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(Investigative Ophthalmology and Visual Science. 2003;44:5228-5234.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0312
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Topical Treatment with Antisense Oligonucleotides Targeting Tumor Necrosis Factor-{alpha} in Herpetic Stromal Keratitis

Susanne Wasmuth,1,2 Dirk Bauer,2 Yanning Yang,3 Klaus-Peter Steuhl,1 and Arnd Heiligenhaus2

1From the Department of Ophthalmology, University of Essen, Essen, Germany; 2Ophtha-Lab, St. Franziskus Hospital, Münster, Germany; and the 3Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan, China.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Tumor necrosis factor (TNF)-{alpha} is a pleiotropic factor that is critical for the development of inflammation. The authors investigated whether topical application of TNF-{alpha}-antagonizing molecules, antisense oligonucleotides (ASON), might be an effective way of modifying the course of immune-mediated herpetic stromal keratitis (HSK).

METHODS. ASON targeting TNF-{alpha} mRNA were examined for their efficiency in interfering with the production of this cytokine in vitro. In vivo uptake was determined by FITC-labeled ASON. HSV-1 corneally infected mice were injected three times with ASON. Mice from the control groups received unrelated control oligonucleotides (CON) or buffer. The clinical course of HSK, the delayed-type hypersensitivity (DTH) reaction, the uptake of [3H]thymidine from cells derived from the spleen, virus-neutralizing antibody titers in the serum, and viral replication in the infected eyes were determined. The eyes were examined histologically. The corneal TNF-{alpha} content was measured by ELISA.

RESULTS. The TNF-{alpha} ASON reduced the lymphocytic cytokine expression in vitro. In vivo, the FITC-labeled molecules were detected in the cornea even after 10 days. In the TNF-{alpha} ASON mice the incidence of HSK decreased, and the severity of the disease was diminished. The corneal content of TNF-{alpha} was reduced, and the number of inflammatory cells was decreased. The other investigated parameters were not significantly altered by TNF-{alpha} ASON treatment.

CONCLUSIONS. The data suggest that TNF-{alpha} ASON diminishes the release of TNF-{alpha} from cultured lymphocytes and from lymphocytes in the HSV-1–infected cornea. This topical treatment mitigates the course of HSK, whereas the systemic antiviral effector functions were not impaired.

Herpes simplex virus type (HSV)-1 can cause a well-defined, immune-mediated, blinding corneal disease in humans and mice, termed herpetic stromal keratitis (HSK).1 There is profound evidence that the corneal inflammation in HSK is orchestrated by CD4+ T cells.2 The corneal infiltration contains abundant polymorphonuclear cells (PMNs). Although PMNs are important for the limitation of virus replication, they are also involved in tissue destruction in the cornea.3 4 Diverse proinflammatory cytokines, such as IL-2 and interferon (IFN)-{gamma}, have been shown to be involved in the disease process.5 6

Tumor necrosis factor (TNF)-{alpha} is produced mainly by T cells, PMNs, dendritic cells, and macrophages and is essential for NF-{kappa}B-dependent activation of several genes coding for subsequent chemotactic factors that attract and activate numerous cellular subsets.7 It increases the activity of inducible nitric oxide synthase (iNOS) and can induce prostaglandins and leukotrienes. TNF-{alpha} triggers granulocytes to internalize complement-coated HSV particles.8

Studies on the mRNA coding for TNF receptors have shown elevated expression in herpes-infected mouse eyes.9 The major sources of TNF-{alpha} that is released into the cornea are neutrophils and macrophages.10 Anti-cytokine antibody treatment in latently infected NIH mice has shown that TNF-{alpha} is one of the key mediators in recurrent HSK and is also released from the damaged corneal cells.11 Chemokines responsible for the recruitment and activation of PMNs and T cells are enhanced after TNF-{alpha} is produced in the HSV-infected cornea.12

As TNF-{alpha} plays an important part in the development of blinding HSK, the elimination of TNF-{alpha} may be a candidate therapeutic approach. There are several possible ways of blocking TNF-{alpha} functions in vivo. The administration of anti-TNF-{alpha} antibodies has the disadvantage of potential systemic side effects due to a developing humoral immune response after repeated application. Alternatively, the administration of TNF-{alpha} antagonists13 or soluble TNF-{alpha} receptors14 has been proposed.

