Downregulation of ten-eleven translocation-2 triggers epithelial differentiation during organogenesis.

DNA methylation of cytosine bases is a major epigenetic modification that regulates gene expression and vertebrate development. The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and active DNA demethylation influences gene expression specific to each developmental stage, although recent reports have shown that TET also has a non-catalytic function. In fetal mice, the epithelium in the submandibular gland (SMG) buds as a derivative of the oral cavity at embryonic day 11 (E11) and, by E15, it begins to differentiate into the salivary epithelium, which expresses water-channel aquaporin 5 (AQP5). The functional differentiation of the SMG epithelium can be regulated epigenetically, but how TET enzymes contribute is largely unknown. Here, we used several techniques, including hydroxymethylated DNA immunoprecipitation qPCR and histological analysis, to examine the changes in 5hmC levels and AQP5 and TET expression during SMG development. We found that 5hmC levels and AQP5 expression increased in the E15 SMG epithelium, while TET2 expression in the terminal buds decreased at E15. In agreement with the in vivo observations, Tet2 inhibition ex vivo led to the upregulation of AQP5 expression in terminal buds of the SMG epithelium. These results suggest that the downregulation of TET2 expression at E15 is a critical epigenetic event that establishes the epithelial fate for functional SMGs during development.

Soluble Lutheran/basal cell adhesion molecule is detectable in plasma of hepatocellular carcinoma patients and modulates cellular interaction with laminin-511 in vitro.

Lutheran (Lu), an immunoglobulin superfamily transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a subunit of laminin-511 (LM-511) that is a major component of basement membranes in various tissues. Our previous study showed that Lu/B-CAM was cleaved by MT1-MMP and released from cell surfaces. In this study we examined the soluble Lu/B-CAM in culture media and in plasma of mice bearing HuH-7 hepatocellular carcinoma (HCC) cells and patients with HCC. Two HCC cell lines, HepG2 and HuH-7, released Lu/B-CAM into the culture media. Although Lu/B-CAM was cleaved by MT1-MMP in HuH-7 cells, HepG2 cells released Lu/B-CAM in a MMP-independent manner. The concentration of Lu/B-CAM released into mouse plasma correlated with tumor size. Moreover the soluble Lu/B-CAM in plasma of HCC patients was significantly decreased after resection of the tumor. Immunohistochemical studies showed that although the expression of Lu/B-CAM was observed in most HCCs, MT1-MMP was not always expressed in tumor tissues, suggesting that a part of Lu/B-CAM in plasma of HCC patients was also released in a MMP-independent manner. In vitro studies showed that the soluble Lu/B-CAM released from HCC cells bound to LM-511. Moreover the soluble Lu/B-CAM influenced cell migration on LM-511. These results suggest that soluble Lu/B-CAM serves as not only a novel marker for HCC but also a modulator in tumor progression.

Tunneling nanotube-driven complete regeneration of murine fetal skin.

This study investigated the three-dimensional (3D) cellular interactions and tunneling nanotubes (TNTs) during fetal mouse skin regeneration on embryonic days 13 (E13) and 15 (E15). We aimed to understand spatial relationships among cell types involved in skin regeneration and assess the potential role of TNTs. Full-thickness skin incisions were performed in E13 and E15 embryos. Wound sites were collected, embedded in epoxy resin, processed for 3D reconstruction (1 µm thickness sections), and subjected to whole-mount immunostaining. We conducted in vitro co-culture experiments with fetal macrophages and fibroblasts to observe TNT formation. To assess the effect of TNTs on skin regeneration, an inhibiting agent (cytochalasin B) was administered to amniotic fluid. Results revealed that E13 epidermal keratinocytes interacted with dermal fibroblasts and macrophages, facilitating skin regrowth. TNT structures were observed at the E13-cell wound sites, among macrophages, and between macrophages and fibroblasts, confirmed through in vitro co-culture experiments. In vitro and utero cytochalasin B administration hindered those formation and inefficient skin texture regeneration at E13 wound sites. This emphasizes the necessity of 3D cellular interactions between epidermal and dermal cells during skin regeneration in mouse embryos at E13. The prevalence of TNT structures indicated their involvement in achieving complete skin texture restoration.

COX-2 expression in stromal fibroblasts self-limits their numbers in lymph node inflammatory responses.

