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J Virol, January 1998, p. 121-132, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Transgenic Mice Expressing Human Immunodeficiency Virus Type
1 in Immune Cells Develop a Severe AIDS-Like Disease
Zaher
Hanna,1,2
Denis G.
Kay,1
Marc
Cool,1
Serge
Jothy,3
Najet
Rebai,1 and
Paul
Jolicoeur1,4,5,*
Laboratory of Molecular Biology, Clinical
Research Institute of Montreal, Montreal, Quebec H2W
1R7,1
Departments of
Medicine2 and
Microbiology and
Immunology,4 Université de Montréal,
Montreal, Quebec H3C 3J7, and
Departments of
Pathology3 and
Experimental
Medicine,5 McGill University, Montreal,
Quebec H3G 1A4, Canada
Received 16 June 1997/Accepted 16 September 1997
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ABSTRACT |
We have constructed transgenic (Tg) mice expressing the entire
human immunodeficiency virus type 1 (HIV-1) coding sequences in cells targeted by HIV-1 infection in humans. These Tg mice developed
a severe AIDS-like disease leading to early death (<1 month). They
developed muscle wasting, severe atrophy and fibrosis of lymphoid
organs, tubulointerstitial nephritis, and lymphoid interstitial
pneumonitis. In addition the expression of RANTES was increased in
various tissues of these Tg mice relative to that in the normal
controls. Disease appearance was correlated with the levels of
transgene expression. The numerous pathologies observed in these mice
are remarkably similar to those observed in human AIDS and, more
specifically, in pediatric AIDS.
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INTRODUCTION |
Several investigators have
constructed transgenic (Tg) mice to model human immunodeficiency virus
type 1 (HIV-1)-induced diseases (32). Expression of the
whole HIV-1 genome in Tg mice led to the development of an AIDS-like
syndrome, but this phenotype was observed in only a single line
(35). More recently, it was found that the expression of the
whole HIV-1 genome harboring a modified long terminal repeat (LTR)
induced cataracts, weeping eye, and wasting in Tg mice (14).
Expression of the 3' half of the HIV-1 genome in Tg mice induced a
severe nephropathy (13, 33). Expression of either Nef
(15) or Tat (57) in Tg mice was found to induce epidermal hyperplasia, while expression of Nef, Gag, or protease in
lens fiber cells was responsible for the development of cataracts (2, 12, 27, 56). Tg mice expressing HIV-1 Nef under the regulation of the T-cell-specific CD3 
promoter-enhancer element (52), the T-cell receptor beta (TCR
) chain
enhancer-promoter (36), or the CD2 regulatory elements
(4) showed various degrees of depletion of CD4+
thymocytes and of peripheral T cells. However, additional important characteristics of AIDS were not observed in these mice, while other
features, such as a large increase of the B-cell population, that are
not seen in human AIDS were observed (36).
In an attempt to develop a more relevant model of HIV-1 infection, we
set out to express HIV-1 gene products in the same cells of Tg mice as
those usually found infected in HIV-1-positive individuals, i.e.,
CD4+ T cells and cells of the dendritic/macrophage lineage.
We used the human CD4 gene promoter sequences flanked by the enhancer of the mouse CD4 gene to express the whole HIV-1 coding sequences in Tg
mice. We have previously shown that these enhancer-promoter sequences
can direct expression of a surrogate gene specifically in
CD4+ CD8+ and CD4+
CD8
thymic T cells, in peripheral CD4+
CD8 T cells, and in macrophages (23). Thus,
expression of HIV-1 with this promoter should mimic more closely the
expression of HIV-1 detected in HIV-1-infected individuals. In this
report, we present evidence that indeed these Tg mice develop a severe AIDS-like disease which is dependent on the levels of HIV-1 expression.
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MATERIALS AND METHODS |
Generation of Tg mice.
The CD4A (12.5-kbp) and CD4C
(14.4-kbp) promoters have been described previously (23).
Each was fused to an 8.8-kbp BssHII-SacI fragment
of the HIV-1 pNL4-3 clone (1) and to simian virus 40 polyadenylation sequences as described before (21) and
cloned in the pBR322 vector to generate CD4A/HIVWT and
CD4C/HIVWT transgenes, respectively. The transgene DNAs
were excised with AatII, purified by agarose gel
electrophoresis, and microinjected into fertilized (C57BL/6 × C3H)F2 oocytes, as described before (21).
Chimeric mice were generated by inoculation of ES cell clones
containing the CD4C/HIVWT transgene into C57BL/6
blastocysts as described elsewhere (42). Mice were bred as
heterozygotes with C3H or CD1 mice obtained from Charles River Canada
(St. Constant, Quebec, Canada). The presence of the transgene was
confirmed by Southern blot hybridization of tail DNA with
32P-labeled total HIV-1 sequences as a probe, as described
previously (21). The Tg mice and their non-Tg littermates
were housed in the same cages.
RNA purification and Northern blot analysis.
RNA was
isolated by the method of Chomczynski and Sacchi (6) from
different tissues, and 10 µg from each sample was electrophoresed on
formaldehyde agarose gels and processed for hybridization with a
32P-labeled 8.8-kbp BssHII-SacI HIV-1
probe, as previously described (21).
Flow cytometry.
