A myristoylated alanine-rich C-kinase substrate (MARCKS) inhibitor peptide attenuates neutrophil outside-in β₂-integrin activation and signaling

ABSTRACT

MARCKS is an actin and PIP2-binding protein that plays an essential role in neutrophil migration and adhesion; however, the molecular details regarding MARCKS function in these processes remains unclear. Neutrophil adhesion and migration also require the cell surface receptors β₂-integrins. We hypothesized that MARCKS inhibition would alter neutrophil β₂-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in β₂-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out β₂-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn²⁺-stimulated static adhesion, cell spreading and β₂-integrin clustering, suggesting a role for MARCKS function in outside-in β₂-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in β₂-integrin activation and signaling.

KEYWORDS:

• Beta2-integrin
• ICAM-1
• MARCKS
• neutrophils
• outside-in

Introduction

Neutrophils play an essential role during the innate immune response. To transmigrate from the vasculature to sites of inflammation and/or microbial infection, neutrophils must be recruited from the bloodstream using specialized cell surface receptors, known as beta2 (β₂)-integrins, that interact with adhesion molecules expressed on the surface of endothelial cells (e.g., intercellular adhesion molecule 1 (ICAM-1)). The details of neutrophil capture, rolling, endothelial adhesion, post-adhesion strengthening, crawling, and transmigration have largely been determined; however, there are still gaps in knowledge regarding specific cell signaling mechanisms [1,2], including β₂-integrins. It is clear that β₂-integrins play an important role in adhesion strengthening, crawling, and transmigration [3], all of which are required processes for normal biological functions and host defense mechanisms. This is evidenced by the severe consequences of lack of functional β₂-integrins seen in patients with leukocyte adhesion deficiency (LAD), which severely weakens the host immune system, resulting in recurrent and/or chronic bacterial infections leading to significant morbidity and mortality [4].

Although critical for survival, neutrophils are considered a double-edged sword. When their destructive powers designed to combat pathogens become dysregulated, excessive neutrophil infiltration or neutrophil activation can cause significant damage to host tissues [5]. In fact, neutrophil derived tissue injury is known to play a role in numerous acute and chronic inflammatory diseases, including myocardial infarction [6], stroke [7], ischemia-reperfusion injury [8,9], rheumatoid arthritis [10,11], severe SARS-CoV-2 [12,13], and sepsis-associated organ damage (i.e., acute lung injury) [14,15]. Several animal models have highlighted the potential and/or ability to target neutrophils by inhibiting β₂-integrins [14,16]; however, the use of monoclonal antibodies targeting β₂-integrins in clinical settings have not been successful [17,18]. Despite this, β₂-integrins remain a desirable target for modulating neutrophil responses [19]. A more detailed understanding of how β₂-integrins are regulated may help identify novel targets for modulating dysregulated neutrophil responses.

B₂-integrins are transmembrane heterodimers that consist of a common β-subunit (CD18), which is non-covalently associated with one of the four known α-subunits (CD11a, CD11b, CD11c, and CD11d) [20]. The two most prominent integrins on neutrophils are CD11a (LFA-1 or α_Lβ₂) and CD11b (Mac-1 or α_Lβ₂). Within circulating, inactive neutrophils, CD11b integrins are primarily contained within the cytoplasm, secondary and tertiary granules, and secretory vesicles. The CD11a integrins that are present on the surface of resting neutrophils exist in an inactive/low affinity state with a ‘bent’ conformation. This combination of low surface expression and inactive conformation are control measures to help prevent nonspecific neutrophil binding and activation, as this could have damaging effects on host tissue and vasculature. When neutrophils receive an activation signal through intracellular signaling pathways, β₂-integrin surface expression is increased (upregulation), and conformational changes take place (‘affinity’). This type of integrin stimulation is termed ‘inside-out’ activation and is driven by intracellular signals received by G protein coupled receptors (GPCRs) and Fc receptors, among others [21]. B₂-integrins can also become activated when the extracellular domain interacts directly with extracellular matrix proteins or other cell surface ligands (ICAM-1, fibrinogen, etc.) and initiates its own signaling, termed ‘outside-in’ activation [1,20,22,23]. Details regarding the downstream signaling pathway from outside-in β₂-integrin activation are incomplete; however, multiple lines of evidence show that this type of activation is linked to neutrophil firm adhesion and cell spreading [23].

Myristoylated alanine-rich C kinase substrate (MARCKS) is a well-known protein kinase C (PKC) substrate and actin-binding protein. It is essential for several integrin-dependent cellular functions, such as adhesion, cell spreading, and migration in numerous cell types [24–26]. In most resting cells, MARCKS protein is associated with the inner leaflet of the plasma membrane due to hydrophobic insertion of an N-terminal myristoyl-moiety into the lipid bilayer, and electrostatic interactions between the MARCKS effector domain (ED) and 3–4 PIP2 molecules [27–29]. The ED of MARCKS is subject to reversible regulation by PKC phosphorylation or Ca⁺⁺/calmodulin binding. Either of these events displace MARCKS from the membrane to the cytosol and releases PIP2. Once dephosphorylated, MARCKS reassociates with the plasma membrane [30–33]. This reversible association with the plasma membrane is known as the ‘myristoyl-electrostatic switch’ and has been shown to play a key role in adhesion and cell spreading in multiple cell types [34,35].

To date, in vitro research has demonstrated a role for MARCKS protein in exo-, endo- and phagocytosis, embryogenesis, growth-cone formation, myoblast spreading and adhesion, mast cell and neutrophil degranulation, tumor cell motility, and directed migration of vascular endothelial cells, fibroblast and neutrophils [24,25,33,36–42]. MARCKS function has been examined in various cell types by a variety of methods including transgenic expression of mutant proteins, RNAi cellular knock-down, knock-out mice (embryonic lethal) and inhibitor peptides designed to mimic either the effector domain or N-terminus amino acid sequence. Derivatives of the N-terminus mimetic peptide known as MANS (myristoylated N-terminal sequence) have entered Phase II clinical trials as potential airway therapeutics [43–45]. Additional mechanistic understanding regarding the effect of MARCKS-targeting peptides on neutrophil function could further inform development of novel anti-inflammatory therapies.

