PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2010 February 15.
Published in final edited form as:
PMCID: PMC2704022
NIHMSID: NIHMS94619

Human Basophils Secrete IL-3: Evidence of Autocrine Priming for Phenotypic and Functional Responses in Allergic Disease1

Abstract

Although IL-3 is commonly recognized for its growth factor-like activity, in vitro studies have long demonstrated a unique capacity for this cytokine to also augment the pro-inflammatory properties and phenotype of human basophils. In particular, basophils secrete mediators that are hallmark in allergic disease including vasoactive amines (e.g. histamine), lipid metabolites (e.g. LTC4) and cytokines (e.g. IL-4/IL-13), which are all markedly enhanced with IL-3 pretreatment. This priming phenomenon is observed in response to both IgE-dependent and IgE-independent stimulation. In addition, IL-3 directly activates basophils for IL-13 secretion and enhanced CD69 expression –two markers that are elevated in allergic subjects. Lymphocytes are commonly thought to be the source of the IL-3 that primes for these basophil responses. However, we demonstrate here for the first time that basophils themselves rapidly produce IL-3 (within 4h) in response to IgE-dependent activation. More importantly, our findings definitively show that basophils rapidly bind and utilize the IL-3 they produce, as evidenced by functional and phenotypic activity that is inhibited in the presence of neutralizing anti-IL-3 receptor (CD123) Ab. We predict that autocrine IL-3 activity resulting from low-level IgE/FcεRI cross-linking by specific allergen represents an important mechanism behind the hyper-reactive nature of basophils that has long been observed in allergic disease.

Keywords: human, basophil, allergy

Introduction

The role of basophils in the pathogenesis of allergic inflammatory diseases is best defined by the fact that these cells secrete three key classes of inflammatory mediators, including vasoactive amines (e.g. histamine), lipid metabolites (e.g. LTC4) and cytokines (e.g. IL-4/IL-13) (1, 2). These substances, which are all hallmark in allergic inflammation, are sequentially released by basophils especially in response to stimuli that cross-link the high affinity IgE receptor (FcεRI) on the surfaces of these cells (e.g. as allergen binds to specific IgE). In addition, there is mounting evidence that components linked to both adaptive and innate immunity influence this so-called IgE-dependent activation, either in a positive or negative manner. In particular, in vitro studies have long demonstrated that IL-3, more so than any other factor known to date, markedly increases basophil responsiveness in the release of these mediators and does so for a variety of stimuli. Moreover, IL-3 directly induces IL-13 production by basophils without the need for co-stimulation (3, 4). in vitro studies using human adult stem cells and in vivo studies in non-human primates have also demonstrated that IL-3 is critical for both basophil development and survival (5, 6). Of course, these activities of IL-3 are mediated through receptors (CD123) highly expressed on basophils, which are retained on these cells throughout their development and maturation from bone marrow precursors. Thus, in light of the importance of IL-3 in regulating essentially every aspect of basophil biology, it seems appropriate to infer that this growth factor/cytokine likewise plays an important role in the pathogenesis of allergic disease.

It has long been thought that activated T cells provide the IL-3 responsible for augmenting the pro-allergic functions of basophils. In particular, T cells secrete IL-3 upon activation through the T cell receptor (TCR) or by agonists that mimic the signaling associated with this mode of activation. Likewise, other hematopoietic cells, including natural killer cells, mast cells, and some megakaryocytic cells have all been reported to secrete this cytokine and therefore may also contribute (7). However, we demonstrate here for the first time that basophils themselves rapidly produce IL-3 when activated through the IgE receptor. More importantly, our findings definitively show that basophils rapidly bind and utilize the IL-3 they produce, as evidenced by functional and phenotypic activity that is inhibited in the presence of neutralizing anti-IL-3 receptor (CD123) antibodies. Overall, we predict that this autocrine activity of IL-3 plays a critical role in the “priming” phenomenon that has long been observed among basophils from allergic individuals.

