A tolerogenic peptide down-regulates mature B cells in bone marrow of lupus-afflicted mice by inhibition of interleukin-7, leading to apoptosis

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease mediated by T and B cells. It is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, designated hCDR1, ameliorated the serological and clinical manifestations of SLE in both spontaneous and induced models of lupus. In the present study, we evaluated the status of mature B cells in the bone marrow (BM) of SLE-afflicted mice, and determined the effect of treatment with the tolerogenic peptide hCDR1 on these cells. We demonstrate herein that mature B cells of the BM of SLE-afflicted (New Zealand Black × New Zealand White)F₁ mice were largely expanded, and that treatment with hCDR1 down-regulated this population. Moreover, treatment with hCDR1 inhibited the expression of the pathogenic cytokines [interferon-γ and interleukin (IL)-10], whereas it up-regulated the expression of transforming growth factor-β in the BM. Treatment with hCDR1 up-regulated the rates of apoptosis of mature B cells. The latter was associated with inhibited expression of the survival Bcl-xL gene and of IL-7 by BM cells. Furthermore, the addition of recombinant IL-7 abrogated the suppressive effects of hCDR1 on Bcl-xL in the BM cells and resulted in elevated levels of apoptosis. Hence, the down-regulated production of IL-7 contributes to the hCDR1-mediated apoptosis of mature B cells in the BM of SLE-afflicted mice.

Introduction

Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease mediated by T and B cells, leading to the increased production of pathogenic autoantibodies against several self antigens. The latter are associated with clinical manifestations in various organs.^1–3 Several strains of mice that spontaneously develop an SLE-like disease have been reported, of which the [New Zealand Black (NZB) × New Zealand White (NZW)]F₁ (BWF₁) female mice are the most widely used.⁴ A peptide, designated hCDR1, based on the complementarity-determining region (CDR) 1 of a human monoclonal anti-DNA antibody,⁵ was designed and synthesized in our laboratory. The hCDR1 was shown to ameliorate the serological and clinical manifestations of SLE in both spontaneous and induced models of lupus.⁶ Furthermore, hCDR1 reduced the secretion and expression of the pathogenic cytokines interferon-γ (IFN-γ), interleukin-10 (IL-10), IL-1β and tumour necrosis factor-α, while up-regulating the immunosuppressive cytokine, transforming growth factor-β (TGF-β).⁶

Apoptosis was found to play an important role in the pathogenesis of autoimmune diseases, including SLE.^7–9 It was reported that apoptotic lymphocytes are capable of accelerating the onset of lupus-like manifestations in BWF₁ mice.¹⁰ Furthermore, SLE patients were reported to manifest an increased rate of lymphocyte apoptosis,¹¹ which correlates with disease activity.¹² These data underscore the role of dysregulated apoptosis in the pathogenesis of SLE.

Abnormal B-cell activation and differentiation to memory or plasma effector cells were found to be involved in the pathogenesis of SLE.¹³ These B cells produce autoantibodies that mediate tissue injury, they function as antigen-presenting cells that present epitopes of self antigens to autoreactive T cells, and they produce soluble mediators involved in the organization of lymphoid tissues and in the initiation and perpetuation of inflammatory processes.^14,15

Primary B-cell development takes place in the bone marrow (BM). However, only 10% of the BM-derived immature B cells exit to the spleen wherein one-third of the latter progress to the mature B-lymphocyte pool.^16,17 Mature B cells migrate repeatedly through the blood and lymph to B-cell areas of lymph nodes, Peyer’s patches and the spleen.¹⁸ In SLE patients, a large increase of oligoclonal plasma cell precursors and pre-germinal centre B cells was reported.¹⁹

During B-lymphocyte development in the BM, IL-7, produced by stromal cells, plays a crucial role in the expansion and survival of precursor B cells.²⁰ Severe lymphopenia occurs in mice that are deficient in IL-7,²¹ thereby demonstrating its fundamental role in lymphopoiesis.

