International Immunopharmacology xxx (xxxx) xxxx

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International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

Airway exposure to Staphylococcal enterotoxin type B (SEB) enhances the

number and activity of bone marrow neutrophils via the release of multiple
cytokines
A.P. Ferreira-Duartea, A.S. Pinheiro-Torresa, W.M. Takeshitaa, V.O. Gushikena,
⁎
I.A. Roncalho-Bucka, G.F. Anhêb, I.A. DeSouzaa,
a
Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai (São Paulo), Brazil
b
Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Background: The lung infections by Staphylococcus aureus are strongly associated with its ability to produce
Staphylococcal enterotoxin B enterotoxins. However, little is known about the mechanisms underlying trafficking of bone marrow (BM)
Bone marrow neutrophils during airway inflammation induced by Staphylococcal enterotoxin B (SEB). We therefore aimed to
Cell trafficking investigate the effects of mouse airways SEB exposure on BM neutrophil counts and its adhesive properties as
Lung inflammation
well as on the release of cytokines/chemokines that orchestrate BM neutrophils trafficking to lung tissue.
MIP-2 and SDF1α
Methods: Male BALB/c mice were intranasally exposed to SEB (1 µg), and at 4, 16 and 24 h thereafter, BM,
circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. BM neutrophils adhesion,
MAC-1 and LFA1-α expressions (by flow cytometry) as well as measurement of cytokine and/or chemokines
levels were assayed after SEB-airway exposure.
Results: Prior exposure to SEB promoted a marked influx of neutrophils to BAL and lung tissue, which was
accompanied by increased counts of BM immature neutrophils and blood neutrophilia. BM neutrophil expres-
sions of LFA1-α and MAC-1 were unchanged by SEB exposure whereas a significant enhancement of adhesion
properties to VCAM-1 was observed. The early phase of airway SEB exposure was accompanied by high levels of
GM-CSF, G-CSF, IFN-γ, TNF-α and KC/CXCL1, while the latter phase by the equilibrated actions of SDF1-α and
MIP-2.
Conclusion: Mouse airways exposure to SEB induces BM cytokines/chemokines release and their integrated
actions enhance the adhesion of BM neutrophils leading to acute lung injury.

1. Introduction major serological types (SEA to SEE) and new types of Staphylococcus
aureus-related toxins (SEG to SEIY) [6]. These toxins are classified as
The gram-positive bacterium Staphylococcus aureus is an opportu- superantigens due their ability to bind to major histocompatibility
nistic pathogen strongly associated with severe hospital-acquired in- complex class-II (MHC class II) molecules without prior antigen pro-
fections, leading to acute lung inflammation, pneumonia and sepsis cessing by antigen-presenting cells, thus promoting strong stimulation
[1,2]. S. aureus often asymptomatically colonizes the upper respiratory of the immune system by T cells proliferation [7].
tract, but the risk of infection is increased when host defenses are Accumulation of pulmonary neutrophils is crucial for the defense of
compromised [3]. The lung infections by S. aureus are strongly asso- the organism against bacterial infections [8]. Neutrophils originate
ciated with its ability to produce several virulence factors such as ad- from the bone marrow (BM) hematopoietic stem cells that differentiate
hesins, collagenases, protein A, coagulases, hemolysins and leukocidins in a neutrophil lineage in response to both extracellular stimulus
[4]. Among these factors, Staphylococcal enterotoxins (SEs) are re- (growth factors and cytokines) and intracellular regulators, such as
cognized as one of the most important classes of virulence factors that transcription factors [9]. At homeostatic conditions, differentiation and
lead to the pathological conditions [5]. SEs belong to a family of maturation of BM myeloid progenitor cells are tightly regulated by low
structurally related heat-stable 25–30 kDa proteins, comprising five serum levels of granulocyte colony-stimulating factor (G-CSF) and

⁎
Corresponding author at: Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), 13202-550 Jundiai (São Paulo), Brazil.
E-mail address: [email protected] (I.A. DeSouza).

