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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Arthritis Rheum. Author manuscript; available in PMC Jun 1, 2012.
Published in final edited form as:
PMCID: PMC3106129
NIHMSID: NIHMS275807

A Signature of Aberrant Immune Responsiveness Identifies Myocardial Dysfunction in Rheumatoid Arthritis

Abstract

Objective

Heart failure is an important cause of mortality in patients with rheumatoid arthritis (RA). Evidence suggests that immune mechanisms contribute to myocardial injury and fibrosis, leading to left ventricular diastolic dysfunction (LVDD). In this study, we sought to identify a signature of LVDD in patients with RA by analyzing the responsiveness of the innate and adaptive immune systems to stimulation ex vivo.

Methods

Subjects (n=212) enrolled prospectively from a population-based cohort underwent echocardiography, and left ventricular function was classified as normal, mild LVDD, or moderate-to-severe LVDD. The release of 17 cytokines by blood mononuclear cells in response to stimulation with a panel of 7 stimuli or in media alone was analyzed using multiplexed immunoassays. Logistic regression models were used to test for associations between a multi-cytokine immune response score and LVDD after adjusting for clinical covariates.

Results

An 11-cytokine profile effectively differentiated subjects with moderate-to-severe LVDD from those with normal LV function. An immune response score (range 0 – 100) was strongly associated with moderate-to-severe LVDD (odds ratio per 10 units: 1.5; 95% confidence interval: 1.2, 2.1) after adjusting for serum IL-6, brain natriuretic peptide, and glucocorticoid use, as well as other RA characteristics and LVDD risk factors.

Conclusion

The major finding of this study is that aberrant systemic immune responsiveness is associated with advanced myocardial dysfunction in patients with RA. The unique information added by the immune response score on the likelihood of LVDD warrants future longitudinal studies of its value in predicting future deterioration in myocardial function.

Heart failure (HF) is an important complication of rheumatoid arthritis (RA) that leads to the premature death of many patients. Until recently, this complication has been overshadowed by the increased risk of coronary heart disease and myocardial infarction in RA. Since 2004, epidemiological studies have confirmed a significantly increased risk of incident HF among people with RA unexplained by traditional cardiovascular risk factors or coronary heart disease (1, 2). HF has a grim prognosis in patients with RA, with up to 35% mortality in the first year after diagnosis—a rate of HF death that is two-fold higher than the analogous rate for persons in the general population (3).

How rheumatoid disease leads to HF is unknown. Several indicators of disease activity or severity predict incident HF, including rheumatoid factor, elevated acute phase reactants, high disability and global severity scores, as well as extra-articular manifestations such as interstitial lung disease, scleritis, and vasculitis (1, 2, 4). A prevailing theory is that chronic, systemic immune activation with elaboration of inflammatory mediators, including cytokines such as TNF-α, IL-1, and IL-6, leads to microvascular dysfunction and ultimately to myocardial remodeling and fibrosis (5). A recent study reported higher expression of adhesion molecules, HLA molecules, and inflammatory cytokines by cardiac endothelial cells and cardiomyocytes in patients with inflammatory rheumatic disease as compared to controls, suggesting immune activation contributes to cardiovascular disease in the RA population (6). Yet, it remains unclear how immune mechanisms conspire in the pathogenesis of myocardial disease in RA.

The unique effect of RA on myocardial function appears to be impairment of diastolic filling, relaxation, or compliance, known as diastolic dysfunction (5). Numerous case-control echocardiography studies have reported an increased prevalence of impaired diastolic function in RA patients even without clinical cardiovascular disease (714). When HF occurs, persons with RA are more likely to have preserved systolic function compared to persons without arthritis (3), suggesting RA-related immune mechanisms incite myocardial injury in a manner that tends to culminate in diastolic dysfunction. Notably, isolated diastolic dysfunction in the general population is associated with increased mortality (15). Thus, improved understanding of the clinical and biological determinants of diastolic dysfunction may explicate the pathogenesis of HF with preserved systolic function and illuminate new targets for therapy, with the ultimate goal of impacting the high mortality of HF in patients with RA.

Further, the identification of biomarkers reflecting immune events early in the pathogenesis of myocardial injury, prior to the development of clinical HF, may enable recognition of patients at future risk for myocardial dysfunction. In attempt to meet this aim, we have devised an approach to identify complex biomarkers based on ex vivo cytokine production, reflecting the responsiveness of the peripheral innate and adaptive immune systems (16). We have shown that profiles of ex vivo cytokine release in response to broad stimulation, summarized as an multi-cytokine prediction score, may have utility in differentiating patients at high risk for disease complications (16). The objective of this study was to identify an “immune signature” of myocardial dysfunction in RA by testing the hypothesis that distinct ex vivo cytokine profiles correlate with the degree of left ventricular diastolic dysfunction (LVDD) after adjusting for cardiovascular risk factors and RA disease characteristics.

Patients and Methods

Study design and participants

We conducted a cross-sectional analysis of baseline data from a prospective study of RA subjects in a community population-based incidence cohort. This study used resources of the Rochester Epidemiology Project, a medical records linkage system providing access to complete medical records for individuals who receive medical attention (17). We identified residents of Olmsted County, Minnesota aged ≥ 18 years who first fulfilled the American College of Rheumatology (ACR; formerly the American Rheumatism Association) classification criteria for RA between January 1, 1980 and December 31, 2007. From this cohort, 266 of 475 eligible subjects alive and residing in Olmsted County, Minnesota, were recruited. The institutional review boards of the Mayo Foundation and Olmsted Medical Center approved this study, which was conducted in accordance with the Declaration of Helsinki. All subjects provided written informed consent.

