Inflamed Glomeruli—Specific Gene Activation that Uses Recombinant Adenovirus with the Cre/loxP System
TAKASHI YOKOO*,,
TOYA OHASHI,,
YASUNORI UTSUNOMIYA*,
HIROAKI SHIBA,
JIN SONG SHEN,
YUTAKA HISADA,
YOSHIKATSU ETO,,
TETSUYA KAWAMURA* and
TATSUO HOSOYA*
*Division of Nephrology and Hypertension, Department of Internal Medicine,
Jikei University School of Medicine, Tokyo, Japan. Department of Gene Therapy, Institute of DNA Medicine, Jikei University
School of Medicine, Tokyo, Japan. Department of Pediatrics, Jikei University School of Medicine, Tokyo,
Japan. Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd, Osaka,
Japan.
Correspondence to Dr. Takashi Yokoo, Division of Nephrology and Hypertension,
Department of Internal Medicine, Jikei University School of Medicine, 3-25-8
Nishi-Shimbashi, Minato-ku, Tokyo, Japan 105-8461. Phone: 813-3433-1111; Fax:
813-3433-4297; E-mail:
tyokoo{at}jikei.ac.jp
Abstract. The authors previously reported that bone marrow-derived
CD11b+CD18+cells could be used as a vehicle to deliver
foreign genes intoinflamed glomeruli and that this vehicle cell (v-cell)
couldretard the progression of nephritis by delivering anti-inflammatory
molecules.As a next step, the authors tried to establish a switching system
bywhich v-cells are activated only at the inflamed glomeruli.A recombinant
adenovirus (Ad) that expressed Cre recombinaseunder the control of the
interleukin-1ß (IL-1ß)promoter (AxIL-1pr/Cre) was constructed and
transfected intov-cells. After confirming that AxIL-1pr/Cre expresses Cre by
lipopolysaccharide(LPS) treatment, AxIL-1pr/Cre was infected together with
anotherAd bearing a switching reporter unit in which the LacZ geneis
activated under the control of the CAG promoter by the Cremediatedexcisional
deletion of interposed stuffer DNA. Only a negligiblenumber of
double-infected (Cre/loxPCAG) cells expressed LacZ.This number, however, was
significantly increased by LPS, whichsuggests that LPS-induced Cre
effectively deletes the stufferDNA, which allows for a complete CAG promoter.
DBA/2j mice werethen transplanted with Cre/loxPCAG cells via a tail vein and
treatedwith anti—glomerular basement membrane (GBM) serum. Totrace the
transplanted cells, marker v-cells, infected withAxCANLacZ to constitutively
express the LacZ gene, were alsoused. Although transplanted cells expressing
LacZ collectedin the spleen independent of anti-GBM treatment, they did not
expressthe LacZ gene in the mice transplanted with Cre/loxPCAG cells.On the
other hand, transplanted cells were recruited in theglomeruli and expressed
the LacZ gene upon anti-GBM treatment.These results suggested that only the
v-cells recruited in theglomeruli could be switched on and activate foreign
genes.
Glomerulonephritis, in most cases, progresses toward endstagedamage,
despite active treatment with drugs such as steroidhormones, and throughout
the world a large number of patientshave had to undergo dialysis. This
suggests the need for a next-generationtherapy against glomerulonephritis
that can reduce the problemsthat are encountered in conventional therapy. We
previouslyused bone marrow cells to establish an inflamed site-specificgene
delivery system (1). Before
transplantation, bone marrowcells were differentiated into monocyte-lineage
cells ex vivoto express CD11b and CD18, both of which are ligands of
intercellularadhesion molecule—1
(2), so that these cells could
berecruited at the site of intercellular adhesion molecule—1
expression.Using this system, we retarded glomerular inflammation duringthe
development of glomerulonephritis by delivering an anti-inflammatorycytokine
(3). However, these genetically
modified cells werealso collected in other tissues, such as spleen, without
anyassociation with adhesion molecules
(3). Therefore, as the next
steptoward clinical application, we tried to establish a switchingsystem by
which vehicle cells are activated only at the inflamedsite.
The proinflammatory cytokine interleukin (IL)-1ß playsan important
role in the initiation and progression of glomerulonephritis
(4,5).
