By
From the * Institute of Experimental Immunology, Department of Pathology, University Hospital of
Zürich, CH-8091 Zürich, Switzerland; and the Division of Hematology, Department of Internal
Medicine, University Hospital of Zürich, CH-8091 Zürich, Switzerland
The hematologic consequences of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were studied in wild-type mice with inherent variations in their interferon (IFN)-/
responder ability and in mutant mice lacking
/
(IFN-
/
R0/0) or
IFN
(IFN-
R0/0) receptors. During the first week of infection, wild type mice demonstrated a
transient pancytopenia. Within a given genetic background, the extent of the blood cell abnormalities did not correlate with the virulence of the LCMV isolate but variations were detected
between different mouse strains; they were found to depend on their IFN-
/
responder phenotype. Whereas IFN-
R0/0 mice were comparable to wild-type mice, IFN-
/
R0/0 mice
exhibited unchanged peripheral blood values during acute LCMV infection. In parallel, the bone
marrow (BM) cellularity, the pluripotential and committed progenitor compartments were up
to 30-fold reduced in wild type and IFN-
R0/0, but remained unchanged in IFN-
/
R0/0
mice. Viral titers in BM 3 d after LCMV infection were similar in these mice, but antigen localization was different. Viral antigen was predominantly confined to stromal BM in normal
mice and IFN-
R0/0 knockouts, whereas, in IFN-
/
R0/0 mice, LCMV was detected in
>90% of megakaryocytes and 10-15% of myeloid precursors, but not in erythroblasts. Although IFN-
/
efficiently prevented viral replication in potentially susceptible hematopoietic
cells, even in overwhelming LCMV infection, unlimited virus multiplication in platelet and
myeloid precursors in IFN-
/
R0/0 mice did not interfere with the number of circulating
blood cells. Natural killer (NK) cell expansion and activity in the BM was comparable on day 3 after infection in mutant and control mice. Adaptive immune responses did not play a major
role because comparable kinetics of LCMV-induced pancytopenia and transient depletion of
the pluripotential and committed progenitor compartments were observed in CD80/0 and
CD40/0 mice, in mice depleted of NK cells, in lpr mice, and in perforin-deficient (P0/0) mice
lacking lytic NK cells. Thus, the reversible depression of hematopoiesis during early LCMV infection was not mediated by LCMV-WE-specific cytotoxic T lymphocyte, cytolysis, or secreted IFN-
from virally induced NK cells but was a direct effect of IFN-
/
.
Viral infections are frequently associated with a transient
reduction of the number of circulating blood cells as a
consequence of bone marrow (BM)1 suppression. If virusinduced dysfunction of the BM is severe, secondary bacterial invasion or bleeding may be lethal for the host (1). The
majority of viruses inducing abnormalities of hematopoiesis
are non- or poorly cytopathic for blood cells or have no
known tropism for blood cell precursors (for review see reference 2). In vivo, few data analyzing virus and/or host mechanisms of suppressed BM function are available. In
vitro, some viruses may interfere with the proliferation and
maturation of hematopoietic progenitors by infecting stromal fibroblasts (e.g., cytomegalovirus [3]) or BM macrophages (e.g., human immunodeficiency virus [4]). Moreover, there is evidence suggesting a critical role of the
host's own immune response in causing BM suppression in several virus infections (5, 6). Lymphocytic choriomeningitis virus (LCMV) is a non-cytopathic RNA virus for
which many aspects of viral immunobiology have been
studied in great detail (7, 8). Although mice are the natural
host and reservoir for LCMV (9, 10), occasional transmission to laboratory workers has been followed by mild abnormalities of the cellular blood count (CBC) (11). Only
very rarely, a fatal hemorrhagic disease has been observed
in humans (12), but it occurs in guinea pigs (9, 10). In several other arenavirus infections (e.g., Machupo, Junin, and
Tamiami viruses), hemorrhagic fever, widespread necrosis in the lymphatic tissue, and a global depression of BM are
consistently found (9, 13). Few studies analyzed the mechanism of LCMV-induced hematopoietic dysfunction (14);
some have speculated that LCMV-induced radioresistant
NK cells caused the hematopoietic abnormalities (17). The
respective role of the effects of soluble cytokines, NK or T
cells or LCMV itself, interfering with BM function, however, were not assessed in these studies.
The control of a primary LCMV infection and successful
virus elimination from infected organs depends on virus
specific CTL (18). These protective CTL have also been
shown to cause immunopathological disease dependent
upon virus and host parameters and according to the dominant early location of the infection, i.e., choriomeningitis after intracerebral infection, hepatitis after systemic infection, and footpad swelling after local subcutaneous infection (19). Therefore, it would be expected that at least
part of the BM suppression seen during LCMV infection
might also be caused by T cell-mediated immunopathology. Type I IFNs (IFN- Therefore, murine LCMV infections offer an experimental model to analyze pathogenesis of BM suppression
and virus-induced hemorrhagic fever. The present study
attempts to explore the respective role of innate resistance
or immune mechanisms versus that of specific immunity in
the dysregulation of hematopoiesis in vivo during the acute
phase of LCMV infection. By comparing wild-type with
mutant mice, which had inactivated IFN- Mice.
Mice with a homozygous inactivation of the IFN- Virus.
