an acute attack of gouty arthritis includes clinical features such as joint inflammation , erythema , fever , and extreme , unbearable pain resulting in reduced mobility of the afflicted joint . the symptoms are caused by monosodium urate monohydrate ( msu ) crystal deposition into the intra - articular joint spaces . crystal precipitation provokes mass polymorphonuclear ( pmn ) leukocyte infiltration into the joints where these cells release inflammatory cytokines which mediate joint damage [ 4 , 5 ] . however , in the absence of clinical intervention , gout is self - limiting in nature and is able to spontaneously resolve over a period of a week [ 69 ] . the exact theory behind the resolution phase of gout remains an enigma although macrophages and their cofactors have been implicated in the mechanism [ 1012 ] . platelet activating factor ( paf , 1 - 0-alkyl-2-acetyl - sn - glycero-3-phosphocholine ) was discovered in 1972 , it is known as one of the most potent phospholipids , involved in platelet aggregation and anaphylaxis . it is produced by a variety of immune cells including monocytes / macrophages , neutrophils , eosinophils , basophils , and platelets [ 14 , 15 ] . there are two main pathways which lead to cellular synthesis of paf , the de nova pathway and remodelling pathway . inflammatory or immune stimuli induce paf primarily via the remodelling pathway whereby membrane bound paf - acetylhydrolase ( paf - ah ) which is an extracellular phospholipase a2 catalyses the transfer of an acetyl residue from acetyl - coa to 1-alkyl - sn - glycero-3-phosphocholine ( lyso - paf ) . however , the full mechanisms and the exact role of various enzymes on paf degradation have not been fully elucidated to date . to exert its effect , paf binds directly to a specific g protein coupled receptor and paf is well regarded as a potent mediator of inflammatory pathologies such as acute pancreatitis , diabetes , and renal failure . however , recent studies suggest paf could also play a crucial role in regulating immune responses . studies have highlighted the necessity of paf receptor activation in deactivation of ultraviolet b ( uvb ) induced suppression of hypersensitivity responses . in addition , paf administration to equine alveolar macrophages increased phagocytic capacity and superoxide anion production . moreover , during immunological defence , paf can play an immunomodulatory role including monocyte or macrophage degranulation , cytokine release , phagocytosis , and cell adhesion [ 25 , 26 ] . lps , antimonoclonal anti - human cd62 , anti - mouse igg fitc , and mouse igg1 were purchased from sigma - aldrich ( poole , uk ) . recombinant human paf - acetylhydrolase was purchased from sigma - aldrich , usa . leucocyte rich cones were obtained from volunteer donors collected from nhs cord blood and transplant bank at colindale , london . the leucocyte rich cells were then spun on histopaque at 1200  rpm for 20 mins . the mononuclear portion was obtained and washed in hanks balanced solution . cells were allowed to adhere for an hour after which nonadherent cells were washed and full media replenished with dulbecco 's media containing ab serum . differentiated macrophages were determined by light microscopy and flow cytometry phenotypic analysis of differentiation markers . cells were washed and fed at days 1 , 3 , 5 , and 7 of culture and stimulated with lps ( 10  g / ml ) or msu crystals ( 0.5  mg / ml ) with or without pretreatment with web2086 for 1624 hours after which supernatants were collected and stored in aliquots at 70c prior to analysis . pretreatment with rhupaf - ah at concentrations of 2.520  g / ml was carried out for half an hour prior to addition of msu crystals to monocyte cells . paf and tnf levels in cultured supernatants were determined by elisa ( purchased from blue gene biotech , china , and r and d systems europe , resp . ) using manufacturer 's recommendations . for paf determination , culture supernatants were pretreated with balance solution provided in the elisa kit , mixed , and then processed according to manufacturer 's instructions . all samples were measured in duplicate , with results expressed as the mean  sem cytokine concentration ( ng / ml ) from at least 4 experiments . indirect flow cytometric analysis was performed as previously described ( 12 ) , using primary antibodies at 10  g / ml and secondary antibodies at 1  :  64 dilution . endothelial cells were detached by trypsin / edta and the trypsin activity was quenched in excess growth medium supplemented with serum . monocyte / macrophage cultures were detached by 15-minute incubation in ice - cold phosphate buffered saline ( pbs ) containing 2.5  mm of edta , followed by scraping and washing in growth medium containing serum . data analysis was performed using a flow cytometer analyser purchased from beckton dickinson ( bd ) , uk . huvec e - selectin expression was calculated by dividing the staining intensity ( mean fluorescent intensity ( mfi ) ) of e - selectin by the staining intensity of class - matched control antibody , thus resulting in a relative fluorescent intensity ( rfi ) of expression ( where an rfi of 1.00 is equivalent to no antigen expression ) . results were expressed as the mean  sd rfi from at least 3 separate experiments . analysis of inhibition by web2086 on tnf secretion and endothelial cell e - selectin expression was carried out using 1-way analysis of variance ( anova ) with dunnett 's correction . this detection of paf occurs at day 5 and peaks at day 7 of monocyte differentiation . the phenotype of monocytes as they differentiate into macrophages has been well characterised and it has previously been postulated that they can acquire an anti - inflammatory phenotype . a similar model showed that at days 57 of monocyte culture , upon stimulation with msu crystals , an anti - inflammatory phenotypic response was enabled . this was corroborated by the detection of tgf1 secretion by human in vitro differentiated macrophages in response to msu crystal challenge . coincubation of day 7 in vitro differentiated macrophages with the paf receptor antagonist ( web2086 ) and msu crystals resulted in a decrease in paf secretion ( figure 2 ) . to investigate whether paf production was autocrine suppressive , msu crystal stimulated macrophages were incubated in the presence of