Immunopathology of atopic keratoconjunctivitis

Conjunctival biopsies from 11 patients with atopic keratoconjunctivitis (AKC) and from 13 age-matched healthy individuals undergoing cataract surgery were analyzed by light microscopy and immunohistochemical techniques. Histology of AKC specimens showed goblet cell proliferation, epithelial pseudotubular formation, eosinophil and mast cell invasion of the epithelium, and pronounced mononuclear cell infiltration of the substantia propria, often with frank granuloma formation. Epithelium of AKC conjunctiva showed significantly more T cells (CD3+, CD5+), T-helper cells (CD4+), macrophages (Mac-1+, CD14+), activated T cells, (CD25+), and dendritic cells (CD1+), and a higher helper/suppressor ratio than did control subjects. In the substantia propria, AKC specimens showed dramatically increased inflammatory cell infiltration with significantly more cells staining, in order of frequency, for T-cells (CD3+, CD5+), T-helper cells (CD4+), T-suppressor/cytotoxic cells (CD8+), macrophages (CD14+, Mac-1+) activated T cells (CD25+), B cells (CD22+), and dendritic cells (CD1+, HLA-DR+). Fifty-three percent of T cells in the substantia propria expressed the interleukin-2 receptor protein (CD25+). These findings indicate that the chronic conjunctivitis of AKC is complex, with activated T-cells and macrophages dramatically participating in the process. Successful long-term control of the potentially binding conjunctival inflammation of this disease is likely to require therapeutic strategies directed toward more than just the mast cell component of the process.     

Ocular rosacea. A histologic and immunopathologic study

Acne rosacea is an idiopathic dermatologic disease that frequently produces conjunctival inflammation. The authors studied the histology and immunopathology of epibulbar conjunctival biopsy specimens from eight patients with ocular rosacea and compared the findings with those from conjunctiva from 13 normal individuals. The conjunctival epithelium in ocular rosacea was attenuated and infiltrated by inflammatory cells, mainly T-helper/inducer (CD4) cells, phagocytic cells, and antigen-presenting (CD14, Mac-1) cells. The difference between the normal control group and the rosacea group in the number of mononuclear cells forming these populations was statistically significant (P less than 0.01). The substantia propria of the rosacea specimens contained large subepithelial infiltrates of chronic inflammatory cells, and in some cases frank granuloma formation was evident. There was an overall mean increase of nearly all cell types, but especially of T-helper cells in the rosacea specimens compared with the controls. Interestingly, T-helper/inducer (CD4) cells, which were outnumbered by the T-suppressor (CD8) cells in the normal conjunctival epithelium (CD4/CD8 = 0.85), outnumbered the CD8-positive cells in the rosacea specimens (CD4/CD8 = 1.6). There also was a 3.5-fold increase of the CD4/CD8 ratio in the rosacea conjunctival stroma compared with the normal specimens. The mechanism involved in rosacea conjunctival inflammation resembles a type IV hypersensitivity reaction.     

Role of enhanced expression of m-CSF in conjunctiva affected by cicatricial pemphigoid

