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The lymph follicle: a hard nut to crack
Immunology Today, VoL 9, Nos 7and8, 1988
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The lymphfollicle: a hard nut to crack 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. If one considers a lymph follicle, everything appears clear at first glance: its cell composition is known and its connection with the humoral immune response is established beyond doubt. However, the mechanisms controlling cell migration, multiplication and differentiation within these lymphoid follicles are far from clear and form a hard nut which has resisted the efforts of numerous investigators for decades.
General features of lymph follicles Lymph follicles are nodules 1-2 mm in diameter. Their central region, the germinal center (GC), is subdivided into a dark and a light zone (Fig. 1). The periphery, corona or mantle zone, either entirely surrounds the GC or forms a crescent-shaped cap over it. Capillaries are always found in the close vicinity of, or inside the follicles. The GC may be absent or highly enlarged; its cell composition also changes as cells ceaselessly migrate, multiply and differentiate. After antigenic stimulation, B-lymphoid cells proliferate inside the GC and then differentiate into either memory or immunoglobulin (Ig)-secreting cells 1.2. Most lymph follicles are found inside the lymph organs (spleen, lymph nodes), or !n the mucosal-associated tissue along the digestive, respiratory or urinary tracts (especially the tonsiis, Peyer's patches and the appendix). Most lymph nodules are thus at the junctions of the lymph circulation (lymph nodes, gut), in direct contact with the blood (spleen) or at sites of intense antigen inundation (digestive and respiratory tracts). What cells make up lymph follicles? B cells form the major component of the follicular cell population: generally, small B cells are located in the corona and large ones in the GC (centroblasts in the dark zone, centrocytes in the light) ~. Many surface features of these B cells have been determined: most corona cells express surface 5'-nucleotidase and bear IgM, IgD and major histocompatibility complex (MHC) class II antigens. Centroblasts and centrocytes can express all Ig isotypes except IgD and are rich in MHC class II antigens 3. It has frequently been suggested that coronal B cells are quiescent, whereas centroblasts are dividing and centrocytes are maturing ceils. A small percentage of these B cells appear as precursors of plasma cells (immunoblasts, plasmoblasts; Ref. 4 and L.J. Simar, PhD thesis, University of Liege, 1973). These cells synthesize cytoplasmic Ig but are apparently not secretory S.
240
Institute of Human Histology, State University of Liege, 20 Rue de Pitteurs, B-4020, Liege,Belgium.
ErnstHeinen,NadineCormannand Cedle Kinet-Denol It must be emphasized that GC B cells are PNA positive and generally bear Fc and C3b receptors. They lack antigens recognized by Mel 14 (Ref. 6) or Hermes-1 (Ref. 7) antibodies, which indicates that they do not recirculate. They may, however, move within the follicles from the dark to the light zone or to the corona, and also to the medullary cords in the lymph nodes. About 5% of the GC cells are T cells3,8: their phenotype is mostly that of helper T cells9. Recently Kroese (PhD thesis, University of Groeningen, 1987) underlined the fact that rat T cells located in GCs are of both the T helper and suppressor cell phenotype, and have the Leu-7 antigen in common with natural killer cells. Moreover, these T cells are at least temporarily sedentary as indicated by the absence of the Mel 14 antigen 6. Velardi et a/. lo showed that lectin-stimulated T cells isolated from GCs do not produce interleukin 2; they are thus neither truly helper nor lyric cells. T cells isolated from GCs are small lymphocytes which establish contacts with B cells, macrophages and follicular dendritic cells (E. Heinen, PhD thesis, University of Liege, 1986). Macrophages also populate the follicles. In the 6C, tingible body macrophages frequently occur 11.12. They intensively phagocytose lymphoid cells (tingible bodies), including cells that are in the S phase of the cell cycle. Along with these macrophages, which do not absorb antigens, GCs also contain normal macrophages laden with antigens brought in from outside or picked up inside the follicles (E. Heinen, PhD thesis, Liege, 1986). Indeed, after injection of gold-labelled antigens in mice, macrophages endocytosing these particles can occasionally be found in the GC of draining lymph nodes. The follicular dendritic cells form an unusual cell type encountered exclusively in the lymph follicles. They represent 2% of the total cell population in the GC 13 and extend long and complex cytoplasmic processes forming a network around lymphoid cells, especially in the light zone of the GCs. Follicular dendritic cells are identified by specific monoclonal antibodies 14. Their origin is unclear: they do not arise from the bone marrow ~s. Most data indicate that they differentiate from reticular cells16, although they share some antigens with macrophages 14.
