Tissue macrophages are a diverse group of specialized cells that are pivotal in host defense, wound healing and immunoregulation [27
]. They are thought to arise from circulating monocytes, which migrate to tissues and develop specific phenotypes such as Kupffer cells in the liver, microglial cells in the central nervous system and OC in bone [27
]. Circulating monocytes are a heterogeneous population characterized by specific cell surface markers with unique proliferative and physiological properties that differentiate into specific effector cells in response to signals present in blood and tissues. Among these effector cells, OC are of particular interest due to the central importance of these cells in pathologic bone resorption in inflammatory arthritis. In previous studies, we demonstrated that the frequency of circulating OCP was greatly increased in a subset of PsA patients particularly those with joint destruction [3
]. Herein, we show that PsA patients have an elevated percentage of CD14+CD16+ pro-inflammatory monocytes in the peripheral blood. Based on the observation of CD16 up-regulation in cells cultured in OC-promoting (M-CSF and RANKL) but not DC-promoting conditions (GM-CSF and IL-4), we examined whether CD16 up-regulation also occurs in vivo
and identified cell subsets that expressed intermediate and high levels of CD16 in PsA. Intriguingly, we found OC arise from circulating CD16+ monocytes in PsA, whereas OC were generated from the CD16- subset in healthy controls. Finally, we showed a positive correlation between the level of CD16 cell surface expression and the extent of bone resorption. These studies indicate that the major reservoir of OCP in PsA is CD16+ cells, a finding that may catalyze the development of susceptibility biomarkers for arthritis in Ps patients and a treatment response marker in PsA patients with erosive arthritis.
In general, the CD14+CD16+ monocyte subset is thought to be more mature than the CD14+CD16- subset [6
monocytes release high levels of pro-inflammatory cytokines and manifest phenotypic and functional characteristics of macrophages and DC [4
]. Our results provide evidence that the CD16+ cells can also differentiate into OC. The existence of CD16int
cells with a lower level of bone resorption suggests that monocytes exhibit a transitional state in OC differentiation. Additional support for the plasticity of CD16 expression in these cells was the successful conversion of freshly sterile-sorted CD16- monocytes into CD16+ cells following overnight culture in media without exogenous cytokines (data not shown). Collectively, these data (Figure ) suggest that monocytes undergo a transition stage with intermediate expression of CD16 before differentiating into OC, presumably following exposure to RANKL and M-CSF in the bone marrow, circulation, and the joint. It is important to note that the clinical significance of CD14+CD16+ expansion may depend on the disease state. For example, although CD14+16+ cells are increased in psoriasis, inflammatory arthritis and sepsis, the differentiation fate of these cells is determined by cytokines and chemokines in the local microenvironment. It is likely that dendritic cells and inflammatory monocytes predominate in the case of sepsis, whereas osteoclastogenic cytokines foster OC differentiation in inflammatory arthritis.
Consistent with results of Komano and colleagues [24
] and Lari and colleagues [29
], our sorting results from HCs indicate that OC are derived from the CD16- monocyte subset. We were puzzled by our contradictory data in PsA subjects where the majority of OC were derived from CD16+ cells. One plausible explanation is that elevated levels of inflammatory cytokines and chemokines in PsA subjects promote the up-regulation of CD16 on monocytes. As a result, monocytes isolated from PsA subjects are more responsive to osteoclastogenic factors than those isolated from healthy individuals. Support for this concept was the finding that CD16 cell surface expression increased when cells were cultured with TNF or osteoclastogenic (RANKL + M-CSF) but not with DC-promoting cytokines (GM-CSF + IL-4) in vitro
. Moreover, intermediate surface expression of CD16 noted in many Ps and PsA subjects may represent a transitional state in which monocytes are primed for osteoclastogenesis in response to environmental signals.
