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The Journal of Immunology, 2006, 177: 1040-1051.
Copyright © 2006 by The American Association of Immunologists

Nonspecific Desensitization, Functional Memory, and the Characteristics of SHIP Phosphorylation following IgE-Mediated Stimulation of Human Basophils1

Donald MacGlashan, Jr.2 and Natalia Vilariño

Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previous studies of secretion from basophils have demonstrated the phenomenon called nonspecific desensitization, the ability of one IgE-mediated stimulus to alter the cell’s response to other non-cross-reacting IgE-mediated stimuli, and a process that would modify phosphatidylinositol 3,4,5-phosphate levels was speculated to be responsible for nonspecific desensitization. The current studies examined the changes and characteristics of SHIP1 phosphorylation as a measure of SHIP1 participation in the reaction. Based on the earlier studies, two predictions were made that were not observed. First, the kinetics of SHIP1 phosphorylation were similar to reaction kinetics of other early signals and returned to resting levels while nonspecific desensitization remained. Second, in contrast to an expected exaggerated SHIP phosphorylation, cells in a state of nonspecific desensitization showed reduced SHIP phosphorylation (compared with cells not previously exposed to a non-cross-reacting Ag). Discordant with expectations concerning partial recovery from nonspecific desensitization, treatment of cells with DNP-lysine to dissociate bound DNP-HSA, either enhanced or had no effect on SHIP phosphorylation following a second Ag. These experiments also showed a form of desensitization that persisted despite dissociation of the desensitizing Ag. Recent studies and the results of these studies suggest that loss of early signaling components like syk kinase may account for some of the effects of nonspecific desensitization and result in a form of immunological memory of prior stimulation. Taken together, the various characteristics of SHIP phosphorylation were not consistent with expectations for a signaling element involved in nonspecific desensitization, but instead one which itself undergoes nonspecific desensitization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Like all receptor-mediated reactions, secretion from basophils and mast cells is a self-limited process. An outline of the down-regulatory control mechanisms taking place in human basophils is now in place although the precise participants or process is still unknown. In the relatively short time frame of typical experiments, down-regulation or desensitization is not the loss of cell surface receptors but instead appears to be the result of changes in the signaling apparatus (1). We have identified two regions of down-regulation occurring during IgE-mediated secretion (2). The earliest process precedes the activation of syk kinase but a second process, occurring at a point just distal to the activation of PI3K, occurs simultaneously with the receptor-proximal process (3). In addition, this more distal process appears associated with the phenomenon previously called nonspecific desensitization. In this context, nonspecific desensitization only refers to IgE-mediated signaling because the response to a variety of non-Fc{epsilon}RI-dependent stimuli (FMLP, C5a, etc.) is unaffected by complete "nonspecific" desensitization. The more distal site of down-regulation appears to alter events associated with the activities of PI3K. In basophils, the activity of p21ras and the downstream raf/MEK/ERK pathway are completely dependent on PI3K activity when cells are activated through Fc{epsilon}RI. For studies in human basophils, the phosphorylation state of Akt was used as an indicator of the presence of phosphatidylinositol, 3,4,5-phosphate (PIP3)3. Because Akt phosphorylation was transient, while the phosphorylation state of the p85{alpha} regulatory subunit of PI3K (and steps preceding PI3K activation) was sustained, one interpretation was that stimulated down-regulation operated on the activity of PI3K or, more likely, on its products. These experiments also demonstrated that the down-regulatory events that alter activity of pathways downstream of p21ras in response to one Ag, also inhibit the pathways downstream of p21ras (but not upstream) when the cells are restimulated with a non-cross-reacting Ag. Therefore, this region of down-regulation may be responsible for the phenomenon of nonspecific desensitization.

It is becoming increasingly clear that there are multiple regulatory influences on the ras-Raf-1 interaction and/or activities. For example, there is a family of rasGAP proteins that regulate p21ras GTPase activity, switching it off (4, 5). There are also a variety of inputs into Raf-1 that modulate its activity (6, 7, 8). However, in the context of a process that regulates PIP3 levels and therefore may regulate the IgE-dependent ERK pathway in basophils, phosphatases such as SHIP (SHIP1 or SHIP2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are also interesting candidates (9, 10, 11). There are a variety of studies using murine mast cells that have suggested that the presence of SHIP determines the ability of the mast cell to secrete (12). Recent studies have identified a subpopulation of human subjects with lower than usual levels of SHIP1 in circulating basophils that is associated with a releasability of these basophils to a cytokine called histamine-releasing factor (HRF) (13). These studies have focused attention on the role of SHIP in regulating the secretion of mast cells and basophils and suggest that characterization of SHIP activity during secretion should be an important first step in understanding its role.

Although SHIP may have several roles in signaling, e.g., acting as an adaptor for recruitment of dok-rasGAP (14, 15, 16), its enzymatic activity appears to have a role in regulating PIP3 levels in the membrane. PIP3 acts as a site of recruitment for a variety of proteins (17) and therefore the ability of SHIP to metabolize PIP3 to phosphatidylinositol 3,4-bisphosphate may provide a means of down-regulating an ongoing signaling cascade that depends on PIP3. These enzymatic activities of SHIP would be consistent with a process of nonspecific desensitization in basophils, i.e., a post-PI3K event that is critical for driving the ras-ERK pathway. However, no studies of SHIP signaling in basophils have been reported and if a role for SHIP (or other similar phosphatase activity) were to be implicated, one would hypothesize that SHIP would have to remain active in desensitized cells.

The initial primary question in the current set of studies focused on the observations that Ag (rather than anti-IgE)-driven stimulation results in a transient activation of all signaling steps thus far examined. One exception is the elevation in cytosolic calcium but due to unresolved technical issues related to the use of fura-2 to detect intracellular free calcium, the precise nature of transience for the calcium response is not well-known (18). Other than this possible exception, the evidence from other signaling elements would predict that SHIP participation would also be transient. However, previous studies noted maximal nonspecific desensitization after 60–80 min of stimulation with the first Ag, sufficient time for many other signaling steps to be near resting levels. If SHIP participation were responsible for nonspecific desensitization, we postulated that unlike other signaling elements, its participation would appear sustained. A related or possibly alternative prediction would be that in an experiment where two non-cross-reacting Ags were added sequentially, SHIP participation would appear more readily, either as a faster or more exaggerated response after the second stimulus was added, leading to a suppressed response downstream of PI3K. There are several other characteristics that might be expected for a molecular species that participates in desensitization and these were examined as well.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

The following were purchased: PIPES, BSA, EGTA, EDTA, D-glucose, NaF, Na4P2O7, Na3VO4, 2-ME, Nonidet P-40, FMLP (Sigma-Aldrich); crystallized human serum albumin (HSA) (Miles Laboratories); FCS and RPMI 1640 containing 25 mM HEPES and L-glutamine (BioWhittaker); Percoll, (Pharmacia); Tris (hydroxymethyl)-aminomethane, Tween 20 (Bio-Rad); leupeptin, DTT, PMSF (Boehringer Mannheim); anti-phosphotyrosine mAb (4G10) and anti-Fc{epsilon}RI{gamma} (Upstate Biotechnology); rabbit anti-phospho-ERK Ab, anti-phospho-Akt (Thr308 specific), anti-Akt, anti-phospho-SHIP, anti-PTEN, anti-phospho-PTEN, anti-phospho-Src (Tyr416) and anti-phospho-Lyn (Tyr507) and biotinylated molecular mass markers (Cell Signaling); anti-syk mAb, 4D10, antiphosphotyrosine mAb (PY20), anti-actin (C-11), anti-Lyn (44), anti-Fyn (FYN3) and anti-SHIP Ab (P1C1) (Santa Cruz Biotechnology); anti-HRP-conjugated donkey anti-rabbit Ig Ab, HRP-conjugated sheep anti-mIg Ab, protein G Sepharose beads (Amersham Life Science). Goat anti-human IgE Ab was prepared as previously described (19). PP1 and LY294002 (Calbiochem), NVP-QAB205 (gift of GlaxoSmithKline) (20). DNP (7)- and benzylpenicilloyl (BPO) (11)-HSA (21) were synthesized as previously described and gp120-OVA provided by Tanox. DNP-GFP (green fluorescent protein) was synthesized by the method used for DNP-HSA using a purified cloned GFP.

