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Macrophages in lungs can be classified into two subpopulations, alveolar macrophages (AMs) and interstitial macrophages (IMs), which reside in the alveolar and interstitial spaces, respectively. Accumulating evidence indicates the involvement of IMs in lung metastasis, but the roles of AMs in lung metastasis still remain elusive. An i.v. injection of a mouse hepatocellular carcinoma (HCC) cell line, BNL, caused lung metastasis foci with infiltration of AMs and IMs. Comprehensive determination of arachidonic acid metabolite levels revealed increases in leukotrienes and PGs in lungs in this metastasis model. A 5-lipoxygenase (LOX) inhibitor but not a cyclooxygenase inhibitor reduced the numbers of metastatic foci, particularly those of a larger size. A major 5-LOX metabolite, LTB4, augmented in vitro cell proliferation of human HCC cell lines as well as BNL cells. Moreover, in this lung metastasis course, AMs exhibited higher expression levels of the 5-LOX and LTB4 than IMs. Consistently, 5-LOX–expressing AMs increased in the lungs of human HCC patients with lung metastasis, compared with those without lung metastasis. Furthermore, intratracheal clodronate liposome injection selectively depleted AMs but not IMs, together with reduced LTB4 content and metastatic foci numbers in this lung metastasis process. Finally, IMs in mouse metastatic foci produced CCL2, thereby recruiting blood-borne, CCR2–expressing AMs into lungs. Thus, AMs can be recruited under the guidance of IM-derived CCL2 into metastatic lungs and can eventually contribute to the progression of lung metastasis by providing a potent arachidonic acid–derived tumor growth promoting mediator, LTB4.

The injection of Clostridium difficile toxin A into the ileal loops caused fluid accumulation with the destruction of intestinal epithelial structure and the recruitment of neutrophils and macrophages. Concomitantly, intraileal gene expression of CX3CL1/fractalkine (FKN) and its receptor, CX3CR1, was enhanced. When treated with toxin A in a similar manner, CX3CR1-deficient (CX3CR1−/−) mice exhibited exaggerated fluid accumulation, histopathological alterations, and neutrophil recruitment, but not macrophage infiltration. Mice reconstituted with CX3CR1−/− mouse-derived bone marrow cells exhibited exacerbated toxin A-induced enteritis, indicating that the lack of the CX3CR1 gene for hematopoietic cells aggravated toxin A-induced enteritis. A heme oxygenase-1 (HO-1) inhibitor, tin-protoporphyrin-IX, markedly increased fluid accumulation in toxin A-treated wild-type mice, indicating the protective roles of HO-1 in this situation. HO-1 expression was detected mainly in F4/80-positive cells expressing CX3CR1, and CX3CR1−/− mice failed to increase HO-1 expression after toxin A treatment. Moreover, CX3CL1/FKN induced HO-1 gene expression by isolated lamina propria-derived macrophages or a mouse macrophage cell line, RAW264.7, through the activation of the ERK signal pathway. Thus, CX3CL1/FKN could induce CX3CR1-expressing macrophages to express HO-1, thereby ameliorating toxin A-induced enteritis.

Macrophages accumulate during the course of corneal neovascularization, but its mechanisms and roles still remain elusive. To address these points, we herein examined corneal neovascularization after alkali injury in mice deficient in fractalkine receptor/CX3CR1, which is normally expressed by macrophages. After alkali injury, the mRNA expression of CX3CR1 was augmented along with accumulation of F4/80-positive macrophages and Gr-1-positive neutrophils in the corneas. Compared with wild-type mice, CX3CR1-deficient mice exhibited enhanced corneal neovascularization 2 wk after injury, as evidenced by enlarged CD31-positive areas. Concomitantly, the accumulation of F4/80-positive macrophages, but not Gr-1-positive neutrophils, was markedly attenuated in CX3CR1-deficient mice compared with wild-type mice. The intraocular mRNA expression of vascular endothelial growth factor (VEGF) was enhanced to similar extents in wild-type and CX3CR1-deifient mice after the injury. However, the mRNA expression of antiangiogenic factors, thrombospondin (TSP) 1, TSP-2, and a disintegrin and metalloprotease with thrombospondin (ADAMTS) 1, was enhanced to a greater extent in wild-type than CX3CR1-deificient mice. A double-color immunofluorescence analysis demonstrated that F4/80-positive cells also expressed CX3CR1 and ADAMTS-1 and that TSP-1 and ADAMTS-1 were detected in CX3CR1-positive cells. CX3CL1 enhanced TSP-1 and ADAMTS-1, but not VEGF, expression by peritoneal macrophages. Moreover, topical application of CX3CL1 inhibited corneal neovascularization at 2 wk, along with enhanced intraocular expression of TSP-1 and ADAMTS-1 but not VEGF. Thus, these observations indicate that accumulation of CX3CR1-positive macrophages intraocularly can dampen alkali-induced corneal neovascularization by producing antiangiogenic factors such as TSP-1 and ADAMTS-1 and suggest the potential therapeutic efficacy of using CX3CL1 against alkali-induced corneal neovascularization.

