Acute gout is an intensely painful form of inflammatory arthritis caused by the formation of monosodium urate monohydrate (MSU) crystals in the joint and connective tissues. An acute attack of gout is characterized by intense pain and swelling and reddening of the skin around the affected area. Without treatment this inflammatory response will resolve naturally over 7–10 days.
Interleukin-1β (IL-1β) has been identified as a pivotal cytokine in gout and MSU crystal–induced inflammation (1–3). More recently, studies have identified IL-1β production following activation of the NLRP3 inflammasome in resident macrophages as a key event in the initiation of an attack of acute gout (4–7). However, there is a growing body of evidence indicating that cells of the monocyte/macrophage lineage play a pivotal role not only in the initiation but also in the progression and resolution of acute gouty inflammation.
It has been proposed that the self-limiting nature of an acute gout attack is linked to the differentiation state of the MSU crystal–recruited monocyte population. In vitro studies indicate that isolated blood monocytes that are differentiated into macrophages switch from producing proinflammatory cytokines to producing the antiinflammatory cytokine transforming growth factor β1 (TGFβ1) in response to MSU crystal stimulation (8–10). These data would indicate that monocytes drive and differentiated macrophages resolve inflammation in gout. However, other studies show that MSU crystals induce a classic proinflammatory response in both primary and bone marrow–derived macrophages (3, 4, 11–14), indicating a key role for macrophages in the initiation of the inflammatory cascade. In addition, there is evidence to suggest that upon recruitment to the site of inflammation, early infiltrating monocytes do not exhibit a proinflammatory phenotype and therefore may not be driving the early phase of inflammation in gout (5). Together, these findings raise questions about the true functional phenotypes expressed by MSU crystal–recruited monocyte/macrophage populations in vivo.
There is extensive evidence illustrating the ability of cells from the monocyte/macrophage cell lineage to express a diverse range of activities in response to changes in their local environment (15). Two general populations of macrophages, M1 and M2, have been classified based on their environmental drivers and their resulting phenotype (16). While M1 macrophages exhibit a proinflammatory phenotype that is classically driven by lipopolysaccharide (LPS) and interferon-γ, M2 macrophages express an antiinflammatory/repair phenotype that is driven by Th2 cytokines and glucocorticoids. The clearly opposing functions of the M1 and M2 macrophage phenotypes highlight the need to provide a physiologically relevant immune environment for the investigation of monocyte differentiation, and emphasize the importance of investigating the differentiation of MSU crystal–recruited monocytes in vivo.
The aim of this study was to profile the differentiation pathway of MSU crystal–recruited monocytes in vivo to determine whether the infiltrating monocyte population differentiates into a proinflammatory (M1-like) or antiinflammatory (M2-like) macrophage phenotype in a murine model of acute MSU crystal–induced inflammation.
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Contrary to the findings of previous in vitro studies, our results indicate that during the early stages of MSU crystal–induced inflammation, the newly recruited monocyte population exhibits a noninflammatory phenotype that develops into a proinflammatory M1-like phenotype in vivo. Increased responsiveness to MSU crystal restimulation is associated with differentiation of the recruited monocyte population (F4/80lowGr-1int7/4+) toward a macrophage phenotype (F4/80highGr-1–7/4–) and morphology, an increase in phagocytic capacity, and an increase in the expression of proIL-1β.
Reports of earlier in vitro studies investigating the effect of monocyte differentiation on responsiveness to MSU crystals propose that proinflammatory monocytes differentiate into noninflammatory or suppressive macrophages. However, those studies have predominantly used panels of monocyte/macrophage cell lines or primary monocytes differentiated in vitro over several days in sterile media (8–10, 19, 20). These in vitro environments differ significantly from the highly complex and dynamic inflammatory environment encountered by MSU crystal–recruited monocytes in vivo. One possible reason for the apparent lack of a proinflammatory macrophage response in earlier in vitro studies is the absence of continual low-level Toll-like receptor (TLR) signaling by TLR ligands that is linked with priming of intracellular stores of proIL-1β in vivo (3, 21). Without this natural priming event, cultured macrophages would be unable to raise a proinflammatory response to MSU crystals in vitro. A key strength of the current study is that MSU crystal–recruited monocyte differentiation occurs during the course of the MSU crystal–induced response in vivo. As such, this approach provides a more accurate representation of the natural process of monocyte-to-macrophage differentiation.
In this study we confirm that early infiltrating monocytes are unresponsive to MSU crystal stimulation ex vivo. This finding provides further evidence that the resident macrophage, rather than the newly recruited monocytes, initiates the primary phase of inflammation in vivo (5). The lack of proinflammatory activity by early-recruited monocytes appears to be primarily associated with low levels of proIL-1β. The activation of the NLRP3 inflammasome and the cleavage of proIL-1β by active caspase 1 to release active IL-1β are important steps in the initiation of MSU crystal–induced inflammation (4–7). As a result, low levels of proIL-1β would significantly block the ability of newly recruited monocytes to raise a proinflammatory response to MSU crystal restimulation, despite the presence of active caspase 1.
The generation of proIL-1β pools has been shown to be impaired in CD14-knockout macrophages (7). It is therefore possible that the low CD14 expression observed on MSU crystal–recruited monocytes plays a key role in blocking proIL-1β accumulation, resulting in a noninflammatory monocyte phenotype. This may be compounded by the poor phagocytic activity of early- recruited monocyte/macrophages. It has been proposed that CD14 rather than phagocytosis of crystals is important for IL-1β production (7). However, it is more likely that a certain degree of codependency exists, whereby CD14 signaling is necessary for proIL-1β production but crystal phagocytosis is required to trigger activation of the NLRP3 inflammasome and release of active IL-1β. Therefore, in the absence of either CD14 expression or phagocytic function, MSU crystal–induced activation of monocyte/macrophages may not occur.
