Basophil priming by neurotrophic factors. Activation through the trk receptor

Basophil Priming by Neurotrophic Factors

Activation Through the trk Receptor'

Beatrice Burgi,* Uwe H. Otten,t Brigitte Ochensberger,* Silvia Rihs,* Klaus Heese,t Patricia B. Ehrhard,t Carlos F. Ibanez,* and Clemens A. Dahinden2*

There is increasing evidence that nerve growth factor (NGF) acts on cells of the immune system, apart from i ts neurotrophic effects. In human basophils, NCF potentiates mediator release and primes the cells to produce leukotriene C, in response to C5a. It is, however, unknown whether other homologous neurotrophins also act outside the nervous system, and whether activation of basophils by NCF requires interaction with trk tyrosine kinase receptors, the low affinity NGF receptor (LNCFR), or both. A triple mutant NCF designed to interrupt binding to the LNCFR was found to activate basophils with equal efficacy as wild-type NCF, demonstrating that the LNCFR is not necessary. Despite a 10 times lower potency of mutant NCF, no LNCFR expression was detected by FACS analysis. Brain-derived neurotrophic factor, which interacts with trkB, was inactive at concentrations up to 1000 ng/ml (>30,000-fold lower potency than NCF), while neurotrophin-3, which is thought to interact with trkC, trkB, and more weakly with trk, induced a threshold effect at 300 ng/ml (-10,000-fold lower potency), demonstrating that 1) the LNCFR cannot deliver a direct signal; and 2) basophils do not express functional trkB and trkC receptors. In agreement with the functional data, basophils (in contrast to other granulocyte types) expressed mRNA for trk, but not trkB or trkC, and no or minimal mRNA for LNGFR. These data demonstrate that human blood basophils express functional trk receptors that do not require the participation of LNCFR, and that, among the neurotrophin family, NCF is unique in priming basophils. Thelournal of Immunology, 1996, 157: 5582-5588.

N eurotrophic factors regulate a variety of steps in the development of the nervous system, such as neuronal survival, differentiation, and transmitter synthesis. The first

recognized and best known neurotrophin (NT)3 is nerve growth factor (NGF), which is required for the development and survival of certain sensory and sympathetic neurons of the peripheral nervous system (reviewed in Refs. 1-4). More recently, several other NTs have been identified, termed brain-derived neurotrophic factor (BDNF) ( 3 , NT-3 (6, 7) and NT-4/5, which share extensive se- quence homologies and structural similarities (5-10). These neurotrophic factors all regulate the survival and differentiation of partly distinct partly overlapping neurons (reviewed in Refs. 11-1 3).

The first identified and cloned NGF binding protein is the low affinity nerve growth factor receptor (LNGFR) (14, 15), which belongs to an increasingly large family of membrane molecules (including TNFRI, TNFRII, TNFRh, Fas, CD40, CD27, and CD30). These receptors regulate, mainly by cell-cell interactions

*Institute of Immunology and Allergology, Inselspital, Bern; 'Department of Physiology, University of Basel, Vesalianum, Vesalgasse, Basel, Switzerland; and *Laboratory of Molecular Neurobiology, Department of Medical Chemistry and Biophysics, Karolinska Institute, Stockholm, Sweden

Received for publication November 14, 1995. Accepted for publication Septem- ber 20, 1996.

The costs of publication of this article were defrayed in part by the payment of

accordance with 18 U.S.C. Section 1734 solely to indicate this fact. page charges. This article must therefore be hereby marked advertisement in

31-41906.94 to C.A.D. and 31-39121.93 to U.H.O.). ' This work was supported by the Swiss National Science Foundation (Grants

' Address correspondence and reprint requests to Dr. Clemens A. Dahinden, Institute of Immunology and Allergology, Inselspital, CH-3010 Bern, Switzerland.

