Sankichi Horiuchi

IL-6 and Its Soluble Receptor Orchestrate a Temporal Switch in the Pattern of Leukocyte Recruitment Seen during Acute Inflammation

During acute inflammation, leukocyte recruitment is characterized by an initial infiltration of neutrophils, which are later replaced by a more sustained population of mononuclear cells. Based on both clinical and experimental evidence, we present a role for IL-6 and its soluble receptor (sIL-6R) in controlling this pattern of leukocyte recruitment during peritoneal inflammation. Liberation of sIL-6R from the initial neutrophil infiltrate acts as a regulator of CXC and CC chemokine expression, which contributes to a suppression of neutrophil recruitment and the concurrent attraction of mononuclear leukocytes. Soluble IL-6R-mediated signaling is therefore an important intermediary in the resolution of inflammation and supports transition between the early predominantly neutrophilic stage of an infection and the more sustained mononuclear cell influx.

The soluble interleukin 6 receptor: mechanisms of production and implications in disease

Interleukin 6 (IL‐6) performs a prominent role during disease and has been described as both a pro‐ and anti‐inflammatory cytokine. A key feature in the regulation of IL‐6 responses has been the identification of a soluble interleukin 6 receptor (sIL‐6R), which forms a ligand‐receptor complex with IL‐6 that is capable of stimulating a variety of cellular responses including proliferation, differentiation and activation of inflammatory processes. Elevated sIL‐6R levels have been documented in numerous clinical conditions indicating that its production is coordinated as part of a disease response. Thus, sIL‐6R has the potential to regulate both local and systemic IL‐6‐mediated events. This review will outline the central role of sIL‐6R in the coordination of IL‐6 responses. Details relating to the mechanisms of sIL‐6R production will be provided, while the potential significance of sIL‐6R during the development of clinical conditions will be emphasized. We want to convey, therefore, that when thinking about the inflammatory capability of IL‐6, it is essential to consider not only the action of IL‐6 itself, but also the effect sIL‐6R may have on cellular processes.—Jones, S. A., Horiuchi, S., Topley, M. Yamamoto, N., Fuller, G. M. The soluble interleukin 6 receptor: mechanisms of production and implications in disease. FASEB J. 15, 43–58 (2001)

Soluble IL-6 Receptor Governs IL-6 Activity in Experimental Arthritis: Blockade of Arthritis Severity by Soluble Glycoprotein 130

Studies in IL-6-deficient (IL-6−/−) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mechanism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6−/− mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. Histopathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasynovial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis.

Differential Regulation of Neutrophil-Activating Chemokines by IL-6 and Its Soluble Receptor Isoforms

Interleukin-6 signaling via its soluble receptor (sIL-6R) differentially regulates inflammatory chemokine expression and leukocyte apoptosis to coordinate transition from neutrophil to mononuclear cell infiltration. sIL-6R activities may, however, be influenced in vivo by the occurrence of two sIL-6R isoforms that are released as a consequence of differential mRNA splicing (DS) or proteolytic cleavage (PC) of the cognate IL-6R (termed DS- and PC-sIL-6R). Using human peritoneal mesothelial cells and a murine model of peritoneal inflammation, studies described in this work have compared the ability of both isoforms to regulate neutrophil recruitment. In this respect, DS- and PC-sIL-6R were comparable in their activities; however, these studies emphasized that IL-6 trans signaling differentially controls neutrophil-activating CXC chemokine expression. In vitro, stimulation of mesothelial cells with IL-6 in combination with either DS-sIL-6R or PC-sIL-6R showed no induction of CXC chemokine ligand (CXCL)1 (GROα) and CXCL8 (IL-8), whereas both isoforms enhanced CXCL5 (ENA-78) and CXCL6 (granulocyte chemotactic protein-2) expression. Moreover, when complexed with IL-6, both isoforms specifically inhibited the IL-1β-induced secretion of CXCL8. These findings were paralleled in vivo, in which induction of peritoneal inflammation in IL-6-deficient (IL-6−/−) mice resulted in enhanced keratinocyte-derived chemokine and macrophage-inflammatory protein-2 (the murine equivalent of CXCL1 and CXCL8) levels, but reduced LPS-induced CXC chemokine (the murine equivalent of CXCL5) expression. Reconstitution of IL-6 signaling in IL-6−/− mice with IL-6 and its soluble receptor isoforms corrected this chemokine imbalance and suppressed overall neutrophil infiltration. These data confirm that sIL-6R-mediated signaling primarily limits neutrophil influx; however, induction of CXCL5 and CXCL6 may regulate other neutrophil responses.

