Download Characterization of Splenic Cells during the Early Phase of Infection

Jpn. J. Infect. Dis., 64, 256-259, 2011
Short Communication
Characterization of Splenic Cells during the Early Phase of
Infection with Neuropathogenic Mouse Hepatitis Virus
Hiromi Kashiwazaki, Fumihiro Taguchi1, Yuzuru Ikehara2, and Rihito Watanabe*
Department of Bioinformatics, Faculty of Engineering, Soka University, Tokyo 192-8577;
of Veterinary, Nippon Veterinary and Life Science University, Tokyo 180-8602; and
2Research Center for Medical Glycoscience, National Institute of
Advanced Industrial Science and Technology, Ibaraki 305-8568, Japan
1Department
(Received February 7, 2011. Accepted April 12, 2011)
SUMMARY: The highly neuropathogenic cl-2 and less virulent srr7 viruses isolated from the neurotropic JHM strain of the mouse hepatitis virus exhibit super acute spread of virus (SAS), a term
applied when rapid viral spread from an organ or part of the initially infected site to another nonadjacent organ or part is detected within 12 h after infection. Herein, we used a cytospin procedure to
confirm SAS in splenic cells derived from mice whose brains were infected with these viruses. The
cytospin procedure enabled effective preservation of the cells on glass slides. With this method, we
could characterize extremely low populations of infected cells in the spleen (less than 0.1z) at 12 h postinoculation with srr7. We observed that all kinds of splenic cells examined were infected, including
B220＋Ly-6C＋ plasmacytoid dendritic cells. The population of viral antigen-positive splenic cells was
only slightly higher in cl-2 infection than in srr7 infection, but the cells showing viral production were
present in numbers significantly higher in cl-2 infection compared with srr7 infection.
The soluble receptor-resistant (srr) mutant 7 (srr7) of
the highly neuropathogenic JHM strain, cl-2 exhibits
slightly lesser virulence than its maternal virus (1).
However, srr7 still is highly virulence, causing the death
of infected mice within 10 days after infection (2).
Histopathologically, initial viral antigens can already be
detected at 12 h post-infection (hpi) either with cl-2 or
srr7 in infiltrating cells that appear in the subarachnoidal space and spleen at the same time, although
viruses were used to inoculate the brain and the viral
titers in the bloodstream remained below the detectable
level during the early phase of infection. We designated
the manner of viral spread observed in these viral infections as super acute spread of virus (SAS) (2). In addition, our recent study using paraffin-embedded and
frozen sections obtained during the initial phase, i.e.,
12–48 hpi with cl-2 and srr7 showed that the two viruses
did not differ in terms of the population of infected
cells, which appeared in the meninges as inflammatory
cells and in the spleen at 12 hpi (2). In that study,
however, we failed to characterize the infected cells, except for those of the F4/80- or CD11b-positive
monocyte/macrophage cell lineages. The technical limitation to characterize the infected cells in vivo was a
problem encountered both in our previous studies (1,2)
and other studies investigating the properties of mouse
cells infected with neuropathogenic mouse hepatitis virus (MHV); this may be partially attributed the down
regulation of the expression of marker proteins in the
infected cells (3). The problems of using paraffin-em-
bedded or frozen materials were overcome to some extent using cell cultures (4,5) or ex vivo experimental
procedures (6).
In this study, we employed a cytospin procedure to
collect and mount leukocytes on glass slides and thereby
achieved improved preservation of the antigenicities of
cell marker proteins and cell architecture and verified
this by immunofluorescent studies of infected cells.
