Abstract
BACKGROUND
The mechanism(s) of intravenous immunoglobulin (IVIG) towards inhibition of monocyte phagocytic activity involves the function and/or the expression of inhibitory FcγRIIb in a murine model. To confirm these findings in human monocytes, we used a human monocyte phagocytic model in vitro to study the effects of IVIG on the phagocytic activity and the expression of FcγR genes.
METHODS
Part A: Monolayer Monocyte Phagocytosis Assay
Normal volunteer’s peripheral blood mononuclear cells (PBMC) were isolated from heparin anticoagulated blood by Ficoll-Hypaque (Pharmacia Biotech) density separation. The PBMCs were washed and the monocytes were purified using a magnetic bead-positive selection method with anti-CD14 antibody (Miltenyl Biotec). 105 monocytes were incubated in a microtiter plate at 37°C for 1 hour before exposed to IVIG 0.5 g/L. Anti-D (WinRho) sensitized Rh positive (R2R2) red cells were added to the monocytes at 0.5 hour and 18 hour post-IVIG treatment. After 1 hour incubation with sensitized RBC, monocytes phagocytic activity is measured by chemiluminescence detection with a LumiCount (Packard). The readings were normalized with maximal chemiluminescence signal achieved by the monocytes without prior exposure to IVIG (positive control). Part B: RT-PCR of FcγRIIa and FcγRIIb
After 18 hours of exposure to two different concentrations of IVIG (0.5 and 5 gm/L), monocytes were collected and total RNA was isolated with TRIzol reagent (Invitrogen). 1 μg of RNA was used to generate first strand cDNA using Superscript II RT kit (Invitrogen). FcγRIIa and IIb were amplified with AmpliTaq Gold DNA polymerase system (Applied Biosystems). The PCR products were evaluated by polyacrylamide gel electrophoriesis.
RESULTS
Part A:
Dose-response curves were generated by plotting normalized chemiluminescence against the concentration of anti-D used to sensitize the red cells. Anti-D sensitized red cells were phagocytosed by monocytes in a dose-dependent manner. There is a time-dependent inhibition of monocyte phagocytosis when monocytes were incubated with IVIG at 0.5 gm/L. (Fig. 1)
Part B:
There is no significant difference in the gene expression of FcRγIIb and FcγRIIa in the adherent monocytes after incubating with either low dose (0.5 gm/L) or high dose (5 gm/L) of IVIG for 18 hours. (Fig 2)
CONCLUSION
Delayed inhibition of phagocytic activity with18-hour exposure to IVIG is not directly mediated via the modulation of FcγRIIb gene expression in human monocytes. Other mechanisms, such as intracellular signalling or receptor coupling, might be involved in the delayed inhibitory effects of IVIG.
