INTRODUCTION

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Lyme disease is a tick-transmitted infection caused by the spirochete Borrelia burgdorferi (17, 25, 42). Illness in humans can be successfully treated with antibiotics; however, in the absence of treatment, arthritis, carditis, neurological complications, and skin abnormalities may develop (42). Arthritis is a late-stage symptom in approximately 60% of individuals who are not treated with antibiotics at the time of infection. The arthritis is associated with the presence of spirochetes in the joint tissue and is characterized by tendonitis, synovitis, and inflammatory cell infiltrate (41, 42).

The mouse model of Lyme disease has provided an opportunity to study the development of arthritis upon infection with B. burgdorferi. In mice, arthritis is coincident with spirochetes in the joint tissues and is characterized by tendonitis, synovial hyperproliferation, and infiltration of neutrophils (7). There is a spectrum of disease with different mouse strains having differing susceptibilities to B. burgdorferi-induced pathology. Infected C3H mice develop severe arthritis with large numbers of bacteria infiltrating joint tissue (8, 12, 52). C57BL/6 mice predominantly develop mild arthritis and yet harbor large numbers of spirochetes that equal those in C3H mice (8, 10, 30). BALB/c mice show a spectrum of disease severity. At low inoculum doses, BALB/c mice develop mild arthritis which becomes more severe as the inoculum dose is increased (30). It appears that there may be more than one mechanism of resistance, since resistance in BALB/c mice correlates with low levels of spirochetes in the joints, while in C57BL/6 mice arthritis is mild even in the presence of large numbers of spirochetes (30).

A mapping project has been carried out in this laboratory using the F₂ intercross generation of C3H/HeNCr and C57BL/6NCr mice (51). Four chromosomal regions were linked to arthritis development by quantitative trait loci (QTL) analysis, confirming that arthritis development is a complex, multigenic trait with multiple mechanisms. Interestingly, ankle swelling and histopathological severity were linked to distinct chromosomal regions in this study. An additional suggestive quantitative trait locus for histopathology was identified in a region of chromosome 11 that contains a cluster of immune and inflammatory regulatory cytokines, including interleukin-3 (IL-3), IL-4, IL-5, and IL-13. This cluster of genes is associated with the regulation of Th cell responses, regulating T-cell and inflammatory responses to infection (22).

Previous studies provide conflicting evidence regarding the involvement of IL-4 in Lyme arthritis. Antibody (Ab)-mediated depletion of IL-4 in BALB/c and C3H mice suggested a protective effect for IL-4 (29, 33), as did experiments in which treatment of C3H/HeN mice with recombinant IL-4 reduced arthritis severity (28). Others have shown that the kinetics of IL-4 production differs in severe and mildly arthritic mice (27). However, in BALB/c mice, interruption of the B7/CD28 costimulatory pathway using an Ab to CD86 prevented T-cell production of IL-4, yet it had no effect on arthritis development (48). Most recently, arthritis-resistant DBA mice that were genetically deficient in IL-4 did not show increased arthritis severity when infected with B. burgdorferi (14). These conflicting results suggest that the regulation, production, and action of IL-4 during B. burgdorferi infection may be more complex than could be examined in previous experiments.

In response to conflicting results in the literature and the suggestive linkage from our mapping study, we have chosen to look at the effects of IL-4 production in Lyme disease by using mice that are genetically altered to prevent the expression of IL-4, the  chain of the IL-4 receptor, or both. These mice enabled us to examine the involvement of IL-4 in the development of Lyme arthritis, regardless of the cell type producing it, location of its production, site of action, or responsive cell type. In other pathogen systems, it has been found that IL-13 signaling through the IL-4R chain can compensate for the absence of IL-4, because the two cytokines have many of the same effects (20, 24, 39, 49). Therefore, the IL-4R knockout with disruption of both IL-4 and IL-13 signaling allowed us to determine whether IL-13 plays a role in the severity of Lyme arthritis and/or is able to compensate for a loss of IL-4 during infection.