PURPOSE: Local proliferation of macrophages has been reported to augment the inflammatory response in various human and experimental diseases. Macrophage accumulation in the submucosa is also an important feature in the pathogenesis of ocular cicatricial pemphigoid (OCP). In the present study, the role of local proliferation of macrophages in conjunctiva affected by OCP and the relationship between local proliferation of macrophages and expression of macrophage-colony-stimulating factor (m-CSF) in such conjunctiva were examined. METHODS: Biopsy specimens from the conjunctiva of 10 untreated patients with active OCP and from 5 normal subjects were studied for the expression of m-CSF, macrophages, and proliferating cell nuclear antigen (PCNA), a cell cycle protein, by immunohistochemistry. Dual staining for CD68 (a cell surface marker for macrophages) and PCNA was also performed to identify proliferating macrophages. In addition, fibroblasts isolated from conjunctiva of normal individuals and from patients with OCP were studied for the expression of m-CSF by immunostaining and real-time PCR. To identify the factors that induce m-CSF in conjunctival fibroblasts, the fibroblasts were incubated with different concentrations of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha, and the levels of m-CSF mRNA were determined by real-time PCR and the amount of m-CSF produced was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Normal conjunctiva showed weak expression of m-CSF in the conjunctival epithelial cells and stroma. Conjunctival expression of m-CSF protein was significantly (P < 0.0001) increased in conjunctival biopsy specimens from patients with OCP. m-CSF was detected in the infiltrating macrophages, stromal cells (presumably fibroblasts), and conjunctival epithelial cells. Compared with normal control conjunctival tissue, a 1.2-fold increase in the expression of mRNA for m-CSF was detected by real-time PCR in the conjunctival tissue obtained from patients with OCP. Increased expression of m-CSF correlated significantly (P < 0.0004) with an increased stromal accumulation of macrophages in conjunctival biopsy specimens of patients with OCP. A number of these accumulated macrophages (CD68-positive) were found to be proliferating (PCNA-positive). In addition, fibroblasts isolated and cultured from conjunctiva of patients with OCP showed significantly increased (1.7-fold) expression of m-CSF compared with normal conjunctival fibroblasts. When conjunctival fibroblasts were treated with IL-1alpha or TNF-alpha, real-time PCR and ELISA detected an increased level of m-CSF. CONCLUSIONS: An increased expression of m-CSF was observed in conjunctiva from patients with active OCP. There was a positive correlation between expression of m-CSF and accumulation of macrophages in conjunctival biopsy sections obtained from patients with OCP. Increased expression of m-CSF, mainly by conjunctival fibroblasts and infiltrating inflammatory cells, may play an important role in the regulation of local proliferation of macrophages in OCP. In the conjunctiva of patients with OCP, this process could augment or enhance the local inflammatory response and tissue injury consequent to it.     

Mooren ulcer: an immunopathologic study

PURPOSE: To determine the pattern and distribution of mononuclear cells, adhesion, and co-stimulatory molecules in the conjunctiva of patients with Mooren ulcer. METHODS: Conjunctival biopsy specimens were obtained from 6 patients with Mooren ulcer and 6 healthy individuals. Immunohistochemistry was performed on frozen sections of the cryopreserved human conjunctivas using monoclonal antibodies directed against CD1alpha, CD3, CD4, CD8, CD20, CD25, CD57, and CD68 cells; the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), very late activation-4 (VLA-4), ICAM-1, and LFA-1; and the co-stimulatory molecules CD28, B7-1, B7-2, and CTLA-4. RESULTS: Differences in expression on the conjunctival epithelium from patients with Mooren ulcer and normal subjects were noted only for VCAM-1, VLA-4, ICAM-1, and LFA-1. The ratio of CD4+/CD8+ cells in Mooren ulcer specimens was significantly higher (3.5-fold). However, in the substantia propria, Mooren ulcer specimens revealed significantly increased numbers of CD1alpha+, CD3+, CD4+, CD20+, CD28+, B7-1+, B7-2+, and CD68+ cells. The ratios of CD4+/CD8+ cells and B7-2+/antigen-presenting cells in Mooren ulcer specimens were significantly higher (5-fold). All tested adhesion molecules showed significant up-regulation in the patients' conjunctivas. Mooren ulcer vascular endothelial cells prominently expressed E-selectin, VCAM-1, VLA-4, and ICAM-1 compared with normal conjunctiva. CONCLUSION: The simultaneous presence of multiple types of inflammatory cells, adhesion, and co-stimulatory molecules in Mooren ulcer conjunctiva suggests that their interaction may contribute to a sustained immune activation as at least part of the pathogenic mechanism of this disorder.     

