Involvement of apoptosis and interferon-gamma in murine toxoplasmosis.

PURPOSE: A murine toxoplasmosis model has been developed that results in central nervous system (CNS) and ocular inflammation characterized by encephalitis with numerous brain tissue cysts and milder inflammation with rare tissue cysts in the eye after 4 weeks of Toxoplasma gondii infection. In this model IFN gamma and inducible nitric oxide (iNO) are protective against T. gondii infection. In this study, the role of apoptosis in the pathogenesis of toxoplasmosis was investigated. METHODS: C57BL/6 (wild-type mice), B6MRL/lpr, and B6MRL/gld (defective Fas or FasL expression, respectively) mice were infected intraperitoneally with 20 to 30 tissue cysts of the ME-49 strain of T. gondii. Mice were killed at days 0, 14, or 28 after infection. The eyes and brains were harvested for histologic, immunohistochemical, and molecular studies. Analysis included immunostaining for Fas, FasL, Bcl-2, and Bax; in situ apoptosis detection (TUNEL assay); RT-PCR amplification for IFN gamma; and measurement of ocular nitrite levels. The control mice were naïve mice of each strain that received no inoculation or injection. RESULTS: Wild-type mice appeared to constitutively express apoptotic molecules at higher levels in the eye than in the brain. Consequently, during T. gondii infection, apoptosis was greater in the eyes than in the brain. Untreated naïve lpr and gld mice showed no expression of Fas and FasL, respectively. After infection, a slightly higher number of tissue cysts (lpr, 11.8 +/- 2.4; gld, 10.3 +/- 3.4) were found in the brains of the mutants than in the control animals (8.8 +/- 2.9). However, no significant differences between the number of apoptotic cells, inflammatory scores, or number of tissue cysts were noted in the eyes. IFN gamma mRNA in control mice was detected at day 28 after infection, whereas in both mutants, mRNA production occurred earlier, at day 14. Ocular nitrite levels were higher in lpr and gld mice than in wild-type mice. CONCLUSIONS: No significant difference in the degree of ocular inflammation and apoptosis was detected between the wild-type and Fas or FasL mutant mice. However, there was an earlier and subjectively greater expression of IFN gamma in the brain and eye and a higher level of nitrite in the ocular tissue of mutant strains than in the wild type. Multiple factors are likely to be involved in the pathogenesis of ocular toxoplasmosis.

Apoptosis in cytomegalovirus retinitis associated with AIDS.

PURPOSE: To determine the role of apoptosis in the pathogenesis of cytomegalovirus retinitis in patients with the acquired immunodeficiency syndrome. METHODS: Forty-three eyes from patients with cytomegalovirus retinitis treated before the introduction of highly active anti-retroviral therapy were examined by routine histopathology and in situ techniques to detect apoptosis (TUNEL assay). Apoptosis was graded on a scale from 0 to 3+ by quantitating the number of TUNEL positive cells per case using a standard grading procedure. Statistical analysis describing the association between apoptosis grade and the proportions of eyes with active CMV infection, with choroidal inflammation and treated with the sustained-release intravitreal ganciclovir implant was performed using the Armitage procedure. RESULTS: Apoptosis in both CMV infected and uninfected retinal cells was detected in 28 of 41 eyes (68%); 13 (45%) with active and 15 (55%) with inactive cytomegalovirus retinitis. The degree of apoptosis was mild (1+) in 14 eyes, moderate (2+) in 6 eyes and severe (3+) in 8 eyes. Apoptosis was not identified in two eyes without CMVR. An increase in apoptosis grade was positively associated with active CMVR (p = 0.014). There was no significant association between apoptosis and choroidal inflammation. The presence and the severity of apoptosis was less in eyes treated with the sustained-release intravitreal ganciclovir implant compared to those treated with systemic anti-viral therapy, however, the difference was not statistically significant. CONCLUSIONS: Apoptosis contributes to retinal cell loss in eyes with cytomegalovirus retinitis associated with AIDS but did not correlate with the progressive loss of retinal cell function in patients with treated, inactive CMVR.

Biphasic ocular inflammatory response to endotoxin-induced uveitis in the mouse.

