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The Journal of Immunology, 2005, 174: 7242-7249.
Copyright © 2005 by The American Association of Immunologists

{gamma}-Amino Butyric Acid Type B Receptors Stimulate Neutrophil Chemotaxis during Ischemia-Reperfusion1

Madhavi J. Rane2,*, David Gozal3,{dagger},{ddagger}, Waseem Butt*, Evelyne Gozal{dagger},{ddagger}, William M. Pierce, Jr{ddagger}, Shang Z. Guo{dagger}, Rui Wu*, Aviv D. Goldbart{dagger}, Visith Thongboonkerd*, Kenneth R. McLeish*,§,|| and Jon B. Klein3,*,§,¶,||

Departments of* Medicine, {dagger} Pediatrics, {ddagger} Pharmacology and Toxicology, § Biochemistry and Molecular Biology, and Core Proteomics Laboratory, University of Louisville, and || Veterans Affairs Medical Center, Louisville, KY 40202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Serine/threonine kinase Akt, or protein kinase B, has been shown to regulate a number of neutrophil functions. We sought to identify Akt binding proteins in neutrophils to provide further insights into understanding the mechanism by which Akt regulates various neutrophil functions. Proteomic and immunoprecipitation studies identified {gamma}-amino butyric acid (GABA) type B receptor 2 (GABABR2) as an Akt binding protein in human neutrophils. Neutrophil lysates subjected to Akt immunoprecipitation followed by immunoblotting with anti-GABABR2 demonstrated Akt association with the intact GABABR. Similar results were obtained when reciprocal immunoprecipitations were performed with anti-GABABR2 Ab. Additionally, GABABR2 and Akt colocalization was demonstrated by confocal microscopy. A GABABR agonist, baclofen, activated Akt and stimulated neutrophil-directed migration in a PI3K-dependent manner, whereas CGP52432 a GABABR antagonist blocked such effects. Baclofen, stimulated neutrophil chemotaxis and tubulin reorganization in a PI3K-dependent manner. Additionally, a GABABR agonist failed to stimulate neutrophil superoxide burst. We are unaware of the association of GABABR with Akt in any cell type. The present study shows for the first time that a brain-specific receptor, GABABR2 is present in human neutrophils and that it is functionally associated with Akt. Intraventricular baclofen pretreatment in rats subjected to a stroke model showed increased migration of neutrophils to the ischemic lesion. Thus, the GABABR is functionally expressed in neutrophils, and acts as a chemoattractant receptor via an Akt-dependent pathway. The GABABR potentially plays a significant role in the inflammatory response and neutrophil-dependent ischemia-reperfusion injury such as stroke.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ischemia-reperfusion injury is the underlying mechanism of common and frequently fatal illnesses such as myocardial infarction, acute renal failure, and stroke. Neutrophils play a central role in ischemia-reperfusion injury (1). Additionally, many neurological insults are accompanied by an acute inflammatory reaction due to neutrophil infiltration, the latter contributing to neuronal damage (2, 3, 4, 5, 6). Neutrophil functions such as chemotaxis, exocytosis, or superoxide burst are initiated in activated neutrophils. Activated neutrophils can contribute to tissue damage by 1) release of proteolytic enzymes, 2) release of reactive oxygen species, and 3) stimulation of proinflammatory cytokines. Therefore, identifying proteins that mediate neutrophil functional responses might allow manipulation of signal cascades and potentially modulate specific neutrophil functions (7, 8, 9, 10).

Participation of neutrophils in inflammation requires the interplay of multiple cell membrane receptors and downstream protein-protein interactions that convey information from cell surface receptors to the cell interior. One protein that is a significant regulator of neutrophil survival and function is the serine/threonine kinase Akt. Akt, also known as protein kinase B, is a cellular homologue of a viral oncogene v-Akt (11, 12, 13). Akt has been shown previously to regulate a number of neutrophil functions including, chemotaxis, respiratory burst, apoptosis, and actin polymerization (14, 15, 16, 17, 18, 19). To better understand the role of Akt in regulating various neutrophil functions, proteomic analysis was applied to Akt immunoprecipitates to identify putative Akt binding proteins. A C-terminal fragment of the {gamma}-amino-butyric acid (GABA)4 type B receptor 2 (GABABR2) was identified as an Akt binding protein. The identification of a fragment of GABABR2 instead of an intact receptor could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor. However, immunoblot analysis demonstrated presence of intact GABABR2 in human neutrophils. Additionally, association and colocalization of intact GABABR2 and Akt was demonstrated by Akt and GABABR2 immunoprecipitations and confocal microscopy, respectively.

