Müller glia as an important source of cytokines and inflammatory factors present in the gliotic retina during proliferative vitreoretinopathy.

Retinal gliosis is characterized by biochemical and physiological changes that often lead to Müller glia proliferation and hypertrophy and is a feature of many neuro-degenerative and inflammatory diseases such as proliferative vitreoretinopathy (PVR). Although Müller glia are known to release inflammatory factors and cytokines, it is not clear whether cytokine production by these cells mirrors the pattern of factors present in the gliotic retina. Lysates from normal cadaveric retina and gliotic retinal specimens from patients undergoing retinectomy for treatment of PVR, the Müller cell line MIO-M1 and four human Müller glial cell preparations isolated from normal retina were examined for their expression of cytokines and inflammatory factors using semi-quantitative dot blot antibody arrays and quantitative arrays. Comparative analysis of the expression of inflammatory factors showed that in comparison with normal retina, gliotic retina exhibited greater than twofold increase in 24/102 factors examined by semiquantitative arrays, and a significant increase in 19 out of 27 factors assessed by quantitative methods (P < 0.05 to P < 0.001). It was observed that with the exception of some chemotactic factors, the majority of cytokines and inflammatory factors were produced by Müller glia in vitro and included G-CSF, MCP-1, PDGF-bb, RANTES, VEGF, and TGFß2. These results showed that a large number of inflammatory factors expressed by Müller glia in vitro are upregulated in the gliotic retina, suggesting that targeting the production of inflammatory factors by Müller glia may constitute a valid approach to prevent neural damage during retinal gliosis and this merits further investigations.

Prospects for the application of Müller glia and their derivatives in retinal regenerative therapies.

Neural cell death is the main feature of all retinal degenerative disorders that lead to blindness. Despite therapeutic advances, progression of retinal disease cannot always be prevented, and once neuronal cell damage occurs, visual loss cannot be reversed. Recent research in the stem cell field, and the identification of Müller glia with stem cell characteristics in the human eye, have provided hope for the use of these cells in retinal therapies to restore vision. Müller glial cells, which are the major structural cells of the retina, play a very important role in retinal homeostasis during health and disease. They are responsible for the spontaneous retinal regeneration observed in zebrafish and lower vertebrates during early postnatal life, and despite the presence of Müller glia with stem cell characteristics in the adult mammalian retina, there is no evidence that they promote regeneration in humans. Like many other stem cells and neurons derived from pluripotent stem cells, Müller glia with stem cell potential do not differentiate into retinal neurons or integrate into the retina when transplanted into the vitreous of experimental animals with retinal degeneration. However, despite their lack of integration, grafted Müller glia have been shown to induce partial restoration of visual function in spontaneous or induced experimental models of photoreceptor or retinal ganglion cell damage. This improvement in visual function observed after Müller cell transplantation has been ascribed to the release of neuroprotective factors that promote the repair and survival of damaged neurons. Due to the development and availability of pluripotent stem cell lines for therapeutic uses, derivation of Müller cells from retinal organoids formed by iPSC and ESC has provided more realistic prospects for the application of these cells to retinal therapies. Several opportunities for research in the regenerative field have also been unlocked in recent years due to a better understanding of the genomic and proteomic profiles of the developing and regenerating retina in zebrafish, providing the basis for further studies of the human retina. In addition, the increased interest on the nature and function of cellular organelle release and the characterization of molecular components of exosomes released by Müller glia, may help us to design new approaches that could be applied to the development of more effective treatments for retinal degenerative diseases.

Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE).

