Effect on Cell Surface Markers following Allergen-Induced Desensitization of Human Whole-Blood Basophils

Abstract

Background: Allergen-specific immunotherapy (SIT) leads to reduced symptoms upon allergen exposure through as yet unresolved mechanisms. Desensitization of basophils to specific allergens during the updosing phase of injection immunotherapy may contribute to the clinical effect of SIT. Here we report a protocol for efficient in vitro allergen-mediated desensitization of basophils in whole blood and the effect of desensitization on the expression of basophil activation markers (CD203c and CD63) as well as histamine release in response to allergen challenge. Methods: Whole blood from grass pollen-allergic subjects was incubated with Phleum pratense extract by stepwise increase of the allergen concentration in the culture from well below to well above the allergen threshold concentration for activation of basophils. Desensitization was determined by measuring the expression of the basophil activation markers CD63 and CD203c by FACS following challenge with high allergen concentrations. Results: The basophil desensitization protocol reported here affected both the expression of the cell-surface markers and the levels of histamine release. Following the stepwise desensitization procedure the whole-blood basophils were not activated when challenged with more than 10-fold increased allergen concentration. Conclusion: We have established a protocol for basophil desensitization. By mimicking the updosing phase of immunotherapy we raised the allergen threshold for basophil activation and obtained efficient desensitization for all donors. We showed that conditions leading to desensitization affect histamine release and expression of different basophil markers alike.

Marked Differences in the Signaling Requirements for Expression of CD203c and CD11b versus CD63 Expression and Histamine Release in Human Basophils

“…If actin polymeriziation did, in fact, determine whether desensitization occurred, then the stimulated expression of CD203c might not show desensitization because expression occurred to the same extent regardless of the presence of actin inhibitors. A recent study by Lund et al [41] demonstrated desensitization of CD203c expression using a sub-threshold method. The signaling requirements for this approach to demonstrating FcεRI-mediated desensitization have not been explored, therefore, to link results for CD203c with our previous signaling studies, FcεRI-mediated desensitization was studied with the calcium free-method using anti-IgE antibody as the stimulus.…”

“…Although desensitization appears to occur for the all the measured endpoints, some caution is reserved for these studies since the rate of desensitization was altered somewhat for CD11b and CD203c, especially at later time points, raising the possibility that the mechanisms for down-regulating these two responses may still differ, in part, from the mechanisms used to turn off histamine release and, by extension, CD63 expression. Recent studies have also demonstrated desensitization of CD203c expression but as noted above, the methodology used low dose escalation to establish some degree of desensitization [41]. Taken together, it seems clear that some aspects of desensitization are operating on the expression of CD203c and CD11b.…”

Antibody repertoire complexity and effector cell biology determined by assays for IgE-mediated basophil and T-cell activation

“…The effects of IL-3 and GM-SCF on basophil reactivity are most extensively described but recently also the influence of IL-33 and IL-18 has been investigated as conditioning factors for basophil activation (Pecaric-Petkovic et al, 2009;Smithgall et al, 2008). Upon IgE-allergen mediated basophil activation the two primary flow-cytometry markers CD63 and CD203c differ with respect to kinetics and type of activation (all-or-none versus graduated increased expression -see (Hennersdorf et al, 2005;Lund et al, 2010) for details), indicating that the two markers represent two different pathways of cellular response. In contrast to CD63, CD203c is constitutively expressed on the surface of basophils and in addition to being up-regulated in response to IgE cross-linking; this marker is also increased by IL-3 alone in the absence of allergen (Buhring et al, 2004;Hauswirth et al, 2007).…”

Sequential allergen desensitization of basophils is non‐specific and may involve p38 MAPK

“…In the literature, several in vitro desensitization experiments performed on cultured mast cells (12,13), purified basophils (17,18) and peripheral mononuclear blood cells (15,16) are described. Furthermore, desensitization in these experiments was often performed with subthreshold allergen doses in the presence or absence of extracellular calcium and did not address allergen concentrations optimal for response (15,(17)(18)(19)(20).…”

“…Furthermore, desensitization in these experiments was often performed with subthreshold allergen doses in the presence or absence of extracellular calcium and did not address allergen concentrations optimal for response (15,(17)(18)(19)(20). Although subthreshold desensitization has been the standard method for investigating desensitization mechanisms, it does not properly reflect the situation of in vivo rapid drug desensitization.…”