For infection, srr7 and cl-2 viruses were propagated
and titrated using DBT cells maintained in Dulbecco's
modified Eagle's minimal essential medium (DMEM)
(Nissui, Tokyo, Japan) supplemented with 5z fetal bovine serum (FBS) (Japan Bioserum, Fukuyama, Japan),
as described previously (7). Under deep anesthesia, 102
plaque-forming unit (PFU) of these viruses were used to
inoculate the right frontal lobe of specific-pathogen-free
(SPF) inbred BALB/c mice purchased from Charles
River (Tokyo, Japan). The mice were maintained according to the guidelines set by the committee of our
university. Before infection, the mice were kept in the
SPF facility at Soka University. The approval of the
committee was obtained, and 7–16-week-old mice were
infected and maintained at our P3-level laboratory.
For the cytospin procedure, the spleen cells were suspended in PBS (Nissui) containing 0.02z ethylenediaminetetraacetic acid tetrasodium salt dihydrate and
0.5z bovine serum albumin (both from Sigma, Tokyo,
Japan). The cytospin technique was used to maximize
the yield of cells from the spleen. For this purpose, 0.1
ml of the fluid sample containing 1 × 106 cells was
added to the chamber of a silane-coated glass slide
(Muto, Tokyo, Japan), and the slide was secured with a
metal clip. Filter cards, sample chambers, and metal
clips were obtained from SHANDON (Pittsburgh, Pa.,
USA). After spinning at 2,000 rpm for 4 min, the slides
were removed from the cytospin chamber, fixed in
*Corresponding author: Mailing address: Department of
Bioinformatics, Faculty of Engineering, Soka University,
1-236 Tangi-chou, Hachioji, Tokyo 192-8577, Japan. Tel
& Fax: ＋81-426-9465, E-mail: rihitow＠soka.ac.jp
256
Fig. 1. The spleen cells obtained from one mouse were studied, and representative data after independent experiments carried out at least two times are shown in (A)–(C). The differences among each experiment did not exceed
the SD variation shown in each figure (data not shown). (A) Infected cell population of splenic cells detected by
immunofluorescence-based counting on glass slides after the cytospin procedure. Viral antigen-positive (V＋) cell
numbers were counted while viewing whole areas mounted on glass slides under a fluorescence microscope (Zeiss,
Frederick, Md., USA), and the total number of splenic cells on each glass slide was estimated from the cell number
counted in 20 randomly selected fields. Data represent three independent experiments with one mouse. Data are
the mean ± SEM (*P º 0.05, t test). (B) Populations of V＋ and cell marker-positive cells among V＋ cells calculated after double immunofluorescent staining for each cell marker indicated in the figure and for viral antigens in
the splenic cells collected and mounted on glass slides for the cytospin procedure. Averaged percentages and SDs
were obtained by counting fluorescence-positive cells in photographs taken from 20 randomly selected fields at
×40 in each glass slide prepared from the spleen from one mouse. The number of total cells per field ranged from
66 to 635 in 660 fields, whereas that of V＋ cells ranged from 1 to 9 per field in 560 fields. (C) The number of splenic cells producing infectious viral particles was estimated by counting plaques produced after co-culture with DBT
cells for 24 h (white bars). For the detection of virus-producing leukocytes in the spleen, 1 × 104–1 × 107, 4 ×
107, or 1 × 108 spleen cells were overlaid on semi-confluent DBT cells seeded in 24- or 6-well plates or a 10-cm
petri dish. After 24-h incubation, the number of clear plaques in DBT cells was counted. By logarithmic dilution
of the spleen cells before adding to the DBT cell culture, the endpoint of a cell population in which no plaque could
be detected in DBT cells was determined (black bars). (a), 4 × 107 spleen cells did not produce any plaque. Data
are the mean ± SEM (*P º 0.01, t test). The number of mice used in each group is shown. (D) Spleen weight
(white bars) and splenic cell populations (black bars). Numbers in parenthesis indicate the numbers of mice used.
Data are the mean ± SEM (*P º 0.005, t test).
cells were infected with srr7 and cl-2 viruses at 12 hpi,
respectively (Fig. 1A). SAS was further investigated by
the co-culture of splenic cells recovered from infected
mice with MHV-susceptible DBT cells (Fig. 1C).
