Stimulation of TNF-α Production from Monocytes by Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia: A Novel Mechanism of Myelosuppression by Autoantibodies.

Although aplastic anemia (AA) is a T-cell mediated disease, recent studies have revealed the presence of antibodies (Abs) specific to proteins derived from hematopoietic progenitor cells in the serum of AA patients. It is as yet unclear whether these auto-Abs play some roles in the pathophysiology of AA. We previously demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 37% of AA patients. Some reports identified moesin-like molecules on the surface of blood cells such as T cells and macrophages. It is therefore conceivable that anti-moesin Ab in AA patients may bind these immune cells and modulate hematopoietic function of AA patients. To test these hypotheses, we first studied the expression of moesin on various types of blood cells using monoclonal Ab specific to moesin (clone 38/87). Flow cytometry detected the expression of the protein recognized by anti-moesin Ab on T cells and monocytes from healthy individuals, acute monocytic leukemia cells lines including U937 and THP-1, and an acute T-lymphoblastic leukemia cell line, Molt-4, but failed to detect the molecule on CD34⁺ cells from healthy individuals and myeloid leukemia cell lines as well as B-lymphoblastic leukemia cell lines. Treatment of THP-1 cells with phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide (LPS) augmented the expression level of moesin. To confirm the expression of the moesin-like protein on the surface of monocytic leukemia cell lines, Molt-4 and THP-1 were treated with sulfo-NHS-SS-biotin, and the cell surface proteins were isolated with avidin-fixed column, and were subjected to Western blotting and peptide mass fingerprinting. Western blotting with anti-moesin monoclonal Abs showed two clear bands of proteins (75 kD and 80 kD); an amino acid sequence compatible with moesin was confirmed in the protein eluted from the 80 kD band. Next, we purified anti-moesin Abs from AA patients’ sera using affinity chromatography with recombinant moesin protein. Western blotting showed binding of the serum-derived Abs to a fraction of surface proteins of Molt-4, U937 and THP-1. When THP-1 cells were incubated in the presence of PMA and LPS with 5 αg/ml of control IgG or anti-moesin Abs derived from an AA patient’s serum, TNF-α production from THP-1 cells stimulated by anti-moesin Abs was 1.9–2.3 times as much as that from the control culture depending on the concentration of LPS. Incubation of THP-1 cells in the presence of monoclonal anti-moesin Abs showed the similar augmentation of TNF-α production. These results indicate that anti-moesin Abs may be involved in the suppression of hematopoiesis of AA patients by stimulating TNF-α production from monocytes.