https://doi.org/10.1016/j.intimp.2019.106009
Received 1 August 2019; Received in revised form 30 August 2019; Accepted 25 October 2019
1567-5769/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: A.P. Ferreira-Duarte, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.106009
A.P. Ferreira-Duarte, et al. International Immunopharmacology xxx (xxxx) xxxx

granulocyte-macrophage colony-stimulating factor (GM-CSF) [10]. airways administration. BAL fluid, blood, BM and lung tissue were
However, in inflammatory conditions, besides the high levels of G-CSF obtained at different times after SEB administration, as detailed below.
and GM-CSF, other mediators such as tumor necrosis factor-α (TNF-α),
interferon-γ (IFN-γ), interleukin-3, stromal cell-derived factor 1-α 2.4. Leukocyte counts in bronchoalveolar fluid
(SDF1-α) and KC (CXCL1) induce a rapid increase in the production of
BM granulocytes, consequently enhancing the number of neutrophils in BAL fluid was obtained at 4, 16 or 24 h after intranasal SEB ad-
peripheral blood [11]. Blood neutrophils adhere to the endothelium, ministration. Briefly, the trachea was cannulated with a polyethylene
allowing their migration to inflammatory sites through interactions of catheter (1 mm in diameter) connected to a syringe. The lungs were
MAC-1 (αMβ2; CD11b/CD18) and lymphocyte function-1 (LFA-1; washed by flushing with PBS solution containing heparin (20 UI/mL).
αLβ2; CD11a/CD18) integrins in neutrophils with ICAM-1 and VCAM-1 The PBS buffer was instilled through a tracheal cannula as a one 1.5-mL
immunoglobulins in endothelial cells [12]. Among the SE, SEA is the aliquot recovered by five 300-mL aliquots. The fluid collected was
most studied due its potent effect on the stimulation of T cells. When centrifuged (500g for 10 min at 20 °C). The cell pellet obtained was
administered orally, SEA produces a non-specific stimulation of intra- resuspended in 2 mL of PBS solution. Total leukocyte counts were
epithelial lymphocytes in the intestinal mucosa which results in am- performed in a Neubauer chamber, while differential counts were car-
plification of normal local immunologic responses, by increased pro- ried out on a minimum of 200 cells using cytospin preparation stained
duction of IFN-γ [13]. Exposure to SEA in the airways of rodents leads with Diff-Quick. The leukocytes were classified as PMN (neutrophils
to acute lung inflammation and massive neutrophil infiltration [14] due and eosinophils) and mononuclear cells based on normal morphological
to the release of multiple pro-inflammatory mediators, including TNF- criteria.
α, interleukins (IL)-1, IL-2, IL-6, IL-8 and IL-10, nitric oxide, arachi-
donic acid metabolites and neuropeptides [15,16]. Alongside SEA-in- 2.5. Histopathological analysis
duced cells infiltration in airways, in BM there is a marked increase of
mature and immature forms of neutrophils [14]. Recently, airway ex- Mouse lungs were removed after intranasal exposure to SEB (or
posure to SEA was shown to induce BM accumulation of mature and PBS) and post-fixed by immersion for at least 24 h with 10% buffered
immature forms of neutrophils which was associated with integrated formalin, after which they were macroscopically examined and cut
actions of the cytokines/chemokines, amplifying the adhesive responses transversally into slices of ~3 mm. For histopathological analysis, only
of neutrophils [17]. SEB is the most potent SE, since at even low con- the middle third of the caudal aspects of both lungs were sent for em-
centrations it can cause multiple organ system failure and death. This bedding in paraffin. Sections of these lung portions, 4–5 μm thick, were
toxin is produced in large quantities by Staphylococcus aureus and stained with hematoxylin–eosin and evaluated under a Nikon Eclipse
possibly the main responsible for the pathological conditions induced E200 microscope adapted with a Nikon Coolpix 995 camera (3 Mpixel).
by this bacterium [18]. However, SEB has been largely neglected re- For each animal, the extent of the lung infiltrate was determined by
garding its pro-inflammatory actions, and no studies have evaluated the establishing the percentage of compromised bronchial and bronchiolar
consequences in BM after lung exposure. Therefore, in the present segments within 30 of such structures, randomly selected at low power
study, we used a model of SEB exposure in the airways to investigate fields (i.e., using a ×4 objective). In addition, using the 40× objective,
the adhesive properties of BM neutrophils as well as the release profile 18 random digital images per group were taken within areas of overt
of cytokines and chemokines that orchestrate trafficking of BM neu- peribronchiolar inflammation. Total inflammatory cell counts were
trophils to lung tissue. determined from these images, using the Imagelab Analysis Software
(version 2.4) (IMAGELAB 2000; Softium, São Paulo, Brazil) and ex-
2. Material and methods pressed as number of cells/mm2. Quantification was focused on peri-
bronchiolar areas, provided these regions were the main sites of in-
2.1. Reagents flammation.

