of malnourished mice bearing Dalton's
Debasis Ghoshal PHD* and Subha
Department of Chemistry* and
Microbiology**, Bose Institute, Calcutta, India.
The immunomodulatory effect of a mouse bone-barrow-derived
cytokine (BIM), (mol wt<10 kd), was studied in mice bearing Dalton'
s lymphoma. It was observed that this factor increased the life-span
of mice malnourished with respect to vitamin B-complex and ascorbic
acid and infected with Daltons' lymphoma, by 40±4 days when compared
to malnourished lymphoma controls while in animals maintained on
balanced diet (BDF) the increase in life-span was just over 11±2
days. In cultured bone marrow cells at different time intervals
after introduction of lymphoma cells it was shown that introduction
of lymphoma cells increased the secretion of BIM. While the lymphoma
developed the secretion of BIM diminished much earlier in malnourished
than in BDF mice. This observation further strengthens our previous
findings that the BIM acted as an immunomodulator much more effectively
in malnourished animals than in animals fed a balanced diet, where
a feed-back inhibitory effect might be present.
Immunodeficiency due to malnutrition paves the
way for the development of many types of lymphoma1. It
is currently posited that functional defect of the lymphoid tissue
compartment, as a consequence of defective interaction between host
and cell, may be one of the possible reasons for lymphoma development2.
If a specific defect lies within the host then reatinal therapy
correcting the microenvironment of the host would be more effective
than eliminating the neoplastic cell. Thus conventional radiotherapy
and chemotherapy are now widely in use. Recently, as part of monoclonal
antibody«technology, immunological techniques including antibody
therapy and biological response modifiers, eg interferons, have
begun to be explored. Bone marrow transplantation in patients who
are otherwise resistant to conventional treatment is now being explored
and this technique may be more effective in the early stage of the
disease3. But bone marrow transplantation is a very expensive
and difficult approach in an already immunocompromised Affected-affected
host. In this paper we discuss a possible immunomodulatory approach
to treating lymphoma by bone marrow cytokine.
Earlier reports4-6 showed that a rodent
bone marrow cell secreted factor immunomodulated malnourished immunosuppressed
mice by not only improving the T and B cell population and functions
in immunocompetent organs, but also the bone marrow cellular compartment
and peripheral blood profile. An increased resistance towards lung
and gastrointestinal infections, which otherwise proves fatal in
untreated malnourished control3, was also observed. To
further study the immunomodulatory activity of this factor i 1000
t was subsequently tested in mice infected with Dalton's lymphoma.
Materials and method
Male Swiss mice (age 30 days; body wt 16±2g)
were maintained on ad libitum balanced diet for 7 days under 12-h
light- dark cycle in suspended wire cages. The mice were then divided
into two batches of 20 each and one batch rendered malnourished
in regard to B-complex vitamins and ascorbic acid4,5.
Development of lymphoma
A Dalton's lymphoma (DL) cell line is maintained
in male Swiss mice at Bose Institute. The lymphoma cells were collected
from the peritoneum in normal saline in an aseptic condition and
pelleted in cold centrifugation (500 rpm for 10 min). The viability
of cells was studied by the trypan blue exclusion method (>95%
viable) and each experimental mouse received 2 x 104
cells, intraperitoneally. The lymphoma was allowed to grow in vivo
for 3 days, in both the balanced-diet-fed (BDF) and B-complex and
ascorbic-acid deficient (D) groups
At different time periods bone marrow cell cultures
were performed in the BDF-DL group by the method described below
and the secretory profile of the bone marrow factors evaluated,
compared and contrasted with uninfected controls.
Preparation of bio-immunomodulator
Unfractionated mouse bone marrow cells, flushed
from the femurs of healthy young animals, were repeatedly aspirated
and ejected from a syringe to obtain single cell suspensions. The
cells were cultured in RPMI 1640 (pH 7.3) serum-free medium at 37°C
for 18 h at a concentration of 3x 106 cells/l. At the
end of the incubation period, the cells were pelleted by centrifugation
(500 rpm) at 4°C and the supernatant fluid was collected6.
The cell-free crude extract was then subjected to membrane filtration
under N2 pressure with continuous stirring and with a
molecular cut-off range at 10 kd (Amicon, USA). Two fractions were
obtained, Fr A (mol wt > 10 kd) and Fr B (mol wt < 10 kd).
