Tryptase Activates the Mitogen-Activated Protein Kinase/Activator Protein-1 Pathway in Human Peripheral Blood Eosinophils, Causing Cytokine Production and Release¹

During allergic inflammatory reactions mast cells and eosinophils can interact when the eosinophils have infiltrated into the tissues in the late phase response or when the inflammation becomes chronic (1, 2). Eosinophil cross-talk is important in regulating the severity, duration, and outcome of the allergic response. Within this framework we demonstrated that human peripheral blood eosinophils induce IgE-desensitized rat peritoneal mast cells to release histamine (3). We previously reported that rat peritoneal mast cells enhance eosinophil survival in vitro by the induction of GM-CSF autocrine production (4, 5). Mast cell-derived TNF-α was found to be the preformed mediator predominantly responsible for this effect (4). Another mast cell preformed mediator tryptase, present in virtually all mast cell types (6), was found to cause the release of eosinophil cationic protein from eosinophils (7) and to act as their chemoattractant (8) as well as to induce IL-8 production by human epithelial cells and neutrophils (9, 10). Tryptase is a serine protease with trypsin-like activity that cleaves several proteins and peptides such as fibrinogen, kininogen, and vasoactive intestinal peptide (11, 12). The cleavage of proteinase-activated receptor (PAR)⁴-2 by tryptase induces receptor-mediated signaling in human vascular endothelial cells (13), leading to inositol 1,4,5-triphosphate production. Furthermore, it has recently been found that human peripheral blood eosinophils from normal and mild asthmatics express PAR-2 (14).

Tissue eosinophilia is a constant feature of allergic inflammation and correlates with high tissue levels of eosinophil granule proteins and eosinophil-derived cytokines (15, 16). Two cytokines with proinflammatory properties, IL-6 and IL-8, are produced by eosinophils (16, 17). IL-8 is a potent granulocyte chemoattractant and can serve as a marker of tissue eosinophilia (18, 19). IL-6, in turn, regulates acute phase protein production (20), B cell proliferation, and final differentiation (21).

The production of these cytokines is regulated by some transcription factors, such as NFAT, NF-κB, and AP-1 (22, 23, 24). The latter is actually a family of transcription factors and is composed of members of the Jun, Fos, and activating transcription factor (ATF) subfamily, which are sequestered in the cytoplasm (25). Upon activation by Jun N-terminal mitogen-activated protein kinase (MAPK), AP-1 is phosphorylated and translocates to the nucleus where it regulates the activity of many genes involved in the inflammatory response (26). The initial enhancement of cytosol calcium by tryptase, as a result of inositol 1,4,5-triphosphate production (13), might be required for the involvement of MAPKs that, in turn, activate AP-1. Therefore, the MAPK/AP-1 pathway could play an important role in mediating mast cell-derived tryptase effects in allergic inflammation.

In the present work we evaluated the ability of mast cells to induce IL-6 and IL-8 production and release by eosinophils. We also investigated the role of tryptase and its signal pathway in this event.

Eosinophils were purified according to a previously published procedure (27) from the peripheral blood of mildly atopic volunteers (20–44 years old, with blood eosinophilia ranging from 4 to 10%) according to the guidelines established by the Hadassah-Hebrew University Human Experimentation Helsinki Committee. None of the volunteers had been taking any medication during the previous 3 mo. Venous blood, collected in heparinized syringes, was subjected to dextran (Pharmacia Biotech, Uppsala, Sweden) sedimentation and leukocytes were centrifuged on Ficoll-Paque (density = 1.077; Sigma-Aldrich, St. Louis, MO) for 25 min at 700 × g. Neutrophils and T cells in the granulocyte-enriched pellet were tagged with micromagnetic beads to anti-CD16 and anti-CD3 Abs, respectively (Miltenyi Biotec, Bergisch Gladbach, Germany). Eosinophils were collected at a purity of >99%, as assessed by Kimura’s staining, and at a viability of >99%, as assessed by trypan blue (Sigma-Aldrich) exclusion test.

Rat mast cells were isolated by a sterile procedure from the peritoneal cavity of “Sabra” rats, an outbred strain of the Hebrew University. Rat peritoneal lavage was performed with Tyrode buffer containing 0.1% gelatin (TG buffer) and mast cells were purified on 22.5% metrizamide (Sigma-Aldrich) in TG buffer. Mast cells were collected at a purity of 97–100%, as assessed by toluidine blue staining (Sigma-Aldrich), and at a viability of >99% (28).

