Scleroderma-specific autoantibodies embedded in immune complexes mediate endothelial damage: an early event in the pathogenesis of systemic sclerosis

Abstract

Background: Consistently with their diagnostic and prognostic value, autoantibodies specific for systemic sclerosis (SSc) embedded in immune complexes (ICs) elicited a pro-inflammatory and pro-fibrotic cascade in healthy skin fibroblasts, engaging Toll-like receptors (TLRs) via their nucleic acid components. The objective of this study was to investigate the pathogenicity of SSc-ICs in endothelial cells.

Methods: ICs were purified from the sera of SSc patients bearing different autoantibody specificities (antibodies against DNA topoisomerase I, centromeric proteins, RNA polymerase, and Th/To), patients with systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS), or healthy controls (NHS) using polyethylene glycol precipitation. Human umbilical vein endothelial cells (HUVECs) were incubated with ICs, positive and negative controls. mRNA levels of endothelin-1 (et-1), collagenIα1 (colIα1), interferon (IFN)-α, and IFN-β were investigated by real-time PCR; et-1 and il-6 mRNA levels were assessed after pre-treatment with bafilomycin. ICAM-1 expression was evaluated by cell ELISA; secretion of IL-6, IL-8, and transforming growth factor (TGF)-β1 in culture supernatants was measured by ELISA. The expression of Fcγ receptors (CD64, CD32, and CD16) was assessed in endothelial cells at FACS analysis. Intracellular signaling pathways culminating with NFκB, p38MAPK, SAPK-JNK, and Akt were assessed by Western blotting. Healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with ICs, and TGF-β1 secretion and mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1, protein expression of α smooth muscle actin (α-SMA), and IL-6 were evaluated by Western blotting; et-1 mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-β inhibitors and stimulated with ATA-ICs.

Results: All SSc stimulated IL-6 secretion; ACA-ICs and anti-Th/To-ICs increased ICAM-1 expression; all SSc-ICs but anti-Th/To-ICs augmented IL-8 levels; all SSc-ICs but ACA-ICs and ARA-ICs upregulated et-1, and all SSc-ICs but ARA-ICs affected TGF-β1 secretion. colIα1, IFN-α, and IFN-β mRNA levels were not affected by any SSc-IC. FcγRII (CD32) and FcγRIII (CD16) were not detectable on HUVECs, while FcγRI (CD64) was minimally expressed. A differential modulation of tlr expression was observed: tlr2, tlr3, and tlr4 were upregulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in tlr9 upregulation. Pre-treatment with bafilomycin did not affect the upregulation of et-1 and il-6 induced by ATA-ICs, ACA-ICs, and anti-Th/To-ICs; a 23% reduction in both genes was reported for ARA-ICs. All SSc-ICs activated p38MAPK and Akt, and all SSc-ICs but ARA-ICs yielded the activation of NFκB; ATA-ICs and ACA-ICs increased the activation rate of both subunits of SAPK-JNK. When healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with SSc-ICs, TGF-β1 secretion, colIα1, α-SMA, and IL-6 expression levels were significantly modulated. Pre-treatment with IL-6 and TGF-β inhibitors prevented et-1 upregulation induced by ATA-ICs by 85% and 77%, respectively.

Conclusions: These data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies on the endothelium. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.

Keywords: Autoantibodies; Endothelial cells; Fibrosis; Immune complexes; Inflammation; Systemic sclerosis; Toll-like receptors.

