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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. MATERIALS and METHODS
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Previous study suggested that MRL-lpr/lpr mice treated with tamoxifen (TAM) had less severe proteinuria, reduced serum titre of anti-dsDNA autoantibodies and an increased survival rate. To investigate further the regulatory mechanisms of TAM on MRL-lpr/lpr female mice, a total dose of 200 µg per mice (5·5 mg/kg) was given every 2 weeks subcutaneously, while the control mice were injected with oil only. After being treated with TAM four times, the mice were killed and cellular functions were evaluated. The TAM-treated groups had smaller sized spleen and lymph nodes. Flow cytometric analysis of splenocytes had a significantly lower percentage of cell number of T cells and double negative T cells (CD4 CD8 T cells). There was no difference in cytokine production (interleukin (IL)-2, IL-4, IL-5, IL-10 and interferon-γ (IFN-γ)) from splenocytes stimulated with concanavalin A (Con A) or cytokines (IL-6) secreted by peritoneal exudate cells when stimulated with lipopolysaccharide (LPS). However, IL-2 from lymph node cells was significantly higher on TAM-treated mice. Finally, splenocytes or purified T cells stimulated with anti-CD3 antibody plus cross-linking immunoglobulin G (IgG) of the TAM-treated group had higher 3H-incorporation of proliferation assay compared with that of control groups. In vitro study further demonstrated that IL-2-activated proliferation of lymph node double negative (DN) T cells can be inhibited by TAM treatment in a dose-dependent manner. Our finding demonstrated that TAM may potentially influence T cells and modulate the immune function, which offers a novel approach to explore the feasibility of hormone therapy for autoimmune diseases.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. MATERIALS and METHODS
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

MRL-lpr/lpr mice spontaneously develop an autoimmune disorder similar to human systemic lupus erythematosus (SLE) characterized by hypergammaglobulinaemia, the production of anti-dsDNA, anti-small nuclear ribonucleoprotein (snRNP) and rheumatoid factor-specific autoantibodies, and immune complex-mediated end organ disease of the kidney and salivary glands. 1–4 A main characteristic of MRL-lpr/lpr, which exists in mouse but not in human SLE, is massive lymphadenopathy caused by a defective Fas molecule,5,6 which causes defective activation-induced cell death of peripheral αβ T cells7,8 and leads to the expansion of double-negative CD4 CD8 B220+ T cells. 9 Most of the MRL-lpr/lpr mice develop a progressive loss of kidney function at 3–6 months of age and with a 50% mortality rate at the age of 6 months. 10

Tamoxifen (TAM) is a synthetic non-steroidal anti-oestrogen triphenylethylene compound; a potent oestrogen antagonist with high affinity for oestrogen receptor11,12 and has been used as a preventive drug for breast cancer disease.13,14 TAM seems to have a plethora of effects, including inhibition of calmodulin,15,16 antioxidant activity, 17 stimulation of transforming growth factor-β (TGF-β) secretion,11,18 induction of apoptosis,19,20 interaction with P-glycoprotein, inhibition of Ca2+-phospholipid-dependent protein kinase C (PKC) 15,21–23 and down-regulation of insulin-like growth factor-I (IGF-I). 24 It has been documented that oestrogen treatment in MRL-lpr/lpr mice or NZB/W F1 mice (castrated males, ovariectomized females, or regular females) can exacerbate the disease.25,26 Recently, several studies have shown that TAM has the beneficial effects on experimental murine lupus model.27,28 However, the role of TAM in the pathogenesis of lupus strain mice has not been well documented.

In this study, we investigated the effect of TAM on disease severity and the change of immune effect cells in autoimmune MRL-lpr/lpr mice. Further understanding the mechanisms of TAM is important for the future application for autoimmune diseases other than breast cancer, as well as for designing new derivatives of the drug with higher potency and less deleterious side-effects.

MATERIALS and METHODS

  1. Top of page
  2. Summary
  3. Introduction
  4. MATERIALS and METHODS
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Experimental animals

Five to 6-week-old female MRL/MpJ-lpr (lpr) (H2K) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in the Animal Center of National Taiwan University College of Medicine. MRL-lpr/lpr mice were followed for the life span and proteinuria to confirm the pathological changes of these mice.