AIMS/HYPOTHESIS: Up-regulation of the receptor for AGEs (RAGE) and its ligands in diabetes has been observed in various tissues. Here, we sought to determine levels of RAGE and one of its most important ligands, S100B, in diabetic retina, and to investigate the regulatory role of S100B and RAGE in Müller glia. METHODS: Streptozotocin-diabetes was induced in Sprague-Dawley rats. RAGE, S100B and glial fibrillary acidic protein (GFAP) were detected in retinal cryosections. In parallel, the human retinal Müller cell line, MIO-M1, was maintained in normal glucose (5.5 mmol/l) or high glucose (25 mmol/l). RAGE knockdown was achieved using small interfering RNA (siRNA), while soluble RAGE was used as a competitive inhibitor of RAGE ligand binding. RAGE, S100B and cytokines were detected using quantitative RT-PCR, western blotting, cytokine protein arrays or ELISA. Activation of mitogen-activated protein kinase (MAPK) by RAGE was determined by western blotting. RESULTS: Compared with non-diabetic controls, RAGE and S100B were significantly elevated in the diabetic retina with apparent localisation in the Müller glia, occurring concomitantly with upregulation of GFAP. Exposure of MIO-M1 cells to high glucose induced increased production of RAGE and S100B. RAGE signalling via MAPK pathway was linked to cytokine production. Blockade of RAGE prevented cytokine responses induced by high glucose and S100B in Müller glia. CONCLUSIONS/INTERPRETATION: Hyperglycaemia in vivo and in vitro exposure to high glucose induce upregulation of RAGE and its ligands, leading to RAGE signalling, which links to pro-inflammatory responses by retinal Müller glia. These data shed light on the potential clinical application of RAGE blockade to inhibit the progression of diabetic retinopathy.

Triamcinolone attenuates macrophage/microglia accumulation associated with NMDA-induced RGC death and facilitates survival of Müller stem cell grafts.

Retinal ganglion cell (RGC) death in glaucoma models is associated with accumulation of activated microglia, a sign of neural degeneration which has been shown to constitute a barrier for transplant cell survival and migration. This study investigated the use of triamcinolone (TA) to control macrophage/microglia accumulation in a model of RGC depletion to create a permissive environment for stem cell grafting. Intravitreal NMDA alone or in combination with TA was used to induce rapid onset of RGC death in 3-4 week old Lister hooded (LH) rat eyes prior to Müller stem cell transplantation into the vitreoretinal space. The effect of NMDA on RGC death and microglial accumulation was assessed immuno-histochemically, whilst electroretinography (ERG) was used to assess RGC function. Post transplantation, survival of grafted cells and their association with microglia were also examined by immunohistochemical methods. Intravitreal injection of NMDA alone resulted in severe macrophage/microglia accumulation associated with extensive RGC death 4-7 days post-treatment. Combination of NMDA with TA significantly reduced microglial numbers in the RGC when compared to NMDA only treated eyes while still depleting the retina of RGC. At the same time, NMDA/TA treatment also caused functional RGC loss as demonstrated by reduction of the scotopic threshold response. Upon transplantation with Müller stem cells, NMDA/TA treatment caused significant reduction in the number of transplant associated macrophage/microglia compared to eyes treated with NMDA alone. On this basis it is proposed that intravitreal injection of TA may be useful as an effective anti-inflammatory agent to control macrophage/microglia accumulation induced by RGC death, thereby creating a retinal environment permissive to cell transplantation studies.

Current prospects for adult stem cell-based therapies in ocular repair and regeneration.

Recent advances in stem cell biology have led to the exploration of stem cell-based therapies to treat a wide range of human diseases. In the ophthalmic field, much hope has been placed on the potential use of these cells to restore sight, particularly in those conditions in which other established treatments have failed and in which visual function has been irreversibly damaged by disease or injury. At present, there are many limitations for the immediate use of embryonic stem cells to treat ocular disease, and as more evidence emerges that adult stem cells are present in the adult human eye, it is clear that these cells may have advantages to develop into feasible therapeutic treatments without the problems associated with embryonic research and immune rejection. Here we discuss the current prospects for the application of various adult ocular stem cells to human therapies for restoration of vision.

Immune function, mutant frequency, and cancer risk in the DNA repair defective genodermatoses xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy.

