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Blood, Vol. 113, Issue 11, 2547-2556, March 12, 2009
Granulocyte/macrophage–colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects
Blood Uchida et al.
113: 2547
Supplemental materials for: Uchida et al
Files in this Data Supplement:
- Document 1. Supplemental results and discussion (PDF, 15.2 KB)
- Table S1. Concentration response of the calibration curve for measuring GM-CSF autoantibody concentration in normal human serum* (PDF, 43.7 KB)
- Figure S1. Detection of GM-CSF autoantibodies in healthy individuals (JPG, 59.1 KB)
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(A) A standard curve for quantification of GM-CSF autoantibodies for the ELISA was generated by serial dilution of highly purified GM-CSF autoantibodies as described in the Methods. Each symbol represents the mean (± SE) optical absorbance of 5 samples determined at each concentration. (B) The specificity of the ELISA was determined by measuring serum from healthy individuals that had been depleted of IgG (No IgG) and then spiked with either GM-CSF autoantibody (GM-CSF autoAb) or with IgG1 – 4 (indicated). Each bar represents the mean (± SE) of five determinations. Asterisks indicate optical absorbance values below the assay sensitivity. (C) Serum GM-CSF autoantibody concentrations in healthy individuals (n = 72) were measured using a GM-CSF capture antigen that was glycosylated (Berlex) or unglycosylated (Invitrogen). Data are shown as whisker plots indicating the interquartile range (upper and lower borders of box), the 90th and 10th percentile (error bars), the 95th and 5th percentile (upper and lower open symbols), the median (solid horizontal line in box), and mean (dashed line in box) values of GM-CSF autoantibody levels.
- Figure S2. Measurement of Fc-receptor–mediated phagocytosis in whole blood (JPG, 59.4 KB)
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Phagocytosis was first measured in isolated neutrophils resuspended in buffered HBSS at various concentrations (A–B) to verify the assay conditions before measuring the endogenous phagocytic capacity of unstimulated neutrophils in fresh whole blood from healthy individuals (C). Fc-receptor mediated neutrophil phagocytosis was measured using flow cytometry as described in the Methods. (A) The coefficient of phagocytosis was calculated as the percentage of neutrophils containing internalized microspheres multiplied by the mean fluorescence intensity of phagocytic neutrophils. Each symbol represents the mean (±SE) of four determinations at the indicated neutrophil concentration. Data for the coefficient of phagocytosis were fit by regression to the equation, f(x) = 2.01 × 108 (1 – exp(– 0.0014×)), resulting in R2 = 0.974 and P < 0.0001. (B) The phagocytic capacity was calculated as the coefficient of phagocytosis multiplied by the neutrophil concentration. Each symbol represents the mean (±SE) of four determinations at the indicated neutrophil concentration. Data for the phagocytic capacity were fit by regression to the following equation: f(x) =9.2665 × exp(– 0.5 ((×-56.1983)/15.1742)2), resulting in R2 = 0.981 and P < 0.0001. (C) The endogenous baseline phagocytic capacity of neutrophils in unstimulated whole blood was measured repeatedly (n = 10) in healthy subjects (n = 8) to determine the intra-subject and inter-subject subject differences. An asterisk indicates the difference between inter and intra-subject values was significant at a P value of < 0.001.
- Figure S3. Impaired phagocytosis of alveolar macrophages in autoimmune PAP (JPG, 126 KB)
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Alveolar macrophages healthy controls (HC) or PAP patients (PAP) were evaluated for phagocytic uptake of various fluorescently-labeled pathogens or particles (red-orange color) as indicated. Phase contrast photomicrographs are included for cell visualization. Autofluorescence (green color) can be seen in some cells. The serum GM-CSF autoantibody level in the PAP patient was 227 µg/ml. Original magnification is 40×; bar represents 40 µm.
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