How do lymph folliclesappear? The development of lymph follicles has been studied during embryogenesis, in neonates and in adults. They are absent from embryos or found only in the form of primary follicles. According to some authors, conjunctive cells increase in numbers before lymphoid cells appear, whereas others claim that lymphoid cells first accumulate locally and induce conjunctive cells to differentiate, for example, into follicular dendritic cells. Williams and Nossa116 studied the retention of bacterial flagellin in rat neonates and concluded that it is fixed only in 10 day old rats; they considered that the capacity to retain antigens © 1988, ElsevierPublications,Cambridge 0167 - 4919/88/$02.00
Immunology Today, VoL 9, Nos 7andS, 1988 ii
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appeared before the arrival of lymphocytes. This contradicts the data of Dijkstra et al. 17 that indicate that antigen retention occurs only in primary follicles, after the arrival of lymphoid cells. Kroese et al. is recently showed that 3 day old rats are unable to mount a GC reaction but that their lymphoid cells can give rise to a response when injected into irradiated adult rats. One may conclude, therefore, that the follicular microenvironment, namely the follicular dendritic cells, must acquire a certain degree of maturation (most probably cell receptors and the capacity to produce cytokines) to allow GC formation. In adults, antigenic stimuli induce an enlargement of lymphoid follicles and an increase in their number 19. GC development undergoes cyclical changes after antigenic stimulation ~. After the introduction of antigen, the follicles undergo an induction phase, barely detectable morphologically: lymphoid cells are stimulated and migrate towards the follicles. Four days after antigen injection, dividing centroblasts are found in primary follicles; 24 hours later tingible body macrophages are seen, while the GC is composed mostly of centroblasts. The typical subdivision into dark (centroblasts) and light (centrocytes) zones appears only after 1-3 weeks. This aspect persists from a few weeks to several months. In the end phase the centroblasts disappear. During a primary response 66% of B cells bear IgM on their surface and 10% IgG; after a second antigenic challenge these proportions are reversed - 30% are IgM positive and 72% IgG positivez°. It must be emphasized, however, that although GCs in tonsils and lymph nodes contain mainly IgG-positive cells, GC B cells in Peyer's patches express mainly IgA2~. In sheep, Peyer's patches contain numerous large follicles which appear during embryogenesis and which produce quantities of lymphocytes during the postnatal nc=ri~rl ~bl IV~I
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cles are apparently equivalent to the bursa of Fabricius, the fetal liver or the bone marrow and produce virgin B cells2z. What is the role of follicular dendritic cells? One characteristic feature of follicular dendritic cells is their capacity to fix considerable numbers of immune complexes on their cytoplasmic extensions23. These immune complexes are retained by Fc or C3b receptors without endocytosis for long periods of time 24.zs. During the induction phase, after primary contact with antigen, follicular dendritic cells do not play a part in lymphocyte activation since they fix antigens only when specific antibodies are present. They thus appear to act at a later stage, either during the formation of memory B cells, or during the control of the secretion of antibodies z4.z6. Indeed, Kunkl e t a / . 27 showed that the injection of immune complexes induces the enlargement of existing GCs, the development of new ones, the appearance of B memory cells and an increase in Ig affinity. According to our results, follicular dendritic cells create a microenvironment where they improve the survival of lymphoid cellszs, favour lymphoid cell multiplication but appear to slow down the differentiation in Ig-secreting cellszg. The mechanisms by which survival and proliferation are ensured, or differentiation is inhibited in lymphoid cells, are unknown. Direct contact between follicu-
Fig. 1. A lymph follicle after antigenic stimulation: a corona (C) surrounds a large germinal center with apparent dark (D) and light (L)zones (x 288). lar dendritic cells and B cells (and perhaps also other cells). . . . . . seems to play a role: isolated follicular dendritic cells appear as clusters, surrounding lymphoid cells with their cytoplasmic extensions3°. Prostaglandins3; and products of the 5'-nucleotidase activity apparently secreted by these follicular dendritic cells3z may act on lymphoid cells, but much further research is required before the accessory role of follicular dendritic cells is fully understood. Where do the follicular B-lymphoid cells come from? Following the initiation of a primary response, centroblasts and centrocytes develop from bone marrowderived cells (IgM +, IgD+). During their circulation, they encounter antigens in sites containing T cells and macrophages33. Cellular phenomena such as transformation of lymphocytes into blast cells during this induction phase have been well studied. However, why these cells home to the GC is unknown. During a secondary resp.onse, the GC cells are mostly...recruited. from B memory cells34.3s. This response is quicker than the primary response, perhaps because the cells able to react are more numerous and more responsive.
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v l W$ GC B cells not only divide but also undergo gene rearrangements leading to the expression of other Ig isotypes (under the control of T or other cells)2o. Ig affinity also increases as a result of a clonal selection of the bone marrow cells, B memory cells and those cells whose antigenic specificity is modified by hypermutation. Indeed, inside the GC, cells pass through phases of hypermutagenicity inducing amino acid changes in the hypervariable zones of their Ig molecules 36. The factors controlling these phenomena are unknown. Not all cells that penetrate the follicles actually divide: according to results obtained by Kroese et aL 37, only a few clones proliferate and occupy the GC. The notion that GC cells are oligoclonal is, however, hardly reconcilable with the fact that GC cells are subjected to hypermutagenicity0 giving rise to new clones of high Ig affinity.
