Even in the absence of an appropriate model or direct evidence, T cells have been hypothesized to exacerbate the manifestations of Lyme disease. To define definitely the role of T cells in Lyme disease, the course of disease in immunocompetent and B cell-deficient mice was compared. By 8 wk postinoculation, immunocompetent mice resolved both carditis and arthritis, whereas foci of myocarditis and severe destructive arthritis characterized disease of B cell-deficient mice. Cell transfer experiments using infected B6-Rag1 knock out mice demonstrated that: 1) innate immunity mediated the initial sequelae of infection, 2) transferring both naive T cells and B cells induced resolution of carditis and arthritis, 3) infected mice reconstituted with T cells developed myocarditis and severe destructive arthritis, and 4) CD4+ T cells were responsible for the observed immune-mediated pathology. These data demonstrate directly the deleterious effect of T cells in Lyme disease.

Lyme disease, a multisystem illness caused by Borrelia burgdorferi (Bb),3 is the most common arthropod-borne infection in the United States, with about 16,000 U.S. citizens being infected each year (1), and about 60% of untreated North Americans developing arthritis (2). Although studies have shown that the humoral response to Bb is important for protection and resolution of Lyme disease (3), the relative participation of T cells in immune-mediated sequelae of Lyme disease remains controversial. Lack of consensus exists for three primary reasons. Early studies using infected SCID mice, which lack T cells and B cells, demonstrated the contribution of non-lymphocyte-based mechanisms, such as inflammatory mediators, in the development of carditis and arthritis (4, 5). The course and intensity of human Lyme disease follows various patterns: short and self-limiting; treatment-sensitive neural, joint, or cardiac manifestations; recurrent episodes of inflammation; and persistent manifestations. There was not a suitable animal model to examine the response of T cells, in the absence of B cells, to Bb.

Although isolation of spirochetes from cardiac tissue (6, 7) and synovium (8, 9) of human patients clearly establish that inflammation is triggered by infection, other host factors clearly regulate the severity and longevity of disease. Early studies revealed high concentrations of proinflammatory cytokines IL-1 (10), IL-6 (11), and TNF-α (12, 13). Because components of both innate and cellular immunity produce such cytokines that differentially regulate inflammation, emphasis was placed on identifying cell populations and subsets activated during infection. Th1 cells were isolated from arthritic joints of patients with chronic Lyme disease, and a higher ratio of Th1 to Th2 cells in synovial fluid was associated with more severe joint disease (14, 15). Similarly, in mice, arthritis susceptibility and severity were correlated with the presence of CD8+ T cells and/or Th1 cells (16, 17, 18, 19), whereas resistance was associated with a Th2 response (15, 16, 19, 20). This hypothesis was supported by the finding that treatment of infected mice with lymphokine-specific mAbs, which activated the opposite T cell subset, and thus modulated disease severity (17, 21). Based on these findings, it has been hypothesized that Th1 cells and CD8+ T cells exacerbated the manifestations of Lyme borreliosis and had an important role in the pathology of persistent Lyme disease. However, in all these studies the B cell compartment of the immune response was intact and detectable Bb-reactive Abs were produced. Thus, analyzing the data becomes difficult, especially in light of recent findings demonstrating that a T cell-independent response mediated by B cells is critical and sufficient for resolution of Lyme disease (22).4

The recent availability of knockout (KO) mice, with selected genes disrupted, provides an appropriate model to assess definitively the role of T cells in exacerbating, attenuating, or resolving Lyme disease-associated carditis and arthritis. In this study, B cell-deficient mice bearing T cells were used to ascertain the consequence of a cell-mediated immune response against Bb. The results clearly demonstrated, for the first time, the deleterious contribution of T cells in the pathogenesis of Lyme disease in mice.

Adult female mice purchased from The Jackson Laboratory (Bar Harbor, ME) were infected with a low passage clonal strain of Bb (cN40) (23). For each experiment, a frozen aliquot of Bb was thawed and expanded at 33°C in modified Barbour-Stoenner-Keller medium (24). Spirochetes were grown to mid-log phase and assessed for viability, and 104 spirochetes were injected intradermally above the shoulders. At the time of necropsy, both the urinary bladder and inoculation site were cultured in medium to confirm infection (25).

Rear limbs and hearts were processed for histology. Arthritis prevalence was determined by examining sagittal sections of knee and tibiotarsal joints from each mouse. Values of arthritis severity are the mean scores from the most inflamed tibiotarsal joint of individual mice in each group ± the SD, assessed on a scale of 0 (negative) to 3 (severe), as described (26). Sagittal sections of the heart through the aortic valve were examined for inflammation and scored as active (+) or inactive (−) carditis, as described (25). In addition, these studies revealed a novel lesion (myocarditis) heretofore not described in the mouse model. Myocarditis consisted of focal or diffuse interstitial infiltrates of mononuclear leukocytes in the ventricular myocardium. Wilcoxon rank-sum test (two-tail probability) was used to evaluate differences in arthritis severity between control and experimental groups of mice.