Staphylococcal enterotoxin B (SEB), and Formyl-Methionyl-Leucyl- 2.6. Leukocyte counts in blood and bone marrow (BM)
Phenylalanine (fMLP) were purchased from Sigma Aldrich Co (St.
Louis, MO, USA). Interleukin-8 (IL-8), ELISA kits for all cytokines, ad- Blood samples were obtained from the abdominal artery after SEB
hesion molecules (VCAM-1 and ICAM-1), fluorescein isothiocyanate- or PBS administration into the mouse airway. The total counts were
conjugated anti-mouse MAC-1 and anti-mouse LFA1-α were obtained made (Neubauer), and blood smears were used for differential counts
from BD Biosciences Pharmingen (San Jose, CA, USA). Fluofort (Enzo (Diff-Quick stain) where a minimum of 300 cells were counted and
Life Sciences International, NY, USA). classified as PMN and mononuclear cells, on normal morphological
criteria. For BM leukocyte counts, femurs from mice were removed
2.2. Animal experimentation guidelines immediately after sacrifice, at 4, 16 and 24 h after exposure to SEB or
PBS. The epiphyses were cut transversely, and BM cells were collected
All animal care and experimental protocols were approved by the by flushing the two femurs with PBS (2.5 mL per femur), and the total
Ethical Principles in Animal Research adopted by the Brazilian College (Neubauer) and differential (Diff-Quick stain) cell counts were made. A
for Animal Experimentation (COBEA), and followed the Guide for the minimum of 300 cells were counted in order to differentiate immature
Care and Use of Laboratory Animals. Four-week-old male BALB/c mice granulocytes (myeloblasts, pro-myelocytes, and neutrophilic/eosino-
were provided by the Central Animal House Services of State University philic myelocytes) or mature granulocytes (neutrophilic/eosinophilic
of Campinas (UNICAMP). Animals were housed three per cage on a 12 h metamyelocytes, neutrophilic/eosinophilic band cell and mature neu-
light–dark cycle, in temperature-controlled rooms and received water trophil/eosinophil).
and food ad libitum until used in the Animal House Services of Faculty of
Medicine of Jundiaí (FMJ). 2.7. Measurement of cytokines in BM

2.3. Intranasal administration of SEB and leukocyte counts in BAL fluid The cytokines GM-CSF, KC/CXCL-1, TNF-α, IFN-γ, SDF1-α, G-CSF
and MIP-2 were measured in the BM supernatant. All measurements
BALB/c mice were intranasally exposed to SEB (0.1, 0.3 or 1 µg/ were carried out using commercially available ELISA kits (Mouse
50 µL) or sterile phosphate-buffered saline (PBS; control group). All SEB DuoSet ELISA Development System), following the instructions of the
solutions was prepared and maintained in sterile conditions until mice manufacturer (R&D, Minneapolis, MN, USA).

