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Departments of
*
Surgery and
Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610; and
Canji, Inc., San Diego, CA 92121
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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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, CpG motifs, or apoptotic cell fragments, the immature
DC will begin a maturation process involving modulation of chemokine
receptor expression, migration to the draining lymph node, increased
processing and presentation of peptides on both class I and class II
MHC molecules, up-regulation of costimulatory molecules such as CD80
and CD86, and increased production of soluble cytokines such as IL-12
(3, 4). Within the lymph node, the mature DC secretes
chemokines such as macrophage-inflammatory protein-3
to attract
large numbers of naive T cells to the area, allowing the presented
peptide:MHC to interact with a wide variety of TCR complexes. Dysregulation of the immune response occurs during sepsis and often leads to the development of multiorgan failure. Although the pathogenesis of the sepsis response to bacterial pathogens has been extensively studied, the mechanisms leading to high morbidity and mortality in patients remain unclear. Possible explanations include an unregulated production of pro- and anti-inflammatory cytokines that leads to endothelial injury as well as increased activation-induced cell death (apoptosis) of T and B lymphocytes that limits acquired immune response-mediated clearance of pathogens (5, 6).
The effects of sepsis on DC function, however, are largely unknown.
Bacteremia, endotoxemia, and generalized peritonitis, all of which
promote extensive inflammatory cytokine release (e.g., TNF-) may
induce unregulated and widespread activation and maturation of DCs. In
the latter phases of sepsis, DCs may globally lose their ability to
produce IL-12 and generate protective Th1 immune responses against the
primary and secondary infectious organisms (7, 8).
Alternatively, extensive activation of DCs at early time points could
promote responses that limit the capacity of these cells to contain the
sepsis response. Controlling the extent of the inflammatory response
and DC activation may provide novel therapeutic approaches to limit the
untoward outcomes of the sepsis response.
The goal of the present study was to determine whether modification of DCs toward a less activated state would influence the outcome of sepsis. Previous studies demonstrated that IL-10 potently inhibits DC maturation, Ag presentation, and IL-12 production and maintains macropinocytosis and endocytosis (9, 10). Furthermore, DCs treated with an anti-IL-10 Ab show an increased capacity to activate and prime naive T cells to a more prominent Th1 polarization, suggesting that autocrine regulation of DCs by this cytokine is important (11).
We hypothesized that transduction of DCs to induce transient expression of human IL-10 (hIL-10) would inhibit DC maturation in the setting of sepsis and might modify the outcome to the septic response. To accomplish this, we considered the use of recombinant viral vectors. DCs have been shown recently to be permissive to adenovirus (Adv) infection in vitro at high particle concentrations (12). Due to the relatively short duration of transgene expression, Adv appears promising as a vector for transient gene therapy (13, 14), appropriate for acute inflammatory processes such as sepsis.
We show in this work that the footpad injection of adenoviral vectors results in efficient transgene expression by DCs in the draining popliteal lymph node but not in other lymphoid organs. Transduction of DCs with adenoviral vectors expressing IL-10 is also associated with reduced DC maturation and reduced CD4+ and CD8+ T cell activation in the draining lymph node. IL-10 gene expression localized to the popliteal lymph node DCs improves the survival of mice challenged with cecal ligation and puncture in a murine model of polymicrobial sepsis. These studies suggest that compartmentalized expression of IL-10 in DCs is effective in improving the outcome in sepsis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free female C57BL/6 mice between 5 and 8 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME). They were maintained on standard rodent food and water ad libitum. The studies were approved by the institutional animal care and use committee at University of Florida College of Medicine before initiation of these studies.
