Aggravation of Anti-Myeloperoxidase Antibody-Induced Glomerulonephritis by Bacterial Lipopolysaccharide (1)
Anti-neutrophil cytoplasmic autoantibodies (ANCAs) comprise a group of autoantibodies directed against proteins contained in the lysosomal compartments of neutrophils and monocytes. An experimental animal model of anti-MPO- induced NCGN was developed that involves the adoptive transfer of mouse MPO-reactive splenocytes into im- mune-deficient mice. These mice developed severe NCGN with pathological features that were remarkably similar to human anti-MPO-associated glomerulonephritis.
Induction of Glomerulonephritis by Passive Transfer of Anti-MPO IgG
C57BL/6 mice received a dose of 100 ug/g body weight of sterile-filtered anti-MPO IgG by intraperitoneal injection. Where stated, groups of mice additionally received a single intraperitoneal injection of 5.0, 0.5, or 0.05 g/g of LPS (Escherichia coli, serotype 026-B6) dissolved in sterile PBS 1 hour after the administration of IgG.
On day 1, no differences were detected in levels of circulating anti-MPO antibodies between the experimental groups. However, on day 6, mice that received the highest dose of LPS (5 ug/g) had a significantly lower anti-MPO titer compared to mice receiving anti-MPO alone.
By day 1, hematuria had developed in all mice that received anti-MPO antibodies with or without LPS, which persisted until the time of sacrifice at day 6. Most of these mice also had proteinuria and leukocyturia at day 6.
Addition of 5.0 ug/g LPS resulted in 20.4% (range, 16 to 26%) crescentic and 22.8% (range, 12 to 36%) necrotic glomeruli, whereas addition of 0.5 g/g LPS resulted in 20.8% (range, 14 to 26%) crescentic and 28.4% (range, 22 to 34%) necrotic glomeruli on day 6.
A further increase in the number of glomerular infiltrating CD45 leukocytes and FA11 macrophages was observed on addition of 5.0 or 0.5 ug/g LPS.
An early glomerular influx of neutrophils was markedly enhanced on addition of 0.5 g/g LPS resulting in large neutrophilic aggregates.
Intraperitoneal administration of 5.0, 0.5, and 0.05 ug/g LPS led to a rise in serum TNF- levels 1 hour after injection. In case of the high (5.0 ug/g) and medium (0.5 g/g) dose of LPS, this was followed by a rise in serum MPO levels at day 1.
Pathological analysis at day 6 revealed that pretreatment with TN3 significantly attenuated glomerular crescent formation.
Immunohistochemical analysis showed that pretreatment with TN3 significantly attenuated glomerular influx of CD45 total leukocytes and FA11 macrophages.
Peritoneal exudate cells (PECs) were obtained by flushing of the peritoneal cavities of three to four WT or Mpo/ mice with sterile Hanks’ balanced salt solution (HBSS) 4 hours after intraperitoneal administration of 1 ml of 3% thioglycollate. When peritoneal exudate cells were pretreated with 10 ng/ml of murine TNF- for 10 minutes, incubation with anti-MPO IgG resulted in a modest but significant increase in superoxide production as compared to normal mouse IgG or buffer alone.
Priming with TNF- is necessary for the induction of a respiratory burst by sera or purified IgG from ANCA- positive patients. doses of LPS that aggravated anti-MPO IgG-induced glomerulonephritis also gave rise to increased levels of circulating MPO. Based on these observations we speculate that circulating MPO, in the presence of high levels of anti-MPO antibodies, may disperse MPO-ANCA-mediated activation to resting neutrophils resulting in amplification of the inflammatory response.