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Antiinflammatory effects of reconstituted high-density lipoprotein during human endotoxemia

High-density lipoprotein (HDL) has been found to neutralize LPS activity in vitro and in animals in vivo. We sought to determine the effects of reconstituted HDL (rHDL) on LPS responsiveness in humans in a double-blind, randomized, placebo-controlled, cross-over study. rHDL, given as a 4-h infusion at 40 mg/kg starting 3.5 h before endotoxin challenge (4 ng/kg), reduced flu-like symptoms during endotoxemia, but did not influence the febrile response. rHDL potently reduced the endotoxin-induced release of TNF, IL-6, and IL-8, while only modestly attenuating the secretion of proinflammatory cytokine inhibitors IL- 1ra, soluble TNF receptors and IL-10. In addition, rHDL attenuated LPS- induced changes in leukocyte counts and the enhanced expression of CD11b/CD18 on granulocytes. Importantly, rHDL infusion per se, before LPS administration, was associated with a downregulation of CD14, the main LPS receptor, on monocytes. This effect was biologically relevant, since monocytes isolated from rHDL-treated whole blood showed reduced expression of CD14 and diminished TNF production upon stimulation with LPS. These results suggest that rHDL may inhibit LPS effects in humans in vivo not only by binding and neutralizing LPS but also by reducing CD14 expression on monocytes.

line and cholesterol, neutralized endotoxin in whole blood more effectively than LDL, VLDL, and natural HDL (15,16). Pretreatment of animals with rHDL reduced endotoxininduced TNF production, leukopenia and lethality (17)(18)(19). The present study was designed to investigate the endotoxin-neutrahzing properties of rHDL (40 mg/kg) in humans in vivo.

Materials and Methods
Human Endotoxemia. Eight healthy male volunteers (mean age 24, range 20-28 yr) were enrolled in this double-blind, crossover, randomized, placebo-controlled study. Written informed consent was obtained from all participants. Medical history, physical and routine laboratory examination, chest x-ray and electrocardiogram were normal. The volunteers did not smoke, did not use any medication and did not have any febrile illness in the month preceding the study. Each participant was studied on two occasions, separated by a wash-out period of six weeks. On one occasion the subject was challenged with endotoxin in combination with placebo, on the other in combination with rHDL. The study was approved by the research and ethical committees of the Academic Medical Center.
The study drug rHDL Lot no. 4.955.006.0 (ZLB Central Laboratory, Bern, Switzerland) was supplied as a pyrogen-free lyophilized product with 91% apoA-1 purity (20). The appropriate amount of rHDL , in a solution containing 2% protein and 10% saccharose, was aspirated into dark-colored Amberlite syringes (Plastipak, Becton-Dickinson, Mountain View, CA) and administered as a 4-h infusion through an intravenous line at a dose of 40 mg/kg. The placebo solution consisted of isotonic saline and was administered in an identical manner.
The Escherichia coli endotoxin preparation used in this study, lot EC-6 (D. Hochstein, Bureau of Biologics, Food and Drug Administration, Bethesda, MD) was administered over one minute in an antecubital vein of the contralateral arm at a dose of 4 ng/kg, 3.5 h after the initiation of the placebo or rHDL infusion. The study was performed at a special research unit under continuous supervision of at least two physicians with emergency and resuscitation equipment immediately available. Blood pressure and heart rate were assessed every 30 min using a Dinamap device (Criticon, Tampa, FL) during the first 8 h after endotoxin challenge; oral temperature and respiratory rate were assessed at the same time points. Adverse events were registered throughout the confinement periods by a clinical symptom score. Adverse events were scored by incidence and severity (0 as absent, 1 as weakly, 2 as moderately, and 3 as severely present).

