Impact of BH3-mimetics on Human and Mouse Blood Leukocytes: A Comparative Study.

BH3-mimetics are small molecule inhibitors that neutralize the function of anti-apoptotic BCL-2 family members. BH3-mimetics have recently gained a lot of popularity in oncology because of their success in cancer treatment. However, BH3-mimetics might have a broader clinical application. Here, we established an ex vivo flow cytometric assay allowing the comparison of the impact of BH3-mimetics (ABT-199, ABT-263, WEHI-539, and S63845) on leukocyte populations of both, healthy human subjects and C57BL/6 J wild type mice. BH3-mimetics were added to freshly drawn blood that was diluted 1/2 in cell medium, and BH3-mimetics-mediated impact on leukocyte count was assessed by flow cytometry. Our results demonstrate that responses towards 1µM of BH3-mimetics can be identical as well as considerably different in leukocytes of humans and mice. For instance, the inhibition of BCL-2 by ABT-199 caused cell death in all types of lymphocytes in mice but was exclusively specific for B cells in humans. Moreover, inhibition of BCL-XL by WEHI-539 affected solely mouse leukocytes while targeting MCL-1 by S63845 resulted in efficient induction of cell death in human neutrophils but not in their mouse counterparts. Our ex vivo assay enables initial identification of analogies and differences between human and mouse leukocytes in response towards BH3-mimetics.

IL-4 enhances survival of in vitro-differentiated mouse basophils through transcription-independent signaling downstream of PI3K.

Interleukin 4 (IL-4) is a critical cytokine implicated with TH2 immune reactions, which are linked to pathologic conditions of allergic diseases. In that context, the initiation of TH2 responses can critically depend on early basophil-derived IL-4 to activate T-cell responses, which then amplify IL-4 secretion. As a pleiotropic cytokine, IL-4 acts on a broad variety of hematopoietic and non-hematopoietic cells. However, the effect of IL-4 on basophils themselves, which are emerging as relevant players in allergic as well as autoimmune diseases, was only scarcely addressed so far. Here we used in vitro-differentiated mouse basophils to investigate the direct effects of IL-4 on cellular viability and surface expression of the high-affinity receptor for IgE, FcεRI. We observed that IL-4 elicits pronounced pro-survival signaling in basophils, delaying spontaneous apoptosis in vitro to a degree comparable to the known pro-survival effects of IL-3. Our data indicate that IL-4-mediated survival depends on PI3K/AKT signaling and-in contrast to IL-3-seems to be largely independent of transcriptional changes but effectuated by post-translational mechanisms affecting BCL-2 family members among others. Additionally, we found that IL-4 signaling has a stabilizing effect on the surface expression levels of the critical basophil activation receptor FcεRI. In summary, our findings indicate an important regulatory role of IL-4 on in vitro-differentiated mouse basophils enhancing their survival and stabilizing FcεRI receptor expression through PI3K-dependent signaling. A better understanding of the regulation of basophil survival will help to define promising targets and consequently treatment strategies in basophil-driven diseases.

BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells.

Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.