Construction of a recombinant Adv expressing hIL-10 or gfp
A derivative of human Adv serotype 5 was used as the source of viral DNA backbone. This modified adenoviral vector backbone contains a deletion of bp 355–4,021, resulting in a deletion of the E1a, E1b, and protein IX polypeptides. In addition, there is a deletion of bp 28,592–30,470, which results in a loss of 1.9 kb of DNA from the E3 region. A recombinant Adv expressing the hIL-10 cDNA transgene was constructed using standard homologous recombination methods, as originally described by Graham and Prevec (15). Briefly, the hIL-10 cDNA containing the full-length translated region (from pDSRG-IL10 plasmid, respectively; obtained from K. Moore, DNAX Research Institute, Palo Alto, CA) was subcloned into the BamHI/XbaI cloning site of the pACN transfer plasmid. The pACN transfer plasmid is based on pBR322 and contains, from 5' to 3', the Adv serotype 5 (Ad5) inverted terminal repeat and packaging signal (Ad5 bp 1–358), the human CMV immediate early enhancer/promoter (CMV), the Adv serotype 2 tripartite leader sequence (PL), a multiple cloning site, and Adv serotype 2 bp 4,021–10,457. This plasmid was cotransfected into 293 cells along with a ClaI-linearized fragment of the plasmid described above containing the modified human Adv serotype 5 vector backbone. Additionally, a recombinant Adv containing an empty expression cassette was constructed for use as a control. All viral constructs were similar, with the exception of the transgene, and the production and purification procedures were identical (16).
Footpad gene therapy
Following anesthesia mice were injected into both hind footpads with 50 µl Adv or buffer using an insulin syringe and a 29-gauge needle (BD Biosciences, Franklin Lakes, NJ). Mice received injections of 2 x 105, 2 x 107, or 2 x 1010 particles of a recombinant Adv construct expressing hIL-10 (Adv/hIL-10), green fluorescent protein (gfp) as a reporter gene (Adv/gfp), or an identical Adv recombinant containing an empty cassette (Adv/empty) in buffer (PBS supplemented with 2 mM MgCl2 and 3% sucrose). Twenty-four hours later mice were euthanized or underwent cecal ligation and puncture.
Detection of gfp expression
Mice were anesthetized, and the hind footpad as well as popliteal lymph nodes were harvested and stored in 30% sucrose until frozen sections were made. The tissues were sectioned at 5 µm and then photographed using a fluorescent microscope (Axioskop wide-field fluorescent microscope; Zeiss, New York, NY).
Flow cytometric analysis of lymph node leukocytes
The popliteal lymph node was harvested 24 h after footpad injection. Lymph nodes were first dissected at 4°C with 30-gauge needles, filtered through a 70-µm pore size cell strainer (Falcon, BD Biosciences), then resuspended in 400 U/ml collagenase D (Roche, Norwich, CT) and incubated for 30 min in a 37°C water bath (17). After washing twice with buffer (1% BSA and 1 mM EDTA (Fisher Scientific, Pittsburgh, PA) and 0.1% sodium azide (NaN3; Sigma-Aldrich, St. Louis, MO) in HBSS without phenol red, calcium, and magnesium (Cellgro; Mediatech, Herndon, VA), cells were resuspended in 4% Hanks’ azide buffer (HBSS without calcium, magnesium, or phenol red, with 4% BSA, 0.1% sodium azide, 0.2% anti-CD16/32, and 1 mM EDTA), followed by staining. DCs were characterized using anti-CD11c and anti-CD8 (clone 53-6.7) Abs (Immunotech, Miami, FL). Maturation of DCs was assessed by the levels of MHC class II and CD86 expression. T cells were identified with anti-CD3, anti-CD4, or anti-CD8 Abs. T cell activation was assessed by the expression of CD69 and CD25. Directly conjugated Abs were used for FITC, PerCP, and PE. Samples were acquired and analyzed on a FACSCalibur instrument (BD Biosciences, San Jose, CA).
