We have previously explored the use of genetically modified keratinocytes/fibroblasts for systemic or local delivery of IL10 and have noted a drop in IL10 secretion when cells are assembled into a bi-layer tissue construct (raft cultures). In this study we examine the basis for this reduction. 

Objectives:

To establish the mechanisms behind the fall in secretion i.e., is it due to loss of cells and/or loss of transgene expression or can the fall be attributed to increased accumulation of transgene product in the cells.           

Methods:

Normal keratinocytes and fibroblast cells were transduced with a bi-cistronic MMLV-retroviral vector encoding for GFP & IL10 (30%+ by GFP flow-cytometry). GFP is used as a control for a non-secreted gene product. Submerged/raft cultures were constructed (as described elsewhere) with these cells. IL10-secretion rates were normalized for cell number. Cell-associated IL10 was measured by treating washed cell pellets with TritonX-100 and then analyzing lysates for amount of IL10. 

Results:

 

The percentage of GFP-positive-cells remains unchanged when cells are placed in raft-cultures.

Conclusions:

1) There is no selective loss of transduced cells in rafts to explain the fall in secretory-rate.  

2) GFP-levels remain essentially unchanged in submerged and raft cultures, suggesting that transgene expression is unchanged in both culture models (see-columns B&C).

3) There is a 10-fold drop in IL10 in raft-cultures primarily from reduced keratinocyte secretion, not fibroblasts (see-columns D&E)

4) This drop in keratinocyte IL10 secretion is accompanied by an increase in cell-associated IL10 as compared to fibroblasts (see-columns F&G).

5) These results suggest that the drop in IL10 secretion in raft cultures results primarily from a reduction in IL10 release from keratinocytes.

Supported by NIH-DEO4511 (to LBT)