Findings in classified rheumatoid arthritis

ACPA and RF are commonly found in RA and indeed are components of the definition of seropositive disease. As a practical matter, ACPA are typically measured using commercial enzyme-linked immunosorbent assay-based tests that are certified and approved for clinical use, and when assessed using these methods are designated anti-cyclic citrullinated peptide (anti-CCP) antibodies. Anti-CCP antibodies have been described in subjects with classified RA as demonstrating a sensitivity of 60% to 70% with a specificity of approximately 98%, improving substantially over the approximate 80% to 85% specificity of RFs. However, the specificity of ACPAs differs by the specific anti-CCP commercial test utilized, the control population against which the test is compared, and the stage of disease being evaluated. In addition to enzyme-linked immunosorbent assays, there are an increasing number of peptide- and protein-based array methods that can be used to refine the epitope specificity of ACPAs, which demonstrate a very wide variety of non–cross-reactive citrullinated peptide specificities in patients with classified RA.

In addition to ACPA and RFs, several other autoantigens are recognized by smaller subgroups of patients with classified RA. These autoantibodies have been reported to recognize carbamylated proteins antigens (anti-CarP) wherein arginine is converted to homocitrulline, mutated citrullinated vimentin, RA-33, and peptidyl arginine deiminase type 4, among others.

Findings in at-risk populations

Using stored serum banks to study the evolution of autoantibodies in the preclinical period, both increase in titers and epitope spreading have been found to occur that encompasses a wider variety of targets as the clinical onset nears. With regard to assessment of subjects in real time, a subset of FDR elaborate RA-related autoantibodies, analyzed as anti-CCP2, anti-CCP3.1, RF by nephelometry, and RF by isotype positivity. When analyzed, a largely non-Hispanic white FDR population was found to exhibit an approximately 16% positivity inclusive of this range of autoantibodies [(23), and updated], and the North American Native population was reported to exhibit anti-CCP antibodies in 17% of FDR, 11% of more distant relatives, and 3% in controls. In FDR within multicase families in Sweden, concentrations and frequencies of anti-CCP and RF isotypes were also significantly increased. Of note, in that population the distribution of IgA and IgM isotypes was higher than IgG in the relatives, whereas the IgG isotype dominated in probands with RA.

FDRs have also been studied to determine whether there are similar increases in epitope spreading associated with an anti-CCP–positive test. One study showed that anti-CCP–positive FDRs elaborate a substantially increased number of ACPA than control FDRs without RA-related autoantibodies, with a subset being positive for 9 or more ACPA in a pattern typical for classified RA. An increasing number of positive ACPAs was also associated with the presence of a tender joint on examination. In another independent study of an FDR population using a 6-member ACPA panel, a high prevalence of ACPAs (48%) and was found as compared with controls (10%), with citrullinated vimentin most commonly targeted. Of note, a high proportion of ACPAs in that study were of the IgA isotype.

Thus, RA-free FDRs show reactivity to multiple ACPA, demonstrating that both an anti-CCP positivity in this population is not likely to be a “false positive,” but rather is similar in nature to subjects with classified RA, and that there is evidence of genetic and/or familial effects within the FDR population. What is as yet unknown is whether other anti-CCP characteristics found in patients with classified RA, such as increased avidity, substantial complement activating potential, and altered G0 carbohydrates that promote IgG effector functions, are also present in samples from these real-time at-risk populations, although these questions are under study. Additionally, because only 3% to 5% of FDRs will ultimately develop RA, the high proportion of immune abnormalities in FDRs must either not be a stable phenotype, reflect early immune dysregulation that progresses to other autoimmune diseases in this population, or that the individuals elaborating the immune dysregulation do not all progress through additional stages of disease. Although limited in scope, evidence for both conclusions is present. Finally, reflecting clinical variability in early arthritis, in subjects enrolled in the Canadian Early Arthritis Cohort who underwent baseline and at least the 12-month follow-up studies, both RF and ACPA were found to fluctuate, with approximately 10% of subjects converting from ACPA negative to ACPA positive, and vice versa.

