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Wegener's granulomatosis (WG) is an autoimmune condition marked by vasculitis of small and medium sized vessels particularly affecting the upper respiratory tract and kidneys. There is a strong mucosal component similar to other autoimmune conditions such as systemic lupus erythematosus and Behçet's disease. While the pathogenesis of WG is not completely known, auto-antibodies such as IgG ANCAs have been implicated in endovascular damage and modulation of neutrophil / monocyte responses by Fc receptor (FcR) signaling. Due to the substantial mucosal involvement in WG (oral, nasal, and upper respiratory tract involvement), it is probable that IgA antibodies (perhaps IgA ANCAs) play a role in disease. Given discrepancies in associating ANCA levels with disease activity, future work should determine if IgA ANCAs are present in WG patients and examine the biology underlying the ANCAs' signaling partners - the FcRs.
A 57 year old Caucasian gentleman hobbles into the exam room during a Monday morning Internal Medicine clinic at the VA. He is obviously in pain, grimacing and bracing against the counter, as he lowers himself into a chair. His daughter grabs his arm to help him sit, but he shrugs her off. Once comfortable, he grabs a Kleenex from the counter and mops up his nosebleed that has begun trickling onto his shirt. When asked if he wants a wet paper towel, there is no response. His daughter chimes in, “I'm sorry, he doesn't hear so well anymore. It's been getting worse the past year and a half; you just have to speak up sometimes.”
When eliciting his chief complaint, he reports feeling “fine”, but his daughter informs that he has been having sinus infections and nose bleeds intermittently for almost two years. “It might be due to allergies though since his eyes get red from time to time.” As he covers his mouth to cough, several small purpuritic lesions come into view on his elbow. When asked about his pain when sitting down, he responds, “Yea, sometimes my hip or knee hurt. I'm just getting old, but I try not to let it keep me from working.” Upon further questioning, the patient admits that the joint pain, sinus problems, and hearing loss all started about the same time 18 months ago, lasted for several months, improved, and are now returning.
This is his first visit to a physician in thirty years since he had a hernia repaired in the late 1970s. He has a 20 pack year smoking history, drinks alcohol occasionally, is widowed, and works at a quarry. He has a brother with rheumatoid arthritis.
On physical exam, his temperature is 98.9°F, and his vital signs are all stable, except for mild hypertension. Both hearing and visual acuity are reduced with conjunctivitis present. His nasal mucosa appears inflamed with brown crusted lesions on his septum. His oral cavity is moist with multiple half-centimeter ulcers seen on his buccal mucosa. There are no cardiac murmurs, but stridor is present over his trachea with diffuse coarse breath sounds in his lungs. His abdominal exam is normal. There are purpuritic lesions on his right elbow and splinter hemorrhages in his nail beds. Both his right knee and hip appear stiff and painful when moving but are not red or warm to touch.
Routine labwork reveals an elevated creatinine, moderately high blood glucose, mild eosinophilia, and high LDL. His total lymphocyte count, hematocrit, and electrolytes are all normal. Urine analysis reveals glucose, microhematuria, and red cell casts with no indication of a urinary tract infection. A routine nose swab is positive for MRSA.
His constellation of symptoms, including respiratory, upper airway, renal, musculocutaneous, ocular, and auditory involvement, prompts additional labwork and a chest x-ray. The radiograph shows multifocal cavitary lesions. A rheumatological panel reveals an equivocal rheumatoid factor and positive PR3-specific anti-neutrophil cytoplasmic antibodies.
Several autoimmune conditions present with mucosal manifestations, such as the oral ulcers, epistaxis, and conjunctivitis mentioned in the clinical vignette. Among others, examples of mucosal autoimmune conditions include systemic lupus erythematosus (SLE) (in which presence of oral ulcers is one of the diagnostic criteria ), Behçet's disease (in which oral ulceration is required for diagnosis ), pemphigus vulgaris (PV) (in which mucosal ulcers are almost always present ), Crohn's disease (in which inflammation can occur in any portion of the gastrointestinal tract), and, the condition described above, Wegener's granulomatosis (WG) . Auto-antibodies, such as anti-nuclear antibodies (ANA) in SLE or anti-desmoglein 3 in PV, are present in several of these conditions providing another immunopathologic similarity between them. Evaluating auto-antibody function in conditions with mucosal manifestations may provide insights for additional research into the pathogenesis of these complex diseases. Given the involvement of upper airway, nasal, and oral mucosa and the established role of auto-antibodies in the disease, WG provides an important model for the role of antibodies in mucosal autoimmunity.