Although the importance of CD16 in immune regulation was emphasized by its critical role in uncontrolled systemic infection and sepsis [16
], the function of CD16 in osteoclastogenesis remains largely unknown. Based on current available data, CD16 might be involved in the regulation of OC development through its ITAM. CD16 is considered an ITAM-bearing molecule due to its association with FcRγ [16
]. The regulation of signaling through Fc receptors such as CD16 is extremely complex. With different affinities to ligand engagement, ITAM-containing Fc receptors generate either activating or inhibitory immune response signals [30
]. Furthermore, cross-regulation and interaction between ITAM-associated receptors greatly magnifies the complexity of this regulation [31
]. We propose a similar complex regulation of osteoclastogenesis by CD16 through its ITAM. To date, except for CD16, many ITAM-bearing surface receptors involved in OC differentiation have been well studied [32
]. Humphrey and colleagues proposed a model whereby signals delivered by surface ITAM-bearing proteins regulate the expression of many genes involved in osteoclastogenesis [33
]. This model provides a mechanism to explain the regulation of OC formation and provides an explanation for the positive correlation between the CD16 surface expression and bone erosion activity shown in this study. Current data suggest that ITAM-bearing proteins might act in concert to program cells into a fusion-competent state [35
], regulate the multinucleation process [36
], and recruit Syk kinase [33
], similar to the model proposed by Humphrey and colleagues [33
]. Understanding of the interactions between CD16 and other ITAM-bearing proteins will likely reveal the contribution of CD16 to osteoclastogenesis at the molecular level.
CD16 regulates the production of TNFα by both direct and indirect mechanisms. Binding of CD16 by Escherichia coli
triggers TNFα secretion [16
], and conversely, a dramatic decrease in TNFα production is observed in FcγIII-deficient mice [37
]. Recently, Kramer and colleagues revealed how CD16 activation regulates TNFα production [38
]. Activation of CD16 induces TNFα through the mitogen-activated protein kinase pathway but at the same time, CD16 activation can also limit TNFα production through phosphoinositide 3-kinase signaling. Of relevance to these findings is the fact that the proinflammatory CD14+CD16+ monocytes are major sources of TNFα, a cytokine that potentiates osteoclastogenesis [13
], and IL-6, a cytokine that promotes OC maturation [39
]. Therefore, it is likely that the elevated TNFα in PsA patients is partially responsible for the increased OCP in these subjects. Currently, we do not know if the expanded CD14+CD16+ cells are a major source of TNFα in PsA; however, it appears that TNFα does not induce CD14+CD16+ expansion through 'autocrine' regulation, because the frequency of CD14+CD16+ cells decreases in the presence of TNFα (Figures and vs. and ). TNFα has a prominent inhibitory effect on CD16 cell surface expression for a subset of CD14+ monocytes (indicated by arrows in Figure and ). This inhibitory effect of TNFα on CD16 expression was observed for all samples we processed including controls. Currently, it is unclear why TNFα blocks CD16 expression in a particular monocyte subset, resulting in two distinct CD14+ populations (Figures and , upper left and right panels). It will be important to determine if the CD14+ cells with high CD16 expression are more likely to differentiate into OC compared with CD14+ cells with an intermediate level of CD16.
In conclusion, OCP derived from PsA patients display several unique properties compared with OCP that arise from HC, providing further support to our previous studies [3
]. We showed that OCP arise from different monocyte populations in PsA subjects and healthy controls. In addition, TNFα upregulation of CD16 cell surface expression on CD14+CD16+ cells was significantly greater in PsA patients than in HC. We also demonstrated an expansion of circulating CD14+CD16+ monocytes in PsA subjects and identified cells that express intermediate levels of CD16. Moreover, surface expression of CD16 correlated with the extent of bone resorption in vitro
for PsA monocytes. Collectively, our results suggest a model in which a subset of CD16- cells undergoes a transition to intermediate CD16 expression in response to inflammation, and subsequently differentiate into CD16+ cells prior to OC formation. Thus, CD16 may be a marker for OCP in PsA patients, although it is highly probable that additional molecules that specifically characterize this population will be identified. From a translational perspective, inhibition of the CD16- to CD16+ transition in circulating monocytes may have clinical applications for the treatment of metabolic and inflammatory bone disorders.