Abbreviations used in this paper: NT, neurotrophin; NCF, nerve growth factor;

tor receptor; LTC,, leukotriene C,; RT-PCR, reverse transcription-polymerase BDNF, brain-derived neurotrophic factor; LNCFR, low affinity nerve growth fac-

chain reaction; rh, recombinant human; wt, wi ld type; mu, mutant; hu, human.

Copyright 0 1996 by The American Association of lrnmunologlsts

through the corresponding ligands, various functions, predomi- nantly in immune cells, such as apoptosis, cell proliferation, and differentiation (15). The LNGFR binds all neurotrophic factors with similar affinity (16, 17). However, it is still unclear whether the LNGFR is involved in mediating the biologic effects of NGF in the nervous system, since more recently, three tyrosine kinase receptors, trk, trkB, and trkC, have been identified that bind with high affinity and partially overlapping selectivities, NGF, BDNF, and NT-3, respectively (18-24). These receptors transduce signals upon cross-linking by the NT dimers, but whether the LNGFR participates in this response is controversial and may depend on the cell type studied (reviewed in Refs. 11-13 and 18-28). Very recent studies even indicate that the LNGFR can by itself transduce signals independently of trk NT receptors (29-31).

There is increasing evidence that NGF also mediates important functions outside the nervous system (5,32-43). In humans, NGF appears to promote the differentiation of myeloid progenitor cells (38-40) and B lymphocytes (36, 37) and to regulate the function of monocytes (42, 43) and basophils (41). Particularly impressive is the effect of NGF on basophil mediator release (41). Pre-expo- sure of mature human basophils to NGF enhances mediator release to all known agonists studied to date and primes the cells for the formation of large amounts of leukotriene C, (LTC,) upon stimulation with IgE-independent agonists that by themselves do not promote lipid mediator formation (41, 44). The low concentration of NGF required to prime basophils indicates that high affinity binding sites exist in this cell type (41, 44). However, it is not known whether basophils express trk receptors, and whether other neurotrophic factors can also affect basophil function. Further- more, it is unclear whether the LNGFR is involved, or even necessary, for the bioactivity of NGF outside the nervous system.

In this study we compared the effect of NGF with those of the homologous NTs BDNF, NT-3, and NT-4/5. Using a mutant NGF, in which three amino acids critical for binding to the low affinity

0022-1 767/96/$02.00

The Journal of Immunology 5583

receptor were exchanged by alanine, we studied the role of the LNGFR in basophil activation. Finally, we demonstrate by RT- PCR the presence of trk mRNA in basophils purified to homogeneity and show that the basophil is the only polymorphonuclear granulocyte type of peripheral blood that expresses trk as a functional signal-transducing receptor for NGF.

Materials and Methods Cytokines and agonists

Human rIL-3 was a kind gift from Sandoz AG (Basel, Switzerland and Vienna, Austria). 29C6, a murine mAb reacting with the a-chain of the high affinity IgE receptor (FceRI) at a site that does not compete with IgE binding, was a generous gift from Drs. Hakimi and Chizzonite (Hoffmann- La Roche, Nutley, NJ) (45). C5a and CSade'A'E were purified from human serum. and were homogeneous according to SDS-PAGE, microzone electrophoresis at pH 8.6, reverse phase HPLC, and amino acid analysis (44). rhNGF (P-dimer), rhNT-3, and rhBDNF were kindly provided by Genen- tech (South San Francisco, CA) (46), and rhNT-4 was obtained from Pep- roTech. Inc. (Rocky Hill, NJ). Recombinant NGF triple-mutated P-dimer (K32A+K34A+E35A), which has lost binding activity for the LNGFR, was produced in baculovirus-infected cells and purified by ion exchange chromatography on an S-Sepharose column (Pharmacia, Uppsala, Sweden) (28 ) . FMLP was purchased from Novabiochem (Laufelfingen, Switzerland).

