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*
Kennedy Institute of Rheumatology Division, and
Division of Medicine (Medicine A), Imperial College School of Medicine, London, United Kingdom;
Fourth Department of Surgery, University of Helsinki Central Hospital, Helsinki, Finland; and
Department of Hepatology, Royal Free and University College Medical School, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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concentrations. Both IL-10
protein and the effects of secreted IL-10 were detectable for 10 wk
after AdvmuIL-10 injection. Furthermore, the immunoregulatory effect of
a single AdvmuIL-10 injection was manifest both by a reduction in
TNF-
, IFN-
, and RANTES release from stimulated splenocyte
cultures, and also by a change in the proportion of
CD45RBhigh/low lymphocytes in the spleen compared with
control mice. The delivery of AdvmuIL-10 resulted in a significantly
diminished host antiadenoviral response compared with control
adenoviral vectors. Thus, gene therapy strategies using adenoviral
vectors encoding immunoregulatory and antiinflammatory cytokines may
prove to be a potent approach for the treatment of chronic inflammatory
disease. Antiinflammatory cytokine expression protects against immune
responses directed at gene vectors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transfer of specific colonic T cell subsets from the
IL-10-/- mice into
RAG-2-/- (lymphocyte-depleted) mice reveal that
the colitis is mediated by a subset of CD4+ T
cells that are predominantly of the Th1 phenotype (7).
Thus, inflamed colonic tissue secretes increased levels of TNF- and
IFN-, but no IL-4 (6). In addition, disease can be
abrogated by the administration of anti-IFN- (6),
anti-IL-12 (8), or anti-TNF- Abs (T. Scheinin,
unpublished observations). Luminal bacteria are essential for disease
as IL-10-/- mice reared in germ-free conditions
remain disease free, while inoculation with specific commensal
bacterial strains induces colitis via an Ag-driven Th1 response
(9, 10).
Mice given a systemic injection of LPS, a major constituent of luminal
Ag, develop endotoxic shock associated with elevated levels of
proinflammatory cytokines (11). LPS injection leads to a
significantly higher mortality in IL-10-/- mice
than wild-type controls (12); this effect being mediated
by a more profound elevation of serum levels of TNF-, IL-1, IFN-,
and IL-12. IL-10 plays a crucial role in regulating the response to
LPS, as the excess mortality induced by LPS in
IL-10-/- mice can be reversed by prior or
concurrent administration of recombinant murine IL-10. Likewise, the
prior administration of anti-IL-10 Abs to wild-type mice increases
the mortality observed with previously sublethal doses of LPS
(12).
Further insights into the role of IL-10 in the control of inflammatory mechanisms in colitis come from study of C.B-17 SCID mice repopulated with wild-type CD4+CD45RBhigh T cells. These mice develop a colitis that is also dependent on luminal bacteria and driven by a Th1 response (13). Cotransfer of the reciprocal CD45RBlowCD4+ T cells with the normally pathogenic CD45RBhigh cells prevents the onset of colitis. IL-10 is essential for the differentiation of this regulatory function, as CD45RBlowCD4+ T cells from IL-10-/- mice are unable to prevent the colitis induced by wild-type CD45RBhigh cells (14). Furthermore, treatment with an anti-murine IL-10R mAb abrogates the inhibition of colitis mediated by wild-type CD45RBlowCD4+ T cells.
The administration of exogenous IL-10 has efficacy in treating
experimental colitis in the
CD4+CD45RBhigh transfer
(15) and dextran sodium sulfate models (16).
Furthermore, in patients with Crohn’s disease, daily injection of
IL-10 generates a modest clinical response (17, 18),
although with lower efficacy than alternative biological therapies such
as anti-TNF- Abs (19). Likewise, while daily
injection of weanling IL-10-/- mice with murine
IL-10 prevents disease onset, similar treatment once colitis is
established is only partially able to suppress disease
(6). The dichotomy in therapeutic efficacy in the
IL-10-/- mouse may reflect the pharmacodynamics
of systemic IL-10 administration, delivery of inadequate IL-10 to
inhibit mucosal Th1 responses and proinflammatory cytokine release, or
the fact that early exposure to IL-10 is required for the
differentiation of immunoregulatory cells.
