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*
Department of Microbiology, University of Western Australia and the Western Australian Institute for Medical Research, Nedlands, Western Australia, Australia; and
Division of Virology, Animal Health Trust, Kentford, Suffolk, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have highlighted the importance of NK cells as early
mediators of host defense against viral infections (reviewed in Ref.
3), including acute infection with MCMV (4).
Once activated, NK cells kill infected targets via effector mechanisms
that vary at different physiological sites. In the spleen NK cells
predominantly regulate MCMV replication via perforin-dependent
cytolysis, whereas in the liver their effector function is mediated
mainly via the production of IFN-
(5). A subset of
NK1.1+ cells has been shown to limit the
replication of MCMV in the spleen and, to a lesser extent, in the liver
via operation of the Cmv1 locus (6).
Cmv1 is an autosomal dominant locus first identified in mice
of the MCMV-resistant C57BL/6J background, but not on the
MCMV-susceptible BALB/c background, hence leading to the designation of
these strains as Cmv1r and
Cmv1s, respectively (7). Mice
congenic to the BALB/c background have been produced to have C57BL/6J
alleles for varying regions of the NK gene complex on mouse chromosome
6 (8). This complex encompasses the Cmv1 locus
and the Ly55c gene that encodes NK1.1. These strains include
the BALB.B6-Cmv1r strain, which has the
NK1.1+, Cmv1r phenotype,
and the BALB.B6-CT6 strain, which is NK1.1+,
Cmv1s. These strains are particularly
useful to study NK cell function during virus infection because of the
NK1.1+ phenotype on both susceptible and
resistant backgrounds.
In contrast to cells such as monocytes and neutrophils, it has been
difficult to study the role of NK cells in situ due to the inability to
use a specific marker for immunohistologic identification. The NK1.1 Ag
is the most widely used murine pan NK marker. PK136, a mAb against
NK1.1, was first described by Koo and Peppard in 1984 (9),
but numerous attempts to detect NK1.1 in situ with this reagent have
been unsuccessful. Several studies have identified NK cells on the
basis of asialo-GM1, IFN-, or perforin expression
(10, 11, 12); however, these markers are not specific for NK
cells and can be detected on several other cell types, including
macrophages and activated T cells. In situ histological analysis of NK
cells therefore has been difficult, as it relies on subtractive
analysis.
In this report, we describe the first demonstration of anti-NK1.1 staining in situ to identify NK cells using a modified in vivo perfusion/fixation method. Using this method, we have compared the acute NK1.1+ cellular response to wild-type MCMV infection in the visceral organs of genetically susceptible intra-NK complex recombinant BALB.B6-CT6 mice with that in resistant C57BL/6J and BALB.B6-Cmv1r mice (all NK1.1+). Expression of viral Ags and the consequences of infection on other cellular subsets have also been analyzed in this study. In susceptible mice the results demonstrate that MCMV infection in the marginal zones of splenic white pulp results in local changes in various cellular constituents, including macrophages and NK cells. In the liver distinct foci of infection were comprised of large numbers of macrophages and NK1.1+ cells surrounding infected cytomegalic cells. In resistant mice, MCMV infection was predominantly in the red pulp of the spleen and was associated with increased NK1.1+ cell and macrophage accumulation at sites of viral infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred, specific pathogen-free C57BL/6J and BALB/c were obtained from the Animal Resources Center (Perth, Australia). The congenic mouse strains BALB.B6-CT6 and BALB.B6-Cmv1r, described previously (8), were bred in-house. All animal experimentation was performed with the approval of the animal ethics and experimentation committee of the University of Western Australia and according to the guidelines of the National Health and Medical Research Council of Australia.
Cells
Primary mouse embryo fibroblasts were grown in MEM (Life Technologies, Grand Island, NY) supplemented with 10% newborn calf serum (Life Technologies). Single-cell suspensions of mouse splenocytes were prepared in RPMI 1640 (Life Technologies) supplemented with 10% FCS (Tissue Culture Grade; CSL, Melbourne, Australia), L-glutamine (10 mM), 2-ME (20 µM), and antibiotics (100 µg/ml penicillin, CSL; 40 µg/ml gentamicin, Pharmacia & Upjohn, Sydney, Australia). To isolate splenocytes, spleens were dissociated in Dounce homogenizers (Kontes, Vineland, NJ), and the dispersed cells were washed once in RPMI 1640 complete medium. Erythrocytes were lysed in NH4Cl, and the nucleated cells were washed an additional two times in complete medium to remove residual lysis buffer and undispersed stromal elements. Splenocytes were suspended in mouse osmolarity-buffered saline (MOBS; 330 mosmol)/5% FCS for analysis by flow cytometry.