	MATERIALS AND METHODS

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Mice. Male and female C3H/HeN mice were obtained from the National Cancer Institute. Male and female C3H/HeJ, BALB/cJ, BALB/cJ-Il-4^tm2Nnt, C57BL/6J, and C57BL/6J-Il-4^tm1Nnt mice were obtained from the Jackson Laboratories (Bar Harbor, Maine). BALB/cJ-IL-4R^/ mice were generated as previously described (40). BALB/cJ-IL-4^/ IL-4R^/ mice were obtained by an intercross between BALB/cJ-IL-4^/ and BALB/cJ-IL-4R^/ mice. The designation C3H is used when data have been pooled, including for experiments performed with both C3H/HeJ and C3H/HeN mice. Mice were housed in the Animal Resource Center at the University of Utah Medical Center, according to the National Institutes of Health guidelines for care and use of laboratory animals.

Bacteria and infection. Mice between 4 and 6 weeks of age were infected by intradermal injection in the shaven back with 2 × 10³ bacteria of the N40 isolate of B. burgdorferi (provided by S. Barthold, University of California at Davis, at passage 3 from an infected mouse) (8), a mode of infection reported to require the fewest spirochetes and to most closely mimic tick transmission (6, 43). In one experiment, mice from 10 to 13 weeks of age were used, with results similar to those for mice 4 to 6 weeks of age. Passage 4 spirochetes were grown in BSK-H medium containing 6% rabbit serum (Sigma, St. Louis, Mo.) for 3 to 5 days prior to injection, enumerated using a Petroff-Hauser chamber, and diluted with sterile medium. Mock-infected animals received intradermal injection of sterile BSK-H medium in the shaven back and tested negative for B. burgdorferi infection by both PCR and serology.

Measurement of ankle joints. Rear ankle joints of mice anesthetized with methoxyflurane (Pitman-Moore) were measured with a metric caliper (Mitutoyo, Tokyo, Japan) during each week of infection. Measurements were taken in the anterior-to-posterior position, with the ankle extended, through the thickest portion of the ankle. Numerous factors can influence normal joint measurements, including age, sex, and strain of mice; therefore, findings are reported as the change in joint measurement relative to that of mock-infected matched animals.

Histopathology of ankle joints. The rear ankle joint displaying the greatest swelling at the time of sacrifice was taken from each mouse for histological analysis. Samples were fixed in 10% formalin, decalcified, and embedded in paraffin, and sections were stained with hematoxylin and eosin. Sections were viewed in a blind fashion and given a score for severity ranging from 0 to 4+, as previously described (30).

Preparation of DNA from infected tissues. Mice were sacrificed at 4 weeks postinfection, and rear ankle joint and heart tissues were prepared as previously described (47). Briefly, tissue specimens were incubated in 0.1% collagenase A (Boehringer Mannheim) solution at 37°C overnight and were then mixed with an equal volume of proteinase K solution (Boehringer Mannheim) and incubated at 55°C overnight. DNA was then recovered by extraction with an equal volume of phenol-chloroform and precipitated with ethanol. After digestion with 1 mg of DNase-free RNase (Sigma) per ml the samples were extracted again, and DNA was recovered by precipitation. The precipitated DNA was resuspended in 1 to 1.5 ml of water, and the DNA content was determined by measuring the absorbance at 260 nm.

Quantification of DNA from infected tissues. The levels of B. burgdorferi DNA in joints and hearts were determined by continuous-monitoring PCR, using the LightCycler (Idaho Technologies, Idaho Falls, Idaho) as previously described (36). Briefly, 200 ng of sample DNA was amplified in a final volume of 10 µl containing 50 mM Tris (pH 8.3), 3 mM MgCl₂, 4.5 µg of bovine serum albumin, 200 µM deoxynucleoside triphosphates, a 1:30,000 dilution of SYBER Green I (Molecular Probes, Eugene, Oreg.), 5 µM each primer, 0.5 U of Taq polymerase (GIBCO BRL, Gaithersburg, Md.), and 110 ng of TaqStart Ab (ClonTech, Palo Alto, Calif.). Forty cycles of amplification were performed, with each cycle consisting of three steps: heating at 20°C/s to 95°C with a 1-s hold, cooling at 20°C/s to 60°C with a 1-s hold, and heating at 1°C/s to 84°C. This technique monitors the cycle-by-cycle accumulation of fluorescently labeled product. The cycle at which the product is first detected was an indicator of relative starting copy number and was calculated by using the LightCycler analysis software. B. burgdorferi was quantified by using the chromosomally encoded recA gene, and values were normalized to 10⁴ copies of a single-copy mouse gene, nidogen. The oligonucleotide primers used to detect mouse nidogen were nido.F (5'-CCA GCC ACA GAA TAC CAT CC-3') and nido.R (5'-GGA CAT ACT CTG CTG CCA TC-3'). The oligonucleotide primers used to detect B. burgdorferi recA were nTM 17.F (5'-GTG GAT CTA TTG TAT TAG ATG AGG CTC TCG-3') and nTM17.R (5'-GCC AAA GTT CTG CAA CAT TAA CAC CTA AAG-3').

