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Department of Microbiology, University of Tennessee, Knoxville, TN 37996
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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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Most investigations on the fate of plasmid DNA and the origin of immunogenic material have focused on systemic administration, especially the i.m. route (13). Our laboratory has focused on mucosal delivery and has observed that this form of plasmid administration results in good immune induction as well as immune modulation (14, 15, 16). Following mucosal inoculation, protein expression was observed in the lung and DLN, but it is not clear how the plasmid disseminates to such sites or how mucosal plasmid DNA delivery results in the systemic immune induction and the immunomodulatory effects observed.
In the present report, we probe the fate of eukaryotic expression plasmid DNA after mucosal delivery and evaluate possible mechanisms by which plasmid DNA expressing IL-10 achieves immunomodulation. Our results indicate that plasmid DNA disseminates widely in the body following mucosal delivery. Distal tissues such as the dermis, bone marrow, and lymphoid tissues remote from the deposition site become seeded with plasmid DNA and, in addition, become accessible to cells presumably transfected at the inoculation site or draining lymphoid tissue. Whereas IL-10 protein can be demonstrated in the blood stream and can additionally be produced by transfected cells at local sites, the modulatory effects of IL-10 plasmid DNA appears mainly to result from central effects in T cell function. Thus T cells appear to be silenced for a period that extends beyond the duration of plasmid DNA gene expression. However, the silenced T cell reactivity was readily recovered by exogenous IL-2 treatment, indicating that the mechanism involved in long-term modulation is induction of an anergic-like state.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five- to six-week-old female BALB/c mice (Harlan Sprague-Dawley, Indianapolis, IN) and 6- to 8-wk-old CB-17 SCID mice (Taconic Farms, Germantown, NY) were used for the study. BALB/c mice were housed conventionally, and SCID mice were housed in sterile microisolator cages in the animal facility. All food, water, bedding, and instruments were autoclaved or disinfected. All manipulations were performed in a laminar flow hood. To prevent bacterial superinfection, all SCID mice received treatment of sulfamethoxazole/trimethoprim (Biocraft, Elmond Park, NY) at the rate of 5 ml/200 ml of sterile drinking water. The investigators adhered to the guidelines set by the Committee on the Care of Laboratory Animal Resources, Commission of Life Sciences, National Research Council. The animal facilities of the University of Tennessee are fully accredited by the American Association of Laboratory Animal Care.
Viruses
HSV-1 KOS and HSV-1.17 strains were grown on a Vero cell monolayer (ATCC cat. no. CCL81), titrated, and stored in aliquots at -80°C until used.
Plasmid DNA preparation
Plasmid DNA encoding IL-10 under SV40 promoter was a gift of Dr. T. Mosmann (Edmonton, Alberta, Canada). A vector DNA without IL-10 gene was made by digestion of IL-10 plasmid DNA with EcoRI followed by ligation of the purified vector fragment. Plasmid DNA encoding ß-galactosidase (pCMVß; cat. no. 6177-1) and plasmid DNA encoding a red-shift variant of green fluorescent protein (pGFP; cat. no. 6084-1) were purchased from Clontech (Palo Alto, CA). Plasmid DNA was purified by polyethylene glycol precipitation as described previously (17). Protein expression of the different plasmids in eukaryotic cells (human embryonic kidney cells, HEK293) was determined as described elsewhere (15).
Immunization with HSV
BALB/c mice were anesthetized with methoxyflurane (Metophane; Pittman-Moore, Mundelein, IL) and received footpad injection of 106 PFU of HSV-1 KOS.
Plasmid DNA administration
For intranasal (i.n.) administration, 3 wk postinfection, 200 µg of plasmid DNA was suspended in 25 µl of sterile PBS and dropped onto the nasal mucosa. The mice inhaled the DNA as they breathed. Intramuscular administration was performed by injecting 100 µg of plasmid DNA in 50 µl of sterile PBS to each biceps muscles.
Adoptive transfer protocol
HSV-infected BALB/c mice received i.n. administration of 200 µg of IL-10 DNA or vector DNA. Around 4 wk later, the splenocytes were obtained from the mice and were enriched for T cells by passing though a nylon-wool column. These T cells were adoptively transferred i.v. to untreated SCID mice by tail injection. In some experiments, 500 U of rIL-2 (cat. no. 906011, Hemagen, Columbia, MD) was i.p injected into SCID mice at days 0, 1, 2, and 3 following adoptive transfer. The enriched populations were analyzed by flow-cytometric analysis that usually consists of >85% of CD3+ T cells (>65% of CD4+ T cells and >25 of CD8+ T cells). The enriched cells were also analyzed for the presence of administered plasmid DNA by PCR that showed no amplified bands for ß-gal or IL-10 plasmid DNA.
