Immunopathogenesis of Behçet’s disease with special emphasize on the possible role of antigen presenting cells
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- Pay, S., Şimşek, İ., Erdem, H. et al. Rheumatol Int (2007) 27: 417. doi:10.1007/s00296-006-0281-6
Behçet’s disease (BD) is a systemic vasculitis with unclear etiology and pathogenesis. Although several viral and bacterial causes have been investigated in detail for several years, of late it is widely accepted that microorganisms may play a role as a trigger, or as a cross-reactive antigens that interfere with self-antigens, such as heat-shock proteins. Genes such as HLA-B51, MICA and TNF, considered to play a crucial role in the pathogenesis of BD, are located in the major histocompatibility complex (MHC). However, it has been accepted that, only HLA-B51 is directly related with pathogenesis of the disease, and others have strong linkage disequilibrium with HLA-B51. Some other genes such as IL1, Factor V and ICAM-1, KIR and eNOS assumed to take a part in the pathogenesis are settled out of the MHC region. In patients with BD, several abnormalities in innate and acquired immunity were detected. Mainly, the hypersensitivity of T lymphocytes to different types of antigens plays a critical role in the pathogenesis. However, it is not clear that whether defective signal transduction or antigen presenting cell (APC) dysfunction is responsible for T cell hypersensitivity. Cytokines and chemokines secreted from APCs and T cells are suggested to cause the neutrophil hyperactivation. Activated neutrophils secrete some cytokines, which prime themselves and also stimulate Th1 cells. These relationships among APCs, Th1 lymphocytes and neutrophils constitute the basis of immune responses in BD. In this review, we focus on the possible role of APC in the pathogenesis of BD with an attempt to take attention of researchers in this field to these cells at the crossroads of innate and adaptive immunity, and discuss other potential contributors including cells of the immune system and mediators.
Behçet’s disease (BD) is a systemic vasculitis that may involve both the arteries and veins of almost any organ. The disease is an inflammatory disorder characterized mainly by recurrent oral and genital ulcers and uveitis. However, it can present with cutaneous, articular, neurological, pulmonary, and intestinal manifestations other than this classical triad. The etiology of BD is mostly unidentified, but generally accepted hypothesis of disease pathogenesis is that an intense inflammatory reaction is elicited by an infectious agent in a genetically predisposed host. The genes that have been suggested to play a role in the pathogenesis of BD such as HLA-B51, MICA, and TNF are located on the major histocompatibility complex (MHC). Among them, only HLA-B51 is the strongest genetic predisposition factor for BD defined so far, whereas the participation of the others has been suggested to be due to linkage disequilibrium with the HLA-B51 itself. Furthermore, a number of genes, including IL-1, Factor V, ICAM-1, KIR, and eNOS, which are located outside the MHC region has been proposed to contribute to the pathogenesis of BD. Several immunological aberrations have been shown to be associated with BD. The pathergy reaction is a distinctive feature of BD and might plainly reflect immunopathogenesis of the disease as indicating the role of both innate and acquired immunity during the course of the disease. Early pathergy reaction at 4 h is characterized by the rapid accumulation of neutrophils without evidence of vasculitis at the needle prick sites . The dermis at 48 h of pathergy reaction was infiltrated mostly by T lymphocytes and monocytes/macrophages, with neutrophils comprising less than 5% of the infiltrating cells . It is therefore likely that increased chemotaxis of neutrophils may contribute to the initiation of the reaction, while activated T lymphocytes are required for the progression of the whole pathergy reaction.
Antigens, superantigens, and autoantigens in the pathogenesis of BD
A number of viruses, including hepatitis viruses, parvovirus B19, and herpes simplex virus (HSV) have been implicated in the etiology of BD. However, only evidence with regard to HSV is found to be suggestive of a possible link. HSV-1 genome and serum antibodies against the virus have been found in a higher proportion of patients with BD than in controls . HSV DNA can be detected in the genital and oral ulcers of patients with BD . Furthermore, inoculation of HSV into the mice has been shown to cause lesions mimicking BD .