Cell preparations

Venous blood from healthy unselected volunteers, anticoagulated with 10 mM EDTA, was mixed with 0.25 vol of 6% dextran, and erythrocytes were allowed to sediment at room temperature. Leukocytes were pelleted by centrifugation (150 X g, room temperature, 20 min), resuspended in HA buffer (20 mM HEPES (Calbiochem-Behring Corp., La Jolla, CA), 125 mM NaCl, 5 mM KCI, O S mM glucose, and 0.025% BSA (Boehringer Mannheim, Mannheim, Germany) and further fractionated by discontinu- ous Percoll (Pharmacia) density gradients as previously described (47). The bands formed at the density interfaces of the gradients ( 1.070/1 .0795 g/ml) were harvested, washed three times (400 X g, I O min, doc), and resuspended in HACM buffer (HA buffer supplemented with I mM CaCI, and I mM MgCI,) for mediator release experiments at a cell density of approximately I O 5 basophils/ml. The purity of basophils ranged from I O to 3070, with contaminating cells consisting mainly of lymphocytes.

Basophil purification for PCR

The basophil fraction from the Percoll gradient was further depleted from contaminating cells by negative selection using a mixture of Ab-coated paramagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) coated by aCD4, aCD8, aCD19, aCD14, and aCD16 at a concentration adapted to the composition of contaminating cells, and the cell separation was performed using a MACS system (Miltenyi Biotec) according to the manufacturer's recommendations, resulting in a basophil purity of 70 to 95% (48). These highly enriched basophils were either used for RNA extraction or further purified to >99% by labeling the cells with FITC-conjugated aFceRI (29C6) at 4°C in a HEPES buffer supplemented with 1 mM EDTA. The FceRI a-chain-positive cells were selected by sorting with a FACStar Plus (Becton Dickinson, San Jose, CA). The sorted cells were collected into a tube placed on ice and immediately used for RNA extraction. The granulocyte fraction obtained at the bottom of the Percoll gradients ( I ,0795 g/ml) containing 12 to 15% eosinophil and 85 to 88% neutrophils with no basophils or mononuclear cells was also used for RNA extraction. Eosinophils of >99% purity were isolated by a combination of Percoll gradients with negative selection using aCD16-coated paramagnetic beads (49).

Mediator release from human basophils

The experiments were performed in a shaking water bath at 37°C. Cells were allowed to warm to 37°C for I O min before adding cytokines or buffer control. After an additional I O min, a basophil trigger or control was added, and the reaction was stopped by placing the tubes in ice water 20 min after stimulus addition, a time period previously found to be sufficient for a complete mediator release reaction. After pelleting the cells by centrifugation (400 X g. 10 min, 4"C), 500 pl of the supernatants was added to 500 yl HACM buffer and 1 ml of 0.8 M HCIO,, mixed, and again centrifuged (600 X g. 15 min, 4°C). This supernatant was either stored at -70°C or used directly for histamine measurement using an automated fluorometric method (50). The other 500 pI of the supernatants was stored at -70°C

before determination of sulfidoleukotrienes release by RIA as described previously (41 ).

Histamine release was expressed as a percentage of the total cellular histamine content determined by cell lysis, and LTC, generation was expressed as picograms of LTC, per 1000 basophils. The number of basophils in the basophil-enriched leukocyte preparation was determined by the total cellular histamine in the lysate, assuming a histamine content of 1 pg histaminehasophil. All experiments were performed in duplicate with a variability of < 10% of the mean. All experiments were repeated at least three times with identical results (e.g., dose-response studies), except that the amounts of mediator released differed among experiments due to the well-known donor variability of basophil mediator release. Therefore, SDS are not shown for the data combined from experiments from different donors. However. even minor differences and the threshold effects of NTs were statistically significant using Student's paired t test.

Biologic activity of NGF and the NGF mutant

Serial dilutions of wild-type (wt) and mutant (mu) NGF of the same preparation used for the basophil experiments were assayed for biologic activity on explanted chick embryonic day 9 sympathetic ganglia, as previously described (28, SI), by comparison to standards obtained with purified mouse NGF, for which 1 biological unit (BU) is equivalent to approximately 5 ng/ml.

