1 First Department of Internal Medicine, Sapporo Medical University, South 1 West 17, Sapporo 060-8543, Japan
2 Kirin Pharmaceutical Laboratory, Takasaki 370-1295, Japan
3 La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA
Correspondence to: T. Nakano, Jikeikai Hospital, 6-50 Asahigaoka 5, Chuo-ku, Sapporo 064-8575, Japan
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Abstract |
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Keywords: aging, malignant tumor, rheumatoid arthritis, Ts cell
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Introduction |
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Bioactive 13 kDa human GIF has been detected in culture supernatants of some human T cell hybridomas specific for bee venom phospholipase A2 (6). Upon stimulation with an anti-TCR ß antibody, some of the GIF-producing T cell hybridomas released GIF having affinity for the specific antigen. However, the antigen-specific human GIF has not been biochemically identified. Molecular cloning of murine and human GIF indicated that GIF of both species are 13 kDa peptides of 115 amino acids, and that human GIF has 90% homology with murine GIF at the amino acid level (7). Unexpectedly, various cell lines, including Th hybridomas and B cell lines, contained mRNA that hybridized with GIF cDNA, and culture supernatants of these cell line cells contained a 13 kDa peptide that bound anti-GIF in immunoblotting. However, the peptide from these cell line cells lacked bioactivity, and only the peptide from Ts hybridomas had the activity (8). Recombinant GIF expressed in Escherichia coli and COS1 cells were also inactive (7). Since the nucleotide sequence of the GIF cDNA from the Th hybridomas and B cell lines was identical to the GIF cDNA from Ts cells, we speculated that the bioactivity of GIF peptides was generated by post-translational modifications of the peptide in Ts cells (8). Indeed, more recent studies have provided evidence that cysteinylation of Cys60 in the inactive GIF, which causes conformational changes in the GIF molecules, is responsible for the generation of bioactive GIF (9,10).
In the present study, we obtained polyclonal antibodies against E. coli.-derived recombinant human GIF and established an ELISA system for the detection of GIF in human sera. The results show that a measurable quantity of GIF is present in most human sera, and indicate that serum GIF in aged normal individuals and in patients with rheumatoid arthritis or malignant tumors represents a 55 kDa molecule having both the GIF determinant and the TCR determinant.
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Methods |
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Rabbits were immunized by i.m. injection of 100 µg of the rhGIF peptide included in complete Freund's adjuvant, followed by seven booster injections of the peptide with incomplete Freund's adjuvant. The IgG fraction of the antiserum was employed to detect GIF by ELISA. The fraction was biotinylated using sulfosuccinimidyl N-(D-biotinyl)-6-aminohexanoate (Dojindo, Kumamoto, Japan) following the manufacturer's protocol.
Anti-human TCR antibody (H-20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The preparation is affinity-purified goat polyclonal antibody raised against a peptide mapping at the C-terminus of TCR
of human origin.
Measurement of the serum GIF level by ELISA
The serum concentration of GIF was measured using a sandwich ELISA system as follows. Each well of a 96-hole microtiter plate (Maxi Sorp; Nunc, Roskilde, Denmark) was coated with 100 µl of the IgG fraction of rabbit anti-rhGIF at 1 µg/ml in 50 mM sodium bicarbonate buffer (pH 9.0) and left overnight at 4°C. The plate was washed 4 times with PBS containing 0.05% Tween 20 (Sigma, St Louis, MO) (PBST) and the wells were rinsed with PBS. The plate was washed between each of the following steps. The wells were blocked with Super Block (Pierce, Rockford, IL) at 4°C overnight. Serum samples were diluted 1/10 with PBS, and these samples (100 µl) were added to the wells and incubated at 4°C overnight. Recombinant hGIF diluted with PBS containing 0.5% BSA was used as a standard. Then, 100 µl of 0.5 µg/ml biotinylated anti-rhGIF in PBST was added and incubated at 4°C overnight. The plates were then incubated with 100 µl of 1/5000 dilution of horseradish peroxidase (HRP)streptavidin (Zymed, San Francisco, CA) for 2 h at room temperature. TMB solution (Dako, Carpinteria, CA) was added to each well to develop the ELISA signals and after incubation for 30 min at room temperature, 1 N HCl/3 N H2SO4 was added to each well to stop the reaction. ELISA signals were determined by absorption at 450 nm and GIF concentration was calculated from a standard curve drawn by using rhGIF of known concentrations.
