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The Journal of Immunology, 1999, 162: 4762-4772.
Copyright © 1999 by The American Association of Immunologists

The Origin and Function of Soluble CD14 in Experimental Bacterial Meningitis1

Anje Cauwels2,*, Karl Frei{dagger}, Sebastiano Sansano*, Colleen Fearns{ddagger}, Richard Ulevitch{ddagger}, Werner Zimmerli* and Regine Landmann3,*

* Division of Infectious Diseases, Department of Research, University Hospitals, Basel, Switzerland; {dagger} Department of Neurosurgery, University Hospital, Zürich, Switzerland; and {ddagger} Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Murine experimental meningitis models induced by either Escherichia coli LPS, live Streptococcus pneumoniae, or Listeria monocytogenes were used to study the origin and potential function of soluble CD14 (sCD14) in the brain during bacterial meningitis. Whereas intracerebral infection caused only a minor and/or transient increase of sCD14 levels in the serum, dramatically elevated concentrations of sCD14 were detected in the cerebrospinal fluid. Reverse-transcriptase PCR and FACS analysis of the leukocytes invading the subarachnoid compartment revealed an active amplification of CD14 transcription and concomitant surface expression. These findings were confirmed by in situ hybridization and immunohistochemical analysis. In contrast, parenchymal astrocytes and microglial cells were shown not to significantly contribute to the elevated levels of sCD14. Simultaneous intracerebral inoculation of rsCD14 and S. pneumoniae resulted in a markedly increased local cytokine response. Taken together, these data provide the first evidence that sCD14 can act as an inflammatory co-ligand in vivo. Thus, during bacterial meningitis, sCD14 is massively released by intrathecal leukocytes, and the sCD14 found in the cerebrospinal fluid can play an important role in the pathogenesis of this disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite antimicrobial therapy, bacterial meningitis remains a severe infection with a high mortality (1, 2) and frequent neurologic sequelae (3, 4). The main pathogens causing acute bacterial meningitis are Escherichia coli and group B streptococci in neonatal meningitis, and Streptococcus pneumoniae and Neisseria meningitidis in children and adults. In contrast, meningitis caused by Haemophilus influenzae has almost completely disappeared with the introduction of an efficacious conjugate vaccine. Intracellular Listeria monocytogenes mainly cause meningitis among newborns, elderly people, and immunocompromised patients (5, 6, 7).

Several animal studies revealed intrathecally produced TNF-{alpha} and IL-1ß as the most potent mediators of cerebral inflammation and blood-brain barrier impairment (8, 9). One of the hallmarks of bacterial meningitis is the development of neutrophilic pleocytosis in the cerebrospinal fluid (CSF).4 Chemokines such as IL-8, growth-related gene product-{alpha}, monocyte-chemotactic protein-1, MIP-1{alpha}, MIP-1ß and MIP-2 (10, 11), and the complement component C5a have been identified as the chemotactic substances responsible for the extensive leukocyte recruitment into the subarachnoid space (12, 13, 14). Chemotaxis of circulating leukocytes into the central nervous system is absolutely required to elicit an efficient local host defense. However, it also actively contributes to the progressive blood-brain barrier breakdown and brain injury via the release of harmful mediators such as proteases, glutamate, reactive oxygen intermediates, and nitric oxide.

The membrane-bound form of CD14 (mCD14) is a GPI-anchored glycoprotein present on myeloid cells as an inflammatory receptor for LPS and Gram-positive or mycobacterial cell wall components (15, 16, 17). The expression of mCD14 on the cell surface can be modulated by various inflammatory stimuli (18, 19). In addition to mCD14, two soluble isoforms of CD14 (sCD14) exist in circulation. In vitro, sCD14 can act as a co-ligand of endotoxin for the activation of cells devoid of mCD14, such as endothelial and epithelial cells, astrocytes, or vascular smooth muscle cells (20, 21, 22, 23). The (patho)physiologic role of sCD14 in vivo, however, remains unclear. Besides its possible proinflammatory function as a co-ligand, sCD14 was shown to prevent cytokine release, and even to protect mice against LPS-induced shock, although this did require higher sCD14 concentrations than are ever encountered in vivo (24, 25). In normal human blood, sCD14 is present at a concentration of 2–3 µg/ml, but increased levels (up to 10 µg/ml) seem to be associated with a wide variety of human inflammatory diseases, whether of infectious (26, 27, 28), autoimmune (29, 30), or even traumatic origin (31, 32).

Since functional sCD14 is present in blood and extravascular fluids, we were interested in the presence of sCD14 in CSF and its possible role in the pathogenesis of meningitis. As described in this study, we observed a marked enrichment of sCD14 in the CSF of patients with bacterial, but not viral meningitis. We therefore decided to analyze the origin and function of sCD14 during experimental bacterial meningitis. To accomplish this, we established several different mouse meningitis models, induced by either LPS from E. coli, live S. pneumoniae, or L. monocytogenes. In this work, we show that the synthesis of CD14 transcripts, de novo surface expression of mCD14, and release of sCD14 into the CSF is a function of the infiltrating leukocytes. Moreover, coinjection of rsCD14 with live pneumococci revealed a proinflammatory rather than antiinflammatory function for the sCD14 released in the brain during the progression of bacterial meningitis. These data provide new insights into the pathogenesis of bacterial meningitis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

The study population consisted of 34 patients with bacterial meningitis and 32 patients with viral meningitis. CSF samples were collected via lumbar puncture from children with infectious meningitis on admission to the University Children’s Hospital Zürich, from October 1988 to May 1993. CSF samples were divided into aliquots for bacteriologic and viral analysis and cytokine determinations, as described (11, 33, 34). In addition, sCD14 levels in CSF of 10 control patients undergoing lumbar puncture for a noninflammatory disease were examined. The results were statistically analyzed with the Kruskal-Wallis test and Spearman’s rank correlation coefficient test.

