Analysis of Human Corneal Igg by Isoelectric Focusing
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Investigative Ophthalmology & Visual Science, Vol. 29, No. 10, October 1988 Copyright © Association for Research in Vision and Ophthalmology Analysis of Human Corneal IgG by Isoelectric Focusing J. Clifford Woldrep,* Robin L. Noe,f and R. Doyle Stulringf Parameters which regulate the localization and retention of IgG within the corneal stroma are complex and poorly understood. Although multiple factors are involved, electrostatic interactions between IgG and anionic corneal tissue components, ie, proteoglycans (PG) and glycosaminoglycans (GAG) may regulate the distribution of antibodies within the corneal stroma. Isoelectric focusing (IEF) and blotting analysis of IgG revealed a restricted pi profile for both central and peripheral regions of the normal cornea. Similar analysis of pathological corneas from keratoplasty specimens in Fuchs' dys- trophy and keratoconus reveal a variable IEF profile. In the majority of keratoplasty specimens from patients with corneal edema or graft rejection, there was generally little or no IgG detectable. These results suggest that in edematous corneas where there is altered PG/GAG in the stroma and modified fluid dynamics, there is a concomitant loss of IgG. These findings may have implications for immuno- logic surveillance and protection of the avascular cornea. Invest Ophthalmol Vis Sci 29:1538-1543, 1988 The humoral immune system plays an important the soluble plasma proteins through ionic interac- role in mediating immunologic surveillance and pro- tions. The PGs and GAGs have long been known to tection of the avascular cornea. Immunoglobin G serve important physiological functions within the (IgG) is the major component of this humoral net- cornea regulating the collagen fiber organization,9 work which is responsible for diverse functional and pH,10 and swelling properties.9" Borcherding et al biological properties within the eye.1 The source of have demonstrated that the concentration of acidic corneal IgG is thought to be diffusion from the limbal GAGs differs from the central to the limbal regions of vessels.2"4 Factors that regulate the diffusion of IgG the cornea.9 Since the different GAGs vary with re- into the corneal stroma are extremely important spect to their numbers of negatively charged sulfate since there is normally no IgG produced locally groups, a subsequent electrostatic gradient within the within the quiescent eye.1"3 The localization and re- corneal stroma might, therefore, indirectly determine tention of IgG in the tissues, including the corneal which species of IgG gain access to the central and stroma, is dependent, in part, on the plasma IgG peripheral regions. Experiments were designed to test concentration, catabolic rate, and diffusion coeffi- this hypothesis through isoelectric focusing and anal- cient.5 Interactions between IgG and tissue compo- ysis of IgG isolated from normal human corneas ob- nents also regulate the tissue distribution of circulat- tained at autopsy and diseased corneas removed at ing IgG.6 Electrostatic factors may be paramount in keratoplasty. regulating the corneal distribution of plasma pro- teins. This is suggested by studies demonstrating the differential distribution of positively charged IgG and Materials and Methods negatively charged albumin within the corneal Ocular Tissues stroma.7 In the corneal stroma, the acidic proteogly- gans (PG) and the glycosaminoglycans (GAG) may Normal human globes and corneas were obtained function as fixed, anionically charged tissue ele- within 24 h of death from the Georgia Lions Eye ments8 which indirectly regulate the distribution of Bank at Emory University Eye Center (Atlanta, GA) and the Lions Eyes of Texas Eye Bank at the Cullen Eye Institute of Baylor College of Medicine (Hous- From the *Baylor College of Medicine, Center for Biotechnol- ton, TX). The corneas and aqueous humor were re- ogy, Division of Vision Research, The Woodlands, Texas, and the moved and frozen at — 80 °C prior to analysis. Kera- fEmory University Eye Center, Department of Ophthalmology, Atlanta, Georgia. toplasty specimens, aqueous humor and serum were Supported by grants EY-07154, 5T-32 EY-07092, and obtained at the time of surgery and were frozen EY-05097 from the National Eye Institute, Bethesda, Maryland. within three hours. Just prior to analysis, 8 mm nor- Submitted for publication: February 8, 1988; accepted April 18, mal corneal buttons were thawed and the central 4 1988. mm was separated from the remaining peripheral 4 Reprint requests: J. Clifford Waldrep, PhD, Baylor College of Medicine, The Center for Biotechnology, Division of Vision Re- mm ring using a trephine. Keratoplasty specimens search, 4000 Research