Published online before print July 16, 2004
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* Comparative and Experimental Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville; and
Department of Cellular Biology, Medical College of Georgia, Augusta
1Correspondence: M409 Walters Life Sciences, 1414 Cumberland Avenue, Department of Microbiology, University of Tennessee, Knoxville, TN 37996. E-mail: btr{at}utk.edu
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Key Words: HSV-1 • cornea • angiogenesis • cytokine • chemokine
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To test our hypothesis that IL-1 represents one of the first mediators to initiate the inflammatory cascade, we blocked its activity using the IL-1 receptor antagonist protein (IL-1 ra), which is a naturally occurring isoform of IL-1 and can bind to IL-1 receptors with high affinity but fails to induce signal transduction [12 ]. The protein is routinely used in treatment of human immunoinflammatory conditions such as rheumatoid arthritis [13 ]. IL-1 ra protein administered systemically down-regulates cytokine production in humans and rats [14 15 16 ]. In the ocular alkali burn murine model, IL-1 ra has been shown to have a therapeutic potential [16 ]. We show, using a combination of systemic and local IL-1 ra administration, that the severity of corneal angiogenesis and HSK is diminished following ocular HSV infection. This was a consequence of a down-regulation of various inflammatory mediators, normally induced by IL-1 in HSV-infected corneas. Thus, blocking IL-1 activity using IL-1 ra protein could represent a valuable, therapeutic approach in managing HSK.
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Virus
HSV-1 RE (obtained from Dr. Robert Hendricks Laboratory, University of Pittsburgh School of Medicine, PA) was used in the present study. The virus was propagated and titrated on a monolayer of Vero cells (American Type Culture Collection, Manassas, VA, Cat. No. CCL81) using standard protocols [17
]. Infected Vero cells were harvested, titrated, and stored in aliquots at –80°C until used.
Corneal HSV-1 infection
Mice were ocularly infected with HSV-1 RE under deep anesthesia induced by intraperitoneal injection of Avertin (Sigma-Aldrich, St. Louis, MO). Mice were lightly scarified on their corneas with a 27-gauge needle, and a 4-µl drop containing the required dose of virus was applied to the eye and gently massaged with the eyelids.
Clinical observations and angiogenesis scoring
The eyes were examined on different days postinfection (p.i.) by a slit-lamp biomicroscope (Kowa Co., Nagoya, Japan), and the clinical severity of keratitis of individually scored mice was recorded as described previously [11
]. Briefly, the clinical lesion score of HSK was described as 0, normal cornea; 1, mild haze; 2, moderate haze, iris visible; 3, severe haze, iris not visible; 4, severe haze and corneal ulcer; and 5, corneal rupture. Angiogenesis severity was measured as described previously [18
]. To quantify the degree of neovessel formation, two primary parameters were used: the circumferential extent of neovessels (as the angiogenic response is not uniformly circumferential in all cases) and the centripetal growth of the longest vessels in each quadrant of the circle. The longest neovessel in each quadrant was identified and graded between 0 (no neovessel) and 4 (neovessel in the corneal center) in increments of 
0.4 mm (radius of the cornea is 1.5 mm). According to this system, a grade of 4 for a given quadrant of the circle represents a centripetal growth of 1.5 mm toward the corneal center. The score of the four quadrants of the eye was then summed to derive the neovessel index (range, 0–16) for each eye at a given time-point.
Subconjunctival (S/c) inoculations
S/c inoculation of recombinant human (rh)IL-1 ra protein (0.5 mg/cornea, 1 mg/cornea; supplied by Amgen Inc., Thousand Oaks, CA) was performed as described previously [8
]. Briefly, S/c inoculations were done using a 2-cm, 32-gauge needle and syringe (Hamilton, Reno, NV) to penetrate the perivascular region of conjunctiva, and the required dose of IL-1 ra protein was delivered into the S/c space. Control mice received 0.2% sodium hyaluronate in phosphate-buffered saline (PBS) only. Mice received rhIL-1 ra 1 day before corneal infection with HSV-1.