Delayed type hypersensitivity (DTH)
At different time points after IL-10 DNA treatment, test Ags in 30 µl of sterile PBS were injected into the ear pinnae of the HSV-infected BALB/c mice. The increase in ear thickness was measured 48 h later with a screw gauge meter (Oditest; H. C. Kroeplin, Schluechtern, Germany) as described previously (18). Test Ags used were UV-inactivated HSV-1 KOS (105 PFU before UV inactivation) or Vero cell extract. In a separate experiment, the ear of vector DNA-treated mice received the injection of 80 ng of IL-10 protein along with HSV Ag. At the same time or 3 h after HSV + IL-10 protein injection, the ear pinnae were injected with 1 µg of anti-IL-10 Ab (PharMingen, San Diego, CA; cat. no. 18140D) or 1 µg of rat IgG1 isotype Ab (PharMingen; cat. no. 20610D). The DTH response of the IL-10 DNA-treated mice was tested as described above. In some animals, at day 28 after i.n. treatment of IL-10 DNA, 1 µg of anti-IL-10 Ab or 1 µg of rat IgG1 isotype Ab were injected into ear pinnae along with HSV Ag. Similarly, SCID mice were tested for the DTH responses on the next day of adoptive transfer.
Virus challenge
On day 3 following adoptive transfer, the SCID mice were challenged with HSV as described elsewhere (19). Briefly, before challenge the left flank of mice was depilated by using a hair clipper (Oster Animal Hair Clipper, Milwaukee, WI) and a chemical depilator, Nair (Cater-Wallace, New York, NY). After depilation, the mice were anesthetized with metophane, and a total of seven scarifications were made on an approximate 5-mm2 area using a 27-gauge needle. A total of 10 µl of 106 PFU (100 LD50) of HSV-1 strain 17 was added to the scarifications and gently massaged. Animals were inspected daily for the development of zosteriform ipsilateral lesions, general behavior changes, encephalitis and mortality. The severity of the lesions was scored as follows: 1 = vesicle formation; 2 = local erosion and ulceration; 3 = mild to moderate ulceration; 4 = severe ulceration, hind limb paralysis, and encephalitis; and 5 = death.
HSV-specific lymphoproliferation
This assay has been described in detail elsewhere (19). Briefly, at days 28 or 65 after plasmid DNA treatment, the splenocytes of vector or IL-10 DNA-treated mice were enriched for T cells by a nylon-wool column and used as responder populations. These T cells were restimulated in vitro with irradiated syngeneic splenocytes infected with UV-inactivated HSV (multiplicity of infection (moi) of 1.5 before UV inactivation) or irradiated naive splenocytes, and incubated for 5 days at 37°C. In some experiments, rIL-2 (10 U/well) was added. Con A (5 µg/ml) was used as a polyclonal positive control and incubated for 3 days. Eighteen hours before harvesting, [3H]thymidine was added to the cultures. In at least five independent experiments, proliferative responses were tested in quadruplicated wells and the results expressed as mean cpm ± SD.
IL-2 assay by ELISA
On day 28 after IL-10 DNA treatment, splenocytes from the IL-10 DNA or vector DNA-treated BALB/c mice were obtained. The enriched T cells (5 x 106 cell/ml) were restimulated in vitro with 5 x 105 of irradiated syngeneic splenocytes infected with HSV-1 KOS or irradiated naive splenocytes for 3 days at 37°C. Similar number of cells were stimulated with 5 µg of Con A as a polyclonal positive stimulator for 48 h. The culture supernatants were screened for the presence of IL-2 by ELISA assay. ELISA plates were coated with anti-IL-2 Ab (PharMingen, cat. no. 18161D) and incubated overnight at 4°C. The plates were washed three times with PBS containing 0.5% Tween-20 and blocked with 3% of nonfat dry milk for 2 h at room temperature. After washing, serially diluted samples and standard were added to the plates in duplicate, and incubated overnight at 4°C. After being washed, biotinylated anti-IL-2 Ab (PharMingen; cat. no. 18000D) was added and incubated 2 h at 37°C. The plates were washed and peroxidase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA; cat. no. 016-030-084) was added. The color was developed by adding the substrate solution (11 mg of 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid in 25 ml of 0.1 M citric acid, 25 ml of 0.1 M sodium phosphate, and 10 µl of hydrogen peroxide). IL-2 concentration was calculated with an automated ELISA reader (SpectraMAX 340, Molecular Devices, Sunnyvale, CA).
