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Plus IFN-
Induce Connexin43 Expression and Formation of Gap Junctions Between Human Monocytes/Macrophages That Enhance Physiological Responses 1
* Departamento de Ciencias Fisiológicas, Pontificia Universidad Católica de Chile, Santiago, Chile; and Departments of
Pathology and
Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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, and IFN-
on gap junctional communication (dye coupling) and on the expression of connexin43 (immunofluorescence, immunoblotting, and RT-PCR) in monocytes/macrophages were studied. Freshly isolated human monocytes plated at high density and treated either with LPS plus IFN- or TNF- plus IFN- became transiently dye coupled (Lucifer yellow) within 24 h. Cells treated with LPS, TNF-, or IFN- alone remained dye uncoupled. In dye-coupled cells, the spread of Lucifer yellow to neighboring cells was reversibly blocked with 18 -glycyrrhetinic acid, a gap junction blocker, but it was unaffected by oxidized ATP or probenecid, which block ionotropic ATP-activated channels and organic anion transporters, respectively. Abs against TNF- significantly reduced the LPS plus IFN--induced increase in dye coupling. In dye-coupled monocytes/macrophages, but not in control cells, both connexin43 protein and mRNA were detected, and their levels were higher in cells with an elevated incidence of dye coupling. In dye-coupled cells, the localization of connexin43 immunoreactivity was diffuse at perinuclear regions and thin cell processes. The addition of 18--glycyrrhetinic acid induced a profound reduction of monocyte/macrophage transmigration across a blood brain barrier model. It also induced a significant reduction in the secretion of metalloproteinase-2 in cells treated with TNF- plus IFN-. We propose that some monocyte/macrophage responses are coordinated by connexin-formed membrane channels expressed transiently at inflammatory sites in which these cells form aggregates. |
Introduction |
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3 days in humans (1). As compared with healthy conditions, in many disease states there is increased bone marrow production of monocytes that have a shorter circulation time and rapidly extravasate (2). Once they reach an inflammatory focus within a tissue, monocytes differentiate into macrophages by growing and increasing their lysosomal content, the amount of hydrolytic enzymes, the number and size of mitochondria, and the extent of their energy metabolism (3, 4). Macrophages are involved in the defense against microorganisms, interaction with lymphoid cells during the immune response, tumor surveillance, disposal of cell debris (5, 6), and wound repair (7, 8). Macrophage clusters are frequent in tissues affected by diverse inflammatory disease states, in which cell-to-cell proximity may allow intercellular contacts necessary to accomplish some relevant functions. Under those conditions, their physical proximity might allow formation of intercellular junctions, such as gap junctions. The latter correspond to channels that communicate the cytoplasm of contacting cells, allowing the intercellular transfer of ions and small compounds, thereby coordinating diverse metabolic and electrical functions of cell communities (9, 10). A gap junction channel is constituted by joining two hemichannels or connexons, each formed by six connexin monomers surrounding a central pore, and each connexon is contributed by one of two contacting cells (11). Connexins are a family of homologous proteins, and each provides permeability and regulatory properties to the channels they form (12).
In agreement with the possibility that monocytes/macrophages may establish direct intercellular communication, it has been reported that J774 cells, a mouse macrophage cell line (13), and foam cells found in the carotid artery contain the mRNA of connexin43 (14). Moreover, connexin43 has been detected in activated peritoneal macrophages (15, 16). Nevertheless, findings of gap junctional communication between homocellular monocyte/macrophage contacts or heterocellular monocyte/macrophage-endothelial cell contacts have been controversial. Although an absence of gap junctional communication between primary monocytes/macrophages (14, 16) or between monocytes/macrophages and endothelial cells (14) has been reported, others have reported morphological and/or functional evidence of gap junctional communication between macrophage-macrophage (17, 18) and macrophage-polymorphonuclear cell or epithelial cell (19, 20). This controversy might be resolved with the identification of factors that regulate the expression of connexins in monocytes/macrophages.
We recently reported that microglia treated simultaneously with TNF- and IFN- express connexin43 and form functional gap junctions (21). In this work, we demonstrate that treatment with TNF- plus IFN- induces gap junctional communication mediated at least by connexin43 between freshly isolated human monocytes/macrophages. This form of homocellular cell-cell communication enhanced the secretion of the metalloproteinase metalloproteinase (MMP)-2,3 but not MMP-9. In addition, connexin43 was detected at cellular interfaces between monocytes/macrophages and endothelial cells of a blood brain barrier (BBB) model, and gap junction blockade reduced the transmigration of monocytes/macrophages across this model.