LCMV-WE was originally obtained from Dr. F. Lehmann-Grube (Hamburg, Germany) (9) and LCMV-Docile (a variant isolated from a LCMV-WE(UBC)-carrier mouse) was obtained from Dr. C.J. Pfau (Troy, NY) (33) as a plaque-purified
second passage virus. The original LCMV-Armstrong isolate was
obtained from Dr. M.B.A. Oldstone (Scripps Clinic and Research Foundation, La Jolla, CA) (34). Second passage virus derived from plaque-purified isolates was propagated on BHK cells
(Armstrong) or L929-fibroblast cells (WE) or MDCK cells (Docile). The viruses were titrated using an immunological focus assay,
which is two to threefold less sensitive than virus titers detected
by in vivo infection (35). LCMV titers of BM cells are presented
as PFU per 106 nucleated BM cells.
Peripheral Blood Values and BM Cytology.
Blood samples (40 µl) were obtained from the retro bulbar plexus of ether-anesthetized mice; they were diluted 1:5 in electro-coated EDTA microtainer tubes (Becton Dickinson, Mountain View, CA) containing DMEM + 5% FCS. CBC were then determined in a hemocytometer using mouse-specific discriminator settings (TECHNICON H1®, Technicon Instruments Corp., Tarrytown, NY), and
the differential white blood cell count was defined microscopically by scoring blood smears stained with May-Grünwald-Giemsa.
Reticulocyte counts were quantitated by flow cytometry (SYSMEX R 3000®, Toa Medical Electronics, Kobe, Japan). BM was
obtained by flushing femora and/or tibiae with a 25-gauge needle; differential counts of 200 cells were determined on MayGrünwald-Giemsa, chloracetate esterase, Pluripotential and Committed Progenitor Assays.
The pluripotential hematopoietic progenitor pool (CFU-S) of LCMV-infected
mice was enumerated as described before (36). Recipient mice
were immunized with 102 PFU LCMV-WE. 2-3 wk later these
mice were lethally irradiated (9 Gy), and within 3 h were reconstituted intravenously with 104 or 105 syngeneic sex-matched BM
cells from acutely infected or uninfected control mice. The rationale in preimmunizing recipient mice was to prevent growth inhibition of the transplanted CFU-S by cotransferred virus from
infected mice, which activates innate effector mechanisms in the
naive unprimed recipient and thus would alter the CFU-S content in question (14). 8 d after irradiation, the recipient mice were
killed and the spleens fixed in Tellesnicky's fixative. Individual
colonies visible on the surface of the spleen were counted under a
dissecting microscope and the numbers of CFU-S per femur were
calculated.
/
) and type II IFNs (IFN-
) play an important role in the early phase of infection. They
inhibit unlimited viral replication before antiviral effector
CTL are induced and thereby prevent exhaustion of virusspecific CTL (20, 21). In parallel with the IFN-
/
response, NK cells become activated, proliferate, and infiltrate infected tissue (22, 23). Effector functions of NK cells
elicited during LCMV infection involve perforin-mediated
cytolysis of infected target cells (24) and/or cytokines produced by NK cells, such as IFN-
(25) or TNF-
(26). In
tissue culture systems, these cytokines have been found to
inhibit hematopoietic progenitor cell growth (27). In one
report, minute amounts of Fas ligand on the surface of freshly isolated murine NK cells were demonstrated; however, there is still contradictory evidence for Fas-mediated
cytotoxicity of NK cells against Fas-expressing targets and
its functional role in vivo remains to be established (28, 29).
/
receptors
(IFN-
/
R0/0) or IFN-
receptors (IFN-
R0/0), the following questions were addressed. First, are IFNs, acting via
IFN R expressed on pluripotential or on lineage-committed hematopoietic progenitors, involved in early LCMVinduced hematopoietic suppression, and what is the respective role of IFN-
/
and that of IFN-
? Second, does the
absence of IFN R expression interfere with LCMV-induced
NK cell activation, and what are the consequences of possibly activated NK cells for hematopoietic cells in vivo? Third, the LCMV-induced effects on circulating blood
cells and blood cell precursors were analyzed under conditions of NK cell depletion with a mAb, absent expression
of Fas on hematopoietic cells, or lack of perforin produced
by NK cells, or of deficiency of CD8+ or CD4+ T cells.
The results show a major role for IFN-
/
but neither of
IFN-
, nor of perforin nor of Fas and neither of NK nor of T cells during the first 7 d of LCMV infection. Importantly, in absence of a functional IFN-
/
R, extensive virus replication of LCMV in megakaryocytes and, to a lesser
extent, in myeloid precursors appeared not to interfere
with maturation and release of differentiated blood cells,
including platelets, into circulation.
/
receptor (IFN-
/
R0/0) and those lacking the IFN-
receptor
(IFN-
R0/0) have been described previously (21, 30). These mutant mice have a pure 129Sv/Ev (H-2b) genetic background and
129Sv/Ev mice were used as controls in all experiments. The antiviral immune response of knockout mice devoid of CD8+
(CD80/0) (31) or CD4+ (CD40/0) (32) T cells or with a disrupted
perforin gene (P0/0) (24) have been reported before. CD80/0 mutants have a pure C57BL/6 (H-2b) background and CD40/0 mice
have been backcrossed four times to C57BL/6 mice. CD40/0,
CD80/0, and P0/0 mice were compared with normal C57BL/6
mice in all experiments. Knockout mice, C57BL/6, DBA/2, and
BALB/c mice were obtained from the breeeding colony at the
Institut für Versuchstierkunde (University of Zürich, Switzerland); CBA/J and C57BL/6 lpr/lpr mice were purchased from
BRL (Füllinsdorf, Switzerland), and C3H/HeJ from Harlan Olac,
Ltd. (Zeist, Netherlands). All mouse strains were kept under specific pathogen-free conditions. Sex-matched mice of 8-12 wk of
age were used. The animal protection law of the Kanton of
Zürich limits numbers of mice to be used in experiments, particularly if disease is severe; therefore, experiments generally were
repeated two times with groups of 2-4 mice.
