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The Journal of Immunology, 2008, 181, 2713 -2722
Copyright © 2008 by The American Association of Immunologists, Inc.
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Ly-6G+CCR2– Myeloid Cells Rather Than Ly-6ChighCCR2+ Monocytes Are Required for the Control of Bacterial Infection in the Central Nervous System1

Alexander Mildner2,*, Marija Djukic2,{dagger}, David Garbe{dagger}, Andreas Wellmer{dagger}, William A. Kuziel{ddagger}, Matthias Mack§, Roland Nau{dagger},¶ and Marco Prinz3,*

* Department of Neuropathology, University of Freiburg, Freiburg, Germany; {dagger} Department of Neurology, Georg-August-University, Göttingen, Germany; {ddagger} Protein Design Labs, Inc., Fremont, CA 94555; § Department of Internal Medicine, University of Regensburg, Regensburg, Germany; Department of Geriatrics, Evangelisches Krankenhaus, Göttingen-Weende, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Myeloid cell recruitment is a characteristic feature of bacterial meningitis. However, the cellular mechanisms important for the control of Streptococcus pneumoniae infection remain largely undefined. Previous pharmacological or genetic studies broadly depleted many myeloid cell types within the meninges, which did not allow defining the function of specific myeloid subsets. Herein we show that besides CD11b+Ly-6G+CCR2– granulocytes, also CD11b+Ly-6ChighCCR2+ but not Ly-6ClowCCR2– monocytes were recruited in high numbers to the brain as early as 12 h after bacterial challenge. Surprisingly, CD11b+Ly-6ChighCCR2+ inflammatory monocytes modulated local CXCL2 and IL-1β production within the meninges but did not provide protection against bacterial infection. Consistent with these results, CCR2 deficiency strongly impaired monocyte recruitment to the infected brains but was redundant for disease pathogenesis. In contrast, specific depletion of polymorphonuclear granulocytes caused elevated local bacterial titer within the brains, led to an aggravated clinical course, and enhanced mortality. These findings demonstrate that Ly-6ChighCCR2+ inflammatory monocytes play a redundant role for the host defense during bacterial meningitis and that predominantly CD11b+Ly-6G+CCR2– myeloid cells are involved in the restriction of the extracellular bacteria.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Streptococcus pneumoniae is a Gram-positive extracellular bacterial pathogen that causes severe bacterial meningitis in industrialized countries, with a mortality rate of one-fourth in adults. Many patients survive with neurological sequelae such as deafness, epileptic seizures, and neuropsychological deficits (1, 2).

In humans suffering from bacterial meningitis and in animal models of this disease, leukocytes, predominantly myelomonocytic cells such as monocytes, macrophages, and neutrophil granulocytes, quickly enter the subarachnoid space in response to local production of cytokines, chemokines, and other chemotactic stimuli (3). Activated myeloid cells play a major role in bacterial clearance and also secrete a variety of inflammatory mediators that may not only kill bacteria, but also cause damage to the CNS (4). Since the cerebrospinal fluid itself lacks cell populations capable of initiating effective immune response against invading pathogens, it remains obscure which cells are initially stimulated by bacteria and bacterial products and trigger leukocyte migration into the CNS.

Local tissue inflammatory responses to microbial challenge are not only characterized by early neutrophil attraction but also by concomitant prolonged monocyte recruitment. Although chemokine release and cell surface display of complementary leukocyte and endothelial/epithelial adhesion molecules are centrally involved in this process, the underlying role of monocyte recruitment during bacterial meningitis is still unclear. In a rat model of pneumococcal meningitis, liposome-based depletion of meningeal and perivascular macrophages aggravated clinical symptoms and led to increased bacterial titers, suggesting a protective role of these cells during bacterial meningitis (5). Depletion of all blood monocytes and macrophages in spleen and liver, and probably also many tissue macrophages including perivascular macrophages, reduced the migration of white blood cells into the cerebrospinal fluid (CSF)4 and strongly inhibited local IL-1β concentrations in infected rats, suggesting a crucial role of monocytes during meningitis (6).

Monocytes, as blood mononuclear cells with bean-shaped nuclei, express CD11b, CD11c, CD14, and CD16 in humans and CD11b and F4/80 in mice, and they lack B, T, and NK markers. However, monocytes are morphologically and phenotypically heterogeneous with different roles. At present, they are subdivided into two main subsets: a short-lived "inflammatory" subset (Ly-6ChighCCR2+CD62L+CX3CR1low) that homes to inflamed tissue, where it can trigger immune response, and a "resident" subset (Ly-6ClowCCR2–CD62L–CX3CR1high) with a longer half-life, which homes to noninflamed tissues (7). Dendritic cells such as Langerhans cells in the skin (8) and tissue macrophages like postnatal microglia in the brain (9), however, were recently shown to arise from the Ly-6ChighCCR2+ monocyte population, indicating a broad role of this myeloid subpopulation for tissue homeostasis during health and disease. Whether the different monocyte populations are already fully differentiated in the bone marrow or whether they can shift into each other in the peripheral blood is still a matter of debate.