DNA isolation and PCR
DNA was isolated from popliteal lymph nodes or footpad tissue using the Qiagen DNeasy Tissue Kit (Qiagen, Valencia, CA). PCR was performed with reagents from the GeneAmp PCR Core Kit (PE Applied Biosystems, Foster City, CA). For each sample, 1 µg DNA was added to a 50-µl PCR containing 2 mM MgCl2, 1x PCR buffer II, 0.2 mM dNTPs, 1.25 U AmpliTaq DNA polymerase, and 50 µmol of specific oligonucleotide primers. The primer sequences for hIL-10 were 5'-CGGCCGCTCGAGTCTAGAC-3' and 5'-GTGGATAGCGGTTTGACTCAC-3'. The PCR temperature profile consisted of a single cycle at 94°C for 2 min; 35 cycles for 1 min at 94°C (denaturing), 1 min at 60°C (annealing), and 2 min at 72°C (extension); and a final cycle at 72°C for 7 min. The PCR products were electrophoresed on a 2% agarose gel containing 0.4 µg/ml ethidium bromide.
Cecal ligation and puncture model of polymicrobial sepsis
For induction of a polymicrobial sepsis, mice were subjected to cecal ligation and puncture as previously described (18). In brief, laparotomy was performed, and the cecum was isolated, ligated, and punctured through and through with a 22-gauge needle. Depending on the experiment, mice were either euthanized at 24 h after surgery, and organs and blood were harvested, or animals were observed for up to 10 days to determine outcome.
Caspase-3 activity assay
Protein extracts were prepared by homogenization of thymus tissues, and caspase-3 activity was determined by a fluorometric assay (Enzyme Systems Products, Livermore, CA). Harvested thymi were homogenized in 1 ml 25 mM HEPES buffer (pH 7.5) containing 5 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 1 µg/ml leupeptin, and aprotinin. After centrifugation at 13,500 rpm for 15 min, the supernatants were collected. Protein concentrations in the supernatant were assayed using a protein assay kit (Bio-Rad, Hercules, CA). Fifty micrograms of the extracted proteins were incubated with the synthetic fluorescent substrate benzylooxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin at a concentration of 20 mM in 0.1 M HEPES buffer (pH 7.4) containing 2 mM DTT, 0.1% 3-[93-cholamidopropyl]dimethylammonio]-1-propane-sulfonate, and 10% sucrose. The kinetics of the proteolytic cleavage of the substrates were monitored in a fluorescence microplate reader using an excitation wavelength of 360 nm and an emission wavelength of 535 nm. The fluorescence intensity was calibrated with a standard concentration of 7-amino-4-trifluoromethylcoumarin, and the caspase activity was calculated from the slope of the recorded fluorescence and expressed as the relative fluorescence intensity.
Cytokine measurements
Murine IL-6 and murine and hIL-10 in plasma were measured by specific ELISA using commercially available reagents. A minikit for murine IL-6 (Endogen, Cambridge, MA) was used, whereas the human and murine IL-10 ELISAs used Ab pairs (hIL-10, clones 9D7 and 12G8 (R&D Systems, Minneapolis, MN); mIL-10, JES052A5 (Endogen)).
Presentation of data and statistics
Results are presented as the mean ± SEM. Differences between experimental groups were considered significant at p < 0.05, as determined by one-way ANOVA. Post-hoc analyses were performed with Student-Newman-Keuls multiple range test. Differences in survival were determined with Kaplan-Meier log-transformed survival analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Particles (2 x 105 or 2 x
1010) of recombinant Adv expressing gfp or
containing an empty cassette were injected into both hind footpads of
mice. Twenty-four hours later, the draining popliteal lymph nodes were
harvested and examined for gfp by either frozen section or flow
cytometry. Frozen sections of the popliteal lymph nodes showed
fluorescent cells distributed in the subcapsular and T cell areas of
the lymph node (Fig. 1
a).
