Beta B1-Crystallin: Identification of a Candidate Ciliary Body Uveitis Antigen

Abstract

purpose. Perineuclear anti-neutrophil cytoplasmic antibody (pANCA), a marker antibody present in 12% of patients with anterior uveitis, recognizes cytoplasmic antigens in the nonpigmented ciliary body epithelium, a probable site of immunologic reactivity in this inflammatory disease. In this study, a recombinantly isolated pANCA monoclonal antibody was used to identify the corresponding antigenic target(s) in the ciliary body.

methods. Proteins from microdissected eye bank ocular ciliary body tissue were used to identify the corresponding ANCA antigen. Parallel two-dimensional protein gels were used for simultaneous identification of candidate antigenic protein spots by Western blot analysis and as a source of material for proteomic analysis. Multiple independent methods including Western blot analysis, confocal microscopy, and RT-PCR were used to provide additional characterization of the candidate protein.

results. Proteomic analysis suggested that beta B1 (βB1)-crystallin is the primary ciliary body antigen. The presence of βB1-crystallin in the human ciliary body was confirmed by Western blot with a βB1 specific anti-peptide antibody. Confocal microscopy revealed colocalization of the antigenic reactivity of both anti-βB1 antibody and monoclonal pANCA. RT-PCR confirmed the presence of βB1-crystallin RNA in the ciliary body tissues.

conclusions. This study identified βB1-crystallin as a new cytoplasmic ciliary body antigenic target of a marker autoantibody associated with uveitis. This characterization of βB1-crystallin outside the lens raises questions about its extralenticular expression, intracellular role, and potential target of inflammation in uveitis.

Immune pathogenesis of uveitis probably results from an antigen-specific immune response and inflammatory cytokines. ^{1 2 3} From a conceptual standpoint, identification of disease-specific antigens may provide clues to understand the specific pathogenesis of either the primary disease target or the secondary complications of disease and may ultimately suggest potential tools to design targeted immunologic therapy. A variety of immune-mediated diseases, thought to result through humoral and/or cellular immunologic pathways, are associated with marker antibodies with corresponding antigen(s) that have been validated as targets for the disease-specific immune response. Perhaps the best characterized examples come from the comprehensive set of T-cell antigens defined with autoreactive serum antibodies of patients with diabetes mellitus. ⁴ Other autoantigens, identified by serum marker antibodies in Wegener granulomatosis, myasthenia gravis, ulcerative colitis, and Behçet syndrome, have been shown to be useful as a marker for serologic testing for diagnostic purposes, to identify clinically meaningful disease subgroups, to determine potential for disease complications, and as the basis for the development of new animal models of autoimmune disease. ^{5 6 7 8 9 10 11 12}  

Anti-neutrophil cytoplasmic antibodies (ANCAs) represent a diverse set of marker autoantibodies characterized by reactivity against human neutrophils in a standard laboratory analysis. ANCAs are found in approximately 30% of patients with anterior uveitis. ¹³ Most uveitis-associated ANCAs are distinct from other previously characterized ANCAs and do not react against known antigens, including lactoferrin, elastase, cathepsin G, myeloperoxidase, and proteinase 3. ¹³ A subgroup of ANCAs, perinuclear (p)ANCAs, have been identified in 12% of patients with anterior uveitis and their presence is independent of underlying risk factors for anterior uveitis, such as HLA-B27 positivity or associated rheumatologic disease. ¹³  

