Proliferative Vitreoretinopathy Lymphocytes in Epiretinal Membranes David G. Charteris, FRCS, Paul Hiscott, PhD, Ian Grierson, PhD, Susan L. Lightman, PhD Background: To investigate the potential contribution of inflammatory and immunemediated processes contributing to the pathogenesis of proliferative vitreoretinopathy (PVR), an immunohistochemical study was undertaken to characterize the infiltrating inflammatory cells in epiretinal membranes surgically removed from the eyes of patients with PVA. Methods: Twenty-one epiretinal membranes obtained surgically from eyes with PVR complicating rhegmatogenous retinal detachment were studied immunohistochemically using the ABC technique and a panel of monoclonal and polyclonal antibodies. Results: T lymphocytes were found in 18 of the 21 specimens and generally constituted a small percentage of the total cell number. CD4+ T cells were found in 14 of the 18 membranes containing T cells. Three of six frozen membranes contained T cells that were positive for the interleukin-2 receptor. In 5 of 16 membranes studied, cells positive for the macrophage/monocyte marker were found. No B lymphocytes or neutrophils were identified, and there were no deposits of complement or immunoglobulins. Positive staining for the class II MHC antigen HLA-DR was found in 7 of the 21 membranes, a result that was more consistent in frozen than in fixed tissues. Conclusion: The study suggests that T lymphocytes are present in PVR epiretinal membranes and may be activated. These cells have the potential to playa role in the pathobiology of PVA. Ophthalmology 1992;99: 1364-1367
Proliferative vitreoretinopathy (PVR) is a process of cellular proliferation and contraction that may complicate rhegmatogenous retinal detachment and is the most common cause of failure of surgical retinal reattachment. 1,2 The pathogenesis ofPVR involves proliferation of various cell types forming contractile membranes on both anterior and posterior surfaces of the detached neuroretina. Retinal pigment epithelium (RPE), glial cells, cells of fibroblastic and myofibroblastic morphology, and inflammatory cells have been demonstrated in pathologic and immunohistochemical studies of PVR membranes. 3,4 Attention has recently been focused on the inflammatory and immunologic processes involved in PVR celOriginally received: October 18, 1991. Revision accepted: March 16, 1992. From the Department of Clinical Science, The Institute of Ophthalmology, London, United Kingdom. Supported by Wellcome Trust grants 17095/1.4R and 030412/11.4R. Reprint requests to David G. Charteris, FRCS, Manchester Royal Eye Hospital, Oxford Road, Manchester MI3 9WH, United Kingdom.
1364
lular activation and membrane formation. 5- 7 Results have suggested that, in addition to the presence of inflammatory cells, there may be immunoglobulin and complement deposition in these membranes 7 and expression of MHC class II antigens. 6•7 This study was undertaken with the aim of characterizing the infiltrating inflammatory cells and investigating the role of these cells in epiretinal membranes (present on the vitreal side of the detached neuroretina) obtained surgically from eyes with PVR. Cellular class II MHC antigen expression and deposition of complement components and immune complexes also were investigated to assess their potential contribution to the pathobiology of PVR membrane formation.
Materials and Methods Specimens A total of 21 epiretinal membranes were available for immunohistochemistry. All specimens were obtained by
Charteris et al . Lymphocytes in Proliferative Vitreoretinopathy surgical removal of epiretinal membrane tissue from eyes with PVR complicating rhegmatogenous retinal detachment. One patient had a previous traumatic intraocular foreign body before development of retinal detachment (this epiretinal membrane was of similar histopathologic appearance to the others in the study). No patients had pre-existing inflammatory eye disease. Fifteen specimens were formalin fixed and paraffin embedded and six were frozen in OCT (Shandon, Runcorn, United Kingdom) and stored at -70 0 C until used.
