Cellular Inflammation in Models of Acute Gout
Gout is a common form of inflammatory arthritis that is caused by the precipitation of monosodium urate crystals (MSU) in the joints. The acute form of gout is associated with sudden painful inflammatory episodes characterised by a large infiltration of neutrophils. The involvement of monocytes and macrophages is also recognised to be important, although the exact roles of monocytes and macrophages in gout need to be clarified. The overall objective of this thesis was to investigate the contributions of neutrophils, monocytes and macrophages to acute gouty inflammation. To determine whether the presence of other cells may be affecting neutrophil activation in gout inflammation, human blood neutrophils were stimulated with MSU as a purified population and in a mixed white blood cell population. The half-life of neutrophils in culture increased from 10h in purified neutrophil cultures to >24h in mixed cell cultures. The increase in viability was associated with large increases in cytokine production (TNF[alpha], IL-1[Beta], IL-6, IL-8) in mixed cell cultures. Exposure of neutrophils to media conditioned by MSU-stimulated mononuclear cells improved both neutrophil viability and stimulated the IL-8 production from neutrophils to a greater extent than direct contact with MSU. Exposure to conditioned media also primed MSU-stimulated neutrophil superoxide responses. High superoxide production was also observed when serum was lowered to <10%. These results indicate that neutrophil activation in gout largely occurs via soluble factors present in the pro-inflammatory environment, rather than by direct contact with MSU alone. Polygodial, a compound derived from the plant, Horopito (Pseudowintera colorata), and a number of structurally related sesquiterpene dialdehydes were tested for their potential to suppress neutrophil activation in gout. Polygodial inhibited MSU- and PMAstimulated neutrophil superoxide production in vitro with IC50 values of 0.78 [micron] and 0.16 [micron] respectively. This activity was largely dependent on the dialdehyde functionality but was enhanced by the presence of ring hydroxyl groups. The removal of hydroxyl groups was also associated with an increased cytoxicity. Polygodial and two other sesquiterpene dialdehyde compounds inhibited neutrophil superoxide production in an MSU model of acute gout, while Polygodial and a second compound also inhibited neutrophil recruitment. These studies confirm Polygodial and two structurally related sesquiterpene dialdehyde as potential anti-inflammatory compounds for potential use in gout. To confirm whether monocytes and/or macrophages were important in the onset of gout, resident macrophages and MSU-recruited monocytes were studied in a peritoneal model of MSU-induced inflammation. Gr-1+,7/4- monocytes were recruited to MSU-induced inflammation within 4h, however high cytokine production (IL-1[beta], TNF[alpha], IL-6, MCP-1) in the peritoneum preceded peak monocyte infiltration. Infiltrating monocytes did not produce high amounts of IL-6 and TNF[alpha], nor were they able to produce proinflammatory cytokines when re-stimulated with MSU ex vivo. However, resident macrophages exhibited production of IL-1[beta], TNF[alpha] and IL-6 following exposure to MSU ex vivo. Depletion of macrophages in vivo by clodronate liposomes led to a reduced recruitment of neutrophils and a lowered production of IL-1[beta] and IL-6 without affecting the recruitment of monocytes. These data identify macrophages, rather than monocytes, as key cells in initiationing and driving of inflammation in acute gout. The lack of responses from MSU-stimulated monocytes led to the question of what function, if any, was being exhibited by monocytes during MSU-induced inflammation. Therefore, the phenotype and corresponding function of monocytes was profiled over the course of MSU-induced inflammation. Newly recruited monocytes were small in size and expressed high levels of Gr-1 and 7/4, low levels of F4/80 and CD80. Over 48h the expression of Gr-1 and 7/4 was lost, while the expression of CD80 increased. These changes were associated with an increase in cell size, phagocytic capacity, and the production of pro-inflammatory cytokines in response to MSU. Changes in phenotype indicated differentiation into immature and then resident-like macrophages, and this was confirmed by PKH26-labelling infiltrating monocytes, which acquired an F4/80hi, resident macrophage phenotype after 3 to 5 days. Differentiation into macrophages was associated with an increased uptake of apoptotic neutrophils both in vivo and following ex vivo incubation. The phagocytosis of apoptotic neutrophils was associated with a reduced production of IL-1[beta] following stimulation with MSU ex vivo. These results indicate that MSU-recruited monocytes are not pro-inflammatory cells in gout, rather, they differentiate into resident macrophages over 3 to 5 days, and may over time aid in the resolution of gouty inflammation through the clearance of apoptotic neutrophils, a process that also suppresses further inflammatory responses. Together, the results of this thesis indicate that macrophages are key pro-inflammatory cells in gout contributing to neutrophil recruitment and cytokine production; that recruited neutrophils are activated by soluble mediators produced by MSU-activated cells, as well as by low serum environments; and that recruited monocytes are capable of both mediating the resolution of MSU-induced inflammation and replenishing the local resident macrophage population. These results provide a more clear model of the cellular events that occur during acute gout in humans that to date have been limited by a lack of in vivo-based studies.