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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2914-2924
Hematopoietic Remodeling in Interferon- -Deficient Mice
Infected With Mycobacteria
By
Peter J. Murray,
Richard A. Young, and
George Q. Daley
From the Whitehead Institute for Biomedical Research, Cambridge; and
the Department of Biology, Massachusetts Institute of Technology,
Cambridge, MA.
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ABSTRACT |
Control of intracellular bacterial infections requires
interferon- (IFN-) both for establishing a Th1 T-cell response
and for activating macrophages to kill the bacteria. Exposure of mice deficient in IFN- to mycobacterial infection produces an immune response characterized by a Th2 T-cell phenotype, florid bacterial growth, and death. We report here that IFN--deficient mice infected with mycobacteria also undergo a dramatic remodeling of the
hematopoietic system. Myeloid cell proliferation proceeds unchecked
throughout the course of mycobacterial infection, resulting in a
transition to extramedullary hematopoiesis. The splenic architecture of
infected IFN--deficient mice is completely effaced by expansion of
macrophages, granulocytes, and extramedullary hematopoietic tissue.
These features coincide with splenomegaly, an increase in splenic
myeloid colony-forming activity, and marked granulocytosis in the
peripheral blood. Systemic levels of cytokines are elevated,
particularly interleukin-6 (IL-6) and granulocyte colony-stimulating
factor (G-CSF). These results suggest that in addition to its central
role in cellular immunity, IFN- may be a key cytokine in coordinate
regulation of immune effector cells and myelopoiesis. This model should
be valuable for deciphering the cross-talk between the immune response
and hematopoiesis during bacterial infection and for improving our understanding of the mechanisms that control chronic infections.
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INTRODUCTION |
ANALYSIS OF THE host response to
mycobacterial infections has shown much about the roles of cytokines in
the regulation of the aquired immune response. One of the most
important cytokines involved in antimycobacterial immunity is
interferon- (IFN-). Several lines of evidence demonstrate that
IFN- has an indispensible role in antimycobacterial immunity. Mice
lacking functional IFN- or IFN- receptor (IFN-R)
genes1-5 are extremely susceptible to infection with both
virulent Mycobacterium tuberculosis1, 3 and
avirulent M bovis BCG strain.2 Humans with null
mutations in IFN-R genes are also susceptible to mycobacterial
infection.6,7
The mechanism of IFN- action in controlling and eliminating
mycobacteria involves at least two functions; the development of a Th1
immune response and the activation of macrophages (reviewed in Cooper
and Flynn8). On recognition of infected macrophages, IFN- production from antigen-specific CD4+ T cells is
initiated to activate macrophages to kill intracellular bacteria. The
mechanism of action involves the activation of downstream molecules
such as the transcription factors STAT1 and IRF-1,9 which
are considered essential for regulating the expression of genes whose
products are required for macrophage effector functions such as
inducible nitric oxide synthase (iNOS). Mice with targeted mutations in
the genes encoding STAT1, IRF-1, and iNOS are susceptible to
mycobacterial infection,10-13as well as other intracellular
pathogens.14,15
The response of an organism to infection entails a carefully
choreographed series of inflammatory events, with macrophages and
neutrophils playing a critical role in the acute cellular phase
followed by the development of aquired immunity specifically directed
against the pathogen. The hematopoietic system must respond to
infection by adjusting the production of the appropriate cell types.
The roles of specific cytokines in the production of different hematopoietic cell types are relatively well understood, but less is
known about the coordinate regulation of the immune response and
hematopoiesis when stressed by infections. In immunodeficient mice
susceptible to chronic infection, this regulation is perturbed, providing a valuable model system.
While doing experimental infections in IFN--deficient mice using an
avirulent M bovis strain (BCG), we observed that mice succumbed
to infection as expected2 and had a total shift to a Th2
response to the bacteria. However, in contrast to wild-type animals,
IFN--deficient mice mounted a pathologic inflammatory response
characterized by expansion of hematopoietic lineages in the spleen and
bone marrow, an increase in precursor activity in these organs, and a
massive granulocytosis in the spleen and blood. The results suggest
that in the absence of IFN- and effective macrophage function, a
pathologic granulocytic reponse is mounted in an attempt by the
organism to combat mycobacterial infection. These data also suggest
that IFN- is a key cytokine in regulating communication between
immune effector cells and the hematopoietic system.