We have cloned a pANCA, Fab 5-3, using antibody phage-display technology and used it as a tool to identify candidate pANCA antigens. ¹⁴ A primary pANCA antigen in the human neutrophil is histone H1, but it is important to note that this monoclonal antibody recognizes multiple antigens in human tissue and bacteria. ^{15 16 17 18} This observation most likely reflects reactivity against a shared epitope. The monoclonal pANCA identifies tissue antigens in the ciliary body, retina, and mast cells. ^{15 16} Reactivity in the ciliary body is particularly intriguing, because inflammation of the ciliary body and iris is observed early in the course of experimental uveitis. ^{19 20 21} It is therefore possible that expression of antigen in the ciliary body microenvironment plays a role in pathogenesis of idiopathic anterior uveitis. ^{22 23} In the ciliary body, the nonpigmented epithelium (NP-CBE) harbors cytoplasmic antigens that are avid binders of the pANCA Fab 5-3 and the major antigen is observed at approximately 31 to 32 kDa. ^{15 24} The purpose of the present study was to identify the natural target ANCA-reactive NP-CBE antigen(s) of the human ciliary body by using the monoclonal pANCA Fab 5-3. Identification of this antigen is an important focus and a first step in developing a model of uveitis pathogenesis in ANCA⁺ patients. 

Methods

Antibodies

Recombinant Fab 5-3, a cloned human UC-pANCA Fab monoclonal antibody, was purified as a hexahistidine-tagged product ^{13 14} and used as a primary antibody in Western blot and immunofluorescence studies. Rabbit anti-bovine beta B1 (βB1)-crystallin peptide antibodies were also used in these studies. The anti-peptide antibody Lap 20 recognizes a highly conserved, 10-amino-acid, C-terminal peptide and was used to identify βB1-crystallin in the human and rabbit (Fig. 1) . The anti-peptide antibody Lap 17 was prepared against the nonconserved bovine N-terminal PAPA region peptide and served as a negative control antibody for identifying human βB1-crystallin (Fig. 1) . 

Ocular Specimens

Human eyes obtained from deceased donors were procured from the University of California Los Angeles (UCLA) Eye Bank 4 to 24 hours after death and processed immediately on arrival. The tenets of the Declaration of Helsinki governed the procurement and management of human tissue. The eyes were stored in a moist chamber at 4°C until dissection. The eyes were bisected in the horizontal plane adjacent to the macula. The anterior segment of the eye was meticulously microdissected to obtain the ciliary body and iris specimen without disruption of the lens capsule. The dissected tissues were snap frozen and stored at −80°C. Frozen sections were obtained from tissues that were frozen and stored in optimal cutting temperature (OCT) embedding solution. 

Proteins

Ciliary body protein extracts were obtained after tissue homogenization in glass in 2% SDS, 100 mM dithiothreitol, and 60 mM Tris (pH 6.8). The homogenized sample was heated to 100°C for 5 minutes. Remaining tissue fragments were removed by centrifugation; the supernatant was frozen at −80°C until use. Total bovine soluble lens proteins were extracted in phosphate-buffered saline. Human, recombinant βΒ1-crystallin was obtained for use in Western blot (kindly provided by author KL). 

Isolation of Nonpigmented and Pigmented Rabbit Ciliary Body Epithelium

Rabbit ciliary body (New Zealand White x Dutch belted cross) was isolated and separated into the nonpigmented and pigmented layers, digested into single cell suspensions, and established as primary cultures according to standard procedures. ²⁵ The nonpigmented cell cultures were more than 99% pure, and the pigmented cell cultures had approximately 20% contamination by nonpigmented epithelial cells. Purity of the cultures has been morphologically assessed by immunohistochemical identification of the H⁺K⁺ATPase, as previously described. ^{26 27}  

Confocal Microscopy

Nonpigmented ciliary body epithelium was cultured on glass coverslips, as described. ^{28 29} Fab 5-3 or the Lap antibodies were used at a dilution of 1:20 and 1:50, respectively, in PBS-0.05% Tween (PBST) and applied to methanol-fixed coverslips for 30 minutes. After three washes in PBST, bound antibody was detected with FITC-conjugated anti-human IgG (Fab′)₂ at a dilution of 1:1000 (Pierce, Rockford, IL) or Texas red-conjugated donkey anti-rabbit IgG (H+L) at a dilution of 1:400 (AffiniPure; Jackson ImmunoResearch, West Grove, PA). Slides were analyzed with a laser scanning confocal microscope (Fluoview; Olympus America, Inc., Melville, NY). Simultaneous detection of the FITC- and Texas red-labeled cells was achieved with excitation with argon (488 nm) and krypton (568 nm) lasers and analysis of emissions at 525 to 540 nm and above 630 nm, respectively. Colocalization was studied in a single X-Y optical section of a thickness of 0.5 μm, viewed under a 60× oil-immersion objective, and merged by using the image-analysis software (Fluoview; ver. 2.1.39; Olympus). 