Drug treatment

Mice were administered subcutaneous (s.c.) injections of 200 µg or 800 µg of TAM (Sigma Chemical Co., St. Louis, MO) dissolved in 50 µl peanut oil, every 2 weeks. Drug treatment was given regularly to observe life span and serological change. For further study the cellular mechanism, the mice were treated four times and killed at 15 weeks of age. Control mice were injected with peanut oil only. This dose of 200 µg TAM was determined according to preliminary experiments demonstrating that MRL-lpr/lpr mice treated with this dose showed less severe proteinuria, reduced serum titre of anti-dsDNA autoantibodies and an increased survival rate.

Determination of proteinuria

Proteinuria was measured in a semiquantitative manner, using a tetrabromphenol paper (Eiken Chemical Co., Tokyo, Japan) on fresh urine samples. This colorimetric assay, which is relatively specific for albumin, was graded 1+ to 4+ and the approximate protein concentrations were as follows: 1+ : 30 mg/dl, 2+ : 100 mg/dl, 3+ : 1000 mg/dl, and 4+ : > 1000 mg/dl. High-grade proteinuria was defined as higher than 2+ (100 mg/dl).

Culture medium

Most of the experiments were performed in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 4 m m l-glutamine, 10 m m HEPES pH 7·3, 50 m m 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin and 0·25 mg/ml amphotericin. For cell proliferation assay, the cells were cultured in serum-free medium (AIM-V, Gibco/BRL, Gaithersburg, MD) supplemented with 2% TCM (mouse serum replacement, Celox Corp., Hopkins, MN).

Determination of anti-ss, -dsDNA immunoglobulin G (IgG) antibodies

For the assay of serum anti-dsDNA IgG or -ssDNA IgG antibody levels, plates were coated with methylated bovine serum albumin (BSA, Sigma). After overnight incubation, plates were washed and 2·5 µg/ml calf thymus dsDNA (Sigma) or ssDNA (dsDNA boiled for 15 min and cooled on ice) in phosphate-buffered saline (PBS) was applied overnight at 4°. The plates were than aspirated and blocked with PBS containing 1 mg/ml gelatin. Sera to be tested were diluted 1/200 in PBS–gelatin. After 2 hr incubation at room temperature, plates were washed three times with PBS containing 0·05% Tween 20, and horseradish peroxidase-conjugated goat anti-mouse γ chain-specific antibodies diluted in PBS–gelatin were added to each well. After 2 hr of incubation at room temperature, following washing, plates were incubated with the substrate, ABTS (2, 2′-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid, Sigma) and read using an enzyme-linked immunosorbent assay (ELISA) reader (Microplate, Bio-Tek Instrument, Inc., New York, NY) at 420 nm. The anti-DNA antibody results are presented as ELISA units (EU/ml) using a standard monoclonal antibody (mAb) 742H.7D (concentration: 74 ± 0·5 ng/ml), specific for dsDNA. 29

Flow microfluorometric (FMF) analysis

Six to eight mice of each group were killed by cervical dislocation. The spleens and lymph nodes were removed, and lysing red blood cells with ammonium chloride–Tris buffer to isolate single cells. Peritoneal exudate cells were isolated by peritoneal lavage and washed three times with Hank's solution before use. Phenotypic analysis of spleen cells and peritoneal exudate cells was done by FMF. 30 Aliquots of cells (2·5–5 × 105 cells) were suspended in 0·1 ml of PBS with 0·1% sodium azide and incubated at 4° for 30 min with predetermined optimal concentration of appropriate fluoroscein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies (PharMingen, San Diego, CA). The cells were washed and resuspended in 0·5 ml of PBS with 0·1% sodium azide and subjected to fluorescence-activated cell sorting (FACScan analysis). A total of 10 000 cells were counted, the frequency of each cell surface marker was determined using appropriate software (FACScan, Becton Dickinson, Mountain View, CA). Controls were cells suspended in medium.