Previous results from our lab and others have shown that inhibition of MARCKS with an N-terminal mimetic peptide known as MANS significantly attenuates functional responses of primary neutrophils, including migration and adhesion [24,26,36,43,46]. Studies by Eckert et al. determined that MANS peptide displaces MARCKS from the membrane to the cytosol in resting neutrophils, but does not alter fMLP-induced MARCKS phosphorylation [26]; however, additional insight into the mechanisms of MANS effect on neutrophil functions have not been elucidated. In other cell types, such as myoblasts and fibroblasts, MARCKS has been implicated in integrin-dependent cellular responses [25,47]. In the current study, we hypothesized that inhibition of MARCKS with the MANS peptide would alter β₂-integrin activation and signaling in primary human neutrophils. We examined the impact of MANS-mediated MARCKS inhibition on β₂-integrins using methods to target both ‘inside-out’ and ‘outside-in’ β₂-integrin activation pathways. Our results show that MANS peptide does not impact fMLP-stimulated inside-out β₂-integrin upregulation or affinity change but does inhibit outside-in β₂-integrin-dependent adhesion, cell spreading and avidity, as well as β₂-integrin-dependent respiratory burst. This study further informs our understanding of the cellular mechanism of MARCKS inhibition with MANS and is the first study to suggest a role for MARCKS protein in outside-in β₂-integrin activation and signaling in primary human neutrophils.

Materials and methods

Isolation of human PMNs

Human blood collection protocols were reviewed and approved by Institutional Research Ethics Committee of NCSU (IRB approval #616). For all neutrophil experiments, 10–30 ml of whole blood was collected using heparinized syringes from healthy human volunteers that provided informed consent for participation. Neutrophils were isolated from whole blood using Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation. Briefly, heparinized whole blood was mixed with 0.6% Dextran with 0.9% NaCl in a 15 mL polypropylene conical and allowed to settle at room temperature for 45–60 minutes. Up to 10 ml of leukocyte-rich plasma was aspirated using a bulb syringe and layered on 5 mL of Ficoll in a separate 15 mL conical tube. Cells were then centrifuged at 1800 rpm for 20 minutes with the brake off. The supernatant was discarded and remaining blood cells within the cell pellet were removed by 60 seconds of hypotonic lysis. Isolated neutrophils were resuspended/washed in sterile HBSS (Life Technologies) without additives. Cell number and viability was quantified using trypan blue exclusion (1:1) and a manual hemocytometer count. Final suspension of cells was in HBSS⁺⁺ chemotaxis buffer [1× HBSS, 1 mM Ca²⁺, 1 mM Mg²⁺, 5% fetal bovine serum (Gibco)] at the indicated concentration for each experiment. All experiments were completed within 4 to 6 hours of blood collection. Neutrophils from individual human donors were used for all time points and treatment conditions for each experiment (i.e., ‘n’ represents a separate human donor).

Peptide treatment

The MyristoylAted N-terminal Sequence (MANS) and Random Nucleotide Sequence (RNS) peptides were synthesized by Genemed Synthesis, Inc. The sequence of MANS is identical to the first 24 amino acids of the human MARCKS protein: myristic acid-GAQFSKTAAKGEAAAERPGEAAVA. The RNS peptide is a randomly scrambled control: myristic acid-GTPAPAAEGAGAEVKRASAEAKQAF. Peptide working solutions were resuspended in sterile PBS. Where indicated, pretreatment of cell suspensions with indicated peptide concentrations occurred at 37°C for 30 minutes.

Fluorescence labeling of neutrophils

For adhesion experiments, isolated neutrophils (1×10⁷/mL in HBSS) were incubated with the fluorescent dye calcein AM (Corning) at 2 μg/mL for 30 minutes at room temperature, protected from light. Cells were then centrifuged at 1200 rpm for 10 minutes and resuspended in HBSS⁺⁺ with 2% heat inactivated FBS to the appropriate final experimental concentration.

Beta2-integrin inhibition

As a positive control β₂-integrin inhibition, isolated human neutrophils were pretreated with 30 μg/mL anti-human CD18 F(ab’)₂ (Ancell Corp) at 37°C for 30 minutes. Anti-human IgG2a F(ab’)₂ (Ancell Corp) was used as an isotype control.

Flow cytometry

To avoid unintended stimulation of isolated neutrophils, hypotonic lysis of red blood cells was not performed prior to flow cytometry. Surface expression assay: Cells were pretreated as specified, stimulated with 100 nM fMLP (Sigma) or vehicle control (DMSO) for 5 minutes, diluted with 1 volume ice cold PBS and placed on ice for 5 minutes, spun at 1200 rpm for 5 minutes, resuspended in sterile PBS with 5% FBS and labeled with PE-conjugated anti-CD11b antibody clone ICFR44 (Novus Biologicals), PE-conjugated anti-CD11b CBRM1/5 antibody (Biolegend), or PE-conjugated IgG1 and IgG2a isotype controls (Biolegend). Flow cytometry was performed using Calibur FACScan. ICAM-1 binding assay: Cells were treated with 50 µM MANS, 50 µM RNS, or PBS for 30 minutes then washed with PBS and resuspended in HBSS⁺⁺ +2% FBS buffer. Cells were incubated with 40 µg/mL ICAM-1/Fc and/or 100 nM fMLP for 5 minutes. Cells were fixed with 2% PFA, washed, resuspended with PE-conjugated anti-Fc F(ab’)₂ fragment (1:100 dilution) for 20 minutes at 4°C, and washed again with PBS prior to acquisition. Flow cytometry was performed using BD LSRII. Flow cytometry experiments were performed in the Flow Cytometry and Cell Sorting facility at North Carolina State University, College of Veterinary Medicine.

