Follicular dendritic cell dedifferentiation reduces scrapie susceptibility following inoculation via the skin

Transmissible spongiform encephalopathies (TSEs) are a group of subacute infectious neurodegenerative diseases that are characterized by the accumulation in affected tissues of PrP(Sc), an abnormal isoform of the host prion protein (PrPc). Following peripheral exposure, TSE infectivity and PrP(Sc) usually accumulate in lymphoid tissues prior to neuroinvasion. Studies in mice have shown that exposure through scarified skin is an effective means of TSE transmission. Following inoculation via the skin, a functional immune system is critical for the transmission of TSEs to the brain, but until now, it has not been known which components of the immune system are required for efficient neuroinvasion. Temporary dedifferentiation of follicular dendritic cells (FDCs) by treatment with an inhibitor of the lymphotoxin-beta receptor signalling pathway (LTbetaR-Ig) 3 days before or 14 days after inoculation via the skin, blocked the early accumulation of PrP(Sc) and TSE infectivity within the draining lymph node. Furthermore, in the temporary absence of FDCs before inoculation, disease susceptibility was reduced and survival time significantly extended. Treatment with LTbetaR-Ig 14 days after TSE inoculation also significantly extended the disease incubation period. However, treatment 42 days after inoculation did not affect disease susceptibility or survival time, suggesting that the infection may have already have spread to the nervous system. Together these data show that FDCs are essential for the accumulation of PrP(Sc) and infectivity within lymphoid tissues and subsequent neuroinvasion following TSE exposure via the skin.     

Skin-derived dendritic cells acquire and degrade the scrapie agent following in vitro exposure

The accumulation of the scrapie agent in lymphoid tissues following inoculation via the skin is critical for efficient neuroinvasion, but how the agent is initially transported from the skin to the draining lymph node is not known. Langerhans cells (LCs) are specialized antigen-presenting cells that continually sample their microenvironment within the epidermis and transport captured antigens to draining lymph nodes. We considered LCs probable candidates to acquire and transport the scrapie agent after inoculation via the skin. XS106 cells are dendritic cells (DCs) isolated from mouse epidermis with characteristics of mature LC cells. To investigate the potential interaction of LCs with the scrapie agent XS106 cells were exposed to the scrapie agent in vitro. We show that XS106 cells rapidly acquire the scrapie agent following in vitro exposure. In addition, XS106 cells partially degrade the scrapie agent following extended cultivation. These data suggest that LCs might acquire and degrade the scrapie agent after inoculation via the skin, but data from additional experiments demonstrate that this ability could be lost in the presence of lipopolysaccharide or other immunostimulatory molecules. Our studies also imply that LCs would not undergo maturation following uptake of the scrapie agent in the skin, as the expression of surface antigens associated with LC maturation were unaltered following exposure. In conclusion, although LCs or DCs have the potential to acquire the scrapie agent within the epidermis our data suggest it is unlikely that they become activated and stimulated to transport the agent to the draining lymph node.     

Determining the role of mononuclear phagocytes in prion neuroinvasion from the skin

Many prion diseases are acquired by peripheral exposure, and skin lesions are an effective route of transmission. Following exposure, early prion replication, upon FDCs in the draining LN is obligatory for the spread of disease to the brain. However, the mechanism by which prions are conveyed to the draining LN is uncertain. Here, transgenic mice were used, in which langerin(+) cells, including epidermal LCs and langerin(+) classical DCs, were specifically depleted. These were used in parallel with transgenic mice, in which nonepidermal CD11c(+) cells were specifically depleted. Our data show that prion pathogenesis, following exposure via skin scarification, occurred independently of LC and other langerin(+) cells. However, the depletion of nonepidermal CD11c(+) cells impaired the early accumulation of prions in the draining LN, implying a role for these cells in the propagation of prions from the skin. Therefore, together, these data suggest that the propagation of prions from the skin to the draining LN occurs via dermal classical DCs, independently of langerin(+) cells.     

