The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kawamoto, K.
Right arrow Articles by Matsuda, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawamoto, K.
Right arrow Articles by Matsuda, H.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CALCIUM COMPOUNDS
*CALCIUM, ELEMENTAL
*FORMALDEHYDE
*PARAFORMALDEHYDE
The Journal of Immunology, 2002, 168: 6412-6419.
Copyright © 2002 by The American Association of Immunologists

Nerve Growth Factor Activates Mast Cells Through the Collaborative Interaction with Lysophosphatidylserine Expressed on the Membrane Surface of Activated Platelets1

Keiko Kawamoto*, Junken Aoki{dagger}, Akane Tanaka*, Atsuko Itakura*, Hiroyuki Hosono{dagger}, Hiroyuki Arai{dagger}, Yasuo Kiso{ddagger} and Hiroshi Matsuda2,*

* Faculty of Agriculture, Department of Veterinary Clinic, Tokyo University of Agriculture and Technology, and {dagger} Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and {ddagger} Faculty of Agriculture, Department of Veterinary Anatomy, Yamaguchi University, Yamaguchi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Effect of nerve growth factor (NGF) on platelet-associated mast cell activation was investigated. Although neither NGF alone nor platelets alone induced significant 5-hydroxytriptamine (5-HT) release from rat peritoneal mast cells, marked 5-HT release was detected when costimulated with NGF and calcium ionophore-activated platelets. This response reached maximal levels as early as 5 min after the initiation of the coincubation and was completely blocked by anti-NGF Ab or by an inhibitor for a tyrosine kinase of the trkA NGF receptor. Paraformaldehyde-fixed platelets activated with either calcium ionophore or thrombin exhibited the collaborative ability, suggesting the possible involvement of some membrane molecules expressed on activated platelets in mast cell activation. Because activation of platelets induced expression of phosphatidylserine (PS) and/or lysoPS on membrane surface, and since lysoPS, unlike PS, initiated the NGF-induced 5-HT release, lysoPS expressed on activated platelets may be involved in the mast cell activation. Moreover, intradermal injection of NGF and activated platelets into the rat skin increased local vascular permeability. These findings suggested that NGF collaboratively worked with membrane lysoPS of activated platelets to induce mast cell activation. Thus, NGF released in response to inflammatory stimuli may contribute to mast cell activation in collaboration with locally activated platelets in the process of inflammations and tissue repair.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nerve growth factor (NGF)3 is a well-characterized neurotrophic factor that is commonly believed to play a crucial role in the development and maintenance of the central and peripheral nervous systems including sympathetic and sensory neurons (1, 2, 3). Two classes of NGF receptors have been identified by their relative affinities; the low-affinity NGF receptor is a 75-kDa glycoprotein, and the high-affinity receptor is a 140-kDa molecule containing a tyrosine kinase domain that is encoded by the trkA proto-oncogene (TrkA) (4, 5). In addition to neurotrophic activity, increasing evidences give rise to possible multifunctional properties of NGF on immunocompetent cells including lymphocytes, monocytes/macrophages, and mast cells through functional NGF receptors (4, 5, 6, 7, 8, 9, 10, 11). In prior studies, we have demonstrated that NGF promotes not only granulocyte differentiation from human PBMC and murine bone marrow cells (12, 13), but also differentiation of connective tissue-type mast cells from murine bone marrow cells and bone marrow-derived culture mast cells (14). NGF is capable of supporting survival of neutrophils (15), eosinophils (16), and mast cells (9) by preventing apoptosis, and enhancing functional properties of neutrophils, eosinophils, macrophages, and mast cells: phagocytosis, superoxide production, matrix metalloproteinase-9 production, and chemotaxis (11, 12, 16, 17, 18, 19, 20). These experimental findings strongly support a possibility that NGF acts as a cytokine that is capable of modulating inflammatory responses and tissue repair. In fact, the topical application of NGF to cutaneous wounds accelerates the rate of wound healing in normal and diabetic mice (21).

Mast cells are often abundant along blood vessels, and generate and release a number of vasoactive mediators including histamine, serotonin (5-hydroxytriptamine; 5-HT), and leukotrienes by cross-linking of Fc{epsilon}RI-IgE with its specific Ag (22). In addition to the immunological stimulation, intradermal or s.c. administration of NGF to rats causes immediate vasodilatory responses characterized by the degranulation of local mast cells (23, 24). In contrast with the in vivo reports, NGF alone is insufficient to induce chemical mediator release from rat peritoneal mast cells (PMC) and the addition of exogenous phosphatidylserine (PS) or lysoPS, a deacylated PS derivative, to NGF is necessary for the mast cell activation (8, 25, 26). However, the mechanisms by which NGF and serinephospholipids induce mast cell activation and their pathophysiological roles have been poorly understood.

PS is a membrane phospholipid component normally distributed at the internal side of the plasma membrane. Activation of platelets by thrombin or calcium ionophore results in loss of membrane asymmetry and expression of PS and/or lysoPS on their cell surface, which is provided to catalyze hemostatic plug formation and blood coagulation (27, 28). In the tissue repair process, circulating platelets rapidly adhere to the subendothelial connective tissue exposed by vascular injury. Following this event, attached platelets are activated by a perivascular matrix (27, 28). Activated platelets most likely provide the phospholipid molecules at the affected sites. Therefore, we speculated that NGF may interact with platelets accumulating at the site of injured blood vessels, where mast cells locate abundantly, resulting in mast cell activation in vivo. To examine this hypothesis, we investigated whether activation of mast cells was modulated by NGF and platelets in vitro, by measuring 5-HT release from rat PMC. We report herein a novel collaborative interaction between NGF and surface lysoPS on activated platelets, thereby leading to mediator release from mast cells in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
NGF and other reagents

2.5S NGF purified from murine submaxillary glands was a gift from A. M. Stanisz and J. Bienenstock (McMaster University, Hamilton, Canada). Neurotrophic activity of the NGF preparations was determined as described previously (12). Cytochrome c and busulfan were purchased from Sigma-Aldrich (St. Louis, MO). PS, lysoPS, lysophosphatidylic acid (lysoPA), lysophosphatydylcholine (lysoPC), lysophosphatidylethanolamine, and lysophosphatidylinositol were purchased from Avanti Polar Lipids (Alabaster, AL). We also used K252a (Calbiochem, La Jolla, CA), PD98059 (New England Biolabs, Beverly, MA), and LY294002 (Calbiochem). PS-specific phospholipase A1 (PS-PLA1) was purified as described previously (29). Unless otherwise indicated, all chemicals and Abs were purchased from Sigma-Aldrich.

