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(The FASEB Journal. 2001;15:768-774.)
© 2001 FASEB

Immunoglobulin and cytokine expression in mixed lymphocyte cultures is reduced by disruption of gap junction intercellular communication

ERNESTO OVIEDO-ORTA*1, PHILIPPE GASQUE and W. HOWARD EVANS*

University of Wales College of Medicine, Department of Medical Biochemistry and
* Wales Heart Research Institute, Heath Park, Cardiff CF14 4XN, Wales, U.K.

1Correspondence: Bristol Heart Institute, Bristol Royal Infirmary, Level 7, Upper Maudlin St., Bristol BS2 8HW, England, U.K. E-mail: e.oviedo-orta{at}bristol.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Connexins (Cx), the protein subunits assembled into gap junction intercellular communication channels, are expressed in primary lymphoid organs and by circulating leukocytes. Human tonsil-derived T and B lymphocytes express Cx40 and 43; circulating human T, B, and NK lymphocytes express Cx43 and directly transfer between each other a low molecular dye indicative that functional gap junctions exist. We now identify specific properties in the immune system underwritten by gap junctions. Mixed lymphocytes cultured in the presence of two reagents with independent inhibitory action on gap junction communication, a connexin mimetic peptide and 18-{alpha}-glycyrrhetinic acid, markedly reduced the secretion of IgM, IgG, and IgA. The secretion of these immunoglobulins by purified B cells was also reduced by the two classes of gap junction inhibitors. Complex temporal inhibitory effects on the expression of mRNA encoding interleukins, especially IL-10, were also observed. The results indicate that intercellular signaling across gap junctions is an important component of the mechanisms underlying metabolic cooperation in the immune system.—Oviedo-Orta, E., Gasque, P., Evans, W. H. Immunoglobulin and cytokine expression in mixed lymphocyte cultures is reduced by disruption of gap junction intercellular communication.


Key Words: connexins • intercellular communication • interleukins • mimetic peptides • interferon gamma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CELL-TO-CELL interactions are crucial for lymphocyte maturation and activation. Intercellular cross-play modulates the expression at the plasma membrane of receptors that mediate antigen and ligand recognition and trigger regulatory intracellular signaling cascades (1 2 3 4 5) . Integrins, cadherins, and selectins are examples of the wide range of membrane molecules ensuring intercellular adhesion and enabling cell–cell signaling during cell migration and activation in the immune system (1 , 6 7 8 9 10) .

Gap junctions are cell-surface specializations that facilitate direct transfer of small molecules and ions of less than 1 kDa between cells. Each intercellular channel unit is generated from a pair of interacting hemichannels (connexons) contributed by each of the cooperating cells. Connexons are constructed of six protein subunits (connexins) arranged around a central pore. Connexins constitute a family of proteins with similar basic topology in the plasma membrane with two extracellular facing loops, four transmembrane domains, one intracellular loop, and the amino and carboxyl termini positioned at the cytoplasmic aspect (11 12 13 14) .

Studies of the functional integrative roles of gap junctions have been advanced by use of inhibitors of direct intercellular communication effected by these organelles (15) . Synthetic connexin mimetic peptides with sequences corresponding to each of the gap facing extracellular loops have been demonstrated to block intercellular dye transfer in a variety of cells (16) , electrical communication (17 , 18) , and the propagation of Ca2+ waves (19) . Connexin mimetic peptides also inhibit cellular interactions underwritten by gap junctional communication, such as smooth muscle-endothelial relaxation of arteries (20 , 21) . Initially, these mimetic peptides were shown to delay the assumption of synchronous contraction by aggregating cardiac myocytes (22) .