Proteomic Identification

Proteins from microdissected human ciliary body (pure ciliary body was microdissected from donor eyes obtained <24 hours after death) were isolated by preparative two-dimensional (2-D) protein electrophoresis (IPGphor isoelectric focusing; Amersham Pharmacia Biotech, Piscataway, NJ). The first-dimension separation allowed multiple identical isoelectric focusing (IEF) strips to be prepared for parallel analysis. Second-dimension resolution of protein spots was performed by separation on a 12% SDS-polyacrylamide gel. Some of the second-dimension gels were used to identify the candidate antigens by Western immunoblot analysis after transfer onto nitrocellulose and probing for Fab 5-3 reactivity. Other second-dimension gels were used for sequence analysis and protein identification of the candidate protein antigen. A 31-kDa protein spot, identified by Fab 5-3 reactivity, was initially identified by multiparameter proteomic analysis with the assistance of the Keck Proteomics Facility at Yale University (New Haven, CT). The analysis revealed βB1-crystallin as the probable reactive ciliary body 31-kDa antigen. 

Western Blot Analysis

Tissue extracts were fractionated by 12% SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes (Amersham Life Sciences, Buckinghamshire, UK) in Tris glycine buffer (National Diagnostics, Atlanta, GA) and verified by ponceau S red staining (Sigma Chemical Co., St. Louis, MO). The membrane was then blocked in 5% nonfat milk in TBS-0.05% Tween 20 (TBST; Pierce). Blots were incubated for 1 hour with Fab 5-3 at a concentration of 2 μg/mL or Lap 17 or Lap 20 (at dilutions of 1:500) in TBST and washed three times in TBST. Alkaline-phosphatase-conjugated goat anti-human IgG (Fab′)₂ (Pierce) or alkaline-phosphatase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) was exposed to the blots at a 1:1000 dilution. Nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate (BCIP/NBT) was used to visualize the location of the bound antibody (Sigma). 

Reverse Transcription-Polymerase Chain Reaction

Isolation of RNA from ciliary body that was manually microdissected, snap frozen, and stored at −80°C was performed with standard techniques. ³⁰ Briefly, the tissue was homogenized for 60 seconds (TRIzol; Gibco/BRL Gaithersburg, MD). Protein was extracted with phenol-chloroform, and the RNA was precipitated with isopropanol, washed with ethanol, and resuspended in RNase-free, HPLC-grade water. The purity of the RNA was ascertained with a ratio of absorbance at 260 nm to absorbance at 280 nm. 

RT-PCR was performed on the ciliary body RNA by using lyophilized beads (Ready To Go RT-PCR; Amersham Pharmacia Biotech). First-strand synthesis was performed with approximately 1 μg RNA in a thermal cycler (model 9700; Applied Biosystems, Foster City, CA) preheated to 42°C, using an oligo dT primer. After 30 minutes of reverse transcription at 42°C, the reaction was heated to 95°C for 5 minutes. The tubes were removed from the thermal cycler and allowed to cool. PCR was performed with gene-specific forward and reverse primers, as well as 1 μL DNA Taq polymerase (Amplitaq; Roche Molecular Biochemicals, Branchburg, NJ). For both GAPDH and βB1, the reactions comprised 35 cycles (95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds, with a final cycle at 72°C for 7 minutes). The sequence of the primers used to amplify βB1-crystallin were 5′-CCATCAAAATGGATGCCCAGGAGCACAAAATCTCC-3′ (forward primer), and 5′-AGGGTTGGGGCAAGGTAGCAGAGTGAGGTGTGG-3′ (reverse primer; GibcoBRL). The sequences of the primers used to amplify GAPDH were 5′-TCACCAGGGCTGCTT TTAACTC-3′ (forward primer), and 5′-GGTCATGAGTCCTTCCACGATA-3′ (reverse primer; GibcoBRL). PCR products were then examined on 1.5% agarose gel with ethidium bromide staining. 

