Follicular dendritic cells in germinal centre development

G E R M I N A L CENTRES munological memory. ImmunoL Rev., 53, 3. Kroese, F.G.M., Wabbena, A.S., Opstelten, D. et al. (1987b), B lymphocyte differentiation in the rat, production and characterization of monoclonal antibodies to Blineage-associated antigens. Europ. J. lmraunol., 17, 921. Kroese, F.G.M., Wubenna, A.S., Seijen, H.G. & Nieuwenhuis, P. (1987a), Germinal centers develop oligoclonally. Europ. J. lmmunol., 17, 1069. Liu, Y.-J., Joshua, D.E., Williams, G.T., Smith, C.A., Gordon, J. & MacLennan, I.C.M. (1989), Mechanisms of antigen-driven selection in germinal eentres. Nature (Lond.), 342, 929-931. Liu, Y.-J., Cairns, J.A., Abbot, S.D., Holder, M.J., Jansen, K., Bonfois, J.-Y., Gordon, J. & MacLennan, I.C.M. (1991), Recombinant 25-kiloDalton

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Follicular dendritic cells in germinal centre development L.H.P.M.

Rademakers

University o f Utrecht, Department o f Pathology, Academic Hospital Utrecht, P.O. Box 85,500, NL-3508 GA Utrecht (The Netherlands)

Follicular dendritic cells (FDC) function as accessory cells and provide a microenvironment allowing proliferation and differentiation of germinal centre B cells. In man, abnormal germinal centres occur in pathological conditions and in germinal centre cell-derived nonHodgkin lymphomas. In such germinal centres, alterations in the framework of FDC and ultrastructural changes are regularly found, suggestive o f a disturbed microenvironment made by FDC. The immunohistochemical and morphological properties of FDC in

the different phases of germinal centre formation may provide information on their functional stage. Such a knowledge contributes to a better understanding o f the genesis of abnormal germinal centres. This paper gives a short overview of the development of the FDC framework and will focus on two main points : (1) the nature o f the precursor cells o f FDC and (2) the heterogeneity o f FDC in relation to their functional stage. During germinal centre fol'mation during the immune response, several developmental phases can

be distinguished (Nieuwenhuis and Lennert, 1981). Initially, (1) large blasts accumulate in primary germinal centres. This is followed by a second phase (2) in which the germinal centre is composed o f centroblasts and starry sky macrophages existing till day 7 o f stimulation. In the third phase, (3) a zonal structure o f a dark zone, mainly composed o f centroblasts, and a light zone containing the whole spectrum o f germinal centre cells including FDC, becames a u parent. Dependent on antigenic stimulation, this phase may last

258 14 days up to several months. Finally (4) the zonal structure diminishes. The germinal centre mainly consists of centrocytes and FDC and may subsequently go into regression. On the development of the FDC framework during the primary response, few data exist. In primary follicles, primitive reticnlum cells, but no mature FDC occur (Veerman, 1975). These cells have the capacity to retain immune complexes (Dijkstra et ai., 1984). In man, similar cells in primary germinal centres are reactive with the DRC-1 antibody which recognizes FDC (Stein et al., 1982). Our own observations in man and rodents and those of others (Heusermann et al., 1980; Imai et al., 1983) indicate that during the initial accumulation of blasts, the reticulum is pushed aside. Primitive reticulum cells are only present at the boundary and around blood vessels. Ultrastructnrally, typical FDC become apparent at the apex of the follicle as the light zone develops and appear to penetrate into the dark zone. During expansion of germinal centres, the number of FDC increases. In human lymph DRC-1 antibody) are present in the majority of the dark zones and always in dense patterns in the light zones of secondary germinal centres (Stein et al., 1982). The frequencies of FDC differ between the dark and the light zone. In man, FDC in the dark zone comprise about 2 % of the cell population; in the light zone the proportion of FDC is 2.5-fold higher. Here, FDC may occupy 10-20 % of the germinal centre volume (Rademakers et ai., 1983). The fate of FDC when germinal centres disappear is unknown. Observations of Hendriks (1981) suggest that they dedifferentiate into the same primitive reticular cells from which they originate. The origin of FDC is not well established and is still controversial. Marker studies have shown that FDC express antigens which

