* Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305-8506, Japan;
Domestic Research Fellow, Japan Society for the Promotion of Science, Tokyo 102-0083, Japan;
CREST, Japan Science and Technology, Kawaguchi 332-0012, Japan; and
Department of Immunology, National Institute of Infectious Diseases, Tokyo 162-0044, Japan
Received January 22, 2003; accepted April 23, 2003
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ABSTRACT |
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Key Words: TCDD; germinal center; high-affinity antibody-forming cell; high-affinity antibody; immune suppression; humoral immunity.
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INTRODUCTION |
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In the primary humoral immune response, B cells differentiate along two distinct pathways in response to protein-based antigens (reviewed by Kelsoe, 2000). After the activation of antigen-specific B cells by antigen-specific T cells in secondary lymphoid tissues such as the spleen, one pathway leads to the differentiation of antibody-forming cells (AFCs) that secrete IgM. Afterward, AFCs producing downstream immunoglobulin (Ig) classes such as IgG1 are generated. These AFCs secrete low-affinity antibodies encoded by variable gene segments that have not yet been mutated. The other differentiation pathway leads to the formation of germinal centers (GCs) and the subsequent generation of high-affinity AFCs. The activated B cells proliferate vigorously and form GCs. In these GCs, B cells expressing Igs with variable affinities for the antigen are generated by somatic hypermutations of the Ig genes. The cells with improved affinities are positively selected and continue on to become high-affinity AFCs in the early GC formation, while memory B cells are generated in the late GC formation. Within these reactions, cells with low-affinity Igs are eliminated by apoptosis. These processes, which increase the affinity of antibodies, are considered to be important for the effective and rapid elimination of an antigen in the primary humoral immune response.
Previous studies have demonstrated the suppressive effects of TCDD on the IgM response to antigen exposure (Harper et al., 1994; reviewed by Holsapple et al., 1991
; Smialowicz et al., 1996
). The fact that the antibody response against a T-cellindependent (TI) antigen is suppressed by TCDD (Harper et al., 1994
; Smialowicz et al., 1996
) suggests that B cells are the direct targets of TCDD toxicity. As regards the specific site of suppression, TCDD has been reported to inhibit IgM secretion by B cells, which are activated by anti-Ig antibody and growth factors, or by a superantigen in vitro; however, TCDD has been shown to exert only a slight effect on the proliferation of these activated B cells (Luster et al., 1988
; Wood and Holsapple, 1993
). These previous studies suggest that TCDD directly inhibits the terminal stage of the B cell response during low-affinity antibody production. Furthermore, Sulentic et al. (2000)
recently proposed, based on their results obtained using CH12.LX cells, that TCDD suppresses µ gene expression as well as IgM secretion by inducing the binding of the AhR to the xenobiotic-responsive element within the Ig heavy chain 3
-enhancer.
On the other hand, in prior studies, helper T-cell function was also suggested as a target of TCDD toxicity in the suppression of antibody production. The IgM response to a T-celldependent (TD) antigen has been reported to be more sensitively suppressed than that to a TI antigen (Kerkvliet et al., 1990). The splenocyte antibody response in nu/nu athymic mice was more resistant than that observed in nu/+ littermates (Kerkvliet and Brauner, 1987
). Since the interaction of helper T cells with antigen-stimulated B cells is indispensable for inducing not only the differentiation of low-affinity AFCs, but also for GC formation and subsequent B-cell differentiation into high-affinity AFCs (Garside et al., 1998
; Han et al., 1995
; Perez-Melgosa et al., 1999
; Takahashi et al., 1998
), the inhibition of T-cell function by TCDD may cause the suppression of high-affinity AFC production. Although previous studies have reported that TCDD suppresses IgG-class antibody production (Harper et al., 1994
; Ito et al., 2002
; Kerkvliet et al., 1996
; Lundberg et al., 1991
; Shepherd et al., 2000
; Warren et al., 2000
), it is not clear if only low-affinity IgG is sensitive to TCDD, or if high-affinity IgG production is also affected by TCDD. In the latter case, high-affinity AFCs may be affected in a mechanism that is different from low-affinity AFCs. To clarify whether and how TCDD affects the high-affinity antibody response to antigens, we carried out this study, focusing on the formation of GC, the generation of high-affinity AFCs, and the production of high-affinity antibodies in the primary humoral immune response of C57BL/6 mice.