OBJECTIVE: To examine the kinetics and mechanisms of endotoxin-induced uveitis in the mouse. METHODS: C3H/HeN mice were injected subcutaneously with 0.3 mg of Salmonella typhimurium lipopolysaccharide (LPS) in 0.1 mL of phosphate-buffered saline solution or phosphate-buffered saline solution alone in 3 separate experiments; mice were killed after 1, 3, 5, and 7 days. In 2 other separate experiments, mice were killed 1, 3, 6, and 24 hours after LPS injection. All eyes were collected for histological examination, immunohistochemical analyses, aqueous protein level determination, and reverse transcriptase-polymerase chain reaction for ocular interleukin (IL)1alpha, IL-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor messenger RNA (mRNA). Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha and IL-6 levels in aqueous and serum samples. RESULTS: Results were consistent for all experiments. Numbers of ocular inflammatory cells and levels of aqueous protein peaked 1 and 5 days after LPS injection. Control mice did not develop inflammation. Serum and aqueous IL-6 and ocular IL-6 mRNA levels peaked at 1 day and subsided at 3 days. However, ocular IL-1alpha, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor mRNA appeared, peaked, and subsided at 3, 5, and 7 days, respectively. Predominant infiltrating cells were neutrophils at 1 day and macrophages at 5 days. Although no ocular inflammatory cells were detected before 24 hours after LPS injection, tumor necrosis factor alpha mRNA was noticed at 1 hour, peaked at 3 hours, and disappeared at 6 hours and granulocyte-macrophage colony-stimulating factor mRNA was spotted only at 3 hours after LPS injection. CONCLUSIONS: The ocular inflammatory response to C3H/ HeN mouse endotoxin-induced uveitis is biphasic for 7 days. The first wave appears at day 1 and subsides by day 3. A second, higher peak appears at day 5. The 2 inflammatory waves are related to the kinetics of the different cytokines released in the eye. This is in contrast to the rat monophasic endotoxin-induced uveitis model, which has only one peak of intense inflammation associated with cytokine release. CLINICAL RELEVANCE: A biphasic inflammatory response associated with cytokine release lasting several days is observed in C3H/HeN mice with endotoxin-induced uveitis. Because human anterior uveitis has a tendency to be recurrent in nature, this might be a better experimental model.

Cytokines and apoptotic molecules in experimental melanin-protein induced uveitis (EMIU) and experimental autoimmune uveoretinitis (EAU).

The cytokine profile and occurrence of apoptosis during experimental melanin-protein induced uveitis (EMIU) were investigated and compared with that of experimental autoimmune uveoretinitis (EAU). EMIU or EAU was induced in Lewis rats. Eyes were collected at different time points after immunization. Cytokine mRNA expression was identified in the inflammatory cells in the uvea of EMIU rats; IL-2, IFN-gamma and IL-12 increased at the peak of the inflammation, and then tapered off as inflammation subsided. IL-4 and IL-10 increased at the peak of ocular inflammation, and persisted with inflammation resolved. Fas and FasL were expressed consistently in ocular resident cells of EMIU, but were elevated in EAU. In EAU, Bcl-2 expression showed a sharp peak in inflammatory cells but not in the resident cells. In EMIU, high levels of Bcl-2 were present and persisted in both ocular resident and inflammatory cells. Expression of Bax was relatively stable in both EAU and EMIU. Cellular DNA fragmentation was detected in the retinal glial cells of EAU and some inflammatory cells of EMIU. In EMIU, the dynamics of Th1 cytokines were consistent with the ocular inflammation, whereas persistent expression of Th2 cytokines was consistent with their known regulatory role. The continuous high expression of Bcl-2 and the high ratio of Bcl-2 to Bax in the eyes of EMIU may possibly contribute to prevention of ocular tissue damage, and of inflammatory cells from undergoing apoptosis, thus resulting in chronic recurrent inflammation.

Inhibition of experimental melanin protein-induced uveitis (EMIU) by targeting nitric oxide via phosphatidylcholine-specific phospholipase C.

