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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Competing interests
  9. References
  10. Supporting Information

Objective

Graves' disease (GD) is a common autoimmune disease involved autoantibody production. Although we previously reported that osteopontin (OPN), a proinflammatory protein, affected development of GD through NF-κB activation, little is known about the role of OPN in regulating immunoglobulin production in GD. CD40 Ligand (CD40L) is expressed on the surface of activated CD4+T cells and costimulates CD40 on B cells, stimulating production of immunoglobulins, a process which has been reported to play a vital role in immunological signalling transduction in several autoimmune diseases. This study sought to characterize the relationship between CD40L and GD development, as well as investigating the role of OPN in modulating immunoglobulin production in GD via CD40L.

Methods

Forty incident patients with GD, twenty-one patients with GD in remission and twenty-seven healthy controls were recruited. Both membrane-bound and soluble forms of CD40L were measured, and their correlations with clinical parameters were studied. In addition, correlation between OPN and CD40L level was also examined. Furthermore, we studied the regulatory effect of OPN on CD40L in CD4+T cells.

Results

We demonstrated that the CD40L levels were enhanced in patients with GD and recovered in patients with GD in remission. CD40L levels correlated with clinical GD diagnostic parameters and OPN concentration. Moreover, human recombinant OPN and plasma samples from patients with GD increased CD40L expression, which could be significantly suppressed by OPN monoclonal antibody. In addition, CD40L antibody blocked the immunoglobulin production augmented by OPN in cultured peripheral blood mononuclear cells (PBMCs), isolated from patients with GD and healthy subjects.

Conclusion

These results indicate that CD40L is induced by OPN and serves as the downstream effector of OPN for immunoglobulin production in GD development.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Competing interests
  9. References
  10. Supporting Information

Graves' disease (GD) is a common organ-specific autoimmune disease in which an ongoing immune response to thyroid-specific antigens, such as the thyrotropin (TSH) receptor, thyroid peroxidase (TPO) and thyroglobulin (Tg), leads to hyperthyroidism and diffuse hyperplasia of the thyroid gland.[1-4] Thus, autoantibodies produced by B cells play a central role in the pathogenesis of GD.[5, 6]

Osteopontin (OPN), a multifunctional extracellular matrix protein, is a potential proinflammatory cytokine and major amplifier of Th1-mediated immune response.[7, 8] The elevation of OPN is reported to be tightly associated with autoimmune disease, such as multiple sclerosis (MS), rheumatoid arthritis (RA), autoimmune hepatitis, atherosclerosis, graft-vs-host disease (GVHD) and experimental autoimmune encephalomyelitis (EAE).[9-13] Besides, we previously reported that the serum OPN level and mRNA expression in peripheral blood mononuclear cells (PBMCs) were enhanced in patients with GD.[14] Interestingly, it is reported that OPN promotes B-cell immunoglobulin production and proliferation.[15, 16] Our previous finding also pointed out strong correlations between OPN levels and autoantibody production in GD, including antibodies: thyrotrophin receptor antibody (TRAb), thyroglobulin antibody (TGAb) and thyroid peroxidase antibody (TPOAb). Thus, these results drive us to study the possible mechanism of OPN regulating immunoglobulin production in GD.

B-cell activation, differentiation and immunoglobulin production are regulated by CD4+ T cells, particularly Th2 cell.[17] Besides, the signal transduction requires interactions between CD40 ligand (CD40L) on activated CD4+ T cells and CD40 on B cells.[18] CD40L is a type II surface protein expressed by CD4+ T cells, basophils, mast cells and eosinophils.[19] CD40L on the surface of activated CD4+ T cells stimulates its cognate receptor CD40 on B cells for proliferation and secretion of immunoglobulins.[20] CD40L exists in membrane-bound (mCD40L) and soluble forms (sCD40L), both of which are shown to be important modulator of immune–inflammatory events in autoimmune diseases.[21] Indeed, CD40L contributes to the enhancement of autoimmune diseases such as SLE, RA, lupus nephritis, multiple sclerosis and autoimmune diabetes.[22-24] Recently, it was reported that the serum ratio of tumour necrosis (TNF) α: sCD40L is a marker for GD status.[25] However, little is known about the relationship between CD40L and GD development and more importantly, the mechanism of modulating CD40L level in GD.