WG, one of the anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV), is an autoimmune condition involving small and medium sized vessels. This disease frequently manifests in the respiratory tract (including upper airways) and kidneys [5; 6]. Over 90% of WG patients present with inflammation of their oral or nasal mucosa. In fact, WG is diagnosed by the presence of two out of the 4 criteria established by the American College of Rheumatology (ACR), which includes nasal or oral inflammation, abnormal chest radiograph, excessive urinary sediment, and granulomatous inflammation on biopsy . The Chapel Hill Consensus criteria for systemic vasculitis are another means of classifying disease; it places more emphasis on biopsy results . While not an ACR diagnostic criteria for enrollment in clinical trials, approximately 93% of WG patients have detectable ANCA titers during active disease . The diagnostic emphasis on oral/nasal inflammation and respiratory tract involvement combined with the frequent presence of ANCA support the use of WG as a model of antibody involvement in mucosal autoimmunity.
Although the specific etiology of WG is not known, WG is a complex immunological disease resulting from certain environmental triggers occurring on the background of genetic predisposition [9; 10]. However, what specific factors contribute to a condition that targets primarily the upper respiratory tract and kidney? What elements lead to a condition with systemic vascular and targeted end-organ effects? While WG serves as an example of auto-antibody involvement in mucosal immunity, examining unique features of this disease allows better understanding of the clinical immunology underlying the disorder and of applications for future research and treatment of this vasculitis.
In this short analytical review, we use WG as a model for examining themes of auto-antibodies and mucosal immunity and for evaluating immunological mechanisms unique to this disease.
WG is predominant in Caucasians (97.7% of cases) yet its prevalence is rare (approximately 1 out of 33,000 Caucasian individuals) [11; 12; 13; 14]. Peak onset occurs between ages 45 and 65 with reports showing either a higher prevalence among men  or no gender bias . The patient described above is typical demographically for WG – a white male in his late fifties. There also appears to be a differential geographic distribution related to WG and other AAVs. WG is more common in Northern Europe with decreasing prevalence when traveling southward towards the equator [9; 11; 12; 13; 16; 17]. In contrast, microscopic polyangiitis, another AAV, is more prevalent in decreasing latitudes . A similar pattern of prevalence related to relative location to the equator has been observed in the Southern Hemisphere as well . This spread may reflect differing environmental exposures that trigger the respective conditions, may reflect genetic admixture among European individuals, or, likely, may result from some combination of the two.
Genes that have been associated with WG susceptibility encode an array of immunologically relevant molecules, many of which are located in the Major Histocompatibility Complex (MHC) . Alleles of HLA-DPB1 and retinoid X receptor β (RXRB), two neighboring genes in the immune-gene dense MHC class II region, confer one of the strongest associations of any genomic region [18; 19]. Polymorphisms in the genes for a protein tyrosine phosphatase, PTPN22 , and a T lymphocyte negative regulator, CTLA4 , associate with WG as well as numerous other autoimmune conditions. A genetic study focusing on interleukin 10 (IL10) suggests this cytokine influences auto-antibody production in WG patients . Other immunologically related molecules, including integrin beta 2 (ITGB2, CD18) , the SERPIN cluster on Chromosome 14q32.1 , and the Fc receptors (FcR) FCGR2A and FCGR3A , are implicated by genomic data in WG pathogenesis.
Data have also revealed specific environmental triggers for disease onset or relapse. Chronic nasal carriage of Staphylococcus aureus, as was seen in the clinical vignette, predisposes to disease relapse [26; 27]. This helps explain why adjuvant therapy with trimethoprim-sulfamethoxazole (an antibiotic also known as co-trimexazole, Septra®, or Bactrim®) reduces the rate of active disease relapse in a subgroup of WG patients  (although it is also possible that this drug is actually treating bacterial sinusitis mistaken for a WG flare). Inhalation of silica dust in an occupational setting, such as mining, quarrying (the profession of the patient described above), or farming, has been shown to influence emergence of AAVs but not necessarily WG . Various agricultural exposures such as solvents and silica also increase the risk of an ANCA-associated vasculitis .
In WG, vascular damage has been largely attributed to excessive activation of neutrophils and monocytes. Such ill-fated immune responses are derived from a combination of genetic and environmental factors such as those mentioned above [10; 31]. Given the role of neutrophils and monocytes in WG pathogenesis, it is not surprising that the most common immunological marker of disease is the presence of ANCAs , as were detected in the patient from the clinical vignette.
