Seiji Mita

Elevation of circulating interleukin 6 after surgery: Factors influencing the serum level

To investigate the effect of surgical trauma and other factors on the postoperative elevation of serum interleukin 6 (IL-6), we examined changes in IL-6 concentration after major thoracoabdominal surgery. Serum IL-6 levels reached the maximum concentration on the first postoperative day in all 38 patients, with peak ranging from 1400.8 +/- 383.4 pg/ml (mean +/- SEM) to 29.8 +/- 3.8 among six groups who underwent surgery at different sites. The IL-6 peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during surgery (r = 0.554, P < 0.01 and r = 0.427, P < 0.01, respectively). The peak concentration of serum IL-6 in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (P < 0.05), despite a similar degree of surgical trauma defined by the operation length and volume of blood loss during surgery. Peak IL-6 concentration observed in a patient who underwent esophagectomy was about 100-fold greater in fluid drained from the thorax than in the peripheral blood. IL-6 mRNA was demonstrated in leukocytes from thoracic and abdominal exudate at 6, 24 and 48 h after surgery. In contrast, IL-6 mRNA could not be detected in leukocytes from the peripheral blood. Similar findings were also observed for interleukin 8 (IL-8). However, interleukin 1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) were detected only once after surgery in the drainage fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

T Cell‐Replacing Factor (TRF)/Interleukin 5 (IL‐5): Molecular and Functional Properties

TRF has originally been defined as a T-cell-derived lymphokine that triggers activated B cells for a terminal differentiation into Ig-secreting cells. HPLC-purified TRF from Sup of a murine TRF-producing B151 cell is an acidic glycoprotein, exerts BCGF II activity and induces expression of IL-2 receptors. It does not show IL-1, IL-2, IL-3, BSF-1/IL-4, or IFN gamma activity. We prepared monoclonal TB13 and NC17 antibodies against HPLC-purified B151-TRF which are specific for and can inhibit TRF as well as BCFG II activity of B151-TRF. Moreover, TB13 as well as NC17 antibody can immunoprecipitate the 46 Kd molecule from B151 Sup which exerts TRF as well as BCGF II activity. Complementary DNA (pSP6K-mTRF23) encoding for murine TRF/IL-5 was cloned and its entire nucleotide sequences were determined. The murine TRF/IL-5 cDNA encodes 133 amino acids including N-terminal strongly hydrophobic regions. Secreted recombinant TRF/IL-5 (apparent m.w. of 46 Kd) has 113 amino acid residues and also comprises homodimers of a molecule with an apparent m.w. of 25 to 30 Kd. TRF/IL-5 mRNA is constitutively expressed in constitutively TRF-producing B151 and is inducible in some T cell lines upon stimulation with PMA or Con A. TRF/IL-5 mRNA is also expressed in Tbc-primed T cells upon the stimulation with PPD, whereas its expression is not effectively induced in non-primed spleen cells by stimulation with Con A or PMA plus calcium ionophore. The translation product of murine TRF/IL-5 cDNA triggers resting as well as activated (DNP-primed or LPS-stimulated) murine B cells for terminal differentiation into Ig-secreting cells (IgM, IgG1, or IgA) accompanied by increased mRNA expression for secreted forms of relevant Ig heavy chain (mu, gamma, or alpha). Among these, increases in the level of mu, and alpha-specific mRNA for the secreted form of IgM and IgA, respectively, are prominent. Moreover, TRF/IL-5 induces maturation of resting B cells into IgM-secreting cells. TRF/IL-5 promotes growth of activated B cells as well as BCL1 cells. TRF/IL-5 is, therefore, a growth as well as a differentiation inducing factor for B cells. Moreover, it induces functional IL-2 receptors on resting as well as activated B cells, besides TRF and BCGF II activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular cloning and expression of the murine interleukin-5 receptor.

Murine interleukin‐5 (IL‐5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL‐5 receptor by expression screening of a library prepared from a murine IL‐5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti‐IL‐5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N‐terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL‐5 with a single class of affinity (KD = 2–10 nM). FDC‐P1 cells transfected with the cDNA for murine IL‐5 receptor showed the expression of IL‐5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL‐5 for proliferation, although parental FDC‐P1 cells did not show any detectable IL‐5 binding. In addition, several cDNA clones encoding soluble forms of the IL‐5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL‐5. Homology search for the amino acid sequence of the IL‐5 receptor reveals that the IL‐5 receptor contains a common motif of a cytokine receptor family that is recently identified.