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are common to macrophages (Gerdes et aL, 1983; Kinet-Denoel et aL, 1985; Parmentier et al., in press), B-cell (Johnson etaL, 1986) and myeloid cell lineages (Schriever et al., 1989). However, a bone marrow origin of FDC is less obvious (Humphrey and Sundaram, 1985). Most studies (Veerman, 1975; Kamperdijk et aL, 1978; Heuserman et aL, 1980; Dijkstra et al., 1984) suggest that FDC transform from fibroblastic reticulum cells (FRC). Contrarywise, Miiller-Hermelink et al. (1981) proposed a different cell lineage for both FDC and FRC. Enzyme histochemical studies on isolated human FDC in our laboratory showed that FDC have an enzyme pattern which differs from that of macrophages. A small proportion of FDC has alkaline phosphatase activity. This property favours a mesenchymal cell lineage. In sections of human tonsils, we localized DRC-l-positive cells having alkaline phosphatase activity around blood vessels in and at the periphery of germinal centres. By electron microscopy, FDC showing alkaline phosphatase activity had the cytology of rather primitive FDC. Moreover, we observed FDC around capillaries in the germinal centre being in direct contact by desmosomal attachments with cells present in the perivascular space. These observations suggest that FDC may transform from perivascular, pericyte-like mesenchymal ceils. Like pericytes, such cells have contractile properties (Herman and D'Amore, 1985). We therefore studied the immunohistochemical distribution of the contractile proteins myosin and a-actin and the intermediate filament proteins vimentin and desmin in the human tonsils. Within the vimentin-positive mesenchymal cells, two subpopulations were present, e.g. a-actin-positive and desmin-positive cells. The localization of a-actin and desmin reactivity was different, as demonstrated in serial sections and by double immunostaining for both markers.

0t-Actin reactivity was observed in interfollicular regions and in the outer part of the mantle zone of, in particular, the large follicles. Desmin reactivity was also present in the interfollicular areas and peripherally in mantle zones. In tonsils composed of large germinal centres, desmin reactivity was scarce. In tonsils containing small germinal centres, desmin reactivity was more abundant. Here it was present within primary germinal centres, in solitary cells located in the light zone, around intrafollicular capillaries, in rather high density patterns in the mantle zone and in interfollicular regions. In such small germinal centres and in mantle zones, desmin reactivity was observed in FDC, as shown by co-localization of desmin and DRC- 1 reactivity after two-colour immunostaining. But, there was no co-localization of ~-actJn and DRC-1 reactivity. We conclude that FDC arise from mesenchymal cells present in the lymphoid stroma which contain vimentin and desmin as intermediate filament proteins. According to their ultrastructure and actin and myosin content, mesenchymal cells in lymphoid tissue are defined as myofibroblasts or myoid cells (Miiller-Hermelink et al., 1981; Toccanier-Pelte et al., 1987). Myofibroblasts also occur in granulation tissue and in other pathological conditions and form a heterogenous cell population with regard to the presence of contractile and intermediate filament proteins (Franke and Moll, 1987; SkaUy et al., 1989). They may share properties with pericytes (Sims, 1986) and are thus able to migrate and have the capacity to proliferate and differentiate rapidly. Thus far, there is no evidence that FDC are able to divide. So the subpopulation of desmin-positivc myofibroblasts forms a stromal reserve pool of mesenchymal cells for generation of FDC during the formation of germinal centres. Desmin-positive cells present in the mantle zones and around intrafollicular capillaries can transform