Recombinant mouse sCD14 and anti-CD14 Abs

The production of the recombinant mouse CD14 and anti-CD14 Abs used in our laboratory is extensively described elsewhere (R. Woelky Bruggmann et al., manuscript in preparation). Briefly, mouse CD14 was truncated at amino acid 344 and linked with a histidine tag into the baculoviral expression vector pAcSG2. Sf9 insect cells were infected with the recombinant baculovirus using BaculoGold virus cotransfection (all components from PharMingen, San Diego, CA). Recombinant His-tagged protein was harvested from BTI-TN-5B1-4 (high five) insect cells (Invitrogen, San Diego, CA) infected with the recombinant virus and purified by Ni-nitrilotriacetic acid agarose (Qiagen, Basel, Switzerland) chromatography using elution with a continuous gradient of imidazole (0–250 mM). The insect cells transfected with a recombinant baculovirus DNA carrying the cDNA encoding the His-tagged rsCD14 protein produced a soluble 48-kDa protein that reacted with both monoclonal and polyclonal anti-CD14 Abs and bound LPS in a cell-free system.

The G5A10 and F5D72 mAbs directed against mouse CD14 were generated in armenian hamsters immunized with the mouse rsCD14. Spleen leukocytes were isolated from hamsters with a positive blood titer for anti-CD14 Abs and fused with myeloma cells to yield anti-CD14-producing hybridoma cells. The mAbs were purified using a protein G column (Pharmacia, Dübendorf, Switzerland). The polyclonal 1617 anti-CD14 Ab, used in a biotinylated form for detection in the mouse sCD14 ELISA, was obtained after immunization of a New Zealand White rabbit with murine rsCD14 and purification of the resulting antiserum via protein G chromatography.

Human and mouse sCD14 ELISA

Concentrations of sCD14 in CSF or serum were determined by ELISA systems developed in our laboratory. For human sCD14, microtiter plates were coated with the anti-CD14 Ab 63D3 and detection occurred via peroxidase-labeled 3C10, as previously described (19). For mouse sCD14, plates were coated overnight at 4°C with 2 µg/ml G5A10 anti-CD14 mAb in 0.1 M bicarbonate buffer, pH 9.3. After washing, nonspecific binding was blocked for 2 h at 37°C with 0.02 M sodium phosphate buffer (pH 7.5) containing 2% BSA and 0.01% Tween. Samples or standard (7.8–500 ng/ml His-tagged rsCD14) were diluted in the blocking buffer and incubated for 2 h at 37°C. After extensive washing, 500 ng/ml biotinylated polyclonal rabbit anti-mouse CD14 (1617) was added and incubated for 1 h at 37°C, followed by additional washing and 1-h incubation with streptavidin-horseradish peroxidase (Zymed Laboratories, South San Francisco, CA; 1/5000 dilution) at 37°C. A final washing step preceded detection with the tetramethylbenzidine chromogen, as described for human sCD14.

Experimental meningitis models

Six- to seven-week-old female 129/Sv agouti mice and seven- to nine-week-old female C57BL/6 mice (used for the L. monocytogenes model only) were obtained from BRL (Füllinsdorf, Switzerland). The mice were anesthetized via i.p. injection of 100 mg/kg Ketamine (Ketalar; Warner-Lambert, Baar, Switzerland) and 20 mg/kg Xylazinum (Xylapan; Graeub, Bern, Switzerland), and subsequently intracerebrally (i.c.) inoculated with 25 µl containing either 0.9% NaCl, 5 µg LPS (E. coli serotype 0111:B4; Sigma, St. Louis, MO), various doses of live S. pneumoniae (clinical isolate of serotype 3), or L. monocytogenes (strain EGD, provided by Dr. R. M. Zinkernagel, University Hospital, Zürich, Switzerland). In case of coinjection with rsCD14, 5 µl of a 5x concentrated bacterial suspension was mixed with either 20 µl NaCl or purified rsCD14 at a concentration of 375, 90, or 22.5 µg/ml. The rsCD14 was tested for the presence of LPS by a chromogenic Limulus assay (Chromogenix, Mölndal, Sweden) and revealed concentrations lower than 10 pg/ml endotoxin. Intracerebral injection of 25 µl of the bacteria-rsCD14 mixture thus delivered 7.5, 1.8, or 0.45 µg rsCD14 into the brain. The infectious dose of bacteria was retrospectively assessed by plating each inoculum. At indicated time points, the animals were euthanized by i.p. injection of 100 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL), blood was taken by cardiac puncture, perfusion was performed with Ringer’s solution (Braun Medical, Emmenbrücke, Switzerland), and CSF was collected by puncture of the cisterna magna, as described by Carp et al. (35). Blood was allowed to clot at 37°C and subsequently centrifuged at 20,000 x g for 10 min to obtain serum. Sera were examined individually, whereas CSF samples of three to six animals were pooled. Pooled CSF samples were serially diluted to assess the bacterial load via plating. After centrifugation, CSF supernatants were stored at -20°C to await further analysis, while the blood cells in the pellets were counted, identified via cytospin, checked for mCD14 surface expression via FACS analysis, and lysed in guanidinium-isothiocyanate-acetate buffer for later RNA isolation. After CSF collection, brains were quickly removed and fixed overnight immersed in 4% (w/v) paraformaldehyde in PBS (pH 7.4). The fixed brains were embedded in paraffin blocks and sectioned with a microtome at 2–3 µm thickness for immunohistochemistry and in situ hybridization.

