Paper 11
SPONDYLOARTHROPATHIES
ANKYLOSING SPONDYLITIS IS CAUSED BY KLEBSIELLA
Evidence from Immunogenetic, Microbiologic, and Serologic Studies
Alan Ebringer, BSc, MD, FRCP, FRACP
From the Division of Biomolecular Sciences, King's College, University of London; and Department of Rheumatology, UCM School of Medicine, Middlesex Hospital, London, England.
DEFINITION OF THE PROBLEM: THE LINK BETWEEN KLEBSIELLA AND ANKYLOSING SPONDYLITIS
Ankylosing spondylitis (AS) is caused by Klebsiella pneumoniae infection. The reason for proposing this particular bacterium, as the causative agent of this disease, is that specific antibodies to this microbe, but not to twelve others, have been demonstrated by two groups, initially by us in England63-44 and subsequently by another group in Finland."
Patients suffering from AS, during active phases of the disease, as defined by elevations in acute phase reactants, have specific antibodies to Klebsiella pneumoniae, but not to other bacteria, such as:
Borrelia burgdorferi
Campylcbacter jejuni
Chlamydia trachomatis
Escherichia coli
Proteus mirabilis
Pseudomonas aeruginosa
Salmonella typhimurium
Shigella sonnei
Staphylococcus aureus
Streptococcus pyogenes
Yersinia enterocolitica
or the yeast Candida albicans.
It was in 1976, that for the first time, an association was suggested between the gram-negative microbe Klebsiella and the chronic, remitting inflammatory arthritic disorder, AS.22
Using three separate approaches, involving immunogenetic, microbiologic, and serologic criteria, this article reviews the background and evidence that links the presence of Klebsiella to the development of recurrent inflammation in AS patients, especially during active phases of the disease. If an association can be clearly established between Klebsiella and AS, then this has important therapeutic implications. It could provide a novel approach to treating or at least controlling severe and active inflammatory phases of the disease, especially in younger patients, before deformities or severe ankylosis of the spine has developed, thereby avoiding or preventing the severe sequelae of AS. Furthermore, a therapeutic response, would by itself, have important theoretical implications in that it could provide additional evidence that Klebsiella is the causative agent of this disease.
The presence of specific anti-Klebsiella antibodies in AS patients, during acute phases of the disease, clearly indicates that these patients have recently been exposed to these specific bacteria, and the speculative proposal is being made that the consequent immune response leads to secondary pathologic complications of "reactive arthritis," especially in a genetically susceptible host, such as one carrying HLA-B27." The test of this theory is clear and relatively simple: removal of anti-Klebsiella antibodies. Elimination of the implicated bacteria, using antibiotics or whatever means, should lead to reduction in inflammation, which can be assessed readily by a drop in levels of acute phase reactants.
If the level of acute phase reactants drops, then the clinical question would arise, whether elimination of specific antibacterial antibodies would lead to patient improvement and arrest in the progression of the disease. It is evident that damage that had already occurred, such as arthritic deformities, contractures, or "bamboo spine," could not be altered by such therapeutic procedures. The clinical end-point that would be expected if this theory is correct is that no new damage would occur in AS patients when specific anti-Klebsiella antibodies had disappeared and acute phase reactants had returned to normal levels.
If the disease continued to progress despite the disappearance of the specific antibodies following antibiotic or dietary therapy, then this would indicate that the specific antibacterial antibodies are interesting epiphenomena occurring in AS, but the bacteria evoking these responses are not the causative agents of this disease.
HISTORICAL BACKGROUND
Although an arthritic skeleton from presumably an AS patient was first described by Connor, in Paris, in 1692, it was not until the end of the 19th century that AS was considered as a distinct inflammatory disorder of the spine and large joints by European physicians such as Bechterew in Russia, Fagge in England, Marie in France, and Strumpell in Germany.10 In the American literature, this condition was thought to be a variant of rheumatoid arthritis (RA) and known as "rheumatoid spondylitis." It was not until the publication of the Rome and New York criteria in 1961 and 1966, respectively, that consensus was achieved, and AS recognized as a distinct entity, quite separate from RA.
Within a few years, however, this clinical separation of AS from RA was eminently confirmed from an entirely unexpected quarter, when it was demonstrated by American57 and European' workers that both diseases occur more frequently in individuals carrying particular HLA antigens. HLA-B27 was found to occur more frequently in AS patients, whereas a significant proportion of RA patients subsequently were found to carry HLA-DR4,5'-60 compared to the frequencies of these HLA antigens found in the healthy, control population. These HLA associations have altered significantly our perception of rheumatology in that no viable model of either of the two diseases, AS or RA, can be proposed without, at the same time, providing some explanation for these immunogenetic observations. Can these immunogenetic observations, such as HLA-B27 in AS, be linked to gram-negative bacteria, such as Klebsiella?
