On Apoptosis in Development

Apoptosis means doom for an individual cell. As such we tend to automatically assume that apoptosis is a Bad Thing, but in reality apoptosis often is quite necessary for normal physiological function at the organism level. In order for our bodies to maintain the homeostasis that defines so many of our cellular processes, we have to sacrifice some cells. As it turns out, we actually wind up sacrificing enormous numbers of cells every day. Worn out red blood cells, dangerously self-reactive lymphocytes, individual columnar epithelial cells and others. These processes are tightly regulated, so much so that most cell types actually require biochemical signals from neighboring cells, tissues, or even distant organs just to tell them to keep living. The anti-apoptotic survival signals can fall below a threshhold value and/or be overridden by pro-apoptotic stimuli, which normally results in swift induction of the apoptotic program. When individual cells develop mutations that deafen them to these signals, they become dangerous proliferation-happy pre-cancerous cells more interested in their own survival than that of their constituent organism.

Figure A: TUNEL histochemical staining in murine liver, brown cell is apoptotic.

The apoptotic program ultimately results in highly oxidative and degradative enzymes (such as proteases) hidden away in the mitochondria being released into the cytoplasm to wreak havoc. Usually the raw material of a dying cell is tidily absorbed by its neighbors to be recycled. I've always imagined mitochondria as pulsing with a low, gentle buzz in normal cellular physiology with occassional metallic pings as statistical flucuations in the net free energy of electrons falling down the electron transport chain is captured in ATP. Following this, I think the sound of caspase-8 et al slicing open the mitochondria would be like the initial panicked braking shriek of a train loaded with Furbies who are quickly drowned out in the self-amplifying roar like a tornado grinding through a gravel pit as the apoptotic effector enzymes set to work dissolving the cell from within.

Apoptosis is absolutely essential not just to adult homeostasis, but also to normal ontogeny. Without apoptosis organs would fail to separate, fingers would remain stuck together, and many other things would go very, very wrong. There are 2 families of intracellular proteins that battle to determine whether or not a cell will become apoptotic: the (generally) pro-apoptotic Bcl-2 family and the (generally) anti-apoptotic IAP family. Conveniently, IAP stands for Inhibitor of Apoptosis Protein. A recent review by Dr. O'Riordan et al discussed the diverse and essential roles for IAP proteins in normal tissue development across a wide range of model organisms. From ablated organ development in the absence of Diap1 in Drosophila larvae to stunted hematopoeitic developmental repertoire in the abscence of Survivin in mice, IAPs seem to be evolutionarily conserved signal transducers that integrate diverse extracellular signals into a coherent cellular action. Developmentally, the IAP proteins seem to be involved in everything from proper vascularization to chromosome stability, although it is important to note that direct modulation of apoptosis in developmental processes has only been established in invertebrates. Lack of any one of several IAPs in higher chordates has not been directly linked to developmental apoptosis, but several abnormal embryonic phenotypes and attenuated adult functional capacities have been demonstrated.

IAPs are grouped by the prescence of BIRs (baculovirus IAP repeats) and many also have RING domains. Both motifs have been found to have zinc-finger conformations and the interaction of different sections of adjacent BIR motifs in some proteins, such as direct inhibition of pro-apoptotic caspases-3 and -7 by BIR2 of XIAP (X-linked inhibitor of apoptosis protein), has been found to modulate a number of diverse effects. These diverse effects are potentiated by the ubiquitin ligase activity that some RING domains have demonstrated. IAPs help the organism balance necessary apoptosis and unnecessary apoptosis, and because apoptosis is required for the homeostasis of most tissues the IAP family has been evolutionarily conserved and biochemically diversified. IAPs remain an active and engaging area of research that holds great promise in the treatment of pathologies from cancer to intracellular bacterial infections and underscore how a little sacrifice for the team by one cell can make a massive impact on the constituent organism's overall fitness.

IAPs have also been found to modulate innate immunity, which will be discussed in another post.

ORIORDAN, M., BAULER, L., SCOTT, F., & DUCKETT, C. (2008). Inhibitor of Apoptosis Proteins in Eukaryotic Evolution and Development: A Model of Thematic Conservation Developmental Cell, 15 (4), 497-508 DOI: 10.1016/j.devcel.2008.09.012

Additional Source: Molecular Biology of the Cell; Alberts et al; 4th ed.; pages 1010-1014

Dendritic Cell Phagocytosis of Infected Apoptotic Cells Favors Development of a TH17 Phenotype

Background: TH17 discussed previously here; previous brief run-down of T-cell development here.