Immunohistochemical Staining Serial sections of the embedded specimens were cut at 5thickness. Paraffin-embedded sections were dewaxed through toluene and graded alcohols; frozen sections were warmed to room temperature and fixed for 5 minutes in acetone. Initial staining was performed using hematoxylineosin. Immunohistochemical staining was performed using the avidin-biotin complex (ABC) method (Vector, Peterborough, United Kingdom). After blocking of endogenous peroxidase activity, slides were washed in phosphate-buffered saline (PBS) pH 7.3, and normal serum was applied for 20 minutes. Excess serum was then removed, and primary antibody was applied for 30 minutes at an appropriate dilution in PBS. Slides were then washed in PBS, and biotinylated secondary antibody was applied for 30 minutes. After washing in PBS, ABC complex was then applied for 45 minutes. After washing, aminoethylcarbazole (B.D.H., Poole, United Kingdom) was used to give a red-pink final reaction product, and slides were counterstained with hematoxylin. The following primary antibodies were used on the paraffin-fixed sections: UCHLl (Dako, High Wycombe, United Kingdom) for T lymphocytes, OPD4 (Dako) for a CD4+ T lymphocyte subset (T helper/inducer), L26 (Dako) for B lymphocytes, HLA-DR alpha chain (Dako, code no. M746) for expression of HLA-DR Class II antigen, Mac 387 (Dako) for monocytes/macrophages, IgG (Serotec, Oxford, United Kingdom) for immunoglobulin G, IgM (Serotec) for immunoglobulin M, and C3c (Serotec) for complement component C3c. On frozen sections, the following antibodies were used: T3 (Dako) for T lymphocytes, T 4 (Dako) for CD4 + T lymphocytes (T helper/inducer), T8 (Dako) for CD8+ T lymphocytes (T cytotoxic/suppressor), IL2R (Dako) for activated T lymphocytes bearing interleukin-2 receptors, HLA-DR (Dako, code no. M704) for expression ofHLADR class II antigen and both anti macrophage (CD68 antigen) (Dako, code no. M718), and Mac 387 for monocytes/macrophages. Two antibodies were used for the detection of MHC class II expression, as the HLA-DR antibody is recommended only for frozen sections, whereas the HLA-DR alpha chain antibody reacts with antigens on formalinfixed, paraffin-embedded tissue. Similarly the anti macrophage antibody (CD68) can only be used on frozen tissue, whereas the MAC 387 antibody can be used on both frozen and fixed material. ~m
Sections of membranes in which aggregations of inflammatory cells were found also were stained with rabbit antisera to cytokeratins (Dako) and glial fibrillary acid protein (GFAP) (Dako) to determine the relation of the inflammatory cells to RPE and glial components of the membranes. In each specimen, estimates were made of (1) total cell number, (2) cells of fibroblastic morphology, and (3) cells of lymphocyte morphology staining positively with T lymphocyte monoclonal antibodies. Specimens also were examined to identify cells of neutrophil polymorphonuclear leukocyte morphology. The percentage of T cells within the total cell population was calculated for each specimen; the ratio of T cells to fibroblast-like cells was calculated to investigate the relationship between the presence of T cells and cells of fibroblastic morphology. Negative control studies were made by omitting the primary antibody on a section from each specimen. Sections of normal human retina, choroid, and sclera were stained using each primary antibody. Positive controls were provided by tissues (inflammatory eyelid or orbital lesions) with known positive staining for each primary antibody.
Results Controls Studies of normal retina, choroid, and sclera showed very occasional UCHLl + and CD4+ T cells, and HLA-DR positive cells in the choroid. All other antibodies were negative on these tissues. Positive-staining cells on the inflammatory lesion controls verified the efficacy of each primary antibody.
Proliferative Vitreoretinopathy Membranes T lymphocytes were found in 18 of the 21 membranes studied. Table 1 documents the T-cell percentage of the total cell population. In general, small numbers ofT cells were seen in the sections studied, but in 3 membranes T cells constituted more than 5% of the total number of cells (Fig 1). The ratio of T cells to fibroblast-like cells is shown in Table 2. In many of the membranes, T cells were found closely related to pigment-containing cells. In some membranes, T cells were related to foci of cytokertin-positive (RPE) cells or GFAP-positive (glial) cells, but, in general, no overall pattern could be established in the relationship of T cells to these cell types. In those specimens in which the approximate clinical duration (7 cases: duration range, 6 weeks to 6 months; mean, 3 months) could be determined, no variation of inflammatory cell content could be detected in relation to clinical "age." In 14 of the 18 T-cell positiv~ membranes, small numbers of CD4 + T cells were identified. In three of the six frozen tissues studied, CD8+ T lymphocytes were identified. Interleukin-2 receptor positive cells also were found
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Ophthalmology
Volume 99, Number 9, September 1992
Table 1. T Lymphocyte Percentage of Total Cell Population T Cell
PVR Membranes (n = 18)
10%
6 9 2 1*
PVR = proliferative vitreoretinopathy. • This membrane contained 13% T lymphocytes.