Antibodies
PE-conjugated anti-NK1.1 (PK136), CD11b (M1/70), CD4
(GK1.5), CD8 (53-6.72), and CD19 (1D3); FITC-conjugated anti-TCR
(H57-596); biotin-conjugated anti-NK1.1 (PK136), biotin-conjugated
anti-CD11c (HL3), and streptavidin-PE were purchased from
PharMingen (San Diego, CA). Anti-asialo-GM1 was purchased from Wako
Pure Chemicals (Osaka, Japan). The anti-MCMV mAb 3B2 has been
described previously (13).
Treatment of mice
Groups of mice used for immunofluorescence analysis of tissue sections (two per group), FACS analysis (three per group), or plaque assays (three per group) were infected i.p. with 104 PFU of salivary gland-propagated stocks of the virulent MCMV strain K181-Perth. Control animals were mock infected with MOBS/5% FCS. Mice were sacrificed at 1, 2, 3, 4, and 6 days postinfection (p.i.), and tissue was collected as described below. Viral titers are expressed as the means from three infected animals.
Removal of tissues and fixation protocols
For in vitro fixation of tissues, mice were euthanized by cervical dislocation, and tissues were covered with Cryo-embed OCT (Tissue-Tek, Torrance, CA), snap frozen in liquid nitrogen, and stored at -80°C. For fixation of liver and spleen in situ, animals were anesthetized with Penthrane and perfused with 20 ml of the appropriate fixative via the left ventricle. Following perfusion/exsanguination, the mice were left at room temperature for 5 min to ensure appropriate penetration of tissues by the fixative. Following systemic perfusion, lungs were fixed by intratracheal injection of 10 ml of fixative. To ensure that lung architecture was maintained, perfusion was performed at a rate of 2 ml/min. Tissues were then removed and stored at -80°C.
Tissue sections taken from unperfused animals were treated as described
in Table I
. In vivo fixation was
performed using either 4% paraformaldehyde (general purpose grade 1;
BDH, Kilsyth, Australia) or the periodate-lysine-paraformaldehyde (PLP)
solution adapted from McLean and Nakane (14).
Paraformaldehyde was prepared as a 4% solution in MOBS and buffered to
pH 7.4 before use. The PLP solution was prepared with the following
modifications. Briefly, 0.15 M lysine-HCl was buffered to pH 7.4 with
0.1 M Na2HPO4 and
subsequently diluted to 0.075 M lysine-HCl with 0.1 M
Na2HPO4. A 2%
paraformaldehyde solution was prepared fresh in deionized
H2O and adjusted to pH 7.4. One part of the
resulting solution was added to three parts of lysine phosphate buffer
supplemented with 2.5 mg/L sodium periodate and 600 µl/L
glutaraldehyde. All solutions were stored at room temperature and used
within 12 h of preparation.
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Sections (6 µm) sections were cut on a cryostat and mounted
onto Silane (ICN Pharmaceuticals, Aurora, OH)-coated slides. Untreated
tissues were fixed as described in Table I
before rehydration in MOBS
for 5 min. Fixed tissues were rehydrated, and after this step both
types of tissue were treated identically. Paraformaldehyde-associated
autofluorescence was quenched by incubation in 50 mM
NH4Cl for 10 min at 4°C before blocking with
5% normal goat serum (NGS) in MOBS containing 3% BSA for 30 min at
room temperature. Primary Abs diluted in MOBS with 3% BSA were then
added for 1 h at 4°C followed by the appropriate second-step
reagent for 30 min at room temperature. Secondary Ab reagents were
diluted in MOBS with 5% NGS and streptavidin-conjugated fluorochromes
were diluted in 5% skim milk powder. Washing between each step was
performed three times with MOBS. Sections were mounted in 50%
glycerol/MOBS and visualized by either standard epifluorescence or on a
confocal microscope (MRC 1000/1024 UV laser scanning confocal
microscope; Bio-Rad, Hemmel, U.K.). A focal series of seven horizontal
planes of section spaced by 0.5 µm was monitored sequentially for
FITC and PE using the 488- and 543-nm laser lines of an argon or a
green helium-neon laser, respectively, a double-dichroic mirror for the
excitation beam, an FITC band-pass 522/35-nm barrier filter, and a
580/32-nm filter for PE. The FITC/PE eight-bit-encoded 768- x
512-pixel images from the same plane of section were superimposed and
visualized with a red/green pseudo color scale on a true color display
monitor before printing using a Codonics NP-1600 printer (Codonics,
Middleburg Heights, OH).
