The pathogenesis of arthritis in Lyme disease: humoral immune responses and the role of intra-articular immune complexes

We studied 78 patients with Lyme disease to determine how immune complexes and autoantibodies are related to the development of chronic Lyme arthritis. Circulating C1q binding material was found in nearly all patients at onset of erythema chronicum migrans, the skin lesion that marks the onset of infection with the causative spirochete. In patients with only subsequent arthritis this material tended to localize to joints where it gradually increased in concentrations with greater duration of joint inflammation. In joints, its concentration correlated positively with the number of synovial fluid polymorphonuclear leukocytes. Despite the prolonged presence of putative immune complexes, rheumatoid factors could not be demonstrated. These observations suggest that phlogistic immune complexes based on spirochete antigens form locally within joints during chronic Lyme arthritis.     

Autoantibodies from synovial lesions in chronic, antibiotic treatment-resistant Lyme arthritis bind cytokeratin-10

Although the causative agent of Lyme disease is definitively known to be the tick-borne spirochete, Borrelia burgdorferi, the etiology of chronic joint inflammation that ensues in a subset of patients remains less well understood. Persistence of arthritis after apparent eradication of the spirochete suggests an autoimmune reaction downstream of the original bacterial infection. We have generated recombinant Ab probes from synovial lesions within affected arthritic joints in an attempt to recapitulate disease-relevant Ag-binding specificities at the site of injury. Using this panel of intra-articular probes, as well as Ab fragments derived from patient peripheral blood, we have identified cytokeratin 10, present in synovial microvascular endothelium, as a target ligand and a putative autoantigen in chronic, antibiotic treatment-resistant Lyme arthritis. Furthermore, there is cross-reactivity between cytokeratin 10 and a prominent B. burgdorferi Ag, outer surface protein A. Release of the self protein in the context of inflammation-induced tissue injury and the resulting in situ response to it could set in motion a feed-forward loop, which amplifies the inflammatory process, thereby rendering it chronic and self-perpetuating, even in the absence of the inciting pathogen.     

CD28 deficiency exacerbates joint inflammation upon Borrelia burgdorferi infection, resulting in the development of chronic Lyme arthritis

Lyme disease, caused by the tick-borne spirochete Borrelia burgdorferi (Bb), is a multisystem illness, affecting many organs, such as the heart, the nervous system, and the joints. Months after Bb infection, approximately 60% of patients experience intermittent arthritic attacks, a condition that in some individuals progresses to chronic joint inflammation. Although mice develop acute arthritis in response to Bb infection, the joint inflammation clears after 2 wk, despite continuous infection, only very rarely presenting with chronic Lyme arthritis. Thus, the lack of an animal system has so far prevented the elucidation of this persistent inflammatory process that occurs in humans. In this study, we report that the majority of Bb-infected CD28-/- mice develop chronic Lyme arthritis. Consistent with observations in chronic Lyme arthritis patients, the infected mutant, but not wild-type mice present recurring monoarticular arthritis over an extended time period, as well as anti-outer surface protein A of Bb serum titers. Furthermore, we demonstrate that anti-outer surface protein A Abs develop in these mice only after establishment of chronic Lyme arthritis. Thus, the Bb-infected CD28-/- mice provide a murine model for studying chronic Lyme arthritis.     

Decline in the frequencies of Borrelia burgdorferi OspA161 175-specific T cells after antibiotic therapy in HLA-DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory lyme arthritis

Synovitis in patients with antibiotic-refractory Lyme arthritis persists for months to several years after antibiotic therapy. This course, which may result from infection-induced autoimmunity, is associated with T cell recognition of Borrelia burgdorferi outer surface protein A (OspA(161-175)) and with HLA-DR molecules that bind this epitope, including the DRB1*0401 molecule. In this study, we used tetramer reagents to determine the frequencies of OspA(161-175)-specific T cells in samples of PBMC and synovial fluid mononuclear cells (SFMC) from 13 DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory arthritis. Initially, three of the six patients (50%) with antibiotic-responsive arthritis and four of the seven patients (57%) with antibiotic-refractory arthritis had frequencies of OspA(161-175)-specific CD4(+) T cells in peripheral blood above the cutoff value of 4 per 10(5) cells. Among the five patients with concomitant PBMC and SFMC, four (80%) had OspA tetramer-positive cells at both sites, but the mean frequency of such cells was 16 times higher in SFMC, reaching levels as high as 1,177 per 10(5) cells. In the two patients in each patient group in whom serial samples were available, the frequencies of OspA(161-175)-specific T cells declined to low or undetectable levels during or soon after antibiotic therapy, months before the resolution of synovitis in the two patients with antibiotic-refractory arthritis. Thus, the majority of patients with Lyme arthritis initially have increased frequencies of OspA(161-175)-specific T cells. However, the marked decline in the frequency of such cells with antibiotic therapy suggests that persistent synovitis in the refractory group is not perpetuated by these cells.     