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MATERIALS AND METHODS |
Mice.
Mice heterozygous for the targeted mutation in the gene encoding
IFN- created by Dalton et al2 were initially obtained from The Jackson Laboratories (Bar Harbor, ME). We used IFN-  / mice on either the Balb/c or C57BL/6 backgrounds
throughout these experiments. The pathology observed and time to death
were essentially the same in either strain, however, some minor
differences were observed, and these are noted in the text, tables, and
figure legends where appropriate. IFN- / mice have
been housed and bred under conventional conditions for over 2.5 years
with no observable consequences to their health. Extensive analysis has been performed on naive IFN- / mice and relevant
results are noted in the text. Most mice used in this study were
obtained by intercrossing / or +/+ mice. In some
experiments, Balb/c or C57BL/6 mice were purchased from The Jackson
Laboratories and used as controls. When genotyping was necessary,
Southern blots of BamHI-digested tail DNA were probed with a
full-length IFN- cDNA.
Mycobacteria.
The Pasteur BCG strain was used in this study. BCG was grown in MH9
broth as described.16,17 In some experiments, recombinant mycobacteria secreting a variety of cytokines16 were also
used to inoculate IFN- / mice.
Infections.
Mice were infected at 8 to 12 weeks of age by either the intravenous
(IV) or intraperitoneal (IP) route with comparable results. A total of
seven independent experiments were performed, using four to 20 mice per
group. Mice were killed at a variety of times postinfection to examine
features of the pathology. For most experiments, mice were inoculated
with 1 × 105 colony-forming units (CFUs) of bacteria.
For time course studies, 1 × 106 CFUs bacteria were
used. This increased the time to morbidity by
approximately 2 weeks. Bacterial numbers injected were
determined by plating serial dilutions of the bacterial suspension on
MH10 plates and counting colonies 21 to 28 days later.
Histology.
Tissues (liver and spleen) were fixed in phosphate-buffered formalin
and embedded. Sections were stained with hematoxylin and eosin or with
the Ziehl-Neelsen stain for acid-fast bacteria and counterstained with
methylene blue. Bone marrow aspirates and spleen cells were analyzed by
cytospin and staining with Leukostat (Fischer Diagnostics, Pittsburgh,
PA).
Blood and cytospins.
Mice were bled from the retro-orbital plexus and blood collected into
heparinized tubes (Vacutainer; Becton Dickinson, Franklin Lakes, NJ).
Smears were made and stained with Leukostat. White blood cell (WBC)
counts were made by diluting blood 1:100 in 2% acetic acid. Numbers
are expressed as WBC per microliter. Plasma was stored at
20°C until required for cytokine analysis.
Dispersed spleen and bone marrow cells (2 × 104) were
centrifuged onto slides and stained with Leukostat. Cell differentials were performed and details are recorded in the table notes and figure
legends. Generally, cells from individual mice were counted (100 to 300 cells). Results are recorded and the averages ± standard devations
(SD).
Cytokine levels.
Cytokine levels in the plasma were determined by enzyme-linked
immunosorbent assay (ELISA) using specific reagents for interleukin-6 (IL-6), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, IL-10 (Pharmingen, San Diego, CA), tumor necrosis factor- (TNF-) and IFN- (Endogen, Boston, MA). Detection
limits for ELISAs were as follows: IL-3 (5 pg/mL), IL-6 (20 pg/mL),
GM-CSF (100 pg/mL), IL-10 (50 pg/mL), TNF- (100 pg/mL), IFN- (20 pg/mL), IL-12 (50 pg/mL). G-CSF levels were assayed using a
G-CSF-responsive cell line (Ba/F3-GR) as described.18
These cells are Ba/F3 cells transfected with a plasmid encoding the
murine G-CSF receptor under the control of a constitutive promoter.