Cell suspensions were prepared from lymphoid
organs and stained with antibodies, as previously described
(23). Fluorescein isothiocyanate (FITC)-coupled anti-mouse
CD4, phycoerythrin-coupled anti-mouse CD8, and FITC-coupled anti-mouse
TCR
antibodies were purchased from Cederlane Laboratories.
Monoclonal antibodies RA3-6B2 (murine anti-B220), kindly provided by R. Coffman (DNAX Research Institute of Cellular and Molecular Biology,
Palo Alto, Calif.), and Mac-1 (Boehringer Mannheim Inc., Montreal,
Quebec, Canada) were used in an indirect assay. The second antibody was
FITC-conjugated anti-rat immunoglobulin G (IgG) (mouse absorbed;
Kirkegaard and Perry, Inc.). Cytometric analyses were performed
with a FACscan (Becton Dickinson) as described previously
(23).
Peritoneal macrophage preparation.
Mice were injected
intraperitoneally with 1 ml of mineral oil 2 days prior to sacrifice.
Peritoneal cells were harvested and plated on petri dishes. The
attached macrophages were washed and processed for in situ
hybridization (ISH) or collected for RNA extraction.
Detection of Igs.
Total Ig levels were measured by
two-antibody assays with affinity-purified goat anti-Ig antibodies as
described previously (24).
Microscopic analysis.
Mice were anesthetized with avertin,
sera were collected, and the animals were exsanguinated with
phosphate-buffered saline. Lymphoid organs were collected and immersion
fixed in paraformaldehyde or periodate-lysine-paraformaldehyde fixative
(38). The remainder of the animal was then perfusion fixed
with paraformaldehyde or periodate-polylysine-paraformaldehyde
fixative. Organs to be assessed were embedded in parraffin, sectioned
at 5 µm, and stained with hematoxylin and eosin, as described
previously (21). Tg and control non-Tg tissues were assessed
blindly by two investigators (S.J. and D.G.K.).
ISH.
ISH was performed on paraffin-embedded tissues, using
35S-UTP-labeled antisense and control sense RNA probes as
described previously (21). A mixture of two probes was used:
the 1.4-kbp HindIII-SacI fragment
(nucleotides 8131 to 9566) of the pNL4-3 clone (GenBank accession
number M19921) and the 627-bp HindIII fragment
(nucleotides 407 to 1034) of the HIV-1 BH102 clone (GenBank accession
number M15654). Tissues from non-Tg control animals hybridized with antisense probes, as well as Tg animal tissues hybridized with sense
probes, failed to exhibit any specific hybridization signal. RANTES
expression was detected by using a 373-bp murine cDNA probe cloned by
reverse transcription-PCR from thymus RNA, with the sense primer
5'-CTCTGCCGCGGGTACCATGAAG and the antisense primer 5'-GTGGCATCCCCAAGCTGGCTAG.
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RESULTS |
Construction of Tg mice and mosaicism of founder mice.
Five CD4C/HIVWT founders (F15564, F17001, F17018,
F17027, and F17086) and four CD4A/HIVWT founders (F21093,
F22194, F22200, and F22410) were produced. Southern blot analysis
indicated that the structures of both transgenes (Fig.
1A) appeared to be grossly intact (data
not shown).
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FIG. 1.
(A) Structure and expression of CD4A/HIVWT
and CD4C/HIVWT transgenes. The enhancer fragment of the
mouse CD4 gene (mCD4 enh.) (vertically hatched bar), the human CD4
promoter (hCD4C prom.) (white bars and boxes), the HIV-1 pNL4-3 DNA
fragment (diagonally hatched bars and boxes), simian virus 40 (SV40)
polyadenylation sequences (black boxes), and the HIV-1 fragments cloned
in GEM-4 (Promega) and used for riboprobes (lower white bars) are
illustrated. Restriction sites: A, AatII; Bs,
BssHII, S, SacI. (B and C) Transgene RNA
expression in CD4A/HIVWT and CD4C/HIVWT mice.
Northern blot analysis was carried out on total RNAs (10 µg)
extracted from different organs of CD4A/HIVWT mice (from
lines F22200, F22194, and F22410) (B) or CD4C/HIVWT mice
(from lines F17018, F17001, and F17086) (C). K, kidney; Ln, lymph node;
Th, thymus; Lu, lung; S, spleen; Lv, liver: Mu, muscle; He, heart; Br,
brain; Mgl, mammary gland of a mouse mammary tumor virus/HIV Tg mouse
(28) used as a positive control. Hybridization was performed
with a 32P-labeled 8.9-kbp SacI fragment from
the HIV-1 genomic sequences. The filters were then washed and
rehybridized with an actin probe.
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CD4C/HIV
WT founders were bred with C3H or CD1 mice for the
first generation (N1). All founder animals produced normal-size N1
litters, but the frequency of Tg animals in these litters was
significantly lower than expected, except for one founder (F17018),
suggesting that these founders were mosaic (Table
1). The CD4C/HIV
WT founder
F17027 produced only non-Tg pups and non-Tg 12- to 19-day-old
fetuses
(data not shown), confirming that this founder was indeed
mosaic. The
CD4C/HIV
WT F17018 male founder died suddenly 3.5 months
after birth, and
no further analysis was carried out due to autolysis.