-naphthyl butyrate esterase, or Sudan black B stained smears.
5
mol/l 2-ME, penicillin-streptomycin, 0.9% methylcellulose, 30% FCS, 1% BSA in the presence of optimized concentrations of growth factors (mIL-3 [30 U/ml] and mGM-CSF [30 U/ml] for CFUGM; mIL-3 [30 U/ml] and hEpo [5 U/ml] for BFU-E; mIL-3
[30 U/ml] and mTPO [500 ng/ml] for CFU-Meg). Recombinant murine IL-3 and GM-CSF were obtained from PharMingen, Inc. (San Diego, CA), recombinant human erythropoietin
was purchased from Cilag AG (Schaffhausen, Switzerland), and recombinant murine thrombopoietin (mTPO) was a gift from Genentech, Inc. (South San Francisco, CA). Total BM cells were
plated at a concentration of 5 × 104 cells/ml in duplicate plates.
The cultures were incubated at 37°C in a humidified atmosphere
containing 5% CO2 for 5-7 d (CFU-Meg), 7 d (CFU-GM), or 8 d
(BFU-E) and individual colonies were scored by morphology.
Colonies were defined as containing at least three large refractile
cells for CFU-Meg (37) and >50 cells for CFU-GM. For the microscopic identification of the cell type present, colonies were periodically picked, spread on glass slides, and stained with MayGrünwald-Giemsa or acetylcholinesterase (megakaryocytes) for
confirmation. Each experiment was performed using individual unpooled BM cells and results from four to eight culture plates were combined for each determination. The total number of colonies per femur was calculated using the cellularity obtained for
the individual BM.
Immunocytochemistry Procedures. BM smears on slides were fixed in acetone for 10 min. The rat anti-LCMV mAb VL-4 was used as primary Ab and detected by an indirect immunoenzymatic staining procedure using peroxidase-labeled goat anti-rat Ig (Tago; diluted 1:30), followed by alkaline phosphatase-labeled donkey anti-goat Ig (Jackson Immunoresearch Laboratories, Inc., West Grove, PA; diluted 1:30). Smears were counterstained with Meyer's hemalum (Kantonsapotheke, Zürich, Switzerland) for 2 min. Percentages of infected BM cells were quantitated microscopically by counting at least 200 cells from one or more smears per individual mouse. The infected cell type was readily defined by the cytomorphology after counterstaining.
Antibody and Poly I/C Treatment.
Mice were treated intraperitoneally with 1 mg of the IL-2R-specific mAb TM-
1 (rat
IgG2b) (38) or control normal rat IgG (Sigma Chemical Co., St.
Louis, MO) in 500 µl of PBS. One single dose of Ab was injected
2 d before virus challenge. Poly I/C (Serva, Heidelberg, Germany) was administered intravenously at a concentration of 200 µg/mouse 20 h before the cytotoxicity assay.
Cytotoxicity Assays.
NK cell activity of spleen and BM cells
was determined by a 51Cr-release assay as described previously
(20). In brief, mice were infected intravenously 3 d previously
with LCMV. Doses and virus strains used are indicated in the results. BM cells (pooled cells from both femora) were resuspended
at 7 × 106/ml in MEM plus 2% FCS. YAC-1 target cells were
labeled with 1 µCi of 51Cr sodium chromate for 1 h at 37° C,
washed twice, and resuspended at 105/ml. Threefold dilutions of
spleen cells were incubated with 100 µl of targets in 96-well microtiter round-bottomed plates for 5 h. A total of 70 µl of the
supernatant was assayed for released 51Cr. The percent-specific
release of 51Cr was calculated as ([experimental release spontaneous release] × 100/[total release
spontaneous release]). LU,
a measure of relative cytolytic activity, was determined according
to established methods to be the number of lymphocytes necessary to lyse 33% of the standard number of target cells during the
standard test duration. LUs per femur were computed by dividing
the total number of lymphocytes per organ by LU.
Flow Cytometry. Single-cell suspensions of spleen or BM were preparated at 4°C in BSS containing 2% FCS and 0.2% NaN3. Cell surface markers were quantified by staining with mAbs conjugated with fluorochromes; the antibodies used were the following: anti-CD4 and anti-CD8 (Becton Dickinson), anti-NK 1.1 (PharMingen), anti-TER 119 (PharMingen). To analyze NK expansion, cells were triple stained with FITC-conjugated anti-CD8, FITC-conjugated anti-CD4, PE-conjugated NK 1.1, a biotinylated anti-erythroid (TER 119) Ab, and a Tricolor-streptavidin conjugate. Multiparameter analysis was performed with a FACScan® (Becton Dickinson) using logarythmic scales. Viable cells were gated by forward and side scatter of light.
Statistics. Hematologic data are presented as means ± SD unless stated otherwise. Virus titers are expressed as log10 of PFU per organ. Comparisons were made using the two-tailed unpaired Student's t test, the analysis of variance (ANOVA), and the Scheffe's post hoc procedure as appropriate. A P value of less than 0.05 was regarded as significant.