Data collection

The subjects completed a questionnaire consisting of a visual analogue scale for pain and the Health Assessment Questionnaire (HAQ) for disability. Medical records were reviewed to ascertain cardiovascular risk factors, including body mass index (BMI; kg/m2), coronary heart disease, diabetes mellitus, dyslipidemia, hypertension, and smoking, as defined previously (14). Medication usage, including conventional and biologic disease-modifying agents, glucocorticoids, and non-steroidal anti-inflammatory drugs, was ascertained by patient questionnaire and verified in the medical records. Laboratory testing included C-reactive protein (CRP; Roche, Indianapolis, IN), anti-citrullinated peptide antibodies (ACPA; Quanta Lite CCP3 IgG ELISA, INOVA Diagnostics, San Diego, CA), rheumatoid factor (RF; Behring Nephelometer II, Dade Behring, Newark, DE), and brain natriuretic peptide (BNP; Biosite Diagnostics, San Diego, CA).

Echocardiography

Two-dimensional and Doppler echocardiography was performed on all subjects in the Mayo Clinic Echocardiography Laboratory as previously described (15). Certified cardiac sonographers performed the studies, and two experienced cardiologists made all interpretations. LVDD was classified according to a standardized algorithm as previously described (15). The categories of diastolic function were: normal (no LVDD); mild LVDD (impaired relaxation without evidence of increased filling pressures); or moderate-to-severe LVDD (impaired relaxation associated with moderate elevation of filling pressures, pseudonormal filling, advanced reduction in compliance, or reversible/fixed restrictive filling). We also included six individuals with prevalent HF in the moderate-to-severe LVDD group. After excluding 54 of 266 subjects with indeterminate diastolic function due to technical factors (e.g., dysrhythmia), the study sample consisted of 212 subjects.

Immune signatures

The methodology used to assess the responsiveness of ex vivo cytokine production by peripheral blood mononuclear cells (PBMC) was recently described (16). Fresh PBMC isolated by Ficoll density gradient centrifugation were immediately cultured in media alone or with one of a panel of 7 stimuli, which was designed to induce broad responses of innate and adaptive immunity. The panel included immobilized anti-CD3 and anti-CD28 monoclonal antibodies (anti-CD3/anti-CD28; Dynabeads ® Human T-Activator, Invitrogen, Carlsbad, CA); bacterial CpG oligonucleotides (CpG); combined CMV and EBV lysates (CMV/EBV; Advanced Biotechnologies, Columbia, MD); human heat shock protein 60 (HSP60; Stressgen, Victoria BC, Canada); PMA with ionomycin (PMA/ionomycin; Sigma, St. Louis, MO); phytohemagglutinin (PHA; Sigma, St. Louis, MO); and staphylococcus enterotoxins A and B (SEA and SEB; Toxin Technology, Sarasota, FL). For each stimulation, 4 × 105 PBMC were cultured in 200 μl of medium (RPMI 1640 + 10% fetal bovine serum + 1× penicillin-streptomycin-L-glutamine) containing the stimulant (or media alone) in quadruplicate wells of a 96-well culture plate. The final concentrations in cell culture were as follows: anti-CD3/anti-CD28, 0.5 × 106 beads per culture well; PHA, 5 μg/ml; SEA, 10 ng/ml, with SEB, 10 ng/ml; CMV lysate, 1 μg/ml, with EBV lysate, 1 μg/ml; CpG, 10 μg/ml; HSP60, 1 μg/ml; and PMA, 1 μg/ml, with ionomycin, 700 ng/ml. The PBMC were incubated at 37°C in 5% CO2 for 48 hours, when the supernatants were harvested and stored at −80°C until analysis.

A panel of 17 cytokines was analyzed using the MSDR 96-Well MULTI-SPOTR Human Cytokine Assays tissue culture kit [Meso Scale Discovery (MSD), Gaithersburg, MD], including: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-10, IL-12, IL-13, IL-17, IFNγ, TNFα, MCP-1 (CCL2), MIP1β (CCL4), G-CSF, and GM-CSF. Cytokine concentrations were determined using the Sector 2400 instrument (MSD) based on a standard curve generated for each plate using manufacturer-supplied reagents and analyzed using the Discovery Workbench Software v2.0 (MSD). The intra- and inter-assay reproducibility is favorable relative to the high level of informative biological variation assessed using this approach (16).

Flow cytometry

Cryo-preserved PBMC were stained and fixed with fluorophore-conjugated primary antibodies to CD3, CD4, CD8, CD14, CD19, CD11c, CD56 and CD123 (Becton Dickinson, Franklin Lakes, NJ) per manufacturer protocol with isotype controls. Data were acquired on a FACScan (Becton Dickinson) instrument and analyzed using the manufacturer’s analysis program, Cell Quest (Becton Dickinson).

