Three-dimensional reconstruction of live adherent human dendritic cells labeled with a fluorescently coupled antibody to CD11C. In this image, tunneling nanotubules can be seen to interconnect individual dendritic cells over large distances.

Immune system cells are connected to each other by an extensive network of tiny tunnels that, like a building’s hidden pneumatic tube system, are used to shoot signals to distant cells.

This surprising discovery, reported by two Pitt School of Medicine researchers in the September issue of the journal Immunity, may explain how an immune response can be so exquisitely swift. The research not only proves that cells other than neurons are capable of long-distance communication but also reveals a hereto-unknown mechanism that cells use for exchanging information.

Blood-derived dendritic cells and macrophages, both antigen-presenting cells, make use of these so-called tunneling nanotubules to relay molecular messages, report Pitt’s Simon C. Watkins and Russell D. Salter. Further research may show there are additional cell types with these microscopic tunnel connections. Thus far, their studies suggest the tunnels do not exist between commonly used fibroblast and tumor cell lines.

If not for a minor mishap while carrying out an experiment, the authors might not have discovered the existence of these physical structures and conducted the studies that revealed their role in intercellular communication.

Using a custom-built, multicamera, live-cell microscopic imaging system, Watkins and Salter found that, in a matter of seconds, dendritic cells and macrophages can send waves of calcium and other small molecules to cells hundreds of micrometers away. Each nanotubule measures 35- to 200-nanometers across—5,000 times smaller than the width of a human hair—and at any given time cells may have up to 75 of these extensions, each of varying lengths.

Russell D. Salter

“Considering their scale, these nanotubules are allowing communication between fairly distant cells,” said Salter, an associate professor of immunology. “If instead of a culture dish we were talking about a large metropolitan area, the distance would be about the equivalent to four or five city blocks. That’s nothing short of amazing.”

Salter and Watkins are the first to explain the function of tunneling nanotubules, structures that were first described in fruit flies in 1998 and subsequently identified in a handful of different types of animal and human cells.

Simon C. Watkins

“It’s one thing to find that this intricate physical network exists but quite astonishing to learn that immune system cells are using it to relay molecular signals to one another,” said Watkins, professor and vice chair of the Department of Cell Biology and Physiology and director of the University’s Center for Biologic Imaging.

While gap junctions—interconnecting molecular bridges that conjoin tightly packed cells—are known to generate signals and transport other molecules between cells, the researchers say the tunneling nanotubules are quite different.

“This is clearly a third form of intercellular communication, distinct from gap junctions and synapses used by nerve cells,” said Watkins, who is also a professor of immunology. “And, it is possible that tunneling nanotubules are essential for the function of the immune system, just as gap junctions are critical for the function of cardiac muscle. Exactly how this is so, we don’t know.”
“Further study may help us better understand how they’re involved in the local inflammatory response of the immune system,” Salter said. “For instance, we may find that dendritic cells use this network to distribute antigens to other cells, and it may be conceivable to follow the entire pathway by tracing the network of tunneling nanotubules.”

The authors’ discovery builds on their recent research showing how dendritic cells respond to stimuli. But, as Watkins and Salter admit in their paper, it was due largely to an accidental observation: that giving just the slightest poke to a single cell can set off a chain reaction whereby cell after cell discharges bursts of calcium.

In their earlier studies, they described how dendritic cells unfurl hidden veils—membranes that are so thin they can barely be imaged—and use these veils to move in on and capture their target. In the presence of E. coli, this occurs so rapidly and with such vigor that in accelerated time-lapse video the cells appear like a pack of wild animals feeding on a carcass.

But two things baffled the researchers. Dendritic cells extended their veils even before making physical contact with E. coli, yet macrophages, cells not normally picky about the antigens they engulf, were completely unresponsive to the bacteria. To understand how dendritic cells first sense the presence of an antigen and why the reaction is cell-specific, the authors decided to look at calcium flux, a well-recognized early measure of stimulation in numerous cell types. The use of a fluorescent dye, which allows direct measurement of calcium levels, would determine if calcium flux occurs before dendritic cells unfurl their veils.

With a microinjection tip, they squirted a mixture of E. coli fragments into a culture dish; indeed, one to two minutes before the appearance of the thin membranes, there were bursts of color indicative of calcium flux. Given their earlier results, the researchers anticipated that by repeating the experiment with macrophages there would be no response. But as luck would have it, the microscopic bacteria sample somehow got clogged inside the tip, and before Watkins realized the need to pull away from the cell, he had already given it a jab.

“On the screen it looked like flash bulbs going off in a dark concert arena,” Salter recalled of the moment when he and Watkins witnessed how that little mishap caused the macrophages to release bursts of calcium.

Returning to dendritic cells, the researchers found that by giving a deliberate poke with an empty microinjection tip it caused the same reaction. But why some cells responded and others did not made Salter and Watkins wonder whether there was some sort of physical structure connecting only those cells that discharged. A literature search turned up a handful of papers describing tunneling nanotubules, and further imaging using the highest magnification possible disclosed their presence in both the dendritic cells and macrophages.

In their most definitive experiment, the researchers placed dendritic cells, macrophages, and a small amount of the E. coli mixture in the same culture dish. The dendritic cells, as would be expected, fluxed calcium in response to the E. coli. But a few seconds later, calcium could also be seen shooting through the tiny tunnels extending from dendritic cells to neighboring macrophages.
“This may solve some of the mystery of how a local stimulus directed at a very small number of cells can be amplified and result in a successful immune response,” explained Watkins.

“Quite possibly, the tunneling nanotubules enable a small number of dendritic cells with captured antigens to reach other dendritic cells in lymph nodes, increasing the number of these cells capable of stimulating T lymphocytes,” added Salter.

The finding that nanotubules play a role in sending molecular signals to other immune system cells calls into question the long-held belief that immune system cells talk to one another solely by secreting substances such as cytokines, the authors say. It now seems clear that intercellular communication is much more complicated. While it would be fascinating to see this interplay inside living tissue, detecting the tiny tubules in such a complex environment may be nearly impossible with current technology.

Salter and Watkins’s research was supported by the National Cancer Institute of the National Institutes of Health.