The transient depression of the peripheral CBC
during the first week of LCMV infection varied according
to the virus dose and the different genetic backgrounds of
the murine host. For all virus isolates tested, maximally decreased blood values were observed between days 3 and 5 after infection and were most prominent in the reticulocyte, granulocyte, and platelet compartments (in decreasing
order of suppression). There was some variation in the absolute CBC of uninfected control mice between the different mouse strains (e.g., mean reticulocyte count: 182 × 106/ml in C57BL/6; 247 × 106/ml in BALB/c mice);
therefore, the relative decrease from baseline values (percent) was compared (as shown for reticulocytes; Table 1).
Within one mouse strain, the extent of suppression of blood cell numbers did weakly correlate with the LCMV isolate,
i.e., all blood cell parameters were comparably reduced on
days 3-5 after infection in Armstrong-, WE-, or Docileinfected mice. However, between different mouse strains the
CBC varied after infection with low LCMV doses (2 × 102
PFU): C57BL/6, CBA/J, and C3H/HeJ had lower relative
reticulocyte (Table 1) and granulocyte counts than DBA/2
and BALB/c mice; less stringently, the same pattern was
observed in the platelet numbers (e.g., CBA/J, 83%; C3H/
HeJ, 49%; and C57BL/6, 37% below baseline after LCMVWE 2 × 102 PFU). The magnitude of these variations between different mouse strains was small but significant and
correlated with the IFN-/
responder status (i.e., IFN-
/
high responders had a lower CBC than IFN-
/
low responders) as reported after infection with LCMV (39, 40).
In contrast, infection with a high dose of LCMV (2 × 106
PFU) induced an extensive decrease of all blood cell parameters, to an extent that was comparable for all the mouse
strains tested (Table 1, granulocytes 90-95%, platelets 78-
95% below baseline). After infection with all three LCMV
isolates, reductions in RBC counts were small. If the halflife of the mouse RBCs is taken into account (around 50 d), it is probable that decreases from baseline were mainly
due to repetitive individual blood sampling. Thus, high
doses of LCMV (2 × 106 PFU) induced a drastic transient
pancytopenia with minimal values 3-5 d after infection, independently of the virus isolate or the genetic background
of mice. To avoid variations, high doses of LCMV-WE
therefore were used for all subsequent experiments.
|
Because the responder phenotype
of mice seemed to influence the severity of LCMV-induced
blood cell abnormalities, peripheral hematologic parameters after LCMV infection were studied in mice with inactive receptors for IFN-/
(IFN-
/
R0/0) or for IFN-
(IFN-
R0/0). The hematologic status of uninfected receptor-deficient and wild-type mice maintained under specific
pathogen-free conditions was identical (data not shown).
Infection of 129Sv/Ev wild-type mice with a high dose of
LCMV-WE (2 × 106 PFU) resulted in the same kinetics of
peripheral pancytopenia as seen in infected mice of other
genetic backgrounds (Table 2). Starting on day 1 after infection, maximal depression of blood cell values was seen
on days 3-5 with predominant abnormalities in the circulating reticulocyte (97% below baseline), platelet (65% below baseline), and neutrophil (72% below baseline) compartments. All peripheral parameters returned near baseline
values on day 7. A similar time course was observed for infected IFN-
R0/0 mice. In contrast, LCMV-WE-infected
IFN-
/
R0/0 mice showed stable hematologic parameters
comparable to that of uninfected controls during the entire
study period. When compared with infected wild-type or
IFN-
R0/0 mice they had a 180-200-fold higher reticulocyte, a three- to fivefold increased platelet, and a two- to
threefold increased neutrophil count on day 3 after infection (Table 2).
|
BM function was evaluated in wild-type and
IFN R mutants after acute infection with LCMV-WE (2 × 106 PFU). Femoral cellularity of wild-type and IFN- R0/0
mice was reduced by half on day 1 after infection and reduced to a minimum of 7% in wild-type mice and 11% of
baseline values in IFN-
R0/0 mice on day 3, respectively
(Fig. 1). Thereafter, total cellularity returned to normal values in IFN-
R0/0 mice by day 7 and in wild-type mice by
day 10. In contrast, IFN-
/
R0/0 mice showed minimal
changes in femoral cell content during the entire study period. In parallel, splenic cellularity decreased considerably until day 3 in wild-type and IFN-
R0/0 mice but remained
unchanged in IFN-
/
R0/0 mice (data not shown). Cytological analysis of BM preparations on day 3 after infection
revealed a marked deficiency of the nonmitotic mature cell
population in wild-type and IFN-
R0/0 mice but not in
IFN-
/
R0/0 mice (Table 3). In wild-type and IFN-
R0/0
mice, LCMV-WE infection resulted in a 2-2.4-fold reduction of erythroid elements, which was more apparent
when absolute cell numbers per femur were compared
(wild type 0.4 × 106; IFN-
R0/0, 0.7 × 106; IFN-
/
R0/0, 4.5 × 106). Polymorphnuclear granulocytes of wildtype and IFN-
R0/0 mice were reduced 3-3.4-fold and
the proportion of early myeloid precursors (blasts, promyelocytes) was significantly increased by a factor of 2.5 when
compared with IFN-
/
R0/0 mice. The relative increase
of marrow lymphocytes in wild-type and IFN-
R0/0 mice
on day 3 after infection had disappeared by day 7; monocytes were slightly increased in both mutants and wild-type
mice 7 d after LCMV-WE infection. The amount of megakaryocytes was determined semiquantitatively and was two-
to fourfold reduced in wild-type and IFN-
R0/0 mice, but
unchanged in IFN-
/
R0/0 mice during the entire study
period.
|
Analysis of committed hematopoietic progenitors 3 d after infection with LCMV-WE showed a distinct effect on
different lineages in the BM of wild-type and IFN- R0/0
mice: the absolute numbers of BFU-E and CFU-GM per
femur were 30-fold below baseline levels and, to some
lesser extent, also the numbers of CFU-Meg were reduced
(20-fold) (Fig. 2). This was in contrast with IFN-
/
R0/0
mice, which, when compared with uninfected control mice,
had normal or slightly increased numbers of all lineagecommitted progenitors 3 d after LCMV-WE infection. Infection with LCMV of wild-type and of IFN-
R0/0 mice
resulted in much smaller sizes of colonies and the relative frequencies of all CFUs per plated BM cells were at least
10-fold reduced as compared with IFN-
/
R0/0 mice.
