Cell Activation and Control

Cell Activation & Control in Scleroderma
By Stuart A. Berger Ph.D., Toronto, Ontario, Canada (Part of 3 year Research Study Report)

Dr. Berger completed this research project with funding from the Scleroderma Society of Ontario

Scleroderma is a remarkably complex disease. When dealing with such a disease that is comparatively rare, often difficult to diagnose, and where the disease course in each patient is unique, the standard questions such as: "What is the original cause? What differences can account for the symptoms? How can the symptoms be controlled, and How can a cure be identified?" become very difficult to address in a systematic way.

That is why scientists interested in diseases such as scieroderma often attempt to simplify the task by studying appropriate models.

Tight Skin (TSK) mouse
In the case of scleroderma, we are fortunate that such a model does exist. This model is called Tlght Skin (TSK) mouse. These mice have a dominant mutation that predisposes them to a systemic fibrosis that shares many characteristics with the human disease. These include excessive growth and thickening of connective tissue, over production of autoantibodies. The cause of the disease in these mice is not known, however the mutation has been linked to a defect in a protein found in the extracellular matrix. Some have speculated that this defect is responsible for the scleroderma-like state.

Mast Cells
Another aspect of scleroderma that these mice share with the human disease is the involvement of cells called mast cells. Mast cells are cells of the immune system that can be found all throughout mucosal tissues such as the lung, skin, and the gastrointestinal system.

Mast cells are best known for their involvement in allergies. When mast cells come in contact with allergens for which they have been sensitized, they undergo a very rapid reaction called 'degranulation'. The consequence of this degranulation reaction is the release of a 'soup' of proinflammatory factors. These factors include histamine which is responsible for many of the immediate symptoms of allergy. Later in the reaction, mast cells also produce and release factors that 'recruit and activate other cells of the immune system.  Consequently, mast cells can be considered as one of the earliest initiators of inflammation.

In the early stages of scleroderma, activated mast cells are often found in high numbers in the affected tissues. Many of the factors released by degranulated mast cells have a direct stimulators effect on cells in the connective tissue. Furthermore, some cells in the connective tissue produce their own factors that can support the growth of mast cells.

Therefore, in scleroderma, there appears to be a mutually stimulators cycle between connective tissue cells and mast cells. Many researchers believe that this 'vicious cycle' of mutual stimulation makes a significant contribution to the initiation and maintenance of scieroderma. Importantly, as the scleroderma-hke disease in TSK mice develops, many activated mast cells can be found in the thickening skin and other affected sites. Genetic studies with mast cell deficient mice and experiments with mast cell 'stabilizing agents have shown that mast cells make a significant contribution to thescleroderma-hke disease.

In our laboratory, we are interested in the factors that control mast cell growth and activation. Our general goals are to understand, at the molecular level, how signals received by mast cells result in these important endpoints. One molecule that we study extensively is a growth factor called Steel Factor (SLF). SLF is produced by fibroblasts and is critical for the support of mast cells in the connective tissue since mice that do not produce SLF have no mast cells. Mast cells are also stimulated to grow by other factors called cytokines. These cytokines are produced by other cells of the immune system and act to recruit and increase mast cell numbers at sites of infection.

Since mast cells appear to play a very important role in scleroderma, we have begun to study the properties of mast cells from TSK mice. Our hypothesis is that there may be differences in the ability of these cells to respond to growth or activating signals and that these differences may contribute to the scleroderma-like characteristics of these mice.

Mast cells, like other cells of the immune system originate in the bone marrow and then migrate to distant tissues. It is possible to generate large numbers of mast cells for cellular and molecular studies by incubating bone marrow cells in the proper growth medium. The most important component of this growth medium is a cytokine called interleukin-3 (IL-3) which stimulates mast cells to grow and differentiate. Under these conditions, greater than 95% of the cells are mast cells, four weeks following the initiation of the cultures. These mast cell cultures can be maintained for upwards of three months.

We have generated mast cell cultures in this manner from normal and TSK ntice and have compared some of their properties. In the short time since initiating these studies, we have found some remarkable differences between the normal and mutant mast cells. We found that the TSK cultures appear to have an increased growth rate compared to normal cells.

We have also compared the ability of TSK mast cells to become activated in response to an allergen for which they have been sensitized. As shown in Figure 3, TSK mast cells degranulate to a greater extent in response to an allergen than normal cells. This observation indicates that TSK cells are more 'sensitized' than normal cells.

A third difference between TSK and normal mast cells is that in the presence of SLF, TSK mast cells appear to differentiate into mature mast cells at a slower rate than normal mast cells.

Summary
Our work with TSK mast cells has revealed very important differences in growth, activation and differentiation properties compared to normal cells. The fact that these cells appear to have an increased growth rate in response to the cytoldne IL-3 may provide an important clue as to why there is an overabundance of these cells in connective tissue. Their increasedsensitivity to degranulation by allergens is consistent with their apparent excessive activation in the connective tissue and may explain why mast cells in TSK niice appear to be overactivated. Finally, their altered differentiation pattern in response to SLF is similar to that observed for mast cells in human scleroderma and provides another important parallel with the human disease.