The increasing evidence for the presence of connexins and functional gap junctions in lymphocytes is highly suggestive that they play a physiological role. We now show that disruption of gap junctional communication influences fundamental facets of lymphocyte function, including immunoglobulin (Ig) secretion and cytokine production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
T and B lymphocytes were purified from tonsils from children with recurrent tonsillitis by using Histopaque-1077® (Sigma, Poole, U.K.). Cell counting was performed in a hemocytometer chamber and viability was assessed by trypan blue staining. B lymphocytes were further purified from the cell suspension after staining with a mouse anti-human CD19 FITC conjugate (Dako, Glostrup, Denmark) and sorted on a fluorescence-activated cell sorter (FACS) flow cytometer (FACS-440, Becton Dickinson, Rutherford, N.J.). B lymphocytes were also purified using a human B cell accessory kit (Biotecx Labs, Houston, Tex.) according to the manufacturer’s instructions. T lymphocytes were identified by staining with a mouse anti-human CD3 FITC conjugate (Dako, Glostrup, Denmark). The purity of isolated cells prepared by both methods was between 95 and 99% as determined by FACS analysis. Lymphocyte cultures were performed with cells from the same donor in order to avoid an allogeneic mixed lymphocyte reaction.

Peptides
GAP27 (SRPTEKTIFII), a synthetic peptide corresponding to a sequence in the second extracellular loop of Cx43, and two control peptides corresponding to cytoplasmic Cx43 protein sequences GAP18 (MGDWSALGKLLDKVQAC, amino terminus) and GAP20 (EIKKFKYGIEEHC intracellular loop) were synthesized by FMOC solid-phase chemistry to 95% purity by Genosys (Cambridge, U.K.) or Severn Biotech (Tewkesbury, U.K.). Peptides were dissolved in a minimum volume of dimethyl sulfoxide and added to cell cultures as described below. No effect of the solvent added on its own was observed.

In vitro lymphocyte activation
A mixed population of T and B lymphocytes (~107 cells/well) was incubated for different periods of time in the presence or absence of 1 µg/ml phytohemagglutinin-L (PHA-L, Boehringer Mannheim, Germany) in RPMI 1640 medium (Gibco-BRL, Paisley, U.K.) supplemented with 10% fetal calf serum, 1 x 105U penicillin, 1 x 105U streptomycin, 250 µg/ml amphotericin B, 25 mM HEPES, and 1 mM L-glutamine. B lymphocytes were cultured with 10 µg/ml of lipopolysaccharide (LPS, Escherichia coli, Serotype 055:B5, Sigma, Poole, U.K.) in RPMI 1640 supplemented as described above. To evaluate the effects of gap junction communication inhibitors, GAP27 peptide (300 µM) and 18-{alpha}-glycyrrhetinic acid (150 µM) were added to lymphocytes cultures at 12 h intervals.

In vitro antibody production and ELISA
Supernatants from lymphocyte cocultures were collected and IgM, IgG, and IgA synthesis was measured by using specific ELISAs. Microtiter plates were coated overnight at 4°C with 3 µg/ml of protein-L (ACTIgen, Cambridge, U.K.) as a capture molecule in 0.01 M Na2CO3/NaHCO3 buffer pH 9.6. Plates were saturated with 1% BSA, 0.05% Tween 20 in phosphate-buffered saline (PBS: 100 mM NaCl, 100 mM Na2HPO4/NaH2PO4, pH 7.5), then incubated for 2 h at room temperature with serial dilutions of standard human IgM, IgG, and IgA (Sigma) and the test samples. Plates were washed twice using 0.05% Tween 20 in PBS and incubated for 1 h at room temperature with 1/5000 of biotin-conjugated goat anti-human µ-, {gamma}-, or {delta}-chain Fc-specific antibody (Sigma). Plates were washed three times in 0.05% Tween 20 in PBS and incubated for 30 min at room temperature with 1/10 000 horseradish peroxidase-labeled avidin (Sigma, Poole, U.K.). After washing three times with 0.05% Tween 20 in PBS, o-phenylenediamine dihydrochloride peroxidase substrate (Sigma) was added. The reactions were stopped by addition of 50 µl of 3M HCl. Optical density at 492 nm was determined using a microtiter plate reader (Bio-Rad, Hercules, Calif.).