Experimental melanin protein-induced uveitis (EMIU) is an autoimmune uveitis induced by immunization with uveal melanin protein. Fas and FasL enhancement is reported in rats with EMIU. Tricyclodecan-9-yl-xanthogenate (D609), a specific inhibitor of phosphatidylcholine-specific phospholipase C, inhibits inducible nitric oxide synthase (iNOS) induction. In two independent experiments, 35 Lewis rats with EMIU received either D609 or PBS daily. The eyes and draining lymph nodes were collected for histology, analyses of nitrite, peroxide, and superoxide dismutase, Fas and FasL immunochemistry, in situ hybridization for iNOS mRNA and in situ apoptosis detection at the peak of the disease. Both experiments showed significant inhibition of EMIU by D609. Decreases in nitrite and peroxide, increase of superoxide dismutase and lower expressions of iNOS mRNA were found in D609-treated, as compared to PBS-treated eyes. There was mild enhancement of Fas and FasL in the eyes and lymph nodes of D609-injected animals. DNA fragmentation was increased in the lymph nodes of D609-treated rats. We conclude that iNOS activation is responsible for NO production in eyes with EMIU. The suppressive effect of D609 on EMIU may result from scavenging NO and activating apoptosis previously inhibited by NO along with other anti-inflammatory effects.

Morphologic and genetic analysis of retinal angioma associated with massive gliosis in a patient with von Hippel-Lindau disease.

We report morphologic and genetic analysis of bilateral retinal angiomas in a 35-year-old patient with von Hippel-Lindau (VHL) disease. Enucleation of both eyes revealed extensive intraocular tumor. Whereas the right eye demonstrated large amounts of retinal angioma tissue, the left eye showed small areas of retinal angioma associated with massive diffuse retinal gliosis. Genetic analysis of the angioma showed allelic deletion of the VHL gene locus, suggesting that the origin of the angiomas was directly related to the patient's underlying VHL disease. Genetic analysis of the pleomorphic glial proliferation showed no allelic VHL gene deletion, which is consistent with the assessment that the glial component represents a reactive process. Apoptosis detected by TUNEL revealed lack of DNA fragmentation in the angioma; in contrast, many positive signals were found in the massive gliosis. We confirmed that the abnormal VHL genes were located in the "stromal" cells of the retinal angioma. Massive gliosis in VHL disease is a true reactive retinal gliosis.

VHL gene deletion and enhanced VEGF gene expression detected in the stromal cells of retinal angioma.

OBJECTIVE: Retinal angioma frequently occurs in von Hippel-Lindau (VHL) disease. However, VHL gene alterations have not been documented in retinal angiomas. METHODS: Using tissue microdissection and polymerase chain reaction amplification, we have analyzed 7 retinal angiomas associated with VHL disease for loss of heterozygosity of the VHL gene. In addition, vascular endothelial growth factor expression was evaluated in these tumors by immunohistochemistry and in situ hybridization. RESULTS: All 6 informative retinal angiomas showed loss of heterozygosity of the VHL gene. Loss of heterozygosity was detected in vacuolated "stromal" cells, but not in vascular cells or reactive glial tissue. Vascular endothelial growth factor protein and messenger RNA were also present in vacuolated "stromal" cells. CONCLUSIONS: These findings suggest that vacuolated "stromal" cells represent the true neoplastic component in retinal angioma. These cells express vascular endothelial growth factor and therefore may be responsible for abundant neovascularization of retinal angioma.

Conjunctival epithelial cells cultured on human amniotic membrane fail to transdifferentiate into corneal epithelial-type cells.

PURPOSE: Previous studies on the use of human amniotic membrane (HAM) in rabbit stem cell deficiency models have found the new epithelium growing over the HAM to express cornea-specific keratins (K3 and K12) in 40% of the cases, suggesting that HAM may have induced conjunctival epithelial cells to transdifferentiate into cornea-type epithelial cells. The current study was performed to determine whether HAM could induce transdifferentiation of conjunctival epithelia] cells when cultured in vitro. METHODS: Conjunctival grafts taken from the fornices of New Zealand white rabbits (6-12 weeks old) were placed over HAMs and lifted to an air-media interface using polypropylene double rings. These cultures were maintained in supplemented hormonal epithelial medium with and without 3T3 feeder cells. Rabbit corneal epithelial cells were cultured similarly using strips of keratolimbal grafts placed over HAM. The cultures were terminated at various times between the 8th and 15th day. The cultured epithelial cells were examined histologically and immunohistochemically using monoclonal antibodies AK-2 (to K12 keratin), AM-3 (to goblet cell mucin), and AE-5 (to K3 keratin). RESULTS: Both conjunctival and corneal epithelial cells cultured on HAMs showed multilayered, differentiated epithelial structures. On immunohistochemical examination, both epithelial cells stained positive for AE-5. None of the cultured conjunctival epithelial cells stained positively for AK-2, while the corneal epithelial cells showed positive staining with AK-2. There were no AM-3-positive goblet cells in either epithelial cell culture. There was no difference in the immunohistochemical patterns between cultures with or without 3T3 feeder cells. However, culture without feeder cells seemed to manifest a more degenerative appearance than those with feeders. CONCLUSION: HAM does not induce transdifferentiation of conjunctival epithelial cells into corneal-type epithelial cells under the in vitro culture conditions used in this study.