GABA is the main inhibitory neurotransmitter in the mammalian central nervous, where it exerts its effects through ionotropic (GABAA/C) receptors to produce fast synaptic inhibition and metabotropic (GABAB) receptors to produce slow, prolonged inhibitory signals. GABABR include two receptor subtypes GABABR1 and GABABR2. The GABABR is composed of dimers and ligand-induced heterodimerization of R1 and R2 subunits have been shown to be essential for GABABR trafficking and function (20, 21). Because Akt associated with the GABABR2 in human neutrophils, and GABABR agonists have been shown to stimulate directed migration in neurons, we addressed the hypothesis that the GABABR2 acts as a neutrophil chemoattractant receptor via an Akt-dependent mechanism. The present study identified for the first time the presence of functional GABABR2 in human neutrophils and its association with Akt. Additionally, baclofen, a GABABR2 agonist, stimulated Akt activation and directed neutrophil migration in a PI3K-dependent manner both in vitro and in a stroke animal model. Thus GABABR may play a significant role in the inflammatory component of ischemia-reperfusion injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

Baclofen, fMLP, paraformaldehyde, cytochrome c, saponin, and anti-GABABR (catalog no. G5915) Ab were obtained from Sigma-Aldrich. Donkey anti-guinea pig Ab (catalog no. 706-005-148) was obtained from Jackson ImmunoResearch Laboratories. Rabbit polyclonal anti-GABABR2 Ab (catalog no. 56311) was obtained from QED Bioscience. LY294002 and wortmannin were obtained from Calbiochem. Tissue culture-treated clear polyester membrane transwells (6.5 mm diameter, 3.0-µm pore size) were obtained from Corning. Hema 3 kit (H&E stain) was obtained from Protocol (Fisher Scientific). Mouse anti-tubulin {alpha}{beta} mixed Ab was obtained from Research Diagnostics. Goat anti-mouse rhodamine red IgG was obtained from Molecular Probes. Goat normal serum was obtained from Vector Laboratories. Anti-Akt PH domain Ab was obtained from Upstate Biotechnology, and anti-formyl peptide receptor (FPR) Ab (sc-13193), isotype control Ab, goat anti-rabbit IgG, and goat anti-mouse IgG Ab were obtained from Santa Cruz Biotechnology. Alexa Fluor 680 donkey anti-goat IgG (catalog no. A21084) was obtained from Molecular Probes. Anti-glutamic acid decarboxylase (catalog no. AB1511) Ab was obtained from Chemicon International. Protein A-Sepharose was obtained from BD Pharmingen. Histone H2B was obtained from Boehringer Mannheim. CGP52432 SKF97541 and bicuculline were obtained from Tocris Cookson. Chambered, cover glass wells for confocal microscopy were obtained from Nalgene Nunc International.

Isolation of neutrophils and culture conditions

Neutrophils were isolated from venous blood obtained from healthy volunteers as previously described (18). Neutrophil preparations routinely contained >95% neutrophils, as determined by morphology, and were >99% viable by trypan blue dye exclusion. Neutrophils were suspended in RPMI 1640 supplemented with 10% FCS, L-glutamine, penicillin, and streptomycin and incubated for the indicated times at 37°C in 5% CO2.

Immunoprecipitation of GABABR2 and Akt

Neutrophils (2 x 107) were prewarmed at 37°C before immunoprecipitation. Cells were pelleted by centrifugation followed immediately by addition of 200 µl of lysis buffer containing Tris-HCl 20 mM pH 7.4, NaCl 150 mM, Triton X-100 1% (v/v), Nonidet P-40 0.5% (v/v), EDTA 1 mM, EGTA 1 mM, sodium orthovanadate 20 mM, p-nitrophenol phosphate 10 µM, NaF 20 mM, PMSF 5 mM, aprotinin 21 µg/ml, and leupeptin 5 µg/ml. Following centrifugation at 15,000 x g for 15 min at 4°C, cleared lysates were incubated with 1 µg of isotype control Ab, or 1 µg of anti-Akt-PH domain Ab or 1 µg of anti-GABABR Ab overnight with continuous rotation at 4°C. Protein A-Sepharose beads (15 µl) were then added and samples were rotated for an additional 2 h at 4°C. Beads were washed once by centrifugation in Krebs buffer, then resuspended in 50 µl 2x Laemmli buffer and boiled for 3 min. Proteins were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, and blocked with 5% milk/TBST for 1 h. Blots of anti-Akt immunoprecipitants were probed with anti-GABABR (1/1000), and blots of anti-GABABR2 immunoprecipitants were probed with anti-Akt (1/1000) antiserum in 5% BSA/TBST (w/v) and peroxidase-conjugated, secondary Ab in 5% milk/TBST (w/v). Products were visualized by chemiluminescence.