The monocyte population in the bloodstream that is recruited to the inflamed meninges during bacterial infection has not yet been identified in vivo. Elucidating the cell types involved could theoretically open new therapeutic options. We therefore set out experiments to characterize the specific monocyte subtype that is recruited to the inflamed brain and then to identify its role for the local immune response within the meninges.

In this report we demonstrate for the first time that concomitant with CD11b+Ly-6G+CCR2– granulocytes also CD11b+Ly-6ChighCCR2+, but not Ly-6ClowCCR2–, monocytes invade the meninges during bacterial infection and are capable of modulating local cyto- and chemokine response. Surprisingly, this cell population does not contribute to the host defense since the absence of inflammatory Ly-6Chigh monocytes does not alter mortality and bacterial replication in the CNS and the spleen. In contrast, our study identifies Ly-6G+CCR2– granulocytes as the major myeloid cell subset that crucially drives defense against S. pneumoniae infection in the CNS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Induction of meningitis and myeloid cell depletion

A strain of S. pneumoniae serotype 3 was used. Bacteria were grown on blood agar plates overnight and harvested with 0.9% saline. Frozen aliquots were used for the experiments and adjusted with saline to the required concentration of 4 or 5 log10 CFU in 25 µl. Meningitis experiments were conducted in male C57BL/6 und CCR2–/– mice using a modification of a previously published model of meningitis based on injection of bacteria into the lumbal cerebrospinal fluid (10). In brief, mice were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). A skin incision was made exposing the lumbar spine. Using a 29-gauge needle, 25 µl of a suspension containing 4 or 5 log10 CFU S. pneumoniae or an equal amount of saline was slowly injected into the spinal canal at the level of L4 or L5. All animals resumed their normal behavior after awaking from anesthesia. For depletion experiments, Abs were injected 12 h prior infection and every 24 h later. Transient neutropenia was achieved by using 10 µg anti-Gr-1 mAb (RB6-8C5; eBioscience). Inflammatory monocytes were depleted with 10 µg mAb MC-21 (9, 11). Rat IgG2b served as control Ab.

During the course of the experiment mice were assessed by a clinical score (12). Mice unable to walk were killed for ethical reasons. For the determination of bacterial load, animals were killed at several time points and the cerebellum and spleen were removed, homogenized in saline (dilution 1/10), and undiluted homogenate as well as serial 1/10 dilutions (10 µl each) were plated on blood-agar plates. The detection limit was 103 CFU/ml. CX3CR1GFP/+ mice were a kind gift of Dan Littman (New York University, New York, NY) (13), and CCR2–/– animals were provided by William A. Kuziel (Protein Design Labs, Fremont, CA) (14).

Cell preparation

For meningeal cell isolations mice were transcardially perfused with PBS. With longitudinal and diagonal incisions through the cranium, cranial bone was removed. Meninges were afterwards carefully removed and rinsed in PBS. For adoptive transfer experiments mice received 2.5 x 105 GFP+ bone marrow cells from CX3CR1GFP/+ mice i.v. 6 h after infection with S. pneumoniae or saline, and 24 h later meningeal cells were isolated and cells were prepared for FACS analysis.

All experiments were performed at the Central Animal Care Facility of the University Hospital Göttingen. The protocol was approved by the district Braunschweig legislation for animal experiments, Germany.

RNA isolation and real-time PCR analysis

RNA was extracted from isolated meningeal cells by using RNeasy Mini kits (Qiagen) following the manufacturer’s instructions. The tissue was flushed with ice-cold HBSS, and RNA was isolated using RNeasy Mini kits following the manufacturer’s instructions. The samples were treated with DNase I (Roche) and 1 µg of RNA was transcribed into cDNA using oligo(dT) primers and the SuperScript II RT kit (Invitrogen). cDNA (2.5 µl) was transferred into a 96-well Multiply PCR plate (Sarstedt) and 12.5 µl ABsolute QPCR SYBR Green master mix (ABgene) plus 9.6 µl double-distilled H2O was added. The PCR reaction was performed as described recently (15).

Flow cytometry

Blood and meningeal cell samples were prepared at 4°C in buffer solution (PBS containing 2% FCS, 1 mM EDTA and 0.2% NaN3) and stained with CD11b, Gr-1, B220, NK1.1, CD45 (all eBioscience), Ly-6C and Ly-6G (BD Pharmingen), or CCR2 (16). After lysis of erythrocytes with FACS lysis solution for blood samples (BD Biosciences), cell suspensions were analyzed on a FACSCalibur (BD Biosciences). Data were acquired with WinMDI.

Tissue preparation and immunohistochemistry

Deparaffinized and hydrated brain tissue was cut into 1-µm-thick sections. Sections were then pretreated with microwaving for 3 x 5 min in citric acid buffer (10 mmol/L (pH 6.0)). After blocking with 10% FCS/PBS for 30 min, primary Abs were applied. For chloracetate esterase staining, longitudinal paraffin sections were incubated for 1 h with a solution containing naphthol AS-D chloroacetate (Sigma-Aldrich), pararosaniline (Merck), and sodium nitrite (Sigma-Aldrich). Granulocytes appeared as red stained cells, providing evidence for metabolism of chloroacetate esterase.

