C. Kinet-Denoel

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. I. Procedure and morphological characterization

Follicular dendritic cells have been isolated from human tonsils and adenoids and characterized at the ultrastructural level. Follicles were dissected and digested with different hydrolytic enzymes. The cells were separated by sedimentation at unit gravity. By this procedure we obtained follicular dendritic cells enveloping lymphocytes with their cytoplasmic extensions in a way analogous to that described for isolated thymic nurse cells. The ultrastructural features of isolated follicular dendritic cells are similar to those observed in situ. Prolonged enzymatic action caused loss of the enveloped lymphocytes.

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.

Retention of immune complexes by Fc receptors on mouse follicular dendritic cells.

Follicular dendritic cells (FDC) are located inside lymph follicles and are mainly characterized by their capacity to retain antigens. We investigated this aspect in mice lymph nodes by using bovine serum albumin (BSA) labelled with 5‐nm colloidal gold particles and homologous anti‐BSA antibodies bound to 20‐nm gold particles. Gold‐labelled BSA injected alone in non‐immunized mice was only rarely found in FDC cytoplasmic interdigitations. Injected in the form of immune complexes, it was retained by FDC. Antigen‐free anti‐BSA antibodies injected under similar conditions as immune complexes were always found in draining lymph nodes in the same locations as BSA‐anti‐BSA immune complexes. F(ab′)2 from mouse immunoglobulins linked to colloidal gold particles were very rarely found between the FDC extensions, whereas it was intensely phagocytosed by macrophages. Our study permitted precise ultrastructural localization between FDC cytoplasmic extensions or inside macrophages and other cells of the lymph nodes, but it also pointed out that homologous antibodies linked to colloidal gold particles might be retained by FDC in the absence of antigens. These observations, carried out with colloidal gold, were checked by using 125I‐labelled anti‐BSA antibodies. Complement activation determinations of gold‐labelled antibodies or immune complexes showed that antibodies or immune complexes fixed on colloidal gold particles do not activate the complement. This observation enabled us to conclude that Fc receptors play a significant part in the retention of gold‐labelled antibodies or immune complexes by FDC of lymph nodes.

Precise localization of antigens on follicular dendritic cells.

SummaryHorse-spleen ferritin or bovine serum albumin conjugated to colloidal gold (BSA-gold) were injected subcutaneously in preimmunized mice. In draining lymph nodes both antigens were located in macrophages or between the cytoplasmic processes of follicular dendritic cells (FDC). Some of the antigens remained trapped on FDC until day 31 after injection. Simultaneous injection of both antigens showed that they were located between the infoldings of the same FDC. These cells are thus able to retain at least two different antigens on their surface. The peculiar arrangement of ferritin between the cytoplasmic infoldings suggests that this antigen is fixed on both cell membranes by specific antibodies. The trapped immune complexes could thus stabilize the FDC membrane system.The antigen retention requires the presence of specific antibodies since BSA-gold or ferritin injected without preimmunization were not found between FDC processes. Nonantigenic materials, such as colloidal gold or carbon particles, are not trapped by FDC, except when injected in large amounts.The antigens were trapped on the surface of FDC, however unfrequently in close contact with lymphocytes. FDC might protect lymphocytes against an excess of immune complexes and act as regulators of contacts between lymphocytes and immune complexes.

Follicular Dendritic Cells in Lymph Nodes after X-irradiation

Follicular dendritic cells (FDC), non lymphoid cells present in lymph follicles, are characterized by numerous cytoplasmic processes retaining antigen-antibody complexes. Their origin, nature and function are unknown. Mice inguinal lymph nodes after 4.5 or 7.5 Gy X-irradiation were depleted of lymphoid cells. Ultrastructural observations during the first few days post-irradiation show that FDC are unaltered and possess dendritic processes enveloping dense material. Furthermore, they show intense metabolic activity. A lamina densa, never observed so well-developed in other lymph node cells, was detected around the nuclear envelope. The localization of junctions between FDC was analysed. FDC preserve their typical cytoplasmic processes even if lymphoid cells are rare. The latter thus seem not to be responsible for the maintenance of FDC integrity or their development. The possible role of this for antibody production is discussed. Irradiated lymph nodes of lymphoid cells are highly convenient for studying FDC. Isolation of FDC from irradiated lymph organs would seem to be possible.

Mouse lymph node follicular dendritic cells: quantitative analysis and isolation.

Follicular dendritic cells (FDC) are peculiar non-lymphoid cells only present in lymph follicles especially in germinal centers. They retain antigens in form of immune complexes on the surface of their membranes (1, 2, 3). Few data are available concerning their nature, origin and function. Their study is difficult as they represent only a low percentage of the cell population inside the germinal centers. Recently we achieved the isolation of FDC from human tonsils and adenoids (4). After isolation human FDC form spherical structures where they envelope lymphoid cells (mainly B cells, some T helper cells) with their cytoplasmic extensions. Isolated tonsillar FDC bear IgM, IgA, IgG and IGE but no IgD on their surface. They share also some antigens with macrophages (HLA-DR and Leu-M3) and express, at high density, C3b and Fc receptors (5).

Colloidal gold, a useful marker for antigen localization on follicular dendritic cells

Bovine serum albumin (BSA) bound to colloid gold particles (BSA-gold; 20 nm diameter) and injected into preimmunized mice was found at ultrastructural level in different locations of the lymph nodes. It was detected particularly in the secondary lysosomes of macrophages and between the cytoplasmic processes of the follicular dendritic cells. Between these processes the gold particles were isolated or grouped in clusters; they were in close contact with cell membranes or embedded in dense material. Colloidal gold injected alone was not retained on these cells. The presence of anti-BSA antibodies in the serum was necessary for trapping of BSA-gold particles on follicular dendritic cells. Injections of BSA alone after BSA-gold had been administered to preimmunized mice eliminated most of the BSA-gold from the dendritic processes. BSA-gold is thus trapped in the form of immune complexes which behave characteristically. BSA-gold is thus a suitable marker for antigen localization. Being small and electron dense it permits more precise location than radioactive markers.

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.