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. These results show that TNF-alpha is produced by infiltrated mononuclear cells (MNC) and PTEC within areas of interstitial infiltrate, and thus may modulate the immune response during interstitial rejection.