G Model
IMLET-4736; No. of Pages 9
ARTICLE IN PRESS
Immunology Letters xxx (2009) xxx–xxx
Contents lists available at ScienceDirect
Immunology Letters
journal homepage: www.elsevier.com/locate/
The drug monosodium luminol (GVT) preserves crypt-villus epithelial
organization and allows survival of intestinal T cells in mice infected
with the ts1 retrovirus
Virginia L. Scofield ∗ , Mingshan Yan, Xianghong Kuang, Soo-Jin Kim, Paul K.Y. Wong
Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park - Research Division, 1808 Park Road 1-C, Smithville, TX 78957, USA
a r t i c l e
i n f o
Article history:
Received 25 July 2008
Received in revised form
13 November 2008
Accepted 1 December 2008
Available online xxx
Keywords:
Retrovirus
GVT
Intestine
Oxidative stress
a b s t r a c t
Of the cytopathic retroviruses that affect mammals, including HIV-1, many selectively infect CD4+ T cells
and cause immunosuppressive syndromes. These diseases destroy both the thymus and the small and
large intestines, after infecting and killing T-lineage cells in both tissues. A mutant of the murine leukemia
retrovirus MoMuLV-TB, called ts1, causes this syndrome in susceptible strains of mice. In FVB/N strain
mice that are infected at birth, thymic atrophy, CD4+ T cell loss, intestinal collapse, body wasting, and
death occur by ∼30–40 days postinfection (dpi). Apoptosis of ts1-infected T-lineage cells, in the thymus,
peripheral lymphoid system and intestines is caused by accumulation of the ts1 mutant viral envelope
preprotein gPr80env , which is inefficiently cleaved into the mature viral proteins gp70 and PrP15E. We
show here that ts1 infection in the small intestine is followed by loss of intestinal epithelial cell (IEC)
thyroid-stimulating hormone (TSH) and cell cycling gradients (along the crypt-villus axes), accumulation
of gPr80env in intestinal cells, apoptosis of developing T cells in the lamina propria (LP), and intestinal
collapse by ∼30 dpi. In infected mice treated with the antioxidant drug monosodium luminol (GVT® ),
however, normal intestinal epithelial cell gradients are still in place at 30 dpi, and IECs covering both the
crypts and villi contain large amounts of the antioxidant transcription factor Nrf2. In addition, no apoptotic
cells are present, and accumulated gpr80env is absent from the tissue at this time. We conclude that GVT
treatment can make ts1 a noncytopathic virus for intestinal lymphoid cells, as it does for thymocytes [25].
As in the thymus, GVT may protect the intestine by reducing oxidant stress in infected intestinal T cells,
perhaps by prevention of gPr80env accumulation via Nrf2 upregulation in the IECs. These results identify
GVT as a potential therapy for intestinal diseases or inflammatory conditions, including HIV-AIDS, in
which oxidative stress is a triggering or exacerbating factor.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
The murine retrovirus MoMuLV causes thymic leukemias by
8–10 months of age in susceptible mice that are infected at birth
[1]. A mutant of MoMuLV, called ts1, causes a fulminant disease
with a much shorter timecourse, leading to death of infected animals by 30 days after infection (dpi) [2]. The ts1 mutant has a
mutation in its env gene, so that productively infected cells contain
an abnormal gPr80env preprotein [3,4]. This abnormality interferes
with the normal cleavage of the gPr80env to the mature viral envelope proteins gp70 and PrP15E. Without efficient cleavage, gPr80env
accumulates in certain infected target cells, including astrocytes in
the central nervous system (CNS), thymocytes and T cells [5,6]. In
∗ Corresponding author at: The University of Texas M.D. Anderson Cancer Center, Science Park - Research Division, Department of Carcinogenesis, PO Box 389,
Smithville, TX 78957, USA. Tel.: +1 512 237 9344; fax: +1 512 237 2444.
E-mail address: vscofi[email protected] (V.L. Scofield).
glial cells of the central nervous system, this accumulation initiates an unfolded protein response, endoplasmic reticulum stress,
mitochondrial stress, oxidative stress and apoptosis [7–18]. Similar
responses occur in T-lineage cells of infected mice, which accumulate gpr80env and die as a consequence of oxidative stress-induced
apoptosis [19].
The systemic consequences of ts1 infection are similar to HIVAIDS at many levels [6,8,20]. Studies in this paper were designed to
identify specific effects of ts1 infection on the mouse small intestine, whose symptomatology is similar to that induced by HIV-1,
the primate retrovirus SIV, and the feline immunodeficiency virus
FIV [21–24]. The intestines are significant in retroviral diseases for
another reason. Like the thymus, they can receive bone marrow Tcell progenitors and host their maturation into mature T cells (see
below). This makes this tissue an important target for retroviral
infection and cellular damage, because production of naïve T cells
normally continues in these tissues throughout life, and because
intestinal lymphoid cells in the LP govern the health and functions of the mucosal epithelium above. We therefore conducted
0165-2478/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.imlet.2008.12.012
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012
G Model
IMLET-4736; No. of Pages 9
2
ARTICLE IN PRESS
V.L. Scofield et al. / Immunology Letters xxx (2009) xxx–xxx
this study in parallel with investigations of thymus tissues in ts1infected mice [25].
Fig. 1 shows that mouse small intestines are composed of fingerlike villi facing the gut lumen and of crypts buried in the connective
tissue. Both regions are covered with a continuous layer of epithelium, with the pluripotent stem cells for the entire epithelium
residing in the lower crypts [26–28]. These stem cells give rise
to several cell types found on the villi: to absorptive enterocytes
covering the villi (the predominant cell type in the mucosa), to
mucus-producing goblet cells and enteroendocrine cells scattered
over the villi between the enterocytes (specific cell types are not
shown in the figure). Crypt stem cells also give rise to defensinproducing Paneth cells, which form a cup at the bottom of the
crypts, and which do not move upward toward the villi [27,28].
The progeny of the crypt epithelial stem cells are transit amplifying cells and differentiating cells, which move upward, as they
divide, toward the crypt-villus junction [CVJ]. Once differentiated,
these cells stop dividing, so that the epithelium covering the villi,
above the CVJ, contains mostly quiescent enterocytes, goblet cells
and enterendocrine cells nudging each other passively upward to
the tips of the villi. When the IECs arrive at the tips of the villi, they
undergo apoptosis and are shed, together with any T cells that have
inserted themselves between the cells (top of Fig. 1). The CVJ thus
is a sharp boundary that separates dividing from quiescent cells.