Enzyme-linked immunospot (ELISPOT) for cytokine producing cells
The detailed methods of ELISPOT assay for the quantification of
cytokine producing cells were described previously (14).
Twenty-eight days following IL-10 gene transfer, the splenocytes of
vector or IL-10 DNA-treated mice were enriched for T cells by a
nylon-wool column. The T cells were analyzed for IL-4 and IFN-
spot-forming cells by ELISPOT. To generate cytokines, the T cells were
stimulated in vitro with enriched dendritic cell populations obtained
by the method of Nair et al. (20). The enriched dendritic
cells were pulsed with UV-inactivated HSV (moi of 5 before UV
inactivation) for the HSV-specific cytokine production. The T cells and
stimulator DC (naive or pulsed) were added at a responder-to-stimulator
ratios of 10:1, 5:1, 2.5:1, and 1.25:1 in 200 µl of RPMI 1640 medium
with 10% fetal bovine serum per well into ELISPOT plates which were
coated with anti-mouse IL-4 (PharMingen; cat. no. 18191D) or
anti-mouse IFN- (PharMingen; cat. no. 18181D). After 96 h
incubation, the plates were washed and biotinylated anti-mouse IL-4
(PharMingen; cat. no. 18042D) or biotinylated anti-mouse IFN-
(PharMingen; cat. no. 18112D) was added. After 1 h of incubation
at 37°C, 100 µl of alkaline phosphatase-conjugated streptavidin in
PBS (1 µg/ml) was added and the plates were incubated for another
1 h. The spots were developed by using nitroblue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate as a substrate and counted 24
h later under a dissecting microscope. Mean cytokine spot forming cells
(SFC) ± SD from five independent experiment was expressed as
results. In some experiments, rIL-2 (10 U) was added to culture
wells.
IL-10 detection in serum after IL-10 gene transfer
Groups of mice received i.m. or i.n. treatment of either 200 µg of IL-10 plasmid DNA or vector. At different days, serum samples were collected from the mice and analyzed for the presence of IL-10 in the serum by ELISA assay. ELISA plates (Dynatech, Chantilly, VA) were coated overnight at 4°C with 2 µg of rat anti-mouse IL-10 (PharMingen; cat. no. 18141D). The wells in the plates were washed with PBS containing 0.05% Tween (PBS-T) and blocked with 3% milk for 2 h at 37°C. The sera samples and the rIL-10 (PharMingen; cat. no. 19281V) at a concentration of 1 ng/ml) were added and serially diluted. The standard and samples were incubated overnight at 4°C. After washing with PBS-T, 1 µg of biotinylated anti-IL-10 Ab (PharMingen; cat. no. 18152D) per ml was added and incubated at 37°C for 2 h. After the wells were washed, peroxidase-conjugated streptavidin (Jackson ImmunoResearch) was added and incubated at 37°C for 2 h. Substrate was added for color development. IL-10 concentration was measured by automated ELISA reader (Spectra MAX 340; Molecular Devices). Each time point contains sera from at least four mice.
Isolation of DNA and RNA
At different time points following administration of pCMVß, pGFP, or vector DNA (data not shown), several tissues including muscle, DLN, spleen, lung, liver, bone marrow, and ear were obtained. The tissue samples were minced into small pieces (or sometimes single cell suspensions of DLN and spleen were made). The preparations were treated with TRI Reagent (Molecular Research Center, Cincinnati, OH). Total RNA and DNA were isolated by manufacturer’s protocol. Briefly, RNA was extracted with chloroform and precipitated with isopropyl alcohol. The contaminating plasmid DNA was removed with RQ-1 RNase-free DNase (Promega, Madison, WI). The absence of plasmid DNA in the RNA preparations was verified by performing PCR on the total cellular RNA without reverse transcription. DNA was isolated by precipitation of interphase and phenol phase with ethanol. The precipitated DNA was washed with 0.1 M sodium citrate followed by 75% ethanol. Anticoagulated blood samples were also obtained at different time points after i.n. administration of pCMVß or pGFP. The blood samples were centrifuged, and plasma was carefully separated from the blood cell pellet. DNA from 0.5 ml of plasma were extracted twice with phenol/chloroform and precipitated with ethanol. The extracted DNA was dissolved with sterile nuclease-free water. Peripheral blood cells (PBC) were washed with DMEM containing 10% FBS and incubated for 90 min to separate adherent and nonadherent cell populations. Each population was treated with TRI Reagent, and total DNA and RNA were extracted as described above. All the procedures including PCR were performed in a separate laminar flow hood to avoid cross-contamination.