In this study, we report that CD40L levels are significantly increased in GD and improved in GD remission. Expression correlated with GD clinical parameters. Moreover, we demonstrate that OPN might enhance immunoglobulin production in GD through stimulating CD40L level. Our results suggest that CD40L may be a potent biomarker for GD and reveal a regulatory role of OPN on CD40L in the inflammatory processes of GD.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Competing interests
  9. References
  10. Supporting Information

Subjects

Forty incident patients with GD and twenty-one patients with GD in remission were recruited from Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. All incident patients with GD, without previous treatment, were newly diagnosed through standard clinical and laboratory examinations. Clinical examinations included patients' history, physical examination and radioactive iodine uptake test. The laboratory examinations included free T3 (FT3), FT4, sensitive TSH (sTSH), TSH receptor antibody (TRAb), thyroglobulin antibody (TGAb) and thyroperoxidase antibody (TPOAb). Patients with GD in remission had been treated with methimazole for at least half a year and maintained a euthyroid state with largely improved parameters compared with initial patients with GD. Twenty-seven age and sex-matched healthy subjects without any past or present history of thyroid disease were enrolled in this study. This study is approved by the ethics review committee from Shanghai Jiao Tong University School of Medicine. The informed written consent was obtained from each participant.

Fresh whole blood was collected into tubes containing EDTA and stored on ice. The plasma was immediately centrifuged at 2000 x g for 10 min at 4 °C to remove platelets as previously described.[26, 27] At the same time, PBMCs were isolated by Ficoll-Paque centrifugation and immediately processed for flow-cytometric analysis or cell culture.

Cell culture and treatment

Fresh isolated PBMCs were cultured in six-well plates at 3 × 106 cells per well in the presence or absence of 1 μg/ml recombinant human OPN (R&D Systems, Minneapolis, MN, USA). For experiments involving plasma treatment, prefiltered plasma samples at a dilution of 1 : 4 were added into the PBMC cultures. For blocking experiments, purified anti-OPN monoclonal antibody (mAb, R&D Systems) or an isotype-matched control antibody at a final concentration of 10 μg/ml was used. After 12 h, cells were harvested for real-time PCR; and after 24 h, cells were harvested for flow-cytometric analysis. For experiments concerning immunoglobulin production, anti-CD40L mAb or control antibody (10 μg/ml; R&D Systems) was used. Cell culture supernatants were collected for ELISA analysis at the end of 10 days.

ELISA

The plasma OPN, SCD40L levels were measured by ELISAs according to the manufacturers' procedures. The ELISA kits for OPN and SCD40L were purchased from R&D Systems. The minimum detectable dose of each kit is 0·006 ng/ml for OPN, 2·1 pg/ml for SCD40L. For analysis of secreted immunoglobulin, ninety-six-well flat bottom ELISA plates (NUNC) were coated with purified goat anti-human IgM and IgG (Bethyl Laboratories, Montgomery, TX, USA). The sensitivities of the specific assays were 4 ng/ml for IgG and 8 ng/ml for IgM.

Flow-cytometric analysis

To determine the CD40L level on CD4+ T-cell surface, two-colour analysis was performed using FITC-conjugated anti-CD40L mAb and PE-labelled anti-CD4 mAb (eBioscience, San Diego, CA, USA). Briefly, after surface staining, cells were washed and fixed in fixation solution and analysed using a flow cytometer (FCM, BD Biosciences, San Diego, CA, USA). The appropriate isotype antibodies were used as controls. The gating for lymphocytes was shown in Fig. 1a.

image

Figure 1. Plasma CD40L and CD4+T cells surface CD40L level in normal controls, initial patients with GD, and patients with GD in remission. (a) Representative dot plot analysis of flow cytometry showing the respective gating for lymphocytes, CD4+T cells (FL2: phycoerythrin (PE)-CD4). (b) CD4+T cells were gated, and CD40L expression on the CD4+T cells was analysed. Data are representative from a healthy subject, an initial patient with GD and a patient with GD in remission. (c) CD4+T cells surface CD40L level was analysed by flow-cytometric analysis from 28 initial patients with GD, 17 patients with GD in remission and 17 healthy subjects. (d) Plasma specimens prepared from 40 initial patients with GD, 21 patients with GD in remission and 27 healthy controls were measured by enzyme-linked immunosorbent assay (ELISA).

