β-chemokine expression and distribution in paraffin-embedded transplant renal biopsy sections:: analysis by scanning laser confocal microscopy

Previous immunohistochemical and in situ hybridisation studies have shown that, in tubulitis associated with acute cellular rejection of human renal allografts, intratubular T cells proliferate and are fully activated in situ. In the immunohistochemical study reported here we have attempted to establish some understanding of the involvement of the beta-chemokines RANTES, MCP-1, MIP-1 alpha and MIP-1 beta in recruiting T cells to the intratubular site. Paraffin-embedded routine biopsy sections were treated for conventional indirect immunofluorescence to detect the selected chemokines. Scanning laser confocal microscopy was used to provide a measure of fluorescence intensity resulting from binding of FITC-labelled secondary antibody. Cells expressing chemokines could be identified and, within the limits of the staining method, it was possible to obtain a semi-quantitative assessment of individual chemokine activity at different points in biopsy sections by constructing a profile of fluorescence intensity. High concentrations of chemokines (especially RANTES, MIP-1 beta and/or MIP-1 alpha) were localised to the basolateral surface of tubular epithelial cells (TEC). MCP-1 was also consistently present but at a lower level than RANTES except in one case identified as BANFF category 3. There was diffuse distribution of chemokines in the interstitial matrix and low intensity fluorescence outlined some endothelial cells of peritubular venules and interstitial fibroblast-like cells. Our results suggest a mechanism for specific chemotactic recruitment of inflammatory cells by TEC-produced chemokines.