TH17 cells are an inflammatory T-cell subtype implicated in acute adaptive immune response as well as chronic autoimmune diseases. We have known for a while now how to create TH17 cells in a dish: just add TGFb and IL-6 (a general T-cell proliferation cytokine such as IL-2 wouldn't hurt either). However, we haven't been very clear on how exactly those kind of conditions would arise in vivo. TGFb is anti-inflammatory and helps slow the adaptive immune response down while IL-6 is pro-inflammatory and revs up the immune system. So how would these 2 molecules be made at the same time? Logically speaking, they wouldn't as it is tempting to think of the immune system as monolithic: capable of one state at a time, either ramping up inflammation or cooling it down. But because TH17 cells have been found in vivo infiltrating tissues and being made in the lymph nodes, and because we haven't yet found any other combination of cytokines that leads to the thorough development of TH17, TGFb and IL-6 must be co-expressed under some condition, somehow.
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Before we get there, I'm going to blather at ya'll about dendritic cells for a moment (although I wish I BlogSpot had the functionality to embed this in a text-wrapping box). Dendritic cells (DCs) are the interface between the innate and adaptive immune systems. DCs are phagocytic, meaning they can gobble up pretty much any cell that they see fit to do so to. When they gobble something up, they put it into a special vacuole and crank it through molecular blenders (called proteasomes) to generate short little peptides that they then port back out to their surfaces on major histocompatibility II proteins (MHCII) that T-cells can bind to and recognize. T-cells can't recognize peptides, called antigens here, without the context of MHCII. Correct peptide-MHCII-CD4-TCR signalling tells the T-cell to do adaptive immune stuff that helps the body fight off disease.

Figure A: A dendritic cell grown in vitro on collagen. Note the tentacley things protruding all over the place, those are the dendritic processes. Not all of the dendritic processes in this picture are from that one cell as other dendritic cell bodies are off screen. Picture somewhat altered for better contrast from original Wikimedia Commons jpeg.

DCs also possess Toll-like receptors that recognize PAMPs (pathogen-associated molecular patterns). PAMPs include dsRNA (TLR3), flagellin (TLR5), single-stranded RNA (TLR7), unmethylated CpG DNA (TLR9) and, most importantly here, LPS (TLR4) [LPS is a constitutive component of Gram-negative bacteria, which shed it. It is also known as endotoxin.]. When a TLR binds its ligand, the DC gets activated and migrates to the lymph node where the naive T-cells are hanging out. Once there it secretes various effector cytokines that have specific actions on the T-cells. These cytokines include IL-2, IL-4, IL-6, IL-12, TGFb, TNFa, and a whole other mess of alphanumeric soup noodles.
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Anyway, back to the conundrum of how inflammatory IL-6 and anti-inflammatory TGFb get co-expressed and make TH17 cells in vivo. It turns out that infected apoptotic cells can lead to the right co-expression. DC processing of apoptotic cells induces TGFb to throttle down inflammation in case the immune system has gotten too aggressive and is killing off the host organism (as occurs in septic shock). DC processing of non-host infectious agents usually induces IL-6. So when a DC processes an apoptotic cell that was infected with an intracellular microbe of some sort, both stimuli are right there and the DC trundles off to the lymph node making both TGFb and IL-6.

The group in the paper below used LPS-loaded apoptotic B-cells and E. coli-infected apoptotic neutrophils to test this out. They mixed the B-cells and neutrophils with DCs and let them stew for a while as the DC chewed thoughtfully and decided what to do. They then collected the culture medium off fo the DCs, which would contain any differentiation and/or signalling cytokines the DCs secreted in response to the apoptotic cell stimulus. Next, this medium was added to cultures of naive CD4+ T-cells and they sat back and watched what happened via flow cytometry, mRNA expression, and production of phenotypic marker cytokines (specifically, IL-10 and IL-17).

Using unloaded apoptotic B-cells or uninfected apoptotic neutrophils didn't do much by way of making TH17 cells as only TGFb was produced (made Tregs, though, see below). However, this was reversible through complementation with exogenous IL-6. Also, DCs exposed just to LPS such that TLR4 was strongly activated did induce lots of IL-6 production, but this didn't lead to much TH17 production unless IFNg was blocked (this consequently blocks the production of TH1 cells).

It turns out that apoptotic cells also induce a Treg response through TGFb. Tregs are the cells that calm the rest of the immune system down when it's gotten too excited. They are characterized by expression of Fox3p and secretion of IL-10. A sizeable portion of the naive T-cells also made IL-10 and flow cytometric analysis revealed that some had become double T-cells, secreting both IL-10 and IL-17. However, this was only during the initial stimulation of the naive T-cells. Later on, after these now-differentiated T-cells had calmed down, re-stimulation with IL-23 increased IL-17 production and downregulated IL-10 mRNA, indicating that this dual expansion may be a transient phenomenon.

The group replicated their findings in vivo. They used a Citrobacter rodentium model of hemorrhagic colitis in which many cells in the gastrointestinal epithelium go apoptotic and inhibited that apoptosis. Inhibited apoptosis led to lower infiltration of TH17 cells than untreated mice. There were also a bunch of good genetic and chemical controls, but I'm not going to discuss all of that here because 1) it's a LOT of detail and 2) I'm more interested in blathering about the implications of this finding.

