Caroline O. S. Savage

Background: Acute interstitial nephritis (AIN) is a common cause of acute kidney injury and is characterised by a dense interstitial cellular infiltrate, which has not been well defined. Previous studies have demonstrated a correlation between Epstein-Barr virus (EBV) infection and AIN. The purpose of our study was to define the nature of the interstitial immune infiltrate and to investigate the possibility of renal infection with EBV. Methods: Seventy-eight patients with AIN were identified from renal biopsy reports in a single centre over an 18-year period. Immunohistochemical staining was performed to define the cellular infiltrate. In situ hybridization and immunohistology were used to detect EBV. Results: A positive correlation between CD68 macrophage infiltration and serum creatinine concentration at presentation was identified. IL-4, eotaxin, CCR3, CCR5 and VCAM-1 were all expressed in biopsies of AIN. Using in situ hybridization and immunohistochemistry, EBV was not detected in any of the AIN sections analysed. Conclusion: This study has assessed the nature of the interstitial infiltrate in AIN. EBV was not detected in the renal biopsies, suggesting that EBV is not a pathogenetic factor in AIN.

Summary: An increased proportion of CD4⁺ CD25⁺ T cells has been reported in Wegener’s granulomatosis (WG) and may represent an accumulation of regulatory T cells (Treg). CD25 is also expressed on recently activated effector T cells. We have determined the relative proportion of these subsets in a large patient cohort. The fraction of Treg in peripheral blood mononuclear cells from patients and healthy controls was determined by assessment of Foxp3 expression on CD4⁺ CD25⁺ T cells. The functional activity of Treg was determined by their ability to suppress proliferation and cytokine production in response to proteinase‐3. Although WG patients demonstrated an increased fraction of CD4⁺ CD25⁺ T cells, the percentage of Foxp3‐positive cells was decreased. In addition, the percentage of Treg was inversely related to the rate of disease relapse. CD4⁺ CD25^hi T cells were able to suppress T‐cell proliferation to proteinase‐3 in healthy controls and anti‐neutrophil cytoplasm antibody (ANCA)‐ negative patients (at time of sampling) but not in ANCA‐positive patients. In patients with active disease, an increased proportion of CD4⁺ Foxp3⁺ cells was associated with a more rapid disease remission. Patients with WG demonstrate abnormalities in the number and function of Treg and this is most pronounced in those with most active disease. This information is of value in understanding the pathogenesis and potential treatment of this disease.

The inflammation of blood vessel walls that is associated with autoimmune disorders characterized by anti-neutrophil cytoplasm antibodies (ANCA) represents dysregulation of normal physiological processes, whereby neutrophils recruited to the vessel wall by cytokine-activated endothelium show destructive behaviours that initiate damage with endothelial apoptosis and denudation. Anti-endothelial cell antibodies may also help to focus and escalate injury. Understanding the molecular mechanisms underlying the interplay between ANCA, aberrant neutrophil behaviour and vascular damage will allow development of more focused therapies.

Anti-myeloperoxidase (anti-MPO) antibodies have been implicated in the pathogenesis of small-vessel vasculitis, but the molecular mechanisms by which these antibodies contribute to disease are unknown. For determination of how anti-MPO antibodies affect inflammatory cell recruitment in small-vessel vasculitis, intravital microscopy was used to monitor leukocyte behavior in the accessible cremasteric microvessels under various experimental conditions. After local pretreatment of the cremaster muscle with cytokines (TNF-α, IL-1β, or keratinocyte-derived chemokine), administration of anti-MPO IgG to wild-type mice reduced leukocyte rolling in favor of augmented adhesion to and transmigration across the endothelium. This led to a decrease in the number of systemic circulating leukocytes and, similar to the early events in the development of vasculitic lesions, an increase in leukocyte recruitment to renal and pulmonary tissue. TNF-α led to the greatest recruitment of inflammatory cells, and IL-1β led to the least. When anti-CD18 was co-administered, anti-MPO IgG did not affect leukocyte rolling, adhesion, or transmigration; similarly, anti-MPO IgG did not produce these effects in Fc receptor γ chain^−/− mice. This study provides direct in vivo evidence of enhanced leukocyte–endothelial cell interactions in the presence of anti-MPO IgG and highlights the critical roles of Fcγ receptors and β₂ integrins in mediating these interactions. In addition, it suggests that neutrophils primed by cytokines in the presence of anti-MPO IgG can have systemic effects and target specific vascular beds.

