Toll-like receptor 2 and toll-like receptor 4 expression on CD64+ monocytes in rheumatoid arthritis: Comment on the article by Iwahashi et al


Toll-like Receptor 2 and Toll-like Receptor 4 Expression on CD64+ Monocytes in Rheumatoid Arthritis: Comment on the Article by Iwahashi et al

To the Editor:

We read with interest the article by Iwahashi et al (Iwahashi M, Yamamura M, Aita T, Okamoto A, Ueno A, Ogawa N, et al. Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis. Arthritis Rheum 2004;50:1457–67), on the expression of Toll-like receptor 2 (TLR-2) and TLR-4 on CD14+ blood monocytes, and TLR-2 expression on synovial macrophages, of patients with rheumatoid arthritis (RA). They report an increased frequency of CD16+,CD14+ cells in RA patients compared with healthy controls, as well as increased surface expression of TLR-2 in CD16+ versus CD16− monocytes of both patients and controls. In contrast, monocytes from RA patients expressed higher levels of TLR-4, independent of CD16 status.

We have similarly been interested in the expression of TLR-2 and TLR-4 in RA and would like to report our findings, which differ quantitatively from those obtained by Iwahashi and colleagues. We found that in RA patients, monocyte/macrophage expression of both TLR-2 and TLR-4 was increased in comparison with control subjects. In addition, we found that the lymphocyte (nonmonocyte) population showed increased expression of TLR-2, but not TLR-4. We performed flow cytometric analysis using antibodies against TLR-2 (phycoerythrin labeled), TLR-4 (allophycoryanin labeled), and CD64 (fluorescein isothiocyanate labeled), on both peripheral blood (PB) and synovial fluid (SF) mononuclear cells isolated by Ficoll-Hypaque density-gradient centrifugation. PB and SF from 7 patients with active RA were studied, as was PB from 9 healthy control subjects.

CD64, the high-affinity receptor for IgG (Fcγ receptor I), is expressed on both macrophages and monocytes and was used as our monocyte/macrophage marker. We found that nearly all CD64+ mononuclear cells (92–99%) were TLR-2+, and this proportion was independent of disease status. However, TLR-2 expression was higher in monocytes from RA patients than from controls (mean fluorescence intensity [MFI] 362.6 ± 80.3 versus 178.7 ± 19.8 [mean ± SEM]; P = 0.025 by Wilcoxon's test) (Figure 1). The MFI was similar in the SF and blood compartments of RA patients (MFI on synovial monocytes 360.1 ± 107.4). CD64+ cells from normal subjects had a low frequency of TLR-4 expression (0.24%) compared with RA blood cells (mean ± SEM 2.98 ± 1.34%) or SF cells (2.52 ± 0.84%). The MFI of TLR-4 expression was also higher in patients than in normal subjects (Figure 1). Finally, we investigated for evidence of TLR-2 expression on lymphocytes, since TLR-2 has also been implicated in the regulation of T cell activation. In the non-CD64 population of mononuclear cells, we did not find expression of TLR-2 in normal subjects, whereas RA patients had 3.7% positivity.

Figure 1.

Difference in CD64+ monocyte staining for Toll-like receptor 2 (TLR-2) in rheumatoid arthritis (RA) patients (black) and healthy controls (white). Results shown are from 1 representative patient and 1 representative control. Numbers at the bottom are the mean fluorescence intensity (MFI) of staining for TLR-2 and TLR-4 (mean values in 7 RA patients and 9 controls). For both TLR-2 and TLR-4, the MFI in blood monocytes was significantly higher in patients than in controls (P = 0.025 and P = 0.005, respectively). FL = fluorescence; PE = phycoerythrin; SF = synovial fluid.

There is no previous report of a comparative study of TLR-2 and TLR-4 expression on mononuclear cells from SF and PB in RA. Our results show that both TLR-2 and TLR-4 are expressed at higher levels in the CD64+ population, but expression does not differ between PB and SF in RA patients. The difference between our results and those obtained by Iwahashi et al could be due to the antibodies used (CD16 versus CD64), which may identify different subpopulations of monocytes in vivo. Our findings suggest that systemic inflammation in RA is capable of enhancing TLR expression on monocytes and macrophages in general, which, in the presence of their cognate ligands, may serve to further enhance the proinflammatory capacity of these cells.

Matthias Frasnelli MD*, Alexander So FRCP, PhD*, * Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.