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*
Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105; and
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 upon stimulation with LPS.
When macIL-10tg mice were challenged with LPS, serum levels of
proinflammatory cytokines were attenuated compared with controls.
Infection with Mycobacterium bovis bacille
Calmette-Guérin resulted in 
10-fold-higher bacterial loads
than in wild-type mice. Normal T and B cell responses were observed in
macIL-10tg mice, suggesting that macrophage-specific overexpression of
IL-10 predominantly acts in an autocrine/paracrine manner, resulting in
chronically deactivated macrophages that manifest an impaired ability
to control pathogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and IL-12
(2, 3, 4). Inhibition of IL-12 production by IL-10
(4) may be a mechanism by which IL-10 weakens the
development of Th1-type T cell responses (5). However,
IL-10 can also have immunostimulatory effects by inducing the
proliferation of mast cells (6) and CD8 T cells
(7). As demonstrated in IL-10 gene-deficient mice,
the absence of IL-10 causes overproduction of inflammatory cytokines
after LPS challenge (8) and development of chronic
inflammatory bowel disease (9). However, IL-10-deficient
mice also show increased resistance to intracellular pathogens as
diverse as Leishmania major (10),
Listeria monocytogenes (11), Chlamydia
trachomatis (12), Mycobacterium avium
(13), and Mycobacterium bovis bacille
Calmette-Guérin
(BCG)3 (14, 15). Thus, IL-10 is required to prevent immunopathology, but it
can also delay or impair protective responses against pathogens.
We previously found that overexpression of IL-10 from T cells in a
transgenic mouse line impairs clearance of BCG (16). T
cells from these mice produced abundant IFN-
and IL-2 in response to
BCG infection, indicating that excess IL-10 inhibited killing of BCG at
the level of the infected macrophage without affecting T cell
responses. The use of T cell IL-10-transgenic mice has also provided
important information on the role of T cell-derived IL-10 in models of
autoimmune diseases (17, 18). However, because IL-10 is
only overexpressed in these mice when T cells become activated, effects
of overproduction of IL-10 by innate immune cells cannot be studied.
The phenotype of mice with a macrophage-specific disruption of STAT3
(19) closely resembles that of IL-10-deficient mice,
suggesting that macrophages are the major target of IL-10 in vivo. For
these reasons, we were interested in creating a mouse model to analyze
the consequences of overexpression of IL-10 from macrophages
themselves. To direct transgene expression to macrophages, promoter
fragments of the CD11b (20), c-fms
(21), scavenger receptor A (SR-A)
(22), lysozyme (23), and MHC class II
(MHC-II) (24) genes have been used. None of these
constructs is ideal for macrophage-specific expression of transgenes,
because they are either not restricted to macrophages or only active in
a subset of these cells. Human CD68 and its murine homolog macrosialin
are expressed in the endosomal compartment of cells of the mononuclear
phagocyte lineage (25, 26, 27). To achieve macrophage-specific
overexpression of murine IL-10 tagged with a Flag epitope in vivo, we
have used an expression cassette that combines 2.9 kb of the CD68 5'
flanking region with the 83-bp first intron of the CD68 gene, which has
been shown to contain a macrophage-specific enhancer (28).
This combination of human CD68 gene sequences directed
macrophage-specific expression of a type III human SR-A in
transgenic mouse lines (29).
In this study, we show that human CD68 sequences can be used to direct constitutive expression of a Flag-IL-10 transgene in vivo specifically in macrophages. Transgenic IL-10 acted on macrophages in an autocrine manner, resulting in deactivated macrophages with impaired capacity to produce inflammatory cytokines when challenged with LPS. Although adaptive immune responses were largely intact, macIL-10tg mice were unable to clear mycobacteria efficiently.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine IL-10 cDNA was amplified by PCR to lack the
endogenous sequence encoding the signal peptide. The PCR product was
digested with MluI and NheI (introduced in the
amplifying oligonucleotides) and cloned into the same sites in
pEF-BOS/s/Flag (gift of Dr. D. Hilton, Walter and Eliza Hall
Institute, Melbourne, Australia). The IL-10 coding region is
fused in frame to DNA encoding the IL-3 signal sequence and the Flag
epitope. To express Flag-IL-10 under control of the CD68 promoter, we
made use of the CD68 promoter construct described recently
(29), which combines the 2940 bp of sequence 5' to the ATG
and the 83-bp first intron of the human CD68 gene in a pcDNA3 backbone
where the CMV promoter had been first deleted. The human SR-A
sequence was removed by digesting the construct with XbaI.