Static adhesion assay

Individual wells of Immulon2HB flat bottom 96-well plates were coated with 10 μg/mL Recombinant ICAM-1 (R&D Systems) or 5% FBS in PBS (for controls). Isolated human neutrophils at 1 × 10⁷/mL in HBSS were labeled with calcein AM (Corning) at a concentration of 2 μg/mL and primed with human GM-CSF (EMD Millipore) at 5 ng/mL for 30 minutes, protected from light. Cells were then centrifuged at 1000 rpm for 8 minutes and then resuspended in HBSS⁺⁺ +2% FBS buffer at 7.0 × 10⁵ cells/mL. Appropriate blocking antibodies and/or peptides were added to cells and incubated for 30 minutes at 37°C. The ICAM-1 coated plate was washed once with 1X PBS before cells were added to individual wells. Cells were added to wells and allowed to settle for 10 minutes at 37°C. 0.5 mM Mn²⁺ was added to individual wells. Plates were incubated for 10 minutes prior to initial fluorescence reading. Cells were gently dumped and washed with PBS, reading fluorescence (485 nm excitation, 535 nm emission) at each wash step. Fluorescence washing was divided by initial fluorescence and multiplied by 100 to calculate percent adhesion. The first wash that demonstrated less than 10% adhesion in the non-stimulated cells (plated on 5% FBS coating) was considered the result. Treatment groups were tested in triplicate.

Immunofluorescence microscopy

Chambered coverslips were either coated with 5% FBS in PBS (unstimulated control) or 10 µg/mL ICAM-1/Fc (R&D Systems) overnight. Isolated primary neutrophils were primed with 5 ng/mL human GM-CSF for 30 minutes at room temperature protected from light. Cells were then centrifuged at 1000 rpm for 8 minutes and then resuspended in the chemotaxis buffer HBSS⁺⁺ +2% FBS buffer at 1.0 × 10⁶ cells/mL. Appropriate concentrations of peptides were added to cells and incubated for 30 minutes at 37°C. The ICAM-1 (or ICAM-1/Fc) coated chamber coverslip was washed once with 1X PBS before cells were added to individual wells. 1.3 × 10⁵ cells were added to the ICAM-1 or ICAM-1/Fc-coated chamber coverslips for 10 minutes. For fMLP stimulated wells, cells were allowed to settle for 10 minutes prior to addition of 100 nM fMLP or vehicle control for 5 minutes. For Mn²⁺ treated cells, 0.5 mM Mn²⁺ was added to individual wells after neutrophils were plated for 10 minutes on ICAM-1. Cells were then fixed to the coverslip, washed with PBS, blocked with rabbit serum, and labeled with AF549-conjugated anti-CD11b antibody clone M1/70 (Biolegend) before imaging with an Olympus I×83 Inverted Microscope. Cells that displayed morphology indicative of spreading were counted and averaged for each stimulation group.

Respiratory burst

Isolated neutrophils were resuspended in HBSS⁺⁺ +2% FBS buffer to a final concentration of 3.0 × 10⁶ cells/mL. Cells were then incubated with indicated treatments at 37°C for 30 minutes prior to each experiment and 100 μL of cells from indicated treatment groups were placed in individual wells of 5% FBS PBS or insoluble immune complex (IIC) coated Immulon2HB plates. Plates were coated overnight at 4°C with BSA (100 μg/mL). To generate IIC substrate, BSA (Sigma)-coated wells were washed three times and recoated with anti-BSA antibody (50 μg/well) and incubated for 2 hours at 37°C. Prior to the addition of cells, all wells were washed three times with sterile PBS. For PMA-stimulated respiratory burst, cells were allowed to settle for 10 minutes prior to the addition of dihydrorhodamine-123 (DHR-123) (Sigma) (10 μM final concentration) and PMA (100 ng/mL final concentration) (Sigma). In the case of IIC-mediated respiratory burst, DHR-123 was added immediately following addition of cells to the well. An fMax fluorescence plate reader (Molecular Devices) was used to measure initial fluorescence (485 nm excitation, 530 nm emission) followed by a fluorescence reading every 15 minutes for 120 minutes. Results are reported as nm fluorescence. Treatment groups were tested in triplicate. Individual time points were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. *p < .05 MANS compared to IIC. #p < .05 anti-CD18 F(ab)’₂ compared to IIC. Staurosporine p < .05 for all time points beginning at 30 minutes.

Cell Lysate stimulation

Prior to the experiment, low density insoluble immune complex was prepared as follows. Wells of a 24 well tissue culture plate was coated with 100 µg/mL bovine serum albumin overnight at 4°C. The wells were washed three times with PBS before the addition of coating with 33.35 µg/mL rabbit anti-bovine albumin antibody (Sigma) for 2 hours at 37°C. The wells were washed three times with PBS prior to the addition of cells. Isolated neutrophils (7×10⁶ were resuspended in HBSS⁺⁺ with 2% FBS and pretreated with PBS, 50 µM MANS, 50 µM RNS, 30 µM 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-D] pyrimidine (PP2) (Sigma), or 30 µg/mL anti-CD18 F(ab’)₂ prior to stimulation with immune complexes for 15 minutes at 37°C. An Eppendorf tube of cells remained unstimulated for a suspension control. Plates were centrifuged at 1200 rpms for 10 minutes then immediately transferred onto ice and lysed with ice cold RIPA buffer [1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 mM sodium pyrophosphate and 50 mM sodium fluoride] containing protease inhibitors and phosphatase inhibitors for 30 minutes shaking on ice. After lysis, cell supernatants were centrifuged for at 1300 rpm for 10 minutes at 4°C. Supernatants were collected and frozen at −80°C until immunoblotting.

