Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0903431 on March 2, 2004

Published online before print March 2, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0903431v1
75/5/928    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vilariño, N.
Right arrow Articles by MacGlashan, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vilariño, N.
Right arrow Articles by MacGlashan, D. W., Jr
(Journal of Leukocyte Biology. 2004;75:928-937.)
© 2004 by Society for Leukocyte Biology

Actin cytoskeleton-dependent down-regulation of early IgE-mediated signaling in human basophils

Natalia Vilariño and Donald W. MacGlashan, Jr1

Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland

1 Correspondence: Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: dmacglas{at}jhmi.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two regions of down-regulation of Fc{varepsilon}RI [high-affinity immunogloublin E (IgE) receptor] signaling have been localized recently in basophils. An early down-regulatory step is located proximal to syk and appears responsible for a transient syk phosphorylation in antigen-stimulated basophils. A second, more distal region appears responsible for the transient activation of the ras–extracellular-regulated kinase (Erk) pathway when syk phosphorylation is sustained in anti-IgE-stimulated basophils. As the actin cytoskeleton has been demonstrated to inhibit the early Fc{varepsilon}RI signaling in rat basophilic leukemia cells, we explored the hypothesis that the actin cytoskeleton was responsible for the transience of syk phosphorylation in antigen-stimulated basophils. The inhibition of F-actin polymerization with latrunculin A induced a sustained syk phosphorylation in basophils stimulated with an optimal dose of the antigen benzyl penicilloyl–human serum albumin. However, in the presence of latrunculin A, Erk phosphorylation remained transient after stimulation with the antigen or anti-IgE. Latrunculin A also increased downstream events such as histamine release, leukotriene C4 release, and the intracellular calcium signal, although some of these effects were not specific for an immunologic stimulus. Our results suggest that the actin cytoskeleton is responsible for down-regulation of Fc{varepsilon}RI signaling at a point located proximal to syk phosphorylation. Moreover, the fact that latrunculin A did not result in sustained Erk phosphorylation supports the presence of a second down-regulatory step between syk and Erk that cannot be overcome by a sustained early signal.

Key Words: antigen • latrunculin • syk • Erk


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All cells need mechanisms to limit and ultimately stop an activating signal. Therefore, every signaling cascade triggered by a stimulus should be endowed with activating and inactivating mechanisms. The inactivating or down-regulating mechanisms can occur at many points in a receptor-driven signaling cascade and may be constitutive or induced. Constitutive mechanisms of down-regulation should be commonplace; e.g., a local phosphatase to counter low-level kinase activities may account for the changes in phosphorylation resulting from exposure of cells to phosphatase inhibitors such as pervanadate. Induced mechanisms of down-regulation may be more interesting in that they offer a means for the cell to dynamically regulate the rate of inactivation and therefore regulate the extent of the cellular response. The loss of a cell-surface receptor by endocytotic mechanisms is a common method of down-regulation that involves numerous regulated steps. For immunoreceptors, such as the high-affinity immunoglobulin E (IgE) receptor, the loss of the cell-surface receptor does not always occur in a time-frame consistent with the transience of downstream signaling steps, suggesting the presence of other mechanisms. Knowing which mechanisms are dominant regulators of signaling and how these various mechanisms interact to bring about regulation of the ongoing response is information that is needed to understand control of the cellular response.

In our cellular model, the human basophil, activation through aggregation of the Fc{varepsilon}RI (high-affinity IgE receptor) triggers a signaling cascade that leads to release of different mediators: histamine, leukotriene C4 (LTC4), and interleukin (IL)-4. The early steps of this cascade include Src family tyrosine kinase activity, phosphorylation of the receptor, and recruitment and activation of the tyrosine kinase syk [1 2 3 ]. After this early signaling, several downstream pathways are activated, among them the calcium signal, phosphatidylinositol 3-kinase (PI-3K), and the ras–extracellular-regulated kinase (Erk) pathway [4 ]. All these signaling steps are part of the cascade of activating events that follow IgE-mediated stimulation; however, not much is known about how this signal is down-regulated. Yet, down-regulation clearly occurs, as the basophil response to an immunologic stimulus reaches a plateau for histamine, LTC4, and IL-4 release, even at optimal concentrations of the stimulus, and this plateau does not represent the maximum capacity of the cell to release [5 ].

Some down-regulatory mechanisms have been described in other related cell types. In rat basophilic leukemia (RBL) cells, internalization of the receptor occurs rapidly after aggregation [6 , 7 ], but this mechanism does not apply to human basophils [8 ]. In murine mast cells, Src homology 2-containing-inositol 5'-phosphatase (SHIP), an enzyme that reduces PI-3,4,5 trisphosphate (PIP3) levels, down-regulates mediator secretion [9 ]. Recent studies of ours further demonstrate that PI-3K activity is sustained following stimulation with anti-IgE antibody and that phosphorylation of SHIP, but not dephosphorylation of phosphatase and tension homologue deleted on chromosome 10 (PTEN), occurs during this reaction. These results strongly suggest that SHIP may be responsible for the transient appearance of PIP3. Other studies suggest that SHIP may have a role in down-regulation of basophil function, and its low expression leads to hypersecreting phenotypes [4 , 10 ]. Also in RBL cells, the actin cytoskeleton seems to be involved in down-regulation of the early IgE-mediated signaling, as inhibitors of actin polymerization increased phosphorylation of syk at an early time-point and prolonged phosphorylation of the ß subunit of the receptor [11 12 13 ]. Some similarities between RBL cells and basophils suggest that the actin cytoskeleton could play a down-regulatory role in basophils: not only do basophils and RBL cells undergo a remodeling of the actin cytoskeleton after Fc{varepsilon}RI aggregation, but inhibitors of actin polymerization induce an increase of IgE-mediated secretion in both cell types [12 , 14 15 16 17 ].

Recent work in our laboratory suggests the location of two down-regulatory regions in the IgE-mediated signaling pathway in basophils. The first identified region of down-regulation was localized in the pathway to LTC4 release downstream of PI-3K and upstream of ras. This down-regulatory mechanism, still to be identified, is responsible for the transient activation of the ras–Erk pathway when earlier signaling, such as syk, shc, Grb2/SOS, and PI-3K activation, is sustained [4 ]. The experiments that revealed the presence of this down-regulatory region were performed using anti-IgE antibody as the Fc{varepsilon}RI-aggregating stimulus. However, later studies showed that aggregation of the receptor with a wide variety of antigens induced a transient activation of early (syk) and late (Erk) signaling, which suggests the presence of another down-regulatory region in the IgE-mediated signaling proximal to syk [18 ].