RT-PCR of Laser-Miscrodissected Tissue

Eyes were procured from the eye bank, the intact lenses were removed, and the eye hemisected before being embedded and frozen in OCT (Sakura FineTek, Torrance, CA). Laser capture microdissection was performed to isolate ciliary body epithelial cell layers on laser capture microscopic (LCM) transfer film (Arcturus, Mountain View, CA), as described by Wadehra et al., ³¹ using an LCM microscope (model 200; Pixcell, Arcturus; located in the UCLA Human Tissue Research Center). One-step RT-PCR was performed directly, by using the transfer film in a first-strand reaction containing the lyophilized beads (Ready-to-Go; Amersham Pharmacia Biotech) in the thermal cycler (model 9700; Applied Biosystems), as previously described. ³¹  

Results

Identification of βB1-Crystallin as a Candidate Ciliary Body ANCA Antigen

With human ciliary body protein extracts resolved by 2-D protein gel electrophoresis followed by Western blot analysis, the monoclonal antibody, Fab 5-3, was used to identify a major corresponding ciliary body antigen at an apparent size of 31 to 32 kDa. Parallel 2-D gels were used, and the candidate protein sent for identification by the Keck Laboratories (Yale University). Results from matrix-assisted laser desorption ionization mass spectrometry (MALD-MS) were obtained by using a tryptic digest of the candidate protein. A ProFound search of the National Center for Biotechnology Information (NCBI) nonredundant database (http://www.ncbi.nlm.nih.gov) identified peptide masses spanning 44% of the predicted βB1-crystallin protein sequence. Use of the PeptideSearch program European Molecular Biology Laboratory (EMBL) nonredundant database (http://www.embl-heidelberg.de/; provided in the public domain by EMBL, Heidelberg, Germany) to search the same peptide masses also confirmed βB1-crystallin as a candidate protein but without definitive identification. Independent confirmation of βB1-crystallin as the reactive protein was performed by immunologic validation. 

Confirmation of βB1-Crystallin as an ANCA Antigen

Independent confirmation of βB1-crystallin as a candidate ANCA antigen was performed with several complementary approaches. First, total soluble bovine lens protein was used as an antigenic target in Western blot analysis (Fig. 2A) . The Lap 20 and Fab 5-3 antibodies demonstrated reactivity with the identical protein bands (lanes 1 and 2, respectively). Confirmation of the Fab 5-3 recognition was performed with recombinant human βB1-crystallin used as an antigenic target in Western blot analysis (Fig. 2B) . Fab 5-3, the monoclonal pANCA, identified the recombinant βB1-crystallin. The expected reactivity of the human βB1-crystallin with the antibody Lap 20 and the absence of reactivity with the antibody Lap 17 were confirmed (Fig. 2B) . Second, similar reactivity at approximately 31 to 32 kDa was observed with both the Fab 5-3 and Lap 20 antibodies against whole protein extracts from human ciliary body-iris specimens (Fig. 3) . The multiple bands observed at 28 to 32 kDa are probably multiple species of βB1-crystallin, in that it is well known for its susceptibility to loss of amino acids from the N terminus. The other candidate Fab 5-3 ciliary body-associated antigens, larger than 67 kDa, have not yet been identified. ^{32 33 34} These results confirm that βB1-crystallin is present in the ciliary body and is an antigen for the monoclonal pANCA Fab 5-3. 

