β-Defensins chemoattract macrophages and mast cells but not lymphocytes and dendritic cells: CCR6 is not involved

Authors

  • Afsaneh Soruri,

    1. Department of Cellular and Molecular Immunology, Georg-August-University Göttingen, Göttingen, Germany
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    • These two authors contributed equally to this work.

  • Jasmin Grigat,

    1. Department of Cellular and Molecular Immunology, Georg-August-University Göttingen, Göttingen, Germany
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    • These two authors contributed equally to this work.

  • Ulf Forssmann,

    1. IPF PharmaCeuticals, Hannover Medical School, Hannover, Germany
    2. Center of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany
    3. Merck KGaA, Darmstadt, Germany
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    • These two authors contributed equally to this work.

  • Joachim Riggert,

    1. Department of Transfusion Medicine, Georg-August-University Göttingen, Göttingen, Germany
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  • Jörg Zwirner Dr.

    Corresponding author
    1. Department of Cellular and Molecular Immunology, Georg-August-University Göttingen, Göttingen, Germany
    • Department of Cellular and Molecular Immunology, Georg-August-University Göttingen, Humboldtallee 34, D-37073 Göttingen, Germany, Fax: +49-551-395-843
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Abstract

β-Defensins are natural peptide antibiotics whose immunomodulatory functions are poorly understood. In the present study, macrophages were found to migrate to human β-defensins (HBD)-1 to -4 using Gαi proteins as well as MAPK ERK, p38 and JNK as signal transducers. In addition, mast cells responded to HBD-1 to -4 with calcium fluxes as well as chemotaxis, which increased upon stimulation with IgE plus antigen or ionomycin. In contrast, human β-defensins were unable to induce migration of memory lymphocytes and dendritic cells (DC). Similar to HBD, the murine β-defensin (mBD)-8 mobilized macrophages and lacked the ability to recruit memory T cells. These findings were unexpected as CCR6 on memory T cells and DC has been previously observed to be a receptor for human β-defensins. In support of our findings, however, RBL-2H3 as well as 300.19 cells stably expressing CCR6 proved to be unresponsive to HBD-2 and -3. Intriguingly, our observation of a PKC-independent homologous desensitization between HBD-1 to -4 suggests a common receptor for HBD. In summary, chemoattraction of macrophages and mast cells is evolutionary conserved within the β-defensin family despite a considerable sequence variation and distinct antimicrobial activities. However, CCR6 is not a functional receptor for β-defensins.

Abbreviations:
Ø:

buffer

HBD:

human β-defensin

mBD:

murine β-defensin

Introduction

Human defensins are short cationic β-sheet peptides with molecular masses ranging from 3 to 5 kDa. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated α- and β-defensins 1, 2. The α- and β-defensins are products of distinct gene families that are thought to have evolved from an ancestral β-defensin gene 3. Despite the considerable sequence variation among β-defensins, a striking similarity on the level of secondary and tertiary structure was found 4.

β-Defensins are expressed by epithelial cells of the skin and the respiratory, urogenital and gastrointestinal tract where they are either constitutively expressed or induced by different stimuli such as cytokines and bacteria 58. The main recognized function of defensin peptides is antimicrobial. Defensins disrupt cell membranes of microorganisms that are rich in negatively charged phospholipids 9. Their spectrum of activity is diverse and includes bacteria, fungi and viruses 10.

Recently, β-defensins have been shown to play significant roles in adaptive as well as innate immunity. They modulate the cytokine response of mononuclear blood cells 11, and promote systemic antigen- or tumor-specific immune responses 1215. In keeping with these findings, human β-defensin (HBD)-1 to -3 as well as murine β-defensin (mBD)-2, -3 and -29 were shown to selectively chemoattract memory T lymphocytes and/or immature DC via CCR6 12, 1618. As some β-defensins additionally induced migration of monocytes and mast cells 1922 that do not express CCR6, further receptors for β-defensins were postulated 23. To this end, studies of the immunomodulatory properties of defensins are still in their infancy despite the well-appreciated importance of these peptides for the host defense 24.

The purpose of the present study was to investigate the impact of β-defensins on cells of the innate and adaptive immune system in order to (i) identify those immune cells that are primary targets for β-defensin-induced immunomodulation and (ii) define receptor usage by β-defensins. According to our results, β-defensin-induced chemoattraction functions through a common receptor on macrophages and mast cells. However, we could not confirm CCR6 to be a functional receptor for β-defensins.

