Balance between IL-3 and type Iinterferons and their interrelationship with FasL dictates lifespan and effector functions of human basophils.

BACKGROUND: In contrast to eosinophils and neutrophils, the regulation of the lifespan of human basophils is poorly defined, with the exception of the potent anti-apoptotic effect of IL-3 that also promotes pro-inflammatory effector functions and phenotypic changes. Type I IFNs (IFN-α, IFN-ß), which are well known for their anti-viral activities, have the capacity to inhibit allergic inflammation. OBJECTIVE: To elucidate whether type I IFNs have the potential to abrogate the lifespan and/or effector functions of human basophils. METHODS: We cultured human basophils, and for comparison, eosinophils and neutrophils, with IL-3, interferons, FasL and TRAIL, alone or in combination, and studied cell survival, effector functions and signalling pathways involved. RESULTS: Despite an identical pattern of early signalling in basophils, eosinophils and neutrophils in response to different types of interferons, only basophils displayed enhanced apoptosis after type I IFN treatment. IFN-γ prolonged survival of eosinophils but did not affect the lifespan of basophils. IFN-α-mediated apoptosis required STAT1-STAT2 heterodimers and the contribution of constitutive p38 MAPK activity. Whereas the death ligands FasL and TRAIL-induced apoptosis in basophils per se, IFN-α-mediated apoptosis did neither involve autocrine TRAIL signalling nor did it sensitize basophils to FasL-induced apoptosis. However, IFN-α and FasL displayed an additive effect in killing basophils. Interestingly, IL-3, which protected basophils from IFN-α-, TRAIL- or FasL-mediated apoptosis, did not completely block the additive effect of combined IFN-α and FasL treatment. Moreover, we demonstrate that IFN-α suppressed IL-3-induced release of IL-8 and IL-13. In contrast to IFN-α-mediated apoptosis, these inhibitory effects of IFN-α were not dependent on p38 MAPK signalling. CONCLUSIONS AND CLINICAL RELEVANCE: Our study defines the unique and granulocyte-type-specific inhibitory and pro-apoptotic function of type I IFNs and their cooperation with death ligands in human blood basophils, which may be relevant for the anti-allergic properties of type I IFNs.

In human basophils, IL-3 selectively induces RANKL expression that is modulated by IgER-dependent and IgER-independent stimuli.

BACKGROUND: Receptor activator of NF-κB ligand (RANKL) is expressed as either surface (hRANKL1, hRANKL2) or soluble (hRANKL3) form. RANKL is involved in multifaceted processes of immunoregulation and bone resorption such as they occur in rheumatoid arthritis (RA). Interestingly, activated basophils, which are effector cells in allergic inflammation, contribute to the progress of collagen-induced arthritis (CIA), a mouse model for RA. Here, we investigate under which conditions human basophils express RANKL. METHODS: Among other stimuli, basophils were cultured with IL-3 alone. Alternatively, as a secondary stimulus, IgER-dependent or IgER-independent agents were added simultaneously either with IL-3 or after prolonged IL-3 culturing. Expression of RANKL protein and mRNA was analyzed by flow cytometry, ELISA, and real-time PCR. A coculture system was applied to investigate biological activity of basophil-derived RANKL. RESULTS: We show that in human basophils, IL-3 but no other stimulus induces de novo expression of soluble and surface RANKL, of which the latter enhances survival of MoDC. Upon simultaneous stimulation, IgER cross-linking reduces surface RANKL expression, while IgER-independent stimuli have no effect. This is in contrast to consecutive stimulation, as triggering with both IgER-dependent and IgER-independent stimuli enhances RANKL expression, particularly in its soluble form. Real-time PCR analysis shows that RANKL expression is mainly regulated at the mRNA level. CONCLUSION: This study identifies IL-3 as a potent inducer of RANKL expression in human basophils, suggesting them to interact with bone physiology and activation of immune cells. IgER-dependent and IgER-independent stimuli modulate the IL-3-mediated RANKL expression in a time- and stimulus-dependent fashion.

IL-33 is a mediator rather than a trigger of the acute allergic response in humans.