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Cell purification

CD4+ T cells (>95% purity) were positively isolated from cultured PBMCs using the human CD4 MicroBeads, in accordance with the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).

Quantitative real-time polymerase chain reaction

Total RNA was isolated from CD4+ T-cell samples using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and 1 mg total RNA was converted into first-strand cDNA with the first-strand cDNA synthesis kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Quantitative RT-PCR was performed using SYBR Master Mix (Takara, Shiga, Japan) on an ABI Prism 7900HT (Applied Biosystems, Foster City, CA, USA). A human GAPDH gene was used as an endogenous control for sample normalization. Results were presented as the fold expression relative to that of GAPDH. PCR primers were as follows: for human GAPDH, forward 5′-TGATGACATCAAGAAGGTGGTGAAG-3′ and reverse 5′-TCCTTGGAGGCCATGTGGGCCAT-3′; for human CD40L, forward 5′-GAGCAACAACTTGGTAACCCT-3′ and reverse 5′-GGCTGGCTATAAATGGAGCTTG-3′.

Statistical analyses

Descriptive data are shown as mean ± SD. P-values less than 0·05 were considered significant. Correlations between the different variables were analysed by simple correlation using Pearson's test. Differences between multiple groups were tested by one-way anova and post hoc comparisons were performed with Tukey's test. Chi-squared test was used to determine the differences of gender between groups. All statistical analysis was performed using the spss version 13.0 (IBM, Chicago, IL, USA). All graphics were performed by Graph Pad Prism 5.0 (Graph Pad Software, La Jolla, CA, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Competing interests
  9. References
  10. Supporting Information

Plasma soluble CD40L and membrane-bound CD40L on CD4+ T cells in patients with GD

Subject characteristics and anthropometric parameters are summarized in Table 1. The incident patients with GD had significantly higher serum FT3, FT4, TPOAb, TRAb and TGAb levels and significantly lower serum sTSH level than normal subjects and patients with GD remission. Flow-cytometric analysis showed largely enhanced mCD40L expressions on CD4+ T cells isolated from patients with GD than those from the other two groups (Fig. 1b,c). Besides, plasma concentration of sCD40L was highest in the initial GD group (Fig. 1d). Moreover, previous study reported that increased sCD40L is released from increased mCD40L[28], and we found a significant correlation between mCD40L and sCD40L level (Fig. S1). Furthermore, mCD40L correlated with majority of clinical GD parameters including FT3, FT4, TSH and TRAb. However, sCD40L strongly correlated with all clinical GD parameters (Table 2). These results indicated that CD40L might be involved in GD development.

Table 1. Clinical characteristics of healthy controls, initial patients with GD, and patients with GD in remission
 Healthy controlsInitial GDGD in remission
  1. TRAb, TSH-receptor antibody; TGAb, thyroglobulin antibody; TPOAb, thyroperoxidase antibody.

  2. a

    < 0·01, initial GD compared with healthy controls.

  3. b

    < 0·01, GD in remission compared with initial GD.

n (male/female)27 (9/18)40 (13/27)21 (7/14)
Age (years)35·92 ± 8·1034·97 ± 12·4334·95 ± 13·24
FT3 (pmol/l)4·41 ± 0·4529·01 ± 14·08a4·24 ± 0·69b
FT4 (pmol/l)14·71 ± 1·6842·50 ± 13·80a12·57 ± 3·33b
sTSH (mIU/l)1·85 ± 1·080·007 ± 0·019a1·61 ± 1·04b
TRAb (IU/l)0·38 ± 0·1617·96 ± 11·73a5·05 ± 5·94b
TPOAb (IU/ml)1·76 ± 5·08455·50 ± 406·73a44·54 ± 81·89b
TGAb (IU/ml)4·51 ± 6·07 255·65 ± 363·34a22·83 ± 32·76b
Table 2. The correlation of CD40L level with classic GD diagnostic parameters
GD parameterssCD40LmCD40L
r P r P
  1. TRAb, TSHreceptor antibody; TGAb, thyroglobulin antibody; TPOAb, thyroperoxidase antibody.

