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Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the production of TNFα and IL-12 by macrophages via H2-receptors
International Immunopharmacology 1 Ž2001. 1867–1875 www.elsevier.comrlocaterintimp
Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the production of TNFa and IL-12 by macrophages via H 2-receptors Yasutaka Azuma) , Mitsuko Shinohara, Pao-Li Wang, Atsushi Hidaka, Kiyoshi Ohura Department of Pharmacology, Osaka Dental UniÕersity, 8-1 Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, Japan Received 18 January 2001; received in revised form 10 May 2001; accepted 4 June 2001
Abstract Histamine is released from stimulated basophils and mast cells, and plays an important role in the pathogenesis of allergic inflammatory processes. In vitro treatment of macrophages with histamine resulted in inhibition of chemotaxis. Moreover, histamine at 10y5 M markedly inhibited the production of superoxide anions by both opsonized zymosan-A and phorbol 12-myristate 13-acetate ŽPMA. stimulated macrophages and histamine at a concentration range of 10y7 to 10y5 M significantly inhibited phagocytosis of Escherichia coli by macrophages. In addition, H 2-selective receptor agonist dimaprit resulted in inhibition of macrophage chemotaxis and markedly inhibited the production of superoxide anion by PMA-stimulated macrophages and phagocytosis of E. coli by macrophages. On the other hand, histamine and dimaprit both resulted in a concentration-dependent inhibition of lipopolysaccharide-induced production of TNFa and IL-12 by macrophages. These results suggest that histamine and dimaprit may inhibit chemotaxis, phagocytosis, superoxide anion production, and the production of TNFa and IL-12 by macrophages via H 2-histamine receptors. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Histamine; Macrophage; Phagocytosis; TNFa ; IL-12; H 2-histamine receptors
1. Introduction Histamine is released from stimulated basophils and mast cells and also plays an important role in the pathogenesis of allergic inflammatory processes as well w1–3x. There is accumulating evidence that histamine acts as a modulator of inflammation and immunological events w4x. For instance, histamine inhibited T-lymphocyte- and natural killer cell-medi-
) Corresponding author. Tel.: q81-72-864-3058; fax: q81-72864-3158. E-mail address: [email protected] ŽY. Azuma..
ated cytotoxicity. In addition, histamine inhalation suppresses T-lymphocyte proliferation in human w5x and histamine is found to depress chemotaxis of neutrophils w6,7x and the production of superoxide anion by fMLP-stimulated neutrophils w1x. Reactive oxygen species derived from the superoxide anion produced by macrophages and neutrophils participate in host defense by killing bacteria. Macrophages and neutrophils are essential for controlling the majority of infections and are mediators of inflammation so that the effects of histamine on these functions may have important implications at sites of inflammation. Macrophages and neutrophils are usually the first cells of the immune system to encounter invading
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 1 1 2 - 6
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pathogens such as bacteria and fungi, and are ready to leave the circulation and mediate an immediate response to the foreign antigen or pathogen. This phagocyte response to infection in vivo is initiated by adhesion to vascular endothelial cells, and progresses to directed cell migration into the extravascular tissue space. The migration of phagocytes results in phagocytosis and intracellular killing of the invading microorganisms by generation of bactericidal reactive oxygen species derived from the superoxide anion radical. These previous findings prompted us to examine possible effects of histamine on macrophage functions. To this end, we simultaneously examined the effect of histamine on the innate host defense functions of rat peritoneal macrophages. The investigation included studies on chemotaxis, phagocytosis and the production of superoxide anion.
2. Materials and methods 2.1. Materials Dimaprit and histamine were supplied by Research Biochemicals International ŽNatick, MA.. A monoclonal antibody against ED2 was provided by Pharmingen ŽSan Diego, CA.. Fluorescein-conjugated Escherichia coli ŽK-12. bioparticles was purchased from Molecular Probes ŽEugene, OA.. Other chemicals used were all of the highest purity commercially available. 2.2. Isolation of macrophages from the peritoneum The protocol employed here meets the guidelines of the Japanese Society for Pharmacology. All efforts were made to minimize animal suffering and to reduce the number of animals used. Macrophages were isolated by peritoneal lavage from adult male Wistar rats weighing 200–250 g, 4 days after injection of 20 ml of 1% glycogen. Lavage was performed by washing the peritoneal cavity with 100 ml of phosphate buffered saline ŽPBS, pH 7.2. supplemented with 20 Urml heparin and 1 mM EDTA. Care was taken not to cause internal bleeding while collecting macrophages in the exudate w8x. For chemotaxis assays, washed macrophages in Dulbecco’s modified eagle medium ŽDMEM., were al-
lowed to adhere to plastic dishes for 30 min at 37 8C in humidified atmosphere containing 5% CO 2 . They were then resuspended in DMEM. For phagocytosis and superoxide anion production assays, washed macrophages in DMEM were used. The purity of the adherent cells exceeded 95% as determined by immunostaining with anti-ED2 antibody Ža monocyter macrophage marker.. 2.3. Chemotaxis assays Chemotaxis assays were performed by a method previously described w9x with minor modifications w10x. In brief, zymosan A-activated serum ŽZAS. was diluted to 5% in medium and placed in the lower wells of a 96-well microplate chemotaxis chambers as the stimulant. Polycarbonate filters with 5-m m pores were placed on the wells. Experiments were initiated by the addition of the mixture containing macrophages at 2 = 10 6rml together with different concentrations of histamine or dimaprit to the upper wells. After incubation for 90 min, the filter was disassembled. The cells on the filter were fixed and stained with a Diff–Quik staining kit. The upper side of the filter was then scraped free of cells. The number of cells migrating to the lower side was determined by measuring optical densities at 595 nm using a microplate spectrophotometer ŽMolecular Devices, Sunnyvale, CA.. When the number of macrophages migrating to the lower side of the filter was measured by direct microscopy Ž=400. in 20 random fields, 5% ZAS in the lower well showed 302 " 19.6 cells, medium alone in the lower well showed 20.0 " 4.0 cells, and 5% ZAS in both well showed 31.0 " 2.6 cells, respectively. 2.4. Phagocytosis assays Phagocytosis assays were performed by a method previously described w11x with minor modifications w12x. In brief, macrophages adjusted to 1 = 10 6rml were allowed to adhere for 1 h. Macrophages were preincubated with histamine or dimaprit at one of four concentrations for 0.5 or 3 h. Following the addition of fluorescein conjugated E. coli ŽK-12. bioparticles at 1 = 10 6rwell, incubation was then initiated. After incubation for 30 min, extracellular fluorescence was quenched by adding 25 m g trypan
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2.6. Cytokine assays Macrophages adjusted to 2.5 = 10 6rml were allowed to adhere to 24-well plates for 30 min. Adherent macrophages were preincubated with histamine or dimaprit at one of four concentrations for 1 h, followed by incubation with lipopolysaccharide ŽLPS. at 1 m grml for 20–21 h. After medium was collected from wells, supernatant concentrations of rat TNFa and IL-12 were measured by commercially available ELISA w15x. 2.7. Data analyses Fig. 1. Effects of histamine on chemotaxis of macrophages. Macrophages were treated with histamine at one of six concentrations for 90 min. The data from 11 separate experiments are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in macrophages treated with medium.
