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* Department of Medicine, Oklahoma University Health Sciences Center, and Veterans Affairs Medical Center, Oklahoma City, OK 73014;
Department of Molecular Cell Biology, Free University Medical Center, Amsterdam, The Netherlands;
Institute of Experimental Dermatology and Department of Dermatology, University of Münster, Münster, Germany;
Department of Dermatology and Allergy, University of Ulm, Ulm, Germany; and
¶ Department of Immunology, Erasmus MC, Rotterdam, The Netherlands
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Abstract |
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actA mutants of L. monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6Chigh subset. In contrast, this was not evident after infection with hly mutants, which neither escape phagosomes nor replicate intracellularly. Importantly, when CD11b+ leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6Chigh monocytes. Kinetic analysis showed a significant influx of Ly-6Chigh monocytes into the brain 2 days after systemic infection. This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression. These data indicate that the Ly-6Chigh monocyte subset transports L. monocytogenes into the brain and establish their role as Trojan horses in vivo. |
Introduction |
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L. monocytogenes is a facultative intracellular bacterium that invades humans via the gastrointestinal tract and causes bacteremia as well as a variety of CNS infections (8). In the mouse model of systemic listeriosis, bacteremia typically precedes CNS infection and is composed of both cell-free bacteria and infected leukocytes (9, 10). Because intracellular and extracellular L. monocytogenes are present together in the circulation, it has been unclear whether extracellular bacteria invade the CNS directly or whether parasitized leukocytes transport them into the CNS. Data supporting the latter mechanism come from histological studies of experimentally infected mice that identified infected phagocytes in the choroid plexus (11). In addition, studies from our laboratory showed that killing extracellular L. monocytogenes in blood with gentamicin did not prevent bacterial infection of the brain (12). This finding suggests that migration of parasitized leukocytes from the bloodstream into the CNS is instrumental in neuroinvasion. Taken together, these data support a central role for infected phagocytes in systemic dissemination and neuroinvasion by L. monocytogenes.
Most L. monocytogenes-infected leukocytes in the blood have been identified morphologically as mononuclear cells (10), but the exact phenotype of the leukocytes that transport these bacteria through the bloodstream and into the brain is not yet known. Moreover, precise identification of parasitized mononuclear phagocytes in the blood during experimental infection of mice is complicated by the fact that no single marker exclusively recognizes mouse monocytes and distinguishes them from granulocytes (13, 14, 15, 16, 17, 18). Recent studies from our group and others indicate that mouse monocytes are a heterogeneous population composed of different subsets that function differentially in steady state and inflammation (18, 19, 20). These data offer an exciting new paradigm for the study of mouse blood monocytes and their roles during infection withintracellular pathogens. To capitalize on this, the experiments reported in this study analyzed mouse monocytes and their subsets during systemic infection with L. monocytogenes. Our results show that a subset of monocytes distinguished by high level expression of Ly-6C (Ly-6Chigh) harbors the majority of L. monocytogenes in the bloodstream. Importantly, we also demonstrate that systemic infection stimulates an influx of these cells, some of which contain bacteria, into the brain coincident with bacterial invasion. The monocyte influx coincides with up-regulation of macrophage chemoattractant protein-1 (MCP-1;4 CCL2) gene expression.
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Materials and Methods |
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CD3-PE, CD11b (M1/70)-PE-Cy5 and -Cy5.5, CD19-PE, CD62L-PE (MEL-14), GR-1-PE (RB6-8C5), Ly-6G-PE (1A8), NK1.1-biotin, and isotype control mAb were purchased from BD PharMingen (San Diego, CA) as direct conjugates. Rat anti-mouse Ly-6C (ER-MP20) was used as hybridoma culture supernatant and as direct FITC conjugate (21).
Bacteria
Bacteria were stored in brain heart infusion broth (Difco, Detroit, MI) at 109 CFU/ml at -70°C. Wild-type L. monocytogenes strains included EGD and 10403s. Gene deletion mutants of L. monocytogenes strain 10403s were obtained from D. Portnoy (University of California, Berkeley, CA) and included the listeriolysin O-deficient (hly) DP-L2161 and actA-deficient (actA) DP-L1942 (22, 23). L. monocytogenes strain NF-L512 containing a chromosomal actA-gfpuv-plcB transcriptional fusion was obtained from N. Freitag (Seattle Biomedical Research Institute, Seattle, WA) (12). For experiments, 0.5 ml of stock culture was diluted in 4 ml of broth and then cultured for 4.5 h at 37°C. Bacteria were diluted in sterile PBS before injection into mice.