The antisense oligonucleotide technique involves the use of a DNA sequence, 12 to 25 mer long, containing a base sequence complementary to a part of the targeted mRNA. The application of this may reduce the level of TNF-{alpha}. Two major limitations of the topical use of these cytokine-inhibiting agents are well known. First, the topical TNF-{alpha} blockade may interfere with the antiviral response, ultimately leading to recurrent HSV disease. Second, the epithelial barrier may limit the passage of these polyanionic molecules into the corneal stroma. On the contrary, topical application may avoid modulation of the systemic immune response to HSV-1, and it is not likely to induce untoward corneal inflammation or systemic side effects. Although there is some controversy about the mechanisms of action of ASON,15 16 17 it is now widely accepted that the antisense technique is a useful tool for specific blockade of the synthesis of a single protein of interest.18 19 20

In this study, we investigated whether the topical administration of ASON targeting TNF-{alpha} is capable of reducing the level of this cytokine in the cell culture supernatants of cultured lymphocytes and in the cornea of mice and whether the course of HSK could be effectively altered by this treatment. The hypothesis that the systemic antiviral immune response is not markedly altered by topical ASON treatment was proved.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
DNA Oligonucleotides
ASON directed to TNF-{alpha} and CON were designed and manufactured by Biognostik (Göttingen, Germany). A full-length backbone modification was used to protect them against enzymatic cleavage by DNases. In these phosphorothioates one of the nonbridging oxygens of the phosphate backbone is replaced by a sulfur atom. This is achieved through postcoupling oxidation with a sulfurizing reagent (EDITH (3-ethoxy-1,2,4-dithiazoline-5-one); PE Applied Biosystems, Weiterstadt, Germany). The ASON (CGA AGT TCA GTA GAC AG) used in the present study is the reverse complement to the bases 300 to 316 of the total sequence of murine TNF-{alpha}, and it targets an exon of the coding region. Controls are obtained by mixing up the bases of the antisense oligonucleotide in a randomized fashion. Cross homologies with other genes were excluded for both sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). ASON and CON (GCT CTA TGA CTC TTC AG) have identical chemical modification.

ASON Uptake Studies In Vitro
Single-cell suspensions from regional lymph nodes and spleens were prepared from HSV-infected mice at days 7 and 14 postinfection (PI).21 The cells were treated with concentrations of 1 or 2 µM FITC-labeled ASON in culture medium (RPMI 1640; Invitrogen-Gibco, Eggenstein, Germany) containing 5% fetal calf serum (FCS; Biochrom, Berlin, Germany), HEPES (Roth, Karlsruhe, Germany), and ß-mercaptoethanol (Sigma-Aldrich, Taufkirchen, Germany). After incubation for periods of between 15 minutes and 48 hours, the cells were washed three times with phosphate-buffered saline (PBS) and apportioned into two groups. The cells of the first group were scattered on slides, fixed in 4% paraformaldehyde (Sigma-Aldrich) in RPMI 1640 medium, dehydrated in an ascending series of alcohol, and examined by fluorescence microscopy for the uptake of ON. The cells of the second group were examined by flow cytometry (FACScalibur; BD Biosciences, Lincoln Park, NJ). For each sample, 20,000 events were measured. Cells without FITC-ASON served as the negative control.

ASON Efficiency on Lymphocytes In Vitro
Single-cell suspensions from regional lymph nodes and spleens were seeded out in 96-well plates (Falcon 3072 Microtest 96; BD Labware Europe, Meylen, France) at 106 cells per well in a volume of 100 µL ASON or CON were added to the culture medium at concentrations of 5, 10, or 20 µM and were incubated for 4 to 6 hours. After incubation, groups of lymphocytes were cocultured with 10 µL of UV-inactivated HSV-1 (concentration 1 x 107 PFU/mL before inactivation), ConA (concentration 5 µg/mL; Sigma-Aldrich), or medium. Twenty-four hours later, the supernatants were harvested and examined for their TNF-{alpha} content by a standard ELISA (PharMingen, Heidelberg, Germany).21 Each experiment was performed in four wells and was then repeated.

Mice
Female BALB/c mice were obtained from Charles River (Sulzbach, Germany). The animals were aged 6 to 8 weeks and were maintained according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and according to the guidelines approved by the institutional animal care and use committee.