Alternative strategies for IL-10 therapy that deliver constant high
levels of the protein to the gastrointestinal tract may be therapeutic
for established disease in the IL-10-/- mouse.
Gene therapy using adenoviral vectors encoding IL-10 has been shown to
provide high-level protein expression and have therapeutic efficacy in
animal models of rheumatoid arthritis (20, 21). The
systemic administration of adenoviral vectors encoding
galactosidase (Advgal) to healthy mice leads to protein expression
mainly within the liver and spleen, but also in the colon
(22). We hypothesized that i.v. administration of
replication-deficient adenoviral vectors encoding murine IL-10
(AdvmuIL-10) would prove efficacious in both the prevention and
treatment of colitis in IL-10-/- mice. In this
paper, we demonstrate that this approach prevented the spontaneous
development of colitis and also reversed the clinical and histological
features of established disease. The systemic manifestations of disease
were diminished, and the enhanced response to LPS characteristic of
IL-10-/- animals was reversed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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General reagents were of research grade and purchased from Sigma (St. Louis, MO). All reagents used for cell culture were determined to be LPS free using a Limulus amebocyte lysate assay (BioWhittaker, Wokingham, Berkshire, U.K.) as directed by the manufacturer (sensitivity, <10 pg/ml) (23).
Animals
IL-10-/- mice on a C57BL/6 background (purchased from Harlan, Oxon, U.K.) were back-crossed for one generation onto DBA/1 mice to increase breeding vigor and disease expression. Progeny from the interbreeding of the heterozygous offspring were used in all experiments. Newly bred mice were screened for homozygote IL-10-/- and wild-type genotype (C57BL/6 x DBA1). Briefly, tail samples were digested in 250 µl PCR lysis buffer (120 µg/ml proteinase K, 50 mM KCl, 10 mM Tris-HCl pH 8.8, 0.1 mg/ml gelatin, 714 µM MgCl2, 0.45% Nonidet P-40, 0.45% Tween) at 56°C overnight. The DNA encoding the IL-10 and neomycin regions were amplified by PCR using the following synthetic oligonucleotide murine primer pairs: 5'-TAGGCGAATGTTCTTCC-3' (IL-10 sense) and 5'-CAGGCAGCATAGCAGTG-3' (IL-10 antisense); IL-10 sense and 5'-CCTGCGTGCAATCCATCTTG-3' (neomycin). After 35 cycles, the products were separated on 1% agarose gels and visualized by ethidium bromide staining. Bands were observed at 1.5 Kb (neomycin) and 1 Kb (IL-10) under UV light. Mice were maintained in specific pathogen-free (SPF) conditions in a laminar flow hood at all times with free access to food and water. All experiments involving animals were approved by the local ethical review process committee and performed under license from the home office.
Preliminary experiments demonstrated that, under standard laboratory conditions, IL-10-/- animals developed a progressive colitis from 4 wk of age. Clinical manifestations of disease included the passage of mucous, diarrhea, rectal prolapse, and weight loss of >5% of total body weight. Mice were examined weekly and given a clinical score that consisted of one point for each of the above signs. Previous work has demonstrated close correlation between the clinical score and the histological severity of colitis (correlation coefficient 0.865; T. Scheinin, unpublished observation).
Adenoviral vectors
The recombinant E1-deleted type 5 adenoviral vectors, encoding
murine IL-10 under the transcriptional control of the rous sarcoma
virus promoter (AdvmuIL-10), -galactosidase under the CMV promoter
(AdvGal), or empty cassette adenovirus (Adv0), were
generously donated by A. P. Byrnes, M. Wood, and H. Charlton
(University of Oxford, Oxford, U.K.). Viruses were propagated in the
293 human embryonic kidney cell line (Quantum Biotechnology, Montreal,
Canada) and purified by ultracentrifugation through two cesium
chloride gradients (Boehringer Mannheim, Lewes, Sussex, U.K.). The
titer of adenoviral vectors was determined by plaque assay on 293 cells
(24). Viral stocks were aliquoted and stored in 10%
glycerol at -80°C until use.
Experimental protocols.
