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Backgrounds: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)¹⁻³. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP⁴⁻⁵, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP. Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF⁶⁻⁸. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition., Objective: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay., Methods: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMRα) with high purity⁹⁻¹³. The recombinant sGMRα was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions¹⁴(,)¹⁵. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMRα than those produced by other insects or yeasts., Results: The cell-free assay system using sGMRα yielded the data with high plasticity and reliability. GM-CSF binding to sGMRα was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMRα is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells., Conclusions: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

The aim of the project is to develop a novel method estimating granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing capacity with high-throughput and good reproducibility. For that purpose, we designed a cell-free receptor binding assay consisting of a solid-phase recombinant soluble GM-CSF receptor alpha (GMRalpha) and a biotinylated GM-CSF (bGM-CSF). Using this system, competitive inhibition of bGM-CSF binding to soluble GM-CSF receptor alpha (sGMRalpha) by GM-CSF autoantibody or IgG fractions from the sera of patients with pulmonary alveolar proteinosis was examined, resulting in excellent reproducibility. Binding inhibition was correlated with growth inhibition of TF-1 cells, a GM-CSF dependent cell line. These results suggest that our cell-free system can be applied to estimate the neutralizing capacity of GM-CSF autoantibodies ex vivo., (2010 Elsevier B.V. All rights reserved.)

Numerous reports have documented the presence of autoantibodies working against naturally occurring cytokines in humans in health and disease. In most instances, their physiological and pathophysiological significance remains unknown. However, recent advances in the methodologies for detecting cytokine autoantibodies and their application in research focused on specific disorders have shown that some cytokine autoantibodies play an important role in the pathogenesis of disease. Additionally, levels of cytokine autoantibodies may also correlate with disease severity and progression in certain infectious and autoimmune diseases but not in others. This suggests that cytokine-specific pathogenic differences exist. While multiple lines of evidence support the notion that high avidity cytokine autoantibodies are present and likely to be ubiquitous in healthy individuals, their potential physiological role, if any, is less clear. It is believed that they may function by scavenging pro-inflammatory cytokines and thereby inhibiting deleterious 'endocrine' effects, or by serving as carrier proteins, providing a 'reservoir' of inactive cytokines and thus modulating cytokine bioactivity. A central hypothesis is that sustained or repeated high-level exposure to cytokines triggers defects in T-cell tolerance, resulting in the expansion of existing cytokine autoantibody-producing B cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

High levels of granulocyte/macrophage-colony-stimulating factor (GM-CSF) autoantibodies are thought to cause pulmonary alveolar proteinosis (PAP), a rare syndrome characterized by myeloid dysfunction resulting in pulmonary surfactant accumulation and respiratory failure. Paradoxically, GM-CSF autoantibodies have been reported to occur rarely in healthy people and routinely in pharmaceutical intravenous immunoglobulin (IVIG) purified from serum pooled from healthy subjects. These findings suggest that either GM-CSF autoantibodies are normally present in healthy people at low levels that are difficult to detect or that serum pooled for IVIG purification may include asymptomatic persons with high levels of GM-CSF autoantibodies. Using several experimental approaches, GM-CSF autoantibodies were detected in all healthy subjects evaluated (n = 72) at low levels sufficient to rheostatically regulate multiple myeloid functions. Serum GM-CSF was more abundant than previously reported, but more than 99% was bound and neutralized by GM-CSF autoantibody. The critical threshold of GM-CSF autoantibodies associated with the development of PAP was determined. Results demonstrate that free serum GM-CSF is tightly maintained at low levels, identify a novel potential mechanism of innate immune regulation, help define the therapeutic window for potential clinical use of GM-CSF autoantibodies to treat inflammatory and autoimmune diseases, and have implications for the pathogenesis of PAP.

Anti-cytokine autoantibodies in healthy individuals have been widely reported but the occurrence is variable and inconstant. We hypothesized that cytokine-binding in vivo may explain their variable and infrequent detection. Therefore, we focused on the detection of the cytokine-autoantibody complexes and found that anti-cytokine autoantibody to IL-2, IL-8, tumor necrosis factor-alpha, vascular endothelial growth factor and granulocyte-colony stimulating factor were present in all 15 individuals evaluated, while those to IL-3, osteopontin and macrophage-colony stimulating factor were not detected in anyone. Autoantibodies against IL-4, IL-6, IL-10, and interferon-gamma were variously detected. Thus, we discovered that anti-cytokine autoantibodies to multiple cytokines are ubiquitous in healthy individuals.

Objectives: During the course of investigating the etiology of idiopathic pulmonary alveolar proteinosis (IPAP), the authors found that the autoantibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) was consistently present in both sera and the lung tissue. The autoantibody completely blocked bioactivity by direct binding with high specificity and avidity. Because of the existence of patients with congenital PAP who lack GM-CSF receptors and the development of PAP in GM-CSF and GM-CSF receptor knock-out mice, the autoantibody is likely to be the causative agent for IPAP. However, this finding posed the question as to why the autoantibody against GM-CSF caused lesions only in the lung., Methodology: To answer this question, the authors investigated, using immunohistochemistry, confocal laser microscopy, and immunoblotting, the expression of PU.1 in tissue macrophages. PU.1 is a critical transcription factor for terminal differentiation of alveolar macrophage (AM), as reported previously in the lung, liver, spleen, kidney, intestine and brain., Results: PU.1 was consistently expressed in the nuclei of normal AM, but faintly or not at all in IPAP-AM. Moreover, it was not expressed in the nuclei of any other normal tissue macrophages tested. Blood monocytes expressed PU.1 in the cytosol but not in the nuclei. These results suggested that the differentiation pathway is different between AM and other tissue macrophages. PU.1 was not expressed in the nuclei of newborn rat lung AM but was gradually expressed during the first 10 days., Conclusions: The authors demonstrated that GM-CSF is crucial for terminal differentiation of AM, but not for that of other tissue macrophages.