Sialyltransferase and Neuraminidase Levels/Ratios and Sialic Acid Levels in Peripheral Blood B Cells Correlate with Measures of Disease Activity in Patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis: A Pilot Study.

OBJECTIVE:We attempted to determine whether the level of enzymes sialyltransferase (ST) and neuraminidase (Neu) and sialic acid (SIA) in patients with systemic lupus erythematosus (SLE) correlates with the SLE Disease Activity Index (SLEDAI) and in patients with rheumatoid arthritis (RA) correlates with the Disease Activity Score28 (DAS28). METHODS:We examined cell-surface levels of ST6Gal-1, Neu1, ST3Gal-1, Neu3, α-2,6-SIA, and α-2,3-SIA by using fluorescent anti-enzyme antibodies, fluorescent-conjugated Sambucus nigra lectin, and fluorescent-conjugated Maackia amurensis lectin on blood cells in SLE and RA patients and assessed correlations of these levels with SLEDAI and with DAS28. Areas under the curve (AUC) were calculated for different variables against SLEDAI. RESULTS:The B-cell ST3Gal-1/Neu3 ratio positively correlated with SLEDAI scores (ρ = 0.409 and P = 0.002, statistically significant after Bonferroni' correction for multiple analyses.). It was supported by the inverse correlation of B-cell Neu3 levels with SLEDAI scores (ρ = -0.264, P = 0.048). The B-cell ST3Gal-1/Neu3 ratio against SLEDAI yielded an AUC of 0.689, which was comparable to that of anti-dsDNA levels at 0.635. In contrast, both ST3Gal-1 and Neu3 levels of RA B cells (r = 0.376, P = 0.013; r = 0.425, P = 0.005, respectively) correlated positively with high disease-activity DAS28 scores. CONCLUSION:B-cell ST3Gal-1/Neu3 ratios in SLE and B-cell ST3Gal-1 and Neu3 levels in RA with high disease-activity DAS28 scores correlated with disease activity measures and may be useful in monitoring disease activities

B-cell α-2,3-sialic acid (SIA) levels in systemic lupus (SLE), healthy controls (Ctrl), and rheumatoid arthritis (RA).

<p>B-cell α-2,3-SIA levels indicated the mean fluorescence intensity of the B-cell’s SIA staining results. (A) Positive correlation between B-cell α-2,3-SIA levels and serum anti-dsDNA antibody levels (WHO unit/ml) (n = 106). Using a staining method similar to that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151669#pone.0151669.g001" target="_blank">Fig 1</a>, the second layer of staining used FITC-conjugated <i>Macckia amurensis</i> lectin to bind cell surface α-2,3-SIA. WHO: World Health Organization. The correlation was ρ = 0.313 and <i>P</i> = 0.001. (B) Comparison of B-cell α-2,3-SIA levels among various groups. SLE (n = 106) vs. Ctrl (n = 31) gave <i>P</i><0.001; SLE vs. RA (n = 157) rendered <i>P</i><0.001; RA vs. Ctrl gave <i>P</i> = 0.011. All were significant when Bonferroni’ correction was done to make a significant <i>P</i>-value at 0.0167.</p

Demographic data of enrolled SLE and RA patients.

<p>Demographic data of enrolled SLE and RA patients.</p

Correlation between the B-cell ST3Gal-1/Neu3 ratios and SLEDAI or and DAS28.

<p>B cells were stained as described in Patients and methods. The B-cell ST3Gal-1/Neu3 ratio indicated the ratio of the mean fluorescence intensity (MFI) of the B-cell’s ST3Gal-1 staining results divided by the MFI of the same B-cell’s Neu3 staining results. (A) Positive correlation between the B-cell ST3Gal-1/Neu3 ratio and SLEDAI. SLEDAI: systemic lupus erythematosus disease activity index; In order to have distinctive circles in the graph, those circles with SLEDAI scores equal to 0, 2, and 4 were adjusted on one-to-two figure(s) to the right of the decimal point either above or below 0, 2, and 4. The correlation was ρ = 0.409 and <i>P</i> = 0.002 (n = 57). It was statistically significant when compared with a significant <i>P</i>-value = 0.0125 after Bonferroni’s correction was done with no significant correlation was found for correlating the ST3Gal-1/Neu3 ratio of T and PMN cells, and monocytes with SLEDAI scores. (B) Correlation between B-cell ST3/Neu3 ratios and DAS28 (disease activity score28) at r = 0.039 and <i>P</i> = 0.631 (n = 157).</p

The association of B cell sialyltransferase and neuraminidase levels with DAS28 scores in RA patients.