The occurrence of oral ulcers as the first symptom of disease in about 70% of the patients and increase in the rate of oral ulcers after dental interventions together with the evidence of decrease in the incidence of some of the symptoms of the disease with Benzathine Penicillin therapy can favor a role for oral flora in the etiology of BD [3, 6, 7]. Furthermore, the proportion of uncommon serotypes of Streptococcus in the oral flora of patients with BD has been shown to be increased and hypersensitivity in the skin tests with these antigens was demonstrated . In vitro production of IL-6 and INF-γ by T cells from patients with BD was found to be stimulated by Streptococcus Sanguis-related antigens. Likewise, these antigens have been shown to up-regulate the γδ T cells in the T cell cultures of BD patients . It has been reported that T cells from BD patients were induced to produce IL-6 and INF-γ with very low concentrations of superantigens derived from Escherichia coli and Staphylococcus . Recently, MALP-404, lipoprotein of mycoplasma fermantase, was detected in the sera of BD patients. It is possible that infection with mycoplasma might be involved in the pathogenesis of the disease, due to homology of some of the peptides found in MALP-404 and HLA-B51, and detection of mycoplasma in mucosal infections as a causative agent .
Several bacteria including mycobacteria, Borrelia Burgdorferi, Sacchoromyces Cerevisiae, and Helicobacter have been investigated extensively as a causative or triggering factor in Behcet’s pathogenesis. However, there is a consensus that BD is not a result of direct infection by these viruses or bacteria.
Heat shock proteins
Heat shock proteins (HSPs) are a family of proteins that are up regulated under conditions of cellular stress and protect cells from severe damage and premature death by preventing denaturation and degradation of cellular proteins. HSP from bacteria, such as streptococcus and mycobacterium, show considerable homology with those of host HSP. HSP60 found in humans corresponds to the HSP65 obtained from the mycobacterium. It has been reported that epidermal expression of HSP65 is notably increased in the active skin lesions, including erythema nodosum and mucocutaneous ulcers of patients with BD . It is thus possible that recognition of certain 65-kDa HSP peptides by γδ T cells might be important in the pathogenesis of BD . Currently, it has become obvious that four peptides included within mycobacterial 65-kDa HSP are responsible for the proliferation of γδ T cells in patients with BD [13, 14]. These peptides demonstrate significant homology with those of human 60-kDa mitochondrial HSP . It has been generally accepted that, cross-reaction between the microbial HSP and human HSP might be the basis for the relation of infection with autoimmunity . Administration of HSP peptides to the mice via subcutaneous route has been shown to induce uveitis without causing other features related with BD . Likewise, oral administration of these peptides induced clinical uveitis in experimental rats . Another interesting study supporting these findings is the increased S. sanguis colonization with the subsequent development of uveitis in rats exposed to heat shock .
These observations may reflect the important role of stress both in the breakdown of mucosal defenses and anti-HSP reactivity. In view of these findings, an immunological model for BD, which is based on the role of HSP65 as a potential T cell antigen has been postulated by Direskeneli et al. [19, 20]. Accordingly, “HSP60 reactive” T cell clones escaping high affinity clonal deletion may be positively selected with low affinity in thymus where HSP60 present as a self-antigen and circulate in an anergic form in the peripheral blood. In oral mucosa and, possibly, in the skin, after non-specific minor injuries and oral ulcer development, streptococcal and human HSP60 expression may be up regulated, which promotes self-HSP60 reactive clones. These T cells might then pass to the ocular section (after non-specific anterior uveitis that breaks down the blood–retina barrier), be further activated by retinal HSP60, and may cause a chronic ocular inflammation with Th1 type cytokine response [19, 20]. Although this model seems to be very comprehensive, it lacks grounds for the explanation of the development of other features associated with BD.
αβ Crystallin and HSP70
Besides the HSP60, other HSPs have been suggested to play a role in the pathogenesis of BD. αβ-Crystallin is a small stress protein, present in the various tissues of vertebrates including, brain, lens, skeletal muscle and kidney. Increased anti-αβ crystallin antibody levels have been demonstrated in the cerebrospinal fluid of BD patients with neuro-parenchymal involvement . Cerebrospinal fluid antibody responses to HSP and αβ-crystallin also showed a marked correlation with each other, which supports the idea that similar immune mechanisms are operative in the generation of both autoantibody responses. Likewise, elevated anti-HSP70 antibodies were found in patients with BD, although their role in BD still needs to be defined .
