Activation of NOD2 in vivo induces IL-1beta production in the eye via caspase-1 but results in ocular inflammation independently of IL-1 signaling.

Nucleotide-binding and oligomerization domain 2 (NOD2) belongs to the emerging Nod-like receptor (NLR) family considered important in innate immunity. Mutations in NOD2 cause Blau syndrome, an inherited inflammation of eye, joints, and skin. Mutations in a homologous region of another NLR member, NALP3, cause autoinflammation, wherein IL-1beta plays a critical role. Here, we tested the hypothesis that IL-1beta is a downstream mediator of NOD2-dependent ocular inflammation. We used a mouse model of NOD2-dependent ocular inflammation induced by muramyl dipeptide (MDP), the minimal bacterial motif sensed by NOD2. We report that MDP-induced ocular inflammation generates IL-1beta and IL-18 within the eye in a NOD2- and caspase-1-dependent manner. Surprisingly, two critical measures of ocular inflammation, leukocyte rolling and leukocyte intravascular adherence, appear to be completely independent of IL-1 signaling effects, as caspase-1 and IL-1R1-deficient mice still developed ocular inflammation in response to MDP. In contrast to the eye, a diminished neutrophil response was observed in an in vivo model of MDP-induced peritonitis in caspase-1-deficient mice, suggesting that IL-1beta is not essential in NOD2-dependent ocular inflammation, but it is involved, in part, in systemic inflammation triggered by NOD2 activation. This disparity may be influenced by IL-1R antagonist (IL-1Ra), as we observed differential IL-1Ra levels in the eye versus plasma at baseline levels and in response to MDP treatment. This report reveals a new in vivo function of NOD2 within the eye yet importantly, distinguishes NOD2-dependent from NALP3-dependent inflammation, as ocular inflammation in mice occurred independently of IL-1beta.

Anterior uveitis accompanies joint disease in a murine model resembling ankylosing spondylitis.

BACKGROUND: Uveitis is often associated with a systemic inflammatory disease such as ankylosing spondylitis. Our understanding of the eye's susceptibility to immune-mediated uveitis as in the apparent absence of infection has been limited by a relative lack of experimental models. Here we sought to assess whether ocular inflammation occurs in a previously described murine model of proteoglycan-induced spondylitis, wherein mice develop progressive spondylitis, sacroiliitis and peripheral arthritis--features common to the clinical presentations of ankylosing spondylitis. METHODS: Using intravital microscopy we examined the ocular inflammatory response after the onset of arthritis in mice that overexpressed the T cell receptor (TCR) specific for a dominant arthritogenic epitope of cartilage proteoglycan [TCR-Tg (transgenic) mice] or BALB/c controls. RESULTS: Immunized TCR-Tg mice showed a significant increase in the number of rolling and adhering cells within the iris vasculature compared to adjuvant control mice. Cellular infiltration within the iris tissue, as assessed by intravital microscopy and histology, was also increased. Our initial temporal analysis has revealed that immunized TCR-Tg mice show a significant increase in intravascular inflammation by 2 weeks after immunization, but it diminishes at 4 weeks after immunization. CONCLUSIONS: Although these data are preliminary, this model has the potential to clarify the mechanisms accounting for the coexistence of eye and sacroiliac inflammation as occurs in patients with ankylosing spondylitis.

Novel production of interleukin-1 receptor antagonist peptides in normal human cornea.

Interleukin-1 receptor antagonist (IL-1ra) is an important modulator of IL-1 activity in a variety of tissues. IL-1ra is differentially produced by different cell types as a 22-26-kD secreted peptide (sIL-1ra) and/or a smaller 16- or 18-kD intracellular peptide (icIL-1ra). This study was undertaken to evaluate the production of IL-1ra in the human cornea. IL-1ra mRNA can be detected in early passage human corneal epithelial cells and corneal stromal fibroblasts and is significantly enhanced by IL-1. Corneal endothelial cells do not express IL-1ra mRNA. Immunohistochemical studies of cultured corneal cells and whole human cornea demonstrate IL-1ra protein production by both the epithelial and stromal cells but not the endothelial cells. Reverse transcriptase polymerase chain reaction, ELISA, and immunoprecipitation studies indicate that corneal epithelial cells are capable of producing both icIL-1ra and sIL-1ra forms of IL-1ra whereas the corneal stromal cells produce only icIL-1ra. In addition to the larger 18-kD icIL-1ra, both corneal epithelial and stromal cells are also capable of producing a smaller recently described 16-kD icIL-1ra. Thus, the differential production of IL-1ra in the human cornea is unique; whereas both epithelial and stromal cells produce icIL-1ra (type 1 and type 2), the epithelial cells appear to also produce sIL-1ra. It is proposed that these IL-1ra proteins may play an important role in regulating IL-1-induced corneal inflammation.