Shedding of the soluble IL-6 receptor is triggered by Ca2+ mobilization, while basal release is predominantly the product of differential mRNA splicing in THP-1 cells

The soluble IL‐6 receptor (sIL‐6R) is generated through either proteolytic shedding of the cognate receptor (PC‐sIL‐6R), or released as the product of differential mRNA splicing (DS‐sIL‐6R). Using monocytic THP‐1 cells, we demonstrate that both mechanisms are independently regulated, and that each process contributes to sIL‐6R production. Shedding of the IL‐6R was activated by the Ca2+ ionophore, ionomycin, and inhibited by the TNF‐α protease inhibitor (TAPI). In contrast, basal sIL‐6R release was unaffected by Ca2+ depletion and largely insensitive to TAPI. Moreover, although IL‐6R shedding was inactivated by serum starvation, non‐stimulated production remained intact. Basal sIL‐6R production via differential mRNA splicing was shown through the inhibitory action of brefeldin A and an enzyme‐linked immunosorbent assay specific for DS‐sIL‐6R. Release of this isoform was unaffected by ionomycin or TAPI, indicating that Ca2+ mobilization activates PC‐sIL‐6R generation, but not DS‐sIL‐6R. The divergent control of these sIL‐6R isoforms indicates that they may independently influence the inflammatory response.

High-level production of alternatively spliced soluble interleukin-6 receptor in serum of patients with adult T-cell leukaemia/HTLV-I-associated myelopathy.

We have previously shown, using human T‐cell lymphocytotrophic virus‐I (HTLV‐I)‐infected cell lines, that soluble interleukin‐6 receptor (sIL‐6R) is generated through an alternative splicing mechanism. In this study, we examined human sera for the presence of alternatively spliced soluble IL‐6R (AS‐sIL‐6R). We produced a monoclonal antibody (mAb) recognizing the unique sequence of AS‐sIL‐6R peptide, generated by an altered reading frame. We also made recombinant AS‐sIL‐6R protein in Spodoptera frugiperda‐9 (Sf‐9) cells carrying baculovirus, which encoded altered sIL‐6R or conventional IL‐6R cDNA. mAbs specifically recognized AS‐sIL‐6R, but not conventional IL‐6R, as demonstrated by Western blot analyses, fluorescence‐activated cell sorter, immunofluorescence analyses and enzyme‐linked immunosorbent assay (ELISA). We adapted an ELISA system and used it for detection of altered sIL‐6R in sera from 23 healthy persons, 12 patients with adult T‐cell leukaemia (ATL) and 13 patients with HTLV‐I‐associated myelopathy (HAM). Serum levels of AS‐sIL‐6R were 6·4 or 6·1 times greater in ATL (28·7±20·4 ng/ml, P<0·0001) and in HAM patients (27·5±12·1 ng/ml, P<0·0001) than in healthy individuals (4·5±2·1 ng/ml). High levels of AS‐sIL‐6R were also observed in plasma from rheumatoid arthritis patients and in persons with elevated levels of alanine aminotransferase (ALT), antinuclear antibody (ANA), or α‐fetoprotein (AFP). However, in human immunodeficiency virus‐1 (HIV‐1), hepatitis B virus (HBV) or hepatitis C virus (HCV)‐infected individuals, AS‐sIL‐6R levels were not elevated. In this study, we confirmed that AS‐sIL‐6R is indeed present in human sera. These observations suggest that alternative splicing of IL‐6R mRNA is of consequence in ATL, HAM and in some autoimmune diseases. The HTLV‐I‐infected T cells appeared to play an important role in AS‐sIL‐6R production.

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.