There is evidence for defective DNA repair in xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy, but for increased cancer risk only in xeroderma pigmentosum. Natural and adaptive immune surveillance and mutant frequency to 6-thioguanine resistance in circulating T-lymphocytes were studied in five patients with xeroderma pigmentosum, two with Cockayne's syndrome, and one with trichothiodystrophy. Forty-eight-hour cutaneous hypersensitivity responses to recall antigens excluded anergy and circulating CD3+, CD4+, CD8+, and CD16+ cell numbers were within normal limits in all patients tested, as were proliferative lymphocyte responses to PHA, except in the trichothiodystrophy patient. Proliferative responses to recall antigens (PPD, SKSD, and Candida) showed that all patients responded to one or more antigens. Direct natural killer cytotoxicity measured against the human erythromyeloid leukaemia cell line K562 using a 4-h 51Cr release assay was significantly reduced in xeroderma pigmentosum (specific cytotoxicity less than mean +/- SD greater than 17.4 +/- 9.4 per cent, with effector:target cell ratio of 50:1) compared to normal controls (45.8 +/- 17.8), but normal in Cockayne's syndrome and trichothiodystrophy. Generation of lymphokine activated killer cell activity was normal in the two xeroderma pigmentosum lines tested. The mutant frequency in the xeroderma pigmentosum donors was significantly increased (p less than 0.01) and was elevated in the two Cockayne's syndrome donors, taking age into account. No mutants were observed from the single trichothiodystrophy donor. These findings suggest that reduced natural killer cell activity may contribute to the greatly increased susceptibility to skin cancer in xeroderma pigmentosum.

Age-dependent variation in metalloproteinase activity of isolated human Bruch's membrane and choroid.

PURPOSE: To characterize and determine the effect of aging on the matrix metalloproteinase (MMP) component of the extracellular matrix-remodeling mechanism of isolated human Bruch's-choroid. METHODS: Immunohistochemical techniques and western blot analysis were used to detect and localize various members of the MMP family of proteolytic enzymes in the Bruch's-choroid complex. Gelatin substrate zymography was used to detect and quantify the levels of MMP-2 and -9 in homogenates of Bruch's-choroid from both macular and peripheral regions of the human fundus. Aging alterations in these enzymes were quantified by densitometric analysis of photographic negatives of the zymography gels. RESULTS: Intact preparations of Bruch's-choroid showed the presence of inactive forms of two gelatinases (MMP-2, 65 kDa, and MMP-9, 92 kDa), interstitial collagenase (MMP-1, 52 kDa) and stromelysin (MMP-3, 57 kDa). MMP-1 and -3 were localized primarily to Bruch's membrane. MMP-9 was distributed evenly in Bruch's membrane with some patchy presence in the choroidal mass. Distribution of MMP-2 was similar to that of MMP-9, but the staining in Bruch's was much fainter. On gelatin zymography, an active form of MMP-2 (58-kDa species) was frequently observed in peripheral samples but only occasionally in macular regions. The levels of MMP-2 and -9 increased with aging in both the macular and the peripheral regions of the fundus (P < 0.05). MMP-2 levels were lower in macular regions than in the periphery but no such variation was observed with MMP-9. Both these inactive gelatinases could be activated in vitro. CONCLUSIONS: A matrix-degrading mechanism essential for extracellular remodeling was shown to be present in Bruch's membrane. In macular regions, increasing levels of inactive forms of metalloproteinase and scarcity of active forms of MMP-2 suggests possible involvement of impaired extracellular degradation in both aging and macular degeneration.

Soluble TNF receptors in vitreoretinal proliferative disease.

