• E-mail
• Print
• Comment
• Font Size
• Digg
• del.icio.us
• Discuss article

Recombinant Perchloric Acid-Soluble Protein Suppresses The Immunoglobulin Production Of Human-Human Hybridoma Hb4c5 Cells

Posted on: Thursday, 26 February 2004, 06:00 CST

SUMMARY

Because perchloric acid-soluble protein (PSP) has been conserved evolutionally in various species from Escherichia coli to humans, it may reflect an involvement in basic cellular regulation. However, the precise function of PSP is currently unknown. In this study, we examined the direct effect of PSP on the production of immunoglobulin (Ig) using human B, HB4C5, NAT-30, and U266 cells because it has been reported that subcutaneous administration of PSP affects rodent immune systems. Suppression of Ig productivity and decrement of the cell viability was recognized only in HB4C5 cells by the addition of PSP into the medium. On the other hand, PSP had no effect on Ig productivity and cell viability in NAT-30 and U266 cells. In addition, PSP was clearly incorporated by HB4C5 but not by the other cells. These results suggest that the Ig production suppressed by PSP, which has been previously reported to inhibit protein synthesis, contributed to the incorporation of PSP into the HB4C5 cells.

Key words: human B cell lines; enzyme-linked immunosorbent assay; laser-scanning confocal microscopic analysis; protein synthesis.

Perchloric acid-soluble protein (PSP) isolated from rat liver encodes 137 amino acid residues and has a molecular mass of 14,149 Da (Oka et al., 1995). Thereafter, PSP homologs were isolated from mice (Samuel et al., 1997), goat (Ceciliani et al., 1996), human (Schmiedeknechl et al., 1996), chicken (Nordin et al., 2001), and pig (Kaneki et al., 2003). The nucleotide sequences of these complementary deoxyribonucleic acids are highly similar to that of a new family (YER057c/YJGF) of small proteins that are conserved evolutionally from prokaryotes to eukaryotes. The high conservation of PSPs suggests that the PSPs play an important role in cellular physiological functions. The characters of PSP have been described as translation inhibitor (Oka et al., 1995; Schmiedeknecht et al., 1996), heat response protein (Samuel et al., 1997), calpain activator (Melloni et al., 1998), corepressor of PurR (Rappu et al., 1999), and regulator of isoleucine synthesis (Berlage et al., 1998). However, the precise biological role of PSP has not been elucidated yet.

It has been reported that the subcutaneous administration of goal PSP homolog, UK114, identified as tumor antigen inhibited the growth of dimethylbenzanthracene-induced rat mammary carcinomas (Racca et al., 1997; Ghezzo et al., 1999). The inhibitory activity of UK114 seems to be due to the UK114 antibody-dependent cell cytotoxicity and the activation of the complement systems (Bartorelli et al., 1994). Furthermore, Funaro et al. have identified a membrane tumor- associated antigen recognized by anti-UK114 antibody (Funaro et al., 1999). Its molecular weight was approximately 220 kDa, as determined by sodium dodecyl sulfate-polyaerylamide gel electrophoresis, and was different from that of PSP. On the other hand, Panerai et al. have studied the effect of cytokine expression on the suppression of tumor growth by UK114. As a result, the administration of UK114 into mice increased the expression of interleukin 4 (IL-4) and decreased the expression of tumor necrosis factor-[alpha], interleron [gamma] (IFN-[gamma]), and IL-2 (Panerai et al., 1999). The pattern of cytokine production seems to be divided by Th1 and Th2 cells. Th1 cells release IL-2 and IFN-[gamma], whereas Th2 cells release IL-4, IL-5, IL-6, and IL-10. Also, Th1 cells activate the macrophages, and Th2 cells enhance the production of antibody including immunoglobulin (Ig) E. Cytokines decide immunocyte-differentiation. Differentiated cells have specialized functions that are related to immuno-reaction. Because the administration of UK114 modulates the Th1-Th2 cytokine profile, UK114 seems to change the production of antibody in vivo. In this study, we examined the effect of PSP on the production of Ig in the human B cells in vitro.

FIG. 1. Effect of recombinant perchloric acid-soluble protein (rPSP) on the immunoglobulin (Ig) productivity of human B cell lines. Human B cell lines, U266 cells (open circle), NAT-30 cells (open triangle), and HB4C5 cells (closed circle) were cultured in 10 g/ml of insulin, 20 g/ml of transferin, 20 g/ml of ethanolamine, and 25 nM of selenite-ERDF medium containing various concentrations of rPSP for 6 h. After cultivation, each Ig concentration was measured by the enzyme-linked immunosorbent assay method. The relative Ig productivity represented the Ig productivity against that in the absence of rPSP. Results are the means SD of three independent measurements.

FIG. 2. Incorporation of fluorescein isothiocyanate (FITC)- conjugated recombinant perchloric acid-soluble protein (rPSP) by human B cell lines. rPSP directly labeled with FITC was used. Human B cell lines were treated with 1 g/ml of FITC-conjugated rPSP for 6 h (A, NAT-30 cells; B, U266 cells; C, HB4C5 cells). The incorporation of rPSP in human B cell lines was investigated by laser-scanning confocal microscopic analysis. Panel D shows the cross sections on the line at panel C.