Several IgE Abs were used in these studies. A penicillin (BPO)-specific IgE was partially purified from the sera of penicillin allergic patients as previously described (21). A gp120-specific monoclonal IgE was the gift of Tanox. This Ab was biotinylated and could be detected by flow cytometry by streptavidin-Alexa 647, which was calibrated with absolute IgE/basophil according to Materials and Methods described elsewhere (22). A mouse DNP-specific IgE-producing hybridoma cell line (H1-DNP-{epsilon}-26) was obtained from D. Katz (formerly at The Scripps Clinic and Research Foundation, La Jolla, CA) and ascites partially purified as previously described.

Buffers

PIPES-albumin-glucose (PAG) buffer consisted of 25 mM PIPES, 110 mM NaCl, 5 mM KCl, 0.1% glucose, and 0.003% HSA. PAGCM was PAG supplemented with 1 mM CaCl2 and 1 mM MgCl2. PAG-EDTA consisted of PAG supplemented with 4 mM EDTA. Countercurrent elutriation was conducted in PAG containing 0.25% BSA in place of 0.003% HSA. Lactic acid buffer; 0.01 M lactic acid, 0.14 M NaCl, 0.005 M KCl (pH 3.9). ESB is Novex electrophoresis sample buffer containing 5% 2-ME. Complete lysis buffer is 20 mM Tris-HCl (pH 7.5), 100 µg/ml aprotinin, 10 mM benzamidine, 1 mM PMSF, 100 µg/ml leupeptin, 50 mM NaF, 1 mM Na3VO4, 1% Nonidet P-40, and 10% glycerol. Incomplete lysis buffer is complete lysis buffer without the protease inhibitors, Nonidet P-40, glycerol, or vanadate. Radioimmunoprecipitation assay buffer consisted of 50 mM Tris (pH7.4), 30 mM NaF, 5 mM EDTA, 0.5% BSA, 1% Nonidet P-40, 0.25% deoxycholate, 1 mM NaVO4), 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 0.05 mM PMSF, 1 mM DTT, 100 µg/ml aprotinin, 100 µg/ml leupeptin. Stripping buffers were either 7 M guanidine hydrochloride or 65 mM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS. The sensitivity of the subsequent blotting to the choice of stripping agent determined which of these two were used.

Basophil purification

For most of these experiments, residual cells of normal donors undergoing leukapheresis were enriched in basophils using a combination of Percoll density gradients and countercurrent-flow elutriation, as previously described (23). The cells were further purified by negative selection using MACS basophil isolation kit (Miltenyi Biotec). More recently, we have used a mixture of Abs for negative selection from Stem Cell Technologies (basophil purification kit) and columns from Miltenyi Biotec. The purity of basophils was determined by Alcian blue staining (24) and basophils purified from leukapheresis packs generally exceeded 99% purity.

Sensitization and sequential stimulation

For many of the experiments, basophils were sequentially challenged with two non-cross-reacting stimuli. The first Ag was usually DNP-HSA and these studies examined two types of second stimuli, Ag or anti-IgE Ab. Previous studies established that goat polyclonal anti-human IgE Ab did not bind to DNP-specific mouse monoclonal IgE. But because there is potential for anti-IgE to engage Fc{gamma}RIIb on basophils (25), many of the studies focused on Ags and in the case of sequential stimulation, the second non-cross-reacting Ag was a gp120 peptide conjugated to OVA. In these studies, the basophils were also then sensitized with a gp120 peptide-specific monoclonal human IgE (hIgE) that in some experiments was also conjugated with biotin for detection by flow cytometry (calibrated to absolute IgE per basophil). In previous studies, none of the Abs used for sensitization (DNP-, gp120- or penicillin(BPO)-specific IgEs) have been observed to induce signaling without Ag present (2). However, to minimize concerns that some low level, but undetectable, signaling might occur during the sensitization, sensitization was performed at 4°C for 1 h, the cells washed once with ice-cold PAG and resuspended in cold PAGCM before rapidly warming for stimulation. To sensitize the cells with both DNP- and gp120-specific IgE, we found in pilot studies that an approximately equal loading could be accomplished by first incubating the cells with 5 µg/ml DNP-specific mouse IgE (mIgE) and after 45 min adding in gp120-specific IgE to yield a final concentration of 5 µg/ml for the remaining 15 min of incubation on ice. For most of the experiments using purified basophils to measure ERK, Akt, or SHIP phosphorylation in response to Ag, the cells were treated with lactic acid briefly (6–10 s) before sensitization to generate many more unoccupied receptors and to increase loading with the Ag-specific mIgE (26, 27). Because of the variation in receptor expression levels and in the ability of this procedure to dissociate IgE, there was a wide range in the ability to subsequently load the cells with Ag-specific IgE.

To calculate nonspecific desensitization, measurements of the response to DNP-HSA, gp120-OVA, or DNP-HSA then gp120-OVA were made. For leukotriene C4 (LTC4) release, percent nonspecific desensitization is: 100 x (1 – (A/B)), where A = response to sequential DNP-HSA/gp120-OVA and B = the sum of the response to DNP-HSA alone + gp120-OVA alone. For signaling elements, 100 x (1 – (A/B)), where A = the magnitude of the gp120-OVA after DNP-HSA response and B = the gp120-OVA alone response.

Phosphorylation of signaling elements

The phosphorylation of ERK, Akt, and SHIP were assessed using phosphospecific Abs. After stimulating basophils (0.5–1 x 106 cells/sample) in PAGCM buffer, reactions were stopped by brief high-speed centrifugation for 5–10 s, supernatant collected and the cell pellet lysed with hot ESB (electrophoresis buffer; Novex) and separated on 8% (for pSHIP) or 10% Tris glycine gels (for Akt and ERK) (Novex). Early experiments tested lane loading for phosphorylated (Y1020) SHIP1 (pSHIP) by stripping and reblotting with anti-SHIP Ab. However, the anti-SHIP Ab is considerably less efficient than anti-pSHIP so after some pilot studies, we switched using either anti-c-cbl or p85, which do not change expression levels during stimulation. ECL films were converted to digital images with a Kodak DC290 camera and the bands analyzed with NIH Image.

Membrane translocation assay

A total of 4–6 million basophils per condition was stimulated, the cells were pelleted at 8000 x g, and 0.25 ml of a hypotonic buffer containing 50 mM Tris, 10 mM EDTA, 5 mM EGTA and 100 µg/ml aprotinin, 10 mM benzamidine, 0.1 mM AEBSF, 100 µg/ml leupeptin, and 1 mM Na3VO4 were added to the cell pellet. This suspension was sonicated for 3 x 5 s with cooling, centrifuged at 300 x g for 5 min to remove "debris" and then centrifuged at 100,000 x g for 10 min in an Airfuge to obtain a membrane pellet. This pellet was dissolved in 20 µl of Tris buffer containing 1% Nonidet P-40 which was then supplemented with 2x ESB and the solution boiled for 5 min. Following gel electrophoresis on 8% Tris-glycine gets, transfer to nitrocellulose, the blots were developed with anti-SHIP (P1C1), secondary anti-rabbit-biotin conjugate, and then streptavidin-HRP.

