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From the Department of Pathology, Dartmouth Medical School, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, IL-6, and tumor
necrosis factor- as early as 1 to 6 hours after
Staphylococcus aureus exposure. Evaluation of chemokine
expression by reverse transcription-polymerase chain reaction
demonstrated enhanced levels of the CXC chemokine KC 24 hours after
bacterial exposure, which correlated with the appearance of
neutrophils in the abscess. In addition, two CC
chemokines, monocyte chemoattractant protein-1 and
macrophage inflammatory protein-1 were induced within 24
hours after S. aureus exposure and preceded the influx
of macrophages and lymphocytes into the brain. Analysis of abscess
lesions by in situ hybridization identified CD11b+ cells
as the source of IL-1ß in response to S. aureus. Both
intercellular adhesion molecule-1 and platelet endothelial cell
adhesion molecule expression were enhanced on microvessels in
S. aureus but not sterile bead-implanted tissues
at 24 and 48 hours after treatment. These results characterize
proinflammatory cytokine and chemokine expression during the early
response to S. aureus in the brain and provide the
foundation to assess the functional significance of these mediators in
brain abscess pathogenesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rat brain abscess model closely mimics human disease in that the abscess progresses through a series of well-defined stages. In the rat, each stage is defined on the basis of predominant cell types and the histological appearance of the abscess.4 In the human, these stages have been identified using CT scanning techniques.1 The early stage, or early cerebritis, occurs from days 1 to 3 and is typified by neutrophil accumulation, tissue necrosis, and edema. The intermediate, or late cerebritis stage, occurs from days 4 to 9 and is associated with a predominant macrophage and lymphocyte infiltrate. The final, or capsule, stage occurs from day 10 onward and is associated with the formation of a well-vascularized abscess wall that sequesters the lesion and protects the surrounding normal brain parenchyma. Myofibroblasts are found associated with the developing abscess wall at this stage and plasma cells predominate the lesion. Currently, it is not clear what cells are responsible for mediating the immunopathological response to S. aureus in the brain.
Chemokines represent a family of low molecular weight chemotactic
cytokines, classified into groups based on the presence and position of
conserved cysteine residues.5-7
CXC or chemokines
function as neutrophil and lymphocyte chemoattractants and prototypical
family members include IL-8, KC (rodent homologue of growth related
oncogene-), interferon-inducible protein-10 kd (IP-10), and monokine
induced by interferon 
(MIG). CC or ß chemokines are chemotactic
for monocytes and T cells and include such members as macrophage
inflammatory protein-1 (MIP-1), monocyte chemoattractant proteins
(MCPs) 1–5, and regulated on activation T cell expressed and
secreted (RANTES). Chemokines play an important role in recruiting
leukocytes into areas of active inflammation. They are synthesized
locally at sites of inflammation and establish a concentration gradient
to which target cell populations migrate. Several studies have examined
the expression of chemokines in response to central nervous system
(CNS) insults such as stab wound injury in the brain8,9
and experimental autoimmune encephalomyelitis (EAE).10-12
In these models, KC, IP-10, MCP-1, and MIP-1 are up-regulated after
injury. However, the signals responsible for the initial recruitment of
inflammatory cells into brain abscesses have not yet been defined.
In this report, we characterize chemokines and cytokines that are
expressed immediately after the introduction of S. aureus
into the brain. Several proinflammatory cytokines, including IL-1,
IL-1ß, IL-6, and tumor necrosis factor- (TNF-), were found to
be induced. Consistent with the observed neutrophil influx into early
abscess lesions, KC expression was enhanced within 24 hours after
S. aureus exposure. In addition, IP-10, MCP-1, and MIP-1
expression were enhanced in abscesses, preceding the observed influx of
macrophages and lymphocytes. In situ hybridization revealed
that CD11b+ cells produce IL-1ß after S. aureus exposure.
