Totally, 35 patients with untreated SSс and 25 control subjects without autoimmune and other chronic diseases matched by age and gender were included in the study. Mean age of SSc and control participants was 51 [43; 59] and 49 [42; 57] years old, respectively, p = 0.358. The study was performed in accordance with the Declaration of Helsinki of 1975 and its revised version of 2013. The study protocol was approved by the Local Ethics Committee of the Nasonova Research Institute of Rheumatology at February, 10, 2022. All study participants provided written informed consent prior the inclusion in the study.
The diagnosis of SSc met the criteria of the American College of Rheumatology / European Antirheumatic League (ACR/EULAR) 2013 [8]. Most patients (86%) were diagnosed with a limited form of SSc. Continued disease survival was calculated from the time of onset the first "non-Reynaud" symptom. Average disease duration was 5 [2; 11] years. Average disease activity index was 1,9 [1,1; 2,6] points. Interstitial lung disease was diagnosed in 20% of patients; pulmonary arterial hypertension was diagnosed in 12% of patients. Anti-topoisomerase I antibodies were detected in 27% of patients; anti-centromere antibodies were detected in 40% of patients.
Table 1 demonstrates the results of measurements of pro-inflammatory cytokine secretion in primary culture of monocytes from control and SSc study participants.
Table 1
Pro-inflammatory cytokine secretion by cultured monocytes of study participants
Cytokine secretion | Control, pg/ml | SSc, pg/ml | Significance, p |
TNF-α |
non-stimulated | 78 [54, 116] | 133 [95, 244] | < 0.01* |
LPS-stimulated | 2785 [1682, 4166] | 4861 [3095, 5614] | < 0.01* |
without re-stimulation | 87 [47, 145] | 96 [78, 113] | > 0.05 |
after re-stimulation | 91 [73, 146] | 100 [85, 133] | > 0.05 |
IL1β |
non-stimulated | 84 [48, 108] | 236 [118, 261] | < 0.01* |
LPS-stimulated | 753 [575, 1039] | 1439 [1089, 2207] | < 0.01* |
without re-stimulation | 91 [74, 99] | 215 [153, 239] | < 0.01* |
after re-stimulation | 89 [71, 101] | 207 [148, 241] | < 0.01* |
CCL2 (MCP1) |
non-stimulated | 1982 [1501, 3737] | 5817 [2698, 13041] | < 0.01* |
LPS-stimulated | 43304 [26313, 59109] | 28861 [12678, 66766] | > 0.05 |
without re-stimulation | 2308 [1670, 3290] | 8657 [3219, 12477] | < 0.01* |
after re-stimulation | 3109 [2307, 4086] | 4917 [2000, 8514] | < 0.01* |
IL8 (CXCL8) |
non-stimulated | 9721 [6804, 20579] | 32022 [12870, 48973] | < 0.01* |
LPS-stimulated | 300147 [145684, 327361] | 536414 [100492, 605849] | > 0.05 |
without re-stimulation | 10139 [7135, 20329] | 18412 [7955, 35379] | > 0.05 |
after re-stimulation | 20108 [14489, 28112] | 30100 [18417, 96664] | < 0.05* |
Data presented as median and quartiles, Me [Q1, Q3].
Basal and LPS-stimulated secretion of pro-inflammatory cytokines were measured to evaluate pro-inflammatory activation of circulating monocytes. It was demonstrated that non-stimulated and LPS-stimulated secretion of TNF-α and IL1β were significantly higher in SSc group than in healthy participants. Basal secretion of chemokines CCL2 and IL8 was also significantly increased in SSc group while LPS-stimulated secretion of CCL2 and IL8 didn’t differ significantly between control and SSc groups. The ratio of LPS-stimulated and basal secretion of cytokines was considered as pro-inflammatory activation of monocytes. The pro-inflammatory activation in terms of TNF-α and IL1β was not significantly different in control and in SSc groups, and was 32.4 [17.4, 61.8] in control group vs. 42.0 [12.7, 54.0] in SSc group, p > 0.05 for TNF-α; 9.9 [6.9, 16.6] vs. 7.9 [5.0, 12.5], respectively, p > 0.05 for IL1β. Activation in terms of CCL2 and IL8 was significantly reduced in SSc patients in comparison with control group, and was 20.9 [4.9, 35.7] in control group vs. 2.7 [1.6, 10.2] in SSc group, p < 0.01 for CCL2, 17.3 [7.6, 54.8] vs. 11.7 [2.7, 23.9], respectively, p < 0.05 for IL8.
On the 7-th day of cultivation, re-stimulated and non-re-stimulated TNF-α secretion wasn’t different in control and SSc groups. Secretion of IL1β and CCL2 was significantly higher in cells of SSc patients. As for IL8 secretion, it was increased in SSc group, but only re-stimulated secretion of IL8 was significantly higher in SSc in comparison with control group. The second stimulation with LPS was conducted to evaluate the response of monocyte on re-stimulation after the first stimulation followed the rest in refreshed medium. To characterize the tolerance of immune response of monocytes, the ratio of re-stimulated and non-re-stimulated secretion was calculated. The ratio of TNF-α and IL1β secretion was the same in control and SSc groups, and was 1.0 [1.0, 1.3] vs. 1.1 [1.0, 1.2], respectively, p > 0.05 for TNF-α; 1.0 [1.0, 1.0] vs. 1.0 [1.0, 1.1], respectively, p > 0.05 for IL1β. The ratio of CCl2 secretion was significantly higher in SSc group, and was 1.8 [1.1, 3.0], compared to 1.4 [1.0, 1.7] in control group, p < 0.05. The ratio of IL8 secretion was very different among SSc patients, and was averaged 1.9 [1.2, 3.4] vs. 1.8 [1.2, 2.2] in control group, so the difference between SSc and healthy groups wasn’t statistically significant, p > 0.05.