HLA-B27 AND GRAM-NEGATIVE REACTIVE ARTHRITIS
It is the association between HLA antigens and arthritic complications occurring after enteric infections that has provided the vital clues to identifying the specific bacteriologic agents involved in AS and related diseases. The three main gram-negative microorganisms associated with reactive arthritis following enteric infections are Salmonella, Shigella, and Yersinia, and most of the affected patients are carriers of HLA-B27.3 These observations provide a possible clue to any putative infectious agent that may be involved in AS. The agent could probably be a gram-negative microbe, related to Salmonella, Shigella, or Yersinia, but not having such overt enteropathic effects. Such a microbe could readily be a member of the normal, commensal bowel flora. Thus it would not be that unexpected for AS to also be a form of reactive arthritis and the microbe should be related to the enteric pathogens, causing HLA-B27-associated reactive arthrindes.
Because reactive arthritis is also present in Reiter's syndrome and rheumatic fever,40 as well as in gram-negative reactive arthritis,2-62 to entertain the possibility that a similar process operates in either AS or RA is not such a novel or revolutionary concept and quite compatible with existing pathologic precedents and examples; however, it should be apparent that the concept of "reactive arthritis" breaks one of Koch's postulates for incriminating a microbe as the cause of an infection. Clearly, the causative microbe is not to be found at the sites of pathologic lesions but somewhere else, and it is immunologic cross-reactivity mediated by antibodies and immunologically competent cells that cause the secondary tissue damage, characteristic of "reactive arthritis" or rheumatic fever. Thus if AS and RA are forms of reactive arthritis, then it would explain why despite an intensive search for causative agents in the joints and synovial fluids over the last 100 years none has so far been unequivocally identified. The concept of reactive arthritis, when applied to AS and RA, predicates that no viral or bacterial agent will be found in the arthritic joints of AS and RA patients, an expectation more or less in agreement with current negative observations.
Where should one look for such a microbiologic trigger factor? In rheumatic fever, the Streptococcus is located in the tonsils and upper respiratory tract; in Reiter's disease, the agent is in the urethra, and in Salmonella. Shigella, and Yersinia reactive arthritis, the microbe is found in the gut. A possible answer to the question of where one should look for the putative viral or bacterial agent may be provided by the immunogenetic dimension-the association of HLA-B27 with AS.
HLA-B27-THE IMMUNOGENETIC DIMENSION
It is well established that HLA-B27 is associated with AS in all racial groups examined, whether they be European, Japanese, or Asians from the Indian subcontinent.41-42 Furthermore, AS is rare in groups in which HLA-B27 is almost absent, such as the African blacks, and many years ago, Morris Ziff6* noted the lower prevalence of AS in American blacks. By contrast, in ethnic groups in which the frequency of HLA-B27 is found to be above 15% or 20%, AS is a common, almost endemic disease, as observed in Eskimos and Athabascan Indians of North America. The highest prevalence of definite AS is observed among Haida Indians, who have the highest frequency of HLA-B27; 50% of the Haida population have HLA-B27 and virtually 100% of the AS patients are HLA-B27 positive.42 Thus prevalence of AS correlates with the presence of HLA-B27 in all ethnic groups, but it must be noted that the predominant majority of HLA-B27 carriers are healthy individuals, free from any disease. Although the frequency of HLA-B27 is high in AS patients, it rarely reaches 100%, and this raises the question of HLA-B27-negative AS.