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis
RNA was obtained using the ULTRASPECTM RNA isolation system (Biotecx Labs, Biogenesis, Poole, U.K.). First-strand cDNA synthesis was carried out using 1 µg of total RNA purified from lymphocytes isolated from tonsils. Reverse transcription was performed in 20 µl final volume containing 50 ng of pd(N)6 (Phamacia Biotec), 0.2 mM dATP, dCTP, dGTP, dTTP, and 10 mM DTT. Each reaction mix was incubated for 10 min at 25°C with 40 U of rRNasin® (RNase inhibitor, Promega, Southampton, U.K.), followed by incubation with 200 U of Moloney murine leukemia virus reverse transcriptase (MMLV, Gibco-BRL) for 40 min at 42°C, followed by 2 min at 99°C. PCR analysis was performed in a final volume of 50 µl by using 2 µg of cDNA in a mix containing 0.02 mM deoxynucleotide triphosphates, 20 pmol of both sense and antisense oligonucleotide primers, 2.5 mM MgCl2, 1 U Taq DNA polymerase (Promega, U.K.), and 5 µl of 10x Taq DNA polymerase reaction buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton® X-100). Amplification of cDNA encoding for interleukin 2 (IL-2), IL-10, and interferon {gamma} (IFN{gamma}) was performed in a single-tube reaction. PCR primer sequences and expected cDNA size bands are shown in Table 1 . Primers designed from a GAPDH sequence were used as an internal control in each PCR reaction. The reaction was carried out using a Perkin-Elmer/Cetus Thermocycler Model (Perkin-Elmer, Norwalk, Conn.) 9600 as follows: hot start at 94°C for 4 min, followed by 35 cycles; 1 min at 94°C, 2 min 60°C, and 2 min at 72°C, with a final extension cycle at 72°C for 5 min. PCR analysis was also carried out on the reaction mix without MMLV reverse transcriptase as a negative control to eliminate the possibility of any genomic contamination. After staining of the agarose gel with ethidium bromide, the relative fluorescence intensity of each cDNA product was analyzed using a SynGene® Gel Imaging System, controlled by a GeneSnap version 2.6 software (SynGene, Cambridge, U.K.).


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  		Table 1. Primers used for RT-PCR analysis of interleukins mRNAs


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of gap junction inhibitors on IgM, IgG, and IgA production
Secretion of IgM, IgG, and IgA by mixed T and B lymphocytes cultured in the absence of a polyclonal stimulator increased rapidly over 12 h (Fig. 1A B C ), followed by a further sustained increase occurring for up to 48 h. When the cells were cultured in the presence of the connexin mimetic peptide GAP27, a significant and sustained decrease in IgM, IgG, and IgA synthesis was observed (P=0.0013, P=0.0224 and P=0.0002, respectively). The connexin mimetic peptide decreased IgM synthesis by 30% compared to a 40–45% decrease of IgG and IgA. Incubation of cells in the presence of 18-{alpha}-glycyrrhetinic acid resulted in 60% reduction in the IgM, IgG, and IgA secreted (P=0.0008, P=0.0045 and P=0.0012, respectively) (Fig. 1D E F ).



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  		Figure 1. Effects of connexin mimetic peptide GAP27 ({blacktriangleup}), 18-{alpha}-glycyrrhetinic acid ({blacksquare}), and no addition ({diamondsuit}) upon secretion of immunoglobulins IgM, IgG, and IgA by unstimulated T and B lymphocytes (A—C, respectively), and by T and B lymphocytes stimulated with PHA-L (D—F, respectively). For further details, see Materials and Methods.

The specificity of action of the connexin mimetic peptide was demonstrated by studying the effects of two short peptides corresponding to intracellular amino acid sequences in connexins (GAP18 and GAP20; see Materials and Methods). In the absence of PHA-L, little effect on the secretion of the three immunoglobulins (Fig. 2 ) was noted. Similar results were obtained with PHA-L stimulated lymphocyte cultures (results not shown).



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  		Figure 2. Effects of control connexin mimetic peptides [GAP18 (•) and GAP20 (*)] and control (no peptide) ({diamondsuit}) on production of immunoglobulins by mixed T and B lymphocytes.