As noted above, the small and large intestines of mammals
can host the differentiation of bone marrow T-precursor cells into
mature T cells [29–32]. The idea that the intestine might host T cell
differentiation was put forward more than 10 years ago (reviewed
in [32]). However, this claim was controversial until recent lasercapture confocal microscopy studies established clonal identity
between early T cell progenitors in aggregates in the crypts, called
cryptopatches (CPs) and mature T cells in the distal villi [33].
Fig. 1 shows T-cell progenitors differentiating in the lamina propria (LP) connective tissue beneath the CMJ, and mature T cells
above it. This reflects the continuous arrival in the crypts of bone
marrow stem cells, which are c-kit-positive (red). These begin differentiation to become mitotic T-cell progenitor progeny in the
crypt LP (purple). The maturing and fully mature descendants of
these intestine-born T cells are CD8-positive T cells that reside in the
LP of the villi [29–32]. CD4+ cells are also present, but these arrive
from the blood as mature T cells that have already completed their
differentiation in the thymus [34]. These are scattered throughout
the crypt and villous LP. As in the thymus, therefore, the CVJ boundary separates distinct epithelia whose adjacent T cells are at distinct
developmental stages.
In the normal thymus, the cortical and medullary epithelial cells
express two distinct cytokeratin types [35]. Unlike the thymus however, the intestinal epithelium is a simple epithelium throughout;
and all of its cells express CK8 only [36], as do cortical epithelial
cells of the thymus. However, two other differences distinguish the
epithelial cells of the intestinal crypts from those of the villi. One
is these is the constitutive expression of TSH by crypt epithelial
cells, and not by epithelial cells of the villi [37–40]. The other is
the presence of dividing epithelial cells exclusively in crypts, but
not in the villi [26]. The presence and maintenance of these two
standing gradients in the epithelial intestine reflects the presence
of normally differentiating T-cell progenitors in the LP beneath, just
as the presence of a healthy corticomedullary epithelial separation does for thymocytes [25]. Like thymocytes, intestinal T cells
undergo sequential developmental checkpoints that depend upon
their being at the right place, at the right time, in relation to the
epithelium above [35].
In mice infected with ts1 (ts1-only), we show here that intestinal epithelial cell gradients are dysregulated, that extensive T cell
Fig. 1. Diagram showing cell types and cytoarchitecture of the normal mouse small intestine. The tissue is arranged as a single-layered epithelium covering a connective
tissue lamina propria (LP) beneath. The epithelium covers upwellings of cells (villi) that face the gut lumen, and valleys of cells (crypts) buried in the connective tissue below.
The epithelial cells of all villi are derived from a single stem cell type at the bottom of each crypt. These cells divide and differentiate until they reach the crypt-villus junction
(shaded plane), at which time they stop dividing and are nudged to the tops of the villi, where they undergo apoptosis and are shed along with lymphoid cells from the LP.
Several T-lineage cell types are present in the LP beneath the epithelium. These are (a) recent bone marrow T cell progenitor emigrants expressing the c-kit+ marker (red), (b)
their immediate differentiated progeny, which are maturing cryptopatch (CP) cells (purple), (c) and mature differentiated mitotic progeny from these CP cells, which express
the ␥␦ T cell receptor and move up into the villi (yellow). One T cell type in the small intestines is already differentiated when it arrives. These cells come into the intestine
from the thymus via the blood. They are mature thymus-derived T cells expressing the ␣␤ T cell receptor (blue). The looped areas at the right show how these regions appear
in cross-section.
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012
G Model
IMLET-4736; No. of Pages 9
ARTICLE IN PRESS
V.L. Scofield et al. / Immunology Letters xxx (2009) xxx–xxx
apoptosis occurs, and that the intestines collapse by 30 dpi. In mice
infected with ts1 and also treated with GVT (ts1-GVT), however,
crypt-villus cytoarchitecture is intact at 30 dpi, intestinal epithelial cell gradients are in place, no T cell apoptosis is occurring, and
the gPr80env protein is not accumulated by any cell type in the
tissue. In the intact small intestines of GVT-protected mice, most
epithelial cells are negative for gp70, and thus do not appear to be
infected, but they do contain large amounts of the transcription
factor Nrf2, which orchestrates protective antioxidant responses
[41,42]. These effects of ts1-GVT treatment are remarkably similar
to those occurring in the thymi of ts1-GVT mice, whose epithelial
cells also become loaded with Nrf2 [25].
2. Materials and methods
2.1. Virus
The ts1 virus, a mutant of MoMuLV, was propagated in TB cells,
a thymus-bone marrow cell line, and titered on 15F cells, as previously described [8].
2.2. Antibodies and reagents
GVT® (sodium ␣-luminol) was provided by Bach-Pharma, Inc.,
North Andover, MA. Goat anti-MoMuLV gp70 was from Microbiology Associates, Burlingame, CA (this antibody recognizes epitopes
shared by the mature gp70 viral envelope protein and the precursor
preprotein gPr80env ). Anti-mouse Ki67 was from Vector Laboratories, Burlingame, CA.
Polyclonal rabbit anti-mouse TSH␤ was from Cell Signaling
(Boston, MA). Biotinylated mouse monoclonal anti-TSH␤ was a
gift from Dr. John R. Klein. Mand rabbit anti-activated caspase-3
was from Cell Signaling. Mouse monoclonal anti-Nrf2 was from
R&D systems (Minneapolis, MN). FITC and Texas Red-conjugated
anti-mouse, rat, rabbit and goat antibodies were from Jackson
ImmunoResearch (West Grove, PA).
2.3. Mice, infection, and drug treatment
FVB/N mice were obtained from Taconic Farms (Germantown,
NY). Breeding FVB/N pairs were housed in sterilized microisolator
cages and supplied with autoclaved feed and water ad libitum. The
microisolators were kept in an environmentally controlled isolation
room. For ts1 infection, 4-day-old mice were inoculated intraperitoneally with 0.1 ml of vehicle (mock infection) or with 0.1 ml of a
ts1 suspension containing 2 × 107 infectious units/ml. Mice infected
by this protocol, and at this virus dose, become paralyzed and die
at 30–40 dpi [8].
For GVT treatment [8], animals from the control and ts1-infected
groups are divided into two groups each at 2 dpi. The infected mice
are then divided again into two groups, one of whose individuals
received 0.9% normal saline, intraperitoneally, five continuous days
a week, followed by two resting days, until the end of the experiment. At the same time, mice from the other half of the infected
animals received freshly prepared GVT, delivered intraperitoneally
at 200 mg/kg body weight/day in 0.9% normal saline. The uninfected
mice were also divided into two groups, one receiving saline alone,
and the other receiving GVT as described above. No toxic effects
were observed at any time at this dose of GVT when it was used
without infection.