Follicular dendritic cells as targets for intervention in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) are often acquired peripherally, for example by ingestion or iatrogenic exposure. After entry, TSE agents, as identified by disease-specific protein accumulation, usually accumulate on follicular dendritic cells (FDCs) in lymphoid tissues long before infection spreads to the brain. Neuroinvasion of TSE agents is significantly impaired in the absence of mature FDCs. Treatments that interfere with the integrity or function of FDCs extend survival time by blocking replication in lymphoid tissues and spread to the brain. The identification of FDCs as critical for TSE pathogenesis provides a cellular target to which therapies can be specifically directed.     

Early and late pathogenesis of natural scrapie infection in sheep

The pathogenesis of scrapie infection was studied in sheep carrying the PrP(VRQ)/PrP(VRQ) genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie-associated prion protein PrP(Sc), a marker for scrapie infectivity The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyer's patches of the caudal jejunum and ileum, the so-called gut-associated lymphoid tissues (GALT). At the same time, PrP(Sc) was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyer's patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non-GALT-related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8-T10, respectively.     

The sequential development of abnormal prion protein accumulation in mice with Creutzfeldt-Jakob disease.

The distribution and sequential development of prion protein (PrP) accumulation in the central nervous system (CNS) and non-neuronal organs of mice infected with Creutzfeldt-Jakob disease (CJD) were investigated immunohistochemically using a new pretreatment method that greatly enhanced the immunoreactivity of PrP. Prion protein accumulation in the CNS was first detected at 30 days after inoculation and then developed near the inoculation site or periventricular area, and later spread to the whole cerebrum and then to the pons. Its staining took some characteristic forms. Among non-neuronal organs, PrP accumulated in the follicular dendritic cells (FDCs) in spleen, lymph node, Peyer's patch, and thymus. FDCs staining appeared in spleen, lymph node, and Peyer's patch at 21 or 30 days after inoculation, and in thymus at 90 days. Germinal centers developed in the thymus of some CJD-infected mice. No PrP staining was detected in any examined organs of age-matched control mice.     

Detection of PrPSc in lymphoid tissues of lambs experimentally exposed to the scrapie agent

Histoblotting and immunohistochemistry were used to detect disease-associated prion protein (PrP(Sc)) in lymphoid tissues of lambs of known PrP genotype infected with the scrapie agent by stomach tube at the age of 2 months. The ileal and jejunal Peyer's patches and retropharyngeal and distal jejunal lymph nodes were studied 1 week, 5 weeks, 5 months and 11 months after inoculation. Other lymphoid tissues examined included superficial cervical lymph node, tonsil and spleen. PrP(Sc) was not detected in any tissue of any lamb at 1 week post-inoculation. At 5 weeks, PrP(Sc) was detected in tissues of lambs of susceptible PrP genotypes (AV(136)QQ(171) and VV(136)QQ(171)), but not lambs of other PrP genotypes (AA(136)QQ(171), AA(136)QR(171) and AV(136)QR(171)). PrP(Sc) was present in the germinal centres of tonsils, distal jejunal and retropharyngeal lymph nodes, and spleen. In the nodules of ileal and jejunal Peyer's patches, only occasional solitary cells showed the presence of PrP(Sc). At 5 months post-inoculation, increased accumulations of PrP(Sc) were detected in ileal and jejunal Peyer's patches, as well as in the retropharyngeal and distal jejunal lymph nodes of a single lamb inoculated with the agent from a sheep of the same susceptible PrP genotype. Eleven months after exposure to the scrapie agent, PrP(Sc) was detected in all lymphoid tissues examined from sheep of susceptible PrP genotypes. These studies show that PrP(Sc) was detectable in lymphoid tissues 5 weeks after exposure to the scrapie agent by stomach tube in lambs as young as 3 months of age and indicate that the PrP genotype is a significant factor for the rapid uptake and spread of the agent through lymphoid tissues.     