Endotoxin upregulates CCR7 and its ligands in the lymphatic-free mouse iris.

PURPOSE: Using time lapse intravital microscopy and histology, we previously reported that we could not detect migration of antigen-presenting cells from the iris to the regional lymph node. Dendritic cells (DC) in other peripheral tissues migrate to lymph nodes in response to chemokines, CCL19 (ELC) and CCL21b (SLC), that activate the CCR7 receptor. We hypothesized that DCs in an inflamed iris might show a different chemokine receptor and ligand profile, thus explaining the DC's inability to migrate. METHODS: Eyes of 35 BALB/c mice were injected intravitreally with 2 mul of 250 ng E. coli lipopolysaccharide (LPS) or phosphate buffered saline (PBS). Five mice served as naïve controls. After 3 and 6 h, the iris-ciliary bodies were dissected and pooled in groups of five. Total RNA was isolated, and reverse-transcriptase polymerase chain reaction (RT-PCR) for chemokine receptor and ligand mRNA was performed. In addition, one eye from each of the three animals was taken 6 h after LPS injection for immunohistology (IHC). RESULTS: The naïve iris, the iris after PBS injection, and the iris after LPS injection contained CCR5 mRNA at approximately equal levels and did not have detectable CCR6 mRNA. No CCR7 mRNA expression was found in the naïve iris, but it was weakly expressed in PBS-injected eyes and was approximately 3.4 fold upregulated after LPS injection. This was confirmed by IHC with no staining for CCR7 in the control iris but positive staining in the inflamed eyes. Transcripts for the CCR7 ligands, CCL19 and CCL21b, were found after LPS or PBS injection but not in naïve iris-ciliary bodies. CONCLUSIONS: The clear upregulation of CCR7 and its ligands in the inflamed iris suggests that another mechanism prevents iris DCs from migrating. Other possibilities include the absence of co-factors, inhibitory substances, the lack of lymphatics inside the eye, or inadequate biologicAL activity of these chemotactic factors and ligands.

Modification of the Woodruff-Stamper assay demonstrates binding of Toxoplasma gondii tachyzoites to retinal vascular endothelium.

In the human host, infection with the protozoan parasite, Toxoplasma gondii, most commonly involves the eye and/or the brain. Previous work indicates a relative susceptibility of the human retinal vascular endothelium to infection with the T. gondii tachyzoite, which may contribute to this tissue localization. To facilitate the investigation of potential adhesive interactions between parasite and endothelium in the retina, we have modified the Woodruff-Stamper assay, originally described to study lymphocytic-endothelial binding. Vascular endothelium was identified in sections of human retina by Alexa Fluor 594-tagged anti-von Willebrand factor antibody. Binding of yellow fluorescent protein-expressing tachyzoites to endothelium under conditions of flow, simulated by rotation on an orbital shaker, was quantified in a masked fashion using imaging software. We observed multiple yellow spots in contact with Alexa Fluor 594-positive retina, indicating binding of T. gondii tachyzoites to retinal vascular endothelium. This modification of the Woodruff-Stamper assay provides an opportunity to evaluate potential host receptors for T. gondii on the retinal vascular endothelium. In addition, the assay suggests a methodology that could be used to examine adhesion of other microbes to microvasculature in different tissues.

Heterogeneity of 125I-labeled epidermal growth factor.