We previously reported the expression of cyclooxygenase (COX)-2 in draining lymph nodes during carrageenin-induced pleurisy of rats. Here, we analyzed histological and immunohistochemical characteristics of COX-2-expressing cells. After carrageenin administration into the pleural cavity of rats, parathymic lymph nodes were enlarged beginning at 8h and peaking from 24 to 48h. Lymphatic follicles disappeared 16h after injection, and numerous macrophages and fibroblasts were observed in the cortical region. COX-2-expressing cells in the cortical region showed characteristic dendritic processes from 16 to 48h and primarily co-localized with stromal fibroblastic reticular cell markers, α-smooth muscle actin (α-SMA), and desmin. Expression of α-SMA increased following COX-2 expression. Nimesulide, a COX-2 inhibitor, increased the dendritic processes of COX-2-expressing cells as well as expression of both COX-2 and α-SMA. These results suggest that COX-2-expressing cells may be stromal fibroblastic cells, which negatively self-regulate their proliferation and modulate tissue remodeling of draining lymph nodes at inflammatory sites.

Dynamics, structure and assembly of the basement membrane in developing salivary glands revealed by an exogenous EGFP-tagged nidogen probe.

Most epithelial tissues rapidly become complex during embryonic development while being surrounded by the basement membrane (BM). Thus, the BM shape is thought to change dramatically as the epithelium grows, but the underlying mechanism is not yet clear. Nidogen-1 is ubiquitous in the BM and binds to various other BM components, including laminin and type IV collagen. To elucidate the behavior of the BM during epithelial morphogenesis, we attempted to live-label the developing BM with recombinant human nidogen-1 fused to an enhanced green fluorescent protein (hNid1-EGFP). Submandibular glands of mouse embryos were cultured in glass-bottomed dishes and incubated in media containing hNid1-EGFP. Subsequent confocal microscopy clearly visualized the BMs surrounding the epithelial end buds. On three-dimensional reconstruction from Z-series confocal sections, the epithelial BM was observed as a thin sheet that expanded continuously around the entire epithelial basal surface. Because the explants continued to grow well in the presence of hNid1-EGFP, time-lapse confocal microscopy was performed to follow the dynamics of the BM. We found that the epithelial BM is an adaptive structure that deforms in accordance with the rapid shape changes of the developing epithelium. Furthermore, hNid1-EGFP was found to be incorporated differently into the epithelial BM compared with that reported for fibronectin or type IV collagen, suggesting that individual BM components assemble in different ways to form the BM.

Cellular dynamics of epithelial clefting during branching morphogenesis of the mouse submandibular gland.

We cultured the rudimental submandibular gland (SMG) of mice with a non-cell-permeable fluorescent tracer, and observed cell behavior during epithelial branching morphogenesis using confocal time-lapse microscopy. We traced movements of individual cells as shadowgraph movies. Individual epithelial cells migrated dynamically but erratically. The epithelial cleft extended by wiggling and separated a cluster of cells into two buds during branching. We examined the ultrastructure of the clefts in SMG rudiments treated with the laminin peptide A5G77f, which induces epithelial clefting. A short cytoplasmic shelf with a core of microfilaments was found at the deep end of the cleft. We propose that epithelial clefting involves a dynamic movement of cells at the base of the cleft, and the formation of a shelf within a cleft cell. The shelf might form a matrix attachment point at the base of the cleft with a core of microfilaments driving cleft elongation.

Amino acid sequence requirements of laminin beta1 chain peptide B133 (DISTKYFQMSLE) for amyloid-like fibril formation, syndecan binding, and neurite outgrowth promotion.

Peptide B133 (DSITKYFQMSLE), derived from mouse laminin beta1 chain (residues 1298-1309), promotes cell attachment, neurite outgrowth, and amyloid-like fibril formation. Previously, we showed that the N-terminal Asp-deleted peptide B133a (SITKYFQMSLE) promotes integrin alpha2beta1-mediated cell attachment and spreading but does not form amyloid-like fibrils, and that the C-terminal Glu-deleted peptide B133g (DSITKYFQMSL) attaches cells without cell spreading and forms amyloid-like fibrils. In this study, we further investigated the amino acid sequence requirements of B133 for biological function using a set of truncated and Ala-substituted peptides. Attachment of cells to B133g was inhibited by only heparin, and Congo Red analysis indicated that the amyloid-like fibril formation activity of B133g was stronger than that of B133. Alanine scan analysis for the B133g peptide indicated that Asp and Ile residues are essential for cell attachment. Additionally, the N-terminal Asp residue was required for neurite outgrowth. Further, amyloid-like fibril formation required Asp and Ile residues. These data suggest that the amyloid-like fibril formation of B133g is required for cell attachment activity. We also evaluated the attachment of cells to the peptides using syndecan- and glypican-overexpressing cells. B133g attached to syndecan-overexpressing cells but not to glypican-overexpressing cells, suggesting that the amyloidogenic peptides promote syndecan-mediated cell attachment. These findings were useful for clarifying the mechanism of amyloid-like fibril formation and biological functions. The B133 peptide promotes amyloid-like fibril formation, syndecan-mediated cell attachment, and neurite outgrowth and has the potential for use as a biomaterial for tissue engineering.