Infusion pump implantation
Alzet (Palo Alto, CA) osmotic pumps were used to deliver IL-1 ra subcutaneously (s.c.) as described before [19
]. Briefly, the micro-osmotic pumps (Model 1007D) were implanted 24 h before ocular infection. The pumps were loaded with IL-1 ra or vehicle as per the manufacturer’s instructions under sterile condition. The mice were weighed, marked, and anaesthetized using Isoflurane (Abbott Laboratories, North Chicago, IL). Mice were placed into dorsal recumbancy, clipped over the shoulder area, and prepared for the surgery. A small, 1-cm incision was made caudal to the shoulder area, the skin was undermined with a hemostat, and then, the pump was placed into the prepared pocket. Skin was then closed with 9 mm autoclips (Braintree Scientific Inc., Braintree, MA). The pump was taken out after 7 days, and the skin was closed again with 9 mm clips. With a mean pumping rate of 0.5 µl/h (mean fill vol of 100 µl), calculated drug infusion rates were 5 mg/kg body weight (b.wt)/h for 7 days, 2.5 mg/kg b.wt/h for 7 days, and 1 mg/kg b.wt/h for 7 days. Control mice received osmotic pumps with vehicle only for the same period of time.
Histopatholgy
For histopathological analysis, eyes were extirpated at day 20 p.i. and fixed in 10% buffered neutral formalin. Staining was performed with hematoxylin and eosin (H&E; Richard Allen Scientific, Kalamazoo, MI).
Immunofluorescence staining
For immunofluorescence staining, at day 20 p.i., eyes were frozen in optimum cutting temperature (OCT) compound (Miles, Elkart, IN). Sections (6 µ-thick) were cut, air dried, and fixed in acetone for 20 min at 4°C. The sections were blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich) containing unconjugated anti-CD16/32 (1:200) antibody (BD PharMingen, San Diego, CA) for 3 h at 37°C. For detection of CD4+ T cells, the sections were incubated with fluorescein isothiocyanate (FITC)-labeled anti-mouse CD4+ antibody (clone RM4-5, BD PharMingen) in 1% BSA at 4°C overnight. Sections were repeatedly washed in PBS and mounted with Vectashield mounting medium for fluorescence with propidium iodide (Vector Laboratories, Burlingame, CA) and visualized under a microscope (Leica, Wetzlar, Germany).
Flow cytometry
Single-cell suspensions were prepared from four corneas at different days p.i., as described elsewhere [20
] with some modifications. Briefly, corneal buttons were incubated with collagenase D (Roche, Mannheim, Germany) for 60 min at 37°C in a humidified atmosphere at 5% CO2. After incubation, corneas were disrupted by grinding with a syringe plunger and passing through a cell strainer. Cells were washed and suspended in RPMI 1640 with 10% fetal bovine serum. Blocking was done with unconjugated anti-CD16/32 (BD PharMingen) for 30 min. Samples were incubated with FITC-labeled anti-Gr-1 antibody (clone RB6-8C5, BD PharMingen), FITC-labeled anti-CD4 antibody (clone RM4-5, BD PharMingen), and isotype controls for 30 min. All samples were collected on a FACScan (BD Biosciences, San Diego, CA), and data were analyzed using CellQuest 3.1 software (BD Biosciences).
Cytokine enzyme-linked immunosorbent assay (ELISA) of corneal lysate
For preparation of corneal lysates, six corneas per time-point were pooled and minced. All procedures were done on an ice bath. Minced pieces were collected in 1 ml Dulbecco’s modified Eagle’s medium without fetal calf serum and homogenized using a tissue homogenizer (PRO Scientific Inc., Monroe, CT) four times, 15 s each, with a gap of 1 min between homogenization to allow the sample to cool. The lysate was then clarified by centrifugation at 14,000 rpm for 5 min at 4°C. The supernatant was collected and used immediately or stored at –80°C until further use. Lysates were analyzed using a standard sandwich ELISA protocol. Anti-IL-6 capture and biotinylated detection antibodies were from BD PharMingen (clone MP5-20F3), and standard recombinant murine (rm)IL-6 was from R&D Systems (Minneapolis, MN). Antimacrophage-inflammatory protein-2 (anti-MIP-2), antivascular endothelial growth factor (anti-VEGF)164 capture, biotinylated detection antibodies, and recombinant standards for mMIP-2 and mVEGF164 were from R&D Systems. For detection of rhIL-1 ra, anti-human IL-1 ra capture, detection antibody, and rhIL-1 ra were obtained from R&D Systems. The color reaction was developed using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (Sigma-Aldrich) and measured with an ELISA reader (Spectramax 340, Molecular Devices, Sunnyvale, CA) at 405 nm. Quantification was performed with Spectramax ELISA reader software version 1.2.