Around 1 and 50 plaques per 1 × 108 splenic cells were
detected at 12 hpi with srr7 and cl-2 viruses, respectively. The infectivity (Fig. 1A) and viral productivity
(Fig. 1C) of cl-2 were higher than those of srr7, at 12
hpi (Fig. 1A and C; t values on Student's test were P º
0.05 and 0.01, respectively). A 24 hpi, the viral produc-
100z alcohol for 2 min and in acetone for 5 min
(Wako, Tokyo, Japan), and then stored at －359C until
use.
The cytospin procedure was followed by the detection
of SAS by means of immunostaining for splenic cells
obtained from mice at 12 hpi with cl-2 or srr7 viruses
(Fig. 1A), when viral antigens are not detectable in the
brain parenchyma, including the right frontal lobe (2),
which is the site of inoculation. Immunostaining revealed that less than 0.1 and 0.3z of the total splenic
257
tivity of srr7 in the spleen had increased more than 10
times, but did not catch up with that of cl-2 (Fig. 1C);
this finding is consistent those indicating the more effective viral spread and production of cl-2 than srr7 in the
brain (1,2). Therefore, SAS does not determine the
difference in virulence between srr7 and cl-2, which
could be attributed mainly to the manner of cell adhesion molecule 1 (CEACAM1)-, a major receptor of
mouse hepatitis virus (MHVR), dependent or independent infection of srr7 and cl-2, respectively (8), in addition to a difference in viral production rates in vivo after
infection with these viruses (1). Furthermore, we do not
consider that SAS is a cause of the virulence. In fact, the
SAS phenomenon may be the result of the failure in exerting innate immunity againt these viruses by responsible immunocompetent cells, such as monocytes, conventional dendritic cells (cDCs), and plasmacytoid dendritic cells (pDCs), as indicated by cytopathy observed
in the infected F4/80-positive cells (2) and the allowance
of infection in these cells, possibly with virus production
leading to carrying infectious viral particles to the spleen
in a short period, as shown and discussed in the following paragraphs. However, both viruses cause the death
of infected mice before acquired immunity becomes apparent, which contributes to neuropathological features
induced by less virulent neuropathogenic MHV (9,10)
compared to our viruses.
The use of the cytospin procedure provides less accurate quantitative analysis of the infected cell population than other methods, as indicated in Fig. 1B, which
shows a large variation in the number of infected cells in
each field of microscopic examination. However, this
method enables the characterization of infected cells,
which is difficult using frozen or paraffin-embedded
sections (2). As shown in Fig. 1B, all types of cell populations investigated with double-colored immunofluorescence for viral antigens and specific cell marker
proteins of immunocompetent cells, such as F4/80 for
macrophages, CD20 for B cells (Fig. 2B2), and TCR for
T cells (Fig. 2B3), were verified to be infected at all time
points between 12 and 48 hpi. In addition, CD11c-positive cells, especially highly marked and large-sized cells
(Fig. 2B1), i.e., cDCs, were verified to be infected. The
targets of infection included Gr-1＋ granulocytes (Fig.
1B) and B220－Ly-6C＋ polynuclear cells (Fig. 2C).
Many B220＋ cells were also infected (Fig. 1B); many of
Fig. 2. (A1–3) HE staining of the spleen infected with mock or srr7 at 12 or 48 hpi, as indicated in the pictures. The
arrowhead and arrow in the inserted picture in A3 indicate fragmented and pycnotic nuclei, respectively. Black
and white bars indicate 250 and 25 mm, respectively. (B–D) Immunofluorescence with double (B) and triple staining (C) and (D) was performed using antibodies against CD11c (AnaSpec, San Jose, Calif., USA), CD20 (Santa
Cruz Biotechnology, Santa Cruz, Calif., USA), TCR (Biolegend, San Diego, Calif., USA), B220 (BD Pharmingen, Franklin Lakes, N.J., USA), Ly-6C (indicated as Ly6C, BMA Biomedicals, Augst, Switzerland), or viral antigens (indicated as J) in splenic cells after the cytospin procedure prepared from mice infected with srr7 or cl-2 at
12 or 24 hpi, as indicated in the pictures. Three pseudo-colors, red, green, and blue, indicated as /r, /g, and /b in
the pictures, respectively, were used. Arrows in B1–3 show double-stained cells. Arrows in (C) and (D) show polynuclear cells. Arrowheads and arrows in (D) show large B220＋Ly-6C＋V＋ cells with a typical pDC morphology,
and an atypical pDC, respectively. The cells in dotted areas of D show B220＋Ly-6C－ B cells. Triple fluorescence
labeling was analyzed with a Keyence fluorescence microscope (Keyence Corp., Osaka, Japan). White bars indicate 25 mm.
