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. 2004;6(5):R433-46.
doi: 10.1186/ar1212. Epub 2004 Jul 19.

Direct Toll-like receptor 2 mediated co-stimulation of T cells in the mouse system as a basis for chronic inflammatory joint disease

Vera Sobek  1 , Nico Birkner, Ingrid Falk, Andreas Würch, Carsten J Kirschning, Hermann Wagner, Reinhard Wallich, Marinus C Lamers, Markus M Simon
Affiliations

Direct Toll-like receptor 2 mediated co-stimulation of T cells in the mouse system as a basis for chronic inflammatory joint disease

Vera Sobek et al. Arthritis Res Ther. 2004.

Abstract

The pathogenesis of chronic inflammatory joint diseases such as adult and juvenile rheumatoid arthritis and Lyme arthritis is still poorly understood. Central to the various hypotheses in this respect is the notable involvement of T and B cells. Here we develop the premise that the nominal antigen-independent, polyclonal activation of preactivated T cells via Toll-like receptor (TLR)-2 has a pivotal role in the initiation and perpetuation of pathogen-induced chronic inflammatory joint disease. We support this with the following evidence. Both naive and effector T cells express TLR-2. A prototypic lipoprotein, Lip-OspA, from the etiological agent of Lyme disease, namely Borrelia burgdorferi, but not its delipidated form or lipopolysaccharide, was able to provide direct antigen-nonspecific co-stimulatory signals to both antigen-sensitized naive T cells and cytotoxic T lymphocyte (CTL) lines via TLR-2. Lip-OspA induced the proliferation and interferon (IFN)-gamma secretion of purified, anti-CD3-sensitized, naive T cells from C57BL/6 mice but not from TLR-2-deficient mice. Induction of proliferation and IFN-gamma secretion of CTL lines by Lip-OspA was independent of T cell receptor (TCR) engagement but was considerably enhanced after suboptimal TCR activation and was inhibitable by monoclonal antibodies against TLR-2.

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Figures

Figure 1
Figure 1
Direct co-stimulation of pre-sensitized T cells via Toll-like receptor (TLR)-2. (a) Unselected splenocytes or fluorescence-activated cell sorting (FACS)-sorted T cells were cultivated on anti-hamster (ha)IgG plus anti-CD3 (3 ng per well) or anti-haIgG coated plates (control) in the presence or absence of Lip-OspA, Met-Asp-Pro (MDP)-OspA (10 μg/ml each), recombinant interleukin-2 (rec. IL-2; 50 U/ml) or lipopolysaccharide (LPS; 1 μg/ml) for 72 h. Proliferation of cells was measured by [3H]thymidine incorporation. Means ± SEM for six different wells are given. Asterisk denotes significant difference (P < 0.05) from control (anti-haIgG or anti-CD3 without supplements). One representative experiment is shown. (b) FACS-sorted T cells were stimulated with anti-CD3 (3 ng per well) and different amounts of Lip-OspA or MDP-OspA (10, 1 or 0.1 μg/ml each) or with 50 U/ml recombinant IL-2. Proliferation of cells was measured by [3H]thymidine incorporation. Means ± SEM for six different wells are given. Asterisk denotes significant difference (P < 0.05) from control (anti-CD3 without supplements). (c) Analysis of splenocytes from C57BL/6 (B6; wild-type), TLR-2-/- and TLR-4def C57BL/10ScNCr mice for different cell populations before and after FACS sorting for T cells (re-analysis). CD11c+ and I-A+ are, in combination, characteristic markers for dendritic cells. Data are given in percentages.
Figure 2
Figure 2
Direct co-stimulation of cytotoxic T lymphocyte (CTL) lines via Toll-like receptor (TLR)-2. CD8+ T cells from CTL lines (generated against BALB/c, sixth stimulation, day 4) were cultivated on anti-hamster (ha)IgG plus anti-CD3 (0.3 or 0.03 ng per well) or anti-haIgG coated plates (control) in the presence or absence of Lip-OspA, Met-Asp-Pro (MDP)-OspA (10, 1 or 0.1 μg/ml each), recombinant interleukin-2 (rec. IL-2; 50 U/ml), concanavalin A (ConA; 5 μg/ml) or lipopolysaccharide (LPS; 1 μg/ml) for 48 h. Proliferation of cells was measured by [3H]thymidine incorporation. Means ± SEM for six different wells are given. Asterisk denotes significant difference (P < 0.05) from control (anti-haIgG or anti-CD3 without supplements). One representative experiment is shown. Phenotypic analysis (fluorescence-activated cell sorting) of C57BL/6 (B6), TLR-2-/- and TLR-4def anti-BALB/c CTL lines (third stimulation, day 4): Thy1.2+, 99.0–99.5%; CD8+, 93–95%, CD4+, 0.7–2%; CD19+ (B cells), F4/80+ (macrophages), NK1.1+ (NK cells) ≤ 0.2%. SI, stimulation index (calculated based on results with anti-haIgG plus anti-CD3 or with anti-haIgG alone, without the addition of supplements).
Figure 3
Figure 3
Direct co-stimulation of cytotoxic T lymphocyte (CTL) lines by Lip-OspA can be inhibited by anti-Toll-like receptor (TLR)-2 monoclonal antibody. CD8+ T cells from C57BL/6 (B6) CTL lines (generated against BALB/c, fifth stimulation, day 4) were cultivated on anti-hamster IgG plus anti-CD3 (0.3 or 0.03 ng per well) in the presence or absence of Lip-OspA (10 μg/ml) with or without varying concentrations of anti-TLR-2 monoclonal antibody (25, 2.5 or 0.25 μg/ml) or the respective isotype control antibody (25 μg/ml) for 24 hours. Proliferation of cells was measured by [3H]thymidine incorporation. Means ± SEM for three to six different wells are given. Asterisk denotes significant difference (P < 0.05) from control (plus Lip-OspA without the addition of anti-TLR-2 monoclonal antibody). One representative experiment (out of two with similar results) is shown. SI, stimulation index (calculated based on results with anti-CD3 without the addition of supplements; white bars).
Figure 4
Figure 4
Explanation for the involvement of Toll-like receptor (TLR)-2 on pre-activated T cells in pathogen-induced chronic inflammatory joint diseases. Any inflammation will cause the induction of chemokine and cytokine production in several tissue-associated cells in the joint, including fibroblasts, macrophages and dendritic cells. Activated T cells and T effector cells of any specificity (also auto-specificities) can respond to these signals, migrate to the joint, breach endothelial barriers, infiltrate the inflamed foci and sustain inflammatory processes by secreting cytokines in response to direct co-stimulation via TLR-2, without the necessity of engagement of the T cell receptor.
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