Isolation of rat PMC

Outbread male Wistar rats (8–16 wk of age) (Clea Japan, Tokyo, Japan) were kept in our laboratory provided with food and water ad libitum >1 wk before they were sacrificed. PMC were purified by Percoll solution (Pharmacia Biotech, Uppsala, Sweden) as described previously (18). Sedimented PMC (>96% purity and 98% viability) were washed twice and resuspended at a concentration of 105 cells/ml in DMEM supplemented with 10% FCS.

Preparation of platelets

Whole-blood anticoagulated with 0.38% of trisodium citrate was collected from the exposed external jugular vein of normal rats under ether anesthesia. Platelet-rich plasma was prepared by centrifugation of blood samples at 120 x g for 13 min. To activate platelets, platelet-rich plasma was incubated with 1 µM calcium ionophore A23187 at 37°C for 30 min without agitation, and washed twice with PBS containing 0.1% BSA and 300 ng/ml PGI2 (Ono Pharmaceutical, Tokyo, Japan). Washed platelets were resuspended at a concentration of 2 x 108 cells/ml in prewarmed DMEM supplemented with 10% FCS. In some experiments, we used platelets fixed with 1% paraformaldehyde in PBS at 4°C for 20 min following the stimulation with 1 µM calcium ionophore A23187 or 0.2 U/ml thrombin. After the fixation, platelets were washed three times with PBS. Platelets showed no aggregation in the process of the treatment indicated above.

[3H]5-HT release from PMC

5-HT release was determined as described previously (9). Briefly, purified PMC (105 cells/ml) resuspended in DMEM supplemented with 10% FCS were incubated at 37°C for 1 h with 1 µCi/ml of [3H]5-HT (specific activity: 26.3 Ci/mM; New England Nuclear, Boston, MA). The cells were washed five times with ice-cold PBS containing 0.1% BSA to remove unincorporated [3H]5-HT. A total of 105 radiolabeled PMC were incubated in 1 ml of DMEM with or without various concentrations of NGF, various numbers of platelets, and/or various aminophospholipids at 37°C for 30 min, unless especially indicated. After centrifugation for 5 min at 800 x g, aliquots (200 µl) of the supernatants were dissolved in 3 ml of a scintillation mixture (Ready Protein; Beckman Coulter, Fullerton, CA) and counted for {beta} emissions in a scintillation counter (LS500; Beckman Coulter). Total incorporated cpm of PMC were obtained from cell pellets lysed with 1 ml of 1% Triton X-100 for 30 min on ice. The percentage of [3H]5-HT release was calculated as: supernatant cpm/total cpm x 100. The percentage of release of unstimulated PMC was <10%.

Treatment of RBC with calcium ionophore

Venous blood obtained from healthy volunteers was diluted with an equal volume of PBS, and centrifuged in Percoll solution as described above. Sedimented RBC were resuspended at a 30% packed-cell volume in buffer containing 70 mM NaCl, 80 mM KCl, and 10 mM HEPES, and 1 mM CaCl2 (pH 7.4), and then stimulated with 10 µM A23187 at 37°C for 2–3 h (30). After stimulation, PS expressed on the surface of RBC was assessed with FITC-conjugated annexin V (BD PharMingen, San Diego, CA) according to the manufacturer’s instruction. By this procedure, >65% cells expressed PS on their surface. To prepare surface lysoPS "positive" RBC, PS-expressing RBC were treated at 37°C for 10 min with PS-PLA1, which is capable of allowing to accumulate lysoPS on the RBC surface (29). The RBC preparations were washed and fixed with 1% paraformaldehyde for 10 min.

Intracellular calcium mobilization

Freshly isolated PMC were incubated with 2 µM fura 2 acetoxymethyl ester (Dojin, Tokyo, Japan) in Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 0.1% BSA, and 10 mM HEPES (pH 7.4)) at 37°C for 1 h, and then resuspended in 500 µl (106 cells) of the same buffer in a stirring cuvette. Following stimulation with 50 ng/ml NGF in the presence of 5 µM lysoPS or 108 activated platelets, cytosolic calcium of PMC was measured by monitoring fluorescence intensity at an emission wavelength of 510 nm, and excitation wavelengths of 340 and 380 nm using a CAF-110 (JACS, Tokyo, Japan) with a 8100 V3.0 software program.

Pretreatment of PMC with signal transduction inhibitors

Before a 5-HT release assay, PMC were preincubated for 1 h with the following inhibitors: 50 ng/ml K252a, a TrkA inhibitor (31); 100 µM PD98059, a mitogen-activated protein kinase (MAPK) kinase inhibitor (32); or 50 µM LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor (33). The PMC were pretreated with inhibitors or a diluent solution, resuspended in DMEM, and allowed to demonstrate activating effect of NGF and platelets.

Western blot analysis of phosphorylated proteins

Autophosphorylation of TrkA, MAPK, PI3K, and phospholypase C-{gamma} (PLC{gamma}) was examined by using a modification of the method described previously (18). Briefly, PMC were suspended in serum-free medium at a density of 2 x 106 cells/ml and followed by stimulation with NGF (50 ng/ml), lysoPS (5 µM), and/or activated platelets (108 cells/ml) at 37°C for 5 or 30 min. Cells were lysed with lysis buffer (1.0% Nonidet P-40, 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, and 1 µg/ml leupeptin) for 5 min, and then frozen and thawed three times. Lysates were centrifuged at 15,000 rpm for 20 min, and supernatants were incubated with anti-PI3K Ab (Upstate Biotechnology, Lake Placid, NY) or anti-PLC{gamma} 1 (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated with protein-A beads at 4°C overnight with gentle rotation. The boiled immunocomplex samples were subjected to 10% SDS-PAGE and electrically transferred to a membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was incubated at 4°C overnight with the anti-phosphotyrosine mAb and followed to reincubate with peroxidase-conjugated second Ab. For detection of phosphorylated TrkA and MAPK, equal amounts of whole-cell lysates were resolved by SDS-PAGE. The phosphorylation of TrkA or MAPK in each sample was assessed using rabbit polyclonal Ab (Cell Signaling Technology, Beverly, MA) that recognize the two different phosphorylated forms of each protein. The immunoreactive bands were visualized with an enhanced chemiluminescent detection reagent (Amersham, Arlington Heights, IL).