The CD4C/HIV
WT F17086 female founder became ill 5 months
after birth from an
intestinal obstruction caused by a large lymphoma
which did not
express the transgene (data not shown). The other three
CD4C/HIV
WT founders appeared to be healthy during a
12-month period of observation
in terms of development, growth, body
weight, and fertility. Tg
lines could not be established from any of
these CD4C/HIV
WT Tg founders. Members of the first
generation (N1) of CD4C/HIV
WT Tg pups either died early or
were sacrificed before attaining
sexual maturity due to the development
of severe disease. Only
two N2 and no N3 Tg mice could be produced. The
diseases arising
in N1 Tg mice of the C3H or CD1 background were
indistinguishable.
CD4A/HIV
WT founders were bred with C3H mice for several
generations, and Tg lines could be established. The
CD4A/HIV
WT founders and their progeny remained healthy
during the 24-month
period of observation.
Clinical phenotype and assessment of CD4A/HIVWT
Tg mice.
No clinical phenotype was observed in any of the 76 CD4A/HIVWT mice observed for a period of up to 24 months.
Macroscopic and histological examinations were also negative for 20 animals sacrificed at 12 to 14 months, except for 1 animal which
exhibited enlarged lymphoid tissues and a lung tumor (data not shown).
Fluorescence-activated cell sorter (FACS) analysis (CD4/CD8 ratio) was
also normal. The expression of the transgene in the thymuses and the
spleens of these mice was barely detectable by Northern blot analysis
(Fig. 1B). The levels of transgene RNA were estimated to be ~20- to 60-fold lower than those in CD4C/HIVWT Tg mice. This low
level of transgene expression may explain the absence of an obvious
phenotype or signs of disease.
Clinical phenotype of CD4C/HIVWT Tg mice.
The time course of disease development was similar in all four
CD4C/HIVWT lines. Over the first 2 weeks of life, no
clinical abnormalities could be detected in the Tg mice compared to
non-Tg controls. In the third week, the Tg mice were readily
distinguishable from their non-Tg littermates by their lower body
weight (Tg, 7.4 ± 0.8 g [n = 8]; non-Tg,
12.4 ± 2.1 g [n = 4]), slow
movements, and hypoactivity. At later stages, the diseased animals
had developed weakness, severe tremors, ruffled hair, and feeding
problems. Some mice also developed diarrhea (Table
2). The hematocrit measured in a few Tg
mice (n = 3) overlapped that seen in control non-Tg mice (n = 3). All affected N1 (n = 25)
and two N2 animals from four different founders showed the same
phenotype. None of the control non-Tg littermates (n = 127) kept in the same cages as the Tg mice exhibited a similar
phenotype or detectable abnormalities, indicating that this phenotype
was transgene specific.
Pathological assessment of CD4C/HIVWT Tg mice.
At
autopsy, macroscopic observation revealed severe wasting, edema,
and abnormalities of several organs (lymphoid organs, kidney, and
lungs) in most Tg mice compared to control non-Tg littermates (Table
2). However, signs of opportunistic infections (such as abcess,
granuloma, or parisitic proliferation) were not observed. Generally,
when pathological changes were severe in one organ, other organs of the
same mouse were also affected.
(i) Wasting.
Severe wasting (loss of both fat and lean body
mass) was observed in all the Tg mice. The muscle mass was atrophic, no
fat was visible, and the bones (ribs) were thin and friable compared to
the case for normal animals of the same size.
(ii) Lymphoid organs.
All lymphoid organs (thymus, spleen, and
lymph nodes) were atrophic; Tg thymuses contained 0.7 × 106 ± 0.7 × 106 cells (n = 5), compared to 137 × 106 ± 72 × 106 cells (n = 7) in control non-Tg
littermates, Tg spleens had 4.2 × 106 ± 2.9 × 106 cells (n = 7), compared to 72 × 106 ± 52 × 106 cells (n = 7) in control littermates, and Tg mesenteric lymph nodes contained
1.4 × 106 ± 0.7 × 106 cells
(n = 5), compared to 28 × 106 ± 19.5 × 106 cells (n = 5) in control
littermates (± equals standard deviation). This atrophy of lymphoid
organs was also observed in two animals kept in a
specific-pathogen-free facility.
Histological examination revealed abnormalities in the spleens
of these Tg mice (Table
2): partial to extensive loss of splenic
architecture, frequent hypocellularity, and occasionally fibrosis
(Fig.
2B). Changes in the thymus, consisting of
loss of architecture
and hypocellularity, were noted (Table
2; Fig.
2D). Finally,
the mesenteric lymph nodes were hypocellular, and their
architecture
was disorganized (Fig.
2F). Lymphoid organs from non-Tg
littermates
exhibited normal histology (Fig.
2A, C, and E), with the
exception
of 1 of 12 spleens assessed, which showed follicular
regression.
Thus, similar if not identical histopathologies were
observed
in the majority of lymphoid organs examined (18 of 24 combined
organs) of Tg mice from three independent CD4C/HIV
WT
founder lines (Table
2). Such extensive destruction of the lymphoid
organs is likely to severely affect the function of the immune
system.
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FIG. 2.