ANCAs bind enzymes located in the primary azurophilic granules of neutrophils and in the lysosomes of monocytes. (It is important to note that regardless of the name ANCA targets are expressed by and can activate monocytes.) There are six identified molecules ANCAs recognize as autoantigens; the two most commonly tested in a clinical setting include cytoplasmic and perinuclear ANCAs (cANCA, pANCA). (Their names are derived from their fluorescence staining appearance in neutrophils [32; 33; 34].)
cANCAs target proteinase 3 (PR3, PRTN3 [19p13.3]), a serine protease. PRTN3 contains a polymorphism in a transcription factor binding site of the promoter region that has been genetically associated with WG susceptibility . One report observed cANCAs in more than 90% of patients with active disease and 40% of patients in remission .
pANCAs target myeloperoxidase (MPO [17q23.1]), which, like cANCAs, has been genetically associated with WG  and are observed in higher levels in patients with active disease . While cANCAs are more common among WG patients, pANCAs are found in higher titers in other vasculitic conditions such as microscopic polyangiitis and Churg-Strauss syndrome .
Even though ANCAs are regularly detected among WG patients , their presence is not required as a diagnostic criteria from the ACR . However, evidence is compelling as to ANCA involvement in WG pathogenesis: 1.) Neutrophils, which express ANCA targets, have long been observed in the renal lesions of WG patients [5; 6; 40]. 2.) Autoantibodies specific to PR3 (cANCAs) contribute to the inflammatory damage in WG by cross-linking target with antibody receptors or directly initiating a signal cascade to activate an inflammatory response [41; 42; 43]. ANCAs' binding (and subsequent immune reactions) are antigen dependent with cANCA and pANCA inducing differential endpoints in neutrophils activation . In terms of WG, recent work has suggested that cANCAs prime CD14-dependent monocytes and neutrophils  to produce cytokines [e.g. tumor necrosis factor (TNF)]. These effects occur by upregulating toll-like receptor (TLR) and nucleotide-binding oligomerization containing molecules (NOD) in response to bacterial antigens . These data also provide a molecular explanation for the relationship of bacterial infection (Staphyloccous aureus) with the incidence of WG . In addition to generating an inflammatory response, TNF-alpha induces an increased surface expression of PR3 on neutrophils , thereby accelerating the impact of ANCAs on immune cell activation. 3.) Once bound to neutrophil surface PR3, the Fc portions of ANCAs are recognized by IgG Fc gamma receptors to initiate a signaling cascade [48; 49]. 4.) ANCA-Fc gamma receptor binding triggers an inflammatory reaction such as release of cytokines and chemotactic factors [50; 51] or by causing direct cytotoxicity on endothelial cells such as those in the vasculature . To date, the TH1 cytokine response has been the most examined in WG ; however, new evidence also points to a role for the TH17 pathway .
As mentioned above, ANCAs may also directly damage endothelial cells in WG. Alveolar cells removed from WG patients after pneumonectomy show a higher level of PR3 expression than alveolar cells removed from non-WG patients. These pneumocytes also showed higher levels of PR3 mRNA expression than alveolar macrophages or neutrophils isolated from the lung tissue  suggesting the mechanism of ANCA-mediated inflammation may be direct interaction with target tissues. A similar finding has been reported in the kidney, with glomerular cell expression level of PR3 being associated with crescent formation . It would be interesting to determine PR3 expression level in mucosal tissues impacted by WG.
Another theory suggests that auto-antibodies form specific to the complementary / anti-sense protein of PR3  and that memory T cells from some ANCA-related vasculitic patients recognize the complementary protein . While the specific mechanisms regarding induction of autoimmunity by anti-sense antibodies are not fully understood, the complementary PR3 antibodies can specifically bind plasminogen  suggesting a relationship between ANCAs and the coagulation cascade.
Antibodies, such as ANCAs, signal via Fc receptors (FcRs) to provide immunoregulatory signals including activation and/or inhibition of immune cells  such as neutrophils and monocytes . By recognizing the constant portion of antibodies and acute phase molecules such as C-reactive protein , FcRs are central to regulating an immune cell's activity in producing an appropriate immune response or an autoimmune reaction. See Figure 1.