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2.8. Neutrophil adhesion assays in ICAM- and VCAM-coated plates storage mobilization. The intracellular calcium levels were calculated
by use of a general formula: [Ca2+]i = Kd X (F-F min)/(F max- F),
BM cells were collected at 4, 16 and 24 h after SEB or PBS intranasal where Kd = (389) is the Fluoforte dissociation constant, according to a
administration, and subsequently placed in plates (100 mm × 20 mm previous study. The F max value was obtained with triton X-100 (0.1%)
dish) for 30 min at 37 °C, 5% CO2. The cell supernatants were collected, in the presence of saturating calcium (CaCl2 1 mM), whereas Fmin value
and the plates were washed with 2 mL of RPMI 1640 medium. The was obtained with the calcium chelator EGTA (10 mM) plus Tris
supernatant was centrifuged (500g for 10 min at 4 °C), and the cell (20 mM; pH = 8)
pellets were resuspended in 2.5 mL of RPMI. The cell number for each
assay was 4 × 106 cells/mL [19]. The final cell suspension contained 2.11. Statistical analysis
about 70% of mature granulocytes, consisting of 60% neutrophils and
10% eosinophils, whereas the remainder was made of 30% of mono- Data were presented as the mean values ± SEM and were analyzed
nuclear cells. The adhesion of neutrophils was evaluated through the by analysis of variance (ANOVA) for multiple comparisons followed by
measurements of specific peroxidase for neutrophils (myeloperoxidase; Bonferroni post-test, using a program package for statistical analysis
MPO), as detailed below. (GraphPad software, version 5.00; San Diego, USA). p < 0.05 was
The adhesion assays were carried out in 96-well plates pre-coated considered significant.
with recombinant mouse VCAM-1 (2.5 µg/mL) or ICAM-1 (2.5 µg/mL)
for 30 min at 37 °C, 5% CO2. For neutrophil adhesion assays, after in-
3. Results
cubation with Iscovés modified medium (spontaneous adhesion) or
with fMLP (10-8 M), non-adhered cells were removed, and the re-
3.1. Characterization of lung neutrophil infiltration induced by airways
maining cells were washed twice with PBS. Neutrophil adhesion was
exposure to SEB
calculated after 30 min by measuring the amount of myeloperoxidase
(MPO) [20]. For this, each well received hexadecyltrimethyl ammo-
SEB-induced airway inflammation was evaluated by measuring the
nium bromide (HTBA) 0.5% in 50 mM of potassium phosphate buffer,
neutrophil infiltration to BAL fluid and lung tissue. Instillation of SEB at
pH 6.0. Thereafter, each well received 200 µL of o-dianisidine solution
0.3 and 1.0 µg produced a dose-dependent cell infiltration in BAL fluid
(o-dianisidine: 0.167 mg/mL; hydrogen peroxide: 0.0005% in 50 mM of
compared with PBS group (Table 1). The dose of 0.1 µg of SEB did not
phosphate buffer, pH 6.0) immediately before absorbance measurement
produce airway cell infiltration (Table 1). For the further assays, we
at 460 nm with a microplate reader (Synergy H1 Hybrid Reader, Biotek,
routinely used SEB at 1 µg.
USA). Neutrophil adherence was calculated using the absorbance values
Histopathological analysis revealed absence of lung neutrophil in-
and the number of neutrophils added in which well.
filtration from PBS-instilled mice. However, airway exposure to SEB
(1.0 µg) markedly enhanced neutrophil (Fig. 1A, B–E) influx into the
2.9. Flow cytometry assays
connective tissue surrounding the bronchial and bronchiolar segments
within 24 h period observation with maximal response at 4 h after SEB.
BM granulocytes from mice subjected to SEB exposure in the airway
