Follicular dendritic cells in TSE pathogenesis

VIEWPOINT I M M U N O L O G Y T O D AY

Follicular dendritic cells in TSE pathogenesis Moira E. Bruce, Karen L. Brown, Neil A. Mabbott, Christine F. Farquhar and Martin Jeffrey The pathogenesis of transmissible spongiform encephalopathies (TSEs)

S

ince it was first described in normal function of these cells is to trap and often includes a replication phase in 1996, variant Creutzfeldt-Jakob retain native antigen, in the form of immune disease (vCJD) has been recogcomplexes, for presentation to B cells6. lymphoid tissues before infection nized in over 70 patients in the spreads to the central nervous UK, two patients in France and one in the system. Recent studies show that the Association of PrP with FDCs Republic of Ireland. By the end of 1997, laA hallmark of the TSEs is the accumulation boratory studies had confirmed the suspifollicular dendritic cells of the in nervous and lymphoid tissues of abnorcion that vCJD is caused by the bovine germinal centres are critical for this mally folded, relatively protease-resistant spongiform encephalopathy (BSE) agent1, replication. These cells are therefore probably acquired by consumption of contaforms of the host prion protein, PrP (Ref. 7). minated meat products. We do not know The abnormal form of PrP (PrPSc) copurifies potential targets for therapy or with infectivity8 and is believed to be a comhow many more people are incubating vCJD prophylaxis in natural TSEs, such as ponent of the transmissible agent in these infection, and there is no practical preclinical variant Creutzfeldt-Jakob disease. diseases, either alone or in combination with diagnostic test for this disorder. There is, other molecules. Studies of transgenic mice therefore, the potential for accidental personin which the PrP gene has been disrupted to-person infection, for example by transplantation of infected tissues or by the use of contaminated surgical have shown that expression of this protein by the host is required for instruments. We urgently need to understand the pathogenesis of TSE agent replication9,10. For some time, FDCs have been suspected vCJD and related diseases in order to assess these risks, to develop di- of sustaining TSE replication, because high levels of PrP are detected agnostic tests and to explore approaches to prophylaxis and therapy. on these cells in both TSE-infected and uninfected mice11–14. Abnormal PrP accumulation is also seen within the germinal centres in vCJD (Ref. 15) and sheep scrapie16, giving us confidence that obserModelling scrapie infection vations in mouse models are relevant to the natural diseases. BSE and vCJD, along with sheep scrapie, are members of the group Within the spleens of uninfected mice, normal host PrP is deof transmissible spongiform encephalopathies (TSEs) or prion dis- tected by immunostaining only on the diffuse networks of FDC eases. Most of our understanding of the biology of the TSEs has processes (Fig. 2a,b). Within weeks of infection with the ME7 strain come from studies of scrapie isolates in rodent models. There are of scrapie, PrP labelling is seen, not only on FDC networks, but also multiple laboratory strains of mouse-passaged scrapie that differ in in the tingible body macrophages (TBMs) of the germinal centres the characteristics of the disease they produce in infected animals2. (Fig. 2c). There are no antibodies that distinguish between normal These scrapie strains are distinguished by their incubation periods PrP and PrPSc, but blotting sections from infected spleens onto nitroand neuropathological profiles in inbred mouse-strains, characteris- cellulose membranes, and treating these with protease before imtics that are highly reproducible and stable over many mouse-to- munolabelling (the ‘histoblot method’) has shown that pathological, mouse passages. The details of pathogenesis may differ according to protease-resistant PrPSc has indeed accumulated in the germinal centhe scrapie strain used. However, following exposure to infection by tres17. Recently, the location of this accumulation has been defined by peripheral routes such as ingestion or through the skin, infectivity immunostaining at the ultrastructural level18. PrP labelling is seen in usually replicates and accumulates to high levels in lymphoid tissues the extracellular spaces between the tightly packed convolutions of long before spreading to the central nervous system (CNS) (Fig. 1)3,4. FDC processes (the site of retention of trapped immune complexes) Furthermore, removal of the spleen before peripheral scrapie chal- and within the lysosomes of the TBMs (Fig. 3). lenge often delays the onset of neurological disease5, showing that replication in lymphoid tissues can be relevant to disease progression. The involvement of different cell types in the lymphoid tissues has Scrapie in immunodeficient mice been investigated using mainly the ME7 and Rocky Mountain Labora- Studies in the ME7 scrapie model demonstrated that sub-lethal tory (RML) strains of mouse-passaged scrapie. These were derived whole body g-irradiation of mice before or after peripheral challenge from different sources (ME7 from natural sheep scrapie; RML from ex- with scrapie has no effect on either agent replication in the spleen or perimental goat scrapie) and have distinct incubation period and neuro- on the incubation period up to clinical disease and death19. This pathological characteristics. Recent studies have indicated that the fol- implies that scrapie pathogenesis depends on long-lived, radiationlicular dendritic cells (FDCs) of the germinal centres of the spleen, resistant cells, a description that applies to FDCs but excludes most lymph nodes and Peyer’s patches are key players in pathogenesis. The lymphocytes and monocytes. Since then, cellular involvement in 0167-5699/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

