Singing the praises of colony stimulating factors

Key text

Colony stimulating factors regulate white blood cell production

White blood cells form the basis of the body's immune system, guarding against attack by viruses and other microorganisms. Colony stimulating factors are proteins that help regulate the production of white blood cells. These cells are short-lived, so we need to produce a lot of them: healthy adults produce about 200 million new white blood cells every hour, mostly in the bone marrow.

White blood cells are not the only cells produced in the bone marrow. Red blood cells (also called erythrocytes) and platelets (which aid blood clotting) are also produced there in large numbers. Astonishingly, all these cells appear to derive from a single type of master (or stem) cell, called the haematopoietic (blood-forming) cell.

For decades, scientists thought that some sort of mechanism must exist to regulate the production of white blood cells from these haematopoietic cells. But, despite many attempts to discover such a mechanism, its eventual detection was an accident.

A lucky breakthrough

In the mid 1960s, scientists in Israel and Australia independently attempted to grow mouse leukaemia cells in nutrient agar plates. Although unable to do this, they observed the spectacular growth of healthy white blood cells in clusters or 'colonies' around other tissue fragments in the agar.

Further experiments showed that the number and size of the white blood cell colonies were dependent on adding other cells or tissue fragments to the cultures. Scientists speculated that some factor contained in this material was stimulating haematopoietic cells to divide and form these colonies. This still unidentified factor was dubbed a 'colony stimulating factor' (CSF).

Searching for CSFs

The circumstantial evidence for the existence of such a factor grew, prompting Metcalf's team to attempt to extract or purify it from tissues. This was not an easy task: while a colony stimulating factor appeared to be present in all organs of the body, it was there only in very small quantities. It was a bit like searching for a needle in a haystack, except that the shape and size of the 'needle' was unknown.

The project to purify CSF lasted 15 years. As the years went by, new technology became available to help in the task. It was also realised that more than one CSF existed: eventually, the project identified four distinct CSFs, each responsible for stimulating the growth of different kinds of white blood cell.

The nature of CSFs was also explored. Scientists determined that they were glycoproteins, molecules that combine a protein and a large sugar molecule (polysaccharide). Studies revealed that CSFs stimulated the division of immature white blood cells and were essential for the continued development of such cells. CSFs were also shown to influence the ability of certain white blood cells to kill microorganisms.

Little by little

By 1984, scientists had purified several human and mouse CSFs in small quantities. But they were gloomy. The hard work of the past decade, in several laboratories, had failed to produce enough purified CSF to test a single laboratory mouse. In fact, calculations showed that at the current rate of purification it would take 250 years to produce enough of the substance to treat a single human patient for 2 weeks.

The race to mass-produce

A new way was needed, and it was at hand. Molecular biologists were already experimenting with techniques such as gene cloning and the artificial production of enzymes, hormones and other proteins using genetic techniques. The biologists thought that by isolating the genes encoding the various CSFs and inserting them into yeast cells, bacterial cells or mammal cells, it should be possible to produce CSFs in much larger quantities.

The potential of mass-produced CSFs to aid the treatment of disease attracted the interest of a number of commercial enterprises. In the years 1984 to 1987 they raced each other to discover - and patent – the genes responsible for CSF production. Success was almost immediate, and mass production of CSFs followed soon after.

A big day in the lab

Despite this rush of activity, the value of CSFs in the treatment of disease was still untested. Professor Metcalf and his team, now armed with relatively large quantities of several CSFs, set out to see what effect the injection of one particular CSF might have on mice.

After a week of injections, white cell counts in blood samples taken from the mice showed virtually no change. According to Professor Metcalf, 'for an hour gloom reigned in the laboratory'. The mood changed dramatically with the next sample, taken from the abdominal cavity: the effect was obvious, even to the naked eye. The fluid was milky, due to the presence of a large number of white blood cells. This contrasted with the clear fluid obtained from mice that were not injected with the CSF.

Dejection in the lab turned to euphoria. The evidence seemed clear: the injection of CSFs resulted in increased production of white blood cells.

