How to differentiate apoptotic from necroptotic cell death? Part II.

Model systems for necroptosis research

Since necroptosis is what happens when caspase-8 fails to activate, the conditional caspase-8 knockout mouse reveals which tissues are susceptible to this form of cell death during development. Primarily endothelial cells, hematopoietic progenitor cells and leukocytes are susceptible to developmental necroptosis in the absence of caspase-8. CD8⁺ T-cells, monocytes and neutrophils require caspase-8 activity to properly develop and expand and are also quite susceptible to TNFa-induced necroptosis. Ischaemia/reperfusion injury also triggers necroptotic cell death in liver and kidney cells but this may represent a different form of necroptosis, distinct from TNFa-induced necroptosis. Necroptosis can be triggered by several other stimuli, such as immune receptor activation, TLR3 ligation, and RIG-I signalling. There may be other stimuli that induce necroptosis, but suffice to say that various tissues and cells under various conditions are susceptible to this form of cell death. Necroptosis can happen any time, you never know where and you never know when it will strike…

Cellular models of necroptosis

The most commonly used model cell line for necroptosis are the murine fibroblast cells L929. These cells are extremely sensitive to TNFa-induced necroptosis in the presence of the broad-spectrum caspase inhibitor z-VAD-fmk. In fact, they’ll also undergo necroptosis with z-VAD-fmk alone because the L929 cells produce low amounts of TNFa (and other cytokines!) constitutively. L929 cells are useful for the screening of anti- or pro-necroptotic compounds but shouldn’t be considered ‘real’ cells. They really respond very oddly in a number of ways and can’t be trusted entirely, in my experience. They express very high levels of RIPK3, the downstream effector of RIPK1 and this is most likely what makes them so susceptible to necroptotic death.

Mouse embryonic fibroblasts (MEFs) are sometimes susceptible to necroptosis as well, but not always. Bear in mind that authors tend to publish their successful experiments, rather than their failures, so when you see a paper in which necroptosis was induced in MEFs, don’t assume that your MEFs will respond the same way. It will work for some MEFs, but not for others and it remains hard to predict how MEFs will respond.

When dealing with human cells, either primary or cell lines, bear in mind that humans express caspase-10 besides caspase-8. Caspase-10 is activated in the same pathways as 8 and caspase-10 expression appears to be sufficient to prevent necroptosis (since patients deficient in either caspase-8 or -10 are quite viable but often develop Acute Lymhoid Proliferation Syndrome; ALPS) even though caspase-10 can’t substitute entirely for caspase-8. Caspase-10 didn’t evolve in humans but is in fact much older, the rodent lineage simply lost the gene (Figure 1). Presumably, rodent caspase-8 has taken over the functions of both caspase-8 and -10 (see also my review on the subject).

Figure 1: Evolution of caspase-8. Bony fish and their ancestors express two caspase-8 variants: the direct precursor to caspase-8 and -10 ('caspase-810') as well as caspase-18. Caspase-810 splits into two distinct genes (caspase-8 and caspase-10) just before tetrapods, while mammals lost caspase-18. Rodents, finally, lost caspase-10 as well. From my review.

Most primary human leukocytes appear to be susceptible to necroptosis, although no reports of a clear comparison has been published. In the older literature, TNFa or Fas-induced cell death in the presence of z-VAD-fmk is mentioned several times (here, here, here and here, for example) and it seems safe to assume that in most of these cases the cells succumbed to necroptotic cell death.

Certain clones of Jurkat cells are susceptible to necroptosis, but not all of them. In my experience, the FADD-deficient clone 5C3 is highly susceptible to necroptotic death induced by TNFa. The ‘wild type’ jurkat cell line A3 is not and the caspase-8 deficient line I9.2 is only mildly susceptible by itself, but, surprisingly, becomes more susceptible upon addition of z-VAD-fmk, suggesting that caspase-8 is not essential to prevent necroptosis in these cells. The RIPK1-deficient jurkat cell line is not susceptible to necroptosis unless reconstituted with RIPK1 harbouring a cleavage site mutation, The parental clone doesn’t undergo necroptosis upon TNFa stimulation in the presence of z-VAD-fmk. However, the RIPK1 deficient cells are extremely susceptible to all forms of apoptosis, suggesting either an important role of RIPK1 in preventing apoptosis or that these cells lack another anti-apoptotic factor in addition to RIPK1. These cells were initially generated by selecting randomly mutated jurkat clones against the ability to activate NF-kB, although later research has shown that RIPK1 is dispensable for NF-kB activation downstream of TNFa signalling.

The monocytic cell line U937 is also extremely susceptible to TNFa/z-VAD-fmk-induced necroptosis. These cells, as mentioned before ,will also produce high amount of TNFa in a RIPK1-dependent manner when stressed with a variety of stimuli in the presence of z-VAD-fmk. Other monocytic cell lines I tried, THP1 and NB4, are not susceptible to necroptotic cell death. Interestingly, in contrast to jurkat cells, U937 cells also undergo necroptosis when stimulated with TRAIL in the presence of z-VAD-fmk (Figure 2), even though jurkat cells are susceptible to TRAIL-induced apoptosis when FADD is expressed. This suggests that TRAIL may signal differently in U937 cells than it does in jurkat cells. I don’t know why this is, but I’ll share my observation and if anyone has an explanation, I’d be happy to collaborate.

Figure 2: TRAIL induces necroptosis in U937 cells.Treating U937 cells with TRAIL induced necrosis (Sytox Red uptake) which could not be prevented by zVAD, unlike TRAIL-induced apoptosis in Jurkat (A3) cells (A). Necrostatin only prevented necrosis in TRAIL+zVAD treated U937s (B). On Western blot, RIP1 cleavage was barely affected by zVAD in U937 cells, whereas it could be prevented in Jurkat cells. PARP cleavage was affected equally in both cell types (C and D).

These are all the necroptosis models that I’m experienced with. I’m sure there are others but you should run some tests to determine whether or not your favourite cell line is susceptible to necroptotic cell death. In the next chapter, I’ll outline several methods for determining cell death that are suitable for necroptosis research.

What determines whether a given cell is susceptible to necroptosis?

The most important factor that determines a cell’s susceptibility to necroptosis is the expression level of RIPK3 as well as expression of the downstream effector MLKL (mixed lineage kinase domain-like). Those cells that are most susceptible to necroptosis, appear to be those that express the highest levels of RIPK3 (such as L929 cells) while RIPK1 levels are normally quite stable among different cells. In fact, ordinary cells such as HeLa cells can be made susceptible to necroptosis by over-expression of RIPK3.

A recent paper in Science indicates that, indeed, RIPK3 kinase activity is required for necroptotic signalling, as mice expressing a kinase death mutant of RIPK3 did not succumb to necroptosis in the absence of caspase-8.  However, in the presence of caspase-8, these mice succumbed to massive caspase-8-dependent apoptosis. Thus, the kinase activity of RIPK3 both induces necroptosis while RIPK3 can act as a scaffold to promote apoptosis in the absence of kinase activity.