PURPOSE: To measure vitreous levels of soluble TNF-receptors (sTNF-Rs) types I and II in eyes with rhegmatogenous retinal detachment (RRD), uncomplicated or complicated with proliferative vitreoretinopathy (PVR), and in eyes with proliferative diabetic retinopathy (PDR). To examine whether there is any relationship between vitreous levels of sTNF-Rs and clinical features of these conditions and between vitreous sTNF-Rs and TNFalpha levels and serum levels of sTNF-RS: METHODS: Vitreous levels of sTNF-Rs and TNFalpha were measured by enzyme-linked immunosorbent assay in 30 eyes with PVR, 30 eyes with uncomplicated RRD, and 29 eyes with PDR. Vitreous from eyes of 10 deceased donors and 9 eyes with macular holes served as control specimens. Serum levels of sTNF-Rs were measured in 17 patients with PDR and 21 patients with PVR. RESULTS: Vitreous levels of sTNF-Rs I and II were increased in eyes with PVR, RRD, and PDR when compared with control eyes (P < 0.002). However, vitreous levels of sTNF-Rs I and II were higher in eyes with PVR than in eyes with RRD (P < 0.01) or PDR (P < 0.03). This contrasted with the findings that serum sTNF-Rs were higher in PDR than in PVR (P < 0.016) and that vitreous levels of TNFalpha were higher in eyes with PDR than in eyes with PVR (P < 0.0005). In PVR, vitreous sTNF-Rs levels were associated with the duration of retinal detachment, number of previous external operations, and grade of severity, whereas in PDR these levels were not related to the type or duration of diabetes or its complication with traction retinal detachment. CONCLUSIONS: These observations suggest the existence of TNF inhibitory mechanisms within the eye during retinal processes of inflammation and angiogenesis. That high vitreous levels of sTNF-Rs relate to severity of retinopathy suggests that these molecules may constitute reactive products of inflammation. Effective control of TNFalpha activity by sTNF-Rs within the retinal microenvironment may determine the outcome and severity of retinal proliferative conditions.

Vascular adhesion molecules in vitreous from eyes with proliferative diabetic retinopathy.

PURPOSE: To investigate whether proliferative vitreoretinopathy (PDR) is associated with a selective increase in vitreous levels of soluble vascular cell adhesion molecules that mediate leukocyte extravasation and interaction with endothelium during processes of inflammation and neovascularization. METHODS: Vitreous from 55 patients undergoing vitrectomy for treatment of PDR complicated by vitreous hemorrhage and/or traction retinal detachment was assayed for the presence of the soluble vascular cell adhesion molecules sICAM-1, sVCAM-1, and sE-selectin using a standard enzyme-linked immunosorbent assays (ELISA). Vitreous from 12 cadaveric eyes matching age and sex of the patients were used as control samples. RESULTS: Vitreous levels of sICAM-1, sVCAM-1, and sE-selectin were significantly higher in eyes with PDR than in control cadaveric vitreous, and levels of all three molecules did not relate to the type or duration of diabetes mellitus. However, eyes with either traction retinal detachment alone or both traction retinal detachment and vitreous hemorrhage exhibited significantly higher levels of sICAM-1 and sE-selectin than eyes with vitreous hemorrhage alone. Vitreous levels of sVCAM-1 were similar in eyes with either vitreous hemorrhage or traction retinal detachment alone. CONCLUSIONS: The present observations suggest that molecular inflammatory mechanisms may contribute to processes of neovascularization and fibrosis observed in PDR, possibly not as the causative event, but as a result of endothelial, Müller, and retinal pigment epithelial cell activation. The results also indicate that retinal detachment amplifies the existing inflammation within the diabetic retina. Identification of any abnormalities in the production and control of specific adhesion molecules could have important implications in the design of new therapeutic regimens to treat and prevent this sight-threatening complication of diabetes mellitus.

Intercellular adhesion molecule-1 in proliferative vitreoretinopathy.

