Peripheral blood mononuclear cells (PBMCs) were isolated from Ficoll-Hypaque gradients of donor blood and cultured for 3 days in RPMI 1640 medium containing 20% fetal bovine serum (FBS), 100 units/ml interleukin-2 (IL-2) and 3 μg/ml phytohemagglutinin (PHA), plus various concentrations of OHR/AVR118 or PBS [5,8].

To generate adherent cells, PBMCs were seeded in adherence medium, RPMI 1640 supplemented with 5% FBS, 1X non-essential amino acids, 0.5X essential amino acids, 50 μM β-mercaptoethanol, 4 mM L-glutamine, 1X pen-strep, 1X gentamicin, 1X sodium pyruvate, and 0.75% glucose, and incubated for 2 h. Non-adherent cells were removed with three washes of Ca⁺²/Mg⁺²-free PBS. Adherent PBMCs were activated by culture for 72 h in medium containing 20% FBS, 0% - 10% OHR/AVR118 or PBS, plus, where indicated, 1 μg/ml E. coli 055:B5 LPS. In each experiment, all conditions were assayed in duplicate.

Protocols for culturing U937 cells (ATCC; Manassas, VA) and for their treatment with OHR/AVR118 have been described [5]. For activation, cells, seeded at 2 × 10⁵/ml, were cultured for 48 h in medium containing 100 nM PMA plus 5% OHR/AVR118 or PBS (control), with a change of medium after 24 h, following which they were cultured for an additional 48 h in fresh medium containing 5% OHR/AVR118 or PBS-control plus 1 μg/ ml E. coli 055:B5 LPS.

Adherent PBMCs were cultured for 8 days in 6-well plates with medium containing 1000 units/ml IL-4 (Pierce/Endogen, Woburn, MA) and 1000 units/ml GMCSF (Genentech, San Francisco, CA), supplemented with 5% and 10% OHR/AVR118 or PBS, replacing the medium every other day.

Conditioned cell culture media were harvested, quickfrozen in liquid nitrogen, and stored until time of assay. Concentrations of human IL-8, IL-10, IL-12, IL-1β, and TNF-α were measured using ELISA kits from Pierce/ Endogen, whereas concentrations of MCP-1, IL-6, and GM-CSF were measured using ELISA kits from R&D Systems (Minneapolis, MN). Each supernatant was assayed for each cytokine concentration in triplicate. Absorbance was measured on a PowerWave 200 Microplate Scanning Spectrophotometer (Bio-Tek Instruments; Winooski, VT); concentrations were determined by extrapolation from four-parameter logistic fit standard curves generated from dilutions of standard protein supplied by the manufacturer.

Phycoerythrin (PE)- or (FITC)-conjugated mouse monoclonal antibodies against human CD86 (B7.2), CD80 (B7.1), CD32 (FcγRII), CD87 (uPA receptor) CD88 (C5a receptor), CD14, CD18, CD54, CD36, CD64, HLA-DR, DQ, DP (MHC-class II) and HLA-A, B, C (MHC-class I), as well as the appropriate isotype controls, were purchased from BD Biosciences/PharMingen (San Diego, CA). Immunofluorescent labeling of cells with antibody conjugates for cytometric analysis and flow cytometry was performed as described [5].

Extractions of total RNA, reverse transcription, PCR amplification, and gel electrophoresis and scanning were performed as described [5,9].

Following PMA-activation of U937 cells for 48 h in the presence of 5% OHR/AVR118 or PBS, the cells were harvested, washed, and re-suspended in ice-cold Binding Buffer (HEPES-buffered saline containing 5% FBS, 1% glucose, and 1mM sodium pyruvate). Preformed, opsonized bacterial complexes were prepared by incubating FITC-Staphylococcus aureus (StaphA) with anti-StaphA antibodies (opsonizing reagent; Molecular Probes, Eugene, OR) in PBS for 1 hour at 37˚C, followed by cooling on ice. For non-specific binding, cells were incubated with 20 mg/ml of yeast mannan (Molecular Probes) for 10 min at 37˚C. PMA-activated U937 were incubated with opsonized or non-opsonized FITCor BoDipyFITC-labeled StaphA (Molecular Probes), at a 30:1 bacteria to cell ratio, in ice-cold Binding Buffer for 1 h on ice. Uptake was initiated by adding pre-warmed Binding Buffer and incubating cells at 37˚C for 10, 30, 60 and 120 min. The cells were cooled by adding an excess of ice-cold PBS containing 1% FBS and 0.02% sodium azide (PBS Wash) and transferred to ice. All further manipulations were performed on ice with cold buffers. Cells were washed thrice with PBS Wash and once with PBS, resuspended in PBS, and analyzed by flow cytometry. In some experiments, the fluorescence from cell surface-bound bacteria from replicate samples was quenched with 4% trypan blue in citrate buffer, 150 mM NaCl, pH 5.1. In other experiments, the amount of surface bound bacteria was measured using cells incubated at 0˚C throughout. Cells were also labelled with propidium iodide to exclude nonviable cells. Flow cytometry analysis of the cell-associated fluorescence was used to determine the percentage of phagocytic cells (i.e. those with positive fluorescence exceeding those of the cold bind or trypan blue controls) and extent of uptake (fluorescence intensity). To confirm the extent of activation, the secretion of IL-8 and MCP-1 and the surface expression of CD86 and CD32 were determined in all phagocytosis experiments.