Flow cytometry
Flow cytometric analysis of splenocytes was performed using the mAbs described above. Cells were resuspended at 1 x 106/ml, and nonspecific reactivity was blocked in MOBS/5% FCS/5% NGS for 30 min on ice. Labeling was performed for 60 min with the appropriate primary mAb, then with the second-step reagent for 30 min. All incubations were performed at 4°C, with three washes in MOBS/5% FCS between steps. Propidium iodide (2 µg/ml) was incorporated into the final wash to exclude dead cells. The labeled cells were then analyzed on a FACScalibur (Becton Dickinson, San Jose, CA), and files of 10,000 events were collected and analyzed using CellQuest software (Becton Dickinson).
In vivo growth of MCMV
Mice were inoculated with the virulent MCMV strain K181-Perth as described above. At the designated times postinoculation, the animals were sacrificed, and spleens and livers were removed. All organs were individually weighed, homogenized in cold MEM/2% newborn calf serum, and centrifuged at 1800 x g for 15 min at 4°C. The supernatant was stored at -80°C, and viral titers were subsequently quantified on mouse embryo fibroblasts using a standard plaque assay (15).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ identification of NK cells using the NK1.1 marker
recognized by mAb PK136 has been elusive to date. To determine whether
the inability to detect NK1.1 may be due to inappropriate fixation of
this Ag, seven different fixation protocols were applied to cryostat
sections of mouse spleen in an attempt to establish a reliable
technique that allows in situ identification of cells expressing the
NK1.1 Ag (Table II). Visualization of the
NK1.1 marker was only possible after perfusion fixation before
snap-freezing and cryostat sectioning. PLP fixation showed superior
results to fixation with 4% paraformaldehyde (Table II), while
fixation ex vivo resulted in no visible staining with PK136. Detection
of other Ags, such as CD11b, was not dependent on the fixation protocol
(Table II). Tissue architecture and Ag preservation were maintained
optimally when fixation was performed for a minimum of 5 min
postexsanguination.
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along with the strain susceptibility
to MCMV infection and the Cmv1 phenotype (8).
In all cases, fluorescent signal was only detected in
NK1.1+ mouse strains. These data were
complemented by dual labeling with anti-asialo-GM1 or TCR and
confocal analysis (Fig. 1). Fig. 1A shows asialo-GM1+ cells that are
NK1.1-, but no NK1.1+
cells that are asialo-GM1-. The ratio of
asialo-GM1+/NK1.1+ to
asialo-GM1+/NK1.1- was
5:1. Fig. 1B shows that NK1.1+
cells can be distinguished from TCR+ and
NK1.1+/TCR+ (NKT) cells.
The distribution and localization of CD11b+ and
NK1.1+ cells identified following MCMV infection
are described below.
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Having devised a method for the specific detection of NK1.1+ cells in situ, immunohistologic studies were undertaken to determine the roles of these and other cell types to the response that follows MCMV infection.
In the spleen.
Titers of MCMV in the spleens of susceptible mice were detectable by
plaque assay on day 2 p.i. and peaked on day 4 (BALB/c day 2,
2.4 x 103 PFU; day 4, 2.1 x
105; day 6, 4.5 x
104; BALB.B6-CT6, see Table IV) with MCMV Ags first detected by
immunofluorescence in the splenic margins 2 days p.i. In susceptible
BALB/c and BALB.B6-CT6 mice, a positive fluorescent signal to an MCMV
intracellular Ag detected by mAb 3B2 was observed in the splenic
margins by day 2 postinfection and became prominent in these areas 3–4
days p.i. (Fig. 2A). By day 6,
the infection appeared to have been resolved, with low levels of
staining for MCMV Ag evident only in the red pulp (Fig. 3A). The temporal appearance
and intensity of staining of MCMV Ag were consistent with the titers of
MCMV detected in the spleen by plaque assay (Table IV).
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B). Thereafter,
NK1.1+ cells were only identified in the red
pulp, where by day 6 p.i. the foci of NK1.1+
cells had markedly increased (Fig. 3B). Similar to
NK1.1+ cells, CD11b+
macrophages were found in the red pulp by 18 h p.i., but were more
detectable in the marginal zones of the white pulp 1 day p.i. (Figs. 2B and 3C). Accumulation of these cells in
discrete foci was noted in both the red and white pulp regions on days
3 and 4 p.i., with an increase in cell numbers in these foci by
day 6 (Table IV and Fig. 3C). Alterations to the number or
distribution of CD4+ and
CD19+ cells was unremarkable during this study.
