This, to our knowledge, is the first study to show an association between RF, IgG levels in SF ICs, and SF IC induced levels of TNF-α in RA. We also report that IC-induced TNF-α production is partly mediated via FcγRIIa with monocytes/macrophages as the main or perhaps only responder cells. These findings support the hypothesis that ICs in joints might provide a direct link to cytokine-dependent inflammation in RA, at least in RF-positive patients.
A stronger association between cytokine induction, IgG levels and RF was apparent for the SF precipitates than for serum precipitates, which is in agreement with the general belief that RF levels in serum reflect inflammation in the joints. RF has been associated with ICs in several diseases other than RA [1
]. RF can also be produced after vaccination in healthy individuals during the time interval when antibodies and antigen form circulating ICs [4
]. RF-producing B cells are present in the inflamed joints of RA patients [22
] and RF measured in serum might therefore mirror the production of RF in RA joints. We also attempted to measure RF in SF, but for technical reasons we only achieved measurable values in 59% of the cases. Nonetheless, in the measurable subgroup of patients there was a considerably stronger association between SF PEG precipitate induced TNF-α production and RF in SF as compared with conventional RF measured in serum. This finding strengthens our hypothesis that serum RF is merely a reflection of RF produced in the inflamed joints in response to IgG-containing ICs with TNF-α-inducing properties. Moreover, our findings of stronger cytokine-inducing properties of ICs obtained from joints of RF-positive RA patients is consistent with the fact that seropositive RA is associated with a more severe disease outcome [2
Anti-CCP antibodies have been shown to be highly specific for RA [23
] and more strongly associated with joint destruction than RF [24
]. As noted in several earlier studies, we saw a positive correlation between RF and anti-CCP in serum. However, we did not find any associations between anti-CCP and IC-induced TNF-α production or IgG levels in the PEG precipitates. Therefore RF per se
and not the RF-correlated anti-CCP levels appear to be associated with IC-induced TNF-α and consequent joint inflammation.
PEG precipitation is a well recognized technique for the isolation of high-molecular-weight ICs. However, earlier investigations showed PEG-precipitated sera to contain uncomplexed immunoglobulins, C3 [25
] and a number of serum proteins including fibronectin and albumin [26
], besides IC containing IgG plus IgA and IgG plus C3. The view that PEG precipitates are composed only of ICs is therefore too simplistic. Because our cross-sectional approach employed a large number of ICs freshly prepared with an aseptic technique, we avoided the use of alternative, time-consuming techniques such as gel filtration and sucrose gradient centrifugation. To further determine IC content in our precipitates we measured IgG content in the precipitates and showed that IC-induced cytokine induction was dependent on binding to FcγRIIa that, because of its low affinity, preferably binds ICs over monomeric IgG [27
Control experiments have shown that PEG precipitation of ultracentrifuged NHS or RF positive sera devoid of preformed ICs do not enhance TNF-α-inducing effects compared with serum added directly to the cell cultures without prior PEG precipitation. PEG precipitates from ultracentrifuged RF-negative NHS or from ultracentrifuged RF-positive sera induce comparable levels of TNF-α when they are added to responder PBMC cultures. These findings imply that neither PEG precipitation nor RF per se induce IC formation when no ICs are present initially. PEG precipitation on the other hand enhances TNF-α production when preformed ICs had been added to ultracentrifued NHS or RF-positive sera before PEG precipitation. An enhancing effect was also seen when nonaggregated IgG (Endobulin®; Baxter, Vienna, Austria) was added to ultracentrifuged RF-positive sera before PEG precipitation, probably because of minute amounts of dimer IgG in the preparation acting as small ICs.
In this cross-sectional study SF and serum samples were collected in association with therapeutic arthrocenthesis. Our finding that IC-induced TNF-α induction in vitro correlates with the number of swollen and tender joints at the time of sampling suggests that IC-induced cytokine levels might reflect a general quality of joint inflammation in individual patients. We are currently studying the cytokine inducing properties of paired SF samples from different joints obtained at the same time point, as well as paired SF samples from the same joint at different time points; in this way we aim to test the hypothesis that RF-associated induction of proinflammatory cytokines by joint ICs is a stable quality over space and time in individual patients with RA.