The role of CSFs in leukaemia treatment

So far this experiment had only been performed on mice. A far more important task was to test the treatment on humans.

José Carreras was one of the first people to be treated with a CSF. He suffered from acute myelogenic leukaemia, a cancer of the blood that is usually fatal within months if untreated. Unlike other cancers, leukaemia doesn't produce tumours but instead causes the rampant overproduction of cancerous white blood cells. Such cells interfere with vital organ functions, including the production of healthy red and white blood cells and platelets.

Often, the only treatment for acute leukaemia is a bone marrow transplant, which usually proceeds in three stages. The first involves intensive chemotherapy or other treatment to bring the patient's cancerous white blood cell count under control. In the second stage, the patient's bone marrow is first destroyed by intensive chemotherapy to avoid rejection of the new marrow and a new marrow from a compatible donor is then inserted.

The third stage, recovery, is often the most dangerous. Until the donor marrow cells start producing new blood, the patient is left with virtually no immune system. This makes infection very likely.

It is at this stage that CSFs play their part. Their injection into a patient has a dramatic effect on the number of white blood cells produced in the early days after a bone marrow transplant, significantly increasing the patient's chance of survival. José Carreras recovered completely, and he now sings the praises of the treatment. Many other bone marrow transplant recipients have since also benefited from treatment with CSFs.

Other roles for CSFs?

CSFs have a potential role to play in the treatment of other cancers. Infection is the most common cause of death among cancer patients undergoing chemotherapy because the production of white blood cells is affected. CSFs can be injected into such patients after chemotherapy, increasing the speed at which white blood cell counts return to normal. This also allows the possibility of increasing the chemotherapy dosage, since loss of immunity may not be such a significant problem.

Other possible uses for CSFs continue to be explored by scientists around the world, including in Australia, where it all began.

Activity 1. Blood cells

Materials (per student)

• prepared slide of stained blood smear
• monocular microscope

Procedure

1. Focus the prepared blood smear under low power and then high power.

2. Identify the red blood cells (erythrocytes) and white blood cells (leucocytes). (With Leishman's stain, the red blood cells appear yellowish-red; the nuclei of the white blood cells stain a purple colour and any granules in these cells may range from orange to black in colour.)

3. Draw and label each of the different cell types. (You may distinguish different white blood cell types according to the shape of the nucleus.)

4. Estimate the approximate ratio of red blood cells to white blood cells. Work out a way to do this that does not involve counting every cell in the field. Combine your results with those of other students for a more accurate result.

Questions

1. Describe the appearance of a red blood cell. Find out why they appear lighter in colour in the middle of the cell.

2. Describe the appearance of a white blood cell. In what ways do white and red blood cells differ?

3. How many different types of white blood cells are visible? How do they differ from each other?

4. What is the function of any platelets that are visible?

Teachers notes

Red blood cells have a characteristic biconcave shape and, unusually for cells, lack a nucleus. (Red blood cells cannot repair themselves if they are damaged. They live for only about four months before being replaced by new red blood cells produced from stem cells in the bone marrow.)

White blood cells contain a nucleus, and are larger than red blood cells. Some white blood cells have granules in their cytoplasm. These cells are made in the bone marrow and three types are recognised – neutrophils (the most common of the white blood cells), eosinophils and basophils. Other white blood cells have no granules in their cytoplasm. These cells mature in the lymph tissue as well as the bone marrow and two types are recognised – monocytes (the largest of the white blood cells) and lymphocytes.

Platelets are much smaller than either red cells or white cells and are not really cells at all. They are formed in the bone marrow when large cells pinch off pieces of their cytoplasm. There are about 250,000 platelets per cubic millimetre of blood and they play a vital role in blood clotting.

The red blood cells are the most numerous of the blood cells - there are about 5 million red blood cells in each cubic millimetre of blood. There are normally 4000 to 11,000 white cells per cubic millimetre of blood. (Leukemias are a range of cancers that involve the production of large numbers of abnormal white blood cells.)

