EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 41, No. 4, 288-295, April 2009 Nickel induces secretion of IFN-γ by splenic natural killer cells Ji-Yeon Kim1, Kyungmin Huh1, antibody. These results suggest that there is an early Ki-Young Lee1, Jun-Mo Yang2 and rapid innate immune response to nickel, which is and Tae Jin Kim1,3 mediated by NK cells and the NKG2D receptor. The sig- nificance of the innate response to nickel is that it may 1Division of Immunology contribute to development of the late T cell-mediated Department of Molecular Cell Biology and delayed type hypersensitivity against nickel. Samsung Biomedical Research Institute Sungkyunkwan University School of Medicine Keywords: hypersensitivity; immunosorbent techni- Suwon 440-746, Korea ques; interferon-γ; killer cells, natural; nickel; NK cell 2Department of Dermatology lectin-like receptor subfamily K Sungkyunkwan University School of Medicine Samsung Medical Center Introduction Seoul 135-710, Korea 3 Corresponding author: Tel, 82-31-299-6161; Nickel is commonly found in coins, jewelry, and Fax, 82-31-299-6179; E-mail, [email protected] many other surrounding materials, but nickel is the DOI 10.3858/emm.2009.41.4.032 most common occupational as well as environ- mental contact allergen (Basketter et al., 1993; Zug Accepted 1 December 2008 et al., 2008). Nickel in metal prostheses used in orthopedic, dental, and other surgeries may also Abbreviations: CDR, complementary determining region; ELISPOT, cause immune responses to nickel, preventing the enzyme-linked immunosorbent spot; NK cell, natural killer cell; RAG, patients from successfully maintaining the prosthe- recombination activating gene; SPF, specific pathogen-free; Th1, sis (Saglam et al., 2004; Hallab et al., 2005). In T helper 1 contact with biological system, nickel metal corro- des and releases nickel ions, the most common and stable form of which is Ni2+ and is mostly Abstract present as NiCl2 or NiSO4, the nickel salts com- monly used in the study of nickel allergy. About Although nickel hypersensitivity is known as a de- 20% of people show the immune responses to layed-type hypersensitivity mediated by nickel-spe- nickel ions in the skin patch test and some of them cific T cells, it is greatly influenced by other immune clinically develop nickel contact dermatitis (Mortz et cells. Here we show that splenic natural killer cells (NK al., 2001; Schafer et al., 2001; de Waard-van der cells) directly or indirectly respond to nickel by secre- Spek and Oranje, 2008). tion of IFN-γ. Using enzyme-linked immunosorbent The immunological mechanisms underlying the spot (ELISPOT) assays, we found that nickel-reactive allergic response to nickel are not well known, but cells readily secreted IFN-γ when splenocytes were cul- the formation of metal-protein complexes are con- tured in the presence of varying concentrations of nick- sidered to elicit immune responses since a nickel el sulfate (NiSO4) for 24 h. However, nickel-reactive ion is too small to be specifically recognized by T IL-2- or IL- 4-secreting cells were infrequent during the and B cell antigen receptors or immune receptors. Similarly to chemical haptens, metal ions interact 24-h culture with NiSO4. Immune responses to nickel with proteins and then become recognizable by T were innate, not adaptive, in nature since the frequency and B lymphocytes (Budinger and Hertl, 2000; of nickel-reactive IFN-γ-secreting cells did not increase Martin, 2004). Whereas the description of nickel- upon previous exposure to NiSO4 and recombination specific antibodies is rare probably due to the activating gene (RAG)-1-deficient mice contained diversity of proteins conjugated to nickel ion, several nickel-reactive IFN-γ-secreting cells. The involvement groups reported the presence of nickel-specific T of NK cells in the innate response to NiSO4 was con- cells (Kapsenberg et al., 1987; Silvennoinen-Kassi- firmed since we could observe a significant reduction nen et al., 1991; Sebastiani et al., 2002; Gamer- of the frequency of nickel-reactive cells in NK cell-de- dinger et al., 2003). Those nickel-specific CD4+ pleted mice. Furthermore, the number of IFN-γ secret- and CD8+ T cells may explain the immunological ing cells was significantly reduced in the ELISPOT as- pathogenesis of nickel contact dermatitis and says when NKG2D was blocked by anti-NKG2D immune response to nickel-containing metal im- NK cell response to nickel sulfate 289 plants since nickel-specific T cells have been re- cells) by depleting NK cells or blocking a repre- peatedly detected in sensitized human and animals. sentative NK receptor, NKG2D. The immune response to nickel is regarded as an example of delayed type hypersensitivity model, especially if nickel was applied subcutaneously Results (Ishii et al., 1995). However, it is still not clear how initial immune Abundant nickel-responsive