To phenotype lymphocytes populating the lymph nodes and spleens of reconstituted mice, single cell suspensions were prepared from the lymph nodes (inguinal, brachial, axial, mesenteric, superficial, and deep cervical) and spleens of all mice in each group. After incubating cells with anti-mouse CD32/CD16 mAb (PharMingen, San Diego, CA), which reduced nonspecific Ab binding, viable lymphocytes were incubated with fluorochrome-conjugated Abs at 4°C in 96-well V-bottom plates. Samples were simultaneously stained with FITC-labeled anti-CD8 mAb, PE-conjugated anti-CD4 mAb, and CyChrome-labeled anti-CD3 mAb (PharMingen) to phenotype T cells. After 30 min, cells were thoroughly rinsed and fixed with 1% paraformaldehyde. Fluorescence analysis of 104 cells was observed with a Becton Dickinson (Mountain View, CA) analyzer. T lymphocytes were found predominantly in the lymph nodes of Rag1 mice reconstituted with T cells. Furthermore, the CD4 to CD8 ratio (∼2.2:1) was unchanged relative to the lymphocytes originally injected.

To monitor Ab production during infection and after reconstituting B6-Rag1 mice with lymphocytes, mice were eyebled weekly and serum was titrated for reactivity to Bb lysates by ELISAs, as described (26).

To evaluate the relative contribution of T cells in the pathology of Bb infection, Lyme disease-associated carditis and arthritis in disease-resistant C57BL/6J (B6) immunocompetent mice (25, 27) were compared with disease in congenic B6-Igh6 KO mice (which lack B cells but bear T cells) and B6-Rag1 KO mice devoid of both T and B cells (Table I). As expected, B6 mice developed mild carditis and arthritis 3 wk postinoculation (p.i.) that were resolved and inactive by week 8 (Table I). B6-Igh6 KO mice developed severe carditis by week 3 (similar to Fig. 1), and by week 8 focal myocarditis was observed (Table I). Even though B6-Igh6 KO mice were on a B6 arthritis-resistant background, they developed significant tibiotarsal arthritis by wk 3, and by wk 8 all ankles and tibiotarsal joints were severely inflamed. In contrast to B6 mice, which produced significant Ab titers against Bb lysates by day 10 that peaked 21 days p.i., Abs were never detected in the sera of B cell-deficient mice bearing T cells (B6-Igh6 KO mice), confirming the functional absence of B cells (data not shown). Carditis and inflammation of the tibiotarsal joints were observed 3 wk p.i in B6-Rag1 KO mice. At wk 8, both carditis and arthritis remained active in B6-Rag1 KO (Table I), but myocarditis was never observed in mice lacking both T cells and B cells. This observation confirmed the necessity of acquired immunity for disease resolution and the importance of a non-immune-mediated mechanism for initiating carditis and joint inflammation (4). As demonstrated previously, B6-TcrβTcrδ KO mice (which lack both αβ+ T cells and γδ+ T cells but bear B cells) developed mild carditis and synovitis that were resolved by wk 8 (Table I), confirming that B cells were necessary and sufficient for inactivation of carditis and resolution of arthritis.4 Most importantly, the results presented above clearly demonstrated, for the first time, that T cells activated by Bb had a deleterious effect on disease.

Table I.

Comparison of disease development among Bb-infected, B6 immunocompetent mice and B6 immunodeficient micea