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them were B cells and the others were pDCs, which bear
the Ly-6C antigen as well (Fig. 2D). Many of the
B220＋Ly-6C＋ and viral antigen-positive (V＋) cells
(arrowheads in Fig. 2D) were larger than the
B220＋Ly-6C－ B cell population (dotted areas in Fig.
2D) and resembled typical pDCs in shape, with a shifted
localization of the nucleus, similar to plasma cells.
Alternatively, some B220＋Ly-6C＋V＋ pDCs (arrows
in Fig. 2D) with a cellular size similar to that of
B220＋Ly-6C－ B cells exhibited the nuclear architecture
of polynuclear cells, which is distinct from the typical
morphological picture of pDCs initially reported (11). A
recent report indicated that standard populations of
pDCs isolated through the use of monoclonal antibodies
contain a subtype, interferon-producing killer dendritic
cells, with distinct functions (12). The cytospin procedure can be an efficient technique for detecting subpopulations of cells by facilitating the cell architecture
among the populations ascertained by cell-marker proteins, especially in the case of small populations, such as
cells infected during the initial phase of infection.
In order to identify the effects of viral infection on
the leukocyte population in the spleen, we measured the
splenic weight and counted the total cell number after
the lysis of the splenic red blood cells of the infected
mice. Although the splenic weight did not change, the
leukocyte population reduced in accordance with the development of infection (Fig. 1D). Histopathologic examination of the spleen showed that the reduction of the
cell population in the later phase of infection was
mainly due to the loss of leukocytes in the subcapsular
and interfollicular areas (Fig. 2A). The interfollicular
area, with a reduced leukocyte population at 48 hpi, was
occupied by red blood cells (Fig. 2A3), which might account for the unchanged splenic weight (Fig. 1C).
Although apparent necrotic areas were not observed in
the infected spleen, shrunken cells with pycnotic or fragmented nuclei were encountered (inserted picture in Fig.
2A3). Because of the very limited population of infected
cells and only a small population of degenerative cells in
the spleen during the early phase of infection, the reduction in the cell number may be due to a disturbance of
leukocyte homing or maintenance in the spleen, since
conduit systems in the spleen are destroyed after viral
infection (13,14).
Among the cells infiltraing the subarachnoidal space,
syncytial giant cells (SGCs) are observed during the early phase of infection (2). These SGCs express F4/80 or
CD11b antigens and viral antigens, indicating that the
viruses exert cytopathic effects on infected cells of the
monocyte lineage, which play an important role in innate immunity. However, SGCs were not detected in the
splenic cells examined using the cytospin procedure
(data not shown) or the paraffin-embedded sections,
although the cells exhibited pycnotic or fragmented
nuclei (insertion in Fig. 2A3), indicating that SGCs were
destroyed immediately in the spleen after formation at a
higher concentration of cytokines rather than in the
subarachnoidal space, or a high density of infected cells
in a narrow space, like the subarachnoidal space, might
be necessary to form SGCs. At 12 hpi, infected cells in
the spleens appear at an extremely low density in the
marginal zone and gradually scatter into the red pulp
and follicular areas (2). This pattern of spread in the
spleen and the existence of viral particle-producing cells
in the spleen at 12 hpi indicate that the cells initially infected in the subarachnoidal space reach the spleen via
the blood stream at 12 hpi, when viral titers are undetectable levels in the peripheral blood (2). This may be
because many infected cells are observed at the same
time in the subarachnoidal space, although there is a
possibility that a few viral particles leak into the blood
stream and reach the spleen at the time of infection.