Recently, evidence was presented that strongly suggests that HLA-B27-positive AS and HLA-B27- negative AS were different entities. In an extensive literature survey of clinical features in B27-positive and B27-negative AS patients, it was found that in B27-negative patients, the mean age of onset was in the thirties, incidence of the disease in both sexes appeared to be the same, and associated Crohn's disease or psoriasis was frequently present.48
Some years ago, it had been observed that there is a greater prevalence of acute-anterior unveitis (AAU) in B27-positive patients in the United States,43 and subsequently, similar observations were made in the Netherlands.49 Furthermore, it is unusual to observe families with two or more first-degree relatives affected with B27-negative primary AS in the absence of psoriasis or inflammatory bowel disease.71 In general, HLA-B27-negative AS patients rarely develop severe disease, at least in Dutch14 and English populations,71 in the absence of inflammatory bowel disease or psoriasis. Clearly, the presence of HLA-B27 is important in defining severe AS, and thus the B27 gene would appear to be the "arthritogenic gene." This can be tested by transferring the HLA-B27 gene into experimental animals and assess whether such transgenic animals develop arthritis if exposed to gram-negative bacteria, such as Yersinia, one of the microbes involved in HLA-B27-associated reactive arthritis. Several groups have reported studies with transgenic mice, all showing some form of arthritis, and when the animals were exposed to gram-negative bacteria, such as Yersinia, then the occurrence of arthritis rose to almost 100%.39-54
Almost two decades ago, it had been suggested that the HLA-B27 gene was only a marker for a second gene present in the vicinity of HLA-B27, which was coding for a putative "ankylosing spondylitis" gene." The AS gene was thought to be in linkage disequilibrium with the HLA-B27 gene, and this was called a "two gene theory"18; however, the lack of dissociation between AS and HLA-B27 in family studies,71 the association of AS with HLA-B27 in many different racial groups,42 the evidence showing that B27-positive and B27-negative AS are different entities,4* and the demonstration by transgenic studies that HLA-B27 is an arthritogenic gene54 clearly indicate that the "two gene theory" is no longer tenable and does not provide an explanation for the association of HLA-B27 with AS.21
The fundamental assumption of "one gene theories" is that only one gene is involved, thereby suggesting that the HLA-B27 gene itself is the predisposing AS gene.18 At the moment, the two main theories vying for the role to explain the association of HLA-B27 with AS are both based on the assumption that the HLA-B27 gene is the arthritogenic gene, which in association with an environmental factor causes the disease. The evidence suggesting the interaction of an environmental factor comes from identical twin studies, which show that the frequency of the second twin developing AS when the first twin has the disease, that is to say the concordance rate, is well below 50%.w
THEORETICAL MODELS TO EXPLAIN THE ASSOCIATION OF HLA-B27 WITH ANKYLOSING SPONDYLITIS
The two main theories trying to explain the association of HLA-B27 with AS are the receptor theory and the molecular mimicry theory. Any theory trying to answer the question of what causes AS, or what causes RA, must provide an explanation for the specific facts known about these diseases:
1. The male/female ratio in AS is 3/1, and in RA, it is the reverse, 1/3.
2. In AS, 96% of the patients possess HLA-B27, whereas in RA, 70% of the patients possess HLA-DR4.
3. The concordance rate in identical twins in AS is around 40%, whereas in RA, it is about 20%.
It is clear that both AS and RA possess different specificity factors, which can be used to test the validity and viability of any theoretical model proposed to explain the association of HLA with disease. Following are the theoretical models that have been proposed.
Receptor Theory
The receptor theory states that a class I major histocompatibility complex (MHC) molecule acts as a receptor cavity that binds some as yet undefined, external, environmental polypeptide, which then somehow increases the susceptibility of the subject to develop the disease. The receptor theory, at the moment, appears to be favored by most researchers.7 It is based on the idea, initially proposed by Zinkema-gel and Doherty,73 that class 1 MHC molecules present antigens. The recent crystallographic studies by Bjorkman and co-workers8 that the HLA-A2 molecule consists of two alpha domains separated by a floor of eight antiparallel beta strands-thereby forming a groove, which would contain or present an antigenic polypeptide-provides a structural basis for the antigen-presenting function of class I MHC molecules (Fig. 1). At this stage, however, the nature of the "arthritogenic" polypeptides present in the HLA-B27 groove have not, as yet, been identified. Until such a polypeptide is found, the validity of the receptor theory will remain untested, and therefore, the ability of this theoretical model to answer the specificity questions relating to AS will be unknown. Clearly, at the moment, the receptor theory does not offer any testable therapeutic possibilities, either in AS or RA.
