CD18,P,E-Selectins, Chemokines, Leukocytes Migration And Corneal Epithelial Wound Healing

Normal vision requires corneas to be transparent. There are all kinds of corneal epithelial wounds in clinics, sometimes causing severe sight damage,especially those caused by alkali burns.Leukocytes migrate into corneas after hours post-wound, and disappear after wound healing. One of important effects of leukocytes is to eliminate dead cells and pathogens, at the same time,they can play important roles in wound healing. Leukocyte migration from blood to tissues is a key step in inflammation. Leukocyte should be able to recognize the inflammational position, and contact with vascular endothelia, then migrate through blood vessels. Selectins, chemokines, and integins are key molecules in this process. CD18 is the mostimportant part of 62 integrin.It is necessary to understand the mechanism of corneal epithelial wound healing for us to find the best way to treat corneal epithelial wounds ,especially those caused by alkali burn.Part Ⅰ CD18 independent migration of neutrophils following corneal epithelial injuryPurposeTo study the relationship between CD18 and neutrophil migration after corneal epithelial wound,and its impacts on corneal epithelial wound healing.Materials and Methods 1. AnimalsWe use CD18 gene knockout mice as test group and C57BL/6 as control. All mice used in this study were 6 to 8 weeks old, weighed 18 to 20 g, and were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, institutional and federal guidelines.2. Wound modelMice were anesthetized with intraperitoneal injection of Nembutal sodium solution. The central corneal epithelium (2 mm diameter) was demarcated with a trephine and then was removed using a diamond blade for refractive surgery under a dissecting microscope. Care was taken not to injury the epithelial basement membrane and stroma. Each of the injured corneas at each time were stained with fluorescein and photographed with a digital camera every 6 h, beginning immediately after wounding to wound closure, to evaluate the re-epithelialization. The size of the remaining epithelial defects was measured and quantified using the Optimus 6.2 Software.3. Wholemount ImmunohistologyWounded corneas with limbus were removed, fixed, permeabilized, and incubated with fluorescein-conjugated monoclonal antibodies . FITC-conjugated Gr-1 antibody (PharMingen) were selected for neutrophils. Radial cuts were made in the cornea so that it could be flattened by a coverslip, and it was mounted in Airvol containing 1 juM 4',6-diamidino-2-phenylindole to assess nuclear morphology and cell division. Digital images were captured and saved into the computer for analysis. To compare the relative level of neutrophils in the different areas, including limbal, peripheral, paracentral and central fields for eachcornea were assessed and counted separately. At least four corneas were examined for immunohistology, and four quadrants were analyzed for each cornea to obtain the average numbers for each field. The limbus was defined as the intervening zone between the cornea and conjunctiva as the most peripheral field.4. Statistical AnalysisData analysis was preformed using t-test. Data are expressed as mean ± SEM.Results1. A biphasic neutrophil infiltration after corneal epithelial injuriesNeutrophil infiltration was measured by counting the number of GR-1-positive cells in the different areas from limbus to limbus through the central wound area and at various time points postinjury. In corneas collected from WT mice and CD18 KO mice without wounding, neutrophils were very rare and only found some in the limbal region. The neutrophils began to migrate from limbus to wound area 2 hour, reached to the wound leading edge at 6 hour after epithelial wounding and wound center at 18 hour in the WT mice.A different pattern of neutrophil migration was noted in the CD 18 KO mice. The biphasic wave of neutrophil infiltration took place in WT mice was not seen in CD 18 KO mice. Only a few neutrophils existed during first wave period, and only had a higher second wave of neutrophil infiltration. Of interest, however, numbers of infiltrated cells in the second wave is over numbers of infiltrated cells in the second wave of WT mice. These results indicated that corneal epithelial injury induced a biphasic neutrophil infiltration. The first wave might be CD18-dependent, the second wave CD18-independent.2. The epithelial basal cell density and dividing cellsThe epithelial basal cell density was assessed across the diameter of the cornea. By 24 h, epithelial basal cells had almost covered all wounded area in control corneas. This was contrasted to CD18-/- null mice where some wounded area was still devoid of epithelial cells. By 96 h after wounding, the density in the previously wounded area in control mice was almost the same as the peripheral density. In contrast, the basal cell density was significantly lower in the central and paracentral fields of corneas from CD18-/- null mice. Thus the migration aspects of corneal epithelial wound healing were significantly inhibited and delayed at both early and late stages in CD18-/- null mice. These results indicated that alternated neutrophil migration to the wound can change the corneal epithelialization during wound healing.To evaluate the proliferative phase of wound healing, the number of dividing cells in the corneal basal cell layer was determined. Dividing cells were mainly located in the limbal and peripheral regions in both groups during wound healing as we and others described before. Like neutrophil infiltration after wounding in WT mice, there is also a similar biphasic response for basal cell proliferation during wound healing in WT mice. The first wave for dividing cell took place 18 hours, second wave 30 hours after wounding. Of interest, however, there is similar pattern for the dividing cells of CD 18 null mice although