Two possible regions of regulation controlling the response in human basophils raise some interesting questions about the relative contribution of the two down-regulatory processes in controlling late events such as secretion. We viewed the issue as a systems control problem. The studies with anti-IgE antibody suggested that a sustained, early signal could not overcome the down-regulatory process that occurs later in the signaling cascade. However, it was possible that the type of signals generated by stimulation with anti-IgE antibody modified the relationship between the two down-regulatory processes. Therefore, it was important to examine the system response under conditions in which a normally transient, early signal was converted to a sustained signal. If the results with anti-IgE were an indication, we predicted that inhibition of the early, down-regulatory process that causes transient phosphorylation of syk following antigen would not overcome the down-regulatory process causing transient activation of the ras–Erk pathway. We tested this prediction by inhibiting the polymerization of actin. In this study, we explore the involvement of the actin cytoskeleton in the down-regulation of early (syk proximal signaling) and late signaling in antigen-stimulated basophils, as well as its specificity for the syk proximal down-regulatory region in the signaling pathways to mediator release. We also explored the activation of the PI-3K–ras down-regulatory region following stimulation by antigen, as well as by anti-IgE antibody, and confirmed its role in transforming a sustained signal into a signal that is transient.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The following were purchased: piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES), bovine serum albumin (BSA), EGTA, EDTA, D-glucose, NaF, Na3VO4, 2-mercaptoethanol (2-ME), Nonidet P-40 (NP-40), formyl-methionyl-leucyl-phenylalanine (fMLP), and phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO); latrunculin A and ionomycin (Calbiochem, La Jolla, CA); crystallized human serum albumin (HSA; Miles Laboratories, Elkhart, IN); fetal calf serum (FCS) and RPMI 1640 containing 25 mM HEPES and L-glutamine (BioWhittaker, Walkersville, MD); Percoll (Pharmacia, Piscataway, NJ); Tris(hydroxymethyl)-aminomethane and Tween-20 (Bio-Rad, Hercules, CA); Fura-2/AM and Oregon Green phalloidin (Molecular Probes, Eugene, OR); phenylmethylsulfonyl fluoride (PMSF; Boehringer Mannheim, Indianapolis, IN); antiphosphotyrosine monoclonal antibody (mAb) 4G10 (Upstate Biotechnology, Lake Placid, NY); rabbit antiphospho-Erk Ab, anti-Erk, antiphospho-Akt (Thr308-specific), rabbit antiphospho-syk Ab (Tyr352-specific), anti-Akt, and biotinylated molecular weight markers (Cell Signaling, Beverly, MA); anti-syk mAb (Santa Cruz Biotechnology, Santa Cruz, CA); and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit Ig Ab, HRP-conjugated sheep anti-mouse Ig (anti-mIg) Ab, and protein G sepharose beads (Amersham Life Science, Arlington Heights, IL). Goat anti-human IgE Ab was prepared as described [19 ]. Anti-benzylpenicilloyl (BPO) IgE was purified from serum of penicillin-allergic patients as described previously [20 ] and was typically used at 5 µg/ml in RPMI buffer containing 0.1 mM BPO–{varepsilon}-aminocaproic acid. BPO–HSA was synthesized for previous studies [20 ]. The clone to generate antidinitrophenyl (anti-DNP) mIgE (H1 DNP–{varepsilon}-26) was a gift of Teruko Ishizaka. Anti-DNP mIgE was partially purified from ascites fluid as described previously [21 ]. DNP7–HSA was prepared as described previously [21 ].

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. Countercurrent elutriation was conducted in PAG containing 0.25% BSA in place of 0.003% HSA. ESB is Novex electrophoresis sample buffer containing 5% 2-ME. Complete lysis buffer (CLB) is 20 mM Tris-HCl, pH 7.5, 100 µg/ml aprotinin, 10 mM benzamidine, 1 mM PMSF, 50 mM NaF, 1 mM Na3VO4, 1% NP-40, and 10% glycerol. Incomplete lysis buffer is CLB without the protease inhibitors NP-40, glycerol, or vanadate. Stripping buffers were 7 M guanidine hydrochloride or 65 mM Tris-HCl (pH 6.7), 100 mM 2-ME and 2% sodium dodecyl sulfate (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 Percoll density gradients and countercurrent flow elutriation, as described previously [22 ]. Basophils were further purified by negative selection with a basophil purification kit (Stem Cell Technologies, Vancouver, BC) and columns from Miltenyi Biotec (Aubum, CA). The purity of basophils was determined by alcian blue staining [23 ] and generally exceeded 99% when basophils were purified from these leukapheresis packs.

F-actin measurement
Intracellular F-actin levels were measured using Oregon Green phalloidin with a few modifications from the method described previously [15 ]. Basophils (0.1–0.2x106) were stimulated at 37°C, and the reaction was stopped with ice-cold fixation buffer [3.2% paraformaldehyde and 0.25% lysophosphatidylcholine in phosphate-buffered saline (PBS)]. After overnight incubation at 4°C, basophils were washed once and incubated with 0.2 µM Oregon Green phalloidin (in PBS containing 1% BSA) for 20 min at 20°C in the dark. The fluorescent dye was washed away, and fluorescence was measured by flow cytometry.

LTC4 and histamine measurements
A radioimmunoassay was performed using 100 µl supernatant to determine LTC4 levels as described previously [24 ]. Histamine was measured by automated fluorimetry [25 ]. The percentage of total histamine release was calculated after subtraction of spontaneous histamine release. Each condition tested was performed in duplicate (0.02x106 basophils per condition) in histamine and LTC4 release experiments when using impure cells, and histamine was measured in 250 µl supernatant diluted up to 1 ml in saline solution. In these experiments, the concentration of latrunculin A was 50 nM, as it is slightly above the IC50 for histamine release. When histamine release was measured in the same cells as protein phosphorylation (1.5–3x106 pure basophils per singlet condition), with a final volume of incubation of 100 µl, a 5 µl aliquot was taken from the centrifuged supernatant and diluted in 1 ml saline solution for histamine detection. To determine the total histamine content, 5 µl of the cell suspension was lysed with 100 µl HClO4, and an appropriate portion (with respect to the cell number used for stimulation) was diluted further for analysis of histamine.