Treatment of activated PBMCs with an early preparation of OHR/AVR118, was found to increase expression of IL-6 and IL-1β mRNA [8]. To confirm and extend these findings, we measured the effect of OHR/AVR118 on secretion of these cytokines under similar conditions. PBMCs were cultured with varying concentrations of OHR/AVR118 for 24 and 48 h in the presence of PHA and IL-2. Consistent with previous findings, OHR/ AVR118 stimulated the secretion of both IL-6 and IL-1β in a doseand time-dependent manner (Figure 1). Since cells of the monocyte/macrophage lineage are major sources of IL-6 and IL-1β [10,11] the effect of OHR/ AVR118 on the secretion of chemokines and cytokines was tested on PBMC-derived adherent monocytes.

The endotoxin LPS stimulates monocytes to produce several cytokines and chemokines, including IL-6, IL-1β, IL-12, TNF-α and GM-CSF [12-14]. To test the effects of OHR/AVR118 on LPS-induced cytokine secretion, PBMC-derived monocytes were cultured in the presence of LPS and varying concentrations of OHR/AVR118 for 72 h. As with total PBMCs, AVR118 stimulated LPSactivated monocytes to secrete increased amounts of IL-6 (Figure 2(A)). In addition, OHR/AVR118 induced a dose-dependent increase in the secretion of GM-CSF

(Figure 2(B)), similar to findings in total PBMCs (data not shown). Interestingly, increasing concentrations of OHR/ AVR118 suppressed the LPS-induced secretion of IL-12 and TNF-α by adherent monocytes (Figures 2(C) and (D)). In contrast to its stimulation of IL-1β secretion by PBMCs, OHR/AVR118 did not alter IL-1β secretion by LPS-activated monocytes (Figure 2(E)) and had no effect on the secretion of IL-10 (Figure 2(F)).

Primary monocytic cells can be driven by GM-CSF and IL-4 to differentiate into dendritic cells in vitro. We tested the effect of OHR/AVR118 on monocytes cultured for 4 - 8 days with GM-CSF and IL-4. Control cells showed a time-dependent increase in IL-12 secretion, which reached a maximum on day 6, but very little secretion of IL-6. When cultured in the presence of OHR/ AVR118 for 8 days, however, monocytic dendritic cells showed a 3.7-fold increase in IL-6 secretion, but a 2.9- fold decrease in IL-12 secretion, compared with control cells (Figure 3).

We previously showed the dual response of resting versus activated primary monocytes to OHR/AVR118 [5]. In addition, animal model studies suggest that OHR/ AVR118 may have alternative effects depending upon the initial state of the immune response (Cahalon L, Cohen I, personal communication). To determine further whether OHR/AVR118 has both proand anti-inflammatory properties, we treated U937 cells with OHR/ AVR118 or PBS during PMA activation for 2 - 3 days. We observed increased per cell expression of various markers characteristic of differentiating macrophages, including the costimulatory marker CD86 (B7.2) and the functional receptors CD87 (uPA receptor) and CD32 (FcγRII), as well as increases in the percentage of cells positive for CD86 and CD87 (Figure 4). AVR118 decreased the per cell expression of the MHC-class I receptors HLA-A, -B and -C, while not altering the percentage of cells positive for CD32 and MHC-class I (Figure 4). Modest increases in the per cell expression of

CD14 and CD18 were also observed, as well as a large increase in the surface expression of CD88 (C5a receptor) (data not shown). RT-PCR showed that OHR/AVR118 enhanced the expression of CD86, CD32, CD54, CD87, CD18, CD88, and CD80 mRNAs, but only slightly increased the expression of CD14 mRNA, in PMA-activated U937 cells (data not shown).

U937 cells further activated with LPS for 48 h in the presence of OHR/AVR118 showed increased overall levels of CD32, CD86, CD87, CD88, and CD14 compared with control activated cells. In contrast, OHR/ AVR118 had no effect on the surface expression of CD54, the MHC-class II receptors HLA-DR, -DQ, and -DP, and CD80, and decreased the surface expression of HLA-A, -B, -C (MHC-class I), CD36 and CD64 (data not shown).

Similar to our results with primary monocytes, we found that treatment for 24 - 48 h with OHR/AVR118 enhanced the PMA-induced secretion of IL-8, MCP-1, GM-CSF, and IL-1β and the expression of each mRNA (data not shown). In addition, OHR/AVR118 enhanced the secretion of the anti-inflammatory cytokine IL-10, after both PMAand LPS-activation (Figure 5). OHR/ AVR118 had a differential effect on IL-6 secretion, stimulating its secretion by PMA-activated U937 cells, but markedly inhibiting its secretion by cells further stimulated with LPS (Figure 6); this differential response to OHR/AVR118 was confirmed by RT-PCR (data not

shown). It should be noted that these cells did not express IL-12 or CD40 mRNA or protein (data not shown).

PMA activation of U937 cells increases their phagocytic capacity, a characteristic of differentiated macrophages. To determine the effect of OHR/AVR118 on this activity, we monitored cellular uptake of FITC-labeled StaphA following culture for 48 h with PMA plus 5% OHR/ AVR118 or PBS. Surprisingly, OHR/AVR118 inhibited phagocytosis of FITC-labeled bacteria by PMA-activated U937 cells, indicating that OHR/AVR118 inhibits the pro-inflammatory responses of highly activated (mature) monocytic cells (Figure 7). OHR/AVR118 reduced the uptake the uptake of opsonized StaphA particles, as well as the percentage cells exhibiting uptake of opsonized and non-opsonized StaphA complexes.