Tuatara: Volume 17, Issue 3, December 1969
Throughout the History of Man, disease has frequently played a dominant role. Plague, smallpox, malaria, tuberculosis, typhoid, cholera, diphtheria — these are only a few of the killing, crippling diseases to which man is subject. The mass effects of some epidemics of disease can be spectacular. During the Second World War, malaria caused five times as many casualties in the Pacific areas as did the actual fighting (Chandler, 1955); in 1916, an epidemic of malaria actually stopped a major war in Macedonia, no doubt to the great annoyance of the commanding generals (Lapage, 1950). The black plague killed 300,000 people in Naples in 1656, and caused an estimated 100,000,000 deaths in the sixth century. A macabre reference to plague is made in at least one nursery rhyme — ‘Ring-a-ring-a-rosy’ refers to the inflamed rosette on the chest which is one of the first symptoms of the plague, ‘a pocketful of posies’ refers to the aromatic herbs which were sniffed at intervals in the pious and totally misplaced hope that the action would keep away the plague, ‘a-tisket, a-tasket’ refers to the sneezing which is symptomatic of pneumonic plague, and ‘all fall down’ is self-explanatory.
Naturally, many different treatments were used in attempts to control and cure these diseases — mostly without conspicuous success. Nevertheless, long before any real insight into the nature of disease existed, some infectious diseases could be controlled by immunisation.
The real breakthrough came in the treatment of smallpox. It had been clear very early that people who had had the disease, and survived, very rarely contracted it again. With this observation in mind, material was taken from the vesicles of mild cases of smallpox and inoculated into people who had not had the disease, in the hope (with fingers crossed) that they would get only a mild local infection, and so gain immunity against the frightful generalised form of the disease which so often resulted in shocking disfigurement and widespread death.
The method of inoculation varied in different parts of the world. In India, children were wrapped in the clothing of smallpox patients, page 96 in Persia, the prepared crusts of the lesions were eaten, and in ancient China, dried crusts of the smallpox scabs were blown down the noses of children through a tube on what was called, with cheerful optimism, a ‘lucky day’. The practice was common in the middle East, and particularly in Turkey, where ladies free from the scars of smallpox could be sold for high prices on the local markets for service in oriental harems (Morris, 1968). In Europe, however, this monetary incentive was lacking, and so the practice was never greeted with wild enthusiasm by the general populace — they apparently considered that the risks of contracting smallpox in the normal way were quite great enough, and that the disease required no assistance whatsoever from the medical profession. Nevertheless, it was a Gloucestershire doctor, Edward Jenner, who developed the first safe immunisation procedure for smallpox, and thus laid the foundations of the science of immunology. The farmers and milkmaids in Gloucestershire during the eighteenth century held a widespread belief that they would never get smallpox because they generally all had cowpox (a very mild infection) instead — hence the song sung by the milkmaid which includes the line
“…‘my face is my fortune, sire’, she said.”
And her face was her fortune, simply because it was not pitted and scarred by smallpox. Jenner checked the observation in a very brief series of experiments, probably the first set investigation in experimental immunology. However, even though these experiments marked a major turning point in the treatment of disease, it is well to remember that the experiments were very scrappily done. In fact, the editors of the Philosophical Transactions of the Royal Society considered that there was not enough evidence for Jenner's sweeping claims, and refused to publish his account. Jenner therefore published his own account, became famous, and spent the rest of his life in controversies over priority and rewards (Burnet, 1963).
The next major contribution was made by Louis Pasteur, who set out to develop effective vaccines against a variety of diseases. His first breakthrough came in the treatment he devised for chicken cholera. The bacterium which causes the disease grows readily in a simple chicken broth, and Pasteur found that the injection of very small doses of pure culture killed off unprotected chickens in very short order. However, when chickens were inoculated from very old cultures which had been allowed to stand untouched for some months, the birds showed very mild symptoms of the disease and then recovered. They were then found to be immune to the virulent culture. Pasteur spent the rest of his career working out similar methods for ‘attenuation’ of other bacteria and viruses, and this approach has continued to yield very satisfactory results up to the present day.
It is clear that this approach is largely one of trial and error. While is has yielded enormous benefits in the treatment of disease, page 97 it has not given any great insight into the essential nature of disease and the response of the body to infection. In fact, it raises more problems than it solves. What is it about a disease-causing organism that causes disease? How does the body react against it? How does the process of attenuation affect the toxicity of the disease-causing organism (or pathogen), and why does the body show immunity to that pathogen after exposure to the vaccine produced by the pathogen's attenuation?