Duringthe progression of glomerulonephritis, macrophages that havemigrated
into the inflamed glomeruli secrete IL-1ß,which initiates and/or
enhances the process of glomerular inflammation
(6).This suggests that
glomerular inflammation alters the microenvironmentto switch on the
IL-1ß promoter in the migrated cells.We therefore focused on the
IL-1ß promoter as a markerof glomerular inflammation so that inflamed
site-specific activationmight be achieved. The use of this promoter may,
however, belimited because the expression level is low
(7) and may notbe sufficient
for effective gene expression to prevent progressionof glomerulonephritis. We
previously used chicken ß-actinpromoter to express IL-1 receptor
antagonist (IL-1Ra). Usingthis promoter, we successfully expressed enough
IL-1Ra to cancelthe IL-1ß action and suppress renal injury during
anti-GBMglomerulonephritis. Therefore, a gene activation system thatis
regulated by the IL-1ß promoter but driven by thestronger promoter
should be developed.
Cre recombinase derived from bacteriophage P1 is a 38-kD proteinthat
mediates excisional deletion of a DNA sequence flankedby a pair of loxP
sites, which is composed of two 13-bp invertedrepeats separated by an 8-bp
spacer region (8). When it
bindsto the inverted repeats, Cre synapses with a second loxP siteand then
cleaves the DNA in the spacer region to initiate strandexchange with the
synapsed loxP partner. The gene-activationstrategy involves the excisional
deletion of "stuffer DNA" thatlies between the promoter and the
coding region and preventsexpression. Cre is an enzyme; therefore, a small
amount of expressedCre may process large amount of molecules. Even though a
weakpromoter regulates the transfected gene, the Cre/loxP systemmay change
it into a stronger promoter, which allows it to enhancethe gene expression.
Sato et al. (9)
recently reported thatthe Cre/loxP system may give about 50-fold higher
expressionthan the single transfection directly driven by the original
promoter.Therefore, we constructed a "regulator" adenovirus that
bearsCre under the control of the IL-1ß promoter and double-infected
vehiclecells with the "target" adenovirus containing a
Cre-activatingreporter unit driven by the strong CAG (cytomegalovirus
enhancer-chickenß-actin hybrid) promoter
(10) and confirmed the
regulationin vitro and in vivo. In this study, we combined
our gene deliverysystem with the Cre/loxP system for sitespecific gene
activationto reduce the unexpected side effects of delivering multipotent
anti-inflammatorycytokine to other tissues.
Experimental Design
We constructed a regulator recombinant adenovirus that producesCre under
the control of the IL-1ß promoter (AxIL-1pr/Cre)
(Figure 1).After confirming
that AxIL-1pr/Cre introduces the Cre geneinto vehicle cells and that its
expression is enhanced by lipopolysaccharide(LPS) treatment using PCR and
Western blot analysis, respectively,we transfected AxIL-1pr/Cre together with
a target adenovirus,Ax-CALNLNZ (Figure
1), which bears a switching reporter unitin which a LacZ gene can
be activated under the control of theCAG promoter by the Cre-mediated
excisional deletion of interposedstuffer DNA
(Figure 2). Cre-mediated gene
activation in vitrowas confirmed by X-gal assay of
double-transfected vehicle cellstreated with or without LPS. These cells were
then infused intoDBA/2j mice preimmunized with adjuvant and rabbit IgG 4 d
earlierand then treated with anti-GBM serum 24 h later. To trace the
transplantedvehicle cells, cells infected with AxCANLacZ
(Figure 1) expressingthe LacZ
gene with an intact CAG promoter were used. Kidneyand spleen tissue sections
from each group of mice were subjectedto X-gal assay to examine the
activation of the reporter genein vivo.
Figure 1. Structures of adenoviruses. AxIL-1pr/Cre expresses Cre recombinase under
the control of the IL-1ß promoter. AxCANCre and AxCANLacZ express the Cre
recombinase and LacZ gene, respectively, under the control of the CAG
promoter. AxCALNLNZ is designed to express the LacZ gene only after
Cre-mediated excisional deletion of stuffer DNA. Solid lines indicate the
recombinant adenovirus genome, and arrows show the orientation of the
transcription. IL-1pr, IL-1ß promoter; pA, rabbit ß-globin poly(A)
site; SpA, SV40 early poly(A) site.