Analysis of the lymph nodes by flow cytometry revealed that 
27% of
CD11c+, MHC class II-expressing cells were gfp
positive, indicating that DCs had been transduced with the recombinant
Adv and were expressing the transgene (Fig. 1b). Additional
flow analysis revealed that there were essentially no gfp-positive
cells in the CD3+, CD4+,
and CD8+ cell populations, confirming that T
cells are not readily transduced by Adv (data not shown). Examining the
DC population in greater detail revealed that lymphoid DCs
(CD8+CD11c+MHC class
IIhighCD86high) were
transduced to a greater degree than were myeloid DCs
(CD8-CD11c+MHC class
IIhighCD86high). Three
percent of all lymphoid DCs in the lymph node were gfp positive
following injection of 2 x 105 particles of
recombinant Adv, whereas nearly 30% were gfp positive following
injection of 2 x 1010 particles of the same
vector. In contrast, 0.5 and 10% of myeloid DCs were also gfp positive
following administration of 2 x 105 and
2 x 1010 particles, respectively (data not
shown).
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Because DCs are activated by Adv in vitro (14), we
examined the status of DC maturation in CD8+ and
CD8- subsets 24 h after footpad
administration of a recombinant Adv containing an empty cassette
(Adv/empty). We also administered Adv expressing hIL-10 (Adv/hIL-10) to
determine whether the expression of this anti-inflammatory cytokine
blocked DC maturation as reported with administration of the
recombinant protein (10). In addition, because DCs can
stimulate T cell activation, we determined whether hIL-10 expression in
DC within the popliteal lymph node would regulate
CD4+ or CD8+ lymphocyte
activation by assessing CD69 or CD25 expression following viral
exposure. Treatment with 2 x 1010 particles
of the Adv containing an empty cassette led to maturation of both
lymphoid and myeloid DCs in the popliteal lymph node, as demonstrated
by up-regulation of CD86 surface Ag (Fig. 2a). Interestingly, treatment
of mice with the recombinant vector expressing hIL-10 at the same dose
(1010 particles) reversed this effect, with CD86
expression below the levels seen in DCs from mice injected with buffer
alone.
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b). Approximately 2% more
CD4+ and CD8+ cells
expressed CD69 after adenoviral injection compared with those after
buffer treatment. Injection of a higher particle dose (2 x
1010) led to a nearly 6-fold increase in the
number of activated CD4+ and
CD8+ T cells. Interestingly, administration of
2 x 1010 particles of the vector expressing
hIL-10 (Adv/hIL-10) reversed the effect, leading to a similar degree of
activation as that seen with the lower particle dose (Fig. 2b). Local vs systemic appearance of IL-10 is dose dependent
One possible advantage of using IL-10 therapies for the treatment
of inflammatory disease is the capacity to achieve only local
expression, as systemic exposure to this cytokine often leads to
unwanted immunosuppressive effects (19). As demonstrated
by Steinhauser et al. (20), systemic administration of
IL-10 suppressed the immune response to Pseudomonas
pneumonia following cecal ligation and puncture. Therefore, one of the
goals of these studies was to establish whether local adenoviral
expression of IL-10 could be obtained. Therefore, we administered
different quantities of Adv expressing hIL-10 into the footpad of
healthy mice to determine whether local production in the draining
lymph nodes, in the absence of systemic appearance in the circulation,
could be achieved. At 24 h we analyzed plasma for hIL-10, and
lymph nodes for hIL-10 cDNA. With a dose of 2 x
105 particles, we could not detect hIL-10 in the
plasma of recipient animals (Fig. 3a). In marked contrast,
higher doses of Adv expressing IL-10 (2 x
107 particles and 2 x
1010 particles, respectively) showed a
dose-dependent increase in the plasma appearance of hIL-10.