Immunoblotting

Frozen samples were thawed on ice and protein concentrations were measured using BCA Protein Assay Reagent (Pierce). Cell lysate was mixed with 5X sample buffer [25% glycerol, 2% SDS, 60 mM Tris-HCL (pH 6.8), 5% β-mercaptoethanol, 0.1% bromophenol blue in diH20] and boiled for 10 minutes. Equal amount of protein was analyzed in 4–12% SDS-PAGE. Resolved samples were transferred to Immobilon-P PVDF membrane (Millipore). Total protein was determined using No-Stain Protein Labeling Reagent (Invitrogen) followed by blocking 1 hour with 5% nonfat dry milk with Tween-20 (TBS/T; 136 μM NaCl, 20 μM Tris-base (pH: 7.6) and 0.1% Tween-20 v/v). Primary antibody (1:1000) was incubated overnight in 5% BSA TBS/T at 4°C. Membranes were washed with TBS/T and incubated with anti-rabbit horseradish peroxidase (HRP) secondary antibody (1:2000) (Cell Signaling Technology) in 5% BSA TBS/T for one hour, then washed three times for 5 minutes, developed using Bio-Rad Clarity Western ECL Substrate, and imaged using a Bio-Rad ChemiDoc. Western blot images were analyzed in Image Lab and results were normalized to total protein detected by the No-Stain Protein Labeling Reagent [48–50].

Statistical analysis

Data are reported as mean ± SD. All statistical tests were performed using GraphPad Prism software (San Diego, CA) with the appropriate test details in the figure legend. P values < 0.05 were considered statistically significant. *Indicates p < .05, ** p < .01, *** p < .001, **** p < .0001.

Results

MARCKS inhibition with MANS peptide does not alter inside-out activation of neutrophil β₂-integrins.

Previous findings in our lab demonstrated that inhibition of MARCKS protein function significantly decreased chemoattractant-induced (e.g., fMLP, IL-8, and LTB4) neutrophil adhesion and migration [24,26]. While these studies did not determine the mechanism for MARCKS in neutrophils stimulated with chemoattractants, Sheats et al. determined that MARCKS may play a role in neutrophil β₂-integrins [24]. Neutrophils within the vasculature are non-adherent and express low numbers of surface β₂-integrins. When neutrophils are stimulated by chemoattractants, there is a significant increase in surface expression of β₂-integrins, as determined by the expression of CD18 and CD11b. To determine whether inhibition of MARCKS with the MANS peptide altered inside-out activation of β₂-integrins, neutrophils were pretreated with MANS or RNS and stimulated with fMLP, and CD11b surface expression was measured by flow cytometry. As shown in Figure 1a–b, fMLP stimulation induced a significant increase in total CD11b on the surface of neutrophils compared to control. MANS peptide treatment had no effect on fMLP-induced surface expression of CD11b. Calcium chelation with EDTA was used as a positive control for inhibition of CD11b upregulation. MARCKS inhibition with MANS also had no effect on fMLP-induced surface expression of CD18 or MHC class I expression (Supplemental Figure 1), consistent with prior observations [26].

Figure 1. MANS peptide treatment does not inhibit fMLP-induced β₂-integrin affinity conformation change of neutrophils. Neutrophils were pretreated with 50 µM MANS, 50 µM RNS, 10 mM EDTA, or PBS (untreated) for 30 minutes then stimulated with 100 nM fMLP or vehicle control (DMSO) for 5 minutes. Flow cytometry was used to measure total CD11b (ICRF44) (a-b) and high affinity CD11b (CBRM1/5) (c-d) as described. Histograms (b & d) show unstimulated (UTC) cells stained with IgG1 isotype control in red, DMSO (vehicle control/unstimulated) treated in blue, fMLP in green, MANS treated cells in orange, RNS treated cells in cyan, and EDTA (positive control for inhibition) in magenta. Data are represented as mean ± SD, n = 4–9. Mixed-effects analysis with Dunnett’s multiple comparisons test. *p < .05, ** p < .01, *** p < .001, **** p < .0001 indicates when compared to fMLP stimulation.

In addition to increasing the number of surface expressed β₂-integrins, stimulation by chemoattractants also cause integrin conformation to shift from low to high affinity conformation, which facilitates ligand binding. Stimulation with fMLP resulted in a significant increase in the surface expression of high affinity CD11b compared to controls, as detected by CBRM1/5 antibody binding (Figure 1c–d). MANS peptide treatment had no effect on fMLP-induced β₂-integrin high affinity conformation surface expression. Calcium chelation with EDTA was used as a positive control for inhibition of high affinity conformation expression. The high affinity conformation is required for β₂-integrin binding to ICAM-1 [51,52]; therefore, we evaluated whether MARCKS inhibition had an effect on neutrophil binding to ICAM-1 upon activation with fMLP. Pretreatment of neutrophils with MANS or RNS had no effect on neutrophil binding to ICAM-1 (Supplemental Figure 2). This evidence demonstrates that inhibition of MARCKS with the MANS peptide does not alter fMLP-induced inside-out activation of neutrophil β₂-integrins.

MARCKS inhibition with MANS peptide attenuates outside-in β₂-integrin mediated neutrophil adhesion.

When neutrophils become activated by chemoattractants through inside-out stimulation, the high affinity conformation promotes β₂-integrin binding to cellular adhesion molecules expressed on endothelial cells, such as intercellular adhesion molecule 1 (ICAM-1) [51,52]. This binding results in ‘outside-in’ activation and signaling. Outside-in activation and signaling controls firm adhesion, which is a necessary step for several neutrophil effector functions [53,54]. Although inside-out and outside-in activation happen together in vivo, we utilized an in vitro model of outside-in adhesion to determine if MANS-mediated MARCKS inhibition altered outside-in activation of β₂-integrins. Static neutrophil adhesion was induced on ICAM-1 coated wells and enhanced with the application of manganese (Mn²⁺) [21]. Stimulation of neutrophils using the divalent cation Mn²⁺ is used to induce the high affinity conformation of β₂-integrins without increasing total β₂-integrin integrin expression or inducing β₂-integrin inside-out signaling [21,55]. This model induced significant static neutrophil adhesion (Figure 2). MARCKS inhibition with MANS peptide significantly attenuated ICAM-1 + Mn²⁺-induced adhesion in a concentration-dependent manner. The addition of anti-CD18 F(ab’)₂ confirmed that ICAM-1 + Mn²⁺ adhesion is β₂-integrin dependent. The RNS control peptide and isotype controls had no effect on neutrophil adhesion. These results indicate that MARCKS protein plays an essential role in outside-in β₂-integrin mediated static adhesion.