Plasma extravasation assay

Rats anesthetized with pentobarbital sodium were intradermally injected with 50 µl of 100 ng/ml NGF with or without 5 x 106 activated or resting platelets fixed with paraformaldehyde into the shaved dorsal skin, followed by i.v. injection of 1.0% Evans blue dye. The same concentration of cytochrome c was served as a same m.w. control. Injection sites were marked on the skin for orientation. Thirty minutes later, the dorsal skin was removed, and OD of dye infiltration were digitalized by using Gel Print 200i/VGA and Basic Quantifier (Genomic Solutions, Ann Arbor, MI).

In vivo pretreatment with busulfan

To induce thrombocytopenia, rats were pretreated with busulfan according to the method reported previously (34). Briefly, rats were injected i.p. with busulfan (20 mg/kg body weight) twice 10 and 13 days before the plasma extravasation assay. The number of platelets in blood collected from the retro-orbital plexus were counted.

Real-time PCR quantification of rat cytokines

A quantitative RT-PCR was used to determine mRNA levels of cytokines in rat basophilic leukemia cells (RBL-2H3 cells; Health Science Research Resources Bank, Osaka, Japan). Total RNA was isolated from 5 x 105 cells stimulated with 50 ng/ml NGF, 5 x 108 activated platelets, and/or 5 µM lysoPS using a TRIzol reagent (Life Technologies, Rockville, MD), according to the manufacturer’s instruction. A total of 1 µg of total RNA was reverse-transcribed into cDNA by using Moloney murine leukemia virus reverse transcriptase (Superscript II; Life Technologies) and an oligo(dT) primer. Specific primers for amplification were based on published sequences for IL-3, IL-4, IL-10, TNF-{alpha}, IFN-{gamma}, GM-CSF, and {beta}-actin (35, 36). A quantitative PCR was performed using a SYBR Green PCR core reagent kit (PE Applied Biosystems, Tokyo, Japan) following the thermal cycling programs: stage 1, 50°C for 3 min; stage 2, 95°C for 10 min; stage 3, 40 cycles of 95°C for 15 s followed by 60°C for 30 s. Fluorescence intensity was measured in real-time during extension steps for a SYBR Green assay by using a Nippon Bio-Rad thermal cycler (iCycler iQ detection system; Nippon Bio-Rad Laboratories, Tokyo, Japan). The no-template control was not amplified in the 40-cycle PCR.

Statistical analysis

Two-tailed Student’s t test was done for statistical analysis of the data, and p < 0.05 was taken as the level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
5-HT release from PMC induced by NGF and activated platelets

We examined 5-HT release from PMC (105) cultured with a suboptimal dose of NGF (50 ng/ml) and platelets (109) for 30 min at 37°C. As shown in Fig. 1GoA, neither NGF nor platelets alone could induce mediator release from mast cells. However, striking augmentation of 5-HT release (63.8%) was observed when PMC were cultured with 109 A23187-activated platelets and 50 ng/ml NGF simultaneously. These data indicated that NGF and platelets had synergistic effect on in vitro mast cell activation without requiring exogenous serinephospholipids. Whereas coincubation of PMC with unstimulated resting platelets showed little enhancement of NGF-induced 5-HT release, the effect of activated platelets was a nearly 3-fold increase than that of resting platelets. Calcium ionophore is well-known to act as a strong secretagogue for mast cells. To eliminate the possibility that the small amount of A23187 was contaminated in the platelet suspension, we examined the releaseability of the second washing from the activated platelets. The supernatants had no effect on 5-HT release from PMC in the presence or absence of 50 ng/ml NGF (3.5 and 4.3%, respectively). Hence, we used activated platelets for following experiments.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 1. 5-HT release from PMC stimulated with NGF and activated platelets. Released [3H]5-HT was measured by a scintillation counter as described in Materials and Methods. Each value represents the mean ± SE of four to six separate experiments. A, A total of 105 PMC were incubated with 109 resting platelets or with 109 platelets activated with 1 µM A23187 in the presence or absence of 50 ng/ml NGF at 37°C for 30 min. *, p < 0.001; when compared with PMC alone. B, A total of 105 PMC were stimulated with various concentrations of NGF in the presence of 109 activated platelets for 30 min. *, p < 0.01; when compared with PMC plus activated platelets. C, A total of 105 PMC were incubated with increasing numbers of activated platelets in the presence of 50 ng/ml NGF containing control Ab or anti-NGF Ab (1/1000 dilution). *, p < 0.001; when compared with PMC plus NGF. {dagger}, p < 0.001; when compared with control Ab. D, A total of 105 PMC were stimulated simultaneously with 50 ng/ml NGF and 109 activated platelets for 0, 5, 15, 30, and 60 min. *, p < 0.001; when compared with PMC alone.

 
When various concentrations of NGF (0.5, 5, 50, and 500 ng/ml) were added to 105 PMC and 109 A23187-activated platelets, significant release of 5-HT was observed in a dose-dependent manner; this effect was observed at concentrations as low as 0.5 ng/ml (Fig. 1GoB). We next examined the influence of numbers of platelets on NGF-induced mast cell activation. 5-HT release was also dependent on the number of activated platelets together with a fixed dose (50 ng/ml) of NGF; the minimal effect of the 5-HT release was detected when coincubated with 107 activated platelets (Fig. 1GoC). Neutralizing Abs against NGF were provided to verify the specificity of the NGF effect on PMC in the presence of activated platelets. Addition of anti-NGF Ab completely abrogated the 5-HT release induced by NGF and activated platelets (Fig. 1GoC). Control Ab showed no positive effect on 5-HT release. As shown in Fig. 1GoD, this 5-HT release dependent on both NGF and activated platelets reached roughly maximal levels as early as 5 min after the initiation of the incubation with 50 ng/ml NGF and 109 platelets.

Effect of fixation of activated platelets on NGF-induced 5-HT release

Activated platelets secrete chemical mediators which are capable of stimulating mast cells. Therefore, we conducted experiments with platelets fixed with paraformaldehyde to clarify whether some soluble factors released from platelets might be involved in mast cell activation in our in vitro system. Platelets were fixed with 1% paraformaldehyde after stimulation with calcium ionophore A23187 or thrombin. The fixed activated platelets to 50 ng/ml NGF led to significant 5-HT release and marked degranulation of mast cells (Fig. 2Go, A and B). Thus, we concluded that the fixation of activated platelets did not substantially alter their ability to modulate the NGF-dependent mast cell activation.