Detection of B. burgdorferi-specific immunoglobulin (Ig) levels. Serum obtained by retro-orbital bleeding of experimental animals at sacrifice was analyzed for B. burgdorferi-specific Ab using an Ab capture ELISA. Eleven columns per 96-well plate were coated with B. burgdorferi sonicate at a concentration of 5 µg of sonicate/ml. The 12th column was coated with polyclonal rabbit anti-mouse immunoglobulin G (IgG) plus IgA plus IgM (Zymed, South San Francisco, Calif.) at a concentration of 5 µg/ml. Serum samples were added to B. burgdorferi sonicate-coated wells and analyzed for isotype-specific Ab content at the following dilutions: IgG1 at a dilution of 1:100, IgG2a at 1:500, IgG2b at 1:1,000, and IgG3 at 1:1,000. Known concentrations of IgG1, IgG2a, IgG2b, and IgG3 were added to the rabbit anti-mouse Ig wells to ensure that results were within the linear range. Following a 2-h incubation with serum samples, unbound sample was removed by washing, and the anti-B. burgdorferi Ab was detected by the addition of alkaline phosphatase-conjugated rabbit anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (Zymed), respectively, for 1 h. Plates were washed and developed by incubation with 1 mg of p-nitrophenyl phosphate per ml in Tris buffer (Sigma) for 30 min, stopped with 3 N NaOH, and read at 405 nm on a 96-well microtest plate spectrophotometer (Molecular Devices). Values are expressed as the optical density at 405 nm (OD₄₀₅).

Statistical analysis. The degrees of statistical significance of the quantitative differences between sample groups were determined by application of Student's t test.

	RESULTS

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Effect of genetic ablation of IL-4 and the IL-4R chain on B. burgdorferi-induced arthritis severity in BALB/cJ and C57BL/6J mice. BALB/cJ mice deficient in IL-4 or the IL-4R chain were developed through gene targeting in a BALB/cJ embryonic stem cell, an approach that ensures a pure background on which to examine the roles of IL-4 and IL-4R (38, 40). Wild-type C3H mice and BALB/cJ mice of these genotypes [wild type, IL-4^/ IL-4R^/, IL-4-IL-4R^/ and IL-4R^+/] were each infected with 2,000 B. burgdorferi cells intradermally in the shaven back. This dose of B. burgdorferi was expected to cause mild to moderate arthritis in wild-type BALB/cJ mice and severe disease in C3H mice (8, 12, 30, 52).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Effect of IL-4 and IL-4R ablation on ankle swelling in B. burgdorferi-infected BALB/cJ mice. Mice were infected with 2,000 B. burgdorferi cells, and joints were measured at 4 weeks postinfection as described in Materials and Methods. Open circles, values for joints with the greatest swelling from each mouse; black bars, average values for the group. The number of mice in each group is represented below the strain name. Results are combined from four individual experiments. The difference in joint swelling between infected mice on the BALB/cJ background and C3H mice was statistically significant (P < 0.001). No statistical difference was seen between mice on the BALB/cJ background.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Ankle swelling in IL-4-deficient C57BL/6J mice throughout the 4-week course of infection. Mice were infected with 2,000 B. burgdorferi cells, and joints were measured as described in Materials and Methods. Data points represent the averages and bars indicate the standard deviations of values for five to seven infected animals.