Measurement of protein kinase B/Akt kinase activity

Protein kinase B/Akt kinase activity was measured by the ability of the immunoprecipitated enzyme to phosphorylate histone H2B as previously described (17). Briefly, 2 x 107/200 µl of neutrophils were prewarmed for 5 min at 37°C and baclofen 10 µM was added. The reaction was terminated by centrifugation at 2500 x g followed immediately by lysis in buffer containing 1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 137 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 5 mM PMSF, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1% (v/v) Triton X-100. Lysates were centrifuged at 15,000 x g for 15 min at 4°C, and supernatants were incubated with 1 µg of anti-Akt-PH domain Ab, rotating continuously for 1 h at 4°C and then protein A-Sepharose beads were added for an additional 1 h. Beads were washed once each in lysis buffer and kinase buffer (20 mM HEPES, 10 mM MgCl2, 10 mM MnCl2) and incubated in a 30-µl reaction mixture containing 5 µCi of [{gamma}-32P]ATP, 1 mM DTT, 85.7 µg/ml histone H2B, and kinase buffer. Reactions were incubated at 25°C for 30 min and terminated by the addition of 6 µl of 6x Laemmli buffer. The samples were boiled for 3 min, the products were resolved by 10% SDS-PAGE, and 32P incorporation was visualized by autoradiography.

Superoxide production

Release of O

{cjs1138}

2 by neutrophils was measured in duplicate samples by cytochrome c reduction at 550 nm in spectrophotometer, as previously described (14).

Chemotaxis assay of neutrophils

Chemotaxis assay was performed as previously described (14). Briefly, neutrophils were washed and resuspended in Krebs with Ca2+ and Mg2+ (Krebs+). Cells were added to polypropylene microtubes (upper chambers) with or without LY294002 (50 µM for 10 min at 37°C) and wortmannin (100 nM for 1 h at 37°C) in the presence and absence of fMLP (0.3 µM final concentration) or baclofen (10 µM final concentration). To differentiate between directed or random migration, fMLP (0.3 µM final concentration) or baclofen (10 µM final concentration) were added to the bottom of the transwells (lower chambers). Transwells were incubated at 37°C with 5% CO2 in the tissue culture incubator for 30 min. Then the polyester membranes were fixed, stained with H&E stain, and dried at room temperature overnight. Membranes were cut and fixed on the glass slides keeping the bottom surface upright and viewed under the light microscope using x100 magnification. Cells within the scale that had passed through the pores and were at the focal plane of the pores were counted. Results were expressed as the mean plus SEM of the number of cells migrating across a 6.5-mm diameter circle of the membrane.

Tubulin staining of neutrophils

Neutrophils were washed and resuspended in Krebs+. After prewarming the cells at 37°C for 5 min they were treated with baclofen (10 µM final concentration) for 1, 2, 5, 10, 15, 30 min and 1 h. At the end of the corresponding time points cells were centrifuged for 20 s at 2500 x g and fixed by the addition of 200 µl of 3.7% paraformaldehyde for 30 min at room temperature. Cells were then washed twice with Krebs+ and permeabilized with 2% saponin for 30 min at 4°C. After 30 min cells were washed twice with Krebs+ and blocked with 200 µl of 5% goat normal serum for 1 h at 4°C. Mouse anti-tubulin {alpha}{beta} mixture Ab (2 µg final concentration) was added to the 200 µl of 5% goat normal serum and cells were incubated at 4°C overnight. Next day cells were washed with Krebs+ and incubated with goat anti-mouse rhodamine Ab (1 µg final concentration) at 4°C for 1 h. Lastly, cells were washed and resuspended in Krebs+ and placed in chambered cover glass wells. Samples were then examined using Zeiss Axiovert 100 microscope and LSM 510 software.

Colocalization of GABABR2/Akt and FPR/Akt by confocal microscopy

Neutrophils (5 x 106/ml) were suspended in Krebs+. Neutrophils (1 x 106/200 µl) were seeded into confocal chambers and incubated at 37°C for 1 h. The supernatant was gently aspirated out without disturbing neutrophils that had adhered to the coverslips of the confocal chambers. Neutrophils were fixed by addition of 200 µl of 3.7% paraformaldehyde solution and incubated for 30 min at room temperature. Neutrophils were washed twice with Krebs+ and permeabilized with 2% saponin for 30 min at 4°C. After 30 min cells were washed twice with Krebs+ and blocked with 200 µl of Krebs+ containing 2.5% goat normal serum and 2.5% BSA for 1 h at 4°C. Mouse anti-Akt (2 µg) and rabbit anti-GABABR2 (2 µg) was added to the 200 µl of Krebs+ containing 2.5% goat normal serum and 2.5% BSA and incubated with neutrophils at 4°C overnight. Alternatively, cells were stained with mouse anti-Akt and anti-goat FPR Ab. Next day, neutrophils stained with appropriate primary Abs were washed with Krebs+ and incubated with either 200 µl of Krebs+ containing goat anti-mouse rhodamine Ab (1 µg) and goat anti-rabbit fluorescence (1 µg) or goat anti-mouse rhodamine (1 µg) and donkey anti-goat Alexa Fluor (1 µg) at 4°C for 1 h. For negative controls neutrophils were stained with isotype-matched nonspecific Ab and goat anti-mouse rhodamine (1 µg) or goat anti-rabbit fluorescence (1 µg) antisera. Neutrophils were washed and resuspended in Krebs+ and samples were then examined using Zeiss Axiovert 100 microscope and LSM 510 software. No staining was detected in isotype-matched nonspecific Ab-stained cells.

