INDUCTION OF RANTES, HLA-DR, AND INTERCELLULAR ADHESION MOLECULE-1 ON HIGHLY PURIFIED DISTAL TUBULAR CELLS FROM HUMAN KIDNEY 1 PATRICK C. BAER, 2,3 JU ¨ RGEN E. SCHERBERICH, 4 JU ¨ RGEN BEREITER-HAHN, 5 AND HELMUT GEIGER 2 Division of Nephrology, Department of Medicine IV, and Cinematic Cell Research Group, Department of Zoology, J. W. Goethe-University, 60590 Frankfurt am Main, and Department of Medicine II, Hospital Mu ¨ nchen Harlaching, 81545 Mu ¨ nchen, Germany Background. Expression of proinflammatory mole- cules by tubular epithelial cells plays an important role in renal allograft rejection and inflammatory kid- ney diseases. Different studies from patients with acute rejection point to the involvement of distal tu- bular segments. At present no in vitro system for the human distal tubule is established. Methods. Human distal tubular cells were isolated immunomagnetically. Cultured cells were stimulated with cytokines (interferon-, tumor necrosis factor-, interleukin-1, or a cytokine mix). Secretion of RAN- TES (regulated upon activation, normal T-cell ex- pressed and secreted) was evaluated with an enzyme- linked immunoassay. Expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 was as- sessed by flow cytometric analysis and immunofluo- rescence studies. Results. Our data clearly indicate that distal tubular cells express RANTES, HLA-DR, and ICAM-1 in re- sponse to a mixture of specific cytokines. Dexametha- sone inhibited the induced expression of RANTES and HLA-DR significantly, but not that of ICAM-1. Conclusions. We demonstrate an appropriate in vitro system for the human distal tubule. The present study proves the involvement of the distal tubular segment during inflammatory kidney diseases. Although the physiological role of tubular epithelial cells has been extensively evaluated, many processes of the tubu- lar segments under pathophysiological conditions are poorly understood. During renal allograft rejection, tubulointersti- tial changes are a prominent feature of the rejection process (1). Expression of chemokines and adhesion molecules on the surface of renal epithelial cells play a pivotal role in chemoat- traction and infiltration by lymphocytes, monocytes, and granulocytes in renal allograft rejection, inflammatory kid- ney diseases, and autoimmune disorders. The chemokine regulated upon activation, normal T-cell expressed and se- creted (RANTES) is involved in specific chemotactic recruit- ment of inflammatory cells like eosinophils, basophiles, lym- phocytes, and monocytes and has been identified recently in the human renal epithelium (2). Recognition of foreign anti- gens by T lymphocytes during immune responses requires the presence of human leukocyte antigens (HLA) on antigen- presenting cells (3). HLA molecules are the major targets of cell-mediated and humoral allograft responses, and up-regu- lation of HLA-DR expression is regarded as a useful marker of rejection. On tubular epithelial cells of rejecting renal allografts, HLA molecules are expressed de novo during re- jection. The immunoglobulin-like intercellular adhesion mol- ecule (ICAM)-1 increases the avidity of T lymphocyte inter- actions with antigen-presenting cells bearing HLA antigens (3). Proinflammatory cytokines like interferon (IFN)-, inter- leukin (IL)-1, or tumor necrosis factor (TNF)-, released by activated mononuclear cells at the site of inflammatory pro- cesses, were shown to induce the expression of different cell surface antigens and soluble factors in their target cells, thus enabling these cells to amplify the inflammatory process. Limited information is available with respect to the in- volvement of the distal tubule segment of the human nephron during inflammatory diseases and allograft rejec- tion (4). In rejected renal allografts, the more frequently infiltrated distal versus proximal tubules were infiltrated by leukocytes (5). To define the role of distal tubular epithelial cells in renal injury, we have established an immunomag- netic method to isolate highly purified distal tubular cells (DTC) from renal tissue after nephrectomies (6). These puri- fied cells were used to establish an in vitro model for “inflam- matory-like responses” of kidney targets. Cultivated DTC release the chemokine RANTES and express human leuko- cyte antigens and adhesion molecules. The data further sug- gest that leukocytes were transferred and accumulated within the renal interstitium through chemokines released by activated DTC. Human DTC were separated using antibody-coated mag- netic beads (Miltenyi, Bergisch-Gladbach, Germany) as de- scribed and characterized previously (6). DTCs were isolated through a monoclonal antibody (mAb) recognizing Tamm Horsfall glycoprotein, an antigen specific for the thick as- cending limb of Henle’s loop and the early distal convoluted tubule. After immunomagnetic isolation, cells were grown in medium 199 (Gibco, Eggenstein, Germany) with 10% fetal calf serum at 37°C and 5% CO 2 in a humidified atmosphere. As a control we used human proximal tubular cells (PTC) (6). The proinflammatory cytokines IFN- (2, 20, or 200 U/ml), IL-1 (25 U/ml), or TNF- (10 ng/ml) (all from Strathman, Hamburg, Germany) were used. Cells were grown in 24-well culture dishes until confluence. For stimulation of RANTES expression, cells were stimulated with one of the cytokines or with a mix of all three cytokines (cytomix: IFN-, 200 U/ml; IL-1, 25 U/ml; and TNF-, 10 ng/ml) for 48 hr in medium 199 without fetal calf serum. For stimulation of HLA-DR and ICAM-1 expression, cells were stimulated for 96 hr. In se- lected experiments, cells were preincubated with dexameth- 1 Part of these data were previously presented in abstract form at the meeting of Deutsche Gesellschaft fu ¨ r Innere Medizin, April 1998. 2 Division of Nephrology, Department of Medicine IV, J. W. Goethe-University. 3 Address correspondence to: P.C. Baer, Division of Nephrology, Department of Internal Medicine IV, J.W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. E-mail: P.Baer@em.uni-frankfurt.de. 4 Department of Medicine II, Hospital Mu ¨ nchen Harlaching. 5 Cinematic Cell Research Group, Department of Zoology, J. W. Goethe-University. 2456