Eosinophil granule proteins expressed in ocular cicatricial pemphigoid

BACKGROUND—Blister formation and tissue damage in bullous pemphigoid have been attributed to the release of eosinophil granule proteins—namely, to eosinophil derived cationic protein (ECP) and major basic protein (MBP). In the present investigation these eosinophil granule proteins were studied in the conjunctiva of patients with ocular cicatricial pemphigoid (OCP).
METHODS—Conjunctival biopsy specimens obtained from patients with subacute (n=8) or chronic conjunctival disease (n=13) were analysed histologically and immunohistochemically using antibodies directed against EG1 (stored and secreted ECP), EG2 (secreted ECP), MBP, CD45 (common leucocyte antigen), CD3 (pan T cell marker), and HLA-DR (class II antigen).
RESULTS—Subepithelial mononuclear cells, mast cells, and neutrophils were detected in all specimens. The number of mononuclear cells, neutrophils, CD45+ cells, CD3+ cells, and the HLA-DR expression were significantly higher in the subacute than in the chronic disease group. Some eosinophils were found in specimens from five of eight patients with subacute OCP, but in none of the patients with chronic disease. The eosinophil granule proteins (ECP and MBP) were found in the epithelium and substantia propria in patients with subacute conjunctivitis.
CONCLUSIONS—Subepithelial cell infiltration in the conjunctiva greatly differs between subacute and chronic ocular cicatricial pemphigoid specimens. The findings suggest that eosinophil granule proteins may participate in tissue damage in acute phase of inflammation in OCP.

     

T cells and trachoma. Their role in cicatricial disease

Frozen sections of tarsoconjunctival biopsies with trachomatous scarring from 14 black adults undergoing corrective surgery for trichiasis, and "normal" tissue from three postmortem controls, were immunohistochemically stained for the major T- and B-cell subsets, and for macrophages and monocytes. T cells outnumbered B cells by 2 to 17 times, and macrophages and monocytes by approximately 20 times in all specimens. Biopsies were categorized as "inflamed" if a cumulative inflammatory score of cellular staining in the substantia propria with CD4, CD8, and OKM1 monoclonal antibodies was greater than that of control tissues. CD4+ lymphocytes predominated over CD8+ lymphocytes in 5 of 7 inflamed biopsies, whereas CD8+ lymphocytes predominated over CD4+ lymphocytes in 5 of 7 noninflamed biopsies. Lymphoid aggregates were present in five inflamed biopsies, but lacked germinal centers, centrally located B cells, or parafollicular T cells typical of the acute stage of trachoma. CD4+ and CD8+ lymphocytes also were observed in the epithelium and lumen of Meibomian glands. These observations indicate that the inflammatory infiltrate of the tarsoconjunctiva in the cicatricial stage of trachoma is comprised predominantly of T cells, and suggests that T cells may be involved in the genesis of tarsal thickening and conjunctival scarring seen in the later stages of trachoma.     

Cell populations and adhesion molecules expression in conjunctiva before and after bone marrow transplantation

We were interested to analyse the composition of the cellular infiltrate and adhesion molecules expression in the conjunctiva before and at least one hundred days after autologous and allogenic bone marrow transplantation (BMT) and its relation with the presence of dry eye. We used immunohistochemistry on cryopreserved human conjunctiva with monoclonal antibodies to T-lymphocytes (CD3, CD4 and CD8), B-lymphocytes (CD19), macrophages (CD14), natural killer cells (NK, CD57), intercellular adhesion molecule 1 (ICAM-1), E-selectin, vascular cell adhesion molecule-1 (VCAM-1), lymphocyte function associated antigen-1 (LFA-1), very late antigen-4 (VLA-4), interleukin 2 receptor (IL2r, CD25) and HLA-DR. Our autologous recipients had no graft-versus-host disease (GVHD) but allogenic patients had chronic GVHD. After autologous BMT the conjunctiva had significantly more: (1) T lymphocytes (CD3+, CD4+, CD8+) in the epithelium; (2) CD4+ and CD14+ cells in the stroma; and (3) VLA-4 expression in the stroma than before BMT. After allogenic BMT, the conjunctiva exhibited a significant increase of: (1) CD3+ and CD14+ cells in the epithelium; (2) T lymphocytes (CD3+, CD4+, CD8+) and CD14+ cells in the stroma; and (3) VLA-4 and LFA-1 expression in the stroma than before BMT. After the engraftment, the comparison between autologous and allogenic recipients revealed that: (1) there were no significant differences in adhesion molecule expression; (2) the epithelium of autologous recipients had significantly more CD3+ cells; and (3) the stroma of allogenic patients had significantly more CD3+ and CD8+ cells. Among allogenic recipients, CD14+ cells were significantly increased both in the epithelium and in the stroma of patients with signs or symptoms of dry eye in comparison with patients without ocular involvement. Additionally, those having keratoconjunctivitis sicca (KCS) had CD4/CD8 ratios significantly higher than those without KCS. In conclusion, in the conjunctiva after autologous BMT a subclinical cell mediated immune reaction seems to take place. The conjunctivitis of chronic GVHD is complex, with T cells and macrophages dramatically contributing to the process.     

Thyroid associated ophthalmopathy: evidence for CD4(+) gammadelta T cells; de novo differentiation of RFD7(+) macrophages, but not of RFD1(+) dendritic cells; and loss of gammadelta and alphabeta T cell receptor expression

AIM: To characterise periorbital immune cells (stages, kinetics) in active and inactive thyroid associated ophthalmopathy (A-TAO; I-TAO). METHODS: In orbital tissue cryosections of patients with A-TAO (n = 15), I-TAO (n = 11), and healthy controls (n = 14), adipose and fibrovascular areas were evaluated for MHC II(+) cells, CD45(+) total leukocytes, myeloid cells (CD33(+) monocytes; CD14(+) macrophages; mature RFD7(+) macrophages; RFD1(+) dendritic cells (DCs)), and lymphoid cells (CD4(+) T cells; alphabeta and gammadelta T cells; CD20(+) B cells). Results are expressed as medians and 5% confidence intervals. RESULTS: In fibrovascular septae, a surge of CD33(+) immigrants clearly correlating with disease activity generated significantly increased (p<0.05) percentages of CD14(+) and RFD7(+) macrophages. Intriguingly, CD4(+) cells were mostly gammadelta T cells, while alphabeta T helper cells were much less frequent. Successful treatment rendering TAO inactive apparently downregulates monocyte influx, macrophage differentiation, and T cell receptor expression. Similar trends were recorded for adipose tissue. Interestingly, RFD1(+) DCs were completely absent from all conditions examined. CONCLUSION: A-TAO coincides with periorbital monocyte infiltration and de novo differentiation of macrophages, but not DCs. The authors discuss a novel potential role for inflammatory CD4(+) gammadelta T cells in TAO. Successful treatment apparently downregulates orbital monocyte recruitment and effects functional T cell knockout.     

Cell subpopulations in failed human corneal grafts

BACKGROUND/AIMS: Inflammatory cells and antigen presenting cells (APC) are not present under normal circumstances in the centre of the healthy cornea. The purpose of this study was to investigate and phenotype the inflammatory cell populations, particularly with reference to T cell subpopulations and macrophages, and to localise dendritic cells (DC) and other MHC class II positive cells in three groups of grafted corneas: rejected non-inflamed, rejected inflamed grafts, and control dystrophic explants. METHODS: 15 corneal buttons removed during keratoplasty from non-inflamed "quiet" previously grafted corneas, five inflamed corneas requiring urgent regrafting for "graft melting" (in "high risk" corneas), and 10 control dystrophic opaque corneas explanted during their first graft procedure were examined. Cryosections of corneas were immunostained with a panel of monoclonal antibodies (mAb) against CD3, CD4, CD8, CD14, CD25, CD68, HLA-DP, and HLA-DR molecules using the StreptABC method. DC were detected by dual immunostaining as CD1a+ and MHC class II+ and CD19-. Cell densities in immunostained tissue sections were evaluated using a scale from 0 to +4. RESULTS: Immunostaining in control dystrophic corneas was negative for all antibodies. A moderate to high density of CD8+, CD14+, and CD68+ cells was observed in the majority of rejected non-inflamed as well as in rejected inflamed corneal buttons. Strong positivity for HLA-DP and HLA-DR molecules in the epithelium, stroma, and endothelium was also demonstrated. Weak positivity for CD4 and CD25 was observed in six of 15 and 11 of 15 rejected corneas, respectively. The presence of dendritic cells in the basal layer of the epithelium and in the stroma was observed in 50% of the grafts. CONCLUSIONS: A high frequency of macrophages, the presence of DC in the explants, and strong expression of HLA-DP and HLA-DR molecules on resident cells are characteristics of rejected corneal allografts, whether actively inflamed or not. The presence of DC in the stroma of the grafted cornea suggests that they may be mainly responsible for T cell activation and graft rejection since DC are known to be a 100-fold more potent than macrophages as APC.     

