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,
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DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA 94304;
Canji Inc., San Diego, CA 92121; and Departments of
Molecular Microbiology and Immunology and
Neurology, University of Southern California School of Medicine, Los Angeles, CA 90033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The complete protection of EAE observed in IL-10-transgenic mice (4) suggested that transfer of an expressible IL-10 gene might be similarly effective, yet would permit evaluation of such variables as the timing and anatomic localization of IL-10 expression. We have used a replication-deficient adenovirus (rAdV) vector for efficient transfer the human IL-10 gene to the brain with minimal inflammation caused by the virus itself. These data demonstrate the effectiveness of IL-10 in the treatment of this model of multiple sclerosis and suggests that gene-transfer provides a way of maintaining effective doses of IL-10 when frequent injection is not feasible.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SJL/J and CSJLF1/J mice were obtained from
The Jackson Laboratory (Bar Harbor, ME). BALB/cAnN were obtained from
Taconic Farms (Germantown, NY). All mice were housed under specific
pathogen-free conditions and were used between 7 and 12 wk of age.
Mouse spinal cord homogenate (MSCH) was prepared from 8- to 12-wk-old
BALB/cAnN mice as previously described (11).
PLP139–151 peptide was synthesized by
Biosynthesis (Lewisville, TX). For active induction of EAE, mice were
immunized intradermally over three right flank sites with a total of
150 µl of an PBS-oil emulsion containing 1.5 mg of MSCH and 75 µg
of heat-killed Mycobacterium tuberculosis (strain H37RA;
Difco, Detroit, MI). Immunized mice were challenged over three left
flank sites with the same MSCH preparation at day 7 again. The
frequency of disease is nearly 100%, and the day of disease onset is
predictably between days 13 and 14 after the initial immunization. For
induction of remitting-relapsing form of EAE, SJL/J mice were immunized
intradermally over four dorsal flank sites with a total of 150 µl of
PBS-oil emulsion containing 40 µg of
PLP139–151 peptide and 75 µg of H37RA stain of
M. tuberculosis. The incidence of primary disease is >95%,
and the subsequent disease relapse frequency is 
85% after a single
PLP peptide immunization. Clinical signs of EAE were scored as
previously described (11). Significance was determined by
the Mann-Whitney U nonparametric statistical analysis.
Differences were considered significant if p 
0.05.
Production of replication-deficient adenovirus expressing human IL-10 (hIL-10-rAdV)
The vectors were constructed using standard DNA manipulation
techniques. The final stage of construction relies on in vivo
recombination between the transfer plasmid and the large fragment of a
ClaI-digested lacZ-containing derivative of Ad5/d1237
(A/C/
-galactosidase). The final vector is replication deficient,
with the E1a, Elb, and the pIX region deleted. The vector backbone also
has a partial deletion of the E3 region (12). The plasmid,
pDSRG-IL10 containing the human IL-10 cDNA sequence was obtained from
Kevin Moore (DNAX, Palo Alto, CA). The plasmid was digested with
HindIII (5' end) and EcoRI (3' end) to produce a
full-length fragment encoding human IL-10, which was ligated with the
plasmid pBK-RSV (Stratagene, La Jolla, CA). The insert was excised with
XbaI (5' end) and BamHI (3' end), and then cloned
into the XbaI BamHI sites of the transfer
plasmid, pACN (12) to generate pACNhIL-10. Linearized
pACNhIL-10 was cotransfected with the large ClaI fragment of
a plasmid encoding a derivative of Ad5 (13) into human
embryonic kidney 293 cells to obtain recombinant adenovirus expressing
hIL-10. The resulting hIL-10-rAdV was produced in 293 cells and column
purified as previously described (14). The empty
expression cassette control vector used a transfer plasmid, where a
stop codon was placed after the CMV promoter portion; otherwise, the
vector construction was identical with that of the hIL-10-rAdV.
IL-10 and hIL-10-rAdV treatment
For CNS delivery, the indicated number of viral particles were suspended in 10 µl of PBS and injected into the right lateral ventricle using a 28-gauge needle. For peripheral delivery, rAdV suspended in 200 µl of PBS was injected into the tail vein. Alternatively, rAdV was suspended in 50 µl of PBS and delivered intranasally into anesthetized mice.
