International Immunology Advance Access originally published online on January 13, 2006
International Immunology 2006 18(3):409-414; doi:10.1093/intimm/dxh380
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The MHC class I-related FcRn ameliorates murine Lyme arthritis
1 Department of Pathology, Tufts University School of Medicine, Boston, MA 02111, USA
2 Department of Pathology, Tufts University School of Veterinary Medicine, Grafton, MA 01538, USA
3 Jackson Laboratory, Bar Harbor, ME 04609, USA
Correspondence to: B. T. Huber; E-mail: brigitte.huber{at}tufts.edu
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Abstract |
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The identification of the neonatal FcR (FcRn) as an IgG homeostasis regulator has led to research aimed at delineating a role for FcRn in humorally mediated disease. FcRn is a class I-related molecule that prolongs the half-life of serum IgG by preferentially binding IgG at low pH and inhibiting its degradation. Its role in protective immunity to infectious organisms is unknown. We investigated the function of FcRn in the murine model of Lyme arthritis, caused by infection with Borrelia burgdorferi. We infected FcRn–/– and wild-type mice with B. burgdorferi and monitored the development of arthritis. Infected FcRn–/– mice demonstrated decreased serum levels of anti-B. burgdorferi antibodies and borreliacidal activity. Moreover, these mutant mice developed increased ankle swelling and joint histopathology following infection. Our data suggest that FcRn ameliorates murine Lyme arthritis by preventing the degradation of protective borreliacidal antibodies.
Keywords: bacterial, Borrelia burgdorferi, inflammation, rodent
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Introduction |
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The neonatal FcR (FcRn) is structurally characterized as an MHC class I-related protein consisting of two subunits, a heavy chain similar to the
chain of MHC class I proteins and a ß2-microglobulin light chain (1). It binds IgG Fc under conditions of reduced pH and inhibits degradation (2). This pH-dependent interaction has been attributed to histidine residues located at the binding site between FcRn and IgG Fc (3). Originally identified as the FcR required for perinatal transport of maternal IgG (4), FcRn is now known to be responsible for IgG homeostasis at all ages (5, 6). FcRn is intracellularly expressed and binds pinocytosed IgG in acidic endosomes (5). The bound IgG is recycled and released from the cell, while unbound IgG is shuttled downstream to lysosomes, where it is degraded. Although FcRn–/– mice appear histologically normal by all screened criteria, their nursing pups have drastically reduced levels of IgG, suggesting impaired trans-intestinal transport of maternal IgG (6). Furthermore, the IgG half-life is several-fold shorter in the FcRn–/– mice relative to wild-type (w.t.) mice, and decreased levels of IgG antibodies are observed in immunized mutant mice (6). These data established the pivotal role of FcRn in IgG homeostasis and launched further studies to explore its function in disease pathogenesis. Earlier studies had shown that mice rendered deficient in all class I molecules, owing to a deficiency in ß2-microglobulin, are protected from systemic lupus erythematosus-like syndromes, including bullous pemphigoid, presumably because they lack functional FcRn molecules to protect pathogenic IgG antibodies from catabolism (7). Moreover, a recent report demonstrated that FcRn–/– mice are protected from experimentally induced arthritis (8), provoked by pathogenic glucose-6-phosphate isomerase antibodies in the K/BXN model for autoimmune arthritis (9). These results indicate that in the absence of the FcRn molecule, pathogenic antibodies are degraded, resulting in reduced autoimmune disease.
In the present study, we utilized an infectious model of disease, Borrelia burgdorferi-induced Lyme arthritis, to demonstrate that FcRn sustains circulating levels of protective IgG. It is well established that hamsters and mice develop arthritis following needle inoculation or tick transmission of B. burgdorferi, the causative agent of Lyme disease, and B. burgdorferi-specific antibodies protect from and mitigate the disease (10–13).
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Methods |
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Mice
Mice with a null allele of FcRn (FcgrtTm1Dcr) have been described (6). MRL/MpJ-FcRn–/– (14) were produced after 10 generations of back-crossing of the FcgrtTm1Dcr allele onto MRL/MpJ. Mice were housed in filter frame cages and fed food and water ad libitum. Mice were sacrificed by carbon dioxide inhalation. The animal research was carried out according to federal guidelines and institutional policies.
Bacteria
The infectious clone D10/E9 of B. burgdorferi N40 was used in these experiments. Spirochetes were cultured until mid-log phase (5 x 107 cells ml–1) in BSK-H medium supplemented with 6% rabbit serum (Sigma Chemical Co., St Louis, MO, USA) at 33°C. Cell densities were estimated by dark-field microscopy.