A great amount of evidence indicates that SLE is characterized by defects in B-cell tolerance and homeostasis in the periphery, although limited data are available about B cells in the BM.²² In the present study, we evaluated the status of mature B cells in the BM of SLE-afflicted mice, and determined the effect of treatment with the tolerogenic peptide hCDR1 on these cells. We demonstrated here that the B-cell population in the BM of SLE-afflicted BWF₁ mice was largely expanded. The typical pattern of cytokine expression, found in spleen cells of SLE-afflicted mice, could be determined in the BM as well, and treatment with hCDR1 inhibited the expression of the pathogenic cytokines (IFN-γ and IL-10), whereas it up-regulated the expression of TGF-β. We further show that treatment with hCDR1 increased the rates of B-cell apoptosis by a mechanism that involves the inhibition of IL-7 production and the down-regulation of the survival gene, Bcl-xL, in the B cells of the BM compartment.

Materials and methods

Mice

Female BWF₁ mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and BALB/c mice were obtained from Harlan (Jerusalem, Israel). The study has been approved by the Animal Care and Use Committee of the Weizmann Institute of Science.

Synthetic peptides

A peptide, GYYWSWIRQPPGKGEEWIG, designated hCDR1, based on the sequence of the CDR1 of a human anti-DNA monoclonal antibody that bears a major idiotype (16/6Id),⁵ was synthesized by Polypeptide Laboratories (Torrance, CA) and was used in this study. A peptide containing the same amino acids as hCDR1, with a scrambled order (scrambled peptide), SKGIPQYGGWPWEGWRYEI, was used as a control.

Treatment of mice with hCDR1

Mice at the age of 6–7 months were divided into three groups (n = 8–12) and injected subcutaneously once a week for 13 weeks with the vehicle (Captisol® sulphobutylether β-cyclodextrin; CyDex Inc., Lenexa, KS), hCDR1 (50 μg/mouse) or with the control scrambled peptide (50 μg/mouse). The effect of treatment with hCDR1 on the anti-dsDNA antibody levels, proteinuria and immune complex deposits in the kidneys was assessed as previously described.⁶ Results are shown in Table 1, which represents one of four experiments performed with similar results. It can be seen that hCDR1 treatment ameliorated all the manifestations measured.

Table 1.

The effects of treatment with hCDR1 on the clinical manifestations in (NZB × NZW)F₁ mice

	Treatment groups1
	Vehicle	P2	hCDR1	P3	Scrambled
Anti-dsDNA (OD)4	1.2 ± 0.1	0.02	0.7 ± 0.1	0.006	1.3 ± 0.1
Proteinuria (g/l)5	7.1 ± 1.9	0.01	1.9 ± 0.5	0.05	5.0 ± 1.8
ICD6	2.4 ± 0.2	0.002	1.5 ± 0.2	0.0006	2.6 ± 0.2

¹Mice (n = 12/group) at the age of 6.5 months were subcutaneously injected once a week with the vehicle, hCDR1, or the scrambled (control) peptide for 13 weeks.

²P-value for the effect of hCDR1 compared to the vehicle-treated group.

³P-value for the effect of hCDR1 compared to the scrambled peptide-treated group.

⁴Results are of sera from mice that were bled at the end of treatment. Dilution of sera 1 : 250.

⁵Proteinuria levels measured at the end of treatment.

⁶Immune complex deposits (ICD) were assessed at killing.

Antibodies and reagents

The following reagents were used in the study: allophycocyanin-conjugated anti-mouse/human CD45R (B220) (clone RA3-6B2), fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin D (IgD; clone 11–26c), (eBioscience, San Diego, CA), phycoerythrin-conjugated rat anti-IgM (clone 1B4B1) (SBA, Birmingham, AL), CD43-biotin, streptavidin-horseradish peroxidase, phycoerythrin-conjugated hamster anti-mouse Fas monoclonal antibody (Pharmingen, San Diego, CA), and recombinant murine IL-7 (Peprotech Inc., Rocky Hill, NJ).

Annexin V/propidium iodide (PI) staining

Cells were analysed using the Phosphatidyl Serine Detection Kit (IQ Products, Groningen, the Netherlands), according to the protocol supplied by the manufacturer.

TUNEL assay

Apoptosis, as manifested by fragmented DNA, was determined using the In Situ Death Detection Kit (Roche, Indianapolis, IN) based on terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technology, according to the protocol supplied by the manufacturer. Each sample was accompanied by a negative control, consisting of UTP labelled with fluorescein, and a positive control, consisting of DNAse I, grade I (Roche) that was added before the TUNEL staining. Cells were analysed by fluorescence-activated cell sorting (FACS), with forward and side scatter gates adjusted to include all cells and to exclude debris.