Activity 2. Blood cells and their functions in the body

1. Write a short paragraph explaining the ways in which red and white blood cells differ in structure and function.

2. Use the following facts to calculate the number of blood cells in a human.

   There are approximately five million red blood cells in each cubic centimetre of blood.

   There are, on average, about 10,000 white blood cells in each cubic centimetre of blood.

   The total volume of blood in the body is about 5 litres.

   • Approximately how many red blood cells are there in the body?
   • Approximately how many white blood cells are there in the body?

3. Organ transplantation, including bone marrow, is becoming more common.
   • Explain what is likely to happen to a transplanted organ if the recipient is not given drugs to suppress the immune system.
   • Suggest possible side effects of taking drugs that suppress the immune system.

Teachers notes

Information about red and white blood cells can be found in the Teachers notes for Activity 1.

Further reading

Useful sites

A brief introduction to the use of CSFs as a replacement for bone marrow transplants.
http://www.csiro.au/promos/ozadvances/Series6csfCancer.html

Colony stimulating factors – helping blood cells grow (Blood and Marrow Transplant Newsletter, USA)

Provides a good overview of CSFs and their use in bone marrow transplants.
http://www.bmtnews.org/newsletters/issue14/colony.html

Biological therapies: Using the immune system to treat cancer (National Cancer Institute, USA)

Summarises the types of immune cells then shows how biological therapies, including CSFs, can stimulate the immune system's natural anti-cancer function.
http://cancerweb.ncl.ac.uk/cancernet/600072.html

Glossary

chemotherapy. Treatment of disease by using chemical compounds. Cancers are commonly treated by administering chemicals that are toxic to malignant cells.

colony. A group of identical cells (clones) resulting from repeated divisions of a single cell. The identical cells form a cluster that lies on the surface of a food source such as a nutrient agar plate.

enzyme. A protein that acts as a catalyst. Every chemical reaction in living organisms is facilitated by an enzyme.

gene. The basic unit of inheritance. A gene is a segment of DNA that specifies the structure of a protein or an RNA molecule.

gene cloning. The process of producing identical copies (clones) of a gene.

hormone. A substance produced in one part of the body and carried by the blood to another part of the body where it causes a response (eg, insulin, produced by the pancreas, that promotes the uptake of glucose by body cells). For more information see The hormones of the human (Kimball's Biology Pages, USA) and The hormones (Center for Bioenvironmental Research, Tulane and Xavier Universities, USA).

leukaemia. Form of cancer resulting in an overproduction of abnormal white blood cells. This overproduction suppresses normal red blood cell and platelet production.

nutrient agar plate. A sterile, enclosed dish with a layer of a jelly-like substance (agar) containing complete food requirements for growth of bacteria, other small organisms or cells. If the bacteria are well-spaced when they are introduced to the plate, each bacterium will produce a colony of bacteria.

protein. A large molecule composed of a linear sequence of amino acids. This linear sequence is a protein's primary structure. Short sequences within the protein molecule can interact to form regular folds (eg, alpha helix and beta pleated sheet) called the secondary structure. Further folding from interaction between sites in the secondary structure forms the tertiary structure of the protein.

Proteins are essential to the structure and function of cells. They account for more than 50 per cent of the dry weight of most cells, and are involved in most cell processes. Examples of proteins include enzymes, collagen in tendons and ligaments and some hormones. More information can be found at Protein structure and diversity (Molecular Biology Notebook, Rothamsted Research, UK).

white blood cells. (Also known as leucocytes.) White blood cells are the immune system cells. They can be divided into many different categories on the basis of their function and appearance. Many are not found in the blood at all and those that are may have the ability to crawl out of blood vessels, squeezing between the cells of the vessel walls. While some produce antibodies, others produce cocktails of destructive chemicals, others kill virus-infected cells by punching holes in them, and a further class control the entire immune response. For more information see White blood cells (Puget Sound Blood Center, Washington).