Reverse transcription-PCR

Total cellular RNA was isolated by the method of acid guanidinium thiocyanate/chloroform extraction (52). Total RNA was first reverse transcribed into cDNA, which, in turn, was subjected to PCR amplification using primers specific for trk and LNGFR (53) as well as trkB and trkC (trkB. 5'-TCACCGAAATTTTCATCGCAAACCAG-3' (sense) and 5 ' - GAATGGAATGCACCAGTGGTGGTC-3' (antisense), located at nucleotides 316-342 and 1066-1090, respectively; trkC, 5'-GGCAGGAGCA GGGGGAGGCCAAGC-3' (sense) and 5'-GGCCATTGATGGTCTGGT TGGCTG-3' (antisense), located at nucleotides 539-563 and 1171-1 195, respectively) (54). PCR amplification of a constitutively expressed mRNA encoding the ribosomal protein SI2 served as the control for RNA input and amplification (53). Negative controls were reverse transcribed either without RNA or without RT. The amplification steps involved denaturation at 94°C for 1 rnin, annealing for 2 min at 55°C with specific primers or at 50°C with S12 primers, and extension at 72°C for 3 min. The PCR reactions (4 pl) were analyzed by electrophoresis in 1.3% agarose gels in the presence of 0.5 pg/ml ethidium bromide, followed by alkaline blotting of the fragments onto nylon membranes and subsequent hybridization with specific digoxigenin-labeled DNA probe (42, 43, 53). Detection was achieved with AMPPD (3-(2'-Spiroadamantan)-4-methoxy-4-(3'phospho- rylyloxy)-phenyl- 12-dioxethan) as chemiluminescent substrate for alkaline phosphatase conjugated to anti-digoxigenin Abs (Boehringer Mannheim) as recommended by the manufacturer.

FACS analysis

Basophils purified by Percoll gradients and negative selection (48) and PC12 cells were incubated at 4°C with 1 to 5 wg/106 cells of the mAb against the LNGFR, aCD18, and isotype-matched control mAbs for 30 min, washed, and stained with FITC-labeled goat anti-mouse IgG. Fluo- rescent staining was assessed using an EPICS flow cytometer (Coulter Electronics, Hialeah, FL). To demonstrate expression of the trk receptor protein, the cells were fixed with paraformaldehyde (4% in HBSS), permeablized with saponin (0.1% saponin, 10% FCS, and 10 mM HEPES in HBSS) for I O min at 4"C, and stained with rabbit polyclonal Abs raised against the intracellular C-terminal peptide of c-trk (amino acids 777-790) obtained from Calbiochem (La Jolla, CA) or as a control nonimmune rabbit IgG, followed by affinity-purified phycoerythrin-labeled goat anti-rabbit IgG F(ab'), (Sigma Chemical Co., St. Louis, MO). The cells were then stained with FITC-labeled mAb 29C6 against the high affinity IgE receptor, which is specifically and strongly expressed in basophils to gate the basophil population.

Results Mutant of huNGF primes human basophils

The anaphylatoxin CSa is a potent basophil agonist that promotes a strong degranulation response but is by itself incapable of inducing LTC, generation (44, 5.5). We have previously shown that preexposure of basophils to NGF for 10 min primes the cells to produce LTC, in response to this agonist (41,44). This qualitative

5584 BASOPHIL ACTIVATION THROUGH THE trk RECEPTOR

I

FIGURE 1. Dose response of wtNGF and triple- mutated NGF for priming human blood basophils. Basophils were pretreated for 10 min with buffer, wtNGF (open circles), or muNGF (filled circles), respectively, at the concentrations indicated on the abscissa and stimulated for 20 min with C5a (lo-' M). The upper panels show histamine release; the lower panels show LTC, generation. Two different preparations of mutant NGF (K32A+K34A+ E35A) were used in the experiments shown in A and 5. The mean values of three different experiments, each performed in duplicate, are shown. The differ- ence in the amounts of mediator released in the experiments shown in A and 5 are due to differences in the responsiveness of the donor's basophils used, which were not the same for A and 6. No mediator release was observed in basophils ex- posed to wtNGF or muNGF alone (not shown).