Rat MAC-3 anti-mouse Ab (1/200, BD, Biosciences) was used for MAC-3 staining for 30 min at room temperature. As secondary Ab, biotinylated rabbit anti-rat lgs (DakoCytomation) were used diluted 1/200 in PBS containing 1% BSA for 60 min. The slides were counterstained with hemalum (Merck) and washed with aqua dest.

Statistical analysis

Statistical differences of clinical scores were evaluated using a non-paired Student’s t test. Differences were considered significant with a p value of <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expansion of different myeloid cells in the peripheral blood during S. pneumoniae infection

We first determined the extent and kinetics of specific myeloid subsets that are proliferating in the peripheral blood of infected mice. For this purpose wild-type (wt) mice were challenged with 105 CFU of S. pneumoniae injected into the lumbal cerebrospinal fluid and different myeloid blood cells were characterized by flow cytometry (Fig. 1). As early as 12 h after infection, a strong increase of CD11b+Ly-6Cint and CD11b+Ly-6Chigh cells was detectable in the peripheral blood (Fig. 1, top panel). Subsequent analysis revealed that these myeloid cells consisted mainly of CD11b+Ly-6G+Ly-6Cint granulocytes (R2 gate) and of CD11b+Ly-6G–Ly-6Chigh (R1 gate) inflammatory monocytes. Comparative analysis to B lymphocytes indicated that Ly-6G+Ly-6Cint granulocytes increased from a 0.33 ± 0.08 (mean ± SD) ratio before infection to 24.77 ± 14.26 36 h after infection, whereas Ly-6G–Ly-6Chigh inflammatory monocytes improved from a 0.07 ± 0.01 ratio at 0 h to 0.85 ± 0.47 after 36 h (Fig. 1, lower panel). Interestingly, the increase of myeloid cells during the first hours after infection was not due to the appearance of bacteria in blood, since we were only able to detect S. pneumoniae after 24 h postinfection (p.i.) (data not shown).


Figure 1
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  		FIGURE 1. Increase of CD11b+Ly-6G–Ly-6Chigh monocytes and CD11b+Ly-6G+Ly-6Cint granulocytes in the peripheral blood during S. pneumoniae infection of the CNS. Inflammatory make-up of the peripheral blood at indicated time points after bacterial infection examined by flow cytometry (A). Myeloid cells were normalized to 20,000 B cells (B220+) and a ratio was calculated (B). Data represent means ± SD. Asterisks indicate statistically significant differences to the noninfected controls (p ≤ 0.05). At least five mice per group were used for quantification.

 
Rapid invasion of distinct myelomonocytic cell populations to the meninges during S. pneumoniae infection

Because not only granulocytes but also CD11b+Ly-6Chigh inflammatory monocytes were mobilized in the blood, we asked whether these cells could play any role during S. pneumoniae infection. For this purpose we first measured the production of the chemokine CCL2, the main ligand of CCR2, in the inflamed brain (Fig. 2A). As early as 12 h after infection a significant increase of CCL2 production from 47.3 ± 2.1 pg/ml in uninfected animals to 160.1 ± 61.2 pg/ml in infected mice could be observed, and the CCL2 levels continued to increase 36 h p.i. to 1926.3 ± 341.2 pg/ml. These data clearly show that the ligand for CCR2 is produced locally in the infected brain.


Figure 2
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  		FIGURE 2. Production of CCL2 in the infected nervous system and recruitment of myelomonocytic cell populations into the meninges. A, Production of CCL2 was measured by ELISA at different time points after infection. At least three mice per group were used. Shown are means ± SD. Statistically significant differences were indicated with asterisks. B, Characterization of meningeal infiltrates 30 h after S. pneumoniae challenge (right) in comparison to healthy control mice (left). Notably, the Gr-1+ staining revealed that infiltrates are dominated by CD11b+Ly-6G–Ly-6ChighCCR2+ monocytes as well as by CD11b+Ly-6G+Ly-6CintCCR2– granulocytes.

 
In the next step we investigated the recruitment of myeloid cells into the inflamed CNS. Importantly, myelomonocytic infiltrates were observed in the meninges of the CNS already at 12 h (see Fig. 4C), and the cellular composition was examined in more detail at peak of disease 30 h p.i. (Fig. 2B). Mice exhibited numerous CD11b+Gr-1+ cells, suggesting that mainly polymorphonuclear leukocytes were recruited into the meninges. However, further division of the Gr-1 Ag into the Ly-6C and Ly-6G epitopes revealed that besides CD11b+Ly-6G+Ly-6CintCCR2– granulocytes also large numbers of CD11b+Ly-6G–Ly-6ChighCCR2+ inflammatory monocytes infiltrated the meninges. These results suggest that the production of CCL2 leads to a strong recruitment of CCR2+ monocytes into the meninges. In contrast, CD11b+Ly-6G–Ly-6ClowCCR2– resident monocytes were not increased in the CNS during S. pneumoniae meningitis.