Highly purified epidermal growth factor (EGF) was iodinated, and the labeled product with the same isoelectric point as underivatized EGF was isolated by isoelectric focusing. When the 125I-labeled EGF was analyzed by reverse-phase chromatography, the resulting profile of 125I activity was much broader than the profile obtained with underivatized EGF. Rechromatography of 125I-EGF fractions indicated that our highly-purified labelled EGF was indeed heterogeneous. Analysis of each HPLC column fraction demonstrated that degradation of EGF had not occurred. The column fractions containing 125I-EGF were pooled into five groups for analysis of cell binding characteristics. Scatchard plot analysis of the five 125I-EGF pools revealed markedly different binding behaviors. In contrast, they had equal potency in stimulating DNA synthesis, within the sensitivity of our assay. Specific activity measurements indicated that the five HPLC pools of 125I-EGF had varying numbers of 125I atoms per EGF molecule. The heterogeneity of the highly purified 125I-EGF and the binding characteristics of the 125I-EGF subfractions pose serious implications for all workers who use iodinated ligands for receptor binding studies.

Digital video-imaging of leukocyte migration in the iris: intravital microscopy in a physiological model during the onset of endotoxin-induced uveitis.

The process of inflammation is accompanied by an alteration of leukocyte-endothelial dynamics. Reciprocal changes in the endothelium and the white cell permit the leukocyte to relinquish its normal free-flowing state in order to roll, arrest, and emigrate through the endothelium. Although intravital microscopy is an established method to observe this process, the eye has been under-utilized for this purpose. Iris vasculature can be videophotographed without the artifact of trauma. We used rhodamine 6G in vivo staining of leukocytes from BALB/c mice in a model of inflammation induced by intravitreally injected endotoxin. Digital video technology was used to record observations at baseline, 2 h, and 4 h after the endotoxin injection. Off-line analysis of microhemodynamic parameters established that the percentage of venules exhibiting rolling increased significantly from 4% at baseline to 34% at 2 h and 82% at 4 h after endotoxin injection. We found a marked increase in leukocyte arrest within 4 h (601+/-119 cells per mm(2) vs. 2+/-1 cells per mm(2) in control animals). Although shear stress differs minimally between iris arterioles and venules, both rolling and arrest occurred preferentially in venules indicating that shear stress is not the dominant factor for determining cell adhesion. Compared to previous reports on intravital microscopy, our methodology includes refinements or advantages in visualizing cells that have transmigrated as well as the avoidance of surgical trauma. The resolution and quantifiable nature of this technique are such that the methodology can be applied to repetitive observation of leukocyte-endothelial dynamics during an immune response.

Ultrastructural immunolocalization of basic fibroblast growth factor in mast cell secretory granules. Morphological evidence for bfgf release through degranulation.

We previously reported that mast cells (MCs) serve as a source of basic fibroblast growth factor (bFGF), a potent angiogenic and mitogenic polypeptide, suggesting that bFGF may mediate MC-related neovascularization and fibroproliferation. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion, and the mechanism(s) for its release remains controversial. Because MCs release a wide spectrum of bioactive products via degranulation, we hypothesized that MC degranulation may be a mechanism of bFGF release and used ultrastructural immunohistochemistry to test the hypothesis. We reasoned that if bFGF is released through degranulation, it should be localized to MC secretory granules. Human tissues with chronic inflammation and rat/mouse tissues with anaphylaxis were studied. In all tissue samples examined, positive staining (or immunogold particle localization) for bFGF in MCs was predominantly in the cytoplasmic granules. Moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles exhibited minimal immunogold particle localization. In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells. In rat/mouse lingual tissue undergoing anaphylaxis, immunogold particle localization for bFGF was found not only in swollen cytoplasmic granules but also in the extruded granules of MCs. Three different anti-bFGF antibodies gave similar immunogold particle localization patterns, whereas all controls were negative. These results provide morphological evidence suggesting that, despite the lack of a classic secretory peptide in its structure, bFGF is localized to the secretory granules in MCs and may be released through degranulation.

Inhibition of leukocyte sticking and infiltration, but not rolling, by antibodies to ICAM-1 and LFA-1 in murine endotoxin-induced uveitis.