At 30 dpi, mice from all groups were sacrificed, and their small
intestines (2 cm from the jejunum) processed for Western blotting of the tissues or for frozen section immunohistochemistry. All
mice were observed daily for clinical signs of disease, and individuals from all groups were sacrificed at 30 dpi. Intestinal tissues to
be used for Western blotting were removed, snap-frozen in liquid
3
nitrogen and stored at −80 ◦ C until use. For immunohistochemistry, intestine segments were snap-frozen in liquid nitrogen in OCT
embedding compound. These animal protocols were approved by
The University of Texas M.D. Anderson Cancer Center Institutional
Animal Care and Use Committee.
2.4. Western blotting
Western blotting analysis was performed as described previously [8]. Briefly, whole cell protein was extracted from whole
intestines in radioimmunoprecipitation assay buffer (NP40, 0.5%
sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.25 mM
phenylmethylsulfonylfluoride, 1 mg/ml aprotinin, leupeptin, and
pepstatin A, 1 mM sodium orthovanadate, and 1 mM sodium fluoride in phosphate-buffered saline, or PBS). Protein concentrations
were measured using the Bio-Rad Dc protein assay reagent (Bio-Rad
Laboratories, Hercules, CA) as per the manufacturer’s instructions.
These lysates (30 ␮g total protein per sample) were electrophoresed
on 10% SDS-PAGE gels, and then transferred to polyvinylidene difluoride membranes (Millipore Corp, Bedford, MA). The membranes
were blocked at room temperature for 1 h in tris-buffered saline, or
TBS, with 5% nonfat milk, and incubated with unconjugated primary antibody for 2 h, followed by three washes with TBS. The
blots were then incubated with species-appropriate horseradish
peroxidase-conjugated secondary antibody for an additional 1 h.
After three washes, immune complexes were detected by chemiluminescence (NEN Life Science Products, Boston, MA). A monoclonal
anti-␤-actin antibody (Sigma, St. Louis, MO) was used as a loading
control. Western blotting experiments were repeated at least three
times, to verify reproducibility of results.
2.5. Immunohistochemistry
Dissected tissues were snap-frozen in OCT medium and kept
at −80 ◦ C prior to cutting of 5 ␮m-thick serial frozen sections. The
sections were placed on microscope slides, and the slides kept at
−80 ◦ C prior to staining. For staining, the sections were thawed at
room temperature for 30 min, fixed in ice-cold acetone for 5 min,
washed, and incubated overnight at 4 ◦ C with optimum dilutions of
primary antibodies. The slides were then washed and incubated in
FITC-conjugated or TxR-conjugated Fab’2 fragments of anti-rabbit,
anti-rat, or anti-goat IgG. Where double immunostaining was done,
it was with polyclonal or monoclonal antibodies raised in cells or
animals from two different species, followed by FITC-conjugated
anti-IgG for one species, and TxR-conjugated anti-IgG for the other.
After incubation in the secondary reagents and washing, the sections were mounted in mounting medium (Vector Laboratories,
Burlingame, CA) for viewing under an Olympus fluorescence microscope. Control sections were incubated (a) in affinity-purified goat,
rabbit or rat IgG, depending upon the host species of the primary
antibody, (b) with appropriate isotype controls, for monoclonal
antibodies, (c) in secondary antibodies alone, without primary
reagents. Optimum staining conditions were developed for each
antibody, so that no specific binding was observed in sections incubated in control reagents, while specific binding was observed with
the primary antibodies under test. Immunostaining studies were
repeated at least twice, to verify reproducibility of results.
3. Results
3.1. Crypt-villus organization and T cell numbers are abnormal in
intestines from ts1-only mice, but not from those of ts1-GVT
animals
Fig. 2 shows frozen sections of small intestines from 30 dpi uninfected, ts1-only, and ts1-GVT mice, stained with hematoxylin and
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012
G Model
IMLET-4736; No. of Pages 9
4
ARTICLE IN PRESS
V.L. Scofield et al. / Immunology Letters xxx (2009) xxx–xxx
eosin (H&E) for cytoarchitecture (A), with anti-gp70 to identify
infected cells (B), and with anti-CD4 or anti-CD8 to identify cells in
the LP of the villi (C). Hyperproliferation of the crypts is a common
intestinal manifestation of infection by other retroviruses, including SIV in nonhuman primates, FIV in cats, and HIV-1 infection
in humans [21–24]. The H&E and gp70-stained ts1-GVT intestine
images in Fig. 2 show apparently normal crypt-villus organization
at 30 dpi, despite the presence of many infected cells in the LP. CD4positive cells are abundant in the LP regions of uninfected intestinal
villi, virtually absent in the ts1-only villi, but present in the LP of
villi in the section from a ts1-GVT mouse. CD8-positive cells are
reduced in number in the ts1-only mouse intestines, but they are
not depleted from the villi as are CD4-positive cells in the ts1-only
mice (C).
3.2. The TSH gradient of the crypt-villus axis is lost in the ts1-only
intestine, but not in intestines from ts1-GVT mice
In the hypothalamo–pituitary–thyroid (HPT) axis, the neurohormone thyrotropin-releasing hormone (TRH) is produced by the
hypothalamus, and delivered to the anterior pituitary gland below
[40]. TRH triggers production of thyrotropin by pituitary thyrotroph
cells (thyrotropin is the thyroid-stimulating hormone, or TSH). TSH
travels through the blood to the thyroid, where binds to its receptor (the TRHr) on the follicle cells, and these follicle cells and the
follicular fluid respond by producing thyroglobulin and iodinated
thyroxine (T3 and T4), and by releasing thyroxine into the blood.
Recent work has disclosed that the small intestinal epithelial
cells of mice, like pituitary thyrotrophs, express genes for the two
Fig. 2. Loss of crypt-villus organization, ts1 infection and depletion of CD4+ cells in the intestines of ts1-only mice, and maintenance of normal parameters and cell numbers,
in 30 dpi uninfected, ts1-only and ts1-GVT mice. (A) Hematoxylin and eosin (H&E)-stained cross-sections of intestines from animals from these three groups, showing profiles
of the crypts and villi (see Fig. 1 for crypt and villus sectional orientation). Original magnifications: for uninfected and ts1-GVT panels: 10×; for ts1-only panel: 20×. (B)
Sections from uninfected, ts1-only and ts1-GVT intestines, stained for their expression of the ts1 viral envelope protein gp70 (green). Original magnifications: 20×. gp70
staining for the ts1-GVT section was dim, and thus the brightness of this image was magnified 100× for this figure. (C) Sections of uninfected, ts1-only and ts1-GVT intestines,
immunostained either for CD4 or for CD8 (red; uninfected and ts1-only panels) and doubly stained for CD4 or CD8 together with gp70 (green; ts1-only insets and ts1-GVT
panels). The ts1 -inset overlay photomicrographs are magnified from the white-boxed rectangles in the wide-angle panels at the left, and their contrast is upregulated 100×
to allow visualization of stained cells. The arrows in the ts1-only and ts1-GVT panels on the right point to infected CD4-positive cells (yellow in the CD4 ts1-only section) and
to uninfected CD8-positive cells (red in the CD8 ts1-only section. By contrast, both infected (yellow) CD4-positive and CD8-positive cells are evident in the ts1-GVT panels
on the right. The arrow in the CD4 ts1-only section in (C) points to crypts and villi in which CD4-positive cells are absent. Original magnifications: Uninfected; 40×. ts1-only;
10× for the wide-angle panels and 20× for the insets; ts1-GVT; 20× for both panels.
