The immune system finally seems to have heard the buzz that in the 21st century, it's all about connectivity.

In the September 2005 issue of the Immunity, Simon Watkins and Russell Salter from the University of Pittsburgh School of Medicine reported on "Functional Connectivity between Immune Cells Mediated by Tunneling Nanotubules." The scientists demonstrated direct long-range connections, in cell culture, between immune system cells.

Communication via nanotubules joins several other methods through which immune system cells coordinate their actions. The well-known presentation of antigens to killer T cells by dendritic cells, macrophages and B cells and the secretion of cytokines are the main ways by which different cell types communicate. Cells that are located directly next to each other also can communicate via so-called gap junctions.

Nanotubules as a communication method greatly expand the potential range of communication between stationary cells, for example in the skin. The researchers' observations suggested connections between groups of roughly a dozen cells located within about half a millimeter of each other. "But a cell that is part of one group might also be connected with another group downstream," said Russell Salter, associate professor of immunology at the University of Pittsburgh School of Medicine. "There's no real limit" to how many cells could theoretically be connected.

The observations reported in the current Immunity paper began when Watkins and Salter noticed that when a population of dendritic cells in culture were exposed to E. coli-derived proteins, the resulting uptake of calcium did not spread evenly from the area of exposure, as would be expected if the cells were stimulated either by E. coli proteins slowly diffusing through the culture or activation being spread through gap junctions from one cell to its neighbors. Instead, it seemed to be dispersed unevenly from one cell to others that were more distant.

While investigating the phenomenon, the researchers managed to capitalize on their accidental observation that what they termed "mechanical stimulation" in the paper - essentially, a poke with the pipette they used to deliver the bacterial proteins - also could set off an irregular calcium flux. Using high-resolution microscopy, the scientists found "fine tubular structures" - previously been described in the literature as tunneling nanotubules - that might be able to propagate such calcium signals.

While the spread of protein-containing medium is hard to control within the culture dish, the mechanical stimulation could be limited to parts of the dish by more mechanical intervention - essentially, scraping across the dish to disrupt the cell layer. The scientist found that the cell networks that responded to a pipette poke did not cross such a mechanical barrier.

Salter said that the cells they used in their research - in addition to dendritic cells, a cultured tumor cell line called THP-1 - "are not necessarily identical to [immune system cells] in the body," but that they "would function somewhat like Langerhans cells in the skin." Langerhans cells are a type of dendritic cell, the major antigen-presenting cells. They obtain antigens in tissues, such as the skin, migrate to the lymph system, and activate T cells. Salter believed that they might form nanotubule-mediated networks in the skin, break off from such networks during migration, and re-form connections once they have migrated to the lymph system.

The scientists found that the tunneling nanotubule-mediated calcium spread was followed by shape changes in the cells typical for phagocytic activity. They also found that when they placed dendritic cells, macrophages and bacterial proteins in the same culture dish, the dendritic cells, as would be expected, fluxed calcium. But calcium also moved from dendritic cells to neighboring macrophages. Because macrophages were not responsive to bacterial proteins in pure macrophage cell cultures, and control experiments ruled out signal transmission via gap junctions, those experiments were strong evidence for the existence of functional nanotubule connections between cells.

"What we find exciting is that we come at this from the functional side," Salter said. "We saw the calcium influx and had to explain how it might be propagated."

Other researchers have reported seeing tunneling nanotubules, but those reports were by and large purely structural. There was only one prior report as to possible functions, which reported that endosomes might be transported through tunneling nanotubules. Salter said his group was originally puzzled by that report, because their own research indicated a size limit of about 70 kilodaltons, which would be too small for endosomes to pass through.

However, his research suggested that the nanotubules might be gated, so that larger proteins could pass through under the right conditions. Such large proteins might include antigens and even whole viruses; Salter specifically mentioned HIV as an example.

"We're certainly interested in whether that could be transported - not that we have any evidence for that yet, but we're interested," he said.

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