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.

Frequent sensitization to Candida albicans and profilins in adult eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is often associated with atopic airway and skin diseases. More than 80% of EoE patients are sensitized to aero- and/or food allergens. Immunoglobulin (Ig)E-mediated immune responses to microbes have been reported to be deleterious in connection with atopic diseases. AIM: The aim of this study was to obtain a comprehensive overview about the sensitization spectrum of adult EoE patients. METHODS: IgE in sera of 35 patients with active EoE were analyzed for reactivity to Candida albicans, as well as to a panel of recombinant and purified natural allergen components, using a microarray. RESULTS: IgE sensitization to Candida albicans was found in 43% of EoE patients. More than 80% of EoE patients were sensitized to aeroallergens and 22% to food-specific allergen components, whereas 69% of the patients exhibited specific IgE to cross-reactive allergens. Among the latter, profilins were identified as most frequent IgE cross-reactive allergen components. Interestingly, dysphagia, the main symptom of adult EoE patients following rice and/or bread ingestion, was associated with sensitization to cross-reactive allergens such as profilins, pathogenesis-related (PR) 10 and lipid transfer proteins (LTP). Intolerance toward meat rarely correlated with sensitization to animal food allergens. CONCLUSION: Candida albicans and cross-reactive plant allergen components, in particular profilins, were identified as frequent sensitizers in adult EoE patients. Specific elimination therapies are suggested to reveal their actual roles in the pathogenesis of EoE.

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.

Human peripheral blood basophils primed by interleukin 3 (IL-3) produce IL-4 in response to immunoglobulin E receptor stimulation.

In contrast to most cytokines, interleukin 4 (IL-4) expression is restricted to T lymphocytes, with the exception of mast cell lines and mast cells, as more recently demonstrated in rodents. Little is known, however, about the capacity of human nonlymphoid cells to produce IL-4. In this study we show that mature human basophils are capable of expressing IL-4 and examine the regulation of IL-4 production in comparison with the lipid mediator leukotriene C4. IL-4 was produced upon immunoglobulin E receptor (IgER) activation of basophils cultured with IL-3, a cytokine previously shown to prime these cells for enhanced release of inflammatory mediators. In some experiments, IL-3 or IgER activation alone also induced IL-4 production close to the detection limit. The effect of IL-3 on IgER-dependent IL-4 expression was dose and time dependent: maximal IL-4 production occurred between 18 and 48 h preexposure of basophils to 3-10 ng/ml IL-3. IgER-induced IL-4 synthesis and release by basophils cultured with IL-3 was rapid and complete after 6 h. In contrast to IL-3, other cytokines (IL-5, granulocyte/macrophage colony-stimulating factor, and nerve growth factor) that also prime basophils for enhanced histamine and leukotriene C4 release did not promote IgER-induced IL-4 synthesis. Basophils appear to secrete a "TH2-like" cytokine profile since no detectable IL-2 or interferon gamma was produced upon IgER activation. Mononuclear cells (depleted of basophils), cultured in parallel, did not release IL-4 in response to IL-3 and/or IgER activation, and produced approximately ten times less IL-4 than basophils upon nonspecific activation by phorbol ester and calcium ionophore. Thus, human basophils are an important cellular source of IL-4, and may, therefore, in addition to their inflammatory effector functions, also regulate the differentiation of T helper cells and B cells, in particular in allergic diseases.

c-kit ligand: a unique potentiator of mediator release by human lung mast cells.

Mast cells (MC) play a central role in extrinsic allergic reactions such as asthma and may participate in other inflammatory and fibrotic processes. However, with the exception of immunoglobulin E (IgE) receptor-dependent stimulation, no secretagogues of human lung MC have yet been described. It is also unclear whether mediator release can be regulated by certain cytokines as demonstrated previously in basophils and other human inflammatory effector cells. Here, we show that the c-kit ligand (KL), a recently identified stem cell growth factor, at concentrations 10-100 times lower than that required to promote cell proliferation, enhances the release of histamine and leukotriene C4 in response to IgE receptor crosslinking of human lung MC. KL does not induce mediator release per se, but increases the sensitivity of MC to anti-IgE receptor stimulation and also enhances mediator release to maximally effective concentrations of anti-IgE receptor antibody. By contrast, a large number of cytokines examined, including the mast cell growth factors/agonists in rodents, interleukin 3 (IL-3), IL-4, IL-9, and nerve growth factor, were ineffective in this respect. These findings suggest a unique role of KL in regulating effector functions of human mucosal MC.

