Reply to Honjo et al.: Functional relevant expression of Toso on granulocytes

In our recent study, we identified Toso expression on granulocytes using an antibody generated in our laboratory (1). Honjo et al. now show unique and interesting data on Toso that several clones of anti-Toso antibodies do not detect Toso on granulocytes and macrophages, and that Toso mRNA is not detectable on sorted granulocytes or Rag-deficient mice (2).

Toso was originally identified as Fas apoptosis inhibitory molecule 3 (3), and has recently been identified as F_C receptor for IgM (4). Using this anti-mouse–Toso antibody we detected relatively weak but reproducible staining of Toso on granulocytes (Fig. 1A) (1). Quantification uncovered that there is stronger expression of Toso on B cells compared with granulocytes (Fig. 1 A and B). Because of the lower expression of Toso on granulocytes, it may be possible that low-affinity antibodies against Toso could miss Toso expression on granulocytes but identify Toso expression on B cells. Although our study did not include RT-PCR analysis of sorted granulocytes, a recent study, conducted independently, observed expression of Toso mRNA in granulocytes (5). The authors show that the ratio of Toso mRNA expression between B cells and granulocytes reflects our data obtained by flow cytometry analysis. Although Honjo et al. (2) used FACS sorting, Choi et al. (5) used MACS sorting, which may differ in their potential to activate sorted granulocytes. Different activation status could explain differences in Toso expression. In fact, Sigruener et al. showed that enzymatically modified LDL exposure raises Toso expression in macrophages (6). In granulocytes, limited stress, such as temperature changes or shear stress, can lead to activation and might also influence Toso expression. Thus, antibody affinity, purification methods, staining protocols, and activation status of innate cells may influence Toso detection.

Fig. 1.

Functional relevant expression of Toso on granulocytes. (A) Toso expression. Splenocytes from WT and Toso^−/− mice were immunostained with anti-CD19 (B cells) and anti-Gr1 (granulocytes) antibody in combination with DAPI and anti-Toso antibody after pretreatment with Fc receptor block. One representative staining of cells gated on Gr1^hi DAPI⁻ cells (Left) or CD19⁺ DAPI⁻ cells is shown (Right). (B) Quantification of FACS stainings derived from A (n = 4/group). (C–E) WT recipient mice were irradiated and reconstituted with BM from WT mice (CD45.1; group 1), Toso^−/− mice (CD45.2; group 2), or a 1:1 mixture of WT and Toso^−/− BM (group 3). At 30 d posttransplantation, peripheral blood cells were stimulated with different concentrations of fMLP (Formyl-Methionyl-Leucyl-Phenylalanine) and granulocytes (Gr1⁺ cells) were analyzed after 30 min for CD45.1 expression (anti-CD45.1), CD45.2 expression (anti-CD45.2), reactive oxygen species production (dihydrorhodamine staining), and degranulation (side scatter). Representative FACS plots are shown for cells gated on Gr1 (C). The amount of granulocytes expressing either CD45.1 (WT, blue) or CD45.2 (Toso^−/−, red) is given in percent of total blood leukocytes (D, n = 8/group). Activated granulocytes (measured by reactive oxygen species production and degranulation; DHR⁺, SSC^lo) are given in percentage of total granulocytes at the indicated concentrations of fMLP treatment (E, n = 8/group).

The combined data provided by Honjo et al. (2), Choi et al. (5), Sigruener et al. (6) and our group is interesting and may help to identify novel functions of Toso and its regulation. Granulocytes could exhibit expression of different splice variants of Toso, which may also differ in function. In addition, different activation stimuli might influence Toso expression. These theory are interesting and certainly should be investigated further.

To analyze intrinsic function of Toso on granulocytes, we generated mixed bone marrow chimeras by injecting either WT (CD45.1), Toso^−/− (CD45.2), or mixed WT/Toso^−/− (ratio 1:1) bone marrow (BM) into lethally irradiated animals. In mixed BM chimeras, WT and Toso^−/− granulocytes developed (Fig. 1 C and D). Toso^−/− granulocytes in these animals showed increased granulocyte activation compared with WT granulocytes (Fig. 1E). This finding suggested that Toso influences the function of granulocytes intrinsically.

In conclusion, we hypothesize that Toso has a cell-intrinsic and physiological role on granulocytes. The unique and important data from Honjo et al. (2) raise more interesting questions on the Toso field. How does Toso mRNA and Toso protein expression change and toward what stimuli? Is Toso internalized or cleaved during activation? Do alternative splice variants exist and what are their functions? More experimental work has to be done to clarify these interesting questions.