Mesenchymal stromal cell neuroprotection of hydrogen peroxide -challenged pheochromocytoma cells through reducing apoptosis and releasing cytokines.

BACKGROUND AIMS: Immunoregulation of mesenchymal stromal cells (MSC) is more efficient at restoring biologic function of injured tissue than transdifferentiation during transplantation. However, the exact mechanisms and characteristics of MSC regarding immunomodulation are still unknown, especially in the damaged niche after hypoxic-ischemic insult. We investigated the anti-apoptotic actions of MSC against the neurotoxicity of hydrogen peroxide (H(2)O(2)) on pheochromocytoma (PC12) cells. METHODS: To mimic hypoxic-ischemic brain injury in vivo, a relatively high H(2)O(2) concentration and short period were used to treat PC12 cells. MSC were co-cultured directly with the injured PC12 cells, and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS), lactose dehydrogenase (LDH) and nitric oxide (NO) assays, intracellular Ca(2+) and resting membrane potential (RMP) were analyzed. Apoptotic-associated genes and cytokine releases were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: After exposure to H(2)O(2), the viability of PC12 cells was significantly decreased, while the levels of LDH and NO increased, resulting in intracellular Ca(2+) accumulation and cell apoptosis. Co-culture of MSC with the H(2)O(2)-treated PC12 cells sustained viability of the PC12 cells, reduced LDH levels and NO release, and improved Ca(2+) influx and cell apoptosis. The injured PC12 cells exhibited lower RMP following co-culture with MSC compared with the injured PC12 cells alone. The mRNA expression levels of Bcl-2 were up-regulated and caspase-3 levels down-regulated in the MSC co-culture groups. The release of interleukin (IL)-10 was gradually reduced by co-cultured MSC, while the release of IL-6 was sharply increased following MSC co-culture. CONCLUSIONS: These findings indicate that MSC have neuroprotective effects on suppressing cell apoptosis via regulation of the H(2)O(2)-impaired microenvironment, partly through IL-6 and IL-10 cytokine production.

Incorporation of monoclonal antibodies in living rat pheochromocytoma PC12 cells. Evidence for the intracellular formation of immune complex between the incorporated antibody and a target protein.

PC12 cells permeabilized with a low concentration of digitonin (5 microM) under controlled conditions were loaded with monoclonal antibodies (MoAb) against the regulatory subunit type II (RII) of cAMP-dependent protein kinase. After digitonin removal from the nutrient medium (DMEM) the loaded cells repaired within 20-30 min and recontinued growth. The inserted MoAb stayed in the repaired cells at least for several hours. MoAb inhibiting the cAMP binding activity of neural RII [Grozdova et al. (1992) Biochem. Int. 27, 811-822; Sveshnikova et al. (1996) Biochem. Int. 39, 1063-1070] were shown to bind the target antigen inside the cells and influence the properties of intracellular protein kinases.

Evaluation of intracerebral grafting of dopamine-secreting PC12 cells into allogeneic and xenogeneic brain.

The PC12 phenochromocytoma tumor cell line is derived from a rat adrenal medullary tumor and secretes dopamine. We have previously reported that grafted microencapsulated PC12 cells using agarose and poly (styrene sulfonic acid) survived in the xenogeneic brain without immunosuppression. To investigate whether unencapsulated PC12 cells form a tumor and how they provoke immunological reaction, PC12 cell suspension was implanted into the striatum of Sprague-Dawley rat (allogeneic graft) or guinea pig (xenogeneic graft) and histological analysis using Nissl stain and immunocytochemical analysis using antityrosine hydroxylase (TH) antibody were performed 1, 2, and 4 wk after transplantation. Host animals were not immunosuppressed. PC12 cells formed a mass 1 and 2 wk after transplantation both in allogeneic and xenogeneic brain. These grafted PC12 cells were immunoreactive to anti-TH antibody. Four weeks after transplantation, however, grafted PC12 cells in the allogeneic brain were only found within the restricted area near the site of implantation. In the xenogeneic brain, only the trace of grafted PC12 cells were found around the site of implantation 4 wk after transplantation. In both allogeneic and xenogeneic animals, a number of lymphocytes were found in and around the grafts at all period investigated. These findings indicate that PC12 cells could survive in the allogeneic or xenogeneic brain for 2 wk and were ultimately rejected by immunological reaction by 4 wk after transplantation. Implantation of encapsulated PC12 cells in the allogeneic or xenogeneic brain is considered a safe and effective method for delivering dopamine into the brain because PC12 cells will not form a tumor in the long-term even if capsules are damaged in some reason.

Polyreactive autoimmune response induced by PC 12 cell grafts into rat striatum.

In order to better characterize the autoantibodies induced by PC12 cells grafted into rat brain, we have tested sera from these animals by immunoblotting with several preparations, including phosphorylated and dephosphorylated neurofilaments, keratins, PC12 cells and proteins from various rat tissues, and by immunofluorescence of rat spinal cord neurons in culture. Sera from grafted rats reacted with several antigens present in all tissues tested and stained in cultured neurons not only NF but also cell bodies and membranous granular structures. These observations suggest either the polyreactivity of autoantibodies or the induction of a polyclonal B cell activation consecutive to the release of central nervous system antigens into the blood stream. These results are discussed with regard to the role of NF autoantibodies in neurodegenerative diseases.

Differential behaviour of PC12 cells grafted into rat hippocampus and striatum.

To investigate the mechanisms involved in graft survival, a rat cell line (PC12) that differentiates into sympathetic-like neurons by exposure to trophic factors has been grafted into rat striatum and hippocampus, two structures which differ in their amounts of trophic factors. Our results show that grafted PC12 cells behave differently depending on the area of implantation; they display a differentiated morphology in the hippocampus and proliferate as a tumor in the striatum. A qualitatively similar immunological reaction occurs in both structures, characterized by the invasion of T and B lymphocytes, macrophage-like cells and by the expression of major histocompatibility complex class I and II antigens around the graft.