Clostridium difficile has emerged as the important causative agent of antibiotics-associated pesudomembranous colitis; especially its toxin A is presumed to be responsible for the colitis. We examined the pathophysiological roles of IFN-γ in toxin A-induced enteritis using IFN-γ knockout (KO) mice. When toxin A of C. difficile was injected into the ileal loops of BALB/c wild-type (WT) mice, massive fluid secretion, disruption of intestinal epithelial structure, and massive neutrophil infiltration developed within 4 h after the injection. IFN-γ protein was faintly detected in some CD3-positive lymphocytes in the lamina propria and submucosa of the ileum of untreated WT mice. On the contrary, at 2 and 4 h after toxin A injection, IFN-γ protein was detected in infiltrating neutrophils and to a lesser degree in CD3-positive lymphocytes. In the ileum of WT mice, toxin A treatment markedly enhanced the gene expression of TNF-α, macrophage inflammatory protein-1α and -2, KC, and ICAM-1 >2 h after treatment. In contrast, the histopathological changes were marginal, without enhanced fluid secretion in the ileum of toxin A-treated IFN-γ KO mice. Moreover, toxin A-induced gene expression of TNF-α, neutrophil chemotactic chemokines, and ICMA-1 was remarkably attenuated in IFN-γ KO mice. Furthermore, pretreatment of WT mice with a neutralizing anti-IFN-γ Ab prevented toxin A-induced enteritis. These observations indicate that IFN-γ is the crucial mediator of toxin A-induced acute enteritis and suggest that IFN-γ is an important molecular target for the control of C. difficile-associated pseudomembranous colitis.

Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria. Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances. Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF). However, it remains elusive which factor activates macrophage in these processes. Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased. Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid. Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P. aeruginosa infection. The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury. In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury. Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner. Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs

The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides −1481 and −546 and the transcription factor NF-κB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-κB consensus DNA. The activation of NF-κB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.

Ligands for peroxisome proliferator-activated receptor γ (PPARγ), such as 15-deoxy-Δ12,14PGJ2 (15d-PGJ2) have been proposed as a new class of antiinflammatory compounds with possible clinical applications. As there is some controversy over the inhibitory effects of 15d-PGJ2 on chemokine gene expression, we investigated whether 15d-PGJ2 itself affected chemokine gene expression in human monocytes/macrophages and two monocytic cell lines. Here we demonstrate that the 15d-PGJ2 can induce IL-8 gene expression. In contrast, monocyte chemoattractant protein-1 gene expression was suppressed by 15d-PGJ2, while the expression of RANTES was unaltered. Furthermore, concomitant treatment of monocytes/macrophages with 15d-PGJ2 (2.5 × 10−6 M) potentiated LPS-induced gene expression of IL-8 mRNA, but suppressed PMA-induction of IL-8 mRNA. In addition, treatment of U937 and THP-1 cells with 15d-PGJ2 also resulted in induction of IL-8 gene expression. Further studies demonstrated that 15d-PGJ2 regulated IL-8 gene expression via a ligand-specific and PPARγ-dependent pathway. Our observations revealed a previous unappreciated function and mechanism of 15d-PGJ2-mediated regulation of cytokine gene expression in monocytes/macrophages.

The present study was designed to investigate the role of IFN-γ in LPS-induced liver injury following priming with Propionibacterium acnes. At 1 week after priming BALB/c mice with P. acnes, a large number of macrophages (Mφ) and lymphocytes predominantly infiltrated the portal area, resulting in the intrahepatic formation of granulomas consisting of epithelioid and lymphoid cells. In comparison, in IFN-γ gene-disrupted BALB/c mice (IFN-γ knockout mice), the number of infiltrated Mφ was decreased, with a significant reduction in the number and size of granulomas. Subsequent elicitation with a low dose of LPS induced massive hepatic necrosis in wild-type BALB/c mice, with a marked increase in the serum levels of TNF-α, IL-12, and IL-18 and subsequently of alanine transferase. In contrast, IFN-γ knockout mice developed scattered focal necrosis of the liver with significantly lower levels of serum alanine transferase as well as drastic decreases in TNF-α, IL-12, and IL-18 production. The administration of an anti-IFN-γ neutralizing mAb at the eliciting phase significantly alleviated liver injury and reduced serum IL-12 and IL-18 levels. Thus, endogenously produced IFN-γ is involved in the pathogenesis of this liver injury model by regulating Mφ infiltration and granuloma formation in the priming phase as well as cytokine production in the eliciting phase.