Statistical analysis
Unless specified, a one-tailed, paired Student’s t-test has been used.
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Figure 1. Following S/c injection, the presence of IL-1 ra was detectable in the cornea for a short period of time. At indicated time-points, six corneas/group were processed for measuring the hrIL-1 ra protein levels. The level of hrIL-1 ra was estimated from supernatants of corneal lysates of mice infected with 5 x 106 plaque-forming units (pfu) HSV-1 RE by an antibody capture ELISA, as outlined in Materials and Methods. Results are expressed as mean ± SD of three separate experiments (six corneas/time-point).
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Figure 2. No difference in HSK and angiogenesis score between locally administered IL-1 ra-treated mice and vehicle-control mice. (A) IL-1 ra protein was administered S/c 24 h before corneal infection. Mean lesion HSK score at day 20 p.i. of mice infected with 5 x 106 pfu HSV-1 RE. Each dot represents the HSK score from one eye. Horizontal bars and figures in the parentheses indicate the mean ± SD for each group. Data are compiled from two separate experiments consisting of eight eyes in each group. *, Statistically significant differences in mean HSK score (P<0.05) were observed between IL-1 ra Tg mice to vehicle control and IL-1 ra-treated eyes. (B) Mice were infected with 5 x 106 pfu HSV-1 RE. Mice were terminated at day 20 p.i., and eyes were processed for paraffin embedding. H&E staining was carried out on 6 µ sections. Original magnification, x200. (C) Angiogenesis scores for individual eyes of IL-1 ra-treated, vehicle control, and IL-1 ra Tg mice infected with 5 x 106 pfu HSV-1 RE at day 20 p.i. Horizontal bars and figures show the mean for each group. Data are compiled from two separate experiments consisting of eight eyes per group. *, Statistically significant differences in angiogenesis score (P<0.05) were observed between IL-1 Ra Tg mice to vehicle control and IL-1 ra-treated eyes.
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A combination of systemic and S/c IL-1 ra administration diminishes HSK severity and corneal angiogenesis
A reason for the inability of S/c administration in modulating HSK severity may be attributed to the short persistence of IL-1 ra in the cornea. To achieve biologically active concentrations for a sustained period in the corneal microenvironment, different doses of IL-1 ra recombinant protein were administered systemically via s.c. osmotic-pump implantation for 7 days. In addition, mice received a S/c injection (1 mg/cornea; the highest concentration that could be achieved by S/c injection). With this approach, the IL-1 ra protein was detectable in the cornea by ELISA until day 7 postinjection (last time-point analyzed; Fig. 3
). In addition, serum levels of the protein in treated mice (768±163 pg/ml) were significantly higher than vehicle-treated animals (54±37 pg/ml).
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Figure 3. hrIL-1 ra protein was detected in mice receiving a combination of systemic and local administration until day 7 postinjection. At indicated time-points, six corneas/group were processed for measuring the hrIL-1 ra protein levels. Level of hrIL-1 ra was estimated from supernatants of corneal lysates of mice infected with 5 x 106 pfu HSV-1 RE by an antibody capture ELISA as outlined in Materials and Methods. Results are expressed as mean ± SD of three separate experiments (six corneas/time-point).
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Figure 4. Mice receiving IL-1 ra systemically in conjunction with local administration demonstrated reduced HSK severity. (A) IL-1 ra protein was administered systemically along with S/c injection 24 h before corneal infection, as described in Materials and Methods. Mean lesion HSK score at day 20 p.i. of mice infected with 5 x 106 pfu HSV-1 RE. Each dot represents the HSK score from one eye. Horizontal bars and figures in the parentheses indicate the mean ± SD for each group. Data are compiled from two separate experiments consisting of eight eyes in each group. *, **, Statistically significant differences in mean HSK score (P<0.05) were observed between mice treated with 5 mg/kg b.wt/h for 7 days S/c + 1 mg/cornea local (group 1) or 2.5 mg/kg b.wt/h for 7 days S/c + 1 mg/cornea local IL-1 ra and vehicle control. (B) Percentage severity of HSK score at day 20 p.i. of mice infected with 5 x 106 pfu HSV-1 RE. Each vertical bar represents number of eyes showing clinically evident lesions (HSK score 3). Data are compiled from two separate experiments consisting of eight eyes in each group. (C) Mice were infected with 5 x 106 pfu HSV-1 RE. Mice were terminated at day 20 p.i., and eyes were processed for paraffin embedding. H&E staining was carried out on 6 µ sections. Original magnification, x200.