FCA和FICA可增强淋巴滤泡捕捉抗原的能力

目的　探讨向体内注入非抗原类物质能否引起反应性淋巴滤泡形成 ,观察体内注入FCA(完全弗式佐剂 )、FICA(不完全弗式佐剂 )对滤泡树突状细胞 (FDC)捕捉抗原的能力有无影响。方法　将FCA、FI CA分别注入小鼠足底 ,数日后取出腘淋巴结 ,应用PNA B法及PAP免疫组化法染色 ,采取三维重塑方法 ,统计淋巴滤泡、FDC细胞群数量。结果　注入FCA、FICA后第 5日出现初级淋巴滤泡 ,第 3 5日数量达到高峰 ,之后逐渐减退。同时 ,滤泡树突状细胞 (FDC)与B细胞聚集同步出现。结论　动物体内注入有丝分裂物质FCA、FICA引起引流淋巴结内次级淋巴滤泡形成 ,初级滤泡形成过程中B细胞聚集与FDC形成是同步发生的     

Clusterin expression in follicular dendritic cells associated with prion protein accumulation

Peripheral accumulation of abnormal prion protein (PrP) in variant Creutzfeldt-Jakob disease and some animal models of transmissible spongiform encephalopathies (TSEs) may occur in the lymphoreticular system. Within the lymphoid tissues, abnormal PrP accumulation occurs on follicular dendritic cells (FDCs). Clusterin (apolipoprotein J) has been recognized as one of the molecules associated with PrP in TSEs, and clusterin expression is increased in the central nervous system where abnormal PrP deposition has occurred. We therefore examined peripheral clusterin expression in the context of PrP accumulation on FDCs in a range of human and experimental TSEs. PrP was detected immunohistochemically on tissue sections using a novel highly sensitive method involving detergent autoclaving pretreatment. A dendritic network pattern of clusterin immunoreactivity in lymphoid follicles was observed in association with the abnormal PrP on FDCs. The increased clusterin immunoreactivity appeared to correlate with the extent of PrP deposition, irrespective of the pathogen strains, host mouse strains or various immune modifications. The observed co-localization and correlative expression of these proteins suggested that clusterin might be directly associated with abnormal PrP. Indeed, clusterin immunoreactivity in association with PrP was retained after FDC depletion. Together these data suggest that clusterin may act as a chaperone-like molecule for PrP and play an important role in TSE pathogenesis.     

Kinetics and localization of IgE tetanus antibody response in mice immunized by the intratracheal, intraperitoneal and subcutaneous routes.

The heterologous adoptive cutaneous anaphylaxis system was used to determine the kinetics of appearance of IgE-producing cells in various lymphoid tissues of mice following intratracheal (i.t.), intraperitoneal (i.p.), or subcutaneous (s.c.) immunization with tetanus toxoid and Bordetella pertussis organisms. Immunization, i.t. and i.p., produced similar patterns of response with the bronchial lymph nodes quantitatively exceeding the responses in other lymphoid tissues. In both cases the splenic lymphocyte response was second only to the bronchial and both appeared to parallel the serum PCA antibody. It is suggested that both responses represent draining lymph node responses since the bronchial lymph node drains both sites of immunization. After s.c. immunization a primary response of low order was found in the draining popliteal lymph node but not elsewhere. Although a dissociation was seen between responses obtained in various lymphoid tissues following s.c. and i.p. or i.t. immunization, no real evidence for a local mucosal response, such as has been reported for IgA, was obtained. These results lend experimental support to the observations that intratracheal and intraperitoneal immunization routes are most effective in production of IgE antibodies.     

Studies of the lymphoreticular system in the pathogenesis of scrapie: the role of spleen and thymus.