View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 2. NGF-induced PMC activation mediated by fixed-activated platelets. A, After being pretreated with 1 µM A23187 or 0.2 U/ml thrombin, platelets were fixed with 1% paraformaldehyde for 20 min on ice. PMC (105) were incubated with or without 109 fixed or unfixed activated platelets in the presence of 50 ng/ml NGF at 37°C for 30 min. Each value represents the mean ± SE of four separate experiments. *, p < 0.001; when compared with PMC alone. B, Giemsa-stained cytospin preparations of PMC stimulated with NGF and fixed-activated platelets. PMC (105) were incubated with or without 109 fixed A23187-activated platelets in the presence of 50 ng/ml NGF. Marked degranulation is observed in PMC incubated with NGF and fixed activated-platelets (lower right panel); no or little morphological change is observed in PMC with medium alone (upper left panel), NGF alone (upper right panel), or fixed-activated platelets (lower left panel).

 
Effect of aminophospholipids on NGF-induced 5-HT release

Previous studies show that NGF-mediated mediator release from mast cells requires the presence of exogenous serinephospholipids such as PS or lysoPS (8, 25, 26). PS is a membrane component that is normally distributed in an inner leaflet of a phospholipid bilayer. Activation of platelets by calcium ionophore leads to not only surface expression of membrane PS, but also accumulation of lysoPS by the subsequent degradation (28, 37, 38). Therefore, we examined the effect of either PS, lysoPA, lysoPC, lysolysophosphatidylethanolamine, lysophosphatidylinositol, or lysoPS on NGF-induced mast cell activation. As shown in Fig. 3Go, lysoPS induced significant 5-HT release from mast cells in the presence of NGF, but the other aminophospholipids did not.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 3. Effect of various aminophospholipids on NGF-induced 5-HT release from PMC. PMC (105) were incubated with various aminophospholipids (1 µM each) in the presence of 50 ng/ml NGF at 37°C for 30 min. Each value represents the mean ± SE of four separate experiments. *, p < 0.001; when compared with NGF alone.

 
To further clarify the possible involvement of serinephospholipids expressed on membrane surface in the mast cell activation, we examined whether PS- or lysoPS-expressing RBC mediated NGF-induced 5-HT release from PMC. Treatment of RBC with calcium ionophore has been reported to cause loss of phospholipid asymmetry resulting in PS expression on their surface (30). Furthermore, Sato et al. (29) have recently identified a new enzyme of the lipase family secreted from rat-activated platelets, termed PS-PLA1, that specifically act on PS and produce lysoPS. By using this enzyme, we prepared two RBC samples that expressed PS or lysoPS on their surface. When RBC were treated with A23187, >65% of them were positive for binding of annexin V, which was slightly higher than activated platelets (Fig. 4Go). However, the addition of 108 PS-expressing RBC to PMC showed no significant release of 5-HT despite the presence of 50 ng/ml NGF (Table IGo). In contrast, the treatment of PS-expressing RBC with PS-PLA1 led to significant NGF-induced 5-HT release from PMC (Table IGo). Thus, we concluded that NGF was capable of inducing 5-HT release from PMC in the presence of lysoPS but not PS.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4. Detection of serinephospholipids on the surface of platelets and RBC stimulated with calcium ionophore A23187. Resting platelets and RBC were stimulated with (filled histograms) or without (open histograms) calcium ionophore A23187, and then stained with FITC-conjugated annexin V. Data expressed are representative of three separate experiments.

 

View this table:
[in this window]
[in a new window]
  		Table I. Effect of serinephospholipids-expressing RBC on NGF-induced 5-HT release from PMC

 
Effect of NGF and platelets on calcium mobilization of PMC

Because NGF-dependent histamine release of mast cells is inhibited by EDTA (25), we measured cytosolic calcium mobilization of mast cells loaded with fura 2 acetoxymethyl ester in response to addition of NGF and activated platelets. A very slight increase of intracellular calcium levels was observed in single addition of 50 ng/ml NGF, 5 µM lysoPS, or 108 activated platelets, respectively (Fig. 5Go). In contrast, simultaneous addition of NGF and A23187-activated platelets led to a marked increase of intracellular calcium levels, which reached the maximum levels within 200 msec; and the combination of NGF and lysoPS also significantly increased calcium influx (Fig. 5Go).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 5. Intracellular calcium mobilization of PMC stimulated with NGF in the presence of lysoPS or activated platelets. After preincubation with 2 µM fura-2 acetoxymethyl ester, 106 PMC were treated with 50 ng/ml NGF in the presence or absence of 5 µM lysoPS or 108 A23187-activated platelets. Arrows indicate the time point when reagents were applied

 
Signal transduction pathways triggered by NGF and activated platelets

The biological effects of NGF on target cells are mediated by specific cell surface receptors with different affinities: p75 and TrkA (4, 5). We recently demonstrated that rat PMC expressed the latter alone, the NGF high-affinity receptor with a tyrosine kinase domain, whose activation prevented apoptosis and initiated chemotactic movement of mast cells through both MAPK and PI3K signal pathways following tyrosine-phosphorylation of TrkA (9, 18). Therefore, we attempted to determine whether 5-HT release from PMC induced by NGF and A23187-activated platelets was influenced by blockage of TrkA, MAPK kinase, and PI3K by using specific inhibitors K-252a, PD98059, and LY294002, respectively. When PMC were pretreated with 50 ng/ml K-252a for 1 h, the NGF-induced 5-HT release was completely inhibited (Fig. 6Go). Pretreatment with 100 µM PD98059 and 10 µM LY294002 reduced the NGF-induced 5-HT release by 69 and 62%, respectively. The combination of both inhibitors markedly suppressed 5-HT release from PMC that was comparable to the inhibitory effect induced by the pretreatment with K-252a (Fig. 6Go). To obtain more insight into the mechanism of NGF-induced mast cell activation in the presence of activated platelets, we next examined the possible involvement of lysoPS or A23187-activated platelets in TrkA, MAPK, PI3K, and PLC{gamma} signaling pathways. NGF treatment induced tyrosine phosphorylation of TrkA, MAPK, and PI3K, but not that of PLC{gamma} (Fig. 7Go). In contrast, no phosphorylation of the tyrosine residues was detected for individual signal molecules after treatment with either lysoPS or activated platelets. Simultaneous addition of lysoPS or activated platelets with NGF did not change the phosphorylation levels of all the signal molecules (Fig. 7Go).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 6. Suppressive effects of signal transduction inhibitors on NGF-induced 5-HT release from PMC. PMC were preincubated with either 50 ng/ml K252a, 100 µM PD98059, 10 µM LY294002, or assay medium for 1 h before costimulation with 50 ng/ml NGF and 109 A23187-activated platelets for 30 min. Each value represents the mean ± SE of four separate experiments. *, p < 0.001; when compared with NGF alone.