Results

Mobilization of macrophages

Published data regarding the chemotactic impact of HBD on monocytes/macrophages are inconsistent 16, 18, 19. We observed HBD-1 to -4 as well as mBD-8 to induce a concentration-dependent, bell-shaped chemotactic response in human monocyte-derived macrophages in vitro (Fig. 1A).

Figure 1.

β-Defensin-induced migration of human macrophages is subject to homologous desensitization. (A) In vitro chemotaxis of human macrophages was measured in response to human β-defensins and CCL20. Mean values (± SEM) of three to five independent experiments each are shown. (B–E) To investigate receptor desensitization, human macrophages were preincubated with human β-defensins, PMA, or buffer (∅) as indicated. Thereafter, in vitro chemotaxis was measured in response to human β-defensins (1000 ng/mL) or C5a (100 ng/mL). Mean values (± SEM) of three independent experiments each are shown. (F) Human macrophages were preincubated in the absence (open bars) or presence (filled bars) of PKC inhibitor GF109 and, subsequently, with different human β-defensins, PMA, or buffer (∅) as indicated. Thereafter, in vitro chemotaxis was measured in response to HBD-2 (1000 ng/mL). Mean values (± SEM) of three independent experiments are shown. (G) In vivo migration of human macrophages was measured in response to HBD-2 and C5a (20 µg each). To investigate receptor desensitization, cells were preincubated as indicated. Four hours later, peritoneal cells were harvested, stained, and analyzed by FACS. Mean values (± SEM) of five independent experiments each are shown.

To address the issue of signaling via one or more chemotactic receptors, functional desensitization between human β-defensins was investigated. We observed that HBD-1 to -4 were able to desensitize each other's chemotactic activities (Fig. 1B–D, F). However, this effect did not extend to other chemotaxins such as the anaphylatoxin C5a (Fig. 1E) or CCL3 (data not shown). Although desensitization can be of homologous or heterologous nature, only the latter is PKC-dependent 25, 26. As demonstrated in Fig. 1F, the general PKC inhibitor GF109 was unable to block desensitization between HBD. This finding indicates that HBD-1 to -4 signal through a common receptor in macrophages resulting in PKC-independent homologous receptor desensitization. Receptors for β-defensins were also subject to heterologous desensitization as shown by the ability of the PKC activator PMA to desensitize β-defensin-induced chemotaxis (Fig. 1B–D, F).

Migration of macrophages was further studied using a SCID mouse model. We have recently shown that accumulation of human macrophages in the peritoneal cavity of SCID mice as a result of chemotactic cell migration was inducible by injection of anaphylatoxins and chemokines 27. We now observed that human macrophages could be mobilized into the peritoneal cavity of SCID mice by i.p. injections of β-defensins (Fig. 1G). Similar to in vitro chemotaxis experiments, in vivo migration of macrophages in response to HBD-2 was abrogated if HBD-2 itself or other members of the β-defensin family were used for preincubation. No inhibitory effect by β-defensin preincubation was observed when C5a was used as a chemoattractant in vivo (Fig. 1G). These experiments confirm the in vivo relevance and the receptor-specific nature of β-defensin-induced chemoattraction.

We also investigated the chemotactic impact of a linear form of HBD-3. Human macrophages were unresponsive to this modified molecule in vitro and in SCID mice (data not shown) which suggests that a conformational and not a linear epitope of HBD-3 is responsible for its chemotactic activity.

To exclude a possible involvement of CCR6 in β-defensin-mediated chemotaxis of human macrophages, we measured their mobilization in response to the sole chemokine ligand of CCR6, CCL20. As expected, macrophages did not migrate in response to CCL20 in vivo (10 µg i.p., n = 3; data not shown) or in vitro (Fig. 1A).

Mobilization of dendritic cells

IL-4, which is commonly used for the generation of DC in vitro, was recently observed to inhibit migration of monocyte-derived macrophages and DC in response to anaphylatoxins 27. In line with these findings, IL-4 treatment also inhibited migration of human monocyte-derived DC in response to β-defensins in vitro and in vivo (Fig. 2A). Our data indicated that macrophages but not DC are primary target cells for β-defensins. To find further support for this hypothesis, we compared migration of murine macrophages versus DC that had been generated from bone marrow precursors in the presence of M-CSF and GM-CSF, respectively. It was found that macrophages (including J774A.1 cells) were preferentially mobilized by β-defensins in vitro (Fig. 2B) and in vivo (Fig. 2C). Conversely, CCL20 exclusively chemoattracted murine DC but not macrophages, in agreement with the expression of its receptor CCR6 in CD34+ precursor cell-derived DC. Confirming the impact of β-defensins on macrophages, HBD-2 and mBD-8 were found to be potent chemotaxins for the macrophage cell line J774A.1.