Intracellular Ca2+ concentration and mediator measurements

Basophils were labeled with 1 µM fura 2-AM and cytosolic calcium determined by digital microscopy as previously described. A radioimmunoassay was performed using 100 µl of supernatant to determine LTC4 levels as previously described (28). Histamine was measured by automated fluorometry (29). For the pharmacological agents used in these studies, 10 min of preincubation was found in previous or pilot (in the case of NVP-QAB205) studies to be sufficient to "equilibrate" the cells and drug.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Characterization of SHIP recruitment

If PI3K remains active throughout the period of stimulation with anti-IgE Ab, then the transient presence of phosphorylated Akt, indirectly indicating the transient presence of PIP3, suggests the activity of a phosphatase such as SHIP or PTEN. As shown previously, basophils express SHIP1 (13) and not surprisingly, PTEN (Fig. 1B). SHIP1 has been in observed in several forms, and we could detect a very weak band at 135 kDa (which does not image well, ~1–5% of the 145 kDa band) as well as the strong band at 145 kDa. As an indirect measure of the participation of these phosphatases, we examined their phosphorylation state (SHIP1) (15) or dephosphorylation (PTEN) (30) during stimulation with either Ags (DNP-HSA or gp120 peptide-OVA in properly sensitized cells), anti-IgE Ab, or FMLP. Fig. 1A shows phosphorylation of SHIP1 as detected by immunoprecipitation with anti-SHIP1 and blotting with anti-phosphotyrosine (the immunoprecipitation is not very efficient and the band intensities difficult to detect with the relatively limited number of cells available, 4 million per condition in this experiment). Fig. 1, C and D, shows a similar result using an anti-phospho-SHIP1 Ab (Tyr1020), direct blot of whole cell lysates. This Ab also detects a stimulation index for the 135-kDa sized species (with this Ab, there are also a variety of other bands showing stimulation indices >1.0 at lower molecular weights). It was notable that FMLP also induces a weak phosphorylation of SHIP (Fig. 1D).


Figure 1
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  		FIGURE 1. SHIP translocation and phosphorylation following stimulation of basophils. A, Following stimulation, SHIP1 was immunoadsorbed with anti-SHIP1 Ab (P1C1) and phosphorylation detected in the Western blot with anti-phosphotyrosine, 4G10 (top blot) (C, nonstimulated control; aIgE, stimulation with anti-IgE Ab). After stripping, the reblot was with anti-SHIP1 (P1C1; bottom blot). B, Basophils were stimulated with 0.2 µg/ml anti-IgE Ab and whole cell lysates blotted with anti-phospho-PTEN. After stripping, the blots were developed with anti-PTEN. C, Whole cell lysates of basophils blotted with anti-phospho-SHIP1. Basophils were sensitized with both DNP-specific and gp120-specific IgE, washed, and then stimulated with either 3 µg/ml DNP(7)-HSA (lane marked DH) or 0.6 µg/ml gp120-OVA (lane marked gp) (C, nonstimulated control). D, Basophils were stimulated with either 0.2 µg/ml anti-IgE Ab (lane marked aIgE) or 1 µM FMLP and whole cell lysates blotted with anti-phospho-SHIP1 (C, nonstimulated control). Cbl was used as the lane loading control. E, Membrane-associated SHIP, basophils were sensitized with gp120-specific IgE, and stimulated with 0.6 µg/ml gp120-OVA for 15 min (lane marked gp120), cellular membranes isolated, and the dissolved membranes blotted for SHIP1 (C, nonstimulated control). F, Membrane-associated SHIP ± with anti-IgE Ab (0.2 µg/ml) for 15 min (lane marked aIgE). IL-3 receptor subunit beta was used as a membrane-loading control.

 
A similar anti-phosphopeptide Ab does not yet exist for SHIP2 and we have been unable to use commercially available Abs to immunoadsorb SHIP2. Fig. 1, E and F, show that stimulation results in translocation of SHIP1 to cell membranes (n = 3, 2.75 ± 0.65-fold relative to nonstimulated cells) while there was little or no detectable increase in membrane SHIP2 (n = 3, 0.92 ± 0.23-fold). There are reports that PTEN phosphorylation is altered during its activation; in this case, dephosphorylation is reported during activation. Using an Ab that detects the phosphorylation state of PTEN (Ser380/Thr382/383), stimulated basophils were examined for changes in PTEN phosphorylation; no changes were detected (Fig. 1B).

Fig. 2A, inset, shows an example Western blot of the kinetics of SHIP1 phosphorylation following stimulation with Ag (DNP-HSA) and Fig. 2A the average of two experiments where pSHIP, pAkt, and pERK were measured in the same lysates (the kinetics of Akt phosphorylation was similar if the Ser473 anti-phospho-Akt Ab was used for detection rather than the Thr308 Ab). Fig. 2B shows similar experiments using anti-IgE Ab. For the entire set of experiments performed in this study, stimulation indices for both Ag and anti-IgE Ab were similar, 4.06 ± 0.48 for Ags and 4.14 ± 0.32 for anti-IgE Ab (see Fig. 3A). Fig. 2D plots the parameter of interest for these kinetic experiments, previous studies that distinguish anti-IgE Ab from Ag noted the contrast in the ratio of the 60-min time point to the 5-min time point. Surprisingly, this contrast also occurs for SHIP phosphorylation. The plot in Fig. 2D shows data obtained from multiple experiments (not all had the early time points used in Fig. 2, A and B) which ended with a 60' time point as well as experiments (see below) where an 80' time point was examined and four extended experiments that included a 6-h time point. The 60'/5' phosphorylation ratio following Ag stimulation is ~0.30, not too dissimilar from previous experience with syk kinase phosphorylation while the ratio for anti-IgE Ab was near 1.00, also as previously found for studies of syk kinase phosphorylation (31). At the 6-h time point, SHIP phosphorylation was the same as unstimulated cells at the same time. Fig. 2C also shows that SHIP translocation had returned to resting levels after 6 h (see below for further discussion of the data for the 6-h time point). The relationship between the stimulation index for SHIP translocation (x) vs SHIP phosphorylation (y), using a linear fit, has a y-intercept of ~2.0 (r = 0.91, n = 6), i.e., SHIP phosphorylation can be observed absent measurable translocation.


Figure 2
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  		FIGURE 2. SHIP phosphorylation kinetics in basophils. A, Basophils were sensitized with DNP-specific IgE, stimulated with 3 µg/ml DNP(7)-HSA and whole cell lysates used for Western blotting with anti-phospho-SHIP Ab (top blot) or anti-phospho-Akt and anti-phospho-ERK. The plot shows the kinetics, relative to the 5-min band intensities for phospho-SHIP (•), phospho-Akt ({circ}), or phospho-ERK ({blacksquare}) (n = 2). B, Same experimental design but the basophils were stimulated with 0.2 µg/ml anti-IgE Ab (n = 2). C, Six-hour response; the upper blots show SHIP1 translocation at 15 and 360 min in cells stimulated with 3 µg/ml DNP(7)-HSA (the membrane protein, IL-3beta, acting as a loading control), the lower blots showing whole cell lysates blotted for either phospho-SHIP1 or phospho-ERK 1/2 (p85 acting as the loading control). The right three lanes of this blot show cells incubated for 6 h ± DNP-HSA followed by a 10-min stimulation with gp120-OVA (labeled gp-10') (see Fig. 8 legend and accompanying text for further discussion of this protocol). D, A 60':5' ratio of pSHIP band intensity (minus the band intensity in nonstimulated cells) derived from multiple kinetic experiments, following stimulation with either anti-IgE Ab or Ag (DH refers to DNP(7)-HSA and aIgE refers to anti-IgE Ab). For the Ag kinetics, the data was derived from different types of experiments that either had 60 min, 80 min, or 6 h as the late time point.

 

Figure 3
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  		FIGURE 3. Relationship between SHIP phosphorylation and other outcome measures. A, Stimulation index for SHIP phosphorylation taken at the 15-min time point from many of the experiments in this manuscript; ({circ}), Ag as the stimulus (3 µg/ml DNP-HSA or 0.6 µg/ml gp120-OVA), (•), 0.2 µg/ml anti-IgE Ab vs histamine release from the same samples. B, Basophils stimulated with five concentrations of anti-IgE Ab, pShc ({circ}) and pSHIP (•) measured from whole cell lysates and histamine release ({blacksquare}) in the supernatants; data are expressed as a fraction of the response at the optimum of 0.2 µg/ml. The measurements of pSHIP were made at 15 min (n = 3). C, After lactic acid stripping, basophils were sensitized with seven-fold dilutions of biotinylated gp120-specific IgE starting at 5 µg/ml. After washing, the cells were stimulated with an optimal concentration of 0.6 µg/ml gp120-OVA or buffer alone for 15 min and whole cell lysates analyzed for pSHIP (•). Supernatants were analyzed for histamine ({circ}) and a sample of the cells analyzed by flow cytometry for the presence of biotinylated IgE. Based on previous calibration of the flow cytometric intensity, the amount of IgE per cell was calculated for each sensitization (n = 3).

 
In previous studies, we did not find any evidence of the interaction of the Fc portion of our goat anti-IgE Ab with Fc{gamma}RIIb known to be present on basophils (2). However, activation of SHIP is expected by co-cross-linking of Fc{gamma}RIIb with Fc{epsilon}RI (25, 32, 33), so we re-examined stimulation with anti-IgE Ab (0.2 µg/ml) in the presence of high concentrations of nonspecific human IgG (previously shown to modify association of mIgG with Fc{gamma}RIIb on basophils (2)). There was no inhibition of SHIP phosphorylation in the presence of 25 µM hIgG vs 25 µM HSA; 5 and 15 min after stimulation the ratio of stimulation index for pSHIP. +hIgG/+HSA, was 1.07 ± 0.32 and 1.40 ± 0.27, respectively (n = 4, p > 0.05), suggesting again that this was not an issue for this stimulus.

Unlike many other signaling elements, there appears to be a readily measurable level of SHIP1 phosphorylation in resting cells (B cells were isolated from peripheral blood using Miltenyi B cell isolation Abs and negative selection columns. The isolated B cells were stimulated ± whole anti-IgM Ab ± 10 µM PP1, the cells pelleted and lysed with 1x ESB (as with basophils). We found that there was also a resting level of pSHIP that was reduced by 90% with PP1 (the stimulation index with anti-IgM was {approx}3.0). The very low residual level of SHIP phosphorylation on treatment with PP1 suggests that the anti-pSHIP Ab does not readily detect nonphosphorylated SHIP). Previous studies have noted that it is unusual to observe resting phosphorylation of elements like syk, Shc, p85{alpha}, MEK, or ERK, so in general, stimulation indices are not calculated for these species (strictly speaking, with the typical exposures used to capture a reasonable intensity for the stimulated bands, the resting bands are most often not visible, which sets an upper limit of ~2–4% for the resting bands, i.e., the lower estimate of a stimulation index of 25–50). The ability to calculate a stimulation index for SHIP1 allows some comparisons that would otherwise be difficult. For example, there was not a statistically significant correlation between the stimulation index and histamine release (Rs = 0.17, n = 36, p = 0.64) when combining data from stimulation with anti-IgE Ab or Ag (Fig. 3A). Fig. 3A also separates the two types of stimulation, Ag or anti-IgE Ab, but the absence of correlation remains for both types of stimulus. Although there is no correlation among donor basophil responses in these two parameters, within a given preparation the phosphorylation of SHIP1 and Shc are essentially coincident with histamine release in a concentration-dependence curve for stimulation with anti-IgE Ab (Fig. 3B). Likewise, if basophils are sensitized with different levels of gp120-specific IgE and stimulated with an optimal concentration of Ag (gp120-OVA), a so-called sensitivity curve (27) is generated like that shown in Fig. 3C; in this case, there also was no clear distinction between the curves for histamine release and pSHIP1 stimulation indices (this curve shows an EC50 for either endpoint of ~3000 molecules of gp120-specific IgE, a value similar to one found in previous studies of the general population (27)).

Using the phosphorylation of SHIP as a marker of its participation in a signaling reaction complex, we examined several other characteristics of its phosphorylation. There are indications from studies of rat basophilic leukemia (RBL) cells that SHIP participation in the reaction complex does not depend on the activity of syk kinase (34). Fig. 4, A and C, show that SHIP phosphorylation is sensitive to an inhibitor of lyn kinase, PP1, with an IC50 similar to that of the inhibition of Shc phosphorylation (at 10 µM, PP1 inhibited SHIP phosphorylation, inhibition of 87 ± 8%, n = 8). PP1 also inhibited the resting level of SHIP phosphorylation (59 ± 10%). However, the activity of syk drives many of the reactions that might also lead to SHIP phosphorylation/recruitment and the current paradigm for Fc{epsilon}RI signaling places lyn kinase upstream of syk activation. In some cells, SHIP interacts with Shc and current evidence suggests that Shc phosphorylation requires the activity of syk kinase (35). To separate the effects of lyn kinase from its effects on the activity of syk kinase, we used one of the new inhibitors of syk kinase, Novartis NVP-QAB205. To examine the role of PI3K in the phosphorylation of SHIP, cells were incubated with LY294002. Each of these inhibitors has been considered reasonably selective for their respective kinase (see Discussion). However, because the Novartis syk inhibitor has not been used in studies of human basophils previously, we first examined several other signaling steps (Fig. 4). Notably, NVP-QAB205 showed an IC50 for inhibition of Shc phosphorylation of 0.1 µM, an IC50 that is expected from previous in vitro studies of this agent’s actions on syk kinase. In contrast, it had no effect on the phosphorylation (activation site) of lyn kinase (see below). To support the conclusion that lyn kinase was not inhibited, we examined the effect of the syk kinase inhibitor on the cytosolic calcium response. Previous studies have shown that inhibition of src family kinases with PP1 at concentrations suboptimal for complete inhibition of the calcium response results in 3- to 4-fold increases in the time lag (quiescent period between the addition of stimulus and the initial rapid increase in cytosolic calcium) and little or no inhibition of the calcium response once the elevation occurs (36). In contrast, general syk and tyrosine kinase inhibitors (staurosporine, Go-6976, genistein, each of which inhibits syk phosphorylation) cause none or modest increases in the time lag while still inhibiting the calcium response. NVP-QAB205 leads to the latter behavior suggesting that it is not acting on a src-family kinase like lyn kinase (data not shown). As expected, NVP-QAB205 does inhibit other signaling steps thought to be downstream of syk kinase, such as ras-ERK pathway elements and mediator release. Fig. 4A shows an example blot from an experiment testing PP1, LY294002, and NVP-QAB205. Notably, LY294002 did not inhibit SHIP phosphorylation (at 10 µM, inhibition of 10 ± 24%) (in previous studies, the IC50 for LY294002 to inhibit phosphorylation of Akt has been below 1 µM. In an experiment not shown, we examined the concentration dependence for inhibition of Akt and SHIP1 phosphorylation. The IC50 for Akt phosphorylation was 0.6 µM while the IC50 for SHIP phosphorylation was 25 µM with no inhibition at 10 µM). Inhibition by NVP-QAB205 was highly variable. For about three-quarters of the preparations, treatment with 1 µM NVP-QAB205 resulted in no or marginal inhibition of SHIP phosphorylation while in the remaining preparations, inhibition was marked (Fig. 4E). Results were similar for both Ags and anti-IgE Ab. There was no relationship to the amount of histamine release (r = –0.15; histamine release vs the percent inhibition of pSHIP, n = 13, p = 0.66) nor was there any relationship to the stimulation index for SHIP phosphorylation (r = 0.27, p = 0.43). A full kinetic curve for anti-IgE stimulation ± NVP-QAB205 did not reveal any meaningful differences at other time points (i.e., percent inhibition was similar at all time points). In those preparations where the syk inhibitor had little or no effect on SHIP phosphorylation, it completely inhibited phosphorylation of ERK. In one experiment, we examined a broader concentration dependence for pSHIP inhibition by NVP-QAB205. The Western blot for this experiment was first blotted with an anti-phospho-Shc Ab, followed by anti-phospho-SHIP and finally, after stripping, with anti-Shc (to determine loading). The inhibition of Shc phosphorylation followed the same curve shown in Fig. 4D and inhibition of SHIP phosphorylation following the dotted line also shown in Fig. 4D. Even under conditions where phosphorylation of SHIP was partially inhibited at 1 µM, the IC50 for Shc and SHIP phosphorylation differed by ~5-fold.