CD 18 null mice are deficient for the first wave of neutrophil migration to the wounds. This indicated that deficient neutrophil migration of first wave in the CD 18 null mice did not alternate the biphasic pattern of corneal epithelial proliferation. However, the total number of dividing cells in the CD18 null mice is significantly lower one of the WT mice at 18, 24, 30 hour after wounding. But the numbers is significantly higher than that of WT mice at late stage, 42 and 48 hour after wounding. Further analysis to different regions show that in the first wave of WT mice dividing cells mainly exist in limbal and paralimbal areas and in the second wave in paralimbal area. But in the CD 18 null mice, both of waves located in paralimbal area. These results indicated that deficient migration of neutrophil to the wound decreased the proliferative ability of cornealepithelium and altered the distribution pattern of dividing cells after wounding.3. Delayed Epithelialization during Wound Healing in CD18 Null Mice.To elucidate the role of CD 18 in the wound healing process, we made 2 mm round standard corneal epithelial wound in the central area in WT and CD 18-/- mice, and the wound healing process was monitored with fluorescein staining every 6 hours. Wound areas were analyzed by image analysis. A significant difference between WT and CD18 null mice was observed in the gross appearance of the wounds at 12, 18, and 24 hours. Re-epithelization was typically complete in controls by 24 hours but not until 36 hours in CD 18 null mice. These results indicate that wound healing is delayed in CD18-/- null mice.ConclusionIn summary, the observations made in this study regarding the inflammatory responses associated with corneal epithelial wound healing support our hypothesis that corneal epithelial wound healing involves complicated inflammatory mechanisms: (1) corneal epithelial wound is associated with biphasic response for neutrophil influx into wounded corneas. First wave is CD18-dependent; the second wave isCD18-independent. (2) The function of first wave might be responsible for the clearance of bacteria and wound debris and provide some growth factors that support epithelial migration and proliferation. The second wave might be responsible for remodeling and reorganization of wounded corneas. Abnormality of any one of two waves will delay the wound healing process.Further investigations focusing on different contribution of biphasic inflammation to normal wound healing might provide insight into the mechanisms and management of corneal wound healing.Part II Selectins, chemokines, leukocytes migration and corneal epithelial wound healingPurposeTo investigate the roles of selectins, leukocytes, chemokines and cytokines in the corneal epithelial wound healing.Methods1. AnimalsAll mice used in this study were 6-8 weeks old, weighed 22-26g, and were treated according to the Association for Research in Vision andOphthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, institutional and federal guidelines. P-selectin gene knockout mice and PE-selectins double knockout mice were purchased from the Jackson Laboratory, and C57BL/6 mice were purchased from the Harlan Company. All animals were housed in the same rooms at the barrier.2.Corneal epithelial wound modelMice were anesthetized with intraperitoneal injection of Nembutal. Central circular (2mm diameter) corneal epithelial wounds were made through the whole epithelium with a trephine. The epithelia within the leision were removed with a diamond blade for refractive surgery under a dissecting microscope , with the basement membrane intact. Care was taken not to injure the corneal stroma.3.Quantification of reepithelialization ratesAt different timepoints after wounding , the injured corneas were stained with fluorescein and photographed with a digital camera. The pictures were input into a computer and analyzed with the software of Microsoft Photo Editor.4.Whole-mount immunohistology for the corneasAt different timepoints after wounding , corneas were excised and then fixed in 2% paraformaldehyde-PBS for 30 minutes, washed with PBS for 5 minutes each time, totally three times, then blocked with 1% BSA in PBS for 20 minutes, then permeabilized with 0.1% Triton X-100 in PBS for 20 minutes, and then incubated with different diluted FITC or phyco-erythrin-conjugated monoclonal antibodies to label different leukocytes.5.ELISA for chemokines and cytokinesAt different timepoints after wounding , corneas were harvested and homogenized in sterile PBS by ultrasonic and three frozen-thaw cycle. Plate Preparation1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein Immediately coat a 96-well microplate6 with 100 mL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature .2. Aspirate each well and wash with Wash Buffer repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 mL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate andblotting it against clean paper towels.3. Block plates by adding 300 mL of Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.Assay Procedure1. Add 100 mL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.2. Repeat the aspiration/wash as in step 2 of Plate Preparation.3. Add 100 mL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.4. Repeat the aspiration/wash as in step 2 of Plate Preparation.5. Add 100 mL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.6. Repeat the aspiration/wash as in step 2.7. Add 100 mL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.8. Add 50 mL of Stop Solution to each well. Gently tap the plate toensure thorough mixing.9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.Statistical analysisData analysis was performed using repeated measures ANOVA with pairwise multiple comparisons (Student-Newman-Keuls). A P value of <0.05 was considered significant. Data are expressed as mean+SEM.Resultsl.Corneal epithelial wound healingCorneal epithelial wound healing was completed within 24 hours after wounding in wildtype mice; Corneal epithelial wound healing was not completed until 48 hours after wounding in P-selectin gene knockout mice;The corneal epithelial wounds were still open at 48 hours after wounding in P,E-selectin double knockout mice.2.The impacts of selectin deficiency on leukocytes migration into wounded corneasAfter wounding, the neutrophil migration in wildtype mice increased rapidly, arrived at a peak during 12-18h, but the neutrophil migration in P-,PE-selectin gene knockout mice didn't arrived at its peak until 24h. Moreover, at most time points after wounding, the comparison of the number of neutrophils in corneas is: wildtype mice>P-selectin gene knockout mice> PE-selectin gene knockout mice.