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*Genes and Gene Therapy
The Journal of Immunology, 2000, 164: 443-451.
Copyright © 2000 by The American Association of Immunologists

TNF-{alpha} Receptor Signaling and IL-10 Gene Therapy Regulate the Innate and Humoral Immune Responses to Recombinant Adenovirus in the Lung1

Rebecca M. Minter*, John E. Rectenwald*, Kunitaro Fukuzuka*, Cynthia L. Tannahill*, Drake La Face{ddagger}, Van Tsai{ddagger}, Iqbal Ahmed{ddagger}, Elizabeth Hutchins{ddagger}, Richard Moyer{dagger}, Edward M. Copeland, III* and Lyle L. Moldawer2,*

Departments of * Surgery and {dagger} Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL 32610; and {ddagger} Canji, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-{alpha} and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. ß-Galactosidase expression in mice receiving intratracheal instillation of Adv/ß-gal (adenovirus construct expressing ß-galactosidase) was transient (less than 14 days), but a significant early increase of ß-galactosidase expression was seen in mice lacking either or both TNF-{alpha} receptors. Absence of TNF-{alpha} or the p55 receptor significantly attenuated the Ab response to both adenovirus and ß-galactosidase. Human IL-10 expression in the lung suppressed local TNF-{alpha} production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with ß-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-{alpha} signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with ß-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with ß-galactosidase, and is nonimmunogenic in the lung.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adenovirus-mediated gene transfer is an efficient delivery system for in vivo gene therapy. Recombinant adenoviral vectors are particularly attractive because they have been shown to be safe, can efficiently transduce a wide variety of tissue types, do not require replicating target cells for transgene expression, and permit high levels of expression of molecules too large and polar to be conventional drugs (1, 2).

Although efficient gene transduction has been reported, transgene expression in immunocompetent animals has been transient and often has been associated with dose-limiting inflammation. The transient nature of adenoviral-mediated gene transfer is strongly associated with the host’s innate and acquired immune responses to adenoviral infection (3, 4, 5, 6, 7). Besides modifying the adenoviral vector to reduce its antigenicity (2, 8), several strategies have been developed to circumvent the host’s immune response against adenoviral vectors. Depletion of CD8+ or CD4+ cells from immune competent mice, as well as administration of anti-CD4+ Ab (GK1.5) and immunosuppressants such as FK506, prolong transgene expression (9, 10, 11). However, FK506, cyclosporine, or dexamethasone treatment does not significantly decrease generation of anti-adenoviral Abs or improve expression following readministration of the adenoviral vector (8).

TNF-{alpha} has been shown to play a major role in the elimination of adenoviral vectors. TNF-{alpha} is a primary initiator of innate immune responses and determines to a great extent the magnitude and direction of acquired immune responses (12). Elkon et al. (13) have demonstrated that following i.v. administration of first generation adenovirus vectors, TNF-{alpha} null mice, but not perforin or FasL null mice, have reduced lymphocytic infiltration of the liver as well as extended transgene expression for periods of 28 days. Zhang et al. (14) have also reported a decreased inflammatory response and prolonged adenoviral expression of ß-galactosidase in the lung and liver following i.v. and intranasal instillation, and simultaneous i.p. delivery of a soluble TNF receptor type I. Benihoud et al. (15) have demonstrated suppression of an IgG1 anti-adenoviral Ab response following systemic delivery of recombinant adenovirus construct expressing ß-galactosidase (AdV/ß-gal)3 in combined TNF-{alpha}/LT{alpha}-/- mice.

Although a role for TNF-{alpha} in the prolongation of adenovirus gene expression and reduced inflammatory responses has been demonstrated, the adenoviral doses employed were much higher on a per weight basis than would be used in clinical trials, and were associated with significant lung and liver injury. Similarly, Elkon and Benihoud did not explore the role for TNF-{alpha} in the clearance of adenoviral vectors from the lung (13, 15). Therefore, it remains unclear whether more physiologically relevant doses of adenovirus delivered to the lungs produce a TNF-{alpha}-dependent inflammatory response and reduced duration of transgene expression.

Furthermore, TNF-{alpha} signaling occurs through two distinct receptors, p55 (TNFR I) and p75 (TNFR II), which are coexpressed on many cell types. In vivo studies have suggested that the p55 receptor is primarily responsible for the proinflammatory properties of TNF-{alpha}, and the p75 receptor (TNFR II) lacks intrinsic proinflammatory properties of its own, but may potentiate the actions of the p55 receptor (16). In contrast, p75 receptor signaling is presumed responsible for TNF-{alpha}-mediated T cell proliferative responses (17, 18).