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Materials and Methods |
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F-12 medium, MEM, DNase I amplification grade, dNTPs, TRIzol, Superscrip II enzyme, FBS, penicillin, trypsin-EDTA, and streptomycin were from Life Technologies (Grand Island, NY). Lucifer yellow-CH (Ly), 18 -glycyrrhetinic acid (AGA), 1-octanol, Formalin-fixed Staphylococcus aureus Cowan (Pansorbin), PMSF, benzamidine, probenecid, oxidized ATP, endothelial cell growth factor, soybean trypsin inhibitor, 
-aminocaproic acid, leupeptin, Na4P2O7, NaF, Nonidet P-40, BSA essentially Ig free, FITC-conjugated goat F(ab')2 fragment of anti-rabbit IgGs, alkaline phosphatase-conjugated goat anti-rabbit IgG Ab, and 4', 6-diamidine-2-phenylindole (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO). An R-PE-conjugated mouse anti-human IgG mAb was obtained from BD PharMingen (San Diego, CA). Premixed human CD45 and CD14 mAbs conjugated to FITC and PE, respectively, were used (Caltag Laboratories, Burlingame, CA). A neutralizing rabbit polyclonal anti-human TNF- Ab, TNF-, and IFN- were obtained from R&D Systems (San Diego, CA). An anti-connexin43 mAb was obtained from Zymed Lab (San Francisco, CA). A rabbit anti-ubiquitin Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A wizard PCR system, preps DNA purification system, and Taq polymerases were purchased from Promega (Madison, WI). Previously characterized rabbit polyclonal anti-connexin43 Ab (22) and mouse anti-connexin32 mAb (23) were kindly provided by E. Hertzberg, Department of Neuroscience, Albert Einstein College of Medicine. A previously characterized rabbit anti-connexin40 serum was used (24). F(ab')2 fragments from IgGs of connexins sera (anti-connexin43 and anti-connexin40 Ab), ascitic fluid (anti-connexin32 Ab), or nonimmune rabbit sera were isolated using immobilized protein G (Pierce, Rockford, IL), and then F(ab')2 fragments were obtained using the preparation kit (Pierce), as recently described (24, 25).
Isolation and culture of monocytes
Anticoagulated (EDTA) blood was obtained from healthy volunteers, and 2 vol of blood were layered over 1 vol of Nycoprep 1.068 (Nycomed Pharma, Oslo, Norway) and centrifuged at 600 x g for 15 min. Monocytes were then isolated following the procedure described by the manufacturer (Nycomed Pharma AS). To determine the enrichment of the cell suspension with monocytes after the purification procedure, cells were incubated for 30 min at 4°C with a Caltag Ab premixture that contains PE-labeled anti-CD14 Ab (a monocyte marker) and FITC-labeled anti-CD45 (a pan lymphocyte marker) in the presence of 1 mM EDTA to dissociate into single cell clusters of monocytes, fixed in 2% paraformaldehyde, and analyzed within 24 h with a FACScan flow cytometer (BD Biosciences, Bedford, MA).
Monocytes were plated either on 60-mm plastic cell culture dishes (Nunc, Roskilde, Denmark) (5 x 106 cells/60-mm culture dish) or onto Nr.1 (12-mm-diameter) glass coverslips (Paul Marienfeld, Lauda-Konigshofen, Germany). Monocytes were cultured for different periods of time in control conditions of high glucose DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (control condition), or after treatment or LPS.
Dye coupling
Gap junctional communication was tested by observing the intercellular transfer of Ly (5% w/v Ly dissolved in 150 mM LiCl) microinjected through glass microelectrodes by brief overcompensation of the negative capacitance circuit in the amplifier until the impaled cell was brightly fluorescent. After dye injection, cells were observed for 1 min to determine whether dye transfer occurred, as described previously (21). The incidence of dye coupling was calculated by dividing the number of injected cells showing dye transfer to more than one neighboring cell by the total number of cells injected in each experiment multiplied by 100. In most experiments, the average number of cells coupled in a successful impalement (coupling index) was calculated by dividing the total number of stained cells when the dye diffused to two or more neighboring cells by the number of injections that revealed dye coupling. In all experiments, dye coupling was tested in a minimum of 10 microinjected cells. Dye coupling was observed in an inverted microscope equipped with xenon arc lamp illumination and a Nikon B filter (excitation wavelength 450–490 nm; emission wavelength above 520 nm).
Immunofluorescence
Cells cultured on glass coverslips were fixed and permeabilized in 70% ethanol for 20 min at -20°C. Cells were incubated in blocking solution (5 mM EDTA, 1% fish gelatin, 1% BSA essentially Ig free, 1% goat serum, and 20% of human serum) for 30 min at room temperature. Then cells were incubated in diluted primary Ab (anti-connexin43 F(ab')2 fragments, 1/2000) overnight at 4°C. Cells were washed with PBS, pH 7.4, four times for 5 min each, and then were incubated with diluted FITC-conjugated goat anti-rabbit IgG secondary Ab or FITC-conjugated goat anti-mouse IgG for 1 h at room temperature, followed by another rinse period of 1 h. Coverslips were mounted using Gelvatol-Dabco and observed under a Nikon (Melville, NY) labophot-2 microscope equipped with epifluorescent illumination or a confocal microscope (see below). The specificity of the immunoreactivity was assessed by replacing the F(ab')2 fragments of the primary Ab with F(ab')2 fragments of IgGs from a nonimmune rabbit serum.
