Yiman Meng

Priming of Glomerular Mesangial Cells by Activated Macrophages Causes Blunted Responses to Proinflammatory Stimuli

Macrophage-mesangial cell interaction plays a crucial role in the pathogenesis of glomerulonephritis. Activated macrophages trigger mesangial cells to express an array of inflammation-associated genes via activation of NF-κB and AP-1. However, this inflammatory response is often transient and subsides spontaneously. We found that mesangial cells activated by bystander macrophages showed blunted responses of NF-κB to subsequent macrophage exposure. It was associated with sustained levels of IκBβ, but not IκBα. The tolerance observed was reversible and reproduced by conditioned media from activated macrophages (macrophage-conditioned medium (MφCM)). In vivo priming of mesangial cells by activated glomerular macrophages also caused the tolerance of mesangial cells. The macrophage-derived tolerance inducers were heat-labile, and multiple molecules were involved. Among inflammatory cytokines produced by macrophages, TNF-α and IL-1β were able to induce mesangial cell tolerance dose-dependently. The mesangial cell tolerance was also observed in activation of the MAPK-AP-1 pathway; i.e., phosphorylation of ERK, JNK, and p38 MAPK by macrophages was blunted when the cells were pre-exposed to MφCM. Induction of c-fos and c-jun was also abrogated in mesangial cells pre-exposed to MφCM, and the suppression was attenuated by blockade of MAPK activation during the first exposure to MφCM. These data elucidated that mesangial cells, once exposed to macrophages, become insensitive to subsequent activation by macrophages and proinflammatory stimuli. This self defense of glomerular cells may play a role in the resolution of macrophage-mediated, acute glomerulonephritis.

Continuous, noninvasive monitoring of local microscopic inflammation using a genetically engineered cell-based biosensor

Using an inflammation-responsive regulatory element as a molecular sensor, we established a cell-based biosensor for continuous, noninvasive monitoring of local microscopic inflammation in vivo. Glomerular mesangial cells were stably transfected with a marker gene encoding secreted alkaline phosphatase (SEAP) under the control of the κB enhancer elements. The established cells secreted SEAP in vitro in response to proinflammatory cytokines as well as to soluble factors produced by inflamed glomeruli. To examine feasibility of using the established cells for in vivo monitoring of local microscopic inflammation, the sensor cells were transferred selectively into rat glomeruli via the renal circulation. After induction of acute glomerulonephritis, the serum level of SEAP was increased transiently in cell-transferred nephritic rats. The kinetics of serum SEAP was closely correlated with the natural course of the inflammation, and the increase in SEAP was attenuated by suppression of inflammation using an immunosuppressive drug, cyclophosphamide. Neither cell-transferred normal rats nor nephritic rats without cell transfer exhibited increase in the serum level of SEAP. When the sensor cells were transferred extrarenally, elevation of serum SEAP was not observed in nephritic rats, confirming that the locally settled sensor cells responded only to local inflammation. These results suggested that, without invasive procedures like tissue biopsies, continuous monitoring of microscopic inflammation is feasible in vivo via locally created, cell-based biosensors.

Profiling of functional phosphodiesterase in mesangial cells using a CRE‐SEAP‐based reporting system

1 Phosphodiesterases (PDEs) are critically implicated in the regulation of mesangial cell function, but profile of functional PDEs in mesangial cells is still unclear. In this study, we investigated roles of individual PDEs in the regulation of mesangial cell behavior by the cAMP pathway. 2 Reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of the cAMP response element (CRE) were exposed to selective PDE inhibitors in the presence or absence of cAMP, and activity of CRE, expression of CRE‐regulated protein, mitogenesis and cell survival were examined. 3 Exposure of reporter cells to cAMP‐elevating agents resulted in time‐ and concentration‐dependent activation of CRE. Treatment of the cells with any PDE inhibitors alone did not induce CRE activation. Under stimulation with 8‐bromo‐cAMP or 8‐bromo‐cGMP, however, inhibitors of PDE2, PDE3, PDE4 and PDE5 enhanced activation of CRE. Inhibition of PDE1 or PDE6 did not affect the CRE activation. 4 Among different combinations tested, only inhibitors of PDE3 and PDE4 cooperatively increased the level of intracellular cAMP, activity of protein kinase A, activation of CRE, and CRE‐regulated protein, connexin43. 5 Concomitant inhibition of PDE3 and PDE4 attenuated mitogen‐induced activation of extracellular signal‐regulated kinases and cell proliferation. Under serum deprivation, combinational inhibition of PDE3 and PDE4 exclusively caused activation of caspase‐3 and apoptosis. 6 The present data elucidated that PDE3 and PDE4 play critical roles in the regulation of mesangial cell function. PDE3 and PDE4 were identified as the novel, antiapoptotic machinery that supports survival of mesangial cells.

Enhancement of NF-κB activity by resveratrol in cytokine-exposed mesangial cells

Resveratrol, a natural polyphenolic phytoalexin, has been considered as a potential anti‐inflammatory agent because of its suppressive effect on nuclear factor‐κB (NF‐κB). However, we recently found that treatment of glomerular mesangial cells with resveratrol significantly and dose‐dependently enhanced NF‐κB activation triggered by proinflammatory cytokines. This finding was evidenced by different reporter assays as well as by expression of an endogenous NF‐κB‐dependent gene, intercellular adhesion molecule‐1. The NF‐κB promoting effect of resveratrol was also observed in renal tubular LLCPK1 cells, but not in HepG2 hepatoma cells. In all cell types tested, treatment with resveratrol alone did not affect NF‐κB activity. The enhanced activation of NF‐κB by resveratrol progressed for at least 24 h and was accompanied by sustained down‐regulation of an endogenous NF‐κB inhibitor, IκBβ, but not IκBα. Although expression of inducible nitric oxide synthase was suppressed by resveratrol, nitric oxide, a negative regulator of NF‐κB, was not involved in the regulation of NF‐κB by resveratrol. These data elucidated, for the first time, that resveratrol may enhance activation of NF‐κB under certain circumstances.