S E P T E M B E R 2 0 0 0 442

Vo l . 2 1

No.9

PII: S0167-5699(00)01696-0

VIEWPOINT I M M U N O L O G Y TO D AY

Log10 scrapie infectivity titre in tissue, measured by bioassay

TSE pathogenesis has been studied in more detail in immunodeficient mice, using the ME7 scrapie strain in our own laboratory and the RML scrapie strain in Adriano Aguzzi’s laboratory in Brain Zurich (Table 1). Severe combined immunodeficiency (SCID) mice carry a mutation that blocks the rearrangement of immunoglobulin and T-cell Spleen receptor genes, resulting in a profound deficiency in both T and B cells20. Because FDCs require signals from B cells for their maturation, SCID mice also lack functional FDCs (Ref. 21). It has been found that SCID mice are much less susceptible than immunocompetent mice to peripheral challenge with TSE agents, although they are fully susceptible when infection is introduced directly into their brains22–24. This resistance is related to an inability of SCID lymphoid Time tissues to support agent replication. Bone-marrow or fetal-liver Inoculation Onset of Death grafts from immunocompetent donors restore T- and B-cell popuclinical signs lations, leading to the maturation of FDCs (Ref. 21). Following such Immunology Today grafting, SCID mice gain the ability to replicate scrapie in their lymphoid tissues and become fully susceptible to peripheral chalFig. 1. The accumulation of infectivity in the spleen and brain, following lenge23,24. These results are consistent with an involvement of FDCs experimental peripheral challenge of mice with scrapie. Infectivity levels and/or lymphocytes in scrapie pathogenesis. rise in the spleen long before the onset of agent replication in the central The separate involvement of T cells, B cells and FDCs has been nervous system and the subsequent development of clinical disease. The explored by a process of elimination, using mice lacking specific total incubation period up to clinical disease in these models is at least components of the immune system. Early studies of ME7 scrapie in six months. neonatally thymectomized mice indicated that T cells are not likely to be important in this model5. Later studies have exploited the In our own studies, we found that TNF-a-deficient mice have a low growing number of transgenic knockout lines in which specific susceptibility to peripheral challenge with ME7 scrapie and fail to acgenes controlling development of the immune system have been in- cumulate infectivity in their spleens, supporting a critical role for activated. These studies indicate that, as for ME7, T cells are not crit- FDCs rather than B cells in agent replication in this model17. ical for RML scrapie25. However, mice deficient in B cells, which also lack FDCs, have a low susceptibility to peripheral challenge with either scrapie strain and do not replicate infection in their spleens (Ref. Scrapie in mice with PrP-chimaeric immune systems A potential problem in the use of immunological knockout mice is 25 and K.L. Brown, unpublished). The effects of deficiencies in FDCs and B cells can be separated in that the genetic defect that has been introduced may have unexmice in which the signalling between the two cell types has been pected effects on cell types other than those of immediate interest. disrupted. For example, the maturation of FDCs depends on tumor necrosis factor (a) (b) (c) (TNF)-a signalling from B cells and mice that are deficient in TNF-a or its receptor, TNFR1, possess T and B cells but lack mature FDCs (Ref. 26). TNFR1 deficient mice have been reported to be fully susceptible to peripheral challenge with RML scrapie25. On the basis of these findings, the authors concluded that B cells, rather than FDCs, are required for neuroinvasion of RML scrapie, although this interpretation has since been modified in the light of further studies. As no Fig. 2. (a) Double immunofluorescent labelling of an FDC network in a spleen section from an uninformation was given about spleen infectiv- infected mouse, stained for PrP (green) and the FDC marker, FDC-M1 (red), showing a close coloity levels for TNFR1-deficient mice with calization (orange) when viewed by confocal microscopy (bar 5 100 mm). (b) Immunostaining of PrP RML scrapie, it remains possible that the (red) on a diffuse network of FDC processes in a spleen section from an uninfected mouse, viewed by high dose used in this study bypassed the light microscopy (bar 5 20 mm). (c) Immunostaining of PrP (red) in the spleen of a scrapie-infected need for replication in lymphoid tissues, as mouse, viewed by light microscopy, showing diffuse labelling of FDC networks and intense labelling has been observed in SCID mice injected pe- of cells with the appearance of tingible body macrophages (bar 5 20 mm). Abbreviations: FDC, ripherally with high doses of ME7 scrapie24. follicular dendritic cell; PrP, prion protein.