The study of the way in which the body responds to disease has gathered momentum over the last decade to become one of the most active areas of science today. Scientists all over the world are slowly piecing together the jigsaw puzzle of the immune response, with varying degrees of success — the outline is now more or less complete, but many of the pieces are still missing, other pieces show an infuriating obstinacy in their refusal to fit into the pattern at all, and still others insist on fitting into all the wrong places. However, the gaps are slowly filling in, and we know far more about the immune response than we did even five years ago. It is now clear that the basis of the immune response to disease, with all its ramifications and complexities, lies in protein chemistry, and in particular in the chemistry of the antigen-antibody reaction.
Antigens, Antibodies, and the Immune Response
A vertebrate animal is a highly complex entity, consisting as it does of a staggeringly diverse array of enormous molecules, each one tailor-made to carry out a particular function. All these different molecules are intricately dovetailed and integrated into an extremely complex and yet dynamically stable system. It seems obvious that the more complex the system, the greater the number of weak points at which it may be attacked by poisons, by viruses, and by bacteria. Of course, these assaults on the integrity of the body cannot be allowed to go unchecked, and the resultant policing of the body is the responsibility of a very capable immune response system, which has several major functions.
First, it must remove foreign particles, such as dead bacteria, fragments of disintegrated cells, and any small pieces of unwanted miscellaneous flotsam and jetsam found in the body.
Secondly, it must be capable of detecting molecules, and in particular protein molecules, which are foreign to the body. These foreign materials are called ‘antigens’ — the word antigen denotes a substance which, when introduced into the tissues of an animal, will cause the production of antibody molecules which neutralise it. Not all foreign proteins are antigens; gelatine, for example, does not trigger off production of antibody. Conversely, not all antigens are proteins; some large polysaccharides have antigenic effects. The important point here is that invading pathogens carry proteins, foreign page 97 to the host body, as an integral part of their structure. By reacting against the protein of the pathogen, the body reacts against the pathogen itself.
Third, the body must respond to the presence of an antigen by manufacturing an antibody which will deal with that antigen specifically, and with that antigen alone. One of the most characteristic features of the antigen-antibody reaction is the very high degree of specificity involved; for every antigen, there is one, and only one, corresponding antibody type, which is incapable of neutralising any other antigen.
Fourth, after the antigen has been introduced, detected, recognised, and dealt with, the body must retain an immunological memory of the whole ghastly experience, so that if a similar antigen appears in the body at a later date, the challenge can be met much more rapidly and effectively.
The Antigen-Antibody Reaction
Where invasion of the body by a pathogenic organism is concerned, the reaction between an antigen and the antibody which is manufactured to neutralise it is the core of the immune response. This reaction has two important characteristics; first, it is highly specific, and second, it is quantitative — a given amount of antibody will neutralise a given amount of antigen.
One question now arises — just how does an antibody molecule react with an antigen to neutralise it?
At this point it is profitable to compare antibodies with enzymes. The activity of an enzyme is determined by its ‘active site’, an area on the surface of the protein molecule which has a definite configuration, and which is designed to ‘lock on’ to a complementary active site on the surface of the molecule with which the enzyme reacts (the substrate molecule). These active sites may be very small, with only two or three amino acids involved out of the many hundreds which make up the molecule, but it is on the precise shape of the active site that the activity of the enzyme depends. Every protein consists of a chain or chains of amino acids, arranged in a definite, predetermined order. The sequence of amino acids in the chain determines the shape of the molecule, and the shape of the molecule determines its properties. The two or three amino acids which form the active site need not be sequential along the chain. For example, in the enzyme alpha—chymotrypsin, the three amino acids involved in the active site are serine, methionine, and histidine. The histidine is on one chain, while the methionine and serine are on another. However, in three-dimnsional orientation the three are close together; they are brought together by folding and coiling of the protein molecule so that they form an active site on its surface (Koshland, 1963).page 99
It appears that the active sites of antibodies are rigidly preformed in much the same way, and operate in much the same way. Antigen molecules also owe their activity to similar active sites on their surfaces. It is these active sites, otherwise known as ‘haptens’ or ‘antigenic determinants’, which the antibody molecule is manufactured to deal with. A hapten, or antigenic determinant group, can be defined as that specific chemical grouping to which a single antibody site conforms and with which it reacts. It is obvious here that an active site which consists of only two or three amino acids on the surface of an antibody can combine only with a determinant group on the antigen which is much the same size and of exactly complementary configuration.
When the nature of these combining sites is taken into account, the specificity of the antigen-antibody reaction becames understandable. Obviously a very wide variety of different hapten configurations is possible, and for each configuration there is only one possible complementary shape for the active site of the antibody. It thus follows that an antibody which is manufactured to neutralise a specific antigenic determinant cannot have any effect on a different antigen.
