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(TNF-) production were higher in macrophages from resistant C57Bl/6 mice than in macrophages from susceptible C3H/HeJ mice. However, TNF- production was observed in lower concentrations in C3H/HeJ (toll-like receptor-4–/–) macrophages than in C3H/FeJ (TLR4+/+) macrophages, suggesting that TLR4 might contribute to the response to B. burgdorferi. A higher cytokine response to B. burgdorferi was associated with cell death in macrophages from resistant C57Bl/6 mice. Understanding variability in the response of macrophages to B. burgdorferi may contribute to understanding Lyme arthritis.
Borrelia burgdorferi, a causative agent of Lyme disease, causes inflammatory arthritis and joint edema in susceptible mouse strains, particularly C3H.1 Susceptibility is dominant and associated with large numbers of spirochetes in tissues.2 Adaptive immunity controls spirochete burden, but even if this response is absent and there are large numbers of spirochetes in the joints, severe inflammatory joint infiltrates do not occur in C57BL/6 mice,3 suggesting a protective effect by the innate immune system. In C57BL/6 beige mice, which have gross defects in innate immunity, severe arthritis follows B. burgdorferi infection.1 Genetic control of acute Lyme arthritis severity in mice maps to a number of chromosomal regions, some of which encode regulators of innate immunity.4 Macrophage function has been proposed to be critical for mice to combat B. burgdorferi infection.1 Borrelia burgdorferi lipoproteins are mitogenic for murine monocytes5 through stimulation of toll-like receptors (TLR) 1 and 2.6 In addition, non–lipopeptide-derived signals induce tumor necrosis factor- (TNF-), chemokines, and interleukin-6 (IL-6) through the Fc
receptor.7 Levels of TNF- are elevated in sera and synovial fluids of Lyme borreliosis patients,8 and lipoproteins induce TNF- production from elicited peritoneal macrophages.5 Interleukin-12, a cytokine required for the induction of cellular immunity in response to infection,9 has been reported in response to B. burgdorferi in human patients10 and in murine macrophages ex vivo.11 Although it was previously shown that peritoneal macrophages engulf B. burgdorferi and degrade the spirochetes in intracellular compartments,12 and produce TNF- in response to lipoprotein stimulation,5 macrophage cytokine production after ex vivo infection with B. burgdorferi has not yet been associated with arthritis susceptibility. We tested the hypothesis that the pro-inflammatory macrophage response to B. burgdorferi is mouse strain-dependent.
Female, six-week-old C57BL/6J, 129S7/J, C3H/HeJ, and C3H/FeJ mice were obtained from the Jackson Laboratories (Bar Harbor, ME) and housed in the Tufts-New England Medical Center Department of Laboratory Animal Medicine (Boston, MA). The maintenance and care of experimental animals complied with the National Institutes of Health guidelines for the humane use of laboratory animals, and all procedures were conducted under an institutional animal care and use committee–approved protocol.
A murine fibroblast line, ATCC CCL 1 (L cells), was cultured in RPMI 1640 medium plus 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 units/mL of penicillin G, and 100 µg/mL of streptomycin sulfate (L/M
medium; Gibco/Invitrogen, Gaithersburg, MD) at 37°C in an atmosphere of 5% CO2. L cell–conditioned medium (a source of macrophage colony stimulating factor) was harvested one day after the cells reached > 90% confluence and filtered through a 0.22-µm filter prior to use.
Primary cultures of bone marrow–derived macrophages were prepared as previously described.11 Marrow from four femurs (two mice) per strain was pooled for each experiment. The cell pellet was resuspended in RPMI 1640 medium supplemented with glutamine, penicillin, and streptomycin as above, 10% FBS, and 30% L cell–conditioned medium (BMM medium). The cells were plated with 4 x 106 cells/ 100-mm untreated plastic dish in 10 mL of BMM medium and incubated at 37°C in an atmosphere of 5% CO2. Plates were fed on day 3 by adding 10 mL of fresh BMM medium and replated on day 5 into L/M medium with 5 x 105 cells/ well (six-well tissue culture plates) or 1.25 x 105 cells/well (24-well plate containing 12-mm round glass coverslips). Plates were incubated at 37°C in an atmosphere of 5% CO2 for two days before infection on day 7.