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Figure 5. Mice receiving a combination of systemic and local administration of IL-1 ra protein showed diminished angiogenesis following ocular infection. (A) IL-1 ra protein was administered systemically along with S/c injection 24 h before corneal infection, as described in Materials and Methods. Kinetics of corneal neovascularization process in mice infected with 5 x 106 pfu HSV-1 RE. Angiogenesis scores were recorded as mentioned in Materials and Methods at indicated time-points. Data are compiled from two separate experiments involving eight eyes per group and are expressed as mean ± SD. *, Statistically significant differences in angiogenesis score (P<0.05) were observed between mice treated with 5 mg/kg b.wt/h for 7 days S/c + 1 mg/cornea local or 2.5 mg/kg b.wt/h for 7 days S/c + 1 mg/cornea local IL-1 ra and vehicle control. (B) Angiogenesis scores for individual eyes of IL-1 ra-treated, vehicle-control, and IL-1 ra Tg mice infected with 5 x 106 pfu HSV-1 RE at day 20 p.i. Horizontal bars and figures show the mean for each group. Data are compiled from two separate experiments. (C) At day 20 p.i., extensive growth of blood vessels can be seen in IL-1 ra-treated and vehicle-control mice infected with 5 x 106 pfu HSV-1 RE. IL-1 ra-treated mice show minimum angiogenic sprouts near the limbal ring. In comparison, vehicle-control eyes demonstrated extensive growth of blood vessels with the same dose.
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Figure 6. Presence of abundant Gr-1 + ve cells in the cornea of vehicle-control mice but not in IL-1 ra-treated mice at 48 h p.i. Single-cell suspensions of corneal cells were prepared from four eyes at 48 h p.i. The cells were counted and stained with FITC-labeled anti Gr-1 antibody, and numbers of Gr-1-positive cells were enumerated by fluorescein-activated cell sorter (FACS). Dot-plot is representative of one of three separate experiments. The number on the upper-right corner represents the percentage of Gr-1+ cells of total corneal cells at 48 h p.i.
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Figure 7. Diminished CD4+ T cell influx in mice treated with IL-1 ra protein. (A) Single-cell suspensions of corneal cells were prepared from four eyes at day 20 p.i. The cells were counted and stained with FITC-labeled anti-CD4+ antibody, and numbers of positive cells were enumerated by FACS. Isotype control was used as negative control. Dot-plot is the representative of one of three separate experiments. Numbers on the upper-right corner represents the percentage of CD4+ T cells of total gated lymphocytes at day 20 p.i. (B) At day 20 p.i., representative eyes were taken and snap-frozen in OCT compound, and 6 µ sections were cut. The sections were stained for CD4+ T cells using a FITC-labeled anti-CD4+ antibody as described in Materials and Methods. Corneas were mounted with Vectashield mounting medium for fluorescence with propidium iodide and visualized under a microscope. The white arrows indicate the CD4+ T cells in the corneas.
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or IL-1ß (data not shown).
