N. Cormann

Isolation and long-term cultivation of human tonsil follicular dendritic cells

SummaryHighly purified follicular dendritic cells (FDC) were isolated from human tonsils and cultivated for up to 150 days. The cell separation method employed produced pure aggregates (FDC-clusters) composed of FDC and germinal center lymphoid cells, useful for the analysis of the relationship between these two cell types and of the behavior of FDC in culture. During the first few days of culture, lymphoid cells located between FDC extensions survived better than those which were free or partly covered by FDC. After 6 days, the lymphoid population degenerated and only the FDC survived. The unique antigenic pattern of FDC (positive for HLA-DR, DRC-1, CD14b, CD21, CD23, CD35) disappeared within a few days of culture. Recombinant interferon-γ ex-erted a positive effect either on retaining HLA-DR expression or on the reexpression of these antigens by FDC. HLA-ABC antigens were traced until the 10th day and desmosomal junctions until the 14th day. Subsequently, FDC presented peculiar features, including oval and rhomboid shapes, one to ten nuclei, fine amoeboid extensions, stress fibers and a radical dense zone in their cytoplasm. FDC possessed actin, tubulin and vimentin, but neither desmin nor cytokeratin. After 40 days of culture, FDC enlarged and were covered with abundant membrane extensions. Even when kept as long as 150 days in vitro, FDC did not proliferate in any of the culture conditions employed.

Isolation of follicular dendritic cells from human tonsils and adenoids. V. Effect on lymphocyte proliferation and differentiation.

Follicular dendritic cells (FDC) are located only in lymph follicles and are characterized by their capacity to retain high amounts of immune complexes on their plasma membranes. As their functions in germinal centres are unknown, we isolated them from human tonsils and cultured them with autologous lymphoid cells. Cultures of lymphoid cells alone or with added macrophages were used as controls. Lymphoid cells incorporated tritiated thymidine only when FDC and lectins were added; this could be shown after several periods of time. However, the Ig secretion by lymphoid cell populations was inhibited by FDC after several days in vitro. In contrast, the supernatants of lymphocytes cultured alone or with macrophages only for the same periods of time contained increasing amounts of immunoglobulins. This inhibitory effect of FDC on immunoglobulin production was observed for all considered isotypes. Our data suggest that FDC stimulate lymphoid cell proliferation but reduce B-cell differentiation. This is the first accessory cell activity definitely shown for FDC in cultures.

The lymph follicle: a hard nut to crack

Abstract Lymphoid follicles are well characterized in terms of their histology and cellular composition. In this article, Ernst Heinen and colleagues address some of the lesser known aspects of lymphoid follicles, and highlight the fact that many questions remain unanswered about these ordered areas of intense immunological activity.

Cytokines produced in lymph follicles.

The events occurring inside lymph follicles during a germinal center reaction are poorly understood. Using B and T lymphoid cell populations prepared from human tonsillar lymph follicles, and enriched or not in macrophages or in follicular dendritic cells, we examined the production of cytokines by these cells in vitro. Interleukin 6 (IL-6) and tumor necrosis factor (TNF) were found in the supernatants of cultures stimulated with phytohemagglutinin or pokeweed mitogen. IL-1 beta was occasionally detected; its secretion apparently depends on the origin of the tonsils, the stimulation, and the cell populations. IFN-gamma and IL-2 were not produced in significant amounts by these lymph follicle cells. IL-4 was only found in very low concentrations in the supernatant of the different cell cultures. The cell populations containing follicular dendritic cells produced more IL-6 and TNF than the others, especially than those composed of only B and T cells.

Isolation of follicular dendritic cells from human tonsils and adenoids. In vitro culture.