Implication 1: Ulcerative colitis
The sick gut cells in ulcerative colitis aren't necessarily infected themselves. But due to the very high native colonization of the gastrointestinal tract by our friendly microbiota, there are always relatively high levels of LPS in the lumen that, presumably, interact with the epithelium. Therefore, at the ulcers, especially those mediated by Helicobacter spp., there is an increased likelihood that responding DCs will encounter both microbial PAMPs and apoptotic cells. This could explain, at least partly, why TH17 cells are heavily involved in the inflammation associated with gastritis.

Implication 2: Cancer
Given that pre-cancerous cells are generally not listening to the cells around them and have higher rates of mutation, it is entirely plausable that some cancer cells with alter the expression patterns of MHCI on their surface, be recognized by CD8+ T-cells, and summarily executed. However, were a DC to encounter this apoptotic body in the abscence of a TLR ligand, it would go to the lymph node promoting development of Tregs for the cancer cell antigens*. While this may be a good thing in preventing autoimmune reactions when T-cells learn the unmutated antigens still present in cancer cells are bad and should be killed, it also brings up the somewhat unsettling prospect that the this leads to immunotolerance of cancerous cells. The immune system is generally the first line of defense against the development of tumors, and if it has specifically learned to not be reactive against them due to Tregs, then the cancer would be allowed to continue growing and mutating without immune system interference. Needless to say, this is ultimately bad for the organism.

Implication 3: Rheumatoid Arthritis
It is known that inflammed tissues produce IL-23, which encourages stable differentiation of TH17 cells and also acts as a chemoattractant for them. I don't think it's unreasonable to think that levels of IL-23, along with NFkB et al, would be elevated in arthritic tissues and that this could lead to infiltration of TH17 cells. Once there, TH17 cells can cause further inflammation, including tissue damage. This in turn would amplify the production of IL-23, which has been shown to decrease IL-10 production and in turn decrease Tregs. This then leads to a dismal scenario in which more and more TH17 cells are being recruited to drive inflammation in arthritic tissue while at the same time stomping on the Tregs that could help break that feedback loop. However, at the same time DCs would be present and processing the apoptotic cells in the arthritic tissues, and then hopefully driving towards TGFb and Tregs.

It should be noted that each and every implication I have put up above could be complete and utter horsefeathers as the immune system is so complex and operates on so many scales that I could very easily be overlooking a key component that makes all of my conjectures seem silly.


Torchinsky, M., Garaude, J., Martin, A., & Blander, J. (2009). Innate immune recognition of infected apoptotic cells directs TH17 cell differentiation Nature, 458 (7234), 78-82 DOI: 10.1038/nature07781

*This happens anyway, MHCI or not. B7.1 and B7.2 (CD80 and CD86, respectively) expression patterns are often altered in chronic myelogenous leukemia and chorionic gonadotropic, which isn't expressed in adults, sometimes pops back up in tumors.

Apoptosis

Trouble was afoot in the sticky wastelands of the cytosol. The plasma membrane sky was turbulent overhead, sphingomyelin rafts swirling faster than normal as ear-splitting explosions rattled down the signal transduction cascades, amplifying as they arced off towards to distant mass hovering on the horizon. Something wasn't right there, either. The nucleus had been hurt badly in the last infection. It's latticed double envelope still bore the oxidative scars of the inflammatory reaction, the translocons feebly trying to properly refold with half of their functional domains blasted away. The mitochondria were rumbling, louder than normal, but perhaps they only seemed the louder because the endoplasmic reticulum and Golgi stacks were uncharacteristically quiet.

It was dark. Almost all the ATP had been hydrolyzed in the Inflammation, and now the mitochondria weren't producing any more--glucose and lipid transport had all but ceased. Even though they were still rumbling away...

This city was in trouble, and it knew it. The nation's dark Bim messengers were gathering quickly, clustering in ominous clouds with their Smacs while the wounded Bcl-ws could only drift aimlessly amid the sticky desert, observing.

Another explosion rattled down the signal transduction cascades, and with it phospolipid translocases began to fall in slow-motion, plunging into the boiling recesses of the peroxisomes. But this explosion didn't head towards the beaten nucleus; instead it flashed right into the hovering Bim/Smac clouds. A critical threshold had been reached...

The gloomy flocks rapidly dispersed and swarmed the mitochondria, diving into their wrinkled surfaces and rending their membranes. The mitochondrial rumbling rose in pitch, becoming a tortured kind of scream as the oxidative hellions broke loose of their matrix prison. Capsase and cytochome c erupted into the cell, colliding chaotically with everything else inside the cell, shattering the fragile secondary structures and motifs with their destructive oxidations.

The city was awash with the death-cries of the wounded. The nucleus gradually imploded, caving inwards and collapsing into the flailing centrioles and endoplasmic reticulum. The integral proteins of the plasma membrane sky plummeted further downward, rending the sticky desert with their cries as massive holes opened up in the sky and neighboring cities began to suck up the rubble, even before the city had been completely destroyed.

Within minutes, it was all over. Everything was gone, and it was as though the city had never existed in the first place...