PURPOSE: Cell-adhesion molecules are critical elements in intravascular rolling and sticking of leukocytes during acute inflammation. In this process, selectins are thought to be involved in initial adhesion and rolling, and integrin-Ig superfamily interactions are believed primarily to mediate stronger adhesion and transendothelial migration. This study clarifies the role of two adhesion molecules, intercellular adhesion molecule (ICAM)-1 and leukocyte functional antigen (LFA)-1, in endotoxin-induced uveitis (EIU). METHODS: Intravital microscopy was used to record the movement and location of leukocytes in the irises of mice with uveitis induced by intravitreal injection of 250 ng Escherichia coli endotoxin. Each mouse concurrently received an intraperitoneal injection of monoclonal neutralizing antibodies for ICAM-1, LFA-1, or both or control irrelevant antibodies. RESULTS: Mice treated with endotoxin and control antibodies had an inflammatory response that was clearly present at the 6- and 24-hour time points and was mostly resolved by 48 hours. Mice that received anti-ICAM-1 or anti-LFA-1 had significantly fewer cells infiltrating their irises at 6 and 24 hours. Detailed analysis of the 6-hour time point recordings revealed that neither anti-ICAM-1 nor anti-LFA-1 significantly reduced the number of leukocytes rolling on venule endothelial surfaces, but the treatments reduced the number of firmly adherent cells. CONCLUSIONS: These data confirm previous reports that ICAM-1 and LFA-1 are important mediators of EIU. The dynamic in vivo images clearly support the hypothesis that integrin-mediated cell adhesion is more critical for the firm adhesion of sticking cells than for leukocyte rolling.

Reduced leukocyte migration, but normal rolling and arrest, in interleukin-8 receptor homologue knockout mice.

PURPOSE: To determine the role of the murine interleukin-8 receptor homologue (mIL-8Rh, neutrophil chemokine CXC receptor 2) in leukocyte migration using intravital microscopy in a standardized model of eye inflammation, endotoxin-induced uveitis (EIU). METHODS: Two hundred fifty nanograms of E. coli endotoxin was injected into the vitreous of knockout mIL-8Rh(-/-) (n = 7) mice or heterozygous littermate mIL-8Rh(+/-) controls (n = 7). Intravital microscopic examination of iris microvasculature was performed at baseline and 6 and 24 hours after endotoxin injection. The numbers of rolling (cells/mm2 endothelial surface/min), sticking (cells/mm2 endothelial surface), and infiltrating cells (cells/mm2 iris tissue) were evaluated by digital off-line quantification. RESULTS: The number of infiltrating cells was significantly reduced in mIL-8Rh(-/-) mice: 406 +/- 77 cells/mm2 at 6 hours and 242 +/- 50 cells/mm2 at 24 hours in mIL-8Rh(+/-) mice versus 14 +/- 4 cells/mm2 at 6 hours and 38 +/- 11 cells/mm2 at 24 hours in mIL-8Rh(-/-) mice (P < 0.001). In contrast, the absence of the IL-8 receptor homologue did not reduce rolling or sticking. CONCLUSIONS: Iris rhodamine angiography allows precise quantification of leukocyte-endothelial dynamics in the absence of surgical trauma. IL-8 and its homologues are known to be potent signals for leukocyte migration. Although IL-8 has previously been implicated in cell adhesion, video imaging in vivo demonstrated that deletion of the IL-8 receptor homologue had minimal effect on rolling or arrest in this model of inflammation.

Interleukin-6 does not mediate endotoxin-induced uveitis in mice: studies in gene deletion animals.

PURPOSE: Interleukin-6 (IL-6) has been strongly implicated in anterior uveitis based on its presence in aqueous humor from infected eyes and its inflammatory effects when injected intravitreally into rats. We used IL-6-deficient mice to test further the hypothesis that IL-6 contributes to the development of endotoxin-induced uveitis. METHODS: Uveitis was scored by histologic analysis of C3H/HeN mice 24 hours after intravitreal injections of up to 200 ng of recombinant murine IL-6. Uveitis was similarly measured in IL-6-deficient mice and congenic controls 24 hours after intravitreal injection of 250 ng of Escherichia coli endotoxin. Reverse transcription-polymerase chain reaction was used to detect mRNAs for several cytokines at 3 hours postinjection. The IL-6 concentration in aqueous humor samples was determined with a bioassay using the murine B9 plasmacytoma cell line. RESULTS: Direct injection of IL-6 did not induce uveitis. Mice genetically deficient in IL-6 developed endotoxin-induced uveitis that was comparable or more severe than congenic control mice. Compensatory changes in the expression of mRNA for other cytokines were not detected in irises from the IL-6-deficient mice. In IL-6-competent mice that received bilateral endotoxin injections, no correlation was found between the number of infiltrating cells in one eye and the IL-6 concentration in the aqueous humor of the contralateral eye. CONCLUSIONS: In marked contrast to previous conclusions with rats, IL-6 was not sufficient for inducing uveitis in mice. Additionally, IL-6 was not necessary for the development of uveitis subsequent to intravitreal injection of endotoxin in mice.