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012
G Model
IMLET-4736; No. of Pages 9
ARTICLE IN PRESS
V.L. Scofield et al. / Immunology Letters xxx (2009) xxx–xxx
5
subunits of TSH [37]. In addition, a subpopulation of small intestinal
T cells (but not peripheral T cells) express surface TSH receptors [37,38]. These findings point to a local epithelial-lymphocyte
hormone loop, involving TSH produced by crypt epithelial cells,
response to this TSH by intestinal T cells, and feedback to the epithelium [37–39].
Intestinal epithelial TSH gradient is disturbed during rotavirus
[38] and reovirus infection [39]. In both conditions, blocks of enterocytes on the villi reinitiate TSH expression, and groups of LP
lymphoid cells aggregate in the LP below them.
To determine whether intestinal epithelial TSH gradients are
dysregulated in ts1 infection, we compared TSH levels in intestines
from uninfected, ts1-only, and ts1-GVT mice. Fig. 3(A) shows a Western blot in lysates of intestines of mice from the above three groups,
stained for TSH. Uninfected intestinal tissues show an ambient level
of TSH expression at 30 days of age, but the ts1-only intestines contain much more TSH, while the ts1-GVT intestines contain what
appear to be normal TSH levels. To look at what is happening to TSH
gradients at the cellular and tissue level, we then prepared sections
of these intestines and stained them with anti-TSH and anti-gp70.
Fig. 3(B and C) shows that TSH expression is limited to the crypt
epithelium in uninfected mouse intestines, that the entire intestinal epithelium, crypts and villi alike, express TSH in the ts1-only
intestine, but TSH expression is limited to the crypts in the ts1-GVT
intestines.
this tissue [26]. The cell cycling marker Ki67 is an intermediate filament protein found only in dividing cells [reviewed in 26]. Below
the crypt-villus boundary of normal intestines, all epithelial cells
covering the villi are dividing. Above this boundary, they are quiescent. When immunostained with antibody against Ki67, epithelial
cells in the crypts are Ki67-positive, while those covering the villi
are Ki67-negative. Like the epithelial cytokeratin gradients of the
thymus [25] and the TSH gradient of the intestine, intact Ki67
expression patterns, distinguishing the crypts and villi, reflect normal T cell differentiation conditions in the LP below the epithelium.
To determine whether ts1 infection affects the crypt-to-villus
Ki67 gradient, and to find out whether GVT treatment prevents
this, we immunostained one frozen section of an uninfected small
intestine with anti-Ki67 alone, and then doubly immunostained
uninfected, ts1-only and ts1-GVT intestine sections with gp70 and
Ki67. The doubly stained sections were photographed under bandpass filters for FITC and then for Texas Red, and a merged image was
made from the two single-filter images for the ts1-only and ts1-GVT
sections. Fig. 4(A) shows that Ki67 staining is confined to basolateral
surfaces of the crypt epithelial cells of normal intestines, as has been
reported by others [26]. In both the ts1-only (B) and ts1-GVT sections (C), but gp70-infected lymphoid cells are present in the LP, and
Ki67 staining is evident in epithelial cells of both the crypts and villi
of the ts1-only intestines (B). However, while the ts1-GVT section
shows Ki67 staining that is limited to the crypts (C), reflecting the
presence of a normal Ki67 gradient in intestines from ts1-GVT mice.
3.3. The distinct cell cycling patterns of the crypt and villus are
lost in the ts1-only intestine, but not in intestines from ts1-GVT
mice
3.4. In ts1-GVT mice, intestinal epithelial cells contain high levels
of the antioxidant transcription factor Nrf2, and their T cells are
protected from apoptosis
A second epithelial cell gradient exists in the healthy small intestine, and this gradient is disrupted in disease conditions affecting
Like many other cell types, intestinal epithelial cells maintain ambient intracellular levels of NFE-2-related factor 2 (Nrf2),
Fig. 3. Loss of the epithelial crypt-villus gradient organization for TSH in 30 dpi ts1-only mice, and maintenance of this gradient in age-matched ts1-GVT mice. (A) Western
blot showing that TSH levels are upregulated over their normal levels (uninfected lane) in ts1-only intestinal tissues, but not in ts1-GVT intestinal tissues. ␤-actin was used as
a loading control. (B and C) Sections from crypts (B) and villi (C) of uninfected, ts1-only and ts1-GVT sections immunostained for TSH. The top and bottom left panels on the
left show crypts (top; TSH-positive crypts are marked a “+”) and villi (bottom; TSH-negative epithelial cells are marked as “−”) of normal intestines, immunostained for TSH
(green in these two panels). The “hotblock” on the left of the panel showing otherwise TSH-negative villi (arrows) is an area of local reactivation of TSH, most likely reflecting
a disturbance in the barrier or a local infection. For details, see text. By contrast, TSH staining (red) is heavy for epithelial cells of the crypts and villi of ts1-only intestines
(middle panels), but is limited to the crypts for ts1-GVT intestines (right panels). Original magnifications: B; 10×; C; left panels, 20×.
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012
G Model
IMLET-4736; No. of Pages 9
6
ARTICLE IN PRESS
V.L. Scofield et al. / Immunology Letters xxx (2009) xxx–xxx
Fig. 4. Dysregulation of epithelial crypt-villus Ki67 expression (cell cycling) in 30 dpi ts1-only intestines, and maintenance of these gradients in age-matched ts1-GVT
intestines. (A) is a section from an uninfected mouse intestine, stained only for KI67 (red) to show normal distribution of this marker in the crypt-villus axis. The antibody
identifies Ki67 on the crypt epithelial cells, but not on epithelial cells of the villi (crypts and villi are marked). Crypts and villi in A, B, and C are labeled. All panels in (B) are
photomicrographs of the same section of a ts1-only mouse intestine, showing gp70-only staining (green), KI67-only staining (red), and merged images for both antibodies
(co-stained cells appear yellow). Both the crypt and epithelial cells are Ki67-positive in this intestinal section. All panels in (C) are from one section of a ts1-GVT mouse
intestine, stained, and photographed as described above for the section in (B). In this intestine, Ki67 staining of the epithelium is limited to the crypts. Original magnification:
(A); 4×. (B and C); 10×.
which is a transcriptional factor that coordinately upregulates
many genes that participate in antioxidant defenses [41–45].