Functional recombinant PSP (rPSP) produced by Escherichia coli was prepared according to the previous report (Oka et al., 1999). Human B cell lines, the IgM-producing human-human hybridoma HB4C5, the IgM-producing Burkitt lymphoma cell line NAT-30, and the IgE- producing myeloma cell line U266 were used in this study. HB4C5 cells were a fusion product of a human lymphocyte from a lung cancer patient and a human fusion partner, NAT-30. NAT-30, U266, and HB4C5 cells were cultured in enriched-HDF (ERDF) medium (Kyokuto Pharmaceutical, Japan) supplemented with 10 g/ml of insulin, 20 g/ ml of transferin, 20 g/ml of ethanolamine, and 25 nM of selenite (ITES-ERDF) at 37 C under humidified 5% CO^sub 2^-95% air (Murakami et al., 1982). Cells were inoculated at 1 10^sup 4^ cells/well in a 96-well culture plate and cultured in 200 l of medium with various concentrations of PSP for 6 h. After the cultivation, each culture medium was transferred to a 1.5-ml tube and centrifuged at 200 g for 5 min. Then, each supernatant was used for the measured amount of Ig secreted by each human B cell line. IgM and IgE concentrations were measured by enzyme-linked immunosorbent assay using anti-human IgM and IgE antibodies (TAGO, Burlingame, CA), as mentioned in a previous report (Sugahara et al., 1994). Viable cells were counted using a hemocytometer in the presence of trypan blue. All examination was measured in triplicate, and data were analyzed by Student's t-test to evaluate the significance of difference. In only HB4C5 cell line was the viable cell number slightly decreased by the addition of PSP, and each relative viable cell number was 84 5.8%, 92 9.3%, 89 6.2%, 103 11.2%, 94 5.8%, and 100 1.8% in the presence of 10-^sup 0^, 10-^sup 1^, 10^sup -2^, 10^sup -3^, and 10^sup -4^ g/ml of PSP and the absence of PSP, respectively. On the other hand, PSP did not affect the viable cell number of NAT-30 and U266 cells in the concentration range measured (data not shown). Ig productivity of each cell line was calculated as follows: Ig concentration was divided by viable cell number, and the relative Ig productivity represented the Ig productivity against that in the absence of rPSP (Fig. 1). Ig productivity oi HB4C5 was dose- dependently decreased by the addition of PSP, and the significant decrement was observed under the 10^sup -3^ g/ml of PSP (closed circle). Ig productivities of NAT-30 (open triangle) and U266 (open circle) cells did not change with the addition of PSP. These results suggest that the addition of PSP decreases the production of Ig, but its effect is specific for HB4C5 cells.

Okamoto et al. (2002) reported that the histone protein increases Ig productivity in HB4C5 but not in NAT-30, U266, and Hmy-2 cells because many histones were incorporated in HB4C5 cells but not in other cells. Although its mechanism is not elucidated in detail, PSP also may be incorporated by HB4C5 cells. Therefore, PSP was labeled with fluorescein isothiocyanate (FITC) to determine the incorporation by human B cell lines, using laser-scanning confocal microscopic (LCM) analysis. In brief, rPSP was directly labeled by FlTC (Sigma Chemical Co., St. Louis, MO). After the labeling reaction, FITC-conjugated PSP was separated from the unconjugated FITC by dialyzation against 10 mM sodium phosphate buffer (phosphate buffered-saline [PBS], pH 7.4) and sterilized by filtration before use. Human B cell lines were cultured in the medium including FITC- conjugaled rPSP (1 g/ml) for 6 h. After cultivation, cells were washed three limes with PBS, fixed for 10 min at room temperature with 50% ethanol, and mounted on slides using Permafluor (Immunotech, Marseille, France) containing 50% glycerol. Samples were observed by an LCM with an argon ion laser (488 nm) attached to an Olympus GB200 microscope (Olympus, Tokyo, Japan) und pictures were taken of the same condition. The strong signals of the FITC- conjugated rPSP were observed in HB4C5 cells (Fig. 2C) but not in NAT-30 (Fig. 2A) and U266 (Fig. 2B). Figure 2D panel shows the cross- section on the line at the panel C and rPSP was recognized in cells. These results suggest tha\t rPSP are incorporated by HB4C5 cells.

Perchloric acid-soluble protein has been reported as the inhibitor of protein synthesis in the rabbit reticulocyte lysate system (Oka et al., 1995), and this effect was due to endoribonuclease activity of PSP (Morishitu et al., 1999). In this study, the suppression of Ig production by rPSP was observed only in the HB4C5 cell line, which incorporated much rPSP. These observations suggest that the suppression of Ig production seems to be attributed to the inhibition of cellular protein synthesis by the incorporated rPSP. If it is assumed that PSP is the functional inhibitor of protein synthesis in vivo, its activity may be needed for cellular function through protein synthesis.