Human mast cell line 1 (HMC-1; a kind gift from Dr. J. Butterfield, Mayo Clinic, Rochester, MN) was cultured in Iscove’s medium supplemented with 10% v/v iron-enriched calf serum, 1.2 mM α-monothioglycerol, 200 U/ml penicillin, 200 μg/ml streptomycin, and 2 mM gentamicin (Biological Industries, Beit Haemek, Israel) at 37°C. The cells were passaged every 5 days (29).

To obtain mast cell sonicate, isolated rat peritoneal mast cells or HMC-1 were resuspended in medium containing RPMI 1640, 200 U/ml penicillin, 200 μg/ml streptomycin, 2 mM gentamicin, 0.1 mM nonessential amino acids, and 5% v/v heat-inactivated FCS (enriched medium (EM); Biological Industries) at a concentration of 5 × 10⁶ cells/ml and were disrupted by continuous sonication in ice for 1 min (W-380 sonicator (Heat Systems Ultrasonics, Farmingdale, NY); duty cycle, 5 s; output power, 50%). The sonicates were microcentrifuged for 5 min and debris-free supernatants were aliquoted and stored at −70°C.

Freshly isolated eosinophils (1 × 10⁵/100 μl EM) were seeded in 96-well plates and cultured at 37°C in a humidified atmosphere of 5% CO₂ for 10 min to 18 h according to the different experimental aims. Eosinophil cultures were incubated with one of the following additions in 100 μl of EM: mast cell sonicate (0.2–2 × 10⁵ cells), 0.1–150 nM human skin recombinant I-β-tryptase (a kind gift of Dr. M. Haak-Frendscho, Promega, Madison, WI), 2.5 ng/ml PMA, 1 μM dexamethasone, 0.1 mg/ml cycloheximide, 10⁻³ M actinomycin D, 1 μM to 1 nM histamine, 1 μM pyrilamine, 1 μM cimetidine (all purchased from Sigma-Aldrich); rabbit anti-human PAR-2 polyclonal Abs with antagonist’s property to this receptor (0.1–15 μg/ml; a kind gift from Dr. H. Kita, Mayo Clinic); and tryptase inhibitors GW-455378A and GW-585361A (1–50 μM; a kind gift of Dr. K. Ray, GlaxoSmithKline, Hertfordshire, U.K.). The specificity of the two inhibitors toward tryptase in comparison with factor Xa and thrombin was tested by enzymatic assays. The IC₅₀ of GW-455378A was >100 μM for factor Xa vs ∼1 nM for tryptase. The IC₅₀ of GW-585361A was 0.09 μM for factor Xa and 0.32 μM for thrombin vs 29 nM for tryptase. In some experiments, eosinophils were preincubated with the following MAPK inhibitors for 30 min at 37°C: 35 μM PD 98059 (Calbiochem-Novabiochem, San Diego, CA), 10 μM SB 202190 (Calbiochem-Novabiochem), 25 μM curcumin (Sigma-Aldrich), or 1 μM cyclosporin A (Sigma-Aldrich), before addition of 50 nM human recombinant tryptase. None of the treatments had toxic effects on eosinophils as assessed after 18 h of incubation by trypan blue exclusion test. For preparation of eosinophil supernatants, eosinophils were incubated at 37°C for 3 days with the different activators or with EM alone (nonactivated cells). Suspensions were centrifuged and supernatants were collected, aliquoted, and stored at −70°C.

In control experiments, eosinophils were incubated at a 1:1 ratio with human PBMC sonicate. PBMC were isolated from the same blood used to purify the eosinophils, as follows: the interface of the Ficoll-Paque gradient containing the PBMC was recovered and washed twice by centrifugation in EM (150 × g for 5 min). The PBMC pellet was resuspended in EM (4 × 10⁶/ml) before sonication. All the experiments were conducted in triplicate for the indicated times.

The human fetal lung fibroblast cell line FHS 738 (HTB-157; American Type Culture Collection, Manassas, VA) was used as a control for some experiments. Fibroblasts were cultured and subcultured in supplemented DMEM (Biological Industries) containing 5% v/v heat-inactivated FCS as described (30).