In WG, the downstream effects of cANCA–FcR signaling result in an oxidative burst leading to inflammatory / vasculitic consequences [42; 62]. cANCAs are known to interact with FCGR3B (CD16B) and FCGR2A (CD32A) resulting in neutrophil activation [49; 63]. ANCA binding with FCGR3B also induces cell adhesion thereby promoting a pro-inflammatory state . While the molecular pathway from ANCA-FcR binding to oxidative burst is not completely elucidated, FcR binding by ANCA is required for the Syk phosphorylation involved in neutrophils activation  providing evidence of the necessity of the ANCA-FcR interaction.
Phosphatidylinositol 3 kinase (PI3K) activation and subsequent recruitment of protein kinase B (PKB), a serine-threonine kinase, have been observed in immune cell activation by Fc gamma receptors [66; 67]. PI3K activation is also observed in neutrophils stimulated by ANCAs supporting a role for Fc gamma receptors in WG. However, the kinetics of PKB activation differed between conventional Fc gamma receptor binding and ANCA-FcR binding in neutrophils suggesting the involvement of other co-modulating receptors in ANCA stimulation .
Even though FCGR2A and FCGR3B have been the focus of research to date, it is possible that other FcRs are involved in ANCA recognition on neutrophils and monocytes especially since other receptors likely participate . Additional FcRs are known to be expressed on neutrophils and monocytes such as the IgA Fc receptor, FCAR (CD89) , and the inhibitory Fc gamma receptor, FCGR2B (CD32B) .
Genetic variations within FcR genes, both structural and allelic, are known to influence susceptibility to disease, which emphasizes the importance of these molecules in WG pathogenesis. Polymorphisms in FCGR2A and FCGR3A have been linked to risk of disease relapse among WG patients by allowing chronic nasal carriage of Staphylococcus aureus and altering interactions with ANCAs . Other studies have not found a relationship between WG and FCGR2A variation . However, due to the low prevalence of WG, studies examining genetic associations have analyzed limited numbers of patients with a corresponding limitation in statistical power.
FCGR3B also contains variants, particularly the NA1 allele, that may influence ANCA-associated vasculitis [71; 72]. Neutrophils from individuals with the FCGR3BNA1 allele are more likely to release alpha-defensins, a chemotactic agent, upon stimulation with ANCAs, providing an example of how FcR genetic variation may impact WG .
Copy number variation (CNV) is the result of an entire gene or gene segment being duplicated or deleted in some individuals. An altered number of genes may lead to a relative overexpression of protein (in the case of higher copy number) or a deficiency of protein (in the case of gene deletion). Compared to healthy controls, FCGR3B CNV has been associated with both SLE and WG . In SLE, disease susceptibility appears to be associated with a gene deletion; in WG, disease susceptibility is associated with a higher copy number. This suggests a role for FcRs in differentiating between systemic and organ-specific autoimmune reactions . FCGR3B copy number has been correlated with FCGR3B expression level on the cell surface and in soluble form, as well as correlated with neutrophil processing of immune complexes .
Additional genotyping of FcR polymorphisms is needed in larger collections to understand more fully the impact of these molecules in WG. Variants among Fc gamma receptors have been associated with other autoimmune conditions characterized by mucosal involvement such as Behçet's disease , Crohn's disease , and SLE [73; 77]. The breadth of associations highlights the importance of FcR genetics in mucosal autoimmunity. It is also possible that FcR variants will help explain particular disease manifestations within WG. For example, it is likely that variants of FCGR3B are related to renal manifestations given the relationship of FCGR3B CNV to glomerulonephritis in rats , lupus nephritis in humans [78; 79], and end stage renal disease in humans . The main barrier in gaining this understanding will be assembling collections with sufficient power to detect associations, especially among clinical subgroups, given the low prevalence of WG.
While it is clear that ANCAs are involved with WG pathogenesis , it is less certain if their presence or titer level corresponds with disease activity. Some reports suggest that ANCA titer at the end of aggressive therapy regimen relates to risk of relapse [82; 83; 84]; however, another study reports no relationship between relapse and cANCA level . It would be useful to gauge the risk of relapse and necessary length of cyclophosphamide therapy, which is the first line pharmacologic treatment for WG, based on an immunological marker (i.e cANCA titer) given the adverse effects of the medication such as risk of bladder cancer, leukemia, and lymphoma . Due to the contradictory results of the above studies, perhaps a marker other than the IgG cANCA would be a more appropriate or adjunct marker of disease activity.
