LONDON -- Macrophages are the body's scavengers, cells that clean up cellular debris, dead cells, dust particles and -- most importantly -- invading microorganisms. They do this vital work by a process called phagocytosis, taking particles into themselves to be broken down and disposed of. Recent research suggests that there are at least two different types of phagocytosis. One type triggers an inflammatory reaction, while the other does not.
Now, two researchers in London have identified a new molecular difference between the two types of phagocytosis. Their work could eventually lead to an explanation of how organisms such as Mycobacterium tuberculosis and Leishmania major are able to invade and survive in host macrophages.
Emmanuelle Caron and Alan Hall, of the Cancer Research Campaign Oncogene and Signal Transduction Group at the Medical Research Council's Laboratory for Molecular Cell Biology at University College London, in the U.K., report their findings in a paper in the Nov. 27 issue of Science. Their paper is titled "Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases."
Phagocytosis is a phenomenon known throughout the animal kingdom, from single-celled amoebae to mammals. It is defined as the recognition and engulfment by a cell of particles larger than 0.5 micrometers in diameter. These particles may be dust particles, apoptotic cells, bacteria or other microorganisms.
Phagocytosis plays an important role in the remodeling involved in development, as well as in fighting infectious diseases. A particle phagocytosed by a macrophage is taken up by the cell, and internalized in a vacuole called a phagosome. The phagosome then fuses with vacuoles called lysosomes. The contents of the lysosomes are acidic. In the case of microorganisms, this change in pH leads to their death. After digestion, protein fragments of the microorganism are displayed on the surface of the macrophage, where they are presented to other cells of the immune system, such as B cells and T cells, triggering a specific immune response.
Usually, before phagocytosis occurs, a microorganism becomes coated either by complement or by immunoglobulins, in a process known as opsonization. Complement-opsonized microorganisms become bound to macrophages via a receptor called complement receptor 3 (CR3), while those covered with immunoglobulin G bind to Fc gamma (Fcg) receptors.
Rho GTPases Focus Of Interest
Stimulation of each of these receptors leads to a different morphological response by the cell: when CR3 is stimulated, the particle seems to stick to the cell and then sink into it until completely engulfed, while when the Fcg receptor is stimulated, the cell membrane protrudes pseudopodia to reach out and "catch" the particle. In the latter case, phagocytosis is accompanied by the production of toxic reactive oxygen species, and inflammatory cytokines such as tumor necrosis factor alpha. But this does not happen in CR3-mediated phagosomes.
Caron told BioWorld International the researchers "wanted to understand the cell biology and signal transduction underlying phagocytosis of microorganisms in macrophages. We knew that, when bacteria such as Salmonella and Shigella enter cells -- such as epithelial cells -- which are unable to perform phagocytosis normally, they activate a group of molecules called the Rho family of guanosine triphosphatases, known as Rho GTPases. This suggested that these molecules were likely candidates for being involved in phagocytosis in macrophages."
The Rho GTPases are molecular switches, active when bound to GTP. Previous work in Hall's laboratory and elsewhere has shown that the Rho GTPases were involved in a signaling cascade, which begins with stimulation of growth factor receptors and ends with changes in the organization of the actin cytoskeleton. Reorganization of the actin cytoskeleton is known to be necessary for the movements of the cell membrane involved in phagocytosis.
Caron and Hall therefore examined the roles of the three main Rho GTPases, which are called Rho, Rac and Cdc42. "We used microinjection and transfection techniques," Caron said. "We knocked out, one by one, each of these three, Rho, Rac and Cdc42. We found that Fcg receptor-mediated phagocytosis was abolished when we knocked out the function of either Cdc42 or Rac, but not Rho. Whereas, for CR3-mediated phagocytosis, Rho activation was absolutely critical. But this type of phagocytosis was not interfered with if Cdc42 or Rac functions were blocked."
New Phagocytosis Classes Proposed
The two researchers also showed that only Rho becomes associated with phagosomes resulting from CR3- mediated phagocytosis -- a process called recruitment -- while Rho, Rac and Cdc42 were recruited to phagosomes formed during Fcg receptor-mediated phagocytosis.
Caron and Hall suggest a new classification of types of phagocytosis based on their observations. In Science, they write: "We propose that the Cdc42/Rac-dependent uptake (typified by Fcg receptors) be termed type I phagocytosis and the Rho-dependent uptake (typified by CR3) be termed type II. The recruitment of Rho, Rac and Cdc42 in type I (but only Rho in type II) phagocytosis suggests a molecular explanation not only for the well known morphological differences observed between Fcg receptor- and CR3-mediated phagocytosis but, more important, for the different associated biological responses."
Next, the researchers plan to find out more about what happens upstream and downstream of GTPase activation. "We want to know how the receptor 'talks' to Rho, Rac and Cdc42, and what are the partners for Rho, Rac and Cdc42 that enable them to 'talk' to actin," Caron said. "In addition, we are interested in looking at other receptors. We looked at two important receptors, but there are dozens of receptors able to mediate phagocytosis. Will all these receptors fall into the type I and type II categories that we have proposed? Is there a type III category?"
The researchers also intend to examine what happens when other phagocytic targets, such as apoptotic cells, are involved. "We know that the uptake of apoptotic cells is not linked to a pro-inflammatory response, so we imagine that they are taken up by a process which resembles the CR3-mediated phagocytosis, which we have called type II," Caron added.
In addition, she said, it was striking to observe that most bacteria that are able to survive and multiply in macrophages, such as Legionella, Mycobacterium tuberculosis and Leishmania, all enter the cell via CR3, too. "This is speculation, but this makes us think that the reason is that this receptor induces less toxic responses," she said. "If we could understand the signal transduction pathway that follows the entry of Mycobacterium into the cell and compare it to what happens normally, we would be able to pinpoint the differences -- if there are any differences with an intracellular pathogen. This could conceivably lead to the development of new drugs against these microorganisms." *