Cytokine mRNA levels in rat ocular tissues after systemic endotoxin treatment.

PURPOSE: An intertwined cascading network of cytokines is believed to direct the development of endotoxin-induced uveitis (EIU). This study investigated mRNA levels of interleukin (IL) 1 alpha, IL-1 beta, IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta 1, and the helper T lymphocyte marker, CD4, during the course of EIU in rats. METHODS: Reverse transcription followed by polymerase chain reaction amplification was used to determine relative mRNA levels in four ocular tissues (iris/ciliary body, cornea, lens, and neuroretina) at 0, 1, 3, 6, 24, and 48 hours after subcutaneous injection of 200 micrograms of Escherichia coli endotoxin. RESULTS: Four general patterns of mRNA expression were observed: (1) constitutively expressed and unaffected by endotoxin; (2) constitutively expressed but further induced by endotoxin, reaching peak levels at 3 hours postinjection; (3) initially undetectable or marginally detectable and induced by endotoxin, with peak levels occurring 3 hours postinjection; and (4) never present at appreciable levels. The most dramatic responses were seen in the mRNA levels of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma in iris/ciliary body. Lesser mRNA level responses were found for IL-1 beta and IL-6 in cornea and for IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma in neuroretina. Little or no changes in mRNA levels were observed for CD4 or TGF-beta 1 in any tissue or for any mRNA examined in lens. CONCLUSIONS: These data show that subcutaneous endotoxin induces cytokine mRNA expression differentially in ocular tissues. These data support the hypothesis that induction of cytokine expression in iris/ciliary body plays a major role in the development of EIU.

Retinal pigment epithelial cells secrete interleukin-6 in response to interleukin-1.

Interleukin-6 (IL-6) is a peptide whose properties include the ability to activate T-lymphocytes, stimulate the secretion of immunoglobulin, induce neuronal differentiation, and trigger the release of acute phase proteins. We have detected IL-6-like activity in conditioned medium from cultured human retinal pigment epithelial (RPE) cells with a bioassay based on the ability of IL-6 to induce the proliferation of murine B-9 plasmacytoma cells. Biologic activity increased approximately 90-fold when the cells were cultured in the presence of IL-1 alpha (30 units/ml). Western blot analysis confirmed that conditioned medium from IL-1 alpha-stimulated RPE cells contained peptides with molecular weights ranging between 19,000 and 30,000 and reactive with antibody to IL-6. Finally, Northern blot analysis indicated that cells cultured in the presence of interleukin-1 contained a 1.2 kilobase transcript that hybridized to a cDNA probe specific for IL-6 messenger RNA. IL-6 peptide on Western blots and mRNA on Northern blots were undetectable unless cells were cultured in the presence of IL-1 alpha. Although IL-6 is synthesized by a variety of cell types, this report is the first to detect its synthesis by an eye-specific cell type. Furthermore, these observations indicate that retinal pigment epithelial cells respond to IL-1, a cytokine that previously has been implicated in ocular inflammation.

Cell adhesion molecule expression in cultured human iris endothelial cells.