In studies of monocyte/macrophage transmigration across the BBB model, the distribution of connexin43 and CD14 was studied by double labeling. The membranes that contain BBB cells and monocytes/macrophages in the process of transmigration were fixed and permeabilized in 70% ethanol for 20 min at -20°C, mounted in OCT, and cut in cryostat in sections of 10 µm. The sections were then simultaneously incubated with the appropriate dilution of F(ab')2 fragments of the polyclonal anti-connexin43 and PE anti-CD14 mAb for 1 h, followed by five washes with PBS. Cells were incubated with FITC goat F(ab')2 fragments of anti-rabbit IgGs for 1 h, followed by five washes with PBS. Preparations were mounted in gelvatol and observed by confocal microscopy (Bio-Rad, Hercules, CA) Radiance 2000 laser scanning confocal microscopy) at the Central Facilities of Albert Einstein College of Medicine. The fluorescence was observed in an inverted confocal microscope equipped (Nikon) with laser illumination and filters to FITC and Rhodamine (Bio-Rad; excitation wavelength 488 and 547 nm, emission wavelength above 500 and 560 nm, respectively). Pictures were acquired by Bio-Rad LaserSharp 2000 program and then were transferred to Adobe Acrobat program.
Western blotting
Monocyte cultures were harvested by scraping with a rubber policeman, pelleted by centrifugation, and washed with ice-cold PBS plus 2 mM PMSF (2 mM CaCl2, pH 7.4) before lysis by sonication (Microson Heat System, Farmingdale, NY) in a solubilization buffer containing protease inhibitors (200 µg/ml soybean trypsin inhibitor, 1 mg/ml benzamidine, 1 mg/ml -aminocaproic acid, and 2 mM PMSF) and phosphatase inhibitors (20 mM Na4P2O7 and 100 mM NaF). Monocytes/Macrophages were cultured in medium without serum, and after 24 h of culture under control conditions or treatment with 1 ng/ml TNF- plus 1 ng/ml IFN- the conditioned medium was collected to measure MMPs (the effect of TNF- plus IFN- on the induction of dye coupling between monocytes was the same in the presence or the absence of FBS in the culture medium; data not shown). Equal volumes of monocyte/macrophage-conditioned medium were centrifuged through a 30-kDa exclusion molecular mass Centricon filter (Amicon S.A., Beverly, MA) to reduce the volume by 20-fold. The concentrated samples were then subjected to Western blot analysis in parallel with aliquots of medium conditioned from monocytes/macrophages treated with 200 nM PMA for 12 h, which were used as a positive control of MMPs.
Proteins were measured in aliquots of cell lysates using the Bio-Rad protein assay. Western blot analyses were performed, as described previously (21). Briefly, 150 µg of protein was resolved in 8% SDS-PAGE. Gels were then blotted onto nitrocellulose and electrotransferred. To control for equal protein loading and blotting, nitrocellulose sheets were stained with Ponceau S and destained with distilled water, as described (26). Blots showing lanes with equal amount of proteins were then incubated in 5% nonfat milk in TBS, pH 7.4, for 30 min at room temperature and then kept overnight at 4°C in rabbit anti-connexin43, mouse anti-connexin43, rabbit anti-MMP-2, or anti-MMP-9 Abs diluted in 5% nonfat milk in TBS. Blots were rinsed repeatedly in TBS and then incubated for 1 h at room temperature in alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG Abs diluted in 5% nonfat milk in TBS. After rinsing in TBS, blots were incubated with alkaline phosphatase substrate (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium tablets; Sigma-Aldrich). Prestained low molecular mass markers and/or aliquots of rat heart homogenate were used to identify the electrophoretic mobility of connexin43-reactive bands.
Detection of connexin43 ubiquitination
Cell pellets were sonicated in 0.1 ml of 1:1 mixture of lysis buffer/buffer A (lysis buffer, 2% SDS, 100 U/ml aprotinin, 200 mg/ml soybean trypsin inhibitor, 1 mg/ml benzamidine, 1 mg/ml -aminocaproic acid, and 2 mM PMSF; buffer A, 5% Nonidet P-40, 20 mM EDTA, 250 mM NaCl, 20 mM Na4P2O7, 100 mM phosphate buffer, pH 7.4). Connexin43 was immunoprecipitated using the rabbit anti-connexin43 Ab following the procedure described previously (27). The immunoprecipitated connexin43 was resuspended in Laemmli’s buffer separated by SDS-PAGE and electrotransferred to nitrocellulose. Connexin43 and ubiquitin were detected in adjacent lanes of the nitrocellulose using a monoclonal or a polyclonal anti-connexin43 Ab and a rabbit anti-ubiquitin Ab, respectively.
RT-PCR detection of connexin43 mRNA
Total RNA from monocytes was isolated using TRIzol reagent. Possible contamination with DNA was avoided by treatment of total RNA (2 µg) with DNase I amplification grade. DNase activity was stopped by addition of 0.5 mM EDTA and heating 15 min at 65°C. Reverse transcription was done using DNase-treated RNA (2 µg) by adding 1 µl oligo(dT) (10 µM). The reaction mixture was heated for 10 min at 70°C, and then 7 µl of a solution containing dNTPs, first strand buffer 5x, and 2 µl DTT (100 mM) was added. The reaction mixture was heated for 2 min at 42°C, and then 1 µl of SuperScript II enzyme was added. Reverse transcription was achieved by heating the reaction mixture for 50 min at 42°C and then 15 min at 70°C. An extra reaction mixture without SuperScript II enzyme was used as control for DNA contamination.