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Immunology Today,Vol. 9, Nos 7andS, 1988
l cells not divide there also? It may simply be that T cells homing to GCs have lost their proliferative capacity, do not encounter adequate mitogens or are eliminated by tingible body macrophages when dividing. It should be interesting to pursue this question particularly since T cells are essential for the GC reaction; there must be a regulatory system allowing a limited number of T cells, probably arising from the adjacent thymus-dependent zones, to specialize into GC T cells. Numerous immune complexes are retained by follicular dendritic cells. How do these complexes gain access to the follicles? Kamperdijk eta/. 39 propose that immune complexes enter the GC by diffusion. However, according to Sainte-Marie and Peng4o lymph does not irrigate the follicles easily. The immune complexes can apparently be imported by B cells (E. Heinen, PhD thesis; University of Liege, 1986). What is the role played by these immune What is the fate of the follicularB cells? complexes? According to Klaus eta/. 24 the C3b compleB cells proliferate at a high rate inside the GC: their cell ment fragment stimulates cell proliferation, but we cycle lasts about 6 hours. One astonishing observation is observed either stimulation or inhibition according to the that many of these cells die and are phagocytosed by Ig isotype used in the complex (unpublished). The imtingible body macrophages. Cytokinetic studies indicate mune complexes are mainly found in the light zone that GCs may be 'graveyards' for many lymphocytes 12. where cells appear to differentiate. Do they effectively What is the reason for this? It is possible that elimination influence cell differentiation and act as feedback regulah:~: occurred of B cells that erroneously arranged the tors of antibody synthesis during the maintenance phase genes for their isotype switch or that no longer recognize "of the immune response as proposed by Tew et a/.26? the antigen after somatic mutations of the hypervariable There is as yet no clear answer to this question. Ig region, or badly replicated their DNA during the very Many B cells bear Fc receptors: Ig fixed on Fc receptors short S phase. These three reasons do not, however, fully is generally considered to be inhibitory to B-cell explain this waste of cells. It may be that, as in the stimulation 41. Since we observed that B cells can transfer thymus, cell-cell interactions occur within the lymphoid immune complexes to follicular dendritic cells, we sugfollicles eliminating cells expressing inadequate recepgest that these B cells are then freed from Ig and so tors. allowed to proliferate. Alternatively, another role should Surviving cells stop multiplying, differentiate and miperhaps be attributed to this intercellular contact. For grate. Many questions surround these events: what example, immune complexes fixed on Fc receptors inhibit directs the differentiation of GC B cells into plasma or the Ig-forming response42; therefore, follicular dendritic memory cells? The observations made by o u r g r o u p 29 cells mav Dresent them ~o B cells ~ncl th~r~hv ~ln~^lrlr~^ln support the view that follicular dendritic cells reduce ;the the differentiation pathway ofthese-ceils.--" . . . . . . . . . . . formation of Ig-secreting cells. This could thus favour the All these cellular actions are strictly controlled inside development of memory cells: indeed, plasma cells are the particular microenvironment of the GC, but very little rarely found in GCs. They mainly differentiate after the is known about the factors involved there and much migration of cells from the GC, for example from work remains to be done in defined culture systems. In immunoblasts in the medullary cords of lymph nodes 5 or addition, the functional relationships between follicles from circulating lymphocytes that have homed to the and the vascular structures and the histological environmucosae (gut, respiratory tract) or the bone marrow. ment (especially the thymus-dependent zones) must be Other workers have noted, however, that plasma cells clarified. As long as our knowledge of GC phenomena are occasionally present inside GCs38. This can be exremains poor, we will not be able to manipulate in a plained if a balance exists between agents that inhibit physiological way the humoral immune reactions in Ig-secreting cell formation and others that favour it. In conditions such as hypo- or hyperimmunoglobulinemia, normal GCs few plasma cells are found, indicating autoimmunity, lymphomas, or other pathological states. I;r~.dominance of follicular dendritic cell action, but other factors (immune complexes, cytokines, vascularization, References etc.) certainly affect this microenvironment and mod1 Lennert, K. (1978) Malignant Lymphomas Other than ulate the differentiation into memory or Ig-secreting Hodgkin's Disease, Springer-Verlag cells. 2 Thorbecke, G.J.,Asofsky, R., Hochwald, G.M. and Siskind, The immunochemical features of B memory cells are G.W. (1962)J. Exp. Med. 116, 295-310 unclear. Many contradictory data exist, perhaps because 3 Tsunoda, R. and Kojima, M. (1987)Acta Pathol. Jpn 37, 575-585 different states of memory exist. In summary, we can say 4 von Gaudecker, B. and Hinrichsen, K. (1965)Z. Zellforsch. 65, that memory cells, according to the microenvironment 139-162 that they encounter, differentiate into plasma cells, and 5 Geldof, A.A., van de Hende, M. and Jandts, E.M. (1983) Cell return to GCs to recommence a cycle or simply to die. Tissue Res. 231, 135-142 6 Reichert, R.A., Gallatin, W.M. and Weissman, L.L (1984) Open questio.s J. Exp. Med. 157, 813-827 GCs are sites where there is great pressure on B cells to 7 Jalkanen, S.T., Bargatze, R.F., Herron, LR. and Butcher, E.C. divide (short cell cycle, high number of divisions). Why do (1986) Eur. J. Immunol. 16, 1195-1202
Immunology Today, Vol. 9, Nos 7and8, 1988
