Abstract
Although aplastic anemia (AA) is a T-cell mediated disease, antibodies (Abs) specific to proteins derived from hematopoietic cells are often detected in the serum of patients with AA. It is not clear whether these auto-Abs play any role in the pathophysiology of AA. A previous study demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 40% of AA patients (Takamatsu H, et al. Blood, 2007) and that anti-moesin Abs derived from AA patients could bind immune cells such as T cells and monocytes and enhance TNF-α secretion from a monocytic leukemia cell line THP-1. To substantiate the ability of anti-moesin Abs to induce the expression of myelosuppressive cytokines through immune cells, the secretion of other cytokines from THP-1 cells in response to binding of anti-moesin Abs was analyzed. THP-1 cells were incubated in the presence of PMA and LPS with 10 μg/ml of anti-moesin Abs purified from AA patients' serum or the same amount of control IgG for 48 hr. Anti-moesin Abs enhanced the secretion of TNF-α and IL-1β 2 to 5 times more than the control Abs. However, anti-moesin Abs did not enhance secretion of IL-2, IL-6 and IL-10. When peripheral blood mononuclear cells (PBMCs) isolated from 10 healthy individuals were incubated in the presence of 5 μg/ml of anti-moesin Abs alone, the amount of TNF-α in the medium was 2 to 10 times more than those of control cultures and was almost comparable to that of the culture stimulated by 100 ng/ml of LPS. Next, in order to determine whether the cytokine secretion induced by anti-moesin Abs requires FcγR cross-linking of the Abs, Fab fragments were prepared from anti-moesin Abs of an AA patient with papain treatment and the effect of the Fab fragment on THP-1 cells was examined. Purified Fab fragments failed to induce TNF-α from THP-1 cells while they moderately inhibited TNF-α secretion induced by anti-moesin Abs. Finally, to determine which signaling pathway is activated by the treatment of THP-1 cells with anti-moesin Abs, the effects of specific inhibitors of kinases were examined, including extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), all of which are involved in the signal transduction required for TNF-α secretion. The inhibition of ERK 1/2 using PD98059 suppressed TNF-α secretion induced by anti-moesin Abs in a dose dependent manner. In contrast, the interruption of JNK or p38 kinase with specific inhibitors JNK I, (L) form or SB202190 significantly enhanced TNF-α secretion in response to anti-moesin Abs in association with the up-regulation of the ERK 1/2 activity. These results indicate that anti-moesin Abs may therefore be involved in the suppression of hematopoiesis in AA patients by stimulating pro-inflammatory cytokine secretion from monocytes and that the cross-linking of the Fc portion of anti-moesin Abs and the activation of the ERK1/2-dependent pathway thus play an essential role in the anti-moesin Abs-induced TNF-α secretion from monocytes.
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