PCR experiments (30 s at 95°C, 30 s at 56°C, and 45 s at 72°C for 30 cycles for connexin43 and 22 cycles for 
-actin; linear amplification was obtained between 26 and 34 cycles for connexin43 and between 18 and 26 cycles for -actin) were done on 2 µl of the reverse-transcriptase product plus 23 µl of PCR mix containing specific primers (4 µM) for human connexin43 (sense, 5'-GGG TTA AGG GAA AGA GCG ACC-3'; antisense, 5'-CCC CAT TCG ATT TTG TTC TGC-3'; databank accession number NM 000165), PCR buffer (1x), MgCl2 (1.5 mM), dNTPs (0.2 mM), and 0.25 U Taq polymerase. An internal positive control was included in each experiment using human -actin-specific primers (sense, 5'-CGG AAC CGC TCA TTG CC-3'; antisense, 5'-ACC CAC ACT GTG CCC ATC TA-3'; databank accession number XM 004814). The connexin43- and -actin-amplified cDNA fragments were resolved in agarose gels (1.5%). Ethidium bromide staining revealed bands of connexin43- and -actin-amplified PCR products that migrated near the predicted values of 247 and 281 bp, respectively, determined by comparison with a 100-bp DNA ladder (Winkler, Santiago, Chile).
The 247-bp PCR product was isolated from a low melting agarose gel and purified using Wizard PCR Preps DNA purification system. Its identity was confirmed to correspond to a fragment from human connexin43 by automated sequencing using an ABI Prism310 sequencer (PerkinElmer, Wellesley, MA), as described by Muscillo et al. (28).
Transmigration assays of monocytes in a model of the human BBB
Human fetal astrocytes and HUVECs were obtained, as described by Weiss et al. (29, 30). Cocultures were previously characterized for their barrier properties (31, 32, 33). Briefly, human fetal astrocytes (1 x 105 cells) were seeded onto the underside of a gelatin-coated 3-µm pore-size tissue culture insert (Falcon; BD Labware, Franklin Lakes, NJ). Cells were allowed to attach for 4–5 h, then inserts were placed upright into a 24-well tissue culture tray, and endothelial cells (1.6 x 104/insert) were seeded onto the upper side of each insert. Cocultures were maintained for 3 days in M199 culture medium supplemented with 10% FBS and 5% human serum; both sera were heat inactivated. A total of 3 x 105 monocytes (pretreated with TNF- plus IFN- in M199 culture medium plus 10% FBS) were added onto the top of each insert, and 24 h later their transmigration in response to monocyte chemoattractant protein-1 (MCP-1) was assayed. After 24 h, the cells from the bottom chamber were collected, and the number of cells that transmigrated was evidenced by FACScan analysis of the cells in the lower chamber culture medium using the premixed human CD45 and CD14 mAbs conjugated to FITC and PE, respectively (29, 30).
Statistical analysis
For statistical analysis of percentage, mean differences of results were tested with the nonparametric Kruskal-Wallis analysis. If a significant F value was obtained, means were compared with the Bofferonni-Dunn multiple comparison test. Student’s one-tailed, paired t test was used to compare the numbers of cells after transmigration and coupling index vs control conditions. A value of p < 0.05 was considered significant.
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Results |
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or TNF- plus IFN-
Freshly isolated monocytes kept in culture for 2 h before treatment with proinflammatory agents (Fig. 1E, time zero in the graph) were dye uncoupled (Fig. 1, B and E). After the 2-h incubation period, cells were treated with LPS (1 µg/ml) plus IFN- (1 ng/ml) for 12, 24, 48, and 72 h. A transient increase in the incidence of dye coupling that reached a maximum value of 43 ± 7% by 24 h was observed, and it decreased almost to control values by 72 h of treatment (Fig. 1, graph in E). An example of dye-coupled cells is shown in Fig. 1D. In a similar manner, the coupling index increased from 0 at 0 h to 2.3 ± 0.8 (n = 11; p < 0.05 as compared with time zero) by 12 h, and 5.2 ± 3.1 (n = 9; p < 0.05 as compared with time zero) by 24 h, and decreased to 2.6 ± 0.47 (n = 10; p < 0.05 as compared with time zero) by 72-h period of culture.
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, or 1 ng/ml IFN-, applied separately, did not increase the incidence of dye coupling measured every 4 h within a 72-h period (data not shown). The observed LPS plus IFN--induced increase in intercellular communication was blocked by coincubation with a neutralizing anti-TNF- Ab (1 µg/ml), suggesting that treatment with LPS plus IFN- induced secretion of TNF-, which in combination with IFN- induced dye coupling between monocytes/macrophages (Fig. 1, graph in E). In agreement with this finding, a similar transient increase in the incidence of dye coupling was observed in cells treated with 1 ng/ml TNF- plus 1 ng/ml IFN- for the same periods of time analyzed for treatment with LPS plus IFN- (Fig. 1, graph in E). Coupling indices of cells treated with TNF- plus IFN- were: 12 h, 2.6 ± 1.8 cells (n = 7; p < 0.05 as compared with time zero); 24 h, 4.3 ± 0.3 cells (n = 7; p < 0.05 as compared with time zero); and 72 h, 1.8 ± 0.9 cells (n = 7; p < 0.05 as compared with time zero).