in three of the six frozen membranes. Sixteen membranes were studied with the antimonocyte/macrophage monoclonal antibodies. In five of these, small numbers of positively staining cells were found. No B lymphocytes or neutrophils were identified in any of the membranes. Of seven membranes examined for deposits of complement component C3c, only one contained small areas of s~a~n ing suggestive of minimal deposition of C3c. No posltlve staining for IgG or IgM was seen in 13 membranes studied. The 15 formalin-fixed, paraffin-embedded membranes were stained with the HLA-DR alpha chain monoclonal antibody. Of these, one contained positively st~ining. fibroblast-like cells. All six frozen membranes stamed Wlth the HLA-DR antibody contained numbers of positively staining fibroblast-like cells and inflammatory cells (Fig 2).
Discussion The potential of T lymphocytes to playa significant ro.le in fibrosis and wound healing has been demonstrated m experimental work. 8- IO Proliferative vitreoretinop~thy can be viewed as a fibrotic wound healing response m a specialized tissue. The demonstration of a population of T lymphocytes in a high percentage of epiretinal membranes
Figure 2. HLA-DR positive cells (arrows) within an epiretinal membrane also containing pigmented cells (arrowhead) (hematoxylin counterstain; original magnification, X480).
suggests that these cells may playa role in promoting the cellular proliferation seen in PVR. We have shown that T cells in PVR membranes may be activated, expressing the interleukin-2 receptor, and, therefore, potentially mediate fibrogenic responses either directly via T cell cytokines such as transforming growth factor-iJ and tumor necrosis factor-iJ (lymphotoxin),8 or via T cell-triggered . d cyto k'meso II release of macrophage-denve Recent work has demonstrated that fibroblasts have the ability to produce a soluble factor that pro~ongs T ~ell survival and increases T cell responsiveness to mterleukin2.12 This mechanism could prolong retention of viable T cells at sites of inflammation and fibrosis, contributing to ongoing immunopathology, and may be of imp?rtance in the interaction between fibroblasts and T cells m PVR membranes. Although T cells were present in relatively small n~m bers in the epiretinal membranes, these may be sufficlent to provide cytokines in adequate local concentration~ to promote cellular proliferation and membrane contractlOn. Although some membranes had foci of T cell~ ~elated to RPE (cytokeratin positive) or glial (GFAP pOSltIve) cells, we found no overall pattern to the T cell distribution. Likewise there was considerable variation in the T cell to fibrobl~st-like cell ratio (Table 2), and it was not possible Table 2. Ratio of T Lymphocytes to Fibroblast-like Cells Ratio
Figure 1. UCHLl-positive T lymphocytes (arrows) within a collagenous epiretinal membrane (hematoxylin counterstain; original magnification, X 400).
1366
1:2-1:5 1:6-1:10 1: 11-1:50 1:51-1:100
Proliferative vitreoretinopathy (PVR) is a process of cellular proliferation and contraction that may complicate rhegmatogenous retinal detachment and is the most common cause of failure of surgical retinal reattachment. 1,2 The pathogenesis ofPVR involves proliferation of various cell types forming contractile membranes on both anterior and posterior surfaces of the detached neuroretina. Retinal pigment epithelium (RPE), glial cells, cells of fibroblastic and myofibroblastic morphology, and inflammatory cells have been demonstrated in pathologic and immunohistochemical studies of PVR membranes. 3,4 Attention has recently been focused on the inflammatory and immunologic processes involved in PVR celOriginally received: October 18, 1991. Revision accepted: March 16, 1992. From the Department of Clinical Science, The Institute of Ophthalmology, London, United Kingdom. Supported by Wellcome Trust grants 17095/1.4R and 030412/11.4R. Reprint requests to David G. Charteris, FRCS, Manchester Royal Eye Hospital, Oxford Road, Manchester MI3 9WH, United Kingdom.
1364
lular activation and membrane formation. 5- 7 Results have suggested that, in addition to the presence of inflammatory cells, there may be immunoglobulin and complement deposition in these membranes 7 and expression of MHC class II antigens. 6•7 This study was undertaken with the aim of characterizing the infiltrating inflammatory cells and investigating the role of these cells in epiretinal membranes (present on the vitreal side of the detached neuroretina) obtained surgically from eyes with PVR. Cellular class II MHC antigen expression and deposition of complement components and immune complexes also were investigated to assess their potential contribution to the pathobiology of PVR membrane formation.