 


View larger version (73K):
[in this window]
[in a new window]
  		FIGURE 7. Western blot analysis for tyrosine phosphorylation of signaling molecules stimulated by NGF. PMC were stimulated with NGF (50 ng/ml), lysoPS (5 µM), and/or A23187-activated platelets (108 cells/ml) at 37°C for 5 or 30 min. Lane 1, Medium alone; lane 2, NGF; lane 3, lysoPS; lane 4, NGF + lysoPS; lane 5, activated platelets; lane 6, NGF + activated platelets.

 
Increased vascular permeability by intradermal injection of NGF and activated platelets

We next attempted to determine a dye extravasation after intradermal injection of NGF to assess a cooperative effect of NGF and activated platelets on mast cell activation in vivo. As shown in Fig. 8GoA, markedly increased extravasation was observed at the injection site of the skin when a mixture of 5 ng NGF and 5 x 106 platelets activated with A23187 or thrombin was administered, whereas injection of NGF alone induced a mild vasodilative response. In controls with PBS vehicle, 5 ng cytochrome c, or activated platelets alone, no or slight response was noted. To evaluate a role of circulating platelets in NGF-induced mast cell activation in vivo, busulfan was injected into rats before intradermal injection with NGF and activated platelets. The injection was induced marked reduction in the number of circulating platelets (<2.0%) 13 days later. Pretreatment with busulfan suppressed the NGF-induced vasodilative response by about half level as compared with that in control rats (Fig. 8GoB).



View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 8. A dye extravasation assay in the skin of rats. Control rats ({square}) and busulfan-pretreated rats ({blacksquare}) were injected intradermally with 50 µl of NGF (5 ng) and/or 5 x 106 fixed-activated platelets, following i.v. injection of 1.0% Evan blue dye. PBS and cytochrome c were served as negative controls. Thirty minutes later, the skin was removed from control rats (A) and busulfan-pretreated rats, and the OD of leaked dye at injected sites was quantified (B). Data expressed are representative of three independent experiments. Lane 1, PBS; lane 2, cytochrome c; lane 3, NGF; lane 4, A23187-activated platelets; lane 5, thrombin-activated platelets; lane 6, cytochrome c + A23187-activated platelets; lane 7, NGF + A23187-activated platelets; lane 8, cytochrome c + thrombin-activated platelets; lane 9, NGF + thrombin-activated platelets.

 
mRNA levels of cytokines

Because NGF increases mRNA levels of several cytokines in mast cells (35), we next examined an effect of activated platelets or lysoPS on NGF-mediated cytokine production by real-time PCR quantification. We used rat basophilic leukemia cell line RBL-2H3 instead of rat PMC. As detected in rat PMC, simultaneous stimulation by NGF and activated platelets caused significant degranulation response in RBL-2H3 cells (data not shown). Treatment with NGF alone induced a marked increase in mRNA expression of IL-3, IL-4, TNF-{alpha}, IFN-{gamma}, and GM-CSF, whereas a slight, but not significant, increase in mRNA expression of IL-10 was detected (Fig. 9Go). However, neither lysoPS nor activated platelets substantially modulated the NGF effect on cytokine gene expression.



View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 9. mRNA expression levels of cytokines in RBL-2H3 cells. Cells were stimulated with NGF (50 ng/ml), A23187-activated platelets (5 x 108 cells), and/or lysoPS (5 µM). Total RNA was isolated, and then reverse-transcribed by using Superscript II and oligo(dT) primer. Quantitative IL-3, IL-4, IL-10, TNF-{alpha}, IFN-{gamma}, and GM-CSF levels were determined by a real-time PCR using an intercalation dye SYBR Green I, and standardized according to respective {beta}-actin mRNA levels. Each value represents the mean ± SE of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mast cells bear specific receptors for IgE and the reaction of cell-bound IgE molecules with multivalent Ag induces release of a variety of chemical mediators and cytokines from mast cells, which greatly contribute to acute and chronic allergic inflammations. In addition to this immunological stimulation, cytokines and growth factors directly modulate mast cell functions (22). Although intradermal injection of NGF has been reported to induce plasma extravasation, how NGF caused the activation of mast cells at the injection site has been conclusively unclear (23, 24). In this study, we clearly demonstrated that NGF led to mediator release from mast cells through the collaborative interaction with activated platelets in vitro. This effect was not substantially influenced by a fixation of activated platelets with paraformaldehyde, suggesting that certain membrane molecules, such as PS or lysoPS (8, 25, 26) expressed on the platelet surface after stimulation with calcium ionophore or thrombin may be involved in NGF-mediated mast cell activation. Because NGF-induced 5-HT release was detected in the presence of lysoPS or lysoPS-expressing RBC, but not PS or PS-expressing RBC, lysoPS rather than PS may act as an alternative potential molecule for NGF-induced mast cell activation. The effect of lysoPS on in vivo histamine release from rat PMC is 1000-fold greater than that of PS (39) and a kind of stimulation leads to PS-PLA1 release from platelets which specifically acts on PS to produce lysoPS, but not on any other lipids (29). These findings support our experimental results that degradation of membrane PS by PS-PLA1 produced lysoPS in the process of their activation and the membrane lysoPS was strongly involved in the NGF-induced mediator release from mast cells. Although some previous reports suggested that the potentiating effect of lysoPS on mediator release of mast cells was required conversion to PS after incorporation into mast cell membrane (40, 41), we ruled out this possibility because the fixation of activated platelets with paraformaldehyde had no influence on 5-HT release from PMC.

Recently, lysophospholipids such as lysoPS, lysoPA, and lysoPC have been reported to induce a transient increase of cytosolic calcium levels in a human T cell line (42), suggesting the presence of the same receptor for the lysophospholipids on the target cell surface. In PMC, lysoPS, unlike the other lysophospholipids, triggered NGF-induced 5-HT release, whereas it led little calcium influx, suggesting that mast cells might express receptors which are capable of recognizing lysoPS. However, because isotope-labeled lysoPS failed to bind to CD36 expressed on rat macrophages (data not shown), a signal of lysoPS might be mediated through some receptors different from CD36.