PURPOSE: To measure vitreous levels of the soluble intercellular adhesion molecule (sICAM-1) in eyes with rhegmatogenous retinal detachment (RRD) complicated or uncomplicated by proliferative vitreoretinopathy (PVR) to investigate whether levels of this molecule related to history of previous retinal surgery or to the duration and severity of PVR. METHODS: The authors measured vitreous sICAM-1 by enzyme-linked immunosorbent assay in 28 eyes with PVR and 35 eyes with uncomplicated RRD. Vitreous from 10 eyes with macular holes and from 12 cadaveric eye donors were used as control specimens. RESULTS: Vitreous sICAM-1 levels were higher in the group with RRD complicated by PVR as a whole than in the group with RRD alone or in the control groups. In patients with no previous retinal surgery, there was no difference in vitreous sICAM-1 levels between the groups with RRD alone and RRD complicated by PVR. However, in patients who had undergone previous external surgery, those with PVR showed higher levels of vitreous sICAM-1 than those with RRD alone. In PVR, raised levels of sICAM-1 were associated preferentially with a history of previous vitrectomy as well as with a longer duration of the condition, although these levels were not related to the grade of PVR. In eyes with RRD alone, the levels of sICAM-1 were not enhanced with the duration of the detachment. Despite showing high vitreous levels of sICAM-1, patients with PVR did not exhibit increased serum levels of this adhesion molecule. CONCLUSIONS: The current observations suggest that those persons in whom PVR develops may have an impairment of the mechanisms that control the inflammatory response to retinal trauma. Persistently raised vitreous levels of sICAM-1 point to the continued operation of cytokine-mediated vascular reactions at the blood-retinal barrier.

Vitreous levels of intercellular adhesion molecule 1 (ICAM-1) as a risk indicator of proliferative vitreoretinopathy.

AIM: To investigate whether high vitreous levels of the soluble intercellular adhesion molecule 1 (sICAM-1) may be related to clinical risk factors of proliferative vitreoretinopathy (PVR) and whether their measurement may serve as an additional risk indicator of this complication in eyes with rhegmatogenous retinal detachment (RRD). METHODS: Levels of sICAM-1 were measured by enzyme linked immunosorbent assays (ELISA) in vitreous from 36 eyes with RRD clinically considered to be at high risk of developing PVR (large retinal breaks, vitreous haemorrhage, long standing RRD, and previous vitreoretinal surgery). Levels of sICAM-1 in this group were compared with those in vitreous from 31 eyes with RRD without clinical risk factors for PVR, 32 eyes with established PVR and 10 eyes with macular holes. RESULTS: Vitreous from eyes with RRD at high risk of developing PVR contained significantly higher levels of sICAM-1 (range 6.1-97.7 ng/ml; Mann-Whitney test, p=0.0002) than those from eyes with RRD at low risk of developing this complication (range 4.8-17.7 ng/ml). Vitreous sICAM-1 levels in eyes with RRD at high risk of developing PVR were significantly lower than in eyes with established PVR (p=0.037), but higher than in eyes with macular holes (p <0.0001). Levels of sICAM-1 >/=15 ng/ml (3 x median of the levels present in control eyes) provide a useful cut off point for a highly specific test (96.7%) with high positive (91.6%) and negative (96.7%) predictive values, despite a relatively low sensitivity (30. 5%). CONCLUSIONS: The present findings suggest that laboratory measurement of sICAM-1 levels in vitreous from eyes with RRD may constitute an additional factor for identifying patients at high risk of PVR. Hence, determination of sICAM-1 levels may aid in the monitoring of patients likely to develop this complication and in the identification of patients who may benefit from adjuvant anti-inflammatory therapy.

Vitreous intercellular adhesion molecule 1 in uveitis complicated by retinal detachment.

AIMS: The vitreous levels of soluble intercellular adhesion molecule 1 (sICAM-1) were investigated in uveitic eyes undergoing vitrectomy for retinal detachment (RD) or other complications, and the presence of this molecule was related to disease activity and vitreous levels of the cytokine tumour necrosis factor alpha (TNF alpha), known to upregulate ICAM-1 expression on various cells. METHODS: Vitreous and serum samples from 23 patients with either active or quiescent uveitis undergoing retinal surgery were examined for the levels of immunoreactive sICAM-1 and TNF alpha by ELISA methods, and for the presence of biologically active TNF alpha. Vitreous from non-uveitic eyes with rhegmatogenous retinal detachment (RRD), macular holes or cadaveric eyes were used as controls. RESULTS: As a whole, vitreous from uveitic eyes complicated or uncomplicated by RRD contained significantly higher levels of sICAM-1 than vitreous from non-uveitic eyes with RRD alone (p < 0.0005), eyes with macular holes (p < 0.0001), or normal cadaveric vitreous (p < 0.0001). The proportion of vitreous containing > 20 ng/ml sICAM-1 (> four times the normal values) was significantly higher in eyes with uveitis complicated by RRD than in those eyes without RRD (Fisher's test, p = 0.02), and although levels of sICAM-1 were higher in eyes with active uveitis than in those with quiet disease (p < 0.02), this could not be dissociated from the increase caused by RRD. There was a relation between the vitreous levels of sICAM-1 and those of immunoreactive TNF alpha (Spearman's correlation coefficient; r = 0.601, p = 0.006), but not between the vitreous levels of sICAM-1 and those of biologically active TNF alpha. CONCLUSION: Increased vitreous sICAM-1 levels and the association of this molecule with the presence of immunoreactive TNF alpha in uveitic eyes confirm the operation of cytokine mediated vascular reactions at the blood-retinal barrier during the development of this condition. The persistence of high vitreous levels of sICAM-1 in eyes with uveitis complicated by RRD despite previous immunosuppression may indicate a low rate of clearance of inflammatory molecules from the vitreous cavity and an exacerbation of the existing inflammatory process by the retinal detachment itself.