GERMINAL CENTRES into FDC and may thus be responsible for an intrinsic increase in the number of FDC during the expansion of germinal centres. In secondary germinal centres, FDC form a heterogenous cell population according to their staining patterns of 5'-nucleotidase and immunostaining pattern for various antigens (Stein et ai., 1982; Carbone et al., 1988; Petrasch et aL, 1989). Some of these properties indicate topographic differences of FDC within the dark and light zones of the follicles. These feature apparently reflect differences in the functional stage of FDC. In the dark zone (Curran et aL, 1982), proliferation of B cells predominates, whereas in the light zone the differentiation of B-cells is concentrated. In our laboratory, we defined the cellular composition of the dark and light zones of human tonsillar germinal centres by means of quantitative and qualitative electron microscopic methods. These zones clearly differed in their content of cleaved blasts, small centroblasts, centrocytes, centroplasmocytoid cells, small lymphocytes and FDC. In tonsils, we distinguished seven ,LfflJ~O . . . . . . e ~ r ~ ~ ,~.~ basis v,^rt,,~.u°:" content in cell organelles, the presence of cellular extensions and of extracellular electron-dense (immune complex) deposits and the occurrence of intermediate filaments. These types were designated as: type 1, primitive FDC; type 2, undifferentiated FDC; type 3, intermediate FDC; type 4, differentiated FDC, type 5, secretory FDC; type 6, pale FDC and type 7, dark FDC. These FDC types showed a different distribution between the dark and light zone of the germinal centre. In the dark zone, type 2 and type 3 predominated, whereas in the light zone, type 4 and type 5 comprised the majority of FDC. Types 6 and 7 are infrequently present in the apical part of the light zone. It can be concluded that FDC in the dark zone are poorly differentiated, physiologically inactive cells. Taken together with a low fre~Jl

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quency of such FDC types in the dark zone, it appears that FDC do not actively participate in the proliferation of germinal centre B cells in the dark zone. FDC in the light zone however, can be regarded as highly differentiated cells, exhibiting a full biologic activity. According to the presence of a well developed rough endoplasmic reticulum, an extensive Golgi complex and numerous cytoplasmic vesicles, types 4 and 5 can be regarded as secretory active cells. In contrast, FDC type 6 and type 7 represent differentiated, but regressive inactive forms, as judged by their poor cytoplasmic organization. Electron-dense depositions presumably associated with antigen presentation are defined to FDC types 4, 5, 6 and 7 in the light zone and not with the undifferentiated FDC types 1, 2, and 3 which have a preferential localization in the dark zone. Our observations on FDC in the light zone suggest that, besides a role in antigen presentation to B cells and clonal expansion of memory B cells (van Rooyen, 1989; Tew et al., 1989), FDC themselves are actively involved in the maintenance of the microenvironment which favours B-cell differentiation. By modulating their secretory activity, FDC are able to stimulate differentiation or allow proliferation of selectively trapped B cells. Such a feature would make the germinal centre susceptible for rapidly operating feedback mechanisms during the immune response. Only a few data exist in which FDC interfere in the sequence differentiation events of memory B cells from centroblasts and centrocytes. Our data on abnormal germinal centre features after HIV1 infection (a condition in which there is evidence that FDC are affected; Parmentier et al., 1990) show a relation of the differentiated FDC types 4 and 5 with the blast-centrocyte content of the germinal centre and the incidence of mitosis. This suggests that these FDC types support the formation of centrocytes from ten-

259 troblasts and have a negative influence on mitosis of germinal centre B cells. However, this is not in line with in vitro studies on isolated tonsillar FDC. Here, the results indicate that FDC stimulate proliferation and inhibit terminal B-cell differentiation into plasma cells (Cormann et al., 1987). In conclusion, FDC arise from a subpopulation of mesenchymal cells, which contain vimentin and desmin as intermediate filament proteins. During germinal centre development, the number of FDC increases, and a heterogeneous population of FDC develops. The morphologically different FDC types have a typical distribution in both the dark and the light zone. The preferential occurrence of highly differentiated FDC in the light zone suggests that FDC have a role in B-cell differentiation. The question is, what factors induce the formation of these highly differentiated FDC types. Data on B-cell lymphomas (Mori et aL, 1988; Scoazek et al., 1989) do not exclude a role of B cells in the formation of FDC. In follicular lymphomas, the antigenic profile of FDC is similar to those in secondary germinal centres (Petrasch et aL, 1990), but ultrastructurally (Rademakers et ai., 1983) they have the appearance of primitive FDC as is observed in the dark zone of germinal centres of tonsils. So additional factors may further stimulate and m a i n t a i n the differentiation of FDC within the light zone. Identification of these factors might be of value for understanding the immunological basis of abnormal germinal centre reactions in man. Such knowledge is also important for studies on isolated FDC and might explain the sometimes contradictory results of "in vivo" and "in vitro" studies on FDC function. References

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260

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