Materials and Methods

Basophil purification

Venipuncture was performed on consenting adults (age range, 21–55 years) using a protocol approved by the Western Institutional Review Board (Seattle, Washington). With the exception of the basophils used in Fig. 3C, donors were not selected based on allergic status. In some instances, preparations also included cells procured from residual cell packs from anonymous subjects undergoing platelet pheresis within the Hemapheresis Unit at Johns Hopkins University. In all cases, mixed leukocyte suspensions were subjected to double-Percoll (1.075/1.081 g/ml) density centrifugation, which produced a basophil-enriched cell (BEC) interface accumulating on top of the 1.081 g/ml density, as previously described (8). After first removing the bulk of cells floating on the 1.075 g/ml Percoll, the BEC interface was then carefully removed, washed once in a Piperazine-N,N’-bis-2-ethanesulfonic acid (PIPES)/albumin/Glucose (PAG) buffer containing 4mM EDTA (PAG-EDTA) and then again in column buffer (PIPES containing 1% BSA and 2mM EDTA). Basophils were purified from the BEC suspensions using the negative selection kit from StemCell Inc., Vancouver, CA, as described (9). In brief, this involved resuspending the BEC suspensions in column buffer and adding first a cocktail of monoclonal antibodies targeting all other leukocytes. After 30 min. incubation on ice, microbeads coupled with anti-mouse immunoglobulin were then added for an additional 30 min. The BEC suspensions were then washed 1x, resuspended in 1 ml column buffer and subjected to magnetic selection through a buffer-primed LS column inserted in a quadroMACs magnet (both from Miltenyi Biotec). Cells not retained in the column (i.e. basophils) were collected, washed in PAG buffer without EDTA, and counted using a Spiers-Levy chamber. Basophil purities exceeded 97% in all preparations, as assessed by Alcian Blue staining.

Fig. 3
Human basophils rapidly generate and secrete IL-3 in response to IgE-dependent stimulation

RNA isolation

Total RNA was isolated from 0.25–1.0 × 106 basophils using the RNA-Bee protocol (Tel-test Inc., Friendswood, TX). Following isopropanol precipitation, RNA was washed with ethanol and nearly dried under vacuum. The RNA was then resuspended in DNase-free water and stored below −70°C.

Quantitative RT-PCR for Detection of IL-3 mRNA

IL-3 mRNA expression in basophils was quantified using real-time RT-PCR, as previously reported (10). Briefly, single-step real-time RT-PCR was performed in an ABI 7300 Thermocycler using Taq Man reagents (Perkin-Elmer). Primer/probe combinations for IL-3 included: forward primer: 5’-TTCTTAAAAATCTCCTGCCATGTCT-3’, reverse primer: 5’-CATTCCAGTCACCGTCCTTGAT-3’ and probe: 5’-CCGCACCCACGCGACATCC-3’. The reporter dye and quencher were FAM and TAMRA, respectively. Expression of IL-3 was normalized to hypoxanthine phosphoribosyl transferase (HPRT), which was determined using the following primer/probe sequences: forward primer: 5-CGGCCGGCTCCGTTA-3’, reverse primer: 5’-TTAGGTATGCAAAATAAATCAAGGTCAT-3’, and probe: FAM 5’-CCGCAGCCCTGGCGTCGT-MGB.

Flow Cytometry

Direct staining of the cell surface activation marker protein CD69 on fixed (buffered 4% paraformaldehyde) basophils was achieved with a PE-conjugated Ab and using appropriate isotype control methods. Single-color flow cytometry was performed using a FACSCalibur machine. Results are reported as median fluorescence intensity (MFI) after subtracting values obtained with isotype control antibodies.

Culture Conditions

For the secretion of cytokines, basophils were cultured in C-IMDM (Iscove’s Modified Dulbeccos Medium, 5% FCS, 1x non-essential amino acids and 5 µg/ml gentamicin), as described (9). Experiments typically involved adding 1.0×105 basophils to the wells of 96-well flat-bottom plates in a volume of 0.125 ml. In experiments investigating IL-3 mRNA expression, cells were instead cultured in opened 1.5 ml polypropylene tubes inserted into 15 ml culture tubes. This facilitated the extraction of RNA without having to first transfer cells from culture plate wells. Cell cultures were initiated by incubating the basophils for 15 min. to equilibrate to 37°C, 5% CO2. An equal volume of stimulus (2X) was then added and the cells cultured for specific time points. A mixture of antibodies against the alpha (clone: 32703; R&D Systems) and beta (clone: EP1037T, Epitomics Inc., Burlingame, CA) subunits of the IL-3 receptor (CD123) was added along with stimulus. This enabled the detection of secreted IL-3, since it effectively blocked this cytokine from binding to its highly expressed receptors on basophils (see Figure 2). By neutralizing bioactivity, the anti-IL-3 receptor Ab mixture (IL-3R Ab) also allowed assessment of whether basophil-derived IL-3 mediated autocrine effects on function and phenotype.