Distribution of TNF alpha and its reactive vascular adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy.

AIMS: This study investigated the presence of the cytokine tumour necrosis factor alpha (TNF alpha) and the vascular adhesion glycoproteins ICAM-1, VCAM-1, E-selectin, P-selectin, and PECAM within fibrovascular membranes of eyes with proliferative diabetic retinopathy (PDR). METHODS: The presence of these molecules was determined by immunohistochemical staining using monoclonal antibodies and the APAAP technique. RESULTS: Staining for TNF alpha was observed on the retinal vascular endothelium of five of 12 specimens, on infiltrating cells within all membranes, and on the extracellular matrix of nine specimens. This staining wa abolished by absorption of the monoclonal antibody with human recombinant TNF alpha. Likewise, ICAM-1 staining was given by infiltrating cells and extracellular matrix of nine membranes and by the endothelium of three of the specimens. VCAM-1, E-selectin, and P-selectin staining was observed on the vascular endothelium of 5/12, 4/12, and 3/12 epiretinal membranes respectively. PECAM was expressed by the endothelium of 4/12 specimens, by infiltrating cells of 8/12 membranes, and also by the extracellular matrix of two of the specimens. CONCLUSION: The widespread distribution of TNF alpha and the nature of the adhesion molecules expressed by vascular endothelial cells in PDR membranes suggest that local activation of TNF alpha and enhanced expression of vascular cell adhesion molecules may play an important role in the development of the proliferative phase of diabetic retinopathy.

Silicone oil concentrates fibrogenic growth factors in the retro-oil fluid.

AIM: To determine whether silicone oil concentrates protein and growth factors in the retro-oil fluid. METHODS: A laboratory analysis of intraocular fluid and vitreous specimens obtained from patients undergoing removal of silicone oil, revision vitrectomy, or primary vitrectomy for macular hole, proliferative vitreoretinopathy (PVR), or retinal detachment. Patients were prospectively recruited from routine vitreoretinal operating lists. Vitreous cavity fluid and vitreous samples were analysed for the presence of transforming growth factor beta (TGF-beta2), basic fibroblast growth factor (bFGF), interleukin 6 (IL-6), and total protein using either commercially available enzyme linked immunosorbent assays (ELISA) or protein assay kits. RESULTS: The median levels of bFGF, IL-6, and protein in the retro-oil fluid were raised (p<0.05) compared to all the other vitreous and vitreous cavity fluid samples. bFGF, IL-6, and protein levels were raised in PVR vitreous compared to non-PVR vitreous. TGF-beta2 levels were not significantly raised in retro-oil fluid or in PVR vitreous. CONCLUSIONS: The concentration of fibrogenic (bFGF) and inflammatory (IL-6) growth factors and protein is raised in retro-silicone oil fluid. This may contribute to the process of retro-oil perisilicone proliferation and subsequent fibrocellular membrane formation.

Matrix metalloproteinases and their natural inhibitors in fibrovascular membranes of proliferative diabetic retinopathy.