Figure 2.

Defensins preferentially mobilize macrophages. (A) Human monocytes were cultured for 7 days with GM-CSF in the absence (macrophages) or presence (DC) of IL-4. Subsequently, in vitro migration in response to HBD-2 (1000 ng/mL) or buffer (∅) was measured. For in vivo studies, cells were injected i.v. into SCID mice together with HBD-2 (10 µg) i.p. After 15–18 h, peritoneal cells were harvested, stained, and analyzed by FACS. (B) Murine macrophages and DC were generated from bone marrow precursors in the presence of M-CSF and GM-CSF, respectively. In vitro chemotaxis of macrophages (filled squares) versus DC (open squares) was measured in response to HBD-2 and murine CCL20. For in vivo studies, macrophages and DC were labeled with PKH26 and injected i.v. into syngeneic BALB/c mice together with HBD-2 (10 µg for macrophages and 20 µg for DC) or murine CCL20 (10 µg) i.p. To investigate receptor desensitization, cells were preincubated as indicated. After 15–18 h, peritoneal cells were harvested and labeled migratory cells identified by FACS analysis. (C) Migration of J774A.1 macrophages in response to HBD-2 and mBD-8 was measured in vitro. (A–C) Mean values (± SEM) of three to four independent experiments each are shown.

Involvement of CCR6

The differential migration of DC and macrophages in response to CCL20 and β-defensins argued against CCR6 being a defensin receptor which is in contrast to previously published data 12, 1618. Therefore, we investigated two cell lines stably expressing human CCR6 which were derived from RBL-2H3 (Fig. 3) and 300.19 cells (data not shown), respectively, by stable transfection. None of these cells migrated towards gradients of HBD-2 and HBD-3 despite a high CCR6 expression level (Fig. 3A) and a rigorous response to CCL20 (Fig. 3B, D). Furthermore, neither HBD-2 nor HBD-3 were able to desensitize CCL20-induced migration (Fig. 3C) or calcium fluxes (Fig. 3E) in CCR6 transfectants. Thus, our data indicate that CCR6 is not a functional receptor for β-defensins.

Figure 3.

RBL-2H3-CCR6 transfectants do not respond to β-defensins. (A) Expression of human CCR6 on RBL-2H3 transfectants was measured by indirect FACS analysis. Cells were incubated with (solid line, empty histogram) or without (filled histogram, no line) anti-human CCR6 mAb followed by a FITC-conjugated secondary Ab. (B) In vitro chemotaxis of RBL-2H3 cells stably expressing human CCR6 was measured in response to CCL20, HBD-2 and HBD-3. Mean values (± SEM) of three independent experiments are shown. (C) To investigate receptor desensitization, cells were preincubated as indicated before in vitro migration against human CCL20 (100 ng/mL) or buffer (∅) was measured. Mean values (± SEM) of three independent experiments are shown. (D, E) RBL-2H3-CCR6 transfectants were used to measure calcium fluxes. Prior to the CCL20 stimuli (100 ng/mL), cells were exposed to either CCL20 (100 ng/mL) or HBD-2 (10 µg/mL) to investigate receptor desensitization. Equal cellular loading with Fluo3/pluronic F127 was controlled by treatment of the cells with 100 nM ionomycin. One representative out of three experiments is shown.

Mobilization of T lymphocytes

HBD-2 has been shown to mobilize memory but not naive T lymphocytes via CCR6 16, 23. According to our results, however, neither memory nor naive human T cells responded to HBD-2, HBD-3 or mBD-8 with chemotaxis (Fig. 4A and data not shown). Furthermore, β-defensins lacked the capacity to desensitize CCL20-induced migration of memory T cells (Fig. 4B). These results confirm that human β-defensins do not signal through CCR6.

Figure 4.

β-Defensins fail to induce migration of memory T lymphocytes. (A) Human CD4+CD45RO+ memory T cells were used to measure in vitro chemotaxis in response to HBD-2, HBD-3, and human CCL20. (B) To investigate receptor desensitization, human memory T cells were preincubated with buffer (∅) or different β-defensins or CCL20. Subsequently, in vitro migration in response to CCL20 (100 ng/mL) or buffer (∅) was measured. (C) In vitro chemotaxis of murine memory T lymphocytes in response to mBD-8 and murine CCL20 was investigated. (A–C) Mean values (± SEM) of three independent experiments each are shown.

