Late phase response of protein secretion; difference between the low and high affinity activation
In order to assess mediator release during mast cell activation by either low- or high affinity stimulation, we analyzed the expression of 91 soluble inflammation markers in the supernatant of mature mast cells after 3, 6 and 24 hours of activation and detected 54 of these at at least one of the four time points sampled. The early response after 3 hours was dominated by cytokines IL-8, IL-13, LIF, HGF and CCL4 produced due to high affinity IgE crosslinking. PD-L1 was induced by both high and low affinity and with no significant difference. A number of shedded surface markers (TNFSF12, TNFSF14, CSF-1, TGF-a) and the cytosolic protein STAMPB, were induced earlier and more persistently by high affinity IgE than by low affinity IgE.
Most effects of signalling through low affinity IgE appeared after 6 hours, and many markers detected after low affinity activation were shared with high affinity signalling. The cytosolic proteins CASP8 and SIRT2 were specifically induced by low affinity IgE at 3 hours. The markers that most notably appeared to be specific for low affinity signalling were shedded CD40, SLAMF4 and CD5. This is consistent with the observations that low affinity IgE signalling has slower onset than high affinity signalling (2). CD40 is involved in the somewhat controversial antigen presentation of mast cells (4). In mast cells, CD5 may not be expressed in the same way as it is on T cells (6), but transcriptional activity at the promoter suggests it is expressed(7). SLAMF4 is expressed as a regulatory molecule on mast cells (5) that may have inhibitory as well as activating functions. Extensive shedding after 6 hours of these molecules as well as IL10-RB and CD6 and significantly more release of CCL3, TGF-b and CASP-8 differentiates the low affinity response of human mast cells from a high affinity response. One may speculate that the phenotype of the mast cell now adapts to the low affinity stimulus. In addition, the membrane receptors released become soluble signals that may modulate the physiologic response.
CSF-1, Fit3L, TGF-a, CD40 and TNFSF11 are known to be targets of ADAM17 that is expressed in mast cells, but there was no clear association of ADAM17 and affinity of IgE or timing of release. CD6 and its ligand CD318 (CDCP1) were shedded after low affinity IgE stimulation at 6 and 24 hours, respectively. While IL-18 and IL-10 were released at 3 and 6 hours under conditions of high affinity IgE, IL-18R1 and IL-10Rb were shedded under low affinity IgE conditions at 6 and 24 hours, suggesting the presence of a feedback loop. Shedding of a receptor may be a method to quickly change the phenotype of the cell by releasing the surface receptor, or to generate a signal molecule, or both (3).