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Beta2-glycoprotein I inhibition of mouse Kupffer cells respiratory burst depends on liver architecture
Comparative Hepatology volume 3, Article number: S43 (2004)
Kupffer cells play important roles in the modulation of immune response, phagocytosis, and senescent cell removal [1, 2]. Hydrolytic enzymes and reactive species produce the killing effects of Kupffer cells and some degree of adjacent tissue damage [1, 3]. Liver macrophages are constantly exposed to antigens from portal circulation, to which development of full inflammatory response is useless and potentially harmful . Neither tissue damage nor inflammation follows senescent cell removal by Kupffer cells, due to the physiological control of inflammation events during antigen processing [2, 5]. Apolipoproteins can modulate macrophage function . Among them, beta2-glycoprotein I (beta2GPI) decreases Kupffer cells respiratory burst while increases efficiency of C. albicans killing . Beta2GPI also binds phosphatidylserine (PS) residues on the surface of senescent cells, targeting them to clearance . In order to get an insight on the role of beta2GPI in the silent antigen removal by Kupffer cells, perfused mouse liver was used as a model of Kupffer cell-dependent phagocytosis and related respiratory burst activity, and results were correlated with those obtained in isolated mouse non-parenchymal cells.
All reagents used were obtained from Sigma (St. Louis, MO), except for beta2GPI that was purified from a pool of human sera . Livers from female CF-1 mice (20á28 g body weight) fed ad libitum were perfused with Krebs-Henseleit bicarbonate buffer pH 7.4, saturated with 95% O2/5% CO2, at 10 mL/min and 37 degrees C, without recirculation . After 15 min equilibration, O2 uptake was measured in the effluent perfusate as it flowed past a Clark-type O2 electrode. Total sinusoidal lactate dehydrogenase (LDH) efflux (in U/g liver) and the respective fractional LDH release (in % of the activity in the tissue) were assessed in the 30á45 min interval as described . Colloidal carbon (C) (0.25 mg/mL; Rotring, Germany) was infused during the 30á45 min interval, either in the absence or presence of 1á30 micrograms beta2GPI/mL, added at 20 min, and rates of C uptake were calculated according to Cowper et al. . Carbon-induced O2 consumption (in micromolar O2/g liver/min) was calculated by subtracting the basal O2 uptake, during the 30á45 min C perfusion interval . Liver samples taken after perfusion with 0.25 mg C/mL in the absence and in the presence of 30 micrograms beta2GPI were fixed in Dubosq Brazil, embedded in Paraplast, and stained with hematoxylin-eosin. Non-parenchymal liver cell preparation was obtained by liver perfusion with collagenase , with viability values higher than 95%. The respiratory burst was evaluated by a luminol-dependent assay  after zymosan stimulation (200 particles/cell) , and results were expressed as relative total light emission or light emission rate.
Results and Discussion
Liver perfusion with C in the absence of beta2GPI led to uptake of C particles and increase in O2 consumption (Fig. 1, Table 1). The latter effect is mainly related to the respiratory burst of Kupffer cells [4, 9], with secondary O2 utilization in mitochondrial respiration for energy supply needed for C phagocytosis (10) and O2 uptake induced in hepatocytes by eicosanoids released from activated Kupffer cells . Both Kupffer cell C uptake (Fig. 1A) and C-induced O2 consumption (Fig. 1B) are inhibited by beta2GPI (1á30 micrograms/mL), with significant (p < 0.05) 23% and 97% decreases being found at 30 micrograms/L beta2GPI, respectively. C-induced O2 consumption inhibition inversely correlates with beta2GPI concentration (r: -0.8455; p= 0.036). In agreement with biochemical data, optical microscopy revealed that C uptake by non-parenchymal cells is diminished by infusion of 30 micrograms/mL beta2GPI (Fig. 2). These effects by beta2GPI are achieved without changes in liver viability, evidenced by comparable fractional LDH effluxes among experimental groups (not shown), and are not mimicked by albumin infusion (Table I). Despite the beta2GPI-induced inhibition of C phagocytosis found in perfused liver, chemiluminescence of isolated non-parenchymal liver cells was insensitive to 30 micrograms beta2GPI/mL (Fig. 3).
Beta2GPI associates with membranes through annexins, PS receptor, lipoprotein receptors, and negatively charged phospholipids such as PS [5, 13]. Interference of beta2GPI with PS availability in the phagocyte membranes may affect cellular responses, such as translocation of protein kinase C (PKC) to cell membranes . This effect could affect PKC and subsequent triggering of PKC-dependent events, including superoxide anion generation and particle uptake . From current data, it is suggested that beta2GPI suppresses the respiratory burst response associated with Kupffer cell phagocytosis, while discretely diminishes particle uptake. The former effect is dependent on intact liver architecture, which allows interactions among different cell-types in the liver .
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This work was supported by grants 7000887/1000887 from FONDECYT, Chile (LAV); 97/02335-5 from FAPESP, Brazil (LFG and VBCJ). Fellowships from FAPESP (99/09505-9 to Paula R. Knox/MS; 01/04379-7 to Karin A. Simon-Giavarotti/PD).
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Gomes, L.F., Knox, P.R., Simon-Giavarotti, K.A. et al. Beta2-glycoprotein I inhibition of mouse Kupffer cells respiratory burst depends on liver architecture. Comp Hepatol 3, S43 (2004). https://doi.org/10.1186/1476-5926-2-S1-S43
- Kupffer Cell
- Respiratory Burst
- Respiratory Burst Activity
- Zymosan Stimulation
- Modulate Macrophage Function