Flow cytometry analysis

Splenocytes and bone marrow cells (1 × 10⁶ cells) were incubated with the relevant antibodies according to the manufacturers’ instructions, and analysed by FACS (Becton Dickinson, Franklin Lakes, NY).

Real-time reverse transcriptase–polymerase chain reaction

Total RNA was isolated from bone marrow and splenic-derived B cells. RNA was then reverse-transcribed to prepare complementary DNA (cDNA) by using Moloney murine leukaemia virus reverse transcriptase (Promega, Madison, WI). The resulting cDNA was subjected to real-time reverse transcriptase–polymerase chain reaction (RT-PCR) according to the manufacturer’s instructions (Roche, Mannheim, Germany). Briefly, a 20-μl reaction volume contained 3 mm MgCl₂, LightCycler HotStart DNA SYBR Green I mix (Roche), specific primer pairs and 5 μl cDNA. The PCR conditions were as follows: 10 min at 95° followed by 35–50 cycles of 15 seconds at 95°, 15 seconds at 60° and 15 seconds at 72°. Primer sequences (forward and reverse, respectively) were as follows: β-actin, 5′-gtgacgttgacatccg-3′ and 5′-cagtaacagtccgcct-3′, Bcl-xL (5′-ggaccgcgtatcagag-3′ and 5′-gcattgttcccgtagag-3′), IFN-γ (5′-gaacgctacacactgc-3′ and 5′-ctggacctgtgggttg-3′), TGF-β (5′-gaacccccattgctgt-3′ and 5′-gccctgtattccgtct-3′), IL-10 (5′-acctcgtttgtacctct-3′ and 5′-caccatagcaaagggc-3′), IL-7 (5′-cgctggatgtattt-3′ and 5′-acgtaggagatgtg-3′). Levels of β-actin were used to normalize the expression levels of the other genes.

Statistical analysis

The unpaired alternate Welch and Student’s t-tests were used for evaluating the significant differences between groups. Values of P≤0·05 were considered significant.

Results

B-cell populations in the BM of BWF₁ mice and the effect of treatment with hCDR1

We analysed the changes in subsets of developing B cells (e.g. pre-pro-B cells, immature B cells and mature B cells) in the BM of SLE-afflicted BWF₁ mice as a result of disease development. To this end, BM cells of mice at the age of 2 months (free of disease), 9 months (SLE-afflicted) and healthy age-matched BALB/c mice were isolated and stained for the B-cell lineage markers (B220, CD43, IgM and IgD). Figure 1(a) demonstrates a representative staining of IgM and IgD on a gated B220⁺ CD43⁻ cell population. The percentage of mature B cells (R3 region, depicting cells expressing both IgM and IgD) was higher (16%) in old SLE-afflicted BWF₁ mice than in young BWF₁ (5%), or in old age-matched BALB/c (7%) mice. Figure 1(b) shows the mean results of staining mature B cells in three experiments. The percentage of mature B cells in the old BWF₁ mice (indicated as 100%) was significantly higher than in young, BWF₁ mice free of disease or in the old age-matched BALB/c (healthy) mice. Consequently, the population of mature B cells is up-regulated in SLE-afflicted mice.

Figure 1.

B-cell populations of the bone marrow (BM) in systemic lupus erythematosus (SLE)-prone mice and the effect of treatment with hCDR1. BM cells of BWF₁ mice at the age of 2 months (free of disease; n = 3), 9 months (SLE-afflicted; n = 3), and of healthy old age-matched BALB/c mice (n = 3), were isolated and stained for B220, CD43, immunoglobulin M (IgM) and IgD. (a) A representative (of three experiments) staining of IgM and IgD gated on B220⁺ CD43⁻ cell population (R1-pre/pro, R2-immature, and R3-mature B cells). (b) Mean percentage (± SD) of mature B cells relative to that in old SLE-afflicted mice (considered as 100%) in three experiments. (c) SLE-afflicted BWF₁ mice (6·5 months old; n = 8 to n = 12/group) were treated weekly with subcutaneous injections of hCDR1 (50 μg/mouse), the scrambled peptide (50 μg/mouse) or the vehicle. At the end of the experiment (after 13 injections), BM cells were harvested and stained for mature B cells. The mean percentage (± SD) of three experiments.

