The effect of leukocyte infiltration on apoptosis in an in vitro thermal injury bioartificial living skin equivalent model.

Abstract

An in vitro bioartificial skin construct (BSC) model was studied to see how inflammatory infiltration affects apoptosis in skin that has been thermally injured. The BSC was used as a target organ. Control BSCs without leukocytes (CON) were burned (BCON) by scalding with phosphate-buffered saline heated to 70 degrees C for 6 seconds, and they were then cooled with room temperature phosphate-buffered saline for 15 seconds. Human alloimmunocytes were added to CON to create rejection cultures (REJ) and to BCON to create burned rejection cultures (BREJ). Slides were stained with hematoxylin and eosin and anti-Lewis antibody. In situ labeling of apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Those sections that were immunostained for Lewis Y were analyzed for intensity stain index (ISI = [sigma P0 x I0]/ total tissue area in pixels). TUNEL was quantified with the following equation: no. of total apoptotic cells / total tissue area. Necrosis and blister formation in the epidermal layer were evident in BCON and BREJ. Pyknosis and nuclear fragmentation-indicators of apoptosis-were also present. Phagocytosis of keratinocytes by leukocytes was seen in REJ and BREJ. Immunostaining showed greater expression of Lewis Y antigen, as determined by ISI, in REJ as opposed to CON (58.2+/-2.3 vs. 36.4+/-2.3, respectively, P<.001), but no significant difference was found between BCON and BREJ (55.0+/-5.7 vs. 60.5+/-3.4, respectively) and REJ and BREJ (58.3+/-2.3 vs. 60.5+/-3.4, respectively). TUNEL staining indicated the presence of apoptosis as follows: REJ versus CON (0.0015+/-0.0002 vs. 0.0003+/-0.0001, respectively, P<.001); BREJ versus BCON (0.0031+/-0.0006 vs. 0.0018+/-0.0004, respectively, P<.05); REJ versus BREJ (0.0015+/-0.0002 vs. 0.0031+/-0.0006, respectively, P = .007). The presence of leukocytes and thermal injury induces apoptosis in BSC. The combination of these two variables results in increased apoptosis as determined by TUNEL. These findings suggest that a common pathway for skin injury may include inappropriate regulation of apoptosis exacerbated by a mechanism that includes inflammatory cellular infiltration.