Figure 2. MANS peptide treatment attenuates outside-in β₂-integrin-mediated neutrophil adhesion. Neutrophils were pretreated with indicated concentrations of MANS and RNS peptides, 30 µg/mL anti-CD18 F(ab)’₂, or 30 µg/mL IgG2a control F(ab)’₂ for 30 minutes prior to application to 96 well plate coated with 10 μg/mL ICAM-1. Cells were allowed to settle for 10 minutes before application of 0.5 mM Mn²⁺ for 10 minutes. Adhesion was quantified as described in the materials and methods section. MANS inhibition of MARCKS demonstrated concentration-dependent inhibition of ICAM-1+Mn-induced adhesion. Data represented as mean ± SD, n = 5–6. Mixed-effects analysis with Dunnett’s multiple comparisons test. * p < .05, ** p < .01, *** p < .001, **** p < .0001 indicates when compared to ICAM-1 + Mn group.

MARCKS inhibition with MANS peptide alters neutrophil cell spreading and β₂-integrin clustering.

Having demonstrated a quantitative effect of MANS-mediated MARCKS inhibition on β₂-integrin outside-in adhesion, we next sought qualitative information regarding the effect of MANS peptide treatment on changes in neutrophil morphology, including cell spreading, and β₂-integrin clustering or avidity using immunofluorescence microscopy staining of CD11b [56]. Unstimulated neutrophils appeared rounded with CD11b staining in the periphery of the cell and relatively homogenous and faint staining in the cell body, indicating no β₂-integrin clustering (Figure 3a). Neutrophils stimulated with Mn²⁺ appeared similar to unstimulated cells with some increased staining of CD11b at the periphery of the cell (Figure 3b). Neutrophils adhered to ICAM-1 exhibited cell spreading and intense staining of CD11b within the body of the cell that is punctate and bright throughout, indicating clustered β₂-integrins (Figure 3c). This response was more robust in neutrophils stimulated with ICAM-1 and Mn²⁺ (Figure 3d). MANS treatment significantly inhibited both cell spreading and β₂-integrin clustering (Figure 3e) to a level that resembled the unstimulated control (Figure 3a). The control peptide RNS had no effect on ICAM-1 + Mn²⁺ neutrophil spreading or β₂-integrin clustering (Figure 3f). We further stimulated neutrophils plated on ICAM-1 + Mn²⁺ with fMLP (Figure 3g) and demonstrated the addition of the inside-out stimulus fMLP did not restore the clustering and spreading defects caused by MANS peptide (Figure 3h). Similar results were observed in neutrophils plated on ICAM-1/Fc (Supplemental Figure 3). We also performed a semi-quantitative analysis of cell spreading to determine the percent spread neutrophils in each condition (Figure 3i), with the white arrows in Figure 3 displaying examples of cells with spreading and β₂-integrin clustering morphology. As the microscopy indicates, MANS peptide decreased neutrophil cell spreading induced by ICAM-1 + Mn²⁺ and the spreading induced by ICAM-1 + Mn²⁺ with fMLP. These qualitative and semi-quantitative data indicate that inhibition of MARCKS with the MANS peptide alters the outside-in β₂-integrin mediated events that follow initial ligand adhesion, including cell spreading and integrin clustering.

Figure 3. MANS peptide treatment alters neutrophil spreading and β₂-integrin clustering on ICAM-1 enhanced with Mn²⁺. fMLP does not restore defects. Immunofluorescence microscopy of CD11b: Neutrophils were pretreated with 50 µM MANS, 50 µM RNS, or PBS for 30 minutes prior to application to ICAM-1 coated chambered coverslip. Cells were incubated for 10 minutes at 37°C. Mn²⁺ was added following the 10 minute incubation and allowed to adhere for another 10 minutes at 37°C. fMLP was applied 5 minutes into stimulation. (a) Neutrophils plated on 5% FBS in PBS. (b) Neutrophils plated on 5% FBS in PBS followed by Mn²⁺. (c) Neutrophils plated on ICAM-1. (d) Neutrophils plated on ICAM-1 and enhanced with Mn²⁺. (e) MANS pretreated neutrophils on ICAM-1 with Mn²⁺. (f) RNS pretreated neutrophils plated on ICAM-1 with Mn²⁺. (g) Neutrophils plated on ICAM-1/Mn²⁺ with fMLP applied 5 minutes into stimulation. (H) MANS pretreated neutrophils plated on ICAM-1/Mn²⁺ with fMLP applied 5 minutes into stimulation. (i) Percent spread cells in each condition. Data are represented as mean ± SD, n = 2.

MARCKS inhibition with MANS peptide attenuates β₂-integrin-dependent, but not β₂-integrin independent, neutrophil respiratory burst

The data presented thus far have shown that inhibition of MARCKS with the MANS peptide alters outside-in activation of β₂-integrins in primary human neutrophils. In addition to being responsible for the physical interactions of neutrophils with ligands, β₂-integrins are also responsible for transmitting intracellular signals to elicit appropriate cellular responses. Therefore, we next evaluated whether MANS-mediated MARCKS inhibition alters signaling of neutrophil β₂-integrins. Reactive oxygen species (ROS) are essential mediators of cellular signaling, so we first examined whether MANS peptide treatment altered respiratory burst using two-different β₂-integrin-dependent and β₂-integrin independent respiratory burst models. Neutrophil respiratory burst was stimulated with either low density insoluble immune complexes (IIC) or phorbol myristate acetate (PMA) and measured using DHR-123, which emits fluorescence once it is oxidized by reactive oxygen species.