B133 (DSITKYFQMSLE), a laminin beta1-derived peptide, contains distinct core sequences for both integrin alpha2beta1-mediated cell adhesion and amyloid-like fibril formation.

The B133 peptide (DSITKYFQMSLE, mouse laminin beta1 chain 1319-1330) promotes cell attachment, and forms amyloid-like fibrils. Here, we evaluated the active core sequences using B133 deletion peptides. B133a, lacking the N-terminal Asp residue, promoted cell spreading via integrin alpha2beta1, whereas B133g, lacking the C-terminal Glu residue, lost the activity. Congo red analysis using the truncated peptides determined that B133g forms amyloid-like fibrils but B133a did not. These results suggest that the N- and C-terminal amino acids contribute to integrin alpha2beta1 binding and to fibril formation, respectively. Further analyses using the truncated peptides showed that the C-terminal eight residues (B133d: KYFQMSLE) are a minimum active sequence for integrin alpha2beta1-mediated cell attachment and the N-terminal nine residues (B133i: DSITKYFQM) are critical for amyloid-like fibril formation. These results suggest that peptide B133 is multifunctional with two different active core sequences: integrin alpha2beta1-mediated cell attachment and amyloid-like fibril formation. Moreover, alanine substitution analysis of B133a indicated that six amino acids, Ile, Thr, Tyr, Phe, Met, and Glu, are important for cell attachment activity. When the Ser residue at the 9th position of B133a was replaced with Ala, the cell attachment activity was enhanced. Further mutation analysis at the 9th position of B133a using various amino acids suggests that hydrophobic amino acids are effective for the integrin alpha2beta1-mediated cell attachment activity. These findings define multifunctional and overlapping sites on the B133 peptide and are useful for designing multifunctional synthetic molecules.

Histamine depolarizes rat intracardiac ganglion neurons through the activation of TRPC non-selective cation channels.

The cardiac plexus, which contains parasympathetic ganglia, plays an important role in regulating cardiac function. Histamine is known to excite intracardiac ganglion neurons, but the underlying mechanism is obscure. In the present study, therefore, the effect of histamine on rat intracardiac ganglion neurons was investigated using perforated patch-clamp recordings. Histamine depolarized acutely isolated neurons with a half-maximal effective concentration of 4.5 µM. This depolarization was markedly inhibited by the H1 receptor antagonist triprolidine and mimicked by the H1 receptor agonist 2-pyridylethylamine, thus implicating histamine H1 receptors. Consistently, reverse transcription-PCR (RT-PCR) and Western blot analyses confirmed H1 receptor expression in the intracardiac ganglia. Under voltage-clamp conditions, histamine evoked an inward current that was potentiated by extracellular Ca2+ removal and attenuated by extracellular Na+ replacement with N-methyl-D-glucamine. This implicated the involvement of non-selective cation channels, which given the link between H1 receptors and Gq/11-protein-phospholipase C signalling, were suspected to be transient receptor potential canonical (TRPC) channels. This was confirmed by the marked inhibition of the inward current through the pharmacological disruption of either Gq/11 signalling or intracellular Ca2+ release and by the application of the TRPC blockers Pyr3, Gd3+ and ML204. Consistently, RT-PCR analysis revealed the expression of several TRPC subtypes in the intracardiac ganglia. Whilst histamine was also separately found to inhibit the M-current, the histamine-induced depolarization was only significantly inhibited by the TRPC blockers Gd3+ and ML204, and not by the M-current blocker XE991. These results suggest that TRPC channels serve as the predominant mediator of neuronal excitation by histamine.

Biologically active sequences in the mouse laminin alpha3 chain G domain.