Findings in classified rheumatoid arthritis

CD4 T cells specific for citrullinated synovial antigens have been identified and quantitated through the use of major histocompatibility class II tetramers, whereas samples from patients with classified RA display an increased frequency of citrullinated peptide (cit)-specific T cells, predominantly of the memory T helper type 1 cell class. Notably, the frequency of cit-specific T cells in RA patients is highest within the first 5 years of disease, and is decreased among patients on biologic therapies independent of disease duration. Autoreactive T cells in patients with RA recognize a combination of citrullinated epitopes carried on class II tetramers, and demonstrate evidence of cellular immunity to citrullinated autoantigens using other techniques. For example, secretion of interleukin (IL)-6 and IL-17 and tumor necrosis factor (TNF)- α by CD4 ⁺ cells was found to be present in normal controls and patients with RA, whereas interferon and IL-10 were specific for RA, with citrullinated aggrecan generating the most robust antigen-specific response.

With regard to B cells, subjects with RA exhibit an increased number of peripheral blood plasmablasts and/or plasma cells, a substantial proportion of which produce ACPAs. Plasma cells producing ACPAs are also commonly found in the synovium. B cells in patients with active RA also exhibit features of dysregulated control of anergy checkpoints during development, as well as signaling alterations in the anergic population.

In addition to antigen-specific lymphocytes, innate lymphoid cells, a novel family of effector cells that are reported to play important roles in the maintenance of lymphoid tissue, tissue repair, and response to infection, are also altered in patients with RA. In this regard, there are several distinct subsets of innate lymphoid cells that secrete a variety of cytokines that are important in RA pathogenesis (IL-5, IL-13, IL-17, IL-22, TNF-α and interferon-γ).

Findings in at-risk populations

Although evidence of dysregulation of autoimmune B and T cells, as well as innate lymphoid cells, is anticipated to be present in the at-risk population, the studies necessary to assess this question are only just underway. In one sense, because antigen-specific T cells are more likely to be found earlier rather than later in disease, systemic high titers of anti-CCP antibodies can also be present in FDRs, and local production of anti-CCP antibodies has been described in the at-risk populations, it is very likely that antigen-specific lymphocytes will be present and detectable. However, major questions include the epitope specificities of this early response, whether there a linked B- and T-cell response that is present, and if there are peptide specificities that can be targeted using antigen-specific immune tolerization approaches.

Findings in classified rheumatoid arthritis

Patients with classified RA exhibit a wide variety of elevated cytokines, with no single cytokine or pattern predominating (eg, see Hueber and colleagues ). There are patterns, however, that are associated with a highly inflammatory state, and IL-1, TNF-α, IL-12p40, and IL-13 have been found to be particularly elevated in early RA.

Findings in at-risk populations

Using retrospective serum studies, it has been found that, shortly before the onset of clinically apparent arthritis, systemic levels of cytokines and chemokines increase in a heterogenous manner, both with regard to the number being elevated in an individual subject and the levels of each factor. With regard to at-risk populations, elevated cytokines are associated with the presence of anti-CCP2 antibody, as well as expanded in FDRs regardless of autoantibody status. Thus, there is substantial evidence that immune dysregulation in at-risk populations is associated with systemic evidence of inflammation. Prospective analyses of this question in at-risk populations, however, have not been reported, and studies of other inflammation-related biomarkers including complement activation fragments are also now underway.

Findings in classified rheumatoid arthritis

There are many genes that associate with RA, including high-risk HLA-DR alleles containing the SE and PTPN22 , as well as more than 100 other linked genes with a modestly elevated relative risk. Notably, a substantial number of these genes encode components of immune-related signaling pathways (reviewed in Bax and co-workers ) and/or also encompass genes expressed in memory effector T cells and associated with CD40, TRAF1, TNFAIP3, and PRKCQ pathways.

Differences in genetic relationships to disease are also present that are based on environmental exposures, such as a relationship between smoking, SE, and the presence of ACPA, and the link between SE and the presence of antibodies to citrullinated enolase.