AIM: To examine epiretinal membranes of proliferative diabetic retinopathy (PDR) for the presence of selective matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), in order to determine whether neovascularisation and fibrosis, characteristic of this complication of diabetes mellitus, are associated with specific anomalies of MMP or TIMP expression. METHODS: The presence of selected MMPs and TIMPs was investigated in 24 fibrovascular epiretinal membranes of PDR, and the findings compared with that observed in 21 avascular epiretinal membranes of proliferative vitreoretinopathy (PVR) and five normal retinas. Specimens were examined for deposition of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), and three tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, and TIMP-3). RESULTS: The results showed that unlike normal retina, which constitutively expresses MMP-1 and TIMP-2, a large proportion of PDR membranes (> 62%) stained for MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and TIMP-3. There were no differences in the expression of these molecules when compared with PVR membranes. A characteristic staining for MMP-9 was observed within the perivascular matrix of PDR membranes, and there was a significant increase in TIMP-2 expression by PDR membranes (p= 0.036) when compared with PVR membranes. CONCLUSIONS: The findings that MMPs involved in degradation of fibrovascular tissue matrix, as well as TIMP-1 and TIMP-2, are found in a large proportion of PDR membranes, and that their expression does not differ from that of PVR membranes, suggest the existence of common pathways of extracellular matrix degradation in pathological processes leading to retinal neovascularisation and fibrosis.

Laser photocoagulation alters the pattern of staining for neurotrophin-4, GFAP, and CD68 in human retina.

AIMS: To investigate the staining pattern of neurotrophin-3 (NT3), neurotrophin-4 (NT4), and brain derived neurotrophic factor (BDNF) as well as glial fibrillary acid protein (GFAP) and CD68 in lasered human retina. METHODS: Retinal laser photocoagulation was performed on four patients (two males, two females) with choroidal malignant melanoma 1-6 days before enucleation. Three other enucleated eyes with malignant melanoma and three normal cadaveric donor eyes were used as controls. Immunohistochemistry was performed to investigate the pattern of staining of NT3, NT4, BDNF, GFAP, and CD68 in 7 mm sections of fixed specimens. RESULTS: Expression of NT4 was detected in the inner and outer nuclear layers of all the retinal sections examined but no NT3 and BDNF staining was seen. NT4 staining was found to be less intense in lasered and melanoma controls compared to normal cadaveric donor retinas. There was an upregulation of GFAP expression in both lasered and control eyes with melanoma in comparison with normal controls. CD68 staining was only observed in retinal pigment epithelium and choroid of lasered eyes. CONCLUSION: NT4 is expressed in inner and outer nuclear layers of normal human retina and its expression is downregulated following laser photocoagulation. This occurs in parallel with an increased expression of GFAP suggesting that reactive changes in Muller cells may be responsible for reduced NT4 staining. Expression of CD68 at the site of laser injury is consistent with a wound healing process as a response to local damage.

Distribution of cytokine proteins within epiretinal membranes in proliferative vitreoretinopathy.

This study reports on the immunohistochemical staining for cytokine proteins of 26 epiretinal membranes obtained from eyes undergoing surgery for the treatment of proliferative vitreoretinopathy. All specimens were investigated for the distribution of staining for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) and interleukin-2 (IL-2). The results showed that 22 of the membranes (85%) stained for TNF alpha not only intracellularly but also in the extracellular matrix. This contrasts with the findings that only 2 membranes stained for IL-1 alpha and that another 3 were positive for IL-1 beta. Staining for the cytokines IL-6 and IFN gamma was also observed in 9 and 7 membranes respectively. None of the specimens investigated stained with antibodies to IL-2 or control antibodies, and none of three normal retinas stained with any of the antibodies used. Pre-absorption of anti-cytokine antibodies with the corresponding human recombinant cytokines abolished staining of cells and extracellular matrix. The present findings support growing evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of proliferative vitreoretinopathy, and draw attention to the possibility that interaction between extracellular matrix-bound cytokine and inflammatory leucocytes or resident cells of the retina may promote the development and perpetuation of this condition.