IL-8 and IL-6 in the eosinophil culture supernatants were quantified using commercial specific ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). The limit of assay sensitivity is 10 and 1.4 pg/ml for IL-8 and IL-6, respectively.

Total RNA was extracted by using the commercial reagent Tri-Reagent (Sigma-Aldrich) based on the acid guanidinium-thiocyanate RNA extraction technique (31). The first-strand cDNA synthesis reaction was catalyzed by SuperScript II RNase H⁻ Reverse Transcriptase and oligo(dT)_12–18 primer (Life Technologies, Rockville, MD) according to the manufacturer’s instructions. The generated complementary DNA was amplified using 1.25 U of Taq DNA polymerase and dNTP mixture and IL-8 (5′-ATGACTTCCAAGCTGGCCGTGGCT and 3′-TCTCAGCCCTCTTCAAAAACTTCTC) primers (Clontech Laboratories, Palo Alto, CA) in the presence of 10% glycerol (Sigma-Aldrich) as a specificity enhancer. Primers for G3PDH, used as a control to test the efficiency of cDNA synthesis, were 5′-ACCACAGTCCATGCCATCACTGCC and 3′-CATGTGGGCCATGAGGTCCACCAC (Clontech Laboratories). The specificity of the primers was confirmed by the manufacturer. DNA templates for IL-8 provided by the manufacturer were used as a positive control. The products, amplified by thermocycler, were electrophoresed on 1.8% agarose gel stained with ethidium bromide (Sigma-Aldrich) and photographed under UV light.

Protein isolation, electrophoresis, and blotting using specific anti-active Abs (for c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK)1/2; Promega) were performed essentially as described (32). Lysis buffer (50 mM Tris, 25 mM KCl, 5 mM MgCl₂, 1 mM EGTA, 1 mM Na₃VO₄) containing a protease inhibitor mixture (Sigma-Aldrich) was added to cell pellets obtained from 8–10 × 10⁶ of eosinophils. Cell debris was removed from lysates by centrifugation (15,300 × g for 10 min) after vortex mixing and sonication (six 10-s bursts at intervals of 10 s using a W-380 sonicator (Heat Systems Ultrasonics) at 50% duty cycles, output 5). All procedures were performed on ice or at 4°C. Protein concentrations were assessed by modified Bradford assay before loading the samples. Samples were analyzed on 10% SDS-PAGE.

The gel was electrotransferred (90 min) to nitrocellulose filter paper blocked in PBS containing 5% BSA and 0.1% Brij (Sigma-Aldrich). The filter was then incubated with rabbit anti-human MAPKs (2–5 μg/ml; AB-255 NA polyclonal Abs; R&D Systems) overnight at 4°C, washed in PBS/Brij, and incubated with secondary peroxidase-conjugated immunopure donkey anti-rabbit Abs (1/5000; Pierce, Rockford, IL) and finally with the reagents of the chemiluminescence system ECL detection kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K.).

EMSA was performed as follows on nuclear extract of eosinophils prepared as described (33). The AP-1 oligonucleotide (5′-GTCTAGAGTGACTCAGCGC-3′, underlined oligonucleotides are representative of AP-1-conserved recognition motif) was labeled in a 20-μl reaction mixture containing 30 ng of the double-stranded oligonucleotide as listed below, 1 μl Klenow DNA polymerase, and 5 μl of 10 μC/μl [α-³²P]dCTP (Amersham). Labeled oligonucleotides (30–100 pg/20 μl/104 cpm) were incubated at 30°C for 30 min, with the nuclear extract (10 μg protein) in a buffer containing 12 mM HEPES (pH 7.2), 60 mM KCl, 0.6 mM Na₂EDTA, 0.6 mM DTT, 5 mM MgCl₂, and 1 μg poly(dIdC). The reaction mixtures were electrophoresed on 4% polyacrylamide gels and photographed under UV light.