Statistical analysis

Patient characteristics, laboratory values, medication usage, and echocardiography data were summarized with descriptive statistics. As previously described (16), the analysis included several steps to account for the data multiplicity, blocking, skewed distributions, and sources of unwanted variability. Mixed effects models were used to normalize log-transformed cytokine concentrations, including fixed effects for age and sex as well as random effects for subject and plate, effectively adjusting for age, sex, and assay effects. We identified a cytokine profile associated with LVDD by selecting stimulated cytokines significantly different between the groups with moderate-to-severe LVDD and normal diastolic function using the 1-degree of freedom (df) test and p-value cutoff of <0.01. This method was based on the premise that the immunological differences associated with RA-related mechanisms of myocardial injury would be clearest between these two groups. For cytokines with significant differences under more than one stimulation condition, the stimulant-cytokine combination with the lowest p-value was used. For descriptive purposes, differences between the normal and mild LVDD groups as well as the normal and moderate-to-severe LVDD groups were tested using the 1-df test from the mixed models. Associations between the frequencies of immunophenotypic markers and either ex vivo cytokine production or LVDD status were analyzed using Spearman correlations or Wilcoxon rank sum tests, respectively.

An immune response score was created as previously described (16). Briefly, the normalized cytokine concentrations for the selected profile were transformed to the z-distribution by subtracting the mean and dividing by the SD of the normal LV function group. The z-scores for each cytokine were added (or subtracted, to account for directionality) to construct the immune response score. To ease interpretation, the score was re-scaled so the cutoff of ≥ 50 optimally discriminated the subjects with moderate-to-severe LVDD from the subjects with normal diastolic function. To assess the utility of this score beyond established determinants of LVDD, we performed multivariate analysis using logistic regression, including the immune response score as the main effect and adjusting for factors associated with LVDD based on univariate analyses of the present study and our previous work (14).

Finally, receiver operating characteristic (ROC) analyses were performed with bootstrap sampling to obtain an estimate of bias for the ROC curve (18). For this analysis, 200 bootstrap samples were drawn from the original data set. An immune response score was developed for each bootstrap sample using methods identical to those used for selecting the original score. Using these scores, an estimate of the expected optimism (a measure of the bias associated with testing the score on the same data used to build the score) was obtained as the mean difference for the area under the curves (AUC) between the original sample and the bootstrap samples (19). The optimism for the bootstrap AUC was subtracted from the original AUC to obtain the bias-corrected estimate.

Results

Characteristics of the study subjects

We studied 212 subjects: 134 with normal diastolic function, 53 with mild LVDD, and 25 with moderate-to-severe LVDD or HF (Table 1). We observed associations between LVDD and clinical characteristics that are known risk factors for myocardial disease, including older age (p<0.001), longer RA duration (p=0.002), and the presence of hypertension (p<0.001), metabolic syndrome (p=0.006), dyslipidemia (p<0.001), and coronary heart disease (p=0.002). Overall, the subjects had low disease activity with a mean CRP of 4.2 ± 6.4 mg/L and low disability with a mean HAQ score of 0.5 ± 0.6. The presence of LVDD was associated with higher serum IL-6 and current use of glucocorticoids. Other RA characteristics were not associated with LVDD. The levels of BNP were higher among subjects with LVDD (p<0.001), highest in the group with moderate-to-severe dysfunction, consistent with elevated filling pressures related to myocardial dysfunction in these individuals.

Table 1
Demographic, cardiovascular, and RA disease characteristics for 212 subjects with RA according to diastolic function category*

A distinct profile of cytokine responsiveness correlated with moderate-to-severe LVDD

Analyses revealed statistically significant differences for 19 of 136 (14%) of the stimulated cytokine concentrations between the groups with moderate-to-severe LVDD or HF and normal diastolic function at the level of α=0.01. The selection process resulted in the following 11-cytokine profile: IL-4, IL-5, IL-12, GM-CSF, and G-CSF release in media alone; IFN-γ, IL-13, and TNF-α release in response to anti-CD3/anti-CD28; IL-8 release in response CpG; IL-10 release in response to PMA/ionomycin; and IL-1β release in response to CMV/EBV stimulation. The distributions of cytokine concentrations for the selected profile were assessed according to category of LV diastolic function (Table 2).

Table 2
Distributions of the cytokine concentrations in stimulated PBMC supernatants according to the category of LV diastolic function*

Next, we tested for differences in the direction and magnitude of cytokine production among the groups (Figure 1). The basal or stimulated ex vivo release of several cytokines typically produced by T cells was significantly decreased in the subjects with moderate-to-severe LVDD or HF. Specifically, the release of IL-4 and IL-5 in media alone and the release of IFN-γ, IL-13, and TNF-α under stimulation with anti-CD3/anti-CD28 were significantly lower in the group with moderate-to-severe LVDD or HF as compared to the subjects with normal diastolic function. The basal release of IL-12, a cytokine involved in Th1 responses though not produced by T cells, and the release of IL-8 in response to CpG were also significantly reduced in the group with moderate-to-severe LVDD or HF.

Figure 1
Analysis of ex vivo cytokine production identifies an immunologic signature of myocardial dysfunction in RA. The profiles of cytokine release by PBMC in response to broad stimulation (see legend) are compared between groups with normal diastolic function, ...