Fig. 1

ICAM-1 expression and IL-6 and IL-8 secretion in HUVECs stimulated with SSc-ICs, PAPS-ICs, SLE-ICs, or NHS-ICs. Endothelial cells were incubated with SSc-ICs, PAPS-ICs, SLE-ICs, or NHS-ICs (1:2 dilution). IL-1β (50 U/ml) and LPS (1 μg/ml) were used as positive controls. Histograms represent mean + standard error of the mean (SEM). *p < 0.01; **p < 0.001; ***p < 0.0001 versus medium. a ICAM-1 expression (O.D.) [mean ± SD]. IL-1β [429.8 ± 151.9] versus medium [2.75 ± 1.71] (p < 0.0001) versus NHS-ICs (p < 0.0001). LPS [979.5 ± 180.3] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [134 ± 16.27] versus medium (p = N.S.) and versus NHS-ICs [34.25 ± 17.63] (p = N.S.). ACA-ICs [297.3 ± 153.7] versus medium (p < 0.001) versus NHS-ICs (p < 0.01). ARA-ICs [157.8 ± 17.61] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [375 ± 42.16] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). PAPS-ICs [311.3 ± 151.7] versus medium (p < 0.001) versus NHS-ICs (p < 0.001). SLE-ICs [216 ± 84.75] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [34.25 ± 17.63] versus medium (p = N.S.). b IL-6 expression (pg/ml) [mean ± SD]. IL-1β [1414 ± 161.2] versus medium [191 ± 0.70] (p < 0.0001) versus NHS-ICs (p < 0.0001). LPS [2900.5 ± 141.4] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [584.5 ± 48.79] versus medium (p < 0.01) and versus NHS-ICs [222.5 ± 31.82] (p = N.S.). ACA-ICs [582 ± 15.56] versus medium (p < 0.01) versus NHS-ICs (p = N.S). ARA-ICs [785.5 ± 210] versus medium (p < 0.001) versus NHS-ICs (p < 0.001). Anti-Th/To-ICs [786 ± 90.51] versus medium (p < 0.001) versus NHS-ICs (p < 0.001). PAPS-ICs [504.5 ± 193] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). SLE-ICs [276.5 ± 40.31] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [222.5 ± 31.82] versus medium (p = N.S.). c IL-8 expression (pg/ml) [mean ± SD]. IL-1β [1186 ± 101.8] versus medium [1.5 ± 0.6] (p < 0.0001) versus NHS-ICs (p < 0.0001). LPS [1659 ± 178.2] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [721.5 ± 108.2] versus medium (p < 0.0001) and versus NHS-ICs [222.5 ± 31.82] (p < 0.001). ACA-ICs [814.5 ± 47.38] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). ARA-ICs [948 ± 69.3] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). Anti-Th/To-ICs [214 ± 22.63] versus medium (p = N.S) versus NHS-ICs (p = N.S.). PAPS-ICs [167 ± 137.2] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). SLE-ICs [434 ± 11.31] versus medium (p < 0.001) versus NHS-ICs (p = N.S.). NHS-ICs [218.2 ± 28.51] versus medium (p = N.S)

Fig. 2

et-1 mRNA expression levels in HUVECs stimulated with SSc-ICs, PAPS-ICs, SLE-ICs, and NHS-ICs; TGF-β1 secretion in HUVECs stimulated with SSc-ICs or NHS-ICs. Endothelial cells were incubated with SSc-ICs, PAPS-ICs, SLE-ICs, or NHS-ICs (1:2 dilution). IL-1β (50 U/ml) and LPS (1 μg/ml) were used as positive controls. *p < 0.01; **p < 0.001; ***p < 0.0001 versus medium. a et-1 (RQ) [mean ± SD]. IL-1β [2.03 ± 0.15] versus medium [1.03 ± 0.06] (p = N.S.) versus NHS-ICs (p = N.S.). LPS [3.13 ± 0.71] versus medium (p < 0.01) versus NHS-ICs (p < 0.001). ATA-ICs [6.32 ± 0.99] versus medium (p < 0.0001) and versus NHS-ICs [0.53 ± 0.59] (p < 0.0001). ACA-ICs [0.80 ± 0.10] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). ARA-ICs [1.07 ± 0.15] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [3.43 ± 1.37] versus medium (p < 0.01) versus NHS-ICs (p < 0.001). PAPS-ICs [1.06 ± 0.27] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). SLE-ICs [10.27 ± 1.86] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). NHS-ICs [0.53 ± 0.59] versus medium (p = N.S.). b TGF-β1 (pg/ml) [mean ± SD]. IL-1β [198.8 ± 48.71] versus medium [21.75 ± 6.24] (p = N.S.) versus NHS-ICs (p = N.S.). LPS [171.5 ± 5.8] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). ATA-ICs [313.5 ± 109.9] versus medium (p < 0.0001) and versus NHS-ICs [22.75 ± 7.59] (p < 0.0001). ACA-ICs [279.5 ± 129.0] versus medium (p < 0.001) versus NHS-ICs (p < 0.001). ARA-ICs [148.5 ± 26.71] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [330.8 ± 177.4] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). NHS-ICs [22.75 ± 7.59] versus medium (p = N.S)