Cytospins of eosinophils (1 × 10⁵ cells) incubated with either 50 nM tryptase or 2.5 ng/ml PMA for 10 min were prepared (3 min at 1000 × g). Cells were fixed in 3.8% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and then rinsed twice in PBS (Sigma-Aldrich). Blocking was performed with 7.5% horse serum (Life Technologies, Glasgow, U.K.) for 30 min. Permeabilized eosinophils were stained directly with mouse monoclonal anti-human c-Jun Abs (Jackson ImmunoResearch Laboratories, West Grove, PA) for 45 min at room temperature (1/200 in PBS) followed by goat anti-mouse Abs (1/100 in PBS for 45 min at room temperature). Propidium iodide (PI; 0.5 μg/ml for 10 min at room temperature; Sigma-Aldrich) was used for counterstaining the eosinophil nuclei. Until analyzed by confocal microscopy, the samples were kept in an antifade solution (90% glycerol, 10% PBS (pH 8) 3% diazabicyclo octane, 0.1% NaN₃; Sigma-Aldrich). Negative controls consisted of slides in which only the second Ab was added.

Slides were examined using a ×63 objective under a Zeiss Axiovert 135 M inverted microscope (Zeiss, Oberkochen, Germany) with 63/1.2 C-Apochromat water immersion lens. The system is equipped with a 25-mW air-cooled argon laser (488-nm excitation line with 515-nm long pass barrier filter for the excitation of green fluorescence). Red fluorescence was excited with the 633-nm internal helium neon laser. Confocal images were converted to a Tif format and transferred to a Zeiss imaging workstation for pseudocolor representation. Brightness and contrast level were achieved using the Zeiss and Adobe Photoshop 3.0 (Adobe Systems, Mountain View, CA) programs.

Freshly isolated eosinophils were rinsed in PBS (3% FCS) and resuspended in 5 × 10⁶/100 μl for staining. Indirect immunofluorescence was performed with rabbit IgG anti-human PAR-2 Abs (10 μg/ml) or with rabbit IgG Abs (10 μg/ml) in PBS 3% FCS for 45 min in ice and then rinsed twice with PBS 3% FCS (1000 × g for 7 min at 4°C). Secondary goat anti-rabbit F(ab′)₂ Abs (1 μg/ml) were added to the eosinophil suspension for 45 min. The eosinophils were then rinsed twice and resuspended with 500 μl of PBS 3% FCS and analyzed by FACS (FACSCalibur; BD Biosciences, Cowley, U.K.) equipped with Consort 32 Lysis version 1.02 software (BD Immunocytometry Systems, San Jose, CA) for data analysis.

For identification of intracellular PAR-2, eosinophils, before staining, were fixed and permeabilized as described above (see Confocal laser microscopy).

Results are expressed as mean ± SEM. Statistical analysis was performed by the Student’s paired t test. A p value of <0.05 was considered statistically significant.

Recently we provided evidence that mast cell sonicate could be useful in the study of biological effects of mast cell preformed mediators (4). Supernatants of sonicated and compound 48/80-activated mast cells similarly affected eosinophil survival and GM-CSF release from eosinophils. As shown in Fig. 1, the rat and human mast cell sonicate induced IL-6 and IL-8 release from human peripheral blood eosinophils in a concentration-dependent manner. Maximal IL-6 and IL-8 release caused by both rat mast cell and HMC-1 sonicate was obtained at an eosinophil:mast cell ratio of 1:1.

To assess whether this effect is specific, human PBMC sonicate was added to the eosinophils. The PBMC sonicate did not release either IL-6 or IL-8 from the eosinophils (n = 3; data not shown).

Different experimental approaches were used to evaluate the involvement of some mast cell preformed mediators on IL-8 release from eosinophils. These mediators comprised histamine, TNF-α, stem cell factor (SCF), and tryptase, all HMC-1 preformed mediators (29, 32, 34), and IFN-γ, which was recently found to be present in the HMC-1 sonicate (94 pg/ml/1 × 10⁶ cells; V. Temkin and F. Levi-Schaffer, unpublished data).

Eosinophils were incubated with HMC-1 sonicate in the presence of the antihistamine drugs pyrilamine (anti-H1) or cimetidine (anti-H2). Both of these compounds, even at the highest used concentration (10⁻⁵ M), had no effect on HMC-1-induced IL-8 release (71.3 ± 4.6 pg/ml IL-8 from eosinophils incubated with HMC-1 sonicate alone vs 67.1 ± 3.1 pg/ml from eosinophils incubated with HMC-1 and pyrilamine and 72.4 ± 5.1 eosinophils incubated with HMC-1 and cimetidine; n = 3). Histamine (10⁻⁹–10⁻⁶ M) failed to induce IL-8 release (14.5 ± 1.4 pg/ml from eosinophils incubated with EM alone vs 16.3 ± 3.4 pg/ml from eosinophils incubated with histamine at 10⁻⁶ M; n = 3).