Middle cerebral artery occlusion (MCAO)

Animal experimental protocols were approved by University of Louisville Institutional Animal Care and Use Committee and are in agreement with the National Institutes of Health guide for the care and use of laboratory animals. All efforts were made to minimize the number of animals used. Male Sprague Dawley rats (250 g; Charles River Breeding Laboratories) were anesthetized; a small cannula was placed in the left lateral ventricle. Osmotic pumps (Alzet) loaded with either baclofen (50 µg/100 µl), CGP52432([3-[(3,4-dichlorophenyl)methyl]amino]propyl-diethoxymethyl-phosphinic acid, 100 µg/100 µl), or vehicle were then connected to the cannula and secured in the cervical region under the skin. Twenty-four hours later, the right MCAO was occluded using a surgical string at its entrance into the lateral base of the skull for 90 min. Reperfusion was then allowed by releasing the occlusion, and re-establishment of blood flow to preligation levels was allowed and verified using laser doppler flowmetry (PeriFlux System 4000; Perimed). For controls, a craniectomy was conducted and the dura opened for vessel exposure but no occlusion was done (sham). After 0, 6, 24, 48, and 96 h, animals were anesthetized and perfused with 2% paraformaldehyde. Brains were removed, and after 24-h fixation with sucrose 25% and paraformaldehyde 2%, serial 5-µm coronal sections were obtained at 100-µm intervals using a cryostat (Leica), and then subjected to immunohistochemical staining with a monoclonal myeloperoxidase Ab (1/1000; LabVision). Unbiased stereological quantification was then performed by an investigator blinded to the various treatments using a Nikon 800F microscope fitted with a motorized stage and a charge-coupled device-based video camera and using Stereo Investigator software (Microbrightfield).

Statistics

Data is shown as mean ± SE. The n value shows the number of separate experiments. Values for p are calculated by the SigmaStat software using Student’s t test. A value p < 0.05 was a priori considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Proteomic analysis of Akt immunoprecipitates

To understand the role of Akt in mediating a number of neutrophil functions we sought to identify Akt binding partners in human neutrophils. Neutrophil lysates were subjected to isotype control or anti-Akt immunoprecipitation. Immunoprecipitated proteins were initially resolved by high-resolution 2D-PAGE as previously described by our group (22, 23). MALDI analysis coupled with BLASTP analysis of Akt immunoprecipitates identified a C-terminal fragment of GABABR2, as an Akt binding protein. The identification of a fragment of GABABR2 instead of an intact receptor could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor. The presence of intact GABABR2 in neutrophils was documented by immunoblotting neutrophil lysates with anti-GABABR2 Ab (Sigma-Aldrich) (Fig. 1, top). These results demonstrated for the first time presence of GABABR2 in human neutrophils.



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  		FIGURE 1. Intact GABABR2 and 65–67 kDa glutamic acid decarboxylase (GAD65/67) is present in human neutrophils. Neutrophil lysates (50 µg) were subjected to 10% SDS-PAGE and immunoblot analysis with anti-GABABR2 (top) and anti-GAD65/67 (bottom) antisera, respectively. Results demonstrate presence of intact GABABR2 and 65–67 kDa glutamic acid decarboxylase in human neutrophils.

 
To indirectly determine whether the neurotransmitter GABA is produced in human neutrophils, we tested the presence of glutamic acid decarboxylase, the critical enzyme that converts glutamate to GABA. Immunoblot analysis of neutrophil lysates demonstrated the presence of 65–67 kDa glutamic acid decarboxylase in human neutrophils (Fig. 1, bottom).

Detection of GABABR2 in human neutrophils by confocal microscopy

To confirm the presence of GABABR2, neutrophils were stained with anti-GABABR2 Ab (QED Bioscience) and visualized by Zeiss Axiovert 100 microscope and LSM 510 software (Fig. 2A). GABABR2 was detected in cytosolic plasma membrane, epicenter of the cell, and in nucleus of neutrophils (Fig. 2A), as opposed to FPR (Fig. 3A), which was expressed in the cytosol and plasma membrane. Colocalization of GABABR2 or FPR and Akt was demonstrated by confocal microscopy (Figs. 2C and 3C). GABABR2 or FPR is represented in green (Figs. 2A and 3A), Akt is represented in red (Figs. 2B and 3B), and colocalization of GABABR2 or FPR and Akt is represented in yellow (Figs. 2C and 3C). Additionally, colocalization was also visualized in a graphic profile of red (Akt) and green (GABABR2 or FPR) intensities across the cell using the Zeiss LSM 510 software (Figs. 2D and 3D). These results show that the GABABR2 and Akt are colocalized in the cytosol, plasma membrane, and in the epicenter of the cell. However, FPR and Akt are colocalized in the cytosol and on the surface of the cell (Figs. 2D and 3D). No staining was detected in isotype matched nonspecific Ab and goat anti-mouse rhodamine or goat anti-rabbit FITC-stained neutrophils (data not shown).