Pathology and transgene expression in lymphoid tissues
from CD4C/HIVWT Tg mice. (A to F) Light micrographs of
various lymphoid tissues. (A and B) Non-Tg and Tg (F17001) spleens,
respectively. (C and D) Non-Tg and Tg (F17018) thymuses, respectively.
Note the small size of the Tg thymus. (E and F) Non-Tg and Tg (F17001)
lymph nodes, respectively. Note the tissue disorganization and
hypocellularity of the Tg organ. (G and H) Thymus from a Tg animal
(F17001). ISH was performed with antisense (G) or sense (H) probes.
Note that in panel G the vast majority of the cells are ISH positive
and are of lymphoid morphology. The lymphoid morphology is best shown
in panel H on an adjacent section of the same tissue in the absence of
a cell-specific hybridization signal when exposed to the sense probe.
Magnifications, ×80 (A to D), ×390 (E and F), and ×320 (G and H).
The counterstain was hematoxylin and eosin.
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(iii) Kidneys.
In most Tg mice, kidneys were markedly atrophic
and paler than those in the control mice. In more severely affected
mice, the kidneys had an irregular cortical surface (Table 2). Five of
these mice also exhibited edema, consistent with renal failure. The
histological changes in 10 of 14 mice examined were severe, consisting
of marked tubular atrophy and interstitial fibrosis associated with
interstitial inflammation (Fig. 3B and
D). The lumen of the atrophic tubules was markedly dilated, and their epithelium was thinned. There were no significant glomerular changes.
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FIG. 3.
Pathology and transgene expression in kidneys of
CD4C/HIVWT Tg mice. (A and B) Light micrographs of kidney,
in longitudinal section, from normal (A) and Tg (F17018) (B) mice. Note
the large number of dilated tubules lined by atrophic epithelial cells.
(C) Low-power, dark-field image showing ISH for transgene expression in
Tg animal F17001 with extensive pathology. Note expression of the
transgene in areas corresponding to glomeruli (arrowheads) as well as
ductal regions. (D) Low-power, bright-field image of ISH-positive
interstitial infiltrating cells (F17001) in kidney. (E and F)
high-power images of ISH-positive interstitial infiltrating cells (E)
and cells in glomeruli (F). (G and H) Regions equivalent to those in
panels E and F, respectively, but from a non-Tg animal hybridized with
an antisense probe. Magnifications, ×80 (A and B), ×200 (C and D),
and ×830 (E to H). The counterstain was hematoxylin and eosin.
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(iv) Lungs.
The lungs of some Tg mice were firm compared to
those of non-Tg mice. Histological examination showed a marked
thickening of alveolar walls by infiltrating mononuclear cells in some
Tg animals (Fig. 4B). Such severe
interstitial infiltration was not observed in non-Tg littermates (Fig.
4A). No evidence of airspace disease was noticed in Tg mice. Two Tg
mice with extensive lung disease were assessed for Pneumocystis
carinii infection by Grocott's stain of lung sections and were
found to be negative.
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FIG. 4.
Pathology and transgene expression in livers, lungs, and
intestines of CD4C/HIVWT Tg mice. (A and B) Non-Tg and Tg
(F17018) lungs, respectively. Note the extensive interstitial
pneumonitis in the Tg lung. (C to E) Tg expression is seen in
interstitial infiltrating cells and alveolar macrophages in the lung
(F17001) (C) (S, alveolar space), in Kupffer cells (liver macrophages)
(F17001) (D), and in cells in the lamina propria of the small intestine
(F17018) (E). Magnifications, ×190 (A to D) and ×400 (insets to
panels C and D panel E). The counterstain was hematoxylin and eosin.
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FACS analysis of lymphoid organs from CD4C/HIVWT Tg
mice.
FACS analysis performed on thymocytes of six Tg mice from
founder F17001 with antibodies against CD4, CD8, and TCR showed dramatic changes: the CD4+ CD8+ and
CD4+ CD8 cells were almost absent in all Tg
mice, while CD4 CD8+ and CD4
CD8 T cells constituted the largest populations (Fig.
5). Interestingly, only a minority of
thymocytes (<0.1%) were found to express TCR, an indication that the
large population of remaining CD4 CD8+
thymocytes did not have the phenotype of mature T cells. They most
likely represent double-positive CD4+ CD8+
immature T cells in which the CD4 molecule may well have been downregulated by HIV-1 gene products, possibly Nef (19). A
similar analysis carried out on spleen and lymph node cells of Tg mice showed a very low percentage of mature CD4+ (0 to 6%) and
CD8+ (1 to 3.8%) T cells and a reduction in the number of
TCR-expressing cells (<3%, versus a control value of ~18%). Other
N1 offspring (n = 2) from other lines (F17018 and
F17086) showed a similar but variable depletion of CD4+ T
cells in peripheral lymphoid organs.
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FIG. 5.
Cytofluorometric analysis of thymocytes and splenocytes
of a young CD4C/HIVWT Tg mouse. Thymocytes and
splenocytes from a Tg mouse (F17001) and a non-Tg control mouse
were analyzed by two-color flow cytometry for the expression of CD4 and
CD8 (A) and TCR (B). The percentages of cells found in each
quadrant are indicated; 104 cells were analyzed.
