How germs meet their opposites - a mystery revealed in real time

Sophisticated microscope technology has made it possible for Australian and American scientists to record previously unknown interactions between two classes of immune cell right at the beginning of the 'antigen transport chain', the apex of the immune response. An important discovery, this takes us one step further towards being able to control disease and infection.

Dr Tri Phan

Media Release: 09 June 2009

Sophisticated microscope technology has made it possible for Australian and American scientists to record previously unknown interactions between two classes of immune cell right at the beginning of the 'antigen transport chain', the apex of the immune response.

Using intravital two-photon microscopy, which allows light to penetrate deep into tissue and processes to be observed as they happen in a live animal, Dr Tri Phan from Sydney's Garvan Institute of Medical Research, while working with Dr Jason Cyster from the University of California San Francisco, captured an intriguing interaction between macrophages and B cells.

Their findings, describing the atypical and highly specialised 'subcapsular sinus (SCS) macrophages' and their interaction with antibody producing B cells, are published online in the prestigious international journal, Nature Immunology.

The lymph system, an exquisitely designed drainage and filtering network, forms the core of the body's immune system. When a bacterium, or other invader, breaches the skin it is carried in lymph vessels to the nearest lymph node to be destroyed by macrophages or dendritic cells (scavenging immune cells), unless first captured by them along the way.

As well as employing a 'seek out and destroy' approach, our immune system has evolved a second more sophisticated function. It stores a memory of invaders. To generate that memory, B cells need to become activated.

"We were interested in what drives B cell activation, and where it occurs," said Tri. "To become activated, B cells must get to know the enemy. They must 'see' the shape of bacterial or viral antigens - their three-dimensional surface structures - so that they can create antibodies to help destroy the invader in the present, as well as form 'memory B cells' which will attack similar invaders in the future."

"To get to know antigens this way, B cells need to meet them in their native state. Until now, we have not been able to figure out how antigen actually gets to a B cell without first being broken down, or destroyed, by a macrophage or dendritic cell."

Lymph vessels around the body drain into channels in and around lymph nodes known as 'sinuses'. These sinuses are lined with endothelial cells and macrophages that appear to form a barrier against antigen. The largest sinus, the one enclosing the lymph node capsule, is known as the subcapsular sinus.

Tri explained that SCS macrophages are embedded within the sinus lining, with their 'heads' capturing antigen from newly arriving lymph on one side, and their 'tails' delivering it to B cells on the other side.

"What we've witnessed through the microscope shows intact antigen being passed through the subcapsular sinus. SCS macrophages behave almost like miniature conveyor belts, passing antigen to B cells waiting at the other side. All this happens within minutes of the antigen's arrival in a lymph node."

"This is a new and very important finding. While other groups have noted the presence of unusual macrophages in the subcapsular sinus, and have also observed the presence of B cells nearby, their relationship to one another has been unclear."

"Each time we characterise, or describe, an essential element of the immune system, it takes us a step further towards being able to control disease and infection."

With the ability to see whole processes in action that two-photon microscopy gives them, medical researchers will be able to build up a picture of the way the immune system functions much more rapidly than would have been conceivable even a decade ago.



Two photon microscopy

Since the mid-1990s, fluorescent proteins have been used as tags, or markers, helping cell biologists track the movements (under the microscope) of their favourite molecules within cells.

Fluorescence is excitation of an electron inside a molecule to a high energy state (the electron moves from one orbit to the next). When it relaxes and changes to its lower energy state, it releases that energy as light.

Until now, scientists have been limited to some extent by the proteins available to them, most of which are excited by shorter wavelengths of light - Green Fluorescent Protein (GFP), for example, fluoresces when stimulated by waves of around 488 nanometers. This is fine for tracking proteins inside cells in culture, but not good for tracking events in living tissue. That is because short wavelengths easily become absorbed or scattered by molecules in their path, so do not pass easily through tissue and bone.

Ideally, you would like an organ to be 'optically transparent'. Within the visible range, it's obviously not transparent because it doesn't let light through. At the longer light wavelengths, however, in the near-infrared and infrared ranges, tissue becomes more optically transparent. So light with those longer wavelengths will penetrate much deeper without getting scattered or absorbed.

A month ago, the team that won the Nobel Prize for developing the GFP published the news that they have developed a protein that glows at wavelengths around 700 nanometers, in the infrared range. This protein will vastly expand the 'reach' of a microscope.

Conventional microscopy uses single photon excitation. The advantage of two-photon microscopy is that you expand your 'reach' by exciting the electron with two photons of half the energy. By making the photons half the energy, you double their wavelength and ability to penetrate tissue, because energy is inversely proportional to wavelength.

In two-photon microscopy, an electron is excited by two photons that arrive in very rapid succession.  The first photon bumps up the electron halfway, the second photon bumps it up the rest of the way.

Recent advances in technology have led to the production of lasers that can consistently produce photons that are so densely packed together that they are only 10-15 seconds apart, allowing 2 photon excitation.

Using GFP and Single photon excitation, a confocal microscope will allow you to see to a depth of around 50 microns. Using GFP with two-photon microscopy, you get much deeper penetration, allowing intra-vital studies such as the one described in this media release.

Combining two-photon microscopy with infrared proteins will allow scientists to see even deeper into tissue. Limits of penetration up to now have been about 500 microns - or roughly .5 millimetre. The new infrared proteins will expand that severalfold.

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