Mixed T and B lymphocyte populations stimulated in vitro by PHA-L were also studied. This mitogen stimulated IgM secretion by twofold in mixed lymphocyte populations (Fig. 1D ), and IgG and IgA secretion was increased by 25% (Fig. 1E, F ). A decrease in IgA values was noted after 12 h despite no significant cell death being detected. With PHA-L-stimulated mixed populations of lymphocytes, the connexin mimetic peptide again reduced IgM synthesis by ~70% (P=0.0098), and IgG and IgA by 40% (P=0.0005 and P=0.1736, respectively). A similar reduction in secretion of the immunoglobulins was observed with 18-{alpha}-glycyrrhetinic acid (Fig. 1D E F ).

To dissect the effect of gap junction inhibitors on signaling cooperation between B lymphocytes, purified B cells were cultured on their own in the presence of LPS and the effects of the two inhibitors on immunoglobulin synthesis examined. Under these conditions the connexin mimetic peptide inhibited the synthesis of IgM, IgG, and IgA at early time points up to 24 h. However, at later time points, the connexin mimetic peptide had little effect on immunoglobulin global synthesis (Fig. 3 ). The production of immunoglobulins by B cells was also inhibited by 18-{alpha}-glycyrrhetinic acid. IgG, and IgM levels were significantly decreased, with lower levels of IgA observed mainly between 12 and 24 h.



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  		Figure 3. Effects on secretion of immunoglobulins: IgM, IgG, and IgA by lipopolysaccharide (LPS) stimulated B lymphocytes of connexin mimetic peptide GAP27 ({blacktriangleup}), 18-{alpha}-glycyrrhetinic acid ({blacksquare}), and no peptide ({diamondsuit}). Cells were stimulated with LPS over 48 h.

Effect of gap junction inhibitors on IL-2, IL-10, and IFN{gamma} mRNA synthesis by lymphocytes
The effects of the two gap junction communication blockers on cytokine production were studied. mRNAs encoding human IL-2, IL-10, and IFN{gamma} were detected in unstimulated mixed lymphocyte cultures. Expression of IFN{gamma} and IL-2 mRNAs was evident after 12 h (Fig. 4A B ) whereas IL-10 mRNA could be detected only after 48 h of culture (Fig. 4C ). Expression of IFN{gamma} mRNA was not affected by exposure of cells to the connexin mimetic peptide (Fig. 4A ), but 18-{alpha}-glycyrrhetinic acid significantly decreased its secretion between 48 h and 84 h. In contrast, the connexin mimetic peptide inhibited expression of IL-2 mRNA between 48 h and 86 h but 18-{alpha}-glycyrrhetinic acid showed only a significant effect on IL-2 mRNA levels after 84 h of culture (Fig. 4B ). IL-10 mRNA was detected after 48 h and peaked at 84 h, followed by a rapid decrease. Both inhibitors completely abrogated the synthesis of IL-10 mRNA by lymphocytes. No significant changes in cytokine synthesis were found when cells were cultured in the presence of a control peptide GAP18 (Fig. 5 ).



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  		Figure 4. Effects of connexin mimetic peptide GAP27 ({blacktriangleup}), 18-{alpha}-glycyrrhetinic acid ({blacksquare}), and control (no peptide) ({diamondsuit}) on production of IL-2 (A), IFN{gamma} (B), and IL-10 (C) by PHA-L stimulated mixed populations of T and B lymphocytes.



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  		Figure 5. Effects of control connexin mimetic peptide GAP18 on IL-2 (A), IFN{gamma} (B), and IL-10 (C) by PHA-L stimulated mixed populations of T and B lymphocytes. Curves represent ({diamondsuit}) control (no peptide) and effect of ({blacktriangleup}) GAP18 peptide.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Intercellular cross-talk underpins cooperative metabolic activities in T and B lymphocytes. The production of immunoglobulins and cytokines is dependent on the interaction and signaling between lymphocytes. In the present work, we studied the effects of two classes of gap junction intercellular communication inhibitors on the secretion by human T and B lymphocytes of immunoglobulins and cytokines.

