Akihisa Harada

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.

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.

Active participation of CCR5+CD8+ T lymphocytes in the pathogenesis of liver injury in graft-versus-host disease

We examined the molecular pathogenesis of graft-versus-host disease-associated (GVHD-associated) liver injury in mice, focusing on the role of chemokines. At the second week after cell transfer in the parent-into-F1 model of GVHD, CD8(+) T cells -- especially donor-derived CD8(+) T cells -- infiltrated the liver, causing both portal hepatitis and nonsuppurative destructive cholangitis (NSDC). These migrating cells expressed CCR5. Moreover, macrophage inflammatory protein-1alpha (MIP-1alpha), one of the ligands for CCR5, was selectively expressed on intralobular bile duct epithelial cells, endothelial cells, and infiltrating macrophages and lymphocytes. Administration of anti-CCR5 antibody dramatically reduced the infiltration of CCR5(+)CD8(+) T lymphocytes into the liver, and consequently protected against liver damage in GVHD. The levels of Fas ligand (FasL) mRNA expression in the liver were also decreased by anti-CCR5 antibody treatment. Anti-MIP-1alpha antibody treatment also reduced liver injury. These results suggest that MIP-1alpha-induced migration of CCR5-expressing CD8(+) T cells into the portal areas of the liver plays a significant role in causing liver injury in GVHD; thus, CCR5 and its ligand may be the novel target molecules of therapeutic intervention of hepatic GVHD.

Peyer's patch is the essential site in initiating murine acute and lethal graft-versus-host reaction.

Acute graft-versus-host disease (a-GVHD) is initiated primarily by immunologically competent cytotoxic T cells (CTLs) that express anti-host specificities. However, the host lymphoid compartment in which these precursor CTLs are initially stimulated remains unclear. Here we show that gut Peyers patches (PPs) are required to activate anti-host CTL responses in a well characterized murine acute graft-versus-host reaction (a-GVHR) model, involving transfer of parent lymphocytes into F1 hybrid recipients. The a-GVHR was prevented when recruitment of donor T cells into PP was interrupted either by disrupting the gene encoding chemokine receptor CCR5 or by blocking integrin α4β7–MAdCAM-1 (mucosal vascular addressin) interactions. Mice deficient for PPs failed to develop a-GVHD in two models of disease induction. Thus, blockade of CTL generation in PPs might offer new strategies for circumventing a-GVHD.

Anti–Monocyte Chemoattractant Protein-1/Monocyte Chemotactic and Activating Factor Antibody Inhibits Neointimal Hyperplasia in Injured Rat Carotid Arteries

Monocyte chemoattractant protein-1 (MCP-1)/monocyte chemotactic and activating factor (MCAF) has been suggested to promote atherogenesis. The effects of in vivo neutralization of MCP-1 in a rat model were examined in an effort to clarify the role of MCP-1 in the development of neointimal hyperplasia. Competitive polymerase chain reaction analysis revealed maximum MCP-1 mRNA expression at 4 hours after carotid arterial injury. Increased immunoreactivities of MCP-1 were also detected at 2 and 8 hours after injury. Either anti-MCP-1 antibody or nonimmunized goat IgG (10 mg/kg) was then administered every 12 hours to rats that had undergone carotid arterial injury. Treatment with 3 consecutive doses of anti-MCP-1 antibody within 24 hours (experiment 1) and every 12 hours for 5 days (experiment 2) significantly inhibited neointimal hyperplasia at day 14, resulting in a 27.8% reduction of the mean intima/media ratio (P<0.05) in experiment 1 and a 43.6% reduction (P<0.01) in experiment 2. This effect was still apparent at day 56 (55.6% inhibition; P<0.05). The number of vascular smooth muscle cells in the neointima at day 4 was significantly reduced by anti-MCP-1 treatment, demonstrating the important role of MCP-1 in early neointimal lesion formation. However, recombinant MCP-1 did not stimulate chemotaxis of vascular smooth muscle cells in an in vitro migration assay. These results suggest that MCP-1 promotes neointimal hyperplasia in early neointimal lesion formation and that neutralization of MCP-1 before, and immediately after, arterial injury may be effective in preventing restenosis after angioplasty. Further studies are needed to clarify the mechanism underlying the promotion of neointimal hyperplasia by MCP-1.

Pivotal role of TARC, a CC chemokine, in bacteria-induced fulminant hepatic failure in mice.

Thymus and activation-regulated chemokine (TARC) is a recently identified lymphocyte-directed CC chemokine which specifically chemoattracts T helper type 2 CD4(+) T cells in human. To establish the pathophysiological roles of TARC in vivo, we investigated whether a monoclonal antibody (mAb) against TARC could inhibit the induction of hepatic lesions in murine model using Propionibacterium acnes and lipopolysaccharide (LPS). P. acnes-induced intrahepatic granuloma formation in the priming phase is essential to the subsequent liver injury elicited by a low dose of LPS. The priming phase appears to be dominated by Th1 type immune responses determined by the profile of chemokine and chemokine receptor expression. TARC was selectively produced by granuloma-forming cells, and CC chemokine receptor 4 (CCR4)-expressing CD4(+) T cells migrated into the liver after LPS administration. In vivo injection of anti-TARC mAb just before LPS administration protected the mice from acute lethal liver damage, which was accompanied by a significant reduction of both CCR4 mRNA expression and IL-4 production by liver-infiltrating CD4(+) T cells. Moreover, both TNF-alpha and Fas ligand expressions in the liver were decreased by anti-TARC treatment. These results suggest that recruitment of IL-4-producing CCR4(+) CD4(+) T cells by granuloma-derived TARC into the liver parenchyma may be a key cause of massive liver injury after systemic LPS administration.