It has been established that a high proportion of extraneural replication of scrapie agent occurs in tissues associated with the lymphoreticular system, such as spleen. The question arises as to which components of the system are responsible for scrapie replication and whether such components can be identified by means of physiological manipulations. A series of experiments in mice showed that splenectomy deprived the host of a significant fraction of its capacity to permit ME7 scrapie agent to replicate, resulting in increases (10 to 30 per cent) of incubation period following intraperitoneal infection. This lost capacity remained for at least 60 days of age after neonatal splenectomy, although a smaller fraction of the total replicative capacity was vested in the spleen of the newborn than in that of mice aged 5 or more days; prolongation of incubation period in splenectomized newborn mice was over 9 per cent irrespective of the interval to injection.Experiments involving thymectomy were performed to discover whether the thymus-dependent portion of the lymphoid system was responsible for some of the spleen's ability to permit scrapie agent to replicate. The results failed to show any involvement of the thymus and its dependent lymphoid system in the replication and pathogenesis of scrapie in mice.     

Correlation between lymphocyte proliferative responses and dendritic cell migration in regional lymph nodes following skin painting with contact-sensitizing agents

We have investigated whether there exists a correlation between the induction of draining lymph node cell (LNC) proliferation in contact allergy and the accumulation of dendritic cells (DC) within such lymph nodes. CBA/Ca mice, which compared with mice of BALB/c strain, mount a more vigorous lymphocyte proliferative response following sensitization, also exhibited a more marked accumulation of DC in draining lymph nodes 24 h following skin painting. Moreover, studies with the skin-sensitizing fluorochromes fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC) revealed that DC-enriched fractions of draining LNC prepared from CBA/Ca mice contained a higher percentage of antigen-bearing cells than did those from BALB/c mice. A relationship between DC migration into lymph nodes and the magnitude of the induced LNC proliferative response was also indicated by experiments performed in BALB/c mice with a variety of contact allergens. It was observed that there was a direct correlation between the vigour of the proliferative response measured 3 days following exposure and the frequency of DC in draining nodes at 24 h. Collectively these data suggest that following skin sensitization the migration of DC into the draining lymph nodes influences quantitatively the primary immune response and the development of contact allergy.     

Interaction between dendritic cells and nerve fibres in lymphoid organs after oral scrapie exposure

In transmissible spongiform encephalopathies (TSEs), the infectious agent, called PrPsc, an abnormal isoform of the cellular prion protein, accumulates and replicates in lymphoid organs before affecting the nervous system. To clarify the cellular requirements for the neuroinvasion of the scrapie agent from the lymphoid organs to the central nervous system, we have studied, by confocal microscopy, the innervations within Peyer’s patches, mesenteric lymph nodes and the spleen of mice in physiological conditions and after oral exposure to prion. Contacts between nerve fibres and PrPsc-associated cells, dendritic cells (DCs) and follicular dendritic cells (FDCs), were evaluated in preclinical prion-infected mice. Using a double immunolabelling strategy, we demonstrated the lack of innervation of PrPsc-accumulating cells (FDCs). Contacts between nerve fibers and PrPsc-propagating cells (DCs) were detected in T-cell zones and cell-trafficking areas. This supports, for the first time, the possible implication of dendritic cells in the prion neuroinvasion process.     

Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis

The levels of protection found in vaccine studies of murine visceral leishmaniasis are significantly lower than for cutaneous leishmaniasis; whether this is due to the high-challenge murine model employed and/or is a consequence of differences required in tissue-specific local immune responses is not understood. Consequently, an intradermal murine model of visceral leishmaniasis has been explored. Intradermal inoculation established a chronic infection in susceptible mice which was associated with a pattern of parasite clearance with time postinfection in the liver and skin; in contrast, parasite persistence and expansion was observed in lymphoid tissue (spleen and draining lymph node). The course of disease found appears to be similar to those reported for subclinical canine and human visceral leishmaniasis. Clearance of parasites from the skin was correlated with an inflammatory response and the infiltration and activation of CD4(+) and CD8(+) T cells. In contrast, in lymphoid tissue (lymph node or spleen), the production of Th1/Th2 cytokines (interleukin-4 [IL-4], IL-10, and gamma interferon) appeared to correlate with parasite burden and pathogenesis. In vaccination experiments employing the Leishmania infantum D-13 (p80) antigen, significantly higher levels of protection were found with the intradermal murine model (29 to 7,500-fold more than naive controls) than were found with a low-dose intravenous infection model (9 to 173-fold). Thus, this model should prove useful for further investigation of disease pathogenesis as well as vaccine studies of visceral leishmaniasis.     

