Ultrastructural Alterations of Platelets and Monocytes Associated with Autoantibodies in Systemic Lupus Erythematosus

Introduction. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a high titer of autoantibodies, and SLE patients are predisposed to thrombosis. Monocytes and platelets are potentially involved in the pathogenesis of SLE. Monocytes are a source of immune modulators for T and B lymphocytes and they increase the risk of thrombosis through exposing tissue factor on their cell surface. Platelets are activated in SLE and may promote thrombotic events by expressing the procoagulant phosphatidylserine. The aim of this work was to investigate the ultrastructural changes of monocytes and platelets in SLE patients in association with titers of autoantibodies.

Methods. SLE patients were included in the study based on the diagnostic criteria of the American College of Rheumatology. Platelets from 5 SLE patients and 3 healthy donors were isolated by gel-filtration on Sepharose 2B from citrated platelet-rich plasma. Monocytes of 6 SLE patients and 3 healthy donors were isolated using density gradient centrifugation of whole citrated blood followed by negative immune-separation. The >96% purity of the isolated monocytes was confirmed using flow cytometry and the viability of the isolated monocytes assessed by exclusion of trypan blue was 95-99%. The ultrastructure of platelets and monocytes from SLE patients and healthy donors was studied with transmission electron microscopy. A total 192 individual platelets and 154 monocytes from SLE patients were analyzed and compared with 60 individual platelets and 22 monocytes from healthy donors.

Results . Most platelets from SLE patients and healthy donors had a normal discoid shape without or with 1-2 short filopodia. Intracellular components (α-granules, dense granules, open canalicular system, lysosomes and mitochondria) were well visualized, albeit somewhat less clear in the SLE platelets. The biggest morphological difference between platelets from healthy subjects and SLE patients was revealed in the platelet plasma membrane. All platelets from healthy donors had normal ultrastructure of the plasma membrane, while the membrane of SLE platelets was very often (in 70% of the cells) shaggy and rough and not as smooth as in normal platelets. About 38% of the SLE platelets were fully bordered with a shaggy plasma membrane, while 32% of the platelets had only partially abnormal plasma membrane. Cells from a patient with anti-cardiolipin and anti-beta-2-glypoprotein antibodies had the most striking membrane abnormalities that were found in 100% of platelets analyzed.

Monocytes with normal ultrastructure (ruffled cell surface, formation of filopodia, an indented nucleus, and uniform chromatin distribution) comprised 64% of the total in the SLE patients and 95% in the healthy donors. Unlike monocytes from healthy subjects, 19% of monocytes from the SLE patients had ultrastructural signs of apoptosis with a smooth cell surface, electron-dense cytoplasm, mitochondrial swelling, and a spherical nucleus with perinuclear accumulation of condensed chromatin or condensation of chromatin into dense spherical particles. About 17% of monocytes from the SLE patients and 5% from the healthy donors had necrotic features, such as smooth cell surface without or with quite a few filopodia, low electron-density cytoplasm, small electron-dense mitochondria, and spherical pyknotic nucleus with more diffuse euchromatin. The SLE patients with a high titer of anti-dsDNA antibodies (>200 IU/ml) had significantly more apoptotic (25%, p<0.01, χ²-test) and necrotic (20%, p<0.05) monocytes than those of the SLE patients with a normal titer of anti-dsDNA antibodies (<25 IU/ml) (6% and 10%, respectively).

Conclusions . In SLE patients, most platelets have abnormal ultrastructure of the plasma membrane (loss of smoothness, shagginess), likely due to deposition of pathological immune complexes. A substantial fraction of monocytes in SLE undergo morphological alternations of apoptotic or necrotic types. Such alterations were more pronounced in patients with an increased level of anti-cardiolipin, anti-beta-2-glypoprotein, and anti-dsDNA autoantibodies. The results confirm the involvement of both platelets and monocytes in the pathogenesis of SLE, including functional and structural alterations associated with increased production of various autoantibodies.

The work was supported by the Program for Competitive Growth at Kazan Federal University.