This concept also explains the quantitative aspect of the antigen-antibody reaction. One antibody active site will neutralise one antigenic determinant — as long as an antigenic deteminant occupies an antibody site, no other antigenic determinant can occupy the same site.
It must always be remembered that the active sites are very small areas compared with the molecule as a whole. Nevertheless, it is these minute active sites which enable the antibody molecule to grasp, fasten onto, bind, and neutralise the corresponding antigen.
The sizes of these active sites have been estimated, and they are minute. They range from a minimum size of 100 Angstrom2 to a maximum size of 2000 Angstrom2 (Day, 1966). (To give some idea of the size of an Angstrom unit — facial hair grows at the rate of about 30 Angstrom units per second).
The Structure of Antibodies
When an antigen is detected, and antibody production starts, the first antibody molecules produced are fairly large. During the early stages of the response the animal produces an antibody molecule with a molecular weight around the 1 million mark, but after a while it switches over to production of a lighter version of the same antibody with a molecular weight of only 150,000. The heavy and light versions of the antibody are respectively referred to as 19S and 7S gamma globulin; the ‘gamma globulin’ refers to that fraction of the blood proteins in which antibodies are found, and the S refers to Svedburg units, which indicate the rate of sedimentation of a page 100 molecule in a centrifugal field, thus indicating its size. The switchover from 19S to 7S gamma globulin apparently occurs in each individual cell which is manufacturing antibody (Nossal, 1964).
The general structure of the antibody molecule has been determined in some animals. For example, rabbit antibody molecules can be broken down into four parts—four straight amino acid chains which are linked together in the complete molecule by disulphide bonds and hydrogen bonds. These chains come in two pairs — a pair of long, heavy chains each of molecular weight 50,000, and a pair of shorter chains, each of molecular weight 25,000. The two heavy ‘A’ chains are identical, and the two lighter ‘B’ chains also match each other. The A chains lie alongside each other, and the B chains flank the paired A chains on either side at one end of the molecule. Each antibody molecule has two identical active sites, carried at the end of the molecule which has the two B chains. This can be inferred from the finding that it is possible to break the A chains halfway along their length with suitable enzymes; this gives two fragments, one of which is inactive, while the other (which carries the two B chains) retains its activity unimpaired. When this fragment is split down the middle by breaking a single disulphide bond holding the A chains together, the resulting two fragments (each consisting of a B chain and half an A chain) can each neutralise on antigen molecule (Nisonoff and Thorbecke, 1964).
To sum up this far, then, the following sequence of events applies. A pathogen which carries antigen molecules enters the body. The antigens are detected by the body's immune response system, and antibody molecules specific for that particular antigen are produced. The antibody molecule carries two identical active sites, which may be contributed to by both the A and B chains of the molecule. Each of these active sites is capable of attaching itself to the antigenic hapten, or determinant group, and thus neutralising the antigen. This action is the core of the immune response, and it is now time to see how it is accomplished.
The Lymphatic System
The arterial blood pressure in mammals is much higher than it is in the earlier, superseded models such as the amphibians. This high blood pressure enables the animal to operate much more efficiently, and confers major advantages, such as more efficient respiration, more economical elimination of waste products, and so on. Nevertheless, the increase in arterial blood pressure does introduce some important problems, which have to be solved. Due to the high pressure of blood in the arteries, a certain amount of fluid, together with some plasma proteins, leaks out through the capillary walls into the body tissues. Obviously, this fluid must be returned to the bloodstream, or the tissues will become flooded and page 101 massive oedematous swelling will result. With blood pressure increasing throughout the evolutionary history of the vertebrates, this problem was solved as it arose by the parallel evolution of the lymphatic system. In essence, the lymphatic system consists of a system of blind-ending, thin-walled tubes, which drain fluid from the tissues and return it to the bloodstream through either the thoracic duct or the right lymphatic duct in the neck. In mammals the movement of fluid in the system is aided by movement of muscles adjacent to the lymphatic vessels, and the vessels are equipped with valves to prevent backflow.
At intervals along the course of these lymphatic vessels, there are well-defined structures called lymph nodes. The fluid in the lymphatic vessels passes through the nodes on its way to the thoracic duct.
These lymph nodes are intimately concerned with the immune response. However, it is important to remember that the immune response system operates on the cellular level, rather than on the organ or organ system level. The cells which mediate the immune response show an extraordinary degree of flexibility, and many are very mobile. A number of different cell types wander through the blood and the body tissues, while others stay anchored in the lymph nodes and emerge into the blood stream only as the result of unusual pathological conditions.
