Direct cell–cell contact of monocytes/macrophages with preactivated T lymphocytes leads to the secretion of high levels of proinflammatory cytokines and has been implied in the disturbed cytokine balance seen in RA [21
]. As shown here and described previously, the most likely candidates responsible for the T cell-dependent TNF-α production are synovial macrophages from patients with RA. However, in some studies an involvement of peripheral-blood monocytes in the process of this chronic disease has also been suggested [30
]. This raises the question of the contribution of monocytes to the increased TNF-α load seen in patients with RA. To address this we used monocytes from patients with RA in the co-culture system, because previous studies have examined monocytes from healthy donors only. The finding that monocytes from patients with RA produced less TNF-α than monocytes from controls was unexpected in view of the proposed contribution of this interaction to the excessive TNF-α levels observed in this disease. Monocytes from patients with RA and controls did not produce any cytokines in the absence of additional stimuli, indicating that peripheral-blood monocytes were not preactivated and that the cell separation techniques used did not lead to artificial ex vivo
stimulation of the monocytes.
The induction of TNF-α unequivocally required direct cell contact of the monocytes with T cells, which excludes the possibility that the stimuli of cytokine production are soluble mediators released from the fixed T lymphocytes. Comparing the TNF-α levels produced by synovial monocytes/macrophages with those produced by peripheral monocytes clearly shows that monocytes are not major contributors to the TNF-α load in RA.
The experiments presented here also indicate that the reduced production of TNF-α is not an intrinsic feature of monocytes from patients with RA, because the monocytes are capable of a full TNF-α response to stimulation with LPS. This is in agreement with previous reports about the LPS response of monocytes from patients with RA [39
], although the evidence is somewhat controversial [41
]. Furthermore, when measuring other cytokines such as IL-1β, IL-8 and IL-10, we observed that monocytes from patients with RA responded equally well to LPS as did monocytes from controls (data not shown).
Because monocytes from patients with RA are fully capable of producing cytokines, the most likely explanation for the suppression of the T cell-dependent TNF-α response is the presence of regulatory serum factors. The serum protein ApoA-1 has been shown to act as a regulator of cytokine production [25
]. The authors found that autologous serum from healthy controls was able to inhibit T cell-dependent TNF-α production in monocytes. They identified ApoA-1 as the molecule blocking the contact-mediated activation of monocytes. ApoA-1 is regarded as a 'negative' acute-phase protein and has been described as being present only in reduced levels in sera from patients with RA [43
] and juvenile idiopathic arthritis [46
], which makes it an unlikely candidate for systemic counter-regulation of cytokine production in RA. High levels of ApoA-1 have been found in the local synovitic environment in RA, where the molecule seems to act as an inhibitory regulator of cytokine production [47
]. In its absence, one would expect cytokines to reach extremely high levels.
The present results confirm that patients with RA do not have an increased serum concentration of ApoA-1 compared with that of healthy controls. Consequently, the strong inhibitory activity of RA sera cannot be explained by an increased ApoA-1 concentration alone, although the result of a significant correlation between ApoA-1 concentration and inhibitory serum activity is remarkable.
The results are best explained by additional inhibitory factors that seem to be present in RA sera and seem to bind to the monocyte cell surface, as indicated by two indirect lines of evidence. First, monocytes from patients with RA cultured in FCS produced less TNF-α in response to activated T cells than those from controls (Fig. ), which indicates that the soluble factor is transferred from the in vivo situation to the co-culture assay. The most likely mode of transfer of the factors would be in cell-bound form on the surface of the monocytes. Second, preincubation of monocytes from healthy controls in RA sera was sufficient to inhibit TNF-α production, and extensive washing did not abolish the inhibitory effect. Again, 'coating' of the monocyte surfaces by the suggested inhibitory factors, which prevents the full interaction between monocytes and T cells, might account for the reduced TNF-α production.
The inhibitory factor(s) described here seem to be specific for RA, and are most pronounced in patients with active disease. It can be proposed that, with increasing disease activity of RA, ApoA-1 (in addition to other factors) becomes upregulated and thus contributes to the downregulation of contact-mediated TNF-α production by monocytes.