Monocyte chemotactic protein 1 is a potent activator of human basophils.

Cytokines belonging to the RANTES/SIS family are highly induced in a number of pathophysiological processes such as autoimmune disorders, cancers, atherosclerosis, and chronic inflammation. However, apart from their chemotactic activity on monocytes and particular lymphocyte types, the biological activities in the human system of this recently discovered cytokine family are largely unknown. Here we report that one family member, described as monocyte chemotactic protein 1 (MCP-1), strongly activates mature human basophils in a pertussis toxin-sensitive manner. MCP-1 causes a rise in the cytosolic free calcium level in basophils and monocytes, but not in other blood leukocyte types, and triggers basophil degranulation at low concentrations (ED50 = 3-10 nM). Thus, MCP-1 is a cytokine capable of directly inducing histamine release by basophils. Furthermore, MCP-1 promotes the formation of leukotriene C4 by basophils pretreated with interleukin 3 (IL-3), IL-5, or granulocyte/macrophage colony-stimulating factor. MCP-1-induced basophil mediator release may play an important role in allergic inflammation and other pathologies expressing MCP-1.

Interleukin 3-dependent mediator release in basophils triggered by C5a.

The anaphylatoxin C5a is a potent trigger for basophil degranulations, but in contrast to IgE-dependent basophil activation, it does not result in the synthesis of sulfidoleukotrienes (leukotriene C4/D4/E4). Thus, degranulation and the generation of lipid mediators are separately regulated cellular responses. Exposure of human blood basophils to the cytokine IL-3 alone does not induce the release of histamine in cells from most donors and never leads to the generation of LTC4, indicating that IL-3 is not a direct agonist for basophil mediator release. However, preincubation of basophils with IL-3 enhances the degranulation response to C5a. Most importantly, IL-3 "primes" basophils to release large amounts of leukotriene C4 after challenge with C5a (mean of 50 gp LTC4 per nanograms cellular histamine), while neither peptide alone is capable of inducing the formation of bioactive lipids. This effect is dose dependent, occurring at IL-3 concentrations considerably lower than are required to stimulate the growth of bone marrow progenitor cells. IL-3 affects the extent but not the time course of basophil degranulation, and leukotriene release of cells sequentially exposed to IL-3 and C5a occurs very rapidly concomitant with degranulation. A preincubation of the basophils with IL-3 is strictly required for C5a-induced LTC4 synthesis, but not for an enhancement of degranulation. Priming for C5a-induced lipid mediator generation occurs rapidly after exposure of the cells to IL-3, starting at 1 min and reaching maximal effects at 5 min, but this altered state of responsiveness is relatively long lasting. Cell fractionation studies indicate that the basophil is the source of lipid mediators and that IL-3 affects the basophil response directly. This study demonstrates that IL-3 is a potent modifier of effector functions of mature basophils; this is possibly of greater in vivo significance than its growth factor properties. The large amounts of LTC4 formed after triggering of IL-3-primed basophils may not only enhance but also qualitatively change the pathophysiological consequences of complement activation, and this might be important in the pathogenesis of immediate type hypersensitivity reactions, shock syndromes, and inflammation.

Deletion of the NH2-terminal residue converts monocyte chemotactic protein 1 from an activator of basophil mediator release to an eosinophil chemoattractant.

Chemotactic cytokines of the CC subfamily (CC chemokines) are considered as major mediators of allergic inflammation owing their actions on basophil and eosinophil leukocytes. The monocyte chemotactic protein (MCP) 1 is a potent inducer of mediator release from basophils but is inactive on eosinophils. To obtain information on the structural determinants of the activities of MCP-1, we have synthesized several NH2-terminally truncated analogues and tested their effects on basophils and eosinophils. Through deletion of the NH2-terminal residue, MCP-1(2-76) was obtained, which was a potent activator of eosinophils, as assessed by chemotaxis, cytosolic free Ca2+ changes, actin polymerization, and that induction of the respiratory burst. In contrast, the activity of MCP-1(2-76) on basophil leukocytes was dramatically decreased (50-fold) compared with that of full-length MCP-1. Deletion of the next residue led to total loss of activity on eosinophil and basophil leukocytes. Analogues with three or four residue deletions, MCP-1(4-76) and MCP-1(5-76), were again active on both cells, whereas all further truncation analogues, MCP-1(6-76) through MCP-1(10-76), were inactive. Thus, a minimal structural modification can change receptor and target cell selectivity of MCP-1. Our observations indicate that the recognition sites of CC chemokine receptors on eosinophils and basophils are similar, although they discriminate between MCP-1 and MCP-1(2-76) and suggest NH2-terminal processing as a potential mechanism for the regulation of CC chemokine activities.

