Signature: Med Sci Monit, 2002; 8(8): BR313-323 PMID: 12165735

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Basic Research

Received: 2002.03.07 Accepted: 2002.07.10 Published: 2002.08.07

Activation of human polymorphonuclear cells induces formation of functional gap junctions and expression of connexins

Authors’ Contribution: A Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection

María C. Bran~es1 abcdefg, Jorge E. Contreras1 abcdef, Juan C. Sáez1,2 abcdefg 1

Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile 2 Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Source of support: This work was partially financed by grants of Fondo Nacional para el Desarrollo de la Ciencia y la Tecnología (FONDECYT-2960002 and 8990008).

Summary Background:

During inflammatory responses activated polymorphonuclear cells (PMNs) adhere to each other and form clusters within the vasculature or injured tissues. We hypothesized that conditions that partially mimic the chemical environment of inflammatory foci induce the expression of functional gap junctions (GJs) between cultured PMNs.

Material/Methods:

Human PMNs were treated with bacterial lipopolysaccharide (LPS), TNF-α, LPS plus medium conditioned by LPS-treated endothelial cells (ECs) or TNF-α plus ECs conditioned medium. Gap junctional communication was evaluated with the dye coupling technique using a permeant and an impermeant GJ fluorescent dye and GJ blockers. The expression of connexins, GJ protein subunits, was evaluated by immunocytochemistry and immunoblotting. Cytochalasin-D and nocodazole were used to evaluate the involvement of cytoskeleton in the induction of dye coupling.

Results:

Treatment with LPS or TNF-α induced the formation of PMN aggregates, but cells were not dye coupled. If the latter protocols occurred in medium conditioned by LPS-treated ECs or resting ECs, respectively, intercellular transfer only of the GJ permeant molecule was observed in most clustered cells. Dye coupling was reversibly inhibited by GJ blockers and prevented by cytochalasin-D, a microfilament disrupter, but not by nocodazole, a microtubule disrupter. Treatments that induced dye coupling also induced connexin43 and connexin40, but not connexin32 immunoreactivity. None of these connexins was detected in circulating cells.

Conclusion:

EC-derived factor(s) and microfilament integrity are required for dye coupling between LPSand TNF-α-treated PMNs. GJ formation between PMNs is correlated with the presence of connexins 43 and 40, but not 32 and requires intact microfilaments.

key words: Full-text PDF: Word count: Tables: Figures: References: Author’s address:

leukocytes • gap junction subunits • dye coupling • endotoxin • TNF-α • glycyrrhetinic acid

http://www.MedSciMonit.com/pub/vol_8/no_8/2590.pdf 4616 1 7 58 Ms. María C. Bran~es, Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile, email: [email protected]

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BACKGROUND

MATERIAL AND METHODS

At inflammatory foci cellular adhesion is promoted by inflammatory agents (e.g., bacterial lipopolysaccharide, (LPS)) and by soluble factors, e.g., cytokines, released by activated leukocytes and endothelial cells (ECs). The arrest of circulating polymorphonuclear cells (PMNs) at a site of inflammation is an early event during the response mediated by the native immune system. The sequential exposure of different adhesion molecules at the surface of both activated PMNs and ECs allows rolling of PMNs along the endothelial surface followed by their firm adhesion to the endothelium [1]. L- and Eselectins are involved in the rolling step [2], and members of the integrin family are responsible for their firm adhesion to the EC surface (e.g, MAC-1 in PMNs binds ICAM-1 in the endothelium) [3]. Cytokines secreted by activated leukocytes and ECs are one component of an inflammatory signaling pathway. In addition, the close contact between PMNs and the endothelium might allow transfer of cell signals through either a ligandreceptor mechanism mediated by cell adhesion molecules located at the cell surface of contacting cells [4] or by a direct cytoplasm-cytoplasm pathway mediated by gap junctions (GJs) [5].

Reagents

In several systems cell adhesion molecules in the surface membrane are essential for the organization of functional GJs [6]. Gap junctional communication is mediated by intercellular channels that result from the interaction of two hemichannels or connexons, one provided by each of two adjacent cells. An hemichannel is an oligomer of six protein subunits named connexins (Cxs), which constitute a family of highly homologous proteins encoded by different genes [7]. GJs are permeable to ions and small molecules, including second messengers such as Ca2+, inositol 1,4,5-trisphosphate and cyclic nucleotides [8,9], which can activate responses in neighboring cells [10–13]. A number of ultrastructural and molecular studies suggest that leukocytes form GJs at heterologous and homologous cellular contacts. Morphological studies have revealed GJs at macrophage-PMNs interfaces in cell aggregates present in the peritoneal exudate of LPS-treated rainbow trout [14]. Other ultrastructural evidence indicates formation of GJ-like structures between PMNs and ECs after ischemia-reperfusion [5]. In addition, Cx43 has been detected in hamster peritoneal macrophages, and hamster peripheral leukocytes activated with LPS, or by ischemia-reperfusion [5]. To our knowledge, it remains unknown whether human activated PMNs forming aggregates establish gap junctional communication. In this paper, we demonstrate that circulating human PMNs activated with proinflammatory stimuli form functional GJs and express at least two different Cxs. LPS as well as its mediator, tumor necrosis factor-alpha (TNF-α), would be involved in developing intercellular communication between PMNs. Preliminary observations have been previously reported [15].