We also investigated chemotaxis of murine naive (data not shown) and memory T lymphocytes in response to mBD-8 and HBD-2. Similar to their human counterparts, murine lymphocytes were not recruited by HBD (data not shown) or mBD, despite their vigorous chemotactic response to CCL20 (Fig. 4C).

Mobilization of mast cells

HBD-2 to -4 have been shown to induce in vitro chemoattraction of rat peritoneal mast cells whereas HBD-1 completely lacked this activity 21, 22. Investigating chemotaxis of murine mast cells generated from bone marrow precursors in the presence of SCF and IL-3, we observed that HBD-1 as well as HBD-2 to -4 are effective chemotaxins in vitro (Fig. 5A). Activation of murine mast cells by antigen-independent mechanisms (ionomycin) or by IgE plus Ag enhanced migration in response to β-defensins (Fig. 5B, C). Studying chemotaxis in vivo, the distinction between β-defensin-induced mobilization of resting and activated mast cells was even more pronounced. PKH26-labeled mast cells were recruited into the peritoneal cavity by HBD-2 exclusively after stimulation with ionomycin or IgE plus Ag (Fig. 5D). In vivo migration was also subject to homologous desensitization since preincubation of murine mast cells with HBD-2 abrogated its subsequent chemotactic effects (Fig. 5D).

Figure 5.

Murine and human mast cells migrate in response to β-defensins. (A) Murine mast cells derived from bone marrow precursor cells were stimulated with ionomycin for 24 h. Subsequently, in vitro migration was investigated in response to human β-defensins (1000 ng/mL each) or buffer (∅). (B) Murine mast cells were cultured for 24 h in the presence (filled symbols) or absence (open symbols) of ionomycin. Subsequently, in vitro migration was measured. (C) Murine mast cells were cultured for 4 h in the absence (∅) or presence of ionomycin or in the presence of albumin-DTT after a 24-h period of IgE loading (IgE/Ag). Subsequently, in vitro migration was investigated in response to HBD-2 (1000 ng/mL) or buffer (∅). (D) Murine mast cells were cultured for 4 h in the absence (∅) or presence of ionomycin or in the presence of albumin-DTT after a 24-h period of IgE loading (IgE/Ag). Subsequently, cells were labeled with PKH26 and preincubated as indicated. Thereafter, cells were injected i.v. into syngeneic BALB/c mice together with HBD-2 (10 µg) i.p. After 15–18 h, peritoneal cells were harvested and labeled migratory cells identified by FACS analysis. (E) In vitro migration of HMC-1 cells was measured in response to human β-defensins. (F) To investigate receptor desensitization, HMC-1 cells were preincubated in the absence (open bars) or presence (filled bars) of PKC inhibitor GF109 and, subsequently, with different β-defensins, PMA or buffer (∅). Thereafter, migration was measured in response to HBD-2 (1000 ng/mL) or buffer. (A–F) Mean values (± SEM) of three independent experiments each are shown.

The cell line HMC-1 is widely used as a model for immature human mast cells. Similar to murine mast cells, HMC-1 migrated in vitro in response to all four human β-defensins tested (Fig. 5E). In analogy to human macrophages, HBD-1 to -4 desensitized each other's chemotactic impact on human mast cells (Fig. 5F and data not shown). Likewise, the general PKC inhibitor GF109 was unable to block desensitization between members of the β-defensin family whereas PMA-induced heterologous desensitization was completely abrogated (Fig. 5F). Thus, human β-defensins appear to signal through a common receptor also in mast cells resulting in PKC-independent homologous receptor desensitization.

Induction of calcium fluxes in immune cells

We were unable to detect β-defensin-induced calcium fluxes in human monocyte-derived macrophages or murine macrophages generated from precursor cells in the presence of M-CSF despite the induction of chemotaxis (data not shown). In contrast, the human mast cell line HMC-1 vigorously responded to HBD-1 to -4 with intracellular calcium fluxes (Fig. 6A–D). Furthermore, complete desensitization occurred between the four HBD (Fig. 6 and data not shown). In contrast, the anaphylatoxin C5a that is a potent inducer of intracellular calcium fluxes failed to desensitize the activity of HBD-2 (Fig. 6E).

Figure 6.