Endothelial cells are injury targets in vasculitis and other diseases. However, their abilities to regulate their own fate are becoming increasingly recognized and may influence their susceptibility to injury in different vascular beds.

Objective To evaluate potential mediators of endothelial cell injury in systemic vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs), we investigated the factors controlling the neutrophil respiratory burst and endothelial release of von Willebrand factor (vWF) during neutrophil–endothelial cell interactions. Methods Superoxide release from neutrophils binding to purified P‐selectin or to tumor necrosis factor–activated endothelial cells was measured under flow or static conditions using the superoxide dismutase (SOD)–inhibitable reduction of ferricytochrome c. Neutrophils were activated with fMLP, normal IgG, or ANCA IgG. Enzyme‐linked immunosorbent assay was used to measure vWF. Serine protease activity was measured enzymatically. Results ANCA IgG or fMLP induced superoxide release when perfused over neutrophils that were rolling over P‐selectin, but not those that were binding to endothelial cells. In static assays, endothelial cells inhibited superoxide production by neutrophils. Adenosine inhibited the respiratory burst, and, in cocultures, adenosine deaminase overcame the inhibitory effects of endothelial cells. Serine proteases were released during activated neutrophil–endothelial cell coculture. There was enhanced release of vWF during activated neutrophil–endothelial cell coculture; this was not inhibited by diphenyleneiodonium or by SOD plus catalase, but was inhibited by diisopropylfluorophosphate. Conclusion Endothelial cells inhibit superoxide generation by fMLP and ANCA‐activated neutrophils. The release of vWF occurs during coculture and is sensitive to serine protease, but not NADPH oxidase inhibition. Serine proteases may play a more important role than reactive oxygen species as mediators of endothelial injury during ANCA‐associated systemic vasculitis.

ANCA (anti-neutrophil cytoplasm antibody)-associated small vessel vasculitis is an inflammatory condition associated with the production of autoantibodies to neutrophil cytoplasmic components. The disorder results in destruction of the microvasculature, infiltration of neutrophils into tissues, which is followed later by mononuclear cells, leading to injury and the formation of granulomatous lesions. Initiators for the disease are undetermined but a pro-inflammatory environment is required. Other influencing factors may include environmental triggers, genetic propensity or infectious agents. The primary cellular event in the condition involves the neutrophils, which are likely to be responsible for the majority of tissue injury. Binding of the autoantibody to neutrophils initiates cell activation via a complex intracellular signalling cascade, culminating in the release of pro-inflammatory mediators, proteolytic enzymes and reactive oxygen species. Adhesion of neutrophils to endothelial cells is observed in vitro and more investigations in this area may explain the focussing of the disease to certain vessels/tissues. Current treatment regimens have substantial toxicity. Although newer developments are an improvement there is still a pressing need for more targeted therapies, which could be provided by extrapolating information emerging from basic scientific research.

Antineutrophil cytoplasm antibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. ANCA are directed against antigens expressed on the surface of cytokine-primed neutrophils. It was shown previously that whole IgG ANCA and its fraction antigen binding [F(ab′)₂] fragment can activate the GTPase p21^ras. This study shows a functional involvement of this molecule in the ANCA activation of neutrophils by inhibiting the production of superoxide with farnesylthiosalicylic acid. Using the ras activation assay, farnesylthiosalicylic acid inhibits p21^ras binding to its substrate at comparable concentrations to those seen for superoxide inhibition. It is also shown that activation of p21^ras by ANCA is transient, peaking at 5 to 10 min and returning to baseline by 30 min. The use of ras isoform–specific antibodies in Western blots established, for the first time, that Harvey-ras is not present in human neutrophils, but both Kirsten-ras (K-ras) and Neuronal-ras are. Stimulation with ANCA is able to differentially activate K-ras without effects on neuronal-ras. The activation of p21^ras by ANCA and its F(ab′)₂ is prevented by inhibition of both Src kinases and phosphatidylinositol-3-kinase, indicating a cooperative role for both molecules in the G protein pathway activated by ANCA F(ab′)₂ upstream of p21^ras. It is concluded that ANCA selectively activates K-ras during induction of a respiratory burst via pathways involving multiple upstream kinases.