Under steady-state conditions, Nrf2 is held in an inactive state
as part of a complex with another protein called Keap-1. When
oxidant stress conditions occur, Keap-1 releases Nrf2, which
then is phosphorylated and transported to the nucleus. When
it arrives there, Nrf2 activates genes that have antioxidant
response element (ARE) sequences in their promoters (reviewed
in [44]).
For some time, it has been assumed that correction of an
abnormal-redox situation, once completed, re-establishes the
Keap1-Nrf2 complex, bringing Nrf2 levels down again [44]. However, studies in our laboratory and by others now show that high
levels of free Nrf2 can be stabilized in the cytoplasm and nucleus
under certain kinds of oxidant stress conditions [8,15,16,46], or
by drugs that inhibit Nrf2 degradation in proteasomes [46]. Other
agents that stabilize Nrf2 in cells others increase Nrf2 mRNA and
protein levels in cells over the long term [47]. Either or both effects
could maintain low-ROS conditions in tissues during extended periods of oxidative stress. We therefore asked with intestines, as we did
for the thymus [25], whether Nrf2 upregulation and stabilization
occur in intestines of ts1-only vs. GVT mice.
The Western blot in Fig. 5(A) shows that Nrf2 levels in intestinal
tissues of ts1-only mice are slightly lower than those of uninfected
intestines, but that that they are significantly upregulated in ts1GVT intestines. To determine which cells are responsible for the
increased Nrf2 levels in ts1-GVT intestines, we then stained uninfected, ts1-only and ts1-GVT thymi at 30 dpi for gp70 and for Nrf2.
Fig. 5(B) shows that the ts1-only section has neither gp70 nor Nrf2
inside any recognizable cells, because the tissues apparently have
collapsed, and few intact epithelial cells remain. By contrast, the
ts1-GVT intestine contains profiles of apparently healthy villi whose
epithelial cells and lymphoid compartments are intact, but whose
epithelial cells show heavy loading with Nrf2, and whole LP cells
are infected.
In the same experiment, we then asked whether high-Nrf2 conditions in the ts1-GVT intestines might be related to reduced or
absent apoptosis in the intestinal tissues of ts1-GVT mice, as they
are in the ts1-GVT thymus. To do this, we compared levels of the
apoptotic protein cleaved caspase-3 in intestines from uninfected,
ts1-only, and ts1-GVT mice. Caspase-3 is the executive enzyme
that completes the many different apoptotic cascades that occur
in most cell types [48]. The presence of cleaved caspase-3 is a specific indicator of apoptotic cell death in tissues. Fig. 5(A) shows that
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012
G Model
IMLET-4736; No. of Pages 9
ARTICLE IN PRESS
V.L. Scofield et al. / Immunology Letters xxx (2009) xxx–xxx
7
Fig. 5. Nrf2 expression and caspase-3-mediated apoptosis in intestinal lysates and sections from uninfected, ts1-only and ts1-GVT mice. (A) Western blot showing Nrf2 and
cleaved caspase-3 expression for tissues from mice of each of these three groups. (B) Sections of small intestines of mice from these three groups, doubly immunostained Nr2
(red) and for gp70 (green), with co-staining appearing as yellow. The uninfected section shows a low ambient level of epithelial Nrf2 expression, typical in this tissue. The
ts1-only section, by contrast, is collapsed, and although much staining is present, little cell-associated Nrf2 or gp70 are evident. In the ts1-GVT section, the mucosal epithelial
cells express Nrf2 (red), and the LP lymphoid cells express gp70 (green). The co-stained epithelial cells in the ts1-GVT panel (yellow) are infected transepithelial dendritic
cells (V.L. Scofield, manuscript in preparation). Original magnifications: 20×.
a low ambient level of cleaved caspase-3 is present in uninfected
intestinal tissues, but that high levels of this enzyme are present in
intestines of ts1-only mice, where many cells are undergoing apoptosis. By contrast, caspase-3 is absent in the intestines of ts1-GVT
mice.
3.5. In ts1-GVT mice, accumulated gPr80env is reduced
In our studies with the thymus (companion paper), we were
surprised to note that accumulated gPr80env , which is always
present in ts1-infected T-lineage cells, is apparently absent in ts1GVT thymocytes. We concluded that the low-ROS conditions in
GVT-treated thymocytes, which can be brought about even without infection with ts1, could promote proper folding and normal
cleavage of gPr80env into gp70 and PrP15E. To determine whether
this might also be true in the intestines, we performed a Western blot of intestines from uninfected, ts1-only, and ts1-GVT mice,
stained with anti-gp70 to identify the molecular weights of gp70immunoreactive proteins in the tissues. Fig. 6 shows that in the
ts1-only intestine, gp70-immunoreactive material is present at
both the 80 kDa (accumulated gPr80env ) and 70 kDa (processed
gp70) positions in the gel. This two-band pattern is characteristic of gp70 Western blots of lysates from astrocytic cells infected in
vitro [15,16]. However, only gp70 was present in the intestinal lysate
from the ts1-GVT mouse, and this was present in small amounts. We
Fig. 6. Western blot of 30 dpi intestinal tissues from uninfected, ts1-only and ts1GVT mice, probed for gp70. The antibody identifies epitopes present in both the
band present at 80 kDa, which is gPr80env (the viral preprotein) and in the faint band
present at 70 kDa, which is one of its cleavage products. The blot shows that gPr80env
accumulation occurs in intestinal tissues of ts1-only mice, but not in intestinal tissues
of ts1-GVT mice.
conclude that gPr80env is not accumulated in cells of the ts1-GVT
intestine.
4. Discussion
Like ts1, other cytopathic retroviruses, including HIV-1, SIV and
FIV, cause damage to the intestines and selective loss of CD4+
cells [6,21–24]. At present, there is still much to learn generally
about intestinal responses to cytopathic retrovirus infection, but
we believe that our work provides information relevant to HIV-1
enteropathy, and that it identifies GVT as a therapeutic agent that
can prevent, maintain, or restore gut epithelial and T cell homeostasis in HIV-AIDS. If this is possible, then repopulation of the intestines
with autologous or transplanted bone marrow T-cell progenitors
might be possible, even though HIV-infected cells are still present,
even under HAART treatment [49].