IFN-γ secreting cells in responses to nickel can appear before the gene- mouse spleen ration of nickel-reactive memory T cells. To avoid inappropriate immune responses to self compo- To investigate the nickel hypersensitivity, we first nents, the induction of T cell immune response assessed the frequencies of nickel-reactive cells requires preceding innate immune responses to among splenocytes from untreated 6 week-old invading microorganisms or tissue damages C57BL/6 mice by ELISPOT assay. Since the (Kroczek et al., 2004). Innate immune responses ELISPOT assay was performed by 24-h stimulation against pathogen- or danger-associated molecular with NiSO4 and naive T cells usually require at patterns provide costimulatory signals to antigen- least a few days for immune response, it was specific T cells (Matzinger, 2002; Akira et al., 2006), expected that only memory nickel-reactive T cells but if the costimulatory signals are not sufficient, could respond to nickel ions and secrete cytokines. antigen-specific T cells do not respond or are swit- We measured the numbers of IL-2-, IL-4-, and ched to become tolerant (Hawiger et al., 2004; IFN-γ-secreting cells since T cells participating in Goodnow et al., 2005). To the best of our kno- the immune response to nickel could be either Th1 wledge, there were no reports that showed innate or Th2 cells (Artik et al., 2001; Sebastiani et al., immune responses against metal ions. In fact, the 2002). Total splenic cells were isolated and then 1 nickel allergy was difficult to be established in the × 105 cells were cultured in each well of ELISPOT mouse model and could be more efficiently indu- plates with complete RPMI 1640 media and varying ced by the concomitant administration of lipopoly- concentrations of NiSO4 for 24 h. Contrary to our saccharide, a bacterial molecular pattern molecule expectation, a high frequency of nickel-reactive (Sato et al., 2007). In the current study, we inten- cytokine-secreting cells were detected upon NiSO4 ded to measure nickel-responsive T cells in the stimulation in non-sensitized mice (Figure 1). mouse spleen by using enzyme-linked immuno- Although there were IL-2- or IL-4-secreting cells in sorbent spot (ELISPOT) analyses and unexpectedly the spleen, the IFN-γ-secreting cells outnumbered found that there were a lot of nickel- reactive IFN-γ IL-2- or IL-4- secreting cells. The number of secreting cells among splenic cells even in the IFN-γ-secreting cells increased in a concentration- non-immunized mice. We further investigated the dependent manner as we assayed with increasing nature of nickel-reactive cells and provided evi- concentrations of NiSO4. Whereas the numbers of dences that they included natural killer cells (NK IL-2- or IL-4-secreting cells decreased in high Figure 1. A high frequency of nick- el-responsive cells in mouse spleen. Single cell resolution of nick- el-induced IL-2, IL-4, and IFN-γ pro- duction in freshly isolated splenic cells as verified by measuring cyto- kine production using the ELISPOT assay. Each well was coated with given anti-cytokine antibodies, plat- ed with 1 × 105 spleen cells, and incubated for 24 h in the presence of 200 μM NiSO4. Each cytokine-se- creting cells were visualized by the biotinylated cytokine detection anti- body, streptavidin-HRP, and HRP substrate. 290 Exp. Mol. Med. Vol. 41(4), 285-295, 2009 concentrations of NiSO4 (≥ 400 μM), the number did not significantly increase in mice immunized of IFN-γ-producing cells remained high in high with NiSO4 for either 2 or 4 weeks. These findings concentrations of NiSO4 (Figure 2). We could further support that nickel-reactive IFN-γ-secreting observe more than 100 IFN-γ-secreting cells per 1 cells shown in naive mice are innate cells and that × 105 cells when splenocytes were cultured in the they are not expanding upon the exposure to concentration of NiSO4 ≥ 200 μM. NiSO4. With these results, we hypothesized that nickel- reactive cells might be innate immune cells such γ as NK cells and γδ T cells, since they have a IFN- -secreting cells are also seen in RAG-1-deficient feature of memory lymphocytes and are respon- mice and reduced in number by depletion of NK cells ding to given antigens without previous sensiti- Although nickel-reactive IFN-γ-secreting cells had zation. Especially, we paid attention to IFN-γ-secre- characteristics of innate cells, there is still a possi- ting cells, which might be early-reacting NK cells. bility that they may be innate T cells such as NKT cells, γδ T cells or intraepithelial T cells. Since these innate T cells are absent from recombination No increase in the number of IFN-γ-secreting cells activating gene (RAG)-1-deficient mice, we assessed upon previous immunization of NiSO4 nickel-reactive IFN-γ- secreting cells in spleens The presence of a high frequency of IFN-γ- from RAG-1-deficient mice (Figure 4).