Endotoxin binding and elimination by monocytes: secretion of soluble CD14 represents an inducible mechanism counteracting reduced expression of membrane CD14 in patients with sepsis and in a patient with paroxysmal nocturnal hemoglobinuria

Abstract

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% +/- 0.8% in healthy subjects to 50.5% +/- 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-alpha) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-alpha release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 +/- 0.4 and 20.1 +/- 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-alpha.

FIG. 1

Specificity and time scale of the EECs of PBMCs from healthy volunteers. Different numbers of PBMCs were incubated with 1 ng of smooth LPS per ml, and changes in EECs determined by the LAL test are expressed. ○, 2 × 10⁶ PBMCs from 15 healthy volunteers; ⧫, 2 × 10⁵ PBMCs from 10 healthy volunteers; •, 2 × 10⁴ PBMCs from 7 healthy volunteers; ▿, 2 × 10⁶ cells of the pancreatic cancer cell line SW1116 (n = 3), ▴, 2 × 10⁶ cells of the colorectal cancer cell line HT29 (n = 3); ◊, 2 × 10⁶ cells of the hepatoblastoma cell line HepG2 (n = 3).

FIG. 2

EEC of a patient suffering from PNH under different clinical states. PBMCs (2 × 10⁶) from a patient suffering from PNH with different clinical statuses were incubated with 1 ng of smooth LPS (E. coli O55:B5) per ml. Changes in EEC determined by the LAL test are expressed. ○, 2 × 10⁶ PBMCs from 15 healthy volunteers; ▴, ▵, and ⧫, 2 × 10⁶ PBMCs from the PNH patient in a septic state, in a state of recovery from acute cholecystitis and in a healthy state, respectively.

FIG. 3

Comparison of levels of binding of FITC-labeled LPS by monocytes from a PNH patient in different clinical states. ○, 2 × 10⁶ PBMCs from eight healthy volunteers; ▾, ▵, and ▪, 2 × 10⁶ PBMCs from the PNH patient in a state of recovery from sepsis, in a state of recovery from cholecystitis, in a healthy state, respectively; ◊, control without any FITC-labeled LPS.

FIG. 4

Expression of mCD14 and HLA-DR on gated monocytes; 10⁶ PBMCs harvested from healthy volunteers (n = 15) (□), patients with sepsis (n = 13) (░⃞), and a PNH patient in a state of sepsis (▪) were stained with RPE-labeled anti-CD14 (Leu-M3) or FITC-labeled anti-HLA-DR. Values are means of percent positive monocytes (P < 0.01 compared to healthy subjects).

FIG. 5

Endotoxin-induced TNF-α release from PBMCs. The levels of TNF-α after stimulation with 1 ng of LPS (E. coli O55:B5) per ml are given. ○ and ▿, 2 × 10⁶ PBMCs from 15 healthy volunteers with and without MEM-18, respectively; • and ▴, 2 × 10⁶ PBMCs from 13 patients with sepsis with and without MEM-18, respectively; ◊, control with no LPS stimulation.

FIG. 6

Spontaneous and endotoxin-induced release of sCD14. The levels of sCD14 after stimulation with 1 ng of LPS (E. coli O55:B5) per ml are indicated. Values are for • and ○, 2 × 10⁶ PBMCs from six healthy volunteers, stimulated with LPS and unstimulated, respectively; ▴ and ▵, 2 × 10⁶ PBMCs from six patients with sepsis, stimulated with LPS and unstimulated, respectively.