blue in 13 mM citrate buffer ŽpH 4.4.. The dye was removed after 1 min and the fluorescence intensity was determined at 485 nm excitation and 535 nm emission wavelengths using a multilabel counter ŽWallac, Turku, Finland..
Results were all expressed as the mean " S.E. and the statistical significance was determined by the Unpaired t-test followed by estimation of the least significant difference.
3. Results 3.1. Histamine The effect of histamine on macrophage chemotaxis was examined after 90 min incubation. First, to
2.5. Superoxide anion production assays Superoxide anion production was detected by a method previously described w13x with minor modifications w14x. In brief, induction was by stimulation of 1 = 10 5 macrophagesr100 m l with 200 nM phorbol 12-myristate 13-acetate ŽPMA. or 100 m grml opsonized zymosan-A ŽOPZ.. The macrophages adjusted to 1 = 10 6rml were allowed to adhere for 1 h. Macrophages were preincubated with histamine or dimaprit at one of four concentrations for 15 min. Following the addition of PMA or OPZ containing 160 m M ferricytochrome c, incubation was then initiated. Superoxide anion production was assessed by inhibition of cytochrome c reduction using 600 Urml superoxide dismutase. The absorbance at a wavelength of 550 nm was measured with the aid of a microplate spectrophotometer ŽMolecular Devices. at different times after the incubation. Results were converted to nmol of cytochrome c reduced by using the extinction coefficient EŽ550., i.e. 2.1 = 10 4r Mrcm.
Fig. 2. Effects of histamine on superoxide anion production by OPZ-stimulated macrophages. Adherent macrophages were preincubated with histamine at one of four concentrations for 15 min, followed by incubation with OPZ. The data from five independent experiments are shown. ) P - 0.05, significantly different from each control value obtained in macrophages stimulated with OPZ alone.
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Fig. 3. Effects of histamine on phagocytosis of E. coli by macrophages. Adherent macrophages were preincubated with histamine at one of four concentrations for 0.5 or 3 h, followed by incubation with E. coli for 0.5 h. The data from nine separate experiments are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in macrophages treated with medium.
Fig. 5. Effects of dimaprit on chemotaxis of macrophages. Macrophages were treated with dimaprit at one of six concentrations for 90 min. The data from five separate experiments are shown. ) P - 0.05, significantly different from each control value obtained in macrophages treated with medium.
evaluate whether histamine has any chemoattractant effects, histamine was placed in the lower chamber well. However, it was found that histamine at concentrations of 10y1 0 to 10y4 M did not affect macrophage chemotaxis Ždata not shown.. In contrast, when macrophages and histamine were placed
in the upper well, with ZAS in the lower well as stimulant, histamine at a concentration range of 10y8 to 10y7 M inhibited chemotaxis markedly ŽFig. 1.. Superoxide anion production by OPZ-stimulated macrophages was unaffected by histamine up to 2 h of incubation, but at 3 h incubation there was nearly 15% more inhibition when histamine was used at a concentration of 10y5 M ŽFig. 2..
Fig. 4. Effects of histamine on superoxide anion production by PMA-stimulated macrophages. Adherent macrophages were preincubated with histamine at one of four concentrations for 15 min, followed by incubation with PMA. The data from nine independent experiments are shown. ) P - 0.05, significantly different from each control value obtained in macrophages stimulated with PMA alone.
Fig. 6. Effects of dimaprit on superoxide anion production by PMA-stimulated macrophages. Adherent macrophages were preincubated with dimaprit at one of four concentrations for 15 min, followed by incubation with PMA. The data from four independent experiments are shown. ) P - 0.05, significantly different from each control value obtained in macrophages stimulated with PMA alone.
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macrophage by histamine is associated with inhibition of phagocytosis of OPZ by macrophages, another stimulant PMA was used in the superoxide production assays. As shown in Fig. 4, histamine at 10y5 M markedly inhibited the production of superoxide anion by PMA-stimulated macrophages at 2 and 3 h incubation time. However, superoxide anion production was not markedly affected after 3 h incubation when the lower concentrations of histamine Ž10y8 to 10y6 M. were used. 3.2. H2-receptor inÕolÕement Fig. 7. Effects of dimaprit on phagocytosis of E. coli by macrophages. Adherent macrophages were preincubated with dimaprit at one of four concentrations for 0.5 or 3 h, followed by incubation with E. coli for 0.5 h. The data from five separate experiments are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in macrophages treated with medium.