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  		FIGURE 2. Colocalization of GABABR2 and Akt in human neutrophils. Neutrophils were fixed and permeabilized and stained with isotype-matched nonspecific Ab (data not shown) or anti-Akt and anti-GABABR2 antisera as described in Materials and Methods. Stained cells were then visualized by confocal microscopy (n = 3). A, GABABR2 staining in neutrophils is depicted in green, in this confocal image. B, Akt staining in neutrophils is depicted in red, in this confocal image. C, A merged image of anti-Akt and anti-GABABR2-stained neutrophils. Yellow staining viewed in this image depicts colocalization of Akt and GABABR2. D, Colocalization of Akt and GABABR2 was also visualized by profiling red (Akt) and green (GABABR2) intensities across a cell using Zeiss Axiovert 100 microscope and LSM 510 software. These results show that GABABR2 was detected in cytosol, plasma membrane, epicenter of the cell, and in nucleus of neutrophils. Additionally, Akt/GABABR2 colocalization was detected in the cytosol, plasma membrane, and in the epicenter of the cell. No staining was detected in isotype-matched nonspecific Ab-stained cells.

 


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  		FIGURE 3. Colocalization of FPR and Akt in human neutrophils. Neutrophils were fixed and permeabilized and stained with isotype-matched nonspecific Ab (data not shown) or anti-Akt and anti-FPR (fMLP receptor) antisera as described in Materials and Methods. Stained cells were then visualized by confocal microscopy (n = 3). A, FPR staining in neutrophils is depicted in green in this confocal image. B, Akt staining in neutrophils is depicted in red in this confocal image. C, A merged image of anti-Akt and anti-FPR-stained neutrophils. Yellow staining viewed in this image depicts colocalization of Akt and FPR. D, Colocalization of Akt and FPR was also visualized by profiling red (Akt) and green (FPR) intensities across a cell using Zeiss Axiovert 100 microscope and LSM 510 software. These results show that FPR was detected in cytosol and plasma membrane of neutrophils. Additionally, Akt/FPR colocalization was detected both in the cytosol and plasma membrane of neutrophils. No staining was detected in isotype-matched nonspecific Ab-stained cells.

 
Confirmation that the GABABR2 is present in a complex with Akt in neutrophils

As the intact GABABR2 was detected in human neutrophils we next determined whether this receptor directly associated with Akt. To this end neutrophil lysates were subjected to anti-Akt-PH domain or isotype control Ab immunoprecipitations followed by immunoblotting with anti-GABABR2 Ab. Fig. 4A demonstrates an interaction between Akt and GABABR2 in anti-Akt but not isotype control precipitates. Reciprocal immunoprecipitations with anti-GABABR2 Ab confirmed association between Akt and GABABR2 (Fig. 4B). These data confirmed that Akt and GABABR were present together in a complex however, failed to demonstrate that they were functionally linked.



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  		FIGURE 4. Association of Akt/GABABR2 in human neutrophils. A, Neutrophil lysates were subjected to isotype control or anti-Akt immunoprecipitations followed by immunoblotting with anti-GABABR2 Ab. Isotype and Akt immunoprecipitates were also probed with anti-Akt Ab to demonstrate that Akt was immunoprecipitated. B, Neutrophil lysates were subjected to isotype control or anti-GABABR immunoprecipitations followed by immunoblotting with anti-Akt Ab. Isotype and Akt immunoprecipitates were also probed with anti-GABABR2 Ab to demonstrate that GABABR2 was immunoprecipitated. Results demonstrate association between Akt/GABABR2 in anti-Akt and anti-GABABR but not in isotype control immunoprecipitations (n = 3).

 
The GABABR is functionally linked to Akt

To determine a functional consequence of Akt/GABABR association, we addressed the hypothesis that GABABR activation would lead to subsequent Akt activation in human neutrophils. To test this hypothesis, neutrophils were stimulated with the GABABR agonist baclofen (10 µM) and subjected to immunoblot analysis with anti-phospho Ser473 Akt Ab. Baclofen induced a rapid and transient Akt Ser473 phosphorylation between 2 and 5 min that was resolved after 10 min, with no changes in overall Akt expression (Fig. 5A). Additionally, pretreatment with a PI3K inhibitor, LY40029 (50 µM) blocked baclofen induced Akt phosphorylation with no changes in overall Akt expression (Fig. 5B). Furthermore, the GABABR agonist, baclofen (10 µM) increased Akt activation (Fig. 5C, lane 2), in a PI3K-dependent manner, as pretreatment with LY294002 inhibited baclofen stimulated Akt kinase activity (Fig. 5C, lane 5). Similar results were obtained with SKF97415(10 µM), a GABABR agonist (data not shown). In contrast, treatment with the GABABR antagonist CGP52432(10 µM) blocked baclofen-induced histone H2B phosphorylation by Akt (Fig. 5D, lane 3).



