The human body is known to produce stones in various locations, most commonly in the kidneys, the bladder, the gallbladder and the appendix. Many of these stones appear without any obvious reason. However, a new study published in the journal Immunity shows the whole storyline for the first time: how gallstones are formed.
Gallstones: what are they?
Gallstones are stones that form within the gallbladder, affecting about 25 million Americans and in Germany, about 6 million. In the USA, about 100,000 people die each year of gallstones and associated complications, and this disease is among the top causes for hospitalization.
Gallstone / Gall bladder stone. Image Credit: eleonimages / Shutterstock
Gallstones are typically composed of cholesterol and calcium crystals. The gallbladder epithelium concentrates bile and increases its acidity, so that cholesterol and calcium salts can dissolve in it to saturation levels and beyond. At such a point, supersaturation and precipitation occurs, leading to the crystallization of cholesterol and calcium salts. However, this is not just a physicochemical process. There is also an unknown aggregating agent that causes gallstones to form first, and then grow. The present study throws light on this factor.
The problem with gallstones is that while most are asymptomatic, some cause extreme colic, or waves of spasmodic pain in the abdomen. More serious if not more painful complications may also arise, including perforation of the gallbladder, peritonitis or infection of the general abdominal cavity, and even death. Surgery is the way out in such a case. Most gallstones are cholesterol crystals, and are more frequent in people who have high cholesterol levels. However, how the microscopic crystals of cholesterol build up into stones as large as several centimeters across is a surprisingly under-researched topic – until now.
The current study used a nontraditional approach to their investigation into how gallstones occur – one which took them on a tour of museums, butcher houses and modern operation rooms. They looked at human gallstones kept on display at the Charité hospital museum in Berlin and did their own tests. They examined pig bile from an abattoir. They even analyzed bile and gallstones from patients who had surgical procedures for gallstone disease.
The first step was taken when they successfully analyzed the composition of biliary sludge, the granular concentrated material within gallbladders containing gallstones. The sludge came from patients on hepatobiliary stents. Fluorescence microscopy was carried out on this material, and the researchers found large clumps of extracellular DNA (ecDNA), and high levels of neutrophil elastase activity.
Secondly, they found that ecDNA was also seen in pig biliary sludge, and in human gallstones. Moreover, they found that ecDNA, neutrophil elastase and histone H3 ( a DNA-associated protein) were all found on the surface of gallstones, indicating the role played by NET in the formation of gallstones. Almost 90% of gallstones also showed evidence of neutrophil elastase activity at the surface, the actively growing part, whether or not active inflammation was present. Thus neutrophil deposition of ecDNA is a key event in gallstone formation.
Subjecting these varied materials to modern investigative techniques, the team found one common factor: all gallstones are covered with traces of a white cell type called neutrophils, part of the granulocyte population in the blood. These immune cells form part of the first frontier against invading bacteria and other harmful particles, including crystals which are not normally found in the blood.
When they encounter cholesterol crystals in the gallbladder, they go into their usual act, attempting to engulf the crystals and dispose of them. However, in the process they undergo severe damage, with lysosomal disruption. The digestive lysosomal enzyme granular cathepsin G enters the cytoplasm, binds to DNA and causes the strands to spread out like a network, and finally extrudes them from the cell. this lysosomal leakage causes the formation of a sticky network of chromatin strands over the surface of the crystals. This is called a neutrophil extracellular trap (NET). The NET wraps around multiple crystals to form clumps, forming increasingly large stones.
To prove this, the team found that when cholesterol and calcium crystals in solution were added to a neutrophil culture and incubated, small crystals were quickly precipitated, and gallstones began to form and grow. Both calcium and cholesterol crystals are found in gallstones, because of the effect of NET on crystals clustered together by the sticky tide of bile within the gallbladder.
The way out
The researchers also tested the effects of temporarily incapacitating neutrophils in vivo, or of NET inhibitors, and found that both reduced gallstone growth. The striking discovery is that “The production of gallstones can be greatly reduced or even stopped if the formation of these nets is inhibited using drugs,” according to researcher Luis Munoz. Using drugs to treat gallstones in this way is a previously unexplored option.
For instance, the drug Metoprolol could be used in this way. This is a simple beta-blocker, so called because it blocks the beta-adrenergic receptors that mediate some effects of chemicals like adrenaline on various body organs and tissues. This drug has thus been in use for many years to treat high blood pressure. However, when used in patients with gallstones, metoprolol inhibits the entry of neutrophils into the circulation from the tissues. This automatically reduces NET formation and thus brings down the chances of gallstone formation. There are also other drugs called PAD inhibitors, which selectively prevent NET formation from neutrophils in experimental setups. These validate the role ascribed to the immune system in gallstone formation in the current study.
The importance of this process is that it applies not only to gallstone formation but also to kidney and salivary gland stones, among others. This means that these can also be prevented by the same drugs, which will provide relief to many sufferers without the need for surgery.
Neutrophil extracellular traps initiate gallstone formation. Luis E. Muñoz, Sebastian Boeltz, Rostyslav Bilyy, Christine Schauer, Aparna Mahajan, Navena Widulin, Anika Grüneboom, Irmgard Herrmann, Edgyda Boada, Manfred Rauh, Veit Krenn, Mona H.C. Biermann, Malgorzata J. Podolska, Jonas Hahn, Jasmin Knopf, Christian Maueröder, Solomiya Paryzhak, Tetiana Dumych, Yi Zhao, Markus F. Neurath, Markus H. Hoffmann, Tobias A. Fuchs, Moritz Leppkes, Georg Schett, & Martin Herrmann. August 15, 2019. DOI:https://doi.org/10.1016/j.immuni.2019.07.002. https://www.cell.com/immunity/fulltext/S1074-7613(19)30318-8