Fig. 3

tlr mRNA expression levels in HUVECs stimulated with SSc-ICs, PAPS-ICs, SLE-ICs, and NHS-ICs. Endothelial cells were incubated with SSc-ICs, PAPS-ICs, SLE-ICs, or NHS-ICs (1:2 dilution). LPS (1 μg/ml), Poly I:C (1 μg/ml) and ODN CpG (5 μM) were used as positive controls. *p < 0.01; **p < 0.001; ***p < 0.0001 versus medium. a tlr2 (RQ) [mean ± SD]. LPS [8.8 ± 0.42] versus medium [1.06 ± 0.08] (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [12.50 ± 3.54] versus medium (p < 0.0001) and versus NHS-ICs [0.75 ± 0.63] (p < 0.0001). ACA-ICs [5.65 ± 0.78] versus medium (p < 0.01) versus NHS-ICs (p < 0.01). ARA-ICs [0.83 ± 0.09] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [0.85 ± 0.23] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). PAPS-ICs [2.25 ± 0.21] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). SLE-ICs [1.15 ± 0.49] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [0.75 ± 0.63] versus medium (p = N.S.). b tlr3 (RQ) [mean ± SD]. Poly I:C [2.30 ± 0.28] versus medium [1.03 ± 0.04] (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [2.65 ± 0.21] versus medium (p < 0.0001) and versus NHS-ICs [1.15 ± 0.07] (p < 0.0001). ACA-ICs [2.5 ± 0.28] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). ARA-ICs [0.85 ± 0.07] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [0.80 ± 1.14] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). PAPS-ICs [1.75 ± 0.21] versus medium (p < 0.01) versus NHS-ICs (p < 0.01). SLE-ICs [0.30 ± 0.14] versus medium (p < 0.01) versus NHS-ICs (p < 0.001). NHS-ICs [1.15 ± 0.07] versus medium (p = N.S.). c tlr4 (RQ) [mean ± SD]. LPS [8.25 ± 0.35] versus medium [1.01 ± 0.02] (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [16.50 ± 2.12] versus medium (p < 0.0001) and versus NHS-ICs [1.15 ± 0.07] (p < 0.0001). ACA-ICs [3.65 ± 0.21] versus medium (p < 0.01) versus NHS-ICs (p < 0.0001). ARA-ICs [0.80 ± 0.14] versus medium (p = N.S.) versus NHS-ICs (p < 0.01). Anti-Th/To-ICs [0.70 ± 0.28] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). PAPS-ICs [1.85 ± 0.21] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). SLE-ICs [0.35 ± 0.17] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [1.21 ± 0.57] versus medium (p = N.S.). d tlr9 (RQ) [mean ± SD]. LPS [2.8 ± 0.14] versus medium [1.12 ± 0.03] (p < 0.001) versus NHS-ICs (p < 0.0001). ODNCpG [2.7 ± 0.28] versus medium (p < 0.001) versus NHS-ICs (p < 0.0001). ATA-ICs [1.25 ± 0.35] versus medium (p = N.S.) and versus NHS-ICs [0.75 ± 0.64] (p = N.S.). ACA-ICs [0.9 ± 0.2] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). ARA-ICs [1.4 ± 0.13] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [2.20 ± 0.42] versus medium (p < 0.01) versus NHS-ICs (p < 0.001). PAPS-ICs [1.4 ± 0.12] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). SLE-ICs [2.70 ± 0.28] versus medium (p < 0.001) versus NHS-ICs (p < 0.0001). NHS-ICs [0.85 ± 0.49] versus medium (p = N.S)