In the current study, we have used a transgenic approach employing mice lacking functional TNFR I and/or TNFR II to explore the role played by each receptor signaling in the response to adenovirus. Recognizing the role TNF-{alpha} plays in the clearance of adenoviral vectors raises the possibility that local expression of an immunomodulant that suppresses TNF-{alpha} could prolong transgene expression without the adverse consequences of systemic immunosuppression. For example, human IL-10, which is produced predominantly by monocytes/macrophages and TH2 cells, promotes immune deviation from a TH1 to a TH2 cell-mediated immune response (19, 20). This immune deviation mediated by IL-10 is due in part to the inhibition of cytokine production by TH1 cells, particularly IFN-{gamma} (21), TNF-{alpha} (22), IL-8 (23), and IL-12 (21), and inhibits APC functions (24, 25). These immunosuppressive properties of IL-10 may provide a means to down-modulate the local anti-inflammatory response, which may lead to prolonged transgene expression following adenoviral vector-mediated transduction in the lung.

Therefore, the purpose of these studies was to determine the role played by individual TNF receptor signaling pathways in the clearance of adenoviral vectors from the lung when administered in quantities presumed to be clinically relevant. Moreover, we assessed whether coexpression of human IL-10 could suppress endogenous TNF-{alpha} production, reduce the immunological response, and extend transgene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free male and female C57BL/6 and B6 x 129 mice were obtained from either The Jackson Laboratory (Bar Harbor, ME) (20–25 g) or the University of Florida Health Science Center Animal Resources Department. Male and female B6 x 129 mice expressing a null form of TNF-{alpha} (tnf-/-), and C57BL/6 mice expressing a null form of the p55 TNF receptor (TNFR I) (p55-/-), a null form of the p75 (TNFR II) (p75-/-), or combined null forms of the p55 and p75 (p55-/-p75-/-) receptors (20–25 g) were bred and maintained in a specific pathogen-free environment at the University of Florida Health Science Center Animal Resources Department. p55-/- and tnf-/- mice were originally obtained from Amgen (Boulder, CO), whereas p75-/- and p55-/-p75-/- mice were obtained from Immunex (Seattle, WA). Mice were housed in a barrier facility in groups of five per cage for the duration of the experiments.

Animal preparation

Mice were anesthetized with 35 mg/kg body weight of i.p. sodium pentobarbital. After induction of anesthesia, a mid-line incision was made in the neck, the fat pad was elevated caudally, and the trachea was visualized. Under direct visualization, the trachea was cannulated with a sterile 30-g needle, and 32 µl of buffer or buffer containing adenoviral vector (1 x 1010 particles per animal) was delivered. In studies examining the inflammatory response to adenoviral vectors, mice received an intratracheal instillation of either 1 x 1010 particles of adenovirus construct expressing ß-galactosidase (Adv/ß-gal) or human IL-10 (Adv/hIL-10). To assess whether coexpression of IL-10 could prolong ß-galactosidase expression, additional mice received simultaneous instillations of 5 x 109 particles of Adv/ß-gal and 5 x 109 particles of either Adv/hIL10 or an identical adenovirus vector with an empty cassette (Adv/empty).

The incision was then closed and the mice were returned to their cages to recover. Mice were sacrificed by cervical dislocation at 1, 3, 5, 7, 9, or 14 days following viral instillation. Blood was collected via a retro-orbital venipuncture utilizing a heparinized capillary tube. The lungs and trachea were removed en bloc and snap frozen in liquid nitrogen.

Construction of a recombinant adenovirus expressing ß-galactosidase or human IL-10

A derivative of human adenovirus serotype 5 (26) was used as the source of viral DNA backbone. The construct was deleted in early region 1, polypeptide IX, and early region 3. Specifically, the vector contains a deletion of bp 355-3325 to eliminate E1a and E1b functions, a deletion of bp 3325–4021 to eliminate protein IX function, and a deletion of bp 28,592–30,470 to eliminate E3 functions (27).

Recombinant adenoviruses were constructed using standard homologous recombination methods as described by Graham and Prevec (28). To generate a recombinant adenoviral vector expressing human IL-10, a cDNA sequence encoding IL-10 was isolated from the pDSRG-IL10 plasmid (obtained from Dr. Kevin Moore, DNAX Research Institute, Palo Alto, CA) (GenBank accession no. M57627) (29).

ß-Galactosidase expression in the lung

ß-Galactosidase activity was detected in the lungs using a chemiluminescent reporter gene assay system (Tropix, Bedford, MA). Baseline ß-galactosidase activity was obtained from the lung of untreated animals analyzed simultaneously, and was subtracted from the ß-galactosidase measurements obtained from treated mice. Baseline ß-galactosidase activity from the lungs of control animals was less than 1% of peak activity seen in lungs from mice instilled with adenoviral vectors delivering ß-galactosidase cDNA.

Serum and lung homogenate cytokine measurements

Bioactive TNF-{alpha} was measured in serum and lung homogenates using the TNF-{alpha}-sensitive WEHI 164 clone 13 murine fibrosarcoma cell line (30). A standard curve was generated with human TNF-{alpha}, and the sensitivity of the assay was 5–25 pg/ml.