Cytokine production

The cytokine production pattern of spleen cells was measured using sandwich ELISA. Spleen cells or lymph node cells with a concentration of 1 × 107 cells/ml were set up in 24-well plates in RPMI-1640 medium supplemented with 10% FCS in the presence of concanavalin A (Con A, 10 µg/ml, Sigma). Peritoneal cells with a concentration of 4 × 106 cells/ml were set up in 24-well plates in RPMI-1640 medium supplemented with 10% FCS in the presence of lipopolysaccharide (LPS, 10 µg/ml, Sigma). To determine the optimal levels of cytokine, the supernatants were collected at different time point and the maximal response was noted at 48 hr. Sandwich ELISA was used to assay the levels of cytokines. Predetermined concentrations of anti-interleukin (IL)-2, interferon-γ (IFN-γ), IL-4, IL-5, IL-6, tumour necrosis factor-α (TNF-α) and IL-10 antibodies (PharMingen) were coated to ELISA plates and incubated at 4° overnight. After washes, supernatant was added to the plates and incubated at room temperature for 2 hr. Biotin-conjugated anti-IL-2, IFN-γ, IL-4, IL-5, IL-6, TNF-α and IL-10 antibodies and horseradish-peroxidase conjugated streptavidin were added subsequently. Enzyme activity was evaluated using ABTS as the substrate.

Proliferative response of splenocytes or splenic T cells

To understand whether the T cells were involved in the modulatory mechanisms of disease, T cells were enriched by the method of nylon wool column. 30 The purity of CD3+ T cells was at least 90% as analysed by flow cytometry. For proliferative assay, 1 × 105 cells/well of purified T cells were cocultured with 5 × 105 cells/well of 3000 rad irradiated splenocytes as antigen presenting cells in the presence of hamster anti-mouse-CD3 antibody (1 µg/ml) cross-linked with anti-hamster IgG antibody (0·5 µg/ml) or 5 µg/ml Con A only. After 2 days, cultures were pulsed with 1 µCi of [3H]thymidine and harvested 8 hr later. The result of proliferative response was expressed as counts per minute (c.p.m.).

Purification of lymph node (LN) CD4+ T cells and double negative T cells

To further understand the effect of TAM on specific subpopulation of T cells in vitro, both CD4+ and double negative T cells (CD4 CD8, DN T cells) of 15-week-old female MRL-lpr/lpr mice were isolated by magnetic separation with a magnetic-activated cell sorting (MACS) column (Miltenyi Biotech, Bologna, Italy) according to a previous report. 31 Briefly, lymph node cells were isolated by lysing red blood cells with ammonia chloride–Tris buffer and washed three times. LN T cells were enriched by nylon wool column as previously described. Adhered cells were depleted by incubation at 37° for 2 hr then the non-adherent cells was harvested for further study. T cells were resuspended in PBS plus 0·5% BSA and 5 m m ethylenediamine tetra-acetic acid (EDTA) (MACS buffer). T cells were then cultured with anti-mouse CD4 (L3T4) MicroBeads conjugated antibody (for positive selection of CD4+ T cells) or combined anti-mouse CD4 and CD8a (Ly-2) MicroBeads conjugated antibody (for negative selection to purify DN T cells) for 15 min at 4° and then washed by MACS buffer. Antibody-coated LN T cells were next run through a specially designed column and separated into positive and negative populations using a high-gradient magnetic field. The purity of both populations was directly assessed by FMF analysis. For proliferative assay, 2 × 105 cells/well of purified LN CD4+ T cells or DN T cells were preincubated with a titrated concentration of TAM (0·1–5·0 µm) for 2 hr, then cells were further activated by adding 1·0 ng/ml of human IL-2. After 3 days, each well was pulsed with 1 µCi of [3H]thymidine and harvested 8 hr later. The result of proliferative response was expressed as stimulation index (SI).