+/

Spirochete numbers in the joint and heart tissues of animals lacking IL-4 or the IL-4R chain. Although IL-4 does not appear to play a role in regulating arthritis severity, it may be involved in the control of B. burgdorferi numbers. In previous experiments, animals which had been treated with an IL-4 Ab yielded higher numbers of spirochetes following culture for several days, suggesting that IL-4 is involved in the control of spirochete numbers (29). To more directly address the numbers of B. burgdorferi in tissues, we performed continuously monitored PCR on DNA collected from tissues at 4 weeks postinfection. Previously, using a different PCR technology, we had shown that, at an inoculum dose of 2,000 B. burgdorferi cells, 5- to 10-fold fewer spirochetes persist in the joint and heart tissues of BALB/cAn than of C3H/HeJ mice (30). In this study, with a newly developed PCR method, fivefold fewer spirochetes were found in the joints of wild-type BALB/cJ mice than of C3H/HeN mice (Fig. 3A). A similar three to fivefold difference in spirochete numbers was seen between the IL-4- and IL-4R-deficient mice and the C3H/HeN mice. Spirochete numbers were threefold higher in the hearts of C3H/HeN mice than in those of the wild-type, IL-4-, or IL-4R-deficient BALB/cJ mice (Fig. 3B). Overall, deficiency in IL-4 or IL-4R had no effect on spirochete numbers in tissues from BALB/cJ mice.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   B. burgdorferi numbers in joint (A) and heart (B) tissues of IL-4- and IL-4R-deficient mice on the BALB/cJ background. Mice were infected with 2,000 B. burgdorferi cells, and tissues were harvested at 4 weeks postinfection. DNA was isolated from tissues and assessed using continuously monitored PCR. Circles, amounts of B. burgdorferi-specific DNA present in individual mouse tissues relative to genomic mouse DNA. Black lines, average values for the animals in each group (n  6 in each group). Uninfected controls were negative for B. burgdorferi. These results are representative of four separate experiments. The difference in B. burgdorferi numbers between infected mice on the BALB/cJ background and C3H mice was statistically significant (P  0.025 for joints and P  0.04 for hearts [two-tailed t test]).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   B. burgdorferi numbers in joint (A) and heart (B) tissues from IL-4-deficient animals on the C57BL/6J background. Mice were infected with 2,000 B. burgdorferi cells, and tissues were harvested at 4 weeks postinfection. DNA was isolated from tissues and assessed using continuously monitored PCR. Circles, amounts of B. burgdorferi-specific DNA present in individual mouse tissues relative to genomic mouse DNA; black bars, average values for the animals in each group (n  4 in each group). Uninfected controls were negative for B. burgdorferi. The difference in B. burgdorferi numbers in heart tissues, between infected mice on the C57BL/6J background and C3H mice, was statistically significant (P  0.001 [two-tailed t test]).

Immunoglobulin isotype profiles in mice lacking IL-4 and IL-4R. Passive immunization of naive mice with serum from animals infected with B. burgdorferi prior to infection has been shown to be effective in preventing infection (9, 11). The importance of antibodies for resolution of Lyme disease has also been confirmed in studies using immune-compromised scid- and rag-deficient mice, which are unable to resolve infection (13, 15, 45, 46). It has not directly been shown that any single isotype of Ab is more effective at clearing B. burgdorferi or influencing the development of severe arthritis. However, it has been inferred from the reported effects of IL-4 depletion that Th2-dependent isotype IgG1 could be more effective than other isotypes at clearing B. burgdorferi (29, 33). Because IL-4 induces class switching to IgG1, we determined by ELISA the relative levels of various IgG isotypes in mice infected with 2,000 B. burgdorferi cells. Table 2 shows a comparison of the relative amounts of Ab produced by wild-type C3H and BALB/cJ mice, as well as the IL-4- and IL-4R-deficient mice on the BALB/cJ background. It is quite interesting that the C3H mice produced greater quantities of all Ab isotypes than the wild-type BALB/cJ mice did. The IL-4- and IL-4R-deficient mice had barely detectable levels of IgG1, which were greatly reduced compared to levels in the wild-type BALB/cJ mice. IL-4- and IL-4R-deficient mice produced increased amounts of IgG2b. IgG2a and IgG3 levels showed little variation between mice of the BALB/cJ background.