Forest Drive, The Woodlands, TX 77381. consisted of one-half of an 8 mm button which was 1538 Downloaded from iovs.arvojournals.org on 10/02/2021 No. 10 IEF OF CORNEAL IgG / Woldrep er ol 1539 trephined into a central 4 mm hemicircle and a pe- dilution of mouse monoclonal antibody to human ripheral portion. IgG (MAB 066, Chemicon International, El Se- gundo, CA) for 60 min. The blots were washed for 15 Isoelectric Focusing (IEF) min with three changes of Tris-Saline and incubated with a 1:100 biotin-conjugated horse anti-mouse IgG Gels were prepared for IEF as described by Hoff- (VectaStain kit). After washing for 15 min with three man and Jump, on GelBond Agarose Film (FMC changes of Tris-Saline, the blots were incubated in a Corporation, Rockland, ME) by an open casting 1:200 dilution of the biotin-avidin ABC peroxidase technique.12 Using pyrogen-free water, 0.64 ml of pi complex (Vectastain kit, Vector Laboratories, Bur- 3-10 ampholytes (FMC) was added to 7 ml of lingame, CA) for 30 min. The blots were then washed 60-65°C melted FMC isogel agarose (1% W/v final for 15 min in Tris-Saline with three changes and de- concentration) containing 1.0 gram sorbitol and 1.0 veloped for 5 min in chloro-1-naphthol (Sigma, St. ml of glycerol. Some gels were supplemented with pi Louis, MO), 30 mg in 10 ml methanol plus 40 ml 8.0-10.5 ampholytes to increase resolution of basic 0.05 M Tris Buffer, pH 7.6, and 5-^1 of 30% H O . proteins. The final volume was adjusted to 10 ml, and 2 2 The developed blots were washed in dH O and stored the gels were poured under a heatlamp onto pre- 2 in the dark to dry, overnight. All antibody dilutions warmed, GelBond film on a level casting platform. were made in BGEN. All incubations and washing After solidification, the gels were cured for a mini- procedures were carried out on a rocker platform. mum of 1 hr at 4°C in a humidified chamber. Sam- The pi range of the serum and corneal IgG was deter- ples were added using applicator wicks (FMC pi 3-10 mined from the migration distances of the pi stan- standards, serum, or aqueous humor) or corneal dards. pieces were applied directly to the gel for direct tissue isoelectric focusing.13 A pH gradient was generated across the gel using a 1.0 N NaOH catholyte solution Results (pH 13.0) and a 0.5 N acetic acid anolyte solution (pH 2.6). An initial current of 1.0 W was used for 10 As an extension of previous quantitative and quali- min to establish the pH gradient. Isoelectric focusing tative analyses of immunoglobulins eluted from was initiated for 30 min and the sample applicators human corneas, we sought to further characterize and corneal pieces removed from the gel surface. Iso- corneal IgG using direct tissue isoelectric focusing electric focusing was continued for an additional 30 (IEF) and blotting onto nitrocellulose. Our assays re- min (600 volt/hours). Water accumulation on the gel quired as little as a 2 X 4 mm piece of corneal tissue for IgG analysis. Initial IEF analysis of normal surface was removed by periodic blotting with filter human corneal IgG revealed that the pi of both cen- paper. The isoelectric point (pi) or the pH at which tral and peripheral corneal IgG is restricted when there is no net mobility of a protein in an electric field compared to the pi of normal human serum IgG was determined using defined pi standards. (Figs. 1, 2). The pis of IgG species within the normal cornea is distributed within a narrower pH range than Blotting that of serum. The average lower limit detected for The IEF gels were rinsed for 60 seconds in distilled central corneal IgG was 5.78 ± 0.11 and the average water with agitation. The IEF gels were then blotted for peripheral corneal IgG was 5.60 ± 0.10 (Table 1). by direct application of pieces of nitrocellulose equili- These lower limit pi values are significantly different brated in 0.05 M Tris-Saline, pH 7.6. A wet piece of from the corresponding lower serum IgG pi (5.27 filter paper was placed onto the surface of the nitro- ± 0.27) (Table 1). The upper pi limit for central cor- cellulose, and the gel was then inverted onto ten neal IgG was 8.73 ±0.14 and for peripheral corneal layers of absorbent paper towels. A glass sheet was IgG was 8.95 ± 0.11 (Table 1). These pi values were then placed against the back of the GelBond surface not significantly different from the corresponding and a 1 kg weight applied for 10 min. Transfer of the upper serum IgG pi (9.4 ± 0.10) (Table 1). Qualita- focussed proteins occurred by diffusion. The nitro- tive IEF profiles of aqueous humor were not obtained cellulose blot was then removed and cut for staining since only trace amounts were detectable on the ni- and analysis. The Standards were stained with amido trocellulose blots due to extensive fractionation of the black or india ink.