As shown in Figure 4, both IIC and PMA stimulated robust and sustained neutrophil respiratory burst. MANS peptide treatment significantly inhibited IIC-induced respiratory burst (Figure 4a), while RNS peptide had no effect. IIC-induced respiratory burst was β₂-integrin dependent, as shown by significant inhibition with anti-CD18 F(ab’)₂ treatment and was PKC dependent, as indicated by staurosporine inhibition of respiratory burst (Figure 4a). MANS peptide treatment had no effect on PMA-induced respiratory burst of neutrophils. PMA-induced respiratory burst was independent of β₂-integrins, as indicated by lack of inhibition seen with anti-CD18 F(ab’)₂ treatment, but was dependent on PKC, as indicated by staurosporine inhibition of respiratory burst (Figure 4b). These results demonstrate that inhibition of MARCKS with the MANS protein attenuates β₂-integrin-dependent respiratory burst, but not β₂-integrin independent respiratory burst. These data led us to hypothesize that MARCKS protein functions upstream of assembly of the NADPH oxidase complex.

Figure 4. MANS peptide inhibits β₂-integrin-dependent neutrophil respiratory burst stimulated by insoluble immune complexes but not PMA-stimulated β₂-integrin independent neutrophil respiratory burst. Neutrophils were pretreated for 30 minutes with 30 µg/mL anti-CD18 F(ab)’₂, 30 µg/mL IgG2a control F(ab)’₂, 50 µM MANS, 50 µM RNS, 100 nM staurosporine, or media alone (control). Respiratory burst was quantified by DHR-123. Cells were stimulated by insoluble immune complexes (IIC) (a) or PMA (b). Data are represented as mean ± SD, n = 2–4. Individual time points were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. *p < .05, ** p < .01, *** p < .001, **** p < .0001 indicates MANS compared to IIC. #p < .05, # p < .01 indicates anti-CD18 F(ab)’₂ compared to IIC. Staurosporine p < .05 for all time points beginning at 30 minutes.

MARCKS inhibition with MANS peptide does not alter IIC-induced activation of p38 MAPK nor Akt

The p38 MAPK is an important cell signaling protein known to play an essential role in neutrophil functions [57–59]. Previous studies have identified a role for p38 MAPK in β₂-integrin-dependent neutrophil adhesion and ROS production [60,61]. Neutrophil stimulation with immune complexes induces activation (phosphorylation) of p38 MAPK, and p38 MAPK inhibition decreases neutrophil ROS production on IIC [62]. Akt is also an important signaling molecule in neutrophils. Akt phosphorylation is observed in neutrophils stimulated by immune complexes, iC3b, ICAM-1, and by CBR LFA-1/2 antibodies [62–64], indicating that Akt plays an important role in β₂-integrin outside-in signaling.

We investigated whether attenuation of IIC-stimulated respiratory burst with the MANS peptide was associated with altered phosphorylation of p38 MAPK and Akt in MANS treated neutrophils stimulated by low density IIC. Isolated neutrophils were treated with either MANS, RNS, PP2, or anti-CD18 F(ab’)₂ prior to stimulation with IICs (Figure 5). The Src kinase inhibitor PP2 was included as a positive control for inhibition of p38 MAPK and Akt phosphorylation in IIC-stimulated neutrophils [62]. MANS and RNS peptides had no effect on p38 MAPK phosphorylation (Figure 5a). Treatment with anti-CD18 F(ab’)₂ also did not inhibit p38 MAPK phosphorylation (Figure 5a). MANS and RNS peptides and anti-CD18 F(ab’)₂ had no significant effect on Akt phosphorylation (Figure 5b). These results indicate that inhibition of MARCKS with the MANS peptide does not alter IIC-induced p38 MAPK or Akt activation in neutrophils.

Figure 5. MANS peptide treatment does not alter IIC-induced p38 nor Akt activation in neutrophils. Neutrophils (7x10⁶) were pretreated with either PBS, 50 µM MANS, 50 µM RNS, 30 µM PP2 (positive control for inhibition), or 30 µg/mL anti-CD18 F(ab)’₂ then stimulated on low density immune complexes for 15 minutes or left in suspension (Sus). Phosphorylation of p38 or Akt was analyzed by Western blotting of whole-cell lysates. Equal loading was confirmed following transfer with Invitrogen No-Stain Protein Labeling Reagent. The percentage of phosphop38 or phosphoAkt was determined based on the total protein in the respective lane and the signal of the phosphop38 or phosphoAkt band. Blot shown is representative of three independent experiments. Data are represented as mean ± SD, n = 3. One-way ANOVA with Dunnett’s multiple comparisons test. *p < .05, *p < .01 indicates when compared to IIC.

Discussion

MARCKS is a natively unfolded, ubiquitously expressed protein that demonstrates reversible actin and PIP2 binding. Interaction of MARCKS with its binding partners is regulated by competing signals from reversible PKC-phosphorylation, and calcium/calmodulin binding, of the effector domain [35,65–68]. MARCKS is known to play a key role in neutrophil adhesion, migration, and respiratory burst. Previous results from our lab and others have shown that inhibition of MARCKS with an N-terminal mimetic peptide known as MANS significantly attenuates functional responses of primary neutrophils [24,26,36,46]; however, details regarding the cellular mechanism of MANS-mediated inhibition of neutrophil functions has not been fully elucidated. In the current study, we hypothesized that inhibition of MARCKS with the MANS peptide would alter β₂-integrin activation and signaling in primary human neutrophils. We examined the impact of MANS-mediated MARCKS inhibition on β₂-integrins using methods to target both ‘inside-out’ and ‘outside-in’ β₂-integrin activation pathways. Our results show that MANS peptide does not impact fMLP-stimulated inside-out β₂-integrin upregulation or affinity change but does inhibit outside-in β₂-integrin-dependent adhesion, cell spreading and avidity, as well as β₂-integrin-dependent respiratory burst. This is the first study to suggest that inhibition of MARCKS with the MANS peptide alters β₂-integrin-dependent neutrophil functions, such as adhesion and respiratory burst, by inhibiting outside-in β₂-integrin activation.