Properties of Pro-Inflammatory Cell Type-Specific Leukocyte Chemotactic Cytokines, Interleukin 8(IL-8) and Monocyte Chemotactic and Activating Factor (MCAF)

Inflammation is a host defensive reaction to tissue injuries caused by various conditions such as trauma, infections, invasion of foreign particles, ischemia-reperfusion syndrome, malignant tumors, and autoimmune diseases. The early reactions of inflammations consist of plasma leakage and leukocyte infiltration into the lesion. The types of infiltrating leukocytes depend upon the kind, degree, and timing of tissue injuries. Although several factors such as C5a, leukotriene B4 and bacteriaderived formyl peptides have been long known as leukocyte chemotactic factors, these factors chemoattract any kind of leukocytes, thus suggesting the existence of cell type-specific leukocyte chemotactic factors (28). Over the past several years, numerous members of a new family of heparinbinding polypeptide cytokines with a molecular weight of 8-10 kDa have been identified (Ref. 25) (Table 1) . These factors have 4 cysteine residues at the wellconserved positions and are presumed to possess a similar structure (25). These cytokines are produced massively and rapidly by various kinds of cells in response to inflammatory stimuli including lipopolysaccharide (LPS) , viruses, interleukin 1 (IL-1), tumor necrosis factor (TNF) and so on [Tables 2 and 3 show the cell sources and inducers of interleukin 8 (IL-8) and monocyte chemotactic and activating factor (MCAF) described below]. Furthermore, it has become evident that several of these cytokines have chemotactic activity for specific type(s) of leukocytes, suggesting that these factors are bona fide chemoattractants involved in the leukocyte recruitment seen in the inflammatory processes. These factors are subdivided into two subgroups depending on whether the first two cysteines are separated by a single amino acid or in an adjacent position (Table 1). The former are called intercrine

IL-4 and a glucocorticoid up-regulate CXCR4 expression on human CD4+ T lymphocytes and enhance HIV-1 replication.

CXCR4 is a key co‐receptor required for the infection of T‐tropic HIV‐1 strain of CD4+ T lymphocytes. The regulation of this chemokine receptor was therefore studied. Th2 polarized cells expressed more CXCR4 than Th1 cells. Among a panel of cytokines and stimulants, a Th2 type cytokine interleukin‐4 (IL‐4) selectively up‐regulated the mRNA level as well as surface protein expression of CXCR4 within 16 h. In addition, CXCR4 was also up‐regulated by a glucocorticoid, dexamethasone. These treated cells became more responsive in transendothelial migration assays to the specific CXCR4 ligand, SDF‐1α. Furthermore, up‐regulation of CXCR4 was also associated with the enhancement of HIV replication in human CD4+ T lymphocytes. This study indicates the enhanced T‐tropic HIV‐1 infection to CD4+ T lymphocytes through up‐regulation of CXCR4 by several immunomodulating agents, IL‐4, and a glucocorticoid. These findings may explain the shift to T‐tropic HIV‐1 dominance during AIDS progression when Th2 comes to predominate. J. Leukoc. Biol. 64: 642–649; 1998.

Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice.

Chronic intermittent injection of carbon tetrachloride (CCI4) for more than 10 weeks induced liver fibrosis in mice, as evidenced by positive Azan staining and increased intrahepatic collagen content. Preceding the onset of liver fibrosis, interleukin‐6 (IL‐6) gene expression was enhanced in liver and immunoreactive IL‐6 was detected in infiltrating inflammatory cells. To delineate the role of IL‐6 in this process, we treated IL‐6‐deficient mice with CCl4 in a similar manner for 12 weeks, after which fibrotic changes were less evident and serum albumin levels were lower in IL‐6‐deficient than wild‐type mice. Moreover, CCl4‐induced expression of transforming growth factor β1 and hepatocyte growth factor genes in liver was significantly reduced in IL‐6‐deficient mice. Thus, IL‐6 may be vitally involved in fibrotic changes and maintenance of serum albumin levels, partly by modulating intrahepatic expression of these cytokines. J. Leukoc. Biol. 66: 601–608; 1999.

Inhibition of neutrophil-mediated acute inflammatory injury by an antibody against interleukin-8 (IL-8)

Abstract. Chemokines are a family of cytokines regulating the migration and functions of leukocytes in a cell-type specific manner. A prototype of C-X-C chemokines, interleukin-8 (IL-8), chemoattracts and activates neutrophils in vitro, and IL-8 concentrations in body fluids are markedly increased in several neutrophil-mediated acute inflammation. Moreover, we previously reported that the administration of a neutralizing antibody to IL-8 prevented neutrophil-mediated tissue injury, as well as neutrophil infiltration, in several animal disease models. These observations implicate IL-8 as a major mediator of neutrophil-mediated tissue injury. Furthermore, we recently showed that an anti-IL-8 antibody effectively prevented two models that are very relevant to clinical situations; endotoxemia-induced acute respiratory distress syndrome (ARDS)-like lung injury and cerebral reperfusion injury. These results raise the possibility that IL-8 is a novel target for therapeutic intervention in neutrophil-mediated acute inflammation.