Figure 4
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  		FIGURE 4. Effect of pharmacological agents on IgE-mediated phosphorylation of SHIP1. A, A representative experiment in which basophils were stimulated with 0.2 µg/ml anti-IgE Ab following a 10-min preincubation in PAGCM alone, 10 µM PP1 (src family kinase inhibitor), 10 µM LY294002 (PI3K inhibitor), or 1 µM NVP-QAB205 (syk kinase inhibitor). Western blotting for either phospho-SHIP1 or phospho-Shc are shown, with lane loading assessed by reblotting with anti-Shc. Samples were measured 15 min after stimulation with anti-IgE Ab. B, Enhancement of SHIP1 phosphorylation during stimulation in the presence of EDTA, basophils were stimulated with 0.2 µg/ml anti-IgE Ab ± 2 mM EDTA (added with the anti-IgE Ab). Samples were measured at 15 min. C, Concentration dependence for PP1, basophils stimulated with 0.2 µg/ml anti-IgE ± PP1 or an equivalent concentration of the DMSO carrier, 10 min with PP1 before a 15-min incubation with anti-IgE Ab. Controls included unstimulated cells ± PP1 at 10, 1, and 0.1 µM PP1. Data were plotted as a fraction of the anti-IgE control, pShc (•), pSHIP ({circ}). D, Compilation of available results for the effects of NVP-QAB205 on various endpoints. Basophils were stimulated with 0.2 µg/ml anti-IgE Ab for 15 min and phosphorylation of Shc ({blacksquare}), lyn (•), Syk ({diamondsuit}), Cbl, ERK (only at the 1 µM concentration) assessed by Western blotting. Elevations in cytosolic calcium ({square}) were assessed in fura-2-labeled basophils and mediators (histamine ({diamond}) and LTC4 release) in supernatants. A concentration dependence for inhibition of SHIP1 phosphorylation by NVP-QAB205 is shown ({circ}) from one experiment. E, Inhibition of SHIP1 phosphorylation by 1 µM NVP-QAB205 in 13 different preparations of purified basophils.

 
Because down-regulation of signaling occurs in the absence of extracellular calcium (a desensitization-style protocol) the implication is that whatever elements mediate the down-regulation, these elements operate adequately without extracellular calcium. We examined SHIP phosphorylation following stimulation with anti-IgE Ab ± simultaneous addition of 2 mM EDTA (i.e., the cells were resting before stimulation in calcium containing buffer). On average, SHIP phosphorylation was 1.80 ± 0.26 (n = 6, p = 0.028) greater in the presence of EDTA (Fig. 4B). The enhancement was generally observed at all times during a kinetic assay although enhancement was somewhat less early in the reaction (data not shown). Loading the cells with the cell permeant calcium chelator, BAPTA, and adding EDTA during stimulation, had little further effect on SHIP phosphorylation (data not shown). This behavior is unlike the effects of EDTA on other signaling elements where there is ~50% inhibition of syk, shc, and ERK phosphorylation when cells are stimulated without extracellular calcium (data not shown).

The data above suggest, that for the most part, SHIP phosphorylation is susceptible to the same influences observed for other early activation elements and that it is also dependent on src-family kinase activation rather than syk. We have not previously shown that lyn activation has behavioral characteristics like syk with respect to Ag vs anti-IgE vs either stimulus plus an inhibitor of actin polymerization. Fig. 5 shows that the activation phosphorylation site of lyn does indeed show these divergent characteristics; sustained with anti-IgE Ab (Fig. 5A), transient with Ag (Fig. 5B), both of which are altered to be more sustained with the addition of latrunculin A. We did not detect changes in the phosphorylation state of the inhibitor site of lyn for any stimulus (data not shown). We were unable to consistently detect {gamma}-chain phosphorylation following stimulation with Ag (although in one experiment, it was transient like lyn phosphorylation) but the Fc{epsilon}RI{gamma} chain following anti-IgE Ab consistently showed steadily increasing phosphorylation, a characteristic that was mimicked for the activation site of lyn when latrunculin A was included. The Ab used to detect the phosphorylation state of the activation site of lyn was a general src-family kinase selective Ab and should have been able to detect changes in fyn phosphorylation as well (as a third band migrating 3 kDa above the p56 of lyn). This was never detected following anti-IgE Ab and only occasionally observed following stimulation with Ag (BPO-HSA) although there was no stimulation index (fyn itself is readily detected by specific Abs in basophil lysates). Fig. 5C shows that latrunculin A also prolonged Ag-induced phosphorylation of SHIP.


Figure 5
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  		FIGURE 5. Kinetics of lyn activation site phosphorylation or Fc{epsilon}RI{gamma} phosphorylation. A, Basophils were stimulated with anti-IgE Ab for the times shown, cells were lysed and analyzed for Fc{epsilon}RI{gamma} phosphorylation (n = 3) or phosphorylation of the activation site of lyn (n = 3). For the measurements of Fc{epsilon}RI{gamma} phosphorylation, the cell lysates were immunoadsorbed with anti-phosphotyrosine (PY20) and the blots developed with an anti-Fc{epsilon}RI{gamma} and the data were plotted as a fraction of the 5-min time point ({blacksquare}) (left ordinate). An anti-phospho-src Ab was used to detect lyn activation site phosphorylation in whole cell lysates (right ordinate); ({diamond}) nonstimulated cells, (•) stimulation with 0.2 µg/ml anti-IgE Ab, ({circ}) stimulation with anti-IgE Ab in the presence of 500 nM latrunculin A. B, Using a similar experimental design, basophils (briefly treated to remove some endogenous IgE) were first sensitized with 5 µg/ml penicillin (BPO)-specific IgE then stimulated with 0.5 µg/ml BPO(11)-HSA ± 500 nM latrunculin A. This plot includes two separate studies, one in which the kinetics of lyn-activating site phosphorylation were studied alone (left ordinate, n = 3) and one in which the effects of 500 nM latrunculin A were examined (right ordinate, n = 3). Phosphorylation of lyn with anti-pSrc was measured in whole cell lysates. For the left ordinate ({blacksquare}), BPO(11)-HSA vs ({diamond}) control, and the right ordinate, BPO(11)-HSA ({circ}) with and (•) without latrunculin A. C, Same design as B but pSHIP measured in whole cell lysates (n = 2); (•) no latrunculin A, ({circ}) with 500 nM latrunculin A. 