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b). In contrast, in only one of three replicates could
hIL-10 cDNA be recovered from the more distant inguinal lymph node. No
hIL-10 cDNA could be recovered from any other organ, as determined by
PCR (data not shown). Local expression of hIL-10 leads to increased survival of septic mice
Having demonstrated that local expression of IL-10 could be
achieved, we next determined whether the compartmentalized expression
of this cytokine in peripheral lymph nodes would affect the survival of
mice developing polymicrobial sepsis following cecal ligation and
puncture. To obtain local expression of IL-10, mice were pretreated
with footpad injections of 2 x 105
particles of Adv expressing hIL-10 or containing an empty cassette,
respectively, 24 h before cecal ligation and puncture. Mice
pretreated with the recombinant expressing hIL-10 had a significant
increase in survival (55%, 11 of 20), whereas animals receiving the
empty vector control (Adv/empty) died at the same rate as mice injected
with buffer (both 25%, 5 of 20; Fig. 4).
Furthermore, we tested the effect on sepsis in mice treated with the
high particle dose of 2 x 1010 of the
respective Adv vectors (Adv/hIL-10, Adv/empty, and buffer control
only), a dose that resulted in systemic exposure of hIL-10 (see Fig. 3). In contrast to the former study, there was no difference in
survival among the treatment groups (Adv/empty, 4 of 20; Adv/hIL-10, 5
of 20; buffer control, 4 of 20).
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Twenty-four hours following cecal ligation and puncture, and thus
48 h after footpad injection of 2 x
105 particles, plasma IL-6 and murine IL-10
levels as well as thymic caspase-3 activity were determined. Earlier
studies had shown that increased levels of these cytokines and
increased thymic apoptosis correlate with an adverse outcome in similar
animal models and in patients with sepsis (21, 22). At
this time point, however, no differences among the three treatment
groups were seen despite significant differences in outcome (Fig. 5). There was, however, a trend toward
reduced plasma IL-6 and increased murine IL-10 levels as well as
decreased thymic caspase-3 activity in the group of mice treated with
footpad injection of Adv expressing hIL-10 (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study also demonstrates a dose effect of Adv on in vivo DC
activation, such that higher particle numbers of Adv
(1010) promote DC maturation, while lower numbers
(105) do not cause significant DC maturation in
vivo. We also found a similar dose effect for activation in in
vitro-derived DC cultures (data not shown). Although only 30% of all
DCs in the lymph node are transduced following administration of
1010 particles, the majority of both
CD8+ and CD8- DC
populations in the popliteal lymph node are mature at 24 h
following injection. In addition, popliteal lymph node T cells are
activated with this dose of Adv. This suggests that at higher doses
recombinant Adv may act as an adjuvant of transduced as well as
uninfected DCs and subsequent T cell activation. DC maturation may be
induced directly by viral DNA or proteins derived from the virions
(2). Alternatively, maturation may result from the
paracrine actions of IFN-
produced as a consequence of
transduction-mediated induction of inflammatory cytokines produced by
infected DCs, macrophages, or activated T cells (2).
Importantly, DC maturation as well as T cell activation are efficiently
blocked in vivo when recombinant Adv expresses hIL-10. Previous in
vitro studies suggest that exposure of immature DCs to IL-10 blocks
maturation and makes them resistant to maturation stimuli applied at
later time points (24). Although we have not determined
that DCs expressing the IL-10 transgene in vivo are resistant to
subsequent maturation stimuli, we have found that in vitro transduction
of DCs with Adv expressing IL-10 will still undergo phenotypic
maturation with LPS. However, the production of IL-12 by Adv/hIL-10
transduced cells in response to LPS-induced maturation remains
suppressed (our unpublished observations). These results, when
considered in the context of the findings of Steinbrink et al.
(24), suggest that the adenoviral vector itself may modify
the DC response to IL-10, and IL-10 may modify the DC response
to adenoviral vector transduction in vivo.
Perhaps one of the most interesting findings is that targeted expression of IL-10 in popliteal lymph node DCs provides significant protection from the lethal effects of generalized peritonitis. Based upon our studies reported here, only 3% of the DCs in the draining lymph node were transduced following injection with 105 particles of Adv, and IL-10 expression was localized to the popliteal lymph node and footpad. Expression of hIL-10 cDNA was not reproducibly observed in any other lymphoid structure or organ, and IL-10 protein was not detected in the circulation.