Subjects
The subjects employed in this study were 362 healthy donors (71 males and 291 females, aged 2099 years), 26 patients with allergic diseases (seven males and 19 females; aged 2085 years; median age, 32 years), 39 patients with malignant tumor (20 males and 19 females; aged 20100 years; median age, 76 years) and 47 patients with rheumatoid arthritis (eight males and 39 females; aged 1974 years; median age, 51 years).
Among the 362 adult healthy donors, most of the donors aged 2069 years were staff workers of hospitals, nursing homes and laboratories, and most of the aged subjects (over 70 years old) were residents of old-age homes. All healthy donors were free of acute infection and immunological diseases. Many of the aged subjects were receiving medication for hypertension, hyperlipidemia, sleeplessness, osteoporosis or angina pectoris. Among the 26 patients with allergic diseases, 14 suffered from allergic rhinitis, 13 suffered from allergic dermatitis and six suffered from bronchial asthma (some patients had two or more symptoms.). Diagnosis of rheumatoid arthritis was made using the 1987 criteria of the American College of Rheumatology (12). Patients were clinically active and did not meet the criteria for clinical remission of rheumatoid arthritis (13). The mean duration of the disease was 6 years ± 1 month (range, 3 months to 28 years). To assess the radiological bone damage in the patients with rheumatoid arthritis, Larsen's damage score, which indicates the degree of bone erosion of 20 joints of the fingers, was employed (14). The rates of progression in Larsen's damage score were calculated by dividing the difference in the damage scores obtained at two different times by the number of months between the scoring. The 39 malignant tumor cases included seven cases of stomach cancer, four cases of lung cancer, four cases of hepatocellular carcinoma, four cases of colorectal cancer, three cases of multiple myeloma, two cases of malignant lymphoma, two cases of pancreas cancer, two cases of cholangiocarcinoma, two cases of renal cell carcinoma, two cases of prostate cancer, one case of esophageal cancer, one case of breast cancer, one case of skin cancer, one case of thyroid cancer, one case of metastatic liver cancer of unknown origin, one case of lung cancer with hepatocellular carcinoma and one case of acute leukemia. All of these cases were in the advanced stage.
Identification of serum GIF by immunoblotting
Serum GIF were analyzed by SDSPAGE and immunoblotting. Serum samples were diluted 1/50 with PBS and 25 µl of each sample, mixed with the same volume of sample buffer, was electrophoresed in an 18% polyacrylamide slab gel under reducing conditions at 20 mA for 45 h. The peptides were then transferred to a PVDF membrane (Immobilon; Millipore, Bedford, MA). After blocking with 2% BSA in PBS for 1 h at room temperature, the membrane was incubated with 1 µg/ml of the IgG fraction of anti-rhGIF antiserum in PBST at 4°C overnight. After washing, the membrane was incubated with a 1/4000 dilution of HRP-conjugated anti-rabbit Ig (Amersham, Little Chalfont, UK) and HRPstreptavidin (Zymed), and the specific signals were visualized using an enhanced chemiluminescence Western blot detection system (Amersham).
The same method was used for the detection of peptides with the TCR chain determinant except for the use of goat polyclonal anti-human TCR
chain antibody and HRP-conjugated anti-goat Ig (Dako).
Affinity chromatography was employed to confirm the presence of peptides with the TCR chain determinant in human serum. Affinity-purified anti-human TCR
chain antibody was coupled to HiTrap NHS-activated Sepharose (Pharmacia Biotech) following the manufacturer's protocol. A serum sample (5 ml) was diluted 1/10 in PBS and applied to 1 ml of a TCR
chain-coupled column and the flow-through fraction was circulated at 4°C overnight through the column. After washing with a 30 column volume of PBS, proteins retained in the column were eluted with 0.1 M glycineHCl buffer (pH 3.0). After adjusting pH to 7.0, the fraction was added with 0.1% SDS and dialyzed against PBS. The sample was then analyzed by SDSPAGE and immunoblotting with anti-rhGIF antibody.
