Download Identification of a novel autoantigen in aplastic anemia

Specific antibodies to moesin, a membrane-cytoskeleton linker protein, in the
serum of patients with aplastic anemia: their incidence and possible role in the
pathophysiology of bone marrow failure.
Shinji Nakao, Hiroyuki Takamatsu. Cellular Transplantation Biology, Kanazawa
University Graduate School of Medical Science
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 recently 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 g/ml of control IgG or anti-moesin Abs derived from
an AA patient’s serum, TNF- 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- production. These results
indicate that anti-moesin Abs may be involved in the suppression of hematopoiesis of AA
patients by stimulating TNF- production from monocytes.