Eosinophil degranulation. An immunologic determinant in the pathogenesis of the Mazzotti reaction in human onchocerciasis

Onchocerciasis patients treated with diethylcarbamazine often undergo a severe inflammatory response, the Mazzotti reaction. To assess the eosinophil's role in the pathogenesis of the Mazzotti reaction, we obtained serial blood, plasma, and skin biopsy specimens from 21 heavily infected patients and 3 endemic controls, both before and during therapy with diethylcarbamazine. Samples were analyzed for blood eosinophils, plasma levels of eosinophil granule major basic protein (MBP) and eosinophil-derived neurotoxin, eosinophil infiltration and eosinophil and mast cell degranulation in the skin. After the first dose of diethylcarbamazine, blood eosinophils fell from a pre-treatment level of 888 +/- 111 to 203 +/- 42 cells/mm3 at 8 h. This decrease was followed by a marked eosinophilia developing over the remaining 7 days of treatment and 14 days of follow-up. Plasma eosinophil-derived neurotoxin levels increased from 56 +/- 4 ng/ml pretreatment to a peak of 82 +/- 9 ng/ml at 8 h and returned to pretreatment levels by 48 h. Beginning at 12 h, plasma MBP levels increased from 730 +/- 74 ng/ml pretreatment to a peak of 1140 +/- 74 ng/ml after 5 days. Pretreatment skin biopsies stained for MBP by immunofluorescence showed a bright fibrillar pattern in the dermis consistent with chronic eosinophil degranulation; the MBP was localized on elastic tissue fibers. After treatment, skin biopsy specimens showed both the pretreatment fibrillar MBP staining pattern as well as focal eosinophil degranulation. Deposition of MBP around microfilariae in the papillary dermis was visible as early as 1.5 h. The lowest blood eosinophil levels and peak plasma eosinophil-derived neurotoxin levels coincided with the infiltration and degranulation of eosinophils in the skin. Mast cell degranulation in the skin was maximal by the first posttreatment biopsy (1.5 h) coincident with the beginning of eosinophil degranulation. Although the pathogenesis of the Mazzotti reaction is clearly complex, our results indicate that eosinophil degranulation is characteristic of the response and that it occurs with a time course suggestive of a role for the eosinophil in determining the clinical and pathologic manifestations of the reaction.     

Localization of the guinea pig eosinophil major basic protein to the core of the granule

The localization of the guinea pig eosinophil major basic protein (MBP) within the cell was investigated by the use of immunoelectron microscopy and by isolation of the granule crystalloids. First, by immunoperoxidase electron microscopy, we found that the MBP of eosinophil granules is contained within the crystalloid core of the granule. Specific staining of cores was present when rabbit antiserum to MBP was used as the first stage antibody in a double antibody staining procedure, whereas staining was not seen when normal rabbit serum was used as the first stage antibody. Second, crystalloids were isolated from eosinophil granules by disruption in 0.1% Triton X-100 and centrifugation through a cushion of 50% sucrose. Highly purified core preparations yielded essentially a single band when analyzed by electrophoresis on polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). The E1%1cm of the core protein was 26.8 +/- 1.0 (X +/- SEM); the E1%1cm for the MBP was 26.3. The core protein could not be distinguished from the MBP by radioimmunoassay (RIA) and essentially all of the protein in the core preparations could be accounted for as MBP. The results indicate that the MBP is contained in the core of the guinea pig eosinophil granule and that it is probably the only protein present in the core.     