Statistical analysis and mouse survival analysis

Data were expressed as mean ± SEM for each group. Significance was determined using two-tailed unpaired Student's t-test. A P-value of less than 0·05 was considered to be significant. For the analysis of survival, the end event was defined as death. All data were analysed from the date of birth on a weekly basis, and survival probabilities were non-parametrically estimated at event times by the Kaplan–Meier method. 33

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. MATERIALS and METHODS
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

MRL-lpr/lpr mice treated with TAM had less severe proteinuria and increased survival rate

In choosing optimal dosage of TAM for the treatment of MRL-lpr/lpr mice, two differential doses (200 or 800 µg of TAM) were injected subcutaneously into mice at an interval of 2 weeks, and control mice were injected with oil only (each group contain 12 mice). Figure 1 suggests that TAM treatment will, at best, delay onset of proteinuria, and prolong survival for a period of 2 months. One-third of the control mice developed proteinuria by week 14. Furthermore, at week 20 the percentage of mice developing proteinuria was lower in TAM-treated mice compared to that of control mice (30% in TAM-treated mice and 80% in control mice, P ≤ 0·05). There was no difference of proteinuria between high-dose (800 µg/mice) and low-dose (200 µg/mice) TAM-treated mice ( Fig. 1a). As previously established 1 the 50% mortality rate for the control mice was at 5 months. In contrast, TAM-treated mice shown a significant increased survival rate ( Fig. 1b) as analysed by the incomplete observation method. 32 It is of note that the low-dose TAM-treated group appeared to have a better survival; therefore 200 µg/mouse was chosen as the dosage for the subsequent experiments. To further demonstrate the mechanism responsible for the observed results, we repeated the animal study by using two groups, the control and 200 µg TAM per mouse-treated group; each group contains 20 mice. Ten mice from each group were followed for their serological characteristics. The remaining mice were killed at 15 weeks of age (by that time, mice had been treated with TAM four times), and assayed for cellular function: including FMF analysis, cytokine production, proliferative response and purified CD4+ T cells or DN T cells for in vitro study.

image

Figure 1. MRL-lpr/lpr mice treated with TAM showed less severe proteinuria (a) and a significant increased survival rate (b). The ‘2+’ means 100 mg/dl protein in urine. The life span data were analysed by incomplete observation methods. 33 Each group contained 12 mice.

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As previously shown, 34 MRL-lpr/lpr mice exhibited marked hypergammaglobulinaemia; the levels of IgG anti-ssDNA and anti-dsDNA antibodies were followed up regularly every month. The data suggested that IgG anti-dsDNA and anti-ssDNA autoantibodies increased with age. However, the level of IgG anti-dsDNA antibody did not show any statistical difference between these two groups. (P = 0·289, data not shown).

Effect of TAM treatment on cellular characteristics

To determine the effects of TAM in MRL-lpr/lpr lymphadenopathy, the weight of spleen or total cervical, axillary, inguinal and mesenteric lymph nodes was determined in control and TAM-treated mice at 15 weeks of age. A significant reduction in spleen weight was detected in TAM-treated mice as 72% of controls (0·36 ± 0·07 g versus 0·26 ± 0·05 g, P ≤ 0·005), while reduction in total lymph nodes weight was insignificant (1·74 ± 0·99 g versus 1·35 ± 0·39 g). FACScan analysis showed that the diminished spleen weight in TAM-treated mice was accounted for by a reduction in the frequency of double negative T cells compared with controls (32·5 ± 3·3% versus 18·9 ± 2·5%, P < 0·006, Table 1). In TAM-treated mice, there was also a proportional increase in CD4+ and CD8+ T cells.

Table 1.  Phenotypic analysis of surface marker expression of spleen, lymph node cells and peritoneal exudate cells *
  % of surface marker expressionTotal cells (×107)
CD69+ TCD4+ TCD8+ TDN T
  • *

    Results are shown as mean ± SEM.

  • The significance of difference among groups was analysed statistically by Student's t -test, differences were considered significant at P < 0·05.

  • ND, not done.