Figure 4
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  		FIGURE 4. Unaltered pathogenesis of S. pneumoniae meningitis in the absence of CCR2. A, CCR2–/– and CCR2+/+ mice were challenged with either 104 (left) or 105 CFU (right) of S. pneumoniae, and survival as well as clinical score were monitored. There were no statistically significant differences observed between the groups. At least 10 animals per group were used. Data represent means ± SD. B, Normal bacterial clearance in the peripheral (spleen) and central compartment (CNS) 12, 24, and 36 h after infection. No statistically significant differences were detectable. At least five animals per time point were used. C, Time-dependent myeloid infiltration of the meninges after intraspinal S. pneumoniae infection. A strong influx of Ly-6ChighLy-6G– monocytes and Ly-6CintLy-6G+ granulocytes was detectable in CCR2+/+ animals. In contrast, CCR2-deficient mice revealed strongly diminished numbers of Ly-6Chigh monocytes during infection, whereas the Ly-6G+ granulocytes appeared largely unchanged. One representative animal out of five is shown. D, Immunohistochemistry of the meninges depicted less MAC-3+ monocytes/macrophages in CCR2–/– mice compared with wild-type controls, whereas chloroacetate esterase staining showed similar amounts of polymorphonuclear granulocytes during S. pneumoniae meningitis. Tissue samples were taken 30 h after infection. Bar = 100 µm.

 
Taken together, these data demonstrate that both CD11b+Ly-6G–Ly-6ChighCCR2+ monocytes as well as CD11b+Ly-6G+Ly-6CintCCR2– granulocytes are strongly attracted to the inflamed meningitis early after infection.

CX3CR1GFP/+ monocytes are efficiently recruited to the inflamed meninges

To assess the ability of GFP-marked monocyte subsets derived from bone marrow to be recruited to the meninges during S. pneumoniae infection, we performed adoptive transfer experiments. We took advantage of the CX3CR1GFP/+ mice, which allow division of the Ly-6Chigh and Ly-6Clow monocyte fractions on the basis of GFP expression levels (7, 9). Respective GFP bone marrow cells were adoptively transferred 6 h p.i. and meningeal cells were prepared 24 h later and examined by flow cytometry (Fig. 3). GFP-labeled cells were clearly visible in the infected CNS but not in the healthy brain. Moreover, these donor-derived attracted cells were clearly positive for CD11b and Ly-6C but negative for Ly-6G, indicating the inflammatory nature of these myeloid cells. Interestingly, we were not able to detect any GFPhighCD11b+Ly-6Clow resident monocytes in the meninges after transfer of CX3CR1GFP/+ bone marrow cells.


Figure 3
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  		FIGURE 3. Adoptively transferred inflammatory GFP+Ly-6Chigh monocytes are efficiently recruited to the meninges during bacterial infection. CX3CR1GFP/+ monocytes were injected 6 h after infection and meningeal cells were recovered 24 h later. Transferred cells were detectable in the CNS of infected but not in healthy control mice. GFP+ cells (R1 gate) express the cell surface markers Ly-6C and CD11b but not Ly-6G.

 
CCR2 is not required for the protection against S. pneumoniae meningitis

The chemokine receptor CCR2 as the cognate receptor for CCL2 has been shown to be crucially involved in the pathogenesis of both sterile autoimmunity (17) as well as of several infections by bacterial and viral germs such as Listeria monocytogenes, Mycobacterium tuberculosis, Theiler’s murine encephalomyelitis virus, and influenza (18, 19, 20, 21).

Because we could detect Ly-6ChighCCR2+ monocytes that infiltrated the meninges in high numbers (Fig. 2B), we decided to study the relevance of CCR2 for the pathogenesis of bacterial meningitis in more detail. CCR2-deficient or -competent mice were challenged with various quantities of S. pneumoniae, and survival time and clinical course were monitored (Fig. 4A). After intralumbal injection into the CSF with a high dose (105 CFU) and lower dose (104 CFU) of S. pneumoniae serotype 3, all treated CCR2–/– and wt mice reached terminal disease with similar kinetics without any statistically significant differences. In both genotypes, increased illness was correlated with typical clinical signs of meningitis such as seizures, paralysis, and neck stiffness (12). Disease score was clearly not different in both groups.

We then determined the ability of bacteria to multiply over time in spleens and brains of infected mice (Fig. 4B). Bacterial load was determined by plating serial dilutions of homogenized tissues on blood-agar plates. Bacterial growth was similarly low at 12 h p.i. in spleens and brains of both groups, but titers in the brain were always higher than those in the spleen. Further measurements at 24 and 36 h after injection confirmed that the bacterial load in wt and CCR2–/– mice were always similar. A lack of CCR2 therefore does not impair the kinetics of S. pneumoniae invasion and multiplication after administration of bacteria into the lumbal CSF.