In this study we used a MARCKS-targeting peptide known as MANS, which stands for MyristoylAted N-terminus Sequence, as a tool for MARCKS inhibition. This peptide is identical to the first 24 amino acids of MARCKS sequence and includes an N-terminal myristoyl-moiety. In vitro and in vivo studies demonstrating the effects of MANS-mediated MARCKS inhibition on cellular migration, adhesion, secretion, degranulation and respiratory burst are numerous [24,25,36,41–43,46,69–72]. A derivative of MANS known as BIO-11006, identical to the first 10 amino acids of MARCKS, has been shown to be suitable for therapeutic use as an aerosolized peptide and has been evaluated in Phase II clinical trials in patients with chronic obstructive pulmonary disease, late-stage non-small cell lung cancer and acute lung injury [43–45,73]. Although these MARCKS inhibitor peptides have been reported on extensively, the cellular mechanisms of inhibition have not been fully elucidated in neutrophils. Given the clinical treatment potential of MARCKS-targeting therapies and the need for novel targets in neutrophil-mediated diseases such as severe neutrophilic asthma and acute lung injury, we would like to fill the knowledge gap regarding how MARCKS inhibition with the MANS peptide alters neutrophil activation and signaling in the hopes that this understanding will inform novel therapeutic approaches to neutrophil-mediated diseases.

MARCKS inhibition with MANS peptide disrupted neutrophil spreading and β₂-integrin clustering on both ICAM-1 enhanced with Mn²⁺ and ICAM-1/Fc substrates (Figure 3e and Supplemental Figure 3c). These findings further inform our understanding of the effect of MANS peptide on neutrophil adhesion. Interestingly, the addition of fMLP as an inside-out stimulus did not restore the defects caused by MANS peptide. While these results are consistent with previous studies that showed that MARCKS plays an essential role in cell spreading in several cell types [47,74–77], they are in contrast to the findings by Eckert et al., whose results showed that MANS peptide treatment did not impact fMLP-stimulated cell spreading and polarization [26]. The reason for this discrepancy is not clear. Similar to our results, Mac-1 deficient neutrophils also display a rounded morphology and lack spreading on immune complexes [78]. Additionally, clustering of β₂-integrins is considered to be an essential part of post-adhesion strengthening and sustained adhesion to ligand [79,80]. Taken together, our results show that inhibition of MARCKS with the MANS peptide disrupts key steps of neutrophil β₂-integrin outside-in activation, including cell spreading and integrin clustering.

Respiratory burst is an important neutrophil effector function and window into neutrophil intracellular signaling. In the current study, we evaluated MARCKS role in both β₂-integrin-dependent (insoluble immune complex – IIC) and independent (PMA) respiratory burst. MANS peptide treatment significantly inhibited IIC-induced respiratory burst but not PMA-stimulated respiratory burst (Figure 4). From these findings, we conclude that MANS treatment does not hinder the assembly and function of the NADPH oxidase complex. Furthermore, because the pan-PKC inhibitor staurosporine blocked both types of respiratory burst, we confirm that PKC is required for neutrophils to undergo successful respiratory burst. Therefore, we suggest that at least one aspect of MARCKS function, which is inhibited by the MANS peptide, must be essential for IIC-stimulated respiratory burst between the level of FcγR-signaling and PKC activation.

Fc receptors and β₂-integrins cooperate in many neutrophil functional responses. Zhou and Brown demonstrated that β₂-integrin and FcγRIII cooperation generates a respiratory burst that is unproductive when each receptor is stimulated separately [81]. The respiratory burst response was also inhibited when either receptor was individually blocked [81]. Other studies have also shown that β₂-integrins are required for neutrophil respiratory burst [82–84], and our results further confirm that β₂-integrins are required for IIC-induced neutrophil respiratory burst (Figure 4a). This finding is in contrast to a previous study showing that superoxide production by immune complex adherent neutrophils was not β₂-integrin dependent [85]. However, superoxide production was only measured 20 minutes into the stimulation and the density of immune complex was not specified. In the current report, anti-CD18 F(ab’)₂ treated cells are significantly different than untreated cells at the 30-minute time point but not at 15 minutes (Figure 4a). It is possible that the limited time point analysis is the reason that the study by Graham et al. did not capture any differences attributed β₂-integrin inhibition [85].

P38 MAPK is known to play an important role in inflammation and several neutrophil functions [58,60,86]. P38 is also involved in neutrophil β₂-integrin mediated adhesion, as either p38 inhibition or Mac-1 receptor blocking antibodies significantly diminish IL-8-induced adhesion to ICAM-1 [87]. Interestingly, similar to our results with inhibition of MARCKS, inhibition of p38 MAPK did not attenuate PMA-stimulated neutrophil adhesion or ROS production [60]. Based on these similarities, we hypothesized that MARCKS could play a role in a pathway involving p38 MAPK and β₂-integrin-dependent adhesion. However, we found that MANS peptide has no effect on p38 MAPK activation in IIC-stimulated primary neutrophils. This is in contrast to previous work that showed MANS treatment significantly diminished both LPS-induced cytokine production and p38 phosphorylation in murine macrophages [88]. P38 is activated by several different pathways, so the differences between our findings and Lee et al. may be explained by different cell types or mechanisms of stimulation (LPS vs IIC).

