 polarization of macrophages into M2 cells in the inflamed glomerulus.
A variety of cytokines, growth factors, and PGs that pleiotropically affect tissue regeneration, cell proliferation, and immune modulation are produced by MSCs. In particular, PGE2 derived from BM-MSCs was recently described as playing a critical role in stimulating IL-10 production by macrophages.12 Therefore, we hypothesized that ASC-derived PGE2 may be a key modulator of macrophage conversion to an immunoregulatory phenotype in our anti-GBM GN experimental model. Despite comparable amounts of 15d-PGJ2, a metabolite of PGD2, in the supernatants of MSCs cultured with and without macrophages, LASCs cultured in the absence of macrophages constitutively secreted abundant amounts of PGE2 compared with BM-MSCs and HASCs (Figure 6A). Secretion of PGE2 by LASCs was even more prominent in cocultures with macrophages (Figure 6B). Moreover, pharmaceutical ablation of LASC-derived PGE2 synthesis by a cyclooxygenase-2 (COX-2) inhibitor or aspirin and blocking of the EP4 receptor (but not the PGD receptor) clearly impaired macrophage conversion to CD163+ cells in the coculture system (Figure 6, C–E). In vivo pharmaceutical ablation of LASC-derived PGE2 synthesis by a COX-2 inhibitor or aspirin also decreased the therapeutic potency of LASCs and the induction of glomerular macrophage polarization to CD163+ cells (Figure 7). Treatment with synthetic PGE2 alone resulted in a significant increase in the conversion of cultured macrophages into CD163+ cells (Figure 6F), but was less efficient than LASC treatment, suggesting that interaction between LASCs and macrophages as well as persistent stimulation or other humoral factors are also required.
LASC-derived PGE2 is an essential soluble factor that promotes polarization of macrophages toward immunoregulatory cells in vitro. (A and B) The concentrations of 15d-PGJ2, a metabolite of PGD, and PGE2 in culture supernatants of MSCs cultured alone (A) or with peritoneal macrophages at a 1:2 ratio (B) are measured. Dotted line indicates the value in medium (A) and in macrophage culture supernatant (B). LASCs constitutively secrete a higher amount of PGE2 than BM-MSCs or HASCs do, and this is enhanced in coculture with macrophages (n=4 in each group). (C and D) The Cox-2 inhibitor CAY10404 (C) and aspirin (D) impaired PGE2 excretion by LASCs cultured with macrophages in a dose-dependent manner. Reduction of LASC-mediated macrophage polarization by CAY10404 (C) and aspirin (D) is also evaluated by flow cytometry as macrophage CD163 expression. Representative histograms and percentage of CD163+ macrophages incubated with CAY10404 or aspirin at indicated concentrations are shown. Dotted line indicates value in cultures containing macrophages alone. (E) The decrease in the CD163+ population is evaluated as the percentage of CD163+ cells cultured with each EP or PGD receptor antagonist compared with CD163+ cells cultured with the respective vehicle control (black column). (F) Macrophage polarization into CD163+ cells after a single stimulation with PGE2. CD163 expression on macrophages incubated for 48 hours with synthetic PGE2 at the indicated concentrations is determined by flow cytometry (n=4–5 per group). All data are mean ± SD. **P<0.05 as determined by ANOVA.
Pharmaceutical ablation of PGE2 synthesis abrogated the therapeutic potency of LASCs. (A–C) LASCs pretreated with 50 μM CAY10404 or 1000 μM aspirin are transferred into anti-GBM GN rats. The 16-hour proteinuria level at day 5 (A), histologic score of glomerular crescent formation at day 7 (B), and glomerular accumulation (C) of CD68+ (left) and CD163+ (right) macrophages at day 7 are evaluated as described in the legend for Figure 2. Dotted lines indicate value in anti-GBM GN rats not subjected to LASC transfer (n=10). All data are mean ± SD. **P<0.05 compared with untreated LASC group as determined by ANOVA (n=7–10 per group).
As discussed above, LASC-derived PGE2 alone was not sufficient to cause conversion of macrophages to an immunoregulatory phenotype. Whereas reports indicate that IL-10, M-CSF, IL-4, and IL-13 promote differentiation of macrophages into M2 cells,31,36 these cytokines were largely absent in LASC supernatants in our study (data not shown). However, secretion of considerable amounts of IL-6 by LASCs was observed (Figure 8A). Interestingly, polarization of macrophages to CD163+ cells was significantly reversed by antibody neutralization of IL-6 in the culture supernatant, and was induced by IL-6 stimulation in vitro (Figure 8, B and C). These data suggest that besides PGE2, LASC-derived IL-6 may also mediate macrophage polarization.
