Mar. 31, 1999 For years, doctors and scientists have sought to understand a complicated mechanism that triggers an over-aggressive immune response in many patients who suffer from severe microbial infections. This immune system overreaction often leads to septic shock, for which no reliable treatments have been found.
Recent data from the Centers for Disease Control and Prevention in Atlanta show that mortality from infectious diseases in the United States has been increasing in recent years, and the number of deaths due to septicemia almost doubled from 4.2 to 7.7 per 100,000, between 1980 and 1992. Infectious diseases now rank third among leading causes of death in the United States, following only cardiovascular diseases and cancer. Furthermore, recent research has linked even cardiovascular diseases and some cancers to infectious diseases.
In a study headed by researchers at Cedars-Sinai Medical Center, scientists have for the first time identified in actual human cells a "receptor" that may be a key component of the process that leads to septic shock, confirming suspicions raised in other recently published studies. In fact, the existence of these receptors -- called Toll-like receptors -- has been the subject of a recent flurry of research.
Data showing this latest evidence was accepted for rapid publication in the March 19 issue of the Journal of Biological Chemistry. Doctors hope this breakthrough may lead to new and more effective approaches for the treatment of severe bacterial infections and endotoxin-associated septic shock that claim thousands of lives each year.
When bacteria from an infection at any site in the body invade the bloodstream, a condition known as septicemia, patients typically experience a variety of symptoms, including chills and fever, and the bacteria are able to travel throughout the body. In 40 to 70 percent of these cases, septic shock follows, characterized by a drop in the blood pressure, multiple organ failure, circulatory collapse and often death.
Particularly perplexing have been cases of septicemia caused by bacteria that fall into the "Gram-negative" classification, based on the common method of staining and identifying bacteria. Gram-negative organisms cause approximately half of the septicemia cases, and contain a toxin, called lipopolysaccharide (LPS) or endotoxin, on their outer membranes. This toxin is released into the blood.
"Interestingly, even tiny amounts of endotoxin shed from these Gram-negative bacteria are recognized by the immune system" said Moshe Arditi, M.D., director of the Division of Pediatric Infectious Diseases at Cedars-Sinai Medical Center, and the leader of the study. In response, the immune system activates various cell types, including the white blood cells to produce inflammatory molecules called cytokines. Cytokines destroy invading pathogens but if the production of cytokines continues unchecked, they actually become toxic themselves. Patients suffering from septicemia who progress to septic shock actually may die as a result of the excessive immune response rather than from the initial bacterial infection alone. In fact, more than 70,000 people in the United States die each year from septic shock resulting from Gram-negative septicemia.
Over the past 15 years, researchers have experimented with medications intended to fight the infection and others designed to control and decrease the inflammatory response and to neutralize the circulating endotoxin or overabundant cytokines. But efforts to control septicemia and prevent it from progressing to septic shock have largely failed, despite multiple clinical trials using these various approaches.
According to Dr. Arditi, researchers have been puzzled by the immune system's ability to recognize minute amounts of endotoxin in the first place. "For many years, several laboratories and several research groups have been looking for the receptor on the cell for this toxin, to determine how this toxin is recognized by the body's immune system," he said. "The goal has been to determine how tiny amounts of bacterial endotoxin are recognized by the cells of the immune system and how they send their signals into the cells to activate them, so that we can develop novel therapeutic approaches to block this often detrimental cascading effect."
Researchers studying fruit flies in the mid-1980s began making seemingly unrelated discoveries that eventually led to a better understanding of the mechanisms contributing to septic shock in humans. First, scientists discovered a gene, called a toll gene, that was crucial to the fruit fly's development. They found that the gene makes a "receptor protein" that delivers developmental signals from the outer portion of the cell to its nucleus. This Toll protein or Toll receptor, it was later learned, also triggers the fruit fly's immune defense, an "innate" immune system which protects it from invading organisms such as bacteria and fungi.
"Unlike the fruit fly, the human body has both innate and adaptive immune systems," said Dr. Arditi. "The innate immune system is the body's first, immediate defense against invading pathogens, providing a brute force, blunt, non-specific response to any invading organism. It immediately releases inflammatory cytokines, then sends a message to the more sophisticated adaptive immune system: 'Gear up. We're going into battle.' The adaptive immune system is much more sophisticated and has more specific weapons, such as antibodies and T-cells."
