Natsuki Motoi

Adult-onset hereditary pulmonary alveolar proteinosis caused by a single-base deletion in CSF2RB

Background Disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signalling causes pulmonary alveolar proteinosis (PAP). Rarely, genetic defects in neonatal or infant-onset PAP have been identified in CSF2RA. However, no report has clearly identified any function-associated genetic defect in CSF2RB. Methods and results The patient was diagnosed with PAP at the age of 36 and developed respiratory failure. She was negative for GM-CSF autoantibody and had no underlying disease. Signalling and genetic defects in GM-CSF receptor were screened. GM-CSF-stimulated STAT5 phosphorylation was not observed and GM-CSF-Rβc expression was defective in the patients blood cells. Genetic screening revealed a homozygous, single-base deletion at nt 631 in exon 6 of CSF2RB on chromosome 22, which caused reductions in GM-CSF dependent signalling and function. Both parents, who were second cousins, showed no pulmonary symptoms, and had normal GM-CSF-signalling, but had a CSF2RB allele with the identical deletion, indicating that the mutant allele may give rise to PAP in an autosomal recessive manner. Conclusions This is the first report identifying a genetic defect in CSF2RB that causes deficiency of GM-CSF-Rβc expression and impaired signalling downstream. These results suggested that GM-CSF signalling was compensated by other signalling pathways, leading to adult-onset PAP.

Direct evidence that GM-CSF inhalation improves lung clearance in pulmonary alveolar proteinosis

BACKGROUND Autoimmune pulmonary alveolar proteinosis (aPAP) is caused by granulocyte/macrophage-colony stimulating factor (GM-CSF) autoantibodies in the lung. Previously, we reported that GM-CSF inhalation therapy improved alveolar-arterial oxygen difference and serum biomarkers of disease severity in these patients. It is plausible that inhaled GM-CSF improves the dysfunction of alveolar macrophages and promotes the clearance of the surfactant. However, effect of the therapy on components in bronchoalveolar lavage fluid (BALF) remains unclear. OBJECTIVES To figure out changes in surfactant clearance during GM-CSF inhalation therapy. METHODS We performed retrospective analyses of BALF obtained under a standardized protocol from the same bronchus in each of 19 aPAP patients before and after GM-CSF inhalation therapy (ISRCTN18931678, JMA-IIA00013; total dose 10.5-21 mg, duration 12-24 weeks). For evaluation, the participants were divided into two groups, high responders with improvement in alveolar-arterial oxygen difference ≥13 mmHg (n = 10) and low responders with that < 13 mmHg (n = 9). RESULTS Counts of both total cells and alveolar macrophages in BALF did not increase during the therapy. However, total protein and surfactant protein-A (SP-A) were significantly decreased in high responders, but not in low responders, suggesting that clearance of surfactant materials is correlated with the efficacy of the therapy. Among 94 biomarkers screened in bronchoalveolar lavage fluid, we found that the concentration of interleukin-17 and cancer antigen-125 were significantly increased after GM-CSF inhalation treatment. CONCLUSIONS GM-CSF inhalation decreased the concentration of total protein and SP-A in BALF, and increase interleukin-17 and cancer antigen-125 in improved lung of autoimmune pulmonary alveolar proteinosis.

IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis

The granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) is the causative agent underlying autoimmune pulmonary alveolar proteinosis (aPAP). It consists primarily of the IgG isotype. At present, information on other isotypes of the autoantibody is limited. We detected serum the IgM isotype of GMAb (IgM-GMAb) in more than 80% of patients with aPAP and 22% of healthy subjects, suggesting that a continuous antigen pressure may be present in most patients. Levels of the IgM isotype were weakly correlated with IgG-GMAb levels but not IgA-GMAb, suggesting that its production may be associated with that of IgG-GMAb. The mean binding avidity to GM-CSF of the IgM isotype was 100-fold lower than the IgG-GMAb isotype, whereas the IC(50) value for neutralizing capacity was 20,000-fold higher than that of IgG-GMAb, indicating that IgM-GMAb is only a very weak neutralizer of GM-CSF. In bronchoalveolar lavage fluid from nine patients, IgG-GMAb was consistently detected, but IgM-GMAb was under the detection limit in most patients, confirming that IgM-GMAb is functionally a bystander in the pathogenesis of aPAP. It rather may be involved in the mechanism for development of IgG-GMAb in vivo.