Analysis of adhesion molecule expression revealed enhanced levels
of both intercellular adhesion molecule-1 (ICAM-1) and platelet
endothelial cell adhesion molecule (PECAM) on vessels associated
with S. aureus but not sterile bead-implanted tissues. These
findings characterize cytokine and chemokine expression in early brain
abscess lesions, and provide a basis for future studies to assess the
functional significance of individual mediators in abscess
pathogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Live S. aureus were encapsulated in agarose beads before implantation in the brain as previously described.4 The use of agarose beads prevents widespread bacterial dissemination or rapid wound sterilization by the host. Briefly, a stock culture of coagulase-positive S. aureus obtained from the microbiology laboratory at Dartmouth Hitchcock Medical Center was grown overnight (16 hours) at 37°C in Luria-Bertani broth (Fisher Biotech, Fair Lawn, NJ). A total of 1 x 109 bacteria were added to a solution of 1.4% low melt agarose (type XII, Sigma, St. Louis, MO) at 40°C. The mixture was then added to rapidly swirling heavy mineral oil (Sigma) pre-warmed to 37°C and cooled to 0°C on crushed ice. Beads were washed four times in 1x DPBS (Mediatech Cellgro, Herndon, VA) to remove mineral oil. Beads with dimensions between 50 and 100 µm, as determined by phase contrast microscopy, were used for implantation into the brain. Sterile beads were prepared in the same manner, without bacteria. The bacterial viability or sterility of bead preparations was confirmed by overnight culture in LB medium.
Animals and Generation of Experimental Brain Abscesses
Adult female Lewis rats were obtained from Charles River Laboratories (Wilmington, MA). The animal use protocol has been approved by the Dartmouth College Institutional Animal Care and Use Committee. Anesthesia was provided by an i.p. injection of 60 mg/kg ketamine and 5 mg/kg xylazine on a body weight basis. A 1-cm longitudinal incision was made along the midline between the ear and the eye to expose the frontal sutures. A burr hole was drilled 5 mm caudal and 0.5 mm lateral to the frontal suture. A Hamilton syringe fitted with flexible tubing and a pulled, fine-tipped glass-fired pipet (diameter < 1 mm) was used to deliver beads into the brain parenchyma. A total of 5 µl of beads were placed 5 to 6 mm deep from the external surface of the calvarium to prevent reflux during injection. Using this approach, bacteria were reproducibly deposited into the head of the caudate or frontal lobe white matter. Control animals were implanted with sterile agarose beads. Previous studies have established that implanting sterile beads into the brain induces minimal inflammation and edema4 (Kielian, unpublished observations). Incisions were closed using sterile wound clips. The mortality rate associated with abscess generation was minimal, with >95% of animals surviving the procedure.
Processing of Tissues for Immunostaining and RNA Analysis
To prepare tissues for immunohistochemistry, animals were perfusion-fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. The brain was removed, postfixed in paraformaldehyde for 30 minutes, and washed in 0.2 mol/L phosphate buffer, pH 7.4, overnight. Tissues were cryoprotected in 30% sucrose and snap-frozen in OCT for immunohistochemistry.
To prepare tissues for RNA isolation, samples of brain tissue containing S. aureus or sterile beads were snap-frozen in liquid N2 and stored at -70°C until RNA extraction was performed.
In Situ Hybridization (ISH) Probe Labeling
ISH was performed using digoxigenin (DIG)-labeled riboprobes specific for rat IL-1ß and MCP-1. IL-1ß and MCP-1 cDNA fragments were amplified by reverse transcription-polymerase chain reaction (RT-PCR) from mRNA isolated from 24-hour abscess tissue using the following primers: IL-1ß sense (5'-TTCAGGAAGGCAGTGTCA-3'), IL-1ß anti-sense (5'-TCTTTGGGTATTGTTTGG-3'), MCP-1 sense (5'-ATGCAGGTCTCTGTCACGCTTC-3'), and MCP-1 anti-sense (5'-AGTTCTCTGTCATACTGGTCAC-3'). The size of products amplified by the IL-1ß and MCP-1 primer pairs were 448 and 445 bp, respectively and verified by sequence analysis. DIG-labeled riboprobes were synthesized from cDNA templates subcloned into the PCR vector pGEM T (Promega, Madison, WI) using the DIG RNA labeling kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. The efficiency and intensity of riboprobe labeling was compared with a DIG-labeled RNA standard using dot blot analysis.