RT-PCR
Total cellular RNA (10 µg/ml) was reverse transcribed using oligo(dT) primers and reverse transcriptase (Promega) according to protocol described previously (21). The contaminating plasmid DNA was removed by treatment with RQ1-RNase-free DNase and verified by PCR using both CMV-1/ß-gal-2 (or GFP-1) primers and ß-gal-1/ß-gal-2 (or GFP-1/GFP-2) primers. The cDNA was made by the reverse transcription reaction incubated at 42°C for 90 min. The cDNA (5 µl) was subject to 35 cycles of amplification as described (22) using ß-gal-1 and ß-gal-2 (or GFP-1 and GFP-2) and ß-actin-1 and ß-actin-2 primers. DNA isolated from the plasma was also amplified by 35 cycles of amplification using CMV-1 and ß-gal-2 (or GFP-2) primers. The primers used follow: CMV-1, 5'-CCATTGACGTCAATGGGAG-3'; ß-gal-1, 5'-ACTTTGACCAACAAGAACGTG-3'; ß-gal-2, 5'-AAATGTGAGCGAGTAACAACC-3'; GFP-1, 5'- TCGTGACCACCCTGACCTAC-3'; GFP-2, 5'-CCTAGGTGGCCTAGATCTATTG-3'; ß-actin-1, 5'-GTGGGGCGCCCCAGGCACCA-3'; ß-actin-2, 5'-CTCCTTAATGTCACGCACGAT-3'.
ß-gal staining
At different days after pCMVß DNA administration, DLN, spleen, lung, and ear tissues were isolated from the mice and fixed in 4% paraformaldehyde in PBS for 1 h at 4°C. The tissues were washed and stained for ß-gal activity as described previously (22). Briefly, the tissues were incubated overnight with the substrate solution containing 2 mg/ml of 5-bromo-4-chloro-3-indolyl ß-D-galactosidase, 20 mM potassium ferrocyanide, 20 mM potassium ferricyanide, 2 mM MgCl2, and 120 µl of 10% Nonidet P-40 and 100 µl of 1% sodium deoxycholate in 20 ml of PBS. After the reaction, the tissues were washed with PBS and fixed with 4% of formaldehyde. The tissues were embedded in paraffin, sectioned at 5 µm, and counterstained with nuclear fast red.
FACS analysis
At day 10 following i.n. pGFP administration, PBC were isolated from anticoagulated blood. Following lysis of RBC, the PBC were incubated in the culture flask for 90 min at 37°C. The adherent cell populations were carefully separated from the nonadherent cells. After washing with DMEM containing 10% FBS, the cells (2 x 106 cells/ml) were immediately fixed with 4% paraformaldehyde. The green fluorescence signals were analyzed by flow cytometry analysis using FL-1 channel. As a positive control, HEK293 cells were transfected with pGFP, and 60 h later the harvested HEK293 cells were fixed. The fluorescent signals from the cells were detected as described above.
Statistical analysis
Significant differences between groups were evaluated using the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were infected systemically with HSV and 3 wk later received
via the i.n. or i.m. routes 200 µg of IL-10 plasmid DNA either
encoding IL-10 or vector DNA. At various days after this exposure, mice
were tested for their cutaneous DTH responses to HSV Ag injected into
the ear pinnae. Suppressed responses in IL-10 DNA-treated mice first
became evident around 5 days posttreatment (Table I
). However, the duration of suppression
differed from i.m. to i.n. treatment. Such suppression by i.m.
injection of IL-10 DNA lasted for 7 wk as reported earlier
(3), whereas suppression had declined by day 45 in i.n.
recipients of IL-10 DNA (Table I). Thus DNA encoding IL-10 administered
mucosally as well as systemically is capable of suppressing Ag-induced
inflammatory responses even when administered after sensitization has
occurred.
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). Both responses are usually
measures of CD4+ T cell function
(19). The responsiveness of the cells to Con A (polyclonal
activator) was equal between IL-10 DNA and vector DNA-treated group.