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Results |
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We speculated that GIF might be present in normal human sera and measured the GIF levels in the sera of the 362 healthy adult donors. As shown in Fig. 2, GIF was detectable in many of the normal serum samples. It was also found that the GIF level in normal serum tends to increase with advancing age. Although no significant differences were found among the 20s, 30s, 40s, and 50s age groups or among the 60s, 70s, 80s and 90s age groups, GIF levels in the 20s and 30s age groups were significantly lower than those in the 60s and older age groups.
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However, we found that the serum GIF levels had markedly increased in some patients with rheumatoid arthritis. Measurement of the levels of GIF in sera from 47 patients with rheumatoid arthritis indicated that the patients had significantly higher serum GIF levels (18.96 ± 2.19 ng/ml) than the age-matched controls of healthy individuals(4.53 ± 0.53 ng/ml) (Fig. 3).
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We also measured the serum GIF concentrations in serum samples from patients with malignant tumor. As shown in Fig. 4, the serum GIF levels in the patients with malignant tumor were higher (13.61 ± 2.93 ng/ml) than those in age-matched healthy donors (5.07 ± 0.56 ng/ml). Four patients with extremely high levels of serum GIF suffered from invasion of cancer and/or metastasis. Indeed, among the 17 patients with a serum GIF level of 10 ng/ml or higher, 11 had metastasis, while only one of the 22 patients with a serum GIF level of less than 10 ng/ml had metastasis.
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We also analyzed several serum samples obtained from healthy aged individuals by SDSPAGE and immunoblotting. In all of the serum samples with a GIF level of 10 ng/ml or higher, the 55 kDa protein with both the GIF determinant and the TCR chain determinant was detected, but the 13 kDa GIF was not detected in any of the samples analyzed (results not shown). We did not pursue the experiments to detect the 55 kDa GIF in the serum samples containing less than 10 ng/ml GIF by ELISA, because non-specific bands appeared in immunoblotting by using a 1:20 dilution of serum samples (see Methods).
To confirm that the 55 kDa GIF in human serum binds to anti-TCR chain antibody, we absorbed a serum of a patient with breast cancer, that had a GIF level of 98 ng/ml, with an anti-TCR
chain antibody-coupled Sepharose column, and the acid eluate of the column was analyzed by SDSPAGE and immunoblotting with anti-GIF. As shown in Fig. 7
, the 55 kDa protein with the GIF determinant was detected in the acid eluate, indicating that the protein possessed both the GIF antigenic determinant and the TCR
chain determinant.
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Discussion |
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In our previous studies using murine Ts hybridomas and homologous transfectants of TCR chain cDNA in the hybridoma (4,5), which formed antigen-specific GIF upon stimulation with anti-CD3, we obtained evidence that the murine 55 kDa GIF is a subunit of antigen-specific GIF and represents a post-translationally formed conjugate of a TCR
chain with a 13 kDa GIF peptide (5). The association of both the GIF determinant and TCR
chain determinant with the human 55 kDa GIF indicates that the human GIF protein detected in the present experiments is a human homologue of the murine 55 kDa GIF.
Previous studies by Thomas et al. (6) on human GIF have shown that human Ts hybridomas specific for bee venom phospholipase A2 constitutively secreted the 13 kDa GIF and produced antigen-specific GIF upon stimulation with anti-CD3. The molecular size of the antigen-specific GIF was estimated to be ~80 kDa by gel filtration, but biochemical identification of the protein was unsuccessful (15). Thus, the present findings were the first demonstration of human 55 kDa GIF, which may represent a subunit of antigen-specific GIF.
