Gerrit Koopman

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.

In the germinal center (GC), B cells are either selected to become memory cells or are eliminated through the process of programmed cell death. FDC which are intimately associated with the GC B cells are thought to be important in this selection process. Previously, we have shown that the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and VLA-4 (CD49d)-VCAM-1 (CD106) adhesion pathways are involved in FDC-B cell interaction. In the present study, we have explored whether these adhesive interactions contribute to the process of B cell selection by studying the effects on apoptosis of GC B cells. Using FDC and B cells derived from human tonsils, we found that only B cells adherent to FDC remain viable: disruption of FDC-B-cell clusters with mAb against LFA-1 alpha (CD11a), VLA-4 (CD49d), ICAM-1 (CD54), or VCAM-1 (CD106) results in apoptosis of the B cells. Furthermore, we found that GC B cells, upon adhesion to plastic-coated purified ICAM-1 (CD54) or VCAM-1 (CD106), show diminished apoptosis. Importantly, we observed that, at low concentration, ICAM-1 (CD54) and VCAM-1 (CD106) act synergistically with anti-IgM, in inhibiting apoptosis. Together, our data strongly suggest that adhesion of B cells via the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) pathway and VLA-4 (CD49d)-VCAM-1 (CD106) pathway contributes to B cell selection.

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors, has been shown to confer metastatic potential to nonmetastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and on non-Hodgkin's lymphomas. Normal lymphohematopoietic cells express barely detectable low levels of variant CD44 glycoproteins, whereas T lymphocytes, upon activation by mitogen or antigen, transiently upregulate expression of specific CD44 variant glycoproteins. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that in the rat confers metastatic capability. It is interesting that overexpression of v6 was also found in several aggressive, but not low-grade, non-Hodgkin's lymphomas.

Presentation of antigen in the form of immune complexes to B lymphocytes by follicular dendritic cells (FDC) is considered to be a central step in the generation of memory B cells. During this process, which takes place in the microenvironment of the germinal center, B cells and FDC are in close physical contact. In the present study, we have explored the molecular basis of FDC-B cell interaction by using FDC and B cells derived from human tonsils. We found that FDC express high levels of the adhesion receptors intercellular adhesion molecule 1 (ICAM-1 [CD54]) and vascular cell adhesion molecule 1 (VCAM-1), while the B lymphocytes express lymphocyte function-associated antigen 1 (LFA-1 [CD11a/18]), very late antigen 4 (VLA-4 [CD49d], and CD44. Furthermore, we established that both the LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways are involved in FDC-B lymphocyte binding, and therefore, these pathways might be essential in affinity selection of B cells and in the formation of B memory cells.