Cells were also tested by ELISPOT for the number of IFN- and IL-4
HSV-specific SFC (Table II). The cell population from IL-10-treated
mice had reduced IFN- SFC, but IL-4 SFC were similar to controls.
Thus the data indicate that IL-10 DNA administration suppresses the
reactivity of CD4+ Th1 T cells.
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The observations above indicate that mucosal delivery of IL-10 DNA results in systemic suppression of T cell reactivity. Several possibilities were considered to explain how mucosally delivered plasmid could result in such suppression. These included the passage of plasmid DNA to remote sites followed by cell transfection and expression at such sites. The plasmid DNA could be transported within cells that had been transfected at the mucosal delivery site or DLN. Alternatively, the IL-10 protein could be expressed locally and enter the blood stream to distribute to distal sites and actively suppress inflammatory responses. Further explanations could be that T cells were silenced in the local or distal lymphoid tissues by exposure to the IL-10 protein or that regulatory cells were induced that inhibited the responses of the Ag stimulated immune T cells.
Detection of plasmid DNA and transfected cells in blood and peripheral sites
To investigate whether plasmid DNA and the expression of encoded
protein was present at the remote sites or in the blood stream,
plasmids encoding ß-gal or GFP were used to avoid the complication of
detecting endogenous cytokine gene expression. Animals were given the
plasmid DNAs or vector control mucosally and then killed at intervals
and multiple tissues, including the separated plasma and PBC, were
collected to detect DNA, RNA, or protein. The results recorded in Table III indicate that plasmid DNA received
i.n. was present in blood by 5 h as detected by PCR using primer
sets that can amplify only the introduced gene and its promoter (Fig. 1, Table III). This DNA persisted for at
least 3 days in plasma and was evident in all tissues analyzed at days
1 and 7. These tissues included the ear pinna and spleen and in the
latter plasmid DNA was detectable until at least day 14. Furthermore,
in a separate experiment, the IL-10 plasmid DNA also disappeared from
most tissues by day 20. The presence of DNA in the plasma means that
early on plasmid DNA can transport via blood as cell-free plasmid DNA
to multiple tissues, but because the technique used did not permit
quantification whether or not the plasma route is more important than
the intracellular route could not be established. Thus plasmid DNA was
also present in separated and washed leukocytes.
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). The RNA
was detectable from day 1 to day 7 and some RNA was still present at
day 14. Even though RNA expression was not detected, the ß-gal and
IL-10 DNA could be still found in nasal tissues at day 21, but had
disappeared by day 28 (data not shown). Regarding protein expression,
230–580 cells per 105 adherent blood cells were
positive for the GFP signal at day 10 but by 21 days protein expressing
cells in the blood were undetectable (see Fig. 2). These data indicate that plasmid DNA
transfection likely occurs at the sites of inoculation and that some
cells enter the blood stream and circulate for prolonged but not
indefinite periods. In addition, PBC might take up cell-free plasmid
DNA within circulation.
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, Table III). In addition, protein-expressing cells were evident in
lungs, DLN and spleen. Furthermore, some cells were also demonstrable
at cutaneous sites (Fig. 2). Regarding the latter, a few
protein-expressing cells could be also demonstrated in the inflammatory
reactions induced at 7 days post-plasmid DNA inoculation but not in
reactions induced at day 21 or beyond. Such data may indicate that the
source of plasmid expression may be recruited cells from the blood
stream as well as cells transfected at cutaneous sites by plasmid DNA
arriving soon after mucosal inoculation. Our data could not distinguish
whether the presence of RNA and protein expression at remote sites
results from direct transfection by plasmid DNA at such sites or the
consequence of seeding by cells transfected by plasmid at inoculation
sites. These issues are under further investigation. However, these
results indicate that mucosal IL-10 DNA delivery leads to wide
dissemination of plasmid DNA to several tissues, and these sites could
serve as a source of IL-10 expression. Active suppression is not involved in long-term modulation of inflammatory response
The initial mechanism considered was that suppression resulted
from the effects of IL-10 protein produced at the sites of plasmid DNA
transfection, and this protein actively inhibited the response at the
tissue site of inflammation. For this mechanism to occur, IL-10 protein
should be present in the bloodstream and tissues. To show that IL-10
protein could inhibit DTH, purified IL-10 was injected into the ear
pinna of HSV-primed mice at the same time as Ag. A single injection of
IL-10 protein to the local inflammatory site was sufficient to mediate
inhibition as observed previously by others (23).