Virus
The HSV-1 (KOS strain) was kindly provided by David Knipe (Harvard Medical School, Boston, MA). The virus was isolated and expanded on Vero cells (CCL 81; American Type Tissue Collection, Manassas, VA) and the virus-containing supernatants were collected. The plaque-forming units were determined in a standard plaque assay.22

Infection
The animals were anesthetized intraperitoneally, and the right cornea was scratched eight times. Then, 105 PFU of HSV-1 was applied to the cornea, as described elsewhere.22

Clinical Score
The animals were examined daily for the development of epithelial and stromal keratitis, as described elsewhere.23 The stromal keratitis was graded on a scale of 0 to 4+ for corneal opacity, corneal neovascularization, edema, and infiltration: 0, not present; 1+, less than 25%; 2+, between 25% and 50%; 3+, between 50% and 75%; and 4+, between 75% and 100%.

Histology
Animals were killed on day 7 or 14 PI. The eyes were removed and fixed for 6 hours in 4% formaldehyde, dehydrated, and embedded in paraffin. The eyes were cut into 5-µm sections and put on 3-aminopropyltriethoxysilane-coated slides (Sigma-Aldrich). The sections were stained with hematoxylin and eosin (HE) according to a standard protocol and mounted (Eukitt; Kindler, Freiburg, Germany). The infiltrating cells in the central cornea were counted by means of bright-field microscopy under a 10 x 10 grid high-power field (x400; two sections per eye, four specimens per treatment). All counts were independently performed by two investigators in a masked fashion.

Experimental Design
The corneas of all mice were infected with 105 PFU of HSV-1 on day 0. The mice belonging to group 1 (n = 30) received ASON on days -1, 1, and 4. The mice in group 2 (n = 30) served as the control for substance-specific bystander effects and therefore received CON on days -1, 1, and 4. The mice in group 3 (n = 31) were given only Tris-EDTA buffer (TE buffer) on days -1, 1, and 4. The animals in group 4 (n = 29) served as an infection control and underwent no further treatment after the corneal HSV-1 infection. Mice were killed either 7 (n = 8 in each group) or 14 (n = 21 to 23 in each group) days PI.

Intracorneal Injection of Study Medication
The 100-nM solution of ASON and CON in TE buffer or buffer alone was injected (2 µL in every case) through a small peripheral corneal incision into the subepithelial cornea by using a subretinal Glaser injector fixed to a 10-µL syringe (Hamilton, Reno, NV).

Studies on the Uptake of ASON into Mouse Cornea In Vivo
The mice received a single subepithelial injection of 2 µL of FITC-labeled ASON. After 24, 48, or 96 hours and 7 or 10 days, the eyes were enucleated, embedded in paraffin, and cut into 5-µm sections. Alternate sections were studied with a standard HE protocol and by fluorescence microscopy.

Homogenates of Corneas
Animals were killed on day 7 or 14 PI. The infected right corneas were removed without any limbal or iris tissue. The corneas were snap frozen in liquid nitrogen and were stored at -80°C until use. They were then thawed and homogenized by manual and ultrasonic treatment. The supernatants were collected after centrifugation at 10,000g.21

HSV-1–Neutralizing Antibody Activity
Serum from the mice in each experimental group was pooled for the analysis of the HSV-specific neutralizing antibody titers.21 Briefly, serial dilutions of the specimens were incubated with 102 PFU of HSV-1 on confluently grown Vero cell monolayers. The plaque-forming units were determined after fixation and crystal violet staining. The dilution that reduced the plaque-forming units to 50% of the corresponding control units was calculated.

Detection of Virus Replication in the Eye
Virus replication was studied with a plaque assay, as reported elsewhere.24 Whole eyes were collected and snap frozen in liquid nitrogen on day 7 PI. The HSV-infected tissue specimens were thawed and homogenized. Serial dilutions of centrifuged supernatants in RPMI were cultured on Vero cell monolayers. After 1 hour of incubation, the supernatants were discarded. Each well was covered with 0.6% RPMI-agarose medium and incubated for 2 to 3 days. The cells were fixed with 37% buffered formalin and stained with 2% crystal violet, and the plaques were enumerated.