To locate the presence of active fractions both the fractions were
screened for antilymphoma activity on BDF mice bearing 6-day-old
lymphoma. Crude Fr A/B was injected ip in three divided doses at
an interval of 5 days to BDF-DL-bearing mice and it was seen, as
will be discussed in detail later, that Fr B contained factor(s)
capable of increasing life-span of Bearing-bearing mice, with a
mean increment of 8 ± 4 days over saline-treated controls.
Studies on Fr B (mol wt < 10
The proteins of the crude filtrate (mol wt <
10 kd) were precipatated by 60% ammonium sulphate cut at 4°C, reconstituted
in 0.9% NaCI, dialized against dd H2O at 4°C overnight
in benzoylated tubing (Sigma, USA), Iyophilized and again reconstituted
in saline. The protein concentration was estimated by Lowry's method7
using BSA as standard. The protein sample (conc.435 mg/ml) was applied
to a Sephadex G-10 (Sigma USA) column (1.8 cm x 27 cm) pre-equilibrated
with 50 mM Tris-HCI buffer, pH 7.2. The flow rate of the column
was maintained at 8.0mSh and 2-ml fractions were collected. The
protein of the collected fractions was measured at 280 nm (Figure
1). The present paper deals with the immunomodulatory effect of
pooled fraction 8-12 under the first peak, hencefonh known as BIM-1
Figure 1. Sephadex G-10 column chromatography
of mouse bone marrow cell secreted factor (mol wt < 10 kd). Pooled
fraction 8-12 under the first peak is BIM-1.
Immunomodulatory effect of BIM-I
Three days after the introduction of lymphoma
in both BDF and D mice, three doses of BIM-1 (days 9, 11 and 23
after introduction of the deficient diet; protein conc 0.3 Fg/dose)
was injected ip, saline being injected into a control mouse. The
mice were weighed every alternative day, their death recorded and
a postmortem (PM) examination along with histopathology was performed.
Statistical evaluation was done using the Kaplan-Meir
probability curve and Students 't'-test.
Figure 2 shows that the deficient mice bearing
Dalton's lymphoma (D-DL) died within 20 days after the onset of
experimental diet, ie within 14 days post-lymphoma introduction
(PLI). Respiratory distress was observed within 9 days PLI and the
deaths, as observed from postmortem (PM) and histopathological findings,
were primarily due to pneumonia of bacterial origin (Figure 3).
The spleen (49 ± 8 mg) and the thymus (2 ± 1 mg) were minimal in
size. There were peticheal haemorrhages on the spleen and histology
showed oedematous fluid, 'stary' appearance of the cOnex and atrophy
of the follicular region. The later observation was also found in
the thymus (Figure 3). The liver was pale, with peticheal haemorrhage
and pus-filled whitish growth on the surface and microscopically
showed infiltration with MN cells (Figure 3). Fatty changes, perivascular
cupping by MN cells, collection of oedematous fluid and some nodules
of the lymphoma cells were also evident. The kidney and intestine
showed signs of haemorrhage.
Figure 2. Immunomodulatory effect of mouse
bone marrow cell secreted BIM1 on mice infected with
Dalton's lymphoma. BDF = Balanced Diet Fed; D = Deficient (8-complex
vitamins and ascorbic acid) diet fed; DL = Dalton's lymphoma; BIM
In contrast, 50% malnourished BIM- I -injected
Bearing-bearing mice showed no respiratory distress till 53 ± 3
days of malnourishment, ie 48 ± 3 days PLI. The abdominal circumference
did not show appreciable growth of the lymphoma in these animals
till 30 ± 8 days PLI (45 ± 6 days of malnourishment) and 22 ± 5
days after the last injection of BIM- 1 (Figure 2).
After 30 ± 8 days of malnourishment 40% of animals
died. Their thymus was rudimentary (wt 2 ± 1 mg) though the spleen
was normal in size (wt 120 ± 20 mg). Histopathological examination
showed collection of oedematous fluid and fibrous changes. The thymus
had giant eosinophilic cells at the corticomedullary junction. There
was haemorrhage of the lungs, collapse of alveoli, infiltration
of MN, and giant eosinophilic and PMN cells in the aveolor cavity
which contained eosinophilic exudation. The liver showed profuse
haemorrhagic spots, oedematous changes, and infiltration by PMN
and MN cells. Giant anaphase stage basophilic cells were found in
clusters that formed nodules (Figure 3). The peritoneum was covered
with the lymphoma cells like a white sheet that was viscous in nature.
Table 1. Effect of a bone marrow-derived
bioimmunomodulator (BIM) on mean organ wt (in mg) of Swiss mice
with Dalton's lymphoma (DL).