Fig. 2
Anti-IL-3 receptor Ab (IL-3R Ab) inhibits the absorption of IL-3 to basophils

Cytokine Measurements

An ELISA protocol for the detection of IL-13 (eBioscience) has been described previously (9). An in-house ELISA was developed to detect IL-3 protein. This involved coating wells of 96-well plates (eBioscience) with 0.100 ml of rat anti-human IL-3 mAb (clone: BVD8-3G11; BD PharMingen) at a concentration of 2.5 µg/ml in PBS for overnight at RT (~23°C). Plates were then washed 4x before blocking with 0.150 ml Assay buffer (eBioscience) for 1h at RT. Wells were once again washed 3x before adding 0.050 ml culture supernatant samples and IL-3 standards (Biosource/Invitrogen) for 2h at RT. Wells were then washed 4x before adding 0.100 ml of biotinylated rat anti-human IL-3 mAb (clone: BVD3-1F9; BD PharMingen) at a concentration of 250 ng/ml in Assay buffer for 1h. Wells were again washed 4x before adding 0.100 ml avidin-HRP (eBioscience) at 1:1000 for 30 min at RT in the dark. Plates were washed 6x before developing with the addition of 0.100 ml TMB substrate for 15 min. at RT in the dark. Reactions were stopped by adding 0.05 ml 2N H2SO4 and read at 450/570 nm using a microplate ELISA reader (model 550, BioRad). The sensitivity of this assay was ~3 pg/ml and had been standardized to a commercial plate (Biosource/Invitrogen).

Histamine Release

Portions of the cell-free supernatants taken from the basophil cultures after 1h incubation were assayed for histamine using automated fluorimetry, as previously described (9). Results are expressed as a percentage of the total histamine content, which was determined by taking a proportionate number of basophils and lysing in 1.6% perchloric acid.

Statistical analysis

Data are presented as mean±SEM unless otherwise indicated. Statistical analysis was performed with Prism4 software (Graphpad Software Inc., San Diego, CA) and involved the use of the paired t-test unless otherwise stated. P values ≤ 0.05 were considered significant.

Results

Human basophils generate IL-3 following IgE-dependent activation

Human basophils produce IL-4 and IL-13 in response to IgE/FcεRI-dependent activation. Therefore, we investigated first whether IL-3 is similarly induced in these cells following stimulation with the same polyclonal goat anti-human IgE Ab reagent (anti-IgE) that has long been used for activating basophils for cytokine secretion (11). Fig. 1A shows real-time RT-PCR data for IL-3 mRNA expression in unstimulated basophils compared to those activated 1h with several doses of anti-IgE. In each instance, mRNA levels were increased relative to HPRT expression, even though responses varied among the 3 pilot experiments (range of ~15 to 1400-fold). As predicted, optimal induction of IL-3 in basophils occurred at a concentration of anti-IgE (~10 ng/ml) that is reported optimal for IL-4 secretion but suboptimal for mediator release (11).

Fig. 1
Human basophils up-regulate IL-3 mRNA expression following IgE-dependent activation

With the optimal anti-IgE dose being narrowed to ~10 ng/ml, we investigated whether there is a relationship between the induction of IL-3 mRNA expression and responsiveness to anti-IgE as it pertain to histamine release. As shown in Fig. 1B, there was a wide range in the induction of IL-3 mRNA (~2 to nearly 9000-fold) to challenge with anti-IgE for 1h among the basophils isolated from 18 different subjects. More importantly, these IL-3 responses were directly related to the percentage of histamine released in the same cultures. For example, IL-3 mRNA levels among basophil suspensions releasing <10% histamine to anti-IgE averaged just 4.8±1.9 -fold above control responses (n=3). In contrast, those releasing between 51–100% histamine in response to stimulation showed a ~1000-fold greater induction (4737±2154, n=3) in IL-3 mRNA.

Initial attempts to detect IL-3 protein in culture supernatants from activated basophils were negative. However, knowing that the IL-3 receptor (CD123) is expressed at very high levels on basophils led us to hypothesize that any IL-3 that is secreted might actually be rapidly absorbed by the basophil and thus be undetected in supernatant. To test this, we spiked cultures (0.250 ml vol.) containing increasing numbers of basophils with recombinant IL-3 (1000 pg/ml). A duplicate set of cultures also received a mixture of anti-IL-3 receptor antibodies (IL-3R Ab) that prevents IL-3 binding to both the alpha and beta subunits of the receptor. After incubating 16h, cell-free supernatants were measured for IL-3 protein content. As shown in Fig. 2A, approximately 700 pg/ml of IL-3 was detected in control cultures devoid of basophils. However, only ~50% of this IL-3 value (~350 pg/ml) remained after culturing with as few as 1×105 basophils, and <20% was recovered in the presence of 5×105 basophils. As predicted, a saturating concentration (2 µg/ml) of IL-3R Ab prevented this apparent absorption of IL-3, with 80–85% of the control levels recovered in the presence of up to 5×105 basophils. An additional experiment shown in Fig. 2B confirmed these observations and further revealed that isotype controls for the IL-3R Ab mix did not prevent basophils from absorbing IL-3.