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Culture media (RPMI 1640, F-10, F-12 and MEM), neomycin, basic fibroblastic growth factor (bFGF), penicillin, streptomycin, fetal bovine serum (FBS), trypsinEDTA, glycine and Tris were from GibcoBRL (Grand Island, NY, USA). NaF, urea and trichloroacetic acid (TCA), were obtained from Merck KGaA (Darmstadt, Germany). Airvol 203 was obtained from Air Products and Chemicals, Inc. (Allentown, CA, USA). Human recombinant TNF-α (hTNF-α) was obtained from Calbiochem-Novabiochem Corp. (San Diego, CA, USA). Histopaque 1077 and 1119, LPS (E. coli serotype 0127(B8), mouse recombinant TNF-α (mTNF-α), puromycin, benzamidine, soybean trypsin inhibitor, ε-amino caproic acid, phenylmethylsulfonyl fluoride (PMSF), leupeptin, sodium pyrophosphate, sodium orthovanadate, octanol, 18α-glycyrrhetinic acid (AGA), dextranFITC (150 kDa), Lucifer yellow-CH (LY), bovine serum albumin (BSA) (fraction V essentially Ig-free), 1,4-diazabicyclo [2.2.2]octane (DABCO), poly-L-lysine (56 kDa), 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) tablets, cytochalasin D, nocodazole, dithiothreitol (DTT) and fish gelatin were obtained from Sigma (St. Louis, MO, USA). Antibodies Goat anti-rabbit IgG antibody conjugated to alkaline phosphatase, F(ab’)2 fragments of goat anti-rabbit IgG labeled with FITC, and F(ab’)2 fragments of goat antimouse IgG labeled with FITC were purchased from Sigma (St. Louis, MO, USA). A rabbit anti-Cx43 serum directed to the C-terminus of the rat protein [16], a rabbit anti-Cx43 serum directed to the extracellular loop-1 of the rat protein [17] and mouse ascites fluid of a monoclonal anti-rat Cx32 [18] were kindly provided by Dr. Elliot L. Hertzberg, Department of Neuroscience, Albert Einstein College of Medicine, New York. A rabbit anti-Cx40 serum was also commercially generated using a synthetic peptide (Bios Chile Ingeniería Genética SA, Santiago, Chile). The sequence of the peptide corresponds to a non-conserved region of the C-terminus mouse Cx40 (amino acids 339–356, KRRLSKASSKARSDDLSV; anti-Cx40/C-terminus antibody) [19]. The peptide was conjugated to Blue Carrier (Hemocyanin from Concholepas concholepas, Biosonda SA, Santiago, Chile) using glutaraldehyde [20]. Immunization of rabbits and collection of sera were performed by Biosonda. Purification of F(ab’)2 fragments IgGs F(ab’)2 fragments were obtained by means of a commercial kit (Pierce). According to the instructions of the provider the IgGs were isolated from each serum, and then F(ab’)2 fragments were obtained by enzymatic digestion and purified using an affinity column. Briefly, for IgGs purification serum was diluted (1:1) in union buffer (0.1 M sodium acetate, pH 5) and then was centrifuged at 1,000 × g during 20 min. Diluted serum

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supernatant was applied over a G protein column, previously equilibrated with union buffer. The column was washed with 6–10 volumes of union buffer until the absorbance at 260 nm reached the base line. IgGs were eluted with 3–5 volumes of 75 mM glycine-HCl buffer (pH 2.6). The eluates were collected in fractions of 1 ml, and their pH was neutralized immediately by the addition of 500 mM Tris (pH 7.6). The absorbance of each fraction was measured at 280 nm and those with the highest value were pooled and dialyzed in semi permeable membranes of 12 kDa molecular weight exclusion (Spectrapor 4, Spectrum Medical Industries, Inc. Terminal Annex, LA, USA), against 0.1 M NH4HCO3. Samples were concentrated using a speed vac concentrator (Integrated SpeedVac System, IS100, Savant Instruments, Inc, Farmingdale, NY, USA). Ten milligrams of IgG were dissolved in 1 ml of digestion buffer (20 mM cystein-HCl in phosphate buffer, pH 10). Papaine coupled to sepharose (0.5 ml) was washed twice with 4 ml digestion buffer, resuspended in 0.5 ml of digestion buffer and added to 1 ml of the IgG solution. The reaction mixture was incubated at 37°C for 5 h or overnight under constant shaking. The digestion products were recovered, mixed with 1.5 ml union buffer, and then passed through a union buffer equilibrated column of protein A coupled to sepharose. The column was washed with 6 ml of union buffer and the collected eluate that contained F(ab’)2 fragments was dialyzed the same as mentioned above. Finally, F(ab’)2 fragments were suspended in 500 µl of 1% BSA in PBS and frozen at –80°C until they were used. Cell cultures HeLa cells stably transfected with rat Cx43, Cx40, or Cx37 DNA were kindly provided by Dr. Klaus Willecke, Institut für Genetik, Universität Bonn, Germany and Dr. Bruce Nicholson, Department of Cell Biology, SUNY at Buffallo, USA. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and selection antibiotics (1 µg/ml puromycin or 300 µg/ml neomycin), as described previously [21]. The RBE4 endothelial cell line derived from the microcirculation of the rat brain cortex [22] was kindly provided Dr. P. O. Couraud, Laboratoire d’Immuno-Pharmacologie Moléculaire, Institut Cochin de Genetique Moléculaire, Paris, France. Cells were cultured on rat tail collagen coated plastic culture dishes in MEM/F10 (1:1) medium supplemented with 10% FBS, 1 ng/ml bFGF, 300 µg/ml neomycin, 100 U/ml penicillin and 100 µg/ml streptomycin. Subconfluent cultures were fed 24 h before collecting the conditioned media (CMEC). LPS- and mTNF-α-induced conditioned media (LPSCMEC and TNF-CMEC, respectively) were obtained from subconfluent cultures fed and treated with 1 µg/ml LPS or 10 ng/ml mTNF-α 24 h before collecting them.