The neutrophil-activating peptide NAF/NAP-1 induces histamine and leukotriene release by interleukin 3-primed basophils.

IgE-independent mediator release from basophils is considered an important event in inflammation, particularly in nonallergic immediate hypersensitivity and in allergic late-phase reactions. This study demonstrates that after exposure to IL-3, basophils release histamine and leukotrienes in response to the neutrophil-activating peptide NAF/NAP-1. Thus, the sequential action of two pure cytokines can promote basophils mediator release. In the presence of IL-3, NAF/NAP-1 functions like a "histamine-releasing factor" and may therefore not only induce cellular infiltration but also provoke symptoms of hypersensitivity reactions.

RANTES and macrophage inflammatory protein 1 alpha induce the migration and activation of normal human eosinophil granulocytes.

The cellular infiltrates of certain inflammatory processes found in parasitic infection or in allergic diseases consist predominantly of eosinophilic granulocytes, often in association with activated T cells. This suggests the existence of chemotactic agonists specific for eosinophils and lymphocyte subsets devoid of neutrophil-activating properties. We therefore examined four members of the intercrine/chemokine superfamily of cytokines (monocyte chemotactic peptide 1 [MCP-1], RANTES, macrophage inflammatory protein 1 alpha [MIP-1 alpha], and MIP-1 beta), which do not activate neutrophils, for their ability to affect different eosinophil effector functions. RANTES strongly attracted normal human eosinophils by a chemotactic rather than a chemokinetic mechanism with a similar efficacy as the most potent chemotactic myeloid cell agonist, C5a. MIP-1 alpha also induced eosinophil migration, however, with lower efficacy. RANTES and MIP-1 alpha induced eosinophil cationic protein release in cytochalasin B-treated eosinophils, but did not promote leukotriene C4 formation by eosinophils, even after preincubation with interleukin 3 (IL-3), in contrast to other chemotactic agonists such as C5a and formyl-methionyl-leucyl-phenylalanine (FMLP). RANTES, but not MIP-1 alpha, induced a biphasic chemiluminescence response, however, of lower magnitude than C5a. RANTES and MIP-1 alpha both promoted identical transient changes in intracellular free calcium concentration ([Ca2+]i), with kinetics similar to those induced by chemotactic peptides known to interact with G protein-coupled receptors. No cross-desensitization towards other peptide agonists (e.g., C5a, IL-8, FMLP) was observed, suggesting the presence of specific receptors. Despite its weaker eosinophil-activating properties, MIP-1 alpha was at least 10 times more potent on a molar basis than RANTES at inducing [Ca2+]i changes. Interestingly, RANTES deactivated the MIP-1 alpha-induced [Ca2+]i changes, while the RANTES response was preserved after MIP-1 alpha stimulation. MCP-1, a potent monocyte chemoattractant and basophil agonist, as well as MIP-1 beta, a peptide with pronounced homology to MIP-1 alpha, did not activate the eosinophil functions tested. Our results indicate that RANTES and MIP-1 alpha are crucial mediators of inflammatory processes in which eosinophils predominate.

Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals.

Neutrophils (PMN) preincubated with recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) for 2 h and then stimulated with the chemotactic factors, C5a or FMLP, produce substantial amounts of the lipoxygenase products 5-Hete, LTB4, and omega-oxidised LTB4 metabolites (4.36 +/- 0.95 (SEM) pM (n = 21) LTB4 and LTB4 metabolites/10(6) PMN). No lipoxygenase metabolites are detected by HPLC and RIA if purified PMN are stimulated by either GM-CSF or chemotactic factors in the absence of exogenous arachidonate. The priming effect of GM-CSF upon chemotactic factor induced generation of lipid mediators is a relatively slow process, clearly evident after 1 h and optimal after 2 h. Leukotriene generation is measurable with 0.8 U GM-CSF/10(6) PMN and is maximal with 80 U (10(-11)-10(-9) M). Upon activation of primed PMN with chemotactic factors, leukotriene synthesis is induced very rapidly. Already 2.5 min after activation the major lipoxygenase metabolites present are 20-OH LTB4 and 20-COOH LTB4. Our study shows that the synthesis of lipoxygenase metabolites from endogeneous AA can be initiated in PMN through receptor mediated processes by the appropriately timed combination of biological soluble inflammatory mediator peptides. Furthermore, these results indicate that GM-CSF not only enhances effector cell functions but can qualitatively change the mediator profile formed after activation with a second triggering signal. Such a mechanism might be important in amplifying inflammatory responses. Alternatively, lipid mediators formed might also have an intracellular or autocoid role and be responsible for the enhancement of other PMN functions like oxygen radical release.