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  		FIGURE 5. GABABR is functionally linked to Akt. A, Neutrophils were incubated with or without 10 µM baclofen at 37°C for 2, 5, and 10 min respectively. Neutrophil lysates generated from control and baclofen-treated samples were subjected to 10% SDS-PAGE followed by immunoblot analysis with anti-phospho-Akt Ser473 (S473) and anti-Akt antisera. Results demonstrate that baclofen (10 µM) induces Akt phosphorylation between 2 and 5 min and is resolved by 10 min (top), without altering the levels of total Akt in every sample (n = 2) (bottom). B, Neutrophils were preincubated with or without 50 µM LY294002 (PI3K inhibitor) for 15 min at 37°C before stimulation with or without 10 µM baclofen for 5 min. Neutrophils were also stimulated with 0.3 µM fMLP. Following stimulation, neutrophil were lysed, and the lysates were subjected to 10% SDS-PAGE and immunoblot analysis with anti-phospho-Akt Ser473 (S473) and anti-Akt antisera. Results demonstrate that fMLP and baclofen (10 µM) induces Akt phosphorylation and baclofen-induced Akt phosphorylation is inhibited by pretreatment with LY294002 (top), without altering the levels of total Akt in every sample (n = 2) (bottom). C, Neutrophils were preincubated with or without 50 µM LY294002 (PI3K inhibitor) for 15 min at 37°C before stimulation with or without 10 µM baclofen for 5 and 10 min, respectively. Following stimulation, neutrophil were lysed, and the lysates were subjected to an in vitro immunoprecipitation kinase assay for Akt using histone H2B as substrate. Kinase reactions were subjected to 12% SDS-PAGE and autoradiography. Results demonstrate that baclofen stimulates Akt kinase activity at 5 min (bottom, lane 2), which is inhibited by preincubation with LY294002 (n = 3) (top, lane 5). Gel was stained with Coomassie blue to document equal amount of substrate (histone H2B) was added in all conditions. D, Neutrophils were untreated or treated with 10 µM baclofen, 10 µM CGP52432(GABABR antagonist), or with 10 µM baclofen plus 10 µM CGP52432for 5 min at 37°C. Neutrophil lysates were subjected to in vitro Akt kinase assay using histone H2B as substrate. Kinase reactions were subjected to 12% SDS-PAGE and autoradiography. Addition of GABABR antagonist, 10 µM CGP52432simultaneously with 10 µM baclofen inhibited baclofen-stimulated Akt kinase activity as demonstrated by loss of histone phosphorylation (n = 2). Gel was stained with Coomassie blue to document equal amount of substrate (histone H2B) was added in all conditions.

 
GABABR stimulation causes reorganization of tubulin

To further explore functional implications of the GABABR-Akt pathway in neutrophils, textual bioinformatic analysis of the proteins identified along with Akt in GABABR2 immunoprecipitates from human neutrophils was performed using CellSpace software (Cellomics). Proteomic analysis identified proteins in the GABABR-Akt signaling module that play a role in microtubule regulation. These proteins included microtubule associated regulatory kinase 4, the dynein H chain, merlin, and focal adhesion kinase, suggesting that stimulation of the GABABR may alter the microtubule cytoskeleton in a PI3K/Akt-dependent manner in neutrophils. To test this hypothesis, human neutrophils were pretreated with or without a PI3K inhibitor followed by stimulation with 10 µM baclofen and stained with an anti-tubulin mAb. Baclofen induced loss of the central microtubule assembly at 15 min in a PI3K-dependent manner (Fig. 6). Additionally, baclofen failed to stimulate respiratory burst in neutrophils (Fig. 7).



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  		FIGURE 6. GABABR stimulation causes reorganization of neutrophil tubulin. Neutrophils were pretreated with or without 50 µM LY294002 (PI3K inhibitor) before stimulation with 10 µM baclofen at varying times. Following stimulation neutrophils were fixed, permeabilized, stained with anti-tubulin Ab, and viewed by confocal microscopy. The results demonstrate that baclofen stimulated tubulin reorganization in a PI3K-dependent manner (n = 3).

 


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  		FIGURE 7. Baclofen failed to stimulate neutrophil superoxide release. The release of superoxide from neutrophils stimulated with 0.3 µM fMLP or 10 µM baclofen was determined. Results are expressed as mean ± SEM in nanomoles of reduced cytochrome c/106 cells for at least five separate experiments. FMLP alone stimulated a significant increase in superoxide release (*, p < 0.0006). In contrast, baclofen failed to stimulate superoxide release in neutrophils.