The maximal neutrophil accumulation in BAL fluid was achieved at
(100 μL of 3 × 106 cells/mL of PBS containing 0.5% BSA and 0.2%
16 h (Fig. 1A). At 24 h, neutrophil counts in BAL fluid and lung tissue
azide; n = 5) were incubated for 20 min AT 4 °C with either with
diminished, returning to basal conditions at 48 h (data no shown,
fluorescein isothiocyanate-conjugated anti-mouse MAC-1, fluorescein
n = 5). These data clearly demonstrate that airways SEB exposure
isothiocyanate-conjugated anti-mouse LFA1-α, phycoerythrin (PE)-
produces a marked lung inflammation.
conjugated for subsequent analysis using a Becton-Dickinson
FACSCalibur Cytometer (San Jose, CA) with CellQuest Pro software.
MAC-1 or LFA1-α fluorescent signals were acquired in the FL-1 and FL- 3.2. Airway exposure to SEB increases number of neutrophils in circulating
2 channels, respectively. Fluorescent events considered for analysis blood and BM
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 We next evaluated the influence of SEB-induced lung inflammation
10,000 events was performed for all samples. Prior to acquisition of on the neutrophil counts circulating blood and BM. The number of
fluorescence, independent groups of cells incubated with either PE- circulating blood neutrophils increased significantly within 24 h after
conjugated mouse IgGk monoclonal isotype control, FITC rat IgG2a (for airway SEB exposure with maximal response at 4 h in comparison with
LFA1-α), IgG2b (for MAC-1) isotype control were analyzed in order to PBS-instilled mice (p < 0.05) (Fig. 2A).
discriminate unspecific fluorescence in FL-1 and FL-2 channels. Surface BM immature neutrophils count increased significantly at 16 h after
content of MAC-1 and LFA1-α was expressed by the mean fluorescence SEB exposure (Fig. 2B), whereas no significant alterations were ob-
obtained from the histogram. served in the number of mature forms (p < 0.05) (Fig. 2C). Lung in-
flammation after SEB exposure is accompanied by increases in both
2.10. Intracellular Ca2+ measurements
Table 1
Dose-response of airways SEB exposure on neutrophil influx to BAL fluid and
Mouse BM granulocytes (2 × 106 cells/ m) obtained as described
BM (immature and mature counts).
above were suspended in MEM in the presence of Fluoforte (3 µM) for
45 min at room temperature protected from light. Thereafter, cells were BAL fluid (×106/mL) Bone Marrow (×106/femur)
centrifuged at 400g for 10 min. The pellets were resuspended
Immature Mature
(1 × 106 cells/mL) in MEM. Aliquots of cell suspension (300 µL) were
dispensed into 96 wells plates (Synergy H1 Hybrid Reader, Biotek, USA) PBS 0.10 ± 0.01 2.04 ± 0.59 14.34 ± 1.81
equipped with a stirring device. To obtain total intracellular calcium SEB (0.1 µg) 0.11 ± 0.01 3.05 ± 0.18 15.83 ± 1.24
levels, the external Ca2+ concentration was adjusted to 1 mM with SEB (0.3 µg) 0.15 ± 0.03* 2.78 ± 0.11 14.09 ± 1.36
SEB (1.0 µg) 0.61 ± 0.11* 2.77 ± 0.17 16.21 ± 0.46
CaCl2, following equilibration for at least 30 s. Next, IL-8 (200 ng/mL)
was added to induce activation of BM granulocytes. The fluorescence of Mice were exposed to SEB at the indicated doses for 4 h before the analysis of
Fluoforte was monitored continuously with monochromator settings of neutrophils counts in BAL and BM cell counts. Control animals were instilled
339 nm (excitation) and 500 mM (emission). The external influx of Ca with PBS. Results are mean ± SEM from 5 mice for each group.
2+
was calculated by subtracting the total Ca2+ levels from the internal *p < 0.05p < 0.05 was considerate significantly compared with PBS group.