The results presented show that gap junctional communication, which was blocked partially by two different inhibitors, is a key component in effecting the secretion of three classes of immunoglobulins. These results also show that the applied reagents blocked IL-10 production and suggested that at 12 and 60 h after commencement of treatment, there was a significant drop in the production of IL-2 and IFN{gamma}. The data show for the first time that cross-talk in the immune system is regulated by gap junctional communication.

Prompting the present work were studies showing that circulating and tissue-derived lymphocytes express Cx43 and 40 (23) , thus extending at the molecular level numerous studies pointing to the presence of gap junctional communication in cells comprising the immune system (24 25 26 27 28) . An antibody generated to extracellular amino acid sequences of Cx43 was used to demonstrate the presence of this connexin on the surface of T and B lymphocytes, and dye transfer between contacting mixed lymphocyte populations was demonstrated by FACS analysis (23) . It is significant that dye transfer between lymphocytes was reduced in the presence of two gap junction inhibitors (23) .

Although a number of reagents have been shown to inhibit gap junction-mediated intercellular communication, especially lipophilic compounds such as octanol, heptanol, and halothane (29 30 31 32 33 34) , the present work used short peptide inhibitors of gap junctional communication, which appear to be more specific in their actions. The connexin mimetic peptides are emerging as benign, specific, and reversible gap junction inhibitors that have been characterized by their actions in blocking dye transfer in HeLa cells (35) , Schwann cells (36) , and lymphocytes (23) . The connexin mimetic peptides reversibly inhibited intercellular calcium propagation in airway epithelial cells (19) and inhibited endothelial smooth muscle interactions in arteries that are underwritten by gap junctions (20 , 21 , 37) . Other connexin mimetic peptides were shown to inhibit electrical and dye coupling of aortic smooth muscle cells (18) . The mechanism of action of connexin mimetic peptides is not fully understood, but is likely to be either directly on gap junction gating after their diffusion into the intercellular gap or by competitive inhibition at the cell surface of docking and accreting connexons. Glycyrrhetinic acid (38 , 39) appears to act in a different manner to the connexin mimetic peptides, possibly by interfering with the phosphorylation of Cx43 (40) . Both the connexin mimetic peptide and glycyrrhetinic acid reduced immunoglobulin secretion, although differences in their actions on cytokine production were evident.

The mechanism of action of the two classes of inhibitors of gap junctional communication on the synthesis by lymphocytes of immunoglobulins and cytokines is not revealed by the present studies. Intercellular contact and cooperation between T and B lymphocytes play crucial roles in isotype class switching, antibody maturation, etc. (41 , 42) . However, clues are provided as to the relative specificity of these two classes of inhibitors of gap junctional communication. In the absence of T cells, B cells markedly reduced secretion of immunoglobulins to levels similar to those seen with unstimulated T and B cell populations. A further small reduction in immunoglobulin synthesis was induced by treatment of B lymphocytes with the connexin mimetic peptide, but this was less than the reduction observed when these cells were incubated with 18-{alpha}-glycyrrhetinic acid, reinforcing the view that the latter acts by different mechanisms and possibly in a less direct manner.

The gap junction inhibitors also influenced the production of cytokines by mixed T and B lymphocytes. Three cytokines were investigated, viz. IL-2, IL-10, and IFN{gamma}. The inhibition of mRNA synthesis encoding IL-2 at 12 h and 60 h by the connexin mimetic peptide may be related to the reduction in IgG and IgM production induced by the peptide, for stimulation of lymphocytes with IL-2 influences the synthesis of these immunoglobulins (43 44 45 46) . Undoubtedly, these inhibitory effects on gap junction intercellular communication are complex, as illustrated by the different time phases of the effect. Furthermore, the major difference between the effects on IL-10 mRNA production by the connexin mimetic peptide and 18-{alpha}-glycyrrhetinic acid may again point to a different mechanism of action of the two classes of inhibitors.