The number of CD8+ cells, especially
CD11b+/CD8+ double-positive
cells, increased on day 6 postinfection, particularly in susceptible
strains (data not shown).
In contrast to the findings in susceptible mice, MCMV Ag was detected
at very low levels in the marginal zones of splenic follicles of
resistant C57BL/6J and BALB.B6-Cmv1r mice (Fig. 3D). This correlated with the significantly lower titers of
MCMV recovered from the spleens of resistant mice at the times and
virus dose used in study (Table IV). Some MCMV-infected cells were also
detected in the red pulp. At 6 days p.i. MCMV Ags were no longer
detected by in situ analysis (Fig. 3D). The low levels of
virus detected correlated with a significant increase in
NK1.1+ cell numbers from day 2 onward (Table IV
and Fig. 3E). In situ analysis showed accumulation of
NK1.1+ cells into foci of the red pulp and in the
margins of the white pulp as early as day 2 p.i. In resistant
strains, NK1.1+ cells were present in
significantly greater numbers from days 2 to 4 p.i. compared with
the susceptible BALB.B6-CT6 mice, consistent with the number of
NK1.1+ splenocytes detected by FACS analysis.
Clusters of CD11b+ macrophages were observed at 2
days p.i. and reached significantly higher levels in the spleen than
those observed for susceptible mice at days 2–4 p.i. (Table IV and
Fig. 3F).
In the liver.
Previous studies have looked at the presence of NK cells in the livers
of MCMV-infected animals. However, the analysis was somewhat
complicated by the use of asialo-GM1 as the marker for NK cells given
the cross-reactivity with other cell types (10, 11, 12, 16, 17). In addition, the analysis was restricted to the
MCMV-resistant C57BL/6J strain. Here, we specifically evaluate the role
of NK1.1+ cells in MCMV-induced liver
inflammation and compare the composition of the inflammatory infiltrate
in NK1.1+-resistant and susceptible mouse
strains. Viral titers in the liver of susceptible (BALB/c;
BALB.B6-CT6) mice were highest at 2 (BALB/c, 4.6 x
104 PFU; BALB.B6-CT6, 2.0 x
104 PFU) and 4 (BALB/c, 6.3 x
103 PFU; BALB.B6-CT6, 4.5 x
104 PFU) days p..i. before decreasing by day
6 (BALB/c, 4 x 103 PFU; BALB.B6-CT6,
1.8 x 103 PFU). Although equivalent titers
were observed in resistant mice (C57BL/6J and
BALB.B6-Cmv1r) at days 2 and 4 p.i.
(2 x 104 and 3 x
103 PFU), infectious virus was undetectable by
day 6 p.i. In susceptible and resistant strains of mice, MCMV Ag
was first identified at 2 days p.i. in numerous foci throughout the
liver parenchyma, with infected cells showing classic signs of
cytomegaly. The foci of infection were frequently associated with NK
cells at this time (Fig. 4). By 4 days
p.i. these cells were surrounded by foci of inflammation consisting of
large numbers of macrophages (CD11b+) and NK
(NK1.1+) cells. Moderate numbers of
CD4+ T cells were observed in tissues from
Cmv1s strains.
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103 PFU at 6 days p.i.
Immunofluorescence analysis with MCMV mAb showed low levels of viral
Ags in the lungs of susceptible mice at 4 days p.i. This finding was
associated with preferential accumulation of macrophages
(CD11b+) and dendritic cells
(CD11c+) at the sites of infection. However, the
clusters of inflammatory cells associated with infection were small
compared with those seen in spleen and liver. By 6 days p.i. a low
level of virus infection was still detectable, and MCMV-positive cells
were found in association with an infiltration of NK
(NK1.1+) cells (Fig. 5). No evidence of infection with MCMV
was identified in the lungs of resistant mice at the virus dose used in
this study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Double labeling with PK136 and anti-asialo-GM1 (Fig. 1A), shows that all NK1.1 cells are asialo-GM1 positive, but
the reverse does not hold true. This is expected, since NK1.1 is
thought to be specific for NK cells, whereas asialo-GM1 has been shown
to be expressed on NK cells, activated T cells, and macrophages
(10, 11, 12, 16, 17). We have also shown that it is possible
to separate NK cells from NKT cells on the basis of TCR expression
(Fig. 1B).
