Oral Prion Infection Requires Normal Numbers of Peyer's Patches but Not of Enteric Lymphocytes
2003; Elsevier BV; Volume: 162; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63907-7
ISSN1525-2191
AutoresMarco Prinz, Gerhard Huber, Andrew J. Macpherson, Frank L. Heppner, Markus Glatzel, Hans‐Pietro Eugster, Norbert Wagner, Adriano Aguzzi,
Tópico(s)Animal Disease Management and Epidemiology
ResumoPrion pathogenesis following oral exposure is thought to involve gut-associated lymphatic tissue, which includes Peyer's patches (PPs) and M cells. Recruitment of activated B lymphocytes to PPs requires α4β7 integrin; PPs of β7−/− mice are normal in number but are atrophic and almost entirely devoid of B cells. Here we report that minimal infectious dose and disease incubation after oral exposure to logarithmic dilutions of prion inoculum were similar in β7−/− and wild-type mice, and PPs of both β7−/− and wild-type mice contained 3–4 log LD50/g prion infectivity ≥125 days after challenge. Despite marked reduction of B cells, M cells were present in β7−/− mice. In contrast, mice deficient in both tumor necrosis factor and lymphotoxin-α (TNFα−/− × LTα−/−) or in lymphocytes (RAG-1−/−, μMT), in which numbers of PPs are reduced in number, were highly resistant to oral challenge, and their intestines were virtually devoid of prion infectivity at all times after challenge. Therefore, lymphoreticular requirements for enteric and for intraperitoneal uptake of prions differ from each other. Although susceptibility to prion infection following oral challenge correlates with the number of PPs, it is remarkably independent of the number of PP-associated lymphocytes. Prion pathogenesis following oral exposure is thought to involve gut-associated lymphatic tissue, which includes Peyer's patches (PPs) and M cells. Recruitment of activated B lymphocytes to PPs requires α4β7 integrin; PPs of β7−/− mice are normal in number but are atrophic and almost entirely devoid of B cells. Here we report that minimal infectious dose and disease incubation after oral exposure to logarithmic dilutions of prion inoculum were similar in β7−/− and wild-type mice, and PPs of both β7−/− and wild-type mice contained 3–4 log LD50/g prion infectivity ≥125 days after challenge. Despite marked reduction of B cells, M cells were present in β7−/− mice. In contrast, mice deficient in both tumor necrosis factor and lymphotoxin-α (TNFα−/− × LTα−/−) or in lymphocytes (RAG-1−/−, μMT), in which numbers of PPs are reduced in number, were highly resistant to oral challenge, and their intestines were virtually devoid of prion infectivity at all times after challenge. Therefore, lymphoreticular requirements for enteric and for intraperitoneal uptake of prions differ from each other. Although susceptibility to prion infection following oral challenge correlates with the number of PPs, it is remarkably independent of the number of PP-associated lymphocytes. Consumption of food contaminated with bovine spongiform encephalopathy (BSE) is believed to cause variant Creutzfeldt-Jakob disease (vCJD) in humans1Bruce ME Will RG Ironside JW McConnell I Drummond D Suttie A McCardle L Chree A Hope J Birkett C Cousens S Fraser H Bostock CJ Transmissions to mice indicate that "new variant" CJD is caused by the BSE agent.Nature. 1997; 389: 498-501Crossref PubMed Scopus (1737) Google Scholar, 2Hill AF Desbruslais M Joiner S Sidle KC Gowland I Collinge J Doey LJ Lantos P The same prion strain causes vCJD and BSE (letter).Nature. 