For in vivo treatment with rhIL-10 (Schering-Plough, Madison, NJ), mice were injected either s.c. with 10 µg in 50 µl of PBS or intracerebrally (i.c.) with 10 µg in 10 µl of PBS using a 28-gauge needle. For daily treatment, rhIL-10 or OVA-protein control was injected into the right and left lateral ventricles on alternating days. CSJLF1/J mice tolerated the daily i.c. IL-10 treatment regiment for 5 days with no apparent signs of stress or weight loss. However, daily OVA or PBS i.c. treatment was not well tolerated.
Histology and immunochemistry
Mice were killed by CO2 asphyxiation, and spinal cords were removed, fixed in 10% formalin, and embedded in paraffin blocks. Sections were stained with hematoxylin and eosin for light microscopy (11). To determine localization of rAdV 2 days after i.c. injection with LacZ-rAdV, frozen tissue sections of the brain and spinal cord were prepared and stained with X-gal reagent and counterstained with hematoxylin and eosin. To determine the levels of hIL-10 after in vivo gene transfer, serum and CNS samples were taken at the days indicated. Brain and spinal cord samples were collected from mice killed by CO2 inhalation and perfused via the heart with ice-cold glucose-containing PBS. The CNS samples were homogenized in glass Tenbrock tissue grinders with 4 volumes of PBS and centrifuged for 15 min at 400 x g. The concentration of hIL-10 in the serum and CNS extract was determined by a sandwich ELISA using JES3-9D7 and biotinylated JES3-12G8 Abs as described (4).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the ability of IL-10 produced within the CNS to influence
the course of EAE, mice were injected with various numbers of
hIL-10-rAdV particles 2–3 days before the predicated onset of disease.
A dose of 3 x 109 viral particles delivered
by the i.c. route fully protected mice from induction of EAE, whereas
3 x 108 viral particles provided partial
protection and 3 x 107 viral particles
showed marginal or no protection and a reduced mortality rate (Fig. 1
A). Control mice not injected
with rAdV showed a hyperacute course of disease, which resulted in an
80% mortality rate. Injection of 3 x 109
particles of an empty control rAdV not expressing hIL-10 had no
measurable effect on the course of disease. Therefore, 3 x
109 viral particles were used for all subsequent
studies. Intravenous injection of 3 x 109
hIL-10-rAdV particles, in contrast, slightly delayed the progression
but did not alter the severity of disease (Fig. 1, B and
C). The inhibition of EAE was transgene specific because
mice injected i.c. with PBS-vehicle control or the empty rAdV particles
had similar kinetics of disease onset and severity, compared with
untreated mice. Thus, neither the i.c. injection procedure nor CNS
infection by the rAdV vector influenced the course of EAE.
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In the present study, the human IL-10 gene was used to construct
the rAdV vector. The activity of human IL-10 is comparable to
endogenous murine IL-10 and has been shown to be active in suppressing
EAE after both s.c. administration (8) and in a transgenic
model of EAE 4 . After i.c. injection of hIL-10-rAdV,
frozen tissue sections of the brain were prepared for
immunohistochemical staining with either a hIL-10-specific mAb or an
isotype control mAb. An intense cytoplasmic staining of hIL-10 was
found in the ependymal cells lining the ventricles (Fig. 2, C and D). This
staining was hIL-10 specific because no hIL-10 immunostaining was
observed after injection of empty rAdV control (data not shown). In
addition, the pattern of staining is very similar to the pattern of
X-gal staining after i.c. injection of LacZ-rAdV vector (Fig. 2, E and F). The brain tissue levels of hIL-10 at
days 4 and 7 after i.c. injection of 3 x
109 hIL-10-rAdV particles ranged from 13 to 155
ng/g of tissue and spinal cord tissue level at day 7 were between 16
and 100 ng/g of tissue, suggesting that hIL-10 secreted into the
cerebral spinal fluid had diffused throughout the CNS (Table I). The CNS levels of hIL-10 began to
decline at day 8 after i.c. injection, and hIL-10 was not detectable
after day 12 (Table I). Human IL-10 also reached high concentrations in
the serum after i.c. injection, although it was no longer detectable by
day 7 (Table I). Intravenous injection of 3 x
109 hIL-10-rAdV particles produced comparable
levels of serum hIL-10 to that achieved after i.c. injection, but no
hIL-10 was detectable in the CNS (Table 1). Thus, the distinction
between the two routes of rAdV delivery was the presence vs the absence
of IL-10 in the CNS, because the IL-10 levels in the serum were
remarkably similar after either route of gene transfer.
