CYTOTOXICITY AND APOPTOSIS IN HUMAN RENAL-ALLOGRAFTS - IDENTIFICATION, DISTRIBUTION, AND QUANTITATION OF CELLS WITH A CYTOTOXIC GRANULE PROTEIN GMP-17 (TIA-1) AND CELLS WITH FRAGMENTED NUCLEAR-DNA

In the present study, we analyzed human renal allografts using immunoh istochemical techniques to determine the site, identity, and frequency of (a) cytotoxic and apoptotic cells, as identified by staining for G MP-17 (TIA-1), a component of cytotoxic granules; and (b) DNA fragment ation in situ, as detected by the TUNEL method. In acute cellular reje ction (n = 15), GMP-17(+) mononuclear cells accounted for 29% +/- 12% of the infiltrating cells in the interstitium (341 +/- 164/mm(2)) and were significantly more concentrated in tubulitis lesions, where they amounted to 65% +/- 14% of the mononuclear cells (96 +/- 61/mm(2)) (p < 0.01 versus interstitium). GMP-17(+) mononuclear cells were also fou nd in sites of endothelialitis. An estimated 80% of the GMP-17(+) lymp hocytes expressed CD8, and 10% to 20% expressed either CD4 or the macr ophage marker CD14. The latter finding led us to analyze normal periph eral blood monocytes by flow cytometry, all of which were found to con tain GMP-17. NK cells and neutrophils, which are known to express GMP- 17, were detected only rarely in allografts. Specimens with cyclospori ne A toxicity (n = 7) or acute tubular necrosis (n = 13) showed fewer GMP-17(+) cells in the interstitium (22 +/- 46/mm(2) and 62 +/- 50/mm( 2), respectively) and tubules (2 +/- 6/mm(2) and 10 +/- 10/mm(2), resp ectively) (all p < 0.01 versus rejection). These differences were due largely to less intense mononuclear cell infiltration. In cyclosporine A toxicity, however, the percentages of GMP-17(+) mononuclear cells w ithin tubules and the interstitium were significantly lower than in re jection (p = 0.02), whereas in acute tubular necrosis significantly to wer percentages were found in the tubules (p = 0.04) but not in the in terstitium. Native kidneys with end-stage diabetic nephropathy (n = 5) had very low proportions of GMP-17(+) cells in interstitial infiltrat es (7% +/- 6%) and in tubules (11% +/- 15%), although the infiltrates were focally intense (517 +/- 355/mm(2)). TUNEL+ cells were found in a cute cellular rejection, predominantly in areas with intense mononucle ar infiltrates and also within lesions of tubulitis and endothelialiti s. Although some TUNEL+ cells were intrinsic renal cells, most appeare d to be infiltrating mononuclear cells, and we were able to detect CD3 in some. In areas of intense cellular infiltration, the percentages o f TUNEL+ cells (range, 0.5% to 4.2%) were comparable to those seen in the rat thymus, indicating a high level of apoptosis. Overall, in the allograft samples, the numbers of GMP-17(+) cells and TUNEL+ cells wer e significantly correlated (r = 0.79; p < 0.01). These data provide ne w evidence that T cell (and possibly macrophage)-mediated cytotoxicity plays an important role in acute renal allograft rejection, particula rly in the case of tubular injury, and furthermore suggest that apopto sis may be a mechanism not only for graft cell destruction, but also f or elimination of activated T cells in the infiltrate.