0.4

0.2 8: 20 lsL2! 0

rTt

0

0- 0.1 1 10 100

Factor Concentration (nglml)

FIGURE 2. Biological activities of purified wild-type and mutant NGF on sympathetic neurons. Serial dilutions of wild-type (open symbol) and mutant (closed symbol) NGF were assayed for their ability to stimulate neurite outgrowth from explanted embryonic day 9 chick sympathetic ganglia. The mean values of duplicate determinations, which varied by <lo% of the average, are shown.

change in the release response to C5a was, therefore, used to compare the effect of wild-type with that of mutant NGF, to investigate whether the LNGFR is necessary for priming the basophils. The triple-mutated NGF was used, which has lost the binding affinity to the acidic LNGFR by changing three critical amino acids (K32, K34, and E35) to alanine, but which is still able to bind trk. Figure 1 shows that the efficacy of mutant NGF to prime basophils was identical with that of the wild-type protein, occurring dose-depen- dently above 1 ng/ml. Maximal effects were already reached at low concentrations of mutant NGF of about 30 ng/ml. However, the potency on a molar basis of mutant NGF was about 1 order of magnitude lower than that of wtNGF, indicating that the LNGFR may be involved, but is certainly not necessary, for the full bioactivity of NGF on human basophils. The same mutant and wild- type NGF preparations were used in the standard neurite outgrowth assay (Fig. 2) . Consistent with a previous study (28), the triple mutation did not significantly affect the bioactivity of NGF in this neuronal assay. We, therefore, attempted to identify the presence of LNGFRs on purified human basophils by labeling with commercially available monoclonal aLNGFR Ab and fluorescein- labeled goat anti-mouse IgG Abs using fluorescent flow cytometry. Monoclonal aCD18 served as a positive control for staining basophils, and PC12 cells served as a positive control for staining LNGFR. However, no specific staining for LNGFR in human ba-

0.1 1 10 0 0.1 1 10 100


60

40

20 NGF GF

0

FIGURE 3. Effects of wtNGF and muNGF on basophil mediator release induced by different IgE-dependent and IgE-independent agonists. Cells were pretreated for 10 min with buffer control (white columns), 10 ng/ml muNCF (hatched columns), and 10 ng/ml wtNGF (black columns), respectively, and then stimulated for 20 min with C5adesArg M), C5a (lo-' M), FMLP M), and aFccRI mAb (29C6; 100 ng/ml), respectively. Histamine (upper panel) and LTC, were measured in the supernatants. The mean values of three experiments performed in duplicate are shown.

sophils could be observed (data not shown), indicating that the expression of LNGFR is below the sensitivity of FACS analysis.

NGF has been shown to enhance or change the release of mediator in response to all IgE-dependent as well as IgE-independent agonists (41, 44, 47). The effect of NGF or the mutant protein on the release response induced by four selected agonists was, therefore, studied: C5a and C5adesArg, which induce degranulation only; FMLP, the only agonist interacting with a seven-transmembrane, G protein-coupled receptor capable of directly inducing LTC, formation; and IgE receptor activation by cross-linking with monoclonal a F c N a-chain Ab. In all cases mediator release was en- hanced, and the effects of maximally effective concentrations of

The Journal of Immunology 5585

t rk trkB trkC LNGFR S12

M 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 n mn-n

I I l l I I I I I I I I I I l l

0 0.1 1 10 100 lo00


FIGURE 4. Comparison of the bioactivities of NGF, BDNF, and NT-3 on human basophils. Basophils were treated for 10 min with buffer control (0) or different concentrations of wtNGF (circles), NT-3 (squares), or BDNF (triangles), respectively, and then stimulated for 20 min with C5a (1 O-' M). The generation of LTC, was measured in the cell-free supernatants. The mean values of three different experiments, each performed in duplicate with cells from different donors, are shown.

wild-type and mutant NGF were identical (Fig. 3). Thus, mutations of the NGF abrogating the interaction with the LNGFR do not affect the bioactivity profile on basophils.

