Staphylococcal enterotoxin A (SEA), eotaxin and ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′ tetraacetic acid (EGTA) were purchased from Sigma Aldrich Co (St. Louis, MO, USA). Iscove's modified Dulbecco's medium (IMDM) was obtained from life technologies (New York, USA). ELISA kits for mouse IFN-γ, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), fluorescein isothiocyanate-conjugated anti-mouse MAC-1, phycoerythrin (PE)-conjugated anti-mouse VLA-4, fluorescein isothiocyanate-conjugated anti-mouse LFA1-α and interleukin-8 (IL-8) were obtained from BD Biosciences Pharmingen (San Jose, CA, USA). Fluoforte was obtained from Enzo Life Sciences International (New York, USA). Interferon-γ was purchased from Boehringer Ingelheim (Berkshire, UK). Antibody anti-MHC class II was obtained from Abcam (Cambridge, UK).

All animal care and experimental protocols were approved by the Ethical Principles in Animal Research adopted by the Brazilian College for Animal Experimentation (COBEA), and followed the Guide for the Care and Use of Laboratory Animals. Four-week-old male BALB/c mice were provided by the Central Animal House Services of State University of Campinas (UNICAMP). Animals were housed three per cage on a 12 h light–dark cycle, in temperature-controlled rooms and received water and food ad libitum until used on the Animal House Services of Faculty of Medicine of Jundiaí (FMJ).

Mouse bone marrow (BM) granulocyte isolation was carried out according to a previous study (Lintomen et al., 2002). Briefly, BM cells were collected and subsequently placed in plates (100 × 20 mm dish) for 30 min at 37°C (5% CO₂). The BM supernatants (non-adhered cells) were collected, washed twice with 2 ml of Iscove's modified Dulbecco's culture medium, and centrifuged (500 g for 10 min at 4°C). The non-adherent BM cell pellets were resuspended in 2.5 ml of culture medium, and the total (Neubauer) and differential (Diff-Quick stain) cell counts were done. The final cell suspension contained about of 70% of mature granulocytes, consisting of 60% neutrophils and 10% eosinophils, whereas the remainder was made of 30% of mononuclear cells. The adhesion and chemotaxis of neutrophils and eosinophils were evaluated through the measurements of specific peroxidases for neutrophils (myeloperoxidase; MPO) and eosinophils (eosinophil peroxidase; EPO), as detailed below.

Cells incubated with SEA (1–30 ng/ml), IFN-γ (10 ng/ml) or sterile IMDM (control group) at 37°C, 5% CO₂ for 30 min to 4 h, after which chemotaxis, adhesion, Ca²⁺ levels and integrin expression were evaluated, as detailed below. In some assays, cells were pretreated with MHC class II blocking antibody, a detailed below.

The number of neutrophils in BM was initially adjusted to 4 × 10⁶ cells/ml. Next, BM cells were incubated with SEA (1–30 ng/ml), IFN-γ (10 ng/ml) or sterile IMDM (control group) at 37°C, 5% CO₂ for 30 min to 4 h. Adhesion and chemotaxis assays for neutrophils were performed as described below. For the adhesion assays, 50 μL of BM cells, treated or not with SEA, were added to 96-well plates pre-coated with recombinant mouse VCAM-1 (2.5 μg/ml) or ICAM-1 (2.5 μg/ml) (Takeshita et al., 2015)_. BM neutrophils were then stimulated with IL-8 (300 ng/ml) for 30 min, and adhesion was calculated by measuring the myeloperoxidase (MPO) activity of adherent cells. Briefly, each well received hexadecyltrimethyl ammonium bromide (HTBA) 0.5% in 50 mM of potassium phosphate buffer, pH 6.0. Thereafter, each well received 200 μl of o-dianisidine solution (o-dianisidine: 0.167 mg/ml; hydrogen peroxide: 0.0005% in 50 mM of phosphate buffer, pH 6.0) immediately before absorbance measurement at 460 nm with a microplate reader (Synergy H1 Hybrid Reader, Biotek, USA). Neutrophil adherence was calculated using the optic density (OD) obtained in MPO assay divided by the number of neutrophils (NE) added in each well.

