AB0135 THE MITOCHONDRIAL-RETICULAR NETWORK (MRN) OF NEUTROPHILIC LEUKOCYTES OF SYNOVIAL FLUID (SF) OF PATIENTS WITH SLE AND RA
Authors: G. V. Kudriavtseva, Y. Malenkov, V. Shishkin, V. V. Shishkin
Journal: Annals of the Rheumatic Diseases (2020)

Background: Objectives: It has been established that in cells, in particular in neutrophilic leukocytes of SF, mitochondria form a mitochondrial-reticular dynamic spatial network (MRN). MRN is the epicenter of apoptosis, reflecting structural and functional changes in the immuno-complex pathology in SLE and RA. Methods: SF was analyzed in patients: 10 SLE (43 ± 2.3 years), 13 RA (45 ± 1.6 years) and 8 donors (42 ± 3.7 years, postmortem). Neutrophilic leukocytes from the SF were isolated by standard methods and resuspended in a composition medium: 70 mM NaCl; 140 mM sucrose; 5.6 mM KCl; 10 mM pyruvate; 8 mM MOPS; pH = 7.4. The cell suspension was centrifuged for 5 min at 800 g. MRN was isolated by centrifuging the resulting supernatant for 15 min at 12 000 g. The resulting MRN fragments were resuspended in citrate-phosphate buffer (pH = 7.4) and used in experiments. The activity of adenosine monophosphate-activated protein kinase (AMPK) was evaluated by Western blotting. Quantitative determination of cytochrome C (Cyt C) was carried out by enzyme immunoassay method using the Human Cytochrome c Platinum ELISA kit (eBioscience, USA). Active forms of oxygen free radicals (AFRF) were registered by EPR. The swelling rate of MRN fragments was determined spectrophotometrically at 540 nm. The electrophoretic mobility (EM) of MRN fragments was determined by the automatic microscope “Parmoquant-2”. Results: MRN of neutrophilic leukocytes of the SF undergoes significant adaptive rearrangements during the development of SLE and RA (tab.1). On average, the expression of biochemical indicators of autophagy (AMPK), apoptosis (Сyt. C), necrosis (level of oxygen free radicals, low-amplitude swelling rate) increases by 2-3 times compared with the conventional norm. Particular attention should be paid to pathological changes in the electrokinetic potential of MRN, which determines the functional state of the SF as a whole as a colloidal system. Obviously, in SLE and RA, depletion of the energy of MRN (a sharp increase in the activity of AMRK), activation of free radical processes, disruption of intracellular ion homeostasis due to an increase in the rate of swelling of MRN as a manifestation of a compensatory-adaptive reaction. It ultimately leads to a decrease in electrokinetic properties of MRN. Thus EM is an integral indicator of physico-chemical properties and architectonics of MRN pointihg to the development of autoimmune pathology. Table 1. EXPRESSION OF INDUCTORS OF AUTOPHAGY, APOPTOSIS, NECROSIS AND ELECTROPHORETIC MOBILITY OF MRN FRAGMENTS OF NEUTROPHILIC LEUKOCYTES OF SF IN SLE AND RA Experience Terms AMPK, cond.unit/mg protein Cyt C, ng/ml AFRF, unit/mg protein Swelling rate of MRN, /min · mg protein EM, m/v · sec Donor (8 ) 0,51±0,05 23,7±5,4 7,3±2,4 0,177± 0,004 1,58± 0,07 SLE (10 ) 1,73±0,04** 49,3±6,5* 21,3±5,1** 0,435±0,005*** 0,35±0,05*** RA (13 ) 1,25±0,07** 47,8±4.8* 15,7±4,3* 0,410±0,007*** 0,41±0,07*** Notes: differences with the control norm: * - p &lt;0.05; ** - p &lt;0.01; *** - p &lt;0.001. Conclusion: Endoplasmic stress occurs in SF cells during the development of SLE and RA, blocking of autophagy and apoptosis leads to a breakdown of neutrophilic leukocyte MRN, accumulation of high molecular products of tissue decay - phlogogens in the intercellular space, among which the expression in the context is characterized by proteins - chaperones Hsp 60-100. These processes are accompanied by a shift in the bioelectric homeostasis of MRN neutrophilic leukocytes, an increase in their swelling rate and a significant decrease in their electrokinetic potential. The described MRN reactions of neutrophilic leukocytes of the SF should be taken into account when developing pharmacologically induced apoptosis as a new approach in the treatment of autoimmune diseases References: [1]Shishkin V. I. et al. Ann Rheum Dis 2017; 76: No 6, p.1077-1078; DOI:10.1136/annrheumdis-2017-eular5364 Disclosure of Interests: None declared