In our results reported here, anti-CD18 F(ab’)₂ did not attenuate p38 activation. This is in contrast with previously published results showing that β₂-integrin blocking antibodies did inhibit p38 activation; however, there are key differences between these previous studies and our current study. In the current report, we utilized low density insoluble immune complexes to stimulate neutrophil activation, which is mediated by both FcγRs and β₂-integrins. The prior studies utilized chemoattractants (e.g., TNFα and IL-8) to stimulate inside-out β₂-integrin responses on a variety of ligands [60,61,87,89]. The differences in cell signaling pathways initiated by these different stimuli could explain differences in our findings. Additionally, p38 has also been implicated in inside-out β₂-integrin activation by studies that showed inhibition of p38 MAPK decreased TNFα-induced upregulation of CD11b in neutrophils [61,90]. We determined in Figure 1 that MARCKS inhibition does not impact inside-out β₂-integrin activation stimulated by fMLP; therefore, MANS peptide treatment is unlikely to affect p38 activation induced by IICs if p38 MAPK activation is linked to inside-out β₂-integrin activation and signaling. Finally, our finding that MANS peptide does not decrease p38 MAPK activation provides further indirect support for MARCKS role in outside-in β₂-integrin activation, as opposed to FcγR signaling. Indeed, several reports have demonstrated that outside-in β₂-integrin activation does not result in p38 phosphorylation [64,91]. Therefore, p38 activation in our IIC-stimulated neutrophils is likely the result of FcγR engagement, and it was not altered in MANS treated neutrophils. This indicates that either MARCKS function is downstream of p38 phosphorylation or that MARCKS function is involved in a separate parallel and essential pathway that leads to IIC-induced respiratory burst in neutrophils.

We also evaluated the effect of MARCKS inhibition on Akt activation due to Akt’s putative role in β₂-integrin outside-in signaling [64]. When localized to the membrane, MARCKS effector domain sequesters PIP2 molecules at a ratio of 1:4 [68]. Displacement of MARCKS from the membrane by calcium/calmodulin binding or PKC-phosphorylation results in increased available membrane PIP2. PIP2 is then converted by phosphoinositide-3-kinase (PI3K) to PIP3, which leads to the increased translocation and subsequent phosphorylation of Akt [92]. Interestingly, previous results in fibroblasts show that MANS inhibition of MARCKS did decrease Akt phosphorylation [25]. This is in contrast to our findings that Akt phosphorylation was not significantly altered in MANS-treated neutrophils. Previous evaluation of Akt activation also utilized PP2 as a positive control for inhibition [62], however, neutrophils treated with PP2 stimulated with IIC resulted in Akt phosphorylation in our study, weakening our conclusions regarding Akt signaling. Although Akt has been associated with outside-in signaling, the signaling events leading to Akt phosphorylation are different than those leading to spreading, which is a primary result of outside-in β₂-integrin activation [92]. While MANS peptide treatment did not inhibit Akt phosphorylation in our study, additional work is needed to better understand how MANS disruption of endogenous MARCKS function impacts other PH-domain containing protein regulators of neutrophil β₂-integrins.

Several lines of evidence have converged to suggest that the ‘root’ function of MARCKS is to regulate PIP2 microdomain availability, upstream of and in response to, PKC and (or) Ca^2±calmodulin signaling pathways and related to the myristoyl-electrostatic switch [33,93]. Studies by Glaser and colleagues showed that physiological concentrations of MARCKS (<10 μM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of PIP2 in phospholipid vesicles by sequestering the acidic phospholipid at a 1:4 molar ratio (MARCKS:PIP2) [68]. In various cell types PIP2 co-localizes with MARCKS in several morphologically active cellular locations, including focal adhesions and membrane ruffles [93–97]. Most recently, studies using live cell fluorescence imaging of macrophages provide convincing evidence of a signaling loop that controls the leading edge of amoeboid cell migration that consists of Ca²⁺-PKC activation of MARCKS leading to release of concentrated pools of PIP2 which are phosphorylated by PI3K to PIP3 [98]. Ultimately, the association between MARCKS and PIP2 influences cellular actin organization [28]. Studies show that alterations in MARCKS ability to shuttle between the cell membrane and cytosol disrupts dynamic cellular events such as adhesion and spreading [47,99]. These data are consistent with findings by Sheats et al., which show that PKC-delta mediated MARCKS phosphorylation, which causes MARCKS to shuttle to the cytosol, is essential for human neutrophil adhesion [100]. In future studies, we will investigate whether MANS disruption of β₂-integrin outside-in activation in neutrophils is associated with differences in distribution or availability of membrane PIP2 microdomains, changes in actin organization, or both.

This is the first study to show that inhibition of MARCKS with the MANS peptide inhibits outside-in, but not inside-out, β₂-integrin activation in neutrophils. A role for MARCKS in integrin-dependent cellular responses has been investigated previously in other cell types, but the molecular details remain incompletely understood. This report expands the understanding of MANS-mediated MARCKS inhibition and implicates a role for MARCKS function in neutrophil responses requiring outside-in neutrophil β₂-integrin activation and signaling. Additional work is needed to determine whether this finding is specific for MANS-mediated peptide inhibition of MARCKS compared to other methods of MARCKS-targeting. In future studies, we will further investigate the mechanism of MANS effect on outside-in activation of neutrophil β₂-integrins by investigating subcellular localization of MARCKS upstream and downstream signaling partners including PI3K, PIP2/PIP3, actin and PH-domain containing actin-binding proteins.

Acknowledgments

We would like to thank Dr. Lauren Schnabel at North Carolina State University for microscope access.

Disclosure statement

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.

Data availability statement

The original contributions presented in the study are included in the article and supplementary material. Data files can also be found at https://doi.org/10.5281/zenodo.8121775. Further inquiries can be directed to the corresponding author at [email protected].

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19336918.2023.2233204

Additional information

Funding

This work was supported by NIH OD/ORIP under grant K01OD015136, North Carolina State University College of Veterinary Medicine, and the Comparative Medicine Institute at North Carolina State University.