β-Defensin-induced calcium fluxes in HMC-1 are subject to homologous desensitization. Upon loading with Fluo3/pluronic F127, HMC-1 cells were monitored for changes of calcium-dependent fluorescence in response to HBD-2 (10 µg/mL). Prior to the HBD-2 stimuli, cells were exposed to HBD-1 (A), HBD-2 (B), HBD-3 (C), HBD-4 (D) (all at 10 µg/mL) or C5a (E) (100 ng/mL) to investigate receptor desensitization. Equal cellular loading with the calcium-sensitive dye was controlled by treatment of the cells with 100 nM ionomycin. One representative out of three independent experiments is shown.

Involvement of MAPK and Gαi

To determine the roles of MAPK in defensin-induced signaling pathways, we used a pharmacological approach. Human macrophages (Fig. 7A) and murine mast cells (Fig. 7B) were pretreated with PD098059, SP600125 or SB203580 that inhibit ERK, JNK and p38 phosphorylation, respectively. We found that all three MAPK contributed to β-defensin-induced chemotaxis.

Figure 7.

MAPK and Gαi contribute to β-defensin-induced chemotaxis. Human macrophages (A) and murine mast cells stimulated with ionomycin for 24 h (B) were preincubated in the absence (∅) or presence of inhibitors for MAPK p38 SB203580, JNK SP600125, ERK PD098059, or pertussis toxin. Subsequently, in vitro chemotaxis was measured in response to HBD-2 (1000 ng/mL). As controls, C5a, CCL3 (100 ng/mL each) or buffer (∅) were used. Mean values (± SEM) of three independent experiments each are shown.

Preincubation of macrophages with pertussis toxin abrogated migration towards HBD-2 (Fig. 7A), indicating that Gαi proteins are instrumental in β-defensin-induced signal transduction.

Discussion

One of the main functions of defensins is the killing or inactivation of microorganisms 10. This capacity renders defensins important players of the innate leg of immunity. Recent evidence also suggests that defensins may be involved in adaptive immunity by attracting immunocompetent cells to the sites of infection and inflammation 23.

HBD-3 and -4 have been demonstrated in the past to chemoattract human monocytes 19, 20 whereas HBD-2 was found to lack this activity 16. Herein, we provide evidence that recruitment of human macrophages is a function common to all four human β-defensins tested as well as to mBD-8. β-Defensin-induced migration of macrophages was confirmed using the murine macrophage cell line J774A.1 or murine macrophages differentiated from bone marrow precursors in the presence of M-CSF. Collectively, mobilization of macrophages represents a common, evolutionary conserved property of the β-defensin family.

Rat peritoneal mast cells have been reported to be chemoattracted by HBD-2 to -4 but not HBD-1 21, 22. This result contrasted with our finding that HBD-1 was a potent chemotaxin for macrophages. Therefore, we reinvestigated this issue. The cell line HMC-1, which is widely used as a surrogate for human mast cells, responded with chemotaxis to gradients of all HBD tested including HBD-1. Murine mast cells differentiated from bone marrow precursors in the presence of IL-3 and SCF also migrated in response to HBD-1 to -4. Intriguingly, murine mast cells increased their chemotactic responsiveness to β-defensins following Ag-independent (by ionomycin) as well as Ag-specific, IgE-mediated stimulation. Thus, our results add another clue to a potential role of defensins in the pathophysiology of allergic reactions 2830.

DC have been demonstrated to migrate in response to gradients of β-defensins 2 and 3 via CCR6 12, 16. According to our results, however, human and murine DC, generated from monocytes and bone marrow precursor cells, respectively, were only marginally mobilized by HBD in vitro and no migration was observed using an in vivo chemotaxis assay. Therefore, we conclude that DC may not represent primary target cells for β-defensin activity. In keeping with this reasoning, IL-4, which is commonly used for the generation of human monocyte-derived DC, was identified as a suppressor of β-defensin-induced chemotaxis. This finding is reminiscent of the inhibitory effect of IL-4 on anaphylatoxin-induced DC migration as a consequence of receptor down-regulation 27.

