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Published on: July 3, 2013
A transient acidification linked to intracellular Ca2+ in anti-mu-stimulated human B-lymphocytes
H Schøyen1, J G Iversen, E B Smeland
1Institute of Physiology, University of Oslo, Norway.
Insights
Anti-mu antibodies trigger rapid intracellular pH and calcium changes in human B-lymphocytes, initiating cell cycle progression. IF5 antibody, targeting CD20, does not induce these signaling events.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Cellular proliferation, particularly in B-lymphocytes, is linked to dynamic shifts in intracellular pH and calcium.
- Understanding these signaling pathways is crucial for deciphering B-cell activation and immune responses.
Purpose of the Study:
- To investigate the specific patterns of intracellular pH and free Ca2+ concentration changes in human B-lymphocytes upon activation.
- To compare the signaling effects of anti-mu antibodies with the CD20-specific antibody IF5.
Main Methods:
- Utilized fluorescent dyes (2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein and indo-I) to monitor intracellular pH and Ca2+ levels.
- Stimulated normal resting human B-lymphocytes with anti-mu antibodies and the IF5 monoclonal antibody.
- Assessed the impact of Ca2(+)-free solutions and the ionophore ionomycin on these responses.
Main Results:
- Anti-mu antibodies induced rapid intracellular acidification followed by alkalinization, and a significant increase in cytoplasmic Ca2+.
- The acidification response to anti-mu was amiloride-resistant, while the subsequent alkalinization was sensitive.
- The CD20-specific antibody IF5 did not elicit changes in intracellular pH or Ca2+.
- Ionomycin mimicked the pH and Ca2+ responses observed with anti-mu.
- Responses to anti-mu were diminished in Ca2(+)-free conditions, indicating reliance on intracellular Ca2+ stores.
Conclusions:
- Anti-mu antibody-induced B-lymphocyte activation involves distinct, rapid changes in intracellular pH and Ca2+.
- These signaling events appear to be mediated through pathways distinct from those targeted by the CD20 antibody IF5.
- Intracellular calcium stores play a critical role in mediating the anti-mu antibody-induced signaling cascade.
Abstract:
Mitogen-induced cellular proliferation is in many cell types preceded by rapid changes in intracellular pH and free Ca2+ concentration. We studied the patterns of pH and Ca2+ changes in normal resting human B-lymphocytes after exposure to anti-mu antibodies and the monoclonal antibody IF5, reactive with the CD20 antigen, both able to activate resting B-lymphocytes to enter the G1 phase of the cell cycle. Monitoring intracellular pH with the pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein, we demonstrated that poly- and monoclonal anti-mu antibodies induced a rapid (maximum change within 2 min) intracellular acidification of 0.06 pH units followed by a slower (10-15 min) alkalinization towards, or slightly above, the resting pH of 6.88. The acidification response was amiloride-resistant, whereas the return to baseline was sensitive. Intracellular free Ca2+ was measured by using the fluorescent Ca2+ dye, indo-I. Exposure of cells to anti-mu resulted in a rapid increase (maximum change within 2 min) in cytoplasmic Ca2+ of 340 nM and a slower decline in fluorescence back to baseline of about 180 nM. In contrast to anti-mu, IF5 caused no change in cytoplasmic Ca2+ and pH. However, the Ca2+ ionophore ionomycin at low concentrations mimicked the Ca2+ response as well as the pH response to anti-mu. In Ca2(+)-free solutions the intracellular Ca2+ stores are usually rapidly depleted and, indeed, the Ca2+ and pH responses to anti-mu were reduced after 5 min and almost abolished after 35 min under such conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
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