We then re-evaluated the capacity of basophils to secrete IL-3 protein by adding IL-3R Ab at the time of activation and for the entire culture incubation. As shown in Fig. 3A, this approach immediately resulted in the detection of IL-3 protein in basophil cultures activated with anti-IgE. In fact, levels were detectable and nearly half-maximal within 2h after activation, had essentially peaked to 91±22 pg/106 basophils by 4h, and remained sustained at 115±43 pg/106 after 16h incubation. Importantly, IL-3 protein was not detected in cultures treated with the IL-3R Ab mix alone, confirming that these antibodies were not simply inducing cytokine production. In addition, increases in IL-3 mRNA preceded the time course observed for the secretion of this cytokine. As shown in Fig. 3B, up to 15-fold greater levels were seen within 15 minutes after activation, compared to basophils cultured in medium alone. By 1h, IL-3 mRNA levels had peaked and averaged more than 1000-fold above those seen with cells cultured in medium alone (n=3).

Basophils were also investigated for IL-3 secretion in response to allergen. Fig. 3C shows results from a representative experiment where basophils isolated from a cat allergic subject secreted detectable levels of IL-3 following stimulation with a cat extract (Holister-Steer). As expected, these basophils also secreted histamine in response to the cat allergen.

Induction of IL-3 in basophils using non-IgE-dependent stimuli

We next determined whether basophils generate IL-3 in response to a variety of stimuli, including common basophil secretagogues (i.e. C5a, FMLP, PMA, ionomycin) but also to the TLR ligands, peptidoglycan (PGN) and LPS. As expected, histamine was released (Fig. 4C) in basophil cultures stimulated with C5a (37±4%), FMLP (53±4%), PMA (64±10%), and ionomycin (31±5%). However, responses to PGN and LPS were no different from values seen with medium control (>3%). As shown in Fig. 4A, IL-3 mRNA levels were the greatest among the cultures stimulated with ionomycin, averaging some 1000±250 fold greater than those in the medium control. In contrast, induction of IL-3 message was typically less than 10-fold above control values with all other stimuli, even those causing histamine release (i.e. C5a and FMLP). Fig. 4B indicates that this was also observed at the level of IL-3 secretion. In this instance, only ionomycin induced detectable levels of IL-3 in cultures stimulated for either 4h (457±27 pg/106 basophils) or 24h (4844±2784 pg/106 basophils) in the presence of IL-3 receptor Ab.

Fig. 4
IL-3 responses in basophils following stimulation with IgE-independent stimuli

Basophil-derived IL-3 mediates autocrine priming for functional and phenotypic responses

Considering the overwhelming evidence that IL-3 influences basophil function, we addressed whether the secretion of this cytokine following IgE-dependent activation mediates autocrine activity that additionally affects basophil function and/or phenotype. With regard to function, we focused on whether using the IL-3R Ab would affect IL-13 secretion –a response that is directly induced in basophils exposed to IL-3. Basophils from 8 different subjects were stimulated with anti-IgE or medium alone in the absence and presence of IL-3R Ab. Cultures were incubated for 16h to optimize for IL-13 secretion. As shown in Fig. 5A, we first investigated whether IL-3 protein was produced under these conditions. Cells from 4 of the 8 subjects stimulated with 10 ng/ml anti-IgE actually secreted detectable amounts of IL-3 without the addition of IL-3R Ab. Nonetheless, adding this reagent markedly increased the levels of IL-3 detected in these cultures (p=0.027). Importantly, IL-3 protein was not produced by basophils cultured with medium alone or with a sub-optimal concentration of anti-IgE, even in the presence of IL-3R Ab. A very different outcome was observed for the IL-13 detected in the same culture supernatants. As shown in Fig. 5B, the addition of IL-3R Ab significantly (p=0.001) but only partially suppressed the IL-13 induced with anti-IgE stimulation, suggesting that autocrine IL-3 was indeed contributing to the IL-13 response. Also, IL-3 levels plotted against the IL-13 secreted in these cultures significantly correlated (r2=0.661, p=0.041), underscoring this finding (Fig. 5C). Importantly, anti-IL-3R completely inhibited the IL-13 response in control cultures stimulated with IL-3 (5 ng/ml) alone (Fig. 5D). Although not shown in figure 5, the IL-4 levels in these experiments averaged 145±32 pg/106 basophils without IL-3R Ab and only 118±26 pg/106 with antibody, or some 19% less (p=0.0078, n=8).

