In the present study we showed that specifically for CCR2 and CCR5 blockade, ligand-induced RA peripheral blood monocyte migration could be blocked by the respective receptor blocking antibody (CCL2: anti-CCR2 or CCL5: anti-CCR5) but not when RA SF was used as chemoattractant. Similarly, combined blockade of CCR2 and CCR5 could not significantly inhibit migration of RA peripheral blood monocytes towards SF in the in vitro chemotaxis model. In contrast, we were able to block CCR1-mediated monocyte migration induced by CCL5/RANTES or SF by using either a CCR1 blocking antibody or a small molecule CCR1 antagonist.
We focused in this study on monocytes, as it has previously been shown that numbers of monocytes/macrophages are related to clinical signs and symptoms in RA 
. Moreover, effective anti-rheumatic treatments in RA induce a decrease in numbers of synovial sublining macrophages, which correlate with clinical improvement independently of the therapeutic strategy (reviewed in 
). If CCR2 or CCR5 blockade would work in RA, it should be at least in part be via an effect on monocyte migration towards the synovial compartment.
Chemokines and their receptors have been shown to participate in a number of various biological processes and due to their diverse role in autoimmune diseases have been considered good therapeutic targets, in particular CCR2 and CCR5 for immune-mediated inflammatory diseases of which RA is a prototype disease 
. In view of these observations, a number of chemokine receptor antagonists (small molecule receptor antagonists and neutralizing antibodies to the receptor) have been designed and tested in animal models and several clinical trials 
. While CCR2 and CCR5 receptor antagonists have shown initial promise in pre-clinical studies 
, blockade of CCR2 
, its ligand CCL2 
, and CCR5 
have failed in clinical trials in RA patients 
. The picture may be different for CCR1 blockade. In a small, randomized clinical trial, patients with active RA were treated for 2 weeks with the CCR1 antagonist CP-481,715 or placebo. In this proof-of-concept study, treatment was administered with a dose of 300 mg given every eight hours. Synovial tissue analysis revealed a marked decrease in the total number of cells, especially in the number of macrophages and CCR1+
cells, after active treatment; only cells capable of expressing CCR1 were affected 
. The biological changes were associated with a trend towards clinical improvement. A larger phase II clinical trial used a reformulated version of CP-481,715 that was dosed twice a day. This study failed to demonstrate clinical efficacy, which may be related to lower drug levels and very high placebo responses in this study 
. Another study comparing the effects of another oral CCR1 antagonist, MLN3897, at a dose of 10 mg once daily also failed to demonstrate clinical efficacy compared to placebo at the levels of receptor occupancy reached in that study 
There may be different explanations for the negative results in the clinical trials. First, we cannot completely exclude the possibility that the levels receptor occupancy needed to effectively block the CCR2 or CCR5 were not achieved in the clinical trials. However, the results presented here suggest that RA peripheral blood monocyte migration towards RA SF cannot be effectively blocked by targeting CCR2 or CCR5, as other chemokine receptors may be more important. Second, CCR5 is expressed by T regulatory cells in humans 
. Therefore, the lack of efficacy of treatment with a CCR5 antagonist could perhaps in part be explained by inhibition of T regulatory cells. This may also be relevant for the observation with the CCR2 antagonists, as CCR2 and CCR5 are very close in homology, and inhibitors often target both 
Apart from these and other mechanisms, it has been suggested that redundancy in the chemokine system may explain the failed trials with CCR2 and CCR5 antagonists. We found that SF-induced monocyte chemotaxis was not affected when one chemokine receptor was blocked, opposed to ligand (CCL2 or CCL5)-induced monocyte chemotaxis. As in RA patients the synovial joint (tissue and SF) contains several ligands for both CCR2 and CCR5 
that are responsible for monocyte recruitment to these compartments (via many receptors such as CCR1 
), this redundancy could have accounted for the observed chemokine receptor blockade failure in both our in vitro
model and in the clinical trials. However, even when CCR2 and CCR5 were blocked simultaneously (similarly at high doses), we were not able to block SF-induced chemotaxis of RA peripheral blood monocytes, suggesting that CCR2 and CCR5 may not be the crucial chemokine receptors promoting monocyte migration towards the inflamed joint. We also might consider other possible explanations for the lack of clinical improvement after blockade of CCR2 or CCR5. In this respect we have showed by monocyte scintigraphy that blocking only the influx of inflammatory cells may be insufficient to induce clinical improvement 
, and it may be important to target chemokine receptors that also interfere with macrophage retention.
Our results suggest that, in contrast to blockade of CCR2 or CCR5, blocking CCR1 may be sufficient to inhibit migration of RA peripheral blood monocytes towards the inflamed synovial compartment in RA. It is conceivable that high levels of CCR1 occupancy at all times are needed to induce clinical improvement in RA, consistent with our original observations using 300 mg of CP-481,715 every eight hours in RA patients 
In summary, CCR2 and CCR5 antagonism may have failed in RA due to redundancy: other chemokine receptors may have substituted for CCR2 and CCR5. In contrast, CCR1 blockade may be sufficient to inhibit migration of RA peripheral blood monocytes towards the synovial compartment in the continuous presence of high levels of receptor occupancy. This notion is supported by recent modeling studies