The immunosuppressive effect of rapamycin is mediated by inhibition of interleukin-2-stimulated T cell proliferation. We report for the first time that rapamycin also inhibits growth factor-induced proliferation of cultured mouse proximal tubular (MPT; IC(50) ~1 ng/ml) cells and promotes apoptosis of these cells by impairing the survival effects of the same growth factors. On the basis of these in vitro data, we tested the hypothesis that rapamycin would impair recovery of renal function after ischemic acute renal failure induced in vivo by renal artery occlusion (RAO). Rats given daily injections of rapamycin or vehicle were subjected to RAO or sham surgery. Rapamycin had no effect on the glomerular filtration rate (GFR) of sham-operated animals. In rats subjected to RAO, GFR fell to comparable levels 1 day later in vehicle- and rapamycin-treated rats (0.25 +/- 0.08 and 0.12 +/- 0.05 ml. min(-1). 300 g(-1), respectively) (P = not significant). In vehicle-treated rats subjected to RAO, GFR increased to 0.61 +/- 0.08 ml. min(-1). 300 g(-1) on day 3 (P < 0.02 vs. day 1) and then rose further to 0.99 +/- 0.09 ml. min(-1). 300 g(-1) on day 4 (P < 0.02 vs. day 3). By contrast, GFR did not improve in rapamycin-treated rats subjected to RAO over the same time period. Rapamycin also increased apoptosis of tubular cells while markedly reducing their proliferative response after RAO. Furthermore, rapamycin inhibited activation of 70-kDa S6 protein kinase (p70(S6k)) in cultured MPT cells as well as in the renal tissue of rats subjected to RAO. We conclude that rapamycin severely impairs the recovery of renal function after ischemia-reperfusion injury. This effect appears to be due to the combined effects of increased tubular cell loss (via apoptosis) and profound inhibition of the regenerative response of tubular cells. These effects are likely mediated by inhibition of p70(S6k).
We have previously shown that lysophosphatidic acid (LPA), an abundant serum lipid that binds with high affinity to albumin, is a potent survival factor for mouse proximal tubular cells and peritoneal macrophages. We show here that BSA also has potent survival activity independent of bound lipids. Delipidated BSA (dBSA) protected cells from apoptosis induced by FCS withdrawal at concentrations as low as 1% of that in FCS. dBSA did not activate phosphatidylinositol 3-kinase, implying that its survival activity occurs via a mechanism distinct from that for most cytokines. On the basis of the following evidence, we propose that dBSA inhibits apoptosis by scavenging reactive oxygen species (ROS): 1) FCS withdrawal leads to ROS accumulation that is inhibitable by dBSA; 2) during protection from apoptosis, sulfhydryl and hydroxyl groups of dBSA are oxidized; and 3) chemical blockage of free sulfhydryl groups or preoxidation of dBSA with H(2)O(2) removes its survival activity. Moreover, dBSA confers almost complete protection from cell death in a well-established model of oxidative injury (xanthine/xanthine oxidase). These results implicate albumin as a major serum survival factor. Inhibition of apoptosis by albumin occurs through at least two distinct mechanisms: carriage of LPA and scavenging of ROS.
Recent evidence indicates that phagocytic clearance of apoptotic cells, initially thought to be a silent event, can modulate macrophage (Mφ) function. We show in this work that phagocytic uptake of apoptotic cells or bodies, in the absence of serum or soluble survival factors, inhibits apoptosis and maintains viability of primary cultures of murine peritoneal and bone marrow Mφ with a potency approaching that of serum-supplemented medium. Apoptotic uptake also profoundly inhibits the proliferation of bone marrow Mφ stimulated to proliferate by M-CSF. While inhibition of proliferation is an unusual property for survival factors, the combination of increased survival and decreased proliferation may aid the Mφ in its role as a scavenger during resolution of inflammation. The ability of apoptotic cells to promote survival and inhibit proliferation appears to be the result of simultaneous activation of Akt and inhibition of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and ERK2 (ERK1/2). While several activators of the innate immune system, or danger signals, also inhibit apoptosis and proliferation, danger signals and necrotic cells differ from apoptotic cells in that they activate, rather than inhibit, ERK1/2. These signaling differences may underlie the opposing tendencies of apoptotic cells and danger signals in promoting tolerance vs immunity.
Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with plasma membrane. Exosomes are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. They are known to carry MHC class I, various costimulatory molecules and some tetraspanins. Recent studies have shown the potential of using native exosomes as immunologic stimulants. Here, we demonstrate a novel means of using exosomes engineered to express a specific tumor antigen to generate an immune response against tumors. We expressed a target tumor antigen, human MUC1 (hMUC1), in 2 MHC typedistinct mouse cell lines, CT26 and TA3HA. Analysis of exosomes purified from these cells revealed that exosomes contained the target MUC1 antigen on their surfaces as well as other welldescribed exosomal proteins, including Hsc70 and MHC class I molecules. In addition, both autologous and allogenic exosomes were able to stimulate the activation of immune cells and suppress hMUC1-expressing tumor growth in a MUC1-specific and doserelated manner. Therefore, these data suggest that exosomes can be engineered from tumor cell lines to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell-free tumor vaccine. © 2004 Wiley-Liss, Inc. Key words: exosomes; membrane vesicles; hMUC1; Hsc70; cell-free tumor vaccine Exosomes were initially described as small membrane vesicles (40 -90 nm in diameter), which are released from reticulocytes in order to eliminate unnecessary proteins, such as transferrin receptor (TfR) or acetylcholine esterase, during the process of their final maturation into red blood cells. [1][2][3][4] These extracellular vesicles are also produced by various kinds of hematopoietic cells, including mast cells, platelets, T lymphocytes, B lymphocytes and dendritic cells (DCs), as well as by intestinal epithelial cells. [5][6][7][8][9][10][11][12][13] Exosomes are known to originate from the inward budding of limiting membrane of multivesicular bodies (MVBs), and these internal vesicles of MVBs are released into the extracellular space by fusion of MVBs with the plasma membrane. 14 Proteomic analysis of DC-or B-cell-derived exosomes revealed selective enrichment of a subset of cellular proteins, including antigen-presenting proteins such as MHC class I and II molecules, heat shock proteins (HSPs), targeting-related MFG-E8 and tetraspanins such as CD9, CD63, CD81 and CD82. [15][16][17][18] In addition, a recent study by Hegman et al. has demonstrated that exosomes secreted by human mesothelioma cells contain a discrete set of proteins associated with antigen presentation, signal transduction, migration and adhesion. 19 The function(s) of exosomes are not well defined but have been reflected by their protein composition and cellular origins. 14,20 Although exosomes are known to discard membrane proteins such as transferrin receptors in reticulocytes, several lines of studies have sug...
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