We demonstrated that the mature B cells constitute the major population of B cells that are affected in BWF₁ old mice, so we wanted to determine the effect of treatment with the tolerogenic peptide, hCDR1, on this population. To this end, BWF₁ mice at the age of 6·5 months were treated with weekly injections (50 μg/mouse) of hCDR1, the control scrambled peptide (50 μg/mouse) or the vehicle. At the end of the experiment (after 13 injections), BM cells were harvested from all mice and stained for the presence of mature B cells. Figure 1(c) presents the mean (three experiments) percentage of mature B-cell staining in the BM. Treatment with hCDR1 significantly down-regulated the percentage of mature B cells in the BM compared with BM-derived mature B cells of either vehicle-treated or scrambled (control) peptide-treated mice. A similar effect of hCDR1 was recently reported by us for mature B cells in the spleen.²³

hCDR1 down-regulates the expression of IFN-γ and IL-10 and up-regulates the expression of TGF-β by BM cells

We reported previously that hCDR1 inhibited the production of the pathogenic cytokines and up-regulated TGF-β in spleen-derived cells.⁶ We therefore determined the expression of cytokines in BM cells. To this end, messenger RNA (mRNA) was prepared from bone marrow cells of BWF₁ mice following 13 weeks of treatment with the vehicle, hCDR1 or the scrambled peptide and analysed for the expression of IFN-γ, IL-10 and TGF-β genes by real-time RT-PCR. Figure 2 presents the mean gene expression of IFN-γ, IL-10 and TGF-β (of two to four experiments), relative to the expression in vehicle-treated mice (considered as 100%). The hCDR1 significantly inhibited the gene expression of IFN-γ and IL-10 in BM cells. In addition, the gene expression levels of the immunosuppressive cytokine TGF-β were up-regulated by hCDR1.

Figure 2.

Treatment with hCDR1 down-regulates interferon-γ (IFN-γ) and interleukin-10 (IL-10) gene expression and up-regulates transforming growth factor-β (TGF-β) gene expression by bone marrow (BM) cells of systemic lupus erythematosus (SLE)-afflicted BWF₁ mice. At the end of the 13-week treatment experiments, messenger RNA was prepared from BM cells of the different treatment groups of BWF₁ mice (n = 8 to n = 12/group) and analysed for the gene expression levels of IFN-γ, IL-10 and TGF-β by real-time reverse transcription–polymerase chain reaction. The means (± SD) of gene expression of IFN-γ (two experiments), IL-10 (two experiments) and TGF-β (four experiments) are shown, relative to the expression determined for the vehicle-treated group (considered as 100%).

Treatment with hCDR1 up-regulates apoptosis of mature B cells in both BM and spleen

Apoptosis was reported to play an important role in lupus.^7,8 Therefore, it was of interest to measure the rate of apoptosis in mature B cells of the BM compared with that in the spleens of mice with SLE and to determine the effect of treatment with hCDR1 on apoptosis in mature B cells. For this reason, BM and spleen cells of BWF₁ mice were harvested following 13 weeks of treatment. The cells were stained for B220, Annexin V, PI, TUNEL and Fas. Messenger RNA was prepared from part of the bone marrow and from B cells that were isolated from the spleens and was analysed for the expression of the anti-apoptotic Bcl-xL gene by real time RT-PCR. Figure 3(a) presents the mean change (two experiments) of staining (relative to vehicle-treated mice) of BM and spleen-derived B cells for Annexin-V, TUNEL and Fas. Treatment with hCDR1 significantly up-regulated the rate of apoptosis of the mature B cells in both organ compartments. To further determine the levels of expression of the survival gene, Bcl-xL, in B cells of mice from the different treatment groups, mRNA was prepared from BM and B cells isolated from spleens. Figure 3(b) shows that the expression of Bcl-xL in BM and in spleen-derived B cells from SLE-afflicted mice treated with hCDR1 was significantly down-regulated. The scrambled control peptide had similar effects; however, the ability of hCDR1 to down-regulate Bcl-xL in the B cells was more substantial.

Figure 3.

Treatment with hCDR1 up-regulates apoptosis of mature B cells in the bone marrow (BM) and spleens of systemic lupus erythematosus (SLE)-afflicted BWF₁ mice. BM and spleens were harvested from mice (n = 8 to n = 12/group) at the end of treatment experiments and stained for propidium iodide (PI) to exclude dead cells. PI⁻ cells were further stained for B220 and Annexin V. (a) Mean (two experiments) change of staining (%) of BM cells and spleen cells for PI⁻ B220⁺ Annexin V⁺, PI⁻ B220⁺ TUNEL⁺, PI⁻ B220⁺ Fas⁺, relative to staining of cells of vehicle-treated mice. (b) Messenger RNA was prepared from part of the BM and from B cells that were isolated from the spleens. It was analysed for gene expression levels of Bcl-xL, by real-time reverse transcription–polymerase chain reaction. Mean change (three experiments) of Bcl-xL gene expression in BM and spleens relative to the expression in the vehicle-treated cells.