Borrelia burgdorferi infection
Low-passage B. burgdorferi N40 were cultured in BSK-H medium supplemented with 6% rabbit serum (Sigma Chemical Co.) at 33°C. Spirochetes were enumerated by dark-field microscopy. Four-week-old MRL/MpJ and MRL/MpJ-FcRn–/– mice were momentarily anesthetized with metofane (Mallinckrodt Vetinary, Mundelein, IL, USA). Then, mice were inoculated with an intra-dermal injection of 5 x 103 B. burgdorferi in 50 µl of BSK-H medium plus 6% rabbit serum into each hind leg. Mock-infected mice received an injection of BSK-H medium alone. Mice were monitored for ankle swelling by measuring the tibiotarsal joint thickness of the extended hindlimbs using a portable thickness gauge (Mahr Federal Inc., Providence, RI, USA).
ELISA for the detection of B. burgdorferi-specific IgG
This protocol was adapted from a technique published by Glickstein et al. (15). Borrelia burgdorferi sonicate (15) was plated in flat-bottomed microplates (Nalge Nunc International, Rochester, NY, USA) at 10 µg ml–1 in 50 µl of coating buffer (0.1 M Na2HPO4, pH 9) and allowed to bind overnight at 4°C. Plates were washed with wash buffer (PBS, 0.5% Tween 20) and then blocked with 200 µl per well of blocking buffer [PBS (pH 7.4), 2% BSA, 0.5% Tween 20] for 45 min at 37°C. Plates were washed again. Then, dilutions of standards and mouse sera (1 : 400) in 100 µl blocking buffer were plated in duplicate at 37°C for 45 min. Following washes, plates were incubated at 37°C for 1 h with alkaline phosphatase-conjugated goat anti-mouse IgG (Promega, Madison, WI, USA) diluted 1 : 2500 in 100 µl of blocking buffer. Plates were washed and treated with p-nitrophenyl phosphate substrate (Pierce, Rockford, IL, USA) in diethanolamine buffer (Pierce) for 10 min. Finally, reactions were stopped by administering 100 µl of 5 N NaOH per well. Microplates were read with a microplate reader at 405 nm.
Extraction of DNA from infected tissue
Ear tissue was dissolved overnight in 500 µl of lysis buffer [100 mM Tris–Cl (pH 8.0), 5 mM EDTA, 0.2% SDS, 200 mM NaCl], supplemented with 10 µl of 20 mg ml–1 proteinase K (Invitrogen, Carlsbad, CA, USA) at 55°C. Then, 500 µl of phosphate-buffered phenol (Invitrogen) was added to each sample. Samples were mixed gently by inverting, transferred to Phase Lock GelTM eppendorf tubes (Eppendorf, Westbury, NY, USA) and spun for 4 min in a microfuge at top speed. Samples were transferred to new Phase Lock GelTM eppendorf tubes, and 500 µl of phenol : chloroform : isoamyl alcohol (25 : 24 : 1, v/v) (Invitrogen) was added to each tube. Samples were mixed well and spun again for 4 min in a microfuge at top speed. The upper layer was transferred to an eppendorf tube. Genomic DNA was precipitated with the addition of 1.4 ml of 95% EtOH and 140 µl of 3 M NaOAc (pH 5.2). Samples were spun for 10 min at top speed in a microfuge. Supernatants were carefully removed, and DNA was re-suspended in 100 µl of H2O. Following re-suspension, samples were treated with 2 µl of 1 mg ml–1 ribonuclease (RNase) A (Qiagen, Valencia, CA, USA) for 1 h at 37°C.
Measurement of spirochetal burden by real-time PCR
PCRs were performed in optical reaction plates (Applied Biosystems, Foster City, CA, USA). First, template DNA was diluted to 100 ng µl–1 in RNase-free water. Then, a master mix was prepared containing 25 µl of QuantiTect SYBR Green PCR Master Mix (Qiagen), 0.75 µl of each primer (20 µM) and 18.5 µl of RNase-free water for each 50 µl reaction. Borrelia burgdorferi-specific RecA was amplified with the following primers: 5'CC AAA GTT CTG CAA CAT TAA CAC CTA AGG and 5' GTG GAT CTA TTG TAT TAG ATG AGG CTC TCG. Mouse genomic DNA was amplified with the following primers: 5' GGA CAT ACT CTG CTG CCA TC and 5' CCA GCC ACA GAA TAC CAT CC. Then, 45 µl of the master mix was dispensed into each well of the reaction plate. A total of 5 µl of template DNA (500 ng) was added to each reaction. The real-time PCR cycler, a GeneAmp® 5700 Sequence Detection System (Applied Biosystems), was programmed with the following conditions: an initial DNA polymerase activation step at 95°C for 15 min, followed by 40 cycles of 94°C for 15 s, 60°C for 30 s and 72°C for 30 s. Standard controls consisting of 10-fold serial dilutions of RecA and nidogen DNA were run in parallel. Sample RecA values relative to the RecA standard curve were determined. Sample nidogen values relative to the nidogen standard curve were also determined. RecA : nidogen ratios were calculated for each sample.