8 Gutman, G. and Weissman, I. (1972) Immunology 23, 465-471 9 Roux, R.V., Ledbetter,J.A. and Weissman, I. (1982) J. Immunol. 128, 2243-2248 10 Velardi, A., Mingari, M.C., Moretta, L. and Grossi,C.E. (1986) J. Immunol. 137, 2808-2813 11 Hoefsmit, E.C.M., Kamperdijk, E.W.A., Hendricks, H.R., Beelen, R.H.J.and Balfour, B.M. (1980) The Reticuloendothelial Syst. 1,417-468 12 Fliedner,T.M. (1967) in Germinal Centersin Immune Responses (Cottier, H., Odartchenko, N., Schindler, R. and Congdon, C.C., eds), pp. 218-224, Springer-Verlag 13 Heinen, E., Kinet-Deno~l,C., Radoux, D. and Simar, L.J. (1985) Adv. Exp. Meal. Biol. 186, 171-183 14 Stein, H., Gerdes,J. and Mason, D.Y. (1982) Clin. Haematol. 11,531-559 15 Humphrey, G.H., Grennan, D. and Sundaram,V. (1984)Eur. J. Immunol. 14, 759-768 16 Williams, G.M. and Nossal, G.J.V. (1966)J. Exp. Med. 124, 47-56 17 Dijkstra, C.D. and Langevoort, H.L. (1982) Cell TissueRes. 222, 69-79 18 Kroese, F.G.M., Wubbena, A.S., Kuijpers, K.C. and Nieuwenhuis, P. (1987)Immunology60, 597-602 19 Laissue,J., Cottier, H., Hess, M.W. and Stoner, R.D.(1971) J. Immunol. 107, 822-831 20 Butcher, E.C., Roux, R.V., Coffman, R.L.etal. (1982) J. Immunol. 129, 2698-2707 21 Matthews, J.B. and Basu, M.K. (1982)Int. Arch. Allergy Appl. Immunol. 69, 21-25 22 Reynolds,J.D. and Morris, B. (1983)Eur. J. Immunol. 13, 627-635 23 Nossal, G.J.V.(1972) Triangle 11, 1-6 24 Klaus, G.G.B., Humphrey,J.H., Kunkl, A. and Dongworth,
Multiple Sclerosis: Immunoloaic. Diaanostic and Therapeut-icA~pects
D.W. (I 980) Immunol. Rev. 53, 3-28 25 Heinen, E., Radoux, D., Kinet-Deno~l,C. etal. (1985)
Immunology 54, 777-784 26 Tew0J.G., Phipps,R.P.and Mandel, T.E.(1980)ImmunoL Rev. 53, 175-201 27 Kunkl, A. and Klaus, G.G.B. (1981)Immunology43, 371-378 28 Cormann, N., Heinen, E., Kinet-Deno~l,C.0 Braun, M. and Simar, L.J.(1986) CR SeancesSoc. Biol. 180, 218-223 29 Corrnann, N., Lesage,F., Heinen, E. etal. (1986)Immunol. Lett. 14, 29-35 30 Lilet-Leclercq,C., Radoux, D., Heinen, E. etal. (1984)J. Immunol. Meth. 66, 235-244 31 Heinen, E., Cormann, N., Braun, M. etal. (1987)Ann. Inst. Pasteur/Immunol. 137,369-382 32 Heinen, E., Kinet-Deno~l,C. and Simar, L.J.(1985) Immunol. Lett. 9, 75-80 33 Heinen, E. and Tsunoda, R. (1987)Immunol. Today8, 142-143 34 Gray, D., MacLennan, I.C.M and Lane, P.J.L (1986) Eur. J. Immunol. 16, 641-648 35 Jelinek,D.F.and Lipsky, P.E.(1987)Adv. Immunol. 40, 1-60 36 Micklem, H.S.(1988)lmmunol. Today9, 1-2 37 Kroese, F.G.M.,Wubbena, A.S., Seijen, H. and Nieuwenhuis, P. (1987) Eur. J. Immunol. 17, 1069-1072 38 Szakal,A.K., Kosco, M.H., Burton, G.F.and Tew, J.G. (1988) Prog. Leukocyte Biol. 7, 281-290 39 Kamperdijk,E.W.A., Dijkstra, C.D. and D6pp, E.A. (1987) Immunobiology 174, 395-405 40 Sainte-Marie,G. and Peng, F.S.(1985) Cell Tissue Res. 239, 31-35 41 Sinclair,N.R.C.(1983) Immunol. Today4, 35-36 42 Uher, F. and Dickler, H.B. (1986) Mol. Immunol. 23, 11771181
quality of science presented from chapter to chapter. The selected topics are representative of the speaker's lectures, but not necessaredited by F. Clifford Roseand RosemaryJones, ily of the central scientific issues John Libbeyand Company,Ltd, 1987. £26.00 of multiple sclerosis. With these (ix + 260 pages)ISBN0 86196 109 9 caveats, there were nonetheless some articles that were of particular interest and might be useful to workers in the field of multiple scleredited by JA. Aarli, M.W.H. Behan and P.O. osis. Behan, B/ackwe//ScientificPublications, 1987. The book is divided into three £60.00 (xviii+ 531 pages)ISBN0 632 01635x sections; 'Immunologic aspects', 'Diagnostic aspects', and 'Therapeutic Multiple Sclerosis is based, according aspects' of multiple sclerosis. The to the book's preface, on a sym- first section on immunology was posium held at Charing Cross hospit- generally well written, though dial in the fall of 1986. The result is a verse in its collection of multiple collection of brief papers by the in- sclerosis related articles. The second vited speakers discussing their re- section on diagnosis presents both spective •topics of interest. The edi- original data and clinical impressions tors themselves point out that there of the various diagnostic tests availare many books on multiple scler- able for multiple .~clerosis.There was osis, and justify this one on the pre- only one article discussing magnetic mise that "few accept the challenge resonance imaging of multiple sclerto deal in depth with the con- osis, which was unfortunate controversies that rage around this sidering the importance most investigators in the field now place on this enigmatic condition". Representing a collection of unre- new technique. The last section was on theraviewed papers by different authors, there is a wide diversity in terms of peutic aspects of multiple sclerosis.
Clinical Neuroimmunology
Whether or not one is a proponent of immunosuppression, including the use of steroids, azathioprine and cyclophosphamide in the treatment of multiple sclerosis, there is a large and ever-increasing literature on this subject. It was somewhat peculiar that in this section there was no mention of these therapies. Instead the collection of articles presented were written about older treatments such as diet supplements and hyperbaric oxygen, which generally have had negative clinical results in carefully controlled studies. In summary, this book will not be generally useful to the reader interested in a broad overview of the current scientific concepts related to multiple sclerosis, particularly in regard to therapy. Nonetheless specific chapters particularly in the first section on immunology present well written reviews and newer data that may be of interest to workers in the field. Clinical immunology is rapidly incorporating many of the advances in cellular immunology into the knowledge base that is useful for the practising physician. The field of
243
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The lymphfollicle: a hard nut to crack 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. If one considers a lymph follicle, everything appears clear at first glance: its cell composition is known and its connection with the humoral immune response is established beyond doubt. However, the mechanisms controlling cell migration, multiplication and differentiation within these lymphoid follicles are far from clear and form a hard nut which has resisted the efforts of numerous investigators for decades.