PURPOSE: To develop a method to isolate human iris microvascular endothelial cells (HIECs) for exploring their constitutive and inflammatory agent-modulated expression of intercellular adhesion molecules (ICAM)-1 and -2, vascular cell adhesion molecule (VCAM)-1, and E-selectin. METHODS: Endothelial cells from collagenase-digested irises were isolated on the basis of their expression of platelet endothelial cell adhesion molecule (PECAM)-1, using antibody-coupled magnetic beads. Cells were characterized as endothelial based on morphologic criteria, their expression of PECAM-1 and von Willebrand factor, their uptake of acetylated low-density lipoprotein, and their ability to form capillary-like networks on a synthetic basement membrane. Constitutive and inflammatory agent-modulated expression of ICAM-1 and -2, VCAM-1, and E-selectin was evaluated by the reverse transcription-polymerase chain reaction, enzyme-linked immunocellular assays (ELICAs), Western blot analysis, and functional studies of leukocyte adhesion to HIEC monolayers. RESULTS: HIECs constitutively expressed mRNA and protein for ICAM-1 and -2, but only low to nondetectable levels of VCAM-1 or E-selectin. When stimulated with endotoxin- or tumor necrosis factor (TNF)-alpha, ICAM-1, VCAM-1, and E-selectin were potently and time- and dose-dependently upregulated at both the message and protein levels. By contrast, ICAM-2 message and protein were slowly downregulated by inflammatory agents over time, but nonetheless remained present and functional. Overall, cytokine- or endotoxin-activation of HIECs resulted in enhanced adhesiveness for leukocytes. CONCLUSIONS: ICAM-1, VCAM-1, and E-selectin have been previously implicated in mediating anterior ocular inflammation. This is a report of the selective isolation of HIECs, with a demonstration of differential expression and regulation of these adhesion molecules in them. In addition, this is the first demonstration of the regulated expression of ICAM-2 in any ocular microvascular cells.

IL-1 and TNF receptor-deficient mice show decreased inflammation in an immune complex model of uveitis.

PURPOSE: To determine the role of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the induction of uveitis by a reverse passive Arthus reaction (RPAR). METHODS: Human serum albumin (HSA) antiserum was injected into the vitreous of "knockout" or "double knockout" mice genetically deficient in IL-1 receptor type I (IL-1RI-/-), TNF receptors p55 and p75 (TNFR p55-/-/p75-/-), IL-1RI and TNFR p55 (IL-1RI-/-/TNFR p55-/-), and controls. Twenty-four hours later, animals were challenged with intravenous HSA. Eyes were enucleated 4 hours after antigen challenge, and inflammation was quantitated by counting cells on histologic sections. Interleukin-6 in aqueous humor was measured with a B9 cell bioassay. The distribution of immune complexes in eyes was observed by immunohistochemical staining for IgG and complement component C3. RESULTS: Four hours after antigen challenge, immune complexes were localized at the ciliary body and iris of receptor-deficient mice. A transient uveitis was most severe at this time. A significant reduction in the median number of infiltrating cells was found in TNFR p55-/-/p75-/- mice (4.8, n = 15), compared with controls (14.2, n = 20, P < 0.05). The median number of infiltrating cells was significantly reduced in IL-1RI-/- mice (knockout 2.6, n = 11; controls 7.4, n = 8, P < 0.005). Interleukin-1RI-/-/TNFR p55-/- mice had a strong reduction in infiltrating cells (knockout 1.6, n = 11; controls 27.3, n = 12, P = 0.002). Interleukin-6 activity in aqueous humor was reduced in IL-1RI-/-/TNFR p55-/- mice (P = 0.03) but not in TNFR p55-/-/p75-/- (P = 0.40) mice. Most IL-1RI-/-mice had no detectable aqueous humor IL-6, but this group was not statistically different from controls. CONCLUSIONS: In contrast to endotoxin-induced uveitis, both IL-1 and TNF appear to have critical roles in RPAR uveitis. When receptors for these cytokines were deleted, the severity of immune complex-induced uveitis was profoundly reduced.

Immunolocalization of integrins in the human retina.