Germinal centers of lymphoid follicles are made of lymphoid cells and of accessory cells. Among these are the follicular dendritic cells or FDC’s. They surround lymphoid cells with fine membrane extensions and we have shown that those closed relations are preserved after isolation of the cells from lymphoid tissues (1,2,3,4). To analyze the interactions that may take place between FDC and lymphocytes in such associations, we wanted keep them alive in vitro. For this purpose, we tried to maintain in culture entire human follicles obtained after dissection under biomicroscope and, secondly, to cultivate isolated human FDC. On an other hand we hoped to maintain pure FDC in culture, that means FDC populations without any accompanying lymphocyte. For this purpose we used a culture technic described by Jordan et al. (5). They obtained a rapid degeneration of mice thymic lymphocytes when the cultures were performed at 24°C.

Immunohistochemical Study on Cultured FDC-C Enriched Lymphoid Cell Populations

The follicular dendritic cell-clusters (FDC-C), which are obtained by mild enzymic digestion from lymph follicles, are now considered as the structural minimum units of the germinal centers in situ (1, 2). One isolated FDC forms a globular structure, engulfing 8 to 9 germinal center B cells. Cultured FDC-C exhibit an accessory function to lymphoid cell proliferation but reduce B-cell differentiation (3). To investigate this accessory function morphologically, we tried to clear the cellular phenomenons happening in the cultured FDC-C enriched populations, especially inside and at the periphery of the FDC-C, which an immunohistochemical method and electron microscopy.

Influence of immunoglobulin isotypes and lymphoid cell phenotype on the transfer of immune complexes to follicular dendritic cells

Follicular dendritic cells (FDC) are located only inside lymph follicles and are characterized mainly by their capacity to retain high amounts of immune complexes by their Fc or C3b receptors. In this work, we examine the influence of immunoglobulin isotypes and the subset of lymphoid cells (B or T) upon the transfer of immune complexes from lymphocytes to FDC. FDC isolated from mice lymph nodes by enzymatic digestion are able to fix, through Fc receptors, gold-labeled immune complexes presented by lymphoid cells. As demonstrated by electron microscopy, this transfer requires the establishment of close contacts between both cell types. Using different cell selection techniques we show that B lymphoid cells take up immune complexes more efficiently than do T lymphoid cells and transfer a larger number of them to FDC. This transfer mechanism is dependent on the immunoglobulin isotype: immune complexes constituted of IgG2a, IgG2b, and IgG1 isotypes are better transferred to FDC than those constituted of IgG3 and IgM.

Follicular Dendritic Cells: Isolation Procedures, Short and Long Term Cultures

The need to isolate and cultivate follicular dentritic cells (FDC) in vitro is becoming increasingly acute in view of the physiological complexity of the germinal centers and the accumulation of data on their pathology.

Interactions between follicular dendritic cells and lymphoid cells.

Circulating B or T cells may home to lymph follicles either into their corona or their germinal center. Little is known about this migration. B cells settling in the follicles are either memory cells or newly formed lymphocytes arising from the bone marrow. Mel 14+ and mostly IgD+ cells accumulate in the corona whereas Mel 14− and IgD− cells are preferently found in the germinal centers where about 5% T helper cells are also present (1).

Lipopolysaccharide suppresses immune complex retention by follicular dendritic cells without cytological alterations

Follicular dendritic cells (FDC) are peculiar cells only located inside lymph follicles and which may be characterized by complex dendritic evaginations retaining high quantities of immune complexes by Fc and C3b receptors. After lipopolysaccharide (LPS) injection in mice the retention of gold-labelled immune complexes was abolished in draining lymph nodes. In order to examine the possibility that the transport of immune complexes to lymph follicles was impaired, we isolated FDC from lymph nodes and incubated them in presence of gold-labelled complexes: no or strongly reduced retention was then observed at the ultrastructural level. This LPS-induced impairment of immune complex fixation by FDC is not due to morphological alteration to the cells but to the inhibition of their Fc and C3b receptors. Further, LPS induces changes in the composition of the lymphocyte population in lymph follicles as higher numbers of blast cells and plasmocytes are observed after treatment.