Fig. 5
IgE-dependent IL-3 secretion by basophils promotes IL-13 secretion through autocrine activity

In a final set of experiments, we investigated whether basophils up-regulate CD69 expression following IgE-dependent activation and if so, whether this results from autocrine IL-3. Although not generally reported on basophils during short-term (i.e. 30 min) activation, we did see increases in CD69 expression on basophils stimulated for ≥1h, with levels peaking between 2-4h and remaining elevated for up to 24h post stimulation (Fig. 6A). The intensity of this response was seemingly dependent on the concurrent histamine release response (percent release in parentheses) following anti-IgE stimulation. Since this time course coincides with IL-3 generation, we sought to determine the contribution of this cytokine by attempting to block CD69 induction with anti-IL-3R Ab. As shown in Fig. 6B, the upregulation of CD69 expression on basophils stimulated with anti-IgE (after 4h and 16h) was significantly inhibited (~35%) with the addition of anti-IL-3R Ab, relative to isotype controls (p<0.003, n=7). Although not shown, the histamine released in the 4h cultures was unaffected with neutralization of IL-3. As expected, the CD69 induced on basophils in control cultures treated with recombinant IL-3 (5 ng/ml) was also suppressed (~66%) with the addition of the IL-3R Ab (p<0.009, n=6).

Fig. 6
CD69 expression is induced on basophils following IgE-dependent secretion and is mediated by autocrine IL-3

The evidence that autocrine IL-3 activity was indeed responsible for CD69 induction was further supported by results from an experiment where basophils were cultured at different densities (i.e. 2-, 0.4-, and 0.08- ×106/ml) for 16h with and without anti-IgE. CD69 levels were not induced on basophils cultured at these densities in medium alone (nMFI=0.20, 0.10, and 0.14, respectively) but were when activated by anti-IgE (nMFI=145.63, 137.32, and 93.06, respectively). Overall, the levels induced were quite comparable and did not reflect differences (up to 25-fold) in cell densities, as would be predicted if CD69 induction was due solely to paracrine IL-3 activity.

Discussion

Overall, the data presented here indicate that human basophils generate IL-3 with parameters identical to those described for their production of IL-4 (11, 12). In particular, increases in IL-3 mRNA were seen as early as 15 minutes following stimulation with anti-IgE Ab, with evidence of IL-3 secretion within the first hour and peaking by 4h after activation. Of course, the rapid secretion of IL-3 only became apparent after blocking its absorption using IL-3R Ab (Fig. 2). It therefore goes to reason that the rapid production of IL-3 plays an important role in allowing for autocrine priming for subsequent effector functions (as discussed below). Another feature of IL-3 production by basophils that bears remarkable similarity to that of IL-4 is its strong link to IgE/FcεRI-dependent activation. Basophils responding well to anti-IgE stimulation (with regard to histamine release) up-regulated IL-3 mRNA by as much as several thousand-fold after 1h and were also more likely to secrete protein for this cytokine (Fig. 1 and Fig 3). In contrast, common basophil secretagogues such as C5a, FMLP and PMA that are all quite capable of activating basophils for mediator release did not induce detectable IL-3 protein, nor did these stimuli greatly affect IL-3 mRNA levels compared to cells cultured in medium alone (Fig. 4). This dissociation of IL-3 secretion from histamine release has long been observed for IL-4 and once again emphasizes that little if any of these two cytokines is pre-formed in basophils and released during degranulation (1).

We predict that changes in cytosolic calcium are important for IL-3 generation (just as they are for IL-4), since ionomycin markedly induced secretion of this cytokine (Fig. 4). In fact, IL-3 secretion exceeded 4000 pg/106 basophils when using this stimulus during a 24h incubation, and likely eliminated the need for IL-3R Ab to block absorption, since receptors were most certainly saturated at these levels. Based on these findings further demonstrating the similarities in the parameters important for IL-3 and IL-4 secretion by basophils, we also predict that IL-3 is controlled pharmacologically much like what has been reported for IL-4, even though studies to definitively show this are yet to be done.