23 ± 2
4 ± 2*
52 ± 6
30 ± 8*
105 ± 10*
49 ± 8*
BDF: Basal diet fed, D: deficient *: P < 0.001.
Fifty per cent of BIM-1 treated mice were alive
after 60 ± 2 days, ie 55 ± 2 days PLI; 45 days after the last injection
of BIM-1. This was in marked contrast to the fate of untreated animals
which died within 20 days of malnourishment, ie 14 days PLI. The
post-mortem and histological findings were similar to those described
The BDF-DL mice showed a steady growth of the
tumour. Fifty per cent of the animals died by 35 ± 4 days PLI. All
the animals were dead by 39 ± 2 days PLI, ie there was only 15 ±
3 increment in life-span of the BDF-DL mice over the malnourished
lymphoma control (P< 0.001). These mice initially showed no signs
of infection; however, after 25 ± 4 days PLI lung infection was
observed. In post-mortem studies thymus and spleen were larger than
those of malnourished lymphoma controls (thymus 4 ± 1 mg, spleen
105 ± 10 mg as against thymus 2 ± 1 mg, spleen 49 ± 8 mg P<0.001,
Table 1). The abdomen was completely filled with y 1000 ellowish-red
ascites fluid and there were cancerous nodules on the intestine
and peritoneal walls. Histological studies of the thymus showed
collection of oedematous fluid and fibrous changes (Figure 3). The
lymphocytes were found to be at different stages of development
and some non-stained cells of various sizes were also present. The
lungs showed collapse of alveoli and infiltration by MN cells, and
PMN cells, with vacuolated cytoplasm, were found in alveolar space
(Figure 3) where there was sometimes basophilic secretion and infiltration
by lymphoma cells. Spleen showed infiltration by DL cells, fibrous
changes and disintegration of pulp. The liver had oedematous changes,
damage to the epithelial lining of the lumen of arteries and veins
and cancer nodules were observed (Figure 3). In some mice, 30 days
PLI, the spleen was found to be enormous (wt 335 ± 50 mg). Histological
studies showed degeneration of Lymphoid follicle and infiltration
by macrophage and lymphoma cells.
Fifty per cent of BDF-DL BIM-treated mice were
dead by 25 ± 2 days PLI. All the animals died by 52 ± 2 days PLI,
thus an increase in lifespan by only 11 ± 2 days over BDF-DL controls
was observed. In contrast DDL-BIM-1-treated mice lived approximately
45 days longer (P 0.001) compared to DL-controls.
No significant difference in longevity was observed
between the BIM-treated groups. The thymus of the BDFDL-BIM mice
weighed 16 ± 5 mg vs BDF-DL controls 4 ± 1 mg (P<0.001) while
the spleen weighed 105 ± 16 mg ( no significant difference from
BDF-DL controls). The spleen was pale and peticheal haemorrhage
was observed. Under the microscope the spleen showed degeneration
of B-cell centres more than T-cell centres. There was infiltration
by lymphoma cells. The thymus showed degeneration of cortex and
oedematous fluid in the follicles. The liver showed fatty degeneration
and evident necrosis. There was infiltration be MN cells and lymphoma
cells were present in nodular formation (Figure 3). The lungs showed
exudatins containing RBC in the alveolar space and rupture of alveolar
wall (Figure 3).
Table 1 shows that in BDF-DL mice with atrophy
of the thymus there was gain in weight and in cellularity (evidenced
by histological studies) after BIM treatment. The spleen showed
initial hypertrophy after lymphoma injection, but there was no change
in gross organ weight following BIM treatment. The malnourished
Bearing-bearing mice showed changes in microscopic thymus structure
(Figure 3) following BIM treatment. Little change in macroscopic
structure was observed. This is in sharp contrast to that observed
in malnourished animals without lymphoma6. The spleen,
on the other hand, showed hypertrophy after lymphoma injection and
again after BIM treatment.
The bone marrow secretory profile of BDF-DL mice
showed an initial overproduction of BIM-1, but as the lymphoma grew
a suppressed production of cytokine was observed (Figure 4).
Reduced socio-economic status, presumably implying
impaired health status, has been reported in association with increased
incidence of lymphoma1. Thus malnourished mice, with
low levels of immune competence have unsurprisingly also exhibited
a rapid spread of lymphoma6,8 - see also Figure 2. However
BIM-I treated malnourished mice survived longer than untreated mice
on a balanced diet while untreated manourished controls died very
early (10 ± 4 days PLI).