Role of inflammatory cells in Chagas' disease. III. Kinetics of human eosinophil activation upon interaction with parasites (Trypanosoma cruzi)

The kinetics of human eosinophil activation and granule secretion initiated by interaction with Trypanosoma cruzi amastigotes was studied by using a monoclonal IgG1 antibody (termed EG2) that is specific for an epitope present only in the secreted forms of both eosinophil cationic protein (ECP) and the eosinophil protein X (EP-X), and hence not detectable in unstimulated resting eosinophils. Studies were carried out by using electron microscopy and indirect immunofluorescence. In the electron microscopy studies, deposits of protein A-gold particles in parasite-containing eosinophils that had been incubated previously with EG2 antibody were first detected 4 hr after initiation of the eosinophil-amastigote interaction. Control tests performed with a monoclonal IgG1 unreactive with eosinophils showed no deposition of protein A-gold particles. EG2 antibody binding was confined to the crystalloid granule matrix, where ECP and EP-X are known to be stored. A similar kinetic pattern of ECP/EP-X solubilization and secretion was confirmed by the results of the indirect immunofluorescence experiments also showing the binding of EG2 antibody after 4 hr of cell-parasite interaction. The kinetics of ECP/EP-X solubilization and secretion paralleled the kinetics of destruction of internalized amastigotes, suggesting a role for these basic proteins in parasite killing. Consistent with this notion was the detection of ECP/EP-X in the fluid of phagocytic vacuoles containing amastigotes and associated with the ingested organisms at the same time as the parasites began to show structural alterations. These results outlined the kinetics of eosinophil activation in terms of the time required for mobilization of two basic proteins associated with eosinophil secretion that are known to be biologically active.     

Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microscopic technique

An immunoelectron microscopic technique using protein A-gold as a specific marker was used for precise intracellular localization of eosinophil granule proteins. Eosinophils from healthy individuals were isolated in metrizamide gradients. Eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were clearly located in the matrix of the large crystalloid-containing granules. In addition, ECP was probably present in the small granules of eosinophils. Major basic protein (MBP) was present in the crystalloid structure of specific granules. This method can be applied in studies of eosinophil degranulation to trace the release of biological effector molecules.     

Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells

Human eosinophils contain several distinctive proteins including eosinophil granule MBP and the membrane-associated CLC protein (lysophospholipase). Human basophils also contain these proteins, indicating biochemical similarities between eosinophils and basophils. To determine whether MBP or CLC protein is present in connective tissue mast cells, we studied human lung and cutaneous mast cells by immunofluorescence by utilizing specific antibodies to CLC and MBP. Cytocentrifuge slides of enriched lung mast cells and mast cells in sections of formalin-fixed, paraffin-embedded cutaneous tissue from urticaria pigmentosa lesions were stained for CLC and MBP. Neither pulmonary nor cutaneous mast cells stained for CLC protein or MBP. In contrast, lung and cutaneous eosinophils in the same preparations showed bright staining for both proteins. The failure to find CLC protein and MBP in mast cells provides additional evidence of dissimilarity between mast cells and basophils, and an immunochemical means to distinguish between them.     

Extracellular killing of Trypanosoma cruzi amastigotes by human eosinophils

Granules released from human eosinophils upon interaction with Trypanosoma cruzi amastigotes in vitro were seen attached to the surface of non-internalized parasites by electron microscopy. Amastigote damage was preceded by the binding of eosinophil granule material to its membrane, and eosinophil granule major basic protein (MBP) bound to the parasite surface was readily detectable. Additional evidence of eosinophil cytotoxicity for extracellular amastigotes was the observation that amastigotes trapped between two eosinophils, without being ingested by either one, were destroyed at the interface. Amastigotes isolated from the spleens of infected mice or grown in culture were similarly sensitive to the lytic effects of purified MBP. These results demonstrate the ability of human eosinophils to lyse T. cruzi amastigotes extracellularly in the absence of antibody and suggest that MBP may be involved in the effect. Thus, eosinophils, known to be capable of destroying phagocytosed amastigotes, could also contribute to the clearance of these parasites through extracellular killing.     

Extracellular Killing of Trypanosoma cruzi Amastigotes by Human Eosinophils1

Granules released from human eosinophils upon interaction with Trypanosoma cruzi amastigotes in vitro were seen attached to the surface of non-internalized parasites by electron microscopy. Amastigote damage was preceded by the binding of eosinophil granule material to its membrane, and eosinophil granule major basic protein (MBP) bound to the parasite surface was readily detectable. Additional evidence of eosinophil cytotoxicity for extracellular amastigotes was the observation that amastigotes trapped between two eosinophils, without being ingested by either one, were destroyed at the interface. Amastigotes isolated from the spleens of infected mice or grown in culture were similarly sensitive to the lytic effects of purified MBP. These results demonstrate the ability of human eosinophils to lyse T. cruzi amastigotes extracellularly in the absence of antibody and suggest that MBP may be involved in the effect. Thus, eosinophils, known to be capable of destroying phagocytosed amastigotes, could also contribute to the clearance of these parasites through extracellular killing.     