We then examined the effect of LCMV-WE infection
on the number of primitive clonal progenitors (CFU-S) depending on their expression of IFN R. Syngeneic lethally
irradiated LCMV-WE immune mice of the 129Sv/Ev wildtype strain were used as recipients in these experiments.
The absolute number of CFU-S per femur 3 d after infection with LCMV-WE was ~10-fold reduced in wildtype and IFN- R0/0 mice as compared with uninfected
control mice of the same genotype (Fig. 3). In contrast,
IFN-
/
R0/0 mice had a normal or even higher frequency of CFU-S after LCMV-WE infection. Obvious
differences in the mean size of the CFU-S obtained were
observed, i.e., spleen colonies of uninfected wild-type and LCMV-WE-infected IFN-
/
R0/0 mice had a three- to
fourfold increase in diameter as compared with infected
wild-type and IFN-
R0/0 mice.
These results indicate that the abnormalities of circulating blood cells observed early in LCMV-WE infection
were caused by a marked deficiency of all functional compartments of the BM. The reduction of the frequency of
the pluripotential and the lineage-committed progenitors
was much greater than that of the cytomorphologically differentiated population. This transient, LCMV-induced suppression of BM was abrogated in mice lacking a functional
IFN-/
R, but not in mice deficient of the IFN-
R.
In vivo susceptibility to LCMV
infection of wild-type and IFN R-deficient hematopoietic
BM cells was evaluated for a high-dose (2 × 106 PFU)
challenge with LCMV-WE. Wild-type and IFN- R0/0
mice showed a similar kinetics of LCMV replication with
slightly higher titers in IFN-
R0/0 mice 7 d after infection
(Fig. 4). In contrast, IFN-
/
R0/0 mice exhibited an enhanced initial replication, i.e., the highest titer in the BM
was reached already on day 1. All three groups showed a
comparable maximal viral load of ~5.6 log10 PFU/femur 3 d
after virus inoculation. Thus, under high-dose conditions, absence of the IFN-
/
R favored uncontrolled viral replication maximally in the initial phase of infection, but
overall maximal viral loads in BM were not affected by the
expression of IFN receptors (note that IFN-
/
R0/0 mice
become persistently infected with LCMV but titers are 1-2 log10 lower in the chronic state than the peak virus titer
measured on day 3 [21, 41]). The kind of BM cell permissive for LCMV replication was characterized in wild-type,
IFN-
/
R0/0, and IFN-
R0/0 mice at different timepoints after infection (Fig. 5 A). Viral antigen was detected
by an LCMV-WE-specific mAb on BM smears; in parallel,
the type of infected hematopoietic cell was identified microscopically by cytomorphologic criteria by counterstaining with Meyer's hemalum. The prevalence of LCMVWE-positive morphologically differentiated hematopoietic cells in wild-type and IFN-
R0/0 mice was low and did
not exceed 20% of megakaryocytes and 0.5% of granulocytes at any timepoint of infection (Fig. 5 B). Virtually all
detectable LCMV-specific antigen in wild-type and IFN-
R0/0 mice was confined to stromal BM cells also on day 3, at times when highest LCMV titers were recovered. In
contrast, in IFN-
/
R0/0 mice >90% of megakaryocytes
were positive for viral antigen 1 d after virus inoculation,
~60% stained positively on day 3 and
50% of all megakaryocytes remained infected during the following week.
In addition, ~12% predominantly immature myeloid precursors (promyelocytes, myelocytes) of IFN-
/
R0/0 BM
exhibited viral antigen peaking on day 3 after LCMV-WE
infection (Fig. 5, A and B). In comparison to wild-type and
IFN-
R0/0 mice, stromal BM cells of IFN-
/
R0/0 mice
were infected to a comparable extent. Interestingly, erythroblasts were resistant to LCMV-WE infection in all three
mouse strains tested, i.e., the frequency of LCMV-positive
erythroblasts in IFN-
/
R0/0 mice was less than 1:800.
NK Activity During Acute Infection with LCMV in IFN R-deficient Mice versus Wild-type Mice.
