Vol 27 No 2 December - The Indian Society for Parasitology

90 CSF production induced by L. donovani amastigote components JPD : Vol. 27 (2), 2003
in the presence of goat anti-mouse TNF-α polyclonal
immunoglobulin G (IgG; R & D Systems). Data in
Table VI show that anti-mouse TNF-α antibody did
not block LDAA-induced CSF production. To
determine the functional properties of LDAA-induced
CSFs in serum and CM, the types of colonies induced
by them were examined. Fig. 2 shows that CSFs from
both the sources formed G, M and GM colonies in the
same proportion of colony types; the GM colonies
were maximum (>60%). To further confirm, as shown
Table VI: Effect of goat anti-mouse TNF-α polyclonal
antibody on the LDAA-induced production of CSFs
by mouse peritoneal MØs, in vitro
a
a
LDAA TNF-α Normal goat Goat anti- CSF activity
b
c
(0.1µg/ml) (5 µg/ml) IgG mouse TNF-α (at 24 h)
b
polyclonal IgG
- - - - 3±1
+ - - - 71±9
- + - - 83±11
- - + - 2±1
- - - + 4±1
+ - + - 68±9
+ - - + 62±8
- + - + 3±1
a 6
MØs (1x10 cells/ml; 3 ml) were incubated with LDAA at 37°C in
5% CO2-air atmosphere for 24 h. Controls received only CDMEM
b
Neutralizing (100%) concentration of goat anti-mouse TNF-α
polyclonal IgG was added to LDAA or TNF-α just before MØ
treatment. Normal goat IgG was used as negative control.
c
The CSF activity was determined in the CM. Data are mean
number of colonies±SD of three separate experiments, run in
triplicate.
Table VII: Neutralization of CSF activity in LDAAa
treated mice serum and MØ CM with specific IgG
Source
b
CSF activity (% inhibition) after treatment with
Medium Anti-G- Anti-M- Anti-GM-
CSF IgG CSF IgG CSF IgG
Serum 140±18 130±16 (7.14) 100±13 (28.57) 50±8 (64.30)
CM 75±10 70±9 (6.66) 54±8 (28.00) 26±4 (65.44)
a
Serum/CM samples were incubated (37°C; 30 min) with an excess
amount of indicated IgG and then assessed for residual CSF
activity. % inhibition was calculated as (no. of colonies preneutralization
- no. of colonies post-neutralization/no. of colonies
pre-neutralization) x 100.
b
The CSF activity was determined in the CM. Data are mean
number of colonies±SD of three separate experiments, run in
triplicate.
No. of colones (%)
250
200
150
100
50
0
Serum
CM
(5) (6.33)
in Table VII, neutralization of the serum and CM
samples with goat anti-mouse GM-CSF IgG led to 64.3
and 65.33% inhibition of the colony formation,
respectively. Similarly, selective neutralization of G-
CSF, in the serum and CM, with goat anti-mouse G-
CSF IgG led to 7.14 and 6.66% inhibition,
respectively, whereas, neutralization with anti-mouse
M-CSF IgG resulted in 28% inhibition of the colony
formation induced by CSFs from both the sources.
LDAA-induced hematopoietic activity in the spleen
and BM of mice: The CFU-GM counts in the spleen
and BM exhibited a maximum of 2.7- and 2.4-fold
increase, respectively, after 24 h of LDAA (0.01-10
mg/kg) administration, compared to those given
control antigen, HI-LDAA or vehicle only (Table
VIII).
DISCUSSION
(28.66) (31.66)
(66.33)
G M GM
Colony types
Fig 2. Composition of the colonies formed in response to LDAAinduced
CSFs. Serum or CM CSF supported colonies were fixed on
glass slides and stained with May-Grünwald-Giemsa solution for
identification. G, granulocyte; M, M0; GM, granulocyte-MØ. Data
are based on an examination of 300 colonies.
Our laboratory is engaged into the research in the
molecular mechanisms of the pathogenesis of VL,
especially at the stimulus/response coupling level of
MØ-amastigote interaction. The results of this study,
apparently for the first time, demonstrate that LDAA
can induce the synthesis and secretion of CSFs both in
vivo and in vitro. Further, a >2-fold increase in the
CFU-GM counts, both in the spleen and BM, of
LDAA-treated mice, suggest the induction of CSFs in
these organs that are the primary sites of L. donovani
(62)