Phosphorylation of syk
Syk phosphorylation was detected with the antiphosphotyrosine Ab clone 4G10 after immunoprecipitation of syk from basophil lysates. After stimulating basophils (1.5–3x106 per condition) in PAGCM buffer at 37°C, the reaction was stopped by adding ice-cold PAG and microfuged for 15 s. The supernatants were recovered for histamine and LTC4 detection, and the pellets were immediately lysed in CLB by vortexing and incubating on ice for 10 min. The lysates were then centrifuged for 3 min at 16,000 g and precleared with protein G sepharose beads for 30 min at 4°C, and the clarified lysates were incubated with anti-syk Ab prebound to protein G sepharose beads (1 µg antibody per 20 µl beads) for 1 h at 4°C. The beads were washed three times with CLB buffer. The immunoprecipitated proteins were eluted by boiling for 5 min in ESB. After electrophoresis and transfer, the membranes were blotted with 4G10 or antiphospho-syk-specific antibody. The membranes were then stripped with SDS buffer and reblotted with anti-syk Ab to determine loading. Enhanced chemiluminescence (ECL) films were converted to digital images with a Kodak DC290 camera, and the bands were analyzed with NIH Image. Data from the antiphosphotyrosine blots were normalized for loading differences using the band intensities from the anti-syk reblot.

Phosphorylation of Erk 1/2 and Akt
The phosphorylation of Erk and Akt was assessed using antiphospho-Erk p42/44 Ab and antiphospho-Akt Ab, respectively. Basophils (0.5–1x106 per condition) were stimulated in PAGCM buffer, and reactions were stopped by adding ice-cold PAG and were microfuged for 15 s. After collecting the supernatants for detection of histamine and LTC4, the pellets were lysed in ESB and boiled for 5 min. The lysates were separated by electrophoresis on 10% Tris-glycine gels (Novex, San Diego, CA) and transferred to a nitrocellulose membrane as described in the product literature. The membranes were incubated overnight at 4°C in Tris-buffered saline/Tween 20 (TBST) containing 3% nonfat dried skim milk (Carnation, Los Angeles, CA) or 4% BSA to block nonspecific binding. Phosphorylated Erk and Akt were detected by a 90-min incubation with the antibodies diluted in TBST containing 3% skim milk or 4% BSA. After washing, the membranes were incubated with HRP-conjugated anti-rabbit Ab for 1 h. The membranes were washed again, and ECL detection was performed as in the product literature (Amersham, Piscataway, NJ). The same membranes were sequentially blotted with antiphospho-Erk Ab and antiphospho-Akt and were reblotted with anti-Akt Ab to assess equal loading after stripping with SDS buffer for 1 h at 50°C. ECL films were converted to digital images with a Kodak DC290 camera, and the bands were analyzed with NIH Image. Erk 1/2 data presented are the result of adding the two bands p42/44.

Cytosolic-free calcium concentration ([Ca2+]i) measurements
Basophils were labeled with 1 µM fura-2/AM for 20 min at 37°C in RPMI 1640 containing 2% FCS (0.3–0.5x106 cells in 200 µl). After washing once with 200 µl PAG, the cells were resuspended in PAG for loading in the microscope observation chamber [26 , 27 ]. Changes in [Ca2+]i were determined by digital video microscopy using techniques described previously in detail [26 , 28 ]. Briefly, 15 µl cells were loaded onto the siliconized coverslip of the microscope and after settling, overlaid with 1 ml PAGCM buffer. After warming to 37°C, monitoring the cells began, and after four frames (each frame is a single measurement of a field of 30–100 cells) of resting [Ca2+]i levels were acquired, the cells were challenged with 1 ml stimulus in buffer. Data were then obtained for 50–150 frames at intervals of 1–10 s to determine the [Ca2+]i response.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To test the prediction that an inhibition of the first down-regulatory region would not overcome the late down-regulatory mechanism, it was necessary to find an inhibitor of the first down-regulatory step. Studies in RBL cells [11 12 13 ] suggested that the cytoskeleton played a critical role in this type of down-regulation. We tested the hypothesis that the cytoskeleton is involved in the early down-regulatory mechanism in human basophils as well as being responsible for the transience of syk phosphorylation after antigen stimulation. If correct, disruption of the actin cytoskeleton will cause a change of syk phosphorylation kinetics from transient to sustained, an observation not yet made in RBL cell studies. First, we characterized the effects of the actin polymerization inhibitor, latrunculin A, on the polymerization state of actin (F-actin) and mediator release in basophils.

Effect of latrunculin A on the actin cytoskeleton of basophils
F-actin was stained with Oregon Green phalloidin after stimulation and fixation of the basophils, and fluorescence was measured by flow cytometry. Latrunculin A disrupted the actin cytoskeleton in basophils, in resting and activated cells. Figure 1A shows examples of the results for one concentration of latrunculin A (left panel, cells without latrunculin A; right panel, cells with drug). Figure 1B shows the concentration dependence of the effect of latrunculin A on the amount of F-actin in resting basophils. Aggregation of the receptor with anti-IgE induced an increase in the amount of F-actin in the cell (light vs. bold tracing in A), as reported previously [15 ]. Figure 1C shows that the anti-IgE-induced increase of F-actin after 5 min of stimulation was also inhibited in the presence of latrunculin A, and this effect has a concentration dependence similar to the inhibition of the resting F-actin.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Effect of latrunculin A on F-actin in basophils. (A) Examples of changes in F-actin content induced by stimulation with anti-IgE Ab ± latrunculin A. The left panel shows control (light tracing) and stimulated (bold tracing, 0.2 µg/ml anti-IgE) flow cytometric distributions of F-actin content after 5 min. Medians for the control and stimulated distributions were 500 and 914, respectively. The right panel shows the distributions for cells incubated with 100 nM latrunculin A for 5 min and 5 min with (bold tracing) or without (light tracing) anti-IgE Ab. Medians for the control and stimulated distributions were 322 and 449, respectively. This protocol was used to generate the data in B and C. (B) Concentration dependence of latrunculin A on the content of F-actin in resting basophils after 10 min of incubation. Data are expressed as percentage of control without drug (n=3; note that the error bars for these studies are smaller than the symbol size). (C) Inhibition of the anti-IgE-induced increase of F-actin by latrunculin A. Basophils were incubated in the absence or presence of 1, 10, 50, 100, 200, and 500 nM latrunculin A for 5 min and incubated ± anti-IgE Ab for an additional 5 min. Without latrunculin A, anti-IgE Ab induces a 1.88-fold increase in F-actin (represented by the dotted line). For each of the concentrations of latrunculin A, the fold change, relative to nonstimulated cells also in the presence of the same concentration of latrunculin A, was calculated and plotted (n=3; *, P<0.05).