Bran~es MC et al – Activation of human polymorphonuclear cells…

Isolation of PMNs Blood samples (6 ml) obtained from normal human volunteers were layered on a discontinuous Histopaque gradient (3 ml Histopaque 1119 under 3 ml Histopaque 1077) and centrifuged (Kubota 8700, Kubota Corporation, Tokyo, Japan) at 600 x g for 30 min at 22°C. The layer of PMNs found right above the red blood cells that sediment to the bottom was carefully aspirated with a Pasteur pipette and diluted in 30 ml of washing solution (0.4% sodium citrate in PBS) and then centrifuged at 400 x g for 10 min at 4°C. The cell pellet was resuspended in 50 ml of lysis buffer (155 mM NH4Cl, 2.7 mM KHCO3 and 3.7 mM EDTA) and maintained 10 min at room temperature followed by centrifugation at 400 x g for 10 min at 4°C. Then, cells were resuspended in cold (4°C) PBS and immediately centrifuged at 400 x g for 10 min at 4°C. Finally, cells were resuspended in RPMI 1640 medium plus 10% FBS. Immunofluorescence Blood smears or freshly isolated human PMNs placed on 1% polylysine coated glass slides were fixed in 70% ethanol for 20 min at –20°C. Samples were incubated in blocking solution (5 mM EDTA, 1% fish gelatin, 1% BSA, 1% goat serum in PBS) for 30 min at room temperature or overnight at 4°C. Then, they were incubated overnight at 4°C in primary antibody, rinsed 10 times in PBS (pH 7.4) for a total of 30 min at room temperature and incubated with FITC-conjugated secondary antibody for 1 h at room temperature followed by another 30 min rinsing period. After free secondary antibodies were washed out, samples were mounted in glycerol containing 16% Airvol 203 dissolved in PBS with DABCO (100 mg/ml). Preparations were then examined in a Nikon Labophot-2 microscope equipped with epifluorescent illumination and photographed using T-Max 400 film (Kodak). Immunoblotting For detection of Cxs cells were suspended in a solution containing 10 mM TCA and 10 mM DTT, pH 7.6, maintained on ice for 30 min and centrifuged for 1 min at maximal speed in an Eppendorf 5415C centrifuge. The supernatant was discarded and the pellet was washed with ether three times for 10 min each. Pelleted proteins were resuspended at 4°C by 1 min sonication in position 1 (Microson ultrasonic cell disrupter, Heat Systems Inc, Farmingdale, NY, USA) in a solution containing 8 M urea, 20 mM Tris pH 8.6, 23 mM glycine, 10 mM DTT, 3 mM PMSF, 10 µg/ml leupeptin and 2 mg/ml soybean trypsin inhibitor. Proteins were measured in aliquots of cell lysates using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA, USA). After protein samples (200 µg) were mixed with Laemmli buffer, they were boiled immediately for 4 min and resolved in 8% SDS-PAGE vertical slab gels in Hoefer Scientific Instruments (San Francisco, CA, USA) mini gel units, in a discontinuous buffer system as described previously [23]. Gels were then blot-

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ted onto nitrocellulose and proteins were electrotransferred to the nitrocellulose sheet at 300 mA for 1.5 h. Blots were incubated in 5% non-fat milk in 0.05 M Trisbuffered saline (TBS) pH 7.4 for 30 min at room temperature or overnight at 4°C and then transferred to an anti-Cx antibody diluted in 5% non-fat milk in TBS. Blots were rinsed repeatedly in TBS and then incubated for 1 h at room temperature in goat anti-rabbit IgG antibody conjugated to alkaline phosphatase diluted 1:2,000 in TBS. After rinsing repeatedly in TBS, blots were incubated in a solution of BCIP/NBT tablets (1 tablet in 10 ml of water). Dot blot Synthetic peptides corresponding to a region of Cx37, Cx40 and Cx43 extracellular loop-1 were dissolved in water, blotted onto a nitrocellulose sheet and fixed for 2 h in a solution that contained 3% Tris-HCl, 14.4% glycine, 1% SDS and 20% methanol. The antigens were detected as described above for immunoblotting. The amino acid sequences of peptides homologous to a region of the extracellular loop-1 of rat Cxs included aminoacids 49–60 of Cx37 (QSDFECNTAQPG), Cx40 (QADFRCDTIQPG) and Cx43 (QSAFRCNTQQPG). Peptides were synthesized in the Macromolecule Center, Washington University, St. Louis, MO, USA. Dye Coupling After the different treatments, cells plated on glass coverslips were bathed with recording medium (F-12 medium free of HCO3– and buffered with 10 mM HEPES, pH 7.2) and the functional state of GJs was evaluated with the dye coupling technique. LY (5% LY in 150 mM LiCl) was microinjected into one cell of a cluster of PMNs attached to a glass coverslip. The dye was injected by repeated 0.1 sec, 0.1 nA current pulses or by oscillations induced by overcompensation of the negative capacitance amplifier until the impaled cell was brightly fluorescent. After injection, cells were observed for 2–3 min on a Nikon Diaphot microscope equipped with xenon arc lamp illumination and a Nikon B filter block (excitation wavelength: 450–490 nm; emission wavelength: >520 nm) in order to determine whether dye transfer occurred. The incidence of dye coupling was scored as the percentage of injections that resulted in dye transfer from the injected cell to more than one neighboring cell. The coupling index was calculated as the mean number of cells to which the dye spread when the spread was observed to more than one cell.

