Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction

Abstract

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo-reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vbeta6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vbeta6-) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vbeta6+ T cells was then determined by FACS analysis, and the division profiles of responder Vbeta6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo-reactive T cells in MLR culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vbeta6+ T cells was significantly reduced in the AM-treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vbeta6+ T cells in cultures with AM than without. In addition, allo-rective T cell synthesis of both Th1 (IL-2 and IFNgamma) and Th2 (IL-6 and IL-10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo-reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT-AMT combination.

Fig. 1

Inhibition by human AM of proliferative responses of murine allo-reactive mixed lymphocyte reaction (MLR). Human AM was added to the transwell of MLR culture containing C57BL/6 responder and DBA/2 stimulator. Lymph node cells (2 × 10⁶ cells/ml) from C57BL/6 mice (as responders) were mixed with gamma-irradiated (20Gly) spleen cells (2 × 10⁶ cells/ml) from histoincompatible DBA/2 mice (as stimulators). These MLR cultures were then cocultured with human AM in the transwell of 24 wells. These culture plates were incubated for 4 days, during the last 8 h of incubation, ³H-thymidine was added. Positive allogenic control was MLR of C57BL/6 responder and DBA/2 stimulator without human AM. Negative syngenic control consisted of responder and stimulator cells from C57BL/6 without human AM. In some experiments, human AM was prefixed with 100% ethanol, dried, and washed with PBS 4 times. This fixed human AM was added to the transwell of allogenic MLR culture containing C57BL/6 responder and DBA/2 stimulator cells. Radioisotope incorporation detected in 4 separate experiments with triplicate wells is presented as mean cpm ± SEM. (*P < 0·001)

Fig. 2

Suppression of responder CD4⁺, V_β6⁺ T cell division by human AM. FACS profile of CD4⁺V_β6⁺ T cells (a) and analysis of responder CD4⁺V_β6⁺ T cell division (b) in murine MLR in the presence or absence of human AM. Lymph node cells (2 × 10⁶) from C57BL/6 mice (as responders) were mixed with gamma-irradiated (20Gly) spleen cells (2 × 10⁶) from histoincompatible DBA/2 mice (as stimulators) in the presence or absence of human AM in the MLR culture transwell. Intracellular fluorescent dye, CFSE, was used to label responder cells before in vitro culture. After 4-day culture, cells were double stained with PE-conjugated anti-V_β6 mAb and APC-conjugated anti-CD4 mAb. Results of mean percentages from 3 independent experiments are presented as mean (%) ±SEM. Data of CFSE histograms represent of 2 separate experiments.

Fig. 3

Inhibition by human AM of Th1 and Th2 cytokine synthesis in murine MLR. Levels of cytokine production in culture supernatants of allogenic MLR with AM (), allogenic MLR without AM (□) and syngenic MLR without AM (▪) were examined by murine Th1 and Th2 cytokine-specific ELISA. Following 2 day incubation, IL-2 synthesis level was measured. In case of IFNγ, IL-6 and IL-10, supernatants harvested from 4 day cultures were subjected to cytokine-specific ELISA. Data represent mean ± SEM from 1 experiment with duplicated wells. (<not detectable; *P < 0·01; **P < 0·001)