Our observation that CCL20, the only known chemokine ligand of CCR6, potently chemoattracted murine DC in vitro and in vivo whereas β-defensins preferentially targeted macrophages necessitated investigation of the interaction between β-defensins and CCR6. Two independent cell lines stably expressing CCR6 upon transfection proved to be unresponsive to HBD-2 and -3 despite vigorous responses to CCL20. CCR6 belongs to the large family of G protein-coupled receptors that have been shown to undergo desensitization upon ligand stimulation 31. In contrast to published data using CCR6-expressing HEK293 transfectants 16, we found no evidence for a desensitizing impact of HBD on CCR6 in RBL-2H3 and 300.19 transfectants. These results were confirmed by our investigation of human memory T lymphocytes that endogenously express CCR6 32. HBD-2 and -3 were neither able to induce chemotaxis nor to desensitize the chemotactic impact of CCL20. Furthermore, murine lymphocytes were unresponsive to mBD-8 as well as to HBD-2. Collectively, these data indicate that CCR6 is not a functional receptor for β-defensins.

The responsiveness of macrophages and mast cells to all tested human β-defensins suggested involvement of a common receptor. To find further support for this hypothesis, we investigated receptor desensitization, which is a regulatory mechanism to prevent the damaging effects of prolonged or excessive activation of G protein-coupled receptors 26. We found β-defensin receptors to be coupled to Gαi proteins due to their sensitivity to pertussis toxin, which confirms previously published observations 16, 21. Heterologous desensitization involving different G protein-coupled receptors is mediated by second messenger-regulated kinases such as PKC and does not require agonist occupancy 25. On the other hand, homologous desensitization affects receptors in the agonist-occupied state and involves phosphorylation by G protein-coupled receptor kinases 26. We found that desensitization between human β-defensins occurred without participation of PKC, indicating that a single receptor is activated by HBD-1 to -4. This receptor for β-defensins was also subject to heterologous desensitization since the PKC activator PMA abrogated β-defensin-induced chemotaxis.

β-Defensin peptide sequences exhibit a large degree of sequence variability, however, the six cysteines are retained. This could imply that disulfide bonding is essential to the functional structure of the molecules. However, in the case of HBD-3, disulfide bonding was relevant only for its chemotactic but not its antimicrobial activities 17, 33. In keeping with these studies, we observed that a linear form of HBD-3 had lost its ability to chemoattract macrophages. Thus, conservation of the three disulfide bonds in β-defensins during evolution is essential for engaging their common chemotactic receptor on immune cells, presumably by determining the tertiary structures which are strikingly similar between different β-defensins 4.

G protein-coupled receptors are known to be connected to the MAPK signaling pathways by classical G protein-stimulated synthesis of second messengers 34. Mammalian cells contain three major classes of MAPK: ERK, JNK, and p38. All three kinases have been shown to play crucial roles in cell migration by distinct mechanisms 35. Our results confirm the importance of EKR, JNK and p38 MAPK also for β-defensin-induced chemotaxis of macrophages and mast cells. In line with these data, p38 and ERK have been observed to participate in the degranulation of mast cells by HBD-3 and -4 22.

In summary, β-defensins are characterized by evolutionary conserved receptor usage in macrophages and mast cells. The existence of a common receptor for β-defensins on immune cells strengthens the view that immunomodulation may represent a principal function of the defensin system.

Materials and methods

Reagents

Pertussis toxin, ERK inhibitor PD098059, JNK inhibitor SP600125, p38 inhibitor SB203580, the PKC inhibitor GF109, and PMA were all from Calbiochem (Merck Biosciences, Darmstadt, Germany). Recombinant (human and/or murine) CCL3, CCL19 and CCL20 were obtained from PeproTech (Cell Concepts, Umkirch, Germany). Recombinant human anaphylatoxin C5a was described elsewhere 27.

Synthetic HBD-1, HBD-2, HBD-3 and HBD-4 have been described elsewhere 19, 20, 36. In addition, a linear form of HBD-3 was synthesized by alkylation of reduced HBD-3 with iodoacetamide 33. In this peptide, all cysteine residues are present as S-carboxamidomethylcysteine to suppress the reactivity of the thiol groups. Commercial sources for defensins were: HBD-2 from PeproTech (Cell Concepts), Bachem (Weil am Rhein, Germany) and The Peptide Institute (PeptaNova, Sandhausen, Germany); HBD-3 and -4 from The Peptide Institute; HBD-1 from PeproTech and The Peptide Institute; mBD-8 from Phoenix Pharmaceuticals (Phoenix Europe, Karlsruhe, Germany). Disulfide pairings of cysteines of all β-defensins (except from PeproTech) were indicated as Cys1-Cys5, Cys2-Cys4, Cys3-Cys6.