The present results emphasize the extended functional consequences of blocking gap junction-mediated interactions and cooperativity in both unstimulated and stimulated populations of T and B lymphocytes. The effects on immunoglobulin synthesis by B lymphocytes are likely to be a consequence of the requirement for direct and protracted cross-talk with T lymphocytes involving, among other mechanisms, the formation of gap junction channels; studies of the directionality of dye transfer in mixed populations of lymphocytes also point to this possibility (23) .

The present results show that a role should be considered for connexins and gap junction intercellular channels in lymphocyte physiology and development. With respect to heterotypic interaction with other cells, the role of gap junctions in the interplay of lymphocytes with endothelial cells is another aspect worthy of exploration, since the two classes of reagents that inhibit gap junction communication also influence such interactions (E. Oviedo-Orta, R. Errington, and W. H. Evans, unpublished data).


   ACKNOWLEDGMENTS
 
We thank the Wales Office for Research and Development and the Medical Research Council for the grant support. This work was supported by grant WS/97/1/013 from the Wales Office for Research and Development.

Received for publication June 27, 2000. Revision received September 18, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Angelopoulou, M. K., Kontopidou, F. N., Pangalis, G. A. (1999) Adhesion molecules in B-chronic lymphoproliferative disorders. Semin. Hematol. 36,178-197[Medline]
  2. Bain, G., Murre, C. (1998) The role of E-proteins in B- and T-lymphocyte development. Semin. Immunol. 10,143-153[Medline]
  3. Bayon, Y., Alonso, A., Hernandez, M., Nieto, M. L., Crespo, M. S. (1998) Mechanisms of cell signaling in immune-mediated inflammation. Cytokines Cell. Mol. Ther. 4,275-286[Medline]
  4. Briscoe, D. M., Alexander, S. I., Lichtman, A. H. (1998) Interactions between T lymphocytes and endothelial cells in allograft rejection. Curr. Opin. Immunol. 10,525-531[Medline]
  5. Freitas, A. A., Rocha, B. (1999) Peripheral T cell survival. Curr. Opin. Immunol. 11,152-156[Medline]
  6. Buckley, C. D., Rainger, G. E., Bradfield, P. F., Nash, G. B., Simmons, D. L. (1998) Cell adhesion: more than just glue. Mol. Membr. Biol. 15,167-176[Medline]
  7. Chia, M. C. (1998) The role of adhesion molecules in atherosclerosis. Crit. Rev. Clin. Lab. Sci. 35,573-602[Medline]
  8. Cooper, E. L., Cossarizza, A., Kauschke, E., Franceschi, C. (1999) Cell adhesion and the immune system: A case study using earthworms. Microsp. Res. Tech. 44,237-253
  9. Fabbri, M., Bianchi, E., Fumagalli, L., Pardi, R. (1999) Regulation of lymphocyte traffic by adhesion molecules. Inflamm. Res. 48,239-246[Medline]
  10. Gonzalez-Amaro, R., Diaz-Gonzalez, F., Sanchez-Madrid, F. (1998) Adhesion molecules in inflammatory diseases. Drugs 56,977-988[Medline]
  11. Goodenough, D. A., Goliger, J. A., Paul, D. L. (1996) Connexins, connexons, and intercellular communication. Annu. Rev. Biochem. 65,475-502[Medline]
  12. Bennett, M. V. L., Barrio, L. C., Bargiello, T. A., Spray, D. C., Hertzberg, E., Saez, J. C. (1991) Gap-junctions—new tools, new answers, new questions. Neuron 6,305-320[Medline]
  13. Evans, W. H. (1988) Gap-junctions—towards a molecular structure. Bioessays 8,3-6[Medline]
  14. Revel, J. P., Yancey, S. B., Nicholson, B. J. (1986) The gap junction proteins. Trends Biochem. Sci. 11,375-377