In addition to describing the first specific identification of NK cells
in situ we report on the inflammatory response that follows infection
with MCMV and compare it in spleen, liver, and lungs of susceptible and
resistant mouse strains. The extent of virus Ag detected by
immunofluorescence correlated with titers of infectious virus recovered
during acute infection. In the spleen sequential section analysis
indicated that MCMV infection was occurring primarily in cells that
were CD11b+ (Fig. 2), consistent with previous
studies that have detected MCMV infection in mononuclear cells of
peripheral tissues (18, 19, 20). Given the rapid accumulation
of CD11b+ cells to the spleen, liver, and lungs,
this population may represent critical players in the early
dissemination of MCMV to peripheral tissues and/or the establishment of
latency. In the spleen monocytes are known to selectively migrate into
the marginal zones (21) in a pattern consistent with that
observed in this study. An increase in the number of
CD11b+/CD8+ cells was noted
at day 6 p.i. McFarland and colleagues have previously shown that
a subset of freshly isolated T cells taken after lymphocytic
choriomeningitis virus infection is also CD11b+
(22). In that study these cells were recognized as the
active effector and virus-specific memory CTLs. Our study focuses on
the acute phase of MCMV infection; hence, the observed
CD11b+/CD8+ cells probably
represent the active stage of CTL proliferation.
In the spleen, both FACS and in situ analysis demonstrated an earlier
increase in NK1.1+ cells in splenocytes from
resistant, compared with susceptible, mice. Nevertheless,
both susceptible and resistant mice showed high levels of
NK1.1+ cells 6 days p.i. (Table IV). At this time
postinfection, the increase in NK1.1+ cells is
partly due to NKT cells. The role of NKT cells in the response of
susceptible and resistant strains of mice to infection with both
wild-type and mutant MCMV (lacking specific open reading frames) has
been analyzed and will be reported separately (E. H. Densley et
al., manuscript in preparation).
In the livers of Cmv1s and
Cmv1r mice, MCMV was first detected in situ at 2
days p.i. Peak titers of virus were equivalent in these strains at day
4 p.i, which was expected, since Cmv1 confers NK
cell-mediated resistance to MCMV infection predominantly in the spleen
and to a lesser extent in the liver (7). In both strains
of mice, foci of infection were associated with prominent infiltration
of NK cells and macrophages by 4 days p.i. (Fig. 4). Dual labeling of
infected livers at days 4–6 p.i. indicated that most
CD8+ cells were also
CD11b+, probably representing activated CTLs.
Although CD4+ T cells were observed in the livers
of susceptible mice, these cells were absent in livers from infected
resistant mice, consistent with previous data demonstrating that T cell
responses are not required for the control of virus replication in the
livers of resistant C57BL/6J mice (23). The absence of T
cells in the livers of resistant mice may be a consequence of the early
control of MCMV by NK cells in the spleen.
Lastly, we studied the inflammatory response to MCMV infection in the lung. Compared with those in spleen and liver, MCMV titers in the lung are lower and are detected later following i.p. infection. Nevertheless, accumulating CD11b+ macrophages, CD11c+ dendritic cells, and NK1.1+ cells were observed surrounding infected cells. Despite the appearance of NK cells, it is unclear whether these cells play a role in restricting early MCMV replication in the lung. Indeed, NK cells are important in preventing the dissemination of MCMV into the lung, but do not appear to play a crucial role with respect to viral clearance at this site (24).
The accumulation of macrophages observed in the lung postinfection with MCMV may be important for the establishment of viral latency at this site. Indeed, MCMV has been identified in macrophages of the lung alveolus 6 mo p.i. (2), and it has been postulated that this may result from viral invasion of alveolar macrophages during the acute stages of infection.
It is now clear that both the host genotype and the presence of viral immune modulators contribute to MCMV pathogenesis and virulence. A better understanding of the NK cellular response evoked by MCMV will improve our understanding of how specific cell types modulate the course of viral dissemination and persistence. Furthermore, identification of NK and other cell types involved in combating wild-type infection will be critical in determining how MCMV evades or subverts the immune response through comparison of cellular responses elicited by MCMV mutants deleted of potential immune modulators. With the in situ detection of NK1.1 reported here, it is now possible to better understand the role of NK cells in viral infection, cancer, and other disease states.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mariapia A. Degli-Esposti, Department of Microbiology, University of Western Australia, Nedlands, Western Australia, Australia.
3 Abbreviations used in this paper: MCMV, murine CMV; MOBS, mouse osmolarity-buffered saline; PLP, periodate-lysine-paraformaldehyde; NGS, normal goat serum; p.i., postinfection.
Received for publication June 19, 2000. Accepted for publication November 3, 2000.
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References |
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(MIP-1)-dependant pathways. J. Exp. Med. 187:1.This article has been cited by other articles:
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