Cellular autoreactivity against heat shock protein 60 in renal transplant patients: peripheral and graft-infiltrating responses

Abstract

Autoreactivity to heat shock protein 60 (Hsp60) has been implicated in the pathogenesis and regulation of chronic inflammation, especially in autoimmune diseases. In transplantation, there is a lack of information regarding the cytokine profile and specificity of cells that recognize self-Hsp60 as well as the kinetics of autoreactivity following transplantation. We studied the cellular reactivity of peripheral and graft-infiltrating lymphocytes against Hsp60 in renal transplant patients. Cytokine production induced by this protein in peripheral blood mononuclear cells indicated a predominance of interleukin (IL)-10 during the late post-transplantation period, mainly in response to intermediate and C-terminal peptides. Patients with chronic rejection presented reactivity to Hsp60 with a higher IL-10/interferon (IFN)-gamma ratio compared to long-term clinically stable patients. Graft-infiltrating T cell lines, cocultured with antigen-presenting cells, preferentially produced IL-10 after Hsp60 stimulation. These results suggest that, besides its proinflammatory activity, autoreactivity to Hsp60 in transplantation may also have a regulatory role.

Fig. 1

Cytokine production induced by heat shock protein (Hsp)60 peptides in peripheral blood mononuclear cells (PBMC) of renal transplant patients in the early (< 6 months) and late (> 1 year) periods post-transplantation. PBMC were cultured in the presence of Hsp60 peptides derived from the N-terminal, intermediate and C-terminal region of the molecule. The number indicates the position of the first amino acid residue in the peptide sequence of the Hsp60 molecule. After 48 h, the supernatant was collected and the cytokines interferon (IFN)-γ and interleukin (IL)-10 were measured by enzyme-linked immunosorbent assay (ELISA). Levels of cytokines induced by Hsp60 peptides are represented as □ (no production); (1–9 pg/ml); ▪ (10–99 pg/ml); ▪ (100–500 pg/ml).

Fig. 2

Cytokine production induced by heat shock protein (Hsp)60 in peripheral blood mononuclear cells (PBMC) of renal transplant patients with different clinical outcomes: clinically stable long-term patients (group 2, LONG: ○, mean 29 years after transplantation, n = 15) and recently diagnosed chronic allograft nephropathy patients (group 3, CAN: ▪, n = 12). PBMC were cultured in the presence of recombinant proteins Hsp60 and its intermediate (I-Hsp60; residues 195–391). After 48 h, the supernatants were collected and the cytokines interleukin (IL)-10 (a), interferon (IFN)-γ (b), transforming growth factor (TGF)-β (c) and IL-4 were measured by enzyme-linked immunosorbent assay. IL-4 was not detected in these two groups of patients. P-values calculated by Mann–Whitney U-test, two-tailed.