Cytokine Content in the Cornea
The content of the cytokine TNF-{alpha} was determined in the homogenized corneas by a standard ELISA (BD PharMingen, San Diego, CA) according to the protocols recommended by the supplier. Briefly, ELISA plates were coated with 100 µL of the diluted capture antibody. After they were blocked with 10% fetal calf serum (FCS) in 200 µL, the specimens were incubated on the plates. After incubation with 100 µL of the diluted and biotinylated detector antibody, avidin-horseradish peroxidase and the substrate containing tetramethylbenzidine (Roche, Mannheim, Germany) dissolved in dimethylsulfoxide (Sigma-Aldrich) and hydrogen peroxide were added. The results were calculated after photometric measurement of optical density at 450 nm by comparison with recombinant cytokine standards.21

Delayed-Type Hypersensitivity Reaction
Delayed-type hypersensitivity (DTH) was measured with a standard method, as described elsewhere.25 In short, each mouse received 50 µL RPMI injected into the left footpad and 1 x 107 PFU HSV-1 in 50 µL RPMI into the right footpad on day 7 or 14. The swelling of the footpads was measured with a micrometer. The HSV-1–specific DTH was calculated as the difference between the swelling in the left and right footpads in millimeters.

Proliferation Assay with [3H]thymidine
Cells isolated from the spleens were cultured in 96-well plates for 3 days at a density of 1 x 105 cells per well. The cells were stimulated with UV-inactivated HSV (2 x 106 PFU before inactivation per well). Negative controls were challenged with medium only, and positive controls were stimulated with 0.5 µg ConA per well. After an incubation with 1 µCi of [3H]thymidine (Amersham Bioscience, Buckinghamshire, UK) per well for 24 hours, the cells were harvested and incorporated radioactivity was measured in a ß-plate reader (Top Count NTX; Packard Bioscience, Meriden, CT).21

Statistical Analysis
Student’s t-test was used to determine the significance of the differences in number of cells in the cornea, DTH, ELISA, neutralizing antibodies, plaque assay, and proliferation assays. Clinical findings were examined by means of the Fisher test. P < 0.05 was considered to indicate a significant difference between two groups.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Uptake of FITC-ON by Draining Lymph Node and Splenic Cells In Vitro
The cultured cells took up the FITC-ASON very quickly. Flow cytometric analysis of cells from the regional lymph nodes demonstrated that more than 67% of the examined cells were fluorescence positive after 15 minutes of incubation with FITC-ASON (Fig. 1) . The fluorescence was pronounced and easily detectable, even after 48 hours. These results were obtained in cells from both the regional lymph nodes and the spleen. These findings were confirmed by fluorescence microscopy, by which a maximum of fluorescence was observed between 8 and 12 hours of incubation.



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  		FIGURE 1. Flow cytometric analysis of cells derived from regional lymph nodes. Left trace: the negative control without FITC; broken trace: events after incubation solely with FITC (0.45% positive); and thick trace: events with FITC-labeled oligonucleotides (67.17% positive). Incubation time, 15 minutes; concentration, 2 µM.

 
TNF-{alpha} ASON-Induced Suppression of TNF-{alpha} Release from Draining Lymph Node and Splenic Cells In Vitro
After incubation with 5 to 20 µM ASON, the level of TNF-{alpha} produced by cells derived from regional lymph nodes was significantly lower than in the control. This pattern was independent of the nature of the antigen stimulus and did not differ between stimulation with UV-inactivated HSV and with ConA (Fig. 2) . Similar results were observed with splenic cells. In contrast, no significant suppression of the TNF-{alpha} protein levels were detected when CON was added to the cells.



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  		FIGURE 2. ELISA data from cell culture supernatants of cells prepared from regional lymph nodes of HSV-1–infected mice. Downregulation of TNF-{alpha} release after 24-hour incubation with ASON. Treatment with CON did not change the TNF-{alpha} content. ({square}) ASON-treated cells with Con A; ({blacksquare}) ASON-treated cells with UV-HSV; () CON-treated cells with ConA; and () CON-treated cells with UV-HSV. *P < 0.05, treated compared with untreated specimens (0 µM).

 
Subepithelial Injection of TNF-{alpha} ASON
Immediately after the subepithelial injection of ASON, CON, or buffer, the cornea became opaque and severely swollen (Fig. 3A) . A few hours after the injection, the cornea had a clinically normal appearance. This reaction was seen in all mice, regardless of what solution had been injected.