Mouse Strain, IntervalArthritis PrevalenceArthritis SeverityInactive CarditisActive CarditisMyocarditisSignificanceb
Week 3       
B6 0.2 ± 0.4 0.2 ± 0.4  5 /5   
B6-Rag1 KO 2.2 ± 0.8 0.4 ± 0.5  5 /5  p < 0.015 
B6-Igh6 KO 3.4 ± 0.6 1.1 ± 0.5  5 /5  p < 0.015 
B6-TcrβTcrδ KO 0 ± 0 0 ± 0 4 /5 1 /5  NSc 
Week 8       
B6 0.2 ± 0.4 .01 ± 0.2 5 /5    
B6-Rag1 KO 4 ± 0 1.6 ± 0.5  5 /5  p < 0.015 
B6-Igh6 KO 4 ± 0 2.3 ± 0.5   4/4 p < 0.015 
B6-TcrβTcrδ KO 0.85 ± 0.8 0.6 ± 0.5 5 /5   NS 
Mouse Strain, IntervalArthritis PrevalenceArthritis SeverityInactive CarditisActive CarditisMyocarditisSignificanceb
Week 3       
B6 0.2 ± 0.4 0.2 ± 0.4  5 /5   
B6-Rag1 KO 2.2 ± 0.8 0.4 ± 0.5  5 /5  p < 0.015 
B6-Igh6 KO 3.4 ± 0.6 1.1 ± 0.5  5 /5  p < 0.015 
B6-TcrβTcrδ KO 0 ± 0 0 ± 0 4 /5 1 /5  NSc 
Week 8       
B6 0.2 ± 0.4 .01 ± 0.2 5 /5    
B6-Rag1 KO 4 ± 0 1.6 ± 0.5  5 /5  p < 0.015 
B6-Igh6 KO 4 ± 0 2.3 ± 0.5   4/4 p < 0.015 
B6-TcrβTcrδ KO 0.85 ± 0.8 0.6 ± 0.5 5 /5   NS 
a

Groups of five infected mice were necropsied 3 and 8 wk p.i. Based upon culture results, all mice were infected. Severity of tibiotarsal joint arthritis was graded on a scale of zero (negative) to three (severe). Inflammation of knee joints was scored as positive or negative, whereas cardiac inflammation was scored as inactive carditis, active carditis, or myocarditis. Results are expressed as the mean ± SD. This experiment is representative of three experiments.

b

Significance of arthritis severity between control B6 mice and experimental groups was determined by Wilcoxon rank-sum test (two-tail probability).

c

NS, not significant compared with arthritis severity in control B6 mice.

FIGURE 1.

Active carditis, severe transmural inflammation, and thrombosis of the aortic root and adventitia at the base of the heart in an infected B6-Rag1 KO mouse reconstituted with T cells 3 wk previously. Note ventricular myocardium (bottom left) was not involved. Hematoxylin/eosin, ×20.

FIGURE 1.

Active carditis, severe transmural inflammation, and thrombosis of the aortic root and adventitia at the base of the heart in an infected B6-Rag1 KO mouse reconstituted with T cells 3 wk previously. Note ventricular myocardium (bottom left) was not involved. Hematoxylin/eosin, ×20.

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Thus, to characterize further the potential of T cells to exacerbate Lyme disease, we utilized cell transfer experiments. For this purpose, B6-Rag1 KO mice were infected with Bb 2 wk before an i.p injection of 2 × 107 splenocytes and 1 × 107 lymph node cells collected from healthy naive B6-Igh6 KO mice. Infected B6-Rag1 KO mice that did not receive lymphocytes and infected B6-Rag1 KO mice reconstituted with lymphocytes from naive B6 mice served as controls. At the completion of each experiment, splenocytes and lymph node cells from all mice were analyzed by flow cytometry to phenotype the populating cells; B cells were not detected in T cell-reconstituted mice either at the time of necropsy or after culturing harvested lymphocytes for 1 wk (data not shown).

By wk 3, actively infected B6-Rag1 KO mice reconstituted with T cells from naive B6-Igh6 KO mice had active carditis (Fig. 1) and significantly inflamed tibiotarsal joints, and 7 wk after cell transfer, mice developed severe myocarditis (Fig. 2) and severe destructive arthritis (Table II). Notably, the myocarditis observed in these mice has not been seen previously in mouse models of Lyme disease. However, in patients infected with Bb concomitant myocarditis and/or pericarditis is not uncommon, and mild left ventricular dysfunction and cardiomyopathy may occur (28). T cell reconstituted mice also developed perivascular and interstitial mononuclear leukocyte infiltrates in their lungs and died 8 wk after cell transfer. Actively infected B6-Rag1 KO mice reconstituted with B and T cells from naive B6 mice had active carditis and arthritis 3 wk after injecting lymphocytes; carditis resolved in three mice but remained active in two mice 7 wk after injecting lymphocytes (Table II). Also, control mice reconstituted with B6 lymphocytes had fewer inflamed joints and arthritis was resolving by wk 7 (Table II). In contrast, infected B6-Rag1 KO mice that did not receive lymphocytes developed carditis (but not myocarditis) and arthritis; disease remained active at all intervals tested (Table II). Because myocarditis has not been previously reported, this approach may provide a suitable model to study the pathogenesis of borrelia cardiomyopathy in humans. We hypothesize that the interaction between T cells and B cells played a critical role in defining the immune response to Bb infection, and suspect that the inflammatory response associated with infection altered the makeup of the T cell response and induced an autoimmune response.