To verify that the observed dye coupling was mediated by gap junctions, we tested the effect of AGA, a gap junction blocker (9, 10), on the increase in the incidence of dye coupling induced by LPS or TNF- plus IFN-. Thirty-five minutes before completion of the 24-h treatment with these proinflammatory agents, cells were treated with 35 µM AGA for 5 min, and then the incidence of dye coupling was evaluated during the following 30 min in the presence of the gap junction blocker. Under these conditions, the LPS or TNF- plus IFN--induced dye coupling was drastically reduced and, after three washes of 5 min each with recording medium, the incidence of dye coupling recovered to values comparable to those found in cells treated only with the proinflammatory mixture for 24 h (Fig. 1F). Treatment with 0.2% DMSO used as vehicle to apply AGA did not reduce the TNF- plus IFN--induced dye coupling observed at all times studied (not shown).
The possibility that the observed cell-cell transfer of Ly resulted from its leakage from the microinjected monocytes, followed by its uptake by neighboring cells, was also studied in cells treated with LPS plus IFN- 24 h. This possible pathway of intercellular dye transfer could include organic anion transporters and ATP ionotropic receptors, such as P2X7, which have been shown to translocate Ly across the plasma membrane (34, 35). After 15-min treatment with 200 mM probenecid, an inhibitor of organic anion transporters, or 2 h with 300 mM oxidized ATP, a blocker of ionotropic ATP receptors, the spread of Ly to neighboring cells was comparable to that observed in cells treated with LPS plus IFN- in the absence of those agents (not shown). Thus, the mechanisms attributed to the known action of those agents did not contribute in the dye transfer described above. Moreover, cells cultured under control conditions or treated with LPS or TNF- plus IFN- for 24 h did not show significant Ly uptake upon the application of the fluorescent dye to the extracellular medium (data not shown).
Immunoreactivity of connexin43 is induced in human monocytes treated with TNF- plus IFN-
In an attempt to identify connexins that could form gap junction channels responsible for the dye coupling observed between monocytes stimulated with proinflammatory agents, we studied the expression of connexin43, connexin40, and connexin32 by indirect immunofluorescence using F(ab')2 fragments of Abs specific to each connexin. All leukocytes (monocytes, lymphocytes, and granulocyte) identified by the appearance of their nuclei stained with DAPI (Fig. 2A) in blood smears of normal humans were immunonegative for connexin43 (Fig. 2B). Similarly, freshly isolated monocytes maintained in culture for 2 h under control conditions did not show connexin43 reactivity (Fig. 2, C or F), but were all positive for CD14 (not shown). In contrast, cells treated with 1 ng/ml TNF- plus 1 ng/ml IFN- for 12, 24, 48, and 72 h were connexin43 positive (Fig. 2, G–J). By confocal microscopy, in subconfluent cultures of monocytes/macrophages, connexin43 was detected in thin processes over the monocytes treated for 24 h with both cytokines (Fig. 2, D and E). No staining was observed by confocal microscopy with F(ab')2 fragments from the preimmune serum on monocytes treated for 24 h with 1 ng/ml TNF- plus 1 ng/ml IFN- (Fig. 2C). Using regular immunofluorescence microscopy, connexin43 reactivity was strong at perinuclear regions and cell processes. At 48- and 72-h postaddition of TNF- plus IFN-, the connexin43-reactive thin processes were diminished, but overall were as intense as at 12 and 24 h after treatment with cytokines (Fig. 2, I and J). Cells cultured under control conditions for 12, 24, 48, and 72 h remained connexin43 negative (data not shown). No staining was observed with F(ab')2 fragments from preimmune serum in monocytes treated for 24 h with 1 ng/ml TNF- plus 1 ng/ml IFN- (Fig. 2F). Previous immunofluorescence studies with F(ab')2 fragment of Abs directed against connexin32 or connexin40 showed labeling at regions of cell appositions in hepatocytes of rat liver sections and HeLa connexin40 transfectants (23, 36). Connexin40 and connexin32 were not detected in human blood smears or freshly isolated monocytes cultured under all conditions described above (data not shown).
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To determine whether the increases in both dye coupling and connexin43 immunofluorescence labeling observed in monocytes treated with 1 µg/ml LPS or 1 ng/ml TNF- plus 1 ng/ml IFN- were associated with changes in total levels of connexin43, relative levels of the junctional protein were determined by Western blot analysis. Monocytes from five volunteers were processed independently. Connexin43 was not detected in total homogenates of cells freshly isolated or cultured for 2 h (Fig. 3A, lanes F and 0, respectively). A distinct band that comigrated with the P2-phosphorylated form of connexin43 present in rat heart homogenate (Fig. 3A, lane H) was detected in monocyte lysates obtained after 12-h treatment with 1 µg/ml LPS plus 1 ng/ml IFN- (Fig. 3A, lane 12). The intensity of the connexin43 band tended to increase progressively after 12, 24, 48, and 72 h of treatment with both proinflammatory agents (Fig. 3A, lanes 12–72). Stimulation of monocytes with 1 ng/ml TNF- plus 1 ng/ml IFN- for 24 h also increased the relative levels of connexin43 (Fig. 3A, last lane, labeled 24). After treatment for 12 h or more with LPS or TNF- plus IFN-, a prominent band of high relative molecular mass (90 kDa as compared with molecular mass standards) was also detected (Fig. 3A). This high molecular mass band was also detected with the anti-connexin43 mAb in Western blot analysis of proteins immunoprecipitated with the polyclonal anti-connexin43 Ab from monocytes treated with LPS plus IFN- by 24 h (Fig. 3B, lane 1). It is likely that the 90-kDa band corresponds to ubiquitinated connexin43 because a band with the same electrophoretic mobility was detected in a sample aliquot of the immunoprecipitated proteins developed with the anti-ubiquitin Ab (Fig. 3B, lane 2). A band with similar intensity and electrophoretic mobility was also detected with a polyclonal anti-connexin43 (Fig. 3B, lane 3). Therefore, the lower intensity of the 90-kDa band detected by immunoblotting of immunoprecipitated material as compared with that detected in total cell homogenates might have resulted from differences in immunoprecipitation efficiency, with the ubiquitinated connexin43 being less efficiently precipitated than the nonubiquitinated connexin43.
