NephrologyClinical Immunology Department, Nantes University Hospital, Nantes, France
Small vessel vasculitis is associated with harmful anti-neutrophil cytoplasmic antibodies directed against sheltered antigens
Wegener's granulomatosis and microscopic polyangiitis are small vessel systemic vasculitides characterized by the presence of anti-neutrophil cytoplasmic antibodies (ANCA) and a high frequency of rapidly progressive glomerulonephritis. The main target antigens for ANCA are proteinase 3 (PR3) and myeloperoxidase (MPO). Both antigens are tightly sheltered from the immune system (cryptic). Indeed, they are stored in cytoplasmic granules of neutrophils, and are very rapidly scavenged after degranulation at the inflammatory site by specific inhibitors: 1-antitrypsin for PR3 [1] and ceruloplasmin for MPO [2]. However, both antigens are exposed at the cell surface after neutrophil priming by a proinflammatory cytokine [tumour necrosis factor
(TNF
)], and this allows neutrophil activation by ANCA in vitro, with production of toxic oxygen species, degranulation of proteolytic enzymes and endothelial cell lysis [3]. This activation process of neutrophils by ANCA is highly regulated, and may be responsible for the vasculitic lesions observed in vivo [3].
Neutrophil apoptosis: saviour or villain?
Neutrophils are suicidal cells that can only choose the way they die. The first possibility is to be recruited to an infected site, and to die while releasing proteases (including PR3) and enzymes producing reactive oxygen species (mainly MPO) [4]. This process is very useful to clean up abscesses, but needs to be tightly controlled to prevent unwanted extension of necrotic activity. The alternative for neutrophil death, i.e. apoptosis, ends up in their clearance without releasing any potentially toxic enzymes. Apoptosis is an active process that is triggered by various stimuli, that leads to inactivation of the intracellular machinery, and specific cell membrane changes that allow recognition and ingestion by professional phagocytes or local resident cells. Therefore, the fate of neutrophils is to die either by necrosis or by apoptosis.
Ageing neutrophils that did not encounter inflammatory battle fields, die by apoptosis. Their life span can be prolonged by pro-inflammatory cytokines or growth factors [5], and this allows more neutrophils to be recruited for combating the insult. Neutrophils adhering to interleukin (IL)-1-activated endothelial cells are protected against apoptosis through engagement of ICAM1 and ß2 integrins [6]. The chemokine, IL-8, as well as the transmigration of LPS-primed neutrophils through an endothelial cell layer, further retard apoptosis [7,8]. Glucocorticoids also delay apoptosis, leading to increased number of neutrophils [9]. However, neutrophils from elderly persons have a decreased ability to be rescued from the apoptotic programme by inflammatory cytokines and growth factors [10,11], and patients with chronic renal failure exhibit accelerated neutrophil apoptosis [12].
For healing to take place, inflammation must be switched off by a co-ordinated programme that includes two main signals: (i) stop recruiting new neutrophils, with return of endothelial cells to a resting state (shedding of adhesion molecules, decreased level of chemokines), and changes in the cytokine balance; and (ii) apoptosis of the neutrophils already present at the inflammatory site. Regulatory loops are very rapidly activated to limit inflammation. For example, adhesion of neutrophils to the extracellular matrix renders them susceptible to TNF-induced apoptosis [13], and reactive oxygen species facilitate neutrophil apoptosis [14]. Neutrophil apoptosis will lead to a progressive loss of function such as chemotaxis, adherence, degranulation and production of reactive oxygen species [15]. CD16 (Fc
RIII) is shed [16] and, despite expression of CD11b and CD11c, adhesion is impaired preventing further activation [15]. Despite increased expression of PR3 and MPO on the surface of apoptotic neutrophils, the capacity of ANCA to induce neutrophil activation is decreased [17]. IL-1ß and TNF
increase phagocytosis of apoptotic neutrophils by macrophages [18]. Macrophages that have ingested apoptotic neutrophils exhibit anti-inflammatory properties [decreased production of IL-1ß, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), TNF
, leukotriene C4 and thromboxane B2 (TxB2) and increased production of transforming growth factor-ß1 favouring scar formation [19]], and also produce soluble Fas that accelerates neutrophil apoptosis [20]. This clearance mechanism is very effective, since even late apoptotic neutrophils are efficiently phagocytosed by macrophages [21]. Steroids also accelerate phagocytosis of apoptotic neutrophils by macrophages as well as by human glomerular mesangial cells, without inducing production of IL-8 and MCP-1 [22].