Furthermore, the subsequent injection (3 h later) of anti-IL-10
abrogated the inhibitory effect of the IL-10 protein (Fig. 3). Abrogation also occurred if the
anti-IL-10 was injected at the same time as IL-10 protein and Ag.
Following mucosal administration of IL-10 plasmid DNA, IL-10 protein
was evident in serum in minimal amounts in some animals by day 2 and
peaked around 8 days postinoculation (Fig. 4). Most animals still had measurable
IL-10 on day 14, but by day 20 IL-10 protein was undetectable.
Therefore, the increase of serum IL-10 level might explain the
inhibition of DTH responses. However, the inhibitory effect of IL-10
DNA administration persisted for at least 2–3 wk beyond the time of
detection of serum IL-10. Furthermore, in other experiments,
anti-IL-10 Ab was injected along with Ag into the ears of
HSV-primed mice that 10 or 28 days previously were given IL-10 DNA
i.n.. The anti-IL-10 treatment failed to affect the extent of the
DTH reaction even though anti-IL-10 could reverse the inhibitory
effects by rIL-10 treatment (Fig. 3). Furthermore, only very few cells
expressing proteins could be found at inflammatory sites at day 14, and
the effects of plasmid DNA modulation persisted beyond the time of
plasmid DNA, RNA, or protein demonstration at peripheral sites (or even
the central lymphoid tissue). Such observations likely mean that the
long-term inhibitory effect on the cutaneous inflammatory reaction was
not the consequence of IL-10 protein expression at the local site. This
observation argues against active suppression as an explanation for
long-term modulation by IL-10 DNA.
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An alternative mechanism of modulation by IL-10 DNA could involve silencing of Ag-reactive T lymphocytes or the induction of regulatory cells in the DLN or spleen by exposure to IL-10 protein. The regulatory effect could result from the action of other sets of T cells (such as Tr1 cells described by others (24)). A series of experiments were designed to evaluate these issues. First, it was supposed that if T cells were silenced as a consequence of exposure to IL-10, their inhibitory activity should be maintained upon transfer to a neutral environment lacking IL-10 protein. This issue was evaluated by adoptively transferring enriched T cells from HSV primed and IL-10 DNA-treated mice (4 wk post-IL-10 DNA treatment) into recipient SCID mice. The transferred cell population lacked adherent cells and was taken at a time when protein and RNA expressing cells were no longer present as detected in parallel experiments in mice given ß-gal marker plasmid DNA. Furthermore, IL-10 DNA could not be demonstrated by PCR in the adoptively transferred cell populations (data not shown). Thus apparently plasmid DNA free lymphocytes were transferred into the SCID mice.
One day after treatment, the SCID mice were tested if the adoptive
transfers could support a DTH response. SCID mice, which received
transfers from HSV-primed mice treated with vector DNA, served as
positive controls for DTH reactions. As shown in Table IV, the recipients of T cells from IL-10
DNA-treated mice showed inhibited inflammatory responses. After testing
animals for DTH reactions, the groups of SCID mice used for adoptive
transfer were challenged on the scratched skin with HSV virus. Animals
were followed for the development of cutaneous zosterification lesions
as described previously (19). As shown in Table IV,
whereas SCID mice that received HSV immune lymphocytes from
vector-treated mice were protected from zoster lesions, those given
cells from IL-10 DNA-treated animals showed severe lesions and died.
Some level of immunity was still evident, however, because animals did
survive longer than control SCID mice that received no adoptive
transfers. The above observations provide evidence of silencing of
effector cells or the presence of inhibitory cells in the transfer
population. Thus in some systems T cells exposed to IL-10 protein can
become inhibitory and also become themselves a source of IL-10
(24). Such cells have been termed Tr1 cells
(24).
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rIL-2 treatment reverses silenced T cell reactivity
Some reports have indicated that inhibitory effects of IL-10 on T
cell functions is reversible by antigenic stimulation in the presence
of exogenous IL-2 (25). To evaluate whether the T cell
silence was affected by IL-2, in vitro and in vivo experiments were
performed on T cells collected 4 wk after mucosal administration of
IL-10 DNA. As shown in Table V, in vitro
stimulation of silenced T cells with Ag in the presence of exogenous
rIL-2 supported both proliferation and IFN- SFC responses.