Spleen cells
Ctrl75·8 ± 2·328·4 ± 4·429·4 ± 3·19·8 ± 1·3 32·5 ± 3·321·0 ± 3·2
TAM67·8 ± 1·8 16·2 ± 1·1 30·5 ± 1·510·2 ± 0·918·9 ± 2·5 14·0 ± 1·0
Lymph node cells
Ctrl87·1 ± 1·742·9 ± 4·513·2 ± 0·63·6 ± 0·664·8 ± 2·7ND
TAM88·8 ± 1·040·0 ± 4·413·6 ± 0·94·7 ± 0·457·9 ± 1·6ND
Peritoneal exudate cells
Ctrl80·5 ± 2·13·1 ± 0·526·0 ± 2·39·4 ± 1·3 46·3 ± 5·60·6 ± 0·1
TAM73·7 ± 2·86·7 ± 4·325·7 ± 1·211·9 ± 1·031·4 ± 4·1 0·6 ± 0·1

Phenotypic analysis of surface marker expression of spleen, lymph node cells and peritoneal exudate cells

The most significant finding was that both the percentage and the absolute number of CD4 CD8 splenic T cells of TAM-treated mice were lower compared with that of the control groups (P < 0·001) ( Table 1). It is suggested that TAM altered immune function. In addition, the percentage of CD4 CD8 T cells was also lower in lymph node cells and peritoneal exudate cells of mice treated with TAM ( Table 1). Generally, phenotypic analysis of surface marker expression of major histocompatibility complex (MHC) class I and B-cell subpopulation showed no difference between these two groups (data not shown). The CD69 molecule is an activated marker for T cells. The expressed level of CD69+ T cells on splenocytes was significantly lower in TAM-treated mice, as summarized in Table 1. However, the data showed no difference in percentage of CD4+ T cells or CD8+ T cells in spleen, lymph node cells and peritoneal exudate cells between these two groups.

Cytokine production by mitogen-stimulated splenocytes, lymph node cells and peritoneal exudate cells

Cytokines secreted from splenocytes such as IL-2, IFN-γ, IL-4, IL-5 and IL-10 produced by mitogen-stimulated T cells were determined ( Fig. 2). The data suggested there was no significant difference in cytokine production between these two groups. In Table 2, the lymph node cells of TAM-treated group produced significantly higher IL-2 (P < 0·01) while stimulated with 10 µg/ml Con A. There was no significant difference in the level of IL-6 between these two groups (P = 0·3210).

image

Figure 2. Cytokine production by mitogen-stimulated splenocytes of control mice (light bar) and TAM-treated (dark bar) mice were analysed after 48 hr culture. MRL-lpr/lpr mice secreted very low levels of IL-2 and IL-4 cytokine no matter whether they received TAM treatment or not. The mean and SEM are shown for the two groups of mice. Between six and eight mice were studied.

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Table 2.   Cytokine analysis of lymph node cells stimulated with Con A or peritoneal exudate cells stimulated with LPS *
 Lymph node cells (ng/ml)Peritoneal exudate cells (pg/ml) IL-6
IL-5IL-10
  • *

    Cytokine was analysis by ELISA as described in Materials and Methods. Results shown as mean ± SEM. Each group contains six to eight mice. IFN-γ and IL-4 were not detectable in lymph node cells stimulated with Con A (10 µg/ml), so does TNF-α and IL-1β on the peritoneal exudate cells stimulated with LPS (10 µg/ml) for 48 hr.

  • The significance of difference among groups was analysed statistically by Student's t -test, differences were considered significant at P < 0·05.

Ctrl3·09 ± 0·050·45 ± 0·052·4 ± 0·3289·4 ± 31·4
TAM1·30 ± 0·51†0·38 ± 0·042·0 ± 0·2239·3 ± 33·2

The proliferative response of T cells was higher in TAM-treated mice

For cell proliferative assay, 1 × 105 cells/well of spleen cells or splenic T cells of 15-week-old control or TAM-treated MRL-lpr/lpr mice were stimulated with either 1 µg/ml anti-CD3 antibody plus 0·5 µg/ml cross-linking anti-IgG antibody or 5 µg/ml Con A. The proliferative response was quantitated by [3H]thymidine after 2 days culture. Splenocytes or splenic T cells of TAM-treated mice showed better proliferative response of 3H incorporation compared to those of normal control mice ( Fig. 3). The data suggested both TAM-treated splenocytes and T cells purified from splenocytes have significantly higher 3H-incorporation ability.