The loss of TSH and cell cycling gradients, in the ts1-only intestine, deserves special mention here. Intestinal enterocytes normally
stop expressing and releasing TSH when they have completed their
differentiation and reached the CVJ [38]. When pathogens infect the
villus epithelium, however, TSH production is re-initiated in localized parts of the villus [37–39]. Once the infection is cleared, the
TSH-re-expressing epithelium is shed and replaced by TSH-cells.
The same sequence of events, this time involving Ki67 expression,
appears to accompany local and global ts1 infection in the mouse
gut [26]. A proportion of all intestinal T cell subsets (CD4+ and CD8+
alike) express the TSH receptor [38]. As suggested previously [38], a
local response loop involving the otherwise quiescent villus epithelium of the intestine may reactivate cell cycling and re-initiate TSH
production, with TSH activating nearby T cells in the LP below, and
the T cells, in turn, maintaining the newly activated epithelium. If
this were to occur on a large scale, and without limitation, it is possible to imagine a scenario in which continued infection/activation
of LP T cells in the intestine could drive the shedding of the epithelium above, and could make this occur too fast for replacement. In
turn, the activated epithelium, as long as it is present, may send
inappropriate signals to the LP T cells, maintaining their ectopic
activation and thereby promoting their infection, and apoptosis. It
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012
G Model
IMLET-4736; No. of Pages 9
8
ARTICLE IN PRESS
V.L. Scofield et al. / Immunology Letters xxx (2009) xxx–xxx
survival, and, especially, how GVT treatment it allows normal processing of gPr80env . We hope that our studies generate interest in
the ts1 mouse as a model for HIV-1-induced thymic and intestinal infection and T cell death. Under conventional HAART therapy
regimens, the intestine can serve as a viral reservoir, allowing gastrointestinal symptoms to continue despite control of viral load in
the blood [50]. If GVT actually re-sets the redox tone in the ts1infected mouse intestine, as it apparently does in the thymus [25],
then repopulation of the intestinal T cell compartment could be
possible, either from autologous bone marrow or allogeneic bone
marrow or stem cell grafting. In addition, GVT treatment may prove
therapeutic for intestinal conditions that cause disease by initiating oxidative stress, because a low-ROS environment would likely
reduce inflammation and maintain normal epithelial cell-lymphoid
cell crosstalk in this tissue [51,52].
Fig. 7. Diagram showing possible mechanisms for protection of 30 dpi ts1-infected
mouse intestines by GVT. The arrows point to processes initiated or promoted by ts1
infection or GVT treatment, and the T-bars identify GVT inhibition or termination of
processes initiated by ts1 infection in T cells. In the ts1-only mouse, both epithelial
and lymphoid compartments of the small intestine are damaged. The result is the
disappearance of epithelial cell gradients, loss of T cells, and intestinal collapse. In
the ts1-GVT mouse, the epithelial and lymphoid cell compartments appear normal,
all intestinal cells survive infection, and ts1 gPr80env does not accumulate in infected
cells. Our data suggest that this could occur either in two ways: via direct antioxidant
activity by GVT, or lowering of T cell intracellular redox setpoints by GSH or by GSH
precursors, both produced and provided by mucosal epithelial cells as a consequence
of their upregulation of Nrf2.
seems likely that this sequence of events contributes to the collapse
of the intestines after cytopathic retrovirus infection.
The results presented here for ts1-GVT mice provide information
in only one context, which is infection and treatment beginning at
the same time (at birth) and continuing until all animals have died.
This setting, however, has shown us that infection of intestinal cells
with ts1 can coexist with apparent health in the intestines, both
in the epithelial and LP lymphoid compartments. These conditions
are accompanied by upregulation of Nrf2, and presumably of redox
defenses in the epithelial cells, which may provide cysteine precursors and glutathione (GSH) to the LP T cells in amounts sufficient
to allow their survival of ts1 infection. If this occurs, it could be also
be responsible for the normal folding of gPr80env in the ts1-GVT
intestine, and this in turn would prevent all of the downstream
consequences of gPr80env accumulation that lead to T cell apoptosis. GVT could also act as a chaperone, preventing misfolding of
gpr80env , or it could act as an antioxidant, detoxifying ROS as they
appear in the infected cells. A diagram summarizing these several
possibilities appears in Fig. 7.
The results shown here are significant in that they provide a
new perspective regarding CD4+ cell loss in HIV-AIDS. We believe
that the ts1-infected mouse is a small-animal model for HIV-AIDS
[20]. Similarities between the two diseases have been identified
in published papers from our laboratory and from others [20,25].
Like HIV-1 in untreated human HIV-AIDS patients, ts1 is a savage
enteropathic retrovirus in untreated mice. However, and remarkably, ts1 is harmless in the intestines of mice treated with GVT. The
ts1 mouse thymus and intestine, described and characterized here,
are now ready for more intensive investigation of ts1 pathogenesis
and the GVT effect. For example, changes in CD4+ and CD8+ T cell
subsets should be followed over time by fluorescence-activated cell
sorting (FACS analysis) in both tissues. This will have to be done at
timepoints earlier than 30 dpi, because thymocyte/intestinal T cell
numbers are so low at this time, and the thymus and villi are both
collapsed, so that the processing of multiple animals for cells is
likely to interfere with the quality of the results.
We also need to find out how GVT causes Nrf2 upregulation and
stabilization in the epithelial cells of these two tissues, how Nrf2
elevation in the epithelia might affect thymic and intestinal T cell
Acknowledgements
We thank Shawna Johnson and Rebecca Deen for assistance in
preparing the manuscripts, Kent Claypool for providing invaluable
help with flow cytometry analysis, and Nancy Otto and Jimi Lynn
Brandon of the Science Park Histology Core for preparing excellent
frozen sections and for advising us regarding immunohistochemical analysis. This work was supported by NIH grants NS43984 and
MH71583 (P.K.Y.), by a Career Re-entry supplement to MH71583
for V.L.S., and by NIH grant MH077470 (V.L.S.). Other support was
provided by NIEHS Center Grant ES07784 and Core Grant CA16672,
both to the M.D. Anderson Cancer Center, Science Park-Research
Division, Smithville, TX, by the Dr. Christian Abee of the Michale E.
Keeling Center for Comparative Medicine and Research, The University of Texas M.D. Anderson Cancer Center, Bastrop, TX, and by
the Longevity Foundation of Austin.
References
[1] Asjo B, Fenyo EM, Klein G. Moloney virus (M-MuLV) leukemogenesis: virus
spread, antibody production and antigenic expression in neonatally virusinoculated young mice. Int J Cancer 1981;28:65–70.