The Flag-IL-10 fragment was released from pEF-BOS/s/Flag-IL-10
with NheI and ligated into the XbaI sites of the
CD68 promoter construct. To express Flag-IL-10 under control of
different fragments of the CD68 promoter, the Flag-IL-10 cDNA was
cloned into a version of pcDNA3.1 lacking the CMV promoter. Fragments
of the CD68 promoter were amplified from the expression cassette and
cloned upstream of the Flag-IL-10 cDNA using EcoRV and
NotI. For injection into male pronuclei of FVB zygotes, the
constructs were digested with the enzymes indicated in Fig. 1
and purified according to standard
procedures, and the concentration was adjusted to 1.5 ng/µl.
Offspring derived from the injections was genotyped by 3-primer PCR
amplifying a 150-bp fragment of the transgene and a 200-bp fragment of
the murine IL-10 gene. Southern blotting of DNA digested with
EcoRI, followed by probing the membrane with the
CD68-Flag-IL-10 fragment labeled with
[-32P]dCTP, confirmed the PCR results and
was used to estimate copy numbers of integrated transgene.
Transgene-bearing founder mice were mated with FVB mice. As described
in the results, only one founder mouse (TG445) produced viable
offspring. This line was designated macIL-10tg and was bred
hemizygously by mating transgene-positive males with wild-type FVB
females.
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Bone marrow-derived macrophages (BMDM) were generated as described previously (16, 30) by differentiation in 15% L cell-conditioned medium as a source of M-CSF for 5–7 days. To obtain peritoneal-derived macrophages (PDM), mice were injected with 3 ml of 4% Brewer’s thioglycolate and were sacrificed after 3 days, and the peritoneal cavity was flushed with 10 ml ice-cold PBS. Cell culture medium for macrophage cultures was DMEM supplemented with 10% FBS, 2-ME (50 µM), and antibiotics. Cell suspensions of spleens, lymph nodes, and thymus were prepared by straining the organs through a nylon mesh (Falcon, Mountain View, CA) and lysis of erythrocytes with NH4Cl followed by two washes with complete medium and counting. These cells were cultured in RPMI 1640 supplemented as above stated for DMEM.
T cell proliferation assay
A total of 5 x 104 splenocytes per well were plated in quadruplicate in 96-well U-bottom plates and stimulated with anti-CD3 mAb. After 40 h, [3H]thymidine (0.25 µCi/well) was added for 8 h, the plates were harvested onto filters, and the amount of incorporated radioactivity was determined with a scintillation counter.
Cytokine detection by ELISA
The concentration of IL-10, Flag-IL-10, TNF-, IFN-, and
IL-12p40 in sera or culture supernatants was determined at appropriate
dilutions by sandwich ELISA using Ab pairs IL-10, IL-12p40, TNF-
(all purchased from BD PharMingen, San Diego, CA), and IFN-
(Endogen, Woburn, MA). For detection of Flag-IL-10, the plates were
coated with anti-Flag M2 Ab (Sigma-Aldrich, St. Louis, MO) to
capture specifically Flag-IL-10; detection was performed using
biotinylated anti-IL-10 Ab.
Flow cytometry
Biotinylated anti-mouse F4/80 Ab was from Serotec (Raleigh,
NC). All other Abs and reagents for flow cytometry were from BD
PharMingen. For staining of cell surface proteins, cells were washed in
PBS with 2% FBS and then incubated for 30 min at 4°C with the
respective combinations of fluorescently labeled Abs. For analytical
purposes, cells were analyzed on a FACSCalibur (BD Biosciences, San
Jose, CA) and sorting was done at the St. Jude Core Facility using a
MoFlo sorter (Cytomation, Fort Collins, CO). To stain for intracellular
cytokines, after the staining procedure for cell surface proteins was
completed cells were fixed in 2% paraformaldehyde for at least 20 min
at room temperature. Cells were then washed two times in staining
buffer (PBS with 0.5% saponin, 0.5% BSA, and 0.05% azide), followed
by a 30-min incubation with PE-labeled anti-TNF- or
anti-IL-10 Ab in staining buffer. After washing again two times,
cells were taken up in PBS and analyzed.