In vitro B. burgdorferi killing assay
Following a published protocol (16), we tested for the viability of spirochetes after incubation with sera from infected mice. Briefly, 2 x 106 B. burgdorferi in PBS supplemented with 5.4 mM glucose, 50% heat-inactivated rabbit serum (Sigma Chemical Co.) and A1956 antibiotic (Sigma Chemical Co.) were aliquoted in duplicate into eppendorf tubes. Five microliters of mouse serum and 10 µl of mouse complement (Sigma Chemical Co.) were added to each tube. Samples were incubated at 33°C for 24 h. For each sample, viable spirochetes were enumerated in five fields, and an average was calculated. Borrelia burgdorferi viability was calculated by dividing the average viability in the treated samples by the average viability in the untreated samples.
Histological analysis of tibiotarsal joints and hearts
Following sacrifice, the tibiotarsal joints and hearts of the animals were removed. Joints were placed in a decalcifying solution (Surgipath, Richmond, IL, USA) and shaken overnight at room temperature. After demineralization, joints were rinsed in water and transferred to 10% buffered formalin (Fisher Scientific, Fairlawn, NJ, USA). Hearts were bisected sagitally and half of each heart was immediately placed in formalin. Joints and hearts were paraffin embedded, sectioned and stained with hematoxylin–eosin at the core histology facility of Beth Israel Deaconess Hospital (Boston, MA, USA). Joints and hearts were examined for arthritis and carditis. Sections were blindly scored on a scale of 0 (no lesion) to 3 (severe inflammation).
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Results |
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Absence of the FcRn results in increased ankle swelling following infection with B. burgdorferi
Previous reports indicated that B. burgdorferi-specific antibodies are associated with protective immunity and resolution of arthritis in the murine model of Lyme infection (11–13). Since FcRn plays a critical role in protecting serum IgG from catabolism (6), we sought to determine the consequences of FcRn in the arthritis caused by infection with B. burgdorferi in early murine Lyme arthritis. To this end, w.t. and FcRn–/– MRL/MpJ mice were infected with low-passage B. burgdorferi N40. Following infection, mice were monitored for arthritis by regular measurements of the tibiotarsal joint diameters.
Both w.t. and FcRn–/– mice developed severe ankle swelling 
12 days following infection (Fig. 1). In three independent trials, FcRn–/– mice had a statistically greater ankle swelling relative to w.t. mice over the course of the experiment. In the representative trial, depicted in Fig. 1, FcRn–/– mice had increased ankle swelling relative to control mice on days 18, 21, 27 and 29. Statistically greater ankle swelling in FcRn–/– mice relative to w.t. control mice was still observed >130 days beyond infection in the longest of the three trials (data not shown). Interestingly, the arthritis severity detected in both the w.t. and the FcRn–/– mice appeared as high as, if not higher than, that which is typically observed in C3H and BALB/c strains (17).
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FcRn–/– mice have reduced serum levels of anti-B. burgdorferi IgG following infection
Since the FcRn molecule salvages IgG from degradation, we asked whether B. burgdorferi-specific IgG was decreased in infected mice that lacked this protective receptor. Following infection, B. burgdorferi-specific IgG serum levels were determined for each mouse using an ELISA. In all trials, the levels of B. burgdorferi-specific IgG were significantly reduced in the FcRn–/– mice compared with the w.t. controls (Fig. 2). These data suggest that the degradation of IgG is indeed greater in the absence of the protective FcRn molecule.
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The presence of FcRn augments the level of borreliacidal antibodies, but not B. burgdorferi burden, in infected mice
The preceding experiments demonstrate that the FcRn–/– mice have increased ankle swelling and reduced B. burgdorferi-specific IgG following infection, suggesting that the number of spirochetes may be higher in these mice. To investigate, we used real-time PCR comparing infected FcRn–/– mice with the w.t. control mice. Following the isolation of DNA from mouse ear tissue, we amplified the B. burgdorferi-specific recA gene and the mouse-specific nidogen gene, using a real-time PCR assay. RecA quantities were normalized relative to nidogen quantities, resulting in a recA : nidogen ratio for each sample. While there was a trend of higher recA : nidogen ratios for the FcRn–/– mice compared with the w.t. controls (Fig. 3A), these cohorts failed to achieve statistical significant difference (P = 0.47, t-test). Real-time PCR products were analyzed by 0.8% agarose gel electrophoresis to verify product specificity (Fig. 3B).