General features of lymph follicles Lymph follicles are nodules 1-2 mm in diameter. Their central region, the germinal center (GC), is subdivided into a dark and a light zone (Fig. 1). The periphery, corona or mantle zone, either entirely surrounds the GC or forms a crescent-shaped cap over it. Capillaries are always found in the close vicinity of, or inside the follicles. The GC may be absent or highly enlarged; its cell composition also changes as cells ceaselessly migrate, multiply and differentiate. After antigenic stimulation, B-lymphoid cells proliferate inside the GC and then differentiate into either memory or immunoglobulin (Ig)-secreting cells 1.2. Most lymph follicles are found inside the lymph organs (spleen, lymph nodes), or !n the mucosal-associated tissue along the digestive, respiratory or urinary tracts (especially the tonsiis, Peyer's patches and the appendix). Most lymph nodules are thus at the junctions of the lymph circulation (lymph nodes, gut), in direct contact with the blood (spleen) or at sites of intense antigen inundation (digestive and respiratory tracts). What cells make up lymph follicles? B cells form the major component of the follicular cell population: generally, small B cells are located in the corona and large ones in the GC (centroblasts in the dark zone, centrocytes in the light) ~. Many surface features of these B cells have been determined: most corona cells express surface 5'-nucleotidase and bear IgM, IgD and major histocompatibility complex (MHC) class II antigens. Centroblasts and centrocytes can express all Ig isotypes except IgD and are rich in MHC class II antigens 3. It has frequently been suggested that coronal B cells are quiescent, whereas centroblasts are dividing and centrocytes are maturing ceils. A small percentage of these B cells appear as precursors of plasma cells (immunoblasts, plasmoblasts; Ref. 4 and L.J. Simar, PhD thesis, University of Liege, 1973). These cells synthesize cytoplasmic Ig but are apparently not secretory S.
240
Institute of Human Histology, State University of Liege, 20 Rue de Pitteurs, B-4020, Liege,Belgium.
ErnstHeinen,NadineCormannand Cedle Kinet-Denol It must be emphasized that GC B cells are PNA positive and generally bear Fc and C3b receptors. They lack antigens recognized by Mel 14 (Ref. 6) or Hermes-1 (Ref. 7) antibodies, which indicates that they do not recirculate. They may, however, move within the follicles from the dark to the light zone or to the corona, and also to the medullary cords in the lymph nodes. About 5% of the GC cells are T cells3,8: their phenotype is mostly that of helper T cells9. Recently Kroese (PhD thesis, University of Groeningen, 1987) underlined the fact that rat T cells located in GCs are of both the T helper and suppressor cell phenotype, and have the Leu-7 antigen in common with natural killer cells. Moreover, these T cells are at least temporarily sedentary as indicated by the absence of the Mel 14 antigen 6. Velardi et a/. lo showed that lectin-stimulated T cells isolated from GCs do not produce interleukin 2; they are thus neither truly helper nor lyric cells. T cells isolated from GCs are small lymphocytes which establish contacts with B cells, macrophages and follicular dendritic cells (E. Heinen, PhD thesis, University of Liege, 1986). Macrophages also populate the follicles. In the 6C, tingible body macrophages frequently occur 11.12. They intensively phagocytose lymphoid cells (tingible bodies), including cells that are in the S phase of the cell cycle. Along with these macrophages, which do not absorb antigens, GCs also contain normal macrophages laden with antigens brought in from outside or picked up inside the follicles (E. Heinen, PhD thesis, Liege, 1986). Indeed, after injection of gold-labelled antigens in mice, macrophages endocytosing these particles can occasionally be found in the GC of draining lymph nodes. The follicular dendritic cells form an unusual cell type encountered exclusively in the lymph follicles. They represent 2% of the total cell population in the GC 13 and extend long and complex cytoplasmic processes forming a network around lymphoid cells, especially in the light zone of the GCs. Follicular dendritic cells are identified by specific monoclonal antibodies 14. Their origin is unclear: they do not arise from the bone marrow ~s. Most data indicate that they differentiate from reticular cells16, although they share some antigens with macrophages 14.