View this table: [in this window] [in a new window]   TABLE 2.   Levels of B. burgdorferi-specific IgG isotypes in sera from IL-4- and IL-4R-deficient BALB/cJ micea

	DISCUSSION

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In this study, we examined the effects of genetic ablation of IL-4 and the IL-4R chain in order to determine the roles that IL-4 and IL-13 play in host defense against B. burgdorferi infection. IL-4 has previously been shown to play a role in resistance to severe arthritis, and its presence correlated with decreased joint swelling during infection (28, 29, 33). Throughout the study we saw variability in the severity of arthritis in BALB/cJ wild-type mice. By doing several experiments, we were able to conclude that the absence of IL-4 or the IL-4R chain did not lead to more severe arthritis or joint swelling when wild-type mice had mild arthritis or to less severe arthritis and joint swelling when wild-type mice had severe arthritis. This conclusion points out the care that must be exercised in using the dose-responsive BALB/c mice and may explain why others using BALB/c mice previously reported a greater effect for IL-4 (28, 29, 33). The results of experiments with mice on the C57BL/6J background were quite clear, showing no differences in arthritis severity or joint swelling between IL-4-deficient and wild-type mice. Our results show that IL-4 and IL-13 are not required for resistance to arthritis upon infection with B. burgdorferi. These results are consistent with the findings for another IL-4-deficient mouse on the resistant DBA background (14).

The role of IL-4 in the host response to B. burgdorferi infection has previously been addressed in the context of Th1 versus Th2 responses in the mouse. A Th1 response including gamma interferon production and isotype switching to IgG2a was suggested to be detrimental to the host, leading to the development of severe arthritis and the inability to control spirochete numbers (28, 29, 33). IL-4 production, as part of a Th2 response, and isotype switching to IgG1 were believed to be crucial for resistance to severe arthritis and control of spirochete numbers (28, 29, 33). Surprisingly, our experiments show that arthritis-susceptible C3H mice make Borrelia-specific Abs of all isotypesIgG1, IgG2a, IgG2b, and IgG3, in larger amounts than those of either of the resistant BALB/cJ or C57BL/6J strains. Although C3H mice are able to produce IgG1 in amounts equivalent to those of BALB/cJ mice, they still harbor fivefold more bacteria in their joints. IL-4- and IL-4R-deficient BALB/cJ mice produce almost no IgG1, yet they are able to control spirochete numbers in both joint and heart tissues, suggesting that IgG1 is not required for host control of spirochete numbers. Interestingly, all of the IL-4- and IL-4R-deficient mice produced more IgG2b than did their wild-type counterparts; however, this change had no effect on spirochete numbers. The C57BL/6J mice that were deficient in IL-4 retained similar bacterial numbers in joints and hearts compared to those retained by wild-type C57BL/6J mice. Overall, the absence of IL-4 and IL-4 receptor signaling had no effect on spirochete presence in tissues; therefore, these cytokines do not appear to be necessary for host control of B. burgdorferi during the first 4 weeks of infection.

A recent study addressed the role of IL-4 in B. burgdorferi infection by using an IL-4-deficient mouse on the DBA background (14). The results presented here confirm and expand on those results by looking at additional mouse strains, as well as examining B. burgdorferi infection in a mouse lacking the IL-4R chain. IL-4 and IL-13 share the IL-4R chain as a common component in their receptor complexes (1, 21, 23, 35), leading to many similar downstream effects (18, 37, 53). For example, granuloma formation in Schistosoma mansoni-infected mice lacking IL-4 was found to be dependent on IL-13 and disappeared when IL-4R^/ mice were infected (19, 20, 24, 34, 44, 49, 50). Through the use of an IL-4R-deficient mouse, the present study has shown that IL-4 is not required for resistance to the development of severe arthritis or for control of bacterial numbers in the joint and heart tissues and that, in BALB/cJ mice, IL-13 is not compensating for the absence of IL-4.

Genes encoding IL-4 and IL-13 can now be eliminated as candidate genes for the region of chromosome 11 with suggestive linkage to arthritis severity (51). Experiments have now shown that the absence of IL-4 on the resistant DBA (14), C57BL/6J, and BALB/cJ backgrounds or the absence of the IL-4R chain on the BALB/cJ background has no effect on arthritis severity. Although IL-4 and IL-13 are not required for resistance to severe arthritis, production of the inflammatory molecules IL-6, IL-11, and IL-12 appears to regulate the host response to B. burgdorferi infection (2-5). Furthermore, the absence of the anti-inflammatory cytokine IL-10 on the C57BL/6 background leads to increased arthritis severity in a resistant mouse strain (16). These results suggest that the balance of pro- and antiinflammatory molecules may be involved in regulating Lyme arthritis severity, but at a level other than the production of IL-4 or through the IL-4-IL-13 signaling pathway.