'Underground' Tunnels Discovered As Means For Communication Between Immune System Cells

Blood-derived dendritic cells and macrophages, bothantigen-presenting cells, make use of these so-called tunnelingnanotubules to relay molecular messages, report Simon C. Watkins,Ph.D., and Russell D. Salter, Ph.D. Further research may show there areadditional cell types with these microscopic tunnel connections. Thusfar, their studies suggest the tunnels do not exist between commonlyused fibroblast and tumor cell lines.

Interestingly, if not for a minor mishap while carrying out anexperiment, the authors might not have discovered the existence ofthese physical structures and conducted the studies that revealed theirrole in intercellular communication.

Using a custom-built, multi-camera live cell microscopicimaging system, they report that, in a matter of seconds, dendriticcells and macrophages can send waves of calcium and other smallmolecules to cells hundreds of micrometers away. Each nanotubulemeasures between 35 and 200 nanometers across -- 5000 times smallerthan the width of a human hair -- and at any given time, cells may haveup to 75 of these extensions, each of varying lengths.

"Considering their scale, these nanotubules are allowingcommunication between fairly distant cells. If instead of a culturedish we were talking about a large metropolitan area, the distancewould be about the equivalent to four or five city blocks. That'snothing short of amazing," remarked Dr. Salter, associate professor ofimmunology at the University of Pittsburgh School of Medicine.

The authors are the first to explain the function of tunnelingnanotubules, structures that were first described in fruit flies in1998, and subsequently, identified in a handful of different types ofanimal and human cells.

"It's one thing to find that this intricate physical networkexists but quite astonishing to learn that immune system cells areusing it to relay molecular signals to one another," said Dr. Watkins,professor and vice chair, department of cell biology and physiology,and director of the Center for Biologic Imaging, University ofPittsburgh School of Medicine.

While gap junctions -- interconnecting molecular bridges thatconjoin tightly packed cells -- are known to generate calcium signalsand transport other molecules between cells, the researchers say thetunneling nanotubules are something quite different.

"This is clearly a third form of intercellular communication,distinct from gap junctions and synapses used by nerve cells. And, itis possible that tunneling nanotubules are essential for the functionof the immune system, just as gap junctions are critical for thefunction of cardiac muscle. Exactly how this is so, we don't know,"added Dr. Watkins, who also is a professor of immunology.

"Further study may help us better understand how they'reinvolved in the local inflammatory response of the immune system. Forinstance, we may find that dendritic cells use this network todistribute antigens to other cells and it may be conceivable to followthe entire pathway by tracing the network of tunneling nanotubules,"said Dr. Salter.

The authors' discovery builds on their recent research showinghow dendritic cells respond to stimuli, but, as they freely admit inthis paper, it was due in large part to an accidental observation, thatgiving just the slightest poke to a single cell can set off a chainreaction whereby cell after cell discharges bursts of calcium.

In their earlier studies, they described how dendritic cellsunfurl hidden veils -- membranes that are so thin they can barely beimaged -- 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 vigorthat in accelerated time-lapse video, the cells appear more like a packof wild animals feeding on a carcass.

But two things baffled the researchers. Dendritic cellsextended their veils even before making physical contact with E. coli,yet macrophages, cells not normally picky about the antigens theyengulf, were completely unresponsive to the bacteria. In order tounderstand how dendritic cells first sense the presence of an antigenand why the reaction is cell-specific, the authors decided to look atcalcium flux, a well-recognized early measure of stimulation innumerous cell types. The use of a fluorescent dye, which allows directmeasurement of calcium levels, would determine if calcium flux occursbefore dendritic cells unfurl their veils.

With a microinjection tip, they squirted a mixture of E. colifragments into a culture dish, and, indeed, one to two minutes beforethe appearance of the thin membranes, there were bursts of colorindicative of calcium flux. Given their earlier results, theresearchers anticipated that by repeating the experiment withmacrophages there'd be no response. But as luck would have it, themicroscopic bacteria sample somehow got clogged inside the tip, andbefore Dr. Watkins realized the need to pull away from the cell, he hadalready given it a jab.

"On the screen it looked like flash bulbs going off in a darkconcert arena," Dr. Salter recalled of that moment, when to both theirgreat surprise the researchers witnessed how that little mishap hadcaused the macrophages to release bursts of calcium.

Returning to dendritic cells, they found that by giving adeliberate poke with an empty microinjection tip it caused the samereaction. But why some cells responded and others did not made Drs.Salter and Watkins wonder if there was some sort of physical structureconnecting only those cells that discharged. A literature search turnedup a handful of papers describing tunneling nanotubules, and furtherimaging using the highest magnification possible disclosed theirpresence in both the dendritic cells and macrophages.

In their most definitive experiment, the researchers placeddendritic cells, macrophages and a small amount of the E. coli mixturein 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 extendingfrom dendritic cells to neighboring macrophages.

"This may solve some of the mystery of how a local stimulusdirected at a very small number of cells can be amplified and result ina successful immune response," explained Dr. Watkins.

"Quite possibly, the tunneling nanotubules enable a smallnumber of dendritic cells with captured antigens to reach otherdendritic cells in lymph nodes, increasing the number of these cellscapable of stimulating T lymphocytes," added Dr. Salter.

The finding that nanotubules play a role in sending molecularsignals to other immune system cells calls into question the long-heldbelief that immune system cells talk to one another solely by secretingsubstances such as cytokines, the authors say. It now seems clear thatintercellular communication is much more complicated. While it would befascinating to see this interplay inside living tissue, detecting thetiny tubules in such a complex environment may be nearly impossiblewith current technology.

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

Immunity Web site: http://www.immunity.com

Download PDF: http://www.eurekalert.org/jrnls/cell/pages/pdf/immunity/watkins.pdf