FIGURE 2.

Myocarditis, mononuclear leukocytes diffusely infiltrated the ventricular myocardium in an infected B6-Rag1 KO mouse reconstituted with T cells 8 wk previously. Hematoxylin/eosin, ×60.

FIGURE 2.

Myocarditis, mononuclear leukocytes diffusely infiltrated the ventricular myocardium in an infected B6-Rag1 KO mouse reconstituted with T cells 8 wk previously. Hematoxylin/eosin, ×60.

Close modal
Table II.

Importance of T lymphocytes in mediating severity of Bb-induced arthritis and carditisa

Donor Cells, IntervalArthritis PrevalenceArthritis SeverityInactive CarditisActive CarditisMyocarditisSignificance,b Severity
Week 3       
2.7 ± 0.5 0.8 ± 0.3  5 /5  p < 0.015 
B+ T cells 1.8 ± 0.4 0.4 ± 0.2  5 /5   
T cells 4 ± 0 1.8 ± 0.8   4 /4 p < 0.015 
Week 7       
3.6 ± 0.5 1.9 ± −.5  5 /5  p < 0.015 
B+ T cells 0.8 ± 1.1 1.1 ± 1.5 3/5 2 /5   
T cells 4 ± 0 3 ± 0   5 /5 p < 0.015 
Donor Cells, IntervalArthritis PrevalenceArthritis SeverityInactive CarditisActive CarditisMyocarditisSignificance,b Severity
Week 3       
2.7 ± 0.5 0.8 ± 0.3  5 /5  p < 0.015 
B+ T cells 1.8 ± 0.4 0.4 ± 0.2  5 /5   
T cells 4 ± 0 1.8 ± 0.8   4 /4 p < 0.015 
Week 7       
3.6 ± 0.5 1.9 ± −.5  5 /5  p < 0.015 
B+ T cells 0.8 ± 1.1 1.1 ± 1.5 3/5 2 /5   
T cells 4 ± 0 3 ± 0   5 /5 p < 0.015 
a

Groups of five B6-Rag1 KO mice were infected 2 wk before an i.p. injection of 2 × 107 splenocytes and 1 × 107 lymph node cells collected from various mouse strains on a B6 background. Donor T lymphocytes were harvested from B6-Igh6 KO mice, and control mice were reconstituted with T and B lymphocytes collected from B6 mice. Three and 7 wk after injecting lymphocytes mice were necropsied, and both splenocytes and lymph node cells from reconstituted mice were analyzed by flow cytometry.

b

Significance of arthritis severity between control mice reconstituted with both T and B lymphocytes and experimental groups was determined by Wilcoxon rank-sum test (two-tail probability).

Because these studies demonstrated that T cells aggravated arthritis and induced myocarditis, which to date has never been observed in mouse models of Lyme disease, the contribution of CD4+ and CD8+ T lymphocytes in mediating myocarditis and severe destructive arthritis was analyzed. For this purpose, B6-Igh6 KO donor mice were depleted of specific T cell subpopulations using serotherapy. Anti-CD4 (GK1.5) and anti-CD8 (2.43) mAb in the form of ascites fluid were administered i.p. to B6-Igh6 KO donor mice, as described (29). Control mice were treated with an irrelevant mAb of the same isotype, D468.3. This treatment schedule caused complete depletion of the appropriate T cell subpopulation, as determined by flow cytometry. Cells were analyzed at both the time of reconstitution and necropsy. Control groups included: 1) infected B6-Rag1 KO mice that were not reconstituted, 2) infected B6-Rag1 KO mice reconstituted with T and B cells from naive B6 mice, 3) infected B6-Rag1 KO mice reconstituted with T cells from B6-Igh6 KO mice not treated with a mAb, and 4) infected B6-Rag1 KO mice reconstituted with cells from B6-Igh6 KO mice treated with isotype-matched irrelevant mAb D468.3. Disease severity was equivalent among mice reconstituted with cells from untreated B6-Igh6 KO mice and from D468.3-treated mice. Although the number of joints inflamed was equivalent among mice reconstituted with T cells and specific T cell subpopulations, only mice reconstituted with unseparated T cells or CD4+ T cells developed severe destructive arthritis by week 7 (Table III). Furthermore, infected B6-Rag1 KO mice reconstituted with CD4+ T lymphocytes or unseparated T cells were moribund and myocarditis was observed at wk 3 in these mice (Table III). These results suggested that CD4+ T cells were the primary mediators of severe arthritis and myocarditis, and confirmed the importance of the interaction between B cells and T cells in shaping the developing cellular immune response. Different T cell subsets probably participated in the immune response generated by B cell-deficient mice and immunocompetent mice because distinct APC populations (i.e., B cells, macrophages, and dendritic cells) differentially regulate the activation of specific T cell subsets (30). Distinct APC types also generate and present different peptide epitopes to T cells (31), thus the T cell repertoire in B cell deficient mice and immunocompetent mice probably differed. Furthermore, expression of additional foreign antigenic epitopes and possibly self-Ags, resulting from the inflammatory response induced by infection, potentially augmented the T cell response to Bb and induced an autoimmune response. Thus, determining the specificity of Bb-reactive T cells will be important for defining the mechanism by which T cells modulate disease severity.

