Effects of the Dialyzable Leukocyte Extracts on Human Limbal Epithelial Cells

Abstract

Purpose: : The dialyzable leukocyte extracts (DLE), is a peptidic fraction of <12KDa obtained from a dialysis of leukocytes with the capacity to transfer the cellular immune response. Despite its mechanism of action is unknown it has been recognized that DLE has an immunomodulatory effect on peripheral blood mononuclear cells. DLE has been used as an adjuvant treatment of patients with herpetic stromal keratitis (HSK), and patients treated with acyclovir plus DLE clinically improved faster than those treated only with acyclovir. It is possible that DLE could induce changes in the microenvironment that support the corneal re-epithelization, the aim of this study was to determine the effect of dialyzable leukocyte extracts on inflammatory cytokine profile of the human limbal epithelial cells.

Methods: : Human Limbal Epithelial Cells (HuLEC) were obtained from sclero-corneal rings from cadaveric donors and cultured in KSFM medium. Culture cells were phenotypically characterized by flow cytometry searching the molecular markers CK19, p63 and Vimentin. After that, HuLEC were stimulated with different concentrations of DLE or PMA/Ionomycine. Culture supernatants were collected at 0, 1 and 24h and then were analyzed by Cytometric Bead Arrays (CBA) to measure the following cytokines IL1b, IL6, IL8, IL10, IL12p70 and TNFa. Results were analyzed by t Test, and p<0.05 was considered as statistically significant.

Results: : We observed that HuLEC produce IL-6 and IL-8 at basal conditions: IL-6, 1h (7.5 pg/mL), 24 h (219 pg/mL); IL-8, 1h (142.2 pg/mL), 24h (1253 pg/mL) we did not observe other cytokines at basal level. When HuLEC were stimulated with DLE we observed a significantly reduction of IL-6 (6.1 pg/mL), 24 h (25.7 pg/mL); IL-8, 1h (11.3pg/mL), 24h (1007.5 pg/mL). DLE stimulation did not induce IL1b, TNFa, or IL12p70.

Conclusions: : DLE diminished the concentrations of pro-inflammatory cytokines (IL-6 and IL-8) produced by HuLEC without induction of IL1b, TNFa or IL12p70. These anti-inflammatory properties could explain the clinical improve observed in patients with HSK; these results suggest that DLE could be used as anti-inflammatory therapeutic tool, nevertheless more studies are necessary to know the real role of DLE in the ocular microenvironment of patients with inflammatory ocular surface diseases.Acknowledgments: This work was partially supported by CONACYT 71291 and CGPI 20082530

Keywords: cornea: basic science • cornea: epithelium • immunomodulation/immunoregulation