1997; 389: 448-450Crossref PubMed Scopus (1218) Google Scholar and ingestion of prions has been implicated in the transmission of other transmissible spongiform encephalopathies (TSE).3Aguzzi A Montrasio F Kaeser PS Prions: health scare and biological challenge.Nat Rev Mol Cell Biol. 2001; 2: 118-126Crossref PubMed Scopus (133) Google Scholar Following experimental intragastric or oral exposure of rodents to scrapie, infectivity and/or disease-associated protease-resistant prion protein (PrPSc) accumulate rapidly in Peyer's patches (PPs), gut-associated lymphoid tissues (GALT), and ganglia of the enteric nervous system4Beekes M McBride PA Early accumulation of pathological PrP in the enteric nervous system and gut-associated lymphoid tissue of hamsters orally infected with scrapie.Neurosci Lett. 2000; 278: 181-184Crossref PubMed Scopus (197) Google Scholar, 5Kimberlin RH Walker CA Pathogenesis of scrapie in mice after intragastric infection.Virus Res. 1989; 12: 213-220Crossref PubMed Scopus (192) Google Scholar long before they are detected in the central nervous system (CNS). Similarly, following experimental oral exposure of non-human primates or sheep to BSE, PrPSc was first detected in lymphoid tissues draining the gastrointestinal tract, long before detection in the CNS.6Bons N Mestre-Frances N Belli P Cathala F Gajdusek DC Brown P Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents.Proc Natl Acad Sci USA. 1999; 96: 4046-4051Crossref PubMed Scopus (168) Google Scholar, 7Foster JD Parnham DW Hunter N Bruce M Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission.J Gen Virol. 2001; 82: 2319-2326PubMed Google Scholar B lymphocytes play a crucial role in peripheral prion pathogenesis: mice devoid of B lymphocytes do not develop disease after intraperitoneal exposure.8Klein MA Frigg R Flechsig E Raeber AJ Kalinke U Bluethmann H Bootz F Suter M Zinkernagel RM Aguzzi A A crucial role for B cells in neuroinvasive scrapie.Nature. 1997; 390: 687-690Crossref PubMed Scopus (447) Google Scholar This is possibly because B lymphocytes induce maturation of follicular dendritic cells (FDCs) by providing tumor necrosis factor-α (TNF-α) and lymphotoxin α/β (LTα/β) trimers to lymphoid organs.9Fu YX Chaplin DD Development and maturation of secondary lymphoid tissues.Annu Rev Immunol. 1999; 17: 399-433Crossref PubMed Scopus (571) Google Scholar Early PrPSc deposition can be detected in FDCs within B cell follicles in lymphoid tissues of patients with vCJD10Hill AF Butterworth RJ Joiner S Jackson G Rossor MN Thomas DJ Frosh A Tolley N Bell JE Spencer M King A Al-Sarraj S Ironside JW Lantos PL Collinge J Investigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples.Lancet. 1999; 353: 183-189Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar and in rodents inoculated with scrapie by peripheral routes.11Brown KL Stewart K Ritchie DL Mabbott NA Williams A Fraser H Morrison WI Bruce ME Scrapie replication in lymphoid tissues depends on prion protein-expressing follicular dendritic cells.Nat Med. 1999; 5: 1308-1312Crossref PubMed Scopus (317) Google Scholar In mouse spleens, mature FDCs have been shown to be crucial for both prion replication and PrPSc accumulation,12Mabbott NA Mackay F Minns F Bruce ME Temporary inactivation of follicular dendritic cells delays neuroinvasion of scrapie (letter).Nat Med. 2000; 6: 719-720Crossref PubMed Scopus (206) Google Scholar, 13Montrasio F Frigg R Glatzel M Klein MA Mackay F Aguzzi A Weissmann C Impaired prion replication in spleens of mice lacking functional follicular dendritic cells.Science. 2000; 288: 1257-1259Crossref PubMed Scopus (323) Google Scholar although prion replication in lymph nodes can occur in the absence of mature FDCs.14Prinz M Montrasio F Klein MA Schwarz P Priller J Odermatt B Pfeffer K Aguzzi A Lymph nodal prion replication and neuroinvasion in mice devoid of follicular dendritic cells.Proc Natl Acad Sci USA. 2002; 99: 919-924Crossref PubMed Scopus (127) Google Scholar In contrast, the role of intestinal B cells in prion pathogenesis following oral challenge is still unclear. Intestinal mucosal immunity provides an important level of defense against foreign pathogens. The ability of B and T lymphocytes to be recruited to the site of infection is critical for an effective immune response. This process is mediated by homing receptors on effector cells with cognate ligands at peripheral or mucosal sites.15Butcher EC Williams M Youngman K Rott L Briskin M Lymphocyte trafficking and regional immunity.Adv Immunol. 1999; 72: 209-253Crossref PubMed Google Scholar Integrin α4β7 plays an important role in the homing of activated lymphocytes to PPs and to the intestinal lamina propria.16Hamann A Andrew DP Jablonski-Westrich D Holzmann B Butcher EC Role of α4-integrins in lymphocyte homing to mucosal tissues in vivo.J Immunol. 1994; 152: 3282-3293PubMed Google Scholar β7 Integrin-deficient (β7−/−) mice suffer from severely-reduced cellularity of PPs (>90% less B and T cells)17Wagner N Lohler J Kunkel EJ Ley K Leung E Krissansen G Rajewsky K Muller W Critical role for β7 integrins in formation of the gut-associated lymphoid tissue.Nature. 1996; 382: 366-370Crossref PubMed Scopus (471) Google Scholar and from impaired intestinal immunity in a variety of disease models.18Artis D Humphreys NE Potten CS Wagner N Muller W McDermott JR Grencis RK Else KJ β7 integrin-deficient mice: delayed leukocyte recruitment and attenuated protective immunity in the small intestine during enteric helminth infection.Eur J Immunol. 2000; 30: 1656-1664Crossref PubMed Scopus (94) Google Scholar, 19Kuklin NA Rott L Darling J Campbell JJ Franco M Feng N Muller W Wagner N Altman J Butcher EC Greenberg HB α(4)β(7) independent pathway for CD8(+) T cell-mediated intestinal immunity to rotavirus.J Clin Invest. 2000; 106: 1541-1552Crossref PubMed Scopus (58) Google Scholar, 20Lefrancois L Parker CM Olson S Muller W Wagner N Schon MP Puddington L The role of β7 integrins in CD8 T cell trafficking during an antiviral immune response.J Exp Med. 1999; 189: 1631-1638Crossref PubMed Scopus (184) Google Scholar With the exception of the GALT, lymphoid organs of β7−/− mice are otherwise normal. These mice are therefore well-suited to dissect the role of mucosa-associated immune tissue, including B cells, in the pathogenesis of enterically-initiated prion disease. In addition, B lymphocytes exert an important organogenic role in the GALT21Debard N Sierro F Browning J Kraehenbuhl JP Effect of mature lymphocytes and lymphotoxin on the development of the follicle-associated epithelium and M cells in mouse Peyer's patches.Gastroenterology. 2001; 120: 1173-1182Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22Golovkina TV Shlomchik M Hannum L Chervonsky A Organogenic role of B lymphocytes in mucosal immunity.Science. 1999; 286: 1965-1968Crossref PubMed Scopus (207) Google Scholar and are likely to be involved in the B cell-dependent development of the follicle-associated epithelium (FAE).23Wu Q Wang Y Wang J Hedgeman EO Browning JL Fu YX The requirement of membrane lymphotoxin for the presence of dendritic cells in lymphoid tissues.J Exp Med. 1999; 190: 629-638Crossref PubMed Scopus (129) Google Scholar However, splenic lymphocytes can acquire prion infectivity,24Raeber AJ Klein MA Frigg R Flechsig E Aguzzi A Weissmann C PrP-dependent association of prions with splenic but not circulating lymphocytes of scrapie-infected mice.EMBO J. 1999; 18: 2702-2706Crossref PubMed Scopus (89) Google Scholar and it is unclear whether their role in prion pathogenesis is restricted to the generation and maintenance of FDCs13Montrasio F Frigg R Glatzel M Klein MA Mackay F Aguzzi A Weissmann C Impaired prion replication in spleens of mice lacking functional follicular dendritic cells.Science. 2000; 288: 1257-1259Crossref PubMed Scopus (323) Google Scholar or whether they may also be involved in prion trafficking.25Kaeser PS Klein MA Schwarz P Aguzzi A Efficient lymphoreticular prion propagation requires prp(c) in stromal and hematopoietic cells.J Virol. 2001; 75: 7097-7106Crossref PubMed Scopus (61) Google Scholar To dissect the organogenetic effects from trafficking components, we administered prions orally to TNFα−/−/LTα−/− mice, which have normal lymphocyte counts but lack the two cytokines TNF-α and LTα,26Eugster HP Muller M Karrer U Car BD Schnyder B Eng VM Woerly G Le Hir M di Padova F Aguet M Zinkernagel R Bluethmann H Ryffel B Multiple immune abnormalities in tumor necrosis factor and lymphotoxin-α double-deficient mice.Int Immunol. 1996; 8: 23-36Crossref PubMed Scopus (196) Google Scholar to B cell-deficient μMT mice,27Kitamura D Roes J Kuhn R Rajewsky K A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain gene.Nature. 1991; 350: 423-426Crossref PubMed Scopus (1548) Google Scholar and to RAG-1−/− mice28Mombaerts P Iacomini J Johnson RS Herrup K Tonegawa S Papaioannou VE RAG-1-deficient mice have no mature B and T lymphocytes.Cell. 1992; 68: 869-877Abstract Full Text PDF PubMed Scopus (2364) Google Scholar which lack all T and B lymphocytes. While there were no recognizable PPs in TNFα−/−/LTα−/− mice, unexpectedly we found that μMT and RAG-1−/− mice had some FDC-M1-positive cells in their atrophic Peyer's patches, but not in spleen nor in lymph nodes. However, these FDC-like structures were not sufficient for enteric prion replication. Here we show that prion replication in the GALT and subsequent neuroinvasion was independent of B cells within the mucosa-associated lymphatic tissue and that the remaining M cells are most likely important for this process. TNFα−/−/LTα−/−, μMT, and RAG-1−/− mice were highly resistant to oral challenge, and their intestines were virtually devoid of prion infectivity at all times after challenge. Therefore, lymphoreticular requirements for enteric and intraperitoneal uptake of prions differ, and the presence of intramucosal B lymphocytes does not appear to be important for prion pathogenesis following oral challenge. C57BL/6, Sv129 × BL/6, μMT,27Kitamura D Roes J Kuhn R Rajewsky K A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain gene.Nature. 1991; 350: 423-426Crossref PubMed Scopus (1548) Google Scholar RAG-1−/−,28Mombaerts P Iacomini J Johnson RS Herrup K Tonegawa S Papaioannou VE RAG-1-deficient mice have no mature B and T lymphocytes.Cell. 1992; 68: 869-877Abstract Full Text PDF PubMed Scopus (2364) Google Scholar and TNFα−/−/LTα−/−26Eugster HP Muller M Karrer U Car BD Schnyder B Eng VM Woerly G Le Hir M di Padova F Aguet M Zinkernagel R Bluethmann H Ryffel B Multiple immune abnormalities in tumor necrosis factor and lymphotoxin-α double-deficient mice.Int Immunol. 1996; 8: 23-36Crossref PubMed Scopus (196) Google Scholar mice were bred at the Institute of Laboratory Animal Science of the University of Zurich and maintained under specific pathogen-free conditions. Wild-type mice were obtained originally from the Jackson Laboratory, Bar Harbor, ME. For detection of alkaline phosphatase (AP) activity, fixed whole PPs were studied. Segments of the small intestine containing one PP were excised, opened, and pinned on dental wax. Tissues were fixed for 10 minutes in Baker formalin calcium medium (pH 6) on ice. At this stage, villi were removed using a binocular microscope, and incubated with 125 nmol/L Tris buffer (pH 9.2) at 37°C for 5 minutes and then incubated in Tris buffer containing naphthol AS-BI phosphate (Sigma, Deisenhofen, Germany) with constant shaking for 10 minutes. The reaction was stopped with ice-cold buffer. Goblet cells were detected by incubating tissue briefly in 1% Alcian blue dissolved in 3% acetic acid. Whole-mount FAEs were then used to enumerate AP-negative M cells as a percentage of the total cell population within the FAE. Statistical significance was determined using the unpaired Student's t-test. Values of P < 0.05 were considered as significant. Mice were fed with logarithmic dilutions of 10% heat- and sarcosyl-treated brain homogenate prepared from mice infected with the Rocky Mountain Laboratory (RML) scrapie strain (passage 4.1). 8 log LD50 of the prion homogenate RML were administered via gastrolavage directly into the stomach. Smaller amounts (see Table 1) were administered by feeding a mixture of brain homogenate with food pellets. For intraperitoneal inoculations, 6 log LD50 of scrapie inoculum was administered. Mice were monitored on alternate days, and scrapie was diagnosed according to standard clinical criteria.Table 1Susceptibility of Mice with Different Alterations of the Gut-Associated Lymphoid Tissue to ScrapieOral infectioni.p. Infection, 6 log LD50Intracerebral infection, 3 × 105 LD508 log LD507 log LD506 log LD50Mouse genotypeGenetic backgroundAttack rateDisease latency (Average ± SD)Attack rateDisease latencyAttack rateDisease latencyAttack rateDisease latencyAttack rateDisease latencyβ7−/−129Sv × BL/66/6230 ± 92/4261,2720/55 × >5106/6217 ± 44/4148 ± 72 × >510RAG-1−/−C57BL/60/55 × >5100/33 × >510ND0/55 × >5105/5139 ± 6μMT−/−C57BL/60/66 > 5100/77 × >510ND0/44 × >5103/3154 ± 2TNFα−/− × LTα−/−129Sv × BL/60/88 × >5100/55 × >5100/55 × >5102/5226,2774/4144 ± 53 × >510Wild typeC57BL/69/10224 ± 14,1/4247,0/55 × >5105/5213 ± 86/6147 ± 91 × >5103 × >510Wild type129Sv × BL/64/4213 ± 102/4250,2560/33 × >5104/4210 ± 125/5151 ± 62 × >510Prnp0/0129Sv × BL/60/6>510NDNDND0/55 × >50β7−/− and wild-type mice developed scrapie upon very high oral prion challenge (8 log LD50) and i.p. and intracerebral inoculation at similar time points, while lower oral concentrations only partially evoked scrapie. All the other strains were protected to varying degrees. Mean incubation time and standard errors were calculated for mice that developed scrapie. The total observation time is indicated for mice that remained disease-free.ND, not determined. Open table in a new tab β7−/− and wild-type mice developed scrapie upon very high oral prion challenge (8 log LD50) and i.p. and intracerebral inoculation at similar time points, while lower oral concentrations only partially evoked scrapie. All the other strains were protected to varying degrees. Mean incubation time and standard errors were calculated for mice that developed scrapie. The total observation time is indicated for mice that remained disease-free. ND, not determined. Bioassays were performed on 1% homogenates of spleens or of PBS-perfused small intestines (containing Peyer's patches). Tissues were homogenized in PBS/BSA with a microhomogenizer and passed several times through 18-gauge and 22-gauge needles. Following preparation of a homogeneous suspension, centrifugation was performed at 500 × g for 5 minutes. Supernatants (30 μl) were inoculated intracerebrally into groups of at least four tga20 mice.29Fischer M Rülicke T Raeber A Sailer A Moser M Oesch B Brandner S Aguzzi A Weissmann C Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.EMBO J. 1996; 15: 1255-1264Crossref PubMed Scopus (785) Google Scholar The titer of the standard inoculum (7.9 log 10 of 50% lethal dose (LD50)/ml, corresponding to 8.9 log LD50/g of brain tissue) was determined by the 50% endpoint calculation method.30Reed J Muench H A simple method of estimating 50% endpoints.Am J Hygiene. 1938; 27: 493-497Google Scholar The relationship y = 11.45 − 0.088 (y, log LD50 × ml−1 of homogenate; x, incubation time in days to terminal disease) was calculated by linear regression.25Kaeser PS Klein MA Schwarz P Aguzzi A Efficient lymphoreticular prion propagation requires prp(c) in stromal and hematopoietic cells.J Virol. 2001; 75: 7097-7106Crossref PubMed Scopus (61) Google Scholar, 31Prusiner SB Cochran SP Groth DF Downey DE Bowman KA Martinez HM Measurement of the scrapie agent using an incubation time interval assay.Ann Neurol. 1982; 11: 353-358Crossref PubMed Scopus (276) Google Scholar Indicator mice were sacrificed after development of terminal scrapie and infectivity titers were calculated. Paraffin sections (2 μm) and cryostat sections (10 μm) from brain, spleen and PPs were stained with hematoxylin and eosin. Immunostaining for GFAP was performed using rabbit GFAP antisera (1:300 dilution; DAKO, Carpinteria, CA) and detected with biotinylated swine anti-rabbit serum (1:250 dilution; DAKO), avidin-peroxidase and diaminobenzidine (Sigma). Antibodies reactive against the follicular dendritic cell marker FDC-M1 (clone 4C11; 1:300 dilution) or the pan-B cell marker CD45R/B220 (BD Pharmingen, San Diego, CA) were used for immunohistochemistry of frozen sections as previously described.32Karrer U Lopez-Macias C Oxenius A Odermatt B Bachmann MF Kalinke U Bluethmann H Hengartner H Zinkernagel RM Antiviral Bcell memory in the absence of mature follicular dendritic cell networks and classical germinal centers in TNFR1-/- mice.J Immunol. 2000; 164: 768-778PubMed Google Scholar Tissue homogenates were adjusted to protein concentrations of 5 mg/ml (brain) or 8 mg/ml (spleen) and treated with proteinase K (20 μg/ml, 30 minutes, 37°C). 50 μg (brain) or 80 μg (spleen) of total protein of each sample were electrophoresed through a 12% SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes by semi-dry blotting. Membranes were blocked with Tris-buffered saline containing 0.1% Tween 20, pH 8.0 (TBST)/5% nonfat milk, incubated with antibodies 6H4 (for brain protein) or 1B3 (for spleen protein), and detected using alkaline phosphatase-linked anti-mouse (6H4) or anti-rabbit (1B3) sera followed by development by enhanced chemiluminescence (ECL; Amersham, Uppsala, Sweden). To evaluate the extent to which the absence of α4β7 integrin, TNF/lymphotoxin, and B and T cells affects the presence of PPs, mice were examined anatomically. PPs of wild-type mice of the C57BL/6 and 129Sv×BL6 genetic background were easily visible to the naked eye, whereas in β7−/−, RAG-1−/−, and μMT mice PPs were undetectable by gross inspection. However, when small intestines were inflated with saline buffer (PBS) and transilluminated under a dissecting microscope (16× magnification), numerous small Peyer's patches could be discerned (Figure 1). The number and location (data not shown) of Peyer's patches in β7−/− mice (n = 6–10, mean 8.0 ± 1.5) were similar to wild-type mice (n = 6–11, mean 7.3 ± 1.5). A comparison with Student's t-test, however, yielded a P value of >0.05, indicating that the number of Peyer's patches in the two groups was not significantly different. In contrast, the number of PPs in B cell-deficient μMT mice were significantly reduced (0–7, mean 2.5 ± 2.2; P < 0.01 if compared to the respective wild-type control). Combined B cell and T cell deficiency in RAG-1−/− mice further reduced the number of PPs (0–5, mean 2.0 ± 1.7; P < 0.01). The absence of TNF-α and lymphotoxin-α (LTα) completely prevented the formation of visible Peyer's patches (P < 0.01). The size of the M cell-containing subepithelial domes of β7-deficient mice were reduced by at least 70% compared to those of wild-type mice (approximately 250 μm versus up to 800 μm for full length of equatorial sections). A similar reduction in the size of FAE was found in B cell-deficient PPs of RAG-1−/− and μMT mice. To determine whether the absence of α4β7 integrin and lymphocytes alters the cellular composition of secondary lymphoid organs, we studied spleen and PPs by immunohistochemistry using antisera reactive against the B lymphocyte marker B220 and with the FDC marker M1 (Figure 2). The germinal center histology of spleens of β7−/− mice was indistinguishable from wild-type spleens. B cell zones were distinct and contained FDC-M1-positive cells of normal shape and localization. In wild-type PPs, FDC networks occupied large regions of the germinal centers. Although there was a significant reduction in the number of B cells in β7−/− PPs, some FDCs were still detectable. In contrast, no PPs were observed in TNF-α/LTα-deficient mice, although a small number of B cells were still present in the small intestine of these mice. Lymphoid organ structure of spleens of TNF-α/LTα-deficient mice was severely impaired, without any separation between T and B cell zones, and FDCs were undetectable. No FDC-M1 positive cells were detected in the spleens of RAG-1−/− and μMT mice, in agreement with earlier reports.22Golovkina TV Shlomchik M Hannum L Chervonsky A Organogenic role of B lymphocytes in mucosal immunity.Science. 1999; 286: 1965-1968Crossref PubMed Scopus (207) Google Scholar Unexpectedly, clusters of cells reactive with FDC-M1 and CD35 (CR1, not shown) were consistently detected in the small atrophic Peyer's patches of RAG-1−/− and μMT mice. Immunostaining also revealed a number of B220 positive cells in spleens and PPs. The majority of these B220-positive cells are most likely NK cells, because the antibody used (CD45R/B220) reacts with an epitope on the extracellular domain of the CD45 glycoprotein, which is expressed on lytically active subsets of lymphokine-activated killer cells such as NK cells and their progenitors in addition to B lymphocytes.33Rolink A ten Boekel E Melchers F Fearon DT Krop I Andersson J A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors.J Exp Med. 1996; 183: 187-194Crossref PubMed Scopus (191) Google Scholar The smaller B220+ cell population in the PPs of RAG-1−/− and μMT mice most likely consist of immature B cells which are still able to produce functional immunoglobulins of the A class even in the absence of surface, membrane-bound IgM or IgD, therefore representing a developmental pro-B cell block in these strains.34Macpherson AJ Lamarre A McCoy K Harriman GR Odermatt B Dougan G Hengartner H Zinkernagel RM IgA production without μ or δ chain expression in developing B cells.Nat Immunol. 2001; 2: 625-631Crossref PubMed Scopus (202) Google Scholar M cells of the FAE are believed to be important antigen sampling sites, and can act as entry sites for pathogens. To determine whether the absence or reduction of lymphocytes alters the composition of the FAE, we performed immunostaining of PPs of wild-type, β7−/−, RAG-1−/− and μMT with antibodies reactive against specific markers for M cells (Figure 3, A–D). M cells, visualized histochemically by their reduced AP activity,35Brown D Cremaschi D James PS Rossetti C Smith MW Brush-border membrane alkaline phosphatase activity in mouse Peyer's patch follicle-associated enterocytes.J Physiol. 1990; 427: 81-88Crossref PubMed Scopus (21) Google Scholar were readily detectable in the FAE of wild-type mice (Figure 3A). In β7−/−, μMT, and RAG-1−/− (Figure 3, B, C, and D, respectively), both PPs and AP-negative M cells were apparent. The presence of PPs and AP-negative M cells was further confirmed by scanning electron microscopy, which allowed visualization of the disorganized brush border of these cells (data not shown). In contrast to PPs of wild-type mice, in which M cells were located at the periphery of the FAE, the distribution of M cells appeared to be random over the entire surface of FAE in PPs of β7−/−, RAG-1−/−, and μMT mice. We then estimated the relative number of intestinal M cells per total epithelial cells within the FAE by counting AP-negative M cells under 400× magnification by light microscopy (Figure 3E). The relative number of M cells was increased in the FAE of β7−/− (mean: 15.4 ± 4.0 vs. 6.7 ± 1.8 for 129Sv × BL/6 mice, P < 0.01), RAG-1−/− (mean: 13.5 ± 2.3 vs. 7.2 ± 2.7 for BL/6, P < 0.05) and μMT (mean: 14.2 ± 2.8 compared to BL/6, P < 0.05) tissues. These data indicate that the differentiation program of the FAE, including M cells, is maintained even in the absence of proper B cell numbers. To determine the contribution of different components of the GALT to the intestinal uptake of orally-delivered prions, mice were challenged with logarithmic dilutions of scrapie inoculum. Attack rate and time to terminal disease were measured. Following high-dose oral infection (8 log LD50) all β7−/− mice, Sv129 × C57BL/6 wild-type mice, and 9 of 10 C57BL/6 wild-type mice developed clinical signs of scrapie with similar incubation times of 213 to 230 days (Table 1). These results indicate that β7−/− mice are highly susceptible to disease via oral administration of prions. Accordingly, accumulation of PK-resistant PrPSc was readily detectable in brains and spleens of clinically sick β7−/− and wild-type mice (Figure 4). Moreover, topography and intensity of spongiosis and gliosis were similar in clinically sick β7−/− mice compared to clinically sick wild-type mice (Figure 5). Importantly, the use of lower prion inocula via the oral route significantly decreased the susceptibility to disease in these mice compared to i.p. challenge. A 10-fold dilution of the scrapie inoculum reduced the scrapie attack rate to 50% in β7−/− and to 50 to 25% in the different wild-type mice. Further dilution of the inoculum did not evoke scrapie in mice of any genotype.Figure 5Morphological changes in the brains of mice orally exposed to prions. Pronounced astrogliosis (GFAP) in the hippocampus of scrapie-sick (terminal disease) β7−/− and wild-type mice was obvious, whereas no gliosis and vacuolization (H&E) was observed in clinically healthy RAG-1−/−, μMT, and TNFα−/− × LTα−/− mice. Original magnification, ×200.View Large Image Figure ViewerDownload Hi-res image Download (PPT) After i.p. prion challenge (6 log LD50) β7−/− and wild-type mice had similar incubation times of 210 to 217 days, indicating normal peripheral prion pathogenesis. As described earlier,8Klein MA Frigg R Flechsig E Raeber AJ Kalinke U Bluethmann H Bootz F Suter M Zinkernagel RM Aguzzi A A crucial role for B cells in neuroinvasive scrapie.Nature. 1997; 390: 687-690Crossref PubMed Scopus (447) Google Scholar, 14Prinz M Montrasio F Klein MA Schwarz P Priller J Odermatt B Pfeffer K Aguzzi A Lymph nodal prion replication and neuroinvasion in mice devoid of follicular dendritic cells.Proc Natl Acad Sci USA. 2002; 99: 919-924Crossref PubMed Scopus (127) Google Scholar B cell-deficient RAG-1 and μMT mice were completely resistant, and TNFα−/−/LTα−/− mice partially resistant, to prions administered i.p. Wild-type mice can be experimentally infected via the i.p. route with prion inocula of less than 3 intracerebral log LD50, yet the minimal dose for or
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