Real-time quantitative RT-PCR
Total RNA from cells and mouse tissues was prepared using
the Absolutely RNA kit (Stratagene, La Jolla, CA), which includes a
DNase I digestion step to remove contaminating genomic DNA. One
microgram of RNA was reverse-transcribed using Superscript II (Life
Technologies, Rockville, MD) and a mixture of oligo(dT) and random
primers. Real-time quantitative PCR was performed on a SDS7700
instrument (PE Applied Biosystems, Foster City, CA). Primers and
probes were designed using PrimerExpress software and had the following
sequences: 
-actin (sense 5'-ACCCACACTGTGCCCATCTAC-3'; antisense
5'-AGCCAAGTCCAGACGCAGG-3'; probe
5'-AGGGCTATGCTCTCCCTCACGCCA-3'), IL-10 (antisense
5'-CCCAAGTAACCCTTAAAGTCCTGC-3'; probe
5'-CCCAAGTAACCCTTAAAGTCCTGC-3', sense
5'-ATAACTGCACCCACTTCCCAGTC-3'), murine IL-10 (sense
5'-ATGCTGCCTGCTCTTACTGACTG-3'), Flag-IL-10 (sense
5'-CAGGACTACAAGGACGACGATGAC-3'). Fluorogenic probes were labeled with
FAM at the 5' and with TAMRA at the 3' end and synthesized by PE
Applied Biosystems. Because primers for -actin and Flag-IL-10
amplified genomic DNA also, controls of RNA samples were included to
control the efficiency of DNase I digestion. To generate standard
curves, PCR products were cloned into pCR2.1 (Invitrogen, Carlsbad,
CA), and miniprep DNA was digested with EcoRI and, after
estimation of DNA quantity on an agarose gel, diluted serially down
from 106 to 1 copies/µl. cDNA samples and
standard dilutions were analyzed in duplicate.
Immunization with OVA
Mice were immunized with 100 µg/footpad OVA (Sigma-Aldrich) in CFA (Pierce, Rockford, IL) followed by a booster injection with 100 µg/footpad OVA in IFA 10 days later. Twenty days after the first injection, mice were sacrificed, blood was collected, and the popliteal lymph nodes were prepared. OVA-specific Ab titers were determined by ELISA. Briefly, plates were coated with OVA (2 µg/ml) and blocked with PBS containing 10% FBS. Sera were diluted down 3-fold serially and allowed to bind. After extensive washing with PBS 0.05% Tween, OVA-specific Ig isotypes were detected by incubation with AP-conjugated isotype-specific goat anti-mouse Abs. Again after extensive washing, substrate was added and the OD450 was measured. The last serum dilution yielding an OD450 value of 0.1 over background was recorded as the endpoint titer for each sample.
LPS challenge
Escherichia coli LPS was purchased from Sigma-Aldrich (catalog no. L4130), dissolved at 5 mg/ml in PBS, and diluted before injection, and a volume of 200 µl was injected i.p. At the indicated time points, mice were sacrificed, blood was collected, and the serum was separated by centrifugation.
Infection with BCG
BCG Pasteur was grown in 40 ml Middlebrook 7H9 broth for 3 days,
washed twice with PBS with 0.02% Tween 80, taken up in 20 ml and
diluted 1/20 in PBS with 0.02% Tween 80, and sonicated in a water bath
sonicator for 15 s to break up clumps. Mice were injected in a
lateral tail vein with 200 µl of this solution, which corresponded to
106 viable bacteria, as determined by plating
of the inoculum. After 2, 5, and 8 wk, groups of mice were sacrificed
and tail DNA was prepared for confirmatory genotyping. The left lung,
upper half of the spleen, and the upper lobe of the liver were used for
determination of CFU. A lobe of the right lung, a piece of the spleen,
and the liver were placed in 4% formaldehyde for histology. The rest
of the spleen was taken for preparation of splenocytes for
restimulation in vitro.
Isolation of CD4 T cells from splenocytes
Splenocytes from groups of six BCG-infected or control mice were prepared as described above and pooled. A total of 2.5 x 108 splenocytes were resuspended in 1.8 ml of ice-cold PBS containing 2% FBS (wash buffer) followed by addition of 200 µl mouse CD4 (L3T4) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were incubated for 30 min at 4°C followed by magnetic separation with a LS positive selection column (Miltenyi Biotec). After washing the column with 3 ml wash buffer, the cell suspension was applied to the column and negative cells were allowed to pass through. The column was washed three times with 3 ml wash buffer. Then the column was removed from the separator and the CD4 T cells were flushed out with 6 ml wash buffer. Cells were centrifuged and resuspended in complete RPMI 1640.
