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IL-4R alpha and gamma chain expression in LPS- and IL-4-stimulated MONO-MAC-6 cells
H Quentmeier1, K Kolsdorf, M Zaborski
1DSM-German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig.
Abstract:
Short-term stimulation of peripheral blood monocytes (PBMo) and cells of the monocytic cell line MONO-MAC-6 with lipopoly-saccharide (LPS) induces high tumor necrosis factor (TNF)alpha mRNA levels. In contrast to the results obtained with primary cells, this effect could not be inhibited by preincubating the cell line with recombinant human interleukin-4 (rh IL-4). This deficiency in response to the cytokine was not caused by a general unresponsiveness of MONO-MAC-6 cells to IL-4. Thus, the expression of the monocyte-associated differentiation markers CD14 and monocyte-specific esterase (MSE), upregulated by long-term stimulation with LPS, could be decreased by IL-4. Long-term LPS treatment apparently induced IL-4 responsiveness of the cell line. While IL-4R alpha mRNA was upregulated about 3-fold, this positive effect was not apparent at the cell surface protein level. In contrast to the constitutive alpha chain expression, the IL-4R gamma chain expression could not be detected with a specific mAb nor by Northern blot analysis. However, reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated the presence of low-level IL-4R gamma chain mRNA in the cell line. We suggest that the low reactivity of the cells to IL-4 might be correlated with the low expression of the gamma chain.
Insights
Peripheral blood monocytes (PBMo) and MONO-MAC-6 cells show high tumor necrosis factor (TNF)alpha mRNA after lipopolysaccharide (LPS) stimulation. Interleukin-4 (IL-4) could not inhibit this in the cell line, suggesting low gamma chain expression limits IL-4 responsiveness.
Area of Science:
- Immunology
- Cell Biology
Background:
- Peripheral blood monocytes (PBMo) and monocytic cell lines are crucial in immune responses.
- Lipopolysaccharide (LPS) is a potent stimulator of monocyte activation, inducing cytokine production.
- Interleukin-4 (IL-4) is a key cytokine modulating immune cell function and differentiation.
Purpose of the Study:
- To investigate the differential response of PBMo and the MONO-MAC-6 cell line to LPS stimulation.
- To determine the effect of recombinant human IL-4 (rh IL-4) on LPS-induced TNFalpha production in these cells.
- To explore the underlying mechanisms of IL-4 unresponsiveness in the MONO-MAC-6 cell line.
Main Methods:
- Short-term and long-term stimulation of cells with LPS.
- Treatment with rh IL-4 to assess cytokine inhibition.
- Analysis of monocyte differentiation markers (CD14, MSE) expression.
- Gene expression analysis of IL-4 receptor alpha (IL-4R alpha) and gamma (IL-4R gamma) chains using Northern blot and RT-PCR.
- Protein expression analysis using monoclonal antibodies (mAb).
Main Results:
- Short-term LPS stimulation induced high TNFalpha mRNA in both PBMo and MONO-MAC-6 cells.
- rh IL-4 failed to inhibit TNFalpha mRNA in the MONO-MAC-6 cell line, unlike in primary PBMo.
- Long-term LPS treatment rendered MONO-MAC-6 cells responsive to IL-4, decreasing CD14 and MSE expression.
- IL-4R alpha mRNA levels increased with long-term LPS, but protein levels did not significantly change.
- Low-level IL-4R gamma chain mRNA was detected in MONO-MAC-6 cells, while protein expression was undetectable.
Conclusions:
- MONO-MAC-6 cells exhibit distinct IL-4 responsiveness compared to primary PBMo, particularly in short-term LPS-stimulated conditions.
- Long-term LPS exposure appears to induce IL-4 responsiveness in the MONO-MAC-6 cell line.
- The limited reactivity of MONO-MAC-6 cells to IL-4 may be associated with the low expression of the IL-4R gamma chain.