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MATERIALS AND METHODS |
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Mouse treatments.
TCDD (purity, 98%) obtained from Cambridge Isotope Laboratory (Andover, MA) was a concentration of 50 µg/ml in nonane. The TCDD/nonane solution was further diluted with corn oil to render a dose volume of 10 µl/g body weight. Ovalbumin (OVA) (Grade VII; Sigma, St. Louis, MO) was dissolved in phosphate-buffered saline (PBS) at a concentration of 1 mg/ml. An equal volume of 9% (w/v) AlK(SO4)2 solution was added to the OVA solution and the pH was adjusted to 6.5 with KOH. After repeated washings with PBS, the alum-precipitated OVA (OVA/alum) was resuspended in PBS at 0.5 mg OVA/ml. The mice were administered a single dose of TCDD (20 µg/kg) or vehicle by gavage and were subsequently immunized intraperitoneally with 100 µg of OVA/alum. On a specified day postimmunization, the animals were sacrificed, and their plasma samples and spleen were examined. In some experiments (4-hydroxy-3-nitrophenyl) acetyl linked to chicken -globulin (NP-CG) (NP:CG conjugation ratio 16:1) (Takahashi et al., 2001
) was used instead of OVA, and on a selected day the plasma samples, spleen, and bone marrow (BM) were examined.
Enzyme-linked immunosorbent assay.
For the determination of OVA-specific IgM levels, 96-well plates were incubated overnight at 4°C after being coated with 1 mg/ml of OVA in PBS and were blocked with 3% BSA in PBS for 1 h at 37°C. The plates were incubated at room temperature for 2 h with serial dilutions (1:10 to 1:100) of each plasma sample. They were then washed and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at 37°C and visualized with o-phenylenediamine dihydrochloride.
OVA-specific IgG1 was measured as described in Nohara et al. (2002) with some modifications. In brief, 96-well plates were coated with 10 µg/ml of OVA, washed, and blocked with BlockAce (Dainippon Pharmaceutical Co., Tokyo). A 100-µl volume of each plasma sample was added to the plates in serial dilutions from 1:500 to 1:2000. After being washed, the plates were incubated with HRP-conjugated anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and visualized as described above. Vehicle control- and TCDD-treated groups were compared in the linear range of absorbance.
Total or high-affinity NP-specific IgG1 was measured by using high-NPhaptenated or low-NPhaptenated bovine serum albumin (NP:BSA conjugation ratio 25:1, NP25-BSA, or conjugation ratio 2:1, NP2-BSA, Biosearch Technologies, Inc., Novato, CA) as the coating antigen according to a previous report (Takahashi et al., 1998) with some modifications. By using this method, the NP25-BSA ELISA detects the total antibodies, and the NP2-BSA ELISA detects the high-affinity antibodies. After 96-well plates were coated with 50 µg/ml of NP-BSA, they were washed and blocked with BlockAce. Then, a 100-µl volume of the diluted plasma samples (1:100 to 1:218,700) or standard (kindly provided by Dr. T Azuma, Tokyo University of Science) was added to the plates, and the bound antibodies were detected as described above. The concentration of plasma samples (diluted 1:2700) was determined from the standard curve.
Cell preparation.
A single-cell suspension was prepared from the spleen as previously described (Nohara et al., 2002). BM cells were isolated by flushing the femurs with 2% fetal calf serum (FCS) (Sigma) in PBS, using a 5-ml syringe with an attached 21-gauge needle. After centrifugation, the cells were hemolyzed by the method used for the splenocytes, and then the cells were washed twice and filtered through a stainless steel mesh for further manipulation.
Enzyme-linked immunospot.
Total AFCs or high-affinity AFCs specific for NP hapten were detected by an enzyme-linked immunospot (ELISPOT), as described in Takahashi et al. (1998). Briefly, nitrocellulose filters (Osmonics, Minnetonka, MN) were coated with 50 µg/ml of NP25-BSA, NP2-BSA, or BSA in PBS and then blocked with 1% BSA in PBS. Splenocytes (6 x105) or BM cells (1.2 x106) were incubated on nitrocellulose filters in 96-well plates at 37°C in 5% CO2 for 2 h. Nitrocellulose filters were washed and stained with alkaline phosphatase (AP)conjugated anti-IgG1 antibody (Southern Biotechnology). The AP activity then was visualized as described in Takahashi et al. (1998)
. The frequency of total AFCs or high-affinity AFCs was determined from the NP25-BSA or NP2-BSAcoated filters by subtracting the background level observed on the BSA-coated filters.