These cells can respond to both IL-3 (Ba/F3 is an IL-3-dependent
pro-B-cell line) as well as G-CSF. Each sample was analyzed for both
IL-3 and G-CSF along with other cytokines. Because plasma IL-3 levels
are generally low (see Fig 7), it is a reasonable assumption that G-CSF
levels are being measured, with the exception of any unknown factors that could stimulate these cells. A microassay was used as
described.18 Briefly, Ba/F3-GR cells were washed three
times with RPMI/10% fetal bovine serum (FBS) and plated in Terasaki
wells at 200 cells per well in a volume of 5 µL. Serially diluted
plasma or IL-3 or G-CSF was added in a volume of 5 µL. After 36 hours, the number of live cells per well was scored visually. Results
are reported as the lowest dilution factor in which there were 100 live
cells per well.
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| Fig 7.
Cytokine levels in the plasma of infected mice. Cytokines
were measured from plasma samples as described in Materials and Methods. Closed symbols: IFN- +/+ mice, open diamonds: IFN- / mice. Values are given in pg/mL except for G-CSF
levels, which are given in the lowest dilution (see basis for this
assay in Materials and Methods). Data representative of multipe
experiments. We consistently detect IL-6, but IL-3 and GM-CSF levels
are commonly at the background level of the ELISA assay.
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Antigen-specific T-cell responses.
Splenocytes were isolated from infected mice and stimulated with
purified protein derivative (PPD; a mycobacterial antigen mixture) as
described.16 Cytokines produced in response to PPD stimulation were measured as described.16,19
Hematopoietic colony-forming potential.
CFUs from the bone marrow or spleen were assessed by plating cells
(15,000 cells per mL) in complete methycellulose media (Stem Cell
Technologies, Vancouver, Canada) containing IL-6, IL-3, erythropoietin
(EPO), and stem cell factor. Colonies (greater than 30 to 50 cells)
were scored 10 days after plating. Results are expressed as described
in the text and figure legend to Fig 5.
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| Fig 5.
Progenitor numbers in the spleen of infected mice. (A)
Spleen CFC counts were performed at different times after infection from wild-type ( ) or IFN- / mice ( ). Results are
reported as total CFCs per spleen. The "0" time point is from
naive mice. (B) Total bone marrow CFC in wild-type () or IFN-
/ mice () from the same experiment shown in (A).
Results are reported as the total CFCs recovered in the leg bones of
each mouse. Each time point is an average of four to six mice per
group. Results were similar in three independent experiments.
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Bacterial CFUs.
Spleen preparations from infected mice were homogenized with a
hand-held blender and serially-diluted in phosphate-buffered saline
(PBS)-0.05% Tween-80. Homogenates were plated onto MH10 plates and
colony numbers scored 21 to 28 days later.
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RESULTS |
General pathological features of infected mice.
Our initial impetus to use the IFN- / mice in
experimental BCG infection was to test the hypothesis that BCG strains
secreting biologically active cytokines16 at the site of
infection could partially rescue the pathology in the mice and delay
death. We performed several experiments using BCG strains secreting
biologically active IL-2, GM-CSF, or IFN-, but none could avert the
extreme pathology in the IFN- / animals (data not
shown). In the absence of effective macrophage activation in
IFN--deficient mice, we anticipated observing a gradual necrosis of
the spleen with a concomitant expansion of bacterial numbers leading to
overwhelming infection and death, as was observed in M
tuberculosis infection.1,3 As expected, IFN-
/ mice were susceptible to BCG infection,2 most mice dying by 6 to 10 weeks postinfection. However, striking pathological changes were observed in the hematopoietic system.
BCG-infected IFN- / mice rapidly developed grossly
enlarged spleens (average weight  0.7 to 1.0 g compared with
wild-type mice average 0.15 g) that became progressively paler in
color as infection proceeded (Fig 1A).
Microscopic inspection of the spleen showed an effacement of the normal
tissue architecture (Fig 1, compare B with C). In contrast to wild-type
animals, IFN- / mice lacked the regular follicular
arrangement of the red and white pulp. The normal lymphocytic
populations of the white pulp of IFN- / mice were
progressively replaced with sheets of leukocytes comprised
predominantly of maturing elements of the myeloid lineage, macrophages,
and neutrophils (Fig 1, compare D and F with E and G and see data
presented below). The splenic red pulp was extensively replaced by
extramedullary hematopoietic tissue (Fig 1H). After 4 to 5 weeks
postinfection, the total number of lymphocytes recoverable from the
spleen dropped to 5% of the control or naive uninfected IFN-
/ mice (data not shown), consistent with a replacement of
the normal cell populations within the spleen. The hematopoietic
alterations observed in the spleen prompted further investigation of
the pathology of BCG infection in IFN- / mice.