For assessment of the effect of AdvmuIL-10 on the development of colitis, 1 x 108 PFU of viral vector (AdvmuIL-10 or Adv0) in 100 µl PBS or vehicle alone was injected into the tail vein of 4- to 5-wk-old IL-10-/- mice under light sedation. To assess the effect of AdvmuIL-10 on established disease, 5 x 108 PFU of viral vector was injected into the tail vein of 10-wk-old IL-10-/- mice with clinical signs of colitis. A total of 100 µl PBS was injected into the tail vein of wild-type littermates as a negative control. At weekly intervals, the clinical score of each animal was assessed as previously described, and stool samples were collected. Animals were sacrificed by cervical dislocation at the indicated time points. Serum was collected via cardiac puncture, spleens were harvested, stool samples were collected, and serial segments of colon, cecum, and ileum were fixed in 10% neutral buffered formalin for histological analysis. In addition, 25 age-matched IL-10-/- mice were injected with 1 x 108 PFU AdvmuIL-10 to assess the sites of IL-10 protein expression. Batches of three mice were randomly selected at two weekly intervals and sacrificed. The liver, spleens, and colons of these animals were weighed and homogenized in 5 µl PBS/mg tissue. Supernatants were harvested after centrifugation and stored at -20°C until assay
Histological analysis.
Samples were routinely processed, sectioned at 5 µm, and stained with hematoxylin and eosin for light microscopic examination. Assessment was performed by an investigator blinded to treatment group. Five segments of colon were examined per mouse and each given a histological score from 0 to 4 as described (6); thus a total score for each mouse from 0 (no change in any segment) to 20 (grade 4 changes in all segments) was obtained. Scores of <5 were deemed to be within normal limits.
Stool samples
Stool samples were collected weekly from all animals and weighed. Samples were emulsified in 500 µl/100 µg stool weight of a solution of 1 mg/ml soy trypsin inhibitor and 1 mg/ml PMSF in PBS. Supernatants were collected after centrifugation at 10,000 x g for 15 min and stored at -20°C.
Spleen cell cultures
After sacrifice, each spleen was placed in RPMI 1640 medium (PAA Laboratories, Yeovil, U.K.) supplemented with 10% FCS, 100 µg/ml penicillin, and 100 µg/ml streptomycin (BioWhittaker). Cell suspensions were obtained by passing tissue through a 200-µm2 nylon mesh. After erythrocyte lysis, cells were washed in HBSS three times before resuspension in medium. Cells were plated at 2 x 106/well in 12-well plates (Falcon; BD Labware, Mountain View, CA) in a final volume of 1 ml of medium with or without 10 µg/ml LPS, 10 ng/ml recombinant murine IL-10 (Schering-Plough, Madison, NJ), 10 µg/ml neutralizing rat anti-murine IL-10 Ab (JES5-2A5, donated by DNAX Research Institute, Palo Alto, CA), or a rat IgG1 isotype control (OX20; American Type Culture Collection, Manassas, VA). Cultures were maintained for 24 h before supernatants were harvested and stored at -20°C .
Measurement of serum antiadenoviral Ab response
The neutralizing antiadenovirus Ab response was analyzed in
serum from untreated or treated IL-10-/- mice
14 days after gene transfer with 5 x 108
PFU of AdvmuIL-10, Advgal, or Adv0 (25). Serum samples
(100 µl) were heat inactivated at 56°C for 30 min and diluted
2-fold in serum-free DMEM. Each dilution was incubated for 90 min at
37°C with 2 x 106 PFU of Advgal and
applied in duplicate to 80% confluent 293 cells on a 96-well plate.
After 1 h at 37°C, 50 µl of DMEM containing 10% FCS was added
to each well, and cells were cultured for a further 36 h. Cell
supernatants were then removed and replaced with 30 µl of 0.25 mM
Tris-HCl, pH 7.8. Plates were vortexed, frozen, and then thawed for
three cycles to detach and lyse cells and centrifuged at 1000 rpm for
20 min. Ten microliters of the supernatant from each well was mixed
with 90 µl of a -galactosidase substrate solution containing1
mg/ml
o-nitrophenyl--D-galactopyranoside,
1 mM MgCl2, 45 mM 2-ME, in 0.1 M sodium phosphate
buffer, pH 7.5. The enzyme reaction was stopped after 5 min with the
addition of 150 µl of 0.1 M
Na2CO3, and plates were
read at 405 nm. The mean OD of serum from four animals for each group
was compared at a dilution that resulted in 50% inhibition of the Ab
response in Advgal-treated mice.