 
Nonspecific desensitization and functional memory

The first prediction–that SHIP participation, as indicated by its phosphorylation state, is relatively sustained–was not clearly observed (see Discussion). The second prediction, or alternate possibility, was that SHIP participation would be enhanced in a sequential two Ag-challenge experiment. Further, if the first Ag used in this type of experiment were to be dissociated from cell bound IgE by addition of monovalent hapten, the effect of the first Ag on the second Ag response should be reduced. This latter prediction comes from the following experiments.

In our previous studies on nonspecific desensitization, the protocol design used a basophil sensitized with a mouse DNP-specific IgE. Following sensitization, the cells were stimulated with DNP(7)-HSA for 80 min and then re-stimulated with anti-human IgE (these studies established the non-cross-reactivity of the anti-hIgE and mIgE). With ambiguity regarding the presence of surface Ag and function during dissociation, cells were incubated with DNP-lysine for times up to 18 h before the addition of anti-IgE Ab (the reaction was performed in RPMI-medium, see methods). In Fig. 6A, one example of this type of experiment is shown. In this experiment, prior stimulation with DNP-HSA led to 35% inhibition of ERK and Akt phosphorylation following the subsequent stimulation with anti-IgE Ab. The addition of DNP-lysine restored some of the response; in this case, there remained 16% inhibition. Fig. 6B shows the average of six of these experiments with ERK and Akt phosphorylation measured. There was little or no dependence of recovery on the amount of time the cells were exposed to DNP-lysine before the addition of anti-IgE Ab, 5 min, 60 min, and 18 h were similar with {approx}40% recovery (see Materials and Methods for this calculation). The extent of recovery from nonspecific desensitization was also a function of how much nonspecific desensitization had occurred. Fig. 6C shows the relationship between the starting extent of nonspecific desensitization and percent recovery for ERK phosphorylation and Fig. 6D shows a similar dependence for LTC4 release. The same results occur if the second stimulus is an Ag, rather than anti-IgE Ab, and also occur if the cells are first treated with DNP-lysine, washed to remove excess Ag (and DNP-lysine), and incubated in medium plus DNP-lysine before rechallenge with anti-IgE Ab (data not shown). Therefore, treatment of the cells to remove Ag aggregates results in some recovery from nonspecific desensitization, albeit only a partial recovery. It should be noted that in the absence of DNP-lysine, nonspecific desensitization persists for hours. In the kinetic experiments described above (Fig. 2), when SHIP phosphorylation was reduced to resting levels at 6 h, nonspecific desensitization of ERK phosphorylation was as marked as observed at 1 h (the Western blot inset to Fig. 3C shows data for the 6 h block of samples, the 1-h data is not shown). To examine whether DNP-lysine did in fact remove bound Ag, similar experiments were performed with a DNP-GFP conjugate, which was demonstrated in pilot studies to have characteristics similar to DNP-HSA (ability to induce nonspecific desensitization, data not shown). A variety of previous studies using either human basophils or RBL cells have noted that dissociation of bound Ag can require extended periods (the reasons for the slow dissociation are not well-understood. Although a highly branched aggregate might require longer to disaggregate, theoretical treatment of this problem does not indicate why the decrease in rate is as extreme as observed.). Fig. 7A indicates that dissociation of DNP(7)-GFP was rapid after only 5 min of binding but considerably slower after 60 min. In both cases, dissociation did not appear complete; the same minimum plateau was reached for both kinetic curves (this will be discussed further below). In data not shown, we found, as expected from previous studies, that addition of DNP-lysine after 80 min of stimulation with DNP(7)-GFP, rapidly reduced residual syk phosphorylation to resting levels. If DNP-lysine was added after 80 min of stimulation with DNP-HSA, SHIP phosphorylation returned to resting levels within 5 min (Fig. 7B). This is consistent with the results for syk phosphorylation.


Figure 6
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  		FIGURE 6. Partial recovery from nonspecific desensitization. A, Basophils were sensitized with DNP-specific IgE, sequentially stimulated with DNP-HSA and anti-IgE, and the extent of ERK or Akt phosphorylation was measured in whole cell lysates. Cells were stimulated with DNP-HSA alone (5' or 80'), anti-IgE alone for 5 min (after an 80' incubation in PAGCM), DNP-HSA plus sequential anti-IgE (after 80') or DNP-HSA for 80' followed by either 5', 60', or 18 h with 1 mM DNP-lysine and then 5' with anti-IgE Ab. B, The average of six experiments where the incubation time with DNP-lysine varied between 5 min, 60 min, or 18 h; ERK and Akt phosphorylation were compared and the extent of nonspecific desensitization calculated (see Materials and Methods). C, Data derived from the experiments used in B, plotted individually to demonstrate the correlation between the extent of nonspecific desensitization and the ability to recover from nonspecific desensitization after disaggregation of the first stimulus with DNP-lysine for 18 h. D, Same type of experiment as shown in B, plotted as in C with LTC4 secretion as the functional readout. 

 

Figure 7
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  		FIGURE 7. Dissociation of DNP-GFP from basophils with DNP-lysine. A, Basophils sensitized with DNP-specific IgE were incubated for various times with an optimal concentration of DNP(8)-GFP (6 µg/ml) and the amount of cellular fluorescence was monitored by flow cytometry. The control fluorescence in these experiments was assessed by incubation of basophils that had not been sensitized with DNP-specific IgE with DNP(8)-GFP for similar periods of time. Median fluorescence minus background fluorescence was calculated for each time point and the fluorescence at each time point calculated relative to the fluorescence just before the addition of DNP-lysine and the experiments averaged (n = 5). After either 5' or 60' of binding with DNP-GFP, 1 mM DNP-lysine was added and the fluorescence followed. B, DNP(7)-HSA was used to stimulate sensitized basophils for 80 min after which 1 mM DNP-lysine added and the amount of pSHIP measured in whole cell lysates. The data are plotted relative to the extent of pSHIP just before the addition of DNP-lysine (n = 3). 

 
The prediction of an exaggerated pSHIP response following addition of a second Ag was then addressed. These experiments were designed as described above. Basophils sensitized with DNP-specific IgE were first stimulated with DNP-HSA, or buffer alone, followed by stimulation with gp120-OVA (in cells sensitized with both DNP-specific and gp120-specific IgE, see Materials and Methods) or anti-IgE Ab. In some tubes, DNP-lysine was added for 30 min before gp120-OVA or anti-IgE Ab. Fig. 8A shows one example of this type of experiment. Fig. 8B summarizes the results; SHIP phosphorylation following addition of the second stimulus was considerably less in cells first stimulated with DNP-HSA. The addition of DNP-lysine did not result in less SHIP phosphorylation, as expected, but no statistically significant change. For these four experiments in addition to four experiments using anti-IgE Ab as the second stimulus, the ratio of 80' DNP-HSA-induced SHIP phosphorylation to its 5' point was 0.26 ± 0.06 (distribution seen in Fig. 2D). The data shown in Fig. 8 were calculated as stimulation indices that were relative to resting cells but there were two ways to calculate the stimulation following sequential stimulation. For example, if there was no prior Ag, the stimulation index was relative to nonstimulated cells (lane 1 from the left in the example blot). If prior Ag was present, there were two ways of calculating the stimulation index: 1) relative to the state of SHIP phosphorylation remaining when the second stimulus was added (lane 3 from the left in the example blot shown in Fig. 8A) or 2) relative to nonstimulated cells. Finally, if both DNP-HSA and DNP-lysine were present, the stimulation index for gp120-OVA was calculated relative to the 80' DNP-HSA + DNP-lysine. Both methods demonstrate a decrease in the stimulation index. For the decrease (middle bars), p = 0.0096 for 5', p = 0.0295 for 15' when calculated relative to the nonstimulated cells (p = 0.0004 for 5', p = 0.0038 for 15' when calculated relative to the 80 min. DNP-HSA measurement). Similar results were found when anti-IgE Ab was the second stimulus (data not shown). We did note in experiments where both phospho-SHIP and phospho-ERK measurements were made in the same lysates, that nonspecific desensitization of the ERK phosphorylation was greater than for SHIP phosphorylation (75 ± 10% vs 24 ± 10%, respectively, n = 4).