Reverse-transcriptase PCR

CSF leukocytes were pelleted, washed, and lysed with guanidinium-isothiocyanate-acetate buffer. Polyadenylated mRNA was isolated using the Quickprep Micro mRNA Purification Kit from Amersham Pharmacia Biotech (Dubendorf, Switzerland), according to the manufacturer’s instructions. The recovered mRNA was reverse transcribed to cDNA using Superscript reverse transcriptase (Life Technologies, BRL) with oligo(dT)12–18 as a primer. This cDNA was analyzed via a new PCR method to quantitate DNA using real-time detection and the 5' nuclease assay, as developed by Perkin-Elmer Applied Biosystems (Foster City, CA). Briefly, a nonextendable oligonucleotide hybridization probe is labeled with a reporter fluorescent dye at the 5' end and a quencher-fluorescent dye at the 3' end. During the extension phase of the PCR cycle, the 5' nuclease activity of the Taq DNA polymerase cleaves the hybridization probe and thus releases the reporter dye from the probe, separating reporter dye from quencher dye. The resulting relative increase in reporter dye emission is proportional to the amount of PCR product accumulated and monitored in real time during PCR amplification using a sequence detector, the ABIPRISM 7700 Sequence detector (PE Applied Biosystems). A computer algorithm calculates the threshold cycle at which each PCR amplification reaches a fixed threshold, the CT value, representing a quantitative measurement of the copies of the target found in any sample (36, 37). CD14 and GAPDH CT values were thus used to calculate the CD14/GAPDH ratio using the formula for comparative CT method (provided by PE Applied Biosystems), 2CT,GAPDH/2CT,CD14, and plotted as such versus time (Figs. 3GoB and 5).



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  		FIGURE 3. sCD14 release (A), CD14 mRNA expression (B), and IL-6 production (C) by primary mouse microglia and astrocytes stimulated with LPS. A total of 25,000 cells were seeded in 100 µl medium and treated with (filled symbols) or without (open symbols) 1 µg/ml LPS. Supernatant was collected after 6, 24, and 48 h and checked for sCD14 (A, detection limit 40 ng/ml) and IL-6 (C, detection limit 150 pg/ml) via ELISA. Data shown are means ± SD from three (microglia) or two (astrocytes) individual experiments. Cells were subsequently lysed for mRNA isolation and quantitative RT-PCR analysis, as described in Materials and Methods. Bar charts in B represent CD14/GAPDH ratios from quadruple RT-PCR determinations.

 
Primer sequences used for mouse CD14 detection were 5'-CAGCCCTCTGTCCCCTCAA-3' forward and 5'-CATCCCCGCGTTACGC-3' reverse. The oligonucleotide hybridization probe, labeled with the fluorescent marker FAM (6-carboxyfluorescein) at the 5' end and the quencher fluor TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) at the 3' end, read 5'-TCCCGACCCTCCAAGTTTTAGCGC-3'. For mouse GAPDH detection, the primer pair consisted of 5'-GGCAAATTCAACGGCACAGT-3' forward and 5'-AGATGGTGATGGGCTTCCC-3' reverse, with internal fluorescent probe (with 5' FAM and 3' TAMRA attached) 5'-AAGGCCGAGAATGGGAAGCTTGTCATC-3'. PCR reagents were obtained from Perkin-Elmer; the primer and fluorogenic probe concentrations were optimized for each primer pair. Finally, the following conditions were used: Buffer composed of 10 mM Tris-HCl (pH 8.3) and 50 mM KCl, 5 mM MgCl2, dNTPs at 300 µM, forward and reverse primers at 1 µM and 450 nM, respectively, internal fluorescent probe at 320 nM, and Taq polymerase at 2.5 U/25 µl reaction volume. Cycle parameters were 95°C for 12 min, followed by 40 cycles of 95°C for 15 s, and 58°C for 1 min.

Flow cytometry

CSF cells were washed in PBS containing 4% FCS and 10 mM sodium azide, treated with normal rabbit serum to block Fc receptors, and then incubated for 30 min at 4°C with the monoclonal hamster anti-mouse G5A10 or anti-trinitrophenol Ab as a control. After washing, the cells were stained for 30 min at 4°C with a FITC-conjugated F(ab')2 fragment of goat anti-hamster IgG (Jackson ImmunoResearch, West Grove, PA). Fluorescence was analyzed in a FACScan with the Cellquest software.