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| Fig 1.
BCG infection of IFN- / causes profound
changes in the spleen. (A) Spleens isolated from infected IFN-
+/+ (top two spleens) or IFN- / (bottom two
spleens) mice 5 weeks after IP administration of BCG show the change in
size and color of the organs. Low power view of spleens sections from
IFN- +/+ (B) or IFN- / (C) mice. Note that the
overall structure of the red and white pulp is intact in IFN-
+/+ mice, but the normal architecture of infected IFN- / is completely effaced (photographed using a 10X
objective). Higher power views of the spleen show that the white pulp
of infected IFN- / mice is extensively infiltrated by
macrophages (E and G), while infected IFN- +/+ mice retain the
normal arrangement and cellular makeup of the white pulp (D and F).
Note that in (E), a small area of lymphocytes remains in the right-hand
portion of the white pulp. Lymphocytes gradually disappeared from the spleen. Magnifications: (D) and (E) (20X objective); (F) and (G) (40X
objective). The red pulp of infected IFN- / mice was
infiltrated with extramedullary hematopoietic tissue (H). Note the
presence of nucleated erythroid cells, cells of the granulocytic
series, and megakaryocytes (40X objective). All sections were stained with hematoxylin and eosin (H&E).
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Granulocytosis in the blood and spleen.
The blood of infected IFN- / mice reflected a marked
leukocytosis, in extreme cases characterized by WBC counts greater than
80,000 cells/µL (Fig 2). The leukocytosis
was most pronounced between days 7 and 17 postinfection (Fig 2).
Microscopic examination of the blood showed that a large expansion of
granulocytes and monocytes had occurred
(Fig 3A). Differential WBC counts
documented granulocytosis and an increase in monocytes
(Table 1).
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| Fig 2.
Expansion of blood leukocytes in infected IFN-
/ mice. Leukocyte counts (in cells per µL) were
measured at different times after infection (abcissa). Closed symbols
are IFN- +/+ mice, open diamonds are IFN- /
mice. Results are typical of numerous experiments and are further
documented in Tables 1 and 2 and Fig 4.
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| Fig 3.
Neutrophilia in the blood, spleen, and bone marrow of
infected IFN- / mice. (A) Peripheral blood (left
panels) or spleen cytospins (right panels) from infected IFN-
+/+ or IFN- / mice (indicated at right) were
stained with leukostat. Note that in the peripheral blood of IFN-
/ mice there are large numbers of mature neutrophils as
well as less mature band forms and numerous monocytes in this smear
(WBC of this mouse was 1.5 × 105/mL). The spleen of
IFN- / also shows cells predominantly of the
granulocyte series of various stages of maturity. All figures were
photographed through a 40X objective). (B) Bone marrow sections (left
panels) or bone marrow cytospins (right panels) from infected IFN-
+/+ or IFN- / mice (indicated at right) were
stained with H&E (sections) or leukostat (cytospins). The bone marrow of infected IFN- +/+ mice displays normal cellularity and
populations of the various cell types. The marrow of IFN-
/ mice has large foci of granulocyte development and the
cell types are largely band forms. Sections were photographed with a
20X objective and cytospins with a 40X objective.
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Examination of the cell types within the spleen (Fig 3B) confirmed that
replacement of the normal lymphoid population with extramedullary
hematopoietic elements had occurred. Enumeration of the cells types
recovered showed a relative expansion of progenitor and maturing
elements of the granulocytic and monocyte-macrophage lineages
(Table 2). Kinetically, granulocyte numbers
increased 7 days postinfection and remained high in the blood until
morbidity (Fig 4, left panels). In the
spleen, mature and immature granulocyte numbers also increased early in
infection (Fig 4, right panels) and declined as morbidity was
established.
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| Fig 4.