Treponematosis and Lyme borreliosis connections: Explanation for Tchefuncte disease syndromes?

A convergence of evidence from macroscopic, radiographic and histologic examination indicates that treponemal infection was present in the 16ST1 Tchefuncte Indian burial population, dated 500 B.C. to 300 A.D. Pattern and nature of lesions suggests that chronic infection induced by variants of the spirochete Treponema pallidum, causing endemic syphilis and/or yaws, resulted in third-stage osseous response. It is suggested that Tchefuncte Indians acquired partial immunity to treponemal infection by exposure to a variant of the related spirochete, Borrelia burgdorferi, the causative agent of Lyme disease. Partial immunity would help explain the relatively mild expression of the treponemal disease process in the 16ST1 skeletal population and the apparent absence of venereal syphilis. Presence of the Borrelia burgdorferi spirochete might be linked to a single incidence of juvenile rheumatoid arthritis. © 1994 Wiley-Liss, Inc.     

The clinical spectrum and treatment of Lyme disease

Lyme disease was recognized as a separate entity because of close geographic clustering of affected children in Lyme, Connecticut, with what was thought to be juvenile rheumatoid arthritis. It then became apparent that Lyme disease is a complex, multisystem disorder. The illness usually begins in summer with erythema chronicum migrans and associated symptoms (stage 1). Weeks to months later, some patients develop neurologic or cardiac abnormalities (stage 2), and weeks to years later, many patients develop intermittent attacks of arthritis (stage 3), which may become chronic, with erosion of cartilage and bone. Patients with severe and prolonged illness have an increased frequency of the B-cell alloantigen, DR2. For patients with early Lyme disease, tetracycline appears to be the most effective drug, then penicillin, and finally erythromycin. High-dose intravenous penicillin is effective for the later stages of the disease.     

In situ diversification of the antibody repertoire in chronic Lyme arthritis synovium

Lyme arthritis is initiated by the tick-borne spirochete, Borrelia burgdorferi. In a subset of patients, symptoms do not resolve in response to standard courses of antibiotics. Chronic joint inflammation may persist despite spirochetal killing, suggesting an autoimmune etiology. The pathogenic mechanisms that sustain chronic Lyme arthritis have not been fully elucidated, although T cells are believed to play a role. The synovial lesion contains elements of a peripheral lymph node, with lymphoid aggregates, plasma cells and follicular dendritic cells. An analysis of activated cells at the site of injury could yield clues regarding the nature of the response and the identity of potential autoantigens. Using laser-capture microdissection, we have isolated plasma cells from the joint tissue of chronic Lyme arthritis patients who underwent synovectomy. Expressed Ig V regions were amplified by RT-PCR. A majority of isolated cells expressed gamma H chains, which is indicative of a class-switched response. There were a large number of nucleotide substitutions from germline, with a higher fraction of replacement mutations in the CDRs, suggesting a process of Ag-driven selection. We have recovered clonal clusters of cells containing identical junctions and V(D)J rearrangements. Sequence analysis reveals a hierarchy of shared somatic mutations between members of a given clone. Intraclonal diversity among plasma cells of close physical proximity points toward an ongoing process of diversification and affinity maturation, possibly driven by the chronic presence of an autoantigen.     

The antibody response in Lyme disease.

We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis.     

Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin

Pathological processes involved in the initiation of rheumatoid synovitis remain unclear. We undertook the present study to identify immune and stromal processes that are present soon after the clinical onset of rheumatoid arthritis (RA) by assessing a panel of T cell, macrophage, and stromal cell related cytokines and chemokines in the synovial fluid of patients with early synovitis. Synovial fluid was aspirated from inflamed joints of patients with inflammatory arthritis of duration 3 months or less, whose outcomes were subsequently determined by follow up. For comparison, synovial fluid was aspirated from patients with acute crystal arthritis, established RA and osteoarthritis. Rheumatoid factor activity was blocked in the synovial fluid samples, and a panel of 23 cytokines and chemokines measured using a multiplex based system. Patients with early inflammatory arthritis who subsequently developed RA had a distinct but transient synovial fluid cytokine profile. The levels of a range of T cell, macrophage and stromal cell related cytokines (e.g. IL-2, IL-4, IL-13, IL-17, IL-15, basic fibroblast growth factor and epidermal growth factor) were significantly elevated in these patients within 3 months after symptom onset, as compared with early arthritis patients who did not develop RA. In addition, this profile was no longer present in established RA. In contrast, patients with non-rheumatoid persistent synovitis exhibited elevated levels of interferon-γ at initiation. Early synovitis destined to develop into RA is thus characterized by a distinct and transient synovial fluid cytokine profile. The cytokines present in the early rheumatoid lesion suggest that this response is likely to influence the microenvironment required for persistent RA.     