RESULTS Endothelial cell-derived factor(s) and microfilaments integrity are required for dye coupling between LPS- and TNF-α-treated PMNs Freshly isolated human PMNs (106 cells/150 µl medium) were placed on glass coverslips. If cells were incubated during 1 h at 37°C in RPMI culture medium they remained single and spherical (Fig. 1A). But, if they were incubated with LPS (1 µg/ml), 10–20% of the cells

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Figure 1. PMNs treated with LPS form clusters. Isolated human PMNs (1×106 cells/150 µl) incubated in RPMI culture medium for 1 h at 37°C onto a glass coverslip remained dispersed (A). But, if PMNs were incubated in the presence of LPS (1 µg/ml), a fraction of them formed clusters denoted by arrows (B). After treatment with LPS several cells that remained as singlets were tightly adhered to the coverslip surface (B, inset). Under control conditions, cells remained round (A, inset). Bar: 150 µm.

formed clusters of variable cell number (Fig. 1B). Isolated spherical cells that were LPS-treated or untreated PMNs were easily washed away by flushing with culture medium. However, after LPS-treatment cell clusters and flattened single cells remained adherent to the surface of the coverslip (Fig. 1, insets). When cellular coupling was tested by microinjecting LY into one cell of a cluster, the dye remained restricted to the injected cell (Fig. 2B). PMNs incubated for up to 3 h in CMEC or LPS-CMEC remained as non-adherent single cells (not shown). Moreover, treatment with 1 (µg/ml LPS in CMEC for 3 h induced formation of cell aggregates but they were not dye coupled (not shown). However, if PMNs were treated with LPS in LPS-CMEC, they clustered as seen after treatment with LPS in RPMI (Fig. 1B) or LPS in CMEC (not shown), but LY spread from the microinjected cell to 3–6 neighboring cells (Fig. 2D and Table 1). LPS-induced cell aggregation was prevented by 30 min preincubation with 5 µg/ml cytochalasin D, a microfilament disrupter, but not by 30 min preincubation with 10 µM nocodazol, a microtubule destabilizer. Nocodazol did not prevent cell aggregation induced by LPS treatment in LPS-CMEC, and dye coupling was comparable to that detected in cells activated in the absence of nocodozole (Table 1). Dye coupling was not evaluated in PMNs treated with cytochalasin D because they did not form clusters. Since PMNs or ECs treated with LPS express and secrete TNF-α that mediates many of the LPS-induced cellular effects [24,25], we studied the effect of mTNF-α on PMNs coupling. Dye coupling was observed after PMNs were stimulated with mTNF-α in CMEC, but not in RPMI medium (Table 1), although under both conditions they formed aggregates (not shown). In RPMI, cells forming aggregates were not tightly adhered to

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Figure 2. Conditioned medium from LPS-treated endothelial cell cultures promotes coupling of LPS-stimulated PMNs. Isolated PMNs treated with LPS 1 µg/ml for 1 h did not show LY transfer to neighboring cells (B). However, in PMNs treated for 1 h with LPS (1 µg/ml) in LPS-CMEC, dye transfer to neighboring cells was observed (D). If these cells were treated for 15 min with 75 µM AGA they were not coupled (F). Fifteen min after AGA was washed out, dye coupling was recovered (H). In PMNs treated with LPS in LPS-CMEC that were bathed for 5 min in LY (5% w/v in culture medium) followed by extensive washes, a spotty and scarce labeling was detected (J), and if dextran-FITC (150 kDa) was microinjected to one cell in the cluster, it was retained in the microinjected cell (L). A, C, E, G, I, and K are phase views of B, D, F, H, J and L, respectively. Bar: 40 µm.

each other and the aggregates were poorly adhered to the coverslip surface difficulting the scoring of dye coupling. Within 1 h of stimulation the incidence of cell coupling remained close to 0% in PMNs treated with 1 ng/ml mTNF-α in CMEC and it was higher in PMNs treated with 10 ng/ml mTNF-α in CMEC than in cells treated with 5 ng/ml mTNF-α in CMEC (Table 1). Incubation in TNF-CMEC promoted cell aggregation comparable to that observed after treatment with mTNF-α in TNF-CMEC, but the incidence of dye coupling was around 20%. Because TNF-α shows species preference for its receptor [26], the possibility that hTNF-α would be more effective in inducing gap junctional communication than mTNF-α was studied. Treatment of freshly isolated PMNs with 10 ng/ml hTNF-α in RPMI for 1 h induced the formation of loose cell aggregates showing an incidence of dye coupling of 15±10 % (n = 4), indicating that hTNF-α is not sufficient to induce gap junctional communication in PMNs.

