Induction of colony-stimulating factors by a 30-kDa secretory protein of Mycobacterium tuberculosis H37Rv.
Colony-stimulating factors (CSFs)-induced increased hematopoietic activity is known to occur in various microbial diseases; however, not much is known during tuberculosis (TB). We investigated the CSF-inducing capability of a Mycobacterium tuberculosis H37Rv component. Swiss mice intravenously injected with purified 30-kDa secretory protein of M. tuberculosis H37Rv (Mtb30; 0.1-10 mg/kg) showed enhanced levels of serum CSFs; maximum response (142 +/- 16 colonies) occurred at 1 mg/kg. In vitro, Mtb30 (1-50 mug/mL) induced mouse peritoneal macrophages (PMs) to elaborate CSFs in the conditioned medium (CM); 25 mug/mL appeared optimal (97 +/- 11 colonies). Both in vivo and in vitro, peak CSF production occurred 24 h after stimulation which levelled-off to background levels by 72 h. Rabbit anti-Mtb30 antibody significantly (p<0.05) reduced CSF production by both Mtb30-stimulated and M. tuberculosis-infected PMs, in vitro. The induced CSFs, both in the serum and CM, appeared to be functionally similar, as they supported the formation of granulocyte (G), monocyte (M) and GM colonies, in similar proportions; the GM colonies were maximum (>79 %). Neutralizing (100%) rabbit anti-mouse interleukin-1 (IL-1) polyclonal antibody did not affect the Mtb30-induced CSF production, indicating it to be IL-1-independent; whereas, CSF production was partly dependent on tumour necrosis factor-alpha (TNF-alpha), as goat anti-mouse TNF-alpha immunoglobulin G only partly inhibited it. Mtb30-induced PM production of CSFs was de novo as it was completely blocked by cycloheximide (50 mug/mL). The CSF-inducing capability of Mtb30 appeared to be proteinaceous in nature as it was heat (70 degrees C; 1 h)-labile, was destroyed by proteases (pronase E and trypsin) and was unaffected by sodium periodate treatment. Further, compared to the controls, Mtb30 induced significantly (p<0.05) high levels of immunoreactive GM-CSF (9+/-1 and 7.5+/-0.8 ng/mL) and M-CSF (4.3+/-0.5 and 3.9+/-0.4 ng/mL) in serum and CM, respectively; G-CSF levels did not increase significantly (p>0.05). Mtb30-treated mice showed a maximum of 2.23- and 2.36-fold increase, in the splenic and femur colony forming unit-GM counts, respectively, as compared to the controls. This is the first report which demonstrates Mtb30-induced production of CSFs that is up-regulated both posttranscriptionally and functionally, and thus adds to our understanding of the molecular pathogenetic mechanisms of TB.
Kaur
Sukhraj
S
National Institute of Pharmaceutical Education and Research, S. A. S. Nagar, India.
Kaur
Harsimrat
H
Singh
Prati Pal
PP
D000818:Animals; D000942:Antigens, Bacterial / Q000008:administration & dosage*; D001426:Bacterial Proteins / Q000008:administration & dosage*; D002478:Cells, Cultured; D003115:Colony-Stimulating Factors / Q000187:drug effects* / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis*; D006410:Hematopoiesis / Q000187:drug effects*; D007275:Injections, Intravenous; D017737:Macrophages, Peritoneal / Q000502:physiology*; D008297:Male; D051379:Mice; D009169:Mycobacterium tuberculosis; D014376:Tuberculosis / Q000097:blood / Q000382:microbiology / Q000503:physiopathology; D015854:Up-Regulation / Q000187:drug effects
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D003115:Colony-Stimulating Factors; D016207:Cytokines