrease in vascular adhesion molecule expression on the BBB.
Therefore, we analyzed the vascular adhesion molecule expression on the
cerebral endothelial cells, including VCAM-1 and ICAM-1, which are
known to play a role in neutrophil extravasation (2, 30, 31) and PECAM. Although in both cases the bacterial infection
resulted in an up-regulation of VCAM in comparison with noninfected
animals (Fig. 7, a vs
b and c), this was clearly enhanced in the MM-
and PVM-depleted meningitis group. This enhancement was visible at both
the level of staining intensity as well as the number of VCAM-positive
vessels. An up-regulation of ICAM-1 and PECAM was also observed, but
this difference was clearly less profound than in the case of VCAM-1
(data not shown). This demonstrates that, despite a significantly
enhanced expression of both relevant vascular adhesion molecules and
chemokines, MM and PVM depletion suppresses leukocyte
extravasation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our present results show that depletion of MM and PVM during
pneumococcal meningitis results in an aggravation of clinical symptoms
(Fig. 3a) demonstrating for the first time that these
macrophages play a protective role during bacterial meningitis. MM and
PVM elimination resulted also in elevated numbers of bacteria, as well
as enhanced levels of inflammatory mediators in both CSF (MIP-2) and
blood (IL-6). Furthermore, a reduced influx of granulocytes and other
leukocytes into the subarachnoid space was observed after MM and PVM
depletion; this occurred despite elevated levels of the relevant
chemokine (MIP-2) and a higher expression of vascular adhesion
molecules (e.g., VCAM-1).
What does this reveal about the role of MM and PVM in the pathogenesis
of bacterial meningitis? Based on the observation that depletion of MM
and PVM, as intrinsic components of the BBB, reduces leukocyte influx
(Fig. 4), it is concluded that MM and PVM somehow support leukocyte
traffic across the BBB. We propose that, among the parameters studied,
this role in leukocyte transmigration constitutes the primary action of
MM and PVM and that this, together with our other observations, can
explain their protective role during meningitis. Although our results
indicate that MM and PVM play a supportive role in the infiltration of
leukocytes across the BBB during bacterial meningitis, the underlying
mechanism remains elusive. One obvious possibility was that in the
early stages of meningitis MM and PVM are activated to produce
mediators (cytokines/chemokines) that induce endothelial activation and
leukocyte migration. We have investigated whether the reduced migration
of leukocytes after MM and PVM depletion was accompanied by a reduced
expression of relevant vascular adhesion molecules (e.g., VCAM-1,
ICAM-1) or chemokines (e.g., MIP-2), and this was clearly not the case.
Instead, enhanced VCAM-1 and MIP-2 levels were found. We believe that
these findings may in fact be the consequence of a local and/or
systemic immune response. An alternative explanation for the role of
PVM and MM may be that they can affect the kinetics of the inflammatory
response during meningitis. A thorough kinetic analysis may provide
further insights.
Because granulocytes in the CSF are considered to eliminate the
majority of bacteria during meningitis (2, 6, 32), a
reduction in granulocytes resulted in increased bacterial numbers in
the CSF (Fig. 3). The higher load of bacteria (and/or their degradation
products) may in turn potentiate leukocyte activation in the CSF, which
will result in enhanced production of various effector molecules,
including reactive oxygen species, that cause neuronal damage
(9). These effector molecules, together with the elevated
systemic cytokine response (e.g., IL-6; Fig. 6), can explain the
observed worsening of the clinical signs (1).
It should be emphasized that our present results cannot demonstrate, or rule out, a possible direct role of MM and PVM in bacterial clearance. Of course, MM and PVM are actively phagocytic (12, 17), and the increase in bacteria in CSF after depletion of these cells is in line with a direct role in clearance. However, considering the concomitant reduction in granulocytes, which likewise represent the major scavengers during meningitis, we cannot presently decide what possible contribution MM and PVM have.
Taken together, these findings demonstrate that MM and PVM play a protective role during bacterial meningitis and suggest that this is, at least in part, due to their supportive role in the influx of leukocytes across the BBB. This also provides the first evidence for a role of MM and PVM during inflammation in the CNS.
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Footnotes |
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2 Abbreviations used in this paper: BBB, blood-brain barrier; PVM, perivascular macrophage; MM meningeal macrophage; CSF, cerebrospinal fluid; BA-hm, blood agar-hemin menadione; MIP, macrophage-inflammatory protein; PECAM, platelet endothelium cell adhesion molecule.
Received for publication May 21, 2001. Accepted for publication August 8, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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during delayed neuronal loss in the facial nucleus of the rat. J. Neurosci. Res. 54:820.[Medline]
attenuates neutrophil recruitment in the central nervous system during experimental bacterial meningitis. Infect. Immun. 67:2590. and -2 are produced intrathecally and mediate chemotactic activity in cerebrospinal fluid of infected mice. J. Immunol. 155:4367.[Abstract]
4 and 1 integrins and bind to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Blood 91:2341. after stimulation with cell walls of Streptococcus pneumoniae and regulate inducible nitric oxide synthase and ICAM-1 expression via autocrine loops. J. Immunol. 163:4308.This article has been cited by other articles:
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