It is the more primitive innate immune system and its overblown response to bacterial toxins such as endotoxin that is responsible for leading to septic shock and frequently to death.
Scientists have found that the Toll mechanism functions in the immune systems of organisms as diverse as fruit flies and tobacco plants, warding off assaults from bacteria and fungi. In insects, the Toll receptors on the surface of the cell and their signaling pathways lead to stimulation of a protein which goes by the name Dorsal, which is responsible for launching an immune response and triggering the production of an antifungal peptide, drosomycin, in the fly.
In the late 1980's, researchers investigating inflammation discovered signaling pathways that converge on a protein called NF-kappa B, which turns on genes that make cytokines and trigger inflammatory responses. In humans, one of the cell surface receptors that pass signals to NF-kappa B is the receptor for the cytokine interleukin-1 (IL-1), which among other things is known to induce fever. Researchers soon realized that the protein Dorsal, activated by Toll receptors in the fruit flies, is structurally similar to NF-kappa B in humans. Soon they also realized that the Toll receptor (in the fruit fly) and the IL-1 receptor (in humans) are also structurally similar.
"There had been a growing belief that the Toll-like receptors, initially observed in the fruit fly, also existed in humans," said Dr. Arditi. "The first real evidence of this came in 1997 from Charles Janeway at Yale University, who identified the first human homologue of the Toll receptors, now called human Toll-like receptor-4 (TLR4). Janeway has also shown that human Toll receptor activates NF-kappa B. Since then, five human Toll-like receptors (TLR1-5) have subsequently been cloned, and even more have been identified."
The work of Janeway and his colleagues was the first indication that these newly found Toll-like receptors were able to elicit a human immune response.
Results of two studies published a few months ago by scientists at Genentech and Tularik, two biotechnology research organizations in San Francisco, further linked the Toll-like receptors to endotoxin and the human innate immune system. "They expressed the Toll-like receptor-2 (TLR2) on the surface of cells that usually do not respond to endotoxin," said Dr. Arditi. "Those cells then started to respond to endotoxin by activating an immune response and making cytokines, suggesting that LPS may be recognized by and send a signal through Toll-like receptors in cells."
At about the same time, scientists experimenting with a curious strain of mice that are unresponsive to endotoxin reported that they identified that the missing gene in these mice was the TLR4 gene, further supporting the concept that Toll-like receptors and TLR4 are essential for endotoxin signaling in cells.
"Still, until now, there were no data showing whether these Toll-like receptors (TLR2 or TLR4) were indeed present in real human cells that respond to endotoxins. It was not known how endotoxin induced the activation of NF-kappa B in cells. There was no proof that the signaling does go through similar pathways used by the toll receptors. That's what we have found," Dr. Arditi said.
The study used human endothelial cells, cells that line the blood vessels, and human monocytes, blood cells that are important in fighting infection. "We found that endotoxin uses the Toll-receptor signaling pathway to induce NF-kappa B in human cells, as part of the conserved innate immunity," said Dr. Arditi. "We went on to show the presence of the Toll-like receptors (TLR) on the surface of the cells. Both the TLR2 and TLR4 were present on the surface of the human cells. This is the first demonstration that these receptors, which may well be the long-sought endotoxin receptors, are present in human cells such as endothelial cells and monocytes, which are critical targets for endotoxin."
According to Dr. Arditi, the study, which was funded by the National Institutes of Health, may prove to be a critical step in the long journey to effectively combating septic shock. "Coming back to the first question: How does the human innate immune system recognize endotoxin to mount that first, blunt immune reaction, which, if unchecked, progresses all the way to sepsis and septic shock? I think this is how it does it ," he said. "It does it in the same way it is done in fruit flies and in plants. That same innate immune response system has been conserved throughout the evolution from plants to fruit flies to vertebrates and humans, to play a role in our immediate reaction to infections. By identifying the receptors and the signaling pathways, perhaps we can soon begin to develop new approaches to stop the cascade of this lethal syndrome."
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