Evidence that both the Fab 5-3 and Lap 20 antibodies recognize the same protein was obtained by confocal microscopy. Rabbit βB1-crystallin protein sequence is not yet available in the database; however, our previous work demonstrated the presence of a cytoplasmic Fab 5-3 antigen in purified rabbit nonpigmented ciliary body epithelium. ²⁴ Nonpigmented rabbit ciliary body epithelium was used as the target in immunofluorescence experiments. Figure 4 demonstrates reactivity of Lap 20 with the cells, confirming the presence of a protein bearing the βB1-crystallin peptide epitope. Lap 17 (data not shown) was not reactive with these cells. Results of the confocal microscopy (Fig. 4) confirmed colocalization of Lap 20 and Fab 5-3 reactivity in these cells, providing additional evidence for the presence of βB1-crystallin in the ciliary body epithelium. Immunostaining results did not distinguish passive, intracellular accumulation of βB1-crystallin from the active production of the protein at this site. 

Expression of βB1-Crystallin in the Ciliary Body

RNA was obtained from human ciliary body-iris specimens and used as a substrate for RT-PCR (Fig. 5) . The quality of RNA was evaluated by its ability to serve as a substrate for RT-PCR of GAPDH. RNA from human lens was used as a positive control for βB1-crystallin RT-PCR. Amplification of the expected product was achieved with βB1-crystallin-specific primers (Fig. 5) . The PCR product was sequenced to confirm its identity as βB1-crystallin. 

To rule out the possibility of lens contamination, additional RT-PCR experiments were performed with ciliary body epithelium obtained through laser capture microdissection (Fig. 6) . βB1-crystallin was identified in the ciliary body epithelium by RT-PCR and was not present in the adjacent regions of the slide (Fig. 6D) , providing independent confirmation that this protein is expressed in the ciliary body. 

Discussion

Anterior uveitis is a major cause of morbidity in young and middle-aged adults and is believed to be responsible for up to 70,000 cases of vision loss in the United States. ³⁵ Marker antibodies are useful in identifying antigenic targets of chronic inflammatory diseases mediated by both cellular and humoral immunity. ^{5 10 17 18 36 37} Previous work defined pANCA as a marker antibody in approximately 12% of patients with anterior uveitis and identified reactivity of a monoclonal pANCA Fab 5-3, by using the ciliary body epithelium, a site of potential immunologic activity in anterior uveitis. ^{24 38} Although the nuclear-associated 32- to 33-kDa pANCA antigen in the neutrophil was defined as histone H1, the major pANCA antigen in the ciliary body was defined as a cytoplasmic antigen that migrated similarly at approximately 31 to 32 kDa. ³⁹ The goal of the present work was to identify major pANCA reactivity in the ciliary body. This study surprisingly revealed βB1-crystallin as the major candidate uveitis-associated ANCA antigen in the ciliary body and provides independent evidence for the extralenticular expression of βB1-crystallin. 

β-Crystallins comprise approximately half the protein mass of human lens crystallins. They exist as soluble aggregates consisting of multiple combinations of six different proteins. The β-crystallins are heterogeneous in molecular mass and charge but have similar secondary and tertiary structures. ⁴⁰ Cytotoxicity experiments suggest that β-crystallins may be associated with the plasma membrane of lens epithelial cells, but their function in this subcellular location is unknown. ^{3 41} Recent interest in the expression of crystallin proteins revealed the extralenticular expression and molecular chaperone function of the α-crystallins. ^{42 43 44} βB2-crystallin, the most abundant β-crystallin, is also expressed extralenticularly, but the function is unknown. ^{45 46 47} βB2 crystallin is highly soluble and heat stable and is expressed in the lens, retina, brain, and testis. ^{45 48} Although the biological function of βB2-crystallin in extralenticular sites is obscure, its potential role in phosphorylation pathways implies an involvement in signal-transduction pathways. ⁴⁵ It is highly likely that other crystallin proteins, including βB1, have important, as yet undefined, nonlenticular biological roles. 