Pathogenesis of rheumatoid arthritis: how early is early?

Studies of cytokine expression in rheumatoid arthritis have provided key insights into the pathogenesis of disease and have offered clues for effective therapy. Patterns of T-cell products in chronic rheumatoid synovitis suggest that T helper type 1 cells contribute to the perpetuation of disease. However, there is no guarantee that the mechanisms of late disease are identical to very early rheumatoid arthritis. Evaluation of the cytokine profile at the earliest time points after onset of symptoms could identify novel targets that prevent progression to chronic arthritis.     

Two-dimensional cryostat section electrophoresis: a novel method and its application to the diseased synovial membrane

Summary A two-dimensional electrophoresis technique for analysing sections of human tissue is described. Cryostat sections, 10 μm thick, are placed on an isoelectric focusing gel and then transfered to an SDS gel in the second dimension. The protein pattern is visualized by silver staining and is thought to represent soluble proteins. The silver-stained proteins were found to be both reproducible and, to the extent tested, organ-specific. This method was used to analyse 43 synovial membranes from patients suffering from rheumatoid arthritis or degenerative joint diseases. The analysis did not reveal any specific protein pattern for rheumatoid arthritis. The protein spot number was not related to the cause of arthritis. However, the total protein spot number was related to the histomorphological synovitis type, with those exhibiting either an exudative or proliferative synovitis pattern possessing significantly higher protein spot numbers than those specimens exhibiting a sero-fibrous or lympho-plasmacytic pattern of synovitis.     

Treatment of persistent knee effusions with intra-articular radioactive gold.

Patients with a total of 112 chronic knee effusions unresponsive to the usual conservative methods of therapy were treated with intra-articular injections of radioactive gold and followed from 6 months to 5 years. Most patients in the study had rheumatoid arthritis but others had ankylosing spondylitis, psoriatic arthritis, intermittent hydrathrosis and undiagnosed synovitis. After 6 months 81% showed improvement. This figure diminished to approximately 70% at 1 and 2 years, 60% at 3 and 4 years and 50% at 5 years. The mose beneficial results were seen in patients with intermittent hydrarthrosis. Twelve patients with a synovial cyst and/or rupture improved. Patients with thin synovia and anatomic stage I or II disease did best, but those with stage III disease also did well. After the injection 33% of the patients had a reactive synovitis. A variable amount of radioactive material escaped from the knee to the regional lymph nodes and general circulation.     

Requirement of IL-17 receptor signaling in radiation-resistant cells in the joint for full progression of destructive synovitis

IL-17 is a proinflammatory cytokine suspected to be involved in inflammatory and autoimmune diseases such as rheumatoid arthritis. In the present study, we report that IL-17R signaling is required in radiation-resistant cells in the joint for full progression of chronic synovitis and bone erosion. Repeated injections of Gram-positive bacterial cell wall fragments (streptococcal cell wall) directly into the knee joint of naive IL-17R-deficient (IL-17R-/-) mice had no effect on the acute phase of arthritis but prevented progression to chronic destructive synovitis as was noted in wild-type (wt) mice. Microarray analysis revealed significant down-regulation of leukocyte-specific chemokines, selectins, cytokines, and collagenase-3 in the synovium of IL-17R-/- mice. Bone marrow (BM) chimeric mice revealed the need for IL-17R expression on radiation-resistant joint cells for destructive inflammation. Chimeric mice of host wt and donor IL-17R-/- BM cells developed destructive synovitis in this chronic reactivated streptococcal cell wall arthritis model similar to wt-->wt chimeras. In contrast, chimeric mice of host IL-17R-/- and donor wt BM cells were protected from chronic destructive arthritis similar as IL-17R-/- -->IL-17R-/- chimeras. These data strongly indicate that IL-17R signaling in radiation-resistant cells in the joint is required for turning an acute macrophage-mediated inflammation into a chronic destructive synovitis.     