Because IFN-/
plays an
important role in NK responses and because NK cells
might be involved in regulation of differentiation of stem
cells, probably dependent upon MHC expression, the various mice used in this study were tested for NK activation
in the BM. Earlier studies had shown that NK activity in
spleens generated in IFN-
/
R0/0 mice was drastically reduced when compared with wild-type or IFN-
R0/0 mice
(21). Primary ex vivo NK-mediated cytolysis was measured on YAC-1 targets. When compared at maximal effector to
target ratios, a vigorous cytolytic NK activity was observed
in the wild-type mice and, to some lesser extent, in the IFN
/
R0/0 and IFN-
R0/0 mice after infection with 2 × 106 PFU LCMV-WE (Fig. 6). Given the three- to fourfold
reduction of marrow cellularity of wild-type and IFN-
R0/0 mice, the absolute number of cytolytic NK cells in the
BM (expressed as LU per femur) was two- to threefold
higher in IFN-
/
R0/0 mice. Interestingly, a comparable
NK response was seen in BM after infection with a low
dose of LCMV-WE (2 × 102 PFU; data not shown) in
both mutants and the wild-type strain, but not in IFN-
/
R0/0 mice treated with poly I/C. Next, we examined the
LCMV-WE-induced NK proliferation in the BM by flow
cytometry. A triple staining of total marrow cells using an
erythroid-specific mAb (TER 119), a NK cell-specific
mAb (NK 1.1), and a mixture of anti-CD8 and anti-CD4
was performed 3 d after infection with 2 × 106 PFU
LCMV-WE. To increase sensitivity and detection levels of NK cells, subset analysis was performed with cells gated
negatively on TER 119; this eliminated the high number
of erythroid cells in uninfected mice. Baseline NK and
CD4/CD8 percentages were comparable for uninfected
wild-type and IFN R-deficient mice (<2%). After infection, the percentage of NK 1.1-positive cells in the BM increased ninefold in wild-type mice, four- to fivefold in
IFN-
/
R0/0 mice, and six- to sevenfold in IFN-
R0/0
mice (Fig. 7). To a lesser extent, an expansion of the CD4/
CD8+ population was observed in the BM of wild-type
and IFN-
R0/0 mice, whereas in the IFN-
/
R0/0 mutant mice no significant increase in the percentage of CD4/ CD8+ cells was detected on day 3 after infection. Thus,
there was no correlation between NK activity in BM and
susceptibility to LCMV-induced BM suppression.
Hematologic Effects in Absence of Perforin- or FAS-mediated Cytolytic Effector Functions of NK Cells or Virus-specific CTL after LCMV-WE Infection.
The correlation between LCMVWE-associated changes of blood cells and NK activity was
evaluated in infected C57BL/6 mice depleted of NK cells
with TM-1 (38), in P0/0 mice, which show no cytolytic T
cell or NK acitvity on susceptible target cells in vitro (24),
and in lpr mutants with no functional Fas-Fas ligand interaction. LCMV-WE-infected (2 × 106 PFU) mice of all
groups showed the same kinetics of blood cell depression as
observed for wild-type C57BL/6 mice. On day 3 after viral
inoculation, all groups had clearly reduced reticulocyte counts (82-98% below baseline), thrombocytopenia (75-
82% below baseline), two- to fivefold reduction of neutrophils, and some lymphopenia (Table 4). Mutant mice deficient for the CD8 or CD4 molecule were included in this
experiment, because LCMV-specific CTL responses in
these mice are either absent (CD80/0) (31) or partially
(CD40/0) (32) reduced. Both of these LCMV-WE-infected
mutant mouse strains showed a comparable pancytopenia
on day 3, as did lpr, P0/0, normal rat IgG-treated mice, and
TM-
1-treated mice. TM-
1 treatment 2 d before
LCMV-WE infection efficiently eliminated NK-mediated cytolytic activity on sensitive YAC-1 targets when compared
with normal rat IgG-treated controls (Table 4).
|
To assess potential effects of perforin- or Fas-dependent
early induced CTL on pluripotential and lineage-committed progenitors, a quantitative analysis of hematopoietic
precursors was performed in CD40/0, CD80/0, P0/0, and lpr
mice infected with 2 × 106 PFU of LCMV-WE (Table 5).
The absolute numbers of BFU-E, CFU-GM, and CFU-Meg
per femur did not differ significantly between the mutant
mouse strains tested on day 3 of infection and all values were in the range of 72-85% below baseline levels. BFU-E
and CFU-GM of these four mouse strains were less affected
by LCMV when compared with infected wild-type 129Sv/
Ev and IFN- R0/0 mice. This finding suggested that non-
MHC-linked genes were important for quantitative differences, because CD80/0, P0/0, and lpr mice are of pure
C57BL/6 origin (H-2b) and CD40/0 mice are four times
backcrossed to C57BL/6, whereas IFN-
R0/0 mutants are
of pure 129Sv/Ev background (H-2b). Accordingly, lethally irradiated LCMV immune C57BL/6 mice were used
as recipients to determine CFU-S numbers of LCMVWE-infected CD40/0, CD80/0, P0/0, and lpr mice. The absolute numbers of CFU-S per femur on day 3 after LCMV
infection were in the same range in these mutants and 10fold lower as compared with uninfected controls (see Table 5).
Thus, CTL or NK effectors are apparently not influencing suppression of hematopoiesis in early LCMV infection.
|
The current study implicates endogenously produced
IFN-/
in causing depletion of the pluripotential and lineage-committed hematopoietic progenitors during the first
week of LCMV infection. LCMV-induced abnormalities
of peripheral blood parameters correlated positively with
the IFN responder phenotype of mice. The key finding was
that in IFN-
/
R0/0 mice, peripheral hematologic parameters and the frequency of blood cell precursors in the BM
were comparable in LCMV-infected and uninfected control mice. Conversely, the decrease in peripheral blood values after infection with LCMV in wild-type, IFN-
R0/0,
CD40/0, CD80/0, P0/0, or lpr mice was associated with a
substantial reduction of the numbers of pluripotential progenitor cells and lineage-committed precursors and, to a
lesser extent, of the morphologically recognizable compartment in the BM. The depletion of the blood cell progenitors was attributable to a direct effect of IFN-
/
and was
independent of contact-dependent cytotoxicity or secreted
factors from NK cells. This is suggested by the NK activity
in BM of wild-type mice and in mice lacking either the
IFN-
/
R or the IFN-
R. Additionally, virus specific
effector T cells generated within the first 7 d of infection
apparently played no critical role in LCMV-induced transient BM aplasia, because knockout mice either lacking peripheral CD8+ or CD4+ T cells were similarly depleted of
pluripotential and committed progenitors. A summary of
the relevant findings in wild type and in the different mutant mouse strains is presented in Table 6.