Our data now show that MSU crystal–recruited monocytes differentiate into an M1-like (proinflammatory) functional phenotype in vivo, as illustrated by the ability of day 3 differentiated monocytes to produce the classic proinflammatory molecules following MSU crystal restimulation ex vivo. Our data also show that significant accumulation of proIL-1β and procaspase 1, in association with increased CD14 expression and phagocytic capacity (22), may be priming the emerging macrophage phenotype for hyperresponsiveness to MSU crystal stimulation.
It is interesting to note that elevated intracellular expression of the active form of IL-1β in MSU crystal–recruited M1-like macrophages did not correlate with elevated IL-1β expression in vivo, indicating that active IL-1β was not being secreted. This provides evidence for the accumulation of a pool of active IL-1β in M1-like macrophages. The availability of this pool of IL-1β for immediate release upon MSU crystal stimulation may facilitate the rapid, exacerbated inflammatory response observed following MSU crystal restimulation in vivo.
Contrary to the development of an M1-like macrophage phenotype, TGFβ1 production by unstimulated MSU crystal–recruited cells was observed ex vivo. However, the absence of the M2 surface marker (CD206) indicates that this TGFβ1 production is not associated with the development of M2 macrophages. Instead, the observed increase in background TGFβ1 production over time is likely associated with an increase in the ability of the differentiating monocyte/macrophages to phagocytose apoptotic neutrophils, a process strongly linked with TGFβ1 production, neutrophil clearance, and resolution of acute inflammation (23–25). Whether TGFβ1 production plays a role in the accumulation of intracellular IL-1β in the recruited M1-like macrophage, possibly by blocking cytokine secretion, remains to be determined.
Interestingly, the ex vivo restimulation data indicate that MSU crystal activation can abrogate TGFβ1 production linked with neutrophil clearance. At this time it is not clear how this occurs. However, it does illustrate that the differentiating recruited macrophage has the capacity to respond to MSU crystals and shut down TGFβ1-driven resolution, independent of the ability to release proinflammatory cytokines. The in vivo rechallenge data provide further evidence that the emerging proinflammatory M1-like macrophage can override the process of resolution and is capable of initiating a second, exacerbated inflammatory insult. Interestingly, features of this secondary response appear to be more strictly regulated, leading to a more rapid shutdown of proinflammatory cytokine production and a significant drop in the accumulation of monocytes and macrophages over time. Nevertheless, it appears that in the first instance both the development of the proinflammatory M1-like macrophage phenotype in vivo and the continued deposition of fresh MSU crystals at the site of inflammation are necessary for overriding resolution and driving ongoing inflammation in an attack of gout.
Historically, neutrophils have been widely viewed as the major driving force for orchestrating inflammation in and resolution of gout attacks. Both MSU crystals and the inflammatory cytokine environment have been shown to prolong neutrophil survival and stimulate neutrophil IL-8 (CXCL8) and superoxide production, factors that can contribute to ongoing inflammation (26–30). In contrast, neutrophil apoptosis and clearance by phagocytic cells are recognized as playing a key part in TGFβ1 production and resolution (24, 26, 28, 31, 32). Based on our findings, the differentiation of recruited monocytes into M1-like macrophages may play an equally important role in directing the progression of inflammation in gout. These macrophages appear to be able to contribute to inflammation through the expression of a functional inflammatory phenotype, but they may also represent the key phagocytic cells involved in neutrophil clearance, thereby facilitating resolution. Although care needs to be taken when directly extrapolating the M1-like functional response ex vivo to the in vivo environment, the in vivo restimulation data provide strong evidence that the observed proinflammatory M1-like macrophage phenotype is functionally relevant in vivo.
The results of this study unveil a new level of complexity in our understanding of the progression of gouty arthritis, whereby recruited monocytes, along with neutrophils, contribute to the progression of inflammation in and resolution of gout (Figure 6). In this model, resident macrophage activation initiates the inflammatory cascade, resulting in the recruitment of neutrophils and monocytes. Early MSU crystal–recruited monocytes exhibit a “nonresponsive” phenotype in vivo. However, during this early phase, recruited neutrophils may respond to both MSU crystals and local mediators of inflammation to produce CXCL8 and drive self-recruitment. Over time, the recruited monocyte population down-regulates expression of markers of hemopoiesis and becomes primed to respond to ongoing MSU crystal deposition via increased phagocytic capacity and expression of CD14, and via the accumulation of proIL-1β, active caspase 1, and intracellular IL-1β, consistent with differentiation into an M1-like macrophage. In the absence of ongoing inflammatory stimulation, these phagocytic macrophages contribute to neutrophil clearance and resolution of the inflammatory response. However, the stimulation of these proinflammatory M1-like macrophages with fresh MSU crystals then overrides TGFβ1-dependent resolution and drives a secondary wave of inflammation in vivo.
Figure 6. Phenotypic profile of in vivo monocyte-to-macrophage differentiation in MSU crystal–induced inflammation (see Discussion). Ly-6C = lymphocyte antigen 6 complex, locus C (see Figure 1 for other definitions).
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This study serves to further emphasize the integral involvement of the monocyte/macrophage phenotype in the inflammation profile of a gout attack. Importantly, our data show for the first time that differentiation of MSU crystal–recruited monocytes into a proinflammatory M1-like macrophage phenotype in vivo, combined with ongoing MSU crystal deposition, may play a significant role in abrogating resolution and perpetuating inflammation in gout.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Harper had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Martin, Harper.
Acquisition of data. Martin, Shaw, Liu, Steiger.
Analysis and interpretation of data. Martin, Shaw, Steiger, Harper.