Acknowledgments This work was financially supported in part by
grants from the Ministry of Education, Culture, Sports, Science and
Technology.
Conflict of interest None to declare.
REFERENCES
1. Matsuyama, S., Watanabe, R. and Taguchi, F. (2001): Neurovirulence in mice of soluble receptor-resistant (srr) mutants of
mouse hepatitis virus: intensive apoptosis caused by less virulent
srr mutant. Arch. Virol., 146, 1643–1654.
2. Takatsuki, H., Taguchi, F., Nomura, R., et al. (2010): Cytopathy
of an infiltrating monocyte lineage during the early phase of infection with murinecoronavirus in the brain. Neuropathology, 30,
361–371.
3. Kyuwa, S., Cohen, M., Nelson, G., et al. (1994): Modulation of
cellular macromolecular synthesis by coronavirus: implication for
pathogenesis. J. Virol., 68, 6815–6819.
4. Godfraind, C., Langreth, S.G., Cardellichio, C.B., et al. (1995):
Tissue and cellular distribution of an adhesion molecule in the
carcinoembryonic antigen family that serves as a receptor for
mouse hepatitis virus. Lab. Invest., 73, 615–627.
5. Nakagaki, K., Nakagaki, K. and Taguchi, F. (2005): Receptor-independent spread of a highly neurotropic murine coronavirus
JHMV strain from initially infected microglial cells in mixed neural cultures. J. Virol., 79, 6102–6110.
6. Zhou, H. and Perlman, S. (2006): Preferential infection of mature dendritic cells by the JHM strain of mouse hepatitis virus.
Adv. Exp. Med. Biol., 581, 411–414.
7. Taguchi, F., Yamada, A. and Fujiwara, K. (1980): Resistance to
highly virulent mouse hepatitis virus acquired by mice after lowvirulence infection: enhanced antiviral activity of macrophages.
Infect. Immun., 29, 42–49.
8. Watanabe, R. and Takase-Yoden, S. (2006): Neuropathology induced by infection with Friend murine leukemia viral clone A8-V
depends upon the level of viral antigen expression. Neuropathology, 26, 188–195.
9. Lavi, E., Suzumura, A., Hirayama, M., et al. (1987): Coronavirus mouse hepatitis virus (MHV)-A59 causes a persistent, productive infection in primary glial cell cultures. Microb. Pathog., 3,
79–86.
10. Anghelina, D., Pewe, L. and Perlman, S. (2006): Pathogenic role
for virus-specific CD4 T cells in mice with coronavirus-induced
acute encephalitis. Am. J. Pathol. 169, 209–222.
11. Grouard, G., Rissoan, M.C., Filgueira, L., et al. (1997): The
enigmatic plasmacytoid T cells develop into dendritic cells with
interleukin (IL)-3 and CD40-ligand. J. Exp. Med., 185,
1101–1111.
12. Vremec, D., O'Keeffe, M., Hochrein, H., et al. (2007): Production of interferons by dendritic cells, plasmacytoid cells, natural
killer cells, and interferon-producing killer dendritic cells. Blood,
109, 1165–1173.
13. Dauner, J.G., Williams, I.R. and Jacob, J. (2008): Differential
microenvironment localization of effector and memory CD8 T
cells. J. Immunol., 180, 291–299.
14. Hsu, K.M., Pratt, J.R., Akers, W.J. et al. (2009): Murine
cytomegalovirus displays selective infection of cells within hours
after systemic administration. J. Gen. Virol., 90, 33–43.
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