We have also previously reported that TLR2 ligands, such as PGN, enhance both IgE-dependent and IgE-independent responses in basophils much like IL-3 (9). We therefore considered that this mode of innate immune stimulation might prime basophils by inducing IL-3 that would then mediate autocrine effects. However, our results indicated that PGN (and LPS) only poorly induced IL-3 mRNA compared to those seen with IgE-dependent activation. In addition, basophils did not secrete detectable IL-3 when cultured with these TLR ligands (Fig. 4). It seems appropriate to conclude that the priming of basophil responses reported for TLR2 agonists is not the result of autocrine IL-3 effects. It is also important to note that we also considered the possibility that IL-5 and GM-CSF could mediate basophil priming by inducing autocrine IL-3. However, IL-3 mRNA was not induced in basophils treated with these cytokines (JTS, unpublished data). Like the TLR2 ligands, these substances appear to augment basophil responses independently of IL-3 generation.

The concept that IL-3 production is linked to IgE/FcεRI-dependent activation was first suggested nearly 20 years ago using murine mast cell lines (13). In those studies, mast cells were reported to up-regulate IL-3 mRNA as well as the expression of message for IL-4, IL-5 and IL-6 when subsequently stimulated through FcεRI or activated with calcium ionophore. A more recent study reports that IL-3 is secreted by mouse mast cells even in response to monomeric IgE alone, which acts in an autocrine fashion to extend survival (14). However, it is important to note that the bone marrow-derived mast cells used in these studies were in fact first exposed to exogenously added IL-3 in order to induce their development from precursor cells. It therefore seems possible that this added IL-3 may have played a role in regulating its own production. Moreover, unlike the mouse data which implicates the importance of IL-3 in mast cell development and function, recent evidence indicates that IL-3 does not affect the differentiation of human mast cells (15). Thus, these issues raise questions regarding the translatability of the two models, particularly as it pertains to the role of IL-3 in mast cell vs. basophil function.

Nonetheless, the results presented in the current study are not unlike those seen with murine mast cells in that they clearly show that human basophils also rapidly generate mRNA and secrete protein for IL-3 within the first 4h following IgE-dependent activation. However, the basophil studies are clearly more relevant to clinical disease in demonstrating that they utilize the IL-3 they produced. For example, in vitro studies have long shown IL-3 to be the most potent cytokine to affect human basophil function and that it does so by binding receptors densely expressed on their cell surface. It is therefore plausible that these cells may very well regulate their own priming in vivo. The importance of this hypothesis is underscored by the long-standing belief that in vitro basophil releasability is clinically relevant (16). In fact, many studies conducted during the past 30 years have shown evidence that basophils from allergic subjects are primed both functionally and phenotypically when compared to cells from normal subjects.

To investigate the possibility that basophils mediate their own priming, we explored whether the IL-13 generated by these cells following IgE-dependent activation is, indeed, secondary to IL-3 secretion. It seemed possible that the prolonged kinetics (~24h) originally reported for optimal IL-13 secretion (17) vs. the time course for IL-4 (~4h) (12) in response to IgE-dependent activation is actually due to the actions of autocrine IL-3. By neutralizing IL-3 activity using IL-3R Ab, we were able to demonstrate that ~30% of the IL-13 secreted during a 16h incubation was dependent on autocrine activity (Fig. 5). Of note, we accurately predicted that neutralizing IL-3 would have less of an effect on IL-4 (~19% reduction) and histamine (no change), since release of these mediators, respectively, occur either simultaneously or after IL-3 is produced. However, it is also fair to conclude that much of the IL-13 produced during IgE-dependent activation is in fact independent of IL-3. This finding thus supports our long-held belief that at least 2 pathways exist in basophils for the production of IL-13 –one that is FcεRI-mediated and one dependent on IL-3 (1). Therefore, the IL-3 produced by basophils likely functions, in part, to augment and/or prolong the secretion of IL-13 that is initiated upon IgE-dependent activation.

Evidence that basophils might mediate their own priming is also supported by the finding that autocrine IL-3 induces the expression of CD69 following IgE-dependent activation. First, it is important to note that Suzukawa, et al. have recently shown that low-level FcεRI-dependent activation up-regulates CD69 expression on basophils, but only under conditions that involved the addition of IL-3 along with the anti-FcεRIα Ab used in these studies (18). Our results confirm that basophils express CD69 following IgE-dependent activation, but additionally show that IL-3 does not need to be added exogenously to do so (Fig. 6A). In observing the relatively slower kinetics for CD69 induction (≥1h) compared to the minutes for the up-regulation of degranulation-specific markers (i.e. CD63 and CD203c), we then hypothesized that the former is regulated indirectly by autocrine IL-3. Indeed, this was our conclusion after confirming that the addition of IL-3R Ab markedly inhibited CD69 induction resulting from IgE-dependent activation (Fig. 6B,C).