When macrophages were pretreated for 0.5 h with histamine at different concentrations, no marked alteration was seen in phagocytosis of E. coli ŽFig. 3, left panel.. In contrast, histamine at concentrations of 10y7 to 10y5 M significantly inhibited phagocytosis by macrophages pretreated for 3 h in a concentration-dependent manner ŽFig. 3, right panel.. It should be noted that the OPZ used in this study activates macrophages via activation of phagocytosis of OPZ by macrophages. To determine whether inhibition of superoxide production in OPZ-stimulated
It has been previously reported that macrophages express H 2-selective histaminergic receptors. To determine their possible involvement in the observed change after incubation with histamine, the H 2-selective agonist dimaprit was examined. Addition of dimaprit at a concentration of 10y8 M to macrophages inhibited chemotaxis to ZAS ŽFig. 5.. Moreover, dimaprit at 10y5 M markedly inhibited the production of superoxide anion at 3 h incubation time ŽFig. 6.. However, dimaprit at the three lower concentrations did not affect superoxide production at different times up to 3 h of incubation. As shown in Fig. 7, dimaprit at concentrations of 10y7 to 10y5 M significantly inhibited phagocytosis by macrophages, pretreated for 3 h, in a concentration-dependent manner Žright panel., while phagocytosis by macrophages was unaffected after 0.5 h pretreatment Žleft panel..
Fig. 8. Effect of histamine and dimaprit on the production of TNFa by macrophages in response to LPS. Macrophages were allowed to adhere to plates for 30 min. Adherent macrophages were preincubated with histamine or dimaprit at one of four concentrations for 1 h, followed by incubation with 1 m grml LPS for 20–21 h. The data from four separate animals are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in LPS-treated macrophages.
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Fig. 9. Effect of histamine and dimaprit on the production of IL-12 by macrophages in response to LPS. Macrophages were allowed to adhere to plates for 30 min. Adherent macrophages were preincubated with histamine or dimaprit at one of four concentrations for 1 h, followed by incubation with 1 m grml LPS for 20–21 h. The data from four separate animals are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in LPS-treated macrophages.
3.3. TNFa and IL-12 productions Macrophages are main cells to secrete inflammatory cytokine TNFa . In addition, macrophages produce IL-12, which bridges the gap between innate and acquired immunity. Indeed, macrophages not only are involved in inflammatory responses through the production of TNFa but also influence the Th1rTh2 balance through the production of IL-12. Therefore, we examined the effect of histamine and dimaprit on the production of TNFa and IL-12 by macrophages stimulated with LPS at 1 m grml. Addition of increasing concentrations of histamine to macrophages resulted in a concentration-dependent inhibition of LPS-induced TNFa production ŽFig. 8, left panel.. Similarly, increasing concentrations of dimaprit blocked the production of TNFa in response to LPS ŽFig. 8, right panel.. In addition, increasing concentrations of histamine and dimaprit both caused a concentration-dependent inhibition of LPS-induced IL-12 production in response to LPS ŽFig. 9..
4. Discussion The essential finding presented in this study is that histamine affects macrophage function via H 2histaminergic receptors. This is the first direct demonstration of interaction of macrophage H 2histaminergic receptors with chemotaxis, phagocytosis and superoxide anion production by macro-
phages. In this study, we found that histamine significantly inhibited chemotaxis to zymosan A-activated serum and phagocytosis of E. coli. Additionally, histamine markedly inhibited the production of superoxide anions by PMA- and OPZ-stimulated macrophages. Since these immunomodulating effects of histamine were also seen in macrophages treated with dimaprit, H 2-histaminergic receptor signaling appeared to be involved in these events. PMA stimulates macrophages by activating protein kinase C and the subsequent potentiation of nicotinamide adenine dinucleotidephosphate ŽNADPH. oxidase to produce superoxide anions w16–18x. NADPH oxidase is assembled through a multistep process from several components including the two subunits of cytochrome b558 Žp22 p h o x and gp91 p h o x ., the small G proteins ŽRac and Rap., and the cytosolic factors Žp47 p h o x , p67 p h o x and p40 p h o x . at the plasma membrane w19–21x. Furthermore, PMA induces phosphorylation of the cytosolic factor p47 p h o x via activation of protein kinase C w22x. The cytosolic components including phosphorylated p47 p h o x then migrate to the membrane, where they associate with membrane-bound components to assemble the catalytically active oxidase w23–25x. On the other hand, the H 2-histaminergic receptor stimulates adenylyl cyclase, resulting in an increase in the intracellular concentration of cyclic AMP. Recently, protein kinase A following activation of cAMP was found to negatively regulate the cytosolic factor p47 p h o x w26x. Therefore, it is likely that the histamine signal decreased the activity of cytosolic factor p47 p h o x via
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an increase in intracellular cAMP by the H 2histaminergic receptor. However, the exact molecular mechanisms underlying the suppression of the production of superoxide anions by histamine still remain to be elucidated. In principle, the longer the duration of superoxide anion production, the more effective would be the sterilization of the invading bacteria at the site of inflammation. Therefore, inhibition of superoxide anion production by histamine may result in inhibition of intracellular killing of microbial invaders. Moreover, at a concentration of 10y8 M histamine inhibited macrophage chemotaxis, without affecting phagocytosis and the production of superoxide anion, at least under the conditions employed. Chemotaxis is directed movement of macrophages in response to chemotactic factors, such as C5a fragments, and is mediated by specific receptors on the surface. In response to chemotactic factors, specific receptors mediate both calcium influx and efflux w27,28x, and an increase in intracellular calcium concentration promotes chemotaxis. The addition of cAMP is reported to inhibit the increase in intracellular calcium concentration and induce the depolymerization of filamentous actin w29x. Hence, it is conceivable that histamine may inhibit macrophage chemotaxis by