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor

Murine interleukin‐5 (IL‐5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL‐5 receptor (IL‐5‐R) is composed of at least two membrane protein subunits and is responsible for IL‐5‐mediated signal transduction. One subunit of the high affinity IL‐5‐R is a 60 kDa membrane protein (p60 IL‐5‐R) whose cDNA was isolated using the anti‐IL‐5‐R monoclonal antibody (mAb), H7. This subunit alone binds IL‐5 with low affinity. The second subunit does not bind IL‐5 by itself, and is expressed not only on IL‐5‐dependent cell lines but also on an IL‐3‐dependent cell line, FDC‐P1. Expression of the p60 IL‐5‐R cDNA in FDC‐P1 cells, which do not bind IL‐5, reconstituted the high affinity IL‐5‐R. We have characterized the second subunit of the IL‐5‐R by using another anti‐IL‐5‐R mAb, R52.120, and the anti‐IL‐3‐R mAb, anti‐Aic‐2. The anti‐Aic‐2 mAb down‐regulated binding of IL‐5 to an IL‐5‐dependent cell line, Y16. Both R52.120 and anti‐Aic‐2 mAbs recognized membrane proteins of 130–140 kDa expressed on FDC‐P1 and Y16 cells. The R52.120 mAb recognized both murine IL‐3‐R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL‐5‐R was reconstituted on an L cell transfectant co‐expressing AIC2B and p60 IL‐5‐R, whereas only the low affinity IL‐5‐R was detected on a transfectant co‐expressing AIC2A and p60 IL‐5‐R.(ABSTRACT TRUNCATED AT 250 WORDS)

Curcumin Inhibits Interleukin 8 Production and Enhances Interleukin 8 Receptor Expression on the Cell Surface Impact on Human Pancreatic Carcinoma Cell Growth by Autocrine Regulation

Curcumin, the yellow pigment in turmeric, has been shown to prevent tumor progression in a variety of tissues in rodents. The authors investigated the effect of curcumin on human carcinoma cell lines to determine whether constitutive interleukin‐8 (IL‐8) production of tumor cells was correlated with nuclear factor κB (NF‐κB) activation and cell growth activity.

Characterization of the human IL-5 receptors on eosinophils

Interleukin 5 (IL-5) receptors on the cell surface of human eosinophils and other hematopoietic cells were characterized using radiolabeled recombinant IL-5. The binding of 35S-labeled murine IL-5 to eosinophils from normal human peripheral blood was rapid and saturable within a 30-min incubation at both 4 and 37 degrees C. The binding of 35S-labeled murine IL-5 to eosinophils was inhibited by an excess of unlabeled murine and human IL-5 or by an anti-murine IL-5 monoclonal antibody (NC17) but not by other human cytokines. Scatchard plot analysis revealed that human eosinophils have a single class of high affinity receptor (Kd 170-330 pM; number of binding sites: 260-380/cell). IL-5 receptors on eosinophils from four patients with eosinophilia displayed similar characteristics. Affinity cross-linking experiments resulted in the identification of human IL-5 receptor on eosinophils with a molecular mass of 55-60 kDa. Among the various cells besides eosinophils and cell lines that we could test, a subline of HL-60 (YY-1 cells) was found to display a significant number of IL-5 receptor. These results suggest that IL-5 may act on limited types of cells in the human system.

Establishment of IL-5-Dependent Early B Cell Lines by Long-Term Bone Marrow Cultures

We established two different IL-5-dependent Ly1+ early B cell lines in long-term bone marrow culture system. One of them (J-87) is stromal cell (ST2) dependent and the other (T-88) is ST2 independent. Both J-87 and T-88 are B220+, Ly1+, sIgM-, Ia-, Thy1-, and IL-2R+, and respond to IL-3 and IL-5 in the presence of ST2. The T-88 can proliferate only in response to IL-5 in the absence of ST2. Southern blot analysis using JH probe revealed that configuration of IgH gene of both cell lines shows rearranged pattern. Binding assay for radiolabeled IL-5 to T-88 demonstrated that T-88 has two classes of IL-5 binding sites (low and high affinity) on the membrane. These data strongly suggest that there are IL-5-sensitive stages at both stromal cell-dependent and stromal cell-independent phases in early B cell development.