The production of several other cytokines was significantly increased (Figure 1). The release of IL-10 in response to PMA/ionomycin and of IL-1β in response to CMV/EBV stimulation was significantly higher in the subjects with moderate-to-severe LVDD or HF as compared to subjects with normal diastolic function. T cells typically produce IL-10 whereas IL-1β is typically produced by myeloid lineages. Additionally, the basal release of GM-CSF and G-CSF (media alone), also typically produced by myeloid cells, was markedly increased in the subjects with moderate-to-severe LVDD or HF. Indeed, the majority of the differences were between the groups with moderate-to-severe LVDD and normal diastolic function. In the group with mild LVDD, the responsiveness of IL-10 to PMA/ionomycin was marginally increased (p=0.046), but no other significant differences were observed as compared to the group with normal diastolic function.

A multi-cytokine immune response score discriminated subjects with moderate-to-severe LVDD

Next, we compared the distributions of the immune response score between the groups with normal diastolic function, mild LVDD, and moderate-to-severe LVDD or HF (Figure 2). The scores were very similar between the groups with normal LV function and mild LVDD. In the group with moderate-to-severe LVDD, 79% of subjects had a score of ≥ 50. Using this cutoff, the sensitivity of the immune response score to classify moderate-to-severe LVDD was 79% (95% CI: 58% to 93%), the specificity was 71% (95% CI: 63% to 79%) and the accuracy was 72% (95% CI: 65% to 79%). Modest correlations of the score were observed with hypertension (r=0.17; p=0.033), coronary heart disease (r=0.17; p=0.035), and duration of RA (r=0.25; p=0.002).

Figure 2
A multi-cytokine immune response score differentiates subjects with moderate-to-severe LVDD or HF. The immune response score was created by transforming the normalized cytokine concentrations of the subjects with LVDD to the z-distribution of the subjects ...

Multivariate analysis of the association between the immune response score and moderate-to-severe LVDD

A 10-unit increase in the immune response score was strongly associated with moderate-to-severe LVDD or HF with an adjusted odds ratio (OR) of 1.5 (95% CI: 1.2, 2.1; p=0.004) (Table 3). Serum IL-6, BNP, and glucocorticoid use were associated with the presence of moderate-to-severe LVDD or HF, respectively, with an adjusted OR of 1.1 per 1 pg/ml increase in IL-6 (95% CI: 1.03, 1.2; p=0.007), adjusted OR of 2.3 per doubling of BNP (95% CI: 1.2, 4.4; p=0.013), and an adjusted OR of 4.1 (95% CI: 1.1, 15.3; p=0.04) for glucocorticoid use. Age, hypertension, female sex, and history of coronary heart disease were associated with clinically meaningful risks of moderate-to-severe LVDD or HF but were not statistically significant in this model. The associations of the immune response score, IL-6, BNP, and glucocorticoid use with LVDD remained significant after additional adjustment for clinical covariates, including cardiovascular risk factors (obesity, diabetes mellitus, dyslipidemia, smoking and metabolic syndrome), RA disease characteristics (disease duration, RF, ACPA, and methotrexate use), and serum CRP.

Table 3
Multivariate analysis of the association between the immune response score and the presence of moderate-to-severe left ventricular diastolic dysfunction or heart failure as compared to normal diastolic function *

To assess the added value of the immune response score beyond the contribution of clinical predictors, we performed ROC analyses for three models: a model with the immune response score alone, a model of clinical variables alone (including serum IL-6 and BNP), and a combined model of clinical variables plus the immune response score (Figure 3). The immune response score demonstrated favorable overall accuracy with uncorrected AUC of 80% (95% CI: 70% to 90%) and bias-corrected AUC of 68%. For the clinical model, which included the same variables as the multivariate analysis (Table 3), the uncorrected AUC was 85% (95% CI: 76% to 93%), and the bias-corrected AUC was 84%. For the combined model, the uncorrected AUC was 88% (95% CI: 80%, 97%), and the bias-corrected AUC was 86%.

Figure 3
Receiver operating characteristics (ROC) analysis of the immune response score. Using the entire sample (n=212), ROC curves are shown for the immune response score (blue line), the clinical model (red line), and the model combining the immune response ...

Correlations of flow cytometric analysis with the cytokine profiles and LVDD

Finally, we performed analyses to determine if the frequencies of particular immune cell subtypes could explain the associations between the cytokine profiles and LVDD. We identified 10 subjects with normal diastolic function, 8 with mild LVDD, and 8 with moderate-to-severe LVDD, selected to represent the spectrum of immune responsiveness for each of the cytokines in our panel. Multi-color flow cytometry was performed to determine the frequency of CD3+, CD4+ and CD8+ T cells, CD19+ B cells, CD14+ monocytes, CD56+ natural killer cells, CD11c+ myeloid dendritic cells, and CD123+ plasmacytoid dendritic cells. Correlations were observed for IL-10 responsiveness to PMA/ionomycin stimulation with CD4+ T cells (r=0.53; p=0.005) and CD11c+ myeloid dendritic cells (r=−0.41; p=0.038). A correlation was also observed for IL-4 production in media alone with the frequency of CD14+ monocytes (p=0.034). Thus, we observed 3 statistically significant correlations. Considering that 88 tests were performed at the significance level of 0.05 (data not shown), up to 5 could be due to chance alone.