ISH
Frozen sections of fixed tissue were mounted onto RNase-free polyL-lysine-treated glass slides and air-dried for 30 minutes at room temperature before use. Tissues were rehydrated in three changes of Tris-buffered saline (TBS), pH 7.4, before proteinase K treatment. Sections were permeabilized with proteinase K (10 µg/ml) in TBS with 2 mmol/L CaCl2 for 15 minutes at 37°C. Slides were rinsed with TBS at 4°C and transferred to a solution of 0.1 mol/L triethanolamine, pH 8.0, for 3 minutes, followed by incubation in 0.5% acetic anhydride for 10 minutes on a stir plate. Following two washes in TBS, tissues were dehydrated through a series of graded alcohols and allowed to air-dry. Before use, DIG-labeled riboprobes (100 ng/ml) were denatured for 5 minutes at 65°C in hybridization buffer (50% deionized formamide, 10% dextran sulfate, 100 µg/ml salmon sperm DNA, 2x standard saline citrate, 0.02% sodium dodecyl sulfate). Riboprobes were added to tissue sections, coverslipped, and incubated overnight (12–16 hours) at 55°C in a humidified chamber. The following day, slides were washed with increasing stringency using standard saline citrate. Next, tissues were treated with blocking reagent (Boehringer Mannheim) for 15 minutes at room temperature before incubation with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (1:500 dilution) for 1 hour at room temperature. Following three washes in TBS, slides were developed using a NBT/BCIP substrate (Boehringer Mannheim) at 4°C in the dark. The color reaction was terminated by extensive washes in ddH2O. Tissues were dehydrated through a series of graded alcohols and xylene before coverslipping.
Immunohistochemistry
Frozen sections of fixed tissue were processed for immunohistochemistry using the avidin-peroxidase method as previously described.13 The following antibodies were used for adhesion molecule analysis: ICAM-1 (TLD-4C9) and PECAM (TLD-3A12; both from Serotec, Raleigh, NC), L-selectin, P-selectin, and vascular cell adhesion molecule-1 (VCAM-1; PharMingen, San Diego, CA), and the isotype control Abs MOPC-21 and UPC10 (mouse IgG1 and IgG2a, respectively, Sigma), and rabbit IgG (Vector Laboratories, Burlingame, CA). Astrocytes and monocytes/microglia were identified on the basis of GFAP (Dako, Carpenteria, CA) and CD11b (OX-42, Serotec) staining, respectively. Biotinylated secondary antibodies included anti-mouse IgG, anti-rabbit IgG (both from Vector Laboratories), or anti-hamster IgG (Caltag, South San Francisco, CA). Slides were developed using the substrate 3,3'-diaminobenzidene (DAB).
Cytokine Immunohistochemistry
Cells producing the proinflammatory cytokine IL-1ß were identified by immunohistochemical staining as previously described.14 Primary mouse anti-rat IL-1ß (Serotec) and mouse IgG1 isotype control antibodies (MOPC-21, Sigma) were used to evaluate IL-1ß protein expression in S. aureus- and sterile bead-implanted tissues. Biotinylated horse anti-mouse IgG (Vector Labs) was used with the ABC peroxidase reagent (Vector Elite ABC kit, Vector Labs) to detect IL-1ß protein. Reaction products were developed using a DAB substrate (Vector Labs).
RNA Isolation and RT-PCR
Total RNA was isolated from brain tissue using the TriZol reagent
(Gibco BRL, Grand Island, NY) according to the manufacturer’s
instructions. Before use in RT-PCR, RNA samples were treated with DNase
I (Gibco BRL). RT-PCR was performed using a GeneAmp RNA PCR kit (Perkin
Elmer, Foster City, CA) with 100 ng of total input RNA. After reverse
transcription, cDNA templates were denatured for 105 seconds at 95°C,
then amplified in 35 cycles of annealing (60°C, 30 seconds),
extension (60°C, 30 seconds), and denaturing (95°C, 15 seconds).