Dye transfer between PMNs is mediated by gap junctional communication Dye coupling was reversibly inhibited by treatment with 75 µM AGA (Fig. 2F, H and Table I) or 500 µM octanol (Table 1), two gap junction blockers [27,28]. The staining pattern of PMNs treated for 1 h with 1 µg/ml LPS in LPS-CMEC followed by 5 min incubation with LY applied extracellularly (5% in culture medium) (Fig. 2J) was radically different (spotty and scarce) from that observed after microinjecting the dye (Fig. 2D). Thus, dye was not spreading by way of extracellular space. Moreover, 150 kDa dextran-FITC (5% in 150 mM LiCl) microinjected into one cell of a cluster was always retained in the injected cell (Fig. 2L and Table 1). Thus, cells were not connected by cytoplasmic bridges. Treatment of freshly isolated PMNs with proinflammatory molecules induces immunoreactivity to Cx40 and Cx43, but not to Cx32 F(ab’)2 fragments of anti-Cx43, -Cx40 and -Cx32 antibodies labeled GJ plaques in rat heart sections (Fig. 3A),

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Table 1. Dye coupling in PMN aggregates under various experimental conditions. Experimental condition LPS in RPMI LPS in LPS-CMEC Octanol Octanol wash out AGA AGA wash out Dextran-FITC LPS in LPS-CMEC + Nocodazol (10 mM) mTNF-α in CMEC (1 ng/ml) mTNF-α in CMEC (5 ng/ml) mTNF-α in CMEC (10 ng/ml) Octanol Octanol wash out TNF-CMEC TNF-α in RPMI (10 ng/ml)

Incidence of coupling (%) 0.0±0.0 90.8±14.6 0.0±0.0 100.0±0.0 20.0±0.0 80.0±0.0 0.0±0.0 85.7±9.8 0.0±0.0 40.0±5.0 87.7±10.0 25.0±3.0 80.0±9.0 20.0±0.0 0.0±0.0

Coupling index 0.0±0.0 4.5±1.2 0.0±0.0 4.0±1.0 2.0±1.0 3.0±1.3 0.0±0.0 3.3±1.4 0.0±0.0 1.0±1.0 3.3±1.4 1.5±0.6 3.5±1.4 3.0±0.8 0.0±0.0

Isolated PMNs were suspended in RPMI, in conditioned medium by EC (CMEC) or in conditioned medium by ECs treated with 1 µg/ml LPS or 10 ng/ml mTNF-a (LPS-CMEC or TNF-CMEC, respectively). Then, PMNs were incubated for 1 h at 37°C with 1 µg/ml LPS or 10 ng/ml mTNF-a and dye coupling was evaluated at room temperature by microinjection of LY within the following 30 min. The effect of 500 µM octanol and 75 µM AGA on cells showing high incidence of coupling, LPS in LPS-CMEC and mTNF-α in CMEC, was also studied. Coupled cells were treated for 2 or 15 min with octanol or AGA, respectively, and dye coupling was tested during the following 20 min. After washing out octanol and AGA by replacing the recording medium 4 times during a total of 5 min, dye coupling was measured during the following 20 min. Dextran-FITC (MW 150 kDa), a non permeant GJ molecule, was microinjected in cells treated with LPS in LPS-CMEC that were coupled to LY. Incidence of coupling and coupling index correspond to the average of 5 different experiments ± S.D. except in octanol, AGA, Dextran-FITC and LPS in LPS-CMEC plus Nocodazole where three experiments were done. In each experiment a minimum of 10 cells were microinjected. Each set of experiments included multiple treatments and PMNs used in each one were from different blood donors.

HeLa cells transfected with Cx40 cDNA (Fig. 3B) and rat liver sections, respectively (Fig. 3D). In support to the specificity of anti-Cx40 antibody its F(ab’)2 fragments did not react with HeLa-Cx43 (Fig. 3C) or -Cx37 transfectants (not shown). In addition, the anti-Cx40 serum recognized a band of about 40 kDa in total homogenates of HeLa-Cx40, but not of -Cx43 and –Cx37 transfectants (Fig. 3, lanes 2, 1 and 3, respectively). Figure 3. Anti-Cx43, -Cx40 and -Cx32 antibodies recognize their antigens in cells or tissues expressing them but do not react with circulating human PMNs. Reactivity to F(ab’)2 fragments of anti-Cx43 antibody directed against the C-terminus in a section of rat heart (A), anti-Cx40 antibody in HeLa Cx40(B) and HeLa Cx43- (C) transfected cells, and anti-Cx32 antibody in a section of rat liver (D) is shown. A single band of about 40 kDa was detected in immunoblots of total homogenates (200 µg of proteins) of HeLa Cx40 transfectants (lane 2), but not of HeLa cells transfected with Cx37 (lane 1) or Cx43 (lane 3). The electrophoretic mobility of BSA (66 kDa), egg ovalbumin (45 kDa) and rabbit muscle glyceraldehyde-3 phosphate dehydrogenase (36 kDa) are indicated in the right. Negative reaction with F(ab’)2 fragments of anti-Cx43 (E), -Cx40 (F) and Cx32 (G) antibodies is shown in PMNs (denoted by arrows) present in smears of freshly drawn peripheral blood. Bar: 15 µm (A, B and C), 50 µm (D), 10 µm (E-G). Observations illustrated in this figure correspond to a representative result from five different experiments carried out independently. Blood samples were from five different donors.