 
Latrunculin A-induced increase of mediator release
Figure 2 demonstrates that latrunculin A markedly enhances histamine release and LTC4 release induced by aggregation of Fc{varepsilon}RI. Latrunculin A enhanced histamine release following stimulation with an optimal concentration of anti-IgE (0.2 µg/ml), with enhancement reaching a maximum at concentrations higher than 100 nM (Fig. 2A) . The kinetics of anti-IgE-induced mediator release in the presence of latrunculin A is similar to the kinetics in the absence of latrunculin A, although histamine release (data not shown) and LTC4 release (Fig. 2B) reach a higher plateau in the presence of the drug. Latrunculin A also increased histamine release and LTC4 release induced by BPO–HSA in basophils sensitized with BPO-specific IgE (Fig. 2C and 2D) .



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Effect of latrunculin A on IgE-mediated histamine and LTC4 release. (A) Concentration dependence of latrunculin A on anti-IgE-induced histamine release. Basophils were incubated and stimulated with 0.2 µg/ml anti-IgE for 45 min in the absence or presence of different doses of latrunculin A for 5 min. The solid line indicates release in the absence of latrunculin A. (B) LTC4 release kinetics in the presence ({circ}) and absence (•) of 50 nM latrunculin A in basophils stimulated with 0.2 µg/ml anti-IgE (impure basophils were used; n=4). LTC4 release in the presence of latrunculin A but no anti-IgE ({diamond}). Asterisks indicate statistically significant enhancement; P< 0.05. (C) Histamine release (n=8) and (D) LTC4 release (n=9) were measured in the experiments included in Figures 3B and 4B . Basophils were sensitized for 20 min with BPO-specific IgE, washed, and preincubated with (shaded bars) or without (open bars) 500 nM latrunculin A for 5 min. Basophils were then stimulated with 0.5 µg/ml BPO–HSA for 5 and 60 min in the presence of calcium. Latrunculin A concentration was maintained throughout the experiment (*, P<0.05).

 
To examine whether the effects of latrunculin A were specific for IgE-mediated stimulation, basophils were stimulated with fMLP, ionomycin, and PMA, stimuli that use signaling pathways that are partially distinct from Fc{varepsilon}RI aggregation. Histamine and LTC4 release induced by these stimuli in the presence and absence of latrunculin A was measured. Latrunculin A induced an increase of LTC4 and histamine release to fMLP; however, it did not have any effect on the response to PMA or ionomycin (Table 1 ). The concentration-response curves for PMA- and ionomycin-induced secretion were not affected by latrunculin A (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of Latrunculin A on IgE- and Non-IgE-Mediated Histamine and LTC4 Release

 
Syk phosphorylation
Basophils were sensitized with BPO-specific IgE and then preincubated for 5 min with latrunculin A and stimulated with an optimal concentration of BPO11.5–HSA. The basophils were harvested at 5 and 60 min after stimulation. Previous studies, where a more complete time-course of syk phosphorylation was examined, established that the 5- and 60-min time-points were optimal for exploring the transience of the signal [18 ]. The ratio of phosphorylation at 60 min to phosphorylation at 5 min (60/5 ratio) was used as an index of the transience of the response (previous studies showing a ratio of 0.1–0.2 for antigen-induced signaling and a ratio of 0.88 for stimulation with anti-IgE antibody). syk phosphorylation induced by BPO–HSA was significantly more sustained in the presence of latrunculin A than in the BPO–HSA control (Fig. 3A and 3B ). The 60/5 ratio of syk phosphorylation was 0.23 ± 0.11 and 0.77 ± 0.25 (P<0.05) for control and latrunculin A-incubated basophils, respectively. Latrunculin A also induced an increase in the peak of syk phosphorylation at 5 min (Fig. 3B) . To be certain that the general syk phosphorylation detected with the antiphosphotyrosine antibody 4G10 reflects syk activation, a specific antiphospho-syk antibody to Tyr352, a phosphorylation site required for phospholipase C{gamma}1 (PLC{gamma}1) activation [29 ], was also assayed. Phosphorylation of Tyr352 of syk was also transient in BPO–HSA-stimulated basophils and sustained in latrunculin A/BPO–HSA-incubated cells (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Effect of latrunculin A on IgE-mediated syk phosphorylation. (A) One example of a Western blot of syk phosphorylation detected with the phospho-Tyr (pTyr) Ab 4G10 in syk-immunoprecipitated lysates. Lower blot shows syk loading. Basophils were sensitized for 20 min with BPO-specific IgE, washed, and preincubated with or without 500 nM latrunculin A for 5 min. Basophils were then stimulated with 0.5 µg/ml BPO–HSA for 5 and 60 min. (B) Syk phosphorylation in BPO–HSA-stimulated basophils. Basophils were sensitized with BPO-specific IgE, washed, and preincubated with ({circ}) or without (•) 500 nM latrunculin A for 5 min. The experiment was done two ways, differing by the presence (n=4) or absence (n=3) of extracellular calcium during the preincubation with latrunculin A. Basophils were then stimulated with 0.5 µg/ml BPO–HSA for 5 and 60 min in the presence of calcium. As the results were similar, the seven experiments were combined for the figure. The ordinate is expressed as the fraction of 4G10 band intensity at the 5-min time-point for cells stimulated with antigen in the absence of latrunculin A. (C) syk phosphorylation in anti-IgE-stimulated basophils. The cells were preincubated with ({circ}) or without (•) 500 nM latrunculin A in the absence of calcium. Basophils were then stimulated with 0.2 µg/ml for 5 and 60 min in the presence of calcium (n=3). (B and C) Results are expressed as fraction (Fx) of syk phosphorylation at 5 min after stimulation in the absence of drug (*, P<0.05).