Flow cytometry.
Splenocytes (1 x 106) were stained with fluorescein isothiocyanate (FITC)-conjugated anti-B220 mAb (Pharmingen, San Diego, CA; clone RA3-6B2) and phycoerythrin (PE)-conjugated anti-CD3 mAb (Pharmingen; clone 1452C11) to detect B cells and T cells. GC B cells, which are known to be detectable with peanut agglutinin (PNA) (Shinall et al., 2000), were stained with FITC-conjugated PNA (Honen Corporation, Tokyo) and PE-conjugated anti-B220 mAb (Pharmingen; clone RA3-6B2). After staining, the cells were treated with 7-aminoactionmycin D (Sigma) to label the dead cells and measured using a FACSCalibur (Becton Dickinson, Mountain View, CA) as described in Nohara et al. (2000)
. The cells were gated to exclude dead cells and cell debris and then were analyzed. Data were collected from 10,000 cells.
Histologic studies.
Serial spleen cryosections (6 µm) and paraffin sections (4 µm) were prepared for histochemical staining. The GCs were visualized by incubating acetone-fixed cryosections with biotin-conjugated PNA (Vector, Burlingame, CA), followed by incubation with HRP-conjugated streptavidin (Vector) and 3,3-diaminobenzidine (DAB) as the substrate. Apoptotic cell death in the GCs was estimated in cryosections by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method using an in situ apoptosis detection kit (TaKaRa Biomedicals, Tokyo), and DAB was used as the chromogen. To co-label TUNEL+ cells with PNA, AP-conjugated streptavidin (Southern Biotechnology) was used instead of HRP-conjugated streptavidin, and the AP activity was visualized as described in Takahashi et al. (1998). Paraffin sections were examined to detect cellular proliferation in the spleen by an immunohistochemical method using anti-Ki-67 Ab (Santa Cruz Biotechnology, Santa Cruz, CA; clone M-19) as described in Weihua et al. (2000)
with a slight modification. After the deparaffinization and subsequent rehydration of the sections, antigen retrieval was conducted by autoclave processing instead of boiling. To visualize Ki-67+ cells, sections were incubated with anti-Ki-67 Ab followed by incubation with HRP-conjugated anti-goat IgG (Sigma) and DAB as the substrate. Then the sections were stained with hematoxylin to identify the white pulp. The stained sections were visualized under a Leitz DMRBE microscope (Leica, Wetzler), and the images were captured by a 3CCD color camera (Leica). Then the area of white pulp, Ki-67+ cell clusters, and GCs were quantified by analyzing the digitized images using Leica Qwin Version 2.2A software. TUNEL+ cells were counted using a Leitz DMRBE microscope. Three random fields per spleen were analyzed at a fivefold magnification for Ki-67+ analysis, and 15 random fields per spleen were analyzed at a 40-fold magnification for TUNEL+ analysis. The means of the values obtained per each spleen were used to calculate the ratio of Ki-67+ area to the area of white pulp or the ratio of TUNEL+ cell number to the GC area.
Statistical analysis.
The results are expressed as the mean ± standard error of 58 mice per group. Significant differences between the vehicle-control group and the TCDD-treated group were determined by two-tailed Students t-test, and a value of p < 0.05 was considered significant.
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RESULTS |
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TCDD Exposure Suppresses High-Affinity AFC Generation in the Spleen and High-Affinity IgG1 Level in the Plasma
Since TCDD exposure suppressed GC formation in the spleen, we used NP-CG to determine whether the generation of hapten-specific high-affinity AFC is reduced by TCDD exposure. High-affinity AFCs were shown to be early products of GCs and subsequently accumulate to BM (Smith et al., 1997). We thus evaluated the frequency of high-affinity AFC in addition to the total (high and low) AFCs in the spleen and BM on days 10 and 14 by ELISPOT (Fig. 5
). As is shown in Figure 5A
, a large number of total NP-specific AFCs was detected in the spleen of the control mice on day 10, and these cells were decreased on day 14. In these control mice, the number of total AFC in the bone marrow was much smaller than that in the spleen. As compared with the vehicle-control group, the number of total AFCs was reduced in the spleen of the TCDD-treated group (by 90 and 64% on days 10 and 14, respectively). In parallel with the total AFCs, high-affinity NP-specific AFCs were abundant in the spleen of the control mice on day 10 and decreased on day 14 (Fig. 5B
). TCDD exposure was clearly shown to reduce the number of high-affinity AFCs on days 10 (by 96%) and 14 (by 65%). These reductions in the high-affinity AFCs by TCDD exposure were more prominent than those observed in the total splenocytes (by 51% on day 10 and by 41% on day 14). The numbers of bone marrow cells in the NP-CGimmunized mice were not altered by TCDD exposure (data not shown).
