Both Fcgamma and complement receptors mediate transfer of immune complexes from erythrocytes to human macrophages under physiological flow conditions in vitro

Abstract

Abnormal clearance by the mononuclear phagocytic system of immune complexes (IC) is important in the pathogenesis of systemic lupus erythematosus (SLE). We have developed an in vitro model to investigate the cellular mechanisms involved in the transfer of soluble IC from erythrocytes to human macrophages under physiological flow conditions. In this assay, erythrocytes bearing fluorescently labelled IC are perfused over monolayers of human monocytes or monocyte-derived macrophages in a parallel-plate flow chamber, and transfer quantified using confocal microscopy and flow cytometry. Using aggregated human IgG as a model IC, we have been able to demonstrate transfer of IC from erythrocytes to macrophages. Blocking studies with specific neutralizing antibodies have shown that both complement and Fcgamma receptors are required for IC transfer. Blockade of CR4 (alpha(x)beta(2) integrin), FcgammaRIIa or FcgammaRIII reduced transfer, while anti-CR3 (alpha(m)beta(2) integrin) had no effect. Blockade of CR3, FcgammaRIIa or FcgammaRIII also reduced the number of adhesive interactions between fluorescently labelled IC-bearing erythrocytes and macrophage monolayers. Taken together with the transfer data, this suggests differing roles for these receptors in the human IC transfer reaction that includes an adhesive function which facilitates IC processing by mononuclear phagocytes. Finally, a functional effect of the FcgammaRIIa R131/H131 polymorphism, important in susceptibility to SLE, has also been demonstrated using this model. Uptake of IgG(2) but not IgG(1)-containing soluble IC was reduced by macrophages from individuals homozygous for the R131 allelic variant of the receptor.

Fig. 1

Soluble immune complexes (IC) are transferred from flowing erythrocytes and internalized by monocytes. Heat-aggregated gamma globulin (HAGG) labelled with Alexa Fluor® 488 was opsonized using 20% human serum and incubated with erythrocytes for 10 min, prior to flow over monolayers of human monocytes at 0·55 ml/min. Cross-sectional confocal images are shown of the monocyte monolayer demonstrating IC that have been internalized by these cells after transfer from the flowing erythrocytes.

Fig. 2

Transfer of fluorescently labelled immune complexes (IC) from erythrocytes to mononuclear phagocytes flow conditions. The flow chamber system was adapted to allow continuous recirculation of erythrocytes bearing IC over monocyte monolayers. (a) Soluble IC were lost progressively from erythrocytes during recirculation: erythrocytes at a 1% haematocrit bearing opsonized heat-aggregated gamma globulin (HAGG) were recirculated over monocyte monolayers at 0·55 ml/min for 30 min. Aliquots were removed at intervals and stained using a fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG antibody and analysed by flow cytometry. Results are expressed as the percentage reduction in the number of cells positive for HAGG relative to the number positive prior to flow (time 0). (b) A representative fluorescence activated cell sorter histogram showing a reduction in fluorescent signal on erythrocytes removed after 10 min of recirculation through the flow chamber.

Fig. 3

Blockade of FcγRIIa, FcγRIII or CR4 reduces uptake by macrophages of immune complexes (IC) transferred from erythrocytes under physiological flow conditions in vitro. Heat-aggregated gamma globulin (HAGG) labelled with Alexa Fluor® 488 was opsonized and bound to erythrocytes. These were perfused at 0·55 ml/min over macrophage monolayers that had been pretreated specific neutralizing antibodies against (a) FcγRI, RIIa and RIII or with unlabelled HAGG, or (b) against CR1, CR3 or CR4 or with the RGD tripeptide. Uptake of labelled HAGG by the macrophages was quantified by confocal microscopy in terms of the percentage cells positive for HAGG. *P = 0·015 **P = 0·003 #P = 0·033.

Fig. 4

Larger immune complexes (IC), formed at a lower antigen : antibody ratio, are transferred from erythrocytes to macrophages more efficiently than smaller complexes and their uptake is inhibited by blockade of CR4 on the macrophage. (a) Fluorescently labelled hepatitis B surface antigen (HBsAg)/anti-HBsAg IC were formed at varying antigen : antibody ratios, opsonized using human serum and bound to erythrocytes. These were perfused over monocyte-derived macrophages, with uptake by measured by confocal microscopy. (b) Erythrocytes bearing fluorescently labelled HBsAg/anti-HBsAg IC were perfused over macrophage monolayers that had been pretreated with neutralizing antibodies against complement receptor type 3 (CR3) or CR4. IC were formed at antigen : antibody ratios of either 1 : 4 or 4 : 1 and their uptake by the macrophages was quantified by confocal microscopy in terms of the percentage cells positive for the IC. *P = 0·028 **P = 0·003 #P = 0·009.

Fig. 5

Blockade of either CR3, FcγRIIa or FcγRIII reduces the number of adhesive interactions between erythrocytes bearing immune complexes (IC) and macrophage monolayers. Erythrocytes were incubated with opsonized heat-aggregated gamma globulin (HAGG), fluorescently labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and perfused over macrophage monolayers that had been preincubated with neutralizing antibodies against (a) and (b) complement receptor type 1 (CR1), CR3 and CR4 or (c, d) FcγRI, FcγRIIa and FcγRIII, or media containing control Ig. The number of (a) and (c) rolling or the number of (b, d) arrested cells in 10 random fields was quantified using Cell Motion Analysis software. Values are means ± s.e.m. (n = 3 independent experiments). *P = 0·028 #P = 0·016 **P = 0·003.

Fig. 6

Uptake of IgG₂ but not IgG₁-containing soluble immune complexes (IC) is reduced by macrophages from individuals homozygous for the R131 allelic variant of FcγRIIa. Monocytes were isolated from the peripheral blood of healthy individuals who had been genotyped for the R131/H131 polymorphism of FcγRIIa. These cells were differentiated into macrophages in vitro to form monolayers and then exposed to either fluorescently labelled (a) aggregated human IgG₂ or (b) hepatitis B surface antigen (HBsAg)/anti-HBsAg IC (which contain mainly IgG₁ antibodies) in the fluid phase under physiological flow conditions in the parallel-plate flow chamber. Uptake was quantified by confocal microscopy in terms of the number of the number of cells positive for IC per field of view. **P = 0·001.