Cytokine analysis
Cytokine concentrations were measured by sandwich ELISA using
paired Abs according to the manufacturers recommendations (IL-10 and
TNF- were purchased from BD PharMingen, San Diego, CA; IFN- was
purchased from Genzyme Diagnostics, Cambridge, MA; IL1- and RANTES
were purchased from R&D Systems, Abingdon, Oxon, U.K.).
SAP measurement
SAP levels were measured in terminal serum samples by ELISA. Briefly, microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) were coated overnight with 4 µg/ml trinitrophenylated keyhole limpet hemocyanin before serial dilutions of sera and standards (murine SAP; Calbiochem-Novabiochem, Nottingham, U.K.) were added. Bound SAP was detected with rabbit anti-mouse SAP (Calbiochem-Novabiochem) followed by peroxidase-labeled donkey anti-rabbit Ig (Amersham Life Sciences, Little Chalfont, U.K.). Samples were developed using the peroxidase substrate system (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and read at 450 nm on a plate reader (Labsystems, Chicago, IL).
Statistical analysis
Data that exhibited a normal distribution were analyzed using a two-tailed t test; for comparison of more than two means, a two-way ANOVA was performed. Data that did not exhibit a normal distribution was displayed as the median value.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-10-/- mice spontaneously develop an
enterocolitis with associated systemic features such as weight loss and
an elevated acute-phase response (6). Daily i.p.
injections of rIL-10 have been shown to prevent disease onset if
commenced from 4 wk of age. To investigate the therapeutic efficacy and
expression duration of adenoviral vectors encoding IL-10, 4-wk-old
IL-10-/- mice received a single systemic
injection of 1 x 108 PFU AdvmuIL-10, Adv0,
or PBS vehicle. Mice that had received PBS or Adv0 developed a
progressive colitis associated with failure to thrive, whereas
AdvmuIL-10-injected mice did not exhibit clinical signs of colitis and
gained weight throughout the 10-wk experiment (Fig. 1
; p 
0.001). After
sacrifice, histological sections of five regions of each colon were
scored for disease activity. The mean histological score of
IL-10-/- mice treated with AdvmuIL-10 was
2.5 ± 0.8, which was significantly lower than the mean score of
mice injected with either Adv0 (8.3 ± 2.3; p
0.025) or saline (12.1 ± 1.8; p 0.001).
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than AdvmuIL-10-treated mice (Fig. 2;
p 0.001). On sacrifice, 10 wk after therapy,
IL-10-/- mice treated with either saline or
Adv0 had significantly elevated SAP levels (1742 ± 439 and
1636 ± 369 µg/ml, respectively) compared with
AdvmuIL-10-treated mice (280.8 ± 58.65 µg/ml; p
0.004 cf saline; p 0.002 cf Adv0; Fig. 2). There
were no significant differences in SAP concentration between
AdvmuIL-10-treated IL-10-/- mice and littermate
C57BL/6 x DBA1 mice (280 ± 58.7 and 167.3 ± 51.7
µg/ml, respectively; data not shown).
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Daily i/p IL-10 injection to 10-wk-old
IL-10-/- mice will only partially abrogate
established colitis (6). In contrast, a single systemic
injection of 5 x 108 PFU AdvmuIL-10 to
10-wk-old IL-10-/- mice with overt disease
returned clinical scores to that of control mice, while 5 x
108 PFU of Adv0 has no effect on disease
progression (Fig. 3). Thus the clinical
score in AdvmuIL-10-treated mice fell rapidly to baseline over the 4-wk
experiment, whereas in Adv0-treated mice it rose from 1.5 ± 0.57
to 2.75 ± 0.5 (p 0.001). This was
reflected in the elevated stool IL-1 concentrations measured in
IL-10-/- mice treated with Adv0 compared with
both AdvmuIL-10-treated IL-10-/- mice
(p 0.02) and saline-treated C57BL/6 x
DBA1 wild-type controls (p 0.01) (Fig. 3).