FT40·771<0·0010·543<0·001
FT30·766<0·0010·564<0·001
sTSH−0·490<0·001−0·380·006
TRAb0·4290·0050·4530·005
TPOAb0·605<0·0010·2220·158
TGAb0·3730·0190·1140·515

Correlation between CD40L level and plasma OPN level

Consistent with our previous findings, plasma OPN level in patients with GD was significantly higher than those from normal controls in our cohort. We further recognized that plasma OPN levels were significantly recovered in patients with GD remission (Fig. S2). Interestingly, as shown in Fig. 2, both the level of CD40L on CD4+ T cells and plasma concentration of sCD40L displayed a significant correlation with the plasma OPN level, which suggested that there might be a connection between OPN and CD40L.

image

Figure 2. Correlation of CD40L level with OPN. Pearson's test shows the correlation of plasma OPN levels with CD4+T cells surface CD40L and plasma CD40L levels. Both correlations shown were significant.

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Induction of CD40L by human recombinant OPN and plasma OPN from patients with GD

We then examined whether OPN modulate CD40L level. Plasma specimens derived from patients with GD, but not those from normal subjects, stimulated CD4+ T-cell surface CD40L expression from 5 new incident GD and 5 normal subjects with PBMC coculture. When plasma OPN was abrogated by an OPN mAb, the induction effect of GD plasma on CD40L expression was significantly suppressed (Fig. 3a). Meanwhile, recombinant human OPN exhibited a similar effect in the induction of CD4+ T-cell surface CD40L production, which confirmed our notion that OPN stimulated CD40L levels (Fig. 3a). Consistent with these data real-time PCR showed that treatment with plasma from patients with GD and recombinant human OPN led to marked increase CD40L mRNA level in CD4+ T cells, which could be blocked by OPN antibody (Fig. 3b). Collectively, these data suggested that pathologically elevated CD40L level in patients with GD might be mediated by OPN.

image

Figure 3. The effect of OPN on induction of CD40L expression. (a) PBMCs isolated from five patients with GD and five normal subjects were cultured for 24 h with recombinant human OPN, normal plasma or patient plasma pretreatment with a neutralizing OPN antibody (Ab) or its isotype control (IgG). mCD40L was then analysed by flow-cytometric analysis. (b) Expression of the CD40L genes was determined by RT-PCR in purified CD4+ T cells after the PBMCs had been cultured for 12 h.

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CD40L monoclonal antibody inhibits the augmentation of immunoglobulin production by OPN

Osteopontin was reported to regulate B-cell activation and immunoglobulin production. Consistently, OPN levels strongly correlated with autoantibodies in GD as we previously reported and demonstrated here (Table. S1). Meanwhile, CD40L plays a vital role in signalling transduction for B-cell activation. As we proved that OPN could induce CD40L, it is possible that OPN increase immunoglobulin production through CD40L. Indeed, treatment of PBMCs from patients with GD and normal subjects with human recombinant OPN augmented productions of IgG and IgM. Moreover, the CD40L mAb significantly block the induction effect of OPN on immunoglobulin production (Fig. 4). Taken together, our findings indicated that the OPN might mediate immunoglobulin production through CD40L induction, thus contributed to GD pathogenesis and development.

image

Figure 4. Anti-CD40L mAb blocks the OPN augmented production of immunoglobulin. PBMCs from patients with GD and normal subjects were cultured for 10 days with OPN in the presence of anti-CD40L mAb or control mAb. Values are presented as percentage vs immunoglobulin amount produced by control cultures with medium alone. Culture medium from patients with GD produced (mean ± SD) 1560 ± 589 ng/ml IgG and 615 ± 331 ng/ml IgM. Culture medium from normal controls produced 999 ± 474 ng/ml IgG and 296 ± 205 ng/ml IgM. n = 5.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Competing interests
  9. References
  10. Supporting Information

