Immunological studies of Crohn's Disease.

Introduction

Immunology is an immensely complex field. The human immune system is the result of hundreds of millions of years of evolution. Humans, and their ancestor organisms, have always been in a permanent state of biological warfare with other organisms in their environment. This escalating "arms race" has led the human body to develop a vastly complex armory of immune cells and chemicals, and to develop an enormous array of tactics and strategies to deal with ever-mutating microorganisms that try to invade and infect the body.

Although, in recent years, great strides have been made in understanding the human immune system, mostly as a result of the development of complex genetic, chemical, and information analysis techniques, the human immune system is still poorly understood. This is true in relation even to the most common of diseases, such as the common cold, flu, hepatitis, etc.

It is beyond doubt that the immune systems of Crohn's patients are very different from those of normal people, though it is still unclear whether this is a cause or a result of the disease.

Micro-organisms that have evolved multiple strategies to evade the human immune system, and thus have a highly complex interaction with it, are among the most poorly understood. Such organisms include viruses, such as HIV (Human Immunodeficiency Virus), and mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae. The fact that all of these micro-organisms are constantly mutating and evolving further complicates matters.

Given all of these facts, it is no surprise that the interaction between the human immune system and Mycobacterium paratuberculosis is very poorly understood. Johne's disease, or Mycobacterium paratuberculosis in animals, is poorly understood, even though the bacterium can be isolated from infected animals in their billions. Also, inadequate funding of research has led to poorly designed and executed studies, which add further to the controversy.

In the words of Dr. RJ Chiodini, in the debate M. paratuberculosis in Foods and the Public Health Implications, held at the 5^th International Colloquium on Paratuberculosis in Madison, Wisconsin, September 29-October 4, 1996

If M. paratuberculosis has an etiologic relationship to Crohn's disease, an immunologic response to this agent should be present: Crohn's disease is defined as an immunologic hyper-responsive condition to an exogenous and/or endogenous antigen. There have been many reports on the immunologic responses of Crohn's disease patients to M. paratuberculosis and/or mycobacterial antigens in general (Table 3). About half of these studies suggest that there is a significant response in Crohn's disease patients while the other half refute these findings. The variety of methodologic and immunologic explanations that may account for the failure to demonstrate an immunologic response in Crohn's disease patients greater than that found in controls have been reviewed elsewhere. Studies that have investigated immune responsiveness to mycobacteria in Crohn's disease have as a whole, lacked rigorous design and standardization. Clinical design (including patient selection), non-state-of-the-art methodologies, and interpretation and analysis of data have been poor or potentially biased. Well designed studies using appropriate methodologies are necessary to determine whether an immune response to M. paratuberculosis is present or absent in Crohn's disease

What are immunological studies trying to achieve?

Essentially the purpose of immunological studies is as follows. If the body has been infected by a bacterium, then there could be a reaction to that bacterium by the immune system. To detect whether or not the bacterium is present in the body, you do not look directly for the organism, but instead look for the immune reaction to it, in the form of antibodies.

Every bacterium presents antigens (proteins) to the body. The immune system produces antibodies to that antigen. Each antibody is able to react only to one specific antigen. Picture an antigen as a lock, and an antibody as a key that fits only that lock. If you look in the blood of the diseased host, and find the "keys", then probably the "locks" are there too, or have been there in the past.

Bacteria present many antigens, all different, corresponding to many different "locks". The immune system needs only to successfully find the structure of one of those "locks" to be able to deal with the bacterium. The purpose of the Acquired Immunity system is to find the structure of bacterial "locks". While it is likely that the immune system will find "keys" to many of the "locks" on invading bacteria, it is unlikely that it will find "keys" to all of the "locks". Thus, it is important that if a study looks for an immune reaction to a particular antigen (protein), then it must be certain that immune system is capable of finding the antibody to that antigen.

Shared antigens between mycobacteria.

