Mechanisms of glomerular leukocyte trafficking

Multiple forms of glomerulonephritis (GN) result from inappropriate recruitment of leukocytes into glomeruli. As such, it is essential that the mechanisms driving leukocyte recruitment to the glomerular vasculature are understood. Using an acute model of GN induced by an antibody against the glomerular basement membrane (GBM), it was previously demonstrated that glomerular leukocyte recruitment is dependent on the initial accumulation of platelets to the glomerulus. However, the molecular mechanisms of platelet recruitment to the glomerulus under these conditions are unknown. In addition, with the use of intravital microscopy, studies have demonstrated that leukocyte recruitment to the inflamed glomerulus can occur without an initial rolling interaction normally observed in post-capillary venules. In contrast, leukocytes undergo immediate arrest within glomerular capillaries. However, the behavior of neutrophils after this initial arrest interaction is poorly understood. Multiphoton confocal microscopy (MPCM) is a form of in vivo imaging which allows continuous visualization of leukocyte behavior over extended periods. Therefore the aims of the work described in this thesis were to further our understanding of the interactions required for induction of leukocyte recruitment upon glomerular inflammation. To achieve these aims, mice were treated with anti-GBM antibody and glomeruli were visualized using in vivo imaging techniques. The first set of studies examined the molecular basis of glomerular platelet recruitment. Platelets were isolated from a donor mouse, labeled with a fluorochrome then infused into a recipient mouse during conventional intravital microscopy experiments. Using this approach, it was observed that platelets lacking the platelet collagen receptor GPVI were reduced in their ability to undergo glomerular platelet recruitment. Similarly, inhibition of the platelet integrin αIIbβ3 or fibrinogen caused significant reductions in anti-GBM antibody-induced platelet adhesion. Using immunohistochemistry, fibrinogen deposition was detected in inflamed glomeruli, occurring via a partially ICAM-1-dependent pathway. Studies in neutrophil-depleted mice undergoing glomerular inflammation demonstrated a role for neutrophils in glomerular platelet accumulation. In contrast, inhibition of adenosine diphosphate-dependent platelet activation did not affect platelet recruitment to the inflamed glomerulus. Taken together, these findings indicate that the combined actions of GPVI and the αIIbβ3/fibrinogen/ICAM-1 pathway contribute to platelet recruitment in the inflamed glomerulus. The aim of the second set of studies was to use in vivo MPCM to investigate leukocyte recruitment to the inflamed glomerulus. This was achieved by visualizing glomerular capillaries and neutrophils by labeling them with specific fluorochromes. Under control conditions, neutrophils were observed to adhere and persist in the glomerular capillaries for an average of 5 minutes (‘dwell time’), with some of these cells remaining static for this period, and others undergoing migration within the vasculature. In contrast, in response to anti-GBM antibody, neutrophil dwell time was significantly increased in the first two hours post anti-GBM antibody treatment, with this change applying to both static and migratory cells. This study extends our understanding of neutrophil behavior following recruitment to the inflamed glomerulus showing that induction of glomerular inflammation does not alter the number of leukocyte recruited to the glomerulus, but increases the duration of their retention. The aim of the next series of experiments was to utilize the MPCM imaging approach to characterize the mechanisms of glomerular leukocyte recruitment. Inhibition of the β2 integrin significantly reduced the dwell time of migrating neutrophils in the first hour of the anti-GBM antibody response, whereas during the second hour, inhibition of the β2 integrin Mac-1 significantly reduced neutrophil dwell time for both static and migratory cells. Anti-Mac-1 treatment also significantly decreased urinary protein excretion, indicating that Mac-1 is a key leukocyte integrin responsible for leukocyte recruitment to inflamed glomerular capillaries. Finally, direct examination of neutrophils during glomerular inflammation in vivo demonstrated that both static and crawling neutrophils generated oxidants in inflamed glomeruli. In summary, these experiments demonstrate that the process of glomerular leukocyte recruitment is more dynamic than previously recognized. Moreover, the application of new imaging technique has revealed novel information about neutrophil behavior in the glomerular vasculature