Reply to Julia Volkmann and Sibylle von Vietinghoff

Julia Volkmann and Sibylle von Vietinghoff raise the interesting idea of transfer of CD177 via extracellular membrane vesicles, and whether such mechanisms could potentially help explain our recently published data showing an accumulation of CD177⁺ neutrophils in gingival crevicular fluid (GCF) of periodontitis patients.¹

It is important to note that the clear-cut distinction of CD177⁺ and CD177^– neutrophils, that is easily detected by flow cytometry in peripheral blood samples (Fig. 1A), is not always seen in GCF samples. In primed neutrophils, that is, cells that have undergone granule mobilization, the MFI of the CD177⁺ population is typically increased as additional CD177 is recruited to the surface from intracellular storage pools.² This could clearly be seen in samples from skin chambers and also in vitro after TNF-priming.¹ In our GCF samples, we noted that not only was the MFI of the CD177⁺ population increased, but the entire histograms were often right-shifted with increased MFI of both the CD177⁺ and CD177^– populations (Fig. 1A). The phenomenon of right-shifted CD177 histograms, as Volkmann and Vietinghoff point out, indeed appears much like the CD177-stainings reported from samples of sternal wound fluid neutrophils.³ However, in the GCF samples used in our study,¹ also the MFI of an unrelated isotype control antibody was increased (Fig. 1B), indicating increased unspecific antibody binding of GCF cells. Due to this, we used the isotype control antibody to set the gates defining the CD177^– and CD177⁺ populations. Had we not used this approach and instead set the gates based on CD177-staining of peripheral blood samples from the same patient, the differences we report in terms of accumulation of CD177⁺ neutrophils in GCF, would have been even bigger. Thus, the right-shifting of the histograms cannot explain the increased proportion of CD177⁺ neutrophils in GCF.

FIGURE 1

Right-shifted histograms of GCF samples stained with anti-CD177- and isotype antibody. Neutrophils from peripheral blood and GCF from periodontitis patients (n = 13) were stained with anti-CD177 and isotype antibody and analysed by flow-cytometry. (A) Histograms of neutrophil CD177 expression in blood and GCF (from one patient out of 13). (B) Histograms of isotype staining in blood and GCF from the same patient.

Open in new tabDownload slide

The idea raised by Volkmann and Vietinghoff is intriguing, and in line with the increasing interest in extracellular vesicles as a means to transfer, even functional, receptors between cells. Even though neutrophil-derived extracellular vesicles containing CD177 have not been (to our knowledge) characterized, we cannot exclude the possibility that such transfer of CD177 between cells takes place in GCF. We welcome any efforts in trying to identify CD177 containing vesicles in GCF and establish mechanistically if and how CD177 transfer occurs.

References