Elevated serum IFN-α was first noted in SLE patients about two decades ago [27
]. More recently, high levels of ISG expression in lupus PBMCs have been widely reported [1
]. This "interferon signature" can be identified by using microarrays or real-time PCR. These approaches have limitations, including the time and labor required to prepare and handle mRNA from clinical samples and their expense. Here, we evaluated the utility of CD64 as a marker for rapidly assessing IFN-I overproduction. Initial suggestions that CD64 is an ISG arose from microarray studies [2
], although it remained unknown whether the increased gene expression was associated with changes at the protein level. Our data show for the first time that CD64 surface-staining intensity on monocytes correlates with ISG expression and disease manifestations in lupus patients and illustrate the potential utility of CD64 measurements for quantifying IFN-I levels in serum or other biologic fluids. We also show that CD64 may be used to monitor the effect of therapy on IFN-I levels.
Compared with real-time PCR and microarrays, CD64 expression on circulating monocytes provides a quick and relatively inexpensive means of assessing a patient's interferon status. One-step staining of whole-blood samples and analysis by using a standard four-color flow cytometer can be completed within 30 to 45 minutes, allowing results to be generated during a patient's clinic visit. Moreover, because MFI from flow cytometry staining is an absolute value, this assay avoids the need to normalize the data to actin or other housekeeping genes, as in real-time PCR assays.
The specificity of CD64 fluorescence intensity for IFN-I levels is suggested by several lines of evidence. CD64 expression was enhanced in a dose-dependent manner by IFN-α and was blocked by the viral IFN-I inhibitor B18R (Figure ). Similarly, TLR7 and TLR9 ligands enhanced monocyte surface CD64 expression in an IFN-I-dependent manner (Figure ). The ability of SLE sera to induce CD64 expression also depended on the presence of IFN-I. CD64 seems unique among the Fc receptors in its regulation by IFN-I. Although IFN-γ can also induce CD64 expression [25
], its contribution to the interferon signature in SLE may be limited, as genes specifically induced by IFN-γ (that is, CXCL9) are not known to be upregulated in lupus patients [26
]. In contrast to the lack of a correlation between CD64 fluorescence intensity and CXCL9 expression, surface CD64 expression correlated with the transcript levels of several ISGs (MX1, IFI44, and Ly6E) (Figure ). In addition, upregulation of monocyte CD64 in the presence of lupus serum was inhibited by the poxviral B18R protein (Figure ), strongly suggesting that IFN-I in SLE sera upregulates CD64 expression. This effect was not seen with blockade of IFN-γ or IL-12 with neutralizing antibodies, despite of the ability of these cytokines to induce CD64 expression [14
Dysregulated CD64 expression may have functional consequences, as this activating FcγR plays important roles in phagocytosis, cytolysis, and induction of inflammatory cytokines [28
]. The balance of activating (CD16, CD32a, CD64) and inhibitory (CD32b) FcγRs on antigen-presenting cells, such as monocytes, determines the response to immune complexes, which are produced abundantly in SLE. In addition, CD64 also is involved in the inflammatory response induced by C-reactive protein [29
]. Elevated expression of CD64 in SLE, therefore, may fuel the chronic inflammation associated with the autoimmune disease. This view is supported by animal studies, as the deletion of Fc receptor γ-chain, a critical signaling component of the activating FcγRs, is sufficient to inhibit the development of glomerulonephritis lupus-prone mice [30
]. The presence of Fc receptor γ-chain on monocytes/macrophages is required for this effect [31
]. Besides monocytes, neutrophils, certain dendritic cell subsets (especially myeloid dendritic cells), and eosinophils also express surface CD64 [32
]. In particular, the fluorescence intensity of CD64 on myeloid dendritic cells from lupus patients was increased and correlated highly with that on monocytes (data not shown). However, because of the paucity of circulating DCs in most SLE patients [5
], they are technically difficult to analyze.
Some limitations to the clinical application of CD64 expression merit consideration, notably in patients with infections. Neutrophil CD64 has been used as a marker for sepsis [32
] and to distinguish between infections with dsDNA and ssRNA viruses [35
]. It has been suggested that neutrophil CD64 expression can be used to aid in the diagnosis of infections in patients with rheumatoid arthritis [37
]. Conversely, in the setting of sepsis, the existence of preexisting autoimmune disease, especially lupus, is a potential confounder. In addition, although CD64 appears to be a surrogate marker of IFN-I activity based on our cross-sectional analysis, longitudinal studies are needed to assess the utility of this marker in monitoring changes in serum IFN-I levels over time.
Recently it was reported that Siglec-1 (CD169) expression on monocytes can be used as a biomarker for IFN-I responses in systemic sclerosis and SLE [16
]. Compared with the two-step flow-cytometry assay for CD169, measuring CD64 on circulating monocytes is simpler, requiring only a single step. Whether CD169 is suitable for bioassays using serum samples has also not been tested. However, similar to the desirability of measuring the expression of more than one ISG with real-time PCR, the use of both CD64 and CD169 staining may be warranted to optimize the reliability of the assay.