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We then directly examined the in vitro borreliacidal activity of the mouse sera from each group of mice. This analysis was carried out in in vitro B. burgdorferi killing assays. Following the protocol of Belperron and Bockenstedt (16), we tested for the viability of spirochetes after 24 h incubation at 37°C with sera from infected mice in the presence of mouse complement (Fig. 4). We found that the viability of the spirochetes was higher in the samples treated with FcRn–/– mouse sera relative to the samples treated with w.t. mouse sera (P = 0.0004, t-test). These data document that the serum from the B. burgdorferi-infected w.t. mice has a significantly higher borreliacidal activity than that from the FcRn–/– mice.
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The severity of the tibiotarsal joint lesions, but not the heart lesions, is greater in infected FcRn–/– mice, as compared with infected w.t. mice
We examined the histopathology of the ankles and hearts of the B. burgdorferi-infected FcRn–/– and w.t. mice. First, tibiotarsal joints and hearts were fixed in formalin and then processed and stained with hematoxylin–eosin (Fig. 5). The stained sections were blindly examined, and the joints and hearts were scored for arthritis and carditis, respectively, on a scale of 0 (negative) to 3 (severe). FcRn–/– mice appear to have increased severity of ankle pathology relative to w.t. mice (Fig. 6A). There appears to be a smaller difference in the heart pathology between the two groups (Fig. 6B).
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Discussion |
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In the murine model of acute Lyme arthritis, antibodies are involved in disease protection and resolution (11–13). Because FcRn protects IgG from degradation, we hypothesized that it is essential for a vigorous antibody response to B. burgdorferi during infection and that its absence would render mice susceptible to heightened disease. The recent development of FcRn–/– mice (6) enabled us to independently evaluate the impact of FcRn deficiency in murine infection with B. burgdorferi. Previously, only ß2-microglobulin–/– mice were available for these analyses. To assess the role of FcRn in acute murine Lyme disease, MRL/MpJ w.t. and FcRn–/– mice were infected with B. burgdorferi and monitored for ankle swelling. Typically, MRL/MpJ mice (with a functional allele of Fas) may exhibit mild autoimmune symptoms, usually occurring late in life (18). MRL/MpJ mice are ancestrally related to C3H mice, a strain known to develop severe arthritis following infection with B. burgdorferi (17).
In this study, we showed that both FcRn–/– and control mice developed severe ankle swelling <2 weeks following infection. However, ankle swelling was statistically higher in the FcRn–/– mice. We corroborated our hypothesis that B. burgdorferi-specific antibodies were decreased in the FcRn–/– mice by using ELISAs to demonstrate that the levels of B. burgdorferi-specific IgG were statistically lower in the FcRn–/– mice. With the preceding evidence, we postulated that the heightened arthritis severity was due to lower levels of antibody-mediated clearance of B. burgdorferi in the FcRn–/– mice. Previously, several reports have associated arthritis severity with increasing numbers of spirochetes in infected tissue. For instance, C3H/HeN mice, a strain that develops severe arthritis, harbor large numbers of spirochetes in infected tissue; additionally, in BALB/c mice, the number of spirochetes in infected tissue is correlated with arthritis severity (17). We proceeded to examine if the spirochetal burden was higher in the FcRn–/– mice, using real-time PCR. Although there was a trend indicating higher bacterial burdens in the FcRn–/– mice relative to the w.t. mice, we could not demonstrate a statistical difference between the groups. This may be attributed to sample sizes in the trial, although it has been noted that in some strains, such as C57BL/6 and DBA/2, arthritis may not be correlated with spirochete numbers (17, 19).
Nevertheless, we still suspected that the efficiency of spirochete killing was higher in the w.t. mice as compared with the FcRn–/– mice due to antibody level differences. Ultimately, the increased borreliacidal activity of the w.t. serum relative to the FcRn–/– serum was successfully demonstrated using in vitro killing assays, confirming our hypothesis. Furthermore, an analysis of the histopathology of infected tissue, including tibiotarsal joints and hearts, revealed a greater degree of infiltrating neutrophils and lymphocytes in the infected FcRn–/– tissue relative to the w.t. tissue. However, there appeared to be no net difference in the carditis between the groups. This is not unexpected since unlike arthritis, carditis resolution is not antibody mediated (13).
Ultimately, our findings have enabled us to contribute to the emerging understanding of the function of FcRn in infectious disease.
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Acknowledgements |
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We are grateful to Jenifer Coburn for helpful discussions. We thank Diana Velez for technical help. The authors do not have a commercial or other association that might pose a conflict of interest. This work was supported by National Institutes of Health (NIH) grants AR45386, NIH DK56597, a grant from the Arthritis Foundation, the Eshe Fund, GRASP and the Alliance for Lupus Research.
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Abbreviations |
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| FcRn | neonatal FcR |
| RNase | ribonuclease |
| w.t. | wild type |
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Notes |
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Transmitting editor: R. S. Geha
Received 3 August 2005, accepted 8 December 2005.
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References |
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