BACKGROUND: IL-33 enhances FcεRI-induced mediator release in human basophils without inducing degranulation itself. In contrast, studies in mice suggested that in the presence of high IgE levels, IL-33 triggers degranulation and anaphylaxis of similar severity as specific allergen. Consistent with this view, sera of atopic patients contain elevated levels of IL-33 after anaphylaxis. In this study, we determined whether IL-33 is potentially anaphylactogenic in humans with high IgE levels by regulating exocytosis independent of FcεRI cross-linking. Furthermore, we investigated whether IL-33 is released upon allergen provocation in vivo. METHODS: In subjects with high serum IgE levels, we measured IL-33-induced histamine/LTC4 in vitro, CD63 translocation ex vivo, and responsiveness of mast cells in vivo by skin prick test (SPT). In asthma patients, release of IL-33 and its correlation with early (tryptase)- and late-phase markers (IL-13 levels, eosinophil numbers) of the allergic response were assessed in bronchoalveolar lavage fluids (BALFs) after allergen challenge. RESULTS: IL-33 itself does not trigger basophil degranulation in vitro and ex vivo, even in subjects with high serum IgE levels, and negative SPTs demonstrate that skin mast cells do not degranulate in response to IL-33. However, in response to allergen challenge, IL-33 is rapidly released into BALFs at levels that do not correlate with other immediate- and late-phase parameters. CONCLUSION: IL-33 is unlikely an independent trigger of anaphylaxis even in subjects with high IgE levels. However, the rapid release of IL-33 upon allergen provocation in vivo supports its role as a mediator of immediate allergic responses.

In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8.

BACKGROUND: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. METHODS: A method is described to massively expand bone marrow-derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. RESULTS: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). CONCLUSION: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.

Robust expression of CCR3 as a single basophil selection marker in flow cytometry.

BACKGROUND: Basophil activation tests (BAT) rely on different combinations of basophil selection and activation markers. Whereas activation markers, especially CD63, are widely validated, the most suitable and robust marker for basophil selection is still a matter of debate. AIMS: Comparison of cell surface expression of two commonly used basophil selection markers (IgE, CD123/HLA-DR) with CCR3 in an unselected group of atopic and nonatopic donors in resting and activated basophils. METHODS: EDTA blood of 94 healthy adults, about half of them atopic by history, was analyzed using two different staining strategies: anti-CD123-PE/anti-HLA-DR-PerCP/anti-lin1-FITC and anti-IgE-FITC/anti-CD3-PerCP/anti-CCR3-PE. Additionally 40 pollen-allergic patients were recruited for the assessment of CCR3 expression after basophil activation. RESULTS: In resting basophils, cell surface expression of the three basophil selection markers was most constant for CCR3. IgE gating strategy showed the highest variation and up to 80% of nonbasophils in the selected gate in certain donors. During basophil activation, a shift of the mean fluorescence intensity for CCR3 toward the lower third of the CCR3-positive population could be demonstrated, but neither were CCR3-positive cells significantly lost for further analysis nor was differentiation between CCR3-positive and CCR3-negative cell populations hampered by this shift. CONCLUSIONS: CCR3 is a stable and highly expressed basophil selection marker, independent of the atopic background or basophil activation state and allows an accurate identification of basophils without need of a second marker. The basophil markers CD123/HLA-DR and IgE showed significantly higher interindividual variability in cell surface expression and are therefore less suited as selection markers.

Flowcytometric analysis of basophil counts in human blood and inaccuracy of hematology analyzers.

BACKGROUND: Differential leukocyte counts are of proven clinical value, but information about basophil counts in normal and disease conditions is scarce although basophils are regarded as key effector cells in allergy. AIMS: To establish and validate flowcytometric methods for counting basophils in peripheral human blood, to determine reference values, and to examine the accuracy of two widely used hematology analyzers. METHODS: Basophils were measured in whole blood by flowcytometry after staining with antibodies against the IL-3-receptor (CD123) or the eotaxin-receptor (CCR3) combined with other markers used for gating or validation purposes. RESULTS: The basophil percentages in 95 healthy adults showed an excellent correlation between the two independent flowcytometric methods, demonstrating that both are accurate and precise. The most robust maker is CCR3, which seems to be sufficient to specifically identify basophils. Normal values of relative and absolute blood basophils counts were 0.22-1.28% and 0.014-0.087 G/L (95% reference intervals), respectively. Basophil counts measured with two hematology analyzers Coulter GEN-S and ADVIA-120 showed no correlation between these instruments. Comparing the data obtained by flowcytometry and the analyzers demonstrate that basophil counts of the GEN-S are erratic, while the ADVIA-120 gives at least an estimation of true basophil numbers. CONCLUSIONS: We provide a solid description and validation of a novel and rapid method for the flowcytometric enumeration of basophil in whole blood. The fact that the most heavily used Hematology autoanalyzer gives completely erroneous results could explain why basophils counts have not yet received recognition as a clinically useful diagnostic marker.