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Figure 8. IL-1 ra-treated mice demonstrated reduced protein levels of IL-6, MIP-2, and VEGF. (A–C) At indicated time-points, six corneas/group were processed for measuring the IL-6, MIP-2, and VEGF protein levels. Levels of IL-6, MIP-2, and VEGF were estimated from supernatants of corneal lysates of mice infected with 5 x 106 pfu HSV-1 RE by an antibody capture ELISA, as outlined in Materials and Methods. Results are expressed as mean ± SD of three separate experiments (six corneas/time-point). *, Statistically significant differences in IL-6, MIP-2, and VEGF level (P<0.05) were observed between IL-1 ra-treated mice and vehicle-control mice.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Molecular and cellular events involved in HSK pathogenesis were down-regulated as a result of the IL-1 ra treatment. Blocking the IL-1 activity resulted in a block to the induction of downstream events and thereby altered the environment normally conducive for the migration of inflammatory cells. IL-1, produced from HSV-infected cells [22 ], helps in the induction of other key cytokines such as IL-6 [11 , 23 ], also shown to be critical in the pathogenesis following ocular HSV infection [8 , 9 ]. Linked with this process is also the production of chemokines such as IL-8 and MIP-2 [9 , 16 , 24 ], thought to be involved in the margination and extravasation of neutrophils at the corneal limbus. These events occur promptly following infection of the corneal epithelium and facilitating neutrophil influx [5 ]. In addition, an autocrine IL-1-feedback loop is possibly involved in tissue remodeling in the cornea through the induction of IL-8 [16 ]. Interfering with these loops, therefore, can alter the overall response following HSV-1 infection and can prevent tissue injury.
In the HSV-infected mouse, cornea peak numbers of neutrophils are attained within 2 days after infection [5 ]. As a result of lack of chemotactic signals resulting from the disruption of the normal IL-1 signaling, neutrophils failed to attain peak numbers. These results are consistent with previous findings [8 , 9 ] including the most recent study conducted with IL-1 ra Tg mice [11 ]. It is clearly evident that neutrophils are the critical, early cellular mediators of HSK pathogenesis, being involved in viral clearance [5 , 6 ] and possibly tissue damage through the release of factors such as nitric oxide [25 ] or remodeling factors such as matrix metalloproteinases (MMPs) [26 ]. In addition, neutrophils have been considered as a source of molecules, facilitating angiogenesis or angiogenic factors themselves such as MMP-9 [26 ] and VEGF [27 ], respectively, and thereby, contribute significantly to the neovascularization process.
Many other cell types can be a potential source of angiogenic factors in the cornea. These include the stromal fibroblast themselves, epithelial cells, and inflammatory cells such as macrophages [21 ]. Possibly with disease progression, there is a shift in the source of angiogenic factors. In addition, apart from VEGF [21 ], other angiogenic factors, such as basic fibroblast growth factor (our unpublished results), or C-X-C chemokines, such as MIP-2 [28 ], may be involved in HSV-induced corneal angiogenesis. The possibilities are innumerable and presently unclear. Whatever the scenario, our results indicate the effectiveness of IL-1 ra in blocking the overall angiogenesis process. Similar results have been obtained in other murine models of corneal angiogenesis [18 , 29 ].
The chronic inflammatory environment is maintained by the angiogenic response, allowing further delivery of inflammatory cells. The development of HSK lesions is dependent on this response, as shown in studies that have targeted the corneal neovascular response early after virus infection [7 ]. One cell type crucial to HSK pathogenesis and presumably delivered by newly formed (and therefore leaky) blood vessels is the CD4+ T cell [3 ]. Thus, mice lacking CD4+ T cells but possessing other components normally required in pathogenesis fail to develop HSK [3 , 4 ]. Consistent with these reports, the lack of blood vessel development in the cornea of IL-1 ra-treated mice prevented the influx of CD4+ T cells. Thus, part of the anti-inflammatory effects observed with IL-1 ra therapy also involved the impairment in the CD4+ T cell response. Hence, IL-1 ra treatment targeted at the early, preclinical events also influenced subsequent clinical episodes in HSK pathogenesis.
Finally and most importantly, results indicate that the activity of IL-1 needs to be kept under continuous check to achieve significant results. The levels of IL-1 in the cornea after a HSV infection remains elevated until approximately day 9 p.i. [10 ]. Thus, the protocol aimed at abrogating IL-1 activity for an extended period of time produced the best results. Thus, our results imply that neutralizing the inflammatory milieu at an early time-point may prove beneficial in reducing the severity of HSK. This could be achieved by blocking IL-1 using IL-1 ra protein. Antagonizing the proinflammatory environment abrogates the cascade of events that culminates in HSK. Thus, targeting IL-1 ra protein proved to be a worthwhile, therapeutic approach for successful management of HSK, a blinding, immunoinflammatory lesion of humans.
Received May 7, 2004; revised June 9, 2004; accepted June 13, 2004.
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