[2] Wong PK, McCarter JA. Genetic studies of temperature-sensitive mutants of
Moloney-murine leukemia virus. Virology 1973;53:319–26.
[3] Kamps CA, Lin Y-C, Wong PKY. Oligomerization and transport of the envelope protein of Moloney murine leukemia virus-TB and of ts1, a neurovirulent
temperature-sensitive mutant of MoMuLV-TB. Virology 1991;184:687–94.
[4] Szurek PF, Yuen PH, Ball JK, Wong PK. A Val-25-to-Ile substitution in the
envelope precursor polyprotein, gPr80env , is responsible for the temperature
sensitivity, inefficient processing of gPr80env , and neurovirulence of ts1, a
mutant of Moloney murine leukemia virus TB. J Virol 1990;64:467–75.
[5] Wong PK, Szurek PF, Floyd E, Saha K, Brooks BR. Alteration from T- to B-cell
tropism reduces thymic atrophy and cytocidal effects in thymocytes but not
neurovirulence induced by ts1, a mutant of Moloney murine leukemia virus
TB. Proc Natl Acad Sci USA 1991;88:8991–5.
[6] Wong PK, Prasad G, Hansen J, Yuen PH. ts1, a mutant of Moloney murine
leukemia virus-TB, causes both immunodeficiency and neurologic disorders
in BALB/c mice. Virology 1989;170:450–9.
[7] Kim HT, Qiang W, Wong PK, Stoica G. Enhanced proteolysis of I␬B␣ and I␬B␤
proteins in astrocytes by Moloney murine leukemia virus (MoMuLV)-ts1 infection: a potential mechanism of NF-␬B activation. J Neurovirol 2001;7:466–75.
[8] Jiang Y, Scofield VL, Yan M, Qiang W, Liu N, Reid AJ, et al. Retrovirusinduced oxidative stress with neuroimmunodegeneration is suppressed by
antioxidant treatment with a refined monosodium a-luminol (Galavit). J Virol
2006;80:4557–69.
[9] Kim HT, Tasca S, Qiang W, Wong PK, Stoica G. Induction of p53 accumulation
by Moloney murine leukemia virus-ts1 infection in astrocytes via activation of
extracellular signal-regulated kinases 1/2. Lab Invest 2002;82:693–702.
[10] Kim HT, Waters K, Stoica G, Qiang W, Liu N, Scofield VL, et al. Activation of
endoplasmic reticulum stress signaling pathway is associated with neuronal
degeneration in MoMuLV-ts1-induced spongiform encephalomyelopathy. Lab
Invest 2004;84:816–27.
[11] Kim HT, Qiang W, Liu N, Scofield VL, Wong PK, Stoica G. Up-regulation of astrocyte cyclooxygenase-2, CCAAT/enhancer-binding protein-homology protein,
glucose-related protein 78, eukaryotic initiation factor 2␣, and c-Jun N-terminal
kinase by a neurovirulent murine retrovirus. J Neurovirol 2005;11:166–79.
[12] Liu N, Qiang W, Kuang X, Thuillier P, Lynn WS, Wong PK. The peroxisome proliferator phenylbutyric acid (PBA) protects astrocytes from ts1 MoMuLV-induced
oxidative cell death. J Neurovirol 2002;8:318–25.
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012
G Model
IMLET-4736; No. of Pages 9
ARTICLE IN PRESS
V.L. Scofield et al. / Immunology Letters xxx (2009) xxx–xxx
[13] Liu N, Kuang X, Kim HT, Stoica G, Qiang W, Scofield VL, et al. Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways
in MoMuLV-ts1-induced apoptosis in astrocytes. J Neurovirol 2004;10:189–
98.
[14] Liu N, Scofield VL, Qiang W, Yan M, Kuang X, Wong PK. Interaction between
endoplasmic reticulum stress and caspase 8 activation in retrovirus MoMuLVts1-infected astrocytes. Virology 2006;348:398–405.
[15] Qiang W, Cahill JM, Liu J, Kuang X, Liu N, Scofield VL, et al. Activation of transcription factor Nrf-2 and its downstream targets in response to Moloney murine
leukemia virus ts1-induced thiol depletion and oxidative stress in astrocytes. J
Virol 2004;78:11926–38.
[16] Qiang W, Kuang X, Liu J, Liu N, Scofield V, Stoica G, et al. Astrocytes survive
chronic infection and cytopathic effects of the ts1 mutant of the retrovirus
Moloney murine leukemia virus by upregulation of antioxidant defenses. J Virol
2006;80:3273–84.
[17] Stoica G, Lungu G, Kim HT, Wong PKY. Up-regulation of pro-nerve growth
factor, neurotrophin receptor p75, and sortilin is associated with retrovirusinduced spongiform encephalomyelopathy. Brain Res 2008;1208:204–
16.
[18] Lungu GF, Stoica G, Wong PKY. Down-regulation of Jab1, HIF-1a, and VEGF by
Moloney murine leukemia virus-ts1 infection: a possible cause of neurodegeneration. J Neurovirol 2008;14:239–51.
[19] Wong PKY, Lynn WS, Lin YC, Choe W, Yuen PH. ts1-MoMuLV: a murine model
of neuroimmunodegeneration. In: Wong PKY, Lynn WS, editors. Neuroimmunodegeneration. Berlin: Springer; 1998. p. 75–93.
[20] Clark S, Duggan J, Chakraborty J. ts1 and LP-BM5: a comparison of two murine
retrovirus models for HIV. Viral Immunol 2001;14:95–109.
[21] Sankaran S, George MD, Reay E, Guadalupe M, Flamm J, Prindiville T, et al. Rapid
onset of intestinal epithelial barrier dysfunction in primary human immunodeficiency virus infection is driven by an imbalance between immune response
and mucosal repair and regeneration. J Virol 2008;82:538–45.
[22] Veazey RS, Tham IC, Mansfield KG, DeMaria M, Forand AE, Shvetz DE, et al.
Identifying the target cell in primary simian immunodeficiency virus (SIV)
infection: Highly activated memory CD4+ T cells are rapidly eliminated in early
SIV infection in vivo. J Virol 2000;74:57–64.
[23] Fukazawa Y, Miyake A, Ibuki K, Inaba K, Saito N, Motohara M, et al. Small
intestine CD4+ T cells are profoundly depleted during acute simian-human
immunodeficiency virus infection, regardless of viral pathogenicity. J Virol
2008;82:6039–44.
