Reduced number and impaired function of circulating progenitor cells in patients with systemic lupus erythematosus

Abstract

Systemic lupus erythematosus (SLE) is associated with premature and accelerated atherosclerosis. Circulating progenitor cells (CPCs) are circulating bone-marrow derived cells that play an important role in the repair of vascular damage that underlies the development of atherosclerosis. The objective of this study was to determine the number and functionality of CPCs in patients with SLE. The study included 44 female SLE patients in an inactive stage of disease and 35 age-matched female controls. CPC numbers in the circulation were determined by FACS with monoclonals against CD14, CD34 and CD133. Peripheral blood-derived mononuclear cell (PBMNC) fractions were cultured in angiogenic medium. The endothelial-like phenotype was confirmed and the colony forming unit (CFU) capacity, migratory capacity and the potential to form clusters on Matrigel were determined. Expression of apoptosis inhibiting caspase 8L was analyzed in PBMNCs and CPCs by gene transcript and protein expression assays. The number of CD34-CD133 double-positive cells (P < 0.001) as well as the CFU capacity (P = 0.048) was reduced in SLE patients. Migratory activity on tumor necrosis factor-alpha tended to be reduced in patient CPCs (P = 0.08). Migration on vascular endothelial growth factor showed no significant differences, nor were differences observed in the potential to form clusters on Matrigel. The expression of caspase 8L was reduced at the transcriptional level (P = 0.049) and strongly increased at the protein level after culture (P = 0.003). We conclude that CPC numbers are reduced in SLE patients and functionality is partly impaired. We suggest these findings reflect increased susceptibility to apoptosis of CPCs from SLE patients.

Figure 1

Number of CD14, CD34, and CD133 positive cells per ml of peripheral blood. The number of peripheral blood derived mononuclear cells from 20 patients and 20 controls that stained single-positive for CD14, CD34 and CD133 and co-stained for CD34 and CD133 were quantified by fluorescence activated cell sorting and are depicted here as the amount of positively stained cells per milliliter of peripheral blood (the line represents the median; *P < 0.05; **P < 0.01; ***P < 0.001).

Figure 2

Adhesion assay and number and morphology of colony forming units (CFU). (a) Peripheral blood derived mononuclear cells (PBMNCs) from ten patients and ten controls were cultured overnight, percentages of attached cells were determined. (b) PBMNCs from ten patients and ten controls were cultured for one week after which CFU were formed and counted. In (a,b), the line represents the mean; *P < 0.05). Representative images of (c) a patient and (d) a control culture (the arrows point to a CFU). Notice the smaller size and the lower number of emerging cells when comparing patient CFU to control CFU.

Figure 3

Migratory response of circulating progenitor cells (CPCs) to tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF). CPCs from ten patients and ten controls were cultured in angiogenic medium for 14 days, detached and placed in the upper chamber of a migration chamber. The lower chamber was filled with medium alone or with medium with either 50 ng/ml VEGF or 50 ng/ml TNF-α. Migration on VEGF and TNF-α was calculated as percentage of migration increase of CPCs compared to spontaneous migration on medium only. The line represents the mean.

Figure 4

Cluster formation on matrigel. Circulating progenitor cells (CPCs) from ten patients and ten controls were cultured in angiogenic medium for 14 days. Cells were detached, resuspended in culture medium and 100,000 cells were added to matrigel coated wells. (a) After 18 hours the number of clusters containing more than ten cells were counted manually in three high power fields per sample. The line represents the mean. (b) A representative image of cluster formation on matrigel. The inset shows an example of a spouting CPC.

Figure 5

Caspase 8(L) expression. The expression of caspase 8(L) was analyzed at the transcriptional and protein expression levels (n = 15 for both groups and n = 5 for both groups, respectively). (a) At the transcriptional level, the uncultured peripheral blood derived mononuclear cells (PBMNCs) of systemic lupus erythemathosus patients showed a reduced caspase 8L:caspase 8 ratio. Representative images from patient and control bands are shown. (b) At the protein level, the ratios of the expression of caspase 8L:caspase 8a+b increased in the systemic lupus erythemathosus patient cells after culture for 14 days when compared to the uncultured PBMNC expression ratios, whereas the expression ratios remained the same in healthy control cells. Representative bands from the western blot are shown.