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  		FIGURE 3. (A) Clinical appearance of a swollen and opaque cornea immediately after subepithelial injection. (B) Paraffin-embedded section of an eye 24 hours after the injection of 2 µL FITC-ASON showing strong fluorescent staining. (C) Paraffin-embedded section of an eye 10 days after injection of 2 µL FITC-ASON showing fading fluorescence. (D) Autofluorescence of an unstained paraffin-embedded section from a normal mouse cornea. Magnification, x200.

 
After a single subepithelial injection of FITC-conjugated ASON, pronounced fluorescent staining of the entire epithelium and stroma was detected by fluorescence microscopy. Figure 3B shows typically pronounced fluorescent staining of a cornea 24 hours after the injection of 2 µL of FITC-ASON. The staining was particularly intense in the superficial stroma. In contrast, the fluorescent staining was weak after 24 hours and undetectable after 48 hours when FITC solution was injected. Even 10 days after FITC-ASON injection, fluorescence staining was still noted in the subepithelial corneal stroma (Fig. 3C) . In contrast, the autofluorescence of a normal mouse cornea is shown in Figure 3D .

Clinical Course of Keratitis after Topical Administration of TNF-{alpha} ASON
After corneal HSV-1 infection, epithelial keratitis developed within 1 day and healed within 5 days. Our observations showed that the course of the epithelial keratitis was not influenced by the ASON injection.

Severe necrotizing keratitis was detected in approximately 60% of the mice in the control group 14 days after corneal infection with HSV-1. On day 14 PI, stromal keratitis in animals treated with ASON was markedly milder than in the infection-only mice (P < 0.01). The severity of stromal keratitis in animals that had received the buffer was similar to that in the mice that had no further treatment at all after HSV infection (Fig. 4) . Compared with the mice that were infected with HSV and received no treatment, the mice in the CON-treated group had slightly milder stromal keratitis, but the difference was not statistically significant. Although 13 of 21 HSV-1–infected mice showed development of HSK, only 3 of 22 mice that were treated with ASON showed signs of HSK (Table 1) .



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  		FIGURE 4. Influence of TNF-{alpha} on stromal keratitis in BALB/c mice 14 days after corneal HSV-1 infection. Stromal keratitis was graded from 0 to 4+, depending on the corneal opacity with neovascularization, edema, and infiltration. Animals treated with CON had less severe keratitis than mice infected with HSV-1 only. Keratitis was nearly absent in mice with ASON treatment. **P < 0.01. Crossbar indicates the mean.

 

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  		TABLE 1. Incidence of Mice with Stromal Keratitis

 
The typical histologic appearance in the various experimental groups on day 14 PI supported and differentiated the clinical results in detail. The animals that had keratitis showed severe stromal edema, heavy epithelial and stromal inflammatory cell infiltration, profound vascularization, necrosis, and occasional ulceration 14 days after corneal HSV-1–infection (Fig. 5A) . Similar histopathological abnormalities of the corneas were found when buffer was injected into the cornea. Compared with these findings, the edema, necrosis, inflammatory cell infiltration and neovascularization were less severe in mice that had also received three injections of CON. In contrast to the other groups of mice, after three applications of ASON only three mice showed mild stromal edema and inflammatory cell infiltration (Fig. 5B) , and the other mice in that group had only very faint signs of inflammation and no tissue destruction.



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  		FIGURE 5. Typical histologic findings in BALB/c mice on day 14 after corneal HSV-1 infection. (A) HSV-1–infected mouse cornea with severe inflammatory cell infiltration and edema. Profound neovascularization. (B) Histology of an eye after HSV-1 infection and three ASON injections. Only few inflammatory cells and mild edema. Magnification, x200.

 
These results were substantiated by the number of infiltrating inflammatory cells. Although corneas of mice from the control groups showed numerous cells, these cells were nearly absent in the corneas of TNF-{alpha} ASON-treated mice (Fig. 6) .



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  		FIGURE 6. Number of inflammatory cells in the corneas in each treatment group, counted in a high-power field. (Magnification, x400). Data are the mean ± SEM, **P < 0.01.

 
Virus Replication in the Eye after Application of TNF-{alpha} ASON
The influence of ASON treatment on HSV-1 replication in the eye was investigated by plaque assay analysis. The findings indicate that HSV-1 was nearly cleared from the HSV-infected eyes within 7 days. After the subepithelial injections of ASON, CON, or buffer we did not detect any change in the virus clearance.