Western blot analysis of macrophage lysates
BMDM were plated in 12-well plates at 2 x 106/well and harvested in 150 µl RIPA buffer containing a protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany) and sodium orthovanadate to inhibit phosphatase activity. After 30 min on ice, lysates were cleared from debris and SDS sample buffer was added. A total of 40 µl lysate was used for SDS-PAGE on 4–15% gradient gels (Bio-Rad). Gels were blotted at 70 V for 2 h onto nitrocellulose membranes. After confirming even transfer by Ponceau staining, the membrane was blocked with 3% nonfat dry milk in PBS with 0.05% Tween 20 for 1 h at room temperature. Primary Abs were diluted in blocking buffer and the membrane was incubated overnight at 4°C. After washing with PBS 0.05% Tween 20, membranes were exposed to peroxidase-conjugated anti-rabbit sera (1/5000 dilution in blocking buffer) followed by three washes in PBS 0.05% Tween 20. Blots were developed using ECL substrate and exposure times between 10 s and 5 min as needed to yield the appropriate signal. Abs against STAT1 and STAT3 and phosphorylated STAT1 and STAT3 were from New England Biolabs/Cell Signaling (Beverly, MA). The anti-suppressor of cytokine signaling (SOCS)3 antiserum was a kind gift from Dr. Y. Takahashi (St. Jude Children’s Research Hospital, Memphis, TN).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Constructs for expression of Flag-IL-10 under control of three
different fragments of the CD68 promoter (Fig. 1
) were injected into
male pronuclei of fertilized FVB oocytes. Six transgenic founder mice
were obtained (Table I). Although all
founder mice developed normally, only TG445 produced viable offspring.
The other transgenic founder mice appeared ill after 6–10 wk of age
and finally died or were euthanized for humane reasons. From two of
these founder mice (TG454 and TG520) serum and tissue samples were
analyzed and very high levels of IL-10 were detected in the serum,
associated with high copy numbers of the transgene as determined by
Southern blotting (Table I and Fig. 1). Although the cause of disease
and death in these mice was unclear, histological findings were
consistent with systemic infection, spreading from pyelonephritis in
one mouse (TG520) and a lumbar soft tissue abscess in another (TG454)
(Table I). Seminiferous tubular degeneration and aspermatogenesis in
founder TG520 explain the infertility observed. The male founder TG445,
harboring only one or two copies of the transgene, showed no signs of
disease and transmitted the transgene in a mendelian fashion. This line
was designated macIL-10tg and is described in this work.
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Although IL-10 was undetectable in the sera of FVB mice, it was
detected at moderate levels in macIL-10tg mice (Fig. 2A). Injection of a
biotinylated anti-IL-10 Ab greatly increased the sensitivity of
detection, as described for IL-4 and IFN- (31), and
revealed that the amount of IL-10 produced in macIL-10tg under normal
conditions is similar to levels observed when FVB mice were injected
with LPS (Fig. 2A). At the mRNA level, real-time
quantitative RT-PCR showed that in spleens of untreated macIL-10tg mice
IL-10 mRNA is 100-fold more abundant than in FVB control spleen
(Fig. 2B). At the protein and at the mRNA level this
difference is entirely due to transgenic Flag-IL-10 (Fig. 2, A and B). When cells from different tissues were
cultured without stimulation, IL-10 could be detected in the
supernatants of macIL-10tg splenocyte and bone marrow cell cultures
and, to a much lesser extent, of lymph node cells but not thymocytes
(Fig. 2C). To define the source of IL-10 more directly,
adherent splenocytes were cultured in the presence of brefeldin A to
block secretion of cytokines and were stained for the
macrophage-specific surface marker F4/80 and intracellular IL-10 (Fig. 2D). IL-10 was detected in the F4/80+
macrophage population in macIL-10tg splenocytes but not in the FVB
counterpart. Similarly, when splenocytes from macIL-10tg mice and FVB
littermate controls were FACS sorted for T cell, B cell, granulocyte,
and macrophage lineage cells, enrichment for macrophages
(Mac1+GR1-) strongly
increased the amount of IL-10 produced from macIL-10tg cells (Fig. 2E). Finally, BMDM from macIL-10tg mice produced high levels
of IL-10 that slightly increased after stimulation with LPS (Fig. 2F), whereas FVB BMDM produced IL-10 only after stimulation
with LPS. Taken together, these experiments show that Flag-IL-10 is
expressed in a macrophage-specific and constitutive manner in
macIL-10tg mice.