  15. Becker, D. L., Evans, W. H., Green, C. R., Warner, A. (1995) Functional-analysis of amino-acid-sequences in connexin 43 involved in intercellular communication through gap-junctions. J. Cell Sci. 108,1455-1467[Abstract]
  16. Chaytor, A. T., Evans, W. H., Griffith, T. M. (1997) Peptides homologous to extracellular loop motifs of connexin 43 reversibly abolish rhythmic contractile activity in rabbit arteries. J. Physiol. (London) 503,99-110[Medline]
  17. Dora, K. A., Martin, P. E. M., Chaytor, A. T., Evans, W. H., Garland, C. J., Griffith, T. M. (1999) Role of heterocellular gap junctional communication in endothelium-dependent smooth muscle hyperpolarization: Inhibition by a connexin-mimetic peptide. Biochem. Biophys. Res. Commun. 254,27-31[Medline]
  18. Kwak, B. R., Jongsma, H. J. (1999) Selective inhibition of gap junction channel activity by synthetic peptides. J. Physiol. (London) 516,679-685[Abstract/Free Full Text]
  19. Boitano, S., Evans, W. H. (2000) Connexin mimetic peptides reversibly inhibit intercellular Ca2+ signaling through gap junctions. Am J. Physiol. 279,623-630
  20. Chaytor, A. T., Martin, P. E., Evans, W. H., Randall, M. D., Griffith, T. M. (1999) The endothelial component of cannabinoid-induced relaxation in rabbit mesenteric artery depends on gap junctional communication. J. Physiol. (London) 520,539-550[Abstract/Free Full Text]
  21. Chaytor, A. T., Evens, W. H., Griffith, T. M. (1998) Central role of heterocellular gap junctional communication in endothelium-dependent relaxations of rabbit arteries. J. Physiol. (London) 508,561-573[Abstract/Free Full Text]
  22. Warner, A., Clements, D. K., Parikh, S., Evans, W. H., Dehaan, R. L. (1995) Specific motifs in the external loops of connexin proteins can determine gap junction formation between chick heart myocytes. J. Physiol. (London) 488,721-728[Medline]
  23. Oviedo-Orta, E., Hoy, T., Evans, W. H. (2000) Intercellular communication in the immune system: differential expression of connexin 40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations. Immunology 99,578-590[Medline]
  24. Hülser, D. F., Peters, J. H. (1972) Contact cooperation in stimulated lymphocytes. Exp. Cell Res. 74,319-326[Medline]
  25. Krenacs, T., Rosendaal, M. (1995) Immunohistological detection of gap-junctions in human lymphoid tissue—connexin 43 in follicular dendritic and lymphoendothelial cells. J. Histochem. Cytochem. 43,1125-1137[Abstract]
  26. Krenacs, T., vanDartel, M., Lindhout, E., Rosendaal, M. (1997) Direct cell/cell communication in the lymphoid germinal center: Connexin 43 gap junctions functionally couple follicular dendritic cells to each other and to B lymphocytes. Eur. J. Immunol. 27,1489-1497[Medline]
  27. Jara, P. I., Boric, M. P., Saez, J. C. (1995) Leukocytes express connexin-43 after activation with lipopolysaccharide and appear to form gap-junctions with endothelial cells after ischemia-reperfusion. Proc. Natl. Acad. Sci. USA 92,7011-7015[Abstract/Free Full Text]
  28. Alves, L. A., deCarvalho, A. C. C., Savino, W. (1998) Gap junctions: a novel route for direct cell–cell communication in the immune system?. Immunol. Today 19,269-275[Medline]
  29. Abou-Hashieh, I., Mathieu, S., Besson, F., Gerolami, A. (1996) Inhibition of gap junction intercellular communications of cultured rat hepatocytes by ethanol: Role of ethanol metabolism. J. Hepatol. 24,360-367[Medline]
  30. Deutsch, D. E., Williams, J. A., Yule, D. I. (1995) Halothane and octanol block Ca2+ oscillations in pancreatic acini by multiple mechanisms. Am J. Physiol 32,G779-G788