Overall, these in vitro findings demonstrating that autocrine IL-3 modulates basophil IL-13 secretion and CD69 expression could help interpret clinical observations. Several studies have shown that basophils produce IL-13 in direct response to IL-3 (3, 4) and that this reaction is greater using cells from allergic vs. normal controls (19). Circulating basophils have also been shown to spontaneously secrete IL-13 following experimental allergen challenge in the nose compared to pre-challenge responses (20). Likewise, Yoshimura, et al. first reported constitutive expression of CD69 on the basophils of asthmatics compared to normal subjects (21). This has since been extended to include basophils prepared from allergic rhinitics and subjects with chronic idiopathic urticaria (22). We have also shown that circulating basophils from subjects allergic to insect venom express greater levels of CD69 following a controlled insect sting compared to pre-sting levels (23). Therefore, it seems feasible that the basophils from these subjects are primed for greater IL-13 secretion and expression of CD69 having come in contact with IL-3. More significantly, the data presented here would imply that the source of this IL-3 could very well be the basophil itself, and that low-level IgE/FcεRI cross-linking resulting from allergen exposure is stimulating this autocrine response at a systemic level. Support for this hypothesis may be forthcoming from studies that investigate whether IgE depletion using omalizumab reduces these markers (i.e. IL-13 and CD69) of basophil priming.

In conclusion, we have shown that human basophils rapidly generate and secrete IL-3 upon IgE-dependent stimulation. Through autocrine activity, this IL-3 is capable of priming basophils for functional and phenotypic properties characteristic of basophils isolated from allergic individuals. We predict that autocrine IL-3 activity resulting from low-level IgE/FcεRI cross-linking by specific allergen represents an important mechanism behind the hyper-reactive nature of basophils that has long been observed in allergic disease.

Acknowledgments

Specific contributions made by authors

JTS designed the research; APB, KLC and JTS performed the research as well as collected and analyzed the data; JTS drafted the manuscript with input from APB and KLC; all authors checked the final version of the manuscript.

Non-standard Abbreviations

BEC
basophil-enriched cells
PIPES
Piperazine-N,N’-bis-2-ethanesulfonic acid
PAG
PIPES/albumin/Glucose
HPRT
hypoxanthine phosphoribosyl transferase
mMFI
median fluorescence intensity
PGN
peptidoglycan

Footnotes

1Supported in part by grant AI 042221 (JTS) from the National Institute of Allergy and Infectious Diseases, National Institutes Health, Bethesda, Maryland

Conflict of interest disclosure

The authors declare no competing financial interests.