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Fig. 1. Neutrophil infiltration in lung tissue and bronchoalveolar lavage (BAL) fluid after mouse airway exposure to Staphylococcal enterotoxin B (SEB). Animals
were exposed to SEB (1 µg) for 4, 16 and 24 h before the analysis of neutrophils counts in BAL and lung tissue (Panel A). Control animals were instilled with PBS
(50 µL, control group). Each column represents mean ± SEM from 5 mice for each group. *p < 0.05 compared with PBS group. Panels B-E: Representative images
from PBS, SEB (4 h), SEB (16 h) and SEB (24 h) groups. Note the marked peribronchiolar infiltrate in mice exposed to SEB when compared with PBS group.
Hematoxylin and eosin; scale bars = 50 μm. Arrows indicate neutrophils.

Fig. 2. Mouse airway exposure to Staphylococcal enterotoxin type B (SEB) increases the number of circulating blood neutrophils (panel A) and immature forms of
bone marrow (BM) neutrophil (panel B). Animals were exposed to SEB (1 µg) at 4, 12 and 24 h. Control animals were instilled with phosphate-buffered saline (PBS;
50 µL). Mature forms of BM neutrophils were also evaluated (panel C). Results are mean values ± S.E.M from 5 mice for each group. *p < 0.05 compared with PBS
group.

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Fig. 3. Levels of bone marrow (BM) GM-CSF (A), G-CSF (B), IFN-γ (C), TNF-α (D), KC/CXCL1 (E), SDF1-α (F) and MIP-2 (G) after mouse airway exposure to
Staphylococcal enterotoxin B; SEB). Mouse BM supernatants were obtained 4, 16 and 24 h post-exposure to SEB (1 µg). Control animals were instilled with PBS
(50 µL). Results are mean values ± S.E.M from 5 mice for each group. *p < 0.05 compared with PBS group.

circulating neutrophils and BM immature cells at 16 h. 3.5. MAC-1 and LFA1-α expression in BM neutrophils

In separate experiments, MAC-1 and LFA1-α expressions in BM

3.3. Levels of cytokines and chemokines in BM neutrophils were evaluated by flow cytometry. Expressions of MAC-1
and LFA1-α in neutrophil remained unchanged in BM neutrophils of the
Cytokines and chemokine levels were assayed in BM supernatant SEB group compared with the PBS group (Table 2).
from SEB-exposed mouse at 4, 6 and 24 h. In the SEB group, increased
levels of G-CSF, TNF-α and KC/CXCL-1 were observed at 4 h when 3.6. Intracellular calcium levels in activated BM neutrophils
compared with PBS-instilled mice (Fig. 3B, D, E). At 16 h post-SEB
exposure, elevated levels of GM-CSF, IFN-γ and KC CXCL-1 were de- We next tested if enhanced Ca2+ mobilization in response to IL-8
tected (Fig. 3A, C, E). Higher levels of G-CSF, TNF-α, SDF1-α and MIP-2 (neutrophils) is affected by prior exposure to SEB. In the PBS group,
were also detected at 24 h post-SEB exposure (Fig. 3B–G). Thus, airways activation of BM neutrophils with IL-8 (200 ng/mL) produced a sig-
SEB exposure induces the release of cytokines and chemokines that may nificant increase in Ca2+ levels (p < 0.05) when compared with basal
orchestrate BM neutrophil trafficking to lung tissue. Ca2+ mobilization, which was further increased when BM neutrophils
had been obtained from SEB-exposed mice (Fig. 5). Higher intracellular
Ca2+ levels found in IL-8-activated BM neutrophils of SEB demonstrate
3.4. Adhesive properties of BM neutrophils for VCAM-1 and ICAM-1 a state of maximal cell activation.