|
The viral and host parameters defining the abnormalities
of peripheral blood cells in the acute phase were influenced
to a minor extent by the LCMV isolate; however, they
correlated with the IFN-/
responder phenotype of the
genetic background of the host. This is documented by the
fact that within a given mouse strain, the extent of the pancytopenia in the acute phase of infection was not determined by the divergent replication kinetics of a rapidly
(Docile), intermediate (WE), or slowly (Armstrong) replicating LCMV isolate. This observation is in accordance
with earlier findings, demonstrating comparable maximal
serum levels of IFN-
/
induced by several LCMV isolates in mice of the same genetic background (40). Between the various mouse strains marked differences in the
amount of IFN-
/
production have been measured, yet in
adult mice no correlation between serum levels of IFN-
/
and susceptibility to lethal choriomeningitis has been demonstrated (40). Evidence for a link between IFN-
/
induction and disease, however, was established in newborn
mice infected with LCMV (39). In these studies, the severity of the delay in organ maturation and liver necrosis as
well as the incidence of death was directly dependent on
the extent of the endogenous IFN-
/
response of the different mouse strains infected with LCMV. Moreover, the
concept of a causal correlation between the amount of endogenously induced IFN-
/
by LCMV and the severity
of BM suppression is in line with in vitro findings, demonstrating IFN-
/
to be a potent inhibitor of proliferation/
maturation of normal BM-derived hematopoietic progenitors (42), as well as of certain leukemic cells in vivo (e.g.,
hairy cell leukemia [43], and CML [44]).
Interestingly, expansion and functional activation of NK
cells in LCMV-infected BM of IFN-/
R0/0 mice was
comparable to that in IFN-
R0/0 and wild-type mice, especially when absolute numbers of NK cells were considered. NK cells are cytolytically activated and proliferate after stimulation with IFN-
/
, IFN-
, or IL-2 in vitro
and in vivo (45, 46) and the peak of NK activity after
LCMV infection parallels the maximal serum concentration of IFN-
/
(22, 23). Apparently, in our experimental
model, NK cells from IFN-
/
R0/0 mice, which cannot
be stimulated by IFN-
/
signaling via the IFN-
/
R,
were comparably activated. This result may hypothetically signal an IFN-
/
effect that is not mediated via the classic IFN-
/
R deleted in these mice. An alternative (i.e., not
IFN-
/
R-dependent) activation pathway of NK cells in
LCMV-infected IFN-
/
R0/0 mice is documented further by the finding, that poly I/C, which is a synthetic
double-stranded RNA and a potent inducer of the IFN-
/
system in vivo (47), did not stimulate NK cells in the BM.
More likely, NK activity in LCMV-infected IFN-
/
R0/0
mice could be due to stimulation by other cytokines (e.g.,
IL-2, IL-12) induced by the overwhelming replication of
LCMV in IFN-
/
R0/0 mice.
There is conflicting data on the role of NK cells inhibiting hematopoiesis in vitro. Under some conditions, human
NK cells suppress myelopoietic (48, 49) and erythropoietic
(50) colony formation. In murine systems, NK cells have
been reported to inhibit growth of syngeneic hematopoietic progenitors (51) or the more primitive CFU-S (52), but
other studies have demonstrated normal hematopoiesis in
vitro in the presence of NK cells (53). Moreover, it has
been suggested that both the antiviral properties (25, 26) as
well as the suppressive effect on in vitro colony formation of NK cells function via the secretion of soluble factors,
such as IFN- or TNF-
(27, 54). As for acute LCMV infection, one report proposed syngeneic rejection of stem
cells by activated NK cells, because treatment with antiasialo GM1 (a rabbit antiserum depleting NK cells but also
partially LCMV-specific CTL nonspecifically [55]) partially
restored the marrow repopulating ability of stem cells from
uninfected syngeneic controls transferred into irradiated LCMV-infected mice (17). In our experimental system,
there was no evidence of NK-mediated suppression of hematopoiesis, either via contact-dependent cytotoxicity or
secreted cytokines, because (a) at the timepoint of maximal
NK activity we found normal precursor and differentiated
blood cell frequencies in the BM of IFN-
/
R0/0, but a
drastic reduction of all hematopoietic compartments in IFN-
R0/0, lpr, and P0/0 mice, in which functionally inactive NK cells proliferate after LCMV infection (24), and (b)
TM-
1, which is a mAb specific for the IL-2 R
chain
and which induces a long-lasting selective depletion of NK
cells, but considerably less of T cells (38, 56), did not abrogate LCMV-induced suppression of peripheral blood parameters. A subtle effect of IFN-
could be observed 7 d
after LCMV infection, at a timepoint when bone marrow
cellularity was restored in IFN-
R0/0 mice, but not in
wild-type mice (Fig. 1). When compared with IFN-
/
,
the efficiency of LCMV-induced endogenous IFN-
in
suppressing BM function seems to be much weaker, however, because the CBC and BM cellularity of IFN-
/
R0/0
mice were not altered at that stage of LCMV infection.