 
Baclofen acts as a neutrophil chemoattractant via a PI3K-dependent mechanism

Microtubule disassembly induces receptor-linked neutrophil chemotaxis (24). Additionally, GABA receptors have been shown to induce directed migration of embryonic cortical cells (25) hence, we hypothesized that GABABR activation may induce neutrophil chemotaxis. In a two-chamber chemotaxis assay, human neutrophils were placed in the top transwell followed by addition of fMLP (0.3 µM; a concentration of fMLP known to induce neutrophil chemotaxis) or baclofen (10 µM; dose determined to be optimal for chemotaxis) were added to the bottom transwell. Both drugs elicited a significant increase in directed migration of neutrophils, which was inhibited in the presence of PI3K inhibitor, 50 µM LY294002 (Fig. 8, lanes 2, 3, 5, and 6). Additionally, we demonstrated that fMLP and baclofen stimulated direct migration of neutrophils, because addition of fMLP or baclofen to neutrophils in the top transwell failed to stimulate neutrophil migration (Fig. 8, lanes 7 and 8). To further establish GABABR specificity for chemotaxis, experiments were conducted with baclofen, 10 µM CGP52432(GABABR antagonist). Baclofen stimulated neutrophil chemotaxis (Fig. 9). Furthermore, CGP52432significantly inhibited baclofen-stimulated neutrophil chemotaxis (Fig. 9, lane 4).



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  		FIGURE 8. Baclofen acts as a neutrophil chemoattractant via a PI3K-dependent mechanism. Neutrophils were untreated or pretreated with 50 µM LY294002 before stimulation with/without 0.3 µM fMLP or 10 µM baclofen (GABABR agonist) and assayed for migration across a polyethylene membrane. Chemotaxis results are expressed as mean ± SEM in number of cells crossing the membrane per 6.5 mm diameter for four separate experiments. fMLP (*, p < 0.003) or baclofen (*, p < 0.008) significantly induced neutrophil chemotaxis, which was significantly inhibited by pretreatment with LY294002 (50 µM) fMLP (*, p < 0.008), and baclofen (*, p < 0.0003). Addition of fMLP (0.3 µM) or baclofen (10 µM) directly to neutrophils failed to stimulate nonspecific neutrophil chemokinesis.

 


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  		FIGURE 9. GABABR antagonist CGP52432blocks baclofen-stimulated neutrophil chemotaxis. Neutrophils were treated with or without 10 µM baclofen (GABABR agonist), 10 µM CGP52432 or CGP52432plus baclofen and assayed for migration across a polyethylene membrane. Chemotaxis results are expressed as mean ± SEM in the number of cells crossing the membrane per 6.5 mm diameter for three separate experiments. Baclofen (*, p < 0.0007) significantly induced neutrophil chemotaxis. Simultaneous addition of CGP52432and baclofen (#, p < 0.014), significantly inhibited baclofen-induced neutrophil chemotaxis.

 
The GABABR functions as a neutrophil chemoattractant receptor

To determine the in vivo role of the GABABR as a neutrophil chemoattractant receptor, osmotic pumps loaded with baclofen (50 µg/100 µl), CGP52432(100 µg/µl), or vehicle were implanted and connected to the left lateral cerebral ventricle of Sprague Dawley rats. Twenty-four hours later, transient right MCAO was performed, and brain tissues examined for myeloperoxidase expressing cells. Substantial increases in the number of myeloperoxidase-positive cells emerged in baclofen-treated animals starting at 24 h after MCAO, whereas pretreatment with CGP52432abolished neutrophil migration to the injured brain area (Fig. 10).



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  		FIGURE 10. GABABR modulates leukocyte influx in ischemic brain. Top, Myeloperoxidase (MPO) staining of rat brain sections subjected to MCAO for 90' and pretreated with baclofen or vehicle (n = 6/group). Bottom, Individual myeloperoxidase-positive (MPO+) cell counts in right hemisphere of rats subjected to MCAO for 90' and pretreated with baclofen, vehicle, CGP52432(CGP), or baclofen/sham surgery (n = 6/group). Marked increases in myeloperoxidase (MPO+) cell influx occurred after baclofen compared with vehicle (p < 0.001) or baclofen/sham surgery (p < 0.001), and were completely abrogated after CGP52432administration (p < 0.0001 vs baclofen; p < 0.001 vs vehicle).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been previously demonstrated that the brain and the immune system share common molecules and receptors (26). Additionally, psychoactive drugs, such as benzodiazepines, have been shown to modulate neutrophil functions including, intracellular calcium release, chemotaxis, and phagocytosis (27, 28). The GABAAR was recently identified as an Akt substrate in the mammalian brain, thereby linking Akt to regulation of synaptic strength (29). However, we are unaware of the association of GABABR with Akt in any cell type. The present study shows for the first time that the GABABR2 is present in human neutrophils and that it is functionally associated with the signaling kinase Akt. Proteomic studies first identified a C-terminal fragment of GABABR2 as an Akt binding protein, instead of the intact receptor. This could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor, however GABABR proteolysis has not been documented. Immunoblotting studies of neutrophil lysates and confocal microscopy of anti-GABABR2-stained neutrophils documented presence of GABABR2 in neutrophils. Additionally, anti-Akt immunoprecipitates demonstrated that the intact receptor was bound to the Akt signal complex. Collectively, these results suggest that GABABR may interact with Akt via its C-terminal tail.