We also analyzed the differences in the frequencies of immune cell subtypes between the patients with moderate-to-severe LVDD and normal diastolic function. The mean differences ranged from 3.7% for CD14+ monocytes to 26.1% for CD11c+ myeloid dendritic cells (p>0.3 for all immunophenotypic markers; data not shown). As the observed differences between the groups in these frequencies were significantly less than the differences in cytokine production (around 50%), in spite of the small sample size for this analysis, our results indicate that this flow cytometric approach is substantially less effective in differentiating the groups with moderate-to-severe LVDD and normal diastolic function.

Discussion

This study is the first to report an association between the responsiveness of the peripheral innate and adaptive immune systems and myocardial dysfunction in patients with RA. We have shown that an 11-cytokine profile differentiated patients with moderate-to-severe LVDD from patients with normal diastolic function. We created an immune response score combining the information from each of the 11 cytokines in the profile to summarize the responsiveness of canonical immune pathways. This score was strongly associated with moderate-to-severe LVDD after adjusting for potential confounders, including age, sex, cardiovascular risk factors, RA characteristics, and immunomodulatory therapies, suggesting it provides unique information above and beyond standard clinical predictors. Neither the differences in ex vivo cytokine production nor the presence of moderate-to-severe LVDD were sufficiently explained by variation in the frequencies of immune cell subsets in the peripheral blood, suggesting that ex vivo functional analysis has the advantage of greater immunological discrimination. We conclude that aberrant systemic immune responsiveness is strongly associated with advanced myocardial dysfunction in patients with RA.

In contrast, the patients with mild LVDD were not significantly different from those with normal LV function. The issue is the extent of continuity along a pathophysiological spectrum from mild to severe LVDD and, ultimately, HF. The immunological findings could represent an epiphenomenon related to established myocardial disease rather than a marker of pre-clinical myocardial injury. However, we favor the alternative hypothesis that the immune response signature is a preclinical predictor of LVDD, for two reasons. First, the significance of “mild” LVDD remains a matter of debate. Whereas severe LVDD is clearly a pre-clinical predictor of future symptomatic HF and mortality, milder LVDD may stabilize or improve in up to 50% of patients (20, 21). Perhaps only a subset of patients with mild LVDD may, over time, be at substantial risk for functional deterioration.

Second, based on our recently published work and that of others, many of our immunological findings are known to occur early in the course of RA, prior to the development of any systemic disease complications (16). The responsiveness of cytokines in T cell immune networks was significantly decreased in the group with moderate-to-severe LVDD. Previous studies have demonstrated that in vitro Th1 (i.e., IFN-γ) and Th2 (i.e., IL-4) cytokine release by stimulated PBMC is significantly decreased in patients as compared to control subjects (2224). Moreover, the responsiveness of several cytokines typically produced by myeloid lineages, including monocytes and dendritic cells, or B cells, was significantly increased among the patients with moderate-to-severe LVDD. Previous studies have also established that immune pathways of myeloid lineages are activated in patients with RA, with respect to induction of Toll-like receptor (TLR) signaling (2527) and proinflammatory cytokine production (28, 29). Immunological profiles similar to ours have previously been associated with disease activity (30, 31) and severity (32, 33), suggesting they might predict higher risk of systemic complications. Further, evidence suggests that disease-modifying therapies, including anti-TNF agents, can favorably modulate the aberrant T cell and innate immune responses (29, 3436).

The immune response score showed considerable overlap between the groups, with over 25% of subjects in both the normal and mild LVDD groups having ‘high’ scores. We speculate that subsets of these patients might be at increased risk for myocardial dysfunction over the course of their disease. Ultimately, the abovementioned issues underscore an important limitation of this study, the cross-sectional design, which precludes assessment of causation. We are planning longitudinal studies of the cohort to address these unanswered questions.

Our findings emphasize that the immune response signature may be most useful in the context of a multivariable risk profile. We showed that the profile of the immune response score, serum IL-6, serum BNP, and glucocorticoid use provided the highest accuracy in identifying individuals with LVDD. Serum IL-6 is an important determinant of myocardial dysfunction and predicts significantly higher risk of incident HF and related mortality in the general population (3740). BNP is a hormone produced by the ventricles in response to pressure or volume overload for the purpose of causing diuresis, natriuresis, and vasodilatation. Serum BNP (or the closely related N-terminal pro-BNP) may be useful in detecting preclinical LVDD, both in the general population and patients with RA (41, 42). BNP and the N-terminal pro-BNP are associated with clinical disease activity and inflammatory biomarkers in RA, including IL-6, suggesting BNP is also a marker of myocardial inflammation (43, 44). In our cohort, serum BNP alone had inadequate specificity to be useful as a screening test for myocardial dysfunction (45), but our findings suggest BNP does add value in the context of a multivariable risk profile. Lastly, therapy with glucocorticoids, particularly high cumulative dose and recent use, is associated with an increased risk of HF (4648).

This study only included patients with RA, so it is uncertain whether the findings are unique to RA or are similar to what occurs among LVDD/HF patients in the general population. A preliminary analysis of the immune response score in 5 non-RA subjects with moderate-to-severe LVDD as compared to 18 non-RA subjects with normal diastolic function revealed no apparent differences. Though consistent with a unique effect of the immune response signature in RA, considering the small numbers of subjects, the question remains open. Further comparison of the immune response signature between LVDD patients with and without RA might reveal distinctions that could ultimately be informative in understanding the pathogenesis of HF that occurs in these different groups.