Murine IL-6, IL-1{alpha}, TGF-ß1, and human IL-10 levels in the lung homogenates, and in the serum where appropriate, were measured by sandwich ELISA using commercially available reagents (murine IL-6 and human IL-10 by Endogen (Woburn, MA), murine IL-1{alpha} by R&D Systems (Minneapolis, MN), and TGF-ß1 by Promega (Madison, WI)).

Myeloperoxidase assay

Pulmonary neutrophil sequestration in the lungs was quantitated by measuring tissue myeloperoxidase content (31). Snap frozen lungs (-70°C) were weighed and homogenized for 1 min in 15 weight:volumes of 0.01 M KH2PO4 with 1 mM EDTA (PE buffer). Following homogenization, the resultant pellet was resuspended in 13.7 mM cetyltrimethylammonium bromide (C-TAB) buffer with 50 mM acetic acid, using the same volume of C-TAB as PE buffer. The resuspended pellet was then sonicated for 40 s at setting 60% on the sonicator (Fisher, Pittsburgh, PA; Sonic Dismembrator, model 300), and centrifuged at 10,000 rpm for 15 min. The resultant supernatant was collected and incubated in a water bath for 2 h at 60°C. Myeloperoxidase activity was then measured in this solution by H2O2-dependent oxidation of 3,3'5,5'-tetramethylbenzidine, which generates a colorimetric reaction. Spectrophotometric absorbance was read at 650 nm and compared with a linear standard curve with a sensitivity of 0.03125 EU.

In situ TUNEL assay of organ apoptosis

In situ TUNEL assay was performed using an in situ apoptosis detection kit (ApopTag, Intergen, Purchase, NY). All steps were performed according to the supplier’s instructions. The fluorescent TUNEL-labeled slides were photographed using a fluorescence microscope. Additional specimens were stained with hematoxylin and eosin for routine histological analysis.

Detection of TNF-{alpha} mRNA by RT-PCR

Total cellular RNA was isolated, and TNF-{alpha} mRNA was quantified as previously described (32). Briefly, total lung RNA was isolated by guanidinium isothiocyanate and acid-phenol extraction (33). The sequence of oligonucleotide primers was: 5'-TNF-{alpha}, ATG AGC ACA GAA AGC ATG ATC; 3'-TNF-{alpha}, TAC AGG CTT GTC ACT CGA ATT; 5'-SOD, GTC TGC GTG CTG AAG GGC GAC; 3'-SOD, TCT CCT GAG AGT GAG ATC ACA. The PCR was performed using 2.5 U AmpliTaq (Perkin-Elmer, Norwalk, CT) for TNF-{alpha}; 27 cycles, SOD; and 21 cycles as follows: 94°C for 1 min (dissociation), 60°C for 1 min (annealing), and 72°C for 2 min (primer extension). The expected fragment lengths were 276 bp for TNF-{alpha} and 314 bp for SOD. Amplicons were visualized using 2% agarose gel electrophoresis. The gels were scanned and the integrated area under the absorbance curves was calculated using a commercial program (SigmaGel; Jandel Scientific, San Rafael, CA). The relative quantities of TNF-{alpha} are presented as the ratio between the intensity of these bands relative to the intensity of the housekeeping gene, Cu/Zn SOD.

Adenovirus and ß-galactosidase Ab measurements

To determine whether a humoral response develops against the expressed ß-galactosidase or human IL-10, a direct ELISA for Abs (IgG) was performed (34). Briefly, recombinant ß-galactosidase (Sigma, St. Louis, MO) or human IL-10 (Schering-Plough Research Laboratories, Kenilworth, NJ) was coated onto 96-well Corning flat-bottom, polystyrene ELISA plates (0.5 µg/ml). After blocking the plates with TBS containing 5% BSA (BSA Sigma Fraction V) and 1% fat-free dry milk (Alba), mouse serum obtained at baseline and at 14 days after intratracheal instillation was diluted from 1/50 through 1/100,000 in TBS/5% BSA, applied to the wells (100 µl), and incubated at room temperature for 2 h. The plates were washed, and 1/3000 diluted HRP-conjugated, goat, anti-mouse IgG Ig (Promega) was added and plates were incubated at room temperature for 1 h. Results were visualized with 3,3', 5,5' tetramethylbenzidine. A positive Ab response was recorded when the absorbance from samples at day 14 was at least 2-fold greater than the maximal absorbance determined from samples at day 0. The quantities of Ab were estimated from the highest inverse dilution of the sample producing a positive signal (2x background).

Measurement of serum-neutralizing Ab response

A functional assay to determine the neutralizing capacity of antisera to prevent the capacity of recombinant adenoviral vectors to infect and transduce HeLa cells (80% subconfluent) was performed in accordance with the following procedure. Antisera were diluted from 1/20 to 1/2560 by a 1/2 serial dilution. The diluted antisera were mixed with 8 x 108 particles per milliliter of a recombinant adenoviral vector expressing the green fluorescent protein (rAdV/GFP) at a 1:1 ratio with serially diluted antisera samples and incubated for 1 h at 37°C. The mixture was then placed on HeLa cells in a 96-well plate and incubated overnight to permit transduction. The plates were then measured for fluorescence intensity at 450 nm using a CytoFluor Multi Reader Series 4000 spectrophotometric plate reader (commercially available from Perspective Biosystems, Cambridge, MA). The resulting fluorescence units were normalized against untreated HeLa cells containing only media and compared with maximal fluorescence (HeLa cells transduced with 4 x 108 particles per milliliter rAdV/GFP). The neutralizing Ab titer was assessed by determining the inverse titer at 50% of normalized maximal absorption.