Figure 8
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  		FIGURE 8. Effect of sequential stimulation on SHIP1 phosphorylation. A, One experiment showing SHIP1 phosphorylation throughout stimulation with 3 µg/ml DNP-HSA followed by incubation ± 1 mM DNP-lysine and then stimulation with 0.6 µg/ml gp120-OVA. The cells were sensitized with approximately equal levels of DNP-specific and gp120-specific IgE. The experimental design is similar to that used in Fig. 5 except that the cells were incubated with DNP-lysine for 30 min (right four lanes) before the addition of gp120-OVA. B summarizes the results for four experiments like those shown in A.

 
Recently published studies demonstrate that syk kinase is slowly lost from basophils following IgE-mediated stimulation (37). In the 80-min time frame, losses of syk should be modest but measurable. In the studies presented in Figs. 6 and 8, we examined changes in syk kinase from the same experiments and found that on average, cells stimulated for 80 min with DNP-HSA expressed 0.82 ± 0.05 (p = 0.008, different from 1.0, n = 8) of the syk in cells incubated for 80 min without DNP-HSA. For the experiments with 6 h time points (shown in Fig. 2), syk in the stimulated cells was 0.76 ± 0.07 of unstimulated cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Following stimulation of basophils with anti-IgE, there is a sustained activation of PI3K and yet, the presence of one of its enzymatic products, PIP3, appears transient (as assessed by the transient presence of phosphorylated Akt). This observation suggests that there are not feedback mechanisms that directly down-regulate PI3K that are operative in this time frame (38). Several phosphatases are known to mediate metabolism of PIP3, SHIP1 is thought to be one. We examined the behavior of SHIP1 by measuring its phosphorylation state. We reasoned that involvement of SHIP1 in a developing reaction complex would be reflected in its phosphorylation state but acknowledge that phosphorylation is not necessarily the equivalent of enzymatic activity. With respect to two other phosphatases that could play a role in PIP3 metabolism, we did not observe significant SHIP2 translocation and did not observe any changes in PTEN phosphorylation. In the absence of an Ab to detect SHIP2 phosphorylation, it is currently difficult to fully interpret the observation regarding the participation of SHIP2 and in the absence of changes in PTEN phosphorylation, there was no evidence of stimulation-mediated participation of PTEN. In both cases, these results do not speak to whether either of these two phosphatases play a constitutive role in PIP3 metabolism.

There were two primary predictions related to a role for SHIP in the phenomenon of nonspecific desensitization: 1) that SHIP phosphorylation kinetics (reflecting is participation in the reaction complex) would be sustained and 2) that SHIP phosphorylation would be greater during stimulation with a sequentially applied second stimulus and that this greater SHIP phosphorylation would be relieved by preincubation with a disaggregating agent. Regarding the first prediction; nonspecific desensitization is a sustained event, without dissociation of the desensitizing Ag, it persists for hours and even with dissociation, there is partial persistence. The results for SHIP1 phosphorylation following stimulation with Ag were intermediate between the prediction of a sustained response like that observed for anti-IgE Ab and the more rapid transience of downstream elements like ras, MEK, or ERK. Previous studies of syk phosphorylation found 60'/5' ratios following Ag to have a range of 0.0–0.5 with a median of ~0.10 (2). In the current studies, the 60':5' ratios for phosphorylation of the activation site of lyn were similar to SHIP1. The relative kinetics of pSHIP1 and pAkt are consistent with SHIP1 playing a role in regulating PIP3 during Ag stimulation; its decay in phosphorylation considerably lags behind Akt phosphorylation. The sustained phosphorylation of SHIP during stimulation with anti-IgE Ab is the pattern we speculated might occur for Ag but thus far, all signaling upstream of p21ras has been sustained following stimulation with anti-IgE. Therefore, these results are not surprising although they do indicate the recruitment of SHIP1 is not susceptible to the same influences that regulate the transience of pAkt or the ras->ERK pathway. It is interesting to note that although Ag and anti-IgE Ab induce similar stimulation indices for SHIP1 phosphorylation (see Fig. 3A) the average amount of histamine release was significantly different (27 ± 4% for Ags, 13 ± 3% for anti-IgE Ab). The sustained recruitment/phosphorylation of SHIP1 following anti-IgE Ab might explain this result. It could be argued that the remaining level of SHIP1 phosphorylation following 80 min with Ag was significant enough to account for cross-desensitization if the localization of SHIP1 were sufficient to alter aggregates created by a non-cross-reacting stimulus. However, nonspecific desensitization was present at 6 h when SHIP phosphorylation and translocation was not distinguishable from resting levels. Without precise knowledge of enzymatics, we cannot draw a strong conclusion from these results, but can state that Ag-induced SHIP phosphorylation follows a kinetic pattern that is consistent with other early signaling elements and it does not need to be observable late in the reaction when nonspecific desensitization remains present.

Regarding the second prediction, the apparent decrease in the extent of SHIP phosphorylation when a second stimulus was added suggests that the simple model that led to the predictions is not adequate. The addition of DNP-lysine did not reduce SHIP phosphorylation (relative to its absence) and perhaps this is not surprising given the relatively small residual SHIP1 phosphorylation following 80+ minutes of stimulation with Ag. These results suggest a behavior that is more similar to an activating element, i.e., SHIP recruitment itself is subject to down-regulatory events on a time scale that is similar to other activating elements. Most notably, the down-regulation that results in nonspecific desensitization seems to apply to SHIP phosphorylation itself. SHIP may well play a role in regulating PIP3 but not be responsible for nonspecific desensitization.

We also showed that transient activation of signaling occurs with lyn activation site phosphorylation during stimulation with Ag, not with anti-IgE Ab. An inhibitor of actin polymerization. Latrunculin A, led to a more sustained kinetic curve for lyn activation site phosphorylation following Ag. Treatment with latrunculin A also led to a kinetic curve for lyn phosphorylation following anti-IgE Ab that resembled the natural kinetic curve for {gamma} phosphorylation following this stimulus. There was an apparent dependence of SHIP phosphorylation on the activity of a src-family kinase rather than syk or PI3K and in this context, SHIP phosphorylation was similarly prolonged by the inclusion of latrunculin A. These results also demonstrate that SHIP phosphorylation is sensitive to down-regulatory mechanisms effecting other early signaling elements rather than showing an independence from this kind of down-regulation. How SHIP participates in an ongoing reaction is not a settled issue. It seems likely that the SH2 domain provides a means of recruitment either to an ITIM motif, such as found on Fc{gamma}RIIb (39), or another receptor (34) or an adaptor phosphorylation site, such as found on shc (40). Once located in the reaction complex, the kinase region may be in a position to mediate metabolism of PIP3 but the C-terminal region may act as an adaptor protein. The C-terminal phosphorylation sites may recruit phosphotyrosine binding or SH2 domain proteins and the polyproline motifs may attract SH3 domains. In this context, the recruitment of a dok protein (15, 16) that itself attracts rasGAP into reaction (5, 41) may have relevance to the down-regulation of the ras->ERK pathway in basophils. If SHIP phosphorylation does indeed mediate the attraction of additional down-regulatory molecules, then the reduction of phosphorylation of SHIP late in the reaction has relevance to regulating the ERK pathway regardless of the significance of SHIP phosphorylation to its enzymatic activity or whether SHIP remains in the vicinity of the plasma membrane.