Suppression of IL-7 by hCDR1 up-regulates apoptosis of B cells in the BM

Interleukin-7 is reported to be essential for B-cell function and survival in the BM;²⁰ for this reason, we determined the levels of IL-7 in mice with SLE and following treatment with hCDR1. Figure 4(a) presents the mean (two experiments) gene expression of IL-7 by BM cells relative to cells of vehicle-treated mice (considered as 100%). The hCDR1 significantly down-regulated the expression of IL-7 as compared with the control peptide, the vehicle-treated, or the untreated SLE-afflicted mice. It was of further interest to determine whether the up-regulated apoptosis of B cells in the BM (Fig. 3) could be related to the lower IL-7 levels observed after treatment with hCDR1. Therefore, we incubated BM cells of hCDR1-treated mice with recombinant IL-7 (10 ng/ml) for 24 hr. The mRNA was prepared from the cells of the different groups and tested for the gene expression of Bcl-xL by real-time RT-PCR. Figure 4(b) demonstrates the mean gene expression levels of Bcl-xL relative to mRNA of vehicle-treated mice (considered as 100%). As shown, the addition of IL-7 to hCDR1-treated cells abrogated the inhibitory effect of hCDR1 on the expression of Bcl-xL. To further test the effect of IL-7 on apoptosis, we incubated BM cells from 10-month-old, SLE-afflicted BWF₁ mice with hCDR1 (25 μg/ml) in the presence or absence of recombinant (r)IL-7 (10 ng/ml) for 24 hr. Cells were then tripled-stained for Annexin V, PI and B220 and analysed by FACS. Figure 4(c) shows the mean results of four experiments, in which the change in apoptotic B cells (defined as Annexin V⁺ PI⁻ B220⁺) was calculated relative to that determined for BM-derived cells of diseased mice incubated in medium alone. It can be seen that the presence of hCDR1 in the medium resulted in enhanced apoptosis of BM-derived B cells, whereas the addition of rIL-7 significantly abrogated this effect.

Figure 4.

hCDR1 suppresses the expression of interleukin-7 (IL-7), leading to apoptosis. At the end of the treatment experiments with hCDR1 (after 13 injections) the following experiments were performed. (a) Messenger RNA was prepared from bone marrow (BM) cells of the experimental mice (n = 8 to n = 12/group) and was analysed for IL-7 gene expression. Mean (two experiments) gene expression of IL-7 by BM cells relative to that in cells of vehicle-treated mice (considered as 100%). (b) BM cells of hCDR1-treated mice were incubated with recombinant IL-7 (10 ng/ml) for 24 hr. Messenger RNA was prepared from the cells and analysed for Bcl-xL gene expression by real-time reverse transcription–polymerase chain reaction. Results are expressed as mean (± SD) Bcl-xL gene expression relative to that in vehicle-treated mice (considered as 100%). (c) BM cells of untreated (10 months) systemic lupus erythematosus (SLE)-afflicted BWF₁ mice (n = 3) were incubated with hCDR1 (25 μg/ml) in the presence or absence of recombinant IL-7 (10 ng/ml) for 24 hr, stained with propidium iodide (PI), Annexin-V and B220 and analysed by fluorescence-activated cell sorting. Mean results of four experiments.

Discussion

The main findings of the present report are that the beneficial effects of treatment with the tolerogenic peptide hCDR1 on the SLE-like disease of BWF₁ mice are associated with the down-regulation of the mature B-cell population in the BM. The hCDR1 reduced the numbers of mature B cells in the BM by inducing apoptosis, as was observed in mature B cells derived from the spleen. Furthermore, hCDR1 down-regulated the expression of the pathogenic cytokines IFN-γ and IL-10 and up-regulated the expression of the immunosuppressive cytokine TGF-β in the BM, as it did in the spleens of SLE-afflicted mice.^6,24,25 The down-regulation of IL-7, which is crucial for B-cell development in the BM, is one of the mechanisms by which hCDR1 decreased the survival of mature B cells.