All mycobacteria are related to some degree, meaning that many of the antigens ("locks") for Mycobacterium paratuberculosis are shared with other mycobacterial species, such as Mycobacterium tuberculosis, and other environmental mycobacteria. Much of the population of developed countries has been vaccinated against Mycobacterium tuberculosis, using the BCG vaccine. Vaccinated persons have already been exposed to all tuberculosis antigens, which means they have also been exposed to whichever paratuberculosis antigens are shared with tuberculosis. If an immunological study looks for an antibody response to a paratuberculosis antigen that is shared with tuberculosis, then both Crohn's and "control" subjects will test positive for that antigen, if they have been vaccinated for tuberculosis. This phenomenon is known as cross-reactivity.

Immune interference.

Another complicating factor is that some mycobacterial species, for example Mycobacterium leprae, are known to interfere with the action of the immune system, thus preventing the immune system from reacting in the expected way. Research to find whether Mycobacterium paratuberculosis is capable of producing similar interference in humans has been inconclusive.

What is known is that the immune reactions of animals to Mycobacterium paratuberculosis is different for each individual animal. Factors which affect the nature of each animals immune reaction are

• Genetic makeup. Some animals seem not to be affected by Mycobacterium paratuberculosis at all, while others rapidly develop a fatal clinical disease condition. One possible explanation for this difference between animals is that some animals have a genetic make-up that predisposes them to developing disease. It must be remembered that the disease symptoms and tissue damage of animals infected with Mycobacterium paratuberculosis are a result of the hosts immune reaction to the bacteria, and do not result from the bacteria producing toxins or consuming host tissue.
• Number of infecting bacteria. Whether or not an individual animal develops a clinical disease condition from a Mycobacterium paratuberculosis infection depends also on the number of bacteria with which the animal is infected. Although only a small number of bacteria is necessary to establish an infection, an infection by a number of infecting bacteria less than this "Minimum Infectious Dose" may be successfully be brought under control by the infected host. The value of this "Minimum Infectious Dose" for Mycobacterium paratuberculosis is unknown, and almost certainly varies from animal to animal.
• Age at time of infection. The younger an animal is at the time of first infection with Mycobacterium paratuberculosis, the more likely it is to develop a clinical disease condition from that infection. Conversely, the older an animal is when first infected, the less likely it is to develop a clinical disease condition.

What is clear is that the ability of Mycobacterium paratuberculosis to cause an infection in a host is dependent on the ability of the host to mount a successful immune reaction to the bacterium. The ability or inability of Mycobacterium paratuberculosis bacteria to interfere with the hosts immune reaction is most likely the factor which determines whether or not the host is able to successfully control the infection.

Autoimmunity.

Some bacterial antigens(proteins) are identical to some proteins that exist in the human body. If the immune system produces antibodies to these antigens, then these antibodies will also react with those proteins of the human body, mistaking them for invading bacteria. This phenomenon is known as autoimmunity. In some people, the immune system does not allow such autoimmune antibodies to exist, because they would damage the body, and destroys them. This may leave those people with no defence against the invading bacteria. Other peoples immune systems allow these antibodies to be formed and to proliferate. This means that they have a defence against the invading bacteria, but will also suffer inflammatory autoimmune symptoms, such as arthritis, uveitis, etc. in sites that may be far away from the main site of infection.

Polar manifestations of mycobacterial diseases.

As discussed on the page "Immune reactions to mycobacteria", mycobacterial diseases present symptoms that fall into two main groups: the controlled and aggressive forms.

• In the controlled form, the sufferer may display only a CMI or T cell reaction.

• In the aggressive form, the sufferer may display only an humoral or B cell reaction.

It has been proposed that Crohn's disease also presents in two polar forms, the aggressive perforating and contained nonperforating forms. See the page "The polar manifestations of mycobacterial disease and Crohn's disease" for more details. It is expected that the subset of Crohn's patients that has the contained nonperforating form will display a "Cell Mediated Immunity" (CMI) reaction to the bacterium, but no "humoral" immune reaction. Those patients with the aggressive perforating form will display an "humoral" reaction, but no CMI reaction. Since most of the studies below look for either a CMI reaction or an humoral reaction, but not both, they are restricting themselves to a partial view of the immune reactions of Crohn's patients. Hence when results are analysed by statistical methods, different immune reactions will be grouped together, most likely giving incorrect or unclear results, or results that indicate partial proof of the hypothesis.