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for 12, 24, and 48 h showed an RT-PCR product between 200 and 300 bp (Fig. 3C, lanes 12, 24, and 48). As an internal control, similar intensities of -actin amplification products were detected under all conditions studied (Fig. 3C, lower panel). The ratio of connexin43/-actin RT-PCR products obtained from the densitometric analysis of two experiments was as follows: control, 0, 12 h: 0.35 ± 0.11, 24 h: 0.48 ± 0.16, and 48 h: 0.76 ± 0.20. Thus, the appearance of connexin43 RT-PCR product at 12 h suggests that treatment with LPS plus IFN--induced connexin43 mRNA transcription and measurements between 12 and 48 h posttreatment indicate a progressive increase in transcript levels. The identity of this product was confirmed as a fragment of connexin43 mRNA by automated sequencing that revealed 100% correspondence with the expected RT-PCR product (data not shown). Gap junctional communication enhances monocyte transmigration across a BBB model
Gap junctions have been observed between polymorphonuclear cells and endothelial cells (15), immortalized macrophages and epithelial cells (18, 20), and lymphocytes and endothelial cells (37, 38), suggesting that heterologous gap junctional communication might play a role in the process of transmigration. To address this possibility, we first identified the presence of connexin43 in monocytes/macrophages transmigrating across a human BBB model (29, 30).
Monocyte transmigration across this model is a process greatly enhanced by chemoattractant molecules, such as MCP-1 (29, 30). To study the possible role of gap junctional communication in the transmigration process, freshly isolated monocytes were added to the top chamber of the BBB model. Monocytes/Macrophages were pretreated for 24 h with TNF- plus IFN- to induce the Cx43 expression and then were added to the top chamber, and we examined their transmigration in response to MCP-1 added in the lower chamber in our model of BBB. The addition of MCP-1 to the lower chamber induced transmigration of monocytes/macrophages pretreated with TNF- plus IFN- (Fig. 4A). To test the functional role of gap junctional communication in the transmigration process, in some cultures cells were subjected to the same treatments described above plus 500 µM octanol or 35 µM AGA to block gap junctions. Both octanol and AGA significantly reduced the number of monocytes that transmigrated under each condition studied (Fig. 4A). Neither ethanol nor DMSO at the final concentration used as vehicle to apply octanol and AGA, respectively, affected the number of cells that transmigrated (not shown). After transmigration, the BBB model was fixed and immunostained for connexin43. The gap junction protein was detected in monocytes, endothelial cells, and astrocytes, and it was more intense at heterocellular contacts (Fig. 4C). Endothelium and monocytes were also CD14 positive (Fig. 4D). The merge of both staining, Cx43 and CD14, is shown in Fig. 4E. Both HUVECs and monocytes/macrophages showed immunoreactivity for CD14. Because HUVECs do not express CD14 (39), this CD14 reactivity is likely to correspond to CD14 molecules or their cleavage product released by activated monocytes that were taken up by the endothelial cells (40).
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MMPs are expressed in inflammatory conditions, and are, as a group, capable of degrading matrix proteins, including components of the BBB such as the endothelial basement membrane, thereby increasing capillary permeability (41). In response to inflammatory mediators and cell activation, many cell types including leukocytes, endothelial cells, astrocytes, and microglia secrete MMPs (41, 42). We studied the possible involvement of LPS plus IFN--induced gap junctional communication in the secretion of MMP-9 and MMP-2 by monocytes/macrophages.
Relative levels of latent (L) and active (A) forms of MMP-9 and MMP-2 were measured in monocytes/macrophages by Western blot analysis of conditioned medium obtained after different treatments. In the medium of nonstimulated cells, levels of each form of MMP-9 and MMP-2 were comparable at 0 (2 h of culture under control conditions) and 24 h of culture (data not shown). Densitometric values of each band detected at 24 h of incubation under treatments that affect gap junctional communication were normalized to the values obtained at time zero. Treatment of monocytes with 1 ng/ml TNF- plus 1 ng/ml IFN- for 24 h increased the levels of both forms of MMP-2 (Fig. 5, A and B). Under the same conditions, levels of both MMP-9 forms were not significantly affected (Fig. 5, A and B). In addition, the TNF- plus IFN--induced increase in both forms of MMP-2 was significantly reduced after 24 h, when cells were simultaneously treated with 35 µM AGA (Fig. 5B). Levels of both forms of MMP-9 found in the culture medium of cells treated simultaneously with AGA, TNF-, and IFN- for 24 h were comparable to those found in the medium of cells treated for the same periods of time with only TNF- plus IFN- (Fig. 5, A and B).