Figure 2. Inflamed site-specific gene activation using the Cre/loxP system. Cre
recombinase expressed by the IL-1ß promoter excises the stuffer DNA from
the AxCALNLNZ genome and consequently generates LacZ expression driven by a
potent CAG promoter.
Animals
Seven-week-old female DBA/2j mice were purchased from NipponCrea (Tokyo,
Japan). All animals used in this study were maintainedin our animal facility
on standard laboratory chow.
Establishment of Vehicle Cells
CD11b and CD18 positive vehicle cells were established as described
elsewhere(1). In brief, bone
marrow cells were harvested from the femur,tibia, and pelvis of 7- to
8-wk-old mice and suspended in Dulbecco'smodified Eagle's medium supplemented
with 10% heat-inactivatedfetal bovine serum, 20% heat-inactivated horse
serum, 20% L-929conditioned medium
(11), 100 U/ml penicillin G,
100 µg/mlstreptomycin, and 0.25 µg/ml amphotericin B. Cells wereseeded
onto unprocessed 10-cm dishes at a concentration of 1x 107
cells per dish and cultured in a humidified atmosphereof 5% CO2
for 1 wk. The population of CD11b and CD18 positivecell was verified to be
homologous by flow cytometry (data notshown).
Preparation and Infection of Recombinant Adenovirus
The replication-defective recombinant adenovirus, AxIL-1pr/Cre,was
constructed essentially according to reports elsewhere
(12,13).
Togenerate AxIL-1pr/Cre, we first cloned IL-1ß promotercDNA (kindly
donated by Dr. C.J. Ballone, St. Louis University
[14])into a cassette cosmid
pAxAwNCre (a gift from Dr. I. Saito,University of Tokyo) that carried an
adenovirus type-5 genomelacking the E3, E1A, and E1B regions to prevent
replication.In this construct, Cre cDNA is located downstream of the
IL-1ßpromoter and is followed by the rabbit ß-globin poly(A)
sequence.The resulting cosmid was cotransfected to 293 cells with the
appropriatelycleaved adenovirus genome lacking the E3 region. Recombinant
viruswas propagated and isolated from 293 host cells. AxCANCre, AxCANLacZ,
andAxCALNLNZ were purchased from RIKEN cell bank (Ibaraki, Japan).Bone
marrow cells were cultivated with Dulbecco's modified Eagle'smedium
supplemented with 10% fetal bovine serum, 20% heat-inactivatedhorse serum,
and 20% L929-conditioned medium for 6 d and theninfected with AxIL-1pr/Cre
and AxCALNLNZ (multiplicity of infection= 100 each), as described elsewhere
(1,3)
Western Blot Analysis
Western blot analysis was performed on the Cre secreted fromtransfected
vehicle cells as described elsewhere
(3). Briefly,after
transfection with AxIL-1pr/Cre and AxCALNLNZ, vehiclecells were cultured for
48 h with or without LPS in a 6-wellplate. Cell were harvested and suspended
in lysis buffer (50mmol/L Tris [pH 7.6], 150 mmol/L NaCl, 1 mmol/L
phenylmethylsulfonylfluoride, 1 mmol/L sodium vanadate, 1 mmol/L DTT, 10
mmol/Lsodium fluoride, 10 µg/ml each of leupeptin and aprotinin,and 1%
Brij-96). Two micrograms of protein were subjected toelectrophoresis in a
12.5% sodium dodecyl sulfate—polyacrylamidegel and then transferred to
nylon membrane. The membrane wasblocked with 5% dried milk, 0.1% Tween 20,
and PBS and incubatedwith an anti-Cre recombinase monoclonal antibody (BabCO,
Richmond,CA). It was then incubated with goat anti-mouse Ig horseradish
peroxidaseconjugate. Antigen-antibody complexes were visualized with
chemiluminescencereagents (Amersham, Buckinghanshire, UK).