Findings in at-risk populations

Despite the increasingly understood relationships between genetic risk and the presence of classified RA, little has been accomplished yet regarding the question of at what point during disease evolution these factors act. With regard to the major risks associated with the SE, in 1 study there was a trend between the presence of ACPA and the SE, but the size of the study apparently limited the ability to definitively answer this question. Thus, this is another question that should be addressed and is indeed under active analysis.

Findings in classified rheumatoid arthritis

In addition to the hardwired genetic contributions, there are genomic and epigenetic changes that are associated with RA and affect lymphocytes, fibroblastlike synoviocytes (FLS) and other cell populations. Epigenetic modifications include DNA methylation, histone methylation, histone acetylation, histone phosphorylation, and expression of microRNAs, and changes are especially prominent in FLS, where they are chronically exposed to an environment rich in proinflammatory cytokines and mediators of oxidative stress. These epigenetic changes represent key means by which environmental factors could influence gene expression and disease heterogeneity, and potentially heritability given that some epigenetic changes can be transmitted to offspring. One important finding is that epigenetic changes in FLS seem to be “imprinted” and durable even after removal from the proinflammatory environment.

Findings in at-risk populations

It is not yet known whether the same epigenetic findings in classified RA are also found in the preclinical disease state, or whether other stage-specific changes are found. One particularly important question is whether FLS exhibit changes that promote the transition from the presence of circulating autoimmunity to the development of local synovitis.

Findings in classified rheumatoid arthritis

Many types of imaging approaches have been applied to the study of patients with classified RA. Perhaps most relevant to the study of preclinical RA and at-risk populations, however, where the question of the presence or absence of changes consistent with inflammatory arthritis is most pressing, are ultrasound (US) and magnetic resonance imaging (reviewed by Tan and colleagues ). Using these techniques, one can detect by US synovitis by identifying within the images effusions, synovial expansion and a power Doppler signal. In addition, through US one can detect bony erosions and measure cartilage thickness. US is useful for tendon assessments and defining tenosynovitis and tendon rupture. Magnetic resonance imaging can also define the same structures, but in addition can visualize bone marrow pathologies not detectable by US, such as bone marrow edema.

Findings in at-risk populations

Studies in at-risk populations have focused on RA-related autoantibody-positive individuals as well as subjects who present to health care settings with arthralgia or early or unclassified inflammatory arthritis. Studies of anti-CCP positive arthralgia patients without clinically detectable arthritis have demonstrated histologic findings without inflammation but with a trend toward CD3 and CD8 T-cell infiltration. Studies of FDR using magnetic resonance imaging of the hands and feet have not demonstrated evidence of synovial inflammation, and analyses of arthralgia/IA populations demonstrate that a subset exhibit US abnormalities. Thus, the situation remains uncertain, but is an important area for prospective investigation, as well as for the development of additional functional imaging approaches.

Findings in classified rheumatoid arthritis

With regard to environmental influences on RA susceptibility, there are several well-accepted exposures that increase or decrease the risk of developing RA, including smoke exposure, hormonal factors, dietary exposures, air pollutants, and silica dust ; however, the exact causal relationships are not understood beyond a potential relationship between smoke exposure and citrullination. In this regard, autoreactivity to citrullinated α-enolase has demonstrated a linkage to both smoking and DR4 SE status. Dietary factors have also been assessed, and particularly compelling findings have included an inverse relationship between omega-3 fatty acid intake and both the risk of developing and severity of RA (reviewed by James and colleagues ).

Findings in at-risk populations

Although limited in scope, studies have demonstrated an inverse relationship between birth control exposure and a direct relationship to smoking with the presence of RF in an FDR at-risk population. In the same population, no association with vitamin D levels was found. In addition, the relationship between air pollution exposure and RA-related autoantibodies in the FDR population has been studied, and demonstrated no relationship. Ongoing studies are evaluating the relationships between omega-3 fatty acid intake and presence of RA-related autoantibodies.

Findings in classified rheumatoid arthritis

In addition to arthritis, other common and clinically significant manifestations of RA include an elevated risk for cardiovascular disease, although the exact time at which this elevated risk is manifest remains uncertain.