Binding of NGF to TrkA on PMC rapidly induces autophosphorylation of tyrosine residue of the receptor, which leads to activation of downstream signal cascades including MAPK and PI3K, thereby resulting in chemotactic movement (9, 18). Simultaneous addition of MAPK kinase and PI3K inhibitors completely blocked NGF-induced 5-HT release from PMC even in the presence of activated platelets, suggesting that 5-HT release may be mediated through both the signal transduction cascades. However, because activated platelets did not influence tyrosine phosphorylation of the individual signal molecules even in the presence of NGF, and because NGF alone was not capable of releasing 5-HT despite leading to phosphorylation of both MAPK and PI3K molecules, the signaling mechanisms by which lysoPS-expressing platelets exert its effect have been unclear.

NGF stimulation in the presence of lysoPS not only induces degranulation of mast cells but also increases the production of several cytokines including TNF-{alpha} (35). Therefore, we examined the effect of activated platelets and lysoPS on NGF-inducible inflammatory cytokine mRNA expression by quantitative real-time PCR. The mRNA levels of IL-3, IL-4, IL-10, TNF-{alpha}, IFN-{gamma}, and GM-CSF were increased in response to NGF, but those were not modulated by addition of lysoPS or activated platelets. Thus, NGF and activated platelets may act collaboratory on 5-HT release, but not on cytokine gene expression. However, as NGF does not significantly increase the production of TNF-{alpha} protein even in the presence of lysoPS (43), the NGF-mediated effect on cytokine production might be limited to the gene expression level.

We confirmed the collaborative action of NGF and activated platelets on mediator release from residential tissue mast cells in rat skin by in vivo extravasation assay. This result implies that the novel activation pathway to mast cells presented here may occur in the pathophysiological condition. NGF alone showed slight effect of vascular permeability. It may be caused by the interaction between circulating platelets probably activated in the injection site and injected NGF because rats with thrombocytopenia manifested significant reduction in NGF-induced vascular permeability. NGF is rapidly released from salivary glands into blood stream in response to fighting stress in rats and mice and serum levels of NGF were increased up to 300 ng/ml (44). In humans, NGF is detected in peripheral blood after parachute diving stress (45). If circulating naive platelets could induce mast cell activation together with NGF, an increase in serum levels of NGF in such conditions would cause fatal systemic shock by massive mast cell degranulation. We consider that the collaborative interaction between NGF and activated platelets demonstrated in this study may occur in the presence of endothelial cell denudation, particularly in the microvasculature at local damaged and inflamed tissues. We found that skin wound led to rapid increase in NGF levels in peripheral blood and affected sites of mice, and that local application of NGF accelerated the wound healing process (21). Correspondingly, increased levels of NGF in local inflammatory tissues or peripheral blood have been found in patients with systemic sclerosis, multiple sclerosis, chronic arthritis, and vernal keratoconjunctivitis (46, 47, 48, 49, 50). Platelets circulating in blood immediately accumulate at the site of injury or hemorrhage leading to their morphological change and biochemical activation. In contrast, mast cells are residential cells adjacent to the endothelium in the normal connective and mucosal tissues. Therefore, NGF locally and/or systemically produced in response to inflammatory stimuli may act with activated platelets recruited to injured sites to modulate the functions of mast cells in vivo. Our results strongly support this hypothesis, and suggest a possible role for NGF in many pathophysiological conditions that lead to mast cell activation. Activation of mast cells induces the release of histamine, leukotrienes, and produces inflammatory cytokines, resulting in the recruitment and activation of circulating leukocytes to the area of allergic and nonallergic inflammation. Mast cells also contribute to innate immunity to bacterial infection (51, 52, 53). The striking cooperative effect of NGF with platelets on mast cell activation may contribute to development of acute and chronic inflammations and wound healing process at damaged tissues.


   Acknowledgments
 
We thank Drs. J. Bienenstock and A. M. Stanisz (McMaster University, Hamilton, Canada) for supplying ultrapurified NGF and for reviewing the manuscript.


   Footnotes
 
1 This work was supported by grants from Pioneering Research Project in Biotechnology and Recombinant Cytokine’s Project (2002-4120) provided by the Ministry of Agriculture, Forestry and Fisheries, Japan. Back

2 Address correspondence and reprint requests to Dr. Hiroshi Matsuda, Laboratory of Clinical Immunology, Faculty of Agriculture, Department of Veterinary Clinic, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. E-mail address: hiro{at}cc.tuat.ac.jp Back

3 Abbreviations used in this paper: NGF, nerve growth factor; TrkA, high affinity receptor for NGF; 5-HT, 5-hydroxytriptamine; PMC, peritoneal mast cell; PS, phosphatidylserine; PA, phosphatidylic acid; PC, phosphatydylcholine; PS-PLA1, PS-specific phospholipase A1; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PLC{gamma}, phospholipase C-{gamma}; RBL-2H3 cell, rat basophilic leukemia cell. Back