To evaluate the potential role of SCF, TNF-α, and IFN-γ on IL-8 release, HMC-1 sonicate was preincubated with different concentrations of neutralizing Abs for these cytokines and then added to the eosinophil cultures. None of these treatments influenced HMC-1-induced IL-8 release (69.7 ± 7.1 pg/ml IL-8 from eosinophils incubated with HMC-1 sonicate alone vs 64.1 ± 4.3 pg/ml from eosinophils incubated with anti-SCF, 73.7 ± 3.4 eosinophils with anti-TNF-α, and 74.8 ± 5.6 pg/ml from eosinophils incubated with anti-IFN-γ).

In contrast, when HMC-1 sonicate was preincubated with the specific tryptase inhibitors GW-455378A or GW-585361A and then added to the eosinophil cultures, inhibition of IL-8 release was observed (see Table I). At an optimal concentration (20 μg/ml), these compounds decreased HMC-1-induced IL-8 release by 89.8 ± 7.6 and 82.5 ± 4.4%, respectively (n = 4; p < 0.001). These data clearly indicate the involvement of tryptase in IL-8 release from human eosinophils.

To investigate the direct role of tryptase in IL-8 production by human eosinophils, the cells were incubated with human recombinant tryptase. As shown in Fig. 2 tryptase induced IL-8 release in a concentration-dependent manner with a maximal release of 81.25 ± 5.2 pg/ml IL-8 at a concentration of 50 ng/ml (n = 6; p < 0.001). Higher tryptase concentrations did not further increase IL-8 release. The tryptase-induced IL-8 release was inhibited to a similar extent by the two tryptase inhibitors GW-455378A and GW-585361A, i.e., by 92.9 and 89.5%, respectively (20 μg/ml; n = 4; p < 0.001). These two tryptase inhibitors did not alter PMA-induced IL-8 release. In fact, eosinophils incubated with PMA (5 ng/ml) released 127.6 ± 8.6 pg/ml while cells incubated with PMA and GW-455378 or GW-585361A (20 μg/ml) released 133.2 ± 12.1 and 124.3 ± 9.1 pg/ml IL-8, respectively (n = 2). In addition, the two compounds did not affect HMC-1-enhanced eosinophil survival (data not shown), an event that is mediated by TNF-α and IFN-γ (35).

To investigate whether the cytokine release induced by HMC-1 and tryptase needs both protein and mRNA synthesis, eosinophil cultures were performed in the presence of cycloheximide or actinomycin D, respectively (Table II). Addition of one of the compounds inhibited both HMC-1- and tryptase-induced IL-6 and IL-8 release from the eosinophils. Moreover, both HMC-1 and tryptase induced the production of mRNA for IL-8 by the eosinophils after an 18-h incubation (Fig. 3).

To study the possible involvement of MAPKs in tryptase-induced IL-8 production by human eosinophils, the cells were cultured for 18 h with tryptase (50 ng/ml) and with 35 μM PD 98059 (mitogen-activated protein/ERK kinase (MEK) inhibitor), 25 μM curcumin (JNK inhibitor), or 10 μM SB 202190 (p38 inhibitor). As shown in Fig. 4, these three compounds partly inhibited the tryptase-induced IL-8 release. When added together they completely abrogated IL-6 and IL-8 release from both tryptase- and medium-incubated eosinophils. In contrast, cyclosporin A had no effect on tryptase-induced IL-8 release but slightly enhanced IL-8 release in medium alone (p < 0.01; n = 4).

Direct tryptase-induced activation of MAPKs was assessed by Western blot using specific anti-active JNK1/2, p38, and ERK1/2 Abs. Tryptase caused phosphorylation of ERK1/2, JNK1/2, and p38 MAPKs in the eosinophils after 3 min of incubation, indicating the specific activation of these MAPKs (Fig. 5).