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When compared with FVB littermates, macIL-10tg mice showed no
major differences in the distribution of T cells and B lymphocytes in
spleen and mesenteric lymph node, except for a slightly reduced
frequency of CD8+ T cells (Table II). The percentage of
Mac1+ cells was slightly but significantly
increased in macIL-10tg spleens. Overall, lymphoid development and the
cellular composition of the peripheral lymphoid organs appeared normal
in macIL-10tg mice. When splenocytes were stimulated with anti-CD3,
proliferation as measured by incorporation of
[3H]thymidine was indistinguishable between FVB
and macIL-10tg splenocytes (Fig. 3A). The ability of macIL-10tg
mice to mount an Ag-specific immune response was tested by immunization
with OVA in CFA (Fig. 3B). The intensity and isotype
distribution of OVA-specific Ab production did not differ between FVB
and macIL-10tg mice, as determined by endpoint titers. In addition to
an unaltered B cell response, macIL-10tg mice developed strong T cell
memory, because restimulation of popliteal lymph node cells with OVA in
vitro resulted in strong production of IFN- (data not shown).
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and IL-12
when stimulated with LPS in vitro
Consequences of overexpression of IL-10 on macrophage function
were first evaluated in vitro using both PDM and BMDM. Compared with
FVB macrophages, stimulation of macIL-10tg macrophages with LPS
resulted in strongly reduced amounts of both TNF- (reduced by
83.6%) and IL-12p40 (71.4%) in the culture supernatants (Fig. 4A). As expected, addition of
exogenous IL-10 strongly reduced the levels of both cytokines in FVB
macrophages but had much less effect on macIL-10tg macrophages.
Importantly, addition of a neutralizing anti-IL-10 Ab to the
cultures restored cytokine production from macIL-10tg macrophages,
demonstrating that the inhibition observed is due to transgenic
Flag-IL-10. Compared with the strong inhibition of TNF- and
IL-12p40, production of NO by macrophages stimulated with LPS was less
affected in macIL-10tg (30.2% reduction)- and IL-10-treated FVB
macrophages (Fig. 4A). In the case of TNF-, the kinetics
of production after LPS stimulation were analyzed by intracellular FACS
staining (Fig. 4B). In FVB macrophages, LPS treatment
induced a sharp increase in the percentage of
TNF-+ cells (45.1% after 30 min and 63.1%
after 75 min). After 150 min, TNF- synthesis had begun to decline,
returning to 16% positive cells by 300 min. The course of TNF-
staining in macIL-10tg macrophages stimulated with LPS was very similar
to that of FVB macrophages exposed to IL-10 plus LPS. There was an
increase in the percentage of TNF-+ cells, but
transgenic and exogenous IL-10 blunted the response by 40% at the
30- and 75-min time points. Furthermore, IL-10 induced a faster
down-regulation of TNF- production, with TNF- staining
approaching baseline levels after 150 min and a >70%
inhibition relative to FVB macrophages.
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We studied whether macIL-10tg mice also produce less TNF- and
IL-12p40 when challenged with LPS in vivo. After i.p. injection of 200
µg LPS, the serum levels of IL-12p40, TNF-, and IFN- were
measured (Fig. 5). In macIL-10tg mice the
response to LPS was dampened with a significant reduction in the levels
of TNF- after 1.5 h, IFN- after 6 h, and both time
points in the case of IL-12p40. Inhibition of cytokine production was
not as strong as in the in vitro culture system (Fig. 4), which is
probably due to the higher relative concentrations of Flag-IL-10 in
pure macrophage cultures compared with in vivo, where the Flag-IL-10
produced will be taken up and removed continuously.
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To investigate the role of macrophage IL-10 production in
mycobacterial infection, macIL-10tg and FVB mice were infected i.v.
with BCG. macIL-10tg mice, as well as FVB mice, controlled the
infection and appeared outwardly healthy. At 2, 5, and 8 wk after
infection, mice were killed and tissue homogenates of spleen, liver,
and lung were plated onto 7H10 plates and the number of CFU per organ
was determined (Fig. 6A). A
difference in CFU was apparent after 2 wk in all three organs but was
stronger at the 5-wk time point. This higher bacterial load was
maintained after 8 wk of infection in liver and spleen but not in the
lung. Histopathological analysis of spleen and liver sections showed
that the number of granulomas in the livers was roughly equal between
macIL-10tg mice and FVB controls at all three time points. Staining for
the presence of acid-fast bacilli revealed that the number of
mycobacteria in the granulomas was higher in macIL-10tg mice than in
FVB mice (Fig. 6B). The development of a BCG-specific T cell
response after infection was assessed by measurement of IFN- in
culture supernatants of splenocytes from infected FVB and
macIL-10tg mice restimulated in vitro with heat-killed BCG (Fig. 7). IFN- production from macIL-10tg
splenocytes was significantly weaker than from FVB cultures 5 wk after
infection, but by 8 wk after infection this difference was no longer
observed and macIL-10tg splenocytes produced even more IFN- than
controls. To dissect the contributions of the T cell and APC components
of the IFN- response, we purified CD4 T cells from BCG-infected mice
as well as control FVB and macIL-10tg mice. We used wild-type and
transgenic BMDM as APC for restimulation in vitro (Fig. 8). As expected, CD4 T cells from
uninfected FVB and macIL-10tg mice did not produce detectable IFN-
when exposed to BMDM pulsed with purified protein derivative
(PPD) or infected with BCG (data not shown). IFN- levels
induced by anti-CD3/Con A in control CD4 T cells were highest for
the combination of FVB CD4 T cells with FVB BMDM and lowest when both
cell types came from macIL-10tg mice. Infection with BCG resulted in
strongly increased IFN- production upon stimulation with
anti-CD3/Con A for all combinations of T cells and APC. Purified
CD4 T cells from BCG-infected macIL-10tg mice showed higher PPD-
and BCG-specific IFN- production than their FVB counterparts. In
contrast, macIL-10tg BMDM induced a much weaker IFN- response than
those from FVB mice. This difference was also evident after stimulation
with anti-CD3/Con A but more pronounced for PPD- or BCG-specific
IFN-. Thus, the use of purified CD4 T cells and defined APC showed
that BCG-specific Th1 T cell development is not impaired in macIL-10tg
mice. Together with normal development of granulomas in macIL-10tg
mice, the main reason for the impaired clearance of BCG appeared to be
an inhibition of intracellular killing by infected macrophages.
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in macIL-10tg macrophages
Increased survival of mycobacteria in macIL-10tg macrophages may
be due to a disturbed response to IFN-. To address this issue, we
chose to analyze the phosphorylation of STAT1, an early event in
IFN- signal transduction, in BMDM from wild-type FVB and macIL-10tg
mice. Macrophages from macIL-10tg mice showed attenuated
phosphorylation of STAT1 when stimulated with titrated amounts of
IFN- (Fig. 9A). Because
inhibition of STAT1 activation by IL-10 has been correlated with
expression of SOCS3 (32), protein levels of SOCS3 were
determined in BMDM (Fig. 9B). SOCS3 was absent in
unstimulated FVB macrophages but was induced 3 h after addition of
IFN-. In contrast, macIL-10tg macrophages contained constitutively
expressed SOCS3, which was further increased by treatment with IFN-,
while neutralization of IL-10 greatly reduced the level of SOCS3.
Overexpression of IL-10 in macIL-10tg macrophages also resulted in
constitutive phosphorylation of STAT3, which was transiently activated
by IFN- in FVB macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophage specificity of CD68 promoter-driven Flag-IL-10 expression
Several lines of evidence indicate that expression of the Flag-IL-10 transgene by the CD68 promoter was restricted to macrophages. First, when cells from different tissues were assayed for Flag-IL-10 production, significantly more IL-10 was detected in supernatants of cultures enriched in macrophages. Second, sorting of splenocytes showed strong enrichment of Flag-IL-10 secretion in the Mac1+GR1- population. Finally, intracellular staining detected IL-10 only in the F4/80+ population within adherent splenocytes of macIL-10tg mice. Overall, our findings confirm the usefulness of the CD68 promoter for macrophage-specific expression in transgenic mice (29). Expression of Flag-IL-10 in macrophages was constitutive and at least as strong as IL-10 secretion induced by treatment with LPS, at both the mRNA and protein levels. In the serum of macIL-10tg mice, IL-10 was present at concentrations comparable to what was induced by LPS in FVB mice.
Several founder mice derived from injection with different fragments of
the CD68 promoter controlling Flag-IL-10 died prematurely without
producing viable offspring. The copy number of integrated transgene was
estimated to be between 20 and 40 in these animals, and very high
levels of Flag-IL-10 were detected in the serum (Table I). Although
there are no published data on toxic effects of IL-10, it cannot be
ruled out that chronic exposure to excessive amounts of IL-10 can have
direct devastating effects on multiple organ systems. In contrast, the
pathological findings we obtained are compatible with systemic
infection, in one case ascending from the genito-urinary tract, in the
other spread from a large abscess. Thus, high chronic IL-10 levels in
these founder mice may have caused paralysis of the immune system
leading to terminal infection.
Comparison of macIL-10tg mice to other models of overexpression of IL-10 in vivo
Overexpression of Flag-IL-10 in macIL-10tg mice did not obviously
interfere with peripheral T and B cell homeostasis. This is consistent
with other transgenic mouse lines overexpressing IL-10 either from
activated T cells (16, 33) or under control of the MHC-II
promoter (24). The induction of Ag-specific adaptive
immunity appeared unaffected despite elevated levels of IL-10 in
macIL-10tg mice (Fig. 3). This is at variance with the recently
reported inhibition of Ag-specific responses by T and B cells in a
transgenic mouse line expressing human IL-10 under the control of the
MHC-II promoter (hIL-10 TG) (Ref. 24 and this study). This
discrepancy is somewhat surprising given that IL-10 is expressed from
APCs in both models, reaching similar steady-state levels in the serum
of mice (Ref. 24 and this study). The cellular source of
IL-10 production directed by the different promoters may be relevant
here. While macIL-10tg B cells did not make Flag-IL-10, B cells from
hIL-10 TG strongly secreted transgenic IL-10 upon stimulation
(24). Also, the expression of IL-10 by dendritic cells can
be expected to differ between the transgenic mouse lines, although
Flag-IL-10 expression by dendritic cells was not tested directly in
macIL-10tg. In addition to spatial differences in IL-10 production, the
temporal context of constitutive or inducible overexpression of IL-10
may explain the differential effects on adaptive immune responses in
macIL-10tg and hIL-10 TG mouse lines.
Macrophage deactivation in macIL-10tg mice affects inflammatory and antimycobacterial responses
Stimulation of macIL-10tg macrophages in vitro with LPS resulted
in similarly down-regulated levels of TNF- and IL-12p40 as were
observed in control macrophages treated with IL-10, and neutralization
of IL-10 increased cytokine production by macIL-10tg macrophages.
Importantly, upon challenge with LPS in vivo, a similar reduction in
the output of TNF- and IL-12p40 was observed. In addition, IFN-
was significantly lower in macIL-10tg, probably reflecting a secondary
effect of decreased IL-12 levels in the transgenic mice. These effects
on the innate response to LPS are consistent with work by others using
exogenous administration of IL-10 (34, 35, 36) or IL-10 gene
transfer (37).
Although down-regulation of cytokine production by macrophages is
desirable and beneficial in autoimmune diseases and septic shock, it
may negatively influence the capacity to fight infections. Infection of
macIL-10tg resulted in 10-fold-higher chronic bacterial loads in spleen
and liver compared with control mice. In its effects on the clearance
of infection with BCG, macrophage-specific overexpression of IL-10 is
similar to what we have found previously for mice overexpressing IL-10
from activated T cells (16), thereby strengthening the
argument that increased production of IL-10 during mycobacterial
infection favors bacterial growth. Because IFN- production by T
cells is essential for defense against mycobacteria (38, 39) and IL-10 can inhibit Th1 responses, one possible mechanism
for increased numbers of bacteria in IL-10tg mice is impaired Th1
development. However, it appears that macIL-10tg mice mount a strong,
if slightly delayed, T cell IFN- response to BCG. Thus,
overexpression of IL-10 does not qualitatively alter IFN- production
from T cells in BCG-infected mice. Depletion of TNF- or the TNF type
I receptor in mice by neutralization with Ab or gene targeting
renders mice highly susceptible to infection with Mycobacterium
tuberculosis (40, 41). Despite induction of a strong
T cell response and recruitment of activated T cells to the lungs of
infected TNF--/- mice, the normally observed
typical granuloma structure failed to develop (41).
Inhibition of TNF- levels by IL-10 might therefore result in
disturbed granuloma formation. However, in BCG-infected macIL-10tg
mice, granulomas developed at normal numbers and with apparently normal
structural characteristics compared with FVB mice. Preliminary data
suggest that TNF- mRNA expression is reduced 2-fold in
BCG-infected macIL-10tg mice (R. Lang, unpublished data), which may not
be enough to cause effects similar to complete depletion by gene
targeting. In contrast, the number of acid-fast bacilli per granuloma
was increased in macIL-10tg mice. We conclude that the killing of the
mycobacteria within infected macrophages is impaired.
IL-10 may inhibit macrophage killing of BCG by various mechanisms. For
efficient control of mycobacteria in macrophages, expression of
inducible NO synthase (iNOS) and production of NO is essential
(42, 43). Treatment of murine macrophages with TNF- and
IFN- potentiates NO generation after mycobacterial infection and
increases antimycobacterial activity (44). Production of
IFN- by splenocytes from macIL-10tg mice was reduced only early in
infection (Fig. 7), and purified CD4 T cells produced high levels of
IFN- (Fig. 8). However, IL-10 could interfere with the signal
transduction of IFN- and consequently inhibit the expression of
iNOS. Inhibition of IFN- signaling by IL-10 has been described and
correlated with the induction of SOCS3 (32). Indeed, when
we compared the ability of IFN- to activate STAT1 in macIL-10tg and
FVB macrophages in vitro, we found a similar inhibition in macIL-10tg
macrophages as Ito et al. (32) had observed when IL-10 was
added to human monocytes. In addition, constitutive activation of STAT3
and expression of SOCS3 were detected in macIL-10tg macrophages. While
these data were obtained in experiments in vitro, preliminary evidence
shows that untreated macIL-10tg mice have slightly (2- to 3-fold)
increased levels of SOCS3 mRNA in tissues (our unpublished
observations), suggesting a possible effect on IFN- signaling also
in vivo. In addition to an iNOS-dependent pathway, TNF- triggers
growth control of BCG in macrophages in a iNOS-independent manner
(45). By decreasing the levels of TNF-, IL-10 may
inhibit iNOS-dependent and -independent bactericidal macrophage
functions. Macrophages from macIL-10tg mice displayed moderate
inhibition of NO production after stimulation with LPS, consistent with
previous reports on inhibition of NO production by IL-10 (46, 47). An important intracellular survival mechanism of pathogenic
mycobacteria is their ability to prevent the maturation of the
phagosome and thereby to evade destruction (48).
Interestingly, in IL-10 gene-deficient mice the percentage of BCG
colocalizing with a marker of acidified phagolysosomes was
significantly higher than in macrophages capable of producing IL-10
(49). Likewise, overexpression of IL-10 may affect the
intracellular trafficking of the bacteria and further inhibit the
maturation of the phagosome. Taken together, IL-10 probably affects
macrophage killing of intracellular BCG at multiple levels.
The macIL-10tg mice we have generated will provide a useful tool to analyze the effects of macrophage-specific overexpression of the immunoregulatory cytokine IL-10. This model should prove valuable not only in studying the immunology of infection but also in other fields where macrophages play a role in pathophysiology, such as tumor immunology and atherosclerosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter J. Murray, Department of Infectious Diseases, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. E-mail address: peter.murray{at}stjude.org
3 Abbreviations used in this paper: BCG, bacille Calmette-Guérin; BMDM, bone marrow-derived macrophage; iNOS, inducible NO synthase; MHC-II, MHC class II; PDM, peritoneal-derived macrophage; PPD, purified protein derivative; SR-A, scavenger receptor A; SOCS, suppressor of cytokine signaling.
Received for publication October 11, 2001. Accepted for publication January 25, 2002.
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) or macIL-10tg (
) were stimulated in 96-well plates with
anti-CD3 mAb at the indicated concentrations and from 40 to 48
h were pulsed with [3H]thymidine. Data shown are mean +
SEM of quadruplicate wells. B, OVA-specific IgM and IgG
subtypes were determined in sera of mice immunized with OVA in CFA as
described in Materials and Methods. Results are mean +
SD of log10 dilution of sera from four mice per
genotype.
) challenged
with LPS. Groups of four mice were injected i.p. with 200 µg LPS.
After 1.5 and 6 h, mice were killed and the serum was analyzed for
cytokines by ELISA. Data are mean + SD; p values from
Student’s t test are indicated.