Effect of tree pollen specific, subcutaneous immunotherapy on the oral allergy syndrome to apple and hazelnut.

BACKGROUND: The efficacy of specific immunotherapy (SIT) in pollen allergy is well established. However, its effect on pollen associated food allergy particularly the oral allergy syndrome (OAS) is not definitely ascertained. OBJECTIVE: The purpose of this controlled prospective study was to investigate whether SIT with tree pollen, mainly birch, has an effect on OAS induced by apple or hazelnut in birch pollen-allergic individuals. METHODS: Twenty-seven birch pollen-allergic subjects with OAS induced by apple or hazelnut underwent open oral provocation tests (OPT) with increasing doses (1 to 128 g) of fresh apple or ground hazelnut 1 year apart. Fifteen of 27 subjects were treated with SIT and 12 were not. Skin-prick test with birch pollen, apple and hazelnut, and specific serum IgE, IgG and IgG4 to rBet v 1, apple and hazelnut were determined. RESULTS: Thirteen of 15 (87%) SIT-treated subjects could eat significantly (P <0.001) more of apple or hazelnut without any symptoms/signs. The average tolerated quantity increased from 12.6 to 32.6 g apple after 1 year in this group. In contrast, only one of 12 (8%) individuals without SIT was able to consume a higher amount without symptoms. On evaluating laboratory parameters, only IgG4 antibodies to rBet v 1 were found to be significantly (P <0.01) increased in the SIT-treated group after 1 year. CONCLUSIONS: The study shows that SIT with extracts containing birch pollen has a positive impact on OAS to apple or hazelnut in birch pollen-allergic individuals. In spite of this outcome, the amount of apple/hazelnut tolerated is still small. Thus, the effect of SIT on the patients' management of OAS remains limited.

Increased coexpression of eotaxin and interleukin 5 in bullous pemphigoid.

While the presence of eosinophils in the skin lesions of bullous pemphigoid is well documented, the chemotactic factors responsible for eosinophil recruitment into the tissue still remain to be defined. In this study, eotaxin and interleukin-5 (IL-5) concentrations were determined in the blister fluid and sera of patients with bullous pemphigoid (acute and remission phase, n=6) in comparison with normal healthy controls (n=6) using the enzyme-linked immunosorbent assay (ELISA) technique. Eotaxin and IL-5 levels were increased in the blister fluid compared with the acute and remission phase sera, as well as compared with the sera of normal controls. In addition, immunoreactivity for eotaxin was predominantly found in the inflammatory cell infiltrate of lesional bullous pemphigoid biopsy specimens. In conclusion, the data provide evidence that co-operation of eotaxin and IL-5 may play an essential role in activating and recruiting eosinophils, which ultimately contribute to the tissue damage in bullous pemphigoid.

Requirements for C5a receptor-mediated IL-4 and IL-13 production and leukotriene C4 generation in human basophils.

Anaphylatoxin derived from the fifth complement component (C5a) in the presence of IL-3 induces continuous leukotriene C4 generation and IL-4 and IL-13 expression in human basophils for a period of 16-18 h. This indicates that the G protein-coupled C5a receptor (C5aR) can induce long-lasting cellular responses. Using anti-N-terminal C5aR Abs, C-terminal C5a hexapeptide analogs, and pertussis toxin, we demonstrate that the putative activation site of the C5aR is both necessary and sufficient for these late cellular responses. Furthermore, continuous pertussis toxin-sensitive G protein-coupled receptor activation and receptor-ligand interaction is ongoing and required during the entire period of product release. However, the late basophil responses have a more stringent requirement for optimal receptor activation. Leukotriene C4 generation appears to be influenced mostly by the way the receptor is activated, because the most active hexapeptide is a superagonist for this response. By contrast, C5adesarg, lacking the C-terminal arginine, induces minimal lipid mediator formation but is fully active to induce IL-4 production and is even a superagonist for IL-13 release. Nevertheless, IL-4/IL-13 synthesis in response to C5adesarg could be blocked by both C-terminal antagonistic peptide as well as anti-N-terminal C5aR Abs, indicating only minor differences of ligand-receptor interactions between C5a and C5adesarg. Taken together, our data demonstrate that long-lasting and continuous signaling occurs through a limited activation domain of the C5aR, which can differentially promote separate basophil functions.