To date, both experimental studies and clinical diagnostic tools focus on the IgG isotype of ANCA as functionally important [81; 87; 88]. This is understandable since IgG is the most common isotype in general, since IgG ANCA have been clinically related to WG disease activity , and since genetic studies have implicated IgG FcRs with WG susceptibility and relapse [25; 73]. However, there may be a role in WG for other immunoglobulin (Ig) isotypes, and another isotype may serve as a marker for disease activity or risk of relapse.
Incubation with an IgM ANCA derived from a patient with severe vasculitis inhibits neutrophil adhesion and increases phagocytosis of apoptotic neutrophils by macrophages, which differ from the pro-inflammatory effects of IgG ANCA incubation . This study focused on an ANCA specific to MPO, which, while present at times, is not the most common ANCA target in WG. The IgM ANCA isotype has also been observed in a patient with pulmonary hemorrhage and acute renal failure , symptoms commonly reported in WG. IgM ANCAs may be present and modulating disease severity in WG.
IgE ANCAs have not been reported in the experimental setting or in a case study isolated from a patient. While unlikely to impact WG pathogenesis, IgE ANCAs could play a role in another vasculitis such as Churg-Strauss syndrome that is known to have positive ANCA titers and high IgE levels. It is not clear if IgE ANCAs have been sought and not found or simply not investigated.
The most likely Ig isotype, other than IgG, to influence WG is IgA. As discussed above (and seen in the clinical vignette), mucosal manifestations are common among WG patients with nasal / oral inflammation and respiratory tract involvement being diagnostic markers of disease . Since IgA is the most prevalent antibody isotype at mucosal surfaces  and since ANCAs are the most prevalent auto-antibodies involved in WG, it is probable that IgA ANCAs are present and impacting disease in WG patients. WG patients frequently manifest glomerular damage , and IgA deposition is the most common cause of glomerulonephritis  providing more circumstantial evidence for IgA ANCA involvement in WG.
IgA ANCAs have been observed in erythema elevatum diutinum , which is a neutrophil-associated vasculitis of the skin. This identification establishes a precedent for IgA ANCA involvement in neutrophil-related and vasculitic diseases. Other conditions with observed IgA ANCAs include cutaneous vasculitis , ulcerative colitis , and Henoch-Schönlein purpura . It needs to be determined if IgA ANCAs can be detected in WG patients and if their presence mediates the disease process.
One possible avenue of investigation is the role of the FcR for IgA (FCAR, CD89) in WG. If IgA ANCAs are present, this molecule would be central to any downstream effects. FCAR is a particularly interesting FcR since it has recently been shown to direct both pro-inflammatory and anti-inflammatory effects [97; 98] in an allele specific manner . The activating or inhibitory effects of IgA are due, at least in part, to differential stoichiometric binding of IgA with FCAR. When two FCAR molecules are complexed together on the cell surface, their close proximity creates a different Fc binding site for IgA that signals activation when engaged. When FCAR remains as a monomer on the cell suface, engagement of the traditional Fc binding site signals the cell to downregulate an immune response . In fact, the binding site of IgA to FCAR impacts immune evasion by Staphyloccus aureus to allow chronic nasal carriage , a risk factor for WG susceptibility . Supposing that IgA ANCAs are present in WG patients, such paradoxical regulation by FCAR could explain a role for ANCAs in the range of severity observed among WG patients [33; 101]. It needs to be determined if these other ANCA isotypes are present in WG patients and what, if any, impact they have on pathogenesis.
WG is a rare autoimmune vasculitic condition characterized in part by mucosal involvement and induced by auto-antibody mechanisms. While the pathogenesis of WG is not completely understood, IgG ANCAs have been implicated in causing direct damage to endovasculature and in modulating neutrophil and monocyte responses via FcR signaling, as supported by both functional and genetic studies. Given the prominent mucosal component in WG (oral, nasal, and upper respiratory tract involvement), it is probable that some form of IgA antibodies are present and play a role in disease. IgA ANCAs are a likely candidate to meet that description. Due to discrepancies in relating ANCA titer with disease activity, future work should focus on identifying alternative ANCAs, such as of the IgA isotype, and evaluating the biology and genetics of the auto-antibody signaling partners, the Fc receptors. Additional research into understanding the role of ANCAs in the mucosal manifestations of WG may provide insight into diseases involving the mucosal immune system as a whole and help explain the oral ulcers and epistaxis observed in the patient above.
The authors received salary support from NIH R01-AR33062, R01-AR42476, P01-AR49084, and T32 AR07450 (NIAMS).
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