T lymphocyte adhesion mechanisms within inflamed human kidney: studies with a Stamper-Woodruff assay

Abstract

Renal inflammatory conditions are characterized by mononuclear cell recruitment to sites of inflammation. We have developed a modified Stamper-Woodruff assay system to analyze mechanisms of functional T cell adhesion to cryostat sections of renal biopsy material from patients with vasculitic glomerulonephritis (GN) and acute allograft rejection. Peripheral blood T cells adhered to intraglomerular, periglomerular, and tubulointerstitial regions of the cortex. Blocking monoclonal antibodies against tissue expressed ICAM-1, VCAM-1, and the CS-1 domain of fibronectin (CS-1Fn) differentially attenuated T cell adhesion. Glomerular adhesion in vasculitic GN and tubulointerstitial adhesion in acute rejection were particularly sensitive to both anti-ICAM-1 and anti-VCAM-1 antibodies, indicating a prominent role for ICAM-1 and VCAM-1 at glomerular sites in vasculitis and at tubulointerstitial sites in rejection. Furthermore, using KL/4 cells (LFA-1 expressing) and Jurkat cells (VLA-4 expressing), we demonstrated specific LFA-1/ICAM-1- and VLA-4/VCAM-1-mediated interactions within glomerular and tubulointerstitial compartments. Jurkat cells also adhered to VCAM-1-free sites, and binding was inhibitable by anti-CS-1Fn antibody, thereby demonstrating a role for VLA-4/fibronectin interactions especially at intraglomerular sites in acute rejection where VCAM-1 is notably absent. We therefore propose a prominent functional role for ICAM-1, VCAM-1, and CS-1 domain fibronectin in T cell recruitment to the inflamed kidney.

Figure 1.

Immunohistochemical localization of VCAM-1 in vasculitic GN (A) and acute rejection (B) in sections of renal biopsies. Magnification, ×50.

Figure 2.

Immunohistochemical localization of ICAM-1 in vasculitic GN (A) and acute rejection (B) in sections of renal biopsies. Magnification, ×50.

Figure 3.

Peripheral blood T cell adhesion to serial sections of renal biopsies in vasculitic GN in the presence of irrelevant control antibody (A) and blocking anti-human ICAM-1 monoclonal antibody; RR1 (B). Magnification, ×100. Adherent cells were visualized using HRP-conjugated anti-CD3 and DAB, and the section was counterstained with Mayer’s hematoxylin as described in Materials and Methods.

Figure 4.

Inhibition of peripheral blood T cell adhesion to glomerular (A) and tubulointerstitial (B) regions of cryostat sections of renal biopsy material from five patients with vasculitic GN using monoclonal antibodies against ICAM-1, VCAM-1, and CS-1Fn as described in Materials and Methods. Data are presented as mean number ± SEM of adherent cells per glomerulus or per field of tubulointerstitium. P values indicate statistical significance of inhibition.

Figure 5.

Peripheral blood T cell adhesion to serial sections of renal biopsies in vasculitic GN in the presence of irrelevant control antibody (A) blocking anti-human VCAM-1 monoclonal antibody, Ig11 (B), anti-CS-1Fn, 90.45 (C), and both antibodies (D). Tissue illustrated here was from a different patient from that in Figure 3 ▶ . Adherent cells were visualized using HRP-conjugated anti-CD3 and DAB, and the section was counterstained with Mayer’s hematoxylin as described in Materials and Methods. Magnification, ×100 (A to C) and ×50 (D).

Figure 6.

Inhibition of Jurkat cell adhesion to glomerular (A) and tubulointerstitial (B) regions of cryostat sections of renal biopsy material from eight patients with vasculitic GN using monoclonal antibodies against VLA-4, VCAM-1, and CS-1Fn as described in Materials and Methods. Data are presented as mean number ± SEM of adherent cells per glomerulus or per field of tubulointerstitium. P values indicate statistical significance of inhibition.

Figure 7.

Inhibition of peripheral blood T cell adhesion to glomerular (A) and tubulointerstitial (B) regions of cryostat sections of renal biopsy material from five patients with acute rejection using monoclonal antibodies against ICAM-1, VCAM-1, and CS-1Fn as described in Materials and Methods. Data are presented as mean number ± SEM of adherent cells per glomerulus or per field of tubulointerstitium. P values indicate statistical significance of inhibition.

Figure 8.

Inhibition of Jurkat cell adhesion to glomerular (A) and tubulointerstitial (B) regions of cryostat sections of renal biopsy material from seven patients with acute rejection using monoclonal antibodies against VLA-4, VCAM-1, and CS-1Fn as described in Materials and Methods. Data are presented as mean number ± SEM of adherent cells per glomerulus or per field of tubulointerstitium. P values indicate statistical significance of inhibition.

Figure 9.

Co-localization of KL/4 cell adhesion (thin arrows) and ICAM-1 expression (thick arrows) (A) and Jurkat cell adhesion (thin arrows) and VCAM-1 expression (thick arrows) (B), using a combined adhesion and immunohistochemical technique. Magnification, ×100 and focused in plane of the leukocyte.