Borrelia burgdorferi strain N40 clone D10/E913 was grown in modified Kelley medium (MKP) supplemented with human serum in place of rabbit serum, aliquoted, and frozen at passage 11 at –80°C until use as previously described.14 Borrelia burgdorferi grown in MKP with human serum are routinely used for infection of mammalian cells in culture because they bind to integrins more efficiently than when grown in Barbour-Stoenner-Kelly medium. Thawed spirochetes were washed twice at ambient temperature in phosphate-buffered saline (PBS) with 0.2% bovine serum albumin (BSA) and resuspended at a concentration of 1.67 x 107 spirochetes/mL (to achieve a multiplicity of infection [MOI] of 100 bacteria per macrophage) in L/m medium without antibiotics. Ten-fold serial dilutions of this culture into L/m medium achieved MOIs of 10 and 1. The L/m medium without antibiotics served as the control (MOI = 0).
Medium was removed from plated macrophages, and the cells were washed once with PBS to remove antibiotics. Spirochetes were added (3 mL/well of each 6-well plate, 0.75 mL/well of each 24-well plate), and plates were centrifuged (540 x g) for 15 minutes at room temperature to maximize bacteria-macrophage contact. The plates were incubated at 37°C in an atmosphere of 5% CO2.
Monensin (Sigma, St. Louis, MO) was added to six-well plates to a final concentration of 3 µM after incubation as described above, and plates were incubated for one hour at 37°C in an atmosphere of 5% CO2. Cells were harvested by washing plates once with PBS, incubating for 5 minutes at 37°C with 0.5 mL of trypsin/EDTA (Gibco/Invitrogen), scraping from the plates, and pooling contents from duplicate wells. Cells (105/well) were aliquoted into a 96-well V-bottom plate for staining using the intracytoplasmic cytokine kit (BD Biosciences Pharmingen, San Diego, CA). Briefly, cells were fixed in Cytofix/Cytoperm (BD Biosciences Pharmingen) for 15 minutes at 4°C, washed once with Perm/Wash (BD Biosciences Pharmingen), and incubated with antibodies to cytokines (1:100 anti–IL-12 phycoerythrin [PE] or anti–TNF- PE in Perm/Wash; BD Biosciences Pharmingen) for 15 minutes at 4°C. Cells were washed twice with Perm/Wash and resuspended in PBS with 2% FBS and 0.1% sodium azide for analysis on a FACSCalibur cytometer (BD Biosciences Pharmingen). Forward and side scatter gating excluded dead cells and debris. The C57Bl/6 and C3H/He mice were compared in four experiments. Results are presented as the mean ± SD for each MOI. Groups were compared by two-way analysis of variance (ANOVA) (Sigma Stat; SPSS, Inc., Chicago, IL). The 129/J and C3H/Fe macrophages were included for comparison in one experiment. Dose-dependent production of IL-12 was secreted from the C57BL/6J and 129-derived macrophages, but at a significantly lower level from the C3H mouse strain macrophages (P = 0.003) (Figure 1A
).
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was induced by B. burgdorferi infection at an MOI of 100 (P = 0.006). Of interest, TNF- was present at a lower level in C3H/HeJ (TLR4–/–) macrophages than C3H/FeJ (TLR4+/+) macrophages (Figure 1B
). Neither TNF- production nor IL-12 production was attributable to endotoxin contamination because the Limulus amebocyte lysate assay (Cambrex Bio Science Inc., Walkersville, MD) showed a low level of endotoxin (10.8 EU/mL) in the media and buffers used, which is below the level of 150 EU/mL reported previously to stimulate macrophages.15 Since B. burgdorferi does not produce lipopolysaccharide, the classic TLR4 agonist, the differences in TNF- production by the C3H/HeJ versus C3H/FeJ cultures suggest a novel TLR4-dependent immediate early macrophage response to some other component of B. burgdorferi. Because a stronger pro-inflammatory cytokine response was paradoxically mounted by macrophages from mouse strains that do not develop significant arthritis, we tested the possibility of mouse strain-dependent susceptibility to B. burgdorferi-induced macrophage death. Infected macrophages were simultaneously incubated with a fluorescent dye that stains nuclei, as well as one that generates a green fluorescent signal only after cleavage by esterases within viable cells, to enumerate live cells as a function of MOI.