How do lymph folliclesappear? The development of lymph follicles has been studied during embryogenesis, in neonates and in adults. They are absent from embryos or found only in the form of primary follicles. According to some authors, conjunctive cells increase in numbers before lymphoid cells appear, whereas others claim that lymphoid cells first accumulate locally and induce conjunctive cells to differentiate, for example, into follicular dendritic cells. Williams and Nossa116 studied the retention of bacterial flagellin in rat neonates and concluded that it is fixed only in 10 day old rats; they considered that the capacity to retain antigens © 1988, ElsevierPublications,Cambridge 0167 - 4919/88/$02.00
Immunology Today, VoL 9, Nos 7andS, 1988 ii
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appeared before the arrival of lymphocytes. This contradicts the data of Dijkstra et al. 17 that indicate that antigen retention occurs only in primary follicles, after the arrival of lymphoid cells. Kroese et al. is recently showed that 3 day old rats are unable to mount a GC reaction but that their lymphoid cells can give rise to a response when injected into irradiated adult rats. One may conclude, therefore, that the follicular microenvironment, namely the follicular dendritic cells, must acquire a certain degree of maturation (most probably cell receptors and the capacity to produce cytokines) to allow GC formation. In adults, antigenic stimuli induce an enlargement of lymphoid follicles and an increase in their number 19. GC development undergoes cyclical changes after antigenic stimulation ~. After the introduction of antigen, the follicles undergo an induction phase, barely detectable morphologically: lymphoid cells are stimulated and migrate towards the follicles. Four days after antigen injection, dividing centroblasts are found in primary follicles; 24 hours later tingible body macrophages are seen, while the GC is composed mostly of centroblasts. The typical subdivision into dark (centroblasts) and light (centrocytes) zones appears only after 1-3 weeks. This aspect persists from a few weeks to several months. In the end phase the centroblasts disappear. During a primary response 66% of B cells bear IgM on their surface and 10% IgG; after a second antigenic challenge these proportions are reversed - 30% are IgM positive and 72% IgG positivez°. It must be emphasized, however, that although GCs in tonsils and lymph nodes contain mainly IgG-positive cells, GC B cells in Peyer's patches express mainly IgA2~. In sheep, Peyer's patches contain numerous large follicles which appear during embryogenesis and which produce quantities of lymphocytes during the postnatal nc=ri~rl ~bl IV~I
F. =f ~^~i.tlrh int,,-~h .+-- ÷l.t~r~=T+~,. TI 1f1,¢,, ,~. ~.¢ ~ h,..,,...I...C^ll-* Ill ll'%ml I IIIV'II.JlT~IIII611~ I . I I ~ , . I I I ~ U I I + . ~ I . l ~ l l l l ,J l l iUlll-
MU5
cles are apparently equivalent to the bursa of Fabricius, the fetal liver or the bone marrow and produce virgin B cells2z. What is the role of follicular dendritic cells? One characteristic feature of follicular dendritic cells is their capacity to fix considerable numbers of immune complexes on their cytoplasmic extensions23. These immune complexes are retained by Fc or C3b receptors without endocytosis for long periods of time 24.zs. During the induction phase, after primary contact with antigen, follicular dendritic cells do not play a part in lymphocyte activation since they fix antigens only when specific antibodies are present. They thus appear to act at a later stage, either during the formation of memory B cells, or during the control of the secretion of antibodies z4.z6. Indeed, Kunkl e t a / . 27 showed that the injection of immune complexes induces the enlargement of existing GCs, the development of new ones, the appearance of B memory cells and an increase in Ig affinity. According to our results, follicular dendritic cells create a microenvironment where they improve the survival of lymphoid cellszs, favour lymphoid cell multiplication but appear to slow down the differentiation in Ig-secreting cellszg. The mechanisms by which survival and proliferation are ensured, or differentiation is inhibited in lymphoid cells, are unknown. Direct contact between follicu-
Fig. 1. A lymph follicle after antigenic stimulation: a corona (C) surrounds a large germinal center with apparent dark (D) and light (L)zones (x 288). lar dendritic cells and B cells (and perhaps also other cells). . . . . . seems to play a role: isolated follicular dendritic cells appear as clusters, surrounding lymphoid cells with their cytoplasmic extensions3°. Prostaglandins3; and products of the 5'-nucleotidase activity apparently secreted by these follicular dendritic cells3z may act on lymphoid cells, but much further research is required before the accessory role of follicular dendritic cells is fully understood. Where do the follicular B-lymphoid cells come from? Following the initiation of a primary response, centroblasts and centrocytes develop from bone marrowderived cells (IgM +, IgD+). During their circulation, they encounter antigens in sites containing T cells and macrophages33. Cellular phenomena such as transformation of lymphocytes into blast cells during this induction phase have been well studied. However, why these cells home to the GC is unknown. During a secondary resp.onse, the GC cells are mostly...recruited. from B memory cells34.3s. This response is quicker than the primary response, perhaps because the cells able to react are more numerous and more responsive.
241
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v l W$ GC B cells not only divide but also undergo gene rearrangements leading to the expression of other Ig isotypes (under the control of T or other cells)2o. Ig affinity also increases as a result of a clonal selection of the bone marrow cells, B memory cells and those cells whose antigenic specificity is modified by hypermutation. Indeed, inside the GC, cells pass through phases of hypermutagenicity inducing amino acid changes in the hypervariable zones of their Ig molecules 36. The factors controlling these phenomena are unknown. Not all cells that penetrate the follicles actually divide: according to results obtained by Kroese et aL 37, only a few clones proliferate and occupy the GC. The notion that GC cells are oligoclonal is, however, hardly reconcilable with the fact that GC cells are subjected to hypermutagenicity0 giving rise to new clones of high Ig affinity.
