Idiopathic pulmonary alveolar proteinosis (I-PAP) is a rare disease of unknown etiology in which the alveoli fill with lipoproteinaceous material. We report here that I-PAP is an autoimmune disease with neutralizing antibody of immunoglobulin G isotype against granulocyte/macrophage colony-stimulating factor (GM-CSF). The antibody was found to be present in all specimens of bronchoalveolar lavage fluid obtained from 11 I-PAP patients but not in samples from 2 secondary PAP patients, 53 normal subjects, and 14 patients with other lung diseases. It specifically bound GM-CSF and neutralized bioactivity of the cytokine in vitro. The antibody was also found in sera from all I-PAP patients examined but not in sera from a secondary PAP patient or normal subjects, indicating that it exists systemically in I-PAP patients. As lack of GM-CSF signaling causes PAP in congenital cases and PAP-like disease in murine models, our findings strongly suggest that neutralization of GM-CSF bioactivity by the antibody causes dysfunction of alveolar macrophages, which results in reduced surfactant clearance.

Previously, we reported the specific occurrence of neutralizing autoantibodies against granulocyte–macrophage colony–stimulating factor (GM-CSF) in the bronchoalveolar lavage fluid from 11 Japanese patients with idiopathic pulmonary alveolar proteinosis (I-PAP). The autoantibody was also detected in sera from all 5 I-PAP patients examined. To determine that the existence of the autoantibody is not limited to the Japanese patients, we examined sera from 24 I-PAP patients in five countries and showed that the autoantibody was consistently and specifically present in such patients. Thus, detection of the autoantibody in sera can be used for diagnosis of I-PAP. To establish a simple and convenient method for diagnosis of I-PAP, we developed a novel latex agglutination test using latex beads coupled with recombinant human GM-CSF. GM-CSF binding proteins isolated from the sera using the latex beads were identified as the autoantibodies of IgG₁ and IgG₂. The titer of the autoantibody determined by this test correlated with that determined by ELISA. Agglutination was positive in 300-fold diluted sera from all 24 I-PAP patients, but negative in sera from four secondary PAP patients, two congenital PAP patients, 40 patients with other lung diseases, and 38 of 40 normal subjects. These results establish that the latex agglutination test is a reliable method for serological diagnosis of I-PAP with high sensitivity (100%) and specificity (98%).

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-{2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl}-3-O-[(R)-3-hydroxy-tetradecanoyl-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 1,4′-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4′ position showed slightly lower activities than the compound with the bisphosphorylated compound(Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumor-promoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.