Retinal S antigen
Retinal S antigen is a protein mostly found in the retina and is probably immunologically protected. An immune response against this protein can only be elicited following tissue destruction due to uveitis. T cell responses against retinal S antigen have been confirmed in various forms of uveitis including BD [19, 22]. Some epitopes of S antigen is found to share homology with particular amino acid regions of HLA-B51 and HLA-B27, which has led to an increased attention to the role of this antigen in the pathogenesis of the disease.
The presence of an elevated IgG antibodies against the α-tropomyosin in BD patients raises the possibility of an autoimmune response against this antigen and might be involved in the pathogenesis of BD. Furthermore, development of ocular and mucocutaneous findings as similar to that seen in BD has been observed after immunization of Lewis rats with this antigen .
Behçet disease is characterized by vascular involvement which is mostly venous. Vasculitis is considered to be central in the pathogenesis of the disease which is responsible for almost all pathologies observed during the course of the disease. Several studies have revealed increased antibodies against endothelial cells in the sera of patients with BD, whereas no correlation was demonstrated between the intensity of antibody response and vascular involvement . It is postulated that these antibodies might cause activation of endothelial cells via binding to α-enolase endothelial antigens. On the other hand, it should be noted that evidence regarding the presence of anti-endothelial antibodies in BD patients is an area of debate [25, 26].
Cytokines and chemokines in the pathogenesis of BD
A large number of studies investigating serum/plasma levels of various cytokines, chemokines and their receptors have been conducted in patients with BD so far. The rationale which prompts researchers for evaluating cytokine levels is not only the investigation of the pathogenesis but also search for a marker that can be used for the determination of disease activity. In this respect, serum/plasma levels of several cytokines, chemokines and cytokine receptors including TNF-α, IFN-γ, IL-1, IL-8, IL-12, soluble IL-2R (IL-2R), and TNF receptor were investigated, in which some of them were reported to be correlated with the clinical activity of BD. Flow cytometric analysis of intracytoplasmic cytokine expression of cells disclosed that the frequency of IL-2 and IFN-γ producing T cells was increased in patients with active BD [27, 28]. Supporting the existence of Th1 type polarization and its possible role in the pathogenesis of the disease, serum IL-2 and IFN-γ levels were also found to be elevated in BD. Frassanito et al.  claimed that increased levels of IL-12, which is produced mainly by antigen presenting cells (APC) is responsible for the development of Th1 type response and, thus APC might play a crucial role in the pathogenesis of BD. Recently, serum levels of another Th1 polarizing cytokine, IL-18, was found to be increased in BD patients, and its levels were shown to be correlated with disease progression. The same researchers also established increased levels of IL-18 mRNA and inducible IFN-γ in the bronchoalveolar lavage of BD patients and stated that IL-18 might be important in the local inflammation of BD [29, 30]. In this regard, we have also demonstrated that serum IL-18 levels markedly increased in BD and correlated with the disease activity but not with the clinical presentations . Those studies also provided evidence for an active role of APC in BD. Contrary to these findings, some studies reported increased serum levels of Th2 type cytokines including, IL-4, IL-10, and IL-13 in BD patients . However, this finding can be explained by compensatory mechanisms in opposition to a strong Th1 type response .