The laminin alpha3 chain is mainly expressed at the skin, and its C-terminal G domain has a critical role in multiple biological functions. We screened for biologically active sites on the mouse laminin alpha3 chain G domain using 107 synthetic peptides on coated plates and conjugated to Sepharose beads with HT1080 human fibrosarcoma cells, HaCaT human skin keratinocyte cells, and human dermal fibroblasts (HDFs). Eleven peptides exhibited cell attachment activity with respect to the peptide-coated plates and/or peptide-Sepharose beads. MA3G28 (WTIQTTVDRGLL) strongly binds to HaCaT cells. Four peptides promoted PC12 cell neurite outgrowth. Heparin inhibited attachment of HDFs to eight peptides on the coated plates. In contrast, EDTA significantly inhibited attachment of HDFs to MA3G27 (NAPFPKLSWTIQ) and MA3G28 but had no effect on the attachment of the other peptides. HDF cells formed well-organized actin stress fibers and focal contacts with vinculin accumulation on MA3G27. Additionally, attachment of HDFs to MA3G27 was inhibited by anti-alpha6 and anti-beta1 integrin antibodies, suggesting that MA3G27 promotes alpha6beta1 integrin-mediated cell adhesion. MA3G57 (NQRLASFSNAQQS) exhibited cell attachment activity only in the peptide bead assay. MA3G57 conjugated to a chitosan membrane promoted HDF attachment and spreading with well-organized actin stress fibers. The anti-beta1 integrin antibody partially inhibited attachment of HDFs to the MA3G57-chitosan membrane, suggesting that the MA3G57 site is involved in beta1 integrin-mediated cell attachment. These active sites are likely important in the biological activities of the laminin alpha3 chain G domain and would be useful for the study of molecular mechanisms of laminin-receptor interactions.

A novel cell-adhesive scaffold material for delivering keratinocytes reduces granulation tissue in dermal wounds.

Novel peptide-conjugated chitosan membranes were fabricated and used to deliver keratinocytes to dermal wounds in mice. Three active peptides of 12 or 13 amino acids each, RLVSYNGIIFFLK (A5G27), ASKAIQVFLLAG (A5G33), and AGTFALRGDNPQG (A99) were selected from a cell-adhesive peptide library of laminin, a major constituent of basement membrane. The peptides were synthesized and coupled to chitosan membranes, and the resulting peptide-chitosan membranes were tested for keratinocyte attachment. Two of the peptides that bind to cell surface heparin-like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Subsequently, A5G27- and A5G33-chitosan membranes were tested as vehicles for keratinocyte delivery in a wound model. We found that keratinocytes were delivered into the full-thickness wound with either membrane. Using the A5G33-chitosan membrane, we further evaluated the activity of the delivered keratinocytes in wound healing. Immunohistochemistry for granulation tissue markers, including tenascin and alpha-smooth muscle actin, showed that keratinocyte delivery by the present peptide-chitosan membranes in the wound bed provided a favorable condition for keratinocyte migration along the wound surface and reduced granulation tissue formation.

Excitatory effect of bradykinin on intrinsic neurons of the rat heart.

The heart receives sympathetic and parasympathetic innervation through the intrinsic cardiac nervous system. Although bradykinin (BK) has negative inotropic and chronotropic properties of cardiac contraction, the direct effect of BK on the intrinsic neural network of the heart is still unclear. In the present study, the effect of BK on the intracardiac ganglion neurons isolated from rats was investigated using the perforated patch-clamp technique. Under current-clamp conditions, application of 0.1 µM BK depolarized the membrane, accompanied by repetitive firing of action potentials. When BK was applied repeatedly, the second responses were considerably less intense than the first application. The BK action was fully inhibited by the B2 receptor antagonist Hoe-140, but not by the B1 receptor antagonist des-Arg9-[Leu8]-BK. The BK response was mimicked by the B2 agonist [Hyp3]-BK. The BK-induced depolarization was inhibited by the phospholipase C inhibitor U-73122. BK evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca2+ markedly increased the BK-induced currents, suggesting an involvement of Ca2+-permeable non-selective cation channels. The muscarinic agonist oxotremorine-M (OxoM) also elicited the extracellular Ca2+-sensitive cationic currents. The OxoM response did not exhibit rundown with repeated agonist application. The amplitude of current evoked by 1 µM OxoM was comparable to that induced by 0.1 µM BK. Co-application of 0.1 µM BK and 1 µM OxoM elicited the current whose peak amplitude was almost the same as that elicited by OxoM alone, suggesting that BK and OxoM activate same cation channels. BK also reduced the amplitude of M-current, while the M-current inhibitor XE-991 affected neither resting membrane potential nor the BK-induced depolarization. From these results, we suggest that BK regulates excitability of intrinsic cardiac neurons by both an activation of non-selective cation channels and an inhibition of M-type K+ channels through B2 receptors.