In the present study we examined the cytokine inducing effects of soluble ICs from RA SF. Collagen type II antibodies occur in a subpopulation of RA patients and these antibodies may form solid phase ICs at the cartilage surface in RA joints. We are currently investigating such ICs to obtain information regarding the similarities and dissimilarities between cytokine responses to soluble ICs (with hitherto unknown antibody specificities) obtained in vivo
and artificial ICs created using well known autoantibodies directed against collagen type II [28
Monocytes/macrophages were shown to be the main or perhaps only responder cells in the induction of TNF-α in our systems. The importance of monocytes in IC-driven joint inflammation is supported by earlier rodent experiments in which synovial macrophages were shown to play a central role in IC-induced arthritis models [6
]. In addition, most disease-modifying drugs in RA are directed at suppressing monocytes and monocyte-derived cytokines [30
]. Recent findings have also highlighted the importance of monocytes/macrophages [9
] and monocyte-derived dendritic cells [10
] in IC-induced cytokine production in RA joints.
Earlier studies conducted by Jarvis and coworkers [31
] demonstrated cytokine-inducing properties of gel filtrated ICs from SF of patients with juvenile RA. Pretreatment of these ICs with native serum decreased subsequent cytokine production as compared with either pretreatment with heat-inactivated serum or no pretreatment [32
]. These findings and our data on FcγRIIa-dependent cytokine production together argue that when ICs become heavily coated with complement, Fc fragments are covered by complement and prevented from interaction with Fc receptors, as was proposed by Nilsson [33
Although complement activation by SF ICs is substantial, for two reasons we chose to study the effect of our ICs in a serum-free cell culture system. PEG precipitated ICs are known to carry covalently bound complement proteins after complement activation in the joint [25
]. The amount of complement proteins on ICs from different joints therefore differs and is dependent on access to the classical complement cascade in the joints of individual patients. By exposing these ICs to a standardized native serum in vitro
, all ICs will induce complement activation and differences between individual patients might diminish or disappear. When screening various cell culture systems we also found that a serum-free medium supplemented with Ultroser®
was superior to serum-containing cell culture media in sustaining IC-induced cytokine production. It was thereby also possible to investigate weak IC-induced responses that were not detected using other cell culture media formulations.
Although according to the literature RA SF may contain higher concentrations of ICs than RA serum [34
], there was no significant difference between TNF-α levels induced by serum or SF precipitates. To be able to precipitate SF samples, hyaluronidase treatment was needed. Also, a number of joint-specific proteins such as partly degraded hyaluronic acid might be co-precipitated with SF ICs in parallel with what has been described for serum proteins [25
]. Because of the experimental setup, we chose not to draw any conclusions from these findings of no difference, but instead we opted to concentrate on differences in cytokine responses between PEG precipitates from body fluids treated equally during the precipitation procedure.
Many studies have reported the importance of Fcγ receptors in the development of experimental arthritis. Thus, knockout mice lacking the activating FcγRIII have been shown to be protected from arthritis [13
] and knockout mice lacking the inhibitory FcγRIIb develop arthritis on a nonarthritis susceptible background [13
]. However, the effect of deleting FcγRIIb has not been consistent [36
]. Rodents lack the primate-specific activating FcγRIIa, which has been shown to be elevated on RA monocytes compared with healthy control individuals [15
]. Arguments are now accumulating that FcγRIIa might be a key activating mediator of IC-induced effects in humans and to act as the functional counterpart of FcγRIII in rodents [14
In the present study, blocking of FcγRIIa resulted in markedly reduced IC-induced TNF-α production, indicating that the IC-induced cytokine production is at least partly mediated via FcγRIIa. We earlier reported that ICs from, for example, patients with systemic lupus erythematosus and artificial ICs can induce cytokine production via FcγRIIa, together with a correlation between IC-induced cytokine production and monocyte density of FcγRII, but not FcγRI or FcγRIII [17
]. We also observed that ICs from patients with cryoglobulinaemia induces cytokine production via FcγRIIa [37
]. Blom and coworkers [9
] recently reported that the expression levels of FcγRII and Fcγ III are elevated on mature RA macrophages and that FcγR expression is correlated with IC induced levels of TNF-α [9
]. Collectively, these data indicate an important role for FcγR expression on monocytes/macrophages in IC-induced inflammation in RA joints, and argue that FcγRIIa blockade is a possible means to suppress IC-driven inflammation in RA. However, a role for FcγRIII can not be excluded for two reasons. The anti-FcγRIII antibody 3G8 used in our studies has been shown occasionally to exert a nonspecific stimulatory effect on cytokine production [17
]. Second, levels of monocyte expression of FcγRIII is low on unstimulated PBMC monocytes. In our earlier report [17
] we investigated FcγR monocyte surface expression in 10 different PBMC populations. Whereas FcγRIII/CD16 exhibited low expression (median fluorescence intensity 39, as comparable with the nonspecific control antibody), levels of FcγRII/CD32 and FcγRI/CD64 were substantially higher (median fluorescence intensities 538 and 1133, respectively; data not shown). The selective importance of FcγRIII on inflammatory macrophages with increased FcγRIII surface expression [9
] must therefore be investigated separately.