Naofumi Mukaida

Essential involvement of interleukin-8 (IL-8) in acute inflammation.

Neutrophil infiltration into inflammatory sites is one of the hallmarks of acute inflammation. Locally produced chemotactic factors are presumed to mediate the sequence of events leading to the infiltration at inflammatory sites. Interleukin‐8 (IL‐8), a novel leukocyte chemotactic activating cytokine (chemokine), is produced by various types of cells upon stimulation with inflammatory stimuli and exerts a variety of functions on leukocytes, particularly, neutrophils in vitro. However, no definitive evidence has been presented on its role in recruiting and activating neutrophils in the lesions of various types of inflammatory reactions. We administered a highly specific neutralizing antibody against IL‐8 in several types of acute inflammatory reactions, including lipopolysaccharide (LPS)‐induced dermatitis, LPS/IL‐1‐induced arthritis, lung reperfusion injury, and acute immune complex‐type glomerulonephritis. Anti‐IL‐8 treatment prevented neutrophil‐dependent tissue damage as well as neutrophil infiltration in these conditions. These results suggest that IL‐8 plays a causative role in acute inflammation by recruiting and activating neutrophils. J. Leukoc. Biol. 56: 559–564; 1994.

Blocking TNF-α in mice reduces colorectal carcinogenesis associated with chronic colitis

The inflammatory bowel disease ulcerative colitis (UC) frequently progresses to colon cancer. To understand the mechanisms by which UC patients develop colon carcinomas, we used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. We found that treating WT mice with AOM and DSS increased TNF-alpha expression and the number of infiltrating leukocytes expressing its major receptor, p55 (TNF-Rp55), in the lamina propria and submucosal regions of the colon. This was followed by the development of multiple colonic tumors. Mice lacking TNF-Rp55 and treated with AOM and DSS showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils, and attenuated subsequent tumor formation. WT mice transplanted with TNF-Rp55-deficient bone marrow also developed significantly fewer tumors after AOM and DSS treatment than either WT mice or TNF-Rp55-deficient mice transplanted with WT bone marrow. Furthermore, administration of etanercept, a specific antagonist of TNF-alpha, to WT mice after treatment with AOM and DSS markedly reduced the number and size of tumors and reduced colonic infiltration by neutrophils and macrophages. These observations identify TNF-alpha as a crucial mediator of the initiation and progression of colitis-associated colon carcinogenesis and suggest that targeting TNF-alpha may be useful in treating colon cancer in individuals with UC.

Molecular mechanism of interleukin‐8 gene expression

A potent leukocyte chemotactic and activating cytokine, interleukin‐8 (IL‐8), is produced by numerous types of cells in response to inflammatory stimuli. Accumulating evidence indicate that the transcription of IL‐8 gene requires the activation of either the combination of NF‐xB and AP‐1 or that of NF‐xB and NF‐IL6, depending on the type of cells. Alternatively, the activation of NF‐xB is indispensable for IL‐8 gene activation in any types of cells examined. On the other hand, an immunosuppressant, FK506, and a glucocorticoid inhibit the gene transcription as well as the production of IL‐8. Molecular analyses of IL‐8 gene repression by these agents revealed that both affected the activity of the transcription factor(s) bound to the NF‐xB site, albeit in different ways, thereby suppressing IL‐8 gene transcription. Collectively, IL‐8 production seems to be controlled mainly at the activation step of the transcription factor(s) bound to the NF‐xB site. J. Leukoc. Biol. 56: 554–558; 1994.

Interleukin-8 (IL-8) and monocyte chemotactic and activating factor (MCAF/MCP-1), chemokines essentially involved in inflammatory and immune reactions

Leukocyte infiltration is a hallmark of inflammation. Knowledge on molecular mechanisms of leukocyte infiltration has advanced rapidly due to the recent elucidation of structures and functions of adhesion molecules and chemokines. Since the discovery of interleukin-8 (IL-8), a prototype of CXC chemokines, in 1987 and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1), a prototype of chemotactic cytokines (CC) chemokines, in 1989, more than 30 members of chemokines have been identified so far. Evidence is accumulating that these chemokines exert overlapping but distinct actions on specific types of leukocytes in vitro through interacting with their specific G-protein-coupled receptors with seven transmembrane domains. However, redundancy at receptor levels has frequently hindered the clarification on the precise physiological or pathophysiological roles of chemokines. Here, we describe the pathophysiological roles of IL-8 and MCAF/MCP-1 in several animal models of neutrophil- and macrophage-mediated inflammation, respectively, by focusing on our recent work using neutralizing antibodies to these chemokines. We discuss further potential roles of these chemokines in T-lymphocyte-mediated immune responses.

Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice.

To clarify interleukin (IL)‐6 roles in wound healing, we prepared skin excisions in wild‐type (WT) and IL‐6‐deficient BALB/c [knockout (KO)] mice. In WT mice, the wound area was reduced to 50% of original size at 6 days after injury. Microscopically, leukocyte infiltration was evident at wound sites. Furthermore, the re‐epithelialization rate was ∼80% at 6 days after injury with increases in angiogenesis and hydroxyproline contents. The gene expression of IL‐1, chemokines, adhesion molecules, transforming growth factor‐β1, and vascular endothelial growth factor was enhanced at the wound sites. In contrast, the enhanced expression of these genes was significantly reduced in KO mice. Moreover, in KO mice, the reduction of wound area was delayed with attenuated leukocyte infiltration, re‐epithelialization, angiogenesis, and collagen accumulation. Finally, the administration of a neutralizing anti‐IL‐6 monoclonal antibody significantly delayed wound closure in WT mice. These observations suggest that IL‐6 has crucial roles in wound healing, probably by regulating leukocyte infiltration, angiogenesis, and collagen accumulation.

Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice

Tumor growth is associated with aberrant myelopoiesis, including the accumulation of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) that have the potential to promote tumor growth. However, the identity, growth, and migration of tumor-associated MDSCs remain undefined. We demonstrate herein that MDSCs at tumor site were composed primarily of bone marrow-derived CD11b(+)Gr-1(hi)Ly-6C(int) neutrophils and CD11b(+)Gr-1(int/dull)Ly-6C(hi) macrophages. Unexpectedly, in vivo bromodeoxyuridine (BrdU) labeling and parabiosis experiments revealed that tumor-infiltrating macrophages were replenished more rapidly than neutrophils. CCR2 deficiency caused striking conversion of infiltrating cellular dominance from macrophages to neutrophils in the tumor with the excessive production of CXCR2 ligands and granulocyte-colony stimulating factor in the tumor without affecting tumor growth. Overall, our data established the identity and dynamics of MDSCs in a tumor-bearing host mediated by chemokines and elucidated unexpected effects of the paucity of macrophages on tumor development.

Blockade of CCR2 Ameliorates Progressive Fibrosis in Kidney

Fibrosis is a hallmark of progressive organ diseases. Monocyte chemoattractant protein (MCP)-1, also termed as macrophage chemotactic and activating factor (MCAF/CCL2) and its receptor, CCR2 are presumed to contribute to progressive fibrosis. However, the therapeutic efficacy of MCP-1/CCR2 blockade in progressive fibrosis remains to be investigated. We hypothesized that blockade of CCR2 may lead to the improvement of fibrosis. To achieve this goal, we investigated renal interstitial fibrosis induced by a unilateral ureteral obstruction in CCR2 gene-targeted mice and mice treated with propagermanium or RS-504393, CCR2 inhibitors. Cell infiltrations, most of which were F4/80-positive, were reduced in CCR2 knockout mice. In addition, dual staining revealed that CCR2-positive cells were mainly F4/80-positive macrophages. Importantly, CCR2 blockade reduced renal interstitial fibrosis relative to wild-type mice. Concomitantly, renal transcripts and protein of MCP-1, transforming growth factor-beta, and type I collagen were decreased in CCR2-null mice. Further, this CCR2-dependent loop for renal fibrosis was confirmed by treatment with CCR2 antagonists in a unilateral ureteral obstruction model. These findings suggest that the therapeutic strategy of blocking CCR2 may prove beneficial for progressive fibrosis via the decrease in infiltration and activation of macrophages in the diseased kidneys.

Constitutive activation of transcription factors NF-?B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines

We previously reported that human head and neck squamous cell carcinomas (HNSCCs) express the pro‐inflammatory and pro‐angiogenic cytokines interleukin (IL)‐1α, IL‐6, IL‐8, and granulocyte‐macrophage colony‐stimulating factor in vitro and in vivo. The promoter region of the genes encoding these cytokines include binding sites for the transcription factors nuclear factor (NF) κB/Rel A, activator protein‐1 (AP‐1), and CCAAT enhancer‐binding protein β (C/EBPβ, or NF‐IL6), which have been reported to contribute to activation of these cytokine genes. In the study presented here, we examined the activation, composition, and function of these transcription factors in HNSCC cell lines that express pro‐inflammatory cytokines, by using electrophoretic mobility shift and reporter‐gene assays. Constitutive activation of NF‐κB, AP‐1, and NF‐IL6 DNA‐binding proteins was detected. Supershift analysis with antibodies specific for NF‐κB, AP‐1, and NF‐IL6 binding proteins showed that the NF‐κB–binding protein included p65/Rel A and p50; AP‐1 activity included c‐jun, junB, junD, and Fra‐1; and NF‐IL6 included C/EBPβ. Mutational analysis of the NF‐κB, AP‐1, and NF‐IL6 sites in the IL‐8 promoter region showed that NF‐κB and AP‐1 sites contributed to constitutive IL‐8 reporter activity in HNSCC. HNSCC lines that exhibited increased IL‐8 secretion relative to simian virus 40–immortalized and primary keratinocyte cell lines also demonstrated a concordant increase in NF‐κB reporter activity relative to nonmalignant keratinocytes. We concluded that the early transcription factors NF‐κB, AP‐1, and NF‐IL6 are constitutively activated in human HNSCC cell lines and that NF‐κB and AP‐1 promote expression of the pro‐inflammatory and pro‐angiogenic cytokine IL‐8 in HNSCC. The demonstration of the activation of these transcription factors will be helpful in defining the identity and role of these and other early gene products that contribute to pathogenesis of the malignant phenotype in HNSCC and in defining potential targets for pharmacologic and molecular therapy of HNSCC. Mol. Carcinog. 26:119–129, 1999. Published 1999 Wiley‐Liss, Inc.

Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration.

To clarify biological roles of tumor necrosis factor receptor p55 (TNF‐Rp55) ‐mediated signals in wound healing, skin excisions were prepared in BALB/c (WT) and TNF‐Rp55‐deficient (KO) mice. In WT mice, the wound area was reduced to 50% of the original area 6 days after injury, with angiogenesis and collagen accumulation. Histopathologically, reepithelialization rate was ~80% 6 days. Myeloperoxidase activity and macrophage recruitment were the most evident 1 and 6 days after injury, respectively. Gene expression of adhesion molecules, interleukin 1α(IL‐1α), IL‐1β, monocyte chemoattractant protein 1, macrophage inflammatory protein 1α(MIP‐1α), MIP‐2, transforming growth factor β1 (TGF‐β1) connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), Flt‐1, and Flk‐1 was enhanced at the wound site. In KO mice, an enhancement in angiogenesis, collagen content, and reepithelialization was accelerated with the increased gene expression of TGF‐β1, CTGF, VEGF, Flt‐1, and Flk‐1 at the wound sites, resulting in accelerated wound healing compared with WT mice. In contrast, leukocyte infiltration, mRNA expression of adhesion molecules, and cytokines were significantly reduced in KO mice. These observations suggest that TNF‐Rp55‐mediated signals have some role in promoting leukocyte infiltration at the wound site and negatively affect wound healing, probably by reducing angiogenesis and collagen accumulation.—Mori, R., Kondo, T., Ohshima, T., Ishida, Y., Mukaida, N. Accelerated wound healing in tumor necrosis factor receptor p55‐deficient mice with reduced leukocyte infiltration. FASEB J. 16, 963–974 (2002)

Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP-1).

We investigated the pathophysiological role of a potent macrophage (Mϕ) chemotactic cytokine (chemokine), monocyte chemotactic and activating factor/monocyte chemoattractant protein‐1 (MCAF/MCP‐1), in an animal model of crescentic glomerulonephritis. Administration of a small dose of nephrotoxic sera induced severe proliferative and necrotizing glomerulonephritis, with crescentic formation in the early phase and glomerulosclerosis in the later phase, in Wistar‐Kyoto rats. MCAF/MCP‐1 protein was detected immunohistochemically in glomeruli, vascular endothelial cells, and tubular epithelial cells in the early phase of injured kidney tissues but not in normal ones. Anti‐MCAF/MCP‐1 antibodies decreased the number of Mϕ in glomeruli, and prevented crescentic formation and the fusion of epithelial cell foot process in nephritic rats, thereby decreasing the excreted amounts of protein to normal levels on days 3 and 6. Furthermore, anti‐MCAF/MCP‐1 antibodies remarkably reduced glomerulosclerosis and improved renal dysfunction as well as proteinuria in the later phase (56 days). These results indicate that MCAF/MCP‐1 essentially participates in the impairment of renal functions associated with crescentic glomerulonephritis by recruiting and activating Mϕ.—Wada, T., Yokoyama, H., Furuichi, K., Kobayashi, K.‐i., Harada, K., Naruto, M., Su, S.‐B., Akiyama, M., Mukaida, N., Matsushima, K. Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP‐1). FASEB J. 10, 1418‐1425 (1996)