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Diabetes Mellitus (DM) is a metabolic disorder which leads to many serious secondary complications like retinopathy, neuropathy, nephropathy, cardiovascular complication, delayed wound healing etc. Diabetics are more prone to infection due to compromised immunity. Hyperglycaemia leads to oxidative and osmotic stress. This short communication is the preliminary study to know the effect of high glucose concentration (mimicking hyperglycemia) and H2O2 (mimicking oxidative stress) on murine macrophage cell line RAW 264.7.
RAW cell line was procured from NCCS Pune and maintained in RPMI 1640 media with 10% Fetal Calf Serum (FCS) and antibiotic (penicillin and streptomycin) at 37 ºC and 5% CO2 in CO2 incubator. 5X104 cells were seeded in each well of 24 well plate, kept in serum free media overnight followed by replacement with serum containing media and treatments (different glucose and H2O2 concentration). Cells were incubated for different time intervals. Supernatant was collected for cytokine estimation by ELISA kit. Viability of cells was estimated by cell MTT assay.
Macrophages play crucial role in connecting innate immunity
(by phagocytosis) and cell mediated immunity by secreting
different cytokines which may have immune stimulatory and
immune suppressive functions. The osmotic stress induced
inhibition in the growth of macrophage cell line, which was
found to be time and concentration dependent (Figure 1).
Lipopolysaccharide (LPS) is the major component of the outer
membrane of Gram-negative bacteria used for the activation of
macrophages. Lipopolysaccharide (LPS) has significant positive
inductive effect on the growth of macrophage cells (Figure 2).
LPS resulted in increased levels of immuno-stimulatory cytokines
IL-4 (Figure 3) and IL-6(Figure 4). No significant change in IL-10
level was seen (Figure 5). Osmotic stress leads to compromised
immunity. The macrophagic cell line when subjected to oxidative
stress showed a considerable decrease in cell count (as observed
by MTT assay) with increase in concentration (from 25μM to
50 μM H2O2 concentration). Cell death was observed at 100 μM
H2O2 concentration (data not shown). Results showed that LPS
had a positive inductive effect on the cell culture and the cell
count increased considerably (Figure 6 & 7). In oxidative stress
the interleukins had an immunomodulatory effect on the cells.
Increased IL-4 (Figure 8), IL-6 (Figure 9) and TNF–α (Figure 10)
suggests immuno-proliferative effect while IL-10 (Figure 11)
suggests immunosuppressive effect. In osmotic stress there was
no significant change in GSH levels in control and stressed cells.
There was a sharp decline in GSH level in presence of H2O2 when
compared to control (data not shown). Moreover, LPS presence
didn’t make any observable change in result. This suggests that
treatment with H2O2 resulted in oxidative stress.
Our findings suggest that upon osmotic and oxidative stress
the levels of immuno-proliferative cytokines (IL-4, IL-6, TNF-α)
decreases while immune-suppressive cytokine IL-10 increases
resulting in macro phage dysfunction. This may be the basis of
immune compromised state in oxidative stress mediated disease
for example Diabetes.