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Hamycin inhibits IL-8-induced biologic response by modulating its receptor in human polymorphonuclear neutrophils
S K Manna1, S Samanta, A K Samanta
1Division of Immunobiology, Indian Institute of Chemical Biology, Calcutta.
Abstract:
IL-8, a neutrophil chemotactic agent, is involved in a large number of neutrophil-driven acute and chronic inflammatory diseases. We have found that hamycin, an antifungal agent, reduces IL-8-induced migration and binding of 125I-labeled IL-8 to neutrophils by 66 and 75%, respectively. Other IL-8-induced biologic functions, such as superoxide generation, intracellular Ca2+ mobilization, and enzyme release were also reduced in hamycin-treated cells by 50 to 75%. Anti-IL-8R Ab (C-X-CR1) and IL-8 itself failed to protect the cells from the effect of hamycin. Scatchard analysis of IL-8 binding data demonstrated that while the normal cells expressed 23,000 +/- 1,704 receptors/cell (Kd = 3.5 nM), the number was reduced to 8,000 +/- 592 receptors/cell (Kd = 3.43 nM) in hamycin-treated cells. Chemical cross-linking of 125I-labeled IL-8 to its receptor followed by 10% SDS-PAGE analysis and autoradiography showed that the signals in hamycin-treated cells were considerably reduced compared with those in controls. In the immunoblot, however, the signals in control and hamycin-treated cells were almost identical. The intensity of the fluorescence emission of diphenyl hexatriene at 430 nm and membrane microviscosity measured by diphenyl hexatriene were considerably reduced in hamycin-treated cells, resulting in a reduced number of functional IL-8R, presumably by conformational change in the receptor. The study suggests that hamycin may be a potent immunomodulator of the IL-8R for alleviation of inflammatory distress.
Insights
The antifungal agent hamycin significantly reduces the binding and migration of Interleukin-8 (IL-8) to neutrophils. This suggests hamycin may modulate IL-8 receptors to alleviate inflammatory conditions.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Interleukin-8 (IL-8) is a key mediator in neutrophil-driven inflammatory diseases.
- Neutrophil migration and activation are critical processes in acute and chronic inflammation.
Purpose of the Study:
- To investigate the effects of the antifungal agent hamycin on IL-8-induced neutrophil functions.
- To elucidate the mechanism by which hamycin influences IL-8 receptor (IL-8R) interactions.
Main Methods:
- Assessing IL-8-induced neutrophil migration and 125I-labeled IL-8 binding.
- Measuring IL-8-stimulated superoxide generation, intracellular calcium mobilization, and enzyme release.
- Utilizing Scatchard analysis to quantify IL-8R expression and affinity.
- Employing chemical cross-linking, SDS-PAGE, autoradiography, and immunoblotting.
- Analyzing membrane microviscosity using diphenylhexatriene fluorescence.
Main Results:
- Hamycin significantly reduced IL-8-induced neutrophil migration (66%) and IL-8 binding (75%).
- Hamycin inhibited other IL-8-induced functions, including superoxide generation, Ca2+ mobilization, and enzyme release (50-75%).
- Scatchard analysis revealed a decrease in IL-8R number (from 23,000 to 8,000 receptors/cell) in hamycin-treated cells, with no change in affinity.
- Chemical cross-linking showed reduced IL-8-IL-8R complex formation, while immunoblotting indicated similar receptor protein levels.
- Hamycin treatment decreased membrane microviscosity, suggesting a conformational change in the IL-8R.
Conclusions:
- Hamycin effectively inhibits IL-8-mediated neutrophil responses.
- The mechanism involves a reduction in the number of functional IL-8 receptors, likely due to conformational changes.
- Hamycin shows potential as an immunomodulator for treating inflammatory diseases by targeting the IL-8 receptor pathway.