1 Department of Bacteriology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
2 Department of Microbiology, Fujita Health University, 1-98 kutsukake-cho, Toyoake, Aichi 470-1192, Japan
3 Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan
4 Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
5 Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri 099-2493, Japan
Correspondence
Keiji Oguma
kuma{at}md.okayama-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB232927.
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INTRODUCTION |
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The mechanism of the adjuvant activity of HA1 and HA3b was investigated by in vitro testing. As Ab production is controlled by many cytokines, the level of cytokines produced by normal spleen cells that had undergone stimulation with HA1 or HA3b was assayed. Of the many cytokines, interleukin 4 (IL4), IL6 and gamma interferon (IFN-) were considered because of the limit of preparation volume and their significance in Ab production: IL4 is known as a B-cell growth factor (Paul, 1991
), IL6 is a pleiotropic cytokine that plays a crucial role in the immune response, including differentiation of B cells into immunoglobulin-secreting cells (Hirano, 1998
), and IFN-
has a role in immunoregulation and macrophage activation, as well as in autoimmunity and cell-mediated cytotoxicity (Bach et al., 1997
). As IL6 production was increased significantly by HA1 and HA3b (as shown in Results), its mRNA transcription level and type of proliferated cells were then determined. Furthermore, as production of IL6 or other cytokines is mediated through several mitogen-activated protein kinase (MAPK) pathways [signal-regulated kinase (ERK) 1/2, p38 MAPK, phosphatidylinositol 3-kinase (PI3-K) and protein kinase A (PKA)], the effects of several inhibitors of these pathways on IL6 production were also observed (Arbibe et al., 2000
; Birkenkamp et al., 2000
; Bode et al., 1998
; Dahle et al., 2004
; Tuyt et al., 1999
).
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METHODS |
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Preparation of recombinant HA subcomponents.
We first cloned the genes for whole HA from type B Lamanna and determined the whole nucleotide sequence (GenBank accession no. AB232927). Thereafter, each HA subcomponent was expressed in Escherichia coli (BL21) as a glutathione S-transferase (GST)-fusion protein. First, recombinant plasmids for each subcomponent were constructed. Templates were generated by PCR with purified DNA from type B Lamanna. All oligonucleotides were synthesized by ESPEC Oligo Service Corporation. The following primer sequences were used: BHA1-F, 5'-CCGGAATTCCGGCATGGAACACTATTCAACAATCCAAAA-3'; BHA1-R, 5'-CCGCTCGAGCGGTTATGGGTTACTCATAGTCCATATC-3'; BHA2-F, 5'-CCGGAATTCCGGCATGTCAGCTGAAAGAACTTTTCTAC-3'; BHA2-R, 5'-CCGCTCGAGCGGTTATATTTTTTCAAGTTTGAACATTTGAT-3'; BHA3b-F, 5'-CGCGGATCCGCGGTTAGTAGCACACAACGAGTATTG-3'; BHA3b-R, 5'-CCGCTCGAGCGGTTAATTAGTAATATCTATATGCAATTTTAT-3'.
The PCR fragments were digested as follows: BHA1, EcoRIXhoI; BHA2, EcoRIXhoI; BHA3b, BamHIXhoI; and cloned into pGEX-6P-3 (Amersham Biosciences) restricted with the same enzymes. The sequences of the constructs were verified by using an ABI PRISM 3100 genetic analyser (Applied Biosystems). The resulting plasmids were then introduced into E. coli and the GST-fusion proteins were expressed and affinity-purified with glutathionesepharose 4B (Amersham Biosciences). After dialysis against a cleavage buffer, GST was removed by a PreScission protease (Amersham Biosciences) treatment. Finally, the purified HA subcomponents were obtained by reapplying to the glutathionesepharose 4B column. From these preparations, contaminating lipopolysaccharides were removed by two successive applications to Detoxi-Gel columns (Pierce). All preparations after this step contained <50 endotoxin units (mg protein)1, as determined in a Limulus amoebocyte-lysate assay (BioWhittaker) (de Haan et al., 2002; Gao & Tsan, 2003
).
SDS-PAGE and immunoblot analyses.