Whole Blood Incubation and In Vitro PBMC Stimulation.
In separate in vitro experiments, blood was collected into pyrogen-free tubes (Falcon 2063; 13ecton Dickinson, Mountain View, CA), containing pyrogen-free heparin (Thromboliquine®; Organon, Oss, the Netherlands, final concentration 30 IU/ml). After incubation of whole blood with rHDL (final concentration 0.5 or 2.0 mg/ml) for 1 h in a 5% CO2 incubator at 37°C, blood was diluted 1:1 in PBS and subsequently human P13MC were isolated by centrifugation over a Lymphopaque density gradient (Ficoll Paque; Pharmacia, Woerden, the Netherlands) at room temperature for 25 rain at 1,000 2-After three washes, P13MC were brought to a concentration of 5 X 106 cells/ml with H13SS and CD14 expression on monocytes was measured using FACScan ® analysis as described below. In parallel experiments, PBMC (5 X 106 cells/ml) were stimulated with LPS (E. coli 0111:134; Sigma Chem. Co., St. Louis, MO; final concentration 10 ng/ml) for 4 h at 37°C in H13SS containing 10% sterile non-acute human serum (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service CLB, Amsterdam, the Netherlands).
After incubation, samples were centrifuged and stored at -20°C until TNF analysis. All experiments were performed four times.
Assays. For leukocyte and differential counts, blood was collected in tubes containing EDTA (K3) (15%) and counted by flow cytometry (Technicon H1 system, Technicon Instruments, Tarrytown, NY). Blood for LPS measurements was collected in pyrogen-free plastic tubes (model 2063; Falcon, Oxnard, CA) containing pyrogen-free heparin (final concentration 30 IU/ml, Thromboliquine®; Organon). For the Limulus assay, platelet rich plasma was prepared by centrifugation of heparinized blood at 180 g for 10 min at 4°C and subsequently stored at -20°C. The Limulus assay was performed as described previously (21 L13P was measured in serum using reagents kindly provided by Dr. S. Carroll (Xoma Corporation, Berkeley, CA), as described previously (22). HDL cholesterol concentrations were assayed us-ing nephelometry (Boehringer Mannheim, Mannheim, Germany).
FACScan ® Analysis. At -3.5, 0, 3, and 24 h relative to endotoxin challenge blood was drawn into tubes containing 15% EDTA (I(3) and placed on ice. After lysis of the erythrocytes with isotonic NH4C1 solution (155 mmol/1 NH4C1, 10 mmol/1 KHCO3, 0.1 mmol/1 EDTA, pH 7.4), samples were centrifuged at 300 2 for 10 min and residual erythrocytes were lysed for 5 min. The remaining cells were washed twice in PBS and subsequently fixed in PBS, containing 1% BSA, 0.3 mmol/1 EDTA, 0.01% sodium azide and 0.1% paraformaldehyde (final concentration 5 X 106 cells/ml). All procedures were performed at 4°C. The following antibodies were used: FITC anti-human CD11b, FITC anti-human CD14, and FITC anti-human CD18 (CLBmon-gran/1,B2, CL13-mon/1,SG3, CLB-LFA-1/1,5D7, respectively; Central Laboratory of the Netherlands Red Cross Blood Transfusion Service CLB, Amsterdam, The Netherlands). As a control FITC IgG 1 (Becton Dickinson) was used. Cells were incubated for 30 min at 4°C after addition of the mAb to the cell suspension and washed twice in ice-cold P13S, containing 0.1% BSA, 0.3 mmol/L EDTA, 0.01% sodium azide. The mean fluorescence intensities (MFI) of labeled granulocytes and monocytes were recorded using FACS ®, after gating the cells using their forward and side scatter properties. Per time point 10,000 granulocytes and 2,500 monocytes were counted. After subtracting control IgG1 fluorescence, specific antibody binding was expressed as percent changes from pretreatment values obtained at 3.5 h before endotoxin challenge. Antibodies against apoA-1 or rHDL in the sera of the volunteers were determined by ELISA before and three months after rHDL infusion. Either rHDL or apoA-1 were used as coats on microtiter plates, and were incubated with volunteer's serum. The assay developed with a sheep antiserum against human apoA-I and an anti-sheep IgG alkaline phosphatase conjugate. Positive controls included tests of the individual sera with a tetanus toxoid coat, and a test with rabbit anti-human apoA-1 on the apoA-1 coat with the corresponding second antibody.
Statistical Analysis. Values are given as mean +SEM. Differences between placebo and rHDL treatment periods in the human endotoxemia model were tested by analysis of variance (ANOVA) for repeated measures using SPSS for Windows. Changes of parameters in time were tested using one-way ANOVA. Differences in CD14 expression and TNF production in the in vitro experiments were analyzed using the Wilcoxon test. A two-sided P value <0.05 was considered significant. Incidence and mean severity of clinical symptoms associated with endotoxemia were tabulated; summary results are given in a descriptive way.

Human Endotoxemia
Clinical Symptoms and Vital Signs. Endotoxin administration elicited clinical symptoms similar to those reported previously (23,24). Briefly, flu-like symptoms were observed including headache, chills, nausea, vomiting, myalgia and backache. All volunteers were symptom free within 24 h after endotoxin challenge.
Infusion of rHDL did not cause any side-effects or changes in routine laboratory parameters. After rHDL treatment circulating HDL cholesterol concentrations increased During the 3-mo follow up period none of the participants developed antibodies against Apo A-1 or rHDL. Furthermore, no seroconversion in HIV 1/2, hepatitis A, hepatitis B surface, hepatitis B core, hepatitis C, and parvovirus B19 status was observed within the 3-mo observation period.
As is shown in Table 1, rHDL reduced the endotoxininduced clinical symptoms. In contrast, rHDL did not influence the febrile response to endotoxin. Peak temperatures were 37.8 +-0.3°C and 37.6 -+ 0.3°C after injection of endotoxin in conjunction with placebo and rHDL, respectively.

In Vitro Whole Blood Incubation and PBMC Stimulation
After incubation of whole blood with 0.5 mg/ml rHDL, a dose corresponding to the in vivo concentrations achieved in this study, CD14 expression on monocytes was reduced to 72.5 -+ 1.0% (P <0.001). The reduction of CD14 expression on monocytes was even greater after incubation of whole blood with 2.0 mg/rnl rHDL (36.0 -+ 0.3%, P <0.001) (Fig. 8 A). PBMC isolated from rHDL-treated whole blood did not produce detectable TNF levels spontaneously. As shown in Fig. 8 B, a dose-dependent reduction in TNF production by endotoxin-stimulated PBMC (isolated from rHDL-treated whole blood) was seen from 3.9 -+ 0.2 ng/ml (control, no rHDL added) to 2.5 + 0.2 ng/ml (0.5 mg/ml rHDL) and 1.4 +-0.3 ng/ml (2.0 mg/ml rHDL) (P = 0.01 for each).

Discussion
The present study is the first to demonstrate the LPSneutralizing capacity of rHDL in humans in vivo. rHDL, given as an intravenous infusion starting before induction of endotoxemia in healthy volunteers, markedly reduced TNF, IL-6, and IL-8, while only modestly reducing the release of the proinflammatory cytokine inhibitors, IL-lra, sTNFr I, sTNFr II, and IL-10. Further, rHDL attenuated endotoxin-induced clinical symptoms and leukocyte activation. These LPS-inhibiting effects appeared in part to be mediated by a rHDL-induced downregnlation of monocyte-bound CD14, the predominant receptor for LPS.
During the rHDL treatment period, endotoxin levels, as measured with the LAL assay, were significantly higher than during the endotoxin/placebo treatment period and endotoxin remained detectable in the circulation longer. These findings are in accordance with a previous study in which patients with high HDL levels exhibited a higher LPS recovery upon ex vivo stimulation with LPS in comparison with patients with low HDL levels (25). It should be noted that the LAL assay includes a dilution and heating step in which endotoxin is recovered from LPS-binding proteins such as LBP and presumably also from HDL. The increased LAL activity after rHDL/endotoxin treatment merely indicates that rHDL indeed bound LPS in vivo. Thus, LAL activities do not necessarily represent bioactive endotoxin levels in humans in vivo.
The reduction in the release of proinflammatory cytokines by rHDL was larger than the reduction in IL-lra, sTNF receptors, and IL-10. Thus, the potency with which rHDL influenced various LPS responses differed. Conceivably, part of the LPS administered did not associate with rHDL. In this context, the threshold dose at which LPS induces fever and release of IL-lra, sTNF receptors, and IL-10 may be lower than the threshold at which it elicits release of proinflammatory cytokines and leukocyte activation.
In this study we demonstrate for the first time that CD14 expression on monocytes was increased in vivo after LPS administration. Previous in vitro studies showed that LPS is able to increase CD14 expression on monocytes (26), while others found that LPS decreased CD14 expression (27). However, clear evidence is given that blocking CD 14 on both monocytes and neutrophils attenuated LPS responses (4,28). An important and new finding is that rHDL induced a downmodulation of CD14 at the surface of monocytes in vivo and in whole blood in vitro. We demonstrated that this reduction in CD14 expression may be biologically relevant, since monocytes isolated from rHDL-treated whole blood produced less TNF upon stimulation with LPS. The mechanism by which rHDL downregulates monocyte CD14 expression remains to be elucidated. If CD14 was shed from the monocyte cell surface by rHDL, one might expect an elevation in sCD14 levels. In our study sCD14 concentrations did not increase during rHDL treatment, but a small change in sCD14 levels by rHDL might have been masked by the already high concentrations of sCD14 constitutively present in the circulation. Nonetheless, our data suggest that it is conceivable that rHDL may reduce endotoxin-induced TNF production not only by binding and neutralizing endotoxin, but also by reducing CD14 monocyte surface expression.
A previous in vitro study demonstrated that activation of granulocyte [32 integrin (CD11b/CD18) on PMN occurred by binding of LPS-LBP complexes on CD14 on PMN and could be blocked by anti-CD14 antibodies (4).Thus the reduction of CD11b/CD18 expression on neutrophils by rHDL in our study might be caused directly by reduced presentation of LPS-LBP complexes. It seems unlikely that the reduction in CDllb/CD18 is caused by rHDLinduced attenuation in CD14 expression on granulocytes because the reduction of LPS-induced increase of CD11b/ CD18 expression by rHDL preceded the rHDL-induced reduction of CD14 expression on granulocytes. As [3"2 integrins participate in the adhesion ofneutrophils to endothelium, the reduction in [32 integrin expression may prevent endotoxin-associated endothelial damage (29,30).
In conclusion, the results of this study clearly demonstrate that rHDL, given as a 4-h infusion at a dose of 40 mg/kg, dramatically reduced the endotoxin-induced inflammatory response as measured by reduced inflammatory cytokines, cell activation, and clinical symptoms in humans. This is partly caused by neutralization of endotoxin by rHDL and by rHDL-induced reduction in CD14 expression on monocytes.
T. van der Poll is a fellow of the Royal Netherlands Academy of Arts and Sciences. We are grateful to the nursing staff of the Special Research Unit for their excellent practical skills and M.O.J.A. Liska for critically reviewing the manuscript.