Interleukin-8 is constitutively and commonly produced by various human carcinoma cell lines

SummaryWe examined the production of interleukin-8 and interleukin-6 by 30 human carcinoma cell lines. Serum levels of interleukin-8 were measured in 14 patients with hepatocellular carcinoma by enzyme-linked immunosorbent assay and Northern blotting. Furthermore, serum interleukin-8 was also investigated in a nude mouse bearing a tumor of the HuH7 hepatoma cell line producing interleukin-8. Of the 30 cell lines, 29 (96.7%) constitutively produced interleukin-8, and 19 of the 29 (65.5%) were high producers (>1 ng/ml culture supernatant). Among the high producers, 4 cell lines released both interleukin-8 and interleukin-6. Interleukin-6 was constitutively produced by 17 of the 30 (56.7%) cell lines, 4 of which (23.5%) were high producers (>1 ng/ml). By Northern blot analysis, mRNAs of interleukin-8 and interleukin-6 were detected in producing cell lines. Of 14 patients with hepatocellular carcinoma, 4 (28.5%) showed increased levels of serum interleukin-8. Furthermore, inoculation of the HuH7 hepatoma cell line which produced the highest amount of interleukin-8 into a nude mouse resulted in tumor production accompanied by an elevated level of human interleukin-8 (646 pg/ml) in the peripheral blood. Thus, interleukin-8 is constitutively and commonly produced by various carcinoma cell lines. The production of interleukin-8 by carcinoma cells may be related to the elevation of serum interleukin-8 in patients with hepatocellular carcinoma. Finally, these cell lines may be valuable for studying the relationship between interleukin-8 and cancer.

The sequence of vessel ligation affects tumor release into the circulation

OBJECTIVE Whether the sequence of pulmonary vein and artery ligation in pulmonary lobectomy for carcinoma affects intraoperative hematogenous cancer cell dissemination is not known. We examined whether vessel ligation sequence affects the presence of circulating cancer cells as reflected by carcinoembryonic antigen messenger ribonucleic acid. METHODS We assayed for the transcripts of carcinoembryonic antigen messenger ribonucleic acid by reverse-transcriptase polymerase chain reaction in peripheral blood taken before, during, and after operation from 30 patients with non-small-cell lung cancer who underwent a curative lobectomy and from six patients with limited-stage small-cell lung cancer who were treated initially with chemotherapy followed by lobectomy. Each patient was randomly assigned before the operation to have either pulmonary vein ligation or pulmonary artery ligation first. Blood taken from 10 patients with interstitial pulmonary fibrosis who underwent an open lung biopsy and 41 healthy subjects served as a control. RESULTS No control samples were positive for transcripts. Sixteen of the preoperative blood samples from the 30 patients with non-small-cell cancers were positive. Of these 16, eight samples remained positive even after lobectomy was performed; the remaining eight samples (four in each ligation group) became negative. Of the 14 initially negative samples (seven in each ligation group), nine samples became positive during the operation. Such conversion during the operation was more common with arterial ligation first (six patients, 85.7%) than with venous ligation first (three patients, 42.9%). Samples from all six patients with small-cell cancer were positive before the operation, and five of six samples remained positive after the operation. CONCLUSIONS Many patients with non-small-cell lung cancer have systemic disease even when they were thought to have resectable tumors. Ligating the pulmonary vein before ligating the artery may lessen intraoperative hematogenous dissemination. Most small-cell lung cancers represent systemic disease even when considered resectable.

Structural comparison of murine T-cell (B151K12)-derived T-cell-replacing factor (IL-5) with RIL-5: Dimer formation is essential for the expression of biological activity

T-cell-replacing factor (TRF)/IL-5 is a T-cell-derived glycoprotein which has pleiotropic activity on lymphoid and myeloid cells. IL-5 polypeptide translated into Xenopus oocytes are heterogeneous in molecular size (40,000 to 60,000 under nonreducing conditions) and yields a monomeric form (Mr of 25,000 to 30,000) under reducing conditions (J. Immun., 140, 1175-1181, 1988). We purified T-cell-derived TRF and rIL-5 using anti-TRF/IL-5 antibody-coupled affinity column from supernatants of a T-cell hybridoma B151K12 and supernatants of HeLa cells, respectively, which had been transfected with murine IL-5 cDNA, and determined their partial N-terminal amino acid sequence (27 residues for B151-TRF and 13 residues for rIL-5). A single amino acid sequence of each sample obtained beginning from methionine that was identical to that predicted from IL-5 cDNA. This finding supports the notion that secreted B151-TRF polypeptide consists of 113 amino acids. Purified B151-TRF supported eosinophilopoiesis of human bone marrow cells as effective as mouse rIL-5 and human rIL-5. B151-TRF competitively inhibited 35S-labeled rIL-5 binding to target cells to the same extent at rIL-5. Treatment of purified rIL-5 and B151-TRF with reducing reagents such as 2-ME, sodium borohydride or dithiothreitol produced a monomeric form of IL-5 which did not exert a biological activity. Reduction and alkylation of rIL-5 caused the loss of binding to its target cells. These results strongly suggest that B151-TRF exists as a homodimer and its primary structure and secondary structures are identical to those of rIL-5. Moreover, the formation of inter-molecular disulfide bond(s) linked by two pairs of cystein residues is essential for the expression of the biological activity of mouse IL-5.