Other limitations of this study include the potential effects of multiple testing on our cytokine analyses, but the use of a stringent threshold for statistical significance minimizes this issue. Clinical factors including disease activity and medication effects likely influence the cytokine responses, and this cross-sectional study is unable to disentangle the influence of these variables on the associations of cytokine profiles with LVDD. However, the findings of our multivariate analysis indicate that the immune response signature provides unique information above and beyond standard CV risk factors. The flow cytometry analyses had small sample size and a limited number of markers, so a different flow cytometric approach to biomarker discovery might be considered in future research. Additionally, the cell-based assays are complex and will be challenging to standardize across laboratories in wider usage. However, the successes of cell-based assays including the T-Spot®. TB and QuantiFERON®-TB-Gold tests provide reassurance that this obstacle can be addressed with assay refinement and technological advances. Finally, although only 56% of eligible subjects participated, bias due to the selection of cases with milder disease is unlikely since non-participants were found to have lesser education, which is a known risk factor for more severe RA.

In conclusion, we have reported the discovery of an immune signature based on the responsiveness of ex vivo cytokine production by peripheral blood cells that is associated with advanced myocardial dysfunction in patients with RA. Future studies should evaluate this signature, perhaps in the context of a multivariable risk profile, for predicting deterioration of myocardial function over time. The findings also inform new hypotheses for the role of aberrant systemic immune function in the pathogenesis of myocardial disease in persons with RA.

Acknowledgments

Grant support: This work was supported by grant R01 R46849 from the National Institute for Arthritis, Musculoskeletal, and Skin Diseases and grant 1 UL1 RR024150 from the National Center for Research Resources (NCRR). Dr. Davis is supported by grant 1 KL2 RR024151 from the NCRR and a New Investigator Award from the Arthritis Foundation. NCRR is a component of the National Institutes of Health (NIH) and the NIH Roadmap for Medical Research. The contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Information on NCRR is available at http://www.ncrr.nih.gov/. Information on Reengineering the Clinical Research Enterprise can be obtained from http://nihroadmap.nih.gov/. Dr. Rizza is supported by grant 1UL1 RR024150.

We thank Dr. Barry L. Karon and Dr. Daniel D. Borgeson for interpreting the echocardiography studies and Dr. Larry R. Pease for helpful discussions on the study design and analysis. We also thank Sherry Kallies for administrative support of this work.