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Discussion |
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plus IFN-, formed small aggregates and became dye coupled through a pathway with pharmacological features of gap junction channels. In addition, monocytes treated with these combinations of proinflammatory agents expressed connexin43 protein and mRNA. Transmigration of monocytes/macrophages across a BBB model was associated with the formation of heterocellular gap junctions, and the addition of gap junction channel blockers reduced the number of monocytes/macrophages that transmigrated across a BBB model. Gap junction blockers also induced a drastic reduction in monocyte/macrophage secretion of MMP-2, but not of MMP-9 forms. Thus, we propose that homo- and heterocellular gap junctional communication might play an important role in coordinating some monocyte/macrophage responses during inflammation.
Under normal conditions, monocytes circulate as nonadherent cells, and thus cannot form intercellular junctions. Consistent with this observation, we found that human monocytes/macrophages freshly isolated or cultured over a period of 72 h under control conditions remained dye uncoupled. Lack of dye transfer between human monocytes/macrophages also has been observed in homocellular cultures not treated with cytokines (14) or between mouse peritoneal macrophages stimulated with either LPS or IFN- (16). In this work, we found that LPS, IFN-, or TNF- alone did not induce dye coupling between cultured human monocytes/macrophages. In agreement with the idea that gap junctional communication between macrophagic cells is not induced by a single humoral factor, it has been recently reported that functional expression of gap junctions between human polymorphonuclear cells requires the action of TNF- plus factors (not yet identified) released by activated endothelial cells (23). Consistent with the concept that monocytes/macrophages found at inflammatory foci respond to several cytokines expressed in concert, induction of dye coupling was observed after treatment with TNF- plus IFN-. Because the increase in dye coupling was associated with an increase in connexin43 immunoreactivity and this dye coupling was drastically reduced upon the application of AGA, the intercellular spread of Ly is likely to have occurred through gap junction channels. The LPS plus IFN--induced dye coupling was blocked with a neutralizing anti-TNF- Ab, and treatment with rTNF- plus IFN- mimicked the effect of LPS plus IFN-. Thus, it is likely that the LPS plus IFN--induced effect on the incidence of dye coupling between monocytes/macrophages was mediated by the autocrine action of macrophage/monocyte-secreted TNF-. TNF- can be secreted by several cell types, including activated endothelial cells (43, 44) and monocytes/macrophages (45), and IFN- can be released by T cells (46). Thus, monocytes/macrophages may express functional gap junctions in vivo in response to the combinations of proinflammatory mediators shown to induce dye coupling in cultured primary cells.
We have observed dye coupling between macrophages of the CNS, microglia, after treatment with LPS or TNF- plus IFN- (21). In both monocytes/macrophages and microglia, the LPS or TNF- plus IFN--induced increase in dye coupling occurred only in 50% of the cells. This might be partially explained by the known phenotypic heterogeneity in each cell population (4, 47), implying that cultures contain cells sensitive and cells less sensitive or insensitive to LPS or TNF- plus IFN-.
Consistent with the lack of dye coupling, we found that circulating monocytes or monocytes/macrophages freshly isolated or cultured for 72 h under control conditions were negative for connexins 32, 40, and 43. Polacek et al. (14) have reported that human monocytes do not contain the mRNAs of connexin32 and connexin43, and we recently demonstrated the absence of connexin43 in most resting rat or mouse microglia (21). We now demonstrated that monocytes/macrophages treated with LPS or TNF- plus IFN-, conditions known to activate macrophages (48), induced the expression of at least connexin43. Gap junction protein subunits have also been detected in peritoneal hamster (15) and mouse (16) monocytes/macrophages. In addition, connexin43 (49) and its mRNA (14) have been detected in foam cells of advanced atherosclerotic plaques in mice and humans. Connexin43 has also been found in macrophages and Kupffer cells of inflamed liver (25, 50) and infiltrated inflammatory cells of inflamed kidney (51), suggesting that connexin43 expression might be a general feature of activated macrophages. Nevertheless, macrophages of early atheromas do not express connexin43 immunoreactivity, and instead are positive for connexin37 (49).
The differential expression of connexins by macrophages might be related to the physiological state of these cells, and different connexins could form channels with different permeability properties to allow intercellular transfer of molecules of different physiological importance. In cultured peritoneal macrophages treated with LPS or IFN-, levels of connexin43 remain as in untreated cells (16). Similarly, we found that none of the proinflammatory agents studied increased the levels of connexin43 when applied alone. Hence, the induction of connexin43 expression might occur in a state of macrophage activation different from that induced by each of these proinflammatory agents. In agreement, we found that treatment with a combination of either LPS or TNF- with IFN-, which acts cooperatively to induce many of the genes involved in inflammation (48), increased the levels of connexin43 protein and mRNA. Although the reactivity of connexin43 detected by Western blot analysis and immunofluorescence and the incidence of dye coupling increased within 48 h of treatment with LPS plus IFN-, later on they showed an inverse relationship. A possible mechanism for dye coupling at later time points could be a reduction in cell adhesion that would destabilize existing, and prevent formation of new gap junction channels, despite the continuous expression of connexin43.