Statistical analysis

Data are presented as the mean ± SEM, and the n for each group is between 6 and 12.The Student t test was used for statistical analysis when two different groups of samples were compared. A one-way ANOVA was used to compare animals at different time points that received the same treatment, and post hoc comparisons were performed using Dunn’s multiple range test. A two-way ANOVA was used to evaluate differences between treatment and time, and a post hoc comparison among the different groups was undertaken with a Student-Newman-Keuls multiple range test. Statistical significance was considered to be achieved if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course response following AdV/ß-gal instillation

C57BL/6 mice underwent intratracheal instillation with either 1 x 1010 particles of AdV/ß-gal in 32 µl of buffer (PBS, pH 7.5, containing 3% (w/v) sucrose, 2 mM MgCl2) or 32 µl of buffer alone at day 0. ß-Galactosidase expression was then measured in the lung on days 1, 3, 5, 7, 9, and 14. Peak expression was seen at days 1 and 5, with return to baseline by day 14 (Table IGo). No ß-galactosidase activity was seen in the lungs of buffer-treated mice.


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  		Table I. Lung ß-galactosidase and lung human IL-10 activities in mice intratracheally instilled with 1010 particles of either AdV/ß-gal or AdV/IL-10, or buffer

 
The TNF-{alpha} response in the lung was biphasic following AdV/ß-gal instillation. There was an initial peak production of TNF-{alpha} on day 1, followed by a second peak of TNF-{alpha} appearance in the lungs at about day 7 (Fig. 1GoA). Although TNF-{alpha} was immeasurable in the serum of these mice at all time points (<5 pg/ml, data not shown), the local expression of TNF-{alpha} in the lungs was verified by RT-PCR (Fig. 2Go). TNF-{alpha} mRNA levels and protein levels in the lung were markedly increased on day 7 when no TNF-{alpha} was detectable in the serum, confirming the local production of TNF-{alpha} in response to adenoviral instillation.



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  		FIGURE 1. Lung cytokine appearance following intratracheal AdV/ß-gal or buffer instillation. TNF-{alpha} appearance was bimodal and peaked on days 1 and days 5–7 (both p < 0.05 vs buffer), with a decline to baseline by day 14 (A). In contrast, IL-1{alpha} and IL-6 concentrations were significantly increased only on day 1 following AdV/ß-gal instillation (B and C), but returned to baseline thereafter and were no longer different from buffer controls.

 


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  		FIGURE 2. Lung TNF-{alpha} mRNA expression on day 7 following AdV/ß-gal instillation. The presence of TNF-{alpha} mRNA in the lungs of mice 7 days following AdV/ß-gal delivery was confirmed by RT-PCR. The intensity of the TNF-{alpha} mRNA and SOD amplicons was determined, and animals receiving AdV/ß-gal had significantly increased levels of TNF-{alpha} mRNA relative to SOD, as compared with controls (buffer alone).

 
In contrast to TNF-{alpha}, IL-1{alpha} and IL-6 concentrations in the lung both peaked on day 1 with marked reductions throughout the remainder of the time course (Fig. 1Go, B and C). There was not a significant increase in myeloperoxidase activity in the lungs of the animals receiving AdV/ß-gal as compared with control animals, indicating that at the doses employed, the infiltration by neutrophils was minimal.

This lack of a significant inflammatory response was confirmed histologically with no evidence of either an early inflammatory response (neutrophil mediated) or a delayed lymphocytic infiltration, as we have seen with higher doses (Fig. 3Go, A and B) (35). Staining for apoptotic lymphocytes or epithelial cells (TUNEL) demonstrated only minimal apoptosis in the lungs of animals that received AdV/ß-gal, and was comparable with controls (Fig. 3Go, D and E). Although histological data are only presented for day 5 (when transgene expression was near its peak and TNF-{alpha} appearance was high), no histological abnormalities were seen in the lungs of adenovirus-instilled mice throughout the 14-day study period.



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  		FIGURE 3. Hematoxylin and eosin (A–C) and TUNEL (D–F) staining 5 days following intratracheal instillation of buffer alone (A and D), AdV/ß-gal (B and E), and AdV/hIL-10 (C and F). Samples were obtained from lungs of mice and were fixed in buffered Formalin. Hematoxylin and eosin staining, as well as fluorescent 3' end labeling of apoptotic nuclei (TUNEL) and propidium iodide counterstaining of total nuclei were performed, as stated in Materials and Methods. Histologically, there was no evidence of pulmonary inflammation or an increase in apoptotic cell death at these doses of adenovirus (1010 particles) as compared with controls (buffer alone). Similar results were seen at days 1, 3, 7, and 14 (data not shown) (magnification x200).