What is known about animal paratuberculosis is that the nature of the host immune response not only differs from animal to animal, it also changes in each individual animal over time. From "Ruminant paratuberculosis:- Current status and future prospects"

Infected animals may experience a cell-mediated response, a humoral response, both a cell mediated and humoral response, or anergy. Initially, there is a cell-mediated response. Later, as the disease progresses, there is a humoral response initiated by the release of bacilli by dying macrophages. There is generally an inverse relationship between cell-mediated and humoral immunity. Early infections are manifested by a cell-mediated immune response (tuberculoid), and as the disease progresses toward the lepromatous stage, humoral immune responses predominate. Ultimately, anergy is reached and cell-mediated and humoral responses can no longer be detected. These immunologic responses occur independent of clinical features, and may appear at any time during the clinical spectrum. The mechanism of anergy during chronic paratuberculosis has not been thoroughly investigated, but may result from the release of soluble factors from macrophages, similar to the proposed mechanism in lepromatous leprosy. A humoral factor that suppresses lymphocyte transformation in vitro has been described in animals chronically infected with M. paratuberculosis.

Mycobacteria can hide their antigens.

Another complication is that pathogenic mycobacteria like to live inside the infected hosts immune cells (macrophages), where they are safe from the attentions of antibodies which can recognise them, i.e. they are not presenting antigen while they are in "sanctuary" inside macrophages.

Also, the antigens presented by spheroplast (this form is "cell wall deficient", i.e. it has no cell wall) forms of mycobacteria are different from the antigens presented by bacillary form ("cell wall intact") forms, because the proteins that make up the center of the bacterium are different from those that make up the cell walls of the bacterium. It is theorised that Crohn's disease is caused by spheroplast forms of M. paratuberculosis.

Antigen selection and preparation.

When selecting a Mycobacterium paratuberculosis antigen to be tested, the following must be fulfilled.

1. The antigen must be unique to Mycobacterium paratuberculosis and not cross reactive with other species of mycobacteria that the patients may already have been exposed to, such as Mycobacterium tuberculosis, Mycobacterium avium or Mycobacterium phlei.

2. The antigen must be one that is immunogenic, i.e. will produce a reaction at least in healthy patients, if not also in sufferers of the disease.

There are many antigens chosen for the studies below. The preparation of antigen falls into two main groups.

• One or more individual antigens. Individual proteins from the target organism are selected, and produced in quantity. This option runs the risk of using a protein that is not immunogenic, i.e. does not elicit any immune response in humans.

• By using dead, whole organisms that have been killed and broken apart, so that all antigens are presented. It is likely that a substantial proportion of these antigens will be cross-reactive with related species of bacteria. Purified Protein Derivative or PPD consists of just such "killed" organisms, as does the BCG vaccine for tuberculosis.

Positive studies.

The following studies all concluded that Crohn's patients had significant immune reactions to the Mycobacterium paratuberculosis antigens tested.

• This paper was published in December 1984. "Possible role of mycobacteria in inflammatory bowel disease. II. Mycobacterial antibodies in Crohn's disease."

• This paper was published in February 1991. It finds clear evidence for immune interference by M. paratuberculosis in humans. "Induction of suppressor cells by Mycobacterium paratuberculosis antigen in inflammatory bowel disease."

• This paper was published in December 1992. It describes finding an elevated antibody response to M. paratuberculosis in 53% of Crohn's patients studied. "Antibodies to Mycobacterium paratuberculosis-specific protein antigens in Crohn's disease."

• This paper was published in March 1994. This study finds that all control patients respond in the same way to M. paratuberculosis antigens (" a monomodal distribution"). However, it finds that the Crohn's patients split into two main groups ("a bimodal distribution"), with 36% having a significantly higher immune response to M. paratuberculosis than controls. "Occurrence, in Crohn's disease, of antibodies directed against a species-specific recombinant polypeptide of Mycobacterium paratuberculosis."

• This paper was published in September 1995. This study finds a significantly higher immune response to M. paratuberculosis in 64.7% of Crohn's patients, as compared with healthy control patients. It also tests five Crohn's patients who underwent surgery for removal of diseased bowel, and finds a dramatic reduction in immune response to M. paratuberculosis 180 days after surgery. "Effect of intestinal resection on serum antibodies to the mycobacterial 45/48 kilodalton doublet antigen in Crohn's disease."