242
Immunology Today,Vol. 9, Nos 7andS, 1988
l cells not divide there also? It may simply be that T cells homing to GCs have lost their proliferative capacity, do not encounter adequate mitogens or are eliminated by tingible body macrophages when dividing. It should be interesting to pursue this question particularly since T cells are essential for the GC reaction; there must be a regulatory system allowing a limited number of T cells, probably arising from the adjacent thymus-dependent zones, to specialize into GC T cells. Numerous immune complexes are retained by follicular dendritic cells. How do these complexes gain access to the follicles? Kamperdijk eta/. 39 propose that immune complexes enter the GC by diffusion. However, according to Sainte-Marie and Peng4o lymph does not irrigate the follicles easily. The immune complexes can apparently be imported by B cells (E. Heinen, PhD thesis; University of Liege, 1986). What is the role played by these immune What is the fate of the follicularB cells? complexes? According to Klaus eta/. 24 the C3b compleB cells proliferate at a high rate inside the GC: their cell ment fragment stimulates cell proliferation, but we cycle lasts about 6 hours. One astonishing observation is observed either stimulation or inhibition according to the that many of these cells die and are phagocytosed by Ig isotype used in the complex (unpublished). The imtingible body macrophages. Cytokinetic studies indicate mune complexes are mainly found in the light zone that GCs may be 'graveyards' for many lymphocytes 12. where cells appear to differentiate. Do they effectively What is the reason for this? It is possible that elimination influence cell differentiation and act as feedback regulah:~: occurred of B cells that erroneously arranged the tors of antibody synthesis during the maintenance phase genes for their isotype switch or that no longer recognize "of the immune response as proposed by Tew et a/.26? the antigen after somatic mutations of the hypervariable There is as yet no clear answer to this question. Ig region, or badly replicated their DNA during the very Many B cells bear Fc receptors: Ig fixed on Fc receptors short S phase. These three reasons do not, however, fully is generally considered to be inhibitory to B-cell explain this waste of cells. It may be that, as in the stimulation 41. Since we observed that B cells can transfer thymus, cell-cell interactions occur within the lymphoid immune complexes to follicular dendritic cells, we sugfollicles eliminating cells expressing inadequate recepgest that these B cells are then freed from Ig and so tors. allowed to proliferate. Alternatively, another role should Surviving cells stop multiplying, differentiate and miperhaps be attributed to this intercellular contact. For grate. Many questions surround these events: what example, immune complexes fixed on Fc receptors inhibit directs the differentiation of GC B cells into plasma or the Ig-forming response42; therefore, follicular dendritic memory cells? The observations made by o u r g r o u p 29 cells mav Dresent them ~o B cells ~ncl th~r~hv ~ln~^lrlr~^ln support the view that follicular dendritic cells reduce ;the the differentiation pathway ofthese-ceils.--" . . . . . . . . . . . formation of Ig-secreting cells. This could thus favour the All these cellular actions are strictly controlled inside development of memory cells: indeed, plasma cells are the particular microenvironment of the GC, but very little rarely found in GCs. They mainly differentiate after the is known about the factors involved there and much migration of cells from the GC, for example from work remains to be done in defined culture systems. In immunoblasts in the medullary cords of lymph nodes 5 or addition, the functional relationships between follicles from circulating lymphocytes that have homed to the and the vascular structures and the histological environmucosae (gut, respiratory tract) or the bone marrow. ment (especially the thymus-dependent zones) must be Other workers have noted, however, that plasma cells clarified. As long as our knowledge of GC phenomena are occasionally present inside GCs38. This can be exremains poor, we will not be able to manipulate in a plained if a balance exists between agents that inhibit physiological way the humoral immune reactions in Ig-secreting cell formation and others that favour it. In conditions such as hypo- or hyperimmunoglobulinemia, normal GCs few plasma cells are found, indicating autoimmunity, lymphomas, or other pathological states. I;r~.dominance of follicular dendritic cell action, but other factors (immune complexes, cytokines, vascularization, References etc.) certainly affect this microenvironment and mod1 Lennert, K. (1978) Malignant Lymphomas Other than ulate the differentiation into memory or Ig-secreting Hodgkin's Disease, Springer-Verlag cells. 2 Thorbecke, G.J.,Asofsky, R., Hochwald, G.M. and Siskind, The immunochemical features of B memory cells are G.W. (1962)J. Exp. Med. 116, 295-310 unclear. Many contradictory data exist, perhaps because 3 Tsunoda, R. and Kojima, M. (1987)Acta Pathol. Jpn 37, 575-585 different states of memory exist. In summary, we can say 4 von Gaudecker, B. and Hinrichsen, K. (1965)Z. Zellforsch. 65, that memory cells, according to the microenvironment 139-162 that they encounter, differentiate into plasma cells, and 5 Geldof, A.A., van de Hende, M. and Jandts, E.M. (1983) Cell return to GCs to recommence a cycle or simply to die. Tissue Res. 231, 135-142 6 Reichert, R.A., Gallatin, W.M. and Weissman, L.L (1984) Open questio.s J. Exp. Med. 157, 813-827 GCs are sites where there is great pressure on B cells to 7 Jalkanen, S.T., Bargatze, R.F., Herron, LR. and Butcher, E.C. divide (short cell cycle, high number of divisions). Why do (1986) Eur. J. Immunol. 16, 1195-1202
Immunology Today, Vol. 9, Nos 7and8, 1988