Expression of hematopoietic cell markers by retinal pigment epithelial cells.

PURPOSE: We investigated the expression of various isoforms of the hematopoietic cell marker CD45 on retinal pigment epithelial cells in relation to their expression of CD68 and the cytokine-reactive intercellular adhesion molecule-1 (ICAM-1). We also determined the effect of the pro-inflammatory cytokines IL-1 beta, TNF alpha and IFN gamma on the expression of these molecules by RPE cells in culture. METHODS: Monolayers of RPE cells between 3rd and 7th passages were cultured in the presence or absence of cytokines, followed by immunohistochemical staining for CD45 (170-220 kD), CD45RA (205 and 220 kD), CD45RO (180 kD), CD68 and ICAM-1, using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. Total (membrane and cytoplasmic) expression of each of the three CD45 isoforms was determined by enzyme-linked immunoassays (ELISA). RESULTS: The majority of RPE cells expressed all isoforms of CD45 on their membranes and the pattern of expression of these molecules was not modified by culture. The greatest intensity of membrane staining was consistently observed with antibodies to CD45RA (205 + 220 kD), while CD45 (170-220 kD) showed to be the predominant isoform within the whole cell, as judged by ELISA assays. Unlike the membrane expression of CD45, only 20% of RPE cells stained for the macrophage surface molecule CD68 following 4 h of culture, but progressive increase in the proportion of CD68 positive cells was observed by extending the culture to 24 and 48 h. Neither the expression of CD68 nor the various isoforms of CD45 were modified by incubation with pro-inflammatory cytokines. Staining for ICAM-1 was observed in 21-25% of RPE cells throughout the 48 h culture. However, incubation with 50 pg/ml of IL-1 beta, TNF alpha and IFN gamma caused a marked increase in the RPE cell expression of ICAM-1 following 4, 24 and 48 h culture. CONCLUSIONS: The observations suggest that hematopoietic cell markers are constitutively expressed on RPE cells and that functions governed by these molecules are not influenced by pro-inflammatory signals. Expression of hematopoietic molecules by RPE cells may influence the macrophage-like properties of these cells and may also aid in the identification of RPE cells during pathological processes, particularly in the proliferative retinopathies, where these cells undergo phenotypic and functional changes.

Cytokines in human intraocular inflammation.

The presence of interleukin 6 (IL-6), interleukin 1 (IL-1), interleukin 2 (IL-2) and tumour necrosis factor (TNF) was investigated in vitreous and aqueous aspirates from eyes undergoing vitrectomy for the treatment of different inflammatory conditions. Cadaveric vitreous from 10 normal subjects were used as controls. IL-6 was observed in 5 specimens from eyes with idiopathic uveitis (range = 26-264 pg/ml), in 2 specimens from eyes with uveitis complicated with retinal detachment (28 and 279 pg/ml, respectively), in 6 samples from eyes with diabetic retinopathy (range = 5-480 pg/ml), in one sample from an eye with phacolytic glaucoma (1190 pg/ml) and in one specimen from an eye with Behçet's disease (366 pg/ml). Although IL-1 was detected in 80% of all the samples investigated, concentrations of this cytokine greater than 3 pg/ml were only observed in 2 specimens from eyes with uveitis (5 and 20 pg/ml, respectively) and 2 samples from eyes with diabetic retinopathy (3 and 31 pg/ml, respectively). TNF was present in 3 specimens from eyes with uveitis (range = 2-24 pg/ml) and 1 sample from eyes with diabetic retinopathy (4 pg/ml), but was not detected in the eyes with phacolytic glaucoma or Behçet's disease. IL-2 (less than 0.1 U/ml) was detected in one sample from an eye with uveitis, one specimen from an eye with uveitis complicated with retinal detachment and 2 samples from eyes with diabetic retinopathy. None of the cytokines measured were detected in any of the control vitreous. The present observations suggest that cytokines, particularly IL-6 and IL-1, may act as local amplification signals in pathological processes associated with chronic eye inflammation.