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The complete prevention of disease with hIL-10-rAdV contrasted
with the partial protection or, in some cases, exacerbation previously
reported with peripherally injected rIL-10 (5, 7, 8). The
ability of hIL-10-rAdV to cause sustained IL-10 production within the
CNS seemed the likely explanation for this difference, but a role for
the rAdV vector itself, or the immune response to it, as a cofactor was
also possible. To test this, immunized CSJLF1/J
mice treated with hIL-10-rAdV were compared with mice given daily
injections of rhIL-10, either by i.c. or s.c. injection. Mice receiving
5 daily i.c. injections of rhIL-10 (day 10–14 after priming) were
completely protected from EAE during the course of the IL-10 treatment,
although 4 of 10 mice began to show signs of disease within 3 days of
the last injection (Fig. 3). Mice
receiving s.c. injections of the same dose of rhIL-10 were not
different from untreated controls or controls injected with OVA (Fig. 3). Thus, the host response to the rAdV vector is not required for
protection and rIL-10 can be effective if given repeatedly by the i.c.
route.
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After immunization with myelin Ag, substantial development of
autoreactive T cells occurs within the first 10 days (15),
suggesting that hIL-10-rAdV injection at day 10 exerted its action by
preventing the pathological effector functions of primed T cells. To
determine whether IL-10 treatment could be equally effective at
preventing priming of autoreactive T cells, mice were injected with
hIL-10-rAdV either 2 days before or 10 days after the initiation of
priming. Because hIL-10-rAdV injection resulted in a transient
expression of IL-10 (Table I
), these time points should preferentially
influence the priming and effector phases, respectively. Mice injected
i.c. with hIL-10-rAdV 2 days before immunization had reduced EAE
severity but were not fully protected, as were mice that received
hIL-10-rAdV 10 days after immunization (Fig. 4). Injection of hIL-10-rAdV at day 10,
however, seems to prevent disease for at least 60 days (Fig. 4B) despite the fact that little, if any, hIL-10 remained
within the CNSs after 10 days (Table I). This suggests that IL-10 may
have long lasting effects on self-reactive T cells.
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). Extensive inflammation
was also observed in EAE-susceptible mice that were given hIL-10-rAdV
i.v. (data not shown). Together, these results suggest that one of the
mechanisms by which IL-10 suppresses EAE is inhibition of cellular
invasion to the CNS parenchyma.
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The complete protection achieved by injection of hIL-10-rAdV just
2–3 days before the onset of disease led us to test its effect on
active disease. At day 15 after immunization, mice were divided into
two groups according to disease severity, one with an average EAE grade
of 1 (Fig. 6A) and the second
with an average grade of 3 (Fig. 6B). When hIL-10-rAdV
treatment was initiated in mice exhibiting an EAE grade of 1, clinical
signs did not progress and mice recovered rapidly. In mice receiving
the control rAdV vector, severe disease progressed unabated with an
eventual mortality rate of >60% (Fig. 6A). In the group of
mice tested with an initial EAE grade of 3, all the mice that received
hIL-10-rAdV showed clinical improvement after progressing to EAE grade
4, whereas the control rAdV-treated mice showed no clinical improvement
and had an eventual mortality rate of 78% (Fig. 6B).
However, hIL-10-rAdV treatment was not effective for mice that had
reached an EAE grade of 5 at the time treatment was initiated (data not
shown). Thus, IL-10 gene transfer during active disease could protect
mice from further disease exacerbation and promote recovery even in
this hyperacute disease model.
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In the majority of patients with multiple sclerosis, clinical
disease follows a remitting-relapsing course. Therefore, practical
therapies must be able to delay or prevent relapses. To address this
issue, expression of IL-10 in the SJL mouse model of
remitting-relapsing EAE was examined. SJL mice were immunized either
with MSCH (Fig. 7A) or
PLP139–151 peptide (Fig. 7B). After
initial remission, groups of mice were given a single injection of
either hIL-10rAdV or control rAdV vector. Mice receiving hIL-10rAdV
were significantly protected from disease relapse, whereas all mice
receiving the control rAdV vector suffered multiple clinical relapses.
