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Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance
Published on: July 22, 2011
Decrease of intracellular fluorescein fluorescence polarization (IFFP) in human peripheral blood lymphocytes
A Eisenthal1, O Marder, D Dotan
1Sourasky Medical Center, Tel-Aviv, Israel.
Insights
Stimulants like phytohaemagglutinin (PHA) and anti-CD3 antibody rapidly decrease intracellular fluorescein fluorescence polarization (IFFP) in human lymphocytes within 40 minutes. This change in IFFP indicates lymphocyte activation and persists for 24 hours.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Lymphocyte activation is crucial for immune responses.
- Intracellular fluorescein fluorescence polarization (IFFP) can reflect cellular changes.
- Specific stimulants trigger distinct lymphocyte activation pathways.
Purpose of the Study:
- To investigate the effect of various stimulants on IFFP in human peripheral blood lymphocytes (PBL).
- To determine the kinetics and duration of IFFP changes following lymphocyte activation.
- To correlate IFFP modulation with specific lymphocyte activation pathways.
Main Methods:
- Detection of IFFP changes in individual lymphocytes using the Cellscan apparatus.
- Stimulation of human PBL with mitogenic concentrations of phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), and anti-CD3 antibody.
- Quantification of lymphocyte activation using a [3H]-thymidine incorporation assay.
Main Results:
- Mitogenic PHA concentrations induced a decrease in IFFP in human PBL within 40 minutes.
- ConA and anti-CD3 antibody similarly decreased IFFP, indicating activation of CD3+ lymphocytes.
- Pokeweed mitogen (PWM) did not affect IFFP at the concentrations used, suggesting specificity for B lymphocyte activation.
- IFFP changes were observed within 20-40 minutes and lasted for up to 24 hours.
Conclusions:
- Certain stimulants rapidly modulate IFFP in human lymphocytes, reflecting cellular activation.
- IFFP serves as a sensitive indicator for early-stage lymphocyte activation, particularly for CD3+ T cells.
- The observed IFFP changes provide insights into the biophysical mechanisms underlying lymphocyte activation.
Abstract:
In the present study we describe the induction of changes in intracellular fluorescein fluorescence polarization (IFFP) in lymphocytes undergoing activation with a variety of stimulants. These stimulants included the lectins phytohaemagglutinin (PHA), concanavalin (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody. Changes in IFFP were detected in individual cells using the Cellscan apparatus. Our results show that by employing mitogenic concentrations of PHA, as revealed in a [3H]-thymidine incorporation assay, a decrease in the IFFP in human peripheral blood lymphocytes (PBL) occurred within 40 min. ConA and anti-CD3 affected similarly IFFP, whereas PWM, a B lymphocyte lectin, had no effect on IFFP at the concentrations employed. Kinetic analysis revealed that changes in IFFP occurred within 20-40 min after exposure to the stimulants and lasted for 24 h. Our results show that stimulants which activate CD3+ lymphocytes caused immediate changes in IFFP, in an enriched population of human PBL. The possible mechanisms involved in IFFP modulation following exposure to selected stimulants are discussed.