PCR for Cre Recombinase
PCR for Cre recombinase was performed as described elsewhere
(15).Briefly, vehicle cells
with or without transfection were harvested,suspended in a lysis buffer (50
mM Tris-HCl [pH 7.5], 100 mMNaCl, and 10 mM ethylenediaminetetraacetic acid)
and incubatedwith proteinase K (100 µg/ml) in 0.1% sodium dodecyl sulfate
for60 min at 50°C. The samples were then extracted with phenol-chloroform
(1:1,vol/vol), and DNA was precipitated with ethanol. The extractedDNA was
used as a template for PCR amplification. The 5' primerwas
5'-GGACATGTTCAGGGATCGCCAGGCG-3', and the 3' primer was
5'-GCATAACCAGTGAAACAGCATTGCTG-3'.The Taq polymerase and
reaction buffer were obtained from Sawady(Tokyo, Japan). PCR conditions were
as follows: an initial denaturationat 92°C for 5 min, then 35 cycles of
amplification (denaturation,92°C for 80 s; annealing, 65°C for 60 s;
and elongation,72°C for 90 s). After electrophoresis in a 2% agarose gel,
amplifiedproducts were visualized with ethidium bromide staining.
Induction of Anti-GBM Glomerulonephritis
Glomerulonephritis was induced with an anti-GBM nephrotoxicserum as
described elsewhere (16). In
brief, GBM was preparedfrom Wister Kyoto rats (Charles River Japan, Inc.,
Kanagawa,Japan) by a differential sieving technique and then by sonication
andcentrifugation. Rabbit anti-GBM nephrotoxic serum was raisedby repeated
immunization of a Japanese White rabbit (KitayamaLabes Co., Ltd., Nagano,
Japan) with particulate GBM. The anti-GBMserum was pooled and decomplemented
at 56°C for 30 min.To induce anti-GBM glomerulonephritis, DBA/2j mice
were intraperitoneallyinjected with 0.5 mg/20 g body wt of normal rabbit IgG
(Cappel,West Chester, PA) and complete Freund's adjuvant (1:1 dilution;
Difco,Detroit, MI). The crossreactivity of this serum with murineGBM was
certified by immunohistochemistry, which detected therabbit IgG on the
glomeruli 3 d after injection (data not shown).
X-gal Assay
X-gal assay was performed to assess the expression of the LacZgene as
described elsewhere (17) with
a small modification.In brief, cells or tissue cryosections (6 µm) were
fixedwith 0.25% glutaraldehyde in PBS for 10 min at 4°C and incubatedat
37°C for 3 h in a reaction buffer that contained 1 mg/mlX-gal
(4-Cl-5-Br-3-indolyl-ß-galactosidase), 5 mMpotassium ferricyanide, 5 mM
potassium ferrocyanide, and 2 mMMgCl2 in PBS. LacZ-positive cells
were stained in blue. To quantifythe recruited vehicle cells in the
glomerulus, >50 glomeruliper mouse were randomly assessed, and glomeruli
containing LacZpositive cell(s) were counted. Values were expressed as the
mean± SEM (%).
Transfection into Vehicle Cells and Regulation of AxIL-1pr/Cre
A recombinant adenovirus that expressed Cre recombinase underthe control
of the IL-1ß promoter (AxIL-1pr/Cre) wasconstructed and infected into
primarily established vehiclecells. DNA was extracted from
AxIL-1pr/Cre-transfected vehiclecells and subjected to PCR specific for Cre
recombinase cDNA.As shown in Figure
3, the 269-bp band, which is specific forthe bacteriophage Cre
recombinase gene (15), was
seen in infectedcells. To confirm that the IL-1ß promoter regulatesthe
expression of Cre recombinase, AxIL-1pr/Cre-transfectedvehicle cells were
treated with or without LPS (3 µg/ml),which was previously confirmed to
activate the IL-1ßpromoter in the vehicle cells
(18). Western blot analysis
revealedthat the expression of Cre recombinase was enhanced significantly
withLPS treatment (Figure 4),
which shows that the expression waseffectively regulated by the IL-1ß
promoter.
Figure 3. Adenoviral transfection of Cre recombinase gene under the control of the
IL-1ß promoter into vehicle cells. Freshly established vehicle cells were
transfected with IL-1pr/Cre and subjected to PCR for the Cre recombinase gene.
The 269 bp gene product corresponds to bacteriophage Cre recombinase.