Intracellular expression and release of Fc epsilon RI alpha by human eosinophils.

Although Fc epsilon R have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, Fc epsilon RI, and Fc epsilon RII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance Fc epsilon R on other cells) failed to induce any detectable surface Fc epsilon R. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for Fc epsilon RI alpha showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR gamma-chain, but no Fc epsilon RI beta. Surface biotinylation followed by immunoprecipitation again failed to detect surface Fc epsilon RI alpha, although surface FcR gamma was easily detected. Since we were able to detect intracellular Fc epsilon RI alpha, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of Fc epsilon RI alpha into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of Fc epsilon RI alpha that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of Fc epsilon RI alpha remains to be determined.     

In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.     

Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.     

Stimulation of basophil and rat mast cell histamine release by eosinophil granule-derived cationic proteins

Major basic protein (MBP), an arginine-rich basic polypeptide that constitutes the crystalloid core of the large specific eosinophil granule, has previously been shown to stimulate noncytolytic histamine release from human basophils and rat mast cells by an IgE-independent mechanism. Two additional basic polypeptides present in eosinophil granules, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), were examined for similar activity in the present study. Acid-solubilized eosinophil granules were fractionated by chromatography on a Sephadex G-50 column. Incubation of basophil-containing human mononuclear cells with the individual column fractions demonstrated that histamine release occurred only with the fractions that contained MBP. The selectivity of the basophil response for MBP was confirmed by using equimolar concentrations of purified MBP, ECP, and EDN. In contrast, both MBP and ECP, but not EDN, stimulated histamine release from purified rat peritoneal mast cells. Reduction and alkylation of the MBP molecule diminished the response of human basophils to MBP but enhanced the potency of the molecule with rat mast cells. The distinct potency of MBP as a stimulus for histamine secretion from human basophils suggests that eosinophil release of MBP may be a specific event in the augmentation of immediate hypersensitivity reactions and other disorders characterized by eosinophilia.     

Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein

Eosinophil granule major basic protein (MBP) is a relatively low molecular weight cationic (pI greater than 10) protein present in the crystalloid core of the eosinophil granule. Amino acid sequence analysis of this protein was undertaken as part of an analysis of the structural basis of the potent cytotoxic activities of MBP on parasites and mammalian cells. Many conventional sequencing strategies were unworkable because of the unusual amino acid composition of MBP and its insolubility in solutions buffered at neutral pH. Less conventional chemical reactions, including cyanogen bromide-induced cleavage at tryptophan and acid-induced cleavage at aspartic acid, were used successfully to obtain peptides which allowed definition of the amino acid sequence of MBP. Characterization of MBP by reverse-phase high pressure liquid chromatography and two-dimensional gel analysis showed no microheterogeneity that might be attributed to post-translational modifications. Comparison of the MBP sequence with a protein sequence data base showed that MBP has no significant sequence homology with other characterized proteins. The basicity (pI 10.9) and hydrophobicity predicted from the MBP sequence are likely responsible for the observed affinity of this cytotoxic molecule for cell surfaces and some serum proteins.     

Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin

The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.     

Release of granule proteins from eosinophils cultured with IL-5.