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DISCUSSION |
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Previous studies have shown that TCDD inhibits IgM production by altering later stages of B-cell maturation into antibody production. That is, TCDD hardly affected the proliferation of activated B cells but inhibited their IgM production (Luster et al., 1988; Wood and Holsapple, 1993
). In contrast, the present paper shows that TCDD suppresses B-cell proliferation. The difference between the previous studies and ours is thought to be dependent on the experimental systems that were used. In the studies by Luster et al. (1998) and Wood and Holsapple (1993)
, cellular proliferation was examined using isolated B cells directly exposed to TCDD. In contrast, in the present paper, the cells were activated and exposed to TCDD in vivo, where many cell types, including T cells, as described above, seems to be involved in the suppression of B-cell proliferation. On the other hand, TCDD has been reported to stimulate the interaction of AhR with p27 kip1 and Rb proteins that are involved in cell-cycle regulation (Kolluri et al., 1999
; Puga et al., 2000
). These direct pathways may also function in B cells activated and affected by other cell types in vivo and may inhibit cellular growth by causing cell cycle arrest. Further studies will be required to fully describe the molecular mechanism of TCDD toxicity in GC formation and the following antibody production.
In this paper, we also demonstrated that TCDD suppresses the high-affinity antibody response. High-affinity AFCs are generated in the GCs of lymphoid organs such as the spleen, and they migrate to the BM, where they survive for several months in mice. In the present paper, we recognized the following time-course: A greater amount of high-affinity AFCs was observed in the spleen on day 10, with a decrease by day 14; in contrast, only a small number of high-affinity AFCs was observed in the BM on day 10, with an increased number observed on day 14 in the vehicle-control mice. However, TCDD greatly suppressed the number of high-affinity AFCs in the spleen on day 10 and also showed a similar suppressive effect on day 14. Since high-affinity AFCs were shown to be early products of GCs (Smith et al., 1997), the inhibition of GC formation from its onset by TCDD seems to be responsible for the decreased high-affinity AFC generation. In the present paper, the levels of high-affinity antibodies in the plasma were also confirmed to be reduced after TCDD exposure. Since the affinity of antibodies is augmented more than 20 times during the affinity maturation (Takahashi et al., 1998
), the suppression of high-affinity antibody production by TCDD is thought to hamper an effective immune reaction.
GCs are also considered to play an essential role for the generation, maturation, and selection of high-affinity memory B cells (MacLennan, 1994). Memory B cells have a long life-span and are responsible for a robust secondary antibody response following re-exposure to an antigen (Ahmed and Gray, 1996
). Actually, the inhibition of GC formation has been shown to result in an attenuation of the secondary antibody response (MacLennan, 1994
). Therefore, the present results suggest that the suppressive effects of TCDD on GC formation during the primary immune response may influence repeated responses to the same antigen over a long period of time. In support of this possibility, we have previously reported that a single-dose oral administration of TCDD followed by OVA/alum immunization attenuated the secondary antibody response of mice boosted with OVA 3 weeks after the primary immunization (Fujimaki et al., 2002
; Nohara et al., 2002
). As an example showing a longer-lasting effect of TCDD on the immune system, perinatal exposure of rats to TCDD is reported to cause persistent suppression of a delayed-type hypersensitivity reaction that was observed until 19 months after birth (Gehrs and Smialowicz, 1999
). The inhibition of GC formation by TCDD that we reported in this paper may inhibit memory B-cell generation and afford another example of a long-lasting effect of TCDD.
In summary, the present paper demonstrates for the first time that TCDD exposure has suppressive effects on the high-affinity antibody response to antigens in the primary humoral immune response. TCDD was suggested to suppress this response by inhibiting the GC formation at an early stage, before the antibody production from high-affinity AFCs could take place.
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ACKNOWLEDGMENTS |
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NOTES |
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