Development of organised conjunctival leucocyte aggregates after corneal transplantation in rats

Abstract

Aim: To investigate the development of lymphoid aggregates in the conjunctiva after corneal transplantation in rats.

Methods: LEW or PVG strain corneas were transplanted orthotopically to PVG rats. Cornea and conjunctiva were examined clinically for up to 42 days. Eyes were removed with attached conjunctiva on days 10 and 15 after transplantation (before and during rejection), together with normal eyes, fixed, paraffin embedded, and examined immunohistochemically.

Results: Clinically, the temporal half of the upper palpebral conjunctiva of recipients of 10/19 allografts and 1/10 isografts developed pronounced swelling, correlating with inflammation and rejection. Histologically, the swelling comprised leucocytic aggregates with an altered overlying epithelium. Aggregates contained granulocytes, macrophages, and cells expressing major histocompatibility complex (MHC) class II, CD4, and CD8, all more numerous in allograft associated conjunctiva. Class II+ cells were more abundant at the surface, whereas macrophages and T cells were more numerous in the deeper stroma. There were few B cells. There was greater CD54 expression by vascular endothelium in allograft associated aggregates. Cells expressing TNFalpha and IFNgamma but not IL1beta were present in stromal and superficial areas.

Conclusions: Corneal transplantation in rats induces the development of organised conjunctival leucocytic aggregates in a fixed location that are significantly more pronounced in recipients of allografts compared with isografts and show characteristics of a Th1 type immune response. These aggregates have characteristics of conjunctiva associated lymphoid tissue and may be sites of presentation of graft antigens and lymphocyte proliferation at the ocular surface.

Figure 1

Schematic diagram of superficial (a) and stromal (b) areas counted for cell infiltrate. “a” had the same width as “b”, but its height varied according to the thickness of the adjacent epithelium. Counts were adjusted to number per 0.04 mm² (the size of the image analysis screen). “b” was 0.04 mm² and located in the area of maximum infiltrate.

Figure 2

(A) Appearance of normal conjunctiva of the temporal half of the upper lid (lid everted with forceps) without clinically evident leucocytic aggregate. Note absence of oedema and normal vasculature (original magnification ×15). (B) ED2⁺ macrophage (arrowheads) and (C) CD8α⁺ cells in normal conjunctiva. (D) Clinical appearance of aggregate on day 10 after transplantation showing hyperaemia, overhanging edge (arrow), and swelling, clinical score 4 (original magnification ×10).

Figure 3

Clinical scores of conjunctiva in allografts (▴, n = 7) and isografts (◊, n = 6) before and during rejection. Broken line indicates score equivalent to histological identifiable lymphoid aggregate.

Figure 4

(A) Location of leucocytic aggregate (arrow) in the palpebral conjunctiva (haematoxylin and eosin). Note goblet cells are absent in adjacent epithelium. m  =  graft margin. (B) Capillary displaying HEV morphology. (C) CD54 expression on basal epithelium, infiltrating cells, and capillary endothelium (inset). (D) Pancytokeratin staining showing lack of epithelium (note pallisading arrangement, arrowheads) overlying the aggregates but occasional cells (arrow) in the stroma. (E) MHC class I staining is not evident in superficial aggregate. (F) Composite picture showing CD4⁺ cells in upper (UL) and lower (LL) conjunctiva of the same section. Infiltration is more dense in UL, between the aggregates (CA) and the cornea (CO), than in LL. (G) NK cell are mainly in the stroma. Note pallisading (arrowheads) of cells in superficial layers. (H) ED2⁺ macrophages in circular arrangement in the aggregate defined by arrows. All unlabelled bars are 100 μm.

Figure 5

Cell numbers in (A) superficial (“a” in Fig 1) and (B) stromal areas (“b” in Fig 1) of five leucocytic aggregates large enough for all cell types to be identified. Closed symbols indicate allografts; open symbols  =  isografts; diamonds  =  day 10; squares  =  day 15 after transplantation. Note lower count of cells in isograft associated aggregates.

Figure 6

(A) MHC class II expressing cells are numerous in superficial leukocytic aggregates. (B) CD4⁺ T cells colocalise with (C) CD25⁺ cells. (D) CD8α staining is greater in stromal areas and expressed on round and dendritic cells. (E) Granulocytes in aggregates within both superficial and stromal areas. (F) IFNγ expression and (G) TNFα expression in leucocytic aggregates. Immunofluorescence double staining showing cells (arrows) positive for (H) IFNγ (red) and CD8α (green) (×20); (I) IFNγ (red) and ED2⁺ macrophages (green) (×40); (J) TNFα (red) and ED2⁺ macrophages (green) (×40). Unlabelled bars are 50 μm.