Differential leukocyte analysis of blood and spleen
populations measured over time in infected animals. Cell counts were
made on blood (left-hand panels) or spleen (right-hand panels)
leukocytes from the time course experiment (see Fig 3). Cells from
+/+ mice are shown in the top panels, while / cells
are in the bottom panels. Time after infection is shown in the
abscissa. Blood differential counts: open bars, lymphocytes; closed
bars, neutrophils and band forms (note that few band forms were
observed in +/+ mice while the number was high in /
mice); striped bars, monocytes. Spleen differential counts: open bars,
lymphocytes; diagonally striped bars, blast forms; closed bars, band
forms; vertically striped bars, mature (fully segmented nucleus)
neutrophils; grey bars, monocytes and macrophages. Eosinophils, which
comprised only a minor fraction of cells in the spleen, and erythroid
series cells, which were detected in comparable numbers, are not
depicted.
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Granulocytosis in the bone marrow.
The bone marrow of infected IFN- / mice developed a
spectacular expansion of granulocytic precursors (mainly band forms, Fig 3B, "/ bone marrow cytospin"), but the total
nucleated cell numbers recovered from the bone marrow at various times
after infection remained similar. The increased myeloid to erythroid ratio was confirmed by differential counting
(Table 3). Interestingly, there was no
apparent anemia in the infected animals, suggesting that the increased
erythropoiesis in the spleen had effectively compensated for any
reduction in bone marrow production. Although spleen erythroid numbers
did not increase in proportion, there was an absolute 10-fold to
15-fold increase in erythroid precursor mass due to splenomegaly (data
not shown).
Expansion of precursor cells on the spleen and bone marrow.
The shift towards myelopoiesis observed in the bone marrow and spleen
of infected IFN- / mice suggested that there would be
an increase in progenitor frequency in both of these organs. To address
this, total myeloid colony forming activity was measured. Relative to
wild-type infected animals, spleen CFCs increased significantly in
IFN- / mice, verifying the large expansion of
hematopoietic progenitors in the spleen following BCG infection (Fig 5A). The increase, as great as two
orders of magnitude, was detectable 3 days after infection and remained
high until morbidity. Analysis of the bone marrow showed that wild-type
animals mounted a distinct hematopoietic response to infection.
Wild-type mice manifested an initial decrease in myeloid progenitor
frequency, perhaps reflecting a mobilization of inflammatory cells, but
subsequently augmented bone marrow cellularity and myeloid progenitor
numbers during the course of infection. In contrast, total nucleated
cells remained relatively constant, as did CFC activity in the bone marrow of IFN- / mice (Fig 5B). Thus, the cellular
response in the spleen and bone marrow differed dramatically between
wild-type and IFN- / animals, with IFN-
/ mice mobilizing a pathologic extramedullary myeloid
response. At baseline in uninfected animals, spleen and marrow CFC
numbers were comparable in wild-type or IFN- / mice,
demonstrating that the dramatic hematopoietic remodeling occurred in
response to mycobacterial infection.
Bacterial numbers.
The organs that carry the greatest burden of mycobacteria following IP
injection, the liver and spleen,1,2,16,19 were analyzed for
bacterial numbers by plating tissue homogenates onto agar selective for
mycobacteria, as well as examing acid-fast-stained organ sections. As
shown in one example (Fig 6), bacterial
numbers in the spleen (after an IP injection of 1 × 106 CFUs BCG) increased in both +/+ and /
mice and were comparable at early time points (days 3 and 7; Fig 6B).
In wild-type animals, bacterial numbers peaked at 17 days and declined
thereafter, but in IFN- / mice, the bacterial load
increased throughout the course of infection. Microscopic examination
of Ziehl-Neelson-stained sections of the spleen and liver showed
comparable numbers of acid-fast bacteria in wild-type and
IFN--deficient mice at early time points. Later in infection,
numerous granulomatous lesions were observed in the livers of infected
IFN- / mice and were associated with florid bacterial
growth and necrosis in both liver and spleen (Fig 6). The expansion of
splenic myeloid progenitors and blood neutrophils in IFN-
/ mice is observed as early as day 7 postinjection,
before expansion of bacterial numbers in the spleen and liver, and
occurs when mycobacterial numbers are comparable in IFN-
/ and wild-type animals, suggesting that the pathologic
myeloid response in IFN- / animals is a consequence of
IFN- deficiency rather than elevated mycobacterial burden.
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| Fig 6.