Received for publication October 5, 2001. Accepted for publication April 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Levi-Montalcini, R., P. U. Angeletti. 1963. Essential role of the nerve growth factor on the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro. Dev. Biol. 7:653.[Medline]
  2. Yankner, B. A., E. M. Shooter. 1982. The biology and mechanism of action of nerve growth factor. Annu. Rev. Biochem. 51:845.[Medline]
  3. Korsching, S., G. Auburger, R. Heumann, J. Scott, H. Thoenen. 1985. Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation. EMBO J. 4:1389.[Medline]
  4. Sutter, A., R. J. Riopelle, R. M. Harris-Warrick, E. M. Shooter. 1979. Nerve growth factor receptors: characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells. J. Biol. Chem. 254:5972.[Abstract/Free Full Text]
  5. Klein, R., S. Jing, V. Nanduri, E. O’Rouke, M. Barbacid. 1991. The trk proto-oncogene encodes a receptor for nerve growth factor. Cell 65:189.[Medline]
  6. Otten, U., P. Ehrhard, R. Peck. 1993. Nerve growth factor induces growth and differentiation of human B lymphocyte. Proc. Natl. Acad. Sci. USA 86:10059.
  7. Ehrhard, P. B., U. Ganter, B. Joachim, U. Otten. 1993. Expression of functional trk proto-oncogene in human monocytes. Proc. Natl. Acad. Sci. USA 90:5423.[Abstract/Free Full Text]
  8. Horigome, K., J. P. Pryor, E. D. Bullock, Jr E. M. Johnson. 1993. Mediator release from mast cells by nerve growth factor: neurotrophin specificity and receptor mediation. J. Biol. Chem. 268:14881.[Abstract/Free Full Text]
  9. Kawamoto, K., T. Okada, Y. Kannan, H. Ushio, M. Matsumoto, H. Matsuda. 1995. Nerve growth factor prevents apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor. Blood 86:4638.[Abstract/Free Full Text]
  10. Torcia, M., L. Bracci-Laudiero, M. Lucibello, L. Nencioni, D. Labardi, A. Rubartelli, F. Cozzolino, L. Aloe, E. Garaci. 1996. Nerve growth factor is an autocrine survival factor for memory B lymphocytes. Cell 85:345.[Medline]
  11. Susaki, Y., S. Shimizu, K. Katakura, N. Watanabe, K. Kawamoto, M. Matsumoto, M. Tsudzuki, T. Furusaka, Y. Kitamura, H. Matsuda. 1996. Functional properties of murine macrophages promoted by nerve growth factor. Blood 88:4630.[Abstract/Free Full Text]
  12. Matsuda, H., M. D. Coughlin, J. Bienenstock, J. A. Denburg. 1988. Nerve growth factor promotes human hemopoietic colony growth and differentiation. Proc. Natl. Acad. Sci. USA 85:6508.[Abstract/Free Full Text]
  13. Kannan, Y., H. Matsuda, H. Ushio, K. Kawamoto, Y. Shimada. 1993. Murine granulocyte-macrophage and mast cell colony formation promoted by nerve growth factor. Int. Arch. Allergy. Immunol. 102:362.[Medline]
  14. Matsuda, H., Y. Kannan, H. Ushio, Y. Kiso, T. Kanemoto, H. Suzuki, Y. Kitamura. 1991. Nerve growth factor induces development of connective tissue-type mast cells in vitro from murine bone marrow cells. J. Exp. Med. 174:7.[Abstract/Free Full Text]
  15. Kannan, Y., K. Usami, M. Okada, S. Shimizu, H. Matsuda. 1992. Nerve growth factor suppresses apoptosis of murine neutrophils. Biochem. Biophys. Res. Commun. 186:1050.[Medline]
  16. Hamada, A., N. Watanabe, H. Ohtomo, H. Matsuda. 1996. Nerve growth factor enhances survival and cytotoxic activity of human eosinophils. Br. J. Haematol. 93:299.[Medline]
  17. Kannan, Y., H. Ushio, H. Koyama, M. Okada, M. Oikawa, T. Yoshihara, M. Kaneko, H. Matsuda. 1991. 2.5S nerve growth factor enhances survival, phagocytosis, and superoxide production of murine neutrophils. Blood 77:1320.[Abstract/Free Full Text]
  18. Sawada, J., A. Itakura, A. Tanaka, T. Furusaka, H. Matsuda. 2000. Nerve growth factor functions as a chemoattractant for mast cells through both mitogen-activated kinase and phosphatidylinositol 3-kinase signaling pathways. Blood 95:2052.[Abstract/Free Full Text]
  19. Tanaka, A., K. Arai, Y. Kitamura, H. Matsuda. 1999. Matrix metalloproteinase-9 production, a newly identified function of mast cell progenitors, is downregulated by c-kit receptor activation. Blood 94:2390.[Abstract/Free Full Text]
  20. Kanbe, N., A. Tanaka, M. Kanbe, A. Itakura, M. Kurosawa, H. Matsuda. 1999. Human mast cells produce matrix metalloproteinase 9. Eur. J. Immunol. 29:2645.[Medline]
  21. Matsuda, H., H. Koyama, H. Sato, J. Sawada, A. Itakura, A. Tanaka, M. Matsumoto, K. Konno, H. Ushio, K. Matsuda. 1998. Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice. J. Exp. Med. 187:287.
  22. Befus, A. D.. 1994. Inflammation: mast cells. L. Pearly, and M. D. Metecky, eds. Handbook of Mucosal Immunology 307. Academic Press, London.
  23. Otten, U., J. B. Baumann, J. Girard. 1984. Nerve growth factor induces plasma extravasation in the rat skin. Eur. J. Pharmacol. 106:199.[Medline]
  24. Marshall, J. S., R. H. Stead, C. McSharry, L. Nielsen, J. Bienenstock. 1990. The role of mast cell degranulation products in mast cell hyperplasia. I. mechanism of action of nerve growth factor. J. Immunol. 144:1886.[Abstract]
  25. Pearce, F. R., H. L. Thompson. 1986. Some characteristics of histamine secretion from rat peritoneal mast cells stimulated with nerve growth factor. J. Physiol. 372:379.[Abstract/Free Full Text]
  26. Tomioka, M., R. H. Stead, L. Nielsen, M. D. Coughlin, J. Bienenstock. 1988. Nerve growth factor enhances antigen and other secretagogue-induced histamine releases from rat peritoneal mast cells in the absence of phosphatidylserine. J. Allergy Clin. Immunol. 82:599.[Medline]
  27. McGregor, J. L.. 1995. The role of human platelet membrane receptors in inflammation. M. Joseph, ed. Immunopharmacology of Platelets 67. Academic Press, San Diego.
  28. Gently, P. A.. 1992. The mammalian blood platelet: its role in haemostasis, inflammation and tissue repair. J. Comp. Pathol. 107:243.[Medline]
  29. Sato, T., J. Aoki, Y. Nagai, N. Dohmae, K. Takio, T. Doi, H. Arai, K. Inoue. 1997. Serine phospholipid-specific phospholipase A that is secreted from activated platelets: a new member of the lipase family. J. Biol. Chem. 272:2192.[Abstract/Free Full Text]
  30. Shiffer, K. A., L. Road, R. K. Emerson, F. S. Kuypers. 1998. Effects of phosphatidylinositol diphosphate on phospholipid asymmetry in the human erythrocyte membrane. Biochemistry 37:3449.[Medline]
  31. Berg, M. M., D. W. Sternberg, L. F. Parada, M. V. Chao. 1992. K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity. J. Biol. Chem. 267:13.[Abstract/Free Full Text]