This result clearly demonstrates that CNS IL-10 gene transfer provides
significant benefit in remitting-relapsing CNS inflammation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The efficacy of hIL-10rAdV given i.c. vs i.v. could not be explained by large differences in the overall production of IL-10 in the brain vs the liver and spleen. Comparable serum IL-10 levels were produced with either route of injection, and the levels in either case were at least 10 times the serum levels measured in fully protected hIL-10-transgenic mice (D. J. Cua and R. L. Coffman, unpublished observations). However, the difference in hIL-10 concentration in the CNS was dramatic between the two routes of injection. Although hIL-10 produced by the ventricular ependymal cells rapidly diffused to the spinal cord and to the serum, hIL-10 produced in the liver and spleen did not reach detectable concentrations in the CNS (the limit of sensitivity of the ELISA was 0.2 ng/g tissue). The presence of hIL-10 in the serum after i.c. injection is primarily due to an unidirectional exit of fluids into the systemic circulation via the arachnoid villi (16). The daily turnover rate of cerebral spinal fluid is nearly 95%, which would result in rapid equilibration of IL-10 from the CNS to the systemic circulation. This is consistent with previous reports demonstrating efficient gene transfer into the ependymal cells lining the brain ventricles and secretion of transduced factors into the cerebral spinal fluid by replication-deficient viral vectors (17, 18, 19, 20). Thus, CNS expression of IL-10 was necessary for inhibition of EAE, but it was not possible to determine whether IL-10 was also required in the periphery for this inhibition. These results may explain the inability of systemic IL-10 treatment to inhibit EAE induced by the transfer of activated T cells (5). Once the T cells are activated and enter the CNS, the impact of IL-10 in the systemic circulation may be limited. Two important features of IL-10 gene transfer contrast it to daily IL-10 injection; steady-state levels of IL-10, rather than wide daily fluctuations in IL-10 concentration, and the mild inflammatory response caused by the virus infection itself. The effectiveness of daily i.c. injections of hIL-10 demonstrates that neither of these parameters is critical for the prevention of EAE by hIL-10rAdV. Thus, the key parameter determining the efficacy of hIL-10 in EAE is the maintenance of effective levels of the cytokine within the CNS.
IL-10 gene transfer just before disease onset was able to completely or
significantly prevent disease in several models of EAE. These include
immunization of CSJLF1/J, SJL, and BALB/c mice
with MSCH or PLP139–151 peptide and C57BL/6 mice
with myelin oligodendrocyte glycoprotein 35–55 peptides (BALB/c and
C57BL/6, data not shown). Intracerebral injection of hIL-10rAdV worked
most effectively when given at day 10, after priming twice with
autoantigen, and a few days before the onset of neurological symptoms.
The reduced effectiveness of IL-10 given at day -2 was likely due to
the transient expression of this vector rather than a counterprotective
effect of early IL-10 expression. Because virally transduced IL-10
production lasted only 8 days in the CNS, it would have ceased in
this group well before the onset of CNS inflammation. Expression of
IL-10 during autoantigen priming only partially suppressed subsequent
disease. Both i.c. (Fig. 4) and i.v. (data not shown) injections of
hIL-10-rAdV 2 days before initial priming, which provided comparable
serum levels of IL-10, only partially inhibited EAE. This is consistent
with a number of studies showing partial, but not complete, reduction
of disease severity after daily systemic administration of soluble
IL-10 starting from the day of immunization (6, 7, 8). These
experiments, taken together, show that hIL-10 delivered by gene
transduction inhibits EAE optimally at the end effector stage within
the CNS. Previous EAE susceptibility studies with mice transgenic for
hIL-10 reached the same conclusion regarding the principal site of
action of IL-10 (4).