8 Gutman, G. and Weissman, I. (1972) Immunology 23, 465-471 9 Roux, R.V., Ledbetter,J.A. and Weissman, I. (1982) J. Immunol. 128, 2243-2248 10 Velardi, A., Mingari, M.C., Moretta, L. and Grossi,C.E. (1986) J. Immunol. 137, 2808-2813 11 Hoefsmit, E.C.M., Kamperdijk, E.W.A., Hendricks, H.R., Beelen, R.H.J.and Balfour, B.M. (1980) The Reticuloendothelial Syst. 1,417-468 12 Fliedner,T.M. (1967) in Germinal Centersin Immune Responses (Cottier, H., Odartchenko, N., Schindler, R. and Congdon, C.C., eds), pp. 218-224, Springer-Verlag 13 Heinen, E., Kinet-Deno~l,C., Radoux, D. and Simar, L.J. (1985) Adv. Exp. Meal. Biol. 186, 171-183 14 Stein, H., Gerdes,J. and Mason, D.Y. (1982) Clin. Haematol. 11,531-559 15 Humphrey, G.H., Grennan, D. and Sundaram,V. (1984)Eur. J. Immunol. 14, 759-768 16 Williams, G.M. and Nossal, G.J.V. (1966)J. Exp. Med. 124, 47-56 17 Dijkstra, C.D. and Langevoort, H.L. (1982) Cell TissueRes. 222, 69-79 18 Kroese, F.G.M., Wubbena, A.S., Kuijpers, K.C. and Nieuwenhuis, P. (1987)Immunology60, 597-602 19 Laissue,J., Cottier, H., Hess, M.W. and Stoner, R.D.(1971) J. Immunol. 107, 822-831 20 Butcher, E.C., Roux, R.V., Coffman, R.L.etal. (1982) J. Immunol. 129, 2698-2707 21 Matthews, J.B. and Basu, M.K. (1982)Int. Arch. Allergy Appl. Immunol. 69, 21-25 22 Reynolds,J.D. and Morris, B. (1983)Eur. J. Immunol. 13, 627-635 23 Nossal, G.J.V.(1972) Triangle 11, 1-6 24 Klaus, G.G.B., Humphrey,J.H., Kunkl, A. and Dongworth,
Multiple Sclerosis: Immunoloaic. Diaanostic and Therapeut-icA~pects
D.W. (I 980) Immunol. Rev. 53, 3-28 25 Heinen, E., Radoux, D., Kinet-Deno~l,C. etal. (1985)
Immunology 54, 777-784 26 Tew0J.G., Phipps,R.P.and Mandel, T.E.(1980)ImmunoL Rev. 53, 175-201 27 Kunkl, A. and Klaus, G.G.B. (1981)Immunology43, 371-378 28 Cormann, N., Heinen, E., Kinet-Deno~l,C.0 Braun, M. and Simar, L.J.(1986) CR SeancesSoc. Biol. 180, 218-223 29 Corrnann, N., Lesage,F., Heinen, E. etal. (1986)Immunol. Lett. 14, 29-35 30 Lilet-Leclercq,C., Radoux, D., Heinen, E. etal. (1984)J. Immunol. Meth. 66, 235-244 31 Heinen, E., Cormann, N., Braun, M. etal. (1987)Ann. Inst. Pasteur/Immunol. 137,369-382 32 Heinen, E., Kinet-Deno~l,C. and Simar, L.J.(1985) Immunol. Lett. 9, 75-80 33 Heinen, E. and Tsunoda, R. (1987)Immunol. Today8, 142-143 34 Gray, D., MacLennan, I.C.M and Lane, P.J.L (1986) Eur. J. Immunol. 16, 641-648 35 Jelinek,D.F.and Lipsky, P.E.(1987)Adv. Immunol. 40, 1-60 36 Micklem, H.S.(1988)lmmunol. Today9, 1-2 37 Kroese, F.G.M.,Wubbena, A.S., Seijen, H. and Nieuwenhuis, P. (1987) Eur. J. Immunol. 17, 1069-1072 38 Szakal,A.K., Kosco, M.H., Burton, G.F.and Tew, J.G. (1988) Prog. Leukocyte Biol. 7, 281-290 39 Kamperdijk,E.W.A., Dijkstra, C.D. and D6pp, E.A. (1987) Immunobiology 174, 395-405 40 Sainte-Marie,G. and Peng, F.S.(1985) Cell Tissue Res. 239, 31-35 41 Sinclair,N.R.C.(1983) Immunol. Today4, 35-36 42 Uher, F. and Dickler, H.B. (1986) Mol. Immunol. 23, 11771181
quality of science presented from chapter to chapter. The selected topics are representative of the speaker's lectures, but not necessaredited by F. Clifford Roseand RosemaryJones, ily of the central scientific issues John Libbeyand Company,Ltd, 1987. £26.00 of multiple sclerosis. With these (ix + 260 pages)ISBN0 86196 109 9 caveats, there were nonetheless some articles that were of particular interest and might be useful to workers in the field of multiple scleredited by JA. Aarli, M.W.H. Behan and P.O. osis. Behan, B/ackwe//ScientificPublications, 1987. The book is divided into three £60.00 (xviii+ 531 pages)ISBN0 632 01635x sections; 'Immunologic aspects', 'Diagnostic aspects', and 'Therapeutic Multiple Sclerosis is based, according aspects' of multiple sclerosis. The to the book's preface, on a sym- first section on immunology was posium held at Charing Cross hospit- generally well written, though dial in the fall of 1986. The result is a verse in its collection of multiple collection of brief papers by the in- sclerosis related articles. The second vited speakers discussing their re- section on diagnosis presents both spective •topics of interest. The edi- original data and clinical impressions tors themselves point out that there of the various diagnostic tests availare many books on multiple scler- able for multiple .~clerosis.There was osis, and justify this one on the pre- only one article discussing magnetic mise that "few accept the challenge resonance imaging of multiple sclerto deal in depth with the con- osis, which was unfortunate controversies that rage around this sidering the importance most investigators in the field now place on this enigmatic condition". Representing a collection of unre- new technique. The last section was on theraviewed papers by different authors, there is a wide diversity in terms of peutic aspects of multiple sclerosis.
Clinical Neuroimmunology
Whether or not one is a proponent of immunosuppression, including the use of steroids, azathioprine and cyclophosphamide in the treatment of multiple sclerosis, there is a large and ever-increasing literature on this subject. It was somewhat peculiar that in this section there was no mention of these therapies. Instead the collection of articles presented were written about older treatments such as diet supplements and hyperbaric oxygen, which generally have had negative clinical results in carefully controlled studies. In summary, this book will not be generally useful to the reader interested in a broad overview of the current scientific concepts related to multiple sclerosis, particularly in regard to therapy. Nonetheless specific chapters particularly in the first section on immunology present well written reviews and newer data that may be of interest to workers in the field. Clinical immunology is rapidly incorporating many of the advances in cellular immunology into the knowledge base that is useful for the practising physician. The field of
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