 
As noted previously, unlike antigen, anti-IgE alone induces a sustained phosphorylation of syk. In the presence of latrunculin A, anti-IgE-induced syk phosphorylation remained sustained (Fig. 3C) . The 60/5 ratios for control and latrunculin A-incubated cells were 1.04 ± 0.17 and 1.53 ± 0.26, respectively (P<0.05). Latrunculin A still induced a statistically significant increase of anti-IgE-induced peak syk phosphorylation, although the relative increase is smaller than in antigen-stimulated basophils.

Erk and Akt phosphorylation
Basophils were stimulated following the protocols described above, and Erk and Akt phosphorylation was measured with specific antibodies. BPO–HSA-induced Erk phosphorylation was transient in the presence or in the absence of latrunculin A (Fig. 4A and 4B ). The values of the 60/5 ratios were 0.09 ± 0.06 and 0.14 ± 0.02 in control and latrunculin A-incubated basophils, respectively (n=3, not statistically significant). Similar results were obtained when the aggregating stimulus was anti-IgE (Fig. 4C ; 60/5 ratios were 0.23±0.12 and 0.40±0.17 for control and latrunculin A-incubated cells, respectively; n=3, not statistically significant). Despite the ability of latrunculin A to cause sustained syk phosphorylation, Erk phosphorylation remained transient. Although any definition of "sustained" might be considered arbitrary, the results for syk phosphorylation are a reasonable guide. Our criterion to define a sustained signal was that phosphorylation at 60 min had to remain higher than 50% of the phosphorylation level at 5 min (this is within 1 SD of the average ratio for syk phosphorylation). The results of Erk phosphorylation, unlike syk, do not fulfill this criterion of a qualitative change of signal from transient to sustained. In studies not shown, we obtained similar results with another actin polymerization inhibitor, cytochalasin D.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 4. Effect of latrunculin A on IgE-mediated Erk and Akt phosphorylation. (A) One example of a Western blot of Erk phosphorylation detected with a specific phospho-Erk Ab in whole basophil lysates. Loading was similar for all lanes. Basophils were sensitized for 20 min with BPO-specific IgE, washed, and preincubated with 500 nM latrunculin for 5 min. Basophils were then stimulated with 0.5 µg/ml BPO–HSA for 5 and 60 min. (B) Erk phosphorylation in BPO–HSA-stimulated basophils. The experiment was performed as in A (n=3), with ({circ}) or without (•) latrunculin A. (C) Erk phosphorylation in anti-IgE-stimulated basophils. The cells were preincubated with ({circ}) or without (•) 500 nM latrunculin A for 5 min and then stimulated with 0.2 µg/ml anti-IgE for 5 and 60 min (n=3). (D) Akt phosphorylation in BPO–HSA-stimulated basophils. The experiment was performed as in A (n=2). (B–D) Results are expressed as fraction (Fx) of protein phosphorylation at 5 min after stimulation in the absence of drug. pErk, Phoshorylated Erk.

 
There was a tendency for latrunculin A to enhance the peak of Erk phosphorylation, although the differences observed in Figure 4B and 4C , are not statistically significant. However, we found that there was a marked difference in the effect of latrunculin A on the peak of Erk phosphorylation among basophil preparations. The peak of Erk phosphorylation was consistently increased by latrunculin A when Erk phosphorylation and mediator release of stimulated controls were weak. However, when Erk phosphorylation and mediator release of stimulated controls were strong, latrunculin A led to little or no increase of Erk phosphorylation at 5 min. There were 21 experiments of Erk phosphorylation ± latrunculin A with a variety of different IgE-dependent stimuli. Using this mixed dataset, a correlation between the magnitude of the peak Erk phosphorylation response to the stimulus versus the magnitude of the increase with latrunculin A yielded an r = –0.53, P= 0.011. (These experiments were not designed to accommodate this kind of post-hoc analysis, as there was no internal control for the variations in Western blotting between experiments. However, the cell number was constant, and the film exposure time used for the analysis was kept constant, so the analysis, although crude, should be reasonably valid. To further validate the analysis, a correlation between the maximum histamine release for a given experiment versus the magnitude of Erk phosphorylation enhancement with latrunculin A was examined and found to be –0.544. Both of these parameters need no internal control, and like peak Erk phosphorylation, maximum histamine release would be a measure of the responsiveness of the cell to stimulation.)

Akt is located in the signaling pathway downstream of PI-3K, and its phosphorylation was used as an index of PIP3 levels. Akt phosphorylation was transient in the presence or absence of latrunculin A in antigen (Fig. 4D) and anti-IgE-stimulated basophils (data not shown). The 60/5 ratios were 0.02 ± 0.02 and 0.07 ± 0.02 for control and latrunculin A-treated basophils, respectively (n=2). In this case, the peak of Akt phosphorylation was greater in the presence of latrunculin A (P<0.001).

Calcium signal
In some of the Erk phosphorylation experiments, latrunculin A (50 and 500 nM) did not induce any increase of Erk phosphorylation at 5 or 60 min in basophils that showed a strong response; however, LTC4 release was more than fivefold higher in the presence of latrunculin A in those very same cells. An increase in the [Ca2+]i response in the presence of latrunculin A might explain the increase in LTC4 release, as cytosolic PLA2 (cPLA2) phosphorylation (as a result of the activity of Erk) acts synergistically with the [Ca2+]i rise to generate the free arachidonic acid used to synthesize LTC4 [30 ]. The effect of latrunculin A on the [Ca2+]i elevation that followed stimulation through Fc{varepsilon}RI was examined. Anti-IgE-activated basophils showed higher elevations in cytosolic calcium in the presence of latrunculin A than control (Fig. 5 ). Latrunculin A alone did not cause any change in basal [Ca2+]i. Two concentrations of latrunculin A were tested, and although only the results with 50 nM latruculin A are shown, 500 nM latrunculin A also induced an increase of the IgE-mediated calcium signal.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. Anti-IgE-induced [Ca2+]i rise in the presence and absence of latrunculin A. Basophils were preincubated for 5 min in the presence ({circ}) or absence (•) of 50 nM latrunculin A. Anti-IgE (0.2 µg/ml) was added at time 0. Latrunculin A did not induce any change in basal [Ca2+]i (n=2).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current studies extend our previous work describing the presence of two regions of down-regulation in the IgE-mediated signaling that leads to LTC4 secretion [18 ]: an early region proximal to syk and a late region distal to syk and PI-3K. In particular, this study indicates that modifying a naturally transient early signal (which in itself might explain a transient later signal) so that it became sustained does not make the later signal sustained. In these studies, antigen is an IgE-dependent stimulus that induces a naturally transient early signal. When the antigen-induced early signaling is forced to be sustained (with latrunculin A treatment), the presence of a down-regulatory mechanism responsible for the transience of late signaling is revealed. In this respect, stimulation with antigen becomes similar to stimulation with polyclonal anti-IgE antibody. No matter what mode of stimulation is used to activate the cell, a sustained, early signal cannot overcome the inhibitory process that makes later signaling transient. Intuitively, we theorized that this might result from the dependence of the downstream activation steps and downstream down-regulatory process on the activation of one of the earlier activating steps.