Transfected cell lines

Transfected murine pre-B 300.19 cells stably expressing human CCR6 have been described previously 37. RBL-2H3 cells were transfected with the human CCR6 cDNA containing expression vector (clone I.D. CCR0600000) from UMR cDNA Resource Center (University of Missouri-Rolla, USA) by electroporation and selected in the presence of G418 (600 µg/mL) for stable expression of human CCR6. CCR6 expression on 300.19 and RBL-2H3 transfectants was verified by FACS analysis as described 27 using an anti-human CCR6 mAb (R&D Systems, Wiesbaden-Nordenstadt, Germany) and FITC-conjugated goat anti-mouse IgG (Dako, Hamburg, Germany).

Human macrophages and dendritic cells

Leukocytes were obtained by leukapheresis from volunteer blood donors in the Department of Transfusion Medicine, University Clinic Göttingen. PBMC were isolated as described 27 and cultured for 1 h at 1 × 107 cells/mL in endotoxin-free RPMI-1640 (Biochrom, Berlin, Germany) supplemented with 5% heat-inactivated autologous serum in flat-bottom plates. After washing off nonadherent cells, adherent mononuclear cells (>90% CD14+ monocytes) were used to generate macrophages or DC. DC were cultured in RPMI-1640 supplemented with 10% FCS (PAN Biotech, Aidenbach, Germany), 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, GM-CSF (300 U/mL) and IL-4 (300 U/mL) (both from R&D Systems). After 7 days, cultured DC expressed high levels of HLA-DR but no CD14 and were characterized as immature due to their moderate expression of CD86 and low expression of CD83 27.

To obtain macrophages, adherent PBMC were cultured in RPMI-1640 supplemented with 5% FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and GM-CSF (100 U/mL). After 1–3 days, cultured macrophages expressed low to moderate levels of HLA-DR but high levels of CD14.

Human lymphocytes

Human peripheral blood CD3+, CD4+/CD45RA+ naive or CD3+, CD4+/CD45RO+ memory T cells were purified from PBMC (see above) by magnetic cell sorting using a negative selection technique according to the manufacturer's recommendations (Miltenyi Biotech, Bergisch-Gladbach, Germany). The purity of T cell subsets was verified by FACS analysis and was always greater than 95%.

Murine lymphocytes

Murine CD4+ T lymphocytes were purified from spleen cell suspensions (BALB/c) by magnetic cell sorting using a negative selection technique according to the manufacturer's recommendations (Miltenyi Biotech). CD4+ naive T cells were further purified by positive selection based on the expression of CD62L (Miltenyi Biotech). CD4+ memory T cells were obtained by depletion of CD62L cells (Miltenyi Biotech). The purity of T cell subsets was verified by FACS analysis and was always greater than 93% for naive and 90% for memory T cells.

Murine bone marrow-derived dendritic cells, macrophages and mast cells

Generation of murine immature DC derived from bone marrow precursor cells has been described previously 27. Briefly, nonadherent bone marrow cells were cultured in RPMI 1640 containing 5% FCS, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-ME, together with 100 U/mL murine GM-CSF in the presence or absence of 100 U/mL murine IL-4 (both from PeproTech) for a total of 6–7 days.

Murine bone marrow-derived macrophages were generated by culturing nonadherent bone marrow cells from BALB/c mice in RPMI 1640 containing 5% FCS, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-ME and murine M-CSF (20 ng/mL; PeproTech) for 6–7 days. Adherent cells were more than 95% macrophages as judged by morphology and homogenous surface expression of C5a and C3a receptors and the absence of CD11c.

Murine bone marrow-derived mast cells were generated by cultivation of nonadherent bone marrow cells from BALB/c mice in RPMI 1640 containing 10% FCS, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-ME, together with murine IL-3 and murine SCF (10 ng/mL each; PeproTech). After 5–10 weeks, more than 95% of nonadherent cells were mast cells as judged by morphology and surface expression of CD117. Mast cells were activated either with ionomycin (750 ng/mL; Sigma-Aldrich, Deisenhofen, Germany) for 4 or 24 h or, in an antigen-specific manner, by preincubation with monoclonal murine IgE anti-DNP (2 µg/mL; Sigma-Aldrich) overnight followed by albumin-DNP (50 µg/mL; Sigma-Aldrich) for 4 h.

