Recent studies indicate that basophils play significant roles in the immune responses induced by helminth infections and allergic diseases [1
]. To date, however, study of their roles in murine models of disease has been hampered by the lack of a direct assay to detect their activation in mice. This study demonstrates that CD200R can be utilized as a marker of murine basophil activation.
Murine basophil surface expression of CD200R, as detected by flow cytometry, increased in response to in vitro activation of whole blood with anti-IgE, ionomycin or fMLP. These results demonstrate that CD200R up-regulation occurs in response to both IgE-dependent and independent pathways of basophil activation. Additionally, incubation of whole blood obtained from mice infected with L. sigmodontis or sensitized against ovalbumin resulted in up-regulation of basophil CD200R surface expression in response to LsAg or ovalbumin once circulating antigen-specific IgE had developed. Further evidence that CD200R is a murine basophil activation marker came from the finding that its expression on basophils increased in proportion to increases in intracellular basophil IL-4 positivity after cellular activation. Finally, basophil CD200R surface expression was found to increase in a dose–response fashion to increasing concentrations of anti-IgE.
One of the advantages of this whole blood assay is that it does not require any basophil priming cytokines or enrichment steps, which in human basophils have been shown in some cases to affect expression of surface activation markers [17
]. If one wanted to conduct such manipulations before assessing basophil activation by surface CD200R expression, however, it would be important to first assess whether such manipulations were altering baseline surface CD200R staining.
CD200R surface up-regulation was found to be rapid and transient, as time course studies demonstrated CD200R surface up-regulation occurs as early as 30 min after IgE-mediated activation, peaks at 1–2 h, and then returns to baseline by 4 h after activation. This time-course is similar to the kinetics of CD63 up-regulation on human basophils, which becomes maximal 60 min after activation, but is slower than that observed for human basophil CD203c expression, which peaks within 15–20 min [18
]. While the reason for different kinetics of human basophil activation markers is not known, it is presumed due to differences in the pathways by which these markers translocate from cytoplasmic compartments to the extracellular space [18
While CD200R has been observed to be present on human basophils in prior studies [11
], to our knowledge this study is the first to demonstrate that CD200R is also present on murine basophils and that its expression on these cells increases in response to cellular activation. As ligation of CD200R inhibits FcERI-mediated activation and degranulation of human basophils and mast cells [8
], it is possible that the transient up-regulation of CD200R we observed on the surface of activated murine basophils represents a negative-feedback mechanism that enables inhibition of further FcERI-mediated activation in the hours immediately following initial cellular activation.
Recently, a number of research groups have discovered that the mouse genome contains a family of CD200R-like proteins with close homology to CD200R (aka CD200R1). These include CD200R2 (CD200RLc), CD200R3 (CD200RLb), CD200R4 (CD200RLa) and CD200R5 [5
]. The antibody we utilized in our studies (anti-CD200R, OX-110) binds to CD200R but not to CD200R3 and CD200R4 [11
]. CD200R, CD200R2, CD200R3 and CD200R4 have all been shown capable of binding to CD200 [5
], a membrane protein expressed by thymocytes, activated T cells, B cells, neurons and endothelial cells [6
]. However, while ligation of CD200R typically results in inhibitory signals, other CD200R family members contain the positively charged amino acid lysine in their trans-membrane regions and thus can associate with ITAM- or YxxM motif-bearing adaptor molecules such as DAP12 that mediate stimulatory signals [5
]. As such, it has been speculated that CD200R and CD200R-like receptors may serve as balancing inhibitory and activating receptors [5
]. With respect to basophils, ligation of CD200R3 molecules has recently been demonstrated to result in activation of murine basophils and mast cells [21
]. As ligation of CD200R inhibits FcERI-mediated activation of these cells [8
], it is possible that releasability of murine basophils is controlled by the relative surface expression of CD200R and other CD200R-like molecules.
In conclusion, we have shown that the inhibitory receptor CD200R is rapidly up-regulated after murine basophil activation. By utilizing this marker and a combination of positive and negative basophil markers, we have developed a flow cytometric assay for detection of basophil activation in whole blood. This assay is straightforward and rapid, taking approximately 6 h for obtainment of blood, in vitro stimulation and flow cytometric analysis. Additionally, given the known inhibitory properties of CD200R ligation on basophils, the finding of CD200R up-regulation in response to FcERI-mediated basophil activation suggests CD200R may function as a negative feedback mechanism in the hours immediately after basophil activation.