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Analysis of B cells with the B220 marker showed the presence of B cells
and indicated that they represented on average a higher
proportion of
the cells of the spleen (54%) (
n = 7) and of the
lymph
nodes (58%) (
n = 4) in these Tg mice than in normal
mice
(40 and 13%, respectively), as expected for a T-cell-depleted
lymphoid organ. Similarly, cells of the monocyte/macrophage/dendritic
lineage, detected with anti-Mac-1, were found to constitute a
higher
proportion of the remaining spleen cells in Tg mice (
n = 3) than in control mice.
B cells in CD4C/HIV
WT Tg mice (
n = 7) did
not appear to function normally, as the levels of serum Igs (1.9 ± 0.9 mg/ml) were
on average only 10% of those observed in non-Tg
control animals
(
n = 6) (24.1 ± 15.5 mg/ml).
Expression of the transgene in CD4C/HIVWT Tg mice.
Northern blot analysis revealed the three main transcripts of HIV-1
(8.8-kb full length, 4.3-kb env specific, and 2.0-kb
multiply spliced) at high levels in the thymus and at moderate levels
in the spleen and lymph nodes (Fig. 1C). These organs are known to support transcription of surrogate genes from this CD4C promoter (23). A weak but detectable signal was also observed in
other organs, such as kidneys, lungs, intestines, and livers (Fig. 1C). This hybridization signal could originate from circulating T lymphoid cells and/or resident macrophages, which are known to support the
expression from this CD4C promoter (23). No expression was detected in testis, skin, muscle, heart, and brain (Fig. 1C).
Transgene expression was further evaluated by ISH with
35S-labeled HIV-1-specific antisense and control sense
riboprobes. On
a per-cell basis, moderate to strong expression of the
transgene
was observed in each of the four Tg lines in various organs
assessed.
In lymphoid tissues, expression was seen in diseased organs
but
was rarely observed in normal-appearing tissues, indicating that
expression of HIV-1 gene products was correlated with development
of
pathological lesions. In the spleen, the numbers of
transgene-expressing
cells varied from several tens to hundreds per
histological section.
Where sufficient splenic architecture was
conserved, transgene
expression was found to be concentrated in the red
pulp areas
(data not shown). In the lymph nodes, again the
disorganization
of tissue architecture made it impossible to determine
the localization
of the expressing cells (data not shown). In the
thymuses whose
architecture was not too distorted, transgene expression
was found
to be concentrated in the cortical region, while fewer
medullary
cells expressed the transgene (Fig.
2G), consistent with an
expression
in CD4
+ CD8
+ (cortical) and
CD4
+ CD8
(medullary) T cells. Interestingly,
in some diseased spleens,
thymuses, and lymph nodes, no transgene
expression could be detected,
suggesting either that the lesions were
induced indirectly or,
more likely, that transgene-expressing cells
were already depleted
in these mice.
Transgene expression was also observed in nonlymphoid tissues. In five
diseased kidneys, cells within the glomeruli (Fig.
3C and F) and
interstitial, most likely infiltrating mononuclear
leukocytes (Fig.
3C
to E) were found to express the transgene.
It was not possible to
determine whether expressing cells within
glomeruli were normal
components of the glomerulus or were infiltrating
cells, although cells
around the periphery of glomeruli, where
epithelial cells are located,
often had elevated levels of transgene
expression. Transgene
expression was undetectable in two other
kidneys with minimal
pathological changes and in one exhibiting
normal morphology. These
results strongly suggested that the kidney
lesions in these mice
were related to the number of cells expressing
the transgene.
Transgene expression was also observed in mononuclear cells in several
other organs, such as the lamina propria of the intestine
(Fig.
4E),
the livers, and the lungs. Liver Kupffer cells (Fig.
4D) and lung
interstitial infiltrating cells and alveolar macrophages
(Fig.
4C) were
positive for transgene expression. In addition,
peritoneal macrophages
from a mouse of line F17001 were found
to express the transgene, as
expected (data not shown). Other
cell types not expected to express the
transgene were negative
by ISH. Thus, the epithelial cells, smooth
muscle and connective
tissue cells of the gastrointestinal tract,
tubular epithelial
cells of the kidney, pneumocytes, hepatocytes,
myocytes of the
heart and skeletal muscle, seminiferous tubules, and
spermatocytes,
as well as vasculature in these organs, were all
negative for
transgene expression. Together, these results are
consistent with
the specificity of the CD4C promoter for
CD4
+ T lymphocytes and for cells of the macrophage lineage.
Studies of CD4C/HIVWT chimeric mice.
Because the
Tg founders which were mosaic survived longer than their nonmosaic Tg
offspring, we decided to generate chimeric CD4C/HIVWT Tg
mice. Possibly, these animals may remain viable for a longer period of
time and thus develop pathologies resulting from a more chronic
exposure to HIV-1 gene products. Cells from two ES cell clones
harboring the transfected CD4C/HIVWT transgene (J1-1838-2
and J1-1838-5) were used to generate chimeric mice. Twelve and 15 of 35 and 21 born mice, respectively, showed coat color chimerism (Table
3). Southern blot analysis with an HIV-1
probe also demonstrated variable contributions of ES cells to multiple
tissues (data not shown). Five of 15 chimeric mice derived from clone
J1-1838-5 exhibited disease early in life and were sacrificed at 18 to
32 days after birth. This disease was similar to that described above
for CD4C/HIVWT Tg mice. The phenotype included low body
weight (6 to 8 g [n = 4], compared to 13 g
for age-matched nonchimeric controls), wasting, slow movement,
hypoactivity, and early death. This phenotype was not observed in
nonchimeric mice, suggesting that it was transgene specific.