Sulfidoleukotriene generation from peripheral blood leukocytes of horses affected with insect bite dermal hypersensitivity.

Sulfidoleukotrienes (sLT) generated in vitro after incubation of equine peripheral blood leukocytes (PBL) with different inducing agents were determined in 18 healthy and 16 insect bite dermal hypersensitivity (IDH)-affected horses. PBL from these 32 horses were stimulated with Concanavalin A, Parascaris equorum, Culicoides nubeculosus and Simulium extracts, and with a six-Grass mix. The cells of all but four horses generated sLT after incubation with Concanavalin A; these four horses did also not produce sLT with the other inducing agents. Of the 28 remaining horses (12 affected with IDH and 16 healthy), all but three generated sLT with the P. equorum extract. The six-Grass mix did not induce sLT production in any of the tested horses. sLT generation with Concanavalin A and Parascaris was statistically not different between IDH-affected and healthy horses. PBL of the diseased horses, however, produced significantly more sLT with the Culicoides (p < 0.01) and Simulium (p < 0.05) extracts than those of the healthy animals. Additionally, sLT generation with the Culicoides extract was measured at different times of the year in one IDH-affected animal and remained high even in winter, when the horse was asymptomatic. sLT and histamine release were determined in 10 horses in parallel. Positive correlations of 0.81 and 0.82 for Concanavalin A and Parascaris (p < 0.01 and p < 0.05, respectively), and of 0.95 and 0.94 for Culicoides and Simulium (p < 0.01) were found between sLT and histamine release. These results indicate that, alike in humans, sLT are released in vitro from equine basophils along with histamine in response to various stimuli and that immediate type hypersensitivity reactions to Culicoides and Simulium are often involved in the pathogenesis of IDH. Thus, sLT generation from equine basophils offers an in vitro diagnostic tool for IDH even in sensitised but asymptomatic horses.

Enhanced production of monocyte chemotactic protein 3 in inflammatory bowel disease mucosa.

BACKGROUND: The beta chemokine monocyte chemotactic protein 3 (MCP-3) has chemoattractant and activating capabilities in monocytes, lymphocytes, eosinophils, and basophils. AIMS: To investigate MCP-3 expression in inflammatory conditions of the human intestinal mucosa. PATIENTS: Forty five colon biopsy specimens from 18 patients with inflammatory bowel disease (IBD; 16 specimens from inflamed and 10 from non-inflamed areas) and 19 control patients were examined. METHODS: Immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) were used for MCP-3 detection in tissue sections. Intestinal epithelial cell lines (HT-29, Caco-2, T-84) were stimulated with interleukin (IL) 1beta, IL-6, and tumour necrosis factor alpha (TNF-alpha) and examined for MCP-3 protein and mRNA expression using immunocytochemistry and RT-PCR, respectively. RESULTS: In tissue sections, MCP-3 protein was detected predominantly in epithelial cells, both in patients with IBD and in controls. MCP-3 staining was particularly pronounced at sites of active mucosal inflammation. The intensity of MCP-3 staining was positively correlated with the extent of epithelial destruction. In intestinal epithelial cell lines, MCP-3 mRNA was expressed, whereas MCP-3 protein was not consistently detected. CONCLUSIONS: Our data show that MCP-3 protein is present in normal and inflamed intestinal tissue. MCP-3 production is substantially enhanced in areas of active inflammation, suggesting an immunoregulatory role of MCP-3 in intestinal inflammation.

Regulation of cytokine expression and leukotriene formation in human basophils by growth factors, chemokines and chemotactic agonists.