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Using the specific reactive F(ab’)2 fragments described above, no Cx43, Cx40 and Cx32 reactivity was detected in circulating PMNs (Fig. 3E-G). The background shown in figure 3E to G was comparable to that observed in blood smears stained with F(ab’)2 fragments of preimmune sera or with the secondary antibody (not shown). Nevertheless, dye coupling between activated PMNs treated with LPS in LPS-CMEC suggested that Cx expression could be induced. Therefore, we examined Cxs 43, 40 and 32 by immunofluorescence after incubation for 1 h at 37°C under various conditions. Freshly isolated PMNs were also immunonegative to Cx43 (Fig. 4A), Cx40 (Fig. 4A) and Cx32 (not shown). After incubation in RPMI (Fig. 4B), or after treatment with mTNF-α (10 ng/ml) in RPMI (Fig. 4C), PMNs remained unreactive to F(ab’)2 fragments from antibodies of each immune serum. PMNs incubated in CMEC (Fig. 4F) or LPS-CMEC (Fig. 4E) also were not immunoreactive to the two Cxs. However, Cx43 and Cx40 were detected in PMNs subjected to treatments that induced dye coupling, that is, both Cxs were detected in PMNs incubated in CMEC and stimulated with mTNF-α (10 ng/ml) (Fig. 4H) and in PMNs incubated in LPS-CMEC and treated with LPS (1 µg/ml) (Fig. 4G). Cells treated with

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Figure 4. LPS and TNF-α induce positive reactivity to Cx43 and Cx40 in PMNs. F(ab’)2 fragments from rabbit anti-Cx43 directed against the C-terminus and anti-Cx40 IgGs were used for immunofluorescent detection of Cxs. Negative immunoreactivity to both Cxs was observed in freshly isolated human PMNs (A), or in PMNs that were incubated for 1 h at 37°C in RPMI culture medium (B), or in PMNs incubated with 10 ng/ml mTNF-α in RPMI (C), in LPS-CMEC (E), or in CMEC (F). Nevertheless, PMNs incubated for 1 h in LPS-CMEC and stimulated with LPS (1 µg/ml) (G), or incubated in CMEC and stimulated for 1 h with 10 ng/ml mTNF-α (H), Cx43 and Cx40 positive immunoreactivity was observed. No label was observed in PMNs incubated in CMEC and stimulated with 10 ng/ml mTNF-α when F(ab’)2 fragments obtained from preimmune sera were used (D). Observations illustrated in this figure correspond to a representative result from five different experiments carried out independently in samples from different donors. Bar: 15 µm.

mTNF-α in CMEC did not react with F(ab’)2 fragments of IgGs of preimmune serum for each Cx (Fig. 4D). PMNs did not show reactivity to Cx32 under any of the above conditions (not shown). The immunofluorescent findings were supported by Western blot analysis. The detection of Cx protein bands with the corresponding electrophoretic mobilities of the Cx43 P2 phosphorylated form (Fig. 5A; lane 2) and Cx40 (Fig. 5B and C, lanes 2) denoted the induction of both Cxs after treatments that induced dye coupling in PMNs. In cells incubated in CMEC (Fig. 5A; lane 1), RPMI medium (Fig. 5B, lane 1) or LPS-CMEC (Fig. 5C, lane 1) no Cx protein bands were detected. Compa-

rable results were obtained in three independent experiments. TNF-α induces translocation of Cx43 to the PMNs surface Since the appearance of dye coupling was accompanied by an induction of Cx expression, the possibility that newly synthesized Cxs were inserted in the plasma membrane was studied in non permeabilized PMNs with the aid of an antibody directed against the extracellular loop-1 of Cx43. This antibody reacted with a peptide corresponding to a segment of the extracellular loop-1 of Cx43 but not of Cxs 37 or 40 (Fig. 6). Consis-

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Figure 5. LPS and TNF-α induce the expression of Cx43 and Cx40 in PMNs. Proteins isolated PMNs were processed for separation by PAGE-SDS and identification by Western blot analysis of Cx40 and Cx43. Cx bands were detected using the antiCx43 antibody directed to the C-terminus or the anti-Cx40 antibody. Lane 1 shows negative reactivity to Cx43 (A) or 40 (B, C) in PMNs incubated for 1 h in CMEC, RPMI medium and LPS-CMEC, respectively. Positive reactivity to Cx43 (panel A, lane 2) and Cx40 (panel B, lane 2) was observed in PMNs treated for 1 h at 37°C with 10 ng/ml mTNF-α in CMEC. Panel C shows the induction of Cx40 in PMNs treated for 1 h with 1 µg/ml LPS in LPS-CMEC (panel C, lane 2). Lanes H correspond to Cx43 reactivity of a protein aliquot (25 µg) of rat heart homogenate used as an electrophoretic mobility standard of the non-phosphorylated (NP), and the phosphorylated forms (P1, P2, and P3) of Cx43, known to run between 40–45 kDa. Cx40 shows an electrophoretic mobility similar to that of the non phosphorylated form of Cx43. Observations illustrated in this figure correspond to a representative result from three different experiments carried out independently in samples from different donors.