  32. Dudley, D. T., L. Pang, S. J. Decker, A. J. Bridges, A. R. Saltiel. 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA 92:7686.[Abstract/Free Full Text]
  33. Vlahos, C. J., W. F. Matter, K. Y. Hui, R. F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:5241.[Abstract/Free Full Text]
  34. Sirois, M. G., M. Simons, D. J. Kuter, R. D. Rosenberg, E. R. Edelman. 1997. Rat arterial wall retains myointimal hyperplastic potential long after arterial injury. Circulation 96:1291.[Abstract/Free Full Text]
  35. Bullock, E. D., Jr E. M. Johnson. 1996. Nerve growth factor induces the expression of certain cytokine genes and bcl-2 in mast cells: potential role in survival promotion. J. Biol. Chem. 271:27500.[Abstract/Free Full Text]
  36. Yin, J. L., N. A. Shackel, A. Zekry, P. H. McGuinness, C. Richards, K. van der Putten, G. W. McCaughan, J. M. Eris, G. A. Bishop. 2001. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I. Immunol. Cell Biol. 79:213.[Medline]
  37. Thiagarajan, P., J. F. Tait. 1991. Collagen-induced exposure of anionic phospholipid in platelets and platelet-derived microparticles. J. Biol. Chem. 266:24302.[Abstract/Free Full Text]
  38. Thiagarajan, P., J. F. Tait. 1990. Binding of annexin V placental anticoagulant protein I to platelets: evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J. Biol. Chem. 265:17420.[Abstract/Free Full Text]
  39. Tamori-Natori, Y., K. Horigome, K. Inoue, S. Nojima. 1986. Metabolism of lysophosphatidylserine, a potentiator of histamine release in rat mast cells. J. Biochem. 103:581.
  40. Hirata, F., J. Axelrod, F. T. Crews. 1979. Concanavalin A stimulated phospholipid methylation and phosphatidylserine decarboxylation in rat mast cell. Proc. Natl. Acad. Sci. USA 76:4813.[Abstract/Free Full Text]
  41. Hirata, F., J. Axelrod. 1980. Phospholipid methylation and biological signal transmission. Science 209:1082.[Abstract/Free Full Text]
  42. Xu, Y., G. Casey, G. B. Mills. 1995. Effect of lysophospholipids on signaling in the human Jurkat T cell line. J. Cell. Physiol. 63:441.
  43. Bissonnette, E. Y., R. Mathison, L. Carter, J. S. Davison, A. D. Befus. 1993. Decentralization of the superior cervical ganglia inhibits mast cell mediated TNF {alpha}-dependent cytotoxicity. 1. Potential role of salivary glands. Brain Behav. Immun. 7:293.[Medline]
  44. Aloe, L., E. Alleva, A. Bohn, U. Otten. 1986. Aggressive behavior induces release of nerve growth factor from mouse salivary gland into the bloodstream. Proc. Natl. Acd. Sci. USA 83:6184.[Abstract/Free Full Text]
  45. Aloe, L., L. Bracci-Laudiero, E. Alleva, A. Lambiase, A. Micera, P. Tirassa. 1994. Emotional stress induced by parachute jumping enhanced blood nerve growth factor levels and the distribution of nerve growth factor receptors in lymphocytes. Proc. Natl. Acad. Sci. USA 91:10440.[Abstract/Free Full Text]
  46. Tuver, M. A., G. Passiu, A. Mathie, L. Aloe. 1993. Nerve growth factor and mast cell distribution in the skin of patients with systemic sclerosis. Clin. Exp. Rheumatol. 11:319.[Medline]
  47. Massaro, A. R., P. Tonali. 1998. Cerebrospinal fluid markers in multiple sclerosis: an overview. Mult. Scler. 4:1.[Abstract/Free Full Text]
  48. Aloe, L., M. A. Tuveri, L. U. Carcassi, R. Levi-Montalcini. 1992. Nerve growth factor in the synovial fluid of patients with chronic arthritis. Arthritis Rheum. 35:351.[Medline]
  49. Lambiase, A., S. Bonini, A. Micera, L. Magrini, L. Bracci-Laudiero, L. Aloe. 1995. Increased plasma levels of nerve growth factor in vernal keratoconjunctivitis and relationship to conjunctival mast cells. Invest. Ophthalmol. Vis. Sci. 36:2127.[Abstract/Free Full Text]
  50. Bonini, S., A. Lambiase, S. Bonini, F. Angelucci, L. Magrini, L. Manni, L. Aloe. 1996. Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma. Proc. Natl. Acad. Sci. USA 93:10955.[Abstract/Free Full Text]
  51. Matsuda, H., N. Watanabe, Y. Kiso, S. Hirota, H. Ushio, Y. Kannan, M. Azuma, H. Koyama, Y. Kitamura. 1990. Necessity of IgE antibodies and mast cells for manifestation of resistance against larval Haemaphysalis longicornis ticks in mice. J. Immunol. 144:259.[Abstract]
  52. Echtenacher, B., D. N. Mannel, L. Hultner. 1996. Critical protective role of mast cells in a model of acute septic peritonitis. Nature 381:75.[Medline]
  53. Malaviya, R., T. Ikeda, E. Ross, S. N. Abraham. 1996. Mast cell modulation of neutrophil influx and bacterial clearance at site of infection through TNF-{alpha}. Nature 381:77.[Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. C. Frasch, K. Z. Berry, R. Fernandez-Boyanapalli, H.-S. Jin, C. Leslie, P. M. Henson, R. C. Murphy, and D. L. Bratton
NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A
J. Biol. Chem., November 28, 2008; 283(48): 33736 - 33749.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. C. Frasch, K. Zemski-Berry, R. C. Murphy, N. Borregaard, P. M. Henson, and D. L. Bratton
Lysophospholipids of Different Classes Mobilize Neutrophil Secretory Vesicles and Induce Redundant Signaling through G2A
J. Immunol., May 15, 2007; 178(10): 6540 - 6548.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
K. S. Park, H.-Y. Lee, M.-K. Kim, E. H. Shin, S. H. Jo, S. D. Kim, D.-S. Im, and Y.-S. Bae
Lysophosphatidylserine Stimulates L2071 Mouse Fibroblast Chemotactic Migration via a Process Involving Pertussis Toxin-Sensitive Trimeric G-Proteins
Mol. Pharmacol., March 1, 2006; 69(3): 1066 - 1073.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
E. Yokoo, Y. Yatomi, T. Takafuta, M. Osada, Y. Okamoto, and Y. Ozaki
Sphingosine 1-Phosphate Inhibits Migration of RBL-2H3 Cells via S1P2: Cross-Talk between Platelets and Mast Cells
J. Biochem., June 1, 2004; 135(6): 673 - 681.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Ahamed, R. T. Venkatesha, E. B. Thangam, and H. Ali
C3a Enhances Nerve Growth Factor-Induced NFAT Activation and Chemokine Production in a Human Mast Cell Line, HMC-1
J. Immunol., June 1, 2004; 172(11): 6961 - 6968.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kawamoto, K.
Right arrow Articles by Matsuda, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawamoto, K.
Right arrow Articles by Matsuda, H.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CALCIUM COMPOUNDS
*CALCIUM, ELEMENTAL
*FORMALDEHYDE
*PARAFORMALDEHYDE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS