Analysis of T cell lines from rejecting renal allografts.

Acute rejection after renal allotransplantation is characterized by the presence of a mononuclear cell infiltrate in the interstitium with involvement of tubuli. In a previous study on renal histology we showed that tubular damage by graft infiltrating cells (GIC) is a sign of clinically significant rejection. We cultured proximal tubular epithelial cells (PTEC) and T cells bearing the interleukin 2 (IL-2) receptor from biopsies after transplantation. In vitro outgrowth of T cells from the biopsy was significantly (P = 0.0014) related to histological signs of graft rejection. Of the T cell lines generated from 25 biopsies, only five lines showed no or low cytotoxicity against donor PTEC. Three cell lines were cytotoxic towards donor PTEC, but not against PHA stimulated donor splenocytes, suggesting tissue specificity of GIC. Treatment of PTEC with interferon (IFN) gamma for 72 hours to upregulate MHC class I and to induce MHC class II expression did not necessarily result in an increased susceptibility to lysis. However, three PTEC lines displayed an increment of susceptibility to lysis after IFN gamma treatment. Analysis of one T cell line from the same graft revealed a high percentage of CD4 positive cells, compatible with a class II restricted cytotoxicity. This was confirmed by blocking experiments using anti-CD4, anti-CD8, anti-class I, and anti-class II antibodies. Blocking experiments were done with 12 of these 25 lines. Anti-CD3 and anti-CD18 antibodies inhibited cytotoxicity in every case, showing that cytotoxicity was T cell mediated.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-1 alpha stimulated TNF alpha production by cultured human proximal tubular epithelial cells.

Tumor necrosis factor alpha (TNF alpha) production by proximal tubuli was studied by immunoperoxidase staining of 20 renal biopsies from transplant patients. A positive staining for TNF alpha on proximal tubuli was seen in nine out of 15 patients with interstitial infiltrate, five without clinical significant rejection and four with moderate to severe interstitial rejection. TNF alpha was only expressed on tubuli within areas of interstitial infiltrate. Expression of TNF alpha in the mononuclear cell infiltrate was seen only in three patients with interstitial rejection. Absence of TNF alpha could be seen in biopsies with no renal abnormalities. To obtain more information on the regulation of TNF alpha production, proximal tubular epithelial cells (PTEC) cultures were established and assessed for production of TNF alpha. Heterogenicity in production of TNF alpha was found in 14 tested PTEC lines cultured under serum free conditions. The presence of IL-1 alpha in the cultures induced a time- and dose-dependent enhancement of TNF alpha production by PTEC. Enhanced production of TNF alpha was not seen after stimulation with other cytokines such as IL-2 or IFN gamma. Inhibition studies with cycloheximide indicated de novo synthesis of TNF alpha. Western blot analysis of supernatants of unstimulated and IL-1 alpha stimulated PTEC indicated a 17 kd product, a size similar to that of recombinant TNF alpha. Northern blot analysis revealed the presence of a 2.0 kb hybridization signal in total RNA of PTEC cultures and up regulation upon treatment of PTEC with 1 ng/ml of IL-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)