Table III.

CD4+ T cell-mediated development of myocarditis in Bb-infected micea

Donor Cells, IntervalArthritis PrevalenceArthritis SeverityInactive CarditisActive CarditisMyocarditisSignificance,b Severity
Week 3       
3.4 ± 0.5 1 ± 0  5 /5   
T cells 4 ± 0 2.4 ± 0.4  1 /5 4 /5 p < 0.015 
CD4+ cells 3.8 ± 0.4 1.8 ± 0.7   5 /5  
CD8+ cells 3.6 ± 0.5 1.2 ± 0.4 1/5 4 /5  NS 
Week 7       
3.8 ± 0.4 1.8 ± 0.4  5 /5   
T cells 4 ± 0 2.8 ± 0.4   5 /5 p < 0.015 
CD4+ cells 4 ± 0 3 ± 0   5 /5 p < 0.015 
CD8+ cells 3.6 ± 0.9 1.5 ± 1  4 /5 1 /5 NS 
Donor Cells, IntervalArthritis PrevalenceArthritis SeverityInactive CarditisActive CarditisMyocarditisSignificance,b Severity
Week 3       
3.4 ± 0.5 1 ± 0  5 /5   
T cells 4 ± 0 2.4 ± 0.4  1 /5 4 /5 p < 0.015 
CD4+ cells 3.8 ± 0.4 1.8 ± 0.7   5 /5  
CD8+ cells 3.6 ± 0.5 1.2 ± 0.4 1/5 4 /5  NS 
Week 7       
3.8 ± 0.4 1.8 ± 0.4  5 /5   
T cells 4 ± 0 2.8 ± 0.4   5 /5 p < 0.015 
CD4+ cells 4 ± 0 3 ± 0   5 /5 p < 0.015 
CD8+ cells 3.6 ± 0.9 1.5 ± 1  4 /5 1 /5 NS 
a

B6-Rag1 KO mice were infected 2 wk before an i.p. injection of donor T cells (2 × 107 splenocytes and 1 × 107 lymph node cells). Donor T lymphocytes were harvested from naive B6-Igh6 KO mice, CD4+ T cells from B6-Igh6 KO mice treated with the mAb 2.43, and CD8+ T cells from B6-Igh6 KO mice treated with GK1.5 mAb. Control infected B6-Rag1 KO mice were not reconstituted with lymphocytes. Three and 7 wk after injecting lymphocytes mice were necropsied, and both splenocytes and lymph node cells from reconstituted mice were analyzed by flow cytometry.

b

Significance of arthritis severity between control B6-Rag1 KO mice that did not receive lymphocytes and experimental groups was determined by Wilcoxon rank-sum test (two-tail probability).

In this study we present unequivocal evidence that elements of both innate and T cell-mediated immunity were responsible for inducing and exacerbating cardiac and joint disease characteristic of Lyme borreliosis. It remains to be determined whether T cell-mediated immunopathology results from the antigenic specificity of T cells, the activation of a specific T cell subset, and/or ability of persisting Ag to induce a hypersensitive response or activate autoreactive T cells in the joint and heart. This model provides the framework for experimental dissection of Lyme disease and for testing therapeutic strategies.

We appreciate the technical assistance of L. Adamson and K. Freet.

1

This work was supported by the National Institutes of Health Grant AI-26815.

3

Abbreviations used in this paper: Bb, Borrelia burgdorferi; B6, C57BL/6J; B6-Tcrβ KO, C57BL/6J-Tcrβtm/Mom129; B6-TcrβTcrδ KO, C57BL/6J-Tcrβtm/Mom129 Tcrδtm/Mom129; B6-Igh-6 KO, C57BL/6J-Igh-6tm/Mom129; B6-scid, C57BL/6-Prkdcscid; KO, knockout; p.i., postinoculation.

4

M.D. McKisic and S.W. Barthold. T cell independent responses to Borrelia burgdorferi are critical for protective immunity and resolution of Lyme disease. Submitted for publication.

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149
:
1905