SDS-PAGE was performed on a 12·5 % separating gel according to the method of Laemmli (1970) and molecular masses were determined by using Perfect Protein Markers (6·5200 kDa; Bio-Rad). Protein bands were visualized by staining with Coomassie brilliant blue (CBB) R-250 (Bio-Rad). For immunoblot analysis, proteins were separated by SDS-PAGE and then electroblotted onto PVDF membranes (Trans-Blot Transfer Medium; Bio-Rad) with a semi-dry blotting apparatus (Nippon Eido). After treating with 10 % skimmed milk in PBS (PBSskimmed milk) at 4 °C overnight and then washing with PBS containing 0·05 % Tween 20 (PBSTween), the membrane was reacted for 1 h at 37 °C with 1 : 1000-diluted rabbit polyclonal Abs. Two different Ab preparations were used. One is anti-16S (toxoid) and the other one is the mixture of three sera against HA1, HA2 and HA3b, which had been prepared by immunization with each GST-free recombinant subcomponent. The membrane was then washed, treated with 1 : 10 000-diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Jackson Immunoreseach) for 1 h at 37 °C, washed again and, finally, the reacted bands were detected on a scientific imaging film (Kodak) by an enhanced chemiluminescenceWestern blotting detection reagent (Amersham Biosciences).
Immunization protocol.
Detoxified NTX, 12S or 16S (2 µg in 0·25 ml in 10 mM PBS, pH 7·4) and NTX mixed with an equal amount(s) of NTNHHA or different HA subcomponents (total, 2 µg in 0·25 ml) were employed as immunogens as listed in Fig. 2. Each preparation was injected subcutaneously six times at 1-week intervals into the dorsal side of ten female BALB/c mice (68 weeks of age; Charles River) that were maintained under specific-pathogen-free conditions in Okayama University vivarium. Also, Alb was used as immunogen alone or in combination with HA1, HA2 or HA3b. The amounts injected were the same as described above, but each preparation was injected into five mice three times at 1-week intervals (Fig. 3
). Six days after the last injection, the mice were sacrificed and blood was collected for serum. This immunization schedule was based on the assumed treatment of patients with dystonia, i.e recurrent injection of a low dose of antigen (toxin).
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Cytokine levels in splenocyte cultures.
Spleen cells were removed aseptically from normal mice and adjusted to 4x106 cells ml1 in RPMI 1640 medium (Gibco) supplemented with 10 % fetal calf serum. Cell suspension (0·1 ml) was distributed into each well of 96-well tissue-culture plates (Cellstar), followed by the addition of 0·1 ml of the different antigens (NTX, 16S, NTNHHA, HA1, HA2, HA3b, HA1+HA3b or HA1+HA2+HA3b) as shown in Figs 4 and 5, previously diluted with the medium to 2, 10, 20, 50 or 100 µg ml1. After incubation for 48 h at 37 °C in a 5 % CO2 incubator, the supernatant of each well was collected separately and amounts of IFN-
, IL4 and IL6 were assayed by quantitative sandwich enzyme immunoassay (AN'ALYZA). Three wells were prepared for each antigen and their readings were averaged. Finally, these were expressed as protein amounts with reference to a standard curve prepared from the results of recombinant mouse cytokines.
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RESULTS AND DISCUSSION |
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Anti-NTX Ab production by immunization with different antigens
Each of the different antigen preparations [toxoids of NTX, 12S and 16S, NTX+non-toxic component (NTNHHA), NTX+HA1, NTX+HA2, NTX+HA3b, NTX+HA1+HA3b or NTX+HA1+HA2+HA3b] were injected into 10 mice and their immunoglobulin and IgG levels against NTX were determined by ELISA. The levels of both immunoglobulin and IgG differed only slightly among the 10 mice immunized with the same antigens and were averaged. As expected, the immunoglobulin level was higher than that of IgG and similar findings were obtained for the other antigen preparations. The mean Ab levels of the mice immunized with 16S or NTX+NTNHHA were significantly higher than those of mice immunized with NTX alone or with 12S, even though the amounts of NTX in the preparations of 16S or NTX+NTNHHA were low (in 16S toxin, the amount of NTX was estimated as approximately one-half to one-eighth that of NTX alone). Similarly, the Ab levels of mice immunized with NTX+HA1, NTX+HA3b, NTX+HA1+HA3b or NTX+HA1+HA2+HA3b were higher than those immunized with NTX alone or NTX+HA2 (Fig. 2a and b). Following immunization with NTX, NTX+HA1, NTX+HA2, NTX+HA3b or 16S (2 µg), end-point ELISA titres were determined. For 16S, NTX+HA1 and NTX+HA3b, end-point titres were quite high (32006400) compared with NTX and NTX+HA2 (approx. 100). These data indicate that the immunogenicity of NTX is increased by HA, especially by HA1 and HA3b. Neither NTNH nor HA2 seemed to have such adjuvant activity. The Ab levels differed only slightly in the sera from mice immunized with NTX+HA1, NTX+HA3b or NTX+HA1+HA3b, indicating that adjuvant activity of HA1 and HA3b is similar and the activity may not be increased dramatically by mixing HA1 and HA3b.