S E P T E M B E R 2 0 0 0 Vo l . 2 1

No.9

443

VIEWPOINT I M M U N O L O G Y T O D AY

T cell

B cell PALS TBM CA

DC

GC Abnormal PrP

FDC

Marginal zone Abnormal PrP Immunology Today

Fig. 3. Distribution of abnormal PrP (red) in the germinal centre (GC) of the spleen from a scrapie-infected mouse. Abnormal PrP is detected between the tightly convoluted processes of the follicular dendritic cells (FDCs) and also within lysosomes of tingible body macrophages (TBMs). Abbreviations: PrP, prion protein; PALS, periarteriolar lymphocytic sheath; CA, central arteriole; DC, dendritic cell. An alternative approach has been to study scrapie pathogenesis in mice carrying the PrP gene in their FDCs but not in their lymphocytes and vice versa. Although the ultimate origin of FDCs and their precursors is controversial27,28, there is general agreement that FDCs are not replaced significantly from the bone marrow in adult mice, but differentiate from resident precursor cells of stromal origin. Bonemarrow grafting into mice depleted of lymphocytes, either genetically (SCID mice) or following lethal doses of g-radiation, leads to the restoration of lymphocyte populations of graft origin. These lymphocytes, in turn, induce the maturation of FDCs that are mainly, if not entirely, of recipient origin27,28. Grafting between PrP-expressing and transgenic PrP-deficient mice therefore produces a mismatch in

PrP genotype between FDCs and surrounding lymphocytes (Fig. 4)14. In these chimaeric mice, PrP immunolabelling of FDC networks is seen only when the FDCs themselves carry a PrP gene and does not depend on the PrP status of the lymphocytes14. This strongly suggests that, in wild-type mice, FDCs themselves are producing high levels of PrP, rather than acquiring it from lymphocytes, which express low levels of the protein on their surfaces29. Furthermore, when the chimaeric mice are challenged with the ME7 scrapie strain, replication of infection in their spleens also depends only on whether the FDCs have a PrP gene14. No replication is seen in the spleens of PrP-deficient mice, even when they are grafted with PrP-expressing bone marrow. This set of experiments provides the strongest evidence to date that a particular cell type, the FDC, is responsible for TSE replication in lymphoid tissues. Similar studies in PrP-chimaeric mice have been carried out using RML scrapie, but here the results are not so clear-cut. Mice with PrPexpressing FDCs are capable of accumulating high levels of RML scrapie in their spleens, whether or not the lymphocytes carry a PrP gene, suggesting that RML, like ME7, can replicate in FDCs (Ref. 30). However, unlike ME7, RML also replicates in the spleens of PrPdeficient mice grafted with PrP-expressing bone marrow, suggesting that other cells might also be involved31. Indeed, for PrP-expressing mice with RML scrapie, infectivity is associated with separated splenic lymphocytes as well as with stroma, but only if the lymphocytes themselves express PrP (Ref. 32). Although a technical explanation for the discrepancy between results for ME7 and RML scrapie has not yet been ruled out, these studies raise the possibility that different scrapie strains target different cell types in the lymphoid tissues.

