Immunohistochemical Analysis of Scarring Trachoma Indicates Infiltration by Natural Killer and Undefined CD45 Negative Cells.

INTRODUCTION: The phenotype and function of immune cells infiltrating the conjunctiva in scarring trachoma have yet to be fully characterized. We assessed tissue morphology and immunophenotype of cellular infiltrates found in trachomatous scarring compared to control participants. METHODOLOGY: Clinical assessments and conjunctival biopsy samples were obtained from 34 individuals with trachomatous scarring undergoing trichiasis surgery and 33 control subjects undergoing cataract or retinal detachment surgery. Biopsy samples were fixed in buffered formalin and embedded in paraffin wax. Hematoxylin and eosin (H&E) staining was performed for assessment of the inflammatory cell infiltrate. Immunohistochemical staining of single markers on individual sections was performed to identify cells expressing CD3 (T-cells), CD4 (helper T-cells), CD8 (suppressor/cytotoxic T-cells and Natural Killer, NK, cells), NCR1 (NK cells), CD20 (B-cells), CD45 (nucleated hematopoietic cells), CD56 (NK and T-cells), CD68 (macrophages/monocytes) and CD83 (mature dendritic cells). The degree of scarring was assessed histologically using cross-polarized light to visualize collagen fibres. PRINCIPLE FINDINGS: Scarring, regardless of clinical inflammation, was associated with increased inflammatory cell infiltrates on H&E and CD45 staining. Scarring was also associated with increased CD8+ and CD56+ cells, but not CD3+ cells, suggestive of a NK cell infiltrate. This was supported by the presence of NCR1+ cells. There was some increase in CD20+ cells, but no evidence for increased CD4+, CD68+ or CD83+ cells. Numerous CD45 negative cells were also seen in the population of infiltrating inflammatory cells in scarred conjunctiva. Disorganization of the normal collagen architecture was strongly associated with clinical scarring. CONCLUSIONS/SIGNIFICANCE: These data point to the infiltration of immune cells with a phenotype suggestive of NK cells in conjunctival trachomatous scarring. A large proportion of CD45 negative inflammatory cells were also present. Future work should seek to understand the stimuli leading to the recruitment of these cells and their role in progressive scarring.

In vivo confocal microscopy and histopathology of the conjunctiva in trachomatous scarring and normal tissue: a systematic comparison.

AIM: To compare in vivo confocal microscopy (IVCM) with the histopathological examination of tissue and cellular changes in normal and diseased conjunctiva. METHODS: Participants underwent clinical examination and IVCM of the tarsal conjunctiva. A biopsy of the upper tarsal conjunctiva was collected and stained with tinctorial stains and by immunohistochemical staining for CD45 and CD83. Connective tissue scarring, inflammatory cell density and the presence of dendritiform cells were quantitatively assessed in a masked manner by both IVCM and histological assessments for comparative analysis. RESULTS: Thirty-four participants with severe trachomatous conjunctival scarring and 33 participants with healthy conjunctiva were recruited. The IVCM connective tissue scarring score was strongly associated with the histological grading of scarring (p<0.001). There was limited evidence of an association between the IVCM inflammatory cell infiltrate and the histological inflammatory cell grade (p=0.05). We did not find any evidence to support the hypothesis that dendritiform cells seen with IVCM are mature, conventional dendritic cells. CONCLUSIONS: The results show that IVCM can be used to robustly quantitate connective tissue scarring and also has a role in measuring the inflammatory cell infiltrate. The discordance between IVCM dendritiform cells and immunohistochemical dendritic cells may be a result of study limitations or may be because these dendritiform structures represent another cell type, such as fibroblasts, rather than dendritic cells.