Materials and Methods Specimens A total of 21 epiretinal membranes were available for immunohistochemistry. All specimens were obtained by
Charteris et al . Lymphocytes in Proliferative Vitreoretinopathy surgical removal of epiretinal membrane tissue from eyes with PVR complicating rhegmatogenous retinal detachment. One patient had a previous traumatic intraocular foreign body before development of retinal detachment (this epiretinal membrane was of similar histopathologic appearance to the others in the study). No patients had pre-existing inflammatory eye disease. Fifteen specimens were formalin fixed and paraffin embedded and six were frozen in OCT (Shandon, Runcorn, United Kingdom) and stored at -70 0 C until used.
Immunohistochemical Staining Serial sections of the embedded specimens were cut at 5thickness. Paraffin-embedded sections were dewaxed through toluene and graded alcohols; frozen sections were warmed to room temperature and fixed for 5 minutes in acetone. Initial staining was performed using hematoxylineosin. Immunohistochemical staining was performed using the avidin-biotin complex (ABC) method (Vector, Peterborough, United Kingdom). After blocking of endogenous peroxidase activity, slides were washed in phosphate-buffered saline (PBS) pH 7.3, and normal serum was applied for 20 minutes. Excess serum was then removed, and primary antibody was applied for 30 minutes at an appropriate dilution in PBS. Slides were then washed in PBS, and biotinylated secondary antibody was applied for 30 minutes. After washing in PBS, ABC complex was then applied for 45 minutes. After washing, aminoethylcarbazole (B.D.H., Poole, United Kingdom) was used to give a red-pink final reaction product, and slides were counterstained with hematoxylin. The following primary antibodies were used on the paraffin-fixed sections: UCHLl (Dako, High Wycombe, United Kingdom) for T lymphocytes, OPD4 (Dako) for a CD4+ T lymphocyte subset (T helper/inducer), L26 (Dako) for B lymphocytes, HLA-DR alpha chain (Dako, code no. M746) for expression of HLA-DR Class II antigen, Mac 387 (Dako) for monocytes/macrophages, IgG (Serotec, Oxford, United Kingdom) for immunoglobulin G, IgM (Serotec) for immunoglobulin M, and C3c (Serotec) for complement component C3c. On frozen sections, the following antibodies were used: T3 (Dako) for T lymphocytes, T 4 (Dako) for CD4 + T lymphocytes (T helper/inducer), T8 (Dako) for CD8+ T lymphocytes (T cytotoxic/suppressor), IL2R (Dako) for activated T lymphocytes bearing interleukin-2 receptors, HLA-DR (Dako, code no. M704) for expression ofHLADR class II antigen and both anti macrophage (CD68 antigen) (Dako, code no. M718), and Mac 387 for monocytes/macrophages. Two antibodies were used for the detection of MHC class II expression, as the HLA-DR antibody is recommended only for frozen sections, whereas the HLA-DR alpha chain antibody reacts with antigens on formalinfixed, paraffin-embedded tissue. Similarly the anti macrophage antibody (CD68) can only be used on frozen tissue, whereas the MAC 387 antibody can be used on both frozen and fixed material. ~m
Sections of membranes in which aggregations of inflammatory cells were found also were stained with rabbit antisera to cytokeratins (Dako) and glial fibrillary acid protein (GFAP) (Dako) to determine the relation of the inflammatory cells to RPE and glial components of the membranes. In each specimen, estimates were made of (1) total cell number, (2) cells of fibroblastic morphology, and (3) cells of lymphocyte morphology staining positively with T lymphocyte monoclonal antibodies. Specimens also were examined to identify cells of neutrophil polymorphonuclear leukocyte morphology. The percentage of T cells within the total cell population was calculated for each specimen; the ratio of T cells to fibroblast-like cells was calculated to investigate the relationship between the presence of T cells and cells of fibroblastic morphology. Negative control studies were made by omitting the primary antibody on a section from each specimen. Sections of normal human retina, choroid, and sclera were stained using each primary antibody. Positive controls were provided by tissues (inflammatory eyelid or orbital lesions) with known positive staining for each primary antibody.