Immunohistochemistry

Immunohistochemical staining was performed on paraformaldehyde-fixed and paraffin-embedded brain sections. Briefly, sections mounted onto Superfrost/Plus slides (Menzel-Gläser, Medite Nunninger, Switzerland) were deparaffinized at room temperature with xylol (4 x 5 min), and rehydrated by immersion in a graded series of ethanol baths. After washing (3 x 5 min with 0.01 M PBS), slides were boiled in the microwave for 10 min in 0.5 M Tris-HCl, pH 10.5, washed again, and treated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity. Another washing step preceded blocking with 1.5% normal goat serum (Vector Laboratories, Burlingame, CA) for 20 min at room temperature, followed by overnight incubation at 4°C with 0.2 µg/ml of the F5D72 hamster anti-CD14 mAb diluted in PBS containing 1% BSA. In control experiments, brain sections were treated with a hamster anti-trinitrophenol mAb (PharMingen) instead of primary Ab. To visualize parenchymal astrocytes and microglia, brain sections were stained with anti-glial fibrillary acidic protein (PharMingen) or F4/80 Abs (kindly provided by Dr. Siamon Gordon, University of Oxford, Oxford, U.K.), respectively. The slides were subsequently washed with 0.05 M Tris-HCl, pH 7.6, and incubated for 30 min at room temperature with biotinylated goat anti-hamster IgG (Vector Laboratories) diluted 1/200 in PBS containing 1.5% normal goat serum. Another washing step with 0.05 M Tris-HCl, pH 7.6, was followed by treatment with the VECTASTAIN Elite ABC reagent (Vector Laboratories) and development with 3-amino-9-ethyl carbazole chromogen (AEC). After rinsing in distilled water, slides were counterstained with hematoxylin for 15 s, rinsed well with tap water, and mounted.

In situ hybridization

Antisense and sense CD14 riboprobes were prepared and used for in situ hybridization of paraffin-embedded brain sections, as previously described in detail (38, 39).

Primary microglia and astrocyte cultures

Primary astrocyte and microglia cultures were prepared from brains of newborn mice, as described in detail before (40). First passage astrocytes and microglia were seeded at 25,000 cells/100 µl culture medium (DMEM containing 20% FCS) and stimulated with LPS (1 µg/ml). After 6, 24, and 48 h, the supernatant was collected for cytokine and sCD14 determination and cells were lysed in guanidinium-isothiocyanate-acetate buffer for later mRNA isolation, cDNA synthesis, and real-time quantitative RT-PCR for both CD14 and GAPDH transcripts.

Cytokine determinations

TNF was measured by the degree of cytotoxicity on WEHI cells in the presence of 1 µg/ml actinomycin D, using mouse rTNF as a standard. The sensitivity of the bioassay per se was less than 0.05 pg/ml, resulting in a detection limit for CSF samples (diluted 1/15) of 0.7 pg/ml. Reagents for the mouse IL-6 ELISA were purchased at PharMingen, and the ELISA was performed as suggested by the manufacturer. The sensitivity of the assay was approximately 15 pg/ml, and detection limit for diluted CSF samples was <=0.5 ng/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
sCD14 in the CSF of meningitis patients

We measured sCD14 in cerebrospinal fluid from children with different types of meningitis. As shown in Fig. 1Go, sCD14 levels in the CSF were significantly higher (p < 0.0001, Kruskal-Wallis test) in patients with bacterial meningitis (mean = 1825 ng/ml, 82% of the samples >400 ng/ml), as compared with those with viral meningitis (mean = 388 ng/ml, 12% >400 ng/ml) or controls (mean = 178 ng/ml, 100% <400 ng/ml). Bacterial meningitis was caused by H. influenzae type b (n = 19), N. meningitidis (n = 10), S. pneumoniae (n = 3), E. coli (n = 1), or Stomatococcus mucilagenosus (n = 1). A correlation between the absolute amount of sCD14 in CSF and clinical outcome was lacking because only 1 of 34 bacterial meningitis patients eventually died. He had a sCD14 concentration of 475 ng/ml. sCD14 levels and TNF or IL-6 concentrations in the CSF were analyzed by nonparametric Spearman’s rank correlation test. There were, however, no associations found between sCD14 and either TNF levels (p = 0.2) or IL-6 concentrations (p = 0.8) (data not shown).



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  		FIGURE 1. sCD14 levels in CSF of patients with bacterial (n = 34) or viral meningitis (n = 32) compared with CSF from control patients without inflammatory disease (n = 10). Human sCD14 concentrations were determined by specific ELISA, as described in Materials and Methods.

 
sCD14 in CSF and serum during experimental mouse meningitis

The specific increase of sCD14 in the CSF of patients with bacterial meningitis prompted us to investigate its origin in the brain. Specifically, we asked whether the elevated sCD14 concentrations in the CSF during bacterial meningitis arise from active local production in the brain or rather from passive leakage from the blood through a progressively disrupted blood-brain barrier. We injected either 5 µg E. coli LPS or various inocula of live S. pneumoniae bacteria or L. monocytogenes (10, 14, 34) i.c. These three experimental models enabled us to evaluate the prevalence and importance of sCD14 release during Gram-negative and Gram-positive bacterial meningitis.