image

Figure 3. Proliferative response of splenocytes or splenic T cells. Reactivity of splenocytes or purified T cells from splenocytes of MRL-lpr/lpr mice was set up with 5·0 × 105 of irradiated spleen cells with stimulation. Microcultures were prepared with 1 × 105 splenocytes or T cells from 15 week-old MRL-lpr/lpr mice s.c. injected with TAM or not previously, in the presence or absence of anti-CD3 antibody plus anti-IgG cross-linked or 5 µg/ml Con A only. After 2 days, the proliferation was quantitated by measuring the incorporation of [3H]thymidine. The result of proliferative response was expressed as counts per minute (c.p.m). Each group contains eight mice.

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Lymph node DN T cells pretreated with TAM can inhibit IL-2-induced proliferation in a dose-dependent manner

To further study the effect of TAM on subpopulation of T cells, we used MACS to separate CD4+ T cells and DN T cells from lymph node cells of MRL-lpr/lpr mice. We found that IL-2-induced proliferative response of DN T cells was inhibited by TAM treatment in a dose-dependent manner. In contrast, the inhibitory effect on LN CD4+ T cells was not observed until a rather high dose of TAM (5·0 µm) was given ( Fig. 4).

image

Figure 4. The effect of TAM on in vitro proliferative response of purified LN CD4+ T cells and DN T cells. The response of purified CD4+ T cells or DN T (2 × 105 cells/well) from lymph nodes of 15 week-old MRL-lpr/lpr mice were preincubated with titrated concentration of TAM (0·1–5·0 µm) for 2 hr, then cells were further activated by adding 1·0 ng/ml of human IL-2. After 3 days, cultures were pulsed with 1 µCi of [3H]thymidine and harvested 8 hr later. The data were presented as mean ± SEM for four animals per time point from one representative experiment of two performed. Significant difference (*P ≤ 0·05) from TAM inhibited proliferative response is indicated.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. MATERIALS and METHODS
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Both clinical and experimental evidence suggests that sex hormones play the critical role in the pathogenesis and disease severity in human SLE and murine models of lupus. 34 Oestrogen has been documented to display potent immunoregulatory properties; however, the exact roles of the sex steroids in autoimmune diseases remain unclear. Many studies have demonstrated aggravation of lupus disease in MRL-lpr/lpr and NZB/W F1 mice upon continuous exposure to physiological levels of oestrogen.25,26 However, Steinberg et al. suggested that MRL-lpr/lpr mice have a small response to gonadal hormone, 25 and autoimmunity in these mice is not strongly influenced by this hormone. In our study, we found that TAM treatment of MRL-lpr/lpr mice had a beneficial effect on both female and male (data not shown) mice. While the association between oestrogen metabolites and autoimmunity has been well documented, the mechanisms by which exogenous oestrogen can affect the disease process remain unclear. Oestrogen has been demonstrated to suppress T-cell function and yet exacerbate humoral immunity. 36 Clearly, the effects of oestrogen and metabolites on autoimmunity are complex, involving multiple cell types, signal pathways and various soluble mediators. The data here demonstrated that TAM, a potent oestrogen antagonist, could alleviate disease severity and decrease DN T-cell numbers in MRL-lpr/lpr mice.