IL-10基因修饰的未成熟树突状细胞在大鼠角膜移植排斥反应中的作用

目的：通过建立大鼠角膜穿透性移植模型,探讨IL－10基因修饰的未成熟树突状细胞在大鼠角膜移植排斥反应中的作用及其作用机制。方法：建立大鼠同种异体穿透性角膜移植模型,将受体SD大鼠随机分为：阳性对照组、GFP－DC组、8－DC组及IL－10－GFP－DC组,分别于角膜移植术前3 d尾静脉注射等量的PBS、供体Wistar 大鼠骨髓源8－DC （培养8d的DC）、转染48h的GFP－DC及IL－10－GFP－DC。术后每天在裂隙灯下观察角膜植片情况,记录排斥反应指数及角膜植片存活时间,在移植术后第14 d行各组角膜组织的病理学检查及免疫组织化学检查。结果：IL－10－GFP－DC组角膜植片存活时间较GFP－DC组、8－DC组比较显著延长（ P＜0．01）。术后第14 d时IL－10－GFP－DC组角膜植片的混浊、水肿、新生血管及排斥指数均显著降低（P＜0．01）。病理组织学检查结果显示各实验组角膜植片的炎症反应较阳性对照组轻,植片中央未见明显新生血管。免疫组织化学结果显示：IL－10－GFP－DC组的CD4＋、CD8＋、CD25＋、IL－2＋、NK＋及NF－κB＋阳性细胞数量较阳性对照组、GFP－DC组、8－DC组减少,差异均具有显著统计学意义（P＜0．01）。结论：经过供体来源未成熟树突状细胞预处理的受体,角膜植片的存活时间显著延长,成功诱导角膜移植免疫耐受。 CD4＋、CD8＋、CD25＋、IL－2＋、NK＋及NF－κB＋阳性细胞参与了同种异体角膜移植排斥反应的调控,IL－10－GFP－DC可降低CD4＋、CD8＋、CD25＋、IL－2＋、NK＋及NF－κB＋阳性细胞的浸润,抑制角膜移植排斥反应的发生。     

The T-lymphocyte chemoattractant Mig is highly expressed in vernal keratoconjunctivitis

PURPOSE: To examine the expression of the three interferon-gamma-inducible CXCR3-binding chemokines, CXCL10/IP-10 (interferon-gamma-inducible protein of 10 KDa), CXCL9/Mig (monokine induced by interferon-gamma), and CXCL11/I-TAC (interferon-inducible T-cell alpha chemoattractant) in the conjunctiva of patients with vernal keratoconjunctivitis (VKC). These chemokines exhibit potent T-lymphocyte chemoattractant activity. DESIGN: Immunohistochemical study. METHODS: Conjunctival biopsy specimens from 16 patients with active VKC and nine control subjects were studied by immunohistochemical techniques using monoclonal antibodies directed against IP-10, Mig, and I-TAC. The phenotype of inflammatory cells expressing chemokines was examined by double immunohistochemistry. RESULTS: In the normal conjunctiva, very weak Mig immunoreactivity was observed on basal epithelial cells and on vascular endothelial cells in the upper substantia propria. There was no immunoreactivity for the other chemokines. In all VKC specimens, strong immunoreactivity for Mig was expressed by epithelial cells, vascular endothelial cells, and inflammatory mononuclear cells. Inflammatory mononuclear cells expressing IP-10 and I-TAC were noted in 10 and nine specimens, respectively. The numbers of Mig(+) inflammatory cells were significantly higher than the numbers of IP-10(+) and I-TAC(+) inflammatory cells (P <.001). Inflammatory cells expressing Mig were CD4(+) T-helper/inducer cells (71.6 +/- 3.2%), CD8(+) T-cytotoxic/suppressor cells (19.5 +/- 1.5%), and CD68(+) monocytes/macrophages (5.3 +/- 5%). All inflammatory cells expressing IP-10 and I-TAC were CD68(+) monocytes/macrophages. CONCLUSIONS: The CXC chemokine Mig is selectively and highly expressed in VKC suggesting a pathogenic role of the chemokine receptor CXCR3 and the ligand Mig in the recruitment of activated T lymphocytes.     

Expression of chemokine receptors in vernal keratoconjunctivitis

BACKGROUND/AIMS: Chemokines are small peptides which are potent activators and chemoattractants for leucocyte subpopulations. Their action is mediated by a family of seven transmembrane spanning G-protein coupled receptors. The aims of this study were to examine the expression of the chemokine receptors CCR1, CCR3, CCR5, CXCR3, and CXCR4 in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and to investigate the phenotype of inflammatory cells expressing these chemokine receptors. METHODS: Conjunctival biopsy specimens from 16 patients with active VKC, and eight control subjects were studied by immunohistochemical techniques using a panel of monoclonal antibodies directed against human CCR1, CCR3, CCR5, CXCR3, and CXCR4. The phenotype of inflammatory cells expressing chemokine receptors was examined by double immunohistochemistry. RESULTS: In the normal conjunctiva, few inflammatory cells expressed CXCR3 in five of eight specimens. There was no immunoreactivity for CCR1, CCR3, CCR5, and CXCR4. In VKC specimens, membranous immunoreactivity for CXCR3 was noted on inflammatory cells in all specimens. Compared with control specimens, VKC specimens showed significantly more inflammatory cells expressing CXCR3 (54.3 (SD 34.3) v 3.3 (5.0); p<0.001). Few CCR1+, CCR3+, CCR5+, and CXCR4+ inflammatory cells were observed in only three of 16 specimens. Double immunohistochemistry revealed that all CXCR3 positive inflammatory cells were CD3 positive T lymphocytes and that 61.7% (3.7%) of the infiltrating T lymphocytes were reactive for CXCR3. CONCLUSIONS: CXCR3 is the predominant chemokine receptor and is expressed abundantly on T lymphocytes in the conjunctiva of patients with active VKC. These data suggest a potential role for CXCR3 receptors in the regulation of lymphocyte recruitment within conjunctiva of VKC patients. New therapeutic strategies that block CXCR3 may inhibit T lymphocyte recruitment and suppress adverse inflammatory reactions.     

Ocular cicatricial pemphigoid associated with hyperproliferation of the conjunctival epithelium

We determined the mitotic rate, measured by evaluating uptake of tritiated thymidine autoradiographically, and the frequency of goblet cells in conjunctival epithelial biopsy specimens from nine normal subjects and from 11 patients (seven women and four men ranging in age from 50 to 80 years) with ocular cicatricial pemphigoid. The mitotic rate of patients with the disease was significantly higher than that of normal subjects, 7.2 +/- 2.2 vs 1.6 +/- 0.2 labeled cells per 100 basal epithelial cells (P less than .01). The goblet cell frequency, however, was significantly less in patients than in normal subjects. This suggests that ocular cicatricial pemphigoid is associated with hyperproliferation of the conjunctival epithelium, with a concurrent failure of normal conjunctival differentiation.     

Scanning electron microscopy of conjunctival surfaces in patients with ocular cicatricial pemphigoid

The conjunctival surfaces of ten patients with active, ocular cicatricial pemphigoid, three patients with drug-controlled ocular cicatricial pemphigoid, and six patients with normal conjunctivas were studied using scanning electron microscopy. A homogeneous granular sheet of amorphous mucin-like material was observed covering extensive areas of the conjunctiva in eight of ten patients with active ocular cicatricial pemphigoid. This sheet of amorphous material was absent on drug-controlled ocular cicatricial pemphigoid and normal conjunctival specimens. Our study demonstrates that patients with active ocular cicatricial pemphigoid possess ocular surface mucus that appears thicker and more continuous than normal ocular mucus when observed with scanning electron microscopy. This observation is in agreement with clinical observations of thick mucus strands in the inferior fornix of patients with active ocular cicatricial pemphigoid.     

Immunophenotypic analysis of the inflammatory infiltrate in ocular cicatricial pemphigoid. Further evidence for a T cell-mediated disease

Ocular cicatricial pemphigoid (OCP) is characterized by the deposition of immunoglobulin and complement along the conjunctival epithelial basement membrane zone (BMZ). In order to further elucidate the cellular populations of the local inflammatory infiltrates, the authors used a panel of monoclonal antibodies in cryostat tissue sections to delineate T cell subsets, B lymphocytes, dendritic cells, and macrophages in six patients with OCP. In comparison with matched controls of the epibulbar conjunctiva, the authors discovered a threefold increase in T lymphocytes within the epithelium and a 20-fold increase within the substantia propria. In contrast with the normal-standing population of conjunctival T lymphocytes, there were activated interleukin 2 receptor (IL-2R)-positive lymphocytes in both the epithelium and the substantia propria. Macrophages were the second most common cells in the substantia propria, accounting for 12.7% of the mononuclear population--a threefold increase over the normal percentage. B cells and plasma cells, normally absent from epibulbar conjunctiva, were the next most prominent populations, constituting 6.9 and 4.6%, respectively, of all mononuclear cells. Dendritic cells which process antigen locally constituted only 1.2% of the mononuclear cell population, but were increased 25-fold over normal controls. By elaborating cytokines that promote fibroplasia, the T cells in OCP may be effector cells along with macrophages and other inflammatory cells in bringing about scarification of the substantia propria, and may furthermore be responsible for an immunoregulatory defect that allows local B lymphocytes to produce autoantibodies to the BMZ.     

Ultrastructural characteristics of conjunctiva in cicatricial pemphigoid

Conjunctival biopsy specimens were obtained from three patients with clinically active ocular cicatricial pemphigoid and from two of these patients after successful immunosuppressive therapy. Compared with specimens from normal subjects, the pretreatment specimens showed profound alteration in interepithelial adhesion as evidenced by a dramatic increase in desmosomes. Unusually prominent tonofilaments and tonofibrils were seen throughout the cytoplasm. The basal lamina showed areas of discontinuity, duplication, and focal thickening. The lamina propria was thickened and demonstrated an infiltration of inflammatory cells and disorganized collagen fibrils. Post-treatment specimens had a relative absence of desmosomes between the widely separated epithelial cells, less prominent tonofilaments and tonofibrils among epithelial cells, and, in one specimen, goblet cells. The enhanced network of desmosome-tonofilament complexes serves to tightly bind epithelial cells, perhaps providing a clue to the pathophysiology of conjunctival surface changes.     

Two cases of cicatricial pemphigoid showing atypical immunofluorescent findings.

We describe two patients with the clinical symptoms of cicatricial pemphigoid (CP). Biopsy specimens of the conjunctiva were taken. Histologic examination revealed subepidermal bullae and infiltration of inflammatory mononuclear cells. Direct immunofluorescent study showed immunoglobulins bound to the basement membrane zone (BMZ) in these patients. The patients also had intercellular immunoglobulin deposition in the conjunctival epithelium. No circulating anti-BMZ antibodies were detected, but one patient had a circulating antiintercellular antibody. Rare cases of CP with atypical immunofluorescent findings are reported.     