Findings in at-risk populations

Reported studies of this important question are limited in nature, and many are ongoing in a number of at-risk populations. Importantly, identifying a risk of cardiovascular disease that precedes clinically apparent articular RA may drive interventions not only to prevent future RA, but cardiovascular disease as well.

Findings in classified rheumatoid arthritis

Loss of bone as manifest by osteopenia and local erosions commonly occurs along with the most severe forms of RA, likely through both local and systemic inflammatory and remodeling processes that alter relative rates of bone formation and degradation by osteoclasts and osteoblasts. ACPA directed to citrullinated vimentin may be particularly important in inducing bone loss in patients with RA.

Findings in at-risk populations

Studies in at-risk populations are limited; however, ACPA-positive individuals without detectable synovitis have been found to demonstrate diminished bone by the use of micro-computed tomography analyses. Thus, it seems that in addition to evidence of systemic inflammation by cytokine elevations, local bone loss may also characterize the RA-related autoantibody at-risk status.

Findings in classified rheumatoid arthritis

Many links between RA and the lung have been made. For instance, cigarette smoke exposure is highly associated with risk for the development of RA, especially in the presence of HLA-expressing the DR4 SE. In addition, through the use of sensitive high-resolution computed tomography, patients with early RA demonstrate a substantial number of pulmonary abnormalities.

Findings in at-risk populations

Notably, at-risk individuals who do not have RA but exhibit high-risk RA-related autoantibodies demonstrate a high rate of inflammatory airway disease that is not associated with a smoking history or other similar exposures. Local production of ACPA and other RA-related autoantibodies has been demonstrated through studies of induced sputa from FDR, where RF and anti-CCP antibodies can be readily demonstrated in the sputum but not in the peripheral blood of a subset of this population. Because of these findings, the lung is now considered to be a primary site of the initiation of RA, although other mucosal sites may also be candidates for initiation of RA.

Findings in classified rheumatoid arthritis

Several lines of investigation suggest that dysbiosis occurs in patients with RA, which may be associated with risk, severity, or targets of injury. For example, an elevated occurrence of severe periodontitis is reproducibly found in patients with RA, and conversely patients with RA who exhibit severe periodontitis have higher disease activity scores. Patients with severe periodontitis also manifest higher titers of IgG and IgM antibodies to Porphyromonas gingivalis , which is important because this organism exhibits enzymatic activity that can citrullinate fibrinogen and enolase in the presence of bacterial gingipains. In addition, though, other studies have found that Prevotella and Leptotrichia , but not P gingivalis , are present in higher numbers in the subgingival microbiota in patients with recent onset untreated RA.

Finally, studies of the gut, a mucosal site where microbes are well known to be able to influence immune responses and where experimental arthritis has been modulated by the exposure in the gut to a single organism, have suggested that change in the microbiota are associated with concurrent RA, especially an expansion of Prevotella copri species.

Findings in at-risk populations

There is strong potential for the mucosa and/or mucosal dysbiosis to be an early driver of RA development. The rationale is based on recent studies strongly suggesting that mucosal inflammation, production of RA-related autoantibodies, and dysbiosis can occur coincidently. In addition, in at-risk subjects, there is a specific elevation of antibodies to P gingivalis but not to related strains. Therefore, substantial efforts are currently being made to further address these relationships in at-risk populations.

Findings in classified rheumatoid arthritis

Measures of environmental and genetic influences include metabolic fingerprints that can be assessed through nuclear magnetic resonance spectroscopy-based metabolomics techniques.

With regard to patients with classified RA, the serum metabolic fingerprint was found to be clearly different than controls, with lactate and lipids as discriminators of the inflammatory burden in early RA; differences were also related to the level of C-reactive protein.

Findings in at-risk populations

As a means by which one can assess a sum of many different endogenous and exogenous influences, metabolomics studies hold promise for helping to understand the biologic underpinnings of RA initiation and early evolution. Because of this, there are ongoing studies to evaluate this question.