means of altering receptor activation, calcium flux, or actin polymerization in pathways related to chemotaxis. It is also likely that the mechanisms related to chemotaxis are more sensitive to treatment with histamine than the other functions tested. Phagocytosis is the process by which cells recognize and engulf large particles and it is important for host defense mechanisms as well as for tissue repair and morphogenetic remodeling. Two of the best characterized phagocytic receptors in macrophages, Fc gamma ŽFcg . receptors and the complement receptor 3 Žalso known as Mac-1., are involved in phagocytosis of microorganisms during infection. Activation of these two receptors induces the reorganization of filamentous actin structures following extracellular stimuli. Recently, histamine was found to inhibit the expression of complement receptor 3 in neutrophils w30x. Therefore, it is likely that histamine inhibits phagocytosis through expression of complement receptor 3 in macrophages. Alternatively, it is conceivable that histamine may affect the flow
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through the membrane and the expression of Fcg receptors. Nonetheless, taken together, these data are suggestive that there may be immunosuppression of the innate host defense system mediated by macrophages. Although the immunological significance of the present findings is unknown, it is possible that histamine-mediated suppression of macrophage functions may have a beneficial effect on type II Žcytotoxic. hypersensitivity or delayed-type hypersensitivity. Namely, macrophages are at least in part associated with the mechanisms of cytotoxic hypersensitivity and delayed-type hypersensitivity. An interesting finding of the present study is that histamine negatively regulates LPS-induced TNFa and IL-12 production by macrophages via H 2-histaminergic receptors. Macrophages and other phagocytic cells are components of innate or natural immune mechanisms, while lymphocytes are components of acquired or specific immune mechanisms. However, innate and specific immunities are not autonomous. This can be mediated through cytokines released by macrophagesrmonocytes that play a critical role in influencing the Th1rTh2 balance which, in turn, dictates the type of immune response generated. Thus, IL-12 activates T-cells and NK cells to proliferate, produce IFNg , and lyse target cells w31x, and also is a central inducer of cell-mediated immunity by promoting the development, proliferation and function of Th1 cells w32x. In fact, macrophages not only are involved in inflammatory responses through the production of TNFa but also influence the Th1rTh2 balance through the production of IL-12. Here we demonstrated that histamine inhibits LPSinduced TNFa and IL-12 production by macrophages. Since IL-12 plays important and contrasting roles in regulating Th1rTh2 immune responses, there is much interest in the factors controlling their production. Our observations suggest that conditions related to increased histamine may play an important role in a wide range of immune reactions including the Th1 and Th2 immune response. These results suggest that activation of histamine receptor may establish a novel paradigm for immunity regulation. It thus appears that histamine inhibits chemotaxis, phagocytosis, the production of superoxide anion production, and LPS-induced TNFa and IL-12 production by macrophages via H 2-histaminergic receptors. Taken together, these findings suggest that
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synthetic histamine-receptor ligands may have therapeutic applications in diseases in which macrophages play prominent pathogenic roles. Elucidation of interactions between histamine and the host defense system needs to be continued in future studies, and will undoubtedly be of great benefit for therapy in humans.
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Acknowledgements This work was performed in part at the Institute of Dental Research, Osaka Dental University.
References
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w1x Seligmann BE, Fletcher MP, Gallin JI. Histamine modulation of human neutrophil oxidative metabolism, locomotion, degranulation, and membrane potential changes. J Immunol 1983;130:1902–9. w2x Owen DAA. Inflammation—histamine and 5-hydroxytryptamine. Br Med Bull 1987;43:256–69. w3x Bury TB, Corhay JL, Radermecker MF. Histamine-induced inhibition of neutrophil chemotaxis and T-lymphocyte proliferation in man. Allergy 1992;47:624–9. w4x Beer DJ, Matloff SM, Rocklin RE. The influence of histamine on immune and inflammatory responses. Adv Immunol 1984;35:209–69. w5x Bury TB, Radermecker MF. Depression of polymorphonuclear chemotaxis and T-lymphocyte proliferation following histamine inhalation in man. Eur Respir J 1989;2:828–33. w6x Rocklin RE. Modulation of cellular immune responses in vivo and in vitro by histamine receptor-bearing lymphocytes. J Clin Invest 1976;57:1051–8. w7x Patrone F, Dallegri F, Lauzi G, Saccheitti C. Reversal by cimetidine of histamine-induced inhibition of true chemotaxis in neutrophil polymorphonuclears. Res Exp Med 1980; 176:201–5. w8x Azuma Y, Shinohara M, Wang PL, Ohura K. Quinolones alter defense reactions mediated by macrophages. Int Immunopharmacol 2001;1:179–87. w9x Azuma Y, Shinohara M, Murakawa M, Endo M, Ohura K. Possible interaction between new quinolones and immune functions in macrophages. Gen Pharmacol 1999;32:609–14. w10x Azuma Y, Ohura K. Comparison of the effect of lidocaineepinephrine and prilocaine-felypressine to alter macrophage functions. Int Immunopharmacol 2001;1:911–23. w11x Azuma Y, Shinohara M, Wang PL, Suese Y, Yasuda H, et al. Comparison of inhibitory effects of local anesthetics on immune functions of neutrophils. Int J Immunopharmacol 2000;22:789–96. w12x Azuma Y, Wang PL, Shinohara M, Ohura K. Differentiation