References

1. Schroeder JT, MacGlashan DW, Jr, Lichtenstein LM. Human basophils: mediator release and cytokine production. Adv Immunol. 2001;77:93–122. [PubMed]
2. Falcone FH, Zillikens D, Gibbs BF. The 21st century renaissance of the basophil? Current insights into its role in allergic responses and innate immunity. Exp Dermatol. 2006;15:855–864. [PubMed]
3. Redrup AC, Howard BP, MacGlashan DW, Jr, Kagey-Sobotka A, Lichtenstein LM, Schroeder JT. Differential regulation of IL-4 and IL-13 secretion by human basophils: their relationship to histamine release in mixed leukocyte cultures. J Immunol. 1998;160:1957–1964. [PubMed]
4. Ochensberger B, Daepp GC, Rihs S, Dahinden CA. Human blood basophils produce interleukin-13 in response to IgE-receptor-dependent and -independent activation. Blood. 1996;88:3028–3037. [PubMed]
5. Kepley CL, Pfeiffer JR, Schwartz LB, Wilson BS, Oliver JM. The identification and characterization of umbilical cord blood-derived human basophils. J Leukoc Biol. 1998;64:474–483. [PubMed]
6. Dvorak AM, Monahan-Earley RA, Estrella P, Kissell S, Donahue RE. Ultrastructure of monkey peripheral blood basophils stimulated to develop in vivo by recombinant human interleukin 3. Lab Invest. 1989;61:677–690. [PubMed]
7. Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, Wang XY, Algate PA, Franklin RA, Oberhaus SM, Steelman LS, McCubrey JA. Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs. Leukemia. 1999;13:1109–1166. [PubMed]
8. Schroeder JT, Saini SS. Assay methods for measurement of mediators and markers of allergic inflammation. In: Detrick B, Hamilton RG, Folds JD, editors. Manual of molecular and clinical laboratory immunology. 7th ed. Vol. 2006. Washington, DC: ASM Press; 2006. pp. 964–974.
9. Bieneman AP, Chichester KL, Chen YH, Schroeder JT. Toll-like receptor 2 ligands activate human basophils for both IgE-dependent and IgE-independent secretion. J Allergy Clin Immunol. 2005;115:295–301. [PubMed]
10. Schroeder JT, Bieneman AP, Xiao H, Chichester KL, Vasagar K, Saini S, Liu MC. TLR9- and FcepsilonRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression. J Immunol. 2005;175:5724–5731. [PubMed]
11. Schroeder JT, MacGlashan DW, Jr, Kagey-Sobotka A, White JM, Lichtenstein LM. IgE-dependent IL-4 secretion by human basophils. The relationship between cytokine production and histamine release in mixed leukocyte cultures. J Immunol. 1994;153:1808–1817. [PubMed]
12. MacGlashan D, Jr, White JM, Huang SK, Ono SJ, Schroeder JT, Lichtenstein LM. Secretion of IL-4 from human basophils. The relationship between IL-4 mRNA and protein in resting and stimulated basophils. J Immunol. 1994;152:3006–3016. [PubMed]
13. Plaut M, Pierce JH, Watson CJ, Hanley-Hyde J, Nordan RP, Paul WE. Mast cell lines produce lymphokines in response to cross-linkage of Fc epsilon RI or to calcium ionophores. Nature. 1989;339:64–67. [PubMed]
14. Kohno M, Yamasaki S, Tybulewicz VL, Saito T. Rapid and large amount of autocrine IL-3 production is responsible for mast cell survival by IgE in the absence of antigen. Blood. 2005;105:2059–2065. [PubMed]
15. Shimizu Y, Matsumoto K, Okayama Y, Sakai K, Maeno T, Suga T, Miura T, Takai S, Kurabayashi M, Saito H. Interleukin-3 does not affect the differentiation of mast cells derived from human bone marrow progenitors. Immunol Invest. 2008;37:1–17. [PMC free article] [PubMed]
16. Marone G, Spadaro G, Patella V, Genovese A. The clinical relevance of basophil releasability. J Allergy Clin Immunol. 1994;94:1293–1303. [PubMed]
17. Li H, Sim TC, Alam R. IL-13 released by and localized in human basophils. J Immunol. 1996;156:4833–4838. [PubMed]
18. Suzukawa M, Komiya A, Yoshimura-Uchiyama C, Kawakami A, Koketsu R, Nagase H, Iikura M, Yamada H, Ra C, Ohta K, Yamamoto K, Yamaguchi M. IgE- and FcepsilonRI-mediated enhancement of surface CD69 expression in basophils: role of low-level stimulation. Int Arch Allergy Immunol. 2007;143 Suppl 1:56–59. [PubMed]
19. Sin AZ, Roche EM, Togias A, Lichtenstein LM, Schroeder JT. Nerve growth factor or IL-3 induces more IL-13 production from basophils of allergic subjects than from basophils of nonallergic subjects. J Allergy Clin Immunol. 2001;108:387–393. [PubMed]
20. Saini S, Bloom DC, Bieneman A, Vasagar K, Togias A, Schroeder J. Systemic effects of allergen exposure on blood basophil IL-13 secretion and FcepsilonRIbeta. J Allergy Clin Immunol. 2004;114:768–774. [PubMed]
21. Yoshimura C, Yamaguchi M, Iikura M, Izumi S, Kudo K, Nagase H, Ishii A, Walls AF, Ra C, Iwata T, Igarashi T, Yamamoto K, Hirai K. Activation markers of human basophils: CD69 expression is strongly and preferentially induced by IL-3. J Allergy Clin Immunol. 2002;109:817–823. [PubMed]
22. Vasagar K, Vonakis BM, Gober LM, Viksman A, Gibbons SP, Jr, Saini SS. Evidence of in vivo basophil activation in chronic idiopathic urticaria. Clin Exp Allergy. 2006;36:770–776. [PubMed]
23. Gober LM, Eckman JA, Sterba PM, Vasagar K, Schroeder JT, Golden DB, Saini SS. Expression of activation markers on basophils in a controlled model of anaphylaxis. J Allergy Clin Immunol. 2007;119:1181–1188. [PubMed]