Leishmania donovani amastigote components-induced colony-stimulating factors production

Abstract

Increased hematopoiesis, driven by colony-stimulating factors (CSFs), is known to occur in infectious diseases. However, whether Leishmania donovani component(s) can directly induce the synthesis and secretion of CSFs is not known. We report that L. donovani amastigote antigens soluble in culture medium (LDAA; 0.01–10 mg/kg), injected intravenously in BALB/c mice, induced the production of serum CSFs; maximum induction (128>16 colonies) occurred at 1 mg/kg. In vitro also, LDAA (0.01–1 mg/ml) induced mouse peritoneal macrophages (MØs) to elaborate CSFs in the conditioned medium (CM); 0.1 mg/ml LDAA appeared optimal (68±9 colonies). Both in vivo and in vitro, the kinetics of CSF production were similar with peak response occurring 24 h after stimulation and return to background levels by 72 h. A predominant ∼12 kDa LDAA protein (LDAA-12) also induced CSF production, both in serum and CM, in a dose-and time-dependent manner . Rabbit anti-LDAA-12 antibody significantly ( p<0.05) reduced both the LDAA-and LDAA-12-induced CSF production, in vitro. Functionally, the LDAA-12-induced CSFs, both in the serum and CM, appeared to be similar as they supported the formation of granulocyte (G), MØ (M) and GM colonies, in vitro, in similar proportion; GM colonies were maximum (>80%). Further, LDAA-12 induced significantly ( p<0.05) high GM-CSF levels both in serum and CM (19±3 and 15±2 ng/ml, respectively), as compared to the controls. Neutralizing (100%) goat anti-mouse tumour necrosis factor-α (TNF-α) immunoglobulin G did not affect the LDAA-12-induced CSF production by MØs, indicating it to be TNF-α-independent. LDAA-12 induced de novo CSF production, as MØs co-treated with LDAA-12 and cycloheximide (50 μg/ml) did not elaborate CSFs. The CSF-inducing capability of LDAA-12 appeared to be heat (70 C; 1 h)-labile, destroyed by proteases (pronase E and trypsin) and was unaffected by sodium periodate treatment. In LDAA-12-treated mice, the splenic and femur colony forming unit-GM counts showed a maximum of 2.2- and 1.9-fold increase, respectively, as compared to the controls. These data are the first to directly demonstrate that L. donovani amastigote components can induce the production of CSFs that may play important role(s) in the pathogenesis of visceral leishmaniasis.