  31. Javid, P. J., Watts, S. W., Webb, R. C. (1995) The gap junctional inhibitor octanol inhibits NO-induced relaxation in vascular smooth muscle. FASEB J 9,A914(abstr.)
  32. Bastide, B., Neyses, L., Ganten, D., Paul, M., Willecke, K., Traub, O. (1993) Gap junction protein connexin 40 is preferentially expressed in vascular endothelium and conductive bundles of rat myocardium and is increased under hypertensive conditions. Circ. Res. 73,1138-1149[Abstract/Free Full Text]
  33. Bastide, B., Herve, J. C., Cronier, L., Deleze, J. (1995) Rapid onset and calcium independence of the gap junction uncoupling induced by heptanol in cultured heart-cells. Pfluegers Arch. Eur. J. Physiol. 429,386-393[Medline]
  34. He, D. S., Burt, J. M., Kurjiaka, D. T. (1998) Halothane reduces the open probability of gap junction channels in A7r5 cells. Mol. Biol. Cell 9,1888
  35. Martin, P. E. M., George, C. H., Castro, C., Kendall, J. M., Capel, J., Campbell, A. K., Revilla, A., Barrio, L. C., Evans, W. H. (1998) Assembly of chimeric connexin-aequorin proteins into functional gap junction channels—reporting intracellular and plasma membrane calcium environments. J. Biol. Chem. 273,1719-1726[Abstract/Free Full Text]
  36. Mambetisaeva, E. T., Gire, V., Evans, W. H. (1999) Multiple connexin expression in peripheral nerve, Schwann cells, and Schwannoma cells. J. Neurosci. Res. 57,166-175[Medline]
  37. Chaytor, A. T., Evans, W. H., Griffith, T. M. (1997) Peptides homologous to extracellular loop motifs of connexin 43 reversibly abolish rhythmic contractile activity in rabbit arteries. J. Physiol. (London) 503,699
  38. Taylor, H. J., Chaytor, A. T., Evans, W. H., Griffith, T. M. (1998) Inhibition of the gap junctional component of endothelium-dependent relaxations in rabbit iliac artery by 18-alpha glycyrrhetinic acid. Br. J. Pharmacol. 125,1-3[Medline]
  39. Davidson, J. S., Baumgarten, I. M. (1988) Glycyrrhetinic acid derivatives—a novel class of inhibitors of gap-junctional intercellular communication structure-activity relationships. J. Pharmacol. Exp. Ther. 246,1104-1107[Abstract/Free Full Text]
  40. Guo, Y. H., Martinez Williams, C., Gilbert, K. A., Rannels, D. E. (1999) Inhibition of gap junction communication in alveolar epithelial cells by 18 alpha-glycyrrhetinic acid. Am. J. Physiol. 20,L1018-L1026
  41. Milstein, C. (1991) Affinity maturation of antibodies. Immunol. Today 12,93[Medline]
  42. Berek, C., Ziegner, M. (1993) The maturation of the immune-response. Immunol. Today 14,400-404[Medline]
  43. Callaghan, M., Whelan, A., Feighery, C., Bresnihan, B. (1993) IL-2 Enhances polyclonal IgM but not IgM rheumatoid factor synthesis by activated human peripheral-blood B-cells. Clin. Exp. Immunol. 93,212-217[Medline]
  44. Kaul, R., Sharma, A., Lisse, J. R., Christadoss, P. (1995) Human recombinant IL-2 augments immunoglobulin and induces rheumatoid-factor production by rheumatoid-arthritis lymphocytes engrafted into severe combined immunodeficient mice. Clin. Immunol. Immunopathol. 74,271-282[Medline]
  45. Miyajima, H., Hirano, T., Hirose, S., Karasuyama, H., Okumura, K., Ovary, Z. (1991) Suppression by IL-2 of IgE production by B-cells stimulated by IL-4. J. Immunol. 146,457-462[Abstract]
  46. Nonoyama, S., Farrington, M. L., Ochs, H. D. (1994) Effect of IL-2 on immunoglobulin production by anti-CD40-activated human B-cells—synergistic effect with IL-10 and antagonistic effect with IL-4. Clin. Immunol. Immunopathol. 72,373-379[Medline]



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