In contrast to the above observations, there were some observations that do distinguish SHIP phosphorylation characteristics from other signaling elements we have studied to date. First, the stimulation index for SHIP phosphorylation has no relationship to the magnitude of histamine release among different donor basophils. Parenthetically, if the anti-phospho-SHIP Ab detected some nonphosphorylated SHIP, the calculated stimulation index would be blunted and such a cross-reactivity would add noise to the calculation. However, as noted in earlier, this cross-reactivity is probably minimal. In previous studies, we found a good correlation among different donors between the cytosolic calcium response and histamine release (42). Therefore, the magnitude of change in at least one activating signal element is a predictor of subsequent responsiveness. If SHIP were simply a negative regulator, one might find an inverse correlation but there are indications from studies in other cell types that SHIP may also act as an adaptor to recruit other signaling elements (43), so that its precise role, positive or negative, is not completely clear at this time. If SHIP’s role is multifaceted, the absence of neither a positive nor negative correlation might reflect this mixed role.

A second interesting characteristic is the augmented phosphorylation in the absence of extracellular calcium (presence of EDTA). This is the first phosphorylated signaling element we have found that shows this characteristic. A typical desensitization protocol is performed in the presence of EDTA and the rate of decay in the activation of various signaling elements is faster without extracellular calcium (18). Any element that participates in desensitization might be expected to be as active, if not more active, in the absence of extracellular calcium. In recent studies of the loss of syk kinase during stimulation, we also found an enhancement in the rate of loss in the absence of extracellular calcium (37), making the observation about SHIP phosphorylation a second process that may be more active in a desensitization-style experiment. In this context, SHIP behavior is consistent with a greater rate of down-regulation in the absence of extracellular calcium and therefore more consistent with the properties of desensitization.

These are the first published studies of a syk kinase inhibitor that has selectivity in basophils. We have shown that piceatannol, a selective syk inhibitor in rodent cells (44), probably does not even function as a syk inhibitor in human basophils except possibly at extremely high concentrations (>>100 µg/ml) (45). The NVP-QAB205 inhibitor is an analog of a current generation of human syk inhibitors that in our studies of IgE-mediated activation of human basophils has an IC50 of 100 nM for inhibition of Shc phosphorylation, a likely direct downstream substrate of syk kinase. In contrast, there was no apparent inhibition of the phosphorylation of the activation site of lyn kinase which presumably results from the activity of lyn itself. The effect on SHIP phosphorylation was therefore, quite confusing. Taken together, it seems unlikely that any observed inhibition of SHIP phosphorylation results from inhibition of syk kinase, the aggregate IC50 is 5- to 30-fold higher than the consistent inhibition of many events thought to be downstream of syk. This absence of dependence of syk, however, is consistent with what was observed in RBL cells (34). Recent studies in murine cells indicate a variable expression of lyn and fyn and possibly opposing functions for the two src kinases (46). In our studies of activation site phosphorylation for src family kinases, we were unable to detect fyn phosphorylation but further study would be necessary to draw conclusions from these observations. The activity of NVP-QAB205 for lyn kinase is known from ex situ studies to be 10- to 100-fold less potent than for syk but its potency for fyn kinase is unknown (20). Perhaps the variable effects observed on SHIP phosphorylation reflect variable participation of fyn kinase in the early activation cascade.

The data do show a consistent lack of inhibition of SHIP phosphorylation in the presence of the PI3K inhibitor, LY294002, suggesting that this enzyme does not regulate SHIP participation.

The studies shown in Fig. 6 suggest that there is functional memory of prior stimulation; dissociating previously bound Ag results in only partial recovery of desensitization. The remaining loss of function may be caused by the actual loss of signaling components. Recent studies have shown that human basophils slowly lose syk kinase during optimal and even suboptimal stimulation (37). In the experiments described in Figs. 6 and 8, there was a statistically significant loss of ~20% of the syk kinase. It is possible that other signaling components are lost by similar mechanisms (possibly by ubiquinylation); the net effect may be greater than can be accounted for by the loss of syk kinase alone. This loss may result in the persistent partial state of desensitization. Nevertheless, some caution must be used in the interpretation of these results because it was not possible to observe complete dissociation of bound multivalent DNP-GFP. A variety of previous studies have noted a similar phenomenon both in RBL cells (47) and human basophils (21), so it is not surprising that there is some residual binding. Because this bound Ag does not appear to induce any signaling, one interpretation is that the multivalent Ag enters a state of monogamous bivalent binding, i.e., one Ag-one IgE with both Fab arms bound to the same Ag. This state would be expected to be extremely stable and difficult to dissociate. However, an alternative explanation for the presence of residual bound Ag has been proposed; some Ag is bound to immobilized IgE/receptor aggregate complexes which also reflects an aggregated state that is unable to signal (48). In extended binding studies to be presented in a future publication, we did not find that performing the dissociation in the presence of latrunculin A or at 4°C altered the fraction remaining bound. Both of these conditions were predicted (48) to eliminate the remaining bound fraction because immobilizing the receptors this way requires cell function and/or a functional actin network. Therefore, the evidence suggests a state of monogamous bivalent binding is a better explanation for this phenomenon. If this remaining DNP-Ag bound to the basophil surface does not represent aggregated receptors, then functional memory due to signaling component loss may be a valid explanation of desensitization that persists after aggregates are removed. Based on extended kinetic studies, we might also expect that progressively greater late nonspecific desensitization would be accounted for by the progressive loss of syk in the first 18 h of stimulation (37). However, it seems unlikely that component loss could account for all the observed nonspecific desensitization.

In summary, several forms of down-regulation appear to occur during IgE-mediated stimulation of human basophils. Previous studies noted down-regulation in two regions of signaling; peri-syk kinase and peri-PIP3. Although a role for SHIP in dynamically regulating PIP3 and therefore causing nonspecific desensitization is not formally excluded by the current studies, it was not expected that recruitment of SHIP into the reaction complex–as evidenced by its phosphorylation–would be sensitive to the same down-regulatory mechanisms associated with other activating elements. Indeed, SHIP phosphorylation undergoes modest nonspecific desensitization. With the exception of its absence of sensitivity to removing extracellular calcium, the characteristics of SHIP phosphorylation were more consistent with activating elements rather than the expectations for desensitizing elements. These studies also demonstrate a third process that accompanies nonspecific desensitization and is persistent, a process that may be related to the loss of signaling components such as syk kinase.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI20253. Back

2 Address correspondence and reprint requests to Dr. Donald MacGlashan, Jr., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail address: dmacglas{at}jhmi.edu Back

3 Abbreviations used in this paper: PIP3, phosphatidylinositol 3,4,5-phosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; HSA, human serum albumin; BPO, benzylpenicilloyl; PAG, PIPES-albumin-glucose; hIgE, human IgE; mIgE, mouse IgE; LTC4, leukotriene C4; pSHIP, phosphorylated CY1020 SHIP; RBL, rat basophilic leukemia. Back

Received for publication October 11, 2005. Accepted for publication May 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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