Basophils stimulated with IL-3 plus C5a selectively express IL-4 and IL-13 and continuously produce leukotrienes (LT) for hours. C5a combined with IL-5 or granulocyte-macrophage colony-stimulated factor was, however, much less effective in promoting cytokine expression and a late continuous phase of LTC4 production, possibly due to lower expression levels of their receptor alpha chains. Basophils also express several chemoattractant receptors, including high levels of C5a receptors, macrophage chemotactic protein (MCP) receptors (CCR2) and eotaxin receptors (CCR3), intermediate levels of CXCR1, CXCR2 and platelet-activating factor receptors, and lower levels of N-formyl-Met-Leu-Phe (fMLP) receptors. However, among the corresponding agonists, only C5a, fMLP and much more weakly MCP1, were found to induce cytokine expression and continuous LTC4 release, and only when combined with IL-3. CCR3, which is highly expressed on basophils and has been shown to mediate strong migratory but weak release responses, does not regulate cytokine expression. The weakly expressed fMLP receptor is an efficient activator of several cell functions including LTC4 formation, while CXCR2 hardly affects basophil function despite considerable expression. Thus, chemoattractant-receptors mediate different cellular responses unrelated to their expression levels.

Circadian elevation of IL-6 levels in Muckle-Wells syndrome: a disorder of the neuro-immune axis?

Muckle-Wells syndrome (MWS) is a rare autosomal dominant hereditary disorder characterized by chronic recurrent urticaria, arthralgia, sensorineural deafness, and in some cases nephropathy due to amyloidosis (AA type). We report a 21-year-old woman and her father, both suffering from this syndrome, in whom elevated serum levels of IL-6 could be documented during the flares of urticaria, and discuss the relevance of this finding for MWS.

High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines.

Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues.

Eotaxin-2, a novel CC chemokine that is selective for the chemokine receptor CCR3, and acts like eotaxin on human eosinophil and basophil leukocytes.

A novel human CC chemokine consisting of 78 amino acids and having a molecular mass of 8,778.3 daltons (VVIPSPCCMF FVSKRIPENR VVSYQLSSRS TCLKAGVIFT TKKGQQ SCGD PKQEWVQRYM KNLDAKQKKA SPRARAVA) was isolated together with three minor COOH-terminally truncated variants with 73, 75, and 76 residues. The new chemokine was termed eotaxin-2 because it is functionally very similar to eotaxin. In terms of structure, however, eotaxin and eotaxin-2 are rather distant, they share only 39% identical amino acids and differ almost completely in the NH2-terminal region. Eotaxin-2 induced chemotaxis of eosinophils as well as basophils, with a typically bimodal concentration dependence, and the release of histamine and leukotriene C4 from basophils that had been primed with IL-3. In all assays, eotaxin-2 had the same efficacy as eotaxin, but was somewhat less potent. The migration and the release responses were abrogated in the presence of a monoclonal antibody that selectively blocks the eotaxin receptor, CCR3, indicating that eotaxin-2, like eotaxin, acts exclusively via CCR3. Receptor usage was also studied in desensitization experiments by measuring [Ca2+]i changes in eosinophils. Complete cross-desensitization was observed between eotaxin-2, eotaxin and MCP-4 confirming activation via CCR3. No Ca2+ mobilization was obtained in neutrophils, monocytes and lymphocytes, in agreement with the lack of chemotactic responsiveness. Intradermal injection of eotaxin-2 in a rhesus monkey (100 or 1,000 pmol per site) induced a marked local infiltration of eosinophils, which was most pronounced in the vicinity of postcapillary venules and was comparable to the effect of eotaxin.

Regulation of cytokine expression by human blood basophils.

IL-4 and IL-13 are key immunoregulatory cytokines because of their ability to induce and amplify Th2-type immune responses and by promoting IgE formation. The basophil is a particularly prominent source of IL-4/IL-13, which are rapidly produced upon Fc epsilonRI cross-linking. Cytokine expression by basophils is unique and distinct from other cell types, since: (1) Basophils are the only cell type constitutively expressing IL-4 and IL-13 mRNA. IL-4/IL-13 message and protein are expressed in a very restricted manner, since neither the mRNAs nor the protein products of most proinflammatory Th1-type, and even Th2-type, cytokines or chemokines are expressed; (2) Basophils secrete IL-4/IL-13 also upon IgE-independent activation (IL-3 plus C5a), and they are thereby potentially capable of initiating a Th2 response. Furthermore, we identified an adjuvant from helminths capable of directly inducing IL-4 formation, and (3) IL-4 expression by basophils is resistant to counter-regulatory effectors inhibiting Th2 development. Studies about the regulation of IgE-dependent and -independent IL-4/IL-13 expression by different cytokines, growth factors and chemokines demonstrate that the different basophil effector functions such as chemotaxis, exocytosis, leukotriene C4 formation and cytokine expression are regulated separately. Thus, our study supports a key immunoregulatory role of basophils in the skewing of immune responses to Th2.