[24] Howard KE, Burkhard MJ. FIV infection induces unique changes in phenotype
and cellularity in the medial iliac lymph node and intestinal IEL. AIDS Res Hum
Retroviruses 2007;23:720–8.
[25] Scofield VL, Yan MS, Kuang X, Kim SJ, Crunk D, Wong PKY. The drug monosodium
luminol (GVT) preserves thymic epithelial cell cytoarchitecture and allows thymocyte survival in mice infected with the T cell-tropic, cytopathic retrovirus
ts1. Immunol Lett 2009, doi:10.1016/j.imlet.2008.12.009.
[26] Potten CS, Martin K, Kirkwood TB. Ageing of murine small intestinal stem cells.
Novartis Found Symp 2001;235:66–79, discussion 79–84, 101–4.
[27] Barker N, van de Wetering M, Clevers H. The intestinal stem cell. Genes Dev
2008;22:1856–64.
[28] van der Flier LG, Clevers H. Stem cells, self-renewal, and differentiation in the
intestinal epithelium. Annu Rev Physiol 2008 [Epub ahead of print].
[29] Guy-Grand D, Azogui O, Celli S, Darche S, Nussenzweig MC, Kourilsky
P, et al. Extrathymic T cell lymphopoiesis: ontogeny and contribution to
gut intraepithelial lymphocytes in athymic and euthymic mice. J Exp Med
2003;197:333–41.
[30] Ishikawa H, Naito T, Iwanaga T, Takahashi-Iwanaga H, Suematsu M, Hibi T, et al.
Curriculum vitae of intestinal intraepithelial T cells: their developmental and
behavioral characteristics. Immunol Rev 2007;215:154–65.
[31] Klein JR. Thymus-independent development of gut T cells. Chem Immunol
1998;71:88–102.
9
[32] Lambolez F, Azogui O, Joret AM, Garcia C, von Boehmer H, Di Santo J, et
al. Characterization of T cell differentiation in the murine gut. J Exp Med
2002;195:437–49.
[33] Podd BS, Thoits J, Whitley N, Cheng HY, Kudla KL, Taniguchi H, et al. T cells
in cryptopatch aggregates share TCR ␥ variable region junctional sequences
with ␥␦ T cells in the small intestinal epithelium of mice. J Immunol
2006;176:6532–42.
[34] Cheroutre H, Lambolez F. The thymus chapter in the life of gut-specific intra
epithelial lymphocytes. Curr Opin Immunol 2008;20:185–91.
[35] Klug DB, Carter C, Crouch E, Roop D, Conti CJ, Richie ER. Interdependence of
cortical thymic epithelial cell differentiation and T-lineage commitment. Proc
Natl Acad Sci USA 1998;95:11822–7.
[36] Wang L, Srinivasan S, Theiss AL, Merlin D, Sitaraman SV. Interleukin-7 induces
keratin expression in intestinal cells: potential role of keratin-8 in interleukin6-induced barrier function alterations. J Biol Chem 2007;282:8219–27.
[37] Wang J, Whetsell M, Klein JR. Local hormone networks and intestinal T cell
homeostasis. Science 1997;275:1937–9.
[38] Scofield VL, Montufar-Solis D, Cheng E, Estes MK, Klein JR. Intestinal TSH production is localized in crypt enterocytes and in villus ‘hotblocks’ and is coupled
to IL-7 production: evidence for involvement of TSH during acute enteric virus
infection. Immunol Lett 2005;99:36–44.
[39] Montufar-Solis D, Garza T, Teng BB, Klein JR. Upregulation of ICOS on CD43+
CD4+ murine small intestinal intraepithelial lymphocytes during acute reovirus
infection. Biochem Biophys Res Commun 2006;342:782–90.
[40] Pazos-Moura CC, Ortiga-Carvalho TM, Gaspar de Moura E. The autocrine/
paracrine regulation of thyrotropin secretion. Thyroid 2003;13:
167–75.
[41] Lindl KA, Jordan-Sciutto KL. Examining the endogenous antioxidant response
through immunofluorescent analysis of Nrf2 in tissue. Methods Mol Biol
2008;477:229–43.
[42] Pickett CP, Nguyen T, Nioi P. The Nrf2-ARE signaling pathway and its activation
by oxidative stress. J Biol Chem 2009 [Epub ahead of print].
[43] Moi P, Chan K, Asunis I, Cao A, Kan YW. Isolation of NF-E2-related factor 2
(Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to
the tandem NF-E2/AP1 repeat of the ␤-globin locus control region. Proc Natl
Acad Sci USA 1994;91:9926–30.
[44] Chen XL, Kunsch C. Induction of cytoprotective genes through Nrf2/antioxidant
response element pathway: a new therapeutic approach for the treatment of
inflammatory diseases. Curr Pharm Des 2004;10:879–91.
[45] Nguyen T, Yang CS, Pickett CB. The pathways and molecular mechanisms regulating Nrf2 activation in response to chemical stress. Free Radic Biol Med
2004;37:433–41.
[46] Gharavi N, Haggarty S, El-Kadi AO. Chemoprotective and carcinogenic effects
of tert-butylhydroquinone and its metabolites. Curr Drug Metab 2007;8:1–7.
[47] Purdom-Dickinson SE, Sheveleva EV, Sun H, Chen QM. Translational control of
Nrf2 protein in activation of antioxidant response by oxidants. Mol Pharmacol
2007;72:1074–81.
[48] Walsh JG, Cullen SP, Sheridan C, Luthi AU, Gerner C, Martin SJ. Executioner
caspase-3 and caspase-7 are functionally distinct proteases. Proc Natl Acad Sci
USA 2008;105:12815–9.
[49] Lau A, Villenueve NF, Sun Z, Wong PK, Zhang DD. Dual roles of Nrf2 in cancer.
Pharmacol Res 2008;58:262–70.
[50] Belmonte L, Olmos M, Fanin A, Parodi C, Bare P, Concetti H, et al. The intestinal mucosa as a reservoir of HIV-1 infection after successful HAART. AIDS
2007;21:2106–8.
[51] Bengmark S, Jeppson B. Gastrointestinal surface protection and mucosa reconditioning. J Parenter Enteral Nutr 1995;19:410–5.
[52] Schmidt W, Wahschaffe U, Schafer M, Zippel T, Arvand M, Meyerhans A, et al.
Rapid increase of mucosal CD4 T cells followed by clearance of intestinal cryptosporidiosis in an AIDS patient receiving highly active antiretroviral therapy.
Gastroenterology 2001;120:984–7.
Please cite this article in press as: Scofield VL, et al. The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization
and allows survival of intestinal T cells in mice infected with the ts1 retrovirus. Immunol Lett (2009), doi:10.1016/j.imlet.2008.12.012