Inflammatory response in the early stages of wound healing after excimer laser keratectomy.

OBJECTIVE: To evaluate the inflammatory response and its potential role in the early stages of corneal wound healing after excimer laser keratectomy. MATERIALS AND METHODS: Lewis rats underwent excimer keratectomy using a 193-nm excimer laser. The central corneas were ablated in 3 depths: group A, epithelium; group B, superficial stroma; or group C, deep stroma. Eyes were harvested 1, 12, 24, and 36 hours, and 1 week after the rats were killed. Immunohistochemistry was used to test frozen sections with monoclonal antibodies of various inflammatory cellular markers. RESULTS: Reepithelialization was observed at 12 hours in group A, and at 24 hours in groups B and C. Regenerated epithelium covered the denuded corneal surface in groups B and C after 1 week. The expression of major histocompatibility complex II antigen was detected in infiltrating cells, corneal epithelial cells, and endothelial cells 1 hour after surgery. Only a few macrophages and Langerhans cells were in the limbus at baseline. Macrophages migrated from the limbus to the corneal ablation zone and increased 2-fold after 36 hours in all 3 groups compared with baseline. Occasional lymphocytic infiltration was identified after 25 to 36 hours. CONCLUSION: Macrophages play an active role in the wound healing after laser keratectomy and may contribute to transient corneal haze.

Increased vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGFbeta) in experimental autoimmune uveoretinitis: upregulation of VEGF without neovascularization.

Experimental autoimmune uveoretinitis (EAU) was induced in Lewis rats and B10.A mice by immunization with S-antigen (S-Ag) to study the potential roles of vascular endothelial growth factor (VEGF) and the beta1 and beta2 isoforms of transforming growth factor (TGFbeta1 and TGFbeta2) during the progression of the disease. VEGF has been implicated as an angiogenic factor in ischemic retinopathies; however, Lewis rats developing EAU have high levels of VEGF in the retina, but no neovascularization. In the present study, immunohistochemical staining for VEGF, TGFbeta1 and TGFbeta2 was performed on the retinas of Lewis rats developing EAU or with oxygen-induced ischemic retinopathy. In rats immunized with S-antigen, a marked upregulation of VEGF was immunohistochemically visualized from the inner nuclear layer to the inner limiting membrane prior to blood-retinal barrier (BRB) failure and lymphocytic infiltration. VEGF is normally induced by hypoxia and its induction leads to neovascularization. Coincident with the increase in VEGF, there was increased immunoreactivity for TGFbeta1 and TGFbeta2 within the same layers of the retina. In contrast, rats with ischemic retinopathy and retinal neovascularization showed only a modest increase in VEGF immunoreactivity, which is largely confined to retinal ganglion cells and inner retinal vessels, and little or no increase in TGFbeta1 or TGFbeta2. In addition, in mice developing EAU, which does not have an abrupt onset as it does in rats and may involve neovascularization, a comparable upregulation of VEGF in the inner retina to that seen in rats developing EAU occurs with no increase in TGFbeta1 or TGFbeta2. Since TGFbeta can inhibit endothelial cell proliferation, it is likely that an increase in TGFbeta may prevent VEGF from exerting its endothelial growth activity in the rat EAU model, but VEGF may be operative in inducing BRB failure. These data suggest that there is a complex interaction among growth factors in the retina and that retinal neovascularization may require an imbalance between stimulatory and inhibitory factors.