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.

Interleukin 5 modifies histamine release and leukotriene generation by human basophils in response to diverse agonists.

Human interleukin 5 (IL-5), known as a selective colony-stimulating factor of the eosinophil lineage and activator of mature eosinophils, also profoundly influences the mediator release profile of human basophils. IL-5 by itself triggers neither granule release nor de novo synthesis of lipid mediators. However, at low concentrations (0.1-10 ng/ml), IL-5 rapidly primes basophils for enhanced histamine release and leukotriene C4 (LTC4) generation in response to all established basophil agonists. LTC4 generation is more strongly affected by IL-5 than histamine release. In particular, IL-5 renders basophils capable of producing large quantities of LTC4 in response to C5a, which, without the cytokine, induces histamine release only. Finally, IL-5 renders basophils responsive to agonists (neutrophil-activating peptide 1 and C3a), which are otherwise inefficient triggers for basophil mediator release. The effects are similar to the recently established bioactivity of IL-3 on basophils, with the exception of its influence on IgE-dependent basophil activation, which is less pronounced. Thus, IL-5 strongly modulates the function not only of eosinophils but also of basophils, the two major effector leukocyte types involved in allergic inflammatory processes, e.g., in asthma.

Monocyte chemotactic protein 3 is a most effective basophil- and eosinophil-activating chemokine.

CC chemokines constitute a novel class of cytokines that attract and activate monocytes and lymphocytes, as well as basophil and eosinophil leukocytes, with distinct target cell profiles, and are believed to be involved in the regulation of different types of inflammation. The action of the recently identified monocyte chemotactic protein 3 (MCP-3) on human basophil and eosinophil function was studied and compared with that of other CC chemokines. In basophils, MCP-3, MCP-1, RANTES, and macrophage inflammatory protein (MIP)-1 alpha all induced cytosolic-free calcium concentration ([Ca2+]i) changes and, with different efficacies, chemotaxis (RANTES = MCP-3 >> MCP-1 > MIP-1 alpha), histamine release (MCP-1 = MCP-3 >> RANTES > MIP-1 alpha), and leukotriene C4 formation, after IL-3 pretreatment (MCP-1 = MCP-3 >> RANTES > MIP-1 alpha). Thus, MCP-3 was as effective as MCP-1 as an inducer of mediator release, and as effective as RANTES as a stimulus of basophil migration. In contrast to MCP-1, MCP-3 was also a stimulus for eosinophils, and induced [Ca2+]i changes and chemotaxis as effectively as RANTES, which is the most potent chemotactic cytokine for these cells. Desensitization of the transient changes in [Ca2+]i was used to assess receptor usage. In basophils, stimulation with MCP-3 prevented responsiveness to MCP-1 and RANTES, but not to MIP-1 alpha. No single CC chemokine (except for MCP-3 itself) affected the response to MCP-3, however, which was prevented only when the cells were prestimulated with both MCP-1 and RANTES. In eosinophils, by contrast, cross-desensitization between RANTES and MCP-3 was obtained. RANTES and to a lesser extent MCP-3 also desensitized eosinophils toward MIP-1 alpha. The desensitization data suggest the existence of three chemokine receptors: (a) a MCP-1 receptor expressed on basophils but not eosinophils that is activated by MCP-1 and MCP-3; (b) a RANTES receptor in basophils and eosinophils that is activated by RANTES and MCP-3; and (c) a MIP-1 alpha receptor that is activated by MIP-1 alpha, RANTES and, more weakly, by MCP-3. This study shows that MCP-3 combines the properties of RANTES, a powerful chemoattractant, and MCP-1, a highly effective stimulus of mediator release, and thus has a particularly broad range of activities toward both human basophil and eosinophil leukocytes.

Receptor-directed inhibition of chemotactic factor-induced neutrophil hyperactivity by pyrazolon derivatives. Definition of a chemotactic peptide antagonist.