Interestingly, tryptase also induced IL-8 release in human fetal lung fibroblasts (745.3 ± 43.1 pg/ml in 50 ng/ml tryptase vs 481.7 ± 36.2 pg/ml in medium alone; n = 3; p < 0.001). Similarly to the eosinophils, the tryptase-induced release of IL-8 from the fibroblasts was abrogated by the MAPK inhibitors PD 98059 (35 μM), curcumin (25 μM), and SB 202190 (10 μM) added together (346.8 ± 21.8 pg/ml in tryptase-incubated cultures and 311.4 ± 41.7 in medium alone; n = 3; p < 0.001).

To investigate whether AP-1 transcription factor mediates the tryptase-induced IL-8 release, eosinophils were preincubated with dexamethasone (10⁻⁶ M) for 45 min and then cultured with tryptase for 18 h. Dexamethasone completely abrogated HMC-1- (from 46.7 ± 5.7 to 4.1 ± 1.2 pg/ml; n = 4; p < 0.001) and tryptase-induced (from 41.3 ± 4.2 to 4.7 ± 3.6 pg/ml; n = 4; p < 0.001) IL-6 release. Similarly, IL-8 release induced by HMC-1 and tryptase was inhibited by dexamethasone, i.e., for HMC-1 from 64.3 ± 6.9 to 1.7 ± 1 pg/ml (n = 4; p < 0.09) and for tryptase from 53.2 ± 4.3 to 4.7 ± 3.6 pg/ml (n = 4; p < 0.001).

Confocal microscopy analysis of eosinophils incubated for 10 min with either tryptase or PMA (as a control) showed the translocation of the c-Jun from the cytoplasm to the nucleus (Fig. 6). In fact, at this time point yellow regions corresponding to overlapping green (FITC anti-p65 Abs) and red images (PI) indicated c-Jun localization in the nucleus (Fig. 6, B and C). In contrast, freshly isolated eosinophils and eosinophils incubated with EM for 10 min displayed a green cytoplasmic staining, indicating of the presence of c-Jun in the cytoplasm (Fig. 6, A and B).

EMSA was used to analyze AP-1 binding activity to DNA. As shown in Fig. 7, the nuclear extracts of eosinophils incubated for 4 h with tryptase (50 nM) showed enhanced DNA binding activity of AP-1 in comparison to eosinophils incubated with EM alone. Moreover, band shift was prevented by preincubation of nuclear extract with a cold DNA competitor. No DNA binding activity of AP-1 was observed in any cytosol fractions.

PAR-2 was found to mediate tryptase effects in many cell types (36). Recently, PAR-2 was found to be expressed by peripheral blood eosinophils obtained from normal healthy and mild asthmatic patients (14). In our study, flow cytometric analyses of human peripheral blood eosinophils incubated with anti-PAR-2 Abs showed a strong staining for this receptor (Fig. 8). No difference in fluorescence was observed in intact eosinophils and in permeabilized eosinophils (data not shown). When added to the eosinophils 30 min before tryptase, anti-PAR-2 Abs, but not control Abs, reduced the tryptase-induced IL-6 and IL-8 release in a concentration-dependent manner. Maximal inhibitory effect was observed at an Ab concentration of 1 μg/ml. At this concentration, tryptase-induced IL-6 and IL-8 release was reduced by 54.7 and 59.4%, respectively (Table III; n = 3; p < 0.001).

Mast cell-eosinophil interactions can take place during allergic inflammatory reactions, especially once the eosinophils have infiltrated into the inflamed tissues. We have previously reported that rat peritoneal mast cells enhance eosinophil survival through their activation to produce and release the autocrine survival cytokine GM-CSF (4, 5). The mediator responsible for this event is mast cell preformed TNF-α. In addition, mast cells caused the release of eosinophil peroxidase and eosinophil adherence to plastic wells. By a proteomics analysis we have recently detected that [³⁵S]methionine-labeled eosinophils are induced by HMC-1 sonicate to produce a large number of proteins. This shows that mast cells are a potent stimulus for eosinophils and that human peripheral blood eosinophils are highly biosynthetically active cells (35). In this study we have further investigated the influence of mast cells on eosinophil activation by assessing whether mast cells could induce IL-6 and IL-8 production and release from eosinophils. Both these proinflammatory cytokines are produced by eosinophils in vitro (16, 17) and participate in the allergic inflammatory responses (15, 20, 21).