Eosinophils isolated from normal individuals were cultured in the presence of human rIL-5 (hrIL-5) for up to 14 days, and the effects of this exposure were determined. First, the hrIL-5-cultured eosinophils were activated and degranulated more readily than freshly isolated eosinophils. For example, eosinophils cultured for 7 days with hrIL-5 released 30 and 10% of granule eosinophil-derived neurotoxin (EDN) when exposed to Sepharose 4B beads coupled to secretory IgA and IgG, respectively, whereas freshly isolated eosinophils released only 19 and 4%, respectively, of their EDN in response to the same stimuli. Degranulation of hrIL-5-cultured eosinophils was not augmented by further exposure to hrIL-5, whereas degranulation of freshly isolated cells to secretory IgA and IgG beads was increased by exposure to hrIL-5. Second, eosinophils cultured with hrIL-5 had prolonged viability in vitro. For example, after four days of culture with 50 U/ml of hrIL-5, 86% of eosinophils were viable compared to 12% in medium alone. Third, hrIL-5-cultured eosinophils became hypodense, and electron microscopy showed that they contained granules with core and matrix lucency and with evidence of granule fusion. Fourth, hrIL-5-cultured eosinophils spontaneously lost 30 to 60% of their EDN, eosinophil cationic protein, and eosinophil peroxidase and about 50% of their eosinophil granule major basic protein content compared to freshly isolated eosinophils, and all four of the granule proteins were released into the culture medium. Fifth, detailed studies of eosinophils cultured in hrIL-5 showed that 89 +/- 10% of the starting quantity of EDN could be recovered at 7 days. Whereas 99 +/- 1% of the EDN at day 0 was cell associated, by 7 days 60 +/- 9% was in the cell supernatants. Thus, hrIL-5 activates eosinophils, increases their viability, decreases their density, and their content of granule proteins and causes release of the granule proteins into culture fluids. The striking loss of granule proteins during culture with hrIL-5 may be an important mechanism for deposition of these cationic toxins in various diseases where IL-5 plays a role.     

Ozone-induced hyperresponsiveness and blockade of M2 muscarinic receptors by eosinophil major basic protein.

Control of airway smooth muscle is provided by parasympathetic nerves that release acetylcholine onto M(3) muscarinic receptors. Acetylcholine release is limited by inhibitory M(2) muscarinic receptors. In antigen-challenged guinea pigs, hyperresponsiveness is due to blockade of neuronal M(2) receptors by eosinophil major basic protein (MBP). Because exposure of guinea pigs to ozone also causes M(2) dysfunction and airway hyperresponsiveness, the role of eosinophils in ozone-induced hyperresponsiveness was tested. Animals were exposed to filtered air or to 2 parts/million ozone for 4 h. Twenty-four hours later, the muscarinic agonist pilocarpine no longer inhibited vagally induced bronchoconstriction in ozone-exposed animals, indicating M(2) dysfunction. M(2) receptor function in ozone-exposed animals was protected by depletion of eosinophils with antibody to interleukin-5 and by pretreatment with antibody to guinea pig MBP. M(2) function was acutely restored by removal of MBP with heparin. Ozone-induced hyperreactivity was also prevented by antibody to MBP and was reversed by heparin. These data show that loss of neuronal M(2) receptor function after ozone is due to release of eosinophil MBP.     

Neutrophils as a novel source of eosinophil cationic protein in IgE-mediated processes

The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with (35)S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against FcepsilonRI and galectin-3 (FcepsilonRI>galectin-3), but not against FcepsilonRII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.     

Characterization of monoclonal antibodies against human parvovirus B19

Eleven hybridoma cell lines producing mouse monoclonal antibodies (mAbs) against human parvovirus B19 were established. Their specificity was as follows. Approximately 5% of fetal erythroid cells inoculated with B19 reacted with all the mAbs and with anti-B19 positive human serum, but not with negative serum by indirect double immunofluorescence staining. All the mAbs recognized both VP-1 (84 kDa) and VP-2 (58 kDa) capsid proteins of B19 virions propagated in vitro and in vivo by Western blotting, and immunoprecipitated B19 virions.     