References

1. Nicola PJ, Maradit-Kremers H, Roger VL, Jacobsen SJ, Crowson CS, Ballman KV, et al. The risk of congestive heart failure in rheumatoid arthritis: a population-based study over 46 years. Arthritis Rheum. 2005;52(2):412–20. [PubMed]
2. Wolfe F, Michaud K. Heart failure in rheumatoid arthritis: rates, predictors, and the effect of anti-tumor necrosis factor therapy. Am J Med. 2004;116(5):305–11. [PubMed]
3. Davis JM, 3rd, Roger VL, Crowson CS, Kremers HM, Therneau TM, Gabriel SE. The presentation and outcome of heart failure in patients with rheumatoid arthritis differs from that in the general population. Arthritis Rheum. 2008;58(9):2603–11. [PMC free article] [PubMed]
4. Nicola P, Maradit Kremers H, Crowson CS, Ballman KV, Gabriel SE. Do Rheumatoid Arthritis (RA) Disease Characteristics Predict Congestive Heart Failure? Arthritis Rheum. 2004;50(9):S551.
5. Giles JT, Fernandes V, Lima JA, Bathon JM. Myocardial dysfunction in rheumatoid arthritis: epidemiology and pathogenesis. Arthritis Res Ther. 2005;7(5):195–207. [PMC free article] [PubMed]
6. Grundtman C, Hollan I, Forre OT, Saatvedt K, Mikkelsen K, Lundberg IE. Cardiovascular disease in patients with inflammatory rheumatic disease is associated with up-regulation of markers of inflammation in cardiac microvessels and cardiomyocytes. Arthritis Rheum. 2010;62(3):667–73. [PubMed]
7. Meune C, Wahbi K, Assous N, Weber S, Kahan A, Allanore Y. Myocardial dysfunction in rheumatoid arthritis: a controlled tissue-Doppler echocardiography study. J Rheumatol. 2007;34(10):2005–9. [PubMed]
8. Gonzalez-Juanatey C, Testa A, Garcia-Castelo A, Garcia-Porrua C, Llorca J, Ollier WE, et al. Echocardiographic and Doppler findings in long-term treated rheumatoid arthritis patients without clinically evident cardiovascular disease. Semin Arthritis Rheum. 2004;33(4):231–8. [PubMed]
9. Levendoglu F, Temizhan A, Ugurlu H, Ozdemir A, Yazici M. Ventricular function abnormalities in active rheumatoid arthritis: a Doppler echocardiographic study. Rheumatol Int. 2004;24(3):141–6. [PubMed]
10. Alpaslan M, Onrat E, Evcik D. Doppler echocardiographic evaluation of ventricular function in patients with rheumatoid arthritis. Clin Rheumatol. 2003;22(2):84–8. [PubMed]
11. Di Franco M, Paradiso M, Mammarella A, Paoletti V, Labbadia G, Coppotelli L, et al. Diastolic function abnormalities in rheumatoid arthritis. Evaluation By echo Doppler transmitral flow and pulmonary venous flow: relation with duration of disease. Ann Rheum Dis. 2000;59(3):227–9. [PMC free article] [PubMed]
12. Montecucco C, Gobbi G, Perlini S, Rossi S, Grandi AM, Caporali R, et al. Impaired diastolic function in active rheumatoid arthritis. Relationship with disease duration. Clin Exp Rheumatol. 1999;17(4):407–12. [PubMed]
13. Corrao S, Salli L, Arnone S, Scaglione R, Pinto A, Licata G. Echo-Doppler left ventricular filling abnormalities in patients with rheumatoid arthritis without clinically evident cardiovascular disease. Eur J Clin Invest. 1996;26(4):293–7. [PubMed]
14. Liang KP, Myasoedova E, Crowson CS, Davis JM, Roger VL, Karon BL, et al. Increased prevalence of diastolic dysfunction in rheumatoid arthritis. Ann Rheum Dis. 2010;69(9):1665–70. [PMC free article] [PubMed]
15. Redfield MM, Jacobsen SJ, Burnett JC, Jr, Mahoney DW, Bailey KR, Rodeheffer RJ. Burden of systolic and diastolic ventricular dysfunction in the community: appreciating the scope of the heart failure epidemic. JAMA. 2003;289(2):194–202. [PubMed]
16. Davis JM, 3rd, Knutson KL, Strausbauch MA, Crowson CS, Therneau TM, Wettstein PJ, et al. Analysis of complex biomarkers for human immune-mediated disorders based on cytokine responsiveness of peripheral blood cells. J Immunol. 2010;184(12):7297–304. [PMC free article] [PubMed]
17. Maradit Kremers H, Crowson CS, Gabriel SE. Rochester Epidemiology Project: a unique resource for research in the rheumatic diseases. Rheum Dis Clin North Am. 2004;30(4):819–34. vii. [PubMed]
18. Efron B. The jackknife, the bootstrap and other resampling plans. Regional Conference Series in Applied Mathematics; Philadelphia: SIAM; 1982. p. 1982.
19. Efron B, Tibshirani R. An introduction to the bootstrap. Boca Raton: Chapman & Hall/CRC; 1998.
20. Achong N, Wahi S, Marwick TH. Evolution and outcome of diastolic dysfunction. Heart. 2009;95(10):813–8. [PubMed]
21. Zhang Y, Safar ME, Iaria P, Agnoletti D, Protogerou AD, Blacher J. Prevalence and prognosis of left ventricular diastolic dysfunction in the elderly: The PROTEGER Study. Am Heart J. 2010;160(3):471–8. [PubMed]
22. Berner B, Akca D, Jung T, Muller GA, Reuss-Borst MA. Analysis of Th1 and Th2 cytokines expressing CD4+ and CD8+ T cells in rheumatoid arthritis by flow cytometry. J Rheumatol. 2000;27(5):1128–35. [PubMed]
23. Allen ME, Young SP, Michell RH, Bacon PA. Altered T lymphocyte signaling in rheumatoid arthritis. Eur J Immunol. 1995;25(6):1547–54. [PubMed]
24. Kusaba M, Honda J, Fukuda T, Oizumi K. Analysis of type 1 and type 2 T cells in synovial fluid and peripheral blood of patients with rheumatoid arthritis. J Rheumatol. 1998;25(8):1466–71. [PubMed]
25. Huang Q, Ma Y, Adebayo A, Pope RM. Increased macrophage activation mediated through toll-like receptors in rheumatoid arthritis. Arthritis Rheum. 2007;56(7):2192–201. [PubMed]
26. Iwahashi M, Yamamura M, Aita T, Okamoto A, Ueno A, Ogawa N, et al. Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis. Arthritis Rheum. 2004;50(5):1457–67. [PubMed]
27. Roelofs MF, Joosten LA, Abdollahi-Roodsaz S, van Lieshout AW, Sprong T, van den Hoogen FH, et al. The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells. Arthritis Rheum. 2005;52(8):2313–22. [PubMed]