sCD14 started to appear in the CSF 24 h after i.c. injection with each of the meningitis-causing agents; high dose (>=105 CFU) pneumococcal challenge resulted in substantial sCD14 concentrations detectable as early as 6 h after inoculation (Fig. 2Go). In the CSF, sCD14 levels peaked either at 48 h in case of the LPS challenge or 24–48 h after pneumococcal infection, and remained high over the whole observation period. Following L. monocytogenes inoculation, sCD14 levels in the CSF typically continued to increase during 72 h. Western blot analysis of CSF samples demonstrated the presence of both the 48- and the 55-kDa isoforms of sCD14 (not shown). The 48-kDa isoform was, however, clearly predominant, indicating that the sCD14 in CSF derived primarily from proteolytic cleavage of the up-regulated mCD14 molecules, but was also directly secreted into CSF after escaping the intracellular GPI-anchoring mechanism (41). Regardless of the initiating stimulus, sCD14 levels in the CSF were high and persisted associated with inflammation. Interestingly, simultaneous analysis of serum revealed much lower and only transient increases in sCD14 levels. This discrepancy between sCD14 concentrations in CSF and serum suggested the possibility of local production and intrathecal release of sCD14 during bacterial meningitis, rather than leakage from the blood through the impaired blood-brain barrier. Possible cellular sources for intrathecal sCD14 include resident brain microglia, astrocytes, or blood-derived leukocytes in the inflammatory exudate.



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  		FIGURE 2. Time-dependent analysis of sCD14 concentrations in serum and CSF from mice after i.c. injection with either NaCl, 5 µg E. coli LPS, live S. pneumoniae (inocula of 1.3 x 105, 2 x 104, or 1 x 103 CFU), or L. monocytogenes (6.3 x 102 CFU). Results are shown from one experiment that was representative for three independent experiments. Serum data are given as means ± SD from three to six mice; CSF sCD14 values were obtained using pooled CSF from the identical three to six mice.

 
CD14 production and release by primary mouse microglia and astrocytes

Astrocytes and microglia are resident cells of the central nervous system parenchyma. Microglial cells are of myeloid origin and therefore important regulators and effectors of the immune response within the brain. Whereas astrocytes lack a GPI-anchored mCD14 (15) and hence depend on the sCD14 form to respond to LPS and Gram-positive inflammatory stimuli (20, 42, 43), conflicting reports can be found on the expression of mCD14 by microglial cells. Ex vivo analysis demonstrated only a marginal mCD14 expression by mouse microglia (44) and no mCD14 detectable on human microglia (45, 46, 47). Other studies document the involvement of mCD14 in the phagocytosis of Mycobacterium tuberculosis and Cryptococcus neoformans by human and swine microglia, respectively (48, 49).

To examine the ability of microglia and astrocytes to produce and release sCD14, we measured their response to LPS in ex vivo primary cultures. As demonstrated in Fig. 3GoA, neither cell type produced any detectable sCD14 (<40 ng/ml), except for a minor release observed in microglial cultures 48 h after stimulus in one of three independent experiments. Real-time quantitative RT-PCR analysis failed to reveal any active synthesis of CD14 mRNA by astrocytes, whereas microglia clearly transcribed the CD14 gene after LPS treatment (Fig. 3GoB). Apparently, the maintenance of primary microglia in tissue culture also resulted in spontaneous activation, as can be judged from the increased CD14 mRNA production after 48 h in control cells. These data are reminiscent of results obtained by others using human ex vivo microglial cultures (50). The lack of a CD14 response after LPS treatment of astrocytes does not reflect a general insensitivity to inflammatory stimuli. These cells demonstrated brisk IL-6 production after LPS challenge (Fig. 3GoC) as well as enhanced TNF release (data not shown). Interestingly, however, the primary mouse microglia did not produce any detectable IL-6 (<150 pg/ml) in our experiments, although TNF could be detected in the supernatant following LPS activation (data not shown).

Active CD14 production by infiltrating CSF leukocytes during experimental mouse meningitis

An important and crucial feature of bacterial meningitis is the striking pleocytosis, i.e., the exudation of both polymorphonuclear and mononuclear leukocytes from the circulation into the CSF. As shown in Fig. 4Go, a strong pleocytosis could be observed in the CSF of LPS-injected animals, peaking at 24 h and declining afterwards. Infection with pneumococci resulted in an even more pronounced pleocytosis, also peaking 24 h after inoculation in case of lower inocula. Injection of >105 CFU S. pneumoniae, however, caused the most dramatic pleocytosis observed as early as 6 h after inoculation. As already demonstrated before (34), pleocytosis in the L. monocytogenes model progressively increased over time. Regardless of the challenge, CSF leukocytes consisted predominantly of neutrophils. Especially in case of L. monocytogenes infection, monocytes also infiltrated the CSF in large numbers, but their proportion was less than half at any time point.



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  		FIGURE 4. Leukocyte counts in CSF from mice after i.c. injection with either NaCl, 5 µg E. coli LPS, live S. pneumoniae (inocula of 1.3 x 105, 2 x 104, or 1 x 103 CFU), or L. monocytogenes (6.3 x 102 CFU), as determined via trypan blue counting and cytospin. Data shown are from one experiment that was representative for at least three independent experiments. Monocyte numbers are shown as hatched bars, neutrophils (PMN) as solid, and lymphocytes as shaded areas.

 
Since we could exclude brain microglia and astrocytes as the predominant source of the sCD14 in CSF during bacterial meningitis (Fig. 3GoA), we investigated whether leukocytes present in the CSF demonstrated increased expression of CD14 mRNA and mCD14 on their surface. The appearance of sCD14 in CSF (Fig. 2Go) shortly after the increase of leukocytes (Fig. 4Go) suggested that infiltrating leukocytes might be a source of sCD14. To characterize and quantitate the de novo expression of CD14, a recently developed real-time quantitative RT-PCR method was employed. mRNA from CSF leukocytes was purified, reverse transcribed into cDNA, and analyzed via PCR using a primer pair combined with a fluorescent internal probe for both CD14 and GAPDH genes. The relative expression of CD14 and GAPDH genes in CSF leukocytes at various time points after infection is provided in Fig. 5Go. The contaminating blood cells present in the CSF of NaCl-injected animals did not significantly transcribe the CD14 gene (CD14/GAPDH ratio <=0.5), while in marked contrast, leukocytes in CSF of mice inoculated with any of the meningitis-causing agents were actively amplifying CD14 mRNA, with peak levels noted 24 h after the i.c. injection. The most potent inducer of de novo CD14 transcription was the pneumococcal infection. Interestingly, the CD14/GAPDH transcription ratio in the CSF leukocytes was similar regardless of the dose of inoculum.



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  		FIGURE 5. Active CD14 mRNA synthesis in leukocytes obtained from the CSF of mice after i.c. injection with either NaCl, 5 µg E. coli LPS, live S. pneumoniae (inocula of 1.3 x 105, 2 x 104, or 1 x 103 CFU), or L. monocytogenes (6.3 x 102 CFU). CD14/GAPDH RT-PCR CT values represented as bar charts were determined as described in Materials and Methods. Data shown are from one experiment that was representative for three independent experiments.

 
We also assessed mCD14 surface expression of the CSF leukocytes by FACS analysis, as shown in Fig. 6Go. Since neutrophils could be identified as the predominant mCD14-positive cell population, the analysis was performed with scatter gates set on this population. One day after a low dose pneumococcal infection (4 x 102 CFU), 80–85% of the neutrophils were considerably positive for mCD14 (with a mean fluorescence intensity value of 10), and this surface expression was enhanced after an additional 24 h (95% positive cells with a mean fluorescence intensity of 24). At 72 h, the mCD14 expression of the infiltrating leukocytes was even more increased (about 85–90% positive cells with a mean fluorescence of 81).



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  		FIGURE 6. mCD14 expression of CSF leukocytes in response to low dose (4 x 102 CFU) i.c. injection with S. pneumoniae. Data shown are from a representative experiment. FACS analysis was performed on the neutrophil population gated on the basis of forward scatter/side scatter characteristics and CD14 expression. Fluorescence histograms are presented for the CSF leukocytes analyzed 24 (A), 48 (B), and 72 h (C) after infection. Controls represent cells stained with a monoclonal hamster anti-trinitrophenol (dotted line) instead of anti-CD14 Ab (solid line).

 
CD14 Ag and mRNA expression in the brain during experimental mouse meningitis

Ex vivo analysis of primary mouse microglial cells, astrocytes, and the infiltrating leukocytes revealed only the latter to be good candidates for the source of sCD14 observed during bacterial meningitis.

To confirm this hypothesis, brain sections of mock-infected and LPS-, S. pneumoniae-, or L. monocytogenes-inoculated mice were analyzed by immunohistochemistry at various time points to evaluate CD14 protein expression or evaluated using in situ hybridization with mouse CD14 antisense probes to visualize CD14 mRNA synthesis. The data were comparable for each of the three different infectious models; in this work, we show data from animals infected with pneumococci (Fig. 7Go). As early as 24 h after the i.c. injection, infiltrating granulocytes and monocytes were identified in the meninges, brain parenchyma, and ventricular space as CD14 positive by both immunohistochemical staining (shown for meninges in Fig. 7GoA and parenchyma in 7B) and in situ hybridization (Fig. 7Go, C and D). Parenchymal astrocytes or microglia, identified via immunohistochemical staining of adjacent brain sections (data not shown) with either glial fibrillary acidic protein (in brain tissue a specific marker for astrocytes) or F4/80 (a monocyte-macrophage marker) Abs, respectively, were clearly negative for CD14 protein expression. However, in situ hybridization revealed a positive CD14 mRNA signal for resident parenchymal cells (Fig. 7GoD), which may be brain microglia based on positive staining with F4/80 Abs (data not shown), and as approved by negative immunocytochemical staining (data not shown) and positive mRNA analysis via RT-PCR (Fig. 3GoB) of CD14 expression in ex vivo primary mouse microglia. Starting at 48 h after inoculation, capillary cells also expressed both CD14 protein and mRNA, especially those in close proximity to the inflamed meninges (data not shown). No infiltrating leukocytes were seen in the brains of NaCl-injected animals at any time point; staining with anti-CD14 Abs (Fig. 7Go, E and F) or hybridization with the CD14 antisense probe did not reveal any signal. Additional controls demonstrated the specificity of the findings since no signals were detected when LPS-, S. pneumoniae-, or L. monocytogenes-infected brain sections were stained with a nonspecific isotype control IgG Ab, when staining was performed with anti-CD14 Abs pretreated with excess rsCD14, or when hybridization was done with a sense instead of an antisense CD14 riboprobe (data not shown).



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  		FIGURE 7. Localization of the CD14 protein and mRNA expression in brain tissue from mice i.c. injected with live S. pneumoniae (ABCD) or NaCl (EF). A and B show immunohistochemical staining of sagittal brain sections from S. pneumoniae-infected animals with F5D72 anti-CD14 mAbs. Granulocytes and monocytes in the meninges (A) and brain parenchyma (B) are clearly positive. C and D show analogous brain sections analyzed by in situ hybridization with an antisense CD14 riboprobe. Positive are infiltrating cells in the inflamed meninges (C) and parenchyma (D). In the parenchyma, however, many positive cells could not be microscopically identified as infiltrated granulocytes (based on the polymorphonucleated morphology), and probably include resident microglia, as could be expected from our ex vivo primary microglial culture experiments. No meningeal or ventricular inflammation can be observed after mock infection; immunohistochemical staining for CD14 (E and F) is obviously negative.

 
Effect of rsCD14 coinjection on experimental pneumococcal meningitis

The role of sCD14 in mediating host responses to pathogens is not fully understood insofar as its function as a pro- or antiinflammatory protein. Therefore, we examined whether coadministration of rsCD14 with an intracerebral pneumococcal challenge modifies the responses to the microbial challenge. To evaluate the inflammatory reaction in the CSF following rsCD14 coinjection, we measured IL-6 and TNF concentrations in CSF at various time points after pneumococcal infection, since these cytokines have been shown to correlate with injury (51, 52, 53, 54, 55, 56). Four independent experiments were performed coinjecting a low dose of S. pneumoniae with 7.5 µg rsCD14, and data from one representative experiment are presented in Fig. 8Go. In addition, coinjection experiments using 1.8 or 0.45 µg rsCD14 were performed and showed analogous effects, even with the lowest rsCD14 dose, indicating the physiologic relevance of our findings (not shown). Coinjection of rsCD14 resulted in the enhanced release of IL-6 into the CSF (Fig. 8GoA). Depending on the bacterial inoculum size, the IL-6 levels 24 h after infection were 2 to 10 times higher in case of rsCD14 coinjection, reflecting an earlier release of IL-6 or an overall enhancement of IL-6 CSF levels, as shown in this work. Similarly, TNF levels in the CSF were substantially higher when exogenous rsCD14 was coinjected with S. pneumoniae (Fig. 8GoB). CSF collected from mice i.c. injected with NaCl or 7.5 µg rsCD14 alone did never contain any detectable IL-6 (<0.5 ng/ml) or TNF (<0.7 pg/ml; data not shown).



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  		FIGURE 8. Analysis of the CSF following coinjection of exogenous rsCD14 with a pneumococcal i.c. challenge. The four panels show CSF levels of IL-6 (A) and TNF (B), leukocyte counts (C), and bacterial load (D). Four independent experiments were performed; data from one representative experiment are shown. In this experiment, mice were infected i.c. with live S. pneumoniae, together with (filled symbols, 4 x 102 CFU) or without (open symbols, 4.7 x 102 CFU) 7.5 µg of rsCD14. IL-6 levels were determined via ELISA with a detection limit for CSF <=0.5 ng/ml. TNF concentrations were measured via bioassay using WEHI cells; the detection limit for diluted CSF was less than 0.7 pg/ml. Data were obtained using pooled CSF from three or four different mice. Control i.c. injections with NaCl or 7.5 µg rsCD14 alone did not cause any detectable IL-6 or TNF release or leukocyte migration into the CSF at any time point.

 
The enhanced IL-6 and TNF response following rsCD14 coinjection was not reflected in an altered leukocyte migration into the subarachnoid space (Fig. 8GoC), nor did it significantly modify the inflammatory responsiveness of these infiltrating blood leukocytes, as judged by FACS analysis of their surface mCD14 expression 24, 48, or 72 h after bacterial challenge (data not shown).

We also examined the bacterial load in the CSF following i.c. infection (Fig. 8GoD). Whereas the bacterial growth more or less reached a plateau in the absence of exogenous rsCD14, a steady increase (to >1 log difference) of bacteria in the CSF 72 h after infection could be observed when rsCD14 was coinjected. This suggests that the presence of excess rsCD14 in the brain somehow stimulates bacterial growth and/or enables the bacteria to escape clearance.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We observed a specific increase of sCD14 in the CSF of children with bacterial, but not viral meningitis, and hence decided to study the source and possible role of sCD14 in the brain during the progression of bacterial meningitis. On that account we established three different experimental mouse meningitis models via the intracerebral injection of either LPS or live Gram-positive bacteria. Regardless of the meningitis-causing stimulus, the sCD14 levels in the CSF were high and persisting, whereas in the circulation sCD14 concentrations were much lower and/or transient. This divergence suggested the local sCD14 production in the brain. Three major cell types were likely candidates for CD14 synthesis in the brain: residential microglia, astrocytes, and infiltrating blood leukocytes.

Ex vivo analysis of primary mouse astrocytes and microglia demonstrated their lack of an appreciable CD14 response upon inflammatory stimulation. Immunohistochemical and in situ hybridization experiments supported these data. The appearance of sCD14 in the CSF shortly after the onset of pleocytosis prompted us to inspect the infiltrating leukocytes for the synthesis of CD14 mRNA and mCD14 surface expression. Via RT-PCR and FACS analysis, we could prove that the neutrophils present in the CSF of mice inoculated with any of the meningitis-inducing agents actively amplified their CD14 transcription and mCD14 surface expression. Again, in situ hybridization and immunohistochemical studies of mouse brain sections could confirm these results.