In conclusion, rPSP suppressed Ig production in HB4C5 cells but did not alter Ig production in the human B cell lines NAT-30 and U266. Once rPSPs are incorporated into cells, they may suppress Ig production through their ability to inhibit protein synthesis. This is the first report on the suppression of Ig production by PSP in cells.

REFERENCES

Bartorelli, A.; Berra, B.; Ronchi, S., et al. Immunochemical reactivity of mammalian liver anligen (UK101) in human tumors and non neoplastic tissues. J. Tumor Marker Oncol. 9:37-47; 1994.

Berlage, J. L.; Langendorf, M. J.; Downs, D. M. Complex metabolic phenotype caused by a mutation in yjgF, encording a member of the highly conserved YER057c/YjgF family of proteins. J. Bacteriol. 180:6519-6528; 1998.

Ceciliani, F.; Faotto, L.; Negri, A., et al. The primary structure of UK-114 tumor antigen. FEBS Lett. 893:147-150; 1996.

Funaro, A.; Horenstein, A. L.; Ghisolfi, G., et al. Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114. monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient. Exp. Cell Res. 247 (Suppl. 2):441-450; 1999.

Ghezzo, F.; Berta, G. N.; Bussolati, B., et al. Perchloric acid- soluble proteins from goat liver inhibit chemical carcinogenesis of Syrian hamster cheek-pouch carcinoma. Br. J. Cancer. 79 (Suppl. 1):54-58; 1999.

Kaneki, K.; Matsumoto, M.; Suzuki, K., et al. Purification characterization and developmental expression of pig liver PSP. Comp. Biochem. Physiol. B 134:571-578; 2003.

Melloni, E.; Michelli, M.; Salamino, F., et al. Molecular and functional properties of a calpain activator protein specific for [mu]-isoforms. J. Biol. Chem. 273:12827-12831; 1998.

Morishita, R.; Kawagoshi, A.; Sawasaki T., et al. Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis. J. Biol. Chem. 274:20688-20692; 1999.

Murakami, H.; Masui, H.; Sato, G. H., et al. Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. Proc. Natl. Acad. Sci. USA 79 (Suppl. 4):1158-1162; 1982.

Nordin, H.; Matsumoto, M.; Suzuki K., et al. Purification, characterization and developmental expression of chick (Gallus domesticus) liver PSP protein. Comp. Biochem. Physiol. B 128:135- 143; 2001.

Oka, T.; Nishimoto, Y.; Sasagawa, T., et al. Production of functional rat liver PSP protein in Escerichia coli. Cell. Mol. Life Sci. 55:131-134; 1999.

Oka, T.; Tsuji, H.; Noda, C., et al. Isolation and characterization of a novel perchloric acid-soluble protein inhibiting cell-free protein synthesis. J. Biol. Chem. 270:30060- 30067; 1995.

Okamoto, T.; Sugahara, T.; Tachibana, H., et al. Increase of immunoglobulin productivity of human-human hybridoma HB4C5 cells by histone. Biosci. Biotechnol. Biochem. 66 (Suppl. 6):1241-1245; 2002.

Panerai, A. E.; Sacerdote, P.; Bianchi, M., et al. Chronic administration of UK-114, a multifunctional emerging protein, modulates the Th1/Th2 cytokine pattern and experimental autoimmune diseases. Ann. NY Acad. Sci. 876:229-235; 1999.

Racca, S.; Di Carlo, F.; Bartorelli, A., et al. Growth inhibition of DMBA-induced rat mammary carcinomas by UK 114. Virchows Arch. 431 (Suppl. 5):323-328; 1997.

Rappu, P.; Shin, B. S.; Zalkin, H., et al. A role of highly conserved protein of unknown function in regulation of Bacillus subtilis purA by the purine repressor. J. Bacteriol. 181:3810-3815; 1999.

Samuel, S. J.; Tzung, S. P.; Cohen, S. A. Hrp12, a novel heat- responsive, tissue-specific, phosphorylated protein isolated from mouse liver. Hepatology. 25 (Suppl. 5):1213-1222; 1997.

Schmiedeknecht, G.; Kekhoff, C.; Orso, E., et al. Isolation and characterization of 14.5 kDa trichloroacetic acid-soluble translational inhibitor protein from human monocytes that is upregulated upon cellular differentiation. Eur. J. Biochem. 242:339- 351; 1996.

Sugahara, T.; Sasaki, T.; Murakami, H. Enhancement of immunoglobulin productivity of human-human hybridoma HB4C5 cells by basic proteins and poly-basic amino acids. Biosci. Biotechnol. Biochem. 58 (Suppl. 12):2212-2214; 1994.

HIROAKI KANOUCHI,1 AYA MATSUO, TATSUZO OKA, HIROFUMI TACHIBANA, AND KOJI YAMADA

Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan (H. K., A. M., H. T., K. Y.) and Department of Veterinary Physiology, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan (T. O.)

(Received 24 July 2003; accepted 4 September 2003)

1 To whom correspondence should be addressed at Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki- city, Okayama 701-0192, Japan. E-mail: kano@med.kawasaki-m.ac.jp

Copyright Society for In Vitro Biology Jul/Aug 2003

More News in this Category