It has been reported that the CCR2-CCL2 axis is required for the egress of bone marrow cells and that the lack of CCR2 results in fewer circulating Ly-6Chigh cells (9, 22). We therefore determined the frequency and kinetics of Ly-6Chigh cells in the meninges of CCR2 knockout and wt mice at different time points after infection. Importantly, the amount of Ly-6ChighLy-6G– monocytes and Ly-6CintLy-6G+ granulocytes was identical in the meninges of CCR2–/– and wt mice before infection (Fig. 4C). After infection, however, the quantity of Ly-6Chigh monocytes peaked in wt mice 24 h p.i., with similar frequency to Ly-6G+ granulocytes, and slightly decreased 6 h later. In contrast, CCR2-deficient mice largely failed to show a significant recruitment of Ly-6Chigh cells into the CNS and only Ly-6CintLy-6G+ granulocytes were detectable there. Further histochemical analysis confirmed the strong reduction of MAC-3+ monocytes/macrophages in the meninges in the absence of CCR2, whereas the amount of chloroacetate esterase-positive granulocytes was virtually indistinguishable between the two genotypes (Fig. 4D).

Ly-6ChighLy-6G– monocytes regulate meningeal cyto- and chemokine production but are not required for the control of bacterial meningitis

Inflammatory Ly-6ChighLy-6G– monocytes are equipped with high levels of receptors that respond to inflammatory cytokines, resulting in migration of the cells to the site of inflammation, where they subsequently contribute to resolution of the inflammatory process and might finally differentiate into dendritic cells (7).

We generated mice lacking inflammatory Ly-6ChighLy-6G– monocytes to determine whether these cells might contribute to protection from pneumococcal infection. Application of the anti-CCR2 Ab MC-21 resulted in an efficient reduction of this specific monocyte subset in the bloodstream, whereas Ly-6CintLy-6G+ granulocytes (Fig. 5A) or Ly-6C–Ly-6G– resident monocytes remained numerically unchanged (data not shown and Ref. 9). Unexpectedly, both groups of infected mice had a similar survival curve, and clinical scores across the entire disease time were almost identical (Fig. 5B). Even secondary lymphoid organs such as the spleen, which is an important bacterial clearance site, contained approximately equal bacterial titers in all animals tested independently for the presence of inflammatory monocytes (Fig. 5C). As expected, the amount of Ly-6ChighLy-6G– monocytes was strongly diminished in the meninges of infected animals after application of the MC-21 Ab (Fig. 5D), pointing to a confined lack of this myeloid cell population. We also determined the total cell numbers in the inflamed meninges of isotype and anti-CCR2 Ab-treated mice by FACS analysis and found, besides the significant reduction of inflammatory monocytes in anti-CCR2 Ab-treated mice, similar numbers of granulocytes (Table I), indicating that the migration of granulocytes is independent of the presence of monocytes.


Figure 5
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  		FIGURE 5. CD11b+Ly-6G–Ly-6Chigh monocytes modulate local CXCL2 and IL-1β production within the CNS but do not provide protection against bacterial infection. A, Application of an anti-CCR2 Ab (MC-21) strongly depleted Ly-6G–Ly-6Chigh monocytes but not Ly-6G+Ly-6Cint granulocytes in the peripheral blood 12 h after injection of 10 µg of the Ab or of the IgG2b isotype control, respectively (left). Only CD11b+ cells were gated in the dot blots. Ratio of Ly-6G–Ly-6Chigh monocytes and Ly-6G+Ly-6Cint granulocytes to B220+ B cells in the peripheral blood (right). At least five animals per group were used. *, p ≤ 0.05. B, Depletion of Ly-6G–Ly-6Chigh monocytes did not modulate the survival of mice (left) and the clinical score (right) of meningitis. Injection of Abs was performed 12 h before infection and every 24 h later. Ten mice per group were used. Data indicate means ± SD. C, Unchanged bacterial loads of S. pneumoniae 30 h after infection in spleen and cerebellum in the absence of Ly-6Chigh monocytes. Data represent means ± SD (n = 5 animals). D, Strong decrease of invading inflammatory monocytes into the meninges after depletion of Ly-6G–Ly-6Chigh monocytes in the peripheral blood. Importantly, the influx of Ly-6G+Ly-6Cint granulocytes remained unchanged. One representative animal out of five is shown. E, RNA was isolated from meningeal cells 30 h p.i. and real-time PCR was performed for CXCL2, lysozyme M, IL-15, and IL-1β. Open bars illustrate control (isotype-treated) animals and filled bars represent Ly-6Chigh-depleted animals. Three mice were used per group. Data show means ± SD. *, p < 0.05.

 

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  		Table I. Quantitative assessment of meningeal infiltrates during S. pneumoniae meningitis after depletion of selective myeloid subpopulationsa

 
The local cytokine and chemokine production was then assessed quantitatively by RT-PCR in Ly-6ChighLy-6G–-depleted animals during meningitis (Fig. 5E). Surprisingly, the enzyme lysozyme that has known antibacterial activity against Gram-positive bacteria was slightly reduced and IL-1β and CXCL2 were significantly increased in the absence of Ly-6Chigh cells. We therefore conclude that the presence of Ly-6ChighLy-6G– inflammatory monocytes might not be a prerequisite for the control and resolution of S. pneumoniae infection within the CNS, but their absence leads to a change of the cytokine repertoire.