(p<0.05)After wounding, the macrophage migration in wildtype mice arrived at a little peak at 6h, then got two large peak at 24h and 42h separately, but the macrophage migration in P-,PE-selectin gene knockout mice didn't arrived at its peak until 18h. Moreover, at most time points after wounding, the comparison of the number of macrophages in corneas is: wildtype mice>P-selectin gene knockout mice> PE-selectin gene knockout mice.(p<0.05)After wounding, the dendritic cell migration in wildtype mice arrived at a peak at 18h, but the dendritic cell migration in P-,PE-selectin gene knockout mice didn't arrived at its peak until 24h. Moreover, at most time points after wounding, the comparison of the number of dendritic cells in corneas is: wildtype mice>P-selectin gene knockout mice> PE-selectin gene knockout mice.(p<0.05)After wounding, the Y 6 T cell migration in wildtype mice andP-selectin gene knockout mice started a peak at 6h, but the Y 5 T cell migration in PE-selectin gene knockout mice didn't arrived at its peak until 18h. Moreover, at most time points after wounding, the comparison of the number of Y 8 T cells in corneas is: wildtype mice>P-selectin gene knockout mice> PE-selectin gene knockout mice.(p<0.05)After wounding, the a PT cell migration in wildtype mice and P-selectin gene knockout mice arrived at a peak at 24h, but the a PT cell migration in PE-selectin gene knockout mice didn't arrived at its peak until 30h. Moreover, at most time points after wounding, the comparison of the number of a PT cells in corneas is: wildtype mice>P-selectin gene knockout mice> PE-selectin gene knockout mice.(p<0.05)3.The roles of chemokines in leukocytes migrationThe role of KC in neutrophil migration: In wildtype mice, KC got its first peak at 12h, accordingly the first peak of neutriphils was at 12-18h. Then KC and neutrophils got their second peaks at 24h and 36h separately. In P-selectin gene knockout mice, KC got its first peak at 12h, the first peak of neutriphils was at 24h. During the increase of neutrophils, the decrease of KC was reduced, then neutrophils increased again at 30h and got another peak at 36h.At the same time,KC increased again at 36h, and neutrophils increased accordingly at 42h. In PE-selectin geneknockout mice, KC got its peak at 18h, accordingly neutrophils got a peak during 18-30h which made KC increase again and get its second peak at 36h, then neutrophils also got the second peak at 42h.The role of JE in macrophage migration: In wildtype mice, JE started to increase at 6h and got a peak at 12h, accordingly macrophage started to increase at 6h, maintained a platform till 18h, then increased rapidly and got the first peak at 24h. During this period,JE stopped decreasing and maintained a platform till 24h. At 30h, macrophage increased again and got its second peak at 42h. In P-selectin gene knockout mice, JE got a peak during 6-12h, accordingly macrophage got its peak at 18h. JE maintained a platform during 18-30h,and macrophage increased again at 30h. In PE-selectin gene knockout mice, JE got a peak at 18h, accordingly macrophage got its peak during 18-24h which reduced the decrease tendency of JE, then macrophage increased again at 30h.The role of MIP-3 in dendritic cell migration: In wildtype mice, MIP-3 started to increase at 6h, and got a peak at 12h, accordingly dendritic cell started to increase at 6h,got a peak at 18h,and maintained the high level till 30h.During this period, MIP-3 stopped decreasing at 18h,maintain this level till 30h which made dendritic cell increase again at 30h and get another peak at 42h, and then MIP-3 increased again at 36h.The similar things also happened in P-,PE- selectin gene knockoutmice.4.The cytokines in wounded corneasThe peak time of IL-lwas covered by the peak time of neutrophil and macrophage; The genetics of IL-1 was similar to that of INF- Y ,and the increase of IL-1 followed the increase of INF- Y closely; When neutrophil increased,IL-6 also increased, then neutrophil would decrease to some extent.Conclusion1. Compared with wildtype mice, the corneal epithelial wound healing was delayed significantly in selectin gene knockout mice.2. Compared with wildtype mice, leukocytes migration into wounded corneas were reduced significantly in selectin gene knockoutmice......not only were the peak time delayed, but the leukocytes numberwere also reduced. It may be one of the reasons of the delay of wound healing in selectin gene knockout animals.3. KC could induce the neutrophil migration into the wounded corneas and activated neutrophils could generate more KC which could induce more neutrophil migrations; JE could induce the macrophage migration into the wounded corneas and activated macrophages could generate more JE which could induce more macrophage migrations; MIP-3 couldinduce the dendritic cell migration into the wounded corneas. 4. The IL-1 generated in the wounded corneas might be mainly produced by activated neutrophils and macrophages; INF-y might induced the production of IL-1; Activated neutrophils might generate IL-6 which could limit the increase of neutrophils through some mechanism and form a negative feedback.