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

An in vitro bioartificial skin construct (BSC) model was studied to see how inflammatory infiltration affects apoptosis in skin that has been thermall y injured. The BSC was used as a target organ. Control BSCs without leukocy tes (CON) were burned (BCON) by scalding with phosphate-buffered saline hea ted to 70 degrees C for 6 seconds, and they were then cooled with room temp erature phosphate-buffered saline for 15 seconds. Human alloimmunocytes wer e added to CON to create rejection cultures (RET) and to BCON to create bur ned rejection culttures (BREJ). Slides were stained with hematoxylin and eo sin and anti-Lewis antibody. fn situ labeling of apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNE L). Those sections that were immunostained for Lewis Y were analyzed for in tensity stain index (ISI = [Sigma P-0 x I-0]/total tissue area in pixels). TUNEL was quantified with the following equation: no. of total apoptotic ce lls/total tissue area. Necrosis and blister formation in the epidermal laye r were evident in BCON and BREJ. Pyknosis and nuclear fragmentation-indicat ors of apoptosis-were also present. Phagocytosis of keratinocytes by leukoc ytes 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 w as found between BCON and BREJ (55.0 +/- 5.7 vs 60.5 +/- 3.4, respectively) and RET and BREJ (58.3 +/- 2.3 vs 60.5 +/- 3.4, respectively). TUNEL stain ing indicated the presence of apoptosis as follows: RET 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 +/- 0.006, respectively, P = .007). The presence of leukocytes and thermal injury induces apoptosis in BSC. The com bination of these two variables results in increased apoptosis as determine d by TUNEL. These findings suggest that a common pathway for skin injury ma y include inappropriate regulation of apoptosis exacerbated by a mechanism that includes inflammatory cellular infiltration.