Laminin peptide-conjugated chitosan membrane: Application for keratinocyte delivery in wounded skin.

Tissue engineering requires the delivery and survival of cells to organ sites needing repair. Previously, we showed that an active laminin peptide (AG73: RKR-LQVQLSIRT)-conjugated chitosan membrane promoted cell adhesion and spreading in vitro. Here, we seeded human keratinocytes onto AG73-chitosan membranes and found that nearly 80% of the cells were attached to the membranes within 2 h. The membranes carrying the keratinocytes were inverted and placed onto exposed muscle fascia on the backs of nude mice. After 3 days, the keratinocytes had migrated from the membrane and established a stratified epidermis-like structure on the fascia. Cells recognize the AG73 through transmembrane proteoglycan syndecans, which recognition system has not previously been tested in tissue engineering applications. We suggest that the AG73-chitosan membrane is useful as a therapeutic formulation and is applicable as a cell delivery system such as delivering keratinocytes to a wound bed.

Laminin-1 peptide-conjugated chitosan membranes as a novel approach for cell engineering.

Laminin, a major component of the basement membrane, has diverse biological activities. Recently, we identified various biologically active sequences on laminin-1 by using a large set of synthetic peptides. Chitosan, a polysaccharide, is biodegradable and has been used as a biomaterial. Here, we conjugated several biologically active laminin peptides onto chitosan membranes and measured the cell attachment activity of peptide-conjugated chitosan membranes with various cell types. The active laminin peptide-conjugated chitosan membranes promoted cell attachment with cell type specificity. A99 (AGTFALRGDNPQG)-chitosan membrane promoted cell attachment with well-organized actin stress fibers. This adhesion was inhibited by EDTA but not by heparin. AG73 (RKRLQVQLSIRT)-chitosan membrane promoted cell attachment with filopodia formation, and this adhesion was inhibited by heparin but not by EDTA. These data suggest that the A99-chitosan membrane interacted with an integrin cellular receptor and that the AG73-chitosan membrane promoted proteoglycan-mediated cell attachment, as previously reported. Furthermore, both AG73-chitosan and A99-chitosan membranes effectively promoted neurite outgrowth with PC12 rat pheochromocytoma cells. We conclude that conjugation on a chitosan membrane is applicable for testing quantitatively the biological activity of synthetic peptides and that these constructs have a potential ability to serve as bioadhesive materials for tissue regeneration and engineering.

Salivary gland morphogenesis and basement membranes.

The basement membrane separates the epithelium from the surrounding mesenchyme and plays an essential role in the development of various epithelial-mesenchymal organs. Among these, the submandibular salivary gland (SMG) has been chosen to review the expression patterns and roles of the epithelial basement membrane and its components, in particular the laminins, during SMG morphogenesis. At the outset, a brief description of SMG development is provided with special reference to changes in the epithelial architecture and the epithelial basement membrane. The restricted expression patterns of various laminin isoforms in the developing SMGs are also summarized. Furthermore, an overview is given of several lines of experimental evidence that indicate significant but distinct roles for laminin-1 and laminin-10, their individual domains and their receptor-mediated signaling in SMG morphogenesis.

Inhibition of hair follicle growth by a laminin-1 G-domain peptide, RKRLQVQLSIRT, in an organ culture of isolated vibrissa rudiment.