Results Controls Studies of normal retina, choroid, and sclera showed very occasional UCHLl + and CD4+ T cells, and HLA-DR positive cells in the choroid. All other antibodies were negative on these tissues. Positive-staining cells on the inflammatory lesion controls verified the efficacy of each primary antibody.
Proliferative Vitreoretinopathy Membranes T lymphocytes were found in 18 of the 21 membranes studied. Table 1 documents the T-cell percentage of the total cell population. In general, small numbers ofT cells were seen in the sections studied, but in 3 membranes T cells constituted more than 5% of the total number of cells (Fig 1). The ratio of T cells to fibroblast-like cells is shown in Table 2. In many of the membranes, T cells were found closely related to pigment-containing cells. In some membranes, T cells were related to foci of cytokertin-positive (RPE) cells or GFAP-positive (glial) cells, but, in general, no overall pattern could be established in the relationship of T cells to these cell types. In those specimens in which the approximate clinical duration (7 cases: duration range, 6 weeks to 6 months; mean, 3 months) could be determined, no variation of inflammatory cell content could be detected in relation to clinical "age." In 14 of the 18 T-cell positiv~ membranes, small numbers of CD4 + T cells were identified. In three of the six frozen tissues studied, CD8+ T lymphocytes were identified. Interleukin-2 receptor positive cells also were found
1365
Ophthalmology
Volume 99, Number 9, September 1992
Table 1. T Lymphocyte Percentage of Total Cell Population T Cell
PVR Membranes (n = 18)
10%
6 9 2 1*
PVR = proliferative vitreoretinopathy. • This membrane contained 13% T lymphocytes.
in three of the six frozen membranes. Sixteen membranes were studied with the antimonocyte/macrophage monoclonal antibodies. In five of these, small numbers of positively staining cells were found. No B lymphocytes or neutrophils were identified in any of the membranes. Of seven membranes examined for deposits of complement component C3c, only one contained small areas of s~a~n ing suggestive of minimal deposition of C3c. No posltlve staining for IgG or IgM was seen in 13 membranes studied. The 15 formalin-fixed, paraffin-embedded membranes were stained with the HLA-DR alpha chain monoclonal antibody. Of these, one contained positively st~ining. fibroblast-like cells. All six frozen membranes stamed Wlth the HLA-DR antibody contained numbers of positively staining fibroblast-like cells and inflammatory cells (Fig 2).
Discussion The potential of T lymphocytes to playa significant ro.le in fibrosis and wound healing has been demonstrated m experimental work. 8- IO Proliferative vitreoretinop~thy can be viewed as a fibrotic wound healing response m a specialized tissue. The demonstration of a population of T lymphocytes in a high percentage of epiretinal membranes
Figure 2. HLA-DR positive cells (arrows) within an epiretinal membrane also containing pigmented cells (arrowhead) (hematoxylin counterstain; original magnification, X480).
suggests that these cells may playa role in promoting the cellular proliferation seen in PVR. We have shown that T cells in PVR membranes may be activated, expressing the interleukin-2 receptor, and, therefore, potentially mediate fibrogenic responses either directly via T cell cytokines such as transforming growth factor-iJ and tumor necrosis factor-iJ (lymphotoxin),8 or via T cell-triggered . d cyto k'meso II release of macrophage-denve Recent work has demonstrated that fibroblasts have the ability to produce a soluble factor that pro~ongs T ~ell survival and increases T cell responsiveness to mterleukin2.12 This mechanism could prolong retention of viable T cells at sites of inflammation and fibrosis, contributing to ongoing immunopathology, and may be of imp?rtance in the interaction between fibroblasts and T cells m PVR membranes. Although T cells were present in relatively small n~m bers in the epiretinal membranes, these may be sufficlent to provide cytokines in adequate local concentration~ to promote cellular proliferation and membrane contractlOn. Although some membranes had foci of T cell~ ~elated to RPE (cytokeratin positive) or glial (GFAP pOSltIve) cells, we found no overall pattern to the T cell distribution. Likewise there was considerable variation in the T cell to fibrobl~st-like cell ratio (Table 2), and it was not possible Table 2. Ratio of T Lymphocytes to Fibroblast-like Cells Ratio
Figure 1. UCHLl-positive T lymphocytes (arrows) within a collagenous epiretinal membrane (hematoxylin counterstain; original magnification, X 400).
1366
1:2-1:5 1:6-1:10 1: 11-1:50 1:51-1:100