The adhesive properties of BM neutrophils were measured in VCAM- 4. Discussion

1 and ICAM-1 pre-coated plates. In the PBS group, activation of BM
neutrophils with fMLP (10−8 M) significantly increased (p < 0.05) cell In the present study, we have demonstrated that neutrophil in-
adhesion to VCAM-1 when compared with spontaneous adhesion filtration in SEB airway-exposed mice impacts the BM environment by
(Fig. 4A). Prior exposure to SEB in the airways after 4 h further in- enhancing immature neutrophils count and the levels of the cytokines/
creased (p < 0.05) fMLP-induced BM neutrophil adhesion to VCAM-1 chemokines; GM-CSF, G-CSF, IFN-γ, TNF-α, KC/CXCL1, SDF1-α and
(Fig. 4A). Similarly, the fMLP-induced neutrophil adhesion to ICAM-1 MIP-2. It was also found that the activation state of BM cells (as mea-
was largely increased (p < 0.05) compared with spontaneous adhesion sured by intracellular Ca2+ levels) was higher in SEB group as well as
(Fig. 4B). However, SEB exposure failed to affect the BM neutrophil the adhesive response to VCAM-1.
adhesion to ICAM-1 (Fig. 4B). Mouse airways exposure to SEB enhance Neutrophils are the most abundant leukocyte produced by BM, and
the BM adhesive properties in VCAM-1, a cell adhesion molecule that the storage pool of mature neutrophils can be rapidly mobilized into
regulates the transendothelial cell migration and its closely associated circulation during infections by a mechanism involving an orchestrated
with different physiopathological conditions, including lung in- production and release of cytokines and chemokines that tightly reg-
flammation. ulate its adhesive and chemotactic properties [12,21]. In the present

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Fig. 5. Mouse airway exposure to Staphylococcal enterotoxin B (SEB) increases

the calcium levels in bone marrow (BM) neutrophils. Mice were exposed to SEB
(1 µg) or instilled with PBS. Isolated BM granulocytes (2 × 106 cells/mL) were
loaded with Fluofort (10 µM) and stimulated with IL-8 (200 ng/mL). Assays
were carried out in the presence of Ca 2+ (1 mM) to yield total influx of Ca 2+.
Results represent the mean values ± SEM (n = 4–5 animals). *p < 0.05
compared to respective basal Ca2+ mobilization #p < 0.05 compared with IL-
8 levels in PBS group.