The individual role of type 1 and type 2 T helper (Th1 and Th2) cell subsets, exhibiting different capacities of cytokine secretion, in the development and acceleration of SLE have not been well defined. Because several cytokines produced by Th2 cells, such as IL-4, IL-5, IL-6 and IL-10, are known to promote antibody production by B cells, 37 it has been speculated that Th2 cells and related cytokines may play an active role in the development of autoantibody-mediated autoimmune diseases such as SLE. Dayan et al. have shown that the beneficial effects of TAM and anti-oestradiol antibody on experimental murine lupus are associated with cytokine modulation, 28 but the data here did not show any difference in cytokine production from splenocytes stimulated with Con A after TAM treatment in MRL-lpr/lpr mice. In contrast to the non-autoimmune BALB/c mice used in Dayan's study, lupus-prone MRL-lpr/lpr mice were used in this study. Many studies have demonstrated multiple cytokine production defects in MRL-lpr/lpr mice, which may be the reason why the cytokine change was not as obvious as in the previous study. TAM may modulate autoimmune response via several different mechanisms. The pathogenesis of autoantibody production in MRL lupus largely requires CD4+αβ T cells, which provide the help to autoreactive B cells;38,39 however, the individual Th1 and Th2 cell subsets in this process remain unclear. The appearance of autoantibody is the hallmark of both human and murine lupus, suggesting a requirement for cytokines produced by Th2 cells in autoreactive B-cell activation. The most interesting finding of the data is that the number of double negative T cells in TAM-treated MRL-lpr/lpr mice was significantly lower than that of control group. Lymphadenopathy in MRL-lpr/lpr mice consists primarily of T cells with unusual phenotypes (CD4 CD8, CD2, IL-2R, TCR-αβ+, CD3+, and B220+). 40 The lpr DN T-cell population is distinct from other T cells with the characteristic that little IL-2 is produced in response to mitogenic stimulation or T-cell receptor (TCR)/CD3 engagement. 41 This extremely low level of IL-2 production by lpr DN T cells was caused by both the increased instability of mRNA and the reduced activation of IL-2 gene promoter. 42 The IL-2 level secreted by lymph node cells was significantly higher in the TAM-treated group, and lymph node cells contain more than 80% of DN T cells, so in vivo treatment of TAM may affect DN T-cell function, including cell numbers and IL-2 production function. Recently, Radavanyi et al. found that IL-2 signalling may prime activated T cells to TCR-mediated apoptosis and that this pathway may not require fas expression, as they chose MRL-lpr/lpr mice as their animal model. 43 Their investigation also revealed that the defect of activation induced apoptosis in T cells from lpr mice was age-related. In our study, we begin to treat mice with TAM at 8 weeks of age; the age that was the oldest group of Radavanyi's study. We have designed a new animal study, and mice will be treated with TAM as early as 5 weeks of age, so we can determine whether there will be a large reduction in lymphadenopathy. In addition, TAM did not interfere with T-cell activation as determined by CD25 and CD69 expression in the presence of Con A stimulation in vitro, and the data is similar to that in human T cells. 22 The data here also demonstrated that DN T cells did respond to IL-2-activated activation but poorly to cross-linking anti-CD3 activity or phytohaemagglutinin (PHA) (data not shown). To further understand the effect of TAM on IL-2-activated proliferation of DN T cells, the T cells were pretreated with TAM and then stimulated with IL-2 (1·0 ng/ml). Interestingly, pretreatment of TAM on DN T cells can effectively inhibit their proliferative response in a dose-dependent manner. In contrast, the inhibitory effect on CD4+ T cells was not observed until a rather high dose of TAM was given. We therefore speculated that TAM might potentially influence DN T cells and modulate the immune function both in vivo and in vitro. After TAM treatment, the cell cycle of activated T cells may show sustained accumulation in S phase, and the cells are then susceptible to death. Another possibility is that the oestrogen levels in control group mice may be much higher than that of TAM-treated mice. Higher oestrogen can inhibit T-cell function; 36 subsequently the ability of thymidine incorporation was much lower in control-group mice.

In conclusion, the present study further demonstrated the beneficial effects of TAM on an animal model of lupus. The exact immunoregulatory mechanisms of TAM remain to be elucidated. Nevertheless, as TAM is a relatively safe drug without serious adverse reactions, it might be effective in the treatment of human SLE and related disorders.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. MATERIALS and METHODS
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This research was supported by a grant, DOH88-HR-835, from the National Health Research Institute of the Republic of China.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. MATERIALS and METHODS
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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