Figure 4. Regulation of Cre recombinase expression by the IL-1ß promoter in the
IL-1pr/Cre transfected vehicle cells. Freshly established vehicle cells were
transfected with IL-1pr/Cre and treated with or without lipopolysaccharide
(LPS), which activates the IL-1ß promoter in vehicle cells. These cells
were subjected to Western blot analysis of the expression of Cre recombinase.
The 38-kD protein corresponds to Cre recombinase.
Regulation of Gene Expression by the Cre/loxP System In Vitro
To elucidate whether the reporter gene is activated by the Cre/loxPsystem
in vitro, AdxIL-1pr/Cre was cotransfected with the
"target"adenovirus AxCALNLNZ bearing a switching reporter unit,
whoseLacZ gene is activated under the control of the CAG promoterby the
Cre-mediated excisional deletion of interposed stufferDNA. As a control,
AxCACre expressing Cre recombinase underthe control of the intact CAG
promoter was also cotransfectedwith AxCALNLNZ into vehicle cells. As shown in
Figure 5, onlya negligible
number of AxIL-1pr/Cre and AxCALNLNZ double-transfectedcells expressed the
LacZ gene, whereas these cell numbers weresignificantly increased by LPS
treatment. On the other hand,the cells that were double-transfected with
AxCACre and AxCALNLNZ,in which Cre recombinase was constitutively induced,
expressedthe LacZ gene independent of LPS treatment. These results indicate
thatLPS-induced Cre recombinase effectively deletes the stufferDNA, which
results in a complete CAG promoter, which inducesthe LacZ gene.
Figure 5. Cre/loxP system—mediated gene expression in vitro. Vehicle
cells were double-transfected with AxIL-1pr/Cre and AxCALNLNZ and treated with
or without LPS. These cells were subjected to X-gal staining to assess LacZ
gene expression. As a control, vehicle cells were transfected with AxCANCre
and AxCALNLNZ by the same protocol.
Regulation of Gene Expression by the Cre/loxP System In Vivo
To examine whether the transgene is activated by the Cre/loxPsystem in
vivo, DBA/2j mice preimmunized with adjuvant and rabbitIgG were injected
with vehicle cells, double-transfected withAxIL-1pr/Cre and AxCALNLNZ via a
tail vein, and treated withanti-GBM serum 24 h later. Three days later, mice
were killedand tissue specimens were subjected to X-gal staining to assess
theLacZ expression. To trace the transplanted vehicle cells,
AxCANLacZ-infectedmarker cells, which constitutively express the LacZ gene
underthe control of the CAG promoter
(Figure 1), were infused to
miceby following the same protocol. In the mice transplanted withmarker
cells, LacZ positive cells were detected in the spleenregardless of anti-GBM
treatment (Figure 6, a and b).
Only anegligible number of LacZ-positive cells were detected in thekidney
(0.4 ± 0.1% of glomeruli contained LacZ-positivecell;
Figure 6c), whereas many cells
were detected in the glomerulus3 d after anti-GBM treatment (85.6 ±
7.7% of glomerulicontained LacZ-positive cells;
Figure 6d). Because
ß-galactosedasewas widely exudated, it is difficult to specify the
locationof LacZ-positive cells; however, it seems to have been in the
mesangialarea (Figure 6, e through
g and Figure 7e through
g). Usingtwo-color immunofluorescence staining with anti-ß
galactosidaseand F4/80, we confirmed that the LacZ-positive cells recruited
inglomerulus still possessed the property of monocyte-lineagecells (data not
shown). These results suggest that transplantedcells accumulated in spleen
independent of inflammation andwere recruited to the inflamed glomeruli upon
treatment withanti-GBM serum. On the other hand, transplanted cells must be
accumulatedin the spleen; however, almost no LacZ-positive cells couldbe
detected in spleen from the mice transplanted with double-transfectedcells.
In contrast, LacZ-positive cells were detected in theglomeruli of these mice
(Figure 7d). The kinetics of
the numberof LacZ-positive glomeruli is shown in
Figure 7h. It peakedat day 3
(79.4 ± 10.9% of glomeruli contained LacZ-positivecells) and decreased
later, which corresponds to our previousdata that
CD11b+CD18+ (Mac-1+) cells were detected in
glomeruli3 d after anti-GBM serum injection but translocated later into
interstitiumuntil 7 d (our unpublished data). Because there are no
significantpathologic changes at this time point (data not shown), we could
notprove the relevance of the severity of glomerular damage andthe number of
infiltrated LacZ-positive cells. However, theseresults suggest that the
vehicle cells that collected in thespleen were not activated, whereas the
cells recruited to theinflamed glomeruli could be switched on to activate the
LacZgene.
