Ron A. Adelman (New Haven, CT, USA)

Advantages:

To develop a culture model of human fetal retinal pigment epithelium (hfRPE) to examine the effects of serum and proinflammatory cytokines on the tight junctions of the outer blood retinal barrier.  In the wet form of age-related macular degeneration (AMD), leaky capillaries grow in the subretinal space, exposing the apical side of the RPE to serum. Further, proinflammatory cytokines are present.  Both pathologic stimuli affect tight junctions in other tissues. Cultured hfRPE were used to examine effects on tight junctional proteins, the claudins and occludin.

Methods:

To examine effects of serum, cells were maintained in a serum-free medium. This medium was supplemented with serum in the apical, basal, or both media chambers. To examine cytokine effects, cells were maintained in growth medium (5.0% or 0.5% serum) or in serum-free medium. TNF-á, IL-1â, or INF-ã was added to both media chambers for 48 hours. Transepithelial electrical resistance (TER) was utilized to monitor permeability of tight junctions. Relative conductance of Na+ and K+ was estimated from TER in buffered NaCl or KCl. Effects on claudins and occludin were measured using real time RT-PCR, immunoblotting, and immunofluorescence. Claudin expression was knocked down using siRNA.

Effectiveness / Safety:

The transepithelial electrical resistance of cells in serum-free medium was ~200 omega-cm2, close to that found in native tissue. With apical, but not basal, serum the TER increased 2-3 fold. Apical serum also affected ion selectivity of tight junctions by increasing the conductance of K+ relative to Na+.  TNF-á decreased TER substantially in all media conditions, while INF-ã led to a smaller decrease; IL-1â had little effect. TNF-á also affected ion selectivity. At the molecular level, Claudin 19 mRNA was the predominant claudin in vivo and in culture. Knockdown of claudin 19 eliminated barrier function. The expression and distribution of claudin 19 was unaffected with the addition of apical serum. By contrast, there was a correlation between TER and the expression level of occludin. In all media, TNF-á decreased the expression of claudin 19, but increased the expression of claudin 2. The other cytokines had minimal effects on tight junctions.

Take home message:

Subretinal serum appears to decrease the permeability of the outer blood-retinal barrier via occludin, a known regulator of permeability. By making the barrier tighter, apical serum may help limit the spread of edema. TNF- á, had the most pronounced effect on RPE tight junctions by partially replacing claudin 19 (makes junctions less permeable to cations) with claudin 2 (makes junctions leakier to cations).