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by in vitro treatment of lidocaine-epinephrine and prilocaine-felypressine in neutrophils. Immunol Lett 2001. in press. Azuma Y, Wang PL, Shinohara M, Okamura M, Inui Y, et al. Comparative studies of modulatory effect to the function of rat peritoneal neutrophils treated with new quinolones. Immunol Lett 1999;69:321–7. Azuma Y, Wang PL, Shinohara M, Ohura K. Immunomodulation of the neutrophil respiratory burst by endomorphins 1 and 2. Immunol Lett 2000;75:55–9. Azuma Y, Shinohara M, Wang PL, Ohura K. 15-DeoxyD12,14-prostaglandin J2 inhibits IL-10 and IL-12 production by macrophages. Biochem Biophys Res Commun 2001;283: 344–6. Gerard C, McPhail LC, Marfat A, Stimler-Gerard NP, Bass DA, et al. Role of protein kinases in stimulation of human polymorphonuclear leukocyte oxidative metabolism by various agonists. J Clin Invest 1986;77:61–5. Tauber AI. Protein kinase C and the activation of the human neutrophil NADPH-oxidase. Blood 1987;69:711–20. Kessels GCR, Krause K, Verhoeven AJ. Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C. Biochem J 1993; 292:781–5. Babior BM. The respiratory burst oxidase. Curr Opin Hamatol 1995;2:55–60. Bokoch GM. Chemoattractant signaling and leukocyte activation. Blood 1995;86:1649–60. Dagher MC, Fuchs A, Bourmeyster N, Jourdan A, Vignais PV. Small G proteins and the neutrophil NADPH oxidase. Biochimie 1995;77:651–60. Tardif M, Rabiet MJ, Christophe T, Milcent MD, Boulay F. Isolation and characterization of a variant HL60 cell line defective in the activation of the NADPH oxidase by phorbol myristate acetate. J Immunol 1998;161:6885–95. Chanock SJ, El Benna J, Smith RM, Babior BM. The respiratory burst oxidase. J Biol Chem 1994;269:24519–22. Lomax KJ, Malech HL, Gallin JI. The molecular biology of selected phagocyte defects. Blood Rev 1989;3:94–104. Nauseef WM. Cytosolic oxidase factors in the NADPH-dependent oxidase of human neutrophils. Eur J Haematol 1993; 51:301–8. El Benna J, Faust LP, Johnson JL, Babior BM. Phosphorylation of the respiratory burst oxidase subunit p47 phox as determined by two-dimensional phosphopeptide mapping. J Biol Chem 1996;271:6374–8. Gallin JI, Rosenthai AS. The regulatory role of divalent cations in human granulocyte chemotaxis: evidence for an association between calcium exchanges and microtubule assembly. J Cell Biol 1974;62:594–609. Naccache P, Freer RJ, Showell HJ, Becker EL, Shaafl RI. Transport of sodium, potassium, and calcium across rabbit polymorphonuclear leukocyte membrane: effect of chemotactic factor. J Cell Biol 1977;73:428–44.
Y. Azuma et al.r International Immunopharmacology 1 (2001) 1867–1875 w29x Hamachi T, Hirata M, Koga T. Effect of cAMP-elevating drugs on Ca2q efflux and actin polymerization in peritoneal macrophages stimulated with N-formyl chemotactic peptide. Biochim Biophys Acta 1984;804:230–6. w30x Francis JW, Todd RF, Boxer LA, Petty HR. Histamine inhibits cell spreading and C3bi receptor clustering and diminishes hydrogen peroxide production by adherent human neutrophils. J Cell Physiol 1991;147:128–37.
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w31x Trinchieri G. Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 1994;84:4008–27. w32x Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, et al. Identification and purification of natural killer cell stimulatory factor ŽNKSF., a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989;170:827–45.
Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the production of TNFa and IL-12 by macrophages via H 2-receptors Yasutaka Azuma) , Mitsuko Shinohara, Pao-Li Wang, Atsushi Hidaka, Kiyoshi Ohura Department of Pharmacology, Osaka Dental UniÕersity, 8-1 Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, Japan Received 18 January 2001; received in revised form 10 May 2001; accepted 4 June 2001
Abstract Histamine is released from stimulated basophils and mast cells, and plays an important role in the pathogenesis of allergic inflammatory processes. In vitro treatment of macrophages with histamine resulted in inhibition of chemotaxis. Moreover, histamine at 10y5 M markedly inhibited the production of superoxide anions by both opsonized zymosan-A and phorbol 12-myristate 13-acetate ŽPMA. stimulated macrophages and histamine at a concentration range of 10y7 to 10y5 M significantly inhibited phagocytosis of Escherichia coli by macrophages. In addition, H 2-selective receptor agonist dimaprit resulted in inhibition of macrophage chemotaxis and markedly inhibited the production of superoxide anion by PMA-stimulated macrophages and phagocytosis of E. coli by macrophages. On the other hand, histamine and dimaprit both resulted in a concentration-dependent inhibition of lipopolysaccharide-induced production of TNFa and IL-12 by macrophages. These results suggest that histamine and dimaprit may inhibit chemotaxis, phagocytosis, superoxide anion production, and the production of TNFa and IL-12 by macrophages via H 2-histamine receptors. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Histamine; Macrophage; Phagocytosis; TNFa ; IL-12; H 2-histamine receptors
1. Introduction Histamine is released from stimulated basophils and mast cells and also plays an important role in the pathogenesis of allergic inflammatory processes as well w1–3x. There is accumulating evidence that histamine acts as a modulator of inflammation and immunological events w4x. For instance, histamine inhibited T-lymphocyte- and natural killer cell-medi-
) Corresponding author. Tel.: q81-72-864-3058; fax: q81-72864-3158. E-mail address: [email protected] ŽY. Azuma..
ated cytotoxicity. In addition, histamine inhalation suppresses T-lymphocyte proliferation in human w5x and histamine is found to depress chemotaxis of neutrophils w6,7x and the production of superoxide anion by fMLP-stimulated neutrophils w1x. Reactive oxygen species derived from the superoxide anion produced by macrophages and neutrophils participate in host defense by killing bacteria. Macrophages and neutrophils are essential for controlling the majority of infections and are mediators of inflammation so that the effects of histamine on these functions may have important implications at sites of inflammation. Macrophages and neutrophils are usually the first cells of the immune system to encounter invading
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 1 1 2 - 6
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pathogens such as bacteria and fungi, and are ready to leave the circulation and mediate an immediate response to the foreign antigen or pathogen. This phagocyte response to infection in vivo is initiated by adhesion to vascular endothelial cells, and progresses to directed cell migration into the extravascular tissue space. The migration of phagocytes results in phagocytosis and intracellular killing of the invading microorganisms by generation of bactericidal reactive oxygen species derived from the superoxide anion radical. These previous findings prompted us to examine possible effects of histamine on macrophage functions. To this end, we simultaneously examined the effect of histamine on the innate host defense functions of rat peritoneal macrophages. The investigation included studies on chemotaxis, phagocytosis and the production of superoxide anion.