Figure 6. Location of vehicle cells after transplantation in mice treated with or
without anti-GBM serum. Vehicle cells were labeled with AxCANLacZ to
constitutively express the LacZ gene and infused to mice. These mice were
treated with or without anti-GBM serum and killed 3 d later. Spleen and kidney
specimens were subjected to X-gal assay. Five mice per group were examined,
and representative pictures are shown. (a) Spleen of the mouse without
anti-GBM serum. (b) Spleen of the mouse with anti-GBM serum. (c) Kidney of the
mouse without anti-GBM serum. (d through g) Kidney of the mouse with anti-GBM
serum. Magnifications: x100 in a through d; x400 in e through
g.
Figure 7. Cre/loxP system—mediated gene expression in vivo. Vehicle
cells were double-transfected with AxIL-1pr/Cre and AxCALNLNZ and infused to
mice. These mice were treated with or without anti-GBM serum and killed at day
0, 1, 3, 5, 7. Spleen and kidney specimens were subjected to X-gal assay.
Representative pictures of day 3 are shown. (a) Spleen of the
double-transfected mouse without anti-GBM serum. (b) Spleen of the mouse with
anti-GBM serum. (c) Kidney of the mouse without anti-GBM serum. (d through g)
Kidney of the mouse with anti-GBM serum. (h) Quantitative analysis.
Percentages of glomeruli containing the LacZ-expressing cells were determined
and described as % LacZ positive glomeruli (%). Magnifications: x 100 in
a through d; x 400 in e through g.
The Cre/loxP system has recently been used for gene activationand
inactivation in transgenic mice
(19,20)
as well as for activationof a transgene located in the adenovirus genome
(21) and ona cell chromosome
(22). In this study, we
applied this geneactivation strategy to the development of an "inflamed
site-molecularswitch" by using an adenovirus that expresses Cre
recombinaseunder the control of the IL-1ß promoter. The Cre-producing
viruswas used to activate the reporter gene under the control ofa potent CAG
promoter in the second adenovirus genome. Doubletransfection of these two
adenoviruses achieved inflamed site-specificactivation, which was maintained
in vitro and in vivo.
It was reported that in vivo gene activation within the glomerulus
couldbe achieved by use of a tetracycline regulatory system
(23)by which expression of
the reporter gene in the cloned mesangialcell trapped in the glomerular
vasculature could be regulatedby oral tetracycline administration
(24). This approach maybe
described as manual regulation; however, during the developmentof chronic
inflammation, the activity for local inflammationis turned on and off and is
difficult to assess by systemicmanifestation for the period of drug
administration. Therefore,an automatic regulatory system by which gene
expression is controlleddepending on the activity for inflammation would be
ideal. Inthis regard, Kitamura and Kawachi
(25) used the promoter of
—smoothmuscle actin to induce a transgene, because the
expression ofthis actin is markedly up-regulated in mesangial cells in awide
range of experimental and human glomerular diseases
(26,27),
andestablished an automatic on/off switching system to expressthe transgene
only where glomerular inflammation occurs anddeactivate it when the
inflammation has subsided
(25). Suchsuccess may
strengthen the rationale for therapeutic gene deliveryfor the treatment of
glomerular inflammation. The use of thissystem, however, is limited because
gene activation by the —smoothmuscle actin promoter is
restricted in glomerular mesangialcells. Also, because stable gene
transfection into residentialmesangial cells in vivo has yet to be
accomplished, it is difficultto apply this system directly to therapeutic
intervention, atleast in its current form. Our gene delivery system is based
onthe genetic manipulation of bone marrow—derived cells,which are
recruited at the site of inflammation in associationwith adhesion
molecule(s). Therefore, we sought another promoterfor automatic gene
activation, which restricted the recruitedinflammatory cells and focused on
the IL-1ß promoter,because macrophages recruited to the inflamed
glomeruli secreteIL-1ß, which initiates and/or enhances the progression
ofglomerular inflammation, suggesting that glomerular inflammationalters the
microenvironment to switch on the IL-1ßpromoter in the migrated cells.
