Cytotoxic Immune Responses May Explain Microvasculature Damage in Post-antibiotic Lyme Arthritis

Patients with Lyme arthritis (LA) who don't respond to oral antibiotics generally receive IV antibiotic therapy, which improves joint swelling considerably. However, some patients develop massive proliferative synovitis that can last months to several years, a condition called post-antibiotic LA.

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The basic pathogenetic feature is an excessive, dysregulated inflammatory immune response in joints during Borrelia burgdorferi infection that persists in the post-infectious period. Several animal and human studies have suggested cytotoxic lymphocytes may play a role in post-antibiotic LA. One example is that obliterative microvascular lesions are present in about half of LA synovia, and their extent correlates directly with autoantibodies to certain vascular-associated antigens.

Allen C. Steere, MD, principal investigator of the Steere Laboratory in the Center for Immunology and Inflammatory Diseases at Massachusetts General Hospital and director of translational research in the Rheumatology Unit, and colleagues have identified lymphocytes with cytotoxic potential that may mediate this feature of synovial pathology. Their report appears in Arthritis & Rheumatology.

Frequencies of Potentially Cytotoxic Cells

The researchers first analyzed paired samples of peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from:

• 15 patients with antibiotic-responsive LA (samples obtained from swollen knees before or soon after the start of oral antibiotic therapy)
• 22 patients with post-antibiotic LA (samples collected close to the time of IV antibiotic therapy when knees were still moderately swollen but few, if any, live spirochetes remained)
• 7 patients with rheumatoid arthritis (RA)
• 15 healthy control subjects (PBMC only)

When the team measured lymphocyte types that are capable of cell-mediated cytotoxicity, the percentages were:

• CD3+CD8+ T cells—Significantly greater in post-antibiotic LA than antibiotic-responsive LA
• CD3+CD4−CD8− double-negative T cells, mostly γδ cells—Significantly greater in post-antibiotic LA than antibiotic-responsive LA
• Natural killer (NK) cells—Significantly lower in all patient groups than in healthy controls

Values were similar in RA and LA patients, but in RA compared with post-antibiotic LA, percentages of CD8+ cells tended to be higher, and those of double-negative cells tended to be lower.

By contrast, percentages of CD4+ cells were significantly greater in antibiotic-responsive LA, probably because these cells help B cells produce antibodies important for controlling B. burgdorferi infection.

Quantifying Cytotoxic Potential

The researchers then measured levels of three cytotoxicity markers (granzyme A, granzyme B, and perforin) and the inflammatory cytokine interferon-γ in the cells of interest in the three patient groups. To assess secretion, they also analyzed protein levels of the three cytotoxicity mediators in synovial fluid and serum.

CD8+ cells, γδ cells, and NK cells showed evidence of cytotoxic or inflammatory potential. The three mediators necessary for cytotoxic responses were found in synovial fluid, suggesting they were probably secreted by cells within the joint.

T cell Receptor Vβ Gene Usage

To identify joint-specific T cell repertoires involved in microvascular damage, the researchers applied Vβ repertoire profiling to T cell subsets from five patients with antibiotic-responsive LA and five with post-antibiotic LA.

They found the same three Vβ gene products were enriched in both CD4+ and CD8+ cell populations. This suggests CD4+ cells facilitate cross-presentation of vascular autoantigens to CD8+ cells, leading to the expansion of cytotoxic lymphocyte populations.

Later-stage Findings

To assess cytotoxic potential late in the course of post-antibiotic LA, the team analyzed synovial tissue from three teenagers who required therapeutic synovectomy 9 to 14 months after nonresponse to IV antibiotics:

• Patient 1 had marked vascular proliferation but all vessels were open
• Patient 2 had some open vessels but many were partially or completely obliterated, complement (C5b–9) deposition was apparent on some partially blocked vessels, and the activating receptor NKG2D was prominently expressed around vessels; this patient had autoantibodies to annexin A2 and apolipoprotein B100
• Both patients 1 and 2 had CD8+ cells prominent in angiocentric locations and tightly intermixed with CD4+ cells around capillaries, although CD4+ cells were not in direct contact with endothelial cells
• In patient 3, the synovial vasculature was almost entirely obliterated, and the cytotoxic process appeared inactive, with only a few CD8+ and CD4+ cells detected; this patient had autoantibodies to endothelial cell growth factor as well as the other two vascular antigens

An issue for future research is whether these autoantibodies cause vascular pathology directly or whether their action is mediated through the engagement of NKG2D receptors on NK cells or CD8+ T cells.

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