Mouse infection
Female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME), 8–16 wk old, were used in all experiments. They were infected by i.p. or i.v. injection of 1–2 LD50 of wild-type L. monocytogenes or 106–107 CFU of other bacteria and then were euthanized at the indicated time with ketamine/xylazine (Vedco, St. Joseph, MO). Blood was collected into PBS containing 10 mM EDTA, and blood leukocytes were isolated as previously described (10, 12). In experiments that required harvesting of bacteria, leukocytes, or RNA from the brain, the animals were perfused with 30 ml of PBS via the left ventricle to remove blood from the brain. Leukocytes were isolated from whole brains by enzymatic digestion with 0.1% collagenase D (Roche, Indianapolis, IN) and 10 µg/ml DNase I (Sigma-Aldrich, St. Louis, MO), followed by immunomagnetic collection of CD45+ or CD11b+ cells on a miniMACS column (Miltenyi Biotech, Auburn, CA) (24). In other experiments the brain was divided lengthwise along the main sagittal fissure, with half the specimen used for quantifying CFU of bacteria in the brain and the other half being processed for real time-PCR (described below).
In some experiments mice were infected with L. monocytogenes strain NF-L512. Eighteen hours later they underwent surgical implantation of Alzet osmotic pumps (Durect, Cupertino, CA) filled with gentamicin (Sigma-Aldrich; 100 mg/ml in PBS) as previously described (12) and were injected i.v. with 0.2 ml of clodronate liposome into the lateral tail vein to eliminate monocytes in vivo (20). Dichloromethylene-bisphosphonate (clodronate) was a gift from Roche (Mannheim, Germany) and was incorporated into liposomes as previously described (25). Repopulating monocytes were labeled 24 h after depletion by i.v. injection of 0.2 ml of PBS-containing liposomes labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) (Molecular Probes, Eugene, OR).
Flow cytometry and cell sorting
Samples of 105 blood leukocytes, or the entire cell pellet from individual brains, were incubated in 96-well microtiter plates with 3% normal mouse serum and anti-CD16/32 mAb (BD PharMingen) for 30 min on ice before addition of isotype-matched control or test mAb. Cells were incubated with mAb for 30 min and then were washed three times with PBS/BSA/azide and postfixed with 1% paraformaldehyde. Flow cytometry was performed on a FACSCalibur (BD PharMingen), whereas cell sorting was performed on a MoStar (DakoCytomation Colorado, Ft. Collins, CO).
Microscopy
Leukocytes were cytocentrifuged onto coverslips, then fixed with 2% paraformaldehyde for 10 min at room temperature and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). Nonspecific Ab binding was blocked using preincubation with PBS plus 5% donkey serum and 2% mouse serum. Then the cells were immunolabeled with CD11b or Ly-6C mAb, followed by fluorochrome-conjugated F(ab')2 of donkey anti-rat secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) and nuclear counterstaining with 4',6-diamido-2-phenylindole hydrochloride (DAPI; Molecular Probes, Eugene, OR). Bacteria were labeled with L. monocytogenes antiserum (Difco), followed by fluorochrome-conjugated F(ab')2 of donkey anti-rabbit secondary Ab (Jackson ImmunoResearch Laboratories). Fluorescence microscopy under oil immersion (x1000) was used to quantify Ab-labeled cells and bacteria. Confocal microscopy was performed on a TNS NT microscope (Leica, Deerfield, IL) with four-laser stimulation and four-channel image collection.
Real-time PCR for MCP-1
Perfused brains from infected and uninfected control mice were flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction was performed with the BD Atlas Pure Total RNA Labeling System (Clontech, Palo Alto, CA). Total RNA was reverse transcribed in 10-µl reactions using TaqMan reverse transcription reagents (PE Applied Biosystems, Foster City, CA) in 96-well optical reaction plates with optical caps (PE Applied Biosystems). Conditions for the reaction consisted of hold steps of 10 min at 25°C and 30 min at 48°C, followed by denaturation at 95°C for 5 min in an ABI PRISM SDS 7700 thermocycler (PE Applied Biosystems). Reverse-transcribed cDNA and RT-negative controls were diluted to 1 ng/µl. Then real-time PCR reactions were run with SYBR Green PCR Master Mix (PE Applied Biosystems), custom-made primers from IDT Technologies (Coralville, IA) for MCP-1 (forward, 5'-CCCAAAGAAGCTGTAGTTTTTGTCA-3'; reverse, 5'-CAGCACAGACCTCTCTCTTGAGC3'), and the housekeeping gene, hypoxanthine phosphoribosyl transferase (forward, 5'-GTTGAAGATATAATTGACACTGGTAAAACA-3'; reverse, 5'-AGCTTGCAACCTTAACCATTTTG-3'), with a forward:reverse primer concentration ratio of 6:1. In other experiments commercial primers for mouse MCP-1 (BioSource, Camarillo, CA) were used to confirm the results. Real-time PCR reactions were run at 50-µl volumes in 96-well optical reaction plates using the ABI PRISM SDS 7700 system. Thermocycling conditions were as follows: hold at 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min, hold at 95°C for 15 s, hold at 60°C for 20 s, ramp to 95°C in 19 min 59 s (for a dissociation curve), hold at 95°C for 15 s.