Basophil priming by NTs

Although there is increasing evidence of important regulatory effects of NGF outside the nervous system, no information is yet available concerning whether other highly homologous NTs also have nonneuronal bioactivities. It was, therefore, of interest to de- termine whether BDNF and NT-3 affect basophil function. Such experiments should also answer the question of whether the LNGFR can by itself transduce signals in basophils, since all NTs interact with the LNGFR with similar affinity. Priming for C5a- induced LTC, formation was used for a sensitive read-out of pos- sible priming effects of neurotrophic factors other than NGF. As illustrated in Figure 4, rhBDNF was devoid of activity up to 1,000 ng/ml and is thus at least 30,000 times less active than rhNGF. Furthermore, rhNT-3 was inactive at physiologically relevant concentrations. However, a significant threshold response was observed at 300 nglml, corresponding to an approximately 10,OOO- fold lower potency of NT-3 compared with that of NGF. rhNT-4/5 obtained from a commercial source was also tested at concentrations up to 100 ng/ml in three separate experiments and was found to be inactive; it is thus at least 1000 times less potent than rhNGF (data not shown).

Basophils express the trk proto-oncogene

All results obtained to date indicate that basophils have a functional trk receptor, but neither trkB nor trkC. We thus analyzed whether basophils express mRNA for the trk receptor family and the LNGFR. Because the basophil is a very rare leukocyte type that cannot be purified in large numbers from peripheral blood, RT- PCR was used for identification of trk, trkB, trkC. and LNGFR mRNA. Figure 5 shows that leukocytes enriched in basophils to a purity of -70% by density gradients and negative selection constitutively expressed mRNA for trk and the LNGFR, but not for trkB and trkC, in agreement with the functional data. This pattern of mRNA expression did not change when the basophil preparation was stimulated with IL-3 and C5a. To avoid false positive results due to contaminating lymphocytes when using this highly sensitive method, basophils were further purified to apparent homogeneity by positive selection of FceRI+ cells using FACS analysis. Figure 6 shows that mature human blood basophil granulo-

653-

298-

FIGURE 5. Expression of NT receptor mRNA in human blood basophils. Total RNA (1.5 Kg) isolated from leukocytes enriched in basophils by negative selection to a purity of 70% (contaminated by 30% lymphocytes) was reverse transcribed and amplified for 35 cycles using trk, trkB, trkC, and LNGFR primers. PCR amplification (25 cycles) of ribosomal 512 cDNA served as a control for RNA input and amplification. PCR products were electrophoresed in agarose gels, blotted onto nylon membranes, and probed with the appropriate digoxigenin- labeled DNA probes. Lane 2, unstimulated basophils; lane 3, basophils stimulated with IL-3 (10 ng/ml) and C5a (10 nM) for 3 h; lane I , PCR products obtained from PC1 2 cells (trk, LNGFR, and S12), SH SY5Y cells (trkB), or rat hippocampus (trkC) used as positive controls; lane M, digoxigenin-labeled DNA molecular size markers VI (Boehr- inger Mannheim).

A M 1 2 3 bP

653 - 298-

4 trk

FIGURE 6. Trk expression by purified basophil and neutrophil/eosinophil granulocytes. Top, RT-PCR for trk was performed as described in Figure 5, using total RNA from PC1 2 cells as a positive control (lane I ) , RNA from basophils purified to homogeneity by FACS (lane Z), or RNA from a mixture of neutrophils and eosinophils (lane 3 ) purified from the same leukocyte preparation. Bottom, PCR amplification of control ribosomal protein S12 for 33 cycles.