Transgene expression was assessed by ISH for several tissues (kidney,
spleen, lymph node, and thymus) of chimeric mice. Five
chimeric mice
derived from clone J1-1838-5 harbored HIV-1-expressing
cells in several
organs, and the level of expression per cell
was relatively high (data
not shown), being comparable to that
observed for Tg animals.
Generally, higher numbers of ISH-positive
cells correlated with
histopathology. In three animals, transgene
expression was observed in
the kidneys, in cells of several glomeruli
as well as in interstitial
mononuclear cells. Two of these mice
showed clear evidence of kidney
disease. The pathology observed
was indistinguishable from that
observed in the CD4C/HIV
WT Tg animals. Here, greater
pathology was observed in the animal
exhibiting the highest number of
ISH-positive cells. With the
exception of one thymus, which exhibited a
loss of corticomedullary
junction, lymphoid tissues from three chimeric
animals with demonstrated
lower numbers of transgene-expressing cells
had no detectable
pathological lesions. Among the eight chimeric mice
derived from
the other clone (J1-1838-2), none showed any evidence of
pathology
in their tissues, and only one animal showed minimal evidence
of transgene expression in lymphoid tissues. However, reverse
transcription-PCR performed on peritoneal macrophages isolated
from
three chimeric animals indicated that there was expression
in these
cells (data not shown), and this was confirmed by ISH
for one animal.
These results suggest that the absence of a phenotype
in these chimeric
mice may be related to the low levels of transgene
expression.
Together, these results extend and confirm our data on the mosaic
founder mice. They show that the presence of high numbers
of cells
expressing moderate to high levels of this transgene
correlates with
pathology.
Overexpression of RANTES in CD4C/HIVWT Tg
mice.
HIV-1 infection has been found to upregulate
-chemokine expression in monocytes in vitro and in vivo
(46). To determine whether the expression of
-chemokines was affected in CD4C/HIVWT Tg mice, we
measured RANTES expression in several tissues of Tg mice by using
ISH. The pattern of RANTES expression mimicked that of
transgene expression. In four of seven Tg animals examined, moderate to
high numbers of RANTES-expressing cells were detected. Expression
was detected in lymphoid tissues, with the highest levels seen in lymph
nodes (Fig. 6A to C). Expression was also detected in cells of the lamina propria of the gastrointestinal tract
(Fig. 6D) and in interstitial infiltrating and glomerular cells of the
kidney (Fig. 6E), as well as in cells infiltrating the lung and in
Kupffer cells (data not shown). In control non-Tg littermates, only
very low levels of RANTES expression (occasional expressing cells)
were detected in lymphoid tissues. These results suggest that
RANTES may play some role in the HIV-1-induced changes seen in
these mice.
View larger version (170K):
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|
FIG. 6.
Elevated levels of RANTES expression in
CD4C/HIVWT Tg mice. Assessment was done by ISH. (A to C)
Lymph node (F17018). Dark-field (A) and bright-field (B) images of the
same field show tissue hybridized with the antisense probe. (C)
Hybridization with the control sense probe. Note the hypocellularity of
the node in panel B. (D and E) ISH-positive cells of the lamina propria
of the intestine (F17001) (D) and in interstitial cells in the kidney
(F17001) (E). Magnifications, ×180 (A to C and E) and ×380 (D). The
counterstain was hematoxylin and eosin.
|
|
| |
DISCUSSION |
Expression of the transgene in target cells for HIV-1
infection.
Our results show that Tg mice expressing the complete
HIV-1 coding sequences under the regulation of the CD4C promoter
develop a severe AIDS-like disease followed by early death. We have
previously reported that this promoter, derived from the human and
mouse CD4 genes, allows expression of surrogate genes in
CD4+ CD8+ and CD4+ thymocytes, in
peripheral CD4+ T cells, and in macrophages
(23). Expression of the transgene in CD4C/HIVWT
mice was found to be apparently in the same cells, namely, T cells and
macrophages. In addition, we have recently documented by further
examination of CD4C/CD4 Tg mice that this promoter is active in spleen
dendritic cells (41a). These represent the specific subsets
of cells which are normally targeted for infection by HIV-1 in humans
(10, 16, 40, 47, 48). Expression of HIV-1 in these specific
cell populations of Tg mice appears to be important to elicit this
AIDS-like disease, since our other mice similarly expressing the whole
HIV-1 genome at high levels in different cell populations and through
different promoters did not develop any apparent abnormal phenotype of
the immune system (28, 55). Although both the T cells and
the dendritic/macrophage cell lineages express the HIV-1 genome in the
CD4C/HIVWT Tg mice, it is not clear whether expression in
each of these lineages is required for induction of each of the
phenotypes observed. In fact, expression of HIV-1 in
dendritic/macrophage cells may be sufficient to elicit several of the
phenotypes seen in these mice. On the other hand, expression of a
specific factor by HIV-1-expressing CD4+ T cells may be an
absolute requirement for development of some or all phenotypes.
Additional work is needed to approach this question.
To date, only mice from one other Tg line expressing the whole HIV-1
genome under the regulation of the HIV-1 LTR have been
reported to
develop an AIDS-like disease characterized by early
death and
pathological changes in several organs (
35). Some
of these
changes (early death, lymphadenopathy, thymus hypoplasia,
and lung
lesions) were similar to those seen in CD4C/HIV
WT Tg mice,
while others (epidermal hyperplasia and absence of detectable
HIV-1 RNA
in spleen by ISH) were different or (kidney disease)
absent. However,
since only one line (no. 13) of Tg mice developed
this disease, an
insertional mutation by the transgene contributing
to the phenotype
could not be ruled out.
Levels of HIV-1 expression and development of diseases.
In
addition to the specific cell populations expressing the transgene,
another parameter which may have influenced the development of disease
in CD4C/HIVWT Tg mice is the level of HIV-1 expression. In
these Tg mice, it appears that a relatively high level of viral RNA
expression was required for the development of the disease. Four of
five Tg founder mice which were mosaic for the transgene, and
presumably harbored lower numbers of HIV-1-expressing cells, failed to
develop disease or developed the disease much later than their
nonmosaic N1 offspring. Similarly, only chimeric mice having the
highest number of HIV-1-expressing cells developed histopathological
lesions. Finally, the CD4A/HIVWT Tg mice, which expressed
HIV-1 at much lower levels, did not develop disease even after a long
period of observation. Therefore, as in human AIDS (25), the
viral RNA load appears to be an important determinant of disease in
CD4C/HIVWT Tg mice. Since no reinfection cycle occurs in
these mice, this viral RNA load is likely to mimic a steady-state load
of virus expression in humans, although more work will be needed to
determine whether these two parameters reflect the same pathogenesis.
AIDS-like pathologies in Tg mice.
Several pathological
changes observed in these CD4C/HIVWT Tg mice are
similar to those found in individuals with AIDS, and especially in
pediatric AIDS (5), consistent with the fact that the
transgene is expressed early in life.
Early death, which constitutes the most dramatic phenotype in these
mice, is also seen in a high percentage of infants (
49)
and
in some adults (
17,
43) with AIDS. Except in some mice
with
a severe lung or kidney disease, the exact cause of death
remains
obscure. Death is unlikely to result from only bacterial
or viral
infections, since no evidence of bacteremia or extensive
viral
infection was observed in these mice. Additionally, early
death was
also observed in two animals kept in a specific-pathogen-free
facility.
Death is also unlikely to be exclusively the result
of the loss of T
cells, since nude mice kept in the same rooms
survived much longer than
these CD4C/HIV
WT Tg mice. The kidney or lung diseases also
cannot account for
all of these early deaths, since severe and
life-threatening kidney
or lung lesions were not present in all mice
which died early.
This early death may be related to the severe wasting
observed
in the majority of these mice. In HIV-1-infected humans,
wasting
has been found to be a strong predictor of survival
(
54).
Wasting indeed represents the second-most-striking phenotype in these
mice. It has been observed in a relatively high percentage
of AIDS
patients (
9,
22), and its pathogenesis is not well
understood but it is thought to be multifactorial (
22). A
similar
cachexia syndrome has been described for other Tg mice
expressing
the whole or the 3' half of the HIV-1 genome (
14,
44). However,
in one of those studies, it was observed only in
mice homozygous
for the transgene (
44) and may have resulted
from an insertional
mutation by the transgene. Therefore, the wasting
seen in the
heterozygous CD4C/HIV
WT Tg mice represents a
very good model of HIV-1-associated wasting
in humans.
The third striking phenotype observed in these Tg mice is the small
size of the lymphoid organs, with the loss of their normal
architecture
accompanied by a severe depletion of thymocytes and
peripheral T cells.
Severe premature involution of the thymus
with depletion of both
lymphoid and epithelial thymic cell populations,
and specifically of
Hassall's corpuscules, is a feature of both
pediatric and adult AIDS
in humans (
20,
29,
50). In the
thymuses of the Tg mice,
CD4
+ CD8
+ immature and CD4
+
CD8
mature T cells appear to be initially preferentially
lost. Human
CD4
+ CD8
+ thymic T cells have been
reported to be infectable with HIV-1
in vitro (
11,
47). The
loss of T cells in peripheral organs
of these mice is likely to reflect
the virtual absence of precursor
T cells in the thymus. Recent evidence
indicates that a reduction
in early life of both CD4
+ and
CD8
+ T cells in HIV-1-infected children (a DiGeorge-like
immunophenotype)
is associated with a rapid progression to AIDS
(
34). The loss
of the architecture of the spleen and lymph
nodes associated with
fibrosis and hypocellularity also mimics the case
for the lymphoid
organs of adults and, especially, children with
advanced AIDS
(
29). Thymic atrophy and the loss of T cells
in peripheral lymphoid
organs have previously been reported to occur in
Tg mice expressing
only the HIV-1
nef gene under the
regulation of T-cell-specific
promoters (
4,
36,
52),
suggesting that the thymic phenotype
in the CD4C/HIV
WT Tg
mice may be caused primarily by the expression of the
nef gene in T cells. The construction of additional Tg mice expressing
mutant HIV-1 genomes is under way to test this hypothesis.
The fourth-most-important pathology seen in these Tg mice affects the
kidneys. Kidney disease in AIDS patients is relatively
frequent
(
41,
51), particularly in children (
53). A broad
spectrum of renal lesions have been described, including focal
and
segmental glomerulosclerosis, mesangial hyperplasia, and
tubulointerstitial
nephritis, but the pathogenesis of these changes is
not well understood
(
26,
41). In the CD4C/HIV
WT
Tg mice, the predominant features of the kidney disease
(tubulointerstitial
nephritis) mimic what has been observed in humans.
Expression
of the HIV-1 transgene in glomerular and tubular epithelial
kidney
cells was not expected to occur with the CD4C promoter used
here.
In fact, most of the cells expressing the transgene in the kidney
appear to be infiltrating interstitial mononuclear cells, some
with a
lymphocyte cell morphology. Cells of unknown identity were
also found
to express the transgene in glomeruli. This is consistent
with the
suggestion that glomerular cells may express CD4 in humans
(
30). Cells of the macrophage lineage which express the
transgene
are also likely to be present in the kidneys. A role for
macrophages
in HIV-1-associated kidney diseases has previously been
postulated
(
3). In addition, the expression of HIV-1 gene
products may
directly affect epithelial kidney cells. A severe kidney
disease
has previously been found to develop in Tg mice expressing the
3' half of the HIV-1 genome under the regulation of the HIV-1
LTR
(
13,
33). The kidneys were larger than those of non-Tg
controls, and most of their glomeruli were severely sclerotic.
In
contrast, in CD4C/HIV
WT Tg mice, the kidneys were smaller
than normal and tubulointerstitial
nephritis was the histological
change most frequently observed.
The identity of the cell population
expressing the 3'-half HIV-1
transgene in these mice was not reported,
and it is unclear whether
the pathogeneses of these two distinct kidney
diseases in these
mice and in the CD4C/HIV
WT Tg mice are
the same or different.
The lung lesions constitute the fifth main phenotype detected in these
Tg mice. This phenotype does not seem to be caused
by
P. carinii, which was not found in these tissues. It resembles
the
pulmonary lymphoid hyperplasia/lymphoid interstitial pneumonitis
observed in children, and rarely in adults, with AIDS (
5,
29,
39), whose origin is unknown but is thought to be immune mediated
(
7).
Finally, the overexpression of RANTES in various tissues of these
Tg mice represents a sixth phenotype which may be relevant
to the human
disease. Although MIP-1
and MIP-1
have been the
main
-chemokines found to be elevated in HIV-1-infected monocytes
(
46), the levels of RANTES and other
-chemokines have
been
found to be elevated in lymph nodes of macaques infected with
the
pathogenic SIVmac239 but not in animals infected with its
nef-deleted derivative (
58) and in brains of
SIVmac251-inoculated
macaques exhibiting encephalitis (
45).
Moreover, these
-chemokines
appear to represent the major soluble
factors responsible for
HIV-1-suppressive effects secreted by
CD4
+ and CD8
+ T cells derived from
HIV-1-infected individuals (
8,
31).
Overexpression of
RANTES has also been reported in inflammatory
diseases
(
37). In addition, high levels of RANTES would be
expected
to compete with the macrophage-tropic HIV-1 virions for the
CCR-5
receptor and thus exert a strong selection pressure for the
emergence
of T-cell variants of HIV-1 utilizing another coreceptor,
such
as CXCR4/fusin (
18). Our results suggest that this may
be amenable
to experimentation in vivo.
In summary, our results show that the expression of HIV-1 gene products
in specific subsets of cells which are normally targeted
for HIV-1
infection in humans can be particularly pathogenic in
mice. These mice
developed a systemic disease characterized by
several pathological
changes strikingly similar to those observed
in human AIDS. Such a
constellation of pathologies has not been
reported for other Tg mice
expressing HIV-1 through different
promoters, except in a single line
derived by Leonard et al. (
35).
Therefore, the
CD4C/HIV
WT Tg mice described here represent the best model
of Tg mice yet
available for human AIDS.
| |
ACKNOWLEDGMENTS |
Zaher Hanna and Denis G. Kay contributed equally to this work.
This work was supported by grants from the Medical Research Council of
Canada to P.J. and from the National Health Research and Development
Program to Z.H. D.G.K. was supported by a career development award
from the Fonds de la Recherche en Santé du Québec.
We gratefully acknowledge Nathalie Gauthier, Karina Lamarre, Ginette
Masse, Benoît Laganière, Michel Robillard, and Michel Ste-Marie for excellent technical assistance. We also thank Sonia Colombi for some of the FACS analysis on CD4A/HIVWT Tg
lines. Finally, we thank Rita Gingras for typing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Clinical
Research Institute of Montreal, 110 Pine Ave., West, Montreal,
Québec, Canada H2W 1R7. Phone: (514) 987-5569. Fax: (514)
987-5794. E-mail: jolicop{at}ircm.umontreal.ca.
| |
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J Virol, January 1998, p. 121-132, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