Twenty-four–well plates containing macrophage monolayers on coverslips were removed from the incubator after three hours hr and washed twice with PBS. CellTracker Green (10 µM 5-chloromethylfluorescein diacetate; Invitrogen, Carlsbad, CA) and 6-diamidino-2-phenylindole dihydrochloride (0.02 µg/mL; Invitrogen) in RPMI 1640 medium were added to the monolayer and incubation was continued for 30 minutes. Plates were then washed twice with PBS, fixed with 0.5 mL paraformaldehyde (3% in PBS; Sigma), washed twice with PBS, and stored overnight at 4°C in 1 mL of PBS with 1% BSA and 3% normal goat serum. Coverslips were rinsed with PBS and mounted onto slides using the ProLong anti-fade kit (Invitrogen) according to manufacturer’s instructions. Adherent cells were scored as live (faint blue nuclei and green cytoplasm) or dead (blue nuclei and unstained cytoplasm) for three fields per coverslip in two independent experiments. To facilitate comparisons between experiments, the number of live cells for each MOI (1, 10, and 100) was normalized to the number at MOI = 0 to obtain percent live cells. The values were compared between groups by two-way ANOVA and pairwise comparisons were made between groups by the Holm-Sidak method (Sigma Stat).
A significant decrease in the proportion of adherent live cells was observed in C57Bl/6 macrophages but not in C3H/ HeJ macrophages at an MOI 
1 (Figure 2). Many rounded-up cells were additionally noted prior to washing the cover-slips in these wells. Thus, B. burgdorferi infection induced cell detachment and death in macrophages from C57Bl/6 mice, but not from C3H/He mice. Cell death has previously been associated with inflammatory cytokine production in macrophages infected with Mycobacterium tuberculosis,16 as well as in T cells, and may be functionally linked.17
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A functional innate immune response is required for arthritis resistance in C57Bl/6 mice, and blocking the macrophage inhibitory cytokines IL-6 and IL-10 exacerbates arthritis in this strain. A beneficial inflammatory response may be self-limited by B. burgdorferi-induced apoptosis, production of anti-inflammatory cytokines, or both. Resistance to Lyme arthritis may therefore depend on an effective early cytokine release by macrophages and temporal limitation of the immune reaction by ensuing macrophage apoptosis. Understanding mouse strain-specific variability in the response of macrophages to B. burgdorferi may contribute to understanding the earliest steps in the development of Lyme arthritis.
Received May 3, 2006. Accepted for publication June 23, 2006.
Acknowledgments: We thank Dr. Linden Hu for helpful discussions, and Dr. Georg Weber for many insightful suggestions on the manuscript.
Financial support: Lisa J. Glickstein was supported by a grant from the Harold G. and Leila Y. Mathers Foundation. Jenifer L. Coburn was supported by a biomedical science grant from the Arthritis Foundation, National Institutes of Health grants AI-40938 and AI-051407, and by Public Health Service grant 1 P30DK39428 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases to the Center for Gastroenterology Research on Absorptive and Secretory Processes at Tufts-New England Medical Center.
* Address correspondence to Lisa J. Glickstein, Massachusetts General Hospital, 149 13th Street, Room 8301, Charlestown, MA, 02129. E-mail: lglickstein{at}partners.org 
Authors’ addresses: Lisa J. Glickstein, Massachusetts General Hospital, 149 13th Street, Room 8301, Charlestown, MA, 02129, Telephone: 617-726-1529, Fax: 617-726-1544, E-mail: lglickstein{at}partners.org. Jenifer L. Coburn, Tufts-New England Medical Center, 750 Washington Street, NEMC Box 41, Boston, MA, 02111, Telephone: 617-636-5952, Fax: 617-636-3216, E-mail: jcoburn{at}tufts-nemc.org.
IIb ß3 mediates binding of the Lyme disease agent Borrelia burgdorferi to human platelets. Proc Natl Acad Sci U S A 90: 7059–7063.This article has been cited by other articles:
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