Fcγ receptor expression on splenic macrophages in adult immune thrombocytopenia

Abstract

Splenic macrophages play a key role in immune thrombocytopenia (ITP) pathogenesis by clearing opsonized platelets. Fcγ receptors (FcγR) participate in this phenomenon, but their expression on splenic macrophages and their modulation by treatment have scarcely been studied in human ITP. We aimed to compare the phenotype and function of splenic macrophages between six controls and 24 ITP patients and between ITP patients according to the treatments they received prior to splenectomy. CD86, human leucocyte antigen D-related (HLA-DR) and FcγR expression were measured by flow cytometry on splenic macrophages. The major FcγR polymorphisms were determined and splenic macrophage function was assessed by a phagocytosis assay. The expression of the activation markers CD86 and HLA-DR was higher on splenic macrophages during ITP compared to controls. While the expression of FcγR was not different between ITP and controls, the phagocytic function of splenic macrophages was reduced in ITP patients treated with intravenous immunoglobulin (IVIg) within the 2 weeks prior to splenectomy. The FCGR3A (158V/F) polymorphism, known to increase the affinity of FcγRIII to IgG, was over-represented in ITP patients. Thus, these are the first results arguing for the fact that the therapeutic use of IVIg during human chronic ITP does not modulate FcγR expression on splenic macrophages but decreases their phagocytic capabilities.

Keywords: Fc receptors; autoimmunity; macrophages; spleen.

Figure 1

Splenic macrophage phenotype between controls and immune thrombocytopenia (ITP) patients. (a) Macrophage phenotype was determined by flow cytometry. Splenocytes were first gated on the expression of CD14. The expression of the different FcγR (CD16, CD32A, CD32B, CD64), of activation markers [CD86, human leucocyte antigen D‐related (HLA‐DR)], of scavenger receptor (CD163) and of mannose receptor (CD206) were measured. Results of one representative ITP patient are depicted in grey shaded histograms. Dashed lines represent control isotype staining. The mean percentage of positive macrophages for each staining in overall patients is given. (b) Expression of CD32b was determined using clone 2B6. Five patients and one control (dashed square) had high median fluorescence intensity (MFI) compared to others that was associated with the presence of the FCGR2C–ORF polymorphism. (c) The expression of the different FcγR, CD86, HLA‐DR, CD163 and CD206 measured as the median fluorescence intensity (MFI) was compared between controls (n = 6) and ITP patients (n = 24), with exclusion of FCGR2C–ORF patients for CD32b and CD32a/CD32b expression. Results are summarized in dot‐plots. The horizontal bar represents the mean with the standard error of the mean. P‐value derived by Student's t‐test; n.s. = non‐significant; *P < 0·05; **P < 0·01.

Figure 2

Splenic macrophage phagocytic functions. To assess their phagocytic capability, macrophages isolated from the spleen of three controls and six immune thrombocytopenia (ITP) patients were incubated with fluorospheres either uncoated or coated with human immunoglobulin (Ig)G at 4°C and 37°C. For some experiments, a FcγR blocking agent was added. (a) The percentage of phagocytosis was determined by flow cytometry. (b) The localization of fluorospheres within macrophages was confirmed by fluorescence microscopy. (c) Phagocytosis index measured is summarized by histograms (mean with standard error of the mean). P‐value derived by Student's t‐test or paired t‐test, as appropriate; n.s. = non‐significant; *P < 0·05. [Colour figure can be viewed at wileyonlinelibrary.com].