The growth of the lymphoma has a suppressive
effect on bone-marrow secretory profile (Figure 4), similar to that
observed during malnourishment (Figure 1). It seems that in the
establishment of lymphoma some factor(s) are involved which directly
or indirectly suppress bone marrow cytokine secretion that is essential
for optimum immune response4-6. This may also explain
the immune suppression observed during parasitism9,10 1000
sup> and in the case of Burkitt's lymphoma11. Whether
this initial upsurge followed by suppression of BIM-1 production
has got anything to do with establishment of infection/tumour remains
to be confirmed. Because secretory products believed to be present
in the serum of Bearing-bearing mice, suppress BIM secretion, injection
of BIM-1was used to find out whether it has any immunomodulatory
effect on mice bearing Dalton's lymphoma whether the mice are well-fed
Table 1 indicates that BIM seems to act differentially
on thymus and spleen depending upon the nutritional status of the
Bearing-bearing host. In BDF animals introduction of lymphoma stimulates
indigenous BIM production (Figure 4) so as to counter the threat
of lymphoma by increasing the production of WBC in circulation6.
We injected BIM in the early stage of lymphoma establishment and
this might trigger a negative feed-back system as suggested earlier6
and as has also recently been observed with LIF12. As
the lymphoma established itself, the bone marrow showed hypoplasia
(observed from cyto-centrifuged smears). This may be one of the
causative factors for suppression in indigenous BIM production.
Figure 3. Histological changes observed
in Swiss mice, (basal diet fed (BDF) and malnourished infected with
Daltons' lymphoma (DL) and treated with BIM1 Giemsa's
stain. a1 = BDF-DL; a2 = BDF-DL-BIM1;
a3 = D-DL; a4 = D-DL-BIM1.
Liver. a1-a3: Infiltration
of neutrophil and mononuclear cells in hepatic tissue, oedema, necrosis
and formation of cancer nodules by lymphoma cells. a4:
Lesser amount of infiltration of WBC in hepatic tissue. Cancer nodules
very small in size and few in number. x 400.
Lung. Bronchopneumonia. a2 and a4
show reduction in microbial load compared to a1 and a3
Thymus. a1 and a3: atrophy
of thymus. a2 and a4: regeneration of thymus
cortex more than medulla x 100.
In malnourished mice, in whom BIM production
was suppressed much earlier6, external BIM injection
seemed to revive immunocompetence more effectively and to be able
to prevent rapid growth of tumour cells.
Earlier studies showed that malignant cells display
elevated Na+-K+-ATPase activity and increased
intracellular (IC) Na+ ion conc13. Sodium
ions now appear to be a favoured candidate for the role of a major
'early' mediator of cell division. Moreover, reduction in the extracellular
(EC) CA2+ ion has also been found to favour continued
growth of malignant cells13.
Our studies on brain lysosmal Na+-K+-ATPase14
and CA2+ - Mg2+-ATPase14 showed
a different effect of BIM in BDF control and malnourished rats.
In BDF animals immunization suppressed the ATPase activity while
in malnourished animals an increase in activity was noticed. BIM
injection in BDF animals showed no significant changes in ATPase
activity, compared to immunized BDF controls, while in malnourished
animals an init 1000 ial suppression was noticed followed by opening
of the ion channel.. This opening of the ion channel, as evidenced
from increased ATPase activity, probably alters the EC/IC ionic
balance of the malignant cells tilting it in favour of the normal
cellular microenvironment. This correction of ionic microenvironment
seems to be able to prevent rapid tumour growth in D-DL-BIM mice,
as stated above, thereby supporting our previous observation6
that D-mice gain more from BIM treatment.
In conclusion, from our previous communication4,6
it was evident that the bone-marrow secreted factor showing immunomodulatory
activity was active in malnourished immunosuppressed animals more
effectively than BDF controls. In this communication similar observations
were also noted. Here the bone-marrow secreted factor worked better
in malnourished lymphoma bearing mice than in controls fed a balanced
diet thus strengthening our previous hypothesis that a physiological
feed-back inhibitory activity might be present in BDF controls at
an early stage of lymphoma development.
Acknowledgement - The authors thankfully
acknowledge the help and encouragement received from Prof. AK Barua,
Department of Chemistry and Prof. SL Chakraborty, Department of
Microbiology, Bose Institute. Research support was provided by the
Indian Council of Medical Research.
Figure 4. BIMI secretion from bone marrow
at different periods after introduction of Daltons' lymphoma (DL)
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