Renal ischemia-reperfusion injury amplifies the humoral immune response

Abstract

Renal transplant recipients who experience delayed graft function have increased risks of rejection and long-term graft failure. Ischemic damage is the most common cause of delayed graft function, and although it is known that tissue inflammation accompanies renal ischemia, it is unknown whether renal ischemia affects the production of antibodies by B lymphocytes, which may lead to chronic humoral rejection and allograft failure. Here, mice immunized with a foreign antigen 24-96 hours after renal ischemia-reperfusion injury developed increased levels of antigen-specific IgG1 compared with sham-treated controls. This amplified IgG1 response did not follow unilateral ischemia, and it did not occur in response to a T-independent antigen. To test whether innate immune activation in the kidney after ischemia affects the systemic immune response to antigen, we repeated the immunization experiment using mice deficient in factor B that lack a functional alternative pathway of complement. Renal ischemia-reperfusion injury did not cause amplification of the antigen-specific antibodies in these mice, suggesting that the increased immune response requires a functional alternative pathway of complement. Taken together, these data suggest that ischemic renal injury leads to a rise in antibody production, which may be harmful to renal allografts, possibly explaining a mechanism underlying the link between delayed graft function and long-term allograft failure.

Figure 1.

Murine IRI is characterized histologically. (A) SUN rises to a maximum at 24 hours and is back to presurgery value by 7 days. Shown are means ± SEM, with n=3 in the IRI-treated group. (B) Body weight changes after the surgery in both IRI- and sham-treated mice and return to presurgery values by 4 days; n=2 in the IRI-treated group. (C) Periodic acid-Schiff (PAS) staining of outer medullary tubules from an IRI-treated mouse at 48 hours reveals necrosis and sloughing of tubular epithelial cells. Original magnification: ×10; ×20 in inset. (D) PAS staining at 48 hours after sham surgery shows intact tubules. Original magnification: ×10; ×20 in inset. (E) PAS staining of IRI-treated mouse at 7 days with resolution of tubular necrosis. Original magnification: ×10; ×20 in inset. (F) PAS staining of sham-treated mouse at 7 days with normal tubules. Original magnification: ×10; ×20 in inset.

Figure 2.

Antigen-specific IgG₁ titers change over time. In all graphs, shown are means ± SEM. (A) Mice that underwent IRI versus sham surgery and then had ip immunization with 25 μg NP-keyhole limpet hemocyanin (KLH) 24 hours after surgery had sustained increases in NP-specific IgG₁ titers (P=0.04 by mixed model analysis beginning at day 35). Data shown are from ELISA; n=5 for IRI-treated group and n=4 for sham-treated group. (B) The effect of reperfusion time on the immune response was tested. Although the mouse is in the recovery phase from IRI at 4 days, the augmentation effect was greatest at that time. Data shown are from day 35 after immunization. Data shown are from ELISA; n=5 for day −7 IRI, n=6 for day −4 IRI, n=5 for day −1 IRI, and n=3 for sham-treated. By ANOVA, P=0.10.

Figure 3.

IRI causes an increase in the number of NP-specific splenocytes. In this experiment, mice were treated with IRI (n=4) versus sham surgery (n=4) on day 0, immunized on day 4, and euthanized on day 22. Unmanipulated animals (n=3) underwent no surgery and were not immunized. Splenocytes were harvested and subjected to FACS. Back-gating was used to exclude CD3^pos cells (which remove all T lymphocytes) and restrict analysis to B220^hi cells (B lymphocytes). IRI and immunization combined raise the number of both antigen-binding B lymphocytes in the spleen after immunization (P<0.05).

Figure 4.

Humoral immune amplification after IRI requires T lymphocyte processing and bilateral IRI. (A) Mice were immunized 4 days after IRI with NP-KLH and treated with daily injections of cyclosporine from day 4 to week 6 post-IRI. The augmentation effect of IRI on NP-specific IgG₁ levels was reduced by cyclosporine treatment, indicating an important role for T cell processing and signaling; n=4 for both groups. (B) Mice were immunized 4 days after IRI with 5 µg NP-ficoll injected ip. No amplification of the antigen-specific IgG₃ titer was seen in mice with IRI compared with sham-treated mice. IgG₃ is the IgG subclass raised by T-independent antigens. Data shown are from ELISA; n=6 for IRI-treated group and n=5 for sham-treated group. (C) Mice were subjected to unilateral renal ischemia and injected with NP-KLH after 24 hours of reperfusion. Mice with unilateral IRI did not develop amplification of the immune response to NP-KLH compared with sham-treated controls. These data would suggest that more severe injury or metabolic renal failure with elevated SUN is required for the observed effect. Data shown are from ELISA; n=3 for unilateral IRI-treated group and n=5 for sham-treated group.

Figure 5.

Mice deficient in the alternative pathway of complement do not exhibit amplified humoral immunity. (A) Mice that have a deletion of the gene for factor B (fB^−/− mice), which causes inactivation of the alternative pathway of complement, do not have any difference in the antigen-specific IgG₁ titers between IRI- and sham-treated groups. Data shown are from ELISA; n=5 for IRI-treated group and n=3 for sham-treated group. (B) Wild-type mice that were treated with a reversible inhibitor of factor B 2 hours before surgery had no significant amplification effect. Data shown are from ELISA; n=5 for IRI-treated group and n=4 for sham-treated group.

Figure 6.

Mice that cannot produce IL-10 do not display the observed effect after IRI. (A) Mice with a targeted genetic deletion of the gene for IL-10 were subjected to renal IRI or sham treatment, and they were immunized with NP-KLH after 24 hours of reperfusion. The IL-10^−/− mice subjected to IRI did not show increased titers of anti-NP IgG₁ compared with sham-treated animals. Data shown are from ELISA; n=3 for IRI-treated group and n=3 for sham-treated group. (B) qPCR data from IRI- versus sham-treated kidneys show that WT mice produce IL-10 within the kidney after renal IRI. The fB^−/− mice produced significantly lower amounts of IL-10 after renal IRI than wild-type animals. Data are reported relative to WT sham-treated kidneys. Primer t test P=0.10.