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Figure 7. TNF-α promotes the insertion of Cx43 into PMNs cell surface. Isolated human PMNs were suspended during 1 h at 37°C in CMEC (A) or in CMEC plus mTNF-α (10 ng/ml) (B and C). Then, cells were fixed with 1% paraformaldehyde (A and B) or were fixed and permeabilized with ethanol (C) and cells were processed for Cx43 immunofluorescent detection using the F(ab’)2 fragments of a rabbit antibody directed to the extracellular loop-1 of Cx43. Fluorescent view of Cx43 reactivity assayed with the F(ab’)2 fragments of the same anti-Cx43 antibody in non-permeabilized rat astrocytes (positive, D) and in an ethanol fixed rat heart section (negative, E). Observations illustrated in A-C correspond to a representative result from three different experiments carried out independently in samples from different donors. Bar: 50 µm (A-C), 10 µm (D) and 15 µm (E).

tent with the presence of hemi-GJ channels exposing the extracellular loops of Cx43 to the external surface of rat astrocytes [29] the F(ab’)2 fragments of the antibody directed against the extracellular loop-1 of Cx43 labeled the surface of unpermeabilized astrocytes (Fig. 7D). In addition and in agreement with the localization of heart Cx43 almost exclusively at myocyte-myocyte contacts (Fig. 3A), the F(ab’)2 fragments of the antibody directed against the extracellular loop-1 of Cx43 did not stain rat heart sections (Fig. 7E), presumably because the reaction with extracellular loop epitopes was physically hindered.

Figure 6. Specificity of antibody directed against the extracellular loop1 of Cx43. Twenty (a) or forty (b) micrograms of synthetic peptide homologous to a region of the extracellular loop-1 of Cx43 (Cx43/loop-1), Cx37 (Cx37/loop-1) or Cx40 (Cx40/loop-1) were blotted onto nitrocellulose and then analyzed using the anti-Cx43 antibody directed against the extracellular loop-1.

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Figure 7 shows that freshly isolated PMNs did not react with the F(ab’)2 fragments of the antibody directed against the extracellular loop 1 of Cx43 after incubation in CMEC (Fig. 7A). But, after treatment with 10 ng/ml mTNF-α in CMEC for 1 h, a condition that induced Cx43 expression and dye coupling, diffuse fluorescent labeling was detected on the surface of clustered PMNs as well as on PMNs that remained as single cells (Fig. 7B). Figure 7C shows the Cx43 reactivity of PMNs treated with the same protocol as in figure 7B, but using permeabilized cells. Under the latter conditions, Cx43 labeling in many cells was more pronounced at perinuclear regions suggesting that in those cells the permea-

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bilization procedure might have reduced Cx43 reactivity found on the cell surface and thus increased the relative intracellular labeling.

DISCUSSION We demonstrated that circulating human PMNs treated with LPS or mTNF-α in the presence of factors secreted by EC form aggregates and communicate between them through a pathway sensitive to GJ blockers, indicating that activated PMNs may communicate to each other through GJs. Those treatments also induced the expression of Cx43 and Cx40, but not Cx32 reactivity. During microinjection, leakage of LY to the extracellular space frequently happens, raising the possibility that dye staining of neighboring cells may occur by mechanisms independent of GJs, such as uptake from the extracellular milieu. The infrequent staining of PMNs in cell clusters bathed in LY rules out the possibility of uptake mechanisms such as organic ionic transporters [30,31], LY permeable channels (e.g. ATP-activated P2Z/P2X7 channels [32] or gap junction hemichannels [29,33–35]). Further indication of dye coupling via GJs was the lack of LY spreading in LPS or mTNF-α-treated PMN clusters, the reversible inhibition of LY spreading by GJ blockers and the absence of neighboring cell staining after microinjection of a GJ impermeant tracer (dextran-FITC). Thus, the most likely pathway for the observed dye spread between neighboring PMNs is gap junctional. The soluble factors produced by endothelium and leukocytes, through paracrine and autocrine action, are important signals for progression of the inflammatory response. It is known that cytokines activate a sequential cascade of other cytokines secreted in a redundant or complementary manner [36]. Thus, it has been described that the degranulation response of PMNs is generated by IL-1β, but not directly, because it needs other factors secreted by the IL-1β-activated endothelium [37]. Similarly, we observed that PMNs formed GJs when they were treated with LPS in LPS-CMEC. One possibility is that the factor present in the conditioned medium is TNF-α. Consistently, we observed that the application of mTNF-α in CMEC increased the incidence of dye coupling. It is possible that the amount or stability of TNF-α secreted by the LPS-activated endothelium was not enough to reach the effective concentration necessary to induce PMN aggregation and coupling. Nonetheless, as human or mouse TNF-α alone did not induce cell coupling, CMEC might contain additional factor(s), different from TNF-α, that act together to induce coupling between PMNs. Among possible factors different from inflammatory cytokines are proteolytic enzymes that modify the cell surface [38] favoring cell adhesion and therefore gap junctional communication. The degranulation response of PMNs depends on transport mechanisms mediated by microfilaments and by microtubules [39]. Incubation with cytochalasin D prevented the formation of PMNs aggregates, probably