Suppressive effect of antiflammin-2 on compound 48/80-induced conjunctivitis. Role of phospholipase A2s and inducible nitric oxide synthase.

Phospholipase A2s (PLA2s) are a family of esterases that initiate the arachidonic acid cascade, which results in the production of numerous inflammatory mediators. We investigated the expression of Group I and II PLA2 proteins and Group II mRNA in normal conjunctivae and in the conjunctivae of mice with compound 48/80-induced conjunctivitis. Conjunctivitis was induced in C57BL/6 mice by topical instillation of compound 48/80 (C48/80). Mice were then treated with corticosteroid (Pred Forte), antiflammin-2 (AF2, a synthetic peptide that inhibits PLA2), or a placebo (Dacriose, an isotonic, buffered, sterile eye irrigating solution). Low levels of PLA2s were detected on the epithelium of normal conjunctivae. One hr after C48/80 instillation, the expression of PLA2s appeared and increased in the substantia propria, peaked at 6 hr, and returned to baseline 72 hr later. Compared to the placebo, the conjunctivitis was moderate in the AF2-treated group and mild in Pred Forte-treated group. The expression of PLA2s was suppressed in mice treated with Pred Forte and AF2. iNOS mRNA was also diminished in the AF2- and Pred Forte-treated groups. The mechanisms by which anti-allergic medications suppress conjunctivitis may involve the inhibition of PLA2s and iNOS.

Recombinant adenovirus encoding gp100 modulates experimental melanin-protein induced uveitis (EMIU).

Experimental melanin-protein induced uveitis (EMIU) is a T-cell mediated autoimmune uveitis induced by immunization with bovine uveal melanin protein. Gp100, a melanocyte lineage-specific protein, is identified as a human melanoma antigen. A recombinant adenovirus construct encoding gp100 (Ad2CMV-gp100) has been used as a vaccine for cancer therapy. This study examines the effect of Ad2CMV-gp100 on EMIU. To induce EMIU, rats were injected intraperitoneally on day 7 before immunization with ad2CMV-gp100, control adenovirus encoding LacZ (Ad2CMV-LacZ), or no virus. On day 21 after immunization, the right eye was processed for histology and the left eye was analysed for cytokines by quantitative reverse transcriptase-polymerase chain reaction. Western blot analysis showed that uveal melanin-protein contains gp100. In three independent experiments, ocular inflammation was significantly suppressed, and expression of ocular IL-12p40 mRNA was much lower in the rats which received Ad2CMV-gp100 before immunization than in those that received Ad2CMV-LacZ or no virus. No abnormalities developed in rats which received Ad2CMV-gp100 or Ad2CMV-LacZ alone. Therefore, Ad2CMV-gp100 injection prevents the development of EMIU, at least in part, through cytokine regulation.

Apoptosis in patients with posterior uveitis.

BACKGROUND: Apoptosis plays a part in the pathogenesis of autoimmune diseases. OBJECTIVE: To investigate the expression of apoptotic markers in the eyes of patients with uveitis. METHODS: With the use of immunohistochemical and in situ apoptotic detection techniques, apoptotic molecules (Fas or Fas ligand [FasL]) and nuclear DNA fragmentation were examined in 8 enucleated eyes with Behçet's disease (1), sarcoidosis (1), subretinal fibrosis and uveitis (1), sympathetic ophthalmia (4), and the Vogt-Koyanagi-Harada syndrome (1); in 5 chorioretinal biopsy specimens with acute retinal necrosis (2), multifocal choroiditis (1), sarcoidosis (1), and subretinal fibrosis and uveitis (1); and in 3 normal control eyes. RESULTS: Fas and FasL were constitutively expressed in the normal human retina, but they were expressed much less in the choroid. Increased expression of Fas and FasL was found in the retina, chorioretinal scar, and choroidal granulomas in uveitic eyes. However, Fas and FasL expression was absent in the biopsy specimens with acute retinal necrosis, and little Fas or FasL was noted on infiltrating lymphocytes. DNA fragmentation was also identified in eyes with chorioretinal scar and gliosis. CONCLUSIONS: Apoptosis occurs in uveitic eyes and may play a regulatory role in limiting ocular inflammation. In uveitic eyes, a dysregulation of the Fas-FasL apoptotic pathway may lead to gliosis and fibrosis.