The final step was incubation for 7 minutes at 42°C. Each
experimental sample was amplified in parallel using primers specific
for ß-actin to assess uniformity in the amount of input RNA and gel
loading. Primers used for PCR amplification are listed in Table 1
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Cytokine expression in brain tissue was examined by RNase
protection assay (RPA) using the RiboQuant RPA kit (PharMingen). The
following multiprobe template sets were used, each specific for rat
cytokines: CK-1, which detects transcripts for IL-1 and
-1ß, TNF-ß, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-, IL-2,
and interferon (IFN)-; and CK-3, which detects IFN-ß, TNF-ß,
granulocyte-macrophage colony stimulating factor (GM-CSF),
transforming growth factor (TGF)-ß1,
-ß2, and -ß3,
lymphotoxin ß (Ltß), TNF-, macrophage migration inhibitory
factor (MIF), and IFN-. Both template sets contained probes
for the housekeeping genes L32 and GAPDH to serve as internal controls
for the assay. Probes were synthesized using
[-33P]UTP (New England Nuclear, Boston, MA)
resulting in an average specific activity of 1 x
106
cpm/µl. The RNase protection procedure was
carried out according to the manufacturer’s instructions using 5 to 10
µg per sample of total RNA. Each assay included normal Lewis rat
brain RNA and yeast tRNA as controls. Products were resolved on a 6%
acrylamide gel, dried, and exposed to film (Kodak BioMax MR, Rochester,
NY).
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Results |
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The signals responsible for the initial recruitment of
inflammatory cells into brain abscesses have not yet been investigated.
The predominant cell type present within early abscess lesions are
neutrophils, whereas macrophages begin to accumulate at day 4 after
bacterial exposure.4
We examined the expression of several
CXC and CC chemokines to define those that may play a role in the
recruitment of neutrophils and macrophages into brain abscess lesions.
Consistent with the observed neutrophil influx into early abscess
lesions, KC expression was enhanced in animals implanted with S.
aureus-containing beads at 24 hours, with levels remaining
elevated up to 4 days (Figure 1A)
. This
finding suggests that KC may be important for neutrophil recruitment
into brain abscesses in vivo. Similarly, the levels of
IP-10, a CXC chemoattractant, were up-regulated in the brains of
animals receiving S. aureus beads within 24 hours (Figure 1A)
. This increase in IP-10 expression preceded the appearance of
macrophages and lymphocytes into the abscess.
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, MCP-1, and
RANTES, by RT-PCR. As shown in Figure 1B
, MCP-1 and MIP-1 were
elevated within 24 hours after S. aureus exposure. This
increase in CC chemokine expression precedes the first detectable
accumulation of macrophages in the abscess, which occurs at days 4 and
5.4
At time points before 24 hours, we found no
significant differences in the levels of chemokine expression between
S. aureus- and sterile bead-implanted tissues (data not
shown). However, chemokine induction in response to sterile beads was
transient, with levels beginning to decline by 24 hours compared to the
continued expression of MCP-1, MIP-1, KC, and IP-10 in S.
aureus-implanted tissues. There was no significant difference in
the amount of MIP-2, RANTES, or fractalkine expression between S.
aureus- and sterile bead-implanted tissues (data not shown).
Importantly, we were not able to detect any of the chemokines examined
here in normal Lewis rat brain (data not shown). These results suggest
that KC, IP-10, MIP-1, and MCP-1 participate in inflammatory cell
recruitment into experimental brain abscesses. In turn, newly recruited
neutrophils and macrophages can amplify the immune response through the
synthesis of proinflammatory cytokines and chemokines. Proinflammatory Cytokines Are Expressed during the Acute Phase of Experimental Brain Abscesses in Vivo
To define the array of cytokines produced during the early phase
of brain abscess development, RNase protection assays (RPA) were
performed on total RNA isolated from S. aureus- and sterile
bead-implanted tissues. Figure 2A
shows
the proinflammatory cytokines that are expressed within 24 hours after
S. aureus exposure. These include IL-1, IL-1ß, IL-6,
and TNF-. We also found elevated levels of TGF-ß1 and lymphotoxin
ß (Ltß) in abscess tissues at all time points examined (Figure 2B)
.
During the acute phase of abscess induction, we did not observe the
expression of any T cell-derived cytokines, such as IL-2 and IFN-.
This finding is consistent with the absence of a significant
lymphocytic infiltrate early in abscess pathogenesis. S.
aureus- and sterile bead-implanted tissues and normal Lewis rat
brain were found to express equivalent amounts of mRNA for
TGF-ß2, TGF-ß3, and MIF
(Figure 2B)
. Collectively, these findings reveal activation of a potent
proinflammatory response to S. aureus in the brain.