Bran~es MC et al – Activation of human polymorphonuclear cells…

because it inhibited the transport of MAC-1 containing vesicles, a microfilament dependent process [39], and thereby impeded cell adhesion and also GJ formation between PMNs. Cx43 GJ channel clustering in the cell membrane [40] and the transport of Cx43 from the endoplasmic reticulum to the plasma membrane [41] are also microfilament dependent. Moreover, the lack of effect of nocodazol on PMN coupling suggests that Cx containing granules are not translocated through a microtubule-dependent mechanism. The detection of Cx43 and Cx40 by Western blot analysis of PMNs treated with mTNF-α or LPS but not of PMNs that were not dye coupled is consistent with an induction of Cx expression. Induction of Cx expression also occurs in hamster leukocytes after a period of ischemia-reperfussion [5]. In lung cells transfected with the Cx43 promoter over a human growth hormone gene as activator of expression, transcription is activated by factors present in the serum of rats previously treated with LPS or directly by LPS or IL-1β [42]. In these studies, the serum with more activity over the Cx43 promotor was that obtained after 1 h LPS administration, time at which high levels of TNF-α are expressed [43]. In our hands, Cx induction in PMNs occurred in cells treated with LPS or mTNF-α plus the factor(s) present in the medium conditioned by ECs suggesting that Cx expression in PMNs might be induced by the same or a similar mechanism as that described by Fernandez-Cobo et al. [42]. Cx hemichannels have been previously demonstrated in the non junctional membrane of diverse cell types [29,33–35]. In agreement, we detected Cx43 in nonjunctional membrane by immunofluorescence using an antibody that specifically recognizes its extracellular loop-1, and its translocation to the plasma membrane of activated PMNs, where it is likely to be in the form of hemichannels that may open without docking with an hemichannel in an apposed membrane. Similarly, immunofluorescence has revealed Cx43 on the surface of subconfluent astrocytes in culture [44]. The phosphorylated forms P2 and P3 of Cx43 have been localized in the plasma membrane of cell lines [45,46], suggesting that most or all the P2 form detected in activated PMNs is on the cell surface. Nevertheless, it can not be ruled out that other forms of Cx43 are also expressed but, because of their sensitivity to PMNdependent proteolysis, were not detected. Cx43 is quickly proteolized by serine proteases inhibited by PMSF [47]. The co-expression of more than one Cx by a single cell type may allow the formation of GJ containing homo or/and heterotypic or/and heteromeric channels [7]. It has been demonstrated that both Cxs found in activated PMNs form homotypic channels (Cx40/Cx40 and Cx43/Cx43) in exogenous expression systems [21,48]. However, Cx40/Cx43 heterotypic channels do not form [21,48]. Nevertheless, it has been demonstrated that cells co-expressing Cx40 and Cx43 form functional heteromeric channels [49]. LY is negatively charged, there-

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fore it is possible that other negative molecules such as IP3, cAMP and cGMP, may also permeate GJ between PMNs. In the case that homotypic Cx43 channels were formed, and as they exhibit little charge selectivity [21], it is possible that Ca2+ and other small cations, such as NAD+, diffuse through these junctions. In various normal tissues, gap junctional communication coordinates metabolic cellular responses [10–12]. Under pathological conditions such as CCl4– or LPSinduced liver necrosis [50,51], brain stab wounds [52], inflammatory renal diseases [53], and atherosclerotic plaques [54,55] it has been shown that fixed tissue and/or infiltrated macrophages gather at inflammatory foci express Cxs. Moreover, dye coupling between cultured microglia [52] and electrical coupling between macrophage colony-forming cells [56] demonstrate that they are able to establish gap junctional communication. Since PMNs aggregation is common in diverse microvascular pathologies, including reperfusion associated injury, acute respiratory distress syndrome, endotoxemia and autoimmune diseases [57,58] it is likely that gap junctional communication between PMNs plays a role in these processes.

CONCLUSIONS The present studies provide the first demonstration that human PMNs are able to communicate to each other through GJs formed, at least, by Cxs 43 and 40, but not by Cx32. TNF-α and factors present in the CMEC as well as microfilaments integrity are required to induce functional expression of GJs between human PMNs. Acknowledgments We are grateful to Dr. M. V. L. Bennett for his comments on the text and to Ms. Gladys Garcés for her technical assistance. We also thank to the blood bank of the Hospital Clínico, Pontificia Universidad Católica de Chile for providing us with normal blood samples. The data in this paper are from a thesis submitted in partial fulfillment of the requirements for the Degree of Doctor in Biological Sciences (M. C. Brañes) in the Pontificia Universidad Católica de Chile.

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