Molecular Mimicry Theory
The molecular mimicry theory states that the HLA antigen has a sequence, which resembles biochemically and immunologically a similar sequence, located in some environmental agent, such as a bacterium.17 The molecular mimicry theory was first proposed in 1976, when it was suggested that several gram-negative microorganisms, such as Klebsiella, Shigella, and Yersinia carry antigens cross-reacting with HLA-B27.24-70 In 1977, increased isolation of Klebsiella from AS patients was reported from London.30
In 1980, anti-B27 allogeneic human tissue typing sera were found to bind preferentially to Klebsiella antigens using three different
Figure 1. Schematic representation of the first two domains of an HLA-B27 molecule. The "molecular mimicry" theory proposes that an anti-Klebsiella antibody binds to part of the hexamer sequence, present on the outer lip of the a-1 domain and when present in high concentration will damage the HLA-B27-positive lymphocyte. The "receptor theory" pro-poses that a bacterial or viral polypeptide, some 20 to 30 amino acids long, fits into the groove between the a-1 and a-2 domains and thereby, probably, through T-cell activation, win produce inflammation and AS.
techniques: hemagglutination, radiobinding assay, and radiolabelled antigen competition assay.4 Subsequently, reports appeared from several different centers, confirming the presence of cross-reactivity between HLA-B27 and gram-negative bacteria.
In 1984, a group from Amsterdam reported that mouse monoclonal anti-HLA-B27 sera showed increased binding for Klebsiella, Shigella, and Yersinia antigens.*5
In 1985, a group from Los Angeles carried out the reverse experiment, when they described an anti-Yersinia monoclonal antibody, which reacted with 12 out of 12 HLA-B27 1ymphoblastoid cell lines, but with only 4 out of 31 B27-negative ones. However, three of the four reactive B27-negarive cell lines carried HLA-B7, an antigen that cross-reacts with HLA-B27.44
In 1986, it was shown that the anri-HLA-B27 (M2) monoclonal antibody bound specifically to a 70 kD component of Klebsiella pneumoniae, whereas no such reactivity was demonstrated with five other monoclonal antibodies.55
In 1987, studies from Oldstone's group identified a hexamer amino acid sequence, Gln-Thr-Asp-Arg-Glu-Asp (QTDRED), which was present in both the HLA-B27.5 molecule (residues 72-77) and Klebsiella pneumoniae nitrogenase reductase enzyme (residues 188-193).** Furthermore, antibodies to this shared sequence were found in the sera of the AS patients." In the case of HLA-B27, this hexamer epitope is located on the outer lip of the alpha-1 domain and therefore accessible to circulating anti-Klebsiella cross-reacting antibodies (Fig. 1). The identification of such an epitope raises the question whether antibodies to it would also recognize HLA-B27-positive cells.
In 1989, Husby et al37 showed that rat antisera raised against Klebsiella nitrogenase enzyme polypeptides, containing the shared QTDRED hexamer, reacted with 13 of 14 synovial biopsies obtained from HLA-B27- positive AS patients, but with none of seven biopsies obtained from HLA-B27-negative individuals suffering from either RA or osteoarthritis.
It would seem that molecular mimicry between HLA-B27 and Klebsiella bacteria has been defined down to the level of similarity of six amino acids, found in both the suspect bacteria and the genetically susceptible population. Thus the term "molecular mimicry" could not be said to be inappropriate when studying cross-reactivity between bacterial antigens and HLA molecules. Furthermore, this molecular mimicry would appear to provide a simple model for the pathogenesis of AS in genetically susceptible individuals.
MOLECULAR MIMICRY AND PATHOLOGY OF ANKYLOSING SPONDYLITIS
If there is cross-reactivity between HLA antigens and bacteria, then infection by such microorganisms will lead to the production of antibodies, which will have both antimicrobial, as well as antiself or autoimmune activity. Only a small proportion or a subset of the antimicrobial antibodies will also have antiself or autoimmune activity. The bacterial antigens, carrying the shared sequences, will be immunogenic, especially around the edges of the shared sequences, because it is at the sites that the immune system will not recognize that it is dealing with a self antigen. If for instance, the bacterial shared sequence is spanning residues 188-193 of the nitrogenase molecule then the edge antigenic pentamer, 187-188-189-190-191, will not be recognized as a self antigen, because of the haptenic effect of the nonself amino acid at position 187.
The antibodies produced against such an edge antigenic determinant, however, may well bind to HLA-B27. There is thus no breakdown of tolerance, and the evocation of bacterial antibodies having antiself or autoimmune activity is part of the normal immune response when encountering partially cross-reacting antigens. A similar process occurs in rheumatic fever.40 When present in small quantities, such antibodies would not activate the complement cascade, and therefore, no cytotoxic event and no inflammation would ensue (Fig. 2A). When such antibodies are present in high concentrations, however, they would activate the complement cascade with consequent stimulation of inflammation that eventually may result in localized tissue damage and fibrosis, especially in the entheses around the lumbar spine and sacroiliac joints6 (Fig. 2B).