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Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils
Published on: November 16, 2015
Expression of low-density lipoprotein receptors in peripheral blood and tonsil B lymphocytes
J B De Sanctis1, I Blanca, H Rivera
1Institute of Immunology, Faculty of Medicine, Central University of Venezuela, Caracas.
Insights
Human B lymphocytes express low-density lipoprotein receptors (LDLR), which are upregulated by IL-2 and pokeweed mitogen stimulation. This suggests a potential role for LDL in B cell function.
Area of Science:
- Immunology
- Cell Biology
Background:
- B lymphocytes play crucial roles in adaptive immunity.
- Low-density lipoprotein receptors (LDLR) are involved in cholesterol metabolism.
- The role of LDLR in B lymphocyte physiology is not well understood.
Purpose of the Study:
- To investigate the expression and function of LDLR on human B lymphocytes.
- To determine the effect of immune stimulation on LDLR expression and LDL uptake by B cells.
Main Methods:
- Purification of B lymphocytes from peripheral blood leukocytes.
- Assessment of LDLR expression using a monoclonal anti-LDLR antibody.
- Measurement of LDL internalization using 125I-LDL and LDL-DiI.
- Analysis of LDL binding kinetics using Scatchard and Lineweaver-Burk plots.
Main Results:
- B lymphocytes express LDLR, with expression increasing upon stimulation with IL-2 and pokeweed mitogen (PWM).
- IL-2 and PWM significantly enhanced LDL internalization by B cells.
- Scatchard and Lineweaver-Burk analyses revealed increased binding affinity and capacity for LDL upon stimulation.
Conclusions:
- Human B lymphocytes express functional LDLR.
- Immune stimulation upregulates LDLR expression and LDL uptake in B cells.
- LDL may play a significant physiological role in B lymphocyte function.
Abstract:
B lymphocytes, purified from peripheral leucocytes from young normolipaemic humans, expressed and internalized low-density lipoprotein receptors (LDLR). The expression was assessed by a monoclonal anti-LDLR. The internalization of LDL was assessed by LDL labelled with 125I (125I-LDL) and 1,1'-dioctadecyl-3,3,3',3' tetramethyl-indocarboxycyanine perchlorate (LDL-DiI). The expression of LDLR, assessed by anti-LDLR, was: 38 +/- 8% (n = 5) for fresh purified cells, 60 +/- 10% (n = 12) for non-stimulated cells, 79 +/- 5% (n = 10) for IL-2 (100 U/ml)-stimulated cells and 95 +/- 5% (n = 8) for pokeweed mitogen (PWM) (1:200 dilution)-stimulated cells. The optimal concentrations of agonist were 100 U/ml of IL-2, and 1:200 dilution of PWM. IL-2 and PWM increased the internalization of LDL-DiI by 1.5-fold. The internalization of LDL-DiI was maximal at 60 microg of protein/ml (48 +/- 8%). Scatchard analysis revealed a Kd of 3.2 +/- 0.22 x 10(-8) M and 2180 +/- 190 binding sites in non-stimulated cells, a Kd of 7.73 +/- 0.36 x 10(-9) M and 12,500 +/- 430 binding sites for IL-2 (100 U/ml)-stimulated cells, and a Kd of 7.2 +/- 0.43 x 10(-9) M and 13,250 +/- 450 binding sites for PWM (1:200 dilution)-stimulated cells. Lineweaver-Burk analysis of LDL binding (LDL-DiI) revealed that the apparent Kd for non-stimulated cells was 1.3 +/- 0.11 x 10(-8) M, and 9.2 +/- 0.2 x 10(-9) M and 7.5 +/- 0.25 x 10(-9) M for IL-2- and PWM-stimulated cells, respectively. B lymphocytes from tonsils also showed a high expression of LDLR assessed with anti-LDLR (70 +/- 6%). The high expression of LDLR and the avid internalization of LDL suggest that LDL may be important for B cell physiological responses.