Besides those studies examining serum cytokine levels, there have been some studies investigating synovial fluid levels of those cytokines in BD in an attempt to understand the factors responsible for the non-erosive character of the Behcet’s arthritis. Ertenli et al.  detected decreased synovial fluid levels of TNF-α, IL-1β, IL-1Ra, and TGF-β in BD patients as compared to RA patients. We have shown that synovial IL-18 levels were lower in BD patients as compared to RA patients . We also studied synovial metalloproteinases (MMP), proteases which are accountable for the erosions seen in chronic synovitis, in the synovial fluid samples of BD patients, and found no difference with regard to the levels of MMP-1, an MMP which behaves as an acute phase reactant, between BD and RA patients. On the other hand, MMP-3 levels of BD patients, an MMP which has been known to play a more potent role in the development of erosions noticed in RA, were found to be decreased as compared to RA patients [35, 36]. These attenuated cytokine profile and consequently decreased MMP expressions may probably contribute to the non-erosive course of Behcet’s arthritis. It has been well known that synovial neutrophils play a pivotal role in the development of erosions in patients with RA. Nevertheless, synovial fluid concentrations of CXC chemokines, including IL-8, GRO-α and ENA-78 were found to be significantly lower in BD patients comparing with those of RA . Moreover, reduced CXCR2 chemokine receptor expression on the surface of the synovial neutrophils was shown in BD patients . These data therefore revealed that chemotactic factors that attract neutrophils to the site of inflammation is relatively diminished in Behcet’s synovitis which might eventually protect the joint from the harmful effects of neutrophils. The role of CXC chemokines in the angiogenesis and pannus formation has previously been shown . In this regard, detection of decreased CXC chemokine in Behcet’s synovitis might contribute to the non-erosive nature of the arthritis.
Cells of the immune system in the pathogenesis of BD
Several ample evidence which indicates the significant role of T cell mediated immune responses in the pathogenesis of BD can be found: (1) Immunopathological studies from active inflammatory sites including aphthous ulcers, and pathergy reactions demonstrated the presence of intense cellular infiltration which mostly consists of T cells. (2) Strong Th1 type polarization was demonstrated and its intensity was shown to be correlated with the disease activity. (3) Therapeutic efficacy of drugs known to suppress T cell functions such as cyclosporine A has been reported in uveitis of BD. Various abnormalities of T cells have been described in patients with BD. Many researchers identified a reduced CD4+/CD8+ T cell ratio resulting from both a decrease in CD4+ T cells and an associated increase in CD8+ T cells. The decrease in the number of CD4+ T cells can be explained by the detection of significantly lower frequency of CD4+CD45RA+ (suppressor-inducer) T cells in the peripheral blood of patients with active BD . The proportion of peripheral blood γδ T cells was consistently reported to be elevated in BD which accounts for 2–5% peripheral blood T cells in healthy subjects. Furthermore, these γδ T cells were shown to express activation markers, such as CD25, CD69, and CD29 and produce inflammatory cytokines including IFN-γ, TNF-α, and IL-8 [40, 41].
In addition to their role in killing target cells via apoptosis, NK and NK-T cells may be involved in the polarization of acquired immune response, since these cells produce various cytokines, including IFN-γ and IL-4 at a very early stage of inflammation. Some investigators reported increased proportion of both NK and NK-T cells in BD, while the others reported normal or even decreased levels of these cells in the peripheral blood of BD patients . Up to now, no firm evidence shows whether NK or NK-T cells are directly taking part in the pathogenesis of BD.
It is suggested that T cells in BD are hypersensitive to a variety of antigens. Hence, Streptococcus-derived antigens were shown to stimulate the production of IL-6, IFN-γ, and peptides that enhance neutrophil functions by T cells from patients with BD. Similarly, E. coli-related peptides stimulated the production of IFN-γ by T cells of BD patients [9, 42]. Furthermore, it has been reported that T cells from BD patients were induced to produce IFN-γ with very low concentrations of staphylococcal enterotoxins that were not able to stimulate T cells from healthy subjects and patients with rheumatoid arthritis .
As it has already been cited in the text, four peptide determinants within the mycobacterial 65-kDa HSP (and the corresponding peptides from human HSP60) induced markedly increased lymphoproliferative responses which mostly confined to γδ T cells in BD [13, 14, 33]. Abnormal signal transduction through T cell receptors might thus elucidate the hypersensitivity of T cells to various antigens .
Neutrophils are the essential elements of the innate immunity. Hyperactivity of the neutrophils is an important aspect of the immunological abnormalities seen in BD. Increased chemotaxis, phagocytosis, superoxide generation and myeloperoxidase expression, as well as enhanced expression of CD11a, CD10, and CD14 on the cell surface have been reported in the neutrophils of BD patients .
