Cell surface expression of intermediate filament proteins vimentin and lamin B1 in human neutrophil spontaneous apoptosis

Abstract

Neutrophils represent an important source of autoantigens for antineutrophil cytoplasmic antibody associated with vasculitis. To date, two cytoskeletal proteins, vinculin and vimentin, have been reported to be expressed on the cell surfaces of activated macrophages, platelets, and apoptotic T lymphocytes. However, such cell surface expression has never been studied in human neutrophils. As we recently demonstrated that different cytoskeletal proteins were cleaved in apoptotic neutrophils, we hypothesized that some of these were expressed on the cell surface of apoptotic neutrophils. Herein, we found that among vinculin, paxillin, gelsolin, vimentin, lamin B1, alpha-tubulin, and beta-tubulin, only the two intermediate filament (INFIL) proteins, vimentin and lamin B1, are expressed on the cell surface of 24-h aged neutrophils [spontaneous apoptosis (SA)]. By monitoring intracellular expression of vimentin and lamin B1 during SA, we found that these two proteins were cleaved and that such cleavage was reversed by the pan caspase inhibitor N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD-fmk). When neutrophil apoptosis was delayed or suppressed by lipopolysaccharide or the cytokines granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, or interleukin-4, the loss of intracellular expression of vimentin and lamin B1 was prevented. The INFIL proteins were absent from the cell surface when neutrophil apoptosis was delayed. Addition of z-VAD-fmk significantly decreased the cell surface expression of vimentin and lamin B1 during SA. This study provides the first evidence that apoptotic neutrophils express cytoskeletal proteins on their surface, opening the possibility that these cells may participate in the development of autoantibodies directed against cytoskeletal proteins, a condition frequently reported in several inflammatory diseases.

Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody

“…Intriguingly, a number of neo-epitopes are modified intracellular proteins that become exposed on the cell surface, as shown here with vimentin in the context of cellular senescence. The increased presence of MDA-modified vimentin on the surface of senescent cells may indeed serve as an "eat me" signal to immunocytes, as vimentin has been detected on the surface of apoptotic neutrophils (43,75) and T cells (76), which ultimately leads to phagocytosis by macrophages. Moreover, another study has suggested that cell-surface vimentin present on neighboring cells may interact with radiation induced O-GlcNAc-modified proteins from apoptotic cells to initiate their engulfment by macrophages (77).…”

“…In addition to playing a fundamental role as an intermediate filament in cytoskeletal assemblies, vimentin has been shown to associate with the nucleus and cell membranes of various cell types (37)(38)(39)(40)(41)(42)(43). Because 9H4 recognizes senescent cells by SACE analysis, which is an assay that requires robust antibody interactions with cell-surface antigens, we performed a cellular fractionation experiment to determine if vimentin was enriched on the surface of senescent NDFs.…”

“…Inflammation is considered to be a major consequence of SASP, which proposes that senescent fibroblasts are primed to become proinflammatory cells. Despite lacking a secretory signal sequence, vimentin is externalized on the cell surface and secreted in various physiological conditions that involve induction of proinflammatory signals, suggesting a possible phenotypic connection to SASP (42,43,50,51). We therefore sought to measure secreted vimentin using our polyreactive 9H4 antibody as a potential readout of senescent cells in vitro and in vivo.…”

The intriguing normal acute inflammatory response in mice lacking vimentin

“…More recently, we demonstrated that among the different cytoskeletal proteins we have tested, paxillin, gelsolin, vinculin, a-and b-tubulin, only the IF protein vimentin and lamin B1 were expressed on the cell surface of apoptotic neutrophils, suggesting that neutrophils may be a source of autoantigens for the development of autoantibodies directed against vimentin and lamin B1 associated with various rheumatic diseases [17]. Furthermore, the presence of vimentin on the cell surface of apoptotic neutrophils may be involved in the recognition process of apoptotic neutrophils by phagocytes for the resolution of inflammation, although this remains to be demonstrated.…”

“…Using the murine air pouch model, we have also demonstrated that vimentin was cleaved when neutrophils harvested from lipopolysaccharide (LPS)-induced air pouches were incubated in vitro with the potent pro-apoptotic plant lectin Viscum album agglutinin-I [18]. Given the importance of vimentin in general immunology [9,[19][20][21][22][23], inflammation and biology of neutrophils [10][11][12][13][14]17], and the fact that a generation of mice totally devoid of vimentin could develop and reproduce normally [3], we decided to investigate the role of vimentin during an inflammatory response in vivo and on neutrophil cell functions in vitro, two situations which have been curiously not or poorly investigated.…”