Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Detection of Expressed IL-32 in Human Stomach Cancer Using ELISA and Immunostaining

  • Seo, Eun-Hee (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kang, Jeong-Woo (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Ki-Hong (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Cho, Min-Chul (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Lee, So-Jung (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Hee-Jong (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Jung-Hee (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Eun-Jin (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Park, Dong-Ki (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Soo-Hyun (Department of Biomedical Science, Konkuk University) ;
  • Choi, Yang-Kyu (College of Veterinary Medicine, Konkuk University) ;
  • Kim, Jin-Man (Department of Pathology, Chungnam National University College of Medicine) ;
  • Hong, Jin-Tae (College of Pharmacy, Chungbuk National University)
  • Published : 2008.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Interleukin (IL)-32 is a recently identified proinflammatory cytokine that is one of the IL-18 inducible genes, and plays an important role in autoimmune and inflammatory diseases. We produced antibodies against IL-32 and studied the expression of IL-32 in human stomach cancer. We detected IL-32 secreted from K-562 cells which were stably transfected with IL-32 and in the sera of stomach cancer patients by a sandwich ELISA using a monoclonal antibody KU32-52 and a polyclonal antibody. In order to optimize a sandwich immunoassay, recombinant IL-32a was added, followed by the addition of a biotinylated KU32-52 into microtiter plate wells precoated with a goat anti-IL-32 antibody. The bound biotinylated KU32-52 was probed with a streptavidin conjugated to HRP. This sandwich ELISA was highly specific and had a minimal detection limit of 80 pg/ml (mean${\pm}$SD of zero calibrator) and measuring up to 3,000 pg/ml. This ELISA showed no cross-reaction with other cytokines such as hIL-1$\alpha$, hIL-1$\beta$, hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-$\alpha$. Intra-assay coefficients of variation were 18.5% to 4.6% (n=10), and inter-assay coefficients were 23% to 9% (n=10). The average IL-32 level in the sera of 16 stomach cancer patients (189 pg/ml) was higher than that of 12 healthy control men (109 pg/ml). Our results indicate that serum IL-32 level can be detected by using an established ELISA, and that this immunoassay and mAb KU32-09 specific for immunohistochemistry can be used in the detection of expressed and secreted IL-32 in stomach cancer patients.