Generation of rat eosinophils by recombinant rat interleukin-5 in vitro and in vivo

The addition of recombinant rat interleukin-5 (IL-5), which was purified from the hemolymph of silkworm Bombyx mori larvae infected with IL-5-expressing recombinant virus, to cultures of rat bone marrow cells resulted in an increase in the number of Luxol-fast-blue staining eosinophils in a time- and concentration-dependent manner. After 6 days culture with 100 pM recombinant rat IL-5, more than 90% of the bone marrow cells were eosinophil. The contents of major basic protein (MBP) in the bone marrow cells determined by Western blot analysis using a polyclonal antibody to rat MBP were also increased by recombinant rat IL-5 (100 pM). Furthermore, intravenous injections of recombinant rat IL-5 twice a day for six consecutive days increased the population of eosinophils in peripheral blood cells and in bone marrow cells. These findings indicate that rat IL-5 induces terminal differentiation and proliferation of progenitor cells to mature eosinophils in rats.     

Substance P-induced airway hyperreactivity is mediated by neuronal M(2) receptor dysfunction

Neuronal muscarinic (M(2)) receptors inhibit release of acetylcholine from the vagus nerves. Hyperreactivity in antigen-challenged guinea pigs is due to blockade of these M(2) autoreceptors by eosinophil major basic protein (MBP) increasing the release of acetylcholine. In vivo, substance P-induced hyperactivity is vagally mediated. Because substance P induces eosinophil degranulation, we tested whether substance P-induced hyperreactivity is mediated by release of MBP and neuronal M(2) receptor dysfunction. Pathogen-free guinea pigs were anesthetized and ventilated. Thirty minutes after intravenous administration of [Sar(9),Met(O(2))(11)]- substance P, guinea pigs were hyperreactive to vagal stimulation and M(2) receptors were dysfunctional. The depletion of inflammatory cells with cyclophosphamide or the administration of an MBP antibody or a neurokinin-1 (NK(1)) receptor antagonist (SR-140333) all prevented substance P-induced M(2) dysfunction and hyperreactivity. Intravenous heparin acutely reversed M(2) receptor dysfunction and hyperreactivity. Thus substance P releases MBP from eosinophils resident in the lungs by stimulating NK(1) receptors. Substance P-induced hyperreactivity is mediated by blockade of inhibitory neuronal M(2) receptors by MBP, resulting in increased release of acetylcholine.     

TNF-alpha potentiates C5a-stimulated eosinophil adhesion to human bronchial epithelial cells: a role for alpha 5 beta 1 integrin.

Cooperative action of inflammatory mediators and adhesion molecules orchestrates eosinophil recruitment during allergic inflammation in the airways. This study investigated the mechanisms involved in increasing eosinophil adhesion to human bronchial epithelial cells (HBEC) following priming and activation of eosinophils with TNF-alpha and complement protein C5a, respectively. Under primed conditions, eosinophil adhesion increased 3-fold from basal (16%), and the effect was significantly greater (p < 0.05) than the increase following stimulation with C5a alone (2-fold). Eosinophil contact with HBEC was essential for priming. In contrast to C5a, adhesion of eotaxin-stimulated eosinophils to HBEC was not primed with TNF-alpha nor IL-5, a known eosinophil-priming agent. Priming caused activation of alpha(M)beta(2) integrin; mAb against either the common beta(2) integrin subunit or its ICAM-1 ligand reduced the primed component of adhesion. Using mAbs against beta(1) or alpha(5), but not alpha(4) integrin subunit, together with anti-beta(2) integrin mAb, reduced stimulated adhesion to basal levels. Cross-linking alpha(5)beta(1) integrin increased alpha(M)beta(2) integrin-dependent adhesion of eosinophils. There are no known adhesion molecule ligands of alpha(5)beta(1) integrin expressed on HBEC; however, fibronectin, the major matrix protein ligand for alpha(5)beta(1) integrin, was detected in association with HBEC monolayers. A mAb against fibronectin, in combination with anti-beta(2) integrin mAb, reduced adhesion to basal levels. In conclusion, alpha(5)beta(1) integrin may provide a contact-dependent costimulus for eosinophil priming that, together with TNF-alpha, potentiated C5a activation of alpha(M)beta(2) integrin and increased eosinophil adhesion to ICAM-1. Fibronectin, associated with HBEC, may act as a ligand for alpha(5)beta(1) integrin. Dual regulation of eosinophil priming may prevent inappropriate activation of eosinophils in the circulation.