PURPOSE: Integrins are cell surface proteins that participate in interactions between cells and with extracellular matrix. Binding of integrins to their ligands influences cell activities including proliferation, migration, and differentiation. Expression of integrin subunits from three different subfamilies were examined in human retina. METHODS: Integrins were detected in frozen sections of two human retinas with an avidin-biotin-complex immunohistochemical technique, using nine different monoclonal antibodies specific for alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha v, beta 1, beta 2, and beta 3. One retina was from a patient who had conjunctival squamous cell carcinoma, and the other was from uninvolved regions of an eye with a choroidal melanoma. RESULTS: All integrins tested were detectable in consistent patterns in two retinas. All except alpha 2 and alpha 4 were stained vibrantly in retinal and choroidal vessels. All alpha subunit staining of vessels showed overlap or close proximity to beta 1 staining. In addition to vessels, beta 1 was also present in the internal limiting membrane; alpha 2, alpha 3, alpha 4, alpha 5, and beta 2 were all found throughout much of the neural retina, albeit with distinctive staining patterns. Other than in association with vessels, alpha 6 and alpha v were not detected in neural retina, and beta 3 was only weakly detected in the nerve fiber layer; alpha 4 and beta 2 were expressed in the retinal pigment epithelium; beta 1 and beta 2 were strongly expressed in drusen present in one of the eyes. CONCLUSION: Nine integrin subunits have been found to have unique distributions in adult human retina. An understanding of the distribution in normal retina can serve as a useful contrast to patterns of staining associated with retinal diseases.

Immunolocalization of integrins in proliferative retinal membranes.

PURPOSE: Integrins are cell surface adhesion molecules that serve as receptors for extracellular matrix components or for other cells. Integrins help regulate processes such as cell proliferation, migration, and differentiation. These processes are thought to have fundamental roles in the pathogenesis of proliferative retinal membranes in diseases such as proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Therefore, the authors sought to determine the expression pattern of integrins in human proliferative membranes. METHODS: Tissue was obtained from two patients with PVR, two with PDR, and one subretinal neovascular membrane from a patient with presumed ocular histoplasmosis. Integrins were detected with an avidin-biotin-complex immunohistochemical technique using nine different monoclonal antibodies specific for alpha subunits 2, 3, 4, 5, 6, and V, and beta subunits 1, 2, and 3. RESULTS: All integrin subunits studied were detectable to varying degrees in proliferative membranes. beta 1 and alpha 6 were especially prominent at the edges of most PVR and PDR membranes. Pigmented cells expressed up to nine different integrin subunits, in contrast to normal RPE cells, which immunostained for only alpha 4 and beta 2. Proliferative diabetic retinopathy vessels expressed all nine integrin subunits examined, including alpha 4, which was poorly expressed in vessels of nondiabetic retinas. CONCLUSIONS: Integrin subunits are readily detectable in pathologic membranes. Both PVR and PDR are associated with altered integrin expression in vascular endothelium and pigmented cells. The distribution of integrins at the edge of a membrane suggests a role in the growth or contraction of the membrane, presumably by participating in the interaction between cells and substances such as vitreous collagen. Therefore, integrin antagonists may hold promise for the treatment of proliferative retinopathies.

Platelet-derived growth factor ligands and receptors immunolocalized in proliferative retinal diseases.

PURPOSE: Platelet-derived growth factor (PDGF) and its receptors could contribute to the development of proliferative retinal membranes, because PDGF is angiogenic and is both mitogenic and chemotactic for retinal pigment epithelial (RPE) and glial cells, components of membranes. The authors sought to determine whether PDGF ligands and their receptors were present in proliferative retinal membranes. METHODS: To localize PDGF ligands and receptors, the authors examined normal postmortem control retinas, intact eyes with proliferative vitreoretinopathy (PVR) or proliferative diabetic retinopathy (PDR), and membranes removed by vitrectomy from patients with PVR, epimacular proliferation, PDR, or PVR with PDR of previous onset. Sections were stained with antibodies specific for each PDGF ligand and receptor, using an avidin-biotin-complex immunohistochemical technique. To correlate PDGF receptor beta (PDGFR beta) and ligand immunostaining, sections were doubled labelled with antibodies specific for either PDGF-A or PDGF-B. RESULTS: Ligands. In the normal retina and choroid, staining for the A-chain was limited to vascular cells. Only the nerve fiber layer and vessels were positive for the B-chain. In diseased tissue, PDGF-A immunoreactivity was detected as intense staining ( ) of all but one of the proliferative retinal membranes; some RPE cells were positive for PDGF-A, especially in the eye with PDR. PDGF-B was also present in many proliferative retinal membranes but not in RPE cells. Receptors. In the normal retina and choroid, both PDGFR alpha and PDGFR beta were detected only in vessels. In proliferative retinal membranes, both receptors were detected in vessels. Long strands of RPE-like cells at the edges of PVR membranes were strongly positive for PDGFR beta but negative or +/-, respectively, for PDGFR alpha. Double-label assays showed that PDGFR beta was often colocalized with each PDGF ligand, especially in pigmented cells. CONCLUSIONS: PDGF ligands and receptors are widespread in proliferative retinal membranes of different origin. Because PDGFR beta and PDGF-B were colocalized in many of the same cells, the potential for autocrine and paracrine stimulation of cell migration and growth exists. These results are consistent with a role for PDGF ligands and receptors in the pathogenesis of different proliferative retinopathies.