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0.001 cf 10-wk-old littermates), which
was not significantly different from age-matched wild-type controls
(0.25 ± 0.25; p 0.39; Fig. 3
). In contrast,
mice treated with Adv0 demonstrated persistent colitis (histological
score of 10.25 ± 3.1; p 0.02 cf AdvmuIL-10
treatment). Likewise, whereas terminal SAP levels were significantly
elevated in Adv0-treated IL-10-/- mice compared
with controls (1716 ± 639 µg/ml cf 189 ± 45 µg/ml;
p 0.05), there was no significant difference in
acute-phase proteins between AdvmuIL-10-treated
IL-10-/- mice and wild-type controls (Fig. 3).
AdvmuIL-10 treatment inhibits TNF- and IFN- release from
LPS-stimulated splenocytes
IL-10-/- mice release elevated levels of
TNF- and IFN- in response to challenge with LPS, a major
component of luminal Ag (12). To determine whether a
single dose of AdvmuIL-10 resulted in persistent suppression of this
inflammatory response, we compared the proinflammatory cytokine release
from LPS-stimulated spleen cells harvested from mice used in the above
experiments. TNF- and IFN- release from unstimulated splenocytes
were below the sensitivity of the ELISA for all animals (<40 pg/ml and
<120 pg/ml, respectively). LPS (10 µg/ml) induced a significantly
greater TNF- and IFN- response in splenocytes harvested from
IL-10-/- mice 10 wk after injection with saline
or Adv0 than AdvmuIL-10 or wild-type controls (Fig. 4a, p 0.005
for TNF- and p 0.05 for IFN-). AdvmuIL-10
treatment also diminished the LPS response in splenocytes from
IL-10-/- mice treated at 10 wk of age and
sacrificed 4 wk later, although this did not reach statistical
significance (data not shown). The addition of IL-10 (10 ng/ml)
decreased both TNF- and IFN- release from all animal groups to
undetectable levels (data not shown).
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The efficacy of AdvmuIL-10 both on the treatment and prevention of colitis and the long-term suppression of LPS-induced responses from cultured splenocytes may be due to the direct immunosuppressive effect of secreted IL-10 throughout the 10 wk of the experiment. Alternatively, early exposure to IL-10 may redress differences in T cell differentiation that have been reported in the IL-10-/- mouse. Levels of IL-10 protein in both the serum and homogenates of liver, colon, and spleen were determined at 2-wk intervals throughout the experiment.
High levels of IL-10 (17 ± 4.8 ng/ml) could be detected in the
serum of AdvmuIL-10-injected IL-10-/- mice for
up to 7 days after injection with adenovirus. Serum levels of IL-10
after this time and in all other groups were below the threshold of the
ELISA. IL-10 protein was detected in homogenates of the liver, colon,
and spleen of IL-10-/- mice for 
10 wk after
injection with 1 x 108 PFU AdvmuIL-10 (Fig. 5). The biological activity of the IL-10
was determined in splenocyte cultures harvested from both
IL-10-/- mice 10 wk after viral injection and
C57BL/6 x DBA1 littermates.
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release (Fig. 4b). As expected, neutralizing IL-10 had no enhancing effect
on TNF- release by splenocytes from IL-10-/-
mice treated with saline or Adv0 (data not shown); in contrast it led
to a 3-fold increase in TNF- release from splenocytes harvested from
IL-10-/- mice treated with AdvmuIL-10 10 wk
previously (preliminary experiments determined that the isotype control
OX20 had no effect on LPS-stimulated TNF- release from splenocytes
harvested from AdvmuIL-10-treated mice). This demonstrates that at
least part of the diminished LPS response of splenocytes from
AdvmuIL-10-treated IL-10-/- mice is
attributable to secreted IL-10 and confirms the biological activity of
the IL-10 detected in splenic homogenates. However, even after secreted
IL-10 has been neutralized, TNF- release from LPS-stimulated
splenocytes from AdvmuIL-10-treated IL-10-/- or
control C57BL/6 x DBA1 mice is still significantly lower than saline
or Adv0-treated IL-10-/- mice, suggesting a
immunoregulatory change independent of the contemporaneous presence of
IL-10.