In vitro chemotaxis

In vitro chemotaxis was assayed using the HTS Transwell-24 system from Corning (Beyer Lab., Düsseldorf, Germany). Cells diluted at 1 × 106/mL in migration buffer (RPMI 1640 with 1% BSA) were placed in the upper wells whereas the various chemoattractants diluted in migration buffer as indicated were added to the lower wells. For chemotaxis of human and murine macrophage and DC as well as RBL-2H3 transfectants, polycarbonate membranes with a pore size of 8 µm were used and incubation was performed at 37°C in a 5% CO2 atmosphere for 90 min. If human or murine lymphocytes were used, pore size was 5 µm and incubation time 4 h; HMC-1 cells: 8 µm and 3 h; murine mast cells: 5 µm and 3 h. Migration was stopped and migrated cells detached by placing Transwell chambers for 15 min on ice. Subsequently, migrated cells in the lower chambers were counted using a hemocytometer. All determinations were performed in duplicate.

To investigate the role of PKC in receptor desensitization, cells (1 × 106 in 1 mL medium) were preincubated in the presence or absence of the PKC inhibitor GF109 (4 µM) at 37°C for 30 min and centrifuged.

For desensitization studies, cells (1 × 106 in 1 mL medium) were preincubated at 37°C for 30 min with selected chemotaxins (200 ng/mL) or PMA (2 µM) and centrifuged.

To study the role of Gαi in signal transduction, human monocyte-derived macrophages were incubated overnight in the presence of pertussis toxin (100 ng/mL) (Calbiochem) before their use in chemotaxis experiments.

To study the role of MAPK in signal transduction, cells were preincubated at 37°C for 30 min with PD098059, SP600125 or SB203580 (20 µM each) and centrifuged.

Migration of human cells in a SCID mouse model

All animal work was conducted in accordance with guidelines for animal welfare and was approved by the government of Lower Saxony, Germany. In vivo migration studies were performed as described elsewhere 27. SCID mice (strain CB-17 SCID of both sexes; weight 19–24 g) were obtained from Charles River (Sulzfeld, Germany). Cells (1 × 107 in 200 µL PBS) were injected into the tail vein of SCID mice together with a chemotaxin (10 or 20 µg in 200 µL PBS), which was injected into the peritoneal cavity. After 4 or 15–18 h, mice were sacrificed and peritoneal cells harvested by lavage. Subsequently, cells were stained with anti-HLA-DR-PE in combination with FITC-anti-CD14 or FITC-anti-CD86 to identify migrated human macrophages and DC, respectively. Absolute numbers of migrated human cells were calculated from the percentage of HLA-DR+ cells as determined by FACS analysis and the total peritoneal cell count.

For desensitization studies, human cells (1 × 107 in 1 mL PBS) were preincubated at 37°C for 1 h with selected chemotaxins (2 µg), washed twice in PBS and resuspended in 200 µL PBS for injection.

Migration of murine bone marrow-derived cells in vivo

In vivo migration studies were performed as described elsewhere 27. Murine bone marrow-derived cells were labeled with the red fluorescent dye PKH-26 (Sigma-Aldrich) according to the manufacturer's instructions. Cells (1 × 107 in 200 µL PBS) were then injected into the tail vein of BALB/c mice (weight 20–24 g; age 8–20 weeks) together with a chemotaxin (10 or 20 µg in 200 µL PBS, as indicated), which was injected into the peritoneal cavity. Approximately 15–18 h later, mice were sacrificed and peritoneal lavage performed. Subsequently, peritoneal cells were counted and analyzed by FACS. Absolute numbers of migrated labeled cells were calculated from the percentage of red fluorescent cells as determined by FACS analysis and the total peritoneal cell count.

For desensitization studies, labeled cells (1 × 107 in 1 mL PBS) were preincubated at 37°C for 1 h with selected chemotaxins (2 µg), washed twice in PBS and resuspended in 200 µL PBS for injection.

Calcium measurements

A total of 106 cells were loaded in 700 µL RPMI containing 5% FCS, 1 µM Fluo3-AM and 0.02% Pluronic F127 (both from Molecular Probes, Invitrogen, Karlsruhe, Germany). Subsequently, the cell suspension was diluted twofold with RPMI 10% FCS and was incubated for 10 min at 37°C. Cells were washed twice with Krebs Ringer solution composed of 10 mM HEPES (pH 7.0), 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM glucose. The changes in fluorescence intensity of Fluo3 were monitored on a LSRII cytometer (Becton Dickinson). Equal loading of the samples was controlled by treatment with 100 nM ionomycin (Sigma-Aldrich).

Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SO 478/1). Transfected pre-B 300.19 cells stably expressing human CCR6 were obtained from B. Moser (Theodor Kocher Institut, University of Berne, Switzerland). The authors would like to thank Olga Walter and Kerstin Eckelmann for excellent technical assistance.

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