Akt functionally interacts with multiple signaling pathways (30, 31, 32). We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPKAPK-2, and heat shock protein 27 (30). Additionally, we demonstrated that a disruption of Akt-heat shock protein 27 interaction resulted in neutrophil apoptosis (17). To identify additional members of the Akt signal complex that regulate neutrophil functions, proteomic analyses of Akt immunoprecipitates in human neutrophils were performed. Proteomic analysis of Akt immunoprecipitates from human neutrophils identified a C-terminal fragment of GABABR2. Presence of an intact GABABR2 in human neutrophils and its association with Akt was demonstrated by immunoprecipitation studies, immunoblot analysis, and confocal microscopy. Additionally, confocal staining identified GABABR2 in the cytosol, plasma membrane, epicenter of the cell, and in the nucleus of neutrophils. Detection of GABABR2 in the nucleus was surprising; however, GABABR2 has been shown previously to interact with nuclear proteins such as transcription factors CREB2 and activating transcription factor x and factor 4 (33, 34).

Akt was shown previously to be activated in human neutrophils by fMLP, leukotriene B4, IL-8, Fc{gamma}R cross-linking, LPS, GM-CSF, and antineutrophil cytoplasmic Abs (18, 30, 35, 36, 37). We report for the first time, that a GABABR agonist, baclofen, stimulates Akt phosphorylation and activation in a PI3K-dependent manner, whereas CGP52432 a GABABR antagonist inhibits baclofen-stimulated Akt activation, suggesting a functional association of Akt and GABABR in human neutrophils.

Akt activation has been shown to regulate various neutrophil functions, including apoptosis, actin polymerization, chemotaxis, and superoxide burst activity (14, 15, 16, 17, 18, 19). In the current study, baclofen failed to stimulate neutrophil superoxide burst in neutrophils. Proteins that play a role in the regulation of the microtubule were identified in the GABABR-Akt signaling module and GABABR activation resulted in disassembly of the tubulin network in a PI3K-dependent manner. In neutrophils, disassembly of the microtubule network has been shown to induce development of polarity, a key feature required for chemokinesis and chemotaxis (24). Recent studies have suggested a role for the GABABR in directed migration of embryonic cortical neuron, and motility of spinal neuroblasts, thus supporting the concept that these receptors can guide directed migration or growth (25). The present study shows that GABABR selectively induces neutrophil chemotaxis via an Akt-dependent mechanism because pretreatment with LY294003 or simultaneous addition of CGP52432inhibited baclofen-stimulated neutrophil chemotaxis. Additionally, pretreatment with LY294003 inhibited baclofen-stimulated Akt phosphorylation and Akt activation. Furthermore, in vivo studies demonstrated increased migration of neutrophils into brain regions subjected to ischemic-reperfusion injury. It is possible that pharmacological inhibition of PI3K may affect multiple signaling pathways other than Akt in human neutrophils. It is also possible that other pathways such as p38 MAPK or Erk MAPK pathways may feed into the Akt pathway or may be down stream of the Akt pathway. However, those studies are beyond the scope of the current study. In human neutrophils we have previously demonstrated that fMLP-stimulated Akt activation is dependent on p38 MAPK activation (30).

These results suggest an important role for GABABR in the extent of the inflammatory response, and ultimately in the magnitude of cell loss in stroke (38, 39, 40). Increased glutamate release occurs in multiple organs subjected to ischemia (41, 42, 43) and glutamic acid decarboxylase, the critical enzyme-converting glutamate to GABA does not require oxygen, and is ubiquitously expressed in tissues such as brain, heart, kidney, and lung (44, 45). In the current study, the presence of glutamic acid decarboxylase in neutrophils was documented by Western blot analysis. Hence, we postulate that induction of GABABR-Akt mediated chemotaxis underlies a major mechanism of inflammatory regulation in tissues undergoing ischemia-reperfusion and thereby provides novel potential interventional targets for palliation of ischemic tissue injury.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants HL66358 (to J.B.K.), HL63912, P50HL60296, and HL69932 (to D.G.), and HL74296 (to E.G.). K.R.M. and J.B.K. are supported by the Department of Veterans Affairs Merit Review. This work is also supported by the American Heart Association Grant SDG 0335278N (to M.J.R.) and the Kentucky Challenge for Excellence Trust Fund. Back

2 Address correspondence and reprint requests to Dr. Madhavi J. Rane, University of Louisville, Baxter Research Building, 570 South Preston Street, Louisville, KY 40202. E-mail address: mrane{at}louisville.edu Back

3 D.G. and J.B.K. contributed equally as senior authors. Back

4 Abbreviations used in this paper: GABA, {gamma}-amino-butyric acid; FPR, formyl peptide receptor; GABABR2, {gamma}-amino-butyric acid type B receptor 2; MCAO, middle cerebral artery occlusion. Back

Received for publication July 28, 2004. Accepted for publication February 23, 2005.


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 Introduction
 Materials and Methods
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 Discussion
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