To explore this reasoning further, we examined a simple, heuristic mathematical model of the signaling cascade. From a systems control perspective, we demonstrate that the behavior described above is consistent with activation and deactivation of the later signaling steps being dependent on the activation of an earlier step. Figure 6 shows one possible heuristic scheme, in which the signaling cascade has been simplified to elements that might be considered essential for the measurements we have made. The scheme roughly represents the signaling cascade that proceeds from Fc{varepsilon}RI receptor aggregation to cPLA2 phosphorylation (Fig. 6A) . Note that in this scheme, activation of P2 and N2 (the later, down-regulatory step) requires activation of an earlier activating step, represented by P1 (Fig. 6B) . [In this model, N2 is activated by P1; the activation of N2 by S was explored, but the kinetic curves of P2 and P3 activation do not resemble the experimental data (the curves are transient but decay to a stable baseline that is significantly above resting levels). However, we have no direct knowledge about which upstream elements activate the second down-regulatory region; thus, the assignment was somewhat arbitrary.] The reactions in this signaling model were assigned kinetics with enzymatic characteristics. There are no experimental data about the amount of substrate, enzyme, and equilibrium constants of these signaling reactions in basophils, but as the model was intended to be largely heuristic, the kinetic constants were assigned values that led to kinetics consistent with the experimental data observed in the absence of latrunculin A, i.e., transient responses for syk, Akt, and Erk. In other words, the dashed lines in Figure 6C and 6D , which intentionally emulate the kinetic behavior of early and late signaling of antigen-stimulated basophils, were fixed to be the starting behavior. Then the model was used to simulate what would happen in case of inhibition of the first down-regulatory step (N1A). If N1A is disallowed or reduced to low levels of activity (i.e., eliminating the first down-regulatory step), the kinetics of activation of P1 change from transient to sustained (Fig. 6C , solid line); however, P3 activation remains transient as a result of the action of the second down-regulatory step N2 (Fig. 6D , solid line). Such a model supports the experimental conclusion that the functional outcome of these two sequential, down-regulatory mechanisms in the cascade of IgE-mediated signaling is always a transient signal with or without sustained, early activation.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. One model of early IgE-mediated signaling. (A) Schematic of IgE-mediated signaling to LTC4 release in basophils. Squares mark regions of down-regulation. MEK, Mitogen-activated protein kinase kinase. (B) A heuristic model: The model has an initial stimulus (S), two positive or activating steps (P1 and P2), and an outcome (P3); S would represent the aggregation reaction (kinetically described by a logarithmic approach to a maximum based on previous studies; ref. [20 ]), P1 the activation of syk, P2 an intermediate step such as the formation of PIP3, and P3 the activation of Erk. Two negative or inhibitory steps (N1 and N2) were included, also induced by the stimulus, which inhibit P1 and P2, respectively, and representing the early and late regions of down-regulation. A third, constitutively active, negative step (N3) down-regulates P3 (some negative regulator of the last step is needed if P3 is to revert to its resting state). The connectors between the various elements do not represent direct activation, just the ordered flow of the signal, so each connector might correspond to several signaling steps in the actual pathway. Each element in the model was considered to have two states, active (A) and inactive (I), and the reactions inducing a state transition were represented by a Michaelis-Menten equation {e.g., dP1A/dt=(V1·S)[P1]/(KMN1+[P1]), where P1A = the active state of P1, P1 = the inactive state of P1, S = stimulus strength, V1 = a rate constant, and KMN1 = a Michaelis-Menten Km-like constant}. (C) Kinetics of P1 activation in the presence (dashed line) and absence (solid line) of an active N1. (D) Kinetics of P3 activation in the presence (dashed line) and absence (solid line) of an active N1.

 
The later, down-regulatory region was first described in anti-IgE-stimulated basophils, which show a sustained activation of early signaling (syk, shc, Grb2-SOS, PI-3K) but a transient activation of later steps (ras-Erk, Akt) [4 ]. As syk phosphorylation in antigen-stimulated basophils is transient, there is no direct evidence that the late, down-regulatory region is also active after antigen stimulation. Indirect evidence that this later down-regulation occurs during antigen stimulation is derived from studies that show that antigen stimulation alters the Akt/Erk response following further stimulation with a noncross-reacting antigen [18 ]. The current results confirm that the late, down-regulatory region is also activated by antigen; when syk phosphorylation was sustained in latrunculin A, antigen-stimulated basophils, Erk phosphorylation remained transient. Akt activation kinetics followed the same pattern as Erk activation. Although Erk and Akt phosphorylation were increased in the presence of latrunculin A, an effect observed at 5 and 60 min, the transience of the response was not significantly affected. The increase of Erk and Akt phosphorylation by latrunculin A could be a direct effect of the actin cytoskeleton, but as Erk and Akt are located downstream in the signaling cascade, it is reasonable that the increase of the early signal affects the magnitude of the late signal to some extent, a prediction of the model in Figure 6 as well (Fig. 6D) .

