Elevation of conjunctival epithelial CD45INTCD11b⁺CD16⁺CD14⁻ neutrophils in ocular Stevens-Johnson syndrome and toxic epidermal necrolysis

Abstract

Purpose: Ocular complications related to Stevens-Johnson Syndrome (SJS)-Toxic Epidermal Necrolysis (TEN) may persist and progress after resolution of systemic disease. This is thought to be related in part to persistent ocular innate-immune signaling. In this study, our aim was to characterize infiltrative conjunctival cellular profiles during acute (<12 months) and chronic (>12 months) disease.

Methods: Consecutive patients presenting with SJS-TEN over a 12-month period were followed for 1 year. Detailed clinical examination and conjunctival impression cell recovery was analyzed by flow cytometry for the presence of intraepithelial leukocytes and compared with healthy controls (n = 21).

Results: Ten patients were recruited of whom six had acute disease and five were classified as TEN (SCORTEN = 1, n = 4). Conjunctival inflammation was graded as absent/mild in a total of nine patients; but despite this, evidence of fornix shrinkage was observed in nine subjects. This inversely correlated with disease duration (P < 0.05). A reduction in percentage of CD8αβ(+) T cells compared with controls (80% vs. 57%; P < 0.01) was associated with a corresponding increase in the number/percentage of CD45(INT)CD11b(+)CD16(+)CD14(-) neutrophils (186 vs. 3.4, P < 0.01, 31% vs. 0.8%, P < 0.001). Neutrophils inversely correlated with disease duration (r = -0.71, P = 0.03), yet there was no absolute change in the CD8αβ(+) or neutrophil populations during the study period (P = 1.0).

Conclusions: These data highlight that a neutrophilic infiltrate is present in mildly inflamed or clinically quiescent conjunctival mucosa in patients with ocular SJS-TEN, where neutrophil numbers inversely correlate with disease duration. Neutrophil persistence endorses the hypothesis of an unresolved innate-inflammatory process that might account for disease progression.

Keywords: SJS; TEN; innate immunity; neutrophils.

Figure 1

Gating strategy to determine cellular populations. Representative plots of a subject with SJS-TEN demonstrating the gating strategy used to identify conjunctival leukocytes. Live leukocytes populations (lymphocytes, monocytes, and granulocytes) were identified in conjunctival cells by gating for CD45⁺ cells that were negative for the dead cell exclusion dye Sytox blue and back-gated to show the forward and side scatter profiles of the CD45⁺ live cells (A). Lymphocyte subsets were identified according to the expression of CD3 and CD56 in one panel (to discriminate T cells [CD3⁺CD56⁻]; natural killer cells [CD56⁺CD3⁻]; NKT cells [CD3⁺CD56⁺]) and B cells [CD19⁺CD20⁺] [B] [CD8⁺ cell populations shown are TCRαβ⁺]). Monocytes were further characterized by the presence of CD14 and CD16 (C). Neutrophils were defined as CD45^INT, CD14⁻, CD11b⁺, and CD16⁺ granulocytes (D). Cell populations of interest are boxed (blue).

Figure 2

The dominant cellular infiltrate in the conjunctival epithelium in SJS-TEN is characterized by an increase in CD45^INTCD11b⁺CD16⁺CD14⁻ neutrophils. Twenty-one healthy subjects were compared with 10 patients with SJS-TEN (six patients in the acute stages, open square; and 4 in the chronic stages of systemic disease, closed square). Representative flow cytometry plots are shown. Statistical comparisons between healthy and SJS-TEN conjunctival leukocytes and lymphocytes (A), CD45⁺CD3⁺CD56⁻CD8αβ⁺ T cells ([B], boxed); CD45^INTCD11b⁺CD16⁺CD14⁻ neutrophil (C); and CD45⁺CD14⁺CD16⁻ monocyte (D) populations were undertaken by the Mann-Whitney U test. NS, not significant. *P > 0.05. **P < 0.01).

Figure 3

CD45^INTCD11b⁺CD16⁺CD14⁻ neutrophils are inversely correlated with disease duration but persist with time. CD45^INTCD11b⁺CD16⁺CD14⁻ neutrophil populations were correlated with disease duration at presentation (0 months; [A]) using Spearman's correlation (*P > 0.05]; **P < 0.05). Nine out of 10 patients with SJS-TEN were sampled at 0 and 12 months (B). Paired analysis of SJS-TEN conjunctival CD45^INTCD11b⁺CD16⁺CD14⁻ neutrophil populations at 0 (closed squares) and 12 months (open squares) were undertaken by the Wilcoxon signed rank test (P > 0.05; [B]).