The mechanism underlying the hyperactivity of neutrophils in BD is not known precisely. Several proinflammatory cytokines, such as TNF-α and GM-CSF are proposed to be accounted for the priming of neutrophils. The crucial roles of T cells in the neutrophil hyperactivation of BD have been demonstrated in previous studies .
Th1 type cytokines and chemokines including IL-17, IFN-γ, IL-8, and TNF-α have been suggested to be the cause of neutrophil hyperactivity. IL-17, which is largely produced by activated CD4+ and CD8+ T cells, has been found to be involved in the recruitment of neutrophils to the site of inflammation .
IL-18, which is mainly produced by APCs, may up regulate the neutrophil functions in addition to its role in the polarization of immune response toward Th1 . Primed neutrophils can be readily activated by cytokines which leads eventually to a considerable change in their characters. These changes observed in neutrophils are characterized by increased life span, expression of surface markers which are more typical of dendritic cells, such as CD40 and CD 83, and production of cytokines including IL-1, IL-1Ra and MIF [46, 47]. Additionally, activated neutrophils in BD patients have been detected to produce significant quantities of IL-12 and IL-18, which accounts for the priming of the neutrophils themselves as well as polarization of the immune response toward Th1 [48, 49].
Antigen presenting cells
It is proposed that abnormalities in T cells, but not those in APCs contribute actively to the pathogenesis of BD. Supporting the central role of T cells in the pathogenesis of the disease, increased proportion of peripheral blood γδ T cells and influence of these cells on Th1 type polarization of the immune response have been reported. Moreover, T cell hypersensitivity in BD to the various bacterial and viral antigens has been demonstrated. Of note, as considered to be the only gene that has been shown to be linked with susceptibility to BD, antigens that could be bound by HLA-B51 can only be presented to cytotoxic T cells, but not to the naïve ones.
On the contrary, some researchers suggested that BD might probably be a disease of APCs, and that T cells may be “innocent bystanders”. In this regard, epidermal Langerhans cells were found to be significantly increased at the site of pathergy reaction . Furthermore, it has been shown that these cells are more active in BD patients as compared to healthy controls [51, 52]. Mege et al.  have reported that cultured monocytes of patients with BD produced more TNF-α, IL-6, and IL-8 either spontaneously or after stimulation with LPS.
IL-12 is a cytokine, which is mostly produced by APCs and plays a crucial role in the skewing of immune response toward Th1. Likewise, IL-18, as a cytokine produced by APCs, contributes to the Th1 type polarization, particularly in association with IL-12. Detection of a correlation between the serum IL-12 levels and Th1 cells, and increased serum IL-18 levels in BD patients, obviating the possibility that APCs might play an important role in the pathogenesis. Furthermore, IL-18 has been shown to up regulate neutrophil functions . In addition, genetic studies disclosed that BD is associated with IL-1A -889 allele and IL-1A-889/IL-1B + 5887 haplotype . Boiardi et al.  further characterized a single-nucleotide polymorphism located at position -607 of IL-18 gene promotor region in BD. It is thus possible that genetically determined hyperactivity of APCs might play a part in the pathogenesis of BD. However, it is not fully determined whether this probable hyperactivity is an intrinsic defect of APCs or secondary to unknown interactions between immune cells. Kötter et al.  have found an increased serum interferon-alpha levels in BD patients with ocular involvement. Interferon-alpha is mainly produced by dendritic cells in response to viral stimuli and increased levels of this cytokine was also demonstrated in diseases characterized by Th2 type immune response such as SLE and type 1 diabetes mellitus . Although these findings were seemingly unexpected in the face of Th1 type polarization seen in BD, they suggested that dysregulation of interferon-alpha genes or dysfunctional interferon-alpha might be an explanation. As similar to the findings of Kötter, we demonstrated increased serum levels of interferon-alpha in BD patients. We also found that serum interferon-beta levels, a cytokine produced by dendritic cells and leading Th2 type immune response, significantly decreased in BD patients as compared to healthy controls . Furthermore, phenotypical analysis of peripheral blood dendritic cells in BD patients disclosed decreased percentage of plasmacytoid dendritic cells which is suggestive of active participation of this cell subset in the pathogenesis of BD . All these findings can be taken as an evidence of functional abnormality of dendritic cells in BD.