Previous studies in RBL cells have shown that inhibition of the actin cytoskeleton alters the very earliest signals, but these same studies have not examined later signaling, such as Akt and Erk phosphorylation. Our current studies extend these observations to include downstream elements and suggest a reason why they do not become sustained. If later signaling is to be made sustained, then inhibition of the later, down-regulatory process is needed. Our previous studies point to a role for a phosphatase such as SHIP 1/2 (we have preliminary evidence that PTEN activity is not altered) in this later down-regulation by reducing PIP3 levels. Numerous studies in other cell types also suggest a role for SHIP in down-regulating this region of the signaling cascade. Although there are no inhibitors of SHIP and no strict knockouts of SHIP in humans available for study, one possible cell source to examine is the histamine-releasing factor (HRF) responder [10 ]. Basophils that respond to HRF with secretion express relatively low levels of SHIP and can be examined for the characteristics of Akt and Erk phosphorylation. Such studies are currently underway by MacDonald and her colleagues [10 ].

As these studies suggest a similar role for the cytoskeleton in modulating IgE-mediated signaling in human basophils and RBL cells, some of the mechanistic studies of this process in RBL cells may apply to basophils. The fact that inhibition of actin polymerization changes syk phosphorylation kinetics from transient to sustained shows that there is an actin cytoskeleton-dependent down-regulation of the activating signal at some point between receptor aggregation and syk activation. Inhibition of actin polymerization was achieved with latrunculin A, which was chosen, as it was reported to have a lesser effect on the response to nonimmunologic stimulus in RBLs than cytochalasin D (another actin polymerization inhibitor) [12 ]. Latrunculin A sequesters G-actin that is released from the F-actin filaments and prevents it from repolymerization into new filaments [31 ]. As expected from its mechanism of action, latrunculin A reduced the resting levels of F-actin as well as the IgE-mediated increase of F-actin. In RBL cells, current evidence suggests that the actin cytoskeleton is regulating early signaling by controlling the structure of lipid rafts or the components that reside in the lipid rafts necessary for coupling Fc{varepsilon}RI aggregation with early signaling kinases such as lyn or fyn [13 ].

The inhibition of actin polymerization with latrunculin A also increased the calcium signal and histamine and LTC4 release. Although the modulation of the calcium signal by latrunculin A could be a result of the effect of the drug on the early signaling, with the information currently available, a variety of explanations is possible. For example, the mediator release results also demonstrate that the regulation of the basophil response by the actin cytoskeleton is not specific for the early IgE-mediated signaling, as disruption of actin filaments also enhanced the response to fMLP and other stimuli [32 33 34 ]. As syk kinase is not activated by fMLP, the implication of these results is that the syk proximal, down-regulatory process is not the only down-regulatory mechanism in the pathways to mediator release controlled by the actin cytoskeleton.

In summary, there are two identified regions of down-regulation during IgE-mediated signaling in basophils. An early down-regulatory region, located between receptor aggregation and syk and responsible for the transience of early signaling, is dependent on the actin cytoskeleton. Another down-regulatory region is located between PI-3K and ras and is responsible for the transience of the ras–Erk pathway. These studies asked whether inhibition of the early, down-regulatory process could significantly overcome down-regulation by the later process, making downstream events less transient. Although there were some increases in the peaks of Akt and Erk activation (as markers of the downstream signals), these signaling steps retained their transient behavior. One interpretation is that the early signals are not only responsible for activation of later signals but also responsible for activating the later, down-regulatory process. The resulting competition between enhanced, late-activating and deactivating signaling cascades results in only modest perturbations in later signaling. Therefore, only events that are independent of later signaling pathways benefit in a significant way by alterations in early down-regulation. This provides a means for the cells to selectively control the various activation cascades, presumably conferring selective alterations in function. Although this study reveals a way in which human basophils down-regulate IgE-mediated signaling, a similar strategy of down-regulation is probably present in other signaling systems and other cell types.


   ACKNOWLEDGEMENTS
 
The National Intitutes of Health Grant AI20253 supported this work. We thank Valerie Alexander for her technical assistance with some of these studies.