Bacterial loads in infected mice. (A)
Histopathology in the liver. Low power (20X objective) views of the
liver of infected IFN- +/+ (top) or IFN- /
(bottom) mice show that numerous granulomatous lesions form in the
liver of IFN- / mice, while IFN- +/+ mice have
few granulomas. Granulomas in IFN- +/+ mice contained no
acid-fast bacteria (right-hand panel) at the time assayed (5 weeks
postinfection), while IFN- / mice contained numerous
bacteria (red rods within the granuloma structure). At the stage shown,
100% of granulomas contained acid-fast bacteria (data not shown) (B)
Quantitation of bacterial numbers in the spleen. Closed symbols:
IFN- +/+ mice, open diamonds: IFN- / mice. Time
after infection is shown in the abscissa. Dilutions of spleen
homogenates were plated onto MH9 media and bacterial colonies measured
21 to 28 days later.
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Levels of cytokines in the peripheral blood.
To investigate the possible mechanisms of the pathologic changes in the
BCG-infected IFN- / mice, we analyzed plasma levels of
a series of cytokines. Levels of IL-6 were increased in infected / mice compared with wild-type mice, while other
cytokines that may be relevant for hematopoietic expansion (such as
IL-3 and GM-CSF) remained low. An example of such an analysis is
depicted in Fig 7. Plasma from individual
mice was examined at four time points after infection, and cytokine
levels were analyzed by ELISA or bioassay. Results are shown for
IFN-, IL-6, IL-3, and G-CSF. We did not detect GM-CSF, TNF-, or
IL-12 in the plasma, and levels of IL-10 were very low. The results
show that in wild-type animals, IFN- levels increase, then fall
systemically as the infection is controlled. In mice lacking IFN-,
however, levels of IL-6 and G-CSF were generally higher at all time
points examined, but increased dramatically when most mice were
moribund (Fig 7). The systemic increase in IL-6 and G-CSF may
contribute to the observed granulocytosis, but the greatest increase in
these factors occurs well after the peak of myeloid activity detected
in the blood and spleen.
Antigen-specific T-cell responses.
Because IFN- is essential for the development of a Th1 response to
mycobacteria, we also tested if the expected shift to a Th2 phenotype
had occurred in the infected IFN- / mice. When splenocytes were stimulated with PPD, readily detectable levels of
Th2-associated cytokines such as IL-3, IL-4, and IL-5 were produced
(Fig 8). These results confirm the findings
of others using IFN- / mice and show that IFN- is
essential for the development of a Th1 response to BCG. In the absence
of IFN-, T-cell activity is shifted to a Th2 phenotype.
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| Fig 8.
In infected IFN- / mice, the T-cell
response to BCG is profoundly shifted to the Th2 phenotype. Splenocytes
from infected IFN- +/+ mice (left side of each panel) or IFN-
/ mice (right side of each panel) were stimulated in
vitro with media alone (open bars) or PPD (filled bars). Cytokine
levels were measured by ELISA after 72 hours stimulation and expressed
in pg/mL. In the experiment shown, mice on the C57BL/6 background were
used. Similar results were obtained using IFN- / on
the Balb/c background.
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DISCUSSION |
Models systems where the normal regulation of cytokine levels is
disrupted are valuable tools to understand hematopoiesis, and in
particular, to probe the mechanisms of leukocyte differentiation and
production. Bacterial, protozoan, and viral infection in these models
can provide additional information about how the immune system
component of hematopoiesis responds to invading microorganisms. In the
absence of IFN-, BCG-infected mice profoundly remodeled their
hematopoietic system to favor increased granulocyte production in the
marrow and spleen. The model described here yielded unexpected results that further defines the critical role of IFN- in regulating both macrophage function and the adaptive response to infection.
Pathological consequences of BCG infection in IFN--deficient
mice.