28. Kowalski ML, Wolska A, Grzegorczyk J, Hilt J, Jarzebska M, Drobniewski M, et al. Increased responsiveness to toll-like receptor 4 stimulation in peripheral blood mononuclear cells from patients with recent onset rheumatoid arthritis. Mediators Inflamm. 2008;2008:132732. [PMC free article] [PubMed]
29. Leirisalo-Repo M, Paimela L, Jaattela M, Koskimies S, Repo H. Production of TNF by monocytes of patients with early rheumatoid arthritis is increased. Scand J Rheumatol. 1995;24(6):366–71. [PubMed]
30. Fabris M, Tolusso B, Di Poi E, Tomietto P, Sacco S, Gremese E, et al. Mononuclear cell response to lipopolysaccharide in patients with rheumatoid arthritis: relationship with tristetraprolin expression. J Rheumatol. 2005;32(6):998–1005. [PubMed]
31. Kawashima M, Miossec P. mRNA quantification of T-bet, GATA-3, IFN-gamma, and IL-4 shows a defective Th1 immune response in the peripheral blood from rheumatoid arthritis patients: link with disease activity. J Clin Immunol. 2005;25(3):209–14. [PubMed]
32. Macht LM, Elson CJ, Kirwan JR, Gaston JS, Lamont AG, Thompson JM, et al. Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60. Immunology. 2000;99(2):208–14. [PMC free article] [PubMed]
33. van Roon JA, Verhoef CM, van Roy JL, Gmelig-Meyling FH, Huber-Bruning O, Lafeber FP, et al. Decrease in peripheral type 1 over type 2 T cell cytokine production in patients with rheumatoid arthritis correlates with an increase in severity of disease. Ann Rheum Dis. 1997;56(11):656–60. [PMC free article] [PubMed]
34. Berg L, Lampa J, Rogberg S, van Vollenhoven R, Klareskog L. Increased peripheral T cell reactivity to microbial antigens and collagen type II in rheumatoid arthritis after treatment with soluble TNFalpha receptors. Ann Rheum Dis. 2001;60(2):133–9. [PMC free article] [PubMed]
35. Kawashima M, Miossec P. Effect of treatment of rheumatoid arthritis with infliximab on IFN gamma, IL4, T-bet, and GATA-3 expression: link with improvement of systemic inflammation and disease activity. Ann Rheum Dis. 2005;64(3):415–8. [PMC free article] [PubMed]
36. Seitz M, Zwicker M, Wider B. Enhanced in vitro induced production of interleukin 10 by peripheral blood mononuclear cells in rheumatoid arthritis is associated with clinical response to methotrexate treatment. J Rheumatol. 2001;28(3):496–501. [PubMed]
37. Haugen E, Gan LM, Isic A, Skommevik T, Fu M. Increased interleukin-6 but not tumour necrosis factor-alpha predicts mortality in the population of elderly heart failure patients. Exp Clin Cardiol. 2008;13(1):19–24. [PMC free article] [PubMed]
38. Bahrami H, Bluemke DA, Kronmal R, Bertoni AG, Lloyd-Jones DM, Shahar E, et al. Novel metabolic risk factors for incident heart failure and their relationship with obesity: the MESA (Multi-Ethnic Study of Atherosclerosis) study. J Am Coll Cardiol. 2008;51(18):1775–83. [PubMed]
39. Karpinski L, Plaksej R, Kosmala W, Witkowska M. Serum levels of interleukin-6, interleukin-10 and C-reactive protein in relation to left ventricular function in patients with myocardial infarction treated with primary angioplasty. Kardiol Pol. 2008;66(12):1279–85. [PubMed]
40. Chrysohoou C, Pitsavos C, Barbetseas J, Kotroyiannis I, Brili S, Vasiliadou K, et al. Chronic systemic inflammation accompanies impaired ventricular diastolic function, detected by Doppler imaging, in patients with newly diagnosed systolic heart failure (Hellenic Heart Failure Study) Heart Vessels. 2009;24(1):22–6. [PubMed]
41. Redfield MM, Rodeheffer RJ, Jacobsen SJ, Mahoney DW, Bailey KR, Burnett JC., Jr Plasma brain natriuretic peptide to detect preclinical ventricular systolic or diastolic dysfunction: a community-based study. Circulation. 2004;109(25):3176–81. [PubMed]
42. Harney SM, Timperley J, Daly C, Harin A, James T, Brown MA, et al. Brain natriuretic peptide is a potentially useful screening tool for the detection of cardiovascular disease in patients with rheumatoid arthritis. Ann Rheum Dis. 2006;65(1):136. [PMC free article] [PubMed]
43. Solus J, Chung CP, Oeser A, Avalos I, Gebretsadik T, Shintani A, et al. Amino-terminal fragment of the prohormone brain-type natriuretic peptide in rheumatoid arthritis. Arthritis Rheum. 2008;58(9):2662–9. [PMC free article] [PubMed]
44. Provan SA, Angel K, Odegard S, Mowinckel P, Atar D, Kvien TK. The association between disease activity and NT-proBNP in 238 patients with rheumatoid arthritis: a 10-year longitudinal study. Arthritis Res Ther. 2008;10(3):R70. [PMC free article] [PubMed]
45. Crowson CS, Myasoedova E, Davis JM, 3rd, Roger VL, Karon BL, Borgeson D, et al. B-type natriuretic peptide is a poor screening tool for left ventricular diastolic dysfunction in rheumatoid arthritis patients without clinical cardiovascular disease. Arthritis Care Res (Hoboken) 2011 Jan 10; [Epub ahead of print] [PMC free article] [PubMed]
46. Davis JM, 3rd, Maradit Kremers H, Crowson CS, Nicola PJ, Ballman KV, Therneau TM, et al. Glucocorticoids and cardiovascular events in rheumatoid arthritis: a population-based cohort study. Arthritis Rheum. 2007;56(3):820–30. [PubMed]
47. Souverein PC, Berard A, Van Staa TP, Cooper C, Egberts AC, Leufkens HG, et al. Use of oral glucocorticoids and risk of cardiovascular and cerebrovascular disease in a population based case-control study. Heart. 2004;90(8):859–65. [PMC free article] [PubMed]
48. Wei L, MacDonald TM, Walker BR. Taking glucocorticoids by prescription is associated with subsequent cardiovascular disease. Ann Intern Med. 2004;141(10):764–70. [PubMed]

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