A central question is why does compartmentalized local expression of IL-10 improve the outcome in sepsis, while systemic production does not. Previous studies examining the role of systemic endogenous IL-10 in the pathogenesis of sepsis are somewhat confounding, but some suggest that systemic expression of this cytokine contributes to untoward immune suppression and mortality following sepsis (20, 25). Other studies with systemic administration of exogenous IL-10 have also demonstrated increased mortality (26). In a recent report we demonstrated that i.v. administration of recombinant Adv expressing IL-10 failed to protect mice from mortality in an identical cecal ligation and puncture model of sepsis. In contrast, when the same vector was administered intrathymically, survival was significantly improved (27, 28). In these same studies intrathymic injection of Adv led to local expression, primarily in DCs, whereas the nonprotective i.v. administration of Adv resulted in hepatocyte transduction and the systemic appearance of IL-10. We are, therefore, led to postulate that local contained expression of IL-10 in certain primary and secondary lymphoid organs may promote host survival during the sepsis response, in contrast to the systemic expression of this cytokine. This hypothesis is consistent with studies where high systemic levels of endogenous IL-10 predict adverse outcome in septic and trauma patients (29), presumably by causing generalized immune suppression and a predominant Th2 response, as seen after thermal injuries and generalized peritonitis (30).
Beyond the issue of local expression, the capacity to target DCs and the ability of IL-10 to maintain an immature DC phenotype in a lymphoid structure may be of critical importance in regulating the sepsis response. Previous studies suggest that DCs are lost from tissues during sepsis (31), perhaps due to activation-induced cell death compromising the acquired immune responses to pathogens. Our in vitro studies suggest that IL-10 expression in Adv/hIL-10 transduced DCs does not suppress increases in CD86 and MHC class II Ags following LPS stimulation, but does limit IL-12 production (our unpublished observations). Therefore, the presence of immature or mature DC without the capacity to produce normal amounts of IL-12 could promote the generation of regulatory T cells (32) that may limit untoward aspects of the acquired or innate immune responses in the setting of sepsis. We are currently examining these possibilities.
As we did not assess the cell phenotype from the footpad of Adv-injected mice, these studies may underestimate the total number of DCs transduced. If tissue myeloid DCs in the footpad were transduced at the site of injection, IL-10 expression may have interfered with the maturation of these cells, limiting their expression of chemokine receptors, and their ability to migrate to the draining lymph node. In fact, Takayama et al. (33) demonstrated that exposure of immature DCs to IL-10 prevents their expression of CCR7 and impairs the homing of these cells to secondary lymphoid tissues. Furthermore, IL-10 exposure of DCs results in their expression of chemokine decoy receptors that fail to signal and elicit migration and serve only as a sink for locally produced inflammatory chemokines (34).
Overall, these studies provide a new in vivo approach to modify murine DC function that allows direct manipulation of the immune response. Our data also demonstrate that gene targeting of DCs with IL-10 may be useful for acute infectious diseases, such as sepsis syndromes. Although the mechanism of action is not resolved, compartmentalized expression of IL-10 in DCs clearly alters the response to sepsis. Further studies will be required to more fully delineate the cellular mechanisms by which compartmentalized modification of DC function impacts host immunity and outcome in sepsis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lyle L. Moldawer, Department of Surgery, University of Florida College of Medicine, Room 6116, Shands Hospital, 1600 SW Archer Road, Gainesville, FL 32610. E-mail address: moldawer{at}surgery.ufl.edu
3 Abbreviations used in this paper: DC, dendritic cell; Adv, adenovirus; gfp, green fluorescent protein; hIL-10, human IL-10.
Received for publication September 21, 2001. Accepted for publication January 25, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), or
buffer (
). Twenty-four hours later, mice underwent cecal ligation
and puncture and were observed for 6 days. *, p
< 0.05, Adv/hIL-10 vs Adv/empty or buffer.