Ly-6CintLy-6G+ myeloid cells are mandatory for the control of S. pneumoniae infection of the meninges

We next studied the impact of Ly-6CintLy-6G+ granulocytes on disease pathogenesis. To do so, Ly-6CintLy-6G+ cells were pharmacologically depleted by using an anti-Gr-1 Ab before disease. Flow cytometry verified almost complete deletion of this myeloid cell population, but Ly-6ChighLy-6G– cells were unaltered (Fig. 6A).


Figure 6
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  		FIGURE 6. CD11b+Ly-6G+Ly-6Cint myeloid cells are essential for the control and clearance of S. pneumoniae meningitis. A, Efficient depletion of Ly-6G+Ly-6Cint granulocytes in the peripheral blood by monoclonal anti-Gr1 Ab RB6-8C5. Ten micrograms of anti-Gr1 led to a complete absence of Ly-6G+Ly-6Cint granulocytes in comparison to isotype control (rat IgG2b). Inserts in the FACS blots (left) showed in the forward and side scatter that the anti-Gr1 treatment did not lead to a down-regulation of Gr1 since the side scatter high cells are disappearing. Ly-6Chigh monocytes were not affected by this protocol. Quantification was performed by counting similar numbers of B220+ cells. Five animals per group were used. *, p < 0.05. B, Depletion of granulocytes led to earlier death (left) of infected mice and a significantly increased clinical score (right). Injection of Abs was performed 12 h before infection and every 24 h later. Ten mice per group were used. *, p < 0.05. C, S. pneumoniae CFU was measured 30 h after infection in isotype and anti-Gr1-treated mice. Elevated bacterial load in the cerebella of anti-Gr1-treated mice (*, p < 0.05). D, FACS analysis of Ab-treated animals revealed the absence of Ly-6G+Ly-6Cint granulocytes in the meninges. One representative animal out of five is shown. E, RNA was isolated from the meninges 30 h p.i. and real-time PCR was performed for CXCL2, lysozyme M, IL-15, and IL-1β. Isotype control animals are shown as open bars and granulocyte-depleted mice as filled bars. Three mice were used per group. Data are shown as means ± SD. *, p < 0.05.

 
Mice were subsequently inoculated with 105 CFU of S. pneumoniae, and incubation times and clinical scores to terminal disease were measured (Fig. 6B). Importantly, Ly-6CintLy-6G+-depleted animals died significantly earlier and exhibited more severe clinical symptoms of disease compared with isotype-treated control animals. We then compared bacterial loads in spleens and brains 30 h after infection (Fig. 6C). Notably, the bacterial concentrations were significantly increased in the CNS but not in the spleens after Ly-6CintLy-6G+ depletion in comparison to immunocompetent animals, thus proving an essential function of these cells for combating S. pneumoniae infection.

FACS analysis confirmed that Ly-6CintLy-6G+ granulocytes were virtually absent from the meninges of Ab-treated animals (Fig. 6D). However, the depletion of granulocytes led to a reduction of total cell infiltrates of ~50% (Table I). Nevertheless, inflammatory monocytes were present in comparable numbers (56,000 ± 13,000 cells) in isotype-treated mice vs 77,000 (±40,000) cells in anti-Gr-1 Ab-treated mice.

Since Ly-6CintLy-6G+ cells might contribute to the bacterial clearance by releasing bacteriotoxic cytokines, proinflammatory peptides in the meninges were measured 30 h p.i. (Fig. 6E). We observed strongly reduced levels of CXCL2, IL-15, and IL-1β, suggesting that bacterial clearance might act via a burst of several chemoattractive and proinflammatory factors.

The results imply that Ly-6CintLy-6G+ cells rather than Ly-6ChighLy-6G– or Ly-6C–Ly-6G– monocytes are required to provide local host resistance to bacterial meningitis induced by S. pneumoniae.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have shown herein that specific myelomonocytic subsets, namely CD11b+Ly-6ChighCCR2+ inflammatory monocytes and CD11b+Ly-6G+CCR2– granulocytes, but not CD11b+Ly-6C–CCR2– monocytes, are efficiently recruited to the meningeal compartment during bacterial meningitis caused by the Gram-positive pathogen S. pneumoniae. Despite the fact that Ly-6ChighCCR2+ monocytes are efficiently recruited into the meninges, the depletion of this monocyte subset or the chemokine receptor CCR2 preserved the ability to limit bacterial growth, while depletion of Ly-6G+CCR2– cells conferred an increased vulnerability to pneumococcal infection. These differential and nonredundant effects of specific myeloid subsets were surprising, since all defects affected cells, which are thought to be crucial for host defense against bacteria.