Another important finding obtained in the present study is that the 55 kDa GIF is formed in vivo. It should be noted that all previous studies on the murine 55 kDa GIF were carried out using Ts hybridomas and their homologous transfectants with the TCR chain cDNA and the protein was detected in the culture supernatant of anti-CD3-stimulated cells. The presence of the human homologue in the sera of healthy aged individuals suggests that the 55 kDa GIF is formed in vivo under physiological conditions.
The association of the TCR chain determinant with the human GIF indicates that the cell source of the 55 kDa GIF is probably Ts cells, which bear TCR
ß. In the murine system, the 55 kDa GIF was formed only after stimulation of the Ts hybridomas with antigen-pulsed antigen-presenting cells or anti-CD3 (4,5), which facilitated translocation of the cytosolic 13 kDa GIF into the endoplasmic reticulum (16). When Th cell clones and Th hybridomas were stimulated with anti-CD3, the translocation of inactive cytosolic GIF to subcellular organelles did not occur and these cells failed to form the 55 kDa GIF. If the same principles apply to the human system, the cell source of the human 55 kDa GIF described here would be Ts cells, which constitutively secrete 13 kDa bioactive GIF.
The results of previous experiments on OVA-specific murine Ts hybridomas and a homologous transfectant of TCR chain cDNA in the hybridoma indicated that the 55 kDa GIF is a product of TCR
chain cDNA (5). When the OVA-specific effector-type Ts hybridoma cells were stimulated by antigen-pulsed APC, essentially all 55 kDa GIF released from the cells was associated with a peptide having the TCR ß antigenic determinant (4). However, the homologous transfectant secreted both the TsF and 55 kDa GIF, the latter of which is not associated with the TCR ß+ peptide (5). The results indicated that association of the TCR ß chain is not required for the secretion of 55 kDa GIF from Ts cells. Studies by Kuchroo et al. (17) on the inducer-type murine Ts hybridomas suggested that the inducer-type TsF, i.e. TsF1, is a single chain molecule having the TCR
determinant but lacking the TCR ß chain determinant. They also showed that the TCR
chain gene, but not the TCR ß chain gene, is required for the formation of TsF1 (18). Further studies are required to determine whether the 55 kDa GIF detected in human sera is associated with the TCR ß chain to form an effector-type TsF or whether it represents a single chain molecule. In any event, it seems likely that the 55 kDa GIF detected in human sera in the present study is a product of antigen-specific Ts cells and may represent TsF or its subunit. Previous studies have shown that the murine 55 kDa GIF is bioactive (5) and that the bioactive GIF has immunoregulatory effects on Ig synthesis (19). If the same principle applies to the human GIF system, it is reasonable to speculate that the 55 kDa GIF in human sera has immunosuppessive activities.
It is surprising that the 13 kDa GIF peptide was not detected in human sera in which 55 kDa GIF was detected. In our previous experiments on murine Ts hybridomas, one half or more of the 13 kDa GIF in culture supernatants of the cells was of an inactive form (20). After stimulation of the cells with anti-CD3 or antigen-pulsed APC, the majority of the bioactive 13 kDa GIF appears to be utilized for the formation of the 55 kDa GIF (5), but culture supernatant of the stimulated cells contained much more inactive 13 kDa GIF than bioactive 55 kDa GIF. Lack of the 13 kDa GIF in human sera suggests that the 13 kDa GIF peptide is easily metabolized in vivo.
In vivo effect of human 55 kDa GIF is not known. However, the increase in the 55 kDa GIF in the sera of patients with malignant tumors suggests that antigen-specific GIF is formed in these patients and plays a role in the immunosuppressive state in such patients. Takahashi et al. (21) reported that Ts cells are generated in melanomas in mice. The 55 kDa GIF may also be involved in the decrease in immune responsiveness in aged individuals. Further studies are required for elucidation of the mechanisms involved in the formation of 55 kDa GIF in vivo, and for identification of its bioactivity.
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Abbreviations |
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APC antigen-presenting cell |
GIF glycosylation inhibiting factor |
HRP horseradish peroxidase |
OVA ovalbumin |
TsF suppressor T cell factor |
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Notes |
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Transmitting editor: M. Taniguchi
Received 21 February 2000, accepted 25 May 2000.
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References |
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