Identification of a mechanism for lung inflammation caused by Mycoplasma pneumoniae using a novel mouse model

Human Mycoplasma pneumoniae (MP) pneumonia is characterized by alveolar infiltration with neutrophils and lymphocytes and lymphocyte/plasma cell infiltrates in the peri-bronchovascular area (PBVA). No mouse model has been able to mimic the pathological features seen in human MP pneumonia, such as plasma cell-rich lymphocytic infiltration in PBVA. To figure out the mechanism for inflammation by MP infection using a novel mouse model that mimics human MP pneumonia, mice were pre-immunized intraperitoneally with Th2 stimulating adjuvant, alum, alone or MP extracts with an alum, followed by intratracheal challenge with MP extracts. The toll-like receptor-2, which is the major receptor for mycoplasma cell wall lipoproteins, was strongly up-regulated in alveolar macrophages in a latter group after the pre-immunization but prior to the intratracheal challenge. Those findings demonstrated that acceleration of innate immunity by antecedent antigenic stimulation can be an important positive-feedback mechanism in lung inflammation during MP pneumonia.

A cell-free assay to estimate the neutralizing capacity of granulocyte–macrophage colony-stimulating factor autoantibodies

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.

Light chain (κ/λ) ratio of GM-CSF autoantibodies is associated with disease severity in autoimmune pulmonary alveolar proteinosis☆☆

Previous studies demonstrated that antigranulocyte colony-stimulating factor autoantibody (GMAb) was consistently present in patients with autoimmune pulmonary alveolar proteinosis (aPAP), and, thus, represented candidature as a reliable diagnostic marker. However, our large cohort study suggested that the concentration of this antibody was not correlated with disease severity in patients. We found that the κ/λ ratio of GMAb was significantly correlated with the degree of hypoxemia. The proportion of λ-type GMAb per total λ-type IgG was significantly higher in severely affected patients than those in mildly affected patients, but the proportion of κ-type was unchanged. The κ/λ ratio was significantly correlated with both KL-6 and SP-D, which have been previously reported as disease severity markers. Thus, the light chain isotype usage of GMAb may not only be associated with the severity of aPAP, but may also represent a useful disease severity marker.

Reduced GM-CSF autoantibody in improved lung of autoimmune pulmonary alveolar proteinosis

To the Editors: Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterised by excessive accumulation of surfactant materials within alveolar spaces [1]. Patients with autoimmune PAP (aPAP) present a high level of granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies (GM-Ab) in the serum as well as in bronchoalveolar lavage fluid (BALF) [2]. GM-Ab neutralise the biological activity of GM-CSF in the lung [3], impairing terminal differentiation of alveolar macrophages and macrophage-mediated pulmonary surfactant clearance [4]. Based on the aetiology, clinical trials of exogenous GM-CSF supplementation have been carried out by a number of physicians with variable response rates ranging from 40 to 62% [5–9]. Previously, we reported that in three patients who received a pilot GM-CSF inhalation therapy, oxygenation was improved and the concentration of GM-Ab in BALF was reduced [7]. Bonfield et al . [8] also reported that the serum titre of GM-Ab was reduced during successful treatment of aPAP with subcutaneously injected GM-CSF. However, our recent phase II trial of GM-CSF inhalation involving 35 patients revealed that serum levels of GM-Ab remained unchanged throughout the therapy, suggesting that GM-CSF inhalation therapy did not affect the production of GM-Ab [9]. Thus, the effect of exogenous GM-CSF administration on GM-Ab levels in the serum remains controversial. This discrepancy may be due to differences in the route of administration and/or the dose of GM-CSF. Aerosolised GM-CSF reaches the lower respiratory tract and may stimulate immature alveolar macrophages directly to promote terminal differentiation and improve the local clearance of the accumulated surfactant and GM-Ab, although it does not affect the production of systemic GM-Ab. To test this hypothesis, …

A cell free assay system estimating the neutralizing capacity of GM-CSF antibody using recombinant soluble GM-CSF receptor.

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.