In cells permanently coupled through connexin43-containing channels, the degradation of the junctional protein is through both lysosomal and proteosomal pathways (52). The proteosomal pathway involves the increase of the apparent molecular mass of the connexin43 to 90 kDa by insertion of polyubiquitin (53, 54). This is the same apparent molecular mass of the band we detected in lysates of monocytes/macrophages treated with LPS or TNF- plus IFN-. Therefore, although we did not evaluate directly the involvement of each pathway in connexin43 degradation, the high levels of ubiquitinated connexin43 in LPS or TNF- plus IFN--treated cells suggest a prominent degradation in proteosomes without ruling out degradation through the lysosomal pathway.
Ultrastructural identification of gap junctions and/or gap junctional communication between macrophages and other cell types such as polymorphonuclear cells (19) and epithelial cells (18, 20) has been reported. In this study, we demonstrated that human monocytes, during the process of transmigration, form connexin43-containing gap junctions with cells of a BBB model. In addition, we found that blockade of gap junctions reduced the number of monocytes that transmigrated across the BBB model, suggesting the importance of gap junctional communication in the efficiency of the transmigration process across a tight endothelium. In support of the possible role of gap junctional communication for leukocyte transmigration, it is known that leukocyte-endothelial cell contact is a prerequisite to elicit oscillation of intracellular Ca2+ concentrations in endothelial cells (55), and blockade of the latter results in an inhibition of monocyte transmigration (56). Moreover, the intercellular transfer of signals that elicit oscillations in endothelium intracellular Ca2+ is blocked with anti-cell adhesion molecule Abs (55, 57) that would also prevent the assembly of gap junction channels (56). The inhibitory effect of the anti-cell adhesion molecule Abs rules out the involvement of a paracrine signaling mechanism, and thus supports the role of a pathway that allows transferring of intercellular signals without dilution in the extracellular space, such as gap junctions. Recently, the formation of heterocoupling between lymphocytes and endothelial cells and a modest reduction in transmigration of lymphocytes across a monolayer of endothelial cells caused by gap junction blockers was reported (58). The discrepancy with our findings might have several explanations, including differences in the type of both migratory and endothelial cells. Related to the latter, an alternative explanation might be whether the endothelial cells express tight junctions. In our system, the expression of tight junctions is higher due to the presence of astrocytes (E. A. Eugenín and J. Berman, unpublished observations). In the absence or low expression of tight junctions, transmigration of inflammatory cells across an endothelium might not require heterocellular gap junctional communication. Further studies will be required to clarify this apparent controversy.
The expression of connexin43 by monocytes/macrophages might not be required solely for transmigration. In fact, in the liver, where the endothelium is fenestrated and therefore it is not a physical barrier for migratory cells, macrophages found in necrotic foci after LPS administration are connexin43 positive (25). Hence, connexin43 might be required for macrophage functions such as removal of cell debris. Accordingly, we observed a dramatic reduction in TNF- plus IFN--induced release of MMP-2. The requirement of gap junctional communication for IL-1-induced release of MMPs by synovial cells has been reported recently (59). In this work, the basal release of MMP-9 was not affected by the blockade of gap junctions, suggesting the involvement of a gap junction-independent process.
Gap junctional communications between members of a cell community allow the coordination of cellular, metabolic, and/or electrical responses (6, 10). Several mechanisms could participate, including intercellular propagation of calcium waves and intercellular transfer of electrotonic potentials that could activate voltage-dependent calcium channels, permitting the influx of Ca2+. Thus, calcium-dependent processes, such as secretion, could be triggered by environmental conditions or extracellular ligands acting in one or few sensitive cells of a cluster and elicit Ca2+-dependent responses in all cells of the cluster communicated to the sensitive ones via gap junctions. This idea is a possible explanation of how gap junction communication regulated the secretion of MMP-2 or transmigration, by recruiting more cells to respond to some specific stimulus. However, it is important to note that all available gap junction blockers, including those used in this work, are not specific (60). Alternative gap junction blockers, such as Abs that recognize domains of the extracellular loops of Cxs, are likely to interfere with cell adhesion molecules known to be essential in several functional macrophage responses, including transmigration (8, 29), and therefore were inappropriate for this study. In addition, inhibitory peptides with sequences identical with regions of the extracellular loops might be processed or digested by macrophages and, in other systems, have been shown to be useful only over minutes to hours (60). This time frame is insufficient for our experiments in human monocytes/macrophages treated with TNF- plus IFN-, because the time course necessary for our experiments is 3 days. Thus, the ultimate demonstration of the role of gap junctional communication in cell transmigration and secretion of MMPs may depend upon more specific experimental approaches, such as inducible tissue ablation of specific Cxs.
The requirement of IFN- plus LPS or TNF- for inducing dye coupling between macrophages/monocytes might be related to the known synergistic action of IFN- with LPS or TNF- on diverse macrophage responses such as antitumor, bactericidal, and antiviral activities (48). We propose that the transient expression of connexin43 by monocyte/macrophage cells allows for the formation of gap junction channels that allow the intercellular conversation required for different cellular responses and functions during inflammation, a common factor of diverse pathologies.
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2 Address correspondence and reprint requests to Dr. Eliseo A. Eugenín, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: eeugenin{at}aecom.yu.edu
3 Abbreviations used in this paper: MMP, metalloproteinase; AGA, 18 -glycyrrhetinic acid; BBB, blood brain barrier; DAPI, 4', 6-diamidine-2-phenylindole; Ly, Lucifer yellow; MCP, monocyte chemoattractant protein.
Received for publication August 19, 2002. Accepted for publication November 25, 2002.
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