Interleukin-10 (IL-10) augments allograft arterial disease: paradoxical effects of IL-10 in vivo

Abstract

Interleukin-10 (IL-10) is an anti-inflammatory helper T cell type 2 (Th2) cytokine that modulates Th1-type cytokine production. Graft arterial disease (GAD) is a vascular obliterative process mediated via the Th1 cytokine interferon-gamma (IFN-gamma); allografts in IFN-gamma-deficient animals do not develop GAD. We investigated the effect of IL-10 and anti-IL-10 on GAD in murine heart transplants and whether anti-IL-10 reestablishes GAD in IFN-gamma-deficient hosts. Major histocompatibility complex class II-mismatched hearts were transplanted for 8 weeks into wild-type or IFN-gamma-deficient mice. In one set of experiments, wild-type hosts received daily administration of phosphate-buffered saline (PBS) or increasing IL-10; in a subsequent set of experiments, wild-type hosts received weekly PBS, rat IgG, or anti-IL-10 monoclonal antibody; IFN-gamma-deficient recipients received weekly PBS or anti-IL-10 monoclonal antibody. Explanted allografts were assessed for parenchymal rejection and GAD, cytokine profiles, and adhesion/costimulatory-molecule expression. Exogenous IL-10 resulted in increased Th2-like cytokine production; nevertheless, it exacerbated parenchymal rejection and GAD and increased CD8(+) infiltration. Anti-IL-10 did not significantly affect the extent of rejection or GAD, cytokine profiles, or immunohistology of the allografts in wild-type hosts. Adhesion molecule (CD54 and CD106) expression was not diminished by IL-10 treatment, and costimulatory-molecule (CD80 and CD86) expression was augmented by administration of exogenous IL-10. Allografts in IFN-gamma-deficient recipients showed mild rejection and no GAD, regardless of anti-IL-10 treatment. IL-10 in vivo thus has markedly different effects than predicted from in vitro experience. Although allografts develop Th2-like cytokine profiles treatment with IL-10 causes exacerbated rejection and GAD.

Figure 1.

Parenchymal rejection (open bars) and GAD (closed bars) of MHC II-mismatched bm12 allografts 8 weeks after transplantation into B6 wild-type or IFN-γKO recipients. A: Bar graphs showing results from a series of experiments in which B6 wild-type recipients received daily subcutaneous 0.5-, 1.0-, and 2.5-μg rmIL-10 or PBS treatment, whereas IFN-γKO recipients received either weekly intraperitoneal anti-IL-10 mAb or PBS treatment. Parenchymal rejection and GAD scores at the highest dosage of IL-10 (2.5 μg/day) are significantly increased in grafts from IL-10-treated B6 recipients compared with the PBS-treated B6 recipients. Parenchymal rejection and GAD are significantly reduced in IFN-γKO recipients compared with B6 recipients with or without anti-IL-10 treatment. B: Results from anti-IL-10 treatment experiments in B6 wild-type recipients. No significant difference was observed between the anti-IL-10 treatment group and control rat IgG-treated group.

Figure 2.

Representative sections of bm12 allografts explanted from either B6 wild-type recipients or IFN-γKO recipients 8 weeks after transplantation. A–D: H&E staining; E–H: Elastic tissue staining. Note that bm12 allografts from B6 recipients show multifocal mononuclear infiltrates (A and B), whereas the graft from IFN-γKO recipients show reduced parenchymal rejection (C and D); coronary vessels of bm12 allografts from B6 recipients show well-developed GAD (E and F), and, in contrast, the vessels of grafts from IFN-γKO recipients show no sign of GAD (G and H). Scale bar = 50 μm.

Figure 3.

Representative flow cytometry analysis of extracted intragraft CD4⁺ and CD8⁺ lymphocytes 8 weeks after transplantation. A: Grafts from PBS-treated B6 recipients; B: Grafts from B6 recipient with 2.5 μg/day IL-10 treatment subcutaneously. IL-10 treatment resulted in a shift to greater relative numbers of CD8⁺ cells.

Figure 4.

A: Primary one-way MLRs with or without 0.1 μg/ml rmIL-10. Irradiated bm12 splenocytes were used as stimulators, and purified CD4⁺ lymphocytes were used as responders (5 × 10 of each). Culture supernatants from quadruplicate wells were collected on day 3. IFN-γ concentration in the culture supernatants was measured by enzyme-linked immunosorbent assay. IL-10 diminished IFN-γ production from 123 ± 19 U/ml to 25 ± 1 U/ml. B: One-way MLRs using whole splenocytes from transplanted B6 wild-type recipients as responders. Proliferation decreased with increasing IL-10 dose; in the 2.5-μg/day IL-I0-treated group, proliferation was significantly decreased compared with the PBS-treated group. *P < 0.05.

Figure 5.

Representative flow cytometry analysis of graft-infiltrating cells gated on CD4⁺ lymphocytes (A and B) or CD8⁺ lymphocytes (C and D) in transplanted hearts. Graft-infiltrating cells were recovered from the allografts and stained using anti-CD8-PE, anti-CD4-PerCP, and either biotinylated anti-IL-4, anti-IL-10, anti-IFN-γ, or isotype-matched control antibody and APC-conjugated streptavidin. The thresholds were adjusted to 5% for background staining in the isotype-matched control antibody staining. A: PBS-treated B6 recipients. 51% of the CD4⁺ lymphocytes were IFN-γ-positive above background, and no positive staining was detected for IL-4 or IL-10. B: B6 recipients receiving 1.0 μg/day of IL-10. CD4⁺ lymphocytes secreted Th1 cytokine IFN-γ as well as Th2 cytokines IL-4 and IL-10. C: PBS-treated B6 recipients. Of the CD8⁺ lymphocytes, 65% were IFN-γ-positive above background, and no positive staining was detected for IL-4 or IL-10. D: B6 recipients receiving 1.0 μg/day IL-10. CD8⁺ lymphocytes also secreted Th1 cytokine IFN-γ, as well as Th2 cytokines IL-4 and IL-10.