Fig. 4

Intra-cellular signaling pathways in HUVECs stimulated with SSc-ICs, PAPS-ICs, SLE-ICs, or NHS-ICs. Endothelial cells were incubated with SSc-ICs, PAPS-ICs, SLE-ICs, or NHS-ICs (1:2 dilution). IL-1β (50 U/ml) was used as a positive control. Results are expressed as the ratio of phosphorylated to non-phosphorylated forms, evaluated using the ImageJ software. Western Blotting images are representative of a single experiment. pNFκB: phosphorylated NFκB; p38MAPK: phosphorylated p38MAPK; pp46SAPK-JNK: phosphorylated p46SAPK-JNK; pp54SAPK-JNK: phosphorylated p54SAPK-JNK; pAkt: phosphorylated Akt. Histograms represent mean + standard error of the mean (SEM). *p < 0.01; **p < 0.001; ***p < 0.0001 versus medium. a pNFκB/NFkB. IL-1β [7.75 ± 0.49] versus medium [1.02 ± 0.05] (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [5.00 ± 0.55] versus medium (p < 0.0001) and versus NHS-ICs [22.75 ± 7.59] (p < 0.0001). ACA-ICs [3.00 ± 1.39] versus medium (p < 0.01) versus NHS-ICs (p = N.S.). ARA-ICs [2.17 ± 1.55] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [2.98 ± 1.75] versus medium (p < 0.01) versus NHS-ICs (p = N.S.). PAPS-ICs [2.13 ± 0.26] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). SLE-ICs [1.65 ± 0.13] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [1.25 ± 0.21] versus medium (p = N.S.). b pp38/p38. IL-1β [31.98 ± 9.37] versus medium [1.4 ± 0.4] (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [26.75 ± 7.3] versus medium (p < 0.0001) and versus NHS-ICs [22.75 ± 7.59] (p < 0.001). ACA-ICs [22.43 ± 9.57] versus medium (p < 0.001) versus NHS-ICs (p < 0.01). ARA-ICs [26.15 ± 5.27] versus medium (p < 0.001) versus NHS-ICs (p < 0.001). Anti-Th/To-ICs [24.65 ± 12.77] versus medium (p < 0.001) versus NHS-ICs (p < 0.001). PAPS-ICs [21.93 ± 7.77] versus medium (p < 0.001) versus NHS-ICs (p < 0.01). SLE-ICs [11.63 ± 9.14] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [3.2 ± 0.85] versus medium (p = N.S.). c pp46SAPK-JNK/p46SAPK-JNK. IL-1β [1.98 ± 0.17] versus medium [1.03 ± 0.05] (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [1.85 ± 0.19] versus medium (p < 0.0001) and versus NHS-ICs [22.75 ± 7.59] (p < 0.0001). ACA-ICs [1.65 ± 0.13] versus medium (p < 0.001) versus NHS-ICs (p < 0.0001). ARA-ICs [1.60 ± 0.48] versus medium (p < 0.01) versus NHS-ICs (p < 0.001). Anti-Th/To-ICs [1.23 ± 0.22] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). PAPS-ICs [1.22 ± 0.09] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). SLE-ICs [1.13 ± 0.19] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [0.88 ± 0.32] versus medium (p = N.S.). d pp54SAPK-JNK/p54SAPK-JNK. IL-1β [20.88 ± 1.56] versus medium [1.05 ± 0.06] (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [7.30 ± 0.63] versus medium [21.75 ± 6.24] (p < 0.0001) and versus NHS-ICs [22.75 ± 7.59] (p < 0.0001). ACA-ICs [3.75 ± 0.59] versus medium (p < 0.001) versus NHS-ICs (p < 0.01). ARA-ICs [2.52 ± 0.41] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [2.02 ± 0.60] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). PAPS-ICs [3.98 ± 0.24] versus medium (p < 0.001) versus NHS-ICs (p < 0.01). SLE-ICs [3.05 ± 2.17] versus medium (p < 0.01) versus NHS-ICs (p = N.S.). NHS-ICs [1.73 ± 0.28] versus medium (p = N.S.). e pAkt/Akt. IL-1β [1.90 ± 0.39] versus medium [1.02 ± 0.05] (p < 0.01) versus NHS-ICs (p < 0.01). ATA-ICs [1.50 ± 0.24] versus medium [21.75 ± 6.24] (p = N.S.) and versus NHS-ICs [22.75 ± 7.59] (p = N.S.). ACA-ICs [1.80 ± 0.48] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). ARA-ICs [1.70 ± 0.14] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [1.90 ± 0.42] versus medium (p < 0.01) versus NHS-ICs (p < 0.01). PAPS-ICs [2.02 ± 0.59] versus medium (p < 0.001) versus NHS-ICs (p < 0.01). SLE-ICs [1.42 ± 0.65] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [1.08 ± 0.10] versus medium (p = N.S)