A novel model of bacterially-induced acute anterior uveitis in rats and the lack of effect from HLA-B27 expression.

BACKGROUND: Humans with the major histocompatibility antigen B27 (HLA-B27) are especially at risk for developing rheumatic disorders such as ankylosing spondylitis and Reiter's syndrome. Acute anterior uveitis (AAU) often occurs in association with these diseases or in HLA B27 positive individuals without joint disease. METHODS: We induced acute anterior uveitis in Lewis rats by a standard model, the intraperitoneal injection of 200 micrograms of Escherichia coli endotoxin. We also developed a novel model of uveitis secondary to gram-negative infection. RESULTS: Transgenic rats that expressed a low copy number of the B27 gene did not differ statistically from litter mate controls in the intensity of anterior uveitis as judged by histology, enumeration of cells in aqueous humor, protein in aqueous humor, or slit lamp examination. The majority of rats exposed to live Salmonella enteritidis or Yersinia enterocolitica 0:3 using either an oral or intravenous route of infection developed anterior uveitis. In contrast to the disease induced by endotoxin that is most intense 24 hours after the endotoxin challenge, uveitis induced by live bacteria usually began 7 to 9 days after exposure to bacterial products, was more often unilateral, persisted for as long as 3 weeks, and was sometimes recurrent. The expression of HLA-B27 did not appear to influence the incidence or severity of uveitis in B27+ low copy heterozygous animals. CONCLUSION: This rat model of AAU should facilitate evaluation of bacterial antigenic component(s) involved in the pathogenesis of live gram-negative bacteria induced AAU.

Immunolocalization of transforming growth factor-alpha and its receptor in the normal and hyperoxia-exposed neonatal rat retina.

PURPOSE: Transforming growth factor-alpha (TGF-alpha) is a mitogenic polypeptide for a variety of different cells types including retinal neurons and glial cells. We have examined the temporal and spatial expression of TGF-alpha and its receptor in the normal and hyperoxia-exposed neonatal rat retina to determine if the expression is consistent with a role in retinal development and response to retinal injury. METHODS: We have used immunohistochemistry to examine TGF-alpha and epidermal growth factor receptor (EGF-R) on postnatal days (1, 5, 10, 14, 18, and 25). To examine TGF-alpha and EGF-R expression after retinal injury we studied the retinas from rats which were exposed to 80% oxygen for 10 days and then recovered in room air. Immunolocalization of type IV collagen was performed to examine the retinal vasculature development after hyperoxia. RESULTS: The pattern of TGF-alpha and EGF-R expression in the neural retina evolved from a diffuse pattern on postnatal day 1 to restricted sites on postnatal day 14. The TGF-alpha immunoreactivity was consistent with localization in Müller cells on postnatal day 14. Both TGF-alpha and EGF-R patterns were altered in the retinas from rats that had been exposed to hyperoxia and recovered in room air for 4 days. The type IV confirmed immunostaining confirmed vaso-obliteration in the deep layer of retinal vessels after hyperoxia. CONCLUSIONS: Our findings of altered expression of TGF-alpha and EGF-R during retinal development suggests a biological function for this growth factor, possibly promoting retinal cell proliferation, differentiation, and survival. The altered immunolocalization of TGF-alpha and EGF-R in the hyperoxia-exposed retina suggest that TGF-alpha is likely involved in the retinal response to injury.