The finding of megakaryocytes and myeloid precursors
expressing LCMV antigen in high amounts in the presence
of activated NK cells, and yet lack of NK-mediated immunopathology in the BM of IFN-/
R0/0 mice, was surprising. One possible explanation might be that LCMV by
itself, in contrast with some other viruses, such as murine cytomegalovirus, does not alter class I MHC expression nor
does it inhibit the ability of IFN-
to upregulate MHC
class I in virus-infected cells (57). Therefore, LCMV-infected
MHC class I-expressing hematopoietic precursors could be
protected from NK cell-mediated lysis because cells expressing high levels of MHC class I are more resistant to
NK cells than those lacking expression of MHC class I (58).
Although there is evidence for Fas ligand expression on the
cell surface of freshly isolated murine NK cells (29), the
role of the Fas pathway in NK killing in vivo is still poorly understood. In vitro studies have demonstrated some Fas
expression on human erythroid and myeloid colonies in
the presence of TNF-
and/or IFN-
(59), as well as upregulation of the Fas antigen by influenza virus-elicited
IFN-
/
on HeLa cells (60). The theoretical possibility,
that Fas-mediated cytotoxicity of NK cells against Fas-expressing BM cells could be involved in LCMV-induced BM
aplasia, is not supported here because NK cells were not
critical in the first place. Furthermore, lpr mice exhibited a
comparable suppression of BM and blood values in the
presence of highly activated NK cells (data not shown) 3 d
after LCMV infection; this documents a minor significance
of the Fas receptor-Fas ligand system of NK cells, because a
nonoperational apoptotic signaling in lpr mice did not revert LCMV-induced transient depression of hematopoiesis.
Mutant IFN-/
R0/0 mice exhibited an intense viral
replication in megakaryocytes and, to a lesser extent, in
myeloid precursors. Interestingly, LCMV by itself did not
interfere with maturation and the release of platelets or
granulocytes into the circulation, because the number of
these blood cells remained unchanged in acutely infected
IFN-
/
R0/0 mice. We do not not know at which stage
of maturation megakaryocytes are infected in IFN-
/
R0/0
mice, but it is conceivable that LCMV might infect morphologically unidentifiable progenitors of the megakaryocytic lineage. A similar finding has been described for HIV
patients, for which it has been proposed that HIV infection of
megakaryocytic or myeloid progenitors by itself does not inhibit their proliferation/differentiation potential in vivo unless antibodies suppressing the growth of these progenitors
are present in the serum (61, 62). We had no evidence of infection of pluripotential hematopoietic progenitors, because
erythroblasts did not express viral Ag in serial BM analysis during the first week of LCMV infection. LCMV seemed
to persist for a longer time period in megakaryocytes than
in myeloid precursors, because immature granulocytes were
virus free on day 7 of LCMV infection, whereas a considerable proportion of megakaryocytes still stained positively
for viral Ag. This finding may be due to differences in the
cellular tropism of LCMV but could simply reflect the fact
that the turnover of myeloid cells is much higher and,
therefore, infected myeloid precursors were released into circulation very efficiently.
Although IFN-/
R0/0 mice are able to mount normal
Th and CTL responses against cytopathic viruses like vaccinia or vesicular stomatitis virus, LCMV specific CTL activity is lacking due to exhaustive activation of virus-specific
CTL (21). Therefore, the excessive viral multiplication in
megakaryocytes and, to a lesser extent, in myeloid precursors was not associated with a CTL-mediated immunopathology, documented by the normal range of these cells in
the BM and normal values of circulating blood cells. In addition, LCMV-activated CD4+ or CD8+ T cells or their
secreted products (e.g., TNF-
, IFN-
) could not account
for BM suppression in acute LCMV infection, since CD80/0
and CD40/0 mutant mice, which have no CD8+ or CD4+
T cells in the periphery (31, 32), respectively, had comparably reduced hematopoietic progenitors and blood cells. It
should be pointed out, however, that the present study analyzed the acute phase of LCMV infection, in which innate
immune mechanisms dominate and the frequency of LCMVspecific CTL is still increasing. Therefore, the pathogenesis
of hematologic abnormalities at later timepoints of infection might be distinct from the one described here and may
involve other immunopathologic mechanisms, e.g., cytolytic activity or secreted cytokines from CTL, such as TNF-
or
IFN-
. The presented results and concepts may be generalizable and offer explanations for mechanisms of hematopoietic disease in noncytopathic viral infections and hemorrhagic fevers caused not only by infection with arenaviruses
such as LCMV or Junin virus but also by Dengue fever virus. For these viruses, high concentrations of IFN-
/
have been measured in the serum, and the magnitude and
the duration of circulating IFN-
/
correlated directly
with the severity and the evolution of the disease (63).
Address correspondence to Daniel Binder, the Institute of Experimental Immunology, Schmelzbergotrasse 12, CH-8091 Zürich, Switzerland.
Received for publication 19 September 1996
Recombinant mTPO was kindly provided as a gift by Genentech, Inc. (South San Francisco, CA).We are grateful to A. Althage, E. Horvath, and R. Rüegg for excellent technical assistance and to N. Wey for processing the microphotographs. We thank M. Aguet, P. Aichele, M. van den Broek, S. Ehl, Paul Klenerman, and K. Maloy for helpful discussions and critical reading of the manuscript.
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