Type 1 Pili Enhance the Invasion of Salmonella braenderup and Salmonella typhimurium to HeLa Cells

The relationship between type 1 pili‐associated adhesion and invasion to HeLa cells by Salmonella braenderup and S. typhimurium was studied. When the clinical isolates of these strains were grown in L‐broth, they showed both type 1 pili formation and mannose‐sensitive adhesion to HeLa cells. On the other hand, the type 1 pili‐defective mutants, which were obtained either by repeated subcultures on L‐agar plates or by the transposon Tn1‐insertion mutagenesis of the S. braenderup and S. typhimurium strains, concomitantly lost mannose‐sensitive adhesion to HeLa cells. When the HeLa cells were incubated with Salmonella, the type 1 piliated strains invaded the HeLa cells with much higher infection rate than did the type 1 pili‐defective strains. The invasion of type 1 piliated strains to HeLa cells was markedly inhibited in the presence of d‐mannose. The infectivity of the strain, which lost type 1 pili but still had mannose‐resistant adhesion, was slightly higher than that of the strains defective in both mannose‐sensitive and mannose‐resistant adhesion. These results suggested that type 1 pili have a role in enhancing the invasion of S. braenderup and S. typhimurium to HeLa cells.

Altered interleukin-2 receptor alpha-chain is expressed in human T-cell leukaemia virus type-I-infected T-cell lines and human peripheral blood mononuclear cells of adult T-cell leukaemia patients through an alternative splicing mechanism.

A polymerase chain reaction (PCR) method was used to detect the interleukin‐2 receptor α‐chain (IL‐2Rα) chain which lacks the conventional transmembrane (TM) domain in mRNA from human T‐cell leukaemia virus type‐I (HTLV‐I) ‐infected cell lines or peripheral blood mononuclear cells (PBMC) isolated from adult T‐cell leukaemia (ATL) patients. Primer pairs encompassing the TM domain were selected to generate a 357‐base pair (bp) fragment. A 146‐bp PCR product was observed consistently in addition to the target 357‐bp PCR product in mRNA from HTLV‐I‐infected cell lines, such as MT‐1, MT‐2, MT‐4 and in PBMC isolated from ATL patients. However, this 146‐bp PCR product was undetectable in HTLV‐I‐negative cell lines. The product was also detected in PBMC from normal individuals if activated in vitro with phytohaemagglutinin but not without stimulation. DNA sequence analyses revealed that exons from 5 to 7, which define a 211‐bp region containing the conventional TM domain, were deleted in the 146‐bp PCR product. The C‐terminal amino acid sequence starting from Gly174 of the 211‐bp‐deleted molecule was distinct from that of conventional IL‐2Rα as a result of an altered reading frame. We identified a 45 000 MW peptide generated from IL‐2Rα mRNA through this exon skip in cell lysate of MT‐1 and MT‐2 by Western blot analyses using an antibody raised against the peptides specific to an altered IL‐2Rα. Our results indicate that an altered IL‐2Rα chain is expressed in HTLV‐I‐infected T lymphocytic cell lines and in ATL patients.

Hodgkin's lymphoma cells are efficiently engrafted and tumor marker CD30 is expressed with constitutive nuclear factor-κB activity in unconditioned NOD/SCID/γcnull mice

As there are very few reproducible animal models without conditioning available for the study of human B‐cell‐type Hodgkins lymphoma (HL), we investigated the ability of HL cells to induce tumors using novel NOD/SCID/γcnull (NOG) mice. Four human Epstein–Barr virus‐negative cell lines (KM‐H2 and L428 originated from B cells, L540 and HDLM2 originated from T cells) were inoculated either subcutaneously in the postauricular region or intravenously in the tail of unmanipulated NOG mice. All cell lines successfully engrafted and produced tumors with infiltration of cells in various organs of all mice. Tumor cells had classical histomorphology as well as expression patterns of the tumor marker CD30, which is a cell surface antigen expressed on HL. Tumor progression in mice inoculated with B‐cell‐type, but not T‐cell‐type, HL cells correlated with an elevation in serum human interleukin‐6 levels. Tumor cells from the mice also retained strong nuclear factor (NF)‐κB DNA binding activity, and the induced NF‐κB components were indistinguishable from those cultured in vitro. The reproducible growth behavior and preservation of characteristic features of both B‐cell‐type and T‐cell‐type HL in the mice suggest that this new xenotransplant model can provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth, and to develop novel anticancer therapies. (Cancer Sci 2005; 96: 466–473)