Basophil priming by neurotrophic factors. Activation through the trk receptor.

There is increasing evidence that nerve growth factor (NGF) acts on cells of the immune system, apart from its neurotrophic effects. In human basophils, NGF potentiates mediator release and primes the cells to produce leukotriene C4 in response to C5a. It is, however, unknown whether other homologous neurotrophins also act outside the nervous system, and whether activation of basophils by NGF requires interaction with trk tyrosine kinase receptors, the low affinity NGF receptor (LNGFR), or both. A triple mutant NGF designed to interrupt binding to the LNGFR was found to activate basophils with equal efficacy as wild-type NGF, demonstrating that the LNGFR is not necessary. Despite a 10 times lower potency of mutant NGF, no LNGFR 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 NGF), while neurotrophin-3, which is thought to interact with trkC, trkB, and more weakly with trk, induced a threshold effect at 300 ng/ml (approximately 10,000-fold lower potency), demonstrating that 1) the LNGFR 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 LNGFR, and that, among the neurotrophin family, NGF is unique in priming basophils.

Human blood basophils produce interleukin-13 in response to IgE-receptor-dependent and -independent activation.

Interleukin-13 (IL-13) is a recently discovered immunoregulatory cytokine. The cellular sources of IL-13 and the regulation of its expression are largely unknown. Here we show that human basophils produce IL-13 in response to IgE-receptor (IgER) crosslinking, IL-3, IL-3 plus C5a, but not C5a alone. Human basophils express IL-13 in a restricted manner since, apart from IL-4, no other cytokines encoded on the cytokine gene cluster (IL-3, IL-5, and granulocyte macrophage-colony-stimulating factor [GM-CSF]), are induced. Highest levels of IL-13 are formed after IgE-independent activation leading to a prolonged secretion of IL-13. The response to IgER-cross-linking is more transient preferentially inducing IL-4, IL-3 is a unique cytokine regulating IL-13 production by human basophils: Among a large number of cytokines tested, only IL-3 is capable of directly inducing IL-13 expression. Furthermore, although some IL-13 is produced in response to C5a in the presence of IL-5, GM-CSF, IGF-1 or IL-1 beta, IL-3 is by far the most effective. IL-13 production was blocked by actinomycin D and cycloheximide and conditions leading to IL-13 release also lead to the induction of IL-13 mRNA. This study supports an important immunoregulatory role of human blood basophils, owing to their capacity to simultaneously express IL-13 and IL-4 in a restricted manner.

Basophil histamine release and leukotriene production in response to anti-IgE and anti-IgE receptor antibodies. Comparison of normal subjects and patients with urticaria, atopic dermatitis or bronchial asthma.

The IgE receptor-dependent in vitro mediator release in basophils is characterized by a large interindividual variability both in normal and atopic subjects. The mechanism and the clinical impact of this finding, however, is largely unclear. The aim of the present study was to examine the role of surface-bound IgE and of response-modifying cytokines such as interleukin 3 (IL-3) as possible factors determining basophil releasability in atopic patients and normal controls. Cells from 30 individuals (6 with urticaria, 7 with asthma, 7 with atopic dermatitis, and 30 healthy controls) were isolated and stimulated for mediator release by IL-3 and different triggering antibodies directed against IgE or IgE receptor. Our data suggest that serum IgE levels and basophil receptor occupancy with IgE are not involved in the mechanism of basophil releasability. Furthermore, IL-3-induced similar effects on mediator release in almost all individuals, rather excluding the possibility that releasability is regulated by cytokine priming of basophils. Interestingly, we found that patients with atopic disease have a reduced capacity of releasing mediators upon activation, the mechanism of which is unclear. In conclusion, our findings support the hypothesis that basophil releasability is dependent on cell-imminent mechanisms in basophils, which may be altered in selected atopic patients.