Impact of CD14 on Reactive Oxygen Species Production from Human Leukocytes Primed byEscherichia coliLipopolysaccharides

Abstract

Lipopolysaccharides (LPS) from Gram-negative bacteria prime human polymorphonuclear neutrophils (PMNs) via multicomponent receptor cluster including CD14 and MD-2·TLR4 for the enhanced release of reactive oxygen species (ROS) were triggered by bacterial derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). In this study, we investigated the impact of CD14 on LPS-induced priming of human PMNs for fMLP-triggered ROS generation (respiratory or oxidative) burst. Monoclonal antibodies against human CD14 (mAbs) as well as isotype-matched IgG2a did not influence significantly fMLP-triggered ROS production from LPS-unprimed PMNs. Anti-CD14 mAbs (clone UCHM-1) attenuated LPS-induced priming of PMNs as it had been mirrored by fMLP-triggered decrease of ROS production. Similar priming activity of S-LPS or Re-LPS from Escherichia coli for fMLP-triggered ROS release from PMNs was found. Obtained results suggest that glycosylphosphatidylinositol-anchored CD14 is the key player in LPS-induced PMN priming for fMLP-triggered ROS production. We believe that blockade of CD14 on the cell surface and clinical use of anti-CD14 mAbs or their Fab fragments may diminish the production of ROS and improve outcomes during cardiovascular diseases manifested by LPS-induced inflammation.

Monoclonal Antibody to CD14, TLR4, or CD11b: Impact of Epitope and Isotype Specificity on ROS Generation by Human Granulocytes and Monocytes

“…Unexpectedly, Ca 2+ mobilization in monocytes exposed to anti-CD14 UCHM-1 Fc mIgG2a antibody has been not associated with O 2 ·− generation (SOD-inhibitable ferricytochrome C reduction) [ 64 ]. Although in our settings the anti-CD14 UCHM-1 Fc mIgG2a antibody caused certain priming effect on fMLP-triggered O 2 ·− /ROS production by human PMNs, we did not observe any statistically significant differences [ 14 ]. The data from other works have suggested that anti-CD14 UCHM-1 Fc mIgG2a antibody is able to elicit in monocytes or PMN sufficient signal for phosphoinositide breakdown and Ca 2+ mobilization but it is not enough to initiate the assembly of NADPH oxidase and O 2 ·− /ROS generation [ 14 , 78 ].…”

“…Although in our settings the anti-CD14 UCHM-1 Fc mIgG2a antibody caused certain priming effect on fMLP-triggered O 2 ·− /ROS production by human PMNs, we did not observe any statistically significant differences [ 14 ]. The data from other works have suggested that anti-CD14 UCHM-1 Fc mIgG2a antibody is able to elicit in monocytes or PMN sufficient signal for phosphoinositide breakdown and Ca 2+ mobilization but it is not enough to initiate the assembly of NADPH oxidase and O 2 ·− /ROS generation [ 14 , 78 ]. Sufficient mobilization of Ca 2+ in all monocytes but only in subset of PMN (40%) has been detected only after CD14 crosslinking by anti-CD14 Mo2 Fc mIgM or anti-CD14 MEM-18/63D3 Fc mIgG1 antibodies followed by secondary F(ab′) 2 fragments.…”

“…All together, these facts indicate CD14 as a promising therapeutic target. The impact of CD14 in TLR4-initiated signaling events has been studied in several works [ 64 , 78 – 82 ] including our own [ 14 ].…”

“…CD14 as evidenced from data presented in Table 1 is involved in LPS-dependent PMN priming [ 103 ]. The relative weak effectiveness of anti-CD14 UCHM-1 Fc mIgG2a antibody as a suppressor of LPS-dependent PMN priming for fMLP-triggered O 2 ·− /ROS production may be explained by its epitope specificity that blocks CD14 incompletely [ 14 ]. However, the inhibitory effectiveness of anti-CD14 UCHM-1 antibodies may be improved by replacing their mIgG2a isotype with mouse or human IgG1.…”

“…Today, a range of specific antibodies against CD14, TLR4, and CD11b are being used as the essential tool to elucidate their role in acute inflammation [ 1 , 5 – 16 ]. We have shown earlier that some certain antibodies such as anti-CD11b ICRF-44 Fc mIgG1 or anti-TLR4 HTA125 Fc mIgG2a are unable to ameliorate significantly the N -formyl-methionyl-leucyl-phenylalanine- (fMLP-) triggered ROS production (luminol) from LPS-primed PMNs [ 12 , 13 ], while anti-CD14 UCHM-1 Fc mIgG2a suppresses LPS priming successfully [ 14 ]. Molecular mechanisms underlying our observations, however, have not been described.…”