We established a serum-free organ culture system of isolated single vibrissa rudiments taken from embryonic day 13 mice. This system allowed us to test more than 30 laminin-derived cell adhesive peptides to determine their roles on the growth and differentiation of vibrissa hair follicles. We found that the RKRLQVQLSIRT sequence (designated AG-73), which mapped to the LG-4 module of the laminin-alpha1 chain carboxyl-terminal G domain, perturbed the growth of hair follicles in vitro. AG-73 is one of the cell-binding peptides identified from more than 600 systematically synthesized 12 amino acid peptides covering the whole amino acid sequence of the laminin-alpha1, -beta1, and -gamma1 chains, by cell adhesion assay. Other cell-adhesive laminin peptides and a control scrambled peptide, LQQRRSVLRTKI, however, failed to show any significant effects on the growth of hair follicles. The AG-73 peptide binds to syndecan-1, a transmembrane heparan-sulfate proteoglycan. Syndecan-1 was expressed in both the mesenchymal condensation and the epithelial hair peg of developing vibrissa, suggesting that AG-73 binding to the cell surface syndecan-1 perturbed the epithelial-mesenchymal interactions of developing vibrissa. The formation of hair bulbs was aberrant in the explants treated with AG-73. In addition, impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in the AG-73-treated-hair matrix epithelium, indicating that AG-73 binding perturbs various steps of epithelial morphogenesis, including the basement membrane remodeling. We also found a region-specific loss of the laminin-alpha1 chain in the basement membrane at the distal region of the invading hair follicle epithelium, indicating that laminins play a part in hair morphogenesis.

Ile-Lys-Val-Ala-Val (IKVAV)-containing laminin alpha1 chain peptides form amyloid-like fibrils.

The Ile-Lys-Val-Ala-Val (IKVAV) sequence derived from laminin-1 promotes cell adhesion, neurite outgrowth, and tumor growth and metastasis. Here, we examined amyloid formation of an IKVAV-containing peptide (LAM-L: AASIKVAVSADR, mouse laminin alpha1 chain 2097-2108). The LAM-L peptide was stained with Congo red and exhibited fibrils in electron microscopy with a characteristic cross-beta X-ray diffraction pattern. Further, infrared spectra of LAM-L suggested a beta-sheet structure. These results indicate that LAM-L forms amyloid-like fibrils. We also examined amyloid-like fibril formation of LAM-L analogs. The neurite outgrowth activity of the LAM-L analogs was closely related to their amyloid-like fibril formation.

Cdk4/cyclin D1 kinase, a universal and critical regulator of apoptosis.

BACKGROUND: We have previously described that overexpression of cdk4/cyclin D1 is the primary and critical mediator of apoptosis in PC12 cells even under otherwise physiological conditions. However, it is unclear whether this phenomenon is specific to neuronal cells or is more universally true. MATERIALS AND METHODS: Cyclins and cdks were transiently overexpressed in a variety of cultured cells, and examined for apoptosis. RESULTS: By flow cytometry, apoptosis was observed in all cell lines overexpressing cdk4 or cyclin D1. Immunofluorescence revealed that cells overexpressing cdk4 or cyclin D1 exhibited nuclear features characteristic of apoptosis. Furthermore, in cells defective for retinoblastoma protein (Rb), apoptosis could be induced by overexpression of any cdks or cyclins. CONCLUSION: Up-regulation of cdk4 kinase activity is the primary and critical mediator of apoptosis regardless of the cell types. In addition, inactivation of Rb renders cells further susceptible to apoptosis by abnormal expression of any of the cell cycle regulators.

IL-18 provided in dying bacterial-infected macrophages induces IFN-γ production in functional T-cell hybridoma B6HO3 through cell conjugates.

We have previously reported that the co-culture of functional T-cell hybridoma B6HO3 with dying J774 macrophage cells infected with Listeria monocytogenes (LM) results in the production of IFN-γ by B6HO3 cells. Here, we explore the mechanism underlying this phenomenon. We found that IFN-γ production was dependent on IL-18, but that the dying LM-infected macrophages produced no more than 100 pg/ml of IL-18, much less than the amount of IL-18 required for stimulating B6HO3 cells to produce IFN-γ. Furthermore, IL-18 binding protein added to the co-culture was unable to easily gain access to IL-18 for neutralisation. B6HO3 cells formed cell conjugates with J774 macrophages, and IFN-γ-producing B6HO3 cells were spatially and temporally associated with LM-infected macrophage cell death that exhibited neither pyroptosis nor pyronecrosis. These results suggest that the IL-18 produced by dying LM-infected macrophages is released to the interface of the cell conjugates, thereby inducing B6HO3 cells to produce IFN-γ. Based on the present and also previous findings, we propose that IL-18 released from macrophages because of cell death caused by bacteria may be the primary cytokine that triggers the innate IFN-γ production that is required for activating the bactericidal functions of macrophages at early stages of bacterial infection.