LASC-derived IL-6 is another soluble factor that promotes polarization of macrophages into CD163+ cells in vitro. (A) IL-6 concentration in the culture supernatants of BM-MSCs, HASCs, and LASCs incubated for 24 hours. (B) Neutralization of IL-6 using an anti-IL-6 antibody results in a dose-dependent decrease in the population of LASC-induced CD163+ macrophages compared with cultures treated with normal goat IgG (black column). (C) Recombinant IL-6 alone promotes the phenotypic switch of macrophages into CD163+ cells (n=5 per group). All data are mean ± SD. **P<0.05 as determined by ANOVA.
Crescent formation is a hallmark of active renal disease and determines the outcome in patients with human GN, including anti-GBM GN, ANCA-associated GN, lupus nephritis, and IgA nephropathy. Although BM-MSC administration has been shown to ameliorate acute tubular injury induced by cisplatin or ischemia reperfusion,37,38 and subcapsular injection of ASCs in rat kidneys has been shown to reverse folic acid–induced acute tubular damage,32 the capacity of MSCs to ameliorate glomerular damage has not been demonstrated until this report. Here, we clearly demonstrate the therapeutic superiority of ASCs to BM-MSCs in ameliorating renal damage in a rat crescentic GN model that recapitulates aspects of human anti-GBM GN. In addition, we demonstrate that LASCs in particular attenuate neutrophil, CD8+ T cell, and CD68+ macrophage recruitment during the initial phases of the disease. We also show that LASCs promote the phenotypic switching of glomerular macrophages to immunoregulatory cells during later phases of anti-GBM GN, and that this switching is dependent on LASC-derived PGE2 and IL-6 (Figure 9). Together, these activities of LASCs reduce formation of the glomerular crescents that lead to progressive renal dysfunction and proteinuria.
Model for LASC-mediated amelioration of anti-GBM GN. LASCs impair recruitment of neutrophils and CD8+ T cells into the glomerulus during the initial phase of anti-GBM GN and promote PGE2-EP4 receptor-dependent phenotypic conversion of infiltrating macrophages to immunoregulatory macrophages during subsequent phases of the disease. Other soluble factors, including IL-6, are also required for this process. The accumulation of a significant number of immunoregulatory macrophages in the glomeruli protect against development of proteinuria and glomerular crescent formation, which is critical for a positive prognosis in anti-GBM GN.
We showed that intravenous administration of LASCs, HASCs, or BM-MSCs significantly reduces total macrophage infiltration in diseased glomeruli. Phenotypic conversion of macrophages to CD163+ cells in diseased glomeruli was demonstrated in the LASC-treated group, but was less prominent after HASC treatment and was minimal in BM-MSC–treated animals. It is well known that CD163+ macrophages represent anti-inflammatory M2 macrophages,30,31,39–41 and we and others have clearly demonstrated colocalization of CD163 and cytosolic IL-10 in glomerular macrophages in anti-GBM GN.28,29 Furthermore, blockage of the angiotensin II receptor or treatment with statins has been shown to attenuate anti-GBM GN, together with augmentation of CD163+ glomerular macrophages.28,29 In our in vitro study, LASC treatment effectively increased the number of CD163+ and CD206+ cells and the level of IL-10 secretion, but only a small population of rat peritoneal macrophages expressed both CD206+ and CD163+ in our coculture system. In addition to our in vitro evidence, we found a greater increase in IL-10 secretion in CD163+ cells than in CD206+ cells in diseased glomeruli after LASC transfer. Therefore, we speculate that LASC-mediated conversion of macrophages to IL-10–producing CD163+ cells ameliorates glomerular injury in rat anti-GBM GN. In mice and humans, CD163+ and CD206+ macrophages are classified as anti-inflammatory M2c cells producing IL-10 and profibrotic M2a-like cells, respectively.30 Interestingly, M2a- or M2c-macrophage transfer has been shown to dramatically attenuate renal injury after mouse adriamycin nephropathy,42–44 suggesting both M2a and M2c macrophages have renoprotective effects. Considering the above evidence, a precise characterization of the M2 cell subtype induced by LASC treatment and an evaluation of the efficacy of adoptive transfer of CD163+ macrophages is needed for a more complete understanding of the therapeutic significance of LASC-mediated conversion of macrophages into CD163+ cells in anti-GBM GN.
However, it remains unclear whether LASC-induced CD163+ macrophages are directly involved in protecting