Fig. 5

TGF-β1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with supernatants from HUVECs incubated with SSc-ICs or NHS-ICs. Fibroblasts were exposed to supernatants from HUVECs incubated with SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) was used as a positive control for collagen and mmp-1 synthesis. Histograms represent mean + standard error of the mean (SEM). *p < 0.01; ***p < 0.0001 versus medium. a TGF-β1 (pg/ml) (mean ± SD). ATA-ICs [223.2 ± 90.60] versus medium [1.08 ± 0.22] (p < 0.01) and versus NHS-ICs [56.25 ± 24.92] (p = N.S.). ACA-ICs [396.20 ± 203.90] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). ARA-ICs [207.40 ± 111.70] versus medium (p < 0.01) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [208.40 ± 97.70] versus medium (p < 0.01) versus NHS-ICs (p = N.S.). NHS-ICs [56.25 ± 24.92] versus medium (p = N.S.). b colIα1 (RQ) [mean ± SD]. TGF-β [3.37 ± 0.42] versus medium [1.05 ± 0.04] (p < 0.0001) versus NHS-ICs (p < 0.0001). ATA-ICs [2.56 ± 0.93] versus medium (p < 0.0001) and versus NHS-ICs [1 ± 0.5] (p < 0.0001). ACA-ICs [2.54 ± 0.35] versus medium (p < 0.0001) versus NHS-ICs (p < 0.0001). ARA-ICs [0.60 ± 0.08] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [0.80 ± 0.28] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [1 ± 0.4] versus medium (p = N.S.). c mmp-1 (RQ) [mean ± SD]. TGF-β [0.51 ± 0.04] versus medium [1.02 ± 0.04] (p = N.S.) versus NHS-ICs (p = N.S.). ATA-ICs [4.91 ± 3.74] versus medium (p = N.S.) and versus NHS-ICs [3.08 ± 2.40] (p = N.S.). ACA-ICs [0.13 ± 1.09] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). ARA-ICs [2.46 ± 2.0] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). Anti-Th/To-ICs [1.09 ± 0.65] versus medium (p = N.S.) versus NHS-ICs (p = N.S.). NHS-ICs [3.08 ± 2.40] versus medium (p = N.S)

Fig. 6

α-SMA and IL-6 protein expression in fibroblasts stimulated with supernatants from HUVECs incubated with SSc-ICs and NHS-ICs. Fibroblasts were exposed to supernatants from HUVECs incubated with TNF-α (10 ng/ml), SSc-ICs, or NHS-ICs (1:2 dilution). TNF-α (10 ng/ml) was used as a positive control. SN, supernatants. Histograms represent mean + standard error of the mean (SEM). *p < 0.01; ***p < 0.0001 versus medium. a α-SMA. TNF-α SN [7.0 ± 0.71] versus medium SN [1.1 ± 0.14] (p < 0.01) versus NHS-ICs SN (p < 0.01). TNF-α [7.70 ± 1.70] versus medium [0.9 ± 0.15] (p < 0.001). ATA-ICs SN [12.15 ± 2.33] versus medium SN (p < 0.0001) and versus NHS-ICs SN [1.15 ± 0.07] (p < 0.0001). ACA-ICs SN [10.58 ± 0.25] versus medium SN (p < 0.0001) versus NHS-ICs SN (p < 0.0001). ARA-ICs SN [15.90 ± 2.70] versus medium SN (p < 0.0001) versus NHS-ICs SN (p < 0.0001). Anti-Th/To-ICs SN [7.30 ± 1.69] versus medium SN (p < 0.01) versus NHS-ICs SN (p < 0.01). NHS-ICs SN [1.15 ± 0.07] versus medium SN (p = N.S.). b IL-6. TNF-α SN [12.41 ± 2.54] versus medium SN [1.05 ± 0.07] (p < 0.0001) versus NHS-ICs SN (p < 0.0001). TNF-α [6.65 ± 1.63] versus medium [0.08 ± 0.28] (p < 0.01). ATA-ICs SN [7.50 ± 2.26] versus medium SN (p < 0.001) and versus NHS-ICs SN [1.71 ± 0.17] (p < 0.01). ACA-ICs SN [3.77 ± 0.66] versus medium SN (p = N.S.) versus NHS-ICs SN (p = N.S.). ARA-ICs SN [10.77 ± 1.22] versus medium SN (p < 0.0001) versus NHS-ICs SN (p < 0.0001). Anti-Th/To-ICs SN [13.78 ± 0.73] versus medium SN (p < 0.0001) versus NHS-ICs SN(p < 0.0001). NHS-ICs SN [1.71 ± 0.17] versus medium SN (p = N.S)