Change in Content of TNF-{alpha} in the Cornea after Application of ASON
To investigate whether the improvement of HSK after ASON treatment may be associated with a modulation of proinflammatory cytokines, the cytokine expression in the cornea was measured by an ELISA technique. The levels of TNF-{alpha} in the corneas were markedly diminished, by approximately 50%, after repeated subepithelial treatment with TNF-{alpha} ASON compared with the other groups of mice (Fig. 7) . With respect to the TNF-{alpha} levels, no significant differences were observed between the mice that underwent HSV-1 infection only and those that were treated with buffer or CON in addition.



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  		FIGURE 7. Influence of TNF-{alpha} ASON in BALB/c mice on day 14 after corneal HSV-1 infection. The corneal TNF-{alpha} content was significantly reduced after TNF-{alpha} ASON treatment compared with the only HSV-infected mice. Treatment with CON or buffer did not have any influence. *P < 0.05. Crossbar indicates the mean.

 
Effect of TNF-{alpha} ASON on the Systemic Immune Response against HSV-1
On day 7 PI, HSV-1–neutralizing antibodies were detected in the serum specimens. The titers measured on day 14 PI were increased. The different treatment regimens did not influence the neutralizing antibody titers (Fig. 8) .



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  		FIGURE 8. Influence of TNF-{alpha} ASON in BALB/c mice after corneal HSV-1 infection. Neutralizing antibody titers in the serum on days 7 and 14 PI. ASON, CON, or buffer had no influence. Data are the mean ± SEM.

 
Compared with the HSV-1–infected mice that received no further treatment, in those that received ASON by subepithelial injection the DTH on day 7 PI was slightly suppressed, but the difference did not reach the level of significance. On day 14 PI, the DTH response did not differ between the mice in the various groups (Fig. 9) .



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  		FIGURE 9. Influence of TNF-{alpha} ASON in BALB/c mice after corneal HSV-1 infection. HSV-1–specific DTH on day 14 PI. ASON, CON, or buffer had no influence. Data are the mean ± SEM.

 
The T cells separated from the spleen on day 14 after corneal HSV-1–infection proliferated when cocultured with HSV antigen or ConA. This response was not altered by the corneal injections of ASON and was not significantly different from that in the groups of mice that were treated with CON or buffer (Fig. 10) .



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  		FIGURE 10. Influence of TNF-{alpha} ASON in BALB/c mice on day 14 after corneal HSV-1 infection. Counts per minute measured as uptake of [3H]thymidine. In cells from the spleen, the various treatments did not influence the proliferation. Data are the mean ± SEM.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The observations recorded in this study indicate that the FITC-labeled ASON were taken up rapidly in vitro by cells taken from the regional lymph node and spleen and in vivo by cells in the cornea. The intracellular fluorescence remained stable for a minimum of 2 days in vitro and 10 days in vivo. It has been observed previously that activated lymphoid cells have an enhanced susceptibility to the uptake of ON.26 Therefore, additional uptake enhancers were not used in our study.

The in vitro results showed a specific downregulation of TNF-{alpha} protein after the administration of ASON but not CON. The clearly suppressed levels of TNF-{alpha} measured in cell culture supernatants by ELISA, even after nonspecific stimulation with ConA, point to the marked effect of ASON. Despite this, the downregulating effect was achieved only at the higher ASON concentrations used. For this reason we decided to inject the drug in vivo repeatedly. The secretion of TNF-{alpha} in the cornea was significantly impaired after ASON treatment given three times in vivo. No further effect was obtained by additional injections.

With respect to the technique for in vivo application of ASON into the cornea, the subepithelial injection method was more effective than the micropocket technique with sucralfate, which has been proposed as a depot for controlled delivery of drugs27 and which we used as another delivery mode for ASON. Subepithelial injection appeared to be a safe method for repeated, reliable, and atraumatic local placement of a precisely measurable amount of ON.

