The expression and clinical significance of different forms of Mer receptor tyrosine kinase in systemic lupus erythematosus

Abstract

Objective: To investigate the expression and clinical significance of trans-membrane MerTK (mMer) on circulating CD14+ monocytes/macrophages and soluble MerTK (sMer) levels in plasma in systemic lupus erythematosus (SLE).

Method: 108 SLE patients and 42 healthy controls were recruited in this study. The expression of mMer on the surfaces of CD14+ monocytes/macrophages was evaluated by flow cytometry (FCM). The sMer levels were measured by ELISA. Real-time quantitative PCR was applied to evaluate the mRNA levels of MerTK and ADAM17.

Results: Both mMer expression on CD14+ monocytes/macrophages and sMer levels in plasma significantly increased in SLE patients compared to healthy subjects. The frequency of anti-inflammatory MerTK expressing CD14+CD16+ monocytes decreased in SLE. mMer expression was positively correlated with CD163 expression on CD14+ cells. Both the mMer expression on CD14+ monocytes/macrophages and sMer levels in plasma were positively correlated with SLEDAI. Furthermore, more elevated mMer and sMer levels were found in patients with higher SLEDAI, presence of anti-SSA, anti-Sm autoantibodies, and lupus nephritis.

Conclusion: Both mMer and sMer levels significantly increased in SLE and positively correlated with disease activity and severity. The upregulation of MerTK expression may serve as a biomarker of the disease activity and severity of SLE.

Figure 1

Comparison of gene expressions in PBMC (26 healthy controls and 35 SLE patients for MerTK; 6 healthy controls and 6 SLE patients for ADAM17) and CD14+ monocytes/macrophages. Relative MerTK expression levels in PBMC and CD14+ are shown in (a) and (b), respectively. (c) and (d), respectively, demonstrated the ADAM17 expression in PBMC and CD14+ monocytes/macrophages. Histograms in solid show the relative gene expression in SLE patients compared with expression in healthy controls (histogram in blank). Vertical lines out histograms show standard errors. PBMC: peripheral blood mononuclear cell; MerTK: Mer tyrosine kinase; ADAM17: A Disintegrin And Metalloproteinases domain 17. *P < 0.05.

Figure 2

Example of quantification of blood CD14+ monocytes/macrophages and CD14+CD16+ macrophage subset. Membrane MerTK and CD163 expression were measured by flow cytometry as mean fluorescence intensity (MFI). Following monoclonal anti-human Abs were used for detection of FITC-conjugated anti-CD14 Ab, APC-conjugated anti-CD163 Ab, and PE-conjugated MerTK Ab. (a) Cell distribution based on the forward-scatter and side-scatter; the monocytes/macrophages population is identified and gated accordingly. The fraction of monocytes/macrophages positive for CD14 is identified and gated. (b) Histogram showing the MFI of CD14+ monocytes/macrophages positive for MerTK in IgG1 isotype (Purple line), healthy controls (Heavy blue line), and SLE patients (Light green line). mMer expression on CD14+ monocytes/macrophages subset was significantly elevated in patients with SLE compared with healthy controls. (c) CD163 expression on the surface of CD14+ monocytes/macrophages shown by histogram was different between SLE patients (Light blue line) and healthy controls (Red line). Bar showed the more expansion of CD163 expression on CD14+ monocytes/macrophages subset in SLE patients than healthy controls. (d) Correlation between CD163 and mMer expression on CD14+ cells. (e) Characterization of the monocytes/macrophages subsets in PBMC from healthy controls and patients with SLE. The dot plot represented the CD14 and CD16 expression on monocytes/macrophages. Percentages of CD14+CD16+ subset among total monocytes/macrophages were significantly reduced in SLE patients. Circles and squares in solid represented CD14+CD16+ cell frequencies of healthy controls and SLE patients, respectively. CD14+CD16+ monocytes/macrophages subset had elevated mMer expression in patients with SLE in comparison with healthy controls. (f) Comparison about sMer levels in plasma between healthy controls and SLE patients. (g) Correlation between sMer in plasma and mMer on CD14+ cells. The mean ± SD of MFI was shown by bars represented for SLE patients in solid and healthy controls in blank. Horizontal lines above bars showed difference and vertical lines showed standard errors. FSC: forward scatter; SSC: side scatter; mMer: membrane Mer tyrosine kinase; FITC: fluorescein isothiocyanate; APC: allophycocyanin; PE: phycoerythrin; PBMC: peripheral blood mononuclear cell. **P < 0.001, ***P < 0.0001.

Figure 3

Correlations of mMer and sMer with different clinical parameters such as SLEDAI score and 24 hours proteinuria excretion in patients with SLE. (a) mMer expression on CD14+ monocytes/macrophages was positively correlated to SLEDAI score. (b) sMer in plasma had positive correlation to SLEDAI score, 24 h: 24 hours proteinuria excretion. mMer: membrane Mer tyrosine kinase; sMer: soluble Mer tyrosine kinase; SLEDAI: SLE disease activity index; Spearman's rank correlation test was used to assess correlations.

Figure 4

mMer and sMer levels according to the clinical manifestations in SLE. (a) mMer expression on CD14+ monocytes/macrophages in patients with a SLEDAI more than or equal to 8, WBC less than 4 × 10⁹/L, and with SSA, Sm or lupus nephritis. (b) Plasma sMer concentrations in patients with a SLEDAI more than or equal to 8, 24 h proteinuria excretion more than or equal to 0.1 g/d, and with SSA, Sm or lupus nephritis. SLEDAI = SLE disease activity index. WBC: leukocytes; SSA: anti-SSA antibody; Sm: anti-Sm antibody. Circles in solid and in blank represented the mMer or sMer levels in the presence and absence of manifestations in SLE with the studied parameters, respectively. Horizontal lines above dots showed difference. *P < 0.05, **P < 0.01, ***P < 0.001.