2. Materials and methods 2.1. Materials Dimaprit and histamine were supplied by Research Biochemicals International ŽNatick, MA.. A monoclonal antibody against ED2 was provided by Pharmingen ŽSan Diego, CA.. Fluorescein-conjugated Escherichia coli ŽK-12. bioparticles was purchased from Molecular Probes ŽEugene, OA.. Other chemicals used were all of the highest purity commercially available. 2.2. Isolation of macrophages from the peritoneum The protocol employed here meets the guidelines of the Japanese Society for Pharmacology. All efforts were made to minimize animal suffering and to reduce the number of animals used. Macrophages were isolated by peritoneal lavage from adult male Wistar rats weighing 200–250 g, 4 days after injection of 20 ml of 1% glycogen. Lavage was performed by washing the peritoneal cavity with 100 ml of phosphate buffered saline ŽPBS, pH 7.2. supplemented with 20 Urml heparin and 1 mM EDTA. Care was taken not to cause internal bleeding while collecting macrophages in the exudate w8x. For chemotaxis assays, washed macrophages in Dulbecco’s modified eagle medium ŽDMEM., were al-
lowed to adhere to plastic dishes for 30 min at 37 8C in humidified atmosphere containing 5% CO 2 . They were then resuspended in DMEM. For phagocytosis and superoxide anion production assays, washed macrophages in DMEM were used. The purity of the adherent cells exceeded 95% as determined by immunostaining with anti-ED2 antibody Ža monocyter macrophage marker.. 2.3. Chemotaxis assays Chemotaxis assays were performed by a method previously described w9x with minor modifications w10x. In brief, zymosan A-activated serum ŽZAS. was diluted to 5% in medium and placed in the lower wells of a 96-well microplate chemotaxis chambers as the stimulant. Polycarbonate filters with 5-m m pores were placed on the wells. Experiments were initiated by the addition of the mixture containing macrophages at 2 = 10 6rml together with different concentrations of histamine or dimaprit to the upper wells. After incubation for 90 min, the filter was disassembled. The cells on the filter were fixed and stained with a Diff–Quik staining kit. The upper side of the filter was then scraped free of cells. The number of cells migrating to the lower side was determined by measuring optical densities at 595 nm using a microplate spectrophotometer ŽMolecular Devices, Sunnyvale, CA.. When the number of macrophages migrating to the lower side of the filter was measured by direct microscopy Ž=400. in 20 random fields, 5% ZAS in the lower well showed 302 " 19.6 cells, medium alone in the lower well showed 20.0 " 4.0 cells, and 5% ZAS in both well showed 31.0 " 2.6 cells, respectively. 2.4. Phagocytosis assays Phagocytosis assays were performed by a method previously described w11x with minor modifications w12x. In brief, macrophages adjusted to 1 = 10 6rml were allowed to adhere for 1 h. Macrophages were preincubated with histamine or dimaprit at one of four concentrations for 0.5 or 3 h. Following the addition of fluorescein conjugated E. coli ŽK-12. bioparticles at 1 = 10 6rwell, incubation was then initiated. After incubation for 30 min, extracellular fluorescence was quenched by adding 25 m g trypan
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2.6. Cytokine assays Macrophages adjusted to 2.5 = 10 6rml were allowed to adhere to 24-well plates for 30 min. Adherent macrophages were preincubated with histamine or dimaprit at one of four concentrations for 1 h, followed by incubation with lipopolysaccharide ŽLPS. at 1 m grml for 20–21 h. After medium was collected from wells, supernatant concentrations of rat TNFa and IL-12 were measured by commercially available ELISA w15x. 2.7. Data analyses Fig. 1. Effects of histamine on chemotaxis of macrophages. Macrophages were treated with histamine at one of six concentrations for 90 min. The data from 11 separate experiments are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in macrophages treated with medium.
blue in 13 mM citrate buffer ŽpH 4.4.. The dye was removed after 1 min and the fluorescence intensity was determined at 485 nm excitation and 535 nm emission wavelengths using a multilabel counter ŽWallac, Turku, Finland..
Results were all expressed as the mean " S.E. and the statistical significance was determined by the Unpaired t-test followed by estimation of the least significant difference.
3. Results 3.1. Histamine The effect of histamine on macrophage chemotaxis was examined after 90 min incubation. First, to
2.5. Superoxide anion production assays Superoxide anion production was detected by a method previously described w13x with minor modifications w14x. In brief, induction was by stimulation of 1 = 10 5 macrophagesr100 m l with 200 nM phorbol 12-myristate 13-acetate ŽPMA. or 100 m grml opsonized zymosan-A ŽOPZ.. The macrophages adjusted to 1 = 10 6rml were allowed to adhere for 1 h. Macrophages were preincubated with histamine or dimaprit at one of four concentrations for 15 min. Following the addition of PMA or OPZ containing 160 m M ferricytochrome c, incubation was then initiated. Superoxide anion production was assessed by inhibition of cytochrome c reduction using 600 Urml superoxide dismutase. The absorbance at a wavelength of 550 nm was measured with the aid of a microplate spectrophotometer ŽMolecular Devices. at different times after the incubation. Results were converted to nmol of cytochrome c reduced by using the extinction coefficient EŽ550., i.e. 2.1 = 10 4r Mrcm.
Fig. 2. Effects of histamine on superoxide anion production by OPZ-stimulated macrophages. Adherent macrophages were preincubated with histamine at one of four concentrations for 15 min, followed by incubation with OPZ. The data from five independent experiments are shown. ) P - 0.05, significantly different from each control value obtained in macrophages stimulated with OPZ alone.