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indicates that the IL-2 treatment
reversed the silencing effects in that animals both developed DTH
reaction and showed resistance levels to HSV comparable to that
mediated by immune T cells from vector-treated animals. Our data
indicate that the T cell silencing effect mediated by IL-10 appears
reversible by antigenic stimulation in the presence of exogenous
IL-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The modulatory
effect likely proceeded by multiple mechanisms, but because suppressed
Ag-specific T cell functions persisted beyond the duration of
detectable plasmid encoded protein and was maintained upon adoptive
transfer of T cells into a plasmid-free environment, one of the
modulatory effects was assumed to involve silencing of T cell
reactivity. Such silenced T cells were not themselves transfected by
plasmid DNA and not a source of IL-10. Moreover, their anergic state
was reversible by exposure to Ag in the presence of exogenous
IL-2. The observation that plasmid DNA was evident in plasma following intranasal inoculation was surprising, although such has been reported after systemic injection (26, 27, 28). How plasmid DNA gains entrance from a mucosal surface into the blood stream was not resolved. Likely routes could be through the highly vascularized nasal cavity or lung alveoli (29). In fact, protein-expressing cells were readily detectable in the lung. Others have reported that the circulation half-life of plasmid DNA is brief and of the order of 5 min to 16 min (26, 27, 30, 31). However, in our studies plasmid DNA was detectable for at least 3 days after a single mucosal administration. This finding may imply that seeding occurs over a lengthy period or that some plasmid DNA may be protected from breakdown by binding to cells or by being bound to some serum protein. Some support for latter idea has been reported by others (27, 32, 33). The presence of free plasmid DNA in the plasma could mean that this is the primary means by which plasmid DNA disseminates widely to distal tissues.
An additional transport mechanism could involve transfected cells,
which were detectable in the blood stream for a longer period than was
free plasmid DNA. Thus by RT-PCR using RNA from washed leukocytes,
signals were detectable for 2 wk, and by using the nonsecreted protein
GFP, transfected cells were evident for at least 10 days. The nature of
the transfected cells was not established but were likely mainly
macrophages and dendritic cells. In other studies, macrophages were
shown to rapidly take up plasmid DNA via their scavenger receptor
following i.v. plasmid DNA administration (27, 34). A
recent report also showed macrophages containing GFP were evident in
blood following i.m. injection of plasmid DNA encoding GFP
(35). Other reports have indicated that plasmid DNA
transport, at least from the site of muscle or skin inoculation to the
DLN, proceeds via transfected dendritic cells (8, 9, 10, 11, 12). It
will be of interest to further identify the nature of transfected blood
cells as well as define the localization of their transfection.
Possibilities include the bloodstream itself, the inoculated nasal
mucosa, or the DLN. We are currently attempting to verify the latter
idea by comparing the number of transfected cells in the circulation of
normal mice with LT
k/o animals that lack lymph nodes (36, 37). It seems likely, however, that not all cells were
transfected at the local lymph node or blood stream because cells of
nonmyeloid type were evident in peripheral tissues. Thus occasional
cells with fibroblast morphology were evident in noninflamed skin
sections in animals inoculated mucosally with ß-gal plasmid DNA.
These observations may mean that plasmid DNA may exit the blood stream
and also transfect cells at tissue sites. Experiments are underway to
further investigate these issues.
Our studies demonstrate that mucosal delivery of plasmid DNA encoding IL-10 provided long-term suppression of an Ag-induced inflammatory reaction orchestrated by CD4+ Th1 T lymphocytes. Accordingly, mice infected with HSV and subsequently given IL-10 DNA had suppressed cutaneous DTH reactions to viral Ags. Suppression took a few days for onset but persisted for 5 wk. Many investigations have demonstrated that IL-10 can modulate Th1 mediated functions with multiple mechanisms likely occurring (2). These include inhibition of cytokine production and action on target cells, as well as inhibition of recruitment of nonspecific cells to inflammatory sites (2, 38). IL-10 protein in lesion sites can mediate such effects as was demonstrated in this communication as well as by others (23). However, whereas direct active suppressive effects of IL-10 protein in lesions might account for part of the suppression at least in the early phase, this mechanism could not provide the complete explanation for our observations. Thus, the suppression persisted well beyond the duration of detectable IL-10 protein in serum (6–14 days) or even protein-producing cells (up to 3 wk) at least as measured by detection of a marker protein. In addition, whereas the modulatory effect of exogenous IL-10 protein could be neutralized by anti-IL-10 injected into inflammatory sites, similar injections were without effect on suppression induced by mucosal plasmid DNA administration. Furthermore, the IL-10 DNA suppressed activity of T cells was retained when such cells were adoptively transferred to an environment lacking IL-10 plasmid DNA.