Graves' disease is an organ-specific autoimmune disease resulting from T-cell recognition of self-autoantigen and abnormal B cells. In GD, both Th1 involved cell-mediated immunity and Th2 involved humoral immunity occur.[29, 30] In the immune responses of the Th1 system, cytokines such as IFN-γ and IL-12 are produced, which induce activation of natural killer cells and cytotoxic T cells. By contrast, in the immune responses of the Th2 type, cytokines such as IL-4 or IL-5 are produced, which promote activation of mast cells or B cells.[31] Although GD is considered as a Th2 dominant disease, cytokine expression profiles in serum and thyroid tissues from patients with GD indicate a mixed Th1/Th2 status at any time.[32] These activated T cells in turn increase the secretion of thyroid-specific antibodies from B cells.[33]

Osteopontin is chemotactic for many cell types including T cells, macrophages and dendritic cells; it also promotes B-cell immunoglobulin production.[34] In inflammatory situations, OPN influences cell-mediated immunity. It was demonstrated that OPN was highly elevated in CD4+ synovial T cells from patients with RA and mediated amplification and perpetuation of rheumatoid synovitis.[12] It was also reported that blockade of OPN reduced alloreactive CD8+T-cell-mediated GVHD.[13] Recently, we first discovered that OPN levels were enhanced in serum and PBMCs from patients with GD, and could affect GD development through NF-κB activation and the subsequent changes in inflammatory status.[14] In this study, we provided a novel role of OPN in promoting B-cell immunoglobulin production in GD development through the induction of CD40L on CD4+ T cell. OPN is thought to be a major amplifier of Th1-mediated immune response. However, we provide evidence that OPN enhances CD40L level on Th2 cell and activates humoral immunity, thus providing a possible regulatory mechanism of crosstalk between Th1 cell and Th2 cell in GD.

CD40-L is a 33-kD glycoprotein which is rapidly and transiently expressed on the surface of activated CD4+ T cells.[20] In several autoimmune and inflammatory diseases, increased sCD40L is released from increased mCD40L.[28] In our study, we demonstrated that CD40L levels were increased in plasma and on CD4+ T cells from patients with GD. Both sCD40L and mCD40L correlated with GD clinical parameters, indicating CD40L might be serve as a biomarker for GD. Besides, sCD40L might be more potent because it showed even stronger correlation than mCD40L. Interestingly, one recent study also found sCD40L levels to be elevated in serum from patients with GD and suggested ratio of TNFα: CD40L as a biomarker for GD. However, they failed to find any correlation between serum sCD40L and clinical parameters.[25] It has been pointed out that CD40L was also expressed on the surface of platelets and was released after platelet activation and thrombus formation.[35] Serum sample preparation leads to in vitro clot formation and activates platelets, and thus may increase non-CD4+ T-cell-associated sCD40L levels. To prevent interference of sCD40L released from platelets, platelet-poor plasma, instead of serum, was largely recommended by previous technique studies and also used in our experiments.[26, 27] Hence, this may be the explanation of different conclusions from our study and the previous report.

In summary, we reported elevations of CD40L in plasma and on CD4+ T cells from patients with GD. CD40L level strongly correlated with GD clinical parameters and OPN level. In addition in vitro assays showed that human recombinant OPN increased CD40L level. Moreover, plasma from patients with GD enhanced CD40L expression, which was suppressed after OPN abrogation. Furthermore, we demonstrated that CD40L mAb could block OPN augmented immunoglobulin production. Our findings indicated that CD40L was involved in GD development and OPN might enhance immunoglobulin production in GD through stimulating CD40L level.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Competing interests
  9. References
  10. Supporting Information

This study was supported by the grants from the National Natural Science Foundation of China (No. 81270872), Shanghai Municipal Natural Science Foundation (No. 11495803400), the Sector Funds of Ministry of Health (No. 201002002, No. 201202008) and The National Key New Drug Creation and Manufacturing Program of Ministry of Science and Technology (No. 2012ZX09303006-001).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Competing interests
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Competing interests
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
cen12229-sup-0001-TableS1-Figs-S1-S2.pdfapplication/PDF201K

Fig S1. Correlation between the level of CD40L on CD4+ T cells and sCD40L level.

Fig S2. Plasma OPN level in normal controls, initial GD patients, and GD patients in remission.

Table S1. The correlation of OPN level with classic GD diagnostic parameters.

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