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Involvement of Calcium Signaling
aSektion Experimentelle Anaesthesiologie and bDepartment of Clinical Anaesthesiology, University Clinic, 89075 Ulm, Germany
Address for correspondence: E.M. Schneider, Sektion Experimentelle Anaesthesiologie, Department of Clinical Anaesthesiology, University Clinic, 89075 Ulm, Germany. marion.schneider{at}medizin.uni-ulm.de Ann. N.Y. Acad. Sci. 1010: 78-85 (2003).
Microvascular endothelial cells (mECs) circulate at higher numbers in patients with severe sepsis and hemophagocytic syndromes. Although these blood mECs might stem from damaged microvasculature, they are perfectly viable and lead to the establishment of cell lines. Such mECs were cultured in low-dose human serum pools (0.5%) and MEM-alpha medium. Antigenic profiling revealed the expression of CD36, factor VIIIa, CD95-ligand, and CD44, but also CD146. We studied the antioxidative effect of the hematopoietic growth factor G-CSF1 after in vitro stimulation with LPS from E. coli 0111:B4; the growth factor appeared to exhibit a protective effect on organ function in patients with SIRS. mECs were stimulated with 1 µg/mL of LPS for 24 h and 48 h with and without G-CSF (3x103 U/mL) preincubation. After 24 h, supernatants of the stimulated mEC were tested for IL-8 by ELISA, and cells were tested for hemoxygenase-1 (HO-1, Hsp32) by immunohistochemistry and flow cytometry using OSA110 (mAb, Stressgene). Stimulation with LPS upregulated IL-8 by a factor of 2 to 10 in mEC. Preincubation with G-CSF markedly downregulated the LPS-induced IL-8 secretion (20-50%), but IL-6 production was not affected. Upon 48 h of LPS stimulation, mECs developed massive signs of apoptosis and concomitant caspase 3 activation. Caspase 3 activity induced by LPS (24 h) or by staurosporin (6 h) was found to be dramatically downregulated by the G-CSF preincubation protocol.
Key Words: G-CSF • microvasculature • sepsis • endothelial cells • systemic inflammatory response syndrome
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