Effects of Using Antibody Labels upon Monomax 6 Cell Line:
The effects of LPS on the expression of the surface antigens ( CD14) by using MM6 cells line were incubated with 1000ng/ml for two different time at overnight and at 3 hours, and the effects of CD14 expression was assessed by using EPICS flow cytometer. This experiment was repeated three times. And the average X mean fluorescence was calculated for each strain.
Table1: CD14 Expression in MM6 Cells after 3Hours Incubation with LPS from Differing Bacterial Species.
The specificity of the LPS effects on CD14 was tested by comparing various LPS preparation on two different times at 3hours and overnight. It was interestingly to know the different between the incubation times with LPS. For this purpose, MM6 cells were exposed to LPS from differing Gram-Negative Bacteria for either three hours or overnight. There was no significant change in mean CD14 fluorescence when the LPS were incubated for three hours with MM6 cells. In contrast, the overnight incubation of MM6 with LPS was sufficient for a significant increase in mean CD14 fluorescence to occur. These observations indicate that a prolonged contact of LPS with cells was necessary for the LPS effects on CD14.
Figure1: shows the expression of cell surface antigen CD14 in MM6 cells after 3Hours Incubation with LPS from Differing Bacterial Species. MM6 cells ( ) were incubated with1000ng/ml of LPS, and the expression of CD14 was measured by flow cytometry. The CD14 expression was reduced in all LPS species expect in Salmonella.typhosa. The x-mean florescence 2.67 was found to be optimal for each LPS type for the expression of CD14.
The specificity of the LPS effects on CD14 was tested by comparing various LPS preparation. The results are shown on table 1 and figure 1. A 3 hours incubation
In table1 & figure 1, results are expressed as change in LPS-treated cells compared with simultaneously untreated cells. The incubation of LPS from Salmonella.typhosa with MM6 cells shows that the level of CD14 was increased after 3hours of incubation, and the level of CD14 expression was reduced when MM6 cells were incubated with LPS from other species. The amount of CD14 expression in untreated MM6 cells and MM6 cells with Mouse IgG1 antibody were similar, as shown in figure 1 and table 1. Figure1 shows that CD14 expression was weakly reduced by LPS treatment for 3 hours in all species expect Salmonella typhosa which showed
The effects LPS from E.coli 0111:B4 and other species on the expression of the cell surfaces antigen CD14 were assessed. In Fig.1, results are expressed as change in LPS-treated MM6 cells compared with stimultaneously with MM6 cells treated with anti human CD14. Results in figure 1 show that the level of CD14 expression in MM6 cells were reduced after 3Hours Incubation with LPS from Differing Bacterial Species expect in Salmonella typhosa which shows slightly increase in the expression of the cell surface antigen CD14.where as in figure 2 the results show that the amount of CD14 expression in MM6 were significantly increase after overnight (18 hours) incubation in all analyzed species. The amount of CD14 expression in the MM6 cells incubated with isotype control-FTIC (Mouse IgG1) were similar to the level of CD14 expression by untreated MM6 cells (blank). However, results in Figure 1 and 2 show that the CD14 expression was weakly reduced by LPS treatment in E.coli 0111:B4, E.coli 0127:B8, E.coli 055:B5 and Salmonella.typhimurium for three hours and strongly enhanced by overnight incubation with LPS from all analyzed species included Salmonella typhosa. To detect very early change, we measured CD14 expression by MM6 cells at three hours incubation.
Table2: shows the overnight incubation of LPS strains with MM6 cell line.
It was interestingly to know the different between the incubation times with LPS. For this purpose, MM6 cells were exposed to LPS from differing Gram-Negative Bacteria for either three hours or overnight. There was no significant change in mean CD14 fluorescence when the LPS were incubated for three hours with MM6 cells. In contrast, the overnight incubation of MM6 with LPS was sufficient for a significant increase in mean CD14 fluorescence to occur. These observations indicate that a prolonged contact of LPS with cells was necessary for the LPS effects on CD14.