角膜移植排斥反应的铺片免疫组化研究

目的 探讨角膜移植排斥反应的发生机制及其参与细胞的表型。方法 制备正常大鼠和穿透性角膜移植鼠的角膜和虹膜睫状体铺片,用８种单克隆抗体,于铺片上进行免疫组化染色。结果 正常周边角膜及角膜缘可见少量的Ｔ细胞（ＣＤ３）、辅助／诱导性Ｔ细胞（ＣＤ４）、抑制／细胞毒Ｔ细胞（ＣＤ８）、巨噬细胞、树突细胞、主要组织相容性复合体Ⅱ类抗原阳性细胞及β转化生长因子阳性细胞;同种角膜移植术后７及１２天角膜和虹膜睫状体中     

Diagnostics and pharmacological treatment of ocular cicatrical pemphigoid

Ocular cicatricial pemphigoid (OCP) is an autoimmune disease characterize by mucous membrane fibrosis and skin changes resulting with scarring. The pathogenic mechanisms of ocular cicatricial pemphigoid are incompletely understood. Antibasement membrane antibodies which lead to subepithelial blistering, granulation tissue and inflammatory infiltrate formation in the substantia propria are thought to be the main pathophysiological mechanisms in cicatricial pemphigoid. It has been found eosinophils and increased collagen type I and III. Human leukocyte antigen HLA-DR2, HLA-DR4 and DQw7 genotypes have been identified as conferring increased susceptibility to the development of this disease. Ocular cicatrical pemphigoid (OCP) is one of the forms of bullous pemphigoid. Initial symptoms of ocular pemfigoid are not characteristic. Conjunctival fibrosis may cause severe entropion, trichiasis, symblepharon, dry eye syndrome, corneal epithelial erosions or ulceration. Secondary glaucoma is one of the most frequent complications. Ocular cicatricial pemphigoid may be chronic, acute, or subacute disease with periodic exacerbation of conjunctival inflammation. The treatment in this disease are topical drops or ointment (lubricants, corticosteroids, antibiotics, antiglaucomatous). Oral dapsone and corticosteroids may control the activity of the disease. In other progressive cases immunosuppressive drugs must be used (azathioprine, cyclophosphamide, methotrexate, mycophenolan mofetil, daclizumab, intravenous immunoglobulin therapy). To make an early diagnosis of ocular cicatricial pemphigoid, biopsy and immunohistochemical analysis of conjunctiva should be performed in every case of persistent conjunctival inflammation.     

Immunohistochemical analysis of orbital connective tissue specimens of patients with active Graves ophthalmopathy

PURPOSE: To explore the immune mechanism of Graves ophthalmopathy (GO) by analyzing infiltrating cells in orbital connective tissue (OCT) specimens of patients with active GO using immunohistochemical methods. METHODS: Five OCT specimens obtained from patients with active GO and five control specimens obtained from forensic cadavers who died from nonmedical reasons were stained with anti-CD3, CD4, CD8, CD45RO, HLA-Dr, CD25, and TNF-alpha monoclonal antibodies. Positively stained cells were counted and results were interpreted as cell counts/mm2. Four of five GO patients had never been treated with any immunomodulating therapy. Only one had received oral prednisolone prior to tissue sampling, but this treatment had ceased 5 months before surgery. RESULTS: The retro-orbital tissue specimens obtained from forensic cadavers did not show any significant positive staining for any monoclonal antibody tested. However, the specimens from GO patients showed positively stained means of 36.66 +/- 4.61 HLA-Dr+, 12.8 +/- 3.42 CD8+, 11.8 +/- 1.78 CD4+, 16.6 +/- 1.81 CD3+, 21.2 +/- 3.12 CD45RO+, 10.4 +/- 2.07 TNF-alpha+, 7.2 +/- 1.48 CD25+, 3.2 +/- 1.09 CD4+CD8+, 4.6 +/- 1.67 CD4+CD45RO+, 2.8 +/- 0.83 CD8+CD45RO+, 1.6 +/- 0.89 CD4+CD25+, and 1.8 +/- 1 0.83 CD8+CD25+ cells/mm2. CONCLUSIONS: Our study supports that most of the infiltrating lymphocytic cells in the active stage of GO are T cells, and a significant proportion of them are CD45RO+ cells. Infiltration of OCT by HLA-Dr+, CD25+, and TNF-alpha cells suggests that Th1-type immune reaction with the interference of proinflammatory cytokine(s) (TNF-alpha) may be important in the pathogenesis of disease. Further studies are needed to understand the disease pathogenesis and may provide a scientific basis for future treatment alternatives for the disease (e.g., anti-cytokine treatment).