Implications of an FDC involvement FDCs are specialized to trap and retain unprocessed antigens, in the form of immune complexes, and to present these to B cells in the course of the selection of clones producing high-avidity antibodies and the generation and maintenance of B-cell memory6. Some conventional viruses, including HIV-1, have also been shown to be trapped and retained by the FDC (Ref. 33). The association of high levels of PrP with FDC networks in uninfected mice suggests that

Table 1. Susceptibility of immunodeficient mice to peripheral challenge with two strains of scrapiea ME7 scrapie

RML scrapie

Deficiency

Immunodeficient mouse

Susceptibility Immunodeficient mouse

T cells T cells, B cells and FDCs B cells and FDCs FDCs

Neonatally thymectomised wild-type SCID mMT TNF-a2/2

High5 Low24 Lowb Low17

CD42/2, CD82/2, b2-m2/2, perforin2/2 SCID, RAG-12/2, RAG-22/2 mMT TNFR12/2

Susceptibility High25 Low25 Low25 High25

Abbreviations: FDCs, follicular dendritic cells; SCID, severe combined immunodeficiency; mMT, disrupted immunoglobulin m chain; TNF-a2/2, tumour necrosis factor a deficient; b2-m2/2, b2-microglobulin deficient; RAG-12/2 and RAG-22/2, V(D)J recombination activation genes 1 and 2 deficient; TNFR12/2, tumour necrosis factor receptor 1 deficient; RML, Rocky Mountain lab strain. bK.L. Brown, unpublished. a

S E P T E M B E R 2 0 0 0 444

Vo l . 2 1

No.9

VIEWPOINT I M M U N O L O G Y TO D AY

this protein may play a part in the trapping function. Furthermore, in TSE-infected spleen, abnormal PrP accumulation is associated with the tight clusters of convoluted FDC processes that are the site of trapped immune complexes. These clusters of processes, which indicate that the FDCs are activated, are abnormally hyperplastic in mice with end-stage scrapie compared with uninfected controls18. It is tempting to speculate that TSE agents, whatever they are made of, subvert the normal function of PrP to focus and amplify infectivity on the FDCs. It may even be the case that natural TSE infections exist only because FDCs normally express high levels of PrP. The identification of FDCs as key cells significantly advances our understanding of the pathogenesis of the TSEs, but raises many questions. We know almost nothing about how infection spreads from the site of challenge to the germinal centres, although this transport may involve mobile dendritic cells (as opposed to nonmobile FDCs, which have a completely different lineage and function). Although very low levels of infectivity have been detected in blood in some rodent TSE models, there is no evidence that infection spreads to the CNS via the bloodstream. Indeed, PrP expression by circulating lymphocytes and other bone-marrow-derived cells appears to be irrelevant to neuroinvasion in both the RML and ME7 models14,30–32. Rather, there is evidence that spread occurs along peripheral nerves4,34, but, as the germinal centres are poorly innervated, there is a missing link between FDCs and nerve endings. Mature FDCs do not move from the germinal centre, but it has been suggested that they shed parts of their membranes carrying immune complexes (iccosomes), which are taken up by B cells35. TBMs scavenge the remains of unselected B cells and, possibly, also fragments of FDC membranes. As infectivity and PrPSc are likely to be associated with these membrane fragments in TSE-infected animals, B cells or TBMs might act as vehicles for exporting infection from the FDCs to more highly innervated regions of the lymphoid tissues.