cytes indeed express trk mRNA, in contrast to other polymorphonuclear granulocyte types (a mixture of eosinophils and neutrophils), consistent with a lack of priming activity of NGF on eosinophils (56) and neutrophils (our unpublished observa- tions). The lack of trk mRNA in eosinophils was further confirmed using RNA from highly purified eosinophils by depleting neutrophils with aCD16-coated beads (data not shown). To compare the expression level of mRNA of the high and low affinity NGF receptors in basophils, mRNA from basophils purified to homogeneity was reverse transcribed, and PCR was performed with 28 cycles using primers for trk and the LNGFR, respectively. As shown in Figure 7, no mRNA of the LNGFR could be detected, in marked contrast to the strong signal for trk message. A faint band of the PCR product for LNGFR became visible only when the cDNA was amplified with 35 cycles (data not shown). Taken to- gether, these experiments demonstrate that basophils express

5586

trk LNGFR s12 n

M 1 2 n n 1 2 1 2

I I I I I I I

bP 653 - 298 -

FIGURE 7. NGF receptor mRNA expression in purified blood basophils. Total RNA isolated from FACS-purified basophils (lane 2) or from PC1 2 cells (lane 1 ) was reverse transcribed and amplified for 28 cycles using trk and LNGFR primers or for 20 cycles using S12 primers, and the amplified products were analyzed as described in Figure 5. The RNA used for the experiments shown in Figures 5, 6, and 7 were from separate leukocyte preparations isolated from different unselected blood donors.

mRNA for trk, but not trkB and trkC, and no or only minimal mRNA for the LNGFR.

As a further means to demonstrate trk expression by basophils and to show that the trk mRNA is translated into the trk receptor protein, we stained permeablized basophils with rabbit anti-trk Abs followed by phycoerythrin-conjugated goat anti-rabbit F(ab'),. The cells were than double-labeled with FITC-conjugated mAb against the FceRI a-chain (29C6) as a specific marker for basophils. Figure 8 demonstrates that indeed the FceRl' basophils expressed trk protein, as indicated by the shift in red fluorescence of cells treated with anti-trk compared with that of control rabbit Abs.

Discussion This study shows that among the highly homologous NTs, NGF, BDNF, NT-3, and NT-4/5, NGF is unique in priming basophils for C5a-induced LTC, formation and at enhancing mediator release in response to other IgE-dependent and independent agonists. Using a triple-mutated NGF analogue that cannot bind to the LNGFR but still retains the ability to interact with trk (28). we demonstrate that the LNGFR is not necessary to activate basophils. Thus, trk appears to deliver all the necessary signals in our experimental system, since wild-type and mutant NGF have equal bioactivities with identical efficacy. The conclusion that the LNGFR does not deliver any direct or necessary signal in basophils is further supported by the inability of BDNF to prime basophils even at very high concentrations and by the only marginal effects of NT-3; both BDNF and NT-3 interact with LNGFRs with similar affinity as NGF (17, 57). The LNGFR belongs to a family of membrane proteins that control a variety of functions in cells of the hemopoietic lineage ( I 6). Therefore, the effects of NGF outside the nervous system, such as those studied here, may have been expected to be mediated via the LNGFR. Indeed, there is increasing evidence of a signaling capacity of the LNGFR, which may be involved in the regulation of apoptosis, cell proliferation, and cellular differentiation (29.30). Most interesting in this regard is the fact that LNGFR appears to activate the sphingomyelin cycle (3 I) , a signal transduction pathway that is shared with other immunoregulatory cytokines, such as TNF-a and IL-Ip (58, 59).

Although the LNGFR does not appear to be necessary for the full bioactivity of NGF on human basophils, we observed an approximately I order of magnitude lower potency of the muNGF protein for priming basophils, in contrast to the similar dose-re-

BASOPHIL ACTIVATION THROUGH THE trk RECEPTOR

A

l o g Fluorescence

FIGURE 8. Trk expression by human blood basophils. Fixed and permeabilized cells were stained with polyclonal rabbit anti-trk peptide Abs (c-trk) or control rabbit Abs (control), followed by phycoerythrin-conjugated goat anti-rabbit IgG F(ab'),. The cells were then stained with FITC-labeled mAb against the FceRI a-chain 29C6, as a specific marker of the basophils. The figure shows the red fluorescence of the FccRI-positive cell population (one representative experiment of three performed).

sponse curves for the wild-type and mutant NGF in the neurite outgrowth assay. A role of the LNGFR in aiding the interaction of NGF with trk is, however, unlikely, since we have been unable to demonstrate the presence of LNGFRs on basophils by FACS analysis, and virtually no mRNA of the LNGFR could be detected by RT-PCR in purified basophils.