Enhanced levels of leukotriene B(4) in synovial fluid in Lyme disease

The purpose of this study was to evaluate the potential role of LTB(4) and cysteinyl leukotrienes in Lyme disease (LD). Therefore, a total number of 34 patients divided into four groups was studied. The patients were classified as having Lyme arthritis (n = 7) or Lyme meningitis (n = 10), and as control groups patients with a noninflammatory arthropathy (NIA) (n = 7) and healthy subjects (n = 10). LTB(4) as well as LTC(4) secretion from stimulated polymorphonuclear leukocytes (PMNL) from all groups of patients showed no statistical differences. LTB(4) levels in synovial fluid were significantly increased in patients with Lyme arthritis (median 142 ng/ml, range 88-296) when compared to the control subjects with NIA (median 46 ng/ml, range 28-72) (p < 0.05). No statistical difference of urinary LTE(4) levels between all the different groups of patients was observed. These results show that cysteinyl leukotrienes do not play an important role in the pathogenesis of LD. In contrast to previous findings in rheumatoid arthritis, LTB(4) production from stimulated PMNL was not found to be increased in LD. However, the significantly elevated levels of LTB(4) in synovial fluid of patients with Lyme arthritis underline the involvement of LTB(4) in the pathogenesis of this disease.     

Positive IgG Western blot for Borrelia burgdorferi in Colombia.

In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF) sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC) for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma), the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.     

Circulating CD4+CD161+ T Lymphocytes Are Increased in Seropositive Arthralgia Patients but Decreased in Patients with Newly Diagnosed Rheumatoid Arthritis

Improved understanding of the immune events discriminating between seropositive arthralgia and clinical synovitis is of key importance in rheumatology research. Ample evidence suggests a role for Th17 cells in rheumatoid arthritis. We hypothesized that CD4+CD161+ cells representing Th17 lineage cells may be modulated prior to or after development of clinical synovitis. Therefore, in a cross-sectional study, we investigated the occurrence of CD4+CD161+ T-cells in seropositive arthralgia patients who are at risk for developing rheumatoid arthritis and in newly diagnosed rheumatoid arthritis patients. In a prospective study, we evaluated the effect of methotrexate treatment on circulating CD4+CD161+ T-cells. Next, we assessed if these cells can be detected at the level of the RA joints. Precursor Th17 lineage cells bearing CD161 were found to be increased in seropositive arthralgia patients. In contrast, circulating CD4+CD161+T-cells were decreased in newly diagnosed rheumatoid arthritis patients. The decrease in CD4+CD161+ T-cells correlated inversely with C-reactive protein and with the 66 swollen joint count. Methotrexate treatment led to normalization of CD4+CD161+ T-cells and reduced disease activity. CD4+CD161+ T cells were readily detected in synovial tissues from both early and late-stage rheumatoid arthritis. In addition, synovial fluid from late-stage disease was found to be enriched for CD4+CD161+ T-cells. Notably, synovial fluid accumulated CD4+CD161+T-cells showed skewing towards the Th1 phenotype as evidenced by increased interferon-γ expression. The changes in peripheral numbers of CD4+CD161+ T-cells in seropositive arthralgia and early rheumatoid arthritis and the enrichment of these cells at the level of the joint predict a role for CD4+CD161+ T-cells in the early immune events leading to clinical synovitis. Our findings may add to the development of RA prediction models and provide opportunities for early intervention.     

Cell-based immunotherapy with suppressor CD8+ T cells in rheumatoid arthritis

The chronic persistence of rheumatoid synovitis, an inflammation driven by activated T cells, macrophages, and fibroblasts causing irreversible joint damage, suggests a failure in physiologic mechanisms that down-regulate and terminate chronic immune responses. In vitro CD8(+)CD28(-)CD56(+) T cells tolerize APCs, prevent the priming of naive CD4(+) T cells, and suppress memory CD4(+) T cell responses. Therefore, we generated CD8(+)CD28(-)CD56(+) T cell clones from synovial tissues, expanded them in vitro, and adoptively transferred them into NOD-SCID mice engrafted with synovial tissues from patients with rheumatoid arthritis. Adoptively transferred CD8(+)CD28(-)CD56(+) T cells displayed strong anti-inflammatory activity. They inhibited production of IFN-gamma, TNF-alpha, and chemokines in autologous and HLA class I-matched heterologous synovitis. Down-regulation of costimulatory ligands CD80 and CD86 on synovial fibroblasts was identified as one mechanism of immunosuppression. We propose that rheumatoid synovitis can be suppressed by cell-based immunotherapy with immunoregulatory CD8(+) T cells.     

Anti-Sa antibodies: prognostic and pathogenetic significance to rheumatoid arthritis

Anti-Sa antibodies are detected in the serum of 20–47% of patients with rheumatoid arthritis. These antibodies have a high degree of specificity for the disease, and appear to identify a subset of early rheumatoid arthritis patients destined to have aggressive and destructive disease. It has recently been confirmed that anti-Sa antibodies are directed to citrullinated vimentin, thus placing them in the anti-citrulline family of autoantibodies. The Sa antigen has previously been shown to be present in synovium. This, along with the demonstration of citrullinated proteins in rheumatoid synovium, suggests that anti-Sa antibodies may play a pathogenetic role in the initiation and/or persistence of rheumatoid synovitis.