Regulation of the receptor for TNFalpha, TNFR1, in human conjunctival epithelial cells

Abstract

Purpose: Previous studies demonstrated that mast cell-derived TNFalpha stimulation is critical to the upregulation of intercellular adhesion molecule (ICAM)-1 on human conjunctival epithelial cells (HCECs), which is an important feature of ocular allergic inflammation. Shedding of TNFR1 by TNFalpha-converting enzyme (TACE) is a primary mechanism for the regulation of TNFalpha-mediated events. This process has not been examined in HCECs. In this study, the authors examined the regulation of TNFR1 expression and shedding by TACE on primary HCECs and the IOBA-NHC conjunctival epithelial cell line.

Methods: Primary human conjunctival mast cells and epithelial cells were obtained from cadaveric conjunctival tissue. HCECs were incubated with and without activators (IgE-activated mast cell supernates, phorbol myristate acetate [PMA; to activate TACE], TNFalpha, and IFNgamma [to upregulate TNFR1]) for 24 hours. Pretreatment with the TACE inhibitor TAPI-2 was used to inhibit shedding of TNFR1. Supernates collected from the incubations were analyzed with ELISA for soluble TNFR1 (sTNFR1). With the use of flow cytometry, cells were harvested from these experiments for analysis of TNFR1 and ICAM-1 receptor expression.

Results: IgE-activated conjunctival mast cell supernates upregulated the expression of TNFR1. TAPI-2 inhibited the PMA-induced release of sTNFR1 receptor and enhanced the surface expression of TNFR1 in HCECs in a dose-dependent manner. Upregulation of TNFR1 expression by priming with TAPI-2 and IFNgamma resulted in enhanced ICAM-1 expression in response to TNFalpha stimulation (significant change in the slope of the dose-response curve).

Conclusions: These results demonstrate that TACE promotes TNFR1 shedding in HCECs and that TNFR1 expression may be a more significant target than TNFalpha for intervention in ocular inflammation.

Figure 1

Representative overlay histograms (n = 3) showing upregulation of staining for TNFR1 (x-axis) on primary HCECs after incubation with supernates from anti-IgE–stimulated purified conjunctival mast cells (black) compared with unstimulated HCECs (gray). MFI was increased by 2 ± 0.4 units (P < 0.05).

Figure 2

Effect of PMA stimulation on sTNFR1 release from unprimed (gray bars) versus TAPI-2–primed (black bars) IOBA-NHC cells (A; n = 4, P < 0.05) and primary HCECs (B; n = 2).

Figure 3

Percentage inhibition of PMA-stimulated release of sTNFR1 by EDTA (dashed line [■]) and TAPI-2 (solid line [◆]) from IOBA-NHC cells (A; n = 2) and primary HCECs (B; n = 2).

Figure 4

Comparison of effect of TAPI-2 and EDTA on release of sTNFR1 (gray bars) versus sICAM-1 (black bars) from IOBA-NHC cells (A; n = 2) and primary HCECs (B; n = 2).

Figure 5

Representative overlay histograms (n = 4) showing upregulation of staining for TNFR1 (x-axis) on primary HCECs after priming with TAPI-2 and IFNγ (black) compared with unstimulated HCECs (gray). MFI was increased by of 2.7 ± 0.9 units (P < 0.05).

Figure 6

Effect of TAPI-2/IFNγ priming on TNFα-mediated upregulation of ICAM-1 expression on IOBA-NHC cells (A) and primary HCECs (B). (A) Dose–response curves to TNFα with and without TAPI-2/IFNγ priming (left y-axis; percentage ICAM-1 expression greater than unstimulated cells; *P < 0.05 for comparisons between unprimed and primed treatments, including slopes; n = 4). Also shown is the lack of effect of TAPI-2/IFNγ priming on TNFα-stimulated release of sICAM-1 into supernates from the same experiments (right y-axis; sICAM-1 concentration [minus unstimulated] in pg/mL). (B) In primary HCECs, TAPI-2/IFNγ priming resulted in a similar enhanced response to TNFα-mediated ICAM-1 upregulation (5 ng/mL concentration of TNFα shown; n = 2).

Figure 7

Schematic representation of regulation of TNFR1 shedding and its role in TNFα regulation. Regulation of the availability of TNFα receptors provides a protective mechanism against excessive TNFα activity. Proteolytic cleavage of TNFR1 is facilitated by TACE, which can be activated in vivo by the release of reactive oxygen species (ROS) and inhibited by TIMP-3. Shedding of TNFR1 results in the downregulation of receptor numbers on the cell surface, which decreases cellular sensitivity to TNFα. Soluble forms of TNFR1 can also bind to circulating TNFα, resulting in neutralization of TNFα-mediated responses. In our in vitro studies, PMA was used to stimulate ROS, and TAPI-2 was used to inhibit TACE. TAPI-2, a derivative of hydroxamic acid, inhibits TACE by interfering with zinc-dependent activation in the catalytic site of the metalloprotease.