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Fig. 3. Effects of histamine on phagocytosis of E. coli by macrophages. Adherent macrophages were preincubated with histamine at one of four concentrations for 0.5 or 3 h, followed by incubation with E. coli for 0.5 h. The data from nine separate experiments are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in macrophages treated with medium.
Fig. 5. Effects of dimaprit on chemotaxis of macrophages. Macrophages were treated with dimaprit at one of six concentrations for 90 min. The data from five separate experiments are shown. ) P - 0.05, significantly different from each control value obtained in macrophages treated with medium.
evaluate whether histamine has any chemoattractant effects, histamine was placed in the lower chamber well. However, it was found that histamine at concentrations of 10y1 0 to 10y4 M did not affect macrophage chemotaxis Ždata not shown.. In contrast, when macrophages and histamine were placed
in the upper well, with ZAS in the lower well as stimulant, histamine at a concentration range of 10y8 to 10y7 M inhibited chemotaxis markedly ŽFig. 1.. Superoxide anion production by OPZ-stimulated macrophages was unaffected by histamine up to 2 h of incubation, but at 3 h incubation there was nearly 15% more inhibition when histamine was used at a concentration of 10y5 M ŽFig. 2..
Fig. 4. Effects of histamine on superoxide anion production by PMA-stimulated macrophages. Adherent macrophages were preincubated with histamine at one of four concentrations for 15 min, followed by incubation with PMA. The data from nine independent experiments are shown. ) P - 0.05, significantly different from each control value obtained in macrophages stimulated with PMA alone.
Fig. 6. Effects of dimaprit on superoxide anion production by PMA-stimulated macrophages. Adherent macrophages were preincubated with dimaprit at one of four concentrations for 15 min, followed by incubation with PMA. The data from four independent experiments are shown. ) P - 0.05, significantly different from each control value obtained in macrophages stimulated with PMA alone.
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macrophage by histamine is associated with inhibition of phagocytosis of OPZ by macrophages, another stimulant PMA was used in the superoxide production assays. As shown in Fig. 4, histamine at 10y5 M markedly inhibited the production of superoxide anion by PMA-stimulated macrophages at 2 and 3 h incubation time. However, superoxide anion production was not markedly affected after 3 h incubation when the lower concentrations of histamine Ž10y8 to 10y6 M. were used. 3.2. H2-receptor inÕolÕement Fig. 7. Effects of dimaprit on phagocytosis of E. coli by macrophages. Adherent macrophages were preincubated with dimaprit at one of four concentrations for 0.5 or 3 h, followed by incubation with E. coli for 0.5 h. The data from five separate experiments are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in macrophages treated with medium.
When macrophages were pretreated for 0.5 h with histamine at different concentrations, no marked alteration was seen in phagocytosis of E. coli ŽFig. 3, left panel.. In contrast, histamine at concentrations of 10y7 to 10y5 M significantly inhibited phagocytosis by macrophages pretreated for 3 h in a concentration-dependent manner ŽFig. 3, right panel.. It should be noted that the OPZ used in this study activates macrophages via activation of phagocytosis of OPZ by macrophages. To determine whether inhibition of superoxide production in OPZ-stimulated
It has been previously reported that macrophages express H 2-selective histaminergic receptors. To determine their possible involvement in the observed change after incubation with histamine, the H 2-selective agonist dimaprit was examined. Addition of dimaprit at a concentration of 10y8 M to macrophages inhibited chemotaxis to ZAS ŽFig. 5.. Moreover, dimaprit at 10y5 M markedly inhibited the production of superoxide anion at 3 h incubation time ŽFig. 6.. However, dimaprit at the three lower concentrations did not affect superoxide production at different times up to 3 h of incubation. As shown in Fig. 7, dimaprit at concentrations of 10y7 to 10y5 M significantly inhibited phagocytosis by macrophages, pretreated for 3 h, in a concentration-dependent manner Žright panel., while phagocytosis by macrophages was unaffected after 0.5 h pretreatment Žleft panel..
Fig. 8. Effect of histamine and dimaprit on the production of TNFa by macrophages in response to LPS. Macrophages were allowed to adhere to plates for 30 min. Adherent macrophages were preincubated with histamine or dimaprit at one of four concentrations for 1 h, followed by incubation with 1 m grml LPS for 20–21 h. The data from four separate animals are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in LPS-treated macrophages.
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Fig. 9. Effect of histamine and dimaprit on the production of IL-12 by macrophages in response to LPS. Macrophages were allowed to adhere to plates for 30 min. Adherent macrophages were preincubated with histamine or dimaprit at one of four concentrations for 1 h, followed by incubation with 1 m grml LPS for 20–21 h. The data from four separate animals are shown. ) P - 0.05, ) ) P - 0.01, significantly different from each control value obtained in LPS-treated macrophages.
3.3. TNFa and IL-12 productions Macrophages are main cells to secrete inflammatory cytokine TNFa . In addition, macrophages produce IL-12, which bridges the gap between innate and acquired immunity. Indeed, macrophages not only are involved in inflammatory responses through the production of TNFa but also influence the Th1rTh2 balance through the production of IL-12. Therefore, we examined the effect of histamine and dimaprit on the production of TNFa and IL-12 by macrophages stimulated with LPS at 1 m grml. Addition of increasing concentrations of histamine to macrophages resulted in a concentration-dependent inhibition of LPS-induced TNFa production ŽFig. 8, left panel.. Similarly, increasing concentrations of dimaprit blocked the production of TNFa in response to LPS ŽFig. 8, right panel.. In addition, increasing concentrations of histamine and dimaprit both caused a concentration-dependent inhibition of LPS-induced IL-12 production in response to LPS ŽFig. 9..