Studies on poxvirus infection in cats

Summary The development of clinical disease and the pathogenesis of cowpox were studied in domestic cats inoculated by a variety of routes. Intradermal titration in two cats demonstrated that as little as five pfu of cowpox virus caused a primary skin lesion. Intradermal inoculation of 10⁵ pfu cowpox virus resulted in severe systemic disease. Large amounts of virus (10³ pfu/g) were isolated from skin lesions and the turbinates of cats killed at eight and 11 days post-inoculation (dpi). Lesser amounts of virus (10² pfu/g) were isolated from lymphoid tissues and the lung, and small amounts of virus were isolated from various other tissues. A white cell-associated viraemia was detected from 5 dpi onwards. Skin scarification with 10³ or 50 pfu cowpox virus enabled reproduction of the naturally-acquired disease. Cat-to-cat transmission was demonstrated from cats inoculated by skin scarification, but caused only subclinical infection in sentinel cats. Oronasal inoculation resulted in transient coryza and milder generalized disease than skin inoculation, and no transmission to sentinel cats. Preliminary investigations showed vaccinia virus (Lister strain) to be of low infectivity in cats while inoculation of ectromelia virus (Mill Hill strain) did not cause any clinical signs.     

Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie.

The scrapie-associated form of the prion protein (PrPSc) accumulates in the brain and lymphoid tissues of sheep with scrapie. In order to assess whether detecting PrPSc in lymphoid tissue could be used as a diagnostic test for scrapie, we studied the localization and distribution of PrPSc in various lymphoid tissues collected at necropsy from 55 sheep with clinical scrapie. Samples collected from the spleen, palatine tonsil, ileum, and five different lymph nodes were immunohistochemically stained for PrPSc. PrPSc was found to be deposited in a reticular pattern in the center of both primary and secondary lymphoid follicles. In addition, granules of PrPSc were seen in the cytoplasm in macrophages associated with the lymphoid follicles. In 54 (98%) of the 55 scrapie-affected sheep, PrPSc was detected in the spleen, retropharyngeal lymph node, mesenteric lymph node, and the palatine tonsil. However, only in the palatine tonsils was PrPSc present in a consistently high percentage of the lymphoid follicles. PrP was not detected in any of the lymphoid tissues of 12 sheep that had no neurohistopathological signs of a scrapie infection. We conclude that the tonsils are the best-suited lymphoid tissue to be biopsied for the detection of PrPSc in the diagnosis of clinical scrapie in living sheep.     

Phenotypic characteristics of antigen-bearing cells in the draining lymph nodes of contact sensitized mice

Following contact sensitization of mice there is a rapid accumulation of dendritic cells (DC) within lymph nodes draining the site of exposure. Previous studies have revealed that cells bearing high levels of contact allergen can also be identified within the low buoyant density fraction of draining lymph node cells, and it has been assumed that the majority of these are DC. The purpose of the present study was to establish the phenotypic characteristics of the antigen-bearing cells which appear in lymph nodes within hours of skin painting with contact allergens, including the contact sensitizing fluorochromes fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC). Indirect immunofluorescence and two-colour immunofluorescence analyses revealed that initially all antigen-bearing cells which arrive in the draining lymph nodes express class II MHC antigens and exhibit a dendritic morphology. Phagocytic cells, cells reactive with F4/80 and anti-Mac-1 antibodies and lymphoblasts are not associated with detectable levels of antigen. In addition, although Thy-1+ cells co-fractionate with lymph node DC they are not dendritic in nature and are not associated with antigen. These data are compatible with the hypothesis that following skin sensitization epidermal Langerhans' cells bind antigen and transport it to the regional lymph nodes. In addition, it is clear that the recently described population of Thy-1+, Ia- dendritic cells within the murine epidermis do not perform a similar function.     

Retention of Ag‐specific memory CD4+ T cells in the draining lymph node indicates lymphoid tissue resident memory populations

Several different memory T‐cell populations have now been described based upon surface receptor expression and migratory capabilities. Here we have assessed murine endogenous memory CD4⁺ T cells generated within a draining lymph node and their subsequent migration to other secondary lymphoid tissues. Having established a model response targeting a specific peripheral lymph node, we temporally labelled all the cells within draining lymph node using photoconversion. Tracking of photoconverted and non‐photoconverted Ag‐specific CD4⁺ T cells revealed the rapid establishment of a circulating memory population in all lymph nodes within days of immunisation. Strikingly, a resident memory CD4⁺ T cell population became established in the draining lymph node and persisted for several months in the absence of detectable migration to other lymphoid tissue. These cells most closely resembled effector memory T cells, usually associated with circulation through non‐lymphoid tissue, but here, these cells were retained in the draining lymph node. These data indicate that lymphoid tissue resident memory CD4⁺ T‐cell populations are generated in peripheral lymph nodes following immunisation.     

Murine scrapie infection causes an abnormal germinal centre reaction in the spleen

Follicular dendritic cells (FDCs) of the lymphoreticular system play a role in the peripheral replication of prion proteins in some transmissible spongiform encephalopathies (TSEs), including experimental murine scrapie models. Disease-specific PrP (PrPd) accumulation occurs in association with the plasmalemma and extracellular space around FDC dendrites, but no specific immunological response has yet been reported in animals affected by TSEs. In the present study, morphology (light microscopical and ultrastructural) of secondary lymphoid follicles of the spleen were examined in mice infected with the ME7 strain of scrapie and in uninfected control mice, with or without immunological stimulation with sheep red blood cells (SRBCs), at 70 days post-inoculation or at the terminal stage of disease (268 days). Scrapie infection was associated with hypertrophy of FDC dendrites, increased retention of electron-dense material at the FDC plasma membrane, and increased maturation and numbers of B lymphocytes within secondary follicles. FDC hypertrophy was particularly conspicuous in immune-stimulated ME7-infected mice. The electron-dense material was associated with PrP Napoli accumulation, as determined by immunogold labelling. We hypothesize that immune system changes are associated with increased immune complex trapping by hypertrophic FDCs expressing PrP Napoli molecules at the plasmalemma of dendrites, and that this process is exaggerated by immune system stimulation. Contrary to previous dogma, these results show that a pathological response within the immune system follows scrapie infection.     

Complement component C5 is not involved in scrapie pathogenesis

During transmissible spongiform encephalopathy (TSE) infections the accumulation of abnormal prion protein within the brain is often accompanied by severe neurodegeneration. Studies have implicated complement, including the membrane attack complex (MAC, C5b-C9), in inducing pathology in some neurodegenerative diseases. Recent studies show the MAC is localized on neurons in the brains of TSE patients implicating complement-mediated cell lysis in TSE neuropathology. To determine the role of the MAC in TSEs, we compared scrapie pathogenesis in C5-deficient and C5-sufficient mice. C5-deficient mice developed clinical scrapie with incubation periods similar to C5-sufficient mice. Furthermore, the severity of the neuropathology was not significantly different between C5-deficient and C5-sufficient mice. These data show that C5, and the MAC, are not involved in TSE pathogenesis.