For the chemotaxis assays, we used a 96-multiwell chemotaxis chamber (Bignold et al., 1990). The wells in the microplate at the bottom compartment were filled with 29 μl of IL-8 (200 ng/ml). A polycarbonate filter (5 μm pore size) was positioned on the loaded microplate and hold in place with corner pins. Next, 25 μl of BM cells were placed directly onto the filter sites. The chamber was then incubated for 2 h at 37°C in humid atmosphere with 5% CO₂. Following incubation, the non-migrating cells on the origin side (top) of the filter were removed by gently wiping the filter with a tissue and the chamber was centrifuged at 200 g for 5 min at 20°C. The filter was removed and the number of neutrophils that migrated into the bottom compartment was determined by measuring MPO activity, as described above. The results were expressed as percent of neutrophils in relation of a standard curve of varying concentration of the original cells suspension.

The number of eosinophils in BM was initially adjusted to 8 × 10⁶ cells/ml. Next, BM cells were incubated with SEA (1–30 ng/ml), IFN-γ (10 ng/ml) or sterile IMDM (control group) at 37°C, 5% CO₂ for 30 min to 4 h. Adhesion and chemotaxis assays for eosinophils were performed as described below. Fifty microliter of BM cells were added to 96-well plates pre-coated with recombinant mouse VCAM-1 (2.5 μg/ml) or ICAM-1 (2.5 μg/ml) (Takeshita et al., 2015)_. BM eosinophils were then stimulated with eotaxin (300 ng/ml) for 30 min, and adhesion was calculated by measuring the peroxidase (EPO) activity of adherent cells. To achieve this, 50 μL of EPO substrate (1 mM H₂O₂, 1 mM o-phenylenediamine and 0.1% Triton X-100 in Tris-buffer pH 8.0) were added to each well. After 30 min of incubation at room temperature, 25 μl of H₂SO₄ (4 M) were added to each well to stop the reaction and absorbance was measured at 490 nM with a microplate reader (Synergy H1 Hybrid Reader, Biotek, USA). Eosinophil adherence was calculated considering the optic density (OD) obtained in EPO assay divided by the number of eosinophils (EOS) added in each well (Conran et al., 2001).

For the eosinophil chemotaxis assays, a 96-multiwell chemotaxis chamber was used (Roviezzo et al., 2004). The wells in the microplate at the bottom compartment were filled with 29 μl of eotaxin (300 ng/ml). A polycarbonate filter (5 μm pore size) was positioned on the loaded microplate and hold in place with corner pins. Next, 25 μl of BM cells were placed directly onto the filter sites. The chamber was then incubated for 2 h at 37°C in humid atmosphere with 5% CO₂. Following incubation, the non-migrating cells on the origin side (top) of the filter were removed by gently wiping the filter with a tissue and the chamber was centrifuged at 200 g for 5 min at 20°C. The filter was then removed and the number of cells that migrated into the bottom compartment was determined by measuring residual EPO activity, as described above. The results were expressed as % of eosinophils or neutrophils in relation of a standard curve of varying concentration of the original cells suspension.

Intracellular Ca²⁺ levels were monitored in non-adherent BM cells (1 × 10⁶ cells/ml) loaded with a fluorogenic calcium-binding dye (FluoForte), according to a previous study (Takeshita et al., 2015). BM cells were incubated with SEA (10 ng/ml), and suspended in a medium containing Fluoforte (3 μM) for 45 min at room temperature protected from light. Thereafter, cell suspension was centrifuged at 400 g for 10 min. Aliquots of cell suspension (300 μl) were dispensed into 96 wells plates (Synergy H1 Hybrid Reader, Biotek, USA) equipped with a stirring device. To obtain total intracellular Ca²⁺ levels, the external Ca²⁺ concentration was adjusted to 1 mM with CaCl₂, following equilibration for at least 30 s. Next, either IL-8 (200 ng/ml; for neutrophils) or eotaxin (300 ng/ml; for eosinophils) was added to induce cell activation. The Fluoforte fluorescence was monitored continuously with monochromator settings of 339 nm (excitation) and 500 mM (emission). The external influx of Ca²⁺ was calculated by subtracting the total Ca²⁺ levels from the internal storage mobilization. The intracellular calcium levels were calculated by use of a general formula: [Ca ²⁺]i = K_d × (F-F_min)/(F_max- F), were Kd = (389) is the Fluoforte dissociation constant, according to a previous study (Grynkiewicz et al., 1985). The F_max value was obtained with triton X-100 (0.1%) in the presence of saturating calcium (CaCl₂ 1 mM), whereas F_min value was obtained with the calcium chelator EGTA (10 mM) plus Tris (20 mM; pH= 8).