increasing concentrations of the paf receptor antagonist and this time tnf production was analysed . inclusion of web2086 at concentrations of 25100  m in these cultures resulted in peak tnf production ( figure 3(b ) ) . in fact , this tnf production was comparable to that released by day 1 monocytes after msu crystal stimulation which had been determined in a previous study . prior investigations have reported that macrophage cell lines and human in vitro differentiated macrophages can ingest msu crystals without releasing inflammatory cytokines ( tnf , il-1 , and il-6 ) [ 11 , 12 ] ; indeed , it was identified that instead macrophages produced anti - inflammatory tgf1 . in addition , it was observed that tgf1 was able to inhibit endothelial cell activation and e - selectin expression . essentially in this study coincubation of endothelial cells with anti - tgf1 and a mixture of day 1 and day 7 msu crystal stimulated supernatants unmasked only a partial degree of endothelial e - selectin reexpression even with using maximum concentrations of neutralising antibody against tgf1 . this suggested that other anti - inflammatory factors could also be released by macrophages upon phagocytosis of msu crystals . another anti - inflammatory model whereby macrophages play an active role in regulating inflammation and resulting in paf mediated activity by macrophages is the phagocytosis of apoptotic neutrophils . indeed , this mechanism also results in a lack of proinflammatory cytokine release and associated inflammation [ 2729 ] . pretreatment of freshly isolated monocytes with rhupaf - ah and msu crystals resulted in partial suppression of tnf release . full suppression of tnf was not expected as manufacturers reference data indicated that rhupaf - ah cleaved the paf analogue by approximately 50% at 10  g / ml , which was observed in figure 3(a ) . it is likely that paf exerts an immunomodulatory effect on msu crystal stimulated macrophages as coincubation in the presence of web2086 unmasked tnf production , implicating that paf may be involved in autocrine and paracrine suppression of macrophages . there are studies that highlight paf 's role in endocrine , autocrine , and intercrine signalling . endothelial activation precedes the inflammatory response by msu crystals which has been observed in vitro and in vivo . an antagonist of paf was able to reduce e - selectin mediated endothelial activation ( figure 4 ) . although paf is well regarded as a potent mediator of inflammation , emerging research suggests that paf can mediate inflammation control [ 3032 ] . impaired anaphylactic responses were reported in mice lacking the paf receptor [ 23 , 26 ] . another study showed that mice stimulated with lps were protected from endotoxin shock when injected with paf systemically . in these mice , a reduction in proinflammatory cytokine secretion , namely , interferon gamma , il-1 , and tnf , was also observed . researchers have highlighted the role of paf mediated interference in microbial associated damage as well . paf was pivotal in the extent of pathological consequences due to h. pylori infection and gastric mucin synthesis . moreover , prophylactic administration of paf antagonist to rats with gastric injury exerted an anti - inflammatory effect reducing mucosal apoptosis , tnf , and nitric oxide synthase 2 activity and resulted in rapid gastric ulcer healing . in fact , paf has been identified as a molecular sensor recognising cellular damage and exerting effects on cytokines and cox-2 transcription which resulted in systemic immune suppression . deposition of msu crystals into intra - articular joints causes joint swelling and joint damage as well as oxidative stress [ 2 , 3 , 5 , 37 ] . hence , it is feasible that a similar sensory mechanism of cell damage detection by paf and paf receptor signalling could be underpinning gouty arthritis . this study identifies macrophage derived paf and paf related molecules in the noninflammatory phagocytosis of msu crystals by human blood derived differentiated macrophages . in this model , the paf receptor antagonist was able to have a number of effects ; it was able to inhibit endothelial cell activation measured by e - selectin upregulation and was also implicated in downregulating tnf secretion by msu crystal stimulated monocyte cells . essentially , the immune response to msu crystals is clearly multifaceted in nature involving a complex interplay by inflammatory and noninflammatory mechanisms involving various leukocyte derived factors which act in coordination during the resolution phase of gouty arthritis . noticeably , little is known about the role that paf - acetylhydrolases play in paf activation and degradation or how paf exerts its effects through its receptors . the model of monocyte , macrophage , and msu crystal induced inflammation could be used to further examine these molecular mechanisms and unravel the associated paf signalling and receptor pathways . in conclusion , this study suggests a role for paf and related molecules in the anti - inflammatory pathway by which macrophages phagocytose msu crystals .
human blood derived in vitro differentiated monocytes or macrophages are a population of cells which have been investigated over the years to determine the role these cells play in the resolution phase of gout . 
 macrophages are able to phagocytose monosodium urate monohydrate ( msu ) crystals without releasing inflammatory factors . 
 this study analysed macrophage platelet activating factor secretion and its possible role in the pathway of gout resolution . 
 analysis of sunatants from in vitro differentiated macrophages stimulated with msu crystals revealed the secretion of platelet activating factor ( paf )  1.54  0.10 mean  sem ;  ng / ml per 106 cells . 
 this secretion was absent in immature monocytes treated similarly . 
 when these monocytes were pretreated with recombinant human paf - acetylhydrolase ( rhupaf - ah ) and msu crystals resulted in tnf suppression . 
 addition of web2086 , a platelet activating factor ( paf ) antagonist , to differentiated macrophages with msu crystals unmasked tnf secretion 0.7  0.06 mean  sem ;  ng / ml per 106 cells . 
 this study identifies a role for paf and the paf receptor antagonist in the pathway by which macrophages ingest msu crystals and resolve the concomitant inflammation .