In human blood, two nucleated cell types, or leucocytes, are predominant. These leucocytes are, of course, heavily outnumbered by the enucleate red blood cells, or erythrocytes, but these cells do not enter into immune reactions. By far the most numerous leucocyte type in human blood is the neutrophil — the nucleus in this cell is an elongate, bent or twisted body with several lobes, and the cytoplasm contains a large number of small granules. These cells are phagocytic; their primary function appears to be the engulfing and subsequent destruction of any foreign particles which appear in the blood or tissues. The other numerous cell type (20-25% of the blood leucocytes in man) is the lymphocyte. The lymphocyte is a spherical cell, with a large, nearly spherical nucleus surrounded by a thin rim of cytoplasm; the cytoplasm normally contains no granules. It is this cell type which has been attracting most attention over the last few years, as far as the immune response in concerned. Other leucocytes are found in the blood, but these are not numerous and their functions are not yet clear.
Situated in the lymph nodes are cells which look like larger versions of the lymphocyte. These ‘plasma cells’ have a greater amount of cytoplasm than do the lymphocytes, and this cytoplasm is well endowed with endoplasmic reticulum. This indicates that these cells are well equipped to synthesise large quantities of protein. Plasma calls almost never leave the lymph nodes. The lymphocytes, on the other hand, circulate freely around the body, through the page 102 blood stream, into the tissues, and back into the blood stream through the lymphatic system (Gowans, 1962).
One striking feature of the lymphocyte is that it appears to have a very long lite span — a life span of 10 years has been established for it, and it could well be even longer (Buckton and Pike, 1964). This indicates that the lymphocyte is in a resting state in its normal condition.
Keeping this background in mind, we can now look at the typical course of events following an antigenic challenge.
Stages of the Immune Response
In any response by a mammal to an antigenic challenge, there are a number of well defined stages, each marked by easily-recognisable changes in the lymphatic system at the cellular level. The first stage of the response centres around recognition of antigen. This appears to be the responsibility of the lymphocytes, but there is also strong evidence that a cell population resident in the lymph nodes is also capable of recognising antigen. When an antigen is injected subcutaneously, the first sign of a response by the host animal is an almost immediate and startling drop in the number of lymphocytes leaving the lymph node which drains the infected area. The lymphocytes detect the antigen, move to the lymph node, and remain there instead of passing through (Hall and Morris, 1965). The cell count in the lymph leaving the node may remain low for as long as 12 to 24 hours, before rising again. The mechanism which causes this unusual lymphocyte behaviour pattern is not yet certain, but it certainly has something to do with antigen recognition. The lymphocyte is a resting cell, inactive but pregnant with celestial fire, and contact with antigen stimulates it to give birth to its full potential. This process of antigen recognition applies also to some of the cells in the lymph node, and in allied structures such as the thymus gland, and these cells show significant changes in the nucleus which are associated with gene activation (Black and Ansley, 1965; Agrell and Molander, 1969). Lymphocytes are also capable of making the same type of response to an external stimulus (Burton, 1968). These changes are first detectable in the histone protein of the nucleus, and this is significant because it is highly likely that the function of histone is to mask those parts of the genetic code which are not required by a particular cell. Obviously, if a cell is going to undergo a marked change in behaviour, genetic information must be made available to it to control the change. Significantly, this change in histone protein is promptly followed by increased RNA synthesis, increased protein synthesis, and consequent growth of the cell leading ultimately to division.
Just how an antigen causes these cellular changes is not yet known. Nevertheless, antigen recognition is a recognisable page 103 phenomenon, and there is little doubt that the circulating small lymphocyte is intimately concerned with this recognition phase.
The next phase in the immune response is one of lymphocyte recruitment. About 24 hours after the antigen makes its first contact with the lymph node, the cell content of the lymph begins to increase. This increase enables the body to bring an enormous number of immunologically competent cells to bear on the antigenic concentration.
This phase of lymphocyte recruitment paves the way for the third stage. At the end of the first 72 hours, the plasma cells in the lymph nodes go into a phase of very active growth and division. A lymph node at this stage is a seething mass of actively dividing cells.
Antibody synthesis on a large scale commences at about this time. Most of the antibody is produced by the plasma cells resident in the lymph nodes — cells which are well equipped with endoplasmic reticulum, and which ordinarily remain in the lymph nodes and never enter the blood stream.