The effects of MHC class II blocking antibody on the adhesion and chemotactic responses of non-adherent BM cells after SEA or IFN-γ incubation were examined. Briefly, cells were incubated for 30 min at 37°C with mouse anti MHC class II blocking antibody (2 μg/ml) before addition of SEA, IFN-γ or IMDM. Next, BM cell adhesion or chemotaxis was assayed in the presence of either IL-8 (neutrophils) or eotaxin (eosinophils).

Cells were incubated with SEA for 2 h (100 μL of 3 × 10⁶ cells/ml of PBS containing 0.5% BSA and 0.2% azide). Next, each cell sample was incubated another 20 min at 4°C with fluorescein isothiocyanate-conjugated anti-mouse MAC-1, phycoerythrin (PE)-conjugated anti-mouse VLA-4, or fluorescein isothiocyanate- conjugated anti-mouse LFA1-α. Subsequent analysis using a Becton-Dickinson FACSCalibur Cytometer (San Jose, CA) with CellQuest Pro software was done. MAC-1, VLA-4, and LF1-α fluorescent signals were acquired in the FL-2 and FL-1 channels, respectively. Fluorescent events considered for analysis were those that appeared on a gate positioned in the region expected to be enriched with granulocytes on SSC vs. FSC dot-plot. Acquisition of 10,000 events was performed for all samples. Prior to fluorescence acquisition, independent groups of cells incubated with either PE-conjugated mouse IgGk monoclonal isotype control, FITC rat IgG2a (for LFA1-α) or IgG2b (for MAC-1) isotype control were analyzed in order to discriminate unspecific fluorescence in FL-2 and FL-1 channels. Surface content of MAC-1, VLA-4, and LFA1α were expressed by the mean fluorescence obtained from the histogram.

The IFN-γ levels were measured in mouse BM cell supernatant using commercially available ELISA kits (Mouse DuoSet ELISA Development System), following the instructions of the manufacturer (R&D, Minneapolis, MN, USA).

Data were presented as the mean values ± SEM and were analyzed by analysis of variance (ANOVA) for multiple comparisons followed by Bonferroni post-test, using a program package for statistical analysis (GraphPad software, version 5.00; San Diego, USA). p < 0.05 was considered significant.

Activation of BM neutrophils with IL-8 (200 ng/ml) markedly enhanced the cell adhesion on both VCAM-1 and ICAM-1 pre-coated plates in comparison with non-stimulated cells (P < 0.05; Figures 1A–D). Prior incubation (2 h) of neutrophils with SEA (3–30 ng/ml) significantly (P < 0.05) decreased the IL-8-induced adhesion, as observed in both ICAM-1 and VCAM-1-coated plates (Figures 1A,B). Inhibition of IL-8-induced neutrophil adhesion by SEA was of the same magnitude at 1 to 4 h incubation (Figures 1C,D).

Prior incubation (2 h) of neutrophils with SEA (1–30 ng/ml) also significantly (P < 0.05) decreased the IL-8-induced chemotaxis compared with non-stimulated cells (Figure 1E). Incubation of neutrophils with SEA (10 ng/ml) for prolonged time periods (4 h) did not produce a further inhibition of cell chemotaxis (Figure 1F).

The spontaneous neutrophil adhesion and chemotaxis (in the absence of IL-8) were not significantly affected by incubation with SEA in any dose and incubation-time used.

MAC-1 and LFA 1α are key integrins responsible for the neutrophil functions (Furze and Rankin, 2008; Nourshargh and Alon, 2014). Therefore, the expressions of both proteins in BM cells were evaluated in the presence and the absence of SEA, using flow cytometry. The expressions of MAC-1 and LFA 1α were not significantly affected by SEA compared with untreated cells (Table 1).

Activation of BM eosinophils with eotaxin (300 ng/ml) markedly enhanced (P < 0.05) the cell adhesion in plates pre-coated with either VCAM-1 (Figures 2A,C) or ICAM-1 (Figures 2B,D) in comparison with non-stimulated cells (spontaneous activity). Prior incubation (2 h) of eosinophils with SEA (1–10 ng/ml) significantly (P < 0.05) reduced the eotaxin-induced adhesion in VCAM-1 and ICAM-1-coated plates in comparison with untreated cells (Control; Figures 2A,B). Maximal inhibition of eosinophil adhesion by SEA (10 ng/ml) on VCAM-1 and ICAM-1 was obtained at 1 h-incubation, maintaining the inhibition by 4 h-incubation (Figures 2C,D).

Similar inhibitory results were obtained in the chemotaxis assays (Figures 2E,F). Eosinophil chemotaxis was markedly increased (P < 0.05) in eotaxin-stimulated cells, which was significantly inhibited by SEA (30 ng/ml) (Figures 2E,F). The spontaneous eosinophil chemotaxis was not significantly affected by SEA in any dose and incubation-time used.

The spontaneous adhesion and chemotaxis of eosinophils were not significantly affected by SEA in any dose and incubation-time used.

VLA-4 is the main integrin responsible for the eosinophil functions (Stone et al., 2010; Nourshargh and Alon, 2014). We have therefore evaluated the expression of this protein in BM cells by using flow cytometry. The expression of VLA-4 was not significantly affected by SEA compared with untreated cells (Table 1).

In BM cells activated with IL-8 (200 ng/ml) the intracellular Ca²⁺ levels was significantly increased (P < 0.05) compared to basal conditions (Table 2). Prior incubation with SEA significantly inhibited the intracellular Ca²⁺ mobilization in IL-8-activated cells. Similarly, in BM cells activated with eotaxin (300 ng/ml), the intracellular Ca²⁺ levels significantly increased (P < 0.05) compared to basal conditions, which was markedly reduced by incubation with SEA (Table 2).

BM cells were incubated with anti-MHC class II antibody (2 μg/ml; 30 min, 37°C, 5% of CO₂) before SEA incubation (10 ng/ml, 2 h). Anti-MHC class II antibody prevented he reductions by SEA of IL-8-induced adhesion (Figures 3A,B) and chemotaxis (Figures 3C) in comparison to non-treated cells.

Likewise, pre-treatment with anti-MHC class II antibody fully prevented the reduction by SEA of the eotaxin-induced eosinophil adhesion (Figures 4A,B) and chemotaxis (Figure 4C) in comparison to non-treated cells.

The levels of INF-γ was greater in the cell supernatant after SEA stimulation (10 ng/ml, 2 h; P < 0.05) when compared with control group (30.6 ± 6.2 and 104.3 ± 28.3 pg/mg protein for control and SEA, respectively). We next evaluated the effects of exogenous INF-γ addition on adhesion and chemotaxis of neutrophils and eosinophils. Figure 5 shows that incubation of neutrophils with IFN-γ (10 ng/ml) significantly reduced (P < 0.05) the IL-8-induced adhesion on VCAM-1 and ICAM-1 coated plates. Prior incubation with anti-MHC class II antibody prevented the reduced IFN-γ-induced adhesion on VCAM-1 (but not ICAM-1)-coated plates (P < 0.05) (Figures 5A,B). Incubation of neutrophils with IFN-γ also significantly reduced the IL-8-induced chemotaxis, which was prevented by prior treatment with anti-MHC class II antibody (Figure 5C).

Incubation of eosinophils with IFN-γ (10 ng/ml, 2 h) significantly reduced the eotaxin-induced adhesion on ICAM-1 (but not VCAM-1), as well as on cell chemotaxis (Figure 6). These reductions by IFN were fully restored (P < 0.05) by prior incubation with anti-MHC class II antibody.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.