The Role of CD68+ Cells in Bronchoalveolar Lavage Fluid for the Diagnosis of Respiratory Diseases

Addressing the critical challenge in the differential diagnosis of severe inflammatory lung diseases, we propose a novel methodology for the analysis of macrophage surface recep-tors, CD68 and CD206, using specific non-antibody ligands. We aimed to develop a non-antibody alternative for the fluorometric detection of CD68+ cells, focusing on mac-rophages as key functional markers in inflammatory processes. Our marker based on di-oleylphosphatidylserine (DOPS), a specific ligand to CD68, incorporated into a liposomal delivery system. The specificity of this DOPS-based ligand can be precisely modulated by the liposome's composition and the polyvalent presentation of the ligand. We synthesized a series of fluorescently-labeled DOPS-based ligands and developed a liposome-based sandwich fluorometric assay. This assay enables the isolation and quantification of CD68 receptor presence from bronchoalveolar lavage fluid (BALF). The results confirmed the specific binding of DOPS/lecithin liposomes to CD68+ cells compared to control lecithin systems. Furthermore, the incorporation of PEGylated 'stealth' liposomes significantly enhanced binding specificity and facilitated the generation of distinct binding profiles, which proved valuable in differentiating various inflammatory conditions. This approach yielded unique binding profiles of PS-based ligands to CD68+ cells, which varied signifi-cantly among a broad range of respiratory conditions, including primary ciliary dyskine-sia, bronchial asthma, bronchitis, bacterial infection, pneumonia, and bronchiectasis. Confocal Laser Scanning Microscopy demonstrated selective binding and intracellular localization of the DOPS-based marker within CD68+ macrophages from BALF samples of patients with bronchitis or asthma. Fourier-transform infrared (FTIR) spectroscopy has revealed changes in characteristic bands of the FTIR spectra upon binding of DOPS-based marker with CD68+ cells, providing a quantitative measure of binding parameters and potentially able to differentiate between CD68+ cells subtype profile. The binding parame-ters of this multivalent composite ligand with the CD68 receptor are comparable to those of antibodies. This suggests that for certain in vitro diagnostic applications (e.g., BALF analysis in bronchial asthma), where the cellular diversity is limited, the inherent binding specificity of phosphatidylserine may offer a sufficient and viable alternative to conven-tional antibodies. Our results demonstrate the remarkable potential of this novel approach for the developing non-antibody-based systems for the differential diagnosis of the respir-atory diseases.