Statistical analysis
Tests performed included one-way ANOVA with Tukey’s multiple comparison test and two-tailed Student’s t test with equal variance (PRISM, GraphPad, San Diego, CA). Both tests used a level of significance set at p < 0.05.
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Results |
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Because subpopulations of mouse monocytes have been identified recently (18, 20), further analysis of monocyte subsets was performed using recently established immunophenotypic criteria (20). Identification of monocytes on the basis of low orthogonal light scatter, as performed in steady state (20), was not possible, because this property was dramatically altered by L. monocytogenes infection (Fig. 1). Therefore, monocytes were distinguished from neutrophils on the basis of their differential expressions of GR-1 or the more neutrophil specific Ly-6G when plotted against the Ly-6C expression of gated CD11bhigh cells. These studies confirmed that monocytes from infected mice were CD11bhigh/Ly-6Gneg-low, expressed a variable amount of Ly-6C, did not express other lineage markers, including NK1.1, CD3, and CD19, and displayed typical monocytic morphology (20) (data not shown). Further analysis of Ly-6Chigh monocytes from infected mice showed that they were GR-1+ and CD62L+ (Fig. 1) and thus correspond to the CX3CR1low/GR-1+ monocytes identified by Geissmann et al. (18).
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actA and hly mutants of L. monocytogenes are avirulent in mice, infection with actA bacteria elicited a percentage of Ly-6Chigh monocytes similar to wild-type bacteria (Fig. 2). In contrast, infection with hly mutants did not do so on a consistent basis, and if present, the shift was always minimal.
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Discussion |
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The Ly-6Chigh subset is expanded by infection with L. monocytogenes or with the protozoan pathogen Leishmania major, resulting in a monocyte left shift toward a predominance of less mature cells that are recently released from the bone marrow (20). Interestingly, avirulent L. monocytogenes hly mutants, which do not produce listeriolysin O and typically neither escape phagosomes nor replicate intracellularly, did not stimulate this shift. By comparison, listeriolysin O-producing actA mutants, which do escape phagosomes and replicate intracellularly, but are avirulent because they lack F-actin-based motility, did elicit a subpopulation shift similar to that of wild-type bacteria. L. major is an obligate intracellular protozoan that resides in modified phagosomes and does not escape from them until the parasitized cell ruptures (28). However, L. major does produce a pore-forming cytolysin at 37°C that is maximally active at pH 5.0–5.5, similar to listeriolysin O (29, 30). Although its role in leishmaniasis is not clear, it is probably produced during intracellular growth within mammalian macrophages. Taken together, this suggests that the hemopoietic growth factor-driven monocyte left shift may be elicited in response to microbial proteins, perhaps hemolysins, that are expressed intracellularly during infection (31, 32).
The finding that Ly-6Chigh monocytes were the main transporters of intracellular bacteria in the bloodstream prompted us to test whether these cells also entered the brain. Indeed, we found that systemic L. monocytogenes infection induced a significant influx of Ly-6Chigh monocytes into the brain. Moreover, nearly 90% of infected CD11b+ leukocytes in the brain were Ly-6Chigh mononuclear phagocytes, consistent with the hypothesis that blood-borne L. monocytogenes enter the brain via phagocyte-facilitated invasion, with Ly-6Chigh monocytes acting as the Trojan horse (7). As Ly-6Chigh monocytes are CCR2+ and migrate to MCP-1 (18, 19), this selective recruitment into the brain is probably mediated at least in part by MCP-1, as we observed a significantly up-regulated expression of this chemokine in the brains of infected mice. MCP-1 has an established role in mediating recruitment of CCR2+ monocytes into the CNS (33, 34). Several different cell types, such as astrocytes, brain endothelial cells, and the newly recruited monocytes themselves, could have produced the MCP-1 mRNA detected in these experiments (35). Nevertheless, it remains to be determined the extent to which MCP-1 provides the initial stimulus for monocyte recruitment or just amplifies an ongoing monocytic influx, and whether other monocyte-attracting chemokines are also involved.