 
ß-Galactosidase expression in p55-/-, p75-/-, p55-/-p75-/-, and tnf-/- mice.

p55-/-, p75-/-, p55-/-p75-/-, and tnf-/- mice underwent intratracheal instillation with 1 x 1010 particles of AdV/ß-gal at day 0. ß-Galactosidase expression was measured on days 5 and 14, as these time periods corresponded to peak expression and loss of expression (Table IGo), respectively, in our initial time course studies. p55-/-p75-/- mice had a significant increase in ß-galactosidase expression at day 5 as compared with C57BL/6 mice (Fig. 4GoA; p = 0.0007). Similarly, the tnf-/- mice expressed ß-galactosidase levels that were also significantly higher (Fig. 4GoB; p = 0.00673). p55-/- and p75-/- mice expressed intermediate levels of ß-galactosidase, but in each case, levels were still statistically higher (both p < 0.05) than seen in the wild-type controls.



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  		FIGURE 4. Peak ß-galactosidase expression 5 days following intratracheal instillation of AdV/ß-gal in C57BL/6, p55-/-, p75-/-, p55-/- p75-/- (A), B6 x 129, and tnf-/- (B) mice (8–12 mice per group). p55-/-, p75-/-, p55-/- p75-/-, and tnf-/- mice had significant increases in peak ß-galactosidase expression on day 5 as compared with appropriate background wild-type controls (p < 0.05 by ANOVA and Student-Newman-Keuls multiple range test). However, by day 14, ß-galactosidase expression had returned to baseline.

 
However, at a dose of 1 x 1010 particles of AdV/ß-gal, ß-galactosidase expression had returned to baseline by day 14 in all strains of mice, regardless of their TNF-{alpha} or TNF receptor status (data not shown).

The presence of neutralizing Abs against the adenovirus was measured in all strains of mice, and mice lacking either a functional TNF-{alpha} (tnf-/-) or both TNF receptors (p55-/-p75-/-) were found to have significantly lower levels of neutralizing adenoviral Abs (p < 0.05) as compared with appropriate wild-type strains (C57BL/6 and B6 x 129) (Fig. 5Go). Although not statistically significant, the reduced Ab response appeared to be mediated in large part through the p55 receptor.



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  		FIGURE 5. Serum titers for neutralizing Abs against adenovirus. A functional assay to measure the ability of mouse serum to prevent Adv/GFP infection and expression in HeLa cells was conducted, as described in Materials and Methods. The presence of elevated GFP fluorescence in the HeLa cells was indicative of low levels of neutralizing Ab. p55-/- p75-/- (A) and tnf-/- (B) mice had significantly increased GFP fluorescence in HeLa cells at low serial dilutions of mouse sera, indicating the relative absence of anti-adenoviral neutralizing Ab.

 
Similarly, tnf-/- and p55-/- null mice had significantly lower levels of total IgG Abs recognizing ß-galactosidase as compared with wild-type animals (both p < 0.05; Fig. 6Go). The reductions in Ab response in the p55-/-p75-/- approached, but did not reach, statistical significance.



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  		FIGURE 6. Presence of total anti-ß-galactosidase IgG Abs in the serum of mice intratracheally instilled with AdV/ß-gal. Abs against ß-galactosidase from day 14 diluted mouse serum were captured with recombinant protein and total IgG visualized with a HRP-conjugated anti-mouse IgG. p55-/- (A) and tnf-/- (B) mice had significantly reduced absorbance at low serial dilutions, which indicates reduced levels of total anti-ß-galactosidase IgG.

 
Time course response following AdV/hIL-10 instillation

C57BL/6 mice underwent intratracheal instillation with either 1 x 1010 particles of AdV/hIL-10 or buffer alone at day 0. Human IL-10 expression was then measured in the lung and serum at days 1, 3, 5, 7, 9, and 14. Peak expression in the lungs was seen at days 1 and 5 (Table IGo). By 14 days, expression had declined, but was still 35% of peak levels seen at day 5. In contrast, ß-galactosidase activity had declined by ~99% within 14 days. Serum levels of human IL-10 in mice treated with AdV/hIL-10 were modest throughout the period when lung expression was peaking (10-fold less than peaking lung levels), with concentrations reaching 870 ± 230 pg/ml on day 5 (vs undetectable in buffer-treated animals).