Rat peritoneal mast cell and the human mast cell line HMC-1 were used as a source of mast cells. Rat peritoneal mast cells are a readily available source of a large number of mast cells with connective tissue phenotype similar in many aspects to human skin mast cells. Even though HMC-1 lacks FcεRI it is still a very useful tool for in vitro studies on a large number of human mast cells of the mucosal phenotype. In fact, it contains mostly β-tryptase and only traces of, if any, chymase (29). In addition, HMC-1, like rat peritoneal mast cells, contains other mediators, such as preformed histamine and heparin, TNF-α, and SCF (29, 32, 34).

In our study, both types of mast cell sonicate caused IL-6 and IL-8 production and release by human peripheral blood eosinophils in a concentration-dependent fashion. This would indicate that both connective tissue and mucosal mast cells can interact with eosinophils and that this interaction is conducted by a common mediator seemingly specific for mast cells because PBMC sonicate was ineffective.

The next aim of our study was to determine this mast cell mediator(s). Among mast cell preformed mediators, histamine, TNF-α, SCF, IFN-γ, and tryptase might be good candidates to cause IL-6 and IL-8 release. Histamine has been shown to stimulate eosinophil superoxide production (37) and, together with PGD₂, to increase their cytosolic calcium (38). In addition, histamine induces IL-6 and IL-8 production in different cell types (39, 40, 41). As we and others have previously reported, TNF-α causes IL-8 and GM-CSF production by human eosinophils via NF-κB activation (5, 42). SCF through c-Kit receptor induces very late Ag-4-mediated eosinophil adhesion to endothelial cells (43). IFN-γ enhances eosinophil survival and eosinophil-mediated cytotoxicity (44), and we recently found that IFN-γ preformed in HMC-1 partially enhances eosinophil survival (V. Temkin and F. Levi-Schaffer, unpublished data). Tryptase, in turn, induces IL-8 production and release by many cell types, such as endothelial cells and neutrophils (9, 10). Moreover, tryptase causes eosinophil degranulation (7) and contraction of bronchial smooth muscle (45). Tryptase inhibitors suppress not only the early phase but also the late phase of allergic inflammation, indicating tryptase influences on infiltrated inflammatory cells and on cytokine/chemokine production (46, 47).

In our study, cimetidine (H1 antagonist), pyrilamine (H2 antagonist), anti-SCF, anti-TNF-α, and anti-IFN-γ neutralizing Abs had no influence on HMC-1-induced IL-8 release. In contrast, the tryptase-specific inhibitors GW-455378A and GW-585361A inhibited the HMC-1-induced IL-8 release. There are at least two tryptase isoforms, α and β, that share a sequence identity of ∼98%. The β-isoform appears to be activated intracellularly and stored in the secretory granules of most human mast cells, including the HMC-1 cell line (29, 48). We found that both the human recombinant skin I-β tryptase and the HMC-1 sonicate induce both IL-8 release and its mRNA production.

Human peripheral blood eosinophils can both store preformed IL-6 and IL-8 (49, 50) and several other cytokines in their secondary granules and synthesize them in response to specific stimuli (16, 50). Therefore, we assessed whether tryptase could induce not only the release of preformed IL-6 and IL-8 but also their mRNA and the protein syntheses. Eosinophils were incubated with tryptase or HMC-1 in the presence of cycloheximide, a protein synthesis inhibitor, or actinomycin D, a mRNA synthesis inhibitor. The IL-6 and IL-8 levels in the eosinophil supernatants were found to be negligible. Moreover, tryptase as well as HMC-1 sonicate were found to induce IL-8 mRNA expression. Therefore, tryptase induces both the release and the de novo production of these cytokines from the eosinophils.

We next characterized the signal transduction pathway involved in tryptase-induced-IL-8 production.

AP-1, one of the transcription factors regulating IL-8 production in many cells (24), is a family of transcription factors composed of homodimers and heterodimers which are members of Jun, Fos, and ATF subfamilies that bind to a common DNA site, the AP-1 binding site (25). MAPKs are upstream activators of AP-1 (26). ERK, p38, and JNK, subfamilies of the MAPK pathway, induce Fos and Jun production by activating different transcription factors such as Elk-1 and ATF. However, only JNK causes Jun phosphorylation, an essential step of AP-1 activation that includes AP-1 translocation to the nucleus and its binding to DNA. We found that tryptase induces phosphorylation of ERK1/2, p38, and JNK1/2 kinases and that MEK1/2 (upstream activators of ERK1/2), p38, and JNK inhibitors strongly decreased tryptase-induced IL-8 release.

