Purpose: Trefoil factor family (TFF) peptides (formerly P-domain peptides; trefoil factor) are small (7-12 kDa) protease-resistant secreted peptides designated pS2 (or TFF1), SP (TFF2) and ITF (TFF3). Human conjunctival goblet cells (GCs) are known to synthesize TFF, but TFF expression by these cells has not been studied in pathological conditions. We quantified trefoil factor family (TFF) gene transcripts in pterygium, and we immunolocalized TFF protein.
Methods: Eleven pterygium specimens were studied, together with 19 biopsy specimens of normal human conjunctiva as controls. TFF1 (pS2), TFF2 (spasmolytic peptide) and TFF3 (intestinal trefoil factor) mRNA expression was semiquantified by means of reverse-transcription polymerase chain reaction amplification (RT-PCR). TFF1, TFF2 and TFF3 mRNA levels were determined individually, relative to beta2 microglobulin housekeeping gene mRNA (internal standard), by coamplification of the target fragments and beta2 microglobulin in the same tube. Five pterygia and five normal human conjunctival biopsy specimens were also analyzed for TFF1 and mucin (MUC5AC) protein expression by immunostaining with monoclonal antibodies. Anti-PS2 (Zymed Laboratories, San Francisco), a mouse monoclonal antibody (MAb) against the 30 C-terminal amino acids of human TFF1, and P2802 (provided by Doctor Marie-Christine Rio, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM, Strasbourg, France), a mouse MAb directed against a synthetic peptide corresponding to the last 28 amino acids of TFF1, were used at 1/20 dilution. A mouse monoclonal antibody directed against the peptidic core of gastric M1 mucin was used as previously described. M1 immunoreactivity is encoded by the MUC5AC gene.
Results: TFF1 and TFF3 mRNA was expressed in all normal conjunctival and pterygium specimens. TFF2 mRNA was not expressed by either sample type, but was expressed by the positive control (human stomach cDNA). TFF1 mRNA expression was stronger in pterygium than in controls (p=0.02). TFF3 mRNA expression was similar in the two sample types (p=0.89). TFF are coexpressed and act in concert with mucins to protect mucous epithelia and trigger wound-healing responses. Inflammation and ulceration of the gastrointestinal tract are associated with increased TFF expression. Conjunctival GCs secrete TFF in both pigs and humans. We found that TFF1 mRNA was overexpressed in pterygium relative to healthy conjunctiva, whereas the TFF1 immunostaining patterns were similar. TFF1 protein expression was confined to goblet cells. However, whereas all GCs were positive for MUC5AC, not all GC were labeled by anti-TFF1 mAbs in either normal conjunctiva or pterygium. The observed TFF1 mRNA overexpression in pterygium was not associated with abnormal TFF1 peptide localization. Increased MUC5AC protein expression would be expected in pterygium, because of increased GC density. Indeed, in conjunctival diseases such as dry-eye syndrome in which GC density is decreased, mucin secretion is also decreased. This could explain the increased expression of TFF1 mRNA in pterygium, although not all GCs expressed TFF1 protein. TFF proteins are copackaged within mucous cell granules; TFF1 preferentially colocalizes with MUC5AC, and TFF3 with MUC2. However, we found some cell granules containing MUC5AC but not TFF1. The proportion of TFF1-negative GCs was similar in pterygium and normal conjunctiva. The normal TFF3 mRNA expression in pterygium was unexpected and suggests that only GCs involved in TFF1 secretion are overrepresented in this pathological tissue. TFF2 mRNA was undetectable in both normal conjunctiva and pterygium, possibly because of its copackaging in mucous cell granules and its preferential cosecretion with MUC6, which is not expressed in the conjunctiva.
Conclusion: As in normal conjunctiva, the TFF1 and TFF3 genes are expressed by conjunctival goblet cells in pterygium, contrary to the TFF2 gene. Only TFF1 gene expression was elevated in pterygium compared to normal conjunctiva.