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Figure 1. PLB maturation was confirmed by flow cytometric analysis of maturation markers. (A) PLB cells were matured with vitamin D3 (Vit D3) to induce a monocytic phenotype or with dimethylformamide to induce a neutrophilic phenotype and analyzed after 4-5 days for FcγRI, FcγRII, FcγRIII and FcαR expression. (B) PLB cells were matured with Vit D3 to induce a monocytic phenotype or with dimethylformamide to induce a neutrophilic phenotype and analyzed after 4-5 days for FcαR, CD11b, CD14 and SIRL-1 expression.

Figure 2. Expression of FcγRs and FcγR-mediated ROS production in PLB cells. (A) SIRL-1 transfected PLB cells were stimulated with 20 μg/ml plate-bound human IgG (hIgG) and 10 μg/ml anti-SIRL-1 or an isotype control mAb. Measurements were corrected for spontaneous H2O2 production. Plots represent the background-corrected cumulative ROS production with and without SIRL-1 cross-linking. Either neutrophilic differentiated or monocytic differentiated PLB cells were used (n=3 in total). The representative example shows ROS production in monocyte-differentiated PLB. (B) Intracellular ROS production using dihydrodichlorofluorescein diacetate (H2DCFDA). Wild type or SIRL-1 transfected PLB cells were stimulated with 10 μg/ml plate-bound human IgA and 25μg/ml anti-SIRL-1 or an isotype control mAb. Percentage of inhibition (for calculation see Materials and Methods) by WT PLB and PLB SIRL-1 after anti-SIRL-1 mAb treatment relative to cIg mAb treated cells is shown. n=2 (neutrophilic) or n=3 (monocytic).

Figure 3. All PLB cell lines show comparable expression of SIRL-1. Expression of SIRL-1 on PLB cells stably transfected with wild type SIRL-1 (YY) and SIRL-1 tyr-phe mutants including mutation of the N-terminal ITIM (FY); the C-terminal ITIM (YF) and both ITIMs (FF). Non-transfected PLB cells were taken as a control. Cells were matured toward either a neutrophilic phenotype or a monocytic phenotype. Cells were analyzed by flow cytometry after staining with anti-SIRL-1 or an isotype control mAb (open histograms and grey histograms respectively).

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