In the lymph, however, a population of cells appears which is capable of synthesising antibody. These are unlike the plasma cells in that they do not have a well defined endoplasmic reticulum (Cunningham, Smith and Mercer. 1966). At the height of the immune response there may be as many as 20,000,000 of these antibody-forming cells being carried away in the lymph every hour from the node where they were formed. However, it may well be that antibody manufacture is not their most important function. It appears that these cells are able to initiate immune reactions in other lymph nodes in the absence of antigen. This mechanism allows all the lymph nodes in the body to become involved in an immune response, thus enormously amplifying the response itself (Hall, Morris, Moreno, and Bessis, 1967).
By this time, any self-respecting pathogen is beginning to wonder if it has bitten off more than it can chew. In fact, as a result of all this frenzied activity on the part of the lymphatic system, the antigenic challenge may well be beaten off. However, this is by no means the end of the story. One of the most important properties of the immune response system is its capacity to respond to a second attack of a particular antigen much more rapidly and effectively than it responded to it the first time. It is clear that some memory of the first, or primary, response is retained by the lymphoid cells after the antigenic challenge is over; the presence of this immuno-logical memory allows the response to a second challenge by the same antigen to be rapid, massive, and effective. It is this property of the immune response which makes immunisation so successful. The vaccine is a very weakened version of the real thing, and it can easily be dealt with by the relatively weak primary immune response. Consequently, when the immunised body is exposed to the full-blooded page 104 pathogen, it is capable of giving it a very hostile reception, and the infection is never allowed to become established.
The nature of this immunological memory is not at all clear, but it appears to be twofold. There is plenty of evidence to show that the circulating small lymphocyte is responsible for a major part of this immunological memory, but there appears to be a sedentary population which has this ability as well. The nature of this ‘residential’ memory is not certain, but for some time after a primary challenge, a stimulated node can respond more vigorously and rapidly than one where antigen had not previously been encountered. (Morris, 1968).
Problems and Applications
There are, of course, a number of problems which have not yet been solved. The precise relationship between lymphocytes and plasma cells is not known, although it seems likely that the lymphocytes, once the antigen is recognised, pass relevant information to the plasma cells. The precise nature of this information transfer is not known, but there is some evidence that RNA is involved (Hashem, 1965). In any case, it seems very likely that antigen, as such, never comes in contact with the antibody-forming cells at all, and the circulating small lymphocytes may act in an intermediary role.
There are other problems as well. So far in this article, only the response to pathogenic organisms has been discussed. However, immune reactions of other types do occur, one being graft rejection. In this reaction, antibody synthesis (if it occurs) appears incidental to some cellular effector mechanism, although all the cellular steps listed above take place in the same way.
Nevertheless, our present understanding of the workings of the lymphatic system has allowed us to make some advances in medical practice which were not possible ten years ago. Knowledge of the steps involved in the immune response, and of the cells which mediate the response, is vitally important in transplant surgery. A transplanted organ is obviously foreign protein introduced on a massive scale, and the body deals with it accordingly. As far as the surgeon is concerned (not to mention the patient) the process of rejection which results must be stopped or minimised. This can be done, at the moment, in two ways.
First, accurate tissue matching is important. If a donor can be found whose proteins match those of the recipient fairly closely, the risk of rejection is reduced. This has proved very successful in kidney transplantation, so much so that now 90% of transplanted kidneys survive at least five years, and the figures are improving all the time.
Second, it is possible to impair the immune response system by injecting antilymphocyte serum (A.L.S.). This serum is produced by injecting human lymphocytes into a horse, or similar animal, page 105 allowing the primary response to pass, and then giving the animal a second dose. The animal thereupon develops a massive secondary immune response against the injected lymphocytes. The anti-lymphocyte factor is isolated from the horse blood, purified in various ways, and then injected into the potential transplant patient. This promptly disposes of his lymphocytes, and at this point the transplant is made. Without his lymphocytes, the recipient is incapable of recognising the transplanted organ as foreign—his circulating antigen-recognition system is virtually non-existent, and periodic injections of ALS make sure that it stays that way.
However, there are major disadvantages involved in the use of this technique.
First, it reduces resistance to disease, in particular viral diseases and those diseases caused by the smaller bacteria. However, resistance to disease is not lost altogether, as the residential immunological memory is still intact.
Second, the technique is not completely effective — some lymphocytes survive, and they commence rejection of the transplant.
Third, the ALS is not pure, and some side effects show up — not only the lymphocytes are attacked by ALS.
Fourth, this type of treatment appears to make the lymphatic system more susceptible to cancers after a time.
It seems possible, however, that some progress will be made in another direction. It is possible to induce a degree of tolerance to introduced foreign materials. The combined use of induced tolerance, ALS, and accurate tissue typing probably holds out the best hope for successful long term organ transplantation in the foreseeable future.
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