Concluding remarks Several lines of evidence implicate FDCs in the replication of TSEs in lymphoid tissues. B cells are also important, but probably mainly because they are required for the maturation of FDCs. The FDC therefore presents a potential target for intervention in the naturallyoccurring TSEs, as treatments that interfere with the integrity or function of these cells are predicted to prevent replication in lymphoid tissues. One such treatment has recently been described: disruption of the signalling between B cells and FDCs by a lymphotoxin b receptor/immunoglobulin fusion protein temporarily de-differentiates FDCs (Ref. 36). This treatment, shortly before or after peripheral challenge with either RML or ME7 scrapie, has also been shown to block replication in the spleen and to delay neuroinvasion37,38. Moira Bruce ([email protected]), Karen Brown, Neil Mabbott and Christine Farquhar are at the Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh UK EH9 3JF. Martin Jeffrey is at VLA Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, UK EH26 0PZ.

Genotype of host

Germinalcentre genotypes

Genotype of bone-marrow donor

Scrapie replication in the spleen

Mature FDC associated with lymphocytes

Stromal precursor cell PrP+/+

PrP+/+

PrP+/+

PrP –/–

PrP –/–

PrP+/+

Immunology Today

Fig. 4. Summary of the study reported by Brown et al.14, showing that ME7 scrapie replication in lymphoid tissues depends on PrP-expressing FDCs. PrP-expressing or PrP-deficient mice that were deficient in lymphocytes (either genetically or following g-irradiation) were grafted with PrP-expressing or PrP-deficient bone marrow. This led to the maturation of FDCs of host origin, in the presence of lymphocytes of graft origin. In scrapie-challenged mice, replication of infectious agent was seen only when FDCs carried a PrP gene. The PrP genotype of the lymphocytes had no influence on this replication. Abbreviations: PrP, prion protein; FDCs, follicular dendritic cells. References 1 Bruce, M.E. et al. (1997) Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 389, 498–501 2 Bruce, M.E. (1993) Scrapie strain variation and mutation. Br. Med. Bull. 49, 822–838 3 Mabbott, N.A. et al. (1998) Involvement of the immune system in TSE pathogenesis. Immunol. Today 19, 201–203 4 Kimberlin, R.H. and Walker, C.A. (1979) Pathogenesis of mouse scrapie: dynamics of agent replication in spleen, spinal cord and brain after infection by different routes. J. Comp. Pathol. 89, 551–562 5 Fraser, H. and Dickinson, A.G. (1978) Studies of the lymphoreticular system in the pathogenesis of scrapie: the role of spleen and thymus. J. Comp. Pathol. 88, 563–573 6 Heinen, E. et al. (1995) Follicular dendritic cells: origin and function. Curr. Top. Microbiol. Immunol. 201, 15–47 7 Bolton, D.C. et al. (1982) Identification of a protein that purifies with the scrapie prion. Science 218, 1309–1311 8 Diringer, H. et al. (1983) Scrapie infectivity, fibrils and low molecular weight protein. Nature 306, 476–478 9 Bueler, H. et al. (1993) Mice devoid of PrP are resistant to scrapie. Cell 73, 1339–1347 10 Manson, J.C. et al. (1994) PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology. Neurodegen. 3, 331–340

S E P T E M B E R 2 0 0 0 Vo l . 2 1

No.9

445

VIEWPOINT I M M U N O L O G Y T O D AY

11 Kitamoto, T. et al. (1991) Abnormal isoform of prion protein accumulates in follicular dendritic cells in mice with CreutzfeldtJakob disease. J. Virol. 65, 6292–6295 12 McBride, P.A. et al. (1992) PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapieinfected mice. J. Pathol. 168, 413–418 13 Ritchie, D.L. et al. (1999) Visualisation of PrP protein and follicular dendritic cells in uninfected and scrapie infected spleen. J. Cell. Pathol. 4, 3–10 14 Brown, K.L. et al. (1999) Scrapie replication in lymphoid tissues depends on prion protein-expressing follicular dendritic cells. Nat. Med. 5, 1308–1312 15 Hill, A.F. et al. (1999) Investigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples. Lancet 353, 183–189 16 van Keulen, L.J.M. et al. (1996) Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. J. Clin. Microbiol. 34, 1228–1231 17 Mabbott, N.A. et al. (2000) Tumor necrosis factor alpha-deficient, but not interleukin-6-deficient, mice resist peripheral infection with scrapie. J. Virol. 74, 3338–3344 18 Jeffrey, M. et al. (2000) Sites of prion protein accumulation in scrapieinfected mouse spleen revealed by immuno–electron microscopy. J. Pathol. 191, 323–332 19 Fraser, H. and Farquhar, C.F. (1987) Ionising radiation has no influence on scrapie incubation period in mice. Vet. Microbiol. 13, 211–223 20 Bosma, G.C. et al. (1983) A severe combined immunodeficiency mutation in the mouse. Nature 301, 527–530 21 Kapasi, Z.F. et al. (1993) Induction of functional follicular dendritic cell development in severe combined immunodeficiency mice: influence of B and T cells. J. Immunol. 150, 2648–2658 22 O’Rourke, K.I. et al. (1994) SCID mouse spleen does not support scrapie agent replication. J. Gen. Virol. 75, 1511–1514 23 Lasmezas, C.I. et al. (1996) Immune system-dependent and -independent replication of the scrapie agent. J. Virol. 70, 1292–1295 24 Fraser, H. et al. (1996) Replication of scrapie in spleens of SCID mice