TNF-{alpha} production was suppressed in the lungs of animals receiving AdV/hIL-10 as compared with those receiving AdV/ß-gal (p < 0.05), whereas IL-1{alpha} levels were unaffected (Fig. 7Go, A and B). In contrast, lung IL-6 and myeloperoxidase activity were significantly increased in the animals receiving AdV/hIL-10 as compared with those receiving AdV/ß-gal (p < 0.01 for both) (Fig. 8Go, A and B). It is important to note that although MPO activity was significantly increased in animals receiving AdV/hIL-10, the inflammatory response was still modest, and histologically there was no evidence of increased neutrophil infiltration of the lung (Fig. 3GoC). The concentrations of the immunomodulatory cytokine TGFß1 were also significantly reduced in the animals receiving AdV/hIL-10 as compared with those receiving AdV/ß-gal (p < 0.05) (Fig. 8GoC). Murine IL-10 was not detected in the lungs of animals receiving AdV/hIL-10 or AdV/ß-gal (data not shown).



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  		FIGURE 7. Lung TNF-{alpha} and IL-1{alpha} levels following instillation of either buffer alone, AdV/ß-gal, or AdV/hIL-10. Samples were obtained at days 1, 3, 5, 7, 9, and 14; however, for the sake of clarity, only time periods when the individual cytokines were at their peak, are values reported. TNF-{alpha} levels on days 1 and 7 were significantly reduced (p < 0.05) in mice receiving AdV/hIL-10 as compared with those receiving AdV/ß-gal (A). In contrast, IL-1{alpha} levels were comparable in mice receiving either AdV/ß-gal or AdV/hIL-10 throughout the study period (B).

 


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  		FIGURE 8. Lung IL-6, myeloperoxidase activity, and TGFß1 levels following instillation of either buffer alone, AdV/ß-gal, or AdV/hIL-10. Lung IL-6 levels were significantly increased (p < 0.05) in animals receiving AdV/hIL-10 as compared with buffer alone or AdV/ß-gal on day 5 and thereafter (A). Similarly, myeloperoxidase activity as a measure of neutrophil infiltration in the lung was also significantly increased at all time points (B) in animals receiving AdV/hIL-10 (B). In contrast, the immunomodulant TGFß1 was significantly decreased in mice receiving AdV/hIL-10 (p < 0.05) as compared with both control and AdV/ß-gal-treated animals (C).

 
In contrast to mice receiving AdV/ß-gal that developed a robust Ab response against both the adenovirus and the ß-galactosidase, Abs against both adenoviral proteins and human IL-10 were either very modest, or undetectable in mice treated with AdV/hIL-10 (p < 0.05 vs AdV/ß-gal) (Fig. 9Go). Similar to animals receiving AdV/ß-gal, the animals treated with AdV/hIL-10 did not demonstrate histologic evidence of pulmonary inflammation, and apoptosis in the lung was comparable with animals receiving buffer only (Fig. 3Go, C and F) despite sustained levels of IL-10 in the lung (3–10 ng/g wet weight).



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  		FIGURE 9. Serum anti-adenovirus neutralizing Abs and total anti-human IL-10 and anti-ß-galactosidase IgG measured 14 days following instillation of buffer alone, AdV/ß-gal, or AdV/hIL-10. There was a significant reduction (p < 0.05) in anti-adenoviral Abs in the AdV/hIL-10-treated mice as compared with AdV/ß-gal-treated mice (A). Anti-adenoviral neutralizing Abs were immeasurable in the buffer-treated animals, and these animals were conservatively given a titer of 20, which was the lowest dilution factor measured. Mice receiving AdV/hIL-10 generated no Ab response to the transgene product (anti-human IL-10 Abs) as compared with animals receiving AdV/ß-gal, which developed significantly higher levels of anti-ß-galactosidase Abs (B).

 
Coexpression of AdV/hIL-10 and AdV/ß-gal

C57BL/6 mice underwent intratracheal instillation of 5 x 109 particles of either AdV/ß-gal and AdV/hIL-10 or AdV/ß-gal and AdV/empty cassette in an effort to determine whether coexpression of IL-10 would extend ß-galactosidase expression. Despite therapeutic levels of IL-10 at day 5 in the lungs of animals receiving AdV/hIL-10, which were not statistically different from the levels of human IL-10 seen with 1010 particles of AdV/hIL-10, there was not a significant increase in ß-galactosidase expression. Peak levels of ß-galactosidase expression (day 5) in mice transfected with both AdV/ß-gal and AdV/hIL-10 were 34.5 ± 6.6 µg/g wet weight (n = 12) vs 32.4 ± 4.2 µg/g wet weight in mice transfected with AdV/ß-gal and AdV/empty (n = 12; p = NS) (Fig. 10Go). ß-Galactosidase expression had returned to baseline by day 14 in both treatment groups. Similarly, coexpression of human IL-10 did not significantly reduce the IgG Ab response to ß-galactosidase when it too was coexpressed (1810 ± 703 vs 2593 ± 604 titer/ml; p = NS), although the Ab response to human IL-10 was undetectable.