The role of βB1-crystallin in the pathogenesis of uveitis is not yet understood. It is clear from our work that βB1-crystallin is expressed in the ciliary body and that it is an antigen for the monoclonal pANCA Fab 5-3. There are several potential explanations for this observation and avenues for future investigation. One explanation is that reactivity against βB1-crystallin occurs as a nonspecific consequence of an activated inflammatory state during anterior uveitis and inflammation in the region of the ciliary body. An alternative hypothesis is that reactivity against βB1-crystallin may drive the inflammatory response in the anterior chamber and may be involved in the disease’s pathogenesis. A third possibility is that reactivity against βB1-crystallin may correlate with the development of such uveitis-associated complications as cataract. 

One of the major complications of uveitis is cataract, thought to have either a primary association with the intraocular inflammation or a secondary association with the use of topical steroids. ⁴⁹ The role of anti-crystallin antibodies in cataract pathogenesis is controversial. Theories range from a primary mechanism in lens epithelial damage to a secondary bystander effect of cataract by autoimmunization through a permeable cataractous lens capsule. ⁴¹ There is some evidence that immune-mediated damage, through an anti-crystallin response, may play a role in cataract pathogenesis. ⁵⁰ Experimental immunization of crystallin proteins in B6C3/F mice results in lens epithelial migration and cortical cataract. ⁵⁰ Additionally, monoclonal antibodies with specificity against an amino acid sequence shared in several microbes and the βA3-crystallin also generates lens epithelial cell (LEC) damage, leading to the hypothesis that chronic or recurrent infection through molecular mimicry could trigger immune-mediated cataract. ⁴¹  

Humoral immunity against lens antigens has also been observed in the setting of human uveitis, although the specific lens protein antigens and the cataract status of these patients were not defined. ⁵¹ In addition, anti-crystallin antibodies have been demonstrated in patients with significant age-related cataract but not in normal individuals with no demonstrable ocular disease. ^{51 52 53} Characterization of anti-crystallin antibodies from 15 patients with mature cataract has revealed significant reactivity against βB1-, βA1-, βA4-, and γ-crystallins. ⁵³ However, it is not clear whether the reactivity is an epiphenomenon secondary to autoimmunization through a leaky lens capsule or is related to the pathogenic mechanism of lens damage. 

The observation that βB1-crystallin is present in the ciliary body and its identification as a candidate tissue pANCA antigen is provocative, because it is possible that local antigen expression, in combination with the ciliary body microenvironment, plays a role in the pathogenesis of uveitis. ³⁸ This experimental trail points to βB1-crystallin as a relevant autoantigenic target of Fab 5-3, a monoclonal pANCA. Additional studies are needed to define disease-associated immunologic reactivity against βB1-crystallin and to determine whether this reactivity plays a role in the pathogenesis of uveitis or in the development of uveitis-associated cataract. 

²  Contributed equally to the work and therefore should be regarded as equivalent senior authors.

 Supported by National Eye Institute Grants EY00360, EY12239, and EY13708. LKG is a James A. Adams Scholar of Research to Prevent Blindness.

 Submitted for publication December 20, 2001; revised July 5, 2002; accepted July 19, 2002.

 Commercial relationships policy: N.

 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.

 

Figure 1.

 

Sequence comparison of bovine and human βΒ1-crystallin. The alignment of published sequences of the human (P53674) and bovine (P07318) βΒ1-crystallin is shown. Peptide sequences used to generate the βΒ1anti-peptide antibodies are identified in italic (Lap17) or bold, underlined print (Lap20).

Figure 2.