The presence of mRNA for trk, but not trkB or trkC, was demonstrated in human basophils using RT-PCR, indicating that basophils constitutively express the trk receptor. Furthermore, the expression of the trk protein by basophils was demonstrated by flow cytometry. These data correlate with the unique bioactivity of NGF compared with those of other NTs. It is very unlikely that basophils express receptors of the trk family other than trk. although the possibility that trkB and/or trkC has been expressed earlier in differentiation cannot be entirely excluded. TrkB and trkC are highly homologous tyrosine kinase receptors, particularly in their intracellular catalytic domain, and there is no indication of differences in signal transducing pathways used by the trk NT receptor family ( 1 1-13. 18-24). Basophils should, therefore, be activated by BDNF, NT-4/5, or NT-3 if expressing trkB or trkC. respectively. BDNF and NT-4/5 are high affinity ligands for trkB unable to interact with trk, while NT-3 has been reported to strongly activate both trkC and trkB (20-24, 60). NT-3 has been shown to interact also with trk (21-23, 60, 61). The weak bioactivity of NT-3 at high concentrations on human basophils, which corresponds to an approximately 10,000-fold lower potency than that of NGF, can thus easily be explained by trk activation in response to NT-3. Assuming that the effect of NT-3 on human basophils is mediated exclusively by trk, its potency was even lower than anticipated from studies with neuronal cells. Since neuronal cells express the different trk receptors generally in a less exclusive manner, the experiments reported here also provide a useful functional assay for the selectivity of trk to the various NTs. Our results demonstrate indeed only minimal and physiologically irrelevant activation of trk by NTs other than NGF.

There are now several lines of evidence that the neurotrophic growth factor NGF also acts on certain leukocyte types (32-43)

The Journal of Immunology

and thus must be considered an immunoregulatory cytokine. It is, however, unknown whether at least some of the effects of NGF on immune cells described in the literature are due in part, or even exclusively, to an interaction with the LNGFR, particularly when higher NGF concentrations are needed to observe an effect. In this regard it is interesting that the LNGFR has been demonstrated to be present on B cells, and that LNGFR expression increased upon cell activation, enhancing the effect of the NGF (62). Further studies are thus needed to examine whether NGF is unique in its ability to act outside the nervous system, or whether neurotrophic factors such as BDNF and NT-3 can also act on certain leukocyte types other than basophils.

Among the different polymorphonuclear granulocyte types, basophils appear to be unique with regard to trk expression. Ba- sophils and eosinophils are particularly closely related cell types. They are generated from a common progenitor, share most of their surface markers, and respond to an almost identical set of growth factors, cytokines, and chemotactic agonist in a very similar manner. The finding that basophils, but not eosinophils, express trk is, therefore, remarkable and may indicate the participation of basophils in inflammatory immune reactions that do not involve eosinophils.

Basophils are important inflammatory effector cells because of their capacity to release the vasoactive mediators, histamine and LTC,, and are thought to participate particularly in allergic late phase reactions and chronic allergic inflammatory processes. The unique ability of NGF to strongly regulate the function of basophils could indicate an interaction between the nervous system and the effector phase of allergic inflammation. Furthermore, NGF has been shown to be present in murine mast cell granules, the primary effector cells of immediate allergic reactions (63). If NGF is also expressed in human mast cells, it could constitute an important link between human mast cell activation and the function of basophils in the following allergic late phase reaction.

In conclusion, we find that among different NTs, NGF is unique in regulating basophil functions. This bioactivity does not require the participation of the LNGFR and is mediated exclusively by activation of trk receptors expressed on human blood basophils.

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