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Journal of Virology, April 2008, p. 3791-3795, Vol. 82, No. 7
0022-538X/08/$08.00+0     doi:10.1128/JVI.02036-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Alteration of B-Cell Subsets Enhances Neuroinvasion in Mouse Scrapie Infection{triangledown}

Christine von Poser-Klein,1 Eckhard Flechsig,1 Tanja Hoffmann,1 Petra Schwarz,2 Harry Harms,1 Raymond Bujdoso,3 Adriano Aguzzi,2,{dagger} and Michael A. Klein1,{dagger}*

Institute of Virology and Immunobiology, University of Würzburg, D-97078 Würzburg, Germany,1 Institute of Neuropathology, Department of Pathology, University Hospital of Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland,2 Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, United Kingdom3

Received 14 September 2007/ Accepted 24 December 2007


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ABSTRACT
 
Acquired forms of prion diseases or transmissible spongiform encephalopathies are believed to occur following peripheral exposure. Prions initially accumulate in the lymphoid system before spreading to the nervous system, but the underlying mechanisms for prion transfer between the two systems are still elusive. Here we show that ablation of the B-cell-specific transmembrane protein CD19, a coreceptor of the complement system, results in an acceleration of prion neuroinvasion. This appears to be due to an alteration of the follicular dendritic cell (FDC) network within the lymphoid tissue, thereby reducing the distance between FDCs and adjacent nerve fibers that mediate prion neuroinvasion.


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TEXT
 
Although prion infections are likely to be acquired orally, animal studies have shown that peripheral infection with prions may occur by a variety of routes (17). These studies have shown that following peripheral inoculation, most prion strains accumulate in the lymphoreticular system. The site for accumulation of prion infectivity and the disease-associated protein, designated PrPSc, during the lymphoreticular phase is the follicular dendritic cell (FDC) network within germinal centers of lymphoid tissue (16, 19). Following accumulation within lymphoid tissue, prions migrate along peripheral nerves to the brain, presumably via the spinal cord (7, 10, 11) or the vagus nerve (2).

The function of FDCs in peripheral prion pathogenesis is regulated in part by B cells, which provide growth and differentiation factors for the stroma-derived cells (1, 6). B cells can be divided into two phenotypically and functionally distinct populations, referred to as B-1 cells (Ly-1) and conventional B cells (B-2) (8, 13). B-1 cells are in a more activated state than B-2 cells (28) and are a major source of antibody production (29).

Signal transduction through a B-cell receptor primarily determines the fate and function of B cells and is regulated by other cell surface receptors such as the CD19/CD21 complex, which serves as a positive regulator for an immune response (21). In contrast, negative regulators such as CD22 are crucial for terminating B-cell-receptor signal transduction to avoid improper immune response against self-antigens (21). These coreceptor-mediated B-cell immune regulatory functions may also extend to an effect upon the FDC network. To test this hypothesis, we investigated prion pathogenesis in mice that lacked either CD19 or CD22. Since CD19–/– mice (24) have a decreased number of B-1 cells whereas CD22–/– mice (20) contain fewer B-2 cells, we reasoned that these experiments would also address the role of different B-cell subsets in peripheral prion pathogenesis.

Following intracerebral challenge of mice with either a low dose (3 x 103.1 50% lethal doses [LD50]) or a high dose (3 x 106.1 LD50) of RML prions, the incubation times to terminal prion disease were similar in wild-type, CD19–/–, and CD22–/– mice (Fig. 1A and B). Surprisingly, the absence of CD19 resulted in a reduced incubation time after peripheral exposure to RML prions which was independent of the injected dose. CD19–/– mice developed terminal disease in a significantly shorter incubation time than did control mice (129Sv) after intraperitoneal (i.p.) inoculation with either a high dose (176 ± 4 days versus 201 ± 5 days [mean ± standard deviation]; n = 7, P < 0.0005 [where n = number of mice per group]) or a low dose (212 ± 5 days [n = 10] versus 246 ± 7 days [n = 8], P < 0.0003) (Fig. 1D). In contrast, CD22–/– mice showed an incubation time similar to that seen with control mice (C57BL/6) after i.p. challenge with either a high dose (199 ± 5 days [n = 9] versus 196 ± 4 days [n = 7]) or a low dose (235 ± 7 days [n = 10] versus 239 ± 5 days [n = 8]) (Fig. 1C). Regardless of the difference in latency upon infection with a low dose, prion titers (Table 1) and histopathological changes in the brains of CD19–/– and CD22–/– mice at the terminal stage of the disease were similar to those found in wild-type controls (data not shown).


Figure 1
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  		FIG. 1. Latency of prion disease in CD19–/–, CD22–/–, and wild-type mice. (A and B) No difference in the incubation time of CD19–/– (B) and CD22–/– mice (A) was found after intracerebral inoculation with RML prions, in comparison to that of wild-type mice on the same genetic background. (C and D) After i.p. inoculation, CD22–/– mice (C) developed prion disease with an incubation time similar to that of wild-type mice regardless of the dose applied, whereas CD19–/– mice (D) showed a significant reduction in the incubation time with both a high dose (P < 0.0005 by log rank test) and a low dose (P < 0.0003) of RML prions. The dose of inoculum, the incubation time (mean number of days ± standard deviation), and the number (n) of infected/number of diseased mice per group are indicated in the survival curves.


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  		TABLE 1. Prion infectivity in spleen cell fractions, total spleens, and brains of mice after i.p. challenge with a low dose of RML prions

To get further insights into the peripheral pathogenesis, spleens of terminally sick CD19–/–, CD22–/–, and wild-type mice were analyzed for prion infectivity, level, and distribution of PrPSc. Titers in spleens were similar, approximately 6 log LD50 units per ml of 10% homogenates (Table 1), as calculated by the incubation time method (5). Comparable levels of PrPSc (Fig. 2A) and similar distributions of PrPSc associated with the FDC networks (Fig. 2B) were found in samples of all the genotypes, as judged by Western blotting and histoblot analysis (26), respectively. Furthermore, the prion titers during the disease progression (at days 34 and 90) were determined by a bioassay (5) of different splenic cell fractions (B cells, T cells, non-B/T cells, and a stromal fraction) isolated by magnetically activated cell sorting as previously described (23). Again, similar levels of prion infectivity were found in cell fractions from CD19–/– and wild-type mice. These findings imply that the reduced incubation time in CD19–/– mice was not the result of an increased accumulation of prions within either the spleen lymphoid cell fraction or the FDC-containing stromal fraction.


Figure 2
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  		FIG. 2. Accumulation of PrPSc in spleens of terminally ill mice after i.p. challenge with RML prions. (A) Western blot analysis of splenic tissue (80 µg) from CD19–/– and CD22–/– mice and corresponding wild-type controls shows similar levels of proteinase K (PK)-resistant PrPSc at the indicated days postinoculation (dpi) and independent of the dose (LD50) given. Signals for PrP were detected with the polyclonal anti-PrP serum SA2124. –, no PK treatment; +, PK treatment. (B) Histoblot analysis of splenic sections from CD19–/–, CD22–/–, and wild-type (129Sv) mice following i.p. inoculation with a low dose shows similar distributions of PrPSc associated with the FDC networks when treated with PK. No PrP signal was detectable in noninfected tissue of wild-type mice [129Sv(no)].

To search for alterations in the microarchitecture of the lymphoid organs responsible for the shorter incubation time of prion disease in CD19–/– mice, spleen sections were analyzed by immunostaining. Neither the number of T cells stained with either anti-CD4 or anti-CD8 antibodies nor the distribution of B cells stained with either anti-CD45R or peanut agglutinin was altered in comparison to that in the wild-type mice (data not shown). Moreover, the numbers of FDC networks in the spleen assessed with antibodies against FDC-M1 and CD21/CD35 appeared to be similar (data not shown), and morphometric measurements revealed similar numbers of FDC networks in spleen sections of CD19–/–, CD22–/–, and wild-type (129Sv) mice (Fig. 3C).


Figure 3
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  		FIG. 3. Location and distance of FDC networks and nerve fibers in spleens of noninfected mice. (A) Immunofluorescent staining for FDCs with CD21/CD35 (green) and nerve fibers with THL (red) shows that the distance between the FDC network and nerve fibers associated with B-cell follicles in the spleens of CD19–/– mice is reduced compared to that of CD22–/– and wild-type control mice. Scale bar, 100 µm. (B) Quantification of the distance between the FDC networks and adjacent nerve fibers in splenic follicles of three mice for each group is shown as individual dots, and mean values are shown as bars. The mean values were 88 ± 15 µm (n = 25) for CD19–/– mice compared to 185 ± 33 µm (n = 23) for CD22–/– mice and 198 ± 43 µm (n = 20) for wild-type (129Sv) mice. (C) Quantitative analysis of FDC networks in spleens of CD19–/–, CD22–/–, and wild-type (129Sv) mice show no difference in total number of CD21/CD35-positive FDC networks. Data represent the number of FDC networks per mm2 (mean ± standard deviation) in serial sections (n = 7) from three mice of each genotype analyzed.

Surprisingly, the FDC networks within the white pulp of spleens of CD19–/– mice seem to be altered with respect to the distance between the FDC networks and innervating nerve fibers visualized by double labeling against CD21/CD35 and tyrosine hydroxylase (THL), respectively. As shown in Fig. 3A, the distance between the FDC network and the dense plexuses of nerve fibers surrounding the central arterioles in spleen sections from CD19–/– mice was reduced compared to that seen in CD22–/– and wild-type (129Sv) mice. However, the typical localization and organization of FDC networks and nerve fibers within the lymphoid follicles from all three lines are unaltered (Fig. 3A). Moreover, the total amount of THL in the spleens of CD19–/– mice seems to be no higher than that in CD22–/– and wild-type mice, as determined by Western blotting (data not shown), excluding the possibility that the shorter latency of CD19–/– mice to peripheral exposure is due to hyperinnervation of the lymphoid organs by sympathetic nerves, as recently shown (7, 10, 11). In order to measure the altered distance between the FDC networks and nerve fibers in the spleens of CD19–/– mice, a morphometric analysis was performed by measuring this distance in follicles of spleen sections from three uninfected mice of each genotype. A reduction of more than 50% in this distance was found in CD19–/– mice, compared to CD22–/– and wild-type mice (129Sv) (Fig. 3B). These data suggest that the reduced incubation time of disease in CD19–/– mice after peripheral application of prions is most likely related to the reduced distance between the FDC networks and innervating peripheral nerve fibers, which enables prions to be rapidly transported to the peripheral nervous system.

The conclusion reached in this study that closeness of the FDC network and the peripheral nervous system in CD19–/– mice may allow a more efficient migration of prions between the two sites is supported by a recent study showing that positioning of the FDCs within the spleen affects prion neuroinvasion (22). However, the accelerated response of these mice is observed only after peripheral challenge with a low dose of infectivity (22), whereas CD19–/– mice display a shorter incubation time after peripheral infection with either a high or a low dose of RML prions. This implies that additional factors may contribute to the phenotype described here. The activity of CD19 often relies on its association with molecules such as CD21 (4) and others (3). Therefore, the loss of CD19 may disturb B-cell activation and function, which, if it exists, may be mediated through the complement system that has been shown to facilitate neuroinvasion of prions (12, 15). Since CD19 represents a costimulatory molecule of the complement systems, it is possible that compensatory mechanisms are up-regulated in the absence of CD19, thereby facilitating neuroinvasion of prions. However, prion titers in the spleens of prion-infected CD19–/– mice were similar to those found in controls, excluding the possibility that the intrinsic capacity of the spleen to retain and replicate prions is altered in CD19–/– mice (Table 1). FDCs may retain prions indirectly through interactions between C3 and CD21/CD35 or between C1q and C1q receptor, thereby bridging the gap between FDCs and peripheral nerves (16, 19). In line with this idea, the reduced incubation time of CD19–/– mice after peripheral challenge is the consequence of the shorter distance between FDCs and peripheral nerves and the compensatory activity of the complement system due to loss of CD19 on B cells.

Alternatively, significant effects on prion pathogenesis may be expected if aberrant signaling in mice devoid of CD19 leads to modulation of the cytokine response by B cells, as suggested by Matsushita et al. (18). Various cytokines have been shown to have an effect on the development of prion disease (27). Since mice devoid of CD19 developed a Th1 cytokine bias with reduced production of Th2 cytokines such as interleukin-10 (18), it is possible that the accelerated response of CD19–/– mice to peripherally applied prions may due to the loss of anti-inflammatory cytokines, rather than a direct effect of loss of CD19 function. Indeed, certain inflammatory conditions are associated with an increase in the spread of prions within the host (14, 25). PrPSc deposition in these inflamed areas is associated with up-regulation of lymphotoxin and induction of FDCs (9). However, the number of FDC networks in mice devoid of CD19 was not altered (Fig. 3C).

The findings reported here support the notion that the distance between the FDC network and the adjacent peripheral nerves contributes to the neuroinvasion of prions. However, additional mechanisms might be involved in this process, which are still unknown in the understanding of peripheral prion pathogenesis.


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ACKNOWLEDGMENTS
 
We thank Nele Lindner for her excellent technical assistance.

This study was supported by the Bavarian research cooperation for prions (FORPRION), by the German Research Foundation (SFB 479, TP-C9), by the Federal Ministry of Education and Research (BMBF, FKZ: 01KO0516), and by funds from the Biotechnology and Biological Sciences Research Council (BBSRC). E.F. is supported by the Emmy Noether-Programm of the German Research Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Virology and Immunobiology, University of Würzburg, Versbacherstr. 7, D-97078 Würzburg, Germany. Phone: 49-931-201-49164. Fax: 49-931-201-49553. E-mail: klm{at}vim.uni-wuerzburg.de Back

{triangledown} Published ahead of print on 16 January 2008. Back

{dagger} These two senior authors contributed equally to this study. Back


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Journal of Virology, April 2008, p. 3791-3795, Vol. 82, No. 7
0022-538X/08/$08.00+0     doi:10.1128/JVI.02036-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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