We interpret such observations to mean that some form of central suppression of lymphoid function had occurred. This notion was further supported by the observation that Ag-specific lymphocyte responses measured in vitro were suppressed especially as regards Th1 cytokine production. We have referred to the inhibited effect as T cell silencing and favor this mechanism over one of inhibition by a separate set of regulatory cells. Thus, in some systems investigators have demonstrated that long-term stimulation of CD4+ T cells in the presence of IL-10 leads to the production of a subset of T cell (Tr1 cells) which secret IL-10 and inhibit the action of Th1 cells (24). Using adoptive transfers of cell mixtures into recipient mice and measuring their DTH reactions, no evidence for regulatory effects was forthcoming. Furthermore, in T cell populations taken from suppressed mice, no evidence for an increase in the number of IL-10 producing cells was observed. Although we lacked positive evidence for the existence of regulatory Tr1-like cells after mucosal IL-10 DNA administration, this issue is being further investigated.
The nature of T cell silencing requires further study, but this is
difficult to accomplish in the system we described because the viral Ag
specific CD4+ T cell frequency is low. However,
the silenced cells would appear to be in a reversible state of anergy,
because in addition to recovering their former function with time,
their Ag-specific function could be recalled by stimulating cells in
the presence of exogenous IL-2. This IL-2 reversible effect was evident
both in vitro as well when the suppressed cells were adoptively
transferred to SCID mouse recipients given IL-2 and Ag. Interestingly,
whereas the adoptive transfer of silenced cells to SCID mice failed to
confer protection against HSV challenge, the effect was reversed in
recipient of IL-2. In other system involving IL-10 protein mediated
unresponsiveness of CD4+ T cells, the effect was
reversible by culture of the cells in vitro with exogenous IL-2
(25). However, in another system unresponsiveness induced
by IL-10 appears to be irreversible by IL-2 (39). It could
be that the IL-10 treatment down-regulates IL-2 receptor -chain on
the T cells, inhibiting IL-2 signaling. In our system, IL-2 could
functionally reverse the Ag unresponsiveness state, indicating that the
signaling pathway through IL-2 receptor was not inhibited by IL-10
treatment. In fact, we could detected no change on the expression of
IL-2 receptor -chain on the T following IL-10 DNA administration
(unpublished data).
In conclusion, mucosal delivery of plasmid DNA results in widespread distribution in the body. This distribution may occur by transport of free plasmid DNA as well as by transfected cells. However, persistence of the plasmid DNA is not indefinite and was far shorter than up to 9 mo reported by some to occur following i.m. plasmid DNA administration (40). Nevertheless, this shorter duration represents a potential advantage of mucosal delivery in some clinical situations. One such instance may be when using gene delivery to express immunomodulators. Thus modulating an unwanted inflammatory immune reaction may concomitantly render recipients more susceptible to infection by agents where defense depends on the function of the type of immune response being suppressed. In fact, in the case of HSV infection, the CD4+ Th1-mediated inflammatory reaction, while undesirable and tissue damaging in certain locations such as the eye (41), is actually the principal means of immune control following HSV infection (19, 41). Suppressing the response renders animals more susceptible to infection and virus can spread to critical tissues such as the brain (42). Indeed, in our studies using adoptive transfer experiments we showed that T cells from HSV primed IL-10 DNA-treated mice were less immunoprotective against HSV challenge than were T cells from untreated animals. Accordingly, modulating immunity using cytokine gene delivery somewhat resembles a Faustian dilemma!
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Barry T. Rouse, Department of Microbiology, M409 Walters Life Sciences Building, University of Tennessee, Knoxville, TN 37996-0845. E-mail address:
3 Abbreviations used in this paper: DLN, draining lymph node; i.n., intranasal; ß-gal, ß-galactosidase; GFP, green fluorescence protein; DTH, delayed type hypersensitivity; ELISPOT, enzyme-linked immunospot; SFC, spot forming cell; moi, multiplicity of infection; PBC, peripheral blood cell.
Received for publication March 17, 1999. Accepted for publication June 10, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient mice: effects on secondary lymphoid organ development and humoral responsiveness. J. Immunol. 155:1685.[Abstract]
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870 bp)
and RNA (