In this study we have also demonstrated that tryptase causes IL-8 release from human fetal lung fibroblasts and that this event is completely abrogated by MEK1/2, p38, and JNK inhibitors. Altogether these findings clearly indicate that tryptase causes IL-8 production through MAPK activation in other cell types.

In addition, dexamethasone, which inhibits AP-1, perhaps by preventing its phosphorylation (51), completely abrogated both HMC-1-induced and tryptase-induced IL-6 and IL-8 release from human eosinophils. It is interesting to point out that it has been reported in other cell systems that dexamethasone can inhibit IL-8 production by inhibition of NF-κB (52).

Tryptase also induced the translocation of c-Jun to the nucleus, an essential step in AP-1 activation. Furthermore, in nuclear extracts of eosinophils incubated with tryptase, the DNA binding activity of AP-1 was strongly enhanced. Maximum binding activity was observed after 4 h and not at the time c-Jun translocation was found to take place (10 min). This would strengthen the assumption that Elk-1 and ATF transcription factor activations that lead to the c-Fos and c-Jun synthesis are required in this step.

Interestingly, Elk-1 can be dephosphorylated and consequently down-regulated by activated calcineurin (53). This phosphatase is probably slightly activated even in freshly isolated eosinophils, because the dephosphorylated pattern of its downstream NFATp was found to be present in freshly isolated, nonactivated peripheral blood eosinophils (V. Temkin, N. Arai, and F. Levi-Schaffer, unpublished data). These facts could explain the slightly stimulatory effect of cyclosporin A, inhibitor of the calcineurin, on IL-8 release from eosinophils cultured with medium alone. Interestingly, calcineurin has been implicated to be involved in the regulation of mast cell and T cell IL-6 and IL-8 production, perhaps via NFAT activation (22, 23). Cyclosporin A had no effect on tryptase-induced IL-8 release from the eosinophils, probably because the strong activation of ERK, JNK, and p38 MAPKs could overcome the inhibitory effect of calcineurin.

Tryptase is known to exert several of its cellular effects through cleavage of PAR-2 (13). We next tried to identify whether tryptase activates human eosinophils through PAR-2 binding/activation. Fibroblasts have been shown to express PAR-2s that mediate tryptase-induced fibroblast proliferation (54), and we presently found that tryptase causes their IL-8 production. Recently, PAR-2 was found to be expressed by peripheral blood eosinophils obtained from normal healthy and mildly asthmatic patients (14). In our study, PAR-2 was also shown to be present on the plasma membrane of eosinophils isolated from the peripheral blood of atopic dermatitis and rhinitis patients. Furthermore, Abs that are antagonists to PAR-2 partly reduced tryptase-induced IL-6 and IL-8 release in a concentration-dependent manner indicating that, at least in part, PAR-2 mediates these tryptase effects on the eosinophils. PAR-2 belongs to the thrombin receptor family. It is a G protein-coupled receptor activated with proteolytic cleavage of the extracellular domain (55). Thrombin cleaves PAR-1, PAR-3, and PAR-4, while PAR-2 is preferentially cleaved by trypsin and tryptase (55, 56) and cleaves, to a lesser extent, PAR-1 (54). Interestingly, mRNA for PAR-3 was recently found to be expressed by eosinophils (14). The finding that PAR-2 mediates tryptase-induced IL-8 production by eosinophils and by fibroblasts is in line with the observations from other cell types such as epithelial cells (9), neutrophils (10), and endothelial cells (57).

It is interesting to point out that in PAR-2 knockout mice the early phase of allergy is not affected, while the onset of inflammation is delayed (58). This could be explained by the fact that various mast cell mediators released at the early phase, such as tryptase, are responsible for inducing cytokine release from eosinophils and other inflammatory cells in the late phase.

In summary, we have provided evidence that tryptase activates the MAPK/AP-1 pathway. This is probably mediated by the cleaving of PAR-2s, which results in the production of IL-6 and IL-8 by human peripheral blood eosinophils.

We believe that these findings further elucidate mast cell-eosinophil interactions and underline the important role of tryptase in this cross-talk as well as in the overall allergic reaction.