Keywords

References

  1. Banda, N. K., A. Vondracek, D. Kraus, C. A. Dinarello, S. H. Kim, A. Bendele, G. Senaldi, and W. P. Arend. 2003. Mechanisms of inhibition of collagen-induced arthritis by murine IL-18 binding protein. J. Immunol. 170: 2100-2105 https://doi.org/10.4049/jimmunol.170.4.2100
  2. Bazan, J. F., J. C. Timans, and R. A. Kastelein. 1996. A newly defined interleukin-1? Nature 379: 591 https://doi.org/10.1038/379591a0
  3. Cagnard, N., F. Letourneur, A. Essabbani, V. Devauchelle, S. Mistou, A. Rapinat, C. Decraene, C. Fournier, and G. Chiocchia. 2005. Interleukin-32, CCL2, PF4F1 and GFD10 are the only cytokine/chemokine genes differentially expressed by in vitro cultured rheumatoid and osteoarthritis fibroblast-like synoviocytes. Eur. Cytokine Netw. 16: 289-292
  4. Chen, Q., H. P. Carroll, and M. Gadina. 2006. The newest interleukins: Recent additions to the ever-growing cytokine family. Vitam. Horm. 74: 207-228 https://doi.org/10.1016/S0083-6729(06)74008-0
  5. Cho, H. J., J. Y. Cho, M. H. Rhee, H. S. Kim, H. S. Lee, and H. J. Park. 2007. Inhibitory effects of cordycepin (3'-deoxyadenosine), a component of Cordyceps militaris, on human platelet aggregation induced by thapsigargin. J. Microbiol. Biotechnol. 17: 1134-1138
  6. Cho, Y., J. Kang, M. Cho, C. Cho, S. Lee, Y. Choe, et al. 2001. Down modulation of IL-18 expression by human papillomavirus type 16 E6 oncogene via binding to IL-18. FEBS Lett. 501: 139-145 https://doi.org/10.1016/S0014-5793(01)02652-7
  7. Dahl, C., R. Schall, H. He, and J. Cairns. 1992. Identification of a novel gene expressed in activated natural killer cells and T cells. J. Immunol. 148: 597-603
  8. Dinarello, C. A. 2004. Therapeutic strategies to reduce IL-1 activity in treating local and systemic inflammation. Curr. Opin. Pharmacol. 4: 378-385 https://doi.org/10.1016/j.coph.2004.03.010
  9. Goda, C., T. Kanaji, S. Kanaji, G. Tanaka, K. Arima, S. Ohno, and K. Izuhara. 2006. Involvement of IL-32 in activationinduced cell death in T cells. Int. Immunol. 18: 233-240 https://doi.org/10.1093/intimm/dxh339
  10. Huh, S., K. Lee, H. S. Yun, D. J. Paik, J. M. Kim, and J. Youn. 2007. Functions of metallothionein generating interleukin- 10-producing regulatory CD4+ T cells potentiate suppression of collagen-induced arthritis. J. Microbiol. Biotechnol. 17: 348-358
  11. Joosten, L. A., M. G. Netea, S. H. Kim, D. Y. Yoon, B. Oppers- Walgreen, T. R. Radstake, et al. 2006. IL-32, a proinflammatory cytokine in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 103: 3298-3303
  12. Kim, S. H., S. Y. Han, T. Azam, D. Y. Yoon, and C. A. Dinarello. 2005. Interleukin-32: A cytokine and inducer of TNF-alpha. Immunity 22: 131-142
  13. Kojima, H., M. Takeuchi, T. Ohta, Y. Nishida, N. Arai, M. Ikeda, H. Ikegami, and M. Kurimoto. 1998. Interleukin-18 activates the IRAK-TRAF6 pathway in mouse EL-4 cells. Biochem. Biophys. Res. Commun. 244: 183-186 https://doi.org/10.1006/bbrc.1998.8236
  14. Kundu, M. and J. Basu. 2006. IL-32: An emerging player in the immune response network against tuberculosis? PLoS Med. 3: e274 https://doi.org/10.1371/journal.pmed.0030274
  15. Mallat, Z., A. Corbaz, A. Scoazec, P. Graber, S. Alouani, B. Esposito, Y. Humbert, Y. Chvatchko, and A. Tedgui. 2001. Interleukin-18/interleukin-18 binding protein signaling modulates atherosclerotic lesion development and stability. Circ. Res. 89: E41-E45 https://doi.org/10.1161/hh1901.098735
  16. Min, C. K., Y. Maeda, K. Lowler, C. Liu, S. Clouthier, D. Lofthus, E. Weisiger, J. L. Ferrara, and P. Reddy. 2004. Paradoxical effects of interleukin-18 on the severity of acute graft-versushost disease mediated by CD4 and CD8 T cell subsets after experimental allogeneic bone marrow transplantation. Blood 104: 3393-3399 https://doi.org/10.1182/blood-2004-02-0763
  17. Nakanishi, K. 2001. Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine Growth Factor Rev. 12: 53-72 https://doi.org/10.1016/S1359-6101(00)00015-0
  18. Nam, K. S., M. K. Kim, and Y. H. Shon. 2007. Inhibition of proinflammatory cytokine-induced invasiveness of HT-29 cells by chitosan oligosaccharide. J. Microbiol. Biotechnol. 17: 2042-2045
  19. Netea, M. G., T. Azam, G. Ferwerda, S. E. Girardin, M. Walsh, J. S. Park, et al. 2005. IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL- 1beta and IL-6 production through a caspase 1-dependent mechanism. Proc. Natl. Acad. Sci. USA 102: 16309-16314
  20. Netea, M. G., T. Azam, E. C. Lewis, L. A. Joosten, M. Wang, D. Langenberg, et al. 2006. Mycobacterium tuberculosis induces interleukin-32 production through a caspase-1/IL-18/interferongamma- dependent mechanism. PLoS Med. 3: e277 https://doi.org/10.1371/journal.pmed.0030277
  21. Okamura, H., H. Tsutsui, S. Kashiwamura, T. Yoshimoto, and K. Nakanishi. 1998. IL-18: A novel cytokine that augments both innate and acquired immunity. Adv. Immunol. 70: 281-312 https://doi.org/10.1016/S0065-2776(08)60389-2
  22. Okamura, H., H. Tsutsui, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto, et al. 1995. Cloning of a new cytokine that induces interferon-gamma. Nature 378: 88-91 https://doi.org/10.1038/378088a0
  23. Plater-Zyberk, C., L. A. Joosten, M. M. Helsen, P. Sattonnet- Roche, C. Siegfried, S. Alouani, et al. 2001. Therapeutic effect of neutralizing endogenous IL-18 activity in the collageninduced model of arthritis. J. Clin. Invest. 108: 1825-1832 https://doi.org/10.1172/JCI200112097
  24. Pomerantz, B. J., L. L. Reznikov, A. H. Harken, and C. A. Dinarello. 2001. Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1beta. Proc. Natl. Acad. Sci. USA 98: 2871-2876
  25. Shioya, M., A. Nishida, Y. Yagi, A. Ogawa, T. Tsujikawa, S. Kim-Mitsuyama, et al. 2007. Epithelial overexpression of interleukin-32alpha in inflammatory bowel disease. Clin. Exp. Immunol. 149: 480-486 https://doi.org/10.1111/j.1365-2249.2007.03439.x
  26. Shoda, H., K. Fujio, Y. Yamaguchi, A. Okamoto, T. Sawada, Y. Kochi, and K. Yamamoto. 2006. Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases. Arthritis Res. Ther. 8: R166 https://doi.org/10.1186/ar2074
  27. Siegmund, B., J. A. Sennello, H. A. Lehr, G. Senaldi, C. A. Dinarello, and G. Fantuzzi. 2004. Frontline: Interferon regulatory factor-1 as a protective gene in intestinal inflammation: Role of TCR gamma delta T cells and interleukin-18 binding protein. Eur. J. Immunol. 34: 2356-2364 https://doi.org/10.1002/eji.200425124