25 26 27

28 29 30 31 32

33

34

35 36 37 38

follows reconstitution with wild-type mouse bone marrow. J. Gen. Virol. 77, 1935–1940 Klein, M.A. et al. (1997) A crucial role for B cells in neuroinvasive scrapie. Nature 390, 687–690 Kosco-Vilbois, M.H. et al. (1997) To ‘B’ or not to ‘B’ a germinal center? Immunol. Today 18, 225–230 Humphrey, J.H. et al. (1984) The origin of follicular dendritic cells in the mouse and the mechanism of trapping immune complexes on them. Eur. J. Immunol. 14, 859–863 Kapasi, Z.F. et al. (1998) Follicular dendritic cell (FDC) precursors in primary lymphoid tissues. J. Immunol. 160, 1078–1084 Mabbott, N.A. et al. (1997) T-lymphocyte activation and the cellular form of the prion protein. Immunology 92, 161–165 Klein, M.A. et al. (1998) PrP expression in B lymphocytes is not required for prion neuroinvasion. Nat. Med. 4, 1429–1433 Blattler, T. et al. (1997) PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 389, 69–73 Raeber, A.J. et al. (1999) PrP-dependent association of prions with splenic but not circulating lymphocytes of scrapie-infected mice. EMBO J. 18, 2702–2706 Racz, P. and Tenner-Racz, K. (1995) Germinal center tropism of HIV-1 and other retroviruses. In Follicular Dendritic Cells in Normal and Pathological Conditions (Heinen, E., ed.), pp. 159–181, R.G. Landes McBride, P.A. and Beekes, M. (1999) Pathological PrP is abundant in sympathetic and sensory ganglia of hamsters fed with scrapie. Neurosci. Lett. 265, 135–138 Tew, J.G. et al. (1989) The alternative antigen pathway. Immunol. Today 10, 229–232 Mackay, F. and Browning, J.L. (1998) Turning off follicular dendritic cells. Nature 395, 26–27 Mabbott, N.M. et al. (2000) Temporary inactivation of follicular dendritic cells delays neuroinvasion of scrapie. Nat. Med. 6, 719–720 Montrasio, F. et al. (2000) Impaired prion replication in spleens of mice lacking functional follicular dendritic cells. Science 288, 1257–1259

Keeping up with the Trends… Immunology in other Trends journals Driving cells into atherosclerotic lesions – a deleterious role for viral chemokine receptors? H. Hengel and C. Weber: Trends in Microbiology 8, 294–296 Cytokines in rheumatoid arthritis: trials and tribulations N.L. Carteron: Molecular Medicine Today 6, 315–323 Xenotransplantation in CNS repair: immunological barriers and strategies for overcoming them T. Brevig, J. Holgersson and H. Widner: Trends in Neurosciences 23, 337–346 Internalizing antibodies and targeted cancer therapy: direct selection from phage display libraries U.B. Nielsen and J.D. Marks: Pharmaceutical Science and Technology Today 3, 282–291 Helminth C-type lectins and host-parasite interactions A. Loukas and R. Maizels: Parasitology Today 16, 333–339

S E P T E M B E R 2 0 0 0 446

Vo l . 2 1

No.9