The two pyrazolon derivatives, phenylbutazone and sulfinpyrazone, selectively inhibit chemotactic peptide-induced effects on neutrophils. As they antagonize the induction of acute neutropenia in vivo and of cellular hyperadhesiveness, lysosomal enzyme release, hexose monophosphate shunt activity, and superoxide production in vitro, these effects occur with a specificity not shared with other prostaglandin biosynthesis inhibition by these drugs resembles the competitive type of antagonism and occurs at concentrations attainable in vivo under clinical conditions. The locomotory machinery, the direction-finding mechanisms, and the basic metabolic machinery of the cell are unaffected. These drugs interfere with specific binding of the formylpeptide to its receptor on neutrophils.

Modulating influence of chemotactic factor-induced cell adhesiveness on granulocyte function.

The importance of adhesion in regulating locomotion and accumulation of polymorphonuclear leukocytes (PMN) has remained vague. We found that the chemotaxis of human PMN resuspended in heat-inactivated plasma was maximal toward 1-10 nM N-formyl-met-leu-phe (f-Met-Leu-Phe), but fell below random motility toward >/= 100 nM. This impressive decrease of motility was paralleled by increased cell adherence on Petri dishes being minimal at 1 nM and maximal at >10 nM f-Met-Leu-Phe (6+/-1 and 37+/-2% [SE] adherent cells, respectively). Checked by phase-contrast microscopy, cells under stimulated adhesion lost the typical bipolar shape of moving PMN and became immobilized and highly flattened. PMN, preexposed to 250 nM f-Met-Leu-Phe and tested after washing, retained increased adhesiveness and showed extremely low random and chemotactic motility. In contrast, preexposure to 1 nM f-Met-Leu-Phe had no effect on chemotaxis. Supporting the concept that immobilizing hyperadhesiveness does not correspond to a general functional hyporesponsiveness of PMN, no depression of the initial ingestion rate was observed in the presence of 250 nM f-Met-Leu-Phe. Moreover, a close correlation was found between the induction of PMN adhesiveness and the stimulation of the hexose monophosphate pathway activity as well as of lysomal enzyme release (r >/= 0.98). Thus, "chemotactic deactivation" and "high-dose inhibition of chemotaxis" by N-formyl peptides is the consequence of increased cell adhesiveness. This phenomenon provides a mechanism for cell trapping at the inflammatory site. Conversely, if operative in circulating blood, e.g., in septicemia, it may impair PMN emigration to such sites.

Role of cell surface contact in the kinetics of superoxide production by granulocytes.

The complement-derived anaphylatoxin C5a and a putative analogue of bacterial chemotactic factor (N-formyl-methionyl-leucyl-phenylalanyl [fMLP]), as well as bacterial lipid A, all stimulate human granulocyte (PMN) adhesiveness and superoxide (O-2) production in a concentration-dependent manner. Since attachment of particulate matter to the PMN membrane is an early event in the triggering of respiratory burst of these cells, we further examined how adherence might modulate the release of O-2 induced by soluble mediators of inflammation. We found that both the quantity and kinetics of O-2 production depend on prior attachment of the cells to a surface. In stirred suspensions of PMN, fMLP induces only a short burst (2.5 min) of O-2 release associated with reversible PMN aggregation. The magnitude, but not the time course, of both these responses depend on the fMLP concentration. Unlike the short respiratory response of cells in suspension, PMN allowed to settle onto stationary petri dishes, then overlaid with fMLP, rapidly spread and attach to the surface where they remain and release O-2 throughout the 60-min test period. Prolonged O-2 release also follows fMLP stimulation in suspensions of PMN pretreated with cytochalasin B, in which case aggregation becomes irreversible during the 20-min burst. If fMLP is slowly infused into a suspension of cells at 37 degrees C or if PMN are challenged at 0 degrees C, and then warmed to 37 degrees C, O-2 release greatly decreases or becomes undetectable. Suspended PMN do not respond to a second challenge by the same stimulus regardless of the rate or temperature at which the first stimulus was added, a phenomenon formerly described as desensitization. However, if PMN challenged with fMLP in suspension undergo the short respiratory response and then are later placed in petri dishes, they adhere and resume production of O-2 without further stimulation. Chemotactic factor-induced adherence and O-2 release of PMN on a surface is entirely independent of either the mode of activation or prior O-2 release during preincubation in suspension. Human C5a also promotes PMN adherence and prolonged O-2 release in petri dishes. Furthermore, lipid A increases O-2 release and adherence of settled PMN, but fails to elicit either response from suspended PMN. These results indicate that cell surface contact plays an essential role in triggering the respiratory burst of PMN activated by soluble stimuli. This long-lasting O-2 release by chemotactic factor-stimulated PMN may play a significant role in inflammatory reactions when PMN become adherent in vivo.