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To define the early kinetics of proinflammatory cytokine induction
in response to S. aureus, RNA was isolated from S.
aureus- or sterile bead-implanted tissues from 1 to 24 hours after
treatment and evaluated by RPA. As shown in Figure 3
, the induction of IL-1 and ß was
immediate, with detectable levels observed as early as 1 hour after
S. aureus exposure. The amount of IL-1 and ß
detected in S. aureus-implanted tissues progressively
increased with time and still remained strong at 4 days (Figures 3 and 2A)
. Sterile beads induced a transient increase in IL-1 and -1ß
that was first evident 3 hours after treatment. However, the amount of
IL-1 and -1ß detected in sterile bead-implanted tissues was
significantly less than that observed after S. aureus
exposure, and the response was short-lived in the former, with
expression disappearing within 18 hours (Figure 3)
. Both TNF- and
IL-6 were induced within 3 to 6 hours after S. aureus
exposure with levels remaining elevated until 4 days (Figures 3 and 2A)
. As expected, normal Lewis rat brain did not express any of these
proinflammatory cytokines (Figure 3)
. The immediate induction of
IL-1 and ß, TNF-, and IL-6 suggest that these mediators play an
important role in the early response to S. aureus in the
brain.
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We have established by RPA that S. aureus leads to the
induction of proinflammatory cytokine and chemokine expression in the
brain. In an attempt to identify the cellular source(s) of IL-1ß, ISH
and immunohistochemical staining of S. aureus-implanted
tissues were conducted. IL-1ß was selected because of its rapid
induction and high levels of mRNA expression based on RPA analysis. As
shown in Figure 4
, IL-1ß-positive cells
were found to colocalize with CD11b (OX-42) staining, suggesting that
they are of the monocyte/macrophage lineage (ie, resident microglia and
infiltrating monocytes) or infiltrating polymorphonuclear leukocytes
(PMNs). Specificity was confirmed by the inability of an IL-1ß
sense riboprobe to react with abscess tissue (Figure 4B)
. MCP-1 was not
detected in abscess lesions by ISH, possibly a result of the decreased
sensitivity of this method compared to RT-PCR (data not shown). To
confirm the presence of IL-1ß protein within the developing abscess,
tissues were stained with an IL-1ß-specific
antibody. As shown in Figure 5
, numerous IL-1ß-positive cells were
associated with the area of active inflammation in S.
aureus-implanted tissues at both 24 and 48 hours. In contrast, no
IL-1ß-positive cells were observed in tissues receiving sterile beads
(Figure 5)
. These results indicate that IL-1ß mRNA and protein are
abundantly expressed within early abscess lesions by CD11b-positive
cells.
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Because proinflammatory cytokines such as TNF- and IL-1 are
known to induce adhesion molecule expression on endothelial
cells15-17
and because these cytokines are induced soon
after S. aureus exposure, we were interested in examining
adhesion molecule expression in S. aureus- and sterile
bead-implanted tissues. The activation-induced adhesion molecules
ICAM-1, P-selectin, and VCAM were evaluated, as were PECAM and the
leukocyte activation marker L-selectin. As shown in Figure 6
, S. aureus induced PECAM
expression on brain vascular endothelial cells at 24 and 48 hours,
whereas vessels associated with sterile bead-implanted tissues were
negative. Small vessels in both S. aureus- and sterile
bead-implanted tissues were found to express detectable levels of
ICAM-1 (Figure 6)
, however, the distribution of ICAM-1-positive
vessels was distinct. ICAM-1-positive vessels in
S. aureus-implanted tissues were distributed diffusely
throughout the injected hemisphere, compared to the restriction of
ICAM-1-positive vessels associated with sterile beads to the vicinity
of the stab wound. Both PECAM and ICAM-1 are important adhesion
molecules for the extravasation of neutrophils and monocytes into
tissues,17,18
the predominant cell types found
infiltrating early brain abscess lesions. Vessels in both S.
aureus- and sterile bead-implanted tissues were negative for VCAM
and P-selectin during the period examined, and L-selectin expression
was not detected on infiltrating leukocytes in either tissue (data not
shown). These results reveal the induction/up-regulation of PECAM and
ICAM-1 expression on brain vascular endothelial cells in response to
S. aureus, and suggest that they are critical to the
evolving pathological process.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within 24 hours after S. aureus implantation, KC, IP-10,
MIP-1, and MCP-1 expression were elevated compared to animals
implanted with sterile agarose beads. We found that the introduction of
sterile beads into the brain induced transient chemokine expression,
which began to decline around 24 hours. This effect may be a
consequence of direct damage to the blood-brain barrier during bead
administration. In contrast, chemokine levels in S.
aureus-implanted brain remained elevated up to 4 days, suggesting
the rapid and persistent establishment of a strong chemotactic gradient
that parallels the continued presence of bacteria and their
by-products. It appears as if the sustained production of chemokines is
required for leukocyte entry into tissues because, although sterile
beads induce transient chemokine expression, there is no accumulation
of inflammatory cells in these tissues4
(Kielian,
unpublished observations). The chemokines we have detected in our brain
abscess model most likely serve as signals for recruitment of
neutrophils and monocytes, cells which are important for effective
bacterial neutralization and eventual abscess resolution. Chemokines
could also serve to facilitate the phagocytic function of glial cells,
aiding in the destruction of S. aureus and eliminating
debris from the abscess site. In addition to serving as
chemoattractants, chemokines can activate target cell populations by
inducing a rapid and transient calcium flux. It is possible that
chemokines induced in response to S. aureus serve to
regulate mediator production in responding cell populations in the
brain, although this remains to be determined.
Pyogenic bacteria, such as S. aureus, elicit a potent
innate immune response typified by the production of proinflammatory
cytokines such as IL-1, IL-1ß, TNF-, and
IL-6.19,20
Indeed, we have detected these proinflammatory
cytokines in the rat brain immediately after S. aureus
exposure. These cytokines can also exert pleiotropic effects on cells
within the CNS. For example, both IL-1ß and TNF- act as potent
stimulators of cytokine and chemokine expression for both astrocytes
and microglia.21-24
TNF- and IL-1 can also stimulate
IL-6 production by astrocytes which, in turn, acts in an autocrine
matter to potentiate IL-6 release.25
Due to this complex
regulation of proinflammatory mediators, it is not unreasonable to
predict that if not tightly controlled, amplification of the immune
response would result in damage to normal host tissue. Indeed, in
addition to bacterial neutralization, the immune response induced by
S. aureus leads to the destruction of a significant portion
of normal brain tissue in proximity to the bacteria. Evidence in
support of this is demonstrated by several studies revealing the
detrimental effects of IL-1ß, TNF-, and IL-6 on the CNS. For
example, in concert with IFN-, TNF- activates murine microglia to
produce toxic reactive nitrogen intermediates.26,27
Exposure of the CNS vasculature to IL-1ß and TNF- leads to
increased adhesion and migration of inflammatory cells into the CNS and
alterations in vascular permeability which may persist for several
weeks.28,29
TNF- has been shown to indirectly mediate
neuronal cell injury30
and is toxic to
oligodendrocytes.31,32
Finally, overexpression of IL-6 in
the CNS can lead to several detrimental conditions including reactive
gliosis, neurodegeneration, breakdown of the blood-brain barrier,
angiogenesis, and chronic expression of C3.33,34
Studies
are ongoing to directly assess the importance of individual
proinflammatory cytokines in the initiation and progression of brain
abscess using in vivo Ab neutralization experiments.
Nevertheless, it is likely that a complex interaction exists between
IL-1, ß, TNF-, and IL-6, bacterial neutralization, and
destruction of normal brain parenchyma.
Previous studies have described chemokine receptor expression on those
cell types associated with brain abscesses in
vivo.35
For example, monocytes express CCR5 whose
natural ligands include MIP-1, MIP-1ß, and RANTES.6,7
Microglia36-38
and astrocytes39,40
have also
been shown to express CCR5, which may contribute, in part, to the
activation of these cells in areas immediately surrounding the
developing abscess. One characteristic of chemokines is their
promiscuity in terms of their ability to bind multiple receptors. In
addition to CCR5, monocytes and microglia express CCR1 which also binds
MIP-1 with high affinity. Both microglia and monocytes express CCR2,
the only known receptor for MCP-1. We do not currently know whether
CCR1, CCR2, and/or CCR5 are important for monocyte recruitment into
evolving brain abscesses. Receptors for the proinflammatory cytokines
TNF- and IL-1 have been shown to be constitutively expressed on some
neurons and glial cells in the rat brain.41
Based on this
pattern of receptor expression, it is intriguing to consider what
effect IL-1 and TNF- might exert on neurons in our brain abscess
model. In this case, receptor expression may be detrimental, leading to
neuronal cell death via signaling through TNFR. In addition to glial
and neuronal cells, TNFR is expressed by monocytes/macrophages which
are found infiltrating abscess lesions.42,43
Activation of
monocytes/macrophages via TNF--dependent signaling leads to the
synthesis of nitric oxide44
and cytokine and chemokine
mediators45-47
which can amplify the immune response. In
the CNS, some neurons and astrocytes express IL-6R. However, the amount
of IL-6R on astrocytes is not sufficient by itself to deliver an
activation signal, and requires the presence of a soluble form of the
IL-6R generated by shedding of the membrane-bound receptor or by mRNA
alternative splicing.25,48
Interestingly, astrocytes and
sympathetic neurons can produce IL-6 and may respond to it in an
autocrine/paracrine manner if sIL-6R is present,25,48,49
a
mechanism for amplifying IL-6 expression in the CNS. We are actively
investigating proinflammatory cytokine receptor expression to identify
which cell populations are competent to respond to the mediators
detected in early brain abscess lesions.
The cell types responsible for proinflammatory cytokine and chemokine
production in experimental brain abscesses remain to be defined. Their
rapid induction suggests that the response is driven by resident CNS
glia or cells associated with the ensuing inflammatory infiltrate (ie,
neutrophils and/or monocytes/macrophages). Results from ISH analysis
revealed that CD11b+ cells are the source of IL-1ß within early brain
abscesses. This population includes resident microglia and infiltrating
monocytes and neutrophils. In agreement with our findings, it has been
shown that human microglia can produce IL-1ß,50,51
and
S. aureus has been shown to directly stimulate IL-1ß
production by neonatal rat microglia in
vitro.24
We have preliminary data (Kielian,
unpublished observations) which demonstrates that S. aureus
can directly induce neonatal mouse microglia and astrocytes to produce
numerous proinflammatory cytokines and chemokines including IL-1ß,
TNF-, IL-6, MCP-1, MIP-1, MIP-1ß, MIP-2, RANTES, and MIP-1.
Studies are ongoing to further identify the cell types within
developing brain abscesses responsible for mediator production.
The question remains, what is the impetus driving abscess formation and
can the progression of the abscess and immunopathological destruction
of surrounding normal brain tissue be minimized by altering the
production of proinflammatory cytokines? The chronic overexpression of
proinflammatory cytokines initially induced in response to S.
aureus may itself have a direct detrimental effect on the
surrounding normal brain parenchyma, ultimately leading to tissue
destruction. The uncontrolled amplification of the immune response to
S. aureus can be explained by the autocrine/paracrine action
of proinflammatory cytokines on resident glial cell populations in the
brain. For example, IL-1 could stimulate astrocyte activation and
production of TNF- and IL-6,21,52-54
whereas activated
microglia produce IL-1 and TNF-.50,51
It is possible
that a tissue toxic microenvironment might evolve from the robust
production of proinflammatory cytokines immediately following exposure
to S. aureus, as shown in this study. This overproduction of
proinflammatory cytokines could result in a detrimental or cytocidal
bystander effect, leading to the damage of surrounding normal brain
parenchyma. Thus, the process of neural tissue necrosis may be underway
before the arrival of large numbers of neutrophils. By controlling the
amount of proinflammatory cytokine production in response to
S. aureus, this could lead to bacterial neutralization
while minimizing tissue destruction due to the chronic overactive
immune response.
In summary, the results presented in this report establish the expression of several proinflammatory cytokines and chemokines within early brain abscess lesions which may participate in cell activation and amplification of the immune response initiated by S. aureus. Characterization of inflammatory mediators produced in response to S. aureus in the brain may lead to novel therapies for the clinical management of brain abscesses.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from the National Institutes of Health/National Institute of NDS (NS-27321) and The Hitchcock Foundation.
Accepted for publication May 19, 2000.
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References |
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