The different cell populations involved in the induction of the inflammatory response in the subarachnoid space during bacterial meningitis have not been fully characterized. For example, it still remains unclear which cells are primarily stimulated by the invading bacteria and bacterial products. A candidate for this initial step could be the meningeal macrophage. However, studies that investigated the role of meningeal macrophages during meningitis by liposomal clodronate depletion of these cells showed inconsistent results (5, 23). Both studies used different modes of clodronate administration, either the intraventricular (5) or the intracisternal (18) route. Because of the direction of CSF flow, the intraventricular route appears to be more efficient in depleting the macrophages of the choroid plexus, ventricular ependyma, and possibly also of the meninges. Intraventricular clodronate strongly reduced meningeal inflammation in a murine model of pneumococcal meningitis (5). This suggests that resident macrophages within the choroid plexus, ventricular ependyma, and the meninges are responsible for the cytokine and chemokine gradient causing leukocyte extravasation in early meningitis.

The contribution of circulating monocytes is less clear. This prompted us to study meningeal inflammation during pneumococcal infection in more detail. We found that in response to intralumbal S. pneumoniae infection both Ly-6ChighCCR2+ inflammatory monocytes and Ly-6G+CCR2– granulocytes rapidly became the predominant cell types in the diseased meninges, as has been similarly shown for the lung in response to i.p. H. influenzae injection (24).

By virtue of their mobility, by being equipped with immune-related receptors, and by their ability to produce antibacterial substances such as NO, IL-1β, and others, CD11b+Ly-6ChighCCR2+ inflammatory monocytes presented a plausible candidate for the uptake and killing of extracellular Gram-positive bacteria. We found a nonessential role of monocytes/macrophages as clearance sites of S. pneumoniae. However, meningeal macrophages may protect brain tissue from invasion by granulocytes and bacteria, thereby preventing neuronal injury during S. pneumoniae meningitis (23). Neuron-specific enolase in CSF as a measure of neuronal tissue damage was elevated in experimental rabbits with S. pneumoniae meningitis after intracisternal clodronate treatment (23). In the present study, we were unable to measure neuron-specific enolase in CSF because of the low CSF volume in mice.

Several groups have employed clodronate-containing liposomes to eliminate the circulating pool of monocytes and macrophages in the blood, spleen, and liver (25). This approach was also used to study the role of blood-derived macrophages in experimental pneumococcal menigitis in rabbits (6) and experimental autoimmune encephalomyelitis in rats (26). In contrast to the present approach, these studies could not distinguish and deplete the different monocyte populations specifically. Clodronate liposomes lead to depletion of all circulating monocytes, including Ly-6Chigh and Ly-6Clow monocytes, as well as tissue macrophages such as Kupffer cells and splenic macrophages (27). A transient loss of NK cell activity after i.v. liposomal clodronate infusion also has been described (28). These data also indicated that the usage of phagocyte depletion by clodronate has a severe impact on the immune system in general by releasing intracellular factors from dying cells. Therefore, i.v. liposomal clodronate does not appear to be suitable for the detailed study of the pathogenic mechanisms of a myeloid-based immune response as in meningitis. To overcome these limitations, we decided to deplete only the invading Ly-6Chigh monocytes, which does not result in increased cytokine levels in the blood (11).

Although it has been shown that IL-1β produced by monocytes/macrophages during pneumococcal meningitis is a key player for the migration of granulocytes in the CSF (29, 30), depletion of Ly-6ChighCCR2+ monocytes in the meninges did not change clinical outcome in our hands. The local IL-1β production within the meninges was not decreased by the lack of this monocyte subtype. Our data imply that local IL-1β production is primarily independent from invading Ly-6ChighCCR2+ monocytes, and other cells can maintain inflammatory cytokine production. Due to our cell-specific depletion, F4/80+ meningeal and perivascular macrophages usually diminished by local liposomal clodronate treatment were still present in the meninges of treated animals. Moreover, meningeal macrophages are not the only source of proinflammatory cytokines in the brain including the CSF. Most TNF-{alpha} and IL-1β in the CSF during meningitis presumably originate from blood-derived cells after migration into the CSF or other cells within the CNS (e.g., glial cells, endothelial cells). Interestingly, local CXCL2 and IL-1β production was even significantly increased in the absence of inflammatory Ly-6ChighCCR2+ monocytes, but changes in cellular compositions within the CSF could lead to alterations of the cytokine pattern.

Our results further show that CCR2, the receptor for CCL2, is not necessary to inhibit the growth of bacteria in pneumococcal menigitis. It has been shown in several infection models that CCR2-dependent monocyte recruitment and activation are essential for host survival, for example, during L. monocytogenes infection (31). However, our results clearly indicate that CCR2 signaling also is not required for the development of inflammation in meningitis caused by the extracellular pathogen S. pneumoniae. These data nicely fit to the redundant role of Ly-6Chigh inflammatory monocytes in our depletion experiments in view of the fact that this myeloid subset is strongly reduced in CCR2-deficient animals, since this chemokine receptor is needed for the egression of monocyte populations from the bone marrow (22).