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  		FIGURE 10. Coexpression of AdV/hIL-10 and AdV/ß-gal. Female C57BL/6 mice (12 per group) receiving AdV/hIL-10 and AdV/ß-gal had peak ß-galactosidase levels on day 5 of 34.5 ± 6.6 µg/g wet weight, which was not significantly increased over peak levels seen in mice receiving AdV/empty vector and AdV/ß-gal, 32.4 ± 4.2 µg/g wet weight. ß-Galactosidase was immeasurable in both groups on day 14.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Adenoviral-mediated gene transfer has had widespread application in a variety of preclinical disease models, including rheumatoid arthritis, wound healing, solid organ transplantation, vascular occlusive disease, cancer, and several genetic disorders (36, 37, 38, 39, 40, 41). The overwhelming limitations of adenovirus-mediated gene therapy in all applications to date have been transient gene expression, dose-dependent inflammation, and the inability to readminister the viral construct due to humoral immune responses to the vector.

In the present study, we have demonstrated the ability to achieve therapeutic levels of transgene expression in the lung (both ß-galactosidase and human IL-10) using a first generation recombinant adenovirus construct that is highly purified and is delivered at a dose that is lower than that utilized in the majority of prior work (usually 1 x 109-1 x 1010 PFU/ml) (14, 42, 43). Using this dose of highly purified recombinant adenovirus, we did not observe any significant histologic evidence of inflammation in the lung, and only a very modest inflammatory response was detected by the presence of proinflammatory cytokines (TNF-{alpha}, IL-1{alpha}, and IL-6). Interestingly, we consistently see a transient reduction in transgene expression between days 1 and 5 following intratracheal instillation of recombinant adenoviral constructs. This biphasic response may be secondary to initiation of the innate immune response. Most investigators performing in vivo studies with adenovirus have used 3 days posttransfection as their initial time point, and therefore may not have observed this increased expression at 24 h (14, 15).

Blockade of TNF-{alpha} has been shown to increase and prolong transgene expression in the lung and liver (13, 14); however, TNF-{alpha} receptor signaling in the lung following adenoviral delivery has not yet been elucidated; nor has the role of TNF-{alpha} in the clearance of adenoviral vectors been defined following low dose delivery of adenovirus to the lung. For example, Zhang administered approximately two logs higher concentrations of adenovirus and detected TNF-{alpha} in the plasma for the first 2 days (14). However, these doses of TNF-{alpha} produced marked lung injury, characterized by an initial neutrophil inflammatory response and subsequent T cell infiltration. Lung TNF-{alpha} production was not evaluated.

In contrast, we have demonstrated that only TNF-{alpha}, and not other proinflammatory cytokines such as IL-1 and IL-6, has a lung-specific, bimodal production during adenovirus gene therapy. The early response (day 1) appears to be a nonspecific inflammatory response to the adenovirus, because the concentrations of IL-1 and IL-6 were similarly increased. However, TNF-{alpha} expression (both mRNA and protein) increased again on days 5 through 9 coincidental with a decline in transgene activity. Using transgenic mice, we have demonstrated that the absence of TNF-{alpha} or both TNF-{alpha} receptors leads to increased early transgene expression. Interestingly, TNF-{alpha} signaling through both receptors (p55-/- and p75-/-) plays a contributory role in the clearance of transgene expression. This latter finding is both novel and unexpected.

Historically, it has been suggested that the p55 TNF receptor is primarily responsible for proinflammatory properties of TNF-{alpha} and that the p75 receptor may function by potentiating the actions of the p55 receptor (16). Our studies clearly demonstrate complementary, independent roles for signaling through both TNF-{alpha} receptors for the clearance of adenoviral vectors from the lung. Tartaglia et al. (17) have demonstrated that signaling through the p75 TNF receptor stimulates the proliferation of peripheral T cells. It is conceivable that TNF-{alpha} signaling through the p75 receptor is required for a T cell proliferative response in the lung, which leads to more rapid clearance of the adenovirally infected cells. Douni et al. (44) have also demonstrated that production of the p75 TNF receptor is associated with increased NF-{kappa}B activity in the PBMC compartment, which may be involved in the clearance of these adenoviral proteins as well.

Interestingly, the development of an Ab response to both the adenovirus and ß-galactosidase appeared to be dependent primarily on p55 receptor signaling. p75-/- null mice were able to generate normal Ab responses against both the adenovirus itself as well as ß-galactosidase. The differences between the levels of Ab developed against adenoviral proteins and ß-galactosidase seen in the p55-/- and p55-/-p75-/- mice suggest different mechanisms of clearance for these viral and transgene proteins. The implications for this Ab response in naive animals remain unclear, however, because there was no direct relationship between the magnitude of the Ab response against either ß-galactosidase or adenovirus, and the magnitude or duration of ß-galactosidase expression in p55-/- or p75-/- mice. Similarly, although the Ab responses were markedly lower in tnf-/- and p55-/-p75-/- mice and peak expression was higher (day 5), duration of transgene expression was unrelated to the magnitude of the Ab response. This is similar to the findings of Benihoud et al. (15), who found that despite suppression of an anti-adenoviral Ab response in TNF-{alpha}/LT{alpha} null mice, there was no prolongation of transgene expression in the liver following systemic delivery of rAdV/ß-gal. The mechanism by which transgene expression is lost in the absence of neutralizing Abs remains unresolved at this time.