However, there may be a darker side to apoptosis since a defect in the effective clearance of apoptotic bodies may lead to autoimmunity, especially systemic lupus erythematosus [23]. The antigens recognized by ANCA are indeed expressed at the surface of apoptotic neutrophils [24,25], and we showed that Brown Norway (BN) rats injected with syngenic apoptotic neutrophils without the use of adjuvant develop ANCA [26].
The development of ANCA positive systemic vasculitis may require a three step pathological process
(i) An exogenous stimulus increases neutrophil and macrophage apoptosis
Uptake and degradation of apoptotic bodies by local resident cells or professional phagocytes is a very rapid process, such that it is a rare observation in random tissue sections. However, apoptotic macrophages are observed in ANCA-positive renal vasculitis [27].
Inhaled substances, including silica, are phagocytosed by macrophages and neutrophils in the upper respiratory tract. Because the cells cannot digest this inorganic substance, they form a foreign body reaction leading to granuloma formation. Silica induces apoptosis of human peripheral blood lymphocyte [28]. Silica also induces Fas-ligand expression in lung macrophages in vitro and in vivo, and promotes Fas-dependent macrophage apoptosis in a murine model of silicosis [29]. Intratracheal instillation of silica in Wistar rats induces dose-dependent apoptosis of alveolar neutrophils and macrophages and granuloma formation in the lung [30]. Furthermore, apoptotic leukocytes have been identified within lung granulomas [30]. Silica is a ligand for scavenger receptor A (SR-A) on the surface of apoptotic phagocytes [31], but its ability to interfere with apoptotic neutrophil clearance by macrophages is unknown. Finally, several case-control studies have shown a strong association between silica exposure and ANCA positive systemic vasculitis [3234, see 35 for review].
Other exogenous agents may trigger both apoptosis and ANCA production. Streptococcus pneumoniae can induce apoptosis as well as necrosis of neutrophils [36]. ANCA-positive systemic vasculitis may be associated with subacute streptococcal endocarditis [37]. Propylthiouracil (PTU) increases thyroid follicular cell apoptosis, but its effect on neutrophils is unknown [38], and PTU treatment may be complicated by ANCA positive glomerulonephritis [39].
(ii) Increased apoptotic neutrophil exposure together with danger signals induces ANCA production
Defective clearance or increased exposure to apoptotic cells is known to induce systemic lupus erythematosus [23]. Dendritic cells can ingest apoptotic cells and may present self-derived antigen if appropriate danger signals are provided. However, dendritic cells that have ingested apoptotic cells may exhibit, in the absence of these danger signals, a diminished capacity to stimulate naive T cells [40]. Nevertheless, we were able to break tolerance to neutrophils and generate ANCA in BN rats by injection of rat syngenic apoptotic neutrophils without the use of adjuvant [26]. The danger signals in our crude apoptotic neutrophil preparation that led to ANCA production in these animals remains to be determined.
Once ANCA are present, they act as amplifying factors. ANCA further increase neutrophil apoptosis [41]. Neutrophils that have been opsonised by ANCA are more rapidly phagocytosed by macrophages, with production of TNF and TxB2 [42], as well as IL-1 and IL-8 [43], promoting recruitment of additional neutrophils and inflammation. This pro-inflammatory environment may favour antigen presentation instead of safe clearance [23].
(iii) Environmental and genetic factors amplify disease expression
After ANCA production, additional events are required to trigger systemic vasculitis. Animal models have shown that ANCA are not sufficient to generate vasculitis: inflammation should be targeted in one organ by reactive oxygen species infusion, ischaemia or mild anti-glomerular basement membrane (GBM) deposits to trigger the disease [44]. This probably was not the case in our original observation, since the control animals treated with anti-GBM antibodies had no signs of glomerulonephritis [26]. In patients with systemic vasculitis, acute infections that may prime endothelial cells, can indeed trigger flares of the disease [45].
Several genetic factors may contribute to disease expression [3]. We previously reported an association between PR3-ANCA-positive vasculitis and 1-antitrypsin deficiency (Pi-Z allele) [1,46,47]. This inherited anti-protease deficit is associated with a more severe disease outcome [48].
Conclusion
Defective clearance or increased exposure to apoptotic neutrophils may be the initiation mechanism for ANCA production and subsequent development of systemic vasculitis. Animal models with defective apoptotic neutrophil clearance may provide insight into the pathophysiology of ANCA-positive vasculitis.
Acknowledgments
Thanks to J. Savill for critically reviewing this paper.
Notes
Correspondence and offprint requests to: NephrologyClinical Immunology Department, Nantes University Hospital, 30 boulevard Jean Monnet, 44093 Nantes, France. Email: vesnault{at}nantes.inserm.fr
References