Anti-Alb Ab production by immunization with different antigens
Mice were immunized with Alb with or without mixing with one of the HA subcomponents (HA1, HA2 or HA3b) and their Ab levels against Alb were determined by ELISA. Each antigen was injected into five mice. The Ab levels of five mice immunized with the same antigens differed only slightly and were averaged. The mean Ab levels of the mice immunized with Alb+HA1 or Alb+HA3b were significantly higher than those of mice immunized with Alb alone or Alb+HA2 (Fig. 3a and b). Therefore, it was concluded that the immunogenicity of Alb is increased, again, by HA1 and HA3b.
Cytokine production from spleen cells stimulated with different antigens
Spleen cells obtained from non-immunized mice were incubated for 48 h in the presence of different antigens (NTX, NTNHHA, 16S, HA1, HA2, HA3b, HA1+HA3b or HA1+HA2+HA3b) in 96-well plates and the levels of IL6, IL4 and IFN- were assayed. The level of IL4 differed only slightly for all of the antigen-stimulated preparations (data not shown), whereas IL6 levels were significantly different: HA1>HA1+HA2+HA3b, HA1+HA3b, 16S, HA3b>NTNHHA, HA2>NTX (Fig. 5
). In the case of IFN-
, production was clearly increased only by stimulation with 16S (Fig. 4
). Therefore, it is strongly suggested that HA1, HA3b and 16S, which has these two HA subcomponents, demonstrate adjuvant activity mainly by increasing the production of IL6 from lymphocytes. The reason why only 16S increased IFN-
production is not clear. This may be attributed to the tertiary structure of 16S having NTNH that is not formed by the preparations of NTX mixed with any HA subcomponents (the combination of NTX and HA does not occur without NTNH).
The transcription levels of mRNA for IL6 in the spleen cells stimulated with NTX, 16S, NTX+HA1 or NTX+HA3b were estimated by RT-PCR with G3PDH as a reference (Fig. 6). The levels of transcription were visibly increased in the presence of 16S (25 µg) and NTX (12·5 µg) mixed with equal amounts of HA1 or HA3b compared with NTX stimulation alone (12·5 or 25 µg). The specificity of IL6 production was further confirmed by observing the effects of signal-transduction inhibitors on IL6 production (Fig. 7
). IL6 production with any stimulants was remarkably inhibited by SB202190, Ly294002 and KT5720, but not by PD98059. Our data show clearly that the IL6 production was mediated by p38 MAPK, PI3-K and PKA, but not by ERK1/2, in spleen cells.
Flow-cytometry analysis
As the production of IL6 in spleen cells was increased by stimulation with 16S, HA1 and HA3b, the type of proliferated cells was analysed by flow cytometry using labelled Abs against CD3 and CD19. The percentage of CD19-positive cells was higher following stimulation by 16S and NTX mixed with HA1 or HA3b compared with stimulation with NTX alone (Fig. 8). The percentage of CD3-positive cells remained unchanged (data not shown).
Based on these results, it is concluded that HA1 and HA3b have adjuvant activity, probably by increasing IL6 production. As both HA1 and HA3b are components of the 16S toxin molecule, this may explain why higher levels of anti-NTX Abs are induced following immunization with 16S than with NTX alone. IL6 production may be provoked either in monocytes that phagocytose HA1 and HA3b or in lymphocytes, or in both. Recently, we found that HA1 has two -trefoil domains similar to the B-chain (lectin) of ricin, and that HA3b has the sialoadhesin (Siglec) family motif. The former binds to galactose and the latter binds to sialic acid of glycoprotein and/or glycolipid of red blood cells and epithelial cells of the small intestine (Fujinaga et al., 2004
; Inoue et al., 2003
). HA1 and HA3b may also bind to lymphocytes. Such binding might provoke the signal to produce IL6, leading to proliferation of Ab-producing cells (CD19-positive cells) and, finally, high levels of Ab production. From the experiment using signal-transduction inhibitors, it became clear that p38 MAPK, PI3-K and PKA are involved in IL6 production. We are now investigating the detailed mechanism of how IL6 is produced by stimulation with HA1 and HA3b.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 9 August 2005;
accepted 11 August 2005.
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