Received September 18, 2003; revised January 14, 2004; accepted January 15, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lavens-Phillips, S., MacGlashan, D. W., Jr (1998) The role of p53/56 lyn and p72syk in IgE-mediated signal transduction in human basophils FASEB J. 12,591a(abstract)
  2. Kepley, C. L., Wilson, B. S., Oliver, J. M. (1998) Identification of the Fc {varepsilon}RI-activated tyrosine kinases Lyn, Syk, and Zap-70 in human basophils J. Allergy Clin. Immunol. 102,304-315[CrossRef][Medline]
  3. MacGlashan, D. W., Jr, Miura, K., Lavens-Phillips, S. (2000) Pharmacology of IgE-mediated desensitization of human basophils: effects of protein kinase C and Src-family kinase inhibitors Biochem. Pharmacol. 60,1717-1727[CrossRef][Medline]
  4. Miura, K., Lavens-Phillips, S., MacGlashan, D. W., Jr (2001) Localizing a control region in the pathway to LTC4 secretion following stimulation of human basophils with anti-IgE antibody J. Immunol. 167,7027-7037[Abstract/Free Full Text]
  5. MacGlashan, D. W., Jr, Laven-Phillips, S., Miura, K. (1998) IgE-mediated desensitization in human basophils and mast cells Front. Biosci. 3,D746-D756
  6. Isersky, C., Rivera, J., Segal, D. M., Triche, T. (1983) The fate of IgE bound to rat basophilic leukemia cells. II. Endocytosis of IgE oligomers and effect on receptor turnover J. Immunol. 131,388-396[Abstract]
  7. Furuichi, K., Rivera, J., Isersky, C. (1984) The fate of IgE bound to rat basophilic leukemia cells. III. Relationship between antigen-induced endocytosis and serotonin release J. Immunol. 133,1513-1520[Abstract]
  8. MacGlashan, D. W., Jr, Mogowski, M., Lichtenstein, L. M. (1983) Studies of antigen binding on human basophils. II. Continued expression of antigen-specific IgE during antigen-induced desensitization J. Immunol. 130,2337-2342[Abstract]
  9. Huber, M., Helgason, C. D., Damen, J. E., Liu, L., Humphries, R. K., Krystal, G. (1998) The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation Proc. Natl. Acad. Sci. USA 95,11330-11335[Abstract/Free Full Text]
  10. Vonakis, B. M., Gibbons, S., Jr, Sora, R., Langdon, J. M., MacDonald, S. M. (2001) Src homology 2 domain-containing inositol 5' phosphatase is negatively associated with histamine release to human recombinant histamine-releasing factor in human basophils J. Allergy Clin. Immunol. 108,822-831[CrossRef][Medline]
  11. Pierini, L., Harris, N. T., Holowka, D., Baird, B. (1997) Evidence supporting a role for microfilaments in regulating the coupling between poorly dissociable IgE-Fc {varepsilon}RI aggregates downstream signaling pathways Biochemistry 36,7447-7456[CrossRef][Medline]
  12. Frigeri, L., Apgar, J. R. (1999) The role of actin microfilaments in the down-regulation of the degranulation response in RBL-2H3 mast cells J. Immunol. 162,2243-2250[Abstract/Free Full Text]
  13. Holowka, D., Sheets, E. D., Baird, B. (2000) Interactions between Fc({varepsilon})RI and lipid raft components are regulated by the actin cytoskeleton J. Cell Sci. 113,1009-1019[Abstract]
  14. Gillespie, E., Lichtenstein, L. M. (1972) Histamine from human leukocytes: studies with deuterium oxide, colchicine and cytochalasin B J. Clin. Invest. 51,2941-2947
  15. MacGlashan, D. W., Jr, Bochner, B., Warner, J. A. (1994) Graded changes in the response of individual human basophils to stimulation: distributional behavior of early activation events J, Leukoc, Biol 55,13-23[Abstract]
  16. Pfeiffer, J. R., Seagrave, J. C., Davis, B. H., Deanin, G. G., Oliver, J. M. (1985) Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells J. Cell Biol. 101,2145-2155[Abstract/Free Full Text]
  17. Marone, G., Kagey, S. A., Lichtenstein, L. M. (1981) IgE-mediated histamine release from human basophils: differences between antigen E- and anti-IgE-induced secretion Int. Arch. Allergy Appl. Immunol. 65,339-348[Medline]
  18. MacGlashan, D. W., Jr (2003) Two regions of down-regulation in the IgE-mediated signaling pathway in human basophils J. Immunol. 170,4914-4925[Abstract/Free Full Text]
  19. MacGlashan, D. W., Jr, Peters, S. P., Warner, J., Lichtenstein, L. M. (1986) Characteristics of human basophil sulfidopeptide leukotriene release: releasability defined as the ability of the basophil to respond to dimeric cross-links J. Immunol. 136,2231-2239[Abstract]
  20. MacGlashan, D. W., Jr, Lichtenstein, L. M. (1983) Studies of antigen binding on human basophils. I. Antigen binding and functional consequences J. Immunol. 130,2330-2336[Abstract]
  21. MacGlashan, D. W., Jr, Dembo, M., Goldstein, B. (1985) Test of a theory relating to the cross-linking of IgE antibody on the surface of human basophils J. Immunol. 135,4129-4134[Abstract]
  22. MacGlashan, D. W., Jr, White, J. M., Huang, S. K., Ono, S. J., Schroeder, J., Lichtenstein, L. M. (1994) Secretion of interleukin-4 from human basophils: the relationship between IL-4 mRNA and protein in resting and stimulated basophils J. Immunol. 152,3006-3016[Abstract]
  23. Gilbert, H. S., Ornstein, L. (1975) Basophil counting with a new staining method using alcian blue Blood 46,279-286[Abstract/Free Full Text]
  24. Hayes, E. C., Lombardo, D. L., Girard, Y. (1983) Measuring leukotrienes of slow-reacting substance of analphylaxis: development of a specific radioimmunoassay J. Immunol. 131,429-440[Abstract]
  25. Siraganian, R. P. (1974) An automated continuous-flow system for the extraction and fluorometric analysis of histamine Anal. Biochem. 57,383-394[CrossRef][Medline]
  26. MacGlashan, D. W., Jr, Hubbard, W. C. (1993) Interleukin-3 alters free arachidonic acid generation in C5a-stimulated human basophils J. Immunol. 151,6358-6369[Abstract]
  27. Miura, K., Hubbard, W. C., MacGlashan, D. W., Jr (1998) Phosphorylation of cytosolic PLA2 (cPLA2) by interleukin-3 (IL-3) is associated with increased free arachidonic acid and LTC4 release in human basophils J. Allergy Clin. Immunol. 102,512-520[CrossRef][Medline]
  28. MacGlashan, D. W., Jr (1989) Single-cell analysis of Ca++ changes in human lung mast cells: graded vs. all-or-nothing elevations after IgE-mediated stimulation J. Cell Biol. 109,123-134[Abstract/Free Full Text]
  29. Law, C., Chandran, K. A., Sidorenko, S. P., Clark, E. A. (1996) Phospholipase C-{gamma}1 interacts with conserved phosphotyrosyl residues in the linker region of syk ad is a substrate for syk Mol. Cell. Biol. 16,1305-1315[Abstract]
  30. Miura, K., MacGlashan, D. W., Jr (2000) Dual phase priming by interleukin-3 for leukotriene C4 generation in human basophils J. Immunol. 164,3026-3034[Abstract/Free Full Text]
  31. Coue, M., Brenner, S. L., Spector, I., Korn, E. D. (1987) Inhibition of actin polymerization by latrunculin A FEBS Lett. 213,316-318[CrossRef][Medline]
  32. Marone, G., Columbo, M., Soppelsa, L., Condorelli, M. (1985) Histamine release from human basophils by pepstatin A Agents Actions 16,306-309[CrossRef][Medline]
  33. Marone, G., Poto, S., Petracca, R., Triggiani, M., de Lutio di Castelguidone, E., Condorelli, M. (1982) Activation of human basophils by staphylococcal protein A. I. The role of cyclic AMP, arachidonic acid metabolites, microtubules and microfilaments Clin. Exp. Immunol. 50,661-668[Medline]
  34. Hook, W. A., Siraganian, R. P. (1977) Complement-induced histamine release from human basophils. III. Effect of pharmacologic agents J. Immunol. 118,679-684[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
D. MacGlashan Jr. and N. Vilarino
Nonspecific Desensitization, Functional Memory, and the Characteristics of SHIP Phosphorylation following IgE-Mediated Stimulation of Human Basophils
J. Immunol., July 15, 2006; 177(2): 1040 - 1051.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0903431v1
75/5/928    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vilariño, N.
Right arrow Articles by MacGlashan, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vilariño, N.
Right arrow Articles by MacGlashan, D. W., Jr

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS