IgE-mediated activation of mast cells and basophils necessarily includes a variety of signaling events that serve to terminate the activation processes. But many negative feedback loops become active only after the cell is stimulated. By and large, it will be these processes that will be discussed in this review.
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To understand deactivation or self-terminating signaling, it is first necessary to explore the steps that occur during activation that lead to cellular functions. This is, in itself, an enormous topic and it should be said that in order for a cell to return to a resting state of activation, there must be many layers of homeostatic mechanisms. For illustrative purposes, consider the simple model of receptor-mediated activation shown in figure 1. In the simplest scheme, figure 1 a shows only the activation events that occur early in the reaction.
In this simple scheme, each step involves a kinase that phosphorylates its substrate, which is also a kinase of some kind. In order for these steps to be controlled, two feedback loops are introduced into the model. In figure 1 , positive or activating steps are designated as P1, P2, etc. But note that in this model, the negative components are activated by positive components. It does not have to be this way, but experimental data suggest that this is a common way of activating or recruiting negative components.
For the cell to settle back to the resting state, there must be another layer, shown in figure 1 c. These components designated CN1, etc. To return to the original resting state, it seems likely that something must stop the original signal. In other words, although the various persistent negative feedback mechanisms could terminate an ongoing reaction, by themselves they could not return the system to its resting state.
P1, P2, etc. In this simple scheme, the next layer consists of phosphatases that are constitutively active CN1, CN2, etc. Only one constitutively active element has been identified with some assurance, PTEN. There are several other known phosphatases that have been identified in mast cells, but their targets and roles remain unknown.
With this abstract basis as a background, the IgE-mediated reaction can be overlaid. In figure 1 d, a few of the pieces have been applied. The reaction starts with aggregation of the IgE receptor. Numerous studies have demonstrated that both activation and deactivation events require the continued presence of aggregates.
Dissociation of the aggregates results in a rapid return of all signaling components, positive and negative, to their resting state, with the provision that this statement only applies to an early time frame.
There are deactivation events that cannot be reversed if sufficient time passes, generating a form of activation memory. The details of what events aggregation induces are still to be discovered, but it seems clear that one early step is the association and activation of a src family kinase.
For murine mast cells, lyn, fyn and hck have been implicated [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ], but the canonical first step is the activation of lyn kinase.
At this point, there are many branching steps. Lyn, fyn and syk mediate the phosphorylation of many proteins that carry the reaction forward.
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This enzyme has a primary target, the phosphorylation of phosphatidyl 4,5-bisphosphate in the membrane, resulting in the generation of phosphatidylinositol-3,4,5-trisphosphate PIP3. In human basophils, PI3 kinase is absolutely required for secretion of all 3 classes of mediators [ 8 , 9 ] prestored mediators like histamine, newly synthesized, rapidly released mediators like LTC4 and newly synthesized, slowly released mediators like IL-4 , suggesting that the generation of PIP3 is a critical second messenger. Stopping at this point, it is possible to add two downregulatory steps.
The first is an early feedback loop that modulates the activity of the earliest steps. This step will be explored below, but is currently unknown. The second feedback step to add is the recruitment of the lipid phosphatase, SHIP SH2-containing inositol phosphatase. In human basophils, it is possible to use a methodological maneuver to demonstrate the engagement of deactivation mechanisms.
First, a simple inspection of the kinetics of histamine release demonstrates the presence of deactivation.
Only rarely can human basophils be induced, under natural conditions, to release all of the preformed granule contents, i. It is not because the granules cannot be secreted, but that there is a self-limiting process that stops the release process and this can be demonstrated by several methods [ 10 ].
This self-limiting process can be demonstrated in another way. Stimulation of the basophil in the absence of extracellular calcium results in no mediator secretion [ 11 ]. If this condition is maintained for 45—60 min and calcium is returned to the reaction, no secretion occurs compared to simply incubating the cells in the absence of extracellular calcium. This operational method has been termed desensitization. The desensitization to the aggregating antigen is termed specific desensitization and the blunting of the response to other antigens is called nonspecific desensitization.
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Notably, these two forms of desensitization refer only to IgE-mediated activation. Activation through other types of receptors is usually intact [ 10 ], although one study found some IgE-mediated blunting of an FMLP-induced G-protein-linked receptor response [ 14 ] if the basophils were treated with wortmannin. But by and large, most experiments suggest that the processes of downregulation that occur apply only to signaling cascades that have relevance to IgE-mediated secretion.
The data are derived from several previously published [ 15 , 25 ] and unpublished studies. Purified human basophils were stimlulated with either goat polyclonal anti-IgE antibody or monoclonal IgM anti-hIgE antibody at a concentration considered optimal or supraoptimal for histamine release. The ratio of the supraoptimal to optimal response is plotted for histamine release or phosphorylation of SHIP-1 as detected on a Western blot. The relationship of signaling steps to these operationally defined forms of desensitization is an area of active investigation. For reasons not yet understood, stimulating human basophils with polyclonal anti-IgE antibody results in a sustained activation of signaling elements like syk and its substrates for at least 1—3 h [ 15 ].
For example, the activity of PI3 kinase is sustained. In contrast, stimulation with antigens for basophils sensitized with the appropriate antigen-specific IgE results in transient activation of all early signaling molecules. In human basophils, both phosphorylation of Akt and activation of the entire p21ras to Erk pathway are transient, with their respective phosphorylation states returning to near resting levels by 60 min [ 10 ].
Therefore, this stimulus highlights a point in the signaling cascade that is experiencing downregulation; some process results in the metabolism of PIP3.
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If basophils are desensitized with antigen i. These two pieces of information lead to a proposal that nonspecific desensitization is operative at the point of PIP3 metabolism. In basophils and mast cells, SHIP1 has been shown to translocate to the plasma membrane during stimulation [ 16 ]. It is present and probably acts to establish a set point for activating the cells, but SHIP is dynamically recruited to the reaction complex.
There are no studies that demonstrate a way to increase the intrinsic enzymatic activity of SHIP, but during stimulation, SHIP is phosphorylated and this phosphorylation site allows SHIP to be recruited to the activation complex [ 17 , 18 , 19 ].
IgE receptor and signal transduction in mast cells and basophils.
In other words, SHIP is one of the dynamically activated negative signaling elements. Mast cells become exquisitely sensitive to IgE-mediated stimulation. There are pathological states in humans that appear to result in basophils with lower levels of SHIP expression and result in basophils that are more sensitive to stimulation [ 21 ]. Studies of the supraoptimal side of the dose response curve for anti-IgE antibody in basophils show that SHIP is more heavily phosphorylated than at optimal levels of secretion [ 23 ].
These results suggest that for this stimulus, overrecruitment of SHIP prematurely shuts down secretion. It should be noted that this is a special property of stimulation with polyclonal anti-IgE antibody. Figure 2 summarizes some of the observations regarding the difference between a polyclonal anti-IgE antibody and antigens [ 24 , 25 ].
By and large, events happen faster but the response dampens more rapidly on the supraoptimal side of the dose-response curve for polyclonal anti-IgE antibody while for antigens, events are slower. The results for antigens are consistent with cross-linking theories for antibody-antigen complexes where supraoptimal antigen concentrations result in less or smaller aggregates than optimal concentrations and therefore should have functional consequences similar to events on the suboptimal side of the dose-response curve [ 26 , 27 ].
The speculation is that for polyclonal anti-IgE antibody, the optimum for histamine release is no longer the optimum for the number and quality of aggregates, as it is for antigen stimulation. Instead, the anti-IgE antibody generates even larger and more effective aggregates as one slides onto the supraoptimal side of the dose-response curve, but downregulatory effects begin to take precedence and mediator secretion suffers despite the presence of more and larger aggregates [ 24 ].