4. Discussion The essential finding presented in this study is that histamine affects macrophage function via H 2histaminergic receptors. This is the first direct demonstration of interaction of macrophage H 2histaminergic receptors with chemotaxis, phagocytosis and superoxide anion production by macro-
phages. In this study, we found that histamine significantly inhibited chemotaxis to zymosan A-activated serum and phagocytosis of E. coli. Additionally, histamine markedly inhibited the production of superoxide anions by PMA- and OPZ-stimulated macrophages. Since these immunomodulating effects of histamine were also seen in macrophages treated with dimaprit, H 2-histaminergic receptor signaling appeared to be involved in these events. PMA stimulates macrophages by activating protein kinase C and the subsequent potentiation of nicotinamide adenine dinucleotidephosphate ŽNADPH. oxidase to produce superoxide anions w16–18x. NADPH oxidase is assembled through a multistep process from several components including the two subunits of cytochrome b558 Žp22 p h o x and gp91 p h o x ., the small G proteins ŽRac and Rap., and the cytosolic factors Žp47 p h o x , p67 p h o x and p40 p h o x . at the plasma membrane w19–21x. Furthermore, PMA induces phosphorylation of the cytosolic factor p47 p h o x via activation of protein kinase C w22x. The cytosolic components including phosphorylated p47 p h o x then migrate to the membrane, where they associate with membrane-bound components to assemble the catalytically active oxidase w23–25x. On the other hand, the H 2-histaminergic receptor stimulates adenylyl cyclase, resulting in an increase in the intracellular concentration of cyclic AMP. Recently, protein kinase A following activation of cAMP was found to negatively regulate the cytosolic factor p47 p h o x w26x. Therefore, it is likely that the histamine signal decreased the activity of cytosolic factor p47 p h o x via
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an increase in intracellular cAMP by the H 2histaminergic receptor. However, the exact molecular mechanisms underlying the suppression of the production of superoxide anions by histamine still remain to be elucidated. In principle, the longer the duration of superoxide anion production, the more effective would be the sterilization of the invading bacteria at the site of inflammation. Therefore, inhibition of superoxide anion production by histamine may result in inhibition of intracellular killing of microbial invaders. Moreover, at a concentration of 10y8 M histamine inhibited macrophage chemotaxis, without affecting phagocytosis and the production of superoxide anion, at least under the conditions employed. Chemotaxis is directed movement of macrophages in response to chemotactic factors, such as C5a fragments, and is mediated by specific receptors on the surface. In response to chemotactic factors, specific receptors mediate both calcium influx and efflux w27,28x, and an increase in intracellular calcium concentration promotes chemotaxis. The addition of cAMP is reported to inhibit the increase in intracellular calcium concentration and induce the depolymerization of filamentous actin w29x. Hence, it is conceivable that histamine may inhibit macrophage chemotaxis by means of altering receptor activation, calcium flux, or actin polymerization in pathways related to chemotaxis. It is also likely that the mechanisms related to chemotaxis are more sensitive to treatment with histamine than the other functions tested. Phagocytosis is the process by which cells recognize and engulf large particles and it is important for host defense mechanisms as well as for tissue repair and morphogenetic remodeling. Two of the best characterized phagocytic receptors in macrophages, Fc gamma ŽFcg . receptors and the complement receptor 3 Žalso known as Mac-1., are involved in phagocytosis of microorganisms during infection. Activation of these two receptors induces the reorganization of filamentous actin structures following extracellular stimuli. Recently, histamine was found to inhibit the expression of complement receptor 3 in neutrophils w30x. Therefore, it is likely that histamine inhibits phagocytosis through expression of complement receptor 3 in macrophages. Alternatively, it is conceivable that histamine may affect the flow
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through the membrane and the expression of Fcg receptors. Nonetheless, taken together, these data are suggestive that there may be immunosuppression of the innate host defense system mediated by macrophages. Although the immunological significance of the present findings is unknown, it is possible that histamine-mediated suppression of macrophage functions may have a beneficial effect on type II Žcytotoxic. hypersensitivity or delayed-type hypersensitivity. Namely, macrophages are at least in part associated with the mechanisms of cytotoxic hypersensitivity and delayed-type hypersensitivity. An interesting finding of the present study is that histamine negatively regulates LPS-induced TNFa and IL-12 production by macrophages via H 2-histaminergic receptors. Macrophages and other phagocytic cells are components of innate or natural immune mechanisms, while lymphocytes are components of acquired or specific immune mechanisms. However, innate and specific immunities are not autonomous. This can be mediated through cytokines released by macrophagesrmonocytes that play a critical role in influencing the Th1rTh2 balance which, in turn, dictates the type of immune response generated. Thus, IL-12 activates T-cells and NK cells to proliferate, produce IFNg , and lyse target cells w31x, and also is a central inducer of cell-mediated immunity by promoting the development, proliferation and function of Th1 cells w32x. In fact, macrophages not only are involved in inflammatory responses through the production of TNFa but also influence the Th1rTh2 balance through the production of IL-12. Here we demonstrated that histamine inhibits LPSinduced TNFa and IL-12 production by macrophages. Since IL-12 plays important and contrasting roles in regulating Th1rTh2 immune responses, there is much interest in the factors controlling their production. Our observations suggest that conditions related to increased histamine may play an important role in a wide range of immune reactions including the Th1 and Th2 immune response. These results suggest that activation of histamine receptor may establish a novel paradigm for immunity regulation. It thus appears that histamine inhibits chemotaxis, phagocytosis, the production of superoxide anion production, and LPS-induced TNFa and IL-12 production by macrophages via H 2-histaminergic receptors. Taken together, these findings suggest that
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synthetic histamine-receptor ligands may have therapeutic applications in diseases in which macrophages play prominent pathogenic roles. Elucidation of interactions between histamine and the host defense system needs to be continued in future studies, and will undoubtedly be of great benefit for therapy in humans.
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Acknowledgements This work was performed in part at the Institute of Dental Research, Osaka Dental University.
References
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