Analysis of harvested splenocytes by flow cytometry revealed no
differences in either the ratio of CD4+ to
CD8+ cells or in the cellular expression of CD62L
between groups (data not shown). However, whereas splenocytes from
IL-10-/- mice treated with saline or Adv0 at
either 4 or 10 wk were predominantly CD45RBlow,
those from AdvmuIL-10-treated IL-10-/- mice or
control C57BL/6 x DBA1 littermates were predominantly
CD45RBhigh (Fig. 6). In addition, there were marked
differences between groups in the level of the T cell-secreted
chemokine RANTES, which acts to recruit circulating leukocytes to sites
of inflammation. Thus, LPS-stimulated splenocytes harvested from
IL-10-/- mice treated with AdvmuIL-10 10 wk
previously secreted significantly lower levels of RANTES (2.4 ±
0.3 ng/ml) than splenocytes from IL-10-/- mice
treated with either Adv0 (10.2 ± 0.68 ng/ml; p
0.0001) or saline (9.7 ± 0.88 ng/ml; p 0.0013;
Fig. 6).
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Previous studies have demonstrated elevated titers of anti
adenovirus Abs in mice treated with adenoviral vectors
(26). This response can be diminished by interrupting Ag
presentation at the time of initial exposure using nondepleting
anti-CD4 Abs (27) or coadministration of soluble
CTLA4Ig (28). We hypothesized that the neutralizing Ab
response to AdvmuIL-10 vectors would be diminished, as the protein
encoded for by this virus will act to suppress T cell activation in
response to the presented Ag. A bioassay was used to detect the
presence of antiadenoviral Abs in the serum of
IL-10-/- mice treated 2 wk previously with
saline, Adv0, AdvmuIL-10, or Advgal. A standard curve was generated
using 2-fold dilutions of the serum from
IL-10-/- mice injected with Advgal. Dilution
to 1:16 led to a 50% reduction in the ability of this serum to
neutralize the exogenous Advgal; thus, the mean OD for each group at
this dilution was compared. Serum from saline-treated
IL-10-/- mice gave a mean OD of 1.42 ±
0.01 representing 100% infection of the 293 cells (25).
There was no significant difference in the ability of serum from Adv0-
and Advgal-injected mice to neutralize exogenous Advgal (mean ODs
of 0.726 ± 0.001 and 0.74 ± 0.13, respectively;
p < 0.9). This suggests that the antiadenoviral Abs
target viral Ags and are not specific to the construct inserted into a
particular virus. In contrast, a 1:16 dilution of serum from
AdvmuIL-10-injected mice had significantly lower ability to neutralize
exogenous Advgal (mean OD 1.2 ± 0.16; p <
0.042 compared with Adv0 and p < 0.044 compared with
Advgal).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our study, a single systemic dose of 1 x 108 PFU AdvmuIL-10 given at 4 wk of age was sufficient to prevent the clinical onset of colitis in IL-10-/- mice throughout the 10 wk of the experiment. In addition, it suppressed the mucosal release of proinflammatory cytokines, diminished the acute-phase response, and led to significantly lower histological scores than Adv0- or saline-treated IL-10-/- mice. Furthermore, in contrast to daily i.p. injections of IL-10, a single i.v. dose of 5 x 108 PFU AdvmuIL-10 administered to 10-wk-old IL-10-/- mice with clinical and histological signs of colitis completely reversed their disease. Four weeks after injection, the histological score and SAP level was not significantly different to littermate C57BL/6 x DBA1 controls. In controls contrast, 10-wk-old IL-10-/- mice treated with Adv0 developed progressive colitis with elevated histological scores and SAP levels.
The colitis in IL-10-/- mice is dependent on an
enhanced Th1 T cell response to the bacterial component of luminal Ag
(7). The differentiation of Th1 cells reactive to luminal
Ag is dependent on both the cognate interaction between naive T cells
and APCs and the stimulatory effects of cytokines such as IL-12
(31). This effect of APC-derived IL-12 is enhanced by the
IFN- release from the developing Th1 lymphocyte (32).