rapidly migrate to peripheral blood to further reach the respiratory

mucosa.
GM-CSF and G-CSF are classically known to regulate the prolifera-
tion, differentiation and maturation of BM myeloid progenitor cells into
neutrophils, facilitating their trafficking to the blood during in-
flammatory diseases [22,23]. Staphylococcal enterotoxins directly and
indirectly elicit GM-CSF production in human blood monocytes and
nasal epithelial cells [24]. In our study, pulmonary neutrophil in-
filtration induced by SEB at 16 h was accompanied by higher levels of
GM-CSF in BM, which may explain the increased counts of immature
Fig. 4. Mouse airways exposure to Staphylococcal enterotoxin B (SEB) en-
hances adhesion of bone marrow neutrophils to VCAM-1 but not to ICAM-1- neutrophils at this time-period. On the other hand, the G-CSF levels in
coated plates. After 4, 16 and 24 h of SEB (1 µg) exposure (or PBS instillation), BM were significantly higher at 4 h and 24 h (but not at 16 h) after SEB
BM was collected and placed on VCAM-1 (panel A) or ICAM-1 (panel B)-coated exposure suggesting a role of G-CSF at the initial phase of BM neu-
plates. BM was stimulated or not with fMLP (10−8 M). After 30 min, the neu- trophil trafficking and in restoring BM homeostatic conditions after the
trophil adhesion was quantified by measurement of myeloperoxidase (MPO) critical stage of SEB-induced lung inflammation.
activity. Results are mean values ± S.E.M from 5 mice for each group. An accurate regulation of the IFN-γ response from the host is re-
*p < 0.05 compared with respective spontaneous adhesion; #p < 0.05 com- quired for effective inflammatory reactions against microbial infections
pared with PBS group after stimulation with fMLP. NE, neutrophils; OD, optical [25]. IFN-γ is known to be a suppressor of hematopoiesis and BM failure
density.
syndromes during chronic inflammation are associated with increased
IFN-γ production [26]. Although, in BM granulocytes from mice, IFN-γ
Table 2 reduces chemotactic and adhesive properties of these cells [27], high
Effects of mouse airway exposure to Staphylococcal enterotoxin B (SEB) on the levels of IFN-γ have been associated with increased expression of en-
MAC-1 and LFA-1α expressions in bone marrow granulocytes. dothelial adhesion molecules essential to leukocyte recruitment [28].
Mean Fluorescence Intensity Exposure of SEB to mice leads to T cell activation and proliferation that
is regulated by high IFN-γ production [29]. In our study, high levels of
MAC-1 LFA 1α IFN-γ in BM were found at 16 h after SEB exposure, indicating a reg-
Control 1216.8 ± 77.7 154.2 ± 7.6
ulatory role of this cytokine on BM neutrophil mobilization after in-
SEB 1209.9 ± 115.6 162.4 ± 9.8 fection.
Tumor necrosis factor-α (TNF- α) is a potent cytokine implicated in
Mice were exposed to SEB (1 µg) or instilled with PBS. BM granulocytes were acute and chronic lung inflammatory diseases [30] and plays a crucial
gated for MAC-1 and LFA1-α population for the expression analysis of these role in the differentiation of BM granulocytes during pulmonary in-
molecules on flow cytometer. Results are mean values ± SEM from 5 mice for flammation by increasing BM myeloid cell production [31]. An asso-
each group. p < 0.05 was considerate significantly.
ciation between TNF-α and the chemokine KC/CXCL1, favoring neu-
trophil influx has been reported in mice [32]. Local release or
study we confirmed that airway exposure of SEB in mice resulted in
exogenous injection of KC/CXCL1 consequentially increases TNF-α
marked neutrophil accumulation in the peribronchiolar and alveolar
production that is prevented by treatment with anti-CXCL1 [33]. Che-
space of the lungs within 24 h. As well as this, in BM, we observed a
mokine KC/CXCL1 itself selectively stimulates rapid mobilization of
significant increase in immature (but not mature) forms of neutrophil,
mature neutrophils from BM to inflammatory sites [34]. Additionally,
suggesting an efficient and rapid progenitor cell differentiation to re-
an effective BM neutrophil evasion into blood is antagonistically
store mature neutrophil stores. The number of neutrophils in the blood
regulated by the chemokine receptors CXCR2 and CXCR4, both of
peaked at 4 h but remained elevated until 24 h after airway exposure of
which are expressed in neutrophils [35]. While CXCR4 retains neu-
SEB. Therefore, our findings that number of the mature neutrophils in
trophils in BM, CXCR2 ligands facilitate their exit into the blood. The
BM is not affected by airway SEB exposure indicates that these cells

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CXCR4 ligand SDF-1α (CXCL12), and the CXCR2 ligands KC (CXCL1) Acknowledgements
and macrophage inflammatory protein MIP-2 (CXCL2) are both con-
stitutively expressed on BM endothelium [36]. In a murine pulmonary Ivani A. Desouza thanks the Fundação de Amparo à Pesquisa do
epithelial cell line, TNF-α enhances both mRNA and protein levels of Estado de São Paulo (FAPESP) for financial support (Grant: 2017/
MIP-2 [37]. In a murine model of acute peritonitis, rapid mobilization 25867-8). Authors also thank Dr. Edson Antunes of the Department of
of BM neutrophils is driven by the coordinated actions of KC, MIP-2 and Pharmacology (Faculty of Medical Sciences of the State University of
G-CSF acting via distinct mechanisms in which G-CSF assumes a reg- Campinas – UNICAMP) for providing reagents and a critical revision of
ulatory role by retaining BM neutrophils via down-regulating CXCR4 the article for important intellectual content.
expression [32]. In our study, the levels of TNF-α and KC were elevated
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