Antigen-specific activation and differentiation of B cells occurs in vivo in germinal centres where naïve B cells are activated and mature into antibody-producing plasma cells, or, alternatively, become memory B cells.²⁶ Because SLE is characterized by the generation of large amounts of autoantibodies directed against a variety of self-antigens, the loss of B-cell tolerance clearly plays a key role in the disease. As demonstrated in animal models, B-cell tolerance is established at multiple checkpoints throughout B-cell development, both in the BM and in the periphery and is enforced largely by negative selection, although positive selection and sequestration into the marginal zone compartments has also been described.²⁷

In SLE patients, peripheral checkpoints were found to be defective, with a further increase in autoreactivity within the mature naïve compartment.^28,29 In this study, we demonstrated that the BM of SLE-afflicted BWF₁ mice possessed an enlarged fraction of mature B cells, as compared with disease-free controls (Fig. 1a,b). However, treatment with the tolerogenic peptide, hCDR1, reversed the expansion of mature B cells (Fig. 1c). Hence, suppression of B-cell maturation by hCDR1 occurs already in the BM. In accordance with this, we demonstrated that treatment of SLE-afflicted BWF₁ mice with hCDR1 down-regulated the maturation of spleen-derived B cells (T1, T2 and marginal zone B cells), an effect that was associated with down-regulation of B-cell activating factor (BAFF).²³

The immunomodulatory effects of hCDR1 on the cytokine profile of the BM cells as well as on the apoptotic process may explain the reduced number of mature B cells observed in the treated mice. We found that the production of IFN-γ and IL-10, two pathogenic cytokines in SLE, was down-regulated in the BM of the hCDR1-treated mice. These effects could contribute to reducing mature B cells because both IFN-γ and IL-10 were previously found to promote differentiation and maturation of B cells.^30,31

Apoptosis plays an important role in maintaining homeostasis and quality control in the renewing cell systems.^7,8 The consequence of genetic dysregulation of B-cell development might be the survival of aberrant B cells. In accordance, it was shown that peripheral B cells taken from SLE patients contain populations that spontaneously produce immunoglobulins and that they can mature into antibody-secreting cells when cultured in vitro even in the absence of B-cell differentiation activators.³² We demonstrated here that the expanded mature B-cell population in the BM of SLE-afflicted mice is decreased following treatment with hCDR1, at least because of increased apoptosis of the cells, in which the expression of the death receptor, Fas, was up-regulated, and the expression of the survival gene, Bcl-xL, was down-regulated (Fig. 3). Similarly, we previously reported that hCDR1 differentially affected the expression of Bcl-xL in spleen-derived T and B cells, in a manner that up-regulated Bcl-xL in T cells and down-regulated it in B cells.³³ Mechanistically, the repression of Bcl-xL in B cells was, at least partially, the result of the ability of hCDR1 to down-regulate the production of IL-7. Indeed, IL-7, which is crucial for B-cell development,²⁰ is produced in vivo by a majority of stromal cells in the BM.³⁴ The ability of hCDR1 to suppress IL-7 production further suggests that this tolerogenic peptide affects the BM microenvironment, which in SLE patients was reported to function abnormally.^35,36 Because the expression of TGF-β in the BM of hCDR1-treated, SLE-afflicted mice was up-regulated, and because TGF-β was reported to down-regulate stromal IL-7 secretion,³⁷ it is likely that the reduced BM production of IL-7 in response to hCDR1 results from the up-regulation of TGF-β. Taken together, these results suggest that the combination of reduced levels of IL-7 and increased apoptosis of mature B cells following treatment with hCDR1 contribute to the amelioration of SLE-associated disease manifestations.

Glossary

Abbreviations:

BM

bone marrow

BWF₁

(New Zealand Black × New Zealand White)F₁

CDR

complementarity-determining region

FACS

fluorescence-activated cell sorting

IFN

interferon

Ig

immunoglobulin

IL

interleukin

NZB

New Zealand Black

NZW

New Zealand White

PI

propidium iodide

r

recombinant

RT-PCR

reverse transcription–polymerase chain reaction

SLE

systemic lupus erythematosus

TGF

transforming growth factor

TUNEL

terminal deoxynucleotidyl transferase dUTP nick end labelling

Disclosures

The authors have no financial conflict of interest.