In the intact gastrointestinal tract, Ag presentation also results in
IL-10 release that acts to antagonize the positive feedback loop toward
Th1 lymphocyte differentiation. Thus in the IL-10-deficient mouse
persistent antigenic stimulation and uncontrolled release of IL-12 and
IFN- may lead to excessive generation and uncontrolled activation of
a set of Th1 T cells reactive to luminal Ag, thus converting protective
immunity to a pathological response (33). This hypothesis
is supported by increased TNF- and IFN- release in
IL-10-/- mice exposed to LPS, a major
constituent of luminal Ag (12). In our experiments there
was no difference in TNF- or IFN- release from cultured spleen
cells isolated from wild-type, IL-10-/-, or
IL-10--/- mice treated with AdvmuIL-10.
However, the addition of LPS to the cultures resulted in significantly
increased release of TNF- and IFN- from splenocytes isolated from
IL-10-/- mice treated with saline than those
treated with AdvmuIL-10 or wild-type mice. This demonstrates that the
therapeutic effect of AdvmuIL-10 is consistent with a reduction in the
inflammatory response to LPS and that this effect persists for at least
10 wk.
It was possible to detect murine IL-10 in the serum of
AdvmuIL-10-injected mice at concentrations ranging from 0.8 to 64
ng/ml for up to 1 wk after adenoviral injection. These results are
comparable to those reported in DBA1 mice given a systemic injection of
1 x 109 PFU of an adenoviral vector
encoding viral IL-10 (21). In contrast, when AdvmuIL-10
was given i.p. to the rat, serum levels of just 12 pg/ml were recorded
for up to 6 days, which may explain the lack of efficacy reported in
established trinitrobenzene sulfonic acid colitis
(29). It has previously been reported that systemic
delivery of adenoviral vectors encoding a marker protein generate high
levels of protein expression in the liver and spleen, but also within
the colon for up to 7 days (22). We have demonstrated
persistent IL-10 expression in the liver, spleen, and colon of
AdvmuIL-10-injected IL-10-/- mice for 10 wk.
Indeed, there was functional evidence for secreted IL-10 in cultures of
splenocytes harvested from animals in the above
experiments. Thus, neutralizing secreted IL-10 with a mAb increased
LPS-induced TNF- release from AdvmuIL-10-treated
IL-10-/- mice as well as control C57BL/6
x DBA1, but had no effect on cultures of spleen cells from
IL-10-/- mice treated with saline. Therefore,
it is possible to detect the sequelae of AdvmuIL-10 therapy for at
least 10 wk after a single injection.
Recombinant IL-10 delivery is able to prevent the colitis induced by
the transfer of
CD45RBhighCD4+ T cells into
SCID mice (15). However, this effect is only transient, as
colitis will develop after the treatment is stopped. In contrast,
long-term protection is achieved by cotransfer of
CD45RBlowCD4+ T cells,
which are dependent on IL-10 for their formation and both IL-10 and
TGF- for their effect (14). The effectiveness of
AdvmuIL-10 therapy could result from immunosuppression due to long-term
secretion of IL-10 from infected cells in the liver, spleen, and
gastrointestinal tract. Alternatively, IL-10 exposure may result in the
differentiation of a group of regulatory T cells that exert their
effect by the release of immunoregulatory molecules such as
TGF-.