CD11b+Ly-6G+CCR2– granulocyte-depleted animals consistently demonstrated in our study earlier death and higher CNS bacterial burdens compared with control animals. This is in conflict to a previous study where induction of systemic leukopenia by nitrogen mustard in experimental rabbits did not cause increased bacterial growth rates within the CSF after intracisternal injection of S. pneumoniae (32). One reason may be the nonselective action of nitrogen mustard, which affected several lines of leukocytes but probably did not fully deplete one class of white blood cells. In this study, S. pneumoniae led to the induction of numerous cyto- and chemokines in the brain, including CXCL2, IL-15, and IL-1β, shortly after bacterial exposure. Granulocytes produce large amounts of these factors in an autocrine fashion, and CXCL2, IL-1β, and IL-15 were clearly down-regulated in Gr-1-depleted animals. Therefore, our data imply that these molecules are required for the granulocyte-mediated host defense in experimental pneumococcal meningitis. These conclusions are in line with previous studies, which showed a critical role of neutrophils and CXCL2 and CXCL1 in the containment and neutralization of bacteria in a brain abscess model (33).

Furthermore, we found that depletion of Ly-6G+CCR2– granulocytes resulted in strongly decreased levels of the chemokine CXCL2. This chemokine has been shown to play an important role in the pathogenesis of meningitis, since its neutralization reduced neutrophil recruitment to the CNS during bacterial meningitis caused by H. influenzae (34). Another study nicely demonstrated that in mice deficient in the murine IL-8 receptor homolog (a receptor for CXCL2), invasion of Ly-6G+ granulocytes into the peritoneal cavity in response to inflammatory stimuli was strongly impaired (35). Conversely, increased levels of CXCL2 accompanied by enhanced granulocytosis in the CSF were not correlated with disease protection in CD14-deficient animals suffering from pneumococcal meningitis (36).

It is well known that the anti-Gr-1 Ab used in our study strongly reacts with Ly-6G and that this Ab also interferes with another Ly-6 family molecule, Ly-6C (37). Ly-6G is expressed on polymorphonuclear leukocytes, namely granulocytes, while Ly-6C is expressed on various mononuclear cells including polymorphonuclear leukocytes and monocytes (38, 39). In the present study, we used an Ab concentration of 10 µg/ml that ensured selective depletion of CD11b+Ly-6G+CCR2– cells but not of CD11b+Ly-6ChighCCR2+ or CD11b+Ly-6C–CCR2– monocytes (11). Under these conditions we were able to dissect the functional properties of Gr-1+ vs Gr-1– cells during bacterial meningitis.

How might granulocytes execute their antibacterial program? In addition to the production of proteolytic enzymes and reactive oxygen intermediates, interleukins are critical for the performance of granulocytes. A key role for IL-15 for the clearance of bacteria was shown recently (24). IL-15 was produced by Gr-1+ granulocytes. It was required for efficient clearance of experimental murine H. influenzae pneumonia, as at 4 days, p.i. lungs from IL-15 knockout mice contained 100-fold more bacteria than did wt mouse lungs. IL-15 may enhance granulocyte activity via the autocrine system and it activates NK cells by binding to IL-15R{alpha} and/or IL-2R on the surface of NK cells and granulocytes (24). IL-15 further plays a pivotal role in the activation and survival of NK cells by close interaction with granulocytes. Secretion of IL-15 by CD11b+Ly-6G+CCR2– cells could therefore result in further activation of NK cells. NK cells also produce IFN-{gamma} and other cytokines such as TNF-{alpha} that are involved in killing of intra- and extracellular pathogens. Accordingly, CD11b+Ly-6G+CCR2– granulocyte-depleted C57BL/6 mice were in our studies more susceptible to pneumococcal meningitis, demonstrating that this myeloid cell population is important for the control of extracellular bacteria as well and that IL-15 may play a pivotal role in this scenario.

In addition to their beneficial effect in bacterial neutralization, granulocytes have the potential to induce tissue damage through the release of soluble factors. Indeed, previous studies have demonstrated that limiting granulocyte influx to the CNS provides some benefit in situations where inflammation accompanies CNS disease (40, 41).

In conclusion, this study clearly demonstrates that neutrophilic granulocytes are able to inhibit the growth of S. pneumoniae in meningitis, thereby supporting antibiotic therapy administered under clinical conditions. Unlike macrophages resident within the CNS, circulating monocytes do not appear to contribute to the containment of infection in meningitis caused by predominantly extracellular encapsulated bacteria. Postinfection strategies aiming at enhancing the phagocytic activity of granulocytes in a specific timeframe might offer some protection in meningitis.


   Acknowledgments
 
We thank Olga Kowatsch for excellent technical assistance.


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


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Fritz-Thyssen-Stiftung, the Gemeinnützige Hertie-Stiftung (to M.P.). The Deutsche Forschungsgemeinschaft (DFG) (to M.D. and R.N.). A.M. is a fellow of the Gertrud Reemtsma foundation. Back

2 A.M. and M.D. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Marco Prinz, Department of Neuropathology, University of Freiburg, Breisacher Strasse 64, D-79106 Freiburg, Germany. E-mail address: marco.prinz{at}uniklinik-freiburg.de Back

4 Abbreviations used in this paper: CSF, cerebrospinal fluid; p.i., postinfection; wt, wild type. Back

Received for publication February 14, 2008. Accepted for publication June 15, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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A. Mildner, M. Mack, H. Schmidt, W. Bruck, M. Djukic, M. D. Zabel, A. Hille, J. Priller, and M. Prinz
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