TNF-α is implicated in the initiation of cytokine cascades in various inflammatory settings. To assess the interactions of multiple cytokines at the level of inflammatory effector cells, we examined the effects of TNF-α on the expression of two IL-8Rs (CXCR1 and CXCR2) on polymorphonuclear leukocytes (PMNs). TNF-α decreased the surface expression of CXCR2 in a dose- and time-dependent manner. In contrast, CXCR1 expression was not affected by TNF-α. The release of CXCR2 into the supernatant of TNF-α-treated PMNs was detected by immunoblotting and immuno-slot-blot analyses, suggesting that the down-regulation of CXCR2 was caused mainly by shedding from the cell surface. The CXCR2 down-regulation was inhibited by PMSF and aprotinin, supporting the hypothesis that the shedding was mediated by serine protease(s). The intracellular Ca2+ mobilization and chemotaxis in response to IL-8 were suppressed by the pretreatment of PMNs with TNF-α, indicating that the decrease in CXCR2 was reflected in the decreased functional responses to IL-8. In contrast, the O2− release, which is mediated by CXCR1, was not suppressed by TNF-α. The treatment of whole blood with TNF-α also caused a significant reduction in CXCR2 and markedly suppressed intracellular Ca2+ mobilization and chemotaxis in response to IL-8, while enhancing the O2− release. These findings suggest that TNF-α down-regulates CXCR2 expression on PMNs and modulates IL-8-induced biologic responses, leading to the intravascular retention of PMNs with an enhanced production of reactive oxygen metabolites.

Th2 cytokines, such as IL-4, IL-10, and IL-13, suppress proinflammatory cytokine production by monocytes/macrophages. Since monocyte chemoattractant protein-1 (MCP-1) is presumed to play an important role in monocyte recruitment and activation during inflammatory and immune responses, we examined here the effects of these Th2 cytokines on MCP-1 production by human blood monocytes and alveolar macrophages. Unstimulated, highly purified blood monocytes did not produce MCP-1 spontaneously, while LPS treatment induced the production of MCP-1 and its mRNA expression. All Th2 cytokines tested suppressed LPS-induced MCP-1 production and its mRNA expression, although the suppressive effect of IL-13 was weaker than that of IL-4 or IL-10. In contrast, IL-10, but neither IL-4 nor IL-13, induced unstimulated peripheral blood monocytes to produce biologically active MCP-1 protein within 4 h, reaching a maximal level at 12 h. IL-10-induced MCP-1 production was reduced by pretreatment of IL-10 with anti-IL-10 Ab, negating the involvement of contaminated endotoxin. Moreover, IL-10 induced MCP-1 mRNA expression in unstimulated monocytes, independent of de novo protein synthesis. Furthermore, human alveolar macrophages produced MCP-1 spontaneously, and the production was inhibited by IL-4 or IL-13, but was augmented by IL-10. These findings suggest that IL-10 regulates MCP-1 production by monocytes/macrophages in a different way from other Th2 cytokines, such as IL-4 and IL-13, and contributes to host defense responses.

Intrathymic negative selection and differentiation of regulatory T (Treg) cells are a cardinal event in central tolerance system. Thymic dendritic cells (DCs) are presumed to be capable of inducing the apoptosis of autoreactive T progenitor cells and of alternatively differentiating them into Treg cells. In spite of the presence of heterogenous DC subsets in thymus, similarly observed on other lymphoid organs such as lymph nodes and spleen, the role of each intrathymic DC subset in the central tolerance is still unclear. Recently, we demonstrated that mice deficient in a CC chemokine receptor, CCR2, exhibited a selective diminution in CD11c+CD11b+CD8-Sirpα+ conventional DC (cDC) subset in the thymus with an accumulation of T cells with reactivity against serum self-antigen in the periphery. Here, we further revealed that Sirpα+ cDCs in the thymus can selectively capture blood-borne antigens due to their unique intrathymic localization nearby small vessels and inside perivascular regions, and then induce the antigen-specific Treg cells and subsequent clonal deletion to an intravenously injected antigen, depending on the intensity of antigen presentation. Finally, we established a novel way to in vivo expand antigen-specific Treg cells via intrathymic antigen presentation by thymic Sirpα+ cDCs, which can dampen aberrant immune reaction in an antigen-specific manner.