These results encouraged us to attempt treatment with hIL-10rAdV after
the onset of disease. In mice with mild or moderate disease, i.c.
injection of hIL-10rAdV halted disease progression and induced rapid
remission, whereas control mice continued to progress and often
required termination. In addition, IL-10 gene transfer after remission
from acute disease also prevented disease relapses for at least 55
days, whereas control mice had two subsequent disease relapses. These
results suggest that the presence of IL-10 for 8 days in the CNS during
events leading to disease onset has significant long-term therapeutic
benefits for CNS inflammatory disorders. It should be noted that CNS
administration of a single 20-µg dose of mouse IL-10 protein after
clinical disease onset had no inhibitory effect on severity of EAE
(21). These results illustrate the importance of
continuous expression of IL-10 within the CNS during the effector phase
of disease for inhibition autoimmune encephalomyelitis. Many recent
studies have begun to address the need for local expression of
therapeutic agents in this disease model. These include local CNS gene
transfer of IL-4, TGF-, CTLA-4-Ig, platelet-derived growth factor-A,
and dimeric p75 TNFR using replication-deficient viral vectors or
genetically engineered cells (17, 22, 23, 24, 25). It should be
mentioned that many different types of immune modulations including
blockade of IL-1-converting enzyme and OX40L/OX40R complex and
administration of 1,25-dihydroxyvitamin D3 as
well as inducible NO synthase inhibitors have been shown to reduce EAE
severity (26, 27, 28, 29); however, relatively few were able to
significantly prevent or reverse disease when given to mice just before
or after the onset of disease symptoms (30, 31).
IL-10 could potentially affect EAE pathogenesis at several points
distal to T cell priming, including recruitment of inflammatory cells
to the CNS and CNS tissue destruction. The absence of visible cellular
infiltrates in mice given hIL-10-rAdV suggests inhibition of macrophage
and granulocyte recruitment and extravasation. IL-10 is known, for
example, to inhibit TNF-
induction of VCAM-1 expression in the CNS
and VLA-4 expression on T cells (32, 33, 34, 35). However, the
lack of effectiveness of systemic IL-10 as well as the ability of CNS
IL-10 to rapidly modulate active disease suggests that IL-10 acts
principally on steps during and after entry into the CNS. During the
acute phase of EAE, as demyelination and axonal degeneration take
place, IL-10 could inhibit both the production and the actions of
proinflammatory cytokines and chemokines, including IL-1, IL-6, IL-12,
GM-CSF, M-CSF, IFN-
TNF-, macrophage inflammatory protein
(MIP)-1, MIP-1, and MIP-2, by both resident microglia and
infiltrating mononuclear cells (36, 37, 38, 39). We are currently
measuring gene expression patterns in hIL-10-rAdV and control mice to
define more precisely the mechanism of action of IL-10 in this disease
model.
The experiments described here demonstrate the therapeutic potential IL-10 in both the prevention and treatment of a Th1-mediated autoimmune disease, provided it can be delivered to the target organ for an appropriate period of time. Although systemic expression or injection of IL-10 allows penetration into many organs, the blood–brain barrier effectively limits the entry of IL-10 to the CNS, despite disruptions to the barrier that might result from CNS inflammation. Gene transfer provides an attractive alternative to recombinant cytokine therapy, especially for prolonged treatment of anatomical sites that are difficult to access. Replication-deficient adenovirus vectors have proven to be convenient experimental tools for cytokine delivery studies in animal models, but their immunogenicity and limited duration of expression make them less than ideal for treatment of chronic conditions, such as multiple sclerosis. These experiments do show, however, that therapy with a vector conferring extended, and perhaps regulated, expression of IL-10 would have promise for the treatment of multiple sclerosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert L. Coffman, 901 California Avenue, Palo Alto, CA 94304.
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; PLP, proteolipid protein; rAdV, replication-deficient adenovirus; MSCH, mouse spinal cord homogenate; hIL-10-rAdV, replication-deficient adenovirus expressing human IL-10; i.c., intracerebral.
Received for publication July 10, 2000. Accepted for publication October 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or the s.c. (
)
route from 10 to 14 days after initial immunization. Control mice
received no i.c. injection (
), 10 µg of OVA (
), or 3 x
109 hIL-10-rAdV (•) particles by the i.c. route at day 10
of immunization. Asterisks indicate a significant inhibition of EAE in
i.c. hIL-10-treated mice, compared with OVA-treated mice
(p 
), or no i.c. injection (