Specificity of LPS effects on CD14:
Discussion
The specificity of the LPS effects on CD14 was tested by comparing various LPS preparations. The results are shown in tables 1 and 2. Three hours incubation with LPS of Salmonella.typhimurium markedly increased CD14 expression in MM6. At the same concentration of LPS from other analyzed Gram-Negative Bacteria, expression of CD14 from the surfaces of MM6 cells was slightly decreased. On the other hand, the expression of CD14 was significantly increased after overnight incubation with all analyzed Gram-Negative Bacteria. Results in Figure 1 and 2 confirm this finding.
How KemF CD14-LPS interaction is around 10-30 nm in the presence of 100 ng / ml ReLPS about 100 000-150 000 molecules of CD14 are busy, but only about 13 000 CD14 required for maximal cell activation. This means that (at high concentrations of LPS), most of the molecules CD14, involved in LPS binding are not necessary for activation. This phenomenon is called "spare receptor effect" was proposed for LPS-CD14-ligand receptor system is more. The concentration of free LPS required to saturate half of the available receptors CD14 (CD) on differentiated THP-1 cells was more than 100 times higher than the concentration of LPS provided half of the maximum IL-8 response (EC50). A large number of additional CD14 may play an important role at low concentrations of LPS, as we have shown by 0.1 and 1 ng / ml LPS, where an increase in the number of available CD14 molecules led to an increase in mRNA TNFα. Thus, these 'spare' receptors may increase the dose sensitivity of these cells, a ligand binding at low concentrations of LPS especially if other cellular or serum derived endotoxin binding molecules compete for LPS.
Further evidence supporting this theory is that in another population of leukocytes, neutrophils, similar to the sensitivity of the LPS-stimuli can be observed in monocytes, neutrophils, although with far less (only 3000-5000) molecule CD14. LPS-dose compared with the effect of the curve is very similar to neutrophils and monocytes Showing saturation of 1-10 ng / ml concentration of LPS. In the presence of LBP is 1-10 ng / ml LPS can cause maximum activation of complement receptor mediated by three potential glue maximum stimulation oxidative burst or secretion peak TNFα, indicating that much less of the molecule CD14, compared with the amount expressed on the surface of monocytes, which are necessary for full LPS-induced cell activation.
High expression of CD14-monocytes may be important when different ligands compete for CD14-binding. CD14 acts as a 'recognition receptors and is responsible for determining not only LPS but also other "pathogen associated molecular patterns. Surface-CD14 expressed binds with high affinity for a large number of molecules of different species and thus meet the first line, the less specific antigen test available microbe. more specific but less sensitive CD14-associated signaling elements (TLRs) interact with only a small portion of the molecule CD14 binding the specific ligand for specific TLR, which leads to the formation of the complex CD14/TLR and in the initiation of the microbe-specific protection mechanisms.
In addition to its role in cell activation at a relatively large number of surface-CD14 expressed may play an important role in the design of LPS, too. CD14-bound and internal LPS molecule undergo chemical changes resulting in detoxification of endotoxin. "Overexpression" of CD14 on monocytes, also seems to be an important role in enhancing CD14-mediated phagocytosis of gram-negative bacteria. Schiff and her colleagues created a CD14-transfected THP-1 cells and cleaved membrane CD14 molecule expressed using different concentrations of the enzyme phosphatidyl inositol specific phospholipase C. Thus, like our current approach, they can create THP-1 cells with graded numbers of CD14 surface and may be a linear relationship between CD14-expression and phagocytosis of BODIPY-labeled E.coli. These data clearly indicate that in contrast to CD14-mediated activation of cell CD14-mediated clearance of gram-negative bacteria does not show saturation, and increases with the available molecules CD14.
Conclusion
In order to evaluate the effect of saturation of CD14-activation of monocytes, mononuclear cells were incubated with anti-CD14 blocking mAb of graded numbers of free molecules of CD14 in monocytes, as described above. Partially blocked cells were incubated with 3% NHS, and 0.1-100 ng / ml ReLPS for 30 min at 37 ° C. The amount of TNFα mRNA quantified, corrected with GAPDH mRNA and compared with blasttransformatsii cells. Origin of TNFα mRNA synthethised in the presence of 10 ug / ml MAB considered CD14-independent, and this value was used in each sample for calculation of the CD14-mediated activation.
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