LPS-stimulated splenocytes isolated from
IL-10-/- mice 10 wk after AdmuIL-10 treatment
released significantly less TNF- and IFN- than Adv0- or
saline-treated IL-10-/- mice. The addition of
anti IL-10 Abs was unable to reverse this effect completely, which may
suggest that there are phenotypic differences between spleen cells
harvested from IL-10-/- mice and those
harvested from IL-10-/- mice treated with
AdvmuIL-10. In support of this hypothesis, Berg et al. have reported
that CD4+ cells isolated from
IL-10-/- mice express low levels of CD45RB
compared with wild-type mice (6). It should be remembered
that whereas wild-type
CD45RBlowCD4+ T cells
protect against colitis in the CD45RBhigh
transfer to SCID mouse model, CD45RBlow cells
isolated from IL-10-/- mice are in fact
pathogenic. We demonstrate that, whereas spleen cells isolated from
IL-10-/- mice expressed low levels of CD45RB,
those isolated from either AdvmuIL-10-treated
IL-10-/- mice or wild-type littermates
expressed high levels. In addition, treatment with AdvmuIL-10 reduced
the levels of RANTES secreted by splenocytes from
IL-10-/- mice. Elevated levels of RANTES, a T
cell-derived chemokine that directs infiltrating leukocytes to sites of
inflammation, have been found in the mucosa of patients with Crohn’s
disease (34, 35). Therefore, in addition to inhibiting
proinflammatory cytokine release, AdvmuIL-10 therapy is likely to
decrease leukocyte recruitment. Levels of IL-5, IL-10, and TGF-
release were below the limit of detection of the assays used (data not
shown). In this study, it was not possible to confirm whether treatment
with AdvmuIL-10 promotes the formation of regulatory T cells.
Studies of adenoviral vectors encoding foreign and nonimmunoregulatory
proteins have demonstrated host antivirus immune responses that limit
the duration of delivered gene expression and prevent retreatment
(26, 36, 37). Both cellular and humoral antiadenoviral
responses can be diminished by coadministration of nondepleting
anti-CD4 Abs or CTLA4Ig (27, 28). It is possible that
administration of adenoviral vectors encoding immunoregulatory proteins
such as IL-10 will also provoke a diminished host response. Thus,
prolonged IL-10 expression from AdvmuIL-10-infected respiratory
epithelium has been reported, whereas the expression of the foreign
protein -galactosidase is curtailed by antiadenoviral host responses
(38). One might predict that
IL-10-/- mice would generate enhanced
antiadenoviral responses, leading to increased adenoviral vector
clearance and reduced tissue expression of the delivered protein.
However, we have demonstrated that both the expression of IL-10 within
tissue homogenates and its immunoregulatory effects persist for at
least 10 wk in this model after systemic administration of AdvmuIL-10.
Furthermore, we have demonstrated that the prolonged efficacy of
AdvmuIL-10 is associated with a diminished antiadenoviral Ab response
compared with administration of Adv0 or Advgal.
Trials of systemic IL-10 in patients with Crohn’s disease have achieved only a modest therapeutic benefit, despite the ability of exogenous IL-10 to inhibit proinflammatory cytokine release by mononuclear cells isolated from Crohn’s disease tissue in vitro (39). In this paper, we have demonstrated the therapeutic advantage of adenoviral vectors encoding IL-10 over systemic IL-10 administration in the prevention and treatment of colitis in the IL-10-/- mouse. In addition, the delivery of an immunoregulatory cytokine gene is shown to minimize the host response to the adenoviral vector and thus allow long-term IL-10 expression. Whether, using alternative delivery systems such as adenoviral vectors, IL-10 could prove effective in patients with Crohn’s disease remains speculative. However, these results highlight the potential of IL-10 gene therapy in the treatment of chronic inflammatory conditions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James O. Lindsay, Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, 1 Aspenlea Road, Hammersmith, London, W6 8LH, U.K. E-mail address: j.lindsay{at}ic.ac.uk
3 Abbreviations used in this paper: SAP, serum amyloid protein; AdvmuIL-10, adenoviral vector(s) encoding murine IL-10; Advgal, adenoviral vector(s) encoding -galactosidase; Adv0, empty cassette adenovirus; SPF, specific pathogen-free.
Received for publication February 9, 2001. Accepted for publication April 12, 2001.
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, plays a major role in sustaining the chronic phase of colitis in IL-10-deficient mice. J. Immunol. 161:3143. interferon-dependent mechanism. Infect. Immun. 66:5157. for Crohn’s disease: Crohn’s Disease cA2 Study Group. N. Engl. J. Med. 337:1029. beta TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN- production is IFN--dependent. Int. Immunol. 5:1119. gene expression in pediatric small bowel Crohn’s disease. J. Pediatr. Gastroenterol. Nutr. 25:371.[Medline]
receptor signaling and IL-10 gene therapy regulate the innate and humoral immune responses to recombinant adenovirus in the lung. J. Immunol. 164:443.This article has been cited by other articles:
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