In contrast to the long-lasting fluorescence in the cornea caused by FITC-labeled ASON, unbound FITC was cleared from the corneas within 48 hours. This indicates that ASON were incorporated by cells in the cornea and remained within the cells. The antisense technique has been used to treat immune-mediated diseases,28 29 and for local application of antiviral medications into the eye. Specifically, fomivirsen (Vitravene; Isis Pharmaceuticals, Carlsbad, CA) was designed for intravitreal administration to treat CMV retinitis.30 31 To the best of our knowledge, we describe for the first time that ASON injection into the cornea can be used for modulating diseases of the anterior part of the eye. Administration by injection permits ASON to have access to all corneal layers.

The course of epithelial keratitis that occurs early after corneal contact with HSV-1 was not impaired by the topical blockade of TNF-{alpha}. The virus replication in the eye was not influenced, as supported by unchanged viral titers in plaque assays. This indicates that the mode of ASON treatment of the cornea is not primarily related to an influence on the replication of HSV-1 in the mouse cornea. There is profound evidence that HSV-induced stromal keratitis with onset late after HSV-1 infection is mediated by the immune response to the virus rather than the cytopathogenic effect of the virus. The observations reported herein show that corneal applications of ASON against TNF-{alpha} markedly reduced the incidence and severity of stromal keratitis in mice. This notion is in agreement with previous observations that TNF-{alpha} is upregulated in the corneas of mice with herpetic keratitis and that anti-TNF-{alpha} antibody treatment abrogates the disease.11

It has been reported that phosphorothioate ON may provoke some nonspecific inflammatory reactions, regardless of their base composition. Our preliminary experiments showed that HSK was not significantly altered and that the secretion of the proinflammatory cytokine TNF-{alpha} in the cornea was not influenced by the intracorneal injection of a randomly designed ON.

It has recently been demonstrated that HSK is minimized or abrogated by treatment with IL-10 protein or naked plasmid DNA.32 33 The improvement of HSK noted after three subepithelial injections of ASON TNF-{alpha} was associated with an elevated content of IL-10 in the corneas (Wasmuth S, et al., unpublished observation, 2002). Therefore, the increased secretion of IL-10 may play a part in amelioration after treatment with ASON.

The corneal treatment with ASON targeting TNF-{alpha} in our experiments did not affect the DTH response. In addition, the injections were not associated with a change in the serum titers of neutralizing antibodies. Taken together, these results suggest that treatment of the cornea with ASON antagonizing TNF-{alpha} may not modulate HSK primarily by affecting the systemic immune response.

There are several other possible mechanisms by which TNF-{alpha} ASON treatment of the cornea may achieve protection against the development of HSK. TNF-{alpha} is known to be involved in leukocyte migration into tissue.34 This cytokine is also known to induce surface expression of ICAM-1.35 Exogenous TNF-{alpha} enhances the release of MIP-1{alpha} in cultured macrophages,36 which, in turn, is critical for the development of HSK.37 TNF-{alpha} also stimulates the production of matrix metalloproteinases by corneal cells,38 which have been shown to be upregulated during the development of ulcerative necrotizing HSV keratitis.39 In addition, TNF-{alpha} also regulates the migration of Langerhans’ cells in the cornea,40 which are crucial for the development of HSK41 and the induction of DTH. Because of the local application of ASON, only the direct microenvironment seems to be affected. Therefore, the influence on the systemic immune response is only slight.

In summary, the results demonstrate that the antisense technique is a useful and safe approach that can be used to reduce the severity of HSK, to achieve a topical antagonism of the proinflammatory cytokine, TNF-{alpha}, and to modify immune-mediated diseases. However, further studies are needed to define the mode of action in more detail.


   Footnotes
 
Supported by Grant He 1877/12-1 from the Deutsche Forschungsgemeinschaft and the Ernst und Berta Grimmke-Stiftung.

Submitted for publication March 26, 2003; revised August 4, 2003; accepted August 5, 2003.

Disclosure: S. Wasmuth, None; D. Bauer, None; Y. Yang, None; K.-P. Steuhl, None; A. Heiligenhaus, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Arnd Heiligenhaus, Ophtha-Lab, Department of Ophthalmology, St. Franziskus Hospital, Hohenzollernring 74, 48145 Münster, Germany; arnd.heiligenhaus{at}t-online.de


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Streilein JW, Dana MR, Ksander BR. Immunity causing blindness: five different paths to herpes stromal keratitis. Immunol Today. 1997;18:443–449.[CrossRef][Medline][Order article via Infotrieve]
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