• This paper was published in November 1995. This study investigates the immune reaction to mycobacterial antigens thought to induce autoimmunity in humans. It finds that a subset of Crohn's patients exhibits "a high intensity antibody reaction" to M. paratuberculosis. "Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis."

Inconclusive studies.

The following studies concluded that although Crohn's patients exhibited an unusual or abnormally high immune reaction to Mycobacterium paratuberculosis, this reaction was not sufficiently strong to conclude that Crohn's patients had an immune reaction to Mycobacterium paratuberculosis, i.e. the data were "statistically insignificant".

• This paper was published in December 1991. This study shows "unexpected positivities for mycobacterial antigens" in a small subset of inflammatory bowel disease patients, but concludes that these are statistically insignificant. "Antibodies to Mycobacterium paratuberculosis in patients with Crohn's disease.".

• This paper was published in February 1992. This study finds no evidence of an immune response to mycobacterial antigens in inflammatory bowel disease patients, but states that an anergy reaction cannot be excluded to explain this. The population size of the study is not mentioned. It concludes that it is likely that some form of bacterial infection is partly responsible for inflammatory bowel disease. "Mucosal cell-mediated immunity to mycobacterial, enterobacterial and other microbial antigens in inflammatory bowel disease."

• This paper was published in March 1992. "Antigen induced suppression in peripheral blood and lamina propria mononuclear cells in inflammatory bowel disease."

• This paper was published in March 1993. This study finds that 25% of Crohn's patients had a significantly elevated immune response to M. paratuberculosis, but concludes that there is no statistically significant difference between patient and control groups. "Antibodies to Mycobacterium paratuberculosis and nine species of environmental mycobacteria in Crohn's disease and control subjects"

• This paper was published in 1994. "Humoral response to mycobacteria in patients with Crohn disease".

Negative studies.

The following studies reach the conclusion that there is no evidence for a significant immune reaction to M. paratuberculosis in patients with Crohn's disease.

• This paper was published in April 1988. "No evidence for antibodies to mycobacterial A60 antigen in Crohn's disease sera by enzyme-linked immunoabsorbent assay (ELISA)."

• This paper was published in May 1990. This study searches for a CMI (T cell) response to Mycobacterium paratuberculosis in each of three groups, one of Crohn's disease patients, one of ulcerative colitis patients, and one of control patients, into two subsets: those with an immune response (responders) and those without (non-responders). Since there is no statistical difference in the ratio of responders to non-reponders between groups, it concludes that there is no role for mycobacteria in the etiology of inflammatory bowel disease. "T-cellular immune reactions (in macrophage inhibition factor assay) against Mycobacterium paratuberculosis, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium avium in patients with chronic inflammatory bowel disease."

• This paper was published in January 1991. This study describes the search for an humoral (B cell) immune response to Mycobacterium paratuberculosis. It finds a "wide variation" in the strength of this immune response in Crohn's patients, but no data that are statistically significant. "Mycobacterium paratuberculosis and Crohn's disease."

• This paper was published in December 1995. This study tests for immune reaction to M. paratuberculosis and M. tuberculosis in patients with Crohn's disease, ulcerative colitis and control patients. It uses "whole, heat-killed bacteria" as antigen. It finds evidence for a reaction to both in "virtually all" patients, with no statistically significant difference between groups. It finds a high correlation between patients reactions to M. paratuberculosis and patient reactions to M. tuberculosis, i.e. patients tended to react to both or to neither. "Peripheral cell-mediated immune response to mycobacterial antigens in inflammatory bowel disease."

• This paper was published in March 1996. This study searches for an humoral (B cell) immune response to a recently discovered protein that is highly specific to M. paratuberculosis. It finds no "statistically significant" difference between patients with inflammatory bowel disease and control patients. It also finds no statistical correlation between Crohn's disease activity (Harvey-Bradshaw index) and immune response to M. paratuberculosis. "Antibodies against Mycobacterium paratuberculosis in Crohn's disease."

Source: http://archive.crohn.ie/immunol.htm
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