Tumor Necrosis Factor Abstracts

Oligodendrocytes (OLs) and their Myelin membranes are the primary targets in the AutoImmune Disease Multiple Sclerosis (MS).

The inflammatory Cytokine Tumor Necrosis Factor-alpha (TNF-) has been implicated as a mediator of OL cell injury.

TNF- is detectable within MS lesions and induces Apoptosis of mature human OLs in vitro.

One possible mechanism by which TNF- mediates cell death is through the activation of c-jun N-terminal Kinase (JNK).

We have previously shown that treatment of human OLs with TNF- leads to activation of JNK.

Here we provide evidence that p53, a regulator of the cell cycle and Apoptosis, is a mediator of TNF--induced Apoptosis of OLs.

Although p53 was undetectable by western blot analysis in adult human OLs, its levels increased within 24 h after TNF- treatment (100 ng/ml).

The induced p53 was Immunolocalized to the Nucleus prior to the appearance of significant numbers of Apoptotic Cells.

Overexpression of p53 by AdenoVirus-mediated Gene transfer into human OLs in vitro resulted in marked Apoptosis as revealed by in situ cleavage of DNA (TUNEL positive), decreased Mitochondrial function, and release of Lactate Dehydrogenase into the culture medium.

These in vitro studies demonstrate that increased p53 levels are associated with Apoptosis of human OLs.

The findings further implicate p53 as a target for the JNK pathway activated during TNF--mediated cell death of human adult OLs.

Oligodendrocytes (OLs) are the primary targets in the AutoImmune Disease Multiple Sclerosis (MS).

Cell Receptors belonging to the Tumor Necrosis Factor Receptor (TNF-R) superfamily, such as TNF receptors and fas, are implicated in signaling the injury response of OLs.

The p75 NeuroTrophin Receptor (p75(NTR)), another member of the TNF-R superfamily, has been reported to mediate Nerve Growth Factor (NGF)-induced Apoptosis in some Neural Systems.

To address the potential relationship between p75(NTR) signaling and OL injury, we assayed adult human OLs cultured under several different conditions for p75(NTR) and Tyrosine Kinase receptor trkA expression, for NGF-mediated Apoptosis.

And for NGF-mediated Jun Kinase (JNK) or Nuclear Factor (NF) kappaB activation.

In the current study, we have found expression of p75(NTR) on cultured adult CNS-derived human OLs but not on other Glial Cells. TrkA was not detected on these OLs in any of the culture conditions tested.

Treatment of the OLs with varying concentrations of NGF over a range of time periods resulted in no significant increase in numbers of terminal transferase (TdT)-mediated d-uridine Tripphosphate (UTP)-Biotin nick-end labeling positive OLs.

Whereas, significant cell death was observed using TNF-.

Furthermore, unlike TNF- treatment, NGF treatment did not significantly activate JNK, although both TNF- and NGF induced Nuclear translocation of NF-kappaB.

These findings contrast with the recent report of NGF-mediated Apoptosis in the OLs of neonatal rats matured in vitro, which express p75(NTR) but not (trkA), and suggest that, at least in humans, p75(NTR) signaling may mediate responses other than Apoptosis of OLs.

Despite what is known about the early signaling events in Tumor Necrosis Factor- (TNF-)-induced Apoptosis, characterization of the downstream events remains largely undefined.

It is now known that a cross-talk exists between the Interferon and TNF- pathways.

This linkage allows recruitment of the cell proliferation suppressor PKR (dsRNA-dependent Protein Kinase) from the Interferon pathway to play a pivotal role in TNF--induced Apoptosis.

In this study, we took advantage of the differential TNF- susceptibilities of human ProMonocytic U937 SubClones, deficient in or overexpressing PKR, to further characterize the role of PKR in Apoptosis.

By reverse transcription-polymerase chain reaction, we demonstrated that TNF- transiently induces the tumor suppressor p53 in U937 cells.

This p53 induction lags behind the TNF- induction of PKR by 1 h.

By Cell Viability Determination, UltraStructural Studies, Apoptotic DNA Laddering, and Antisense Techniques, it was shown that inhibition of p53 expression in PKR-overexpressing U937 cells abrogates the TNF--induced Apoptosis in these cells.

Conversely, overexpressing wild type p53 in PKR-deficient U937 cells confers the susceptibility of these cells to TNF--induced Apoptosis.

This latter result indicates that p53 induction is an event downstream of TNF--induced up-regulation of PKR.

Thereby, further establishing the critical role of p53 in TNF--induced Apoptosis in U937 cells.

PKR-overexpressing U937 cells were found to possess a constitutively higher level of p53, which partly explains why these cells spontaneously undergo Apoptosis even without TNF- treatment.

Finally, a model is presented on the interplay between PKR and p53 in effecting TNF--induced Apoptosis in U937 cells.

Cell numbers are controlled by a homeostatic mechanism between cell growth, arrest and programmed cell death (Apoptosis) in normal and Cancerous tissues.

One of the tumor suppressor Genes, p53, functions as a Transcription Factor or transcriptional regulator through DNA and protein binding properties, and plays an important role in regulating cell cycle and induction of Apoptosis.

Although there are two Apoptotic Pathways, p53-independent and p53-dependent, the latter will be emphasized and discussed in this section.

Since p53 is often inactivated due to mutation in human Cancers, understanding the p53-dependent Apoptotic Pathway is extremely important.

Analysis of p53-dependent Apoptosis as well as Apoptosis caused by other p53-related Genes should provide a clue to a new strategy for Cancer therapy.

Oligodendrocytes (OLs) and their Myelin membranes are the apparent injury targets in the putative human AutoImmune Disease Multiple Sclerosis. The basis for this selective injury remains to be defined.

OLs in vitro have been shown to be susceptible to both Tumor Necrosis Factor (TNF) and non-TNF-dependent Immune Effector mechanisms.

The former involves initial Nuclear injury (Apoptosis); the latter, when mediated by activated T-Cells, involves initial cell membrane injury (Lysis).

In the current study, we determined whether human adult CNS-derived OLs could be protected from the above Immune Effector mechanisms (Inflammation).

By selected NeuroTrophic Factors (CNTF, BDNF, NGF, NT-3, and NT-4/5) or Cytokines demonstrated to protect from human or experimental AutoImmune DeMyelinating Diseases (Interferon-ß [IFN-ß], IL-10, and TGF-beta).

Nuclear injury was assessed in terms of DNA fragmentation using a DNA nick-end-labelling technique; cell membrane injury was assessed by Lactate Dehydrogenase or Chromium 51 release.

MTT and cell counting assays were used to assess cell viability and cell loss, respectively.

Among the NeuroTrophic Factors and Cytokines tested, only CNTF significantly protected the OLs from TNF-mediated injury.

CNTF also protected the OLs from Serum deprivation-induced Apoptosis. CNTF, however, did not protect the OLs from injury induced by activated CD4⁺ T-Cells.

CNTF also did not protect human fetal Cortical Neurons from Serum deprivation or TNF-induced DNA fragmentation.

Nor did it protect the U251 human Glioma Cell Line from DNA fragmentation induced by a combination of TNF and reduced Serum concentration in the culture media.

Our results indicate that potential protective effects of NeuroTrophic Factors or Cytokines on Neural cell populations can be selective both for cell type involved and mechanism of Immune-mediated injury.