The (patho)physiologic function of sCD14 in vivo generally remains an enigma. On the one hand, physiologic concentrations of sCD14 were shown in vitro to enable LPS and various Gram-positive components to bind and stimulate mCD14-negative cells (20, 21, 22, 23). On the other hand, supraphysiologic concentrations of sCD14 could attenuate LPS-induced TNF release both in vitro and in vivo, and could even provide protection in a murine model of endotoxic shock (24, 25). To examine a possible pro- or antiinflammatory role for sCD14 in the progression of bacterial meningitis, we coinjected live pneumococci with rsCD14 and evaluated the subsequent inflammatory reaction and bacterial expansion in the CSF. Although leukocyte infiltration clearly remained unaffected by rsCD14, both IL-6 and TNF levels in the CSF were considerably higher when exogenous rsCD14 was simultaneously administered with the bacteria. Cells lacking the mCD14 surface receptor, such as astrocytes and endothelial cells, were shown to be strictly dependent on sCD14 to respond to various bacterial inflammatory stimuli in vitro (20, 21, 22, 42, 43). Hence, it is reasonable to conclude that the amplified cytokine release following rsCD14 coinjection primarily resulted from sCD14-dependent stimulation of brain cells by the bacteria. These data provide the first evidence that sCD14 functions as a co-ligand for bacterial components to induce an inflammatory reaction in vivo, in the setting of active disease.

Another interesting observation following intracerebral coinjection of rsCD14 with S. pneumoniae was the progressive growth of the pneumococci in the CSF at a time point when bacterial growth after injection of S. pneumoniae alone reached a plateau. This observation is in agreement with the reduced bacterial load in blood and lungs of E. coli-challenged CD14-deficient mice (57), and suggests the existence in the brain of some rsCD14-induced mechanism(s) to escape bacterial clearance and/or stimulate bacterial expansion. Surface-exposed mCD14 has been reported to play a role in the phagocytosis of various pathogens (48, 49, 58, 59, 60) and apoptotic cells (61). In addition, both Gram-negative, Gram-positive, and mycobacterial components were shown to physically bind to sCD14 (17, 62, 63). It is therefore conceivable that the binding of sCD14 to bacteria competitively hinders phagocytosis via mCD14 surface receptors. On the other hand, complexing of sCD14 to the bacterial surface could possibly compete with complement- or IgG-mediated opsonization, and thus compromise phagocytosis and clearance via CR3 or Fc receptors, respectively. However, since opsonic activity is absent or barely detectable in both normal and infected CSF (64, 65), the relevance of this latter hypothesis can be questioned. sCD14 could also participate in the reduced bacterial load by modulating the bacterial growth either directly or indirectly. Cytokines such as TNF, IL-1, IL-2, granulocyte-macrophage CSF, and IL-6 have been shown to promote the growth of various bacterial pathogens (66, 67, 68, 69). Therefore, we tested rsCD14, several cytokines, and chemokines present in CSF during bacterial meningitis for their growth-modulating properties on pneumococci in vitro, and found a clear dose-dependent growth-stimulating effect of IL-6 on the bacteria (not shown). The enhanced intrathecal cytokine production resulting from rsCD14 coinjection might therefore indirectly be the cause or contribute to the related expansive growth of the pneumococci in the CSF.

Further studies are required to determine the exact (direct or indirect) rsCD14-induced mechanism(s) that enables the striking bacterial expanse in the CSF. However, it seems obvious that sCD14 released in the brain during the course of bacterial meningitis, by its ability to augment the intrathecal cytokine release and bacterial growth, acts as a pro- rather than antiinflammatory mediator.

In conclusion, sCD14 released by intrathecal leukocytes during bacterial meningitis is clearly capable of amplifying the local IL-6 and TNF release and can influence subarachnoid bacterial growth and/or clearance. These data provide the first evidence that sCD14 acts as a co-ligand for bacterial components to evoke an inflammatory response in vivo. In contrast to a murine model of systemic endotoxic shock, in which an excess of exogenous rsCD14 could moderate the secretion of TNF in circulation and protect against the LPS-induced lethality (25), sCD14, released in the brain during the course of bacterial meningitis, may exacerbate the disease.


   Acknowledgments
 
We thank Dr. David Nadal (University Children’s Hospital, Zürich, Switzerland) for providing cerebrospinal fluid samples from patients with infectious meningitis and controls. Dr. Peter Nelböck (Hoffmann-La Roche, Basel, Switzerland) is greatly acknowledged for the use of the ABIPRISM 7700 Sequence detector to perform real-time quantitative RT-PCR.


   Footnotes
 
1 This work was supported by Swiss National Science Foundation Grants Nrs 31-5243.97 and 4038-04398612. Back

2 Current address: University of Gent, Plandeis Interunisity Institute for Biotechnology; K. L. Ledeganckstraat 35, B-9000 Gent, Belgium. Back

3 Address correspondence and reprint requests to Dr. Regine Landmann, Division of Infectious Diseases, Department of Research, University Hospitals, Hebelstrasse 20, CH-4031 Basel, Switzerland. E-mail address: Back

4 Abbreviations used in this paper: CSF, cerebrospinal fluid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; i.c., intracerebral; m, membrane; MIP, macrophage-inflammatory protein; s, soluble. Back

Received for publication October 22, 1998. Accepted for publication January 13, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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