Furthermore, because the IL-1ßgene is inactivated in most resting
mononuclear phagocytes anda stimulus was required to induce its expression
(28,29,30),
basalactivation of the IL-1ß promoter without a stimulusmay be quite
low, as is suggested by Figures
3 and
4. One majorproblem with the
use of this promoter was the low-level inductionafter stimulation
(7), which may not be
sufficient for genedelivery to cancel the inflammatory cytokine(s). Our
previousstudy confirmed that the chicken ß-actin promoterinduces enough
IL-1Ra to cancel IL-1ß action duringthe development of
glomerulonephritis (3).
Therefore, a geneactivation system, which is regulated by the IL-1ß
promoterbut driven by the CAG promoter, was established by combiningthe
IL-1ß promoter and Cre/loxP system. In this study,we showed that
regulation is achieved in the vehicle cells invitro and in
vivo.
This system, however, has several disadvantages compared withthe previous
gene activation systems. First, the ideal systemshould be switched on at the
initiation of inflammation andswitched off at its cessation. The Cre/loxP
system combinedwith the IL-1ß promoter is a one-way activation system
andcould not switch off the gene expression even after the IL-1ß
promoterwas deactivated. In this regard, Kruth et al.
(31) reportedthat
transplanted macrophages disappear within 4 d of theirrecruitment to inflamed
glomeruli. Furthermore, we used an adenovirusto introduce both a regulator
and a target gene, because otherconventional methods that have used liposome,
calcium phosphatecoprecipitation, and retrovirus could not effectively
transduceforeign genes into vehicle cells (unpublished data). Gene
expression,therefore, may be high but transient. We believe that such natural
eliminationmay overcome the disadvantage of this system. Second, as shownin
Figure 7, only a small number
of vehicle cells could be recruitedin the glomeruli even after anti-GBM serum
administration whenthis gene delivery system was used. In this regard, we
previouslyreported that delivery of IL-1Ra, which is driven by chicken
ß-actinpromoter, may suppress the glomerular inflammation in the
anti-GBMglomerulonephritis even if only a few vehicle cells may be recruited
bythis insult (3), which
suggests that therapeutic efficacy doesnot depend on the number of recruited
cells but on the amountof secreted transgene. Because adenovirus may
transduce multiplecopies of the foreign gene and because those may be driven
bythe strong promoter, the anti-inflammatory molecules secretedfrom a small
number of vehicle cells may cancel the proinflammatorycytokine from native
cells. This fact strengthens the rationaleof using adenovirus-mediated
Cre/loxP system with our gene deliverysystem because this may provide the
strong gene activation whilemaintaining inflamed-site specificity.
In conclusion, we succeeded in that inflamed glomeruli-specificgene
activation was achieved when the Cre/loxP system was combinedwith the
IL-1ß promoter. We recently modified thissystem and established
"vehicle cell—producing tissue"by repetitive injection of
retrovirally modified hematopoieticstem cells into sublethaly irradiated mice
to prolong the restrictedtime window of the current system
(32). We have only just begun
thisresearch, and many problems must be solved before any majorinvestment in
clinical use can be made. We still believe thatthese trials represent the
next step in research aimed at thediscovery of a novel therapeutic strategy
for glomerulonephritis.
Acknowledgments
We thank Dr. I Saito (University of Tokyo) for providing cosmids,Dr.
Clifford J. Bellone (St. Louis University) for providingIL-1ß promoter
gene, and Ms. T. Murata for immunohistochemicalanalysis.
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Received for publication November 30, 2000.
Accepted for publication May 12, 2001.
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[Abstract][Full Text][PDF]
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Abstract
Introduction
—smooth muscle actin to induce a transgene, because the
expression of this actin is markedly up-regulated in mesangial cells in a wide
range of experimental and human glomerular diseases
(26,27),
and established an automatic on/off switching system to express the transgene
only where glomerular inflammation occurs and deactivate it when the
inflammation has subsided
(25). Such success may
strengthen the rationale for therapeutic gene delivery for the treatment of
glomerular inflammation. The use of this system, however, is limited because
gene activation by the 