We anticipated that experimental infection of IFN- /
mice with BCG would expose the strain's faulty immunity. As expected, the mice became visibly moribund about 4 to 5 weeks after a standard IP
administration of BCG and died several weeks later. However, in our
attempts to purify splenic T cells to analyze the Th2 response, we were
intrigued by the pathologic changes observed. Despite gross
splenomegaly, T cells were virtually absent from the spleen 4 weeks
after infection of IFN- / mice, and histologic
examination showed effacement of normal red and white pulp. The red
pulp contained numerous hematopoietic elements and superficially
resembled bone marrow. The white pulp was tightly packed with
macrophage-like cells and granulocytes, giving the spleen the
pathologic appearance of a myeloproliferative disorder. Quantitation of
the cell types in the spleen showed a diminution in the percentage and
absolute numbers of lymphocytes and an expansion of granulocytic
elements. The expansion of granulocyte numbers in the spleen was also
reflected in an increased blood leukocyte count, comprised almost
exclusively of neutrophils and band forms. The bone marrow also showed
a large increase in the percentage of cells in the granulocytic series. The pathologic changes resembled a severe leukemoid reaction or a
myeloproliferative disorder. This was in marked contrast to what was
observed in infected wild-type animals; the leukocyte count remained
steady at all times after infection, although the percentage of
neutrophils increased during the transient phase of active infection
(see Fig 4). On successful resolution of infection, the relative
percentage of neutrophils in the blood returned to normal. Cell
populations in the spleen and marrow of wild-type mice remained steady
in all experiments.
Examination of cytokine levels in the plasma showed that in infected
wild-type mice, IFN- levels were easily detectable early in the
infection (see for example Fig 7), presumably as a consequence of
increased T-cell and macrophage activity. Increased IL-6 levels were
recorded in most mice, but particularly late in IFN- / mice, suggesting that IL-6 may have played a role in driving the pathologic granulocytosis in IFN- / mice. In addition,
G-CSF levels were greatly elevated in IFN- / mice,
particularly towards death. Because G-CSF is essential for regulating
neutrophil production18 (and see below), any increase in
levels of this cytokine may be a significant factor in expansion of
granulocytes in infected mice.
Our attempts to reverse the extreme phenotypes observed in
IFN--deficient mice by administration of a BCG strain secreting active IFN- failed, despite data that adequate levels of IFN- were produced by the mycobacteria to activate macrophages and induce
IRF-1 and iNOS expression in vitro16 (and data not shown). Attempts by Flynn et al3 to reconstitute M. tuberculosis-infected IFN--deficient mice with exogenous IFN-
similarly failed to rescue lethality caused by infection. The context
in which IFN- is used by the immune system appears essential in
controlling mycobacterial infection.
Comparison to other models of disregulated hematopoiesis.
Some previously described murine models of perturbed hematopoiesis have
exhibited hematopoietic parameters similar to those described here;
namely increased spleen EMH, increased granulocyte production, and decreased cellularity in the bone marrow. Continuous administration of the IL-4-like Th2 cytokine IL-13 induces
extra-medullary hematopoiesis (EMH) and splenomegaly with markedly
elevated numbers of hematopoietic CFUs, a pathology mimicked by
infection with Nippostrongylus brasiliensis, which induces a
strong Th2 response.20 Mice lacking the IL-8 receptor show
a deficiency in neutrophil migration and recruitment to sites of
infection and consequently develop a compensatory neutrophilia and
excessive EMH.21 Overexpression of cytokines including
IL-6,22 G-CSF,23 and IL-524 has
been shown to induce grossly elevated levels of neutrophils or
eosinophils. Enhanced granulopoiesis due to genetic changes that
increase the pool of progenitors has been documented in the max41
transgenic mouse25 and following retroviral transduction of
the bcr-abl oncogene.26 The model described by Johnson et
al27 in which bone marrow cells transduced with a
GM-CSF-expressing retrovirus are allowed to repopulate a
lethally-irradiated recipient has similar features to BCG-infected
IFN- / mice. These mice develop a lethal
myeloproliferative syndrome with progressive loss of cells in the bone
marrow, enhanced neutrophil and monocyte/macrophage production, spleen
hypercelluarity, and a possible decrease in lymphocyte numbers.
Does unregulated cytokine production play a role in the hematopoietic
abnormalities observed in BCG-infected IFN- / mice? Our results show that IL-6 and G-CSF levels are indeed elevated in
infected IFN- / mice. Interestingly, the highest
levels were observed late in infection and did not correlate with the expansion of spleen progenitor numbers or blood leukocytes. Because our
assays determine plasma cytokine levels and not local production in the
marrow or spleen, our analysis of the role of cytokines is limited.