<p>ST3: ST3Gal-1, sialyltransferase 3; Neu3: neuraminidase 3; DAS28: disease activity score 28; RA: rheumatoid arthritis. B cell ST3 and Neu3 levels indicated the mean fluorescence intensity of the B-cell’s ST3Gal-1 and Neu3 staining results. Correlation coefficients and <i>P</i>-values for: (A) r = 0.165, <i>P</i> = 0.047; (B) r = 0.155, <i>P</i> with a trend (n = 146 of RA patients in non-remission in A and B); (C) r = 0.170, <i>P</i> with a trend; (D) r = 0.201, <i>P</i> = 0.026 (n = 123 of RA patients with moderate and high disease activity in C and D); (E) r = 0.376, <i>P</i> = 0.013; (F) r = 0.425, <i>P</i> = 0.005 (n = 43 of RA patients with high disease activity in E and F).</p

Sialic-Acid-Related Enzymes of B Cells and Monocytes as Novel Markers to Discriminate Improvement Categories and to Fulfill Two Remission Definitions in Rheumatoid Arthritis

The enzymes α-2,6-sialyltransferase 1 (ST6Gal1), neuraminidase 1 (Neu1), α-2,3-sialyltransferase 1 (ST3Gal1), and neuraminidase 3 (Neu3) are known to affect immune cell function. However, it is not known whether the levels of these enzymes relate to remission definitions or differentiate American College of Rheumatology (ACR), European League Against Rheumatism (EULAR), and Simplified Disease Activity Index (SDAI) responses in patients with rheumatoid arthritis (RA). We measured the ST6Gal1, Neu1, ST3Gal1, and Neu3 levels of B cells and monocytes in RA patients and correlated the cells’ enzyme levels/ratios with the improvement in the ACR, EULAR and SDAI responses and with the two remission definitions. The difference in the B-cell Neu1 levels differed between the ACR 70% improvement and non-improvement groups (p = 0.043), between the EULAR good major response (improvement) and non-good response groups (p = 0.014), and also between the SDAI 50% or 70% improvement and non-improvement groups (p = 0.001 and 0.018, respectively). The same held true when the RA patients were classified by positive rheumatoid factor or the use of biologics. The B-cell Neu1 levels significantly indicated 2005 modified American Rheumatism Association and 2011 ACR/EULAR remission definitions (area under the curve (AUC) = 0.674 with p = 0.001, and AUC = 0.682 with p < 0.001, respectively) in contrast to the CRP and ESR (all AUCs < 0.420). We suggest that B-cell Neu1 is superior for discriminating ACR, EULAR, and SDAI improvement and is good for predicting two kinds of remission definitions

Univariate analysis of correlations between independent markers and measures of clinical arthritic activity.

*<p>Spearman’s correlation. All other correlations (r) are Pearson’s correlation coefficients for 111 patients with rheumatoid arthritis and r (<i>P</i>) values are shown. MCP-1, macrophage chemotactic factor-1; PTX3, pentraxin 3; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; SJC, swollen joint count; TJC, tender joint count; GH, visual analog scale for general health; DAS28, disease activity score 28 [ESR included]; DAS28-MCP-1, disease activity score 28 [MCP-1 included instead of ESR]; DAS28-CRP, disease activity score 28 [CRP included].</p

Multivariate analysis of correlations between independent markers and measures of clinical arthritic activity for RA patients with blood ESR ≥28 mm/hr.

<p>Correlation was analyzed by estimating Pearson’s correlation coefficients for 74 patients with rheumatoid arthritis (RA); <i>P</i> values (CI) are shown. Abbreviations are defined in the footnotes for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055346#pone-0055346-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055346#pone-0055346-t003" target="_blank">3</a>.</p

Demographic, laboratory, and clinical characteristics of enrolled RA^* patients.

*<p>RA, rheumatoid arthritis; Mean [SD], mean ± standard deviation; RF, IgM rheumatoid factor; Anti-CCP, anti-cyclic citrullinated peptide 3 antibody; MCP-1, macrophage chemotactic protein-1; PTX3, pentraxin 3; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; GH, visual analog scale for general health [maximum score  = 100]; DAS28, disease activity score 28 [ESR included]; DAS28-MCP-1, disease activity score 28 [MCP-1 included instead of ESR]; DAS28-CRP, disease activity score 28 [CRP included]. +Data shown are median [25th percentile, 75th percentile]. Serum rheumatoid factors were examined at diagnosis (newly diagnosed RA patients) and were not subsequently re-checked, however, 33% of regularly treated RA patients had RF re-checked on enrollment due to loss of the data; anti-CCP antibodies were measured on enrollment for all RA patients.</p