Human IL-10 is generally considered a powerful anti-inflammatory cytokine that suppresses IFN-{gamma}, TNF-{alpha}, and IL-1 production, and suppresses the effector functions of macrophages and TH1 T cell-mediated immune responses (19, 20). IL-10 is also known to down-regulate Ag presentation. Therefore, we anticipated that expression of human IL-10 in the lung following intratracheal delivery of AdV/hIL-10 would be increased due to TNF-{alpha} suppression, and possibly prolonged secondary to other IL-10-induced anti-inflammatory properties, as compared with ß-galactosidase expression. We also hypothesized that perhaps coexpression of human IL-10 with another transgene (ß-galactosidase in this case) would lead to increased and prolonged expression of the coexpressed vector. Although human IL-10 expression suppressed local production of TNF-{alpha}, this translated into only modest increases in the duration of human IL-10 expression, and no changes in ß-galactosidase production when coexpressed. Surprisingly, lung levels of IL-1{alpha} were unaffected by IL-10 expression, and IL-6 levels and myeloperoxidase content were actually increased in animals treated with AdV/hIL-10. It should be noted that this biochemical evidence of an inflammatory response to AdV/hIL-10 delivery to the lung was very modest, as there was no histological evidence of lung injury or an increase in apoptotic cell death.

The inability of human IL-10 to extend ß-galactosidase expression in the lung differs from the findings of Qin et al., who observed that coexpression of viral IL-10 increased ß-galactosidase expression (45). There are several potential explanations for these different findings, one being the differences in the route of administration and another being the varying biological activities of viral and human IL-10. Although both viral and human IL-10 share the ability to suppress macrophage activation and Ag presentation, human IL-10, particularly at high concentrations, stimulates IL-2-dependent NK cell proliferation and NK cytotoxicity (46). Furthermore, under some experimental conditions, human IL-10 may have unexpected proinflammatory properties, and this is thought to explain its ability to suppress tumor growth (47).

Although we could not demonstrate the ability to extend ß-galactosidase expression with the coexpression of human IL-10, we can clearly demonstrate that levels of human IL-10 in the lung can be achieved well within its therapeutic range (1–10 ng/g wet weight), without a significant systemic appearance (<10% of lung concentrations). Additionally, we have shown that AdV/hIL-10 delivered to the lung at these doses does not induce a systemic anti-adenoviral or anti-human IL-10 Ab response. This is in marked contrast to the intratracheal delivery of AdV/ß-gal, which leads to increased Ab production against both the adenoviral proteins as well as the reporter gene. These findings are also in contrast to the studies of David et al. (48), who observed strong Ab responses to systemically administered adenovirus vectors expressing IL-10, similar to that seen against adenovirus-expressing ß-galactosidase or empty cassettes. In our studies, Abs to adenoviral proteins were much more modest when AdV/hIL-10 constructs were compared with AdV/ß-gal. These differences in Ab responses to AdV/IL-10 may be due to the site of adenoviral delivery. In our experience, anti-adenoviral Ab production following delivery of recombinant adenovirus containing human IL-10 differs significantly, depending on whether virus is administered intratracheally or systemically. These observations may suggest differing immune responses to adenovirus, dependent upon the route of administration.

These findings taken together suggest that human IL-10 coexpression is unlikely to be helpful in reducing the inflammatory response or extending transgene expression to the lung delivery of adenoviral vectors. However, adenoviral vectors expressing human IL-10 can be delivered independently to the lungs without a significant inflammatory response in the lung or significant systemic IL-10 levels, can result in local expression for periods up to 2 wk, can suppress local TNF-{alpha} and TGF-ß production, and are not associated with a systemic Ab response. Thus, targeted delivery of IL-10 offers a potential approach for the treatment of acute lung diseases associated with inappropriate or excessive TNF-{alpha} or TGF-ß production, in which local administration of IL-10 is recommended.


   Footnotes
 
1 This work was supported in part by Grant P30 HL-59412, awarded by the National Heart, Lung and Blood Institute; Grants GM-40586 and GM-53252, awarded by the National Institute of General Medical Sciences; and a contract with Canji, Inc. R.M.M. and J.E.R. are currently supported by a research fellowship (T32-GM-08721) in burns and trauma, awarded by the National Institute of General Medical Sciences. Back

2 Address correspondence and reprint requests to Dr. Lyle L. Moldawer, Department of Surgery, Room 6116, Shands Hospital, University of Florida College of Medicine, Gainesville, FL 32610. E-mail address: Back

3 Abbreviations used in this paper: AdV/ß-gal, adenovirus construct expressing ß-galactosidase; AdV/hIL-10, adenovirus construct expressing human IL-10; GFP, green fluorescent protein; SOD, superoxide dismutase; LT{alpha}, lymphotoxin {alpha}. Back

Received for publication July 9, 1999. Accepted for publication October 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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