 

Immunologic reactivity of the monoclonal pANCA Fab 5-3 against βΒ1-crystallin. (A) Whole soluble bovine lens protein was used as an antigenic target in Western blot analysis. The protein was separated on a 12% SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and analyzed for reactivity with Fab 5-3 and Lap 20. Lane 1: proteins identified by reactivity with Lap 20, an anti-peptide antibody that recognizes the C terminus of βΒ1-crystallin; lane 2: the reactivity of Fab 5-3 against bovine lens protein; Lane 3: total protein on the gel, as detected by Coomassie stain. Secondary antibody alone for either the Lap 20 or Fab 5-3 antibody did not show reactivity (not shown). Right: molecular weight (MW) markers. (B) Reactivity of Fab 5-3 against a purified, recombinant, human βΒ1-crystallin was evaluated by Western blot analysis. The protein was separated on a 12% SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and analyzed for reactivity with Fab 5-3, Lap 20, and Lap 17. Secondary antibody alone (lane 1) was the control for the Lap 20 and Lap 17 primary antibodies (AP-conjugated goat anti-rabbit antibody). Lap 17 (lane 2) and Lap 20 (lane 3) were used as primary antibodies and detected with the AP-conjugated goat anti-rabbit antibody and colorimetric reaction. Reactivity of the Fab 5-3 antibody (lane 4) as detected with an AP-conjugated goat anti-human antibody with colorimetric reaction. secondary antibody control for Fab 5-3 was nonreactive (lane 5).

Figure 3.

 

The monoclonal pANCA Fab 5-3 and Lap 20 recognizes a 31- to 32-kDa protein in human ciliary body extracts. Human ciliary body extracts were prepared and separated on a 12% SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and analyzed for reactivity. Samples were applied to the gel in equal protein concentration as judged by Coomassie stain. Right: molecular weight (MW) markers. Both the Fab 5-3 (lane A) and the Lap 20 (lane B) primary antibodies showed reactivity with the ciliary body proteins, as revealed with the appropriate secondary antibody and colorimetric reaction. In contrast, Lap 17 (lane C) did not react with human ciliary body. Secondary antibody control for Fab 5-3 (lane D) was nonreactive.

Figure 4.

 

Colocalization of Fab 5-3 and Lap 20 reactivity in cultured, nonpigmented rabbit ciliary body epithelium. Rabbit ciliary body was harvested, separated into the nonpigmented and pigmented epithelium, cultured on coverslips, fixed, and stained by indirect immunofluorescence. (A) Fab 5-3 reactivity was detected with an FITC-conjugated anti-human IgG (Fab′)₂, and (B) Lap 20 reactivity was detected with Texas red-conjugated donkey anti-rabbit IgG (H+L). (C) The merged colocalization pattern. (D) Secondary antibody control experiments were performed with FITC-conjugated anti-human IgG (Fab′)₂ and (E) Texas red-conjugated donkey anti-rabbit IgG (H+L). (F) Merged control image.

Figure 5.

 

RT-PCR identification of βΒ1-crystallin in human ciliary body-iris tissue. RNA extracts from human ciliary body-iris (lanes 1 and 2) and lens (lane 3) were used as a template for RT-PCR. The control is also shown (lane 4). Primers specific for βΒ1-crystallin and GAPDH were used. No product was obtained without template or without added reverse transcriptase (not shown).

Figure 6.

 

RT-PCR identification of βΒ1-crystallin in laser-microdissected ciliary body. (A) Frozen sections of human ciliary body were cut, stained with hematoxylin-eosin, and evaluated with a laser capture microscope. (C) The ciliary body epithelium was dissected and used directly as a template for RT-PCR (bottom, lanes 1 and 2). (B) The ciliary body after laser dissection. (D) Laser capture of material adjacent to the ciliary body epithelium was used as a negative control (bottom, lane 3). The corresponding RT-PCR results are shown. No product was obtained without template or without added reverse transcriptase (not shown). Magnification: (A, B) ×40; (C, D) ×100.

The authors thank Madhuri Wadehra for help in confocal microscopy and Maria Avina, UCLA Human Tissue Research Center, for expert assistance in laser capture microdissection.