Current studies are aimed at addressing the exact roles of these
factors through a genetic approach by intercrossing mutant strains of
mice to generate animals lacking both IFN- and IL-6 or IFN- and
G-CSF. We will also need to determine if ectopic production of other
cytokines such as GM-CSF or IL-3 play a role in the pathology. Because
IFN- / mice have a strong Th2 response, T-cell
production of IL-3 and GM-CSF is elevated (Fig 8 and P.J.M.,
unpublished observations). It would also be informative to test BCG
infection in mice which lack IFN- and T cells by intercrossing
IFN- / with RAG2 / or nu/nu
mice. A similar strategy was used by Sharara et al28 to
show that T cells drive EMH in mice laking the common chain of the
receptors for IL-2, IL-4, IL-7, IL-9, and IL-15.
Comparison to other infection models of IFN--deficient mice.
Pathogenesis in IFN--deficient mice has been studied in several
other well characterized models. M tuberculosis-infected IFN- / mice die rapidly with unchecked bacterial
growth and caseous necrosis in the liver and other
organs.1-3 These investigators described some granulocytic
infiltration in the lungs, but other hematopoietic parameters were not
noted. Toxoplasma gondii infection of IFN- /
mice was particularly revealing.29 These mice fail to
control the infection, but develop a robust IL-12 response. Significantly, the mice had granulomatous infiltration of the peritoneum (the site of innoculation) characterized by excess neutrophils and eosinophils. Examination of other hematopoietic parameters in T gondii-infected IFN- / mice
may show similarities with the model described here. Finally,
Leishmania infection of IFN- / mice has been
described by several groups. L major is lethal to these mice,
which develop a prominent Th2 response.30 In contrast,
L donovani infection provided a contrary example of a parasite
whose growth was initially unchecked, but surprisingly began to resolve
3 months after infection.31 Taylor and Murray31 speculate that, in the absence of IFN-, IL-12 drives an
anti-Leishmanial pathway that depends on TNF- to activate macrophage
effector function. Thus IFN- / mice have been useful
in defining absolute requirements for IFN- action (eg, in all
mycobacterial infection) as well as IFN--independent effector
functions. It will be interesting to determine if hematopoietic
remodeling occurs in these other models.
Possible mechanisms of hematopoietic expansion in infected IFN-
/ mice.
A fascinating pathologic process is initiated in BCG-infected IFN-
/ mice that leads to a profound dysregulation of normal hematopoiesis. The lessons learned from this system help demonstrate the role of IFN- in normal immunity and expose compensatory
mechanisms that arise under conditions of immune deficiency. There are
several mechanisms that might account for the pathologic changes we
observed in the IFN--deficient mice. The most provocative
hypothesis to arise from our observations is that IFN- normally
plays a critical role in downregulation of the acute inflammatory
response and modulation of the humoral (neutrophilic) and cellular
(macrophage) arms of the immune response; loss of the negative
regulatory function of IFN- itself predisposes to pathologic
neutrophilia. This hypothesis is supported by observations on the
activity of IFN- on hematopoietic colony assays in vitro and various
clinical observations.
IFN- is a key activator of macrophage function, but it is also a
potent suppressor of hematopoiesis. IFN- has been shown to have
potent inhibitory effects on a diversity of myeloid and lymphoid
progenitors and long-term culture initiating cells (LTC-IC) when
expressed in stromal cells in long-term bone marrow
cultures32 or in transgenic mice.33 IFN-
binds directly to receptors on erythroid precursors and suppresses
erythroid colony formation in vitro.34 IFN- appears to
be a central effector of aplastic anemia,35 the anemia of
chronic disease,36 and acquired immunodeficiency syndrome
(AIDS)-related cytopenias.37,38 IFN- inhibits the neutrophil-stimulating activity of IL-8 and ENA-7839; the
absence of IFN- function may thus lead to unopposed neutrophil activation. The precise role of IFN- in hematopoietic regulation is
complex, as some in vitro data argue that IFN- may promote modest
expansion of the earliest CD34+ hematopoietic
precursors40,41and may stimulate platelet production by
effects on megakaryocytes.42 Other work on the direct
effects of IFN- on single cell colony assays of CD34+
bone marrow progenitors show that IFN- stimulates monocytic colonies
and inhibits granulocytic colonies, effects that are antagonized by the
Th2 cytokine IL-4.43
Genetically deficient strains of mice provide insights that complement
the in vitro data on the role of interferons in hematopoiesis. Mice
deficient in |
Abstract
Introduction
Abstract