Data from the mouse model of systemic L. monocytogenes infection suggests that peripheral infection initiates a cascade of events causing recruitment of Ly-6Chigh monocytes, and the bacteria they contain, into the brain. Key components of this cascade probably include translocation of NF-
B in cerebral vessels (36) and up-regulation of adhesion molecules, including P-selectin, ICAM-1, and VCAM-1, on brain endothelial cells (7, 37). In vitro data suggest that once infected cells arrive in the CNS, bacteria from monocytes can invade a variety of cells, including neurons and endothelial cells, by cell-to-cell spread (10, 38, 39, 40). How these events relate to human infection is not completely clear. In this light, Hertzig et al. (41) recently reported that normal human serum contains IgG against the L. monocytogenes invasion protein InlB, thus inhibiting direct bacterial invasion of human brain microvascular endothelial cells. These data support a role for phagocyte-facilitated invasion of the CNS in humans as well.
A key finding of the present study is that a relevant leukocyte influx occurs before significant bacterial invasion. This is similar to recent data in systemic listeriosis in mice showing that Listeria-specific T cells also enter the brain in the absence of CNS infection (42). Our finding that monocytes enter the brain before neutrophils contrasts with the sequence reported by others. Lopez et al. (37) used immunohistochemistry to study leukocyte recruitment in the brains of s.c. infected mice and found that the first leukocytes to enter the CNS were neutrophils. These discrepant results may be attributable to differences between the infection models or mouse strains, as well as to the relative sensitivities of immunohistochemistry of brain sections vs flow cytometry of whole brain isolates for detecting and identifying small numbers of cells. In addition, recent data show that the mAb used to identify neutrophils by immunohistochemistry also reacts with GR-1+ monocytes (17). Thus, newly recruited cells identified as neutrophils could have been Ly-6Chigh monocytes. After intracranial inoculation of L. monocytogenes, neutrophils are the first leukocytes recruited into the brain (43, 44, 45). There are obvious differences between systemic and intracranial routes of infection, in particular, the fact that leukocyte recruitment is triggered by the presence of bacteria and bacterial products in the CNS after intracranial inoculation.
An interesting point in the context of this study is that we observed in preliminary experiments that the same Ly-6Chigh subpopulation of blood monocytes also can harbor the phylogenetically unrelated intracellular pathogen, L. major (our unpublished observation). L. major disseminates in susceptible mice to visceral organs, including liver, spleen, and bone marrow (46, 47). The fact that some, albeit few, Ly-6Chigh monocytes contain L. major is remarkable for several reasons: firstly, because L. major is thought to disseminate mostly through lymphatics; secondly, because this Ly-6Chigh monocyte subset is apparently permissive for different microbes; and thirdly, because this subset shows the strongest increase in size in the course of infection. Although the percentage of infected Ly-6Chigh blood monocytes was very low (0.15%), they may add a novel aspect to dissemination of L. major. Now that it has become possible to correlate more accurately mouse and human monocyte subsets (18, 20), our data indicate that the Ly-6Chigh monocyte subset in the mouse and its human counterpart, identified as CD14+CD16-, represent a pathway for microbes to disseminate within a mammalian host that is conserved among a wide variety of intracellular pathogens.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Douglas A. Drevets, Veterans Affairs Medical Center 111/c, 921 NE 13th Street, Oklahoma City, OK 73014. E-mail address: douglas-drevets{at}ouhsc.edu
3 C.S. and P.J.M.L. contributed equally to this work.
4 Abbreviations used in this paper: MCP-1, macrophage chemoattractant protein-1; clodronate, dichloromethylene-bisphosphonate; DAPI, 4',6-diamido-2-phenylindole hydrochloride; F-actin, filamentous actin; GFP, green fluorescence protein; DiD, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate.
Received for publication September 26, 2003. Accepted for publication January 21, 2004.
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References |
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B and upregulation of adhesion molecules and chemokines. Mol. Microbiol. 31:1709.[Medline]
and -2 are produced intrathecally and mediate chemotactic activity in cerebrospinal fluid of infected mice. J. Immunol. 155:4367.[Abstract]
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) and brain (
) were determined by serial dilution and plating. B, Histograms show the Ly-6C expression of CD11bhigh cells from representative control and infected animals. C, The CD11bhighLy-6Cmed-high cell population was selected, and numbers of monocytes and neutrophils were determined based on differential staining of GR-1. Data shown are the mean ± SEM Ly-6Cmed-high monocytes (
) and neutrophils (